Progress in Systemic Lupus 2007 Nova Medical Books

PROGRESS IN SYSTEMIC LUPUS
ERYTHEMATOSUS RESEARCH
PROGRESS IN SYSTEMIC LUPUS
ERYTHEMATOSUS RESEARCH
TOMAS I. SEWARD
EDITOR
Nova Biomedical Books

New York
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Progress in systemic lupus erythematosus research / TomasI. Seward (editor).
p. ; cm.
Includes bibliographical references and index.
ISBN-13: 978-1-60692-743-4
1. Systemic lupus erythematosus. I. Seward, tpmas I.
[DNLM: 1. Lupus Erythematosus, Systemic. WR 152 P964 2008]
RC924.d.L85P76 2008
616.7’7--dc22
2007030893
Published by Nova Science Publishers, Inc. New York

CONTENTS
Preface vii
Expert Commentary Therapeutic Plasmapheresis in the Treatment of

Complicated Systemic Lupus Erythematosus
Claudia Stefanutti, Fabio Mazza and Valeria Riccieri
Expert Commentary Pregnancy, a Challenge in Patients with

Systemic Lupus Erythematosus 9
Javier A. Cavallasca and Maria del Rosario Maliandi
Short Communications Influence of Exercise on the Peripheral Circulation in

Patients with Systemic Lupus Erythematosus and
Systemic Sclerosis 17
Etsuko Maeshima and Kanako Furukawa
Chapter 1 New Frontiers in the Research of Cardiovascular

Disease Associated with Systemic Lupus Erythematosus 29
Laura Gonzalez-Lopez, J. I. Gamez-Nava and Arnulfo Nava
Chapter 2 Comorbidity in Systemic Lupus Erythematosus: Aspects

of Cardiovascular Disease, Osteoporosis and Infections 77
Irene E. M. Bultink, Ben A. C. Dijkmans and
Alexandre E. Voskuyl
Chapter 3 Severe Tissue Trauma Triggers Lupus

Autoimmune Disease 105
Khairul Anam, Mihret Amare, Shruti Naik,
Kathleen A. Szabo and Thomas A. Davis
Chapter 4 Immunotherapy with Ig-Derived Peptides in SLE:

Current Status and Directions 131
Antonio La Cava and Bevra H. Hahn
vi Contents
Chapter 5 Autoantibodies as Prognostic or Diagnostic Markers of

Psychiatric Manifestations in SLE 155
Paola Margutti, Federica Delunardo, Tania Colasanti,
Ettore Piro and Elena Ortona
Chapter 6 High-Dose Immunosuppression with Autologous

Stem Cell Transplantation in Severe Refractory
Systemic Lupus Erythematosus 177
Igor A. Lisukov, Vera V. Sergeevicheva,
Svetlana A. Sizikova, Alexander D. Kulagin,
Irina V. Kruchkova, Andrey V. Gilevich, Alexey E. Sizikov,
Ludmila P. Konenkova, Elena R. Chernykh, Olga Y. Leplina
and Vladimir A. Kozlov
Chapter 7 MR Spectroscopy, Diffusion and Diffusion Tensor

Imaging in Systemic Lupus Erythematosus 193
Pia C. Sundgren, Patricia Cagnoli and William McCune
Chapter 8 Personalized Medicine for Systemic Lupus

Erythematosus: A New Challenge for the Near Future 211
Ana M. Bertoli and Luis M. Vilá
Chapter 9 Therapeutic Potential of HMG-CoA Reductase

Inhibitors (Statins) in Systemic Lupus Erythematosus 227
Przemyslaw J. Kotyla and Bogna Sliwinska-Kotyla
Index 245
PREFACE
This new book is devoted to leading-edge research developments in lupus which is a

condition of chronic inflammation caused by an autoimmune disease. Autoimmune diseases
are illnesses that occur when the body's tissues are attacked by its own immune system. The
immune system is a complex system within the body that is designed to fight infectious
agents, for example, bacteria, and other foreign invaders. One of the mechanisms that the
immune system uses to fight infections is the production of antibodies. Patients with lupus
produce abnormal antibodies in their blood that target tissues within their own body rather
than foreign infectious agents. Because the antibodies and accompanying cells of
inflammation can involve tissues anywhere in the body, lupus has the potential to affect a
variety of areas of the body. Sometimes lupus can cause disease of the skin, heart, lungs,
kidneys, joints, and/or nervous system. When only the skin is involved, the condition is
called discoid lupus. When internal organs are involved, the condition is called systemic
lupus erythematosus (SLE).
Chapter 1 - Cardiovascular disease is a major cause of mortality in patients with systemic
lupus erythematosus (SLE). Around 52% of the autopsies performed in SLE in our centre
have shown cardiac involvement and most of these disorders were not diagnosed pre-mortem
[1].
Current topics for research are accelerated atherosclerosis, pulmonary hypertension,
development of valvular disease and new other forms of cardiac involvement. Most of the
patients deceased by cardiovascular disease have an accelerated atherosclerosis. Some
authors reported a substantial increase in risk of fatal vascular event in women with SLE
compared with matched controls. New methods such as electron-beam computed tomography
have demonstrated a highly prevalence of coronary disorder even in asymptomatic patients.
Additionally, high resolution carotid ultrasonography has shown a high prevalence of carotid
lesions. Both cellular and molecular mechanisms of the accelerated atherosclerosis are
complex and not entirely explained by the traditional cardiovascular risk factors, therefore,
the research targets on nontraditional factors.
Pulmonary hypertension is relevant by its morbidity and mortality. Previously considered
as rare entity; new non-invasive studies have detected patients with earlier involvement; our
group have reported a prevalence of 18% in a series of 204 patients assessed by Doppler
echocardiography. A follow-up study in our cohort observed an increase of 4 times greater of
viii Tomas I. Seward
risk for cardiac failure in patients with asymptomatic pulmonary pressure >40 mmHg.
Strategies for the treatment of pulmonary hypertension include immunosuppressive therapy,
prostanoids, phosphodiesterase inhibitors or endothelin receptor antagonists. Most of the
current information related with the response to these treatments proceeds from short-term
studies with a wide variability in the outcome measures making necessary additional research
in this area.
There is also a renovate interest in the valvular disease, recent studies show association
between antiphospholipid antibodies and mitral valve nodules or mitral regurgitation, the
potential effects of these antibodies on the endothelial activation require to be evaluated.
Cardiac dysfunction is another important area for research; several studies have shown a
high prevalence of left ventricular diastolic dysfunction. In the authors series this
manifestation is present in around 63%. Nevertheless, to date no follow-up studies have been
done to evaluate the importance of diastolic dysfunction on the morbidity or mortality.
In summary cardiovascular disease in systemic lupus represents a very exciting area for
research being necessary to increase the number of long-term prospective cohorts and well-
designed controlled trials in order to improve the clinical care of the patients with this
involvement.
Chapter 2 - Over the last decades, the survival of patients with systemic lupus
erythematosus (SLE) has improved dramatically. Having improved treatment for active lupus
disease, the challenge is now to understand and prevent the long-term complications of the
disease, which may be due to the disease itself or the therapies used. To date, long-term
complications of SLE are now considered to be important, including cardiovascular disease,
osteoporosis and infections.
Cardiovascular disease in patients with SLE, including coronary artery disease, ischemic
cerebrovascular disease, and peripheral vascular disease, is the result of premature
atherosclerosis. Besides the traditional risk factors (like hypertension, hypercholesterolaemia
and smoking), renal insufficiency, raised homocysteine levels, and the presence of anti-
phospholipid, antibodies have been recognized as additional risk factors for cardiovascular
disease in SLE. Recent studies have demonstrated that the nitric oxide pathway and its
endogenous inhibitor asymmetric dimethylarginine may also be involved in the pathogenesis
of cardiovascular organ damage in SLE. The metabolic syndrome and insulin resistance in
SLE patients are current topics of research in this field.
Several studies have demonstrated a high prevalence of low bone mineral density in
patients with SLE, especially in females. In the last few years, more attention is paid to
osteoporotic fractures, one of the items of the organ damage index for SLE, and likely the
most preventable form of musculoskeletal organ damage in SLE patients. Recent studies have
demonstrated an increased frequency of symptomatic vertebral and nonvertebral fractures in
patients with SLE. Moreover, a high prevalence of mostly asymptomatic vertebral fractures
in patients with SLE was detected. These vertebral fractures were associated with previous
use of intravenous methylprednisolone. The importance of identifying vertebral fractures in
SLE patients is illustrated by the observed association between prevalent vertebral fractures
and reduced quality of life as well as an increased risk of future vertebral and nonvertebral
fractures in the general population.
Preface ix
Infection imposes a serious burden on patients with SLE. In case series, infectious
complications were found in 25% to 45% of SLE patients, and infection as primary cause of
death has been demonstrated in up to 50% of SLE patients. Defects of immune defence and
treatment with corticosteroids and other immunosuppressive agents are supposed to play a
role in the pathogenesis of infections in SLE. Recently, research has focused on the role of
the lectin pathway of complement activation in the occurrence of infections in SLE.
In this review the results of recent studies on cardiovascular disease, osteoporosis and
infectious complications in SLE will be discussed.
Chapter 3 - Systemic lupus erythematosus (SLE) is a chronic, complex autoimmune
disease characterized by high levels of non-organ-specific, self-reactive antibody production
leading to immune complex formation. The etiology of this autoimmune disease remains
elusive. The disease results in multiple health problems including increased infection, renal
and skin disorders, neurological complications, osteoporosis, rheumatoid arthritis,
osteoarthritis, and fibromylagias. Tissue damage associated with severe injury can result in
profound immune dysfunction that involves suppressive cell types and a cascade of
inflammatory and tissue reparative mediators. Mice from MRL strains have been used as
models to study SLE pathogenesis. Wild type MRL/MpJ mice exhibit SLE autoimmune
disorders but symptoms are manifested much later in life (70-90 weeks) compared to
MRL/MpJ-Faslpr mice (17-22 weeks) which possess a lymphoproliferative mutation. Based
on our preliminary findings, the authors hypothesize that following traumatic tissue injury,
the activation of specific cell types, cytokines and other mediators involved in wound healing
and repair processes may be critical in triggering lupus-like disease. The authors investigated
the role of a severe (15% total body surface area) full-thickness cutaneous burn on the early
onset of lupus-like autoimmune disease in young adult, lupus prone wild type MRL/MpJ
mice, and control BALB/c mice. MRL/MpJ mice develop autoimmune disease 4-15 weeks
post injury, manifested by skin lesions, vasculitis, epidermal ulcers, cellular infiltration,
splenomegaly, lymphadenopathy, hypergammaglobulinemia, elevated autoantibodies, and
renal pathologies including proteinuria, glomerulonephritis, and immune complex deposition.
Post-injury survival rate of injured MRL/MpJ mice is significantly reduced due to
autoimmune related complications. In contrast, neither uninjured MRL/MpJ mice nor burned
BALB/c displayed signs of autoimmunity or premature death. The authors analyzed mRNA
expression of numerous cytokines at the wound margins in the skin at days 1-7 post injury.
The authors results do not reveal an early or clear Th1 or Th2 cytokine expression pattern
during the early wound repair process but demonstrate a correlation between the pathogenic
effects of dysregulated interleukin-beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor
alpha (TNF-α) and prostaglandin-E2 (PGE2) synthesis and the early onset of lupus-like
disease. Interestingly, the authors found that normal skin isografts transplanted onto the
dorsum of MRL/MpJ mice with healed wounds (30- 40 days post burn injury) are rejected
within 7 days after transplantation. In contrast, skin grafts transplanted onto uninjured age-
matched MRL/MpJ achieved long-term survival. Collectively, these findings suggest that
traumatic injury exacerbates inflammatory skin disease and the early onset of severe multi-
organ SLE pathogenesis.
Chapter 4 - Understanding the mechanisms that control autoimmune reactivity is an
essential step to improve management of autoimmune diseases including systemic lupus
x Tomas I. Seward
erythematosus (SLE). In SLE, the interaction between hyperactive T cells and B cells causes
dysregulated production of autoantibodies that by themselves or in immune complexes fix to
tissue and cause organ damage. The immune cell subsets that take part in this process have
come under intense scrutiny in the past few years, and new information has been acquired on
how they interact to induce and/or modulate disease. This information has also led to the
development of autoantibody-derived peptide therapies that can effectively influence murine
SLE. This chapter describes the rationale, current experimental evidence, preclinical models,
and future directions for the use of autoantigen- and Ig-derived peptides in SLE. Considering
that autoantibodies in lupus patients have amino acid sequences similar to those of murine
antibodies, and at similar locations, it is likely that some of those strategies will be potentially
useful in the therapy of human SLE.
Chapter 5 - In the course of Systemic Lupus Erythematosus (SLE), a variety of
psychiatric disturbances are reported, including mood disorders (depressive symptoms),
psychosis and anxiety. The reported prevalence of psychiatric disorders in SLE varies widely,
ranging from 17% to 75%, but the diagnosis of these syndromes is difficult and depends on
the exclusion of complications due to an iatrogenic effect of drugs, to metabolic
abnormalities or to infections. Moreover, the diagnosis requires a careful psychiatric
evaluation to exclude merely reactive psychological disturbances.
It has been suggested that several autoantibody specificities play a role in the
pathogenesis of neuropsychiatric SLE. Potential pathogenic relevance has been attributed to,
among others, antineuronal, antiphospholipid, antiganglioside, anti-DNA, anti-ribosomal P
protein and anti-endothelial cell antibodies. However, particularly regarding psychiatric
syndromes, conflicting results have been reported on the association between serum
autoantibodies and symptoms.
The diagnostic and/or prognostic role of autoantibodies associated to psychiatric
disorders in SLE is discussed.
Chapter 6 - Carry out a prolonged studies to elucidate the role of high dose
immunosupression therapy (HDIST) with autologous hematopoietic stem cells
transplantation (SCT) in the treatment of severe and refractory autoimmune disease. In this
study the authors analyzed single center experience of HDIST followed by SCT for refractory
SLE.
The 13 patients with SLE, who had disease progression despite previously treatment with
pulse Cy intravenous, prednisolone (standard doses and pulse therapy), oral Cy, azathioprine
or metotrexate, were enrolled in the clinic of our Institution from 1998 to 2007. All patients
were seriously ill, with SLE disease activity indices (SLEDAI) of 6-30, including cases with
central nervous system lupus, lung vasculitis, carditis, nephritis with nephrotic syndrome and
cytopenia. Autologous haemopoietic stem cells were collected from bone marrow (n=4) or
mobilized from peripheral blood with Cy (3g/m2/once) and granulocyte colony-stimulating
factor (G-CSF) (n=9). Pretransplant conditioning regimens included BEAM ± ATG (n=2),
Melphalan 140 mg/m2 + Etoposid 1600 mg/m2 (n=2), Cy 200 mg/kg ± ATG (n=2) and
Melphalan 120 mg/m2 (n=1), Cy 140 mg/kg (n=6). Transplanted CD34+ MNC were more
then 2x106/kg. Median time to an absolute neutrophil count (ANC) greater than 0,5x109 /l
and platelet count greater than 50x109/l was 11 and 15 days, respectively. Three patients died
on days 11, 22 and 63 due to transplant-related complications. All of the patients who died
Preface xi
due to transplant-related complications had long history of the corticosteroids treatment,

multiple and severe episodes of infections pre-SCT and received more then one anticytoststic
drugs or ATG as conditioning regimen. All of the alive transplanted patients, who recovered
hematopoiesis, showed improvement in disease activity: 8 (CR) and 2 (PR). The median
follow up is now 14 month. One patient died due to lung vasculitis progression after 8 years
after SCT. We conclude that HDIST followed by SCT for refractory SLE is feasible,
effective and achievement of prolonged, corticosteroid-free remissions is a reality.
Chapter 7 - Although clinical assessment is the cornerstone of SLE and NPSLE
diagnosis; it can be difficult to make and is frequently presumptive. MR findings play an
important role in supporting a clinical diagnosis and findings include volume loss, focal
white matter hyperintensity, diffusion abnormalities due to ischemic injuries, evidence of
prior hemorrhage or infarct, and meningeal enhancement. Nevertheless, several previous
studies have shown that conventional pre-and post-contrast enhanced brain MR appear
normal in approximately one third of both symptomatic and asymptomatic NPSLE patients
and even more frequently in SLE patients. It has been suggested that other modalities such as
MR spectroscopy (MRS), diffusion weighted imaging (DWI) and diffusion tensor imaging
(DTI) could be additional tools in the evaluation and monitoring of SLE and NPSLE patients.
This chapter will describe these new MRI techniques, recent research results and trends, and
discuss if these techniques may add any valuable information that may further elucidate the
pathogenesis of NPSLE. These techniques may possibly aid in diagnosing and differentiating
NPSLE from other diseases with similar acute clinical symptoms as well as in monitoring
disease progression.
Chapter 8 - Systemic lupus erythematosus (SLE) is a chronic disease with higher
morbidity and mortality when compared to the general population. Disease outcome varies
among patients, in part, because they do not respond the same to a given treatment modality.
There is, therefore, a clear need for drug treatments to be selected according to the
characteristics of an individual patient, in order to improve efficacy and reduce the number
and severity of adverse drug reactions. Personalized medicine means the prescription of
specific therapeutics best suited for an individual. This novel treatment approach should be
regarded as an ongoing progression of healthcare which is advancing with genomic tools.
Although still a translational challenge into the practice of rheumatologists, data for
immunosuppressive drugs (mainly coming from transplant patients) is now available and
waiting to be introduced in the field of autoimmune diseases such as SLE. Examples of that
are genetic studies of drug metabolizing systems that can affect the efficacy, pharmacokinetic
and tolerability profile of drugs such as azathioprine, cyclosporin A, mycophenolate mofetil,
cyclophosphamide and glucocorticoids, among others. Although quite attractive,
implementation of pharmacogenomics may not be so simple. Ideally the genetic trait should
be accomplished with a well characterized patient from the socio-demographic, cultural and
ethnic background as well as co-morbidities and drug exposure history, variables known as
being modifier factors for drug efficacy and tolerability. Major efforts should also be made to
systematically evaluate the patient; in this sense, validated outcome measures of disease
activity, accumulated damage and health-related quality of life could be very valuable to
quantify drug response. Worldwide integrated genomic information should also be an
unequivocal subject for the future. Although quite attractive, personalized medicine is not
xii Tomas I. Seward
without some drawbacks; cost, infrastructure and worldwide networking are not minor issues
to overcome. Finally, personalized medicine must be strongly interpreted from the ethics
perspective and regulated by the law.
Chapter 9 - Systemic lupus erythematosus is a connective tissue disorder of unknown
origin and a relatively bad prognosis. In the last fifty years the big progress in the treatment
of the condition has been made, resulting in the improvement of prognosis and prolongation
of the life span. Nowadays we are prepare enough to manage the acute form of the disease,
treat acute vasculitis lupus nephritis, decrease inflammation. As the result of extension of the
life span the lupus face has been changed. Today a premature atherosclerosis is considered
the main risk factor for increased mortality among lupus patients. The premature
development of atherosclerosis is a well known clinical phenomena since the work of
Urowitz and colleagues who described the term bimodal peak of mortality and attributed the
second peak of mortality to premature development of atherosclerosis and its fatal
complications. For almost two decades the mechanism of the atherosclerosis in lupus had not
been explained satisfactory. In the early nineties of the last century Ross suggested that
atherosclerotic process is an inflammation in its nature. Now atherosclerosis is considered a
chronic inflammatory condition of the vessel wall. In the recent years many immunological
mechanisms in the development and progression of the atherosclerotic lesions have been
recognized. Since the atherosclerosis and atherosclerosis in lupus patients share the same
pattern of inflammation it would be reasonable to treat the patients with a drug that cools the
inflammation on one side and halts progression of atherosclerosis on the other.
Statins, inhibitors of HMG-CoA reductase, a key enzyme in the pathway of cholesterol
synthesis have been commonly used in the therapy of ischemic coronary events and the
control of hypercholesterolemia. In last years evidence was accumulated suggesting that
action of statins go far beyond lipid lowering properties and may affect function of immune
system cells. An array of anti-inflammatory effects has now been identified, including
reduced production of pro-inflammatory cytokines, chemokines and adhesion molecules;
reduced expression of inducible major histocompatibility complex (MHC) class II molecules
by antigen presenting cells (APC); lowering of C-reactive protein levels and reduced
production of reactive oxygen species. The effect that is separated from lipid lowering
properties is sometimes called pleiotropic action of statins. The beneficial effect is often seen
before the normalization of lipid profile suggesting that action of statins comprises at least
two mechanism – cholesterol synthesis inhibition and direct effect on arterial wall and
endothelium.
Such pleiotropic effect may benefit patients with autoimmune rheumatic diseases,
including lupus.
The repertoire of drugs used in the treatment of systemic autoimmune disorders is
characterized by unfavorable influence on lipid profile in patients treated, thus may
perpetuate the development of atherosclerosis evoked by the inflammatory process itself.
In this regard statins may act on two main fields; being a concomitant therapy for
autoimmune disorders and normalize the lipid profile abnormalities in patients with
connective tissue disorders.
This overview assesses the evidence for using statins in patients with systemic lupus
erythematosus.
In: Progress in Systemic Lupus Erythematosus Research ISBN 978-1-60021-861-3
Editor: Tomas I. Seward, pp. 1-8 © 2007 Nova Science Publishers, Inc.
Expert Commentary
THERAPEUTIC PLASMAPHERESIS IN THE

TREATMENT OF COMPLICATED SYSTEMIC
LUPUS ERYTHEMATOSUS
Claudia Stefanutti∗, Fabio Mazza and Valeria Riccieri

Department of Clinical and Medical Therapy – Plasmapheresis Unit and
Rheumatology Laboratory – University “La Sapienza”of Rome
‘Umberto I‘ Hospital. Rome, Italy
ABSTRACT
Systemic Lupus Erythematosus (SLE), can be complicated by nephritis and various
forms of vasculitis. The treatment is aimed at reducing the autoimmune reaction,
responsible for vascular damage, and is usually done with immunosuppressive agents.
The therapeutic approach for immune complex-mediated vasculitis, as with most
systemic autoimmune diseases, is aimed at reducing the pathological immune complexes
and autoantibodies. However, conventional drug treatment is not always successful and
has a high side-effects potential, especially during long-term therapy. It is also well-
known that is not always effective to avoid the development of complications. Such
severe cases need definitely of innovative therapeutic approaches, because of their poor
prognosis. Indications for the use of therapeutic plasmapheresis in SLE have undergone
several modifications since the first treatment in 1974. Once thought to be a promising
treatment for selected aspects of SLE, conventional apheresis has been limited as a co-
therapeutic adjunct in managing the disorder. Conventional plasmapheresis techniques
such as plasma-exchange have considerable disadvantages. The non-selective removal of
plasma constituents and the necessary substitution with foreign plasma or human albumin
∗
Corresponding Author: Stefanutti Claudia, M.D., Ph.D., Professor of Internal Medicine. Specialties: Emergency
Surgery; Liver and Metabolic Diseases. Head of Plasmapheresis Unit. Clinical and Medical Therapy
Department. University of Rome “La Sapienza”. Policlinico “Umberto I”. Viale del Policlinico 155, 00161
Rome. Italy (EU). Tel. +390649970578 ; Fax. +39064440290 ; +390649970141 ; Email: [email protected];
[email protected]
2 Claudia Stefanutti, Fabio Mazza and Valeria Riccieri
is associated with a certain range of possible side-effects (anaphylactoid reactions,

infections). More recently, a new technique called Immunoadsorption Apheresis (IA)
was introduced. IA is different from traditional plasmapheresis in that the plasma is
filtered through dextran sulphate membranes and then reinfused. A clinical evaluation of
the effect of conventional plasmapheresis and IA, with the additional pharmacological
therapy in the management of patients affected by SLE, who are non responsive to drugs
was performed in our Unit. The outcome of the above mentioned clinical study
confirmed the opportunity of a combined therapeutic approach using pharmacological
and non pharmacological treatment of Lupus Nephritis. The double association treatment
might be potentially extended to other complications of the disease, such as vasculitis. It
is reasonable to infere that the acceptable clinical result obtained, appears to be most
probably related to the use of a combined therapeutic strategy, rather than to one
treatment only. The above reported experience should be clearly confirmed by increasing
the clinical sample. A case-controlled, randomized study should be the target of further
investigations. On the other side prominent, conflicting ethical considerations, such as
the controversial role and complexity of the so called sham-apheresis, as ‘placebo’,
should be taken into account. The therapeutic potential of IA in combination with
immunosuppressive drugs should be furtherly investigated not only in SLE, but also in
other autoimmune diseases, with circulating pathological immune complexes.
Immunoadsorption treatment using dextran sulfate membranes showed to be effective in
the removal of anti-DNA antibodies, that are closely associated with pathogenicity in
SLE, improving progressively the renal function. In conclusion, it is argued that
syncronised therapy is useful at least in inducing a faster remission in patients with
proliferative lupus nephritis, non responsive to the immunosupressive agents alone.
INTRODUCTION
Autoimmune diseases are of considerable medical and economic importance since the
age- and sex- adjusted prevalance rate as in the first half of the 90’s was approximately 1.22
per 1000 (US white population), and can sometimes follow a complicated course. Within the
frame of autoimmune diseases, Systemic Lupus Erythematous (SLE) is recognized to be
undoubtedly a serious disease with involvement of several organ(s) and systems. The goal of
treatment is to relieve symptoms and protect organs by decreasing inflammation and/or the
level of autoimmune activity. Patients with mild symptoms may need only intermittent
courses of antiinflammatory treatment. Those with involvement of internal organ(s) may
require high doses of corticosteroids in combination with other drugs that suppress the
immune system. Nonsteroidal antiinflammatory drugs (NSAIDs) are generally used in
attempt of reducing inflammation and pain in muscles, joints, and other tissues.
Corticosteroids are more effective than NSAIDs in reducing inflammation and restoring
function when the disease is active. Hydroxychloroquine is an antimalarial agent that
demonstrated to be particularly effective for patients with fatigue, skin, and joint disease.
Immunosuppressive medications (methotrexate, azathioprine, cyclophosphamide) are used
for treating patients with more severe manifestations of SLE with involvement of internal
organ(s). Patients with SLE can develop different combinations of symptoms and organ
involvement developing severe complications such as nephritis, peripheral neuropathy, and
various form of vasculitis. The immunosuppressive pharmacological treatment is the first line
Therapeutic Plasmapheresis in the Treatment of Complicated Systemic Lupus… 3
intervention of the above mentioned complications. However, it is relatively common to

observe the failure of the pharmacological approach in terms of avoiding even severe
complications in SLE. Furthermore, particularly on long-term course, the conventional
treatment based on immunosuppressive drugs may be complicated by the occurrence of mild
to severe side effects. In patients with severe neurological or renal disease, therapeutic
plasmapheresis (plasma-exchange) (PE) was used to remove antibodies and other immune
substances from the blood to suppress immunity. Plasmapheresis has also been used to
remove cryoglobulins that can lead to vasculitis, after precipitation. It is widely recognized
that therapeutic apheresis might be a useful tool in the therapeutic management of certain
rheumatic diseases when their clinical evolution includes life-threatening complications not
responding to the conventional immunosuppressive treatment alone. Apheresis ‘per se’
cannot exclude the pharmacological treatment that should be associated in a combined
therapeutic strategy designed in order to allow the best possible approach to attain a
favourable clinical outcome. The dextran sulfate cellulose column immunoadsorption system
(Selesorb® Kaneka Corporation, Osaka, Japan) (IA), can selectively remove circulating anti-
DNA antibody, anti-cardiolipin antibody, and immune complex from the bloodstream. “In
vitro” experimental study on plasma of patients with SLE showed that Selesorb® has high
adsorbent capacity of anti-DNA antibody, anti-cardiolipin, and immune complexes, but not
for total proteins, albumin, immunoglobulins, and complement. Rather than the unselective
removal of plasma, adsorption procedures allows the selective or specific removal of
immunoglobulins and circulating immune complexes, which seems to be a more reasonable
and pathogen-removal oriented approach. In SLE in which antibodies and immune complexes
play significant roles, corticosteroids and other immunosuppressive drugs have been used to
suppress antibody production to favourably interfere with disease activity. A common clinical
experience confirmed that immunosuppressive drugs such as methotrexate azathioprine and
cyclophosphamide, and/or PE, may be beneficial in the above mentioned illness. However,
controlled studies to assess definitely their efficacy are not available. The primary advantage
of IA to plasmapheresis is that fluid substitution is not necessary and thus, the risk of allergic
reactions and potential transmission of infections is definitely reduced. However, the
potential disadvantage inherent in more specific elimination might be insufficient in
removing the pathological substances underlying the disease. In autoimmune diseases, the
columns used are mostly those with adsorbers with different binding capacities for
immunoglobulins and/or circulating immune complexes. In this pivotal study, we report our
clinical experience on patients with SLE, with an immunoadsorption apheresis technique,
where immunoglobulins and immune complexes were selectively removed from the plasma
by columns containing dextransulfate on cellulose (Selesorb®).
METHODS
Immunoadsorption Plasmapheresis: (Selesorb®)
Selesorb® (Kaneka Corp., Osaka, Japan) is a chemical affinity technique able at

removing anti-double-stranded DNA antibodies (anti-dsDNA), selectively from the blood of
patients with immune disorders and it is based on immunoadsorption with a dextran sulphate
cellulose column (figure 1, 2). The process is thought to work via cross-reactivity of anti-
dsDNA with dextran sulphate, which is negatively charged (polyanion). A Kaneka MA-01
(Kaneka Corp., Osaka, Japan) system was used as plasma separator. The total volume of
plasma to be processed through the columns was determined from the titer of anti-DNA
antibody, and body weight. A plasma volume of 45 ml/kg or from 2.5-3.5 l was processed,
and the treatment interval ranged from two to three days. The disease activity was also taken
into account as defined by the recurrence or remission of symptoms related to the
complication responsible for the extracorporeal treatment of the patient.
Patients
We treated a total of 6 patients, 5 females and 1 male, (mean age: 39.5 years) with SLE,
submitted to therapeutic plasmapheresis using Selesorb® immunoadsorption system (Table
1). Informed consent was obtained from all patients. The majority of the above mentioned
patients showed at least one severe complication related to the pre-existing immunological
disorder, not responding to the conventional medical approach. The mean blood volume
processed per session, was of approximately 3000 ml.
Results
Patients affected by SLE improved after treatment with IA. Vasculitis was completely
recovered in one patient although nephritis symptoms were substantially unchanged. The
complete remission of nephritis was observed in the other five patients (table 1). The mean
serum creatinine values in patients with SLE before and after IA apheresis are reported in
table 2. The progressive significant decline of proteinuria after 1-year treatment with
apheresis in one patient belonging to the above mentioned group is reported in figure 3. The
progressive decrease of blood nitrogen and creatinine levels in a second patient along with a
relatively significant faster reduction of proteinuria was obtained after no.12 sessions of IA
apheresis (figures 4, 5, 6). Plasma total protein and albumin levels, before and after
immunoadsorption apheresis, did not show statistically significant variations. There were no
short-term or long-term changes of clinical importance in electrolytes, hepatic and renal
function tests, albumin and coagulation parameters. In conclusion, 5 patients out 6 showed an
overall improvement. It is to be underlined, that the patients enrolled in this study were
definitely identified as to be very poor or none-responders to the pharmacological treatment,
prior to be submitted to the combined treatment with therapeutic apheresis.
DISCUSSION
Autoimmune diseases include a broad and heterogeneous group of diseases characterized

by inflammation and damage to the blood vessels, thought to be induced by an autoimmune
response. Vasculitis may occur alone or in combination with other diseases, and may be
confined to one organ or involve several organ systems. Individual disease range from the
mild to the severe. Symptoms vary widely, even within the same disease. Although there is
no definite cure for SLE, for the vast majority of people with the disease, effective treatment
can reduce the most painful symptoms, and decrease inflammation processes. Medications
often are prescribed for people with SLE, depending on which organ(s) are involved, and the
severity of involvement. Commonly prescribed medications include: nonsteroidal anti-
inflammatory drugs, such as aspirin and ibuprofen, usually recommended for muscle and
joint pain, and arthritis. Acetaminophen and corticosteroids, are used to reduce inflammation
and suppress activity of the immune system. Antimalarials are prescribed for skin and joint
symptoms. In general, it may take a long-time before these drugs demonstrate a beneficial
effect. Immunomodulating pharmacological agents suppress activity of the immune system
and biologic drugs block the production of specific antibodies, like those against DNA, or act
by suppressing the synthesis of antibodies through other mechanisms. We know from the
literature that Azathioprine reduced all cause mortality but did not improve renal outcomes.
Cyclophosphamide reduced the risk of increasing serum creatinine but not the mortality.
Induction with cyclophosphamide and steroids, as far as it is known, is probably the best
acceptable therapy, particularly because of the benefit found in terms of preservation of renal
function. Until now the therapeutic approach of cyclophosphamide-steroids is probably the
best pharmacological treatment to be associated to PE. Plasmapheresis, is also performed for
severe symptoms. Although there are no controlled studies, uncontrolled observations
suggest that this therapy may obtain a reduction in the plasma creatinine concentration in 55
to 87 percent of patients. Renal function can be improved or at least stabilized with the above
described therapeutic approach, although death from active vasculitis remains a major
clinical event. A reasonable plasmapheresis prescription is to exchange one plasma volume
three times weekly for two to three weeks. However, in 1992, a randomized, controlled trial
of Lewis EJ et al. comparing a standard-therapy regimen of prednisone and
cyclophosphamide with a regimen of standard therapy plus plasmapheresis in 86 patients
with severe lupus nephritis in 14 medical centers failed to improve the clinical outcome in
patients with SLE and severe nephritis, as compared with the standard regimen alone.
Photopheresis, gamma globulin, interferon, and plasmapheresis are all more recent treatment
options which mechanisms of action are not completely understood, but have been reported
as to be at least effective in slowing or interrupting the progression of the disease. They may
not be a cure, but have put patients in a state of remission, without the side effects of
depressing the immune system. Absolute recognized SLE indications of plasmapheresis
include hyperviscosity, cryoglobulinemia, pulmonary hemorrhage and Thrombotic
Thrombocytopenic Purpura. However, it may also be useful in cyclophosphamide-resistant,
serious, organ-threatening disease. Refinements in apheresis technology may expand the
indications. In most cases, our patients trated with IA obtained relief of symptoms and
complications has been obtained. However, the therapeutic potential of IA in combination
with immunosuppressive drugs shuld be investigated further not only in SLE, but also in
other autoimmune diseases, with circulating pathological immune complexes. In this study
immunoadsorption showed to be able at removing with high efficiency a great amount of
immunoglobulins from the bloodstream. Notwithstanding, it is to be underlined that
conventional PE removes antibodies and other plasmatic factors to about 50 to 75%, although
the evidence of therapeutic effects is mainly derived from case observation, uncontrolled case
series, and prospective controlled trials have been rarely performed even with conventional
plasmapheresis. Even other biochemical parameters related to inflammation and local organ
involvement such as creatinine and creatinkinase concentrations were reduced on average. As
far as the role of IA in the treatment of autoimmune diseases is concerned, the reasonable
question could be: when is to be suggested? In our opinion, in severe, aggressive forms,
poorly sensitive to the usual pharmacological approach (poor- none- responder patients). In
patients who cannot be submitted to the conventional treatment because of intolerance and
high incidence of side effects or adverse reactions. We guess that a careful selection of
candidates is mandatory. In this clinical pivotal study no adverse reactions or complications
were noted. So far, standardized recommendations regarding frequency, intensity and
duration of treatment, and combining immunosuppressive treatment cannot be given. This
preliminary clinical experience showed that the use of a “combined” therapeutic approach is
probably a better model strategy in the management of patient affected by immunological
disorders non responsive to conventional therapy. The therapeutic contribution of IA in
combination with immunosuppressive drugs in the treatment of refractory SLE needs deeper
investigations, increase in the number of subjects to be studied, a case-control randomized
design, and the evaluation of a long-term follow-up. To our knowledge trials like above using
most refined apheretical techinique such as IA apheresis are lacking. In our opinion this is an
area that needs futher investigation, comparison to existing therapeutic regimens, and in
particula, well-designed studies on the proposed combined approach: innovative selective
apheresis and immunosuppressive drugs.
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a new extracorporeal immunoadsorption system, in patients with systemic lupus
erythematosus. Arthritis and Rheumatism 1991; 34(12): 1546-52.
[2] Hashimoto H, Tsuda H, Kanai Y, et al. Selective removal of anti-DNA and
anticardiolipin antibodies by adsorbent plasmapheresis using dextran sulfate columns
in patients with systemic lupus erythematosus. J. Rheumatol. 1991; 18:545-51.
[3] Lewis EJ, Hunsicker LG, Lan SP, Rohde RD, Lachin JM. A controlled trial of
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[4] Ishizuka T, Suzuki K, Hara M, et al. Successful use of immunoadsorption therapy to
treat diffuse proliferative glomerulonephritis in a patient with systemic lupus
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[5] Matsuki Y, Suzuki K, Kawakami M, et al. High-avidity anti-DNA antibody removal

from the serum of systemic lupus erythematosus patients by adsorption using dextran
sulfate cellulose columns. Journal of Clinical Apheresis 1996; 11: 30-35.
[6] Funauchi M, Ikoma S, Enomoto H, et al. High-Affinity anti-DNA antibody parallels
clinical course of immunoadsorption therapy for systemic lupus erythematosus.
Internal Medicine 1996; 35(5): 367-72.
[7] Jones JV, Cumming RH, Bucknall RC, et al. Plasmapheresis in the management of
systemic lupus erythematosus? Lancet 1996; i: 709-711).
[8] Funauchi M, Ikoma S, Imada A, Kanamaru A.Combination of Immunoadsorption
Therapy and High-dose Methylprednisolone in Patients with Lupus Nephritis;
Possible Indications in Patients with Early Stage. J. Clin. Lab. Immunol. 1997; 49:
47-57.
[9] Funauchi M, Imada A, Horiuschi A, et al. Effect of immunoadsorption therapy in
systemic lupus erythematosus. Jpn. J. Apheresis 1997; 16(1): 192-3.
[10] Matsuki Y, Suzuki K, Kawakami M, Ishizuka T, Hidaka T, Nakamura H. Adsorption
of anaphylatoxins from the plasma of systemic lupus erythematosus patients using
dextran sulfate cellulose columns. J. Clin. Apheresis. 1998; 13(3):108-13.
[11] Kutsuki H, Takata S, Yamamoto K, Tani N. Therapeutic selective adsorption of anti-
DNA antibody using dextran sulfate cellulose column (Selesorb) for the treatment of
systemic lupus erythematosus. Ther Apher 1998; 2(1):18-24. Review.
[12] Schneider KM: Plasmapheresis and immunoadsorption: different techniques and
their current role in medical therapy. Kidney Int. Suppl. 1998; 64:S61-5. Review.
[13] Nakamura Y, Yoshida K, Itoh S, et al. Immunoadsorption plasmapheresis as a
treatment for pregnancy complicated by systemic lupus erythematosus with positive
antiphospholipid antibodies. American Journal of Reproductive Immunology 1999;
41: 307-11.
[14] Uramoto KM, Michet CJ Jr, Thumboo J, Sunku J, O'Fallon WM, Gabriel SE.Trends
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[15] Hidaka T, Suzuki K, Matsuki Y, Takamizawa-Matsumoto M, Kataharada K,
Ishizuka T, Kawakami M, Nakamura H, Yabuki T, Kutsuki H: Evaluation of
adsorption selectivity dextran sulfate bound cellulose beads for the removal of anti-
DNA antibodies. Ther. Apher. 1999; 3(1):75-80.
[16] Korbet SM, Lewis EJ, Schwartz MM, Reichlin M, Evans J, Rohde RD. Factors
predictive of outcome in sever lupus nephritis. Lupus nephritis collaborative study
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[17] Braun N, Bosch T: Immunoadsorption, current status and future developments.
Expert Opin. Investig. Drugs 2000; 9(9): 2017-38.
[18] Suzuki K: The role of immunoadsorption using dextran-sulfate cellulose columns in
the treatment of systemic lupus erythematosus. Ther. Apher. 2000; 4(3): 239-43.
[19] Wallace DJ. Apheresis for lupus erythematosus: state of the art. Lupus 2001;
10(3):193-6. Review.
[20] Nakaji S: Current topics on immunoadsorption therapy. Ther. Apher. 2001; 5(4):301-
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[21] Kamijo Y, Kaneko Y, Ichikawa T, Kobayashi N, Koyama T, Kakegawa T,

Kamijo H, Kono K, Minami S, Tanaka N, Arakura H, Hirata M, Higuchi M,
Kiyosawa K, Hora K. A case of nephrotic syndrome due to lupus nephritis which was
controlled with low-density lipoprotein apheresis. Ther. Apher. 2002; 6(6):459-62.
[22] Avenhaus B, Avenhaus W, Schneider M, Domschke W, Gaubitz M. Development of
an in vitro miniature model to simulate immunoadsorption in patients with systemic
lupus erythematosus. J. Clin. Apheresis 2002;17(4):183-9.
[23] Blake JS, Butani L. Rapidly progressive lupus glomerulonephritis and concomitant
microangiopathy in an adolescent. Lupus 2002;11(8):533-5.
[24] Danieli MG, Palmieri C, Salvi A, Refe MC, Strusi AS, Danieli G. Synchronised
therapy and high-dose cyclophosphamide in proliferative lupus nephritis. J. Clin.
Apheresis 2002;17(2):72-7.
[25] Braun N, Junger M, Klein R, Gutenberger S, Guagnin M, Risler T. Dextran sulfate
(Selesorb) plasma apheresis improves vascular changes in systemic lupus
erythematosus. Ther. Apher. 2002; 6(6):471-7.
[26] Cagnoli L; Italian Society of Nephrology.Instructions and implementations for
percutaneous renal biopsy. Guidelines for the therapy of glomerular nephropaties. G.
Ital. Nefrol. 2003; 20 Suppl 24: S3-47.
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GPAnet, 2004: 21-38.
[28] Kaoru Sugimoto, Ken Yamaji, Kwang-Seok Yang, Yoshinori Kanai, Hiroshi
Tsuda, Hiroshi Hashimoto Immunoadsorption Plasmapheresis Using a Phenylalanine
Column as an Effective Treatment for Lupus Nephritis Therapeutic Apheresis and
Dialysis 2006; 10 (2), 187–192.
[29] Stefanutti C Stefanutti C, Di Giacomo S, Mareri M, Ceccarelli F, and Valesini G.
Cyclophosphamide and Immunoadsorption Apheresis Treatment of Lupus Nephritis
Nonresponsive to Drugs Therapy Alone Biodrugs 2005; 19 (2):129-133.
[30] Micheloud D, Nuño L, Rodríguez-Mahou et al. MEfficacy and safety of Etanercept,
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lupus diffuse proliferative nephritis complicating pregnancy. Lupus Volume
15, Number 12, December 2006, pp. 881-885(5).
Editor: Tomas I. Seward, pp. 9-15 © 2007 Nova Science Publishers, Inc..
Expert Commentary
PREGNANCY, A CHALLENGE IN PATIENTS WITH

SYSTEMIC LUPUS ERYTHEMATOSUS
Javier A. Cavallasca and Maria del Rosario Maliandi

Division of Rheumatology, Hospital de Clinicas “Jose de San Martin”,
School of Medicine, University of Buenos Aires, Buenos Aires, Argentina
INTRODUCTION
Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease that typically

affects women of childbearing age. The peaks incidence of SLE occurs between the ages of
15 and 40 years, with an estimated female to male incidence of 9:1. Most studies analyzing
the relationship between SLE and pregnancy, observed an increased of fetal and maternal
risks, notably when pregnancy occurs in active SLE. Also the frequent association between
SLE and antiphospholipid syndrome (APS) represent another risk situation for this patients
and the product of conception.
FERTILITY
Fertility in SLE patients is not impaired in the absence of severe disease, end stage renal
disease and cytotoxic medications. The main risk factor for premature ovarian failure is the
use of cyclophosphamide, and is closely related to age and total dose received. The use of
azathioprine, cyclosporine and methotrexate is not associated with ovarian failure.
10 Javier A. Cavallasca and Maria del Rosario Maliandi
INFLUENCE OF PREGNANCY ON SLE
Normal pregnancy is associated with palmar and facial erythema that can mimic a lupus
rash , difusse arthralgias and joint efussions which may be confused with lupus arthritis, hair
loss, mild anemia because of hemodilution and thrombocitopenia.
Whether lupus flares are more frequent during pregnancy remains controversial. While
some authors hold that pregnancy is not a cause of disease exacerbations, other researchers
have found exacerbations in 74% of cases. This may be due to the differences in study
populations, the number of patients included in the series, methodological differences in the
study design, the existence or lack of a control group, and the definition of flare that is being
used.
Flares can be present during all trimesters and in the puerperium period, generally they
involve minor organ manifestations (e.g. cutaneous), however major organs manifestations
(e.g renal flares) can occur.
According to the services of Rheumatology and Obstetrics of the Hospital de Clínicas
José de San Martin, University of Buenos Aires, which includes 72 pregnancies in 61 SLE
patients followed between 1986 and 2004 , fourteen disease exacerbations were identified
(19%). The majority of flares were mild, skin was the most common manifestation (27%) and
there were five cases of renal exacerbations (7%).The third trimester was the one with most
exacerbations and during puerperium they were observed in three patients (4%).
Flare rates have consistently been highest in patients with poorly controlled SLE,
particularly these with lupus nephritis (LN), renal failure and hypertension.
Therefore patient should be advised to become pregnant when the disease is inactive, and
for at least 6 months if they have history of renal involvement.
INFLUENCE OF SLE ON PREGNANCY:

OBSTETRIC AND FETAL OUTCOME
Historically lupus pregnancy was associated with a high rate of obstetric and fetal
complications. These include spontaneous abortion, late miscarriage, intrauterine growth
retardation, preterm delivery and prematurity. With the widespread use of careful monitoring
and treatment schedules of these patients many improvements in both fetal and maternal
pregnancy outcomes have occurred, with an overall fetal loss as high as 50% in unplanned
pregnancies versus 13% in planned pregnancies. In our series there were 61 live births, 85%,
(one twin birth), six stillbirth (8%) and 5 spontaneous abortions (7%).
Reported incidences for preterm delivery have ranged between 17-49% in recent studies,
and lupus activity, hypertension and corticosteroid use are the strong predictors of preterm
birth. In our experience forty six percent of 72 pregnancies ended in preterm deliveries.
Significantly more women in the preterm delivery group were taking ≥ 10 mg/day of
prednisone compared to the term delivery group.
Pregnancy, a Challenge in Patients with Systemic Lupus Erythematosus 11
LUPUS AND HYPERTENSIVE PREGNANCY

COMPLICATIONS
Lupus pregnancy is associated with an increased risk of pre-eclampsia of 13-32%,

especially in the setting of lupus nephritis. Pre-eclampsia and eclampsia can mimic lupus
with both presenting as edema, thrombocytopenia, hyperuricemia, anemia, hypertension,
proteinuria, hematuria and seizures in eclampsia.
Pre-eclampsia is often difficult to distinguish from active LN. Investigations of serum
complements C3, anti DNA antibodies and urinary sediment can help to differenciate
between both diseases. We have seen in our study, gestational hypertension in 15 pregnancies
(21%) and preeclampsia in 8 pregnancies (11%). No eclampsia or HELLP syndrome
(hemolysis, elevated liver enzymes, low platelets) occurred.
LUPUS NEPHRITIS (LN)
On the other hand, the activity of LN at conception greatly impacted on fetal losses
which ranged between 25-57% in women with active LN versus 8-12.5% in those with stable
renal disease. In our report patients whose disease manifested as nephritis had live births in
70% of pregnancies, however, no association with abortions or stillbirths was seen.
ANTIPHOSPHOLIPID SYNDROME (APS)
The APS confers increased risks of abortions, fetal intrauterine growth retardation and
death, thrombosis in the mother and severely early onset of preeclampsia. Two studies have
reported that if an APL positive lupus patient left untreated, would only have a live birth rate
of 20%, but with the use of low dose aspirin, the live birth rate increased to 42-44%, and with
combined low dose low molecular weight heparin(LMWH) live birth rates doubled to 71-
80%. In our series, 85% of pregnancies in patients with APS resulted in live newborns, 4 of
which (30%) also had low birth weight.
NEONATAL LUPUS ERYTHEMATOSUS
All lupus patient contemplating pregnancy should have an anti-Ro/SSA, anti-La/SSB

status determined, these antibodies are present in 30-50% of SLE patients. They cross the
placenta and cause neonatal lupus in 1% of babies born to these mothers, and subsequent
children have a 25% risk. The majority of the affected babies suffer a transient and often mild
lupoid rash lasting 3-6 months, but the most severe complication is congenital heart
block(CHB), which is diagnosed by fetal bradycardia at 16-23 weeks of gestation , with a
mortality of 20% in the neonatal period.
The first fetal echocardiogram should be obtained at 16 weeks of gestation and then
weekly for high risk infants (prior fetus with CHB) or every 2 weeks in lower risk settings.
In our study, there was only one infant with CHB in an anti-Ro/SSA positive mother.
Although intrauterine dexamethasone was administered, the infant did not survive.
DRUGS IN PREGNANCY AND BREASTFEEDING
SLE in women in their reproductive years may need drugs treatment during pregnancy ,
puerperium and in the breastfeeding period , to control maternal disease and to ensure
successful pregnancy outcome.
Because only drugs considered safe can be studied in pregnant or lacting women, the
number of controlled studies is small, and only information on the safety of drugs during
these periods is derived from experimental and precilinical studies.
NON STEROIDAL ANTINFLAMMATORY

DRUGS (NSAID)
Cox-1 and Cox-2 are involved in ovulation and implantation. Several case reports and
small series have described transient infertility after treatment with non-aspirin NSAID, such
as indomethacin, diclofenac, piroxicam and naproxen, also some studies in animals and
humans have shown that these drugs can inhibit the rupture of the luteinized follicle.
Non selective Cox inhibitors are not teratogenic and can be continued during the first and
second trimesters, but all NSAID (except aspirin add less than 100 mg/day) after gestational
week 20 th can cause constriction of the ductus arteriosus and impair fetal renal function.
They should be withdrawn at gestational week 32.
In relation to low dose aspirin (LDA) there is no consensus on when to stop it before
delivery. Some advice cessation of the treatment one week before a planned delivery with
epidural anesthesia. Other experts do not stop LDA in patients with APS.
Most NSAID are excreted in very small quantities into breast milk. The American
Academy of Pediatrics considers ibuprofen, indomethacin, diclofenac, piroxicam, naproxen,
mefenamic acid, tolmetin, and flufenamic acid to be compatible with breastfeeding.
At present there are no reliable data on selective Cox-2 inhibitors.
COSTICOSTEROIDS
11-β hydroxi steroid dehydrogenase in the placenta converts cortisol and costicosterone
to inactive forms. On the other hand fluorinated corticosteroids (betamethasone and
dexamethasone), are less well metabolized by the placenta. High dose (> 30 mg/day)
glucocorticoid therapy is associated with major maternal complications including
hyperglicemia, diabetes mellitus and hypertension.
Stress doses of hydrocortisone at delivery are recommended in patients on corticosteroids

long term therapy.
Breast feeding is allowed with moderate doses of corticosteroids.
ANTIMALARIAL DRUGS
Chloroquine and hidroxichloroquine cross the placenta with no significant difference in

the mean concentration in maternal and cord blood.
Different articles did not find an increase in congenital malformations or cardiac
conduction disturbances in children exposed antenatally to these drugs.
The suppression of this drugs may produce a SLE flare so, when indicated continue
antimalarials during pregnancy and lactation.
Methotrexate
Methotrexate is contraindicated during pregnancy and in the breastfeeding period . It

must be withdrawn three months before a planned pregnancy.
Cyclophosphamide (CYC)
CYC is gonadotoxic in both women and men. Cryopreservation of sperm and sperm
banking is the method of choice in men, preservation of gonadal function in women is best
done with a gonadotrophin-releasing hormone agonist.
This drug is contraindicated during pregnancy and the breast feeding period. As MTX, it
must be withdrawn three months before a planned pregnancy.
Azathioprine (AZA)
It does not adversely affect the fertility of both women and men. It can be used during
pregnancy at daily dose not exceeding 2 mg/kg/day.
Nursing is not recommended by the American Academy of Pediatrics.
Cyclosporin A (Cs A)
It can be used in pregnancy at the lowest effective dose, with close control of maternal
blood pressure and renal function during therapy. Breastfeeding is not recommended.
Mycophenolate Mofetil (MMF)
MMF is contraindicated during pregnancy. The drug should be stopped at least 5 six
weeks before a planned pregnancy. Breastfeeding is not allowed.
Leflunomide
Leflunomide is contraindicated during pregnancy and breastfeeding. Discontinue 2 years

prior to conception. Consider the use of cholestiramine to enhance elimination from the body
until plasma levels of leflunomide are undetectable.
Tacrolimus
It may be maintained during pregnancy at the lowest possible dose. Nursing is possible.
Intravenous Immunoglobulin
It can be used in pregnancy and in the breastfeeding period.
CONCLUSION
Pregnancy in a lupus patient continues to be a major challenge for the physician and it
should be considered as a high – risk situation. However, if it is planned when the disease is
stable and under close supervision by a multidisciplinary team, we could expected for the
mother and her baby a good outcome.
REFERENCES
Clowse ME, Magder LS, Witter F, Petri M. The impact of increased lupus activity on
obstetric outcomes. Arthritis Rheum. 2005 ;52:514-21.
Lockshin MD. Pregnancy does not cause systemic lupus erythematosus to worsen. Arthritis
Rheum. 1989;32:665-70.
Nossent HC, Swaak TJ. Systemic lupus erythematosus. VI. Analysis of the interrelationship
with pregnancy. J. Rheumatol. 1990; 17:771-6.
Khamashta MA, Hughes GR. Pregnancy in systemic lupus erythematosus. Curr. Opin.
Rheumatol. 1996 ;8:424-9.
Cervera R, Font J, Carmona F, et al. Pregnancy outcome in systemic lupus erythematosus:
good news for the new millennium. Autoimmun Rev. 2002;1:354-9.
Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with

heparin and low-dose aspirin is superior to low-dose aspirin alone. Am. J. Obstet.
Gynecol. 1996 ;174:1584-9.
Rai R, Cohen H, Dave M, Regan L. Randomised controlled trial of aspirin and aspirin plus
heparin in pregnantwomen with recurrent miscarriage associated with phospholipid
antibodies (or antiphospholipid antibodies) BMJ. 1997 ;314:253-7.
Norella C T Kong. Pregnancy of a lupus patient- a challenge to the nephrologist. Nephrol.
Dial Transplant 2006;21:268-272.
Moroni G, Ponticelli C. The risk of pregnancy in patients with lupus nephritis. J. Nephrol.
2003 ;16:161-7.
Meyer O. Making pregnancy safer for patients with lupus. Joint Bone Spine. 2004 ;71:178-
82.
Dhar JP, Sokol RJ. Lupus and pregnancy: complex yet manageable. Clin. Med. Res.
2006;4:310-21.
Ostensen M, Khamashta M, Lockshin M,et al. Anti-inflammatory and immunosuppressive
drugs and reproduction. Arthritis Res. Ther. 2006;8:209-.
Short Communications
INFLUENCE OF EXERCISE ON THE PERIPHERAL

CIRCULATION IN PATIENTS WITH SYSTEMIC
LUPUS ERYTHEMATOSUS AND SYSTEMIC
SCLEROSIS
Etsuko Maeshima∗ and Kanako Furukawa**

*
Department of Health and Sport Management,
Osaka University of Health and Sport Sciences, Japan
**
Third Department of Internal Medicine,
Wakayama Medical University
811-1 Wakayama-city, Wakayama, 641-0012 Japan
ABSTRACT
We evaluated the influence of exercise on the peripheral circulation in patients with
systemic lupus erytematosus (SLE) and systemic sclerosis (SSc). Six patients with SLE
(SLE group), 10 patients with SSc (SSc group), and 11 members of their families or
medical staff (control group) were studied. After listening to a 1-hour lecture while
seated, the subjects performed stretching exercise. The blood pressure, pulse rate, and
skin temperature were measured prior to exercise and after 10 minutes of rest following
the exercise session. The skin temperature at the fingertip was used as an index of the
peripheral circulation. There were no significant changes of blood pressure and pulse rate
after exercise in any of the 3 groups. Although there was no significant change of skin
temperature after exercise in the SLE group, the post-exercise skin temperature was
significantly lower than the pre-exercise temperature in the SSc group, (P<0.05).
Moreover, the post-exercise skin temperature of the SSc group was significantly lower
than that of the other two groups. Exercise is likely to cause exacerbation of peripheral
∗
1-1 Asashirodai, Kumatori-cho, Sennan-gun, Osaka 590-0496, Japan. e-mail:[email protected]. Fax: +81-724-
53-8818. Tel:+81-724-53-8845
18 Etsuko Maeshima and Kanako Furukawa
circulatory disturbance in SSc, even when patients perform low-intensity exercise.

Therefore, it is necessary to monitor the peripheral circulation by measuring the skin
temperature when patients with such diseases perform exercise.
Keywords: systemic lupus erythematosus, systemic sclerosis, exercise, peripheral

circulation.
INTRUDUCTION
The beneficial effects of exercise include a decrease of sympathetic nervous tone,

dilation of the peripheral blood vessels, increased dermal blood flow, and mental relaxation,
as well as improvement of lifestyle-related diseases [1]. Recently, more and more people in
Japan have begun to incorporate exercise into their daily lives for health maintenance and
promotion, as well as for the prevention of various diseases. It is expected that patients with
autoimmune diseases may benefit from daily exercise in terms of improvement of their
peripheral circulation, and exercise may also help to alleviate problems caused by their
medications, such as hypertension, diabetes, and osteoporosis. There have been reports that
exercise is beneficial for lupus fatigue [2-4] and that it improves the aerobic capacity, quality
of life (QOL), and depression in patients with systemic lupus erythematosus (SLE) [5]. In
patients with systemic sclerosis (SSc), it is thought that exercise capacity is a useful index of
pulmonary function [6-8]. However, the effect of exercise on the peripheral circulation of
such patients have not yet been examined. Accordingly, we investigated whether exercise
improved the peripheral circulation of patients with SLE or SSc.
SUBJECTS
Among patients with autoimmune diseases who underwent consultations for patients
with intractable diseases at a health center in Wakayama Prefecture, 16 patients (3 men and
13 women) were studied. They did not have Raynaud's phenomenon, cyanosis, ulcers, or
finger defects at the time of the consultation, and they were all willing to cooperate with the
investigation. In addition, 11 members of their families and some of the medical staff (4 men
and 7 women) were studied as a control group, making a total of 27 subjects. The
autoimmune disease was SLE [9] in 6 patients (SLE group) and SSc [10] in 10 patients (SSc
group). All of the patients were well-controlled and were being treated as outpatients.
METHODS
After listening to a 1-hour lecture while seated, the subjects performed stretching
exercise involved moving the upper or lower extremities while seated for 30 minutes. The
blood pressure, pulse rate, and skin temperature were measured before exercise and after 10
minutes of rest following the completion of exercise. The blood pressure and pulse rate were
Influence of Exercise on the Peripheral Circulation in Patients… 19
measured in the right arm (brachial artery) using an automated sphygmomanometer (Terumo
Electronic Sphygmomanometer, Terumo Corporation, Japan, Tokyo). The skin temperature
was employed as an index of the peripheral circulation, and was measured at the palmar
aspect of the tips of the right and left index fingers using an instantaneous dermatherm
(Scalar Corporation, Tokyo). A "decreased skin temperature" was defined as a decrease of
skin temperature on either the right or left side by 0.5 degrees or more, while an "increased
skin temperature" was defined as an increase by 0.5 degrees or more. The patients also
evaluated their own symptoms (pain, paresis, and cold sensation of the hands, fingers, and
feet) on a three-point scale ("improved," "unchanged," or "aggravated"), and filled in
questionnaires.
The lecture was held at the health care center for patients treated at institutions both
inside and outside Wakayama Prefecture, so a detailed medical history or information about
current medications was not obtained.
Results were subjected to statistical analysis using the Wilcoxon signed-rank test, the
Kruskal-Wallis test, and the chi-square test.
RESULTS
1. Age
The age of the patients ranged between 32 and 82 years (48.5 ± 18.4 years) in the SLE
group, 46 and 76 years (63.2 ± 9.4 years) in the SSc group , and 30 and 74 years (53.2 ± 15.8
years) in the control group, with no significant differences among the three groups.
2. Blood Pressure
Systolic Pressure
The pre-exercise systolic blood pressure ranged from 90 to 156 mmHg (115.7 ± 24.8
mmHg) in the SLE group, 110 to 156 mmHg (130.1 ± 17.5 mmHg) in the SSc group, and 99
to 150 mmHg (124.9 ± 15.0 mmHg) in the control group, showing no significant differences
among the three groups.
The post-exercise systolic pressure ranged from 100 to 154 mmHg (120.7 ± 22.8 mmHg)
in the SLE group, 104 to 176 mmHg (132.0 ± 25.5 mmHg) in the SSc group, and 105 to 186
mmHg (131.2 ± 23.1 mmHg) in the control group, again showing no significant differences.
There was also no significant change of systolic pressure after exercise in any of the groups
(Figure 1).
Diastolic Pressure
The pre-exercise diastolic blood pressure of the SLE, SSc, and control groups ranged
from 62 to 88 mmHg (75.2 ± 10.1 mmHg), 58 to 91 mmHg (74.4 ± 12.8 mmHg), and 65 to
87 mmHg (75.1 ± 6.8 mmHg), respectively, with no significant differences among the three
groups. The post-exercise diastolic pressure ranged from 64 to 98 mmHg (76.2 ± 14.2
mmHg), 58 to 101 mmHg (77.1 ± 15.0 mmHg), and 58 to 94 mmHg (78.4 ± 9.8 mmHg),
respectively, and there were also no significant differences among the groups. Furthermore,
there was no significant change of blood pressure after exercise in any group (Figure 2).
Figure 1. Systolic blood pressure. There were no significant changes after exercise in any group.
Figure 2. Diastolic blood pressure. There were no significant changes after exercise in any group.
3. Pulse Rate
The pre-exercise pulse rate of the SLE, the SSc, and the control groups ranged from 57 to
88 bpm (74.3 ± 11.9 bpm), 60 to 85 bpm (70.5 ± 9.5 bpm), and 43 to 109 bpm (74.0 ± 17.8
bpm), respectively, with no significant differences among the three groups. Post-exercise
pulse rates ranged from 54 to 79 bpm (71.7 ± 10.0 bpm), 65 to 87 bpm (73.7 ± 6.8 bpm), and
50 to 120 bpm (74.5 ± 19.8 bpm), respectively, again showing no significant differences.
There was also no significant change of the pulse rate with exercise in any of the groups
(Figure 3).
Figure 3. Pulse rates. There were no significant changes after exercise in any group.
4. Skin Temperature
Right Index Finger

The pre-exercise skin temperature of the right index finger ranged from 25.9 to 32.9°C
(31.4 ± 2.7°C), 22.1 to 32.1°C (28.9 ± 4.0°C), and 22.8 to 34.6°C (30.7 ± 3.9°C) in the SLE,
SSc, and control groups, respectively, with no significant differences among the groups. The
post-exercise skin temperature ranged from 26.7 to 34.9°C (32.1 ± 2.9°C), 21.8 to 33.4°C
(27.8 ± 4.4°C), and 22.9 to 34.7°C (31.8 ± 3.4°C), respectively, showing differences among
the three groups. There was a significant difference between the SLE group and the SSc
group (P<0.05), as well as between the SSc group and the control group (P<0.05) (Figure.4).
Left Index Finger

The pre-exercise skin temperature of the left index finger ranged from 24.9 to 33.4°C
(31.1 ± 3.2°C), 21.1 to 32.0°C (28.2 ± 3.8°C), and 23.1 to 34.6°C (30.5 ± 4.2°C) in the SLE,
SSc, and control groups, respectively, showing no significant differences.
Figure 4. Pre-exercise and post-exercise skin temperature of the right index finger. The pre-exercise
skin temperature showed no significant differences among the three groups. However, the post-exercise
skin temperature showed variation among the groups. There was a significant difference between the
SSc group and the SLE group (P<0.05), as well as between the SSc group and the control group
(P<0.05).
Figure 5. Pre-exercise and post-exercise skin temperature of the left index finger. The pre-exercise skin
temperature showed no significant differences among the three groups. However, the post-exercise skin
temperature showed variation among the groups (P<0.05). There was a significant difference between
the SSc group and the SLE group (P<0.05), as well as between the SSc group and the control group
(P<0.05).
Post-exercise skin temperature ranged from 24.2 to 35.0°C (31.9 ± 3.9°C), 20.2 to 32.6°C
(27.3 ± 4.0°C), and 22.9 to 34.2°C (31.4 ± 3.4°C), respectively, showing variation among the
three groups. There was a significant difference between the SLE group and the SSc group
(P<0.05), as well as between the SSc group and the control group (P<0.05) (Figure. 5).
When the skin temperature of each group was compared between before and after
exercise, there were no significant changes in the SLE group or the control group. In the SSc
group, however, the post-exercise skin temperature of the right finger showed a significant
decrease compared with the pre-exercise value (P<0.05) (Figure. 6A, B).
Figure 6A. Skin temperature of the right finger.
Figure 6B. Skin temperature of the left finger.
Figure 6A, 6B. Comparison of the skin temperature before and after the exercise in each group. There
were no significant changes in the SLE and control groups, but the SSc group had a significantly lower
post-exercise skin temperature of the right finger compared with the pre-exercise value (P<0.05).
5. Post-Exercise Interview
Among the 6 patients in the SLE group, one patient stated that his/her symptoms (pain,
pareisis, or cold sensation) were "aggravated" (the skin temperature increased). On the other
hand, 5 patients reported that their symptoms were "unchanged" or "improved" (3 of them
had an increased skin temperature and 2 (33.3%) had a decreased skin temperature). In the
SSc group, 2 patients answered that their symptoms were "aggravated," and both of them had
a decrease of skin temperature with exercise. On the other hand, although 8 patients answered
that their symptoms were "unchanged" or "improved," only 1 of them had an increase of skin
temperature and 6 (60.0%) of them had a decrease of temperature. In the control group, all of
the subjects answered that their symptoms were either "unchanged" or "improved." Among
them, 7 subjects had an increase of skin temperature and 4 had a decrease. There were no
significant differences among the three groups (Table 1).
Table 1. Subjective symptoms and the skin temperature
DISCUSSION
Peripheral Circulation
There have been some previous reports about the effects of exercise in patients with
autoimmune diseases. The effect of bicycle ergometer aerobic exercise for eight weeks was
assessed in 23 SLE patients, showing that exercise did not exacerbate their disease and that it
actually improved their aerobic capacity and fatigue [2]. Ramsey-Goldman et al. divided 10
SLE patients into two groups, which were an aerobic exercise group and a range of
motion/muscle strengthening group, and examined the influence and safety of these different
kinds of exercise on fatigue and functional status [3]. Neither type of exercise exacerbated
disease activity, while both improved fatigue, functional status, cardiovascular fitness, and
muscle strength, as well as increasing bone turnover. Tench et al. also reported the use of
appropriately graded aerobic exercise for the management of fatigue in SLE patients [4].
These authors concluded that exercise was effective for improvement of fatigue, depression,
and aerobic capacity, but the influence of exercise on the peripheral circulation was not
examined. It has also been reported that exercise is useful for evaluation of lung function in
SSc [6-8], but the influence on the peripheral circulation has not been examined in SSc
patients either.
No significant changes of the blood pressure and pulse rate were observed after exercise
was performed in this study, and there were also no significant differences among the groups.
Thus, this study assessed low-intensity exercise that did not affect the blood pressure and
pulse rate. However, the digital skin temperature (used as an index of peripheral circulation)
decreased significantly after exercise in the SSc group. Skin temperature is regulated by
contraction and dilation of the cutaneous blood vessels. An increment cutaneous blood flow
increases the skin temperature and a decline of blood flow decreases the skin temperature.
During exercise, the cutaneous vessels dilate and cutaneous blood flow increases, which
causes the skin temperature to rise [1]. In this study, the skin temperature after exercise was
significantly lower in the SSc group than in the other two groups, and the skin temperature
after exercise lower than the temperature before exercise. On the other hand, there were no
significant changes of the skin temperature after exercise in the SLE and control groups.
Paroxysmal vasospasm of the fingers, or Raynaud’s phenomenon, is a frequent
abnormality in patients with SSc, mixed connective tissue disease, and SLE. Raynaud’s
represents dysregulation of the neuroendothelial control of vascular tone and it is essentially
an exaggerated vasospastic response to cold or emotion. The prevalence of Raynaud’s
phenomenon in SLE takes the middle range between the rate of 95% found in patients with
scleroderma and the level of 3% seen in those with rheumatoid arthritis [11]. Raynaud’s
phenomenon is nonspecific and may be present for years before the development of other
changes due to SLE, SSc, or dermatomyositis. Such vasospasm is regarded as the main cause
of the disturbance of peripheral circulation in many autoimmune diseases, including SLE
[11]. However, patients with SSc not only have abnormal vascular function (vasospasm), but
also develop changes of vessel structure and the nervous system, an imbalance between
vasodilation and vasoconstriction, abnormal platelet function, and abnormal erythrocyte
deformation [12-14]. Generally, vasospasm due to Raynaud’s phenomenon rarely leads to
permanent damage, although small ulcers on the fingers can occur following prolonged and
frequent attacks. However, disturbance of the digital circulation is more prominent in SSc
compared with other autoimmune diseases, and ulcers at the tips of the digits are included in
the criteria defining this disease. Thus, it seems that peripheral circulatory disturbance is
more severe in SSc compared with other autoimmune diseases.
When recommending exercise for patients with SSc, we should emphasize that it is
important to undergo assessment of the peripheral pulses or skin temperature measurement.
Interview Findings
The results of the interviews showed no significant differences among the three groups.
In the SLE group, 2 patients answered that their symptoms were “unchanged” or “improved,
although their skin temperature decreased. Even in the SSs group, in which the pre-exercise
skin temperature was significantly lower than that of the other groups and the post-exercise
temperature was even lower, approximately 80% of the patients answered that their
symptoms were "improved" or "unchanged." This shows the difficulty of evaluating a
decrease of skin temperature during or after exercise only on the basis of patients' symptoms.
The reason for the discrepancy between the changes of symptoms and the actual skin
temperature is unclear. It is possible that activity had a beneficial influence such as a
refreshing feeling, which may make it difficult for patients to notice the deterioration of their
peripheral circulation. The refreshing effect of exercise or decreased transmission of pain is
often discussed in association with β-endorphin, which is secreted by the pituitary gland and
immunocompetent cells [15,16]. However, previous reports indicate that exercise above a
certain intensity threshold is required to promote the secretion of β-endorphin, and it is
unlikely that secretion of this hormone was increased by the low-intensity exercise performed
in our study.
Among patients whose skin temperature decreases after exercise, it can be one of the risk
factors for exaceerbation of digital ulcers or necrosis. Therefore, when patients with
peripheral circulatory disturbance perform exercise, it is necessary to monitor the status of
their peripheral circulation by skin temperature measurement.
CONCLUSION
During low-intensity exercise, there were no significant differences of blood pressure and
pulse rate between patients with autoimmune disease and the healthy control group. A
decrease of peripheral blood flow was not observed in the SLE patients. However, SSc
patients were likely to show deterioration of peripheral circulation even with low-intensity
exercise. Therefore, it is necessary to monitor the peripheral circulation by assessing the
peripheral pulses or skin temperature when patients with such diseases perform exercise.
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M, Riemekasten G, Emery P, Chadha-Boreham H, Charef P, Roux S, Black CM,
Seibold JR. (2007). Submaximal exercise testing in the assessment of interstitial lung
disease secondary to systemic sclerosis: reproducibility and correlations of the 6-min
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[9] Tan EM, Cohen AS, Fries JF, Masi AT, McShane DJ, Rothfield NF, Schaller JG,
Talal N, Winchester RJ. (1982). The 1982 revised criteria for the classification of
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Allegar N, Varga J, Massa M. (1993). Skin thickness score in systemic sclerosis: an
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1892-1896.
[11] Wallace DJ. (1997). Clinical and laboratory features in systemic lupus erythematosus:
Cutaneouse and cutaneovasucular manifestation of systemic lupus erythematosus. In:
Wallace DJ, Hahn HB (Eds.), Dubois’ Lupus Erythematosus (5th edition, pp.693-721).
Baltimore, Maryland: William and Wilkins.
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(1996). Raynaud’s phenomenon and systemic sclerosis. Ann. Ital. Med. Int., 11, 125-
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[13] Herrick AL. (2005). Pathogenesis of Raynaud’s phenomenon. Rheumatology, 44,
587-596.
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Psychoneuroendocrinology 2000; 25: 551-562.
Chapter 1
NEW FRONTIERS IN THE RESEARCH OF

CARDIOVASCULAR DISEASE ASSOCIATED WITH
Laura Gonzalez-Lopez,∗ J. I. Gamez-Nava,** and Arnulfo Nava**

*
Department of Internal Medicine - Rheumatology,
Hospital General Regional 110
Prolongacion Circunvalacion-Oblatos 2208,
Guadalajara, Jalisco, México, 44340. Prostgraduate Program of Public Health Sciences
Centro Universitario Ciencias de la Salud Universidad de Guadalajara
**
Division of Musculoskeletal and Autoinmune Diseases,
Clinical Epidemiology Research Unit
Hospital de Especialidades del Centro Medico Nacional de Occidente
Belisario Domínguez 1000, Guadalajara,
Jalisco, México, 44320
ABSTRACT
Cardiovascular disease is a major cause of mortality in patients with systemic lupus
erythematosus (SLE). Around 52% of the autopsies performed in SLE in our centre have
shown cardiac involvement and most of these disorders were not diagnosed pre-mortem
[1].
development of valvular disease and new other forms of cardiac involvement. Most of
the patients deceased by cardiovascular disease have an accelerated atherosclerosis.
Some authors reported a substantial increase in risk of fatal vascular event in women
∗
Correspondence to: L. Gonzalez-Lopez, Salto del Agua 2192, Col. Jardines del Country. Guadalajara, Jalisco,
Mexico, 44210. Email: [email protected]; [email protected]. Telephone/ Fax: +52-33-38-54-
13-69.
30 Laura Gonzalez-Lopez, Jorge I. Gamez-Nava and Arnulfo Nava
with SLE compared with matched controls. New methods such as electron-beam
computed tomography have demonstrated a highly prevalence of coronary disorder even
in asymptomatic patients. Additionally, high resolution carotid ultrasonography has
shown a high prevalence of carotid lesions. Both cellular and molecular mechanisms of
the accelerated atherosclerosis are complex and not entirely explained by the traditional
cardiovascular risk factors, therefore, the research targets on nontraditional factors.
Pulmonary hypertension is relevant by its morbidity and mortality. Previously
considered as rare entity; new non-invasive studies have detected patients with earlier
involvement; our group have reported a prevalence of 18% in a series of 204 patients
assessed by Doppler echocardiography. A follow-up study in our cohort observed an
increase of 4 times greater of risk for cardiac failure in patients with asymptomatic
pulmonary pressure >40 mmHg. Strategies for the treatment of pulmonary hypertension
include immunosuppressive therapy, prostanoids, phosphodiesterase inhibitors or
endothelin receptor antagonists. Most of the current information related with the response
to these treatments proceeds from short-term studies with a wide variability in the
outcome measures making necessary additional research in this area.
There is also a renovate interest in the valvular disease, recent studies show
association between antiphospholipid antibodies and mitral valve nodules or mitral
regurgitation, the potential effects of these antibodies on the endothelial activation
require to be evaluated.
Cardiac dysfunction is another important area for research; several studies have
shown a high prevalence of left ventricular diastolic dysfunction. In our own series this
manifestation is present in around 63%. Nevertheless, to date no follow-up studies have
been done to evaluate the importance of diastolic dysfunction on the morbidity or
mortality.
In summary cardiovascular disease in systemic lupus represents a very exciting area
for research being necessary to increase the number of long-term prospective cohorts and
well-designed controlled trials in order to improve the clinical care of the patients with
this involvement.
Cardiovascular disease is a major cause of mortality in patients with systemic lupus

erythematosus (SLE). Several studies have found increase in cardiovascular involvement in
autopsies. Around 52% of the autopsies performed in SLE in our centre show cardiac
involvement and most of these disorders were not diagnosed pre-mortem [1].
development of valvular disease and new other forms of cardiac involvement. All the cardiac
structures can be involved in the heart including pericardium endocardium, myocardium,
valves and conduction tissue. Also are involved the arteries including both pulmonary as
coronaries.
PERICARDITIS
Pericarditis is one of the most well documented cardiac involvements in SLE and is
included among the manifestations described in the ARA/ACR classification criteria.
Pericarditis has been demonstrated by echocardiography between 11-54% of the patients with
New Frontiers in the Research of Cardiovascular Disease… 31
SLE [2]. This manifestation can be asymptomatic or with severe symptoms and even life-
threatening, severe complications as cardiac tamponade, constrictive pericarditis and purulent
pericarditis are fortunately rare. Effusions are associated with disease activity. The diagnosis
of pericarditis or pericardial effusion frequently is unsuspected or the symptoms are only mild
and transitory. Figure 1 shows a case of pericarditis observed in an autopsy from a patient
with SLE in our centre. Note the thickening of the pericardium and the mild inflammatory
infiltrate.
Figure 1. Shows a case of lupus pericarditis observed in autopsy from our hospital. The presence of
thickening of pericardial and hyperplasia of the mesothelial cells can be observed (black arrow). A mild
to moderate inflammatory infiltrate is present. Hematoxylin-Eosin staining 40 x. (Photo courtesy Dr.
Maria R. Flores-Marquez, Department of Pathology, CMO IMSS, Guadalajara, Mexico).
From a study in China from 310 patients retrospectively evaluated only 18 had
pericarditis or pericardial effusion reported in their charts clinical, from these the main signs
for suspicion were precordial pain in 7 (39%), dyspnea in 11 (61%), pericardial rub in only
one patient, typical abnormalities in the ECG were found in 7 (39%), and cardiomegaly on
the chest radiograph in 8 (44%), although in 17 (94%) the pericarditis was documented by the
echocardiography [3]. The treatment for pericarditis and/or pericardial effusion depends of
the severity of the involvement. For mild cases usually the treatment with nonsteroidal anti-
inflammatory drugs and corticosteroids (prednisone 0.5 mg / kg / day or equivalent) are
sufficient [2]. In severe cases such as tamponade or associated myocarditis this complication
must be managed with the patient hospitalized. Some patients have a good response with
intravenous bolus of methylprednisolone (1 g daily for three days) [2, 3]. Cardiac tamponade
is developed when fluid accumulation in the pericardial space raise the pressure surrounding
the heart impairing the cardiac filling, resulting in elevated venous pressure and impaired
cardiac output leading to shock. This constitutes an emergency that can be fatal if not treated
opportune and appropriateness. Echocardiography is the best method for the diagnosis of
tamponade showing a diastolic collapse of the free wall of the right atrium and/or the right
ventricle, the collapse is exaggerated during expiration because the right heart filling is
reduced [4]. There is a lack of controlled studies evaluating immunosuppressive drugs in
severe pericardial involvement. The “corner stone” of the treatment for cardiac tamponade is
drainage of the pericardial effusion. Medical treatment is usually ineffective and only
constitute an adjuvant therapy while is available the pericardial drainage [4]. The use of
inotropic agents to treat the shock caused by cardiac tamponade is usually ineffective and the
mechanical ventilation without pericardial drainage may produce a sudden drop in blood
pressure with impairment of cardiac filling [4].
Algorithm for the treatment of pericarditis in SLE
Pericarditis and pericardial

effusion associated with SLE
Mild involvement Moderate or large

pericardial effusion
Anti-inflammatory
Cardiac tamponade
drugs, low doses of
corticosteroids
Yes No
Drain effusion plus High doses of

high doses of corticosteroids +
corticosteroids + immunosuppressive
immunosuppressive drugs
drugs
Pericardial effusion
solved
Yes No
Consider other
etiologies
MYOCARDITIS
Several studies have reported that myocarditis is an infrequent finding in lupus. A recent
report describes a small series of 11 patients diagnosed with acute myocarditis associated
with lupus [5]. The following symptoms were present at the time of the admission: dyspnea
10/11, fever 6/11, orthopnea 5/11, paroxysmal nocturnal dyspnea 4/11, chest pain 4/11, leg
swelling 3/11 and palpitations 2/11 [5]. The clinical signs are unspecific showing basal
crackles on chest auscultation 8/11, raised jugular venous pressure 6/11, tachycardia 6/11,
gallop rhythm 5/11, peripheral oedema 4/11, new murmur 3/11, or irregular pulse rate 1/11.
Some series have found association between myocarditis with positive anti-dsDNA antibody
or low C3 or C4 levels [5]. Acute myocarditis usually is associated with other organ
involvement including nephropathy, lymphopaenia, leukopaenia, thrombocytopenia or
anaemia [5]. Only one-third of the patients have elevated creatine kinase. Lupus
anticoagulant or anticardiolipin antibodies are not useful markers for myocarditis in SLE [5].
Histological findings of myocarditis include small foci of fibrinoid necrosis with
infiltrates of plasma cells, lymphocytes and foci of myocardial fibrosis [2].
Immunocomplexes and complement deposits can be found in the myocardium and their blood
vessels [2]. Figure 2 and 3 shows a case of myocarditis by SLE observed in autopsy in our
hospital. Figure 2 shows a 40X image showing foci of inflammation composed by
lymphocytes with some macrophage and plasmatic cells. Figure 3 shows a 60x image
showing associated fibrinoid necrosis associated with the inflammatory cells.
Noninvasive myocardial imaging techniques for myocarditis include echocardiography,
nuclear imaging with gallium or indium labeled antimyosin antibodies and magnetic
resonance imaging [6].
Figure 2. Shows a case of lupus myocarditis observed in an autopsy from our centre. Note the presence
of inflammatory infiltrates in the myocardium (black arrows). Hematoxylin-Eosin staining 40 x. (Photo
courtesy Dr. Maria R. Flores-Marquez, Department of Pathology, CMO IMSS, Guadalajara, Mexico).
Figure 3. Details of the inflammatory infiltrate observed in myocarditis. Note the predominance of
mononuclear cells (thin arrows). Also note the necrosis of some myocardial fibers (wide arrow).
Hematoxylin-Eosin staining 60 x. (Photo courtesy Dr. Maria R. Flores-Marquez, Department of
Pathology, CMO IMSS, Guadalajara, Mexico).
Echocardiography in Myocarditis
Echocardiography is the method most amply used in the clinics for support the diagnosis
of myocarditis. Findings in the echocardiography include left and/or right ventricular
dysfunction, segmental wall motion abnormalities (such as hypokinetic, akinetic or diskinetic
wall) [7]. These findings in the echocardiography are not conclusive, and other confirmatory
techniques should be used when possible. Echocardiography can be used as a method to
evaluate the response to the therapy; most of the abnormalities in acute myocarditis improve
or even may disappear. Recent techniques such as tissue Doppler imaging and myocardial
velocity measurements are useful to evaluate the changes in function of the therapy.
Scintography
Scintography studies have recovered renovated interest in myocarditis specially indium

monoclonal antibody fragments that are directed against myosin. These autoantibodies bind
to cardiac myocytes that have exposed myosin to extracellular fluid space. Therefore, these
autoantibodies are markers of myocyte necrosis, and reflect the extent of this necrosis. This
method differs from the traditional use of gallium that detects only myocardial inflammation
[6]. Some authors have evaluated the performance of the antimyosin antibodies in patients
with suspected myocarditis. In a series of patients who were evaluated by endomyocardial
biopsy and their histopathologic results were used “as gold standard”. The antimyosin
antibodies showed a high sensitivity (91-100%) and high negative predictive value (93-
100%), but very low specificity (31-44%) or positive predictive value (28-33%) [8].
Therefore, the antimyosin imaging can be considered as useful test for exclusion of
myocarditis in case of negative results but is not very useful to confirm myocarditis in case of
positive findings and further confirmatory procedures should be performed.
Magnetic Resonance
Contrast-enhanced magnetic resonance is another useful technique used for the diagnosis
of myocardial inflammation [6]. Myocarditis is associated with myocyte injury tissue edema
and cellular swelling. Therefore, the assessment of T1 and T2 relaxation times are useful for
detection of myocarditis, as well as, the following of this complication [9]. MRI can help to
difference patients with myocardial infarction from those with myocarditis [10]. Patients with
myocardial infarction have a segmental early subendocardial defect with a delayed high
enhancement predominantly anteroseptal. Instead, patients with myocarditis have no early
defect and they present focal or diffuse nonsegmental, nonsubendocardial delayed
enhancement predominantly in an inferolateral location [10]. In a recent study were
compared 6 patients with active SLE (2 of them with myocarditis) versus 5 patients inactive
and 5 healthy volunteers with the aim to determine differences in myocardial T2 relaxivity in
MRI [11]. Active SLE patients, independently of myocardial involvement or not had
significantly higher T2 relaxation times compared with inactive SLE and controls. Also, 3/6
active patients (two of them with myocarditis) had abnormal focal areas of increased signal
intensity and wall hypokinesis [11]. Focal areas of contrast intake were detected in these
patients. This intriguing report concludes that active SLE patients had high myocardial T2
relaxation times, even without a myocardial involvement. Further studies need to be
performed to evaluate the role of MRI using endomyocardial biopsy as gold standard in
patients with suspicion of myocarditis associated to SLE.
Endomyocardial Biopsy
Endomyocardial biopsy is an invasive method that confirms the presence of myocarditis,

although may cause severe complications, therefore their utilization is limited. Some
indications of the endomyocardial biopsy have been proposed [12] including: the exclusion
of common etiologies of dilated cardiomyopathy, subacute or acute symptoms of heart failure
refractory to standard treatment, worsening of ejection fraction refractory to therapy,
development of arrhytmias affecting the hemodinamics specially progressive heart block or
ventricular tachycardia, heart failure with concurrent rash fever or peripheral eosinophilia or
suspicion for giant cell myocarditis [12]. Small series of patients have been evaluated with
endomyocardial biopsy in SLE. Sakaguchi et al, evaluated 14 endomyocardial biopsies in
lupus where the main finding is fibrosis, whereas, only 4/11 patients showed moderate cell
interstitial infiltration [13]. Immunofluorescence of these biopsies shows immunoglobulin G,
and fibrinogen deposits in the membrane of myocytes and interstitium.
Figure 4. Shows a substitution of the myocardial fibers by fibrosis (Masson staining).

This image can be observed in cases of chronic inflammation by increase in the collagen
synthesis.
Figure 4. Masson staining showing extensive fibrosis between the myocardial fibers (blue color)
observed in response to chronic inflammation of the myocardium. (Photo courtesy Dr. Maria R. Flores-
Marquez, Department of Pathology, IMSS, Guadalajara, Mexico).
Treatment of Myocarditis
All the patients with myocarditis by SLE should be hospitalized and followed with
supportive care in a similar way to other forms of myocarditis [14]. These supportive
measures include diuretics to decrease ventricular filling pressures, and angiotensin
converting-enzyme inhibitors to diminish vascular resistance. Some patients may require
most potent vasodilators such as sodium nitroprusside. Digoxin should be used with extreme
precaution because can increase proinflammatory cytokines. Inotropic agents or use of a
ventricular assist device can be required for some patients; sometimes can be required
antiarrhythmic agents in case of atrial or ventricular arrhythmias. Due to acute myocarditis
constitute a severe manifestation of disease activity in SLE this should be treated with
corticosteroids and immunosuppressive drugs, although most of the evidence of the effect of
this drugs in SLE is obtained from case-series. Currently, there are not controlled trials to
evaluate the efficacy of immunosuppressive drugs in myocarditis by SLE. In a case-series of
11 patients with myocarditis (5), all received high dose of systemic corticosteroids including
prednisone, hydrocortisone or in more severe cases intravenous methylprednisolone. Most of
the half of them [7/11], were treated with intravenous cyclophosphamide associated with
corticosteroids, although two of them deceased after the first infusion of cyclophosphamide
secondary to infections [5].
ATHEROSCLEROSIS IN SLE
Most of the patients with SLE who have deceased by cardiovascular disease had an
accelerated atherosclerosis. Atherosclerosis can be considered a chronic inflammatory
condition on the vessel wall characterized by lipids (cholesteryl esters mainly) accumulated
within the macrophages (foam cells) and smooth muscle cells in the intimal layer [15]. The
lesions progress into atherosclerotic plaques composed by extracellular matrix component
smooth muscle cells, macrophages, connective tissues, lymphocytes and a fibrous cap over a
pool of extracellular lipid [15]. Current theories indicate that in atherosclerosis coexist an
active inflammation and an autoimmune process [15, 16].
Premature or accelerated atherosclerosis in SLE was initially described in 36 autopsies
performed by Bulkley and Roberts in 1975 [17]. In 1976 Urowitz et al, reported a bimodal
mortality pattern in SLE, calling the attention to the consequences of atherosclerosis
particularly to the high incidence of myocardial infarction as a major cause of death in those
patients with longer disease duration [18]. Many groups have confirmed the high frequency
of accelerated atherosclerosis in SLE. The two main consequences of atherosclerosis are
coronary and cerebrovascular disease. Some authors have reported a cumulative prevalence
of coronary artery disease in SLE of 6 to 10%, although subclinical disease may occurs in 35
to 40% of the patients [19].
Traditional risk factors explain only partially the high frequency of atherosclerotic
complications. Recently, a number of “new” risk factors for the development of
atherosclerosis in SLE have been proposed. These include autoantibodies such as anti-B2
glycoprotein I, antiphospholipid antibodies, anti-oxidized LDL (anti-Ox-LDL), and anti-heat
shock protein 60/65 (anti-HSPs). Currently, is also revalorized the role of some traditional
risk factors such as homocysteine and C-reactive protein (CRP). Finally, some studies have
investigated the role of cytokines, factors that participate in cell-to-cell interactions, and some
infections in the genesis of atherosclerosis. The table 1 describes the risk factors associated
with accelerated atherosclerosis.
Table 1. Risk factors for atherosclerosis in SLE
Traditional risk factors Nontraditional risk factors
(a) Dyslipidemia Antiphospholipid antibodies (aPLs)

(b) Hypertension Anti-oxidized low-density lipoproteins (anti-OxLDL)
(c) Inflammation (CRP) Anti heat shock proteins (anti-HSPs)
(d) High levels of homocysteine Proinflammatory Cytokines
(e) Smoking Infections
(f) Utilization of corticosteroids
(g) Lifestyle
(h) Disease activity
Traditional Risk Factors
The traditional risk factors for development of atherosclerosis have been extensively
studied in SLE. New clues have been found about the mechanisms and effects of these risk
factors for SLE patients.
Dyslipidemia in SLE
Although dyslipidemia is highly prevalent in SLE and their role on the atherogenesis
seems to be one of the most studied, some authors have shown evidence that this risk factor is
not frequently assessed in the ordinary clinical practice. Al-Herz et al, observed in a
retrospective charts review that serum lipid profiles were measured only in 37 from 60
patients (62%) in a lupus clinic and in 19 from 123 (15%) patients obtained from private
practice [20]. In 56 patients where the lipid profile was obtained, 31 (55%) of them had
dyslipidemia underlying the importance of to assess this risk factor in the lupus clinics.
There has been described a typical pattern of dyslipidemia in SLE that is characterized by
low high-density lipoprotein (HDL), raised triglycerides (TGs), and mild or no elevation of
low-density-lipoprotein, with a raising of lipoprotein (a) (Lp(a)) [21, 22]. There is a close
relationship between disease activity and dyslipidemia in these patients [23]. Some
mechanisms are supposed to participate in the association between dislypidemia and disease
activity. There is considered that an impairment in the activity of lipoprotein lipase (enzyme
responsible for the catabolism of chylomicrons), is responsible for the elevated levels of
triglyceride found in SLE. One study detected in 47% from 105 patients with SLE
autoantibodies directed against lipoprotein lipase with capacity for inhibition of this enzyme
[24].
Hypertension and Smoking
Elevated high blood pressure is highly prevalent in SLE. Recent studies showed that this
risk factor has a prevalence that ranges from 12 to almost 30% of the patients [25, 26]. Both,
smoking as systemic hypertension constitute two well documented risk factors for coronary
and cerebrovascular disease. Both factors increase the levels of tissue factor (formerly known
as thromboplastin), that is a key initiator of the coagulation cascade that leads to fibrin
formation [27]. Tissue factor is involved in the pathogenesis of atherosclerosis by promoting
thrombus formation and to induce migration and proliferation of vascular smooth muscle
cells [27]. Smoking has multiple other effects on the atherogenesis producing vascular
inflammation and abnormalities in the oxidative stress. Different studies have shown that
smokers have higher leucocytes counts in blood, increase in C-reactive protein (CRP),
interleukin-6 and higher serum levels of soluble intercellular adhesion molecules [28].
Smoking may increase the expression of metalloproteinases and plasmin. Metalloproteinases
are expressed in the atherosclerotic plaques where may contribute to instability of the plaque
and the rupture stimulating neovascularization via generation of angiogenic peptides [28]. In
patients with SLE, smoking has been found consistently as a major risk factor for
atherosclerosis and cardiovascular events. In a recent study Urowitz reported in an extensive
number of patients surged from a cohort that smoking was one of the major factors associated
with atherosclerotic vascular events in SLE [29]. In a multiethnic cohort of patients with SLE
performed in the United States smoking also was found as one of the strongest predictors for
vascular events, together with elevated levels of CRP and antiphospholipid antibodies [30].
These data suggest the need to discourage smoking in patients with SLE and to inform the
patients the increase of risk for a vascular event in case of persistence of this behavior.
Homocysteine in SLE
Homocysteine is an aminoacid containing sulfur produced during the metabolism of

methionine with multiple effects on the vascular endothelium. High levels of homocysteine
are prothrombic, increased collagen production and decrease the availability of nitric oxide.
Elevated homocysteine has been proposed as cerebrovascular and cardiovascular risk factor
in SLE. One of the earliest observations about the possible role of homocysteine in the
atherosclerosis of SLE was made in a prospective cohort study by Petri et al [31]. They
observed a prevalence of plasmatic hyperhomocysteinaemia (>14.1 mumol /l) in 15% of their
patients. After adjusting by other traditional risk factors hyperhomocysteinaemia increased
the risk for stroke (OR=2.44, 95%CI 1.04-5.75, p=0.04) and arterial thrombotic events
(OR=3.9, 95%CI 0.97-12.54, p=0.05). Further studies have confirmed the association
between elevated levels of homocysteine and thrombosis [32, 33]. However, these findings
are not consistently reported [34].
Homocysteine levels can be decreased by the utilization of folic acid and vitamin B
supplementation. Statins used for treatment of hyperlipidemia can also decrease the
hyperhomocysteinaemia. Nevertheless, although homocysteine is a potentially modifiable
factor for cardiovascular events in SLE, currently there is a lack of controlled trials to
evaluate the effect of a normalization of the levels of homocysteine on the rate of
cardiovascular events in SLE. These trials are required to define if hyperhomocysteinemia is
truly a risk factor that requires be treated or constitutes only an epiphenomenon of the
systemic inflammation in SLE.
Corticosteroids
Corticosteroids are associated with increase of risk factors for atherosclerosis including
the redistribution of body fat, modifications in blood pressure, and effects on the plasmatic
glucose. The alterations on the lipid profiles are related with the doses of corticosteroids.
More than one decade ago, MacGregor et al observed that prednisone dose lower than 10 mg/
daily did not affect the lipid profile, whereas, higher dose can increase the levels of
triglycerides and some apolipoprotein [35]. Other authors have subsequently confirmed this
strong association of lipid levels and corticosteroids [36].
Although, the role that play the corticosteroids in the accelerated atherosclerosis in SLE
is not simple. Intriguingly, the corticosteroids may also improve the “protective factors” for
atherogenesis. Corticosteroids can suppress the inflammation and some of their components
that are related with atherosclerosis. These facts explain in part some of the controversial
findings reported in the literature. Roman et al. [37] observed that those patients with carotid
atherosclerosis had received a lower total cumulative dose of corticosteroids compared with
patients without carotid plaques. Corticosteroids therefore, have a complex role in the
pathogenesis of atherosclerosis; strategies to decrease the doses using other drugs such as
antimalarials can contribute to improve the rates of accelerated atherosclerosis in SLE.
Inflammation
Atherosclerosis has an important inflammatory component, examination of plaques of

atherosclerosis shows infiltration of T cell lymphocytes and macrophages observed mainly in
early stages. Several substances facilitate the infiltration by inflammatory cells including the
monocyte chemotactic protein-1 (MCP-1), and different adhesion molecules. Inflammatory
mechanisms contribute to instability and rupture of plaques. Interferon (IFN) has been found
in the area surrounding atherosclerotic plaques. IFN contribute to the instability of a plaque
and rupture, through to reduce collagen synthesis in conjunction with increase in the
synthesis of matrix metalloproteinases leading to destabilization of the plaque, fissuring it
and provoking the rupture [38, 39].
High levels of C-reactive protein (CRP), detected by high-sensitivity immunoassay are
considered strong predictors for coronary artery disease. CRP is synthesized in the liver in
response to different cytokines including interleukin-6. Studies in vitro have shown that CRP
activates the complement system, induces the production of MCP-1, increase the expression
of cellular adhesion molecules (CAM), stimulate the monocyte production of tissue factor
and are mediators in the macrophage uptake of LDL [40]. High levels of CRP also produce
an endothelial dysfunction mediated through their effects in to suppress vascular endothelial
cell expression and the activity of endothelial nitric oxide synthase [40]. This endothelial
dysfunction is considered nowadays an important component of the initiation of
atherogenesis. CRP has other important actions implicated on the atherogenesis including
promote a procoagulant state mediated by their effects in inducing of plasminogen activator
inhibitor-1, and increase the erythrocyte adhesiveness and subsequent aggregation [40].
TNF-α and IL-1
Different studies have been observed evidence of the participation of TNF-α and IL-1 in
the atherosclerotic plaques. Both cytokines have a number of effects on the atherogenesis
including to stimulate the smooth muscle proliferation, induce local inflammation in blood
vessels, activation of macrophages, promote expression of cell adhesion molecules and
induce secretion of matrix metalloproteinases [41]. Both IL-1 and TNF-α, are powerful
inhibitors of lipoprotein lipase leading to increase the levels of triglycerides. TNF-α also
exerts other metabolic effects related with atherosclerosis including redistribution of adipose
tissue and impairment in the insulin sensitivity [41]. Some authors have described in women
with SLE and history of cardiovascular disease an increase in plasmatic levels of TNF-α and
soluble receptors of TNF1 and TNF2 compared with those patients without cardiovascular
disease [42]. Patients with cardiovascular disease also had a positive correlation between
levels of TNF-α and cholesterol or triglycerides [42].
Autoantibodies
a) Anti-Oxidized Low Density Lipoproteins (Anti-Oxldl Antibodies)

After the migration from the blood circulation to the subendothelial layer of the artery,
the low-density proteins (LDL) particles suffer an oxidative modification which is
atherogenic. These oxidized LDL promote proliferation of smooth muscle of the arteries,
activate monocyte-macrophages, increase the foam cell formation, leukocyte recruitment and
interfere with endothelium mediated relaxation [43-45].
The oxidation of LDL transform these lipoproteins in antigenic. Autoantibodies reacting
with oxidized LDL have been found in the atherosclerotic plaques. In animal models, the
titers of antibodies against oxidized LDL (anti-Ox-LDL) correlates with the progression of
atherosclerosis and in humans, the presence of anti-Ox-LDL is independent risk factor for
progression of carotid plaques [15].
b) Antiphospholipid Antibodies
Beta 2-glycoprotein I, is a molecule located on the surface of endothelial cells, in
subendothelial regions and the intimal of the arteries. Antibodies directed against to Beta 2-
glycoprotein I (anti-B2-GPI) have been associated with the development of atherosclerosis.
B2-GPI can bind Ox-LDL resulting in the complexe denominated B2-GPI-Ox-LDL, a very
stable complex that may accelerate the process of atherosclerosis [43]. Antibodies directed
against this complexe increases LDL uptake by macrophages contributing to the foam cell
formation. Anti-B2-GPI also binds endothelial cells inducing the expression of adhesion cell
molecules increasing the adhesion of monocytes to the endothelium [46]. Interestingly,
further evidence in several studies considered functional differences in the classes of
immunoglobulin that are compound of the antibodies anti-B2-Ox-LDL. Whereas, IgG
autoantibodies directed against B2-GPI-ox-LDL are atherogenic, the IgM anti-ox-LDL seems
to be protective [46, 47].
Anticardiolipin antibodies have a controversial association with cardiovascular events. In
a study performed in middle-age-men, the frequency of anticardiolipin antibodies was greater
in those patients who developed myocardial infarction or death associated to cardiac event
compared with patients who did not developed cardiac disease [48]. However, a more recent
study failed to find a positive association between high titres of antibodies anticardiolipin
antibodies, anti-Ox-LDL and myocardial infarction [49]. In patients with SLE the presence of
antiphospholipid antibodies is associated with coronary disease. In a prospective cohort of
166 patients with SLE after the adjustment by other important risk factors, the presence of
lupus anticoagulant increase the probability to develop ischemic coronary disease and stroke
[50]. Other cohort studies have also confirmed the increase in risk for coronary artery disease
in SLE patients with antiphospholipid antibodies [51, 52].
Anti-Heat Shock Proteins
Heat shock proteins (HSPs) have been found in atherosclerotic plaques and there is some
evidence regarding an association between plaque inflammation and higher levels of
antibodies directed against these HSPs [53]. The role of this anti-HSP in atherosclerotic
lesions can be explained as an effect in response to the injured endothelium that expresses
HSPs, or instead, be a cross-reaction originally directed against some microorganisms that
infected the arterial walls where HSPs may be expressed as response to these infections. In
support of the second hypothesis, experimental models have observed that the rabbit vascular
cells infected by Chlamydia pneumoniae (infection that has been linked to atherosclerosis),
express the HSP-60 leading to mitogenic effects with increase in the number of vascular
smooth muscle cells that is a step in the development of atheroma [54]. Anti-HSPs 65/60 are
experimentally involved in endothelial cytotoxicity [55]. Higher titres of anti-HSP65 have
been associated with advanced carotid atherosclerotic lesions and an increase in the risk for
5-year mortality [56]. Anti-HSP65 antibodies have been also associated with coronary
calcification assessed by electron-beam computed tomography [57].
Infections
Chlamydia Pneumoniae is an infectious agent associated with the development

atherosclerosis. Lipopolysaccharides of this microorganism are associated with increase in
the levels of C-reactive protein, and HSP-60 both involved in the development of
atherosclerosis and the inflammation of the vascular bed. Molecular evidence of Chlamydia
infection has been found in some patients with SLE and coronary artery calcification
evaluated by electron beam computed tomography [58]. Although, many confounding factors
can be implied in these findings and other studies should be performed to confirm this
possible association. Therefore, in the case of be confirmed the role of infections in
atherosclerosis further therapeutic strategies should be evaluate to treat these infections to
reduce the risk of coronary disease in SLE.
Cohort Studies and Risk Factors for Atherosclerosis
A number of cohort studies have recently evaluated the effect of different risk factors on
the development of vascular events. These studies are important because may proportionate
important clues for evaluation of causal associations. In the lupus cohort of Toronto
University from 1,087 patients included the frequency of atherosclerotic vascular events was
10.9% [29]. A number of factors were associated with cardiovascular events particularly was
interesting the observed association with neuropsychiatric involvement [29]. LUMINA, is a
multiethnic cohort performed in United States. In this cohort 546 patients were assessed for
risk factors associated with the subsequent development of vascular events including
myocardial infarction or angina, stroke and peripheral arterial involvement. The frequency of
vascular events was 6.2%, being more frequently observed the cerebrovascular involvement
(18 events), followed by cardiovascular (13 events) and peripheral vascular (5 events). The
risk factors associated with vascular events were older age, current smoking, longer follow-
up time, elevated serum levels of C-reactive protein and antiphospholipid antibodies (30). In
the Hopkins Lupus Cohort, 380 patients with SLE were followed in order to assess the
development of atherosclerosis and/or coronary artery disease. Patients with lupic
anticoagulant developed a higher frequency of myocardial infarction (22%), but
antiphospholipid antibodies were not associated with subclinical atherosclerosis including
carotid plaques or coronary calcification [51].
Whereas, an important number of cohort studies have been developed to assess
atherosclerosis in adults patients with SLE. The information proceeding from follow-up
studies in pediatric population is limited. The incidence and risk factors for atherosclerosis in
pediatric patients are not well known. In a recent prospective cohort, 139 children were
followed in order to determine the role of active disease and therapy on the abnormalities of
lipid levels [59]. A reduction in the prednisone dose during the follow-up was associated with
decrement in the levels of cholesterol and triglycerides, whereas, those patients with
proteinuria developed increase in cholesterol, triglycerides and LDL levels [59].
Diagnostic Methods in Atherosclerosis
The approaches to study atherosclerosis can be classified in two different forms: Those
that the outcomes rely on clinical events (or primary endpoints) including myocardial
infarction, stroke or death caused by atherosclerotic complications, being these events
presented late in the disease. The second approach is based in to study surrogate measures
that detect atherosclerosis earlier, where the impact of preventive strategies can modify the
natural course of the disease. Therefore, for this second approach the useful of noninvasive
techniques for the detection of atherosclerosis even in asymptomatic patients can be utilized
in order to establish opportune therapeutic measures. Overall, the assessment of
atherosclerosis may involve noninvasive techniques (B-mode ultrasound or electron beam
computed tomography), semi-invasive techniques or invasive techniques (coronary
angiography).
Coronary Angiography
Coronary angiography is the gold standard technique to assess the severity of stenosis of
coronary arteries caused by atherosclerosis. Angiitis of the coronary vessels, thrombosis
caused by hypercoagulable states, and coronary spasm that are entities to be considered in
SLE, can also be assessed by coronary angiography [60]. Figure 5 shows a partial obstruction
of the coronary artery caused by atheroma in the coronary angiography.
An “old concept” in coronary disease, is that in order to produce a myocardial infarction

is required a significant occlusion of the coronary artery usually higher than 50%. However,
more recent evidence suggests that the rupture of an atherosclerotic plaque can produce
myocardial infarction even without significant coronary stenosis. Vulnerable atherosclerotic
plaques that are associated with mortality have been studied in their histopathology.
Figure 5. Coronary angiography showing the presence of atheroma that produces an incomplete
obstruction of the vascular lumen in a patient with systemic lupus erythematosus.
In general around 60% of the plaques susceptible of rupture have a thrombogenic core of
lipids, macrophages, T cells, neovascular formation and calcium deposits. Presumably the
ruptures of these types of plaque are caused by the macrophages digestion and the apoptosis
of smooth muscle cells induced by cytokines. Whereas, in another 30-40% of patients
without coronary stenosis there are coronary thromboses overlie plaques with significant
luminal inflammation [61]. In SLE patients inflammatory cells infiltrate may be considered a
major marker of plaque vulnerability even independently of plaque structure or stenosis.
Therefore, major efforts need to be done in order to determine by other techniques the
coronary involvement without the limitations of coronary angiography.
Intracoronary Ultrasonography
This technique can be useful to identify atheroma in arteries with normal angiography
providing insights into the extent and distribution of atherosclerotic plaque and evaluating
both the vessel wall and plaque morphology [62].
Intracoronary ultrasound is useful to evaluate the plaque core. A calcified plaque is

echoreflective, whereas, a fibrous plaque is hyperechoic and a plaque with a lipid-rich core is
hypoechoic. The reliability of this technique is higher to distinguish calcifications in the
plaques or fibrous plaques but decreases significantly to distinguish lipid-rich plaques. Since
the epidemiological point of view, this technique is limited by their invasiveness and their
complications. To date, there are no published reports evaluating the role of this technique in
lupus patients.
Electron Beam Computed Tomography and Helical Computed Tomography
The evaluation of coronaries by electron beam computed tomography and helical

computed tomography constitute two related techniques to evaluate calcification in coronary
arteries using computed tomography. Pathological studies have shown a good correlation
between the severity of calcification in atherosclerotic plaques and the severity and extend of
coronary heart disease [63]. Under this basis, the assessment of coronary calcification
constitutes a valuable surrogate measure to detect early atherosclerosis. Currently, there is
some studies assessing the prevalence of coronary calcification in SLE assessed by electron
beam computed tomography. In an interesting study, the performance of the Framingham risk
score for increased cardiovascular risk was evaluated in asymptomatic women with SLE.
These patients were also evaluated by electron beam computed tomography in order to detect
coronary atherosclerosis [64]. There were no statistical differences in the Framingham scores
between patients with SLE and controls, although; the prevalence of coronary calcification
was significantly higher in SLE (19.4% versus controls 6.2%, p=0.02). This study shows two
important aspects: the prevalence of asymptomatic coronary disease is high in SLE and the
performance of Framingham risk score to predict coronary calcifications in SLE is low and
can underestimate the true risk for coronary disease. Another recent study evaluated the
variables associated with coronary artery calcification by electron beam computed
tomography in SLE. Higher age, homocysteine concentrations and disease duration were
associated with coronary artery calcification [65]. The association between
hyperhomocysteinaemia and coronary artery calcification is relevant because is a modifiable
risk factor and future studies should evaluate the effectiveness of therapies used to decrease
this and other risk factors in order to reduce the incidence of coronary calcifications. Future
studies with electron beam computed tomography of the coronary arteries also should
demonstrate their utility as predictors of hard outcomes in SLE patients.
Magnetic Resonance Coronary Angiography (MRCA) and Positron Emission

Tomography
MRCA can provide a 3 dimensional of the coronary arteries being useful to detect large
stenoses. Although, small stenoses can be not detected constituting an important limitation in
terms of its utility for the prevention of coronary disease complications. Positron emission
tomography (PET) can be used to evaluate coronary flow and flow reserve. The main
disadvantage is its inability to detect coronary stenosis lower 50%. Some authors found in
patients with hypercholesterolemia show that myocardial blood flow can be improved after
lipid lowering therapy suggesting a promissory role in the research of results of lipid-
lowering therapy [66]. So far the evaluation of these strategies in SLE requires additional
studies.
B-Mode Ultrasound
B-mode ultrasound constitutes a noninvasive technique for the visualization of intima-

media thickness (IMT) in the lumen of the carotid, aortic or femoral arteries [67]. Carotid
IMT measurement correlates adequately with coronary atherosclerosis and is an independent
risk factor for coronary heart disease in general population. Patients with SLE have a higher
prevalence of carotid plaques shown by this technique compared with controls [68].
A potential of this technique is to monitor the changes in IMT in response to therapy.
Strategy that requires be evaluated in future studies of prevention for coronary heart disease
in SLE.
Myocardial Perfusion Scintigraphy
Some recent series have showed that a high proportion of patients with SLE had
abnormalities in scintigraphy. One study found a prevalence of 28% of abnormalities
evaluated with scintigraphy with single photon computed tomography with technetium 99-
sestamibi. None of these patients had angina or antecedents suggestive of coronary
involvement. Factors associated with abnormalities in the scintigraphy in this study were
associated with diabetes mellitus, lower levels of HDL or concurrent vasculitis [69]. Issues
regarding to specificity and predictive values of this technique in SLE should be solved in
future researches.
Coronary Flow Reserve Evaluated by Doppler
Coronary flow reserve (CFR) measurement is used to assess epicardial coronary stenoses.
CFR also, can be utilized to examine the integrity of microvascular circulation. Transthoracic
Doppler echocardiography is a useful technique to measure the blood flow velocity. This
technique is reliable to measure the blood flow velocity in the distal and middle segment of
left anterior descending coronary artery, circumflex coronary artery and right coronary artery.
Currently, the use of contrast enhancement combined second harmonic-imaging technique
increased the feasibility of the transthoracic Doppler echocardiography [70]. The clinical
applications of the transthoracic Doppler to assess CFR include: functional assessment of
coronary stenosis, to detect severe stenoses, assessment of the wall motion, evaluate the
impairment of microvascular circulation. Doppler echocardiography to assess CFR also can
be used to refer a patient for a therapeutic procedure (such as percutaneous transluminal
coronary angioplasty), to monitor coronary restenosis or assessment of a reperfused

myocardial infarction. The utility of this method in SLE to predict patients who will develop
coronary artery disease require to be evaluated by prospective cohorts.
Treatment of Atherosclerosis in SLE
The treatment for accelerated atherosclerosis in SLE can be classified as a) preventive

(oriented to modify in asymptomatic patients the risks for a hard outcome such as myocardial
infarction or death), focused to decrease the progression and complications in a patient with a
previous cardiovascular event (such patients who already developed angina or myocardial
infarction) that is also known as “secondary prevention”.
The preventive treatment of atherosclerosis requires the early recognition of a patient in
risk. Risk factors such as smoking, obesity, diabetes mellitus, or physical inactivity should be
treated. Bruce et al reported that a significant proportion of their patients with SLE who
developed acute coronary events had a previous inadequate management of some potentially
reversible risk factors particularly hypercholesterolemia, smoking and obesity [71]. A more
recent study of this group reported that only 285 of their patients with hypercholesterolemia
were treated with lipid lowering treatment [72]. These two studies underline the importance
to treat adequately the risk factors for atherosclerosis in SLE. Patients with abnormalities in
the studies that show atherosclerosis including electron beam computed tomography or B-
mode ultrasound of the carotid should be considered candidate for preventive treatment in
order to decrease the risk of a hard outcome such as myocardial infarction or stroke.
Antimalarial Drugs in Atherosclerosis
Several reports have found a significant benefit of antimalarial drugs in decrease the
levels of cholesterol [73-75]. Together with their effects in cholesterol, antimalarial drugs
exerts effects in increase the levels of HDL and decrease levels of LDL. Recently, it has been
demonstrated an increase in the clearance of LDL cholesterol in patients with SLE who
receive chloroquine [76]. Antimalarial drugs should be considered in patients with SLE that
have abnormalities in their lipid profile, as a strategy to prevent atherosclerotic
complications. These drugs can be useful for reduce the abnormalities in the lipid profile in
patients who receive prednisone [77]. However, there is a lack of studies evaluating the effect
of these drugs using subrogate measures of atherosclerosis such as B-mode ultrasound or
electron beam computed tomography. This constitutes an interesting are for further research
in atherosclerosis.
Statins in SLE
Statins inhibit 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase. The drug

has effects decreasing the levels of lipids and affecting immune responses having a
pleiotropic pharmacological activity. Statins decrease the levels of C-reactive protein [78],
decrease the levels of some proinflammatory cytokines such as interleukin-6 and TNF-α [79].
Statins has immunomodulatory properties reducing the inflammatory component of the
atherosclerotic plaque [80]. Statins should be used with careful in SLE: is well established
the effect of these drugs on lipid metabolism, although statins may increase the B cell
reactivity by enhancing the Th2 immune response [80]. Statins have been associated with
lupus like syndrome and develop of antinuclear antibodies in patients without rheumatic
disorders with this treatment [80]. On the opposite side of these findings, in animal models of
lupus, statins may have benefits. Atorvastatin for example administered in murine models of
SLE reduce titres of anti-dsDNA antibodies, decrease the deposit of immunoglobulin on the
glomeruli, and decrease the proteinuria and azotemia [80]. Therefore, is important a deeper
evaluation of the effects of statins in patients with SLE. Unfortunately, there are some
barriers to evaluate statins in controlled clinical trials in patients with SLE. A recent paper
was unable to perform a randomized controlled trial of pravastatin in SLE caused by the lack
of participation, and high rates of dropouts [81]. These authors informed subsequently a
decrement in the total cholesterol and LDL observed in the group of patients who received
pravastatin compared with those who did not receive the drug, although the utilization of
corticosteroids decreases the effectiveness of the statin [82]. Patients with SLE usually
receive a multiplicity of medications that increase the risk of side-effects and the willingness
of the patients to receive new drugs. Patients who are nonparticipants have more concerns
about their health status, the medication used in the trial and the possible inclusion in the
placebo group compared with those who accept their participation [83]. Trials evaluating
preventive strategies should take into account the possible high rates of no-participation of
these patients. A double-blind multicenter trial is required in order to raise a sufficient sample
of patients to evaluate the effect of statins in SLE and give a relevant information to support
the clinical decision making.
Aspirin Therapy
The lack of evidence from controlled trial about the benefits of aspirin in the prevention
of myocardial infarction in SLE is intriguing, because aspirin is an old-drug with recognized
benefits to decrease the rates of coronary disorders in other populations. An important
number of randomized controlled trials have shown that low-dose aspirin decrease the risk
for myocardial infarction in men of the general population. However, a randomized trial was
unable to demonstrate benefits in healthy postmenopausal women in order to decrease the
rates of nonfatal myocardial infarction or death from cardiovascular causes [84]. In SLE there
is a lack of controlled studies to evaluate if aspirin decrease or not the risk for myocardial
infarction. This is an important issue that requires to be evaluated in controlled trials, because
these patients constitute a high-risk group for cardiovascular events by the factors enunciated
previously. Although, other factors should be considered, among these factors is quite
relevant that a high proportion of patients with SLE are concurrently treated with
nonsteroidal anti-inflammatory drugs (NSAIDs). There is evidence in the literature that the
concomitant regular utilization of NSAIDs interferes with the benefits of low-dose aspirin in
the prevention of myocardial infarction [85]. This finding is particularly important in patients
with SLE that are chronic consumers of NSAIDs and further studies should be performed to
solve if aspirin can be protective in SLE even with the concomitant administration of
NSAIDs.
Angiotensin-Converting–Enzyme Inhibitors
Angiotensin converting-enzyme inhibitors (ACEI) are frequently used to treat the

hypertension in SLE patients, although, to date there are no controlled studies have been
performed to evaluate their role on the reduction of the cardiovascular mortality in these
patients. The evidence of the literature on other populations shows that ACEI reduces the
rates of death from cardiovascular causes, myocardial infarction and stroke in high-risk
patients for the development of coronary heart disease including among these diabetes
mellitus [86, 87]. But there is no information regarding the long term effects on the protection
of coronary heart disease in SLE with the use of these drugs. Therefore, although the
evidence in other populations point-out to additional benefits with these drugs in
atherosclerosis, is currently required to evaluate the effect of ACEI inhibitors on the primary
and secondary prevention of atherosclerotic complications in SLE.
General Measures Used for Atherosclerosis
Because of the evidence that moderate and high doses of corticosteroids contribute to the
development of coronary heart disease in patients with SLE, it is recommended the utilization
of these drugs at the lowest dosage guided by the severity of the disease activity, and
reducing these doses when possible. An adequate evaluation of the disease activity is
mandatory to take appropriate measures regarding to the corticosteroid dosage. Because the
evidence described above also denote that corticosteroid can be also protective if they are
used adequately in patients with chronic inflammation.
Treatment of Atherosclerotic Complications
In 2004 Ward reviewed in a state hospitalization database the outcomes of all the
hospitalizations for myocardial infarctions and strokes in patients with SLE and compared
them with the outcomes in controls without SLE [88]. No significant differences were
observed between SLE and controls in the following outcomes: risk of in-hospital mortality
or congestive failure, although men with SLE had longer length of stay compared with
controls [88]. A number of case-reports have been published describing the results of
percutaneous transluminal coronary angioplasty and coronary stent in SLE, although; there is
a lack of information of follow-up studies to evaluate the rates of coronary restenosis in these
patients. Being an interesting issue to be evaluated in future cohort studies because the
restenosis could be expected be higher in these patients due to the high number of coronary
risk factors that they present.
Conclusions in the Management of Risk Factors for Atherosclerosis
In an excellent paper Bruce summarize some of the recommendations to treat the risk
factors of atherosclerosis [89]. The strategy recommended is to select targets that should be
achieved by individual patients. These targets should be take into account the main risk
factors for coronary heart disease such as blood pressure, levels of lipids, other comorbid
diseases (such as diabetes mellitus or obesity), smoking, etc. Patients with SLE can be
beneficiated with aspirin if they have additional risk factors such as a previous event of
coronary heart disease, or presence of antiphospholipid antibodies. ACE inhibitors are
recommended for patients with heart failure, diabetes mellitus or left ventricular hypertrophy
[89]. Currently, there is adequate evidence to recommend antimalarial drugs in those patients
with an abnormal lipid profile, although the data for take a position about statins is waiting
for controlled studies. Strategies in order to decrease homocysteine levels in SLE are still
without be evaluated by controlled trials and therefore is uncertain their benefit in SLE.
PULMONARY HYPERTENSION
Definition
Pulmonary arterial hypertension (PAH) is an elevation of the pulmonary vascular

pressure caused by an increase in pulmonary arterial with or without increase in the venous
pressure. Hemodynamically, PAH can be defined as an increase of the pulmonary artery
pressure at resting greater than 25 mm Hg at rest or greater than 30 mm Hg during exercise
measured invasively with a pulmonary artery catheter. Although, other definitions are based
in a measure systolic pulmonary artery pressure greater than 30 mm Hg [90].
The prevalence of pulmonary hypertension in SLE depends of the method employed for
the assessment. In the clinical context PAH can be identified in various scenarios: a) during a
directed assessment for the pulmonary pressure in a symptomatic patient, b) during the
screening of patients at risk or c) be discovered incidentally during the evaluation of an
asymptomatic patient.
Prevalence Studies
Prevalence of PAH reported in SLE depends of the design of the study and the method
used for the diagnosis. PAH in SLE was considered previously as a very rare entity and with
very high-mortality at the short term of follow-up, this because that in earlier studies PAH
was diagnosed almost exclusively during a cardiac catheterization performed in patients with
severe symptomatology. Similar findings are found in studies based in diagnostic obtained
from clinical charts where PAH seems to be observed infrequently in SLE. A more recent
retrospective study found only 46 patients with PAH from a lupus cohort of 786 patients [91].
Nevertheless, one of the main disadvantages of this study is the lack of a systematic
assessment to exclude PAH in the remaining 740 other patients [91], limiting the validity of
their conclusions.
Nowadays, the utilization of non-invasive methods particularly the Doppler
echocardiography has allowed the early detection in asymptomatic population. In patients
evaluated by Doppler echocardiography the prevalence of PAH in SLE goes from 11% to
43% [92-94]. In a prospective study we systematically assessed 204 outpatients with SLE
from two rheumatology clinics using Color-Doppler echocardiography technique, obtaining a
prevalence of PAH of 18% (defining PAH as a finding of systolic pulmonary artery pressure
>30mmHg) [95]. However, there are important concerns about the specificity of Doppler
compared with catheterism and the reproducibility of the Doppler studies when they are
evaluated sequentially. Recent reviews have described that the performance of Doppler to
measure PSAP has been increased with an appropriate training of the evaluators, the
development of the new technology of the equipments and protocols for the measurement of
pulmonary artery pressures.
Postmortem Studies
There is a lack of studies of prevalence of PAH based in findings of autopsies. When the
pathologist is oriented to evaluate changes suggestive in the pulmonary arteries, the
prevalence of these changes seems to be increased. Our group has found a high frequency of
histological changes compatible with moderate or severe pulmonary hypertension in a group
of autopsies of patients with SLE from a tertiary-care center. Interesting in only a minimum
number of patients was suspected cardiovascular disease although, autopsies showed a high
proportion of cardiovascular abnormalities.
An important determinant of the lower frequency of the “antemortem” diagnosis of PAH
is the relative absence of symptoms in earlier stages. Most of the patients included in our
cohort with pulmonary artery pressure between 30 to 40 mm Hg described only mild
symptoms or they were asymptomatic [95]. Symptoms described by the patients with
moderate or severe pulmonary hypertension include dyspnea, dyspnea during the
performance of activities or exercise, fatigue, syncope, or an unspecified chest pain. Clinical
but not early findings suggestive of PAH are data of heart failure (oedema in legs,
hepatomegaly, raised jugular venous pressure, etc.). A loud pulmonic heart sound was
observed in around the half of the patients with PAH in a study based in a retrospective chart
review [91].
Pathogenesis
The specific mechanisms that cause pulmonary hypertension in SLE remain unknown.
Although pathological findings are very similar to the primary form of pulmonary
hypertension and the response to some therapies suggest similar mechanisms of development
between both entities. In both, PH associated with SLE and primary PAH, the characteristic
of a severe disease observed in the pulmonary vessels is the plexiform lesion that is
constituted by an abnormal proliferation of endothelial cells, smooth muscle thickening and a
remodeling of the adventitia (see figure 6). In SLE genetic polymorphisms, environmental
factors and inflammatory mechanisms seems to participate in the pathogenesis of PAH. Some
similitudes are observed between PAH associated to SLE and the primary form of PAH. In
cases of the familial PAH, a variety of the primary form of PAH, there is found mutations in
the bone morphogenetic protein receptor 2 (BMPR2), that is a member of the transforming
growth factor- beta (TGF-β) superfamily that play a central role in the programmed cell
death. Abnormalities in the regulation of this apoptosis could promote the angiogenesis
developing the classical proliferation of the vascular endothelium [96, 97]. Primary PAH
present abnormalities in the production and degradation of nitric oxide (NO). Increase in the
expression of vascular endothelial growth factor (VEGF), angiopoietin-1, 5-lipoxygenase, 5-
lipoxygenase activating factor, and endothelin-1, have been found in lungs from patients with
severe pulmonary hypertension [98].
Figure 6. A. Obliteration of the pulmonary vascular lumen (thin arrows) in a case of pulmonary
hypertension secondary to SLE. B. Plexiform lesions (wide arrow) in another case of pulmonary
hypertension in SLE. Hematoxylin-Eosin staining 40 x (Photo Courtesy of Dr. Jose Ornelas-Aguirre,
and Dr. Gonzalo Vazquez-Camacho. Department of Pathology, Centro Medico de Occidente IMSS,
Guadalajara, Mexico).
In systemic lupus erythematosus, high levels of molecules that participate in the

pathogenesis of primary PAH have been also found. High levels of endothelin-1 have been
observed in patients with SLE and PAH [92] suggesting a common participation of
endothelin in the genesis of both primary form and pulmonary hypertension associated with
SLE. High levels of VGEF and its receptors have been found in active SLE [99]. Although,
these levels have not been investigated in PAH in SLE and further studies should be
performed to evaluate if VGEF is associated with this entity.
In both primary and secondary forms of PAH including those associated with SLE the
pathological changes observed in the pulmonary arteries include medial hypertrophy,
concentric intimal fibrosis, in situ thrombosis, pulmonary arteritis and typical plexiform
lesions [100]. Inflammatory infiltrates with macrophage and lymphocytes in the lesions are
observed in SLE suggesting that inflammatory cells and their products may cause these
lesions. Therefore, this is the theoretical basis for the utilization of immunosuppressive
therapies in PAH associated to SLE. Some cytokines such as tumor necrosis factor alpha
(TNF-α), interleukin-6, interferon gamma and interleukin-2 have been associated with other
forms of PAH but their role in the PAH in SLE, still have not been sufficiently evaluated.
Around a decade ago was observed that patients with SLE and PAH had higher levels of IL-6
compared with controls but their effect on the pathogenesis of the disease remain unsolved
[101].
Some autoantibodies have been associated with PAH associated to SLE among these the
presence antiphospholipid antibodies have been with certain frequency reported by almost 2
decades in patients with SLE and PAH [102, 103]. However, a recent study did not find
association of PAH with these autoantibodies [104].
Antiendothelial cell antibodies were reported increased in patients with SLE with PAH
by most of a decade ago [105]. However, these have been reported in a reduced number of
patients with SLE and there is a lack of studies by other authors that support these findings.
Most recently the possible role for these autoantibodies has been investigated in patients with
idiopathic PAH and the form associated with scleroderma [106]. In SLE patients these
autoantibodies could participate in the release of endothelin-1 as response to the vascular
injury [107], being an interesting hypothesis to be explored in SLE with PAH.
Diagnostic of PAH
Most of the diagnostic methods show only suggestive findings that are not
pathognomonic of PAH. Therefore, the gold standard for the diagnosis continues being the
invasive assessment of pulmonary pressure. There are some findings in obtained with the
non-invasive methods that require further evaluation for PAH in SLE.
Chest radiograph: may show cardiomegaly with prominent pulmonary artery segments
and oligaemic lung fields (see figure 7).
Electrocardiography: may show changes of right ventricular hypertrophy that are
nonspecific or enlargement of the atrium.
Figure 7. Plain radiograph of a woman with pulmonary artery hypertension and SLE. Note the
prominence of the pulmonary arteries. The cardiac catheterization identified a pulmonary pressure of
108 mmHg.
Bidimensional-echo. The findings on the 2D-echo are unspecific and only can be used
for suspicion of PAH but not confirm the presence of this entity. Most patients with
symptomatic PAH present enlarged right-side chambers. In moderate - severe cases as
response to pressure overload they may have a reduced right ventricular systolic function
accompanied or not by a systolic flattening of the interventricular septum and thickness of the
septum wall. The ratio: interventricular septum / posterior left ventricular wall is increased
(>1). Usually the systolic global function is preserved excepting in cases with myocardial
failure that predicts a poor prognosis.
Color-Doppler Echocardiography
Doppler echocardiography is the best non-invasive method in order to estimate the

systolic pulmonary artery pressure (SPAP). Most of the patients with PAH have tricuspid
regurgitation. This findings is useful to compute the sPAP using the Bernoulli formula which
consist in: multiply 4 (maximum velocity tricuspid regurgitation)2 + right atrial pressure.
Different formulas have been used to calculate the right atrial pressure that ranges usually
from 4 to 10 mmHg. As other measures of echocardiography the accuracy of the
measurement of pulmonary artery pressure depends importantly of the experience of the
personnel who perform the echocardiography. The correlation between Doppler and cardiac
catheterization findings of the SPAP is considered as good [108]. There are 3 main
advantages of Doppler for PAH: a) Doppler echocardiography the most reliable non-invasive
method to measure PAP in patients under suspicion of PAH, b) can be useful for prevalence
studies in patients with mild or absent symptomatology, c) seriated measures can be

performed in order to measure response to therapy or progression of the disease. A series of
studies have been now performed in order to detect PAH in SLE using Doppler
echocardiography [92, 94, 95, 104, 109-112]. Interestingly, only few reports have been
published about the clinical value of the measurement of PAP by Doppler-echocardiography
in order to predict the evolution. Winslow et al, more a decade ago shows that the frequency
of patients who develop PAH in SLE increase during the following [94]. Our group reported
that patients with SLE with higher values of pulmonary systolic pressure evaluated by
Doppler echocardiography develop a high incidence of cardiac failure in the following 3
years of the diagnosis [113]. Recent advances to improve the accuracy of the techniques have
been developed these include the development of new ultrasound imaging equipment, use of
pulsed-wave tissue Doppler imaging, contrast, tridimensional and intracardiac
echocardiography. Particularly, contrast echocardiography is a useful means of enhancing the
faint Doppler tricuspid flow signal that is essential for determining PAP.
Right Heart Catheterization
This method provides information about 3 important elements: a) Confirms the presence
of elevated pressure with absence of pulmonary venous hypertension, b) can predict survival
depending of the severity of PAH, the affectation of the right atrial pressure, and cardiac
index, c) finally right heart catheterization can be useful to evaluate the response to treatment.
Unfortunately this invasive method may have complications, and mortality has been observed
in some patients, also requires be performed in-hospital by trained personnel and is costly.
However, continue being the gold standard for the measurement of PAP and should be
performed in those patients with an echocardiogram suggestive of moderate – severe PAH.
Advances in the Treatment of Pulmonary Hypertension
Several therapeutic strategies have been evaluated in SLE these strategies include
calcium channel blockers, prostanoids, endothelin receptor antagonists, phosphodiesterase
type 5 inhibitors, immunosuppressive drugs and combination therapy.
Table 2 synthesizes the strategies employed for the treatment of patients with PAH in
SLE.
Table 2. Drug Therapy in Pulmonary Hypertension: Current and potential therapies

for pulmonary hypertension in SLE
Group of drugs
1. Vasodilators 2. Immunosuppressive drugs
Calcium channel antagonists a) Cyclophosphamide
a) Nifedipine
Prostacyclin therapy 3. Anticoagulants
a) Intravenous Epoprostenol a) Warfarin
b) Beraprost
c) Treprostinil 4. Conventional management
a) Oxigen therapy
Endothelin Antagonists b) Inotropic agents
a) Bosentan c) Diuretics
b) Sitaxsentan
c) Ambrisentan
Nitric Oxide
Phosphodiesterase Inhibitors
a) Sildenafil
Calcium Channel Blockers

Vascular tone is regulated by the entry and exit of calcium from the smooth muscle.
Calcium channel blockade with calcium antagonists produces vasodilatation by decreasing
calcium entry into vascular smooth muscle. Interesting there is no studies evaluating the
effects of the new calcium channel blockers in PAH associated with SLE. Nifedipine has
been considered as a therapeutic option although this information proceeds from case-series
and still further studies are required to evaluate their benefits as concomitant therapy.
Prostanoids
Intravenous epoprostenol was one of the first drugs with substantial efficacy showed in
primary PAH, thereafter, other prostanoids were subsequently developed. Other prostanoids
include iloprost, and treprostinil. Prostanoids activate the cell surface prostacyclin receptors
producing vasodilation. Some of the effects of prostanoids may be mediated by activation of
the peroxisome proliferator-activated receptor , producing antiremodeling effects [114]. In
SLE with PAH) only some case-series and case-reports have been described In one of these
series 6 patients with moderate-severe PAH associated with SLE, the utilization of
epoprestenol was associated with a decrement of both mean pulmonary artery pressure and
pulmonary vascular resistance in 4 patients with an associated improvement in the New York
Heart Association functional class, suggesting that this therapy can be effective in PAH
secondary to SLE, although the lack of comparative studies is an important limitation in the
understanding of the efficacy of the drug for these patients. Iloprost has been also only
evaluated in case-reports of patients with SLE and PAH. Controlled studies with these drugs
are required to demonstrate its efficacy for these patients and a careful examination of their
side-effects and long- term results should be performed before to establish a conclusion of the
benefits of these therapies.
Endothelin Receptor Antagonists

Bosentan is an endothelin A/B receptor antagonist. Several studies have showed clinical
efficacy of bosentan in patients with primary PAH or associated with connective tissue
disease [115, 116]. Recently, it has been developed ambrisentan an oral endothelin A
receptor-selective antagonist. This drug has been evaluated in a randomized, placebo-
controlled trial made in patients with primary PAH. Showing an improvement in exercise
capacity, WHO functional class, 6-min walk distance, and hemodynamic parameters [117].
Sitaxsentan, is another selective endothelin A receptor antagonist showing benefits in patients
with PAH [118]. There is no information currently available about whether a selective
endothelin A receptor antagonists offer or not advantages over a nonselective endothelin A/B
antagonists in patients with PAH, and data from comparative studies with both drugs are still
lacking.
Phosphodiesterase Type 5 (PDE5) Inhibitors

There is currently a relevant interest about the efficacy of Sildenafil in the treatment of
PAH [119]. Sildenafil has effects as vasodilator but also has antiproliferative actions on
pulmonary vascular smooth muscle cells by reducing cell proliferation and inducing apoptosis
[120]. In a randomized trial different doses of sildenafil (20, 40, or 80 mg three times daily)
were compared with placebo showing significant improvement in 6-min walk distance,
functional class and hemodynamic parameters [119]. The final results of the study showed
non-significant differences in efficacy between doses, therefore, currently the recommended
doses for treatment of PAH is 20 mg three times daily. In an outstanding paper Wilkins
published the first double-blind study comparing bosentan versus sildenafil in patients with
PAH including both the idiopathic form and the associated with connective tissue disease
[121]. Although, their results showed no significant differences between both groups, there
was a trend in higher improvement for the 6-min walk distances, plasma brain natriuretic
peptide levels, and right ventricular muscle mass determined by magnetic resonance imaging
in favor of sildenafil. Further controlled trials comparing both drugs are required to support
the reliability of these findings.
Immunosuppressive Drugs
There is a lack of information from controlled studies using immunosuppressive drugs
for the treatment of pulmonary hypertension in SLE. Most of the information proceed from
case-series or not controlled trials. Our group reported the results of monthly administered
intravenous cyclophosphamide versus oral enalapril and standard care measures in patients
with pulmonary hypertension [95]. A significant improvement in functional class and a
decrement in the pulmonary pressure (measured by Doppler echocardiography) were
observed for patients in the arm of cyclophosphamide compared with enalapril. According to
our results when the pulmonary systolic pressure is ≥ 35 mm Hg only 2 patients are required
to be treated for obtaining an additional improvement over enalapril. Additional studies with
immunosuppressive drugs are required including a comparison head to head with
phosphodiesterase type 5 inhibitors or endothelin antagonists to determine the role of

immunosuppressive drugs in PAH associated with SLE. Also is currently ignored if these
immunosuppressive drugs are useful to increase the response when combined with bosentan
or sildenafil. To date, the utilization of immunosuppressive drugs constitutes a strategy that
should be considered in patients with moderate or severe pulmonary hypertension in SLE.
Table 2 shows a summary of the drugs used for PAH.
Combination Therapy
There is considered that combination therapy of PAH with prostanoids, endothelin
receptor antagonists, and PDE5 inhibitors will be an effective strategy for PAH when solid
evidence will become available. Unfortunately, most of the current evidence of the
effectiveness of combination therapy comes from uncontrolled studies and case-series. A
recent study was unable to show significant differences with the combination of inhaled
iloprost plus bosentan versus monotherapy [122]. The combination of bosentan and sildenafil
seems to be a reasonable option in those patients without a satisfactory response with
monotherapy and randomized controlled trials are required to evaluate their efficacy as first
therapeutic option in patients with severe PAH.
OTHER FORMS OF CARDIAC INVOLVEMENT
Left Ventricular Diastolic Dysfunction
The cardiac cycle encompasses systole and diastole. The systolic function reflects the
ability of the ventricle to contract and eject the blood to the pulmonary or systemic
circulation. Instead, diastolic function reflects the ability of the ventricle to relax and fill.
Diastolic dysfunction constitutes a disturbance in ventricular relaxation, distensibility or
filling [123].
Diastolic dysfunction can be asymptomatic or be accompanied of symptoms or heart
failure. Also patients with diastolic dysfunction can have associated or not impairment in the
ejection fraction. When the patients have a preserved ejection fraction but symptoms such as
dyspnea associate with efforts, venous congestion, oedema and the finding of diastolic
dysfunction these patients are classified as having a diastolic heart failure. Diastolic function
also depends of numerous interrelated contributing factors: including heart rate, loading
conditions and contractility.
Doppler echocardiography is a noninvasive technique that provides the means to assess
specific abnormalities of diastolic function. A series of indices have been provided to assess
diastolic function by Doppler echocardiography. The most usually used are the mitral inflow
velocities. The E wave corresponds to early flow during left ventricle relaxation whereas the
A wave corresponds to the wave originated by the late ventricle filling following the atrial
contraction. Under normal conditions E wave exceeds A wave velocity. In case of impaired
relaxation, A wave velocity can exceeds the E wave velocity given an abnormal relation E:A,
and a prolonged deceleration of the E wave. However, when the left ventricular diastolic
pressure is highly increased, the contribution of the atrial contraction to the ventricular filling
decrease being E wave predominant but with a rapid deceleration. These abnormalities in the
contraction lead to a restrictive pattern with high E wave velocity, and an abnormal increment
in the E:A relation.
Diastolic Dysfunction in SLE
Diastolic dysfunction is highly prevalent in patient with SLE. Our group has found a
prevalence of diastolic dysfunction of 63% of 97 patients studied. This diastolic dysfunction
was not associated with age, disease duration, disease activity or anticardiolipin antibodies
[124].
In a recent report, patients with SLE and primary antiphospholipid syndrome were
evaluated using Doppler echocardiography [125]. Patients with antiphospholipid syndrome
primary or associated with SLE have impairment in the diastolic function compared with SLE
without antiphospholipid syndrome.
Currently, there are no studies to assess the incidence and consequences of diastolic heart
failure in SLE. Criteria for the diagnostic of diastolic heart failure have been proposed by the
European Society of Cardiology. These are the presence of signs and symptoms of congestive
heart failure, normal or mild abnormality of left ventricular systolic function and evidence of
abnormal left ventricular relaxation, filling, diastolic distensibility or diastolic stiffness
[126].To date there is no controlled trials to evaluate the treatment the diastolic heart failure
in SLE. The objectives of the treatment for diastolic heart failure for any patient include the
improvement of hemodynamic conditions, improving preload and afterload therefore,
therapeutic strategies should be directed to these targets [127]. Angiotensin converting
enzyme (ACE) inhibitors and angiotensin inhibitors have a beneficial because they reduce
both preload and afterload, induce regression of left ventricular hypertrophy and decrease of
myocardial interstitial fibrosis. Alderosterone antagonists including spironolactone and
canrenone also decrease the myocardial fibrosis. B-blockers and calcium antagonists such as
verapamile are useful to reduce tachycardia improving the diastolic filling.
Current recommendations for the treatment of patients with diastolic heart failure of any
etiology are presented in the table 3.
Table 3. Current recommendations for the treatment of diastolic heart failure of any
etiology
Drugs
Diuretics: furosemide, clortalidone, spironolactone
Long acting nitrates
ß adrenergic blockers (atenolol, carvedilol, nebivolol)
Calcium channel blockers: verapamile,
ECA-inhibitors (perindopril, rampiril)
Angiotensin II receptor blockers (irbesartan, candersartan)
Aldosterone antagonists (spironolactone)
THROMBOSIS AND AUTOANTIBODIES
Occurrence of thrombotic phenomenon in SLE patients has been recognized, as well as

its close relationship with autoantibodies, mainly those so called antiphospholipid (aPL), that
mainly encompass but not restricted to, anticardiolipin antibodies (aCL), anti-β2GPI
antibodies, and the lupus anticoagulant. However, aCL remains an ancillary stone for the
routine evaluation of SLE patients regarding serum antiphospholipid reactivity, and of
interest is that several cardiovascular manifestations associates with aCL positive status even
in absence of thrombotic phenomenon.
In a concise report [128] an evaluation was performed for the association of mannose-
binding lectin gene polymorphisms and cardiac manifestations in SLE patients, founding that
although the prevalence of cardiovascular disease in SLE patients carrying MBL-deficient
genotypes was 3.3 times higher than in patients with non-deficient genotypes, the association
was in fact with the coexistent antiphospholipid syndrome that these patients displayed.
Anti-Lamin B1 Autoantibodies as Major Determinant of Thromboprotection

in SLE Patients Displaying Antiphospholipid Antibodies
Autoantibodies to lamins, the major polypeptide components of the nuclear lamina, have
been reported in selected sera from patients with autoimmune diseases, including anti-lamin
B in systemic lupus erythematosus (SLE) and anti-lamins AC in autoimmune chronic active
hepatitis (CAH). In a carefully performed study, Senecal JL et al [129] studied the frequency,
specificity, and isotype of autoantibodies to major and minor lamins by immunoblotting on
purified rat liver lamins in 190 sera from normal controls (n = 62), rheumatic disease controls
(n = 42), and autoimmune disease patients (n = 86). The frequency of anti-lamin in normal
controls was 85.5%, and ranged from 77 to 100% in the other groups. In particular, anti-
lamin B was not more common in SLE than in normal sera. Anti-lamin isotype were IgG
and/or IgM. The highest end point titers (greater than or equal to 1:3200) were observed with
chronic autoimmune hepatitis (CAH), SLE, and rheumatoid arthritis (RA) sera with IgG anti-
lamins AC, B, or ABC, or with IgM anti-lamins ABC. None of these SLE and RA patients
had evidence of liver disease. Reactivity with minor lamins was more frequent in CAH. They
conclude from those initial works that anti-lamin autoantibodies were present in sera from
most individuals and that the highest titers are found in sera from patients with autoimmune
diseases and probably those patients displaying a highest titer anti-lamin antibodies could
represent a special subgroup. However, the non-specificity of these western-blot detected
anti-lamin antibodies was evident, and this notion was enforced by the findings of other
groups [130] that identified this non-specific IgG isotype anti-lamin reactivity using
immunofluorescence and western-blot techniques in sera of 60 patients with chronic fatigue
syndrome. These authors speculate that occurrence of autoantibodies to a conserved
intracellular protein like lamin B1 provides new laboratory evidence for an autoimmune
component in chronic fatigue syndrome, however, the anti-lamin reactivity in these patients
was at low titers, and it was already known that clinically healthy subjects could display this
reactivity.
Research on aLB1 reached an important advance once an specific solid-phase enzyme-

linked immunoassay using recombinant human LB1 was developed [131], solving the
discrimination step between unspecific low-titer reactivity and the High Titers aLB1, as
evidenced in the work by Senécal et al [131] determining the frequency and clinical
significance of high titers of IgG autoantibodies to nuclear lamin B1 in a large number of
unselected and well-characterized systemic lupus erythematosus (SLE) patients, disease
controls, and normal healthy controls.
Antibodies anti lamin B 1(aLB1) and Frequency of Thrombotic Events
In this cross-sectional study [131], detection of anti-lamin B1 autoantibodies with the

new immunoassay was performed on serum samples obtained at first evaluation of 238
consecutive French Canadian adults: 61 healthy control subjects, 20 patients with
osteoarthritis, 22 with ankylosing spondylitis, 11 with autoimmune hepatitis, 30 with
rheumatoid arthritis, and 94 with SLE. SLE patients were studied for 57 disease
manifestations. A case-control study was performed to analyze the relationship between anti-
lamin B1 status and thrombotic manifestations between SLE onset and last follow-up. High
titers of anti-lamin B1 were strikingly restricted to a subset of 8 SLE patients (8.5%). The
mean anti-lamin B1 titer was higher in this subset than in the other SLE patients or any
control group (P<0.001). By univariate analysis and stepwise multiple logistic regression, the
most striking association of anti-lamin B1 was with lupus anticoagulant (LAC) antibodies (P
= 0.00001). Although LAC were significantly associated with thrombosis in SLE patients,
anti-lamin B1 was not. The frequency of thrombosis in SLE patients expressing both LAC
and anti-lamin B1 was similar to that in patients without LAC (P = 1.0). However, patients
expressing LAC without anti-lamin B1 had a greater frequency of thrombosis (p = 0.018).
High titers of IgG anti-lamin B1 autoantibodies are highly specific for a subset of SLE
patients whose clinical characteristics include the presence of LAC and other laboratory
manifestations of the antiphospholipid syndrome. The presence of LAC without anti-lamin
B1 may define a subset of SLE patients at greater risk for thrombosis.
aLB1 and Thromboprotection
The fore mentioned findings introduced the concept that the expression of clinical
features associated to antiphospholipid antibodies may be co-modulated, at least from the
statistical approach. This hypothesis was of clinical relevance, because if confirmed, a new
clinical subset of patients displaying antiphospholipid antibodies may arise, and with the
presence of High-Titer aLB1 been the major determinant for thromboprotection.
The same group of researchers performed a cross-sectional study using serum samples
obtained at first evaluation of 259 English Canadian and French Canadian patients from SLE
registries at 3 hospitals [132] in order to demonstrate the association between autoantibodies
to nuclear lamin B1 (aLB1) and protection against thrombosis (“thromboprotection”) in
patients with SLE, and to elucidate the mechanism by which aLB1 cause thromboprotection
in vivo. Since a number of autoantigens in SLE have been localized specifically to the
external surface of apoptotic blebs, it was hypothesized that circulating aLB1 may block the
procoagulant effect of apoptotic blebs by binding to LB1 displayed at the external bleb
surface. In a subanalysis restricted to lupus patients the relationship between aLB1 and lupus
anticoagulant (LAC) status with thrombotic manifestations between onset of disease and last
follow-up were evaluated. Reactivity of aLB1 with Jurkat or endothelial cells, which had
been induced to undergo apoptosis, was determined by indirect immunofluorescence.
Localization of LB1 in apoptotic cells and blebs was analyzed by con focal microscopy and
surface labeling of cell membrane proteins. High-titer aLB1 was restricted to a subset of SLE
patients (46 patients), with an overall frequency of 17.8% (range 11.6-24.3% in the 3
centers). LB1 antibodies were significantly associated with LAC but not with antibodies to
cardiolipin (aCL) or beta(2)-glycoprotein I (anti-beta(2)GPI). The frequency of thrombosis
differed markedly depending on aLB1 and LAC status, as follows: presence of LAC and
absence of aLB1 50%, presence of both LAC and aLB1 22.7%, absence of both LAC and
aLB1 25.5%, absence of LAC and presence of aLB1, 20.8%. Further subclassification of
patients based on aCL and anti-beta(2)GPI status revealed that, in the presence of LAC but in
the absence of aCL, anti-beta(2)GPI, and aLB1, the frequency of thrombosis was 40%,
whereas in the presence of aLB1, it decreased strikingly, to 9.1%. LB1 was found to be
translocated into surface membrane blebs during apoptosis and to be entirely enclosed within
the apoptotic bleb plasma membrane of Jurkat and endothelial cells. The presence of aLB1 in
SLE patients with LAC essentially nullifies the strong prothrombotic risk associated with
LAC. Hence, aLB1 is associated with thromboprotection. Reactivity of aLB1 with apoptotic
blebs does not seem to play a direct role in mediating this protection, since LB1 is buried
within apoptotic blebs and inaccessible to circulating aLB1. The mechanism by which aLB1
confers thromboprotection in SLE remains to be elucidated.
Relationship between thrombophilia and cardiac manifestations in SLE are mainly
restricted to those involving intracardiac thrombus formation whether as free thrombus or
those aggregated over a cardiac structural abnormality.
Autoantibodies and Cardiac Involvement
Patients with SLE may display cardiac manifestations, and, in fact, several
autoantibodies can mediate cardiac damage: this is the case with antiphospholipid antibodies
(aPL), anti-Ro/SS-A antibodies, anti-endothelial cells antibodies, and so on [133].
Autoantibodies can directly affect heart tissue or, alternatively, can trigger mechanisms
able to cause heart damage. For instance, aPL can contribute to cardiac damage enhancing
atherosclerosis phenomena, causing thrombosis of coronary arteries or starting an immune
complexes mediated reaction and deposition at the valves level.
The consequence of autoantibodies damage have been reported in several heart
structures, such as valves, myocardium, pericardium, conduction tissue and cardiac arteries,
in patients suffering from systemic lupus erythematosus (SLE), antiphospholipid syndrome
(APS), Sjögrens syndrome and other connective tissue diseases.
VALVULAR ABNORMALITIES
Anatomical and functional valvular abnormalities have been described in SLE.

Libman.Sacks endocarditis, also termed “atypical verrucous endocarditis”, is the most
characteristic lesion [134]. In a recent study [135] a frequency of 1.5% for aortic valve
calcification in 199 SLE studied patients was described. Anatomical lesions were observed in
15 – 75% in necroscopy studies (136), in 40 – 50% of cases with the transthoracic
echocardiography (TTE) and in 50 – 60% with transesophageal echocardiography (TEE)
[137]. Therefore, TEE is more sensitive than TTE in revealing valve abnormalities.
Anatomical abnormalities are generally found in the mitral and aortic valves [137].
Histological studies have shown two types of verrucae: 1) active lesions which consists of
fibrin clumps, focal necrosis and mononuclear cells infiltrates, more frequently observed in
young patients with recent disease onset which rarely lead to a hemodynamically significant
valvular lesion; and 2) healed lesions characterized by vascularised fibrous tissue sometimes
associated with calcifications [17] found in patients with long-standing disease and with
long-term corticosteroids use and frequently associated with functional valve abnormalities,
especially valvular insufficiency. An association between valvular abnormalities and
antiphospholipid antibodies (aPL) has also been reported, [138] but this association remains a
matter of controversy. In a recent study [104], 200 SLE patients were studied to evaluate the
association of aPL with cardiovascular manifestations of SLE. Antiphospholipid antibodies
were present (defined as IgG or IgM anticardiolipin >40 IU/ml or the presence of lupus
anticoagulant) in 42 patients (21%), mitral valve nodules and moderate-to-severe mitral
regurgitation were more common in aPL-positive patients (both 14.3% versus 4.4%; P
<0.02). Thirty-one percent of patients with high titers of IgG aPL (>80 IU/ml) had mitral
valve nodules, compared with 20% of patients with mildly to moderately elevated levels of
IgG aPL (16–80 IU/ml) and 4% of patients without IgG aPL (overall P < 0.001). Levels of
soluble tumor necrosis factor receptors were higher in the presence of both aPL and mitral
valve nodules. LV dimensions, systolic function, and carotid artery stiffness as well as
prevalences of Raynaud’s phenomenon, pulmonary hypertension, and atherosclerosis were
similar in aPL-positive and aPL-negative patients. It is clear from this large cohort, that SLE
patients with aPL (particularly IgG aCL) are more likely to have mitral valve lesions and
consequent significant mitral regurgitation. The association of both aPL and soluble TNF
receptors with mitral nodules suggests a possible pathogenic link between those antibodies,
endothelial cell activation, and inflammation in the development of these lesions. In this
population, aPL are not associated with abnormalities in myocardial or large artery structure
and function, preclinical or clinical atherosclerosis, or other vascular manifestations such as
Raynaud’s phenomenon, pulmonary hypertension, or systemic hipertensión.
Several aspects of pathogenetic hypotheses involving potential association between
valvular abnormalities and antiphospholipid antibodies are reviewed in a recent works by
different groups [2, 133], contributing to understand the development of valvular
abnormalities in SLE patients: 1) aPL and anti-endothelial antibodies bind to and activate
endothelial cells, leading to platelet aggregation with thrombus formation; and 2) deposition
of immunocomplexes between the endothelium and the basal membrane, leading to an
infiltration of inflammatory cells.
In a work by other group verrucous endocarditis is generally asymptomatic and only

occasionally leads to a cardiac murmur. In fact, the verrucae are near the edge of the valves
and therefore tend not to deform the closing line, even when they are very large and protrude
into the cardiac chambers. Complications due to verrucous endocarditis are rare, although
embolic events can occur. Lesions are hemodynamically significant in only 3–4% of SLE
patients, requiring surgical treatment in half of them. Infectious endocarditis was observed in
7% of patients with valvular disease, and stroke or peripheral embolism in 13%. Rupture of
chordae tendinae was rarely observed. The risk of endocarditis is increased by dental surgical
treatments. Appropriate antibiotic prophylaxis should be considered in all SLE patients with
valvular abnormalities, especially if they are taking immunosuppressive drugs. Jensen-Urstad
et al [139] recently reported a close association between valvular abnormalities and
cardiovascular disease (CVD) as well as raised levels of homocysteine and triglycerides in
SLE patients. Therefore, patients with valvular disease should be screened for clinical and
subclinical atherosclerotic features.
Since Libman–Sacks endocarditis is clinically silent in the majority of cases, generally it
is not treated. When it is found in an early active stage, corticosteroids (prednisone 1
mg/kg/day) are recommended, especially if antiphospholipid antibodies and lupus
anticoagulant are negative. It has been reported that valvular abnormalities frequently
resolved over time. When endocarditis is detected at a later stage during the course of the
disease, careful clinical surveillance is necessary and, if the lesion becomes hemodynamically
significant, valve surgery is needed.It is necessary to carefully evaluate the type of surgical
treatment: valve repair, replacement with mechanical valve or bioprosthetic porcine graft.
Anatomic valve repair does not need anticoagulation but often leads to repeated surgery and
later valve replacement. Bioprosthetic porcine valves have also been hindered by
complications such as valvulitis relapse. Therefore, mechanical valve replacement seems to
be the best choice in lupus patients.
RHYTHM AND CONDUCTION TISSUE

ABNORMALITIES
Several reviews included these issues in their scope [2, 133]. Sinus tachycardia is the
most frequent rhythm abnormality and is quite common in SLE patients. Atrioventricular
block and bundle branch block are also observed. However, they are rare in adults and occur
in only 2% of children born from mothers with anti-Ro/SSA antibodies. Rhythm and
conduction abnormalities are mostly asymptomatic or may lead to some mild complaints such
as palpitation or fatigue. In some cases syncope may occur. Sinus tachycardia can be due to
fever, anaemia or cardiac abnormalities including pericarditis and myocarditis. Conduction
defects can also be the result of antimalarial use. In SLE patients with rhythm and conduction
abnormalities, electrolyte balance and thyroid hormonal status should be investigated. The
treatment is based on the use of common antiarrhythmic drugs and in the most severe cases
the implant of a pacemaker is necessary.
Heart Conduction Tissue and Anti-Ro/SSA Antibodies
The mothers of children born with complete heart block (CHB) are generally positive for
autoantibodies, namely anti-SSA/Ro and anti-SSB/La.32. This observation is consistent with
the assumption that maternal autoantibodies could affect the heart conduction tissue of the
newborn.
The transplacental transfer of maternal IgG autoantibodies seems to play a major role in
the pathogenic mechanism of CHB, although some questions are still debated. The facts in
favour of the direct pathogenic role of anti-SSA/Ro and anti-SSB/La autoantibodies in CHB
are: 1) the presence of maternal autoantibodies in fetal/neonatal circulation; 2) the elution of
anti-SSA/ Ro antibodies from cardiac tissues of affected fetuses; 3) the complete AV block in
pups of mice passively injected with anti-SSA/Ro 52 antibodies; and 4) the fact that anti-
SSA/Ro IgG inhibits, in vitro, L-type CA2þ channel in rat and rabbit heart. On the other
hand, against the role of Ro and LA antibodies in CHB is that: 1) CHB is exceptionally rare
in adults with these antibodies; 2) there is a discordance of CHB in twins; and 3) there are
few babies of Ro/La mothers affected by CHB.
Electrocardiographic abnormalities other than those found in CHB, have been reported in
association with anti-SSA/Ro antibodies, such as bradycardia, atrioventricular (A-V) blocks
of various degrees and prolongation of the QT interval. The abnormalities much closer to a
direct involvement of the cardiac conduction tissue are the incomplete A-V blocks and QT
prolongation. The QT interval prolongation was found to be transient, in fact it normalizes
within the first year of life when maternal anti Ro/SSA antibodies disappear from neonatal
circulation. In contrast with these findings, a recent case control study, comparing the
electrocardiographic features of children born to anti-Ro/SSA positive and negative mothers
all affected by connective tissue diseases, does not find any significant difference, showing
that some interpretation problem is not yet solved. In a recent work [140], an evaluation of
the QT dispersion in SLE patients without overt cardiac involvement was performed,
founding that QT dispersion is significantly increased in this patients but without no
correlation between QT dispersion and duration of SLE, SLEDAI-K score, corticosteroid
usage, and presence of anti-SS-A antibody. More work is needed to identify if this prolonged
QT dispersion can be a useful noninvasive method for early detection of cardiac involvement
in SLE patients.
CONCLUSION
In summary cardiovascular disease in systemic lupus represents a very exciting area for
future research being necessary to increase the number of long-term prospective cohorts and
well-designed controlled trials in order to improve the clinical care of the patients with this
involvement. Controlled studies evaluating the effect of modification of risk factors for
atherosclerosis and other cardiovascular complications on the morbidity and mortality of the
patients with SLE are still required. An adequate assessment of patients with SLE should be
taken into account these risk factors in order to modify the prognosis of the patients. Also an
early detection with the non-invasive methods of cardiac complications can lead to develop
new strategies for the treatment. Application of new drugs and newer indications for the use
of old drugs should be evaluated in controlled trials and prospective cohorts with the
objective of decrease the morbidity and mortality of the patients with these complications.
ACKNOWLEDGMENT
The authors of this chapter thank to Dr. María Rosa Flores-Márquez, Dr. Gonzalo
Vazquez-Camacho, and Dr. Jose Manuel Ornelas-Aguirre from the department of pathology
in the Centro Medico Nacional de Occidente IMSS Guadalajara Mexico by the histological
images for this chapter. Authors also thank Dr. Julia Dolores Sanchez-Hernandez and Dr.
Alberto Muñoz-Rocha by their valuable suggestions for the pathogenesis of atherosclerosis.
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Chapter 2
COMORBIDITY IN SYSTEMIC LUPUS

ERYTHEMATOSUS: ASPECTS OF
CARDIOVASCULAR DISEASE, OSTEOPOROSIS
AND INFECTIONS
Irene E. M. Bultink∗, Ben A. C. Dijkmans and

Alexandre E. Voskuyl
Department of Rheumatology, VU University Medical Center,
Room 4A49, PO box 7057, 1007 MB Amsterdam, The Netherlands
ABSTRACT
Over the last decades, the survival of patients with systemic lupus erythematosus
(SLE) has improved dramatically. Having improved treatment for active lupus disease,
the challenge is now to understand and prevent the long-term complications of the
disease, which may be due to the disease itself or the therapies used. To date, long-term
complications of SLE are now considered to be important, including cardiovascular
disease, osteoporosis and infections.
Cardiovascular disease in patients with SLE, including coronary artery disease,
ischemic cerebrovascular disease, and peripheral vascular disease, is the result of
premature atherosclerosis. Besides the traditional risk factors (like hypertension,
hypercholesterolaemia and smoking), renal insufficiency, raised homocysteine levels,
and the presence of anti-phospholipid, antibodies have been recognized as additional risk
factors for cardiovascular disease in SLE. Recent studies have demonstrated that the
nitric oxide pathway and its endogenous inhibitor asymmetric dimethylarginine may also
be involved in the pathogenesis of cardiovascular organ damage in SLE. The metabolic
syndrome and insulin resistance in SLE patients are current topics of research in this
field.
∗
Tel: +31-20-4443432; Fax: +31-20-4442138.
78 Irene E. M. Bultink, Ben A. C. Dijkmans and Alexandre E. Voskuyl
Several studies have demonstrated a high prevalence of low bone mineral density in
patients with SLE, especially in females. In the last few years, more attention is paid to
osteoporotic fractures, one of the items of the organ damage index for SLE, and likely
the most preventable form of musculoskeletal organ damage in SLE patients. Recent
studies have demonstrated an increased frequency of symptomatic vertebral and
nonvertebral fractures in patients with SLE. Moreover, a high prevalence of mostly
asymptomatic vertebral fractures in patients with SLE was detected. These vertebral
fractures were associated with previous use of intravenous methylprednisolone. The
importance of identifying vertebral fractures in SLE patients is illustrated by the
observed association between prevalent vertebral fractures and reduced quality of life as
well as an increased risk of future vertebral and nonvertebral fractures in the general
population.
Infection imposes a serious burden on patients with SLE. In case series, infectious
complications were found in 25% to 45% of SLE patients, and infection as primary cause
of death has been demonstrated in up to 50% of SLE patients. Defects of immune
defence and treatment with corticosteroids and other immunosuppressive agents are
supposed to play a role in the pathogenesis of infections in SLE. Recently, research has
focused on the role of the lectin pathway of complement activation in the occurrence of
infections in SLE.
In this review the results of recent studies on cardiovascular disease, osteoporosis
and infectious complications in SLE will be discussed.
INTRODUCTION
Systemic lupus erythematosus (SLE) is a chronic autoimmune disorder that usually

affects multiple organ systems and may be associated with considerable morbidity and
mortality.
Over the last 3 decades, the survival of patients with SLE has improved dramatically. A
60% decrease in the standardized all-cause mortality rate (SMR) from 1970-1979 (SMR 4.9)
to 1990-2001 (SMR 2.0) has been demonstrated in a large international multicenter study [1].
The improved health outcome in patients with SLE has been attributed to earlier and better
diagnostic methods as well as better immunosuppressive treatment strategies. Despite the
improved survival, mortality rates in patients with SLE are still significantly higher than
those in the general population, across countries, and throughout the course of the disease, up
to 20 years disease duration [1]. The most important causes of death in patients with SLE are
disease activity (complicated by organ system involvement), treatment related complications
such as infections, and long-term disease complications (particularly cardiovascular disease).
From 1970 to 2001, a change in death causes in SLE patients has been observed [1-3]. In this
period of time, a decrease in the rate of death primarily due to disease activity has occurred
[1, 4], while death due to cardiovascular disease remained unchanged [4] or slightly
increased, [1] and cardiovascular death is currently probably the commonest cause of death in
lupus patients in the Western world [1, 4]. Despite improvements in immunosuppressive
treatment strategies for active disease, infections are still an important cause of death in SLE
patients, both early and late in the disease course [1-3, 5-7].
Comorbidity in Systemic Lupus Erythematosus… 79
Apart from mortality, prognosis in SLE has also aspects with regard to morbidity. The
prolonged survival of patients with SLE is also associated with considerable morbidity due to
long-term complications of the disease. These long-term complications in SLE patients may
be due to the disease itself, the therapies used, or comorbid diseases, and are associated with
functional limitations and a reduced quality of life.
In this review, recent research in the field of three important long-term complications in
patients with SLE will be discussed. First atherosclerotic cardiovascular disease, which is the
major cause of cardiovascular, neuropsychiatric, and peripheral vascular organ damage in
SLE patients, as assessed with the Systemic Lupus International Collaborating
Clinics/American College of Rheumatology (SLICC/ACR) damage index score [8].
Secondly, osteoporosis and fractures, which contribute to damage in the most frequently
involved system in SLE patients: the musculoskeletal system. In the third item, results of
recent research on infectious complications in SLE will be highlighted.
CARDIOVASCULAR DISEASE IN SLE
Over the last decades, accelerated atherosclerosis and premature cardiovascular disease
(CVD), including coronary heart disease [9], ischaemic cerebrovascular disease [10], and
peripheral vascular disease [11] has been identified as a major contributor to the morbidity
and mortality of patients with SLE. In female SLE patients, the risk of myocardial infarction
is 10-fold increased [12], the risk of stroke 8 to 10-fold [12, 13] and the risk of heart failure
may be increased as much as 13-fold [13] as compared to age- and sex-matched healthy
controls. Atherosclerotic events also tend to occur at younger ages in patients with SLE. In
patients with SLE, the first myocardial infarction occurs at a mean age of 49 years as
compared to 65 to 74 years in the general population, which illustrates the premature
occurrence of CVD in SLE [9].
In addition, several studies have demonstrated a high prevalence of subclinical
atherosclerosis in 17% to 40% of SLE patients, as measured by myocardial perfusion scans,
presence of atherosclerotic plaques in the carotid artery and assessment of coronary artery
calcifications by computer tomography [14-16]. Endothelial dysfunction, as defined by
impaireded flow-mediated dilatation of the brachial artery, was present in 55% of SLE
patients as compared to 26% of healthy persons in a recent study [17]. Data from an autopsy
study performed in patients with SLE demonstrated that 52% of patients had moderate to
severe atherosclerosis at the time of death, regardless of the cause of death [2].
Risk factors for CVD in SLE
The mechanisms underlying the accelerated atherosclerosis in SLE are not fully
understood, but endothelial dysfunction and/or atherosclerosis in combination with
prothrombotic factors are supposed to be important contributors to this process [18] . In
recent years, both traditional and non-traditional risk factors have been identified, which play
a role in the pathogenesis of CVD in SLE.
1. Traditional Risk Factors

Patients with SLE have an increased prevalence of several traditional risk factors for
atherosclerosis, such as hypertension [12, 19, 20], diabetes mellitus [19], obesity [19, 20],
inactivity [19-21], hypercholesterolaemia [20] and hypertriglyceridaemia [19] and these risk
factors are, also in SLE patients, associated with an increased risk for CVD. However, even
after adjustment for the presence of the traditional Framingham risk factors, the risk for
cardiovascular events in patients with SLE is still 7 to 17-fold increased [12, 22]. Therefore,
additional metabolic, inflammatory, medication related and lifestyle factors are suggested to
contribute to the development of atherosclerosis and vascular disease in SLE.
2. Antiphospholipid Antibodies
The occurrence of a secondary antiphospholipid antibody syndrome in SLE patients is
common and is characterized by both arterial and venous thrombosis [23].
The presence of lupus anticoagulant has been recognized as an independent risk factor
for myocardial infarction [24] and cerebral arterial occlusion [25] in SLE patients. These
findings support the idea that thrombosis plays a role in the increased risk of CVD in SLE.
Studies of the relationship between the presence of lupus anticoagulant and atherosclerosis in
SLE demonstrate conflicting results. In a Swedish study, the presence of lupus anticoagulant
was associated with an increased intima-media thickness of the carotid artery [26], but in a
recent study in the USA the presence of lupus anticoagulant or anticardiolipin (aCL)
antibodies was not associated with coronary or carotid atherosclerosis [27].
The role of aCL and anti-ß2-glycoprotein 1 (ß2GP1) antibodies in the development of
CVD in SLE is not completely clear. In the general population, the presence of aCL
antibodies has been associated with an increased risk for future myocardial infarction [28,
29], but this finding has not been confirmed in SLE yet. In the study of Svenungsson and
colleagues, both aCL antibodies and anti-ß2GP1 antibodies tended to be associated with
arterial disease in lupus patients, but these associations were not statistically significant [26].
However, several other studies have suggested that aCL and anti-ß2GP1 antibodies
might play a role in the atherosclerotic process in SLE [30-33]. The low-density lipoprotein
(LDL) particle is susceptible to oxidative modification, which accounts for its atherogenic
properties. Oxidized LDL (ox-LDL) contributes to the formation of foam cells in
atherosclerotic lesions [30]. Ox-LDL can bind anti-ß2GP1, which results in ß2GP1-ox-LDL
complexes [31]. Antibodies against these complexes are supposed to increase LDL uptake by
macrophages, which contributes to foam cell formation, a process considered important in
atherosclerotic plaque formation [33]. Moreover, it was demonstrated that binding of these
antibodies to ß2GP1 on endothelial cells induces expression of adhesion molecules, which
further enhances leukocyte adhesion to the endothelium [32]. The role of antibodies to ox-
LDL in the pathogenesis of atherosclerosis in SLE is still under debate. The occurrence and
high titres of these antibodies have been associated with the extent of atherosclerosis and
CVD in reports, but other studies suggested that antibodies to ox-LDL play a protective role
in the atherosclerotic process [33, 34]. Future research might provide more insight in the
subtle interplay between thrombogenesis and atherogenesis and in the role of
antiphospholipid antibodies in the pathogenesis of CVD in lupus.
3. SLE Related Risk Factors

Several diseases related factors have been recognized as independent risk factors for
CVD in SLE.
In SLE patients, longer disease duration [9, 20, 21] and premature menopause [19] have
been associated with an increased risk of CVD.
In addition, renal disease is an important contributor to CVD in lupus. Nephritis is a
highly frequent complication in SLE and may cause proteinuria and renal failure. Nephrotic
syndrome and proteinuria are associated with un unfavourable lipid profile [35] and
atherosclerosis in SLE [36, 37]. Moreover, an elevated serum creatinine or history of renal
disease are associated with atherosclerosis [38, 39].
Dyslipidaemia in SLE is characterized by a reduced high-density lipoprotein (HDL)
cholesterol as well as an increased LDL cholesterol, VLDL cholesterol, triglycerides [40] and
increased lipoprotein a (Lp(a)) [26, 41]. Dyslipidaemia occurs especially in active disease
[40] and is associated with atherosclerosis and cardiovascular events [19, 26, 36]. The
mechanisms underlying the dyslipidaemia in SLE are not well known, but tumor necrosis
factor-α (TNF-α) induced de novo hepatic lipogenesis and inhibition of LDL are two possible
mechanisms [18]. This hypothesis is supported by the strong associations found between
elevated circulating levels of TNF-α, high triglycerides and low HDL levels in SLE patients
[42]. Recently, the presence of anti-lipoprotein lipase antibodies (anti-LPL) in association
with dyslipidaemia and increased inflammatory parameters have been demonstrated in
patients with SLE [43]. Further research is necessary to unravel the role of these antibodies in
the pathogenesis of atherosclerosis in lupus.
The role of antibodies against endothelial cells (aEC) in the development of CVD in SLE
is not clear. These antibodies have been associated with vasculitis and disease activity [44]
and direct stimulation of endothelial cells in SLE [45], but no correlation between aEC
antibodies and CVD was found in one study [46].
Atherosclerosis can be considered to have an important inflammatory component and
several inflammatory factors are supposed to promote atherosclerosis in SLE. In the general
population, raised serum concentrations of C-reactive protein (CRP) are a strong predictor of
future coronary events [47]. In line with this finding, raised serum levels of CRP were
associated with subclinical atherosclerosis [39] and cardiovascular arterial events in SLE
patients [48]. Moreover, serum levels of complement increase in response to inflammation
and studies in the general population have demonstrated associations between high
complement C3 levels and traditional cardiovascular risk factors and coronary heart disease
[49, 50]. In lupus patients, increased complement C3 levels were correlated with increased
vascular stiffness of the aorta [39] and with coronary calcifications in SLE [36]. Furthermore,
we found an association between high C3 levels and metabolic syndrome score in female
lupus patients [51]. This finding is in line with the results of a study by Chung et al,
demonstrating an association between higher levels of inflammation and prevalence of the
metabolic syndrome in patients with SLE [52]. In lupus, the systemic inflammatory response
is accompanied by systemic complement activation and immune complex deposition in
specific tissues. Complement activation induces endothelial cell activation, the release of
monocyte chemoattractant protein-1 (MCP-1), and the release of IL-6 from vascular smooth
muscle cells, which promote recruitment of leukocytes and atherosclerosis [53, 54]. Recently,
Asanuma and colleagues found increased concentrations of IL-6 and MCP-1 in lupus patients
and these cytokines were associated with inflammation, disease activity, body mass index and
low HDL levels [14]. Moreover, IL-6 concentrations were correlated with coronary
calcification [14]. Inflammation in SLE might also contribute to the dyslipidaemia associated
with lupus by IL-6 mediated inhibition of lipoprotein lipase (LPL), the key enzyme in the
metabolism of LDL and VLDL [15]. Reduced LPL activity has been demonstrated in lupus
patients and was strongly associated with high triglyceride levels [55].
Recently, decreased binding of annexin V to endothelial cells has been proposed as a
possible mechanism contributing to atherosclerosis in SLE [56]. Annexin V has anti-
thrombotic properties and decreased binding of this plasma protein to endothelial cells,
caused by antiphospholipid antibodies, might promote atherosclerosis. Associations between
annexin V binding and carotid intima media thickness in lupus patients with a history of
CVD have been demonstrated [56]. Further research is necessary to elucidate the role of
annexin V and other inflammatory factors in the atherosclerotic process in lupus.
4. Oxidative Stress
Several studies have demonstrated increased oxidative stress in patients with SLE [57,
58]. In lupus patients with a history of cardiovascular events, raised plasma levels of ox-LDL
have been demonstrated [26]. The contribution of ox-LDL to foam cell formation in
atherosclerotic plaques has been debated. However, ox-LDL levels have also been associated
with renal manifestations in SLE, and it is well known that nephritis and renal failure are risk
factors for CVD. Therefore, the exact role of ox-LDL in the atherosclerotic process in SLE is
still under debate and will be subject of more research.
The hypothesis that oxidative stress contributes to the atherosclerotic process in SLE is
supported by a study demonstrating reduced activity of the antioxidant enzyme paraoxanase
in SLE [59].
Recently, the role of the nitric oxide pathway and its endogenous inhibitor asymmetric
dimethylarginine (ADMA) has been investigated in SLE. In the general population, high
plasma ADMA levels are associated with endothelial dysfunction [60]. Moreover, high
ADMA levels are a predictor of acute coronary events [61] and a risk factor for
cardiovascular events and mortality in patients with end stage renal disease [62]. In patients
with SLE, AMDA levels were significantly higher in patients with a history of cardiovascular
events than in patients without a CVD history [63]. Moreover, high ADMA levels in SLE
were associated with disease activity, high titers of anti-dsDNA antibodies [63, 64] and with
coronary calcification [64]. The observed association between high ADMA levels and high
titers of anti-dsDNA antibodies is in line with results from in vitro studies. Anti-dsDNA
antibodies were shown to be reactive with the arginine-glycine-rich domains in recombinant
heterogeneous nuclear ribonucleoprotein A2 (hnRNP A2) [65]. These domains are also
preferred sites for the methylation of arginine to ADMA by type 1 protein arginine
methyltransferase (PRMT1) [66]. In the presence of anti-dsDNA, methylation of hnRNP A2
by PRMT1 was increased to 3.5 times that of the control level. Therefore, anti-dsDNA
antibodies might be a trigger for increased ADMA production by up regulating methylation
of arginine residues by PRMT1. Furthermore, anti-dsDNA monoclonal antibodies were
demonstrated to augment the inflammatory reaction by the release of proinflammatory
cytokines from mononuclear cells [67]. These studies suggest that anti-dsDNA antibodies
might play a role in the development of CVD in SLE by enhancing ADMA production and
by enhancing the inflammatory reaction. However, a prospective study is required to answer
definitively the question of whether high ADMA levels are an independent risk factor for
future cardiovascular events in SLE.
5. Metabolic Risk Factors

In a prospective study in 337 patients with SLE from the Hopkins lupus cohort, raised
plasma homocysteine levels were found in 15% of the patients and high homocysteine levels
were an independent risk factor for stroke and arterial thrombosis [68]. This finding was
confirmed by a Dutch study, reporting an increased risk for arterial thrombosis in SLE
patients with high homocysteine levels [69].
The metabolic syndrome is a condition characterized by the clustering of cardiovascular
risk factors, including hypertension, obesity, insulin resistance and dyslipidaemia, and is
associated with an increased risk of diabetes mellitus and cardiovascular mortality in the
general population, especially in women [70, 71]. Recently, research has focused on insulin
resistance and the associated metabolic syndrome in SLE. In patients with SLE, an increased
prevalence of insulin resistance was demonstrated [72, 73]. A study in the United Kingdom
reported that 18% of female SLE patients, as compared with 2.5% of healthy female controls,
fulfilled the criteria of the National Cholesterol Education Program (NCEP) metabolic
syndrome [72]. A study by Chung et al in the United States reported that the metabolic
syndrome was present in 29.4% of male and female SLE patients and 19.8% of healthy
controls, using the NCEP definition [52]. In a recent study in 141 Dutch female SLE patients,
the prevalence of the metabolic syndrome was 16% and a high metabolic syndrome score was
associated with previous treatment with intravenous methylprednisolone, renal insufficiency,
older age, higher erythrocyte sedimentation rate and higher C3 levels [51]. Moreover, the
mean metabolic syndrome score was significantly higher in SLE patients with a history of
cardiovascular events than in those without a previous cardiovascular event [51]. In both the
Dutch and the USA study, associations were found between the metabolic syndrome and high
levels of inflammation. Therefore, the metabolic syndrome might provide a link between
inflammation and the increased vascular risk in SLE. A prospective study is necessary to
investigate whether the metabolic syndrome is a predictor of cardiovascular events and
mortality in patients with SLE.
6. The Role of Anti-Rheumatic Drugs

The role of corticosteroid treatment in the development of CVD in SLE has been
investigated in many studies. Corticosteroids have a negative effect on blood pressure,
glucose metabolism and body fat distribution. On the other hand, corticosteroids have an anti-
inflammatory effect, which might be beneficial with respect to the atherosclerotic process.
The evaluation of the effect of corticosteroids on CVD in SLE is complicated, since
corticosteroids are usually prescribed in patients with more active disease. Therefore, a
history of corticosteroid treatment might reflect a higher inflammatory disease state, which is
supposed to be an important risk factor for CVD per se [47]. Associations between increased
exposure to corticosteroids and atherosclerosis and cardiovascular events have been reported
in patients with SLE [9, 20, 38], but other studies failed to find such an association [16, 74].
The benefit or harm of corticosteroids might be dose-dependent. In SLE patients, a daily dose
of < 10 mg corticosteroids did not have an adverse effect on lipid levels [75], but a dose of >
10 mg daily was associated with increased cholesterol and triglyceride levels [75, 76]. The
conflicting results of the studies on this subject illustrate the dual role of corticosteroid
therapy with respect to the development of CVD in SLE.
Antimalarial drugs reduce the atherosclerotic risk in SLE by several mechanisms. First,
antimalarials have an anti-inflammatory effect in mild to moderate disease activity. Secondly,
a beneficial effect of antimalarials on the dyslipidaemia in SLE, especially in case of
concomitant treatment with corticosteroids, has been reported in several studies [77-80]. In
the third place, antimalarials may have a beneficial effect on insulin sensitivity, because these
drugs prolong the half-life of the active insulin-receptor complex through the inhibition of
insulin dissociation from its receptor [81]. However, the effect of antimalarials on insulin
resistance has not been investigated in SLE patients yet.
Despite the great number of studies performed in the context of CVD and its risk factors
in SLE, little is known about the role of other antirheumatic drugs. However, a negative
association between previous cyclophosphamide use and the prevalence of carotid plaque
was demonstrated in one study [16], suggesting that tight control of disease activity might be
beneficial in the prevention of atherosclerosis in SLE.
Prevention and Treatment of CVD in SLE
Traditional risk factors, disease-related factors and the associated metabolic changes,
immunologic factors, anti-phospholipid antibodies, lifestyle factors and use of antirheumatic
drugs all contribute to the accelerated atherosclerosis and premature CVD in subgroups of
patients with SLE. Prospective studies in large patient groups are necessary to evaluate the
relative influence of each of these factors. In the meantime, physicians should use a proactive
approach to suppress disease activity and other modifiable risk factors for atherosclerosis in
these high-risk patients. Screening for risk factors in SLE patients is very important for
several reasons. First, in patients with SLE, a high prevalence of multiple risk factors has
been demonstrated [19, 20]. Secondly, a recent quality improvement study in Canada has
demonstrated that hypertension and especially hypercholesterolaemia are not managed
adequately in the majority of SLE patients [82]. In the third place, many of the risk factors
mentioned may be relatively easily recognized and managed in clinical practice by lifestyle
advices (stop smoking, physical activity, weight reduction) or medication (anti-hypertensive
treatment, lipid lowering agents, anti-diabetic medication, anticoagulants, supplementation
with folic acid and vitamin B12 in case of increased homocysteine levels). It was suggested
by Bruce, that the diagnosis of SLE can be regarded as a risk factor for CVD itself, like
diabetes, which has implications for the ideal targets for blood pressure, lipid levels and
glucose levels in SLE patients [15].
The identification of biological markers of disease activity associated with
atherosclerosis might be a tool to improve therapy and prevent cardiovascular complications
in SLE.
In addition, physicians eagerly await the definitive results of current large-scale

prospective studies evaluating the effect of different intervention strategies to reduce the risk
for CVD in SLE. The preliminary results of a Canadian intervention study are promising
[83].
OSTEOPOROSIS AND FRACTURES IN SLE
In recent years, osteoporosis and fractures have been recognized as important disease
complications in patients with SLE. The growing interest in these unfortunate disease
complications is justified for several reasons. First, in studies from 1990 to 2007, a high
prevalence of low bone mineral density (BMD) in patients with SLE has been demonstrated,
especially in female patients [84-101]. Secondly, osteoporotic fractures are an item of the
SLICC/ACR organ damage index for SLE. In the third place, it was demonstrated in studies
in the general population, that osteoporotic fractures are associated with a reduced quality of
life [102], an increased risk of future fractures [103, 104], and an increased mortality rate
[103].
Osteoporosis in SLE
A reduced BMD, as measured by Dual Energy X-ray absorptiometry (DEXA), is highly

frequent in patients with SLE. Osteopenia is reported in 25-74% of SLE patients [93, 95,
101] and osteoporosis, defined as a T score less than -2.5 SD, is reported in 1-23% [84, 96,
98, 100]. The great majority of studies of BMD performed in patients with SLE had a cross-
sectional study design [84, 86, 88-95, 97-100, 105-112]. Only a few longitudinal studies have
been published [85, 113-116]. The prevalence of osteopenia and osteoporosis reported in
studies in lupus patients differs widely as a consequence of differences between study
populations with respect to size, mean age, ethnic background, menstrual status, disease
severity and treatments used. The aetiology of bone loss in SLE is supposed to be
multifactorial, involving both non-disease related and disease related factors.
Non-Disease Related Risk Factors for Osteoporosis in SLE
Higher age [84, 90, 93, 96, 101], low body-mass index [84, 86, 93], postmenopausal
status [84, 86, 93, 94, 101], and non-Afro-Caribbean race [101] have been recognized as
significant non-disease related risk factors for reduced BMD in patients with SLE. Two small
studies in men demonstrated no increased prevalence of osteoporosis in male SLE patients
[106, 109]. Several studies have evaluated the effects of smoking and alcohol use, but no
significant associations with low BMD in patients with SLE have been reported [86, 90, 93,
101, 112, 113]. Data on other possible risk factors for low BMD (e.g. family history of
osteoporosis and reduced mobility) are generally not available.
Disease Related Risk Factors for Osteoporosis in SLE
Disease duration, inflammation, renal failure, deficiency of 25(OH) vitamin D, reduced

physical activity, hormonal factors and medication related factors have been suggested to
play a role in the development of osteoporosis in SLE.
In contrast to two studies [98, 105] reporting an association between disease duration and
BMD in SLE patients, most studies did not find such an association [84, 86, 88, 90, 94, 108,
112].
Disease activity is supposed to play a role in the development of osteoporosis in SLE
[117]. Serum levels of pro-inflammatory cytokines, such as TNF-α, IL-1 and IL-6, are
increased in patients with SLE [118] and production of bone-resorbing lymphokines by B-
cells in SLE patients has been described [119]. These findings suggest that disease activity
might be a risk factor for osteoporosis in SLE. Indeed, Petri and colleagues reported that
disease activity, as measured by low serum C4 levels, was an independent predictor of low
BMD in the lumbar spine [120]. However, no association between disease activity and BMD
was found in all recent studies, including studies using cumulative measures for disease
activity over time [86, 90, 94-96, 98, 100, 105, 106, 109, 115].
The relationship between BMD and organ damage in patients with SLE has been
investigated in several studies, with conflicting results. In four studies [94, 98, 105, 111],
higher organ damage score was significantly associated with low BMD in patients with SLE,
but in four other studies [86, 90, 91, 93] no association between BMD and organ damage
score in SLE patients was found. The reasons for this discrepancy are unclear.
Renal failure might contribute to the development of low BMD in patients with SLE
through several mechanisms. Renal damage due to SLE may result in an impairment of
vitamin D 1-hydroxylation by the kidneys, secondary hyperparathyroidism and increased
osteoclastic bone resorption. However, no significant association between renal function and
BMD was reported in recent studies [86, 90, 94], which might be explained by the relatively
small number of patients with renal failure included in most recent studies and exclusion of
patients with renal failure in several other studies [88, 92, 95, 98, 100, 106, 109, 110, 112].
Deficiency of 25(OH) vitamin D might be regarded as a non-disease related as well as a
disease related risk factor for osteoporosis in SLE. However, the increased prevalence of
25(OH) vitamin D deficiency reported in patients with SLE, as compared to healthy controls
[121] is usually ascribed to the disease related conscious avoidance of exposure to sunlight
and/or the use of sunscreens [99, 105, 106, 121]. In a study in 107 Dutch SLE patients,
deficiency of 25(OH) vitamin D was significantly associated with low BMD in the lumbar
spine, but not at the hip [86].
Reduced physical activity is considered an important risk factor for the development of
osteoporosis in several rheumatic disorders [117]. Bhattoa et al [84] reported a significant
negative association between Steinbrocker functional classification and lumbar spine and hip
BMD in SLE patients, but other studies failed to demonstrate an association between physical
activity and BMD in SLE patients [86, 90, 91, 115].
Several hormonal factors may be involved in the pathogenesis of low BMD in lupus.
SLE is characterized by enhanced hydroxylation of estradiol, increased oxidation of
testosterone and relatively low plasma levels of androgens, such as dehydroepiandrosterone
sulphate (DHEAS). In addition, hyperprolactinaemia has been reported in patients with SLE,
which may further decrease serum levels of estradiol and androgens [122]. In a study in 37
premenopausal SLE patients, a significant positive relationship between serum DHEAS
levels and BMD was demonstrated [89]. In a small study in 20 male SLE patients, no
association between hyperprolactinemia and low BMD was found [109]. Premature ovarian
dysfunction as a result of active disease or caused by medication, is common in female SLE
patients and contributes to bone loss by a reduction of overall estrogen exposure. In a study
by Gordon et al [91], significantly decreased BMD values in SLE patients with a disordered
menstrual history were found. These findings support the viewpoint that hormonal factors
contribute to the development of low BMD in SLE.
The role of corticosteroids in the development of osteoporosis in SLE is still under
debate. Various studies reported a relationship between corticosteroid use and low BMD [84,
85, 90, 91, 93, 94, 98, 100, 110], but several other studies did not find a significant
relationship between corticosteroid treatment and BMD in SLE patients [86-89, 92, 96, 105,
106, 108, 109, 111-113]. The reasons for this discrepancy are unclear, but may be related to
differences in patient populations in size, mean age and menstrual status, as well as
differences between centers in treatment strategies for osteoporosis and use of corticosteroids.
Moreover, corticosteroids might play a dual role with respect to the development of
osteoporosis in SLE. On the one hand corticosteroid therapy may induce bone loss, but on the
other hand corticosteroids might have a beneficial effect on bone mass by suppressing
inflammation. Bone loss in patients with SLE might also be induced by other drugs which are
commonly used in these patients. Cyclophosphamide may induce osteoporosis by induction
of premature ovarian failure. In addition, long-term exposure to oral anticoagulation, and to a
lesser extent, low-molecular-weight heparin, has been associated with increased bone loss in
the general population [123], but this has not been investigated in patients with SLE.
Fractures in SLE
In contrast to the large number of studies on BMD, only a few studies on osteoporotic
fractures in SLE have been published. In 4 studies, symptomatic vertebral and peripheral
fractures occurring since disease onset were reported in 9-16.5% of SLE patients [84, 91, 93,
124]. The incidence of symptomatic vertebral and peripheral fractures was demonstrated to
be 5 times higher in female SLE patients as compared to age-matched healthy women, and
higher age and prolonged use of corticosteroids were identified as the most important risk
factors for fractures [124]. In this large study, 10% of the symptomatic fractures were
reported to be vertebral fractures.
Only two studies on prevalent vertebral fractures in SLE patients, using a standardized
method to assess vertebral fractures, have been performed and in these studies at least 1
vertebral fracture was detected in 20-21% of the patients [86, 107]. In both studies, vertebral
fractures were defined as a reduction of ≥ 20% of the vertebral body height. The frequency of
vertebral fractures (20%) in 90 Dutch SLE patients with mean age 41 years, was high in
comparison to the 12% prevalence of vertebral fractures in the general population of Europe
with an age between 65 and 69 years [125]. Interestingly, in both studies on prevalent
vertebral fractures in SLE, no association between oral corticosteroid use and vertebral
fractures was found, but in the Dutch study, previous use of intravenous methylprenisolone
was associated with prevalent vertebral fractures [86]. In both studies, BMD was not different
between SLE patients with and without prevalent vertebral fractures [86, 107]. This finding is
in line with results of a previous study in the general population, which reported that the
proportion of fractures attributable to osteoporosis is modest, ranging from 10% to 44%
[126]. These data suggest that bone quality contributes more to bone strength than bone
quantity and point to the limited value of BMD measurement by DEXA in the assessment of
future fracture risk. The high frequency of both peripheral and vertebral fractures
demonstrated in SLE patients highlights the need to develop better strategies for the
prevention and treatment of osteoporosis and fractures as important disease complications in
lupus patients. The need for better prevention and treatment strategies is also illustrated by
the relative high frequency of undertreatment of SLE patients with manifest osteoporosis or
at high risk of the development of osteoporosis, as demonstrated in the Dutch study [86].
Prevention and Treatment of Osteoporosis in SLE
Prevention strategies directed toward SLE patients at risk for osteoporosis and fractures
include advice for maintaining a normal body weight and daily performance of weight-baring
physical activity. In addition, physicians should encourage a healthy diet and strongly advice
against smoking. Moreover, adequate calcium and vitamin D supplementation should be
prescribed. We advocate to perform BMD measurement by DEXA in SLE patients treated
with corticosteroids and/or in postmenopausal patients with SLE. Moreover, as a
consequence of the high prevalence of vertebral fractures demonstrated in SLE patients,
physicians should consider to perform spine X-rays (analyzed using a standardized method
for assessing vertebral deformities) next to BMD measurement of the spine and hip in the
assessment of osteoporosis and future fracture risk. Postmenopausal women with
osteoporosis and/or fractures and women on corticosteroids should be treated with
appropriate antiosteoporosis medication, eg bisphosphonates. Results of a recent study in
postmenopausal women in the general population have demonstrated a significant beneficial
effect on BMD and a significantly reduced risk for symptomatic vertebral fractures (but not
for other fractures) in patients continuing bisphosphonate therapy for 10 years as compared to
patients who discontinued bisphosphonates after 5 years [127]. Moreover, the safety of long-
term treatment with bisphosphonates up to 10 years was demonstrated in this study [127].
Bisphosphonates are contraindicated in patients with renal failure and in premenopausal
lupus women planning pregnancy. Bisphosphonates are maintained in bone for a long period
of time even after discontinuation of therapy, and fetal abnormalities due to bisphosphonates
were demonstrated in animal studies [128]. Hormonal replacement therapy may be useful in
postmenopausal women without an increased risk of thrombosis.
INFECTIONS IN SLE
Importance of Infections in SLE
Infections are an important cause of morbidity and mortality in patients with SLE.
Infections occur in 25% to 50% of SLE patients in case series [129-132] and a third of the
infections in SLE patients were demonstrated to be major infections for which hospital
admission is required [129, 130, 133]. Infection as primary cause of death has been reported
in up to 50% of patients with SLE [5, 7].
Spectrum of Infections in SLE
SLE patients suffer from a broad spectrum of infections caused predominantly by

community-acquired bacteria [129-131, 133], but also opportunistic infections are reported
[5]. Among Indian SLE patients, tuberculosis was demonstrated to be the commonest
infection [134]. Moreover, a high incidence (up to 21%) of Herpes zoster infection was found
in several studies in SLE patients [129, 130, 135-139].
Risk Factors for Infections in SLE
Treatment with corticosteroids and immunosuppressive agents and defects in immune

function (which may result from the disease itself and its therapy) are supposed to play a role
in the pathogenesis [5], but also disease activity [7, 133, 140-142], disease duration [129,
143], active nephritis [135, 144] and decreased renal function [142] have been linked to an
increased risk of infection in lupus patients.
The role of corticosteroid use as an independent risk factor for infections in SLE was
demonstrated in several studies [130-132, 135, 145], but other studies failed to find such an
association [129, 133, 140-142]. The impact of immunosuppressive drugs, such as
cyclophosphamide, azathioprine and methotrexate, on the immune system is well established.
Use of immunosuppressive drugs was identified as an independent risk factor for infections
in some studies in SLE [131, 132, 142, 145], but not in other studies in patients with SLE [7,
86, 133, 140]. Some studies failed to demonstrate an additive effect of cytotoxic drugs in
increasing the overall infection rate, but showed an increased incidence of Herpes zoster
infections in patients using cyclophosphamide [136, 146] or azathioprine [135]. Since
corticosteroids and immunosuppressive drugs are used mostly in patients with more severe
disease, it is likely that both disease activity and drug therapy contribute to the increased
infection risk in SLE. In a recent study, a strong negative association between
hydroxychloroquine use and the occurrence of major infections was demonstrated [129]. This
finding might be explained by the frequent use of hydroxychloroquine in the treatment of
patients with mild lupus disease activity. Another explanation might be protection against
infections due to the antimicrobial properties of hydroxychloroquine. However, antimalarials
act against pathogenic organisms that are very uncommon in Western European countries
[129].
Defects in the Complement System and Infections in SLE
Several defects in both humoral and cellular immunity have been described in lupus
patients, which might contribute to an inadequate immune defence [5]. The potential role of
macrophage defects, polymorphonuclear cell defects, defects in number and function of
natural killer cells, T cells and B cells, immunoglobulin defects and dysfunction of the
reticuloendothelial system in the pathogenesis of infections in SLE has been extensively
described by Iliopoulos and Tsokos [5]. This chapter is restricted to the results of recent
research on the contribution of the complement system to the increased infection risk in SLE.
The complement system plays an important role in host defence against microorganisms
and the increased infection rate in SLE patients has been attributed in part to defects in the
complement system [5]. A strong association between genetic deficiencies of early
components of the classical pathway of complement activation and the development of SLE
has been demonstrated [147]. In particular, deficiency of C1q is a major risk factor for the
development of SLE. Moreover, deficiency of C1q might play a role in the susceptibility to
infections in SLE, since C1q plays a role in the recognition and clearance of apoptotic
material [148] and binds predominantly to antibodies and protein structures on bacteria and
viruses, resulting in complement activation. The consumption of complement proteins by
circulating and tissue-fixed immune complexes might also limit the amount of complement
that is available for host defence against invading pathogens.
Deficiency of Mannose-Binding Lectin and Infections in SLE
Recently, the lectin pathway of complement activation has also been suggested to play a
role in the pathogenesis of SLE [149] and in the occurrence of infections in SLE patients
[150-152]. Mannose-binding lectin (MBL) is a serum protein that shares many features with
C1q [153]. In contrast to C1q, which is a recognition molecule in the classical pathway of
complement activation, MBL serves as one of the recognition molecules in the lectin
pathway of complement activation [154]. MBL may activate complement through the lectin
pathway by interacting with MBL-associated serine proteases (MASP). In addition, MBL can
directly opsonise pathogenic microorganisms and enhance the activity of phagocytes [154].
In a meta-analysis of all available case-control studies, homozygosity for variant MBL
alleles was demonstrated to be a minor risk factor for the presence of SLE [155]. A
significant association between MBL codon 54 variant B and SLE was reported in that study.
In two Danish studies [150, 151] and a Japanese study [152], SLE patients homozygous
for MBL variant alleles were at an increased risk for serious infections in comparison with
patients who were heterozygous or homozygous for the normal allele. In contrast, in a study
in Dutch SLE patients, no association between the biological activity of MBL and the
occurrence of infections was found [129]. In this study, functional MBL activity was
determined using three different assays: 1) an assay measuring functional MBL serum levels
which is dependent only on the amount of functional protein, 2) an assay measuring activity
of the MBL/MASP complex by assessing MBL-induced C4 deposition and 3) an assay
measuring the complete MBL pathway activity in serum, which is sensitive to defects in all
components of the MBL pathway. None of these assays demonstrated an association between
deficient MBL activity and the occurrence of infections or major infections in patients with
SLE [129]. There are two possible explanations for the discrepancy between the results of the
genetic and the phenotypic studies. First, functional activity of MBL in serum not only is
determined by mutations in the gene encoding MBL, but is also dependent on promoter
polymorphisms, MASP activity, serum levels of other complement factors, and
environmental factors [156, 157]. Therefore, measurement of functional MBL activity is
supposed to be a better estimation of the in vivo situation than assessment of MBL genotypes
when evaluating the association between the MBL pathway of complement activation and the
occurrence of infections in SLE. Secondly, the discrepancy between the genotypic and
phenotypic data could be explained by unidentified linkages between mutations or
polymorphisms in the MBL gene with mutations or polymorphisms in other genes, which
might influence the genetic approach. The results of this study suggest that deficiency of
functional MBL activity does not play a role in the susceptibility to infections or major
infections in SLE. However, this does not exclude an influence of defects in the complement
system on the occurrence of infections in SLE. Therefore, future research will be focused on
the role of defects of other complement factors and complement inhibitors in the
pathogenesis of infections in SLE.
Diagnostic and Therapeutic Considerations
It is often difficult to distinguish infection from disease flare in febrile patients with SLE.
As a consequence of an inadequate inflammatory response, signs and symptoms of infection
may be subtle or absent, especially in patients treated with immunosuppressive drugs. On the
other hand, clinical symptoms of lupus activity may simulate infection. Physicians should be
aware that false-positive serological tests for several infections (e.g. Lyme disease,
toxoplasmosis, syphilis), which are caused by polyclonal hypergammaglobulinaemia, are
often found in SLE patients [5]. In addition, skin tests may be false-negative in case of
corticosteroid use [5]. Chills and leukocytosis are markers favouring infection. Moreover, in
a study in SLE patients, high C-reactive protein levels were associated with infection, in the
absence of serositis [158]. Another study demonstrated increased serum procalcitonine levels
in lupus patients with bacterial or fungal infections as compared to patients with viral
infections or with lupus flare [159]. The presence of low complement levels and high serum
anti-DNA antibody titers suggests lupus flare.
Strategies to reduce the morbidity and mortality related to infections in SLE include
hygienic measures, prophylactic use of antimicrobial agents and immunisations.
Prophylaxis against Pneumocystis carinii, using a regimen of low-dose
trimethoprim/sulfamethoxazole or inhaled pentamidine should be considered in selected SLE
patients being treated with immunosuppressive agents [160]. Moreover, prophylaxis with
isoniazide is recommended in SLE patients with positive tuberculin skin test requiring high
dose corticosteroids [161].
Although initial reports suggested that pneumococcal vaccinations in patients with SLE
might induce disease exacerbations [162], recent studies demonstrated that vaccinations
against influenza en pneumococcus are safe in this patient group [163, 164], although
influenza vaccination was less effective in SLE patients than in controls [163].
Early diagnosis and treatment of infections in patients with SLE remains a difficult
challenge for physicians. More research is necessary to develop laboratory tests that will
improve differentiating between infections and disease exacerbations in patients with SLE.
CONCLUSION
The importance of CVD, osteoporosis, and infections as long-term complications in

patients with SLE, can not be over emphasized. The aetiology of these complications in SLE
is supposed to be multifactorial, including risk factors that also apply to the general
population, disease-related and treatment-related risk factors. The relative contributions of
these risk factors are not fully understood. Ongoing research should attempt to further
unravel the pathogenic mechanisms, applying especially to SLE patients. Physicians treating
patients with SLE should be aware of the long-term complications that might develop in their
patients. In addition, SLE patients themselves need to understand how to modify lifestyle
factors that increase the risk of premature atherosclerosis, osteoporosis, fractures and
infectious complications. Treatment regimens will benefit from the results of large scale
prospective studies evaluating the effect of different intervention strategies to reduce the risk
of CVD, osteoporosis and infections in patients with SLE.
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Chapter 3
SEVERE TISSUE TRAUMA TRIGGERS LUPUS

AUTOIMMUNE DISEASE
Khairul Anam1, Mihret Amare1, Shruti Naik1,

Kathleen A. Szabo2 and Thomas A. Davis1,∗
1
From the Regenerative Medicine Department at the Naval Medical Research Center
2
Department of Diagnostic Pathology at the Walter Reed Army
Institute of Research, Silver Spring, 20910-7500
ABSTRACT
Systemic lupus erythematosus (SLE) is a chronic, complex autoimmune disease
characterized by high levels of non-organ-specific, self-reactive antibody production
leading to immune complex formation. The etiology of this autoimmune disease remains
elusive. The disease results in multiple health problems including increased infection,
renal and skin disorders, neurological complications, osteoporosis, rheumatoid arthritis,
osteoarthritis, and fibromylagias. Tissue damage associated with severe injury can result
in profound immune dysfunction that involves suppressive cell types and a cascade of
inflammatory and tissue reparative mediators. Mice from MRL strains have been used as
models to study SLE pathogenesis. Wild type MRL/MpJ mice exhibit SLE autoimmune
disorders but symptoms are manifested much later in life (70-90 weeks) compared to
MRL/MpJ-Faslpr mice (17-22 weeks) which possess a lymphoproliferative mutation.
Based on our preliminary findings, we hypothesize that following traumatic tissue injury,
the activation of specific cell types, cytokines and other mediators involved in wound
healing and repair processes may be critical in triggering lupus-like disease. We
investigated the role of a severe (15% total body surface area) full-thickness cutaneous
burn on the early onset of lupus-like autoimmune disease in young adult, lupus prone
wild type MRL/MpJ mice, and control BALB/c mice. MRL/MpJ mice develop
autoimmune disease 4-15 weeks post injury, manifested by skin lesions, vasculitis,
∗
Address correspondence and reprint requests to: Thomas A. Davis, Ph.D.1,3 Regenerative Medicine Department,
Naval Medical Research Center, room 2A10, 503 Robert Grant Avenue, Silver Spring, MD USA 20910. Tel.
301-319-9528, Fax 301-319-7210, E-mail: [email protected]
106 Khairul Anam, Mihret Amare, Shruti Naik et al.
epidermal ulcers, cellular infiltration, splenomegaly, lymphadenopathy,

hypergammaglobulinemia, elevated autoantibodies, and renal pathologies including
proteinuria, glomerulonephritis, and immune complex deposition. Post-injury survival
rate of injured MRL/MpJ mice is significantly reduced due to autoimmune related
complications. In contrast, neither uninjured MRL/MpJ mice nor burned BALB/c
displayed signs of autoimmunity or premature death. We analyzed mRNA expression of
numerous cytokines at the wound margins in the skin at days 1-7 post injury. Our results
do not reveal an early or clear Th1 or Th2 cytokine expression pattern during the early
wound repair process but demonstrate a correlation between the pathogenic effects of
dysregulated interleukin-beta (IL-1β), interleukin-6 (IL-6), tumor necrosis factor alpha
(TNF-α) and prostaglandin-E2 (PGE2) synthesis and the early onset of lupus-like
disease. Interestingly, we found that normal skin isografts transplanted onto the dorsum
of MRL/MpJ mice with healed wounds (30- 40 days post burn injury) are rejected within
7 days after transplantation. In contrast, skin grafts transplanted onto uninjured age-
matched MRL/MpJ achieved long-term survival. Collectively, these findings suggest that
traumatic injury exacerbates inflammatory skin disease and the early onset of severe
multi-organ SLE pathogenesis.
Keywords: SLE, autoimmunity, lupus, trauma, and burns.
INTRODUCTION
Autoimmune disease is estimated to affect at least 2% of the population in the United

States [1]. Systemic lupus erythromatosus (SLE) is a chronic, complex, multi-organ,
autoimmune disease characterized by high levels of non-organ-specific, self-reactive
antibodies directed to cellular DNA, RNA, and histone components [2, 3]. Although SLE
primarily afflicts pre-menopausal females (ages 12-40) it can occur in males, and
disproportionately affects African Americans [4, 5]. Several genes have been implicated in
the development of lupus; however, not all individuals with a genetic predisposition develop
SLE. Extreme physical and emotional stress, psychosocial, physiologic, hormonal, and other
factors are likely involved as triggers for SLE [3, 6-8]. Such factors have been linked to the
manifestation of Gulf War Illness, a lupus-like condition [9-14]. Moreover, exposure to
chemicals, vaccines, medications, drugs, UV radiation, and other ubiquitous environmental
factors may promote SLE in otherwise healthy individuals [15-18]. A number of studies have
suggested that the immune response to infectious agents and foreign antigens (bacterial, viral,
and allergen) play a key role in triggering the activation of autoreactive T and B lymphocytes
and induction of anti-DNA responses [19-21]. Immune reactivity may cause tissue damage
without the classical signs of an autoimmune disease.
Individuals diagnosed with SLE are at increased risk of significant health problems,
including deteriorating renal dysfunction, skin pathologies, increased infections, seizures and
other neurological complications, and chronic musculoskeletal pain syndromes such as
osteoporosis, rheumatoid arthritis, osteoarthritis, and fibromyalgia [2, 22]. Many of these
individuals require long-term clinical care and systemic immunosuppressive/immunotherapy.
Warfighters, for example, experience exposure to extreme environments and as a result
endure physical and emotional stress making them particularly susceptible to SLE.
Severe Tissue Trauma Triggers Lupus Autoimmune Disease 107
Furthermore, an increase in the number of women in combat situations could lead to

increased incidence of lupus in the U.S. fighting force, because SLE afflicts women on
average more than men [23]. Warfighters with SLE may be unable to continue their military
service, therefore, greatly impacting U.S. military force strength.
Tissue trauma is a leading cause of disease experienced by military personnel and as
accidents in civilian populations. The time course of wound repair and the amount of scar
tissue formed depend on several factors including the type of wound, the extent of the tissue
damage, inflammation, the presence of devitalized tissue and nonviable foreign tissue, and
infection. More specifically, repair of damaged tissue involves a well-orchestrated sequence
of events, initiated by platelet aggregation and fibrin polymerization, followed by infiltration
of leukocytes [24-26]. Among the earliest cues for initiation of the wound repair response is
the release of chemokines and growth factors by infiltrating degranulating platelets,
keratinocytes, resident mast cells, and damaged tissue. Neutrophils, macrophages, and other
inflammatory cells are recruited to the area of damaged tissue in response to chemokines and
growth factors released at the site of the wound [27]. Leukocyte infiltration is also essential
for removal of necrotic cellular debris, whereas many cell types participate in the removal of
apoptotic cells and in subsequent reparative and remodeling processes [27-29].
Tissue damage associated with severe tissue injury can result in profound immune
dysfunction and induction of adaptive immune response [30, 31]. While inflammatory
cytokines are indispensable in wound healing and the restoration of homeostasis, it is often
the excessive activity of either proinflammatory or anti-inflammatory cytokines that causes
injury to the host or renders the host immunocompromised. Aberrant shifts in immune
reactivity can result in delayed wound healing and in serious systemic complications such as
hypermetabolism and catabolism leading to peripheral protein waste, systemic inflammatory
response syndrome (SIRS), multiple organ dysfunction (MOD), sepsis, shock, and even death
[32-35].
The adaptive immune response has evolved to recognize and attack foreign agents while
maintaining tolerance to self-antigens. Adaptive immunity is induced by activation of T
lymphocytes. Loss of T helper cell function and cytokine production after severe tissue
damage is evident in the switch from Th1 inflammatory responses (augmented IL-2, TNFα
and IFNγ production) towards counterinflammatory Th2-reactivity (IL-4 and IL-10) mediated
by macrophages, CD4+ and CD8+ T cells [36]. Dysregulation of proinflammatory cytokines
has been shown to play a pivotal role in SLE etiology [37-39].
Wild type MRL/MpJ mice exhibit SLE autoimmune disorders but symptoms are
manifested much later in life (70-90 weeks) compared to MRL/MpJ-Faslpr mice (17-22
weeks) which possess a lymphoproliferative mutation [40,41]. In this study, we demonstrate
accelerated development of lupus-like autoimmune disease in young adult, wild type
MRL/MpJ mice following tissue trauma (cutaneous burn wound). Results from our studies
indicate that these mice develop severe SLE, 10-15 weeks post injury, with characteristic skin
lesions, cellular infiltration, hypergammaglobulinemia, anti-DNA autoantibodies, immune
complex formation, glomerulonephritis, and lymphadenopathy. Our results also demonstrate
a correlation between the pathogenic effects of dysregulated cytokine production (IL-1β, IL-
6, TNF-α, PGE2) and the early onset of SLE. Taken together, results from this study suggest
that traumatic injury exacerbates inflammatory skin disease and the early onset of severe
multi-organ SLE pathogenesis.
MATERIAL AND METHODS
Animals
Five to six-week-old female MRL/MpJ mice and BALB/c mice were purchased from The
Jackson Laboratory (Bar Harbor, ME) and maintained at the Armed Forces Radiobiology
Research Institute or the Walter Reed Army Institute of Research (WRAIR, Silver Spring,
MD) animal facility, which are both accredited by the Association for the Assessment and
Accreditation of Laboratory Animal Care International. All procedures were conducted using
facilities and protocols approved by the Animal Care and Use Committee of AFRRI (#2004-
02-001) and WRAIR (protocol #K06-005). Mice were housed five animals per cage prior to
surgery or any treatment, and individually caged post burn injury in standard micro-isolator
polycarbonate caging. Mice were used for experimentation at 8 to 12 weeks of age. Animal
rooms were maintained at 21° ± 2°C with 50% ± 10% humidity on a 12-hr light/dark cycle.
Commercial rodent ration (Harlan Teklad Rodent Diet 8604) was available freely, as was
acidified (pH=2.5) water to control opportunistic infections.
Experimental Design
At 12 weeks of age, mice received either 15% TSBA full-thickness burn or were sham-
treated. Two sets of experiments were conducted; the first set (twenty-one animals for each
experimental and control group) was for examination of survival rate, urine proteinuria, and
the development of “lupus like” cutaneous lesion formations on the ears, neck and dorsum
until the mice reached 9 months of age (6 months post injury). At days 1, 3, and 7 post injury,
skin biopsies from another cohort of mice (n = 3 mice per group at each time point) were
excised from the wound margin and screened using a custom made RT-PCR microarrays
(Applied Biosystems Foster City, CA) containing oligo sequences for 184 inflammatory
cytokine and wound repair gene transcripts. Mice that developed severe skin lesions and/or
those with proteinuria levels of > 500 dm/dl were euthanized by C02 inhalation followed by
cervical dislocation. Immediately, post euthanasia, blood samples were collected by cardiac
puncture for examination of serum IgG levels. Spleen and kidneys were removed for
evaluating splenomegaly, and immunopathology, respectively. Skin lesions and adjacent
normal skin were excised, fixed with 10% formalin, embedded in paraffin, and sectioned.
After deparaffinization and rehydration, 5 µm sections were washed (3x) with PBS and
stained with hematoxylin and eosin (HandE). In the latter set of experiments (n=5 mice per
group), isogeneic skin graft experiments were conducted on mice 30-40 days post burn injury
or sham-treatment. Graft survival was examined three times weekly for 1 month. Photographs
of skin lesions, skin grafts, and histological sections were taken with a digital Fuji Finepix
Camera or a Nikon DXM 1200 Digital Camera mounted on a Nikon Eclipse E800
microscope. Images were imported into Adobe Photoshop CS2 for reproduction.
Burn Injury Model
Mice were anesthetized using either an intraperitoneal injection of ketamine (75 mg/kg),
xylazine (15 mg/kg), acepromazine (2.5 mg/kg) or isoflurane inhalation. After shaving the
dorsum, the exposed skin was washed gently with room temperature sterile water and
prepped with Betadine (a 10% povidone-iodine solution for skin disinfection). The Betadine
solution from the prepped area was wiped off using 3 series of sponge gauzes containing 70%
isopropyl alcohol. In a few selected studies, mice were further treated with a depilatory agent
(Nair) to remove remaining hair stubble. Using a surgical skin marker, a 15mm diameter
circular area along the dorsal midline region was outlined. A full thickness burn (10-15%
total body) was introduced with an electrocautery bovie (370-400oC for 1.5 sec: Bovie Aaron
Medical, St. Petersburg, FL). This protocol causes a well demarcated, full thickness, injury in
anesthetized mice that is nonlethal with < 0.5% mortality. Wounds became covered with
eschar, and there was no macroscopic evidence of infection. Wounds were topically treated
with Bacitracin immediately after the burn and left uncovered without a dressing. Once mice
recovered from anesthesia, they were placed in separate cages and maintained under standard
conditions in the animal facility. With the exception of pain medication (Buprenorphine 0.1
mg/kg SC BID; Reckitt Benckiser Pharmaceuticals, Richmond, VA) for the first two days
post burn, no other treatment or topical wound care was administered. At various time points
post injury mice were euthanized by CO2 inhalation followed by cervical dislocation.
Skin Lesions, Splenomegaly and Lymphadenopathy
Following wounding and sham treatment, mice were observed weekly for skin lesions
and protruding lymph nodes (cervical, brachial and inguinal). At the time of death or
euthanasia, skin lesions were scored by gross pathology using the following scale: 0= none,
1= small and localized to one site (face or ears), 2 = moderate, more than one site involved, <
2 cm (face, ears, dorsum); and 3 = severe, > 2 cm (face, ears and dorsum). Spleens were
weighed and enlarged lymph nodes scored on a scale of 0-3 (0= none; 1 = small, at one site; 2
= moderate, more than one site; and 3 = large, more than two sites).
Proteinuria
Urine was tested for proteinuria using commercially available kits (Multistix, Bayer ,
Elkhart, IN). Proteinuria scored as 0 (negative), <30mg/dl (trace 0.5+), 30 mg/dl (1+), 100
mg/dl (2+) and >500 mg/dl (3+). Animals were considered to have proteinuria if they scored
2+ for two consecutive urine samples.
Measurement of Serum IgG Antibodies
Total serum IgG, IgG1, IgG2a, IgG2b and IgG3 isotype concentrations were determined
by ELISA. Polystyrene plates precoated with goat anti-mouse Fc specific IgG capture
antibody and blocked were commercially purchased (RandD Systems, Minneapolis, MN).
100 µl of Ig standards (Southern Biotechnology Associates, Birmingham, AL) were added
per well in a series of 2-fold dilutions (125ng/ml – 3.9ng/ml), and serum Ig concentrations
were assessed at a 1:200,000 dilution (100 µl per well). After two hours of incubation at
room temperature, the plates were washed three times with phosphate-buffered saline
solution (PBS) containing 0.05% Tween-20 (wash buffer). Bound Ig was detected with 100
µl per well of appropriately diluted horseradish peroxidase conjugated anti-IgG (Chemicon,
Temecula, CA), IgG1, IgG2a, IgG2b, and IgG3 antibodies (Southern Biotechnology
Associates, Birmingham, AL). Secondary antibodies were added to the plates for one hour at
room temperature, followed by 3 washes. Then, 100 µl per well of freshly prepared substrate
solution containing equal volumes of 0.4g/L 3,3’,5,5’ tetramethylbenzidine and 0.02%
hydrogen peroxide was used to develop the assay (Pierce, Rockford, IL). Reaction was
stopped with 100 µl per well of 2N sulfuric acid (Sigma, St Louis, MO) and the absorbance
was measured at 450 nm using a 680 Microplate reader (BioRad, Hercules, CA). Results are
denoted as the Ig concentration (mg/ml) at various time points.
Measurement of Autoantibodies
Ig class specific anti-DNA antibodies were measured by ELISA. Polystyrene covalink

96-well microtitre plates (Fisher, Pittsburgh, PA) were coated with 50 µl per well of 10 µg/ml
Calf Thymus DNA (Sigma, St Louis, MO) and allowed to incubate overnight at 4oC. After
washing three times with wash buffer, 300 µl of blocking solution (3% bovine serum
albumin, BSA, in PBS) were added per well and incubated for two hours at room
temperature. The plates were washed three times with wash buffer and 100 µl of diluted sera
were added to each well, (dilutions ranged from 1:50 to 1:100,000). After two hours of
incubation at room temperature, plates were washed three times with wash buffer. Then, 100
µl of appropriately diluted horseradish peroxidase conjugated anti- IgG (Chemicon,
Temecula, CA), IgG1, IgG2a, IgG2b, and IgG3 antibodies (Southern Biotechnology
Associates, Birmingham, AL) were added per well to the plates for one hour, and followed by
three washes. 100 µl per well of freshly prepared substrate solution containing equal volumes
of 0.4g/L 3,3’,5,5’ tetramethylbenzidine and 0.02% hydrogen peroxide were used to develop
the assay (Pierce, Rockford, IL). Reaction was stopped with 2N sulfuric acid (Sigma, St
Louis, MO) and the absorbance was measured at 450 nm using a 680 Microplate reader
(BioRad, Hercules, CA). Results are denoted as the OD450 at various dilutions.
Renal Pathological Assessment
Mice were euthanized by CO2 inhalation followed by cervical dislocation, and the
kidneys were removed. One kidney was fixed with buffered formalin for > 48 hr, embedded
in paraffin blocks, sectioned and stained with hematoxylin and eosin (HandE) or periodic
acid Schiff (PAS) by standard methods. Glomerular pathologies were evaluated
morphometrically by light microscopy. The glomerular lesion (mesangial hypercellularity,
increase in mesangial matrix, crescent formation, and necrosis) was graded on a
semiquantitative scale from 0 to 3 (0 = normal, 1 = mild, 2 = moderate, 3 = severe) for more
than 20 glomeruli per mouse. Scores assigned to each of these elements were added together
to yield a mean renal score. Values were reported as the mean + standard deviation (SD) of
seven specimens. For immunofluorescence studies of deposition of Ig’s, the second kidney
was embedded in optimal cutting temperature (OCT) compound (Miles Inc, Elkhart, IN) and
snap-frozen in a solution of 2-methylbutane and dry ice. Tissue samples were stored at -80oC.
Immunoflorescence Staining
Snap frozen kidneys were cut into 3µm thick cryosections and frozen tissue sections
mounted on glass slides. A DAKO Autostainer Plus Universal Staining System (DAKO,
Carpenteria, CA) was used for the immunohistochemical and immunofluorescent staining.
Immunofluorescent detection of IgG was performed on sections of frozen blocks of mouse
kidney using a FITC-conjugated goat-antimouse Ig antibody (Jackson Immunoresearch
Laboratories Inc., West Grove, PA ), incubated for 30 minutes at room temperature using a
1:250 dilution prepared with background reducing antibody diluent (DAKO),and visualized
by dark field microscopy. Immunohistochemical detection of C3 was performed on sections
of frozen blocks of mouse kidney using a labeled polymer (EnVision plus rabbit, DAKO,
Carpenteria, CA) for visualization by light field microscopy. Rabbit polyclonal antibody for
C3 (Abcam, Cambridge, MA) was used at a dilution of 1:10 with background reducing
antibody diluent (DAKO) and incubated for 30 minutes at room temperature. The chromogen
3,3’ Diaminobenzidine (DAKO) was used. The sections were counterstained with
Hematoxylin (DAKO) and then cover-slipped. Negative tissue controls included normal
mouse kidney. Negative reagent controls consisted of a serial section (the second unstained
frozen slide), processed identical to the first unstained frozen slide, but normal rabbit serum
was substituted for the primary antibody in every assay.
RNA EXTRACTION
Mice were euthanized by CO2 inhalation followed by cervical dislocation on days 1, 3

and 7 post-burn injury. Total RNA was extracted from skin excised from the wound margin
and stored in RNAlater (Ambion, Austin, TX). Briefly, skin tissue was homogenized in
Trizol reagent (Invitrogen, Carlsbad, CA) and total RNA was isolated using Qiagen RNeasy
Lipid Tissue Mini Kit (QIAGEN Inc. Valencia, CA) according to manufacturer’s
instructions. RNA’s were resuspended in 30 μl of 10 mM Tris buffer, pH 7.5. Sample purity,

quantity, and quality was assessed by determining the A260/280, A260/230 ratio on an Nanodrop
Spectrophotometer (NanoDrop Technologies Inc. Wilmington, DE) and by measuring
28S/18S ribosomal RNA ratio and RNA Integrity Number (RIN) using a Agilent 2100
BioAnalyzer (Agilent Technologies Inc. Santa Clara, CA). All Agilent RNA integrity values
were > 8.5. Reverse transcription was performed with Roche 1st Strand Synthesis kit (Roche
Diagnostics Corporation, Indianapolis, IN). Briefly, 2.5 μg of RNA sample was added to a
master mix containing 1x reaction buffer, 5 mM MgCl2, 1 mM deoxynucleotide mix, 6.4 μg
random primers, 100 units RNase inhibitor, and 40 units AMV reverse transcriptase. 10 mM
Tris buffer, pH 7.5 was used to reach 40 μl final reaction volume. Then, final reaction
mixture was subjected to a single reverse-transcription cycle of 25 °C for 10 min, 42 °C for
60 min, 99 °C for 5 min, and 4 °C for at least 10 min.
Real-Time quantitative PCR (RT-PCR) Gene Profiling for Proinflammatory

Transcripts
Quantitative real-time polymerase chain reaction (RT-PCR) was performed using the
ABI Prism 7900HT Sequence Detection System (Applied Biosystems, Foster City, CA).
Custom designed ‘Wound Repair’ TaqMan® Low Density Array (TLDA) cards (Applied
Biosystems, Foster City, CA) were used to assess gene expression. The set of TLDA cards
were comprised of 184 individual target assays (including respective forward and reverse
primers and a dual labeled probe (5’-6-FAM; 3’-MGB)) in quadruplicate on a 384-well card
(96 genes per card). Amplification parameters were as follows: one cycle of 50 oC for 2 min
and 95 oC for 10 min followed by 40 cycles of 95 oC for 30 sec and 60 oC for 1 min. Two
samples were processed on each card.
RT-PCR Data Analysis
RT-PCR data was analyzed using the Sequence Detection System version 2.1 included
with the ABI Prism 7900HT SDS and using Microsoft Excel. The threshold cycle (Ct) for
each sample was manually set to 0.2 and the baseline was set between 3 and 15 cycles. 18S
ribosomal RNA was used as an endogenous housekeeping control gene for normalization and
− ΔΔCT
the comparative Ct method was used to calculate the relative fold expression by 2 [42,
43]. Assays with Ct values greater than 35 cycles were excluded from analysis.
Skin Transplantation
Full-thickness skin specimens for transplantation were obtained from naïve female
MRL/MpJ mice, 8-10 weeks of age, and grafted onto syngeneic naive (8-10 weeks of age)
and experimental (15-17 weeks of age) recipient female MRL/MpJ mice which had fully
recovered from a previous 15% full-thickness TSBA burn injury. Briefly, full thickness donor
skin (3 cm x 3 cm) was harvested from the dorsal skin of euthanized and shaved MRL/MpJ
donor mice, and the underside was gently scraped with a scalpel to remove fat and muscle.
Skin was immersed in cold PBS prior to transplantation. The dorsal surfaces of anesthetized
(i.p. injection of 150 mg/kg ketamine) recipient mice were shaved and washed with 70%
ethanol. A graft bed was prepared with fine curved scissors by removing an area of epidermis
and dermis down to the level of the intrinsic muscle. Grafts, 3 cm2 in area, were fitted to the
prepared bed without suturing and then covered with an adhesive plastic bandage. After 7
days, the adhesive bandage was removed. Graft survival was then followed by daily visual
inspection. Rejection was defined as complete necrosis and loss of viable skin tissue.
Statistics
Mann-Whitney's U test was used to determine the statistical significance of differences

between groups. Survival, incidence of proteinuria, and skin graft rejection-survival was
analyzed by the Kaplan-Meier method and the Log-rank test was used to determine the
statistical significances. P-values less than 0.05 were considered significant.
RESULTS
Severely Injured MRL/MpJ Mice Develop a “Lupus-Like” Syndrome
To assess the role of wound trauma on SLE-like disease development in MRL/MpJ mice
and control BALB/c were subjected to either a full-thickness 15% TSBA burn on the dorsum
or sham-treated. Mice were examined at least twice a week for 6 months the development of
gross skin rashes, ear necrosis, or lymphadenopathy. Results illustrated in Figures 1A-1C
show that within a 1-2 months timeframe post burn injury MRL/MpJ mice started to
spontaneously develop a lupus-like phenotype characterized by severe, excoriating
dermatitis-vasculitis in the dorsum and scapular regions + ear necrosis. Histological sections
of MRL/MpJ skin lesions demonstrate mixed acute and chronic inflammatory cell infiltrates
extending from the epidermis to the subcutis with abnormal hair follicle proliferation (data
not shown). In contrast, no such lesions were observed in either sham-treated or wounded
BALB/c mice.
To assess the effect of burn injury on renal disease development, we monitored urine
protein levels on a weekly basis as an index of proteinuria. Mice were considered to have
proteinuria if they scored >100 mg/dl (>2+) for two consecutive urine samples within a 2
week timeframe.The cumulative incidence of proteinuria (> 100 mg/dl) for each group of
mice is shown in Figure 2A. The incidence and severity of urinary protein scores (Figure 2B)
increased in injured MLR/MpJ mice over time compared to sham-treated MRL/MpJ mice,
wherein only 1 mouse developed severe proteinuria at day 135 post treatment. In contrast,
minimal protein levels were detected in the urine collected from burn injured or sham-treated
BALB/c mice throughout the study interval (data not shown).
As shown in Figure 1C, the observed occurrence of severe skin lesions and proteinuria
results in death 1-2 months later. MRL/MpJ presenting with a SLE lupus-like syndrome died
significantly earlier, with a median survival rate of 103 days, when compared to the 100%
survival of sham-treated BALB/c mice during the 6 month evaluation time period. Six
months after the severe burn injury (mice 8 months of age) only 2 of 21 of the burned
MRL/MpJ mice were alive and showed no gross macroscopic evidence of cutaneous
autoimmune disease.
B C
100 3
Incidence of skin lesions
Skin lesion score
2.5
75
2
50 1.5
1
25
0.5
0 0
0 20 40 60 80 100 120 140 160 180 1-2 mo 3-6 mo
Days post burn injury Time post burn injury
Figure 1. Burn trauma augments SLE development in lupus-prone MRL/MpJ mice. A. Photographs of
typical skin and ear lesions in MRL/MpJ mice exhibiting lupus-like symptoms at 2-6 months post burn
injury. In contrast, no lesions were observed in burned BALB/c mice or age-matched sham-treated
MRL/MpJ mice. B. Cumulative incidence burn injured MRL/MpJ mice [■] and sham-treated MRL/MpJ
mice [●] exhibiting skin lesions. C. Mean skin lesion score (see Material and Methods) of burninjured
mice [■] and sham-treated mice [□].
As depicted in Figures 1C, a biphasic survival response ensued with a cohort of

MRL/MpJ mice that displayed autoimmunity within 1-2 months post burn injury and a
separate cohort of MRL/MpJ mice that developed cutaneous lupus-like lesions 4-6 months
post burn injury.
For this reason, data from these two groupings were pooled and evaluated separately. In
sharp contrast, 19 of 21 sham-treated age-matched control MRL/MpJ mice survived to
greater than 36 weeks of age and showed no incidence of cutaneous disease and minimal
proteinuria during the same observation period. One sham-treated MRL/MpJ mouse
spontaneously died at 3 month of age and the other at 5 month of age with unknown causes
of deaths. Sham-treated (21 of 21) and burn injured BALB/c mice (21 of 21) appeared normal
and healthy throughout the study period, showed no signs of proteinuria or premature death
and thus were not evaluated rigorously. Notably, at the time of euthanasia the comparison of
spleen weights between wounded MRL/MpJ mice at 4-6 months post injury and sham-treated
mice at 6 months post injury, revealed a mild splenomegaly (~1.7-fold increase) in mice
exhibiting lupus-like disease (393 + 90mg, n=10 versus 231 + 60 mg, n=13, P< 0.05, Figure
3A). Similar increases were noted in the size of some of the cervical, brachial and inguinal
lymph nodes. Approximately, 30% of the mice with skin lesions had enlarged lymph nodes at
4-6 months post burn injury, while sham-treated MRL/MpJ mice did not exhibit visible signs
of enlarged lymph nodes (Figure 3B). In comparison, no significant differences in spleen
weight and lymph nodes size between sham-treated and wounded BALB/c mice was
observed (data not shown).
A B
100 4
Incidence of proteinuria (%)
3.5
Proteinuria score
75 3
2.5
50 2
1.5
25 1
0.5
0 0
0 20 40 60 80 100 120 140 160 180 1-2 mo 3-6 mo
Days post burn injury Time post burn injury
C 100
80
Percent survival
60
40
20
0
0 20 40 60 80 100 120 140 160 180
Days post burn injury
Figure 2. Wounded lupus-prone MLR/MpJ develop proteinuria and have marked decrease survival in
comparison either to age-matched sham-treated MRL/MpJ mice or control BALB/c mice (data not
shown). (A) Cumulative incidence of proteinuria (> 100mg/dl) (B) Mean proteinuria score (see
Material and Methods) of burn injured mice [■] and sham-treated mice [□]. (C) Percent survival rate of
burn injured MRL/MpJ mice (■) and sham-treated MRL/MpJ mice [●].
A B
500 3
2.5
Lymph node score

Spleen weight (mg)
400
2
300
1.5
200
1
100 0.5
0 0
1-2 mo 3-6 mo 1-2 mo 3-6 mo
Time post burn injury Time post burn injury
Figure 3. Spleen weights (A) and lymph node scores (B) in burn injured mice [■] and sham-treated
mice [□] MRL/MpJ lupus prone mice. * p< 0.05.
Serum Hypergammaglobulinemia and Anti-DNA Antibodies
Hypergammaglobulinemia and elevated levels of autoantibodies, such as anti-dsDNA

antibody, in the serum are characteristic of autoimmune disease in lupus-prone mice. To
determine whether burn injury affected serum Ig concentrations in MRL/MpJ mice, we
measured serum IgG1, IgG2a, IgG2b and IgG3 isotype specific anti-dsDNA antibodies by
ELISA at 0-1, 2-3 and 4-6 months post burn injury and in sham-treated mice at 6 months (end
of study). As shown in Table 1, burn injury in MRL/MpJ mice induced a significant elevation
(up to threefold increase) of serum IgG1, IgG2a, IgG2b and IgG3 isotypes in comparison to
Ig levels in the serum of sham-treated MRL/MpJ mice after 6 months of time. Isotypic
analysis of serum anti-dsDNA antibodies revealed significant elevations in serum anti-
dsDNA IgG2a, IgG2b and IgG3 levels were differentially induced between wounded
MRL/MpJ mice and sham-treated MRL/MpJ mice at 4-6 months post wounding (Figure 4).
The ratio of the anti-dsDNA IgG2a to anti-dsDNA IgG1, a parameter of Th1/Th2 balance,
was significantly increased in the wounded MRL/MpJ mice at 4-6 months post injury.
Notably, the frequency of IgG2a, IgG2b and IgG3 anti-dsDNA antibodies was significantly
lower in sham-treated MRL/MpJ mice. As expected, no significant difference in serum IgG
isotypes and IgG specific anti-DNA antibodies were detected in either burned or sham-treated
BALB/C mice at the end of the study period (data not shown). Thus indicating wound trauma
promotes production of anti-dsDNA autoantibodies in lupus-prone mice.
Table 1. Serum IgG subclasses in sham-treated controls and burned MRL/MpJ mice at
the time of euthanasiaa
Sham-treated Burned
IgG1 0-2 wks ND 0.43 + 0.259

2-3 mo ND 5.52 + 0.77*
4-6 mo 1 .46 + 0.18* 4.41 + 1.88*
IgG2a 0-2 wks ND 0.14 + 0.13

1-2 mo ND 1.59 + 0.34*
3-6 mo 0.64 + 0.29* 2.16 + 0.51*
IgG2b 0-2 wks ND < 0.014

1-2 mo ND 0.17 + 0.6*
3-6 mo 0.09 + 0.03 0.27 + 0.08*
IgG3 0-2 wks ND < 0.01

1-2 mo ND 0.17 + 0.06*
3-6 mo 0.01 + 0.01 0.27 + 0.07*
a
Sera IgG isotypes were measured by ELISA at 1:200,000 dilution. Results are expressed as the Ig
concentration in mg/ml + SEM.
*
p < 0.05 versus 0-2 week post burn measurements.
Renal Histopathology
Glomerulonephritis is a characteristic pathological feature of murine SLE. To investigate

the effects of burn injury on renal disease progression, kidney sections obtained at the time of
necropsy were examined by standard histopathological and immunohistochemical techniques
for evidence of glomerular inflammation and immune complex deposition.
The photomicrographs in Figure 4 are representative glomeruli from a wounded
MRL/MpJ mouse exhibiting lupus-like syndrome 90 days post injury and glomeruli from an
age-matched sham-treated control MRL/MpJ mouse. PAS stained glomeruli from mice
presenting with lupus like syndrome typically showed a marked increase in glomerular
cellularity with histopathological evidence of diffuse proliferative glomerulonephritis,
segmented glomeruli, proliferative changes in mesangial and endothelial cells of the
glomeruli, increase in mesangial matrix, capillary basement membrane thickening,
mononuclear cell infiltrates in interstitium, and often the presence of intratubular
proteinaceous casts. All these findings are indicative of glomerular dysfunction. Kidneys
from age-matched sham-injured MRL/MpJ mice revealed glomeruli with normal cellularity,
mesangium, and glomerular basement membranes. The average renal lesion score in mice
exhibiting lupus-like syndrome was significantly greater than that of uninjured age-matched
control MRL/MpJ mice (Figure 4B).
1 1.4
Anti-dsDNA IgG1 Anti-dsDNA IgG2a
1.2
0.8
1
A450
0.6
A450
0.8
0.6
0.4
0.4
0.2
0.2
0 0
50 100 200 1000 10000 50 100 200 1000 10000
Reciprocal serum dilution Reciprocal serum dilution
1.4 Anti-dsDNA IgG2b 1

Anti-dsDNA IgG3
1.2 0.8
1
0.6
0.8
A450
A450
0.6 0.4
0.4
0.2
0.2
0 0
50 100 200 1000 10000 50 100 200 1000 10000
Reciprocal serum dilution Reciprocal serum dilution
Figure 4. Serum anti-dsDNA antibody titer levels of different IgG subclasses between burn injured
MRL/MpJ mice ( ● 1-2 weeks post burn; ■1-2 months post burn; ▲ 3-6 months post burn) and sham-
treated MRL/MpJ mice (♦ 6 months). Reactivity of diluted serum with calf thymus DNA was
determined by ELISA. Values are the mean + SD absorbance values at 450nm (4-8 serum samples per
time point).
Glomeruli from sham-treated or burn injured BALB/c mice showed no evidence of

glomerular disease. Consistent with this observation, immunofluorescence staining against
total Ig showed intense positive staining of the peripheral capillary loops of the glomeruli
from wounded MRL/MpJ mice. Immunostaining against C3 showed comparable immune
complex deposition. Such deposits were found mainly within the affected glomeruli. In sharp
contrast, immunofluorescence and immunostaining analysis of sham-treated MRL/MpJ
kidneys demonstrated minimal Ig and C3 deposition. Together these findings suggest that
wound trauma promotes glomerulonephritis in lupus-prone mice.
Expression of Cytokine mRNA in Skin Post Burn Injury
Abnormalties in cytokine production have been shown to contribute to the development

of autoimmune disease in lupus-prone mice.
PAS IgG C3
A
Sham-
treated
Burned
18
B 16
Renal lesion scores

14
12
10
8
6
4
2
0
1-2 mo 3-6 mo
Time post burn injury
Figure 5. Accelerated glomerulonephritis and immune complex deposition in lupus-prone MRL/MpJ

mice following burn injury trauma. (A) At the time of sacrifice, the kidneys were removed and then
sectioned before staining with PAS, FITC-conjugated anti-mouse IgG, or anti-mouse C3.
Representative photomicrographs of glomeruli from burn injured and sham-treated MRL/MpJ mice are
shown (400x magnification; scale bars = 25 μm). In burn injured mice immunofluorescence for IgG
was diffusely, globally and strongly present as intraglomerular deposits in the mesangium and capillary
wall. C3 staining was present multifocally or globally as intraglomerular deposits in the cytoplasm of
the mesangium and capillary wall. C3 deposits resulted in a thick appearance of the glomerular
capillary loops. These IgG and C3 deposits resulted in a thick appearance of glomerular capillary loops.
The level of Ig and C3 immunostaining was strong in comparison to age-matched sham-treated mice.
(B) Kidney sections were graded for glomerular inflammation, cellular infiltration, proliferation,
crescent formation, and necrosis. Scores from 0 to 3+ were assigned to each of these elements and then
added together to yield a mean renal score (n= 5 mice; 10-15 glomeruli per kidney section were
counted, 2-3 sections per mouse).
To determine whether accelerated lupus onset in burn injured MRL/MpJ mice is related
to aberrant expression of mediators that play a role in the early inflammatory response, we
measured the transcript levels of 184 genes (cytokines, chemokines, growth factors, wound
repair response mediators) using custom made taqman cDNA arrays. We identified that
transcripts for IL-1β, TNF-α and PGE2 were elevated in burned MRL/MpJ mice skin for the
first 7 days (Figure 5) when compared to the expression levels of these mediators in the
wound margins of burned BALB/c mice, which fail to develop cutaneous and renal
autoimmunity. There was no significant difference in the expression of both Th1 and Th2
cytokine and other inflammatory gene transcripts between MRL/MpJ and BALB/c mice (data
not shown).
Skin Isograft Rejection
To determine whether injured MRL/MpJ mice developed T-cell mediated autoimmunity

following severe burn injury, we evaluated whether these mice could mount an isograft
rejection response. We transplanted syngeneic naïve skin onto the dorsum of MRL/MpJ mice
30-40 days post burn injury. Graft survival was determined and compared with that of non-
injured age-matched control MRL/MpJ mice. As shown in Figure 6, MRL/MpJ mice that
were previously subjected to severe wound trauma promptly rejected the naïve MRL/MpJ
syngeneic skin, with a mean survival time of 8 days (n = 5). Histological analysis of the
isografts revealed heavy lymphocytic infiltration and extensive tissue damage (data not
shown).
1000
day-1
day-3
day-7
Relative gene expression
100
10
1
IL-1β IL-6 PGE2 TNFα IL-1β IL-6 PGE2 TNFα
BALB/c MRL/MpJ
Figure 6. Quantitative analysis of IL-1β, IL-6, TNF-α and PGE-2 transcripts in MRL/MpJ and BALB/c
wound margin skin tissue at days 1, 3, and 7 days post burn injury. The results represent the mean + SD
(n= 6) relative gene expression level of transcripts in comparison to those levels present in naïve skin.
In contrast, skin graft sites (n = 5) on sham-treated MRL/MPJ mice were uniformly

healed by 2-weeks post transplantation. The graft integrity remained intact throughout the 30-
day observation period without any gross visible evidence of rejection. Microscopic
evaluations of these grafts failed to identify any inflammatory lesions and revealed normal
epidermis and dermis architecture.
Burned MRL Sham-treated MRL

A
D-7
D-14
100
B
Graft survival (%) 75
50
25
0
0 5 10 15 20 25 30
Days post skin transplantation
Figure 7. Burn injury in lupus-prone MRL/MpJ mice results in the loss of tolerance to self. Severely
injured MRL/MpJ mice reject skin isografts from naïve MRL/MpJ donors whereas skin grafts were
uniformly healed within 2-weeks and accepted for more than 30 days after transplantation in sham
treated age-matched control MRL/MpJ recipient mice (n= 5). (A) Photographs of skin isografts at day 7
and 14 post transplantation. (B) Graft survival was determined and presented as a Kaplan-Meier plot
(n= 5 per group, p < 0.05). Burn injured MRL/MpJ mice (■) and sham-treated MRL/MpJ mice [●].
CONCLUSION
SLE is an autoimmune disease with a complex etiology and is characterized by

multiorgan autoreactivity leading to the production of autoantibodies [2, 3, 6]. In this study,
we directly demonstrate that severe trauma can be added to the list of triggering events that
promote the manifestation of SLE autoimmune disease in lupus-prone MRL/MpJ mice.
Unlike wounded BALB/c mice (non-lupus prone), wounded MRL/MpJ mice show
accelerated lethality, or had to be euthanized, within 2-6 months post-wounding due to
complications associated with spontaneous development of severe vasculitis,
lymphoadenopathy, or renal injury. Wounded MRL/MpJ mice (18 of 20 mice; 90%), unlike
sham-treated mice or wounded BALB/c control non-lupus mice, developed severe skin
lesions, hypergammaglobulinemia, increased circulating levels of anti-dsDNA antibodies,
proteinuria, and renal pathologies. Light, immunohistochemical and immunofluorescent
microscopic examination of glomeruli showed mesanglial proliferation, diffuse thickening of
the basement membrane, inflammatory cell infiltration, and marked Ig and C3 deposition in
glomeruli. Even more striking, we observed that MRL/MpJ mice, at 30-40 days after injury
recovery, rejected syngeneic skin grafts at a time when disease onset was first evident at the
macroscopic level. In contrast, sham-treated mice differ in their incidence of autoimmune

disease progression, anti-dsDNA antibodies, immune complex glomerulonephritis and
survival (1 out of 20 mice died < 6 months post burn injury) rate.
Severe injury has been shown to lead to pronounced defects in immune function,
including increased proinflammatory cytokine production, decreased antigen recognition,
increased Th2 cytokine production, and altered antibody production [30, 33, 36, 44-48].
Among other factors, imbalances in pro and anti-inflammatory cytokine production is thought
to play a major role in the progression of autoimmune SLE disease [49-54]. Despite strain
differences in expression, we found that IL-1β, IL-6, TNFα, and PGE2 transcripts were
consistently elevated in MRL/MpJ wounds compared to BALB/c wounds. However, the
degree of over expression differed greatly with IL-1β and PGE2 most markedly elevated.
These factors are produced primarily by activated macrophages during an inflammatory insult
and have potent stimulatory effects on T and B lymphocytes, NK cells and neutrophils,
increasing PGE2 synthesis and acute phase protein production by these cells [55-59]. In
major burns there is an abundant release of proinflammatory mediators that contribute to
macrophage hyperstimulation [57]. After several days the hyperstimulated macrophages
become globally inhibitory and induce elevated production of IL-10, which enhances Th2
responses [56-58, 60]. Our findings are consistent with studies suggesting potent local and
systemic roles for proinflammatory mediators (IL-1β, IL-6, TNFα) in promoting the
differentiation of Th2 autoimmune responses in both SLE patients and lupus-prone mice [38,
52-54, 61-67] . On the other hand, we found no evidence for increased expression of IL-1α,
IL-2, IL-4, IL-5, IL-10, and TGF-β, transcripts in MRL/MpJ wounds when compared to
BALB/c wounds. Furthermore, Voronov et al [68] and Liang et al [69] reported that IL-1β
deficient mice and anti-IL-6 antibody treated mice are resistant to SLE induction,
respectively. In our study, despite the marked increase in cytokine levels in the adjacent
wound tissue at days 1-7 post burn injury the absolute levels of IL-1, IL-2, IL-4, IL-5, IL-6,
IL-8, IL-10, IL-12, TNFα, and IFNγ in the serum were unaltered when compared to sham-
treated control mice (data not shown). On the other hand, we did not monitor circulating and
tissue levels of cytokines throughout disease progression. Therefore, the systemic kinetics of
early Th1 versus Th2 responses following burn injury in MRL/MpJ mice and their correlation
link with increasing autoimmune disease severity remains unclear.
The over expression (hyperstimulation) of IL-1 in MRL/MpJ wounds is intriguing given
that marrow derived and peritoneal macrophages from SLE-prone mice have been shown to
under express IL-1β and other multiple cytokines in response to LPS stimulation, apoptotic
cells, other macrophage activators and danger signals due to a cell membrane signaling defect
[70-73]. IL-1β can stimulate Ig production directly or synergize with other B-cell stimulatory
factors [61,74]. Post severe burn injury, macrophage overstimulation has been shown to lead
to global down modulation of inflammatory cytokine production and elevated anti-
inflammatory IL-10 and PGE2 synthesis, which have been shown to augment Th2 immune
responses. Elevated production of Th2-dependent Ig autoantibody subclasses in the serum of
wounded MRL mice strongly suggests a skewing of the Th1/Th2 balance toward a Th2
response. MRL/MpJ wounded mice had significantly elevated serum levels of anti-dsDNA
antibodies of the IgG1, IgG2b, and IgG3 isotypes, isotype switching which is known to be
dependent on Th2 cytokines [64, 65]. Our results are consistent with the model that
glomerulonephritis in autoimmune kidney disease is predominantly dependent upon IgG2b

and IgG3 Th2-dependent nephritogenic autoantibodies, deposition [75, 76]. Recently,
Shimizu et al [77] reported that the nature of Th1/Th2 response is critical in determining the
type and severity of glomerulonephritis that develops in lupus prone mice.
Mice from the MRL strain are prone to develop systemic lupus erythematosus and have
demonstrated accelerated wound healing and scarless tissue regeneration, however many of
the mechanisms involved in these clinically relevant pathologies are unclear [78-80].
Notably, the nature of the antigen may be important in driving autoimmune pathology in
lupus-prone mice. In burn wounds, clearing a plethora of self-antigens in necrotic tissue is an
essential role of the macrophage and contributes to their hyperstimulated state [31, 60].
Macrophages from lupus-prone strains have been shown to have an apoptotic-dependent
autoimmune phenotype which includes aberrant cytokine expression [73]. Importantly, non-
autoimmune mice do not demonstrate this defect. Dysregulated functional activity (decreased
phagocytosis, errors in self-Ag recognition and processing) and aberrant signaling events
(cytokines, apoptotic ligands and receptors) involved with the clearance of apoptotic cells are
thought to predispose an individual to autoimmune disease [15, 73, 81-85]. Rapid clearance
of apoptotic cells is essential to prevent intracellular leakage of toxic cell contents, additional
inflammatory cascades and the shift from tolerance to immunity [86-88].Thus, effective
clearance of apoptotic cells might be an active process of immune tolerance following a
traumatic injury or “danger signal” minimizing exposure to self antigens and the expansion of
self-reactive effector T cells.
Autoimmunity coincides with the loss of tolerance to the self [1, 20]; it is thought of as a
persistent failure of an integrated fabric of components rather than the adverse consequence
of a specific "forbidden clone" [89, 90]. In comparison to uninjured MRL/MpJ mice, where
skin acceptance and healing in the gross appeared to be complete at 14 days with a full pelt of
hair by day 21, syngeneic skin grafts in previously wounded MRL/MpJ mice were uniformly
rejected in 7-10 days. Thus, isografts were accepted in tolerant mice and rejected in mice
exhibiting immunological reactivity against self (autoimmune). At this time, it is unclear
whether the antigen-driven isograft rejection response is primarily T-cell or B-cell (humoral)
mediated or both, although we detected high serum levels of autoimmune antibodies. No
doubt, specific factors associated with severe trauma and the subsequent wound healing
processes in lupus-prone mice play a role in triggering latent autoimmune responses. The
relationship between the factors that trigger the early onset and development of autoimmune
disease following traumatic injury remain to be defined. Our central hypothesis is that “the
activation of specific cell types and the expression of genes, cytokines, and other mediators
affecting wound healing and repair processes may be critical in the breakdown of self versus
non-self recognition triggering autoimmune diseases following traumatic tissue injury”.
In summary, our research shows that traumatic injury can activate the SLE disease
processes. The link between traumatic injury and the manifestation of SLE, along with the
increasing numbers of female U.S. military personnel deployed to theatre, make autoimmune
diseases, like SLE, highly relevant to military populations. Improved understanding of the
mechanisms triggering SLE and disease progression could lead to diagnostic and prevention
strategies that would reduce the negative impact of SLE not only on individual Warfighters,
but the hundreds of thousands of civilians stricken with this debilitating (and potentially life-
threatening) disease.
ACKNOWLEDGEMENTS
This work was supported by ONR work unit 601153N.4508.519.A0508.

The authors are employees of the U.S. Government. This work was prepared as part of
their official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title
is not available for any work of the United States Government.’ Title 17 U.S.C §101 defined
a U.S. Government work as a work prepared by a military service member or employees of
the U.S. Government as part of that person’s official duties. The opinions or assertions
contained in this paper are the private views of the authors and are not to be construed as
reflecting the views, policy or positions of the Department of the Navy, Department of the
Army, Department of Defense nor the U.S. Government. The experiments reported herein
were conducted in compliance with the Animal welfare Act and in accordance with the
principles set forth in the current edition of the Guide for Care and Use of Laboratory
Animals, Institute for Laboratory Animal Resources, National Research Council, National
Academy Press, 1996.
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Chapter 4
IMMUNOTHERAPY WITH IG-DERIVED PEPTIDES

IN SLE:
CURRENT STATUS AND DIRECTIONS
Antonio La Cava∗ and Bevra H. Hahn

Division of Rheumatology, Department of Medicine,
David Geffen School of Medicine at the University of California Los Angeles,
Los Angeles, California 90095-1670
ABSTRACT
Understanding the mechanisms that control autoimmune reactivity is an essential
step to improve management of autoimmune diseases including systemic lupus
erythematosus (SLE). In SLE, the interaction between hyperactive T cells and B cells
causes dysregulated production of autoantibodies that by themselves or in immune
complexes fix to tissue and cause organ damage. The immune cell subsets that take part
in this process have come under intense scrutiny in the past few years, and new
information has been acquired on how they interact to induce and/or modulate disease.
This information has also led to the development of autoantibody-derived peptide
therapies that can effectively influence murine SLE. This chapter describes the rationale,
current experimental evidence, preclinical models, and future directions for the use of
autoantigen- and Ig-derived peptides in SLE. Considering that autoantibodies in lupus
patients have amino acid sequences similar to those of murine antibodies, and at similar
locations, it is likely that some of those strategies will be potentially useful in the therapy
of human SLE.
∗
Corresponding author. Tel.: (310) 267 4975; Fax: (310) 206 8606; E-mail: [email protected]
132 Antonio La Cava and Bevra H. Hahn
INTRODUCTION
The functionality of the immune system relies upon an intricate network of interactions
among soluble mediators and lymphocyte subpopulations that have both regulatory and
effector functions. The fine balance among the components of this network constitutes what
is known as immune homeostasis, which is the ability of the immune system to maintain a
condition of internal equilibrium even when faced with (mostly external) challenges. If one
or more of the mechanisms of immune homeostasis become impaired and unable to control
lymphocytes that are autoreactive (reactive to self), autoimmunity can ensue [1-2].
Although autoreactivity is an intrinsic feature of the immune system and is the result of a
diverse repertoire protecting individuals from most pathogens, it carries nonetheless the
potential risk to harm the host. To prevent such a possibility, several mechanisms are in place
to limit the number and/or activity of autoreactive clones. During ontogeny, most of the self-
reactive T cells are eliminated in the thymus, and B cells that react against self-determinants
are deleted in the bone marrow. However, a large number of autoreactive lymphocytes escape
those “central” processes of selection and form a peripheral pool of potentially
autoimmunity-inducing lymphocytes (which could become activated in the presence of self-
antigens released after destruction of tissue, injury, or inflammation). To avoid the
pathological consequences of autoimmune reactivity, several “peripheral” tolerance
mechanisms operate throughout life.
Schematically, those mechanisms can be divided into the following: 1)
hyporesponsiveness (anergy) to antigenic stimulation; 2) activation-induced cell death
(apoptosis), when lymphocytes receive strong activating signals that lead to a cascade of
events resulting in cell death; 3) ignorance to antigens that are sequestered in locations not
directly exposed to immune surveillance; 4) phenotypic skewing of cytokine production; 5)
suppression. The last category includes the action of specialized regulatory/suppressor T
lymphocytes that keep autoimmunity at bay via functional or physical inactivation of
autoreactive cells. As seen later, those lymphocyte subsets may represent a major target of
immune modulation by peptide therapy.
RATIONALE FOR USING PEPTIDE-BASED IMMUNOTHERAPY

IN AUTOIMMUNE DISEASE
Autoimmunity is highly influenced by genetic and environmental factors. A widely

accepted view of the pathogenesis of autoimmune diseases is that an abnormal immune
response of genetically susceptible individuals to environmental agents can lead to
development of autoimmune reactivity, which then is ineffectively controlled by
immunoregulatory mechanisms [5]. Once established, the dysregulated activity, function and
signaling of immune cells can promote tissue and organ damage concomitantly with an
ongoing poorly regulated production of pro-inflammatory soluble mediators [6].
T cells need two signals to become activated. Signal one is given by the engagement of
the T cell receptor on the T cell and the major histocompatibility complex (MHC)/peptide
Immunotherapy with Ig-Derived Peptides in SLE… 133
complex on the antigen presenting cell (APC). Signal two involves the binding of a
costimulatory molecule on the T cell with its ligand on the APC. Increased costimulation in
conjunction with a lowered threshold for T-cell activation is common in autoimmunity and
contributes to abnormal regulatory and effector immune cell functions [7].
Preclinical studies using various animal models of autoimmune diseases have
demonstrated that blocking costimulation (e.g. CTLA-4/CD80/CD86, CD40/CD40 ligand)
[8-9] and/or effector CD4+ T cells may help to control autoreactivity [10]. Considering that
this approach blocks T cell activation in general, it lacks specificity. Therefore, dose and
duration of treatment need to be balanced against the risk of side effects. Other therapeutic
targets and alternative options that have emerged include soluble receptors, monoclonal
antibodies and molecular mimetics or biologicals that can target defined disease pathways
[11]. In SLE, candidate molecules for therapies have included antagonists of B-cell-activating
molecules such as B lymphocyte stimulator (BLyS), of pro-inflammatory cytokines such as
interferon (IFN)-α, and of receptors such as FcγRIIB receptor or the toll-like receptors [12-
14]. Selective targeting of selected immune cell subsets (i.e. dendritic cells, CD20+ B cells)
has also been evaluated with encouraging preliminary results, particularly for B-cell
depletion [15-16] (Figure 1).
Figure 1. Schematic representation of the current targets of immunotherapy in SLE. After presentation
of autoantigen by APC, T cells can provide help to B cells for the production of autoantibodies that
cause tissue and organ damage. The targets of immunotherapy are indicated in red and include – in
addition to the autoantigen- or Ig-derived peptides described in the chapter – soluble molecules such as
IFN-α (targeted by anti-IFN-α antibodies), and BLyS (and/or its receptor(s), targeted by anti-BlyS
antibodies or by TACI Ig). Target surface molecules include CD20 (targeted by anti-CD20 antibody),
CD22 (targeted by anti-CD22 antibody), and CD28/B7 (targeted by CTLA4 Ig). Other
immunotherapeutics not included in the figure are mycophenolate mofetil (which targets intracellular
pathways common to T and B cells), and LJP 394 (abetimus sodium), an oligonucleotide of four
dsDNA epitopes coupled to an inert platform. The term Tregs refers to regulatory/suppressor T cell
subsets with different phenotypes.
The benefits of those approaches vary in degree depending on the disease status, the
heterogeneous clinical characteristics of patients, and concomitant therapies. Some of these
novel approaches are constrained either by their limited specificity or by concomitant side
effects caused by the broad targeting of disparate immune cell subsets and pathways.
To induce immune tolerance with a more specific, selective targeting of antigen-reactive
cells (including helper T cells, regulatory t cells and B cells), another approach uses peptides
to eliminate only deleterious cells while sparing the host from adverse side effects. The
rationale behind this approach is that if a peptide is immunogenic – i.e. it can help to provoke
disease – it is then possible that it can also prevent disease when administered at certain
“tolerogenic” doses and under defined routes and circumstances [17]. In other words,
immunogenic peptides derived from disease-associated antigens that modulate cell functions
and/or interactions might replace autoantigens for the modulation of T cell autoreactivity
(and thus pathology).
CONSIDERATIONS FOR USING PEPTIDES TO INDUCE

TOLEROGENIC IMMUNE RESPONSES
The form in which an antigen is administered has a crucial role in influencing the
immune system and in inducing an immunogenic response or not, since antigen in aggregated
form or mixed with adjuvant provoke immunogenic responses, while soluble monomeric
antigens induce tolerance.
To inhibit T-cell activation, soluble synthetic peptides are typically administered using
the oral, intravenous, intraperitoneal, or intranasal routes. The mechanisms underlying the
induction of tolerance after administration of soluble peptides remain debated and may
involve one or more of the mechanisms of peripheral tolerance, with different cells being
anergised, killed, regulated, or being functionally diverted during the process of tolerisation.
Bystander suppression may be an important additional feature of suppression to several
antigens, capable of “spreading” tolerance to additional epitopes and thus helping
compensate for the problem that responses to individual antigens may wax and wane over
time, even in the same individual [18].
Peptide Selection
The selection of the most appropriate epitopes in the design of therapeutic peptides is
crucial for success. An aspect to consider is that a peptide can bind to one or more MHC
alleles and not to others. In mice, the use of inbred strains of animals provides major help in
the identification and choice of peptides for therapy.
In humans, the polymorphism of the MHC genes represents an important challenge in
peptide design. However, certain associations of defined HLA (the human MHC) with
autoimmune diseases provide a natural platform for peptide design, once target antigens have
been identified [19], to obtain theoretically maximal coverage for most disease-associated
alleles in the general population [20].
Peptide Dosage
It may often be difficult to identify the ideal dose of peptide for induction of immune
tolerance, considering that both high and low doses may be effective yet responsible for
significantly different protective mechanisms. In mice, doses used to induce immune
tolerance have ranged from a few micrograms [21] to milligrams [22]. In general, high dose
regimens seem to cause substantial deletion of antigen-specific cells [23] and/or T cell anergy
[24-25], while both high- and low-dose models seem to induce populations of T cells with
immunoregulatory activities [26-28].
Route of Administration
Another crucial aspect in determining the active dose and activity of a peptide is the
route of administration, in addition to peptide solubility, half-life, vehicle of administration,
and structural characteristics.
The route of administration is central in the activity of a peptide because it can either
determine tolerogenicity or immunogenicity (also depending on dosage, as mentioned above).
Although intradermal or intravenous administration may require the lowest doses of peptide
to achieve tolerogenicity, the inconvenience of injection may favor in some cases the choice
of highly effective high-dose regimens - i.e. via intranasal delivery [29]. For example, nasal
instillation of lupus-prone SNF1 female mice with a histone-derived peptide H471-93 induced
a dose-dependent nasal tolerance to the peptide before or after peptide sensitization of those
mice. The effect was associated with increased production of interleukin (IL)-10 and the
suppression of T helper (Th)1-type cytokines by lymph node cells. Chronic nasal instillation
of mice with the H471-93 peptide not only suppressed the development of autoantibodies, but
also altered the severity of glomerulonephritis in lupus-prone (SWR x NZB)F1 (SNF1) mice
[30].
Duration of Tolerance
The duration of tolerance has considerable therapeutic implications in the clinical use of
peptides. In general, peptide immunotherapy is more effective in preventing rather than
reversing or halting disease.
For example, intranasal [31] or intraperitoneal [32] administration of a single dose of a
soluble form of a myelin basic protein (MBP) peptide corresponding to the dominant epitope
of autoimmune encephalomyelitis is effective in preventing disease for 1−6 weeks [33].
However, from 8 weeks onward, autoreactive responsiveness slowly recovers in euthymic,
but not adult, thymectomised mice, suggesting that peptide-induced tolerance is a peripheral
phenomenon which can be reversed by new effector T cells exported from the thymus. A
consequence of this phenomenon is that sustained suppression by peptide therapy may
require repeated administration of peptide, as suggested by many studies [34].
Mechanisms of Peptide-Induced Tolerisation: The Role of APCs
The binding of a peptide to the MHC has to mimic the naturally processed epitope of the
autoantigen and also has to lack an associated inflammatory innate immune response [35]. In
normal conditions, APC encounter antigens in a proinflammatory environment of cytokines
and innate immune responses that are triggered by pathogen-derived molecules. After
encounter, APC differentiate and migrate to draining lymph nodes where they stimulate Th1
or Th2 responses. If an antigen is given in the form of peptide – which means in the absence
of proinflammatory cytokines and innate triggers – when APC migrate to the draining
lymphatics, they will generate immunoregulatory responses.
The APC that seem to best induce tolerance after soluble peptide administration are the
dendritic cells (DC), to which cells soluble peptides are known to bind directly [36-38],
following which the DC activate regulatory T cells. Indeed, repeated injections of self-
peptide−loaded immature DC can protect from autoimmune disease through the generation of
IL-10−producing CD4+ T cells [39].
TYPES OF IMMUNOTHERAPEUTIC PEPTIDES
A limited knowledge of the T-cell epitopes that are critical to the pathogenesis of an
autoimmune disorder is the most significant obstacle for using peptides as surrogate native
antigens capable of inducing immune tolerance.
For certain diseases, target autoantigens are relatively well defined, such as the
acetylcholine receptor (AchR) in myasthenia gravis [40] and thyroglobulin in autoimmune
thyroid disease [41]. For other diseases, the knowledge of the role of candidate autoantigens
in the disease pathogenesis is a necessary prerequisite before embarking in the design of any
therapeutic approach aimed at modulating immune responses to those autoantigens. Only
once these premises are met, the synthesis of peptides can possibly target pathogenic T-cell
autoreactivity.
Peptides can be synthesized to be structurally similar to their native form or they can be
designed as altered peptide ligands [42-44] (APL – which are analogue peptides with defined
substitutions at individual residues causing lack of stimulation of antigen-specific T cells but
retaining ability to bind the MHC). Peptides can also be prepared as multiple antigenic
peptides [45] (MAP – which are multiple, usually identical, peptide sequences attached to a
core of branching lysine residues to yield a multivalent construct). In SLE, MAP have only
been used as immunogens capable of inducing lupus-like disease in non-autoimmune mice
[46] and will therefore not described further. For APL, different subtypes exist. The APL that
give equivalent responses are referred to as agonist peptides; those capable of stimulating at
reduced doses are called superagonists; and those that do not stimulate T-cell clones but
inhibit antigen-specific activation are called antagonist peptides.
In general, APL-based vaccination is complicated by factors including the broadness of
the T-cell repertoire and their often unpredictable effects (i.e. hyperreactivity), yet several
groups have investigated – often with success – the use of APL antagonists as potential
vaccines for autoimmune disease [47].
A recent, unconventional approach to inactivate autoreactive T cells that drive pathology

using peptide delivery is represented by gene therapy with minigenes that encode self
antigen-derived peptides [48]. This strategy seems to promote tolerance by inducing
cytotoxic/suppressive T CD8+ cells after endogenous expression of peptide in the context of
MHC class I molecules [49-50]. With gene therapy, however, the possible release of
cytokines or apoptotic bodies associated with cell lysis should be pondered in view of
possible (even if temporary) exacerbation of disease [51].
PEPTIDE-BASED THERAPY IN AUTOIMMUNITY
Several studies in mice have provided useful insights into the therapeutic use of
immunogenic peptides to delay onset and/or progression of autoimmune disease. These
approaches have enabled the evolution from the traditional pharmacologic strategies of
immune suppression to therapies that attack the pathologic process at a different level,
arguably more directly, via selective control of certain aspects of the inflammatory process
accompanying the autoimmune response. While these studies have given impetus to the
development of alternative strategies of disease management, they have shown in the
meantime significant limitations, e.g. inability to restore long-term immune homeostasis and
superiority in preventing rather than curing inflammation. The new challenge is to overcome
those difficulties by improving current protocols for longer-lasting, possibly curative effects.
OVERVIEW OF PEPTIDE IMMUNOTHERAPY IN AUTOIMMUNITY
To increase the chances of clinically relevant treatment of autoimmunity, it may be

preferable, at least theoretically, to focus on antigens that participate in the mechanisms that
control and sustain inflammation.
In the case of multiple sclerosis, desensitization of T cells specific for the autoantigen
myelin basic protein (MBP) can be achieved via the therapeutic use of a random amino acid
copolymer (glatiramer acetate or Copaxone) – which competes with MBP epitopes for
presentation to T cells, and induces Th2 responses [52].
In experimental autoimmune encephalomyelitis (EAE), an animal model of multiple
sclerosis caused primarily by Th1 cells, glatiramer acetate effectively suppressed the disease.
Copaxone has been approved by health agencies for treatment of multiple sclerosis in
humans, and it has measurable but limited benefits in some patients [53].
However, while bias toward Th2 responses is beneficial in EAE - and possibly in
multiple sclerosis - the same may not be said for SLE, a disease in which both the Th1 and
Th2 arms of the adaptive immune system are hyperactive and affected by the disease [54]. In
the case of SLE, experimental evidence in animal models has revealed the potential of self
antigen-based peptide therapies using mechanisms that in addition to modulation of cytokine
profiles also involve the functional activity of T cells that regulate/suppress production of
autoantibodies [55-56].
Other explored therapies in systemic autoimmune diseases include the neutralization of

pathogenic autoantibodies (anti-β2GP1 antibodies) with peptides in the treatment of the
antiphospholipid syndrome [57].
In organ-specific autoimmunity, different routes of peptide administration have been
tested with encouraging results [58]. Effectiveness of mucosal tolerance induced by peptides
of the AChR in autoimmune myasthenia gravis is under consideration as a therapeutic
modality for patients with myasthenia gravis [59], and intraperitoneal injections of a peptide
derived from glutamic acid decarboxylase in one trial delayed the onset and incidence of
type-1 diabetes in mice [60].
Two studies with peptides derived from heat-shock proteins for the treatment of diabetes
and rheumatoid arthritis have also provided encouraging results. Individuals with newly
diagnosed type-1 diabetes treated by subcutaneous injections at three time points (at study
entry and after 1 month and 6 months) with 1 mg of a peptide derived from the heat-shock
protein hsp60 maintained islet cell function ten months after the first injection, in association
with a modulation toward Th2-type responses [61].
In individuals with rheumatoid arthritis, a peptide derived from the bacterial heat-shock
protein dnaJ administered orally resulted in an increased percentage of T cells producing IL-
4 and IL-10 as well as reduced production of interferon (IFN)-γ and tumor necrosis factor
(TNF)-α, also suggesting an induction of regulatory T cells after therapy [62].
Last but not least, the activation of suppressive CD8+ T cells may represent an additional
way to regulate autoantibody production. Administration of plasmid DNA encoding MHC
class I-binding peptide T-cell epitopes from the VH regions of anti-DNA antibodies induced
specific CD8+ T cells that killed anti-DNA-producing B cells in lupus-prone (NZB x
NZW)F1 (BWF1) mice [49]. The vaccination with those CTL-activating epitopes inhibited
anti-DNA antibody production, delayed development of nephritis, and prolonged survival of
the treated lupus-prone mice [49].
RATIONALE FOR USING IMMUNOTHERAPEUTIC

PEPTIDES IN SLE
The current pharmacologic treatment of SLE is not curative and may often result in
nonspecific immune suppression, with complications including infection and the disruption
of natural immune regulation. In general, to suppress the autoimmune response in SLE,
treatments use drugs such as corticosteroids, cyclophosphamide and mycophenolate mofetil –
highly effective in reducing mortality, yet associated with several important adverse side
effects, particularly infection. Alternative approaches of molecular intervention, using
peptides, can interfere with the production of specific pathogenic autoantibodies and
modulate the activity of autoreactive B and/or T cells, thus avoiding many of the effects of
those classical non-specific therapeutic agents.
As for other autoimmune disease, the strategies used to develop peptide treatment in SLE
have mostly consisted of identification of sequences that could stimulate autoreactive T cells
ex vivo, followed by the synthesis of the corresponding relevant peptides for administration to
lupus mice. In general, different protocols have tested efficacy using different routes of
delivery, doses and frequency of peptide administration in different spontaneous or induced

murine lupus models. After that step, initial translational studies in humans have used either
T cell clones or peripheral blood mononuclear cells in vitro or ex vivo for tests of peptide
validity before embarking into clinical trials. At the time of this writing, one such peptide
(Edratide – a peptide from a CDR region in an antibody to DNA) has been studied in patients
with SLE, with phase II/III trials near completion.
CONSIDERATIONS FOR PEPTIDE-BASED

IMMUNOTHERAPY OF SLE
The ability to modify pathogenic responses via tolerisation with synthetic peptides is
currently under evaluation in SLE. The approach typically follows the validation from
mechanistic studies that have confirmed the value of targeting certain defined antigenic
specificities rather than others. Subsequently, tolerogenic regimens are designed in
accordance with the concept discussed above that if tolerisation to an antigen in an animal
models leads to clinical control of autoimmunity, this control could possibly be translated to
humans using the same approach. However, for SLE there are several limitations in this
assumption. First, the antigens that trigger the autoimmune process in SLE probably include a
series of antigens that are temporally, quantitatively and qualitatively involved in different
ways and/or in the diverse phases of the pathogenesis of the disease (for example histones
associated with nucleosomes induce ANA and anti-DNA antibodies, mimotopes in Epstein-
Barr virus may induce anti-Sm antibodies, β2GP1 can induce anti-cardiolipin antibodies;
immunization with one of these may induce responses that spread to include the others).
Second, even if identified in murine or human SLE, antigens might differ in the two species.
Third, by the time of immunotherapeutic intervention, an initiating antigen may be less
relevant in a process that is self-sustaining through other mechanisms. Despite these
important limitations, peptide-based therapy holds great promise in SLE, and significant
progress has been made. On the other hand, given the above premises, immunotherapy with
peptides in SLE should not rely on a single peptide because of the multiple autoantigens
involved in the pathogenesis of the disease and because of the diversity of patients and their
clinical status. Since we cannot tolerise T cells against all known and unknown epitopes
involved in the pathogenesis of SLE, the suppression of responses to multiple epitopes after
administration of a single peptide would be particularly appealing. In practice, the current
situation seems to lead toward that possibility, considering that often the peptides derived
from self-epitopes can be promiscuously presented and recognized by lupus T cells in the
context of diverse MHC alleles. These findings have opened up practical new possibilities to
develop tolerogenic peptides for the therapy of human SLE.
Many of the experiments in SLE have been performed using high doses of peptides
administered to lupus mice in the absence of adjuvant, to minimize the development of an
immune response. One reason for the requirement of high doses of peptide has been the
limited stability of peptides, an aspect that might possibly be partly overcome using peptide
analogues of higher stability [63].
Table I. Prevention of lupus-like disease manifestations following administration of

Ig-based peptides in murine SLE*
Mouse Peptide Autoantigen Dose R oute R ef.
BWF1 p34+p58+p84 VH from anti-DNA Ig 5 x 300 g i.v. 67
BWF1 pCDR3 anti-DNA Ig (16/6 Id) 4 x 50 g i.p. 96
BWF1 pCDR3 anti-DNA Ig (16/6 Id) 10 x 100-500 g i.v., s.c. 80
BWF1 pCons (artificial, Ig consensus) 1 mg monthly i.v. 79
BWF1 pCD.III (minigene) VH from anti-DNA Ig 3 x 50 g i.d. 49
BWF1 pIgCons (minigene) (artificial, Ig consensus) 6x10*5 transfected B cells i.v. 50
___________________________________________________________________________________________
*The Ig-derived peptide was given to premorbid mice at the doses indicated. The protective effects
consisted of delayed onset of proteinuria and/or prolonged survival of the mice.
Table II. Therapy of murine lupus-like disease using Ig-based peptides*
Mouse Peptide Autoantigen D ose Route Ref.
BWF1 pCons (artificial, Ig consensus) 1 mg monthly i.v. 79
BWF1 pCDR3 anti-DNA Ig (16/6 Id) 10 x 100-500 g i.v., s.c. 81
*The Ig-derived peptide was given to nephritic mice at the doses indicated. The therapeutic effects were
represented by prolonged survival of the mice and/or delayed progression of renal disease.
PROCEDURES OF PEPTIDE SELECTION IN SLE
Peptides Eluted from MHC Molecules
To identify T cell epitopes in autoimmune mice and patients, a strategy has been to elute
and sequence self peptides bound to MHC class II molecules on the APC.
This approach has allowed characterization of I-Ak and I-Ek-associated self-peptides
from lymph nodes of lupus-prone MRL/lpr mice, including peptides from histone H2A, the
RNA splicing factor SRp20, and different ribosomal proteins that were absent in similar
preparations from non-autoimmune C3H mice (which have the same MHC haplotype of
MRL/lpr mice) (64). Among a set of eleven peptides that stimulated proliferation of T cells
from both lupus and normal peptide-immunized mice, only four peptides (i.e. peptides 4–23
of SRp20, 84–103 of H2A, 42–59 of β2-microglobulin, and 110–129 of the MHC class II
molecule I-Akβ) induced proliferation of T cells from I MRL/lpr mice (65). Using a similar
approach, Datta and coworkers identified a peptide eluted from MHC class II molecules of a
murine APC line (fed with chicken chromatin) that corresponded to residues 22–42 of the
chicken histone H5 (homologous to H1′) [66]. This naturally-processed peptide was

recognized by B and T cells of SNF1 mice, stimulated helper functions of several
nucleosome-specific pathogenic T helper clones, and the antibodies against the peptide cross-
reacted with DNA and nucleosomes and accelerated nephritis in SNF1 mice [66].
Peptides Derived from the VH Region of Anti-DNA Antibodies
Using more than 400 overlapping peptides representing the entire VH region of four anti-
DNA antibodies, Singh, Hahn and coworkers isolated four Ig peptides that induced
proliferation of splenocytes from young BWF1 mice and that were able to stimulate IgG anti-
dsDNA Ab production, when cultured in the presence of spleen B and T cells from BWF1
lupus mice [67-68].
Peptides Recognized by T Cells from Both Lupus Mice and Patients
Datta and coworkers identified dominant nucleosomal epitopes involved in cognate

interactions between autoimmune SLE T helper cells and anti-DNA Ig-producing B cells. By
scanning overlapping synthetic peptides, and by mass spectrometry of naturally processed
peptides, five major epitopes in nucleosomal histones were localized, namely H1’22-42, H2B10-
33, H385-105, H416-39, and H471-94 [69-70, 66]. Mice and patients with SLE had in their
peripheral blood T cells as well as B cells which recognized these epitopes, and with age,
autoantibodies against those epitopes reacted with nuclear autoantigens. Tolerogenic therapy
with a single histone peptide epitope could stop the progression of established
glomerulonephritis in lupus-prone mice which by tolerance spreading inactivated
autoimmune T and B cells. Importantly, those histone peptides could bind multiple human
HLA-DR motifs and behaved as promiscuous epitopes [71].
Another peptide of interest is the aminoacid sequence 83–119 of the spliceosomal SmD1
protein (a D1 protein of the Smith (Sm) antigen, part of the small nuclear ribonucleoprotein),
which encompasses a B cell epitope in lupus mice and patients [72-73] and is capable of
binding dsDNA and nucleosomes [74]. This peptide provides CD4+ T cell help to anti-
dsDNA antibodies in BWF1 mice and in one third of lupus patients [75].
High-dose intravenous tolerance to SmD1183-119 (0.6-1 mg/month) delayed production of
autoantibodies, postponed onset of nephritis, and prolonged survival of BWF1 lupus-prone
mice [76]. Of note, one week after SmD1183-119 tolerance induction in prenephritic mice, a
decrease of IFN-γ and IL-4 expression were observed concomitantly with an increased
expression of transforming growth factor (TGF)-β1 [76]. The immune tolerance to SmD1183-
+
119 could be adoptively transferred by CD90 T cells, which were capable of reducing T-cell
help for autoreactive B cells in vitro. The increased frequency of regulatory CD4+ type-1
regulatory T cells in this system associated with the prevention of autoantibody generation
[76].
Finally, Muller and coworkers identified an epitope located within the peptide 131–151
of the spliceosomal U1-70 K protein that stimulated proliferation of and IL-2 release by
CD4+ T cells from MRL/lpr and BWF1 lupus mice [77]. These authors showed that a
phosphorylated analogue of peptide 131–151 (called P140) was a promiscuous epitope
recognized by CD4+ T cells and antibodies from MRL/lpr mice as well as antibodies from
SLE patients [78].
PEPTIDE-BASED IMMUNOTHERAPY OF SLE
Unlike animal models, it is not yet feasible in humans to begin prophylactic treatment at
the early stages of an autoimmune process (i.e. before it manifests clinically). This aspect
may represent a problem for peptide immunotherapy, considering that it has been frequently
shown that peptide-based therapies in animal models are less effective at inhibiting or
reversing an established disease rather than at preventing it.
Only a few studies were successful in mice that have already developed disease. In one
of those studies, administration of H416–39 peptide to SNF1 mice with established
glomerulonephritis prolonged survival and halted the progression of renal disease [18]. A
similar result was obtained after monthly intravenous administration into 20-week-old BWF1
mice of the peptide pCons, an artificial consensus peptide based on the VH regions of several
BWF1 IgG anti-DNA antibodies that had T-cell stimulatory activity [79]. Last, a peptide
derived from the sequence of the complementary-determining region 3 (pCDR3) of an anti-
DNA monoclonal antibody that expressed the 16/6 Id was used successfully in the therapy of
20–28-week-old BWF1 mice [80-81].
ANTI-DNA IG-DERIVED PEPTIDE FOR THE IMMUNOTHERAPY

OF SLE
pCons Peptide
Several autoantibodies to DNA – the hallmark antibodies of SLE – contain amino acid
sequences that induce in vitro T cell proliferation and cytokine secretion [82-83].
Identification of several such T cell epitopes encoded within the VH regions of murine
autoantibodies to DNA indicated that these immunogenic sequences have similar location
and amino acid content in several anti-DNA antibodies [79].
An algorithm was derived to design a 15-mer synthetic consensus peptide (pCons) which
described most of the self-Ig-derived stimulatory sequences in the CDR1/FR2 region of VH
regions from several murine anti-DNA antibodies, for possible use as tolerogen in BWF1
mice. A non-stimulatory peptide with analogous I-Ed binding motifs, yet violating the
consensus motif, was synthesized as a negative control and was called pNeg [79]. Monthly
intravenous injection of 1 mg of pCons was effective in delaying appearance of multiple
autoantibodies and nephritis and in prolonging the survival of treated BWF1 mice beginning
at 10 weeks of age (healthy) or at 20 weeks of age (diseased)[79]. Tolerance was induced by

pCons but not by pNeg, in spite of their strikingly similar molecular structure (Figure 2).
Figure 2. Molecular structure of pCons and pNeg peptides. Despite the similarity, only the Ig-based
peptide pCons can protect BWF1 mice from lupus-like disease, whereas pNeg has virtually no effects
on disease (see text for details).
Since pCons contained amino acid sequences that bound both the MHC class II molecule
and the MHC class I molecules, tolerisation by pCons affected both CD4+ and CD8+ T cell
compartments. Both CD8+ T suppressors (Ts) and CD4+CD25+ regulatory T cells (Tregs)
expanded in vivo in pCons-treated mice, and were fully capable of suppressing anti-DNA
production from B cells [84-85].
CD8+ suppressor T (Ts) cells mainly operated via soluble factors such as cytokines (as
indicated by transwell experiments) to block B cell-mediated production of anti-DNA
antibodies [84].
Previous data had suggested that CD8+ Ts became physiologically defective in aging
BWF1 mice that developed SLE [86]. Therefore, the data seemed to indicate that it could still
be possible to call up these cells and have them be functionally active under appropriate
conditions. Studies aimed at defining the mechanisms by which pCons-induced CD8+ Ts

blocked anti-DNA antibody production and nephritis suggested that the protection exerted by
these cells both in vitro and in vivo depended in part on the upregulation of TGF-β and Foxp3
molecules, and on an increased resistance to apoptosis of the Ts (as compared to similar cells
from naïve mice) [84].
Importantly, tolerisation with pCons not only affected CD8+ Ts, but also the number and
function of peptide-reactive CD4+CD25+ Tregs (85). Although the protocol of tolerisation did
not associate with significant expansion of CD4+CD25+ Tregs when considered in toto, the
relative percent and absolute number of Tregs specific for pCons expanded considerably after
tolerisation [85]. The pCons-induced peptide-reactive Foxp3-expressing Tregs suppressed
anti-DNA antibody production via cell contact mechanisms that included engagement of
membrane-bound TGF-β and glucocorticoid-induced TNF-receptor (TNF-R) family member
18 (GITR), since blockade of these surface molecules on Tregs partly abrogated their in vitro
suppressive capability on the B cells [85].
Overall, the suppressor and regulatory T cells raised by tolerisation with the anti-DNA-
based peptide pCons appeared to use different yet complementary mechanisms to skew the
hyperactive state of lupus B cells towards a reduced production of autoantibodies. One
mechanism was a direct suppression on B cells, and another was an interference with the help
provided by CD4+ T cells to the B cells [87].
Another consideration is that since the pCons-reactive Tregs mainly required cell contact
while the Ts needed soluble factors to operate, the Ts were likely influenced by the local
milieu to suppress in a broader fashion than the Tregs, for which antigen specificity was
necessary [88].
hCDR1 Peptide
The peptide hCDR1 – which is based on the complementarity-determining region

(CDR)1 of a human monoclonal anti-DNA autoantibody that bears the 16/6 idiotype
(common to mice and humans) – also ameliorates the serological and clinical manifestations
of lupus in both spontaneous (BWF1 mice) and induced (BALB/c mice) SLE models [89].
Ten subcutaneous weekly injections of hCDR1 (50 or 200 μg/mouse) mitigated
proteinuria and kidney damage, reduced anti-dsDNA antibody titers and proinflammatory
cytokines, and upregulated production of TGF-β [89]. The therapeutic potential of hCDR1
was also tested in a model of lupus of severe combined immunodeficient (SCID) mice
engrafted intraperitoneally with peripheral blood lymphocytes from patients with SLE. SCID
mice that were treated with hCDR1 (50 μg/mouse) once a week for 8 weeks developed lower
quantities of human dsDNA antibodies than mice treated with a control peptide. Furthermore,
treatment with hCDR1 resulted in reduced proteinuria, and reduced glomerular deposition of
human IgG immune complexes, and of murine complement C3 than in controls [90].
One molecular mechanism that associated with the amelioration of SLE after hCDR1
therapy was a diminished expression of phosphorylated JNK kinase in the T cells, which also
exhibited a reduced rate of apoptosis (most likely because of the down-regulation of the
activity of the pro-apoptotic JNK kinase) [91]. Another mechanism induced by hCDR1 was
the down-regulation of the cell adhesion receptors LFA-1 and CD44 [92]. Since these
receptors operate as accessory molecules in mediating APC-T cell interactions, the reduced
proliferation of autoreactive T cells could be possibly ascribed, at least in part, to this specific
action of hCDR1 on the T cells. Importantly, the beneficial effects induced by hCDR1 could
be transferred to diseased lupus BWF1 mice, in which the adoptive transfer of hCDR1-treated
cells downregulated the disease manifestations [93]. The beneficial effects observed included
an increase in the number of regulatory CD4+CD25+ T cells expressing cytotoxic T
lymphocyte antigen 4 (CTLA-4) and Foxp3 (93). The central role of Tregs in the mechanisms
of protection were confirmed by experiments in which the depletion of the CD25+ cells
diminished significantly the therapeutic effects of hCDR1, whereas administration of
enriched CD4+CD25+ T cells was highly beneficial in diseased mice [93]. Finally, the
amelioration of disease manifestations was associated with a down-regulation of the
cytokines IFN-γ and IL-10 and an upregulation of TGF-β, a molecule that substantially
contributed to the suppression of T-cell autoreactivity [93].
SAFETY CONSIDERATIONS IN THE USE OF

IMMUNOTHERAPEUTIC PEPTIDES
In the use of immunotherapeutic peptides, at least in the early stages of clinical

development (phase I/II clinical trials), relevant information on the efficacy and safety of the
treatment should be recorded together with the use of biomarkers of immunological relevance
(helpful in defining response patterns, thus identifying surrogate markers of efficacy). These
approaches are in place in studies with the only Ig-based peptide currently in phase II/III
clinical trials in SLE patients, the peptide developed in the Mozes’ laboratory called Edratide.
Tolerisation of humans with nucleosomal-associated histone peptides might offer an
advantage over the Ig-derived peptides, in that histone peptides are widely conserved in
ontogeny and thus unlikely to provoke allergic reactions. The Ig-based peptides currently
under study are either artificial or present in some but not all human anti-DNA antibodies,
and therefore could be foreign to some patients. However, these possibilities are theoretical
and remain to be tested in human trials.
CONCLUSION
Despite our growing understanding of the immune mechanisms elicited by Ig-derived

peptide immunotherapy in SLE, there are still many obstacles in fully exploiting the potential
of these therapeutic agents in clinical practice. The data available in the mouse are
encouraging because similar peptide sequences exist in murine and human anti-DNA
antibodies, and, as mentioned before, insights into the mechanisms of immune dysfunction in
lupus mice may have the potential for rapid translational therapeutic use in human SLE [94-
95].
Our current knowledge seems to indicate that many, if not most, of the beneficial effects
of peptide immunotherapy may result from specific elimination or modulation of autoreactive
T cells and/or the expansion of regulatory immune responses. Despite this information,
additional work is required to establish peptide sequences with optimal effects in most
patients, such as peptides based on promiscuous epitopes with broad specificity, capable of
providing long-lasting immune tolerance, and peptides likely to tolerise against more than
one antigenic specificity. In any case, combination with standard therapeutic agents will most
likely be still needed to obtain maximal benefits in the clinical use of peptides in SLE.
ACKNOWLEDGMENTS
A.L.C. and B.H.H. are supported in part by grants from the National Institutes of Health
(AR53239 and AI63515 to A.L.C., and AI/AR46776 to B.H.H.), the Arthritis Foundation
Southern California Chapter, and gifts from the Dorough Foundation, the Horchow Family
Foundation, and Jeanne Rappaport.
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Chapter 5
AUTOANTIBODIES AS PROGNOSTIC OR
DIAGNOSTIC MARKERS OF PSYCHIATRIC
MANIFESTATIONS IN SLE
Paola Margutti, Federica Delunardo, Tania Colasanti,

Ettore Piro and Elena Ortona∗
Dipartimento di Malattie Infettive, Parassitarie e Immunomediate,
Istituto Superiore di Sanità, Rome, Italy.
ABSTRACT
In the course of Systemic Lupus Erythematosus (SLE), a variety of psychiatric
disturbances are reported, including mood disorders (depressive symptoms), psychosis
and anxiety. The reported prevalence of psychiatric disorders in SLE varies widely,
ranging from 17% to 75%, but the diagnosis of these syndromes is difficult and depends
on the exclusion of complications due to an iatrogenic effect of drugs, to metabolic
abnormalities or to infections. Moreover, the diagnosis requires a careful psychiatric
evaluation to exclude merely reactive psychological disturbances.
It has been suggested that several autoantibody specificities play a role in the
pathogenesis of neuropsychiatric SLE. Potential pathogenic relevance has been attributed
to, among others, antineuronal, antiphospholipid, antiganglioside, anti-DNA, anti-
ribosomal P protein and anti-endothelial cell antibodies. However, particularly regarding
psychiatric syndromes, conflicting results have been reported on the association between
serum autoantibodies and symptoms.
The diagnostic and/or prognostic role of autoantibodies associated to psychiatric
disorders in SLE is discussed.
∗
Address correspondence to: Elena Ortona, Department of Infectious, Parasitic and Immune-Mediated Diseases;
Section of Immune-Mediated Diseases; Istituto Superiore di Sanità, V.le Regina Elena 299, 00161 Rome Italy.
Phone: +39.06.49902760; Fax.+39.06.49387112; E-mail [email protected]
156 Paola Margutti, Federica Delunardo, Tania Colasanti et al.
INTRODUCTION
Neuropsychiatric involvement in patients with Systemic Lupus Erythematosus (SLE),

first mentioned by Kaposi more than 100 years ago, still remains one of the main challenge
facing rheumatologists and other physicians [1]. The American College of Rheumatology
recently proposed a classification of 19 different neurological and psychiatric syndromes of
SLE [2, 3]. These case definitions encompass neurological syndromes, involving the central
and peripheral nervous system as well as psychiatric disorders, cognitive deficits and acute
confusional states (Table). However, major problems are still related to low specificity of
some of these syndromes such as headache, cognitive impairment or mood disorders [4].The
diagnosis of neuropsychiatric SLE (NPSLE) is complex not only because of the considerable
prevalence variation (14-80%) but also because of the wide spectrum of neuropsychiatric
manifestations. Different neuropsychiatric manifestations result from a variety of mechanisms
including antibodies, vasculitis, thrombosis, hemorrhages and cytokine-mediated damages.
Table 1. American College of Rheumatology Classification of Neuropsychiatric

Manifestations in SLE
Neurological manifestations
Myasthenia gravis
Acute inflammatory demyelinating polyradiculoneuropathy
(Guillan-Barrè syndrome)
Demyelinating syndrome
Myelopathy
Headache
Neuropathy, cranial
Aseptic meningitis
Mononeuropathy (single/mutiplex)
Plexopathy
Autonomic disorder
Polyneuropathy
Cerebrovascular disease
Movement disorder (chorea)
Seizure and seizure disorders
Psychiatric manifestations
Acute confusional state

Cognitive dysfunction
Anxiety disorder
Mood disorder
Psychosis
Autoantibodies as Prognostic or Diagnostic Markers… 157
Of note, despite the dramatic clinical manifestations, too often changes at the
morphological neuroimaging techniques are minimal and non specific. The diagnosis of
NPSLE remains largely one of exclusion and is approached in individual patients by
thorough clinical evaluation, supported when necessary by autoantibody profiles, diagnostic
imaging, electrophysiologic studies and objective assessment of cognitive performance [5].
Autoantibodies associated to NPSLE may have a diagnostic and/or prognostic role. This
chapter describes and discusses the role of the autoantibodies reported to be associate to
NPSLE.
AUTOANTIBODIES IN NPSLE
Anti-Neuronal Antibodies
Anti-neuronal antibodies are a group of antibodies that react to neuronal components.

Several studies, using different techniques to detect antibodies, demonstrated that anti-
neuronal antibodies are detected predominantly in sera of patients with neuropsychiatric SLE
than in patients without neuropsychiatric manifestations [6-13]. Competition assays showed
that the binding of anti-neuronal antibodies was blocked by mycobacterial glycolipids,
suggesting a link between mycobacterial infection and neuropsychiatric SLE [9]. Hanson and
coworkers showed that a 50-kDa protein may be an important target for these autoantibodies,
preponderantly found in NPSLE patients and that the antigen may play a role in the
pathogenesis of some neurological manifestations in SLE [8]. To validate the hypothesis that
some neuropsychiatric manifestations of SLE are mediated by the direct effect of antibody
binding to neuronal membranes, one study of Bluestein and coworkers showed that anti-
neuronal antibodies were significantly more concentrated in cerebrospinal fluid (CSF) of
patients with SLE and central nervous system diseases than in CSF of SLE patients without
neurological involvement [13].
Brain Reactive Antibodies

Brain reactive antibodies (BRA) are a subgroup of anti-neuronal antibodies that bind to
integral membrane proteins of the brain. BRA correlates with psychosis and/or seizures in
SLE patients [14].
Anti-Microtubule-Associated Protein 2 Antibodies

Microtubule-associated proteins (MAPs) interact with the microtubules of the cellular
cytoskeleton. Other specific immunological markers of neuropsychiatric SLE are the
autoantibodies to microtubule-associated protein 2. This cellular protein is restricted to
neurons and is important in the control of cytoskeletal integrity and other neuronal functions
[15].
Anti-Neurofilament Antibodies
Antibodies to cytoskeletal neurofilament protein antigens (ANFA) had an increased
incidence in patients with neuropsychiatric SLE compared with SLE patients without
neuropsychiatric symptoms. ANFA were found to be directed against the 205 and 160 kDa
proteins of the neurofilament triplet [16].
Anti-Ganglioside Antibodies
Ganglioside is a compound composed of a glycosphingolipid with one or more sialic
acids linked on a hydrophilic sugar chain that contains antigenic determinants. It is a
component of the cell plasma membrane which modulates cell signal transduction events.
The gangliosides most commonly recognized by neuropathy-associated autoantibodies are
GM1, asialo-GM1, GD1a, GD1b, GM2 and GQ1b. Persistent high titer of anti-ganglioside
(AGA) IgM is associated with several diseases, particularly neuropathies. The role of AGA
IgM is to eliminate the immunosuppressive gangliosides shed from tissues during ageing,
degeneration of neural and extraneural tissues and tumor growth and necrosis. In addition, in
vitro observations with human and murine monoclonal antibodies suggest that they are
capable of complement dependent cytotoxicity and apoptosis. AGA IgM can cause leakage of
the blood brain barrier (BBB) in a concentration-dependent and complement-independent
manner, bind to neuronal gangliosides to create a neuromuscular block and serve as a marker
of axonal damage in neuropathies such as multiple sclerosis [17]. Several studies
demonstrated the association between AGA and neuropsychiatric SLE and one study
demonstrated that AGA may have predictive value [18], but these autoantibodies are not
specific of NPSLE and are present also in sera of patients with different neuropathies. In a
study, Pereira and coworkers showed a significant correlation between IgG AGA in the CSF
and IgM AGA in the serum of NPSLE patients. Some NPSLE patients had AGA in the CSF
but not in the sera, suggesting that intrathecal antibody production can result in the
development of this manifestations [19]. Galeazzi and coworkers, in a study of a large cohort
of European patients with SLE, showed an association between AGA IgG and migraine,
dementia and peripheral neuropathy and an association between AGA IgM and depression
[20]. In another study, Chen and coworkers demonstrated the presence of AGA in the CSF of
children with NPSLE, and showed a clear association between AGA and brain computerized
tomography scan. This study suggested that the detection of AGA in the CSF have a
predictive value for the onset of a neuropsychiatric flare [21].
Anti-Triosephosphate Isomerase Antibodies

Sera from patients with NPSLE were screened for antibodies to mouse choroid plexus
cell line ECPC-4 by Western blotting. A 29-kDa protein band detected in NPSLE sera was
identified as triosephosphate isomerase (TPI). TPI plays an important role in glycolysis and is
essential for efficient energy production. Using western blotting technique, Watanabe and
coworkers demonstrated IgG specific to TPI in sera and CSF of patients with NPSLE. The
serum anti-TPI IgG index was higher in the NPSLE than in other autoimmune diseases,
demonstrating a high specificity for the diagnosis of NPSLE [22]. In CSF anti-TPI IgG form
immunocomplexes and contribute to the pathogenesis of NPSLE by activating the
complement system [23].
Anti-Glial Fibrillary Acidic Protein Antibodies

Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system in
astrocyte cells. It is involved in cell structure and movement, in cell communication and in
the functioning of the BBB. A high positive predictive value of anti-GFAP antibodies for
NPSLE has been described [24]. Recently, Trysberg and coworkers showed increased levels
of GFAP in the cerebrospinal fluid of patients with NPSLE. Moreover, successful therapy
with cyclophosphamide in NPSLE patients resulted in significantly decreased CSF levels of
GFAP [25]. On the contrary, Conti and coworkers found no correlation between anti-GFAP
antibodies and psychiatric morbidity [26].
Serum Lymphocytotoxic Antibodies

An important place in the immune network is reserved for specific interactions between
regulatory antibodies and their ligands on T and B lymphocytes. Lymphocytotoxic antibodies
(LCA) are detected in a great majority of patients with SLE. Several studies show or claim a
relationship between the presence of LCA and neurological manifestations in SLE patients;
the results, however, remain questionable due to the difference in detection methods as well
as in definition of central nervous system (CNS) involvement [27]. Analysing frequency of
HLA class II antigens (DR and DQ) and LCA in patients with NPSLE, Silva and Donadi
showed that HLA-DR4 may have a protective role for the development of NPSLE whereas
HLA-DR9, in addition to HLA-DR3 and to LCA, may predispose to neuropsychiatric
abnormalities [28]. Analysis of LCA serially in SLE patients revealed fluctuations in LCA
associated with NPSLE relapses or remissions [29].
Anti-CD4 Antibodies
Several lines of evidence indicate that the CD4 glycoprotein may be recognized by
autoantibodies. Lenert and coworkers evaluated the presence of serum anti-CD4 antibodies in
patients with SLE. The antibody reactivity has been analyzed both on native CD4 (by
immunofluorescence) and on recombinant CD4 (by ELISA and Western blot). The results of
this study showed that the anti-CD4 reactivity occurred in SLE patients with active disease
and, as measured by the SLEDAI, was associated with particular clinical manifestations,
including neuropsychiatric disease [30].
Anti-SSA/Ro Antibodies
The SSA/Ro antigens are nuclear and cytoplasmic polypeptides which serve as
autoantigens in SLE and Sjögren's syndrome. They contain two major isoforms of 60 and 52
kDa. Initially, these antibodies were thought to be an epiphenomenon of autoimmune
diseases. Recent studies have shown that they are associated with specific clinical
manifestations and disease subsets [31]. To identify factors predictive of NPSLE, a large
cohort of SLE patients was followed over 11 years. The presence of anti-SSA/Ro antibodies
was demonstrated to be one independent predictor of significant neuropsychiatric damage
[32]. Anti-SSA/Ro antibodies were detected also in CSF of patients with NPSLE [33]. In a
clinical observation regarding suicide attempts in NPSLE patients, all harbored elevated titers
of anti-SSA/Ro antibodies [34]. On the other hand, Shimojima and coworkers investigating
the predictive value of distinct clinical factors in patients with NPSLE, demonstrated that
anti-SSA/Ro antibodies were not significant for NPSLE and they did not predict the
development of NPSLE [35]. In accordance with the previous study, Conti and coworkers did
not find any significant correlation between the presence of anti-SSA/Ro autoantibodies and
psychiatric involvement [26].
Anti-Sm Antibodies
Anti-Sm antibodies are directed against 7 proteins (B/B', D1, D2, D3, E, F, G) that
constitute the common core of U1, U2, U4 and U5 small nuclear ribonucleoprotein (snRNP)
particles [36]. Winfield and coworkers, in a serologic study on patients with NPSLE, found a
higher incidence of anti-Sm antibodies in the patients with CNS dysfunction compared with a
large group of patients without neuropsychiatric disease, suggesting an association of anti-Sm
with CNS disease in SLE [37]. On the contrary, results of another study found no association
between anti-Sm antibodies and neuropsychiatric features [38].
Anti-DNA Antibodies Cross-Reactive with NMDA Receptor
Many clinical manifestations of SLE are mediated by anti-DNA antibodies that in

particular correlate with disease activity and cause kidney damage [39]. The anti-DNA
antibodies may react directly with DNA or cross-react with non-DNA tissue antigens. In a
study to determine the distinct antigens that anti-DNA antibodies recognize, a phage display
library was screened with a monoclonal antibody specific to double stranded DNA and a
pentapeptide consensus sequence (DWEYS) was revealed [40]. This consensus sequence is
contained in N-methyl-D-aspartate (NMDA) receptors NR2A and NR2B (residues 283-287).
NR2A and 2B are polypeptide chains that associate with the NR1 polypeptide to form
NMDA receptors. NR2 receptors bind the neurotransmitter glutamate and play a role in
learning and memory [41]. DeGiorgio and coworkers demonstrated that a subset of anti-DNA
antibodies cross-react with NMDA receptors and, through an excitotoxic mechanism, can
induce neuronal apoptosis both in vitro than inoculated directly in mouse brain [42]. These
anti-DNA antibodies are present in CSF of patients with SLE who experienced cognitive
decline, but it is not yet known whether they are produced in situ in the brain or cross the
BBB.
BALB/c mice are a strain of mice which not present spontaneous brain pathology,
permitting the analysis of antibody-mediated brain pathology and cognitive dysfunction. To
study whether the anti-dsDNA/NR2 antibodies could cause brain damage when present in
systemic circulation, BALB/c mice were immunized with the peptide to develop cross-
reactive anti-DNA and anti-NR2 antibodies [43]. The administration of LPS to mice breaking
the integrity of BBB, allowed to antibodies to pass from circulation to the brain. In fact,
neuropathology requires a breach in the integrity of BBB and bacterial infection leads to a
loss of the BBB function. The anti-NR2 antibodies showed a preferential binding in the
hippocampus and consequent loss of hippocampal neurons. The loss of neurons in
hippocampus leaded to an impairment in memory function. Mice receiving LPS and
intravenously human antibodies specific to DNA and NMDA receptors extracted from sera of
patients with SLE, elicited cognitive impairment [44]. To determine whether anti-NR2
antibodies exist in the brain of patients with SLE, IgG was eluted from patients’ brain. The
eluted IgG showed binding to DNA and NR and induced neuronal apoptosis when injected
into mouse brain.
The breakdown of BBB may occur not only as infection, but also as cerebral vasculitis,
stress, nicotine exposure, etc. The rise of epinephrine induced by stress is known to increase
cerebral blood flow and to break the integrity of BBB, preferentially in the amygdale. In the
mouse model immunized with the pentapeptide, the administration of epinephrine leaded to
apoptosis of neurons of the lateral amygdale, resulting in behavioral disorders [45]. So the
same antibody specificity can cause either a cognitive or an emotional disorder depending on
the agent used to break the BBB. The presence of anti-DNA, anti-NR2 antibodies are found
in 25-50% of patients with SLE [46]. Several studies yielded conflicting data about the
correlation between the neuropsychological function and serum anti-NR2 antibody levels, but
brain dysfunctions seem clearly correlate to the presence of antibody to the pentapeptide in
CSF and symptom severity correlate with antibody titer [47]. An emerging quantitative
magnetic resonance imaging technique offers the promise of a quantitative physiological
measure of cellular integrity. A recent study using this technique showed that a small group
of patients with SLE and anti-NR2 antibodies presented alteration in amygdale compared
with patients without anti-NR2 antibodies [48].
Non-competitive inhibitors of NMDA receptors, such as MK801 and memantine, are
promising therapeutic tools to protect mice from neuronal injury caused by the direct
injection of anti-NR2 antibodies into the mouse brain. The use of the D-isoform of the
consensus peptide could also lead to neuronal sparing binding to anti-NR2 antibodies and so
preventing their binding to NR2 [45].
Anti-Ribosomal P Antibodies
Anti-ribosomal P antibodies recognize 3 specific ribosomal proteins P0, P1 and P2 [49].
One of the major points of interest of anti-ribosomal P antibodies derives from their high
specificity for NPSLE [50,51]. Elevated titers of anti-ribosomal P antibodies are mainly
detected in SLE patients immediately before and during active disease [52] and may be
associated with particular clinical manifestations, including lupus nephritis, hepatitis [53-55]
and neuropsychiatric involvement [5, 56-60].
In particular, the clinical association between elevated titers of anti-ribosomal P
antibodies and psychosis was originally described by Bonfa and coworkers in patients with
psychosis secondary to SLE [61]. Several studies showed a strong association between
elevated titers of serum anti-P antibodies and NPSLE, predominantly psychosis and severe
depression [54, 61-73]. One study of West and coworkers, examining a cohort of SLE
patients with and without psychiatric manifestations over a period of 10 years, demonstrate
the relationship between psychosis and depression and anti-ribosomal P antibodies [68]. On
the contrary, some reports failed to find any relationship between anti-ribosomal P antibodies
and NPSLE [26, 54, 74-78]. In particular, in a recent study the association between the
presence of anti-ribosomal P antibodies and either lupus psychosis or depression, among a
large cohort of patients with SLE, was not observed [79]. There are conflicting reports on the
importance of anti-ribosomal P antibodies in the CSF. In a recent study, elevated titers of
anti-ribosomal P antibodies were measured in the CSF and in the serum samples of a large
cohort of patients with SLE. Patients were divided into 4 groups: patients with neurologic
syndromes of the CNS; patients with diffuse psychiatric/neuropsychological syndromes;
patients with complex presentations (neurologic syndromes of the CNS and diffuse
psychiatric/neuropsychological syndromes); patients without NPSLE based on ACR
diagnostic criteria [2]. In patients with NPSLE the frequency of CSF anti-ribosomal P
antibodies was significantly higher than in patients without NPSLE. These results suggest
that the presence of IgG anti-ribosomal P antibodies in CSF of SLE patients may be involved
in the appearance of NPSLE, expecially in complex presentations [62].
Anti-ribosomal P antibodies bind and penetrate cells in culture. The cellular receptor
appears to be a membrane form of the P0 38 kDa phosphoprotein, which mediates the
binding and penetration of anti-ribosomal P antibodies. Following penetration, anti-ribosomal
P antibodies were found to localize in the cytoplasm and in the nucleus [80]. Anti-ribosomal
P antibodies are potent inhibitors of protein synthesis and via this pathway mediate cellular
dysfunction. The ability of anti-ribosomal P antibodies to bind and penetrate into living cells
was shown to induce the production of pro-inflammatory cytokines and to increase apoptosis
of penetrated cells. This raises the possibility that these antibodies are of importance in the
pathogenesis of NPSLE [52].
Anti-Endothelial Cells Antibodies

Anti-endothelial cells antibodies (AECA) are a heterogeneous group of autoantibodies
directed against antigens in the membrane of endothelial cells. Originally described in 1971,
a number of targets have now been identified for these autoantibodies [81]. They have been
detected in several autoimmune diseases and have been associated with nephritis and
vasculitis in SLE patients [82]. Recent data suggest their implication in endothelial
dysfunction and a pathogenic role of AECA in SLE [82, 83]. AECA are capable of causing
up-regulation of pro-inflammatory markers and apoptosis in endothelial cells [84]. Song and
coworkers showed a clinical association of AECA with disease activity and neuropsychiatric
manifestations in SLE [85]. Meroni and coworkers found AECA positivity in a high
percentage of SLE patients with involvement of the CNS [86]. In a recent study Conti and
coworkers, investigating the possible correlation of psychiatric manifestations in SLE and the
reactivity of autoantibodies to different autoantigens (endothelial cells, cardiolipin, beta2
glycoproteinI (beta2-GPI), SSA/Ro, La, glial fibrillary acidic protein, ribosomal P protein,
dsDNA and nucleosomes), demonstrated a significant association between AECA and
psychiatric involvement in SLE patients [26, 87]. In this study, patients were categorized as
either with or without psychiatric disorders on the basis of the clinical psychiatric
examination. Patients were considered with psychiatric manifestations only when presented a
severe psychopathology such as psychosis and mood disorders (recurrent major depressive
disorders, dysthymic disorder, and depressive disorder not otherwise specified). Anxiety and
mild depression were not included since these disorders are frequently detected in SLE
patients and are predominantly psychoreactive [3, 88].
Identifying endothelial autoantigens involved in the autoimmune processes during
neuropsychiatric SLE could help to explain the pathogenetic mechanisms involved in the
initiation and progression of psychiatric symptoms.
Anti-Nedd5 Antibodies
With the aim of seeking and characterizing molecules that behave as autoantigens in
NPSLE, Margutti and coworkers provided evidence that the C-terminal region of Nedd5
(Nedd5 C-ter) is a novel autoantigen with a role in neuropsychiatric manifestations [89]. In
fact, the percentage of patients with anti-Nedd5 C-ter serum IgG was higher in group of
patients with neuropsychiatric manifestations than in patients without these disorders. Nedd5
is a mammalian septin known to associate with actin-based structures, such as the contractile
ring and stress fibers [90, 91]. The septins are a family of cytoskeletal GTPases that play an
essential role in cytokinesis, in yeast and mammalian cells [92]. Interestingly, Nedd5 is
predominantly expressed in the nervous system and may contribute to the formation of
neurofibrillary tangles as integral constituent of paired helical filaments in Alzheimer’s
disease [93, 94]. Margutti and coworkers demonstrated that Nedd5 presented an intracellular
redistribution on the cell surface during apoptosis, which may be in part responsible for its
immunogenicity. Indeed, apoptosis may play an important role in by-passing tolerance to
intracellular autoantigens. The specific modification of autoantigens and their redistribution
into blebs at the surface of apoptotic cells may contribute to the induction of autoimmune
responses [95, 96]. Moreover, apoptotic defects and impaired removal of apoptotic cells
could contribute to an overload of autoantigens in the circulation or in target tissues that
could become available to initiate an autoimmune response [97]. In susceptible individuals,
this can lead to autoantibody-mediated tissue damage. Interestingly, the C-terminal region of
Nedd-5 displays a coiled-coil domain. Several autoimmune autoantigens are characterized by
the presence of such domain [98]. Coiled-coil proteins may be exposed to the immune system
as surface structures in aberrant disease states associated with unregulated cell death and
could become autoimmune targets [98]. The unanswered question is whether anti-Nedd5 C-
ter antibodies can cause direct damage, thus contributing to the pathogenesis of psychiatric
manifestations, or whether they are an epiphenomenon of these disorders.
Anti-Phospholipid Antibodies
Anti-phospholipid (aPL) autoantibodies are directed against anionic phospholipids or
protein-phospholipid complexes. The negatively charged phospholipid targets include
phosphatidyl inositol, phosphatidyl glycerol, phosphatidyl serine, phosphatidyl acid, and
cardiolipin. Neutrally charged autoantigen targets include phosphatidyl ethanolamine,
phosphatidyl choline, platelet activating factor and sphyngomyelin. By far, aPL antibodies, in
particular anti-cardiolipin (aCL) antibodies and lupus anti-coagulant (LAC), have been the
most widely investigated antibodies in NPSLE. The association of aPL antibodies in NPSLE
has been reviewed [99]. This class of antibodies are reported to associate with focal
neurological disease such as stroke, transient ischaemic attacks and transverse myelitis [99].
In a study on a large cohort of SLE patients, elevated titers of aPL antibodies were an
independent risk factor for the development of cerebrovascular disease, seizures, and
headache [100].
Anti-Cardiolipin Antibodies
A strong correlation between aCL antibodies and the overall frequency of
neuropsychiatric manifestations was reported in many studies [101-103], but refuted in others
[104,105]. In pediatric SLE, elevated titers of aCL antibodies are also frequently
encountered, and aCL IgG are often associated with CNS involvement [103]. The frequency
of aCL antibodies (IgG isotype) was significant for patients with cognitive dysfunction,
chorea and cranial neuropathy [32]. In an open pilot study of children with NPSLE
manifested as encephalopathy with or without grand mal seizures, focal seizures with
depression or hallucinations, optic neuritis with transverse myelitis and psychosis with
audiovisual hallucinations, a high percentage of these patients had elevated aCL IgG [106].
Paired measurements of aCL antibodies, in the serum and CSF, were performed using the
ELISA method in SLE patients and in controls with other diseases. High titers of CSF aCL
IgG were detected in NPSLE patients with lupus headache, acute psychosis, cognitive
impairment, high cortical dysfunction and altered consciousness. Intrathecal synthesis
occurred in these NPSLE patients, rather than the diffusion of aCL IgG from serum to CNS
compartments [107]. In another study, aCL antibodies were studied in the serum and CSF of
patients with SLE admitted for the assessment of NP disease. Patients with active
neuropsychiatric complaints had positive aCL antibodies in the CSF, some of these patients
presented simultaneous presence of antibodies in their sera and in their CSF. The assessment
of the Q-albumin index revealed abnormal values in a subset of patients with active
neuropsychiatric changes who exhibited positive CSF aCL antibodies, suggesting that
impairment of the BBB function may lead to a leakage of intrathecal aPL antibodies from the
systemic circulation. Additionally, a few patients revealed normal Q-albumin values with a
high IgG-CSF index, suggesting increased intrathecal synthesis of antibodies. The study of
aCL antibodies in CSF was useful in detecting active NPSLE [108]. In another small study,
aCL antibodies were not detected in any CSF samples [109].
Experimental models show direct evidence for the pathogenicity of aCL antibodies and
cognitive dysfunction. In one study, BALB/c mice were immunized with a pathogenic
monoclonal aCL antibody and developed clinical and neurological manifestations [110]. In
another mouse model, polyclonal aCL antibodies purified from pooled serum samples of
patients with SLE had inhibitory effects on cultured normal rat brain astrocytes (RBA-1
cells). These results suggest that aCL Abs have an inhibitory effect on brain cells and elicit
thrombus formation in brain vessels, both of which play a role in NPSLE [111].
Lupus Anti-Coagulant
One study evaluated the relationship between LAC and cognitive dysfunction in SLE
patients. LAC-positive patients were 2 to 3 times more likely than LAC-negative patients to
be designated as cognitively impaired. This study suggests that LAC positivity is associated
with subclinical nervous system compromise and a pattern of deficits compatible with
subcortical involvement, perhaps related to microthrombotic events or vasculopathy [112]. In
a large study, utilizing MEDLINE from 1966 to 1989 to evaluate the validity of LAC in SLE
patients, LAC was significantly associated with neuropsychiatric manifestations[113]. The
findings of an association between LAC with seizures, cerebrovascular accidents and
cognitive dysfunction were also corroborated in another study [32]. The association between
neuropsychiatric manifestations and LAC was reported in many other studies [100, 101], but
refuted in others [105].
Chapman and coworkers in a study on the LAC mechanism, showed that these antibodies
may be involved in the pathogenesis of NPSLE by nonischemic mechanisms, including the
inhibition of astrocyte proliferation and the nonspecific permeabilization and depolarization
of synaptoneurosomes [114].
Anti-Phosphatidyl Ethanolamine Antibodies

In one study, Kamorchkine and coworkers investigating the presence of anti-
phosphatidyl ethanolamine (aPE) antibodies in a population of SLE patients, showed that
neurological involvement was present in most of the patients with aPE antibodies [115].
Other Autoantibodies
The antigenic targets of other autoantibodies demonstrated associated to NPSLE are the
microfilament protein L-fimbrin, the nuclear protein DA1, the galactocerebrosides and the
serine proteinase 3 [24, 116-118]. Further studies are necessary to assign to these
autoantibodies a role as immunological markers of NPSLE.
CONCLUSION
Several published data reported the association between neuropsychiatric manifestations

in SLE and the presence of autoantibodies, although in some cases contrasting data are
reported. The high variability among different studies is probably related to differences in the
populations of patients studied and the laboratory tests used to detect serum antibodies. Some
autoantibodies had specificity to brain constituents such as neurotransmitter receptors, raising
the possibility that autoimmune mechanisms could interrupt or modulate the
neurotransmission or could signal neuronal death through an excitotoxic mechanism (Figure
1). Other autoantibodies could react against self molecules that cross-react with brain
components, with the above described pathological effect. Other autoantibodies reacted
against endothelium. These antibodies could react with BBB, breaking down the barrier
integrity by induction of apoptosis and expression of adhesion molecules (Figure 2). In
conclusion, further studies are necessary to better characterize the specific antigens or
epitopes recognized by autoantibodies associated with NPSLE and to evaluate their potential
use in the diagnosis of NPSLE. The characterization of the target molecules might help
defining the precise role that specific autoantibodies may play in the autoimmune
mechanisms, underlying psychiatric manifestations, and might let us investigate new and
more effective therapeutical strategies.
Figure 1. Access of antibodies to the central nervous system may occur through a disrupted blood-brain
barrier or through de novo synthesis in nervous system. Autoantibodies, binding molecules exposed on
the surfaces of neurons, lead to a neuro-toxic effect.
Figure 2. Effects of anti-endothelial cell antibodies. Anti-endothelial cell antibodies may activate the
endothelium inducing the synthesis of pro-inflammatory cytokines and chemokines and expression of
adhesion molecules, lead to the permeability of the blood-brain barrier.
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Chapter 6
HIGH-DOSE IMMUNOSUPPRESSION WITH

AUTOLOGOUS STEM CELL TRANSPLANTATION
IN SEVERE REFRACTORY SYSTEMIC LUPUS
ERYTHEMATOSUS
Igor A. Lisukov∗, Vera V. Sergeevicheva, Svetlana A. Sizikova,

Alexander D. Kulagin*, Irina V. Kruchkova, Andrey V. Gilevich,
Alexey E. Sizikov, Ludmila P. Konenkova, Elena R. Chernykh,
Olga Y. Leplina and Vladimir A. Kozlov
Institute of Clinical Immunology RAMS SB, Novosibirsk, Russia
*
Novosibirsk State Medical University
ABSTRACT
Carry out a prolonged studies to elucidate the role of high dose immunosupression
therapy (HDIST) with autologous hematopoietic stem cells transplantation (SCT) in the
treatment of severe and refractory autoimmune disease. In this study we analyzed single
center experience of HDIST followed by SCT for refractory SLE.
The 13 patients with SLE, who had disease progression despite previously treatment
with pulse Cy intravenous, prednisolone (standard doses and pulse therapy), oral Cy,
azathioprine or metotrexate, were enrolled in the clinic of our Institution from 1998 to
2007. All patients were seriously ill, with SLE disease activity indices (SLEDAI) of 6-
30, including cases with central nervous system lupus, lung vasculitis, carditis, nephritis
with nephrotic syndrome and cytopenia. Autologous haemopoietic stem cells were
collected from bone marrow (n=4) or mobilized from peripheral blood with Cy
(3g/m2/once) and granulocyte colony-stimulating factor (G-CSF) (n=9). Pretransplant
conditioning regimens included BEAM ± ATG (n=2), Melphalan 140 mg/m2 + Etoposid
∗
Address correspondence to: Prof. Igor A. Lisukov, Institute of Clinical Immunology RAMS, Yadrintsevskaya str.
14, Novosibirsk 630099, Russia. Telefon 3832-28-59-56, fax 3832-25-05-22; E-mail: [email protected]
178 Igor A. Lisukov, Vera V. Sergeevicheva, Svetlana A. Sizikova et al.
1600 mg/m2 (n=2), Cy 200 mg/kg ± ATG (n=2) and Melphalan 120 mg/m2 (n=1), Cy 140
mg/kg (n=6). Transplanted CD34+ MNC were more then 2x106/kg. Median time to an
absolute neutrophil count (ANC) greater than 0,5x109 /l and platelet count greater than
50x109/l was 11 and 15 days, respectively. Three patients died on days 11, 22 and 63 due
to transplant-related complications. All of the patients who died due to transplant-related
complications had long history of the corticosteroids treatment, multiple and severe
episodes of infections pre-SCT and received more then one anticytoststic drugs or ATG
as conditioning regimen. All of the alive transplanted patients, who recovered
hematopoiesis, showed improvement in disease activity: 8 (CR) and 2 (PR). The median
follow up is now 14 month. One patient died due to lung vasculitis progression after 8
years after SCT. We conclude that HDIST followed by SCT for refractory SLE is
feasible, effective and achievement of prolonged, corticosteroid-free remissions is a
reality.
Keywords: stem cell transplantation, systemic lupus erythematosus.
INTRODUCTION
Some of the patients with SLE damaged severely or fatally from the uncontrolled disease
progression, and conventional treatment are not effective. High-dose immunosuppression and
SCT has been proposed as an investigational therapy for patients with severe refractory
autoimmune diseases including SLE [1-5]. This approach is supported by hematopoietic SCT
in animal autoimmune disorders [7-10] and in patients undergoing transplantation for
hematologic disease who also had a coincidental autoimmune disease [11-12]. The rationale
for using SCT to treat autoimmune disease is to maximally suppress or ablate the immune
system and then rescue the patient from prolonged cytopenia by infusing autoulogous stem
cells. Eradication of autoreactive cells by the conditioning regimen, redistribution of an
altered cellular and humoral immunological network or thymic re-education are discussed as
potential mechanisms for response (“reprogramming of the immune system”) [13-17]. The
EBMT/EULAR Registry has collected retrospective data on autoSCT from over 460 patient
with autoimmune diseases [18]. Transplantation-related mortality (TRM) for all autoimmune
disease has been reported to be 8,6% [17-19]. TRM was 12% for 51 patients undergoing
transplantation for SLE [18]. Remission of disease activity was seen in 33/50 (66%)
evaluable patients by six months, of which 10/31 (32%) subsequently relapsed after six
months [18].
We describe 13 patients with primary severe SLE who received HDIST and auto-SCT in
our Institution from 1998 to 2007 (Table 1). Six patients were registered in the
EBMT/EULAR database.
Table 1. Results of autologous SCT in patients with SLE
Sex/Age Duration of Previous treatment Organ Regimen Number of infused Results Follow-up
SLE, years involvement cells
Patient 1 F/21 3 oral corticosteroids, Lung, Renal BEAM+ATG 2,4x108/kg (BM) Complete 5 years
azathioprine, remission
Cy pulse (1)
Patient 2 F/29 14 oral corticosteroids, Renal, BEAM 1,2x108/kg (BM) Died on day
azathioprine, Cy pulse (7), oral Serositis 11 (sepsis)
Cy (7g)
Patient 3 F/21 7 oral corticosteroids, CNS, Renal Melphalan 120 1,3x108/kg (BM) Partial 3,3 years
azathioprine, mg/m2 + response
Cy pulse (6) etoposide 1600
mg/m2
Patient 4 F/15 5 oral corticosteroids, Renal Melphalan 140 1,3x108/kg (BM) Died on day
azathioprine, Cy pulse (3), oral mg/m2 + 22 (sepsis)
Cy (20,8g) etoposide 1600
mg/m2
Patient 5 F/19 9 oral corticosteroids, Cy pulse (9) Renal, Cy 200 mg/kg 20,6x106/kg Died on day
Cytopenia + ATG (PBSC) 63 (CMV)
Patient 6 F/25 4 oral corticosteroids, Renal, CNS, Cy 200 mg/kg 18x106/kg (PBSC) Complete 3 years
azathioprine, Cytopenia remission
Cy pulse (7)
Patient 7 F/42 7 oral corticosteroids, Lung, Heart Cy 120 mg/kg 7x106/kg (СD34+ Complete 2.5 years
azathioprine, (carditis) PBSC) remission
Cy pulse (9) Renal
Table 1. (Continued)
Sex/Age Duration of Previous treatment Organ Regimen Number of infused Results Follow-up
SLE, years involvement cells
Patient 8 F/34 8 oral corticosteroids, Renal, CNS Melphalan 120 3.8x106/kg Partial 1 year
azathioprine, mg/m2 (СD34+ remission
Cy+methylprednisolone pulse PBSC)
(6).
Patient 9 F/24 1 oral corticosteroids, Renal Cy 120 mg/kg 4,3x108/kg Complete 1 year
azathioprine, (СD34+ remission
Cy+methylprednisolone pulse PBSC)
(6)
Patient 10 F/15 8 oral corticosteroids, Cy pulse Renal, Lung, Cy 120 mg/kg 4.3x106/kg Complete 6 months
(4), oral Cy (24g) Heart (СD34+ remission
(coronariitis) PBSC)
Patient 11 F/25 2.5 oral corticosteroids, mtx, Cy+ Renal, Lung, Cy 130mg/kg 5,9x106/kg Complete 4 months
methylprednisolone pulse pulse Heart (carditis) (СD34+ remission
(4), cyclosporin A. PBSC)
Patient 12 F/36 3 oral corticosteroids, Cy pulse Renal Cy 120 mg/kg 2,6x106/kg Complete 3 months
(4), cyclosporin A cytopenia (СD34+ remission
PBSC)
Patient 13 F/34 4.5 oral corticosteroids, Cy pulse Renal, CNS Cy 120 mg/kg 3,6x106/kg Complete 3 months
(9) (PBSC) remission
High-Dose Immunosuppression with Autologous Stem Cell Transplantation… 181
Patient Selection
From April 1998 through February 2007 thirteen females aged between 15 and 41 years
and suffered from SLE were enrolled in an high dose immunosupression (HDIST) with
autologous hematopoietic stem cells transplantation (SCT) study at a single medical center.
Patient eligibility depended on a refractory to standard immunosupression therapies and
either organ- or life-threatening visceral involvment. The inclusion criteria were: not
controlled with pulse therapy Cy glomerulonephritis (World Health Organization (WHO)
class III-IV), central nervous system (CNS) lupus, vasculitis involving the lung or heart, or
transfusion-dependent autoimmune cytopenias. Evaluation of eligible patients by two
independent rheumatologists and transplant physicians, informed consent and approval by the
ethical committee were part of the protocol [13, 16].
Hematopoietic Stem Cell Procurement
Autologous hematopoietic stem cells were collected from bone marrow only for first four
patients (n=4) under general anaestesia and all subsequent patients underwent peripheral
blood hematopoietic stem cells collection (n=9). Three patients, whom bone marrow harvests
was performed, has been achieved suboptimal MNC count (<2x108). PBSC mobilized from
peripheral blood with Cy 4g/m2 and granulocyte colony-stimulating factor (G-CSF)
(filgrastim) at 10 μg/kg subcutaneous daily with leukapheresis initiated when the white blood
cells count reached 1x109/l (n=9). Apheresis was continued daily until the number of
harvested progenitor cells reached a minimum of 2x106 CD34+ cells/kg body weight. All of
patients have successful collection after 2-3 leukapheresis session. The harvests were not
manipulated. Stem cell products cryopreserved at the –80C for at least 2 week after
collection.
Conditioning Regimen
The conditioning regimen was not started until at least 14 days after PBSC apheresis.
Pretransplant conditioning regimens included BEAM (BCNU 300 mg/m2 on day -7, Etoposid
800 mg/m2 and Ara-C 800 mg/m2 on days -6, -5, -4, -3, Melphalan 120 mg/m2 on day -2)
with antithimocyte globulin (ATG) in dose 30mg/kg infused over 10 hours on day 0 (n=1),
BEAM (n=1), Etoposid 1600 mg/m2 on day -2 with Melphalan 140 mg/m2 on day -1 (n=2),
Cy 200 mg/kg in divided doses of 50 mg/kg/day intravenously on days -6, -5, -4, -3 with
ATG (total doses 60 mg/kg) infused over 10 hours in divided doses of 30 mg/kg/day on day
+1, +2 (n=2), Melphalan 120 mg/m2 (n=1), and Cy 200-140 mg/kg intravenously in divided
doses for 4 days -6, -5, -4, -3 (n=6).
Supportive Care
Before, during and for 24 hours after treatment with high dose cyclophosphamide patient
received hyperhydratation with forced diuresis and urometexan (Mesna) infusion for the
prevention of haemorrhagic cystitis. Patients were treated in a single room without air
filtration. All patients followed a standardized supportive care protocol including antiemetic
therapy, analgesia for mucositis, transfusion support, and venoocclusive disease prophylaxis.
A low microbial diet, oral daily fluoroquinolone (1 g/d) changed to intravenouse cefepime on
neutropenic fever, fluconazole (400 mg/d) and acyclovir (10 mg/kg/d) and aerosolized
amphotericin B (10 mg daily twice) were started upon admission and discontinued when the
ANC rebounded to 0,5x109 /l. All patients continued to receive prednisolone 15-30 mg/d.
Methylprednisolone (1g) was administered intravenously 30 minutes before each dose of
ATG. All of patient underwent BMT, received subcutaneous G-CSF (filgrastim) 5 μg/kg was
started the day of hematopoietic stem cell infusion and continued untill the ANC was greater
than 1x109 /l for 3 consecutive days. Irradiated with 25 Gy platelets and red blood cells were
given to maintain platelet count greater than 20 000/μL and hemoglobin level greater than 8.0
g/dL. For the first 6 months after transplantation, patients were treated with daily oral
fluconazole and trimethoprim/sulfamethoxazole orally three times a week.
Table 2. Characteristics of the patients before SCT
Anti-dsDNA, IU/mlc C3 / C4, g/ld ESR, mm/hre SLEDAI score

P1 580 0,42 / 0,1 58 18
P2 160 0,7 / 0,1 25 14
P3 740 0,74 /<0,1 65 30
P4 860 0,4 / 0,16 46 22
P5 107 0,66 /0,11 40 6
P6 1084 0,48 /<0,1 40 15
P7 13 0,3 / 0,1 27 2
P8 34 0,7 / 0,12 50 26
P9 260 0,7 /<0,1 29 12
P10 170 0,4 / 0,2 25 5
P11 50 0,55 /0,1 23 21
P12 18 0,9 /0,2 6 18
P13 98 0,72 /<0,1 25 14
Anti-dsDNA, antibodies to double stranded DNA were determined according to the manufacturer`s
instructions (IMMCO Diagnostics Inc., USA) by enzyme linked immunosorbent assay (ELISA).
Results were expressed in IU/ml, using Wo/80 as ultimate standard (negative result <50 IU/ml,
positive result >60 IU/ml);
C3/C4, levels of the 3rd and 4th complement components were determined in immunoassay (BECKMAN
ArrayR 360 system). The normal ranges were 0,8-2,0 g/l for C3 and 0,16-0,47 g/l for C4;
ESR, erythrocyte sedimentation rate, mm/hr
Assessment of Disease Status [18-22]
Patients returned for scheduled follow-up at 1, 3, 6 and 12 months and then yearly
thereafter. If a patient was not able to return for follow-up, medical records and laboratory
blood testing results were collected from local hospital.
Outcome was based on assessments before SCT (Table 2) and at 1, 3, and 6 months and
yearly after transplantation. Early clinical response is evaluated using local interphysician
vignettes, according the American College of Rheumatology Response criteria for SLE
clinical trail: responder index for lupus erythematosis (RIFLE) criteria and improvement is
revealed after mobilization regimen, including Cy [20].
Outcome was based on physical examination, serology (antibodies to double stranded
DNA, antinuclear antibodies (ANA), anticardiolipin antibodies, C3 component level and
lupus anticoagulant), lupus disease activity index (SLEDAI) [21], and response of
pretransplant abnormalities in involved organ systems (eg, serum creatinine, 24-hour urine
protein and creatinine clearance in nephritis, chest radiograph, and necessary imaging status,
and pulmonary function tests in pneumonitis, evaluation of neurologic deficits in CNS lupus).
Antibodies to double stranded DNA (anti-dsDNA) were determined according to the
manufacturer`s instructions (IMMCO Diagnostics Inc., USA) by enzyme linked
immunosorbent assay (ELISA). Results were expressed in IU/ml, using Wo/80 as ultimate
standard (negative result <50 IU/ml, positive result >60 IU/ml). The presence of ANA was
determined by the HEp-2000 indirect fluorescent antibody test system in accordance with the
manufacturer`s instructions (IMMCO Diagnostics Inc., USA). Improvement was defined as a
50% improvement in any baseline parameter with no deterioration in any objective
parameter. Criteria for complete remission requires that SLEDAI ≤3, prednisolone dose <10
mg daily and absence other immunosuppressive therapy.
RESULTS
Patient data before SCT are summarized in Table 1. All patients had been treated
unsuccessfully with Cy (intravenous pulse and oral) and other immunosupressive drugs. The
reinfused bone marrow (n=4) contained median nucleated cell counts of 1,55x108/kg (range
1,2-2,4x108/kg). The reinfused peripheral blood stem cell harvest (n=9) contained median
4,34x106/kg (range 2,6-20x106/kg) CD34+ cells. Median time to an absolute neutrophil count
(ANC) greater than 0,5x109 /l and platelet count greater than 50x109/l was 11 and 15 days,
respectively. Three patients died on days 11, 22 and 63 due to transplant-related
complications. All of the alive transplanted patients, who recovered hematopoiesis, showed
improvement in disease activity: 8 (CR) and 2 (PR). The median follow up is now 15 month.
One patient died due to SLE relapse: lung vasculitis progression after 8 years after SCT.
Transplant-Related Mortality (Toxity)
Main causes of death in patients with transplant related mortality before engrafment were
infections (mucositis, pneumonia, sepsis) and hemorrhage (gastrointestinal bleeding) during
the severe postchemotherapy cytopenia. All of the patients who died due to transplant-related
complications had long history of the corticosteroids treatment, multiple and severe episodes
of infections pre-SCT and received more then one anticytoststic drugs or ATG as
conditioning regimen. One the patient developed secondary cytopenia due to virus-induced
engrafment failure after hematopoiesis recovery. Lethal cases we presented in detail below.
Patient 1. The 29-year-old woman had a 14-year history of SLE, including arthritis,
pericarditis, WHO class III glomerulonephritis, and elevated titers of ANA. The symptoms
responded to corticosteroids, azathioprine and Cy. In November 1994 the patient developed
nephrotic syndrome with proteinuria 3,3 g/l, acute renal failure (ARF), abdominal syndrome,
ascite, anasarca and high titers of ANA (1:320). Pulse Cy intravenous (3 g in one month) in
combination with pulse methylprednisolone intravenous was given with improvement. The
disease was controlled by continuous therapy of oral prednisolone (minimum dose 20 mg
daily) and azathioprine 150 mg/d and was complicated by frequent infections, including
bacterial pneumonia, pyelonephritis, varicella zoster virus infection. In May 1998 relapse of
the nephrotic syndrome with ARF has been diagnosed, and the remission of SLE was not
achieved despite treatment with pulse Cy and methylprednisolone. The patient was not on
dialysis. In November 1998 the patient underwent HDC (BEAM) followed by unmanipulated
auto-BMT (1,2x108/kg of nucleated cells). Treatment was managed according to a standard
supportive care protocol. The patient developed severe mucositis (grade 4), sepsis followed
by multiorgan failure, severe hemorrhage (gastric bleeding) and died on day +11.
Patient 2. The 15-year-old female patient had suffered from SLE since 1995. SLE
symptoms were arthritis, discoid rash, malar rash, photosensitivity, fever, elevated titers of
ANA (1:160) and elevated titers of anticardiolipin antibodies. The treatment with 1 mg/kg/d
prednisolone and 400 mg Cy weekly was started but did not induce complete response.
Discoid rash, arthralgias and serological symptoms were uncontrolled. Eight months later,
prednisolone was reduced to 20 mg/d because of side effects. In January 1998 lupus nephritis
has been diagnosed. The treatment with oral Cy (at May 1998 total dose was 20,8g) with
addition of penicillamine, azathioprine and increased corticosteroids was continued without
significant improvement. Therefore three pulses of 1g Cy intravenous were given at monthly
intervals. However, patients condition deteriorated, lupus nephritis progressed, disease
activity remained high (SLEDAI 22). The option of HDC was discussed with the patient’s
parents and performed after approval by the ethics committee. In April 2000 unmanipulated
non-cryopreserved autologous marrow (1,32x108/kg of nucleated cells) was reinfused after
conditioning of the recipient with a combination of etoposide 1600 mg/m2 on day -2 and
melphalan 140 mg/m2 on day -1. In the posttransplantation period the patient developed
severe mucositis and enteropathy (day +5), sepsis followed by multiorgan failure (day +10),
severe hemorrhage (gastrointestinal bleeding), pneumonia (day +12) and died on day +22.
ANC reached 0,5x109/l on day +15.
Patient 3. The 19-year-old woman had suffered from SLE (arthritis, malar rash, carditis,
pneumonitis, immunologic disorder) since 1993. The treatment with 1 mg/kg/d prednisolone
induced complete response that lasted till 1999. Relapse of SLE with lupus erythema and
elevated titers of ANA was diagnosed in January 1999. In August 2000 the patient presented
severe cutaneous vasculitis, cytopenia (platelets 45x109/l, leukocytes 1,2x109/l), fever,
nephritis despite treatment with oral prednisolone 0,5 mg/kg/d. There was no sustained
improvement with pulse intravenous Cy (7,4 g in 9 months) in combination with oral
prednisolone (60-25 mg daily) and pulse intravenous methylprednisolone (total dose 5 g).
The disease activity decreased, but cutaneous vasculitis, cytopenia and serological symptoms
were uncontrolled. The functions of major organs were adequate. She was selected for high-
dose immunosuppression and autologous SCT. Priming was with Cy 4 g/m2 followed by G-
CSF 480 μg/d subcutaneously. Significance regress of symptoms of cutaneous vasculitis and
absence of cytopenia were achieved. Prednisolone was reduced to 10 mg/d. Two months
later, conditioning with Cy 50 mg/kg daily for 4 days with ATG 30 mg/kg daily for 3 days
was given. Transplantation of 20,8x106/kg CD34+ autologous stem cells was performed on
October 2002. Engraftment occurred on day 10 (leukocytes 9,0x109/l, platelets 80x109/l). On
day 20 the patient developed fever, abnormal liver function (ALT 4,1 mmol/l (normal<0,68),
AST 2,0 mmol/l (normal<0,68), the total bilirubin level 6,08 mg/dl (normal<1,2)) and severe
pancytopenia (leukocytes 0,2x109/l, platelets 30x109/l, haemoglobin 7 g/dl), on day 30 severe
enteropathy. On the basis of clinical signs, positive results in polymerase chain reaction
assay, detection of Cytomegalovirus (CMV) inclusions in bone marrow the CMV disease was
diagnosed. We started treatment with intravenous ganciclovir 10 mg/kg daily in combination
with intravenous immunoglobulin 0,5 g/kg for five consecutive days and thereafter once
weekly maintenance was given. After two weeks, the liver function returned to normal and
the symptoms of enterocolitis regressed. Unfortunately, the patient remained pancytopenic
(despite the administration of G-CSF), developed sepsis, pneumonia and died on day 63 from
multiorgan failure.
OVERALL AND DISEASE-FREE SURVIVAL
All of the transplanted patients, who recovered stabile hematopoiesis, showed rapid
improvement in clinical disease activity. The median follow up is now 15 month. The
probability of disease-free survival or complete remission defined as requiring no SLE-
activity and immunosuppressive medication, except physiologic doses of prednisolone (<10
mg/day), however the time of entering remission was several month, often because of long-
term high-dose pre-SCT corticosteroid therapy that had to be withdrawn gradually to avoid
abstinent syndrom. Long time of achieving improvement or normalization of serology (ANA,
anti-ds DNA) occurred in patient entering clinical remission (mean 6 month). We not
exclude, there are because cyclophosphomaide-resistant specific memory B-cell prolong
persistent.
The partial remission was defined in two patient, who suffered from CNS-lupus and mild
symptoms are remain after SCT. One patients, admitted to our hospital in November 1999
was severely disabled (lower paraplegia, failure of the pelvic function due to myelitis) with
SLEDAI 30. The total observation time after transplantation is now 74 months. The functions
of bladder and bowel returned to normal, but mild lower paraplegia and serology activity are
remain, that require prolong immunosuppression therapy with steroid. Second SLE patient
presented with organic encephaloneuropathies: abnormalities in neurocognitive function,
such as memory, intellect; reflectory tetraplegia, sensitive ataxia in pretrasplantation period,
present with mild neurological symptoms now (after 1 year after SCT): headache, depression
and transitory psychosis. Patient need in symptomatic, antipsychotic treatment.
Immunosupression by sreroid and cyclosporin is continuing.
The longest continuous duration of SLE remission has been 8 years. Disease progression-
related mortality despite initial response was occurred once after long term stabile clinical
and immunological remission. Case report is as followed.
Patient. The 21-year-old woman with SLE developed lung vasculitis in 1995. She was
treated with corticosteroids 0,5 mg/kg over 10 weeks with some clinical improvement.
Prednisolone was reduced to 10 mg/d. In 1997 lupus nephritis has been diagnosed, pulse Cy
intravenous was given but withdrawn because of allergic dermatitis. The treatment with
prednisolone 1 mg/kg and azathioprine was started but did not produce any effect. In March
1998 the patient was admitted to our hospital with dyspnea, progressive pulmonary
hypertension (Doppler echo showed estimated sistolic pulmonary artery pressure (sPAP) of
49 mm Hg), arthritis, proteinuria (5,3 g/24 hour), renal failure (creatinine clearance 50
ml/min), elevated titres of ANA (1:160). In April 1998 HDC according to the BEAM
protocol followed by unmanipulated autologous bone marrow transplantation (BMT) was
applied. On day 0, ATG (“ATGAM” 30 mg/kg) was given for in vivo T cell-depletion. The
patient developed moderate mucositis and enteropathy (grade 2), neutropenic fever (FUO)
with response to the ceftriaxone 4 g/d + tobramycin 240 mg/d, genital herpes infection with
response to the famciclovir 750 mg/d. Haemorrhage was mild. The time to achieve an ANC
exceeded 0,5x109/l and platelet count exceeded 50x109/l was respectively 16 and 19 days.
After six months complete remission of SLE has been established. The SLEDAI decreased
from 18 to 0, pulmonary and renal function was recovered, ANA were negative. The oral
prednisolone was withdrawn after 10 months of BMT. The follow up is now over five years.
The patient was in stable clinical and immunologic remission and returned to work. At the
November 2006 patient admitted in the hospital with symptoms of respiratory insufiency.
Physical and laboratory examination was revealed progression of lung vasculitis (Doppler
echo showed high sPAP, CT scanning revealed bullous pneumanitis) and 8 weeks of
gestation. After therapeutic abortion, treatment with cyclosporine 150 mg and prednisolon 30
mg daily once was started with transitory clinical improvement, but patient died due to
repeating spontaneously pneumothorax after 8 years after SCT.
The aim of high-doses immunosupression is to destroy self-reactive lymphocytes. We
evaluate the efficacy and toxicity of ASCT with specific attention to long-term immune
reconstitution. Immunological features were assessed before ASCT and at 1, 3, and 6 months
and yearly after transplantation. HDIT induced profound lymphocytopenia. Mean absolute
lymphocyte counts were back to baseline level at the 6 month. The CD4/CD8 ratio
significantly decreased at 1 month after ASCT due to reduced proportions of CD4 T-cells and
remained stable in 6 months. There were no significant changes from pretherapy in the
proportions of CD4+DR+ and CD8+DR+ lymphocytes. At 1 month posttransplant the
number of naïve CD4+45RA+ cell have been reduced significantly as well as CD8+45RA+
cells, but these subpopulations recovered at 6 month after ASCT completely. CD8+45RO+
cells decreased steadily during the posttransplant follow-up. Before conditioning in vitro T-
cell mitogenic responses were decreased in most of all patients with a trend toward a baseline
level at 3 month after ASCT.
CONCLUSION
Patients with refractory and active lupus involving multiple organ systems despite a
relatively young age traditionally have a high disease related mortality rate. Continuing
failing therapy for such patients is problematic but necessary to confirm that the increased
risk HDIST with auto-SCT would be offset by better disease control and induced long-term
remission or partial response with clinical improvement in all refractory alive SLE patients
underwent SCT in our center.
Three patients died due to serious infections in the early posttransplantation period.
Among them, the nucleated cell counts in the reinfused graft product in the two fatal cases
(case1 and case 2) were less than 2x108/kg recipient body weight, and probably it was not
enough for stable hematopoiesis recovery. Although there are no universal agreement,
recommended optimal СD 34+ cell dose (2-5x108 /kg) for successful engraftment after
autologous myeloablative SCT for patient with malignancies has been accepted by most
clinicians [23]. Non-myeloablative conditioning regimen used in most our patient carries
virtually no risk of non-engraftment after SCT. However, our results (data no shown)
demonstrated correlation between infused CD34+ cell dose and faster WBC and platelet
engraftment supporting the role of autologous SC in shortening the period of neutropenia and
critical trombocytopenia. Thus, for all patients in whom HSC collection was unsuccessful in
achieving an adequate number of CD 34+ cells, repeat bone marrow harvest or mobilization
should be performed [23, 24]. In our study, G-CSF in combination with CY did not cause a
disease flare [25]. Most of patients showed an improvement in disease activity after high dose
Cy and G-CSF for PBSC mobilization before pretransplant conditioning [5]. Only one
patient, receiving Cy-mobilization, experienced severe complication and developed
pneumonia and skin infection during pretransplantation period.
Culture negative neutropenic fever, although common during early posttransplantation
regimen, was transient and easily controlled with empiric antibiotic therapy.
All of the patients who died due to transplant-related complications received more then
one anticytostatic drugs in myeloablative doses or ATG as conditioning regimen. The
conditioning regimen for autoimmune disease used to eliminate self-reactivity lymphocytes
within the patient has been designed depending investigator to either specically target
lymphocytes (lymphoablative i.e. non-myeloablative regimen) or to destroy the entire
hematopoietic bone marrow compartment (myeloablative regimen). In cancers, autologous
HSCT regimens are designed to be myeloablative as the rationale is to destroy leukemic
cancer-causing stem cells. [1]. The goal of autologous HSCT for autoimmune diseases is to
generate new self-tolerant lymphocytes after elimination of self- or auto-reactively
lymphocytes (i.e. lymphoablation), rather than ablate and reconstitute the entire
hematopoietic compartment myeloablation). Non-myeloablative regimens are often

considered safer owing to lower organ toxicity, the more rapid engraftment following non-
myeloablative HSCT demonstrated herein also supports of non-myeloablative regimens
compared to myeloablative regimens. Therefore, in SLE, there are no reports comparing
engraftment and responses after non-myeloablative compared to myeloablative regimens for
autologous HSCT [1, 4].
Consistent with previously reports, we think that intensification of the chemotherapy is
not a justified risk for SLE patients and we stoped to use myeloablative conditioning
regimens specific for malignant disease. Now we used a dose-escalated Cy for conditionig, a
standart lupus medication [4, 5]
One of the most important complications was cardiotoxicity, possibly related to direct
CY toxicity and hyperhydration. The patient selection is important to reduce TRM and
complete cardiological assessment before transplantation is highly recommended [26].
Careful patient selection, especially cardiac function evaluation, which is often
underestimated in SLE-patient, may to avoid mortality [26].
A few patients developed CMV disease after AutoSCT [27]. We did not routinely
monitor or of CMV viremia. One patient, who was severely immunosupressed owing to
chronic use of high-dose steroids more then 9 years, was found to have fatal CMV-disease
after ATG-including regimen, that more characterized postallotransplant complication. It may
be cause for recommendation to use standart CMV pretrasplant prophylaxis and CMV-
monitoring for SLE patients with long-term history of immunosuressive therapy. Positive
CMV surveillance assay are the grounds for prompt treatment with ganciclovir [28]. Also,
compared with patients with cancer, patients with lupus have a long history of chronic
immunosuppression and appear unusually prone to infections during mobilization and after
transplantation, mandating aggressive preemptive antimicrobial (i.e. antifungal and antiviral)
prophylaxis [29].
Addition of ATG for SCT for SLE treatment further maximizes immunosupression,
because specific target T-cells [30]. Equine ATG caused acute toxicities that mimicked active
SLE flares and did not target memory B cells.[27,30] Because we obtained significant
clinical response and low rate of severe infection, when treating with Cy along, ATG seem
not to be necessary for the conditioning [5].
We did not found differences in T-cell recovery between patients with complete and
partial response or between patients treated with different regimens. But we noted more rapid
numerous and functional T-cell recovery in SLE patients treated with ASCT compared with
lymphoma patients (data not shown).
Other method of immunoablation, such as anti-CD20 monoclonal antibody (Rituximab),
may be an important therapeutic approach for the treatment of patients intolerant to HDIST
with auto-SCT. Because Rituximab is generally well tolerated and selectively depletes B
cells, its role in immune mediated diseases is now also being explored [31-34]. Future SLE
transplant protocol should consider substitute ATG with rituximab, or alemtuzumab, targets
both T and B-cells[35,36]. We did not incorporate ATG into the conditioning regimen from
2003 year.
Thus, the complication of autologous SCT is connected with patient selection, the choise
of conditioning regimen, and supportive care during and after transplantation.
There are two of our groupe of patients, who developed symptoms of SLE during
pregnancy (1 patient during fatal progression of the disease and 1 patient at the onset of the
disease). Patients with SLE have normal fertility and should not be discouraged from having
children. However, given the high frequency of SLE flares during pregnancy, SLE
pregnancies should be regarded as high risk and close monitoring for disease activity is
mandatory throughout the pregnancy. [37,38].
The involvement of the central nervous system (CNS) is one of the major causes of
morbidity and mortality in systemic lupus erythematosus (SLE) patient [3,39]. Among the
neurologic manifestations of SLE, the most common are the organic encephalopathies, which
basically comprise all potential variations of acute confusion, lethargy, or coma; chronic
dementias; depression, mania, or other affective disturbances; or psychosis; cranial nerve
abnormalities (most prominently, optic neuritis), stroke, peripheral neuropathy et sta. Acute
or subacute mental status changes may be secondary to diffuse cerebritis but should be
differentiated from focal cortical dysfunction resulting from thromboembolic cerebrovascular
accident (CVA) or from diffuse changes resulting from electrolyte or metabolic derangements
(accentuated by concomitant renal failure) [39].
Spinal cord involvement in SLE patient is rare but devastating. Transverse myelitis,
subacute-to-chronic demyelinating syndromes may caused severe invalidity [40,41].
Management of central nervous system (CNS) involvement still remains one of the most
challenging and using of HDIST doesn’t solve problems completely. Our two patient with
CNS involvment did not achieved Cr after SCT but dramatically improved. Recently, the use
of intrathecal methotrexate and dexamethasone has been reported in a small series of patients,
with a good outcome in patients with severe CNS manifestations.
It should be mentioned that all of our patients had experienced multiple and severe
episodes of infections pre-SCT and long-term history of corticosteroids therapy (3-14 years).
The remissions were achieved despite a previous history of refractory or relapsing disease
indicating the potential efficacy of this procedure. It was anticipated control of clinical lupus
activity was reflected by marked falls of anti-ds DNA antibodies, normalization of
complement levels and reduction or withdrawal of corticosteroids.
In concert with the consensus report from EULAR and EBMT [16], patients should be
selected for autoSCT who have severe disease and are refractory to conventional therapy. In
selecting patients, consideration should be given to the balance between disease severity and
organ damage. For patients with SLE, in contrast with malignancies, since organ compromise
is due to lupus, impaired visceral organ function is not a ultimately contraindication, but is
one of the major indication for SCT. While this makes the transplant procedure more
complicated, marked organ improvement, particularly of the lung, the kidney and the central
nervous system has occurred following Cy-contained mobilization and SCT. Thus preexisting
dysfunction of kidneys, liver, and lung is associated with increased mortality from transplant
regimens. Treatment of such patients remains a difficult clinical problem. The presence of
poor prognostic indicators would provide additional support in patient selection for future
SCT studies. Recently, prognostic indicators suggesting a poor outcome in SLE have been
proposed [43]. There are renal dysfunction, hypertension, anemia, low C3, central nervous
system involvement and thrombocytopenia. Patients with these prognostic features have a
considerably shortened life span. Thus, the 10-year survival of patients without features is
estimated to be 86% compared with 60% of patients with these features. The quality of
response to conventional therapy may be a useful criterion in selection of patients earlier in
disease. Pulse Cy (500-1000 mg/m2) is generally considered the standard of care. Eligible for
SCT patients with nonrenal visceral involvement need only fail corticosteroids and 3 months
of pulse Cy. For patients in whom the indication is nephritis, active disease must be present
despite at least 6 cycles of monthly pulse Cy [17].
Judicious selection of patients earlier in disease or in remission, but with a high risk of
relapse or further progression, will improved the results of treatment and transplantation-
related complication.
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Curr. Opin. Rheumatol. 2003;15:528-534.
[30] Young N, Speck B. Antithymocyte and antilymphocyte globulins: clinical trials and
mechanism of action. Prog. Clin. Biol. Res. 1984;148:221-226.
[31] Arzoo K, Sadeghi S, Liebman H. Treatment of refractory antibody mediated
autoimmune disorders with an anti-CD20 monoclonal antibody (rituximab). Ann.
Rheum. Dis. 2000; 61: 922-924.
[32] Looney R.J. Treating human autoimmune disease by depleting B cells. Ann. Rheum
Dis. 2002; 61: 863-866.
[33] Eisenberg R. SLE: rituximab in lupus. Arthritis. Res. Ther. 2003;5:157-159.
[34] Anolik JH, Barnard J, Cappione A, et al. Rituximab improves peripheral B cell
abnormalities in human systemic lupus erythematosus. Arthritis. Rheum.
2004;50:3580-3590.
[35] Willis F, Marsh JC, Bevan DH, et al. The effect of treatment with Campath-1H in
patients with autoimmune cytopenias. Br. J. Haematol. 2001; 114:891-898.
[36] Cohen Y, Polliack A, Nagler A. Treatment of refractoryautoimmune diseases with
ablative immunotherapy using monoclonal antibodies and/or high dose chemotherapy
with hematopoietic stem cell support. Curr. Pharm. Des. 2003;9:279-288.
[37] Georgiou P.E., Politi E.N. et al. Outcome of lupus pregnancy: a controlled study
Rheumatology 2000;39:1014-1019.
[38] Mok CC, Wong R W S Pregnancy in systemic lupus erythematosus Postgrad Med. J.
2001; Vol:77:157-165.
[39] Sanna1 G, Bertolaccini ML, Mathieu A Central nervous system lupus: a clinical
approach to therapy. Lupus (2003) 12, 935–942.
[40] Schantz V, Oestergaard LL, Junker P. Shrinking spinal cord following transverse
myelopathy in a patient with systemic lupus erythematosus and the phospholipid
antibody syndrome. J. Rheumatol. 1998; 25: 1425–1428.
[41] Yazawa S, Kawasaki S, Ohi T et al. Development of severe longitudinal atrophy of
thoracic spinal cord following lupus-related myelitis. Intern. Med. 2001; 40: 353–357.
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[43] Hahn B.H. The potential role of autologous stem cell transplantation in patients with
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Chapter 7
MR SPECTROSCOPY, DIFFUSION AND

DIFFUSION TENSOR IMAGING IN
Pia C. Sundgren1, Patricia Cagnoli2 and

William McCune2
Departments of Radiology1 and Rheumatology2,
University of Michigan Health Systems,
Ann Arbor, MI 48109, USA
ABSTRACT
Although clinical assessment is the cornerstone of SLE and NPSLE diagnosis; it can
be difficult to make and is frequently presumptive. MR findings play an important role in
supporting a clinical diagnosis and findings include volume loss, focal white matter
hyperintensity, diffusion abnormalities due to ischemic injuries, evidence of prior
hemorrhage or infarct, and meningeal enhancement. Nevertheless, several previous
studies have shown that conventional pre-and post-contrast enhanced brain MR appear
normal in approximately one third of both symptomatic and asymptomatic NPSLE
patients and even more frequently in SLE patients. It has been suggested that other
modalities such as MR spectroscopy (MRS), diffusion weighted imaging (DWI) and
diffusion tensor imaging (DTI) could be additional tools in the evaluation and monitoring
of SLE and NPSLE patients. This chapter will describe these new MRI techniques,
recent research results and trends, and discuss if these techniques may add any valuable
information that may further elucidate the pathogenesis of NPSLE. These techniques
may possibly aid in diagnosing and differentiating NPSLE from other diseases with
similar acute clinical symptoms as well as in monitoring disease progression.
194 Pia C. Sundgren, Patricia Cagnoli and William McCune
INTRODUCTION
Neuropsychiatric systemic lupus erythematosus (NPSLE) is a severe and life-threatening

condition and has been reported to be a leading cause of morbidity and mortality in systemic
lupus erythematosus (SLE) patients [1, 2]. The reported prevalence using the American
College of Rheumatology (ACR) nomenclature from 1999 is 37-95 % [3-7].
Clinically, NPSLE can present in a variety of manners, with involvement of the central
nervous system such as septic meningitis, cerebrovascular disease, demyelinating syndrome,
headache, movement disorder, Guillain-Barré syndrome, myelopathy, seizure disorders, acute
confusional state, anxiety disorder, cognitive dysfunction, mood disorder, and psychosis.
NPSLE can also present with peripheral nervous system manifestations such as autonomic
neuropathy, mononeuropathy, myasthenia gravis, cranial neuropathy, plexopathy and
polyneuropathy [8].
Although clinical assessment is the cornerstone of the neuropsychiatric systemic lupus
erythematosus (NPSLE) diagnosis, this diagnosis can be difficult to make and is frequently
presumptive. Some of these clinical features are usually diagnosed promptly and reliably.
However, the attribution of individual neuropsychiatric events to SLE remains a challenge
and NPSLE may occur in the absence of serologic markers or CSF abnormalities. Although a
wide range of neuroimaging tools have been used to evaluate CNS involvement in NPSLE no
single technique has been proven to be definitive for most of the NPSLE syndromes and
attribution is determined on the basis of exclusion using the best available clinical,
laboratory, and imaging data [9].
Findings on magnetic resonance imaging (MRI) play an important role in supporting a
clinical diagnosis of NPSLE. These findings include volume loss, white matter
hyperintensity, diffusion abnormalities due to ischemic injuries, evidence of prior
hemorrhage or infarct, and meningeal enhancement [10-12] (Figure 1). Nevertheless, studies
have shown that conventional pre- and post-contrast enhanced brain MR appear normal in
approximately one third of both symptomatic and asymptomatic NPSLE patients, leading to a
delay in diagnosis and treatment of NPSLE [10-12]. In addition, conventional MRI, although
the best available technique to study anatomical lesions in NPSLE, unfortunately has limited
specificity and cannot reliably distinguish lupus-related lesions from those produced by other
mechanisms. Previous studies have shown that MRI findings do not reliably predict clinical
course or response to therapy [12]. Still, MRI provides superior clinical correlation to CT,
with a high concordance of focal neurologic abnormalities on physical examination with
localized abnormalities in the brain particularly in the periventricular and subcortical white
matter, which can be in the territory of a major cerebral blood vessel [13-15] but also seen in
small vessel disease. However, computed tomography (CT) remains a useful tool in the
emergency setting for detecting large infarct cerebral hemorrhage, massive brain edema, and
for excluding confusing disorders including brain abscess and mass lesions, or when MRI is
unavailable, not tolerated (claustrophobia) or contraindicated.
The prevalence of MRI abnormalities in SLE patients without neuropsychiatric disease
has been described [16] with ranges between 19% [13] to 70% [17]; and varies with age,
severity of SLE or previous documented neurologic involvement [13, 16-19].
MR Spectroscopy, Diffusion and Diffusion Tensor Imaging… 195
It has been suggested that other imaging modalities such as MR spectroscopy (MRS),
single proton emission tomography (SPECT), positron emission tomography (PET), and
magnetization transfer imaging (MTI) could be additional tools in the evaluation and
monitoring of patients with NPSLE [10,11,20-34].
Figure 1 a-c. Axial fluid attenuated inversion recovery (FLAIR) (a), T2-weighted (b) and post contrast
enhanced T1-weighted images (c) of the brain in a 42-year-old female with SLE. Focal non contrast
enhancing areas of increased T2 hyperintensity in the deep and subcortical white matter (arrows) more
in the right hemisphere than left present on axial FLAIR and T2 weighted images.
Of late, it has been suggested that other modalities such as diffusion imaging (DWI)
[35,36,37] and even evaluation with more sophisticated techniques, such as diffusion tensor
imaging (DTI), may further elucidate the pathogenesis of NPSLE and possibly play a role in
diagnosing and differentiating NPSLE from other diseases having similar acute clinical
symptoms and monitoring disease progression [38].
This chapter will focus on a few of these techniques namely MRS, DWI and DTI as
possible imaging tools in the evaluation of SLE and NPSLE patients.
MAGNETIC RESONANCE IMAGING TECHNIQUES
MR Spectroscopy
MR spectroscopy (MRS) is a non-invasive MRI technique that allows the biochemical

metabolites in the brain tissue to be quantified. Most commonly used is 1H (proton) MRS due
to high natural abundance of protons and their high absolute sensitivity to magnetic
manipulation, better spatial resolution, and relative simplicity of technique. Different
biomolecules can be separated by their different chemical shift properties and can be
quantified by the signal obtained at their specific frequencies and the information displayed
as a spectrum. Metabolites in the CNS that are commonly evaluated with MRS in SLE and
NPSLE patients include N-acetylaspartate, choline, creatine/phosphocreatine, myoinositol,
the presence of lactate and lipids, and potentially glutamate/glutamine.
MRS can be performed using different techniques. Most commonly, in the clinical
setting, spectra can be acquired using single voxel spectroscopy (SVS) with a spatial
resolution in the order of one to eight cm3 or the multiple voxel techniques also referred to as
chemical shift imaging (CSI) or magnetic resonance spectroscopic imaging (MRSI), allowing
the derivation of metabolite maps. Both techniques are commonly used in evaluation of SLE
and NPSLE patients. Even if SVS allows evaluation of only small volumes of tissue, it is
time-efficient and allows the acquisition of quantitative data. The CSI allows examination of
a larger volume of tissue, which can then be evaluated using multiple smaller voxels within
the investigated volume. These volumes can be as small as 1 cm3. The two-dimensional CSI
(2D-CSI or MRSI) technique still requires longer acquisition and post-processing time
despite new improvements by the manufacturers. There are several limitations for MRS
regardless of technique, and the spatial resolution of in vivo 1H MRS is limited. MRS cannot
be performed in or adjacent to tissue with high differences in magnetic susceptibility
compared with brain tissue such as bone, air, large vessels, and hemorrhagic lesions. This is
due to the artifacts arising from these structures and the resultant difficulty in obtaining a
homogenous magnetic field, which is essential for good quality MRS study. In addition,
susceptibility artifacts from metal and shunts may obscure the spectra.
The selection of appropriate MRS techniques, including measurement parameters such as
echo time (TE), and repetition time (TR), depends on the clinical question. Long echo-time
(TE) (135-270 msec) is sufficient for detection of the major metabolites such as N-
Acetylaspartate (NAA), Choline (Cho) and creatine/phosphocreatine (Cr) and lactate/lipids
(LL). N-Acetylaspartate (NAA) serves as a measure of normal healthy neuronal tissue.
Choline (Cho) has been implicated as a marker of cellularity and cell turnover, and therefore,
may be used to infer a neoplastic process. Under most conditions, creatine/phosphocreatine
(Cr), present in glial tissue and neurons, and is involved in phosphate transport, is a relatively
stable metabolite used by most investigators as an internal control for quantifying other
metabolites (i.e. metabolite ratios often include creatine as the denominator) [39]. Short echo
times (TE=20-30 ms) evaluations are required when there is need for detection of metabolites
with short relaxation times, such as glutamine, glutamate, myo-inositol and some amino-
acids. Myoinositol is found in glial tissue and is thought to be involved as a second or third
messenger for neurotransmitters and can be evaluated with MRS using a short TE [40].
Altered myoinositol have been shown to be associated with inflammatory injury [41, 42].
2a
2b
Figure 2a-b. Normal magnetic resonance spectroscopy (MRS) spectrum using intermediate TE (144ms)
showing the main metabolic peaks: N-Acetyl aspartate (NAA), choline (Cho), and creatine (Cr) (a).
Normal magnetic resonance spectroscopy (MRS) spectrum using short TE (30ms) showing the main
metabolic peaks of N-Acetyl aspartate (NAA), choline (Cho), creatine (Cr), glutamine/glutamate (Glx),
and myoinositol (mI) peaks (b).
Lactate and macro-lipids are not present on a MRS spectrum performed in a normal
brain. These metabolites are shown to increase in anaerobic metabolism. They have been
demonstrated in cerebral necrosis and in some brain tumors but rarely in association with
SLE and considered, by many, less relevant to the work-up for NPSLE. All the above
metabolites have been reported in the SLE and NPSLE literature [10, 20-24, 29, 43-47].
Intermediate TE and short TE magnetic resonance spectroscopy with normal metabolic
spectra are presented in Figure 2.
Diffusion Weighted Imaging – DWI
Diffusion weighted imaging (DWI) is a well established sequence used commonly in the
detection of early signs of ischemia. It is well known that DWI measures the diffusivity of
water molecules [48]. In routine DWI, water mobility is assessed in only three orthogonal
directions. While this is sufficient to measure the scalar parameter, the apparent diffusion
coefficient (ADC), routine DWI is unable to characterize the directionality of diffusion
(Figure 3).
Figure 3 continued on next page.

Figure 3 a-d. Axial fluid attenuated inversion recovery (FLAIR) (a), T2-weighted (b), diffusion
weighted (DWI) (c) images, and apparent diffusion coefficient map of the brain in 62-year-old male
demonstrating increased T2 hyperintensity in left cerebellum (a, b) and increased signal on the diffusion
weighted images (trace) (c) and low signal on ADC map (d) consistent with acute stroke.
Diffusion Tensor Imaging
Diffusion tensor imaging DTI yields quantitative measures for tissue water mobility as a
function of the direction of water motion. The diffusion of water molecules is characterized
by Brownian motion. DTI of water is probed by application of diffusion-sensitization
gradients in multiple directions [49]. Appropriate mathematical combination of the
directional diffusion weighted images provides quantitative measures of water diffusion for
each voxel via the apparent diffusion coefficient (ADC), as well as the degree of diffusion
directionality, or anisotropy which can be measured by fractional anisotropy (FA) [50]. The
diffusion is called isotropic when the motion is equal in all directions such as in simple fluids.
FA has a value of 0 in isotropic tissues and approaches the value of 1 in highly anisotropic
environments where water is constrained to move along a primary direction. The diffusivity
as measured by ADC and the anisotropy as measured by FA represent just a part of the
information available from the diffusion tensor. Examination of individual eigenvalues which
reflect the diffusivity in longitudinal or transverse directions with respect to fiber tracts may
add additional information and help in tissue characterization. By choosing the eigenvector
associated with the largest eigenvalue, the principal diffusion direction of the brain structure
to be examined can be encoded with color, resulting in color-coding maps or directionally
encoded FA maps (DEC FA maps). In these color-encoded maps the fibers have been given
different colors (red, green and blue) depending on their different diffusion directions [51].
The magnitude of the anisotropy in the tissue, such as FA, can be used as an illumination
factor of the calculation of a directionally encoded color image [51] (Figure 4).
Imaging Findings in SLE and NPSLE Using MRS
Several studies utilizing MRS in the evaluation of SLE and NPSLE patients have been
published over the last couple of years. However, there are large variations in the
spectroscopy technique used i.e. single voxel spectroscopy (SVS) [10,21,22,24,52] or
multivoxel chemical shift imaging (2D-CSI) [29,43,46], in patient selection, in the time of
initial MR scanning, and in the inclusion of interval follow-up, making it difficult to compare
the published data. For example, patient selection may vary from NPSLE patients with acute
neurological symptoms [29] to reports that have focused mainly on NPSLE patients with
major or minor symptoms regardless of when the MR study was performed with respect to
initial symptoms, [21,22,46] or which have focused on SLE patients without any acute
neuropsychiatric symptoms [10,20]. These differences may account for some of the
discrepancies between the presented results.
Most reports demonstrate a decline in the NAA/Cr ratio and an increase in the Cho/Cr
ratio in periventricular white matter and basal ganglia [21, 22, 29]. Other reports demonstrate
more widespread significant decrease in the NAA/Cho ratio and a significant increase in the
Cho/Cr ratio [10, 21, 22, 4, 29]. Such a decline in NAA has been suggested to be a sign of
neuronal loss, whereas an increase in the choline compounds was assumed to be a sign of
increased metabolic turnover/activity [22].
Figutr 4 continued on next page.

Figure 4 a-c. Apparent diffusion coefficient (ADC) map (a), fraction anisotropy (FA) map (b), and
directionally encoded FA map (DEC FA maps) (c). In these color-encoded maps the fibers have been
given different colors (red, green and blue) depending on their different diffusion directions.
Studies have demonstrated a reduction in NAA in injured areas of the brain in SLE
patients as well as in normal appearing white and grey matter. Decreased NAA has been
found in patients with active disease, cerebral atrophy, generalized seizures, psychosis,
neurocognitive dysfunction or confusional states [10, 20, 21, 25, 26, 28, 43, 44, 53].
Decreased NAA/Cho ratios [47] and elevated Cho/Cr ratios [54] have been demonstrated in
patients with cognitive impairment [54]. Both stroke, epilepsy and elevated IgG
antiphospholipid antibodies (aPL) are more commonly seen in SLE patients with
antiphospholipid antibody syndrome (aPLS) than those without (aPLS) [45] and the reduction
of NAA/Cr was more closely related to the IgG-aPL than the presence of stroke or aPLS.[45].
Cho/Cr ratios have been found to be elevated in active NPSLE. Absolute choline levels
have shown to be elevated in correlation with activity, stroke, inflammation and chronic
white matter lesions but also in normal appearing white matter. [10, 20, 21, 24, 26, 46] and
may, therefore, be regarded by some investigators as a poor quality indicator of prognosis
[20, 21, 44, 47].
Few studies have followed patients over time [24, 27, 29]. Two of these studies [24,29]
demonstrated a continued decrease in NAA over time, supporting the theory that diffuse
permanent neuronal loss occurs in many or most NPSLE patients regardless of the clinically
apparent severity of disease. In one study, 8 NPSLE patients with undergoing medical
treatment for acute neurological symptoms where followed 3 and 6 months after initial
examination [29]. Interval-significant decrease in the NAA/Cr ratios was demonstrated
between the first and second examination and insignificant difference between the second
and third examination. In contrast to a previous study demonstrating reversible changes with
inactivity [27] the decline in NAA did not reverse over time [29]. In accordance with a few
previous studies [22, 24] the mean Cho/Cr ratios were significantly increased in these 8
NPSLE patients and remained increased over the follow-up time compared to the normal
controls at the time of presentation.
These findings may suggest that this decline will continue or remain permanent
regardless of medical treatment and support the assumption that neuronal damage, seen as a
decline in NAA, might be complete and irreversible even if the patient receives the
appropriate treatment. Whether the decrease in NAA indicates cytotoxic injury, vasculitis,
microinfarct due to microthrombosis, or the presence of additional processes resulting in
brain injury has yet to be determined.
We have to keep in mind that reduced NAA changes and decreased NAA/Cr ratios can
be seen in many other conditions and are not specific to NPSLE. Alterations in NAA and
NAA/Cr ratios may become a measure of NPSLE severity and outcome rather than a measure
of NPSLE activity. Larger longitudinal studies are needed to evaluate metabolite changes in
response to treatment.
Interestingly, despite the suggestion that ischemia plays a role in the pathophysiology of
NPSLE, obvious elevation of lactate levels (an indicator of anaerobic metabolism) has not
been found in some earlier studies [20,21]. In a more recent study, alteration in LL/Cr ratio
was present and the authors speculated as to whether it was due to an increase in lactate [29]
or an increase in marco-lipids as suggested by others [20]. Most likely it is a combination of
anaerobic/hypoxic injury at time of acute symptoms as well as indicating a host response to
injury, such as an inflammatory reaction, membrane activation or demyelination [21]. The
theory that demyelination is one component is supported by recent findings using diffusion
weighted and diffusion tensor imaging.
An important role of imaging in NPSLE is deducing the etiology of acute focal (stroke-
like) neurological deficits. The differential diagnoses vary and accurate assessment is of
crucial importance, as the treatment for these alternative diagnoses differs. One of the
parameters involved in the differential diagnostic process is the presence of antiphospholipid
antibodies (APL-Ab) [55-57]. While there are correlations between MRI findings and the
presence of APL-Ab there remains some controversy regarding the relationship between the
presence of antiphospholipid antibodies and MRS findings in SLE patients. A few previous
studies have demonstrated no significant differences in metabolic ratios between those with
APLl-Ab and those without [29,46], while another study found significant differences in
metabolic ratios between APL-Ab positive-and APL-ab negative patients [45].
Myoinositol (mI) has been found to be elevated early in the course of an active flare of
NPSLE prior to changes on MRI. It can be speculated that determination of mI in
combination with NAA levels can aid in determining the severity of NPSLE. Since increase
mI with reduction of NAA is found in severe disease while elevated mI with normal NAA
has been described with minor manifestations such as headaches and mild cognitive
impairment [24].
There are potential benefits with the use of 2D-CSI in the work-up of NP-SLE patients.
The CNS involvement in NP-SLE can be diffuse and maximum metabolic abnormalities may
not necessarily be in areas that produce the dominant clinical symptoms or show the presence
of abnormal MRI findings. The 2-D-CSI spectroscopy is able to cover larger areas of the
brain and include both the areas of signal abnormalities or infarcts as well as areas that
appear normal on conventional MR images. Individual voxels within the volume of interest
(VOI) can be placed in different regions of the brain including grey matter or white matter
separately, and will facilitate comparisons between findings obtained from the small regions
of interest (ROI) that can be used for measurements on ADC and FA maps. However, to
investigate and measure a metabolite such as myoinositol short echo time (TE) on the order
of 30ms are required and in those circumstances the SVS technique is still the method of
choice. It will allow for quantification and the use of commercial fitting models such as LC
model [58].
Imaging Findings in SLE and NPSLE Using DWI and DTI
It has previously been demonstrated that NPSLE patients have increased whole brain
diffusivity compared to normal controls [35, 59-61]. This was true for both NPSLE patients
with diffuse and chronic symptoms [35] and those with acute onset of neurological symptoms
[60, 61]. The later study demonstrated differences separately in grey and white matter
compared to normal controls [60] (Figure 5). These findings are suggestive of the presence of
subtle and widespread damage in the brain parenchyma not confined to one tissue
compartment only. Selective gray matter damage in normal appearing gray matter on
conventional MRI in NPSLE patients has also been demonstrated with magnetization transfer
imaging [33]. Reduced structural integrity in the brain, such as axonal loss and/or
demyelination, is a possible etiology for the widespread damage [62, 63].
It is well-known that ADC values decrease and FA values increase when the motion of
water molecules is directionally restricted, for example, by the boundaries of myelin sheets.
Alternatively, loss of structural brain integrity would allow interstitial water molecules to
move in a more unrestricted environment thus resulting in an increase in ADC and decrease
in FA.
A few studies have measured the ADC and FA values both in lesions, and in normal
appearing gray and white matter in SLE patients both with and without NPSLE [36-38, 64].
Signal hyperintensity in cerebral cortex associated with acute ischemic changes has been has
been demonstrated with resolution after treatment [36, 37].
Figure 5 continued on next page.

Figure 5 a-c Comparison of whole brain (excluding CSF) ADC histograms between NPSLE patients
(shadow area) and normal controls ( dashed lines) (a), Histograms of ADC in grey matter only (b) and
of white matter only (c). The NPSLE patients demonstrate significantly increased mean ADC compared
to normal controls and significant differences can also be seen between the groups in both grey and
white matter.
Recent studies have demonstrated that diffusivity in different parts of the normal
appearing brain can be higher in SLE and NPSLE patients compared to normal controls [38,
64]. Regional diffusion differences with increased diffusion in frontal lobe, splenium,
anterior and posterior limbs of the internal capsule was found in a recent study [64]. They
also found significantly decreased FA in the genu and splenium of corpus callosum compared
to normal controls [64]. An increase in ADC and decrease in FA values in normal appearing
white matter have also been demonstrated in a small group of NPSLE patients compared to
normal controls [38]. Similar findings have also been demonstrated in a recent study of SLE
patients compared to normal controls from the same research group [unpublished data]. We
found significant increase in ADC values in normal appearing brain in SLE patients
compared to normal controls [unpublished data]. Moreover, when we compared the ADC and
FA values in a small group of NPSLE patients with those obtained in the same regions in a
group of SLE patients, statistically significant differences (p<0.05) were found in both ADC
and FA values in the internal capsule between the two groups. There were also significant
increased FA values in the amygdala, insular cortex, orbitofrontal cortex, thalamus, and
cingulate cortex in the SLE patients compared to NPSLE patients, and significant differences
in ADC values in amygdala, insular cortex, and cingulate gyrus were also noted when
comparing the two populations [65]. The NPSLE patients demonstrated significant increase
in ADC in insular cortex compared to SLE (p<0.05) and decrease compared to normal
controls. Significantly decreased ADC was present in the amygdala, internal capsule and
cingulate gyrus between NPSLE compared to SLE patients and normal controls (p<0.05).
These findings are, in part, in accordance with a previous report in which they demonstrated
significant decrease in ADC in the amygdala in both SLE and NPSLE compared to normal
controls, but without any significant differences between the two patient groups [66]. They
also demonstrated that the decrease in ADC was more severe in patients with anti-NMDAR
antibodies. These findings are similar to those found in mouse models [67]. These findings of
possible effect on the limbic system in SLE patients that might result in altered emotions
needs to be evaluated in larger studies in the future but is a indicator of limbic involvement
that could in part explain some of the patients’ emotional symptoms.
It seems that when following NPSLE patients over time with repeated MR imaging
persistent and evolving alterations in their normal appearing brain parenchyma occur as
demonstrated by recent unpublished data from our research group demonstrating statistically
significant increase in FA values (p<0.005) in the internal capsule, orbitofrontal cortex, and
insular cortex when comparing follow-up and initial examinations in a small number of
NPSLE patients from the same cohort as mentioned above. There was also a general but non
significant decrease in the ADC values in the same patients over time.
The pathogenesis of NPSLE is complex. The predominant pathologic process in NPSLE
found at autopsy is small vessel disease in the absence of vasculitis or thrombosis, or a “bland
vasculopathy”. It is unclear whether these findings represent previous sites of inflammation
or vaso-occlusion. Immune complex disease-mediated vasculitis, leukoagglutination, direct
antibody mediated injury to vessels or neurons, antibody (aPL) - mediated thrombosis and
direct injury endothelial injury by aPL antibodies or anti-endothelial may all play a role as
well. In addition, some authors have suggested that superimposed demyelination may play a
role in NPSLE [4, 11, 29, 38, 46]. This hypothesis that demyelination is a part of the
pathogenesis is supported by histopathological studies that have described demyelination in
brain parenchyma in patients with NPSLE [68]. This hypothesis can be supported by recent
DTI study evaluating diffusivity parallel (λ║) to the long axis, a measure of axonal injury,
and perpendicular (λ┴) to the long fiber axis, a measure of demyelination, in normal
appearing white matter in 8 NPSLE patients compared to 10 normal controls [38]. The λ┴
was significantly higher in internal capsule, corpus callosum, thalamus, and in the parietal
and frontal white matter bilaterally NPSLE patients compared to healthy and was suggested
to be the result of demyelination. A breakdown of the myelin sheets as in demyelination
would result in less restricted movement of the water molecules perpendicular to the fiber
tracts.
Whole brain tractography performed in SLE patients and normal controls has shown
generally less trackable fibers in the whole brain in SLE patients as compared to normal
controls, which again points to global white matter damage in SLE [64].
CONCLUSION
Neuroimaging has made an important contribution to the understanding of NPSLE.

Conventional MRI with gadolinium is considered the gold standard imaging technique at the
moment, and the more sensitive tool to determine the anatomic location and extension of the
lesions. The newer MRI functional approaches such as MRS, DWI and DTI that have been
described above extend their scope beyond mere diagnostic tools of a structural abnormality
with the potential to provide insight into the underlying disease mechanism and provide a
reliable tool for objective clinical follow up in NPSLE.
Further research is still needed to gain knowledge of the limitations and advantages of
each method and to determine the best combination to diagnose NPSLE.
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Chapter 8
PERSONALIZED MEDICINE FOR SYSTEMIC

LUPUS ERYTHEMATOSUS:
A NEW CHALLENGE FOR THE NEAR FUTURE
Ana M. Bertoli and Luis M. Vilá∗

From the Department of Medicine (Division of Rheumatology),
The University of Puerto Rico Medical Sciences Campus,
San Juan, Puerto Rico
ABSTRACT
Systemic lupus erythematosus (SLE) is a chronic disease with higher morbidity and
mortality when compared to the general population. Disease outcome varies among
patients, in part, because they do not respond the same to a given treatment modality.
There is, therefore, a clear need for drug treatments to be selected according to the
characteristics of an individual patient, in order to improve efficacy and reduce the
number and severity of adverse drug reactions. Personalized medicine means the
prescription of specific therapeutics best suited for an individual. This novel treatment
approach should be regarded as an ongoing progression of healthcare which is advancing
with genomic tools. Although still a translational challenge into the practice of
rheumatologists, data for immunosuppressive drugs (mainly coming from transplant
patients) is now available and waiting to be introduced in the field of autoimmune
diseases such as SLE. Examples of that are genetic studies of drug metabolizing systems
that can affect the efficacy, pharmacokinetic and tolerability profile of drugs such as
azathioprine, cyclosporin A, mycophenolate mofetil, cyclophosphamide and
glucocorticoids, among others. Although quite attractive, implementation of
pharmacogenomics may not be so simple. Ideally the genetic trait should be
accomplished with a well characterized patient from the socio-demographic, cultural and
∗
Address for correspondence: Luis M. Vilá, MD. Division of Rheumatology, Department of Medicine, University
of Puerto Rico Medical Sciences Campus, PO Box 365067, San Juan, PR 00936-5067. Telephone: 787-758-
2525, ext. 1825. Fax: 787-764-6839. E-mail: [email protected]
212 Ana M. Bertoli and Luis M. Vilá
ethnic background as well as co-morbidities and drug exposure history, variables known
as being modifier factors for drug efficacy and tolerability. Major efforts should also be
made to systematically evaluate the patient; in this sense, validated outcome measures of
disease activity, accumulated damage and health-related quality of life could be very
valuable to quantify drug response. Worldwide integrated genomic information should
also be an unequivocal subject for the future. Although quite attractive, personalized
medicine is not without some drawbacks; cost, infrastructure and worldwide networking
are not minor issues to overcome. Finally, personalized medicine must be strongly
interpreted from the ethics perspective and regulated by the law.
INTRODUCTION
Systemic lupus erythematosus (SLE) is a disease characterized by multi-organ

involvement and an unpredictable course that, sometimes, swings independently from the
treatment. Moreover, as a chronic disease, SLE can impact patients’ life; quality of life (QoL)
is usually worse [1], while morbidity [2; 3] and mortality [4] are higher when compared to
the general population.
Although a growing body of literature has focused in SLE, this disease still represents a
clinical challenge. Remarkably, one of the most difficult and misunderstood aspects of SLE is
its treatment. It is well recognized that the long-term outcome varies among patients from
different ethnic populations [5-8] and that patients do not respond the same to a given
treatment modality [9]. In this sense, many epidemiological studies have introduced the
nature versus nurture argument highlighting the necessity to integrate existing genetic and
environmental information available for this disease [10; 11].
While the biomedical research engine continues to fill the pipeline of drugs for the
treatment of SLE, biomarkers for efficacy and tolerability remain, nevertheless, to be
unraveled. There is a clear need for drug treatments to be selected according to the
characteristics of an individual patient, improving, in this way, efficacy and reducing the
number and severity of adverse drug reactions. Personalized medicine aims at this goal and it
simply means the prescription of specific therapeutics best suited for an individual.
In this chapter, we will be addressing the following topics: 1) The Concept of
Personalized Medicine, 2) Rationale for Personalized Medicine in SLE, 3) Implementing
Personalized Medicine in SLE and 4) Future Directions.
THE CONCEPT OF PERSONALIZED MEDICINE
Although pharmacokinetics is an useful tool to guide drug dosing and, eventually,

predicting outcome, some studies have failed to fully explain the relationship between a
given drug, the clinical response and adverse reactions merely based on the drug
pharmacokinetic profile. This may indicate that pharmacotherapy should rely not only in the
general approach of pharmacokinetics, but also in individual genetic variations. The latter can
result in disparities in drug absorption, distribution, metabolism or pharmacological action. It
Personalized Medicine for Systemic Lupus Erythematosus… 213
is estimated that genetics can account for 20 to 95 percent of variability in drug kinetic and
effect [12].
Personalized medicine is the use of detailed information about the patient's genotype and
clinical data in order to select a medication, therapy or preventative measure that is
particularly suited to that individual patient [13]. This novel treatment approach should be
regarded as an ongoing progression of healthcare which is advancing with the use of
nanobiotechnology (an area that applies the tools and processes of nano/microfabrication to
build devices for studying biosystems).
Identifying particular alleles that contribute to variations in the effect and safety profile
of a given drug treatment is the main challenge. Genomics, proteomics and metabolomics
refer to the search of variations in genes that can cause disease, abnormal protein patterns and
abnormal metabolite patterns, respectively [14]. These areas of expertise are the main source
of technology development and knowledge advance in this field. The potential is enormous
for pharmacogenetics (the study of individual genes) and pharmacogenomics (the study of the
genome) to yield a powerful set of molecular diagnostic methods or markers that should
become widely available to clinicians in order to select medications and dose for the
individual patient [15]. As explained by Evans et al [16], “pathways” of genes may be more
significant than individual “candidate” genes as the effect of several genes (and their
polymorphisms) acting together are those responsible of a unique phenotype.
The main advantage of this new treatment approach is that a patient's genotype needs to
be determined only once for any given gene, because, except for rare somatic mutations, it
does not change. Other benefits of this approach are its accuracy, efficacy, safety and speed.
Genotyping methods are improving so rapidly that it will be soon possible to analyze a panel
of genotypes and test for those that are important determinants of drug disposition and effects
[17]. However, medicine should not merely rely on these tests, but to take advantage of them,
bringing all the genetic information together with a well clinically characterized patient.
Although the concept of pharmacogenomics applied to the individual patient sounds
relatively simple and, at the same time, appealing there are many issues to accomplish before
these “theragnostics” (the fusion of therapeutics and diagnostic medicine with the goal of
providing individualized pharmacotherapy) [18] tests become widely available. First, the
development of new automated technologies that allows rapid scoring of microsatellite alleles
and single nucleotide polymorphisms are warranted. Indeed, recent expansions in this field
show promise to achieve this goal [15; 19]. Second, healthcare infrastructure supporting the
integration of genomic data and clinical records is necessary. The latter would involve
physicians, hospitals, healthcare systems, pharmaceuticals and diagnostic-technology
companies [20]. Third, financial outlays should be cost-effective. The implementation of
these technologies may depend not only in the healthcare expenditures but also in the
healthcare system prevailing in a given region; decision driven by the market may also
impact this accomplishment. Although advances in molecular technology hold promise for
reducing the cost and effort involved in genotyping individuals, these techniques remain
expensive and specialized, hindering their widespread use [21]. Finally, personalized
medicine must be strongly interpreted from the ethics standpoint and regulated by the law.
Regulatory, ethical and privacy policies are still matters of concern and the perception of the
public/consumers on personalized medicine remains yet to be elucidated.
The Personalized Medicine Coalition [20] has emerged as a necessity to accomplish the
above mentioned issues. This is a non-profit organization composed by pharmaceutical,
diagnostic, biotechnology and information technology companies as well as academic
institutions and governmental agencies intended to provide consensus on public policy issues
and to serve as a forum for debate and education.
RATIONALE FOR PERSONALIZED MEDICINE IN SLE
Many reasons can be stated regarding the importance of implementing the personalized
medicine approach to the care of lupus patients. First, SLE is one of the most common
autoimmune diseases with a prevalence as high as 241 cases per 100,000 inhabitants [22]. It
has also been recognized in all five continents; however, its distribution is not homogenous,
e.g. patients from minority populations, mostly of African ancestry whether living in the US,
the Caribbean Islands, the United Kingdom, or Continental Europe, tend to show a higher
SLE incidence and prevalence rates compared to those living in Africa [23]. The latter could
be possibly explained by the interplay genetic and environmental factors have in the
occurrence of the disease. Second, these genetic (inherited) and non-genetic (acquired)
factors are known not only to predispose to, but also to modulate the course and outcome of
the disease. Ethnicity is perhaps the most representative feature implicated in the disease
outcome [24-26]. For example, patients from minority populations in the U.S. and Europe, as
a group, tend to have more abrupt disease onset, more severe disease manifestations and an
overall higher degree of disease activity. They also tend to accrue more damage and show
higher mortality rates when compared to Caucasians [27]. Although ethnicity is a broad
defined construct, the genetic trait within certain ethnic group cannot be ignored. Lastly,
although the management of SLE has improved in the past 50 years, there is still a 3–5-fold
increased mortality compared to the general population [28], with major organ damage,
infection and cardiovascular disease as the major challenges for the coming decade. These
facts emphasize the need to better understand the factors predictive of poorer outcome in
patients with lupus, treatment response and toxicity included.
IMPLEMENTING PERSONALIZED MEDICINE IN SLE
Medicine has always attempted to personalize patient-physician relationship and the

patient healing approach has historical and cultural roots. Thanks to the genome project, we
are now facing with renewed excitement the prospect of personalized medicine. Contrary to
the “one size-fits all” model and the “blockbuster drug model” [29], pharmacogenetics and
pharmacogenomics effort is focused in a new model to achieve health.
During the past decade we have witnessed impressive advances in the field of
rheumatology [30] as many new drug treatments have dramatically changed patient’s
prognosis. While the biomedical research engine continues to fill the pipeline of drugs for the
treatment of SLE, biomarkers for efficacy and tolerability are not, nevertheless, yet
developed. In this enthusiasm of improving patients’ outcome, rheumatologists should not
forget the risks these new treatment modalities entail and assure that aggressive treatment is
justified.
Balancing efficacy and the occurrence of adverse events of different drugs is not always
easy to attain. This is particularly difficult for drugs with narrow therapeutic index, such as
the immunosuppressants commonly used in SLE. For these drugs the difference in the
concentrations exerting therapeutic benefit and those causing adverse events is small [31]. On
the other hand, many other drugs show a wide interindividual variability regarding efficacy
and safety; this is particularly remarkable for glucocorticoids, which are still one of the
mainstay drug therapies in the treatment of SLE.
Pharmacogenomics would then, take place to better characterize not only individual
patients’ traits but also groups of patients who have been systematically treated and evaluated
to make possible the assessment of the efficacy and adverse events profile of a certain drug.
Although data built from drug studies in SLE are yet limited, advantage can be taken on
important data from other fields of medicine, such as oncology and transplantation, in which
immunosuppressive drugs are widely used.
More than 1.4 million single-nucleotide polymorphisms were identified in the initial
sequencing of the human genome [32] with over 60,000 of them in the coding region of
genes. Every gene contains some level of polymorphism, with single nucleotide
polymorphisms (SNPs) occurring every 1000–3000 base pairs throughout the human genome.
The current challenge is to elucidate which of these polymorphisms have relevance to the
treatment of rheumatic diseases. Most drug effects are determined by the interplay of several
gene products that influence the pharmacokinetics and pharmacodynamics of medications,
including inherited differences in drug targets (i.e. receptors) and drug disposition (i.e.
metabolizing enzymes and transporters). Genetic polymorphisms with indirect effects on drug
response (neither direct targets of medications nor involved in their disposition) have also
been shown to alter the response to treatment in certain situations [33].
Response to immunosuppressants can be related to several factors including age, sex,
concomitant diseases, drug-drug interactions as well as inheritance. Another observation is
that related to ethnic differences in dose requirements for immunosuppressants. However,
ethnicity could be rather a crude marker for genotype [34; 35]. Although still a translational
challenge in the practice of rheumatologists, data for immunosuppressive drugs are now
available and awaiting to be introduced in the field of autoimmune diseases such as SLE.
Genetic studies of metabolizing systems that can affect the efficacy, pharmacokinetic and
tolerability profile of drugs commonly used in SLE such as azathioprine, cyclosporin A,
mycophenolate mofetil, cyclophosphamide and glucocorticoids, among others are already
available [36] (Table 1). However, only few studies have exclusively addressed the
relationship between a specific genotype and the treatment of lupus.
Table 1. Influence of different genotypes in the pharmacokinetics and pharmacodynamics of drugs commonly used in systemic
lupus erythematosus (SLE)
Drug Gene/allele Affected Affected pathway Consequence Distribution

enzyme/protein
Azathioprine TPMT* coding TPMT Metabolism Less effectiveness 90% of the population has
[37;69-74] (SNPs†) (drug inactivation) More adverse events normal activity
In SLE, only homozygous
deficiency was associated with
myelosuppression
HPRT‡ coding HPRT Metabolism Less effectiveness Less effectiveness
(SNPs) (drug activation)
Mycophenolate UDP-GT¶ coding UDP-GT Metabolism Bioavailability alteration Variable depending on the
mofetil (SNPs) (drug inactivation) Higher toxicity? SNP tested
[46-51] Usually low frequency
MDR§2 Multi-drug resistance Transporter C-24T SNP is associated with a
(SNP) protein 2 (bioavailability) lower oral clearance
IMPDH **coding IMPDH Drug target Less effectiveness
(mutations)
Cyclosporine A MDR1 P-glycoprotein Transporter From no changes to higher 45% to 80% in Caucasians
[52-59] (SNP) (bioavailability) concentrations depending the
SNP tested
CYP††3A4 CYP3A4 Metabolism From no changes to higher SNPs found in 70% of
coding (drug elimination) concentrations depending the Caucasians vs. 4% in
(SNP) SNP tested African Americans
CYP3A5 coding CYP3A5 Metabolism From no changes to higher 75% of Caucasians and
(SNP) (drug elimination) concentrations depending the 50% of African American
SNP tested show genetic inability of
express functional
CYP3A5
Drug Gene/allele Affected Affected pathway Consequence Distribution
enzyme/protein
Cyclophosphamide CYP2B6-coding CYP2B6 Metabolism Higher likelihood of end-stage 12% in patients from a
[38;39] (SNP) (drug activation) renal disease in SLE multiethnic SLE cohort
Lower likelihood of renal
remission
CYP2C19-coding CYP2C19 Metabolism(drug Lower likelihood of renal 25% in patients form a
(SNP) activation) remission multiethnic SLE cohort
Higher likelihood of Lower likelihood of ovarian
end-stage renal disease failure
in SLE
GST‡‡ coding GST Metabolism GSTP1 codon 105 polymorphism
(polymorphisms) (drug inactivation) increases the risks of
myelosuppression and
gastrointestinal toxicity in SLE
patients
Glucocorticoids Glucocorticoid- Glucocorticoid Drug target Mutations can be silent, Up to 60% in Caucasians
[60-68] receptor coding receptor associated with resistance or
(mutations) hypersensitivity, the latter
displaying features of metabolic
syndrome
GST coding GST Pharmacodynamics Drug resistance Up to 50% depending on
(polymorphisms) the genotype;
similar distribution in
Caucasians and African
Americans
*Thiopurine S –methyltransferase; †single nucleotide polymorphism; ‡hypoxanthine phosphoribosyltransferase; ¶uridine diphosphate glucuronosyltransferase;
§ multi-drug resistance-1 gene; ** inosine monophosphate dehydrogenase; ††member of the cytochrome P-450 family; ‡‡glutathione-S-transferase.
The relationship between single nucleotide polymorphisms of thiopurine S –

methyltransferase (TPMT, an enzyme involved in drug inactivation), and azathioprine
toxicity was examined by Naughton et al. [37]. In that study, they found that azathioprine
was generally well tolerated in SLE patients and that only the TPMT homozygous deficiency
was associated with myelosuppression. Owing that the homozygous deficiency was only
found in one out of 120 patients, the authors concluded that TPMT genotyping would
supplement, but not replace, regular blood monitoring in order to estimate the drug safety
profile.
Another example would be that of cyclophosphamide, a drug extensively used in the
treatment of severe clinical manifestations of SLE, such as lupus nephritis.
Cyclophosphamide is a pro-drug that requires metabolic activation by cytochrome P450
(CYP) enzymes to 4-hydroxycyclophosphamide. Multiple CYPs have been involved in this
activation and their polymorphisms implicated in the efficacy and the risk for adverse events.
In the study by Takada et al [38], the authors found that SLE patients with renal involvement
who were homozygous for CYP2B6*5 or CYP2C19*2 had a higher probability to evolve into
end stage renal disease, while patients either heterozygous or homozygous for CYP2C19*2
had a lower risk of developing premature ovarian failure. These findings are of outmost
clinical importance since SLE mainly affects young women in their reproductive years of life,
therefore, they could help making individualized treatment decisions. Cyclophosphamide
metabolites are mainly detoxified by multiple glutathione S-transferases (GSTs). The
relationship between GSTs mutations and the use of cyclophosphamide in SLE was studied
by Zhong et al [39]. In that study the authors concluded that the GSTP1 codon 105
polymorphism increases the risks of cyclophosphamide toxicity in SLE patients.
Data for other immunosuppressant drugs such as mycophenolate mofetil and
cyclosporine A are also available. Although these data come from non-lupus populations,
they can, nevertheless, be very valuable in SLE patients. Similar to what it has been observed
in the lupus population [40-42], African-American renal allograft recipients continue to
exhibit poorer prognosis compared to Caucasian patients [43]. Thus, ethnic differences in the
pharmacokinetics of immunosuppressants are a potential key factor in the differences
observed. The latter are most likely mediated via several non-genetic as well as genetic
factors, including known genetic variations that impair transporter/enzyme activity of genes
such as CYP3A4, CYP3A5, multi-drug resistance 1 and 2 [44]. One of the most outstanding
examples of such ethnic disparities is in the mycophenolate dose requirements in African-
American compared to Caucasians; African-American patients require higher doses to attain
equal response rates [45]. Many genetic variations of the enzymes responsible for
mycophenolate inactivation as well as for the entero-hepatic recirculation have been
described [46-51], however, up to date none of these gene polymorphisms can fully explain
the response discrepancies between ethnic groups. Many gene polymorphisms mainly
affecting the pharmacokinetics of Cyclosporine A, have also been described [52-59].
Although these genetic polymorphisms seem to be highly prevalent in the population, their
impact in drug metabolism is very variable, with no evidence at this moment suggesting the
necessity of any screening before treatment initiation.
Finally, recent findings in the glucocorticoid gene receptor mutations sound very
valuable for their clinical application [60-68]. Although these mutations can be silent, they
have also been associated with resistance or hypersensitivity, the latter displaying features of
metabolic syndrome. Therefore, their implication in the daily clinical practice sound suitable
if we are going to recognize a priori which patients are more likely to respond to
glucocorticoids or, which is also very important in the lupus population, which of them are
more prone to accrue, upon treatment, more cardiovascular risk factors.
FUTURE DIRECTIONS
Despite an increasing data on the variability of drug response phenotypes and their
genetic basis, the challenge now is the translation into the practice of rheumatology. A first
step toward achieving this goal should be minimizing spurious positive findings due to
chance associations between genotypes and phenotypes. Therefore, logical strategies to
identify candidate genetic polymorphisms and their application to the daily rheumatology
practice are needed. These strategies should be directed to the following topics: 1) clinical
areas where to translate and prove the applicability of pharmacogenomics, 2) conceding the
value to the specialty care, 3) infrastructure development and 4) setting the role of public
health and founding sources.
Ideally the genetic trait should be accomplished with a well characterized patient from
the socio-demographic, cultural and ethnic background, co-morbidities and drug exposure
history, as well as variables known as modifying factors for drug efficacy and tolerability.
Major efforts should also be carried on to systematically evaluate the patient; in this sense,
prospective cohort studies with nested designs focusing in drug treatment are an exceptional
source of information regarding the interrelation between environment, socio-demographic,
drug exposure history and the patient genetic trait. Another source of invaluable information
is controlled clinical trials. The setting provided by this type of studies is ideal to better
define the applicability of the genetic knowledge. During controlled clinical trials patients are
well characterized, uniformly treated and systematically evaluated to objectively assess the
drug response and safety profile.
As above mentioned, SLE represents a clinical challenge which requires the
rheumatologist expertise, not only for treatment decisions but also for adequate patient
follow-up. Outcome measures, which are increasingly being used in the routine clinical
practice, can be merged with genomic and genetic data to provide new ways to choose
therapy. The true value of any outcome measure is its ability to show the impact the disease
has on the patient and to enable the clinician to follow the disease course, to evaluate the
effect of therapeutic strategies and to better advise the patient. These outcome measures
should assess at least four domains: disease activity, organ damage, health-related quality of
life (HR-QoL) and survival [75; 76]. Among the different instruments used to evaluate these
domains, rheumatologists should choose among those best suited for their clinical practice,
both in terms of feasibility of its use and the relevance of the data obtained. Indexes to
measure disease activity such as the British Isles Lupus Assessment Group (BILAG),
European Consensus Lupus Activity Measurement (ECLAM), Systemic Lupus
Erythematosus Disease Activity Index (SLEDAI), Systemic Lupus Activity Measure (SLAM)
and Lupus Activity Index (LAI) have been validated prospectively and their reproducibility,
validity and sensitivity to change compared [77]. Critical factors in the use of these indexes
are the physician/investigator training and consensus among clinicians about how these
indexes should be applied. Equally important is to understand the strengths and limitations
each of these instruments have. Health status of lupus patients is not only related to disease
activity but also to damage resulting from the disease itself, concomitant morbidities and
treatment toxicity. The Systemic Lupus International Collaborating Clinics/American College
of Rheumatology Damage Index has been validated to measure irreversible damage [78] and
found to predict mortality [79]. A very valuable advantage of this index is that can reflect the
positive and negative long-term effect of treatment. HRQoL instruments, such as the SF-36,
are also available; the latter has been widely used to measure this domain although there are
disease specific instruments under development [80].
Infrastructure development is also a first requirement if the gap between discovery and
clinical development is to be bridged. Worldwide integrated genomic information should be
an unequivocal subject for the future and multinational clinical trials could be a great
opportunity to accomplish this. Networks supporting all the patient information and
electronic medical records, among others, are important topics of concern for which founding
sources are needed, especially for disadvantaged regions of the world. Lastly, the integration
of public health with personalized medicine has yet to be accomplished [81; 82]. The next
step is to set the role public health is going to play in this new treatment era.
CONCLUSION
Personalized Medicine should be sought as a new perspective to overcome the

embarrassing dichotomy between the body of pharmacogenetic knowledge and its clinical
application. The main objective of this novel “specialty” is to provide better margins of drug
treatment efficacy and safety as new diagnostic tests become available. Although quite
attractive, there are many important issues to be accomplished before this new treatment
perspective can be widely used; infrastructure, financial outlays, a redirection of the public
health and the public opinion as well as the ethics and law standpoints are matters of debate.
Lupus treatment armamentarium is not effective for all patients and, at the same time,
encompasses a wide range of unpredictable adverse effects. Therefore, this disease is the
perfect setting to apply all the knowledge gained through pharmacogenomics. If this is the
future of medicine, scientists and physicians should collaborate all together in the difficult
task of translating genetics and genomics into the clinical practice. There is a necessity,
however, of being proficient enough to properly interpret these evolving concepts and
discoveries in order to offer our patients the best results with the least cost in terms of adverse
effects and financial outlays.
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Chapter 9
THERAPEUTIC POTENTIAL OF HMG-COA

REDUCTASE INHIBITORS (STATINS) IN
Przemyslaw J. Kotyla and Bogna Sliwinska-Kotyla

Department of Internal Medicine and Rheumatology
Medical University of Silesia, Katowice, Poland
ABSTRACT
Systemic lupus erythematosus is a connective tissue disorder of unknown origin and
a relatively bad prognosis. In the last fifty years the big progress in the treatment of the
condition has been made, resulting in the improvement of prognosis and prolongation of
the life span. Nowadays we are prepare enough to manage the acute form of the disease,
treat acute vasculitis lupus nephritis, decrease inflammation. As the result of extension of
the life span the lupus face has been changed. Today a premature atherosclerosis is
considered the main risk factor for increased mortality among lupus patients. The
premature development of atherosclerosis is a well known clinical phenomena since the
work of Urowitz and colleagues who described the term bimodal peak of mortality and
attributed the second peak of mortality to premature development of atherosclerosis and
its fatal complications. For almost two decades the mechanism of the atherosclerosis in
lupus had not been explained satisfactory. In the early nineties of the last century Ross
suggested that atherosclerotic process is an inflammation in its nature. Now
atherosclerosis is considered a chronic inflammatory condition of the vessel wall. In the
recent years many immunological mechanisms in the development and progression of the
atherosclerotic lesions have been recognized. Since the atherosclerosis and
atherosclerosis in lupus patients share the same pattern of inflammation it would be
reasonable to treat the patients with a drug that cools the inflammation on one side and
halts progression of atherosclerosis on the other.
Statins, inhibitors of HMG-CoA reductase, a key enzyme in the pathway of
cholesterol synthesis have been commonly used in the therapy of ischemic coronary
events and the control of hypercholesterolemia. In last years evidence was accumulated
228 Przemyslaw J. Kotyla and Bogna Sliwinska-Kotyla
suggesting that action of statins go far beyond lipid lowering properties and may affect
function of immune system cells. An array of anti-inflammatory effects has now been
identified, including reduced production of pro-inflammatory cytokines, chemokines and
adhesion molecules; reduced expression of inducible major histocompatibility complex
(MHC) class II molecules by antigen presenting cells (APC); lowering of C-reactive
protein levels and reduced production of reactive oxygen species. The effect that is
separated from lipid lowering properties is sometimes called pleiotropic action of statins.
The beneficial effect is often seen before the normalization of lipid profile suggesting
that action of statins comprises at least two mechanism – cholesterol synthesis inhibition
and direct effect on arterial wall and endothelium.
Such pleiotropic effect may benefit patients with autoimmune rheumatic diseases,
including lupus.
The repertoire of drugs used in the treatment of systemic autoimmune disorders is
characterized by unfavorable influence on lipid profile in patients treated, thus may
perpetuate the development of atherosclerosis evoked by the inflammatory process itself.
In this regard statins may act on two main fields; being a concomitant therapy for
autoimmune disorders and normalize the lipid profile abnormalities in patients with
connective tissue disorders.
This overview assesses the evidence for using statins in patients with systemic lupus
erythematosus.
INTRODUCTION
Systemic lupus erythematosus is an autoimmune disorder of unknown background that

serves as a prototype for the other autoimmune disease. It affects mainly young people,
especially women, a population that is not at risk of atherosclerosis development. In the
former time acute course of lupus with lupus nephropathy, hematological complication and
infection had been attracted the attention of physicians. However, with an improvement of
the lupus management we are now able to control well acute phase of disease that contributes
significantly to the improvement of the prognosis and prolongation of the life span. Many
years after the onset of lupus survivals from acute lupus phase face the problem of
unexpected premature cardiovascular incidents.
Epidemiology
The problem of premature atherosclerosis in young lupus patients was described for the
first time by Urowitz and colleagues [1]. They coined the term “bimodal mortality” and
explained that the first peak of mortality is caused by acute phase of disease, but the last one
was attributed to the fatal complication of atherosclerosis. These observations were
confirmed by Bulkey and Roberts, who in series of autopsies of young lupus female patients
found significant atherosclerotic lesions at least in one coronary artery in majority of cases
[2]. These findings were substantiated by subsequent studies that additionally showed
incidence of myocardial infarction being 5 times as high in lupus patients as in general
population. Moreover, the specific target population for the disease was a group of young
Therapeutic Potential of HMG-CoA Reductase Inhibitors (Statins)… 229
women where a age- specific incidence increased by a factor as much as 50 has been
observed [3, 4].
The epidemiological studies showed prevalence of cardiovascular events in SLE at the
level 6-26% [5,6]. With the use of novel non-invasive techniques the ratio of subclinical
atherosclerosis was substantially higher and was established as high as 17-40% [7, 8].
Atherosclerosis and Inflammation
The pathogenesis of accelerated atherosclerosis in SLE is not completely understood. It

is believed that atherosclerosis in SLE is the result of combination of traditional risk factors,
treatment, and inflammation [9, 10]. However, the traditional risk factors are failed to fully
explain the process of accelerated atherosclerosis that suggests the main role of inflammatory
process in beginning and progression of atherosclerotic lesions among the patients with SLE.
Moreover, the role of corticosteroid therapy and subsequent development of atherosclerosis
in SLE is still subject of controversies and the results obtained from the studies gave
contradictory results [4, 11]. Since the traditional risk factors, corticosteroid use are enable to
fully explain the burden of accelerated atherosclerosis, SLE alone (but also the other
connective tissue disorders) should be recognized as novel independent risk factor.
The phenomena of atherosclerosis in patients with autoimmune disease was not
explained well. In nineties of the last century Ross [12] explained that atherosclerosis is equal
to inflammation. In recent years this new understanding of the pathogenesis of atherosclerosis
has included insights in the role of inflammatory mediators in cardiovascular disease, and
characterization of immunologic phenomena that is present in coronary vascular disease but
also may be found in classic autoimmune diseases [13-15]. This contributes to recognition of
atherosclerosis in systemic lupus erythematosus as a chronic inflammatory disease
characterized by circulating autoantibodies, activated T lymphocytes, immune complexes and
proinflammatory cytokines. The level of inflammation in the course of SLE is high enough to
start and progress the atherosclerosis in the patients.
The Role of Endothelium
The anatomic substrate for development of atherosclerosis is endothelium. Besides the

cytokine and immunocompetent cells, the endothelium is the third major player in
atherosclerosis. Expression of functionally important endothelial cell surface is influenced by
circulating cytokines and other stimuli [16]. On the other side the endothelium is the source
of anti–inflammatory and anti-thrombotic compounds that posse the ability to neutralize
strong atherogenic stimuli. This protective role of endothelium is only observed with
endothelium untouched. Under the stimulation of many diverse agents which include
cytokines, autoantibodies, antiphospholipid antibodies, and modified LDL the endothelium
can express a series of changes that allow it to participate in inflammation and immune
response [17]. To characterize better these changes the endothelium activation term was
introduced by Willms-Kretschmer [18] and than reintroduced by Pober, who showed the role
of cytokines in the activation of endothelium [19, 20]. Endothelium activation consists of five
main changes- loss of vascular integrity, expression of leukocyte adhesion molecules, change
in phenotype from anti-thrombotic to pro-thrombotic, cytokine production and up-regulation
of HLA molecules [21-25].
Loss of endothelial integrity promotes the influx the fluid from intravascular space into
subendothelium. Up-regulation of leukocyte adhesion molecules, such as E-selectin, ICAM-1
and VCAM-1 allows leukocyte to interact with endothelial surface and than to penetrate into
the tissue [26]. This transmigration is driven by several factors with monocyte
chemoattractant protein-1 (MCP-1) being the most prominent compound that is synthesized
by endothelial and smooth cells layers [27].
Endothelium activation and subsequent dysfunction has been implicated in acute
inflammatory response and it has been correlated with elevated level of C-reactive protein
[28, 29]. It also occurs in patients with SLE suggesting the involvement of the same
mechanism in spite of disease origin [30]. Moreover the patients with SLE are characterized
by higher nitric oxide production and larger baseline diameter of peripheral artery. The extent
of nitric oxide production, expression of nitric oxide synthase in endothelium and baseline of
brachial artery diameter correlates well with lupus activity but also with the progression of
atherosclerosis [31].
Endothelium is also the site where expression of MHC class II molecules takes place.
Being not a classical antigen presenting cells (APC) the endothelial cells may also express
MHC molecules, that occurs in particular after endothelial stimulation with activated T cells,
interferon gamma (IF- γ) and lymphokine [32-34]. The role of MHC molecules in
atherosclerotic processes has been intensively debated. The main area of interest is premature
atherosclerosis in transplanted organs [35, 36]. Accelerated atherosclerosis in this particular
clinical states correlates well with increased expression of MHC molecules in graft organs,
being at least partially responsible for subsequent graft rejection [37].
COX/Metalloproteinases Role
In last few years the role of metalloproteinases MMP-2 and MMP-9 has been intensively
discussed. Both enzymes are highly expressed in human atherosclerotic plaques and
represents the principal mechanism of collagen breakdown that leads directly to plaque
instability and subsequent thrombus formation [38, 39]. Macrophages are the main source of
MMP-2 and MMP-9, which synthesis is regulated via PGE2 / dependent pathway cAMP
[40]. Several inflammatory stimuli including IL-1, TNF alpha and CD40L induce PGE 2
synthase that in turn activates COX-2 leading to increased biosynthesis of PGE 2 leading
eventually to activation of MMPs [41, 42]. Taking into account expression of COX-2 it is
speculated that cyclooxygenenase-2 might play a role in lipid accumulation in lesional
smooth muscle cells (SMC) and macrophages and favoring formation of foam cells within
atheroma. [43].
Cytokine Involvement
Systemic lupus erythematosus is a disease that is characterized by production of

autoantibodies that is supported by Th2 response. The T helper cells can differentiate into
two TH1 and Th2 subgroups [44]. Th1 response promotes cell mediated immunity and
inflammation via T cell expansion and monocyte activation while Th2 response is
characterized by humor activity with antibody production. In SLE it is believed that
imbalance between TH1 and TH2 response exists favouring Th2 activity and B cell-
dependent autoantibody production [45]. B cell function is orchestrated by a family of Th2
dependent cytokines ( (IL-4, Il-5, Il-6 and Il-10) that work together with released from
monocytes and macrophages IL-1,IL-6 and TNF alpha [46,47]. The latest contributed
significantly to the development of atherosclerosis being present in atherosclerotic plaques,
affecting smooth cells proliferation and inducing the local inflammation in vessel wall and
activation of macrophages [48].
The role of TNF alpha in SLE is controversial. Some studies show a positive correlation
between disease activity and TNF alpha especially with increased triglycerides and low HDL
[49]. However others have found a higher level in inactive disease postulating a protective
role [50]. The others have found no difference in basal levels of TNF alpha in healthy
controls versus SLE [51] Other papers postulate serum TNF-alpha and IL-6 as sensitive
markers of SLE disease activity. They may play a role as independent markers for prediction
of SLE disease activity and to differentiate normal subjects from those having SLE [52].
Being a marker of disease activity TNF alpha may also contribute to the development of
atherosclerosis and play a role in atherosclerotic plaque breakdown via activating MMPs. It
also influences lipid profile disturbances by elevation of triglyceride level [41, 42, 53].
ANTIATHEROSCLEROTIC PROPERTIES OF STATINS
Statins a 3- hydroxymethyl-3-methylglutaryl coenzyme A (HMG-CoA) reductase

inhibitors belong to a class of drugs that characterize by inhibition a key enzyme in the
pathway of cholesterol synthesis [54, 55]. Strong correlation between total and LDL
cholesterol levels and atherosclerotic disease gave the background for use the statins in
primary and secondary prevention of coronary artery disease [55]. Data from angiographic
studies, however showed only minimal changes of atherosclerotic lesions in patients treated
with statins as compared to the control. On the other hand despite of weak influence upon
vessel wall morphology subjects on statins therapy dramatically benefit from the treatment.
This gave the strong suspicion that statins as a class of drug may work at least on two fields;
suppress cholesterol biosynthesis on one side and directly influence the atherosclerotic
process via cholesterol independent way [56].
Statins therapy causes regression of atherosclerotic plaque that may contribute
significantly to the clinical benefits observed in patients on statins therapy [54]. Moreover
therapy with statins decreases the accumulation of cholesterol in arterial wall that is believed
to be a marker of atherosclerosis [57]. The Scandinavian Simvastatin Survival Study (4S) was
the first that showed that cholesterol lowering treatment decreases significantly all-cause
mortality in patients with coronary heart disease and myocardial infarction [58]. The
promising results form this study gave background for subsequent studies. West of Scotland
Coronary Prevention study was the first primary prevention study that showed significantly
reduction of MI and cardiac death [59].
Statins not only reduced morbidity and mortality from coronary heart disease but also
prolonged life span in patient with hyper and normocholesterolemia. The latest suggest that
action of statins is at least partially independent from lipid lowering properties of HMG-CoA
reductase inhibitors [55].
The role of cholesterol in the pathogenesis of atherosclerosis is well established. Some
doubts exist regarding relation between cholesterol level and development of cerebrovascular
disease. Several studies addressed this question and some data obtained from these studies
indicate that similarly to the cases of coronary heart disease therapeutic potential of statins
can be seen in central nervous system vessels [60-63].
Atherogenesis and plaque formation begin with accumulation of low-density lipoproteins
cholesterol in the endothelial compartment of blood vessel followed by its oxidation.
Oxidated lipoproteins accumulated within endothelial space are the main target for
monocytes and macrophages. Enzymes release from monocytes/macrophaes break down
fibrous cap at the top of the plaque and lead to the rupture [64, 65]. Statins posse ability to
stabilize atherosclerotic plaques. Several clinical studies showed potential of statins to
decrease evolution rate of atherosclerotic lesion towards rupture. The one of the possible
mechanism involved into this process is suppression of COX-2 and microsomal prostaglandin
synthase-1 (mPGES-1) by statins. As the result of this process plaque stabilization is
occurred, that prevents its rupture and subsequent thrombus formation that is the main
mechanism leading toward acute myocardial infarction [43, 66].
Platelets activation and aggregation is a key step in thrombus formation. Since they are
already activated they release potent factors involved into amplification platelets aggregation
and recruitment, and thrombosis [67, 68]. Platelets can be activated by different stimuli
including thrombin, adenosine diphosphatate and proinflammatory cytokines (including Il-1
and interferon gamma) [69]. Platelets are also important source of thromboxan A2 (TXA2),
one of the most potent vasoconstrictor in the body. Statins may minimize platelet aggregation
and thromboxan release through partial inhibition of ADP and ATP release from activated
platelets and exerting antioxidant effects [70].
Fibrinogen, an important factor in coagulation exerts its proatherosclerotic action by
stimulation of proliferation, and migration of the smooth cells. These processes are realized
via affecting permeability of vascular endothelium and function of platelets,
monocyte/macrophages as well as exerting proinflammatory and pro-coagulatory effects
[71,72]. The results obtained from the studies indicate that effect of statin on the level of
fibrinogen is relatively mild [73, 74]. However in patients suffering from connective tissue
diseases fibrinogen level may reflect the level of inflammation. This is why fibrinogen is well
known acute phase marker with its level raised significantly in the course of inflammation
[75]. Statins may act on this field by cooling the inflammation following decreasing
fibrinogen level.
Another potent coagulation factor that is found in atherosclerotic plaques is tissue factor
(TF) [76].It is synthesized by damaged or stimulated monocytes, macrophages smooth and
endothelial cells. After binding to activated factor VII, TF promotes coagulation [77]. Statins
reduce level and activity of TF in human monocytes cultures activated but also non-activated
by inflammatory stimuli [78, 79]. This effect is not only restricted to the mononuclear cells
but also can be seen in smooth muscle cells of artery wall as well as endothelial cells [80,
81].
The several studies have addressed the influence of statin upon thrombin formation. The
special emphasis was put on some thrombin generation parameters including such as
thrombin fragment (F1+2), fibrinopeptide A (FPA), and thrombin-antihrombin III (TAT)
complexes [75]. According to data obtained from the studies simvastatin inhibits conversion
of prothrombin to thrombin [82]. Additionally in ex vivo studies simvastatin decreased
thrombin generation and the total activity of the enzyme in patients with significantly high
cholesterol level [83]. Statin are also shown to inhibit platelet-dependent formation of
thrombin. The mechanism of this activity that is supposed to be involved is improvement of
impaired interaction between platelets and coagulation factors, since platelets activity and
aggregability do not change during the treatment [84].
PLEIOTROPIC PROPERTIES OF STATINS
Statin were designed as potent cholesterol lowering drugs. Within many years passed
they proven their efficacy in normalization lipid profile disturbances in secondary and
primary prevention of cardiovascular events [58, 59, 85]. Large trials have demonstrated that
one of the mechanism involved is lowering serum lipid levels, specifically low density
lipoproteins, that is one of the most important risk factors for coronary artery disease [86].
The primary pharmacological function of statins is to suppress HMG-CoA reductase in the
hepatic cells that decreases significantly de novo cholesterol synthesis in the liver. As the
result of this inhibition overexpression of low density lipoprotein receptors on the
hepatocytes surface occurs [87]. By upregulating LDL receptor expression hepatocytes
increase cholesterol clearance. Increased cholesterol utilization and decreased LDL
production contribute enormously to the decreased cardiovascular morbidity and mortality
[88]. Decreasing cholesterol level is really of great patophysiological importance and exerts
clinical benefits to the patients treated. However most studies suggest that non-cholesterol
lowering properties of statin may be of the special biological importance [89-92].
These diverse properties which are apart from lipid lowering potential are sometimes
called pleiotropic effect (Gk. Pleio – meaning many, tropos meaning manner). The term is a
little controversial, since in vivo it is difficult to differentiate what effects are really
independent from lowering cholesterol, as the decrease cholesterol level itself may exert
many biological consequences. Statins block cholesterol synthesis at the level of mevalonate
formation. Mevalonic acid is not only a substrate for subsequent cholesterol synthesis but
also provides substrate for farnesylpyrophosphate and geranylgeranylpyrophosphate. These
compounds are important for isoprenylation of cellular small molecular weight G-proteins of
the Ras superfamily (Ras Rac Rho).G-proteins are involved in regulation of growth, cell-to-
cell interaction and apoptosis [91, 93,]. Isoprenylation of small G proteins is essential for
those proteins to achieve new regulatory functions [94]. Equilibrium between activity of
these proteins provides strong positive stimuli upon endothelium and promotes to exert
vasorelaxation [95]. Influence of cardiovascular risk factors on the cascade of small G
proteins results with activation of small RhoA protein, and Rho kinase (ROCK) leading to
destabilization of nRNA for eNOS synthase [96]. In the same moment strong positive
regulatory effect on prepro ET-1 synthesis could be observed. It provides imbalance between
NO synthesis and overproduction of ET-1. As the result vasoconstriction of vessel is
observed. Endothelial and muscle smooth cells also express another small G protein Rac1
that is the major component of NAD(P)H oxidase that serves in many pathological states as
the source of free radical species. Free radical species can interact with NO and decrease its
bioavailability leading to endothelial dysfunction [95]. Another biological function of RhoA
and ROCK proteins is regulation of endothelial barrier dysfunction by increasing
phosphorylation of myosin light chain. Rho A plays also the role in signal transduction to
muscle and non-muscle myosin II [97]. All those events eventually lead to the blood vessel
contraction and promote development of atherosclerotic plaque. Statins may interfere with
isoprenylation of small G protein and block their patophysiological function. It is suggested
that this is one of the main mechanism that explains well pleiotropic properties of HMG-CoA
Lessons derived from experimental models suggest that statins upregulate eNOS
expression and inhibit smooth muscle proliferation via inhibition Rho-ROCK pathway. The
effect is enhanced by cotemporary inhibition of Ras. Animal models showed that Ras plays
the significant role in formation of atherosclerotic plaques, which was shown in
apolipoprotein knock-out mice [98].
Many studies have proven that statins may posse immunomodulatory properties that
might be of the special benefit in the treatment of autoimmune disorders. Statin may inhibit
nuclear factor κB (NFκB) resulting with inhibition of endothelial activation [99, 100]. Statins
regulate Class II Transactivator that reduce induced by interferon gamma (IFγ) expression of
MHC class II molecules on antigen – presenting cells (APS). MHC molecule expression on
the surface of APC is required for antigen presentation, thus antigen is recognized by T cells
in the context of MHC II antigens. Decreased expression is resulting in antigen tolerance and
down regulation of T cell response [101, 102]. Interaction with the process of MHC II
presentation contributes to the shift of T cell from Th1 response to the protective Th2
response [103, 104]. Statins may also interfere with second signal transmission, since they are
able to decrease expression of CD80/86 molecule on APC (mainly on B cells). Co-
stimulation is essential to evoke full activation of T cells; in the absence of strong Co-
stimulation signal T cell may develop immune tolerance and subsequent lack of activity. In
this model treatment with atorvastatin stopped the progression of lupus in lupus-prone New
Zeland F1 mice [105]. This model may also explain good therapeutic response to statin
therapy observed by Abud-Mendoza in patients with refractory SLE and lupus nephropathy
[106]. In another study four weeks treatment with simvastatin in patients with SLE resulted
with marked suppression of TNF alpha level and decreased lupus activity in SLEDAI scale
[107].
The therapeutic and immunomodulatory effects of statins could also be observed in

collagen-induced arthritis that represents an animal model of rheumatoid arthritis. In this
study simvastatin suppresses significantly articular inflammation and decreases pro-
inflammatory cytokine synthesis, IFNγ, tumor necrosis factor α (TNFα) and interleukin-12
(IL-12) [103]. This phenomenon could be also observed in patients with refractory
rheumatoid arthritis, where administration of HMG-CoA reductase inhibitor - simvastatin
resulted in suppression of inflammation [106]. This effect was confirmed in a larger double
blind, randomized, placebo-controlled trail with atorvastatin. According to the data obtained
from the study atorvastatin has a significant effect on disease activity and reduces the markers
of inflammation [108]. In another study Yokota et al reported inhibition of production of IL-
6, IL-8 and TNFα in fibroblast like synoviocytes from patients with RA [109].
Systemic lupus erythematosus is often complicated by antiphospholipid antibody
syndrome that is believed to at least partially responsible for thrombotic episodes observed
among the patients with SLE. Endothelium plays an essential role in the beginning and
progression of thrombotic changes in the SLE patients. Under influence of stress factors such
as pro-inflammatory cytokines, anti-phospholipid antibodies the phenotype of endothelium is
changed from anti-adhesive toward pro-inflammatory and pro-adhesive. Statins showed their
potential as a anti-inflammatory drugs in antiphospholipid antibody syndrome where they can
inhibit activation of endothelium [110]. In animal model statins are shown to minimize
prothombotic and pro-inflammatory influence of the antiphospholipid antibodies [111]. In
another study monocytes from patients with APS treated with fluvastatin, showed markedly
suppression of tissue factor. Moreover the effect was seen to six month after the end of the
treatment [112].
In the beginning of 21st century we recognize SLE as a complex systemic disease with
late complication from cardiovascular system. Since statins proven their efficacy in treatment
of atherosclerosis in population not affected by connective tissue diseases and showed non-
cholesterol lowering properties it is reasonable to use this group of compounds in primary
and secondary prevention of cardiovascular diseases among the patients with SLE. This
problem was recently addressed by Wajed and colleagues who proposed very tight control of
lipid and cholesterol levels in patients with SLE [113]. It comes form understanding the SLE
as coronary heart disease equivalent, that explains why total and in more extent LDL
cholesterol levels should be as low as in patients with coronary heart disease. The ideal
control of lipid risk factors is to maintain the LDL-cholesterol at the level of 2,6 mmol/l. This
can be easily achieved by the use of statins. The usage of latest may also provide some
additional benefits effects that are driven from immunomodulatory activity of HMG-CoA
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INDEX
age, ix, 2, 4, 9, 19, 41, 43, 45, 59, 79, 83, 85, 87,
A
106, 108, 112, 114, 115, 117, 119, 120, 121, 128,
141, 143, 187, 194, 215, 229
abortion, 186
ageing, 158
accidents, 72, 107, 164, 236
agent, 2, 42, 109, 161
accuracy, 54, 66, 213
aggregation, 40, 232
acetylcholine, 136
aging, 143
achievement, xi, 178
agonist, 13, 136, 148
acid, 12, 111, 142, 163, 224, 233, 242
albumin, 1, 3, 4, 164
activation, viii, ix, 30, 40, 56, 63, 78, 81, 90, 91,
alcohol, 85, 109
105, 106, 107, 123, 129, 132, 133, 134, 136, 138,
alcohol use, 85
149, 150, 151, 202, 216, 217, 218, 229, 230, 231,
algorithm, 142
232, 234, 235, 237, 238, 242
allele, 90, 216, 217, 224
acute confusional state, 156, 194
allergic reaction, 3, 145
acute coronary syndrome, 68, 96, 237, 240
ALT, 185
acute lymphoblastic leukemia, 225
altered peptide ligand, 136, 147, 148
acute renal failure, 184
alternative, 133, 137, 202
adaptive immune system, 137
alters, 127
ADC, 198, 199, 201, 203, 204, 205, 209
Alzheimer’s disease, 163, 173
adenosine, 232
amino acid, x, 131, 137, 142, 143, 149, 151
adhesion, xii, 40, 41, 80, 95, 165, 166, 228, 230,
amygdala, 204
237, 242
anaemia, 33, 64
adipose, 41
ANC, x, 178, 182, 183, 184, 186
adipose tissue, 41
androgens, 86
adjustment, 41, 80
anemia, 10, 11, 189
ADP, 232, 240
angina, 43, 46, 47, 93, 236
adsorption, 3, 7
angiogenesis, 52
adults, 43, 61, 64, 65, 70, 101
angiography, 43, 44
adventitia, 52
angioplasty, 47
adverse event, 125, 215, 216, 218
animal models, 41, 48, 133, 137, 139, 142
aerobic exercise, 24
animals, 12, 108, 134
aetiology, 85, 92
anisotropy, 199, 201, 208, 210
Africa, 214
ankylosing spondylitis, 61
African American(s), 106, 216, 217, 224
Annexin V, 82
antagonism, 147
anthrax, 125
246 Index
antibiotic, 64, 187 assessment, 25, 27, 35, 43, 45, 46, 50, 51, 53, 65, 71,
antibody, ix, 3, 4, 6, 7, 15, 33, 65, 80, 91, 103, 105, 79, 88, 91, 157, 164, 174, 188, 202, 215, 221
110, 111, 116, 118, 122, 126, 133, 138, 139, 144, astrocytes, 164
146, 153, 157, 158, 159, 160, 161, 164, 167, 168, asymptomatic, vii, viii, xi, 30, 31, 43, 45, 47, 50, 51,
169, 170, 171, 183, 192, 201, 205, 206, 231, 235, 58, 64, 70, 78, 193, 194
236 ataxia, 186
anticardiolipin, 6, 33, 41, 59, 60, 63, 68, 75, 80, 95, atherogenesis, 38, 40, 69, 80, 237
97, 168, 174, 175, 183, 184, 206 atherosclerosis, vii, viii, xii, 29, 30, 37, 38, 39, 40,
anticoagulant, 33, 43, 80, 94, 101 41, 42, 43, 45, 46, 47, 49, 50, 62, 63, 65, 66, 67,
anticoagulation, 64, 87 68, 69, 70, 71, 72, 77, 79, 80, 81, 82, 83, 84, 92,
anticytoststic drugs, xi 93, 94, 95, 96, 97, 221, 227, 228, 229, 230, 231,
antigen, xii, 122, 123, 126, 133, 134, 135, 136, 137, 232, 235, 236, 237, 238, 239, 240, 242
139, 141, 144, 145, 148, 150, 157, 228, 230, 234 atherosclerotic plaque, 37, 38, 40, 41, 42, 44, 45, 48,
antigen-specific cells, 135 70, 79, 80, 82, 230, 231, 232, 233, 234, 238, 240
anti-inflammatory, xii, 5, 31, 49, 83, 84, 107, 122, ATP, 232
228, 235, 237, 239, 242 atrium, 53
anti-inflammatory drugs, 5, 31, 49, 235 atrophy, 192, 201, 210
antimalarial agents, 71 attacks, 25, 163
antimalarials, 13, 40, 84, 89 attribution, 194, 206
antinuclear antibodies, 48, 183 auscultation, 33
antioxidant, 82, 232 Austria, 210
antiphospholipid antibodies, viii, 7, 15, 30, 37, 39, autoantibodies, ix, x, 1, 34, 37, 38, 41, 53, 60, 61,
41, 43, 50, 53, 61, 62, 63, 64, 68, 73, 80, 82, 94, 62, 65, 75, 95, 106, 107, 116, 121, 123, 127, 131,
97, 173, 174, 201, 202, 206, 209, 229, 235, 242 133, 135, 137, 138, 141, 142, 144, 146, 151, 155,
antiphospholipid syndrome, 9, 59, 60, 61, 62, 69, 74, 157, 158, 159, 160, 162, 163, 165, 167, 168, 169,
75, 96, 138, 150, 174, 209, 242 170, 172, 174, 229, 231
antipsychotic, 186 autoantigens, 62, 134, 136, 139, 141, 151, 159, 162,
anxiety, x, 155, 194 163, 170
anxiety disorder, 194 autoimmune disease, vii, ix, x, xi, 1, 2, 6, 9, 18, 24,
aorta, 81, 95 25, 26, 60, 71, 75, 105, 106, 107, 114, 116, 118,
aortic valve, 63 121, 122, 123, 129, 131, 132, 133, 134, 136, 137,
APC, xii, 133, 136, 140, 145, 228, 230, 234 138, 146, 147, 148, 150, 158, 159, 162, 177, 178,
APL, 11, 136, 202 187, 190, 191, 192, 211, 214, 215, 228, 229
apoptosis, 44, 52, 57, 62, 132, 144, 152, 158, 161, autoimmune diseases, ix, xi, 1, 2, 6, 18, 24, 25, 60,
162, 163, 165, 234 71, 75, 123, 131, 132, 133, 134, 138, 146, 147,
apoptotic cells, 62, 102, 107, 122, 123, 128, 129, 148, 150, 158, 159, 162, 178, 187, 190, 191, 211,
163, 173 214, 215, 229
Argentina, 9 autoimmune disorders, ix, xii, 67, 105, 107, 178,
arginine, 82, 97 192, 228, 234
argument, 212 autoimmune hepatitis, 61
arterial hypertension, 50, 72, 73, 74 autoimmunity, ix, 67, 69, 70, 106, 114, 119, 120,
arteriography, 209 125, 129, 132, 133, 137, 138, 139, 146, 147, 148,
arteritis, 53 150, 152, 173
artery(ies), 19, 30, 41, 42, 43, 44, 45, 46, 50, 51, 53, autologous bone marrow transplant, 186, 190
54, 62, 63, 70, 74, 79, 80, 94, 95, 97, 228, 230, autonomic neuropathy, 194
233 autopsy, 31, 33, 79, 205
arthritis, 5, 10, 184, 185, 186, 190, 235 autoreactive T cells, 137, 138, 145, 146, 148
aseptic, 102 autosomal dominant, 224
aspartate, 160, 169, 197 availability, 39
aspirin, 5, 11, 12, 15, 48, 50, 71, 72, 241 avoidance, 86
Index 247
azathioprine, x, xi, 2, 9, 89, 177, 179, 180, 184, 186, blood vessels, 5, 25, 33, 40
211, 215, 218, 223, 225, 226 blood-brain barrier, 166, 175
Azathioprine, 5, 13, 216 bloodstream, 3, 6
azotemia, 48 body composition, 225
body fat, 39, 83
body mass index, 82, 225
B
body weight, 4, 88, 181, 187
bone marrow, x, 132, 177, 181, 183, 185, 187, 190
B cells, ix, 90, 101, 131, 132, 133, 134, 138, 141,
bone marrow transplant, 190
143, 144, 147, 149, 150, 188, 192, 234
bone mass, 87, 99, 100
BAC, 96
bone mineral content, 101
bacteria, vii, 89, 90
bone resorption, 86
bacterial infection, 160
bowel, 186
banking, 13
bradycardia, 11, 65
barriers, 48
brain, xi, 57, 157, 158, 160, 161, 164, 165, 167, 172,
basal ganglia, 200
174, 193, 194, 195, 196, 198, 199, 201, 202, 203,
base pair, 215
204, 205, 206, 207, 208, 209, 210
basement membrane, 117, 121
brain abscess, 194
BBB, 158, 159, 160, 161, 164, 165
brain damage, 160
BD, 70, 101, 172, 208
brain structure, 199
behavior, 39, 170
brain tumor, 198
behavioral disorders, 161
branching, 136
beneficial effect, xii, 5, 18, 71, 84, 87, 88, 98, 145,
breakdown, 123, 161, 205, 230, 231
146, 228
breast feeding, 13
bias, 137
breast milk, 12
bilirubin, 185
breastfeeding, 12, 13, 14
binding, 3, 60, 62, 75, 80, 82, 90, 96, 101, 102, 103,
Brownian motion, 199
133, 136, 138, 141, 142, 148, 149, 151, 157, 160,
buffer, 110, 112
161, 162, 166, 170, 174, 233
bundle branch block, 64
bioavailability, 216, 234
burn, ix, 105, 107, 108, 109, 111, 112, 113, 114,
biological activity, 90
115, 116, 117, 118, 119, 120, 122, 123
biological consequences, 233
biological markers, 84
biomarkers, 68, 145, 212, 214 C
biomolecules, 196
biopsy, 8, 34, 35, 67 calcification, 42, 43, 45, 63, 70, 75, 82, 94
biosynthesis, 230, 231 calcium, 44, 55, 56, 59, 88, 97, 98
biotechnology, 214 calcium channel blocker, 55, 56
birth, 10, 11 California, 131, 146
birth rate(s), 11 Canada, 84
birth weight, 11 cancer, 187, 188, 222
births, 10, 11 candidates, 6, 147
bladder, 186 capillary, 117, 118, 119
bleeding, 184 capsule, 204, 205
blocks, 65, 111, 133 cardiac catheterization, 51, 54
blood, vii, 3, 4, 5, 13, 17, 18, 20, 25, 26, 32, 33, 38, cardiac involvement, vii, 29, 30, 65
39, 40, 41, 46, 50, 58, 83, 84, 108, 158, 166, 175, cardiac myocytes, 34
181, 182, 194, 218, 225, 232, 234 cardiac output, 31
blood flow, 18, 25, 46 cardiac tamponade, 31, 32
blood pressure, 13, 17, 18, 20, 25, 26, 32, 39, 50, 83,
84, 225
248 Index
cardiovascular disease, vii, viii, ix, 29, 30, 37, 41, childbearing, 9
51, 60, 64, 65, 67, 68, 69, 70, 71, 75, 77, 78, 79, childhood, 225
93, 94, 95, 98, 214, 229, 235, 239, 242 children, 11, 13, 43, 64, 65, 158, 164, 168, 189
cardiovascular morbidity, 233 China, 31
cardiovascular risk, vii, 30, 39, 45, 81, 83, 93, 96, Chinese, 99, 100, 102, 173
97, 219, 234, 240 chloroquine, 47, 71, 98
cardiovascular system, 235 cholesterol, xii, 41, 43, 47, 70, 71, 81, 84, 98, 225,
Caribbean, 85, 93, 214 227, 231, 232, 233, 235, 239, 241
Caribbean Islands, 214 chorea, 164
catabolism, 38, 107 choroid, 158
catheter, 50 chronic active hepatitis, 60
Caucasians, 214, 216, 217, 218 chronic autoimmune hepatitis, 60
CD34, x, 178, 181, 183, 185, 187, 191 chronic fatigue syndrome, 60, 75
CD34+, x, 178, 181, 183, 185, 187, 191 chronic illness, 221
CD44, 145, 152 circulation, 17, 18, 19, 25, 26, 41, 46, 65, 160, 163
CD8+, 107, 137, 138, 143, 144, 152, 186 classes, 41
cDNA, 119, 173 classification, 27, 30, 86, 156
CE, 50, 223 claustrophobia, 194
cell, ix, x, 35, 37, 40, 41, 48, 52, 53, 56, 57, 62, 63, clinical assessment, xi, 193, 194, 226
69, 73, 80, 81, 82, 90, 95, 105, 107, 113, 117, clinical diagnosis, xi, 193, 194
121, 122, 123, 125, 126, 128, 129, 131, 132, 133, clinical symptoms, xi, 91, 193, 196, 202
134, 137, 138, 141, 142, 143, 144, 145, 146, 147, clinical trials, 48, 139, 145, 192, 219, 220
148, 149, 150, 151, 152, 155, 158, 159, 163, 166, clone, 123, 238
167, 168, 172, 178, 181, 182, 183, 185, 187, 188, closure, 128
190, 191, 192, 197, 229, 231, 233, 234, 237, 241, clustering, 83
242 CNS, 159, 160, 162, 164, 167, 170, 172, 174, 179,
cell adhesion, 40, 145 180, 181, 183, 185, 189, 194, 196, 202, 208
cell death, 52, 132, 163 CO2, 109, 111
cell growth, 69 coagulation, 4, 38, 232, 233, 240
cell line, 158 coagulation factor(s), 233
cell membranes, 241 coding, 199, 215, 216, 217
cell surface, 56, 125, 163, 167, 229 codon, 90, 217, 218
cell transplantation, 178, 190, 191, 192 coenzyme, 48, 231, 241
cellular adhesion, 40 cognition, 170
cellular immunity, 90 cognitive deficit(s), 156, 207
cellulose, 3, 4, 7 cognitive dysfunction, 160, 164, 170, 194
central nervous system, x, 157, 159, 166, 167, 168, cognitive function, 167, 174
169, 171, 174, 177, 181, 189, 190, 192, 194, 206, cognitive impairment, 156, 160, 164, 170, 201
207, 208, 209, 232 cognitive performance, 157
cerebellum, 199 cohort, vii, 30, 39, 41, 42, 43, 50, 51, 63, 67, 68, 69,
cerebral blood flow, 161 71, 73, 83, 93, 94, 95, 97, 99, 101, 102, 103, 108,
cerebral cortex, 203 114, 124, 158, 159, 161, 163, 168, 169, 171, 205,
cerebral hemorrhage, 194 217, 219, 221, 223, 236
cerebrospinal fluid, 157, 159, 168, 171, 172, 174 collagen, 36, 39, 40, 230, 235
cerebrovascular disease, viii, 37, 38, 77, 79, 93, 163, coma, 189
194, 232 combination therapy, 55, 58
channel blocker, 59 communication, 159
chemokines, xii, 107, 119, 166, 228 community, 89
chemotherapy, 188, 191, 192 complement, ix, 3, 33, 40, 78, 81, 90, 91, 96, 102,
chicken, 140 144, 158, 183, 189
Index 249
complement components, 183 coronary heart disease, 45, 46, 49, 50, 68, 69, 72, 79,
complement inhibitors, 91 81, 93, 94, 97, 232, 235, 237, 239, 241
complement system, 40, 90, 91, 158 corpus callosum, 204, 205, 210
complementarity, 144, 151, 152 correlation(s), ix, 27, 45, 54, 65, 81, 106, 107, 122,
complexity, 2 152, 158, 159, 160, 161, 162, 164, 169, 170, 187,
compliance, 124 194, 201, 202, 224, 231, 239
complications, viii, ix, x, xii, 1, 2, 5, 10, 12, 31, 35, cortex, 204, 205
37, 43, 45, 47, 49, 55, 64, 65, 77, 78, 79, 84, 85, corticosteroid therapy, 67, 84, 87, 185, 229, 236
88, 92, 98, 101, 105, 106, 107, 121, 138, 155, corticosteroids, ix, x, 2, 5, 12, 13, 31, 36, 37, 39, 40,
178, 183, 184, 187, 188, 192, 227 48, 49, 63, 64, 78, 83, 84, 87, 88, 89, 92, 100,
components, 40, 60, 90, 91, 106, 123, 132, 157, 165 101, 138, 178, 179, 180, 184, 186, 189, 190
compounds, 200, 229, 233, 235 cortisol, 12
comprehension, 125 coverage, 134
computed tomography, vii, 30, 42, 43, 45, 46, 47, CPC, 158
174, 194, 236 cranial nerve, 189
concentration, 5, 13, 93, 96, 103, 110, 117, 152, 158, C-reactive protein, xii, 37, 38, 40, 42, 43, 48, 68, 71,
237 81, 91, 95, 103, 228, 230, 237
conception, 9, 11, 14 creatine, 33, 196, 197
concordance, 194 creatinine, 4, 5, 81, 183, 186
conditioning, x, 26, 177, 178, 181, 184, 185, 187, cross-sectional study, 61, 85
188 CRP, 37, 38, 40, 71, 81
conduction, 13, 30, 62, 64, 65 CSF, x, 157, 158, 159, 160, 161, 164, 177, 181, 182,
confusion, 189 185, 187, 194, 204
congestive heart failure, 59 CT scan, 186
Congress, iv, 210 cues, 107
connective tissue, xii, 25, 37, 57, 62, 65, 227, 228, culture, 162
229, 232, 235 curing, 137
consciousness, 164 CVD, 64, 79, 80, 81, 82, 83, 84, 85, 92
consensus, 12, 74, 142, 149, 151, 152, 160, 161, 189, cyanosis, 18
191, 214, 220, 226 cycles, 112, 190
consent, 4 cyclooxygenase, 238, 240
constrictive pericarditis, 31 cyclooxygenase-2, 238, 240
consumers, 49, 213 cyclophosphamide, xi, 2, 5, 8, 9, 36, 57, 72, 84, 89,
consumption, 90 101, 138, 146, 159, 182, 211, 215, 218, 223
contact dermatitis, 237 cyclosporin, xi, 180, 186, 211, 215, 224
control, ix, xii, 6, 10, 12, 13, 17, 18, 19, 21, 22, 23, cyclosporine, 9, 186, 218, 224, 225
24, 25, 26, 61, 65, 68, 82, 84, 90, 102, 103, 105, cystitis, 182
108, 112, 113, 114, 115, 117, 120, 121, 122, 125, cytochrome, 217, 218, 224
131, 132, 133, 137, 139, 142, 144, 146, 148, 149, cytokines, ix, xii, 36, 37, 40, 44, 48, 53, 82, 83, 86,
157, 187, 189, 197, 227, 228, 231, 235 93, 105, 107, 119, 122, 123, 125, 126, 128, 129,
control group, 10, 17, 18, 19, 21, 22, 23, 24, 25, 26, 133, 135, 136, 137, 143, 144, 145, 146, 150, 152,
61, 108 162, 166, 228, 229, 231, 232, 235, 237, 238, 239
controlled studies, 3, 5, 12, 49, 50, 57 cytokinesis, 163, 173
controlled trials, viii, 6, 30, 36, 48, 50, 57, 58, 59, 65 cytomegalovirus, 192
conversion, 233 cytoplasm, 119, 162
cooling, 232 cytoskeleton, 157
coronary angioplasty, 47, 50 cytotoxicity, 42, 69, 158
coronary arteries, 43, 45, 62
coronary artery disease, viii, 37, 40, 42, 43, 47, 69,
71, 77, 93, 98, 231, 233, 236, 238, 240
250 Index
diffusion, xi, 164, 193, 194, 196, 198, 199, 201, 202,
D
204, 208, 209, 210
diffusion-weighted imaging, 209
danger, 122, 123, 147
diffusivity, 198, 199, 203, 204, 205, 208, 210
data analysis, 98
digestion, 44
database, 49, 178
dilated cardiomyopathy, 35, 67
death(s) ix, 5, 11, 37, 41, 43, 47, 48, 49, 78, 79, 89,
dilation, 18, 25
92, 107, 109, 114, 132, 184, 232
diluent, 111
decision making, 48
dimethylarginine, viii, 77, 82, 96, 97
decisions, 218, 219
directionality, 198, 199
defects, 18, 64, 89, 90, 91, 122, 129, 163
discordance, 65
deficiency, 86, 90, 91, 216, 218
discrimination, 61
definition, 10, 83, 159
disease activity, x, xi, 3, 4, 25, 31, 36, 38, 49, 59, 67,
deformation, 25
78, 81, 82, 84, 86, 89, 95, 97, 100, 102, 126, 160,
degenerate, 149
162, 177, 178, 183, 184, 185, 187, 189, 191, 212,
degradation, 52
214, 219, 226, 231, 235, 239
dehydroepiandrosterone sulphate, 87, 98
disease progression, x, xi, 117, 122, 123, 177, 178,
delivery, 10, 12, 13, 135, 137, 139
193, 196
dementia, 158
disease-free survival, 185
demyelinating disease, 147
disinfection, 109
demyelination, 202, 203, 205
dislocation, 108, 109, 111
dendritic cell, 133, 136, 148
disorder, vii, xii, 1, 4, 30, 78, 136, 162, 185, 194,
density, viii, 38, 41, 67, 78, 81, 85, 94, 95, 96, 97,
227, 228
98, 99, 100, 233, 239
dispersion, 65, 75
Department of Defense, 124
disposition, 213, 215, 222
depolarization, 165
dissociation, 84
deposition, 62, 63, 81, 91, 106, 111, 117, 118, 119,
distribution, 44, 83, 127, 148, 212, 214, 217, 236
121, 123, 144, 169
diversity, 139, 224
deposits, 33, 35, 44, 118, 119, 127
DNA, x, 2, 3, 4, 5, 6, 7, 11, 91, 97, 106, 107, 110,
depression, 18, 25, 158, 161, 162, 164, 186, 189
116, 118, 128, 138, 139, 141, 142, 143, 144, 145,
depressive symptoms, x, 155
149, 151, 152, 153, 155, 160, 161, 169, 182, 183,
dermatitis, 113, 186
185, 189
dermatomyositis, 25
donors, 121
dermis, 113, 120
Doppler, vii, 30, 34, 46, 51, 54, 57, 58, 59, 71, 73,
desensitization, 137
75, 186
destruction, 132
dosage, 49, 135
detection, 35, 43, 51, 61, 65, 70, 94, 103, 111, 158,
dosing, 212, 223
159, 185, 196, 198
down-regulation, 126, 144, 152
deviation, 150
drainage, 32
diabetes, 12, 18, 46, 47, 49, 50, 72, 80, 83, 84, 138,
drug interaction, 215
150
drug metabolism, 218, 223
diabetes mellitus, 12, 46, 47, 49, 50, 80, 83
drug reactions, xi, 211, 212
diagnostic criteria, 162
drug resistance, 216, 217, 218
dialysis, 184
drug safety, 218
diastole, 58
drug targets, 215, 222
diastolic blood pressure, 19
drug therapy, 89
diastolic dysfunction, viii, 30, 58, 59, 74
drug treatment, xi, 1, 211, 212, 213, 214, 219, 220
diastolic pressure, 19, 58
drugs, x, xi, xii, 2, 5, 12, 13, 36, 40, 47, 48, 49, 50,
diet, 88, 182
56, 57, 64, 66, 71, 72, 84, 87, 89, 98, 102, 106,
differentiation, 122, 149
Index 251
138, 155, 178, 183, 184, 187, 211, 212, 214, 215, enterocolitis, 185
216, 218, 222, 228, 231, 233, 240 enthusiasm, 214
dry ice, 111 environment, 136, 203, 219
ductus arteriosus, 12 environmental factors, 52, 91, 106, 132, 214
duration, 6, 37, 45, 59, 65, 70, 78, 81, 86, 89, 99, enzyme(s), xii, 36, 38, 49, 59, 61, 72, 82, 182, 183,
133, 135, 152, 186, 190 215, 216, 217, 218, 224, 227, 230, 231, 233, 241
duties, 124 enzyme inhibitors, 36, 49
DWI, xi, 193, 196, 198, 199, 203, 205 enzyme-linked immunoassay, 61
dyslipidemia, 38, 67, 96 eosinophilia, 35
dyspnea, 31, 33, 51, 58, 186 epidemiology, 124, 222
dysthymic disorder, 162 epidermal ulcers, ix, 106
epidermis, 113, 120
epilepsy, 201
E
epinephrine, 161
epithelial cells, 238
EAE, 137
Epstein-Barr virus, 139
ears, 108, 109
equilibrium, 132
echocardiogram, 12, 55
equipment, 55
edema, 11, 35, 194
erythrocyte sedimentation rate, 83, 183
education, 178, 214
erythrocytes, 167
Education, 83
Escherichia coli, 69
effusion, 31
ESR, 182, 183
eigenvalue, 199
estradiol, 86
eigenvector, 199
estrogen, 87
electrocautery, 109
ethanol, 113
electrolyte, 64, 189
ethanolamine, 163, 165
electron, vii, 30, 42, 43, 45, 47
ethics, xii, 184, 212, 213, 220
ELISA, 110, 116, 117, 118, 159, 164, 168, 171, 182,
ethnic background, xi, 85, 212, 219
183
ethnic groups, 171, 218, 221, 222
ELISA method, 164
ethnicity, 214, 215, 221
embolism, 64
etiology, ix, 59, 105, 107, 121, 202, 203
emission, 45, 174, 195, 236
EU, 1
emotion(s) 25, 170, 205
Europe, 87, 214
emotional disorder, 161
euthanasia, 108, 109, 115
employees, 124
evolution, 3, 55, 137, 232
encephalomyelitis, 135, 242
examinations, 205
encephalopathy, 164
exclusion, x, 35, 86, 155, 157, 194
encoding, 91, 138, 149
exercise, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
endocarditis, 63, 64, 75
50, 51, 57
endocardium, 30
expenditures, 213
endothelial cells, 41, 52, 62, 63, 73, 80, 81, 82, 95,
experimental allergic encephalomyelitis, 149
96, 117, 162, 172, 230, 233, 238, 240, 242
experimental autoimmune encephalomyelitis, 137,
endothelial dysfunction, 40, 79, 82, 93, 96, 162, 234
147, 148
endothelin receptor antagonists., viii, 30
expertise, 213, 219
endothelium, xii, 39, 41, 42, 52, 63, 80, 165, 166,
exposure, xi, 83, 86, 87, 106, 123, 125, 161, 212,
172, 228, 229, 230, 232, 234, 235, 237, 238
219, 224
end-stage renal disease, 96, 217, 223
extracellular matrix, 37
energy, 100, 158
engagement, 132, 144
England, 168
enlargement, 53
252 Index
gene(s), 60, 75, 91, 102, 103, 106, 108, 112, 119,
F
120, 123, 126, 127, 134, 137, 149, 168, 172, 213,
215, 217, 218, 224, 225, 226, 238, 240, 242
fabric, 123
gene expression, 112, 120, 126, 127, 225
failure, viii, 3, 9, 30, 35, 49, 54, 55, 58, 59, 74, 86,
gene promoter, 224
87, 123, 126, 184, 185, 217, 218
gene therapy, 137
family, 85, 125, 144, 163, 217, 231
generalized seizures, 201
family history, 85, 125
generation, 38, 136, 141, 233, 240, 241
fasting, 71
genetic factors, 218
fat, 113
genetic information, 213
fatigue, 2, 18, 24, 26, 51, 60, 64, 167
genetics, 102, 103, 213, 220
feet, 19
genital herpes, 186
females, viii, 4, 78, 106, 181
genome, 213, 214, 215
fertility, 13, 189
genomics, 220
fetal abnormalities, 88
genotype, 103, 213, 215, 217, 224
fetus, 12
gestation, 11, 12, 186
fever, 33, 35, 64, 182, 184, 185, 186, 187
glass, 111
fibers, 34, 36, 163, 199, 201, 205
glatiramer acetate, 137
fibrin, 38, 63, 107
glomerulonephritis, ix, 6, 8, 106, 107, 117, 118, 119,
fibrinogen, 35, 95, 232, 240
122, 123, 128, 135, 141, 142, 146, 181, 184
fibrinolysis, 240
glucocorticoid receptor, 225
fibroblasts, 73, 238
glucocorticoids, xi, 211, 215, 219, 225
fibromyalgia, 106
glucose, 39, 71, 83, 84
fibrosis, 33, 35, 36, 53, 59
glucose metabolism, 83
fibrous cap, 37, 232
glutamate, 160, 169, 196, 197
fibrous tissue, 63
glutamic acid, 138, 150
filtration, 182
glutathione, 217, 218, 223, 225
fitness, 25
glycerol, 163
fluctuations, 159
glycine, 82, 97
fluid, 3, 31, 34, 167, 195, 199, 230
glycolysis, 158
focal seizure, 164
glycoprotein, 37, 41, 62, 69, 80, 94, 96, 150, 159,
focusing, 219
216, 224
folic acid, 39, 84
gold, 34, 35, 43, 53, 55, 205
follicle, 12
grants, 146
Ford, 209, 239
grey matter, 201, 202, 203, 204
fractures, viii, 78, 79, 85, 87, 88, 92, 98, 99, 101
groups, 17, 19, 21, 22, 23, 24, 25, 26, 37, 57, 60, 63,
fragility, 99
84, 113, 136, 162, 204, 215
frontal lobe, 204
growth, 72, 107, 119, 125, 233
functional approach, 205
growth factor, 72, 107, 119, 125
fungal infection, 91
growth factors, 107, 119, 125
fusion, 171, 213
guanine, 226
guidelines, 67, 242
G
gadolinium, 205 H
gallium, 33, 34
haemoglobin, 185
gamma globulin, 5
hair follicle, 113
gastrointestinal bleeding, 184
hair loss, 10
half-life, 84, 135
hallucinations, 164
Index 253
hands, 19 humidity, 108

harm, 84, 132 hybrid, 128
harvesting, 191 hydrocortisone, 13, 36
Hawaii, 222 hydrogen, 110, 207
HDL, 38, 46, 47, 81, 82, 231 hydrogen peroxide, 110
headache, 156, 163, 164, 186, 194 hyperactivity, 127
healing, 107, 123, 125, 214 hypercholesterolemia, viii, xii, 46, 47, 68, 71, 77, 80,
health, ix, xi, 18, 19, 26, 48, 78, 98, 105, 106, 124, 84, 96, 98, 227, 239, 241
125, 137, 212, 214, 219, 220, 222, 223 hypergammaglobulinemia, ix, 106, 107, 121
health care, 19, 125 hyperhomocysteinemia, 39
health problems, ix, 105, 106 hyperlipidemia, 39, 241
health status, 48 hyperparathyroidism, 86
heart block, 11, 35, 65 hyperplasia, 31
heart disease, 49, 50, 235, 236 hyperprolactinemia, 87
heart failure, 35, 50, 51, 58, 59, 74, 75, 79 hyperreactivity, 136
heart rate, 58 hypersensitivity, 217, 219, 237
heart,, vii, 181 hypertension, vii, viii, 10, 11, 12, 18, 30, 38, 49, 52,
heat, 37, 69, 70, 138, 150 54, 55, 57, 63, 71, 72, 73, 77, 80, 83, 84, 98, 189
heat shock protein, 37, 69 hypertrophy, 50, 53, 59, 236
height, 87 hyperuricemia, 11
hematopoiesis, xi, 178, 183, 184, 185, 187 hyponatremia, 208
hematopoietic stem cells, x, 177, 181 hypothesis, 42, 53, 61, 69, 81, 82, 123, 157, 205, 222
hematuria, 11, 146
hemoglobin, 182
I
hemorrhage, xi, 5, 184, 193, 194
hemostasis, 173
iatrogenic, x, 155
hepatic encephalopathy, 208
ibuprofen, 5, 12
hepatitis, 161
ICAM, 230, 237
hepatocytes, 233
identification, 84, 134, 138
hepatomegaly, 51
idiopathic, 53, 57, 73, 74
herpes, 102, 125
IFN, 40, 122, 126, 133, 141, 145, 235
herpes zoster, 102, 125
IFNγ, 107
high blood pressure, 38
IL-6, ix, 53, 81, 86, 97, 106, 107, 120, 122, 231, 235,
hip, 86, 88
239, 242
hippocampus, 160
IL-8, 97, 122, 235, 242
histogram, 204, 209
illumination, 199
histone, 106, 135, 140, 141, 145
images, 66, 195, 199, 202, 208
histopathology, 44
imaging, xi, 33, 34, 35, 46, 55, 66, 73, 157, 183, 193,
HLA, 134, 141, 147, 159, 168, 172, 221, 230
194, 195, 196, 198, 199, 200, 202, 205, 206, 207,
homeostasis, 107, 132, 137, 241
208, 210, 236
homocysteine, viii, 37, 39, 45, 50, 64, 68, 77, 83, 84,
imaging modalities, 195
97
imaging techniques, 33
Hong Kong, 102
imbalances, 122
hormone, 13, 26
immune complex deposition, ix
hospitalization, 49
immune disorders, 4
hospitals, 61, 213
immune function, 89, 122
host, 90, 107, 132, 134, 202, 238
immune reaction, 69
HSCT, 187
immune regulation, 69, 138, 149
human brain, 208, 209
immune response, 48, 106, 107, 122, 126, 129, 132,
human genome, 215, 223
136, 139, 146, 149, 151, 229, 238
254 Index
immune system, vii, xii, 2, 5, 89, 103, 132, 134, 146, inflammation, vii, xii, 2, 5, 33, 34, 35, 36, 37, 38, 39,
163, 178, 228 40, 42, 44, 49, 63, 68, 69, 71, 81, 83, 86, 87, 95,
immunity, 3, 68, 107, 123, 126, 146, 147, 169, 210, 107, 117, 119, 126, 132, 137, 201, 205, 227, 229,
231 231, 232, 235, 237, 239
immunization, 103, 139 inflammatory arthritis, 242, 243
immunocompetent cells, 26, 229 inflammatory cells, 33, 40, 44, 53, 63, 107
immunocompromised, 107 inflammatory disease, 83, 229, 236
immunogenicity, 135, 163 inflammatory mediators, 229
immunoglobulin, 35, 41, 48, 90, 128, 149, 185 inflammatory response, 81, 91, 107, 119, 230
immunoglobulins, 3, 6 inflammatory responses, 107
immunomodulation, 150, 152 information technology, 214
immunomodulator, 242 informed consent, 181
immunomodulatory, 48, 103, 234, 235 infrastructure, xi, 212, 213, 219, 220
immunopathogenesis, 238 inguinal, 109, 115
immunosuppression, 127, 178, 185, 186, 188, 223 inheritance, 215
immunosuppressive agent, ix, 1, 78, 89, 91 inhibition, xii, 38, 74, 81, 82, 84, 152, 165, 169, 228,
immunosuppressive drugs, xi, 2, 3, 6, 15, 32, 36, 55, 231, 232, 233, 234, 235, 240, 242
57, 64, 89, 91, 211, 215 inhibitor, viii, 40, 72, 77, 82, 112, 235, 240, 242
immunosuppressive therapies, 53, 192 inhibitory effect, 164, 240
immunotherapy, 106, 133, 135, 139, 142, 145, 146, initiation, 40, 107, 125, 162, 218
147, 148, 150, 192 injections, 136, 138, 144, 148
implementation, xi, 8, 211, 213 injuries, xi, 193, 194
in situ, 53, 160, 240 inositol, 163, 197, 208
in situ hybridization, 240 insight, 80, 205
in vitro, 8, 40, 65, 82, 126, 129, 139, 141, 142, 144, instability, 38, 40, 68, 230, 238
152, 158, 160, 172, 187 institutions, 19, 214
in vivo, 62, 91, 103, 128, 143, 144, 147, 149, 186, instruction, 148
196, 209, 224, 225, 233, 238, 242 instruments, 219
incidence, 6, 7, 9, 37, 43, 45, 55, 59, 87, 89, 93, 97, insulin, viii, 41, 77, 83, 84, 97, 98, 225
107, 113, 114, 115, 122, 138, 158, 160, 206, 214, insulin resistance, viii, 77, 83, 84, 97
221, 224, 228, 236 insulin sensitivity, 41, 84
inclusion, 48, 181, 200 integration, 213, 220
India, 102 integrity, 46, 112, 120, 157, 160, 161, 165, 203, 230
indication, 189 intellect, 186
indicators, 189 intensity, 6, 18, 25, 26, 35, 70
indices, x, 58, 177 interaction(s), ix, 37, 94, 97, 131, 132, 134, 141,
indirect effect, 215 145, 151,159, 233, 234, 237, 240, 241
indium, 33, 34 intercellular adhesion molecule, 38, 237
individualization, 223 interference, 144
indomethacin, 12 interferon, 5, 53, 127, 133, 138, 147, 230, 232, 234,
inducer, 146 237, 238, 242
induction, 69, 87, 106, 107, 122, 128, 134, 135, 138, interferon (IFN), 133, 138
141, 147, 163, 165, 190 interferon gamma, 53, 230, 232, 234
infants, 12 Interleukin-1, 127, 128
infarction, 35, 41, 43, 44, 47, 48, 79 interleukin-8, 237
infection, ix, 42, 69, 70, 78, 89, 90, 91, 101, 102, interleukin-beta, ix, 106
103, 105, 107, 109, 126, 138, 161, 186, 187, 188, interstitial lung disease, 27
214, 228 interval, 4, 65, 113, 200, 224
infertility, 12 intervention, 3, 85, 92, 138, 139
intervention strategies, 85, 92
Index 255
intima, 46, 80, 82 limbic system, 205

intrauterine growth retardation, 10, 11 limitation, 45, 56
intravenously, 160, 181, 182 lipase, 38, 40, 67, 81, 82, 95, 96
inversion, 195, 199 lipid metabolism, 48
inversion recovery, 195, 199 lipids, 37, 44, 48, 50, 68, 71, 97, 98, 196, 198, 202
iodine, 109 lipoproteins, 41, 94, 232, 233
irradiation, 190 lipoxygenase, 52
ischemia, 198, 202 listening, 17, 18
isotope, 236 liver, 11, 40, 60, 185, 189, 233
Italy, 1, 155 liver disease, 60
liver enzymes, 11
location, 35, 142, 205
J
locus, 226
longitudinal study, 103, 172
Japan, 3, 4, 17, 18, 19
Los Angeles, 131
joint pain, 5
lovastatin, 241
joints, vii, 2
low-density lipoprotein, 8, 37, 69, 71, 80, 94, 232
LPS, 122, 160
K lumbar spine, 86, 100
lumen, 44, 46, 52
keratinocytes, 107 lung, x, 25, 53, 73, 177, 181, 183, 186, 189
kidney(s), vii, 86, 108, 111, 117, 118, 119, 123, 127, lung function, 25
144, 147, 160, 189 lungs, vii, 52
kinetics, 122 lupus, iv, vii, viii, ix, x, xi, xii, 2, 5, 6, 7, 8, 9, 10, 11,
14, 15, 17, 18, 26, 27, 30, 31, 33, 35, 38, 41, 42,
L 45, 48, 51, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69,
70, 71, 73, 75, 77, 78, 80, 81, 82, 83, 85, 86, 88,
labeling, 62 89, 90, 91, 93, 94, 95, 97, 98, 102, 105, 106, 107,
lack of control, 32, 39, 48 108, 113, 114, 115, 116, 117, 118, 119, 121, 122,
lactate level, 202 123, 124, 125, 127, 128, 129, 131, 135, 136, 138,
lactation, 13 139, 140, 141, 142, 143, 144, 145, 146, 147, 148,
LCA, 159 149, 150, 151, 152, 153, 161, 163, 164, 167, 168,
LDL, 37, 40, 41, 43, 47, 48, 69, 80, 81, 82, 94, 229, 169, 170, 171, 172, 173, 174, 177, 181, 183, 184,
231, 233, 235 185, 186, 187, 188, 189, 190, 191, 192, 194, 206,
leakage, 123, 158, 164 207, 208, 209, 210, 211, 212, 214, 215, 216, 218,
learning, 160, 169 219, 220, 221, 222, 223, 227, 228, 230, 231, 234,
left ventricle, 58 235, 236, 238, 242
lesions, vii, ix, xii, 30, 37, 42, 52, 53, 63, 80, 105, lupus anticoagulant, 41, 60, 61, 62, 63, 64, 69, 75,
107, 108, 109, 113, 114, 115, 120, 121, 128, 194, 80, 94, 174, 183
196, 201, 203, 205, 206, 207, 227, 228, 229, 231 lupus erythematosus, iv, ix, xi, xii, 7, 9, 14, 27, 68,
lethargy, 189 78, 93, 95, 102, 105, 124, 125, 167, 168, 170,
leukemia, 190 171, 174, 194, 206, 207, 209, 211, 212, 216, 221,
leukocytes, 81, 107, 185 222, 227, 228, 231, 235, 236
leukocytosis, 91 lymph, ix, 106, 107, 109, 113, 115, 116, 127, 135,
LFA, 145, 152 136, 140
life span, xii, 189, 227, 228, 232 lymph node, 109, 115, 116, 127, 135, 136, 140
lifestyle, 18, 80, 84, 92 lymphadenopathy, ix, 106, 107, 113
lifetime, 98 lymphocytes, 33, 37, 40, 53, 106, 122, 132, 144,
ligand(s), 123, 128, 133, 146, 149, 159 152, 159, 167, 186, 187
likelihood, 217 lymphoma, 188
256 Index
lymphoproliferative mutation., ix membranes, 2, 117, 157

lysine, 136 membranoproliferative glomerulonephritis, 127
lysis, 137 membranous nephropathy, 127
memory, 160, 169, 185, 186, 188, 210
men, 13, 18, 41, 48, 49, 69, 85, 94, 95, 96, 99, 100,
M
107, 236, 237, 239, 241
meningitis, 194
macrophages, 37, 40, 41, 44, 80, 94, 107, 122, 128,
menopause, 81
129, 230, 231, 232, 233, 238, 240
mental status change, 189
magnetic field, 196
mental status changes, 189
magnetic resonance, 33, 35, 57, 66, 161, 168, 194,
messenger RNA, 173
196, 197, 198, 206, 207, 208, 209
meta-analysis, 90, 103, 239
magnetic resonance imaging, 33, 57, 66, 161, 168,
metabolic changes, 84
194, 207, 209
metabolic syndrome, viii, 77, 81, 83, 96, 97, 217,
magnetic resonance scanning, 206
219
magnetic resonance spectroscopy, 197, 198, 207,
metabolism, 39, 82, 99, 100, 101, 198, 202, 212
208, 209
metabolites, 196, 198, 218
magnetization, 195, 203, 208
metabolizing, xi, 211, 215, 224
magnetization transfer imaging, 195, 203, 208
metalloproteinase, 238
major depressive disorder, 162
methionine, 39
major histocompatibility complex, xii, 132, 149,
methylation, 82, 97
228, 238, 242
methylprednisolone, viii, 31, 36, 78, 83, 180, 184,
males, 106
185
mammalian cells, 163
Mexico, 29, 31, 33, 34, 36, 52, 66
management, ix, 2, 3, 6, 7, 25, 47, 56, 67, 131, 137,
MHC, xii, 132, 134, 136, 137, 138, 139, 140, 143,
214, 228, 239, 242
148, 150, 228, 230, 234, 238
mania, 189
MHC class II molecules, 140, 230, 234
manipulation, 196
mice, ix, 65, 105, 107, 108, 109, 112, 113, 114, 115,
manners, 194
116, 117, 118, 119, 120, 121, 122, 123, 127, 128,
manufacturer, 182, 183
129, 134, 135, 136, 137, 138, 139, 140, 141, 142,
mapping, 209
143, 144, 145, 147, 148, 150, 151, 152, 153, 160,
market, 213
161, 164, 234, 242
marrow, 122, 128, 181, 184, 190, 191, 192
microfabrication, 213
Maryland, 27, 169
micrograms, 135
mass spectrometry, 141
microorganism(s), 42, 90
mast cells, 107
microscope, 109
matrix, 40, 111, 117, 238
microscopy, 62, 111
matrix metalloproteinase, 40, 238
Microsoft, 112
MBP, 135, 137
microtubules, 157, 173
MCP, 40, 81, 230
migraine, 158
MCP-1, 40, 81, 230
migration, 38, 41, 232, 237
measurement, 25, 26, 46, 51, 54, 55, 88, 91, 103, 196
mild cognitive impairment, 202
measures, viii, xi, 30, 36, 43, 47, 49, 54, 57, 86, 91,
military, 107, 123, 124
198, 199, 212, 219
milligrams, 135
mechanical ventilation, 32
minority, 214, 221
media, 46, 80, 82
miscarriage, 10, 15
median, xi, 114, 178, 183, 185
mitogen, 241
medication, 48, 80, 84, 86, 87, 88, 109, 185, 188,
mitral regurgitation, viii, 30, 63
213
mitral valve, viii, 30, 63
medicine, xi, 211, 212, 213, 214, 215, 220, 221, 222,
MMP, 230
226, 241
Index 257
MMP-2, 230 myasthenia gravis, 136, 138, 147, 148, 150, 191, 194
MMP-9, 230 mycobacterial infection, 157
MMPs, 230, 231 mycophenolate mofetil, xi, 133, 138, 211, 215, 218,
mobility, 85, 198, 199 223
models, ix, x, 42, 48, 105, 126, 131, 135, 139, 142, myelin, 135, 137, 147, 203, 205
144, 151, 164, 203, 234 myelin basic protein, 135, 137, 147
molecular mechanisms, vii, 30, 68, 72, 149 myocardial infarction, 35, 37, 41, 43, 44, 47, 48, 49,
molecular mimicry, 125 66, 69, 72, 79, 80, 93, 94, 228, 232, 236, 237, 239
molecular structure, 143 myocarditis, 31, 33, 34, 35, 36, 64, 66, 67
molecular weight, 11, 233 myocardium, 30, 33, 36, 62
molecules, xii, 40, 41, 53, 80, 90, 125, 133, 136, myocyte, 34, 35, 241
137, 143, 144, 145, 148, 152, 163, 165, 166, 198, myosin, 34, 234, 241
199, 203, 205, 228, 230, 237
monoclonal antibody, 34, 128, 142, 147, 152, 160,
N
188, 192
monocyte chemoattractant protein, 81, 96, 230
NAD, 234
monocyte chemotactic protein, 40, 237
National Institutes of Health, 146
monocytes, 41, 231, 232, 233, 235
National Research Council, 124
mononuclear cells, 34, 63, 83, 97, 233
natural killer cell, 90
monotherapy, 58, 240
necrosis, 26, 33, 34, 63, 67, 95, 102, 111, 113, 119,
mood, x, 155, 156, 162, 172, 194
158, 198, 239
mood disorder, x, 155, 156, 162, 172, 194
neovascularization, 38
morbidity, vii, viii, xi, 30, 65, 78, 79, 89, 91, 92, 93,
nephritis, x, xii, 1, 2, 4, 5, 6, 7, 8, 10, 11, 15, 82, 89,
102, 189, 194, 211, 212, 232
95, 102, 127, 128, 138, 141, 142, 144, 146, 147,
morphology, 44, 96, 231
149, 150, 161, 162, 170, 172, 177, 183, 184, 185,
mortality, vii, viii, xi, xii, 5, 7, 11, 29, 30, 37, 42, 44,
186, 190, 218, 223, 227
49, 51, 55, 65, 67, 69, 78, 79, 82, 83, 85, 89, 91,
nephrologist, 15
92, 96, 99, 109, 138, 178, 184, 186, 187, 188,
nephropathy, 33, 228, 234
189, 191, 194, 211, 212, 214, 220, 221, 227, 228,
nephrotic syndrome, x, 8, 177, 184
232, 233, 236
nervous system, vii, 25, 102, 163, 164, 166, 167,
mortality rate, 78, 85, 187, 214
168, 189, 192, 206
mothers, 11, 64, 65
Netherlands, 77
motion, 24, 34, 46, 199, 203
network, 132, 159, 178, 238
mouse model, 126, 128, 161, 164, 174, 205
networking, xi, 212
movement, 159, 194, 205
neuroblastoma, 167
MRI, xi, 35, 66, 174, 193, 194, 196, 202, 203, 205,
neurofibrillary tangles, 163, 173
206, 207, 209
neurofilament, 158
mRNA, ix, 106, 118, 127
neuroimaging, 157, 194
MTI, 195
neuroimaging techniques, 157
multiple sclerosis, 137, 149, 158
neurological deficit, 202
multiplicity, 48
neurological disease, 163
murmur, 33, 64
neuronal apoptosis, 160, 161
muscle cells, 37
neuronal cells, 167
muscle mass, 57
neuronal death, 165
muscle strength, 24
neurons, 157, 160, 161, 166, 197, 205
muscles, 2
neuropathy, 158, 164, 194
musculoskeletal pain, 106
neuroscience, 208
musculoskeletal system, 79
neurotransmission, 165
mutant, 226
neurotransmitter(s), 160, 165, 173, 197
mutations, 52, 91, 216, 105, 107, 217, 218, 224
neutropenia, 187
258 Index
neutrophils, 122
P
New York, iii, iv, 56
New Zealand, 129, 152
pain, 2, 19, 24, 26, 31, 33, 51, 109, 167
nicotine, 161
palpitations, 33
nitrates, 59
paralysis, 242
nitric oxide, viii, 39, 40, 52, 77, 82, 127, 230, 237,
parameter, 116, 183, 198
241
parenchyma, 203, 205
nitric oxide synthase, 40, 127, 230, 237, 241
parents, 184
nitrogen, 4, 127
paresis, 19
NK cells, 122
paroxysmal nocturnal dyspnea, 33
NMDA receptors, 160, 161, 170
particles, 41, 151, 160, 173
NMR, 209
PAS stain, 117
nodes, 109, 115
pathogenesis, viii, ix, x, xi, 38, 40, 52, 53, 66, 68, 77,
nodules, viii, 30, 63
78, 79, 80, 81, 86, 89, 90, 91, 97, 105, 108, 125,
noninvasive tests, 70
127, 132, 136, 139, 148, 152, 155, 157, 158, 162,
normal development, 173
163, 165, 193, 196, 205, 229, 232, 237, 238, 240
NSAIDs, 2, 49
pathogens, 90, 132
nucleosome, 141, 151
pathologist, 51
nucleosomes, 139, 141, 151, 162
pathology, 66, 109, 123, 134, 137, 160, 206
nucleus, 162
pathophysiology, 202
nutrition, 222
pathways, 127, 133, 134, 213, 240, 241
PCR, 108, 112, 126
O pediatric patients, 43, 171
peptides, x, 38, 131, 133, 134, 135, 136, 137, 138,
obese patients, 225 139, 140, 141, 143, 145, 146, 147, 148, 150, 151,
obesity, 47, 50, 80, 83 152, 153
observations, 5, 39, 158, 228 perception, 213
obstruction, 43, 44 perfusion, 66, 70, 79, 207
occlusion, 44, 80 pericardial effusion, 31
oedema, 33, 51, 58 pericardial rub, 31
optic neuritis, 164, 189 pericarditis, 31, 32, 64, 184
orbitofrontal cortex, 204, 205 pericardium, 30, 31, 62
organ, viii, ix, x, 2, 5, 10, 33, 77, 78, 79, 85, 86, 97, peripheral blood, x, 18, 26, 139, 141, 144, 152, 177,
99, 105, 106, 107, 108, 126, 131, 132, 133, 138, 181, 183, 190, 191, 226
181, 183, 187, 188, 189, 212, 214, 219, 223 peripheral blood mononuclear cell, 139
organization, 214 peripheral nervous system, 156, 194
orientation, 209 peripheral neuropathy, 2, 158, 189
orthopnea, 33 peripheral vascular disease, viii, 77, 79, 172
ossification, 101 permeability, 166, 232
osteoarthritis, ix, 61, 105, 106 Persian Gulf, 125
osteoporosis, viii, ix, 18, 77, 78, 79, 85, 86, 87, 88, Persian Gulf War, 125
92, 99, 101, 105, 106 pertussis, 127
outpatients, 18, 51, 101 PET, 45, 195
overload, 54, 163 PGE, 120, 230
overproduction, 234 pH, 108, 112
ovulation, 12 phage, 160
oxidation, 41, 86, 232, 240 phagocytosis, 123, 128, 129
oxidative stress, 38, 82, 96 pharmaceuticals, 213
pharmacogenetics, 213, 214, 223
Index 259
pharmacogenomics, xi, 211, 213, 214, 219, 220, 222, polymorphisms, 52, 60, 75, 91, 103, 213, 215, 217,
223, 226 218, 219, 223, 224, 225, 226
pharmacokinetics, 212, 215, 216, 218, 222, 223, 224, polypeptide(s), 60, 159, 160
225 poor, 1, 4, 6, 54, 189, 201
pharmacological treatment, 2, 4, 5 population, viii, xi, 2, 43, 46, 48, 51, 63, 74, 78, 79,
pharmacotherapy, 212, 213 80, 81, 82, 83, 85, 87, 88, 92, 99, 101, 102, 103,
phenotype(s), 70, 113, 123, 126, 128, 133, 172, 213, 106, 134, 148, 165, 167, 206, 211, 212, 214, 216,
219, 230, 235, 242 218, 219, 224, 228, 235, 236
phosphocreatine, 196 positive correlation, 41, 231
phosphodiesterase inhibitors, viii, 30 positive relation, 87
phospholipids, 163 positive relationship, 87
phosphorylation, 234 positron, 70, 195, 207
photomicrographs, 117, 119 positron emission tomography, 70, 195, 207
photosensitivity, 184 postmenopausal women, 48, 88, 99
physical activity, 84, 86, 88 precipitation, 3
physical exercise, 27 prediction, 95, 231
pilot study, 26, 95, 164 predictors, 10, 39, 40, 45, 68, 95, 99, 101, 147
pituitary gland, 26 prednisolone, x, 177, 182, 183, 184, 185, 186
placebo, 48, 57, 74, 235, 239, 242 prednisone, 5, 10, 31, 36, 39, 43, 47, 64, 98, 102
placenta, 11, 12, 13 preeclampsia, 11
planning, 88 pregnancy, 7, 8, 9, 10, 11, 12, 13, 14, 15, 88, 189,
plaque, 38, 40, 42, 44, 45, 68, 70, 84, 94, 96, 230, 192
232, 238, 240 premature death, ix, 106, 115
plasma, 1, 3, 4, 5, 7, 8, 14, 27, 33, 57, 62, 71, 82, 83, prematurity, 10
86, 96, 97, 158, 239 premenopausal, 87, 88, 98, 99, 100
plasma cells, 33 premenopausal women, 98, 99, 100
plasma levels, 14, 82, 86, 96, 97 pressure, viii, 17, 18, 19, 25, 30, 31, 33, 50, 51, 53,
plasma membrane, 62, 158 54, 55, 57
plasmapheresis, 1, 3, 4, 5, 6, 7, 8 preterm delivery, 10
plasmid, 138 prevention, 18, 45, 46, 47, 48, 49, 70, 71, 84, 88, 98,
plasminogen, 40 123, 141, 182, 192, 231, 232, 233, 235, 240, 241,
platelet activating factor, 163 242
platelet aggregation, 63, 107, 232, 240 privacy, 213
platelet count, x, 178, 182, 183, 186 private practice, 38
platelets, 11, 107, 182, 185, 232, 233 probability, 41, 185, 218
plexus, 158 probe, 112
PM, 70, 71, 95, 237 processing pathways, 173
pneumococcus, 92 production, vii, ix, x, xii, 3, 5, 39, 40, 52, 82, 86,
pneumonia, 103, 184, 185, 187 101, 105, 107, 116, 118, 121, 122, 126, 128, 129,
pneumonitis, 183, 185 131, 132, 133, 135, 137, 138, 141, 143, 144, 147,
pneumothorax, 186 149, 150, 152, 158, 162, 228, 230, 231, 233, 235,
Poland, 227 237, 242
polycarbonate, 108 profit, 214
polymer, 111 progenitor cells, 128, 181, 191
polymerase, 112, 127, 185 prognosis, xii, 1, 54, 65, 79, 92, 102, 191, 201, 206,
polymerase chain reaction, 112, 127, 185 214, 218, 227, 228
polymerization, 107 prognostic value, 93
polymorphism, 97, 102, 103, 134, 215, 217, 218, program, 27, 98
224, 225 programming, 241
260 Index
pro-inflammatory, xii, 86, 132, 133, 162, 166, 172, P-value, 113
228, 235 pyelonephritis, 184
proliferation, 38, 40, 41, 52, 57, 68, 69, 113, 119,
121, 128, 140, 141, 142, 145, 165, 174, 231, 232,
Q
234, 241, 242
promoter, 91, 224, 225
QT interval, 65
prophylactic, 91, 142
QT prolongation, 65
prophylaxis, 64, 91, 182, 188
quality improvement, 71, 84, 98
prostanoids, viii, 30, 55, 56, 58
quality of life, viii, xi, 18, 27, 78, 79, 85, 99, 212,
proteases, 90, 125
219, 221
protective factors, 40, 95
questionnaires, 19
protective mechanisms, 135
protective role, 80, 159, 229, 231
protein(s), x, 3, 4, 41, 42, 52, 60, 62, 69, 70, 82, 90, R
91, 97, 102, 107, 113, 122, 126, 138, 140, 141,
142, 149, 150, 151, 155, 157, 158, 159, 160, 161, race, 85, 221
162, 163, 165, 167, 168, 170, 171, 172, 173, 174, racial differences, 223
183, 213, 216, 217, 224, 233, 234, 238, 241 range, 2, 5, 24, 25, 62, 183, 194, 220
protein structure, 90 rash, 10, 11, 35, 184, 185
protein synthesis, 162 Raynaud’s phenomenon, 25, 27, 63
proteinase, 165 reactive oxygen, xii, 228
proteinuria, ix, 4, 11, 43, 48, 81, 106, 108, 109, 113, reactivity, ix, 4, 48, 60, 61, 106, 107, 123, 131, 132,
114, 115, 121, 140, 144, 184, 186 150, 151, 159, 162, 187
proteomics, 213 reality, xi, 69, 170, 178
prothrombin, 233 receptors, 41, 53, 56, 63, 72, 73, 123, 133, 145, 147,
pro-thrombotic, 230 160, 165, 215, 233
protocol(s), 51, 108, 109, 137, 138, 144, 181, 182, recognition, 47, 90, 97, 122, 123, 148, 151, 229
184, 186, 188 recovery, 121, 148, 184, 187, 188
protons, 196 recurrence, 4
prototype, 228 red blood cell(s), 182
psychiatric disorders, x, 155, 156, 162 redistribution, 39, 41, 163, 178
psychiatric morbidity, 159 reduction, 4, 5, 43, 49, 87, 147, 189, 201, 202, 232,
psychopathology, 162 240, 241
psychoses, 167 refractory, x, xi, 6, 35, 177, 178, 181, 187, 189, 190,
psychosis, x, 155, 157, 161, 162, 164, 170, 171, 172, 192, 234, 235, 242
186, 189, 194, 201 regeneration, 123, 125, 128
public health, 219, 220 regression, 59, 61, 124, 231
public opinion, 220 regression analysis, 124
public policy, 214 regulation, 52, 67, 95, 126, 128, 129, 145, 149, 162,
puerperium, 10, 12 230, 233, 234, 237, 238, 239
Puerto Rico, 211 rehydration, 108
pulmonary arteries, 51, 53, 54 rejection, 113, 120, 123, 224, 226, 230
pulmonary artery pressure, 50, 51, 54, 56, 186 relapses, 127, 159
pulmonary circulation, 73 relationship, 9, 38, 60, 61, 62, 80, 86, 87, 93, 123,
pulmonary function test, 183 159, 161, 164, 167, 173, 174, 202, 212, 214, 215,
pulmonary hypertension, vii, viii, 27, 29, 30, 50, 51, 218, 225, 226
52, 53, 56, 57, 63, 72, 73, 74, 186 relaxation, 18, 35, 41, 58, 59, 197
pulmonary vascular resistance, 56 relaxation times, 35, 197
pulse(s), x, 17, 18, 21, 25, 26, 33, 72, 177, 179, 180, relevance, x, 61, 145, 155, 171, 215, 219, 224
181, 183, 184, 185, 186, 190, 223 reliability, 45, 57
Index 261
remission, 2, 4, 5, 170, 179, 180, 183, 184, 185, 186, scleroderma, 25, 27, 53
187, 190, 191, 206, 217 sclerosis, 137
renal dysfunction, 106, 189 scores, 45, 70, 113, 116
renal failure, 10, 81, 82, 86, 88, 186, 189, 221 SCT, x, 177, 178, 179, 181, 182, 183, 184, 185, 186,
renal function, 2, 4, 5, 12, 13, 86, 89, 186 187, 188, 189
repair, ix, 64, 105, 107, 123 search, 213
reproduction, 15, 109 secretion, 26, 40, 128, 142, 147
residues, 82, 136, 140, 160 sediment, 11
resistance, 36, 83, 126, 144, 217, 219, 224, 225 seizure(s), 11, 106, 157, 163, 164, 167, 194
resolution, vii, 30, 196, 203 selecting, 189
respiratory, 186 selectivity, 7
responsiveness, 135, 148 sensation, 19, 24
restenosis, 47, 50 sensing, 73
retinitis, 224 sensitivity, 34, 40, 196, 220, 225
retinitis pigmentosa, 224 sensitization, 135, 199
reverse transcriptase, 112 sepsis, 107, 125, 179, 184, 185
rheumatic diseases, xii, 3, 100, 101, 149, 215, 228, septum, 54
242 sequencing, 215
rheumatoid arthritis, ix, 25, 60, 61, 98, 100, 105, series, vii, viii, ix, 6, 10, 11, 12, 30, 33, 34, 35, 36,
106, 124, 138, 150, 235, 242 46, 55, 56, 57, 58, 66, 78, 89, 109, 110, 139, 189,
rheumatologist, 219 228, 229
rhythm, 33, 64 serine, 90, 163, 165
ribosomal RNA, 112 serology, 183, 185, 186
right atrium, 31 serum, x, 4, 5, 7, 11, 38, 43, 60, 61, 69, 72, 81, 86,
right hemisphere, 195 87, 90, 91, 96, 101, 103, 108, 110, 111, 116, 118,
right ventricle, 32 122, 123, 126, 155, 158, 159, 161, 163, 164, 165,
risk, vii, viii, xii, 3, 5, 9, 11, 12, 14, 15, 26, 29, 30, 167, 174, 183, 231, 233
37, 38, 39, 41, 42, 43, 45, 46, 47, 48, 49, 50, 61, serum albumin, 110
62, 64, 65, 67, 68, 69, 70, 71, 72, 77, 78, 79, 80, severity, xi, 5, 27, 31, 43, 45, 49, 55, 85, 103, 113,
81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 92, 93, 94, 122, 123, 135, 161, 189, 194, 201, 202, 211, 212
95, 96, 97, 98, 99, 101, 102, 103, 106, 125, 126, sex, 2, 79, 215
132, 133, 163, 187, 188, 189, 190, 192, 218, 221, shares, 90
223, 227, 228, 229, 233, 235, 236, 241, 242 shock, 31, 42, 69, 70, 107, 138, 150
risk factors, viii, 26, 37, 38, 39, 41, 42, 43, 45, 47, sialic acid, 158
50, 65, 67, 68, 69, 70, 71, 77, 79, 80, 81, 82, 84, side effects, 3, 5, 133, 134, 138, 184, 222
85, 87, 92, 93, 94, 95, 97, 98, 102, 126, 192, 221, signal transduction, 158, 234
229, 233, 235, 236, 241 signals, 122, 132
rituximab, 188, 192 sign(s), ix, 31, 33, 59, 91, 106, 115, 185, 198, 200
RNA, 97, 106, 111, 140 similarity, 143
RNA splicing, 140 single photon emission computerized tomography,
ROI, 203 207
room temperature, 109, 110, 111 single-nucleotide polymorphism, 215, 224
Russia, 177 skin, vii, ix, 2, 5, 10, 17, 18, 21, 22, 23, 24, 25, 26,
91, 92, 105, 106, 107, 108, 109, 111, 112, 113,
114, 115, 119, 120, 121, 123, 125, 187
S
skin diseases, 125
smokers, 38
sacrifice, 119
smoking, viii, 38, 43, 47, 50, 77, 84, 85, 88
safety, 8, 12, 24, 88, 145, 213, 215, 219, 220
smooth muscle, 37, 38, 40, 41, 42, 44, 52, 56, 57, 69,
sample, 2, 48, 112
81, 230, 233, 234, 241
scar tissue, 107
262 Index
smooth muscle cells, 37, 38, 42, 44, 57, 69, 81, 230, substitution, 1, 3, 36, 149
233 suffering, 62, 232
SNP, 216, 217 sugar, 158
sodium, 36, 133 suicide, 159
solubility, 135 suicide attempts, 159
somatic mutations, 213 sulfate, 2, 3, 6, 7, 8
sounds, 213 sulfur, 39
species, xii, 139, 228, 234 sulfuric acid, 110
specificity, 35, 46, 51, 60, 133, 134, 144, 146, 156, superiority, 137
158, 161, 165, 170, 194 supervision, 14
spectroscopy, xi, 193, 195, 196, 197, 200, 202, 206, suppression, 13, 127, 132, 134, 135, 137, 138, 139,
207, 208 144, 145, 147, 148, 150, 151, 152, 232, 234, 235
spectrum, 89, 92, 156, 196, 197, 198 surface area, ix, 105
speed, 213 surface structure, 163
sperm, 13 surveillance, 64, 132, 188
sphygmomanometer, 19 survival, viii, ix, 55, 77, 78, 79, 106, 108, 113, 114,
spinal cord, 192 115, 120, 121, 122, 138, 140, 141, 142, 189, 219,
spine, 88 222, 226
spleen, 115, 141 survival rate, ix, 106, 108, 114, 115
splenomegaly, ix, 106, 108, 115 susceptibility, 90, 91, 103, 169, 196, 225
spontaneous abortion, 10 swelling, 33, 35
sprouting, 173 switching, 122
St. Petersburg, 109 symptom(s), ix, x, 2, 4, 5, 19, 24, 26, 31, 33, 35, 51,
stability, 139 58, 59, 91, 105, 107, 114, 155, 158, 161, 162,
stabilization, 232, 240 184, 185, 186, 189, 200, 201, 202, 203, 205, 210
stages, 40, 51, 70, 142, 145, 150 syndrome, 11, 48, 59, 60, 62, 80, 81, 83, 96, 102,
standard deviation, 111 107, 114, 117, 125, 127, 159, 184, 192, 194, 201,
standards, 110 225, 235, 236
statin, 48, 71, 232, 233, 234 synthesis, ix, xii, 5, 36, 40, 99, 106, 122, 127, 136,
statistical analysis, 19 138, 164, 166, 169, 227, 230, 231, 233, 235, 240
stem cells, x, 177, 178, 181, 185, 187, 191 syphilis, 91
stenosis, 43, 44, 46 systemic circulation, 58, 160, 164
stent, 50 systemic lupus erythematosus, iv, vii, viii, ix, xii, 6,
sterile, 109 7, 8, 14, 18, 27, 29, 30, 44, 53, 60, 61, 62, 66, 67,
steroids, 5, 98, 99, 188 68, 69, 70, 71, 72, 73, 74, 75, 77, 92, 93, 94, 95,
stillbirth, 10 96, 97, 98, 99, 100, 101, 102, 103, 123, 124, 125,
strain, 122, 123, 129, 160 126, 127, 128, 129, 131, 146, 147, 148, 149, 150,
strategies, x, 40, 42, 43, 46, 48, 55, 59, 66, 78, 87, 151, 152, 167, 168, 169, 170, 171, 172, 173, 174,
88, 123, 131, 137, 138, 165, 219 175, 178, 189, 190, 191, 192, 194, 206, 207, 208,
strength, 25, 88, 107 209, 210, 221, 222, 223, 226, 228, 229, 236, 237,
stress, 27, 96, 106, 124, 126, 161, 163, 235 239, 242
stress factors, 235 systemic sclerosis, 17, 18, 27, 73
stretching, 17, 18 systems, xi, 2, 5, 78, 183, 187, 211, 213, 215, 226
stroke, 39, 41, 43, 47, 49, 64, 79, 83, 94, 163, 189, systolic blood pressure, 19
199, 201, 202, 240 systolic pressure, 19, 55, 57
structural changes, 208
structural characteristics, 135
T
subacute, 35, 189
subcutaneous injection, 138
T cell, ix, 40, 44, 90, 107, 123, 126, 127, 129, 131,
subgroups, 84, 231, 241
132, 133, 134, 135, 136, 137, 138, 139, 140, 141,
Index 263
142, 143, 144, 146, 147, 148, 149, 150, 151, 152, TNF, ix, 40, 48, 53, 63, 69, 81, 86, 97, 106, 107,
153, 186, 226, 230, 231, 234, 237, 238 119, 120, 122, 138, 144, 230, 231, 234, 235, 239,
T lymphocyte(s), 107, 132, 145, 149, 229 242
tachycardia, 33, 59, 64 TNF-alpha, 69, 97, 231, 239
target population, 228 TNF-α, 40, 48, 53, 81, 86, 107, 119, 120
targets, vii, 30, 50, 59, 84, 133, 162, 163, 165, 188, total cholesterol, 48
215 toxic effect, 166
T-cell(s), 68, 120, 123, 126, 133, 134, 136, 138, 141, toxicity, 186, 188, 191, 214, 216, 217, 218, 220, 223
142, 145, 147, 148, 186, 188, 236 toxin, 127
TCR, 146 toxoplasmosis, 91
technetium, 46 TPI, 158
technology, 5, 51, 213 training, 27, 51, 220
temperature, 17, 18, 21, 22, 23, 24, 25, 26, 110, 111 traits, 215
territory, 194 transcription, 112
testosterone, 86 transcripts, 108, 119, 120, 122
Texas, 241 transduction, 241
TGF, 52, 122, 129, 141, 144, 145, 152, 153 transforming growth factor, 52, 141
thalamus, 204, 205 transfusion, 181, 182
T-helper cell, 126 translation, 219
theory, 201, 202 transmission, 3, 26, 234
therapeutic agents, 138, 145, 146 transplant recipients, 192, 224, 226
therapeutic approaches, 1 transplantation, ix, x, 106, 112, 120, 121, 126, 177,
therapeutic targets, 133, 241 178, 181, 182, 183, 186, 188, 190, 191, 215, 223,
therapeutics, xi, 211, 212, 213, 222 237
therapy, viii, x, xii, 1, 5, 6, 7, 8, 12, 13, 30, 32, 34, transport, 173, 197
35, 43, 46, 55, 56, 58, 69, 70, 71, 73, 74, 84, 88, transthoracic echocardiography, 63, 75
89, 98, 100, 101, 126, 131, 132, 134, 135, 137, trauma, 106, 107, 113, 114, 116, 118, 119, 120, 121,
138, 139, 141, 142, 144, 147, 149, 159, 177, 178, 123, 124, 125, 126
181, 182, 183, 184, 186, 187, 188, 189, 191, 192, trend, 57, 187
194, 213, 219, 222, 223, 225, 227, 228, 231, 234 trial, 5, 6, 15, 48, 57, 71, 101, 102, 138, 150, 190,
threshold, 26, 112, 133 226, 239, 242
thrombin, 232, 233, 240, 241 triggers, 106, 126, 136
thrombocytopenia, 11, 33, 189 triglycerides, 38, 39, 40, 43, 64, 67, 81, 95, 231, 239
thrombosis, 11, 39, 43, 53, 61, 62, 68, 69, 71, 80, 83, tuberculosis, 89
88, 94, 156, 205, 232 tumor, ix, 53, 63, 67, 81, 95, 106, 127, 138, 147,
thrombus, 38, 62, 63, 164, 230, 232 148, 158, 235, 239, 242
thymus, 118, 132, 135, 148 tumor growth, 158
thyroglobulin, 136 tumor necrosis factor, ix, 53, 63, 67, 81, 95, 106,
thyroid, 64, 136, 148 127, 138, 147, 148, 235, 239
time, x, 5, 18, 33, 43, 64, 78, 79, 86, 88, 99, 107, turnover, 25, 100, 126, 197, 200
108, 109, 110, 112, 113, 114, 115, 116, 117, 118, twins, 65
119, 120, 121, 123, 126, 134, 138, 139, 178, 183, type 1 diabetes, 150
185, 186, 196, 200, 201, 202, 203, 205, 213, 220, type 2 diabetes, 97
221, 228
tin, 1
U
tissue, ix, x, 27, 30, 34, 35, 38, 40, 55, 62, 65, 73, 90,
105, 106, 107, 111, 113, 120, 122, 123, 131, 132,
UK, 103
133, 148, 160, 163, 174, 196, 199, 203, 230, 233,
ultrasonography, vii, 30
235, 240, 242
ultrasound, 43, 45, 46, 47, 55
United Kingdom, 83, 214
264 Index
United States, 39, 43, 83, 101, 106, 124, 191, 222
W
universe, 126
unplanned pregnancies, 10
water diffusion, 199
unstable angina, 236
weight reduction, 84
urine, 108, 109, 113, 183
welfare, 124
UV, 106
western blot, 158
UV radiation, 106
Western Europe, 90
white blood cells, 181
V white matter, xi, 193, 194, 195, 200, 201, 202, 203,
204, 205, 209, 210
vaccinations, 92 WHO, 57, 181, 184
vaccine, 125 wild type, ix, 105, 107
Valencia, 111 withdrawal, 189
validation, 93, 139, 168 women, vii, 9, 10, 11, 12, 13, 18, 29, 41, 45, 68, 70,
validity, 51, 139, 164, 220, 226 71, 83, 87, 88, 93, 94, 95, 96, 97, 98, 99, 100,
values, 4, 46, 55, 87, 112, 118, 164, 203, 204, 205, 101, 107, 228, 229, 236, 239, 241
208, 210 World Health Organization, 181
variability, viii, 27, 30, 165, 213, 215, 219 wound healing, ix, 105, 107, 123, 125, 127, 128
variable(s), xi, 45, 212, 218, 219 wound repair, ix, 106, 107, 108, 119, 128
variation, 22, 23, 103, 156, 223 writing, 139
vascular endothelial growth factor (VEGF), 52
vascular wall, 239
Y
vasculitis, ix, x, xii, 1, 2, 5, 46, 81, 95, 105, 113,
121, 156, 161, 162, 172, 177, 181, 183, 185, 186,
yang, 240
202, 205, 227
yeast, 163
vasoconstriction, 25, 234
yield, 111, 119, 136, 213
vasoconstrictor, 232
yin, 240
vasodilation, 25, 56
young women, 218, 229
vasodilator, 57
vaso-occlusion, 205
vasospasm, 25
VCAM, 230, 237
vector, 149
velocity, 34, 46, 54, 58
ventricle, 32, 58
ventricular arrhythmias, 36
ventricular tachycardia, 35
vessels, 18, 25, 43, 52, 164, 196, 205, 232
veterans, 124, 125
viral infection, 91
virus infection, 184
viruses, 90
visualization, 46, 111
vitamin B1, 84
vitamin B12, 84
vitamin D, 86, 88
vitamin D deficiency, 86
VLDL, 81, 82
vulnerability, 44

Progress in Systemic Lupus 2007 Nova Medical Books

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PROGRESS IN SYSTEMIC LUPUS

Nova Biomedical Books

NOTICE TO THE READER

LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

Published by Nova Science Publishers, Inc. New York

Expert Commentary Therapeutic Plasmapheresis in the Treatment of

Expert Commentary Pregnancy, a Challenge in Patients with

Short Communications Influence of Exercise on the Peripheral Circulation in

Chapter 1 New Frontiers in the Research of Cardiovascular

Chapter 2 Comorbidity in Systemic Lupus Erythematosus: Aspects

Chapter 3 Severe Tissue Trauma Triggers Lupus

Chapter 4 Immunotherapy with Ig-Derived Peptides in SLE:

Chapter 5 Autoantibodies as Prognostic or Diagnostic Markers of

Chapter 6 High-Dose Immunosuppression with Autologous

Chapter 7 MR Spectroscopy, Diffusion and Diffusion Tensor

Chapter 8 Personalized Medicine for Systemic Lupus

Chapter 9 Therapeutic Potential of HMG-CoA Reductase

This new book is devoted to leading-edge research developments in lupus which is a

due to transplant-related complications had long history of the corticosteroids treatment,

THERAPEUTIC PLASMAPHERESIS IN THE

Claudia Stefanutti∗, Fabio Mazza and Valeria Riccieri

is associated with a certain range of possible side-effects (anaphylactoid reactions,

intervention of the above mentioned complications. However, it is relatively common to

Immunoadsorption Plasmapheresis: (Selesorb®)

Selesorb® (Kaneka Corp., Osaka, Japan) is a chemical affinity technique able at

Autoimmune diseases include a broad and heterogeneous group of diseases characterized

[1] Suzuki K, Hara M, Harigai M, et al.Continuous removal of anti-DNA antibody, using

[5] Matsuki Y, Suzuki K, Kawakami M, et al. High-avidity anti-DNA antibody removal

[21] Kamijo Y, Kaneko Y, Ichikawa T, Kobayashi N, Koyama T, Kakegawa T,

PREGNANCY, A CHALLENGE IN PATIENTS WITH

Javier A. Cavallasca and Maria del Rosario Maliandi

Systemic lupus erythematosus (SLE) is a multi-system autoimmune disease that typically

INFLUENCE OF PREGNANCY ON SLE

INFLUENCE OF SLE ON PREGNANCY:

LUPUS AND HYPERTENSIVE PREGNANCY

Lupus pregnancy is associated with an increased risk of pre-eclampsia of 13-32%,

LUPUS NEPHRITIS (LN)

ANTIPHOSPHOLIPID SYNDROME (APS)

NEONATAL LUPUS ERYTHEMATOSUS

All lupus patient contemplating pregnancy should have an anti-Ro/SSA, anti-La/SSB

DRUGS IN PREGNANCY AND BREASTFEEDING

NON STEROIDAL ANTINFLAMMATORY

Stress doses of hydrocortisone at delivery are recommended in patients on corticosteroids

Chloroquine and hidroxichloroquine cross the placenta with no significant difference in

Methotrexate is contraindicated during pregnancy and in the breastfeeding period . It

Mycophenolate Mofetil (MMF)

Leflunomide is contraindicated during pregnancy and breastfeeding. Discontinue 2 years

It can be used in pregnancy and in the breastfeeding period.

Kutteh WH. Antiphospholipid antibody-associated recurrent pregnancy loss: treatment with

INFLUENCE OF EXERCISE ON THE PERIPHERAL

Etsuko Maeshima∗ and Kanako Furukawa**

circulatory disturbance in SSc, even when patients perform low-intensity exercise.

Keywords: systemic lupus erythematosus, systemic sclerosis, exercise, peripheral

The beneficial effects of exercise include a decrease of sympathetic nervous tone,

Right Index Finger

Left Index Finger

Figure 6A. Skin temperature of the right finger.

Figure 6B. Skin temperature of the left finger.

Table 1. Subjective symptoms and the skin temperature

Laura Gonzalez-Lopez,∗ J. I. Gamez-Nava, and Arnulfo Nava