Diagnostic testing and interpretation of tests for autoimmunity.

Laboratory testing is of great value when evaluating a patient with a suspected autoimmune disease. The results can confirm a diagnosis, estimate disease severity, aid in assessing prognosis and are useful for following disease activity. Components of the laboratory examination include a complete blood count with differential, a comprehensive metabolic panel, measurement of inflammatory markers and autoantibodies, and flow cytometry. This chapter discusses these components and includes a discussion about organ-specific immunologic diseases for which immunologic laboratory testing is used. Comprehensive laboratory evaluation of a suspected autoimmune illness in conjunction with a thorough clinical evaluation provides a better understanding of a patient's immunologic disease.

A randomized, double-blind, placebo-controlled trial of infliximab in refractory polymyositis and dermatomyositis.

OBJECTIVE: To investigate in a pilot study the safety and efficacy of infliximab in patients with refractory dermatomyositis (DM) and polymyositis (PM). METHODS: A randomized, double-blind, placebo-controlled trial including subjects with active DM or PM. Participants had stable doses of immunosuppressive medication and prednisone (≤0.5mg/kg/day), and exhibited clinical signs of muscle weakness for at least 4 weeks prior to study entry. Participants received infusions of either placebo or infliximab 5mg/kg at 0, 2, 6, and 14 weeks in blinded manner. The primary outcome was a ≥15% manual muscle strength (MMT) improvement at week 16 compared to week 0. The secondary outcome measures were improvement defined by the International Myositis Assessment and Clinical Studies Group (IMACS) criteria. At week 16, responders in each arm had the option of either continuing the same treatment or changing to the non-responder treatment for that study arm. Non-responders in the 5mg/kg infliximab arm were increased to infliximab 7.5mg/kg for weeks 22, 30, and 38. Non-responders in the placebo arm at week 16 received infliximab 5mg/kg at weeks 16, 18, 22, 30, and 38. Outcomes were reassessed at week 40. RESULTS: Twelve subjects completed the study to week 16. Six of the 12 subjects received infliximab treatment at the dose of 5mg/kg with only one subject meeting the responder criteria at that dose. Of the remaining five subjects on infliximab, three crossed over to the infliximab 7.5mg/kg dose. One of those three subjects responded. All six patients in the placebo arm crossed over to the 5mg/kg dosing regimen after week 16, and two of those responded to infliximab. CONCLUSIONS: Infliximab therapy for patients with refractory PM and DM was well tolerated and may benefit a subset of patients.

Prevalence of obsessive-compulsive disorder in patients with systemic lupus erythematosus.

BACKGROUND: The goal of this pilot study was to investigate the prevalence of obsessive-compulsive disorder (OCD) in a group of patients with systemic lupus erythematosus (SLE). METHOD: Fifty adult patients enrolled in out-patient SLE studies at the National Institute of Arthritis and Musculoskeletal and Skin Diseases (February 1995-October 1996) completed a self-report questionnaire adapted from the Yale-Brown Obsessive Compulsive Scale and an in-person psychiatric clinical interview with a psychiatrist or psychiatric clinical nurse specialist. DSM-IV lifetime diagnosis of OCD was determined by clinical interview. RESULTS: Sixteen subjects (32%) met DSM-IV lifetime diagnostic criteria for OCD and an additional 5 (10%) met criteria for subclinical OCD. Mean +/- SD number of symptoms reported on the self-report questionnaire was significantly higher among subjects diagnosed with OCD on clinical interview (40.7 +/- 23.2) compared with those without OCD (8.9 +/- 11.7; t = 5.8, df = 27, p <.001). CONCLUSION: Obsessive-compulsive disorder was 10 to 15 times more common in this cohort of patients with SLE compared with those in community-based studies of OCD. The use of an OCD self-report rating scale proved helpful in the identification of OCD symptoms among patients with SLE. Results suggest that further studies of OCD in patients with SLE are needed and may provide new insight into the pathophysiology of both disorders.

Diagnosis and treatment of inflammatory myopathy: issues and management.

The idiopathic inflammatory myopathies include polymyositis (PM), dermatomyositis (DM) and inclusion body myositis (IBM). The specific etiologies of these muscle diseases are not well known and are thought to involve components of the humoral and cellular immune system as well as other nonimmune factors. Diagnosing these myopathies involves a laboratory evaluation, imaging studies, multidisciplinary consultations, histologic examination and potentially genetic studies. Despite all that we currently know about inflammatory muscle disease with these studies, we find that our current concept of muscle disease is changing. In the cases of immune-mediated necrotizing myopathy and inclusion body myositis, the concept of inflammation needs to be rethought. Moreover, the classification schemes for these idiopathic myopathies may need updating to include current research findings that relate to pathogenesis. With ongoing discoveries, classification and appropriate treatment is becoming increasingly challenging. This paper discusses the inflammatory myopathies, the challenges to diagnosis, classification controversies and potential treatment options.

Plasma proteomic profiles from disease-discordant monozygotic twins suggest that molecular pathways are shared in multiple systemic autoimmune diseases.

INTRODUCTION: Although systemic autoimmune diseases (SAID) share many clinical and laboratory features, whether they also share some common features of pathogenesis remains unclear. We assessed plasma proteomic profiles among different SAID for evidence of common molecular pathways that could provide insights into pathogenic mechanisms shared by these diseases. METHODS: Differential quantitative proteomic analyses (one-dimensional reverse-phase liquid chromatography-mass spectrometry) were performed to assess patterns of plasma protein expression. Monozygotic twins (four pairs discordant for systemic lupus erythematosus, four pairs discordant for juvenile idiopathic arthritis and two pairs discordant for juvenile dermatomyositis) were studied to minimize polymorphic gene effects. Comparisons were also made to 10 unrelated, matched controls. RESULTS: Multiple plasma proteins, including acute phase reactants, structural proteins, immune response proteins, coagulation and transcriptional factors, were differentially expressed similarly among the different SAID studied. Multivariate Random Forest modeling identified seven proteins whose combined altered expression levels effectively segregated affected vs. unaffected twins. Among these seven proteins, four were also identified in univariate analyses of proteomic data (syntaxin 17, α-glucosidase, paraoxonase 1, and the sixth component of complement). Molecular pathway modeling indicated that these factors may be integrated through interactions with a candidate plasma biomarker, PON1 and the pro-inflammatory cytokine IL-6. CONCLUSIONS: Together, these data suggest that different SAID may share common alterations of plasma protein expression and molecular pathways. An understanding of the mechanisms leading to the altered plasma proteomes common among these SAID may provide useful insights into their pathogeneses.

Gene expression profiles from discordant monozygotic twins suggest that molecular pathways are shared among multiple systemic autoimmune diseases.

INTRODUCTION: The objective of this study is to determine if multiple systemic autoimmune diseases (SAID) share gene expression pathways that could provide insights into pathogenic mechanisms common to these disorders. METHODS: RNA microarray analyses (Agilent Human 1A(V2) 20K oligo arrays) were used to quantify gene expression in peripheral blood cells from 20 monozygotic (MZ) twin pairs discordant for SAID. Six affected probands with systemic lupus erythematosus (SLE), six with rheumatoid arthritis (RA), eight with idiopathic inflammatory myopathies (IIM), and their same-gendered unaffected twins, were enrolled. Comparisons were made between discordant twin pairs and these were also each compared to 40 unrelated control subjects (matched 2:1 to each twin by age, gender and ethnicity) using statistical and molecular pathway analyses. Relative quantitative PCR was used to verify independently measures of differential gene expression assessed by microarray analysis. RESULTS: Probands and unrelated, matched controls differed significantly in gene expression for 104 probes corresponding to 92 identifiable genes (multiple-comparison adjusted P values < 0.1). Differentially expressed genes involved several overlapping pathways including immune responses (16%), signaling pathways (24%), transcription/translation regulators (26%), and metabolic functions (15%). Interferon (IFN)-response genes (IFI27, OASF, PLSCR1, EIF2AK2, TNFAIP6, and TNFSF10) were up-regulated in probands compared to unrelated controls. Many of the abnormally expressed genes played regulatory roles in multiple cellular pathways. We did not detect any probes expressed differentially in comparisons among the three SAID phenotypes. Similarly, we found no significant differences in gene expression when comparing probands to unaffected twins or unaffected twins to unrelated controls. Gene expression levels for unaffected twins appeared intermediate between that of probands and unrelated controls for 6535 probes (32% of the total probes) as would be expected by chance. By contrast, in unaffected twins intermediate ordering was observed for 84 of the 104 probes (81%) whose expression differed significantly between probands and unrelated controls. CONCLUSIONS: Alterations in expression of a limited number of genes may influence the dysregulation of numerous, integrated immune response, cell signaling and regulatory pathways that are common to a number of SAID. Gene expression profiles in peripheral blood suggest that for genes in these critical pathways, unaffected twins may be in a transitional or intermediate state of immune dysregulation between twins with SAID and unrelated controls, perhaps predisposing them to the development of SAID given the necessary and sufficient environmental exposures.

CD146+ T lymphocytes are increased in both the peripheral circulation and in the synovial effusions of patients with various musculoskeletal diseases and display pro-inflammatory gene profiles.

Twenty-eight synovial effusions (SE) were obtained from 24 patients, paired samples of peripheral blood (PB) from 10 of these patients, and PB from 36 healthy individuals for analysis of CD146 on T-lymphocytes by flow cytometry. CD146+ or CD146- T-lymphocytes were sorted from three SE to study gene expression profiles and selected genes revalidated using QPCR assays. We found more CD3+CD146+ and CD4+CD146+ T-lymphocytes in PB from patients compared with PB of healthy individuals (4.71% +/- 2.48% vs. 2.53% +/- 1.08%, P = 0.028) and (6.29% +/- 2.74% vs. 2.41% +/- 0.96%, P = 0.0017), respectively, whereas CD8+CD146+ T-lymphocytes were not significantly different (2.55% +/- 1.65% vs. 3.18% +/- 2.59%, P = 0.5008). SE displayed CD146 staining on 16.32% +/- 6.06% of CD3+ cells. This expression was skewed toward CD4+ T-lymphocytes, with CD146 present on 24.06% +/- 8.20% of the CD4+ T-lymphocytes compared with 6.19% +/- 5.22% of the CD8+ T-lymphocytes. CD146 on CD3+, CD4+ and CD8+ T-lymphocytes in SE was significantly higher compared with PB in patients (P < 0.0001, P < 0.0001 and P = 0.0036, respectively). Gene expression profiles of sorted CD146+CD4+CD3+ vs. CD146-CD4+CD3+ T-lymphocytes (n = 2) and CD2+CD146+ vs. CD2+CD 146- (n = 1) from SE, displayed increased CD146, LAIR2, CXCL13, CD109, IL6ST, IL6R, TNFRsf18, and TNFRsf4 genes, whereas decreased CCR7, CCL5, and cytotoxicity-associated genes including granzymes b, h, and k, perforin were found with the CD146- T-lymphocytes. By QPCR higher mRNA expression of CXCL13, CD146 and CD109 was also noted in the CD146+ subset, compared with the CD146- subset, in PB of healthy individuals and in PB and SE from patients. Our study establishes increased CD146+ T-lymphocytes in diseases with joint effusions, and demonstrates pro-inflammatory gene profiles in these cells.

Mechanisms of disease: Environmental factors in the pathogenesis of rheumatic disease.

Most rheumatic diseases are complex disorders for which pathogenetic mechanisms are poorly understood. Nonetheless, increasing evidence suggests that many of these illnesses result from one or more specific environmental exposures in genetically susceptible individuals. Although much progress has been made over the past few decades in advancing our knowledge of the genetics of rheumatic diseases, few studies have assessed environmental features and understanding of which exposures are important in pathogenesis remains limited. In this article, we review the difficulties inherent in deciphering the interacting environmental and genetic risk factors for rheumatic diseases, the current state of knowledge of infectious and noninfectious risk factors, possible mechanisms by which environmental exposures might induce pathologic processes and future directions. The advances in technologies and statistical approaches, development of collaborating consortia and focused resources that have resulted in the explosion of genetic information must now be applied to environmental studies so we can eventually interrupt pathogenesis before the onset of disease and transform the practice of medicine from curative to pre-emptive paradigms.

Antisense cyclic adenosine 5'-monophosphate response element modulator up-regulates IL-2 in T cells from patients with systemic lupus erythematosus.

The cAMP response element modulator (CREM) has been shown to bind specifically to the -180 site of the IL-2 promoter in vitro. CREM protein is increased in T cells of patients with systemic lupus erythematosus (SLE), and it has been considered responsible for the decreased production of IL-2. In this work we show that transcriptional up-regulation is responsible for the increased CREM protein levels and that CREM binds to the IL-2 promoter in live SLE T cells. Suppression of the expression of CREM mRNA and protein by an antisense CREM plasmid, which was force expressed in SLE T cells by electroporation, resulted in decreased CREM protein binding to the IL-2 promoter and increased expression of IL-2 mRNA and protein. Our data demonstrate that antisense constructs can be used to effectively eliminate the expression of a transcriptional repressor. This approach can be used therapeutically in conditions where increased production of IL-2 is desired.

Defective production of functional 98-kDa form of Elf-1 is responsible for the decreased expression of TCR zeta-chain in patients with systemic lupus erythematosus.

Systemic lupus erythematosus (SLE), the prototypic autoimmune disease, is characterized by defective expression of TCR zeta-chain. Elf-1 (E-74-like factor) is a member of the Ets (E-26-specific) family and is crucial for the basal transcription of TCR zeta-chain in Jurkat cells. We previously demonstrated that Elf-1 exists in the cytoplasm mainly as 80-kDa form and after phosphorylation and O-glycosylation it moves to the nucleus as a 98-kDa which binds DNA. We now demonstrate that Elf-1 is crucial for the transactivation of TCR zeta-chain promoter in normal and SLE T cells. Defective expression of TCR zeta-chain in SLE T cells is associated with two distinct molecular defects in the generation of the 98-kDa DNA binding Elf-1 form. In the first, the levels of the 98-kDa form were either decreased or absent. In the second, the apparent levels of the nuclear Elf-1 form were normal but included only two of the three bands into which the nuclear Elf-1 form separated in isoelectric focusing gels. Because both the transcription and the translation processes of Elf-1 gene are normal in SLE T cells, our data demonstrate that abnormal posttranslational mechanisms of the Elf-1 protein result in defective expression of functional Elf-1, and consequently, the transcriptional defect of TCR zeta-chain in patients of SLE.

Direct transfer of p65 into T lymphocytes from systemic lupus erythematosus patients leads to increased levels of interleukin-2 promoter activity.

The recent identification of a number of molecular defects in T cells from patients with systemic lupus erythematosus (SLE) has raised expectations for gene replacement therapy as an option in the treatment of these diseases. In this report, we have adapted an electroporation-based technique to transfer successfully DNA to peripheral blood T cells from normal individuals and patients with systemic lupus erythematosus and rheumatoid arthritis. Transfection efficiency, judged by the percentage of live cells expressing green fluorescence after transfection with a pGFP (green fluorescence protein), reached 32 +/- 3% in normal, 13 +/- 3% in SLE, and 17 +/- 13% in RA T cells. The transfection efficiency was slightly higher in CD8+ than in CD4+ cells, and the cells maintained acceptable (75%) viability up to the fourth post-transfection day. SLE T cells have been shown to display low levels of the p65 subunit of the NF-kappaB transcription factor and decreased production of IL-2. Since NF-kappaB contributes to the transcriptional regulation of the IL-2 promoter, the effect of the forced replenishment of p65 on IL-2 transcription was tested. The low level of interleukin-2 promoter activity in SLE T cells increased to normal levels following transfection with cDNA encoding the NF-kappaB p65 subunit. Taken together, these results demonstrate the feasibility of transfection of T cells from SLE patients by electroporation and the reversal of decreased interleukin-2 promoter activity in SLE T cells, and are an early step toward gene therapy as a method of treatment for these individuals.

Pilot clinical trial of intravenous doxycycline versus placebo for rheumatoid arthritis.

OBJECTIVE: To screen for potential efficacy and assess the feasibility of intravenous (IV) doxycycline as a treatment for rheumatoid arthritis (RA). METHODS: The study was a (stratified, block) randomized, double blind, 12 week, pilot trial of IV doxycycline 300 mg/day versus identical appearing IV placebo given over 2 h for 14 days. The primary comparison was to a hypothesized placebo rate of 20% as described by Paulus. If a total of 14 consecutive subjects receiving doxycycline treatment did not respond, it would be considered futile to proceed to a Phase III trial. We planned a placebo group of 14 subjects to verify the placebo response rate and estimate sample size required for a definitive Phase III trial, if such a trial was warranted based on the pilot study. American College of Rheumatology (ACR) RA response criteria were used. After 23 subjects entered, the study was closed due to recruitment difficulties. RESULTS: At baseline, mean (SD) tender joint count was 37 (11.9), swollen joint count 30 (9.6), morning stiffness 317 (319) min, and erythrocyte sedimentation rate 72 mm/h (27.5). Randomization resulted in 10 subjects receiving doxycycline and 13 receiving placebo. Treatment was stopped in 8 subjects: in 6, treatment was ineffective (one taking doxycycline, 5 placebo), and in 2, rashes occurred (one taking doxycycline, one placebo). Only one subject met ACR response criteria in the doxycycline group and none in the placebo group. Having no responders in the placebo group was consistent with placebo response rate of 20% or less. Several patients required peripherally inserted central catheters for venous access. CONCLUSION: The efficacy of IV doxycycline as a treatment for RA could not be ruled out. However, as the proportion of responders was small, it is unlikely that potential efficacy of IV doxycycline would outweigh potential disadvantages of IV administration.