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Rheumatoid arthritis (RA) is a chronic, inflammatory joint disease of unknown etiology and pronounced interpatient heterogeneity. To characterize RA at the molecular level and to uncover pathomechanisms, we performed genome-wide gene expression analysis. We identified a set of 1,054 genes significantly deregulated in pair-wise comparisons between RA and osteoarthritis (OA) patients, RA and normal donors (ND), or OA and ND. Correlation analysis revealed gene sets regulated identically in all three groups. As a prominent example secreted phosphoprotein 1 (SPP1) was identified to be significantly upregulated in RA compared with both OA and ND. SPP1 expression was found to correlate with genes expressed during an inflammatory response, T-cell activation and apoptosis, suggesting common underlying regulatory networks. A subclassification of RA patients was achieved on the basis of proteoglycan 4 (PRG4) expression, distinguishing PRG4 high and low expressors and reflecting the heterogeneity of the disease. In addition, we found that low PRG4 expression was associated with a more aggressive disease stage, which is in accordance with PRG4 loss-of-function mutations causing camptodactyly-arthropathy-coxa vara-pericarditis syndrome. Altogether we provide evidence for molecular signatures of RA and RA subclasses, sets of new candidate genes as well as for candidate gene networks, which extend our understanding of disease mechanisms and may lead to an improved diagnosis.

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BACKGROUND: In HIV patients, disorders in glucose metabolism seem to be side effects of highly active antiretroviral therapy (HAART) which may be favoured by obesity, abdominal fat accumulation and familial disposition for diabetes mellitus (DM). The aim of our study was to identify patients at high risk for abnormalities in glucose metabolism taking into account HAART, familial disposition for DM and anthropometric parameters. METHODS: Plasma glucose, insulin, c-peptide and insulin resistance (homeostasis model assessment, HOMA) were determined in 44 HIV patients [16 without HAART, 19 with protease inhibitors (PI), 9 without PI (non-PI)] and in 11 healthy subjects. Glucose tolerance was determined by standard procedures. Body mass index (BMI), triceps skin fold thickness and waist circumference were measured and the waist-to-hip-ratio was calculated. Familial disposition for DM was assessed by questionnaire. RESULTS: Impaired fasting glucose was observed in 28% of HAART-treated patients (21% with PI, 7% non-PI), in 13% of HAART-naive but none in healthy controls. 58% of PI, 44% of non-PI, 38% of HAART-naive and none of healthy controls had a HOMA-index > 2.5 which indicates insulin resistance. HAART-treated patients had significantly higher fasting glucose levels (PI: 97 +/- 11 mg/dL, p = 0.048; non-PI: 109 +/- 58 mg/dL, p = 0.009) compared to healthy controls (72 +/- 8 mg/dL). HOMA-Index was higher in PI treated patients (3.74 +/- 3.08) than in HIV negative controls (0.95 +/- 0.28, p = 0.018). The duration of HAART (p = 0.045), overweight and familial disposition for DM (p = 0.017) significantly affected fasting glucose among PI users. Waist circumference affected c-peptide (p = 0.046) concentration in these patients. CONCLUSION: HIV patients on long-term PI therapy with overweight and familial disposition for DM are at high risk to develop abnormalities of glucose metabolism. Thus, measurements of HOMA-Index, BMI and waist circumference should be routinely done especially in PI medicated patients.

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BACKGROUND: The SmD1(83-119) peptide is a major target of the B cell response in patients with systemic lupus erythematosus (SLE). OBJECTIVE: To investigate the T cell response directed against this peptide, its disease specificity, and possible impact on SLE pathogenesis. METHODS: Peripheral blood mononuclear cells derived from 28 patients with SLE and 29 healthy and disease controls were stimulated by the SmD1(83-119) and the recombinant (r)SmD1 protein, and [3H]thymidine incorporation was measured. Patients with SLE were simultaneously tested for autoantibodies, disease activity, clinical symptoms, and medical treatments. RESULTS: T cell reactivity against the SmD1(83-119) peptide was detected in 11/28 (39%) patients with SLE and against the rSmD1 protein in 10/28 (36%) patients. In contrast, only 2/29 (7%) controls exhibited SmD1 reactivity. An analysis of proliferation kinetics showed that SmD1 reactive T cells are activated in vivo, as additionally confirmed by cytometric analysis. Addition of mammalian dsDNA to rSmD1 enhanced the rSmD1-specific T cell response. SmD1(83-119)-specific T cell reactivity was significantly more common in patients with cardiac and pulmonary symptoms. No correlation between T and B cell responses and disease activity was seen. CONCLUSION: SmD1(83-119) is a major T cell epitope of SmD1, commonly recognised by T cells from patients with SLE and much less commonly found by healthy or disease controls. This strong T cell reactivity as well as the high frequency and specificity of anti-SmD1(83-119) antibodies in SLE suggest a possible role in SLE pathogenesis, at least in a subset of patients.

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OBJECTIVE: The ubiquitously expressed intracellular protein formerly designated p68 has been identified as autoantigen at both the antibody and the T cell level in rheumatoid arthritis (RA). METHODS: We used 2 independent approaches, Edman degradation and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry, to characterize p68, and we compared its features with those of the endoplasmic reticulum stress protein BiP. RESULTS: In synovial sections from RA patients, BiP was highly overexpressed as compared with control sections. Under in vitro stress conditions, BiP was found to translocate to the nucleus and the cell surface. BiP-specific autoantibodies were present in 63% of 400 RA patients, in 7% of 200 patients with other rheumatic diseases, and in none of the healthy subjects. Thus, BiP-specific autoantibodies represent a new diagnostic marker in RA. Furthermore, we found that BiP-specific T cell reactivity was altered in RA. In healthy individuals and patients with other rheumatic diseases, BiP-reactive T cells were undetectable. In RA, overt T cell reactivity to BiP was observed or could be induced by specifically blocking antigen presentation to potentially regulatory T cells. CONCLUSION: Since overexpression of BiP has been shown to decrease the sensitivity of cells to killing by cytotoxic T cells, BiP overexpression and BiP-specific autoimmunity may be involved in the pathogenesis of RA.

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The interaction of activated CD44 with its ligand, low m.w. hyaluronan, is involved in inflammation, but no role has been identified for this interaction in the regulation of an immune response to infection. In these studies, infection of C57BL/6 mice with Toxoplasma gondii resulted in increased expression of CD44 on T cells, B cells, NK cells, and macrophages, and a small percentage of CD4(+) T cells express an activated form of CD44. Administration of anti-CD44 to infected mice prevented the development of a CD4(+) T cell-dependent, infection-induced inflammatory response in the small intestine characterized by the overproduction of IFN-gamma. The protective effect of anti-CD44 treatment was associated with reduced production of IFN-gamma, but not IL-12, in vivo and in vitro. Furthermore, the addition of low m.w. hyaluronan to cultures of splenocytes or purified CD4(+) T cells from infected mice resulted in the production of high levels of IFN-gamma, which was dependent on IL-12 and TCR stimulation. Together, these results identify a novel role for CD44 in the regulation of IFN-gamma production by CD4(+) T cells during infection and demonstrate a role for CD44 in the regulation of infection-induced immune pathology.

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Autoreactivity plays a major role in the pathogenesis of RA. The rheumatoid factor has been and still is for now more than 50 years the only autoreactivity that is clinically applied in the diagnosis of RA. This well reflects the current way of thinking that a single antigen or a single cause drives an individual into disease. Although by now many other autoantigens and autoreactivities have been described, their discovery was always on the search for the one and only autoreactivity that causes RA. This includes also immune reactivities directed against xenogenic antigens. But, none of the known RA-associated autoreactivities is present in all RA patients and none of them occurs exclusively in RA. Thus, the observed sensitivities and specificities are well below 100%. Therefore, RA has often been postulated to consist of various immunological subentities with similar clinical symptoms. Nevertheless, none of the autoreactivities correlates with a distinct clinical feature or course of disease. It is about time to say good-bye to the idea that a single antigen or immunoreactvity causes and maintains rheumatoid arthritis. In this paper we present RA as the clinical outcome of an immune system that has shifted from a healthy to an autoimmune steady state. This is accomplished by many different reactivities and autoreactivities that occur either in parallel or one after the other. The entirety of the known RA-associated reactivities and (auto)antigens is presented in detail. The major RA-relevant autoantigens comprise BiP, citrulline, the Sa-antigen, hnRNP A2, p205, IgG, calpastatin, calreticulin, collagen and the shared HLA-DR epitope. The accumulation of factor--involving autoreactivities, cytokines, environmental and genetic factors--that challenge the normal regulatory mechanisms of the immune system lead to a regulatory catastrophe. In individuals developing the clinical features of RA the immune system has been regulated to a new--autoimmune--steady state. This attractor "rheumatoid arthritis" has many features of what has originally been described by Irun Cohen as the immunological homunculus: The healthy immune system is configured such as to direct its attention to major self-antigens. Thus it creates an autoreactivity to many autoantigens as a prerequisite for regulatory mechanisms that are sufficient to control them. The shift from the normal to rheumatoid attractor involves the inflammatory cytokines TNF-alpha, IL-1 and IL-6, autoreactive T- and B cells directed at a variety of synovial and systemic antigens, activated dendritic cells and macrophages, tissue destruction and genetic factors such as the association with shared epitope. Environmental factors involved may also, but do not necessarily, include infection. With the appearance of clinical features of RA, naive, potentially autoreactive T cells infiltrate the synovial compartment and become activated by dendritic cells and other APCs. The autoantigenic peptides that are presented to these T cells are derived from inflammatory cell and tissue destruction as well as from tissue repair and remodeling processes. These T cells proliferate and either provide help to B cells with the specificity to the same antigens or cause direct cytopathic tissue damage. Thereby, more and novel antigens are generated, released and presented again to naive or primed autoreactive T cells. These processes involving cytokines, tissue destruction and autoreactive T cells are sufficient to maintain RA even without the permanent presence of a triggering agent. The recursive autoimmune processes are well consistent with the finding of the many different autoreactivities in RA and their respective sensitivities and specificities. The massive influx of T cells into the arthritic joint is accompanied by the anergization of over 90% of T cells in this compartment--which further substantiates the concept of the RA attractor within the self-regulating immune system. Thereby, the RA-attracted immune system is not able to completely downregulate the inflammation and the local tissue damage/repair. Thus, the immune system is permanently stimulated and suddenly by chance shifts to a stable state different from the healthy system--reaching the wide fields of rheumatoid arthritis which in itself is self-sustaining as the healthy state before disease onset.

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OBJECTIVE: Double-stranded DNA (dsDNA) is a well-known target of autoantibodies in systemic lupus erythematosus (SLE). The majority of these autoantibodies are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. T cell responses to autoantigens, including DNA, have been reported only incidentally in SLE patients. Nevertheless, in murine SLE, naked DNA and complexed DNA (nucleosomes) are known to be recognized by T cells. This study aimed to characterize the antinucleosome response and its clinical impact on human SLE. METHODS: Nucleosomes were prepared from chicken erythrocytes. Sera from SLE and control patients were investigated by enzyme-linked immunosorbent assay (ELISA) for nucleosome-specific antibody responses. Peripheral blood mononuclear cells (PBMC) from SLE and control patients were analyzed by a kinetic T cell proliferation assay. PBMC were subsequently analyzed for nucleosome-specific T cell proliferation. RESULTS: Of 136 SLE patients, 56% were seropositive for antinucleosome antibodies. In contrast, only 3% of 309 control patients (with rheumatoid arthritis, mixed connective tissue disease, undifferentiated connective tissue disease, Lyme borreliosis, scleroderma, Sjögren's syndrome, ulcerative colitis, hepatitis B virus infection, or human immunodeficiency virus infection) were seropositive. Thus, the antinucleosome ELISA had a sensitivity of 56%, a specificity of 97%, and a diagnostic confidence of 90% when applied to SLE. It was therefore superior to an anti-DNA ELISA that demonstrated a 69% diagnostic confidence in the same population. Antinucleosome reactivity in SLE patients correlated significantly with disease activity (P < 0.0001), nephritis (P < 0.002), and psychosis (P < 0.02). When proliferation assays were applied, 14 of 26 SLE patients (54%) were positive for nucleosome-specific T cells that proliferated in response to their cognate antigen. A suppressed response was elicited in 3 SLE patients (12%); in these patients, the PBMC response to nucleosomes was lower than the proliferation of PBMC in the presence of culture medium only. PBMC from the remaining 9 SLE patients (35%) were nonresponsive to nucleosomes in either way. Responding, nonresponding, and suppressed populations differed from each other significantly (P < 0.0001). None of the PBMC from 7 healthy donors and 10 control patients could be stimulated with nucleosomal antigens. CONCLUSION: We present evidence that nucleosomes are major autoantigens in human SLE and that antinucleosomal antibodies are highly specific for the disease. The antinucleosome ELISA has been shown to be superior to the anti-dsDNA ELISA and may thus be a significantly better tool for diagnosing SLE. Nucleosome-specific T cells in SLE patients may help B cells class switch to IgG and undergo affinity maturation.

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OBJECTIVE: The p205 autoantigen and interleukin-2 (IL-2) function synergistically to stimulate T lymphocytes from patients with rheumatoid arthritis (RA), and a p205-derived amino acid sequence is identical to an immunoglobulin sequence located within a domain that is reactive with rheumatoid factors (RF). This study was conducted to analyze in detail the T cell immune response against p205 and to investigate whether immunity to p205 may play a role in T cell-mediated immunopathology in active RA. METHODS: Cibachron blue, protein A-Sepharose, and gel filtration on Sephacryl were used successively to enrich p205 from synovial fluid (SF). T lymphocytes from RA patients were isolated from the peripheral blood (PB), lymph nodes, and SF, and p205 and peptides derived from known sequences were assessed by T cell proliferation assays in the presence of IL-2. RESULTS: P205-specific proliferation of T cells was observed in PB as well as in SF. When p205 was isolated from RA SF, proliferation of RA T cells peaked on day 3. With p205 purified from SF from trauma patients, there was a significant shift of the maximum T cell proliferation to day 8. T cells were of CD4 or CD8 phenotype, and B cells did not proliferate to a significant degree. The T cell response to p205 was always higher for SF mononuclear cells (SFMC) compared with PBMC (P < 0.001). In 1 RA patient who underwent repeated leukapheresis, this led to a reproducible decline in p205-specific T cell proliferation to control levels. PB T cells specifically proliferating in response to p205 were detected in 20 of 32 RA patients (63%). Of 26 patients with other inflammatory rheumatic diseases, only 1 showed a minor response to p205, while normal donors did not demonstrate a significant T cell proliferation. A synthetic p205-derived peptide, with an amino acid sequence identical to an immunoglobulin sequence located in the area where RF binds, was reactive with T cells from RA patients. CONCLUSION: P205 appears to be a major target of autoreactive T cells in RA. P205-specific T cells are primed and more abundant at the site of inflammation. As a T cell target in RA, p205 may well be an antigen involved in the initiation of RF production.

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Dendritic cells (DCs) are professional antigen-presenting cells (APCs), and classical DCs, such as Langerhans cells (LCs) or interdigitating DCs (IDCs) are known to be the most potent stimulators of T lymphocytes. Earlier, several groups described the generation of DCs from monocytes, starting with peripheral blood mononuclear cells (PBMCs), adherent cells or magnetic bead-purified CD14+ cells. Although modifications of the original protocols have already been described, some questions relevant to clinical application and basic studies have not yet been addressed. Granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL4) appear to be necessary, but are not sufficient for the differentiation of monocyte-derived dendritic cells (MoDCs), as indicated by the failure to generate such cells under serum-free conditions. Using adherence purified monocytes, we first investigated the amount of GM-CSF and IL4 required for the differentiation of DCs. Consecutive kinetic studies during the differentiation period were designed to demonstrate how monocytes acquire the phenotype and function of DCs. The results showed that small amounts of GM-CSF and IL4 were required to generate MoDC which acquired their phenotype and function within 4 days. IL13 may substitute for IL4, whereas IL10, TNF alpha or IFN gamma inhibited the generation of MoDCs.

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OBJECTIVE: The autoantigen p68 is a target of autoantibodies as well as autoreactive T cells with a high specificity in rheumatoid arthritis (RA). The binding characteristics of the autoantibodies to their antigen were now analysed biochemically and cytologically. METHODS: Deglycosylation techniques as well as lectin and sugar competition experiments were performed to p68 to discover if the antibodies detected a glycoepitope, Its antigenicity was investigated applying anti-p68 antibodies derived from RA patients in comparison with polyclonal rabbit anti-p68 antibodies. RESULTS: p68 specific antibodies from RA patients did not to bind to p68 that had been deglycosylated by alkaline beta-elimination, O-glycosidase or periodate treatment. In contrast, binding of p68 specific antibodies raised in rabbit was unaffected by either deglycosylation protocol. Furthermore, lectins specific for the carbohydrate N-acetylglucosamine competed with p68 specific antibodies from RA patients for antigen bindings. N-acetylglucosamine by itself also competed with patient derived anti-p68 antibodies for p68 binding. Again, rabbit and anti-p68 antibodies did not elicit these competitive effects. Applying cytoimmunofluorescence, p68 was present in the cytoplasm or endoplasmic reticulum and also in low abundance on the cell surface. Under heatshock conditions, p68 was detectable in the nucleus. CONCLUSIONS: Autoimmunity to p68 during RA is carried by anti-carbohydrate autoantibodies. The carbohydrate modification of p68 appears to be N-acetylglucosamine, which may reflect the regulation of intracellular localisation of the antigen. It is hypothesised that a shift in glycosylation pattern accompanied by an unphysiological localisation of the antigen could trigger antigenicity of p68 during the pathogenesis of IRA.

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OBJECTIVE: A 68k autoantigen has been identified by specific antibodies from patients with rheumatoid arthritis (RA). This study considered whether or not this antigen is a target for T cells and thus may play a part in T cell mediated immunopathology of active RA. METHODS: The 68k antigen was isolated and used in a nitrocellulose bound form to stimulate T cells. Proliferation of T lymphocytes of peripheral blood as well as synovial fluid was measured. RESULTS: Peripheral blood T cells specifically proliferating against the 68k antigen were detected in 19 of 27 patients with RA (70%). For T cells isolated from peripheral blood, proliferation peaked on day 10. When T cells were isolated from actively inflamed synovial fluid, the proliferation kinetics shifted to a peak on day 3. Blockade of HLA class II antigens resulted in an increase of proliferation in the case of HLA-DP. Applying HLA-DP specific antibodies capable of inhibiting antigen presentation mediated by this molecule, T cells of 17 of 27 RA patients (63%) proliferated to a higher extent than with the 68k antigen alone. The phenomenon that an increased proliferation occurred upon blockade of a particular HLA class II family member was also demonstrated for DQ and DR: the 68k antigen likewise stimulated T cells restricted for DP or DQ, respectively. CONCLUSIONS: The novel 68k antigen is a target of both T and B cellular immune responses and as such could play a part in the immune dysfunction of RA. The finding that blocking of certain HLA class II molecules functioning in antigen presentation (for example, via HLA-DQ) results in a higher instead of lower proliferation in vitro, may argue for the presence of antigen specific suppressive T cells.

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Despite commonly applied clinical criteria, the early diagnosis of rheumatoid arthritis (RA) often remains difficult, thus delaying on suitable early treatment. In search for a test furthering the early and reliable diagnosis of RA, we have screened for novel disease specific autoantibodies. To this end proteins were isolated from synovial membranes and other tissues following a special protein purification protocol, and these were separated electrophoretically. Western blots were then used to screen sera of RA patients and of individuals suffering from other rheumatic diseases for antibodies to any of these proteins. The most prominent RA specific immunoreaction was with a 68k antigen, occurring in 110 of 167 RA patients (sensitivity is 66%). The antibody could also be identified in seronegative RA patients but not in healthy individuals (55 tested), in only 1 SLE patient of a group of 98 patients with other rheumatic diseases and in 1 out of 22 HIV patients, resulting in a specificity of 99%. Moreover, the anti-68k antibody could be correlated with a more severe course of RA. 13 out of 20 anti-68k positive RA patients (58%) had subcutaneous nodules, while only 2 out of 11 anti-68k negative (20%) did. The mean sedimentation rate of these antibody positive patients was 51 mm/h and 26 mm/h for the negative respectively. The 68k antigen was shown to be present in all human tissues investigated and is probably ubiquitously expressed. It is either located in the endoplasmatic reticulum or cytoplasm or both. Its isoelectric point is 5.1. It proved to be O-glycosylated and contains only one or a few sugar residues as the untreated and the deglycosylated antigen identical electrophoretical mobilities. The patient derived anti-68k antibodies were directed against the sugar residue: deglycosylation of the antigen completely abolished its immunoreactivity. N-acetylglucosamine competes with the antibody for binding the 68k antigen. The antigen physicochemical data of the 68k antigen argue against identity with one of the autoantigens in this molecular mass range already known to be associated with RA or other autoimmune diseases. It is neither identical to the 62k human antigen (EBNA-1) nor to RA33 (A2hnRNP), the 50k Sa antigen or the Hsp70 class of heats-hock proteins. It is argued that the particular method of protein purification applied in combination with separation via SDS-PAGE in the presence of urea, made it possible to detect a hitherto unidentified antigen. Considering the striking disease specificity of the anti-68k antibody it is now worthwhile to look for corresponding autoreactive T cells in order to analyse its role in the pathogenesis of RA.

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OBJECTIVE: To improve the understanding of the pathogenesis of rheumatoid arthritis (RA) by identifying novel, disease specific autoantibodies. METHODS: Total protein preparations from synovial membranes were separated electrophoretically and immunoblotted. Sera from RA patients were screened for predominant immunoreactions by blotting. A 68 kDa antigen target of the most predominant reaction was detected and further characterised. RESULTS: The dominant immunoreaction in most of the RA sera tested was with a 68 kDa antigen. The antigen is probably ubiquitously expressed. It has an isoelectric point of 5.1, is O-glycosylated, and is located in the endoplasmic reticulum, the cytoplasm, or both. Antibodies to the 68 kDa autoantigen were present in 64% of 167 RA patients tested, and could also be detected in seronegative RA patients, but were present in only 1% of 98 patients with other rheumatic diseases. They could not be detected in 55 healthy controls. CONCLUSIONS: Because of its high sensitivity (64%) and specificity (99%), the anti-68 kDa autoantibody not only provides another valuable parameter for diagnosis, but also represents an antibody that may be involved in the pathological mechanisms leading to RA. This hypothesis can be tested by investigating if 68 kDa specific T cells are present in RA patients.

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Changes in choices of preferred playmates by 3- and 4-year-old children were observed over the course of a school year. All of the children in this study participated in planned, fully mainstreamed, same-age preschool classes. Sociometric assessments were obtained from all children without disabilities in October, February, and May of the school year; peer nominations of three "best friends" were obtained at the beginning and end of the school year. The 3-year-old children showed a general decline in the ratings given to all of their peers over the course of the school year. Four-year-old children showed significant preferences for same-sex peers without disabilities as playmates. The implications of these findings for integrated programs are discussed.

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Screening of a cDNA library (prepared in lambda gt11) of the blood stages of Plasmodium chabaudi chabaudi (AS) with immune serum has revealed an antigen the elicits a strong antibody response in infected mice. The clone (clone 6) expressing that antigen contains a 0.7 kb insert and produces a beta-galactosidase fusion protein of about 150 kDa. In Western blot analysis performed on parasite extracts, monoclonal antibodies and polyclonal sera prepared against the fusion protein revealed that the fusion protein contains part of a malarial protein of 93 kDa. Northern hybridization with clone 6 insert as probe detected a plasmodial RNA of about 3.2 kb, which could well code for a protein of this size. The insert hybridized to a single EcoRI fragment and a single HindIII fragment in genomic Southern blotting, suggesting that the gene is present in one copy in the P. chabaudi genome. The DNA sequence of clone 6 insert predicts a hydrophilic, acidic polypeptide consisting of seven repeats of 23-34 amino acids rich in lysine (24%) and aspartic acid (17.5%).

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We describe a flow cytometric method for the quantitation of small numbers of cells that facilitates proliferation studies in microcultures. A constant number of fluorescent latex microspheres/sample is added to single cell suspensions prepared from the cultures. By flow cytometric analysis, cells are easily distinguishable from the microspheres and can be quantitated on the basis of their light scattering and fluorescence properties is contour plots. As the number of microspheres/sample is known, the relative proportions of cells and microspheres, respectively, can be converted into absolute cell numbers. This quick method is useful for any type of studies where cell numbers less than 1 x 10(5) in a small volume are to be determined.

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