Elevated IgG Antibody Levels to the Mycobacterial 65-kDa Heat Shock Protein Are Characteristic of Patients with Rheumatoid Arthritis

We have previously demonstrated raised levels of IgG and IgA antibody to the mycobacterial 65-kDa heat shock protein (hsp) in the sera of patients with rheumatoid arthritis (RA). We have now attempted to determine whether this phenomenon is specific for RA, and whether it is seen only with the mycobacterial homologue of this particular hsp gene family. We therefore screened antibody levels to the mycobacterial and Escherichia coli hsp 65, and the mycobacterial, E. coli, and human hsp70, in sera from RA, systemic lupus erythematosus (SLE), tuberculosis (TB), ankylosing spondylitis (AS), Crohn's disease, and control donors. RA sera show the greatest increase in IgA binding to the mycobacterial hsp65, but no increase in IgA binding to the E. coli homologue. Similarly, only RA and TB sera show increased IgG binding to the mycobacterial hsp65, and we have shown previously that the titre is greater in RA. In contrast, the use of mycobacterial and E. coli hsp70 preparations as control bacterial hsp gene products has shown that RA patients do not differ from TB or SLE patients in their antibody binding to these proteins. Moreover, neither IgA nor IgG antibody to the human hsp70 in RA sera were higher than in TB, and the IgA binding was not higher than in SLE. These findings suggest that elevated IgG antibody levels to the mycobacterial hsp65 shows some disease specificity, and further studies with the human homologue and at the T-cell level are required.     

Circulating antibodies to heat-shock protein 60 in Crohn''s disease and ulcerative colitis.

Heat-shock proteins (HSPs) are highly conserved immunogenic intracellular molecules that are induced by inflammatory mediators and may induce autoimmune phenomena in vivo. We have recently demonstrated the increased expression of HSP-60 in the colonocytes of patients with ulcerative colitis. To study further the role of HSP-60 in inflammatory bowel disease, we have now measured antibodies to recombinant mycobacterial HSP-65 (a member of the HSP-60 family) in patients with Crohn's disease, ulcerative colitis, healthy volunteers and, as disease controls, patients with confirmed bacterial diarrhoea. In comparison with healthy controls (n = 20; median level of 89 ELISA units; range 24-292), serum IgA HSP-60 antibodies were elevated in Crohn's disease (n = 21; 157; 57-364; P < 0.05) and active ulcerative colitis (n = 16; 188; 58-373; P < 0.01) but not bacterial diarrhoea (n = 10; 106; 51-285). Increased IgA HSP-60 antibody levels in patients with inflammatory bowel disease may occur as the result of HSP release from injured gut epithelium; alternatively, increased intestinal permeability could facilitate mucosal access of luminal antigens and the generation of cross-reactive anti-bacterial HSP antibodies.     

Circulating antibodies to the 60-kD heat shock protein (hsp) family in patients with Helicobacter pylori infection

Whilst the mechanism by which Helicobacter pylori causes different gastroduodenal diseases is uncertain, strains producing the cytotoxin-associated protein (CagA) have greater pathogenicity. Hsps are immunogenic molecules induced by inflammatory mediators. The aim of this study was to assess pathogenicity of hsp antibodies in H. pylori-infected patients. ELISA techniques were used to assay sera of H. pylori-positive patients with gastritis, gastric atrophy, duodenal or gastric ulcer, and H. pylori-negative controls, for antibodies to CagA and to human, mycobacterial, and in 20 sera, H. pylori (hspB) 60-kD hsp. IgA antibodies to mycobacterial hsp60 in atrophy patients were elevated compared with patients with gastritis (P < 0.05) and with H. pylori-negative controls (P < 0.0005). IgA antibodies to human hsp60 in gastric atrophy patients were elevated compared with H. pylori-negative controls (P < 0.05). Patients with atrophy (P < 0.0005) and gastritis (P < 0.05) who were CagA-positive had raised titres of anti-mycobacterial hsp60 IgA antibodies compared with controls. IgA antibody levels to hspB were positively correlated with those to mycobacterial hsp60 (mhsp60) (P < 0.05) and human hsp60 (hhsp60) (P < 0.005). IgA antibodies to hsp60 are associated with gastroduodenal disease, particularly gastric atrophy, in H. pylori-infected patients. Increased humoral responses to hsp60 could either contribute to gastric atrophy or result from greater gastric mucosal damage induced by CagA-positive strains of H. pylori.     

Antibodies against the human heat shock protein hsp70 in patients with severe coronary artery disease

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n = 99) and healthy subjects (n = 99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400-1141) vs. 630 (326-1152) AU/mL, P = 0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p < 0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60kDa hsps.     

Peptide recognition, T cell receptor usage and HLA restriction elements of human heat-shock protein (hsp) 60 and mycobacterial 65-kDa hsp-reactive T cell clones from rheumatoid synovial fluid.

A commonly held postulate regarding the etiology of rheumatoid arthritis (RA) is that of antigenic mimicry. Recent interest has focused on the mycobacterial 65-kDa heat-shock protein (hsp) as a putative causal agent. The 65-kDa hsp has over 40% sequence homology with the human hsp 60, and elevated synovial T cell responses to both antigens have been demonstrated in RA and juvenile rheumatoid arthritis patients. Such T cells should, therefore, be specific for shared epitopes on the two antigens. To investigate this, we screened synovial fluid mononuclear cells from two early RA patients with peptides of the 65-kDa hsp which have the greatest homology with the human hsp 60. We also raised a panel of T cell clones from one of the patients with the 65-kDa hsp. The synovial T cell population from both patients and one of the T cell clones recognized a peptide representing the amino-acid sequence 241-255. This clone also responded to the peptide of the equivalent human sequence, and was restricted by HLA-DQ. A second T cell clone recognized an adjacent epitope (amino acid sequence 251-265) which is also highly homologous with the human sequence, but this clone was restricted by HLA-DR. The clones utilized different V beta gene segments but the same D beta and J beta gene elements, and both exhibited specific cytotoxicity against autologous antigen-pulsed macrophages. Our findings, therefore, do not disagree with the postulate that autoimmune disease could possibly be triggered by bacterial epitopes with homology to self protein. However, it is also noted that there are alternative interpretations of this data.     

Relationship between disease severity and responses by blood mononuclear cells from patients with rheumatoid arthritis to human heat-shock protein 60

The hypothesis that T-cell responses to the 60 000 MW family of heat-shock proteins (hsp) may be related to the severity of rheumatoid arthritis (RA) was examined. Peripheral blood mononuclear cells (PBMC) from most normal individuals and both early and established RA patients proliferated in vitro in response to human hsp 60 and mycobacterial hsp 65 as well as tetanus toxoid (TT) and mycobacterial purified protein derivative (PPD). PBMC from some patients with established RA gave responses to hsp 60 that were above the normal range and/or peaked earlier than PBMC from normal individuals. The responses of PBMC from established RA to hsp 65, but not PPD or TT, were also higher than those from normal individuals, but the peak responses to all three antigens appeared delayed. Thus a selective increase in responsiveness to hsp 60 develops with disease duration in many RA patients. Six assessments of disease activity and severity were made but apart from rheumatoid factor titre, they were unrelated to the proliferative response. Similarly, disease activity and severity did not differ between those RA patients whose hsp 60 stimulated cells produced interferon-gamma and those who did not, although patients whose hsp 60-stimulated T cells produced interleukin-4 (IL-4) and/or IL-10, appeared to have less disease activity and severity than those who did not. Significant negative correlations were found between IL-10 production by hsp 60-stimulated cells and disease assessments. It is considered that RA is less severe in those patients whose hsp 60-stimulated cells produce T-helper 2 type cytokines.     

Heat shock protein 70 (hsp70) as a major target of the antibody response in patients with pulmonary cryptococcosis

Cryptococcus neoformans causes infection in individuals with defective T cell function, such as AIDS, as well as without underlying disease. It has been suggested that humoral as well as cellular immunity might play an important role in the immune response to C. neoformans infection. We have recently shown, using immunoblotting, that the 70-kD hsp family of C. neoformans was the major target molecule of the humoral response in murine pulmonary cryptococcosis. In this study we also used immunoblotting to define the antibody responses in the sera of 24 patients with pulmonary cryptococcosis: 21 proven and three suspected diagnoses. Anti-C. neoformans hsp70 antibody was detected in 16 of 24 (66.7%) patients with pulmonary cryptococcosis. Fourteen of 17 (82.3%) patients with high antigen titres (> or = 1:8) and two of seven (28.6%) patients with low titres (< or = 1:4) had detectable levels of anti-hsp70 antibody. Sera from patients positive for anti-hsp70 antibody showed high titres in the Eiken latex agglutination test for the detection of serum cryptococcal antigen. Our results indicate that the 70-kD hsp family from C. neoformans appears to be a major target molecule of the humoral response, not only in murine pulmonary cryptococcosis, but also in human patients with pulmonary cryptococcosis.     

Antibodies against human heat-shock protein (hsp) 60 and mycobacterial hsp65 differ in their antigen specificity and complement-activating ability.

Although complement activation appears to have an important role both in the early and late phases of atherosclerosis, the exact mechanism of the initiation of this activation is still unknown. Since injuries of the endothelial cells are known to result in increased stress-protein expression we tested the complement-activating ability of recombinant human 60 kDa heat-shock protein (hsp60). Human hsp60 was found to activate the complement system in normal human serum in a dose-dependent manner. Activation took place through the classical pathway. The lack of complement activation in agammaglobulinemic serum indicates that the classical pathway is triggered by anti-hsp60 antibodies. Hsp60 activated complement in the sera of 74 patients with coronary heart disease as well, and a strong positive correlation (r = 0.459, P < 0.0001) was found between the extent of complement activation and the level of anti-hsp60 IgG antibodies but there was no correlation to the level of anti-hsp65 IgG antibodies. Further distinction between anti-hsp60 and anti-hsp65 antibodies was obtained from competitive ELISA experiments: binding of anti-hsp60 antibodies to hsp60-coated plates was inhibited only by recombinant hsp60 and vice versa. Our present findings indicate that anti-hsp60 and anti-hsp65 antibodies are distinct, showing only partial cross-reactivity. Since complement activation plays an important role in the development of atherosclerosis and the levels of complement-activating anti-hsp60 antibodies are elevated in atherosclerosis-related diseases, our present findings may have important pathological implications.     

Serological markers to differentiate between ulcerative colitis and Crohn's disease.

AIM--To assess prospectively the value of three serological tests for differentiating between ulcerative colitis and Crohn's disease, used either alone or combined. METHODS--Coded serum samples from 63 patients with ulcerative colitis and 67 patients with Crohn's disease were analysed. Detection assays for the presence of perinuclear antineutrophil cytoplasmic antibodies (pANCA), serum agglutinating antibodies to anaerobic coccoid rods, and specific IgG antibodies against a Kd-45/48 immunological crossreactive mycobacterial antigen complex (ImCrAC) were studied. Sensitivity, specificity, pre- and post-test probabilities, likelihood ratios, and predictive values of each of these serological tests were determined. RESULTS--The sensitivity and specificity of the pANCA test for the diagnosis of ulcerative colitis were 61 and 79%, respectively. The serum agglutination test for anaerobic coccoid rods had a sensitivity of 42% and a specificity of 89% for a diagnosis of Crohn's disease. The sensitivity of specific IgG antibodies against Kd-45/48 ImCrAC in diagnosing Crohn's disease was 70% and specificity 60%. Although 100% specificity was achieved by combining all three tests in a small group of patients with Crohn's disease (n = 20), combining two or more tests had no additive clinical value. No correlation was found between the presence of any one of these antibodies and disease activity, duration, or localisation of disease. Surgery or medical treatment did not influence the presence of antibodies or the antibody titre. CONCLUSIONS--The value of these tests in the differential diagnosis between ulcerative colitis and Crohn's disease is limited, but the high predictive values and specificities of different tests for both diseases suggest that these tests may be of help in studying disease heterogeneity and in defining different subgroups of patients with different pathogenesis.     

T helper type-1 (Th1)/Th2 profiles of peripheral blood mononuclear cells (PBMC); responses to antigens of Chlamydia trachomatis in subjects with severe trachomatous scarring

The 65-kD hsp from Mycobacterium tuberculosis has been reported to induce an autopathogenic subset of T cells in at least two animal models of autoimmune disease. Reports of increased expression of human hsp60 in the inflamed synovial tissue of rheumatoid arthritis (RA) patients, increased proliferation of RA synovial fluid T cells to mycobacterial hsp65, and increased levels of anti-mycobacterial hsp65 antibody in synovial fluid, have suggested that the highly homologous human (hu) hsp60 may be recognized as an autoantigen in RA patients. In the present study, we have examined by ELISA the serum IgG antibody levels to mycobacterial hsp65 and hu hsp60, as well as to the Escherichia coli hsp60, groEL, in patients with RA, systemic lupus erythematosus (SLE), Reiter's syndrome, active tuberculosis, and normal controls. In all these groups, the levels of anti-groEL and anti-hu hsp60 were significantly higher than the anti-mycobacterial hsp65. Anti-hu hsp60 was positively correlated with anti-groEL, but not with anti-mycobacterial hsp65. Anti-hu hsp60 was competitively inhibited by either soluble groEL or hu hsp60, but little or none by mycobacterial hsp65. Reiter's sera were found to have somewhat higher levels of anti-groEL and anti-hu hsp60 than did normal controls. We conclude that IgG anti-hu hsp60 autoantibodies arise primarily as a consequence of the humoral immune response to E. coli groEL through the recognition of cross-reactive epitopes.     

ANTIBODIES AGAINST THE HUMAN HEAT SHOCK PROTEIN hsp70 IN PATIENTS WITH SEVERE CORONARY ARTERY DISEASE

Heat shock proteins (hsps) play complex role in the function of the immune system, they can activate both humoral and cellular immune response, as well the complement system. Although autoimmunity to hsp70 was implicated in certain autoimmune diseases and other conditions, the exact role of anti-hsp70 antibodies is not known. It was demonstrated by our previous work and other's findings that antibodies against the 60 kDa hsps are strongly associated with coronary atherosclerosis and carotis disease. It is also known that there is increased hsp70 expression at different sites of atherosclerosis. Therefore our aim was to study whether level of anti-hsp70 antibodies correlate with the presence of severe coronary artery disease (CAD). We measured and compared anti-hsp70 IgG antibody levels in CAD patients (n=99) and healthy subjects (n=99) with ELISA. The frequency of these antibodies was high in both groups and there was no significant difference in the median level of anti-hsp70 antibodies between patients with severe CAD and controls (653 (400–1141) vs. 630 (326–1152) AU/mL, P=0.337). Adjustment for age, sex, BMI and lipid parameters did not change this result. Furthermore we did not find a correlation between anti-hsp70 antibody levels and certain risk factors of CAD (age, lipid parameters, body mass index, C-reactive protein, gender, smoking, diabetes and anti-hsp60 antibodies). By contrast, in accordance with our previous findings, anti-hsp60 and anti-hsp65 antibody levels were significantly higher in CAD patients, compared to this control group (p<0.0001 for both variables). We did not find any correlation between the levels of anti-hsp70 and anti-hsp60 or anti-hsp65 antibodies either in the patients or the controls. The exact role of hsp70 in atherosclerosis is controversial, but we suggest that humoral immunity against human hsp70 does not contribute to coronary atherosclerosis in contrast to antibodies against 60 kDa hsps.     

Antibody responses to the chlamydial heat shock proteins hsp60 and hsp70 are H-2 linked.

The effects of both H-2 and non-H-2 genes on antibody responses to two Chlamydia trachomatis heat shock proteins (hsp60 and hsp70) were investigated. These chlamydial proteins are homologs of Escherichia coli GroEL (hsp60) and DnaK (hsp70) and are highly sequence conserved between bacterial and mammalian sources. Antibody responses among 17 different strains of mice immunized with C. trachomatis serovar B and serovar C elementary bodies were evaluated by immunoblot, radioimmunoprecipitation and enzyme-linked immunosorbent assay. Antibody responses to the two proteins displayed host genetic restriction. Of six distinctive H-2 haplotypes, only H-2d generated high antibody responses to hsp70. Five of the six H-2 haplotypes, i.e., H-2a, H-2d, H-2k, H-2q, and H-2s, produced high antibody responses to hsp60. Only the H-2b-bearing strain had low antibody responses to hsp60. By using congenic and H-2 recombinant strains, the genes responsible for regulating antibody responses to hsp70 and hsp60 were mapped to the K-IA region of the H-2 locus. In F1 hybrid crosses between high and low responders, high responses to hsp60 and hsp70 were dominant traits. Other genes outside the H-2 locus also influenced antibody responses to hsp60 and hsp70, since inbred strains of identical H-2 but different background genes displayed variable antibody responses to the proteins. The genetic control of murine immune responses to C. trachomatis hsp60, a putative chlamydial immunopathologic antigen, suggests that a similar genetic mechanism may also exist in humans, and this observation may help to explain the observed variability in the spectrum of chlamydial diseases seen in humans.     

Nucleotide sequence analysis and seroreactivities of the 65K heat shock protein from Mycobacterium paratuberculosis.

Mycobacterium paratuberculosis is the causative agent of Johne's disease, a chronic enteritis in ruminants. It has also been implicated as a possible cause of Crohn's disease, an inflammatory bowel disease of unknown etiology. The mycobacterial 65K heat shock proteins (hsp-65K) are among the most extensively studied mycobacterial proteins, and their immunogenic characteristics have been suggested to be the basis for autoimmunization in chronic inflammatory diseases. In this context, we isolated and sequenced the hsp-65K-encoding gene from our M. paratuberculosis PTB65K genomic library. A high degree of identity was found between the open reading frame (ORF) of the PTB65K gene and those of Mycobacterium tuberculosis (89.6%), Mycobacterium leprae (86.6%), and Mycobacterium avium 18 (98.8%). The amino acid sequence alignment of the PTB65K protein with the hsp-65K homologs revealed that the M. tuberculosis and M. leprae proteins each differed by 36 amino acid residues and that the M. avium 18 protein differed by 8 residues. We also investigated the humoral immune responses of animals with Johne's disease and patients with Crohn's disease against the recombinant PTB65K antigen. Immunoblot analysis showed that sera from only 3 of 10 clinically ill and 5 of 25 subclinically ill cows reacted with PTB65K. In addition, sera from two of two sheep and one of two goats with clinical symptoms of Johne's disease also reacted with PTB65K; 0 samples from 10 normal cows reacted. In humans, sera from 7 of 13 patients with Crohn's disease, 3 of 4 with tuberculosis, 5 of 6 with leprosy, 5 of 12 with non-inflammatory bowel disease, and 0 of 4 with ulcerative colitis reacted with the recombinant PTB65K antigen. These results indicate that this PTB65K heat shock protein is uninformative when used for serodiagnosis of Johne's disease in animals. However, in humans, the high intensity of antibody reactions of some sera from Crohn's disease patients compared with that from noninflammatory bowel disease patients showed a positive correlation with mycobacterial diseases.     

A solid-phase radioimmunoassay for measurement of circulating antibody titres to wheat gliadin and its subfractions in patients with adult coeliac disease

A solid-phase radioimmunoassay for the measurement of circulating antibody titres to wheat gliadin is described. Using this assay, we have measured antibody titres to unfractionated gliadin in normal healthy controls, in coeliac patients on a gluten-free or a normal diet, and in patients with ulcerative colitis and Crohn's disease. High titres of antibodies to unfractionated gliadin were observed only in the patients with untreated coeliac disease. Antibody titres to alpha, beta, gamma and omega gliadin subfractions were measured in patients with untreated coeliac disease and compared with titres in normal controls. Patients with untreated coeliac disease had higher antibody titres to the gliadin subfractions. No specific pattern of circulating antibody titres to gliadin subfractions was observed in the untreated coeliac patients which would provide a diagnostic profile. These results suggest shared antigenicity between the gliadin subfractions.     

Specificity of antibodies induced after immunization of mice with the mycobacterial heat shock protein of 65 kD.

We have previously shown in mice and monkeys that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a strong in vivo helper effect when conjugated to synthetic peptides or bacterial oligosaccharides and given in the absence of any adjuvants. Considering the degree of homology existing in the phylogeny among hsp belonging to the same family, we studied whether antibodies induced in mice with this protocol of immunization with the mycobacterial 65-kD hsp (hsp65) would cross-react, and to what extent, with hsp homologues from other origins, notably with the Escherichia coli GroEL protein and with the human homologue (hsp60). The results obtained show that antibodies to the mycobacterial hsp65 cross-reacted with the E. coli GroEL protein, both in ELISA and Western blot experiments, but not with the human hsp60. In competitive ELISA experiments, the binding of these antibodies to solid-phase hsp65 was very effectively inhibited by low concentrations of the mycobacterial hsp65; however, for human hsp60, 100 times higher concentrations were required in order to obtain similar patterns of inhibition. Finally, murine antibodies to the mycobacterial hsp65 always failed to give positive results in Western blot experiments using extracts of murine cells. Taken together, these data suggest that, after immunization of mice with the mycobacterial hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, anti-hsp65 antibodies are induced which cross-react well with hsp homologues from other prokaryotes (e.g. E. coli GroEL), but which weakly bind the human hsp homologue. These results may have implications for the potential use of microbial hsp molecules in the design of conjugated vaccine constructs.     

A synthetic 10-kD heat shock protein (hsp10) from Mycobacterium tuberculosis modulates adjuvant arthritis

The heat shock protein, hsp10, is an abundant protein in Mycobacterium tuberculosis (Mtb), its nucleotide sequence encoding a protein of 99 amino acids with a molecular mass of 10±7kD. This sequence is phylogenetically conserved, being represented by the GroES homologue of Escherichia coli. Hsp 10 and GroES are members of the chaperonin 10 family of molecular chaperones, and GroES is necessary for the optimal activity of GroEL, a member of the chaperonin 60 family and the E coli homologue of mycobacterial hsp65. Since hsp65 has been implicated in both experimental and human rheumatoid arthritis, we aimed to assess the immunomodulatory effects of its co-chaperonin, hsp10, in experimental arthritis. Our results show that an aqueous solution of a mycobacterial hsp10 delayed the onset and severity of adjuvant-induced arthritis in rodents when administered after disease induction but before joint involvement occurred. This biological activity was specific for the hsp10 of Mtb, since neither GroES nor the rat homologue was effective. Using synthetic hsp10 fragments, the activity was localized to the N-terminal region of the molecule. Assessment of circulating antibody levels to mycobacterial hsp10 and hsp65 indicated that all arthritic rats had increased litres to both hsp10 and hsp65: hsp10-treated rats showed further elevation of this humoral response not only to hsp10 but also to hsp65 when compared with the untreated arthritic control. This is the first report of the immunomodulatory activity of mycobacterial hsp10 in experimental arthritis, and exhibits a potential role for this co-chaperonin in pathophysiological situations.     

Immunogenicity of the meningococcal stress protein MSP63 during natural infection.

Acute- and convalescent-phase sera from 40 patients with meningococcal disease were evaluated for immunoreactivity with the meningococcal member of the hsp60 stress protein family. The IgG response was measured by ELISA, using bacterial cell lysate of the corresponding patients' strain, and purified hsp60 proteins from Neisseria meningitidis (MSP63), Escherichia coli (GroEL) and Mycobacterium bovis BCG (65K) as antigens. Analysis of the antibody responses revealed that 24/35 patients (69%) with elevated anti-meningococcal titres, generated anti-MSP63 antibodies during the time course of infection. Twelve of these patients generated antibodies specific for MSP63, in six patients anti-MSP63 levels exceeded anti-GroEL/65K antibodies. In the remaining six patients, equal levels of anti-MSP63 and anti-GroEL/65K were measured. We conclude that MSP63 is expressed and immunogenic during natural meningococcal infection, and that individual subjects have a restricted response to the antigen, resulting in the recognition of Neisseria-specific hsp60 epitopes and/or cross-reactive hsp60 determinants.     

Secretory granule autoantigen in insulin-dependent diabetes mellitus is related to 62 kDa heat-shock protein (hsp60).

Recent studies in NOD mice suggest that cellular and humoral responses against beta cell protein(s) cross-reactive with mycobacterial heat-shock protein, hsp60, are implicated in the development of autoimmune diabetes. However, this putative, hsp60-related autoantigen has not yet been identified nor have the preceding events triggering the autoimmunity against it. Our recent studies show that antibodies to the mammalian hsp60 bind specifically to the 62 kDa protein located to insulin secretory granules and mitochondria of pancreatic beta cells of healthy mice [1]. In islets of prediabetic NOD mice affected by insulitis, the cellular distribution of this hsp60-related antigen was found to be altered. In the present report, we have examined whether this endogenous hsp60-related protein of secretory granules serves as an autoantigen in type I diabetes. The results of Western blot analysis indicate that diabetic mice sera show reactivity to a 62 kDa islet cell antigen. The NOD mice sera that were positive in detection of the 62 kDa islet cell antigen were also able to recognize the recombinant human hsp60. Immunogold electron microscopy revealed that diabetic NOD mouse sera, cross-reactive to human recombinant hsp60, recognize the antigen located in secretory granules of beta cells. Double-immunogold labelling demonstrated that antigens recognized by both diabetic NOD mice sera and monoclonal hsp60 antibodies co-localized in the same secretory granules of beta cells. Preincubation of islet cell sections with one type of antibody blocks subsequent binding of the other, indicating that epitopes recognized by both antisera on these proteins are shared. Moreover, preadsorption of diabetic sera with the recombinant human hsp60 abolished labelling of secretory granules. These results indicate that the hsp60-related protein of beta cell secretory granules is an autoantigen in type I diabetes in NOD mice.     

Antibody Response of Restricted Isotype to Heat Shock Proteins in Juvenile Chronic Arthritis

Synovial fluid and peripheral blood mononuclear cells from juvenile chronic arthritis (JCA) patients have previously been shown to exhibit substantial proliferative responses to both human and mycobacterial heat shock protein (hsp) 65. We investigated the nature of the antibody response to mycobacterial and E. coli hsp 65 and human and E. coli hsp 70 in 56 JCA patients using an ELISA. Elevated levels of antibodies to both human and E. coli hsp 70 were demonstrated. With hsp 65, raised levels of antibodies to the mycobacterial but not the E. coli protein were detected. Overall, 48% of patient serum samples contained antibodies of at least one isotype to mycobacterial hsp 65. These antibodies were predominantly of IgG and IgM isotype, a finding in contrast to adult rheumatoid arthritis, where IgA and IgG isotypes are most often detected.     

Factor XIII and tissue transglutaminase antibodies in coeliac and inflammatory bowel disease

Tissue transglutaminase (tTg) has been identified as an autoantigen in coeliac disease (CD). There is a marked homology between different forms of transglutaminase, such as tTg and coagulation factor XIII. We compared titres of both IgA- and IgG-antibodies against these two antigens in 20 CD patients, 20 endomysial antibody (EMA)-negative controls and a group with inflammatory bowel disease (34 with Crohn's disease and 23 with ulcerative colitis). IgA-antibodies against tTg correlated with EMA titres and had high sensitivity and specificity in screening for CD. Only in two CD patients were high titres found of IgA-antibodies against factor XIII, non-reactive with tTg. Both lacked bleeding tendency. The presence of IgG-antibodies against tTg, in contrast, had low sensitivity and specificity in screening for CD and were frequently seen in inflammatory bowel disease. Similarly, factor XIII IgG-antibodies displayed a non-specific pattern with modestly elevated titres in patients with Crohn's disease and in both EMA-negative and positive patients. Despite a marked homology with tTg, the occurrence of high titre IgA-antibodies against factor XIII is infrequent in CD, but may-when present-be the result of epitope spreading. The presence of IgG-antibodies in CD and inflammatory bowel disease illustrates the complexity of autoantibody reactions in gastrointestinal disease.