Rheumatoid arthritis patients have elevated antibodies to cross-reactive and non cross-reactive antigens from Proteus microbes.

BACKGROUND AND OBJECTIVE: Although a large number of independent studies have shown a paramount role for Proteus mirabilis in the aetiopathogenesis of rheumatoid arthritis (RA), this hypothesis is still controversial among rheumatologists. The main obstacle to its acceptance is the impression that increased Proteus antibodies in RA patients is a secondary phenomenon, occurring as the result of cross-reactivity between bacterial and self-antigens. To shed light on this problem, we examined the link between antibodies to various cross-reactive and non cross-reactive antigenic peptides from P. mirabilis and analysed the relationship between these antibodies and disease severity in patients with RA. METHODS: Using the ELISA method, serum samples from 70 RA patients and 20 healthy controls were screened for total and class-specific antibodies against three human cross-reactive and non-crossreactive synthetic peptides from P. mirabilis haemolysin, urease C and urease F enzymes. An antibody index, which comprised the total concentration of antibodies against these peptides in each sample, was correlated with the biochemical parameters of disease activity and/or severity, such as the erythrocyte sedimentation rate (ESR), C-reactive protein (CRP) and rheumatoid factors (RF). Furthermore, anti-peptide antibody indices were evaluated among RA patients with different levels of disease activity as defined by ESR and CRP. RESULTS: Significantly elevated levels of total and class-specific IgG antibodies against the 3 Proteus peptides were observed among RA patients compared to healthy controls (p < 0.001). Active RA patients had elevated IgM antibodies against all peptides compared to healthy subjects (p < 0.001). However, no such elevation was observed in IgA anti-peptide antibodies in RA patients. A positive correlation was observed between the antibody indices and ESR (p < 0.001) and CRP (p < 0.01) concentrations, but not the RF status or disease duration. Furthermore, more than 90% of active RA patients showed positive values for the Proteus anti-peptide indices. CONCLUSION: The elevated levels of antibodies against Proteus antigenic epitopes (which are cross-reactive or non cross-reactive with human tissue antigens) observed indicates that this enhanced bacterial immune response in RA patients is specifically triggered by Proteus microbes. Furthermore, the correlation of anti-peptide antibody indices with the biochemical markers of disease activity indicates that these antibodies exert damaging cytotoxic effects on joint tissues during the course of the disease.

HLA and disease.

Many human diseases are associated with HLA class I, class II and class III antigens. It appears that the class III antigen disease associations can be explained by a direct defect operating at the level of either the class III gene or its gene product. The mechanism underlying class I and class II antigen disease associations is at present unknown. In this review we have considered thirty diseases which have been ranked according to their relative risk as defined by the frequency of a given HLA antigen in patient and control populations. The chronic inflammatory disorder, ankylosing spondylitis and its association with HLA B27 has been used as a model to study the HLA linked diseases. We have suggested that the disease may be caused by the Gram-negative microorganism Klebsiella which has antigenic similarity to HLA B27. It is proposed that some antibodies made against Klebsiella bind to HLA B27, thereby acting as autoantibodies leading to the pathological sequelae of chronic inflammatory arthritis. This is the crosstolerance hypothesis or molecular mimicry model and it has been compared to the receptor model. It is further suggested that the crosstolerance hypothesis can be utilised as a general theory to explain the association of other diseases with the class I and class II antigens, and offer a possible explanation for the polymorphism of HLA.

Molecular mimicry and ankylosing spondylitis: possible role of a novel sequence in pullulanase of Klebsiella pneumoniae.

Molecular mimicry has been shown between two sequences of Klebsiella pneumoniae pulD secretion protein (DRDE) with HLA-B27 (DRED) and pulA (pullulanase) enzyme (Gly-X-Pro) with types I, III and IV collagen respectively. IgG antibody levels in AS patients were elevated against 16mer synthetic peptides of HLA-B27 and pulD by enzyme immunosorbent assay (ELISA) compared to controls (P < 0.001). ELISA assays against K. pneumoniae grown in the absence and presence of pullulan demonstrated significant levels of IgA antibody in AS patients compared to controls (P < 0.001). Increased IgA and IgG antibody levels to pulA and types I and IV collagen were observed in AS patients compared to controls (P < 0.001). These observations could be relevant in the sequence of molecular events in AS.

Molecular mimicry between HLA-DR alleles associated with rheumatoid arthritis and Proteus mirabilis as the Aetiological basis for autoimmunity.

Molecular mimicry is one of the pathological mechanisms proposed to explain the association between microorganisms and autoimmune diseases. This review deals with the association between bacteria and rheumatic diseases with a special emphasis on rheumatoid arthritis where upper urinary tract infection by Proteus mirabilis is the possible cause of this severe, arthritic condition. Prospective trials involving anti-Proteus therapy should be carried out.

Cross-reactivity between related sequences found in Acinetobacter sp., Pseudomonas aeruginosa, myelin basic protein and myelin oligodendrocyte glycoprotein in multiple sclerosis.

To investigate the possible role of molecular mimicry to bacterial components in multiple sclerosis (MS) pathogenesis we examined antibody responses to mimicry peptide sequences of Acinetobacter, Pseudomonas aeruginosa and myelin components. Antibodies to mimicry peptides from Acinetobacter (p<0.001), P. aeruginosa (p<0.001), myelin basic protein (MBP) (p<0.001) and myelin oligodendrocyte glycoprotein (MOG) (p<0.001) were significantly elevated in MS patients compared to controls. Antisera against MBP (residues 110-124) reacted with both Acinetobacter and Pseudomonas peptides from 4- and gamma-carboxymuconolactone decarboxylase, respectively. MOG (residues 43-57) antisera reacted with Acinetobacter peptide from 3-oxo-adipate-CoA-transferase subunit A. The role of these bacteria in MS is unclear but demonstrates that molecular mimicry is not restricted to viruses suggesting bacterial infections could play a role in MS pathogenesis. Further work is required to evaluate the relevance of these cross-reactive antibodies to the neuropathology of MS.

Bovine spongiform encephalopathy: is it an autoimmune disease due to bacteria showing molecular mimicry with brain antigens?

Bovine spongiform encephalopathy (BSE) could be an autoimmune disease produced following exposure of cattle to feedstuffs containing bacteria showing molecular mimicry between bacterial components and bovine tissue. Analysis of molecular sequence databases (Genbank and SwissProt) shows that three bacteria (Acinetobacter calcoaceticus,Ruminococcus albus, and Agrobacter tumefaciens) share sequences with the encephalitogenic peptide of bovine myelin, while three molecules in Escherichia coli show molecular mimicry with host-encoded prion protein. Immune responses against these bacteria at both T and B cell levels may cause neurological tissue injury resembling BSE. The role of these bacteria in BSE, if any, merits further investigation.

Ankylosing spondylitis is caused by Klebsiella. Evidence from immunogenetic, microbiologic, and serologic studies.

Ankylosing spondylitis is a form of reactive arthritis following Klebsiella infection, usually occurring in an HLA-B27-positive individual. This conclusion is based on evidence obtained from several disciplines: immunogenetic studies show that there is molecular mimicry between HLA-B27 and Klebsiella; increased isolation of fecal Klebsiella has been reported in both Europe and North America; and finally, antibodies to Klebsiella have been demonstrated in ankylosing spondylitis patients in England and Finland. It is suggested that therapeutic trials should be set up with the aim of eliminating Klebsiella microbes, in an endeavor to test the validity of this theory.

HLA molecules, bacteria and autoimmunity.

It has been well established that many diseases are linked to HLA antigens. Two of the most interesting HLA associations may provide some insight into the pathogenesis of rheumatic inflammatory conditions. In ankylosing spondylitis (AS), 96% of patients possess HLA-B27, whilst the frequency of this marker in the general population is c. 8%. In rheumatoid arthritis (RA), >90% of patients possess either HLA-DR1 or some subtypes of HLA-DR4, whilst the frequency of this marker in the general population is c. 35%. The association between HLA-B27 and reactive arthritis (ReA) has also been well established. Furthermore, it has been shown that ReA is triggered by infection via the gastrointestinal tract due to Yersinia, Salmonella or Campylobacter spp. and in the genitourinary tract due to chlamydia. In a similar way, microbiological and immunological studies have revealed an association between Klebsiella pneumoniae in AS and Proteus mirabilis in RA. This article reviews the possible pathological implications of the associations between HLA-B27, K. pneumoniae and AS, as well as HLA-DR1/DR4, P. mirabilis and RA.

Crossreactivity as a factor in the immune response to Salmonella typhimurium in CBA and BALB/C mice.

The immune response to Salmonella typhimurium was investigated in CBA and BALB/C mice, by an arithmetic Widal agglutination assay. High antibody titres were obtained in CBA and low titres in BALB/C mice after secondary immunisation. A rabbit antiserum raised against S. typhimurium was tested for agglutinating activity after absorption with spleen- and lymph-node cells obtained from unimmunised CBA or BALB/C mice. BALB/C cells consistently removed more anti-S. typhimurium antibodies than did CBA cells, whilst the quantities of Fc receptor-bearing cells were found to be similar in both strains when measured by the erythrocyte-antibody rosette technique. It is suggested BALB/C mice give a low antibody response because their cell-surface antigens crossreact to a greater degree with S. typhimurium than do CBA cell-surface antigens.

Uveitis, vitreous humour, and klebsiella. II. Cross-reactivity studies with radioimmunoassay.

Radioimmunoassay with calf and cow vitreous humour-I125 and rabbit antivitreous humour serum has been employed to investigate the immunological cross-reactivity of vitreous humour with bacterial and mammalian tissue antigens. Klebsiella ultrasonicate preparation at a dose fo 10 000 micrograms/ml was found to inhibit the binding of vitreous humour by 25-100% (p less than 0.001), compared with an inhibition of 5-30% by a similar quantity of E. coli ultrasonicate preparation. Equivalent amounts of Streptococcus pyogenes antigen, bovine haemoglobin, and hyaluronic acid had no inhibitory effect, while horse spleen ferritin was found to inhibit vitreous humour binding between 0 and 10%. These results indicate that klebsiella micro-organisms have antigens which partially resemble some eyeball components. It is suggested that acute anterior uveitis of ankylosing spondylitis may be produced by anti-Gram-negative bacterial antibodies binding to cross-reacting eye antigens.

Uveitis, vitreous humor, and klebsiella. I. Binding studies with rabbit antisera.

Uveitis occur in a proportion of patients with ankylosing spondylitis, and an increased faecal isolation of the Gram-negative micro-organism Klebsiella pneumoniae has been reported from such patients. Immunological cross-reactivity between K. pneumoniae and bovine vitreous humour has been studied by 2 different antibody binding techniques: I125-labelled antigen binding assay with and without carrier, and beta-galactosidase enzyme-immunoassay. Sera from rabbits immunised with whole klebsiella micro-organisms or klebsiella extracts were found to bind labelled vitreous humour antigens to a greater extent (p less than 0.001) than sera from rabbits immunised with Escherichia coli, Streptococcus pyogenes, and phi X 174 virus or sera from the same rabbits before immunisation. It is suggested klebsiella micro-organisms may carry antigenic determinants which resemble vitreous humour antigens.

Genetic diversity in Proteus mirabilis isolates found in the urinary tract of rheumatoid arthritis patients.

OBJECTIVE: Elevated levels of anti-Proteus antibodies but not antibodies to E. coli have been reported in patients with rheumatoid arthritis (RA). The suggestion has been made that P. mirabilis may have a role in the aetiopathogenesis of rheumatoid arthritis. The aim of this study was to determine whether there are differences at the genetic level inisolates of P. mirabilis obtained from controls and RA patients. METHODS: A blind study was performed whereby P. mirabilis isolates obtained from urinary cultures of RA patients and controls were analysed using RAPD PCR. Isolates were then grouped on the basis of their DNA band profile after agarose gel electrophoresis, thereby allowing the composition of the Proteus population in the urinary tract to be analysed at the genetic level. RESULTS: Fourteen different DNA band profiles were obtained from the 93 isolates tested: 70% of these isolates fell into only five of the 14 groups and approximately 25% of all isolates fell into one group. No differences were observed in the frequency of isolates from either control or RA subjects. CONCLUSIONS: There is genetic diversity in P. mirabilis populations found in the urinary tract, but there are no differences in the frequency of these bacteria between RA patients and controls.

Antibodies to prion and Acinetobacter peptide sequences in bovine spongiform encephalopathy.

An amino acid sequence homology has been identified between the bovine prion sequence (RPVDQ) and the Acinetobacter calcoaceticus enzyme, uridine-diphosphate-N-acetyl glucosamine-1-carboxy-vinyl-transferase which also contains (RPVDQ). Class-specific IgA, IgG and IgM antibodies against synthetic peptides containing the structurally related sequences present in bovine prion and A. calcoaceticus were measured in 189 bovine spongiform encephalopathy (BSE) positive cattle, 127 BSE negative cattle and 87 healthy control animals using an ELISA technique. Class-specific IgA, IgG and IgM antibodies against the structurally related synthetic peptides were significantly elevated in BSE positive cattle when compared to BSE negative cattle (P < 0.001) and healthy control animals (P < 0.001). These autoantibodies may have a role in the pathogenesis of BSE.

The use of a low starch diet in the treatment of patients suffering from ankylosing spondylitis.

The majority of ankylosing spondylitis (AS) patients not only possess HLA-B27, but during active phases of the disease have elevated levels of total serum IgA, suggesting that a microbe from the bowel flora is acting across the gut mucosa. Biochemical studies have revealed that Klebsiella bacteria, not only possess 2 molecules carrying sequences resembling HLA-B27 but increased quantities of such microbes are found in fecal samples obtained from AS patients and such patients have Crohn's like lesions in the ileo-caecal regions of the gut. Furthermore AS patients from 10 different countries have been found to have elevated levels of specific antibodies against Klebsiella bacteria. It has been suggested that these Klebsiella microbes, found in the bowel flora, might be the trigger factors in this disease and therefore reduction in the size of the bowel flora could be of benefit in the treatment of AS patients. Microbes from the bowel flora depend on dietary starch for their growth and therefore a reduction in starch intake might be beneficial in AS patients. A "low starch diet" involving a reduced intake of "bread, potatoes, cakes and pasta" has been devised and tested in healthy control subjects and AS patients. The "low starch diet" leads to a reduction of total serum IgA in both healthy controls as well as patients, and furthermore to a decrease in inflammation and symptoms in the AS patients. The role of a "low starch diet" in the management of AS requires further evaluation.

Proteus IgG antibodies and C-reactive protein in English, Norwegian and Spanish patients with rheumatoid arthritis.

The distribution of Proteus antibody levels was compared in English, Norwegian and Spanish patients with rheumatoid arthritis (RA). Using an indirect immunofluorescence method, the IgG antibody titre against Proteus mirabilis was measured in the sera of 27 English, 53 Norwegian and 34 Spanish patients with RA and divided into active and inactive disease groups according to the serum C-reactive protein (CRP) level (> or = 10 mg/l). Serum samples were also collected from 25 English, 30 Norwegian and 14 Spanish healthy individuals who served as controls. The levels of Proteus IgG antibodies were significantly higher in the sera of active RA patients (p<0.001) when compared with the corresponding healthy controls, whether these groups belonged to the English, Norwegian or Spanish populations. Furthermore, active RA patients from each country showed significantly higher levels of Proteus antibodies when compared with inactive English (p<0.01), Norwegian (p<0.001) or Spanish (p<0.001) RA patients. Finally, a significant correlation was observed between Proteus IgG antibody levels and the CRP concentrations in RA patients whether each population was tested individually or all together (p<0.001). The increased levels of Proteus antibodies in RA patients from three different European countries support the concept of a possible aetiopathogenetic role for Proteus microorganisms in the development of RA.

Molecular mimicry: the geographical distribution of immune responses to Klebsiella in ankylosing spondylitis and its relevance to therapy.

The discovery that HLA-B27 is linked to ankylosing spondylitis (AS) and HLA-DR1/DR4 to rheumatoid arthritis (RA) has provided new approaches to the study of the possible causation of these diseases. Several theories have been proposed to explain these associations but only one, namely "molecular mimicry", has provided a specific aetiological agent for each of these diseases. Molecular mimicry between HLA-B27 and two molecules in Klebsiella microbes: nitrogenase and pullulanase D has been reported whilst in Proteus microbes, the haemolysin molecule shows sterochemical similarity to HLA-DR1/DR4. Elevated immune responses to Klebsiella microbes have been demonstrated in AS patients from 10 different countries and this wide geographical distribution suggests that the same aetiological agent is probably acting in producing this condition. Furthermore RA patients show similar immune responses to Proteus microbes. Whether AS or RA are caused by these bacteria can only be resolved by tissue typing all rheumatological patients early, in the course of their disease and then assessing their response to antibiotic chemotherapy in longitudinal studies involving double-blind crossover trials. It is possible that in the future, the course of AS or even RA could be modified by adequate antibiotic chemotherapy or even diets which affect the substrates on which these bacteria grow.

Antibacterial and antipeptide antibodies in Japanese and Finnish patients with rheumatoid arthritis.

It has been suggested that Proteus infection may be involved in the pathogenesis of rheumatoid arthritis (RA). Bacterial and peptide immune responses in patients with RA and other control subjects were investigated in two geographically different populations. Serum samples from Finnish patients with early ( n=72) and advanced ( n=27) RA and 30 Finnish healthy controls, as well as from Japanese RA patients from two different locations: Tokyo ( n=30) and Otsu ( n=30), 18 patients with systemic lupus erythematosus (SLE) and 23 Japanese healthy controls were all screened for the total, and class-specific (IgG, IgA and IgM) antibodies against Proteus mirabilis, Escherichia coli and Serratia marcescens by indirect immunofluorescence assay. These samples were also tested for the determination of levels of isotypic antibodies against the shared epitope involving 16-mer synthetic peptides containing the EQRRAA or ESSRAL sequences and compared to scrambled control peptide by using an enzyme-labeled immunosorbent assay method. Significantly elevated levels of IgG and IgM antibodies to P. mirabilis and antibodies against both EQRRAA and ESSRAL peptides were detected in sera of Finnish patients with early and advanced RA, and in Japanese patients from Otsu or Tokyo compared to their corresponding control groups. In contrast, no difference either in the total or in any of the isotypic antibodies were observed between these groups when serum samples were screened against each of E. coli and S. marcescens or against the control peptide. Furthermore, there was a significant correlation between the antibody levels against Proteus bacteria only and both EQRRAA and ESRRAL peptides. Our findings support the possibility for specific involvement of P. mirabilis in the etiopathogenesis of RA even in early cases.

The autoimmune model of schizophrenia.

Schizophrenia is of mysterious causation. It is not infectious, not congenital, but shows familial aggregation, the Mendelian genetics indicating involvement of multiple codominant genes with incomplete penetrance. This is the pattern for autoimmune diseases, such as Graves' disease of the thyroid, where forbidden clones of B lymphocytes develop, and cause thyrotoxicosis by secreting autoantibodies that react with the thyroid gland's receptor for thyroid-stimulating hormone from the pituitary gland. In 1982, Knight postulated that autoantibodies affecting the function of neurons in the limbic region of the brain are a possible cause of schizophrenia. Today, this is even more probable, with genes predisposing to schizophrenia having being found to be immune response genes, one in the MHC and two for antibody light chain V genes. Immune response genes govern the immune repertoire, dictating the genetic risk of autoimmune diseases. The simplest test for an autoimmune basis of schizophrenia would be trial of immunosuppression with prednisone in acute cases. The urgent research need is to find the microbial trigger, as done by Ebringer for rheumatoid arthritis and for ankylosing spondylitis. This could lead to prophylaxis of schizophrenia by vaccination against the triggering microbe.