Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia lipopolisacharydes and Yop proteins by ELISA

The antibodies against the somatic antigens of Y. enterocolitica O3, O8, O9, O5,27,Y. pseudotuberculosis I, and released proteins Yop were detected using the ELISA in 1634 serum samples and 84 synovial fluids collected from 1290 persons suspected for yersiniosis, as well as 200 serum samples from healthy individuals (blood donors). The presence of antibody in diagnostically significant titres for somatic antigens of Yersinia were detected by ELISA in 20.5% and 50.6%, antibodies for released proteins Yop in 11.5% and 28.4% respectively of blood donors and patients suspected for yersiniosis. The antibody against the O3 antigen of Y. enterocolitica was the most frequently detected antibody while the most infrequent was the antibody for the antigen from the 08 serologic group. The results of the study showed that the humoral response picture to Yersinia antigens in the course of yersiniosis in humans is dependent on the age and sex of the patient, duration of the infection, and clinical manifestations. Most frequently the elevated antibody levels were detected among patients with erythema nodosum and patients with gastrointestinal symptoms. The frequency of occurrence of antibodies for most antigens of Yersinia, together with age increased reaching its peak, on the average, among individuals aged 21 - 40 years. Analysis of individual cases showed that by the end of the first week of infection, elevated levels of antibodies for somatic antigens of Yersinia are evident. On the other hand, antibodies for released proteins Yop as a matter of rule appear in the second week from the onset of clinical symptoms. Within this early phase of infection immunoglobulins of the A and M classes dominate reaching their highest level in the second to third week of the infection. In majority of the individuals studied antibodies of the IgG class reached their highest level much later in relation to those of the IgA and IgM classes. Significant differences were found in IgA antibody detection among individuals with clinical manifestations of stomachaches and arthritis. Nevertheless, among individuals with clinical symptoms of stomachaches, these immunoglobulins as a matter of principle disappear with a period of 2-3 months from the onset of clinical symptoms. In individuals with arthritis however the aforementioned immunoglobulins maintained at considerable levels even after a year. In joint-fluid samples obtained from patients with arthritis antibodies for Yersinia antigens were detected in similar levels just as obtained simultaneously serum from those individuals.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to Yersinia Yop proteins by Western-blot

The results obtained with the use of the western-blotting showed that antibodies for released proteins YopD (33-36 kDa) were the most frequently detected antibodies in serum samples from patients suspected for yersiniosis. Reactions between serum samples studied and the YopD protein were very intense, suggesting that protein is the strongest immunogen among the utilised, released proteins Yop of Yersinia. Antibodies IgM were more often diagnosed in patients with abdominal pain in the contrary to antibodies IgA which were characteristic to patients with reactive arthritis. Detailed analysis of the results of western-blotting on serum samples obtained several times from individuals with yersinosis during the course of infection in this investigation have showed also that antibodies of the IgA class hold longer in serum of individuals with arthritis compared with individuals with yersinosis not complicated by arthritis. In joint-fluid samples obtained from patients with arthritis antibodies for particular released proteins Yop were detected in the same class of immunoglobulins like in serum samples obtained from those individuals.     

The role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis. I. Detection of bacteria, bacterial DNA, lipopolysaccharide and ECA antigen in synovial fluid or blood

To investigate the role of Salmonella and Yersinia in the pathogenesis of spondyloarthropathies and rheumatoid arthritis synovial specimens from 92 patients were analysed for the presence of bacterial DNA with the use of polymerase chain reaction and for the presence of lipopolysaccharide and enterobacterial common antigen (ECA) with the use of Dot-ELISA. In addition, peripheral blood samples were available for PCR analysis from 68 patients. Salmonella and Yersinia chromosomal DNA was not found in any of the synovial specimens and blood samples from the patients. All of the synovial fluids were also culture-negative. Salmonella LPS antigens were observed in 8 (8.6%), Yersinia in 20 (21.7%) and ECA antigens in 32 (34.9%) synovial specimens. Our findings revealed the presence of bacterial degradation products, but not bacteria from the genus Salmonella and Yersinia or their DNA in the synovial fluid or blood of patients with spondyloarthropathies and rheumatoid arthritis.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. I. Occurrence of antibodies to enterobacterial common antigen (ECA)

The antibodies against the Enterobacterial Common Antigen (ECA) were detected using the ELISA in 293 serum samples collected from 185 persons suspected for yersiniosis, as well as 115 serum samples from healthy individuals (blood donors). The presence of IgA antibody in diagnostically significant titres for ECA were detected by ELISA in 3.5%, IgG in 13.0%, and IgM in 5.2% of blood donors. Statistical analysis showed that the frequency of detecting antibodies for ECA among the patients with yersiniosis was significantly higher (p < 0.05) in relation to the blood donors. Most frequently the elevated antibody levels were detected among patients with reactive arthritis (IgA 29.2%, IgG 35.4%, IgM 16.7%) while the most infrequent among patients with abdominal pain in acute phase of yersiniosis (IgA 14.9%, IgG 25.3%, IgM 19.5%). The level of antibodies for ECA, together with age increased reaching its peak, on the average, among individuals aged 41 - 60 years. In majority of the individuals studied antibodies of the IgG class reached the level much higher in relation to those of the IgA and IgM classes. The obtained results showed that the detection of antibodies to ECA may be useful in serodiagnosis of Yersinia infections.     

Comparison of usefulness of lipopolysaccharides extracted by phenol and trichloroacetic acid from Salmonella Typhimurium and Enteritidis for serodiagnosis of salmonelosis

Salmonella Typhimurium and Salmonella Enteritidis are the two predominant serogroups, responsible for about 80% of all human cases of salmonelosis in Poland. Therefore we compared the usefulness of lipopolysaccharides antigens extracted by phenol (Westphal method) and trichloroacetic acid (Boivine method) from Salmonella Typhimurium and Enteritidis in ELISA method for the determination of antibodies. We used one home - made LPS antigen and two others commercially available antigens from SIGMA - Aldrich. Our study showed that the presence of antibodies was found in 35 (74.5%) sera from 47 samples from patients with suspected salmonelosis. There was no significant statistical differences of frequency of appearance of antibodies to all three Salmonella antigens in sera from patients with salmonelosis and in sera from control group. This study showed that all three antigens are useful for determination of IgA, IgG, IgM antibodies for Salmonella serogroup B and D in routine serological diagnosis of salmonelosis. However, it should be considered possibility of cross-reaction between LPS antigen of Salmonella and antibodies to Yersinia enterocolitica which could be correlated with similarity between somatic antigens of these two pathogens.     

Serum immunoglobulin IgG subclass distribution of antibody responses to Yop proteins and lipopolysaccharide of Yersinia enterocolitica in patients with yersiniosis.

To assess the humoral immunological responses at the IgG subclass level in yersiniosis specific antibody responses against lipopolysaccharide of Yersinia enterocolitica 03 (LPS) and Yersinia Yop proteins were analyzed by ELISA. Thirty five patients with arthritis and forty nine patients with uncomplicated yersiniosis were included in the study. Analysis of the IgG subclass responses to the LPS revealed that the subclass distribution for both groups of patients was IgG2>IgG1>IgG3. The concentration of IgG4 was below detection level. The predominant antibody responses to Yop proteins were IgG1>IgG3>IgG2>IgG4 but the frequency of detection of particular IgG subclass antibodies were dependent on the age of patients. Generally, the frequency of occurrence of IgG2 antibodies for Yop proteins of Yersinia together increased with age reaching its peak among individuals aged above 40 years. On the other hand, IgG1 for Yop proteins and IgG3 for Y. enterocolitica LPS were diagnosed more often in serum samples obtained from children than from adults. We also found significantly higher frequency of IgG4 to Yop proteins of Y. enterocolitica in men than in women.     

Humoral response to selected antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis in the course of yersiniosis in humans. IV. Specificity of antigens

The specificity of the lipopolisacharydes and released proteins (Yop) of Yersinia was tested using the sera of rabbits immunised with pathogenic and non-pathogenic strain of Y. enterocolitica and Y. pseudotuberculosis as well as selected sera of patients. The results of this study showed a cross-reactions between the different serotypes of Y. enterocolitica with the strongest reactions between the pathogenic serotypes O:3 and O:9 and pathogenic serotype O:5,27 and non-pathogenic serotype O:5. Sera positive for B. burgdorferi and from patients with Graves' disease showed a slight cross-reactivity with Yop proteins of Yersinia. However, the higher cross-reactivity was observed between the LPS of Yersinia and Salmonella spp. Due to the evidence of cross-reactivity the results of serological investigations should be interpreted with caution.     

Utilization of released proteins (RPs) of Yersinia enterocolitica for serologic diagnosis of yersiniosis. III. Western-blot

The usefulness of the immunoblotting using released proteins (Yersinia outer proteins-Yop) as the antigen for the serological diagnosis of yersiniosis was estimated. The IgA, IgG, and IgM antibody responses of patients with yersiniosis and healthy blood donors were studied by western-blot prepared in our laboratory, and two commercial assays. The results indicate that antibodies of all three classes are most consistently directed against the proteins of YopD, YopM and YopE. Good correlations between the three western-blots were obtained for all proteins except the protein V-AG. Patients with yersinia-triggered reactive arthritis have IgA class antibodies against the YopD more often and for longer period than the non-arthritic patients with yersiniosis.     

Yersinia Yop-Specific IgA antibodies in Hungarian blood donors

Sera of 112 healthy Hungarian blood donors were tested for the presence of Yersinia enterocolitica and Y. pseudotuberculosis-specific agglutinins by tube agglutination, and for that of yersinia outer membrane protein (Yop)-specific IgA antibodies by ELISA. The positive results of this latter assay were confirmed by immunoblot. Only one sample gave a positive agglutination reaction with Y. enterocolitica antigen (group 03) and four exhibited an equivocal reaction with Y. pseudotuberculosis antigens (groups II and IV). Contrary to the low incidence of agglutinins, 15.1% of the samples showed a positive Yop-specific IgA reaction, while further 5.3% samples fell into the equivocal range by ELISA (17 and 6 specimens, respectively). Eleven of these samples (9.8% of all specimens tested) were also positive by immunoblot for the presence of Yop-specific IgA antibodies. These data suggest a higher incidence of yersinia infections than the 1.0-1.4 per 10(5) population predicted on the basis of stool culture results.     

Characterization of outer membrane proteins of Yersinia pestis and Yersinia pseudotuberculosis strains isolated from India

The majority of virulence factors including the 12 Yersinia outer membrane proteins (Yops), 29 Yop secretion proteins (Ysc) and few specific Yop chaperone (Syc) are contributed by the 70 kb LCR middle plasmid of Yersinia pestis. Yersinia pestis isolates recovered during 1994 plague outbreak and rodent surveillance samples of Southern states of India were studied for the presence of important Yops by the conventional procedure of partially purifying outer membrane proteins (Omps) after cultivation in calcium deficient media. Prominent bands numbering 4-5 between 34-42 kDa region corresponding to important Yops were seen in all the isolates as well as in other Yersinia and non-Yersinia species by SDS-PAGE. Western blotting with the polyclonal antisera raised against these Omp preparations revealed few immuno-reactive bands that appeared to be shared among Y. pestis, Y. pseudotruberculosis, Y. enterocolitica, Y. fredrocksenii, Y. intermedia, Y. kristensenii and E. coli. Three recombinant Yop proteins namely, YopM, YopB and LcrV were produced and antisera to these proteins could reveal presence of these Yops in the Y. pestis Omp preparations. In order to further characterize the important Yops among Omps, attempts were made to generate monoclonal antibodies against Omp preparation. Three of the 4 stable reactive clones that were obtained, when tested, had extensive cross-reactions among pathogenic Yersinia species, Y. pestis and Y. pseudotuberculosis isolates, other Yersinia species and the members of Enterobacteriaceae in dot-ELISA and Western blotting. One of the monoclonal antibodies, YP1, exhibited reaction to all the pathogenic Yersinia species and the isolates, with restricted cross-reactivity to Y. intermedia, Y. kristensenii, K. pneumoniae. None of the 4 monoclonal antibodies had reactions with the 3 recombinant Yop proteins. It appears that under low calcium response, the Y. pestis not only activates secretion of Yops but also a large number of other proteins, which as per the present observations are cross-reactive within the family Enterobacteriaceae.     

Diagnostic value of dengue virus-specific IgA and IgM serum antibody detection

The diagnostic value of dengue virus (DV)-specific immunoglobulin A (IgA) serum antibody detection, by an indirect immunofluorescence assay (IFA) was evaluated. For this study, the kinetics of DV-specific IgA serum antibodies was analysed in two experimentally immunised macaques, paired samples from 35 patients suspected of a primary or secondary DV infection, paired sera from patients with high levels of IgA specific antibodies against influenza virus (n = 15), sera from patients with other viral infections (n = 40) and healthy blood donors (n = 10), which served as controls. The presence of DV-specific IgA serum antibodies in humans and in monkeys was compared with that of DV-specific IgM demonstrated in a capture enzyme-linked immunosorbent assay (ELISA). The development of DV-specific IgA and IgM antibodies in macaques proved to be similar to that observed in humans with a DV infection. In sera obtained from suspected primary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 1/6 (17%) and 6/6 (100%), whereas IgM was detected in 4/6 (67%) and 5/6 (83%), respectively. In sera from suspected secondary DV patients during the acute phase and convalescent phase, DV-specific IgA was detected in 18/29 (62%) and 28/29 (97%), whereas IgM was detected in 20/29 (69%) and 28/29 (97%), respectively. The control group consisted of five paired serum samples from yellow fever vaccinated individuals and a patient with acute tick-borne encephalitis, 15 paired serum samples from patients with high levels of IgA antibodies specific for influenza virus and 40 serum samples from patients with specific IgM antibodies against other viruses. Ten serum samples from healthy blood donors were included. Among the control serum samples, in one patient, both DV-specific IgA and IgM antibodies were present, and in three sera DV-specific IgM antibodies could be demonstrated. These data suggest that detection of DV-specific IgA serum antibodies by IFA may have additional value for the diagnosis of DV infection.     

Rapid decay of Salmonella flagella antibodies during human gastroenteritis: a follow up study

An indirect enzyme-linked immunosorbent assay (ELISA) based on Salmonella re-polymerized flagella was employed to measure levels of immunoglobulin (Ig) G, IgM and IgA antibodies in sera from 303 Danish patients diagnosed with either Salmonella enteritidis or Salmonella typhimurium. The antibody-levels were assessed at one, three and six months after onset of salmonellosis, and sera from a control-group of 170 healthy blood donors were additionally analysed in order to establish cut-off values for the analysis. Cross-reactions to other Salmonella serotypes, as well as to Escherichia coli, Yersinia enterocolitica, Campylobacter jejuni, Campylobacter coli and Helicobacter pylori were observed. At one month after onset of symptoms, 70% of the patients recovering from a S. enteritidis infection carried detectable levels of anti-flagella antibodies, as did 77% of the patients recovering from S. typhimurium infection. Three months after onset of symptoms these detection rates had decreased to 46% and 40%; and six months after onset of symptoms the detection rates were 34% and 38%. This rapid decrease in the serum levels of flagella antibodies is in conflict with the "common knowledge" statement of a long-lasting anti-flagella immunoresponse. The present study suggests that such a tenacious statement is (or may be) inaccurate.     

The serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis

The techniques of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting were evaluated for the serodiagnosis of human infections with Yersinia enterocolitica and Yersinia pseudotuberculosis. Lipopolysaccharide (LPS) was prepared from strains comprising four serogroups of Y. enterocolitica and five serogroups of Y. pseudotuberculosis, tested against 200 sera submitted to the Laboratory of Enteric Pathogens for routine serodiagnosis, and shown to contain antibodies to Yersinia LPS by agglutination. Forty four sera were found to contain antibodies that bound to one of the LPS preparations used in the immunoassay. Thirty five of the sera contained antibodies to the LPS of Y. enterocolitica O3, whilst three contained antibodies to the LPS of Y. enterocolitica O5, 27 and Y. enterocolitica O9 LPS respectively. Two sera had antibodies to the LPS of Y. pseudotuberculosis II and a single serum contained antibodies to Y. pseudotuberculosis IV. The SDS-PAGE-immunoblotting procedure described proved to be a reliable procedure for the serodiagnosis of infections with Y. enterocolitica and Y. pseudotuberculosis.     

Composition of immunoglobulins in brucellosis patients using DEAE-cellulose column chromatography.

The composition of immunoglobulins in patients with brucellosis was studied. The method of ion-exchange chromatography on DEAE-cellulose columns was used to define more precisely the physico-chemical character of cysteine-resistant antibodies. The study of IgM, IgA and IgG fractions obtained from the patients sera showed the IgG fraction to possess the greatest serological activity in the agglutination reaction, in the passive haemagglutination reaction and in Coomb's test. Specific antibodies in the remaining 2 fractions (IgA and IgM) were found only in single patients in low titres, mainly in Coomb's test (incomplete antibodies). The study of IgM, IgA and IgG serum fractions before and after cysteine treatment revealed cysteine-resistant antibodies to be usually IgG globulins. The presence of specific IgG antibodies in the sera of patients was found to correlate with active clinical manifestations of brucellosis.     

Detection of autoreactive antibodies to serum IgG and IgA in amoebic liver abscess cases.

Assessment of autoreactive antibodies in response to healthy human serum IgA and IgG was performed by indirect haemagglutination assay on serum samples from 81 amoebic liver abscess cases for IgA and 70 for IgG. Appropriate controls were taken simultaneously. IgA, IgG were isolated and purified from a healthy human serum through Sephadex G-200 and protein A CL 4B sepharose chromatography. These immunoglobulins were used for the detection of its own antibodies in amoebic liver abscess cases. This revealed that 43.20% and 48.50% of the cases were positive for IgA and IgG respectively, where as only 19.35% and 28.30% of the controls were in positive category (IgA and IgG respectively). The mean titres with standard deviation of the autoreactive antibodies to serum IgA both in ALA cases and controls shows a highly significant difference between tests and controls (P less than 0.001). Similarly the mean titres with standard deviation both in ALA and controls for the serum IgG differed significantly (P less than 0.001). This suggests the presence of autoreactive antibodies against serum IgA and IgG in amoebic liver abscess cases.     

Application of released proteins (RPs) of Yersinia Enterocolitica for serological diagnosis of yersiniosis. I. Antigen for ELISA

The usefulness of the released proteins (RPs) prepared in our laboratory from the strain Y. enterocolitica for the serological diagnosis of yersiniosis was estimated. Yersinia enterocolitica 0:8 was cultured under calcium deficient conditions and the virulence factors were isolated from the culture supernatant. The purified proteins were solubilized using sodium dodecyl sulfate. The results of ELISA using released proteins obtained in our laboratory as the antigen were compared to the results of commercial ELISA-Mikrogen and ELISA-Euroimmun. One hundred serum samples obtained from patients suspected in clinical investigations for jersiniosis were tested. Comparison of the frequency of detecting in particular ELISA antibodies to Yersinia showed a high (> 85%) agreement of the results in all of the immunoglobulin classes. The highest sensitivity (97.4%) and specificity (95.4%) was displayed by the ELISA with our antigen in relation to the ELISA-Mikrogen when determining antibodies of IgM class.     

Effects of Yersinia enterocolitica O:3 derivatives on B lymphocyte activation in vivo

The potential sequelae of intestinal infection with Yersinia enterocolitica include reactive arthritis, erythema nodosum, Reiter's syndrome and other autoimmune diseases. The role of the immune response in the pathogenesis of these diseases has not been fully defined, but autoimmune manifestations may be a consequence of the increase in autoantibodies as a result of polyclonal B-cell activation induced by Yersinia. We investigated the effects of Y. enterocolitica O:3 derivatives on B lymphocyte activation in vivo. Groups of five specific pathogen free (SPF) Swiss mice were inoculated with bacterial cell extract, Yersinia outermembrane proteins (Yops) or lipopolysaccharide (LPS) obtained from Y. enterocolitica O:3 and their immunoglobulin-secreting spleen cells were detected by isotype-specific protein A plaque assay. The presence of specific anti-Yersinia antibodies and autoantibodies was determined in mouse sera by ELISA. In all experiments a marked increase in the number of secretory cells of different isotypes was observed as early as the third day after inoculation. IgG and IgM anti-Yersinia antibodies were detected in the sera of all inoculated mice, and autoantibodies against myosin in the sera of those inoculated with bacterial cell extract. The sera from animals stimulated with LPS reacted with myelin, actin and laminin, while the sera from mice inoculated with Yops reacted with myelin, thyroglobulin and cardiolipin. These results suggest that SPF Swiss mice inoculated with any one of the Y. enterocolitica derivatives tested exhibited polyclonal activation of B lymphocytes as a result of stimulation by various bacterial components and not only LPS stimulation.     

A comparison of immunogenicity and protective immunity against experimental plague by intranasal and/or combined with oral immunization of mice with attenuated Salmonella serovar Typhimurium expressing secreted Yersinia pestis F1 and V antigen

We investigated the relative immunogenicity and protective efficacy of recombinant X85MF1 and X85V strains of DeltacyaDeltacrpDeltaasd-attenuated Salmonella Typhimurium expressing, respectively, secreted Yersinia pestis F1 and V antigens, following intranasal (i.n.) or i.n. combined with oral immunization for a mouse model. A single i.n. dose of 10(8) CFU of X85MF1 or X85V induced appreciable serum F1- or V-specific IgG titres, although oral immunization did not. Mice i.n. immunized three times (i.n. x 3) with Salmonella achieved the most substantial F1/V-specific IgG titres, as compared with corresponding titres for an oral-primed, i.n.-boosted (twice; oral-i.n. x 2) immunization regimen. The level of V-specific IgG was significantly greater than that of F1-specific IgG (P<0.001). Analysis of the IgG antibodies subclasses revealed comparable levels of V-specific Th-2-type IgG1 and Th-1-type IgG2a, and a predominance of F1-specific Th-1-type IgG2a antibodies. In mice immunized intranasally, X85V stimulated a greater IL-10-secreting-cell response in the lungs than did X85MF1, but impaired the induction of gamma-interferon-secreting cells. A program of i.n. x 3 and/or oral-i.n. x 2 immunization with X85V provided levels of protection against a subsequent lethal challenge with Y. pestis, of, respectively, 60% and 20%, whereas 80% protection was provided following the same immunization but with X85MF1.     

IgM rheumatoid factors in mice injected with bacterial lipopolysaccharides.

Bacterial lipopolysaccharides (LPS) induced the formation of IgM rheumatoid factors (RF) in several strains of mice including athymic C57BL/6 nude mice, but not in the LPS-resistant C3H/HeJ mice. The RF induced by LPS reacted not only with murine IgG but also with IgG from cows, goats, guinea pigs, and humans. The kinetics of this RF response to injection of LPS were similar to those of antibody response against DNA and a hapten, dinitrophenyl (DNP), and to those of total IgM production. In addition, the RF activity of individual serum samples correlated significantly with levels of anti-DNA and anti-DNP antibodies and of IgM. Therefore, it is concluded that the induction of RF results from polyclonal antibody synthesis by B cells stimulated with LPS. This observation suggests that LPS or LPS-like substances may help to generate RF in patients with rheumatoid arthritis or with some infectious diseases.     

Antibodies to Yersinia enterocolitica serotype 3 in autoimmune thyroid diseases

The prevalence of increased titres of antibodies to Yersinia enterocolitica (serotype 3) has been studied in sera from patients with various thyroid diseases. In contrast to the low prevalences of the antibodies in healty subject (24.3%), titres (greater than 10) of anti-Yersinia enterocolitica (anti-Yersinia) were found more frequently in patients with thyroidal disorders, especially in Graves' disease (70.0%). Furthermore, high titres of the antibodies (greater than or equal to 160) were found only in patients with Graves' disease. There was no significant correlation between the titers of anti-Yersinia antibodies and those of anti-TSH receptor antibodies in sera from patients with Graves' disease. In seven individual samples of sera, the anti-Yersinia antibody titer was high before treatment, but the decrease in the anti-TSH receptor antibody titer following treatment was associated with a simultaneous decline in anti-Yersinia antibodies in all of them. A highly positive and significant correlation between the titers of anti-TSH receptor antibodies and anti-Yersinia antibodies was obtained in each of them. These findings could be merely a reflection of the measurement of the cross-reaction of anti-Yersinia antibodies with anti-TSH receptor antibodies but the possibility of an association between Yersinia infection and the production of anti-TSH receptor antibodies in at least some patients with Graves' disease cannot be ruled out.