Antibodies to Yersinia enterocolitica in immunogenic thyroid diseases

In 1976 Shenkman et al. revealed that in patients with thyroid disorders antibodies against Yersinia enterocolitica could be demonstrated in increased frequency. In 1983 Ingbar et al. first established that the gram-negative bacterium Yersinia enterocolitica shows on its surface saturable binding sites for thyrotropin (TSH). If such binding sites resemble immunologically human TSH receptors this would indicate that TSH receptor antibodies could be produced in selected individuals having been infected with bacteria showing TSH receptors. The aim of our study was to compare the incidence of antibodies against Yersinia enterocolitica in two groups of thyroid disorders which are either immunogenic (Graves' disease and Hashimoto thyroiditis) or non-immunogenic (toxic adenomas, endemic goitre). In our series of 111 patients antibodies against Yersinia enterocolitica were demonstrated in a significantly higher percentage (36.3%) in patients suffering from immunogenic than in patients with non-immunogenic thyroid disorders (19.6%). The antibody titres were mainly directed towards Yersinia subtypes 8 and 3. It may, therefore, be assumed that the gram-negative bacterium Yersinia enterocolitica may have an active part in triggering immunogenic thyroid diseases such as Graves' disease or Hashimoto thyroiditis.     

Cell-Mediated Immunity to Yersinia enterocolitica Serotype 3 in Patients with Thyroid Diseases

The cellular immunity to Yersinia enterocolitica serotype 3 and crude human thyroid extract in 64 patients with thyroid diseases and 25 controls was studied by the leucocyte migration test. In the patient group as a whole and in patients with Graves' disease and nontoxic diffuse goitre a significantly reduced leucocyte migration towards Yersinia was found when compared with the controls. In controls the migration index was not related to the presence or titre of circulating yersinia antibodies, whereas the migration index of patients with yersinia antibodies was lower than the migration index of patients without yersinia antibodies as well as that of the controls. The leucocyte migration inhibition in two patients with recent yersiniosis was normal during the recovery phase.
In the presence of thyroid extract leucocyte migration inhibition differed only significantly in Graves' disease. However, a significantly positive correlation between inhibition of migration by thyroid extract and by Yersinia was found, while no correlation could be demonstrated in the controls.
The cell-mediated immunity towards Yersinia in thyroid diseases thus demonstrated adds further evidence to the association between Yersinia and thyroid disease.     

Humoral immune response to individual Yersinia enterocolitica antigens in patients with and without reactive arthritis.

The IgG, IgA and IgM antibody responses to individual Yersinia enterocolitica antigens were analysed by immunoblotting in 10 patients with reactive arthritis and 11 patients with enterocolitis caused by Y. enterocolitica serotype 0:3. The IgG antibody response was directed against partly different antigens compared with IgA. The expression of these antigens varied with bacterial culture conditions. The IgG and IgM antibody responses in the early stages of infection (less than or equal to 3 weeks) were directed against a large number of antigens. The IgA response was more selective, with antibodies against 240, 48 and 34.5 kD antigens dominating. IgA antibodies against eight antigens ranging in molecular weight from 52.5 to 105 kD were seen exclusively among arthritic patients. When following the IgA antibody response in the two groups of patients for up to 993 days, antibodies tended to persist to selective antigens. However, these antigens varied to some extent between patients, but the persistence was seen both among arthritic and non-arthritic patients. Persistent antibodies to 240, 48 and 34.5 kD antigens were most common. IgA antibodies to a 112 kD antigen were more prevalent and persisted longer in patients with arthritis compared to patients with uncomplicated infection.     

Yersinia enterocolitica: its isolation by cold enrichment from patients and healthy subjects.

Routine culture and cold enrichment were compared in a prospective study on the isolation of Yersinia enterocolitica from patients with intestinal disease. Healthy controls were examined with the cold enrichment method only. Y enterocolitica was isolated from 5.9% of 1635 patient stools, 3.4% of 206 appendices, and 4.0% of 555 control stools. Serotypes 0:3 and 0:9 were eight times more prevalent in patients than in controls. Other serotypes were twice as prevalent in controls than in patients. Cold enrichment did not significantly increase the recovery of serotypes 0:3 and 0:9 in acute enteritis, but it was responsible for all isolates of the other serotypes. Evidence is presented that the other serotypes are not pathogenic. In patient stools, Y enterocolitica was demonstrated less frequently than Salmonella (9.1%), and more often than Campylobacter jejuni (1.8%) and Shigella (0.1%).     

Yersinia enterocolitica and thyroid autoimmunity

The concordance rate of Graves' disease in pairs of identical twins of 30 to 60% points to the influence of environmental factors, and infections have often been incriminated in the pathogenesis. More than 15 years ago we demonstrated an increased frequency of antibodies to Yersinia enterocolitica (Y. ent.) serotype 3, which later was confirmed by others, suggesting a link between infection with Y. ent. and autoimmune thyroid diseases.     

Indirect immunofluorescence staining of human thyroid by antibodies occurring in Yersinia enterocolitica infections.

In the diagnostic routine for tissue antibodies, using indirect immunofluorescence on cryostat sections of human thyrotoxic thyroid, rat stomach and kidney, ninety-six out of 48,388 sera showed a marginal staining of the membrane region of thyroid epithelial cells but no other reaction. Twenty-six of these sera were from patients with acute Yersinia enterocolitica serotype 3 infection but without signs of thyroid disease. Fifty of sixty-three sera with agglutinins against Y. enterocolitica serotype 3 and three out of four sera with agglutinins against Y. enterocolitica serotype 9 also showed this reaction on thyroid sections. It was due to antibodies, mostly of the IgG class, occurring in low titre, which react with intracytoplasmic antigens in thyroid, as staining of live thyroid epithelial cells was negative. The pattern of immunofluorescence on thyroid sections caused by these antibodies could not be distinguished from that caused by smooth muscle antibodies by appearance only. However, smooth muscle antibodies react also on other tissue sections and extracts of contractile proteins which absorb out these, did not change the reaction on thyroid of Y. enterocolitica sera. Absorption with sonicated Y. enterocolitica 3 and 9 antigens, but not with heat-killed whole bacteria, extinguished the reaction on thyroid. This indicates the presence of a cross-reactivity between antigens in these bacteria, different from the O antigen, and antigens in thyroid epithelial cells. Knowledge of this pattern of immunofluorescence on thyroid sections can be of diagnostic significance.     

Antibody activity against lipopolysaccharides, lipid A and proteins from Enterobacteriaceae in patients with chronic inflammatory liver diseases

These studies are concerned with detection of circulating antibodies against various defined enterobacterial antigens in patients with chronic inflammatory liver diseases such as chronic hepatitis type B (n = 46), chronic active hepatitis (CAH) of autoimmune type (n = 10), alcoholic cirrhosis (n = 24) and primary biliary cirrhosis (PBC) (n = 24) as well as in healthy individuals (n = 39). Anti-LPS and anti-lipid A were determined by hemolytic and hemagglutination assay. Immunoblot technique was used to investigate the antibody activity against plasmid encoded proteins from Yersinia enterocolitica. Persistent titers of anti-LPS up to serum dilution 1:32.768 were found with hemolytic and hemagglutination assay in patients with alcoholic cirrhosis or PBC and in healthy control. In contrast nearly 50% of patients with chronic hepatitis B had no hemolytic antibodies against the two LPS E. coli serotypes at the time of liver biopsy. Anti-lipid A was detectable in 58% of patients with alcoholic cirrhosis but in low titers in less than 10% in the other groups (p less than 0.001). Alcoholic cirrhosis was also associated with a high frequency of IgG and IgA antibodies against plasmid encoded proteins from Yersinia enterocolitica. The data indicate that the O-polysaccharides as strong antigens are physiologically exposed to the immune system while lipid A and enterobacterial proteins are solely immunogenic under abnormal conditions.     

Increase in peripheral natural killer cell activity in patients with autoimmune thyroid disease.

Changes in the activity and number of natural killer (NK) cells in peripheral blood in patients with autoimmune thyroid disease were examined. NK activity was measured in a 4-hr 51Cr-release assay and the number of NK cells was analyzed with FITC-conjugated monoclonal antibodies by use of an automated flow cytometer. NK activity in patients with untreated Graves' disease (n = 25, 39.7 +/- 13.5%, P less than 0.05) and Hashimoto's thyroiditis (n = 18, 41.0 +/- 14.2%, P less than 0.05) was high compared to the activity in non-pregnant controls (n = 61, 32.6 +/- 15.0%). NK activity in patients with postpartum Graves' thyrotoxicosis (n = 11, 48.6 +/- 18.9%) was markedly increased compared to the activity in non-pregnant controls (P less than 0.01) and in postpartum controls (n = 29, 33.8 +/- 15.2%, P less than 0.05), although the mean ages of each group did not differ significantly. Moreover, NK activities in the thyrotoxic state were significantly higher than those in the euthyroid state in the same patients with postpartum Graves' thyrotoxicosis or with postpartum destructive thyrotoxicosis. The number of CD16 positive cells increased in patients with postpartum Graves' thyrotoxicosis. However the number of CD16 and CD57 positive cells were normal in all other groups of patients. These results indicate that an increase of NK activity is associated with exacerbation of autoimmune thyroid disease both in Hashimoto's thyroiditis and in Graves' disease and suggest that NK cells might have an important role for the control of disease activity in autoimmune thyroid disease.     

Response of synovial fluid T cell clones to Yersinia enterocolitica antigens in patients with reactive Yersinia arthritis.

From synovial fluids of two patients with reactive arthritis following Yersinia enterocolitica infection, T lymphocyte clones were obtained that showed proliferative responses to Y. enterocolitica. The responses required autologous T-cell-depleted peripheral blood mononuclear cells as antigen presenting cells. Three clones were studied in detail; two of them showed a marked and specific response to Yersinia antigens alone, the other one recognized both Yersinia and Salmonella typhimurium antigens. The antigen-specific proliferation of the clones could be completely blocked by monoclonal antibodies to HLA-DR. These experiments show that synovial T lymphocytes presumably involved in the in situ immune response to microbial antigens triggering reactive arthritis can be cloned directly from the site of inflammation.     

Comparison of 2 methods of hemagglutination tests for antibodies against strains of Yersinia enterocolitica and Yersinia pseudotuberculosis

The authors compare results of the assessment of antibodies against bacterial strains of Yersinia enterocolitica and Yersinia pseudotuberculosis. The examinations were made by the haemagglutination technique. In the reaction of the antigen human and ram erythrocytes were used as carriers. The work comprised 462 examinations of patients with an assumed Yersinia aetiology of the disease and 200 healthy controls. The values obtained during parallel examinations with antigens of Yersinia enterocolitica and Yersinia pseudotuberculosis linked to both types of blood cells were processed by statistical methods. It was revealed that when human and ram erythrocytes are used, the antibody levels correlate but are not identical. In the conclusion the authors recommend to unify the methods of different laboratories to facilitate comparison of results.     

Immunoblot analysis of human IgM, IgG and IgA responses to plasmid-encoded antigens of Yersinia enterocolitica serovar O3

Human IgM, IgG and IgA responses after infection with Yersinia enterocolitica serovar O3 were studied by immunoblotting sera against whole-cell homogenates of a plasmid-containing strain of Y. enterocolitica O3 and a plasmid-free strain derived from it; each strain was grown in conditions expressive for the plasmid. The antibodies observed were directed against several plasmid-encoded polypeptides. The response against different bacterial components decreased uniformly with time and the persisting antibody production was directed against several epitopes. Strong reactions to the prominent plasmid-specified antigens of mol. wts (10(3] 26, 34, 45 and 52.5 were found more often with IgG-class antibodies than with IgM or IgA; the latter immunoglobulins recognised, respectively, antigens of mol. wt (10(3] 26 and 45 (IgM) and 26, 34 and 52.5 (IgA). Immunoblotting of sera from patients with yersinia-triggered reactive arthritis did not reveal any antigens that were involved additionally or specifically. However, IgA-mediated recognition of certain antigens of mol. wts (10(3] 26, 34 and 52.5 tended to persist longer in the arthritic patients.     

Comparative study on IgG and IgA antibodies against human thyroid and eye-muscle antigens in Graves' ophthalmopathy.

Circulating IgG and IgA anti-thyroid and anti-eye muscle antibodies were investigated in 87 patients with Graves' disease (60 cases with ophthalmopathy). The ELISA method was used. Both IgG and IgA antibodies were demonstrated against human thyroid and eye-muscle membrane or cytosol antigens. Anti-eye-muscle antibodies of the IgA type were observed more frequently than those of the IgG type (25 cases vs. 18 were demonstrated with membrane antigens and 37 cases vs. 23 with cytosol antigens). The respective distributions for thyroid antigens the cytosol fraction were 55 cases vs. 13 and 18 cases vs. 36. A significant difference was observed in the anti-thyroid IgG levels and the anti-eye-muscle membrane or cytosol levels between the patients with Graves' disease and those in control group (P less than 0.001). The difference in the IgA antibody to thyroid and eye-muscle antigens was significant between the patients with and without ophthalmopathy (P less than 0.002). The strong correlation between the levels of IgA antibodies to thyroid and those to the eye-muscle cytosol fractions might be connected with the theory of the common aetiology of the thyroid and eye diseases in Graves' ophthalmopathy (P less than 0.001). Circulating IgA anti-human thyroid and eye-muscle antibodies seemed to have a diagnostic relevance in the development of ophthalmopathy in Graves' ophthalmopathy.     

Cross-reactions between Yersinia enterocolitica serogroup 0:3 and other serogroups of the same species, as well as thirty-four other bacterial species

Cross-reactions between antigens from Yersinia enterocolitica serogroup 0:3 and 5 other members of the same species as well as 34 other bacterial species were studied by means of quantitative immunoelectrophoretic methods. A sonicated Y. enterocolitica antigen preparation and corresponding purified rabbit antibodies were used in a reference system that presented 58 regularly visible immunoprecipitates. One antigen was identified as specific for the Y. enterocolitica 0:3 serogroup and two antigens for the species Y. enterocolitica. Y. enterocolitica antigens cross-reacted widely with antigens from other Enterobacteriaceae, but only a few cross-reactions were registered with Gram-negative bacteria outside the Enterobacteriaceae. A partial cross-reaction between all Gram-positive bacteria included in our study and two Y. enterocolitica antigens was demonstrated.     

Yersinia enterocolitica infection does not confer an increased risk of thyroid antibodies: evidence from a Danish twin study

Understanding of the aetiological basis of thyroid autoimmunity may be gained by studying the early stages of the disease process. We aimed to (1) investigate the relationship between thyroid antibody status and Yersinia enterocolitica (YE) infection in euthyroid subjects and (2) explore the relative importance of genetic and environmental risk factors in the acquisition of YE infection. The association between thyroid antibody status and YE infection was explored using a case-control design. Furthermore, thyroid antibody-positive twins were compared with their thyroid antibody-negative co-twin. In 468 twins, IgA and IgG antibodies to virulence-associated outer-membrane proteins (YOPs) of YE were measured. Of these, 147 were thyroid antibody-positive (cases). A total of 147 age- and gender-matched twins were chosen as controls. The prevalence of YOP antibodies was lower among thyroid antibody-positive individuals than among controls. Yersinia infection was not associated with a positive thyroid antibody status: the odds ratio (with 95% CI) for YOP IgA-ab was 0.66 (0.42-1.05), P = 0.078 and for YOP IgG-ab it was 0.95 (0.60-1.50), P = 0.816. Within discordant twin pairs, the thyroid antibody-positive twin did not have an increased risk of Yersinia infection compared to the thyroid antibody-negative co-twin [odds ratio: YOP IgA-Ab: 0.94 (0.49-1.83), P = 0.866, and YOP IgG-Ab: 1.35 (0.72-2.53), P = 0.345]; 41% (95% CI 10-67% of the liability of being YOP antibody-positive was due to genetic effects. In conclusion, Yersinia infection does not confer an increased risk of thyroid antibodies. The genetic contribution in the acquisition of Yersinia infection is modest.     

A microagglutination test for Yersinia enterocolitica infection: recognition of multiple serotypes in experiments with stained suspensions of serotype O5,27.

When suspensions of Yersinia enterocolitica were stained with triphenyl-tetrazolium chloride (TTC) their antigenic specificity, as determined by tube and microtitre-plate agglutination tests, was altered. Thus, a TTC-stained suspension of serotype O5,27 detected antibodies to nine O serotypes of Y. enterocolitica in sera from experimentally infected animals but did not cross-react with antisera to organisms of five other unrelated genera including Brucella. The same suspension did, however, cross-react weakly with antisera to four serotypes of Yersinia pseudotuberculosis. The value of this antigen preparation in detecting antibodies to the wide range of Y. enterocolitica serotypes likely to cause infection in man should now be assessed.     

Immune response to plasmid- and chromosome-encoded Yersinia antigens.

The immune response of humans and mice to temperature-specific, plasmid- or chromosome-encoded proteins of yersinia pestis and Yersinia enterocolitica was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting. Extracts from Y. pestis and Y. enterocolitica strains with and without the virulence plasmids pYV019 and pYV8081, respectively, were resolved by denaturing electrophoresis, and the major antigens were visualized with sera from convalescing plague patients, individuals immunized with plague vaccine, and mice and rabbits immunized with avirulent live yersiniae. The Y. pestis grown in vitro in this study did not express detectable amounts of plasmid-encoded antigens. The sera from plague patients recognized Y. pestis and Y. enterocolitica antigens ranging from 15 to 72 kilodaltons (kDa), whereas sera from immunized subjects recognized four antigenic components in Y. pestis ranging from 17 to 64 kDa and five antigens in Y. enterocolitica ranging from 16 to 68 kDa. Sera from mice reacted with 7 antigens in Y. pestis and 12 antigens in Y. enterocolitica ranging from 14 to 68 kDa, and sera from rabbits reacted with 7 and 10 antigens in Y. pestis and Y. enterocolitica, respectively. All of the plague patient sera, as well as the sera from immunized mice and rabbits, reacted with a 22-kDa Y. enterocolitica plasmid-associated polypeptide, and five of the patient sera also recognized a Y. enterocolitica plasmid-associated 31-kDa protein. The results indicate a common immune response to at least these two plasmid-specified Yersinia outer membrane proteins. Y. pestis apparently expresses these components only in vivo, and in vitro, Y. enterocolitica expresses a greater number of plasmid-associated antigens than does Y. pestis.     

Ankylosing spondylitis,HLA-B27 and Klebsiella. I. Cross-reactivity studies with rabbit antisera.

Sera from rabbits immunized with HLA-B27 lymphocytes showed increased activity against klebsiellal enterobacter antigens using immunodiffusion, bacterial agglutination (P less than 0.025), haemagglutination (P less than 0.001) and radiobinding assays (P less than 0.001). Immunoprecipitin lines were also produced by these antilymphocyte sera against extracts from Yersinia enterocolitica and Shigella sonnci microorganisms. Rabbit anti-klebsiella sera had lymphocytotoxic activity against HLA-B27 lymphocytes obtained from patients with ankylosing spondylitis (P less than 0.001). These results suggest partial cross-reactivity between some antigens found in several Gram-negative microorganisms and HLA-B27 lymphocytes.     

Prevalence of Yersinia plasmid-encoded outer protein (Yop) class-specific antibodies in patients with Hashimoto's thyroiditis

Objective   To investigate the prevalence of class-specific antibodies (IgG, IgA) to Yersinia enterocolitica plasmid-encoded outer proteins (Yops) in patients with diagnosed Hashimoto's thyroiditis.
Methods   Seventy-one patients with Hashimoto's disease, 464 healthy blood donors and 250 patients with non-postinfectious rheumatic disorders (matched controls) were tested for class-specific antibodies to Yops. Anti-Yop antibodies were determined by ELISA and Western blot.
Results   The prevalence of class-specific antibodies to Yops as determined by ELISA was 14-fold higher (20 of 71; 28.2%) in people with Hashimoto's thyroiditis than in the two control groups. These results were confirmed by the Western blot, with 16 positive sera, three equivocal and one negative.
Conclusions   There is strong clinical and seroepidemiologic evidence for an immunopathologic causative relationship between Yersinia enterocolitica infection and Hashimoto's thyroiditis. Further investigation concerning the mechanisms involved and the possible effects of antibacterial chemotherapy on the outcome of Hashimoto's disease is warranted.     

Yersinia enterocolitica isolated from two cohorts of young children in Santiago, Chile: incidence of and lack of correlation between illness and proposed virulence factors.

Yersinia enterocolitica was isolated from children in two cohorts in Santiago, Chile. In a cohort containing a cross section of children aged 0 to 4 years, Y. enterocolitica was isolated from stool samples of 1.1% of children with diarrhea and 0.2% of age-matched control children. In a subgroup of this cohort from which weekly stool samples were obtained from all children irrespective of clinical status, 6% of children had asymptomatic Yersinia infections. In a birth cohort (with a greater representation of children less than 1 year of age and a significantly higher rate of diarrhea), Y. enterocolitica was isolated from 1.9% of children with diarrhea and 0.6% of controls (P = 0.05). Biogroup 1A strains (which lacked traditional phenotypic and molecular markers for pathogenicity) were isolated from seven children with diarrhea but from no control children in the birth cohort (P = 0.02). All other isolates, including all isolates from asymptomatic children, were "pathogenic" strains in biogroup 4, serogroup O3; no association between these isolates and occurrence of disease was found. Y. enterocolitica is found among young children in Santiago, with asymptomatic infections not uncommon occurrences. However, questions about the association between previously described virulence factors and diarrheal illness remain.     

Characterization of circulating Yersinia-specific immune complexes in patients with yersiniosis

The size of immune complexes (ICs) containing Yersinia enterocolitica antigens was studied by size exclusion high-pressure liquid chromatography and sucrose density gradient ultracentrifugation in sera of patients with recent yersiniosis. The ICs detected were relatively small, i.e., of equal size to or slightly larger than the corresponding anti-Yersinia antibodies. The size of the ICs was equal in the patients with Yersinia-triggered reactive arthritis and in those recovering without complications. No changes were observed during a follow-up. The equal size of ICs in the patients with and without arthritis also suggests that antigens and antibodies involved are similar in both patient groups. Taken together with our earlier findings indicating occurrence of high concentrations of Yersinia IgM ICs in the arthritic patients, the present results suggest that Yersinia--IgM ICs have a role in the pathogenesis of Yersinia-triggered reactive arthritis.