Tryptic peptides of human thyroglobulin: I. Immunoreactivity with murine monoclonal antibodies.

Human thyroglobulin (Tg) was treated with trypsin at different concentrations of trypsin/Tg for various incubation times at 37 degrees C using non-reducing conditions. A ratio of trypsin to Tg of 1:100 (w/w) was optimal to release small peptides that were reactive to murine MoAbs to human Tg. Most peptides were released after only 1 h incubation with trypsin, but these peptides were further degraded at longer incubation times. However, a few small peptides, the largest of which with an apparent molecular weight (MWap) of 40 kD, resisted tryptic digestion up to at least 12 h of incubation. These resistant peptides were further degraded by trypsin at 18-24 h of incubation. Tryptic peptides of Tg, released at 1 h and 4 h of incubation, were analysed for their immunoreactivity to 16 well characterized anti-Tg MoAbs by Western immunoblot. Patterns of peptide recognition of these MoAbs were generally unique. Eight MoAbs reacted with peptides of MWap of 10-25 kD and above. Four other MoAbs reacted with peptides of MWap of 25-43 kD and above, and the remaining four reacted with peptides of MWap > 43 kD. Nine of these MoAbs failed to recognize peptides after reduction, suggesting that the MoAbs bind conformation-dependent epitopes. The above information will promote the development of models relating the structure of Tg to the autoimmune process, and may provide an understanding of those regions of Tg responsible for the induction of autoimmune thyroiditis.     

Pituitary-cell autoantibody diversity in sera from patients with untreated Graves' disease

Sera from 22 untreated patients with recently diagnosed Graves' disease (GD) were screened in an immunocytochemical tissue assay for presumptive pituitary IgG autoantibodies, as defined by the presence of immunoreaction with rat and swine pituitary cell types. Forty four patients with Hashimoto's thyroiditis (HT) and 97 healthy subjects were also studied. Anti-pituitary antibodies were found in 14 of the 22 GD sera (64%). Of these, 6 sera reacted with cytoplasmic components of growth hormone (GH) cells, 3 with prolactin (PRL) cells, and 5 with both GH and PRL cells. Yet, none of the immunoreactive sera reacted with human GH, bovine PRL or TSH in dot-blot assays and absorption studies. Anti-pituitary antibodies also occurred in 4 of the 44 HT patients (9.1%) and in 9 of the 97 healthy subjects (9.2%). The frequency of sera revealing anti-pituitary antibodies was significantly higher in patients with GD compared to the groups of HT patients (P less than 0.00005), and healthy subjects (P less than 0.00005). Healthy subjects and patients with HT had a similar frequency of anti-pituitary antibodies (P = 1.0000). These data demonstrate that in thyroid autoimmune conditions antibodies reactive with cytoplasmic components of pituitary GH/PRL cells, may be present in sera from patients with GD. The pathological importance of this observation is at present unknown.     

Autoantibodies in autoimmune thyroid disease promote immune complex formation with self antigens and increase B cell and CD4+ T cell proliferation in response to self antigens

B cells are centrally involved as antigen-presenting cells in certain autoimmune diseases. To establish whether autoantibodies form immune complexes (IC) with self-antigens in autoimmune thyroid disease (AITD) and promote B cell uptake of self-antigen, sera from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls were incubated with human thyroglobulin (Tg) before adding normal peripheral blood mononuclear cells. The deposition of immunoglobulins and C3 fragments on B cells was then assessed. Inclusion of Tg in serum from HT patients promoted B cell capture of IgG and C3 fragments. Furthermore, the binding of Tg to B cells in preparations of normal blood cells was higher in HT serum than in serum from controls and correlated positively with the serum anti-Tg activity, as did the B and CD4+ T cell proliferation. Disruption of the three-dimensional structure of Tg by boiling reduced the proliferative responses. The data indicate that anti-Tg antibodies associated with AITD facilitate the formation of complement-activating Tg/anti-Tg complexes, binding of IC to B cells, and the subsequent proliferation of B and T cell subsets. This represents a novel mechanism for the maintenance of autoimmune processes in AITD and links autoreactive T cell responses with the presence of autoantibodies.     

Evidence for intramolecular B-cell epitope spreading during experimental immunization with an immunogenic thyroglobulin peptide

Thyroglobulin (Tg) is a target autoantigen in autoimmune thyroid diseases, such as Graves' disease (GD) and Hashimoto's thyroiditis. In a previous study we identified three 20mer Tg peptides bearing epitopes of autoantibodies associated with GD (TgP15, TgP26 and TgP41: sequences 2339-2358, 2471-2490 and 2651-2670 of human Tg, respectively). In the present study, we investigated the antigenicity of the above peptides in experimental immunization with Tg, the immunogenicity of antigenic peptides and the possibility of intramolecular B-cell epitope spreading during peptide immunization. For this purpose, two rabbits were injected with human Tg in CFA six times, every three weeks. Two control animals were injected only with CFA. Testing of antisera and of affinity-purified antibodies, by ELISA against the three peptides, revealed reactivity only to TgP41. This synthetic peptide was subsequently administered to two rabbits, in its free form (100 micro g in CFA six times, every two weeks). A strong serological response was developed not only against TgP41, but also to intact human and rabbit Tg. Immunization with TgP41 induced intramolecular B-cell epitope spreading, i.e. production of antibodies to sites on Tg other than that corresponding to TgP41, as revealed by immunoadsorption and competitive ELISA. Histopathological studies did not reveal any infiltration in thyroid glands. We conclude that peptide TgP41 encompasses not only an epitope of disease-associated autoantibodies, but also a dominant immunogenic epitope of experimentally induced Tg-specific antibodies, able to drive B-cell epitope spreading.     

Specificities of anti-neutrophil autoantibodies in patients with rheumatoid arthritis (RA)

The objective of this study was to characterize antigens recognized by neutrophil-specific autoantibodies from patients with RA. Sera from 62 RA patients were screened by indirect immunofluorescence (IIF). Positive sera were further tested by ELISAs for antibodies against various granule proteins and by immunoblotting of electrophoretically separated cell, granule or nuclear extracts. Forty-two sera (68%) reacted with ethanol-fixed neutrophils. In the ELISAs 32% of the 28 medium to strongly IIF-positive sera were negative, while 43% were weakly positive for more than one antigen. Immunoblots of whole neutrophils showed IgG reactions at 25–35 kD, in the 55-kD region, at 80 kD, and at 110kD. Most sera reacted with more than one band. Except for the 55-kD antigen, none of the antigens appeared in lymphocytes. The most notable reactivity in subcellular fractions was with lactoferrin and with bands of 25–35 kD from nuclei. In conclusion, anti-neutrophil autoantibodies from RA patients recognize different antigens in the cytoplasm and in the nucleus. Lactoferrin is one of the common antigens recognized, but also unknown nuclear antigens of 25–35 kD mol. wt are involved.     

Antibodies to acetylcholinesterase cross-reacting with thyroglobulin in myasthenia gravis and Graves's disease.

In the present study we analysed by ELISA the ability of sera from 50 patients with myasthenia gravis (MG), 20 with Hashimoto's thyroiditis (HT), 53 with Graves' disease (GD) and 36 healthy controls (CR) to react with acetylcholinesterase (AChE) from Electrophorus electricus and human thyroglobulin (Tg). Significantly increased anti-AChE activity was exhibited by a high proportion of MG (IgG 36%) and GD (IgG 21%) sera, while increased anti-Tg activity was detected in all three patient groups (MG, IgG 26% and IgA 26%; HT, IgG 85% and IgA 40%; and GD, IgG 51%). Interestingly, a significant proportion of MG and GD sera exhibited both IgG anti-AChE and anti-Tg activities (MG, 18%; P < 0.001; and GD, 15%; P < 0.001, versus CR, 0%). This bi-reactivity was exhibited by anti-AChE antibodies cross-reacting with Tg (anti-AChE/Tg activity); (i) serum anti-AChE activity was effectively inhibited by soluble Tg, and (ii) affinity-purified anti-Tg antibodies cross-reacted with AChE. Cross-reactivity seems to be a property of pathological (auto)antibodies; induced (rabbit) antibodies to AChE or Tg were highly monospecific. Analysis of clinical data showed that increased IgG anti-AChE/Tg activity was well associated with: (i) overlapping GD in MG (P < 0.02), and (ii) ophthalmopathy in GD (P < 0.01). In contrast, no correlation was noted in MG between anti-AChE activity units and anti-Tg activity units or acetylcholine receptor antibody titres. The clinical significance of anti-AChE/Tg antibodies remains to be elucidated.     

Pituitary-cell autoantibody diversity in sera from patients with untreated graves' disease

Sera from 22 untreated patients with recently diagnosed Graves' disease (GD) were screened in an immunocytochemical tissue assay for presumptive pituitary IgG autoantibodies, as defined by the presence of immunoreaction with rat and swine pituitary cell types. Forty four patients with Hashimoto's thyroiditis (HT) and 97 healthy subjects were also studied. Anti-pituitary antibodies were found in 14 of the 22 GD sera (64%). Of these, 6 sera reacted with cytoplasmic components of growth hormone (GH) cells, 3 with prolactin (PRL) cells, and 5 with both GH and PRL cells. Yet, none of the immunoreactive sera reacted with human GH, bovine PRL or TSH in dot-blot assays and absorption studies. Anti-pituitary antibodies also occurred in 4 of the 44 HT patients (9.1%) and in 9 of the 97 healthy subjects (9.2%). The frequency of sera revealing anti-pituitary antibodies was significantly higher in patients with GD compared to the groups of HT patients (P < 0.00005), and healthy subjects (P < 0.00005). Healthy subjects and patients with HT had a similar frequency of anti-pituitary antibodies (P = 1.0000). These data demonstrate that in thyroid autoimmune conditions antibodies reactive with cytoplasmic components of pituitary GH/PRL cells, may be present in sera from patients with GD. The pathological importance of this observation is at present unknown.     

Antibodies to TSH-receptor in thyroid autoimmune disease interact with monoclonal antibodies whose epitopes are broadly distributed on the receptor

The hyperthyroidism of Graves' disease (GD) is caused by TSH-receptor (TSH-R) stimulating autoantibodies (TSAb), leading to overproduction of thyroid hormones. We present evidence for TSAb interaction with three distinct regions of the TSH-R, one in immediate vicinity of the carboxy terminal serpentine. Three murine monoclonal antibodies (MoAbs 28.1, A9 and 31.7) directed to amino acids 36-40, 147-228 and 382-415 were labelled and tested for their binding to human recombinant TSH-R on solid phase. All MoAbs bound to TSH-R with a K(d) of 8-12 nm and showed no competition among themselves. We tested 114 sera from euthyroid controls, 118 TBII positive sera from patients with GD (containing TSAb confirmed by bioassays), 16 TBII positive sera from patients with autoimmune thyroid disease (AIT), who were hypothyroid and had TSH blocking antibodies (TBAb), and 20 patients with AIT, who were hypothyroid but negative for all TRAb. Mid-regional MoAb A9 tracer achieved the highest sensitivity in the GD group (72.0%), whereas C-terminal MoAb 31.7 found most sera positive in the AIT group (87.5%). Surprisingly, the N-terminal MoAb 28.1 had the lowest sensitivity in the GD (10.4%) and AIT group (43.8%). Using a mixture of all three tracer MoAbs did not increase the sensitivity in the GD or AIT group, compared to the best single MoAb alone. Median inhibition of MoAb A9 was significantly (P < 0.001) higher than inhibition of MoAbs 28.1 or 31.7 in the group of GD patients but not in other groups. Almost all patient sera with positive reactivity in the MoAb tracer assays had TBII values in the higher range. However, there were many highly TBII positive sera, which did not show a displacement of the MoAb tracers. We conclude that, contrary to some reports, the binding of TSAb and TBAb to the TSH-R is not restricted to distinct and distant epitopes. The middle part of the TSH-R seems to be more relevant for TSAb binding than the N-terminal part, while a proportion of TSAb autoantibodies also binds to a C-terminal epitope of the TSH-R. The method described here is a TSH independent competitive assay for the detection of TSH-R autoantibodies.     

Autoantibodies against complement C1q correlate with the thyroid function in patients with autoimmune thyroid disease

Autoantibodies against complement C1q (anti-C1q) have been well described in patients with systemic lupus erythematosus, where they correlate with the occurrence of severe lupus nephritis. However, data on anti-C1q in organ-specific autoimmune diseases are scarce. In order to determine the prevalence of anti-C1q in patients with autoimmune thyroid disorders (AITD) and a possible association with thyroid function, we measured prospectively anti-C1q in 23 patients with Graves' disease (GD) and 52 patients with Hashimoto's thyroiditis (HT). Anti-C1q levels were correlated with parameters of thyroid function and autoantibodies against thyroperoxidase, thyroglobulin and thyroid stimulating hormone (TSH) receptor. Twenty-one patients with multi-nodular goitre and 72 normal blood donors served as controls. We found elevated concentrations of anti-C1q more frequently in patients with AITD than in controls: seven of 23 (30%) patients with GD and 11 of 52 (21%) patients with HT, compared with one of 21 (5%) patients with multi-nodular goitre and six of 72 (8%) normal controls. Anti-C1q levels did not correlate with thyroid autoantibodies. However, in GD absolute levels of anti-C1q correlated negatively with TSH and positively with free thyroxine (FT4) and triiodothyronine (FT3). In contrast, in HT, anti-C1q correlated positively with TSH levels. No correlation between TSH and thyroid autoantibodies was found. In conclusion, we found an increased prevalence of anti-C1q in patients with AITD and their levels correlated with the thyroid function in both GD and HT. This correlation seems to be independent of thyroid autoantibodies. Therefore, anti-C1q might point to a pathogenic mechanism involved in the development of AITD that is independent of classical thyroid autoantibodies.     

Study of induction of activation of human peripheral blood mononuclear cells with a non-activating form of anti-CD3 MoAb in autoimmune thyroid disease (AITD).

Anti-CD3 (OKT3) MoAb is a mitogenic agent which activates lymphocytes. We have studied the effects of murine anti-human OKT3 MoAb (IgG1) alone or in combination with IL-2, human thyroglobulin (Tg) and thyroperoxidase (TPO) antigens on the proliferation of whole peripheral blood mononuclear cells (PBMC) (including monocytes) or subtypes (T, CD4+, CD8+, B) as measured by tritiated thymidine (3H-TdR) incorporation. B cell differentiation was studied by measuring numbers of IgG-secreting cells and specific anti-TPO/anti-Tg-secreting cells by SPOT ELISA. PBMC or lymphocyte subtypes, obtained from 45 patients with Hashimoto's thyroiditis (HT), 40 Graves' disease (GD) and 51 normal controls were cultured in 96 microtitre plates for 6 days in the presence of OKT3 MoAb at final concentrations 25-250 ng/ml, IL-2 15 U/ml, Tg and TPO (1 micrograms/ml). Then cultures were pulsed with 0.2 microCi 3H-TdR/well and incorporation was measured after 18 h. IgG and anti-TPO/Tg-secreting cells were detected at 7 days. Higher proliferative responses from whole PBMC preparations in response to any of the combinations including OKT3 MoAb were observed in the HT preparations, while the basal values were the lowest. IL-2 alone increased these responses markedly, but equally in all groups. IL-2 in combination with OKT3 had an additive effect on proliferation, with higher responses in HT. Tg and TPO antigens did not change these responses. Most HT preparations responded with their maximum proliferation to the lowest concentration of OKT3 MoAb (25 ng/ml), whereas in GD and control preparations of PBMC these responses were shifted to higher concentrations (250 ng/ml); even with those, proliferation was not so enhanced in controls when compared with HT and GD preparations. In contrast, the proliferative responses of T cells alone and subpopulations of CD8+ suppressor/cytotoxic cells were decreased in HT preparations compared with controls. Monocytes were necessary for proliferation. In the subpopulation of B cells (> 95% pure) and CD4+ helper/inducer cells, differences did not reach significance. In spite of the effect on proliferation, OKT3 MoAb only mildly but significantly increased the numbers of IgG-secreting cells in HT and GD preparations and did not stimulate synthesis of specific antibodies. Our data suggest that the increased proliferative responses of whole PBMC to OKT3 MoAb in HT preparations might be due to insufficient activation of T suppressor/cytotoxic cells.     

A common autoepitope near the carboxyl terminus of the 60-kD Ro ribonucleoprotein: Sequence similarity with a viral protein

The Ro ribonucleoprotein is composed of hY RNA and a 60.7-kD peptide that is antigenic for autoantibodies produced by many patients with systemic lupus erythematosus or Sjögren's syndrome and mothers of newborns with complete congenital heart block. A major immunoreactive fragment (13 kD) of the 60-kD Ro is bound by 28 of 45 (62%) of the anti-Ro sera tested. Amino acid sequence analysis localizes this fragment to the carboxyl end of the 60-kD Ro peptide. All possible overlapping octapeptides of this 13-kD peptide of 60-kD Ro have been assessed for antigenicity. Sera that bind the 13-kD peptide fragment in immunoblot generally also bind the octapeptides of Ro spanning the sequence AIALREYRKKMDIPA (P<0.01). Inhibition studies with synthetic peptides and purified Ro have established specificity for reference serum antibody binding to an antigenic octapeptide, EYRKKMDI, from this region. The closely related sequence EYRKKLMD is found in the nucleocapsid protein of vesicular stomatitis virus and may portend an immunologic link to this or a related viral antigen. These results also demonstrate that despite fine specificity variation between human sera, there are recurring patterns of anti-Ro binding shared by some patients who have precipitating anti-Ro autoantibodies.     

Expression,characterization, processing and immunogenicity of an insulin-dependent diabetes mellitus autoantigen,IA-2, in Sf-9 cells

Autoantibodies to a 64-kD protein and a 40-kD tryptic fragment from pancreatic islets have been detected at high frequency in the sera of patients with insulin-dependent diabetes mellitus (IDDM). IA-2, a newly isolated transmembrane protein tyrosine phosphatase, is a major islet cell autoantigen in IDDM and the precursor of a 40-kD tryptic fragment. To express large quantities of recombinant IA-2 protein and analyse post-translational modifications we expressed full-length human IA-2 in baculovirus-infected Sf-9 cells. IA-2 expression was analysed by Western blot and by immunoprecipitation of ³⁵S-methionine-radiolabelled proteins with rabbit antisera or IDDM sera. A 120-kD IA-2 protein was detected during the early, but not the late, phase of the infection. Pulse-chase experiments showed that the 120-kD protein was processed into fragments of 64 kD and smaller fragments of ≈ 50 kD, 38 kD and 32 kD. The 64-kD fragment appeared as a doublet. Tunicamycin and PNGase F treatment down-shifted the 120-kD protein and the 64-kD doublet into lower molecular weight bands, suggesting that both were glycosylated. Trypsin treatment converted the 120-kD protein and the 64-kD doublet into a 40-kD fragment. Baculovirus-expressed IA-2 was as sensitive or slightly more sensitive than in vitro translated IA-2 in detecting autoantibodies to IA-2: 66% of sera from newly diagnosed IDDM patients reacted with baculovirus-expressed IA-2 compared with 59% of the same sera which reacted with in vitro translated IA-2. It is concluded that baculovirus-expressed IA-2 is a good source of autoantigen and that a number of lower molecular weight fragments with which IDDM autoantibodies react are derived from the 120-kD full-length IA-2 molecule.     

Profile of autoantibodies in the serum of patients with tuberculosis, klebsiella and other Gram-negative infections

Significant binding of thyroglobulin (Tg) autoantibodies was restricted to primate Tg, although some sera showed weak cross-reactivities with other species at high concentrations. In contrast rabbit anti-human Tg bound to all other animals' Tg in addition to human and this was not due to crossreactions with the thyroxyl residues. Mouse antihuman Tg monoclonal antibodies (MoAb) showed at least three different patterns of cross-reactivities. Group 1 MoAb, like Tg autoantibodies, were primate specific; two out of six MoAb bound only human Tg. Group 2 MoAb also bound a few other animals' Tg in addition to human. Of the three MoAb in group 3, two reacted with most or all of eight animal Tg tested while the other bound to all eight. However the binding of thirteen out of the fourteen MoAb against human Tg including all of the primate Tg specific group 1 MoAb was not inhibited by any of the nine Tg autoantibodies. Reactions of MoAb with heat-treated human Tg varied. Examples of decreased, increased or no change in reactivity could be demonstrated. On this basis at least five or possibly six separate human-specific epitopes could be defined by group 1 MoAb. Monoclonal antibody 1D6 (group 2) was inhibited by six out of nine Tg autoantibodies at high concentrations. This MoAb, similarly to the eight Tg autosera tested, had reduced reactivity with heat treated Tg. These studies demonstrated that human Tg has immunogenic structures conserved in the Tg of many species. The protein also has, however, primate and human-specific antigenic sites but at least five or possibly six of these are not related to autoantibody binding.     

Production of interleukin (IL)-5 and IL-10 accompanies T helper cell type 1 (Th1) cytokine responses to a major thyroid self-antigen, thyroglobulin, in health and autoimmune thyroid disease

Tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma exert detrimental effects in organ-specific autoimmune disease, while both destructive and protective roles have been demonstrated for interleukin (IL)-10, IL-4 and IL-5. We examined the production of these cytokines by peripheral blood mononuclear cells (PBMC) from patients with Hashimoto's thyroiditis (HT), Graves' disease (GD) and healthy controls, upon exposure to a thyroid self-antigen, human thyroglobulin (Tg), in the presence of autologous serum. Initially, TNF-alpha and IL-2 were produced in all three groups, accompanied by IL-10. Release of IFN-gamma, IL-4 and, notably, IL-5 ensued. Both patient groups exhibited increased TNF-alpha, IL-2, IFN-gamma and IL-10 responses, and PBMC from HT patients secreted lower amounts of IL-5 than male, but not female, controls. Enhanced TNF-alpha production by HT cells also occurred in the presence of pooled normal sera, indicating a dependency on intrinsic cellular factors. Conversely, higher production of TNF-alpha and IL-5 occurred in the presence of autologous sera than in the presence of pooled normal sera in both patient groups, indicating a dependency on serum constituents. Complement appeared to promote the production of IL-2 and particularly IL-5, the levels of which were reduced by neutralization of complement by heat- or zymosan treatment. The production of IFN-gamma and IL-2 of the three groups together correlated directly with the serum anti-Tg activity. Moreover, TNF-alpha, IFN-gamma, IL-5 and IL-10 responses were markedly inhibited by partial denaturation of Tg by boiling. We hypothesize that autoantibodies and complement may promote mixed Th1/Th2 cell cytokine responses by enhancing the uptake of autoantigens by antigen-presenting cells.     

Iodination of human thyroglobulin (Tg) alters its immunoreactivity. I. Iodination alters multiple epitopes of human Tg

Human Tg, the site of synthesis of thyroid hormones, thyroxine (T4) and triiodothyronine (T3), is one of the major autoantigens in autoimmune thyroiditis. The degree of iodination of Tg may have a major impact on its immunological properties by changing its antigenicity with respect to antibody binding. We have previously prepared a panel of MoAbs that bind to different epitopes of the Tg molecule. In the present study, we show that iodination alters the conformation of Tg molecule in such a way that it is recognized differently by different MoAbs. Monoclonal antibody 137C1 recognizes Tg regardless of its iodine content. Monoclonal antibody 42C3 recognizes Tg only if the Tg is iodinated either in vitro or in vivo. Monoclonal antibody 133B1 recognizes both in vivo iodinated Tg and non-iodinated Tg, but this MoAb did not recognize Tg following in vitro iodination. Monoclonal antibody 41A5 recognizes intact Tg and tryptic peptides of normal (in vivo) iodinated and non-iodinated Tg, but did not bind the tryptic peptides of artificially (in vitro) iodinated Tg. From the results of these experiments, we conclude that iodination of Tg by either in vivo or in vitro methods changes its conformation in such a way that some natural epitopes are ‘lost’ and some ‘new’ epitopes are generated. The generation of new epitopes may be important in the generation of autoimmune responses leading to autoimmune disease.     

Murine and human B cell epitope mapping of the Mycobacterium tuberculosis 10-kD heat shock protein using overlapping peptides.

The human immune response to the 10-kD M. tuberculosis protein was studied by a competition ELISA using monoclonal antibody (MoAb) SA-12. Twenty-five per cent of the sera from 20 patients with tuberculosis and none from 21 control subjects inhibited binding of SA-12 to the 10-kD antigen. To characterize the antigenic parts of the 10-kD antigen, overlapping decapeptides according to the amino acid sequence of the 10-kD protein were synthesized. In total, 91 sequential decapeptides, with an overlap of nine amino acids, were tested in ELISA with MoAb SA-12, human and murine sera (PEP scan). SA-12 recognized the amino acid sequence WDEDGEK (amino acid 50-56). All human sera, from patients with tuberculosis as well as from control subjects, gave almost identical undulating patterns of reactivity with the decapeptides. No relationship was found between the ability of the patients' sera to inhibit binding of MoAb SA-12 and the binding of these sera to the decapeptides comprising the epitope recognized by SA-12 in the PEP scan. Apparently, antibodies in patients' sera against the 10-kD protein are predominantly directed against discontinuous epitopes and, consequently, the continuous epitopes as presented in the PEP scan are not suitable to discriminate between patients with tuberculosis and control subjects. In the PEP scan, sera from BALB/c mice, both non-immunized and immunized with either live M. tuberculosis or the 10-kD protein gave similar patterns of reactivity, albeit different from the patterns obtained with the human sera. However, after immunization of the mice, clearly increased levels of antibodies to primary structures of the 10-kD protein were observed.     

Thyroglobulin polymorphisms in Tunisian patients with autoimmune thyroid diseases (AITD)

The thyroglobulin (Tg) gene was reported to be linked and/or associated to autoimmune thyroid diseases (AITD) development in European Caucasian populations. Here, we attempt to replicate this finding and to evaluate the contribution of the Tg gene in the genetic susceptibility of AITD in the Tunisian population. We examined the genomic region (11.5cM) containing the Tg gene by genotyping seven microsatellite markers and four SNPs located respectively at exon 10 (Ser715Ala), exon 12 (Met1009Val), exon 21 (Ala1483Ala) and exon 33 (Arg1980Trp) in 15 Tunisian multiplex families affected with AITD including Graves' disease (GD) and Hashimoto's thyroiditis (HT) (members: 87; patients: 27 GD and 16 HT). A case-control study was performed by genotyping the Tgms2 intragenic microsatellite marker (intron 27) and four intragenic SNPs on 108 unrelated patients affected with GD and 169 normal controls. Analysis of family data did not show linkage of the thyroglobulin gene with AITD nor did analysis of case-control data show association of Tgms2 or SNPs with GD. In contrast to the European Caucasian population, we failed to detect any contribution of Tg gene in the genetic component of Tunisian AITD.     

Epitope mapping with synthetic peptides of 52-kD SSA/Ro protein reveals heterogeneous antibody profiles in human autoimmune sera.

The reactivity of autoantibodies present in the sera of 489 patients with Sjögren's syndrome (SS), systemic lupus erythematosus (SLE) and other autoimmune diseases was investigated by ELISA using recombinant 52-kD SSA/Ro protein (rRo52) and 39 overlapping synthetic peptides representing the entire sequence of Ro52. We report that IgG antibodies reacting with rRo52 were present in the sera of a large number of patients with SS (67% of patients with primary SS and 46% of patients with SS associated with SLE), whereas they were less frequent (10-25%) in SLE, rheumatoid arthritis (RA), juvenile chronic arthritis (JCA) and mixed connective tissue disease (MCTD), and absent in scleroderma. Among the 39 peptides tested, five were recognized by sera from 30-65% of patients with SS, namely peptides representing residues 2-11, 107-122, 107-126, 277-292 and 365-382. Patients with JCA had raised levels of IgG antibodies reacting with peptides 2-11 and 365-382, and 51% of patients with MCTD had raised levels of IgG antibodies reacting with peptide 365-382. None of the five peptides was recognized by more than 20% of sera from patients with SLE and RA. Interestingly, and of importance in the field of diagnostic tests based on peptides, the reactivity of antibodies to the Ro52 synthetic peptides varied greatly according to the origin of sera. Inhibition experiments using either patients' sera or antibodies induced in rabbits against Ro52 peptides showed that the four domains 2-11, 107-122, 277-292 and 365-382 are accessible on the surface of the Ro52 protein. These regions may thus be involved in the induction of specific antibodies in autoimmune patients.     

The expression and distribution of S-100 protein and CD 83 in thyroid tissues of autoimmune thyroid diseases

To investigate the expression and distribution of S-100 protein and CD83 in the thyroid tissues of autoimmunethyroid diseases(ATDs),and to study the role of the dendritic cells in the pathogenesis of ATDs,immunohistochemical staining was used on pathological tissues of 20 patients with Hashimoto's thyroiditis(HT)and 20 patients with Graves' disease(GD) to check the expression and distribution of S-100 protein and CD83.Compared with control group(20 cases of thyroid follicular adenoma,TFA),the higher expressions of S-100 inHT(139.38±5.92 vs 59.47±11.69) and GD(119.42±14.48 vs 59.47±11.69) were observed respectively(p<0.001).The increased positive expressions of CD83 which is known as a marker of mature and activated DCs inHT(22.58±13.96 vs 5.19±8.08) and GD(29.92±14.43 vs 5.19±8.08) were also found respectively(p<0.001).Serum TPO antibody(TPO-Ab,67.3±11.6%) and Tg antibody(Tg-Ab,59.8±10.1%) in HT were higher thanthose in GD(28.4±5.7%,23.1±4.9%) and TFA(6.1±3.4%,7.2±4.6%)(p<0.01).Serum TR-Ab in GD(16.3±5.6 U/L) was higher than those in HT(4.8±2.3 U/L) and TFA(2.5±1.2 U/L)(p<0.01).Our findings suggestthat the high expression of DCs' markers may be related to the pathogenesis of HT and GD.The upregulationof both the number and the matured functions of DCs,may lead to present more antigens and to produce moreauto-antibodies(such as Tg-Ab and TPO-Ab in HT,TR-Ab in GD),which may be involved in pathogenesis ofthe autoimmune thyroid diseases.Cellular & Molecular Immunology.2004;1(5):378-382.     

Recognition of synthetic peptides of Sm-D autoantigen by lupus sera.

The reactivity of autoantibodies present in the serum of patients with systemic lupus erythematosus (SLE) was investigated by ELISA using seven overlapping synthetic peptides representing the entire sequence of the polypeptide D component of 'Sm antigen'. Of the 165 SLE sera tested, 59% were found to contain IgG antibodies able to bind to peptide 1-20, while 37% of the sera reacted with peptide 44-67. All sera reacting with peptide 44-67 also reacted with peptide 1-20. These two peptides were only seldom recognized by the sera of 187 patients with other rheumatic autoimmune diseases or by 53 sera of normal individuals. In a parallel study using sera that reacted with the D band in immunoblotting, most of the sera recognized peptides 44-67 (89%) and 1-20 (67%), while 33% of them reacted with peptide 97-119. The use of these synthetic peptides in ELISA may be of considerable help for detecting anti Sm autoantibodies.