Significance of thyroglobulin antibodies cross-reactive with thyroperoxidase (TGPO antibodies) in individual patients and immunized mice.

Thyroglobulin (TG) and thyroperoxidase (TPO), both involved in thyroid hormone synthesis, represent major autoantigens in thyroid autoimmune disease. Despite numerous studies, the emergence, pathophysiological significance and role of autoantibodies to TG and TPO remain elusive. The recent identification of a new category of thyroid-specific autoantibody interacting with both TG and TPO (TGPO autoantibodies) offers a new opportunity in the study of thyroid autoimmunity. To gain a better insight into the significance of these TGPO autoantibodies, measurement in individual samples appeared necessary. The unique property of TGPO autoantibodies, simultaneous binding to TG and TPO, was used to set up a sandwich method which combined coated TG and radio-iodinated TPO. This method was found to be strictly specific for TGPO autoantibodies and sensitive enough to assay TGPO autoantibodies in serum. In humans, TGPO autoantibodies were found in most of the sera with high TG and TPO autoantibody titres, but not in sera negative for TG autoantibodies, whatever the TPO autoantibody titre. Furthermore, high TGPO autoantibody titres were found in sera strongly cytotoxic for cultured porcine thyroid cells. However, significant correlation of TGPO autoantibody titre was observed neither with TG and TPO autoantibody titres (n = 48) nor with complement-dependent cytotoxicity (n = 50). TGPO antibody assay was also performed in individual plasma of CBA/J mice immunized with either human TG (n = 6) or human TPO (n = 6). Immunization with TG induced high levels of not only TG but also TGPO antibodies, which exhibited a strong reactivity for TPO and whose binding to TG and TPO was fully inhibited by TG. In contrast, immunization with TPO induced high levels of only specific TPO antibodies accompanied by low levels of specific TG antibodies. In this case TGPO antibodies were not detected. Of note, TG- and TPO-immunized mice mounted an immune response against their own TG, but did not exhibit histological signs of thyroiditis. Large panels of TG and TPO MoAbs were also investigated with this method: 18/25 TG MoAbs and only 1/13 TPO MoAbs were found cross-reactive. Taken together, these data provide evidence that TGPO antibodies are effectively present in individual patients and TG-immunized mice, are different from specific TG and TPO antibodies, and may derive from natural B cell repertoire by autoimmune processes involving TG and not TPO.     

Genetic and epitopic analysis of thyroid peroxidase (TPO) autoantibodies: markers of the human thyroid autoimmune response

TPO autoantibodies, the hallmark of human autoimmune thyroid disease, are of IgG class and are associated with thyroid destruction and hypothyroidism. Using the immunoglobulin gene combinatorial library approach, a panel of human monoclonal TPO autoantibodies (expressed as Fab) has been generated from thyroid tissue-infiltrating B cells. TPO-specific Fab closely resemble patients' serum autoantibodies in terms of L chain type, IgG subclass, affinities for TPO as well as epitopes recognized by > 80% of TPO autoantibodies in an individual's serum. TPO autoantibody V region genes are not unique; H chain V genes are usually mutated, while L chain V genes are sometimes in germ-line conformation. The autoantibodies recognize an immunodominant region involving conformational, overlapping epitopes in domains A and B. Finally, TPO autoantibody epitopic fingerprints are distinctive for individual sera, are not associated with hypothyroidism, but are conserved over time (indicating a lack of B cell epitope spreading). Evidence for conservation as well as inheritance of the fingerprints in some families, together with V_H gene polymorphisms, may provide insight into the genetic basis of human autoimmune thyroid disease. Furthermore, monoclonal human TPO autoantibodies will be invaluable for B cell presentation of TPO to determine the T cell epitopes involved in TPO autoantibody production.     

Autoimmune response to the thyroid in humans: thyroid peroxidase--the common autoantigenic denominator

Autoimmunity to thyroid peroxidase (TPO), manifest as high affinity IgG class autoantibodies, is the common denominator of human thyroid autoimmunity, encompassing patients with overt hyper- or hypothyroidism as well as euthyroid individuals with subclinical disease. The identification and cloning of TPO (the "thyroid microsomal antigen") provided the critical tool for analyzing B and T cell reactivity to this major thyroid autoantigen. In particular, the availability of immunoreactive TPO permitted the isolation of essentially the entire repertoire of human monoclonal antibodies, a feat unparalled in an organ-specific autoimmune disease. These recombinant autoantibodies (expressed as Fab) provide insight into the genes encoding their H and L chains as well as the conformational epitopes on TPO with which serum autoantibodies interact. Analyses of TPO autoantibody epitopic "fingerprints" indicate a lack of epitope spreading as well as a genetic basis for their inheritance. Limited data are available for the responses and cytokine profiles of T cells to endogenously processed TPO. Moreover, the role of thyroid cells in initiating the autoimmune response to TPO, and of B cells in expanding and/or modulating the response of sensitized T cells, has yet to be established. Finally, because autoantibody (and likely T cell) responses to TPO parallel those to TSH receptor and thyroglobulin, manipulation of T and B cell responses to TPO may provide the basis for the development of immunospecific therapy for autoimmune thyroid disease in general.     

Anti-thyroglobulin autoantibodies in sera from patients with chronic thyroiditis and from healthy subjects: differences in cross-reactivity with thyroid peroxidase.

A significant portion (about 12.7%) of healthy subjects was found to contain anti-thyroglobulin (anti-Tg) antibodies in their sera. We compared the binding activities of these antibodies and of anti-Tg autoantibodies from sera of patients with chronic thyroiditis with human thyroid peroxidase (TPO). The results obtained by ELISA indicated that out of 10 healthy subjects with anti-Tg antibodies, only four had anti-Tg antibodies capable of binding to TPO, whereas anti-Tg autoantibodies from almost all patients with chronic thyroiditis possessed high binding activities to TPO. By use of the immunoprecipitation method, it was also shown that although all anti-Tg autoantibodies from patients precipitated TPO, a majority of anti-Tg antibodies from healthy subjects could not precipitate TPO. Such findings cannot be ascribed to the differences in levels of anti-Tg autoantibodies and anti-TPO autoantibodies in sera and the differences in avidities of anti-Tg antibodies in sera between healthy subjects and patients with chronic thyroiditis. Thus, it can be concluded that anti-Tg antibodies from healthy subjects differ from those of patients with chronic thyroiditis with respect to TPO binding, probably due to difference in fine specificities of these anti-Tg antibodies.     

Autoimmune Response to the Thyroid in Humans: Thyroid Peroxidase-The Common Autoantigenic Denominator

Autoimmunity to thyroid peroxidase (TPO), manifest as high affinity IgG class auto-antibodies, is the common denominator of human thyroid autoimmunity, encompassing patients with overt hyper-or hypothyroidism as well as euthyroid individuals with subclinical disease. The identification and cloning of TPO (the “thyroid microsomal antigen”) provided the critical tool for analyzing B and T cell reactivity to this major thyroid autoantigen. In particular, the availability of immunoreactive TPO permitted the isolation of essentially the entire repertoire of human monoclonal antibodies, a feat unparalled in an organ-specific autoimmune disease. These recombinant autoantibodies (expressed as Fab) provide insight into the genes encoding their H and L chains as well as the conformational epitopes on TPO with which serum autoantibodies interact. Analyses of TPO autoantibody epitopic “fingerprints” indicate a lack of epitope spreading as well as a genetic basis for their inheritance. Limited data are available for the responses and cytokine profiles of T cells to endogenously processed TPO. Moreover, the role of thyroid cells in initiating the autoimmune response to TPO, and of B cells in expanding and/or modulating the response of sensitized T cells, has yet to be established. Finally, because autoantibody (and likely T cell) responses to TPO parallel those to TSH receptor and thyroglobulin, manipulation of T and B cell responses to TPO may provide the basis for the development of immunospecific therapy for autoimmune thyroid disease in general.     

Rarity of autoantibodies to a major autoantigen, thyroid peroxidase, that interact with denatured antigen or with epitopes outside the immunodominant region.

The nature of the autoantibody repertoire to the dominant autoantigen in human autoimmune thyroid disease is controversial. There is evidence that autoantibodies to thyroid peroxidase (TPO) interact with overlapping conformational epitopes in an immunodominant region and binding to denatured (DN) protein is decreased. Contrary data demonstrate TPO autoantibody reactivity with DN-TPO or polypeptide fragments. However, none of the TPO-specific, human monoclonal autoantibodies isolated to date preferentially recognize denatured autoantigen. We therefore searched an immunoglobulin gene phage display library for human autoantibodies that bind TPO denatured by reduction and alkylation (DN-TPO). Thyroid-infiltrating B cells from a typical TPO autoantibody-positive patient were the source of mRNA for library construction. Surprisingly, the library enriched after panning on DN-TPO, as well as a panel of individual clones, preferentially bound native (N)-TPO. Of 13 clones selected using DN-TPO or N-TPO, 12 clones recognized the TPO immunodominant region. Moreover, regardless of selection with N-TPO or DN-TPO, their heavy and light chains were encoded by similar VDJ and Vkappa combinations. One clone (DN4), isolated using DN-TPO, did not interact with the TPO immunodominant region and its H chain derives from a different VH gene. Although DN4 binds specifically to TPO, its affinity is low, unlike the high affinities of other human TPO autoantibodies. In conclusion, human monoclonal autoantibodies that preferentially recognize denatured TPO could not be isolated from an immunoglobulin gene library despite selection with denatured protein. Our findings demonstrate the bias of the human B cell repertoire towards recognition of an immunodominant region on the conformationally intact form of a major thyroid autoantigen.     

Heterogeneity of thyroid autoantigens identified by immunoblotting

Autoimmune thyroid disease in man is commonly associated with autoantibodies against thyroglobulin, microsomes, and the TSH receptor, and the character and specificity of these antithyroid antibodies have been extensively utilized in investigating these conditions. In the present study we have asked whether other thyroid-related antigens exist, against which autoantibodies may be directed. A crude thyroid extract was separated by polyacrylamide gel electrophoresis followed by immunoblotting with serum obtained from patients with Graves' disease or Hashimoto's thyroiditis. Antibodies in sera from patients with Graves' disease and Hashimoto's thyroiditis reacted with many antigenic determinants in immunoblots of the thyroid membrane preparation (2000g supernatant). These determinants were disease specific in that sera from normals and patients with Addison's disease and rheumatoid arthritis did not react, but there was no difference between the patterns of reactivity with Graves' disease or Hashimoto's thyroiditis sera. Thyroglobulin produced two predominant bands of reactivity at 320 and 200 kDa, whereas purified microsomal antigen produced a triplet of bands around 105 kDa, when these preparations were reacted with appropriate autoimmune sera. Nonetheless, some sera produced additional bands with the microsomal antigen blots, indicating that some of the antigens which were detected using crude thyroid membrane remained in the microsome preparation to produce multiple antibody binding reactivities. We were unable to inhibit any of the antibody binding with TSH. Purification of individual thyroid antigens on the basis of their molecular weights should standardize current antibody assays and permit more detailed evaluation of the cellular immune responses in Graves' disease and Hashimoto's thyroiditis.     

Detection, epitope-mapping and function of anti-Fas autoantibody in patients with silicosis

Dysregulation of apoptosis through the Fas-Fas ligand pathway is associated with the onset of autoimmune disease. Since autoantibodies directed against unknown antigens are present in the sera of these patients, sera samples were examined for the presence of autoantibodies directed against the Fas molecule. Using Western blotting and a ProteinChip analysis, autoantibodies against Fas were detected in patients with silicosis, systemic lupus erythematosus (SLE) and systemic sclerosis (SSc), and weakly detected in healthy individuals. Using epitope mapping employing 12-amino-acid polypeptides with the SPOTs system, a minimum of four epitopes and a maximum of 10 epitopes were found. Several amino acid residues involved in binding FasL, such as C66, R87, L90, E93 and H126, were presented within the epitopes. Serum containing a large amount of anti-Fas autoantibody from silicosis patients inhibited the growth of a Fas-expressing human cell line, but did not inhibit the growth of a low Fas-expresser nor a Fas-expresser in which the Fas gene had been silenced by small interference RNA. All epitopes in the intracellular region of Fas were located in the death domain. The possible roles of anti-Fas autoantibody detected in healthy volunteers and patients with silicosis or autoimmune diseases are discussed here.     

Immunoprecipitation of steroidogenic enzyme autoantigens with autoimmune polyglandular syndrome type I (APS I) sera; further evidence for independent humoral immunity to P450c17 and P450c21

Three steroidogenic P450 cytochromes, steroid 17α-hydroxylase (P450c17), steroid 21-hydroxylase (P450c21) and side-chain cleavage enzyme (P450scc), have been described as autoantigens in APS I. In this study we report an immunoprecipitation assay for the detection of autoantibodies to these three enzymes using in vitro³⁵S-labelled antigens. Overall, 33 out of 46 (72%) patients with APS I had autoantibodies to at least one of the three proteins and each protein was recognized by patient sera with equal frequency. A higher rate of autoantibody positivity was observed in APS I patients with Addison's disease compared with patients without Addison's disease (85% versus 39%). All 11 patients with ovarian failure had anti-P450c17 or anti-P450scc antibodies. The immunoprecipitation results with P450c17, P450c21 and P450scc correlated well with the results obtained by immunoblotting assays. In addition, the steroidogenic enzymes 11β-hydroxylase (P450c11β), aromatase (P450arom), 3β-hydroxysteroid dehydrogenase (3βHSD) and adrenodoxin were studied by immunoprecipitation assay, but no reaction was found either with 46 APS I or with 26 healthy control sera. To study the suggested immunological cross-reactivity between P450c17 and P450c21 enzymes, nine APS I patient sera were preabsorbed with bacterially expressed P450c17 or P450c21 and subsequently used in immunoprecipitation assay. The absorption experiments clearly indicated that the preincubation inhibited only the reactivity of corresponding antigen, suggesting independent autoantibody response to the two enzymes. Our results suggest that the immune response to some but not to all steroidogenic enzymes is a specific feature of APS I that may be pathogenically significant.     

Anti-thyroid peroxidase antibody activity in sera of patients with systemic lupus erythematosus.

Although patients with SLE have autoantibodies to thyroid peroxidase (TPO), IgG from sera of SLE patients does not inhibit TPO activities, in contrast with IgG from sera of patients with thyroid disorders. This finding suggests that the specificities of anti-TPO autoantibodies in SLE are different from those in cases of thyroid disorders. These autoantibodies to TPO should be considered when searching for associations between SLE and autoimmune thyroid disorders.     

Autoantibodies to thyroid peroxidase in patients with chronic thyroiditis: effect of antibody binding on enzyme activities.

Using thyroid peroxidase (TPO), which was purified from the thyroid of patients with Graves' disease, we attempted to determine whether sera from patients with chronic thyroiditis contained antibodies to the enzyme. When the binding was tested by ELISA, sera from patients with chronic thyroiditis revealed high binding activities to TPO. When TPO was incubated with IgG from sera followed by treatment with protein A-Sepharose and centrifugation, the remaining TPO activities in the supernatant fraction were lower in most of the patients, as compared to normal controls. Moreover, IgG purified by DEAE-cellulose chromatography from sera in patients interfered with the TPO activities. Titres of anti-TPO antibodies correlated well with those of anti-microsome antibodies. These results indicate the presence of autoantibodies to TPO in sera of most patients with chronic thyroiditis and that TPO may be one component of microsome antigen complexes recognized by the autoantibodies. Studies on the inhibition of TPO by IgG isolated from sera of patients using guaiacol and iodide assays revealed that at least three epitopes of TPO molecule were recognized by autoantibodies and that the antigenic determinants on TPO molecule recognized by autoantibodies could be heterogeneous in patients.     

Analysis of carbohydrate residues on human thyroid peroxidase (TPO) and thyroglobulin (Tg) and effects of deglycosylation, reduction and unfolding on autoantibody binding.

The contribution of carbohydrate residues and peptide chain conformation to autoantibody binding sites on human thyroid peroxidase (TPO) and thyroglobulin (Tg) has been investigated. In addition the nature of carbohydrate residues associated with human TPO has been studied. 125I-labelled human TPO and Tg were treated with the following glycosidases: EndoD, EndoH, neuraminidase, O-glycanase, neuraminidase followed by O-glycanase and PNGaseF. Thereafter binding to different sera containing TPO autoantibodies and Tg autoantibodies was assessed using solid phase protein A to separate antibody-bound and free labelled antigens. In addition, labelled Tg and TPO were treated with reducing agent (dithiothreitol) or sodium acetate buffer pH 7.5, 5.5 and 3.2 (followed by neutralisation with 2 M Tris pH 8.3) prior to antibody binding studies. Furthermore, the effect of deglycosylation and treatment with acid buffers on TPO enzyme activity was studied. The nature of carbohydrate residues associated with hTPO was analysed by assessment of the effects of different glycosidases on 125I-TPO mobility on SDS-PAGE followed by autoradiography and by the use of lectins. Deglycosylation of labelled Tg and TPO had no clear effect on Tg and TPO autoantibody binding. Reduction of labelled Tg and TPO resulted in almost complete loss of autoantibody binding with all sera studied. Furthermore, adjusting the pH of labelled TPO or Tg transiently to pH 5.5 lowered autoantibody binding in the case of all the sera and the effect was more marked at pH 3.2. TPO enzyme activity (guaiacol assay) of unlabelled TPO was decreased after treatment with EndoH but not with other glycosidases. The low pH buffers affected unlabelled TPO enzyme activity measured by iodide assay. Treatment of 125I-labelled TPO with EndoH, neuraminidase and PNGaseF caused marked changes in the double band pattern characteristic of TPO on analysis by SDS gel electrophoresis (TPO doublet). Analysis of changes in the mobility of the 2 bands of the doublet after treatment with different glycosidases and binding studies with lectins indicated that both high mannose and complex type sugar residues were associated with hTPO. The high mannose type residues were associated mostly with the lower band of the hTPO doublet whereas complex type residues were associated mostly with the upper band. Overall, our studies indicate that (1) the major autoantibody binding sites on hTPO and hTg are conformational, (2) sugar residues do not appear to be important in forming the autoantibody binding sites on hTPO and hTg, and (3) both high mannose type and complex type sugar residues are associated with hTPO.     

Proportion of antibodies to the A and B immunodominant regions of thyroid peroxidase in Graves and Hashimoto disease

Autoantibodies to thyroid peroxidase (TPO) predominately recognise conformational epitopes, restricted to two immunodominant regions (IDR) -A and -B. We have estimated the proportion of IDR-A and -B autoantibodies in 75 Hashimoto and 68 Graves patients sera. There were no statically significant differences between Hashimoto and Graves patients sera in the IDR-A and -B autoantibodies level, despite great differences between individual patients sera. In 75 Hashimoto patients sera, the mean value of IDR-A was 24.26%, IDR-B--52.26%, IDR (A + B)-76.66%, and to non-A, non-B regions--23.5%. In 68 Graves disease patients, the mean value of IDR-A was 24.87, IDR-B--54.29, IDR (A + B)--79.07, non-A, non-B--20.92%, at a single autoantibodies concentration 5IU/ml. At high autoantibodies concentration (50 IU/ml) the proportion of autoantibodies to the IDR (A + B) diminished to 58% and to the non-A, non-B TPO regions increased to 42%. The autoimmune response to TPO regions outside the IDR (A + B) epitopes is stronger then previously assumed and this response is also conformation dependent.     

Production and characterisation of a human monoclonal thyroid peroxidase autoantibody.

A human-mouse hybridoma has been produced by fusion of Hashimoto thyroid lymphocytes with the mouse myeloma line X63-Ag8.653. The cloned hybridoma secreted 2.5 micrograms per 10(6) cells per day of an IgG kappa thyroid peroxidase (TPO) autoantibody (2G4) with high affinity (2.5 x 10(9) molar-1) and specificity for human TPO. 2G4 did not react with lactoperoxidase, horseradish peroxidase or human myeloperoxidase or with porcine TPO or with human thyroglobulin. Plastic tubes coated with 2G4 bound about 50% of 125I-labelled human TPO added and the binding was inhibited by IgGs prepared from 18/18 TPO autoantibody-positive sera. This indicated that all 18 sera contained autoantibodies which recognised the same (or closely related) epitope as 2G4. Plastic tubes coated with IgGs from different TPO autoantibody-positive patient sera also bound 125I-labelled TPO but inhibition by 2G4 in this system was not complete. This suggested that the sera contained at least 2 types of TPO autoantibodies, with only one type of autoantibody reactive with the same epitope as 2G4.     

Mapping of autoantigenic epitopes on recombinant thyroid peroxidase fragments using the polymerase chain reaction.

Cloned cDNA templates of thyroid peroxidase (TPO) have been used in conjunction with the polymerase chain reaction (PCR) to express selected segments of the thyroid microsomal/peroxidase antigen (TMA/TPO) as recombinant protein in E. coli. Six small, different recombinant fragments averaging 120 amino acid residues and one large fragment (269 amino acids) of TPO which together encompass 80% of the extracellular region of the molecule have been produced and autoantibody (aAb) binding sites analysed by immunoblotting. A minimum of six independent, sequential antigenic determinants have been localized on the recombinant proteins and these map to the amino terminal, the central core region and the carboxyl terminal of the TPO molecule. More accurately, the six antigenic sites reside on overlapping recombinant TPO preparations termed R1a + R1b (residues 1 to 160) R1c (residues 145 to 250), R2b (residues 457 to 589), R3a (residues 577-677), R3b (residues 657-767) and R3c (residues 737-845). The large fragment of TPO termed R3 (residues 577-845) encompassing R3a, R3b and R3c also reacts with the aAbs. Different sera from patients with autoimmune thyroid disease contain antibodies to TMA/TPO which differ in their fine specificity. The use of recombinant molecular biological techniques together with PCR to prepare small segments of a large autoantigen as recombinant protein will now allow studies to progress on autoepitope mapping of the precise amino acid sequences of the TPO molecule with the use of synthetic peptides.     

Anti-soluble liver antigen (SLA) antibodies in chronic HCV infection

Hepatitis C infection is associated with autoimmune disorders, such as the production of autoantibodies. Anti-LKM1 and anti-LC1, immunomarkers of type 2 autoimmune hepatitis, have been previously associated with a HCV infection. Anti-Soluble-Liver-Antigen autoantibodies (SLA) are specifically associated with type 1 and type 2 autoimmune hepatitis and more closely related to patients who relapse after steroid therapy. The recent molecular cloning of the soluble liver antigen provides the opportunity to develop more specific tests for the detection of antibodies against it. The aim of this work is to characterize anti-soluble-liver autoantibodies in sera from patients chronically infected by HCV. A recombinant cDNA from activated Jurkat cells coding for the full length tRNP(Ser)Sec/SLA antigen was obtained. ELISA, Western Blot and immunoprecipitation tests were developed and used to search for linear and conformational epitopes recognized by anti-SLA antibodies in sera from patients chronically infected by HCV. Anti-soluble liver antigen antibodies were found in sera from 10.4% of HCV-infected patients. The prevalence was significantly increased to 27% when anti-LKM1 was also present. Most anti-SLA reactivity was directed against conformational epitopes on the antigen. The means titers by ELISA were lower than those obtained in type 2 AIH. The result of autoantibody isotyping showed a subclass restriction to IgG1 and also IgG4. This study shows the presence of anti-SLA antibodies in approximately 10% of HCV infected patients. The prevalence of SLA autoantibodies in HCV infected patients increases when LKM1 autoantibodies are also present. The relationship between the prevalence of this characteristic autoimmune hepatitis autoantibody and the implication of an autoimmune phenomenon in the liver injury of patients chronically infected by HCV needs further investigation.     

Centrosome Proteins: A Major Class of Autoantigens in Scleroderma

Autoantibodies to intracellular antigens are a hallmark of autoimmune diseases, although their role in disease pathogenesis is unclear. Centrosomes are organelles involved in the organization of the mitotic spindle and they are targets of autoantibodies in systemic sclerosis (SSc). We used recombinant centrosome autoantigens, centrosome-specific antibodies, and immunoassays to demonstrate that a significant proportion of SSc patients exhibited centrosome reactivity. Two centrosome proteins cloned in our laboratory were used to screen 129 SSc sera by Western blotting. The same sera were screened by immunofluorescence using centrosome-specific antibodies to distinguish centrosomes from nuclear speckles commonly stained by SSc sera. Using these criteria, 42.6% of SSc patients were autoreactive to centrosomes, a larger percentage than reacted with all other known SSc autoantigens. Most centrosome-positive sera reacted with both centrosome proteins and half were negative for other routinely assayed SSc autoantibodies. By these criteria, we have identified a novel class of SSc autoreactivity. Only a small percentage of normal individuals and patients with other connective tissue diseases had centrosome reactivity. These results demonstrate that centrosome autoantibodies are a major component of autoreactivity in SSc and thus have potential in disease diagnosis. Centrosome autoantigens may be useful in studying the development of autoantibodies and chronic inflammation in SSc and perhaps other autoimmune diseases.     

Onset and regulation of anti-lamin B autoantibody production is independent of the level of polyclonal activation

Anti-lamin B autoantibodies are associated with both systemic lupus erythematosus (SLE) and autoimmune liver disease. We examined the possibility that the underlying clinical feature in patients with anti-lamin B autoantibodies might be chronic autoimmune liver disease, and whether the hypergammaglobulinemia present in both disorders is involved in generating anti-lamin B autoantibodies. A lamin B fusion protein (MLB1), consisting of amino acids 77-533 of lamin B fused to TrpE, was used to screen sera from 84 patients with SLE for anti-lamin B autoantibodies. 3/4 prototype human lamin B antisera, 5/84 SLE sera (6%), and 0/30 sera from healthy individuals reacted with MLB1 on immunoblots at a 1:500 dilution. Of the 9 anti-lamin B autoantibody positive patients studied, all but 1 fulfilled at least four ARA criteria for SLE. None of the patients displayed evidence of chronic autoimmune liver disease, suggesting that autoimmune liver disease is not strongly associated with anti-lamin B antibodies in SLE. In SLE, as in "lupoid hepatitis", anti-lamin B autoantibodies are often produced transiently during periods of increased disease activity. Although polyclonal hypergammaglobulinemia is also associated with increased activity of both diseases, anti-lamin B autoantibody production in 2 patients was independent of total immunoglobulin levels, antibodies to irrelevant proteins, and production of some other autoantibodies. Thus, polyclonal activation is insufficient to explain either the initiation or regulation of anti-lamin B autoantibody production, supporting the hypothesis that antinuclear antibodies are antigen-selective.     

Anti-LC1 autoantibodies in patients with chronic hepatitis C virus infection

Various autoantibodies have been reported in patients chronically infected by hepatitis C virus. 2% to 10% of theses patients have anti-liver-kidney microsome type 1 (anti-LKM1) autoantibodies. In type 2 autoimmune hepatitis, anti-LKM1 autoantibodies are frequently associated with anti-liver-cytosol type 1 (anti-LC1) autoantibodies. AIMS: To determine the prevalence of anti-LC1 autoantibodies in a hepatitis C-positive population and characterize their reactivity. METHODS: 146 patients suffering from liver diseases, of which 99 were chronically infected by hepatitis C virus, were tested by Western blotting and immunoprecipitation to detect and characterize anti-LC1 autoantibodies. RESULTS: 12% of this hepatitis C population had anti-LC1 autoantibodies. LC1 positivity by Western blotting was 30% of LC1+ sera. Epitopes were found throughout the protein but linear epitopes were situated in the 395-541 amino acid region of formiminotransferase cyclodeaminase. Three putative conformational epitopes were identified by phage display. CONCLUSIONS: Anti-LC1 autoantibodies are as prevalent as anti-LKM1 autoantibodies in patients infected with hepatitis C virus and their production is not dependent of anti-LKM1 autoantibodies formation. Autoantibody reactivity against the anti-LC1 antigen is different in hepatitis C than in type 2 autoimmune hepatitis. Anti-LC1 autoantibodies can now be regarded as a serological marker of autoimmunity in chronic hepatitis C infection.