Autoimmune targeted disruption of the pituitary-ovarian axis causes premature ovarian failure

Premature ovarian failure (POF) is characterized by amenorrhea and high serum levels of follicle-stimulating hormone (FSH). POF causes female infertility and represents a substantial women's health risk affecting 1% of women by age 40. Although ovarian autoimmunity has been associated with POF, the identity of ovarian Ags recognized is unknown. In this study, we show that autoimmune-targeted disruption of the pituitary-ovarian axis leads to POF. Immunization of SWXJ female mice with the p215-234 peptide derived from mouse inhibin-alpha activates CD4(+) T cells and induces experimental autoimmune oophoritis with a unique biphasic phenotype characterized by an early stage of enhanced fertility followed by a delayed stage of POF. Affected mice show high serum levels of inhibin-alpha-neutralizing Abs that prevent inhibin-mediated down-regulation of activin-induced pituitary FSH release. The loss of activin/FSH down-regulation leads to prolonged metestrus-diestrus, superovulation, increased numbers of mature follicles, increased offspring, accelerated depletion of primordial follicles, and ultimately premature infertility. Thus, inhibin-alpha-targeted experimental autoimmune oophoritis is initiated by CD4(+) Th1 T cells that stimulate B cells to produce inhibin-alpha-neutralizing Abs directly capable of mediating POF and transferring disease into naive recipients. Our inhibin-alpha autoimmune model of POF shows how premature infertility may develop in the context of elevated FSH levels thereby closely mimicking the hallmark features of human POF.     

Specific and sensitive immunoassays detect multiple anti-ovarian antibodies in women with infertility.

Serum anti-ovarian antibodies (AOAs) have been shown in autoimmune premature ovarian failure and in vitro fertilization-embryo transfer (IVF-ET) cases. The specificity of assays detecting these antibodies has been questioned. Researchers have used several techniques (e.g., ELISA and indirect immunofluorescence). Few have reported on the non-specificity and the type of molecular and cellular targets. We reported earlier on the presence of naturally occurring anti-albumin antibodies as the likely factor for non-specificity. Having developed a novel blocking recipe, we show substantial elimination of this non-specificity. With these standardized tests, we hereby report multiple targets at protein and histological levels. In our study group, 15 of 50 (30%) patients with premature ovarian failure and 13 of 50 (26%) IVF-ET patients showed the presence of AOAs. Western blotting showed a large number of patients making AOAs to a 90-kDa protein, followed by 97- and 120-kDa proteins. Histochemically, it was evident that the sera of these patients predominantly react with the oocyte; other somatic cellular targets are also involved. The specific non-invasive test developed by us was found to be useful because it could carry out a reliable diagnosis of an autoimmune etiology that would be very helpful to select patients in whom immune-modulating therapy could be recommended, which in turn may restore ovarian function and fertility.     

Application of antigen retrieval by heating for double-label fluorescent immunohistochemistry with identical species-derived primary antibodies.

Double-label fluorescent immunohistochemistry (IHC) is frequently used to identify cellular and subcellular co-localization of independent antigens. In general, primary antibodies for double labeling should be derived from independent species. However, such convenient pairs of antibodies are not always available. To overcome this problem, several methods for double labeling with primary antibodies from identical species have been proposed. Among them are methods using monovalent secondary antibodies, such as Fab fragments. Soluble immune complexes consisting of primary and monovalent secondary antibodies are first formed. After absorption of the excess secondary antibody with nonspecific immunoglobulin, the immune complexes are applied to sections. By this procedure, unwanted cross-reaction between false pairs of antibodies is avoidable. However, soluble immune complexes often show reduced or no immunoreactivity to antigens on sections. I noted that antigen retrieval (AR) of tissues by heating often but not always showed improved immunoreactivity for soluble immune complexes. Here I demonstrate the examination of conditions for this soluble immune complex method using AR-treated sections and show examples of double-label fluorescent IHC with identical species-derived primary antibodies.     

Anti-ovary antibodies after attempts at human in vitro fertilization induced by follicular puncture rather than hormonal stimulation.

Anti-ovary antibodies (AOA) have been detected in serum samples of women undergoing in vitro fertilization (IVF). High concentrations of these antibodies have been found in women who have had several IVF attempts and they appear to correlate with reduced chances of pregnancy. In this paper, AOA were assayed sequentially in a series of 140 IVF candidates to investigate the respective roles of hormonal stimulation and follicular puncture in inducing the autoimmune response. Serum was obtained 8 days after the beginning of ovarian human menopausal gonadotrophin (hMG) stimulation, then 15 days after follicular puncture. Significantly higher concentrations of IgG (P < 0.0001) AOA were observed in the second series of samples than in the first, suggesting that ovarian trauma and not hormonal stimulation is responsible for triggering antibody production. In the whole group, there was a negative correlation between IgM levels after puncture and oocyte numbers (P < 0.05). Among 'immune-responder' women, the concentrations of IgA AOA (P = 0.01) in the first sample, and of IgG (P = 0.01) or IgA AOA (P < 0.05) in the second, correlated with fewer oocytes after stimulation. There was no variation in the mean concentrations of AOA in women who achieved pregnancy.     

Human placenta‐derived mesenchymal stem cells inhibit apoptosis of granulosa cells induced by IRE1α pathway in autoimmune POF mice

Previous studies have shown that the ovarian failure in autoimmune‐induced premature ovarian failure (POF) mice could be improved by the transplantation of human placenta‐derived mesenchymal stem cells (hPMSCs); however, the protective mechanism of hPMSCs transplantation on ovarian dysfunction remains unclear. Ovarian dysfunction is closely related to the apoptosis of granulosa cells (GCs). To determine the effects of hPMSCs transplantation on GCs apoptosis, an autoimmune POF mice model was established with zona pellucida glycoprotein 3 (ZP3) peptide. It is reported that the inositol‐requiring enzyme 1α (IRE1α) and its downstream molecules play a central role in the endoplasmic reticulum (ER) stress‐induced apoptosis pathway. So the aim of this study is to investigate whether hPMSCs transplantation attenuated GCs apoptosis via inhibiting ER stress IRE1α signaling pathway. The ovarian dysfunction, follicular dysplasia, and GCs apoptosis were observed in the POF mice. And the IRE1α pathway was activated in ovaries of POF mice, as demonstrated by, increased X‐box binding protein 1 (XBP1), up‐regulated 78 kDa glucose‐regulated protein (GRP78) and caspase‐12. Following transplantation of hPMSCs, the ovarian structure and function were significantly improved in POF mice. In addition, the GCs apoptosis was obviously attenuated and IRE1α pathway was significantly inhibited. Transplantation of hPMSCs suppressed GCs apoptosis‐induced by ER stress IRE1α signaling pathway in POF mice, which might contribute to the hPMSCs transplantation‐mediating ovarian function recovery.     

Analysis of complex autoantibody repertoires by surface-enhanced laser desorption/ionization-time of flight mass spectrometry

Normal sera contain a large number of naturally occurring autoantibodies which can mask important disease-associated ones. Western blotting has evolved as the most important tool to demonstrate autoantibodies in autoimmune diseases, because of its ability to simultaneous screening for a wide spectrum of different antigens. In previous studies we have shown the diagnostic potential of the analysis of autoantibodies in autoimmune diseases by means of multivariate statistics and artificial neural networks. However, the Western blotting procedure remains very time-consuming and is also limited in sensitivity. Therefore, we used an on-chip approach for the analysis of autoantibodies. This ProteinChip system uses ProteinChip arrays and SELDI-TOF MS (surface-enhanced laser desorption/ionization-time of flight mass spectrometry) technology for capturing, detection, and analysis of proteins without labelling or without the need of chemical modification. The microscale design of the arrays allows the analysis of very small quantities of proteins. In the present study, we used arrays with biologically activated surfaces that permit antibody capture studies. Protein-A-Chips were incubated with sera of patients (n = 12). After washing, the chips were incubated with a complex solution of autoantigens and subsequently washed again. If the Protein-A bound autoantibodies recognized their antigens, these proteins could be separated by their molecular masses and were to be detected by mass spectrometry. Previous studies using monoclonal antibodies have demonstrated that the detection limit is in the attomole level. Furthermore, all sera were analyzed by conventional Western blotting for direct comparison. In the present study, we have shown complex on-chip antibody-antigen reactions. At higher molecular weights (> 30 kDa) the detection sensitivity of this on-chip method was comparable to conventional Western blotting. At lower molecular mass, the Western blot technique is easily exceeded by the on-chip method. Considering that this on-chip procedure is quite easy to use, is much less time-consuming than Western blotting, and is much more sensitive at least in the low molecular weight range, the SELDI-TOF technology is a very promising approach for the screening of autoantibodies in autoimmune diseases. Due to its versatility, this on-chip technology could allow the large-scale screening for complex autoantibody distributions for diagnostic purposes and early detection of autoimmune diseases might be possible.     

Analysis of HIV-induced autoantibodies to cryptic epitopes on human CD4.

Antilymphocyte antibodies, including autoantibodies to CD4, have been reported in AIDS patients and are postulated to contribute to T cell depletion and immunologic dysfunction. In this paper, we characterize and localize binding sites of human anti-CD4 autoantibodies from a number of HIV+ patients. Epitope mapping by ELISA and Western blotting, together with cross-competition experiments, showed that common autoepitopes were localized to at least two topographically separate sites on the fourth domain of sCD4. These sites were partially dependent on the carboxyl terminus of the soluble molecule and were not exposed on full length membrane CD4, even under denaturing Western blotting conditions. Peptide screening identified peptides from the fourth and third domains that were recognized by several, but not all, anti-CD4 serum samples. Soluble CD4 affinity-purified antibodies were predominantly IgG1 and were not induced to bind mCD4 after gp120 binding to T cells. Analysis of HIV seroconversion panels showed that the appearance of anti-CD4 antibodies followed HIV seroconversion by 6 to 12 months and paralleled anti-gp120 reactivity. This suggested a correlation between immune reactivity to envelope and anti-CD4 antibody production. Together, the data indicate that human anti-CD4 antibodies recognize cryptic conformational and linear epitopes on a cleaved form of CD4. These findings suggest that HIV may induce abnormal cleavage of full length CD4, thereby exposing immunogenic self epitopes normally hidden from humoral and cellular immune interactions. This model of abnormal processing of self Ag has general implications for autoantigen exposure in other autoimmune disorders.     

Association of Maedi Visna virus with Brucella ovis infection in rams

Maedi Visna Virus (MVV) is the etiological agent of a systemic disease of sheep, which causes lesions in lungs, the central nervous system, joints, and mammary glands. It has been speculated that the association with Brucella ovis may lead to the venereal shedding of the virus. In this work, samples of epididymis from ten rams positive for MVV and infected experimentally with Brucella ovis, were subjected to liquid-phase PCR, immunohistochemistry (IHC) and in situ PCR tests, aimed at identifying the pathogens in a tissue context. IHC was carried out using a monoclonal antibody raised against p28 MVV protein and a polyclonal antibody to B. ovis. Liquid phase- and in situ PCR were designed to amplify a portion of MVV proviral DNA Pol sequence. In the animals showing B. ovis-related histopathological changes, IHC clearly demonstrated a positivity for B. ovis and MVV in interstitial and epithelial ductal cells. In situ PCR assessed the presence of MVV proviral DNA in macrophages and elements inside the epithelium. The unaffected and reagent control samples constantly gave negative results. Taken together, these data demonstrate that MVV may affect ovine epididymis, apparently taking advantage of the concurrent infection by B. ovis. The tropism of MVV for the epididymal epithelial cells, may be responsible for its excretion with the semen.     

Physical mapping of nine Xq translocation breakpoints and identification of XPNPEP2 as a premature ovarian failure candidate gene

Women with balanced translocations between the long arm of the X chromosome (Xq) and an autosome frequently suffer premature ovarian failure (POF). Two "critical regions" for POF which extend from Xq13-->q22 and from Xq22-->q26 have been identified using cytogenetics. To gain insight into the mechanism(s) responsible for ovarian failure in women with X;autosome translocations, we have molecularly characterized the translocation breakpoints of nine X chromosomes. We mapped the breakpoints using somatic cell hybrids retaining the derivative autosome and densely spaced markers from the X-chromosome physical map. One of the POF-associated breakpoints in a critical region (Xq25) mapped to a sequenced PAC clone. The translocation disrupts XPNPEP2, which encodes an Xaa-Pro aminopeptidase that hydrolyzes N-terminal Xaa-Pro bonds. XPNPEP2 mRNA was detected in fibroblasts that carry the translocation, suggesting that this gene at least partially escapes X inactivation. Although the physiologic substrates for the enzyme are not known, XPNPEP2 is a candidate gene for POF. Our breakpoint mapping data will help to identify additional candidate POF genes and to delineate the Xq POF critical region(s).     

IgG anti-DNA autoantibodies within an individual autoimmune mouse are the products of clonal selection

Although mice from almost all inbred strains produce IgM anti-DNA antibody in response to B cell mitogens, only (NZB x NZW)F1 mice and mice from other strains that are genetically predisposed to autoimmunity spontaneously produce anti-DNA antibody of the IgG isotype. Because (NZB x NZW)F1 mice display marked B cell hyperactivity, anti-DNA antibody production in these mice has been thought to result from spontaneous, polyclonal B cell activation. Although this may be true for IgM anti-DNA antibodies, our results demonstrate that IgG anti-DNA antibodies are not polyclonal. Rather, IgG anti-DNA autoantibodies within an individual autoimmune mouse are oligoclonal and somatically mutated. These results demonstrate that IgG anti-DNA autoantibodies are the products of clonally selective B cell stimulation and exhibit the same characteristics as secondary immune antibodies to conventional immunogens: they are IgG, they are clonally restricted, and they are somatically mutated.     

Autoantibodies against aggrecan in systemic rheumatic diseases

OBJECTIVE: This study was undertaken to investigate the presence of autoantibodies against the main cartilage proteoglycan, aggrecan, in systemic rheumatic disease sera, and to identify substructure(s) responsible for the autoimmune response. METHODS: Sera were obtained from 86 patients with various systemic rheumatic diseases, 14 with osteoarthritis (OA), 18 with cancer and 40 healthy individuals. The presence of autoantibodies against aggrecan was examined by a solid phase assay and by Western blotting, using proteoglycan aggregates treated with proteolytic enzymes. The positive bands were subjected to nanohigh performance liquid chromatography (nanoHPLC)-MS, in order to identify the aggrecan substructures involved in the autoimmune response. RESULTS: Autoantibodies against aggrecan were identified in all systemic rheumatic disease sera at a high titre, almost three times that observed in healthy controls. OA and cancer sera produced a reaction equal to that of the healthy. Western blotting analysis of aggrecan proteolytic fragments revealed the presence of a triple band, reacting with the patients' sera, of about 37 kDa, which also reacted with a polyclonal antibody against hyaluronan-binding region. NanoHPLC-MS analysis suggested that this band belonged to the G2 domain of aggrecan. CONCLUSION: At least a part of the autoimmune reaction to aggrecan, displayed by the systemic disease sera, involves the G2 domain. The significant difference observed between these sera and those from other diseases, especially cancer, may suggest a possible discriminatory role of anti-aggrecan antibodies. This may help in the differential diagnosis in complicated clinical cases. However, for this to be confirmed, studies in larger cohorts of patients should be performed.     

Optimization,validation, and identification of two reliable antibodies for immunodetection of WNT5A

WNT5A is a secreted, noncanonical WNT signaling protein that has been reported to promote progression of several types of cancer, including oral squamous cell carcinoma. Many WNT5A antibodies are available commercially for immunohistochemistry (IHC) and western blot analysis. Validation of the primary antibodies, however, is often neglected. We characterized antibodies for detecting WNT5A by IHC and western blot analysis. We evaluated one polyclonal and three monoclonal commercially available WNT5A antibodies. After optimization of the IHC assay, all four antibodies showed cytoplasmic WNT5A expression in tissue samples; in contrast, only one antibody detected WNT5A in western blots. A pre-absorption test with recombinant WNT5A showed that AF645 and 3A4 antibodies specifically detected WNT5A in different assays. We suggest that the monoclonal 3A4 antibody is the most appropriate for use with IHC, while the polyclonal AF645 antibody is the best for western blot analysis.     

Thyroid and other autoantibodies in British and Japanese women: an epidemiological study of breast cancer.

To define the role of asymptomatic autoimmune thyroiditis in the cause of breast cancer, the presence of circulating thyroid autoantibodies was studied in two populations, one with a high risk of breast cancer (British women) and one with a low risk (Japanese women). Ostensibly healthy women and patients with breast cancer from both countries were studied. There was no difference in the incidence of thyroid autoantibodies between women with breast cancer and healthy women in either race. The incidence of thyroid autoantibodies in healthy British women, however, was two to three times that in healthy Japanese women. The incidence of reticulin antibodies, was considerably higher in both groups of Japanese women. No remarkable differences in the incidence of antinuclear, smooth-muscle, antimitochondrial, gastric parietal cell, or liver-kidney microsomal antibodies were found between women with breast cancer and healthy women or between the two races. Only the incidence of antinuclear antibodies was marginally higher in Japanese patients with advanced cancer. These results indicate that asymptomatic autoimmune thyroid disease is more prevalent among British than among Japanese women, but they fail to provide direct evidence that autoimmune thyroid disease is associated with breast cancer. Prospective studies of women with autoimmune thyroiditis and studies of young women from low-risk and high-risk populations are needed.     

Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.     

Mining the human autoantibody repertoire: Isolation of potent IL17A-neutralizing monoclonal antibodies from a patient with thymoma

Anti-cytokine autoantibodies have been widely reported to be present in human plasma, both in healthy subjects and in patients with underlying autoimmune conditions, such as autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED) or thymic epithelial neoplasms. While often asymptomatic, they can cause or facilitate a wide range of diseases including opportunistic infections. The potential therapeutic value of specific neutralizing anti-cytokine autoantibodies has not been thoroughly investigated. Here we used mammalian cell display to isolate IL17A-specific antibodies from a thymoma patient with proven high-titer autoantibodies against the same. We identified 3 distinct clonotypes that efficiently neutralized IL17A in a cell-based in vitro assay. Their potencies were comparable to those of known neutralizing antibodies, including 2, AIN457 (secukinumab) and ixekizumab that are currently in clinical development for the treatment of various inflammatory disorders. These data clearly demonstrate that the human autoantibody repertoire can be mined for antibodies with high therapeutic potential for clinical development.     

Mouse PSP94 expression is prostate tissue-specific as demonstrated by a comparison of multiple antibodies against recombinant proteins

Prostate tissue-specific gene expression is crucial for driving potentially therapeutic genes to target specifically to the prostate. Prostate secretory protein of 94 amino acids (PSP94), also known as beta-MSP (microseminoprotein), is one of the three most abundant secretory proteins of the prostate gland, and is generally considered to be prostate tissue-specific. We have previously demonstrated that the expression of the rat PSP94 gene is strictly prostate tissue-specific by an antibody against a recombinant rat PSP94. In order to study prostate targeting utilizing the PSP94 gene in a mouse pre-clinical experimental model, we need to establish antibodies against mouse PSP94 to confirm if it is prostate tissue-specific as well. In this study, firstly we raised a polyclonal antibody against a recombinant glutathione-S-transferase- (GST-) mouse mature form of PSP94. However, it showed very poor immunoreactivity against prostate tissue PSP94 as tested in Western blotting experiments. Neither antibodies against rat PSP94 nor mouse PSP94 showed significant cross-reactivity. Thus a second antibody was established against a recombinant mouse mature PSP94 containing N-terminal polyhistidines, and stronger immunoreactivity against mouse prostate tissue PSP94 was identified in Western blotting experiments. Both of these antibodies showed immunohistochemical reactivity, while the latter showed stronger reactivity in IHC when tested with different fixatives. By studying tissue distribution, we demonstrated that, as with rat PSP94, mouse PSP94 is strictly prostate tissue-specific in experiments of both Western blotting and immunohistochemistry (IHC). This conclusion was also derived from a comparison among antibodies against human, rat, and mouse PSP94, showing very different immunoreactivities in Western blotting and IHC. Finally, a competitive assay between different species was performed. We demonstrated that antibodies against PSP94 from different species (human, primate, rodents) have poor cross-reactivities. These observations also indicate that the PSP94 gene is a rapidly evolving gene in all species. Results from this study have led to the possibility of utilizing PSP94 as a targeting agent specifically to the prostate in a mouse experimental model.     

Specificity of monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice

Antibodies reactive with left-handed Z-DNA arise spontaneously in the sera of patients with SLE and rheumatoid arthritis and in autoimmune MRL mice. However, the precise specificity of these autoantibodies has not been established. In this report, we have characterized four monoclonal anti-Z-DNA antibodies from unimmunized MRL/Mp-lpr/lpr mice that do not cross-react with B-DNA and can discriminate between different types of left-handed helices. Two of the monoclonal antibodies (Za and Zi) behaved similarly in that they bound to two forms of Z-DNA (Br-poly(dG-dC).poly(dG-dC) and AAF-poly(dG-dC).poly(dG-dC) but not to two other Z-form DNA (poly(dG-5BrdC).poly(dG-5BrdC) or poly(dG-5MedC).poly(dG-5MedC)). Neither antibody (Za or Zi) bound significantly to B-DNA or to denatured DNA. A third antibody (Ze) exhibited similar binding characteristics for the Z-DNA preparations, but also recognized denatured DNA. In contrast, a fourth antibody (3-7.3) bound preferentially to poly(dG-5BrC).poly(dG-5BrdC) in Z conformation. These results provide the first evidence for anti-Z-DNA autoantibodies in autoimmune mice that do not cross-react with native or denatured DNA and indicate that these antibodies exhibit considerable heterogeneity in their fine binding specificity.     

Postpartum thyroid dysfunction in Mid Glamorgan

A high prevalence of postpartum thyroid dysfunction has been reported in several countries, but there have been no systematic studies of its prevalence in Britain. Among a group of 901 consecutive, unselected pregnant women thyroid autoantibodies were detected in 117 (13%) at booking. The clinical course of postpartum thyroid dysfunction, factors associated with its development, and its likely prevalence were defined in 100 of these women with thyroid antibodies and 120 women with no such antibodies who were matched for age. None of the women had a history of autoimmune thyroid disease. Normal reference ranges for thyroid function during pregnancy and post partum were established in the 120 women negative for thyroid antibodies. On the basis of these observations postpartum thyroid dysfunction was observed in 49 (22%) of the 220 women studied, and the prevalence in the total group of 901 women was estimated to be 16·7%. Thyroid dysfunction, mainly occurring in the first six months post partum, was usually transient and included both destruction induced hyperthyroidism and hypothyroidism. The development of the syndrome was significantly related to smoking more than 20 cigarettes a day and the presence of thyroid microsomal autoantibodies at booking. Of the 16 women with a family history of thyroid disease in whom thyroid microsomal autoantibody activity was detectable at booking, 11 developed thyroid dysfunction. Age, parity, presence of goitre at presentation, duration of breast feeding, and the sex and birth weight of the infant were not associated with the development of postpartum thyroid dysfunction.The mood changes experienced by women post partum may in part be associated with altered thyroid function during this time.     

Screening for associated autoimmunity in type 1 diabetes mellitus with respect to diabetes control

As an autoimmune disease, type 1 diabetes mellitus (DM) can be associated with other autoimmune disorders. The aim of this study was to detect subclinically associated autoimmune thyroid disease, coeliac disease, and Addison's disease. The presence of autoantibodies was evaluated with special regard to the control of diabetes and to the clinical status of the patient. Fifty-one type 1 diabetic patients (22 men, 29 women, mean age 37+/-11 years, mean duration of diabetes 16+/-13 years) were included into this study. Specific antibodies to islet antigens--glutamic acid decarboxylase (GAD65), protein thyrosine phosphatase IA-2alpha, and to thyroid autoantigens--thyroid microsomal peroxidase (TPO) and thyroglobulin (TG) and also thyroid stimulating hormone (TSH) were measured by RIA. Autoantigens of the small intestine--tissue transglutaminase autoantibodies (ATTG), IgA and IgG antibodies to gliadin (AGA-IgA, AGA-IgG) were evaluated by ELISA. Endomysial autoantibodies (EMA) and adrenal cortex antibodies (ACA) were detected by indirect immunofluorescence microscopy. Eleven new cases of thyreopathy (22 % of patients) were detected by the assessment of thyroid autoantibodies and TSH. Two new cases of thyreotoxicosis were diagnosed during the study. Coeliac disease was diagnosed in at least two cases. Addison's disease was not diagnosed, although the ACA were positive in two patients. No influence of single or combined autoantibody positivity on the control of diabetes was found if normal organ function was preserved. In both patients with thyreotoxicosis the control of diabetes was worsened and improved after treatment. The screening of autoantibodies in type 1 diabetic patients could reveal subclinical cases of AITD or coeliac disease. Subclinical forms of these disorders have no influence on diabetes control. However, impaired organ function may be associated with the worsened control of diabetes as we demonstrated on two newly diagnosed cases of thyreotoxicosis. We suggest the need for the follow-up of patients with positive autoantibodies because further deterioration of the respective organs can be expected.     

Pediatric autoimmune liver diseases: the molecular basis of humoral and cellular immunity

Pediatric autoimmune liver disease is mainly represented by two similar liver disorders: autoimmune hepatitis (AIH) and autoimmune sclerosing cholangitis (ASC), both characterized by hypergammalobulinemia, interface hepatitis and the presence of a wide range of circulating autoantibodies. Although similar features are seen in AIH and inflammatory bowel disease, histological biliary changes are more common in ASC. In addition to their role as diagnostic markers, autoantibodies, such as anti-extractable nuclear antigen (ENA) antibodies and liver kidney microsomal antibody type 1 (LKM1) may be involved directly in inducing aggressive liver diseases. Although the cellular immune response in pediatric autoimmune liver disease has been less intensively investigated than humoral immunity, the importance of antigen specific T cells has been explored. Both alphabeta and gammadelta T cells derived from either peripheral blood and liver biopsies have highly heterogeneous TCR gene usage and cytolytic activity has been demonstrated. There have been attempts to seek triggers of liver autoimmunity and several sequences shared in common between autoantigens and hepatotropic viruses, namely hepatitis B, C and cytomegalovirus have been identified. The presence of cross-reactivity between homologous sequences, especially between HCV and cytochromes, supports the possibility that molecular mimicry plays a role in the induction of autoantibodies and autoreactive cytotoxic T cells.