Microsomal Antigen Research Articles

Polyendocrine deficiency as a diagnostic challenge - A retrospective clinical study

Polyglandular polyendocrine syndromes (PSs) are an assorted collection of infrequent ailments considered by autoimmune motion contrary to more than one endocrine organ, though non-endocrine organs can be affected. The deficiency of parathyroid is quite rare without surgery. This article summarises clinical, epidemiological, and serological, data of patients with PSs. Multifaceted diagnostic difficulties and treatment choices required that the patient be admitted to the hospital for a rather long period, namely for six weeks. Three diabetic patients with various thyroid disorders such as lymphocytic thyroiditis involving parathyroid glands manifesting with severe hypothyroid and hypo-parathyroid status were studied with an occurrence of dengue viral aetiology as a precipitating factor. Further, the patients were investigated for the determination of antibodies against thyroglobulin and microsomal antigens by chemiluminescent microparticle immunoassay. They were also tested for Parathyroid Hormone and 25-hydroxy vitamin D. After three months, all the three hormonal status were brought to normal with substitution and maintenance therapy, and patients were reviewed after six months were found to maintain with the dose. To conclude, thyroiditis may progress during the progression of dengue fever and should be counted as a manifestation of expanded dengue syndrome.

Use of Specific Inhibitory Antibodies for P450 Reaction Phenotyping of Investigational Drugs

Identifying the CYP450 enzymes underlying metabolism of investigational drugs (i.e., P450 reaction phenotyping; PRP) is critical to the development of effective therapeutics. Information gained from such studies can: a) provide insight into structural modifications of test drugs in order to improve metabolic stability and; b) specify an estimate of drug‐drug interaction potential as well as that of interindividual and species differences in oxidative metabolism. CYP450 chemical inhibitors (e.g., a‐naphthoflavone and sulfaphenazole) are often used in reaction phenotyping studies but the outcome is sometimes equivocal due to the non‐specific effects of these chemical agents plus their capacity to undergo P450‐dependent catabolism. In contrast, inhibitory P450 antibodies offer high specificity and irreversibility, and may thus represent a superior approach for characterizing individual P450 enzyme involvement in drug metabolism. We recently developed a compilation of highly‐specific polyclonal and monoclonal antibodies to the CYP450s comprising the major oxidative drug‐metabolizing enzymes in human liver, including CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (aka CYP ImmunoInhibit antibodies). As shown here, each of these antibodies proved capable of eliciting > 80% inhibition of probe drug metabolism by the cognate P450 antigens in human liver microsomes (HLMx), while giving insignificant effects on different CYP450s (Figure 1). The inhibition observed with each of the six CYP450 antibodies was not only highly‐specific but was also dose‐dependent, with maximal inhibition (80–90%) achieved at IgG:microsomal protein ratios ranging from 0.1 mg/mg to 3 mg/mg (Figure 2). Control (pre‐immune) antibodies had no effect on substrate metabolism. Such results show that inhibitory antibodies coupled with HLMx represent an alternative and powerful approach to chemical inhibitors for evaluating involvement of specific P450 enzymes in the hepatic metabolism of investigational drugs. Interestingly, studies ongoing with CYP ImmunoInhibit antibodies and MetMax™ cryopreserved human hepatocytes, which are liver cells that have been permeabilized and cryopreserved, have given analogous results to those achieved with HLMx. The utilization of inhibitory antibodies with HLMx represents an accurate model for PRP of new therapeutics due to the specificity of these immunoreagents, and the irreversible nature of the inhibition achieved, an important benefit when working with metabolically‐stable test agents.Specific Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 1Dose‐Response Inhibition of Microsomal Drug Metabolism by CYP ImmunoInhibit AntibodiesFigure 2

Novel radioassay for anti-thyroperoxidase autoantibodies using protein A coupled magnetizable cellulose particles as an immunoadsorbent

Autoantibodies against thyroid peroxidase (TPO); described initially as thyroid microsomal antigen, are found in most of the patients with autoimmune thyroid disorders. The present study comprised of development, optimization and validation of ‘sandwich’ radioassay for the measurement of TPOAb in human serum. The standardized TPOAb radioassay has maximum binding of 25–30%, non-specific binding of < 0.4%, analytical sensitivity of approximately around 1 IU/ml, acceptable intra- and inter-assay %CV, with a standard working range up to 1400 IU/ml. The in-house developed assay is simple, robust and cost-effective, and therefore can be used for routine measurement of TPOAb in clinical samples.

Schistosomiasis: an epidemiological update on Brazil's southernmost low endemic area in Esteio.

INTRODUCTION: Brazil’s southernmost state, Rio Grande do Sul (RGS), was considered schistosomiasis-free until 1998 when a low endemic focus was identified in Esteio, a city located next to the capital of RGS. In the last two decades, the control interventions applied in the region have been apparently successful, and the absence of new cases indicated the possibility of interrupted schistosomiasis transmission. The objective of this study was to update the clinical and epidemiological data of schistosomiasis in Esteio. METHODS: We reviewed all 28 individuals diagnosed with the infection since 1997 and a survey was applied to a group of 29 school-aged children residing in Vila Pedreira, one of the most affected neighborhoods. RESULTS No eggs were detected in fecal samples using the Helmintex method, and all samples were negative for serum antibodies on examination by the western blot technique using the Schistosoma mansoni microsomal antigen (MAMA- WB). In contrast, 23 individuals (79%) tested positive for the cathodic circulating antigen with the point-of-care immunochromatographic test (POC-CCA) on urine samples. Of the 28 formerly infected individuals, only eight were located, of which four tested positive, and four tested negative for serum antibodies using the MAMA-WB technique. CONCLUSIONS: Current adverse conditions for S. mansoni transmission in Esteio and the absence of a confirmed diagnosis suggests that there is (i) a lack of specificity of the POC-CCA test in low endemic settings, and (ii) a high probability that interruption of schistosomiasis has been achieved in Esteio.

Investigation of antinuclear antibodies in chronic hepatitis B patients

Antinuclear antibodies (ANA) facilitate the diagnosis and evaluation of patients in many systemic autoimmune conditions. Besides, ANA may also be detected in chronic infectious diseases. Although a number of investigations associated with autoantibody positivity in patients with chronic hepatitis C were reported, autoantibody positivity in patients with chronic hepatitis B remain rarely addressed in the literature. The aim of this study was to evaluate the antinuclear antibody (ANA), antimitocondrial antibody (AMA), anti-smooth muscle antibodies (ASMA) and anti-liver-kidney microsomal antigen (LKM) antibodies in chronic hepatitis B patients. Serum samples were obtained from adult patients with chronic hepatitis B diagnosis according to "European Association for the Study of the Liver (EASL)" criteria. Samples were taken from 47 patients (22 female, 25 male) with treatment-naive, histologically-proven chronic hepatitis B. Cases co-infected with HCV and/or HIV or that also had systemic autoimmune diseases were excluded. As a control group, 30 healthy blood donors were included in the study. Autoantibodies, including ANA, AMA, ASMA and LKM were detected with indirect immunofluorescence (IIF) method (Euroimmune, Lubeck, Germany) and evaluated by fluorescence microscope (Eurostar III plus, Germany). Positive results were graded into 4 levels ( "+", "++","+++" and "++++") from weak to strong Positive samples were studied with a immunoblotting method (ANA Profile 3, Euroimmun, AG) for the detection of extractable nuclear antigen (ENA). The positive results were detected in 8 (17%) of the HBV patients while all the samples were negative in the control group. The difference between the groups was significant (p< 0.05). Among the 47 serum samples tested, none of the patients were positive for AMA, ASMA, LKM. ANA was present in eight of the serum samples in which six were female and two were male patients. Among the IIF patterns of ANA positivity, one mixed pattern (homogeneous and nucleolar) and one cytoplasmic anti-golgi antibody pattern were detected. Positivity grade was ''++''. Other positive patterns were nucleolar (two patients), granular (two patients), ribozomal (one patient) and homogeneous (one patient) and positivity grade was ''+''. ENA was detected in three samples. Two of them was granular pattern positive samples. SS-A was borderline (±) in one and SS-B was borderline (±) in one of the samples. In the mixed pattern positive sample, histon was detected as ''+''. Autoantibody positivity between the patient and control groups were statistically significant (p< 0.05). The difference between autoantibody positivity and gender/age was not statistically significant. In conclusion, autoimmune manifestations may be detected in patients with chronic hepatitis B. Low level titer of antibodies such as ANA, AMA, ASMA or LKM may be present in such patients. An increased frequency of these autoantibodies may be associated with non-autoimmune conditions such as chronic viral infection even in treatment-naive patients.

Investigation of liver autoantibodies in anticentromere antibody positive patients: A preliminary research

Background: Anticentromere antibody (ACA) is regarded to be a serological marker specific to CREST (calcinosis, Raynaud’s phenomenon, esophageal dysmotility, sclerodactylia, and telangiectasia) syndrome. ACA is also found in the sera of patients with autoimmune liver disease. In the present work, anti-soluble liver antigenliver pancreas antigen (anti-SLALP), anti-liver cytosolic antigen 1 (anti-LC1), anti-liver kidney microsomal antigen 1 (anti-LKM1), and anti-mitochondrial antibody M2 (AMA-M2) were evaluated in the patients who had positive anticentromere antibody. Material and Methods: A total of 39 patients who were positive anticentromere antibody were enrolled in this study undertaken in the Izmir Katip Celebi University, Ataturk Training and Research Hospital, Microbiology laboratory between January and September 2015. Positive anticentromere antibody and liver autoantibodies were analyzed. Anticentromere antibody and liver autoantibodies were studied by indirect immunofluorescence method (IIF) and immunoblotting method (IB), respectively. The patients who had negative anticentromere antibody were used as a control group. Results: According to the study’s results, positivity was detected in 3 of 39 patients (%7.6) in terms of liver autoantibodies, all of which were AMA-M2. There was no statistically significant difference between ACA and autoimmune liver autoantibodies. Conclusion: In this study, we reported our preliminary experience to provide evidence for the detection of various autoantibodies as potential diagnostic or prognostic tests. Further studies that contain a broad range of patients may contribute to the field.

Hépatite auto-immune associée à une granulomatose septique chronique chez une fille de 2 ans

Chronic granulomatous disease is a rare inherited primary immune deficiency disease characterized by recurrent infection and an increased susceptibility to autoimmunity disorders. We report on the case of a girl with autoimmune hepatitis in chronic granulomatous disease to describe the clinical and biological features and treatment implications for patients with chronic granulomatous disease associated with autoimmune disorders. An 18-month-old girl was referred to our department for investigation of hepatomegaly. She was the third child of non-consanguineous parents. Her two elder sisters had died from infectious diseases at an early age. She had elevated liver transaminase levels with a normal gamma globulin concentration. Negative results were found for all autoimmune markers (antinuclear antibody, anti-smooth muscle, anti-liver-kidney microsomal, anti-liver cytosol and anti-soluble liver antigen). Her liver biopsy showed features of interface hepatitis with portal fibrosis. The diagnosis of seronegative autoimmune hepatitis was established. Treatment with corticosteroids and azathioprine led to clinical improvement with normalization of transaminases. Six months after initial presentation, at the age of 2 years, she was readmitted for fever. Staphylococcus aureus bacteremia was identified with multiple foci of infection (skin infection, arthritis of the right elbow, pneumonia, buttock abscess). The immunological workup revealed chronic granulomatous disease. The course was marked by a fatal outcome despite appropriate antibiotics and intensive care. Early diagnosis of the association between chronic granulomatous disease and autoimmune disorders allows for appropriate treatments, improves the quality of life for affected patients, and reduces the risk of mortality.

PO-1016 Phenotype Variety In Patients With Selective Iga Deficiency

<h3>Background</h3> Selective IgA deficiency (SIgAD) is the most frequent primary antibody deficiency. <h3>Aims</h3> Authors emphasise the phenotype diversity among SIgAD patients included in our clinic casuistry. <h3>Methods</h3> Authors analysed clinical and biochemical features of patients that fulfilled SIgAD diagnosis criteria. They’ve been evaluated concerning previous respiratory and gastro-intestinal diseases severity (necessity for intravenous antibiotics and/or intensive care measures). Serology investigations performed: complete blood count and evaluations for immunoglobulin isotype, IgG subclass, total IgE, specific IgE for cow’s milk precipitins. Autoimmune status estimation included antibodies against: red blood cells, basement membrane, smooth muscle, thyroglobulin, thyroid microsomal antigens, transglutaminase IgG/endomysium and nuclear proteins. Authors accomplished microbiological tests for patients with sinopulmonary infections and analysed stools in order to exclude Giardia/ Cryptococcus infections. Susceptibility for brochiectasis justified chest X-ray. <h3>Results</h3> Among 8 SIgAD patients followed in our clinic, 3 patients were considered as asymptomatic. The others were diagnosed with: recurrent knee arthritis in context of Epstein-Barr virus reactivation confirmed by quantitative PCR for DNA-EBV in serum (1 patient), celiac disease (2 patients), giardiasis (2 patients), asthma and allergic rhinitis (1 patient), cow milk allergy (1 patient). Sinopulmonary infections/ purulent otitis were identified for all symptomatic cases. One patient was diagnosed with tonsil phlegmon. No immunological thrombocytopenic purpura or autoimmune hemolytic anaemia were confirmed. <h3>Conclusions</h3> 1. In spite of small number of SIgAD patients, authors revealed a quite large range of clinical manifestations: from asymptomatic status to recurrent arthritis due to EBV reactivations; 2. Three patients presented more than 1 clinical feature.

Autoimmunity due to RAG deficiency and estimated disease incidence in RAG1/2 mutations

Autoimmunity due to RAG deficiency and estimated disease incidence in RAG1/2 mutations

Does OM-85 BV prophylaxis trigger autoimmunity in IgA deficient children?

BackgroundIgA deficiency (IgAD) is the most common primary antibody deficiency. Although two-third of the cases are reported to be asymptomatic, some IgAD children may have frequent infections that urge the clinicians to search for prophylactic measures. OM-85 BV is one of these agents that is known to stimulate mucosa associated lymphoid tissue, and upregulate Th-1 response. This study was performed to determine a possible role of OM-85 BV in triggering autoimmunity in IgAD children within a four-year-follow up period. MethodsSixty-three children (34 males (54%), 29 females (46%)) with sporadic IgAD and recurrent febrile infections were included. Patients were carefully screened for autoimmunity both on admission and in follow-up: Those with autoimmune features or under immunosuppressant treatment were excluded. Patients were randomly divided into two groups: Group I received bacterial lysate propylaxis (OM-85 BV) (n:37), and Group 2 received no prophylactic regimen (n:26). Development of clinical autoimmune findings or autoantibodies (anti-nuclear antibody (ANA), ANA profile (14 antigens), anti-cytoplasmic antibodies (ANCA), anti-cardiolipin antibodies IgG and IgM (aCL), rheumatoid factor (RF), direct Coombs test, anti-thyroglobulin (anti-T) and anti-thyroid microsomal antigen (anti-M)) were evaluated. ResultsMean age of the study group, age at the onset of infectious symptoms and at admission were 102.9±42.2, 27.1±24.9, and 55.2±25.1months, respectively. Follow-up duration of the whole study group was 48.3±23.1months. Number of infections was 6.2±2.7 per year in the whole study group. Sixteen patients (25.4%) of the whole study group showed ANA positivity in different patterns and titers. Frequency of ANA, ANCA and RF positivity was 24.3%, 5.4%, 2.7% in Group 1, and 26.9%, 11.5%, 3.8% in Group 2, respectively. Statistical comparisons revealed no significant difference between the two groups. ConclusionSignificant clinical or laboratory markers for autoimmunity in follow-up were not observed between receivers or non-receivers of OM-85 BV. Frequency of ANA positivity was comparable to the previously reported values in IgAD children which was not affected by OM-85 BV usage. Possible effect of triggering autoimmunity with repeated cures of bacterial lysates needs to be further clarified. Side effects requiring the cessation of treatment were not recorded.

National Serologic Survey of Haematobium Schistosomiasis in Morocco: Evidence for Elimination

The Moroccan Health Ministry launched a Process of Eliminating Schistosomiasis in 1994. During 2005-2009, the epidemiologic status showed a clear interruption of disease transmission at the national level; only a few residual cases were recorded. Our present study is the first systematic serologic survey to evaluate the transmission status in remaining disease-endemic foci. A study population of 2,382 children born after the date of the last autochthonous cases were selected from provinces with histories of high schistosomiasis transmission (Tata, Chtouka Ait Baha, Errachidia, El Kelaa Des Sraghna, and Beni Mellal). To identify the presence of disease, specific antibodies directed against Schistosoma haematobium adult worm microsomal antigens were detected by using an enzyme-linked immunoelectrotransfer blot assay. The results showed an absence of antibodies in all serum samples. Consequently, our findings confirm either a low transmission status or an interruption of schistosomiasis transmission within the last disease endemic foci.

Improving the detection limit of quantitative diagnosis of anti-S. haematobium antibodies using Falcon Assay Screening Test (FAST) ELISA by developing a new standard curve

Immunodiagnosis of schistosomiasis are currently based on parasitological examinations of stool and urine for egg detection, which is laborious and lacks sensitivity. There are many assays that detect the anti-schistosomal antibodies in patient sera. One of these assays is the Falcon assay screening test (FAST) ELISA that uses adult worm microsomal antigen for Schistosoma haematobium and Schistosoma mansoni, HAMA, MAMA antigen, respectively. This assay depends on quantitative detection of anti-schistosome antibodies, using a standard curve, but the detection limit of FAST-HAMA assay is low due to the small range of the standard curve. In our study, a new wide range (0-80 μl/μl) of standard curve for FAST-HAMA assay was constructed, and the cut-off value of the assay, using the new curve, was determined to be 1.2 units/μl. Screening of 41 S. haematobium-infected sera with FAST-HAMA, using the new constructed curve, showed a sensitivity of 95%. The purity of HAMA antigen and highly specific Abs for S. haematobium lends the FAST-HAMA with the new constructed wide range standard curve as a diagnostic assay with high detection limit for S. haematobium infection.

Genistein Aglycone Does Not Affect Thyroid Function: Results from a Three-Year, Randomized, Double-Blind, Placebo-Controlled Trial

Genistein aglycone positively affects postmenopausal symptoms. However, questions about its long-term safety on the thyroid gland still remain. The parent study was a randomized, double-blind, placebo-controlled trial involving 389 osteopenic, postmenopausal women for 24 months. A subcohort (138 patients) continued therapy for an additional year. Patients received ambulatory care. Participants received 54 mg of genistein aglycone daily (n = 71) or placebo (n = 67), plus calcium and vitamin D(3) at therapeutic doses. Circulating thyroid hormones (TSH, free T(3), free T(4)) and autoantibodies (thyroid peroxidase, thyroglobulin, and thyroid microsomal antigen) were assessed in 40 genistein and 37 placebo subjects who completed 3 yr. Thyroid hormone receptor (THRalpha and THRbeta) and retinoid receptor (RARalpha, RARgamma, and RXRalpha) expression from peripheral blood monocytes was also evaluated at baseline, 12, 24, and 36 months in all 3-yr completers. Genistein administration over 3 yr did not affect serum thyroid hormones or autoantibodies. In addition, there were no differences in THRalpha, THRbeta, RARalpha, RARgamma, or RXRalpha mRNA expression between groups. These data suggest that genistein aglycone intake does not significantly increase the risk of clinical or subclinical hypothyroidism at the dose of 54 mg/d.

Clinical implication of thyroid peroxidase antibody detection

Thyroid peroxidase (TPO) , originally described as thyroid microsomal antigen, is present on the apical surface of thyroid follicular cells and is an antigen involved in cell mediated cytotoxicity. TPO evokes high-affinity, IgG-class autoantibodies (TPOAbs) and TPO-specific T cells that are markers of thyroid infiltration or implicated in thyroid destruction, respectively. Enzyme-linked immunosorbent assay is used most frequently for TPOAb detection and quantification. The other conditions associated with TPOAbs include pernicious anemia, connective tissue disorders, diabetes, inflammatory bowel disease, mood disorders, and fertility-related problems such as miscarriage, infertility, in vitro fertilization failure, pre-term delivery, and postpartum thyroiditis. The detection of TPOAhs is recommended in the investigation of goitre, diagnosis of Graves' disease and Hashimoto's thyroiditis, and the prediction of risk of developing hypothyroidism during subclinical thyroid disease. Key words: Thyroid peroxidase antibody; Autoimmune thyroid disease; Hashimoto disease; Detection(

Increased percentages of autoantibodies in immunoglobulin A-deficient children do not correlate with clinical manifestations

IgA deficiency (IgAD) is frequently associated with autoimmune phenomena. The aim of this study is to evaluate the frequency of 22 different autoantibodies in 60 patients with IgAD and to examine the physical and other laboratory findings of the suspected cases for autoimmune diseases. The evaluated autoantibodies were Anti-nuclear antibody (ANA) profile (autoantibodies against RNP/Sm, SS-A, Ro-52, SS-B, Scl-70, Pm-Scl, Jo-1, centromere B, PCNA, dsDNA, nucleosomes, histones, ribozomal P-protein, AMA-M2), anti-cardiolipin IgG and IgM, anti-neutrophilic cytoplasmic antibodies (ANCA), rheumatoid factor (RF), anti-thyroglobulin (anti-T) and anti-thyroid microsomal antigen (anti-M) and direct cooms test. Forty-one healthy children were included as a control group. ANA titers ≤ 1:80 were accepted as normal and titers ≥ 1:80 are accepted as positive. In ANA screening, 14 patients showed positivity in different titres. Seven of them were equal to or below 1:80. The other seven patients (11.6%) had positive ANA titers (>1:160) whereas three of them had anti-dsDNA, anti-histon and anti-centromer antibodies. These patients did not have any clinical and laboratory signs of autoimmune diseases. ANA positivity was found higher in IgA deficient children (p < 0.05) compared to controls. RF and pANCA were found positive during follow-up of two different selective IgAD patients. IgG and IgM antibodies against cardiolipin, direct coombs, anti-T and anti-M tests were not found positive in any subjects. In conclusion, increased frequency of autoantibodies in IgAD patients may often be observed. However, the detection of autoantibodies do not show or predict whether this patient will develop an autoimmune disease.

THYROID STATUS PARAMETERS AT CHILDREN’S EXPOSED TO IODINE RADIONUCLIDE’S DURING IN UTERO DEVELOPMENT

There was analyzed the status of thyroid system parameters and frequency of individual values prevalence of antibody - carriage at children’s born in May 1986 - February 1987 exposed to iodine radio nuclides in intra-uterine period of development from 4 regions of Gomel Oblast (Gomel, Loev, Rechitsa, Khoiniki). At comparison of prevalence frequency of antibody - carriage to microsomal antigen among young men and girls it is shown that the given parameter statistically significantly is defined more often in the basic group at comparison with the conditional control; also there was marked statistically significant difference in Gomel region among young men and girls. The parameters of thyroid system status (thyrotropic hormone, free thyroxin) at children’s exposed to irradiation during intra-uterine development are within physiological values.

Clonal analysis of liver-infiltrating T cells in patients with LKM-1 antibody-positive autoimmune chronic active hepatitis

Autoantibodies against microsomal antigen of liver and kidney (LKM-1) are diagnostic markers for a subgroup of autoimmune chronic active hepatitis (AI-CAH). Cytochrome P450dbl, now classified as cytochrome P450 IID6, is the major antigen of LKM-1 antibodies. Immunohistological studies suggest that hepatic injury in AI-CAH is mediated by liver-infiltrating T cells. In the present study the specificity and function of liver-infiltrating T cells was analysed at the clonal level. Phenotypical characterization of 189 T cell clones isolated from four liver biopsies of LKM-1 antibody-positive patients showed an enrichment of CD4+ CD8- T cell clones proliferated specifically in the presence of recombinant human LKM-1 antigen (rLKM). This reaction was restricted to autologous antigen-presenting cells and to HLA class II molecules. In order to see whether rLKM was also recognized by peripheral blood T lymphocytes (PBL) we tested the proliferative response of PBL from several individuals. PBL from three of the four patients with LKM-1 antibody-positive AI-CAH proliferated to rLKM, whereas no response was seen with PBL from patients with LKM-1 antibody-negative chronic liver diseases and from healthy blood donors. These data demonstrate that the LKM-1 antigen is recognized by liver-infiltrating T cells in LKM-1 antibody-positive AI-CAH. For further functional characterization, liver-derived T cell clones were tested for their cytotoxic activity. In the presence of phytohaemagglutinin 24 out of 26 CD4- CD8+ but also 20 out of 63 CD4+ CD8- T cell clones lysed autologous as well as allogenic EBV-transformed B cell lines or K562 cells. Five CD4- CD8+ T cell clones lysed autologous but not allogenic B cell lines spontaneously in a HLA class I-restricted manner. Although the antigen specificity of these clones is still unknown the data show the presence of autoreactive T cells at the site of inflammation that could contribute in the pathogenesis of AI-CAH.

Analytical aspects of thyroid antibodies estimation

The search for antibodies that will reliably diagnose, predict and monitor the autoimmune thyroid diseases is a quest that is beset with difficulties. This review will describe the various antigens involved in autoimmune thyroid disease, the development of a humoral response to these antigens and some of the technical difficulties involved in trying to detect and then measure their concentrations.Multiple antigen configurations of thyroglobulin (TG) are produced when it is iodinated resulting in functionally active but immunologically distinct molecules. Thyroid peroxidase (TPO), originally described as thyroid microsomal antigen, is present on the apical surface of thyroid follicular cells and is the antigen most closely involved in cell-mediated cytotoxicity. Multiple B cell reactive epitopes exist each giving rise to different antibodies. The third antigen is the TSH receptor (TSHR) which is a two subunit glycoprotein. The extracellular A subunit is recognised by thyroid stimulating antibodies whilst those antibodies recognising the B subunit, located much nearer the cell surface, appear to function as blocking antibodies.The aetiology and mechanics of the autoimmune cellular and antibody responses involves a combination of HLA linkage, genetics and environmental factors to determine the initial and subsequent stages of the development of autoimmune thyroid disease.Starting off with immunofluorescence or passive tanned red cell agglutination assays, we now favour more sensitive TPO antibody quantitative immunoassays which are standardised using MRC 66/387. The apparent incidence of these antibodies is influenced by the techniques used to detect them and the significance of those positive antibody concentrations detected using assays that report very high incidence rates in the “normal” population remains to be proven using longitudinal studies.Older immunofluorescence and passive tanned red cell agglutination methods have been improved upon to give the current, competitive and non-competitive immunoassays that are much more sensitive and specific for anti-TG antibodies. However, because a wide-range of methods are still being used in clinical laboratories, the sensitivity and specificity of available methods will vary depending on the method used. There are still assays that are calibrated with purified or crude preparations of TG antibody by pooling patient sera or blood donor material. These various secondary standards are often, but not always, calibrated against the primary standard (MRC 65/93). All requests for TG estimation in thyroid carcinoma patients should have TG antibody estimated at the same time because of the possibility of interference in the tumour marker assay by the antibody.Two methods are widely used for the estimation of TSHR antibodies. The first involves bioassays based on cultured cells to measure the stimulating class of antibodies and the second involves receptor assays based on 125I-labeled TSH. The most widely used assay uses detergent-solubilized porcine TSHR with TSHR antibodies to inhibit the TSHR-125I-TSH interaction, and polyethylene glycol to separate receptor-bound and free labelled-TSH by precipitation. TSHR antibody heterogeneity can coexist within an individual patient and change over time is one reason why it has been difficult to develop diagnostically accurate TSHR antibody tests.

Autoimmune liver serology: Current diagnostic and clinical challenges

Liver-related autoantibodies are crucial for the correct diagnosis and classification of autoimmune liver diseases (AiLD), namely autoimmune hepatitis types 1 and 2 (AIH-1 and 2), primary biliary cirrhosis (PBC), and the sclerosing cholangitis variants in adults and children. AIH-1 is specified by anti-nuclear antibody (ANA) and smooth muscle antibody (SMA). AIH-2 is specified by antibody to liver kidney microsomal antigen type-1 (anti-LKM1) and anti-liver cytosol type 1 (anti-LC1). SMA, ANA and anti-LKM antibodies can be present in de-novo AIH following liver transplantation. PBC is specified by antimitochondrial antibodies (AMA) reacting with enzymes of the 2-oxo-acid dehydrogenase complexes (chiefly pyruvate dehydrogenase complex E2 subunit) and disease-specific ANA mainly reacting with nuclear pore gp210 and nuclear body sp100. Sclerosing cholangitis presents as at least two variants, first the classical primary sclerosing cholangitis (PSC) mostly affecting adult men wherein the only (and non-specific) reactivity is an atypical perinuclear antineutrophil cytoplasmic antibody (p-ANCA), also termed perinuclear anti-neutrophil nuclear antibodies (p-ANNA) and second the childhood disease called autoimmune sclerosing cholangitis (ASC) with serological features resembling those of type 1 AIH. Liver diagnostic serology is a fast-expanding area of investigation as new purified and recombinant autoantigens, and automated technologies such as ELISAs and bead assays, become available to complement (or even compete with) traditional immunofluorescence procedures. We survey for the first time global trends in quality assurance impacting as it does on (1) manufacturers/purveyors of kits and reagents, (2) diagnostic service laboratories that fulfill clinicians' requirements, and (3) the end-user, the physician providing patient care, who must properly interpret test results in the overall clinical context.

Heritability of levels of autoantibodies to thyroid antigens using the method of plotting regression of offspring on midparent (ROMP)

Only a few methods can be applied in a simple manner to estimate the genetic control of autoimmunity in humans. Here we examined the heritability of autoantibodies to two thyroid antigens; thyroglobulin (Tg) and thyroperoxidase (TPO, formerly known as thyroid microsomal antigen), using methods of regression of offspring on mid-parental values (ROMP). With the data sets available, affected and unaffected siblings were compared by this rapid screening method using results determined by hemagglutination (HA). The presence of both types of autoantibodies showed positive heritability in patients with Graves' thyrotoxicosis (TT), but it was not observed in chronic lymphocytic or Hashimoto's thyroiditis (CLT) patients. Since these assays have been extensively used over the years by most diagnostic and research laboratories, they should provide some insight as to which quantifiable parameters may be usefully accumulated to help select groups of patients and their families for further genetic study. ROMP may also be useful to determine the sequential appearance of different types of antibody in predicting disease onset in other family members, and in distinguishing maternal and paternal effects on imprinting. The method may be extended to study epitope spreading and other measures of disease progression.