Collagen XVII/BP180 protein expression in squamous cell carcinoma of the skin detected with novel monoclonal antibodies in archived tissues using tissue microarrays and digital microscopy.

Collagen XVII/BP180, a hemidesmosomal adhesion protein, is lost during normal keratinocyte maturation; however, it may be reexpressed upon malignant transformation. In this work, highly sensitive monoclonal antibodies 6D1 and 9G2 were produced, characterized, and used for the detection of collagen XVII in a tissue microarray series of archived samples of nonmelanocytic epithelial neoplasias, including 5 verruca vulgaris, 14 seborrheic keratosis, 38 actinic keratosis, 38 basal cell carcinoma (BCC), 15 basosquamous carcinoma, 58 squamous cell carcinoma (SCC), and 9 normal skin. Digital microscopy and a new tissue microarray software linking image and patient data allowed easy and validated evaluation and quality archiving of stained samples. In normal skin and benign epidermal lesions, collagen XVII protein was restricted to basal keratinocytes. However, possibly as a sign of undifferentiated/transformed state, it was widely expressed in SCC showing elevated levels around invasive tumor fronts with some staining in tumor adjacent stroma, endothelium, and histiocytes. Collagen XVII immunostaining of atypical keratinocytes in most actinic/solar keratosis supports the view of their malignancy and common origin with SCC. Squamous component of basosquamous carcinoma showed moderate reaction, whereas islets of BCC were mainly negative reflecting the diverse genotype and phenotype, and pathogenesis of SCC and BCC. These results suggest that collagen XVII neoexpression may be associated with early atypia/malignant transformation of keratinocytes. Further investigations are under way to analyze the potential of these antibodies for tracing progression and metastatic potential of skin tumors.

Demonstration of autoantibody binding to muscarinic acetylcholine receptors in the salivary gland in primary Sjögren's syndrome.

A significant pathogenetic role of antimuscarinic acetylcholine receptor-3 (anti-m3AChR) autoantibodies in primary Sjögren's syndrome (pSS) has been suggested. However, the binding of these antibodies to the receptors in the target tissues has not yet been demonstrated. In this study, the binding characteristics of pSS sera and anti-m3AChR-monospecific sera affinity-purified from pSS patients to labial salivary gland samples from healthy subjects were studied with light- and electron microscopy. Furthermore, the ultrastructural localisation of in vivo deposited antibodies in pSS salivary glands was also investigated. Light microscopic immunohistochemistry revealed the binding of the anti-m3AChR-specific sera to the membrane of acinar cells. Similar reaction end-products were observed in the pSS salivary gland epithelial cell membranes. With electron microscopy, the autoantibody binding was observed to be localised to the junctions of epithelial cell membranes with nerve endings, both in normal and pSS glands. The results indicate that anti-m3AChR antibodies bind to the receptors in the salivary glands.

Comprehensive regression analysis of hepatitis B virus X antigen level and anti-HBx antibody titer in the sera of patients with HBV infection.

Although the pathogenetic significance of hepatitis B virus x protein (HBxAg) in chronic hepatitis, liver cirrhosis, and primary hepatocellular carcinoma has already been studied, the comparative analyses of both the actual serum HBxAg levels and antibody production against various HBx epitopes have been examined to lesser extent. We have simultaneously investigated the relationship between antibody production (IgG and IgM) against the HBxAg fragments and HBxAg level in the sera of patients with acute (14) or chronic hepatitis (80) and symptomless carriers (12). A recently developed sandwich-type ELISA was used for the quantitative measurements of HBxAg. Overlapping recombinant and synthetic antigens were used to map the fine epitope specificities of circulating anti-HBx antibodies. In acute hepatitis, we have found high and homogenous correlation in the IgM type immune responses against all the examined HBxAg regions. Moreover, strong correlation has been observed between IgG type immune responses to a characteristic C-terminal region (C1: 79-117) and the longest fragment (X: 10-143). Moderate correlation has been found between HBxAg concentration and the IgG type anti-HBx antibody levels against C-terminus of HBxAg in patients with chronic hepatitis. In the case of symptomless carriers, there were also demonstrable associations in the immune responses against the C-terminal sequences; however, significant correlations were found for antibody production against the N-terminal region as well. The examinations show that the C-terminal sequence, responsible for transactivation, promotes an efficient IgG antibody response in all three groups of patients, whereas the negative regulator N-terminal part of the HBxAg molecule for the most part does not trigger antibody production. This suggests that the immune responses against various - biologically active - epitopes of the HBxAg may have a different role in the pathogenesis of hepatitis and may be used as prognostic markers in human HBV infections.

Sandwich type ELISA and a fluorescent cytometric microbead assay for quantitative determination of hepatitis B virus X antigen level in human sera.

The hepatitis B virus X protein (HBxAg) is responsible for severe complications of HBV infections including primary hepatocellular carcinoma. A sandwich type ELISA and a flow cytometric microbead assay for quantitative determination of serum levels of Hbx-Ag are introduced. We have previously developed monoclonal antibody families against well-conserved epitopes on HbxAg, characterized by different immunohistochemical and immunoserological techniques. Special selection of the antibody pairs provided highly sensitive and highly specific tools for quantitative immunoassay development. The resulting assays were tested on human sera (208 samples) collected from patients suffering from different clinical forms of HBV infection. The sensitivity range of the sandwich type ELISA was between 4 and 2000 ng/ml as measured on both the recombinant antigen and the sera of chronic hepatitis patients. A further flow cytometric microbead assay was established and tested in parallel with the ELISA. The quantitative results of these two immunoserological techniques were in strong correlation and they were found to be highly specific and sensitive on clinical samples. The HBxAg ELISA technique is applicable for routine clinical laboratory measurements, and our HBxAg microbead technique is recommended for complex multiparametric measurements combined with other markers.

Autoantibodies to human alpha6 integrin in patients with bullous pemphigoid.

Bullous pemphigoid (BP) is characterized immunologically by tissue-bound and circulating autoantibodies targeting the hemidesmosomal proteins BP230 and BP180. Recent evidence suggests a pathophysiological role for autoantibodies against alpha6 integrin in the subepidermal blister formation of oral pemphigoid. The objective of our study was to investigate the presence of anti-alpha6 integrin antibodies in patients with classical BP. The autoantibody profiles of 30 patients with BP, 10 patients with pemphigus vulgaris, and 20 healthy persons were identified. With the use of PeptideStructure and PlotStructure software, four different antigenic epitopes for alpha6 integrin were predicted, and their fusion recombinant constructs were prepared in an E. coli expression system. Sera were tested for alpha6 integrin autoantibodies by an ELISA technique. Altogether, 52% of the patients with BP displayed circulating antibodies against at least one recombinant protein. Our findings provide the first evidence for the presence of anti-alpha6 integrin antibodies in patients with classical BP.

Experimental bullous pemphigoid generated in mice with an antigenic epitope of the human hemidesmosomal protein BP230.

Bullous pemphigoid (BP) is an IgG-mediated autoimmune blistering disease that targets the hemidesmosomal proteins BP230 and BP180. To investigate the pathogenic role of anti-BP230 antibodies, rabbit polyclonal antibodies were generated against an antigenic sequence of the human BP230 antigen (BPAG 1, 2479-2499), which shows 67% homology in the human and the mouse BP230. Purified IgG from the rabbit anti-serum was transferred subcutaneously into the dorsal skin of neonatal isogeneic CBA/Ca (CBA) mice in a dose of 5 mg (n=7) or 1.2 mg IgG/50 microl (n=16). After 24 h, 1 of the mice injected with 5 mg IgG exhibited blisters, but the dorsal skin of all 7 of them was erythematous, and gentle friction produced a fine persistent wrinkling of the epidermis in 4 mice. The mice injected with 1.2 mg IgG developed less severe symptoms. Immunohistological examinations revealed linear rabbit IgG and mouse C3 depositions along the basement membrane of the perilesional skin and subepidermal blister formation. An intradermal inflammatory reaction (granulocyte infiltration) was also detected. None of these symptoms was seen in mice injected with IgG from a control rabbit anti-serum. These findings demonstrate that antibodies against BP230 can elicit the clinical and immunopathological features of BP in neonatal mice, suggesting that anti-BP230 antibodies may possibly play a pathogenic role in this disease.

A peptide of human muscarinic acetylcholine receptor 3 is antigenic in primary Sjogren's syndrome.

To evaluate the antigenicity of a peptide representing a part of the second extracellular loop of the human muscarinic acetylcholine receptor 3 (m3AChR) with autoimmune sera from primary Sjogren's syndrome (pSS), enzyme-linked immunosorbent assays (ELISAs) were developed. On the basis of the computer-predicted data, a 16-mer synthetic peptide KRTVPPGECFIQFLSE (KRSE213-228) was produced by solid-phase peptide synthesis. cDNA coding for the KRSE peptide was chemically synthetized and utilized to express the recombinant glutathione S-transferase (GST)-KRSE fusion protein. The immunoreactivities of the two antigens were tested in ELISAs with the sera of 40 pSS patients and 40 healthy controls. The specificity of the reaction was confirmed by inhibition assays and immunoblottings. The pSS sera resulted in significantly higher mean optical densities than those of the healthy controls (KRSE: 0.4149 vs 0.1494, p<0.0001; GST-KRSE 0.4765 vs 0.1764, p<0.0001). The immunological recognition with the recombinant fusion antigen was significantly better than that for the free peptide (p=0.0068). The sensitivities of the assays were 77.5% (KRSE) and 97% (GST-KRSE). The results of the concentration-dependent inhibition assays by the two systems of peptide presentation indicated that the KRSE sequence is specific for pSS sera. This is the first demonstration of the antigenicity of a novel peptide fragment of the human m3AChR in pSS. The analysed peptide could be of diagnostic relevance.

Modeling of main characteristics of bullous pemphigoid antigen-2 (BPAG2) peptide structure in serological recognition by autoantibodies.

The serum level of autoantibodies against autoantigens of the bullous pemphigoid peptides 1 and 2 (BPAG1 and BPAG2) is a relevant diagnostic marker. Twelve representative sera of BP were tested against the RSILPYGDSMDRIEKDRLQMAP amino acid sequence that is an epitope fragment of the NC16A domain of BPAG2 (AC Q02802; 507-528) to find the most suitable antigenic form for specific detection of autoantibodies of BP patients' sera by quantitative ELISA system. The antigenic epitope sequence was presented as an antigen in a carrier free form of dimeric peptide (BP22), dimeric peptide fused to glutathione S-transferase (GST-BP22) or dimeric peptide chemically conjugated to polyLys(Ser-DL-Alam) (SAK-BP22). The intensity of ELISA reaction was highest against the recombinant fusion antigen GSTBP22; the chemically conjugated SAK-BP22 performed less well than the free dimeric form of the peptide. In the case of the GST-BP22 antigen, the (GST-BP22)-(GST)492nm optical density values were determined. There was no significant difference between the mean ODs of the GST-BP22 and the SAK-BP22 (0.888 vs. 0.892, p= 0.9726). Conjugating the epitope peptide with the synthetic carrier SAK was advantageous, as it abrogated cross-reactivity with GST carrier protein. Consequently, the SAKBP22 conjugate appears to be the most reliable assay component, avoiding cross-reactivity with GST and simplifying the detection and evaluation of BP autoantibodies in routine ELISA diagnostic system.

Determination of the fine epitope specificity of an anti-hepatitis B virus X protein monoclonal antibody using microanalytical and molecular biological methods.

The recombinant form of the 17kDa, highly hydrophobic and disulfide-bonded hepatitis B virus X protein (HBX) was used for developing a set of monoclonal antibodies (Mab). Our present goal was to determine the fine epitope specificity of our anti-HBX Mab. Based on computer analysis two sequences (amino acids 22-31 and 100-114) were predicted for possessing high immunogenity while the anti-HBX Mab did not recognized them. Limited proteolysis and mass spectroscopic analysis suggested another possible sequence (amino acids 14-26), which also proved to be negative using an immunoserological test. Subsequently, we performed a screen of a phage displayed random peptide library, by which we could localize the epitope to amino acids 88-93. This finding was confirmed using three overlapping fusion peptides spanning amino acids 77-142. Their testing in ELISA assigned the epitope to amino acids 77-95, which supports the result obtained by screening the phage displayed library. Our results suggest the necessity of a complex application of current molecular biological and immunological techniques in fine structure mapping. This approach will be useful to study the prognostic relevance of different antigenic sites on HBX during the development of chronic hepatitis and primary hepatocellular carcinoma.