Contact hypersensitivity to epoxy resin, methyldibromoglutaronitrile, tixocortol pivalate and budesonide in Hungary: a study in 1448 patients.

AIM: In order to map the frequency of contact hypersensitivity (CH) to epoxy resin, methyldibromoglutaronitrile (MDBGN), tixocortol pivalate (TP) and budesonide patch tests were carried out. METHODS: The tests were performed in 1448 patients. Most patients belong to the allergic and irritative contact dermatitis groups. The tests were administered with the allergens epoxy resin 1%, MDBGN 0.3%, TP 1% and budesonide 0.1%, applied on the back. Reactions were evaluated at 40 min, on day 2 (D2), day 3 (D3) and day 4 (D4). In the patients of the Dept. of Dermatology, Venerology and Dermatooncology of Semmelweis University (patients number =1073) reactions were evaluated on day 7 as well. RESULTS: Epoxy resin elicited immediate reactions in 1 patient at 40 min. Further evaluations showed no difference on D3, D4 and D7 with a frequency of CH of 1.03%. Patch testing for MDBGN did not provoke immediate reactions, evaluations showed an increasing hypersensitivity rate (D2: 0.93%; D7:1.77%). Patch tests with TP yielded no immediate reactions, the frequency of CH increased from 0.47% (D2) to 2.01% (D7). No immediate reactions were observed by budesonide; an increase was seen in frequency of CH (D2:0.93% to D7:3.84%). CH to the studied allergens was observed mostly in allergic contact dermatitis group, to budesonide in irritative contact dermatitis and in atopic dermatitis groups as well. CONCLUSION: The data of the present study are the first results about this four allergens in Hungary and to our knowledge from our region as well.

Antineutrophil cytoplasmic antibodies in patients with systemic lupus erythematosus.

We determined the prevalence of antineutrophil cytoplasmic antibodies (ANCAs) in patients with systemic lupus erythematosus (SLE) and evaluated the correlation between ANCA positivity and clinical features. Forty-one patients with SLE and two control groups were examined. One of the control groups consisted of 15 patients with systemic vasculitis, and the other of 12 healthy blood donors. A quantitative enzyme-linked immunosorbent assay technique was used to measure the serum cytoplasmic ANCA (cANCA) and perinuclear ANCA (pANCA) levels. cANCA positivity was found in three patient samples, and pANCA positivity in 10 SLE patients. The occurrence and titres of both ANCA types in SLE patients were similar to those in healthy controls and significantly lower than those in patients with systemic vasculitis. The clinical picture and antibody profile were similar in ANCA-positive and ANCA-negative SLE patients. We conclude that measurement of ANCAs does not provide any additional diagnostic or prognostic data in SLE.

Conformational consequences of coupling bullous pemphigoid antigenic peptides to glutathione-S-transferase and their diagnostic significance.

Recombinant epitopic peptides BP1 and BP2 representing the Bullous pemphigoid autoantigens of BP230 and BP180 bound to the fusion partner glutathione-S-transferase (pGEX-4T-2, Pharmacia) have been previously shown to increase the efficacy of diagnosis of the disease. Using glutathione-S-transferase-bound monomer peptides, the sensitivity of the immunological reaction exceeded that of the free synthetic epitopes and was further increased with the number of epitopic blocks in the multimer fusion products. This has been explained by the avidity effect of the fusion partner dimer formation and the high ligand affinity due to the tandem repetitions of epitopic sequences. However, a beneficial conformation of the bound epitopic peptides might also contribute to the above phenomenon. Circular dichroism (CD) and Fourier transform infrared (FTIR) absorption spectroscopic studies revealed the importance of glutathione-S-transferase to induce and stabilize ordered secondary structures of the epitopic peptides. The free monomer and multimer peptides in aqueous buffer were present as a mixture of unordered and beta-sheet conformation, while binding them to the fusion partner the proportion of ordered secondary structures increased in parallel with the number of antigenic epitopes. The most prominent changes in the conformational state of the monomers in the fusion form were the increase of alpha-helical and beta-sheet and the decrease of unordered conformation, while in the case of oligomeric peptides the adoption of a helical conformation was accompanied by the decrease of beta-sheet structure. An outstanding alpha-helix content (46%) was detected in the case of the trimeric BP1 in its recombinant fusion form.

Development of a system for detection of circulating antibodies against hemidesmosomal proteins in patients with bullous pemphigoid.

Specific antibodies directed against special hemidesmosomal proteins are involved in the pathogenesis of bullous pemphigoid (BP), and detection of these antibodies is crucial for a correct diagnosis. As the BP autoantigen primary structures are known, the question was addressed as to whether it is possible to demonstrate circulating antibodies against BP autoantigens (BPAG1 and BPAG2) by means of an ELISA system, using antigenic epitopes. With the help of the programs Peptidestructure and Plotstructure, antigenic epitopes of BP antigens were predicted, chemically synthesized and screened using serum from 43 proven BP patients. The coding sequences of the best antigenic epitopes were then chemically synthesized and inserted as monomer and homo- or hetero-oligomer forms into fusion-expression plasmids (PGEX-4T, Pharmacia) in-frame to the C-terminus of glutathione-S-transferase. Fusion products were expressed and purified from Escherichia coli cells by affinity chromatography. The recombinant proteins were used for the detection of antibodies in the serum of 43 BP patients and of 60 controls (including 30 healthy persons, 22 patients with pemphigus vulgaris and 8 patients with other bullous dermatoses). Use of the homo- and hetero-oligomers of the recombinant fusion peptides increased the sensitivity of the disease-specific antibody detection. When a mixture of the best recombinant fusion proteins was used, the sensitivity of the ELISA assays in the case of the BP patients' serum was 0.90. This system could form the basis of a rapid and simple system for the diagnosis of BP.

Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes: a long and a short form.

We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular fragments of BP180. By BLAST homology search alignments we extended the 3' untranslated region (3'UTR) of the known BP180 mRNA sequence by 877 bases to completion. Three of 20 cDNAs identified by BLAST searches contained a 610 bp deletion in this new 3'UTR sequence. Northern blot analysis with a probe complementary to this deleted sequence showed binding only to the larger mRNA. The deletion of 610 nucleotides in the smaller mRNA was verified by reverse transcription-PCR and sequencing. Genomic PCR showed the new sequence to be an extension of exon 56 of the COL17A1 gene which suggests that the second mRNA is generated by differential splicing. In normal keratinocytes the level of the smaller transcript was 5-15% of that of the larger transcript whereas in a squamous cell carcinoma cell line this ratio was reversed, the smaller mRNA being three times more abundant than the larger mRNA. The biological significance of this newly identified transcript in protein synthesis and tissue expression or in cell differentiation, proliferation or adhesion is as yet unknown.

Effects of the neuropeptides substance P, calcitonin gene-related peptide and alpha-melanocyte-stimulating hormone on the IL-8/IL-8 receptor system in a cultured human keratinocyte cell line and dermal fibroblasts.

The neuropeptides substance P (SP), calcitonin gene-related peptide (CGRP) and alpha-melanocyte-stimulating hormone (alpha-MSH) are known to be able to regulate the production of cytokines in the skin. Since IL-8 plays an important role in cutaneous inflammation, the effects of SP, CGRP and alpha-MSH on the IL-8/IL-8 receptor (IL-8RA) systems of these cell types were studied. Cultures of human dermal fibroblasts and an immortalized keratinocyte cell line HaCaT were treated with 10-8 M SP, CGRP or alpha-MSH. The results demonstrated that these neuropeptides have different effects on the IL-8 and IL-8RA expressions of the cells. SP and CGRP upregulated the IL-8RA mRNA expression in HaCaT cells, but had no influence on their IL-8 production, whereas, alpha-MSH had no effect on either the IL-8 or the IL-8RA mRNA expression in HaCaT cells. In contrast, alpha-MSH resulted in a time-dependent induction of the IL-8 mRNA expression in dermal fibroblasts. This induction was already detectable after 6 h, and after 12 h there was a 5-fold change in comparison with the controls. The IL-8 content of the supernatant was also increased, with a maximum at 48 h after alpha-MSH treatment. The data established in the present study support the notion that neuropeptides can directly modulate the IL-8/IL-8RA system of keratinocytes and fibroblasts.

[New diagnostic possibilities in dermatologic diseases]. / Uj diagnosztikai lehetöségek a börgyógyászati kórképekben.

The author reviews the latest developments regarding the most important immunological methods for the diagnosis of dermatological diseases. The salt-split-skin, fluorescence overlay antigen mapping and immunoblot techniques are useful methods for the differentiation of autoimmune bullous skin diseases. The use of recombinant fusion products of antigenic epitopes in patients with pemphigus and bullous pemphigoid affords a possibility for the differentiation or diagnosis of the disease by means of a simple ELISA technique. In situ hybridization and the polymerase chain reaction (PCR) technique are very useful in many dermatological diseases to demonstrate infective agents or genetic abnormalities.

Scleromyxedema.

Scleromyxedema is a sclerotic variant of papular mucinosis, in which lichenoid papules and scleroderma-like features are present. We describe a patient with scleromyxedema with IgG type lambda chain paraprotein, a systemic sclerosis-like illness, and myositis. The patient's serum contained Scl 70 antibodies, characteristic of scleroderma. Electromyography showed signs of acute myositis and the creatine phosphokinase (CPK) level was elevated. Multiply passaged fibroblasts from the patient's skin lesions showed altered growth response in vitro. The patient was treated with cyclosporin (4 mg/kg/day) with improvement.

[Food allergy in patients with respiratory allergy. Diagnostic possibilities and problems]. / Légúti allergiás betegek étalallergiája. Diagnosztikai lehetöségek és problémák.

UNLABELLED: In the past two years the authors examined 28 patients with abdominal complaints and allergic respiratory symptoms. Detailed internal, gastroenterological, allergological examinations were made. METHODS: 1 skin Prick-test (SPT) with inhalative and nutritive panel 2. measuring of food-specific (gliadin, alpha-lactalbumin beta-lactoglobulin, ovalbumin) IgG-antibody level detecting with ELISA method, 3. leukocyte migration inhibition (LMI) test against the same foodstuffs, 4. histological examination of the stomach and the duodenum especially for mucosal mastocytes (MMCs). RESULTS: 1. SPT was positive in 23/28 patients for inhalative, but in the 5 cases we did not identify any inhalative allergen. The SPT for the main foodstuffs were positive in 18 patients while in 3 other patients there was urtica only for the other antigens. 2. The food-specific IgG-antibody level was increased in 18/27 patients against one or more antigens. The SPTs and the antibody determination showed identity in 8/18 cases. 3. The LMI tests were positive against one or more main food-products in 23/27 cases. There was common positivity in respect of antigens (between LMI test and antibody identification) in 17 cases. Pathological immunological reactions were presented against the same main foodstuffs with at least two methods for flour in 11, for egg in 10 and for milk in 12 patients. Endoscopic examinations were performed in 27 cases. The number of the MMCs were increased in 22/27 patients. After a specific elimination diet open-food challenges were performed and they confirmed the results of the in vitro and in vivo examinations. CONCLUSION: It is common that the respiratory allergic symptoms in atopic patients accompanied with food allergy for the main foodstuffs caused not only more severe respiratory symptoms, but abdominal complaints too. In respect to the many positive LMI tests the late-type hypersensitivity have important pathogenetical role in it. This three methods together define well the main food-products, which can be antigens as well. The examination of the MMCs supports the local disturbance in the immunoregulatory system.

Juvenile pemphigus foliaceus.

A 7-year-old girl with generalized erythematous, scaling plaques and vesiculobullous lesions on the extremities was diagnosed as having pemphigus foliaceus. Lesional direct immunofluorescence revealed intercellular IgG, IgA and C3 deposition. The patient's serum gave positive reactions against one epitope of desmoglein 3 and the epitope of desmoglein 1 in enzyme-linked immunosorbent assays, but the blood sample for indirect immunofluorescence did not display any circulating antibodies. The patient was successfully treated systemically with prednisolone and dapsone. Currently, she is taking dapsone, 12.5 mg daily. She has been free of lesions for the last 3 years.

The use of skin substrates deficient in basement membrane molecules for the diagnosis of subepidermal autoimmune bullous disease.

A case is presented of subepidermal, autoimmune bullous disease in which the initial examinations suggested the combination of epidermolysis bullosa acquisita and bullous pemphigoid. The diagnosis of epidermolysis bullosa acquisita was made by indirect immunofluorescence microscopy: the patient's serum bound to normal skin substrate but not to type VII collagen-deficient skin substrate derived from a patient with mutilating dystrophic epidermolysis bullosa. The use of skin substrates deficient in basement membrane molecules also excluded the presence of concomitant, circulating bullous pemphigoid autoantibodies in our patient. The diagnosis of epidermolysis bullosa acquisita was confirmed by split mapping, fluorescence overlay antigen mapping and Western blot.

Pyodermatitis-pyostomatitis vegetans.

A 43-year-old woman developed annular and pustular cutaneous lesions preceded by tiny yellow pustules coating the surface of the oral mucosa. The clinical, histological and immunopathological evidence clearly showed that the patient had pyodermatitis-pyostomatitis vegetans. It is suggested that this disease is a distinct entity which should be differentiated from pemphigus vegetans.

[Food allergy in patients with chronic urticaria]. / Etelallergia krónikus urticariás betegekben.

Basic food allergens (bread, milk and egg) were investigated in patients with idiopathic chronic urticaria. In 29 of 43 patients, sensitivity could be shown against one or more antigens. The results suggest not only the anaphylactic IgE-mediated reaction in same patients, but also an antibody or cell-mediated mechanisms in the development of the disease. Besides the Prick test, therefore, testing of other circulating antibodies by means of the ELISA technique and the leukocyte migration test is recommended to establish the causative agents in patients with chronic urticaria.

Identification of different circulating autoantibodies in patients with bullous pemphigoid and pemphigus vulgaris by means of immunoblotting.

Immunoblotting studies with salt-split human epidermis were performed on sera from 15 patients with pemphigus vulgaris and 20 patients with bullous pemphigoid by using peroxidase-labelled antihuman IgG and IgA. Eleven sera of pemphigus vulgaris antigen. Most of the sera gave additional specific bands at 210 and 80 kD, with lower intensity. The sera of 4 patients, 3 of them were in clinical remission, did not yield specific bands. Seventeen sera of the 20 bullous pemphigoid patients yielded a 220-230 kD protein band against the major bullous pemphigoid antigen and 4 of them gave another specific band at 160-180 kD. Five sera produced multiple bands (220, 130, 100 and 75 kD). IgA antibodies against the major bullous pemphigoid antigen were demonstrated in 2 cases with IgA deposits along their basement membrane, as revealed by direct immunofluorescence. The immunoblot patterns correlated only weakly with the clinical findings in bullous pemphigoid. There was a considerable diversity in both clinical findings and immunoblot patterns.

Detection of bullous pemphigoid antibodies by means of synthetic peptides as antigenic epitopes.

In an earlier study, the circulating autoantibodies in patients with bullous pemphigoid (BP) were demonstrated in higher frequencies by means of an immunoblot technique then via indirect immunofluorescence. In the present work with the help of Peptide Structure software, two matched antigenic epitopes were chosen and synthesized. The sera of 34 patients with BP were investigated in parallel by an immunoblot technique using a human epidermal extract, and by an ELISA technique with synthetic peptides. The sera of 16 healthy persons and 10 patients with other bullous diseases served as controls. Among the 34 patients with BP, 23 sera proved positive for at least one synthetic peptide. Positive reactions with the major BP antigen (230 kD) were found in 22 patients by immunoblot technique and in 16 patients by the ELISA technique with the synthetic peptide, whereas with the minor BP antigen (180 kD), positive reactions were found in 9 patients by the immunoblot technique and 18 patients by the ELISA technique using the synthetic antigenic epitope of the minor BP antigen. All control sera were negative for both antigens in ELISA investigations. In 3 patients with pemphigus vulgaris, characteristic bands against pemphigus vulgaris antigen were identified by means of the immunoblot technique, but all other cases were negative. It is suggested that the development of this technique may lead to an opportunity for the rapid and simple diagnosis of BP.

[Effectiveness of immunoblotting in the diagnosis of blistering skin diseases]. / Az immunoblot technika eredményessége a hólyagos börgyógyászati kórképek diagnosztikájában.

Results obtained with the immunoblot technique and indirect immunofluorescence on the specific circulating autoantibodies in 20 patients with bullous pemphigoid and in 15 patients with pemphigus vulgaris were compared. In 17 of the 20 patients with bullous pemphigoid, autoantibodies against the 230 kD major antigen could be demonstrated, and in 9 of these 17 patients more specific autoantibodies against "salt-split" epidermis antigen (220, 180, 130, 100 and 75 kD) were shown. Indirect immunofluorescence were positive only on the sera of 5 of the 20 patients with bullous pemphigoid. The sera of 11 of the 15 patients with pemphigus vulgaris yielded autoantibodies against specific antigen (130 and 85 kD protein). To summarize the results, the immunoblot technique is an excellent method to demonstrate circulating autoantibodies in patients with autoimmune bullous skin diseases and its sensitivity is better than that of the indirect immunofluorescence, procedure. Moreover, it gives a possibility to differentiate among different clinical types.

[Analysis of steroids and binding proteins in the blood of women with androgenetic alopecia]. / Szteroidok és kötöfehérjék vizsgálata androgen alopeciás nök szérumában.

Specific and sensitive radioimmune methods were used to study the steroid levels and binding capacities of their binding proteins in the serum of 30 women with androgenetic alopecia, in order to establish the proportions of the patients in whom hyperandrogenism or hypercorticism can be detected. It was found that the serum testosterone, dehydroepiandrosterone sulphate and cortisol levels did not differ from those for healthy women except dehydroepiandrosterone which was high. In 5 cases (16.6%), the binding capacity of the "sex hormone-binding globulin" was pathologically low, while in 6 patients (20%) the "free androgen index" was elevated. The binding capacity of the "corticosteroide-binding globulin" was pathologically low in 4 of 21 cases (19%) while the "free cortisol index" was high in 5 of these 21 patients (23.8%). Diane treatment (2 mg cyproterone acetate and 50 micrograms ethinyl-oestradiol)--was administered during 2-5 cycles (8 cases), the pathological dehydroepiandrosterone and dehydroepiandrosterone sulphate levels normalized, the testosterone level decreased to the lower limit of the normal range, the binding capacity of the "sex hormone-binding globulin" increased considerably, and the "free androgen index" fell below the normal range. Diane treatment, an essential improvement was observed in the condition of the patients: the hair became less greasy and its tendency to fall out was likewise moderated.

[Functional study of the regeneration of nerve fibers after keratinocyte transplantation]. / Idegrostok regenerációjának funkcionális vizsgálata keratinocyta transzplantáció után.

The functional regeneration of sensory nerves was investigated in 5 patients with skin defects (caused by leg ulcers, a burn or fasciitis necrotisans) after transplantation with a keratinocyte suspension. In this area, first the pain sense and soon after wards the cold and heat sense returned, followed finally by the flare reaction caused by the mustard oil test. No sweat production could be observed during the study, because sweat glands were not found after the keratinocyte transplantation. The reinnervation course is roughly the same for skin defects covered with split skin graft or with a keratinocyte suspension.