ELISA in serological diagnosis of autoimmune bullous diseases (ABS)

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Society meeting SM6. Itnttlultoden,lafoloK~~ Group

at the N-terminal 45 amino acids of the NC16A domain. In PG, antibodies recognize the same immunodominant region of NC16A. In CP, autoantibodies are directed to both the NC16A domain and the C-terminus of BP180 that projects into the lamina lucida/lamina densa interface of the BMZ. In LPP, in contrast to BP, autoantibodies react with an epitope located at the C-terminus of NC16A. Finally, the 97-kD autoantigen of LAD has been identified as a portion of BP180 and a subset of patients with LAD reacts with the BP180NC16A domain. These findings demonstrate that different clinical phenotypes are associated with an immune response to the same autoantigen yet the immunoglobulin subclass of the autoantibody and the epitope that is recognized may be different.

SM6-8 Biochemical detection of antigens recognized by bullous dermatoses autoantibodies Hendri H. Pas. Dept. of Dermatology, University Hospital, Groningen, The Netherlands Immunoblotting is a simple tool to determine the antigen-specificity of sera of patients with autoimmune bullous dermatoses. This way autoantigens recognized by IgG. IgA and recently IgE have been identified in a number of diseases which include bullous and cicatricial pemphigoid, epidermolysis bullosa aquisita, linear IgA dermatosis, various forms of pemphigus and a variety of more rare immunobullous disorders. A number of protein substrates containing the various antigens may be used and when immunoblotting is used as a diagnostic tool, the choice of the right substrates, containing the suspected auto-antigen(s) in an appropriate concentration, is important. Also technical aspects of signal-development. like incubation time and temperature, type of second antibody/conjugates, and proper blocking of nqn-specific interaction will influence the final result. In evalu@ion of the resulting immunoblot pattern calibration tools may greatly facilitate the correct interpretation of the molecular weight of the observed antigens, since often sera will show more bands which may be close to the molecular weight of the suspected autoantigen. A drawback of immunoblotting is that the antigens have to be SDS-denatured prior to use which may result’in loss of conformational epitopes and therefore in loss of immunoblot sensitivity like reported in pemphigus. However in other diseases, like bullous pemphigoid, the correlation between serum immunofluorescence and immunoblot appears to be very high.

1SM6-9 Fluorescence overlay antigen mapping and the use of antigen-deficient substrates in the fine diagnosis of auto-immune bullous dermatoses

epidermolysis bullosa acquisita (EBA) anti-collagen type VII autoantibodies by the salt split skin technique. Recently, we developed two new techniques that allow such a differentiation by IF. In 1) Fluorescence Overlay Antigen Mapping (FOAM) the autoantibody binding is compared with a topographical marker, i.e. monoclonal antibody against type VII collagen. FOAM indeed allowed differentiation between floor binding sera. The use of 2) antigen-deficient knockout substrates in indirect IF allows further diagnosis whether the serum contains combined autoantibodies due to epitope spreading. For instance, EBA autoantibodies do not bind to type VII collagen defcient skin from patients with mutilating recessive dystrophic epidermolysis bullosa whereas anti-laminin 5 autoantibodies do not bind to laminin 5 deficient skin from patients with Herlitz junctional epidermolysis bullosa. The advantage of knockout IF substrate above immunoblot is that the substrate contains native antigens.

SM6-10 Electron microscopy in the diagnosis of autoimmune bullous disorders John A. McGrath. Department of Cell and Molecular Pathology, St. John’s Institute of Dermatology, St Thomas’s Hospital, London, UK The recent development of a number of well-characterised monoclonal antibodies against certain skin structural proteins and the advent of molecular cloning for several of the corresponding genes have both had a significant impact on the value of immunoelectron microscopy as an investigative tool in the diagnosis of the autoimmune bullous dermatoses. Nevertheless, immunoelectron microscopy is still a useful asset. Not only is it able to provide direct information linking autoantibody target antigens with specific structural skin components, but in the assessment of autoimmune sera with unusual immunoblotting characteristics, it may be helpful in defining the nature of the targeted epithelial adhesion junctions, and may provide vital clues to the characterisation of new proteins involved in adhesion. Much of the usefulness of immunelectron microscopy, however, relates to basic research rather than clinical application. For example, recent studies have helped characterise the configuration of type VII collagen-containing anchoring fibrils beneath the lamina densa, the extracellular distribution of type XVII collagen, and the network of adhesive proteins and glycoproteins that comprise hemidesmosomes and desmosomes. Given the simplicity of modem immuohistochemical methods, the role of electron microscopy in the diagnosis of the autoimmune blistering diseases is now less fundamental, but still useful under certain circumstances. SM6-11

ELISA in serological diagnosis of autoimmune bullous diseases (ABS)

M.F. Jo&man, M.C.J.M. de Jong. Dept. Dermatology, University Hospital, Gnmingen, the Netherlands

M. Herd. C. Haase, S. Christophoridis, L. Biidinger. Depf. of Dermatology, RWTH Aachen, Germany

The use of 1.0 M salt split skin for indirect immunofluorescence microscopy (IF) has greatly enhanced the differential diagnosis of subepidermal auto-immune bullous dermatoses. However, the split skin technique does not allow differentiation between sera with identical floor biding. For instance, cicatricial pemphigoid (CP) anti-laminin 5 autoantibodies cannot be differentiated from

A hallmark of most ABS is the presence of circulating autoantibodies (Ab) against intraepidermal and dermoepidermal adhesion structures which can be exploited as a diagnostic tool for tbe identification of distinct ABS. Recent evidence strongly suggests that the ELISA may be superior to western blot (Wp) analysis in the detection of circulating auto-Ab in ABS. Native

Society

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SM7. European

Society for Cosmetic

Ag immobilized in the ELISA more likely provides conformational epitopes recognized by these auto-Ab than denatured Ag of the WB. We have established ELISAs with two recombinant baculoproteins consisting of the extracellular domains of desmoglein 3 (Dsg3) or BPAG2 for the detection of circulating auto-Ab in patients with pemphigus vulgaris (PV) and bullous pemphigoid (BP), respectively. 35/39 BP sera and lO/lO pemphigoid gestationis sera were reactive to BPAG2 compared to O/15 PV sera and 1118 normal sera. Of 16 BPAG2-reactive sera in the ELISA, only six were BPAGZreactive in the WB of the identical baculoprotein. In the ELISA, IO/10 patients with PV and 3/6 patients with drug-induced PV had circulating auto-Ab to Dsg3 while O/12 normals specifically reacted with Dsg3. In addition to being superior to WB analysis, the employed ELISAs utilizing eukaryotic baculovirus-derived proteins may be more sensitive than ELISAs with prokaryotic recombinant proteins because they may provide the genuine conformational epitopes recognized by pathogenic auto-Ab.

SM6-13 Evaluation of the safety and efficacy of potential topical corticosteroid in the treatment of bullous pemphigoid A. Claudy. Hfipital

Edouard

Herriot,

Lyon,

France

Bullous pemphigoid (BP) is a disease with high mortality in the elderly due to long term administration of systemic corticosteroids (CS). We conducted on open label prospective study for safety and efficacy of topical CS in twenty consecutive BP patients. The patients were applied with clobetasol propionate once daily on unaffected skin. Only patients with mild forms of BP were included. The initial dose of CS was 12 mg/m’/day with a tapering off period based on skin symptoms over months. The results showed a healing rate of 63% in mild BP patients with a follow-up period of 11 months. A maintenance therapy was carried out with less potent CS. The side effects were cutaneous infections, dermal atrophy and mild transitory biological anomalies, mainly related to systemic absorption of CS. We concluded that topical potent CS produce rapid healing of bullous lesions in mild form of BP with minor adverse effects. Tapering off period of CS is highly variable and discontinuation of treatment may be difficult in some patients.

1SM6-14 1 Tetracycline and nicotinamide in treatment of bullous pemphigoid George Ch. Chaidemenos. Sfafe Dermatology Thessaloniki,

Chic,

Greece

In consequence to their reported efficacy since 1986, Nicotinamide (N) and Tetracycline (T) have been considered as an alternate to prednisone therapy in some autoimmune bullous diseases and especially Bullous Pemphigoid (BP). So far, from a total of 35 published cases of BP patients receiving N and T in doses of 1500-2500 mg/d, only three failures were reported. The mechanism of action of these drugs on BP is probably by inhibiting neutrophil and eosinophil chemotaxis, migration and histamine release. Mortality that in the past was caused by BP itself and the general debility of the elderly patients is now attributed to its treatment, especially corticosteroids. The combination of N and T is very promising because they are

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effective, at least in selected patients. An exclusion of patients with moderate renal insufficiency might prevent from serious complications. In our hands, Tetracycline 1500 mg/d was effective ‘as a steroid-sparing factor in 4 out of 5 patients with BP refractory to moderate doses of Corticosteroids. The regimen of either T or T and N, is gradually withdrawn after 6 months in complete remission and therapy usually lasts for one year or two in a decreased dose schedule however. SM6-17

Autoreactive T cells as potential targets for immunotherapy of autoimmune bullous diseases (ABD)

M. Hertl, L. Biidinger, R. Riechers. Department Dermatology,

RWTH

Aachen,

of

Germany

There is strong evidence that circulating auto-antibodies (Ab) targeting intraepidermal or dermoepidermal adhesion molecules are critical in the pathogenesis of most ABS. Hence, current therapeutic concepts aim at modulating the production of these auto-Ab. Generally, T cells provide help to the B cells that produce Ab. We thus studied autoreactive T cell responses to desmoglein 3 (Dsg3). the auto-antigen (Ag) of pemphigus vulgaris (PV) and to BPAG2, the putative major auto-Ag of bullous pemphigoid (BP). PV patients with active disease exhibited CD4+ T cell responses to Dsg3, so did normals who carried HLA II alleles similar or identical to those prevalent in PV. These T cells recognized various epitopes of the extracellular portion of Dsg3. Patients with BP had T helper 2 cells (which produced IL-5 and IL-13) specific for the extmcellular portion of BPAGZ. In contrast, healthy carriers of the HLA-DQ/?1*0301 allele, which is prevalent in BP, exhibited T helper 1 cell responses (which produced IFN-y) to BPAGZ. Upon identification of the T cell epitopes of these auto-Ag, immunomodulatory peptides will be generated that are capable of anergizing autoreactive T cells. This strategy will hopefully lead to a more specific modulation of the T cell-dependent production of pathogenic auto-Ab in ABS.

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European Society for Cosmeticand Aesthetic Dermatology

1SM7-2 1 A new skin type is proposed -

“Vasoreactive” C. O’Doherty, C.M.E. Rowlaynd-Payne, J. Kassab. QEI 1 Hosp, WG.C. Her& Wales, Bangor; UK

Royal

Marsden

Hosp,

London;

Univ

N.

In the initial examination of the skin patient, a dermatologist first notices the skin type. This observation is useful in the understanding of skin reactions and may influence the diagnosis and management of skin disease. One of the most widely used classifications of skin type,is the Fitzpatrick model based on the pigmentation of the skin. Another characteristic type is seen in Atopy. Atopics have a skin type characterised by dryness and lichenification. We recognise another skin type, occurring in any ethnic group, neither related to pigment, nor dryness,, but to the