Anti-basement Membrane Zone Antibodies Research Articles

Evaluation of ELISA BP180 and BP230 Autoantibodies in Blister Fluid and Serum in the Diagnosis of Bullous Pemphigoid.

Bullous pemphigoid (BP) is a chronic subepidermal immunobullous disorder. Studies have demonstrated the presence of antibasement membrane zone antibodies (BP180 & BP230) in the blister fluid using enzyme-linked immunosorbent assay (ELISA). To detect and compare BP 180 and BP 230 autoantibodies in the blister fluid and serum of patients with BP by ELISA method. A total of 30 patients diagnosed as BP and not on treatment were included in the study. Blister fluid and serum were subjected to ELISA, and the results were compared. The sensitivity of ELISA BP 180 was found to be 95.8% in the blister fluid and 88.4% in the serum. The sensitivity of ELISA BP 230 in the blister fluid and serum was 20% and 16.6%, respectively. Association between ELISA antibodies done in blister and serum was analysed using Chi-square test and found to be statistically significant with P value <0.05. Blister fluid is an effective alternative to the serum in detecting BP 180 and BP 230 antibodies, especially in uncooperative and elderly patients with poor venous access.

Une présentation rare du syndrome d’intolérance au greffon renal : la pemphigoïde bulleuse

Bullous pemphigoid is an autoimmune bullous cutaneous disease. We report the case of a 60 year-old male patient whose kidney allograft failed and was on hemodialysis for the previous 16 months. After tapering immunosuppressive medication, he presented simultaneous bullous eruption and kidney allograft intolerance syndrome. Investigation showed a positive BP180 anti-basement membrane zone antibody and skin biopsy was consistent with bullous pemphigoid. The patient was treated with corticotherapy and bullous pemphigoid resolved. The development of new onset diabetes and concerns over long term immunosuppression, halted the decision to continue corticotherapy and the patient underwent graft nephrectomy, with resolution of the kidney allograft intolerance syndrome without recurrence of the bullous disease. The occurrence of bullous pemphigoid in patients with failed renal allograft is rare, with only eleven cases reported in literature. This case illustrates how graft nephrectomy can provide a definitive cure to bullous pemphigoid in this setting.

070 Lesional IL-31 correlates with local eosinophil counts in bullous pemphigoid but not other eosinophil rich dermatoses

IL-31 is a pruritogenic cytokine that is elevated in certain dermatoses such as bullous pemphigoid (BP), and atopic dermatitis (AD). In BP, eosinophils appear to be the major source of IL-31. Whether this is specific to bullous pemphigoid, or other eosinophil rich dermatoses is unclear. We evaluated whether(if) IL-31 levels exhibit a correlation with lesional eosinophil levels in several eosinophil rich dermatoses. A retrospective immunohistochemical analysis of IL-31 expression was performed on samples consistent with BP, AD, drug rash, arthropod assault, and non-BP eosinophilic spongiosis. Basal and spinous epidermal staining was graded separately by two independent investigators on a scale of 0 to 3, and a gradient was calculated by subtracting the spinous from the basal score. A Kruskal-Wallis test was used to compare mean basal, spinous, or gradient scores between each disease. And a Spearman-Rank correlation was used to correlate mean basal, spinous, or gradient scores with eosinophil counts with significance defined as P<0.05. Basal epidermal IL-31 scores correlated with local eosinophil counts in BP (R=0.706, P=0.003), though spinous levels did not (R=0.266, P=0.336). Amongst the diseases investigated, basal and spinous keratinocyte levels, as well as, the IL-31 gradient did not show any correlation with lesional eosinophils. Although, the epidermal IL-31 gradient showed a moderate correlation with lesional eosinophil counts (R=0.408, P = 0.003), with removal of the BP cases, this finding was shown to be no longer significant (R=0.246, P=0.188). Herein, we demonstrate that epidermal levels of IL-31 correlate with lesional eosinophilia in BP, but not in other eosinophilic dermatoses. The disease specific microenvironment, with anti-basement membrane zone antibodies, and the cytokine milieu that follows may be the driving force behind eosinophil chemoattraction and local release of IL-31.

016 Brunsting-Perry pemphigoid after remission of previous bullous pemphigoid related to epitope spreading from BP180 to BP230

Brunsting–Perry pemphigoid is a localized form of cicatricial pemphigoid. Here, we report a case of Brunsting–Perry pemphigoid after remission of previous bullous pemphigoid. A 63-year-old Chinese male presented to our department with a three-month history of pruritic lesions in August 2013. Tense vesicles and bullae appeared on his hands, upper limbs and trunk. The skin biopsy showed subepidermal bulla with eosinophil infiltrations in upper dermis. The enzyme-linked immunosorbent assay (ELISA) for IgG antibodies against the BP180 NC16A domain was 90.44 and for the BP230 was 33.75. The patient was diagnosed as BP and treated with prednisone 30mg per day. The lesions improved quickly. Then the dose of prednisone was tapered down and kept at 5mg daily. Four years later, he revisited our department with a seven-month history of head lesions. Clinical examination revealed crusts, and trophic scars on the parietal scalp region. IIF using 1 mol L-1 NaCl-split healthy human skin sections revealed that the anti-basement membrane zone (BMZ) antibodies bound to the roof of the split. The ELISA index for the BP180 NC16A was 25.45 and for the BP230 was 110.82. From these findings, we diagnosed this patient as Brunsting–Perry type pemphigoid. He received oral prednisone (10 mg/d) but the improvement was limited. He later had the crusts debrided and 0.1mg mL-1 dexamethasone wet packing. The lesions healed gradually. The patient continuously had oral prednisone 10 mg daily in the following 9 months and was in stable condition. In this case, we observed a rare transition to Brunsting–Perry type pemphigoid from previous BP. As far as we know, it is firstly reported. And the dominant IgG anti-BMZ antibody switched from anti-BP180 NC16A to anti-BP230. But the mechanism still need further study.

Detection of Anti-basement Membrane Zone Antibodies in the Blister Fluid in Subepidermal Autoimmune Bullous Diseases.

Background:Subepidermal autoimmune bullous diseases (sAIBD) are diverse of conditions with clinicopathological overlap. Circulating autoantibodies in the serum can be demonstrated using indirect immunofluorescence (IIF) microscopy. Artificially split normal human skin is considered as an optimum substrate for the demonstration of anti-basement membrane zone (BMZ) antibodies using IIF in sAIBD; it not only helps to detect the presence of circulating antibodies in the serum but also helps to subclassify these conditions into “roof” and “floor” binding disorders.Aim:In this study, we evaluated the utility of IIF to detect anti-BMZ antibodies in the blister fluid of patients with sAIBD.Materials and Methods:Twenty-two patients with a clinical diagnosis of sAIBD were enrolled in the study. IIF of serum and blister fluid were done simultaneously using salt-split skin as a substrate.Results:Anti-BMZ antibodies could be detected in the blister fluid using IIF in all patients in the study group.Limitation:We could not do enzyme-linked immunosorbent assay of blister fluid. This would have given us the quantitative data of circulating antibodies in the blister fluid.Conclusion:Blister fluid offers an alternate source for the detection of autoantibodies in patients with sAIBD. It may be of particular help in children and in elderly with poor venous access.

Generalized vesiculobullous eruption in an 11-year-old male

An 11-year-old boy with an unremarkable medical history presented with 2-month history of progressive vesiculobullous eruption. Intermittent low-grade fever and arthralgia were also noted. The physical examination revealed multiple erythematous tense bullae and erosions over the face, neck, trunk, bilateral axillae, and wrists (Fig 1). Laboratory tests revealed positive antinuclear antibodies. No mucosal involvement or signs of infection or visceral organ impairment was disclosed. Anti–basement membrane zone antibodies were also detected by indirect immunofluorescence studies.

Épidermolyse bulleuse acquise et réaction du greffon contre l’hôte

Epidermolysis bullosa acquisita (EBA) is a rare acquired blistering disorder caused by production of auto-antibody directed against type-VII collagen. Autoimmune disorders can occur after allogenic bone marrow transplantation as manifestations associated with chronic graft-versus-host disease (GVHD). To date, there have been 10 cases reported in the literature concerning autoimmune blistering diseases following allogenic stem-cell transplants. Herein we describe a new case involving EBA. A 46-year-old woman developed EBA 4 years after allogenic cord blood transplantation for non-Hodgkin T-cell lymphoma complicated by acute digestive and cutaneous GVHD. At physical examination, she had some cutaneous blisters on the abdomen, arms and face, as well as numerous erosions in the buccal cavity. Direct immunofluorescence microscopy revealed linear IgG and C3 deposits along the dermal-epidermal basement membrane zone. Indirect immunofluorescence showed weak IgG G4 anti-basement membrane zone antibodies, which reacted with the dermal side of 1M NaCl-split skin; the autoantibodies were directed against type-VII collagen. This second case of EBA was evocative of a GVHD blistering disease. After the therapeutic failure of dapsone and of combined mycophenolate-prednisone, treatment with rituximab proved effective. EBA may form part of the autoimmune signs associated with chronic GVH. The destruction of basement membrane and of epidermal basal cells that occurs in GVH could give rise to autoimmune bullous disease. However, in our patient, in whom manifestation of chronic GVH was restricted to the lungs, it is difficult to rule out the fortuitous onset of EBA, which presented at a sizeable interval after acute GVH.

Three Cases of Linear IgA/IgG Bullous Dermatosis Showing IgA and IgG Reactivity With Multiple Antigens, Particularly Laminin-332

Linear IgA/IgG bullous dermatosis (LAGBD) is a relatively rare autoimmune bullous disease characterized by both IgA and IgG antibodies to epidermal basement membrane zone. The heterogeneity and pathogenesis of the LAGBD autoantigens have not been fully elucidated. We report 3 Japanese cases of LAGBD (ages 81, 88, and 64 years; 1 woman and 2 men). The patients showed bullous and erosive lesions on the trunk and extremities with minimal mucosal lesions. Histopathological analysis revealed a subepidermal blister with neutrophilic infiltration with eosinophils in 2 cases. Direct and indirect immunofluorescence studies disclosed IgG and IgA antibasement membrane zone antibodies. In immunoblot analyses of various antigen sources, all cases showed IgG and IgA antibodies to various subunits of laminin-332, in addition to IgG and IgA reactivity with type VII collagen, laminin-γ1, and BP230 and BP180 recombinant proteins. Our studies revealed that the 3 LAGBD cases showed prominent IgG and IgA reactivity with laminin-332, which was only rarely reported. In addition, all cases showed IgG and IgA reactivity with other multiple antigens, indicating the role of epitope-spreading mechanisms initiated from laminin-332. The significance of IgA antibodies to laminin-332 should be studied in larger cohorts of both LAGBD and linear IgA bullous dermatosis.

Successfully treated case of pemphigoid gestationis with post-partum intravenous and oral steroids.

Pemphigoid gestationis (PG) is a rare autoimmune blistering disease of pregnancy. PG has an estimated prevalence of 1 case in 50,000–60,000 pregnancies. It is characterized by pruritic, urticarial plaques with the development of tense vesicles and bullae within the lesions. PG has been associated with premature delivery, small-for-gestational-age infants. Recurrences with subsequent pregnancies are often more severe. Oral glucocorticoids are the mainstay of therapy. Differentiation of PG from pruritic urticarial papules and plaques of pregnancy is essential because management and outcomes differ. In instances in which clinical diagnosis is difficult, direct immunofluorescence tests, immunoblots, or ELISA studies of anti-basement membrane zone antibodies are useful in establishing the diagnosis. Signs and symptoms of the disorder typically present during the second or third trimester of pregnancy, but may occur at any time during the pregnancy or anywhere up to six weeks after giving birth. Most patients develop antibodies against 2 hemidesmosomal proteins, BP180 (BPAG2, collagen XVII) and less frequently BP230 [1]. The trigger for the development of autoantibodies in persons with PG remains elusive. Cross-reactivity between placental tissue and skin has been proposed to play a role. PG has a strong association with HLA-DR3 (61–80 %) and HLA-DR4 (52 %), or both (43–50 %), and virtually all patients with a history of PG have demonstrable anti-HLA antibodies. The placenta is known to be the main source of disparate (paternal) antibodies and can thus present an immunologic target during gestation.

Fluorescence overlay antigen mapping using laser scanning confocal microscopy differentiates linear IgA bullous dermatosis from epidermolysis bullosa acquisita mediated by IgA

Linear IgA bullous dermatosis (LABD) and epidermolysis bullosa acquisita (EBA) mediated by IgA antibodies belong to the group of autoimmune subepidermal bullous diseases mediated by IgA autoantibodies. Early and correct diagnosis is crucial because the management and prognosis of the diseases are different. To determine whether fluorescence overlay antigen mapping using laser scanning confocal microscopy (FOAM-LSCM) is helpful in the differentiation between these diseases. FOAM-LSCM and immunoblot studies were performed in 19 patients with disseminated tense blisters who presented with in vivo bound and circulating IgA antibasement membrane zone (BMZ) antibodies on immunofluorescence. Using FOAM-LSCM, in vivo bound IgA above type IV collagen, which is characteristic for LABD, was seen in 14 of the 19 cases, whereas five of the 19 cases had IgA deposits below type IV collagen, typical for EBA. Immunoblot studies showed that IgA antibodies in 11 of the 14 patients with deposits above type IV collagen reacted with different epitopes on BP180, mainly with LAD-1, which is a target antigen in LABD. Among the five patients with deposits below type IV collagen, one showed IgA antibodies to the 200-kDa laminin γ-1 and one had antibodies to the 290-kDa type VII collagen, EBA antigen. Additionally, enzyme-linked immunosorbent assay with recombinant type VII collagen was positive in three of the five cases who presented with IgA deposits below type IV collagen on FOAM-LSCM. The results using FOAM-LSCM were consistent with those obtained on immunoblotting. FOAM-LSCM is useful in routine diagnostics in cases with undetectable circulating anti-BMZ antibodies, and can differentiate LABD from IgA-EBA, the former with in vivo bound IgA above type IV collagen and the latter with IgA deposits below type IV collagen.

Unilateral bullous pemphigoid without erythema and eosinophil infiltration in a hemiplegic patient

In this report, we describe an 88-year-old male stroke patient with unilateral bullous pemphigoid limited to the hemiplegic side. Physical examinations revealed multiple tense bullae with clear and/or bloody contents without apparent erythema on the right thigh and lower leg, accompanied by erosions on the right chest. Histopathologically, no eosinophils were infiltrated into and around the subepidermal bullae. Immunofluorescence revealed deposited and circulating immunoglobulin (Ig)G anti-basement membrane zone antibodies. Immunoblot assays using various antigen sources and enzyme-linked immunosorbent assay revealed that IgG antibodies in this case reacted with unique epitopes between NC16a and C-terminal domains on the 120-kDa LAD-1, the extracellular truncated form of BP180. Three observations were unique in our case. First, the distribution of bullae in our patient was limited to the hemiplegic side. Second, there was no apparent erythema clinically and no eosinophilic infiltration histopathologically. Third, the patient achieved remission without the use of oral corticosteroids. The unusual epitopes in this case may contribute to these phenomena.

Usefulness of BP230 and BP180-NC16a Enzyme-Linked Immunosorbent Assays in the Initial Diagnosis of Bullous Pemphigoid

To investigate the diagnostic value of commercially available BP230 and BP180-NC16a enzyme-linked immunosorbent assays (ELISAs) in routine practice in patients with bullous pemphigoid (BP). Single-center retrospective study. French academic dermatology department. The study population comprised 138 patients, who were admitted from January 1998 through December 2008. Sera samples were analyzed by ELISA; clinical and immunopathological data were recorded from the patients' medical charts. BP230 and BP180-NC16a ELISA scores were evaluated with respect to clinical characteristics (number of blisters, mucosal involvement, localized or generalized disease, and outcome) and routine indirect immunofluorescence (IF). Of the 138 study patients, 81 (59%) had a positive BP230 ELISA result and 119 (86%) had a positive BP180 ELISA result. There was no relationship between a positive ELISA BP230 result and the disease extent at diagnosis or the presence of mucosal involvement. Serum anti-basement membrane zone autoantibodies (indirect IF) were more frequently detected when the BP230 ELISA result was positive (P < .001). The median anti-basement membrane autoantibody titer as detected by indirect IF was higher in patients with a positive BP230 result (P < .001). The BP180 ELISA result was associated with disease extent at diagnosis as estimated by both the percentage of patients with extensive BP (P = .01) and the mean number of blisters (P = .03) but was not associated with mucosal involvement. The currently available BP230 ELISA is a reliable although less-sensitive test than BP180 ELISA in BP, and its diagnostic added value compared with BP180 ELISA alone is approximately 5%. Our results support the predominant contribution of the BP230-specific autoantibodies to anti-basement membrane zone antibody titer as detected by indirect IF.

Rare Cause of Dysphagia

Purpose: Introduction: Bullous diseases of the esophagus are rare causes of dysphagia.These diseases are often autoimmune and present with concomitant skin lesions. Patient Presentation: A 43-year-old woman presented with a skin rash that started one month previously. It spread from the right thigh to involve the whole body. Two weeks later the patient developed odynophagia to solids.She was started on valacyclovir with no improvement. Thus, she was admitted to the hospital for further management. At the time of admission she was on a tapering dose of cyclosporine secondary to failed kidney transplant. Examination revealed multiple flaccid vesicles containing clear fluid, with areas of crusting and hemorrhage. Systemic examination was unremarkable. A skin biopsy performed, showed bullous dermatitis with viral effects on keratinocytes: however cell culture and polymerase chain reaction for herpes simplex virus were negative. EGD showed a large area of ulceration and friability in the mid esophagus. A vesicular blistering lesion was seen in this area with the passage of the scope.Multiple biopsies were taken which revealed squamous mucosa with subepidermal blisters. A direct immunofluorescence test on the skin biopsy showed IgG/C3 linear deposits in the epidermal basement membrane. The patient's clinical picture was most consistent with Epidermolysis Bullosa Acquisita (EBA) and she was treated with steroids and cellcept with significant improvement in her symptoms. She is still being followed up to avoid long-term sequel, such as esophageal strictures. Discussion: EBA is a cutaneous subepidermal autoimmune blistering disorder that results from the effects of IgG autoantibodies directed against collagen VII. Patients present with tense blisters within non-inflamed skin.EBA may lead to mucosal damage and bullous formation in the esophagus from the trauma of food or reflux. These subsequently lead to formation of webs and strictures leading to dysphagia. During endoscopy there may be spontaneous bullous formation from manipulation. In severe cases there may be total mucosal desiccation of the esophagus. Biopsies however diagnostic, may lead to further trauma. Linear IgG/C3 deposits in the basement membrane at the dermal epidermal junction from both skin and esophagus help with the diagnosis. Circulating anti-basement membrane zone antibodies that bind to the floor of the blister on Direct Immunoelectron Microscopy are confirmatory. Linear IgA deposits on skin biopsy correlate with mucous membrane involvement. EBA has shown to be associated with inflammatory bowel disease, in particular crohn's disease. Treatment involves immunosuppresion with steroids, immunomodulators, intravenous immunoglobulins, dapsone or colchicine.

Pseudoporphyria in a haemodialysis patient.

A 72-year-old female with end-stage renal disease due to chronic glomerulonephritis had been treated with conventional haemodialysis for 6 years. She was referred three months ago for management of blisters and cutaneous fragility on the dorsa of both hands (Figure 1a and b). The patient denied extensive sun exposure, sun beds, or use of medications other than calcium carbonate, vitamin B complex, folic acid or erythropoietin. Haemodialysis was performed tri-weekly, 4 h per session, via an arteriovenous fistula over the left forearm, using a cellulose acetate dialyser (FB170, Nipro, Japan). The blood and dialysate flow rates were 200 and 500 cm3/min, and delivered Kt/V (Daugirdas) and urea reduction rates were 1.89 and 0.79, respectively. Blood tests were negative for anti-nuclear, anti-intercellular substance and anti-basement membrane zone antibodies. Urine was also negative for porphyrin and coproporphyrin (spectrophotometry). Skin biopsy (Figure 1c) found a subepidermal vesicle with tombstone-like protruding papillary dermis, whereas immunofluorescence analysis revealed smooth linear vascular staining by IgG (Figure 1d) and IgA. Pseudoporphyria was diagnosed. The blistering lesions gradually improved after initiation of high-flux haemodialysis with a polysulphone dialyser (F80, Fresenius, Germany). At the time of follow-up, the blood and dialysate flow rates were 280 and 500 cm3/min, and delivered Kt/V (Daugirdas) and urea reduction rates were 2.40 and 0.83, respectively. Fig. 1 Pseudoporphyria in a haemodialysis patient. (a, b) Blisters (white asterisk) and cutaneous fragility found on the dorsa of both hands. (c) Haematoxylin and eosin staining found a subepidermal vesicle with tombstone-like protruding papillary dermis and ... Pseudoporphyria is an uncommon blistering disorder with clinical and histological similarities to porphyria cutanea tarda, but lacks urine and serum porphyrin elevations [1]. Pseudoporphyria has been linked with numerous aetiologies including renal failure, ultraviolet A radiation and photosensitizing medications (naproxen, furosemide, nalidixic acid, bumetanide, tetracyclines, amiodarone, etc.) [1]. It is possible that when multiple contributing factors are present in haemodialysis patients, including commonly used photosensitizing medications, the risk of pseudoporphyria may be further enhanced [2]. The mechanism for induction of disease is not understood. Discontinuation of the suspected photosensitizing medication and strict ultraviolet A protection are the mainstay of treatments. Successful treatment of pseudoporphyria following N-acetylcysteine and high-flux haemodialysis has been reported previously [3]. Conflict of interest statement. None declared.

Serological Studies in Bullous Pemphigoid: A Literature Review of Antibody Titers at Presentation and in Clinical Remission

Bullous pemphigoid is associated with antibodies to a 230 kDa and a 180 kDa protein. In a literature review we investigated the role of auto-antibodies as detected by different serological assays. Nine reports containing data on 143 patients were analyzed. Pre-treatment data showed that indirect immunofluorescence, enzyme-linked immunosorbent assay (ELISA) and immunoblotting offer an 82.2% or greater probability of being positive. At the end of the study period, all patients had clinically improved, whether or not they were on therapy. Auto-antibodies were present in 29% of patients evaluated by monkey esophagus immunofluorescence and 75% of those evaluated by human skin immunofluorescence. Positive titers were also reported in 67.6% of patients evaluated by ELISA. In 100% of patients in whom immunoblotting was performed the titers became negative. In 3 patients (5.3%) using human skin immunofluorescence and in one patient (1.4%) using ELISA the titers were increased at the end of the study period. The correlation between anti-basement membrane zone antibodies and the clinical course of bullous pemphigoid requires further and long-term studies.

Circulating IgA and IgE autoantibodies in antilaminin-332 mucous membrane pemphigoid

Antilaminin-332 mucous membrane pemphigoid (MMP) is a chronic autoimmune bullous disease that is often associated with internal malignancy. IgG autoantibodies against laminin-332 in patients with MMP are well documented; however, IgA and IgE autoantibodies against laminin-332 have not yet been described. To characterize IgA and IgE autoantibodies binding to laminin-332 in sera from patients with antilaminin-332 MMP. Sera and skin samples from four patients who met the following criteria were used: (i) subepidermal blistering lesions present on the mucous membranes; (ii) in vivo deposition of IgG along the epidermal basement membrane zone of sampled skin; (iii) circulating IgG antibasement membrane zone antibodies that react with the dermal side of salt-split normal human skin; and (iv) circulating IgG autoantibodies that do not show positivity against type VII collagen or 200-kDa protein (p200 antigen) in immunoblot analysis using dermal extracts. Circulating IgG/IgA/IgE class autoantibodies against laminin-332 were determined by immunoblotting. Circulating IgG autoantibodies against the gamma2, alpha3/gamma2, alpha3 and alpha3/beta3/gamma2 subunits of laminin-332 were demonstrated in sera from four patients, respectively. Serum from one of the four patients showed IgA reactivity with the alpha3/beta3/gamma2 subunits of laminin-332. Serum from one of the four patients showed IgE reactivity with the gamma2 subunit of laminin-332. The control sera failed to display IgG/IgA/IgE reactivity to laminin-332. In addition to IgG autoantibodies, circulating IgA and IgE autoantibodies against laminin-332 are detectable in a subset of patients with antilaminin-332 MMP.

Cicatricial pemphigoid vegetans

A 58-year-old woman with ulcerative colitis of 5 years' duration was first seen in March 2002 with a 5-month history of widespread erosions on the palate and gingiva, subtle scarring of the conjunctiva, and xerostomia accompanying a flare of ulcerative colitis. No skin lesions were observed. Direct immunofluorescence study performed on the oral mucosa showed a linear distribution of immunoglobulin G (IgG) and immunoglobulin A (IgA) at the dermal-epidermal junction. Indirect immunofluorescence demonstrated the presence of circulating IgG anti-basement membrane zone antibody. This patient was successfully treated with sulfasalazine 3 g/day. Two years later, however, she developed extensive, well-defined pustules and vegetating erosions on the skin, symmetrically localized in the inguinal and axillary folds and on the upper inner thighs (Fig. 1). At that time, mucous membrane involvement, except for subtle scarring conjunctivitis, was not observed. Laboratory findings were normal, except for colonoscopy, which disclosed cobblestoning and histologically well-circumscribed granulomas, confirming the diagnosis of ulcerative colitis. On the basis of the clinical features and the flare of coexistent ulcerative colitis, this patient was suspected of having pyoderma gangrenosum. Skin biopsy specimens showed acanthosis, papillomatosis, pseudo-carcinomatous hyperplasia, and subepidermal blisters overlying an infiltrate of leukocytes and eosinophils, which also extended into the lower epidermis (Fig. 2). Direct immunofluorescence of the perilesional skin demonstrated a linear distribution of IgG and IgA at the dermal-epidermal junction. In vivo-bound IgG within the basement membrane zone was documented using laser scanning confocal microscopy, which disclosed the presence of immunoglobulins above collagen type IV and below laminin-5 (Fig. 3). Indirect immunofluorescence study showed the presence of circulating IgG anti-basement membrane zone antibody reacting with the roof and floor of salt-split skin. Characterization of the target antigen (courtesy of Professor Hashimoto of Kurume University, Kurume, Japan) was unsuccessful, as Western immunoblot study on both epidermal and dermal extracts was negative, as was immunoblot with purified laminin-5 and enzyme-linked immunosorbent assay with the NC16a epitope of BP180. Laser scanning confocal microscopy confirmed the diagnosis of cicatricial pemphigoid. Therapy consisting of prednisone 40 mg each morning and sulfasalazine 1.0 g three times daily led to rapid improvement of the skin lesions, but did not affect the conjunctival lesions. After 4 weeks, prednisone was tapered and sulfasalazine was continued at a reduced dose of 0.5 g three times daily. At that time, only post-inflammatory hyperpigmentation and atrophic scars were evident.

Antigen Specificity in Subsets of Mucous Membrane Pemphigoid

Mucous membrane pemphigoid (MMP) has several subsets based on target antigens recognized by their sera. MMP and ocular cicatricial pemphigoid (OCP) sera recognize beta4 integrin subunit, oral pemphigoid sera recognize alpha6 integrin subunit, and anti-epiligrin cicatricial pemphigoid sera recognize laminin 5. Our aim is to determine if autoantibodies in the sera of patients with MMP, OCP, and oral pemphigoid (OP) recognize only their target antigens, and to see if this specificity is maintained throughout the clinical course. An immunoblot assay using bovine gingival lysate was used as substrate. Fifteen MMP patients, eight with OCP, and 15 OP patients were studied before therapy and at multiple intervals during the clinical course. Absorption and blocking studies were performed to determine binding specificity. Sera of patients with MMP and OCP recognize only beta4 integrin subunit, and sera of OP patients recognize alpha6 integrin throughout the clinical course. The sera of patients in the subsets of MMP described in this report show adherence and selectivity to target antigen during the entire clinical course, without crossover, interaction, or change. Hence, these subsets of MMP provide an excellent model to study clinical correlation with antigen and antibody specificity, in autoimmunity.

Childhood Bullous Pemphigoid Successfully Treated with Diaminodiphenyl Sulfone

Bullous pemphigoid (BP) is an acquired autoimmune blistering disease which predominantly affects the elderly. It is rare in children and infants. We reported a 14-year-old girl presenting with a month history of relapsing tense bullae on the face and extremities. Histopathological examination of the lesional skin revealed a subepidermal bulla with infiltration of eosinophils, neutrophils, and lymphocytes. Direct immunofluorescence showed linear deposits of IgM and C3 at the basement membrane zone. Indirect immunofluorescence using normal human skin sections as a substrate detected IgG anti-basement membrane zone antibodies in the patient's serum and that using 1M NaCl split skin sections showed that the patient's antibodies bound to the epidermal side of the split skin. Immunoblot analysis using normal human epidermal extracts demonstrated the presence of autoantibodies against the 230-kDa BP antigen. Furthermore, the patient's serum reacted with the recombinant protein of the NC16a domain of the 180-kDa BP antigen by immunoblot analysis and enzyme-linked immunosorbent assay. Our patient showed significant improvement of the skin lesions with systemic administration of diaminodiphenyl sulfone.

Alterations of basement membrane zone in autoimmune subepidermal bullous diseases

Pemphigoid (BP), mucous membrane pemphigoid (MMP) and epidermolysis bullosa acquisita (EBA) are characterized by the presence of in vivo bound and circulating anti-basement membrane zone (BMZ) antibodies directed against different antigens localized in the upper part of the lamina lucida, lamina lucida-lamina densa border and in sublamina densa region, respectively. The alterations of BMZ lead to blister formation. The aim of the present study was to compare the alterations of various regions of the BMZ in BP, MMP and EBA using laser scanning confocal microscopy (LSCM). Biopsy specimens taken from perilesional patients' skin were cut into 40mum thick slides, followed by double immunofluorescence labeling with antibodies against different BMZ structures and anti-human IgG antibody. Three-dimensional reconstruction (3DR) of various regions of BMZ and of in vivo bound IgG was performed by computer program integrated with LSCM. In BP and MMP, LSCM studies revealed numerous invaginations of BMZ, most pronounced at the level of lamina lucida. Integrity of BMZ was preserved at the level of lamina lucida, lamina densa and sublamina densa. In EBA, continuity of lamina lucida was preserved and numerous invaginations were present. More pronounced invaginations of BMZ were observed at the level of lamina densa, whereas at the level of the sublamina densa region the staining along BMZ was discontinuous. Moreover, large clumps composed of collagen VII vertically oriented to the dermal-epidermal junction extending into the mid dermis were observed. The study showed that alterations of BMZ in BP and MMP occur mainly in the lamina lucida, and in EBA in the sublamina densa region. These alterations in various regions of BMZ in BP, MMP and EBA could be responsible for the differences in the level of blister formation and in the clinical course of the diseases.