Kailash C. Bhol

The Autoantibodies to α6β4 Integrin of Patients Affected by Ocular Cicatricial Pemphigoid Recognize Predominantly Epitopes Within the Large Cytoplasmic Domain of Human β4

This study was undertaken to characterize the antigenic determinants recognized by the autoantibodies of patients with ocular cicatricial pemphigoid (OCP). OCP is a subepithelial, blistering, autoimmune disease that mainly affects the conjunctiva and other mucous membranes. We previously demonstrated that a cDNA clone, isolated from a keratinocyte expression library by using immunoaffinity-purified OCP autoantibody, encoded the cytoplasmic domain of β4 integrin subunit. Our subsequent studies showed that sera from all the OCP patients that were tested recognize the human β4 integrin subunit. To identify the prevalent epitopes of the anti-β4 autoantibodies of OCP, we have used cell lines transfected with vectors encoding a wild-type β4 subunit, a tailless β4 subunit, or a β4 subunit lacking the extracellular domain. Nontransfected cell lines were used as controls. Lysates from these cell lines were analyzed with OCP sera, IgG fractions from OCP sera, and immunoaffinity-purified OCP autoantibodies. Abs to extracellular and cytoplasmic domains of human β4 integrin were used as positive controls, whereas normal human sera and normal human IgG fractions were used as negative controls. The reactivity of OCP Abs was determined by using immunoblotting, immunoprecipitation, and FACS analysis. The results of this study indicate that OCP sera, OCP IgG fractions, and immunoaffinity-purified OCP autoantibodies react with the intracellular and not the extracellular domain of human β4 integrin subunit. In vitro cell culture experiments demonstrated that OCP autoantibody binds to the cytoplasm of the cells. The relevance of these findings to the pathogenesis of OCP is discussed.

Treatment of oral pemphigoid with intravenous immunoglobulin as monotherapy. Long-term follow-up: influence of treatment on antibody titres to human α6 integrin

Oral pemphigoid (OP) is a chronic autoimmune disease, involving the oral cavity, characterized by a homogenous linear deposition of immunoglobulins, complement, or both along the basement membrane zone (BMZ) and a subepithelial blister formation. The α6/β4 heterodimer is an integrin family of adhesion receptors, which mediates basal cell to matrix interactions. Recent evidence suggests a pathophysiologic role for antibodies against human α6 integrin in blister formation in OP, in organ culture studies. Fifty percent of OP patients have been reported to experience disease progression to involve other mucosal tissues, including the eye and larynx. To prevent this extension of disease, systemic therapy with systemic corticosteroids, dapsone, and immunosuppressive agents has been recommended. The use of intravenous immunoglobulin (IVIg) in the treatment of pemphigoid has been recently described. In this study, we present the use of IVIg, in a group of seven patients, with severe OP, in whom systemic conventional treatment was contraindicated. To determine the influence of treatment on antibodies to human α6 integrin in OP, seven patients with OP treated with IVIg therapy and a comparable control group of seven patients with OP, treated with conventional therapy, were evaluated at monthly intervals, for a 12 consecutive month treatment period. An effective clinical response was observed in all seven patients treated with IVIg therapy, after a mean treatment period of 4·5 months. IVIg therapy induced a prolonged and sustained clinical remission in all seven patients after a mean treatment period of 26·9 months. A statistically significant difference was observed in the quality of life pre‐ and post‐IVIg therapy (P < 0·001). Both the study and the control groups had a very similar initial serological response to treatment. A statistically significant reduction in the antibody titres was observed after four months of treatment, in both groups (P = 0·015). Thereafter, patients treated with IVIg therapy had a faster rate of decline in the antibody titres, and the difference in the rate of decline between the study and control groups became statistically significant after six months of treatment (P = 0·03). The use of IVIg therapy resulted in reduction of antiα6 antibody titres and in inducing and maintaining both a sustained, clinical and serological remission.

Linear IgA bullous dermatosis in a patient with chronic renal failure: Response to intravenous immunoglobulin therapy☆☆☆★

Linear IgA bullous dermatosis is a blistering disease with a heterogeneous clinical manifestation, characterized by deposition of IgA along the basement membrane zone of perilesional skin on direct immunofluorescence. We describe a patient with chronic renal failure who experienced linear IgA bullous dermatosis. Long-term administration of intravenous immunoglobulin therapy was associated with clinical remission lasting more than 12 months.

Autoantibodies to Human α6 Integrin in Patients with Oral Pemphigoid

Mucous membrane pemphigoid or cicatricial pemphigoid is a mucocutaneous blistering disease characterized by autoantibodies to different molecules in the basement membrane zone. Our objectives were to identify the target antigen recognized by sera from 20 untreated patients with pemphigoid disease limited to the oral cavity, and to determine the pathogenicity of autoantibodies in oral pemphigoid, with an organ culture model. We conducted indirect immunofluorescence, immunoblot, and immunoprecipitation assays, with accompanying absorption experiments, using normal human skin, conjunctiva and gingiva, bovine gingiva and a tumor cell line, which were reacted with sera from patients with oral pemphigoid, anti-α6 antibody, and control sera. Sera of oral pemphigoid patients selectively and specifically bound to human a6 integrin, a 120-kDa protein present in gingiva and the tumor cell line. Oral pemphigoid sera and anti-a6 antibody produced separation of epithelium from basement membrane (blister formation) of normal human buccal mucosa, after 48 hours, in organ culture.

Linear IgA bullous disease limited to the eye: a diagnostic dilemma: response to intravenous immunoglobulin therapy.

PURPOSE To report on a diagnostic dilemma and treatment challenge in a patient with chronic cicatrizing conjunctivitis without involvement of skin and other mucous membranes persisting for 6 years and not responding to topical and systemic steroids. DESIGN Interventional case report. METHODS We performed direct immunofluorescence of the conjunctiva with fluorescein-conjugated rabbit antihuman antibodies against immunoglobulin A, G, and M, complement 3 component, and fibrinogen. To investigate the presence of circulating antibodies in patients serum, indirect immunofluorescence using normal human conjunctiva, normal human skin, and monkey esophagus as substrate was done. In addition, we did immunoblot analysis using normal human epidermis as substrate to determine the molecular weight of an antigen. The patient was treated with intravenous immunoglobulin (IVIg). The correlation between the titer of circulating antibodies and the activity of conjunctival inflammation at various intervals during the course of IVIg therapy was demonstrated by immunoblot assay with serial dilutions of the patients serum. The highest dilution at which the binding was visible was considered the titer. RESULTS Direct immunofluorescence of the conjunctiva and indirect immunofluorescence with both salt split skin and conjunctiva as substrate disclosed linear deposition of immunoglobulin A (IgA) at the epithelial basement membrane. Immunoblot analysis demonstrated the presence of IgA circulating antibodies in patients serum directed against a 97kDa protein in human epidermis. A continuous decrease in the titer of these antibodies correlating to improvement of clinical symptoms was observed during IVIg therapy. CONCLUSIONS Use of a nonconventional diagnostic tool (immunoblot analysis), in addition to conventional immunohistologic studies, might be helpful in establishing the diagnosis of patients with chronic cicatrizing conjunctivitis. On the basis of results of these laboratory tests and clinical presentation, we believe that this patient has linear IgA bullous disease limited to the eye. IVIg therapy decreased the titer of circulating antibodies and induced a remission in this patient.

Influence of intravenous immunoglobulin therapy on serum levels of anti-ß4 antibodies in ocular cicatricial pemphigoid: A correlation with disease activity

Purpose. To determine any correlation between activity of ocular cicatricial pemphigoid and titer of anti-s4 antibodies, and any effect of intravenous immunoglobulin (IVIg) therapy on serum levels of anti-s4 antibodies followed over a 12 month period, using the specific immunoblot assay (IBA). Patients and methods. Eight patients diagnosed with OCP and treated with IVIg as monotherapy were included in the study. Each patient was treated with at least two immunosuppressive agents prior to the institution of IVIg. The presence of anti-s4 antibodies in the patients’ sera was detected by IBA using bovine gingival lysate (BGL) or tumor cell line lysate (TCL) as substrates. The activity of OCP was graded based on the conjunctival injection using a scale of zero to four in increments of 0.5 at monthly intervals. To determine the correlation between serum levels of circulating autoantibody and the patients’ conjunctival disease activity, the titer of anti-s4 antibodies was determined at monthly intervals during t...

Tacrolimus (FK 506)

Objective The focus of this review is to summarize the mechanism of action, animal and clinical studies, and adverse effects of tacrolimus (FK 506) in treatment after solid organ transplantation and in treatment of autoimmune diseases. Data Sources A detailed search of the literature was done. Both human and animal studies considered relevant and important were used. Study selection Material was taken only from peer reviewed journals. Results FK 506 is a macrolide lactone antibiotic with potent immunosuppressive activity. It acts primarily on CD4+ T helper lymphocytes by inhibiting the production of lymphokines, which are required for cell growth and differentiation, principally interleukin-2, at the transcriptional level. It was first clinically used in patients after liver transplantation. Tacrolimus-based immunosuppression has been proven to be beneficial in prolonging the survival of liver, kidney, heart, lung, pancreas, pancreatic islet, and intestinal allografts. Autoimmune diseases in which the immunosuppressive efficacy of tacrolimus has been tested experimentally include arthritis, systemic lupus erythematosus, uveitis, type-1 diabetes, thyroiditis, autoimmune renal disorders, experimental autoimmune encephalomyelitis, myocarditis, myasthenia gravis, colitis, and alopecia areata. Tacrolimus has been used for treatment of selected human diseases, such as psoriasis, uveitis, corticosteroid-resistant nephrotic syndrome, recalcitrant pyoderma gangrenosum, new onset type 1 diabetes, autoimmune chronic active hepatitis, pediatric autoimmune enteropathy, Crohns disease and atopic dermatitis. Conclusion Tacrolimus is a potent immunosuppressive drug in treatment of patients after solid organ transplantation and in selected autoimmune diseases. Further studies are necessary to better understand intracellular mechanism of action and specify therapeutic indications.

Diagnostic features of pemphigus vulgaris in patients with pemphigus foliaceus: detection of both autoantibodies, long-term follow-up and treatment responses

There are several studies that describe the simultaneous presence and conversion of pemphigus foliaceus into pemphigus vulgaris and vice versa. We describe eight patients with clinical, histological and immunopathological features of pemphigus foliaceus, at the time of the initial diagnosis. After a mean period of 2·5 years, additional serological features of pemphigus vulgaris were observed. During a long‐term follow‐up, systemic therapies, their durations and treatment outcomes were recorded. These patients did not respond to conventional systemic therapy and developed multiple side‐effects from these drugs. Hence, they were treated with intravenous immunoglobulin therapy (IVIg). Prior to the initiation of IVIg therapy, different assays were performed to detect the presence of autoantibodies, including indirect immunofluorescence (IIF), immunoblot assay using bovine gingival lysate, and ELISA. Twenty‐five healthy normal individuals, 12 patients with pemphigus vulgaris, and eight patients with pemphigus foliaceus served as controls for comparison of serological studies. At the time of initial diagnosis, the sera of all eight study patients also demonstrated binding on an immunoblot assay to a 160‐kDa protein (desmoglein 1) only. This is typically observed in pemphigus foliaceus. Prior to staring IVIg therapy, binding was observed to both the 160 kDa and 130 kDa (desmoglein 3) proteins on an immunoblot assay which was characteristic of pemphigus vulgaris. The antidesmogleins, 1 and 3 autoantibodies, were predominantly of the IgG4 subclass in all eight patients studied. IVIg therapy induced remission in four patients and control in four of the eight patients. The total follow‐up period ranged from 2·6 to 9·5 years (mean 5·3 years). It is difficult to determine the exact time at which these patients with pemphigus foliaceus developed pemphigus vulgaris. It is possible that the disease was nonresponsive to conventional immunosuppressive therapy owing to the simultaneous presence of two autoantibodies.

DIFFERENCES IN THE ANTI-BASEMENT MEMBRANE ZONE ANTIBODIES IN OCULAR AND PSEUDO-OCULAR CICATRICIAL PEMPHIGOID

PURPOSE Ocular cicatricial pemphigoid (OCP) is a chronic autoimmune cicatrizing disease which affects the conjunctiva and other squamous epithelium, resulting in a scarring process. A similar process, limited only to the conjunctiva, observed in some patients using eye drops for the treatment of glaucoma, is called pseudo-ocular cicatricial pemphigoid (P-OCP). Immunofluorescence studies demonstrate deposition of immunoglobulins and complement components in the basement membrane zone (BMZ) of the conjunctiva and an anti-basement membrane zone antibody in the serum of patients. A striking association between OCP and MHC class II gene DQB1*0301 has been observed. The purpose of this study was to determine some of the differences in the binding of OCP and P-OCP sera to different lysate in an immunoblot assay, in an attempt to partially characterize the OCP and P-OCP antigens. Furthermore, we wanted to determine if the MHC class II gene association of P-OCP is similar to that of OCP. METHODS We studied sera from 11 patients with active ocular cicatricial pemphigoid and seven patients with pseudo-ocular cicatricial pemphigoid and controls. Indirect immunofluorescence (IIF) studies were done using monkey esophagus and salt split normal human skin as substrate. A sensitive immunoblot assay (IBA) was developed using normal human epidermis, dermis and conjunctiva as substrate. Typing for MHC class II genes was performed on eight pseudo-ocular cicatricial pemphigoid patients by dot-blot analysis and compared to 38 matched controls. RESULTS Weak staining of the basement membrane zone was observed in nine of ten ocular cicatricial pemphigoid sera and five of seven pseudo-ocular cicatricial pemphigoid sera in the IIF assay using monkey esophagus. Using salt split human skin as substrate, ten of eleven ocular cicatricial pemphigoid sera demonstrated low titer weak binding to the epidermal side of the split. No consistent pattern of staining was seen with pseudo-ocular cicatricial pemphigoid sera. Ten of the 11 ocular cicatricial pemphigoid sera demonstrated binding to 230, 205, 160 and 85 kDa proteins in the IBA using normal human epidermis and conjunctiva lysates. When the lysates were first reacted with BP sera and then immunoblotted with ocular cicatricial pemphigoid sera, the 230, 160, and 86 kDa bands disappeared, and only the 205 kDa band persisted. The sera of five of seven pseudo-ocular cicatricial pemphigoid patients bound to 290, 230, 205, 180, 97, and 85 kDa proteins in the epidermis and conjunctiva. However, the 230, 205, 180, and 85 kDa proteins are depleted when the lysates are first reacted with BP and ocular cicatricial pemphigoid sera. In the dermal lysate, the pseudo-ocular cicatricial pemphigoid sera recognize 400, 290, 150 and 45 kDa proteins. None of these are absorbed by BP, ocular cicatricial pemphigoid or pemphigus vulgaris or epidermolysis bullosa acquisita sera. The 290 kDa proteins identified in the dermis and epidermis are distinct from each other. No binding was seen with control sera with the 3 lysates. Statistically, dot-blot analysis did not demonstrate a significant increase in the frequency of the MHC DQB1*0301 gene. CONCLUSIONS Patients with ocular cicatricial pemphigoid and pseudo-ocular cicatricial pemphigoid produce several autoantibodies. However, there are similarities and differences between them. The MHC class II genes associated with pseudo-ocular cicatricial pemphigoid are different from those with ocular cicatricial pemphigoid. This provides a new model system to study the immune abnormalities in idiopathic and drug-related organ specific autoimmunity.

Influence of intravenous immunoglobulin therapy on autoantibody titres to BP Ag1 and BP Ag2 in patients with bullous pemphigoid.

Background   Bullous pemphigoid (BP) is a subepidermal autoimmune blistering disease, which is characterized by blisters on the skin. Autoantibodies to components of the basement membrane zone are usually observed in the sera of patients with BP. Autoantibodies to the bullous pemphigoid antigens (BP Ag1, 230‐kDa desmoplakin protein, and BP Ag2, 180‐kDa hemidesmosomal protein) are present in the sera of BP patients.