Biological therapy of autoimmune blistering diseases.

INTRODUCTION: Autoimmune blistering skin diseases are a group of disorders subdivided according to the location of blister formation: intraepidermal blistering in the pemphigus group and subepidermal in the pemphigoid group. These conditions are clinically heterogeneous and are treated with systemic corticosteroids and/or other forms of immunosuppression on the basis of clinical subtype and disease severity. These approaches may not be effective for the induction and maintenance of clinical response or need to be stopped because of intolerable side effects. AREAS COVERED: Biological therapies can represent a valid alternative strategy in various autoimmune blistering disorders and this review article will address this issue with a special focus on pemphigus vulgaris and bullous pemphigoid. These biological approaches are designed to target B cells, autoantibodies, complement proteins, and several cytokines. EXPERT OPINION: Innovative strategies for the treatment of autoimmune blistering conditions primarily depend on the use of drugs with a high degree of specificity targeting crucial steps in the immunopathology of these disorders. Novel biological agents offer treatment alternatives to patients with autoimmune blistering conditions by targeting B cells, pathogenic autoantibodies, complement and cytokines.

A prospective analysis of anti-desmoglein antibody profiles in patients with rheumatoid arthritis treated with thiol compounds.

BACKGROUND: Drug-induced pemphigus is mainly caused by drugs containing sulfhydryl (thiol) groups in their molecules. OBJECTIVES: To understand the serial alteration of anti-desmoglein (Dsg) antibody profile in patients with rheumatoid arthritis (RA) receiving thiol compounds. METHODS: Anti-Dsg1 or -Dsg3 antibodies were analysed twice in a 1.6-year interval, in the same series of RA patients. RESULTS: Eleven of 204 serum samples (5.4%) and 6 of 139 samples (4.3%) from the same RA group showed a positive reaction against Dsg1 or Dsg3 in the first and second screening tests, respectively. The positive rates were higher than those in patients with collagen diseases including systemic lupus erythematosus, Sjögren syndrome, mixed connective tissue disease, and systemic sclerosis. In comparison with the results in the first and second screening tests, one RA patient newly gained anti-Dsg3 antibody, and at least 4 patients lost the antibodies in 1.6 years. Three patients produced antibodies to Dsg1 and/or Dsg3 in a similar fashion as did in the first screening tests. Only one RA serum sample exhibited an intercellular reactivity to human skin or monkey esophagus by immunofluorescence, and another sample bound to a 130 kDa protein suggestive of Dsg3 by immunoblotting. Most anti-Dsg antibodies in RA patients recognized EDTA-resistant epitopes of Dsg different from EDTA-sensitive epitopes recognized by pemphigus sera. CONCLUSION: RA patients receiving any of the thiol compounds may gain autoantibodies to non-conformational epitopes of either Dsg1 or Dsg3, and that such autoantibodies alone are not capable of inducing acantholysis.

Analysis of thromboembolic risk related to high-dose intravenous immunoglobulin treatment: a preliminary clinical study of 10 patients with autoimmune mucocutaneous blistering diseases.

BACKGROUND: Intravenous immunoglobulin (IVIg) treatment is a well-known treatment that has been used successfully in a broad spectrum of autoimmune diseases. Currently no data are available in the literature about the role of IVIg in the pathogenesis of thromboembolic events in patients with autoimmune blistering diseases refractory to conventional immunosuppressive treatment. AIM: To determine the relationship between IVIg and thromboembolism in patients with autoimmune blistering diseases and to establish a protocol to deal with the thromboembolic risk. METHODS: In our preliminary clinical study, 10 patients with autoimmune blistering diseases underwent IVIg cycles to a total of 133 cycles in all (total number of infusions in the patient group: 399), at a standard dose of 2 g/kg/infusion accompanied by an accurate and a complete clinical and laboratory screening for thromboembolism. Preventive measures, such as hydration before and after IVIg, and administration of 100 mg of acetyl salicylic acid (aspirin) or 1000 IU of subcutaneous heparin calcium per day for 3 weeks, were introduced to reduce the thromboembolic risk. RESULTS: Throughout the 2 years of IVIg treatment, no patient developed a superficial and/or deep venous or arterial thrombosis, even though some of the patients had underlying thromboembolic risk factors and had tested positive for some congenital and acquired thrombophilia markers. CONCLUSIONS: Our results indicate that thromboembolic events are uncommon, despite the presence of risk factors. However, as these disorders are very rare and the percentage of nonresponder patients is very low, further investigations are needed to better understand whether IVIg alone is able to trigger these fatal events in blistering disorders.

Levamisole and Chinese medicinal herbs can modulate the serum interleukin-6 level in patients with recurrent aphthous ulcerations.

BACKGROUND: Recurrent aphthous ulcerations (RAU) are common oral inflammatory lesions. Interleukin-6 (IL-6) is a pro-inflammatory cytokine that has effects on cellular and humoral immunities. Previous studies have shown that the high serum IL-6 levels in some RAU patients can be reduced by drug treatment. This finding suggests that IL-6 may be a useful marker in evaluating therapeutic effects of RAU. METHODS: In this study, we used a solid phase, two-site sequential chemiluminescent immunometric assay to determine the baseline serum levels of IL-6 in a group of 228 patients with RAU, erythema multiforme (EM), traumatic ulcers (TU), oral submucous fibrosis (OSF), pemphigus vulgaris (PV), or Sjögren's syndrome (SS), and in 77 normal control subjects. Some RAU patients were treated with levamisole plus Chinese medicinal herbs or levamisole only for 0.5-5 months and their serum IL-6 levels were measured after treatment. RESULTS: We found that about 99% of the normal control subjects and the patients with EM, TU, or OSF had a serum IL-6 level within the normal limit of 5.0 pg/ml. However, 24% (48/197) RAU patients, 14% (1/7) EM patients, 43% (3/7) PV patients, and 100% (6/6) SS patients had a serum level of IL-6 greater than 5.0 pg/ml. The mean serum level of IL-6 in patients with RAU (3.6 +/- 3.5 pg/ml, P < 0.001), minor type RAU (2.7 +/- 2.0 pg/ml, P < 0.05), major type RAU (5.2 +/- 4.6 pg/ml, P < 0.001), or herpetiform type RAU (4.1 +/- 3.8 pg/ml, P < 0.01) was higher than that in normal control subjects. The mean serum level of IL-6 in major type (P < 0.001) or in herpetiform type RAU patients (P < 0.05) was higher than that in minor type RAU patients. The mean reduction of serum IL-6 level (10.0 +/- 7.1 pg/ml) in RAU patients after treatment with levamisole plus Chinese medicinal herbs was significantly higher than that (5.1 +/- 3.7 pg/ml) in RAU patients after treatment with levamisole only (P < 0.005), suggesting that the combination therapy is superior to the single therapy of levamisole only. CONCLUSION: We conclude that levamisole and levamisole plus Chinese medicinal herbs can modulate the serum IL-6 level in RAU patients. Although the therapeutic effect of RAU can be assessed by a decrease in the frequency, duration and number of the oral ulcerations, it can also be monitored by a reduction of serum IL-6 level in RAU patients.

Intravenous immunoglobulin therapy in the treatment of patients with pemphigus vulgaris unresponsive to conventional immunosuppressive treatment.

BACKGROUND: Severe pemphigus vulgaris (PV) is conventionally treated with high-dose oral prednisone, usually in combination with an immunosuppressive agent (ISA). Some patients experience significant side effects, which are sometimes fatal, from prolonged immunosuppression. OBJECTIVE: Intravenous immunoglobulin (IVIg) was administered to 21 patients with severe cutaneous and mucosal PV who had not responded to the prolonged use of oral prednisone and multiple ISAs. METHODS: A preliminary dose-determination study tested 7 additional volunteers to ascertain the optimal IVIg dose of 2 g/kg per cycle. Parameters to assess clinical outcome were recorded before and after IVIg therapy. Variables tested were highest dose, total dose, and duration of prednisone and ISAs, their side effects, frequency of recurrence and relapse, duration of IVIg therapy, clinical response, induction and duration of remission, number of hospitalizations, total days of hospital stay, and quality of life. RESULTS: Use of IVIg monotherapy resulted in effective control of disease and produced a sustained remission in the 21 patients. The patients became free of lesions and remained so after finishing IVIg therapy. IVIg had a steroid-sparing effect and produced a high quality of life. Serious side effects from the use of IVIg were not observed. IVIg needs to be gradually withdrawn after achievement of clinical control. CONCLUSION: In patients with PV who do not respond to conventional immunosuppressants, IVIg appears to be an effective treatment alternative. Its early use is of significant benefit in patients who may experience life-threatening complications from immunosuppression. IVIg is effective as monotherapy.

Pemphigus vulgaris antibody identifies pemphaxin. A novel keratinocyte annexin-like molecule binding acetylcholine.

Because pemphigus vulgaris (PV) IgGs adsorbed on the rDsg3-Ig-His baculoprotein induced blisters in neonatal mice, it was proposed that anti-desmoglein 3 (Dsg 3) autoantibody causes PV. However, we found that rDsg3-Ig-His absorbs autoantibodies to different antigens, including a non-Dsg 3 keratinocyte protein of 130 kDa. This prompted our search for novel targets of PV autoimmunity. The PV IgG eluted from a 75-kDa keratinocyte protein band both stained epidermis in a pemphigus-like pattern and induced acantholysis in keratinocyte monolayers. Screening of a keratinocyte lambdagt11 cDNA library with this antibody identified clones carrying cDNA inserts encoding a novel molecule exhibiting approximately 40% similarity with annexin-2, named pemphaxin (PX). Recombinant PX (rPX-His) was produced in Escherichia coli M15 cells, and, because annexins can act as cholinergic receptors, its conformation was tested in a cholinergic radioligand binding assay. rPX-His specifically bound [(3)H]acetylcholine, suggesting that PX is one of the keratinocyte cholinergic receptors known to be targeted by disease-causing PV antibodies. Preabsorption of PV sera with rPX-His eliminated acantholytic activity, and eluted antibody immunoprecipitated native PX. This antibody alone did not cause skin blisters in vivo, but its addition to the preabsorbed PV IgG fraction restored acantholytic activity, indicating that acantholysis in PV results from synergistic action of antibodies to different keratinocyte self-antigens, including both acetylcholine receptors and desmosomal cadherins.

Graft-versus-host disease-like immunophenotype and apoptotic keratinocyte death in paraneoplastic pemphigus.

Paraneoplastic pemphigus (PP) is an autoimmune disease, which is frequently associated with non-Hodgkin's lymphoma. Autoantibodies against components of the cytoplasmic plaque of epithelial desmosomes are usually present in the sera and are believed to play a major pathogenic part in acantholysis and suprabasal epidermal blistering. However, another typical histological feature of PP, interface dermatitis with keratinocyte dyskeratosis, is shared with skin diseases that involve epithelial damage mediated by T cells. Here, we present the detailed characterization of the cutaneous T-cell response in a patient with PP and demonstrate a selective epidermal accumulation of activated CD8+ T cells together with an increased local production of interferon-gamma and tumour necrosis factor-alpha, and a strong expression of HLA-DR and ICAM-1 on keratinocytes. Apoptosis was identified as a key mechanism of keratinocyte death, and appeared independent of the FAS/FAS ligand (FAS-L) pathway, as epidermal expression of FAS was not increased compared with normal skin, and FAS-L was undetectable on the protein and mRNA level. Triple therapy with high-dose corticosteroids, cyclophosphamide and intravenous immunoglobulins reduced levels of pemphigus-like autoantibodies and reversed the cutaneous inflammatory reaction leading to long-standing clinical remission. Our findings support the concept of a major contribution of cytotoxic T lymphocytes to the immunopathology of paraneoplastic pemphigus.

Induction of blister-causing antibodies by a recombinant full-length, but not the extracellular, domain of the pemphigus vulgaris antigen (desmoglein 3).

Pemphigus vulgaris (PV) is mediated by autoantibodies to desmoglein 3, the pemphigus vulgaris antigen (PVA). PVA and an extracellular domain of PVA-Ig fusion protein (PV-Ig) can completely adsorb the blister-causing Abs from PV patient sera, suggesting that the extracellular segment of PVA might be sufficient to induce pathogenic Abs. To test this, we immunized rabbits with either PVA or its extracellular domain (EPVA) expressed in insect cells in our laboratory. When Igs were passively transferred from these rabbits into neonatal mice, anti-PVA, but not the anti-EPVA, induced blisters. To understand the basis for their differential pathogenic effects, we examined the properties of these sera. Both sera showed comparable ELISA titers and indirect immunofluorescence reactivity against monkey esophagus, a source of native PVA. Moreover, EPVA, like PVA adsorbed blister-causing Abs from sera of PV patients and rabbits immunized with PVA. In contrast, when IgG preparations were incubated with fura-2-AM (acetyloxymethyl ester)-loaded human keratinocytes in culture, only IgG from anti-PVA serum induced intracellular calcium mobilization. These data showed that PVA but not EPVA can elicit Abs that induced blisters in neonatal mice and mediate intracellular signaling through calcium mobilization.

A case of diet-related pemphigus.

An experimental investigation has lately shown that certain allyl compounds of garlic are able to provoke acantholysis in normal human skin cultured in vitro. The acantholytic effect has been more prominent in the samples from DR4+ donor. We here report a case of superficial pemphigus which appeared spontaneously in a DR4,14+, 49-year-old man and which ran a course that proved to be affected by dietary factors, in particular by the consumption of garlic. In the absence of a conventional treatment and on a garlic-free diet only, the disease ceased for several months. Soon after an unintentional dietary test with a strongly and presumable garlic-spiced fish meal, the pemphigus recurred. Nutritional factors should be added to the ever-growing list of exogenous factors capable of inducing or perpetuating pemphigus in genetically predisposed individuals.

Treatment of autoimmune disease with extracorporeal photochemotherapy: pemphigus vulgaris--preliminary report.

Extracorporeal photochemotherapy is a new form of immunotherapy which involves the extracorporeal photoinactivation of peripheral blood cells by 8-methoxypsoralen in the presence of ultraviolet A irradiation, followed by readministration of the cells. To explore the efficacy of this therapy in the treatment of autoimmune disease, four patients with a lengthy history of corticosteroid and immunosuppressive drug-resistant pemphigus vulgaris were initiated on extracorporeal photochemotherapy. Three patients experienced a complete remission in cutaneous disease expression, permitting discontinuation of medications in two and a substantial decrease in the third. Significant reductions in serum antiepidermal cell antibody titers occurred in all four patients. The treatments were well tolerated without the occurrence of adverse events. These results in a small number of patients suggest that extracorporeal photochemotherapy may prove to be a useful tool in the treatment of aggressive autoimmune disease.

Calcium enhances the sensitivity of immunofluorescence for pemphigus antibodies.

Indirect immunofluorescence (IF) to detect pemphigus and pemphigoid autoantibodies is commonly performed with monkey esophagus (ME) as substrate and phosphate-buffered saline (PBS) as a diluent. The purpose of this study was to evaluate comparative IF titers using human skin (HS) as substrate with variations in the buffers employed. Substrates (ME or HS) were incubated in PBS, Tris-acetate-buffered saline (TAS), TAS with 5 mM CaCl+2 (TAS-Ca+2), and PBS or TAS with 1 mM EDTA, prior to incubation with pemphigus or pemphigoid sera for indirect IF. We examined sera from 11 patients with pemphigus vulgaris (PV), 10 patients with Brazilian pemphigus foliaceus (BPF), and 4 patients with bullous pemphigoid. In 20 of 21 pemphigus sera, endpoint indirect IF titers were highest on normal skin with TAS-Ca+2. Six sera (2 PV and 4 BPF) had endpoints that were 5 double dilutions higher than the endpoints obtained with ME and PBS. Six sera (3 PV and 3 BPF) were 4 double dilutions higher, 7 sera (3 PV and 4 BPF) were 2-3 double dilutions higher, and 2 PV sera were equivalent with both substrate/buffers. Preincubation of either tissue with EDTA prior to indirect IF abolished PV and BPF antibody binding completely. Exposure to EDTA after the tissue was incubated with PV or BPF sera did not affect indirect IF titers. In the presence of Ca+2, the antigen was resistant to trypsin in concentrations of 0.001%; however, in the absence of added Ca+2 it was destroyed by 0.0001% trypsin. These differences were not observed with bullous pemphigoid sera; all 4 sera had similar endpoint indirect IF titers. This study shows a significant increase in the sensitivity of indirect IF assays for pemphigus autoantibodies by the use of Ca+2-supplemented buffers on human skin. This finding may also have implications for procedures designed to purify and/or detect pemphigus antigens.

The effect of retinoic acid on the expression of pemphigus and pemphigoid antigens in cultured human keratinocytes.

Vitamin A and its derivatives (retinoids) have both profound effects on epidermal differentiation and beneficial therapeutic effects in various dermatologic diseases. In order to understand these effects, much work has been done with cultured keratinocytes, which show specific morphologic, cellular, and biochemical changes modulated by retinoids. In an attempt to further define specific molecular effects of retinoids in cultured human keratinocytes, we studied the expression of pemphigus (P) and pemphigoid (BP) antigens by human keratinocytes cultured with retinoic acid (RA) in concentrations which modulated differentiation. Cultures of human keratinocytes in medium with 10% delipidized fetal bovine serum (vitamin A-depleted medium) demonstrated areas of extensive differentiation with flattened stratifying cells, keratohyaline granules, and an anucleate stratum corneum-like superficial layer. These cells also synthesized a 67 kd keratin, characteristic of well-differentiated epidermis. In contrast, cultures of human keratinocytes in the same medium supplemented with (10(-7) M, 3 X 10(-7) M, or 10(-6) M) RA demonstrated less differentiated small cuboidal cells that were stratified but did not form an anucleate layer or keratohyaline granules, and did not synthesize the 67 kd keratin. In order to detect P and BP antigens in these cultures, we used indirect immunofluorescence. In vitamin A-depleted cultures, P antigen either was not detected or was seen focally on the cell surface of basal cells. BP antigen was seen on the basal pole of the basal cells, approximating its in vivo location. In RA-treated cells, P antigen was seen on the cell surface of most of the cells, and BP antigen was seen throughout the cytoplasm of the basal cells. In order to study the expression of newly synthesized antigens, we radiolabeled cultures with 14C-amino acids and quantitatively immunoprecipitated the antigens, which were then identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis. We detected a major decrease in newly synthesized P antigen precipitated from extracts of vitamin A-depleted cells compared to RA-supplemented cells, whereas amounts of newly synthesized BP antigen were about the same. Taken together these data demonstrate that RA, at concentrations that decrease differentiation of cultured human keratinocytes, increases the expression of P antigen and changes the subcellular location of BP antigen.

Pemphigus erythematosus. Presentation of four cases and review of literature.

Four patients with pemphigus erythematosus, three male and one female, are presented and the literature is reviewed. One of the patients had penicillamine-induced pemphigus erythematosus. The mean age of onset was 66 years. Direct immunofluorescent examination of perilesional skin demonstrated immunoglobulin and/or complement deposition in both the intercellular cement substance (ICS) of the epidermis and at the dermoepidermal junction (DEJ) in all four patients. The lupus band test was positive in three of the four patients when biopsies from uninvolved skin were studied. Indirect immunofluorescent examination of the sera for anti-ICS antibodies was positive in all patients. All four patients had positive antinuclear antibody tests (ANA), but anti-deoxyribonucleic acid (DNA) and anti-extractable nuclear antigens (ENA) were negative. One patient had a positive rheumatoid factor. Serum creatinine and total complement were normal in all four patients. The patients were treated with various combinations of oral corticosteroids, topical corticosteroids, dapsone, and oral therapy, which consisted of hydrogen peroxide, benadryl elixir, and decadron elixir. Three patients were in complete remissions and the fourth patient was in incomplete remission after 4 to 10 months of follow-up (mean, 6 months). Patients with pemphigus erythematosus require significantly lower doses of systemic corticosteroids for control of disease. Dapsone is an important agent for adjuvant therapy and facilitates use of lower doses of systemic corticosteroids.