Topical nanocrystalline silver cream suppresses inflammatory cytokines and induces apoptosis of inflammatory cells in a murine model of allergic contact dermatitis.

BACKGROUND: Nanocrystalline silver has both antimicrobial and anti-inflammatory properties. However, the exact mechanisms underlying these activities are not known. OBJECTIVES: The objectives of this study were to assess the anti-inflammatory effects of nanocrystalline silver using a murine model of allergic contact dermatitis, compare the effects with those of tacrolimus and a high potency steroid, and to relate the effects to modulation of pro-inflammatory cytokines and apoptosis of inflammatory cells. METHODS: Dermatitis was induced on the ears of BALB/c mice using dinitrofluorobenzene. Topical treatment, including vehicles, 1% nanocrystalline silver cream, tacrolimus ointment and a high potency steroid, was applied once a day for 4 days. Ear swelling was measured and the erythema was evaluated daily. After 4 days of treatment the mice were killed and samples from the ears were collected for histological and immunohistochemical examination, terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labelling (TUNEL) staining and extraction of total RNA for reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Significant reductions of ear swelling, erythema and histopathological inflammation in mice ears were observed after 4 days of treatment with 1% nanocrystalline silver cream, tacrolimus ointment or a high potency steroid with no significant difference among them. Both RT-PCR and immunohistochemical staining of sections from ear biopsies demonstrated that nanocrystalline silver, tacrolimus and steroid significantly suppressed the expression of tumour necrosis factor (TNF)-alpha and interleukin (IL)-12. TUNEL staining demonstrated a significant increase in the numbers of apoptotic cells in material from the group treated with nanocrystalline silver when compared with that from groups treated with vehicle, tacrolimus or steroid. CONCLUSIONS: This study demonstrates that nanocrystalline silver inhibits allergic contact dermatitis in mice, similar to steroid and tacrolimus. Nanocrystalline silver suppresses the expression of TNF-alpha and IL-12 and induces apoptosis of inflammatory cells; mechanisms by which nanocrystalline silver may exert its anti-inflammatory effects.

Anti-inflammatory effect of topical nanocrystalline silver cream on allergic contact dermatitis in a guinea pig model.

The anti-inflammatory activity of topical nanocrystalline silver cream was assessed and compared with the effects of topical steroids and currently available immunosuppressants using a guinea pig model of allergic contact dermatitis. Dermatitis was induced with dinitrochlorobenzene and treated with different concentrations of nanocrystalline silver, medium and high potency steroids, tacrolimus and pimecrolimus, or appropriate vehicles once daily for 5 days. Erythema was evaluated daily (on a score of 0 to 4, from absent to very severe) and histopathology of the skin biopsies was evaluated after 5 days of treatment. Prior to treatment, the average scores of erythema in all the groups were in the range of 3(+) to 4(+). In the no treatment and vehicles groups these scores remained at about this level for the subsequent 5 days of the study. Nanocrystalline silver reduced erythema within 1 day of treatment in a concentration-dependent manner with significant reduction at silver concentrations of 0.5% and 1% (P < 0.05) and this reduction progressed throughout the study period. Steroids and immunosuppressants produced similar decreases in erythema, with no significant differences compared to 0.5% and 1% nanocrystalline silver. In skin biopsies scored for degree of inflammatory response, effects of treatments mirrored erythema results. This study suggests that nanocrystalline silver cream may have therapeutic potential for topical treatment of inflammatory skin diseases.

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. OBJECTIVE: The objective of this study was to report the influence of intravenous immunoglobulin (IVIg) therapy on autoantibody titres to BP Ag1 and BP Ag2. METHODS: In this prospective study, we measured autoantibody titres to both BP Ag1 and BP Ag2, in 10 patients with severe BP, over a period of 18 consecutive months on each patient, using an immunoblot assay. RESULTS: Prior to the initiation of IVIg therapy, the sera of nine patients demonstrated the presence of high autoantibody titres to both BP Ag1 and BP Ag2. One patient had autoantibodies to BP Ag1 only. A statistically significant decline in the autoantibody titres to both BP Ag1 and BP Ag2 was observed after 3 months of receiving the first cycle of IVIg therapy. This gradual decline in autoantibody titres continued until patients were observed to have non-detectable titres to BP Ag1 after 11 months and to BP Ag2 after 10 months of receiving IVIg therapy. Once patients achieved non-detectable titres, these patients were considered to be in a serological remission. This serological remission was sustained for an additional 7 months of observation. CONCLUSION: Autoantibody titres to BP Ag1 and BP Ag2 can be used to monitor the serological response to treatment in patients with BP. Patients with severe BP who are treated with IVIg therapy, as described in our protocol, achieve a long-term serological remission.

Influence of IVIg therapy on autoantibody titers to desmoglein 1 in patients with pemphigus foliaceus.

Pemphigus foliaceus (PF) is an autoimmune skin disease characterized by the presence of a pathogenic autoantibody to desmoglein 1, an epidermal cadherin molecule. Antibody titers to the desmoglein 1 protein can be used to monitor disease activity and severity in patients with PF. The purpose of this study is to report the influence of IVIg therapy on anti-desmoglein 1 antibody titers, in eight patients with severe PF, over a period of 18 consecutive months on each patient. This prospective study consisted of eight patients with severe widespread active PF at the time of entry into the study. The levels of autoantibody to desmoglein 1 were measured by an ELISA, at monthly intervals. Sera of all eight had high titers of the autoantibody to desmoglein 1, prior to initiating of IVIg therapy. A statistically significant reduction in the autoantibody titer index to desmoglein 1 was seen after 4 months of IVIg therapy. All eight patients were observed to have a progressive decline in autoantibody titer index while they were receiving IVIg. Patients on IVIg therapy had nondetectable titers after a mean period of 13 months and continued to remain in a serological remission for an additional observation period of 5 months. In the context of this study, autoantibody titers to desmoglein 1 can be used to monitor the serological response to treatment in patients with PF. Patients receiving IVIg therapy achieve serological remission.

Treatment of oral pemphigoid with intravenous immunoglobulin as monotherapy. Long-term follow-up: influence of treatment on antibody titres to human alpha6 integrin.

SUMMARY: 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 alpha6/beta4 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 alpha6 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 alpha6 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 antialpha6 antibody titres and in inducing and maintaining both a sustained, clinical and serological remission.

Autoantibodies to human alpha6 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-alpha6 antibody, and control sera. Sera of oral pemphigoid patients selectively and specifically bound to human alpha6 integrin, a 120-kDa protein present in gingiva and the tumor cell line. Oral pemphigoid sera and anti-alpha6 antibody produced separation of epithelium from basement membrane (blister formation) of normal human buccal mucosa, after 48 hours, in organ culture.

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.

Interleukin 1 components in cicatricial pemphigoid. Role in intravenous immunoglobulin therapy.

Interleukin (IL-)1 is an important mediator of inflammatory responses and plays an important role in the pathogenesis of various autoimmune diseases. Cicatricial pemphigoid (CP) is a multisystem autoimmune inflammatory disease. We have studied the role of IL-1 in its pathogenesis. We have investigated the serum levels of IL-1 components (IL-1alpha, IL-1beta, and IL-1Ra), and determined the role of intravenous immunoglobulin (IVIg) therapy in patients with CP. Serum levels of IL-1alpha and beta were significantly higher in untreated patients with active disease compared to levels in patients in prolonged clinical remission and normal human controls (P<0.0001). The serum levels of IL-1Ra were higher in patients in prolonged clinical remission compared to patients with active disease (P=0.002). Hence elevated levels of IL-1alpha and beta and low levels of IL-1Ra correlate with disease activity. The levels of IL-1alpha and beta were statistically significantly higher in sera of CP patients with active disease pre-IVIg therapy compared to post-IVIg therapy (P<0.0001). Statistically significantly higher levels of IL-1Ra were present in post-IVIg treatment serum samples when compared to levels in pre-IVIg treatment (P<0.0001). In the in vitro experiments, the levels of IL-1alpha and beta produced by the peripheral blood mononuclear cells (PBMC) isolated from patients before IVIg therapy were significantly higher when compared to the PBMC isolated from post-IVIg patients (P<0.0001). Significantly higher levels of IL-1Ra were observed in the supernatants of PBMC collected from pre-IVIg patients and cultured with exogenously added IVIg, when compared to the levels of PBMC to which IVIg was not added (P<0.0001). IL-1 may be an important cytokine involved in the pathogenesis of CP. The regulation of IL-1 could be one of the mechanisms, amongst others, by which IVIg may exert its beneficial effect in the treatment of CP.

Pemphigus vulgaris: the role of IL-1 and IL-1 receptor antagonist in pathogenesis and effects of intravenous immunoglobulin on their production.

Intravenous immunoglobulin (IVIG) is increasingly being used for the treatment of autoimmune diseases. In the present report, the role of IVIG on in vivo and in vitro production of IL-1 and IL-1 receptor antagonist (Ra) was studied in patients with pemphigus vulgaris (PV). Serum samples from 20 untreated patients with active PV prior to initiation of systemic therapy, 20 patients receiving IVIG treatment, 20 patients in clinical remission after conventional therapy, and 20 normal human controls were studied to determine the serum levels of IL-1alpha, IL-1beta, and IL-1Ra. The in vitro production of these cytokines was measured in the culture supernatant of peripheral blood mononuclear cells (PBMC) from 10 PV patients immediately before and after IVIG therapy and from age and sex-matched 10 healthy donors simultaneously. Elevated levels of IL-1alpha and IL-1beta were detected (i) in the serum of untreated PV patients with active disease prior to systemic therapy and (ii) before IVIG infusions in patients receiving IVIG therapy. These increased levels are statistically significant when compared to the levels in healthy controls (P < 0.01). A marked reduction of IL-1alpha and IL-1beta was detected (i) in the serum of patients in prolonged clinical remission and (ii) immediately after IVIG infusion in those patients on IVIG therapy. Increased level of IL-1Ra was detected in PV patients in prolonged clinical remission and after IVIG infusion in those receiving IVIG therapy. These differences were statistically significant when compared to the levels in normal controls and to the levels in the sera of patients with active disease (P < 0.01) or just before the beginning of IVIG infusion (P < 0.01). Similar differences in the levels of IL-1alpha, IL-1beta, and IL-1Ra were found in the culture supernatant of PBMC isolated from the PV patients pre and post IVIG therapy. These observations suggests that, compared to normal controls, patients with active PV have reversed levels of IL-1alpha, IL-1beta, and IL-1Ra. IVIG therapy may down-regulate production of IL-1alpha and IL-1beta and enhance production of IL-1Ra, in vivo and in vitro. This might be one of the important mechanisms by which IVIG produces its early therapeutic effects in pemphigus vulgaris.

Simultaneous presence of mucous membrane pemphigoid and pemphigus vulgaris: molecular characterization of both autoantibodies.

There are several reports in the literature describing the coexistence of features of pemphigus vulgaris and pemphigoid in the same patient. We describe 15 patients with clinical, histological, and immunopathological features of mucous membrane (cicatricial) pemphigoid at the time of initial diagnosis. All 15 patients failed to respond clinically to conventional systemic agents over a mean period of 7.2 years. Hence, IVIg therapy was used. Prior to initiating IVIg therapy, features of mucous membrane pemphigoid and pemphigus vulgaris were demonstrated by various serological tests. Different assays were performed to identify molecular characteristics of these two autoantibodies. Twenty-five healthy normal individuals, 22 patients with mucous membrane pemphigoid, 17 patients with pemphigus vulgaris, and 12 patients with pemphigus foliaceus served as controls for comparison of serological studies. On indirect immunofluorescence, using monkey esophagous as substrate, sera of all 15 patients had demonstrable levels of anti-intercellular cement substance (ICS) or anti-keratinocyte cell surface antibody. Sera of 14 patients on salt split skin bound to the epidermal side of the split, which was consistent with mucous membrane pemphigoid. Sera of all 15 patients demonstrated binding to a 205-kDa protein (human B4 integrin) and a 130-kDa protein (desmoglein 3) on immunoblot. In a sample of sera from each of the 6 patients with mucous membrane pemphigoid and pemphigus vulgaris, the anti-ICS antibody was of the IgG4 subclass. The IgG4 subclass is a characteristic feature associated with pathogenic autoantibodies in pemphigus vulgaris. Hence, in such patients, a dual diagnosis should be considered and confirmed by various serological assays. It is possible that the presence of two pathogenic autoantibodies in these patients could have contributed to the lack of response to conventional immunosuppressive therapy.

Identification of ocular cicatricial pemphigoid antibody binding site(s) in human beta4 integrin.

PURPOSE: To identify specific site(s) on human ss4 molecule to which sera from ocular cicatricial pemphigoid (OCP) patients bind and to determine its role in the process of blister formation. METHODS: Clone the fragments representing the extracellular and intracellular domain of ss4 molecule from normal human conjunctival mRNA into an expression vector; map the region to which sera from OCP patients bind by Western blot analysis. Determine the role of the immunodominant region in pathogenesis by demonstrating the ability of the rabbit antibody to the immunodominant region to produce separation of basement membrane zone (BMZ) from the basal epithelial layer when incubated with normal human conjunctiva in an in vitro organ culture model. RESULTS: Majority of the OCP sera tested bound to the C-terminal end of the intracellular domain (IC3.0) of the human ss4 integrin. Further subcloning of IC3.0 demonstrated that a smaller fragment extending from 1489 aa to 1572 aa (IC3.4) was responsible for this binding. This region may have multiple antibody binding sites. Antibody to human IC3.0 and IC3.4 produced in rabbit, resulted in BMZ separation, histologically identical with that observed when normal human conjunctiva was cultured with OCP sera in an human conjunctival organ culture model. CONCLUSIONS: These observations identify IC3.4 as the antibody binding site for sera of OCP patients and suggest a possible role for it in blister formation. Indirectly it highlights certain important aspects of the structural and functional dynamics of the biology of the hemidesmosomes and basement membranes.

In vitro organ culture model for mucous membrane pemphigoid.

Mucous membrane pemphigoid (MMP), also known as cicatricial pemphigoid, is a rare vesiculobullous disease of mucosal tissues, which involves the oral, ocular, and other mucous membranes. We have studied a group of patients with histologically and immunopathologically proven pemphigoid disease involving predominantly the conjunctiva and oral mucosa in addition to other mucosae. The purpose of our study was to (i) demonstrate the specific binding of autoantibodies present in the sera of patients with MMP to normal human oral mucosa by indirect immunofluorescence (IIF) and (ii) to study the role of these autoantibodies in the pathogenesis of subepithelial blister formation using normal human buccal mucosa in organ culture. Serum and IgG fractions from MMP patients showed homogeneous smooth linear binding along the basement membrane zone (BMZ) of the normal buccal mucosa on IIF. Serum from pemphigus vulgaris patients showed intercellular or keratinocyte cell surface staining. BMZ separation developed at 48 h after incubation of normal human buccal mucosa in organ culture, with serum or IgG from patients with MMP but not after addition of normal human serum. Addition of pemphigus vulgaris serum to the in vitro culture of normal human buccal mucosa showed acantholysis. This preliminary report suggests that circulating autoantibodies may have an important role in the pathogenesis of MMP. This in vitro organ culture model will facilitate enhancing our understanding of various molecular events during the process of blister formation in MMP and in the study of other mucosal diseases.

Analysis of current data on the use of intravenous immunoglobulins in management of pemphigus vulgaris.

BACKGROUND: Systemic corticosteroids, with or without the addition of immunosuppressive adjuvant agents, are frequently used in treating patients with pemphigus vulgaris (PV). The severe, catastrophic, and potentially fatal side effects of these agents highlight the need for the development of safe alternatives for PV therapy. Intravenous immunoglobulin (IVIG) therapy has recently been reported to be effective in the treatment of PV. OBJECTIVE: Our purpose was to do a retrospective analysis of the available literature on the use of IVIG in the treatment of PV. We also wished to determine whether the cumulative evidence permits making preliminary conclusions regarding the potential role of IVIG in the overall management of PV. METHODS: A review of the English-language, peer-reviewed literature was conducted for reports on IVIG use in treatment of PV. The available information on 21 patients was used to assess different dimensions of clinical efficacy. RESULTS: A minimum dose of 2 g/kg per cycle given at regular monthly intervals for a minimum of 3 cycles seems be effective in inducing a rapid clinical remission in patients with severe, recalcitrant PV. However, this should not be perceived as a "standard" dose. Tapering and eventual discontinuation of other agents were possible in many patients. Long-term follow-up was not provided to examine the influence of IVIG on the clinical course of disease, its efficacy as monotherapy, and the benefit of using it as maintenance therapy to keep the patient in prolonged clinical remission. In 4 of the 21 patients (19%), use of IVIG was of no clinical benefit. This failure of efficacy was primarily due to inadequate use. IVIG demonstrated beneficial effect in 17 of 21 patients (81%). CONCLUSION: IVIG may be a safe and effective agent in the management of severe, recalcitrant PV. Multicenter controlled studies, using different dose regimens, with lengthy follow-up periods are necessary to clearly define the emerging beneficial role of IVIG.

The autoantibodies to alpha 6 beta 4 integrin of patients affected by ocular cicatricial pemphigoid recognize predominantly epitopes within the large cytoplasmic domain of human beta 4.

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 beta 4 integrin subunit. Our subsequent studies showed that sera from all the OCP patients that were tested recognize the human beta 4 integrin subunit. To identify the prevalent epitopes of the anti-beta 4 autoantibodies of OCP, we have used cell lines transfected with vectors encoding a wild-type beta 4 subunit, a tailless beta 4 subunit, or a beta 4 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 beta 4 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 beta 4 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.

Bovine gingival lysate: a novel substrate for rapid diagnosis of autoimmune vesiculo-bullous diseases. A preliminary observation.

Immunoblot assays have been developed to characterize the autoantigens and to detect autoantibodies in muco-cutaneous autoimmune vesiculo-bullous diseases using different substrates. However the results have been inconsistent, because availability and standardization of different substrates has been a major problem. The aim of this study was to develop an immunoblot assay using bovine gingival lysate as substrate because it is easily and readily available as well as inexpensive. Sera from patients with different vesiculo-bullous diseases were studied. These included 25 patients with pemphigus vulgaris (PV), 8 with paraneoplastic pemphigus (PNP), 12 with pemphigus foliaceus (PF), 25 with bullous pemphigoid (BP), and 22 with cicatricial pemphigoid (CP). Serum samples from 40 normal human volunteers were also studied. The autoantibody titers were determined based on the binding pattern of each disease and compared to those obtained by routine indirect immunofluorescence (IIF). Our observations suggest that the titers from immunoblot assays were significantly higher than titers obtained by IIF (P<0.0001). When the autoantibody titers were compared using bovine gingival lysate and human epidermal lysate as substrate, statistically significant differences were not observed. The use of bovine gingival lysate as a substrate will facilitate the rapid and early serological diagnosis of patients with vesiculobullous diseases. It may also be of benefit to laboratory investigators studying these autoantibodies.

Presence of interleukin 10 in the serum and blister fluid of patients with pemphigus vulgaris and pemphigoid.

Interleukin 10 (IL-10) is an immunoregulatory cytokine produced by T lymphocytes and macrophages. Recently, it has been suggested that IL-10 may be involved in the pathogenesis of various inflammatory and autoimmune diseases. Using an ELISA we investigated the presence of IL-10 in the serum and blister fluid of pemphigus vulgaris (PV) patients with active disease and those in prolonged clinical remission compared with normal controls. Sera from patients with bullous pemphigoid (BP), ocular cicatricial pemphigoid (OCP), oral pemphigoid (OP) and blister fluid from five patients with BP were also studied. Increased levels of IL-10 were detected in the sera of 87.5% of patients with active PV and were statistically significant (P=0.0003) when compared with levels in normal human serum. Lower levels of IL-10 were detected in 12.5% PV patients in remission and were statistically significant (P=0.0001) when compared with levels in patients with active disease. Levels of IL-10 were detected in sera of 4.6% (1 of 24) of the normal controls. The levels of IL-10 were approximately four times higher in blister fluids than levels in the serum in the same PV patients. This difference was highly statistically significant (P=0.0008). A correlation was observed between serum levels of IL-10 and titres of pemphigus autoantibodies and with disease severity. Elevated level of IL-10 was detected in the blister fluid from five BP patients. Levels of IL-10 in the sera of patients with BP, OCP and OP were not significantly increased. These preliminary data suggest that IL-10 in concert with other cytokines may play an important role in the pathogenesis of PV and BP.

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.

Zymodeme alteration of Entamoeba histolytica isolates under varying conditions.

Cysts of Entamoeba histolytica obtained from 2 asymptomatic subjects were cloned in vitro and the isoenzyme patterns and virulence of the cultures derived from them were determined. Incubation in Diamond's TYI-S-33 medium with Crithidia sp. for 80 d resulted in a change from zymodeme III to zymodeme II, with consistent virulence. Axenization by replacing Crithidia sp. with crithidial lysate had no further effect on the zymodeme but the virulence of the amoebae was attenuated. A reversal to zymodeme III, together with augmentation of virulence, was observed on the 20th day after transfer of axenic amoebae to Diamond's TYSGM-9 medium containing the original bacterial flora. The possibility that the isoenzyme pattern determining the zymodeme and the virulence behave as 2 independent variables in E. histolytica is discussed.