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Understanding the acantholytic pathways leading to blistering in pemphigus vulgaris (PV) is a key to development of novel treatments. A novel paradigm of keratinocyte damage in PV, termed apoptolysis, links the suprabasal acantholytic and cell death pathways to basal cell shrinkage rendering a ‘tombstone’ appearance to PV lesions. In contrast to apoptolysis, the classic keratinocyte apoptosis mediating toxic epidermal necrolysis causes death and subsequent sloughing of the entire epidermis. Apoptolysis includes five consecutive steps. (1) Binding of autoantibodies to PV antigens. (2) Activation of EGF receptor, Src, mTOR, p38 MAPK and other signalling elements downstream of ligated antigens, elevation of intracellular calcium and launching of the cell death cascades. (3) Basal cell shrinkage due to: (i) collapse and retraction of the tonofilaments cleaved by executioner caspases; and (ii) dissociation of interdesmosomal adhesion complexes caused by phosphorylation of adhesion molecules. (4) Massive cleavage of cellular proteins by activated cell death enzymes leading to cell collapse, and tearing off desmosomes from the cell membrane stimulating secondary autoantibody production. (5) Rounding up and death of acantholytic cells. Thus, the structural damage (acantholysis) and death (apoptosis) of keratinocytes are mediated by the same cell death enzymes. Appreciation of the unifying concept of apoptolysis have several important implications: (i) linking together a number of seemingly unrelated events surrounding acantholysis; (ii) opening new avenues of investigation into the pathomechanism of pemphigus; and (iii) creating new approaches to the treatment of pemphigus based on blocking the signalling pathways and enzymatic processes that lead to blistering. [ABSTRACT FROM AUTHOR]

Background: Intraveneous immunoglobulin (IVIg) is increasingly used to treat pemphigus vulgaris (PV). The mechanism by which it does so is not known. The following study was conducted to confirm the effectiveness of IVIg for the acute control of active PV and to elucidate the mechanism by which it does.Methods: Twelve patients with active and severe PV unresponsive to conventional therapy with high doses of systemic steroids together with or without a cytotoxic drug were treated with a single dose of IVIg (400 mg/kg/day for 5 days). All patients were concurrently given cyclophosphamide or azathioprine of not already on one of these two drugs. The primary end-points were healing of skin lesions, changes in serum levels of intercelular (IC) autoantibodies and in steroid doses one to 3 weeks after initiation of IVIg.Results: Within 1 week of initiating IVIg the activity of PV was controlled in most cases. Within 3 weeks the average baseline dose of systemic steroid was reduced by 40%. Serum levels of IC antibodies rapidly declined by an average of 59% within 1 week of initiating IVIg and by 70% within 2 weeks. The decrease was selective, as the average serum levels of antibody to varicella-herpes zoster did not decrease in the 4 patients in whom they were measured. The decrease in IC antibodies was inversely related to serum levels of total inmmunoglobulin (IgG). The decrease in IC antibodies was not due to blocking factors in the IVIg preparation and was too rapid to be due to suppression of IgG synthesis, suggesting that it resulted from increased catabolism.Conclusions: IVIg can rapidly control active PV unresponsive to conventional therapy by causing a selective and very rapid decline in the autoantibodies that mediate the disease. We believe it does so by increasing the catabolism of all serum IgG antibodies, and that this results in a selective decrease in only abnormal autoantibodies as catabolized normal anti bodies are replaced by those present in the IVIg preparation. IVIg is the first treatment that achieves the ideal therapeutic goal in auto-antibody diseases, the selective removal of the pathogenic antibodies without affecting the level of normal antibodies. [ABSTRACT FROM AUTHOR]

Background Since 1967 dermatology has used the classic technique of indirect immunofluorescence (IFI) for the detection of autoantibodies against antigens of the skin in diseased people with endemic pemphigus foliaceus. Thirty years later enzyme-linked immunosorbent assays – ELISA (rDsg1 and rDsg3) appeared as a viable option. A group of highly recognized researchers have concluded that ELISA is a simple, sensitive and highly specific method, allowing for diagnostic differentiation between pemphigus vulgaris (PV) and endemic pemphigus foliaceus (EPF). Scientific literature certifies that both ELISA and IIF bear high sensitivity in spite of the fact that a direct comparison between the ELISA and IIF tests has never been performed. Objectives This study was conducted to compare the sensitivity of these tests in detecting antibodies in the EPF. Material and methods Thirty-two serum samples were collected from patients with EPF. The control serum of 15 healthy individuals was tested to detect the presence of antibodies of EPF by indirect immunofluorescence and ELISA (rDsg1 and rDsg3). The IIF was performed, taking human skin as a substrate. Results Antibodies in patients with EPF were detected more commonly by the ELISA (rDsg1) (91%) compared with IIF (81%). Conclusions The ELISA (rDsg1) is slightly more sensitive than IIF in detecting antibodies related to EPV. However, according to our results, we do not currently possess a test with 100% accuracy in differentiating EPF from PV. Although previous studies have associated Dsg3 with PV, the tests performed during this study showed that 12% (4/32) of patients with EPF (cutaneous diseases only) also had Dsg3 antibodies. [ABSTRACT FROM AUTHOR]

Abstract: A major challenge in the development of more effective vaccines for cancer and other diseases is the development of potent adjuvants that can strongly, simply and safely enhance vaccine immunogenicity. Adjuvants that preferentially enhance Th1 type of responses are particularly desirable, as these responses are believed to play the major role in immune resistance to cancer. This study describes the ability of topical application of imiquimod to act as a potent, safe and simple vaccine adjuvant in mice. Groups of C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1mg/dose) weekly×4. Imiquimod in a 5% cream formulation was rubbed into the skin over the injection site for 15s to give a dose of ∼1mg/treatment following each immunization. Control mice were immunized with OVA alone, with irradiated E.G7-OVA cells (that express ovalbumin), with OVA encapsulated in liposomes, or to PBS. Topical imiquimod enhanced anti-OVA antibody responses 100-fold and markedly increased cellular responses compared to mice not given imiquimod. The responses were shifted towards a Th1 phenotype, with marked enhancement of IgG2a, IgG2b, and CD8+ T cell responses and concomitant suppression of IgM and IgG1 responses. More frequent topical applications of imiquimod further enhanced both antibody and cellular responses. There was no detectable local or systemic toxicity associated with treatment. These results indicate that topical imiquimod can safely and strongly enhance both antibody and CD8+ T cell response to OVA immunization, and suggest that it may provide a simple, safe and effective way to enhance the immunogenicity of vaccines in general. [Copyright &y& Elsevier]

The article presents information on the utility of intravenous immunoglobulin (IVIg) in the treatment of pemphigus. Contrary to the uncertain mechanism of action of IVIg in most autoimmune diseases, in pemphigus it seems to work by rapidly, dramatically, and selectively lowering serum levels of pemphigus antibodies. The mechanism of action of IVIg is very similar to that of plasmapheresis. Both procedures rapidly remove circulating pemphigus antibodies from the circulation and do so at about equal rates. However, IVIg has the major advantages over plasmapheresis that the pemphigus antibodies are removed selectively, whereas plasmapheresis as usually performed removes all circulating immunoglobulins, the good with the bad. Furthermore, IVIg appears to be safer.

Summary Pemphigus is a rare autoimmune disease that results in blistering of the skin and oral cavity. It is caused by autoantibodies directed against cell-surface antigens on keratinocytes, which when targeted lose their cellular adhesion properties and separate from one another to form blisters within the epidermis. Differences in the particular antigens targeted by the antibodies and in the distribution of these antigens in the different regions of the body and in the separate layers of the epidermis result in different clinical manifestations of the disease. The disease is diagnosed based on its clinical manifestations (flaccid blisters and erosions on skin and oral mucosa), histology (epidermal acantholysis), and immunological abnormalities (circulating and tissue-fixed antibodies against keratinocyte surface antigens). Pemphigus, which if left untreated is almost always fatal, is generally managed with topical, oral, or intralesional corticosteroids. Other options include plasmapheresis and intravenous immunoglobulin (IVIg), coupled with cytotoxic drugs. Immunosupressants, anti-inflammatory drugs, and antibiotics are used as adjuvants, but apart from IVIg, these therapy options are non-specific and more research is needed to develop treatments with improved side-effect profiles. [ABSTRACT FROM AUTHOR]

In this study, a human melanoma vaccine induced antibody responses in mice that varied significantly from animal to animal. BALB/c mice were immunized to a xenogenic human polyvalent melanoma vaccine that has been used in phase II clinical trials in over 600 patients. Mice were bled biweekly for up to 6 weeks to measure antibody responses. IgG antibody responses to the melanoma vaccine components were detectable within 2 weeks but were much stronger at 4 and 6 weeks. When the pooled sera were further analyzed by Western blot, a complex pattern of antigens was detected. When individual sera from identically immunized mice were assayed by Western blot, a consistent, reproducible pattern of antigen recognition was not seen. Rather, we found significantly different antibody responses among the mice. Both the intensity of antibody responses and the pattern of antigens recognized varied from animal to animal. Although there appeared to be immunodominant antigens that produced antibody responses in most mice, no single antigen induced antibody responses in all mice. These results demonstrate that polyvalent vaccines induce heterogeneous antibody responses in mice treated identically. Analysis of the response of selected melanoma patients immunized to the same vaccine revealed similar antibody responses to the antigens in the melanoma vaccine. Heterogeneity may hamper interpretation of vaccine immunogenicity and relevant tumor antigens in humans. [ABSTRACT FROM AUTHOR]

Abstract Background Two assays are available to detect anti-skin antibodies in patients with fogo selvagem (FS): indirect immunofluorescence (IIF) and immunoblotting (IB). This study was conducted to compare the sensitivity of these assays in detecting FS antibodies. Design Eighty-nine serum samples of 48 patients with FS and control serum from 15 normal individuals were tested concurrently for the presence of FS antibodies by IIF and IB. IIF studies were conducted using four different substrates: human skin, monkey and guinea pig esophagus, and bovine tongue. Results FS antibodies were detected much more commonly by IIF than by IB, i.e. in 71% vs. 28% of serum samples respectively. By IIF, the antibodies reacted most strongly against human skin. Conclusions IIF is a more sensitive assay than IB for detecting antibodies associated with FS. The sensitivity of the test is maximized by using human skin as a substrate. [ABSTRACT FROM AUTHOR]

Background: Antibodies to desmoplakins have been associated with paraneoplastic pemphigus. Here, we report a case of non-neoplastic pemphigus foliaceous with antibodies to desmoplakins. Observation: A 61-year-old woman with pemphigus foliaceous by clinical, histological and immunofluorescent criteria was found to have circulating antibodies to 250 and 215 kDa antigens identified as desmoplakin I and II by immunoblotting and immunoprecipitation. The patient had no evidence of cancer during a follow-up period of 3 years. Conclusion: Although antibodies to desmoplakin I and II have previously been associated with paraneoplastic pemphigus, their occasional finding in erythema multiforme and bullous pemphigoid indicates that they can also occur in non-neoplastic pemphigus. [ABSTRACT FROM AUTHOR]

Immunoglobulin G (IgG) antibodies reactive with intracellular components of transformed cells were detected in 26/35 sera from patients with melanoma using immunofluorescence and/or Western blotting. By extracting cellular proteins with either sodium dodecyl sulphate or moderate concentrations of salt (400 mM NaCl), the protein antigens were partially characterized by immunoblotting procedures. Although considerable heterogeneity in the molecular weights of the protein antigens was observed, two common groups were delineated. The anti-Pol antibodies reacted with 30 kd cytoplasmic protein and the anti-Ca antibodies recognized acidic high molecular weight (75 - 95 kd) proteins. These antigens were detected in all transformed cell lines tested, but were not restricted to them. Anti-Ca and anti-Pol antibodies were not found in sera from patients with other solid tumors or in systemic lupus erythematosus. [ABSTRACT FROM AUTHOR]

The immunologic specificity of bullous pemphigoid (BP) antigens expressed in human skin was studied by reacting the sera of 34 patients with BP with several specimens of normal skin. Most sera with basement membrane zone (BMZ) antibodies reacted to antigens expressed in all specimens of skin examined. A minority of pemphigoid sera reacted to BP antigens expressed in some specimens of skin but not in others. The pattern of cross-reactivity suggests that BP antigens are heterogeneous and that two different groups can be defined serologically. The first is a universal BP antigen(s) which is expressed in all individuals and to which antibodies are formed in most patients with BP. The other group consists of a family of BP antigens whose expression varies from individual to individual. Several immunologically distinct BP antigens can be expressed concurrently in the same specimen of skin. Lastly, the apparent absence of BMZ antibodies in some patients with BP is occasionally the result of antibodies directed to "minor" BP antigens with restricted expression. [ABSTRACT FROM AUTHOR]

Direct immunofluorescence studies were performed on hairy and alopecic areas of scalp in patients with alopecia areata, alopecia totalis and male pattern alopecia. Abnormal deposits of C3 and occasionally of IgG and IgM were found in 92% of 12 patients with alopecia areata and in 21% of patients with male pattern alopecia. No abnormalities were seen in 4 patients with alopecia totalis. In both alopecia areata and male pattern alopecia, the deposits were most common along the basement zone of the inferior segment of hair follicles and occurred with equal frequency in alopecic and normal scalp. These observations suggest that immune factors may play a role in the pathogenesis of alopecia areata. [ABSTRACT FROM AUTHOR]

Cytoplasmic antigens of 3 different specificities are expressed concurrently in the epidermis of normal human skin. They differ by their location in distinct layers of the epidermis. One type is present in all epidermal cells, another only in cells of the superficial layers, and the third only in basal cells. All 3 antigens were present in 10 specimens of normal human skin. The superficial and basal cell antigens were absent in 4 basal cell epitheliomas and 3 squamous cell carcinomas. This finding, together with the stratification of the antigens in different layers of the skin, suggests epidermal cytoplasmic antigens are differentiation antigens. [ABSTRACT FROM AUTHOR]

The reproducibility of the indirect immunofluorescent test for antibodies to human malignant melanoma cytoplasmic antigens was investigated by testing panels of melanoma and normal sera against cells of each of several different melanomas. Tests were performed in replicate, read blindly by two observers, and repeated on different days. Different observers agreed in the interpretation of replicate assays in approximately 80 to 90% of cases. Variability increased considerably when assays were repeated on different days or when different melanomas were used as substrate. Under these circumstances, similar results were obtained in as few as 21% of test. Thus, the results of the indirect immunofluorescent test for melanoma cytoplasmic antibodies must, at the present time be interpreted with great caution. [ABSTRACT FROM AUTHOR]

Antibodies to epidermal cytoplasmic antigens were present in 25 to 34% of 32 normal persons and in 34 to 50% of 53 patients with melanoma. There were at least two different types of cytoplasmic antibodies. The most common reacted to cytoplasmic antigens present only in the upper layers of the epidermis. This type of antibody occurred with equal frequency in patients with melanoma and in normal persons. The increased incidence of cytoplasmic antibodies in melanoma was due to antibodies to cytoplasmic antigens present throughout the epidermis. These antibodies were 2½ times more common in patients with melanoma than in normal persons. The presence of different cytoplasmic antigens in distinct strata of the epidermis suggests they result from epidermal cell differentiation. The epidermal cytoplasmic antigens present throughout the epidermis appear to partially cross-react with cytoplasmic antigens in melanoma cells. [ABSTRACT FROM AUTHOR]

The growth of fresh and I issue cultured B-16 malignant melanoma cells in C57BL/6 mice was quantitated, using as parameters the latent time to tumor appearance, tumor incidence, and mortality rate. The tumor was antigenic, as evidenced by the appearance of humoral antibodies to melanoma-associated antigens during the natural growth of this tumor and by the induction of resistance to lethal doses of tumor by immunization with repeated injections of irradiated, or sublethal doses of viable, melanoma cells. This immunity was not entirely specific, since mice immunized to B-16 melanoma were slightly resistant to challenge with BW10232 mammary adenocarcinoma and, conversely, mice immunized to mammary adenocarcinoma were slightly resistant to melanoma. [ABSTRACT FROM AUTHOR]

The diagnosis of autoimmune blistering skin disease is increasingly based on the skin antigen(s) that is the target of the autoimmune process. Some patients with bullous diseases have unusual basement membrane autoantibodies that react to antigens present in both the epidermal and dermal sides of skin split with 1 M NaC1. This combined staining pattern is due to antibodies directed in part to a 160-kilodalton (kD) basement membrane antigen present on the epidermal side of the split skin and to different antigen(s) present on the dermal side. The identity of the current antigen(s), and the significance of this unusual type of combined staining antibody response is not known. Sera of five patients with combined staining antibodies, 19 patients with other types of basement membrane antibodies (15 directed only to the epidermal and four only to the dermal side of split skin), and 23 patients without basement membrane antibodies were tested by Western immunoblot for reactivity to 4% sodium dodecyl sulfate + 2 M urea extracts of dermal and epidermal side of salt-split skin. Three (60%) of the combined staining sera had antibodies to an 82- 84-kD antigen (p84) reproducibly present in dermal, but not epidermal, extracts of normal human skin. Antibodies to this antigen were absent in the 42 control sera. Western blot affinity-purified antibody to p84 bound only to the basement membrane, on the dermal side of 1 molar salt-split skin. By co-migration experiments, p84 differed from other basement membrane antigens including the major and minor bullous pemphigoid, cicatrizing pemphigoid, and epidermolysis bullosa acquisita antigens, types III and IV collagen, laminin, and epiligrin. Thus, combined staining antibodies are directed in part to an 82-84-kD antigen (p84), a normal but previously undescribed component of the basement membrane of human skin. This antigen differs from the basement membrane antigens recognized by autoantibodies in other subepidermal bullous diseases. The antibody response to p84 provides a specific marker for a novel autoimmune subepidermal blistering skin disease. [ABSTRACT FROM AUTHOR]

The article discusses the involvement of anti-desmoglein antibodies in the pathogenesis of pemphigus vulgaris. This section examines how anti-desmoglein antibodies can cause acantholysis, why is the acantholysis restricted to the basal cell layer, and can other autoantibodies cause acantholysis. The section also offers various reports with regards to the involvement of anti-desmoglein antibodies.

We present a 69-year-old white woman with pemphigus vulgaris limited to the oral mucosa who presented with oral pain and difficulty swallowing of 2 days duration, followed by multiple irregular ulcers arising from normal mucosa with no grouping of individual lesions-herpes simplex should be considered in the differential diagnosis of lesions that appear suddenly in patients with PV, particularly if the lesions fail to respond to an increased dose of corticosteroids. 1999 Elsevier Science B.V. All rights reserved. [ABSTRACT FROM AUTHOR]

The expression of U-CYT antigens, tissue-specific cytoplasmic antigens of keratinocytes that are markers of the terminal differentiation of these cells, was studied in normal skin adjacent to malignant epidermal tumors. Ten basal-cell carcinomas were studied. In all cases, it was found that the expression of U-CYT antigens in normal epidermis gradually decreased with increasing proximity to the tumors and finally disappeared completely within the tumor tissue itself. This finding suggests that basal-cell carcinoma can alter the normal differentiation of adjacent nonmalignant cells. [ABSTRACT FROM AUTHOR]

There is considerable indirect and some direct evidence that immune mechanisms can play an important role in controlling the growth of melanoma. In mice, melanoma can be prevented with vaccines. Consequently, even though naturally occurring immunosurveillance may not be sufficiently potent to prevent melanoma, it is possible that the prophylactic augmentation of immunity to melanoma with vaccines may eventually permit this cancer to be prevented in humans. [ABSTRACT FROM AUTHOR]

The article compares the mortality of bullous pemphigoid (BP) in Europe and the US. The reported mortality of BP is much greater in Europe than in the U.S. Mortality 1 year after initiation of therapy ranges from 19% to 41% in Europe but is only 6%12% in the U.S. Studies in the U.S. are generally smaller than those conducted in Europe. However, the consistency of the reports, i.e., mortality in all European studies, is higher than in all U.S. studies, and the clear nature of the end-point (death) suggest the reported differences between European and U.S. studies are real.

A letter to the editor is presented in response to an article on mortality rates among patients with bullous pemphigoid in the January 31, 2002 issue.

A letter to the editor is presented in response to the article "The Problems With Punitive Damages in Lawsuits Against Managed-Care Organizations," by D.M Studdert and T.A. Brennan, previously published in the January 27, 2000 issue of the "New England Journal of Medicine."

Presents an abstract of a study which examines the frequency, significance and possible cause of the presence of tissue-fixed deposits of immunoglobulin in the cytoplasm of epidermal keratinocytes. Design; Subjects; Measurement and results.

A letter to the editor is presented in response to the article "CDKN2A Mutations in Multiple Primary Melanomas," by Jose Monson, Ling Liu, Herbert Brill, Alisa M. Goldstein, Margaret A. Tucker, Lynn From, John McLaughlin, David Hogg, and Norman J. Lassam in the March 26, 1998 issue.

Presents a response by the author to a letter to the editor about epidermal cytoplasmic antigens.

High-dose intravenous immunoglobulin (IVIg) is being increasingly utilized as an off-label therapy for a variety of autoimmune and inflammatory conditions across various specialties. Numerous reports have shown that it is an effective treatment for autoimmune skin blistering disorders. Unlike most therapies for blistering disorders, IVIg is not immunosuppressive and has a favorable side effect profile. This has allowed its use to expand dramatically over the last decade. However, due to the rarity and severity of autoimmune skin blistering diseases, well-designed prospective trials are generally lacking. This work highlights major research developments and the best evidence to date regarding the treatment of autoimmune pemphigus, bullous pemphigoid, mucous membrane pemphigoid, epidermolysis bullosa acquisita, pemphigoid gestationis, and linear IgA dermatosis with IVIg, providing an update on its efficacy, proposed mechanisms of action, side effect profile, and indications for use. [ABSTRACT FROM AUTHOR]

A major obstacle in performing multicenter controlled trials for pemphigus is the lack of a validated disease activity scoring system. Here, we assess the reliability and convergent validity of the PDAI (pemphigus disease area index). A group of 10 dermatologists scored 15 patients with pemphigus to estimate the inter- and intra-rater reliability of the PDAI and the recently described ABSIS (autoimmune bullous skin disorder intensity score) instrument. To assess convergent validity, these tools were also correlated with the Physician's Global Assessment (PGA). Reliability studies demonstrated an intra-class correlation coefficient (ICC) for inter-rater reliability of 0.76 (95% confirdence interval (CI)=0.61–0.91) for the PDAI and 0.77 (0.63–0.91) for the ABSIS. The tools differed most in reliability of assessing skin activity, with an ICC of 0.39 (0.17–0.60) for the ABSIS and 0.86 (0.76–0.95) for the PDAI. Intra-rater test-retest reliability demonstrated an ICC of 0.98 (0.96–1.0) for the PDAI and 0.80 (0.65–0.96) for the ABSIS. The PDAI also correlated more closely with the PGA. We conclude that the PDAI is more reproducible and correlates better with physician impression of extent. Subset analysis suggests that for this population of mild-to-moderate disease activity, the PDAI captures more variability in cutaneous disease than the ABSIS. [ABSTRACT FROM AUTHOR]

Cancer vaccines, while theoretically attractive, present difficult challenges that must be overcome to be effective. Cancer vaccines are often poorly immunogenic and may require augmentation of immunogenicity through the use of adjuvants and/or immune response modifiers. Toll-like receptor (TLR) ligands are a relatively new class of immune response modifiers that may have great potential in inducing and augmenting both cellular and humoral immunity to vaccines. TLR7 ligands produce strong cellular responses and specific IgG2a and IgG2b antibody responses to protein immunogens. This study shows that a new TLR7 ligand, 3M-019, in combination with liposomes produces very strong immune responses to a pure protein prototype vaccine in mice. Female C57BL/6 mice were immunized subcutaneously with ovalbumin (OVA, 0.1 mg/dose) weekly 4×. Some groups were immunized to OVA plus 3M-019 or to OVA plus 3M-019 encapsulated in liposomes. Both antibody and cellular immune responses against OVA were measured after either two or four immunizations. Anti-OVA IgG antibody responses were significantly increased after two immunizations and were substantially higher after four immunizations in mice immunized with OVA combined with 3M-019. Encapsulation in liposomes further augmented antibody responses. IgM responses, on the other hand, were lowered by 3M-019. OVA-specific IgG2a levels were increased 625-fold by 3M-019 in liposomes compared to OVA alone, while anti-OVA IgG2b levels were over 3,000 times higher. In both cases encapsulation of 3M-019 in liposomes was stronger than either liposomes alone or 3M-019 without liposomes. Cellular immune responses were likewise increased by 3M-019 but further enhanced when it was encapsulated in liposomes. The lack of toxicity also indicates that this combination may by safe, effective method to boost immune response to cancer vaccines. [ABSTRACT FROM AUTHOR]

A critical element in improving the potency of cancer vaccines, especially pure protein or peptide antigens, is to develop procedures that can strongly but safely increase their ability to induce immune responses. Here, we describe that encapsulation of a pure protein antigen and interleukin-2 (IL-2) together into liposomes significantly improves immune responses and tumor protection. Groups of C57Bl/6 mice were immunized weekly ×4 with –0.1 mg of ovalbumin (OVA) injected subcutaneously in PBS or encapsulated in liposomes with or without human recombinant IL-2. Control groups included mice immunized to irradiated E.G7-OVA cells (that express ovalbumin), or to PBS. Sera were collected and pooled by immunization group at baseline and at weeks 2 and 4 to measure antibody responses to OVA by ELISA. Splenocytes obtained at week 4 were tested for anti-OVA cellular responses by ELISPOT. Mice were then challenged to a lethal dose of E.G7-OVA cells to measure tumor-protective immunity. IL-2 liposomes caused no detectable toxicity. Antibody, CD8+ T cell, and tumor-protective immune responses were markedly enhanced in mice immunized to OVA + IL-2 in liposomes compared to mice immunized to OVA, either alone or encapsulated into liposomes without IL-2. These results indicate that IL-2 liposomes enhance antibody, cellular, and tumor-protective immune responses to immunization with a soluble protein. This may provide a simple, safe, and effective way to enhance the immunogenicity of vaccines that consist of pure protein antigens. [ABSTRACT FROM AUTHOR]

Macromolecules expressed on the external surface of human keratinocytes in tissue culture were radioiodinated by the lactoperoxidase technique and solubilized by lysing the cells in nonionic detergent. Soluble labeled proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The pattern of proteins expressed by keratinocytes was distinct for these cells. The major labeled components had Mrs of 28K, 40K, 68K, 72K, and 77K. The expression of several macromolecules changed with the length of time the cells were in culture, suggesting that their expression was linked to epidermal differentiation. Lastly, many but not all of the macromolecules expressed on the surface of keratinocytes were selectively and rapidly released into medium by the cell. [ABSTRACT FROM AUTHOR]

The expression of upper cytoplasmic (U-CYT) antigens which are expressed only in the superficial layers of the epidermis and are markers of epidermal cell differentiation in vivo and of basement zone (BMZ) antigens reacting with bullous pemphigoid serum was studied in keratinocytes in tissue culture. The cells were cultured at an acid pH (5.6-5.8) similar to that of skin and without feeder cells, dermal tissue, or collagen. It was found that the expression of U-CYT antigens decreased markedly in culture. These antigens were expressed in 45-65% of epidermal cells prepared from fresh skin, but in only 5-10% of cells which had been grown in primary culture over 1 mo, and in no cells in secondary or tertiary culture. By contrast, BMZ antigens continued to be expressed in culture. These antigens were expressed by 20-35% of epidermal cells prepared from fresh tissue and by 15-35% of keratinocytes in primary, secondary or tertiary culture. These findings indicate that U-CYT and BMZ antigens can be used to type subpopulations of human keratinocytes in suspension, and suggest that the differentiation of these cells in vitro differs from that which occurs in vivo. [ABSTRACT FROM AUTHOR]

We report a method to establish long-term cultures of melanocytes derived from human hair follicles. Normal human scalp was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by collagenase treatment. Hair-follicle cell suspensions were prepared by trypsin/ethylenediamine tetraacetic acid treatment and cultured in a mixture of Eagle's minimum essential medium (supplemented with 12-O-tetradecanoyl-phorbol-13-acetate and cholera toxin) and keratinocyte serum-tree medium. After contaminating fibroblasts and keratinocytes were removed, cells with two distinct morphologies remained. These Included large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and small bipolar cells, which initially were unpigmented, proliferated very rapidly, and became pigmented after the addition of 3-isobutyl-1-methylxanthine to the culture medium, Both cell types were melanocytes as confirmed by electron microscopy and by staining with antibodies to S-100, GD3, and melanosomal antigens. The availability of cultured hair-follicle melanocytes will facilitate investigations of the role of these cells in normal and abnormal hair biology. [ABSTRACT FROM AUTHOR]

Patients with vitiligo have been found to have circulating antibodies to pigment cells. To evaluate the functional activity of these antibodies, a highly sensitive europium release assay was used to compare complement-mediated cytolysis of human melanocytes by sera of 56 patients with vitiligo (20 with active disease, 25 with inactive disease, 11 with unidentified disease activity) and 47 control individuals. Significant melanocyte lysis was mediated by 32 (57%) of the patients with vitiligo but by only three (6%) of the control sera (p < 0.00 1), and by 17 (85%) of 20 patients with active vitiligo versus 11(44%) of 25 patients with inactive disease (p < 0.025). Mean melanocyte lysis by vitiligo sera was 24% versus 6% by control sera (p <0.0001). A subset of 12 vitiligo sera with high titers of cytolytic antibodies to melanocytes (34% mean cytolysis) reacted minimally (<2% mean cytolysis) to a panel of control cells that included human and murine melanomas, human fibroblasts, lung carcinoma, and rhabdomyosarcoma. These findings indicate that antibodies present in patients with vitiligo have the functional ability to selectively kill melanocytes and are more common in active disease. These observations support, but do not prove, the hypothesis that vitiligo is an autoimmune disease and that antipigment cell antibodies have a role in inducing the disease. [ABSTRACT FROM AUTHOR]

Some individuals have basement membrane zone (BMZ) antibodies that react to both the epidermal and dermal side of skin split with 1 M NaCl. To examine the significance of this combined staining pattern, we tested sera from 185 different, sequential, patients with BMZ antibodies for reactivity to normal human skin split with 1 M NaCl. Six sera (3.2%) stained both the epidermal and dermal sides of split skin, 173 (93.5%) stained only the epidermal side, and 6 (3.2%) only the dermal side. All six sera with a combined staining pattern yielded the same pattern when tested with three different specimens of skin, indicating that this pattern is reproducible. By immunoblot analysis, five (83%) of the six combined staining sera reacted to a 160-kD antigen present only in epidermal extracts of normal skin, one reacted in addition to a 230-kD epidermal antigen, and one did not react to either epidermal or dermal extracts. In contrast, five (83%) of the six sera with dermal staining reacted to a 290-kD antigen present only in dermal extracts. Eighteen (90%) of twenty representative epidermal staining sera reacted to a 230-kD epidermal antigen and seven (35%) sera (five with the 230-kD antibody and two without) also reacted to the 160-kD epidermal antigen. Affinity purified antibody to the 160-kD antigen defined by combined staining sera reacted to the BMZ of normal human skin. These results indicate that the combined staining pattern on 1 M NaCl split skin is due to the presence of a distinctive antibody response directed predominantly to a 160-kD BMZ antigen located on the epidermal side of the split skin and to an as yet unidentified BMZ antigen located on the dermal side. [ABSTRACT FROM AUTHOR]

The antigens in normal human skin defined by antibodies in patients with bullous pemphigoid (BP) were studied by Western immunoblots. Eighteen (90%) of 20 BP sera reacted to a 230-kD antigen. Seven (35%) of the sera reacted to a 160-kD antigen. Two of these reacted only to the 160-kD antigen and five also reacted to the 230-kD antigen. Antibodies to the 160-kD antigen were not present in 25 control sera obtained from normal individuals or patients with other bullous diseases. The 160-kD antigen was present in epidermal extracts of four different specimens of normal human skin but not in dermal extracts or extracts of control cells including melanoma, fibroblasts, lung carcinoma, and colon carcinoma. Monospecific sera with antibodies to either the 230-kD or to the 160-kD antigen reacted solely to their respective target antigens, but not to both, in extracts of epidermis that contained both antigens. The 1 60-kD antigen broke down to a 140-kD fragment, while the 230-kD antigen was unchanged in the absence of protease inhibitors. Western blot affinity purified antibody to the 160-kD antigen bound only to the basement membrane zone on the epidermal side of 1M NaC1 split skin. These results indicate that a 160-kD antigen is a normal component of the basement membrane zone of human skin. The antigen is located on the epidermal side of skin split with 1M NaCl. It is a minor BP antigen, antibodies to which are present in some patients with BP. [ABSTRACT FROM AUTHOR]

Our laboratory has recently reported that over 80% of patients with common vitiligo have circulating antibodies to cell-surfacer antigens on normal human melanocytes. The slow growth rate of these cells limits the assays that can be performed for antibody detection. We now have found that the antigens defined by vitiligo sera on melanocytes are also expressed on FF cells, a revertant line of hamster melanoma cells. These antigens can be detected both by indirect immunofluorescence and specific immunoprecipitation assays. The presence of "vitiligo" antigens on hamster FF cells will aid further study of the abnormal immune response in vitiligo. [ABSTRACT FROM AUTHOR]

The expression of bullous pemphigoid (BP) antigen in different locations on the body was estimated from the highest dilution of BP sera giving a positive reaction at a site by indirect immunofluoresconce. Three sera with high titers (640-1280) of BP antibodies against monkey and guinea pig esophagus were reacted with 46 specimens of normal human skin. There was marked variation in the expression of BP antigen in skin obtained from different sites. The greatest expression of BP antigen was in skin obtained from the flexor surfaces of the arm, leg, and thigh. All specimens from these sites reacted with high dilutions (1:640-1:2560) of BP antibodies. BP antigen was expressed least in skin specimens from scalp, face, and extensor surface of the arm. No BP antigen could be demonstrated in 17% of specimens from these locations and the remainder reacted only with low dilutions (1:40-1:160) of BP antibodies. Skin from trunk, knee, and extensor thigh had intermediate amounts of BP antigen. Similar results were seen with all 3 BP sera. Similar reactions were observed in multiple skin specimens obtained from different sites in the same individual. Of note is that the greatest expression of BP antigen was in areas commonly involved in this disease (flexural surfaces), whereas the least was seen in areas (scalp, face, extensor arm) which are rarely involved. The apparent correlation between the expression of BP antigen and the location of lesions in bullous pemphigoid, suggests that BP antigen expression may play a role in the distribution of skin lesions in this disease. [ABSTRACT FROM AUTHOR]

Immunoprecipitation was used to assay for antibodies to normal human melanocytes in the sera of 12 patients with common vitiligo and 12 normal individuals. The procedure is based on the specific immunoprecipitation using protein A-sepharose of antibodies binding to detergent-soluble, radioiodinated macromolecules of normal human melanocytes grown in culture. Antibodies to melanocytes were found in all 12 patients with vitiligo but in none of the normal sera. None of the sera reacted specifically to normal human fibroblasts or to human melanoma cells radioiodinated in a similar manner. These observations suggest that antibodies to melanocyte-associated antigens are present in common vitiligo. [ABSTRACT FROM AUTHOR]

Patients with vitiligo often have antibodies to pigment cells. To examine whether is relation between the presence of such antibodies and disease activity, sera of 24 patients with vitiligo (10 with active and 14 with inactive disease) and 19 normal individual were tested for antibodies to pigment cell surface antigens using a live cell enzyme-linked immunoabsorbent assay. IgG pigment cell antibodies were present in 80% (eight of 10) of patients with active vitiligo but in none of those with inactive disease or in normal individuals. The antibody level of patients with active vitiligo (mean binding index [BI] 3.3 ± 0.59) was significantly higher than in patients with inactive disease (BI 0.96 ± 0.04) or normal individuals (BI 1.0 ± 0.04, p < 0.001). Antibodies present in eight patients with high titers of pigment cell antibodies reacted to three of four pigment cells but to only one of six unrelated cells. These findings indicate that a correlation exists between the incidence and level of pigment cell antibodies and the activity of vitiligo, and support the hypothesis that vitiligo is an autoimmune disease mediated by an immune reaction to pigment cells. [ABSTRACT FROM AUTHOR]

A letter to the editor is presented which responds to the study by I.J. Vergilis and colleagues entitled "Presence and prognostic significance of melanoma-associated antigens CYT-MAA and MNW-MAA in serum of patients with melanoma," previously published in the "Journal of Investigative Dermatology".

Brazil A 2.5-month-old male infant (born by cesarean section, weighing 3650 g) received one dose of DPT vaccine (against diphtheria, whooping cough, and tetanus), one dose of SABIN vaccine, and one dose of Vaxgrip, a vaccine of purified inactivated myxovirus influenza on September 6, 1996. Five days later, he developed symmetric, pruritic, vesiculobullous lesions which began on the trunk and, over five days, progressively spread to the upper and lower extremities, hands, feet, and face. The clinical appearance is illustrated in Fig. 1. Laboratory studies performed at the time revealed a white blood cell (WBC) count of 8800, with 6% eosinophils, and negative serologic tests for syphilis and urine culture. Seventeen days following the onset of the eruption, tense blisters of varying size on erythematous bases, and occasionally in a rosette distribution at the periphery of urticarial plaques, appeared on the hands and feet and then spread to the torso and extremities. There was no mucosal involvement or systemic manifestations. The blister fluid was yellowish and cultured positive for Staphylococcus aureus. The WBC count was 34,600/mm3 with 35% eosinophils. Serum levels of immunoglobulin G (IgG) were 52 mg% (normal, 678–1714 mg%), IgA = 62.1 mg% (normal, 73–422 mg%), IgM = 81.1 mg% (normal, 54–296 mg%), and IgE = 248.7 U/ml (normal, 4.1 U/ml). Three days later, the WBC count had further increased to 55,400 with 48% eosinophils. The biopsy of a trunk lesion showed a subepidermal blister containing large numbers of leukocytes and eosinophils. The cleavage was just below the basal layer. There was a variable degree of hydropic degeneration of the basal cells. In the epidermis, there was slight spongiosis in the spinous layer and exocytosis of leukocytes, neutrophils, and eosinophils. On the epidermal side of the blister, the interpapillary crests were generally reduced, as were dermal papillae on the floor of the blister. The... [ABSTRACT FROM AUTHOR]

Presents a letter to the editor related to melanoma cells, published in the February 1986 issue of the "The Journal of Investigative Dermatology."

Background: In the development of an antimelanoma vaccine, a critical factor is the identification of antigens that induce a strong immune response in humans and that are expressed by melanoma cells in vivo. The aim of this study was to identify candidate antigens for such vaccine.Methods: Sixty-nine patients with surgically resected melanomas (American Joint Commission on Cancer [AJCC] stage III) were immunized with a polyvalent vaccine containing multiple melanoma antigens. Antimelanoma antibodies generated in the patients' sera were used as probes to identify the melanoma antigens that are immunogenic in humans and that are expressed on the tumor tissue in vivo. Such responses were determined by an immunoblotting assay that employed an antigen source prepared from membrane fractions of freshly excised melanoma tissue.Results and Conclusions: Vaccine treatment stimulated antibody responses in 35 (51%; 95% confidence interval [CI] = 39%-63%) of 69 sequentially enrolled patients. The antibodies were directed to one or more antigens with molecular masses of 45, 59, 68, 79, 89, 95, and/or 110 kd. The most immunogenic antigens were p110 and p68, which induced responses in 33% (95% CI = 22%-44%) and 25% (95% CI = 15%-35%) of patients, respectively. Both antigens were commonly expressed on different melanomas, but they were absent on autologous normal tissue and on an unrelated allogeneic tumor. All the above antigens are attractive candidates for vaccine construction. [ABSTRACT FROM AUTHOR]

Human vitiligo is a common depigmenting disorder that is often associated with polyendocrinopathies. The etiology of vitiligo is unknown although there is indirect evidence of a strong association between antimelanocyte antibodies in animal and human vitiligo. We report direct evidence that vitiligo patients' sera containing antimelanocyte antibodies can lyse cultured human melanocytes by both complement activation and antibody-dependent cellular cytotoxicity (ADCC). Melanocyte cytotoxicity was measured using an ethidium bromide/acridine orange viability assay, after 4 and 16 h incubation with sera from vitiligo patients and from normal controls. Significant melanocyte cytotoxicity was seen with vitiligo patients' sera as an antibody source with both complement-mediated cytotoxicity (p < 0.01) and ADCC (p < 0.05) as effector mechanisms. Nine of 11 vitiligo patients' sera produced cytotoxicity by complement-mediated lysis or ADCC. No cytotoxicity was seen using fibroblast targets and vitiligo patients' sera. The lysis of human melanocytes by vitiligo patients' sera by two different effector mechanisms provides direct support for the autoimmune hypothesis of human vitiligo. [ABSTRACT FROM AUTHOR]

The indirect immunofluorescent (IF) reactivity of the pemphigus antibodies in sera of 21 cases of pemphigus vulgaris (PV), 15 cases of pemphigus foliaceus (PF), and 14 cases of Brazilian PF (BPF) was compared on 2 substrates, notably monkey esophagus (ME) sections and guinea pig esophagus (GPE) sections. The IF reactions of the pemphigus antibodies of PV could be distinguished from those of PF or BPF by differences in their reactivity on ME and GPE sections in 98% of the cases examined in this study. In most cases, the pemphigus antibodies of PV cases gave higher titers and stronger IF staining reactions on ME sections, while those of PF and BPF cases gave stronger reactions on GPE sections. In addition, most (13 of 21) PV sera react with the lowest 3-4 cell layers of ME sections, while most (13 of 15) PF sera failed to do so but did react with the upper layers of the sections. Importantly, in 8 of the 50 cases examined by IF, the choice of substrate affected the detectability of the pemphigus antibodies, i.e., 4 of 15 PF and 2 of 14 BPF sera reacted only with GPE and 2 of 21 PV sera reacted only on ME. These research findings point to the need for an evaluation of the combined use of ME and GPE in routine diagnostic studies of pemphigus antibodies. [ABSTRACT FROM AUTHOR]

We have previously described spontaneous but reversible hair loss that clinically and histologically resembles human alopecia areata in a colony of C3H/ HeJ mice. Alopecia areata in humans is associated with antibodies to hair follicles. This study was conducted to determine whether C3H/HeJ mice with hair loss have a similar abnormal antibody response to hair follicles. Eighteen C3H/HeJ mice with alopecia, 12 unaffected littermates, and 15 control mice were examined for circulating antibodies to C3H/HeJ anagen hair follicles by indirect immunofluorescence and against extracts of isolated C3H/HeJ and human anagen hair follicles by immunoblotting. Using both procedures, antibodies to anagen hair follicles were present in all C3H/HeJ mice with alopecia but in none of the control mice. The antibodies were also present in some unaffected C3H/HeJ littermates but were absent in mice of an unrelated strain with inflammatory skin disease and alopecia, indicating that their appearance did not result from the hair loss. These antibodies reacted to hair follicle-specific antigens of 40-60 kDa present in murine and human anagen hair follicles. These antigens were also reactive with human alopecia areata antibodies. Some of the antibodies in both C3H/HeJ mice and humans with alopecia areata reacted to antigens of 44 and 46 kDa, which were identified as hair follicle-specific keratins. This study indicates that C3H/HeJ mice with hair loss have circulating antibodies to hair follicles similar to those present in humans with alopecia areata. These findings confirm that these mice are an appropriate model for human alopecia areata and support the hypothesis that alopecia areata results from an abnormal autoimmune response to hair follicles. [ABSTRACT FROM AUTHOR]