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The majority of causative variants in familial breast cancer remain unknown. Of the known risk variants, most are tumor cell autonomous, and little attention has been paid yet to germline variants that may affect the tumor microenvironment. In this study, we developed a system called the Consomic Xenograft Model (CXM) to map germline variants that affect only the tumor microenvironment. In CXM, human breast cancer cells are orthotopically implanted into immunodeficient consomic strains and tumor metrics are quantified (e.g., growth, vasculogenesis, and metastasis). Because the strain backgrounds vary, whereas the malignant tumor cells do not, any observed changes in tumor progression are due to genetic differences in the nonmalignant microenvironment. Using CXM, we defined genetic variants on rat chromosome 3 that reduced relative tumor growth and hematogenous metastasis in the SS.BN3IL2Rγ consomic model compared with the SSIL2Rγ parental strain. Paradoxically, these effects occurred despite an increase in the density of tumor-associated blood vessels. In contrast, lymphatic vasculature and lymphogenous metastasis were unaffected by the SS.BN3IL2Rγ background. Through comparative mapping and whole-genome sequence analysis, we narrowed candidate variants on rat chromosome 3 to six genes with a priority for future analysis. Collectively, our results establish the utility of CXM to localize genetic variants affecting the tumor microenvironment that underlie differences in breast cancer risk. Cancer Res; 74(22); 6419–29. ©2014 AACR.

In the event of a radionuclear attack or nuclear accident, the skin would be the first barrier exposed to radiation, though skin injury can progress over days to years following exposure. Chronic oxidative stress has been implicated as being a potential contributor to the progression of delayed radiation-induced injury to skin and other organs. To examine the causative role of oxidative stress in delayed radiation-induced skin injury, including impaired wound healing, we tested a synthetic superoxide dismutase (SOD)/catalase mimetic, EUK-207, in a rat model of combined skin irradiation and wound injury. Administered systemically, beginning 48 hours after irradiation, EUK-207 mitigated radiation dermatitis, suppressed indicators of tissue oxidative stress, and enhanced wound healing. Evaluation of gene expression in irradiated skin at 30 days after exposure revealed a significant upregulation of several key genes involved in detoxication of reactive oxygen and nitrogen species. This gene expression pattern was primarily reversed by EUK-207 therapy. These results demonstrate that oxidative stress has a critical role in the progression of radiation-induced skin injury, and that the injury can be mitigated by appropriate antioxidant compounds administered 48 hours after exposure.

Salen Mn complexes, including EUK-134, EUK-189, and the cyclized analog EUK-207, are synthetic SOD/catalase mimetics that have beneficial effects in many models of oxidative stress. As oxidative stress has been implicated by some investigators in delayed radiation injury, we are investigating whether these compounds can mitigate injury to normal tissues caused by ionizing radiation. This review describes some of this research, focusing on several tissues of interest, including the lung, kidney, and skin. These studies have demonstrated suppression of delayed radiation injury in animals treated with EUK-189 and/or EUK-207. While an antioxidant mechanism of action is postulated, it is likely that the mechanisms of radiation mitigation by these compounds in vivo are complex and may involve non-redox-related mechanisms and differ in various target tissues. It is notable, however, that indicators of oxidative stress are increased in lung and skin radiation injury models, and suppressed by salen Mn complexes. Furthermore, histological, gene expression, and other assessments suggest that salen Mn complex treatment promotes a more normalized tissue environment in irradiated animals, including preservation of microvasculature in the skin and lung. In certain experimental models, including irradiated cell cultures, salen Mn complexes have shown “mito-protective” properties, that is, attenuating mitochondrial injury. In summary, salen Mn complexes could be useful to mitigate delayed radiation injury to normal tissues following radiation therapy, accidental exposure, or radiological terrorism. Optimizing their mode of delivery and other key pharmaceutical properties, as well as gaining more understanding of their mechanism(s) of action as radiation mitigators, are key issues for future study.

Skin grafts from mice expressing human bullous pemphigoid antigen 2 (hBPAG2) in epidermal basement membrane elicit hBPAG2-specific IgG and graft loss in wild-type (Wt) recipients. Graft loss was dependent on CD4+ T cells and correlated with the production and tissue deposition of hBPAG2-specific IgG. To explore the role of CD40/CD40 ligand (CD40L) interaction in this model, Wt mice grafted with transgenic (Tg) skin were treated with hamster anti-CD40L mAb MR1. In contrast to grafted Wt mice treated with equivalent doses of control IgG, 22 of 23 MR1-treated Wt mice did not develop hBPAG2-specific IgG or graft loss foror=60 days. MR1-treated mice also accepted a second Tg skin graft without durable production of hBPAG2-specific IgG or graft loss. Moreover, splenocytes and enriched CD4+ T cells from MR1-treated graft recipients transferred un- or hyporesponsiveness to hBPAG2 to other mice and demonstrated a dominant tolerant effect over cotransferred naive splenocytes following adoptive transfer to Rag2-/- mice. Successful inhibition of hBPAG2-specific IgG production and Tg graft loss following CD40:CD40L co-stimulatory blockade in this model provides opportunities to study mechanisms of peripheral tolerance and generate antigen-specific regulatory CD4+ cells-issues of relevance to patients with pemphigoid as well as individuals undergoing gene replacement therapy for epidermolyis bullosa.

Background Antiepiligrin cicatricial pemphigoid is a mucosal-predominant subepidermal blistering disease associated with an increased relative risk of cancer. In contrast to prior reports showing that anti-laminin (L)-332 autoantibodies are a reliable marker for patients with antiepiligrin cicatricial pemphigoid, a recent report suggested that as many as 40% of patients with bullous pemphigoid (BP) have IgG reactive with this laminin isoform. Objective We sought to determine whether patients with BP possess circulating IgG anti-L-332 autoantibodies. Methods Sera from 100 adults with BP were analyzed by indirect immunofluorescence testing of intact skin, immunoblot studies of human keratinocyte (HK) extracts, and a new L-332 enzyme-linked immunosorbent assay. Sera showing reactivity suggestive of anti-L-332 autoantibodies in these assays were further analyzed in immunoblot studies of HK extracellular matrix and immunoprecipitation studies of biosynthetically radiolabeled HK extracts. Results IgG from all patients with BP bound intact epidermal basement membrane by indirect immunofluorescence microscopy and immunoblotted bullous pemphigoid antigen-1, -2, or both in HK extracts. None of these sera immunoblotted L-332 in HK extracts, although 13 did score above the cut point of a new IgG 4 L-332 enzyme-linked immunosorbent assay (sensitivity = 0.91, specificity=0.98, Youden index=0.89). Further analysis of sera from these 13 patients found: (1) all had IgG that bound the epidermal side of 1 mol/L NaCl split skin by indirect immunofluorescence microscopy; (2) none immunoblotted L-332 purified from HK extracellular matrix; and (3) none immunoprecipitated L-332 from biosynthetically radiolabeled HK extracts. Limitations The basis of false-positive enzyme-linked immunosorbent assay determinations for anti-L-332 IgG among patients with BP is unknown. Conclusion Anti-L-332 autoantibodies remain a reliable marker for patients with antiepiligrin cicatricial pemphigoid.

Anti-epiligrin cicatricial pemphigoid (AECP) is a chronic, autoimmune, subepidermal blistering disease characterized by circulating anti-basement membrane autoantibodies to laminin 5. Recent studies have shown that patients with this form of cicatricial pemphigoid have an increased relative risk for malignant solid tumors. The mechanism underlying this association of AECP and cancer is unknown, but there is accumulating evidence that laminin 5 plays a central role. In this article we report a patient with AECP and co-associated cutaneous T cell lymphoma and summarize all to date reported cases of AECP associated with malignancies. In addition we provide a review of the biology of laminin 5 and its potential role in cancer development.

Pemphigus vulgaris (PV) is an acquired immunobullous disorder. At the early stage of the disease (mucosal PV), patients display only autoimmunity to desmoglein (Dsg) 3 and develop mucosal blisters; while at the later stage of the disease (mucocutaneous PV), patients exhibit non-cross-reactive autoimmunity to both Dsg3 and Dsg1 and acquire cutaneous as well as mucosal blisters. At these two disease stages, Dsg3 autoantibodies exhibit different tissue-binding patterns and pathogenic activities, suggesting that they may recognize distinct epitopes. To test this hypothesis and to investigate the mechanism underlying the disease transition, we studied Dsg3 autoantibody epitopes from mucosal PV patients and patients exhibiting disease transition to mucocutaneous PV. We demonstrated that autoantibodies from the majority of mucosal PV patients target epitopes at the COOH-terminal portion of the Dsg3 ectodomain. Interestingly, only autoantibodies against the Dsg3 NH2-terminal epitope(s) are able to bind human skin. Moreover, we discovered that the intramolecular epitope spreading from Dsg3(87-566) to Dsg3(1-88) is a critical step that precedes the intermolecular epitope spreading from Dsg3 to Dsg1. During disease transition, this mechanism dictates the development of Dsg3 autoantibodies that recognize human skin and lead to expression of cutaneous PV lesions.

Background Anti-epiligrin cicatricial pemphigoid (AECP) is a subepidermal blistering disease characterized by circulating anti-basement membrane autoantibodies to laminin 5. Objective To evaluate the relative sensitivity of immunoblotting and immunoprecipitation techniques for the detection of anti-laminin 5 antibodies, comparative studies using reference laminin 5 antiserum as well as sera from patients with AECP, other immunobullous diseases, and normal volunteers were performed. Methods Equivalent amounts of protein from five different substrates were studied by immunoblotting; immunoprecipitation experiments examined biosynthetically radiolabeled human keratinocyte (HK) extracts. Results HK extracellular matrix (ECM) was the most sensitive substrate for detection of antibodies to laminin 5; extracts of HKs, A-431 cells and HaCat cells represented alternative test substrates (though the later required higher amounts of protein input). Sera from patients with AECP immunoblotted laminin 5 in HK ECM at end titers exceeding those identified in indirect immunofluorescence microscopy studies of 1 M NaCl split skin. Immunoprecipitation studies found that a 10,000-fold dilution of reference laminin 5 antiserum retained the ability to identify laminin 5. Maximal dilutions of sera from AECP patients retaining the ability to immunoprecipitate laminin 5 ranged from 500 to 5,000. Conclusion Immunoprecipitation was the most sensitive technique for detection of anti-laminin 5 antibodies, while immunoblotting of HK ECM or HK extracts represented practical alternatives.

We report the clinical and immunopathologic findings in a cohort of 35 patients with anti-epiligrin cicatricial pemphigoid (AECP). These patients have a mucosal predominant subepithelial blistering disease that is clinically indistinguishable from other forms of cicatricial pemphigoid. The mucosal surfaces of the mouth and eye are most commonly involved. The skin is also involved in most patients, but usually this is less severe than mucosal involvement. AECP is characterized by the binding of circulating IgG autoantibodies to the dermal side of 1M NaCl split human skin on indirect immunofluorescence microscopy. These IgG antibasement membrane autoantibodies target laminin 5, a heterotrimeric protein consisting of alpha3, beta3, and gamma2 subunits. IgG autoantibodies predominantly target the G domain within the alpha subunit. The presence of circulating IgG autoantibodies are specific for the diagnosis of AECP and are not seen in patients with other autoimmune blistering diseases or normal volunteers. Furthermore, we expand on data previously reported on the finding of an increased relative risk for solid cancer in patients with AECP, especially in the first year after blister onset. The majority of cancers documented in a cohort of 35 patients assembled over 12 years of study were adenocarcinomas that were at an advanced stage at their time of detection. This circumstance is thought to account for a high incidence of mortality among AECP patients who develop an associated cancer. AECP patients also demonstrate a significant risk for mortality as a consequence of treatment with systemic immunosuppressives. The current longitudinal study suggests that only a minority of AECP patients go into remission.

This study characterized the specific reactivity, IgG subclass, and complement fixing ability of anti-laminin-5 IgG from 12 patients with anti-epiligrin cicatricial pemphigoid. Circulating IgG from all patients bound the dermal side of 1 M NaCl split skin, immunoprecipitated laminin-5 produced by biosynthetically radiolabeled human keratinocytes, and (in 10 of 12 cases) immunoblotted the laminin-α3 subunit. Analysis of the distribution of IgG subclasses in these patients' circulating anti-laminin-5 autoantibodies by semiquantitative indirect immunofluorescence microscopy using the HP series of subclass-specific monoclonal antibodies revealed: (i) IgG 4 predominant autoantibodies in seven of 11 sera; (ii) IgG 1 and IgG 2 at substantially lower levels in a smaller number of sera; and (iii) no specific IgG 3 anti-laminin-5 autoantibodies in any patients. The same IgG 4 -dominant profile of anti-laminin-5 autoantibodies was found in enzyme-linked immunosorbent assay studies of purified human laminin 5. Direct immunofluorescence microscopy of six skin biopsies from three patients found that IgG 4 was also the predominant subclass of IgG in epidermal basement membranes in situ . Consistent with these findings, sera from 11 of 11 patients with anti-laminin-5 IgG autoantibodies did not fix C3 to epidermal basement membranes in vitro . These immunochemical studies suggest that complement activation does not play a major role in the pathophysiology of this disease and that subepidermal blisters in these patients may develop via a direct effect of anti-laminin-5 IgG itself.

Basement membrane abnormalities have often been observed in kidney cysts of polycystic kidney disease (PKD) patients and animal models. There is an abnormal deposition of extracellular matrix molecules, including laminin-α3,β3,γ2 (laminin-332), in human autosomal dominant PKD (ADPKD). Knockdown of PKD1 paralogs in zebrafish leads to dysregulated synthesis of the extracellular matrix, suggesting that altered basement membrane assembly may be a primary defect in ADPKD. In this study, we demonstrate that laminin-332 is aberrantly expressed in cysts and precystic tubules of human autosomal recessive PKD (ARPKD) kidneys as well as in the kidneys of PCK rats, an orthologous ARPKD model. There was aberrant expression of laminin-γ2 as early as postnatal day 2 and elevated laminin-332 protein in postnatal day 30, coinciding with the formation and early growth of renal cysts in PCK rat kidneys. We also show that a kidney cell line derived from Oak Ridge polycystic kidney mice, another model of ARPKD, exhibited abnormal lumen-deficient and multilumen structures in Matrigel culture. These cells had increased proliferation rates and altered expression levels of laminin-332 compared with their rescued counterparts. A function-blocking polyclonal antibody to laminin-332 significantly inhibited their abnormal proliferation rates and rescued their aberrant phenotype in Matrigel culture. Furthermore, abnormal laminin-332 expression in cysts originating from collecting ducts and proximal tubules as well as in precystic tubules was observed in a human end-stage ADPKD kidney. Our results suggest that abnormal expression of laminin-332 contributes to the aberrant proliferation of cyst epithelial cells and cyst growth in genetic forms of PKD.

We have recently identified patients with a form of cicatricial pemphigoid who have IgG anti-basement membrane autoantibodies directed against epiligrin, a laminin isoform closely related if not identical to laminin 5. These patients' autoantibodies bind the lower lamina lucida of human epidermal basement membrane and immunoprecipitate this laminin isoform from extracts and media of biosynthetically radiolabeled human keratinocytes. Immunoblot studies show that these patients' autoantibodies specifically bind the alpha subunit of this laminin (i.e., laminin subunit alpha 3). We have found no evidence of these autoantibodies in normal volunteers or patients with other bullous skin diseases (including those with other forms of CP). These studies have identified a group of patients with an acquired, autoimmune, subepidermal bullous disorder who have disease-specific autoantibodies directed against the alpha subunit of epiligrin/laminin 5. These findings correlate with prior reports showing that a monoclonal antibody directed against this laminin subunit induces detachment of keratinocytes from extracellular matrix in vitro as well as epidermis from human skin in situ. Together, these findings suggest that this laminin mediates attachment of basal keratinocytes to epidermal basement membrane and that autoantibodies directed against it may be pathogenic. Moreover, recent studies showing that subunits of this laminin isoform are mutated in some patients with Herlitz's junctional epidermolysis bullosa indicate that acquired or inherited abnormalities in this adhesion ligand are associated with skin diseases characterized by separation of epidermis from epidermal BM.

Generalized atrophic benign epidermolysis bullosa (GABEB) is a nonlethal form of junctional epidermolysis bullosa characterized by generalized skin and mucosal blisters that heal with atrophy; other features include alopecia, nail dystrophy, large melanocytic nevi, and autosomal recessive inheritance. The specific aim of this study was to identify an abnormality in epidermal basement membrane adhesion molecules in well characterized GABEB patients that would explain why these subjects' epidermis separates from their epidermal basement membrane. Cryostat sections of nonlesional skin from 8 GABEB patients in 5 different families as well as skin from normal volunteers (controls) were studied by indirect immunofluorescence microscopy using rabbit antiserum directed against a BPAG1 fusion protein or monoclonal antibodies directed against the extracellular domain of BPAG2 (HD18 and 233), epiligrin (P1E1), laminin 5 (GB3), types IV and VII collagen, or integrin subunits alpha 2, alpha 3, beta 1, alpha 6, or beta 4. In these studies, monoclonal antibodies HD18 and 233 showed no reactivity and diminished reactivity, respectively, to the epidermal BM of all GABEB patients. Interestingly, in one patient, the absent or diminished reactivities of monoclonal anti-BPAG2 antibodies were limited to well demarcated portions of an otherwise intact epidermal basement membrane. Moreover, BPAG1, epiligrin, laminin 5, types IV and VII collagen, and all integrin subunits under study were expressed in the same manner in both GABEB and normal human skin. These findings identify an abnormality in the extracellular domain of BPAG2 in the skin of GABEB patients. BPAG2 (type XVII collagen) is a transmembrane, hemidesmosome-associated molecule whose extracellular domain resides at the exact level where blisters develop in the skin of patients with GABEB.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin exposure to ionizing radiation affects the normal wound healing process and greatly impacts the prognosis of affected individuals. We investigated the effect of ionizing radiation on wound healing in a rat model of combined radiation and wound skin injury. Using a soft X-ray beam, a single dose of ionizing radiation (10-40 Gy) was delivered to the skin without significant exposure to internal organs. At 1 h postirradiation, two skin wounds were made on the back of each rat. Control and experimental animals were euthanized at 3, 7, 14, 21 and 30 days postirradiation. The wound areas were measured, and tissue samples were evaluated for laminin 332 and matrix metalloproteinase (MMP) 2 expression. Our results clearly demonstrate that radiation exposure significantly delayed wound healing in a dose-related manner. Evaluation of irradiated and wounded skin showed decreased deposition of laminin 332 protein in the epidermal basement membrane together with an elevated expression of all three laminin 332 genes within 3 days postirradiation. The elevated laminin 332 gene expression was paralleled by an elevated gene and protein expression of MMP2, suggesting that the reduced amount of laminin 332 in irradiated skin is due to an imbalance between laminin 332 secretion and its accelerated processing by elevated tissue metalloproteinases. Western blot analysis of cultured rat keratinocytes showed decreased laminin 332 deposition by irradiated cells, and incubation of irradiated keratinocytes with MMP inhibitor significantly increased the amount of deposited laminin 332. Furthermore, irradiated keratinocytes exhibited a longer time to close an artificial wound, and this delay was partially corrected by seeding keratinocytes on laminin 332-coated plates. These data strongly suggest that laminin 332 deposition is inhibited by ionizing radiation and, in combination with slower keratinocyte migration, can contribute to the delayed wound healing of irradiated skin.

Anti-epiligrin cicatricial pemphigoid is an autoimmune subepithelial blistering disorder of mucous membranes and skin. By immunoblot analyses, sera of most patients with antiepiligrin cicatricial pemphigoid have been shown to react specifically with the alpha3 chain of laminin 5. We describe the first patient with anti-epiligrin cicatricial pemphigoid in whom circulating IgG autoantibodies directed against the beta3 and gamma2-chains of laminin 5 were detected. Treatment with oral prednisolone was beneficial in controlling the disease.

Nitric oxide (·NO) generation from conversion of l-arginine to citrulline by nitric oxide synthase isoforms plays a critical role in vascular homeostasis. Loss of ·NO is linked to vascular pathophysiology and is decreased in chronically inflamed gut blood vessels in inflammatory bowel disease (IBD; Crohn's disease and ulcerative colitis). Mechanisms underlying decreased ·NO production in IBD gut microvessels are not fully characterized. Loss of ·NO generation may result from increased arginase (AR) activity, which enzymatically competes with nitric oxide synthase for the common substrate l-arginine. We characterized AR expression in IBD microvessels and endothelial cells and its contribution to decreased ·NO production. AR expression was assessed in resected gut tissues and human intestinal microvascular endothelial cells (HIMEC). AR expression significantly increased in both ulcerative colitis and Crohn's disease microvessels and submucosal tissues compared with normal. TNF-α/lipopolysaccharide increased AR activity, mRNA and protein expression in HIMEC in a time-dependent fashion. RhoA/ROCK pathway, a negative regulator of ·NO generation in endothelial cells, was examined. The RhoA inhibitor C3 exoenzyme and the ROCK inhibitor Y-27632 both attenuated TNF-α/lipopolysaccharide-induced MAPK activation and blocked AR expression in HIMEC. A significantly higher AR activity and increased RhoA activity were observed in IBD submucosal tissues surrounding microvessels compared with normal control gut tissue. Functionally, inhibition of AR activity decreased leukocyte binding to HIMEC in an adhesion assay. Loss of ·NO production in IBD microvessels is linked to enhanced levels of AR in intestinal endothelial cells exposed to chronic inflammation in vivo.

Cutaneous squamous cell carcinoma (cSCC) is the second most common skin malignancy and it presents a therapeutic challenge in organ transplant recipient patients. Despite the need, there are only a few targeted drug treatment options. Recent studies have revealed a pivotal role played by microRNAs (miRNAs) in multiple cancers, but only a few studies tested their function in cSCC. Here, we analyzed differential expression of 88 cancer related miRNAs in 43 study participants with cSCC; 32 immunocompetent, 11 OTR patients, and 15 non-lesional skin samples by microarray analysis. Of the examined miRNAs, miR-135b was the most upregulated (13.3-fold, 21.5-fold; p=0.0001) in both patient groups. Similarly, the miR-135b expression was also upregulated in three cSCC cell lines when evaluated by quantitative real-time PCR. In functional studies, inhibition of miR-135b by specific anti-miR oligonucleotides resulted in upregulation of its target gene LZTS1 mRNA and protein levels and led to decreased cell motility and invasion of both primary and metastatic cSCC cell lines. In contrast, miR-135b overexpression by synthetic miR-135b mimic induced further down-regulation of LZTS1 mRNA in vitro and increased cancer cell motility and invasiveness. Immunohistochemical evaluation of 67 cSCC tumor tissues demonstrated that miR-135b expression inversely correlated with LZTS1 staining intensity and the tumor grade. These results indicate that miR-135b functions as an oncogene in cSCC and provide new understanding into its pathological role in cSCC progression and invasiveness.

PURPOSE. To report an anti-epiligrin cicatricial pemphigoid (AECP) patient with severe ocular involvement and to provide a practical approach to distinguishing AECP patients from those with other subepidermal blistering diseases. METHODS. Techniques included direct and indirect immunofluorescence microscopy, Western blot and immunoprecipitation studies, as well as interdisciplinary examinations of mucous membranes and skin. RESULTS. This study describes a patient with clinical features of cicatricial pemphigoid, circulating anti-basement membrane zone IgG antibodies, and subepidermal blisters. Histopathology and immunofluorescence analysis suggested the diagnosis of a cicatricial pemphigoid-like type of epidermolysis bullosa acquisita. However, Western blot and immunoprecipitation studies demonstrated that the patient’s serum contained autoantibodies against laminin 5 α3 subunit, leading to the diagnosis of an AECP. CONCLUSIONS. Since patients with AECP have an increased relative risk for malignant tumors, it is important to distinguish this entity within the spectrum of cicatricial pemphigoid patients by additional studies such as Western blot or immunoprecipitation. (Eur J Ophthalmol 2006; 16: 867-9)

We describe a patient with widespread skin lesions and circulating IgG autoantibodies to both type VII collagen and laminin 5. Although autoantibodies to type VII collagen belonged to IgG2, IgG3, and IgG4 subclasses, laminin 5 was targeted exclusively by IgG3 autoantibodies. Interestingly, despite the presence of IgG3 autoantibodies, the patient's serum failed to fix complement to the dermoepidermal junction. In addition, these autoantibodies did not recruit and activate leukocytes or induce dermoepidermal separation in skin sectioned by cryostat. We report a most unusual case of an autoimmune subepidermal blistering with an exclusive IgG3 reactivity to laminin 5.

Anti-epiligrin cicatricial pemphigoid (AECP) is a mucosal-predominant, subepithelial blistering disease characterized by IgG anti-basement membrane autoantibodies to laminin 5 (alpha3beta3gamma2). This and prior studies found that autoantibodies from most patients recognize the alpha-subunit of this laminin isoform. Accordingly, sera from 10 representative patients were tested against prokaryotic recombinants of this polypeptide in epitope mapping studies. cDNAs spanning the full length of the alpha-subunit were generated by PCR, directionally cloned into the pGEX-4T-3 vector, and expressed as glutathione-S-transferase fusion proteins of appropriate size and immunoreactivity. Sera from 9 of 10 AECP patients immunoblotted fusion proteins corresponding to subdomains G2, G3, G4, and G5 at the carboxyl terminus of the laminin 5 alpha-subunit. Serum from 1 patient (and that from normal volunteers) showed no reactivity to any fusion proteins; no sera bound recombinant glutathione-S-transferase alone. Immunoadsorption of patient sera with fusion proteins corresponding to the G domain substantially reduced basement membrane autoantibody titers. IgG from patients with this form of cicatricial pemphigoid recognize the portion of laminin 5 thought to play a key role in promoting keratinocyte adhesion to epidermal basement membrane.

Patients with one form of cicatricial pemphigoid have IgG autoantibodies directed against laminin 5 (alpha3beta3gamma2), an adhesion protein in epidermal basement membrane. Anti-laminin 5 autoantibodies are not found in patients with other skin or mucosal diseases and hence serve as a specific marker for this autoimmune blistering disorder. The demonstration that experimental and patient anti-laminin 5 IgG are pathogenic in animal models indicated that such autoantibodies are central to disease pathophysiology. To investigate further the role of antibody valence and complement in triggering lesion formation in vivo, rabbit anti-laminin 5 (or normal, control) Fab fragments were passively transferred to neonatal BALB/c mice. Mice receiving anti-laminin 5 Fab fragments developed, in a dose-related fashion, circulating anti-basement membrane antibodies, deposits of immunoreactive rabbit IgG (but not murine C3) in epidermal basement membranes, and subepithelial blisters of skin and mucous membranes. Such alterations were not observed in mice treated with equivalent concentrations of normal rabbit Fab fragments. These studies demonstrated that neither complement activation nor cross-linking of laminin 5 in epidermal basement membranes was required for induction of subepidermal blister formation in this animal model of a human autoimmune bullous disease.

Cicatricial pemphigoid is a chronic subepithelial autoimmune blistering disease of mucous membranes and skin. Recently, a subtype of cicatricial pemphigoid with autoantibodies to epiligrin was identified. We describe a Taiwanese patient who presented with ocular, oral, and cutaneous involvement. Direct immunofluorescence showed IgG and C3 deposition in epidermal basement membrane; indirect immunofluorescence showed circulating IgG autoantibodies reactive with the dermal side of 1 mol/L sodium chloride–split skin. Immunoblotting of laminin 5 isolated from the extracellular matrix of cultured human keratinocytes showed no specific reactivity. In contrast, with immunoprecipitation of the conditioned culture media from biosynthetically radiolabeled human keratinocytes, this patient's serum clearly reacted with a series of disulfide-linked polypeptides that correspond to laminin 5(α3β3γ2) and laminin 6(α3β1γ1). This is the first confirmed case of a patient of Chinese ancestry with this disease entity. (J Am Acad Dermatol 2000;42:841-4.)

Background Antiepiligrin cicatricial pemphigoid (AECP) is a chronic autoimmune subepidermal blistering disease characterized by autoantibodies to laminin 5 and clinical features of cicatricial pemphigoid. Only a few patients with AECP have been described to date. The aim of the present study was to analyze the relative frequency of AECP among patients with the clinical phenotype of cicatricial pemphigoid. Observations Serum from 16 consecutive patients with the clinical phenotype of cicatricial pemphigoid were included in this study. Nine patients had circulating IgG autoantibodies by indirect immunofluorescence on sodium chloride–split skin; patients' IgG bound to the epidermal side (n=2), dermal side (n=5), or both sides (n=2) of this test substrate. Interestingly, all 5 cases with dermal binding immunoprecipitated laminin 5 from extracts and media of cultured keratinocytes, and 4 of these serum samples reacted with the α3 subunit of laminin 5 by immunoblotting. None of the patients with dermal binding of IgG demonstrated autoantibodies to type VII collagen. Conclusion Our data suggest that, among patients with the clinical phenotype of cicatricial pemphigoid, AECP may be more frequent than previously assumed.

We report a 39-year-old woman with antiepiligrin cicatricial pemphigoid (CP) in association with non-small cell carcinoma of the lung. At presentation, mucosal lesions showed minimal response to combined systemic immunosuppressive agents. Following the diagnosis of non-small cell lung carcinoma and subsequent treatment with gemcitabine (a second-line chemotherapeutic agent), a significant reduction in both tumour mass and mucosal blistering was observed. Metastatic disease was subsequently associated with recurrent oral erosions. We believe this patient represents the first reported case of paraneoplastic CP.

Bullous pemphigoid antigen 2 (BPAG2) is a 180 kDa type II transmembrane protein associated with hemidesmosomes (HDs) in basal keratinocytes. To better understand how BPAG2 promotes keratinocyte adhesion to epidermal basement membrane (BM), purified IgG against a baculovirus-encoded recombinant was used to localize its carboxyl terminus in human skin by immunogold electron microscopy (IEM). A 2.1-kb BPAG2 cDNA encoding the distal extracellular domain and carboxyl terminus of BPAG2 was used in a baculovirus expression system to create virus that produced a 70-kDa recombinant form of BPAG2 (BV4). BV4 was purified, characterized, and used to raise high-titer specific rabbit IgG. Purified anti-BV4 IgG bound the epidermal side of 1 M NaCl split skin and bound only BPAG2 on immunoblots containing extracts of human keratinocytes. In IEM studies of pre- and post-embedded skin, the distal ectodomain of BPAG2 localized beneath HDs in basal keratinocytes; there was no evidence of BPAG2 beneath melanocytes. Anti-BV4 IgG extensively bound anchoring filaments on the epidermal side of 1 M NaCl split skin; this staining extended along anchoring filaments to their ends. In post-embedded skin, the carboxyl terminus of BPAG2 was localized within the lamina densa, 41 nm (mean of 400 determinations) beneath plasma membranes of basal keratinocytes. BPAG2 thus extends from the intracellular HD plaque of basal keratinocytes to the lamina densa of human epidermal BM.

Patients with anti-epiligrin cicatricial pemphigoid have anti-basement membrane autoantibodies that immunoprecipitate a set of disulfide-linked human keratinocyte polypeptides that co-migrate in sodium dodecyl sulfate polyacrylamide gel electrophoresis with the same complex identified by monoclonal anti-epiligrin (P1E1) and monoclonal anti-nicein/kalinin (GB3) antibodies. In an attempt to further compare the reactivity of patient autoantibodies, P1E1 and GB3, these reagents were tested against the tumor nest basement membranes of 7 papulonodular basal cell carcinomas. These studies found that all of these reagents showed markedly decreased or no reactivity against this substrate. Though their concordant lack of reactivity failed to distinguish these antibodies, these studies did identify a significant defect in papulonodular basal cell carcinoma tumor nest basement membranes. Similarly, integrin subunits alpha 6, beta 4, alpha 3, and alpha 2 as well as bullous pemphigoid antigens 1 and 2 (all potential receptors for the extracellular matrix ligands epiligrin and nicein/kalinin) were also reduced in these tumor nest basement membranes. These findings signify an extensive impairment in the lamina lucida of this neoplasm's basement membrane. Related comparative studies of normal human keratinocytes and transformed human epithelial cell lines (specifically, A-431 and HaCat cells) showed that epiligrin production is markedly decreased in the latter. Decreased expression of epiligrin and nicein/kalinin in papulonodular basal cell carcinoma tumor nest basement membranes in vivo and transformed epithelial cells in vitro indicate that this complex is a transformation-sensitive cell adhesion ligand.

Indirect immunogold electron microscopy studies of cryofixed, freeze-substituted, and post-embedded normal human skin were performed to localize precisely the ultrastructural binding site of circulating autoantibodies from two groups of patients with cicatricial pemphigoid. One group of patients had circulating IgG autoantibodies that bound the dermal side of 1 M NaC1-split skin and immunoprecipitated epiligrin. The other group of patients had circulating IgG autoantibodies directed against the epidermal side of 1 M NaCl-split skin and showed no specific reactivity to any keratinocyte polypeptide by iminunoprecipitation. IgG autoantibodies from all patients with anti-epiligrin cicatricial pemphigoid bound the lowermost aspect of the lamina lucida at its interface with the lamina densa; the greatest staining was seen beneath and beside hemidesinosomes, In contrast, IgG from cicatricial pemphigoid patients whose autoantibodies bound the epidermal side of 1 M NaCl- split skin localized to hemidesmosomes and the junction between hemidesmosoines and the plasma membranes of basal keratinocytes. Although the latter staining pattern is similar to that observed with anti-BPAG2 autoantibodies, sera from our patients with cicatricial pemphigoid did not bind BPAG2 in immunoprecipitation studies of radiolabeled human keratinocyte extracts or show immunoblot reactivity to a fusion protein corresponding to the immunodominant epitope of this polypeptide. These studies demonstrate the following: 1) Autoantibodies from patients with anti-epiligrin cicatricial pemphigoid consistently bind the lower lamina lucida at its interface with the lamina densa; and 2) other patients with the same phenotype may have IgG autoantibodies against yet-unknown epitopes in basal keratinocytes.

The authors investigated 26 regular sexual female partners of 24 men with squamous cell carcinoma of the penis. Neoplasms were diagnosed in 6 (23.1%) of the total of 26 women. STDs were found in 6 (23.1%) women. In 2 the cytologic findings on the cervix were classified as PAP III (PAP IIIa-CIN I, PAP IIIb-CIN III). Squamous cell carcinoma of the uterine cervix was found in 2 women (1 case PAP IV-CIN III; 1 case PAP V-suggestive of invasive carcinoma). Endometrial adenoacanthoma, mammary adenocarcinoma, squamous cell carcinoma of the right hand and non-Hodgkin's malignant lymphoma (centroblastic) were found in 1 case each.

CD200 (OX-2) is a transmembrane glycoprotein that transmits an immunoregulatory signal through the CD200 receptor (CD200R) to attenuate inflammatory reactions and promote immune tolerance. CD200 expression in the skin has not been described previously. We now report that freshly isolated cells of the murine epidermis contain a subpopulation of major histocompatibility complex (MHC) class II-negative, CD3-negative keratinocytes that are CD200-positive. CD200 expression was accentuated in keratinocytes comprising the outer root sheath of the murine hair follicle (HF). When syngeneic skin grafts were exchanged between gender-matched wild-type (WT) and CD200-deficient C57BL/6 mice, significant perifollicular and intrafollicular inflammation was observed, eventually leading to the destruction of virtually all HF (alopecia) without significant loss of the CD200-negative grafts. Minimal and transient inflammation was observed in WT grafts, which persisted long term with hair. There was a 2-fold increase in graft-infiltrating T cells in CD200-deficient skin at 14 d. Alopecia and skin lesions were induced in CD200-deficient hosts by adoptive transfer of splenocytes from WT mice previously grafted with CD200-negative skin, but not from mice grafted with WT skin. Collectively, these results suggest that the expression of CD200 in follicular epithelium attenuates inflammatory reactions and may play a role in maintaining immune tolerance to HF-associated autoantigens.

Patients with a recently identified subepithelial blistering disease have IgG anti-laminin 5 autoantibodies. To determine if such antibodies can be pathogenic in vivo, we developed and characterized rabbit anti-laminin 5 IgG, and passively transferred these antibodies to neonatal mice. Immune rabbit IgG specifically bound human and murine epidermal basement membranes, immunoblotted and immunoprecipitated all laminin 5 subunits from extracts of human and murine keratinocytes, and showed no reactivity to other keratinocyte proteins or epithelial basement membranes that do not contain laminin 5. Mice (n = 29) receiving purified anti-laminin 5 IgG developed, in a dose-related fashion, circulating anti-laminin 5 antibodies, deposits of rabbit IgG and murine C3 in epidermal basement membranes, and subepidermal blisters of skin and mucous membranes. No alterations developed in controls (n = 14) receiving identical amounts of normal rabbit IgG. Passive transfer of anti-laminin 5 (but not control) IgG to neonatal C5- (n = 3) or mast cell-deficient (n = 3) mice produced subepidermal blisters with the same clinical, histologic, and immunopathologic features as those documented in BALB/c mice. These studies establish an animal model of a human blistering disease that can be used to define disease mechanisms and treatment modalities.