Zelmira Lazarova

Passive Transfer of Anti-Laminin 5 Antibodies Induces Subepidermal Blisters in Neonatal Mice

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.

Anti-epiligrin cicatricial pemphigoid and relative risk for cancer

It is not known whether patients with anti-epiligrin cicatricial pemphigoid (AECP) have an increased risk of malignancy. We calculated the expected numbers of cancers in a cohort of 35 such patients based on respective incidence rates for all cancers in the National Cancer Institutes Surveillance, Epidemiology, and End Results (NCI SEER) Registry. Ten patients in this cohort had solitary solid cancers; eight patients developed cancer after onset of AECP (seven within 14 months). The relative risk (RR) for cancer in this cohort was 6.8 (95% confidence intervals [CI]: 3.3-12.5). AECP seems to be associated with an increased relative risk for cancer.

Anti-epiligrin cicatricial pemphigoid: clinical findings, immunopathogenesis, and significant associations.

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 α3, β3, and γ2 subunits. IgG autoantibodies predominantly target the G domain within the α 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.

Antiepiligrin cicatricial pemphigoid represents an autoimmune response to subunits present in laminin 5 (α3β3γ2)

Sera from 20 patients with antiepiligrin cicatricial pemphigoid were studied to define the specific reactivity of their IgG autoantibodies. IgG from all patients bound exclusively to the dermal side of 1 mol/L NaCl split skin and immunoprecipitated laminin 5 (α3β3γ2) from extracts of human keratinocytes (HKs). Immunoblot studies on purified laminin 5 subunits demonstrated that patient IgG bound α3 alone in 16 patients. In two patients, IgG autoantibodies were directed predominantly to the γ2 subunit, yet showed trace reactivity to α3 as well. Sera from two patients did not immunoblot any laminin 5 subunits, their IgG presumably immunoprecipitating laminin 5 via a conformational epitope. Sera from patients with α3 subunit‐specific IgG immunoprecipitated all subunits of laminin 5 as well as polypeptides of 190 and 200 kDa from the conditioned media of HKs. Preclearance studies and experiments utilizing affinity‐purified patient IgG demonstrated that the latter signified laminin 6 (α3β1γ1) that was bound by cross‐reactive α3 subunit‐specific patient IgG. Sera from patients with γ2 subunit‐specific IgG showed no reactivity to laminin 6, except for faint reactivity provided by low levels of their α3 subunit‐specific IgG. Taken together, these findings indicate that antiepiligrin cicatricial pemphigoid signifies an autoimmune response to subunits present in laminin 5.

IgG anti-laminin-332 autoantibodies are present in a subset of patients with mucous membrane, but not bullous, pemphigoid.

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.

A Synthetic Superoxide Dismutase/Catalase Mimetic EUK-207 Mitigates Radiation Dermatitis and Promotes Wound Healing in Irradiated Rat Skin.

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 h 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 plays a critical role in the progression of radiation-induced skin injury, and that the injury can be mitigated by appropriate antioxidant compounds administered 48 h after exposure.

The 120-kDa soluble ectodomain of type XVII collagen is recognized by autoantibodies in patients with pemphigoid and linear IgA dermatosis

Background Type XVII collagen promotes adhesion of basal keratinocytes to epidermal basement membrane, and is the target of disease in patients with certain inherited or acquired blistering diseases. Two forms of type XVII collagen are produced by cultured human keratinocytes: a 180‐kDa full‐length, transmembrane protein, and a recently identified 120‐kDa soluble fragment that corresponds to its collagenous ectodomain. Objectives We aimed to determine the incidence and pattern of reactivity of autoantibodies against the 180‐ and 120‐kDa forms of type XVII collagen in sera from 40 patients with bullous pemphigoid (BP), pemphigoid gestationis or cicatricial pemphigoid (CP), as well as six patients with linear IgA dermatosis (LAD). Methods Various immunochemical techniques were used. Results These studies found that the 120‐kDa fragment of type XVII collagen was bound by circulating autoantibodies in 13 of 38 patients with BP or CP and all six patients with LAD. While many pemphigoid sera had specific reactivity against one but not both forms of this protein, autoantibodies from patients with LAD bound only the soluble ectodomain. Conclusions These findings are consistent with the presence of both neoepitopes and cross‐reactive epitopes on the ectodomain of type XVII collagen. The finding that sera from patients with LAD showed specific reactivity to epidermal basement membrane suggests that such neoepitopes are present in human skin and that their targeting by autoantibodies may contribute to disease pathogenesis.

Anti-epiligrin cicatricial pemphigoid with IgG autoantibodies to the β and γ subunits of laminin 5

Abstract Anti-epiligrin cicatricial pemphigoid is an autoimmune subepithelial blistering disorder of mucous membranes and skin. By immunoblot analyses, sera of most patients with anti-epiligrin cicatricial pemphigoid have been shown to react specifically with the α3 chain of laminin 5. We describe the first patient with anti-epiligrin cicatricial pemphigoid in whom circulating IgG autoantibodies directed against the β3 and γ2-chains of laminin 5 were detected. Treatment with oral prednisolone was beneficial in controlling the disease. (J Am Acad Dermatol 1999;40:637-9.)

Laminin 332 Deposition is Diminished in Irradiated Skin in an Animal Model of Combined Radiation and Wound Skin Injury

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.

Epiligrin is decreased in papulonodular basal cell carcinoma tumor nest basement membranes and the extracellular matrix of transformed human epithelial cells.

Abstract 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 α6, β4, α3. and α2 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 neoplasms 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.