Wound Trauma Exacerbates Acute, but not Delayed, Effects of Radiation in Rats: Mitigation by Lisinopril.

The goal of this study is to understand and mitigate the effects of wounds on acute radiation syndrome (ARS) and delayed effects of acute radiation exposure (DEARE), for preparedness against a radiological attack or accident. Combined injuries from concomitant trauma and radiation are likely in these scenarios. Either exacerbation or mitigation of radiation damage by wound trauma has been previously reported in preclinical studies. Female WAG/RijCmcr rats received 13 Gy X-rays, with partial-body shielding of one leg. Within 2 h, irradiated rats and non-irradiated controls were given full-thickness skin wounds with or without lisinopril, started orally 7 days after irradiation. Morbidity, skin wound area, breathing interval and blood urea nitrogen were measured up to 160 days post-irradiation to independently evaluate wound trauma and DEARE. Wounding exacerbated morbidity in irradiated rats between 5 and 14 days post-irradiation (during the ARS phase), and irradiation delayed wound healing. Wounding did not alter delayed morbidities from radiation pneumonitis or nephropathy after 30 days post-irradiation. Lisinopril did not mitigate wound healing, but markedly decreased morbidity during DEARE from 31 through 160 days. The results derived from this unique model of combined injuries suggest different molecular mechanisms of injury and healing of ARS and DEARE after radiation exposure.

The microRNA landscape of cutaneous squamous cell carcinoma.

Cutaneous squamous cell carcinoma (cSCC) is a keratinocyte-derived skin tumor. It is the second-most-common cancer affecting the Caucasian population and is responsible for >20% of all skin-cancer-related deaths. The estimated incidence of non-melanoma skin cancer in the USA is >1000000 cases per year, of which roughly 20-30% are squamous cell carcinoma. To better understand and treat this challenging cancer, current research focuses on development of novel strategies to improve the understanding of tumor biogenesis on an individual basis. microRNAs are becoming important biomarkers in the diagnosis, prognosis and treatment of cSCC. This review describes the current knowledge on miRNA expression in cSCC and its role as a biomarker for personalized medicine.

MicroRNA-135b Regulates Leucine Zipper Tumor Suppressor 1 in Cutaneous Squamous Cell Carcinoma.

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.

CXM: a new tool for mapping breast cancer risk in the tumor microenvironment.

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.BN3(IL2Rγ) consomic model compared with the SS(IL2Rγ) 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.BN3(IL2Rγ) 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.

Enhanced survival from radiation pneumonitis by combined irradiation to the skin.

PURPOSE: To develop mitigators for combined irradiation to the lung and skin. METHODS: Rats were treated with X-rays as follows: (1) 12.5 or 13 Gy whole thorax irradiation (WTI); (2) 30 Gy soft X-rays to 10% area of the skin only; (3) 12.5 or 13 Gy WTI + 30 Gy skin irradiation after 3 hours; (4) 12.5 Gy WTI + skin irradiation and treated with captopril (160 mg/m(2)/day) started after 7 days. Our end points were survival (primary) based on IACUC euthanization criteria and secondary measurements of breathing intervals and skin injury. Lung collagen at 210 days was measured in rats surviving 13 Gy WTI. RESULTS: After 12.5 Gy WTI with or without skin irradiation, one rat (12.5 Gy WTI) was euthanized. Survival was less than 10% in rats receiving 13 Gy WTI, but was enhanced when combined with skin irradiation (p < 0.0001). Collagen content was increased at 210 days after 13 Gy WTI vs. 13 Gy WTI + 30 Gy skin irradiation (p < 0.05). Captopril improved radiation-dermatitis after 12.5 Gy WTI + 30 Gy skin irradiation (p = 0.008). CONCLUSIONS: Radiation to the skin given 3 h after WTI mitigated morbidity during pneumonitis in rats. Captopril enhanced the rate of healing of radiation-dermatitis after combined irradiations to the thorax and skin.

Aberrant expression of laminin-332 promotes cell proliferation and cyst growth in ARPKD.

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,ß3,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.

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 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.

Antioxidantes, un paradigma en el tratamiento de enfermedades / Antioxidants, a paradigm for diseases treatment

Los antioxidantes son sustancias que pueden proteger a la célula de los efectos nocivos de los oxidantes o radicales libres y contrarrestan, de una manera directa o indirecta, los efectos de los mismo. Al existir una disminución de los niveles de antioxidantes o una inhibición de las enzimas antioxidantes se desencadena un estrés oxidativo, produciendo daño celular por la oxidación a macromoléculas como proteínas, lípidos y ácido desoxirribonucleico. Estudios previos correlacionan el estrés oxidativo con la patogénesis de numerosas enfermedades como Parkinson, enfermedad de Alzheimer, cáncer e infertilidad. El uso de la administración suplementaria de antioxidantes para prevenir algunas enfermedades ha sido estudiado ampliamente. Las sustancias antioxidantes desempeñan una función fundamental en la prevención de enfermedades crónicas no transmisibles, por lo que es importante el consumo de sustancias antioxidantes no enzimáticas o la innovación de nuevos productos químicos enzimáticos que puedan lograr un efecto positivo en la salud del paciente.

Antioxidants are substances that may protect cells against the effects of free radicals. Low levels of antioxidants, or inhibition of the antioxidant enzymes, cause oxidative stress and may damage occurs causing oxidize of the proteins, lipids and Deoxyribonucleic Acid. Previous studies correlate the relationship between oxidative stress and the pathogenesis of numerous diseases such as Parkinson’s disease, cancer and infertility. The use of antioxidant supplements to prevent some diseases has been studied. Antioxidants play an important role in prevention of chronic diseases. The consumption of such compounds can be formed either by enzymatic, non enzymatic or the innovation of chemical compounds with enzymatic antioxidant activity supporting the positive effects on patient health.

Salen Mn complexes mitigate radiation injury in normal tissues.

Salen Mn complexes, including EUK-134, EUK-189 and a newer cyclized analog EUK-207, are synthetic SOD/catalase mimetics that have beneficial effects in many models of oxidative stress. As oxidative stress is implicated in some forms of 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 therapeutic interest, namely kidney, lung, skin, and oral mucosa. 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 differ in the various target tissues. Indicators of oxidative stress are increased in lung and skin radiation injury models, and suppressed by salen Mn complexes. The role of oxidative stress in the renal injury model is unclear, though EUK-207 does mitigate. In certain experimental models, salen Mn complexes have shown "mito-protective" properties, that is, attenuating mitochondrial injury. Consistent with this, EUK-134 suppresses effects of ionizing radiation on mitochondrial function in rat astrocyte cultures. In summary, salen Mn complexes could be useful to mitigate delayed radiation injury to normal tissues following radiation therapy, accidental exposure, or radiological terrorism. Optimization of their mode of delivery and other key pharmaceutical properties, and increasing understanding of their mechanism(s) of action as radiation mitigators, are key issues for future study.

Transient anti-CD40L co-stimulation blockade prevents immune responses against human bullous pemphigoid antigen 2: implications for gene therapy.

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 for >or=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.

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.

Orf-induced immunobullous disease: A distinct autoimmune blistering disorder.

BACKGROUND: Many complications have been reported after orf infection, including lymphadenopathy, secondary bacterial infection, and erythema multiforme. Rare associations with papulovesicular eruptions, including a bullous pemphigoid-like eruption, have also been described. OBJECTIVES: Our purpose was to clinically, histologically, and immunologically characterize two cases of orf-induced blistering disease, and to determine whether this condition represented a novel disease entity distinct from known immunobullous diseases. METHODS: Two patients were clinically described and skin biopsy specimens were collected for routine histology, direct immunofluorescence studies, and polymerase chain reaction analysis to detect orf viral DNA. Patients' sera were assessed for autoantibodies by indirect immunofluorescence studies using normal-appearing human salt-split skin, by Western blot analysis using keratinocyte extracts, dermal extracts, and recombinant type VII collagen, and immunoprecipitation studies of extracts from biosynthetically radiolabeled human keratinocytes. RESULTS: Two distinctive cases of severe, diffuse blistering eruptions after orf infection are described. In one patient, orf virus DNA was detected in the inciting orf lesion, but not in blistered skin, ruling out disseminated orf infection as a cause of the blisters. In both cases, histology revealed subepidermal blisters with mixed inflammatory cell infiltrates containing neutrophils and eosinophils, direct immunofluorescence microscopy studies demonstrated IgG and C3 deposited at the dermoepidermal junctions of perilesional skin, and indirect immunofluorescence studies demonstrated circulating antibasement membrane IgG that bound the dermal side of salt-split skin. Extensive immunoblot and immunoprecipitation studies failed to reveal a consistent, identifiable autoantigen. LIMITATIONS: We describe only two cases. The autoantigen recognized by circulating autoantibodies was not identified. CONCLUSIONS: Orf-induced immunobullous disease is a unique disease entity that is clinically and immunologically distinct from bullous pemphigoid, epidermolysis bullosa acquisita, and other known immunobullous conditions.

A widening perspective regarding the relationship between anti-epiligrin cicatricial pemphigoid and cancer.

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 presenting in a radiation portal.

We report a case of mucocutaneous pemphigus vulgaris in a patient with squamous cell carcinoma of the lung. The cutaneous involvement was limited to the skin within his therapeutic radiation portal. The diagnosis of pemphigus vulgaris was confirmed by histopathology and immunologic studies. Direct immunofluorescence demonstrated IgG and C3 in the intercellular spaces and indirect immunofluorescence was positive on monkey esophagus at a titer of 1:160. Enzyme-linked immunosorbent assay of the patient's serum detected autoantibodies only to desmoglein (Dsg)3, with no reactivity to Dsg1. Immunomapping of perilesional skin from the irradiated field illustrated decreased Dsg1 expression compared with a control sample from an area that was not exposed to radiation. This case provides support for the Dsg compensation hypothesis and may also suggest a mechanism by which irradiation may induce skin lesions.

Increased arginase activity and endothelial dysfunction in human inflammatory bowel 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-alpha/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-alpha/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.

IgG autoantibodies to type VII collagen and an exclusive IgG3 reactivity to the laminin alpha3 chain in a patient with an autoimmune subepidermal blistering disease.

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.

Role of intramolecular epitope spreading in pemphigus vulgaris.

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.