Hydrogen sulfide donor micelles protect cardiomyocytes from ischemic cell death.

Hydrogen sulfide, an important gaseous signaling molecule in the human body, is known to protect cardiomyocytes from ischemia, a condition characterized by insufficient oxygen supply to the cells. Here we show that a nanosized H2S donor micelle releases H2S intracellularly and prevents cardiomyocyte apoptosis in an in vitro ischemia model.

Regenerative medicine approach as an alternative treatment to islet transplantation.

Islet transplantation is considered to be one of the most promising treatment for type I diabetes mellitus (TID). Development of the Edmonton protocol opened the possibility of insulin independence for the patients with TID. However, there is the problem of the donor shortage. Herein we have discussed recent approaches to overcome the problem. It is neccessary to develop a new cellular source for donor islets and to achieve a high engraftment rate. One advantage in TID therapy is that allogeneic islet transplantation is allowed to avoid autoimmunity. That opens broad candidates for the beta-cell source. To achieve a high engraftment rate, is several attempts have sought to develop an appropriate site for transplantation and to modify beta-cells for long-term survival. It is also important to achieve early onset of blood perfusion after transplantation by prevascularization of the islets in vitro. These multiple approaches will bring a milestone in diabetes therapy.

Early intra-amniotic gene transfer using lentiviral vector improves skin blistering phenotype in a murine model of Herlitz junctional epidermolysis bullosa.

Mutations of the LAMB3 gene cause a lethal form of junctional epidermolysis bullosa (JEB). We hypothesized that early intra-amniotic gene transfer in a severe murine model of JEB would improve or correct the skin phenotype. Time-dated fetuses from heterozygous LAMB3(IAP) breeding pairs underwent ultrasound guided intra-amniotic injection of lentiviral vector encoding the murine LAMB3 gene at embryonic day 8 (E8). Gene expression was monitored by immunohistochemistry. The transgenic laminin-ß3 chain was shown to assemble with its endogenous partner chains, resulting in detectable amounts of laminin-332 in the basement membrane zone of skin and mucosa. Ultrastructually, the restoration of ∼60% of hemidesmosomal structures was also noted. Although we could correct the skin phenotype in 11.9% of homozygous LAMB3(IAP) mice, none survived beyond 48 h. However, skin transplants from treated E18 homozygous LAMB3(IAP) fetuses maintained normal appearance for 6 months with persistence of normal assembly of laminin-332. These results demonstrate for the first time long-term phenotypic correction of the skin pathology in a severe model of JEB by in vivo prenatal gene transfer. Although survival remained limited due to the limitations of this mouse model, this study supports the potential for treatment of JEB by prenatal gene transfer.

The expression of nectin-1alpha in normal human skin and various skin tumours.

BACKGROUND: A novel cell-cell adhesion system that consists of nectin and afadin has been identified at cadherin-based cell-cell adherens junctions. Nectin is a Ca2+-independent homophilic and heterophilic cell adhesion molecule that belongs to the immunoglobulin superfamily. Nectin has recently been shown to serve as an alpha-herpesvirus entry and cell-cell spread mediator. In spite of the ubiquitous expression of nectin-1alpha, its detailed localization in human skin has not been examined so far. OBJECTIVES: To investigate the localization of nectin-1alpha in normal human skin and the alteration of its expression in malignant skin tumours. METHODS: Immunohistochemistry was employed to determine the expression of nectin-1alpha and other adhesion molecules. RESULTS: We detected nectin-1alpha in normal human epidermis, follicles and eccrine ducts. Nectin-1alpha was colocalized with E-cadherin at cell-cell adherens junctions of the epidermis. The concentration of the nectin-afadin system at cell-cell adherens junctions was reduced in the early stage of malignant transformation of keratinocytes, such as in basal cell carcinomas and squamous cell carcinomas, where the cadherin-catenin system was preserved. Nectin-1alpha at cell-cell adherens junctions was reduced in human epithelial cancer cells located at the advancing border of the tumour. CONCLUSIONS: Our results showed that nectin-1alpha is located at cell-cell adherens junctions in human skin and that reduction of nectin-1alpha at cell-cell adherens junctions may be involved in the invasion of squamous cell tumours.

Immunohistochemical study of inducible nitric oxide synthase in skin cancers.

BACKGROUND: Nitric oxide (NO) is synthesized from the amino acid L-arginine by NO synthase (NOS). Experimental evidence suggests that increased express of inducible NOS (iNOS), which is an NOS isoform and calcium independent, is related to various pathological processes, such as inflammation and cancer. METHODS: In this study, we used immunohistochemistry to investigate iNOS expression in a series of basal cell carcinomas (BCC), Bowen's disease, squamous cell carcinomas (SCC), extramammary Paget's disease (EPD) and metastatic tumors of the skin. RESULTS: Only 1 of 16 BCC cases was positive for iNOS and the intensity of staining was weak. In most of the 10 cases of Bowen's disease, iNOS was weakly expressed and there was a wide range in the percentage of positive tumor cells. Twelve of the 16 cases of SCC were positive for iNOS and the extent of positivity was greater than in Bowen's disease. Two of the 7 cases of EPD were positive for iNOS, and 12 of the 15 cases of metastatic cancer were positive. Well-differentiated adenocarcinomas were diffusely positive, whereas poorly-differentiated ones showed strong and heterogeneous staining. CONCLUSIONS: These results indicated that the expression of iNOS may reflect the proliferation of tumor cells and that a heterogeneous distribution of iNOS may correlate with a wide variety of biological behavior of tumor cells.

Immunohistochemical expression of cyclooxygenase-2 in skin cancers.

BACKGROUND: Cyclooxygenase (COX), also known as prostaglandin endoperoxide synthase, catalyses the conversion of arachidonic acid to prostanoids. There are two different isoforms of COX, referred to as COX-1 and COX-2. Overexpression of COX-2 has been demonstrated in various neoplasms, such as experimentally promoted tumors, gastrointestinal cancers and breast tumors. METHODS: In this study, we used immunohistochemistry to investigate COX-2 expression in a series of basal cell epitheliomas (BCE), Bowen's disease, squamous cell carcinomas (SCC) and metastatic tumors of the skin. RESULTS: Four of 16 BCE showed a positive reaction for COX-2 and the adenoid type of BCE was the most strongly positive. In Bowen's disease, the extent of positive staining for COX-2 was even higher than that in BCE. Eleven of 15 SCC showed a positive reaction for COX-2 and the pattern of staining was heterogeneous with more intense staining in the center of the tumor nests. In metastatic tumors, the percentage of COX-2-positive tumor cells and the intensity of their staining was low compared with Bowen's disease and SCC. CONCLUSIONS: These results indicate that the intensity of COX-2 staining and its heterogeneous distribution are related to the degree of cellular differentiation and the various phenotypes of tumor cells, but the extent of COX-2 staining did not correlate with the degree of malignancy.

An ultrastructural study of lipodystrophia centrifugalis abdominalis infantilis, with special reference to fibrous long-spacing collagen.

Lipodystrophia centrifugalis abdominalis infantilis (LCAI) in a 2.5-year-old Japanese girl is reported. Clinically she had a curved cutaneous depression with a slightly elevated erythematous border on the left abdomen beyond the left groin. The regional lymph node was palpable. Histologic examination showed a decrease of the fat, an inflammatory infiltrate mainly composed of lymphocytes, and septal fibrosis in the subcutaneous tissue. Immunohistochemical analysis found that infiltrating lymphocytes were mainly positive against CD4. Ultrastructurally nuclei in the adipocytes were crescent shaped and located toward the periphery of the cell. Some banded structures with a periodicity of 150 nm, and with intraperiodic bands, referred to as fibrous long-spacing collagen (FLSC), were observed in the septal area of the fatty tissue. These results indicated that FLSC was correlated with the breakdown of fibrillar collagens in LCAI.

Immunohistochemical localization of ornithine decarboxylase in skin tumors.

BACKGROUND: Ornithine decarboxylase (ODC) plays an important role in the biosynthesis of polyamines. Induction of ODC and polyamine synthesis has been demonstrated in neoplastic tumors and is thought to be related to the degree of malignancy. METHODS: In this study, we investigated a series of basal cell epitheliomas (BCE), Bowen's disease, squamous cell carcinomas (SCC), and metastatic tumors of the skin using an antibody against ODC for immunohistochemistry. RESULTS: Eight of 12 cases of BCE failed to show a positive reaction for ODC. In Bowen's disease, 5 of 13 cases diffusely showed positive reaction for ODC. Fourteen of 15 cases of SCC showed ODC expression, the intensity of which was decreased in the peripheral layer. At higher magnification, the distribution of ODC in the positive SCC cases showed granular and heterogenous patterns. Ten of 14 cases of metastatic skin tumors exhibited positive reactions, and well-differentiated adenocarcinomas tended to show more strongly positive than poorly-differentiated ones. CONCLUSIONS: These results support the conclusion that the intensity and the incidence of positive immunohistochemical staining for ODC correlate with the degree of cellular differentiation, and furthermore, that heterogenous distribution of ODC staining may be associated with heterogeneity of tumor cells.

Remission of antiepiligrin (laminin-5) cicatricial pemphigoid after excision of gastric carcinoma.

PURPOSE: To describe a case of antiepiligrin cicatricial pemphigoid with unusual ocular manifestations and its remission after surgical removal of gastric carcinoma. METHODS: We describe a 61-year-old Japanese man with antiepiligrin cicatricial pemphigoid. RESULTS: He presented with conjunctival injection and discharge preceded by a 6-month period of erosive lesions in the oral mucosa and the truncal skin. An advanced gastric carcinoma was found and his serum immunoprecipitated laminin-5. Despite topical treatment with betamethasone, ofloxacin, and artificial tear solutions, serious symblepharon along the Meibomian line developed with little shortening of the inferior conjunctival sac. Following radical gastrectomy, the ocular and cutaneous lesions turned completely quiet. CONCLUSION: The present case differed from past cases by lacking inferior conjunctival sac shortening and by showing erosive lesions solely at the mucocutaneous junctions. The ocular involvement in this case correlated very well with the severity of gastric carcinoma.

An ultrastructural study of pigmented purpuric dermatitis with special reference to fibrous long-spacing collagen.

A case of pigmented purpuric dermatitis (PPD) in a Japanese man aged 59 years is reported with an interesting ultrastructural finding. Clinically, the lesions, which consisted of telangiectatic puncta and pigmentation, were irregular in shape and occurred predominantly on the lower legs without pruritus. Histologically, lymphocytic perivascular infiltrates and extravasation of red blood cells were observed in the papillary dermis. Ultrastructurally, endothelial cells with ovoid nuclei showed swelling and the lumen of the capillary became narrowed. Several banded structures, so-called fibrous long-spacing collagen (FLSC), were observed in the cytoplasm. They were spindle shaped, about 5 microm in length, and showed crossbands of 300-nm-wide intervals with fine intraperiodic bands. These structures were not observed in dermal connective tissue and fibroblasts. These results suggested that FLSC was synthesized in endothelial cells rather than being phagocytosed by endothelial cells, which might be helpful in investigating the etiology of PPD.

Enhanced expression of SCF in the dermis is a prognostic factor for the regression of urticaria pigmentosa.

Urticaria pigmentosa (UP) is a disorder of mast cell proliferation that occurs in cutaneous tissue. Most patients whose skin manifestations appear in infancy or childhood, experience a resolution of the disease by adolescence. In order to elucidate the relationship between mast cell character and UP prognosis, we used an immunohistochemical approach to examine the expression of stem cell factor (SCF) and c-Kit in the skin of patients with UP. The results revealed intercellular SCF expression throughout the dermis in improving cases. On the other hand, in cases with a tendency to worsen, dermal SCF was recognized only partially or not at all. Regardless of the clinical course, intracellular SCF immunoreactivity of the entire epidermis increased in cases of child onset UP. The c-Kit expression of mast cells in all UP patients showed no relation to clinical features. These findings suggest that SCF in the dermis promotes the differentiation of mast cells infiltrating in UP, and might be an attractive candidate to induce the remission of UP.

Calcitonin gene-related peptide upregulates melanogenesis and enhances melanocyte dendricity via induction of keratinocyte-derived melanotrophic factors.

It has recently been shown that cutaneous axon terminals and epidermal melanocytes make contact via chemical synapses in human skin and that calcitonin gene-related peptide (CGRP) induces melanocyte proliferation. To further clarify the effect of neuropeptides on the biology and morphology of melanocytes, especially with respect to melanogenesis and melanocyte dendricity, organ cultures of normal human skin and cultured melanocytes were exposed to various neuropeptides present in intraepidermal nerve endings. Of the neuropeptides examined, skin exposed to CGRP in organ culture showed increases in melanocyte number, epidermal melanin content, melanosome number, and degree of melanization. CGRP alone had no significant effect on melanogenesis of cultured melanocytes, whereas the addition of medium conditioned by CGRP-stimulated keratinocytes (CGRP-KCM) induced melanogenesis as indicated by biochemical assays of tyrosinase activity and melanin content. Furthermore, CGRP-KCM significantly enhanced melanocyte dendricity, a crucial factor affecting epidermal pigmentation. These findings suggest that keratinocytes produce and secrete some melanotrophic factors following stimulation with CGRP, which modulate growth, melanin synthesis, and dendricity of melanocytes. These data demonstrate intimate interactions between the cutaneous nervous system and melanocytes within the epidermal environment.

Phytol is a novel tumor promoter on ICR mouse skin.

Phytol is a branched, long-chain aliphatic alcohol which has various biological effects. In this study, we examined phytol as a tumor promoter in a mouse skin initiation-promotion model, and compared its promotion activity with that of 12-O-tetradecanoyl phorbol-13-acetate (TPA). Female ICR mice, 7 weeks of age, were initiated with 100 microg of 7,12-dimethylbenz(a)anthracene, and were then topically promoted twice a week for 16 weeks with 100 mg of phytol or with 2.5 microg of TPA. In this model 95% of animals treated with phytol developed skin tumors within 16 weeks. The average number of lesions per mouse treated with phytol was significantly lower than that in mice treated with TPA, and this significant difference continued up to 16 weeks after the end of promotion treatment. Characterization of hyperplasia 48 h after topical application of agents showed that epidermal thickness and vertical thickness following topical application of phytol were significantly increased compared with vehicle controls, but were significantly smaller than in animals treated with TPA. Ornithine decarboxylase (ODC) activity following topical application of phytol was increased in a dose-dependent manner and showed a weak, delayed induction (which was maximal 11-12 h after treatment) as compared with the case of TPA. The specific binding of [3H]phorbol-12,13-dibutyrate (PDBU) by JB6 cells was not inhibited by phytol at concentrations up to 1 mM. These results indicate that phytol has a weak tumor promoter activity compared to TPA and is a non-TPA-type tumor promoter in this model of mouse skin carcinogenesis.

Antiepiligrin (laminin 5) cicatricial pemphigoid associated with an underlying gastric carcinoma producing laminin 5.

Although bullous pemphigoid and cicatricial pemphigoid are sometimes associated with malignancy, it remains uncertain whether such an association is pathogenetically related or just a coincidence attributable to the advanced age of the patients. We report a 61-year-old patient with antiepiligrin (laminin 5) cicatricial pemphigoid (AeCP) associated with an advanced gastric carcinoma. The gastric carcinoma cells in this patient were shown to produce laminin 5 by immunofluorescence microscopy, and the patient's serum contained autoantibodies directed against laminin 5 on immunoprecipitation. Furthermore, the blistering symptoms and the titre of antibasement membrane zone antibodies coordinately changed with the resection and subsequent relapse of the gastric cancer. These observations suggest that the gastric carcinoma producing laminin 5 may have induced the production of autoantibodies to this laminin, which were pathogenic to the skin and mucous membranes in this patient. This report demonstrates a link between this autoimmune subepithelial blistering disease and malignancy. It is of interest and potential great importance to examine other cases of AeCP for such a potential association.

A specific cutaneous lesion revealing myelodysplastic syndrome.

We report on an 86-year-old man with an ulcerated nodule on his left lower leg. Peripheral blood examination and bone marrow findings were compatible with the refractory anemia with an excess of blasts in transformation (RAEB-T) which is typical of the myelodysplastic syndrome (MDS). Because histological examination showed an infiltration of atypical cells of myeloid origin, this lesion was diagnosed as a specific lesion of MDS. Sometimes, only a subjective symptom, such as a skin lesion, precedes the diagnosis of MDS.

Extent of laminin-5 assembly and secretion effect junctional epidermolysis bullosa phenotype.

Junctional epidermolysis bullosa (JEB) is an autosomal recessive skin blistering disease with both lethal and nonlethal forms, with most patients shown to have defects in laminin-5. We analyzed the location of mutations, gene expression levels, and protein chain assembly of the laminin-5 heterotrimer in six JEB patients to determine how the type of genetic lesion influences the pathophysiology of JEB. Mutations within laminin-5 genes were diversely located, with the most severe forms of JEB correlating best with premature termination codons, rather than mapping to any particular protein domain. In all six JEB patients, the laminin-5 assembly intermediates we observed were as predicted by our previous work indicating that the alpha3beta3gamma2 heterotrimer assembles intracellularly via a beta3gamma2 heterodimer intermediate. Since assembly precedes secretion, mutations that disrupt protein-protein interactions needed for assembly are predicted to limit the secretion of laminin-5, and likely to interfere with function. However, our data indicate that typically the most severe mutations diminish mRNA stability, and serve as functional null alleles that block chain assembly by resulting in either a deficiency (in the nonlethal mitis variety) or a complete absence (in lethal Herlitz-JEB) of one of the chains needed for laminin-5 heterotrimer assembly.

Gamma 2 chain of laminin-5 is recognized by monoclonal antibody GB3.

Herlitz junctional epidermolysis bullosa is an autosomal recessive disorder characterized by generalized blistering at the lamina lucida of the cutaneous basement membrane. The monoclonal antibody GB3 has been used as a diagnostic probe because of its lack of reactivity in patient skin. The antigen recognized by GB3 has been identified as laminin-5, a glycoprotein consisting of three subunits (alpha 3, beta 3 and gamma 2). To identify the laminin-5 protein chain that contains the epitope recognized by GB3 and to determine if chain assembly is required for antibody recognition, we expressed a gamma 2 protein constructed from a full-length gamma 2 cDNA. Radioimmunoprecipitation of the culture medium from 293 cells revealed that both GB3 and anti-gamma 2 polyclonal antibodies were capable of directly precipitating recombinant gamma 2 without coprecipitation of other proteins. In immunodepletion experiments, each antibody removed most of the protein that was reactive with the other antibody. The epitope recognized by GB3 is present only when the complex is in the native conformation because GB3 reacted only with the non-reduced laminin-5, but not the reduced laminin-5 in immunoblots. Moreover, because GB3 reacted with laminin-5 of SCC25 cells (gamma 2 in the heterotrimer) but not recombinant gamma 2 in 293 cells (gamma 2 alone) during indirect immunofluorescence staining, this epitope may be dependent upon a less stable conformation of gamma 2. We conclude that GB3 recognizes the gamma 2 chain of laminin-5 and that the epitope is entirely contained in the native form of the gamma 2 chain.

The assembly of laminin-5 subunits.

Laminin-5 is a heterotrimer composed of alpha 3, beta 3, and gamma 2 chains, produced by keratinocytes and the human squamous cell carcinoma line (SCC-25), and is one of the candidate proteins for the genetic lesion in junctional epidermolysis bullosa. Two-dimensional SDS-polyacrylamide gel electrophoresis (first dimension, nonreducing conditions; second dimension, reducing conditions) revealed that the immunoprecipitated laminin-5 from a SCC-25 cell fraction consisted of alpha 3, beta 3, and gamma 2 monomers, a beta 3 gamma 2 heterodimer, and an alpha 3 beta 3 gamma 2 heterotrimer. The presence of the beta 3 gamma 2 heterodimer, but not heterodimers containing an alpha 3 chain and any of the other chains, was suggestive of assembly of laminin-5 proceeding from a beta 3 gamma 2 heterodimer to an alpha 3 beta 3 gamma 2 heterotrimer. We showed, by cotransfection experiments using full-length recombinant beta 3 and gamma 2 chains in a human cell line devoid of endogenous laminin-5, that stable heterodimers can be formed in the absence of alpha 3 chain expression. In the SCC-25 cell fraction, the alpha 3 monomer pool was the smallest of the monomers. Pulse-chase experiments using the cell fraction also indicated that the heterotrimer was assembled after a 10-min pulse and was nearly absent after a 24-h chase. These results are consistent with the synthesis of alpha 3 being limiting for heterotrimer assembly, with rapid association of the alpha 3 chain with beta 3 gamma 2 heterodimers to form complete heterotrimers. Treatment with tunicamycin reduced the size of each of the laminin-5 subunits, indicating that all chains are glycosylated, but that N-linked glycosylation is not necessary for chain assembly and secretion.