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.

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.

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.

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.

Gene transfer into intact plant cells by electroinjection through cell walls and membranes.

Tobacco mosaic virus (TMV) RNA was introduced directly into mesophyll cells of Nicotiana tabacum var. Samsun using electric-field pulses (electroinjection). The injected gene was successfully expressed in the recipient cells as judged by the assay for the virus coat protein using immunofluorescence and by the virus infectivity assay of the homogenate of the electroinjected cells for local lesions on tobacco leaves. As much as 50% of the cells that survived 24 days after electroinjection showed immunofluorescent specks.

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.

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.

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.

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.

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.

The development-associated increase in the hepatic levels of the intrinsic components of the chicken glycine cleavage system.

The hepatic glycine cleavage system in the chicken was examined at different stages of development to study regulation of its biosynthesis. Embryonic levels of polypeptide and mRNA for glycine decarboxylase, one of the three intrinsic components of this enzyme system, were approximately 10% of their adult levels and were rapidly increased following hatching. Those of H-protein, another intrinsic component, were somewhat higher and were more slowly increased throughout the course of development. The change in activities of the two components went with the increase in their polypeptide levels. Moreover, both relative levels of mRNAs for the two components and relative efficiencies of transcription of their genes were not constant during development. T-protein biosynthesis appeared to follow a course similar to that of H-protein. These observations imply that during development, the production of H-protein and T-protein is regulated by similar mechanisms. Further, it is likely that the same mechanisms do not direct the regulatory processes in the biosynthesis of glycine decarboxylase and that embryo and adult livers possess different regulatory modes regarding the biosynthesis of this enzyme system. The shift to the adult mode appeared to occur at birth. The glycine cleavage activity that was three times higher than that in 12-day-old embryo was observed in adult liver mitochondria.

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.

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.