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.

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.

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.

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.