Novel compound heterozygous mutations in the PLEC gene in a neonate with epidermolysis bullosa simplex with pyloric atresia.

Epidermolysis bullosa (EB) is a heterogeneous group of inherited disorders characterized by the blistering of the skin and mucous membranes. Although the molecular basis of EB has been significantly elucidated, the precise phenotypes of the lethal types of EB have not been completely characterized. Herein, we report a severe case of EB with pyloric atresia (PA). The patient was a Japanese boy who not only had skin lesions but also various complications such as PA, dysphagia, hypotonia, infectious keratitis with corneal ulcer, obstructive uropathy and protein-losing enteropathy. Genetic analysis led to the identification of two novel compound heterozygous mutations in the last exon of the plectin (PLEC) gene. Based on this finding, EB simplex with PA was diagnosed. Immunostaining with anti-plectin antibodies revealed truncated plectin proteins lacking the C-terminus in the patient's skin. We also conducted a prenatal diagnosis in subsequent pregnancy. Our report further highlights the crucial role of plectin in many organs and provides valuable information regarding the phenotypes resulting from mutations in the PLEC gene.

Dry preserved multilayered fibroblast cell sheets are a new manageable tool for regenerative medicine to promote wound healing.

This study investigated the therapeutic effects of dry-preserved multi-layered fibroblast cell sheets (dry sheets) on cutaneous ulcers. Dry sheets were prepared by air-drying multi-layered fibroblast cell sheets (living sheets) to cease their life activities. Before in vivo application, we tested the release of growth factors into the medium to examine the mechanisms of dry sheets in wound healing. Vascular endothelial growth factor (VEGF) and hepatocyte growth factor (HGF) were released from both dry and living sheets, while high levels of fibroblast growth factor-2 (FGF-2) and high mobility group box 1 (HMGB1) protein were only from dry sheets. An in vitro fibroblast proliferation assay revealed that the dry sheet eluate significantly enhanced cell proliferation and VEGF and HGF production compared with living sheet eluate. FGF-2-neutralizing antibodies significantly blocked this proliferative response. In wounds created on diabetic mice, the dry sheet-treatment groups using autologous or allogeneic cells showed significantly accelerated wound closure compared with that in the no-treatment group. The storage stability of the dry sheet was better at refrigeration temperature than at room temperature and remained stable for at least 4 weeks. Our data indicated that allogeneic dry sheets represent a promising new tool for regenerative medicine that promotes wound healing.

Freezing of cell sheets using a 3D freezer produces high cell viability after thawing.

In cell therapy, transplanting an appropriate number of cells to the target site is crucial. One way to achieve this is to transplant cell sheets. Transplantation of cell sheets has already been utilized for various diseases in clinical practice. However, reducing the cost of cell sheet utilization is essential so as to facilitate the spread of regenerative medicine. Several ways to reduce costs are available, one of which is the use of allogenic cells. Another alternative is the use of cell sheets, which necessitates the development of methods for freezing cell sheets. This is the first study to report the use of a 3D Freezer for freezing cells. 3D Freezers have been used in the field of food processing and technology for a long time. The 3D Freezer freezes objects using cold air at a uniform temperature from all directions. In this study, we analyzed the cooling speed of human fibroblast sheets in 11 cell preservation solutions using a 3D Freezer and a Program Freezer. The cooling speed was -2 °C per min in the 3D Freezer. Supercooling in 10 cell preservation solutions was lower in the 3D Freezer than in the Program Freezer. Cell viability after freeze-thaw of the cell sheets using 3D Freezer was more than 70% in five cell preservation solutions. The levels of hepatocyte growth factor and transforming growth factor-ß1 were the same not only in the fibroblast sheets frozen using the five cell preservation solutions but also in the non-frozen fibroblast sheets. These results suggest that the 3D Freezer can freeze implantable cell sheets immediately after thawing.

Non-androgen secreting adrenocortical carcinoma in preadolescence: a case report and literature review.

Adrenocortical carcinoma (ACC) is a rare malignancy in childhood. Affected children with ACC mostly present with virilization, but not the pure form of Cushing's syndrome. A 9-year-old Japanese girl was hospitalized, because of the unstable emotions and excessive weight gain. She was diagnosed as having Cushing's syndrome and a left adrenal tumor. The adrenalectomy led to the pathological diagnosis of ACC without metastasis. There was no mutation of PRKACA in the tumor-derived DNA, or p53 in peripheral blood-derived DNA. Testosterone and dehydroepiandrosterone sulfate (DHEA-S) levels were normal throughout the clinical course. On the other hand, these levels were elevated in all five reported cases of preadolescent ACC children with isolated Cushing's syndrome. The exceptional secretory behavior of ACC gave a diagnostic precaution of the rare pediatric cancer.

Hypoxically preconditioned human peripheral blood mononuclear cells improve blood flow in hindlimb ischemia xenograft model.

Transplantation of peripheral blood mononuclear cells (PBMNCs) is a promising therapeutic approach for the treatment of hindlimb ischemia. However, insufficient angiogenesis in ischemic hindlimb after cell transplantation reduces the importance and practicality of this approach. Previously, we demonstrated using mouse models that hypoxic preconditioning augmented the cellular functions of rodent PBMNCs, such as increased cell adhesion capacity and accelerated neovascularization in ischemic hindlimb. To test the clinical application of this therapeutic strategy in this study, we investigated whether the protocol of hypoxic preconditioning, which was established in a condition of 2% O2 for 24 h, can be made available for human PBMNCs (hPBMNCs). In addition, we grafted preconditioned hPBMNCs in a hindlimb ischemia mouse model. Hypoxic preconditioning enhanced cell adhesion capacity and oxidative stress resistance in hPBMNCs. We also observed an up-regulation of platelet endothelial cell adhesion molecule-1 (PECAM-1) in hPBMNCs by hypoxic preconditioning. Furthermore, preconditioned hPBMNCs significantly recovered limb blood flow in ischemic mice after transplantation. These results indicate that our established preconditioning protocol is available for hPBMNCs to effectively reinforce multiple cellular functions. Taken together with our series of study, we believe that this simple but powerful therapeutic strategy will be helpful in curing patients with severe hindlimb ischemia.

Hypoxic preconditioning reinforces cellular functions of autologous peripheral blood-derived cells in rabbit hindlimb ischemia model.

Peripheral blood mononuclear cell (PBMNC) is one of powerful tools for therapeutic angiogenesis in hindlimb ischemia. However, traditional approaches with transplanted PBMNCs show poor therapeutic effects in severe ischemia patients. In this study, we used autograft models to determine whether hypoxic pretreatment effectively enhances the cellular functions of PBMNCs and improves hindlimb ischemia. Rabbit PBMNCs were cultured in the hypoxic condition. After pretreatment, cell adhesion, stress resistance, and expression of angiogenic factor were evaluated in vitro. To examine in vivo effects, we autografted preconditioned PBMNCs into a rabbit hindlimb ischemia model on postoperative day (POD) 7. Preconditioned PBMNCs displayed significantly enhanced functional capacities in resistance to oxidative stress, cell viability, and production of vascular endothelial growth factor. In addition, autologous transplantation of preconditioned PBMNCs significantly induced new vessels and improved limb blood flow. Importantly, preconditioned PBMNCs can accelerate vessel formation despite transplantation on POD 7, whereas untreated PBMNCs showed poor vascularization. Our study demonstrated that hypoxic preconditioning of PBMNCs is a feasible approach for increasing the retention of transplanted cells and enhancing therapeutic angiogenesis in ischemic tissue.

Single-incision laparoscopic splenectomy and splenic autotransplantation for an enlarged wandering spleen with torsion.

A wandering spleen is a rare condition in which the spleen is not located in the left upper quadrant, but instead is found in the lower abdomen or in the pelvic region because of the laxity of the peritoneal attachments. The unusually long pedicle is susceptible to twisting, which can lead to ischemia, and eventually to necrosis. We herein report a case of an enlarged wandering spleen with torsion, successfully treated by single-incision laparoscopic splenectomy and autotransplantation. The transplanted splenic tissues could be identified on a spleen scintigram obtained 3 months after the surgery. Howell-Jolly bodies were not observed in blood specimens. This procedure is able to prevent an overwhelming postsplenectomy infection, and leads to satisfactory cosmetic results.

Hypoxic preconditioning enhances angiogenic potential of bone marrow cells with aging-related functional impairment.

BACKGROUND: Hypoxic preconditioning of bone marrow cells (BMCs) from young healthy individuals can enhance the cells' therapeutic potential. Considering that the response to hypoxia may differ according to the quality of the cells, we assessed the effect of hypoxic preconditioning on BMCs from aged mice and compared the difference in response between BMCs from aged and young mice. METHODS AND RESULTS: BMCs from young (3 months) and aged (20-22 months) mice were subjected to hypoxic preconditioning by culture for 24 h in 2% O2. Compared with BMCs from young mice, those from aged mice showed significantly fewer CD34- or c-kit-positive stem cells, higher expression of p53, and lower telomerase activity. Adhesion, survival and angiogenic potency were also lower in BMCs from aged mice, indicating an aging-related impairment. Hypoxia-preconditioned BMCs from aged mice showed enhanced adhesion, survival, and angiogenic potency with the in vitro assessments, as well as the in vivo implantation into ischemic hindlimbs. All the enhancements by hypoxic preconditioning were comparable between BMCs from aged and young mice, although the angiogenic potential of BMCs with and without hypoxic preconditioning was lower in old mice compared with young mice. CONCLUSIONS: Similar responses to hypoxia by BMCs from both aged and young mice suggest that hypoxic preconditioning could be a useful method of enhancing the angiogenic potential of BMCs.

Congenital segmental dilatation of the duodenum: report of a case.

We herein report what, to our knowledge, is only the fourth known case of segmental dilatation of the duodenum. Antenatal ultrasonography revealed an intraabdominal cyst in the fetus, but the exact location of the segmental dilatation was difficult to find preoperatively. Moreover, even using computed tomography, it was not possible to make a diagnosis prior to surgery. The anatomic characteristics of duodenal dilatation made it difficult to perform the usual resection techniques. In fact, the surgical procedure was different from the previously reported cases. We performed a partial resection of the duodenum followed by a tapering procedure to preserve the ampulla of Vater. The infant had an uneventful postoperative course, and sufficient growth and development has been achieved.

Fatty liver caused by portal vein thrombosis after living donor liver transplantation: a case report.

Portal vein thrombosis (PVT) is a rare complication that occurs after liver transplantation: however, it cannot be ignored as a cause of graft loss and death. We herein report a pediatric case of PVT that caused a fatty change in the graft after living donor liver transplantation. The portal vein was successfully reconstructed using the left great saphenous vein of the same donor. Moreover, the fatty liver recovered after the operation. Our case suggests that the finding of fatty liver is an important marker of PVT and immediate portal reconstruction is performed.

Effect of liver transplantation on multiple bone fractures in an infant with end-stage biliary atresia: a case report.

Osteodystrophy is frequently found in children with chronic cholestatic liver disease. We herein report an end-stage case of biliary atresia that was associated with multiple bone fractures and severe growth retardation. The patient, an 8-month-old female, underwent a living-related liver transplantation and thereafter showed a dramatic improvement in growth and decrease in bone fractures. A correction of the liver function is therefore considered to be a key factor in treating osteodystrophy that is related to chronic cholestatic liver disease. It is also essential to perform liver transplantation at the most appropriate time to enhance and support the growth of these patients.