Regeneration of anterior cruciate ligament by biodegradable scaffold combined with local controlled release of basic fibroblast growth factor and collagen wrapping

The objective of this study was to increase the therapeutic efficacy of anterior cruciate ligament (ACL) surgery using an artificial ligament material developed through a combination of tissue engineering technologies. A poly-L-lactic acid (PLLA) scaffold of plain-woven braid was incorporated with a gelatin hydrogel for controlled release of basic fibroblast growth factor (bFGF) and wrapped with a collagen membrane to allow space for ligament regeneration. For the ACL reconstruction surgery, the PLLA braid scaffold combined with the gelatin hydrogel incorporating bFGF and the collagen wrapping was applied to a tunnel prepared in the femur and tibia of rabbits. The hydrogel was placed in the bone, whereas the portion of the braid inside the joint cavity was wrapped with the membrane. As controls, the PLLA scaffold was applied with the hydrogel or the membrane, or without either material. Bone regeneration in the tunnel and ACL tissue regeneration in the joint cavity were histologically evaluated, and the mechanical strength and collagen content of the regenerated ACL were assessed. When the PLLA scaffold was integrated with both the hydrogel and the membrane, bone and ACL tissues were regenerated in the corresponding sites, in marked contrast to the control groups. Combination of bFGF-controlled release resulted in enhanced mechanical strength of the regenerated ACL tissue. In the joint cavity, it is possible that the local bFGF release inside the membrane enhanced the cell migration and collagen production, and that the surrounding PLLA scaffold results in the biological regeneration of ligament-like tissue. Additionally, significant bone regeneration around the scaffold was observed in the bone tunnel. It is therefore possible that the local controlled release of bFGF near the PLLA braid induced both osseointegration and intrascaffold cell migration in the bone tunnel and joint cavity, respectively, resulting in an overall increase in the mechanical strength of the regenerated ACL.     

Uni-directional differentiation of mouse embryonic stem cells into neurons by the neural stem sphere method

We previously showed that our neural stem sphere (NSS) method promotes the neuronal differentiation of mouse, monkey and human embryonic stem (ES) cells. Here we analyzed changes in expression of marker genes and proteins during neuronal differentiation. When cultured in astrocyte-conditioned medium (ACM) under free-floating conditions, colonies of ES cells formed floating cell spheres, which, within 4 days, gave rise to NSSs. In the spheres, the expression of ES cell marker genes was consistently down-regulated, while expression of an epiblast marker was transiently up-regulated, beginning on day 2, and the expression of neuroectoderm, neural stem cell and neuron markers was up-regulated, beginning on days 3, 4 and 6, respectively. The expression of the marker genes was consistent with that of marker proteins. The time course of expression of these markers in the spheres resembled that of neuronal differentiation from the inner cell mass (ICM) cells of blastula. In contrast, the expression of endoderm, mesoderm, epidermis, astrocyte and oligodendrocyte markers was low and not up-regulated during differentiation. Only a small number of apoptotic cells were present in the spheres. These results suggest that mouse ES cells uni-directionally differentiate into neurons via epiblast cells, neuroectodermal cells and neural stem cells.     

Continuous monitoring of glucose levels in the hepatic vein and systemic circulation during the Pringle maneuver in beagles

Intraoperative continuous glucose monitoring revealed that liver ischemia/reperfusion causes a rapid and profound transition in glucose concentration. We hypothesized that the washout of the glucose stored in the liver leads to a rapid transition in blood glucose concentration. Six female beagles were studied. A portosystemic shunt was established, and the glucose levels in the jugular, hepatic, and portal veins were continuously monitored. All beagles were stabilized for 30 min, and, subsequently, the hepatic artery and portal vein were clamped (the Pringle maneuver). After 30 min of warm hepatic ischemia, the clamp was removed in order to initiate hepatic reperfusion. The endpoint of the experiment was 60 min after the onset of reperfusion. The glucose levels in the abovementioned veins were recorded continuously. The glucose level in the hepatic vein increased 10 min after the start of the Pringle maneuver and was significantly higher than that in the jugular vein and portal vein just before unclamping. The glucose level in the hepatic vein peaked at 2 min after unclamping and that in the portal and jugular veins started to increase after reperfusion. The glucose level in the hepatic vein was significantly higher than that in the jugular and portal veins between 9 min after clamping and 8 min after reperfusion. In conclusion, our study among beagles showed that glucose release from the hepatic vein and sinusoid leads to a rapid elevation in systemic blood glucose levels after liver ischemia/reperfusion. This knowledge might help in the development of new strategies for blood glucose management during hepatectomy.     

Streamlined human antibody generation and optimization by exploiting designed immunoglobulin loci in a B cell line

Monoclonal antibodies (mAbs) are widely utilized as therapeutic drugs for various diseases, such as cancer, autoimmune diseases, and infectious diseases. Using the avian-derived B cell line DT40, we previously developed an antibody display technology, namely, the ADLib system, which rapidly generates antigen-specific mAbs. Here, we report the development of a human version of the ADLib system and showcase the streamlined generation and optimization of functional human mAbs. Tailored libraries were first constructed by replacing endogenous immunoglobulin genes with designed human counterparts. From these libraries, clones producing full-length human IgGs against distinct antigens can be isolated, as exemplified by the selection of antagonistic mAbs. Taking advantage of avian biology, effective affinity maturation was achieved in a straightforward manner by seamless diversification of the parental clones into secondary libraries followed by single-cell sorting, quickly affording mAbs with improved affinities and functionalities. Collectively, we demonstrate that the human ADLib system could serve as an integrative platform with unique diversity for rapid de novo generation and optimization of therapeutic or diagnostic antibody leads. Furthermore, our results suggest that libraries can be constructed by introducing exogenous genes into DT40 cells, indicating that the ADLib system has the potential to be applied for the rapid and effective directed evolution and optimization of proteins in various fields beyond biomedicine.     

5-azacytidine, a chemotherapeutic drug, induces TRAIL-mediated apoptosis in mouse thymocytes in vivo

5-azacytidine (5AzC) is a cytidine analogue with two main effects on cellular conditions; DNA damage, resulting in apoptosis, and DNA hypomethylation, restoring normal growth control and differentiation. However, the molecular mechanism of 5AzC-induced apoptosis is not fully understood. The aim of the present study is to clarify this mechanism in mouse thymocytes in vivo. Ten-week-old, male C57BL/6J mice were injected with 5AzC (100 mg/kg) intraperitoneally, and thymuses were examined for apoptotic changes. In the 5AzC-treated thymus, increases of TUNEL-positive thymocytes and cleaved caspase-3 protein, both biochemical features of apoptosis, were detected. 5AzC-induced apoptosis was observed even in the thymuses of mice deficient in p53, a critical factor in the intrinsic apoptotic pathway, and mice with mutated Fas, a death receptor. Furthermore, levels of p53 and Fas proteins were unchanged in the thymus following 5AzC-treatment in wild-type mice. In the 5AzC-treated thymus, the level of cleaved caspase-8 protein, an initiator of the extrinsic apoptotic pathway, increased with the cleavage of its target protein, Bid. Moreover, the level of TRAIL protein, which induces apoptosis through the cleavage of caspase-8, robustly increased in the thymus treated with 5AzC. In conclusion, the 5AzC-induced apoptosis of thymocytes in vivo is implemented through the extrinsic pathway with the activation of TRAIL.     

Vascular endothelial growth factor in the early stage of skin incision wounds in cyclophosphamide-induced leukocytopenic rats

Wound healing evaluation is important in forensic pathology. We have already shown that vascular endothelial growth factor (VEGF) is produced in the rat skin incision wounds. In this study, we used cyclophosphamide hydrate (CPM) to induce leukocytopenia in rats, and measured VEGF in the skin incision wound area to assess the involvement of leukocytes in the early production of VEGF. Male Sprague–Dawley (SD) rats were intraperitoneally administered CPM 75 mg/kg body weight on day 0 and 5, and dorsal skin incision wounds were made on day 5. One and 3 days after the skin incision, leukocytes counts were determined and skin specimens from the wounds were collected for measurements of total proteins, VEGF proteins, and semi-quantification of VEGF mRNA. VEGF immunohistochemistry and in situ hybridization for VEGF mRNA were also performed. VEGF proteins were smaller in the amount statistically significantly in the 1- and 3-day-old wounds of CPM-induced leukocytopenic rats, whereas VEGF mRNA was increased compared with saline-treated control rats. Immunohistochemically, VEGF was positive in leukocytes and mesenchymal cells including fibroblasts and endothelial cells in the 3-day-old wound of saline-administered control rats, while a few fibroblasts and endothelial cells were positively stained in CPM-administered rats. In situ hybridization showed the localization of VEGF mRNA in mesenchymal cells including fibroblasts and endothelial cells in the 1-day-old wound of CPM-administered rats, whereas saline-administered control rats also showed VEGF mRNA positivity in leukocytes. Our study indicates that leukocytes may be the major source of VEGF in the early stage of the rat skin incision wound.     

Multifocal metanephric adenoma in childhood

Metanephric adenoma is the most commonly occurring member of the metanephric tumor family, which also includes metanephric adenofibroma and metanephric stromal tumor. According to the World Health Organization classification, however, it is not commonly multifocal. Reported herein is the case of a 9-year-old boy with multifocal metanephric adenoma. Histologically, surgical sections showed multifocal proliferation of small rounded and uniform cells with smooth nuclear contours, scant pale-staining cytoplasm, dark-staining nuclei, and inconspicuous nucleoli: the cells were arranged in sheets and acinal, ductal, glomeruloid, and papillary structures. On immunohistochemistry the tumor cells were positive for vimentin, cytokeratins (CAM5.2, AE1/AE3, and CK18), and WT1, but negative for cytokeratin 7 (CK7) and epithelial membrane antigen (EMA). The Ki-67 labeling index was <1%. In addition, cytogenetic analysis indicated a normal karyotype (46XY). Other histologically similar tumors are papillary renal cell carcinoma and nephroblastoma, and it is necessary to distinguish metanephric adenoma from those tumors because of malignancy. In contrast to those tumors, metanephric adenoma has inconspicuous nucleoli, loss of CK7 and EMA expression, and no mitotic figures. Thus, the histological and immunohistochemical features of the present case were compatible with metanephric adenoma.     

Change of mouse CD5(+) B1 cells to a macrophage-like morphology induced by gamma interferon and inhibited by interleukin-4

The in vitro effects of gamma interferon (IFN-gamma) on the mouse CD5(+) B1-cell line, TH2.52, a hybridoma between mouse B lymphoma and mouse splenic B cells that expresses a series of B1 markers, were investigated. A significant number of macrophage-like cells appeared in the cultures of TH2.52 cells exposed to IFN-gamma, these adhering to plastic dishes and exhibiting phagocytic activity. Positive for esterase staining, the macrophage-like cells returned to the original TH2.52 morphology upon removal of IFN-gamma. The change was prevented by treatment with SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase and by transfection of a p38 MAP kinase dominant-negative mutant. Further, interleukin-4 (IL-4) inhibited IFN-gamma-induced phosphorylation of p38 MAP kinase and the appearance of macrophage-like cells. IFN-gamma and IL-4 exhibited contradictory actions on morphological change of CD5(+) B1 cells into macrophage-like cells. Differential regulation of CD5(+) B1 cells by IFN-gamma, a Th1 cytokine, and IL-4, a Th2 cytokine, may have clear immunological significance.     

Overexpression of MDM2 in a sclerosing epithelioid fibrosarcoma: genetic, immunohistochemical and ultrastructural study of a case

Sclerosing epithelioid fibrosarcoma (SEF) is an extremely rare soft-tissue neoplasm. Here, we describe the molecular genetic alterations and histological, immunohistochemical and ultrastructural features of a primary SEF arising in the retroperitoneum. The tumor consisted of uniform small round to ovoid epithelioid cells, arranged in nests and cords and surrounded by a prominent hyalinized collagenous matrix. The tumor cells expressed only vimentin. Ultrastructurally, the tumor cells showed features of fibroblasts, with an abundant rough endoplasmic reticulum in the cytoplasm. Neither p53 gene mutations nor p53 protein overexpression were detected, but more than 70% of all tumor cells showed strong immunoreactivity with murine double minute 2 (MDM2). Our results suggest that MDM2 overexpression is likely to play a role in tumorigenesis in this lesion in p53-dependent or p53-independent pathways. To our knowledge, the present study is the first molecular genetic study of this rare lesion. Further studies will be necessary to clarify the molecular basis of tumorigenesis of this rare lesion.