Yoshiya Asano

Monoclonal antibody to human midkine reveals increased midkine expression in human brain tumors

We produced a rat IgG2a monoclonal antibody against the carboxyl terminal region of human midkine (MK), a novel growth factor. This monoclonal antibody was used in immunohistochemical studies to compare the expression of MK, proliferating cell nuclear antigen (PCNA) and p53 protein in 133 primary brain tumors and 21 carcinoma metastases to the central nervous system. Approximately half of the glioblastomas multiforme (GBMs) (19/32), medulloblastomas (8/14), primitive neuroectodermal tumors (PNETs) (5/11), breast carcinoma metastases (Br-Mts) (6/10) and lung carcinoma metastases (L-Mts) (5/11) as well as some astrocytomas (2/14) had tumor cells that expressed MK; however, oligodendrogliomas, ependymomas, schwannomas, meningiomas, and pituitary adenomas did not express MK. The values of the PCNA-labeling index were statistically higher in GBMs, medulloblastomas, PNETs, Br-Mts, and L-Mts that expressed MK than in those that did not (Wilcoxon rank-sum test, p < 0.05). There was no correlation between MK and p53 protein in all tumor types. Normal and non-neoplastic brain tissues were negative for MK, PCNA, and p53 protein. We conclude that primary and metastatic tumors of the brain express MK and that the MK expression in brain tumors may depend, in part, on the proliferating potential.

Age-Related Accumulation of non-Heme Ferric and Ferrous Iron in Mouse Ovarian Stroma Visualized by Sensitive non-Heme Iron Histochemistry

Sensitive non-heme iron histochemistry—namely, the perfusion-Perls method and perfusion-Turnbull method—was applied to study the distribution and age-related accumulation of non-heme ferric iron and ferrous iron in mouse ovary. Light and electron microscopic studies revealed that non-heme ferric iron is distributed predominantly in stromal tissue, especially in macrophages. By contrast, the distribution of non-heme ferrous iron was restricted to a few ovoid macrophages. Aged ovaries exhibited remarkable non-heme iron accumulation in all stromal cells. In particular, non-heme ferrous iron level was increased in stromal tissue, suggestive of increased levels of redox-active iron, which can promote oxidative stress. Moreover, intense localization of both non-heme ferric and ferrous iron was observed in aggregated large stromal cells that were then characterized as ceroid-laden enlarged macrophages with frothy cytoplasm. Intraperitoneal iron overload in adult mice resulted in non-heme iron deposition in the entire stroma and generation of enlarged macrophages, suggesting that excessive iron accumulation induced macrophage morphological changes. The data indicated that non-heme iron accumulation in ovarian stromal tissue may be related to aging of the ovary due to increasing oxidative stress.

Differentiation of Mouse Induced Pluripotent Stem Cells Into Alveolar Epithelial Cells In Vitro for Use In Vivo

Alveolar epithelial cells (AECs) differentiated from induced pluripotent stem cells (iPSCs) represent new opportunities in lung tissue engineering and cell therapy. In this study, we modified a two‐step protocol for embryonic stem cells that resulted in a yield of ∼9% surfactant protein C (SPC)+ alveolar epithelial type II (AEC II) cells from mouse iPSCs in a 12‐day period. The differentiated iPSCs showed morphological characteristics similar to those of AEC II cells. When differentiated iPSCs were seeded and cultured in a decellularized mouse lung scaffold, the cells reformed an alveolar structure and expressed SPC or T1α protein (markers of AEC II or AEC I cells, respectively). Finally, the differentiated iPSCs were instilled intratracheally into a bleomycin‐induced mouse acute lung injury model. The transplanted cells integrated into the lung alveolar structure and expressed SPC and T1α. Significantly reduced lung inflammation and decreased collagen deposition were observed following differentiated iPSC transplantation. In conclusion, we report a simple and rapid protocol for in vitro differentiation of mouse iPSCs into AECs. Differentiated iPSCs show potential for regenerating three‐dimensional alveolar lung structure and can be used to abrogate lung injury.

Autolysin amidase of Listeria monocytogenes promotes efficient colonization of mouse hepatocytes and enhances host immune response

Listeria monocytogenes is an intracellularly growing pathogen which is able to infect and to spread from cells to cells. It produces several virulence factors required for invasion and intracellular niche colonization. Endogenous peptidoglycan hydrolases which are important for survival of bacteria have been shown to be involved in pathogenesis. An autolysin amidase (Ami)-deficient mutant of L. monocytogenes (Δami) is attenuated in virulence as evidenced by a reduction in mortality of infected mice. We showed that Ami is not essential for bacterial growth and protein secretion. Histopathological analysis suggests that Ami promotes bacterial colonization of hepatocytes. By using cultured eukaryotic cells, we present evidence that a critical function of Ami in pathogenesis is to promote an efficient listerial adherence and internalization into mouse hepatocytes. Simultaneously, the peptidoglycan hydrolase activity of Ami linked to the release of immunologically active cell wall components enhances production of tumor necrosis factor (TNF)-α and interleukin 6. In the early phase of infection, interferon-γ and TNF-α production of Δami-infected mice is significantly less than that of wild-type controls, suggesting a contribution of Ami to enhance the host innate immune response to listerial infection.

Single Axon Branching Analysis in Rat Thalamocortical Projection from the Anteroventral Thalamus to the Granular Retrosplenial Cortex

The granular retrosplenial cortex (GRS) in the rat has a distinct microcolumn-type structure. The apical tufts of dendritic bundles at layer I, which are formed by layer II neurons, co-localize with patches of thalamic terminations from anteroventral (AV) thalamic nucleus. To further understand this microcolumn-type structure in the GRS, one of remaining questions is whether this structure extends into other layers, such as layers III/IV. Other than layer I, previous tracer injection study showed that AV thalamic nucleus also projects to layer III/IV in the GRS. In this study, we examined the morphology of branches in the GRS from the AV thalamus in single axon branch resolution in order to determine whether AV axon branches in layer III/IV are branches of axons with extensive branch in layer I, and, if so, whether the extent of these arborizations in layer III/IV vertically matches with that in layer I. For this purpose, we used a small volume injection of biotinylated dextran-amine into the AV thalamus and reconstructing labeled single axon branches in the GRS. We found that the AV axons consisted of heterogeneous branching types. Type 1 had extensive arborization occurring only in layer Ia. Type 2 had additional branches in III/IV. Types 1 and 2 had extensive ramifications in layer Ia, with lateral extensions within the previously reported extensions of tufts from single dendritic bundles (i.e., 30–200 μm; mean 78 μm). In type 2 branches, axon arborizations in layer III/IV were just below to layer Ia ramifications, but much wider (148–533 μm: mean, 341 μm) than that in layer Ia axon branches and dendritic bundles, suggesting that layer-specific information transmission spacing existed even from the same single axons from the AV to the GRS. Thus, microcolumn-type structure in the upper layer of the GRS was not strictly continuous from layer I to layer IV. How each layer and its components interact each other in different spatial scale should be solved future.

Suppression of starvation-induced autophagy by recombinant toxic shock syndrome toxin-1 in epithelial cells.

Toxic shock syndrome toxin-1 (TSST-1), a superantigen produced from Staphylococcus aureus, has been reported to bind directly to unknown receptor(s) and penetrate into non-immune cells but its function is unclear. In this study, we demonstrated that recombinant TSST-1 suppresses autophagosomal accumulation in the autophagic-induced HeLa 229 cells. This suppression is shared by a superantigenic-deficient mutant of TSST-1 but not by staphylococcal enterotoxins, suggesting that autophagic suppression of TSST-1 is superantigenic-independent. Furthermore, we showed that TSST-1-producing S. aureus suppresses autophagy in the response of infected cells. Our data provides a novel function of TSST-1 in autophagic suppression which may contribute in staphylococcal persistence in host cells.

Transplantation of three‐dimensional artificial human vascular tissues fabricated using an extracellular matrix nanofilm‐based cell‐accumulation technique

We have established a novel three‐dimensional (3D) tissue‐constructing technique, referred to as the ‘cell‐accumulation method’, which is based on the self‐assembly of cultured human cells. In this technique, cells are coated with fibronectin and gelatin to construct extracellular matrix (ECM) nanofilms and cultured to form multi‐layers in vitro. By using this method, we have successfully fabricated artificial tissues with vascular networks constructed by co‐cultivation of human umbilical vein‐derived vascular endothelial cells between multi‐layers of normal human dermal fibroblasts. In this study, to assess these engineered vascular tissues as therapeutic implants, we transplanted the 3D human tissues with microvascular networks, fabricated based on the cell‐accumulation method, onto the back skin of nude mice. After the transplantation, we found vascular networks with perfusion of blood in the transplanted graft. At the boundary between host and implanted tissue, connectivity between murine and human vessels was found. Transmission electron microscopy of the implanted artificial vascular tubules demonstrated the ultrastructural features of blood capillaries. Moreover, maturation of the vascular tissues after transplantation was shown by the presence of pericyte‐like cells and abundant collagen fibrils in the ECM surrounding the vasculature. These results demonstrated that artificial human vascular tissues constructed by our method were engrafted and matured in animal skin. In addition, the implanted artificial human vascular networks were connected with the host circulatory system by anastomosis. This method is an attractive technique for engineering prevascularized artificial tissues for transplantation. Copyright

Nonheme ferric and ferrous iron accumulation in macrophages of rats and cats

Abstract Perfusion-Turnbull and perfusion-Perls methods, which were recently developed in our laboratory, demonstrated that macrophages densely accumulated nonheme iron in both ferrous (Fe(II)) and ferric (Fe(III)) forms. In the spleen treated with bacterial lipopolysaccharide (LPS), which stimulates macrophages to produce cytotoxic nitric oxide (NO), the macrophages in the red pulp heavily loaded with nonheme Fe(II) and Fe(III) survived, while iron-free lymphocytes and less heavily iron-loaded macrophages in the white pulp and marginal zone were seriously affected. These findings suggested that dense nonheme iron shelter could protect macrophages from a NO-rich environment.

Transplantation of artificial human lymphatic vascular tissues fabricated using a cell‐accumulation technique and their engraftment in mouse tissue with vascular remodeling

Transplantation of engineered tissues with microvascular structure is advancing towards therapeutic application to improve the flow of blood and/or lymphatic fluids. In lymphatic disorders, transplantation of tissue‐engineered lymphatic grafts can be an ideal treatment for draining excessive lymphatic fluid. In this study, we examined the transplantation of 3‐dimensional artificial human lymphatic network tissue (AHLT) fabricated by the cell accumulation technique into the subcutaneous tissue and fascia of mice. At 2 weeks after transplantation, the AHLT showed engraftment of artificial lymphatic vessels immunopositive for human CD31 and human podoplanin. Notably, we also observed the generation of blood vessel‐like structure comprising endothelial cells immunopositive for human CD34 and mural‐like cells immunopositive for human CD90 and αSMA, which were considered as myofibroblasts. In the fabrication of AHLT in vitro, the sporadic emergence of human CD34‐positive/Prox‐1‐negative sites was observed, followed by the formation of blood vessel‐like structure in the graft within 7 days after transplantation. The fine structure of engrafted AHLT observed by transmission electron microscopy showed that the engrafted artificial lymphatic vessels possess the specific structures of native lymphatic capillaries such as loose interendothelial connections and anchoring filaments. In contrast, blood vessel‐like structure showed tight interendothelial connections, thick basement membranes, and layers of mural‐like cells, which resemble small blood vessels. These results suggested the remodelling of artificial lymphatic network to form blood vessel‐like structure associated with mural‐like cells along with AHLT fabrication and engraftment.

Changes in non-heme iron histochemistry in the ischemic brains of cats and Mongolian gerbils

Abstract We studied the changes in non-heme iron histochemistry in the ischemic brains of the cat and Mongolian gerbils. For the histochemical visualization of non-heme ferric (Fe(III)) and ferrous (Fe(II)) iron, perfusion-Perls and perfusion-Turnbull methods were used. Deeply anesthetized animals were transcardially perfused with the solution containing 1% HCl, 1% potassium ferrocyanide (perfusion-Perls method) or 1% potassium ferricyanide (perfusion-Turnbull method), and 10% formalin. In the non-ischemic animals, perfusion was performed in the cephalic direction through the abdominal aorta or through the ascending aorta within 2 min after thorachotomy and cardiotomy—incision of the right auricle and left ventricle. In the ischemic animals, perfusion was performed through the ascending aorta 10, 20 and 30 min after cardiotomy. Frozen sections of the brain were treated for 3,3′-diaminobenzidine tetrahydrochloride (DAB) intensification of Perls and Turnbull reaction product. It was demonstrated that cytoplasmic Fe(III) was reduced to Fe(II) in oligodendrocytes of ischemic brains, and that Fe(II) was concentrated in the neuronal and glial cell nuclei regardless of the presence or absence of blood supply impairment.