清水 一彦

Several complications may occur following iliac bone grafting, one of the common sites for autologous bone harvesting. Of these, it is difficult to localize the damage in neurological complications due to the presence of several nerves in a similar distribution area with variations among individuals. To minimize these complications, conventional clinical anatomical studies using normal human cadavers have estimated the theoretical neurological damage area corresponding to the surgical intervention area. We report a case of neuromuscular damage in a 93-year-old woman who had an iliac crest defect after a bone graft, based on the virtual and physical dissections with histological confirmations.In this study, the patient was confirmed to have severe neuromuscular complications with major complications including a hernia protruding through the iliac defect. One of the two ilioinguinal nerves was extracted with the hernia sac through the iliac defect, and its distal part was completely damaged. The iliohypogastric nerve, which was far from the defect foramen, also showed remarkable fibrosis and demyelination, affected by the degeneration of the transversus abdominis muscles.The present anatomical findings show that the area of eventual neuromuscular damage should be estimated to larger than the conventionally predicted area of direct nerve damage, which is usually concomitant with the surgical intervention area.

N-methyl-N-nitrosourea (MNU) is known to cause apoptosis of photoreceptor cells and changes in retinal pigment epithelium (RPE). However, the changes in choriocapillaris, which nourishes photoreceptor cells by diffusing tissue fluid through RPE, have not been reported in detail. Therefore, we studied the ultrastructural transformation in and around the choriocapillaris to characterize the interdependence between choriocapillaris and surrounding tissue components in a mouse model. Seven-week-old male C57BL/6 mice were given a single intraperitoneal injection of MNU (60 mg/kg of body weight). Perfusion-fixed eyeballs were examined chronologically using immunohistochemistry and electron microscopy until the photoreceptor cells were lost. Sequential ultrastructural changes were observed in photoreceptor cells, RPE, Bruch's membrane, choriocapillaris, and choroidal melanocytes after an MNU injection. The lumens of the choriocapillaris narrowed following dilation, and the vascular endothelium showed structural alterations. When the photoreceptor cells were completely lost, the choriocapillaris appeared to be in a recovery process. Our results suggest that transport abnormality through Bruch's membrane and structural changes in the choroid might have influenced the morphology of choriocapillaris. The thin wall of the choriocapillaris appears to be the cause of the vulnerability with its altered morphology.

FOXC2, a forkhead transcriptional factor, is a candidate gene for congenital heart diseases and lymphedema-distichiasis syndrome and yellow nail syndrome; however, there are no reports on Foxc2 and the development of the lung. We have identified lung abnormalities in Foxc2-knockout embryos during investigation of cardiac development. The aim of this study was to clarify the morphological characteristics during lung development using ICR-Foxc2 knockout lungs. Mutant fetuses at embryonic days 10.5-18.5 were obtained from mating of Foxc2(+/-) mice and then analyzed. Notably, Foxc2-knockout lungs appeared parenchymatous and much smaller than those of the wild-type littermates. In the Foxc2 knockout lungs, the capillary beds remained distant from the alveolar epithelium until the late stages, the number of type2 alveolar cells per alveolar progenitor cell was lower and the type1 alveolar cells were thicker in Foxc2 knockout mice. In contrast, Foxc2 expression was only detected in the mesenchyme of the lung buds at E10.5, and it disappeared at E11.5 in Foxc2-LacZ knockin mice. Furthermore, the expression of Lef1 was significantly inhibited in E11.5 lungs. All of these results suggest that the abnormalities in Foxc2 knockout mice may involve maldifferentiation of alveolar epithelial cells and capillary vessel endothelial-alveolar epithelial approach as well as lymph vessel malformation. This is the first report about relationship between Foxc2 and lung development. This animal model might provide an important clue for elucidating the mechanism of lung development and the cause of respiratory diseases.

T cell activation is regulated by a balance between phosphorylation and dephosphorylation that is under the control of kinases and phosphatases. Here, we examined the role of a non-receptor-type protein tyrosine phosphatase, PTP-PEST, using retrovirus-mediated gene transduction into murine T cells. Based on observations of vector markers (GFP or Thy1.1), exogenous PTP-PEST-positive CD4(+) T cells appeared within 2 days after gene transduction; the percentage of PIP-PEST-positive cells tended to decrease during a resting period in the presence of IL-2 over the next 2 days. These vector markers also showed much lower expression intensities, compared with control cells, suggesting a correlation between the percent reduction and the low marker expression intensity. A catalytically inactive PIP-PEST mutant also showed the same tendency, and stepwise deletion mutants gradually lost their ability to induce the above phenomenon. On the other hand, these PIP-PEST-transduced cells did not have an apoptotic phenotype. No difference in the total cell numbers was found in the wells of a culture plate containing VEC- and PIP-PEST-transduced T cells. Moreover, serine/threonine kinase Akt, but not the anti-apoptotic molecules Bc1-2 and Bcl-XL, reversed the phenotype induced by PTP-PEST. We discuss the novel mechanism by which Ala interferes with PIP-PEST. (C) 2014 Elsevier Inc. All rights reserved.

Background: Vasohibin-2 (VASH2) has been identified as an endogenous and vascular endothelial growth factor (VEGF)-independent angiogenic factor that is highly expressed in tumor cells. In the present study, we aimed to determine whether pre-existing vascular changes can be used to predict tumor transformation as benign or malignant. We sought to characterize microvascular changes and tumor development in the intestinal tract of Apc(Min/+) mice and Apc(Min/+)/Vash2(-/-) mice. Methods: Apc(Min/+) mice provide a unique orthotopic model for the development of spontaneous adenomatous polyposis and subsequent carcinomas, a phenomenon termed the adenoma-carcinoma sequence. Apc(Min/+) mice were mated with Vash2(-/-) mice with a mixed C57BL/6 background and the resulting pups were screened for the Min mutation and for the Vash2(-/-) gene by PCR. Intestinal tumors from Apc(Min/+) mice and Apc(Min/+)/Vash2(-/-) mice were removed and either frozen or epon-embedded for subsequent analyses. For 3-dimensional imaging using confocal laser-scanning microscopy and transmission electron microscopy, cryosections were made, and immunofluorescent staining for various markers was performed. Results: We found that structural abnormalities in tumor vessels from benign tumors resembled those in malignant tumors. In addition, a novel angiogenic factor, vasohibin-2 (VASH2) protein, was detected around tumor blood vessels in late-stage adenomas and adenocarcinomas, but was absent from early-stage adenomas in Apc(Min/+) mice. Tumors used to examine endogenous VASH2 (derived from CMT93 colon carcinomas) were less vascularized in Vash2(-/-) mice and were more regular than those seen in wild-type (WT) mice. In addition, tumors in Vash2(-/-) mice were smaller than those in WT mice. Furthermore, cross-breeding of mice homozygous for a deletion of Vash2 with mice heterozygous for the APC mutation resulted in animals that showed a significant decrease in the number of polyps in the small intestine. Conclusion: We propose that VASH2 may modulate the onset of tumors in the gastrointestinal tract by regulating tumor angiogenesis.

A single intravitreal injection of erythropoietin (EPO) (50 ng/eye) or phosphate-buffered saline was administered to 5-week-old Sprague-Dawley rats at the onset of diabetes mellitus (DM) to determine and evaluate the protective effect of EPO on retinal microvessels. DM was induced by an intraperitoneal injection of streptozotocin (STZ; 60 mg/kg body weight). Morphological changes in microvessels in flat retinal preparations were evaluated during the subsequent 4 weeks by three-dimensional imaging of all blood vessels stained with fluorescein isothiocyanate-conjugated tomato lectin, following immunofluorescence techniques. No marked differences were observed in the shape or density of retinal vessels and the number of retinal capillary branches of the four groups [control, EPO, DM, and DM/EPO] up to 4 weeks after STZ administration. We also observed unique type IV collagen-positive filamentous structures that lacked both cellular elements and blood circulation (lectin-/type IV+ acellular strands), suggesting regressed vessel remnants. The lectin-/type IV+ acellular strands were detected soon after the onset of DM in the diabetic rats, and the number of these structures increased in the DM group (P < 0.01). A single intravitreal injection of EPO caused a significant reduction in the number of lectin-/type IV+ acellular strands to levels observed in the control group. However, the lectin-/type IV+ acellular strands were observed in the central area of the retina near the optic disc in all four groups. Intravitreal injection of EPO resulted in downregulation of the EPO receptor, vascular endothelial growth factor (VEGF), and VEGF receptor at 4 weeks. We conclude that EPO may play a primary role against the progression of diabetic retinopathy by reducing blood vessel degeneration at a very early disease stage. (C) 2012 Elsevier Ltd. All rights reserved.

Background: Non-alcoholic fatty liver disease (NAFLD), including non-alcoholic steatohepatitis (NASH), appears to be increasingly common worldwide. Its histopathology and the effects of nutrition on liver function have not been fully determined. Aim: To elucidate the cellular mechanisms of NAFLD induced by a methionine-choline-deficient (MCD) diet in mice. Particular focus was placed on the role of phagocytic cells. Methods: Male C57BL/ 6 mice were fed an MCD diet for 30 weeks. A recovery model was also established wherein a normal control diet was provided for 2 weeks after a period of 8, 16, or 30 weeks. Results: Mice fed the MCD diet for >= 2 weeks exhibited severe steatohepatitis with elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) levels. Steatohepatitis was accompanied by the infiltration of CD68-positive macrophages (Kupffer cells). The severity of steatohepatitis increased in the first 16 weeks but was seen to lessen by week 30. Fibrosis began to develop at 10 weeks and continued thereafter. Steatohepatitis and elevated serum hepatic enzyme concentrations returned to normal levels after switching the diet back to the control within the first 16 weeks, but fibrosis and CD68-positive macrophages remained. Conclusions: The histopathological changes and irreversible fibrosis seen in this model were caused by prolonged feeding of an MCD diet. These results were accompanied by changes in the activity of CD68-positive cells with temporary elevation of CCL-2, MMP-13, and MMP-9 levels, all of which may trigger early steatohepatitis and late fibrosis through phagocytosis-associated MMP induction.

Because the progression and metastasis of solid tumors depend on their local microcirculation, we sought to characterize tumor angiogenesis three dimensionally in a highly metastatic mouse melanoma model, B16BL6 (B16), injected with Matrigel into the subcutis in the skin on the back of syngeneic C57BL/6 mice. We found that B16 with Matrigel grew significantly faster than B16 alone and had altered tumor angiogenesis. Tumor vessels apparently grew vigorously in the opposite direction of the tumor without invading the tumor mass until at least day 10 of injection. In addition, vascular branching resulted not only from sprouting as was seen in B16 without Matrigel but also from vascular splitting, either because of compression from outside the vessels or from septum formation by endothelial cells. This phenomenon was characteristic of B16 cells, but not of other tumor cells, including Lewis lung carcinoma and ASH-1 hybridoma cell lines, both of which were tested under the same conditions. The reduction in various angiogenic factors in Matrigel did not affect the angiogenic patterns and tumor growth. We hypothesize that tumor vessels may vigorously alter their angiogenic patterns in response to the local microenvironment.

The hematopoietic microenvironment has been investigated and well defined in the bone marrow. However, there is a lack of studies on the extramedullary hematopoietic milieu such as in the liver, to which hematopoietic stem cells migrate and there commence hematopoiesis under pathological conditions such as bone marrow failure. We induced extramedullary hematopoiesis by phenylhydrazine in the adult mouse liver and investigated the immunohistochemical, ultrastructural, and molecular changes within this organ. Using an intravital lectin injection technique, we found numerous monocytes attached to the central vein prior to hematopoietic foci formation. These cells were later incorporated into the hematopoietic foci. An increase in the mRNA expressions of the monocyte attracting chemokine CCL-2 (MCP-1) was noted in the central vein region as well as in cells within the hematopoietic foci. Together with local liver components, we regard these monocytes as components of the extramedullary hematopoietic milieu. We conclude that the recruitment of extra-hepatic monocytes is an important event during extramedullary hematopoiesis in the liver and that these monocytes participate in the liver hematopoietic microenvironment.

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP(+)) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP(+) cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.

The vascular endothelia express a variety of structural and biological phenotypes. We used an intravital injection method of plant derived lectins (Lycopersicon esculentum lectin (LEL), Ricinus communis Agglutinin-I (RCA-I), Ulex europaeus Agglutinin-I (UEA-I) and Concanavalin A (ConA)) to elucidate the morphology and function of the sinusoidal endothelium of the liver and bone marrow. All four lectins stained the sinusoidal endothelia of the liver and bone marrow in a patchy granular pattern which differed from the uniform and smooth staining pattern of non-sinusoidal vessels in other organs. By transmission electron microscopy, the granular pattern was identified as internalization of these lectins and subsequent accumulation within the endothelial cells, suggesting their active endocytosis. The endocytosis of these lectins emphasizes the fact that sinusoidal endothelial cells of the liver and bone marrow belong to the reticuloendothelial system (RES), a cell system characterized by internalization of foreign material. We introduce this intravital lectin injection as a useful technique to discriminate sinusoidal endothelial of the liver and bone marrow from other vascular endothelia.

日本産ジョロウグモ（Nephila clavata）の牽引糸遺伝子のcDNAをクローニングし、その塩基配列を初めて報告した。塩基配列から推定される日本産ジョロウグモ牽引糸タンパク質のアミノ酸配列をカイコのフィブロインH鎖や他のクモ牽引糸のものと比較した。牽引糸の繰り返し配列においてAn、（GA）nやGGX配列など共通の特徴を確認できた。クモ牽引糸タンパク質の繰り返し配列のコドン使用頻度をカイコのH鎖フィブロインと比較すると、同様の傾向があった。しかしながら、親水性と疎水性の分布は両者で大きな違いが見られた。引張り強度試験では、日本産ジョロウグモ牽引糸がカイコ繭糸よりも明らかに強いことが分かった。これらのことから、クモ糸を吐くカイコを作出してカイコH鎖フィブロインをクモ牽引糸タンパク質と置き換えること、あるいは両者の混合糸を作らせることにより、糸の強さや性状の異なる新しいシルクの創出が期待される。

We produced two novel rat monoclonal antibodies (LA102 and LA5) to identify mouse lymphatic vessels and blood vessels, respectively. We characterized the two antibodies as to the morphological and functional specificities of endothelial cells of both types of vessels. The antibodies were produced by a rapid differential immunization of DA rats with collagenase- and neuraminidase-treated mouse lymphangioma tissues. LA102 specifically reacted with mouse lymphatic vessels except the thoracic duct and the marginal sinus of lymph nodes, but not with any blood vessels. In contrast, LA5 reacted with most mouse blood vessels with a few exceptions, but not with lymphatics. LA102 recognized a protein of 25-27 kDa, whereas LA5 recognized a molecule of 12-13 kDa. Neither antibody recognized any currently identified lymphatic or vascular endothelial cell antigens. Immunoelectron microscopy revealed that the antigens recognized by LA102 and LA5 were localized on both luminal and abluminal endothelial cell membranes of each vessel type. Interestingly, LA102 immunoreactivity was strongly expressed on pinocytic or transport vesicle membrane in the cytoplasm of lymphatic endothelium. Besides endothelial cells, both antibodies also recognized some types of lymphoid cells. Since, the LA102 antigen molecule is expressed on some lymphoid cells, it may play important roles in the migration of lymphoid cells and in some transport mechanisms through lymphatic endothelial cells.

Steroid sulfatase (STS) is a microsomal enzyme catalysing the hydrolysis of steroid 30-sulfates, including estrogen sulfate, at organ target sites. Steroid sulfatase activity, as well as the activity of various estrogens, has been reported to influence the development of organs. In the kidney, progenitor cells of nephrons develop from mesenchymal cells and finally attach to the ureteric duct at the region termed the cone-shaped projection. However, the mechanisms of this process have not been fully investigated. In the present study, STS-related apoptosis occurring between the renal vesicle and ureteric duct was examined using immunohistochemical techniques. Immunoreactivity to STS was demonstrated at the cortical metanephric mesenchyme surrounding new vessels and was pronounced at the junction between the renal vesicle and ureteric duct (cone-shaped junction). Steroid sulfatase-transfected LLC-RK1 cells (rat proximal cell line) were also examined in vitro. Apoptosis occurred when an estrogen precursor (E1S) was added at concentrations between 10(-4) and 10(-2) mol/L. These results indicate that STS is synthesized in the progenitor cells of proximal tubules. Moreover, STS may be related to apoptosis occurring at the junction between the renal vesicle and ureteric duct in a manner proportional to the availability of STS precursors.

バキュロウィルス発現系を用いてジョロウグモ横糸タンパク質を昆虫細胞で発現させた。予想される分子量を持つ横糸タンパク質の発現が、大腸菌で見られた変則的な発現を伴わず、可溶性分画中に確認された。沸騰水中での10分間の熱処理で発現産物は変性沈殿することなく安定だった。この熱安定性は、横糸タンパク質の簡単で効率的な精製法として利用できることを示した。横糸が200％にも及ぶ伸び率を持つのは、横糸タンパク質のGPGGXモチーフがβ-スパイラル構造というタンパク質の二次構造をとるためと予想されている。我々は昆虫細胞で発現させた日本産ジョロウグモの1エクソン分の横糸タンパク質がβ構造をとり得ることを確認した。このことは、昆虫細胞で発現させた横糸タンパク質が、クモによって出糸される横糸の性状を有している可能性を示唆するものである。

日本産ジョロウグモの横糸タンパク質の特徴を明らかにするため、横糸タンパク質の反復モチーフ配列をコードする1エクソン分の遺伝子をクローニングして、塩基配列を決定し、北米ジョロウグモやマダガスカルジョロウグモの1エクソン分の横糸タンパク質との類似性を比較検討した。それは、(1)GPGGXの44回繰り返し領域、(2)GGXの7回繰り返し領域、(3)スペーサー領域（IIEDLDIIDGADGPIISEELISGS）、そして(4)GPGG（X）nの28回繰返し領域という4つのモチーフ領域から構成されていた。この構成は、北米ジョロウグモやマダガスカルジョロウグモのものと同じであった。北米ジョロウグモやマダガスカルジョロウグモと体長や特徴の異なる日本産ジョロウグモで、横糸遺伝子の繰返しユニット構成における高い保存性が認められたことは、4つのモチーフ領域がジョロウグモ横糸の共通した物理化学的特徴に関わる重要な役割を果たしていることを示すと考えられる。

大腸菌で横糸タンパク質を発現させた場合、主要バンドより分子量の高い域にある多数のラダー状タンパク質が出現した。このほとんどが発現中にフレームシフトを起こし、横糸タンパク質本来のアミノ酸配列を示していないものと推定された。横糸タンパク質のC末端に6×His-agを付加しておくことにより、多数のラダー状タンパク質の中から、横糸mRNAのコドンを忠実に翻訳したと思われる横糸タンパク質のみを、培地1000mlから約50mg精製・回収できることを明らかにした。C末端の6×His-agを利用した精製法で、十分に満足できる収量の横糸タンパク質が大腸菌から得られることを明らかにした。