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[Purpose] The purpose of this study was to clarify the relationship between cardiorespiratory fitness and metabolic markers, including leptin, in healthy young adults. [Subjects] The subjects were 51 healthy young men (mean age, 21.6 years; range, 18-31 years). [Methods] Ventilatory threshold and maximal oxygen uptake were measured on a cycle ergometer. The metabolic markers, HDL-cholesterol, triglyceride, fasting glucose, fasting insulin and leptin, were also measured, as well as and body composition, body height, body weight, percentage fat mass, waist circumference, and hip circumference. Blood pressure levels were recorded at rest. [Results] Our results show that lower cardiorespiratory fitness was significantly associated with higher storage of fat, lipid markers of developing atherosclerosis, and circulating leptin levels. Higher circulating leptin levels were also associated with metabolic risk markers such as obesity and insulin resistance. [Conclusion] Our results suggest that appropriate cardiorespiratory fitness may improve circulating metabolic markers even in healthy young men. It is therefore important that all persons should have high cardiorespiratory fitness from an early period of life for the effective prevention of cardiovascular disease.

The development and tissue differentiation of the adrenal cortex of rats were studied by giving experimental treatments to the fetus in vivo. A low-AGTH-condition was given to the fetus by administrating Dexamethasone through its mother, or directly into the subcutaneous tissue of the fetus, or by decapitation, brain aspiration or hypophysectomy. When the fetus was given the low-ACTH-condition for the last 5 days or more, the hypoplastic adrenal cortex developed specifically. It had nearly normal zona glomerulosa (ZG), and poorly differentiated zona fasciculo-reticularis (ZF-R) without differentiated ZF-R. The observing day was settled at 21 1/2 days old. The shorter the period of low-ACTH-condition was, the more thickened the differentiated zone (ZF-R) grew. On the contrary, the poorly differentiated layer became thinner in proportion to the period of low-ACTH-condition. The poorly differentiated cortical cell had ACTH-reactivity. It seems that the development and tissue differentiation of the ZF-R depend on fetal own ACTH, and poorly differentiated cortical cells appear below the ZG, and differentiate by ACTH and were gradually pushed inside. The zona glomerulosa develops and differentiates without dependence of ACTH. ACTA PATH. JAP. 27: 759-773, 1977.

The pathogenesis of corticosteroid-induced femoral head necrosis is assumed to be related to lipid metabolism. Mature fat cells are believed to play a central role in lipid metabolism. The purpose of this study was to investigate the size of mature fat cells in the human femoral head after steroid treatment. Cancerous bone tissue was obtained from the femoral heads of 20 women who had undergone total hip arthroplasty. This bone tissue was subsequently incubated in a medium containing 10(-7) or 10(-5) M dexamethasone for 5 days. Mature fat cells from the bone marrow were observed by scanning electron microscopy, and the largest diameter of individual fat cells was measured. The size of the mature fat cells in human bone marrow increased after high-dose steroid treatment. The largest fat cell volume after steroid treatment was one and one-half times larger than that observed in the control. Steroid-induced osteonecrosis is known to sometimes occur after high-dose steroid treatment. These findings may indicate the pathogenetic factors in the early stage of steroid-induced osteonecrosis.

Objective Ectopic gastric mucosa of the cervical esophagus, referred to as inlet patch (IP), is considered to be a common development abnormality of the esophagus. We report here a case of hypopharyngeal squamous cell carcinoma bordering on IP of the cervical esophagus. Methods A 44-year-old female underwent partial pharyngectomy, total laryngectomy, cervical esophagectomy, and bilateral neck dissection under the diagnosis of hypopharyngeal carcinoma. Results Resected specimens of the hypopharynx revealed a well-differentiated squamous cell carcinoma in the ulcerative tumorous lesion (3.5 cm × 0.5 cm). A brown patch (2 cm × 1.5 cm) bordering the anal aspect of the tumor comprised ectopic gastric mucosa of fundic type epithelium. Immunohistochemistry revealed the surface mucosal cells of this lesion were strongly positive for human gastric mucin (HGM) and the fundic cells were positive for gastrin. Conclusion The hypopharyngeal squamous cell carcinoma likely developed in association with chronic irritation due to gastric acid from the IP. To our knowledge, this is the first reported case of squamous cell carcinoma related to IP.

The thyroid is composed of many ball-like structures called thyroid follicles, which are supported by the interfollicular extracellular matrix and a dense network of fenestrated capillaries. The follicles consist of many thyrocytes and a few parafollicular cells (C cells). In this microenvironment, the component thyrocytes are highly integrated in their specific structural and functional polarization. In conventional monolayer and floating culture systems, thyrocytes are unable to organize themselves into the follicles with normal cellular polarity. By contrast, in three-dimensional collagen gel culture, thyrocytes easily form stable follicle structures with normal polarity. However, these culture systems cannot reconstruct follicle structures that consist of both thyrocytes and C cells. To overcome this issue, we developed a new organotypic culture system of thyroid tissue fragments, which contain the two cell types, using three-dimensional collagen gel culture with air–liquid interface. Our thyroid tissue-organotypic culture system retains three-dimensional follicle structures with both thyrocytes and C cells for a long term. In relation to our method, we propose a new approach to the cell type-specific culture systems on the basis of in vivo microenvironments of various cell types. We review here four culture systems above to study thyroid biology.

OBJECTIVE To assess whether adipocytes (mesenchymal stromal cells thought to affect the proliferation and differentiation of epithelial cells) affect the behaviour of prostate cancer cells in vitro, as prostate cancer metastasizes to the bone, which is an adipocyte-rich environment. MATERIALS AND METHODS The human bone-metastatic prostate carcinoma cell line PC3 was cultured with or without adipocytes in a three-dimensional collagen gel matrix. Histological and immunohistochemical assays were used to evaluate the proliferation and differentiation of PC3 cells. The cytokine expression of this culture assembly was also examined by reverse transcription-polymerase chain reaction (RT-PCR). RESULTS The proliferation and differentiation of cancer cells were clearly changed on co-culture with adipocytes compared with the control cultures. The mean (range) bromodeoxyuridine (BrdU) indices estimated (according to uptake) to evaluate the growth of the cultured cells were 36.3 (8.55)% in the co-culture and 26.95 (10.50) in the control (P

Interaction between cancer and stromal cells is critical for tumour cell growth and invasion. It has recently been suggested that mature adipocytes, a specific type of stromal cell that is abundant in the breast, affect the biological behaviour of some epithelial cell types. However, the nature of the interaction between mature adipocytes and breast cancer cells remains unclear. The present study has examined the effects of mature rat adipocytes, as the main cell type in adipose tissue, on the growth, apoptosis, and cell adhesion of breast carcinoma cell lines [human oestrogen receptor (ER)-positive cell lines MCF-7, ZR75-1, and T47-D; and the ER-negative mouse cell line MMT 060562] by analysing bromodeoxyuridine (BrdU) uptake, apoptotic indices, and immunohistochemical expression of E-cadherin. As a reference, the effects of rat preadipocytes (immature adipocytes) on the behaviour of the cell lines were examined. The cell lines were cultured in a three-dimensional collagen gel matrix with mature adipocytes or preadipocytes. Mature adipocytes significantly promoted BrdU uptake by all cell lines other than MMT 060562 cells. Preadipocytes decreased the uptake of T47-D cells and had no effect on that of MCF-7, ZR75-1 or MMT 060562 cells. Mature adipocytes or preadipocytes did not affect the apoptotic indices of any of the cell lines. Mature adipocytes did not influence E-cadherin expression in any of the cell lines, but preadipocytes clearly promoted E-cadherin expression by MCF-7 and T47-D cells, but not by R75-1 and MMT 060562 cells. These data suggest that mature adipocytes may be involved in the mechanisms regulating the growth of breast cancer mass through their growth-promoting effect on ER-positive tumour cells.

Thyroid follicles embedded in extracellular matrix (ECM) seem to be supplied enough oxygen by a dense network of capillaries in vivo. Air exposure (AE) causes cells to increase oxygen availability in vitro. We speculated that three-dimensional (3D) environment of ECM together with AE may be applied to a thyroid tissue-organotypic culture, simply simulating such a microenvironment of follicles. To address the issue, we performed 3D collagen gel culture of minced thyroid tissues with or without AE. Most follicles in the tissues without AE died within 7 days. In culture with AE, most of the follicles with calcitonin-positive C cells were kept for over one month. Immunohistochemistry showed that thyrocytes displayed thyroglobulin, thyrotropin receptor, thyroid transcription factor-1 (TTF-1), and pendrin, which are all crucial for thyroid function. C cells expressed calcitonin gene-related peptide and TTF-1. Our study is the first demonstration that 3D collagen gel culture with AE retains 3D thyroid follicles with C cells for a long term. This suggests that ECM and oxygen supply together may be crucial for maintenance of 3D follicle structure and function. Our method will possibly open a new path to the study of thyrocyte-C cell interaction and thyroid biology.

Morphogenesis of MDCK cells in a collagen gel matrix culture under stromal adipocyte-epithelial cell interaction . Background The stromal-epithelial cell interaction is essential for epithelial morphogenesis. Recently, the specific stromal cell type adipocytes, which abundantly exist in perirenal adipose tissue, have been suggested to affect the biological behavior of some epithelial cell types. However, adipocyte-renal epithelial cell interaction remains unclear. We thus examined the effects of adipocytes on the morphogenesis of renal epithelial cells. Methods The renal epithelial cell line, Madin-Darby canine kidney (MDCK), cells were cultured in three-dimensional collagen gel matrix with or without mature unilocular adipocytes. Cultures cells were examined by histochemistry, immunohistochemistry, and electron microscopy. Results Adipocytes extensively promoted the tubule formation of MDCK cells in two different manners. In the first type, after approximately 20% of MDCK cells actively adhered to adipocytes; they organized double-cell structured tubules between the adipocytes and the gel, contacting directly with the entire surface of the adipocytes. In the second type, approximately 70% of MDCK cells apart from adipocytes also formed tubules that had no contact with adipocytes. The component cells of both tubule types at the apical side showed microvilli and peanut agglutinin lectin-positive stain. These cells at the basal side had the basal lamina and type IV collagen-positive stain. Conclusions These results indicate that the specific stromal cell type adipocytes cause MDCK cells to organize the well-polarized tubular structures in two different manners according to their direct and indirect interactions, suggesting that adipocytes may be involved in the regulatory mechanism of renal epithelial morphogenesis.

The thyroid gland is composed of many ball-like structures called thyroid follicles, which are supported by the interfollicular extracellular matrix (ECM) and a capillary network. The component thyrocytes are highly integrated in their specific structural and functional polarization. In conventional monolayer and floating culture systems, thyrocytes cannot organize themselves into follicles with normal polarity. In contrast, in 3-D collagen gel culture, thyrocytes easily form stable follicles with physiological polarity. Integration of thyrocyte growth and differentiation results ultimately in thyroid folliculogenesis. This culture method and subacute thyroiditis are two promising models for addressing mechanisms of folliculogenesis, because thyroid-follicle formation actively occurs both in the culture system and at the regenerative phase of the disorder. The understanding of the mechanistic basis of folliculogenesis is prerequisite for generation of artificial thyroid tissue, which would enable a more physiological strategy to the treatment of hypothyroidism caused by various diseases and surgical processes than conventional hormone replacement therapy. We review here thyrocyte integration, and thyroid folliculogenesis and tissue regeneration. We also briefly discuss a perspective for thyroid tissue regeneration and engineering.

In order to clarify the incidence and types of cardiovascular malformations in Japanese trisomy 21 fetuses, seventeen cases were investigated at around 20 weeks of pregnancy. Cardiovascular malformations were observed in 8 of 17 cases (47.1%). The incidence of cardiovascular malformations was not greatly different from the estimated incidence (50%) in Japanese children with Down syndrome. The preferential elimination would not exist in trisomy 21 fetuses around 20 weeks relevance to cardiovascular malformations. Atrioventricular (AV) septal defect, tetralogy of Fallot and Ebstein's anomaly were observed in one case each. Bicuspid aortic valve and abnormal branching of the aortic arch were present in three and two cases, respectively. AV septal defect, which is perceived as a specific malformation of trisomy 21, was not recognized in high frequency in the present fetal study. A 13-week-old fetus showed multiple malformations; AV septal defect, tetralogy of Fallot and dysplastic bicuspid aortic valve. This case displayed an early morphology of AV septal defect which has rarely been reported in trisomy 21 fetuses. Possible pathogenesis of AV septal defect was discussed in relation to animal models of human trisomy 21. The present study indicated

Background: Prostatic epithelial cells organize functional acinus structures under epithelial– extracellular matrix and epithelial–stromal cell interactions. Recently, the adipose tissue, which surrounds and even exists within the prostate, has been suggested to affect the differentiation and proliferation of some cell types. Therefore, tissue fragments, which consist mainly of epithelial and fibromuscular stromal cells, were cultured in three-dimensional collagen gel matrix culture with adipocytes. Methods: Tissue fragments of rat dorsal prostate, including both epithelial and fibromuscular stromal components, were cultured in collagen gel with or without adipocytes. Epithelial cell differentiation was evaluated with the reconstruction of acinus-like structures and with immuno-histochemistry of rat dorsal prostate-specific proteins, dorsal protein-1 and probasin. The proliferation was examined by uridine uptake. Results: Under coculture of the fragments and adipocytes, epithelial cells reconstructed more differentiated acinus-like structures surrounded by fibromuscular stromal cells than tissue fragment culture without adipocytes. Dorsal protein-1 and probasin expressions of epithelial cells in this coculture system were the same as in rat prostate in vivo. In the coculture, epithelial cells had a higher proliferation activity. Conclusion: These results indicate that adipocytes promote proliferation and differentiation of prostatic epithelial cells. Our new culture model with adipocytes suggests the importance of cell–cell interactions, including those of epithelial cells and adipocytes.

The inflammatory-mechanistic basis of subacute thyroiditis remains unclear. To elucidate the roles of vascular endothelial cell growth factor (VEGF), basic fibroblast growth factor (bFGF), platelet-derived growth factor-BB (PDGF), transforming growth factor-beta1 (TGF-beta1) and epidermal growth factor (EGF) in the inflammatory process, their immunoexpression was examined in biopsy specimens of ten cases. At the granulomatous stage, all cases expressed VEGF, bFGF, PDGF, and TGF-beta1 in monocytes/macrophages infiltrating into follicle lumina, and in both epithelioid histiocytes and multinucleated giant cells of the granulomas. In fibroblasts and endothelial cells around the granulomas, all cases displayed VEGF, bFGF, and PDGF, but TGF-beta1 was detected only in fibroblasts in two cases. No cases expressed EGF in any of the above cell types. At the regenerative stage, all cases expressed VEGF, bFGF, and EGF in regenerating thyrocytes, whereas three and no cases displayed PDGF and TGF-beta1, respectively. Ten, seven and six cases expressed PDGF in fibroblasts, endothelial cells, and monocytes, respectively. In these cell types, all cases expressed VEGF and bFGF, whereas no cases displayed TGF-beta1 and EGF. To estimate the roles of these growth factors in thyroid tissue regeneration, their effects on thyroid folliculogenesis and angiogenesis were examined using collagen gel culture of thyrocytes and endothelial cells, respectively. Cell proliferation was also studied by bromodeoxyuridine (BrdU) uptake. EGF decreased follicle formation and TGF-beta1 drastically inhibited it, but the others had no effect. VEGF showed the greatest effect on vessel formation, although all of the others promoted it. EGF and VEGF or bFGF caused the highest BrdU uptake in thyrocytes and endothelial cells, respectively. The data suggest firstly, that at the granulomatous stage of subacute thyroiditis, growth factor-rich monocytes/macrophages infiltrating into follicle lumina trigger the granulomatous reaction, and VEGF, bFGF, PDGF, and TGF-beta1 produced by the stromal cell types tested mediate the reaction; secondly, that at the regenerative stage, EGF serves follicle regeneration through its mitogenic effect on thyrocytes, although some cofactors with EGF are involved in folliculogenesis and the decreased expression of TGF-beta1, a fibrogenic factor, contributes to thyroid tissue repair; and thirdly, that VEGF and bFGF are more responsible for the angiogenesis at both stages than the other factors studied.

Fat cells contribute not only to systemic lipid metabolism, but also to osteogenesis and hemopoiesis in the bone marrow. The present study represents the first attempt to culture mature unilocular fat cells of the bone marrow. Two methods devised in our laboratory were employed: one is the "ceiling culture method" that utilizes the floating property of the cells; the other, a three-dimensional collagen gel matrix culture that captures unilocular fat cells in the gel matrix. Using these methods, the proliferation of unilocular fat cells from the bovine metacarpal bone marrow was demonstrated. First, we confirmed the proliferative ability of unilocular fat cells derived from the bone marrow, using autoradiography to study 3H-thymidine incorporation into the nuclei. The unilocular fat cells de-differentiated into fibroblast-like fat cells and then proceeded to proliferate. When they underwent a contact inhibition of growth, re-differentiation from fibroblast-like fat cells into unilocular fat cells occurred at a high rate. A specific enzymatic marker of the fat cell, alpha-glycerophosphate dehydrogenase activity related to lipogenesis, was then demonstrated in the cultured fat cells. We examined the functional reactivity of the fat cells by treatment with insulin and cyclic-AMP, and both lipogenesis and lipolysis were also confirmed in them. We concluded that unilocular fat cells from the bone marrow de-differentiated, proliferated and re-differentiated in culture. The present results may help to clarify the various functions of fat cells in the bone marrow.

Transforming growth factor-β1 (TGFβ1) induces a mesenchyme-like cell shape in some epithelial cell types. To clarify the role of TGFβ1 in the morphological regulation of thyrocytes, we performed collagen gel culture of porcine thyrocytes with serum-free medium. TGFβ1-nontreated cells organized follicles. In contrast, the cells treated with 10 ng/ml TGFβ1 became spindle shaped, i.e. they resembled mesenchymal fibroblasts, and did not form follicles. To characterize the spindle-shaped cells, we examined the fine structures and expression of thyroglobulin (Tg) and cytoskeletal proteins using electron microscopy, immunohistochemistry, and immunoblotting. TGFβ1-nontreated cells had microvilli at the apical side facing follicle lumen and had basal lamina at the basal side in contact with collagen gel. TGFβ1-treated cells showed both microvilli and basal lamina at the basal side. TGFβ1-nontreated cells expressed Tg, whereas TGFβ1-treated cells showed no expression. TGFβ1-nontreated cells barely expressed vimentin, but they expressed enough cytokeratin. TGFβ1-treated cells extensively displayed vimentin along with the change in shape to become spindle-like and retained a decreased expression of cytokeratin. TSH (10 mU/ml) did not essentially influence any TGFβ1 effects on the cells. These results indicate that TGFβ1 induces a mesenchyme-like cell shape accompanied by cytoskeletal molecular change and the loss of both epithelial polarization and a function in thyrocytes, and that it results in inhibiting thyroid folliculogenesis with or without TSH.

A rare case of primary choriocarcinoma of the lung in a male is described with immunohistochemistry for human chorionic gonadotropin (hCG), epidermal growth factor (EGF) and EGF-receptor. The extragonadal trophoblastic origin of this pulmonary carcinoma was definitely confirmed by an autopsy examination, and hCG-production and hCG-positive staining of the tumor cells. Furthermore, the tumor cells clearly expressed EGF and its receptor which play an important role in the proliferation and differentiation of normal and neoplastic trophoblasts of the uterus. Our present case suggests that EGF may act in an autocrine manner in the tumor cells of primary pulmonary choriocarcinoma.

当院救急部に搬入された内因性DOAの24症例について原因検索のために病理解剖を行い,これらの臨床推定診断および既往症と剖検診断とを比較検討した。死亡確認がなされた時点における臨床推定診断としては,急性心不全が14例,急性呼吸不全が4例,解離性大動脈瘤破裂が3例,下血が1例,脳内出血が1例および甲状腺機能低下症が1例であった。これらのDOA症例を病理解剖して判明した心停止原因は,急性心筋梗塞9例,解離性大動脈瘤4例,呼吸器疾患による窒息死3例,誤嚥性肺炎2例,およびうっ血性心不全,出血性胃潰瘍,脳梗塞,甲状腺機能亢進症,急性腎不全,成人T細胞性白血病の各1例と,医療の進んだ現在でも剖検で死因が明確ないし訂正された症例は高率の71% (17例)であり,DOA症例に病理解剖の必要性が再認識された。これらのほとんどの症例は病理解剖の原因検索に多大な労力を要する急性心不全であり,死因と高い比率(75%)で因果関係を有していた既往症の聴取が心停止原因の剖検検索に助けとなると思われた。それに対して,解離性大動脈瘤や呼吸器疾患によるDOAについては,臨床診断と剖検診断とが比較的高率に一致していることが認められた。

Absent semilunar valve was found in three fetal cases with cystic hygroma. Two cases which simultaneously showed absent aortic and pulmonary valves (AAV and APV, respectively) had double-outlet right ventricle. The third case, which lacked only the aortic valve, had atrioventricular septal defect and anomalous origin of the right subclavian artery. Two of the three cases had a markedly hypoplastic thymus. Fifteen AAV and 179 APV cases, including the above-mentioned cases and others reported elsewhere, were discussed with special reference to the pathogenesis of absent semilunar valve. Of the 15 AAV cases, hypoplasia of the left heart was observed in 11 cases (73.3%), double-outlet right ventricle in 5 (33.3%), and aortic arch malformations in 6 (40.0%). In the 179 APV cases, there were 111 tetralogy of Fallot (62.0%) and 44 right-sided aortic arch (24.6%). DiGeorge anomaly was found in one AAV and eight APV cases. These results indicate a pathogenesis that is possibly related to hemodynamic abnormalities or abnormal neural crest cells. Further investigation will be needed to elucidate a more definite pathogenesis of absent semilunar valve.

Some follicle cells organize thyroid follicles through proliferation of single cells in collagen gel culture. The aim of this study was to clarify whether two or more (plural) cells can form follicles through aggregation and linkage in this culture system. To address this question, we performed collagen gel culture of porcine follicle cells, using cell labeling with PKH 2 dye. A mixture of dye-labeled and nonlabeled cells was cultured in collagen gel. Organized follicles consisted of both dyed and nondyed cells. This suggested that plural cells reconstructed follicles through aggregation and linkage. To further confirm this finding, cells embedded in collagen gel were cultured in inhibition of cell proliferation with 2 micrograms/ml aphidicolin. Forty to 60 percent of the cells formed follicles, which did not grow larger. Electron microscopy showed that intracytoplasmic cavities appeared in the cells. In contrast, 20-30% of the embedded cells developed into cavity-embracing single cells, which remained signet rings. These results indicate that follicle cells organize follicles in collagen gel culture by two means: through cell division of cavity-embracing single cells and through aggregation and linkage of plural cells.

Thp/+ mouse is known to have differing phenotypes depending on gender of the ThP parent. In the present study, fetuses with maternally [Group A (until day 18 of pregnancy) and Group C (day 19)] and paternally [Group B] inherited ThP were examined with particular reference to the developmental abnormalities of hearts. In addition, a small number of fetuses with maternally inherited twLub2 on day 19 were compared with Thp. Group A ThP fetuses had greater body weight, possibly larger body size, generalized edema (100%), marked enlargement of the hearts (100%) and hypoplastic pulmonary trunk (73.7%). There were no such malformations in Group B ThP fetuses or in the controls (+/+) for both groups. The bilaterally thickened ventricular wall of fetal hearts in Group A and C ThP fetuses bulged into ventricular cavity. The pulmonary valve was also thickened. The labeling indices of the ventricular myocardial cells by BrdU were inclined to be higher in Group A ThP than in the control (+/+) fetuses. Fetuses of twLub2 had abnormalities of the cardiovascular system similar to Group C Thp. The results suggest that cardiac lesions in fetuses with maternally inherited ThP and twLub2 correspond to cardiomyopathy. Overgrowth and enlarged heart indicate the possibility of ThP as an animal model for Wiedemann-Beckwith syndrome.

The precise role of bone marrow adipose tissue (BMAT) in the marrow remains unknown. The purpose of the present study was therefore to describe a novel method for studying BMAT using 3-D collagen gel culture of BMAT fragments, immunohistochemistry, ELISA and real-time reverse transcription-polymerase chain reaction. Mature adipocytes and CD45+ leukocytes were retained for >3 weeks. Bone marrow stromal cells (BMSC) including a small number of lipid-laden preadipocytes and CD44+/CD105+ mesenchymal stem cell (MSC)-like cells, developed from BMAT. Dexamethasone (10 micromol/L), but not insulin (20 mU/mL), significantly increased the number of preadipocytes. Dexamethasone and insulin also promoted leptin production and gene expression in BMAT. Adiponectin production by BMAT was

Subcapsular small cortical cells (SC cells) and subcapsular large cortical cells (LC cells) of bovine adrenal cortex were cultured separately after purification by unit gravity sedimentation, and then compared with inner half layer cells (IL cells) prepared by the same method. Both SC and LC cells were polygonal in shape and their mitochondria were elongated with lamellar cristae. SC cells became as large as LC cells on day 6 of culture with increased cytoplasmic lipid droplets, whereas IL cells showed no change in size. IL cells were spindle-shaped and had mitochondria with tubulovesicular cristae. Both SC and LC cells produced 11β deoxycorticosterone, corticosterone, aldosterone and small amounts of 17α hydroxy progesterone (17 α OH Prog) and Cortisol (F). IL cells produced much more 17α-OH Prog and F than SC or LC cells. When stimulated with ACTH, cortical cells in each group showed cellular retraction and their mitochondria became spherical. The amounts of 17. OH Prog and F increased in all groups, especially in IL cells. These results show that LC cells have similar characteristics to SC cells in both morphology and function, and that they differ from IL cells, which correspond to classical fasciculata reticularis cells. Acta Pathol Jpn 41: 428–436, 1991.

論文(Article), Adipose tissue that consists of mature and immature adipocytes is suggested to contain mesenchymal stem cells (MSCs), but a culture system for analyzing their cell types within the tissue has not been established. Here we show that three-dimensional collagen gel culture of rat sc adipose tissue fragments maintained viable mature adipocytes for a long term, producing immature adipocytes and MSC-like cells from the fragments, using immunohistochemistry, ELISA, and real time RT-PCR. Bromodeoxyuridine uptake of mature adipocytes was detected. Adiponectin and leptin, and adipocyte-specific genes of adiponectin, leptin, and PPAR-gamma were detected in culture assembly, whereas the lipogenesis factor insulin (20 mU/ml) and inflammation-related agent TNF-alpha (2 nm) increased and decreased, respectively, all of their displays. Both spindle-shaped cell types with oil red O-positive lipid droplets and those with expression of MSC markers (CD105 and CD44) developed around the fragments. The data indicate that adipose tissue-organotypic culture retains unilocular structure, proliferative ability, and some functions of mature adipocytes, generating both immature adipocytes and CD105+/CD44+ MSC-like cells. This suggests that our method will open up a new way for studying both multiple cell types within adipose tissue and the cell-based mechanisms of obesity and metabolic syndrome.

Thyroid follicles, an essential functional unit of the thyroid, are ball-like structures and exist in the extracellular matrix in vivo. Thus far, the follicles have not been reconstructed in any culture system. The presumed reason for that was that the in vitro environment for the follicle cells in monolayer culture markedly differed from their environment in vivo. We, therefore, considered that isolated follicle cells had to be localized in a three-dimensional environment of extracellular matrix, specifically collagen, to reconstruct thyroid follicles in vitro. At first, follicle cells were completely isolated. These cells were cultured in the three-dimensional collagen gel. An intracytoplasmic cavity first developed in individual cells. A single cell with the cavity then underwent cell division, and the follicle consisting of two cells was reconstructed. This gradually grew to be a large ball-like structure through proliferation of the component cells, and they exhibited morphological polarity specific for thyroid follicle cells. In addition, these cells clearly produced thyroid hormones. To the best of our knowledge, this report is the first instance of reconstruction of thyroid follicles in an in vitro culture system. This culture system is more useful than the monolayer culture system in that this system provides a more physiological environment for investigations of differentiation of follicle cells. Further experiments using this method will probably provide a new clue to the mechanism of thyroid folliculogenesis.

An 82-year-old woman with type 2 diabetes had been treated with recombinant human insulin for 16 years. She developed large swellings in both sides of her lower abdomen. The masses were soft, painless, and located around her insulin injection sites. Based on the history and clinical features, a diagnosis of insulin-induced lipohypertrophy was made. Total resection revealed that the lesions were composed entirely of fatty tissue. Microscopic examination showed nests of mature adipocytes expanding toward the dermal reticular layer. The hypertrophic adipocytes were twice as large as those from normal subcutaneous areas and contained numerous small lipid droplets. Electron microscopic analysis also revealed a minor population of small adipocytes, suggesting active differentiation or proliferation. Thus, the possible in vivo effects of insulin on adipocytes were clearly observed in this case of insulin-induced lipohypertrophy. To our knowledge, this is the first report of insulin-induced lipohypertrophy with detailed histological examinations.

In the thyroid, new follicle formation from preexisting follicles (mother follicle-derived folliculogenesis) has been poorly understood. To address this issue, we analyzed mother follicle-derived folliculogenesis, using a thyroid tissue-organotypic culture that retains three-dimensional follicles with both thyrocytes and C cells over a long period. Three types of mother follicle-derived folliculogenesis occurred only at the periphery of the tissue fragments embedded in collagen gel: (1) solid nest, (2) budding and (3) lumen-dividing types. Immunohistochemistry showed that neogenic follicles rarely had calcitonin-positive C cells. Electron microscopy showed that their component thyrocytes expressed normal polarity. In growth, bromodeoxyuridine uptake of thyrocytes at the tissue periphery was about 5 times the uptake that occurred at the center. C cells had no uptake. Thyrotropin (TSH, 10 mU/ml) and free calcium (0.17~1.95 mM), which are associated with the biological behavior of thyrocytes and C cells, respectively, did not affect the events described above. The data indicate that thyrocytes, but not C cells, actively undergo growth and three types of mother follicle-derived folliculogenesis at the tissue periphery in a TSH- or free calcium-independent manner. This suggests that the tissue periphery, which may escape from contact inhibition of cell growth, is the regenerative site.

Using rat skin, we studied the influence of fat cells on the proliferation and differentiation of organoid hair follicle cells in a three-dimensional collagen gel matrix culture system. We cultured organoid hair follicles embedded in collagen gel under each of the following three conditions: cell-free collagen gel for control experiments (condition 1); co-culture with fat cells in close apposition (condition 2); and co-culture with fat cells in spatial separation (condition 3). Outgrowths of epithelial cells from the organoid hair follicles associated with perifollicular proliferation of fibroblasts were observed under conditions 1 and 3. Under condition 2, proliferation of both organoid hair follicle cells and fibroblasts was inhibited, but differentiation of the hair follicle cells appeared to be accelerated. Fat cells are considered to have an inhibitory effect on the proliferation of perifollicular fibroblasts, which might have resulted in the inhibition of hair follicle cell proliferation and also in the better maintenance of normal follicular structure and integrity, allowing for hair-type differentiation to proceed. A direct accelerating effect of fat cells on hair follicle differentiation may also have been responsible. In a physiological state (co-culture with keratinocytes on the collagen gel), similar results were observed under conditions 1 and 2. The different findings under conditions 2 and 3 may be due to either of two possibilities: either the concentration gradient of the soluble factors released from fat cells, acting on either the hair follicle cells or the perifollicular fibroblasts as an inhibitor of proliferation, caused the difference in the results, or direct contact between the organoid hair follicle cells and fat cells may have influenced the accelerating effect of fat cells on the differentiation of hair follicle cells.

Summary Extracellular matrix is indispensable for cell differentiation in vivo. The aim of this study is to examine effects of extracellular matrix on adrenocortical cells embedded in collagen gel in terms of cellular proliferation and differentiation. Dissociated human or bovine adrenocortical cells were embedded in collagen gel; the gel was then immediately thin-coated on the bottom of culture dishes. The cortical cells were thus trapped in collagen and cultured in monolayer, a suitable means for observation of culture cells. To the authors' best knowledge, this “collagen gel-embedded” monolayer culture is described here for the first time. Cortical cells were spherical in shape and had the activities of proliferation and steroid production in collagen gel matrix of this culture method. Aldosterone production in particular tended to be maintained much longer than in conventional monolayer culture. This method seems to provide a physiological environment in which to study the differentiation of cortical cells.

Thyroid follicles, an essential functional unit of the thyroid, exist in the extracellular matrix of the tissue in vivo. Therefore, in any monolayer culture system, the follicles cannot be reconstructed. Our previous study adopting three-dimensional collagen gel culture showed that isolated porcine follicle cells reconstructed thyroid follicles specific for the thyroid gland in vivo. To elucidate whether this culture system is also applicable to human follicle cells, and furthermore to provide a culture system for investigations of the pathogenesis of human thyroid diseases, we tried to culture isolated human follicle cells of normal thyroid tissue and of Basedow's disease in three-dimensional collagen gel. In this culture system, they apparently reconstructed thyroid follicles. The component cells of the reconstructed follicles exhibited structural polarity specific for human thyroid follicle cells and produced thyroid hormones. In addition, the cells responded to a TSH-stimulation in terms of morphological and functional differentiation, and they presented HLA-DR antigen with an interferon-gamma-stimulation. This report is a first instance of reconstruction of human thyroid follicles and HLA-DR antigen induction in three-dimensional follicle structures in vitro. This culture system provides a more physiological environment in vitro for biological and pathogenetic investigations of human thyroid follicle cells than the monolayer culture system. Further experiments using this method will probably provide new clues to the pathogenetic mechanisms of human thyroid diseases.

In addition to the morphological examination, the measurement of the content of corticosteroids was done in aldosterone-producing adenoma. Histologically, the adenoma consists of four types of cells. The major component of the adenoma was clear-type cells. In this type of cells, the activities of 3 beta-hydroxysteroid dehydrogenase and glucose-6-phosphate dehydrogenase showed weakly positive. The fine structure was characterized by numerous lipid vacuoles and poor organellae. On the contrary, intermediate- and compact-type cells revealed higher than moderate activities of these enzymes. Moreover, marked development of smooth endoplasmic reticulum and mitochondria was noted. Zona glomerulosa-type cells were observed only in a small part. The content of aldosterone and corticosterone in aldosterone-producing adenoma was significantly larger than those of the other type of adenoma. A tendency of positive correlation between the increase of compact-type cells and content of aldosterone was found. The cell origin of this aldosterone-producing adenoma and functional role of clear- and compact-type cells were also discussed.

A 73-year-old woman suffering from colicky pain with cholelithiasis was subjected an upper GI series, and early gastric cancer in the gastric angle and a deep depressive lesion in the gastric body were found. Diffuse heterotopic glands were observed in the gastric submucosa and malignant changed glands were found in these glands by postoperative histological examination of the resected stomach. Histological examination of the gastric angle lesion disclosed signet-ring cell carcinoma, and cancer cells were noted within the mucosal layer. On the other hand, malignant changes in heterotopic glands of the gastric body were diagnosed histologically as well-differentiated adenocarcinoma. It is said that the gastric gland, which should be essentially found in the lamina propria, is rarely observed in the submucosa. Such a condition can be called “heterotopia” of the stomach. It has been noticed that the stomach with the submucosal heterotopic gland is frequently combind with gastric cancer. However, it is rare that the heterotopic gland itself turns malignant. In this respect, the present case was very significant.

The development and differentiation of the fetal adrenal cortex was studied in the rat by lipid and enzyme histochemistry.The adrenal cortex began to have small amounts of lipid droplets at 171/2 days by oil red O stain and the amounts increased gradually. Cholesterol by Schultz reaction became clear at 201/2 days. At 151/2 days weak activity of glucose 6-phosphate dehydrogenase (G-6-PDH) was already present in the inner cortex, which was followed a day later by weakly positive reaction of 3β-hydroxysteroid dehydrogenase (3β-HSD). At 181/2 days positive activity of secondary alcohol dehydrogenase (SAD) was obvious in the inner cortex. Activity of alkaline phosphatase (Al-Pase) was observed in the subcapsular zone from day 181/2 on. Enzymatic activities of the cortex increased gradually after their appearance as development proceeded. These findings show that histochemical activities of such principal enzymes as G-6-PDH and 3β-HSD precede appearance of lipid droplets.As a conclusion, zona reticularis is not distinguishable from zona fasciculata during the fetal stage, zona fasciculo-reticularis differentiates histochemically in a 171/2 day old fetus, and finally differentiation of zona glomerulosa occurs at 201/2 days in gestation.

A case of malignant cystosarcoma phyllodes with metastasis in the interpectoral lymph node (Rotter's) is presented in this paper. To the author's best knowledge, this is the first case in Japan on this disease, with lymph node metastasis. Although the surgical management of the disease has not yet been standardized, radical or modified radical mastectomy is thought to be the appropriate procedure.

Effects of several conditions known to stimulate the zona glomerulosa of the adrenal cortex in the adult were examined on the fetal adrenal cortex of rats histochemically and electron microscopically. The stimulating conditions were the following; administration of Na restricted diet to the mother, administration of KCl or angiotensin II to the fetus, spironolactone or insulin to the mother. In these, only an intraperitoneal injection of KCl solution to the fetus affected the fetal zona glomerulosa, in which increased activity of glucose-6-phosphate dehydrogenase and hyperfunctional alteration of the mitochondria and the smooth endoplasmic reticulum were observed. This fact shows the fetal glomerulosa cells can react to orae of the specific stimuli on the adult glomerulosa cells., Acta Histochemica et Cytochemica, 13(4), pp.324-329; 1980

Mouse trisomy 16, which was induced by mating males of Rb(16.17)7 Bnr/Rb(9.16)9Rma with three different strains of female, C57BL/6, C57BL/10 and C3H/He, was examined in fetuses and newborns in terms of incidence of occurrence, malformations and sex ratio. The incidence of trisomies tended to be higher in C3H/He than in C57BL/6 and C57BL/10. Trisomies could not survive beyond term in C57BL/6, which was most thoroughly examined in the three strains. Trisomy-associated generalized edema, beginning to appear on day 14 of pregnancy on fetus's back, reached the maximum on day 16 in all strains. On cardiovascular anomalies, persistent common atrioventricular canal was observed in virtually all trisomies from C57BL/10 and C57BL/6, while in about 80% from C3H/He. In conotruncal region, all trisomies from former two crossing exhibited double outlet right ventricle or persistent truncus arteriosus. In addition to these anomalies, riding aorta was found in C3H/He. Hydroureter and hydronephrosis developed from day 17 of pregnancy in trisomies other than those in C57BL/10. The histological study of gonads revealed that trisomy 16 tended to be predominant in males of each crossing. The anomalies encountered in mouse trisomy 16, such as edema, persistent common atrioventricular canal, and hydroureter and hydronephrosis appeared to be in common with human trisomy 21 embryos and fetuses. The tendency of predominance of mouse trisomies in males may provide further homology to human trisomy 21. It is supposed that genetic background by different female strains may affect phenotypic expression of malformations. The extremely high incidence of cardiovascular anomalies is regarded as suitable material for elucidation of pathogenesis.

Rats were fed on low potassium diets in order to observe the effect of dietary low potassium on the adrenal cortex. The authors clarified morphological changes of the hypofunctional glomerular zone and compared these changes with those of the hyperfunctional glomerular zone. Three weeks after or 2 months after the start of a low potassium diet, slight narrowing of the glomerular zone of the adrenal cortex was observed followed by miniaturization of cells, presence of binuclear cells and an increase of lipid with enlarged lipid drops. Electron microscope mainly disclosed changes of mitochondrial cristae consisting of markedly reduced, enlarged and irregularly dilated cristae with shortening or elongation. Granules appeared in mitochondria. Lysosomes or dense bodies were enlarged. The Golgi's apparatus was atrophied but endoplasmic reticulum did not show remarkable changes. These changes were directly opposite to those of the hyperfunctional glomerular zone noted after a potassium load or seen in sodium deficiency. Consequently, these changes were considered to be the changes of the hypofunctional glomerular zone associated with decrease of aldosterone production. ACTA PATH. SAP. 29: 351–362, 1979.

Recently it has been stressed that catecholamines play a significant role in the pathogenesis of hypertension in Spontaneously Hypertensive Rats (Okamoto and Aoki) (SHR). In this report electron microscopical observation in male SHR was made on the adrenal medulla through the processes of the hypertension. The mild hyperfunctional state of norepinephrine-secreting cells in the initial stage of hypertension was expected on the feature of increased population of norepinephrine-secreting cells, its prominent Golgi complex and rough-surfaced endoplasmic reticulum and increased degranulation in the cell. Moreover, it may be assumed a certain functional change of epinephrine-secreting cell system on the base of increased population of dark epinephrine-secreting cells which are regarded as premature cells., Acta Histochemica et Cytochemica, 5(1), pp.39-44; 1972

In the site of steroid production, the histochemical activity of secondary alcohol dehydrogenase on isopropanol as its substrate with NAD as its coenzyme will reflect the inversion of 20α-22R-dihydroxycholesterol in the biosynthesis of steroid hormone, We applied this method on organs from all parts of the body of Wistar strain rats and observed positive reaction in the adrenal cortex, ovary and testis. In the adrenal cortex of mature rats, the zona glomerulosa exhibited a weakly positive activity, the outer to middle portions of the zona fasciculata an intensely positive activity, the inner portion of the zona fasciculata a weakly positive or negative activity, and the zona reticularis a positive activity. A proportional relationship between secondary alcohol dehydrogenase activity and the amount of lipid was present in the adrenal cortex of mature rats. In addition, the specific distribution of young and old rats was described., Acta Histochemica et Cytochemica, 2(3), pp.119-123; 1969

The tissue differentiation of the zona glomerulosa of the fetal adrenal cortex of rats was studied by giving experimental treatments to the fetus in vivo. A low-glucocorticoid-condition was given to the fetus by bilateral adrenalectomy of pregnant rats for removing exogenous glucocorticoids from the fetus, and by brain aspiration of the fetuses for removing the fetal pituitary gland (ACTH) and endogenous glucocorticoids. When the fetus was placed under a low-glucocorticoid-condition for the last couple of days of gestation, poor differentiation of the zona glomerulosa occurred specifically in the fetal adrenal cortex. The degree of the poor differentiation seemed to be proportional to the duration of the low-glucocorticoid-condition. Supplemental administration of glucocorticoids could prevent this poor differentiation of the zona glomerulosa. These results indicate that the tissue differentiation of the zona glomerulosa of the fetal adrenal cortex depends much on glucocorticoids.

ACTH was administered subcutaneously to rat fetus directly at the late stage of fetal development and acute reaction on the fetal adrenal cortex was observed histochemically and electron microscopically. By administration of ACTH the adrenal cortex became remarkably hyperemic and there were swollen cells in all layers, particularly in the middle and inner layers (corresponding to the zona fasciculata and reticularis in adult rat). Marked reduction of lipid, enlarged mitochondria with increased vesicular cristae and increased smooth-surfaced endoplasmic reticulum (SER) were characteristic. The alterations of mitochondria preceded the change of SER, and thereafter mitochondria showed rapid degeneration. The outer layer (corresponding to the zona glomerulosa in adult rat) also showed similar changes by ACTH to those of the other two layers. These results indicated that the fetal adrenal cortex of rats was exogenous ACTH-reactive and its reaction which was different from that of adult cortical cells, seemed to be speciflcally related to the development and differentiation of the cells. ACTA PATH. JAP. 27: 477–484, 1977.

Freezing has been shown to damage pancreatic islets and to disrupt their insulin release, probably because of intracellular ice formation. We compared frozen islets with fresh ones and with others stored at temperatures above freezing from a standpoint of insulin release response to glucose and transplantation. Group A islets, isolated from rats and immersed in 10% dimethyl sulfoxide in RPMI 1640, were stored at -2 degrees C, and group B islets at -196 degrees C, for 7 days. As for group B, the islets were cooled at 1 degree C/min from room temperature to -40 degrees C, subsequently at 3 degrees C/min to -80 degrees C and then put into liquid nitrogen to be rapidly frozen to -196 degrees C. The control islets were fresh. In vitro, basal release at 3.3 mM glucose was similar in group A to that in the controls, but was higher in group B than group A. Stimulated release against 16.7 mM glucose was lower in group A than group B. However, insulin responsiveness, i.e., the ratio of insulin release at 16.7 mM glucose to that at 3.3 mM glucose, was lost in group B. Freezing also caused damage to the group B cells visible under the light and electron microscopes, while group A islets were largely intact. In vivo, after 600 islets were transplanted into streptozotocin-induced diabetic rats, group A was better able to lower fasting blood glucose than was group B, and remained so for 4 weeks. Above sub-zero preservation in the non-frozen state thus seems adequate for the short-term storage for 7 days.

Mature white fat tissue consists primarily of unilocular fat cells. Clearly, the study of the biology of these cells would be most helpful for the elucidation of the mechanism of obesity. We describe a new method termed 'ceiling culture' for culturing in vitro unilocular fat cells obtained from humans and rats. These cells can be maintained in culture for long periods of time and, under such conditions, continue to exhibit specific functions such as lipogenesis and lipolysis. Under the culture conditions described, unilocular fat cells change into multilocular fat cells or cells with a fibroblast-like appearance. These cells then proliferate, form a cell monolayer attached to the substratum, and after becoming confluent, exhibit accumulations of intracytoplasmic lipid droplets. These attached dedifferentiated cells continue to exhibit lipogenesis and lipolysis.

The effects of SU-4885 on the rat adrenal cortex by oral administration of 50mg/kg, single or daily for 10 days, were morphologically studied particularly with histochemical methods for the enzyme and lipid. Lipid depletion with fragmentation of droplets throughout the zona fasciculata in rats with single administration conformed to the pattern of adreno-cortical response to an acute moderate stimulus. Reduced activity of secondary alcohol dehydrogenase, fragmentation of lipid droplets, and acidophilic and trabecular changes in the cell cords were observed in the inner fasciculata following a single or daily administration for 10 days. The medication apparently brought about hypersecretion of adrenocortical steroids, which was similar to the effect of ACTH administration. Reduced activity of secondary alcohol dehydrogenase, which reflected the transformation of cholesterol in the steroid biosynthesis, was always observed in the zona fasciculata following the administration of SU-4885 or ACTH. The result might indicate that the administration inhibits some decrease in the steroid biosynthesis from cholesterol possibly through the ‘short route’.

Observations were made in dogs to determine whether X-irradiation affects the secretion of androgens by the testes, and their responses to exogenous gonadotrophin. The secretory activity was, under pentobarbital anesthesia, determined by measuring 17-oxosteroids in spermatic vein blood. The radiation was applied with 200 or 2, 000 R only to the testes, but it failed to stimulate or significantly alter the secretion of 17-oxosteroids in a period of up to 4 hr after exposure. However, at 200 R an increased secretion of testicular 17-oxosteroids into the spermatic vein became evident within 1-6 days, whereas after 2, 000 R it was observed within 13 days only in a few cases. During the post-irradiation period of 30 days, irradiated testes responded to pregnant mare serum in doses of 40iu/kg by producing larger amounts of 17-oxosteroids than normal, the maximum response occurring within a few days following irradiation. In connection with these experiments, enzyme histochemical studies were carried out on the activity of secondary alcohol dehydrogenase in the testicular tissue. At 200 R, the enzyme activity in the Leydig cell areas was increased considerably and attained to the maximum in a faw days after irradiation, and was then progressively reduced.

A 72-year-old patient with von Recklinghausen's disease showed akinetic mutism within 6 months of the onset of dementia. The findings of diffuse cerebral atrophy on CT and periodic synchronous discharges (PSDs) in EEG suggested Creutzfeldt-Jakob disease. However, autopsy findings of diffuse softening of the subcortical white matter and marked arteriosclerotic changes of the subcortical arterioles with sparing of the cortex in the cerebrum confirmed a diagnosis of Binswanger's disease. Binswanger's disease should be included in the differential diagnosis of dementia showing PSDs in EEG.

Histological findings of the thyroid gland in SHR-control showed that the process from synthesis of thyroglobulin to pinocytosis of iodinated thyroglobulin was actively metabolized under the increased stimulation of TSH. Therefore, it was suggested that a decreased secretion rate of thyroxine was mainly resulted from an impairment of hydrolysis of iodinated thyroglobulin or that of following metabolic process.By administration of thyroxine, the thyroid gland of SHR showed less atrophic changes. From the morphological finding of TSH secreting cells of the anterior pituitary gland in SHR, this result would be due to less suppressed TSH secretion.