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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

We report an autopsy case of HTLV-I-associated myelopathy (HAM) showing clinical features indistinguishable from amyotrophic lateral selerosis (ALS). A Japanese man developed bulbar palsy and generalized neurogenic muscular atrophy with symmetrical hyperreflexia at the age of 57 and died 4.5 years after the onset. He had an increased titer of anti-HTLV-I antibodies in serum and CSF. At autopsy, the leptomeninges were thickened and infiltrated with inflammatory cells. The brain and spinal cord were atrophic. The pyramidal tracts and anterior horn cells of spinal cord were severely degenerated but degenerative changes were also found in the basal ganglia, thalamus, brainstem tegmentum, and the dorsal columns of spinal cord. In all degenerative areas, prominent infiltrations of inflammatory cells were found. The present case indicates that HAM may produce clinical features indistinguishable from ALS.

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.

The skin comprises three layers: epidermis, dermis, and hypodermis. We report here on a skin, reconstructed in vitro, that is composed of all three layers. The top-most layer, epidermis, was exposed to air by a new method. The exposure induced an extensive proliferation, and differentiation, i.e. keratinization was eventually observed in the cultured epidermal cells. Skin thus cultured will be a useful graft of transplantation and provide an ideal model system in which to study diseases of the skin.

The influence of extracellular matrix on the proliferation and differentiation of glioma cells in vitro was investigated by culturing glioma cells in collagen gel and on collagen and laminin films. Rat C-6 glioma cells extended thin cytoplasmic processes, proliferated, and differentiated in collagen gel and on collagen film. The cells were stellate with multiple thin processes like the astrocyte in vivo. The intracellular content of cyclic adenosine-monophosphate in rat C-6 glioma cells on collagen film increased approximately four fold over the control level. In laminin film culture, rat C-6 glioma cells extended thin cytoplasmic processes with many knotty structures and proliferated. These findings confirm that extracellular matrix induces the differentiation of glioma cells.

The irradiated fibroblast-induced response of non-irradiated neighboring cells is called 'radiation-induced bystander effect', but it is unclear in non-irradiated human squamous cell carcinoma (SCC) cells. The present study shows that irradiated fibroblasts promoted the invasive growth of T3M-1 SCC cells, but not their apoptosis, more greatly than non-irradiated fibroblasts, using collagen gel invasion assay, immunohistochemistry and Western blot. The number of irradiated fibroblasts decreased to about 30% of that of non-irradiated fibroblasts, but irradiated fibroblasts increased the growth marker ki-67 display of SCC cells more greatly than non-irradiated fibroblasts. Irradiated fibroblasts did not affect the apoptosis marker ss-DNA expression of SCC cells. Irradiated fibroblasts enhanced the display of the following growth-, invasion- and motility-related molecules in SCC cells more greatly than non-irradiated fibroblasts: c-Met, Ras, mitogen-activated protein kinase (MAPK) cascade (Raf-1, MEK-1 and ERK-1/2), matrix metalloproteinase-1 and -9, laminin 5 and filamin A. Irradiated fibroblasts, but not non-irradiated ones, formed irradiation-induced foci (IRIF) of the genomic instability marker p53-binding protein 1 (53BP1) and expressed transforming growth factor-beta1 (TGF- beta1). Irradiated fibroblasts in turn enabled SCC cells to enhance 53BP1 IRIF formation more extensively than non-irradiated fibroblasts. Finally, effects of irradiated fibroblasts on growth and apoptosis of another HEp-2 SCC cell type were similar to those of T3M-1. These results suggest that irradiated fibroblasts promotes invasion and growth of SCC cells by enhancement of invasive growth-related molecules above through TGF- beta1-mediated bystander mechanism, in which irradiated fibroblast-induced genomic instability of SCC cells may be involved.

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.

A 79-year-old man was referred to this department due to the presence of extrahepatic bile duct carcinoma with a tumor at the left chest wall. The lesion was suspected to be a metastasis of bile duct carcinoma to the left wall, however, computed tomography (CT) revealed no regional lymph node or liver metastases. In addition, cytological and pathological examinations did not show malignancy. At the time of admission, the white blood cell count was 21 460 cells/μL (neutrophils, 18 240 cells/μL) and this elevated to 106 040 before death. In addition, serum granulocyte colony-stimulating factor (G-CSF) was elevated. At 28 d after admission, the patient died. An autopsy showed a poorly differentiated adenocarcinoma with sarcomatous change, which had slightly invaded into the pancreas around the bile duct, and was found in the distal bile duct with multiple metastases to the chest wall, lung, kidney, adrenal body, liver, mesentery, vertebra and mediastinal and para-aortic lymph nodes, without locoregional lymph node and liver metastasis. The cancer cells showed positive immunohistochemical staining for anti-G-CSF antibody. This is believed to be the first report of an extrahepatic bile duct carcinoma that produces G-CSF. Since G-CSF-producing carcinoma and sarcomatous change of the biliary tract leads to poor prognosis, early diagnosis and treatment are needed. When infection is ruled out, the G-CSF in serum should be examined. In addition, examinations such as bone scintigraphy and chest CT should also be considered for distant metastasis.

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.

Spindle epithelial tumor with thymus-like differentiation (SETTLE) is a very rare thyroid tumor. It is one of a family of tumors arising either from ectopic thymus or remnants of branchial pouches that retain the potential to differentiate along the thymic line. Herein is reported a case of SETTLE in a 2-year-old girl. The patient underwent right thyroid lobectomy for a tumor of the right thyroid lobe. The resected specimen of this tumor revealed a whitish and solid mass. On microscopy, the tumor exhibited an area of spindle cells, glandular epithelium, and mucinous cystic lesions. The following findings were obtained on immunohistochemistry: the spindle cell area was diffusely positive for cytokeratin AE1/3 and vimentin, and partially positive for alpha-smooth muscle-specific actin. The glandular structures consisted of columnar cells and the cystic area was also positive for cytokeratin AE1/3. All three components of the tumor were negative for thyroglobulin, thyroid transcription factor-1, S-100 protein, carcinoembryonic antigen, somatostatin, synaptophysin, and chromogranin A. On the basis of the aforementioned findings, SETTLE was diagnosed. The patient remains disease free to date, 2 years after surgery with no additional treatment. To the best of the authors' knowledge the present SETTLE patient is the youngest yet reported.

Publisher Summary This chapter presents the organ culture method for thyroid tissue. Thyroid follicles in vivo are embedded in an inter-follicular extracellular matrix (ECM) supported by a dense network of fenestrated capillaries. The culture cells can be stimulated by various reagents added to the culture medium of the outer dish. In addition, exposing the cellular layer to various concentrations of oxygen permits the embedded cells to be supplied those of oxygen. Culture cells can be observed by phase-contrast microscopy. The collagen gel layer containing viable cells is scraped easily from the culture assembly. The microenvironments of many cell types of the body are subdivided mainly into the following three types. The first is that of parenchymal and stromal cell types of solid organs such as thyroid, adrenal, and liver. The second is that of surface-lining cell types on which sufficient liquid is not overlayed such as those of the skin, cornea, respiratory, and digestive tracts.

Inorganic pyrophosphatase (PPase) controls the level of inorganic pyrophosphate produced by biosynthesis of protein, RNA, and DNA. Thus, PPase is essential for life. PPase expression is unclear in the thyroid. We cloned a new human PPase, phospholysine phosphohistidine inorganic pyrophosphate phosphatase (LHPPase), and established a rabbit polyclonal anti-LHPPase antibody. This is the first study to determine the PPase expression by immunohistochemistry and Western blot. Intranuclear LHPPase expression of thyrocytes was enhanced most prominently in Graves' disease and autonomously functional thyroid nodule. To estimate a regulating factor of subcellular localization of LHPPase, we examined its expression of Graves' disease-derived thyrocytes in vitro with the disease-originated serum. Nuclear expression of LHPPase was lost in cultured thyrocytes even with the serum, while its cytoplasmic expression was retained. The data suggest that increased expression of LHPPase is associated with hyperthyroidism. Intranuclear expression of LHPPase may not be regulated by Graves' disease-derived serum factors.

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.

Adipose tissue consists of mature adipocytes and endothelial cells, which are all supported by the extracellular matrix. Adipose tissue development is closely associated with angiogenesis. However, the adipocyte-endothelial cell interaction is unclear. To address this issue, we examined the effects of endothelial cells on the growth, apoptosis, and differentiation of mature adipocytes in three-dimensional collagen gel culture of the adipocytes with or without rat lung endothelial (RLE) cells. Spindle-shaped preadipocytes, an immature type of adipocyte, developed more actively around the adhesion sites of RLE cells to mature adipocytes in the coculture (rate of preadipocytes: 18.9+/-4.3%) than in the culture of adipocytes alone (2.0+/-5.1%). With respect to growth, RLE cells induced about a three-fold increase in bromodeoxyuridine uptake of mature adipocytes alone, while RLE cells did not influence the uptake of preadipocytes. RLE cells also did not affect the apoptotic indices by immunohistochemistry for single-stranded DNA in mature adipocytes or preadipocytes. These phenomena were not reproduced by RLE cell-conditioned medium, or by certain endothelial cell-produced cytokines. Our in vitro study is the first demonstration that endothelial RLE cells promote the active development of preadipocytes together with increased growth of mature adipocytes. These results suggest that endothelial cells are involved in the enlargement mechanism of adipose tissue mass through their direct adhesion to mature adipocytes.

Osteosarcoma is one of the neoplasms that may occur following exposure to radiation. A case of osteosarcoma arising in the craniofacial bone with a short latency period of 3 years after radiotherapy for maxillary squamous cell carcinoma is described. A 64-year-old-man underwent right partial maxillectomy and chemoradiotherapy due to squamous cell carcinoma of his right maxillary sinus. Histologically, the tumor was composed of moderately differentiated squamous cell carcinoma with a component of spindle cell carcinoma. Three years later, osteosarcoma developed in the craniofacial bone within the irradiation field of the first tumor. Detailed histological examination demonstrated that there was no component of osteosarcoma in the first tumor or squamous cell carcinoma in the second tumor. Radiation-induced osteosarcoma usually occurs after a long latency period of more than 10 years after the radiotherapy. In this case, osteosarcoma was possibly a radiation-induced osteosarcoma with a short latency period of 3 years.

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.

Carcinoma-stromal cell interaction and air-liquid interface (ALI) constitute a specific microenvironment that modulates the biological behavior of carcinoma cells of the upper aerodigestive tract. Although radiotherapy is a useful tool for treating carcinomas of this organ, effects of irradiation on carcinoma cells under carcinoma-stromal cell interaction and ALI are unclear. To address this issue, we examined the effects of irradiation on the proliferation and apoptosis of squamous cell carcinoma cell lines (KB and HEp-2), using three-dimensional collagen gel culture with both carcinoma-stromal cell interaction and ALI. During the second week after irradiation with or without the two factors mentioned above, bromodeoxyuridin (BrdU) uptake and apoptosis of KB, and HEp-2 cell types decreased and increased, respectively. After this stage, the carcinoma cells with these two factors actively re-proliferated together with increased BrdU uptake and decreased apoptosis, whereas the magnitude of these parameters was considerably lower in culture without these factors. We applied our method to carcinoma tissues obtained from several clinical cases. At the same stage, the irradiated carcinoma cells replicated the phenomena observed in cell lines. The data indicate that carcinoma-stromal cell interaction and ALI together promote the re-proliferation of irradiated carcinoma cells and their decreased apoptosis, suggesting that our method is a possible model for testing radiosensitivity of carcinomas in a more physiological condition.

A 58-year-old woman who suffered from a heterozygous Fabry’s disease and immune complex crescentic glomerulonephritis (GN) is reviewed. The diagnosis was made on the basis of the pathologic findings and peripheral leukocyte α-galactosidase activity. Light microscopy revealed a vacuolization of epithelial cells and electron microscopy showed myelin figures in the cytoplasm of visceral epithelial cells typical of Fabry’s disease at the first renal biopsy. During the following 4 months she developed progressive renal failure and a second renal biopsy disclosed the formation of cellular crescents in 7 of 11 glomeruli observed. A rare case of combined Fabry’s disease and crescentic glomerulonephritis is discussed.

F9 teratocarcinoma stem cells treated with retinoic acid (RA) differentiate into visceral endoderm, and this process affects the expression of some extracellular matrix (ECM) proteins. As the direct influence of ECM molecules on the differentiation of F9 stem cells has not been studied, we investigated the effects of various ECM proteins on the early differentiation of these cells. Monolayers of F9 stem cells were cultured on dishes coated with various ECM, such as type I or IV collagen, fibronectin, or laminin. They aggregated and formed spheroid bodies in the absence of RA only on type IV collagen. The outer layer cells of the spheroid bodies exhibited numerous microvilli, junctional complexes and mature cell organelles. Alpha-fetoprotein was positive in the outer layer cells. A small amount of laminin was detected in the matrix of the spheroid bodies. These data suggest that type IV collagen promoted the early stage of the differentiation of F9 stem cells without RA. The other ECM molecules failed to induce them to form spheroid bodies. The reversibility of the structure of the spheroid bodies to a monolayer was also examined. When the spheroid bodies were reseeded only on fibronectin- or laminin-coated culture dishes, they broke down and the cells spread to the surface in the absence of RA. The differentiation of F9 stem cells induced by type IV collagen seemed to be reversible and to take place at an early stage of their morphogenesis. These findings suggest that type IV collagen plays an important role in early embryogenesis.

Purpose: First, to confirm that corneal endothelial cells in the confluent state have the capability to form cellular covering. Second, to establish a method to study the combined effect of extracellular matrices (ECMs) and growth factors on the biological response in corneal endothelial cells in culture. Methods: Bovine corneal endothelial cells were cultured inside a cylinder set on a plastic dish. They formed a confluent cell nest on the dish coated with type I or type IV collagen, laminin, or fibronectin. After the removal of the cylinder, hepatocyte growth factor (HGF), epidermal growth factor, transforming growth factor-α or transforming growth factor-β1 was added to the cultures. Each confluent cell nest enlarged outward, and its increased area size was measured. Cellular response in the nest, including cellular proliferation, was analyzed. Results: The size of the increased area of the culture on type IV collagen plus HGF was the largest of all the combinations of ECMs and growth factors. The responses of component cells in the increased area consisted of cellular hypertrophy, proliferation, migration and giant cell formation. The treatment with type IV collagen plus HGF clearly promoted all the above responses. Conclusions: The biological response of corneal endothelial cells was regulated by ECMs and growth factors.

The localization and biological roles of the multifunctional cell type mast cells remain unclear in subacute thyroiditis that is characterized by both epithelioid granuloma formation and thyroid tissue repair. We examined their immunolocalization with tryptase of a mast cell marker, using the biopsy specimens from 12 cases. In the epithelioid granuloma, no mast cells were detected in any of the cases, although a small number of them (4.6 +/- 2.4) were seen at the fibrous stroma around the granuloma in all cases. By contrast, in all cases, increased mast cells (28 +/- 7.2) localized at the thyroid tissue-regenerative site where both thyroid folliculogenesis and angiogenesis take place. To elucidate possible roles of mast cells in the disease, we also examined their immunoexpressions 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), which affect thyroid folliculogenesis and angiogenesis. In all 12 cases, mast cells displayed all of these growth factors in a manner not specific to the infiltrating site. The data suggest that growth factor-expressing mast cells may play crucial roles in the thyroid tissue repair of subacute thyroiditis, modulating thyroid folliculogenesis and angiogenesis; and that the multifunctionality of the cells may be partly dependent on their expressions of various growth factors.

Subepithelial tissue cell types in vivo are separated from air by the surface-covering epithelial layer of various organs, e.g., the skin, cornea, and respiratory and upper alimentary tracts. The epithelial defect caused by inflammatory, traumatic or surgical injury would be expected to expose the subepithelial tissue-localized fibroblasts to influx air. However, it is unclear what effects air stimulation elicits in fibroblast growth, which is critical for wound healing. To address this question, we examined the proliferation of 3T3 fibroblasts with bromodeoxyuridine (BrdU) uptake, using fibroblast-embedded collagen gel culture with or without air exposure. The BrdU intake of air-exposed fibroblasts was about 6 times that of air-nonexposed cells. To further characterize this fibroblast growth, we examined the expression of mitogen-activated protein kinase (MAPK) cascade, which plays a key role in the growth-signaling pathway of various cell types. Immunohistochemistry and Western blotting showed that air exposure increased MAPK cascade expression of the cells more strongly than air nonexposure. The data indicate that air exposure promotes MAPK cascade-associated fibroblast growth, suggesting in turn that in wound repair air stimulation itself may be involved in the basic mechanisms of subepithelial fibroblast proliferation and that it may be related to the pathogenesis of excessive fibroplasia through fibroblast overgrowth.

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.

We describe a rare case of papillary carcinoma with extensive proliferation of stromal cells. The stromal cells were immunocytochemically positive for vimentin, alpha-smooth muscle actin and desmin, but negative for cytokeratin, epithelial membrane antigen, S-100, thyroglobulin and CD34. These results and the ultrastructure of the stromal cells, which exhibited the characteristics of both fibroblasts and smooth muscle cells, indicated an origin from myofibroblasts. We conclude that myofibroblastic proliferation may contribute to the stromal response in the slow growth of the papillary carcinoma.

Objective: To prepare an in vitro reconstruction of the porcine laryngeal mucosa, for use in cell biological investigations and as a model for the study of laryngeal disease. Design: Using separately obtained epithelial cells and fibroblasts from porcine laryngeal mucosa, we reconstructed the laryngeal mucosa in a three-dimensional collagen gel matrix culture with air-liquid interface. Subjects: Porcine larynges were from 6-month-old pigs obtained from a local abattoir. Results: We successfully reconstructed the laryngeal mucosa in vitro, including epithelium and lamina propria. The epithelial cells showed five to eight cells in thickness and were well differentiated on the reconstructed lamina propria. The differentiation-specific cytokeratin was positive. Conclusions: To our knowledge, this is the first report of a reconstruction of the laryngeal mucosa in a three-dimensional collagen gel matrix culture. Fibroblasts and air-liquid interface treatment exert a great influence on the proliferation and differentiation of the cultured epithelial cells. This culture system will help to provide an appropriate physiologic environment to study the differentiation and disease of the larynx. (Arch Otolaryngol Head Neck Surg. 1996;122:649-654)

We report an autopsied case of paraneoplastic necrotizing myelopathy. The patient had bilateral blindness, quardriplegia, and dyspnea of acute onset and died without remission 7 weeks later. The severe tissue necrosis and demyelination were found in the optic chiasm and from the medulla oblongata throughout the whole length of spinal cord. A papillary carcinoma was found in the thyroid gland at autopsy. In the present case IgG, myelin basic protein and activated helper T cells were increased in the CSF at onset, suggesting a mechanism of autoimmune demyelination for the condition.

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.

We report a case of primary CNS B-cell lymphoma indistinguishable from multiple sclerosis (MS). MRI of the head showed the spontaneous disappearance of the white matter lesions and the progressive cerebral atrophy. The brain biopsy failed to make a diagnosis of CNS lymphoma but rather suggested MS. Although the primary CNS lymphoma was suspected at autopsy, the immunohistochemical study showed the CNS-infiltrating lymphoid cells comprising both T-cells and B-cells. Analysis of the immunoglobulin and T-cell receptor gene rearrangements first provided evidence of primary CNS B-cell lymphoma.

Malignant testicular tumors occurring in non-twin brothers are reported. Both of the brothers suffered from amentia and epilepsy and were the product of a consanguineous marriage. One brother presented with teratocarcinoma and the other seminoma. With a review of the literature, genetic roles in the etiology of testicular neoplasia are discussed.

The effect caused by the double condition of potassium loading (K loading) and simultaneous sodium restriction (Na restriction) on the glomerular zone of rats is reported. After 2-week-diet of K loading and simultaneous Na restriction an increase of width of glomerular zone and decreases and micronization of fat granules were seen. Enzymic activity (glucose-6-phosphate dehydrogenase, steroid 3beta-ol-dehydrogenase) was increased. After 3-week-diet of the same condition the width of glomerular zone became 1/4 to 1/5 of the whole cortex. The cells in the glomerular zone demonstrated a trabecular arrangement. Ultrastructurally, hypertrophy of cells became marked and the size of cell was about 1.5 times that of normal cells. Dense body increased in number and became irregular in shape. In the widened cytoplasm, smooth endoplasmic reticulum and Golgi apparatus were well developed with numerous coated vesicles. Changes of mitochondria such as increase in number, transformation into round shape and vesicular cristae were characteristic. The severity of these changes was proportional to the duration of K loading and simultaneous Na restriction. After 8-week-diet the increase of large-sized lipid vacuole and dense body, and intramitochondrial deposition were observed in parallel with the above-mentioned changes. The changes observed in this study appeared earlier and stronger than those in the single-conditioned group of only K loading or Na restriction. The cause of these changes were considered to be a result of accelerated function of the glomerular cells.

To obtain immature fat cells in vitro, we used a primary culture of undigested mature fat tissue fragments. The immature fat cells, i.e., fibroblast-like fat cells, proliferated extensively from the fat tissue and differentiated after reaching confluence. The process of differentiation was assumed by the development of intracytoplasmic lipid droplets and by the triglyceride content in the cells. Cellular differentiation was induced in high percentages (over 70-80%) of the cells in the medium containing high glucose concentrations (200 mg/dl) supplemented with 10-20% newborn calf serum. The intracellular accumulation of triglyceride was also enhanced by insulin administration. In these cells, a reciprocal relationship was observed between proliferation and differentiation. Fibroblast-like fat cells derived from mature fat tissue in this simple culture system are suitable for the study of the proliferation and differentiation of immature fat cells.

The epidermis from 11-day-old chick embryo shank skin was cultured with 11-day-old chick embryo extract. The growth and the differentiation of the epidermis in culture were studied histologically, electron microscopically and with polyacrylamide gel electrophoresis of keratin proteins. The epidermis cultured with the chick embryo extract proliferated and stratum structures developed simultaneously with the increase in epidermal cell layers. Finally, a keratinized layer was observed after 10 days in culture. Electron microscopic observations revealed that tonofilaments were produced after 2 days in culture and increased thereafter with culture time, becoming condensed with desmosomes. Keratohyaline granules were observed in 7-day cultures. These keratinization characteristics occurring during culture showed the general characteristics of the alpha stratum observed in the skin of in ovo embryos during the early stages of development. However, the development of peridermal and subperidermal granules was poor and the stratum granulosum, which develops at the late stages between the stratum intermedium and the stratum corneum, was not observed. Polyacrylamide gel electrophoresis of S-carboxymethylated keratin proteins showed that the keratin protein band patterns of the culture differed from those of in ovo skin epidermis.

We had two rare cases of Malignant Lymphoma which occurred from the leg with swelling, redness and pain. One case was intramuscular lymphoma and the other case was cutaneous malignant lymphoma. Both patients died within two or three months after diagnosis was confirmed.They had following features;1) Initial symptoms were acute inflammatory state.2) Emergency operation was performed for the compartment syndrome of the leg.3) General condition after operation became worse.4) The diagnosis of malignant lymphoma was difficult to make in early stage.

Chondrocytes from young rabbit articular cartilage were cultured in synovial fluid without newborn calf serum. Chondrocytes proliferated extensively and produced cartilage matrix. The effect of fluid for cell proliferation was dose dependent. When the concentration of synovial fluid is above 50%, the proliferation was greater than the case of control medium containing 10% newborn calf serum. The present findings suggest that synovial fluid contains a factor that regulates the growth and differentiation of chondrocytes.

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.

Polypoid squamous cell carcinoma of the larynx consists of two components, namely, squamous cell carcinoma and sarcomatoid spindle cells. To further investigate the histogenesis of the spindle cell component, we studied two cases of polypoid squamous cell carcinoma of the larynx by immunohistochemistry and ultrastructural analysis. Positive staining for ras oncogene p21 and cytokeratin was demonstrated in both squamous cell carcinoma and spindle cell component. Only the latter component was positive for vimentin. Electron microscopic examination showed well-developed desmosomes in spindle cells. These results suggest that the spindle cell component is epithelial in origin and malignantly neoplastic; it originates from mesenchymal metaplasia or squamous cell carcinoma.

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.