Macrophage populations and expressions of regulatory proinflammatory factors in the rat meninx under lipopolysaccharide treatment in vivo and in vitro.

Macrophages play important roles in host defense mechanisms. In the brain, besides microglial cells, meningeal macrophages are present. However, the pathobiological characteristics of meningeal macrophages in rats remain to be investigated. In normal meninx, immunohistochemically, macrophages reacting to CD163 (macrophage scavenger receptor) and major histocompatibility complex (MHC) class II-expressing cells (involving activated macrophages or dendritic cells) were sporadically seen without age-dependent changes. Injection of lipoplysaccharide (LPS) (5 microg; Escherichia coli) into the cerebrum increased the number of anti-CD68-positive macrophages (with greater phagocytic activity) in the meninx, with a peak at 12 h during observation period until 48 h; MHC class II-expressing cells showed a gradual increase in number from 3 h after injection; however, anti-CD163-positive macrophages did not show significant change. In in vitro studies, LPS (0, 0.02, 0.05, 0.5, 5, 50 and 100 microg/ml) was added to KMY-1 or KMY-2 cells, both of which had been established from a rat malignant meningioma. KMY-1 originally reacted to CD163, but LPS addition at 0.5 microg/ml and greater concentrations decreased the anti-CD163-positive cell number and instead increased the anti-CD68-positive cell number. LPS-treated KMY-2 increased the anti-CD163-positive cell number at 0.05 and 0.5 microg/ml. By RT-PCR methods, LPS (0, 0.5, 5, 50, and 100 microg/ml)-treated KMY-1 and KMY-2 showed an increase in mRNA of monocyte chemoattractant protein-1 (MCP-1, a chemokine), and LPS-treated KMY-2 increased mRNA of nerve growth factor (NGF, an immunological effecter). Collectively, under LPS treatment, macrophages with heterogeneous functions appear in rat meninx; rat meninx-forming cells may be involved in pathogenesis of meningeal inflammation by expressing different immunophenotypes and by producing regulatory proinflammatory factors such as MCP-1 and NGF.

Cutaneous rhabdoid tumor in a cat.

Rhabdoid tumor is a highly aggressive neoplasm of unknown cellular origin in humans, usually occurring in the kidney and central nervous system of infants or children. In older patients, it occurs rarely in other organs, including the skin and soft tissues. A subcutaneous mass in a 13-year-old male mixed-breed cat was composed of nests or sheets of round to polygonal cells with glassy eosinophilic cytoplasmic inclusions. Immunohistochemically, many neoplastic cells expressed vimentin, localized to the cytoplasmic inclusions, whereas the cytoplasm of some neoplastic cells was diffusely positive for neuron-specific enolase, neurofilament, or S-100 protein. By electron microscopy, the cytoplasmic inclusions were found to be composed of aggregates of intermediate filaments. These findings are quite similar to the histologic, immunohistochemical, and ultrastructural features of human rhabdoid tumors and the few rhabdoid tumors reported in animals.

Reduction of mucin-1 gene expression associated with increased Escherichia coli adherence in the canine uterus in the early stage of dioestrus.

The relation between adherence of Escherichia coli and expression of mucin-1 (Muc1: an integral membrane mucin) mRNA in the endometrium was studied in beagle bitches at different stages of the oestrous cycle and in those with cystic endometrial hyperplasia/pyometra complex (pyometra). The number of E. coli adhering to the endometrium was low at pro-oestrus and oestrus and increased at the early stage (day 10) of dioestrus, corresponding to the implantation period; it declined thereafter. Adhesion of the organisms to endometrial epithelial cells collected at day 10 of dioestrus was inhibited by the addition of D-mannose. When endometrial epithelial cells collected at pro-oestrus were treated with hyaluronidase, an enzyme that digests mucins, the numbers of E. coli adhering to the cells tended to increase. With polymerase chain reaction analysis it was possible to detect Muc1 gene transcripts in the endometrium at all stages of the oestrous cycle, although the level of Muc1 mRNA decreased by day 10 of dioestrus. The levels of Muc1 mRNA in bitches with a clinical stage of pyometra were low and comparable to those at day 10 of dioestrus. The number of E. coli adhering to the endometrium and Muc1 mRNA levels in the endometrium were inversely correlated (r=-0.77, P<0.01). Immunohistochemical analysis showed little staining for Muc1 in the endometrial epithelia at day 10 of dioestrus and in bitches with pyometra. These results suggest that reduction of Muc1 expression is associated with increased E. coli adherence in the canine uterus at the early stage of dioestrus, possibly facilitating the development of pyometra.

Effect of co-culturing with embryonic fibroblasts on IVM, IVF and IVC of canine oocytes.

We studied the effects of mouse embryonic fibroblasts (MEF) and canine embryonic fibroblasts (CEF) on IVM, IVF and IVC of canine oocytes. Cumulus-oocyte complexes were harvested from ovaries by slicing, and in vitro maturation was evaluated in three different conditions: culture media only (control), co-culture with MEF, or co-culture with CEF. The oocytes were cultured for 48 or 72 h. Only oocytes larger than 100 microm in diameter with a homogeneous dark cytoplasm and two or more layers of cumulus cells were used. The culture medium was TCM 199+10% fetal bovine serum (FBS) with 100 IU/mL penicillin and 100 microg/mL streptomycin. After 48 h of IVM, the oocytes were fertilized in vitro with fresh canine spermatozoa that had been selected by a swim-up method, and the oocytes and spermatozoa were co-cultured in modified Krebs-Ringer bicarbonate solution (TYH) for up to 20 h in 5% CO2 in air at 38.5 degrees C. After insemination, oocytes were transferred to three different conditions (the same as for IVM) and were cultured. After 48 or 72 h of maturation in vitro, the maturation rate of MII oocytes cultured in co-culture of MEF and CEF was higher than for oocytes cultured in control (P<0.05). Although the rate that reached the MII stage was not different in the 48 and 72 h cultures, the percentage of degenerated oocytes was greater at 72 h in all three treatment groups. The proportion of monospermic and polyspermic oocytes was not different among the three treatment groups. Cleavage rates were higher in the MEF and CEF treatment groups than in the control group (P<0.05). Co-culture with CEF developed the embryo up to the 16-cell stage, and with MEF up to morula stage. In conclusion, co-culture of embryonic fibroblast cells enhanced nuclear and cytoplasmic maturation of canine oocytes.

Use of an incontinent end-on colostomy in a dog with annular rectal adenocarcinoma.

An 11-year-old, mixed-breed dog with dyschezia, tenesmus and haematochezia was presented. A rectal stricture caused by an adenocarcinoma was diagnosed. Following the failure of a rectal 'pull-through' procedure, which dehisced seven days later, an incontinent end-on colostomy was performed, allowing amputation of the colorectum with the anus and perineal skin. A two-piece device consisting of a flange and a drainable pouch was used for postoperative faecal evacuation and collection. Mild peristomal dermatitis was the only complication. Patient management was easily carried out by the owner at home, and the dog survived for four months with a satisfactory quality of life. Incontinent end-on colostomy may prove to be a useful treatment for canine annular colorectal tumours.

Space-occupying leiomyoma in the pelvic canal of a dog.

A large space-occupying mass was found in the pelvic canal of an 11-year-old, entire female shih tzu with rectal tenesmus and dyschezia. Computed tomography revealed an extraluminal mass located on the dorsal aspect of the rectum, causing complete rectal obstruction. Histopathological examination of biopsy tissue identified it as a leiomyoma. The tumour was successfully removed by blunt dissection, via a dorsal approach to the rectum, and there were no complications after surgery.

Characterization of newly established tumor lines from a spontaneous malignant schwannoma in F344 rats: nerve growth factor production, growth inhibition by transforming growth factor-beta1, and macrophage-like phenotype expression.

Transplantable tumor (KE) and clone cell (KE-F11) lines were established from a spontaneous malignant schwannoma found in an aged F344 rat. The primary tumor and KE tumors consisted of oval or spindle cells arranged in ill-defined bundles. Cultured KE-F11 cells exhibited polygonal or spindle configurations. Immunohistochemically, neoplastic cells in KE and KE-F11 reacted to vimentin, S-100 protein, neuron-specific enolase, myelin basic protein, and glial fibrillary acidic protein in varying degrees, indicating neurogenic features; occasional cells reacted to alpha-smooth muscle actin. Cells positive for lysosomal enzymes (acid phosphatase and non-specific esterase), and ED1 (rat macrophage specific) were observed in KE-F11, and electron microscopically, cells with many lysosomes were frequently present, indicating expression of macrophage-like phenotypes. Bioassay analysis revealed that KE-F11 cells produced high levels of nerve growth factor. DNA synthesis was inhibited by addition of transforming growth factor-beta1 (TGF-beta1), and Northern blot analysis revealed that expression of c-myc, a cell cycle-related immediate early gene, was depressed by TGF-beta1. Likely, TGF-beta1 is a factor capable of inhibiting cellular growth of Schwann cells. mRNA expression of the low-density lipoprotein receptor-related protein (LRP) was seen in KE-F11 cells by Northern blot analysis, and the level was decreased by lipopolysaccharide (LPS) treatment. LRP may be attributable to regulation of Schwann cell functions. KE-F11 cells seeded on laminin-coated dishes exhibited more extended cytoplasmic projections than on collagen type I-coated dishes. The present study provides evidence that biological properties of malignant schwannoma-derived cells might be affected by exogenous factors such as TGF-beta1, LPS and laminin. These tumor lines may be useful for studies on pathobiological characteristics of Schwann cells.

Malignant fibrous histiocytoma (MFH) cells and macrophages/histiocytes have a common antigen recognized by a monoclonal antibody risen against a rat MFH-derived cloned cell line.

A monoclonal antibody (B9) was generated by using a rat malignant fibrous histiocytoma (MFH)-derived cloned cell line (MT-8) as the immunogen. Immunohistochemically, B9 reacted specifically with a cytoplasmic antigen of MT-8 cells. Furthermore, B9 immunolabeled another MFH-derived cloned cells (MT-9) and histiocytic sarcoma cells, as well as macrophages/histiocytes in normal and diseased tissues of rats. These findings suggest the presence of a common antigen recognized by B9 between MFH cells and macrophages/histiocytes. This suggests that MFH cells may express histiocytic nature.

Distribution of cells labelled by a monoclonal antibody (A3) against a cloned cell line derived from a rat malignant fibrous histiocytoma.

To pursue the histogenesis of malignant fibrous histiocytoma (MFH), of which the cell of origin is still debated, a monoclonal antibody (A3) was produced against a rat MFH-derived cloned cell line (MT-8). Antigen recognized by A3 was around 80 kDa in molecular weight and was seen on the cytoplasmic membrane of MT-8 cells by immunoelectron microscopy. A3 reacted specifically with MT-8 cells, with another rat MFH-derived cell line (MT-9) and with their induced tumours in syngeneic rats, but not with other rat tumours such as fibrosarcoma, histiocytic sarcoma, malignant meningioma, uterine leiomyosarcoma, endometrial stromal sarcoma, mononuclear cell leukaemia and malignant schwannoma. These findings indicate that A3 has a high specificity for rat MFH cells. In fetuses on gestation days 15, 18 and 20 and in postnatal rats aged 1, 4 and 8 days, A3 reacted with primitive mesenchymal cells in visceral organs and around arteries and bronchi, as well as in the lamina propria of intestinal mucosa, renal interstitium, meninges and perineurium. There were no A3-positive connective tissue cells in organs or other sites in adult rats more than 10 weeks old. It is therefore likely that MFH cells share antigens with primitive mesenchymal cells, which may be multipotent for mesenchymal differentiation. The present study suggests that MFH consists of a population of primitive, undifferentiated mesenchymal cells. A3 also immunolabelled endothelial cells of arteries, venules and pulmonary capillaries in fetal, postnatal and adult rats; vascular endothelial cells in chemically induced hepatic and renal lesions also reacted strongly with A3. However, the significance of endothelial immunoreactivity with A3 remains to be elucidated.

Macrophage populations and apoptotic cells in the liver before spontaneous hepatitis in Long-Evans Cinnamon (LEC) rats.

The inbred mutant strains of Long-Evans Cinnamon (LEC) rats spontaneously develops acute hepatitis as a result of abnormal copper accumulation, followed by chronic hepatitis, cholangiofibrosis and hepatocellular carcinoma. To shed some light on the role of macrophages in the liver failure, immunohistochemical methods were used to investigate the kinetics of macrophage populations in the liver of male LEC rats, in relation to the appearance of myofibroblastic cells and hepatocyte apoptosis. Rats examined at 24 weeks of age and moribund rats killed at 22-25 weeks of age had increased serum concentrations of aspartate aminotransferase and alanine aminotransferase, with jaundice and histological changes indicative of hepatic failure, whereas rats examined at 8, 12, 16 or 20 weeks old showed no such abnormal findings. Immunolabelling with ED1 (a monoclonal antibody recognizing rat macrophages) and ED2 (a monoclonal antibody specific for rat resident macrophages) revealed that numbers of blood monocyte-derived macrophages and Kupffer cells began to increase markedly at 16 weeks of age (before the onset of hepatitis). However, alpha-smooth muscle actin (SMA)-positive myofibroblastic cells (modulated perisinusoidal cells) and hepatocyte apoptosis, demonstrable by the TUNEL method, were rarely seen at 8, 12, 16, 20 or 24 weeks. There was no close relationship between macrophage expansion and the appearance of myofibroblastic cells or hepatocyte apoptosis. In moribund rats, only a few SMA-positive cells were seen in the periportal zones; hepatocytes undergoing apoptosis increased in number, and macrophages engulfing apoptotic bodies were observed occasionally, suggesting that apoptosis was related to hepatic failure as an early event. In addition, immunohistochemical examination demonstrated abnormal deposits of laminin along the sinusoids from 20 weeks, as an initial extracellular matrix protein in LEC rat livers.

Influence of progesterone and oestrogen on growth and morphology of a transplantable rat uterine smooth muscle tumour (SMT-Y).

Tumours of uterine smooth muscle are poorly understood neoplasms in which the effects of steroid sex hormones are complex. The influence of progesterone and oestrogen on a transplantable rat uterine smooth muscle tumour line (SMT-Y) was investigated. Female F344 rats given subcutaneous transplants of tumour fragments developed tumours, 1.5-2 cm in diameter, and were then treated with progesterone (10 mg/rat) or 17 beta-oestradiol (50 mg/rat). Tumours in treated groups were compared with those in untreated controls. During a 9-week observation period after treatment, progesterone promoted tumour growth from 4 weeks, with increased numbers of proliferating cells. In contrast, oestradiol inhibited tumour growth from 6 weeks; the degraded tumours, consisting mainly of vacuolated neoplastic cells, had decreased numbers of proliferating cells and increased numbers of apoptotic cells, demonstrable by in-situ terminal deoxyribonucleotide transferase (TdT)-mediated dUTP nick labelling. Immunohistochemically, tumours in control and progesterone groups were labelled positively for alpha-smooth muscle actin (SMA) and desmin but not for vimentin, whereas the degraded tumours in the oestradiol group had reduced reactivity for SMA and desmin but an increased reactivity for vimentin. These results indicate that progesterone may act as a promoter for uterine smooth muscle tumour growth by stimulating mitotic activity, whereas oestrogen may have suppressive effects on tumour growth, accompanied by morphological changes.

Establishment and characterization of cell lines derived from a transplantable rat malignant meningioma: morphological heterogeneity and production of nerve growth factor.

A cell line (KMY-J) was established from a transplantable tumor (MM-KMY) derived from a spontaneous malignant meningioma arising in an aged F344 rat, and three cloned cell lines (KMY-1, KMY-2 and KMY-3) were induced from the parent KMY-J. Morphologically, KMY-J and tumors induced in syngeneic rats by KMY-J showed cell pleomorphism. All neoplastic cells in KMY-J and its tumors were immunoreactive to vimentin; occasional cells reacted to ED1 (rat macrophage/histiocyte-specific antibody) and alpha-smooth muscle actin (alpha-SMA), indicating expression of histiocytic or myofibroblastic immunophenotypes of meningioma cells. In contrast, KMY-1, KMY-2 and KMY-3 consisted of a uniform cell population differing from each other. KMY-1-induced tumors were similar histologically to meningeal fibrosarcomas. Dendritic cells seen in KMY-2 cultures gave an appearance of arachnoid trabecular cells. In KMY-3 and its tumors, large round cells and multinucleated giant cells were predominant. Cells of these cloned cell lines also reacted to vimentin, but were negative for ED1 and alpha-SMA. By the bioassay using PC12 cells and reverse transcription-polymerase chain reaction for nerve growth factor (NGF) mRNA, production of NGF was demonstrated in the parent and cloned cell lines. The present cell lines may prove useful for studying the histological features of meningeal tumors and the bioactive factors produced by meningeal cells.

Morphological characteristics of a transplantable histiocytic sarcoma (HS-J) in F344 rats and appearance of renal tubular hyaline droplets in HS-J-bearing rats.

A transplantable tumour (HS-J) was established from a spontaneous histiocytic sarcoma found in a 24-month-old male F344 rat. Serial transplantations (seven generations) were made in syngeneic male and female rats by means of intraperitoneal or subcutaneous implants, with a 100% take rate. Rats given HS-J implants developed large nodules locally, with metastasis to distant organs. HS-J tumours consisted mainly of round to oval cells with abundant cytoplasm, arranged in a compact sheet. Enzyme- and immuno-histochemical examination showed that neoplastic cells reacted with ED1 (rat monocyte/macrophage-specific antibody), lysozyme, alpha 1-antitrypsin and lysosomal enzymes (acid phosphatase and non-specific esterase), indicating derivation from cells of the monocyte/macrophage lineage. The majority of neoplastic cells were negative for ED2 (rat tissue macrophage-specific antibody). Abnormal accumulations of hyaline droplets in the proximal renal tubular epithelial cells were seen in HS-J-bearing rats. The droplets were faintly immunopositive for lysozyme, but negative for alpha-2u globulin and albumin. It was considered that excessive production of the protein by tumour cells might lead to subsequent overload in renal tubules. HS-J may prove beneficial for studying the biological behaviour of monocyte/macrophage-derived tumours in the rat.

Characteristics of a rat fibrosarcoma-derived transplantable tumour line (SS) and cultured cell lines (SS-P and SS-A3-1) showing myofibroblastic and histiocytic phenotypes.

A transplantable tumour line (SS) was established in syngeneic rats from a spontaneous fibrosarcoma that had arisen in the submandibular salivary gland of a 24-month-old male F344 rat. A cell line (SS-P) was induced from SS, and a cloned cell line (SS-A3-1) was isolated from SS-P. The primary tumour consisted of oval to spindle-shaped cells arranged in bundles with abundant collagen fibres; ultrastructurally, neoplastic cells exhibited fusiform morphology with prominent rough endoplasmic reticulum. SS tumours showed marked interlacing fascicle and herring-bone growth patterns. SS-P and SS-A3-1 were similar morphologically to each other, consisting of oval, spindle or polygonal cells and occasional multinucleated giant cells. Tumours induced by SS-P and SS-A3-1 were histologically similar to SS tumours. Immunohistochemically, all cells in the primary tumour, SS tumours and tumours induced both by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures gave a positive reaction to vimentin. Interestingly, neoplastic cells reacting to ED1 (rat macrophage/histiocyte-specific antibody) and alpha-smooth muscle actin (alpha-SMA) appeared in SS tumours and tumours induced by SS-P and SS-A3-1 and by SS-P and SS-A3-1 cultures. Cells with histiocytic fine structures and myofibroblastic cells with cytoplasmic actin-like microfilaments were also observed by electron microscopy. The present rat fibrosarcoma-derived transplantable tumour line (SS) and cell lines (SS-P and SS-A3-1) might express myofibroblastic and histiocytic phenotypes, probably depending on the surrounding conditions. These cell lines may prove useful for studying the mechanisms of phenotypic plasticity in neoplastic fibroblasts.