Foregut cyst of the oesophageal wall in a cynomolgus monkey (Macaca fascicularis).

Congenital oesophageal cysts of foregut origin are rare in animals and human beings. This report describes a case in a 4-year-old cynomolgus monkey with no clinical symptoms. The cyst, which was located within the oesophageal submucosal tissue near the mid-point of the oesophagus, was lined with pseudostratified ciliated epithelium and had a thin layer of submucosal tissue. The cyst was surrounded by a smooth muscle layer which was partly intermingled with the circular muscle layer of the oesophagus. The muscularis mucosae of the oesophagus was not shared with the cyst wall. Simple tubular glands were present, opening into the cyst lumen. No communication between the cyst lumen and the oesophagus was observed. Cartilaginous tissue, which is a diagnostic feature of bronchogenic cysts, was not identified in the cyst wall. On the basis of the histopathological features, a foregut cyst of the oesophagus was diagnosed.

Ferro-type orbital state in the Mott transition system Ca2-xSrxRuO4 studied by the resonant x-ray scattering interference technique.

We have succeeded in detecting ferro-type orbital states in Ca2-xSrxRuO4, which is the first outcome in a 4d Mott transition system by the resonant x-ray scattering interference technique. For x=0 (Mott insulator), the resonant signal for d(xy) orbital ordering is observed even at room temperature, in which the Jahn-Teller distortion is negligible. The signal disappears near the metal-insulator transition. On the other hand, in a metallic phase for x=0.5, orbital polarization with d(yz/zx) character dominates. With lowering temperature, the magnitude of the resonant signal slightly decreases owing to the additional influence of the gamma band with d(xy) character.

Participation of different macrophage populations and myofibroblastic cells in chronically developed renal interstitial fibrosis after cisplatin-induced renal injury in rats.

To shed some light on the mechanisms behind renal fibrogenesis, the present study immunohistochemically investigated the participation of different macrophage populations and myofibroblastic cells in rat renal interstitial fibrosis developed chronically after repeated injection of cisplatin (2 mg/kg body weight, once weekly for 7 weeks). During the 19-week recovery period after the final injection, fibrotic lesions progressively developed in the corticomedullary junction, with the greatest level at post-final injection (FPI) week 5, and then the lesions were gradually repaired by PFI week 19, indicative of a healing process. In conformity with the development of fibrotic lesions, the number of myofibroblastic cells reacting with an anti-alpha-smooth muscle actin antibody was increased, with a peak at PFI week 3, and collagens (types I, III, and IV), fibronection, and laminin were excessively accumulated in these areas. Interstitial cells forming the fibrotic lesions showed mitotic activity at the early stages, whereas they disappeared by apoptosis in the healing process. A large number of cells reacting with an antibody of ED1 (for exudate macrophages), ED2 (for resident macrophages), or OX6 (for major histocompatibility complex class II-presenting macrophages and interstitial dendritic cells) had already appeared at PF1 week 1, and then their numbers increased, with a peak at PFI weeks 7, 3, and 9 in ED1-, ED2-, and OX6-positive cells, respectively. Thereafter, the number of ED1- and ED2-positive cells decreased, whereas the number of OX6-positive cells persisted at a high level until PFI week 19. In the healing process, clusters of lymphocytes were present, the development of which might have been related to OX6-positive cells. The present study demonstrated that chronically developing rat renal interstitial fibrosis might be produced by the complicated mechanisms evoked by interactions between different macrophage populations and myofibroblastic cells, because macrophages show heterogeneous functions depending on microenvironmental factors.

Humoral hypercalcemia of malignancy in female F344 rats implanted with a transplantable rat pulmonary carcinoma line (IP).

A transplantable rat pulmonary carcinoma line (IP) in F344 rats is a useful animal model for humoral hypercalcemia of malignancy (HHM). The present study analyzed the degree of HHM by implanting IP into F344 female rats aged 6, 20 or 45 weeks. IP-bearing females developed elevated plasma parathyroid hormone-related protein levels, hypercalcemia and increased osteoclastic activity as well as calcification in various organs. The severity of such HHM differed depending on age; particularly, calcification showed age-dependent reduction. HHM development in IP-bearing females may be influenced by age-related factors.

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.

Immunohistochemical analysis of macrophages, myofibroblasts, and transforming growth factor-beta localization during rat renal interstitial fibrosis following long-term unilateral ureteral obstruction.

Renal interstitial fibrosis was induced in rats by chronic unilateral ureteral obstruction (UUO). To identify the mechanisms behind the fibrosis, macrophage influx, myofibroblast involvement, and the localization of transforming growth factor-beta (TGF-beta, a fibrogenic cytokine) were investigated immunohistochemically in rats euthanatized at 0 (controls), 3, 6, 9, 12, and 15 days after UUO. The number of alpha-smooth muscle actin-positive myofibroblasts began to increase significantly in the medulla from day 3, and the development of medullary fibrosis was confirmed from day 6 by morphometric analysis. From day 9, papillary fibrosis also developed in association with an increased number of myofibroblasts. These myofibroblasts showed a parallel orientation to the mucosal surface of the pelvis. In the medulla and papilla, from day 6 the number of ED1 (primary antibody)-positive macrophages began to increase significantly. There appeared to be a relationship between macrophage influx and myofibroblast involvement. By contrast, in the cortex there was no marked increase in myofibroblasts nor development of fibrotic tissues, regardless of increased number of macrophages from day 6. Immunohistochemically, no staining for TGF-beta was found in infiltrating macrophages or myofibroblasts. However, TGF-beta was localized on some cortical proximal renal tubules both of normal control and obstructed kidneys in the early stages on days 3, 6, and 9, suggesting that the possible origin of TGF-beta may be renal epithelia. However, the staining intensity for TGF-beta on the renal epithelia tended to be weakened in advanced obstructed kidneys on days 12 and 15. The likely contribution of TGF-beta to the advanced stages of UUO-induced renal fibrosis remains to be determined.

Relationship between vimentin expressing renal tubules and interstitial fibrosis in chronic progressive nephropathy in aged rats.

We investigated the relationship between regenerating renal tubular epithelial cells and myofibroblast development in chronic progressive nephropathy (CPN) of aged male F344 rats. We used established criteria to classify disease in rats with CPN as grade 1 (n=9), grade 2 (n=10), grade 3 (n=7) and grade 4 (n=4). Five young rats served as controls (grade 0). The ratio of fibrotic tissues per unit area, assessed in collagen type III-immunostained sections by morphometric analysis, increased significantly with advancing grade of CPN. Vimentin-expressing, regenerating renal tubules were found from grade 1 and continued to increase in number up to grade 3, decreasing slightly, however, in grade 4. Similar kinetics were seen for the number of alpha-smooth muscle actin-positive myofibroblasts, and there was a significant correlation between the number of regenerating renal tubules and myofibroblast development (correlation coefficient=0.83, P<0.01). The myofibroblasts developed in close association with the fibrotic areas seen in grades 1-4; the cells also reacted to desmin or vimentin, indicating the activated state. Immunohistochemistry for platelet-derived growth factor (PDGF)-BB and transforming growth factor (TGF)-beta revealed that vimentin-positive renal tubules were positive for PDGF-BB, but negative for TGF-beta, and that interstitial reactive cells showed no positive reactions for both factors. The present studies on rat CPN showed that regenerating renal tubules may be a major source of a fibrogenic growth factor, PDGF-BB, and that the PDGF-BB might induce the development of fibrogenic cells, myofibroblasts, culminating in progressive interstitial fibrosis.

Macrophages, myofibroblasts, and extracellular matrix accumulation in interstitial fibrosis of chronic progressive nephropathy in aged rats.

Progressive renal fibrosis is considered to be the final common pathway leading to chronic renal failure. Macrophages are thought to play a role in the induction of the myofibroblasts that produce extracellular matrix (ECM) proteins in renal interstitial fibrosis. We immunohistochemically investigated the relationship between infiltrating macrophages and myofibroblast development in chronic progressive nephropathy (CPN) in 24 month-old male F344 rats, and we also analyzed components of ECM proteins using immunofluorescence microscopy. According to histomorphologic criteria for severity, described elsewhere, rats with CPN were divided into grade 1 (n = 20), grade 2 (n = 34), grade 3 (n = 10), and grade 4 (n = 6). The ratio of fibrotic tissues per unit area, determined by morphometric analysis, was increased with advancing grade of nephropathy. The number of interstitial macrophages continued to be increased gradually, with a peak in grade 4. Alpha-smooth muscle actin-positive myofibroblasts developed, surrounding the regenerating renal tubules in conjunction with the fibrotic areas. The number of the myofibroblasts was also increased, with a peak in grade 3, but in grade 4, it was slightly decreased. There was a significant relationship between the number of infiltrating macrophages and the degree of interstitial fibrosis (r = 0.802; P < 0.05). These observations suggest that macrophages and myofibroblasts might be key cells in fibrogenesis in CPN. However, there was no significant correlation between the numbers of macrophages and myofibroblasts (r = 0.198; P > 0.05), although a significant relation between these cells has been reported in the early stages of experimental rat renal fibrosis. Immunostaining for collagen type IV demonstrated increased expression in thickened tubular basement membranes. Abnormal depositions of collagen types I and III, fibronectin, and tenascin were also observed in fibrotic areas adjacent to dilated or atrophic tubules with thickened basement membranes. These ECM proteins were increased in conjunction with the grade of nephropathy, suggesting that ECM accumulation might contribute to progression of renal interstitial fibrosis.

Lysozyme-containing renal tubular hyaline droplets in F344 rats bearing a rat fibrosarcoma-derived transplantable tumor.

Renal tubular hyaline droplets developed in male and female F344 rats bearing a rat fibrosarcoma-derived transplantable tumor (SS). The droplets accumulated exclusively in the proximal renal tubular epithelia as eosinophilic granules of various sizes in hematoxylin and eosin-stained sections. The granules stained bright red with azan-Mallory stain. Immunohistochemically, the droplets were positive for lysozyme to various degrees but were negative for alpha 2u-globulin, albumin, and alpha 1-antitrypsin. These findings indicated the involvement of lysozyme, a low-molecular-weight protein, in the droplet formation. The morphological and immunohistochemical findings of the hyaline droplets bore a close resemblance to those reported in rats as a secondary lesion to spontaneous histiocytic sarcomas. Others have speculated that renal tubular hyaline droplets in histiocytic sarcoma-bearing rats are formed in lysosomes through cellular overload of lysozyme secreted excessively by the tumor cells. However, neoplastic cells of SS tumors were negative to lysozyme. The pathogenesis of renal hyaline droplets appearing in SS tumor-bearing rats remains to be investigated.

A possible immunosuppressant, cycloprodigiosin hydrochloride, obtained from Pseudoalteromonas denitrificans.

Cycloprodigiosin hydrochloride (cPrG.HCl), a member of the prodigiosin family, is a red pigment obtained from the marine bacterium Pseudoalteromonas denitrificans. cPrG.HCl markedly suppressed 3H-thymidine incorporation by concanavalin A stimulated murine splenocytes but had little effect on lipopolysaccharide dependent 3H-thymidine incorporation, indicating that cPrG.HCl acts as a selective inhibitor of T cell proliferation in the same way as other members of the prodigiosin family. cPrG.HCl inhibited the proliferation of the PMA stimulated Jurkat cells through an apoptotic process. Intriguingly, cPrG.HCl inhibited the H+ translocation by vacuolar type ATPase in chromaffin granule membranes without any effect on either its ATPase activity nor on the membrane conductance of phospholipid bilayers, suggesting that cPrG.HCl selectively uncouples H+ translocation from the ATPase reaction rather than acting as a non-specific ionophore. Since crystalline cPrG.HCl is highly stable, it raises the possibility of its therapeutic use as an immunosuppressant.

In vivo responses of macrophages and myofibroblasts in the healing following isoproterenol-induced myocardial injury in rats.

To clarify the relation between macrophage and myofibroblast involvement in various myocardial diseases, the authors investigated the kinetics of these cells in the healing (scar tissue formation) following isoproterenol-induced myocardial injury in rats. Alpha-smooth muscle actin (alpha-SMA) expressing myofibroblasts were seen at the border of the affected area and appeared in the greatest numbers on days 3-7 post-injection, followed by a gradual decrease by day 35. The peak on day 3 was consistent with the timing of the highest proliferative activity of myofibroblasts. The number of ED1-positive macrophages began to increase as early as day 1, reaching a peak on day 3 within the injured myocardium. The expansion of ED1-positive macrophages preceded an increased number of alpha-SMA-positive myofibroblasts suggesting that myofibroblast proliferation and activation may be mediated by factors released by ED1-positive macrophages in response to myocardial injury. The number of ED2-positive tissue-fixed, resident macrophages gradually, increased from day 3 post-injection, and peaked on day 14, but the number of ED2-positive macrophages was consistently fewer than that of ED1-positive macrophages during the 35 day-observation period after the injection. The labelling index of the ED2-positive cells was maximal on day 14, indicative of local proliferation of resident macrophages. In the healing process after myocardial injury, ED1-positive macrophages increase markedly in the early stages: ED2-positive macrophages appear later.

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.

Immunohistochemical study of rat renal interstitial fibrosis induced by repeated injection of cisplatin, with special reference to the kinetics of macrophages and myofibroblasts.

Progressive renal interstitial fibrosis occurs following tissue injury, resulting in chronic renal failure. In the fibrogenesis, macrophages are speculated to induce myofibroblasts producing matrix protein. The kinetics of these cells in renal fibrosis induced in rats by repeated injection of cisplatin (CDDP) (2 mg/kg body weight, once weekly) was investigated immunohistochemically. During the 10-wk injection period, epithelial damage of the proximal renal tubules in the corticomedullary junction was seen, followed by cystic dilation of the affected tubules. During the 8-wk recovery period following the seventh injection, the size of the dilated lumina was diminished and atrophic tubules lined by regenerating epithelial cells appeared. Morphometrical analysis revealed that fibrosis began to develop around the dilated renal tubules in the injection period and was more advanced in the following recovery period. Coinciding with development of fibrotic tissues, the number of alpha-smooth muscle actin-positive myofibroblasts was significantly increased in the areas compared to that of controls. In the injection period, despite a significant increase in myofibroblast number, an elevation of ED-1 (primary antibody)-positive macrophage number was not observed. In the recovery period, however, a significant elevation of macrophage number was noticed in markedly advanced interstitial fibrosis. This suggests that rapid expansion of the macrophage population, probably resulting from release from myelosuppression due to CDDP, might contribute in part to development of myofibroblasts, leading to the augmented fibrosis in the recovery period.

Immunohistochemical observations on the kinetics of macrophages and myofibroblasts in rat renal interstitial fibrosis induced by cis-diamminedichloroplatinum.

It has been speculated elsewhere that growth factors produced by macrophages in response to tissue damage induce a modulation of pre-existing fibroblasts into myofibroblasts, leading to fibrosis. The development of these cells in cis-diamminedichloroplatinum (CDDP)-induced rat renal interstitial fibrosis was observed immunohistochemically. In the cortico-medullary junction, nuclear changes and epithelial necrosis of the proximal renal tubule (mainly the P3 segment) were seen on days 1 and 4 after a single dose (6 mg/kg body weight) of CDDP, and regenerating epithelium appeared on day 7. Gradually developing fibrosis was observed around the affected tubules on days 14 and 28. The increase in fibrosis was confirmed by histometrical analysis. The number of ED-1 (primary antibody) positive macrophages reached a peak in the affected cortico-medullary junction on day 7 and this was accompanied by an increase in muscle actin-positive myofibroblasts. On days 14 and 28, macrophages had declined in number, but the number of muscle actin-positive myofibroblasts in the fibrotic area was still high as compared with control values. Cytoplasmic myofilaments were observed in myofibroblasts by electron microscopy. These findings suggest that the myofibroblasts participate in renal interstitial fibrosis in the rat, and that their appearance may be related to macrophage infiltration in response to tubular injury, at least in the early stages of fibrosis.

Immunohistochemical observations of macrophages and perisinusoidal cells in carbon tetrachloride-induced rat liver injury.

In rat liver injured by carbon tetrachloride, ED-1-positive monocytes and resident macrophages significantly increased in number in damaged perivenular zones on days 2 and 4 after single or double dosing, peaking on day 2 and being two-fold more after the 2nd dosing. ED-2-positive resident macrophages significantly increased in number only on day 4 after the single dosing and on days 2, 4 and 6 after the 2nd dosing with a peak on day 2. A significant increase in number of muscle actin-positive perisinusoidal cells was seen on day 4 and on days 2 and 4 after the 1st and 2nd dosings, respectively, reaching a peak on day 4 and being more remarkable after the 2nd dosing.

High-performance liquid chromatography/chemiluminescence determination of biological thiols with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide.

The peroxyoxalate chemiluminescence detection of biological thiols combined with high-performance liquid chromatography (HPLC) is described. SH groups of the thiol compounds including glutathione (GSH), cysteine, N-acetylcysteine, cysteamine, and D-penicillamine were labelled with N-[4-(6-dimethylamino-2-benzofuranyl)phenyl]maleimide (DBPM), a specific fluorogenic reagent for SH group. The labelling reaction was carried out at 60 degrees C for 30 min and at pH 8.5 and a sample of the resulting reaction mixture was subjected to HPLC. Five kinds of labelled thiols were separated within 12 min on ODS-80 column (150 x 4.6 mm ID; 5 microns) and detected in the ranges from 500 fmol to 2 pmol/100 microL (cysteamine and N-acetylcysteine), to 3 pmol/100 microL (cysteine) and to 5 pmol/100 microL (GSH and D-penicillamine). The lower detection limits were from 7 fmol (cysteamine) to 113 fmol (GSH) per 100 microL (S/N = 2). The method was applied to the determination of thiols in a rat liver. The amounts of glutathione and cysteine were 1.23 +/- 0.15 mumol/g (n = 5) and 0.15 +/- 0.04 mumol/g (n = 5), respectively.

High-performance liquid chromatography-fluorometry for the determination of thiols in biological samples using N-[4-(6-dimethylamino-2-benzofuranyl) phenyl]-maleimide.

The selective determination of thiols in biological samples was investigated by high-performance liquid chromatography using N-[4-(6-dimethylamino-2-benzofuranyl)phenyl] maleimide, which was found to give fluorescent products when treated with certain thiols. Six kinds of thiol (reduced glutathione, cysteine, N-acetylcysteine, cysteamine, homocysteine and coenzyme A) could be separated simultaneously within ca. 12 min and determined at final level of sensitivity. The method was successfully applied to the determination of thiols in rat tissues and plasma and in human normal serum.