To clarify the origin and nature of "histiocytic" cells in malignant fibrous histiocytoma (MFH), immunoreactivities to rat macrophage/histiocyte-specific monoclonal antibodies (ED1 and ED2) and monocyte chemoattractant protein-1 (MCP-1) production were investigated using rat transplantable MFH-derived cloned cells (undifferentiated MT-8 and fibrohistiocytic MT-9). In different cultures, the positive rates for ED1 and ED2 ranged from 2.5% to 26.0% in MT-8, and from 6.0% to 40.0% in MT-9. In homotransplants, ED1-positive cells were frequently observed in MT-9 tumors, but barely seen in MT-8 tumors. In both MT-8 and MT-9 tumors, ED2-positive cells were not detected within tumors. Immunophenotypic characteristics of MT-8 and MT-9 to these antibodies seemed to be easily altered depending on in vitro and in vivo conditions. By ELISA, MCP-1 was hardly detected in culture supernatants of MT-8 and MT-9, but its level was very high in sera of MT-8- and MT-9-tumor-bearing rats. In rats with these tumors, the number of circulating monocytes was significantly increased and the presence of factors stimulating macrophage proliferation was demonstrated by the colony formation assay usint rat bone marrow cells. Histiocytic cells in MT-8 and MT-9 tumors that were produced in nude mice reacted only with antibody to mouse macrophage-associated antigen. This suggests the existence of non-neoplastic, infiltrated macrophages of host origin that were induced probably by MCP-1 and factors stimulating macrophage proliferation. The present studies indicate the presence of heterogeneities in the origin and immunophenotypes of "histiocytic" cells in rat MFH.

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Effects of differences in calcium (Ca) intake on bone metabolism under metabolic acidosis were examined by bone histomorphometry in rats. Rats were divided into 5 diets; low Ca (0.02%) diet (LCD), moderate low Ca (0.3%) diet (LmCD), standard Ca (0.62%) diet (SCD), moderate high Ca (1%) diet (HmCD) and high Ca (3%) diet (HCD). Each diet rats were subdivided into the acidotic group given a 1.8% ammonium chloride solution as drinking water and control group given deionized water. Blood and double labeled bone were collected 30 days later. Arterial blood pH was significantly lower, and plasma ionized Ca level and urinary Ca excretion were higher in the acidotic groups than those in the control groups in all diets. Breaking force in femur and bone volume in tibial proximal metaphysis were significantly lower in the acidotic groups than those in the control groups in LCD, LmCD and SCD. In the acidotic groups, osteoid thickness was significantly higher in LCD than that in LmCD, HmCD and HCD. Mineral apposition rate in the acidotic groups was significantly higher in LCD and LmCD than that in HmCD and HCD. These results suggest that bone mineral loss and bone fragility under metabolic acidosis may be accelerated by high turnover of bone metabolism due to insufficient dietary Ca intake, but can be prevented by adequate supplementation of Ca.

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A radioimmunoassay (RIA) system for quantification of bovine β₂-microglobulin (β₂-M) in serum and urine was developed. The protein isolated from bovine colostrum showed a single band in SDS-PAGE, and its molecular weight was approximately 11, 600. Amino acid sequences for the first 24 residues and the amino acid composition of the protein were in agreement with those in the bovine β₂-M of previous research works. In an Ouchterlony test, a single precipitation line was formed between the protein and the antiserum made by the protein. From these results, it was confirmed that the protein isolated from the colostrum was pure bovine β₂-M. For creation of an RIA calibration curve for urine, a urine void of β₂-M, as much as possible (β₂-M-free urine), and a PBS were used as diluents. Intraassay (n=10) and interassay (n=3) variances were 1.7-4.6% and 7.1-11.5% in the PBS dilute method, and were 1.4-5.1% and 12.3-13.5% in the β₂-M-free urine dilute method, respectively. Mean recoveries were 160±19% (mean±S.D.) and 98.4±7.9% in PBS and β₂-M-free urine, respectively. It was found that the method using the β₂-M-free urine as a diluent was more accurate than using PBS. The β₂-M concentrations in serum and urine of healthy Holstein cows measured by this RIA system showed a logarithmic normal distribution for urine and a normal distribution for serum. The mean β₂-M concentrations were 0.0305^+0.0043_-0.0210mg/l (Geometric mean ±S.D., n=43) in urine and 2.87±0.45 mg/l (Arithmetic mean ±S.D., n=26) in sera. Further, we could not observe the particular tendency of daily variation in urinary β₂-M concentrations of healthy cows (Holstein, n=3×2 days).

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Cytotoxic effect of progesterone on the neoplastic cells was investigated using POS cells, a cell line established from a spontaneous osteosarcoma in a dog. The 20α-HSD activity of POS cells was 0.85±0.26 NADPH nmol/min mg protein, demonstrating that a considerable activity was present in this tumor cell. When progesterone was added to the medium and cultured for 48 hr, progesterone dose-dependently inhibited the cell growth, showing that progesterone was cytotoxic on this osteosarcoma cells in vitro. In order to assure the role of 20α-HSD on the cell growth, various concentrations of four types of steroid derivatives, STZ 20, 23, 25, and 26, potent inhibitors of 20α-HSD, were added to the medium with 0.1μM progesterone. Then, the POS cell growth was more strongly inhibited, which may suggest that the cytotoxicity of progesterone was enhanced by inactivation of 20α-HSD by STZs. From these results, POS osteosarcoma cells have 20α-HSD, which might play an important role in tumor cell growth against the cell toxicity of progesterone.

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The pathogenesis of poikilocytosis in calves was investigated. Nine calves 1 to 2 months old exhibiting poikilocytosis (poikilocytic calves), were examined. The proportion of poikilocytes in their blood was more than 70% in two calves (severely poikilocytic), while it averaged 30% in the remaining seven calves (moderately poikilocytic). All of the polikilocytic calves exhibited mild anemia. The concentration of plasma iron in 8 of them was 20-30% of that in normal calves, but it increased as the calves aged. The polikilocyte count in moderately poikilocytic calves decreased in proportion to the increase of the plasma iron concentration, though a high poikilocyte count was observed in a severely poikilocytic calf even after the plasma iron concentration reached the normal level. Column chromatography of a hemoglobin preparation from all the calves examined revealed 5 distinct peaks. Three peaks were embryonic, fetal and adult hemoglobins, respectively. The other peaks were tentatively designated Hb-2 and Hb-3, respectively. The amount of Hb-2 in the two severely poikilocytic calves was about three times that in the other calves including those moderately poikilocytic, and it decreased as the calves aged. The decrease of Hb-2 in one severely poikilocytic calf was related to a reduction of the proportion of poikilocytes in its peripheral blood. There was no significant difference in age-dependent changes of hemoglobin types between moderately poikilocytic calves and normal calves. Furthermore, SDS-PAGE analysis of red cell membrane revealed that protein 4.2 consisted of two polypeptides in the two severely poikilocytic, and three moderately poikilocytic calves, while it consisted of a single polypeptide in the remaining calves examined. These results indicated that the abnormalities of hemoglobin composition and protein 4.2 in the erythrocyte membrane observed in the severely poikilocytic calves might be responsible for the morphological changes in the erythrocytes, whereas iron deficiency was thought to be the predominant cause of poikilocytosis in the moderately poikilocytic calves.

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The present study was conducted to investigate the influence of aging of recipient oocyte on the developmental ability of reconstituted mouse embryos produced from the cytoplast of oocytes and single blastomeres of early or late 2-cell stage embryos by electrofusion. Oocytes were obtained at 14 (newly ovulated oocytes), 18, 22 (oocyte that time passed after ovulation; aged oocyte) hr after hCG injection and oocyte cytoplast was produced by manual enucleation using a fine glass needle under the dissecting microscope. The aging of the oocytes significantly influenced on the fusion rate of the reconstituted embryos (14hr: 42.7-47.2% vs. 22hr: 75.3-77.6%). Similarly, the cleavage rate of reconstituted embryos increased with aging of the oocytes (14hr: 50.8-56.3% vs. 22hr: 82.2-90.7%). The percentage of reconstituted embryos produced from cytoplast of aged oocytes (22hr post hCG) and single blastomeres of late 2-cell stage embryos developing to the blastocyst (20.8%) was significantly higher than that of reconstituted embryos produced by other combinations (2.0-8.2%: P<0.01). Although cell cycle stage of donor nuclei influenced to developmental ability of reconstituted embryos, these results are probably related to the aging of the oocytes since aged oocytes can be activated more easily by electrical stimulation than newly ovulated oocytes.

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The cat pancreas has a complex neural supply comprising adrenergic, cholinergic and peptidergic innervations. To elucidate the localization of neuropeptides and enzymes in neural elements of the cat pancreatic islets, single or double immunohistochemical staining was performed using avidin-biotin-peroxidase complex and/or peroxidase-antiperoxidase methods. In intrapancreatic ganglia, numerous galanin (Gal)- or vasoactive intestinal polypeptide (VIP)-immunoreactive (IR) ganglion cells were seen. Many nitric oxide synthase (NOS)- or cholecystokinin (CCK)-IR and a few bombesin (Bom)- or substance-P (SR)-IR ganglion cells were also observed. Intrapancreatic ganglion cells were surrounded by numerous VIP- or SP-IR, many Bom- or neuropeptide Y (NPY)-IR, several calcitonin gene-related peptide (CGRP)- or peptide histidine isoleucine-IR and a few NOS- or CCK-IR nerve terminals. In pancreatic islets, many VIP-IR nerve terminals were located predominantly around insulin (Ins)-IR cell masses and many tyrosine hydroxylase-IR nerve terminals were demonstrated in the core of Ins-IR cell masses as well as around them. Gal-, NPY-, SP- or CGRP-IR nerve terminals were found much more frequently within Ins-immunonegative cell areas than in Ins-IR cell masses. These observations may provide a morphological basis for the possible transmitting or modulating role of neuropeptides in pancreatic islets of the cat.

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Aggregation properties of platelets were examined in Japanese Black cattle with Chediak-Higashi syndrome (CHS) and normal control cattle. Platelet aggregation induced by collagen was decreased in platelets of the cattle with CHS, but not ADP (10-20 μM), thrombin (0.5-1.0 U/ml) and phorbol-12-myristate 13-acetate (PMA, 3.2 μM). The aggregation response induced by collagen in CHS platelets lacks the change in shape which usually occurs in normal platelets. Simultaneous stimulation by collagen (10 μg/ml) + ADP (10 μM) is effective in restoring collagen-induced aggregating response in CHS platelet, although pretreatment of ADP (10 μM) could not restore the collagen (10 μg/ml)-induced aggregating response, suggesting that there is a certain threshold of stimulation intensity above which collagen-induced aggregation of CHS platelet can begin. Control normal platelets, previously exposed to ADP (10 μM) and collagen (10 μg/ml), showed no further response to exposure to a third aggregating agent (arachidonic acid, 5 mM). On the other hand, the final agent was able to elicit aggregating responses in CHS platelets, suggesting that the arachidonate aggregating system may be suppressed in CHS cattle, but fully activated in control animals. Furthermore, normal platelets showed a significant decrease in aggregating response to collagen when pretreated with a cyclooxygenase inhibitor, indomethacin (10^-5 M), whereas CHS platelet was insensitive to indomethacin. This indomethacin-treated normal platelet mimicked the CHS collagen-induced aggregation pattern. These data suggest that a signal transduction process from receptor-operated events to arachidonate metabolism is suppressed in collagen-induced CHS platelet aggregation.

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Renal transplantation was performed on 6 cats with chronic renal failure. Clinical signs and the blood chemistry findings (BUN, Cr, IP) improved one week after renal transplantation. Renal anemia also improved 3-4 weeks after surgery. Two cases had the same complications, hydronephrosis and hydroureter within 1 or 2 weeks of surgery, evidenced by marked elevations of BUN and Cr, suggesting that ureteral obstruction at the site of ureterocystostomy is the main complication of renal transplantation in cats. As a new resolution for this problem, we transplanted the ureter with the intact ureteral opening of the bladder from the donor in the remaining 4 cats. Ureteral obstruction was not observed in them, thus the procedure was considered to be useful for avoiding this complication. Two cats died of pneumonia and other complications within 3 months. There were no acute rejections or side effects from the immunosuppressant during the observation period in the remaining four cats.

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A three-year-old, male Yorkshire terrier was presented with blindness, circling, hind limb weakness, and convulsive seizure for the past 3 months. Characteristic clinical findings were chronic, progressive neurological signs involving cerebrum and brain stem, an elevation of brain-type isoenzyme of serum creatine kinase, appearance of high voltage slow activity in electroencephalogram, and multifocal lesions in the cerebral hemispheres on magnetic resonance imaging. Necrotizing encephalitis of Yorkshire terrier was diagnosed after postmortem pathological examination. This is the first case report of the disease in Japan.

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A morphological examination was performed on megakaryocytic emperipolesis containing neutrophils in the rat bone marrow induced by intravenous administration of lipopolysaccharide (LPS). Ultrastructurally, the engulfed neutrophils appeared to lie in the demarcation membrane system (DMS) of megakaryocytes. Intact neutrophils generally migrated from DMS into the lumen of vascular sinus across the endothelium of sinus wall. However, some neutrophils showed characteristics of apoptosis, and DNA fragmentation was detected in these cells by the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling method. Thus, the cell death of neutrophils engulfed by megakaryocytes was thought to be apoptosis. The present results suggest that megakaryocytes contribute to random selection of an excess of neutrophils in the bone marrow of rats treated with LPS.

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Rhodococcus equi is an emerging opportunistic pathogen of human immunodeficiency virus-infected patients. However, little is known about the distribution of virulent and intermediately virulent R.equi in human environment. In the present study, R.equi was isolated from 173 of 234 (73.9%) samples collected from soil and sand on 115 parks and 49 yards in Japan. The numbers of R.equi from soil and sand ranged from 2.5×10¹ to 1.2×10⁵ per gram of sample. None of 1, 294 isolates from those samples showed virulence-associated 15- to 17-kDa antigens and a 20-kDa antigen. These results suggest that avirulent R.equi is widespread in parks and yards, but the human environment has not been contaminated with virulent and intermediately virulent R.equi strains yet.

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A female Maltese dog with patent ductus arteriosus (PDA) showing left congestive heart failure and moderate pulmonary hypertension was evaluated for pulmonary vasodilatory capacity using low concentrations of inhaled nitric oxide (NO) in comparison with oxygen during preoperative cardiac catheterization. Increasing the inspired oxygen concentration (FiO₂) to 1.0 without adding NO did not reduce the mean pulmonary arterial pressure (mPAP). However, inhalation of NO at FiO₂ 1.0 reduced mPAP rapidly without changing other hemodymamic and gas exchange parameters. From these results, inhaled NO caused selective pulmonary vasodilation without producing systemic vasodilation, which may provide a safe and effective mean of evaluating the pulmonary vasodilatory capacity of dogs with PDA.

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A crude extract of the 2nd generation schizonts (2GS) of Leucocytozoon caulleryi and chicken serum containing serum-soluble antigens were applied to an immunoaffinity column using immunoglobulin of chickens recovered from L.caulleryi infections. The fractionated antibody-bound proteins showed several bands between 70 kDa and 35 kDa by the electrophoretic analysis. By immunization of chickens, the fractionated proteins conferred the protection at varying degrees comparable to that conferred by the unfractionated original materials. These results indicated that antiserum against 2GS can recognize the proteins involved in protective immunity.

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The complete genomic sequence of chicken anaemia virus (CAV) TK-5803 strain was determined. Comparisons of sequence data showed 37 nucleotide differences between TK-5803 and Cux-1, 38 nucleotide differences between TK-5803 and 26P4, and 48 nucleotide differences between TK-5803 and 82-2. There were 65 nucleotide differences in the largest open reading frame (ORF3) between TK-5803 and one Australian isolate. Base changes introduced amino acid changes at 6 positions in the C-terminal half of VP3, at 4 positions in the C-terminal quarter of VP2 and at 17 positions in VP1 among the strains. These indicate that the N-terminal half of VP3 and the N-terminal three quarter of VP2 are well conserved, and might sustain essential function of these proteins. The amino acid changes in VP1 which is thought to be the capsid protein may influence the antigenic character of different VP1s.

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Canine pericardium which had been treated with polyepoxy compounds (Denacol [○!R] EX-313) was used as a patch graft for the correction of experimentally-created diaphragmatic defects in five dogs belonging to the same litter. Clinical, macroscopic and histological examinations were conducted every month up to five months after suturing of the patch graft. Clinical examination of the patch graft showed no apparent abnormalities. Macroscopic examination conducted during autopsy showed that the patch graft maintained adequate elasticity for five months after suturing, the surface of the patch graft was covered with a thin membrane and neovascularization was observed. Histological examination showed that the surface of the patch graft was covered with a thin membrane. Inflammatory tissue reactions were observed at one month, but gradually decreased from the second month onwards. In addition, the patch graft had excellent tissue affinity.

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A total of 4, 111 chicken sera collected from 1985 to 1995 at 137 farms in 36 prefectures were subjected to an enzyme-linked immunosorbent assay (ELISA) to test for antibodies against turkey rhinotracheitis (TRT) virus. The antibodies to TRT virus were detected in the chicken sera collected from 1988 to 1995, but the antibodies to TRT virus were undetectable in the sera of chickens collected before 1987. The antibody positive rates each year ranged from 2.2% (1988) to 54.2% (1995). Recently, the TRT virus has spread over the flocks of commercial broiler and layer chicken throughout the country. The serological findings indicated that the TRT virus had invaded the chickens before 1988, and has been widespread thereafter in Japan.

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New cell lines, designated MeMS and MeFS, were established from the epithelial type of mesotheliomas occurring spontaneously in aged male and female Fischer 344 rats, respectively. Morphologically, MeMS and MeFS expressed the epithelial phenotype. Coexpression of keratin and vimentin in both cell lines was confirmed by the immunocytochemistry, one-dimensional gel electrophoresis and immunoblotting for intermediate filament proteins. Hyaluronic acid synthesis of both MeMS and MeFS was demonstrated by the analysis using high-performance liquid chromatography. MeMS and MeFS seem to be useful models for studying the biological nature of rat mesotheliomas.

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Early focal myocardial lesions in rats induced by five cardiotoxic compounds were histopathologically observed 1 hr and 4 hr after a single intravenous injection with 1/10 LD₅₀ and LD₅₀. The lesions were observed 1 hr and 4 hr after the treatment with LD₅₀ of isoproterenol (ISP), 4 hr with 1/10 LD₅₀ of ISP, 4 hr with LD₅₀ of hydralazine (HYD), caffeine (CAF) and cyclophosphamide (CYC), but not with adriamycin (ADR). The lesions consisted of homogeneously intensely eosinophilic staining, contraction band formation and fragmentation of cardiac muscle fibers. The lesions were interspersed in the inner one third of the left ventricular walls including the papillary muscles with ISP, HYD and CAF, and were all over the ventricular myocardium with CYC.

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A primary extranodal lymphosarcoma causing recurrent episodes of dyspnea and exercise intolerance is described in a 13-year-old cat. The affected segment of the tracheal wall was markedly thickened due to transmural infiltration of monomorphic neoplastic lymphoid cells and the lumen was severely compressed. The diagnosis was based on light microscopic studies, positive immunostaining for feline IgA, and special stains.

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Twenty monoclonal antibodies (MAbs) were produced against the virulent ALD or vaccinal GPE strains of classical swine fever (CSF) virus. The MAbs were divided into 7 groups on the basis of the difference in the reaction spectrum to various CSF and bovine viral diarrhea-mucosal disease (BVD-MD) viruses. The panel of the MAbs could classify 20 CSF viruses into 6 types, and could discriminate between not only CSF and BVD-MD viruses but also the vaccinal GPE strain and the field isolates of CSF virus. The results indicated that the MAbs were useful for diagnosis and investigation of CSF viruses.

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A large number of non-pigmented Staphylococcus chromogenes were isolated from the skin of piglets with exudative epidermitis and healthy pigs. Their characteristics were homologous with S.chromogenes type strain, except for pigment production. Strains of non-pigmented S.chromogenes exhibited high levels of DNA homology with S.chromogenes type strain. The colony morphology and pigmentation of non-pigmented S.chromogenes was very similar to S.hyicus type strain, but their characteristics differ in hyaluronidase production, heat stable DNase, Tween 80 hydrolysis and bacitracin resistance. Further, DNA homology drew a distinction between non-pigmented S.chromogenes and S.hyicus type strain.

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