Effect of a Large Dose of Di (2-ethylhexyl) phthalate (DEHP) on Hepatic Peroxisome in Cynomolgus Monkeys (Macaca Fascicularis)

To elucidate the effect of a large dose of di (2-ethylhexyl) phthalate (DEHP), a plasticizer and peroxisome proliferator-activated receptor-α (PPARα) agonist, on hepatic peroxisomes, we orally administered 1,000 mg/kg/day, once daily, to 3 male and 4 female cynomolgus monkeys for 28 days consecutively. Light-microscopic and electron microscopic examinations of the liver were carried out in conjunction with measurement of the hepatic fatty acid β-oxidation system (FAOS), carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (CPT) activities, which are peroxisomal and/or mitochondrial enzyme activities. Electron microscopically, enlargement of the mitochondria was observed with lamellar orientation of the cristae along the major axis. Although the number of peroxisomes showed a tendency to increase when compared with those in a biopsied specimen before treatment, no abnormality in morphology was observed. A slight increase in CPT activity was noted at termination. No changes were noted in hepatic FAOS or CAT activity. In conclusion, although repeated oral treatment of cynomolgus monkeys with a large dose of DEHP induced a subtle increase in the numbers of peroxisomes with slight enlargements of the mitochondria, this low-sensitivity response to peroxisome proliferators in cynomolgus monkeys was considered to be closer to the response in humans than that in rodents.     

Seroepidemiological evidence for the possible presence of Babesia (Theileria) equi and Babesia caballi infections in donkeys in western Xinjiang, China

The prevalence of Babesia (Theileria) equi and B. caballi infections in donkeys in western Xinjiang China was investigated. In total, 93 serum samples were randomly taken from donkeys in the Kashi and Ili areas, and examined for B. equi and B. caballi infections by enzyme-linked immunosorbent assays using recombinant antigens. Of the 93 samples, 9 (9.6%) and 36 (38.7%) samples were positive for B. equi infection and B. caballi infection, respectively. In addition, 2 (2.2%) samples were positive for both B. equi and B. caballi infections. These results indicate that equine babesiosis might be extensively prevalent in donkeys in western Xinjiang.     

Phenotypic analysis of hepatic T lymphocytes in a dog with chronic hepatitis

We evaluated hepatic T lymphocyte phenotypes in a dog with chronic hepatitis. Before treatment, numerous CD3+ lymphocytes were demonstrated in the liver, and the ratio of CD4+/CD8+ was remarkably high (2.96; reference range, 0.33+/-0.12). After treatment, CD3+ lymphocyte infiltration in the liver was reduced, and the ratio of CD4+/CD8+ decreased to 0.31. Therefore, hepatic T lymphocytes, especially CD4+ lymphocytes, might play a central role in the pathogenesis of this dog with chronic hepatitis.     

Cynomolgus macaque CYP4 isoforms are functional, metabolizing arachidonic acid

Cytochrome P450 (CYP) is important for metabolism of not only xenobiotics such as drugs, but also endogenous compounds including arachidonic acids. CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 have been identified in cynomolgus macaque, an animal species widely used for investigation of drug metabolism due to its evolutionary closeness to human. However, their metabolic functions have not been investigated. In this study, proteins were heterologously expressed in Escherichia coli and characterized by metabolic assays using arachidonic acids as substrates that are metabolized by CYP4 isoforms in human. The results showed that all four CYPs metabolized arachidonic acids. Therefore, cynomolgus macaque CYP4A11, CYP4F3v2, CYP4F11, and CYP4F45 are functional enzymes.     

A novel culture method of canine peripheral blood lymphocytes with concanavalin a and recombinant human interleukin-2 for adoptive immunotherapy

This study was designed to develop a novel culture method for the efficient proliferation of canine peripheral blood lymphocytes (cPBL) for adoptive immunotherapy. When cPBL were cultured in the presence of concanavalin A (Con A), proliferation of cPBL was induced and expression of interleukin-2 receptor (IL-2R) which enables to respond to exogenously added IL-2 was upregulated. And then, when cPBL were cultured with recombinant human interleukin-2 (rhIL-2) in addition to Con A, proliferation was accelerated and increased to about 10-fold after 1 week. The phenotypic analysis showed that the main population of the cultured cPBL was consisted of CD8+ positive lymphocytes. Among them, CD4+CD8+ double positive (DP) lymphocytes had significantly increased, and the ratio of CD4+ single positive (SP) lymphocytes to CD8+ SP lymphocytes (CD4+SP/CD8+SP) was decreased as compared to before culturing. To evaluate the cytotoxic activity of cPBL cultured with Con A and rhIL-2, furthermore, cytotoxic assay was carried out against xenogeneic melanoma cell line (MeWo), which resulted in MHC-unrestricted cytokilling. These results suggest that the culture method of cPBL by the use of Con A and rhIL-2 may be useful for generating lymphokine activated killer cells, and also this may be beneficial for adoptive immunotherapy of tumor-bearing dogs.     

Porcine MHC classical class I genes are coordinately expressed in superantigen-activated mononuclear cells

The expression of the major histocompatibility complex (MHC) classical class I genes is important for the adaptive immune response to target virus-infected cells and cancer cells. The up-regulation of the MHC is achieved by hormonal/cytokine signals including IFN-γ-inducible elements. The swine leukocyte antigen (SLA), the MHC class I region of pigs, consists of the duplicated classical class I genes, SLA-1, SLA-2 and SLA-3, but the molecular mechanisms involved in their up-regulation after T cell stimulation have not been fully elucidated. In order to better understand some of the putative regulatory mechanisms of SLA class I gene expression in activated T cells, we examined the coordinated expression of the SLA classical class I, IFN-γ and interferon regulatory factor-1 (IRF-1) genes in the peripheral blood mononuclear cells (PBMCs) of SLA homozygous Clawn miniature swine stimulated for 72h with either IFN-γ or an enterotoxin produced by Staphylococcus aureus. This enterotoxin, toxic shock syndrome-1 (TSST-1), is known to act as a superantigen (sAG) to activate the T cells in various vertebrate species. We showed by using mAbs and flow cytometry that the CD4(+)CD25(+) cell number of swine PBMCs was also increased by TSST-1 and to a lesser degree by IFN-γ. Time course analyses of the expression of the IFN-γ, IRF-1 and the three classical class I genes, SLA-1, SLA-2, and SLA-3, in PBMCs by quantitative real-time PCR revealed a transitory response to TSST-1 or IFN-γ stimulation. The IFN-γ mRNA levels in the PBMCs were continuously up-regulated over the first 48h by TSST-1 or IFN-γ. In contrast, SLA class I expression moderately increased at 24h and then decreased to a baseline level or less at 72h of IFN-γ or TSST-1 stimulation. The three classical SLA class I genes showed similar expression kinetics, although SLA-3 mRNA level was consistently lower than those of SLA-1 and -2. The expression of IRF-1, a modulator of SLA expression, showed similar kinetics to those of the three classical SLA class I genes. The expression profiles detected by flow cytometry of the SLA molecules on the cell surface of PBMCs were maintained at a consistently high level during cell stimulation with either TSST-1 or IFN-γ, which was distinct from the kinetics of mRNA expression. These results showed that miniature swine SLA class I mRNA expression was effectively and equally up-regulated among the three loci and coordinately with IRF-1 gene expression after stimulation of T cell activation by sAG or IFN-γ.     

Molecular characterization of an H1N2 swine influenza virus isolated in Miyazaki, Japan, in 2006

Swine influenza virus (SIV) was isolated from a farm in Miyazaki Prefecture in Japan in July 2006. An isolate was genetically subtyped as H1N2 and was designated A/swine/Miyazaki/1/2006 (H1N2). The nucleotide sequences of all eight viral RNA segments were determined, and then phylogenetic analysis was performed using the neighbor-joining method. All segments were shown to be closely related to those of Japanese SIV H1N2 isolates, which have been circulating since the 1980s. The results indicate the persistence of the SIV H1N2 subtype in the Japanese pig population for more than two decades and emphasize the importance of continuous surveillance for SIV.