Expression of fibrosis-related genes in canine chronic hepatitis

Molecular regulation of fibrosis in chronic canine hepatitis is poorly understood. The authors employed quantitative polymerase chain reaction (PCR) to determine the expression levels of genes reported to be related to fibrosis in other species (human, mouse, and rat) and to elucidate the relationship of these genes with the degree of fibrosis and the presence or absence of ascites and/or jaundice in dogs with hepatitis. Nine fibrosis-related genes were assayed: PDGFB, PDGFD, MMP2, TIMP1, THBS1, COL1A1, COL3A1, TGFB1, and TGFB2. Liver samples of 15 dogs with chronic hepatitis and 4 healthy control dogs were obtained via laparoscopic biopsy and subjected to histologic and quantitative PCR analyses. The expression of all 9 genes showed significant positive correlation (P<.01, r>.70) with the degree of fibrosis. Furthermore, the expression levels of all genes except TGFB1 were significantly higher (P<.05) in dogs with hepatic failure-related symptoms (ascites/jaundice). Results suggest that these 9 genes are integral to the development of fibrosis in canine chronic hepatitis.     

The hypocholesterolemic activity of transgenic rice seed accumulating lactostatin, a bioactive peptide derived from bovine milk β-lactoglobulin

Lactostatin is a novel pentapeptide (IIAEK) derived from bovine milk β-lactoglobulin with greater hypocholesterolemic activity than β-sitosterol, the drug commonly used to treat hypercholesterolemia. We developed transgenic rice expressing lactostatin as a fusion protein with seed storage protein (SSP) glutelins under the control of three different endosperm-specific promoters. Lactostatin accumulated in transgenic rice seed at approximately 1.6 mg/g seeds (dry seeds) without any apparent influence on seed traits such as endogenous SSP expression levels or alterations in the intracellular structures of endosperm cells. Short-term (three day) oral administration of the glutelin fraction containing lactostatin (namely three times of 300 mg/kg body weight/day) extracted from transgenic rice seeds resulted in hypocholesterolemic activity in rats; namely, the serum low-density-lipoprotein (LDL) cholesterol level was significantly reduced accompanied by a significant increase in beneficial serum high-density-lipoprotein (HDL) cholesterol.     

A rapid and simple method to obtain canine peripheral blood-derived macrophages

Macrophages play an important role in a variety of situations, including pathogen elimination, inflammation, and tissue repair. However, these cells are not fully studied in dogs, in part, due to the difficulty of efficiently isolating and culturing them in vitro. In this study, we cultured canine peripheral blood mononuclear cells (PBMCs) with 10 ng/ml of phorbol 12-myristate-13-acetate (PMA) for 5 days to obtain macrophages. A high number of round-adherent cells were obtained without the addition of any cytokine. These cells showed active phagocytic activity and a cell surface antigen profile different from dendritic cells. Our method facilitates a high yield of macrophages in a short cultivation period compared with previous studies. This method might be a powerful tool to study macrophage functions in dogs.     

Screening of therapeutic targets for canine mast cell tumors from a variety of kinase molecules

Options of systemic treatment for canine MCT have been still limited and most canine cases with MCTs eventually undergo relapses even after achievement of a remission. Thus additional therapies are required to establish for the tumor. To identify the novel candidate therapeutic targets for canine MCT, the mRNA expression and phosphorylation statuses of several receptor or non-receptor kinases as well as the inhibitory effect of 95 specific inhibitors on the growth were assessed in three canine MCT cell lines (HRMC, VIMC1 and CMMC1). Among the 14 targets, the mRNAs of 11, 7 and 7 kinases were amplified in HRMC, VIMC1 and CMMC1, respectively. The mRNAs of VEGFR3, PDGFRα, SRC, YES, LCK and FYN were detected in all cell lines. The phosphorylation of 12, 8 and 7 kinases was observed by using specific antibody arrays in HRMC, VIMC1 and CMMC1, respectively. DTK, EPHB6, AMPKα1, CREB, STAT5a and STAT5b were phosphorylated in all cell lines. The 10, 9 and 17 inhibitors exhibited the biological activity against the growth of HRMC, VIMC1 and CMMC1, respectively. Only three inhibitors such as SB218078 (for Chk1), PDGF RTK inhibitor IV (for PDGFR) and radicicol (for Hsp90) suppressed the growth of all three cell lines. The present study indicated that several kinases, such as Chk1, PDGFR and Hsp90, could be used as therapeutic targets in the treatment for canine MCT. Further studies and clinical trials are warranted to apply the inhibitors for the treatment of the tumor.     

Therapeutic effect of anti-TNF-α antibody and levofloxacin (LVFX) in a mouse model of enterohemorrhagic Escherichia coli O157 infection

The ability of an anti-TNF-α antibody to confer protection against enterohaemorrhagic Escherichia coli (EHEC) O157 was investigated in germfree IQI mice. The use of an antibiotic levofloxacin (LVFX) alone or with the antibody was also studied. Protection included an increase in survival rate. Treatment with the anti-TNF-α antibody inhibited the histological signs associated with EHEC infection but did not prevent the colonization of EHEC or production of Shiga toxin (Stx). No clinical signs were observed and EHEC was completely eliminated in the mouse model receiving both anti-TNF-α antibody and LVFX. Anti-TNF-α antibody suppressed inflammatory cytokine response in the mouse kidney and brain by EHEC infection.     

A new pyrethroid-type ester with strong knockdown activity

Thirteen pyrethroid-type esters of substituted 1(or 3)-hydroxymethylimidazolidine-2, 4-dione were synthesised and their knockdown activities against houseflies, mosquitoes and cockroaches were examined. Knockdown activities of 2,4-dioxo-1-prop-2-ynylimidazolidin-3-ylmethyl esters in oil solutions were higher than those of known knockdown pyrethroids; three of the compounds also possessed strong knockdown and flushing-out activities against cockroaches.     

A retrospective study and gene analysis of canine sterile panniculitis

In this study, a retrospective analysis was conducted to assess the current aspects and predisposing factors of canine sterile panniculitis. Miniature dachshund, neutered, and younger dogs appeared to be predisposed. In addition, histories of previous surgery and injection were associated in 46.5% of the cases, with several types of surgical suture materials used. About 88% of the dogs had multifocal lesions, frequently with signs of systemic illnesses. Whereas systemic immunosuppressive therapy was effective in most dogs, surgical excision of lesions was rarely curative. In order to prevent recurrences, over 65% of the cases required prolonged immunosuppressive therapy. Polymorphism of canine alpha(1)AT gene was investigated as a candidate gene for sterile panniculitis. Eight polymorphisms were discovered in seven Miniature dachshunds by direct nucleotide sequencing, which included a 12-bp deletion, three non-synonymous single nucleotide polymorphisms, and four silent substitutions. Genotyping of the two polymorphisms, c.109_120del12 and c.483A>C, which identified at high incidence in the dachshunds, was conducted in 83 dogs of 6 popular breeds. The frequencies of neither polymorphism differed between Miniature dachshunds and other breeds, suggesting that neither is responsible for developing panniculitis.     

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene

OBJECTIVE: To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION: Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES: The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS: P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE: Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.     

Cesium radioactivity in peripheral blood is linearly correlated to that in skeletal muscle: Analyses of cattle within the evacuation zone of the Fukushima Daiichi Nuclear Power Plant

The accident at the Fukushima Daiichi Nuclear Power Plant (FNPP) released a large amount of radioactive substances into the environment. Furthermore, beef contaminated with radioactive cesium above the 500 Bq/kg safety standard was circulated in the food chain in 2011. Japanese consumers remain concerned about the safety of radioactively contaminated food. In our previous study, we detected a linear correlation between radioactive cesium (¹³⁷Cs) activity in blood and muscle around 500 to 2500 Bq/kg in cattle. However, it was unclear whether the correlation was maintained at a lower radioactivity close to the current safety standard of 100 Bq/kg. In this study, we evaluated 17 cattle in the FNPP evacuation zone that had a ¹³⁷Cs blood level less than 10 Bq/kg. The results showed a linear correlation between blood ¹³⁷Cs and muscle ¹³⁷Cs (Y = 28.0X, R² = 0.590) at low radioactivity concentration, indicating that cesium radioactivity in the muscle can be estimated from blood radioactivity. This technique would be useful in detecting high‐risk cattle before they enter the market, and will contribute to food safety.     

Leptospiral lipopolysaccharide stimulates the expression of toll‐like receptor 2 and cytokines in pig fibroblasts

Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L‐LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L‐LPS. Here, we stimulated pig fibroblasts by L‐LPS and then quantitatively measured gene and protein expression levels of two toll‐like receptors (TLRs), TLR2 and TLR4, by real‐time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up‐regulated within 24 h after L‐LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL‐6) and IL‐8 gene expressions were markedly up‐regulated by L‐LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L‐LPS stimulation, thereby inducing expression of cytokines.