Identification of c-kit mutations-independent neoplastic cell proliferation of canine mast cells

Gain-of-function mutations in the proto-oncogene c-kit have been considered the molecular mechanism of neoplastic proliferation of mast cells. However, the importance of c-kit gene mutations is not well evaluated in canine mast cell tumors (MCTs). In the present study, we established and characterized a mast cell line, HRMC, derived from a dog with MCT. We also examined c-kit mutations in HRMC cells and assessed an inhibitory effect of a tyrosine kinase inhibitor, STI571, on HRMC cells. HRMC cells had cytoplasmic metachromatic granules, chymase and tryptase, and expressed both KIT and FcepsilonRI on the cell surface. HRMC cells contained histamine and released beta-hexosaminidase through FcepsilonRI cross-linking and calcium ionophore stimulation. Nucleotide sequence analysis demonstrated no mutations in an open reading frame of c-kit cDNA and genomic DNA of the juxtamembrane domain of c-kit in HRMC cells. STI571 did not show any inhibitory effects on the proliferation of HRMC cells. These findings clearly demonstrated the existence of c-kit mutations-independent neoplastic canine mast cell proliferation. The growth factor-independent mast cell line established in this study might be valuable to explore novel mechanisms of c-kit mutations-independent neoplastic proliferation of mast cells in dogs.     

Measurement of telomerase activity in bovine leukaemia virus infected cows

Telomerase adds new telomeric sequences to the end of chromosomal DNA in order to overcome the end-replication problem. The upregulation of telomerase activity in tumours has been reported in humans and some mammals and is considered to be a tumour marker; however, such activity has not been investigated in cows. Therefore, we investigated telomerase activity in bovine leukaemia, the most common tumour in cows and its relationship with the bovine leukaemia virus (BLV) infection, which is the major cause of leukaemia. Telomerase activity was detected in 25 of 29 bovine leukaemia tissue samples. In peripheral blood lymphocytes (PBL) from BLV-infected cases that did not develop the tumour, telomerase activity was detected in 11 of 71 cases (15.5%). When these cases were classified based on serological tests and the peripheral blood lymphocyte count, the telomerase activity was observed to be the highest in the seropositive, non-lymphoproliferative (PBL<8000 microl(-1)) cases (three of seven cases, 42.9%), and not observed in the lymphoproliferative cases (PBL<16,000 microl(-1)) except in one case. Although the precise pathogenesis of BLV-related diseases remains obscure, persistent lymphocytosis is considered as a pre-neoplastic state. In contrast, our results suggested that given the fact that telomerase activity indicates tumour development, the aleukaemic stage could be defined as the 'pre-neoplastic state'. In conclusion, similar to many tumours in humans, telomerase activity was detected in bovine leukaemia; further, this activity can be a potentially useful prediction marker for tumour development and/or a good therapeutic target.     

Decreased blood cell counts were not observed in cattle living in the “difficult‐to‐return zone” of the Fukushima nuclear accident

White blood cells, especially lymphocytes, are susceptible to radiation exposure. In the present study, red blood cell, total white blood cell, and lymphocyte counts were repeatedly measured in cattle living on three farms located in the “difficult‐to‐return zone” of the Fukushima nuclear accident, and compared with two control groups from unaffected areas. Blood cell counts differed significantly between the two control groups, although almost all the values fell within the normal range. The blood cell counts of the cattle in the “difficult‐to‐return zone” varied across sampling times even on the same farms, being sometimes higher or lower than either of the two control groups. However, neither a statistically significant decrease in blood cell counts nor an increase in the rate of cattle with extremely low blood cell counts was observed overall. The estimated cumulative exposure dose for the cattle on the most contaminated farm was within a range of 500–1000 mSv, exceeding the threshold for the lymphopenia. Because of the low dose rate on these farms, potential radiation damages would have been repaired and have not accumulated enough to cause deterministic effects.     

Prevalence of equine herpesvirus type 1 strains of neuropathogenic genotype in a major breeding area of Japan

A single non-synonymous nucleotide substitution of guanine (G) for adenine (A) at position 2254 in the viral DNA polymerase gene (encoded by open reading frame [ORF] 30) of equine herpesvirus type 1 (EHV-1) has been significantly associated with neuropathogenic potential in strains of this virus. To estimate the prevalence of EHV-1 strains with the neuropathogenic genotype (ORF30 G(2254)) in the Hidaka district--a major horse breeding area in Japan--we analyzed the ORF30 genomic region in cases of EHV-1 infection in this area during the years 2001-2010. Of the 113 cases analyzed, 3 (2.7%) were induced by ORF30 G(2254) strains. This prevalence is lower than those observed in the U.S.A. (10.8-19.4%), Argentina (7.4%), France (24%), and Germany (10.6%).     

Isolation and pathogenic characterization of an OB1 variant of Babesia rodhaini which has a glycophorin A-independent pathway to murine red blood cells

Recent studies using several Babesia spp. have demonstrated that these species commonly recognize host sialic acids of red blood cells (RBCs) for their invasion. Glycophorin A (GPA), which is a major carrier of the sialic acids on RBCs, is a possible invasive receptor for Babesia parasites. In the present study, a variant of Babesia rodhaini was successfully isolated from a GPA homozygous knockout (GPA^−/−) mouse infected with an Australian strain of B. rodhaini which had originally been unable to replicate in GPA^−/− mice. The isolated parasite (designated as an OB1 variant) caused lethal infection to wild-type mice, as in the case of the parent Australian strain. However, although the growth of the OB1 variant in GPA^−/− mice was comparable with that in wild-type mice at 1–4 days after infection, the growth was significantly inhibited from day 5 onward, leading to the eventual survival of the GPA^−/− mice. Resistance of GPA^−/− mice against OB1 infection was lost by splenectomy, although the cytokine responses to the infection in the sera of GPA^−/− mice were similar to those of wild-type mice. The autoantibody levels to GPA-defective RBCs in the sera of GPA^−/− mice were depressed at a lower level at 0–2 days after infection than those of wild-type mice, while the levels of GPA^−/− mice progressively increased and reached comparable levels to those of wild-type mice at day 3 or later. These results indicate that the isolated OB1 variant has a GPA-independent invasion pathway into murine RBCs and suggest that the resistance of GPA^−/− mice against infection with the OB1 variant may be attributed to the effective clearance of the parasitized RBCs lacking GPA in the spleen, possibly mediated by preferential autoantibody binding to the RBC membrane.     

Preimplantation development of embryos in labrador retrievers

Preimplantation development of canine embryos is not well understood. To understand the timing of preattachment embryogenesis relative to the luteinizing hormone (LH) surge, early embryonic development was examined in Labrador Retrievers after artificial insemination. The embryos migrated from the oviduct to the uterus beginning on day 11 after the LH surge. This transport must be completed within 24 h. By day 13 after the LH surge, all of the embryos had moved and were localized in the uterus. The embryos developed to the morula stage within 11-13 days and to the blastocyst stage within 14 days after the LH surge, respectively. These findings add to the current understanding concerning the physiology of preimplantation development and should help further develop assisted reproductive techniques in canine species, such as cryopreservation and subsequent embryo transfer.     

The antagonist activity of lipid IVa on the stimulation by lipid A of TNF-alpha production from canine blood mononuclear cells

Lipid A, the active component of lipopolysaccharide (LPS), exists in the outer membrane of Gram-negative bacteria and binds to the Toll-like receptor 4 (TLR4) and MD-2 complex. On the other hand, the synthetic precursor of Escherichia coli lipid A, tetraacylated lipid IVa, is an agonist for TLR4 and MD-2 complex in murine, equine and feline cells but is an antagonist for lipid A in human cells. The aim of the study was to examine the function of canine Toll-like receptor 4 (TLR4) and MD-2 complex on canine blood mononuclear cells (BMC), by analyzing lipid A- or lipid IVa-induction of TNF-α production from these cells in order to understand canine innate immune system. After 5-h culture of canine BMC with lipid A (lipid A culture) or lipid IVa (lipid IVa culture), the TNF-α, as determined by ELISA, had increased in the supernatants of the lipid A cultures in a dose-dependent manner, whereas the TNF-α was undetectable in supernatant of lipid IVa-treated cultures. The TNF-α was statistically significantly different between the lipid A and lipid IVa cultures (100 and 1000 ng/ml). TNF-α production from canine BMC was inhibited, in a lipid IVa-dose-dependent manner, when the BMC were pre-cultured with lipid IVa for 60 min and then cultured with lipid A for 5h, while in control BMC cultures production if TNF-α was unchanged. These results indicate that the TNF-α production stimulated by lipid A was competed out by pre-exposing the BMC to lipid IVa. Thus, lipid A is an agonist for TNF-α production in canine BMC, whereas lipid IVa appears to be an antagonist against this lipid A stimulation of canine BMC.