Preparation of positive control platelets for detection of canine platelet surface-associated IgG, IgM and complement (C3) by flow cytometry.

An assay for detection of platelet surface-associated (PSA-) IgG, IgM and/or complement (C3) in dogs was modified by preparation of artificial positive control platelets. Flow cytometry of fluorescein isothiocyanate (FITC)-conjugated anti-dog IgG, anti-dog IgM and anti-dog C3 antibodies was used to detect the PSA proteins. IgM single, IgM/C3 double and IgG/IgM/C3 triple positive platelets were prepared. FITC-conjugated anti-IgG antibody bound strongly only to the triple positive platelets. Binding of FITC-conjugated anti-IgM or anti-C3 antibody to the double and triple positive platelets was specifically blocked by preincubation with the respective non-FITC-conjugated same-origin antibodies. These results confirm that FITC-conjugated antibodies specifically detect PSA proteins and that the control platelets prepared in this study are appropriate positive controls for detection of PSA proteins by flow cytometry.

Canine bone marrow cells differentiate into hepatocyte-like cells and placental hydrolysate is a potential inducer.

Hepatocyte growth factor (HGF) can stimulate human and rat bone marrow (BM) cells to differentiate into hepatocytes. A human placental hydrolysate (hPH) stimulates proliferation of hepatocytes, but its role as a potential inducer of BM cells to form hepatocytes is unclear. To determine if canine BM cells stimulated with HGF or hPH differentiate into hepatocyte-like cells, BM cells were cultured with HGF or hPH. The cultured cells underwent morphological examination, expression of albumin and cytokeratin 18 (CK18), hepatic function tests including uptake of low-density lipoprotein (LDL) and cytochrome P (CYP) 450 activity. Albumin mRNA and protein expression of albumin and CK18 proteins were detected in cultures with HGF and hPH. Furthermore, these cells demonstrated LDL uptake and CYP450 activity. These results indicate that canine BM cells can differentiate into hepatocyte-like cells when stimulated by both HGF and that hPH may be an effective inducer of hepatic differentiation.

A dog with acute myelomonocytic leukemia.

A three-year-old dog with marked leukocytosis, lymphadenopathy, and diarrhea showed an increase in unidentified blasts in the peripheral blood, and they were proliferated in the bone marrow. The dog was diagnosed with myelomonocytic leukemia (M4) because the blast cells were demonstrated by cytochemical staining to be both myeloid and monocytic cells. Although the dog was treated with a multi-combination chemotherapy and induction therapy using vitamin K2, it died on day 47 after the first admission. This case is the first report of M4 in Japan.

A case of atypical canine lymphoma with oral mass and multiple osteolysis.

An 8-year-old female Golden Retriever had an oral mass and lameness. Multiple osteolysis of the systemic skeleton without monoclonal gammopathy was shown on electrophoresis of serum and urine samples. Cytological and histopathological examinations of the oral mass revealed atypical polymorphic cells similar to myeloid cells, and bone marrow aspiration indicated that these abnormal cells also might have invaded the bone marrow. These cells were negative to peroxidase and non-specific esterase staining, and clonal expansion of B lymphocytes could be detected by polymerase chain reaction (PCR) assay for antigen receptor gene rearrangement. The case was diagnosed as atypical lymphoma and treated by multi-drug chemotherapy. On the 142nd day after the first admission, the case had remission and the oral mass and multiple osteolysis were improved.

Alpha-fetoprotein in serum and tumor tissues in dogs with hepatocellular carcinoma.

Serum alpha-fetoprotein (AFP) concentrations were measured before and after surgical removal of tumor masses in four dogs with hepatocellular carcinoma (HCC). Localization of AFP was also examined immunohistochemically in tumor tissues. In three cases, the serum AFP concentration was 10 to 20 times higher than that of normal dogs. One to two months after surgery, the serum AFP concentration had decreased to normal range. AFP was localized in the tumor tissues in these three cases. One case, which had a low serum AFP, did not show AFP localization in tumor tissue.

Establishment and characterization of primary canine hepatocellular carcinoma cell lines producing alpha-fetoprotein.

A primary cultured cell line named CHKS was established from a hepatocellular carcinoma (HCC) of a dog showing a high level of serum alpha-fetoprotein (AFP). CHKS secreted a 66 KDD AFP into the growth medium regardless of the presence or absence of fetal bovine serum (FBS). Cloning CHKS with limiting dilution produced 4 clones, CHKS-1, -2, -3, and -4, which secreted 826, 471, 70, and less than 10 ng/ml, respectively, of AFP into the culture medium. In culture, these cell lines were similar in morphology and proliferation pattern to epithelial cells and positive to periodic acid-Schiff (PAS) staining. The presence of mRNA for canine albumin was demonstrated by nested PCR. The doubling times of the clone cell lines were 21, 45, 36, and 35 h, saturation densities 34, 18, 22, and 24 x 10(4)/cm(2), and plating efficiencies 18, 45, 46, and 45%, respectively. Chromosome analysis of these cell lines showed near triploidy. These results show that CHKS and its clones have hepatic cell functions and are useful for carcinogenetic and clinical studies of canine HCC.

Immunohistolocalization of carbonic anhydrase isoenzymes (CA-I, -II and -III ) in canine epididymis.

The immunolocalization of the efferent duct and the epididymis in canine was firstly examined using an the immunohistochemical method with the canine carbonic anhydrase (CA) -I, CA-II and CA-III antisera. The efferent duct was immunonegative for all present canine CA antisera. However, some slender shaped epithelial cells in the head and body segments of the epididymal duct were intensely reacted to the CA-II antiserum. These results suggested that the CA-II might be controlled in the luminal environment in the head and body segments of the canine epididymis by the proton and bicarbonate balance for the maintenance of the spermatozoal stability and movement.