Distribution of T-lymphocyte subpopulation in blood and spleen of normal cattle and cattle with enzootic bovine leukosis.

Immunocytochemical and flow cytometry techniques were used to examine T-lymphocyte subpopulations in peripheral blood lymphocytes (PBLs) and spleen from cases of enzootic bovine leukosis (EBL) in adult cattle, and from normal cattle (adult and young), with a panel of monoclonal antibodies against bovine leucocyte differentiation molecules. Both in PBLs and spleen, the percentages of T-lymphocyte subpopulations (CD3+, CD4+, CD8+, and WC1 + gamma delta T lymphocytes) of EBL-affected and normal adult cattle were significantly lower than those of normal young cattle. The percentages of these T-lymphocyte subpopulations in the PBLs of adult cattle with EBL were lower than those of normal adult cattle, but the converse was true in the spleen. It is suggested that tumour immunity occurred in the spleen. Histological examination revealed no follicular hyperplasia in the spleen, and the proliferation of neoplastic cells began in the red pulp. It is concluded that the spleen is not the organ initially responsible for the transformation of EBL lymphoma and that neoplastic cells migrating from peripheral blood are metastatic.

A new method for evaluation of motor deficits in 3-acetylpyridine-treated rats.

We established a novel method for quantitative evaluation of the motor deficits induced by 3-acetylpyridine (3-AP) in rats. 3-AP (75 mg/kg, i.p.) was injected in a time-controlled manner by a following injection of niacinamide (NIA; 300 mg/kg, i.p.). Changes in the motor function were evaluated by measuring the maximal height of vertical jump (MHVJ) to escape from an electrical shock on the foot, as well as ataxic gait. The MHVJ decreased and reached a minimum value 5-7 days after the 3-AP treatment. The close correlation of the MHVJ and the incidence of ataxic gait indicates that the decrease of MHVJ is a useful quantitative index of motor deficits.

Analysis of a polyadenine tract of the transforming growth factor-beta type II receptor gene in colorectal cancers by non-gel-sieving capillary electrophoresis.

We developed a method to analyze a polyadenine tract, the (A)10 repeat, within the cysteine-rich domain of the transforming growth factor-beta (TGF-beta) type II receptor gene using a non-gel-sieving capillary electrophoresis technique and applied it to the DNA diagnosis of colorectal cancers. This method consists of single-strand DNA amplification of the (A)10 repeat by an asymmetric PCR technique and capillary electrophoresis. A higher concentration of dATP in the PCR reaction mixture led to more specific amplification of the (A)10 repeat. Under the optimal electrophoretic conditions, one nucleotide difference could be determined in 8 to 32 nucleotides. One or two base deletions of the (A)10 repeat in colorectal cancers could be detected under these conditions within 30 min, and the results coincided with those obtained on DNA sequencing analyses. According to a sensitivity study, we could detect the deleted sequence if it was present in 12.5% or more of the wild-type allele. The reproducibility of this technique was satisfactory because the intraassay imprecision (CV) (n = 10) was 1.4%. These results indicate that capillary electrophoretic analysis of small repeated sequences results in easier handling and more feasible automation, compared with conventional gel electrophoretic analysis.

Tumor-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.

A monoclonal antibody, (MAb) c143, that recognizes a tumor-associated antigen (TAA) that is upregulated on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. Immunohistochemical examination of neoplastic tissue and lymph nodes from animals with EBL, revealed three morphologically definable stages of change in the architecture of lymph nodes associated with infiltration and proliferation of neoplastic cells: 1) presence of c143 positive cells at the marginal sinus with no apparent changes in lymph node architecture at the earliest stage of neoplastic cell accumulation, 2) presence of positive cells extending into and distorting the architecture of the lymph node with clear evidence of proliferation, and 3) presence of positive cells throughout the lymph node with total disruption of lymph node architecture. The results indicated that, at the earliest stages of EBL, neoplastic cells in peripheral blood may accumulate at the marginal sinus area and subsequently proliferate and infiltrate pressing follicles leading to the development of clinical signs of lymphosarcoma.

[Reproductive and developmental toxicity studies of taltirelin hydrate (4) perinatal and postnatal study in rats by oral administration].

Perinatal and postnatal study of taltirelin hydrate, a thyrotropin releasing hormone analogue, was carried out in Sprague-Dawley rats. Female rats were given taltirelin hydrate at a dose of 0 (control), 0.15, 1.5, or 15 mg/kg from day 17 of gestation to day 20 after delivery. All pregnant rats were allowed to deliver spontaneously and their offspring were examined. In the 15 mg/kg group, the dams showed the central nervous effects such as wet dog shaking during gestation periods. No adverse effect of taltirelin hydrate on the body weight gain, food consumption and reproductive performance was observed in this group. In the 0.15 and 1.5 mg/kg groups, the drug did not have any adverse effects. Taltirelin hydrate did not have any adverse effects on viability, growth, physical differentiation, functional and behavioral development (coordinated activity, auditory function, emotionality, learning ability, and spontaneous motor activity), and reproductive performance of F1 offspring, and development of F2 fetuses. These results show that the no-toxic dose levels of taltirelin hydrate are 1.5 mg/kg for general toxicity in dams, and 15 mg/kg for reproductive function of dams and for their offspring.

B-1a, B-1b and conventional B cell lymphoma from enzootic bovine leukosis.

In order to characterize the phenotypes of tumor cells and to clarify from which B cell lineage the lymphomas were derived, ten cows with enzootic bovine leukosis were examined by means of immunohistologic staining and flow cytometry. The tumor cells expressed mainly major histocompatibility complex (MHC) class II+ (10/10), BoCD11b+ (9/10), IgG1+ (8/10), B-B2+ (8/10) BoCD5+ (7/10), and lambda light chain+ (7/10). Tumor cells from only one animal expressed sIgM+ (1/10). Tumor cells from all ten animals were negative for IgG2, BoCD3, BoCD4, BoCD8, WC1-N2, and IL-2R alpha. The phenotypes of these tumor cells were all slightly different, suggesting that bovine leukemia virus (BLV)-induced lymphoma expresses phenotypic diversity. Moreover, tumor cells from seven cattle coexpressed BoCD5 and BoCD11b (B-1a cells). On the other hand, tumor cells from two of them only expressed BoCD11b (B-1b cells), and those from one were negative for both BoCD5 and BoCD11b (conventional B cells). Therefore, we concluded that BLV-induced lymphoma cells can be derived from B-1a, B-1b and conventional B cells.

Changes in the distribution of cells expressing tumour-associated antigen in lymph nodes during the progression of enzootic bovine leukosis.

A monoclonal antibody (MAb), c143, that recognizes a tumour-associated antigen that is "upregulated" on neoplastic B cells in cattle with enzootic bovine leukosis (EBL), was used as a marker to study disease progression. An immunohistochemical examination of neoplastic tissue and superficial cervical lymph nodes from 14 animals with EBL revealed three morphologically definable stages of change in the structure of lymph nodes, associated with the distribution of c143-positive cells: (1) the presence of c143-positive cells at the marginal sinus with no apparent changes in lymph node structure; (2) the presence of positive cells extending into and distorting the architecture of the lymph node, with clear evidence of proliferation before overt changes (enlargement of lymph nodes) were evident; and (3) the presence of positive cells throughout the lymph node with total disruption of lymph node structure when clinical signs of lymph node enlargement were evident. The results indicated that the bovine leukaemia virus-transformed lymphocytes or neoplastic cells in peripheral blood accumulate in the marginal sinus area at the earliest stages, and subsequently proliferate and infiltrate into follicles, leading to the development of clinical signs of lymphosarcoma.

Maternal placenta-like endometrial hyperplasia in a beagle dog (Canine deciduoma).

Three thumb-sized segmental swellings were found in both uterine horns of a 15 month-old, non-pregnant and non-pseudopregnant female beagle dog. Histopathological examination of the uterus revealed a marked proliferation of the endometrium that was characterized by two distinct parts, an inner tightly-folded banded layer and an outer dilated spongy layer, quite similar to the maternal placenta except for the fetus and chorion in the lumen. Because the plasma level of progesterone was unusually high at autopsy, this hormonal disorder might be related to the pathogenesis of the endometrial hyperplasia in the present case.

Histological and topographical characteristics of canine granulomatous leptomeningitis.

Histological and topographical characteristics of granulomatous leptomeningitis were studied in 14 laboratory beagle dogs (12 males, two females), 8 to 19 months old. The dogs, which were either saline-treated controls or drug-treated animals in four different toxicity studies, remained clinically normal during the experimental period, and granulomatous leptomeningitis was identified as an incidental finding by routine histopathological examination. There were no macroscopical lesions in the brain or other organs. Microscopical lesions of the central nervous system were of three types, namely (1) a diffuse inflammatory type, characterized by diffuse infiltration of macrophages, (2) a granulomatous type, characterized by non-caseating granulomas, or (3) a fibrotic type, featuring dense connective tissue. Each of the 14 cases fell into one of three disease phases, early, fulminating and healing. In the early phase, only diffuse inflammatory-type lesions were seen. In the fulminating phase, all three types of lesion were present. In the healing phase, discrete granulomas were relatively few and fibrosis was prominent.