Large granular lymphocytic leukaemia complicated with histiocytic sarcoma in a dog.

A 10-year-old castrated male Golden retriever, weighing 36.3 kg was referred for evaluation owing to a decline in general condition. Findings from the complete blood count revealed a marked lymphocytosis (113000/ml). Examination of Wright-Giemsa-stained films of peripheral blood revealed the presence of large granular lymphocytes (LGL). Seventy-two per cent (81360/ml) of the lymphocytes were found to be 12-17 microm in diameter, containing nuclei with mature clumped chromatin and abundant lightly basophilic cytoplasm with a variable number of fine azurophilic granules. Based on these findings this case was diagnosed as LGL leukaemia. As a result of multiple-agent chemotherapy, the markedly elevated levels of lymphocytes gradually decreased to 7500/ml on day 122 and the patient maintained a good quality of life for the following 3 months. However, on around day 237, a soft, raised, bosselated mass on the labial region was noted. The dog was diagnosed as having histiocytic sarcoma based on cytological and histological examination of the mass. Shortly after diagnosis, the dog developed sudden onset of central nervous system signs and died on day 270. A common outcome of canine LGL is the development of acute blast crisis or lymphoma. However, this case was notable for complication with histiocytic sarcoma from another origin.

Capsid protein genetic analysis and viral spread to the spinal cord in cats experimentally infected with feline calicivirus (FCV).

We investigated primitively the molecular basis of the neural spread of a feline calcivirus isolate (FCV-S) from the spinal cord of a cat that died after manifesting excitation. Experimental infections of cats with three clones from parent virus isolate FCV-S, isolated based on plaque size, were performed, and virus recovery from the spinal cord and the nucleotide and predicted amino acid sequences of the viral capsid protein region (ORF2) were compared. In the experimental infection with the one-time cloned virus (C1L1) isolated from a large plaque, the C1L1 was recovered from the spinal cord. In contrast, seven-times cloned C6L7 (from large plaque) and five-times cloned C5S2 (isolated from small plaque) were not recovered from the spinal cord. Genetic analysis of the capsid protein gene of the three viral clones revealed that four bases were different and two amino acids were different at positions 34 (Val in C6L7 and Ala in C1L1 and C5S2) and 46 (Leu in C6L7 and Pro in C1L1 and C5S2) between C6L7 (with large plaque) and C5S2 (with small plaque). The amino acid at position 434 of C1L1 was different from those of C6L7 and C5S2 (Gly in C1L1, D (Asp) in C6L7 and C5S2). From these results, the plaque size seemed not to be related to the spread of virus to the spinal cord. Clone C1L1, which spread to the spinal cord, had a difference of one amino acid from the other two clones, which may be related to the ability to spread to the spinal cord.

Properties of a calicivirus isolated from cats dying in an agitated state.

In June 1993, two of five pet cats kept in Yokohama city in Japan suddenly became agitated and died. Feline calicivirus (FCV) was isolated from them. One strain (FCV-S) was isolated from the spinal cord, lung and tonsil of cat 1, another (FCV-B) from the ileum, medulla oblongata and cervical spinal cord of cat 2, and a third (FCV-SAKURA) from the oral cavity of one of the three surviving cats which showed no clinical signs. These three strains were equally resistant to pH 3.0 and serologically similar to each other, but distinct from strain F9. A genetic analysis, using a 208 base pair fragment from region E of the capsid, showed that FCV-Ari had a 70.4 per cent nucleotide and 77.3 per cent amino acid homology and FCV-F9 had a 68.6 per cent nucleotide and 73.9 per cent amino acid homology with the three strains, indicating that these two strains were genetically distinct from the three new isolates. Unvaccinated cats and cats which had been vaccinated against FCV-F9 developed watery diarrhoea but did not become agitated after the administration of FCV-S. The FCV-S strain did not induce signs of excitability after it was administered orally to specific pathogen-free cats.

Phylogenetic analysis of field isolates of feline calcivirus (FCV) in Japan by sequencing part of its capsid gene.

The molecular epidemiology of the infectious disease caused by feline calcivirus (FCV) in Japan was investigated by analysing the phylogenetic relationship among 21 Japanese field isolates, including the F4 strain, and 30 global isolates. Parts of the capsid gene (B-F) of the isolates were amplified by RT-PCR, and the amino acid sequences were compared with those from the global isolates. Thirty-seven and 14 out of a total of 51 isolates were clustered into two distinct genogroups, I and II respectively, by UPGMA and NJ analysis. Seven of the 21 Japanese isolates (33%) fell into group I together with 30 global isolates, while the other 14 Japanese isolates (67%) belonged to group II. The bootstrap repetition analysis of groups I and II formed by the NJ method gave a value of 99.00%. The 14 latter Japanese isolates were clearly separated from the isolates in group I, and they were different from any previously known FCV, forming a new genogroup, which implies that this lineage has been confined to Japan. Comparing the amino acid sequences shared by groups I and II, the amino acid at position 377 in B region was asparagine (Asn or Asp (NH2)) in group I, while it was lysine (Lys) in all the strains in group II. Similarly, the amino acid at position 539 in the F region was alanine (Ala) or proline (Pro) in group I, while it was valine (Val) in group II; glycine (Gly) at position 557 in group I was serine (Ser) in Group II; and phenylalanine (Phe) or leucine (Leu) at position 566 in genogroup I was tyrosine (Tyr) in group II.

Treatment of mouse melanoma cells with phorbol 12-myristate 13-acetate counteracts mannosylerythritol lipid-induced growth arrest and apoptosis.

Mannosylerythritol lipid (MEL), an extracellularglycolipid from yeast, induces the differentiation ofHL-60 promyelocytic leukemia cells towardsgranulocytes. We show here that MEL is also a potentinhibitor of the proliferation of mouse melanoma B16cells. Flow-cytometric analysis of the cell cycle ofMEL-treated B16 cells revealed the accumulation ofcells in the sub-G(0)/G(1) phase, which is a hallmark ofcells undergoing apoptosis. Treatment of B16 cellsfor 24 h with phorbol 12-myristate 13-acetate (PMA),an activator of protein kinase C (PKC), did notinterfere with the growth and survival of the cells,but it effectively counteracted the MEL-induced growtharrest and apoptosis. The activity of PKC was reducedin B16 cells treated with MEL at a concentration atwhich MEL induced apoptosis. However, incubation withPMA in addition to MEL reversed this reduction in theactivity of PKC. These results suggest thatconverging signaling pathways are triggeredindependently by MEL and PMA and that the signalsmight both be mediated by PKC.

Overexpression and genetic abnormality of p53 in parathyroid adenomas.

To study the significance of p53 abnormality in parathyroid tumors, 32 parathyroid adenomas and 22 hyperplastic glands from 14 cases of secondary hyperparathyroidism were analysed using immunohistochemistry, polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), single-strand conformation polymorphism (SSCP) and DNA sequencing. Immunohistochemical study revealed p53 overexpression in four parathyroid adenomas, of which two showed diffuse and one showed focal nuclear pleomorphism. Genetic analysis disclosed allelic loss in one, and a point mutation (R290H) and a polymorphism (L257 L) in another of the two other adenomas with diffuse nuclear pleomorphism. No abnormalities were discovered in the other two adenomas, although one had a R72P polymorphism in exon 4. There was no evidence of malignancy of the four tumors in either clinical or pathological terms. None of the 22 hyperplastic glands showed p53 overexpression. These results demonstrate that p53 abnormality can occur in benign parathyroid adenomas and is more prevalent in those with nuclear pleomorphism than in those without.

An enzyme-linked immunosorbent assay using nuclear antigen for detection of feline herpesvirus 1 antibody.

To detect antibody against feline herpesvirus 1 (FHV-1) in the sera of cats, the sensitivity and specificity of an enzyme-linked immunosorbent assay (ELISA) using nuclear antigen was investigated. The standardized optical density readings (ODs) of the ELISA obtained by the 1-step serum dilution (1:80) method were compared with the serum neutralization test (SNT) results, with a correlation of 0.993, and with the hemagglutination inhibition (HI) test results, with a correlation of 0.851. The ODs for the ELISA titers were obtained using the serial serum dilution method and were compared with the SNT results, with a correlation of 0.933, and with the HI test results, with a correlation of 0.987. In the experimental infection of 4 specific-pathogen-free cats, the results of different serologic tests (SNT and HI) and the ELISA using the serial serum dilution method revealed rapid production of antibodies after inoculation, whereas the ELISA using the one-step serum dilution method indicated that titers increased more slowly. These results indicate that with the present ELISA using nuclear antigen, there are fewer demands on time and labor, making the method convenient for monitoring FHV-1 infection.

Determination of growth fraction index in mammary carcinoma using MIB-1 monoclonal antibody: estimation of whole tumor proliferative potential using biopsy specimens.

We labeled the Ki-67 antigen in mammary carcinomas, using a MIB-1 monoclonal antibody, to evaluate the usefulness of MIB-1 Ki-67 Growth Fraction Indices (GFI; number of Ki-67 positive cells/total number of cells) of biopsy specimens in estimating the proliferative potential of the carcinomas. Formalin-fixed paraffin sections prepared from biopsy material, primary tumors, and axillary lymph nodes of ten invasive mammary carcinomas were chosen for immunohistochemical study. Bound antibody was detected using the avidin-biotin-complex peroxidase method. The GFI of the resected mammary carcinomas was similar to the estimated values based on the GFI of the biopsy specimens. The GFIs of the metastatic nodes in seven of the carcinomas were similar to those of the primary carcinomas, whereas two carcinomas yielded significantly different GFIs in the metastatic foci. These results suggest that the GFI of a mammary carcinoma biopsy specimen may reflect the proliferative ability of the whole carcinoma.

[Properties of a hemolysin produced by group C streptococci isolated from hemolytic streptococcal infection in formosan squirrels].

The hemolysin produced by group C streptococci (GCH) has an isoelectric point (pI) of 5.6 and a molecular weight of 32,000 and shows hemolytic activity in the absence of 2-mercaptoethanol (2-ME). The hemolytic activity of GCH was compared to that of streptolysin S (SLS). These hemolysins did not differ with respect to the binding and release of hemoglobin (Hb). GCH was bound to phospholipids on the membranes of target erythrocytes, followed by the rapid released of K+ and slow Hb release after a relatively long lag time. GCH showed a lower hemolytic efficiency than SLS, reflecting the fact that these hemolysins destroy erythrocytes by slightly different mechanisms.

Application of biotinylated oligonucleotide probes to the detection of pituitary hormone mRNA using northern blot analysis, in situ hybridization at the light- and electron-microscope levels.

There have been many reports on radioisotopic in situ hybridization (ISH) studies for the demonstration of pituitary hormone mRNAs in normal pituitary gland and pituitary adenomas. Recent studies have revealed that non-radioisotopic ISH has several advantages over the radioisotopic method. Using ISH with biotinylated oligonucleotide probes, we have been able to localize various pituitary hormone mRNAs in paraffin wax or frozen sections of rat normal pituitary gland and human pituitary adenomas. For control studies we used ISH with sense probes, ISH without probes, pretreatment with ribonuclease, ISH with a probe for beta-actin and Northern blot hybridization. Using biotinylated probes, gene transcripts of rat growth hormone and prolactin were detected by Northern blot hybridization. The same biotinylated probes were used not for light microscope ISH but also for the electron microscopical demonstration of rat growth hormone and prolactin mRNAs on the polysomes of the rough endoplasmic reticula. It is emphasized that biotinylated oligonucleotide probes are useful for the analysis of pituitary endocrine function because they are applicable to the three hybridization methods, namely, Northern blot hybridization and ISH at the light and electron microscope levels.

[Studies on hemolytic streptococcal infection: 1). Outbreak of group C hemolytic streptococcal infection in Formosan squirrels].

Between mid-October to mid-November 1992, of 500 freely-ranging Formosan and striped squirrels kept at Garden Y in the suburbs of Kanagawa Prefecture, 414 (82.8%) suddenly died one after another by bleeding from the nasal and oral cavities after developing a mild facial swelling. Isolation of microbes including viruses were carried out from the Formosan squirrels that had suddenly died. Various organs from these animals were histologically examined. 1. In bacteriological tests, beta-hemolytic streptococcal strains were isolated in a pure culture from 5 (83.3%) of 6 Formosan squirrels that had died suddenly. By serological analysis, 14 isolated strains were serotyped as group C according to the classification of Lancefield. From their biochemical characteristics, these were identified as Streptococcus equi subsp. zooepidemicus. A drug sensitivity test revealed that ABPC, PCG, SBPC, CMX and CPZ are highly sensitive against the isolates. 2. In the virological test, the viral isolation was applied for three blind passages by primary cultured kidney cells of Formosan squirrels, but no evidence of CPE was obtained. 3. At autopsy, a pathological change was detected mainly in the lungs. Histopathological examinations revealed severe hypertrophic changes of the alveolar wall in the entire pulmonary lobe. Severe congestion, hemorrhagic pneumonia, neutrophils and macrophages infiltration were observed in the hypertrophic alveolar wall. In the other cases, thrombi were observed in the branches of the pulmonary artery. Other organs demonstrated no remarkable histopathological changes. 4. Streptococcal strains were not isolated from the pharynx in all of the employees working at this garden.

Isolation of a metastasizing cancer cell line from an aflatoxin B1-induced rat liver tumor.

An attempt was made to isolate cancer cell lines from liver tumors that had been induced by aflatoxin B1 (AFB1) in rats. A clonal cell line named AFB-1 was isolated from a liver tumor that was histologically diagnosed as hepatocellular carcinoma. When AFB-1 cells were inoculated into the subcutaneous tissue at the dorsal region of syngenic animals, they metastasized from the site of inoculation into the abdominal cavity to form many tumor nodules throughout the serous membrane and metastatic foci in the kidney and pancreas. They also metastasized into the thoracic cavity to form metastatic foci in the lung. This is the first instance where a metastasizing AFB1-induced cancer cell line has been isolated.