Yasuyuki Momoi

Establishment and characterization of a canine T-lymphoblastoid cell line derived from malignant lymphoma

A canine lymphoma cell line (CL-1) was established in culture from tumor cells found in the pleural fluid of a 7-year old female Japanese terrier with thymic form lymphoma. The CL-1 cells were positive for CD45 and MHC class II and negative for CD4, CD5, CD8, Thy-1 and B-cell specific antigen and surface immunoglobulin. The CL-1 cells had a rearranged T-cell receptor beta-chain gene and a germ-line form immunoglobulin gene, indicating that the CL-1 cells represented a monoclonally expanded population of canine alpha beta T-cell lineage.

Detection of enhancer repeats in the long terminal repeats of feline leukemia viruses from cats with spontaneous neoplastic and nonneoplastic diseases

Enhancer duplication in the long terminal repeat of feline leukemia virus (FeLV) was examined in primary cells from naturally FeLV-infected cats with various neoplastic and nonneoplastic diseases using the polymerase chain reaction. In all cases, a 170-bp band, corresponding to a standard exogenous FeLV with one copy of enhancer, was detected. Repeated enhancer sequences were found in all 8 cases of thymic-form lymphosarcoma, in some cases of lymphosarcoma of other forms (3/8) and myeloid tumors (2/3), and in only 1 of 6 cases with nonneoplastic diseases. The copy number of FeLV proviruses with a repeated enhancer seemed higher than that of those with one copy of enhancer in 3 cases of thymic form lymphosarcoma. In 5 cases of thymic form lymphosarcoma and in 1 case of erythroleukemia, coexistent FeLVs with double and triple enhancers of different sizes were found. Of the enhancer elements, only the SV40 core binding site was found in all the enhancer direct repeats of these FeLVs. All the provirus clones with single and duplicated enhancer sequences from a single tumor showed mutations or deletions characteristic to that tumor, indicating that enhancer repeats may arise in individual animals after infection with a single virus clone. The present findings indicate that FeLV with enhancer repeats generated in the cat is associated with the induction of neoplastic diseases in natural conditions.

Genetic heterogeneity of env gene of feline immunodeficiency virus obtained from multiple districts in Japan

Feline immunodeficiency virus (FIV) infection is widespread in many countries. FIV isolates have been classified into five distinct subtypes, A, B, C, D and E based on their env gene sequences. Several reports indicate that most of the FIVs isolated in Japan belong to subtype B which includes the first Japanese isolate, TM2 strain. To examine the distribution of FIV subtypes in Japan, proviral DNA sequences of the env gene were directly amplified by nested PCR from FIV-infected cats that had been kept in multiple districts throughout Japan. Phylogenetic analysis of the 11 strains showed that four FIV subtypes, A, B, C and D, were present in Japan. Among these subtypes, subtypes B and D were the two most common subtypes in Japan, and they were mainly distributed in the eastern and western parts of Japan, respectively. The present study provides information that is fundamental for development of a vaccine to protect against FIV infection in cats.

MOLECULAR CHARACTERISTICS OF MALIGNANT LYMPHOMAS IN CATS NATURALLY INFECTED WITH FELINE IMMUNODEFICIENCY VIRUS

Neoplastic disease, especially malignant lymphomas, are often observed in cats infected with feline immunodeficiency virus (FIV). In order to clarify the characteristics of lymphoma cells and to investigate the pathogenesis in FIV-infected cats, we examined the lymphoma tissues developed in five cats naturally infected with FIV by Southern blot analyses using feline immunoglobulin (Ig), T-cell receptors (TCR) and FIV probes. All of the five cases were serologically positive for anti-FIV antibody and negative for feline leukemia virus antigen. Of these five lymphoma samples, two displayed rearrangement of the Ig heavy chain gene and deletion of the Ig light (kappa) chain gene, indicating that the tumor cells were committed to B-cell development. One tumor sample was identified as a T-cell lymphoma because of the presence of a rearranged TCR beta-chain gene. The other two cases were considered to be non-T non-B cell lymphoma because they did not show any rearrangement of the Ig and TCR genes. Therefore, no consistent tumor type was found in lymphoma cases infected with FIV. Clonal integration of FIV provirus was not detected in any of the five lymphoma samples obtained from FIV-infected cats using Southern blot analysis, although FIV proviral genome was detected in the genomic DNA of all the lymphoma samples by using a polymerase chain reaction (PCR). These results indicated that FIV might not play a direct role in tumorigenesis of lymphoma in cats.

Detection of apoptosis induced in peripheral blood lymphocytes from cats infected with feline immunodeficiency virus

SummaryPeripheral blood lymphocytes (PBL) from cats infected with feline immunodeficiency virus (FIV) were examined for the occurrence of apoptosis after short-term culture. In the PBL from FIV-infected cats, changes in flow-cytometry scattergram, morphological characteristics of apoptosis and nucleosomal DNA fragmentation were observed. Percentages of apoptotic cells by flowcytometry analysis in PBL from FIV-infected cats (22.4%±9.4%) were significantly higher than those in PBL from uninfected control cats (9.2%±3.5%). The lymphocytes which underwent apoptosis included CD5+, CD4+ and sIgM+ cells, indicating that induction of apoptosis was not restricted to a special subset of lymphocytes. These findings provide evidence of the apoptotic state of PBL in cats with FIV infection.

Molecular characterization of feline immunodeficiency virus genome obtained directly from organs of a naturally infected cat with marked neurological symptoms and encephalitis

SummaryFeline immunodeficiency virus (FIV) was first isolated from cats with immunodeficiency syndrome. Recently, neurological abnormalities and brain lesions were shown in cats infected with FIV. To investigate the FIV genome associated with central nervous system (CNS) lesions, proviral DNA sequences from the V3–V6 region of the FIVenv gene were directly amplified from uncultured necropsy tissues of a 2-year-old naturally FIV-infected cat with marked neurological symptoms and encephalitis. By in situ hybridization, FIV RNA was detected mainly in the astrocytes. Fifteen clones isolated from cerebrum, bone marrow and lymph node samples showed only a small number of mutations or deletions in this region. A representative clone, JN-BR1, was distantly related to the previous Japanese strain (TM2) belonging to the subtype B. However, it was relatively close to the Petaluma strain which is known to infect feline brain-derived culture cells and induce brain lesions in inoculated cats. By phylogenetic analysis, the JN-BR1 strain was placed in subtype A that included Petaluma strain and several other American and European strains. The JN-BR1 strain derived from brain with encephalitis in this study and the Petaluma strain may share a common genetic structure that is related to their neuropathogenicity.

Detection of feline immunodeficiency virus proviral DNA in feline peripheral blood mononuclear cells by the nested two-step polymerase chain reaction

The polymerase chain reaction (PCR) was applied to detect feline immunodeficiency virus (FIV) proviral DNAs in primary peripheral blood mononuclear cells (PBMC). Suitable conditions for PCR amplification were examined to obtain highly sensitive and specific results by simple staining in agarose gel. Specific amplification of FIV proviral DNA in PBMC DNA of FIV-infected cats was achieved by a nested two-step PCR that amplified the DNA first with outer primers and then with inner primers nested within the first primers. PCR amplification using different primers indicated that those based on the gag sequence of the FIV/TM2 strain isolated in Japan were suitable for the detection of FIV genomes in naturally infected Japanese pet cats. By the nested two-step PCR with mixed gag primers of TM2 and Petaluma, isolated in the USA, we could detect FIV genomes in all 11 primary PBMC samples from FIV-seropositive cats tested. The PCR protocol developed here is sensitive and specific for molecular detection of FIV infection in cats.

Quantitative analysis of Fas and Fas ligand mRNAs in a feline T-lymphoid cell line after infection with feline immunodeficiency virus and primary peripheral blood mononuclear cells obtained from cats infected with the virus.

Apoptosis is frequently observed in feline lymphocytes in association with feline immunodeficiency virus (FIV) infection. In this study, to investigate the mechanism of FIV-induced apoptosis, levels of Fas and Fas ligand mRNAs were measured by real-time reverse transcription-PCR. In a feline T-lymphoid cell line the amounts of Fas ligand mRNA increased along with the induction of apoptosis after in vitro infection with FIV. In PBMC collected from 10 cats naturally infected with FIV, Fas ligand mRNA levels were significantly higher than those in PBMC from five uninfected cats. These results indicate that the increased expression of Fas ligand may be involved in the induction of apoptosis of lymphocytes in FIV infection.

Clinicopathological and immunological characteristics of six cats with granular lymphocyte tumors

Clinical and immunological characteristics were investigated in six cases of feline granular lymphocyte (GL) tumor. The ages of the affected cats were relatively old, ranging from 4 to 13 years of age. Gastrointestinal signs were commonly observed in these cases. Only one of the six GL tumor cases was positive for feline leukemia virus (FeLV) antigen. Phenotypic analysis revealed that the GL tumor cells from all of the six cases lacked the T- or B-cell markers. These GL tumor cells were examined by Southern blot analysis using feline immunoglobulin (Ig) and T-cell receptor (TCR) gene probes. GL tumor cells obtained from two cases were identified as cells of T-cell lineage by the presence of a rearranged TCR beta gene, whereas those from the other four cases were considered to be derived from non-T- non-B-cell lineage because of the absence of rearrangement of these genes. These findings indicated that feline GL tumors can be considered as a specific disease entity in feline lymphomas because the cases examined in this study showed onset at an older age, a low incidence of FeLV infection and frequent involvement of gastrointestinal lesions, which are not found in typical FeLV-associated lymphomas. Although no specific phenotypes was observed by phenotypic analysis, the feline GL tumor cells were divided into two consistent genotypes of T-cell or non-T- non-B-cell lineages.

Molecular cloning of feline Fas antigen and Fas ligand cDNAs

The Fas antigen (FasA) and Fas ligand (FasL) are key molecules which mediate apoptosis. For investigation of apoptosis in cats, we isolated molecular clones of feline FasA and FasL cDNAs by using the polymerase chain reaction (PCR) method to amplify cDNAs from feline lymphoma cell lines. These feline FasA and FasL clones contained complete open reading frames encoding 314 and 280 amino acids, respectively. These feline FasA and FasL cDNA clones had structures characteristic of the tumor necrosis factor (TNF) receptor family and TNF family, respectively. The deduced amino acid sequence of feline FasA and feline FasL, respectively showed 45.0%-60.0% and 75.0%-90.0% similarity with their human, mouse and bovine counterparts. These data will be helpful for investigating the role of the FasA and FasL system in apoptosis and for studying the various diseases associated with the deregulation of apoptosis in cats.