Hajime Tsujimoto

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.

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.

Cloning and mapping of cat (Felis catus) immunoglobulin and T-cell receptor genes

Abstractu2003Molecular cloning and chromosomal mapping of the cat immunoglobulin (Ig) and T-cell receptor (TcR) genes were carried out to provide basic information for genetic analysis of immunologic diseases including leukemias and lymphomas in cats. We cloned two Ig constant genes, IGHM and IGHG and three TcR constant genes, TRAC, TRGC, and TRDC, by polymerase chain reaction (PCR) amplification of cDNA from cat peripheral blood mononuclear cells. For chromosomal mapping of the Ig and TcR loci including the IGK, IGL, and TRB on the cat genome, we performed PCR screening of DNAs from 37 cat × rodent somatic cell hybrids by using specific primers for the given genes. Consequently, three loci for IGH, TRA, and TRD, and two loci for TRB and TRG were found to be syntenic and assigned to cat chromosomes (FCA) B3 and A2, respectively. Further, IGK and IGL loci were mapped on FCA A3 and D3, respectively. These findings support the notion that the genetic linkages between the Ig and TcR genes are extensively conserved between humans and cats.

Molecular cloning and functional expression of equine interleukin-1 receptor antagonist

Equine interleukin-1 receptor antagonist (IL-1ra) was molecularly cloned to establish a basis for cytokine therapy of acute and chronic inflammatory diseases in the horse. cDNA clones encoding the whole coding sequence of equine IL-1ra were isolated from equine peripheral blood mononuclear cells (PBMC) that had been stimulated with lipopolysaccharide (LPS). The equine IL-1ra cDNA obtained in this study contained an open reading frame encoding 177 amino acid residues. The predicted amino acid sequence of equine IL-1ra shared 75.7, 75.3 and 76.3% similarity with sequences of human, murine and rabbit IL-1ras, respectively. An N-glycosylation site and five cysteine residues conserved in human, murine and rabbit IL-1ras were also found at the corresponding positions in equine IL-1ra. Recombinant glutathione S-transferase (GST)-equine IL-1ra fusion protein produced by Escherichia coli was purified. This protein was shown to inhibit the cytostatic or cytotoxic activity of IL-1 on A375S2 cells, indicating that the equine IL-1ra cDNA obtained in this study encodes biologically active equine IL-1ra.

The fit -1 common integration locus in human and mouse is closely linked to MYB

The fit-1 locus was originally identified as a common insertion site for feline leukemia virus (FeLV) in thymic lymphosarcomas induced by FeLV-myc recombinant viruses, suggesting that it harbors a gene that cooperates with Myc in T-cell leukemogenesis. We have previously mapped the fit-1 locus to feline Chromosome (Chr) B2. We have now identified conserved sequences that allow the mapping of the murine homolog using the European Interspecific Backcross (EUCIB). This shows that fit-1 is located on mouse Chr 10, 1cM proximal to Ahi-1, a murine retroviral integration locus that is closely linked to Myb. Moreover, the physical linkage to MYB is maintained in the human genome, as shown by cloning of the human homolog of fit-1 from a Chr 6 cosmid library and a series of overlapping PAC clones. Generation of a contig map around the human homolog of fit-1 reveals that it is approximately 100-kb upstream of MYB. In addition to fit-1 and Ahi-1, two other common insertion sites, Mis-2 and Mml-1, have also been mapped adjacent to Myb on mouse Chr 10. Previous analysis of tumors carrying insertions at fit-1, Mml-1, Mis-2 and Ahi-1 showed no obvious abnormalities in Myb expression. However, the cluster of viral insertion loci in this region suggests either the presence of a closely linked activation target or that subtle effects on Myb have been overlooked.

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.

Gammopathy with two M-components in a dog with IgA-type multiple myeloma

A 12-year neutered male mixed-breed dog was referred to hospital for evaluation of chronic diarrhea. Cellulose acetate electrophoresis of its serum revealed two monoclonal peaks in the gamma-globulin fraction. On immunoelectrophoretic analysis, the two monoclonal peaks in the gamma-globulin region were strongly precipitated with anti-dog IgA serum. On sodium dodecyl sulfate-polyacrylamide gel electrophoretic analysis, the fractions corresponding to these two peaks were shown to be dimer and trimer or tetramer of immunoglobulin consisting of heavy and light chains. These results indicated that the studied dog had gammopathy with two M-components with dimer and trimer or tetramer of IgA. Accumulations of large amounts of these immunoglobulins with very high molecular weight in the serum were concluded to induce the hyperviscosity syndrome in this dog in the terminal stage.

Chromosomal translocations in two feline T-cell lymphomas

Two feline malignant lymphoma cell lines, FT-1 and FT-G, established from cats naturally infected with the feline leukemia virus were analyzed for chromosomal aberrations. Both FT-1 and FT-G cells had a modal number of 38 which is the normal diploid (2n) chromosome number of the domestic cat. G-banding-analysis showed that FT-1 had a translocation involving the short arms of chromosome A2 and D3--t (A2;D3) (p-;p+), and FT-G had a translocation involving the short arms of chromosomes A2 and B2--t (A2;B2) (p-;p+). Our data suggest that the chromosomal translocations were closely associated with the tumorigenesis in malignant lymphoma in cats.

Molecular cloning and functional expression of feline thrombopoietin

Feline thrombopoietin (TPO) was molecularly cloned to establish a basis for cytokine therapy of thrombocytopenia in cats. cDNA clones covering the whole coding sequence of feline TPO were isolated from feline liver. The feline TPO cDNA obtained in this study contained an open reading frame encoding 349 amino acid residues. The predicted amino acid sequence of feline TPO shared 78.7, 69.9, 72.9 and 83.0% similarity with sequences of human, murine, rat and canine TPO, respectively. Four cysteine residues and two of four N-glycosylation sites that are conserved among species were also found at the corresponding positions in feline TPO. The feline TPO cDNA fragment encoding the whole amino acid coding region was recloned into an expression vector, and the resulting vector was transfected into 293T cells using the calcium phosphate method. The supernatant of the transfected 293T cells stimulated the proliferation of a human megakaryoblastic leukemia cell line (UT-7/TPO) cells in a dose dependent manner, indicating that the feline TPO cDNA obtained in this study encodes biologically active feline TPO.

Molecular cloning of feline CC–chemokine cDNAs

cDNA clones of feline chemokines, MIP-1alpha, MIP-1beta and RANTES, were molecularly isolated with the purpose of using these sequences for future investigation of the inhibitory effects on lentivirus entry and their role in immunological functions. The feline MIP-1alpha and MIP-1beta cDNA clones spanned their entire coding regions encoding 93 and 92 amino acids, respectively. The amino acid sequences of feline MIP-1alpha and MIP-1beta compared to those of their human, mouse and rat counterparts showed similarities of 75.3-79.6% and 73.9-88.0%, respectively. Feline MIP-1alpha and MIP-1beta had four conserved cysteines with a structure made up of the first two cysteines that are characteristic of the CC-chemokine subfamily. The amino terminal of these MIP-1alpha and MIP-1beta sequences was distinctly hydrophobic, suggesting that they may function as signal peptides. A partial cDNA clone consisting of 193 bp was obtained for feline RANTES, and it also showed a high degree of sequence similarity to those of other species and contained the characteristic structure made up of adjacent cysteines. These molecular clones of feline chemokines will be useful in the examination of their inhibitory effect on the cellular entry of feline immunodeficiency virus.