Characterization of monocyte-derived dendritic cells from cats infected with feline immunodeficiency virus.

Dendritic cells (DCs) are professional antigen presenting cells (APCs) that possess an extraordinary capacity to stimulate naïve T cells and initiate a primary immune response. To develop a DC-based immunotherapy for feline immunodeficiency virus (FIV) infection, we carried out a study to characterize DCs from FIV-infected cats and compared the observations with those obtained from healthy controls. DCs were derived from adherent peripheral blood mononuclear cells that had been cultivated with recombinant feline interleukin 4, granulocyte macrophage colony-stimulating factor and heat-inactivated autologous plasma. Various parameters, such as cell morphology, surface phenotype, endocytosis and mixed leukocyte reaction (MLR), were analyzed to characterize feline DCs. Monocyte-derived DCs from FIV-infected cats as well as those from healthy controls showed a dendritic appearance and expressed an APC-like phenotype (CD1c(+), CD80(+) and MHC class II(+)). However, the expression level of CD1a was variable in the DCs derived from FIV-infected cats, although this was not the case in the DCs derived from the healthy controls. DCs from the FIV-infected cats retained the ability to take up dextran via the mannose receptor and also showed an apparent MLR, indicating that these cells could be useful in immunotherapy. In this study, monocytes obtained from FIV-infected cats could differentiate into functional DCs, suggesting that they might be used in a DC-based immunotherapy against FIV infection.

Disseminated histiocytic sarcoma with excessive hemophagocytosis in a cat.

A 10-year-old Japanese domestic cat was presented with anorexia and weight loss. Severe anemia and thrombocytopenia were detected. Abdominal radiography and ultrasonography revealed the presence of multiple masses in the spleen. Cytological analyses of the masses revealed several atypical histiocytic cells and considerable hemophagocytosis. A splenectomy was performed, and the mass was diagnosed as histiocytic sarcoma on the basis of histopathological, cytochemical and immunohistochemical analyses. Further, abnormal hemophagocytosis was observed in the bone marrow. The cat was administered prednisolone and lomustine, and it survived for 107 days after admission. An autopsy revealed the presence of neoplastic histiocytic cells in the bone marrow, liver, pancreatic lymph node and glomeruli. This is the first case of histiocytic sarcoma in a cat to be reported in Japan.

Inhibitory effect of newly developed CXC-chemokine receptor 4 antagonists on the infection with feline immunodeficiency virus.

CXC-chemokine receptor 4 (CXCR4) functions as a receptor for feline immunodeficiency virus (FIV). Although we previously found that a CXCR4 antagonist, T140, inhibited the FIV replication in vitro, it was not effective in cats infected with FIV because of its low stability in feline serum. To resolve this problem, several T140 derivatives have been developed. Here, we examined the efficacy of T140 analogs, TF14016 and TF14013, on the inhibition of FIV infection. These compounds were shown to significantly inhibit the syncytia formation in CXCR4-expressing cells after co-cultivation with FIV-infected cells and the replication of FIV in a feline lymphoid cultured cell line. These results indicated that TF14016 and TF14013 could be useful as antiviral drugs for cats infected with FIV.

Quantitative assessment of minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction.

Lymphoma is the most common hematopoietic malignancy in dogs. Although a large proportion of dogs with lymphoma can achieve clinical remission by initial chemotherapy, most dogs die as a consequence of tumor relapse. We established a quantitative detection system for minimal residual disease (MRD) in canine lymphoma by using real-time polymerase chain reaction (PCR). A canine T-cell lymphoma-derived cell line, namely, UL-1, was used to examine the specificity and sensitivity of the MRD detecting system. Allele-specific oligonucleotide primers and probes were designed based on the sequence of T-cell receptor gamma chain (TCRgamma) gene fragment of UL-1 cells in conjunction with its downstream sequence, which were obtained from the dog genome database. The real-time PCR system for plasmid DNA containing the TCRgamma gene derived from UL-1 cells and the genomic DNA of UL-1 cells revealed that the system was accurate for 10-100,000 copies per reaction and its sensitivity was 1 cell per 10,000 cells. In order to monitor the kinetics of tumor cell number in canine lymphoma, we quantified the level of MRD in the peripheral blood of 7 dogs with lymphoma under chemotherapy. Since the lymphoma cells from the 7 patients were shown to be B-cell origin from the finding of clonal rearrangement of immunoglobulin heavy chain (IgH) gene, allele-specific oligonucleotide primers and probes were prepared based on the sequence of rearranged IgH gene in each case. The number of peripheral blood tumor cells measured by the real-time PCR was comparable to that estimated by conventional hematological examination in 2 cases of stage V lymphoma. MRD in the peripheral blood was detectable in all 7 cases, even in the complete remission (CR) phase. In the 7 lymphoma dogs, changes in the MRD levels of peripheral blood generally paralleled with the changes in the volumes of lymph nodes. Molecular CR, in which the MRD level was below the detection limit, was not observed in any of these 7 patients under chemotherapy. The MRD level detected by the real-time PCR method described here would be useful for investigating the kinetics of tumor cell growth and its regression in canine lymphoma patients.

Inhibition of the replication of feline immunodeficiency virus by lentiviral vector-mediated RNA interference in feline cell lines.

RNA interference (RNAi) is a sequence-specific RNA degradation process. To inhibit feline immunodeficiency virus (FIV) replication by RNAi, we generated a lentiviral vector expressing a short hairpin RNA (shRNA) that targeted the gag gene of FIV (shGag). shGag transfer significantly inhibited viral replication in cell lines that were chronically infected with FIV, i.e., the 3201/UK8 low, 3201/UK8 high, FL4, and CRFK/FIV cell lines. Moreover, 3201 cells were transduced with the lentiviral vectors and then inoculated with FIV. Although the amount of FIV proviral DNA in shGag-transduced cells was similar to that in the cells transduced with unrelated shRNA or mock-transduced cells, the amount of reverse transcriptase (RT) activity was significantly reduced in the culture supernatant of shGag-expressing cells from 15 to 27 days after inoculation. Thirty days after inoculation, no significant difference was observed in the RT activities but virus with a mutation in the target region of shGag was detected in approximately 21% of the replicated viruses. Therefore, abolishment of the silencing effect of shGag may be due to reasons other than the emergence of escape mutants. These results are useful for developing an RNAi-based gene therapy strategy for controlling FIV infection.

Induction of chemoresistance in a cultured canine cell line by retroviral transduction of the canine multidrug resistance 1 gene.

OBJECTIVE: To induce chemoresistance in a normal canine cell line through the transduction of the canine multidrug resistance 1 gene (mdr1). SAMPLE POPULATION: Madin-Darby canine kidney (MDCK) epithelial cell line. PROCEDURES: The full-length canine mdr1 cDNA clone isolated in our laboratory was inserted into a Moloney murine leukemia virus-based vector to construct the retroviral vector, pLNC-cMDR1. After retroviral transduction of pLNC-cMDR1 into MDCK cells, the expression and function of the P-glycoprotein, a product of mdr1, were assessed by immunoblotting, measurement of rhodamine123 (Rh123) retention, and drug sensitivity assays. RESULTS: P-glycoprotein was strongly expressed in cells transduced with pLNC-cMDR1. This P-glycoprotein was fully functional, as demonstrated by the decreased Rh123 retention and the increased resistance to chemotherapeutic drugs. Measured as 50% inhibitory concentrations, resistance increased 59 times to vincristine and 25 times to doxorubicin in MDCK cells after transduction of pLNC-cMDR1. CONCLUSIONS AND CLINICAL RELEVANCE: Transduction of canine mdr1 is an effective method for inducing chemoresistance in normal canine cells. This system may be applicable to the induction of drug resistance in hematopoietic cells.

Application of RNA interference for inhibiting the replication of feline immunodeficiency virus in chronically infected cell lines.

RNA interference (RNAi) is a process in which double-stranded RNA induces the post-transcriptional sequence-specific degradation of homologous messenger RNA. The present study was carried out to apply the RNAi technology to inhibit the replication of feline immunodeficiency virus (FIV). Four small interfering RNAs (siRNAs) homologous to the FIV gag gene were synthesized and transfected into a feline fibroblastic cell line chronically infected with FIV (CRFK/FIV). These synthetic siRNAs efficiently inhibited the replication of FIV. Next, we examined the effect of retroviral vector-mediated transfer of FIV-specific short hairpin RNA (shRNA) on the replication of FIV in a feline T-cell line chronically infected with FIV (FL4). The retroviral vector-mediated transfer of FIV-specific shRNA was shown to markedly inhibit the replication of FIV in the FL4 cells. These results provide useful information for the development of RNAi-based gene therapy strategy to control FIV infection.