Molecular characterization of swine leukocyte antigen (SLA) genes is important for elucidating the immune responses between swine-donor and human-recipient in xenotransplantation. Examination of associations between alleles of SLA class I genes, type of pig genetic modification, porcine endogenous retrovirus (PERV) viral titer, and PERV subtypes may shed light on the nature of xenograft acceptance or rejection and the safety of xenotransplantation. No significant difference in PERV gag RNA level between transgenic and non-transgenic pigs was noted; likewise, the type of applied transgene had no impact on PERV viremia. SLA-1 gene profile type may correspond with PERV level in blood and thereby influence infectiveness. Screening of pigs should provide selection of animals with low PERV expression and exclusion of specimens with PERV-C in the genome due to possible recombination between A and C subtypes, which may lead to autoinfection. Presence of PERV-C integrated in the genome was detected in 31.25% of specimens, but statistically significant increased viremia in specimens with PERV-C was not observed. There is a need for multidirectional molecular characterization (SLA typing, viremia estimation, and PERV subtype screening) of animals intended for xenotransplantation research in the interest of xeno-recipient safety.
Alleles ; Animals ; Endogenous Retroviruses ; Genes, MHC Class I ; Genes, MHC Class II ; Genome ; Heterografts ; Leukocytes ; Mass Screening ; Recombination, Genetic ; Retroviridae ; RNA ; Swine ; Transgenes ; Transplantation, Heterologous ; Viremia

Human T-cell leukemia virus type 1 (HTLV-1) is a retrovirus demonstrated to be associated with human disease. Infection by the HTLV-1 can cause T-cell leukemia (ATL) in adults. HTLV-1 bZIP factor (HBZ) is a viral protein encoded by the minus strand of the HTLV-1 provirus. Among the regulatory and accessory genes of HTLV-1, HBZ is the only gene that remains intact and which is expressed consistently in all patients with ATL. Moreover, HBZ has a critical role in the leukemogenesis of ATL. Here, we review the function of HBZ in the oncogenesis of HTLV-1 and its molecular mechanism of action.
Animals ; Basic-Leucine Zipper Transcription Factors ; genetics ; metabolism ; Carcinogenesis ; HTLV-I Infections ; pathology ; virology ; Human T-lymphotropic virus 1 ; genetics ; metabolism ; Humans ; Leukemia, T-Cell ; pathology ; virology ; Retroviridae Proteins ; genetics ; metabolism

Multidrug resistant genes are highly expressed in hepatocellular carcinoma that seriousty affects the effect of chemotherapy. Screening of resistant genes from HCC cells and studying its mechanism of drug resistance will be helpful to improve the effecacy of chemotherapy for hepatocellular carcinoma. Here we described an alternative method called cyclical packaging rescue (CPR). First we constructed a retrovirus cDNA library of hepatoma cells and used it to infect fibroblasts. Then we added drugs to screen survival cells. The survival cells, stably integrated helper-free retroviral libraries, were recovered rapidly after transfection with plasmids expressing retroviral gag-pol and env genes. Through this method, retroviral RNAs were directly repackaged into new infectious virions. Recovered retroviral supernatant was then used to reinfect fresh target cells. When performed in concert with selection using functional assays, cDNAs regulating functional responses could be identified by enrichment through multiple rounds of retroviral library recovery and retransmission. Using CPR, we obtained several cDNAs. After a preliminary detection, we found Ribosomal protein S11 (RPS11), Ribosomal protein L6 (RPL6), Ribosomal protein L11 (RPL11), Ribosomal protein L24 (RPL24) possibly had drug resistant function.
Carcinoma, Hepatocellular ; genetics ; pathology ; Cell Line, Tumor ; DNA, Complementary ; Drug Resistance, Neoplasm ; genetics ; Gene Library ; Genetic Vectors ; Humans ; Liver Neoplasms ; genetics ; pathology ; Plasmids ; Retroviridae ; Ribosomal Proteins ; genetics ; metabolism ; Transfection

Kidney cells of canine embryos were separated into single cells using collagenase and dispase. Primary culture was conducted using these cells. To remove fibroblasts, these cells were treated with edetate disodium dihydrate (Na2EDDA), and pure epithelial cells were separated. Recombinant retrovirus particles that manifest teromerase were produced and inoculated into primary culture cells to produce immortalized canine cell strains (JNUCK-1 and JNUCK-2). To examine the characteristics of the produced cell strains, the growth curve, maximum cultured households, and expressed proteins (keratin) were identified. The JNUCK-1 and JNUCK-2 cell lines showed division ability until the 30th generation without growth retardation. JNUCK-1 and JNUCK-2 cell lines clearly expressed telomerase until the 25th generation. The canine distemper virus (CDV) was inoculated into the JNUCK-1 and JNUCK-2 cell lines, as well as in the Madin-Darby canine kidney (MDCK) cell line. The maximum titer of CDV from the JNUCK-1 cell strain was about 200 times higher than that from the MDCK cell strain. However, the JNUCK-2 cell strain produced a lower titer than the MDCK cell strain. We established a new canine kidney epithelial cell line (JNUCK-1) that could produce CDV with high titer.
Cell Line* ; Collagenases ; Distemper Virus, Canine* ; Embryonic Structures ; Epithelial Cells ; Family Characteristics ; Fibroblasts ; Kidney* ; Madin Darby Canine Kidney Cells ; Retroviridae ; Telomerase

OBJECTIVETo investigate the effect of catalase (CAT) on engraftment of human hematopoietic stem cells (HSC) by co-transplanting umbilical cord-derived mesenchymal stem cells (UC-MSC) with over-expressed CAT and human HSC into NOD/SCID mice.

METHODSThe UC-MSC cultured in vitro were transfected by the retrovirus containing green fluorescent protein (GFP) and GFP-CAT genes respectively. MSC-GFP and MSC-GFP-CAT cell lines were sorted by flow cytometry. Co-culture and co-transplant experiments were performed to detect the effects of CAT on expansion and engraftment of human HSC.

RESULTSThe percentage of GFP(+) cells were approximately 97.6% and 96.8% after sorting. The mRNA expression of CAT in MSC-GFP-CAT was 23.9-fold higher than that in UC-MSC. The activity of CAT in UC-MSC, MSC-GFP, MSC-GFP-CAT cells were 19.5, 20.3 and 74.1 Unit respectively. There was no significant differences in the percentage of CD34(+) cells between 3 groups in co-culture experiment. And the percentage of human CD45(+) cells in NOD/SCID mice were (3.22 ± 3.1)%, (4.26 ± 3.56)% and (7.37 ± 4.51)% respectively.

CONCLUSIONMSC-GFP-CAT significantly improves the engraftment of human HSC in NOD/SCID mice, whereas co-culture with the MSC-GFP-CAT can not promote the expansion of HSC in vitro.

Animals ; Catalase ; Coculture Techniques ; Flow Cytometry ; Hematopoietic Stem Cells ; Humans ; Mesenchymal Stromal Cells ; Mice ; Mice, Inbred NOD ; Mice, SCID ; Retroviridae ; Transfection ; Umbilical Cord

OBJECTIVETo improve the MigR1-CD19-CAR (chimeric antigen receptor) that contains a single chain variable region (scFv) which targeted to CD19 through a retroviral vector transduction efficiency of T-lymphocytes.

METHODSInsert the CD19-CAR fragment into the retroviral vector (MigR1) through recombinant DNA technology, after transfecting plat-A packaging cell lines, viral supernatant was collected to transduce K562 cell line and activated human T-lymphocytes. We used flow cytometry to determine the transduction efficiency and RT-PCR to confirm the transcription of CD19-CAR gene. The ability of the transduced T cells to produce IFN-γ and TNF-α in a CD19-specific manner was measured in an enzyme-linked immunosorbent (ELISA) assay.

RESULTS(1)Using MigR1-CD19-CAR retroviral vector to produce the high titer retrovirus. (2)MigR1-CD19-CAR transduction efficiency of K562 cell line was significantly higher than human T-lymphocytes (P<0.01). (3)120 min centrifugation could significantly improve transduction efficiency of T-lymphocytes to (54.5±14.6)%. (4)Transduction efficiency could be improved by deciding transduce time according to T-lymphocytes proliferation fold in vitro individually, and the highest transduction efficiency in the study was 69.3%. The CD19-CAR gene sequence was transcripted specificly with high efficiency. (5) IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased to (13 230±1 543) pg/ml and (4 217±211) pg/ml when coculture with CD19-K562 cells.

CONCLUSIONWe have successfully constructed a second generation CAR which targeted to CD19 through a retroviral vector called MigR1 (MigR1-CD19-CAR). Deciding transduce time according to T-lymphocytes proliferation fold in vitro individually and 120 min centrifugation could improve the CAR transduction efficiency of T-lymphocytes. RT-PCR confirmed that the CD19-CAR gene was specificly transcripted with high efficiency. IFN-γ and TNF-α released by CD19-CAR transduced T-lymphocytes significantly increased when activated by target cells.

Antigens, CD19 ; Cell Proliferation ; Flow Cytometry ; Genetic Vectors ; Humans ; K562 Cells ; Recoverin ; Retroviridae ; T-Lymphocytes ; Transfection

Pulmonary tuberculosis (PTB) is a common infectious disease worldwide. However, mediastinal tuberculous lymphadenitis complicated by oesophageal involvement and oesophago-respiratory fistula is now uncommon due to improved anti-tuberculous regimes and better general awareness. The overall incidence of acquired oesophago-respiratory fistula due to infection is low, and therefore, the lesion is not often a frontrunner in differential diagnosis. Still, tuberculous oesophago-respiratory fistulae can potentially occur in patients with retroviral disease, as they tend to have atypical and more virulent manifestations. In this study, we report the case of multiple oesophago-respiratory fistulae in a patient with PTB and retroviral disease, and highlight the computed tomography features of these lesions as an atypical presentation of PTB in retroviral disease. Clinicians should suspect oesophago-respiratory fistulae if patients present with Ono’s sign, and remain particularly vigilant for patients with underlying PTB and retroviral disease, as early diagnosis and treatment could help to reduce mortality.
Adult ; Diagnosis, Differential ; Esophagus ; physiopathology ; Fistula ; diagnosis ; Humans ; Lung ; pathology ; Male ; Radiography, Thoracic ; Retroviridae ; metabolism ; Tomography, X-Ray Computed ; Trachea ; physiopathology ; Treatment Outcome ; Tuberculosis, Lymph Node ; Tuberculosis, Pulmonary ; complications ; diagnosis

This study was aimed to construct the mouse VCAM-1 expression vector, to establish the stably transfected MSC line and to investigate the effect of VCAM-1-modified mesenchymal stem cells (MSC) on the immunological characteristics of MSC. The cDNA of murine VCAM-1 gene was amplified by RT-PCR from the total RNA isolated from the mouse spleen; then the cDNA was inserted into the retrovirus vector PMSCVmigr-1; the recombinant plasmid was confirmed by restriction endonuclease experiments and sequencing, then designated as PMSCVmigr-1-mVCAM-1; the recombinant plasmid PMSCVmigr-1-mVCAM-1 was transfected into 293 cells by lipofecamin and the supernatant was collected to transfect MSC cell line (C3H10T1/2). Moreover, VCAM-1 expression on MSC was evaluated by FACS. Furthermore, the inhibitory effect of VCAM-1-MSC on lymphocytic transformation was tested by (3)H-TdR incorporation assay. The results indicated that the successful construction of recombinant retroviral expression plasmid of mouse VCAM-1 was confirmed by digesting and sequancing. After transfection of MSC with retroviral supernaptant, the high expression of VCAM-1 on MSC could be detected by flow cytometry. The MSC high expressing VCAM-1 could significantly inhibit the proliferation of Con A-inducing lymphocytes in dose-depentent marrer. It is concluded that recombinant retroviral encoding VCAM-1 (PMSCVmigr-1-mVCAM-1) has been successfully constructed and mouse VCAM-1 has been stably expressed in C3H10T1/2. MSC over-expressing VCAM-1 show more potent immunosuppressive effect on cellular immune reaction in vitro. Our data laid a foundation for the subsequent studying the effect of VCAM-1 transfecting into MSC on immune related disease study.
Animals ; Cell Line ; DNA, Complementary ; Genetic Vectors ; Mesenchymal Stromal Cells ; metabolism ; Mice ; Retroviridae ; Reverse Transcriptase Polymerase Chain Reaction ; Transfection ; Vascular Cell Adhesion Molecule-1 ; genetics

This study aims to construct a eukaryotic expression system for envelope gene of Jaagsiekte sheep retrovirus, observes its localization in 293T cells, and investigates the potential in inducing malignant transformation of NIH3T3 cells. By RT-PCR, the full-length cDNA of envelope gene of Jaagsiekte sheep retrovirus (exJSRV-env) was amplified from the extract of naturally infected sheep lung. The clone of target gene was sub-cloned into eukaryotic expression system pEGFP-C1, and validated by PCR, restriction endonuclease, and sequencing. Bioinformatic analysis concerning biological function and cellular localiza tion of exJSRV-env was also performed. The recombinant clone of exJSRV-env was transfected into 293T cells and NIH3T3 cells by Lipofectamine LTX. The expression and celluar localization in 293T cells were validated by confocal microscopy. Soft agar colony formation assay was employed to test the anchorage-independent growth of NIH3T3. DNA sequencing and restriction enzyme digestion with Kpn I and Hind III indicated the correct construction of the recombinant plasmid, which was named pEGFP-C1/exJSRV-env. Amino acid sequence alignment of exJSRV-env with reference sequences found 85%-100% homogeneity. A YRNM motif was discovered at the cytoplasmic tail of envelope gene, which is exclusively found in exogenous viruses. Phylogenetic tree analysis showed that our clone of exJSRV-env clustered closely with pathogenic exogenous Jaagsiekte sheep retroviruses. Fluorescence microscopy indicated typical membrane localization of exJSRV-env protein. NIH3T3 cells transfected with exJSRV-env lost contact inhibition, and acquired colony forming ability in soft agar. This study indicated that envelope protein of Jaagsiekte sheep retrovirus can induce malignant transformation of mouse fibroblast cell NIH3T3. Discoveries of this study provide a basis for further structural and functional research on Jaagsiekte sheep retrovirus envelope protein.
Amino Acid Sequence ; Animals ; Betaretrovirus ; chemistry ; classification ; genetics ; physiology ; Cell Transformation, Viral ; Green Fluorescent Proteins ; genetics ; metabolism ; Mice ; Molecular Sequence Data ; NIH 3T3 Cells ; Phylogeny ; Retroviridae Infections ; veterinary ; virology ; Sequence Alignment ; Sheep ; Sheep Diseases ; virology ; Transformation, Genetic ; Tumor Virus Infections ; veterinary ; virology ; Viral Envelope Proteins ; chemistry ; genetics ; metabolism

In order to study T-bet function in mouse cells, a novel retroviral vector expressing mouse T-bet and reporter gene Thy1.1 was constructed. Retrovirus particles were then produced by transfection of the recombinant retroviral plasmid into a packaging cell line Platinum-E. The recombinant retrovirus played considerable infection ability. T-bet expression was then identified by FACS after infection of CD4+ primary T cells from T-bet knockout mouse with recombinant retrovirus. To determine if exogenous expressing T-bet has normal function, we checked the expression level of T-bet target gene, Ifng. IFN-y expression was upregulated in the T-bet knockout T cells infected with recombinant retrovirus. In conclusion, we successfully constructed an effective mouse T-bet recombinant retroviral vector.
Animals ; Cell Line ; Genetic Vectors ; Interferon-gamma ; metabolism ; Mice ; Mice, Knockout ; Recombinant Proteins ; biosynthesis ; Retroviridae ; T-Box Domain Proteins ; biosynthesis ; T-Lymphocytes ; metabolism ; Transfection