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1.
Electron. j. biotechnol ; 41: 56-59, sept. 2019. tab, graf
Article in English | LILACS | ID: biblio-1087166

ABSTRACT

Background: Chinese hamster ovary (CHO) cells are the most dependable mammalian cells for the production of recombinant proteins. Replication-incompetent retroviral vector (retrovector) is an efficient tool to generate stable cell lines. Multiple copies of integrated genes by retrovector transduction results in improved recombinant protein yield. HEK-293 and their genetic derivatives are principal cells for retrovector production. Retrovectors packaged in HEK-293 cells pose a risk of infectious agent transmission, such as viruses and mycoplasmas, from serum and packaging cells. Results: In this report, retrovectors were packaged in CHO cells cultured in chemically defined (CD) media. The retrovectors were then used to transduce CHO cells. This method can block potential transmission of infectious agents from serum and packaging cells. With this method, we generated glucagon-like protein-1 Fc fusion protein (GLP-1-Fc) stable expression CHO cell lines. Productivity of GLP-1-Fc can reach 3.15 g/L. The GLP-1-Fc protein produced by this method has comparable bioactivity to that of dulaglutide (Trulicity). These stable cell lines retain 95­100% of productivity after 40 days of continuous culture (~48­56 generations). Conclusions: Suspension CHO cells are clean, safe, and reliable cells for retrovector packaging. Retrovectors packaged from this system could be used to generate CHO stable cell lines for recombinant protein expression.


Subject(s)
Retroviridae , Recombinant Proteins/metabolism , CHO Cells/metabolism , Immunoglobulin Fc Fragments , Cell Line , Chromatography, Gel/methods , Disease Vectors , Glucagon-Like Peptide 1 , Tandem Mass Spectrometry , Batch Cell Culture Techniques
2.
Pesqui. vet. bras ; 39(7): 516-522, July 2019. tab, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1040716

ABSTRACT

Oral lesions are common problems in feline medicine worldwide, and may be associated with different causes, such as infectious agents. There are only a few studies reporting the chief oral diseases and the results for retrovirus tests in shelter cats in Brazil, especially in the South region. This study aimed to identify the main inflammatory oral lesions in shelter cats and verify the test results for feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) infections. Forty-three felines from private shelters in the central region of Rio Grande do Sul state (RS) that presented clinically evident oral lesions, regardless of age, breed, sex, and neuter status, were used in this survey. Serological tests for FIV and FeLV were performed in all cats, and data regarding the rearing system were collected. Sixteen cats (37.2%) were reared in a free system, whereas 27 (62.8%) were kept under a restrict system. Of the 43 cats with oral lesions, 29 (67.44%) presented only one type of lesion, characterized as periodontitis (n=22, 51.16%), followed by gingivitis (n=6, 13.95%), and stomatitis (n=1, 2.32%). Concomitant stomatitis and periodontitis were found in the 14 remaining cats (100%). With respect to the test results for retrovirus infections, nine (20.93%) of the 43 felines were positive for FIV alone. Co-infection with both viruses was observed in seven cats (16.28%). No cat was seropositive for FeLV valone. None of the six cats that presented gingivitis was positive for FIV and FeLV; one cat with stomatitis was positive for FIV and FeLV; of the 22 cats with periodontitis, six (27.27%) were FIV positive and two (9.09%) were FIV/FeLV positive; and of the 14 cats that presented stomatitis and periodontitis, three (21.43%) were FIV positive and four (28.57%) were FIV/FeLV positive. As for diagnosis, 28 cats (65.1%) presented solely periodontal disease (PD), one cat (2.32%) had feline chronic gingivostomatitis (FCG) alone, and 14 (32.5%) had both PD and FCG. The results obtained show that the main oral lesions found in shelter cats in the central region of RS were gingivitis, stomatitis, and periodontitis. Periodontitis, in association or not with stomatitis, was the most frequently observed oral cavity lesion in FIV- and/or FeLV-positive cats. Other factors may contribute to installation of inflammatory oral diseases in shelter cats because most cats with oral cavity lesions tested negative for retrovirus infections.(AU)


As afecções orais são problemas comuns em medicina felina em diferentes locais do mundo e podem estar relacionadas a diferentes causas, como agentes infecciosos. Poucos estudos foram encontrados no Brasil sobre o levantamento das principais doenças orais e dos resultados de testes para retrovírus em gatos de abrigos, principalmente na região Sul. Diante disso, o objetivo deste artigo foi identificar as principais afecções orais inflamatórias em gatos de abrigos e verificar os resultados dos testes para o vírus da imunodeficiência felina (FIV) e o vírus da leucemia felina (FeLV). Foram incluídos 43 felinos provenientes de abrigos privados localizados na região central do Rio Grande do Sul (RS) que apresentavam lesões orais clinicamente evidentes, independente de idade, raça, gênero e estado reprodutivo. Em todos os gatos foram realizados testes sorológicos para FIV e FeLV e obtidas informações referentes ao sistema de criação. Em 16 gatos (37,2%), o sistema de criação era livre, enquanto em 27 (62,8%) era restrito. Dos 43 gatos com lesões orais, em 29 (67,44%) foi verificado somente um tipo de lesão, caracterizado como periodontite (n=22, 51,16%), seguido de gengivite (n=6, 13,95%) e estomatite (n=1, 2,32%). Lesões concomitantes de estomatite e periodontite foram encontradas nos 14 gatos (100%) restantes. Quanto aos resultados dos testes para retrovírus, nove (20,93%) dos 43 felinos testados, foram positivos somente para FIV. Em sete gatos (16,28%) foi observada coinfecção pelos dois vírus. Em nenhum gato foi observado soropositividade somente para FeLV. Dos seis gatos com gengivite, nenhum foi positivo para FIV e FeLV; um gato com estomatite foi positivo para FIV e FeLV; dos 22 gatos com periodontite, seis (27,27%) foram FIV positivos e dois (9,09%) FIV/FeLV positivos; e dos 14 com estomatite e periodontite, três (21,43%) foram FIV positivos e quatro (28,57%) FIV/FeLV positivos. Quanto ao diagnóstico, em 28 gatos (65,1%) foi observada somente doença periodontal (DP), em um (2,32%) somente gengivoestomatite crônica felina (GECF) e em 14 gatos (32,5%) DP e GECF. Diante dos resultados obtidos, pode-se concluir que as principais lesões orais encontradas em gatos de abrigos da região central do RS foram gengivite, estomatite e periodontite; a periodontite associada ou não a estomatite foi a lesão oral mais frequente nos gatos positivos para FIV e/ou FeLV. Acredita-se que outros fatores possam contribuir na instalação de doenças orais em gatos de abrigos, já que houve predomínio de gatos com resultados negativos nos testes para os retrovírus.(AU)


Subject(s)
Animals , Cats , Retroviridae/isolation & purification , Stomatitis/veterinary , Leukemia/veterinary , Gingivitis/veterinary , Periodontal Diseases/veterinary , Brazil/epidemiology , Housing, Animal , Immunity
3.
Braz. J. Vet. Res. Anim. Sci. (Online) ; 56(1): e146687, jun. 2019. tab
Article in English | LILACS, VETINDEX | ID: biblio-1007804

ABSTRACT

Feline Immunodeficiency Virus (FIV) and Feline Leukemia Virus (FeLV) are important etiologic agents of immunosuppressive diseases in felines. The objective of the present study was to determine the prevalence of these retroviruses in domestic cats in Fortaleza, Ceará and the epidemiological factors associated with these infections. Between 2015 and 2016, 138 blood samples were collected and tested for FIV and FeLV by the enzyme immunoadsorption assay (ELISA). Parameters such as breed, gender, age, reproductive status, multi-cat environment, outdoor access and clinical manifestations were evaluated. The results showed that 12.32% were positive for FIV, 5.80% for FeLV and 1.45% for co-infection (FIV/FeLV). FIV+ animals were mostly mixed breed, neutered male adult cats, with indoor lifestyle and living in a multi-cat household. The most common clinical manifestation observed was disorders of the oral cavity. Factors found to increase the risk for FeLV seropositivity include mixed breed, young, spayed female cats, indoor lifestyle living in a multi-cat household were the most common epidemiological factors observed. The most common clinical manifestation was anorexia and apathy. The prevalence of these viruses were relatively high, compared with other region of Brazil. This study demonstrated that mixed breed, castrated, multi-cat environment and indoor lifestyle animals are of greater relevance for FIV and FeLV infection diseases. Factors related to cat demographics and health such as age, sex and type of household are important predictors for seropositive status to FeLV or FIV in Fortaleza. High prevalence of FeLV or FIV observed in our study is of concern, in view of the immunosuppressive potential of the two pathogens.(AU)


O Vírus da Imunodeficiência Felina (FIV) e o Vírus da Leucemia Felina (FeLV) são importantes agentes etiológicos de doenças imunossupressoras em felinos. O objetivo do presente estudo foi determinar a prevalência desses retrovírus em gatos domésticos em Fortaleza, Ceará e os fatores epidemiológicos associados a essas infecções. Entre 2015 e 2016, foram coletadas 138 amostras de sangue e testadas para FIV e FeLV pelo ensaio de imunoadsorção enzimática (ELISA). Parâmetros como raça, gênero, idade, estado reprodutivo, ambiente com vários gatos, acesso ao ar livre e manifestações clínicas foram avaliados. Os resultados mostraram que 12,32% foram positivos para o FIV, 5,80% para o FeLV e 1,45% para a co-infecção (FIV/FeLV). Os animais FIV+ eram na sua maioria gatos machos castrados, adultos, de raça mista, com estilo de vida dentro de casa e vivendo em um ambiente com vários gatos. A manifestação clínica mais comum observada foi distúrbios da cavidade oral. Os fatores encontrados que aumentam o risco de soropositividade para FeLV incluem gatas fêmeas castradas, jovens, de raça mista, com estilo de vida dentro de casa e vivendo em um ambiente com vários gatos, foram os fatores epidemiológicos mais comuns observados. A manifestação clínica mais comum foi anorexia e apatia. A prevalência desses vírus foi relativamente alta em comparação com outras regiões do Brasil. Este estudo demonstrou que os animais de raça mista, castrados, vivendo em um ambiente com vários gatos e estilo de vida dentro de casa são de maior relevância para as doenças infecciosas por FIV e FeLV. Fatores relacionados à demografia e à saúde do gato, como idade, sexo e tipo de domicílio, são importantes preditores do estado soropositivo para FeLV ou FIV em Fortaleza. A alta prevalência de FeLV ou FIV observada em nosso estudo é preocupante, tendo em vista o potencial imunossupressor dos dois patógenos.(AU)


Subject(s)
Animals , Cats , Cats/immunology , Seroepidemiologic Studies , Immunodeficiency Virus, Feline/pathogenicity , Retroviridae
4.
Article in English | WPRIM | ID: wpr-758815

ABSTRACT

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.


Subject(s)
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
5.
Acta neurol. colomb ; 33(2): 115-118, abr.-jun. 2017.
Article in Spanish | LILACS | ID: biblio-886432

ABSTRACT

RESUMEN Se reporta un caso de mielopatía por HTLV-1 en paciente afrodescendiente en su sexta década de vida, procedente de Popayán, Cauca. Presentó sintomatología no típica desde años atrás, pero en los últimos meses aparecieron síntomas característicos y por ello se realizó diagnóstico de HTVL-1 por Elisa. La enfermedad progresó sin posibilidad de detención y solo se le pudo ofrecer manejo analgésico, terapia física y medidas de confort. Se discute la importancia de hacer un diagnóstico temprano para HTLV-1 y la necesidad de intervención antes de que la enfermedad sea incapacitante. Encontramos dos estudios, uno en Perú y otro en Brasil donde se descubren dos formas de identificar objetivamente cambios tempranos en pacientes seropositivos para este virus.


SUMMARY A case of HTLV-I myelopathy is reported in an Afro-descendant patient in his sixth decade of life, from Popa-yán Cauca Colombia. It has not been typical symptomatology since years, but in the last months characteristic symptoms appear and it is when diagnosis of HTVL-1 is made by Elisa (9.51 positive). The disease progresses without the possibility of arrest and can only be offered analgesic management, physical therapy and comfort measures. The importance of making an early diagnosis for HTLV-1 and the need for intervention before the disease is disabling is discussed. We found two studies, one in Peru and one in Brazil, where two ways of objectively identifying early changes in seropositive patients.


Subject(s)
Retroviridae , Spinal Cord Diseases , Paraparesis , Muscle Spasticity
6.
Chinese Journal of Biotechnology ; (12): 204-211, 2016.
Article in Chinese | WPRIM | ID: wpr-242300

ABSTRACT

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.


Subject(s)
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
7.
Rev. méd. (La Paz) ; 22(1): 5-12, 2016. ilus
Article in Spanish | LILACS | ID: lil-797309

ABSTRACT

INTRODUCCIÓN: el virus linfotrópico humano tipo 1 (HTLV-1) está relacionado con leucemia y linfoma de células T del adulto y la paraparesia espástica tropical. Su transmisión se realiza por sangre y fluidos orgánicos. OBJETIVOS: determinar la prevalencia de HTLV y otras enfermedades de transmisión sexual en donantes de sangre del Hospital Nacional (Itauguá, Paraguay). METODOLOGÍA: estudio observacional descriptivo retrospectivo realizado en el tamizaje de donantes de sangre durante los años 2013 al 2015. RESULTADOS: entre 16.100 donaciones se encontraron 61 resultados reactivos para HTLV, lo que da una prevalencia de 0,37%. Las características demográficas de los casos positivos para HTLV son: edad media 37 ± 12 años (rango 19-67 años), leve predominio del sexo masculino 35 casos (57%). La asociación con otras enfermedades de transmisión sexual pudo detectarse en 11 de los 61 pacientes positivos para HTLV, de los cuales el 63% era portador de sífilis. CONCLUSIONES: la prevalencia de HTLV en Banco de sangre del Hospital Nacional es 0,37%. Los afectados por HTLV eran también portadores de serología sifilítica en 63%.


INTRODUCTION: the human lymphotropic virus type 1 (HTLV-1) is related to adult T cell leukemia and lymphoma and the tropical spastic paraparesis. The transmission is by blood and body fluids. OBJECTIVES: to determine the prevalence of HTLV and other sexually transmitted diseases in blood donors in the National Hospital (Itauguá, Paraguay). METHODOLOGY: descriptive retrospective study by screening blood donors between 2013 to 2015. RESULTS: between 16,100 donations we found 61 reactive results for HTLV, giving a prevalence of 0.37%. The demographic characteristics of positive cases for HTLV are: mean age 37 ± 12 years (range 19-67 years), slight predominance of males 35 cases (57%). The association with other sexually transmitted diseases could be detected in 11 of the 61 patients positive for HTLV-1, of which 63% were carrying syphilis. CONCLUSIONS: the prevalence of HTLV in the blood bank of the National Hospital is 0.37%. The affected by HTLV-1 carriers also syphilis serology in 63%.


Subject(s)
Humans , Blood Donors , Human T-lymphotropic virus 1 , Retroviridae/pathogenicity
8.
Chinese Journal of Hematology ; (12): 331-336, 2015.
Article in Chinese | WPRIM | ID: wpr-282039

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>Insert 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.</p><p><b>RESULTS</b>(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.</p><p><b>CONCLUSION</b>We 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.</p>


Subject(s)
Antigens, CD19 , Cell Proliferation , Flow Cytometry , Genetic Vectors , Humans , K562 Cells , Recoverin , Retroviridae , T-Lymphocytes , Transfection
9.
Article in Chinese | WPRIM | ID: wpr-259617

ABSTRACT

<p><b>OBJECTIVE</b>To 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.</p><p><b>METHODS</b>The 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.</p><p><b>RESULTS</b>The 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.</p><p><b>CONCLUSION</b>MSC-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.</p>


Subject(s)
Animals , Catalase , Coculture Techniques , Flow Cytometry , Hematopoietic Stem Cells , Humans , Mesenchymal Stem Cells , Mice , Mice, Inbred NOD , Mice, SCID , Retroviridae , Transfection , Umbilical Cord
10.
Arq. Inst. Biol ; 82: 1-4, 2015. tab
Article in Portuguese | LILACS, VETINDEX | ID: biblio-1026492

ABSTRACT

A leucose enzoótica bovina (LEB) é uma enfermidade causada por um retrovírus e pode apresentar um quadro de linfocitose persistente ou linfossarcomas em bovinos adultos. Este estudo teve como objetivo realizar um levantamento sorológico, por meio do teste de imunodifusão em gel agar (IDGA), de bovinos de corte e de leite, bem como de animais puros de origem (POs), criados nos municípios de Sinop e Sorriso, a fim de se conhecer a distribuição e a ocorrência de anticorpos antivírus da LEB na referida região. De acordo com os resultados obtidos, foi verificado que a prevalência acumulada nas três categorias foi de 11,78% de soropositivos, sendo 12,19% para bovinos de corte, 11,20% para o rebanho leiteiro e 9,09% em animais POs. Dessa forma, este estudo demonstrou que a LEB se faz presente no estado de Mato Grosso, sendo possível verificar que a LEB apresenta índices de prevalência abaixo dos encontrados em outras regiões do país.(AU)


The enzootic bovine leukosis (EBL) is a disease caused by a retrovirus and can be presented as a persistent lymphocytosis, or bovine lymphosarcomas in adult cattle. This study aimed to conduct a serological survey of beef cattle, dairy herd and in purebred cattle raised in the cities of Sinop and Sorriso, in order to understand the distribution and occurrence of antibodies anti-EBL virus in that region. According to the results obtained, the prevalence in the three categories was 11.78% of serum positives, where 12.19% for beef cattle, 11.02% for the dairy herd and 9.09% in purebred cattle animals. Thus, this study demonstrated that EBL is present in Mato Grosso State, and EBL's prevalence rates lower than others reported in several regions of the country.(AU)


Subject(s)
Animals , Cattle , Retroviridae , Enzootic Bovine Leukosis/epidemiology , Immunodiffusion/veterinary , Animal Diseases
11.
Article in English | WPRIM | ID: wpr-65508

ABSTRACT

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.


Subject(s)
Cell Line , Collagenases , Distemper Virus, Canine , Embryonic Structures , Epithelial Cells , Family Characteristics , Fibroblasts , Kidney , Madin Darby Canine Kidney Cells , Retroviridae , Telomerase
12.
Rev. bras. hematol. hemoter ; 36(2): 152-158, Mar-Apr/2014. tab
Article in English | LILACS | ID: lil-710194

ABSTRACT

The Retrovirus Epidemiology Donor Study (REDS) program was established in the United States in 1989 with the purpose of increasing blood transfusion safety in the context of the HIV/AIDS and human T-lymphotropic virus epidemics. REDS and its successor, REDS-II were at first conducted in the US, then expanded in 2006 to include international partnerships with Brazil and China. In 2011, a third wave of REDS renamed the Recipient Epidemiology and Donor Evaluation Study-III (REDS-III) was launched. This seven-year research program focuses on both blood banking and transfusion medicine research in the United States of America, Brazil, China, and South Africa. The main goal of the international programs is to reduce and prevent the transmission of HIV/AIDS and other known and emerging infectious agents through transfusion, and to address research questions aimed at understanding global issues related to the availability of safe blood. This article describes the contribution of REDS-II to transfusion safety in Brazil. Articles published from 2010 to 2013 are summarized, including database analyses to characterize blood donors, deferral rates, and prevalence, incidence and residual risk of the main blood-borne infections. Specific studies were developed to understand donor motivation, the impact of the deferral questions, risk factors and molecular surveillance among HIV-positive donors, and the natural history of Chagas disease. The purpose of this review is to disseminate the acquired knowledge and briefly summarize the findings of the REDS-II studies conducted in Brazil as well as to introduce the scope of the REDS-III program that is now in progress and will continue through 2018.


Subject(s)
Humans , Blood Safety , Hematologic Diseases , Retroviridae Infections/epidemiology , Retroviridae , Blood Transfusion/standards
13.
Article in Chinese | WPRIM | ID: wpr-312580

ABSTRACT

<p><b>OBJECTIVE</b>To investigate the cytotoxicity of normal CD8(+) T lymphocytes retrovirally transduced with WT1 peptide-specific T-cell receptor (TCR) genes against human lung cancer cells.</p><p><b>METHODS</b>HLA-A*2402-restricted and WT1 peptide-specific TCR-α/β genes were cloned from a cytotoxic T lymphocyte clone and inserted into a retroviral TCR expression vector. The cytotoxicity of normal peripheral CD8⁺ T cells transduced with the WT1-TCR genes against human lung cancer cells was evaluated using a standard ⁵¹Cr release assay.</p><p><b>RESULTS</b>The WT1-TCR gene-modified T cells recognized the peptide-pulsed target cells but not the non-pulsed cells. TCR-redirected CD8⁺ T cells lysed WT1-overexpressing human lung cancer cells in an HLA-A*2402-restricted manner, but did not kill normal cells positively expressing HLA-A*2402.</p><p><b>CONCLUSION</b>These data demonstrate the feasibility of adoptive immunotherapy with TCR-redirected T cell for the treatment of lung cancer.</p>


Subject(s)
CD8-Positive T-Lymphocytes , Cell Biology , Cell Line, Tumor , Genes, T-Cell Receptor , Humans , Immunotherapy, Adoptive , Lung Neoplasms , Pathology , Peptides , Receptors, Antigen, T-Cell, alpha-beta , Genetics , Retroviridae , T-Lymphocytes, Cytotoxic , Cell Biology , Transduction, Genetic , WT1 Proteins , Genetics
14.
Chinese Journal of Biotechnology ; (12): 1586-1593, 2014.
Article in Chinese | WPRIM | ID: wpr-345565

ABSTRACT

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.


Subject(s)
Animals , Cell Line , Genetic Vectors , Interferon-gamma , Metabolism , Mice , Mice, Knockout , Recombinant Proteins , Retroviridae , T-Box Domain Proteins , T-Lymphocytes , Metabolism , Transfection
15.
Acta Pharmaceutica Sinica ; (12): 30-36, 2014.
Article in Chinese | WPRIM | ID: wpr-297975

ABSTRACT

APOBEC3 is a class of cytidine deaminase, which is considered as a new member of the innate immune system, and APOBEC3G belongs to this family. The research about APOBEC3G is a new direction of innate immune defense mechanism against virus. APOBEC3G has the restrictive activity on many viral replications, which deaminates dC to dU in the viral genome and then induces extensive hypermutation. APOBEC3G can also interrupt viral replication at several phases such as reverse transcription, replication, nucleocapsid and so on by non-deamination mechanisms. However, virus can encode viral proteins to counteract the restriction activity of APOBEC3G. Elucidation of the antagonistic interaction between APOBEC3G and the virus will be contributed to development of new antiviral drugs in the future.


Subject(s)
APOBEC-3G Deaminase , Animals , Cytidine Deaminase , Genetics , Metabolism , DNA Replication , Deamination , HIV-1 , Physiology , Hepacivirus , Genetics , Physiology , Hepatitis B virus , Genetics , Physiology , Humans , Paramyxoviridae , Genetics , Physiology , Retroviridae , Physiology , Virus Replication , vif Gene Products, Human Immunodeficiency Virus , Metabolism
16.
Journal of Experimental Hematology ; (6): 1396-1401, 2014.
Article in Chinese | WPRIM | ID: wpr-340490

ABSTRACT

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.


Subject(s)
Animals , Cell Line , DNA, Complementary , Genetic Vectors , Mesenchymal Stem Cells , Metabolism , Mice , Retroviridae , Reverse Transcriptase Polymerase Chain Reaction , Transfection , Vascular Cell Adhesion Molecule-1 , Genetics
17.
Singapore medical journal ; : e104-6, 2014.
Article in English | WPRIM | ID: wpr-337828

ABSTRACT

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.


Subject(s)
Adult , Diagnosis, Differential , Esophagus , Fistula , Diagnosis , Humans , Lung , Pathology , Male , Radiography, Thoracic , Retroviridae , Metabolism , Tomography, X-Ray Computed , Trachea , Treatment Outcome , Tuberculosis, Lymph Node , Tuberculosis, Pulmonary , Diagnosis
18.
Article in Korean | WPRIM | ID: wpr-162996

ABSTRACT

Cyclic guanosine monophosphate adenosine monophosphate (cGAMP) synthase (cGAS) detects human immunodeficiency virus (HIV) and produces cGAMP to induce cytokines. Reverse transcribed DNA of HIV is critical for triggering innate immune responses as inhibitor of HIV reverse transcriptase blocked the induction of interferon-beta by the virus. Furthermore, knockout of cGAS in human or mouse cell lines abrogated the production of cytokines by HIV infection highlighting the essential role of cGAS in detection of HIV and other retroviruses.


Subject(s)
Adenosine Monophosphate , Animals , Cell Line , Cytokines , DNA , Guanosine Monophosphate , HIV Infections , HIV Reverse Transcriptase , HIV , Humans , Immunity, Innate , Interferon-beta , Mice , Retroviridae
19.
Article in English | WPRIM | ID: wpr-819732

ABSTRACT

OBJECTIVE@#To study the expression of TRPC6 among prostate cancer cells, establish high expression cell lines of TRPC6, and to provide potential cell mode for prostate cancer oncogenesis and development.@*METHODS@#Occurrence and development of prostate cancer cells, PC3, PC-3 m DU145, 22 rv1, LNCaP and normal prostate epithelial cells in the PrEC TRPC6 expression level were detected by QPCR method. Calcium phosphate transfection method was used to package retrovirus pLEGFP-N1-TRPC6 and pLEGFP-N1-vector and infect the prostate cancer cells, a stable high expression of TRPC6 prostate cancer cells. Sable cell lines of TRPC6, matrix metalloproteinase (MMP) 2, MMP9 expression was detected by QPCR and Western blot. Change of cell invasion ability was detected by Transwell.@*RESULTS@#The expression level of prostate cancer cells TRPC6 were higher than control group PrEC cells. Among TPRC6 the expression of cell line PC 3 transfer potential wre the lowest, and high transfer cell line PC-3M express was the highest. Real-time fluorescent quantitative PCR and western blot results showed that after filter, the seventh generation of cell TRPC6 protein and mRNA expression levels were higher than the control group obviously. Transwell experimental results showed that the overexpression of TRPC6 could promote the invasion ability of PC3 prostate cancer cells.@*CONCLUSIONS@#TRPC6 expressed in prostate cancer cells is in disorder, and its action may be associated with the invasion and metastasis of prostate cancer cells; successful establishment of stable high expression of TRPC6 prostate cancer cells primarily confirm the invasion-trigger ability of TRPC6 on prostate cancer, and lay down the Foundation for exploring the TRPC6's role in the occurrence and development of prostate cancer mechanism.


Subject(s)
Cell Line, Tumor , Humans , Male , Matrix Metalloproteinase 2 , Genetics , Metabolism , Matrix Metalloproteinase 9 , Genetics , Metabolism , Neoplasm Invasiveness , Genetics , Prostatic Neoplasms , Genetics , Metabolism , RNA, Messenger , Genetics , Metabolism , Retroviridae , TRPC Cation Channels , Genetics , Metabolism , TRPC6 Cation Channel
20.
Article in English | WPRIM | ID: wpr-820640

ABSTRACT

OBJECTIVE@#To compare the expression levels of pluripotent genes among incomplete reprogrammed colonies and induced pluripotent stem cells (iPSCs), to explore the relationship between the expression of pluripotent genes and incomplete reprogramming.@*METHODS@#Four genes (Oct4, Sox2, Klf4, C-Myc) were introduced into human foreskin fibroblasts (HFFs) by retroviruses. The HFFs were induced to reprogramming. Different forms of colonies were picked up, analyzed, and compared with iPSCs from different aspects, including the morphology of clones, alkaline phosphatase (AP) staining, immuno-fluorescence, and Q-PCR.@*RESULTS@#In the reprogramming process, different colonies were emerged, some of them exhibited typical human embryonic stem cell morphology (eg., compact colonies, high nucleus-to-cytoplasm ratios, and prominent nucleoli). However, these colonies couldn't maintain these characters after passage. There was an intermediate state, named partially reprogramming. Through analysis and identification, AP staining results were weakly positive, compared with iPSC colonies. The immuno-fluorescence staining demonstrated these colonies just expressed pluripotent protein Oct4. Q-PCR indicated that the expression of exogenous transcription factors was inappropriate, either at a high level or at a low level. Most of the endogenous pluripotency genes were expressed at a low level.@*CONCLUSIONS@#It may be one of the causes of incomplete reprogramming that the exogenous pluripotent gene is low-expressed or over-expressed, and successful reprogramming may depend on a specific stoichiometric balance of Oct4, Sox2, Klf4 and c-Myc.


Subject(s)
Animals , Cell Line , Cells, Cultured , Cellular Reprogramming , Genetics , Child , Fibroblasts , Humans , Induced Pluripotent Stem Cells , Cell Biology , Physiology , Male , Mice , Mice, Inbred ICR , Octamer Transcription Factor-3 , Genetics , Metabolism , Retroviridae , Genetics , Stage-Specific Embryonic Antigens , Genetics , Metabolism , Transfection , Methods
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