HTLV-1 affects the adipogenic differentiation of human mesenchymal stem cells

Financial support: CTC, INCTC, FAPESP, FUNDHERP and CNPq

Viral metagenomics in blood donors with post-donation diseases and negative tests for dengue and Zika viruses RNA detection during a major outbreak of arboviruses in Sao Paulo State in 2016

Not Applicabl

CD3e expression in HTLV-1-infected individuals is associated with proviral load and Tax expression

Financial support: FUNDHERP, CTC, INCTC, FAPESP, CNPq and CAPES

Distribution of QPY and RAH haplotypes of granzyme B gene in distinct Brazilian populations

Introduction: The cytolysis mediated by granules is one of the most important effector functions of cytotoxic T lymphocytes and natural killer cells. Recently, three single nucleotide polymorphisms (SNPs) were identified at exons 2, 3, and 5 of the granzyme B gene, resulting in a haplotype in which three amino acids of mature protein Q48P88Y245 are changed to R48A88H245, which leads to loss of cytotoxic activity of the protein. In this study, we evaluated the frequency of these polymorphisms in Brazilian populations. Methods: We evaluated the frequency of these polymorphisms in Brazilian ethnic groups (white, Afro-Brazilian, and Asian) by sequencing these regions. Results: The allelic and genotypic frequencies of SNP 2364A/G at exon 2 in Afro-Brazilian individuals (42.3% and 17.3%) were significantly higher when compared with those in whites and Asians (p < 0.0001 and p = 0.0007, respectively). The polymorphisms 2933C/G and 4243C/T also were more frequent in Afro-Brazilians but without any significant difference regarding the other groups. The Afro-Brazilian group presented greater diversity of haplotypes, and the RAH haplotype seemed to be more frequent in this group (25%), followed by the whites (20.7%) and by the Asians (11.9%), similar to the frequency presented in the literature. Conclusions: There is a higher frequency of polymorphisms in Afro-Brazilians, and the RAH haplotype was more frequent in these individuals. We believe that further studies should aim to investigate the correlation of this haplotype with diseases related to immunity mediated by cytotoxic lymphocytes, and if this correlation is confirmed, novel treatment strategies might be elaborated.Center for CellTherapy (CTC)Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Fundacao de Amparo a Pesquisa do Estado de Minas Gerais (FAPEMIG)Fundacao Hemocentro de Ribeirao Preto (FUNDHERP

DC-SIGN (CD209) gene promoter polymorphisms in a Brazilian population and their association with human T-cell lymphotropic virus type 1 infection

This study evaluated four polymorphisms located in the DC-SIGN (CD209) gene promoter region (positions −336, −332 −201 and −139) in DNA samples from four Brazilian ethnic groups (Caucasians, Afro-Brazilian, Asians and Amerindians) to establish the population distribution of these single-nucleotide polymorphisms (SNPs) and correlated DC-SIGN polymorphisms and infection in samples from human T-cell lymphotropic virus type 1 (HTLV-1)-infected individuals. To identify CD209 SNPs, 452 bp of the CD209 promoter region were sequenced and the genotype and allelic frequencies were evaluated. This is the first study to show genetic polymorphism in the CD209 gene in distinct Brazilian ethnic groups with the distribution of allelic and genotypic frequency. The results showed that −336A and −139A SNPs were quite common in Asians and that the −201T allele was not observed in Caucasians, Asians or Amerindians. No significant differences were observed between individuals with HTLV-1 disease and asymptomatic patients. However, the −336A variant was more frequent in HTLV-1-infected patients [HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP), 80 %; healthy asymptomatic HTLV-1 carriers, 90 %] than in the control group (70 %) [P=0.0197, odds ratio (OR)=2.511, 95 % confidence interval (CI)=1.218–5.179). In addition, the −139A allele was found to be associated with protection against HTLV-1 infection (P=0.0037, OR=0.3758, 95 % CI=0.1954–0.7229) when the HTLV-1-infected patients as a whole were compared with the healthy-control group. These observations suggest that the −139A allele may be associated with HTLV-1 infection, although no significant association was observed among asymptomatic and HAM/TSP patients. In conclusion, the variation observed in SNPs −336 and −139 indicates that this lectin may be of crucial importance in the susceptibility/transmission of HTLV-1 infections

Evaluation of the immunomodulatory properties of human mesenchymal stromal cells on HTLV-1 infected T lymphocytes

As características das células estromais mesenquimais multipotentes (MSC) podem ser influenciadas em microambientes inflamatórios. No entanto, o comportamento das MSC frente às infecções virais e a exata contribuição da infecção para disfunção das MSC continuam a ser elucidadas. Neste trabalho, avaliamos o efeito imunossupressor de MSC em linfócitos T infectados pelo HTLV-1 e a susceptibilidade da MSC à infecção por este retrovírus. Os ensaios de co-cultivo utilizando MSC e linhagens de linfócitos infectados pelo HTLV-1 resultaram em diminuição na expressão do gene viral tax e do antígeno p19 do HTLV-1. A redução na expressão do gene tax e da proteína p19 foi relacionada com a maior secreção de IL-6 e aumento na expressão dos genes PGE2, IDO e VCAM-1. Para confirmar a influência da imunorregulação das MSC sobre linfócitos T infectados, comparamos a proliferação de linfócitos T isolados de indivíduos infectados pelo HTLV-1 e indivíduos controles cultivados na presença de MSC. Foi observado que as MSC inibem a linfoproliferação de forma similar em amostras controle e na infecção pelo HTLV-1; e este efeito foi mediado pela expressão de PGE2 e IDO. Além disso, a expressão do gene pol e da proteína p19 do HTLV-1 foi menor após o co-cultivo com MSC, indicando que a imunorregulação pelas MSC também atua nas células infectadas pelo HTLV-1. Em seguida, para investigar as alterações provocadas pelo HTLV-1 nas MSC, realizamos análises morfológicas e ultraestruturais em MSC expostas ao HTLV-1 in vitro. Os resultados revelaram que o HTLV-1 induziu o aparecimento de vesículas intracelulares e a expressão das moléculas de superfície VCAM-1, ICAM-1 e HLA-DR. Os níveis de VCAM-1 e HLA-DR também foram mais expressos em MSC cultivadas na presença de PBMC isoladas de indivíduos HAM/TSP. O HTLV-1 não alterou o processo de diferenciação das MSC em osteócitos e adipócitos. No entanto, o contato direto in vitro das MSC com células infectadas pelo HTLV-1 proporcionou uma eficiente infecção das MSC. As partículas virais isentas de células não foram capazes de causar a infecção em MSC. Por fim, para certificar a existência biológica de MSC infectadas pelo HTLV-1, avaliamos a medula óssea de seis indivíduos acometidos por esta infecção. Foi observado um infiltrado de linfócitos T CD4+ na medula óssea de indivíduos HTLV-1+ e a análise do DNA proviral revelou a presença do provírus integrado nessas células T CD4+. O número de unidades formadoras de colônia fibroblastóide (CFU-F) foi menor em indivíduos infectados pelo HTLV-1 quando comparado com o grupo controle. A expressão dos marcadores de superfície e o potencial de diferenciação in vitro em adipócitos e osteócitos foram similares nas MSC obtidas de indivíduos HTLV-1 e indivíduos controle. Foi demonstrada a presença do DNA proviral e da proteína p19 do HTLV-1 nas MSC isoladas de pacientes HTLV-1+. A comparação do perfil de expressão gênica global entre MSC isoladas de HAM/TSP e indivíduos assintomáticos para o HTLV-1 revelou que os genes da catepsina B e da proteína ribossomal L10 foram diferencialmente expressos. Em conclusão, este trabalho demonstra a importância das MSC na imunomodulação de linfócitos infectados pelo HTLV-1 e que a infecção pelo HTLV-1 altera características biológicas das MSC.The characteristics of human multipotent mesenchymal stromal cells (MSC) can be influenced by the inflammatory microenvironment. However, the activity of the MSC against viral infections and the exact contribution of the infection to MSC dysfunction remain to be elucidated. We evaluated the immunosuppressive effect of MSC on HTLV-1 infected T lymphocytes and the susceptibility of MSC for this retroviral infection. Assays using co-culture of MSC and HTLV-1+ T lymphocyte lineages resulted in a decrease of tax gene expression and HTLV-1 p19 antigen. The reduction of the tax gene expression and the HTLV-1 p19 were associated with increased IL-6 secretion and higher PGE2, IDO and VCAM-1 gene expression. To confirm if MSC immunoregulation can influence the proliferation of HTLV-1 infected T lymphocytes, we compared the proliferation of HTLV-1+ individuals and healthy individuals cultured in the presence of MSC. It was observed that the lymphoproliferative inhibition by MSC in infected lymphocytes was similar to the control cells, and this effect was mediated by the expression of IDO and PGE2 genes. Furthermore, the pol gene and the HTLV-1 p19 protein were less expressed after co-culture assay with MSC, suggesting that the immunoregulation by MSC is effective in HTLV-1 infected T cells. In order to investigate the changes caused by HTLV-1 in MSC, we performed morphological and ultrastructural analysis of MSC exposed to HTLV-1 in vitro. The contact with HTLV-1 induced an increase of the intracellular vesicles, in addition the MSC cell surface molecules VCAM-1, ICAM-1 and HLA-DR were upregulated. The expression levels of VCAM-1 and HLA-DR molecules were increased in MSC cultured in the presence of PBMC isolated from HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP) individuals. The MSC differentiation process into osteocytes and adipocytes was not impaired by HTLV-1. In addition, MSCs were efficiently infected by HTLV-1 in vitro due to the direct contact with the HTLV-1-infected cells. However, cell-free virus particles were not capable of causing productive infection. Finally, to ensure the biological function of MSC in HTLV-1 infected patients, we investigated bone marrow (BM) cells from HTLV-1 asymptomatic carriers (HAC) and HAM/TSP individuals. Initially, we observed an infiltration of CD4+ T-cell lymphocytes in BM from HTLV-1 infected individuals and the detection of provirus revealed HTLV-1 integration. The number of colonies of fibroblast progenitor cells (CFU-F) was lower in HTLV-1 infected individuals compared to control. HTLV-1 MSC isolated showed surface molecules expression and differentiation into adipogenic and osteogenic cells similar to control MSC. Proviral DNA and HTLV-1 p19 protein were detected in MSC from HTLV-1 patients. The comparison of global gene expression profiles between MSC isolated from HAM/TSP and HAC individuals revealed that cathepsin B and ribosomal protein L10 were differentially expressed. In conclusion, this study suggests the importance of MSC immunomodulation on HTLV-1 infected T lymphocytes and describe that HTLV-1 infects and alters the biological characteristics of MSC

Inhibition of Expression of HTLV-1 Structural Genes Mediated by Short Hairpin RNA In Vitro

Background: Human T-lymphotropic virus 1 (HTLV-1) is associated with the T-cell malignancy known as adult T-cell leukemia! lymphoma (ATLL) and with a disorder called HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Currently, the treatment of these diseases is based on symptom relief. RNA interference (RNAi) technology has been described as an efficient mechanism for development of new therapeutic methods. Thus, the aim of this study was to evaluate the inhibition of HTLV-1 structural proteins using short hairpin RNAs (shRNAs) expressed by non-viral vectors. Materials and Methods: Reporter plasmids that express enhanced green fluorescent protein-Gag (EGFP-Gag) and EGFP-Env fusion proteins and vectors that express shRNAs corresponding to the HTLV-1 gag and env genes were constructed. shRNA vectors and reporter plasmids were simultaneously transfected into HEK 293 cells. Results: Fluorescence microscopy, flow cytometry and real-time PCR showed that shRNAs were effective in inhibiting the fusion proteins. Conclusion: These shRNAs are effective against the expression of structural genes and may provide an approach to the development of new therapeutic agents.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Centro de Terapia Celular/Fundacao Hemocentro de Ribeirao Preto (CTC/FUNDHERP)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq

T cell receptor signaling pathway is overexpressed in CD4 + T cells from HAM/TSP individuals

ABSTRACT Human T-lymphotropic virus type 1 (HTLV-1) is a human retrovirus related to the chronic neuroinflammatory disease HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). CD4+ T cells activation appears to play a key role on HTLV-1 infection. Here we investigated the expression of genes associated to T cell activation CD3e molecule, epsilon (CD3?), lymphocyte-specific protein tyrosine kinase (LCK), vav 1 guanine nucleotide exchange factor (VAV1), and zeta-chain (TCR) associated protein kinase 70 kDa (ZAP70) on T lymphocytes of HTLV-1-infected individuals and compared to healthy uninfected individuals (CT). We observed that CD3?, LCK, ZAP70, and VAV1 gene expression were increased in CD4+ T cells from HAM/TSP group compared to HTLV-1 asymptomatic patients (HAC). Moreover, ZAP70 and VAV1 were also upregulated in HAM/TSP compared to CT group. We detected a positive correlation among all these genes. We also observed that CD3?, LCK, and VAV1 genes had a positive correlation with the proviral load (PVL) and Tax expression. These results suggest that PVL and Tax protein could drive CD3?, LCK, and VAV1 gene expression in CD4+ T cells, and these genes function on a synchronized way on the CD4+ T cell activation. The elucidation of the mechanisms underlying T cell receptor signaling pathway is of considerable interest and might lead to new insights into the mechanism of HAM/TSP