Evandra Strazza Rodrigues

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.

Knops blood group haplotypes among distinct Brazilian populations

BACKGROUND: The Knops blood group system consists of antigens encoded by exon 29 of complement receptor 1 (CR1) gene. To better elucidate the complexity of Knops group system, the frequency of six single‐nucleotide polymorphisms (SNPs) in three Brazilian populations is determined.

Distribution of human immunodeficiency virus type 1 subtypes in the State of Amazonas, Brazil, and subtype C identification

Few studies have reported the molecular epidemiological characterization of HIV-1 in the Northern region of Brazil. The present study reports the molecular and epidemiological characterization of 31 HIV-1 isolates from blood donors from the State of Amazonas who donated blood between April 2006 and March 2007. Serum/plasma samples from all donors were screened for HIV antibodies by ELISA and the results confirmed by Western blot analysis. Genomic DNA was extracted from the buffy coat using the Super Quik-Gene-DNA Isolation kit. Nested PCR was performed on the env, gag, and pol regions of HIV-1 using the Gene Amp PCR System 9700. Sequencing reactions were performed using the inner PCR primers and the DYEnamic™ ET Dye Terminator Kit, and phylogenetic analysis was performed using the gag, pol, and env gene sequences. We collected samples from 31 blood donors who tested positive for HIV-1 in confirmatory experiments. The male:female ratio of blood donors was 3.4:1, and the mean age was 32.4 years (range: 19 to 61 years). Phylogenetic analysis showed that subtype B is the most prevalent among Northern Brazilian HIV-1-seropositive blood donors. One HIV-1 subtype C and one circulating recombinant form (CRF_BF) of HIV-1 were identified in the State of Amazonas. This is the first study showing the occurrence of a possible “homogenous” subtype C in this region of Brazil. This finding could contribute to a better characterization of the HIV-1 strains that circulate in the country. Key words: HIV-1; Subtypes; Phylogenetic analysis; Blood donors; Molecular and epidemiological characterization

Immunological correlates of favorable long-term clinical outcome in multiple sclerosis patients after autologous hematopoietic stem cell transplantation

High dose immunosuppression followed by autologous hematopoietic stem cell transplantation (AHSCT) induces prolonged clinical remission in multiple sclerosis (MS) patients. However, how patient immune profiles are associated with clinical outcomes has not yet been completely elucidated. In this study, 37 MS patients were assessed for neurological outcomes, thymic function and long-term immune reconstitution after AHSCT. Patients were followed for a mean (SD) of 68.5 (13.9) months post-transplantation and were retrospectively clustered into progression- and non-progression groups, based on Expanded Disease Status Scale (EDSS) outcomes at last visit. After AHSCT, both patient groups presented increased regulatory T-cell subset counts, early expansion of central- and effector-memory CD8(+)T-cells and late thymic reactivation. However, the non-progression group presented early expansion of PD-1(+)CD8(+)T-cells and of PD-1-expressing CD19(+) B-cells. Here, we suggest that along with increased numbers of regulatory T-cell subsets, PD-1 inhibitory signaling is one possible immunoregulatory mechanism by which AHSCT restores immune tolerance in MS patients.

Zika virus RNA detection in asymptomatic blood donors during an outbreak in the northeast region of São Paulo State, Brazil, 2016: ZIKA VIRUS IN BRAZILIAN BLOOD DONORS

In 2015, there was a large Zika virus (ZIKV) outbreak in Brazil. The proportion of asymptomatic infections is very high, and it is possible for transfusion‐transmitted ZIKV (TT‐ZIKV) infection to occur. The prevalence of asymptomatic ZIKV infection among Brazilian blood donors during this epidemic outbreak is unknown.

HTLV-1 infects human mesenchymal stromal cell in vitro and modifies their phenotypic characteristics☆

The typical characteristics of mesenchymal stem cells (MSCs) can be affected by inflammatory microenvironment; however, the exact contribution of HTLV-1 to MSC dysfunction remains to be elucidated. In this study, we demonstrated that MSC cell surface molecules VCAM-1 and ICAM-1 are upregulated by contact with HTLV-1, and HLA-DR was most highly expressed in MSCs co-cultured with MT2 cells. The expression levels of VCAM-1 and HLA-DR were increased in MSCs cultured in the presence of PBMCs isolated from HTLV-1-infected symptomatic individuals compared with those cultured with cells from asymptomatic infected individuals or healthy subjects. HTLV-1 does not impair the MSC differentiation process into osteocytes and adipocytes. In addition, MSCs were efficiently infected with HTLV-1 in vitro through direct contact with HTLV-1-infected cells; however, cell-free virus particles were not capable of causing infection. In summary, HTLV-1 can alter MSC function, and this mechanism may contribute to the pathogenesis of this viral infection.

Silencing of HTLV-1 gag and env genes by small interfering RNAs in HEK 293 cells.

Abstract Since the discovery of RNAi technology, several functional genomic and disease therapy studies have been conducted using this technique in the field of oncology and virology. RNAi-based antiviral therapies are being studied for the treatment of retroviruses such as HIV-1. These studies include the silencing of regulatory, infectivity and structural genes. The HTLV-1 structural genes are responsible for the synthesis of proteins involved in the entry, assembly and release of particles during viral infection. To examine the possibility of silencing HTLV-1 genes gag and env by RNA interference technology, these genes were cloned into reporter plasmids. These vectors expressed the target mRNAs fused to EGFP reporter genes. Three small interference RNAs (siRNAs) corresponding to gag and three corresponding to env were designed to analyze the effect of silencing by RNAi technology. The plasmids and siRNAs were co-transfected into HEK 293 cells. The results demonstrated that the expression of the HTLV-1 gag and env genes decreased significantly in vitro. Thus, siRNAs can be used to inhibit HTLV-1 structural genes in transformed cells, which could provide a tool for clarifying the roles of HTLV-1 structural genes, as well as a therapy for this infection.

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

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 (CD3ɛ), lymphocyte-specific protein tyrosine kinase (LCK), vav 1 guanine nucleotide exchange factor (VAV1), and zeta-chain (TCR) associated protein kinase 70kDa (ZAP70) on T lymphocytes of HTLV-1-infected individuals and compared to healthy uninfected individuals (CT). We observed that CD3ɛ, 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 CD3ɛ, 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 CD3ɛ, 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.

Overexpression of hsa-miR-125b during osteoblastic differentiation does not influence levels of Runx2, osteopontin, and ALPL gene expression

Multipotent mesenchymal stromal cells (MSCs) were first isolated from bone marrow and then from various adult tissues including placenta, cord blood, deciduous teeth, and amniotic fluid. MSCs are defined or characterized by their ability to adhere to plastic, to express specific surface antigens, and to differentiate into osteogenic, chondrogenic, adipogenic, and myogenic lineages. Although the molecular mechanisms that control MSC proliferation and differentiation are not well understood, the involvement of microRNAs has been reported. In the present study, we investigated the role of miR-125b during osteoblastic differentiation in humans. We found that miR-125b increased during osteoblastic differentiation, as well as Runx2 and ALPL genes. To study whether the gain or loss of miR-125b function influenced osteoblastic differentiation, we transfected MSCs with pre-miR-125b or anti-miR-125b and cultured the transfected cells in an osteoblastic differentiation medium. After transfection, no change was observed in osteoblastic differentiation, and Runx2, OPN, and ALPL gene expression were not changed. These results suggest that the gain or loss of miR-125b function does not influence levels of Runx2, OPN, and ALPL during osteoblastic differentiation.

Genes related to antiviral activity are differentially expressed in CD4+ T cell in HAM/TSP patients

CD4+ T cells play a central role in HTLV-1 infection. We investigated the global gene expression profile of circulating CD4+ T cells in HTLV-1-infected individuals. The microarray platform was performed using 12 individual RNA samples: healthy control (CT, n=4), asymptomatic HTLV-1 carrier (HAC, n=4) and HAM/TSP group (n=4). Proviral load (PVL), Tax expression and the percentage of CD4+Foxp3+ cells were analyzed. Hierarchical clustering analysis showed that CT and HTLV-infected groups clustered separately. We identified 25 differentially expressed genes in common between CT vs. HAM/TSP and HAM/TSP vs. HAC analyses and we observed their participation in granzyme A (GZMA) signaling pathways. GZMA, GZMB and PRF1 were validated by qRT-PCR. GZMA and PRF1 gene expression was significantly increased in HAM/TSP group compared to CT and HAC groups. No difference was observed in gene expression level of GZMB. Regulatory T cells (Treg) cells have cytolytic capacity and perforin/granzyme pathways are required for this activity. Foxp3 gene expression was evaluated and it was significantly increased in HAM/TSP group compared to CT and HAC groups. GZMA, GZMB, and PRF1 genes were positively correlated to Foxp3 gene expression. PRF1 and Foxp3 genes were positively correlated with Tax expression and PVL. The percentage of CD4+Foxp3+ cells revealed a significant increase in HAC and HAM/TSP groups compared to CT. Our results suggest that Treg cells may use the perforin/granzyme pathway as a system to suppress the immune cells and may contribute to immunodeficiency, which is observed in HTLV-1 infection.