Comparative metavirome analysis in polytransfused patients.

Due to the high transfusion volume, polytransfused patients with sickle cell disease (SCD) and beta-thalassemia are constantly exposed to parenterally transmitted infections. Currently, we have little information about the virome of such patients and how the virological composition might be influenced by the hemotherapy procedures that these patients receive. The objective of this study was to compare the viral diversity between these two groups with respect to the viral abundance and how it might be affected by the specific conditions of these groups. We sequenced by next-generation sequencing (NGS) and compared the virome of 30 patients with beta-thalassemia major, 45 with SCD, and 16 blood donors from the Blood Center of Ribeirão Preto, Brazil. Predominantly, commensal viruses including Torque teno virus (TTV) genotypes and human pegiviris-1 (HPgV-1) were identified in each group. Strikingly, while HPgV-1 reads were dominant in the SCD group, thalassemic patients showed high TTV abundance, expressed both in viral reads and genotypes. We speculated that the commensal virome of polytransfused patients might be influenced by the transfusion frequency and disease characteristics and that commensal viruses might be used as important genetic biomarkers for these hematological disturbances. Nevertheless, more specific studies are necessary to confirm a relationship between blood virome and transfusion treatment.

Comparative metavirome analysis in polytransfused patients

Due to the high transfusion volume, polytransfused patients with sickle cell disease (SCD) and beta-thalassemia are constantly exposed to parenterally transmitted infections. Currently, we have little information about the virome of such patients and how the virological composition might be influenced by the hemotherapy procedures that these patients receive. The objective of this study was to compare the viral diversity between these two groups with respect to the viral abundance and how it might be affected by the specific conditions of these groups. We sequenced by next-generation sequencing (NGS) and compared the virome of 30 patients with beta-thalassemia major, 45 with SCD, and 16 blood donors from the Blood Center of Ribeirão Preto, Brazil. Predominantly, commensal viruses including Torque teno virus (TTV) genotypes and human pegiviris-1 (HPgV-1) were identified in each group. Strikingly, while HPgV-1 reads were dominant in the SCD group, thalassemic patients showed high TTV abundance, expressed both in viral reads and genotypes. We speculated that the commensal virome of polytransfused patients might be influenced by the transfusion frequency and disease characteristics and that commensal viruses might be used as important genetic biomarkers for these hematological disturbances. Nevertheless, more specific studies are necessary to confirm a relationship between blood virome and transfusion treatment.

Serological evidence of Borrelia circulation among blood donors in the São Paulo state, Brazil.

OBJECTIVE: The objective of this study was to examine the Borrelia seroprevalence among blood donors in Southeast Brazil. BACKGROUND: There is evidence that Borrelia spirochetes are circulating in Brazil; however, there are no studies that characterise these bacteria and investigate their seroprevalence in the Brazilian population. Such a situation, combined with a recent outbreak of tick-borne Rocky Mountain spotted fever in the São Paulo state demonstrates the increasing role of ticks as arthropod vectors in Brazil. METHODS: For the purpose of the study, 452 blood donors from Ribeirão Preto city, São Paulo state were tested using anti-Borrelia immunoglobulin G (IgG) assay. The positive results were also confirmed by Western blot for anti-borrelia IgM/IgG. RESULTS: The anti-Borrelia IgG enzyme-linked immunosorbent assay (ELISA) showed nine positive and nine borderline reactive samples, giving a total seroprevalence of 2·0% of anti-Borrelia IgG among Brazilian blood donors. The confirmation of the seropositive and borderline samples by Borrelia Western blot was demonstrated by IgG-positive results in 16 samples (a seroprevalence of 3.5%). Anti-Borrelia IgM antibodies were also detected in one sample. CONCLUSIONS: Our results demonstrate that Borrelia-like spirochetes may be circulating among blood donors from the São Paulo State and that the ticks have an important epidemiological role as vectors of bacterial infections in this Brazilian region. These results not only alert us to possible actions that might be undertaken in order to completely characterise the aetiological agents of Lyme-like syndromes in Brazil but also the possible impact that these bacterial agents might have on haemotherapy practices.

Human pegivirus-1 (HPgV-1) RNA prevalence and genotypes in volunteer blood donors from the Brazilian Amazon.

OBJECTIVES: The objectives of this study were to evaluate the prevalence of Human Pegivirus-1 (HPgV-1) viremia and genotype diversity among healthy blood donors from the Eastern Brazilian Amazon (city of Macapá, State of Amapá). There is little information for prevalence and circulation of HPgV-1 in this remote Brazilian region. MATERIALS AND METHODS: We conducted a study evaluating the HPgV-1 RNA prevalence and circulating genotypes in 431 volunteer blood donors originating from the Eastern Brazilian Amazon. The obtained HPgV-1 positive samples were submitted to sequencing and genotyping analysis in order to examine the genotype diversity of this virus in the Brazilian Amazon. RESULTS: Our results demonstrated a prevalence of HPgV-1 RNA in 9.5% of the tested blood donors. The phylogenetic analyses of the detected positive samples showed the presence of HPgV-1 genotypes 1, 2 and 3. The most frequently detected genotype was 2 (78.0% of the cases) represented by sub-genotypes 2A (39.0%) and 2B (39.0%). At lower rates, genotypes 1 (14.6%) and 3 (7.4%) were also detected. CONCLUSION: Our results revealed the presence of genotypes with European, Asiatic and African endemicity in Amazonian blood donors, probably due to the complex miscegenation processes that took place in this Brazilian region. More investigations, including information for the prevalence of HPgV-1 RNA in blood donors from other Latin American countries are needed to estimate the viremic rates and genotype distribution of this virus in a highly diverse continent like South America.

Expression differences of genes in the PI3K/AKT, WNT/b-catenin, SHH, NOTCH and MAPK signaling pathways in CD34+ hematopoietic cells obtained from chronic phase patients with chronic myeloid leukemia and from healthy controls.

PURPOSE: The fusion gene BCR-ABL has an important role to the progression of chronic myeloid leukemia (CML) and several signaling pathways have been characterized as responsible for the terminal blastic phase (BP). However, the initial phase, the chronic phase (CP), is long lasting and there is much yet to be understood about the critical role of BCR-ABL in this phase. This study aims to evaluate transcriptional deregulation in CD34+ hematopoietic cells (CD34+ cells) from patients with untreated newly diagnosed CML compared with CD34+HC from healthy controls. METHODS: Gene expression profiling in CML-CD34 cells and CD34 cells from healthy controls were used for this purpose with emphasis on five main pathways important for enhanced proliferation/survival, enhanced self-renewal and block of myeloid differentiation. RESULTS: We found 835 genes with changed expression levels (fold change ≥ ±2) in CML-CD34 cells compared with CD34 cells. These include genes belonging to PI3K/AKT, WNT/b-catenin, SHH, NOTCH and MAPK signaling pathways. Four of these pathways converge to MYC activation. We also identified five transcripts upregulated in CD34-CML patients named OSBPL9, MEK2, p90RSK, TCF4 and FZD7 that can be potential biomarkers in CD34-CML-CP. CONCLUSION: We show several mRNAs up- or downregulated in CD34-CML during the chronic phase.

Mesenchymal stromal cell infusion to treat steroid-refractory acute GvHD III/IV after hematopoietic stem cell transplantation.

Acute GvHD (aGvHD) is a life-threatening complication of hematopoietic stem cell transplantation. Frontline therapy for aGvHD consists of corticosteroid administration. However, ∼25% of the patients have a steroid-refractory disease, a sign of poor prognosis. An alternative therapy for steroid-refractory aGvHD is infusion of mesenchymal stromal cells (MSCs). Herein, we report the results of 46 patients treated with MSC infusion as salvage therapy for steroid-refractory aGvHD III/IV (78% grade IV). Patients received a median cumulative dose of MSCs of 6.81 × 106/kg (range, 0.98-29.78 × 106/kg) in a median of 3 infusions (range, 1-7). Median time between the onset of aGvHD and the first MSC infusion was 25.5 days (range, 6-153). Of the patients, 50% (23/46) presented clinical improvement. Of these, 3 patients (13%) had complete response, 14 (61%) had partial response and 6 (26%) had transient partial response. The estimated probability of survival at 2s year was 17.4%. Only 2 patients (4.3%) presented acute transient side effects (nausea/vomiting and blurred vision) during cell infusion. No patient had late or severe side effects because of MSC infusion. These results suggest that this therapeutic modality is safe and should be considered for steroid-refractory aGvHD, especially in countries where other second-line agents are less available.

Bone marrow-derived cells are recruited by the melanoma tumor with endothelial cells contributing to tumor vasculature.

PURPOSE: Tumor expansion is dependent on neovascularization, a process that requires sustained new vessel formation. Although the critical role of angiogenesis by endothelial sprouting in this process, controversy still prevails on whether angiogenesis involving bone marrow-derived endothelial cells, does contribute to this process. This study aims to evaluate the recruitment of bone marrow-derived cells by the melanoma tumor, including endothelial cells, and if they contribute to angiogenesis. METHODS: A chimeric mouse model of GFP bone marrow was used to induce melanoma tumors derived from murine B16-F10 cell line. These tumors were evaluated for the presence of myeloid cells (CD11b), T lymphocytes (CD3, CD4 and CD8) and endothelial cells (VEGFR2 and CD31) derived from bone marrow. RESULTS: Mice transplanted with GFP+ cells showed significant bone marrow chimerism (90.9 ± 0.87 %) when compared to the GFP transgenic mice (90.66 ± 2.1 %, p = 0.83) demonstrating successful engraftment of donor bone marrow stem/progenitor cells. Analysis of the murine melanoma tumor showed the presence of donor cells in the tumors (3.5 ± 1.7 %) and interestingly, these cells represent endothelial cells (CD31+ cells; 11.5 ± 6.85 %) and myeloid cells (CD11b+ cells; 80 ± 21 %), but also tumor-infiltrating lymphocytes (CD8+ T cells, 13.31 ± 0.2 %; CD4+ T-cells, 2.1 ± 1.2 %). Examination of the tumor endothelium by confocal microscopy suggests the presence of donor CD31+/GFP+ cells in the wall of some blood vessels. CONCLUSION: This study demonstrates that bone marrow-derived cells are recruited by the murine melanoma tumor, with myeloid cells and CD4 and CD8 T lymphocytes migrating as antitumor immune response, and endothelial cells participating of the tumor blood vessels formation.

Defective expression of apoptosis-related molecules in multiple sclerosis patients is normalized early after autologous haematopoietic stem cell transplantation.

Defective apoptosis might be involved in the pathogenesis of multiple sclerosis (MS). We evaluated apoptosis-related molecules in MS patients before and after autologous haematopoietic stem cell transplantation (AHSCT) using BCNU, Etoposide, AraC and Melphalan (BEAM) or cyclophosphamide (CY)-based conditioning regimens. Patients were followed for clinical and immunological parameters for 2 years after AHSCT. At baseline, MS patients had decreased proapoptotic BAD, BAX and FASL and increased A1 gene expression when compared with healthy counterparts. In the BEAM group, BAK, BIK, BIMEL , FAS, FASL, A1, BCL2, BCLXL , CFLIPL and CIAP2 genes were up-regulated after AHSCT. With the exception of BIK, BIMEL and A1, all genes reached levels similar to controls at day + 720 post-transplantation. Furthermore, in these patients, we observed increased CD8+ Fas+ T cell frequencies after AHSCT when compared to baseline. In the CY group, we observed increased BAX, BCLW, CFLIPL and CIAP1 and decreased BIK and BID gene expressions after transplantation. At day + 720 post-AHSCT, the expression of BAX, FAS, FASL, BCL2, BCLXL and CIAP1 was similar to that of controls. Protein analyses showed increased Bcl-2 expression before transplantation. At 1 year post-AHSCT, expression of Bak, Bim, Bcl-2, Bcl-xL and cFlip-L was decreased when compared to baseline values. In summary, our findings suggest that normalization of apoptosis-related molecules is associated with the early therapeutic effects of AHSCT in MS patients. These mechanisms may be involved in the re-establishment of immune tolerance during the first 2 years post-transplantation.

Different expression patterns of LGALS1 and LGALS3 in polycythemia vera, essential thrombocythemia and primary myelofibrosis.

Despite all the knowledge, the cellular and molecular mechanisms involved in myeloproliferative neoplasm (MPN) pathophysiology remain unclear. Authors have shown galectin-1 (Gal-1) and 3 playing roles in tumour angiogenesis and fibrosis, which were correlated with poor prognosis in patients with MPN. In the present study LGALS1 and LGALS3 were differently expressed between polycythemia vera, essential thrombocythemia (ET) and primary myelofibrosis (PMF) diseases. Increased LGALS3 expression was associated with a negative JAK2 V617F status mutation in leucocytes from PMF but not in patients with ET without this mutation. However, a positive Janus kinase 2 (JAK2) V617F cell line established from patients with ET (SET-2 cells) when treated with JAK inhibitor presented high levels of LGALS3. Additionally, high LGALS1 expression was found in CD34(+) cells but not in leucocytes from patients with PMF, in absence of JAK2 V617F mutation, and also in SET-2 cells treated with JAK inhibitor. Thus, our findings indicate that differential expression of LGALS1 and/or LGALS3 in patients with MPN is linked with JAK2 V617F status mutation in these diseases and state of cell differentiation.

Haematological recovery in poor and good haematopoietic stem cell mobilisers.

OBJECTIVES: Evaluate whether poor mobilisers had delayed haematopoietic (neutrophil and platelet) recovery despite receiving similar cell dose as good mobilisers. BACKGROUND: Autologous haematopoietic progenitor cell (HPC) transplantation is indicated to treat some haematological malignancies. This procedure requires HPC mobilisation from bone marrow to peripheral blood. Cell dose is important for a fast haematological recovery. Despite being poor mobilisers, some patients can collect enough cell numbers for transplantation. RESULTS: Fifteen poor mobiliser patients (peak of CD34+ cells ≤10 µL(-1) in peripheral blood) were transplanted at our institution. Haematological recovery (neutrophil ≥ 500 µL(-1) ) in this group was compared to that observed in the group of 16 patients of good mobilisers (peak of CD34+ cells ≥20 µL(-1) in peripheral blood) who received similar cell dose (2·637 ± 0·1744 × 10(6) kg(-1) vs 2·727 ± 0·1746 × 10(6) kg(-1) ; P = 0·7177). The poor mobiliser group had neutrophil and platelet recovery later than the good mobiliser group (on day 12, range 9-14 vs day 10, range 9-22, P = 0·0381 for neutrophil, and on day 22·89 ± 11·16 and 14·08 ± 4·821, P = 0·0193 for platelet). Mortality rates and transfusion requirements were not different between the groups. CONCLUSION: Poor mobilisers have delayed neutrophil and platelet recovery after autologous HPC transplantation despite having received the same cell dose as good mobilisers.

Overview of Zika virus (ZIKV) infection in regards to the Brazilian epidemic.

Zika virus (ZIKV), a mosquito-borne flavivirus, belongs to the Flaviviridae family, genus Flavivirus. ZIKV was initially isolated in 1947 from a sentinel monkey in the Zika forest, Uganda. Little clinical importance was attributed to ZIKV, once only few symptomatic cases were reported in some African and Southeast Asiatic countries. This situation changed in 2007, when a large outbreak was registered on the Yap Island, Micronesia, caused by the Asian ZIKV lineage. Between 2013 and 2014, ZIKV spread explosively and caused many outbreaks in different islands of the Southern Pacific Ocean and in 2015 autochthonous transmission was reported in Brazil. Currently, Brazil is the country with the highest number of ZIKV-positive cases in Latin America. Moreover, for the first time after the discovery of ZIKV, the Brazilian scientists are studying the possibility for the virus to cause severe congenital infection related to microcephaly and serious birth defects due to the time-spatial coincidence of the alarming increase of newborns with microcephaly and the Brazilian ZIKV epidemic. The present review summarizes recent information for ZIKV epidemiology, clinical picture, transmission, diagnosis and the consequences of this emerging virus in Brazil.

Overview of Zika virus (ZIKV) infection in regards to the Brazilian epidemic

Zika virus (ZIKV), a mosquito-borne flavivirus, belongs to the Flaviviridae family, genus Flavivirus. ZIKV was initially isolated in 1947 from a sentinel monkey in the Zika forest, Uganda. Little clinical importance was attributed to ZIKV, once only few symptomatic cases were reported in some African and Southeast Asiatic countries. This situation changed in 2007, when a large outbreak was registered on the Yap Island, Micronesia, caused by the Asian ZIKV lineage. Between 2013 and 2014, ZIKV spread explosively and caused many outbreaks in different islands of the Southern Pacific Ocean and in 2015 autochthonous transmission was reported in Brazil. Currently, Brazil is the country with the highest number of ZIKV-positive cases in Latin America. Moreover, for the first time after the discovery of ZIKV, the Brazilian scientists are studying the possibility for the virus to cause severe congenital infection related to microcephaly and serious birth defects due to the time-spatial coincidence of the alarming increase of newborns with microcephaly and the Brazilian ZIKV epidemic. The present review summarizes recent information for ZIKV epidemiology, clinical picture, transmission, diagnosis and the consequences of this emerging virus in Brazil.

Late emergence of A594V and L595W mutations related to ganciclovir resistance in a patient with HCMV retinitis and long-term HIV progression.

The emergence of ganciclovir (GCV) resistance during the treatment of human cytomegalovirus (HCMV) infection is a serious clinical challenge, and is associated with high morbidity and mortality. In this case report, we describe the emergence of two consecutive mutations (A594V and L595W) related to GCV resistance in a patient with HCMV retinitis and long-term HIV progression after approximately 240 days of GCV use. Following the diagnosis of retinitis, the introduction of GCV did not result in viral load reduction. The detected mutations appeared late in the treatment, and we propose that other factors (high initial HCMV load, previous GCV exposure, low CD4+ cell count), in addition to the presence of resistance mutations, may have contributed to the treatment failure of HCMV infection in this patient.

Autologous hematopoietic SCT normalizes miR-16, -155 and -142-3p expression in multiple sclerosis patients.

Autologous hematopoietic SCT (AHSCT) has been investigated in the past as a therapeutic alternative for multiple sclerosis (MS). Despite advances in clinical management, knowledge about mechanisms involved with clinical remission post transplantation is still limited. Abnormal microRNA and gene expression patterns were described in MS and have been suggested as disease biomarkers and potential therapeutic targets. Here we assessed T- and B-cell reconstitution, microRNAs and immunoregulatory gene expression after AHSCT. Early immune reconstitution was mainly driven by peripheral homeostatic proliferation. AHSCT increased CD4(+)CD25(hi)FoxP3(+) regulatory T-cell counts and expression of CTLA-4 and GITR (glucocorticoid-induced TNFR) on CD4(+)CD25(hi) T cells. We found transient increase in exhausted PD-1(+) T cells and of suppressive CD8(+)CD28(-)CD57(+) T cells. At baseline, CD4(+) and CD8(+) T cells from MS patients presented upregulated miR-16, miR-155 and miR-142-3p and downregulated FOXP3, FOXO1, PDCD1 and IRF2BP2. After transplantation, the expression of FOXP3, FOXO1, PDCD1 and IRF2BP2 increased, reaching control levels at 2 years. Expression of miR-16, miR-155 and miR-142-3p decreased towards normal levels at 6 months post therapy, remaining downregulated until the end of follow-up. These data strongly suggest that AHSCT normalizes microRNA and gene expression, thereby improving the immunoregulatory network. These mechanisms may be important for disease control in the early periods after AHSCT.

ApoptomiRs expression modulated by BCR-ABL is linked to CML progression and imatinib resistance.

BACKGROUND: Chronic myeloid leukemia (CML) is a myeloproliferative disease characterized by the presence of Philadelphia chromosome (Ph) leading to expression of a BCR-ABL1 fusion oncogene. The BCR-ABL protein has a constitutive tyrosine kinase activity which is responsible for CML pathogenesis by promoting cell apoptosis resistance; however, the cellular and molecular mechanisms associated with BCR-ABL expression and apoptosis impairment in CML leukemic cells have not been fully elucidated. METHODS: This study evaluated apoptomiRs and their predicted apoptotic genes in BCR-ABL(+) cells from patients in different phases of CML treated with tyrosine kinase inhibitor (TKI) according to their imatinib (IM) response by qPCR. Phosphotyrosine and c-ABL expressions in HL-60.BCR-ABL cells treated with TKI were done by Western blot. RESULTS: We found that dasatinib (DAS) modulated miR-let-7d, miR-let-7e, miR-15a, miR-16, miR-21, miR-130a and miR-142-3p expressions while IM modulated miR-15a and miR-130a levels. miR-16, miR-130a and miR-145 expressions were modulated by nilotinib (NIL). We observed higher miR-15a, miR-130b and miR-145; and lower miR-16, miR-26a and miR-146a expressions in CML-CP in comparison with controls. CML-AP patients showed low miR-let-7d, miR-15a, miR-16, miR-29c, miR-142-3p, miR-145, and miR-146a levels in comparison with CML-CP. We noted that the miR-26a, miR-29c, miR-130b and miR-146a expressions were downregulated in IM resistant patients in comparison with IM responsive patients. CONCLUSIONS: This study showed the modulation of apoptomiRs by BCR-ABL kinase activity and the deregulation of apoptomiRs and their predicted apoptotic target genes in different CML phases and after treatment with TK inhibitors. ApoptomiRs may be involved in the BCR-ABL(+) cell apoptosis regulation.

Novel polymorphisms in the promoter region of the perforin gene among distinct Brazilian populations and their functional impact.

Cytotoxic T lymphocytes and natural killer cells play a crucial role in eliminating tumour and virus-infected cells. The perforin is a key part of the arsenal that these cells use to destroy their targets. In this study, we characterized single-nucleotide polymorphisms (SNPs) located in the promoter region of the perforin gene among distinct Brazilian ethnic groups. The study was carried out by sequencing this region in three groups: European, African and Asian descents. We demonstrated for the first time the occurrence of three new polymorphisms in the promoter region of gene PRF1: 494A/G (rs78058707), 720G/A (rs75925789) and 1176C/T (rs75183511). Three other SNPs already described in the literature 63A/G (rs35401316), 112A/G (rs10999428) and 1012C/T (rs35069510) were also detected. The SNPs are distributed differently in the ethnic groups studied. The 112G allele was observed at high frequency, especially among Asian descents (48.1%). The 1012T allele was detected only among European descents, the 494G allele only among Asian descents and 1176T allele only in African descents. Based on the association between the polymorphisms described, ten new haplotypes were originated. In functional analysis, we noticed that SNPs present in most common haplotypes cannot induce significant differences in expression levels of perforin alone. In conclusion, this study demonstrates for the first time the existence of three new polymorphisms in perforin promoter and, contrary to what was stated, the presence of these SNPs does not alter the levels of protein expression.