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.

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.

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.

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.

Forced expression of OCT4 influences the expression of pluripotent genes in human mesenchymal stem cells and fibroblasts.

Genetic reprogramming of adult cells to generate induced pluripotent stem (iPS) cells is a new and important step in sidestepping some of the ethical issues and risks involved in the use of embryonic stem cells. iPS cells can be generated by introduction of transcription factors, such as OCT4, SOX2, KLF4, and CMYC. iPS cells resemble embryonic stem cells in their properties and differentiation potential. The mechanisms that lead to induced pluripotency and the effect of each transcription factor are not completely understood. We performed a critical evaluation of the effect of overexpressing OCT4 in mesenchymal stem cells and fibroblasts and found that OCT4 can activate the expression of other stemness genes, such as SOX2, NANOG, CMYC, FOXD3, KLF4, and ßCATENIN, which are not normally or are very weakly expressed in mesenchymal stem cells. Transient expression of OCT4 was also performed to evaluate whether these genes are affected by its overexpression in the first 48 h. Transfected fibroblast cells expressed around 275-fold more OCT4 than non-transfected cells. In transient expression, in which cells were analyzed after 48 h, we detected only the up-regulation of FOXD3, SOX2, and KLF4 genes, suggesting that these genes are the earlier targets of OCT4 in this cellular type. We conclude that forced expression of OCT4 can alter cell status and activate the pluripotent network. Knowledge gained through study of these systems may help us to understand the kinetics and mechanism of cell reprogramming.

Breeding of transgenic cattle for human coagulation factor IX by a combination of lentiviral system and cloning.

Recombinant coagulation factor IX must be produced in mammalian cells because FIX synthesis involves translational modifications. Human cell culture-based expression of human coagulation factor IX (hFIX) is expensive, and large-scale production capacity is limited. Transgenic animals may greatly increase the yield of therapeutic proteins and reduce costs. In this study, we used a lentiviral system to obtain transgenic cells and somatic cell nuclear transfer (SCNT) to produce transgenic animals. Lentiviral vectors carrying hFIX driven by 3 bovine ß-casein promoters were constructed. Bovine epithelial mammary cells were transduced by lentivirus, selected with blasticidin, plated on extracellular matrix, and induced by lactogenic hormones; promoter activity was evaluated by quantitative PCR. Transcriptional activity of the 5.335-kb promoter was 6-fold higher than the 3.392- and 4.279-kb promoters, which did not significantly differ. Transgenic bovine fibroblasts were transduced with lentivirus carrying the 5.335-kb promoter and used as donor cells for SCNT. Cloned transgenic embryo production yielded development rates of 28.4%, similar to previous reports on cloned non-transgenic embryos. The embryos were transferred to recipient cows (N = 21) and 2 births of cloned transgenic cattle were obtained. These results suggest combination of the lentiviral system and cloning may be a good strategy for production of transgenic cattle.

Up-regulation of fas and fasL pro-apoptotic genes expression in type 1 diabetes patients after autologous haematopoietic stem cell transplantation.

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by T cell-mediated destruction of pancreatic ß cells, resulting in insulin deficiency and hyperglycaemia. Recent studies have described that apoptosis impairment during central and peripheral tolerance is involved in T1D pathogenesis. In this study, the apoptosis-related gene expression in T1D patients was evaluated before and after treatment with high-dose immunosuppression followed by autologous haematopoietic stem cell transplantation (HDI-AHSCT). We also correlated gene expression results with clinical response to HDI-AHSCT. We observed a decreased expression of bad, bax and fasL pro-apoptotic genes and an increased expression of a1, bcl-x(L) and cIAP-2 anti-apoptotic genes in patients' peripheral blood mononuclear cells (PBMCs) compared to controls. After HDI-AHSCT, we found an up-regulation of fas and fasL and a down-regulation of anti-apoptotic bcl-x(L) genes expression in post-HDI-AHSCT periods compared to pre-transplantation. Additionally, the levels of bad, bax, bok, fasL, bcl-x(L) and cIAP-1 genes expression were found similar to controls 2 years after HDI-AHSCT. Furthermore, over-expression of pro-apoptotic noxa at 540 days post-HDI-AHSCT correlated positively with insulin-free patients and conversely with glutamic acid decarboxylase autoantibodies (GAD65) autoantibody levels. Taken together, the results suggest that apoptosis-related genes deregulation in patients' PBMCs might be involved in breakdown of immune tolerance and consequently contribute to T1D pathogenesis. Furthermore, HDI-AHSCT modulated the expression of some apoptotic genes towards the levels similar to controls. Possibly, the expression of these apoptotic molecules could be applied as biomarkers of clinical remission of T1D patients treated with HDI-AHSCT therapy.

Interleukin-18 and interferon-gamma polymorphisms are implicated on proviral load and susceptibility to human T-lymphotropic virus type 1 infection.

Interleukin-18 (IL-18) and interferon-gamma (IFN-γ) exert important functions in both innate and adaptive immune responses against intracellular pathogens and viruses. Previous studies suggested that host genetic factors, including cytokines gene polymorphisms, could be involved in the pathogenesis of human T-cell leukemia virus type 1 (HTLV-1)-associated myelopathy/tropical spastic paraparesis (HAM/TSP). Thus, we analyzed -137C/G and -607A/C of the IL-18 promoter and +874T/A of the IFN-γ in DNA samples from 98 HTLV-1-infected individuals exhibiting or not clinical symptoms and 150 healthy control individuals. The IL-18 promoter -607CC genotype was significantly lower in HTLV-1 asymptomatic carriers (HAC) and HTLV-1-infected individuals (HAC + HAM/TSP) than healthy control group. In contrast, the -607AC genotype was significantly higher in HAC and HTLV-1-infected individuals group compared to the healthy control group. The -137G/-607A IL-18 haplotype was higher in infected group than healthy control group, and the -137C/-607C IL-18 haplotype was increased in the healthy control group compared to the others. Finally, the IFN-γ polymorphism analysis showed that the HTLV-1-infected individuals with +874AT genotype presented higher proviral load than +874AA genotype. These data indicate that the IL-18-607AC genotype and -137G/-607A haplotype could be a risk factor for HTLV-1 infection, whereas the protective effect could be conferred by -607CC genotype and -137C/-607C haplotype. Also, the IFN-γ could be implicated on the proviral load levels.

Production of human factor VIII-FL in 293T cells using the bicistronic MGMT(P140K)-retroviral vector.

Hemophilia A is the most common X-linked bleeding disorder; it is caused by deficiency of coagulation factor VIII (FVIII). Replacement therapy with rFVIII produced from human cell line is a major goal for treating hemophilia patients. We prepared a full-length recombinant FVIII (FVIII-FL), using the pMFG-P140K retroviral vector. The IRES DNA fragment was cloned upstream to the P140K gene, providing a 9.34-kb bicistronic vector. FVIII-FL cDNA was then cloned upstream to IRES, resulting in a 16.6-kb construct. In parallel, an eGFP control vector was generated, resulting in a 10.1- kb construct. The 293T cells were transfected with these constructs, generating the 293T-FVIII-FL/P140K and 293T-eGFP/P140K cell lines. In 293T-FVIII-FL/P140K cells, FVIII and P140K mRNAs levels were 4,410 (±931.7)- and 295,400 (±75,769)-fold higher than in virgin cells. In 293T-eGFP/P140K cells, the eGFP and P140K mRNAs levels were 1,501,000 (±493,700)- and 308,000 (±139,300)-fold higher than in virgin cells. The amount of FVIII-FL was 0.2 IU/mL and 45 ng/mL FVIII cells or 4.4 IU/µg protein. These data demonstrate the efficacy of the bicistronic retroviral vector expressing FVIII-FL and MGMT(P140K), showing that it could be used for producing the FVIII-FL protein in a human cell line.

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.

Frequency distribution of XbaIG > T and HaeIIIT > C GLUT1 polymorphisms among different Brazilian ethnic groups.

GLUT is the major glucose transporter in mammalian cells. Single nucleotide polymorphisms (SNP) at GLUT1 promoter and regulatory regions have been associated to the risk of developing nephropathy in different type 1 and type 2 diabetic populations. It has been demonstrated that differences in allelic and genotypic frequencies of GLUT1 gene (SLC2A1) polymorphisms occur among different populations. Therefore, ethnic differences in distribution of GLUT1 gene polymorphisms may be an important factor in determining gene-disease association. In this study, we investigated the XbaIG > T and HaeIIIT > C polymorphisms in six different Brazilian populations: 102 individuals from Salvador population (Northern Brazil), 56 European descendants from Joinville (South Brazil), 85 Indians from Tiryió tribe (North Brazil) and 127 samples from Southern Brazil: 44 from European descendants, 42 from African descendants and 41 from Japanese descendants. Genotype frequencies from both sites did not differ significantly from those expected under the Hardy-Weinberg equilibrium. We verified that the allele frequencies of both polymorphisms were heterogeneous in these six Brazilian ethnic groups.

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.

An enhancer/promoter combination strengthens the expression of blood-coagulation factor VIII in non-viral expression vectors.

We explored the potential of fusion of hepatic locus control region 1 (HCR-1) with HCR-2 to express B-domain-deleted human factor VIII (FVIII) in four cell lines. B-domain-deleted human FVIII expression was controlled by HCR-1/HCR-2, followed by liver specific and ubiquitous promoters. Chimera enhancer HCR-1/HCR-2, followed by cytomegalovirus (CMV) promoter, gave 2-fold more FVIII expression in all cell lines (105.6 +/- 2.8 for Hek-293, 68.8 +/- 3.8 for HepG2, 34.8 +/- 1.3 for CHO, and 27.2 +/- 1.6 ng x mL(-1) x 10(6) cells(-1) for L.N.) when compared to the vector with CMV alone (54.8 +/- 3.3 for Hek-293, 32.4 +/- 1.2 for HepG2, 18.6 +/- 1.1 for CHO, and 10.1 +/- 1.7 ng x mL(-1) x 10(6) cells(-1) for L.N.). Elongation factor 1-alpha gene and human CMV promoters were more efficient than the promoters from the human alpha-1-antitrypsin gene, and fviii was less efficient in hepatic cell lines. HCR-1/HCR-2, followed by strong promoters, increases FVIII expression in vitro. Our results underscore the importance of cis sequences for enhancing in vitro FVIII expression; this may be helpful for designing new strategies to improve heterologous expression systems.

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.

Recombinant expression of coagulation factor VIII in hepatic and non-hepatic cell lines stably transduced with third generation lentiviral vectors comprising the minimal factor VIII promoter.

BACKGROUND: Lentiviral vectors have the capacity to transduce stably non-dividing, differentiated and undifferentiated cells of various tissues, including liver. To obtain high-level expression of transgenes, vectors often rely on viral promoters. However, recent data suggest that the supraphysiologic expression from ubiquitous viral promoters may not be beneficial and harbor the risk of oncogene activation. Therefore this study explored the lentiviral-mediated expression of human coagulation factor VIII (FVIII) driven by the physiologic FVIII gene promoter (FVIII-p), the liver-specific human alpha-1-antitrypsin gene promoter (hAAT-p), the ubiquitous but non-viral EF1alpha promoter (EF1alpha-p) and the viral CMV promoter. METHODS: Hepatic and non-hepatic cell lines were stably transduced with lentiviral vectors encoding FVIIIdelB and EGFP. To compare the different promoters, lentiviral vectors were cloned to drive FVIII expression from FVIII-p, EF1alpha-p, hAAT-p and CMV-p. RESULTS: As expected, the strong viral CMV-p and the ubiquitous EF1alpha-p resulted in the highest FVIII expression in all cell lines tested (CMV-p 1.85 IU/mL/10(6) cells for 293T, 3.15 for HepG2, 5.03 for SK-Hep, 0.91 for Hepa1-6; EF1-alpha promoter 0.30 IU/mL/10(6) cells for 293T, 0.04 for HepG2, 2.75 for SK-Hep, 0.46 for Hepa1-6). While the hAAT-p resulted in low FVIII levels (0.10 IU/mL/10(6)cells in HepG2 and 0.04 in Hepa1-6), the FVIII promoter gave reasonable expression levels in hepatic cells (0.47 IU/mL/10(6)cells in Hepa1-6 and 0.44 in SK-Hep). DISCUSSION: These results indicate the potential usefulness of the FVIII-p for hemophilia A gene therapy.