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.

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.

Effects of FTO and PPARγ variants on intrauterine growth restriction in a Brazilian birth cohort.

Intrauterine growth restriction (IUGR) is related to a higher risk of neonatal mortality, minor cognitive deficit, metabolic syndrome, and cardiovascular disease in adulthood. In previous studies, genetic variants in the FTO (fat mass and obesity-associated) and PPARγ (peroxisome proliferator-activated receptor-gamma) genes have been associated with metabolic disease, body mass index, and obesity among other outcomes. We studied the association of selected FTO (rs1421085, rs55682395, rs17817449, rs8043757, rs9926289, and rs9939609) and PPARγ (rs10865710, rs17036263, rs35206526, rs1801282, rs28763894, rs41516544, rs62243567, rs3856806, and rs1805151) single-nucleotide polymorphisms (SNPs) with IUGR, through a case-control study in a cohort of live births that occurred from June 1978 to May 1979 in a Brazilian city. We selected 280 IUGR cases and 256 controls for analysis. Logistic regression was used to jointly analyze the SNPs as well as factors such as maternal smoking, age, and schooling. We found that the PPARγ rs41516544 increased the risk of IUGR for male offspring (OR 27.83, 95%CI 3.65-212.32) as well as for female offspring (OR=8.94, 95%CI: 1.96-40.88). The FTO rs9939609 TA genotype resulted in a reduced susceptibility to IUGR for male offspring only (OR=0.47, 95%CI: 0.26-0.86). In conclusion, we demonstrated that PPARγ SNP had a positive effect and FTO SNP had a negative effect on IUGR occurrence, and these effects were gender-specific.

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.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant.

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34%) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

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.

Effects of FTO and PPARγ variants on intrauterine growth restriction in a Brazilian birth cohort

Intrauterine growth restriction (IUGR) is related to a higher risk of neonatal mortality, minor cognitive deficit, metabolic syndrome, and cardiovascular disease in adulthood. In previous studies, genetic variants in the FTO (fat mass and obesity-associated) and PPARγ (peroxisome proliferator-activated receptor-gamma) genes have been associated with metabolic disease, body mass index, and obesity among other outcomes. We studied the association of selected FTO (rs1421085, rs55682395, rs17817449, rs8043757, rs9926289, and rs9939609) and PPARγ (rs10865710, rs17036263, rs35206526, rs1801282, rs28763894, rs41516544, rs62243567, rs3856806, and rs1805151) single-nucleotide polymorphisms (SNPs) with IUGR, through a case-control study in a cohort of live births that occurred from June 1978 to May 1979 in a Brazilian city. We selected 280 IUGR cases and 256 controls for analysis. Logistic regression was used to jointly analyze the SNPs as well as factors such as maternal smoking, age, and schooling. We found that the PPARγ rs41516544 increased the risk of IUGR for male offspring (OR 27.83, 95%CI 3.65-212.32) as well as for female offspring (OR=8.94, 95%CI: 1.96-40.88). The FTO rs9939609 TA genotype resulted in a reduced susceptibility to IUGR for male offspring only (OR=0.47, 95%CI: 0.26-0.86). In conclusion, we demonstrated that PPARγ SNP had a positive effect and FTO SNP had a negative effect on IUGR occurrence, and these effects were gender-specific.

The induction of DNA puff BhC4-1 gene is a late response to the increase in 20-hydroxyecdysone titers in last instar dipteran larvae.

The characterization of DNA puff BhC4-1 expression was extended and its response to 20-hydroxyecdysone investigated in Bradysia hygida and in transgenic Drosophila carrying the BhC4-1 gene. In both organisms the activation of BhC4-1 in salivary glands occurs at the end of the larval stage coinciding with the peak in ecdysone titers which induces metamorphosis. Injections of 20-hydroxyecdysone into mid-fourth instar larvae of B. hygida show that the induction of BhC4-1 expression, as well as amplification and puff C4 expansion, are late events induced by the hormone. This late response of BhC4-1 expression was also observed in transgenic salivary glands cultivated in the presence of 20-hydroxyecdysone. In vitro studies using transgenic Drosophila indicate that both repressor and activator factors regulate the timing of BhC4-1 expression in salivary glands.

The DNA puff BhB10-1 gene encodes a glycine-rich protein secreted by the late stage larval salivary glands of Bradysia hygida.

We present the molecular characterization of a gene of Bradysia hygida DNA puff B10 whose temporal expression in the salivary gland correlates with the puff expansion. The transcription unit of this gene, named BhB10-1, was mapped in a 2-kb EcoRI genomic fragment that is amplified in the salivary gland of late fourth instar larvae. Its 1.3-kb transcript undergoes poly-A tail shortening during development, indicating that post-transcriptional controls as well as transcription activation are involved in the temporal regulation of the BhB10-1 gene. Analysis of the deduced amino acid sequence from the cDNA indicates that the BhB10-1 protein is a glycine-rich secretory protein. A BhB10-1-fusion protein expressed in bacteria was used to raise polyclonal antibodies. Using an immunopurified antibody, we identified the product of the DNA puff BhB10-1 gene as a 23-kDa polypeptide that is produced mainly by the salivary gland regions S1 and S3 and is present in the saliva of late larvae. This is the first direct identification of a protein encoded by a DNA puff amplified gene.

Cloning of a B10 DNA puff sequence developmentally amplified and expressed in the salivary gland of Bradysia hygida.

A recombinant clone carrying a 2-kb fragment was isolated from a mini-library of the B10 DNA puff of Bradysia hygida. This fragment was amplified in the salivary gland during the period of DNA puff formation. Amplification started when DNA puff anlage was formed and continued to increase, reaching a maximum of about 10-fold 28 h later. Northern blot hybridization experiments showed that this 2-kb fragment was complementary to two RNA species of about 1.3 kb and 1.1 kb, which are developmentally regulated in the salivary gland. Maximum amounts of these messages were present when the B10 puff is fully expanded.

Developmental regulation of an instability element from the Drosophila fushi tarazu mRNA.

The Drosophila fushi tarazu (ftz) mRNA is one of the shortest-lived metazoan mRNAs, and its instability is crucial for proper development of the embryo. Previously, we identified two cis-acting elements that are required for ftz mRNA degradation, one within the 5' one-third and another in the 3'UTR of the message. Here we focus on the 3'UTR element termed FIE3 (ftz instability element in the 3'UTR). To investigate the developmental regulation of the FIE3-dependent degrading activity we measured the abundance of an FIE3-containing mRNA in ovaries, unfertilized eggs, and different larval and adult tissues. We found that FIE3-degrading activity is present at all developmental stages and tissues examined, except in the ovary. Activation of the FIE3-dependent mRNA decay is independent of fertilization because it could be triggered by egg activation. Finally, we provide evidence that mutation of conserved elements within FIE3 had no effect on mRNA instability.

Molecular characterization of an 18 kb segment of DNA puff C4 of Bradysia hygida (Diptera, sciaridae).

The data presented here are an extension of the molecular characterization of DNA puff C4 of Bradysia hygida. A cDNA related to a gene amplified in this puff and expressed when puff C4 expands was cloned and sequenced. Analysis of the amino acid sequence deduced from the open reading frame present in the cDNA indicate that the encoded protein is secreted and comprises mostly alpha-helical coiled-coil. An 18 kb genomic segment containing the transcription unit of this gene was also cloned and the structure and expression of the 1.4 kb mRNA was determined. Quantitative slot blot hybridization of DNA complementary to the transcription unit shows that this gene is amplified about 21 times in the salivary gland, confirming data previously obtained. Fragments upstream of the 5' end, and beyond the 3' end, of the gene transcription unit were also analysed and shown to be amplified at least eight and five times, respectively. Based on these data we discuss how amplification could occur at DNA puffs.

[Therapeutic of septic children with purpuric presentation with two antibiotic schedules] / Terapêutica de crianças com sepse de apresentação purpúrica com dois esquemas antibióticos.

OBJECTIVE: The authors evaluate the therapeutic efficacy of two antibiotic schedules, ceftriaxone alone and the combined use of ampicillin and chloramphenicol, in the treatment of septic children with purpuric presentation.METHODS: A randomized open clinical trial was conducted including septic children with purpuric presentation treated at a pediatric intensive care unit from April 1988 to June 1992. All cases with systemic purpura standing for less than a week were included in one of two groups, except for those recently hospitalized or with previous hemorrhagic disturbs. Patients in group A received ampicillin and chloramphenicol and those in group B were given ceftriaxone. Quantitative parameters were adopted to compare the efficacy of the two antibiotic schedules: sensitivity of bacteria isolated at blood and liquor cultures, complications, therapeutic procedures, period of hospitalization, and sequelae.RESULTS: 19 cases were included in the group A and 16 in group B, both homogenous on clinical-laboratorial aspects. The parameters evaluated did not show different efficacy between the two antimicrobial schedules tested, except for the number of complications observed during hospitalization, which was higher among the children that received ampicillin and chloramphenicol. The overall mortality for the patients treated was 13.8%, excluded the undernourished. CONCLUSIONS: The authors verify similar clinical therapeutic efficacy with the combined use of ampicillin and chloramphenicol or ceftriaxone, as observed previously. It must be pointed that the number of complications detected during hospitalization were higher in the group that received the combined antibiotic schedule. Low mortality in the present study may be attributed to the early diagnosis and therapeutic measures adopted at the pediatric intensive care unit.

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-mL-1-106 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-mL-1-106 cells-1 for L.N.). Elongation factor 1-α gene and human CMV promoters were more efficient than the promoters from the human α-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.

Mesenchymal stem cells from patients with chronic myeloid leukemia do not express BCR-ABL and have absence of chimerism after allogeneic bone marrow transplant

Bone marrow is a heterogeneous cell population which includes hematopoietic and mesenchymal progenitor cells. Dysregulated hematopoiesis occurs in chronic myelogenous leukemia (CML), being caused at least in part by abnormalities in the hematopoietic progenitors. However, the role of mesenchymal stem cells (MSCs) in CML has not been well characterized. The objectives of the present study were to observe the biological characteristics of MSCs from CML patients and to determine if MSCs originate in part from donors in CML patients after bone marrow transplantation (BMT). We analyzed MSCs from 5 untreated patients and from 3 CML patients after sex-mismatched allogeneic BMT. Flow cytometry analysis revealed the typical MSC phenotype and in vitro assays showed ability to differentiate into adipocytes and osteoblasts. Moreover, although some RT-PCR data were contradictory, combined fluorescence in situ hybridization analysis showed that MSCs from CML patients do not express the bcr-abl gene. Regarding MSCs of donor origin, although it is possible to detect Y target sequence by nested PCR, the low frequency (0.14 and 0.34 percent) of XY cells in 2 MSC CML patients by fluorescence in situ hybridization analysis suggests the presence of contaminant hematopoietic cells and the absence of host-derived MSCs in CML patients. Therefore, we conclude that MSCs from CML patients express the typical MSC phenotype, can differentiate into osteogenic and adipogenic lineages and do not express the bcr-abl gene. MSCs cannot be found in recipients 12 to 20 months after BMT. The influence of MSCs on the dysregulation of hematopoiesis in CML patients deserves further investigation.

Cloning of a B10 DNA puff sequence developmentally amplified and expressed in the salivary gland of Bradysia hygida

A recombinant clone carrying a 2-kb fragment was isolated from a mini-library of the B10 DNA puff of Bradysia hygida. This fragment was amplified in the salivary gland during the period of DNA puff formation. Amplification started when DNA puff anlage was formed and continued to increase, reaching a maximum of abouth 10-fold 28 h later. Northern blot hybridization experiments showed that this 2-kb fragment was complementary to two RNA species of about 1.3 kb and 1.1 kb, which are developmentally regulated in the salivary gland. Maximum amounts of these messages were present when the B10 puff is fully expanded