Death switch for gene therapy: application to erythropoietin transgene expression

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)The effectiveness of the caspase-9-based artificial 'death switch' as a safety measure for gene therapy based on the erythropoietin (Epo) hormone was tested in vitro and in vivo using the chemical inducer of dimerization, AP20187. Plasmids encoding the dimeric murine Epo, the tetracycline-controlled transactivator and inducible caspase 9 (ptet-mEpoD, ptet-tTAk and pSH1/Sn-E-Fv'-Fvls-casp9-E, respectively) were used in this study. AP20187 induced apoptosis of iCasp9-modified C2C12 myoblasts. In vivo, two groups of male C57BI/6 mice, 8-12 weeks old, were injected intramuscularly with 5 mu g/50 g ptet-mEpoD and 0.5 mu g/50 g ptet-tTAk. There were 20 animals in group 1 and 36 animals in group 2. Animals from group 2 were also injected with the 6 mu g/50 g iCasp9 plasmid. Seventy percent of the animals showed an increase in hematocrit of more than 65% for more than 15 weeks. AP20187 administration significantly reduced hematocrit and plasma Epo levels in 30% of the animals belonging to group 2. TUNEL-positive cells were detected in the muscle of at least 50% of the animals treated with AP20187. Doxycycline administration was efficient in controlling Epo secretion in both groups. We conclude that inducible caspase 9 did not interfere with gene transfer, gene expression or tetracycline control and may be used as a safety mechanism for gene therapy. However, more studies are necessary to improve the efficacy of this technique, for example, the use of lentivirus vector.437634644Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Regulation of the GATA3 promoter by human T-cell lymphotropic virus type I tax protein

The Human T-cell leukemia virus type 1 (HTLV-1) non-structural protein Tax plays a crucial role incellular transformation. It activates the transcription factors of various cellular genes and interacts with cellular proteins. There is limited data available on the interaction between specific T-cell transcription factor GATA3 and Tax. Implications for the significance of GATA3 in T-cell development and function, T helper2 (Th2) differentiation, and a role of GATA3 during the immune response have been reported. To determine the effect of the Tax protein on GATA3 gene expression, we investigated the interaction between this protein and the GATA3 promoter and repressor regions. Results demonstrated an interaction between Tax and the GATA3promoter via the transcription factor Sp1 and a role for Tax in the negative regulation of GATA3 expression, through its interaction with the repressor ZEB. This interaction may be involved in the pathophysiology of adult T-cell leukemia/lymphoma (ATL) and tropical spastic paraparesis/HTLV-1-associated myelopathy (TSP/HAM). (C) 2004 Wiley-Liss, Inc.9361178118

Decreased GATA3 mRNA expression in human T-cell lymphotropic virus type 1 (HTLV-1) infection

GATA3 is a specific T-cell transcription factor involved in the expression of T-cell receptor (TCR). In order to characterize the relationship between HTLV-I infection, which has been reported to he associated with down-regulation of genes belonging to the TCR/CD3 complex, and the transcription factor GATA3, we evaluated, by semi-quantitative RT-PCR, the expression of GATA3 gene in HTLV-1 carriers and individuals with related diseases. The study included 4 asymptomatic carriers, 2 patients with adult T-cell leukaemia/lymphoma (ATLL), 1 patient with HTLV-1 associated myelopathy (HAM)/tropical spastic paraparesis (TSP) and 7 healthy blood donors. A considerable decrease in the expression of the GATA3 mRNA was observed in all subjects infected by HTLV-1 and no expression of GATA3 mRNA was observed in 1 subject with ATLL and in 1 with HAM/TSP.32216116

The association of cytokine gene polymorphisms with febrile non-hemolytic transfusion reaction in multitransfused patients

Cytokines are associated with inflammatory responses including febrile non-hemolytic transfusion reactions (FNHTR). Moreover, there are some polymorphisms of these cytokine genes associated with different levels of gene expression. The aim of the present study was to investigate the association of inflammatory cytokine gene polymorphisms with the occurrence of FNHTR in multitransfused patients. We studied two groups of transfused patients: one presenting FNHTR before 20 transfusions of red blood cells concentrates and the other which never presented FNHTR even after 20 transfusions. The gene polymorphisms studied were IL1B - 511C/T and +3953C/T, IL1RN ( intron 2, variable number tandem repeat), IL6-174G/C, IL10 - 1082G/A and - 819C/T, TNF-308G/A and LTA+253G/A using polymerase chain reaction and restriction digestion or sequencing methods. An association of IL1RN*2.2 genotype with the occurrence of precocious FNHTR ( P < 0.025) was detected. This allele and this genotype have been related with higher serum levels of interleukin (IL)-1 beta in vivo and higher promoter activity. No other association was demonstrated. The association of gene polymorphisms related with the increase of inflammatory cytokine gene expression may be a relevant factor in FNHTR and requires confirmation.16318419

alpha-Cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform

The band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue-specific isoforms area I so expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two-hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C-terminal region of the a-cardiac actin. Immunoprecipitation of whole rat heart using an anti-actin antibody, immunoblotted with anti-human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with a-cardiac actin in cardiomyocyte, and we suggest that the binding occur 'in situ,' in the intercalated disc, a site of cell-cell contact and attachment of the sarcomere to the plasma membrane. (C) 2003 Wiley-Liss, Inc.8961215122

Molecular characterization of glucose-6-phosphate dehydrogenase deficiency in Brazil

Molecular characterization of glucose-6-phosphate dehydrogenase (G6PD) variants was carried out in 150 unrelated G6PD deficient blood donors from the region of Campinas, Brazil. By allele specific oligomer hybridization or digestion of exon 4 of the G6PD gene with the restriction endonuclease NlaII, we detected the 202 G-->A mutation in 146 individuals. This mutation was associated with the 376 G-->A substitution and only one haplotype was observed in these individuals. Digestion of exon 6 with the restriction enzyme MboII showed the presence of the Mediterranean variant in three individuals. Haplotype analysis showed, in all three samples, a T at nt 1311 and the C at nt 13 in intron 11, suggesting a European origin of this variant. By SSCP analysis and direct sequencing we detected the mutation nt 1003 G-->A (335 Ala-->Thr) in one blood donor. This mutation was previously described in a boy of Indian ancestry and the variant was denominated G6PD Chatam. The case described here has no Indian ancestry; thus, we presume that the mutations have arisen independently, although we do not know the haplotype of the Indian patient. The haplotype of our case was the most common observed in our population (PvuII, BspHI+, PstI+, 1311C, NlaIII-). Thus, our data-indicate that G6PD A- with the 202 G-->A mutation is the most frequent G6PD deficiency in the population of southeastern Brazil. The remaining variants had a Mediterranean origin. These results are in agreement with the origin of the Brazilian population.471172

Expression of inducible nitric oxide synthase is increased in acute myeloid leukaemia

Significant activity of inducible nitric oxide synthase (iNOS) has been reported in tumour cells, including chronic lymphoid leukaemic cells. In this study, we analysed the expression of NOS in 15 untreated patients with acute myeloid leukaemia (AML) and in 7 normal controls. Using flow cytometry and immunocytochemistry, we demonstrated that patients with AML had a high expression of NOS when compared to controls. There was no correlation between the expression of NOS and the expression of p53 and K, H, and N-ras mutation and expression, suggesting that the high expression of NOS is independent of these proteins and could be the result of transcription factors expressed in AML. Copyright (C) 2001 S. Karger AG, Basel.1063959

G6PD sumare: A novel mutation in the G6PD gene (1292 T->G) associated with chronic nonspherocytic anemia

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RED-CELL MEMBRANE-PROTEIN ABNORMALITIES IN HEREDITARY SPHEROCYTOSIS IN BRAZIL

We studied 14 kindred and nine unrelated patients from southeastern Brazil with the typical form of hereditary spherocytosis. Diagnosis was made on the basis of clinical features, presence of spherocytes on the peripheral blood smears and an abnormal osmotic fragility test. By densitometric tracing of SDS-PAGE stained by Coomassie blue, we detected isolated deficiency of spectrin in 39% of our patients, combined spectrin and ankyrin deficiency in 13%, and deficiency of band 3 in 13%. One of our patients presented ankyrin deficiency without spectrin reduction. Our data suggest that, despite ethnic differences among the Brazilian and European or North-American populations, these biochemical abnormalities in HS patients may be similar.88229529

Abnormal antioxidant system in erythrocytes of mercury-exposed workers

To investigate the effects of chronic exposure to mercury we studied the red cell antioxidant system in mercury-exposed workers through the evaluation of reduced glutathione, catalase and superoxide dismutase systems. Of these workers, some were being exposed at the time and had presented urinary mercury levels considered safe for occupational exposure for at least 3 months prior to the initiation of this study, and others had been on leave for at least 6 months because of intoxication symptoms. Reduced glutathione levels were lower and catalase activity was higher in the workers which were still being exposed, compared to those on leave and controls. No differences were observed between the workers on leave and controls.17422523