Biomedical subjects
Publications and source records attributed to F Farzaneh.
The 2005 International Society for Cell and Gene Therapy of Cancer (ISCGT) Congress was held in Shenzhen, China (www.iscgtchina2005.com) from December 9th-11th 2005. Here, we describe a representation of the most seminal presentations providing an overview of the progress in the field of cancer gene therapy including the successful introduction of the first approved gene therapy drug.
The role of cytokines in protecting against human papillomavirus (HPV) and HPV-associated disease is not fully understood. We compared the frequency of the interleukin (IL)-10 polymorphism (G allele) at position --1082 and the distribution of GG/GA/AA genotypes among 116 HPV-positive women, grouped according to their cervical cytological profiles, with 119 HPV-negative controls with normal smears. No difference was observed in genotype frequency between the groups. Among women in the HPV-positive, smear-normal group, who were re-tested for HPV after 12 months, there was a significant inverse association between presence of at least one variant G allele (high activity) and HPV persistence (OR per G allele = 0.082 [95% CI 0.009-0.73], P= 0.001; after controlling for ethnicity). This association remained significant after controlling for age, smoking and hormonal contraception (OR = 0.028 [95% CI 0.001-0.66], P= 0.001). This preliminary study suggests that higher levels of IL-10 may prevent cervical neoplasia through their role in eliminating HPV.
Murine bone marrow-derived dendritic cells (DC) can be generated by culture in the presence of granulocyte/macrophage colony-stimulating factor (GM-CSF) alone or GM-CSF in conjunction with interleukin-4 (IL-4). However, these two culture methods result in the production of heterogeneous DC populations with distinct phenotypic and stimulatory properties. In this study, we investigated the properties of DC generated under serum-free conditions in the presence or absence of IL-4 and compared their yield and phenotype to that of DC generated in the presence of fetal calf serum (FCS) (+/-IL-4). We did not observe a significant difference in the total cell yield between these four culture conditions, although the proportion of CD11c+ DC in cultures that received FCS was higher than that of their counterparts generated under serum-free conditions. Also, the four culture conditions generated CD11c+ DC with comparable levels of major histocompatibility complex (MHC) class II, CD40, CD80 and CD86 expression, with the exception of cells cultured under serum-free conditions in the absence of IL-4, which displayed suboptimal levels of these markers. Moreover, we compared the functional and stimulatory properties of DC generated under serum-free conditions in the presence or absence of IL-4. DC cultured in the presence of IL-4 were stronger stimulators of allogeneic splenocytes in a primary mixed lymphocyte reaction (MLR) and of naive antigen-specific OT-II transgenic T cells when pulsed with the class II ovalbumin (OVA)323-339 peptide or whole OVA protein than DC cultured in the absence of IL-4. However, both DC populations displayed a similar capacity to take up fluorescein isothiocyanate (FITC)-albumin by macropinocytosis and FITC-Dextran by the mannose receptor and to secrete IL-12 in response to stimulation with lipopolysaccharide (LPS) or an agonistic anti-CD40 monoclonal antibody. Therefore, we conclude that although both DC culture methods result in the production of DC with similar functional abilities, under serum-free conditions, DC cultured in GM-CSF and IL-4 show an increased stimulatory potential over DC cultured in GM-CSF alone. This is an important consideration in the design of experiments where DC are being exploited as immunotherapeutic vaccines.
OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (epsilon and zeta) to fetal (gamma) hemoglobins accompanying the erythroblastosis in fetuses of diabetic mothers, and whether the increased erythroblast count in the fetal blood is associated with an increase in fetomaternal cell trafficking. MATERIALS AND METHODS: Fetal and maternal blood samples were obtained from 11 fetuses and five pregnant women in pregnancies complicated by maternal diabetes mellitus. Blood samples were also taken as controls from 35 fetuses and 33 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with anti-CD71 antibody. Fluorescent antibodies were used to immunostain for gamma (gamma), epsilon (epsilon) and zeta (zeta) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization for X and Y chromosomes was also carried out, to confirm the presence and proportion of enriched fetal cells from the maternal blood. RESULTS: In both fetal and maternal blood the median percentages of erythroblasts positive for gamma-globin, epsilon-globin and zeta-globin chains were significantly higher in fetuses of diabetic mothers compared to controls (fetus, gamma-globin, 76 vs. 64%, p < 0.0001; epsilon-globin, 4 vs. 0%, p < 0.0001; zeta-globin, 4 vs. 0%; p < 0.0001; mother, gamma-globin, 14 vs. 1%, p < 0.0005; epsilon-globin, 0.25 vs. 0%, p < 0.0003; zeta-globin, 0.2 vs. 0%, p < 0.0003). The median percentage of cells with Y signals in maternal blood was also higher in diabetic pregnancies compared to normal controls (7.5 vs. 1%, p < 0.002). CONCLUSIONS: The findings suggest that the fetal erythroblastosis in diabetic pregnancies is accompanied by a delay in the switch from embryonic to fetal hemoglobin chains. In addition, it is associated with an increase in fetomaternal cell trafficking.
The aim of this study was to determine the efficacy of cell separation with single density and triple density-gradient techniques in the yield of foetal erythroblasts isolated from maternal blood. Maternal blood was obtained from 20 singleton pregnancies at 11-14 weeks of gestation immediately before foetal karyotyping by chorionic villus sampling. In each woman, the blood sample was divided into two portions; one portion was used for single density-gradient separation and the other, for triple density-gradient separation. Magnetic cell sorting (MACS) was subsequently performed with anti-CD71/antiglycophorin-A. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the gamma, epsilon and zeta globin chains. The percentage of foetal cells positive for each stain was calculated. Fluorescence in situ hybridization (FISH) for X- and Y-chromosomes was also performed. Comparison was made in the proportion of enriched foetal cells between the two separation methods for each CD71 and glycophorin-A (GPA) antibody. The percentage of erythroblasts enriched from maternal blood that stained positive for gamma, epsilon and zeta globin chains and with Kleihauer-Giemsa was significantly higher in the triple density-gradient separation fractions compared with the single density-gradient fractions with both anti-CD71 and GPA MACS. FISH analysis for the Y-chromosome confirmed the increase in foetal cell proportion in the triple density-gradient samples. Isolation of foetal erythroblasts from maternal blood using triple density-gradient separation and MACS is more effective with regard to foetal cell yield and purity than single density-gradient separation and MACS.
Functional expression cloning strategies are highly suitable for the analysis of the molecular control of apoptosis. This approach has two critical advantages. Firstly, it eliminates prior assumptions about the properties of the proteins involved, and, secondly, it selectively targets proteins that are causally involved in apoptosis control and which affect the crucial cellular decision between survival and death. The application of this strategy to the isolation of cDNAs conferring resistance to dexamethasone and gamma-irradiation resulted in the isolation of a partial cDNA for the catalytic subunit of protein phosphatase 4 (PP4). Cells transfected with this partial cDNA in an expression vector downregulated PP4 and were resistant to both dexamethasone and UV radiation, as demonstrated by both membrane integrity and colony-forming assays. These observations suggest that PP4 plays an important proapoptotic role in T lymphocytes.
OBJECTIVES: To determine the distribution of fetal erythroblasts in the maternal circulation at different gestations. MATERIALS AND METHODS: Maternal blood was obtained from 152 normal singleton pregnancies at 11-40 weeks of gestation. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the zeta (zeta), epsilon (epsilon) and gamma (gamma) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescence in situ hybridization for X and Y chromosomes was also performed. Comparison was made in the proportion of positive fetal erythroblasts among the different gestational ages. RESULTS: The proportion of erythroblasts stained positive with gamma-globin chain and Kleihauer-Giemsa decreased with gestation from a median of 2% at 11 weeks to 0.5% at 40 weeks. Similarly, there was a decrease in the percentage of Y-signal-positive cells from 1% at 11 weeks to 0.3% at 40 weeks. The proportion of enriched fetal erythroblasts stained positive with zeta- and epsilon-globin chains decreased exponentially from respective medians of 0.6% and 1.5% at 11 weeks to zero after 19 weeks and 24 weeks. CONCLUSION: In normal singleton pregnancy the percentage of fetal erythroblasts enriched from maternal blood decreases with gestation.
The ability of intestinal mucosa to absorb dietary ferric iron is attributed to the presence of a brush-border membrane reductase activity that displays adaptive responses to iron status. We have isolated a complementary DNA, Dcytb (for duodenal cytochrome b), which encoded a putative plasma membrane di-heme protein in mouse duodenal mucosa. Dcytb shared between 45 and 50% similarity to the cytochrome b561 family of plasma membrane reductases, was highly expressed in the brush-border membrane of duodenal enterocytes, and induced ferric reductase activity when expressed in Xenopus oocytes and cultured cells. Duodenal expression levels of Dcytb messenger RNA and protein were regulated by changes in physiological modulators of iron absorption. Thus, Dcytb provides an important element in the iron absorption pathway.
Bloom syndrome (BS) is an autosomal recessive disorder characterized by a high incidence of cancer and genomic instability. BLM, the protein defective in BS, is a RECQ-like helicase that is presumed to function in mammalian DNA replication, recombination, or repair. We show here that BLM, but not the related RECQ-like helicase WRN, is rapidly cleaved in cells undergoing apoptosis. BLM was cleaved to 47- and 110-kDa major fragments, with kinetics similar to the apoptotic cleavage of poly(A)DP-ribose polymerase. BLM cleavage was prevented by a caspase 3 inhibitor and did not occur in caspase 3-deficient cells. Moreover, recombinant BLM was cleaved to 47- and 110-kDa fragments by caspase 3, but not caspase 6, in vitro. The caspase 3 recognition sequence (412)TEVD(415) was verified by mutating aspartate 415 to glycine and showing that this mutation rendered BLM resistant to caspase 3 cleavage. Cleavage did not abolish the BLM helicase activity but abolished BLM nuclear foci and the association of BLM with condensed DNA and the insoluble matrix. The results suggest that BLM, but not WRN, is an early selected target during the execution of apoptosis.
Three strategies have been designed to concentrate infectious retroviral vectors from the supernatants of human- (HT1080) and murine- (NIH 3T3) based packaging cells. Streptavidin-conjugated paramagnetic particles in conjunction with (i) antibodies directed against murine fibronectin, (ii) biotinylated lectins, or (iii) biotin-modified packaging cell-surface proteins allow affinity-mediated magnetic concentration of retroviral vectors. Retroviral titers (assayed by colony formation of human myeloid K562 cells) are increased by 1-4 x 10(3)-fold after volume reductions of only 125-fold. Using these procedures, preparations of 5 x 10(8) cfu/ml are routinely made from relatively low-titer (2-5 x 10(5) cfu/ml) starting material. High-titer (paramagnetic) retroviral vector preparations can be used for magnetic field-dependent retroviral infection in vitro. Magnetic field-dependent localization such as this may enable the in vivo administration of formulations that concentrate retroviral infection to the required target tissues and organs.
Protein transduction, an emerging technology with potential applications in gene therapy, can best be described as the internalisation of proteins into the cell, from the external environment. This process relies on the inherent property of a small number of proteins and peptides of being able to penetrate the cell membrane. The transducing property of these molecules can be conferred upon proteins which are expressed as fusions with them and thus offers an alternative to gene therapy for the delivery of therapeutic proteins into target cells. This review describes the three most commonly used protein transduction vehicles; the antennapedia peptide, the herpes simplex virus VP22 protein and HIV TAT protein transduction domain. The future prospects for the application of this technology in gene therapy are also discussed.
BACKGROUND AND OBJECTIVES: To determine the distribution of embryonic and fetal hemoglobin chains in fetal erythroblasts isolated from maternal blood in the first trimester of pregnancy and establish the feasibility of using these chains as markers for fetal cell identification. DESIGN AND METHODS: Maternal blood was obtained from 187 singleton pregnancies at 11-14 weeks of gestation immediately before fetal karyotyping by chorionic villus sampling. In all cases included in this study the fetal karyotype was normal. Fetal erythroblasts were isolated using triple density gradient separation and anti-CD71 magnetic cell sorting techniques. The enriched erythroblasts were stained with Kleihauer-Giemsa and with fluorescent antibodies for the zeta (z), epsilon (e) and gamma (g) globin chains. The percentage of fetal cells positive for each stain was calculated. Fluorescent in situ hybridization (FISH) for X and Y chromosomes was also performed. Comparison was made with the percentage of cells with positive Y-signal FISH in pregnancies with male fetuses. RESULTS: The percentage of fetal erythroblasts stained positive was 37% for the z and 95% for both e and g globin chains, as well as the Kleihauer-Giemsa staining. There was a significant association between the Kleihauer-Giemsa stained cells and those stained with e and g globin chains. There was also an association between cells with Y-signals and those stained with e and g globin chains. INTERPRETATION AND CONCLUSIONS: Embryonic hemoglobin chains can be detected in the enriched fetal erythroblasts, with higher percentages of the e rather than the z globin chains. These chains are therefore potentially unique markers to be used in the identification of cells of fetal origin from maternal blood for prenatal diagnosis of genetic and chromosomal abnormalities.
BACKGROUND AND OBJECTIVES: To determine whether there is a delay or reversal in switch mechanisms from embryonic (e and z) to fetal (g) hemoglobins accompanying the erythroblastosis of anemic fetuses and whether an increased erythroblast count in fetal blood is associated with an increase in feto-maternal cell trafficking. DESIGN AND METHODS: Fetal and maternal blood samples were obtained from 10 cases with rhesus isoimmunization and 2 cases with maternal Parvo-B19 virus at 19-33 weeks' gestation. Blood samples were also taken as controls from 61 fetuses and 86 mothers. Fetal erythroblasts were isolated by triple density gradient centrifugation and magnetic cell sorting with CD71 antibody. Fluorescent antibodies were used to immuno-stain for zeta (z), epsilon (e) and gamma (g) hemoglobin chains. In the maternal samples, fluorescence in situ hybridization (FISH) for X and Y chromosomes was also carried out to confirm the presence and proportion of the enriched fetal cells from maternal blood. RESULTS: In both fetal and maternal blood the percentage of erythroblasts positive for g-globin chain was significantly higher in the anemic fetuses compared to the controls (fetal blood, p<0.001, R=0.91; maternal blood, p<0.001, R=0.56), but there was no significant difference in expression of the e and z-chains. The percentage of cells with Y-signals was also higher in the maternal samples of anemic fetuses compared to normal controls (p<0.001, R=0.56). INTERPRETATION AND CONCLUSIONS: These findings suggest that the erythroblastosis of anemic fetuses is not accompanied by a delay or a reversal in switch from embryonic to fetal hemoglobin chains. Severe fetal anemia is associated with an increase in feto-maternal cell trafficking.
Protein transduction can be described as the direct uptake by the cell of exogenous proteins/peptides or protein/peptide:chemical complexes, as a result of a specific property of the protein/peptide component. In this review, the three most widely studied protein transducing activities are described, with particular emphasis on the TAT protein transduction domain. Current progress in protein transduction technology suggests the potential development of a variety of molecular and cell biology tools that will enable researchers to by-pass conventional genetic routes for modulating the cells' biological activity, thus negating many of the problems associated with genetic intervention. The potential application of this class of molecule in the development of tools for the study of senescent populations is discussed.
The effect of N-acetylcysteine (NAC) on levels of hepatocyte growth factor/scatter factor (HGF/SF) gene transcripts was investigated in the human lung embryonic fibroblast cell line, MRC-5. NAC increased expression of HGF/SF mRNA, in a dose- and time-dependent fashion, by a mechanism independent of glutathione synthesis but sensitive to oxidant stress induced by H(2)O(2). Using actinomycin D to block RNA synthesis, it was observed that NAC had no effect on the stability of the HGF/SF mRNA transcripts. NAC increased HGF/SF promoter activity in cells transiently transfected with chloramphenicol acetyltransferase (CAT) reporter genes driven by HGF/SF gene 5'-flanking sequences. Primer extension analysis demonstrated that NAC enhanced the expression of HGF/SF mRNA transcribed from the main transcription initiation site. Although the 5' flanking region of the HGF/SF gene contains a sequence at -1019 to -1011 with homology to the NF-kappaB response element, electrophoretic mobility shift assay demonstrated that this site did not bind nuclear factors in MRC-5 cells in the presence or absence of NAC. In contrast to the effect on HGF/SF mRNA, NAC did not increase HGF/SF protein production by MRC-5 cells.
Iron absorption by the duodenal mucosa is initiated by uptake of ferrous Fe(II) iron across the brush border membrane and culminates in transfer of the metal across the basolateral membrane to the portal vein circulation by an unknown mechanism. We describe here the isolation and characterization of a novel cDNA (Ireg1) encoding a duodenal protein that is localized to the basolateral membrane of polarized epithelial cells. Ireg1 mRNA and protein expression are increased under conditions of increased iron absorption, and the 5' UTR of the Ireg1 mRNA contains a functional iron-responsive element (IRE). IREG1 stimulates iron efflux following expression in Xenopus oocytes. We conclude that IREG1 represents the long-sought duodenal iron export protein and is upregulated in the iron overload disease, hereditary hemochromatosis.
Modification of murine K1735 melanoma cells to express the immune costimulator B7-1 had no effect on tumor formation in syngeneic mice. In contrast, <40% of mice inoculated with K1735 cells modified to secrete murine interleukin-2 (IL-2) formed tumors, and no tumors formed when the K1735 cells coexpressed both murine IL-2 and B7-1. However, administration of systemic recombinant human IL-2 had no detectable effect on the formation of tumors by the B7-1-expressing K1735 cells. By contrast, admixtures of IL-2-secreting and B7-1-expressing K1735 cells formed fewer tumors than either cell type alone. Murine IL-2 was effective only when secreted locally, because the IL-2-secreting cells inoculated into the right flank did not affect the growth of the B7-1-expressing cells inoculated into the opposite flank.