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Biomedical subjects

F Farzaneh

Publications and source records attributed to F Farzaneh.

At least 37 records · Page 2Linked to original sources
PubMed2000

Low-speed centrifugation of retroviral vectors absorbed to a particulate substrate: a highly effective means of enhancing retroviral titre.

For many gene therapy applications the effective titre of retroviral vectors is a limiting factor both in vitro and in vivo. Purification and concentration of retrovirus from packaging cell supernatant can overcome this problem. To this end we have investigated a novel procedure which involves complexing retrovirus to a dense and particulate substrate followed by a short low-speed centrifugation. The study reported here uses heat-killed, formaldehyde fixed Staphylococcus aureus (Pansorbin) absorbed to PG13 derived retrovirus. This complex was then used to harvest retrovirus from packaging cell supernatant: centrifugation and washing of this complex allows the retrovirus to be both purified and concentrated. This procedure increases the effective titre of retrovirus by up to 7500-fold after an only 200-fold reduction in volume. The affinity of Pansorbin for retrovirus allows concentration regardless of its encoded genes and makes this protocol applicable to other popular packaging cells and envelope proteins. Possible explanations for the marked increase in titre of concentrated virus and the mechanism governing the complexing of retrovirus to Pansorbin are discussed.

Animals↗
PubMed2000

Fusagene vectors: a novel strategy for the expression of multiple genes from a single cistron.

Transduction of cells with multiple genes, allowing their stable and co-ordinated expression, is difficult with the available methodologies. A method has been developed for expression of multiple gene products, as fusion proteins, from a single cistron. The encoded proteins are post-synthetically cleaved and processed into each of their constituent proteins as individual, biologically active factors. Specifically, linkers encoding cleavage sites for the Golgi expressed endoprotease, furin, have been incorporated between in-frame cDNA sequences encoding different secreted or membrane bound proteins. With this strategy we have developed expression vectors encoding multiple proteins (IL-2 and B7.1, IL-4 and B7.1, IL-4 and IL-2, IL-12 p40 and p35, and IL-12 p40, p35 and IL-2 ). Transduction and analysis of over 100 individual clones, derived from murine and human tumour cell lines, demonstrate the efficient expression and biological activity of each of the encoded proteins. Fusagene vectors enable the co-ordinated expression of multiple gene products from a single, monocistronic, expression cassette.

Animals↗
PubMed2000

Fetal and embryonic hemoglobins in erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation.

BACKGROUND AND OBJECTIVES: During fetal development a change in erythropoiesis from hepatic to medullary site occurs. In chromosomally abnormal fetuses this change is delayed. Hemoglobin production also undergoes developmental switches from embryonic to fetal hemoglobins in the first trimester of pregnancy. The aim of study was to determine the proportion of embryonic and fetal hemoglobins in fetal erythroblasts of chromosomally normal and abnormal fetuses at 10-40 weeks of gestation. DESIGN AND METHODS: Fetal blood was obtained from 93 chromosomally normal and 19 abnormal fetuses at 10-40 weeks of gestation. 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 (zeta), epsilon (epsilon) and gamma (gamma) hemoglobin chains. RESULTS: The percentages of the positively stained cells were calculated. In chromosomally normal fetuses the percentage of erythroblasts expressing the zeta chain was 25% at 10 weeks but this decreased exponentially with gestation to less than 1% by 17 weeks. Similarly, the percentage of cells expressing the epsilon chain decreased from 97% at 10 weeks to less than 1% by 25 weeks. In contrast, expression of the gamma chain increased from about 30% at 10 weeks to 90% by 16 weeks and decreased thereafter to 60% at 40 weeks. In the abnormal fetuses, the percentage of erythroblasts expressing the zeta chain and the epsilon chain decreased to less than 1% by 23 and 28 weeks respectively, while maximum expression of the gamma chain was at about 22 weeks. INTERPRETATION AND CONCLUSIONS: In the chromosomally abnormal group the pattern of change in the expression of the various hemoglobin chains during gestation was similar to that in the normal fetuses but was delayed by three to six weeks. These findings suggest that in fetuses with chromosomal abnormalities there is a developmental delay in the switch from embryonic to fetal hemoglobin chains.

Adult↗
PubMed1999

The human and mouse GATA-6 genes utilize two promoters and two initiation codons.

GATA-6 has been implicated in the regulation of myocardial differentiation during cardiogenesis. To determine how its expression is controlled, we have characterized the human and mouse genes. We have mapped their transcriptional start sites and demonstrate that two alternative promoters and 5' noncoding exons are utilized. Both transcript isoforms are expressed in the same tissue-specific and developmental stage-specific pattern, and their ratio appears similar wherever examined. The more upstream noncoding exon showed a substantial degree of homology between the two mammalian species, suggesting a conserved regulatory function. Moreover, in transfection assays we show that elements within this exon act to promote its transcription. Positive regulatory elements that effect transcription from the more downstream exon were not apparent in this assay, revealing a regulatory distinction between the two promoters. We also demonstrate alternative initiator codon usage in both the human and mouse GATA-6 genes. Both isoforms of the protein are synthesized in vitro regardless of which 5' noncoding exon is present in the RNA, although the larger protein has greater transcriptional activation potential in transfection assays. Thus, GATA-6 function in the cell is controlled by a complex interplay of transcriptional and translational regulation.

Alternative Splicing↗
PubMed1999

Effect of costimulation and the microenvironment on antigen presentation by leukemic cells.

Costimulatory signals supplied by genetically modified tumor cells can enable T-cell recognition of tumor-associated antigens that were previously silent when presented by unmodified tumor cells. Although the mechanism of the CD80/CD28 costimulation has been studied extensively in the normal T-cell/antigen-presenting cell (APC) interactions, it is unclear how expression of CD80 by tumor cells mediates its effect. We demonstrate here that optimal CD80 expression on a leukemic cell enhances T-cell recognition of alloantigen primarily by lowering the level of T-cell receptor (TCR) stimulation required for activation. CD80 expression by leukemic cells leads to increased survival of activated T cells by inducing upregulation of the antiapoptotic protein BCL-2, but not BCL-X(L). The cytokine microenvironment in which T cells are activated is crucial in determining their differentiation and consequently the nature of the immune response generated. Many tumor cells produce immunosuppressive cytokines that may not favor the induction of cell-mediated immunity. In this study, the presence of CD80 on leukemic cells increased T-cell activation in vitro, but this did not result in the production of Th1 cytokines. We show that this is due to a leukemia-derived soluble factor that inhibits the production of Th1 cytokines. Optimal expression of a costimulatory molecule, therefore, enhances the ability of leukemic cells to present antigen by amplifying TCR signals, but the microenvironment generated by leukemic cells may suppress the immune response required for their eradication. Thus, strategies aimed at inducing antileukemic immunity by providing leukemic cells with costimulatory functions must ensure the presence of an appropriate microenvironment.

Antigen Presentation↗
PubMed1999

Investigation of maternal blood enriched for fetal cells: role in screening and diagnosis of fetal trisomies.

Prenatal diagnosis of chromosomal abnormalities relies on assessment of risk followed by invasive testing in the group with highest risk. Assessment of risk by a combination of maternal age and fetal nuchal translucency and invasive testing in the 5% of the population with the highest risk would identify about 80% of trisomy 21 pregnancies. Preliminary reports suggest that chromosomal abnormalities can also be diagnosed by fluorescent in situ hybridization (FISH) in maternal blood enriched for fetal cells. This study examines the potential role of this method on the prenatal diagnosis of fetal trisomies. Maternal blood was obtained before invasive testing in 230 pregnancies at 10-14 weeks of gestation. After enrichment for fetal cells, by triple density centrifugation and anti-CD71 magnetic cell sorting, FISH was performed and the proportion of cells with positive signals in the chromosomally normal and abnormal groups was determined. Fetal karyotype was normal in 150 cases and abnormal in 80 cases, including 36 with trisomy 21. Using a 21 chromosome-specific probe, three-signal nuclei were present in at least 5% of the enriched cells from 61% of the trisomy 21 pregnancies and in none of the normal pregnancies. For a cut-off of 3% of three-signal nuclei the sensitivity for trisomy 21 was 97% for a false positive rate of 13%. Similar values were obtained in trisomies 18 and 13 using the appropriate chromosome-specific probe. Examination of fetal cells from maternal blood may provide a noninvasive prenatal diagnostic test for trisomy 21 with the potential of identifying about 60% of affected pregnancies. Alternatively, this technique can be combined with maternal age and fetal nuchal translucency as a method of selecting the high-risk group for invasive testing. Potentially, 80% of trisomy 21 pregnancies could be identified after invasive testing in less than 1% of the pregnant population.

Adolescent↗
PubMed1999

Expression of a variant of CD28 on a subpopulation of human NK cells: implications for B7-mediated stimulation of NK cells.

The ability of NK cells to kill tumor cells is controlled by a balance between activating and inhibitory signals transduced by distinct receptors. In murine tumor models, the costimulatory molecule B7.1 not only acts as a positive trigger for NK-mediated cytotoxicity but can also overcome negative signaling transduced by MHC class I molecules. In this study, we have evaluated the potential of human B7.1-CD28 interaction as an activating trigger for human blood NK cells. Using multiparameter flow cytometric analysis and a panel of different CD28 mAbs, we show that human peripheral blood NK cells (defined by CD56+, CD16+, and CD3- surface expression) express the CD28 costimulatory receptor, with its detection totally dependent on the mAb used. In addition, the level of CD28 varies among individuals and on different NK cell lines, irrespective of CD28 steady-state mRNA levels. By performing Ab binding studies on T cells, our data strongly suggest that binding of two of the anti-CD28 Abs (clones 9.3 and CD28.2) is to a different epitope to that recognized by clones L293 and YTH913.12, which is perhaps modified in the CD28 molecule expressed by the NK cells. We also show that B7.1 enhances the NK-mediated lysis of NK-sensitive but not of NK-resistant tumor cells and that this increased lysis is dependent on CD28-B7 interactions as shown by the ability of Abs to block this lysis. Coculture of the B7.1-positive NK-sensitive cells also led to the activation of the NK cells, as determined by the expression of CD69, CD25, and HLA class II.

Adult↗
PubMed1998

Modulation of Fos-mediated AP-1 transcription by the promyelocytic leukemia protein.

The growth and transformation suppressor function of promyelocytic leukemia (PML) protein are disrupted in acute promyelocytic leukemia (APL) as a result of its fusion to the RARalpha gene by t(15;17) translocation. There is significant sequence homology between the dimerization domain of PML and the Fos family of proteins, which imply that PML may be involved in AP-1 activity. Here we show that PML can cooperate with Fos to stimulate its AP-1-mediated transcriptional activity. Cotransfection of PML with GAL4/Fos strongly induced Fos-mediated activation of GAL4-responsive reporters, indicating a functional interaction between Fos and PML in vivo. Deletion analysis of Fos and PML demonstrated that the intact C-terminal domain of Fos (containing the dimerization domain), and the RING-finger, B1 box and nuclear localization domains of PML are involved in the cooperative activity of Fos and PML. Immunoprecipitation and electrophoretic mobility shift assay showed that PML is associated with the AP-1 complex. PMLRARalpha was also found to enhance the transcriptional activity of GAL4/Fos. The addition of retinoic acid abrogated the PMLRARalpha, but not PML-induced stimulation of GAL4/Fos activity in a dose-dependent manner. This study demonstrated that PML is involved in the AP-1 complex and can modulate Fos-mediated transcriptional activity, which may contribute to its growth suppressor function.

Animals↗
PubMed1998

Thymidine phosphorylase activity and prodrug effects in a three-dimensional model of angiogenesis: implications for the treatment of ovarian cancer.

Platelet-derived endothelial cell growth factor/thymidine phosphorylase (PD-ECGF/TP) is associated with angiogenesis and the progression of human ovarian cancer. The enzyme converts thymidine to thymine and 2'-deoxyribose-1-phosphate and can also metabolize the prodrug 5'-deoxy-5-fluorouridine (Furtulon) to 5-fluorouracil and 5'-deoxy-D-ribose-1-phosphate. The aim of this study was to obtain information about the activities of Furtulon in an established three-dimensional model of angiogenesis. The plan was to study partial and complete effects of Furtulon (in the absence and presence of PD-ECGF/TP or ovarian cancer cyst fluids) on the formation and destruction of microvessels from cultured segments of rat aorta in serum-free media. The endpoint was the number and form of microvessels compared with controls after 4, 7, 11, and 14 (and sometimes 17) days in culture. Furtulon (10 micromol/L) gradually reduced the size and number of microvessels over 17 days of culture (100 micromol/L significantly reduced the number by day 4). PD-ECGF/TP (10 ng/ml) and ovarian cancer cyst fluids (2% in medium, v/v) stimulated the production of microvessels. The culture of explants with Furtulon and PD-ECGF/TP or ovarian cancer cyst fluids (from day 1 or day 11 of culture) enhanced the vasoclastic activity of the drug. The effect of Furtulon at the highest dose (1000 micromol/L) or at a lower dose (100 micromol/L) in the presence of ovarian cancer cyst fluid was not reversible after culture day 11.

Adult↗
PubMed1998

Collagen type VI gene expression in the skin of trisomy 21 fetuses.

OBJECTIVE: To determine whether the mechanism for the retention of interstitial fluid in trisomy 21 fetuses presenting with nuchal translucency at 10-14 weeks' gestation is an alteration in the composition of collagen type VI, which is normally a triple helix formed of three single chains, alpha1, alpha2, and alpha3. The genes responsible for the alpha1 and alpha2 chains, COL6A1 and COL6A2, are located on chromosome 21 and therefore may be overexpressed in trisomy 21, whereas COL6A3 is located in chromosome 2. METHODS: Skin tissue was obtained after termination of pregnancy at 11-16 weeks' gestation in five fetuses with trisomy 21 and five normal controls. Total RNA was extracted and the steady-state levels of COL6A1 and COL6A3 mRNA expression of the gene transcripts were determined. Additionally, the distribution of collagen type VI in the skin of trisomy 21 and normal fetuses was analyzed using an immunohistochemical method. RESULTS: The ratio of the normalized densitometric scores for the mRNA expression of COL6A1 to COL6A3 in the skin of trisomy 21 fetuses was twice as high as in normal fetuses. Immunohistochemistry demonstrated that in trisomy 21 fetuses collagen type VI formed a dense network extending from the epidermal basement membrane to the subcutis, whereas in normal fetuses dense staining was confined to the upper region of the dermis. CONCLUSION: The distribution for collagen type VI is different from normal in the skin of trisomy 21 fetuses, and there is overexpression of COL6A1 compared with COL6A3.

Adult↗
PubMed1998

Changes in antigen expression on differentiating HL60 cells treated with dimethylsulphoxide, all-trans retinoic acid, alpha1,25-dihydroxyvitamin D3 or 12-O-tetradecanoyl phorbol-13-acetate.

We describe changes in antigen expression on HL60 cells with differentiation into granulocytes induced by all-trans retinoic acid (ATRA) or dimethylsulphoxide (DMSO), into monocytes by alpha1,25-dihydroxyvitamin D3 (D3), or into macrophages by 12-O-tetradecanoyl phorbol-13-acetate (TPA). Undifferentiated cells expressed CD13, CD14 (at a low level), CD15, CDw17, CD32, CD33, CD49e, CD63, CD64, CDw65, CD71 and CD87 antigens and bound the unclustered mAb D171 and Mo5. Differentiated and undifferentiated cells were negative for CD16, CD34, CD61, CD66abcde, CD68, CD88, CDw90 and CD93. Four panels of markers were identified whose expression changes significantly following differentiation. CD15, CD49e, CD63, CDw65, CD71 and mAb D171 and IGR-2,1A6 for DMSO; CD13, CD15, CDw17, CD49e, CD63, CDw65, CD71, CD87, CDw92 and mAb D171 and IGR-2,1A6 for ATRA; CD14, CD31, CD35, CD71, CD87, CDw92 and mAb D171 and BRIC18 for D3; CDw12, CD13, CD15, CD31, CD35, CD49e, CD71, CD87, CDw92 and mAb D171 for TPA. These will be useful for analyzing the pathways that regulate differentiation, whether the observed changes are consequences of differentiation or more direct effects of the inducers. HL60 cells provide a model for investigating the regulation of these antigens.

Antigens, CD↗
PubMed1998

p53 alterations and HPV infections are common in oral SCC: p53 gene mutations correlate with the absence of HPV 16-E6 DNA.

To examine the association between HPV infections and p53 gene aberrations, a panel of 28 oral squamous cell carcinomas (SCC) and 12 potentially malignant oral mucosal lesions were analysed for p53 mutations in exons 2-9. p53 protein was analysed by immunocytochemistry using DO7 antibody. The same panel was also examined for the possible presence of HPV infection. p53 overexpression was detected in 13/26 (50%) malignant and 2/9 (22%) premalignant lesions. Mutations in the coding region of the p53 gene were found in 10 malignant samples. None of the premalignant lesions were shown to have p53 mutations. The total number of p53 mutations in 10 samples were 14 of which 12 (85%) were in exon 5 suggesting the presence of hot spots in exon 5 for carcinogens involved in the transformation of oral epithelial cells. The presence of HPV DNA was first screened with consensus primers to the L1 region and nested PCR approach. HPV 6 and HPV 16 were detected in 14/28 (50%) oral SCC and 4 of 12 (33%) precancerous lesions, 7 tumours harboured both types. The samples were then examined for the presence of E6 oncogenic sequence of HPV16 using E6 specific primers. 7/27 (26%) SCC and 5/9 (55%) premalignant lesions harboured E6 DNA of which 6 (3 SCC and 3 premalignant) were negative with L1 primers suggesting possible integration of the specific viral genes or loss of other viral DNA sequences after integration of larger viral fragments. 9/10 (90%) SCC with p53 mutations were negative for E6 DNA. Our results show that both p53 alterations and HPV infection may be important etiological factors in the development of oral cancer. However, there is: i) No concordance between p53 mutations and its overexpression. ii) the presence of HPV capsid DNA (L1) does not necessarily indicate the presence of HPV oncogenic genes. iii) p53 gene mutations, but not overexpression, correlate with the absence of HPV 16-E6 and not L1 gene.

Aged↗
PubMed1998

Cardiac gene expression of GATA-4 transcription factor in human trisomy 21 fetuses with increased nuchal translucency.

This study examines GATA-4 gene expression in cardiac tissue from fetuses with trisomy 21 presenting with increased nuchal translucency thickness at 10-14 weeks of gestation. mRNA was extracted from cardiac tissue after termination of pregnancy at 10-18 weeks of gestation in ten trisomy 21 fetuses and 29 normal controls. Northern and slot blots were performed and densitometric analysis of slot blots was used to determine the steady-state levels of expression of GATA-4. GATA-4 transcript levels were also compared with ANP and BNP, which have previously been measured in the same panel of samples. GATA-4 expression increased significantly with gestation but there was no significant difference between fetuses with trisomy 21 and controls. There was no significant association between GATA-4 expression and the steady-state level of transcripts for the natriuretic peptides.

Abortion, Induced↗
PubMed1997

Transcriptional repression by the promyelocytic leukemia protein, PML.

Acute promyelocytic leukemia is characterized by the presence of a t(15; 17) chromosomal translocation which results in the expression of a chimeric gene product, PMLRAR alpha, consisting of an N-terminal-truncated retinoic acid receptor-alpha fused to a C-terminal-truncated PML. Several structural features, and regions of homology to known transcription factors, suggest that PML may be involved in the regulation of gene expression. In this study we have analyzed the transcriptional regulatory activity of PML using chimeric GAL4/PML constructs and GAL4-responsive reporter plasmids. The data presented demonstrate that PML, when fused to the DNA-binding domain of GAL4 (GAL4/PML), inhibits transcription from GAL4-responsive promoters. The magnitude of this repression is cell type and promoter dependent, and deletion studies show that the putative coiled-coil and part of the serine-rich regions of PML are required for this activity. These regions are also shown to be responsible for the repression of transcription activity from the EGFR promoter. The data presented also demonstrate that GAL4/PML can recruit PMLRAR alpha resulting in the retinoid-inducible transcriptional activation of a GAL4-responsive promoter, a function dependent on the presence of the coiled-coil region of PMLRAR alpha.

Animals↗
PubMed1997

Molecular cloning of human GATA-6 DNA binding protein: high levels of expression in heart and gut.

Human GATA-6 has been cloned from foetal heart by a combination of PCR-based methods and cDNA library screening. The 3.8 kbp cDNA has a coding sequence of 1347 bp the 449 aa protein is virtually identical in the two zinc-finger binding domains to other human GATA sequences, but varies considerably in the amino and carboxy terminal regions. The sequence shows greatest similarity to GATA-6-like sequences from rat, mouse, chicken and Xenopus. Northern analysis and in situ hybridisation show that GATA-6 is expressed at high levels in human adult and foetal heart as well as in gut derivatives. It is postulated that GATA-6, in concert with GATA-4, plays a crucial role in the regulation of cardiac differentiation.

Amino Acid Sequence↗