Moosa Rezwani; Abdulbaset Mazarzaei; Zahra Abbasi-Malati; Ali Akbar Pourfathollah
Abstract
Background: An issue that hinders researchers’ access to Natural Killer (NK) cells is their low proportion in peripheral blood leukocytes. This issue is currently addressed by methods involving a series of differentiation and expansions that are time-consuming and expensive.Objective: We have investigated ...
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Background: An issue that hinders researchers’ access to Natural Killer (NK) cells is their low proportion in peripheral blood leukocytes. This issue is currently addressed by methods involving a series of differentiation and expansions that are time-consuming and expensive.Objective: We have investigated whether the used leukocyte reduction filters, a by-product in the blood transfusion practice that currently is considered waste, can be utilized as a source of the NK cells.Methods: Following the blood donation of 46 donors based on the Iranian Blood Transfusion Organization’s protocols, a sample of peripheral blood of each donor and the leukocyte reduction filter used in their donation procedure have been obtained. The entrapped cells were flushed back from the leukocyte reduction filters. Both groups of samples were analyzed using an automatic hematological analyzer. NK cell isolation was done by the MACS negative selection method. The samples have been comparatively analyzed utilizing flow cytometry data of NK cells’ subpopulation compositions, viability, degranulation patterns, and cytotoxic capacity against the K562 cell line.Results: Every major leukocyte population was abundant in the samples extracted from the used leukocyte reduction filters. The NK cells extracted from leukocyte reduction filters did not show any statistically meaningful differences (P<0.5) from peripheral blood samples in terms of subpopulation composition, viability, degranulation potency, and cytotoxic capacity.Conclusion: Used leukocyte reduction filters can be considered an economic, easy to obtain, and robust source of abundant research-grade NK cells.
Sayeed Bayanolhagh; Mahtab Alinezhad; Kooroosh Kamali; Maryam Foroughi; Hamid Reza Khorram Khorshid; Minoo Mohraz; Fereidoun Mahboudi; Ali Akbar Pourfathollah
Volume 7, Issue 3 , September 2010, , Pages 162-176
Abstract
Background: Numerous evidences indicate that in some HIV-1 positive patients, the humoral and cellular immune responses are induced against HIV-1 proteins and this is inversely related to the progress of infection. Objective: The aim of this study was the evaluation of the Adenovectors containing HIV ...
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Background: Numerous evidences indicate that in some HIV-1 positive patients, the humoral and cellular immune responses are induced against HIV-1 proteins and this is inversely related to the progress of infection. Objective: The aim of this study was the evaluation of the Adenovectors containing HIV genes in induction of immune responses in mice. Methods: The HIV-1 genes including gag p24, rev, nef and exon-1 of tat were amplified from HIV-1 RNA (clade-A). The cDNA of each gene was cloned into a transfer vector. The transfer vector was then co-transformed into E. coli strain BJ5183 together with pAdenovector ΔE1/E3. The recombinant adenoviral construct was transfected into QBI-293A cells. Recombinant viruses were purified and titrated on 293 cell plates. Expression of transgenes was evaluated using western blotting. Then 1012 viral particles were injected into 15 groups of 5 mice and all patterns of combination of these 4 HIV-1 genes were evaluated. After 2 weeks, humoral and cellular immune responses were evaluated using ELISA, cell proliferation and ELISpot (IL-2, IL-4 and IFN-γ) assays, consecutively. Results: It was demonstrated that each gene was expressed. The response targets were mostly toward Th1, though several Th2 responses were also observed. Single injection in our study induced a good cellular response but the humoral responses were not as strong as the cellular ones. Conclusion: Considering and comparing all results and evaluating the various possible interactions revealed that simultaneous injection of tat and gag has enhanced the humoral and cellular responses.
Padideh Ebadi; Mohammad Hossein Karimi; Ali Akbar Pourfathollah; Saheb Ghadam Lotfi; Zahra Soheila Soheili; Shahram Samiee; Smerdis Hajati; Fatemeh Nadali; Bita Geramizadeh; Seyyed Mohammad Moazzeni
Volume 6, Issue 1 , March 2009, , Pages 1-11
Abstract
Background: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides ...
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Background: Dendritic cells (DCs) are ideal accessory cells in the field of gene therapy. Delivery of DNA and siRNA into mammalian cells is a useful technique in treating various diseases caused by single gene defects. Selective gene silencing by small interfering RNAs (siRNAs) and antisense oligodeoxynucleotides (ODN)s is an efficient method for the manipulation of cellular functions. An efficient, functional delivery system with no toxicity problems would be attractive. Objective: We compared two commercially available cationic lipids, Lipofectamine and FuGENE6, in the delivery of both siRNA and antisense ODNs into mice spleen-derived DCs. Methods: Cellular uptake was measured by the means of fluorescein-labelled non-silencing siRNA and antisense ODNs as a model system using flow cytometry. Cytotoxicity of the two delivery systems was compared with propidium iodide and annexin-V staining, and quantified with flow cytometry. The efficiency of our oligonucleotide delivery systems was compared by measuring CD40 expression by flow cytometry. Results: CD40 expression in DCs was 38%. After siRNA transfection by Lipofectamine, CD40 expression decreased to 13%, and after transfection by FuGENE6, it decreased to 18%. The difference was statistically significant. CD40 down regulation in DCs transfected with the two different antisense sequences by Lipofectamine was 21% and 23%, and down regulation after transfection by FuGENE6 was 19% and 18%, respectively. The differences were not statistically significant. The effects of siRNA and antisense ODNs were specific. Conclusion: Lipofectamine was a more potent delivery system in siRNA effect, followed by FuGENE6. There was no significant difference between Lipofectamine and FuGENE6 as a delivery system of antisense ODNs.
