Transplantation of allogenic hematopoietic stem and progenitor cells (HSPCs) with C-C chemokine receptor type 5 (CCR5) Δ32 genotype generates HIV-1 resistant immune cells. CCR5 gene edited autologous HSPCs can be a potential alternative to hematopoietic stem cell transplantation (HSCT) from HLA-matched CCR5 null donor. However, the clinical application of gene edited autologous HSPCs is critically limited by the quality of the graft, as HIV also infects the HSPCs. In this study, by using mobilized HSPCs from healthy donors, we show that the CD34+CD90+ hematopoietic stem cells (HSCs) express 7-fold lower CD4/CCR5 HIV receptors, higher levels of SAMHD1 anti-viral restriction factor, and possess lower susceptibility to HIV infection than the CD34+CD90- hematopoietic progenitor cells. Further, the treatment with small molecule cocktail of Resveratrol, UM729 and SR1(RUS) improved the in vivo engraftment potential of CD34+CD90+ HSCs. To demonstrate that CD34+CD90+ HSC population as an ideal graft for HIV gene therapy, we sort purified CD34+CD90+ HSCs, treated with RUS and then gene edited the CCR5 with single sgRNA. On transplantation, 100,000 CD34+CD90+ HSCs were sufficient for long-term repopulation of the entire bone marrow of NBSGW mice. Importantly, the gene editing efficiency of ~90% in the infused product was maintained in vivo, facilitating the generation of CCR5 null immune cells, resistant to HIV infection. Altogether, CCR5 gene editing of CD34+CD90+ HSCs provide an ideal gene manipulation strategy for autologous HSCT based gene therapy for HIV infection.
Background:: Antiretroviral therapy (ART) has led to a decline in autoimmune diseases but lacks studies on its effect on autoantibodies. Methods: A cross-sectional study with archived samples from 100 paired HIV-1 infected ART naïve and experienced individuals and 100 prospectively collected matched blood-donor controls. Antinuclear antibody, IgG anticardiolipin antibody, IgM and IgG β2 glycoprotein-1 antibodies, and total IgG levels were detected. Results are expressed as mean with standard deviation (SD), median, percentage positivity, and a p<0.05 is considered significant. The study was approved by the Institutional Review Board. Results: The median viral load of the treatment naïve samples was 4.34 Log copies/mL while all were virally suppressed post ART with a median duration of treatment for 12 months (range: 3-36 months). The percentage positivity of antinuclear antibody was 5% among ART naïve and controls with a decrease to 2% post ART (p= 0.441). The positivity for anti-cardiolipin antibody was 15% among ART naïve while none of the ART experienced or controls were positive (p<0.05). IgM β2 glycoprotein-1 was 4%, 1% and 3% among ART naïve, treated and controls respectively (p<0.05). IgG β2 glycoprotein-1 was 2% among ART naïve while none of the treated and controls were positive (p<0.05). The mean total IgG level among ART naïve, experienced, and controls were 21.82 (SD 6.67), 16.91 (SD 3.38), 13.70 (SD 2.24) grams/Litre respectively (p<0.05). Conclusion: ART has a significant effect on IgG anti-cardiolipin antibody and total IgG but only a marginal effect on ANA, IgM, and IgG β2 glycoprotein-1 antibodies.
CD4؉ T cell count estimations are subject to high variations; hence, in this study, the previous day's tested samples were included routinely as the internal quality controls. The percentages of variation of the 2-day values were analyzed for 280 observations and the mean variation for CD4؉ and CD3 ؉ T cell counts ranged from 5.21% to 9.66%. This method is a good internal quality control (IQC) procedure for the estimation of CD3 ؉ and CD4 ؉ T cell counts in resource-poor settings.T he absolute CD4 ϩ T-cell count is an important laboratory tool for monitoring HIV-infected individuals (5). Frequent monitoring of CD4 ϩ T lymphocytes is essential to assess the immune suppression and the disease progression of HIV-infected individuals (10, 3). This also helps physicians to decide when to initiate antiretroviral therapy (ART) (4, 2) and when to change therapy due to immunologic failure in resource-poor settings (7, 11). There are several methods used to estimate the absolute CD4 ϩ T-cell count, with the standard technique used being flow cytometry (1). There are numerous factors that are associated with variation in the absolute lymphocyte subset counts estimated (6). The reported mean variation of CD4 ϩ T cell counts in a "singleplatform" methodology is about 13.7% (range, 10% to 18.3%). The variation reported for "double-platform" systems ranges from 14.5% to 43.4% (mean, 23.4%) (1).Since the CD4 ϩ T cell estimation is subject to a large amount of variation, it is very important that daily quality control measures are carried out in the laboratory. In its guidelines, the National AIDS Control Organization (NACO), India, has suggested for all the laboratories to do daily quality control testing that incorporates two samples (the samples with the lowest and highest CD4 ϩ cell counts) that had been tested the previous day.In this study, we investigated the performance of CD4 ϩ T cell count estimation using the FACSCount system (Becton, Dickinson) by retrospectively analyzing the results of the two daily quality control samples.The CD3 ϩ and CD4 ϩ T cell estimations were carried out using the FACSCount system (Becton, Dickinson) as reported earlier (10). In our laboratory, the testing was carried out from Monday through Friday and two internal quality control (IQC) samples were run every day except Mondays. The previous day's samples with the lowest and the highest CD4 ϩ T cell counts were included as the controls. The samples were stored at room temperature (between 20 and 28°C). The IQC values of a given day were compared with the previous day's values, and percent variation values were calculated. Percent variation was calculated as follows: (count on the first day/count on the second day) Ϫ 1 ϫ 100. The data from July 2010 through January 2012 were analyzed. In the last month of the study, the laboratory also started using the Multi-Check control (Becton, Dickinson, San Jose, CA) for routine quality control testing. The data were also analyzed. A sample showing more than 20% variation from the previous day's value w...
Our preliminary findings suggest that IL-28 polymorphisms could influence both immunological recovery and therapeutic response in HIV infection. Hence, functional studies are warranted to understand the mechanistic basis of IL-28-mediated host genetic influence on HIV therapeutic response.
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