Hematopoietic cell transplantation (HCT) using CCR5-Δ32/Δ32 stem cells from an adult donor has resulted in the only known cure of HIV infection. However, it is not feasible to repeat this procedure except rarely because of the low incidence of the CCR5-delta 32 allele, the availability of only a small number of potential donors for most patients and the need for a very close HLA match between donor and recipient. In contrast, cord blood (CB) transplants require significantly less stringent HLA matching. Therefore, our hypothesis is that cure of HIV infections by HCT can be accomplished much more readily using umbilical cord blood stem cells obtained from a modestly sized inventory of cryopreserved CCR5-Δ32/Δ32 CB units. To test this hypothesis we have developed a screening program for cord blood units and are developing an inventory of CCR5-Δ32/Δ32 cryopreserved units available for HCT. 300 such units are projected to provide for Caucasian pediatric patients a 73.6% probability of finding an adequately HLA matched unit with a cell dose of ≥ 2.5 x 107TNC/kg and a 27.9% probability for Caucasian adults. With a cell dose of ≥ 1 x 107TNC/kg the corresponding projected probabilities are 85.6% and 82.1%. The projected probabilities are lower for ethnic minorities. Impetus for using CB HCT was provided by a transplant of an adult with acute myelogenous leukemia who was not HIV infected. The HCT was performed with a CCR5-Δ32/Δ32 CB unit, and post-transplant in vitro studies indicated that the patient’s peripheral blood mononuclear cells (PBMCs) were resistant to HIV infection.
Background and ObjectivesMesenchymal stem cells (MSCs) are self-renewing, non-specialized cells used clinically in tissue regeneration and sourced from bone marrow, peripheral blood, umbilical cord blood and umbilical cord tissue (UCT). To demonstrate an alternative method for MSC detection, cryopreserved UCT and expanded MSC were screened for MSC markers CD73, CD90, CD105 and CDH-11 by RT-PCR.Methods and ResultsHuman UCT were washed, sectioned, cryopreserved with 10% DMSO and stored in the vapor phase of liquid nitrogen. Fresh and frozen UCT samples were expanded for MSC. UCT and MSC were processed for RNA and screened for CD73, CD90, CD105 and CDH-11 mRNA by RT-PCR. CD73, CD90 and CD105 were detected by flow cytometry and CDH-11 was detected by Western blotting. Short and long-term frozen UCT shows a loss of mRNA expression for CD73 at 33.2±34.0%, CD90 at 6.2±8.2%, CD105 at 17.7±21.6% and CDH-11 at 30.1±26.7% but was not statistically significant to indicate the deterioration. Expanded MSCs from fresh UCT expressed 0.09±0.07-fold CD73, 0.17±0.11-fold CD90, 0.04±0.06-fold CD105 and 0.14±0.08-fold CDH-11. Expanded MSCs from frozen UCTs expressed 0.09±0.06-fold CD73, 0.13±0.06-fold CD90, 0.04±0.05-fold CD105 and 0.11±0.06-fold CDH-11 and confirmed by flow cytometry and Western blotting.ConclusionCD73, CD90, CD105 and CDH-11 were detected by RT-PCR in cryopreserved UCT and MSC expansion. CDH-11 appears as a useful single target MSC marker for quick screening.
Direct detection of the PCR, or DD-PCR is proposed as an efficient method for performing PCR assays. Following the PCR reaction, ethidium homodimer dye is added to the reaction mixture and read by fluorescence. The dye step circumvents the necessity of running reactions on agarose gel electrophoresis, which is the current standard. This simple modification should find wide application for assays utilizing the PCR reaction. Here we show the ready detection of HLA class II polymorphism.
HIV-1 infection afflicts more than 35 million people worldwide, according to 2014 estimates from the World Health Organization. For those individuals who have access to antiretroviral therapy, these drugs can effectively suppress, but not cure, HIV-1 infection. Indeed, the only documented case for an HIV/AIDS cure was a patient with HIV-1 and acute myeloid leukemia who received allogeneic hematopoietic cell transplantation (HCT) from a graft that carried the HIV-resistant CCR5-∆32/∆32 mutation. Other attempts to establish a cure for HIV/AIDS using HCT in patients with HIV-1 and malignancy have yielded mixed results, as encouraging evidence for virus eradication in a few cases has been offset by poor clinical outcomes due to the underlying cancer or other complications. Such clinical strategies have relied on HIV-resistant hematopoietic stem and progenitor cells that harbor the natural CCR5-∆32/∆32 mutation or that have been genetically modified for HIV-resistance. Nevertheless, HCT with HIV-resistant cord blood remains a promising option, particularly with inventories of CCR5-∆32/∆32 units or with genetically modified, human leukocyte antigen-matched cord blood.
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