Morphometric analysis in ethnic neonates from multiple substance exposure.

In the United States, approximately 10% of newborn infants are exposed prenatally to alcohol and/or illicit substances. However, no studies have evaluated the compounding effects of multiple illicit substances exposure in utero as potential teratogen (s). The potential teratogenic effects of nicotine and illicit substances (e.g. cocaine, marijuana and heroin) have previously been studied but there has been no documentation of facial landmark dislocation (s). Our goal is to investigate whether morphometric analysis could differentiate facial landmark dislocations in neonates of African descent, when exposed to alcohol, nicotine and illicit substances, either singly or in combination. Craniofacial features from a cohort of 493 African-American neonates less than 48 hours of age were analyzed by Multivariate Hotelling's T2 analysis of 99 relevant facial landmark triangles. Morphometric analysis discriminated unique asymmetries in groups of certain illicit exposure(s). Neonates with multiple prenatal exposures had fewer facial landmark dislocation(s) compared to single exposures. Deviation from normal facial features has the potential to be used as a screening tool for prenatal exposure to some illicit substances.

RNA Trans-Splicing Targeting Endogenous ß-Globin Pre-Messenger RNA in Human Erythroid Cells.

Sickle cell disease results from a point mutation in exon 1 of the ß-globin gene (total 3 exons). Replacing sickle ß-globin exon 1 (and exon 2) with a normal sequence by trans-splicing is a potential therapeutic strategy. Therefore, this study sought to develop trans-splicing targeting ß-globin pre-messenger RNA among human erythroid cells. Binding domains from random ß-globin sequences were comprehensively screened. Six candidates had optimal binding, and all targeted intron 2. Next, lentiviral vectors encoding RNA trans-splicing molecules were constructed incorporating a unique binding domain from these candidates, artificial 5' splice site, and γ-globin cDNA, and trans-splicing was evaluated in CD34+ cell-derived erythroid cells from healthy individuals. Lentiviral transduction was efficient, with vector copy numbers of 9.7 to 15.3. The intended trans-spliced RNA product, including exon 3 of endogenous ß-globin and γ-globin, was detected at the molecular level. Trans-splicing efficiency was improved to 0.07-0.09% by longer binding domains, including the 5' splice site of intron 2. In summary, screening was performed to select efficient binding domains for trans-splicing. Detectable levels of trans-splicing were obtained for endogenous ß-globin RNA in human erythroid cells. These methods provide the basis for future trans-splicing directed gene therapy.

Apoptotic Effects of Novel Dithiocarbamate Analogs of Emetine in Prostate Cancer Cell Lines.

BACKGROUND/AIM: Prostate cancer is one of the leading causes of death in American males. Emetine, a naturally-derived alkaloid from the Ipecacuanha plant, has been shown to have potential for anti-tumorigenic effects for cancer treatments. The objective of this study was to characterize novel emetine dithiocarbamate (EMTDTC) analogs for potent anti-tumorigenic activity with minimal toxicity to normal prostate cells and identify targeted apoptotic regulatory genes. The leading key compounds, EMTDTC-55 and EMTDTC-56 were studied. MATERIALS AND METHODS: Established methods of cell flow cytometry were used to analyze apoptotic potential in prostate cancer cell lines (DU145, PC3 and LNCaP) and real time-polymerase chain reaction (PCR) for identifying key genes mediating apoptosis. RESULTS: The effect of EMTDTC-55 on DU145, LNCaP and PC3 revealed significant anti-tumorigenic activities. Both compounds showed highly significant apoptotic potential on days 3 and 5 in the prostate cancer cells. Key apoptotic genes were differentially regulated suggestive of cell-cycle arrest and apoptotic induction in androgen-independent cell lines, DU145 and PC3, by both compounds. However, in the androgen-dependent cell line LNCaP, cells were marginally affected by EMTDTC-55, but significant apoptosis was observed by EMTDTC-56 leading to cell-cycle arrest. CONCLUSION: Both dithiocarbamate compounds EMTDTC-55 and EMTDTC-56 have significant chemotherapeutic potential in moderately metastatic DU145 and highly metastatic PC3 cells.

Efficient transduction of human hematopoietic repopulating cells with a chimeric HIV1-based vector including SIV capsid.

Innate immune factors, such as TRIM5α and cyclophilin A (CypA), act as a major restriction factor of retroviral infection among species. When HIV1 infects human cells, HIV1 capsid binds to human CypA to escape from human TRIM5α restriction. However, in rhesus cells, the mismatch between HIV1 capsid and rhesus CypA is recognized by rhesus TRIM5α to reduce HIV1 infectivity through proteasomal degradation. To circumvent this block, we previously developed a chimeric HIV1 vector (χHIV) that substituted HIV1 capsid with SIV capsid, and it significantly increased transduction efficiency for nonhuman primate cells. In this study, we evaluated whether the χHIV vector efficiently transduces human cells, and the transduction efficiency might increase by a CypA inhibitor (cyclosporine) and a proteasome inhibitor (MG132). The χHIV vector could transduce human CD34⁺ cells, as efficiently as the HIV1 vector, in vitro and in xenograft mice, even in the mismatch between SIV capsid and human CypA. Cyclosporine decreased transduction efficiency with the HIV1 vector, whereas it slightly increased transduction efficiency with the χHIV vector in human CD34⁺ cells. MG132 increased transduction efficiency with both χHIV and HIV1 vectors in the same manner. However, MG132 was toxic to human CD34⁺ cells at high concentrations, and both drugs had a small range of effective dosage. These findings demonstrate that both χHIV and HIV1 vectors have similar transduction efficiency for human hematopoietic repopulating cells, suggesting that the χHIV vector escapes from TRIM5α restriction, which is independent of human CypA.

Chicken HS4 insulators have minimal barrier function among progeny of human hematopoietic cells transduced with an HIV1-based lentiviral vector.

Position effects limit the curative potential of gene transfer strategies for the hemoglobinopathies by inducing clonal variability of transgene expression. We evaluated the mitigating effects of the chicken hypersensitivity site 4 (HS4) insulator among lentiviral vector-transduced human hematopoietic cells. We constructed various lentiviral vectors using a green fluorescent protein (GFP) reporter under the control of a reverse-oriented murine stem cell virus (MSCV)-long-term repeat (LTR) promoter or a reverse-oriented ß-globin expression cassette. A full-length HS4, a tandem HS4 core, and a single core insulator were inserted into the 3' LTR in both forward and reverse orientation. All but the reverse single core insulator significantly decreased titers. All reduced %GFP without increasing mean fluorescence intensity (MFI) among erythroid progeny of transduced human CD34(+) cells. A lower coefficient of variation (CV) was observed only among progeny of the full-length vector-transduced cells, yet a fivefold reduction in transduction efficiency was observed. In xenografted mice, the single core insulator decreased both the %GFP and the MFI at 4 and 8 weeks after transplantation with no difference in CVs. These data demonstrate that the inclusion of HS4 insulator elements lowers viral titers, reduces efficiency of transduction, and produces minimal effects on transgene expression among human hematopoietic cells in vitro and in vivo.

5% dimethyl sulfoxide (DMSO) and pentastarch improves cryopreservation of cord blood cells over 10% DMSO.

BACKGROUND: Cell number and viability are important in cord blood (CB) transplantation. While 10% dimethyl sulfoxide (DMSO) is the standard medium, adding a starch to freezing medium is increasingly utilized as a cytoprotectant for the thawing process. Similar to hetastarch, pentastarch has the advantages of faster renal clearance and less effect on the coagulation system. STUDY DESIGN AND METHODS: We compared a lower DMSO concentration (5%) containing pentastarch with 10% DMSO and performed cell viability assay, colony-forming units (CFUs), and transplantation of CB cells in NOD/SCID IL2Rγ(null) mice. RESULTS: CB cells in 5% DMSO/pentastarch had similar CD34+, CD3+, and CD19+ cell percentages after thawing as fresh CB cells. CB cells in 5% DMSO/pentastarch had higher viability (83.3±9.23%) than those frozen in 10% DMSO (75.3±11.0%, p<0.05). We monitored cell viability postthaw every 30 minutes. The mean loss in the first 30 minutes was less in the 5% DMSO/pentastarch group. At the end of 3 hours, the viability decreased by a mean of 7.75% for the 5% DMSO/pentastarch and 17.5% for the 10% DMSO groups. CFUs were similar between the two cryopreserved groups. Frozen CB cells engrafted equally well in IL2Rγ(null) mice compared to fresh CB cells up to 24 weeks, and CB cells frozen in 5% DMSO/pentastarch engrafted better than those in 10% DMSO. CONCLUSION: Our data indicate that the lower DMSO concentration with pentastarch represents an improvement in the CB cryopreservation process and could have wider clinical application as an alternate freezing medium over 10% DMSO.

Development of a human immunodeficiency virus type 1-based lentiviral vector that allows efficient transduction of both human and rhesus blood cells.

Human immunodeficiency virus type 1 (HIV-1) vectors transduce rhesus blood cells poorly due to a species-specific block by TRIM5alpha and APOBEC3G, which target HIV-1 capsid and viral infectivity factor (Vif), respectively. We sought to develop a lentiviral vector capable of transducing both human and rhesus blood cells by combining components of both HIV-1 and simian immunodeficiency virus (SIV), including SIV capsid (sCA) and SIV Vif. A chimeric HIV-1 vector including sCA (chiHIV) was superior to the conventional SIV in transducing a human blood cell line and superior to the conventional HIV-1 vector in transducing a rhesus blood cell line. Among human CD34(+) hematopoietic stem cells (HSCs), the chiHIV and HIV-1 vectors showed similar transduction efficiencies; in rhesus CD34(+) HSCs, the chiHIV vector yielded superior transduction rates. In in vivo competitive repopulation experiments with two rhesus macaques, the chiHIV vector demonstrated superior marking levels over the conventional HIV-1 vector in all blood lineages (first rhesus, 15 to 30% versus 1 to 5%; second rhesus, 7 to 15% versus 0.5 to 2%, respectively) 3 to 7 months postinfusion. In summary, we have developed an HIV-1-based lentiviral vector system that should allow comprehensive preclinical testing of HIV-1-based therapeutic vectors in the rhesus macaque model with eventual clinical application.