Hematopoietic-stem-cell-based gene therapy for HIV disease

Although combination antiretroviral therapy can dramatically reduce the circulating viral load in those infected with HIV, replication-competent virus persists. To eliminate the need for indefinite treatment, there is growing interest in creating a functional HIV-resistant immune system through the use of gene-modified hematopoietic stem cells (HSCs). Proof of concept for this approach has been provided in the instance of an HIV-infected adult transplanted with allogeneic stem cells from a donor lacking the HIV coreceptor, CCR5. Here, we review this and other strategies for HSC-based gene therapy for HIV disease.     

Dog ownership during pregnancy, maternal activity, and obesity: a cross-sectional study

The Avon Longitudinal Study of Parents and Children (ALSPAC) is an observational study of 14,273 UK pregnant singleton mothers in 1990/1991. We examined outcomes of self report of strenuous activity (hours per week) at 18 and 32 weeks of gestation, hours spent in leisure-time physical activities and types, and pre-pregnancy body mass index (BMI); overweight status was defined as pre-pregnancy BMI≥25 and obesity BMI≥30. Pet ownership and activity data were reported for 11,466 mothers. Twenty-five percent of mothers owned at least one dog. There was a positive relationship between participation in activity at least once a week and dog ownership (at 18 weeks, Odds ratio 1.27, 95% confidence interval 1.11-1.44, P<0.001). Dog owners were 50% more likely to achieve the recommended 3 hours activity per week, equivalent to 30 minutes per day, most days of the week (1.53, 1.35-1.72, P<0.001). Dog owners were also more likely to participate in brisk walking activity than those who did not have a dog (compared to no brisk walking 2-6 hrs per week 1.43, 1.23 to 1.67, P<0.001; 7+ hrs per week 1.80, 1.43 to 2.27, P<0.001). However, no association was found with any other types of activities and there was no association between dog ownership and weight status. During the time period studied, pregnant women who had dogs were more active, through walking, than those who did not own dogs. As walking is a low-risk exercise, participation of pregnant women in dog walking activities may be a useful context to investigate as part of a broader strategy to improve activity levels in pregnant women.     

Effect of cell density on metabolism in isolated rat hepatocytes

Freshly isolated rat hepatocytes show many changes in metabolic activities as a function of cell density in the incubation flask. Fatty acid synthesis, cholesterol synthesis, general protein synthesis, and rates of accumulation of pyruvate, lactate, citrate, acetyl-CoA, acetoacetate and beta-hydroxybutyrate decrease as the cell density increases between 1 mg/ml and 60 mg/ml. Glucose release only decreases between 1-5 mg/ml and the concentration of ATP does not vary at any density. There is a small increase in the lactate/pyruvate ratio and a dramatic decrease in the beta-hydroxybutyrate/acetoacetate ratio with increasing cell concentration. When cells at 8 or 28 mg/ml were incubated with added lactate and pyruvate, or alanine, a two fold increase in fatty acid synthesis and 50% decrease in cholesterol synthesis were observed as compared to rates with endogenous substrate. With added glucose the synthetic rates were similar to those obtained with endogenous substrate. However, regardless of the type of substrate used, the less dense cells gave rates up to three times greater than that of the more dense cells. When conditioned medium isolated after incubation of cells at high density was added to the less dense cells, a decrease in the rates of fatty acid synthesis and cholesterol synthesis was observed in the less dense cells; however, when medium from the less dense cells after incubation for the same period was added to the more dense cells, there was no significant change in fatty acid or cholesterol synthesis. These results suggest that a factor may be released into the medium of incubating hepatocytes that progressively inhibits certain metabolic processes as the cell density increases.     

Human T cells in the SCID-hu mouse are phenotypically normal and functionally competent

SCID-hu mice are heterochimeric animals that are constructed by transplanting human fetal thymus (Thy), liver (Liv), and/or lymph nodes into congenitally immunodeficient C.B-17 scid/scid (SCID) mice. Sensitive and specific two-color flow cytometric assays were used to evaluate human lymphocytes from peripheral blood of SCID-hu mice. Kinetic studies presented in this report show long term T lymphopoiesis in SCID-hu mice. Approximately one-half of SCID-hu mice constructed with Thy and Liv tissue develop detectable levels of circulating human T cells by 4 mo after transplantation. The average level of circulating human cells in SCID-hu mice is generally less than 2% of the total lymphoid cells in the peripheral blood of these mice. Some SCID-hu mice with as high as 13% human lymphocytes, however, have been detected. Nearly all human cells in the peripheral blood of SCID-hu mice are CD3+ cells that express TCR-alpha beta. The percentages of gamma delta+, CD4+, CD8+, CD25+, CD69+, and Leu-8+ cells among CD45+ cells in SCID-hu blood are similar to the levels found in adult peripheral blood. On average, 74% of SCID-hu T cells express CD45RA and 18% express CD29. Functional studies demonstrate that cells from SCID-hu Thy/Liv grafts or human T cells from SCID-hu peripheral blood are functionally competent to respond to mitogens or allogeneic human cells in vitro. They are similar to fetal thymocytes or adult T cells, respectively, in these responses. These studies demonstrate that the SCID-hu mouse is a useful model for the analysis of human immune differentiation and function in vivo.     

Complex regulation of TGF beta expression by retinoic acid in the vitamin A-deficient rat

We report the results of a histochemical study, using polyclonal antipeptide antibodies to the different TGF beta isoforms, which demonstrates that retinoic acid regulates the expression of TGF beta 2 in the vitamin A-deficient rat. Basal expression of TGF beta 2 diminished under conditions of vitamin A deficiency. Treatment with retinoic acid caused a rapid and transient induction of TGF beta 2 and TGF beta 3 in the epidermis, tracheobronchial and alveolar epithelium, and intestinal mucosa. Induction of TGF beta 1 expression was also observed in the epidermis. In contrast to these epithelia, expression of the three TGF beta isoforms increased in vaginal epithelium during vitamin A deficiency, and decreased following systemic administration of retinoic acid. Our results show for the first time the widespread regulation of TGF beta expression by retinoic acid in vivo, and suggest a possible mechanism by which retinoics regulate the functions of both normal and pre-neoplastic epithelia.     

The effect of Ku on telomere replication time is mediated by telomere length but is independent of histone tail acetylation

DNA replication in Saccharomyces cerevisiae proceeds according to a temporal program. We have investigated the role of the telomere-binding Ku complex in specifying late replication of telomere-proximal sequences. Genome-wide analysis shows that regions extending up to 80 kb from telomeres replicate abnormally early in a yku70 mutant. We find that Ku does not appear to regulate replication time by binding replication origins directly, nor is its effect on telomere replication timing mediated by histone tail acetylation. We show that Ku instead regulates replication timing through its effect on telomere length, because deletion of the telomerase regulator Pif1 largely reverses the short telomere defect of a yku70 mutant and simultaneously rescues its replication timing defect. Consistent with this conclusion, deleting the genome integrity component Elg1 partially rescued both length and replication timing of yku70 telomeres. Telomere length-mediated control of replication timing requires the TG(1-3) repeat-counting component Rif1, because a rif1 mutant replicates telomeric regions early, despite having extended TG(1-3) tracts. Overall, our results suggest that the effect of Ku on telomere replication timing results from its impact on TG(1-3) repeat length and support a model in which Rif1 measures telomere repeat length to ensure that telomere replication timing is correctly programmed.     

Composition and Function of T Cell Subpopulations Are Slow to Change Despite Effective Antiretroviral Treatment of HIV Disease

The ability to reconstitute a normal immune system with antiretroviral therapy in the setting of HIV infection remains uncertain. This study aimed to characterize quantitative and qualitative aspects of various T cell subpopulations that do not improve despite effective ART. CD4∶CD8 ratio was evaluated in HIV-infected subjects with viral loads >10,000 copies/µl (“non-controllers”, n = 42), those with undetectable viral loads on ART (“ART-suppressed”, n = 53), and HIV-uninfected subjects (n = 22). In addition, T cell phenotype and function were examined in 25 non-controllers, 18 ART-suppressed, and 7 HIV-uninfected subjects. CD4∶CD8 ratio in non-controllers, ART-suppressed, and HIV-uninfected subjects was 0.25, 0.48, and 1.95 respectively (P<0.0001 for all comparisons). The increased ratio in ART-suppressed compared to non-controllers was driven by an increase of CD4+ T cells, with no change in the expanded CD8+ T cell population. Expansion of differentiated (CD28−CD27−CD45RA+/−CCR7−) T cell subpopulations persisted despite ART and minimal changes were noted in naïve T cell frequencies over time. Increased number of CD8+CD28− T cells and increased CD8+ CMV-specific T cell responses were associated with a decreased CD4∶CD8 ratio. Measures of T cell function demonstrated persistence of high frequencies of CD8+ T cells producing IFN–γ. Lastly, though all CD8+ subpopulations demonstrated significantly lower Ki67 expression in ART-suppressed subjects, CD4+ T cell subpopulations did not consistently show this decrease, thus demonstrating different proliferative responses in the setting of T cell depletion. In summary, this study demonstrated that CD4∶CD8 ratios remained significantly decreased and naïve T cell numbers were slow to increase despite long-term viral suppression on ART. In addition, there is a evidence of differential regulation of the CD4+ and CD8+ T cell subpopulations, suggesting independent homeostatic regulation of the two compartments.     

The Immunologic Effects of Mesalamine in Treated HIV-Infected Individuals with Incomplete CD4+ T Cell Recovery: A Randomized Crossover Trial

The anti-inflammatory agent, mesalamine (5-aminosalicylic acid) has been shown to decrease mucosal inflammation in ulcerative colitis. The effect of mesalamine in HIV-infected individuals, who exhibit abnormal mucosal immune activation and microbial translocation (MT), has not been established in a placebo-controlled trial. We randomized 33 HIV-infected subjects with CD4 counts <350 cells/mm³ and plasma HIV RNA levels <40 copies/ml on antiretroviral therapy (ART) to add mesalamine vs. placebo to their existing regimen for 12 weeks followed by a 12 week crossover to the other arm. Compared to placebo-treated subjects, mesalamine-treated subjects did not experience any significant change in the percent CD38+HLA-DR+ peripheral blood CD4+ and CD8+ T cells at week 12 (P  = 0.38 and P  = 0.63, respectively), or in the CD4+ T cell count at week 12 (P  = 0.83). The percent CD38+HLA-DR+ CD4+ and CD8+ T cells also did not change significantly in rectal tissue (P  = 0.86, P  = 0.84, respectively). During the period of mesalamine administration, plasma sCD14, IL-6, D-dimer, and kynurenine to tryptophan ratio were not changed significantly at week 12 and were similarly unchanged at week 24. This study suggests that, at least under the conditions studied, the persistent immune activation associated with HIV infection is not impacted by the anti-inflammatory effects of mesalamine.

Trial Registration

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