Hematologic and immunologic functions were examined in 19 HIV-negative infants of HIV-positive mothers and 19 control infants of HIV-negative mothers. Control infants were selected to match for gestational age, weight, and mode of delivery. Cord blood was obtained from all infants and used for flow cytometric determination of lymphocyte subsets, including the naive CD4 count. Furthermore, to determine thymic output, cord blood mononuclear cells were used for determination of T-cell receptor excision circles (TRECs). Evaluation of progenitor cell function was IntroductionVertical transmission of HIV from an HIV-positive mother to her infant occurs in 15% to 25% of pregnancies if no precautions are taken. However, the risk of vertical transmission of HIV has been dramatically reduced with the introduction of antiretroviral treatment in combination with delivery by elective cesarean section and avoidance of breastfeeding. 1,2 Although infants of HIV-positive mothers are rarely HIVinfected, they may have been exposed to HIV proteins or even HIV particles during fetal life, as indicated by the presence of HIV-specific T cells, immune activation, and positive HIV polymerase chain reaction (PCR) found in HIV-exposed infants. [3][4][5][6][7] Thus, a recent study demonstrated high frequencies of HIV-specific CD4 ϩ cells and a lower frequency of HIV-specific CD8 ϩ cells, indicating transplacental diffusion of HIV-soluble proteins. 8 HIV particles as well as HIV proteins are known to inhibit progenitor cell function and to cause progenitor cell apoptosis which, in turn, would lead to both hematologic and immunologic deficiencies in the infants. [9][10][11][12][13][14][15][16][17][18] Furthermore, cytokine imbalance between Th1-and Th2-type cytokines has been suggested in HIV-positive individuals. [19][20][21] Such an imbalance in pregnant HIV-positive women might also cause cytokine imbalance in the fetus, resulting in immunologic deficiencies. Finally, pregnant HIV-positive women are commonly treated with antiretroviral therapy including zidovudine (AZT), and AZT is known to inhibit bone marrow functions. 22 The present study was conducted to determine if HIV-negative infants of HIV-positive mothers have immune deficiencies as determined by CD4 and CD8 counts in cord blood. Furthermore, thymic output was evaluated by determination of CD4 ϩ and CD8 ϩ cells with naive phenotype (coexpression of CD45RA) and determination of T-cell receptor excision circles (TRECs). Evidence of reduced thymic output was found and, to determine if impaired progenitor cell function might contribute to this, colony-forming cell (CFC) assays were performed to examine the function of myeloid progenitors, and fetal thymic organ cultures (FTOCs) were done to examine the function of T-cell progenitors. Recently, correlation between lymphocyte proliferation and expression of the early activation marker CD69 has been shown. 23,24 To determine if immune activation in infants of HIV-positive mothers might contribute to the lower level of naive CD4 ϩ cel...

Sorting of live infected cells can be performed safely and with no deleterious effects on vector expression using the modified FACS Vantage instrument.

This randomized, placebo-controlled trial examine the long-term effect of granulocyte colony-stimulating factor (G-CSF) on absolute numbers of CD34+ progenitor cells and progenitor cell function in human immunodeficiency virus (HIV)-infected patients. G-CSF (300 microg filgrastim) or placebo was given three times weekly for 12 weeks to 30 HIV-infected patients that had been treated with HAART for at least 24 weeks and not yet achieved CD4 counts above 350 CD4+ cells/microl. Blood samples were collected at weeks 0, 2, 4, 8, and 12, and again 12 weeks after termination of the G-CSF treatment. Significant increase in absolute numbers of circulating CD34+ cells was detected in the treatment group (P = 0.006). The function of progenitor cells was examined in vitro using a colony-forming unit (CFU) assay, and increase in the number of CFU/ml was detected (P = 0.005). In order to estimate the effect of G-CSF on in vivo function of progenitors the white-blood count was determined. Significant increase in white-blood count was found (P < 0.001), while hemoglobin and platelet count decreased (P = 0.001 and P = 0.013, respectively). Significant increase in the CD4 count occurred, but correlation between the numbers of progenitors and the CD4 count was not found. These data suggest that G-CSF mainly increases the number and differentiation of myeloid progenitors.

Before stem cell gene therapy can be considered for clinical applications, problems regarding cytokine prestimulation remain to be solved. In this study, a retroviral vector carrying the genes for the enhanced version of green fluorescent protein (EGFP) and neomycin resistance (neo(r)) was used for transduction of CD34+ cells. The effect of cytokine prestimulation on transduction efficiency and the population of uncommitted CD34+CD38- cells was determined. CD34+ cells harvested from umbilical cord blood were kept in suspension cultures and stimulated with combinations of the cytokines stem cell factor (SCF), FLT3 ligand, interleukin-3 (IL-3), IL-6, and IL-7 prior to transduction. Expression of the two genes was assessed by flow cytometry and determination of neomycin-resistant colonies in a selective colony-forming unit (CFU) assay, respectively. The neomycin resistance gene was expressed in a higher percentage of cells than the EGFP gene, but there seemed to be a positive correlation between expression of the two genes. The effect of cytokine prestimulation was therefore monitored using EGFP as marker for transduction. When SCF was compared to SCF in combination with more potent cytokines, highest transduction efficiency was found with SCF and IL-3 and IL-6 (5.05% +/- 0.80 versus 2.66% +/- 0.53 with SCF alone, p = 0.04). However, prestimulation with SCF in combination with IL-3 and IL-6 also reduced the percentage of CD34+ cells (p = 0.02). Then, prestimulation with SCF and FLT3 ligand was compared. Significant difference in transduction efficiency was not found. Interestingly, FLT3 ligand seemed to preserve the population of CD34+CD38- cells compared to SCF (16.56% +/- 2.02 versus 9.39% +/- 2.35, p = 0.03). In conclusion, prestimulation with potent cytokine combinations increased the transduction efficiency, but reduced the fraction of CD34+ cells. Importantly, the use of FLT3 ligand seemed to preserve the population of uncommitted cells.