Depletion of CD34+ CD4+ cells in bone marrow from HIV-1-infected individuals.

Pancytopenia as a consequence of bone marrow abnormalities is commonly seen in HIV-infected individuals. To examine the effect that HIV-1 has on hematopoietic cells, we compared hematopoietic properties of bone marrow samples from HTV+ patients at various stages of disease with bone marrow samples from uninfected donors. While the absolute number of recovered CD34+ cells and the cloning efficiency of these cells did not differ significantly in HIV+ donors, the percentage of CD34+ CD4+ cells was significantly depleted in late-stage HIV+ patients. We observed a direct correlation between the numbers of CD34+ CD4+ cells in the bone marrow and the peripheral CD4 count. Further characterization of the CD34+ CD4+ subpopulation demonstrated that these cells expressed lower levels of HLA-DR on their surface compared with CD34+ CD4- cells, suggesting an immature phenotype. We also found evidence for expression of HIV-1 coreceptors CXCR-4 and CKR-5 message and protein in CD34+ bone marrow cells. While this finding suggested that hematopoietic cells might be susceptible to HIV infection at an early stage of maturation, thus affecting different cell lineages as they matured, we did not find any evidence for infection of HIV in these cells. These data suggest that HIV affects early hematopoietic progenitor cells either directly or indirectly, and in particular CD34+ CD4+ cells. This finding has important implications for disease pathogenesis and for application of gene therapy approaches that use CD34+ hematopoietic cells.

Comparison of retroviral-mediated gene transfer into cultured human CD34+ hematopoietic progenitor cells derived from peripheral blood, bone marrow, and fetal umbilical cord blood.

Genetic alteration of stem cells ex vivo followed by bone marrow transplantation could potentially be used in the treatment of numerous diseases and malignancies. However, there are many unanswered questions as to the best source of hematopoietic cells for long-term reengraftment and the most effective way to introduce foreign genes into this target cell. We have compared retroviral-mediated gene transfer into CD34+-enriched cells derived from peripheral blood (PB), bone marrow (BM), or fetal umbilical cord blood (CB). Cells from all three sources that had been expanded ex vivo in the presence of stem cell factor (SCF), interleukin-3 (IL-3), IL-6, and granulocyte colony-stimulating factor (G-CSF) showed transduction efficiencies ranging from 5-45%, as measured by acquisition of G418 resistance. The average efficiencies of gene transfer from multiple experiments for PB, BM, and CB were not statistically different. To determine the effect of ex vivo expansion on gene transfer into CB CD34+ cells, we compared the transduction efficiencies of cells exposed to virus immediately after harvest and CD34 selection or after 6 days of culture CD34+ CB cells were more effectively transduced after expansion in culture, showing gene transfer efficiencies 3- to 5-fold higher on day 6 compared with day 0. Last, we examined retroviral transduction via spinoculation of CB CD34+ cells and found it to be approximately as effective as our standard transduction with no significant loss of cell viability as measured by colony formation in semi-solid medium.

Evaluation of hyperbaric oxygen as a chemosensitizer in the treatment of epithelial ovarian cancer in xenografts in mice.

BACKGROUND: Resistance to chemotherapy is common in bulky hypoxic tumors such as epithelial ovarian cancer. Hyperbaric oxygen (HBO) oxygenates hypoxic tissues and promotes neovascularization. These unique properties of HBO may help overcome chemotherapy resistance by increasing both tumor perfusion and cellular sensitivity. This study was undertaken to determine if HBO increases the response of epithelial ovarian cancer to cisplatin chemotherapy. METHODS: In Phase I, 64 nu/nu mice were divided into four groups and subcutaneously inoculated with cells from the A2780 human epithelial ovarian cancer cell line. Group 1 served as controls. Group 2 received weekly intraperitoneal cisplatin (3.15 mg/kg). Group 3 was exposed to HBO (dives) at 2.4 atmospheres absolute pressure for 90 minutes, 7 days a week. Group 4 received both cisplatin and HBO. In Phase II, 72 mice were divided into two groups and similarly inoculated. Both groups received weekly intraperitoneal cisplatin (2.5 mg/kg). Group 1 was not exposed to HBO. Group 2 was exposed to HBO for 5 days a week. RESULTS: Dramatic tumor neovascularization was found in tumors of mice exposed to HBO (P = 0.0001). There was significant (P = 0.014) tumor growth retardation in Phase I for mice receiving both cisplatin and HBO compared with those treated with cisplatin alone. This significance was noted after just two doses of cisplatin but subsequently lost due to reduced numbers of mice. In Phase II, neovascularization was detectable after 10 HBO treatments (2 weeks) and was maximal after 15 treatments (3 weeks). CONCLUSIONS: Hyperbaric oxygen increases vascularity in bulky tumors such as epithelial ovarian cancer. There appears to be a relationship between increased vascularity and enhanced response to chemotherapy that merits further investigation.

In vitro growth effects of colony-stimulating factors in ovarian cancer.

Human recombinant colony-stimulating factors may be used to treat or prevent neutropenia caused by marrow toxic chemotherapeutic agents administered to patients with cancer. Despite their common clinical use, little is known about the potential adverse effects that these cytokines may have on the growth of malignant cells. Indeed, several in vitro reports have indicated that colony-stimulating factors may act as stimulating growth factors in some human malignancies. To evaluate these effects in ovarian cancer, we investigated the possible growth effects of granulocyte colony-stimulating factor (G-CSF/Filgrastim) and granulocyte-macrophage colony-stimulating factors (GM-CSF/Sargramostim) on four established ovarian cancer cell lines, as well as five primary ovarian cancer cultures over a wide range of pharmacologic doses. Cell viability was measured by an ATP bioluminescence assay and expressed as a percentage of untreated control cultures. G-CSF showed no growth-stimulating effects in any of the four established cell lines tested. In the OVCAR-3 cell line, a decrease in growth (> 10%) was seen at 10, 100, and 1000 ng/ml after 5 days of continuous treatment. In the same cell line, GM-CSF caused an increase (> 10%) in growth at the same doses. However, these changes did not demonstrate statistical significance in a dose-dependent fashion. In the five primary cultures treated with G-CSF, only one demonstrated statistically significant increases in growth in a dose-dependent manner. GM-CSF treatment had no significant growth alterations in these same five primary cultures. These results would suggest that colony-stimulating factors may act as growth factors in some but not all ovarian cancer cells. Further investigations into the receptor status of ovarian cancer cells for these cytokines are underway to clarify this issue.