ALBERT

Description: The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

Description: In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV- 1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.

Description: Granulocyte colony-stimulating factor (G-CSF) has been shown to be effective in clinical trials for reducing the period of neutropenia after chemotherapy. In this study, we compared the timing for initiating G-CSF administration after chemotherapy with the duration of neutropenia and hematopoietic regeneration. Nonhuman primates treated with high-dose chemotherapy (mechloroethamine, 1.5 mg/kg, intravenously) and not administered G-CSF therapy experienced 8 days of neutropenia (absolute neutrophil count [ANC] less than 1,000/mm3) and had an ANC nadir of 124 +/- 64/mm3 at day 7. Monkeys receiving G-CSF (5 micrograms/kg/d, subcutaneously) began treatment on either days 1, 3, 5, or 7 after chemotherapy. Monkeys treated with G-CSF had an earlier ANC recovery and the number of days with an ANC less than 500/mm3 and ANC less than 1,000/mm3 was reduced by approximately 50% in all treatment strategies. All G-CSF-treated animals, irrespective of the time that G-CSF was initiated, reached an ANC of 10,000/mm3 on day 13 +/- 1 day after chemotherapy. These results demonstrated that the duration of G-CSF therapy was almost twice as long for monkeys treated on day 1 as it was for monkeys that received therapy beginning on day 7. A comparison of the results for all treated monkeys identified a distinct difference in the responses of monkeys treated on day 1 from that of animals treated with G-CSF at later times. G-CSF initiated 1 day after chemotherapy led to an earlier onset of neutropenia and a more rapid and augmented recovery of myeloid progenitor cells in the peripheral blood when compared with control and delayed therapy groups. This study demonstrates that neutropenia due to a single dose of mechloroethamine can be equally reduced with both early and delayed initiation of G-CSF. Further, initiating G-CSF therapy after 7 days required approximately 50% less days of therapy to reach an appropriate termination point. The applicability of these findings to other chemotherapy regimens and for repeated cycles is uncertain and needs to be further evaluated. This is a US government work. There are no restrictions on its use.

Description: In this study, we examined the potential role of the human immunodeficiency virus (HIV) tat protein in causing the hematopoietic abnormalities frequently observed in HIV-infected individuals. Recombinant tat (r-tat) protein, at concentrations up to 10 micrograms/mL, did not display any stimulatory or inhibitory effect on the survival/proliferative capacity of CD34+ hematopoietic progenitor cells, purified from normal bone marrow (BM). However, exposure of r-tat protein (at concentrations between 10 ng/mL and 10 micrograms/mL) to enriched normal BM macrophages induced the production of a factor(s) in conditioned media that inhibited the in vitro growth of CD34+ cells in liquid cultures and of immature hematopoietic progenitors (day 14 colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) in semisolid assays. Pre-exposure of r-tat protein with a monoclonal neutralizing anti-tat antibody completely abrogated the inhibitory activity present in BM macrophage culture supernatants. The main factor responsible for this suppressive activity was transforming growth factor-beta 1 (TGF-beta 1), as shown by the ability of a polyclonal anti-TGF-beta 1 neutralizing antibody to almost completely reverse the suppressive effect of BM macrophage supernatants on CD34+ cells. TGF-beta 1 bioassays showed that exposure of r-tat protein to BM macrophages significantly increased the levels of both active and latent forms of TGF-beta 1. These results indicate that the production of TGF-beta 1, one of the most potent negative regulator of hematopoiesis, is increased by HIV tat protein and that such increase could contribute to the derangement of the hematopoietic system in HIV-infected individuals.

Description: The human CD34 surface antigen is selectively expressed on hematopoietic stem/progenitor cells, suggesting that it plays an essential role in early hematopoiesis. Using a 1.5-kb partial human CD34 cDNA sequence, RNA-polymerase chain reaction (PCR), and rapid amplification of cDNA ends (RACE) methods, we cloned and sequenced the full-length (2.65 kb) cDNA. The cDNA encodes a type I transmembrane protein with no obvious homology to other known proteins. The entire CD34 gene of 28 kb was cloned, and the coding sequences mapped to eight exons. Mapping of the 5′ termini of mRNAs by 5′-RACE and RNAase protection analyses has indicated that the human CD34 gene uses multiple transcription initiation sites. Analysis of the upstream regulatory sequences revealed the absence of TATA and CAAT box sequences, and the presence of myb, myc, and ets-like DNA binding motifs. We have identified significant homology between human and mouse CD34 genes in 5′ and 3′ untranslated regions, amino acid coding sequences, and 5′ flanking sequences. This investigation of the CD34 gene should facilitate study of the function and regulation of this stem cell antigen.

Description: Granulocyte colony-stimulating factor (G-CSF) has been shown to be effective in clinical trials for reducing the period of neutropenia after chemotherapy. In this study, we compared the timing for initiating G-CSF administration after chemotherapy with the duration of neutropenia and hematopoietic regeneration. Nonhuman primates treated with high-dose chemotherapy (mechloroethamine, 1.5 mg/kg, intravenously) and not administered G-CSF therapy experienced 8 days of neutropenia (absolute neutrophil count [ANC] less than 1,000/mm3) and had an ANC nadir of 124 +/- 64/mm3 at day 7. Monkeys receiving G-CSF (5 micrograms/kg/d, subcutaneously) began treatment on either days 1, 3, 5, or 7 after chemotherapy. Monkeys treated with G-CSF had an earlier ANC recovery and the number of days with an ANC less than 500/mm3 and ANC less than 1,000/mm3 was reduced by approximately 50% in all treatment strategies. All G-CSF-treated animals, irrespective of the time that G-CSF was initiated, reached an ANC of 10,000/mm3 on day 13 +/- 1 day after chemotherapy. These results demonstrated that the duration of G-CSF therapy was almost twice as long for monkeys treated on day 1 as it was for monkeys that received therapy beginning on day 7. A comparison of the results for all treated monkeys identified a distinct difference in the responses of monkeys treated on day 1 from that of animals treated with G-CSF at later times. G-CSF initiated 1 day after chemotherapy led to an earlier onset of neutropenia and a more rapid and augmented recovery of myeloid progenitor cells in the peripheral blood when compared with control and delayed therapy groups. This study demonstrates that neutropenia due to a single dose of mechloroethamine can be equally reduced with both early and delayed initiation of G-CSF. Further, initiating G-CSF therapy after 7 days required approximately 50% less days of therapy to reach an appropriate termination point. The applicability of these findings to other chemotherapy regimens and for repeated cycles is uncertain and needs to be further evaluated. This is a US government work. There are no restrictions on its use.

Description: In this study, we examined the potential role of the human immunodeficiency virus (HIV) tat protein in causing the hematopoietic abnormalities frequently observed in HIV-infected individuals. Recombinant tat (r-tat) protein, at concentrations up to 10 micrograms/mL, did not display any stimulatory or inhibitory effect on the survival/proliferative capacity of CD34+ hematopoietic progenitor cells, purified from normal bone marrow (BM). However, exposure of r-tat protein (at concentrations between 10 ng/mL and 10 micrograms/mL) to enriched normal BM macrophages induced the production of a factor(s) in conditioned media that inhibited the in vitro growth of CD34+ cells in liquid cultures and of immature hematopoietic progenitors (day 14 colony-forming unit-granulocyte-macrophage, burst-forming unit-erythroid, and colony-forming unit-megakaryocyte) in semisolid assays. Pre-exposure of r-tat protein with a monoclonal neutralizing anti-tat antibody completely abrogated the inhibitory activity present in BM macrophage culture supernatants. The main factor responsible for this suppressive activity was transforming growth factor-beta 1 (TGF-beta 1), as shown by the ability of a polyclonal anti-TGF-beta 1 neutralizing antibody to almost completely reverse the suppressive effect of BM macrophage supernatants on CD34+ cells. TGF-beta 1 bioassays showed that exposure of r-tat protein to BM macrophages significantly increased the levels of both active and latent forms of TGF-beta 1. These results indicate that the production of TGF-beta 1, one of the most potent negative regulator of hematopoiesis, is increased by HIV tat protein and that such increase could contribute to the derangement of the hematopoietic system in HIV-infected individuals.

Description: In this report the role played by human immunodeficiency virus type-1 (HIV-1) in the pathogenesis of HIV-1-related thrombocytopenia was investigated. CD34+ hematopoietic stem/progenitor cells were purified from the bone marrow (BM) of HIV-1(+) thrombocytopenic patients, HIV- 1(+) nonthrombocytopenic individuals, HIV-1(-) patients with immune thrombocytopenic purpura, and HIV-1(-) normal donors. CD34+ cells from HIV-1(+) thrombocytopenic individuals alone showed a reduced capacity to give rise to megakaryocytic colonies (CFU-Meg) and also a progressive and significant decline in cell number when placed in liquid culture containing recombinant human interleukin-3 (rIL-3). This decline involved not only megakaryocyte but also erythroid and granulocyte/macrophage progenitors. The defects in megakaryocyte colony formation and CD34+ cell growth did not result from a productive HIV-1 infection of CD34+ cells. Moreover, HIV-1 DNA was absent from CD34+ cells in 10 of 12 thrombocytopenic patients examined. On the other hand, the decreased survival/proliferation of CD34+ cells in liquid culture, within the HIV-1(+) thrombocytopenic patients, was correlated with the presence of HIV-1 p24 antigen in BM plasma. These results demonstrate an impairment of CD34+ cells in HIV-1(+) individuals presenting thrombocytopenia as the only hematologic manifestation. Furthermore, these findings suggest that increased viral replication in the BM microenvironment may cause this impairment and possibly contributes to HIV-induced thrombocytopenia.