ALBERT

Beschreibung: Tumor necrosis factor alpha (TNF alpha), as a modulator of hematopoiesis, interacts with many growth factor receptors, such as interleukin-3, granulocyte-macrophage colony-stimulating factor (CSF), and granulocyte-CSF receptors. Here, we studied the interactions between TNF alpha and the stem cell factor (SCF) receptor, c-kit, in normal CD34+ hematopoietic progenitors and their leukemic counterpart, ie, acute myeloid leukemic (AML) CD34+ cells coexpressing c-kit antigen. The results showed that (1) incubation of normal bone marrow mononuclear cells with 200 U/mL rhTNF alpha for 20 hours induced a diminution of 31.2% +/- 5.2% of CD34+ cells coexpressing c-kit; (2) the same decrease was observed using purified CD34+ cells and, furthermore, their proliferative response to SCF was inhibited by 31.5% +/- 7.3% after exposure to TNF alpha; (3) similar experiments performed on CD34+ c-kit+ AML cells from 11 patients gave comparable results. Further analysis at the mRNA level indicated that TNF alpha decreased c-kit mRNA transcripts. Moreover, using monoclonal antibodies against the two types of TNF alpha receptors, p75 and p55, we showed that the downregulation of c-kit proto-oncogene product by TNF alpha, on normal and leukemic CD34+ cells, was exclusively mediated by the TNF alpha p55 receptor. Therefore, we conclude that TNF alpha acts as a downregulator of the SCF receptor expression.

Beschreibung: The diagnostic value in polycythemia of the presence of endogenous erythroid colonies derived from bone marrow cells (EECs) was assessed in a prospective study on 108 patients referred for polycythemia (Hb greater than g/dL in men, greater than 16 g/dL in women) with normal plasma volume by comparison with the standard criteria, the bone marrow grade, and the serum erythropoietin (Epo) level. Total red cell volume (TRCV) was high (greater than 36 mL/kg in men, 32 mL/kg in women) in 87 cases (group A) and slightly increased in 21 cases (group B). Standard criteria were applicable in 63 of 108 cases (57%); 46 were PV and 17 were secondary polycythemia (SP). Standard criteria were nonapplicable in 45 cases. EECs were present in 65 cases (60%) with a ratio of EEC/Epo-stimulated colonies of 39.5% +/- 18% (extremes 10% to 80%). EECs were noted in 43 of 46 polycythemia vera (PV) and 0 of 17 SP. Among the 45 unclassified cases, EECs were noted in 22: 18 of 29 cases from group A (10 with 2 major and 1 minor criteria; 8 with 2 major criteria) and 4 of 16 cases from group B (with variable standard criteria, 2 belonging to a PV family). In group A, there was a positive significant correlation between EECs and the presence of two major and 1 minor criteria (P less than .0001). In group B, there was a positive significant correlation between EECs and the presence of at least 1 major criterion and 2 minor criteria or a family background (P less than .0001). The unclassified polycythemias with EECs in the bone marrow are characterized by a bone marrow grade and a mean serum Epo level not different from that of patients with PV and an active course of the disease. The unclassified polycythemias without EECs in the bone marrow are a heterogeneous group corresponding in some cases to SPs of unknown origin (slightly increased bone marrow grade and/or high serum Epo level), and in others cases to spurious polycythemias (normal bone marrow grade and/or normal Epo level). In conclusion, EECs were of great value in differentiating PV from SP (P less than .001), and in allowing the diagnosis of PV in the absence of all the standard criteria even when TRCV was slightly increased. In our study, EEC improved the classification of polycythemia by 22%. The recommended diagnostic steps for the evaluation of polycythemia must be reconsidered.

Beschreibung: The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, Seraspenide; Ipsen- Biotech, Paris, France), an inhibitor of murine spleen colony-forming units reduces the number and the percentage in DNA synthesis of progenitors from human unfractionated bone marrow. To determine whether AcSDKP may directly affect the growth potential of purified progenitors even at the most primitive level, CD34+HLA-DRhigh and CD34++HLA-DRlow cells were highly purified by cell sorting. Then, CD34+ subsets were stimulated in liquid culture with combinations of growth factors (GFs) and AcSDKP was added for 20 hours or 6 days and cells plated in methylcellulose. After a 20-hour incubation, we show that AcSDKP (at 10(-10) mol/L) significantly inhibits the colony formation of both CD34+ subsets. Moreover, when added daily for 6 days, AcSDKP: (1) reduces the proliferation of both CD34+ cell fractions stimulated by 3 or 7 GFs, and (2) decreases the number of progenitors generated from the CD34+HLA-DRhigh and CD34++HLA-DRlow cell fractions. Furthermore, we show for the first time, using both high proliferative potential cell and long-term culture initiating cell assays, that AcSDKP inhibits the most primitive cells contained in the CD34++HLA-DRlow subpopulation. Finally, by using limiting dilution assays we demonstrated that AcSDKP acts directly at a single cell level and that its inhibitory effect is reversible and dose dependent.

Beschreibung: The diagnostic value in polycythemia of the presence of endogenous erythroid colonies derived from bone marrow cells (EECs) was assessed in a prospective study on 108 patients referred for polycythemia (Hb greater than g/dL in men, greater than 16 g/dL in women) with normal plasma volume by comparison with the standard criteria, the bone marrow grade, and the serum erythropoietin (Epo) level. Total red cell volume (TRCV) was high (greater than 36 mL/kg in men, 32 mL/kg in women) in 87 cases (group A) and slightly increased in 21 cases (group B). Standard criteria were applicable in 63 of 108 cases (57%); 46 were PV and 17 were secondary polycythemia (SP). Standard criteria were nonapplicable in 45 cases. EECs were present in 65 cases (60%) with a ratio of EEC/Epo-stimulated colonies of 39.5% +/- 18% (extremes 10% to 80%). EECs were noted in 43 of 46 polycythemia vera (PV) and 0 of 17 SP. Among the 45 unclassified cases, EECs were noted in 22: 18 of 29 cases from group A (10 with 2 major and 1 minor criteria; 8 with 2 major criteria) and 4 of 16 cases from group B (with variable standard criteria, 2 belonging to a PV family). In group A, there was a positive significant correlation between EECs and the presence of two major and 1 minor criteria (P less than .0001). In group B, there was a positive significant correlation between EECs and the presence of at least 1 major criterion and 2 minor criteria or a family background (P less than .0001). The unclassified polycythemias with EECs in the bone marrow are characterized by a bone marrow grade and a mean serum Epo level not different from that of patients with PV and an active course of the disease. The unclassified polycythemias without EECs in the bone marrow are a heterogeneous group corresponding in some cases to SPs of unknown origin (slightly increased bone marrow grade and/or high serum Epo level), and in others cases to spurious polycythemias (normal bone marrow grade and/or normal Epo level). In conclusion, EECs were of great value in differentiating PV from SP (P less than .001), and in allowing the diagnosis of PV in the absence of all the standard criteria even when TRCV was slightly increased. In our study, EEC improved the classification of polycythemia by 22%. The recommended diagnostic steps for the evaluation of polycythemia must be reconsidered.

Beschreibung: To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.

Beschreibung: Tumor necrosis factor alpha (TNF alpha), as a modulator of hematopoiesis, interacts with many growth factor receptors, such as interleukin-3, granulocyte-macrophage colony-stimulating factor (CSF), and granulocyte-CSF receptors. Here, we studied the interactions between TNF alpha and the stem cell factor (SCF) receptor, c-kit, in normal CD34+ hematopoietic progenitors and their leukemic counterpart, ie, acute myeloid leukemic (AML) CD34+ cells coexpressing c-kit antigen. The results showed that (1) incubation of normal bone marrow mononuclear cells with 200 U/mL rhTNF alpha for 20 hours induced a diminution of 31.2% +/- 5.2% of CD34+ cells coexpressing c-kit; (2) the same decrease was observed using purified CD34+ cells and, furthermore, their proliferative response to SCF was inhibited by 31.5% +/- 7.3% after exposure to TNF alpha; (3) similar experiments performed on CD34+ c-kit+ AML cells from 11 patients gave comparable results. Further analysis at the mRNA level indicated that TNF alpha decreased c-kit mRNA transcripts. Moreover, using monoclonal antibodies against the two types of TNF alpha receptors, p75 and p55, we showed that the downregulation of c-kit proto-oncogene product by TNF alpha, on normal and leukemic CD34+ cells, was exclusively mediated by the TNF alpha p55 receptor. Therefore, we conclude that TNF alpha acts as a downregulator of the SCF receptor expression.

Beschreibung: To study the frontiers between pluripotent stem cells and committed progenitors and to further define the B-cell pathway in adult bone marrow (BM), CD34+ subpopulations and CD34- B-lineage cells were analyzed by multiparameter flow cytometry, studied by light and electron microscopy, and in short-term and long-term cultures (LTC). While the total CD34+ cells represent 4.9% +/- 0.8 of BM mononuclear cells within the lymphoid-blast window, 73.8 +/- 3.5%, 14.4 +/- 1.8% and 8.8 +/- 2.9% of them were CD34+ CD10- CD19-, CD34+ CD10+ CD19+, and CD34+ CD10+ CD19-, respectively. CD34+ CD10+ CD19+ cells represent a smal homogeneous TdT4 c micro-blast population. Although expressing CD38 and high level of HLA-DR antigens, like myeloid committed progenitors, they did not generate LTC, myeloid, and T lymphoid colonies suggesting that the CD34+ CD10+ CD19+ population represents exclusively B lymphoid committed progenitors. By contrast, all myeloid progenitors and LTC-initiating cells were found in the CD34+ CD10- CD19- cell fraction. This fraction appeared more heterogeneous and contained CD38- HLA-DRlow small cells, larger blasts, and promonocyte-like cells exhibiting small peroxidase-positive granules. Interestingly, CD10 was also present on CD34+ CD19- cells. This population mainly coexpressed CD33 and gave rise to macrophagic colonies.

Beschreibung: The tetrapeptide Acetyl-N-Ser-Asp-Lys-Pro (AcSDKP, Seraspenide; Ipsen- Biotech, Paris, France), an inhibitor of murine spleen colony-forming units reduces the number and the percentage in DNA synthesis of progenitors from human unfractionated bone marrow. To determine whether AcSDKP may directly affect the growth potential of purified progenitors even at the most primitive level, CD34+HLA-DRhigh and CD34++HLA-DRlow cells were highly purified by cell sorting. Then, CD34+ subsets were stimulated in liquid culture with combinations of growth factors (GFs) and AcSDKP was added for 20 hours or 6 days and cells plated in methylcellulose. After a 20-hour incubation, we show that AcSDKP (at 10(-10) mol/L) significantly inhibits the colony formation of both CD34+ subsets. Moreover, when added daily for 6 days, AcSDKP: (1) reduces the proliferation of both CD34+ cell fractions stimulated by 3 or 7 GFs, and (2) decreases the number of progenitors generated from the CD34+HLA-DRhigh and CD34++HLA-DRlow cell fractions. Furthermore, we show for the first time, using both high proliferative potential cell and long-term culture initiating cell assays, that AcSDKP inhibits the most primitive cells contained in the CD34++HLA-DRlow subpopulation. Finally, by using limiting dilution assays we demonstrated that AcSDKP acts directly at a single cell level and that its inhibitory effect is reversible and dose dependent.

Beschreibung: We successfully infected fluorescence-activated cell-sorted CD34+ cells from normal cord blood by the human cytomegalovirus (HCMV) laboratory strain Towne. An inhibitory effect of HCMV on clonogenic myeloid progenitors was observed in primary methylcellulose cultures. After an initial 7-day liquid culture of CD34(+)-infected cells, this inhibition was further amplified in secondary methylcellulose cultures, then involving both the myeloid and erythroid lineages. Under these conditions, viral DNA was detected both in erythroid and myeloid colonies using the polymerase chain reaction (PCR), but reverse transcription PCR (RT-PCR) failed to detect viral RNA. In contrast, when CD34(+)-infected cells were maintained in liquid suspension, both immediate, early, and late transcripts were detected as soon as day 3. In addition, viral production was demonstrated in the culture supernatants, thus confirming that a complete viral cycle occurred under liquid conditions. Furthermore, by resorting cells into CD34+ and CD34- fractions, we showed by RT-PCR that viral replication took place in cells still expressing CD34 antigen, whereas no RNA was found in more differentiated cells that had subsequently lost their CD34 antigen. These findings suggest that HCMV replication can occur at the early steps of progenitor differentiation and may be involved in the viral-induced myelosuppression.