Generation of normal human red cell volume, hemoglobin content, and membrane area distributions by "birth" or regulation?

Using flow cytometry and osmotic lysis measurements, we document here the means and coefficients of variation of the following red cell (RBC) properties: hemoglobin (Hb) content, volume, Hb concentration, and relative lytic tonicity distributions in populations of normal human RBCs, before and after density fractionation. The distributions showed a pattern characterized by much larger coefficients of variation of the Hb content and volume distributions than of the Hb concentration and relative lytic tonicity distributions. From analysis of the factors that determine those RBC properties, the patterns were interpreted as reflecting previously unrecognized statistical proportionalities between cell osmolyte content, Hb content, and membrane area. The possible origin of these statistical links was analyzed by considering alternative models with and without the participation of regulatory processes during cell maturation. A model was shown to be feasible in which mature RBC variability with proportional volume, area, and Hb content arises solely from cell size variability at the last erythroid cell division.     

The role of inheritance and environment in predisposition to vascular disease in people of African descent.

OBJECTIVES: This study sought to compare vascular reactivity and carotid intima media thickness (CIMT) between Afro-Caribbean people in the United Kingdom (UK) and the West Indies and Afro-Caribbean and Caucasian people in the UK. BACKGROUND: Attenuated vascular reactivity and increased CIMT in black patients is seen as evidence for predisposition to vascular disease, but no comparisons exist between Afro-Caribbean people in different settings, which can provide insight into non-inherited determinants of increased ethnic susceptibility. METHODS: A representative community sample of 81 healthy Afro-Caribbean people and 101 Caucasian people in the UK was compared with 197 matched Afro-Caribbean people in Jamaica. Small vessel reactivity was assessed by measuring the absolute change from baseline in the reflection index (RI) of the digital volume pulse during intravenous infusion of albuterol (5 microg/min, DeltaRI(ALB)) and glyceryl trinitrate (5 microg/min, DeltaRI(GTN)). The CIMT was measured ultrasonographically in the distal 1 cm of the common carotid artery. RESULTS: Mean DeltaRI(ALB) was 4.2 percentage points (95% confidence interval [CI], 2.3 to 6.1, p < 0.001) lower in UK Afro-Caribbean people compared with Jamaican Afro-Caribbean people and 2.6 percentage points (95% CI, 0.4 to 4.7, p = 0.02) lower compared with Caucasian people, after adjusting for vascular risk profile. Adjusted mean CIMT of UK Afro-Caribbean people was 0.13 mm (95% CI, 0.08 to 0.17, p < 0.001) greater compared with Jamaican Afro-Caribbean people and 0.05 mm (95% CI, 0.01 to 0.10, p = 0.02) greater compared with Caucasian people. CONCLUSIONS: Healthy UK Afro-Caribbean people have greater and Jamaican Afro-Caribbean people have less impairment of vascular reactivity and intima media thickness compared with UK Caucasian people, suggesting that potentially modifiable environmental interactions may contribute to excess vascular disease in Afro-Caribbean people.     

Murine Hertwig's anemia: premature death after normal bone marrow transplantation is radiation dose-dependent

Marrow transplantation therapy in mice with heritable blood disorders usually leads to rapid blood cell normalization, but is sometimes followed by pancytopenia and premature death. This is especially true in mice with Hertwig's anemia (an/an). Unlike the +/+ recipients, 100% of whom survive for over a year, 66% of the mutant mice die by 6 months posttransplantation, and the rest die soon thereafter. It is not clear whether premature death is due to the radiation dose (10 Gy) or to the fact that the F1 mutant mice receive parental-type cells known to induce hybrid resistance. In the present report, experiments were designed to determine whether the F1-an/an host is more sensitive to radiation and/or resistant to continued expansion of the parental-type +/+ cells. The mutant mice are, indeed, more sensitive to irradiation, with an LD100/30 of 7 Gy as compared with an LD100/30 of 10 Gy for the +/+ mice. The times of anemia onset and death for mutant mice implanted with +/+ cells postirradiation is also radiation dose-dependent. Further evidence that death is due to host radiation damage rather than F1 hybrid resistance was provided by transplanting cells from three morbid 10 Gy-irradiation recipients into unirradiated, anemic, stem cell-deficient, F1-W/Wv secondary hosts. All recipients were repopulated by the original parental cells, were cured of their anemia, and survived for 52 weeks posttransplantation. The an/an mouse's heightened susceptibility to radiation damage appears to be the major factor in early death after transplantation therapy.     

Expression of bcr-abl abrogates factor-dependent growth of human hematopoietic M07E cells by an autocrine mechanism

The introduction of a retrovirus vector expressing p210bcr-abl (P210) into the human factor-dependent cell line M07E resulted in the rapid outgrowth of factor-independent cells. Early after infection, four factor-independent clones were isolated and analyzed in greater detail along with mass populations obtained from separate infections. High levels of P210 tyrosine kinase activity were measured in the factor- independent cells. The mass populations and three of the four clones remained responsive to exogenous growth factors. Concentrated conditioned media isolated from the factor-independent populations and from all clones contained biologically active granulocyte-macrophage colony-stimulating factor (GM-CSF); interleukin-3 (IL-3) was detected at low levels in the mass population and in two of the clones. Neutralizing antibodies to IL-3, GM-CSF, and mast cell growth factor inhibited proliferation of the factor responsive clones by 60% to 90%. These results indicate that the growth autonomy of the P210-expressing M07E cells was acquired via an autocrine mechanism. In addition to factor-independent growth, P210-expressing M07E cells readily acquired a more mature megakaryocytic phenotype compared with control M07E cells. These data provide experimental evidence that expression of P210 tyrosine kinase in human hematopoietic cells induced growth factor secretion resulting in a pleiotropic effect on growth factor dependence and differentiation.     

Cytokine production by primary bone marrow megakaryocytes

Primary human bone marrow megakaryocytes were studied for their ability to express and release cytokines potentially relevant to their proliferation and/or differentiation. The purity of the bone marrow megakaryocytes was assessed by morphologic and immunocytochemical criteria. Unstimulated marrow megakaryocytes constitutively expressed genes for interleukin-1 beta (IL-1 beta), IL-6, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha), by the polymerase chain reaction (PCR) and Northern blot analysis. At the protein level, megakaryocytes secreted significant amounts of IL-1 beta (53.6 +/- 3.6 pg/mL), IL-6 (57.6 +/- 15.6 pg/mL), and GM-CSF (24 +/- 4 pg/mL) but not TNF-alpha. Exposure of human marrow megakaryocytes to IL-1 beta increased the levels of IL-6 (87.3 +/- 2.3 pg/mL) detected in the culture supernatants. Transforming growth factor- beta was also able to stimulate IL-6, IL-1 beta, and GM-CSF secretion, but was less potent than stimulation with phorbol-12-myristate-13- acetate (PMA). The secreted cytokines acted additively to maintain and increase the number of colony-forming unit-megakaryocytes colonies (approximately 35%). These studies demonstrate the production of multiple cytokines by isolated human bone marrow megakaryocytes constitutively or stimulated in vitro. The capacity of human megakaryocytes to synthesize several cytokines known to modulate hematopoietic cells supports the concept that there may be an autocrine mechanism operative in the regulation of megakaryocytopoiesis.     

Administration of pegylated recombinant human megakaryocyte growth and development factor to humans stimulates the production of functional platelets that show no evidence of in vivo activation

This report describes the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG-rHuMGDF) on platelet production and platelet function in humans. Subjects with advanced solid tumors received PEG-rHuMGDF daily for up to 10 days. There was no increase in circulating platelet count at doses of 0.03 or 0.1 microgram/kg/d by day 12 of study. At doses of 0.3 and 1.0 microgram/kg/d there was a threefold median increase (maximum 10-fold) in platelet count by day 16. The platelets produced in vivo in response to PEG-rHuMGDF showed unchanged aggregation and adenosine triphosphate (ATP)-release responses in in vitro assays. Tests included aggregation and release of ATP in response to adenosine diphosphate (ADP) (10, 5, 2.5, and 1.25 mumol/L), collagen (2 micrograms/mL), thrombin-receptor agonist peptide (TRAP, 10 mumol/L) and ristocetin (1.5 mg/mL). Administration of aspirin to an individual with platelet count of 1,771 x 10(3)/L resulted in the typical aspirin-induced ablation of the normal aggregation and ATP-release response to stimulation with arachidonic acid (0.5 mg/mL), collagen, and ADP (2.5 and 1.25 mumol/L). There was no change in the expression of the platelet-surface activation marker CD62P (P-selectin) nor induction of the fibrinogen binding site on glycoprotein IIb/IIIa as reported by the monoclonal antibody, D3GP3. An elevation of reticulated platelets was evident after 3 days of treatment with PEG-rHuMGDF and preceded the increase in circulating platelet count by 5 to 8 days; this reflected the production of new platelets in response to PEG-rHuMGDF. At later time points, the mean platelet volume (MPV) decreased in a manner inversely proportional to the platelet count. Levels of plasma glycocalicin, a measure of platelet turnover, rose 3 days after the initial increase in the peripheral platelet count. The level of plasma glycocalicin was proportional to the total platelet mass, suggesting that platelets generated in response to PEG-rHuMGDF were not more actively destroyed. Thus, the administration of PEG-rHuMGDF, to humans, increased the circulating platelet count and resulted in fully functional platelets, which showed no detectable increase in reactivity nor alteration in activation status.     

Nitric oxide-induced p53 accumulation and regulation of inducible nitric oxide synthase expression by wild-type p53.

The tumor suppressor gene product p53 plays an important role in the cellular response to DNA damage from exogenous chemical and physical mutagens. Therefore, we hypothesized that p53 performs a similar role in response to putative endogenous mutagens, such as nitric oxide (NO). We report here that exposure of human cells to NO generated from an NO donor or from overexpression of inducible nitric oxide synthase (NOS2) results in p53 protein accumulation. In addition, expression of wild-type (WT) p53 in a variety of human tumor cell lines, as well as murine fibroblasts, results in down-regulation of NOS2 expression through inhibition of the NOS2 promoter. These data are consistent with the hypothesis of a negative feedback loop in which endogenous NO-induced DNA damage results in WT p53 accumulation and provides a novel mechanism by which p53 safeguards against DNA damage through p53-mediated transrepression of NOS2 gene expression, thus reducing the potential for NO-induced DNA damage.     

Predictive value of abdominal obesity cut-off points for hypertension in blacks from west African and Caribbean island nations

BACKGROUND: Waist circumferences (WC) >/=94 cm for men and >/=80 cm for women (action level I) and >/=102 cm for men and >/=88 cm for women (action level II) have been suggested as limits for health promotion purposes to alert the general public to the need for weight loss. In this analysis we examined the ability of the above cut-off points to correctly identify subjects with or without hypertension in Nigeria, Cameroon, Jamaica, St Lucia and Barbados. We also determined population- and gender-specific abdominal adiposity cut-off points for epidemiological identification of risk of hypertension. METHODS: Waist measurement was made at the narrowest part of the torso as seen from the front or at midpoint between the bottom of the rib cage and 2 cm above the top of the iliac crest. Sensitivity and specificity of the established WC cut-off points for hypertension were compared across sites. With receiver operating characteristics (ROC), population- and gender-specific cut-off points associated with risk of hypertension were determined over the entire range of WC values. RESULTS: Predictive abilities of the established WC cut-off points for hypertension were poor compared to the specific cut-off points estimated for each population. Different values of WC were associated with increased risk of hypertension in these populations. In men, WC cut-off points of 76, 81, 80, 83 and 87 cm provided the highest sensitivity for identifying hypertensives in Nigeria, Cameroon, Jamaica, St Lucia and Barbados, respectively. The analogous cut-off points in women were 72, 82, 85, 86 and 88 cm. CONCLUSIONS: The waist cut-off points from this study represent values for epidemiological identification of risk of hypertension. For the purpose of health promotion, the decision on what cut-off points to use must be made by considering other additional factors including overall impact on health due to intervention (e.g. weight reduction) and potential burden on health services if a low cut-off point is employed. There is a need to develop abdominal adiposity cut-off points associated with increased risks for cardiovascular diseases in different societies, especially for those populations where the distribution of obesity and associated risk factors tends to be very different from those of the technologically advanced nations. International Journal of Obesity (2000) 24, 180-186     

Hematopoietic growth factors for graft failure after bone marrow transplantation: a randomized trial of granulocyte-macrophage colony- stimulating factor (GM-CSF) versus sequential GM-CSF plus granulocyte- CSF

Delay in hematologic recovery after bone marrow transplantation (BMT) can extend and amplify the risks of infection and hemorrhage, compromise patients' survival, and increase the duration and cost of hospitalization. Because current studies suggest that granulocyte- macrophage (GM) colony-stimulating factor (CSF) may potentiate the sensitivity of hematopoietic progenitor cells to G-CSF, we performed a prospective, randomized trial comparing GM-CSF (250 micrograms/m2/d x 14 days) versus sequential GM-CSF x 7 days followed by G-CSF (5 micrograms/kg/d x 7 days) as treatment for primary or secondary graft failure after BMT. Eligibility criteria included failure to achieve a white blood cell (WBC) count > or = 100/microL by day +21 or > or = 300/microL by day +28, no absolute neutrophil count (ANC) > or = 200/microL by day +28, or secondary sustained neutropenia after initial engraftment. Forty-seven patients were enrolled: 23 received GM-CSF (10 unrelated, 8 related allogeneic, and 5 autologous), and 24 received GM- CSF followed by G-CSF (12 unrelated, 7 related allogeneic, and 5 autologous). For patients receiving GM-CSF alone, neutrophil recovery (ANC > or = 500/microL) occurred between 2 and 61 days (median, 8 days) after therapy, while those receiving GM-CSF+G-CSF recovered at a similar rate of 1 to 36 days (median, 6 days; P = .39). Recovery to red blood cell (RBC) transfusion independence was slow, occurring 6 to 250 days (median, 35 days) after enrollment with no significant difference between the two treatment groups (GM-CSF: median, 30 days; GM-CSF+G- CSF; median, 42 days; P = .24). Similarly, platelet transfusion independence was delayed until 4 to 249 days (median, 32 days) after enrollment, with no difference between the two treatment groups (GM- CSF: median, 28 days; GM-CSF+G-CSF: median, 42 days; P = .38). Recovery times were not different between patients with unrelated donors and those with related donors or autologous transplant recipients. Survival at 100 days after enrollment was superior after treatment with GM-CSF alone. Only 1 of 23 patients treated with GM-CSF died versus 7 of 24 treated with GM-CSF+G-CSF who died 16 to 84 days (median, 38 days) after enrollment, yielding Kaplan-Meier 100-day survival estimates of 96% +/- 8% for GM-CSF versus 71% +/- 18% for GM-CSF+G-CSF (P = .026). These data suggest that sequential growth factor therapy with GM-CSF followed by G-CSF offers no advantage over GM-CSF alone in accelerating trilineage hematopoiesis or preventing lethal complications in patients with poor graft function after BMT.(ABSTRACT TRUNCATED AT 400 WORDS)