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.     

Modulation of megakaryocytopoiesis by thrombopoietin: the c-Mpl ligand

We have further characterized the biological activities, mechanism of action, and target cell populations of recombinant human and murine thrombopoietin (rhTPO and rmTPO) in in vitro human and murine model systems. Alone, hTPO or mTPO stimulated the maturation of immature murine megakaryoblasts as measured in a single cell assay. The combination of hTPO or mTPO and interleukin-6 (IL-6) resulted in a further increase in megakaryocyte differentiation in this system. Murine TPO stimulated mouse megakaryocyte progenitor development. Human megakaryocyte progenitor development was potentiated by hTPO alone and further augmented in the presence of the early-acting cytokines (IL-3) or kit ligand/stem cell factor (KL/SCF). To further define the mechanism of action of TPO, neutralization studies were performed with antisera to IL-3, granulocyte-macrophage colony-stimulating factor (GM- CSF), IL-1 beta, and IL-11. No diminution in TPO activity was observed in the presence of these antisera. Moreover, because adhesive interactions are known to modulate hematopoiesis, we studied whether hTPO might alter such interactions between human bone marrow (BM) megakaryocytes and human BM stromal fibroblasts. No changes were observed in either megakaryocyte expression of the surface molecules lymphocyte function-associated antigen-1, very late activation antigen- 4, or intercellular adhesion molecule-1 or the adhesion of megakaryocytes to stromal fibroblasts after treatment with the growth factor. Furthermore, no induction of secretion of the cytokines IL-1 alpha, IL-1 beta, GM-CSF, IL-6, granulocyte-CSF, tumor necrosis factor- alpha, transforming growth factor-beta 1, or transforming growth factor- beta 2 by primary human BM megakaryocytes was noted after treatment of the cells with hTPO. To address whether TPO affects very primitive hematopoietic progenitors, we studied the residual cells from the BMs of mice treated with high doses of 5-fluorouracil. Although no effect of mTPO alone was noted on the viability or replication of such primitive murine progenitor populations, the triple combination of IL-3 + KL/SCF + TPO stimulated growth of megakaryocyte progenitors. These results indicate that TPO is a highly lineage-specific growth factor whose primary biological effects are likely to be direct modulation of the growth and maturation of committed megakaryocyte precursors and immature megakaryoblasts.     

Ectopic expression of rhombotin-2 causes selective expansion of CD4-CD8- lymphocytes in the thymus and T-cell tumors in transgenic mice

Although the proto-oncogene rhombotin-2 (RBTN-2) is widely expressed in most tissues, it is not expressed in T cells. We investigated the potential for overexpression of RBTN-2 to cause tumors in T cells and other tissues by constructing transgenic mice that expressed RBTN-2 under control of the metallothionein-1 promoter. Despite overexpression of RBTN-2 in all tissues, transgenic mice developed T-cell tumors only, thus indicating that tumorigenesis caused by RBTN-2 is T-cell-specific. Thymic tumors were found between 37 and 71 weeks and were invariably associated with metastasis to nonlymphoid organs. Thymuses from apparently healthy transgenic mice were also examined. In some mice there was an 10-fold increase in the CD4-CD8- thymocyte subset, yet the total number of thymocytes was the same as that in wild-type mice. Thymic homeostasis was maintained by a compensatory reduction in the CD4+CD8+ subset. The expansion of CD4-CD8- thymocytes was associated with increased expression of RBTN-2 and with increased cell proliferation. No differences were found in the proportion of thymocytes undergoing apoptosis in transgenic mice. Furthermore, RBTN-2- induced expansion of CD4-CD8- cells did not block differentiation of these cells. Thymuses with 30% CD4-CD8- cells were essentially monoclonal, indicating that all thymic immunophenotypes were derived from a single clone. Overall, our data are consistent with the following scenario: (1) RBTN-2 expression in T cells causes selective and polyclonal proliferation of CD4-CD8- thymocytes accompanied by a compensatory decrease in other thymocyte subsets; (2) a clone with growth advantage and differentiation potential is selected and populates the thymus; and (3) this clone eventually breaches homeostasis of the thymus, accompanied or followed by metastasis to other organs.     

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)     

幽门螺杆菌cagⅡ对胃上皮细胞IL-8基因转录的影响及机制

目的探讨HpcagⅡ对胃上皮细胞IL-8基因转录的影响及信号传导机制。方法构建 cagⅡ基因位点缺失Hp突变株及带有IL-8报告基因的人胃癌细胞系L5F11,用液体闪烁计数仪测定荧光素酶(IL8转录)活性,用ELISA法测定IL8蛋白浓度。结果所有Hp突变株诱导荧光素酶活性与IL8蛋白浓度较亲代菌株26695均降低[(0．13±0．01)×cpm比(0．59±0．05)×(P<0．01);(0．73±0．13)ng／ml比(2．22±0．65)ng／ml,(P<0．05)]。PTK抑制剂herbimycinA不仅抑制Hp诱导的荧光素酶活性[(0．71±0．18)×cpm比(1．51±0．23)×cpm,(P<0．05)],而且抑制IL-8蛋白表达[(0．83±0．41)ng／ml比(3．22±0．59)ng／ml,(P<0．05)],但herbimycinA对TNFα诱导的荧光素酶活性及IL8蛋白表达均无影响(P均>0．05);PKA抑制剂H7抑制TNFα诱导的荧光素酶活性[(0．74±0．16)×cpm比(2．62±0．26)×cpm,(P<0．001)]及IL8蛋白表达[(1．45±0．38)ng／ml比(4．12±0．43)ng／ml,(P<0．01)],而对Hp诱导的荧光素酶活性无影响(P>0．05)。结论HpcagⅡ中的多基因能够调节胃上皮细胞IL-8基因转录,且这一作用主要经蛋白酪氨酸激酶途径。     

Comparative Efficacy Evaluation of Dicationic Carbazole Compounds, Nitazoxanide, and Paromomycin against Cryptosporidium parvum Infections in a Neonatal Mouse Model

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).     

Effective removal of copper by plasma exchange in fulminant Wilson's disease

BACKGROUND: Patients who present with fulminant hepatic failure due to Wilson's disease may develop hemolytic anemia and renal insufficiency. In this entity, acute hepatocellular necrosis triggers the release of copper ions into the circulation, which leads to toxic effects on red cell metabolic pathways and hemolysis. STUDY DESIGN AND METHODS: The utility of therapeutic plasma exchange to rapidly remove copper and reduce toxic serum copper levels was studied in two patients with fulminant Wilson's disease. RESULTS: Intensive plasma exchange using fresh-frozen plasma replacement removed substantial amounts of copper from the hypercupremic patients, resulting in a rapid reduction in serum copper levels and decreased hemolysis. The net copper removal was proportional to the serum level, ranging from 7,000 to 11,800 micrograms per procedure in one patient and from 3,700 to 6,800 micrograms in the other. CONCLUSION: Plasma exchange allows a rapid reduction in elevated serum copper levels in patients with fulminant Wilson's disease. This leads to an amelioration of hemolytic anemia and provides clinical stabilization until liver transplantation can be performed.     

Translocation breakpoints are clustered on both chromosome 8 and chromosome 21 in the t(8;21) of acute myeloid leukemia

The t(8;21)(q22;q22) is consistently associated with acute myeloid leukemia (AML) M2. Recent data have suggested that breakpoints on chromosome 21 are clustered within a single intron of a novel gene, AML1, just downstream of a region of homology to the runt gene of D melanogaster. In this report, we confirm rearrangement at the same location in at least 12 of 18 patients with t(8;21). Furthermore, we have isolated recombinant clones spanning the breakpoint regions on both the der(8) and the der(21) from one patient. By using a chromosome 8 probe derived from these clones, we show that t(8;21) breakpoints are also clustered on chromosome 8.