Autoantibodies against the platelet glycoproteins (GP) IIb/IIIa, Ia/IIa, and IV and partial deficiency in GPIV in a patient with a bleeding disorder and a defective platelet collagen interaction

To evaluate the physiologic importance of the different collagen receptors on platelets, we screened 806 patients admitted to the hospital because of hemorrhagic diathesis for eventual laboratory evidence of a pathologic platelet collagen interaction, and found 5 patients with an isolated deficiency in collagen-induced platelet aggregation. Four of these five patients had a partial defect, one had a complete defect. The structural and functional analysis of the platelets from the patient with a complete defect showed a deficiency in glycoprotein (GP) IV and autoantibodies against GPIIb/IIIa, GPIa/IIa, and GPIV. Patient plasma had only a minimal effect on normal control platelets and Naka-negative platelets. The analyses of the defect in the patient and of the data in the literature suggest that a single defect may not result in clinical bleeding (GPIV-deficient patients do not bleed), but may become symptomatic in combination with another defect such as the autoantibodies against GPIa/IIa, GPIV, and/or GPIIb/IIIa, all of which are involved in platelet collagen interactions (three of four of our immune thrombocytopenic purpura patients with anti-GPIV and anti-GPIIb/IIIa autoantibodies had a bleeding disorder). We hypothesize that it is the synergism of two abnormalities that results in the defective function, a mechanism that is in agreement with earlier studies on platelet collagen interaction that suggests that a double defect in platelet collagen interactions is required to become clinically apparent.     

Therapy of chronic myelogenous leukemia with recombinant interferon- gamma

Recently, we reported that recombinant interferon-alpha (rIFN-alpha) can induce hematologic remissions and cytogenetic improvement in newly diagnosed Philadelphia (Ph)-positive chronic myelogenous leukemia (CML) patients. Although IFN-gamma is a structurally distinct molecule, this agent suppresses in vitro hematopoietic progenitor cells in a fashion similar to that of IFN-alpha. Therefore, we initiated a study of rIFN- gamma at doses of 0.25 to 0.5 mg/m2/d intramuscularly in patients with Ph-positive benign-phase CML. Twenty-six of 30 patients entered in the study were evaluable. Six patients have achieved a complete hematologic response; four, a partial hematologic response. The median follow-up period of patients who are in complete remission is 7.5 months (range, 5 to 12 months). No relapses have occurred among the complete responders. So far, five patients have had cytogenetic improvement with emergence of 5% to 45% diploid cells in the bone marrow. Fever and flulike symptoms were the most common side effects, with partial tolerance often developing after about 1 week. The majority of patients tolerated therapy with minimal change in performance status. In conclusion, rIFN-gamma has demonstrated clinical activity in CML. On the basis of these observations and the in vitro synergistic growth- inhibitory effects of IFN-alpha and IFN-gamma, we have started trials of combination IFN therapy in CML patients.     

Recombinant human interleukin-6 induces a rapid and reversible anemia in cancer patients

Initial studies have shown that recombinant human interleukin-6 (rhIL- 6) induces anemia. Until now, the pathophysiologic mechanism of this induced anemia has been unknown. To unravel the underlying mechanism, we examined 15 cancer patients receiving rhIL-6 as an antitumor immunotherapy in a phase II study. rhIL-6 was administered subcutaneously at 150 micrograms once daily for 6 consecutive weeks. Various hematologic and biochemical parameters were measured weekly during rhIL-6 treatment and 4 weeks after rhIL-6 discontinuation. To determine plasma volume and red blood cell (RBC) volume, radioisotope dilution assays with labeled autologous RBCs and with human serum albumin were performed before rhIL-6 administration and on day 8 of rhIL-6 therapy. Hemoglobin levels decreased (mean change +/- SE) 7% +/- 1.5% within 3 days after the start of rhIL-6 therapy (P < .0001) and 19% +/- 2% at week 4. Levels had normalized at follow-up. The plasma volume increased 18% +/- 5% during the first week of rhIL-6 administration (P < .003), whereas RBC volume remained unaffected. The mean RBC corpuscular volume remained unchanged for 2 weeks and then began to decrease slowly, reaching its nadir at week 6 (5% +/- 1%; P < .01). Serum iron levels decreased 65% +/- 12% at week 4 (P < .002) and then returned to initial baseline values. Erythropoietin levels increased rapidly up to 68% at week 3 (P < .0001) and had normalized 4 weeks after rhIL-6 therapy. Levels of serum albumin, prealbumin, and transferrin decreased (P < .0001, P < .003, and P < .0001, respectively), whereas levels of serum amyloid A (P < .003), C-reactive protein, haptoglobin, and alpha-1-antitrypsin (P < .0001) increased during rhIL-6 treatment. All levels returned to pretreatment values after discontinuation of rhIL-6. No alterations in reticulocyte counts, serum lactic dehydrogenase levels, and bilirubin levels were observed. A 6-week regimen of subcutaneous rhIL-6 results in a rapid dilution anemia, caused by an acute and significant increase in plasma volume and followed by hypoferremia. This anemia is reversible after the cessation of rhIL-6 treatment.     

A murine cytokine fusion toxin specifically targeting the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) receptor on normal committed bone marrow progenitor cells and GM-CSF-dependent tumor cells

A fusion protein was synthesized consisting of the murine granulocyte- macrophage colony-stimulating factor (mGM-CSF) gene spliced to a truncated form of the diphtheria toxin (DT390) gene coding for a molecule that retained full enzymatic activity, but excluded the native binding domain. The DT390-mGM-CSF hybrid gene was cloned into a vector under the control of an inducible promoter and the protein expressed in Escherichia coli. After induction, a protein was purified from inclusion bodies in accord with the deduced molecular weight of DT390 mGM-CSF. Cell-free studies of the adenosine diphosphate-ribosylating activity of DT390 mGM-CSF showed results that were similar to those of native DT. The DT390 mGM-CSF immunotoxin inhibited FDCP2.1d, a murine myelomonocytic tumor line expressing the GM-CSF receptor with an IC50 (concentration inhibiting 50% activity) of 5 x 10(-11) mol/L. The fusion toxin was specifically cytotoxic and directed by the GM-CSF portion of the molecule because addition of a monoclonal antibody directed against GM-CSF inhibited its ability to kill the cell line. Cell lines that do not express GM-CSF receptor were not inhibited by the fusion toxin. DT390 mGM-CSF was also able to specifically inhibit normal committed bone marrow (BM) progenitor cells as measured in clonal colony-forming unit granulocyte-macrophage assays. Together, these findings indicate that DT390 mGM-CSF may be useful as a novel tool for purging BM of contaminating leukemia cells or in vivo for eliminating residual leukemia cells. Also, it can be used to determine whether committed and/or noncommitted BM progenitor cells express the GM-CSF receptor.     

Recovery of HLA-restricted cytomegalovirus (CMV)-specific T-cell responses after allogeneic bone marrow transplant: correlation with CMV disease and effect of ganciclovir prophylaxis

Protection from cytomegalovirus (CMV) disease in immunocompromised hosts has been shown to correlate with recovery of the host virus- specific CD8+ T-cell response. The administration of ganciclovir to immunosuppressed transplant recipients as antiviral prophylaxis has reduced the early risk of CMV disease, but late disease is observed with increased frequency, suggesting that recovery of the CMV-specific T-cell responses necessary for protective immunity may be delayed in these patients. Therefore, we evaluated reconstitution of CMV-specific T-cell responses in 47 bone marrow transplant (BMT) recipients entered on a randomized placebo-controlled study of ganciclovir. The study drug was initiated at a mean of 24 days after BMT. At day 30 to 40, a minority of patients had recovery of T-cell immunity to CMV and the frequency of reconstitution was equivalent in patients randomized to ganciclovir or placebo. The failure of ganciclovir to effect early reconstitution may reflect the short duration of treatment. Early recovery was associated with the infusion of BM from a CMV seropositive donor (P = .07 for CD8+ cytotoxic T cell (CTL), P = .04 for CD4+ Th). Between day 40 and day 90, recovery of deficient CD8+ and CD4+ CMV- specific T-cell responses occurred in the majority of individuals that received placebo, but in a minority of ganciclovir recipients. Two cases of late-onset CMV disease occurred in ganciclovir recipients. In all patients, the presence of a CTL response to CMV conferred protection from subsequent CMV disease (P = .005), and these protective CTL responses are shown to be specific for structural virion proteins similar to the responses in immunocompetent CMV seropositive individuals. These data confirm the importance of CMV-specific T-cell responses and suggest that a delay in recovery of these responses as a result of ganciclovir prophylaxis may contribute to the occurrence of late CMV disease.     

大鼠心肌梗死后的原癌基因表达

目的本实验旨在探讨心肌梗死后左心室重构的分子生物学机制。方法用地高辛标记的原位杂交方法观察了大面积心肌梗死后存活心肌细胞的原癌基因c-myc。c-fos及c-jun的mRNA水平的动态变化，并用巯甲丙脯酸进行干涉。大鼠随机分为三组：（1）单纯心肌梗死组；（2）梗塞给药组；（3）假手术组。梗塞给药组于手术前3d开始饲以巯甲丙脯酸（2g／L）。结果左室非梗塞区心肌的c-myc，c-fos及c-jun的mRNA水平手术后24h达峰值，c-myc于手术后10d，c-jun于手术后7d恢复到假手术组水平，但c-fos于术后持续高表达。在梗塞给药组，c-myc，c-fos于手术后7d、10d，c-jun于手术后各时间点其mRNA水平达假手术组程度。结论上述基因在非梗塞区心肌均有过量表达，持续时间较长，转换酶抑制剂对其有所抑制。     

Adult bone marrow-derived pluripotent hematopoietic stem cells are engraftable when transferred in utero into moderately anemic fetal recipients

We have used W41/W41 (C57BL/6-Ly 5.1, Gpi-1b) anemic mice and a newly developed double congenic donor strain (C57BL/6-Ly 5.2, Gpi-1a) to determine if adult bone marrow (BM) injected in utero could provide stem cell engraftment. Of 38 fetuses injected intraperitoneally on day 13/14 of gestation with donor BM cells, 17 (47%) were live-born. On day 6, 12% had erythroid engraftment. On day 59, in 50% (8/16) of mice, 50% to 75% of erythroid cells, 42% of T cells, 5% of B cells, and 26% of granulocytes in the peripheral blood (PB) were derived from the in utero-injected donor BM. At 141 days, thymic, splenic, lymph node, BM, and PB chimerism studies showed that 57% to 80% of T cells, 10% to 15% of B cells, and 27% to 43% of granulocytes were of donor origin. At this time, BM was injected into irradiated secondary recipients. On day 104 posttransfer, a mean 23% of T cells, 8% of B cells, and 40% of granulocytes were derived from the in utero donor BM. These data indicate that adult BM has hierarchical engraftment capabilities in W41/W41 mice and prove that stem cells are engraftable in utero.     

Identification and culture of Kaposi's sarcoma-like spindle cells from the peripheral blood of human immunodeficiency virus-1-infected individuals and normal controls [see comments]

We examined 26 patients with human immunodeficiency virus-1 (HIV-1)- associated Kaposi's sarcoma (KS), and 76 HIV-1-infected (HIV-1+) people without KS or uninfected (HIV-1-) controls for the presence of circulating KS-like spindle cells. Adherent cells that had spindle morphology and several characteristics of spindle cells of KS lesions (KS cells) were identified in the peripheral blood mononuclear cell fraction only after culture in the presence of conditioned medium (CM) from activated lymphocytes. The peripheral blood-derived spindle cells (PBsc) expressed a variety of endothelial cell markers, such as Ulex europaeus I lectin, EN4, EN2/3, EN7/44, CD13, CD34, CD36, CD54, ELAM-1, and HLA-DR. However, they were negative for CD2, CD19, PaIE, and factor VIII-related antigen. The PBsc produced angiogenic factors as evidenced by the ability of CM from these cells to promote growth of normal vascular endothelial cells. In addition, subcutaneously injected PBsc stimulated angiogenesis in vivo in athymic nude mice. We determined that the number of PBsc grown from the peripheral blood of HIV-1+ patients with KS or at high risk to develop KS were increased by 78- fold (P = .0001) and 18-fold (P = .005), respectively, when compared with HIV-1- controls. The number of spindle cells cultured from the HIV- 1+ patients at low risk for developing KS, eg, HIV-1+ injection drug users, showed no statistical increase when compared with HIV-1- controls. The presence of increased PBsc with characteristics of KS cells in HIV-1+ KS patients or patients at high risk for developing KS gives insights into the origin of KS cells and may explain the multifocal nature of the disease. In addition, this may be useful in predicting the risk of KS development.