抗肿瘤药羟喜树碱对Wnt通路抑制剂FrpHE的表达作用

目的研究加入羟喜树碱后Wnt通路抑制因子FrpHE(frizzled relatd protein)在人肿瘤细胞株中的表达调控作用。方法选择人肝癌HepG2(HepG2,含野生型p53;Hep3B,p53缺失)和人大肠癌(Lovo,含野生型p53),人神经胶质瘤细胞(U251,p53突变)细胞株为模型,观察抑制剂FrpHE以及反映Wnt通路功能变化的-βcatenin的表达。以RT-PCR技术检测Wnt通路抑制因子FrpHE表达的调节作用,以流式细胞术检测肿瘤细胞中Wnt通路的关键调节因子-βcatenin的表达。结果加入羟喜树碱24h后FrpHE mRNA表达水平在人肝癌细胞(HepG2,含野生型p53;Hep3B,p53缺失)中与对照组相比表达水平显著增加。在人大肠癌细胞(Lovo,含野生型p53)和人神经胶质瘤细胞(U251,p53突变型)中,未见FrpHE mRNA表达。-βcatenin的阳性细胞百分比强度和平均荧光量强度与对照组相比,表达水平降低。结论羟喜树碱在人肝癌细胞中能明显诱导抑制剂FrpHE mRNA的表达。     

The Tall R Wave in Lead V1 in Posterior Myocardial Infarction: A Reciprocal Sign or a His-Purkinje Conduction Disturbance?

The significance of the tall R wave in lead V1 with an R/S ratio greater than or equal to 1 in posterior myocardial infarction (PMI) was investigated in 28 patients during programmed electrical stimulation. The patients had been admitted with acute PMI documented by electrocardiogram and proven by enzymatic increase. Electrophysiological study was performed 3 weeks after acute PMI. In 17 of the 28 patients (group 1), the tall R wave in V1 disappeared during stimulation: In 13 of them a premature atrial extrastimulus was responsible for an abrupt normalization of QRS complex in V1 related to an increase in AH or HV interval. In the 4 remaining patients the disappearance of the tall R wave in V1 was related to a sinus pause. In 14 patients of group 1, a different prematurity in atrial stimulation induced a right or left bundle branch block (BBB). In 11 of the 28 patients (group 2) the tall R wave in V1 was unchanged but a premature atrial extrastimulus induced a right BBB in 5 patients and a left BBB in 6. In conclusion, the normalization of QRS complex in lead V1 during atrial stimulation or alterations in cycle length suggests that the tall R wave in V1 in PMI is not a simple reciprocal sign of leads V8 V9. Its association with different varieties of BBB and changes in AH or HV intervals could suggest a relationship with a His-Purkinje conduction disturbance in some patients.     

Metabolic, membrane, and functional responses of human polymorphonuclear leukocytes to platelet-activating factor

The phospholipid mediator of anaphylaxis, platelet-activating factor (PAF) is chemotactic for polymorphonuclear leukocytes (PMN). We have examined this agent's effects on several other PMN functions. Human PMN were prepared from heparinized venous blood by Ficoll gradient. Metabolic burst was examined by measurement of O2 use and O2.- production in the presence or absence of PAF (10(-6)--10(-9) M). Unless cells were treated with cytochalasin-B (5 micrograms/ml), no significant respiratory burst was demonstrated. However, pretreatment with PAF (10(-7) M) enhanced approximately threefold the O2 utilization found when cells were subsequently stimulated with 10(-7) M FMLP. PAF also stimulated arachidonic acid metabolism in 14C-arachidonic acid- labeled PMN. Thin-layer chromatography analysis of chloroform-methanol extracts showed substances that comigrated with authentic 5- hydroxyeicosatetraenoic acid had a marked increase in radioactivity following PAF stimulation at 10(-7) M. PAF failed to stimulate release of granule enzymes, B-glucuronidase, lysozyme, or myeloperoxidase unless cytochalasin-B were added. PAF from 10(-6) M to 10(-10) M affected PMN surface responses. PMN labeled with the fluorescent dye, chlorotetracycline, showed decreased fluorescence upon addition of PAF, suggesting translocation of membrane-bound cations. Further, the rate of migration of PMN in an electric field was decreased following PAF exposure, a change consistent with reduced cell surface charge. PMN self-aggregation and adherence to endothelial cells were both influenced by PAF (10(-6) M--10(-9) M). Aggregation was markedly stimulated by the compound, and the percent PMN adhering to endothelial cell monolayers increased almost twofold in the presence of 10(-8) M PAF. Thus, PAF promotes a variety of PMN responses: enhances respiratory burst, stimulates arachidonic acid turnover, alters cell membrane cation content and surface charge, and promotes PMN self- aggregation as well as adherence to endothelial cells.     

Interleukin-6 synergizes with M-CSF in the formation of macrophage colonies from purified human marrow progenitor cells

We examined the in vitro stimulative effects of recombinant human interleukin-6 (IL-6, or interferon-beta 2) on purified human bone marrow progenitor cells. IL-6 alone or in combination with erythropoietin (Epo), IL-3, GM-CSF, or G-CSF did not induce colony formation. However, IL-6 strongly synergized with M-CSF in stimulating macrophage colony formation (colony numbers and size). The magnitude of IL-6 synergism with M-CSF was dose dependent; maximal potentiation of M- colony formation was evident at approximately 100 to 1,000 U/mL IL-6. When the addition of IL-6 to M-CSF-supplemented cultures was delayed for more than one day after the beginning of culture, enhancement of macrophage colony formation was lost. IL-6 stimulation of M-CSF- responsive colony formation was not apparent when nonpurified marrow cells were plated, most likely due to endogenous IL-6 release. These observations suggest that IL-6, in addition to playing a role in B- lymphocyte proliferation can potentiate the human immune defence mechanism by stimulating monocyte-macrophage development as well.     

Granulocyte colony-stimulating factor (G-CSF) production and G-CSF receptor structure in patients with congenital neutropenia

Congenital neutropenia (Kostmann's syndrome [KS]) is an autosomal recessive syndrome that is characterized by profound neutropenia, resulting in major clinical infections and death. Since the neutropenia and symptoms in KS improve in response to exogenous administration of granulocyte colony-stimulating factor (G-CSF), we studied bone marrow cytokine (G-CSF, granulocyte-macrophage CSF [GM-CSF], and interleukin- 6) production under both basal and stimulated conditions. No differences in G-CSF, GM-CSF, or IL-6 gene expression were found in bone marrow stromal cells between normal controls and KS patients, and all three cytokines were detected by enzyme-linked immunosorbent assay (ELISA) in medium conditioned by bone marrow stromal cells from normal donors and patients with KS. Each KS patient tested had detectable, functional G-CSF in their own serum before exogenous G-CSF administration. Since G-CSF production appeared normal in KS patients, we then asked whether we could detect structural defects in the signaling portion of G-CSF receptor genes. Polymerase chain reaction (PCR) amplification of the G-CSF receptor transmembrane region alone, and of the transmembrane plus cytosolic portions of the receptor, yielded the size products predicted from the sequences of the normal G- CSF receptor. Single-strand conformational polymorphism (SSCP) analysis of G-CSF receptor PCR products demonstrated no variance in structural conformation between KS patients and normal subjects. These results demonstrate that bone marrow stromal cells in patients with KS secrete normal concentrations of functional G-CSF and suggest that the neutropenia in KS patients is caused by an inability of neutrophilic progenitor and precursor cells to respond to normal, physiologic levels of G-CSF. Such a defect, clinically responsive to pharmacologic doses of G-CSF, might be caused by defects in the post-G-CSF receptor signal transduction pathway.     

Detection of Bm86 antigen in different strains of Boophilus microplus and effectiveness of immunization with recombinant Bm86

The control of tick populations by using conventional strategies poses several problems, including the appearance of organophosphate resistant strains, among others. The possibility of using alternative strategies such as vaccination with tick antigens has been suggested by several authors. One particular antigen (Bm86) has been described and shown to be able to induce a protective immunity against the cattle tick Boophilus microplus. In this paper we demonstrate by means of immunohistochemical staining that this antigen is conserved among several strains of this species. These results correlate with those showing that animals vaccinated with a preparation of recombinant Bm86 were protected against challenge with the four different strains tested, including one resistant to organophosphates. These results favour the immunization with recombinant Bm86 for the control of the cattle tick B. microplus.     

Transfusion of donor buffy coat cells in the treatment of persistent or recurrent malignancy after allogeneic bone marrow transplantation

BACKGROUND: Patients who experience relapse after allogeneic bone marrow transplantation have a poor prognosis. However, preclinical and clinical data have strongly suggested the existence of an immune- mediated anti-tumor effect of allogeneic bone marrow transplantation. This effect, termed graft-versus-leukemia, may be harnessed purposefully in patients with posttransplant relapses by the administration of immune cells obtained by leukapheresis of the original bone marrow donor. STUDY DESIGN AND METHODS: Thirteen patients with persistent or recurrent hematologic malignancy after HLA-matched sibling-donor allogeneic bone marrow transplantation were treated with transfusion of buffy coat cells collected from the original bone marrow donors. Mononuclear cell dose ranged from 1.18 to 4.28 × 10(8) per kg. Alpha-interferon (1.5-3 × 10(6) U/m2 3-5x/week) was given to seven patients. Patients were observed for the development of graft-versus- host disease and disease response. RESULTS: Three of five patients with chronic myelogenous leukemia had complete remissions. One of five patients with active acute leukemia attained complete remission. A sixth acute leukemia patient treated with buffy coat transfusion after the induction of remission with chemotherapy relapsed 12 months later. One patient with myeloma had a complete but transient response. A patient with Hodgkin's disease did not respond. Four patients remain in remission 4, 16, 17, and 29 months after attaining complete remission. Graft-versus-host disease occurred in eight patients, including all of those with a complete response. One patient developed transient pancytopenia. CONCLUSION: The transfusion of donor buffy coat cells has significant anti-tumor activity in patients with relapsed hematologic malignancy after allogeneic bone marrow transplantation. This effect is strongly associated with graft-versus-host disease.