Activity of platelet-activating factor (PAF) acetylhydrolase in plasma from healthy habitual cigarette smokers

Summary The activity of platelet-activating factor acetylhydrolase, a specific metabolizing enzyme of platelet-activating factor (PAF), in plasma of 39 male subjects was determined radiochemically by the method of Stafforini et al., (1987) J Biol Chem 262: 4223–4230, to clarify to what extent PAF affects atherosclerotic disorders induced by habitual smoking. The subjects examined included 18 habitual smokers (mean age: 62±10.6 years) and 21 age-matched nonsmokers. Plasma PAF acetylhydrolase activity was higher in the smokers than in the nonsmokers. There was no difference between smokers and nonsmokers in platelet aggregability in response to PAF or ADP. A higher apoprotein B/apoprotein A-I ratio in smokers as compared to nonsmokers was the only manifestation of abnormal lipoprotein metabolism in the former group. In the smokers, plasma PAF acetylhydrolase activity was directly proportional to total cholesterol, LDL-cholesterol, triglyceride, apoprotein B, LDL-cholesterol/HDL-cholesterol or apoprotein B/apoprotein A-I, and inversely proportional to HDL-cholesterol or apoprotein A-I. The results obtained suggest that alterations in PAF acetylhydrolase levels result from a slightly abnormal lipoprotein metabolism. Determination of plasma PAF acetylhydrolase activity is useful to study the role of PAF in atherosclerotic changes induced by smoking.     

Analysis of acute myeloid leukemia cells by flow cytometry,introducing a new light-scattering classification

A combined flow-cytometric evaluation of light scattering and the immunophenotype of acute myeloid leukemia (AML) cells from 71 newly diagnosed consecutive patients was conducted. Light-scattering characteristic of AML cells examined by flow cytometry and multiple surface markers were also analyzed using the same samples, to enable a comparison with the French-American-British (FAB) classification. Our AML cases could be classified into three light-scattering classification (LSC) types according to their physical properties on flow cytometry. These were type A, where forward light scattering (FSC) of the leukemic cell population was larger than that of lymphocytes, while side light scattering (SSC) was the same or larger than that of lymphocytes but smaller than that of monocytes; type B, where FSC of the leukemic cell population was larger than that of lymphocytes and SSC spread toward that of monocytes; and type C, where both FSC and SSC of the leukemic cell population spread beyond those of monocytes. Although a clear relationship between the FAB classification and LSC classification by the light-scattering profile of AML was not established, we observed the following findings. The majority of cases were classified as type A (58%), while type B comprised 25% and type C comprised 17%. While CD7 expression on AML cells is considered to be an immature characteristic, CD7 was expressed more frequently among LSC type A cases. Furthermore, all but one of the FAB M1 cases were classified as type A. On the other hand, CD7 was not expressed on type C leukemic cells. The percentage of cases in which more than 60% of leukemic cells possessed another immature surface antigen, CD 34ö, was 13/18 (72%) among FAB M1 cases, much higher than among FAB M2 (35%) or FAB M4 (27%) cases. A negative correlation was observed between mature antigen CD33 and CD34 among the FAB M2 cases. The frequency of CD7 expression was 25% among the total cases, and CD7-positive cases were frequent among FAB M1 and M2, but not among FAB M3 cases. These findings concerning LSC and immunophenotyping indicate that the scattergram pattern analysis may contribute towards more precise immunophenotyping, in that it reflects the maturation stage of each AML case.Abbreviations AML acute myeloid leukemia - LSC light-scattering classification - FAB French-American-British classification - CD cluster of differentiation Partly supported by grants in aid from the Ministry of Education, Science and Culture of Japan (03670325, 04247102, 04454572 and 05670916) and from the Fukuoka Anti-cancer Society     

Analysis of the cellular DNA and RNA content in acute leukemias by flow cytometry

Summary In the present study bone marrow samples from 573 patients with newly diagnosed acute myeloid (AML) and lymphoblastic or undifferentiated leukemias (ALL/AUL), were analysed for their cellular DNA und DNA/RNA content, respectively, by means of flow cytometry. From 237 patients with AML 35.4% revealed aneuploid DNA stemlines. While no relation of DNA aneuploidy with other pretherapeutic parameters, including FAB subtype, white blood cell count, lactate dehydrogenase, S-phase index and percentage of blasts in the bone marrow, was observed, cases with aneuploid DNA stemlines revealed a tendency towards longer remission duration. In ALL/AUL 21.8% of 280 patients expressed DNA aneuploidies, which were less frequently found in T-cell ALL (11.1%) as compared to common(C)-ALL (21.4%) or null-cell(null)-ALL (23.5%). DNA aneuploidy was not related with other clinically defined risk factors such as age, white blood cell count, and rapid achievement of remission. Patients with DNA indices <1.0, however, tended to have shorter remissions. A significant difference in RNA indices was observed between AML and ALL/AUL with median values of 14.4 and 10.1, respectively (P<0.05). These data indicate the usefulness of flow cytometric analyses of cellular DNA and RNA content for the characterization and classification of acute leukemias, complementing the identification of clinical risk factors, immuno-phenotyping and cytogenetics.Abbreviations AML acute myeloid leukemia - ALL acute lymphoblastic leukemia - AUL acute undifferentiated leukemia - T-ALL, C-ALL and Null-ALL T-cell, common and Null-cell ALL     

心力衰竭患者血小板活化因子及其受体表达的变化

目的 :探讨心力衰竭患者血浆血小板活化因子 (PAF)、血小板活化因子乙酰水解酶 (PAF AH)活性的变化 ,以及外周血中单个核细胞 (PBMC)PAF受体 (PAF R)的表达。方法 :对 30例心力衰竭患者采用生物法检测血浆中PAF含量 ,酶水解底物显色法测定PAF AH活性 ,流式细胞仪检测PBMC上PAF R的表达。并与 30例健康体检者对照。结果 :心力衰竭患者血浆中PAF含量 (11.12± 1.80 ) μg L ,PAF AH活性 (2 4 .5 0± 7.86 ) μmol (min·L)、PBMC上PAF R的表达与健康体检者比较差异有非常显著性意义 (P <0 .0 1)。结论 :PAF、PAF AH、PAF R三者在心力衰竭发生和发展中起重要作用     

Analysis of heat-shock protein expression in myeloid leukaemia cells by flow cytometry

Expression of heat-shock proteins (hsp) was analysed in the leukaemic cells of 12 patients with acute myeloid leukaemia (AML) and nine patients with chronic myeloid leukaemia (CML). Using monoclonal antibodies to hsp70, hsp90 and hsp60 (ML30, a mycobacterial antigen with homology to human hsp60), we measured hsp levels by flow cytometry of permeabilized cells. Mononuclear cells from 10 healthy volunteers were also examined. The results demonstrate that hsp expression is significantly increased (P<0'01) in the circulating cells of patients with AML compared with cells from CML patients, and compared with normal peripheral blood mononuclear cells. This increased pattern of expression was found for all three heat-shock protein families included in this study. Mononuclear cells from leukaemic patients showed a heterogenous pattern of hsp expression, between different patients, between cells from individual patients, and between the different hsp proteins examined. It is possible that hsp expression relates to the differentiation state or proliferative potential of these leukaemic cells.     

Tyrosine phosphorylation of proteins in primary human myeloid leukemic cells stimulated by macrophage colony-stimulating factor: Analysis by disease type and comparison with normal human hematopoietic cells

We investigated tyrosine phosphorylation of proteins in primary human leukemic cells stimulated by macrophage colony-stimulating factor (M-CSF) in 60 patients with acute myeloid leukemia (AML) and 5 patients with chronic myelomonocytic leukemia and compared the findings for leukemic cells with those of normal human monocytes and bone marrow immature hematopoietic cells. M-CSF induced tyrosine phosphorylation of p140-200, p110, p60, p44, and p42 frequently, and that of p95 and p55 less frequently, in primary myeloid leukemic cells, whereas M-CSF-induced phosphorylation of proteins was not detected in the normal human hematopoietic cells tested. Of these phosphoproteins, p140-200 was phosphorylated in all patients who responded to M-CSF and was considered to be almost identical to Fms, a product of the c-fms proto-oncogene. M-CSF-induced tyrosine phosphorylation was observed frequently (89%) in AML of French-American-British class M4 and infrequently in all other subtypes of AML, including M5. In chronic myelomonocytic leukemia, M-CSF-induced protein phosphorylation was prominent in blast crisis but was not detected in the chronic phase. Both bone marrow immature cells and mature monocytes showed low responsiveness to M-CSF. These findings for responsiveness to M-CSF were correlated with the amount of Fms in each type of cell. We also identified tyrosine phosphorylation of Vav, Shc, and extracellular signal-regulated kinase by M-CSF in some cases. These findings clarified an M-CSF-specific pattern of protein tyrosine phosphorylation and the responsiveness to M-CSF of primary human myeloid cells. Particularly, enhanced phosphorylation responses to M-CSF and increased amounts of Fms protein were observed in restricted human hematopoietic cells with a premature myelomonocytic character.     

流式细胞术分析慢性髓性白血病细胞酪氨酸磷酸化水平

目的：探讨慢性髓性白血病（CML）患者骨髓细胞酪氨酸磷酸化水平（p-Tyr），探讨甲磺酸伊马替尼（商品名：格列卫）治疗对p-Tyr水平的作用和影响。方法：对38例CML患者（其中16例接受格列卫治疗）及5例健康对照者的骨髓细胞进行细胞膜CD45和细胞质内PTyr（PY99）荧光标记，应用流式细胞术（FCM）分析不同病期CML各群细胞p-Tyr水平。结果：①未经甲磺酸伊马替尼治疗的慢性期（CP）、加速期（AP）患者骨髓各群细胞p-Tyr水平高于健康对照者，而CP与AP之间p-Tyr水平差异无统计学意义；②CP患者应用甲磺酸伊马替尼治疗〉12个月者的骨髓细胞p-Tyr水平低于用药〈12个月者；AP期用药≥40个月者的幼稚细胞群p-Tyr水平亦低于用药〈12个月者；③随CML病程延长，幼稚细胞群p-Tyr水平增高，但无明显影响；年龄对p-Tyr水平无明显影响。结论：①应用FCM测定细胞p-Tyr水平可作为快速、敏感的方法检测BCR-ABL（＋）细胞；②CML各期细胞的p-Tyr水平均高于正常对照；③甲磺酸伊马替尼对慢性期白血病细胞p-Tyr水平的降低程度随用药时间延长而增强。     

The role of Clofarabine in the treatment of adults with acute myeloid leukemia

The therapeutic scenario available for adult patients with acute myeloid leukemia (AML) has shown only partial progresses over the last few years. This is especially true for refractory and relapsed AML whose outcome is still extremely disappointing. In this context Clofarabine has offered new promising perspectives within first and second line protocols. This review will firstly describe the initial development in monotherapy, considering then the different potential combination strategies which include both polichemotherapeutic regimens and less conventional approaches with new generation drugs. The potential use of Clofarabine as induction treatment for patients candidate to stem cell transplantation and within conditioning regimens will be finally evaluated.     

Recombinant human granulocyte colony-stimulating factor (G-CSF) combined conditioning regimen for allogeneic bone marrow transplantation (BMT) in standard-risk myeloid leukemia

We previously suggested that using a combined conditioning regimen including rhG-CSF with allogeneic BMT in refractory AML and CML in blast crisis might reduce the rate of relapse and improve disease-free survival, without any major side effects. In this study, we used the same protocol for 10 AML patients in complete remission (CR) and 6 CML patients in the chronic phase (CP). We compared disease-free survival as well as toxic side effects of the regimen with 6 AML patients in CR and 6 CML patients in CP treated with chemoradiotherapy without G-CSF. The conditioning regimen consisted of TBI and high-dose AraC. RhG-CSF was infused continuously at a dose of 5 μg/kg/day, starting 24 hr before the initial dose of total body irradiation (TBI) until the end of AraC therapy. In all 28 cases, there were no early stage deaths due to regimen-related toxicity (RRT). None of the 10 AML cases treated with the G-CSF combined regime relapsed. In 6 AML cases treated conventionally without G-CSF, one patient died of infection and another relapsed. There were no relapses in either CML group. In the combined G-CSF group, one patient died of interstitial pneumonitis 48 days after BMT, while the rest of the CML cases are still alive. There were no relapses with rhG-CSF and no serious adverse effects in terms of RRT, acute graft vs. host disease (GVHD), or leukocyte recovery. Am. J. Hematol. 57:303–308, 1998. © 1998 Wiley-Liss, Inc.     

Novel monoclonal antibody-based therapies for acute myeloid leukemia

There has been long-standing interest in using monoclonal antibodies to improve outcomes of people with acute myeloid leukemia (AML). While several candidate therapeutics have failed at various stages of clinical testing, improved survival of some patients receiving the CD33 antibody-drug conjugate gemtuzumab ozogamicin has provided first evidence that monoclonal antibodies have a role in the armamentarium against AML. Over the last several years, work to improve the success of monoclonal antibody-based therapies in AML has focused on the identification and exploration of new antigen targets as much as on the development of novel treatment formats such as use of unconjugated engineered monoclonal antibodies and conjugated antibodies, delivering highly potent small molecule drugs or radionuclides to AML cells. Here, we will provide a brief overview of current efforts with such investigational monoclonal antibody-based therapeutics.     

Role of endogenous platelet-activating factor in the regulation of pancreatic blood flow during caerulein stimulation

The role of endogenous platelet-activating factor (PAF) in the control of pancreatic blood flow during caerulein stimulation was investigated. Pancreatic blood flow in anesthetized rats was measured continuously by laser Doppler flowmetry for 2h during the intravenous infusion of caerulein (0.25 μg/kg per h). Pancreatic blood flow showed a gradual, consistent, and significant increase, reaching 114.2±2.3% of the basal value after 120 min. Changes in pancreatic blood flow induced by caerulein were completely inhibited by a cholecystokinin (CCK) antagonist (loxiglumide, 5 mg/kg per h, i.v.) and by a specific PAF antagonist (CV-6209, 1 mg/kg, i.v.-bolus). Systemic blood pressure remained stable in all groups. These results suggest an important role of endogenously yielded PAF in regulating pancreatic blood flow during caerulein stimulation to the pancreas.     

流式细胞术应用于金黄色葡萄球菌体外药敏试验的初步研究

目的　建立金黄色葡萄球菌体外流式细胞荧光法药敏试验新方法。方法　选用碘化丙啶作为荧光染料 ,用流式细胞术检测 2株标准株和 5 6株金黄色葡萄球菌临床菌株对苯唑西林的敏感性。根据细菌培养物在不同浓度药物作用后所检测到的荧光强度来判断细菌存活率 ,从而推断MIC值。并与微量稀释法和VITEK仪检测结果进行比较。结果　 5 8株金黄色葡萄球菌的流式细胞荧光法药敏试验结果与VITEK仪和微量稀释法结果完全一致。结论　所建立的金葡菌流式细胞荧光法药敏试验 ,具有快速、准确、客观等优点。     

参芪扶正注射液对急性髓系白血病患者树突状细胞作用的体外研究

目的研究参芪扶正注射液对完全缓解期急性髓系白血病患者树突状细胞的影响,并探讨其调节免疫功能的机制。方法分离培养20例急性髓系白血病患者的树突状细胞,与不同浓度参芪扶正注射液共同培养,MTT法检测其增殖率,流式细胞术检测CD1a、CD83、CD80和CD86的表达,酶联免疫吸附法检测其分泌IL-12的水平,RT-PCR法检测吲哚胺2,3双加氧酶（IDO）的表达。结果参芪扶正注射液对急性白血病患者树突状细胞的增殖和凋亡无明显影响。加入参芪扶正注射液后树突状细胞表达CD80和CD86增多,分泌IL-12明显增加,而表达IDO减少,且具有剂量依赖关系。结论参芪扶正注射液对完全缓解期急性白血病患者的树突状细胞细胞具有明显的影响,提示其可能临床应用于完全缓解期急性白血病患者的免疫调节治疗．     

GM-CSF stimulates proliferation of clonal leukemic bone marrow cells in acute myeloid leukemia (AML) in vitro

 Granulocyte-macrophage colony-stimulating factor (GM-CSF) is known to stimulate granulocytes, monocytes, and macrophages. We studied the effect of GM-CSF on (clonal) bone marrow (BM) cells obtained from AML patients after 7 days of culture in vitro: BM samples were obtained from 19 AML patients at diagnosis (DIA), from two patients with persisting disease (PERS), from eight patients in complete remission (CR), and from 12 healthy donors. Flow-cytometric comparison of differentiated, CD 15-positive cells or of CD34-positive blast cells before and after cultivation showed that the proportion of CD15-positive cells was increased in nine of 12 healthy BM samples, in 14 of 19 cases at DIA, in one of three cases during PERS, and in five of six cases in CR of AML. The proportion of CD34-positive cells was increased in one of 12 healthy BM samples, in seven of 19 cases at DIA, in one of two cases during PERS, and in three of seven cases in CR of AML. Southern blot analysis (SBA) performed in six cases during the course of AML, before and after cell culture, showed that clonal DNA increased after GM-CSF treatment in three of five cases studied at DIA, in six of nine cases studied in CR, in the one case studied at PERS, and in the one studied at relapse (REL). In one case of trisomy 8 at DIA a normal karyotype was demonstrated in CR. However, after 7 days of cultivation of the cells in GM-CSF the trisomy 8 was detected in two of 17 metaphases isolated from colony-cells from methylcellulose cultures. Our data show that a 7-day treatment of BM cells with GM-CSF induced a differentiation of healthy and leukemic BM cells in the great majority of cases. An enrichment of CD34-positive cells was not achieved in healthy BM samples. However, in 70% of the cases in CR and in 30% of the cases at DIA of AML, clonal CD34-positive cells were enriched. This means that GM-CSF stimulates ('primes') leukemic cell growth in vitro. Received: August 17, 1998 / Accepted: April 28, 1999     

36例献血者献血后网织红细胞检测的结果分析

目的探讨急性髓细胞白血病(AML)细胞尿激酶型纤溶酶原激活物受体CD87(uPAR)的表达及其在诊断急性白血病分型、指导预测预后方面的意义。方法应用流式细胞术(FCM)检测了41例初治的AML患者(包括M12例,M217例,M45例,M517例),应用t检验及χ2检验分析CD87阳性与CD87阴性患者在临床表现、染色体异常及完全缓解率间的差异,应用logistic回归分析影响患者完全缓解的具有统计学意义的因素。结果41例AML患者,26例CD87阳性表达,CD87阳性与CD87阴性AML患者比较肝肿大比例分别为73%与40%,(P<0.05),而淋巴结肿大、脾肿大比例无明显的统计学意义(P>0.05);出血倾向比例分别为77%与46%,(P<0.05);染色体异常的比例分别为73%与40%,(P<0.05);1疗程完全缓解率分别为40%与73%,(P<0.05),CD87阳性是导致患者完全缓解率低的独立因素。结论CD87表达有助于AML的诊断,CD87阳性与疾病进展包括浸润、出血、缓解率低等不良预后相关。