A New Method to Collect Granulocytes Using a Low Dose of Hydroxyethyl Starch

A new granulocyte collection method employing discontinuous flow centrifugation and low donor dose of hydroxyethyl starch (HES) is presented. Following the procedure, the increase in serum polysaccharide concentration was 100 mg/dl, about 20% of that seen after standard discontinuous flow leukapheresis. The average efficiency of granulocyte collection by this method is about 64%. The average yield of WBC from 2,600 ml of donors' whole blood is 1.64 +/- 0.54 x 10(10) (mean +/- 1 SD) and the average granulocyte yield is 1.33 +/- 0.51 x 10(10) (mean +/- 1 SD). The granulocytes were shown to migrate to the sites of infection in vivo. This approach is an improvement over standard discontinuous flow leukapheresis because of the high efficiency of granulocyte collection and because multiple procedures are possible without exposing donors to more HES than is currently given during a single procedure.     

Collection of Large Quantities of Granulocyte/Macrophage Progenitor Cells (CFUc) in Man by Means of Continuous-Flow Leukapheresis

Continuous-flow leukapheresis using the AMINCO Celltrifuge® was performed in 35 adult volunteers for the procurement of large quantities of granulocyte/macrophage progenitor cells (CFUc) as part of the mononuclear leucocytes. The donor blood volume processed was 12 1 at a flow rate of 50 ml per min. The centrifuge speed was 800 rpm. The mean yield of mononuclear cells (MNC) per run was 12.0 times 10⁹ and that of CFUc, 8.7 times 10⁵. The leukapheresis did not much affect the donor blood cell concentration except the platelet concentration; this must be considered as a limiting factor for leukapheresis. The mean granulocyte contamination of the leukapheresis-derived leucocytes was only about 10%. Successive leukaphereses (with short-term intervals) in 7 donors led to significant increases in CFUc yield. The possible therapeutic application of blood stem cells in the treatment of haemopoietic failure in man is discussed.     

Fasting (Acute Energy Deprivation) in Man: Effect on Polymorphonuclear Granulocyte Functions,Plasma Iron and Serum Transferrin

The effect of 10 days of total fasting (energy deprivation) on blood polymorphonuclear granulocyte functions, leukocyte numbers, iron and transferrin levels was evaluated in 14 healthy, normal-weight males. Granulocytes from 7 of the subjects were tested in vitro. A statistically significant depression was noted in their bactericidal capacity against Staph. aureus. The 14 subjects showed a marked decrease in the stainable activity of granulocyte alkaline phosphatase and decreases were noted in plasma iron and serum transferrin levels. The iron saturation of serum transferrin was unchanged. Thus, impairment of granulocyte bactericidal functions may occur secondarily to short-term total energy deprivation, in the absence of iron deficiency.     

中分子羟乙基淀粉治疗急性脑梗死的临床疗效观察

目的:探讨中分子羟乙基淀粉治疗急性脑梗死的临床疗效.方法:将68例急性脑梗死患者随机分成治疗组和对照组,每组各34例,对照组给予常规治疗,治疗组在常规治疗同时加用中分子羟乙基淀粉500ml静脉滴注,每天1次,连用7d;分别于治疗前和治疗后7d、治疗后14d进行NIHSS评分和BI评分.结果:治疗后7d、14d治疗组NIHSS评分较对照组明显下降;BI明显上升(P＜0.01).结论:中分子羟乙基淀粉治疗急性脑梗死疗效显著.     

The Effects of Streptokinase and Hydroxyethyl Starch on in vitro Clot Disruption by Ultrasound

Background: In vitro studies showed that low-frequency ultrasound (US) causes blood clot dissolution. This effect is augmented with thrombolytics, microbubbles and microparticles. However, in animal models of transcutaneous delivery, US alone is not effective, probably due to attenuation of US energy by overlying skin. When combined with thrombolytics or microbubbles, transcutaneous US is highly effective. Purpose: To assess the synergistic effect of low-intensity low-frequency US and saline, hydroxyethyl starch (HAES) (a non-gas filled microparticle containing solution), streptokinase (STK), and their combination on blood clot disruption. Methods: Human blood clots from 4 healthy donors, 2–4 hours old, were immersed for 0, 15, or 30 min in 37°C in 10 ml of the above-mentioned solutions, and then were randomized to 10 sec of 20 kHz US or no US. The % difference in weight was calculated. Results: Immersion for 30 min without US resulted in 13.8 ± 1.2% clot lysis in saline, and 22.0 ± 1.3%, 21.7 ± 2.1%, and 23.2 ± 1.9% in STK, HAES, and STK + HAES, respectively (p = 0.002). US augmented clot lysis in all groups and at all time points. With low-intensity US, HAES was not better than saline. However, the combination of HAES + STK with US resulted in larger clot disruption at 15 sec incubation time (46.7 ± 3.2%) than with saline (29.6 ± 2.1%), HAES (29.6 ± 2.5%), and STK (32.8 ± 3.6%) (p < 0.001). Conclusion: low-frequency, low-intensity US combined with HAES and STK resulted in greater clot disruption at short incubation times. This combination may assist in achieving faster reperfusion in in vivo models.     

Generalized Shwartzman Reaction: Role of the Granulocyte in Intravascular Coagulation and Renal Cortical Necrosis

The protective effect of nitrogen mustard-induced neutropenia on the development of the GSR was studied. No coagulation defect was found in neutropenic rabbits and simultaneous administration of Thorotrast and endotoxin did not produce intravascular coagulation or renal cortical necrosis. Infusion of neutropenic rabbits with suspensions of viable granulocytes derived from peritoneal exudates reinduced susceptibility to both intravascular coagulation and renal cortical necrosis. It is concluded that the presence of granulocytes is essential for the production of intravascular coagulation by endotoxin.     

羟乙基淀粉130联合呋塞米治疗急性肺损伤的疗效观察

目的:探讨羟乙基淀粉130联合呋噻米治疗急性肺损伤和/或急性呼吸窘迫综合征(ALI/ARDS)的临床疗效。方法:设治疗组40例ALI/ARDS应用羟乙基淀粉130联合呋噻米治疗,对照组14例单独应用呋噻米治疗,治疗组观察治疗前、静脉滴注羟乙基淀粉130后及接着静脉推注呋噻米后1h的氧合指数(PaO2/FiO2)、PaCO2、R、HR指标。对照组观察治疗前后的上述指标。结果:治疗组患者应用羟乙基淀粉130联合呋噻米治疗后,氧合指数(PaO2/FiO2)、R、PaCO2、HR较前明显改善(P<0.05),且两者合用较单独应用羟乙基淀粉130效果更加明显。结论:应用羟乙基淀粉130联合呋噻米治疗ALI/ARDS能明显改善症状,并且改善肺功能,减轻肺水肿。     

Granulocyte colony-stimulating factor (G-CSF) accelerates reendothelialization and reduces neointimal formation after vascular injury in mice

OBJECTIVE: Neointimal formation following percutaneous coronary intervention (PCI), termed restenosis, limits therapeutic revascularization. Since reendothelialization is one of the determinant factors for the development of neointimal formation, we examined the effects of granulocyte colony-stimulating factor (G-CSF) on reendothelialization and neointimal formation after vascular injury in mice. METHODS AND RESULTS: Wire-mediated vascular injury was produced in the femoral artery of C57BL/6 mice. G-CSF pretreatment significantly accelerated reendothelialization and decreased neointimal formation following vascular injury; however, this inhibitory effect of G-CSF was diminished when G-CSF was started following the injury. Flow cytometry analysis revealed that G-CSF treatment increased the number of endothelial progenitor cells (EPCs: CD34+/Flk-1+) in the peripheral circulation. Vascular injury was also produced in 2 types of mice whose bone marrow was replaced with that of enhanced green fluorescent protein- and Tie2/LacZ-transgenic mice. In the reendothelialized artery of these mice, few bone marrow-derived EPCs were detected. Furthermore, G-CSF treatment reduced the serum level of interleukin (IL)-6. CONCLUSION: G-CSF treatment accelerated reendothelialization and decreased neointimal formation following vascular injury, although there was little contribution of bone marrow-derived EPCs to the reendothelialization of the artery. These results suggest that G-CSF pretreatment has a therapeutic potential for prevention of restenosis following PCI.     

Azelnidipine and Amlodipine Anti-Coronary Atherosclerosis Trial in Hypertensive Patients Undergoing Coronary Intervention by Serial Volumetric Intravascular Ultrasound Analysis in Juntendo University (ALPS-J)

Purpose

Many trials have shown that calcium channel blockers (CCBs) can reduce the cardiovascular (CV) events in patients with coronary artery disease (CAD). The mechanisms of this effect could be associated with plaque regression due to the anti-atherosclerotic properties of CCBs. The goal of this study is to determine the effects of CCB on volumetric quantitative changes of coronary plaques accessed by intravascular ultrasound (IVUS). To confirm this hypothesis, a multicenter randomized trial of CCBs treatment with azelnidipine or amlodipine will be conducted in hypertensive CAD patients undergoing elective percutaneous coronary intervention (PCI).

Methods and Results

Patients who have hypertension and are scheduled for PCI will be enrolled. Subjects will be randomized to azelnidipine or amlodipine and observed for 48 weeks. The primary endpoint will be the percent change of coronary plaque volume. The secondary endpoint will include inflammatory markers, antioxidant activity, and incidence of composite cardiovascular events.

Conclusions

In this study, we will investigate the improvement of coronary plaque with IVUS by treatment with two dihydropyridine CCBs in hypertensive patients undergoing elective PCI. This result will lead to the discovery of more effective drug therapy for inhibition of coronary events.     

Serial volumetric (three-dimensional) intravascular ultrasound analysis of restenosis after directional coronary atherectomy

Objectives. We report the use of three-dimensional (volumetric) intravascular ultrasound (IVUS) analysis to assess serial changes after directional coronary atherectomy (DCA).

Background. Recent serial planar IVUS studies have described a decrease in external elastic membrane (EEM) area following catheter-based intervention as an important mechanism of late lumen renarrowing.

Methods. Thirty-one patients with de novo native coronary lesions treated with DCA in the Serial Ultrasound Restenosis (SURE) Trial and in Optimal Atherectomy Restenosis Study (OARS) were enrolled in this study. Serial IVUS was performed before and after intervention and at 6 months’ follow-up. In a subgroup of 18 patients from the SURE trial, IVUS was also performed at 24 h and at 1 month postintervention. Segments, 20-mm-long (200 image slices), were analyzed using a previously validated three-dimensional, computerized, automated edge-detection algorithm. The EEM, lumen, and plaque+media (P+M = EEM−lumen) volumes were calculated.

Results. At follow-up, lumen volume was smaller than at postintervention (159 ± 69 mm³ vs. 179 ± 49 mm³, p = 0.0003). From postintervention to follow-up, there was a decrease in EEM volume (377 ± 107 to 352 ± 125 mm³, p < 0.0001), but no change in P+M volume (p = 0.52). The Δ lumen volume correlated strongly with ΔEEM volume (r = 0.842, p < 0.0001), but not with ΔP+M volume. In the 18 patients from the SURE Trial, the decrease in lumen and EEM volumes occurred late, between 1 month and 6 months of follow-up.

Conclusions. Volumetric IVUS analysis demonstrated that late lumen volume loss following DCA was a result of a decrease in EEM volume. This was a late event, occurring between 1 and 6 months’ postintervention.     

Granulocyte modulation of endotoxin-stimulated colony-stimulating activity (CSA) production

Incubation of human macrophages with endotoxin resulted in significantly enhanced colony-stimulating activity (CSA) production by these cells. Preincubation of endotoxin with mature granulocytes abolished this stimulatory effect. The stimulatory effect of endotoxin on macrophage CSA production was not abolished, however, by preincubation with NaF-treated granulocytes, granulocyte membranes, or nonphagocytic cells (lymphocytes or erythrocytes). These data suggest that mature granulocytes may play a role in the modulation of CSA production and granulopoiesis by inactivation of stimulatory materials such as endotoxin.     

Expression of Granulocyte Colony Stimulating Factor (G-CSF) and Granulocyte/Macrophage Colony Stimulating Factor (GM-CSF) mRNA upon Stimulation with Phorbol Ester

Stimulated human peripheral blood mononuclear cells (MNC) have been shown to express both G-CSF and GM-CSF. Furthermore, G-CSF is expressed by monocytes but not lymphocytes, whereas GM-CSF is expressed largely by T lymphocytes and at low levels in monocytes/macrophages. Here we present the effect of TPA (12-O-tetradecanoyl phorbol-13-acetate) on G-CSF and GM-CSF expression in stimulated human MNCs and T lymphocytes. We observed that TPA (30nM) decreased G-CSF mRNA levels in MNCs, while ionomycin increased G-CSF in a dose-dependent manner. TPA and ionomycin individually increased GM-CSF mRNA levels in T-lymphocytes and MNCs. Further, GM-CSF was induced synergistically by TPA plus ionomycin, whereas this combination markedly decreased G-CSF mRNA levels in MNCs.These data suggest at least two signaling pathways by which G-CSF and GM-CSF mRNA levels are modulated in a mixed population of monocytes and T lymphocytes, namely protein kinase C (PKC) and calcium. These signals seem to act synergistically in lymphocytes to increase GM-CSF, and not G-CSF mRNA levels specifically. It would also appear these signals act on MNCs in an opposing manner to decrease G-CSF mRNA levels, indicating that activation of PKC and the calcium signaling pathway lead to a cell-type specific modulation of individual cytokines and precise regulation of granulocyte production.     

Intravascular ultrasound assessed incomplete stent apposition and stent fracture in stent thrombosis after bare metal versus drug-eluting stent treatment the Nordic Intravascular Ultrasound Study (NIVUS)

Background

This prospective multicenter registry used intravascular ultrasound (IVUS) in patients with definite stent thrombosis (ST) to compare rates of incomplete stent apposition (ISA), stent fracture and stent expansion in patients treated with drug-eluting (DES) versus bare metal (BMS) stents. ST is a rare, but potential life threatening event after coronary stent implantation. The etiology seems to be multifactorial.

Methods

124 patients with definite ST were assessed by IVUS during the acute ST event. The study was conducted in 15 high-volume percutaneous coronary intervention -centers in the Nordic–Baltic countries.

Results

In early or late ST there were no differences in ISA between DES and BMS. In very late ST, ISA was a more frequent finding in DES than in BMS (52% vs.16%; p = 0.005) and the maximum ISA area was larger in DES compared to BMS (1.1 ± 2.3 mm² vs. 0.1 ± 0.5 mm²; p = 0.004). Further, ISA was more prevalent in sirolimus-eluting than in paclitaxel-eluting stents (58% vs. 37%; p = 0.02). Stent fractures were found both in DES (16%) and BMS (24%); p = 0.28, and not related to time of stent thrombosis occurrence. For stents with nominal diameters ≥ 2.75 mm, 38% of the DES and 22% of the BMS had a minimum stent area of less than 5 mm²; p = 0.14.

Conclusions

Very late stent thrombosis was more prevalent and associated with more extensive ISA in DES than in BMS treated patients. Stent fracture was a common finding in ST after DES and BMS implantation.     

Granulocyte colony-stimulating factor (G-CSF) induces the production of cytokines in vivo

Granulocyte colony-stimulating factor (G-CSF) is a haematopoietic growth factor required for the proliferation and differentiation of haematopoietic precursors of neutrophil granulocytes and is now used to overcome congenital and acquired neutropenia. In addition to increasing the numbers of neutrophils in vivo and modulating neutrophil functions, G-CSF may induce the production of cytokines such as tumour necrosis factor alpha (TNF-alpha). In the present study, the plasma levels of granulocyte-macrophage colony-stimulating factor (GM-CSF) in six healthy volunteers given G-CSF at 10 microgram/kg once daily for 6 d were measured and found to be elevated. The elevated levels (P < 0.05) were detected on day 2, peaked on days 6-7 and returned to baseline on day 12. In vitro, G-CSF did not enhance the secretion of TNF-alpha and GM-CSF from mononuclear cells, whole blood or endothelial cells. However, in the co-presence of whole blood and endothelial cells, the secretion of TNF-alpha was significantly enhanced by G-CSF at low concentrations. The GM-CSF secretion, however, was unaltered. G-CSF pretreatment of whole blood suppressed lipopolysaccharide (LPS)-induced secretion of TNF-alpha and GM-CSF in a dose-dependent manner. These results together with our previous findings suggest that G-CSF induces the production of TNF-alpha and GM-CSF in vivo, and that this production may be due to the co-effects of endothelial cells and whole blood under the influence of G-CSF through an as yet unknown network of cells and cytokines. Treatment of whole blood with G-CSF suppresses LPS-induced secretion of TNF-alpha and GM-CSF.     

Granulocyte colony stimulating factor (G-CSF) in diagnosis and monitoring of non-small-cell lung cancer (NSCLC)

Granulocyte-colony stimulating factor (G-CSF) is one of the glycoproteins called colony-stimulating factors (CSFs). It has been shown that the target of the actions of CSFs are not limited to hematopoietic cells but can also affect the proliferation of nonhematopoietic cells. Some clinical investigations have shown the presence of cell surface receptors for G-CSF in lung cancer cells and autologous production of G-CSF in various human cell lines derived from non-small-cell lung cancer (NSCLC). The purpose of this investigation was to compare serum levels of G-CSF in NSCLC patients to a control group, to assess pre- and post treatment levels of G-CSF in relation to levels of commonly accepted tumour markers such as carcinoembryonic antigen (CEA) and cytokeratin fragment 19 (CYFRA 21-1), and to define the sensitivity of G-CSF in NSCLC. In this study, the serum levels of tumour markers were measured in 34 patients with NSCLC and in 20 healthy subjects. Serum samples were drawn before surgery and 10, 30, 90, 180 and 270 days after surgery. G-CSF and CEA were assayed using ELISA system and CYFRA 21-1 was measured by radioimmunoassay (RIA). Preoperative level of G-CSF was significantly increased in cancer patients relative to the control group. Concentrations of G-CSF and CYFRA 21-1 were decreased on the 10th day, but CEA on the 30th day after operation. The diagnostic sensitivity of G-CSF was 66%, CEA--62% and CYFRA 21-1--51%. Combined use of two markers increased the sensitivity in comparison to the use of G-CSF only. These results suggest that G-CSF may be useful in diagnostic and monitoring of NSCLC, but they need further studies.