APO-1-induced apoptosis of leukemia cells from patients with adult T- cell leukemia

The 48-Kd cell-surface protein APO-1 is a new member of the nerve growth factor (NGF)/tumor necrosis factor (TNF) receptor superfamily. APO-1 is expressed on various cells, including activated T and B cells and some lymphoid and nonlymphoid cell lines. Triggering of APO-1 by the monoclonal antibody anti-APO-1 induces programmed cell death (apoptosis) in APO-1-expressing cells. APO-1 is also present on T-cell lines derived from patients with adult T-cell leukemia (ATL). Therefore, we investigated APO-1 expression and APO-1-mediated induction of apoptosis ex vivo in cells from patients with ATL. Fresh leukemic cells from nine patients with ATL were assayed for APO-1 expression by two-color immunofluorescence. The leukemic cells from all patients strongly expressed APO-1. Incubation of ATL cells with anti- APO-1 in vitro inhibited spontaneous and cytokine-mediated DNA synthesis. Furthermore, DNA isolated from cells treated with anti-APO-1 exhibited polynucleosomal DNA fragmentation (DNA ladder) characteristic for apoptotic cell death. The analysis of APO-1-mediated apoptosis may represent a new approach to the study of growth control in lymphoid malignancies. In addition, induction of apoptosis by administration of anti-APO-1 may represent a new therapeutic approach for aggressive T- cell malignancies such as ATL.     

Treatment of acute graft-versus-host disease with humanized anti-Tac: an antibody that binds to the interleukin-2 receptor

Humanized anti-Tac is a genetically engineered human IgG1 monoclonal antibody specific for Tac, the alpha subunit of the interleukin-2 (IL- 2) receptor, and blocks IL-2-dependent activation of human T lymphocytes. The safety, pharmacokinetics, and immunosuppressive activity of humanized anti-Tac were evaluated in 20 patients who developed acute graft-versus-host disease (GVHD) after allogeneic marrow transplantation. Patients had developed acute GVHD at 5 to 26 (median, 14) days after transplantation and had failed to respond to primary therapy with glucocorticoids. Sequential groups of 4 patients each received a single 1-hour infusion of antibody in escalating doses of 0.5, 1.0, or 1.5 mg/kg; 8 additional patients were then treated with 1.5 mg/kg. A second infusion of antibody was administered after 11 to 48 (median, 16) days in 8 patients who had transient improvement of GVHD after the first infusion. Acute side effects, limited to chills in 1 patient and diaphoresis in another, were observed during or shortly after the antibody infusion. Overall improvement of acute GVHD occurred in 8 patients, 6 of whom were treated with a single antibody infusion and 2 with two infusions. Four responses were complete and 4 were partial. Three additional patients had improvement in one organ but progression in another. Responses occurred in 9 of 16 cases with skin disease, 3 of 15 with liver disease, and 6 of 12 with gastrointestinal disease. Two patients survive at 529 and 645 days after antibody treatment. Two patients died after relapse of leukemia. Sixteen patients died of infection or organ failure between 5 and 211 (median, 55) days. The terminal elimination half-life of the antibody was 44 to 363 hours, with a harmonic mean of 79, 88, and 94 hours, respectively, for the three doses studied. Absolute peripheral blood T-lymphocyte counts remained unchanged during the 56 days after infusion of the antibody. A fraction of circulating T cells expressed the alpha chain of the IL-2 receptor that, in some patients, was bound by antibody in vivo up to 28 days after treatment. No patient developed a measurable antibody response to humanized anti-Tac. Humanized anti-Tac has a long half-life after intravenous injection in humans, superior to any rodent monoclonal antibody specific for human T cells, and does not appear to induce antibody formation in recipients of marrow transplants. Improvement of steroid-refractory GVHD in 40% of patients after only one or two antibody infusions indicates that humanized anti-Tac is immunosuppressive.     

Cerebrospinal Fluid from Subarachnoid Haemorrhage Patients Causes Excessive Oxidative Metabolism Compared to Vascular Smooth Muscle Force Generation

Summary  Cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients can stimulate vascular smooth muscle to generate force in vitro. CSF from SAH patients suffering from delayed ischaemic neurological deficits due to cerebral vasospasm can generate near maximal force in vitro and previous experiments have ascribed this generation of force to be a calcium mediated event. The intracellular calcium concentration has been demonstrated to rise during the vasospastic process. Calcium also stimulates oxidative metabolism as does adenosine diphosphate (ADP), the product of adenosine triphosphate (ATP) hydrolysis. Significant alteration in high energy metabolites such as ATP, ADP and phosphocreatine have also been demonstrated in various models of SAH mediated vasospasm. Vascular smooth muscle predominantly uses oxidative metabolism for force generation and reserves glycolytic metabolism for ion homeostasis. A decrease in oxidative metabolism during force generation would imply failing mitochondria and increased glycolytic high-energy phosphate supply. Increased oxidative metabolism would imply a decreased efficiency of the contractile apparatus or mitochondria.  The aim of this study was to see if SAH CSF stimulation of porcine carotid artery oxidative metabolism was altered during force generation when compared with incremental calcium stimulation with potassium chloride depolarisation. CSF from patients (n=10) who had subarachnoid haemorrhage stimulated force generation but with a significant `right shift' in oxygen consumption. This `right shift' is indicative of an increased energy cost for contractile work. These results suggest that vascular smooth muscle contractile apparatus, when stimulated by subarachnoid cerebrospinal fluid, is consuming excess adenosine triphosphate during force generation.     

The Stimulation of Vascular Smooth Muscle Oxidative Metabolism by CSF from Subarachnoid Haemorrhage Patients Increases with Fisher and WFNS Grades

Summary  The purpose of this paper is to present an in vitro method for examining cerebral vasospasm after subarachnoid haemorrhage (SAH) which correlates to the patients' condition. The O₂ consumption of the porcine carotid artery was monitored, using an oxygen electrode, after exposure to cerebrospinal fluid (CSF) from patients who had a SAH. The vessels were exposed to CSF from SAH patients at a 1 in 30 dilution. Force measurements were carried out using freeze-dried CSF, reconstituted in the organ bath equivalent to undiluted CSF. These observations were then compared to the patients' condition.  We divided the patient CSF samples into those that stimulated oxygen consumption above 0.4 μM/min/g dry wt, and those that did not. It was found that there was a correlation between the stimulation of oxygen consumption and the Fisher grade as well as the World Federation of Neurosurgeons Grading System (WFNS) for the patients. Of the CSF tested, 24 stimulated oxygen consumption above our cut off, and 8 did not (0.84±0.34, n=24 compared with the rate of 0.27±0.1 μmol/min/g dry wt, respectively; SD n=8) at 180 minutes. We then examined the Fisher Grades of these two groups, the results were 3.21±0.88 vs 2.25±0.83 respectively (SD p≤0.01). When examining the WFNS System we found a similar difference between the groups that stimulated respiration and those who did not (WFNS Grades of 2.64±1.1 vs. 1.43±0.53; p≤0.01). The observed stimulation of oxygen consumption also correlated with tension generation in vitro.  The CSF from subarachnoid haemorrhage patients stimulates the oxygen consumption of the porcine carotid artery. This stimulation correlated to the WFNS and Fisher Grades of the patients and can be performed using 1:30 dilution of CSF. We conclude that the metabolic changes that occur in the vessels during vasospasm are important parameters for assessing cerebral vasospasm.     

In vitro therapy with dobutamine, isoprenaline and sodium nitroprusside protects vascular smooth muscle metabolism from subarachnoid haemorrhage induced cerebral vasospasm

Summary  Background. The cerebrospinal fluid (CSF) from subarachnoid haemorrhage (SAH) patients with cerebral vasospasm stimulates vasoconstriction and oxygen consumption in the porcine carotid artery in vitro. Stimulation of oxygen consumption has been used as an in vitro model of vasospasm to assess the relative benefits of nimodipine, isoprenaline, dobutamine, and sodium nitroprusside (SNP).  Method. Samples of human CSF were obtained from SAH patients and applied to de-endothelialised porcine carotid artery. Stimulation of oxygen consumption (as an in vitro marker for a stimulation of the vessels) was monitored and the effects of SNP, isoprenaline, dobutamine or nimodipine were measured.  Findings. The CSF from SAH patients with evidence of vasospasm stimulated oxygen consumption to 0.91±0.17 (μ M O₂/min/g dry wt, ± SD p≤ 0.01) and CSF from SAH patients without vasospasm did not significantly stimulate oxygen consumption 0.27±0.02, with 0.23±0.03 (μ M O₂/min/g dry wt) being an unstimulated rate of respiration for the porcine carotid artery. SNP, isoprenaline or dobutamine significantly (p≤ 0.01) decreased the stimulation of oxygen consumption of the porcine carotid artery whereas nimodipine did not. In a cohort of 41 SAH patients who received nimodipine alone or nimodipine and dobutamine, the in hospital mortality rate of the patients who received only nimodipine was 42% as compared to an in hospital mortality rate of 17% in the nimodipine plus dobutamine group P≤ 0.076).  Interpretation. The in vivo data on the 41 patients is not statistically significant, so further studies are required to determine if the differences are important. SNP, isoprenaline and dobutamine significantly decreased oxygen consumption of the porcine carotid arteries exposed to CSF from SAH patients who had vasospasm whereas nimodipine did not. Our in vitro results suggest that these compounds require further study in patients with SAH who are at risk for vasospasm because they may have a direct benefit for the vasospastic arteries.     

缺血性卒中的高血糖治疗

高血糖能加重急性脑梗死的脑损伤。研究者测试了急性脑梗死时静脉胰岛素强化治疗与普通护理相比的可靠性和耐受性，开展了一个12h内脑梗死的随机多中心双盲实验，其血糖水平≥8．3mmol／L，美国国立卫生研究所脑卒中评分（NIHSS）在3～22分。患者以2：1的比例被随机分配至连续性静脉输注胰岛素或规定每日4次皮下注射胰岛素的强化治疗组。强化治疗组的目标血糖水平〈7．2mmol／L，     

纳米活性炭吸附氟尿嘧啶在胃癌荷瘤裸鼠体内释放及抑瘤作用的观察

目的:观察自制的5-氟尿嘧啶(5-FU)-纳米活性炭(ACNP)在胃癌荷瘤裸鼠体内的释放和抑瘤效果。方法:在BALB/c/nu裸鼠腹腔内移植人胃癌细胞系SGC7901,建立转移模型并随机分组。A组:腹腔内注入5-FU;B组:5-FU-AC-NP;C组:纯生理盐水。1)于不同时间点内眦静脉采血,高效液相色谱检测血浆中5-FU浓度;2)观察各组小鼠的存活状况,比较各组腹腔内瘤结节的质量和大小。结果:5-FU-ACNP组与5-FU组比较,前者血中的5-FU吸收、消除半衰期延长,峰浓度减低且变化平稳,P<0.05;裸鼠腹腔内瘤结节较轻、较少,P<0.05,裸鼠的中位存活时间延长,P≤0.01。结论:自制的ACNP结合5-FU经腹腔给药后,药物入血缓慢,血浆中的浓度低,半衰期延长,毒副反应减轻,对裸鼠腹腔移植瘤有较强抑制作用。     

Cryopreservation of single human spermatozoa

A procedure is described that allows cryopreservation and efficient post-thaw recovery of either a single or a small group of human spermatozoa. This is achieved by injecting them into cell-free human, mouse or hamster zonae pellucidae before the addition of cryoprotectant. The method involves a combination of physical micromanipulation procedures and glycerol-mediated cryoprotection. Zonae were tracked by positioning them in straws between two small air bubbles prior to freezing. Spermatozoa from poor specimens were cryopreserved and their fertilizing ability after thawing was compared with that of fresh spermatozoa from fertile men. Human eggs used for fertilization testing were either 1 day old or in-vitro matured. Only 2% of the frozen zonae were lost and >75% of spermatozoa cryopreserved in this manner were recovered and prepared for intracytoplasmic sperm injection. The feasibility of cryopreserving a single spermatozoon was assessed. Fifteen motile spermatozoa were frozen in 15 zonae, of which 14 were recovered after thawing. Ten were injected into spare eggs, of which eight became fertilized. Spermatozoa recovered mechanically from human zonae fertilized the same proportion of oocytes as fresh fertile control spermatozoa. The recovery and fertilization rates with spermatozoa frozen in animal zonae were 87 and 78% respectively. The fertilization rate was marginally higher (P < 0.05) than that for spermatozoa frozen in human zonae, perhaps because the latter may have acrosome reacted more frequently. The zona pellucida appears to be an ideally suited sterile vehicle for storage of single spermatozoa.