Quantitation of white cell subpopulations by polymerase chain reaction using frozen whole-blood samples. Viral Activation Transfusion Study

BACKGROUND: Previous methods for processing whole blood (WB) for nucleic acid analyses of white cells (WBCs) required fresh blood samples. A simple protocol that involves the freezing of WB for quantitative polymerase chain reaction (PCR) analyses was evaluated. STUDY DESIGN AND METHODS: Controlled studies were conducted in which paired fresh and frozen WB preparations were analyzed. The integrity of WBCs in the frozen WB samples was first assessed by flow cytometry using CD45 fluorescence, and calibration beads to quantitate recovery of WBC subsets. PCR of an HLA-DQ-A sequence was used to quantitate residual WBCs in a double-filtered red cell (RBC) component spiked with serial dilutions of WBCs, as well as in 51 filtered RBCs and 19 filtered platelet concentrates. Y-chromosome-specific PCR was used to quantitate male WBCs in five female WB samples spiked with serial dilutions of male WBCs and in serially collected frozen WB samples from four females transfused with male blood components. RESULTS: By flow cytometry, all major WBC subpopulations in frozen-thawed WB were quantitatively recovered and immunologically intact, although they were nonviable. HLA-DQ-A PCR quantitation of a dilution series from 8 to 16,700 per mL of WBCs spiked into double-filtered RBCs showed linear correlation of the results with both fresh and frozen preparations of the expected WBC concentrations (r2 = 0.98, p<0.0001 for both), without significant difference between observed and expected values (p>0.05). Y- chromosome-specific PCR results in female WB samples spiked with male WBCs were not significantly different in fresh and frozen preparations over a 3 log10 range of male cells. The results of WBC survival studies on frozen WB samples were consistent with previous observations in fresh blood samples. CONCLUSION: Direct freezing of WB enables subsequent recovery of WBCs for quantitative PCR analyses, with results comparable to those of fresh preparations. This protocol should facilitate wider implementation of nucleic acid-based analyses for quality control of WBC-reduced components, as well as for prospective clinical studies of microchimerism in transfusion and transplant recipients.     

Introduction of a novel self-injector for sumatriptan. A controlled clinical trial in general practice

A novel self-injector for the administration of subcutaneous sumatriptan in the treatment of migraine attacks was tested in 138 patients recruited by family physicians in Denmark; 108 patients completed the initial double-blind, crossover part of the study. Sumatriptan 6 mg s.c. was significantly better than placebo at 30, 60, 90 and 120 min after injection in relieving moderate or severe headache to mild or none as well as relieving any headache to none. At 60 min after injection, the treatment response rate was 61% for sumatriptan and 6% for placebo. During the following open-phase trial of four attacks treated with sumatriptan, treatment response rates were 68–74%. During the total of 538 attacks treated, 12 attempts at using the self-injector failed. In the double-blind and open phases, 81% and 90% of patients respectively found the device easy or very easy to use. Adverse effects were benign and short-lasting, but led seven patients to discontinue the study. In conclusion, subcutaneous sumatriptan administered with a novel self-injector is an effective treatment for migraine compared to placebo in patients treated by their family physician.     

Effect of intra-cisternal application of kainic acid on the spinal cord and locomotor activity in rats

Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease of idiopathic etiology. Glutamate excitotoxicity is one of the proposed hypotheses causing progressive death of motor neurons. We aimed to develop an experimental animal model of this disease to enhance the knowledge of pathophysiological mechanism of ALS. Male Wistar rats were infused with Kainic acid (KA) intra-cisternally for 5 days at the dosage of 50 fmol/day and 150 fmol/day. Locomotor activity, sensory function and histological changes in cervical and lumbar sections of spinal cord were evaluated. Glial Fibrillary Acidic Protein (GFAP) and Neurofilament Protein (NFP) were used as immunohistochemical marker for reactive astrogliosis and neuronal damage respectively. Specific Superoxide Dismutase (SOD) activity of spinal cord was estimated. The locomotor activity in the parameter of observed mean action time remained reduced on 14^th day after administration of KA. Spinal motor neurons under Nissl stain showed pyknosis of nucleus and vacuolation of neuropil. GFAP expression increased significantly in the lumbar section of the spinal cord with high dose of KA treatment (p<0.05). NFP was expressed in axonal fibres around the neurons in KA-treated rats. A significant increase in specific SOD activity in both cervical and lumbar sections of the spinal cord was found with low dose of KA treatment (p<0.05). This study concludes that spinal cord damage with some features similar to ALS can be produced by low dose intra-cisternal administration of KA.     

Platelet aggregation by fibrinogen polymers crosslinked across the E domain

There is evidence that platelet interactions with artificial surfaces are mediated by plasma proteins, especially fibrinogen, adsorbed on the surfaces. Multiple site interactions between fibrinogen molecules adsorbed in high concentration and receptors in the unactivated platelet may be sufficient for platelet adhesion and subsequent activation. To examine this hypothesis, we prepared soluble polymers of fibrinogen. Polymers produced by interaction of fibrinogen with Fab'2 fragments of antibodies against fibrinogen's E (central) domain (Fg- Fab'2(E] induced, in gel-filtered platelets, aggregation and serotonin release, which were blocked by monoclonal antibodies against the GPIIb/IIIa complex, by Fab fragments against the D domain, and by metabolic inhibitors; aggregation was attenuated but not abolished by enzymatic removal of ADP (with CP/CPK) or by blockage of ADP binding sites (with FSBA), and when secretion was inhibited by aspirin. Fg- Fab'2(E) also induced a dose-dependent elevation in cytoplasmic Ca2+ (measured by Aequorin luminescence) which was attenuated by CP/CPK and by FSBA, and was eliminated by metabolic inhibitors and by anti- IIb/IIIa antibody. Fibrinogen complexes crosslinked with dimethylsuberimidate or Factor XIII neither aggregated gel-filtered platelets nor inhibited platelet aggregation by ADP and fibrinogen, probably because of inaccessibility of lysine residues in the D (terminal) domain of fibrinogen, which are thought to be required for platelet binding. Thus, soluble complexes of fibrinogen having multiple available platelet receptor recognition sites activate gel-filtered platelets and may provide a useful model for platelet-surface interactions mediated by adsorbed fibrinogen.     

Mast cell growth factor modulates CD36 antigen expression on erythroid progenitors from human bone marrow and peripheral blood associated with ongoing differentiation

To study the differentiation process of erythroid progenitors from normal human bone marrow and peripheral blood, CD34/CD36 sorted cells were cultured in the presence of Erythropoietin (Epo) and Epo plus mast cell growth factor (MGF). The CD34+/CD36- cell fraction from bone marrow supported 74 +/- 33 erythroid burst forming units (BFU-E)/10(4) cells (mean +/- SD, n = 4) in the presence of Epo, which increased 2.1- fold by coculturing with MGF. However, erythroid colony-forming units (CFU-E) were not cultured from the CD34+/CD36- cell fraction. In contrast, the CD34-/CD36+ cell fraction supported CFU-Es in the presence of Epo (152 +/- 115/10(5)) or Epo plus MGF (180 +/- 112/10(5)), whereas BFU-Es were hardly noticed. However, the transition of the BFu-E to CFU-E was observed by incubating CD34+/CD36- cells (10(4)/100 microL) in suspension with Epo plus MGF for 7 days followed by Epo in the colony assay. This was reflected by the appearance of CD34-/CD36+/Glycophorin A+/CD14- cells. In addition high numbers of CFU- Es (1,000 +/- 150, n = 4) were cultured from this cell fraction. In contrast to bone marrow erythroid progenitors, no peripheral blood CFU- Es were cultured from either the CD36+ or CD36- fraction, whereas BFU- Es were predominantly present in the CD36+ fraction. However, the CD34+ progenitor cell from peripheral blood did have intrinsic capacity to differentiate to CFU-Es because CD34+/CD36- cells incubated with Epo plus MGF for 7 days and followed by Epo in the colony assay, supported high numbers of CFU-Es (1,200 +/- 400, n = 3). To study whether additional growth factors have similar effects on erythroid progenitors, experiments were performed with interleukin 1 (IL-1), IL- 3, and IL-6. IL-1 and IL-6 did not modulate the Epo supported proliferation and differentiation. In contrast, IL-3 in the presence of Epo did support CFU-Es, from CD34+/CD36- cells after 7 days in suspension culture. However, flow cytometry analysis showed that Epo plus IL-3 not only supported CD34-/CD36+/Glycophorin A+ cells but also CD36+/CD14+ cells, indicating the differentiation along different cell lineages. In summary, the data show a phenotypic distinction between bone marrow and peripheral blood erythroid progenitors with regard to CD36 expression. In addition, the results suggest that Epo plus MGF or IL-3 and preincubation in suspension culture are prerequisites for the transition of the BFU-E to the CFU-E.     

急性肺损伤患者肺复张手法的应用评价

背景和目的:对于急性肺损伤(ALI)患者应用肺复张手法的安全性和有效性尚存争议,本研究旨在对成人ALI患者接受肺复张手法出现的生理学效应和不良事件进行系统评价.方法:对Au病例的系列、观察性研究和随机临床试验的结果进行分类综述.结果:40项研究的1185例符合选择标准.31项研究的636例应用肺复张手法后氧合显著增     

口服芬戈莫德治疗复发缓解型多发性硬化的安慰剂对照试验

背景:芬戈莫德是1-磷酸鞘氨醇受体调节剂,可以阻止淋巴细胞移行出淋巴结.在Ⅱ期和Ⅲ期临床试验中已经证实,芬戈莫德与安慰剂或肌肉注射β干扰素相比均能显著减少多发性硬化(MS)患者的复发率,并且改善MRI预后指标.方法:本研究为期24个月,采用双盲随机设计,纳入18～55岁,扩展的神经功能障碍量表(EDSS)评分在0～5.5分,且入选前1年内复发至少1次或2年内复发至少2次的复发缓解型MS(RRMS)患者.