Functional reconstitution of the phagocyte NADPH oxidase by transfection of its multiple components in a heterologous system

The phagocyte NADPH oxidase system, as previously defined by cell-free reconstitution, is comprised of five essential components, three of which are produced during late phagocytic differentiation--namely, two cytosolic proteins, p47- and p67-phox--and the large subunit of cytochrome b558, gp91-phox. To confirm that these are the only phagocyte-specific components necessary for oxidase activity in whole cells, the recombinant NADPH oxidase was reconstituted in a heterologous cell line. An undifferentiated multipotent leukemic cell line, K562, which expresses endogenous Rac and the small subunit of the flavocytochrome b558 (p22-phox), was cotransfected with episomal expression vectors containing cDNAs for the three other oxidase components. After 4 days of selection, the complete oxidase system was functionally reconstituted in transfected cells stimulated with phorbol myristate acetate or calcium ionophore. These easily transfected cells provide an ideal model system in which several oxidase components can be genetically manipulated and readily expressed. This system can be used to test the effects of mutations associated with any of the genes affected in chronic granulomatous disease and will facilitate studies on structure-function relationships within several oxidase components. This system will also aid in delineation of upstream regulators functioning through various signaling pathways.     

经桡动脉冠状动脉介入治疗中5F与6F导引导管的对比研究

目的比较5French(5F)及6French(6F)导引导管在经桡动脉冠状动脉介入治疗(TRI)患者中的安全性及有效性。方法共纳入2009年2月至2010年3月患者,收集相关资料录入数据库,包括患者基线临床资料、导引导管的尺寸、靶血管、靶病变的特点、手术的成功率、手术失败原因、经桡动脉冠状动脉介入治疗手术的成功率及失败原因、患者住院期间主要不良心血管事件率及术后桡动脉闭塞率。结果连续纳入患者共185例,接受195次经桡动脉冠状动脉介入治疗术,平均年龄(57±11)岁(33～81岁);其中54例患者纳入6F导引导管组,共进行56次手术,治疗89处病变;138例患者纳入5F导引导管组,共行146次手术,治疗231处病变。AHA B2/C型病变比例在两组间差异无统计学意义(5F组43.7%/29.0%比6F组46.1%/34.6%,P>0.05),但慢性闭塞性病变、分叉病变、钙化病变5F组显著少于6F组(5.6%比14.6%,P=0.005;23.4%比37.1%,P=0.012;9.5%比47.2%,P<0.001);组间的手术时间[(45±21)min比(46±19)min)]、手术X线曝光时间[(15±12)min比(16±13)min]、使用造影剂量[(140±45)ml比(156±56)ml]差异均无显著统计学意义(P>0.05),但是5F组造影剂用量有减少的趋势(P=0.066);组间住院时间[(1.40±1.26)d比(1.29±0.69)d]和手术成功率(95.2%比94.6%)也差异无统计学意义(P>0.05);5F组1例患者术后桡动脉闭塞,6F组无患者术后桡动脉闭塞(P=1.0),5F组1例发生卒中。结论经桡动脉冠状动脉介入治疗,即使是复杂及高危冠脉病变,5F导引导管有效、安全,手术成功率不低于常规使用的6F导引导管;换用5F导引导管进行冠状动脉介入治疗是一种有吸引力的选择。     

Relationship between skin fold thickness and insulin resistance in the essential hypertensive patients in Vietnam

Background Previous studies reported a close relationship between obesity and insulin resistance in the essential hypertensive patients. Objective In this study, we examined the relationship between the skin fold thickness and insulin resistance then developed a formula to estimate the insulin resistance index according to the skin fold thickness in the essential hypertensive patients. Subjects and Methods Medical records of 80 patients (37 males, 43 females) were reviewed and the data were tabulated. Anthropometric indexes (including height, weight, waist circumference, hip circumference, and skins fold thickness at 5 fatty difference points on the Erdheim diagram), fasting plasma glucose and insulin concentration were recorded. The mean age was 57.0 ?9.2 years. The insulin resistance index was calculated following the Homeostasis Model Assessment (HOMA) formula. Results Compared with the group with BMI < 23 kg/m2, the group with BMI≥23 kg/m2 had higher fasting insulin concentration (8.85±4.97 pmol/L vs 15.60±8.70 pmol/L, P<0.001 ) and higher insulin resistance index in ( 2.15±1,24 vs 3.76±2.22, P<0.001). No significant difference in fasting plasma insulin concentration, insulin resistance index between male and female was observed (P > 0.05). There was a positive correlation between skin fold thickness and the fasting insulin concentration and insulin resistance index. The skin fold thickness at point A8 had the best coefficient correlated with fasting plasma insulin(r=0.79, P < 0.001) and insulin resistance index (r= 0.79, P < 0.001). A formula to estimate the insulin resistance index by skin fold thickness at point A8 as: Insulin resistance index = 0.12×[skin fold thickness at A8 point (mm)] - 1. Conclusion: In the essential hypertensive patients, the formula to estimate insulin resistance index as 0.12×[skin fold thickness at A8 point (mm)]-1 may predict accurately the level of insulin resistance. (J Geriatr Cardiol 2005; 2(4):228-232 )     

The CD11b promoter directs high-level expression of reporter genes in macrophages in transgenic mice [published erratum appears in Blood 1995 Apr 1;85(7):1983]

CD11b is the alpha chain of the Mac-1 integrin and is preferentially expressed in myeloid cells (neutrophils, monocytes, and macrophages). We have previously shown that the CD11b promoter directs cell-type- specific expression in myeloid lines using transient transfection assays. To confirm that these promoter sequences contain the proper regulatory elements for correct myeloid expression of CD11b in vivo, we have used the -1.7-kb human CD11b promoter to direct reporter gene expression in transgenic mice. Stable founder lines were generated with two different reporter genes, a Thy 1.1 surface marker and the Escherichia coli lacZ (beta-galactosidase) gene. Analysis of founders generated with each reporter demonstrated that the CD11b promoter was capable of driving high levels of transgene expression in murine macrophages for the lifetime of the animals. Similar to the endogenous gene, transgene expression was preferentially found in mature monocytes, macrophages, and neutrophils and not in myeloid precursors. These experiments indicate that the -1.7 CD11b promoter contains the regulatory elements sufficient for high-level macrophage expression. This promoter should be useful for targeting heterologous gene expression to mature myeloid cells.     

Characterization of the p67phox gene: genomic organization and restriction fragment length polymorphism analysis for prenatal diagnosis in chronic granulomatous disease

The genetic defect in the p67phox-deficient form of chronic granulomatous disease (CGD) follows an autosomal recessive pattern of inheritance. When genomic DNA from normal individuals is digested with HindIII and probed with p67phox cDNA an allelic restriction fragment length polymorphism (RFLP) of 4.0 kb or 2.3 kb is detected. We cloned and characterized the p67phox gene using the cDNA and sequenced the exon/intron boundaries, mapping 16 exons on the 40-kb gene. The polymorphic region was then sequenced to identify the inheritance pattern of amniocentesis-derived fetal cells by genomic amplification. The proband, a 9-year-old female patient with p67phox-deficient CGD, and her phenotypically normal mother are homozygous for the RFLP marker, whereas the father and two brothers are heterozygous. The fetus was shown to be heterozygous as well, showing it had inherited at least one normal p67phox gene from the father and that it was predicted to have a normal phenotype. Cord blood samples at birth showed normal oxidative function. Amplification allows rapid detection of the inheritance pattern for fetal diagnosis in informative families. We report the genomic structure of p67phox and an amplification-based method for detection of the marker on chromosome 1q25, used here for prenatal diagnosis of CGD.     

Acceleration of idiopathic pneumonia syndrome (IPS) in the absence of donor MIP-1 alpha (CCL3) after allogeneic BMT in mice

Idiopathic pneumonia syndrome (IPS) is a significant cause of morbidity and mortality after bone marrow transplantation (BMT) in humans. We developed a murine IPS model in which lethal pre-BMT conditioning and allogeneic T cells results in the recruitment of host monocytes and then donor T cells into the lung by day 7 after BMT, concomitant with development of severe lung dysfunction. We reported the T cell-dependent production of the T cell-attracting chemokine macrophage inflammatory protein-1 alpha (MIP-1 alpha) in the lungs of such recipient mice. We reasoned that MIP-1 alpha might be a critical mediator of IPS. Lethally conditioned mice received transplants of major histocompatibility complex-disparate marrow and either wild-type (MIP-1 alpha(+/+)) or knockout (MIP-1 alpha(-/-)) spleen cells. Recipients of MIP-1 alpha(-/-) cells exhibited accelerated mortality and a decrease in specific compliance that appeared earlier than in recipients of MIP-1 alpha(+/+) cells. Donor CD4(+) and CD8(+) T cell expansion was increased in the spleens of recipients of MIP-1 alpha(-/-) cells. Lungs of recipients of MIP-1 alpha(-/-) cells had earlier recruitment of both T-cell subsets by day 3 after BMT, concomitant with the influx of cells expressing the cytolysins granzymes A and B. Monocyte recruitment was not altered. Levels of inflammatory cytokines were not increased and levels of T cell-attracting chemokines were decreased. The level of the anti-inflammatory cytokine interleukin 13 (IL-13) was lower in the serum and lungs of recipients of MIP-1 alpha(-/-) cells, indicating a skewing toward a more inflammatory T helper cell type 1 (Th1) cytokine milieu. Donor-derived MIP-1 alpha may play a role in allogeneic-induced IPS by limiting aggressive expansion of CD4(+) and CD8(+) T cells.     

A prospective study of in-line radiofrequency ablation in hepatic parenchymal transection--its efficacy and complications

Background. Bleeding during liver transection remains a potential hazard. This study aims to report the efficacy and complications of in-line radiofrequency ablation (ILRFA)-assisted liver resection. Patients and methods. The blood loss of 25 consecutive patients who underwent ILRFA-assisted liver resection was obtained by weighing swabs and measuring suction jar contents during liver resection and calculated in ml per cm² of the transection surface area. Postoperative complications were recorded. Five clinical variables, which might affect blood loss, were analyzed. Results. The mean blood loss during parenchymal dissection for the ILRFA group was 3.4±3.2 ml/cm². Three patients had intra-abdominal collections, including one patient with bile leakage after ILRFA-assisted liver resection. Age, gender, extent of liver resection, liver quality and Pringle maneuver did not demonstrate significant impact on blood loss. Conclusions. This study showed that ILRFA-assisted liver resection was associated with very low blood loss. This is likely to improve the operative safety of liver resection for hepatic tumors. There were no significant postoperative sequelae.     

Replantation in children

The authors have replanted 162 parts in 120 children over the past 15 years. The youngest patient, undergoing successful replantation, was aged 7 months, 3 weeks. Unlike an adult, any child suffering a traumatic amputation should be considered for a possible replantation. Replantation should consist of minimal bone shortening to preserve epiphyseal plates, with repair of all severed structures. Longitudinal K-wires usually provide adequate fixation. Our survival rate for complete replantation in children under the age of 16 years is 77%. Long-term study showed that continued skeletal growth occurred and the digit attained 81% of normal longitudinal length at maturity. Recovery of sensibility in the replanted digit is nearly as good as for isolated digital nerve repair. Patient and parent satisfaction is high when replantation is successful, with uniform approval of the extensive effort required.     

In vivo behavior of human radioiodinated antithrombin III: distribution among three physiologic pools

It has recently been shown that antithrombin III (AT) distributes between plasma, a noncirculating vascular-associated pool and an extravascular pool in rabbit. Study of the in vivo behavior of autologous human 131I-AT demonstrates that in humans AT also distributes among three pools that are analogous to those found in rabbit. From the in vivo kinetic behavior of the 131I-labeled AT, the fractions of total-body AT in the plasma, noncirculating vascular- associated, and extravascular pools were calculated to be 0.393 +/- 0.015, 0.109 +/- 0.016, and 0.496 +/- 0.014, respectively. From three- exponential plasma radioactivity disappearance curves, an average plasma fractional catabolic rate, j3, of 0.576 +/- 0.034 day-1 was obtained for five healthy young men. This is almost identical to the result obtained if plasma 131I-AT disappearance is assumed to fit a two- exponential curve (0.546 +/- 0.038), where the constant C2 from *Ap(t) = C1e-a1t + C2e-a2t is assumed to be equal to 1 - C1. The fraction of the total vascular AT catabolized daily, j3.5, was calculated to be 0.457 +/- 0.034, and the fractional catabolic rate of total-body AT, jT, averaged 0.2271 +/- 0.0176. The results give further support to a model of in vivo behavior in which the vascular AT distributes between plasma and an endothelial receptor. Thus, the latter may serve to mediate activation of AT for its reaction with coagulation proteases and to mediate its entrance into the endothelial cell, where it is either transported to the extravascular fluids or is catabolized.