Prolonged procoagulant activity on overstretch-injured coronary arteries in pigs

Summary.  This study was designed to assess the time course and nature of the vascular procoagulant response after 1.5-fold balloon overstretch injury of the coronary arteries in pigs. Arteries were excised for chromogenic assay of bound factor (F)Xa and thrombin at 24 h, 3 days, 1 week, or 2 weeks after injury. FXa at the site of injury remained elevated for 1 week (4.9 ± 5.9 µg cm⁻², n  = 10), compared with non-injured control arteries (0.4 ± 0.2 µg cm⁻², n  = 18, P  = 0.00025), while thrombin was increased only at 24 h. Tissue factor protein was abundant in non-injured coronaries (10 ± 6 ng µg⁻¹ total protein, n  = 9) and levels were unchanged by injury (13 ± 11 ng µg⁻¹, n  = 6) or 24-h administration of tissue factor pathway inhibitor (16 ± 6 ng µg⁻¹, n  = 6). Persistent tissue factor-mediated procoagulant activity may explain the need for prolonged anticoagulation to attenuate neointimal formation after balloon-induced coronary injury.     

Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.     

白介素与溃疡性结肠炎

近年来对白介素(interleukin,IL)和溃疡性结肠炎(ulcerative colitis,UC)的研究取得了很大进展,我们通过总结整理以前有关IL和UC的文献,概括出IL的产生和在UC发病及病理变化中的作用机制:IL-1直接介导了UC初期阶段炎症的发生:IL-8、IL-6直接促进炎性细胞过度分泌和/或抑制了炎性细胞的凋亡,IL-2分泌减少导致免疫系统内细胞间网络调节失衡, 使局部炎症介质和自由基释放,引起细胞毒作用,IL主要通过影响机体整体和/或局部免疫系统的功能介导UC的产生,并与UC的迁延难愈和反复发作有关．     

Prospective epidemiological study of the prevalence of human leukocyte antigen (HLA)‐B*5701 in HIV‐1‐infected UK subjects

Objectives

Human leukocyte antigen (HLA)‐B^*5701 is strongly associated with developing a hypersensitivity reaction to abacavir (ABC) in White and Hispanic subjects. Across the UK, limited data exist on HLA‐B^*5701 prevalence in HIV‐1‐infected subjects. We determined HLA‐B^*5701 prevalence in the general HIV‐1‐infected population and in specific ethnic groups, particularly Black Africans who, in general, exhibit greater genetic diversity. We also compared HLA‐B^*5701 results obtained from local laboratories with those from a central provider.

Design and methods

Multi‐centre, observational study. All HIV‐1‐infected adult individuals receiving care at participating centres were eligible, irrespective of treatment status or prior exposure to ABC. Subjects provided samples for HLA‐B^*5701 assessment by both local (blood) and central laboratories (buccal swabs). HLA‐B^*5701 prevalence was adjusted to represent the ethnic group composition of the general UK population, and by main ethnic group.

Results

From eight UK centres, 1494 subjects [618 (41%) White, 770 (52%) Black] were recruited. Eighty‐nine per cent of Black subjects reported an immediate country of origin in Africa. Overall adjusted HLA‐B^*5701 prevalence was 4.55% [95% confidence interval (CI) 3.49% to 5.60%]. Among White subjects, prevalence was 7.93% (CI 5.80% to 10.06%). Among Black subjects, only two (both Ugandan) were HLA‐B^*5701 positive giving a rate of 0.26% (CI 0.07% to 0.94%).

Conclusions

HLA‐B^*5701 prevalence was similar to previously reported rates in White HIV‐infected subjects but considerably lower than that reported in Black HIV‐1‐infected subjects, as a result of the large proportion of Black African subjects.     

肿瘤干细胞和抗肿瘤药物研发思路

目前有些抗肿瘤药物尽管有较好的近期疗效,然而肿瘤患者单纯依赖化疗通常难以达到长期生存的目的。本文试图从肿瘤干细胞理论解释抗肿瘤药物近期疗效和远期生存之间存在的矛盾,并探讨抗肿瘤药物的研究发展方向。     

Methicillin-resistant Staphylococcus aureus, Clostridium difficile, and extended-spectrum beta-lactamase-producing Escherichia coli in the community: assessing the problem and controlling the spread

Although health care-associated methicillin resistant staphylococcus aureus and clostridium difficile strains are primarily a risk to hospital patients, people are increasingly concerned about their potential to circulate in the community and the home. They are thus looking for support in order to understand the extent of the risk, and guidance on how to deal with situations where preventing infection from these species becomes their responsibility. A further concern are the community-acquired MRSA and C. difficile strains, and other antibiotic resistant strains circulating in the community such as the Extended-spectrum beta-lactamase (ESBL) Escherichia coli. In response to concerns about such organisms in the community, the International Scientific Forum on Home Hygiene has produced a report evaluating MRSA, C. difficile, and ESBL-producing E. coli from a community viewpoint. The report summarizes what is known about their prevalence in the community, their mode of transmission in the home, and the extent to which they represent a risk. It also includes "advice sheets" giving practical guidance on what to do when there is a risk of infection transmission in the home.     

Metabolic Activity Determines Efficacy of Macroautophagic Clearance of Pathological Oligomeric α-Synuclein

Macroautophagy is an essential degradative pathway that can be induced to clear aggregated proteins, such as those found in Parkinson’s disease and dementia with Lewy bodies, a form of Parkinsonism. This study found that both LC3-II and beclin were significantly increased in brains from humans with Dementia with Lewy bodies and transgenic mice overexpressing mutant α-synuclein, as compared with respective controls, suggesting that macroautophagy is induced to remove α-syn, particularly oligomeric or mutant forms. Aged mutant animals had higher autophagy biomarker levels relative to younger animals, suggesting that with aging, autophagy is less efficient and requires more stimulation to achieve the same outcome. Disruption of autophagy by RNA interference significantly increased α-syn oligomer accumulation in vitro, confirming the significance of autophagy in α-syn clearance. Finally, rotenone-induced α-syn aggregates were cleared following rapamycin stimulation of autophagy. Chronic rotenone exposure and commensurate reduction of metabolic activity limited the efficacy of rapamycin to promote autophagy, suggesting that cellular metabolism is critical for determining autophagic activity. Cumulatively, these findings support the concept that neuronal autophagy is essential for protein homeostasis and, in our system, reduction of autophagy increased the accumulation of potentially pathogenic α-synuclein oligomers. Aging and metabolic state were identified as important determinants of autophagic activity. This study provides therapeutic and pathological implications for both synucleinopathy and Parkinson’s disease, identifying conditions in which autophagy may be insufficient to degrade α-syn aggregates.Parkinson’s disease (PD) is a major neurodegenerative disease that affects approximately 1 million people in the United States. The disease affects the motor system due to loss of dopaminergic neurons. The common pathological hallmark of the disease includes the formation of intracellular inclusions called Lewy bodies,^1,2,3,4 principally composed of the protein α-synuclein (α-syn). The protein misfolds and accumulates, initially as oligomers and ultimately as aggregates.^5,6,7,8 The first mutation linked to PD was found in the α-synuclein gene at position A53T,⁹ and mutant variants of the protein are more prone to aggregate compared with wild-type protein.^10,11,12,13Other genes linked to PD include Parkin,¹⁴ UCHL1,¹⁵ DJ-1,¹⁶ PINK1,^17,18 LrrK2,^19,20 and ATP13a2.²¹ Mutations in these genes are not all associated with synucleinopathy however. Some involve mitochondrial dysfunction (PINK1, parkin, DJ-1), aberrant proteasomal activity (UCHL1), or altered autophagic or lysosomal function (LrrK2 and ATP13a2).^21,22 Recently, mutations of the glucocerebrosidase gene, encoding a lysosomal enzyme, have been shown to be an important risk factor for PD^23,24,25,26 and dementia with Lewy bodies (DLB), another form of Parkinsonism.^24,27 The mechanism by which glucocerebrosidase mutations cause disease is currently unknown but alterations in glucocerebrosidase may affect lysosomal degradation and increase the likelihood of aberrant α-syn accumulation into Lewy bodies, and neurodegeneration.Previously, wild-type α-syn was shown to undergo chaperone-mediated autophagy,²⁸ a lysosomal process that degrades 40% of all proteins, while the mutant variant blocks this lysosomal action.^29,30 As a result, mutant forms of α-synuclein (especially the A53T variant) are more prone to oligomerization and aggregation and are less readily degraded than the wild-type. Thus, they are more likely to accumulate as pathological intracellular inclusions due to decreased lysosomal degradation.Macroautophagy (autophagy) is critical to neuronal health as impairment leads to aberrant protein accumulation, neuronal dysfunction, and cell death.^31,32 In addition to its constitutive activity, macroautophagy is inducible and dually regulated by the mTOR³³ and PI3kinase/beclin/vsp34^34,35 signaling pathways. Induction of autophagy results in the recruitment of multiple Atg proteins^36,37 that initiate formation of double-membrane, autophagic vacuoles (AVs) that envelop proximal cytoplasmic material including aggregated proteins and organelles.³⁸ The AVs subsequently acquire lysosomal hydrolases or fuse with lysosomes to complete the degradative process.^39,40 Normally, this constitutive process is highly efficient in neurons.Up-regulation of autophagy has been previously reported to reverse the pathogenic accumulation of intracellular inclusions. In Huntington’s disease, induction of autophagy promotes the clearance of polyglutamine huntingtin aggregates that are a hallmark of the disease.^41,42 However, induction is not always a beneficial process. In Alzheimer’s disease, it has been suggested that the accumulation of AVs results in increased production of the toxic peptide β-amyloid and may be attributed to reduced fusion or lysosomal degradation.^43,44 Thus, although it appears that cells may up-regulate autophagy to attenuate dysfunction, it cannot be assumed that this is the case and the opposite may, in fact, be true.These studies provide comprehensive pathological and biochemical evidence that macroautophagy is essential for maintaining the homeostasis of α-synuclein levels. Our studies show that autophagy was stimulated in human DLB, and in mutant α-syn-expressing mice to clear aberrant α-syn. In addition, this work provides evidence that blockage of autophagy results in the promotion of the pathogenic α-syn oligomers,^45,46 particularly the mutant variant of α-syn. Finally, clearance of α-synuclein via small molecule induction of macroautophagy is dependent on the metabolic state of the cells, as cells were able to clear oligomeric α-syn when not metabolically compromised following acute exposure to rotenone, but with chronic exposure to rotenone the autophagic capacity to degrade α-synuclein was significantly diminished.