Quantitation of CD62, soluble CD62, and lysosome-associated membrane proteins 1 and 2 for evaluation of the quality of stored platelet concentrates

BACKGROUND: Platelets become activated during storage, which results in secretion of granules, vesiculation of microparticles, secretion of protein, and a number of other biochemical and morphologic processes that decrease the utility of platelet concentrates stored for transfusion. STUDY DESIGN AND METHODS: To evaluate the quality of stored platelet concentrates, the cell surface expression of specific activation-dependent antigens (CD62 and lysosome-associated membrane proteins 1 and 2 [LAMP-1, LAMP-2]) on platelets stored in a hospital blood bank over a 7-day period was examined. Relative microparticle counts and the expression of CD62 by microparticles, as well as platelet concentrate supernatant levels of soluble CD62, were determined. RESULTS: The percentage of platelets expressing CD62 increased significantly from Day 1 to Day 5 (p < 0.05) of storage; the mean fluorescence values for CD62 did not. In contrast, the mean fluorescence values of LAMP-1 and LAMP-2 rose significantly (p < 0.01 and p < 0.05, respectively) between Days 1 and 5. Significant declines in CD62, LAMP-1, and LAMP-2 percent expression and mean fluorescence were seen on Day 6 of storage (p < 0.001). Microparticle numbers increased significantly during storage and correlated with levels of CD62 protein (free and membrane-bound) (r = 0.95 vs. Day 2, p < 0.05; r = 0.88 vs. Day 5, p < 0.05). CONCLUSION: Flow cytometric evaluations of the expression of cell surface CD62, LAMP-1, and LAMP-2 are complementary tests that, especially when used in conjunction with the quantitation of CD62 protein, provided a simple and effective means of evaluating the quality of platelet concentrates stored for transfusion.     

骺板软骨细胞的体外培养及鉴定

目的:建立体外培养及鉴定骺板软骨细胞的方法。方法:实验于2005-03/2006-05在苏州大学附属儿童医院骨科实验室完成。选用3只生后14～28d的新西兰幼兔,空气栓塞处死,暴露股骨下端和胫骨上端,分别取2处的骺板组织,将其剪切成1～3mm3的小块,经胰蛋白酶消化,接种于含150g/L牛血清的1640培养基中,饱和湿度培养,传代。①细胞接种后3h在倒置显微镜下观察细胞生长情况,见细胞贴壁后每天观察2次。②第2代细胞达到80%～90%左右汇合时采用常规苏木精-伊红染色,光镜观察细胞爬片情况。③第3代细胞爬片至细胞达到80～90%左右汇合时,采用苏木素染色3min,3%亮绿染色5min,蕃红花“O”染色5min,光镜观察细胞产生蛋白聚糖情况。④采用PCR检测细胞Ⅱ型胶原的表达。⑤采用四唑盐MTT比色法检测细胞活性。结果:①骺软骨细胞刚接种后呈大小不等之圆形悬浮于培养液中,3h后见大部分细胞贴壁,24h后贴壁细胞呈短梭形、圆形、三角形和不规则形,见细胞分裂相。48h后见细胞伸展明显,细胞分裂相每高倍镜视野可见多个。经隔日换液细胞生长至第5天,细胞呈聚集生长,达到汇合状态,将细胞传代。接种后第1代细胞2d后呈梭形,培养4d传至第2代,第2代细胞长满瓶底后,90%呈胞膜较厚的圆形,10%为梭形,第3代、第4代亦如此。传至第5代见肥大细胞增多,细胞松散,折光性减弱,呈凋亡状态。②细胞爬片后观察骺软骨细胞形态以梭形居多,同时也有圆形、三角形和不规则形。可见细胞分裂及细胞中的分泌小泡。③见细胞呈红色,无绿色,证明蕃红花“O”-亮绿染色阳性,显示所培养的细胞可以分泌蛋白聚糖。④PCR检测术所养细胞含有Ⅱ型胶原,电泳带在440bp上。⑤四唑盐MTT比色法检测显示,第3代骺软骨细胞的生长曲线近似倒“S”形,在第4,5,6天细胞呈对数生长,约在7,8,9,10d达平台期,至第12天细胞出现生长抑制。结论:建立了骺板软骨细胞的体外培养方法,并证实所培养出的细胞分泌蛋白聚糖和Ⅱ型胶原,具有软骨细胞的共同特点。     

ThromCat血栓切除导管系统在急性冠状动脉综合征介入治疗中的应用

目的:评价ThromCat血栓切除导管系统(ThromCat Thrombectomy Catheter System,TTC)在急性冠状动脉综合征(ACS)介入治疗应用中的安全性、工作性能及有效性.方法:入选14例ACS患者进行经皮冠状动脉介入治疗(PCI),分析TTC操作的成功率、TTC应用的临床成功率、术中并发症和术后随访30 d心脏不良事件.结果:TTC操作成功11例,TTC临床成功10例,PCI成功13例,并不伴有心脏不良事件.在PCI中,心肌梗死溶栓治疗(TIMI)血流由使用TIC前的0.5(2.0)升高至使用后的2.0(1.0),心肌灌注分级(MBG)由使用前的1.0(2.0)升高至使用后的2.0(1.0),差异均有统计学意义(均P<0.01).结论:在ACS患者中行PCI,TTC能安全有效地去除非严重钙化的冠状动脉内的血栓.并发症少,操作简单易行,减少PCI手术引起的并发症.     

The addition of high manganese to a copper-deficient diet further depresses copper status and growth of cattle

A study was conducted evaluating the effect of long-term Cu deficiency, with or without high Mn, on growth, gene expression and Cu status of beef cattle. Twenty-one Angus calves were born to cows receiving one of the following treatments: (1) 10 mg supplemental Cu/kg DM (+Cu); (2) no supplemental Cu and 2 mg Mo/kg DM ( - Cu); (3) - Cu diet plus 500 mg supplemental Mn/kg DM ( - Cu+Mn). Calves were weaned at approximately 183 d of age and individually fed throughout the growing and finishing phases. Plasma Cu was lower (P < 0.01) in - Cu calves compared with +Cu calves while high dietary Mn further depressed (P < 0.01) plasma Cu in - Cu+Mn calves v. - Cu calves. Liver Cu concentrations in +Cu calves were greater (P < 0.01) than in - Cu calves, with no differences between - Cu and - Cu+Mn calves. The daily body-weight gain of +Cu calves was greater (P < 0.01) than - Cu calves during the period from birth to weaning, but did not differ during the growing phase. - Cu+Mn calves gained less (P < 0.05) than - Cu calves during the growing phase. DM intake was lower (P < 0.01) in - Cu+Mn calves v. - Cu calves, and did not differ among +Cu and - Cu calves. The relative gene expression of cytochrome c oxidase in the liver was lower (P < 0.05) in - Cu calves compared with +Cu or - Cu+Mn calves. In conclusion, feeding a Cu - deficient diet in combination with high Mn negatively affected the growth and Cu status of beef cattle.     

Gene expression profiling of chondrocytes from a porcine impact injury model

OBJECTIVE: To identify differentially expressed genes between axially impacted and control articular cartilage taken from porcine patellae maintained in organ culture for 14 days. METHODS: Porcine patellae were impacted perpendicular to the articular surface to create an impact injury. Intact patellae (control and impacted) were maintained in culture for 14 days. Total RNA was then extracted from the articular cartilage beneath the impaction and used to prepare two Serial Analysis of Gene Expression (SAGE) libraries. Approximately 42,500 SAGE long tags were sequenced from the libraries. The expression of select genes was confirmed by quantitative real-time PCR analysis. RESULTS: Thirty-nine SAGE tags were significantly differentially expressed in the impacted and control libraries, representing 30 different annotated pig genes. These genes represented gene products associated with matrix molecules, iron and phosphate transport, protein biosynthesis, skeletal development, cell proliferation, lipid metabolism and the inflammatory response. Twenty-three of the 30 genes were down-regulated in the impacted library and five were up-regulated in the impacted library. Quantitative real-time PCR follow-up of four genes supported the results found with SAGE. CONCLUSION: We have identified 30 putative genes differentially expressed in a porcine impact injury model and validated these findings for four of these genes using real-time PCR. Results using this impact injury model have contributed further evidence that damaged chondrocytes may de-differentiate into fibroblast-like cells and proliferate in an attempt to repair themselves. Additional work is underway to study these genes in further detail at earlier time points to provide a more complete story about the fate of chondrocytes in articular cartilage following an injury.     

AZD7762, a novel checkpoint kinase inhibitor, drives checkpoint abrogation and potentiates DNA-targeted therapies

Insights from cell cycle research have led to the hypothesis that tumors may be selectively sensitized to DNA-damaging agents resulting in improved antitumor activity and a wider therapeutic margin. The theory relies on the observation that the majority of tumors are deficient in the G1-DNA damage checkpoint pathway resulting in reliance on S and G2 checkpoints for DNA repair and cell survival. The S and G2 checkpoints are regulated by checkpoint kinase 1, a serine/threonine kinase that is activated in response to DNA damage; thus, inhibition of checkpoint kinase 1 signaling impairs DNA repair and increases tumor cell death. Normal tissues, however, have a functioning G1 checkpoint signaling pathway allowing for DNA repair and cell survival. Here, we describe the preclinical profile of AZD7762, a potent ATP-competitive checkpoint kinase inhibitor in clinical trials. AZD7762 has been profiled extensively in vitro and in vivo in combination with DNA-damaging agents and has been shown to potentiate response in several different settings where inhibition of checkpoint kinase results in the abrogation of DNA damage-induced cell cycle arrest. Dose-dependent potentiation of antitumor activity, when AZD7762 is administered in combination with DNA-damaging agents, has been observed in multiple xenograft models with several DNA-damaging agents, further supporting the potential of checkpoint kinase inhibitors to enhance the efficacy of both conventional chemotherapy and radiotherapy and increase patient response rates in a variety of settings.     

Big birds and their brains: paleoneurology of the New Zealand moa

The moa (Dinornithiformes: Aves) are an extinct group of ratites from the North and South Islands of New Zealand. The ancestors of both the moa and the kiwi were isolated from other Gondwanan fauna as much as 80 million years ago and evolved in the absence of large mammalian predators. As such they represent a natural experiment in the removal of mammalian predation pressure on the encephalization of these two groups of ratites. We have used endocranial and skull morphometry in conjunction with high resolution CT scanning of the skulls of 8 species of moa to assess encephalization and brain morphology in moa and compare these features with extant ratites. Absolute brain size among the moa ranged from 17.0 ml for Euryapteryx curtus to 60.0 ml for female Dinornis giganteus. Values for encephalization quotients (EQ) of moa ranged from 0.205 for Euryapteryx gravis of the southern North Island to a mean (+/- SD) of 0.475 (+/- 0.026) for Anomalopteryx didiformis, partially overlapping values for extant non-New Zealand ratites (emu: 0.402 +/- 0.042; rhea: 0.496 +/- 0.016; ostrich: 0.474 +/- 0.084). Nevertheless, mean +/- SD EQ for all moa examined (0.379 +/- 0.065) was significantly lower than EQ for extant non-New Zealand ratites (0.539 +/- 0.141). Bending of the endocranial axis was much less among moa than either the kiwi or non-New Zealand ratites, consistent with the caudal position of the foramen magnum and the horizontal carriage of the head and upper neck during life. Endocranial morphology of the moa species examined was similar to that for non-New Zealand ratites, with proportionally similar sizes of the olfactory bulb, Wulst, vagal and maxillomandibular foramina, suggesting that the moa occupied similar diurnal niches with comparable sensory specializations to the emu, rhea and ostrich. No evidence of olfactory specialization (i.e., enlarged olfactory bulbs and increased surface area of the olfactory nasal cavity or cribriform plate) was evident in any of the moa skulls, in contrast to the remarkable nasal and olfactory bulb specializations evident in the skull and brain of the little spotted kiwi (Apteryx owenii). We cannot exclude that isolation in the absence of highly encephalized mammalian predators might have contributed to the lower EQ among moa, but it certainly did not lead to any significant reduction in EQ for kiwi; rather the kiwi embarked on a remarkable path of neurological specialization, which allowed them to exploit a niche usually occupied elsewhere by mammals.     

Development of the olfactory system in a wallaby (Macropus eugenii)

We used carbocyanine dye tracing techniques in conjunction with hematoxylin and eosin staining, immunohistochemistry for GAP-43, and tritiated thymidine autoradiography to examine the development of the olfactory pathways in early pouch young tammar wallabies (Macropus eugenii). The overarching aim was to test the hypothesis that the olfactory system of newborn tammars is sufficiently mature at birth to contribute to the guidance of the pouch young to the nipple. Although GAP-43 immunoreactive fibers emerge from the olfactory epithelium and enter the olfactory bulb at birth, all other components of the olfactory pathway in newborn tammars are very immature at birth, postnatal day (P0). In particular, maturation of the vomeronasal organ and its projections to the accessory olfactory bulb appears to be delayed until P5 and the olfactory bulb is poorly differentiated until P12, with glomerular formation delayed until P25. The lateral olfactory tract is also very immature at birth with pioneer axons having penetrated only the most rostral portion of the piriform lobe. Interestingly, there were some early (P0) projections from the olfactory epithelium to the medial septal region and lamina terminalis (by the terminal nerve) and to olfactory tubercle and basal forebrain. The former of these is presumably serving the transfer of LHRH(+) neurons to the forebrain, as seen in eutherians, but neither of these very early pathways is sufficiently robust or connected to the more caudal neuraxis to play a role in nipple finding. Tritiated thymidine autoradiography confirmed that most piriform cortex pyramidal neurons are generated in the first week of life and are unlikely to be able to contribute to circuitry guiding the climb to the pouch. Our findings lead us to reject the hypothesis that olfactory projections contribute to guidance of the newborn tammar to the pouch and nipple. It appears far more likely that the trigeminal pathways play a significant role in this behavior because the central projections of the trigeminal nerve are more mature at birth in this marsupial.     

Cortical cyto- and chemoarchitecture in three small Australian marsupial carnivores: Sminthopsis macroura, Antechinus stuartii and Phascogale calura

The cyto- and chemoarchitecture of the cerebral cortex has been examined in three small (mouse-sized) polyprotodont marsupial carnivores from Australia (the stripe-faced dunnart, Sminthopsis macroura; the brown antechinus, Antechinus stuartii; and the red-tailed phascogale, Phascogale calura) in order to compare the cortical topography of these marsupials with that of diprotodontids, didelphids and eutherians. All three species studied had similar cortical cytoarchitecture. The isocortical surface was dominated by primary somatosensory (S1) and visual (V1) areas. Putative secondary sensory areas (S2, V2M, V2L) were also identified. The primary somatosensory cortex demonstrated clumps of granule cells in the presumptive mystacial field, whereas the primary visual area showed a distinctive chemical signature of intense calbindin immunoreactivity in layer IV. On the other hand, the primary auditory area was small and indistinct, but flanked by a temporal association area (TeA). A cytoarchitecturally distinct primary motor cortex (M1) with prominent pyramidal neurons in layer V and poor layer IV was identified medially to S1, and at rostral levels a putative secondary motor area was identified medial to M1. Transitional areas between isocortex and allocortical regions showed many cyto- and chemoarchitectural similarities to those reported for eutherian (and in particular rodent) cortex. Medially, two cingulate regions were found at rostral levels, with dysgranular and granular 'retrosplenial' areas identified caudally. Laterally, granular and agranular areas surrounded the rostral rhinal fissure, to be replaced by ectorhinal and perirhinal areas caudally. The findings indicate that the cyto- and chemoarchitectural features which characterize the iso- and allocortex in these small marsupial carnivores are similar to those reported in didelphids and eutherians and our findings suggest the existence of putative dedicated motor areas medial to the S1 field.