The relationship of human platelet density to platelet age: platelet population labeling by monoamine oxidase inhibition

The relationship between platelet density and platelet age appears to vary between species with relatively few labeling studies in humans reported. In this study, irreversible monoamine oxidase (MAO) inhibitors were used to biochemically label the circulating platelet population in 15 humans. Platelet samples were then isolated during the 15 days after drug ingestion. The platelets were separated by density on continuous linear Percoll gradients and the density distributions were divided into five fractions containing approximately equal numbers of platelets. Baseline MAO activity was strongly correlated with platelet density. Twenty-four hours after a single dose of tranylcypromine, platelet MAO activities in the density subpopulations were reduced to 14% to 17% of the baseline values. During the first five days after inhibition, the rates of recovery of MAO activity (percentage per day) were inversely proportional to platelet density. The recovery rates in the two most dense fractions were initially slow but increased after five days. Percentage recovery of MAO activity in the least dense fraction was significantly greater than the percentage recovery in the most dense fraction on days 2, 3, 5, and 8 (P less than .01, sign test). These results support the hypothesis that normal human platelets show a small increase in density with age, but they do not exclude the additional possibility that human platelet lifespan is positively correlated with platelet density.     

Assessing the delivery of neutrophils to tissues in neutropenia

Studies of neutrophil kinetics in neutropenic individuals, as well as clinical observations of variability in the occurrence of infection among patients with neutropenia, have suggested that blood neutrophil counts may not uniformly reflect the effective delivery of neutrophils to extravascular tissues where the cells perform their principal host defense functions. To evaluate this possibility we developed a sensitive, reproducible method of measuring the extravascular delivery of neutrophils to a normal mucosal site of neutrophil turnover. This method is based upon the quantification of neutrophils recoverable from saline mouth wash specimens. Twenty-five mL specimens, obtained in a controlled manner from neutropenic patients and normal subjects, were centrifuged and the sediments resuspended in 1.0 mL Hank's buffer with 2 micrograms acridine orange, incubated at 37 degrees C for 15 minutes, and then examined in a hemocytometer chamber by fluorescence microscopy. Neutrophils could be clearly distinguished by their characteristic fluorescence and were counted. With this method as few as 1,500 neutrophils were detected reliably in mouth wash specimens. Mucosal neutrophil counts varied less than 10% with repeated sampling of individual subjects over 5-day periods and were consistently greater than 1.3 X 10(5)/specimen in non-neutropenic individuals. Although profound neutropenia was generally reflected by lower than normal oral mucosal neutrophil counts, these counts were significantly higher in individuals with chronic severe neutropenia (blood neutrophils less than 300/mm3) than in patients with acute neutropenia of comparable severity that had developed following chemotherapy. Also, in individuals recovering from profound neutropenia, neutrophils usually reappeared earlier in mouth wash specimens than in blood, and oral mucosal neutrophil counts attained recovery levels more rapidly than did blood counts. This phenomenon was particularly evident in an individual with cyclic neutropenia. Moreover, mucosal neutrophils could occasionally be detected in profoundly neutropenic patients when neutrophils were not present in blood samples. These findings indicate that mucosal neutrophil counts in individuals with neutropenia provide information about the delivery of neutrophils to tissues that may not be apparent from blood neutrophil counts alone.     

地高辛抗血清对老龄大鼠心肌缺氧复氧损伤影响的体外实验

目的:观察内洋地黄素水平在老龄大鼠心肌细胞缺氧复氧损伤中的变化以及地高辛抗血清的保护作用。方法:实验于2005-04/05在皖南医学院病理生理教研室完成。取10只24月龄和10只6月龄雄性普通级SD大鼠,分别制备青年和老龄大鼠心肌细胞匀浆,老龄和成年大鼠为两大组,每组分为7小组,即每只大鼠心肌随机分到各小组中,共计14小组,每组10支试管。正常对照组:给予CO2和O2的混合气体(1∶19)通气40min;缺氧复氧组:CO2,O2,N2混合气体(5∶4∶91)通气20min后换成CO2和O2的混合气体(1∶19)通气20min;阴性对照组:同缺氧复氧组,但于再给氧前加入0.1mL的非特异性灭活兔血清;地高辛抗血清组:同缺氧复氧组,但于再给氧前加入0.1mL的非特异性地高辛抗血清(分别为1∶90000,60000,30000,10000)。观察大鼠心肌细胞钠-钾-三磷酸腺苷酶活性和线粒体内钙聚集程度,分析其剂量-效应关系。结果:①缺氧复氧时,青年组和老龄组大鼠心肌分泌内洋地黄素均显著升高,但老龄组显著低于青年组[(0.081±0.03),(0.153±0.06);(0.074±0.04),(0.125±0.05)ng/g;P<0.05]。②缺氧复氧时,老龄组与青年组心肌细胞钠-钾-三磷酸腺苷酶活性显著受抑制[(0.239±0.015),(0.778±0.050);(0.350±0.047),(0.836±0.044)μkat/g;P<0.05],老龄组与青年组相比,其抑制效应显著增强(P<0.05)。③缺氧复氧时,老龄组线粒体内钙与青年组比较明显增强[(0.082±0.011),(0.495±0.095);(0.075±0.008),(0.412±0.084)mmol/L,P<0.05]。④老龄组和青年组相比,地高辛抗血清呈剂量依赖性的恢复钠-钾-三磷酸腺苷酶活性(r=0.695,0.797,n=5,P<0.05),减轻线粒体内钙聚集(r=-0.565,-0.649,n=5,P<0.05);经直线回归分析发现,老龄鼠回归系数大于青年组(酶活性抑制K=1.50,0.94,线粒体内钙K=-7.43,-6.46)。结论:心肌细胞缺氧复氧时,老龄鼠损伤较青年大鼠更显著,其机制与老龄大鼠心肌细胞钠-钾-三磷酸腺苷酶对内洋地黄素敏感性增加有关,地高辛抗血清对老龄大鼠心肌细胞缺氧复氧保护作用更有效。     

Estimation of obscure ictal epileptic activity in scalp EEG

Summary We have produced a method to estimate ictal localized epileptic activity hidden among the background in scalp EEGs. When the visually completely different waveforms of the epileptic and background activities are nearly orthogonal, epileptic activity may be approximately extracted from the EEG data matrix by singular value decomposition with subsequent orthogonal rotation to match the distribution of one component with that of the epileptic source. A simulation study was carried out using a matrix mimicking the scalp EEG with an inconspicuous ictal epileptic activity from a dipole source. This hidden epileptic activity was approximately recovered by matching the dipole of interest with the epileptic dipole, even when the simulated waveforms of the epileptic and background activities were not exactly orthogonal. High linear correlation between these two types of waveforms hampered the recovery of the epileptic activity. In another simulation study employing two epileptic dipoles producing activities with the same waveform and a brief time lag, it was indicated that the temporal relationship between the epileptic activities could be also estimated using the cross-correlation function. In the preliminary clinical application of this method to the ictal EEGs of complex partial seizures, rhythmic activities with seemingly epileptic waveforms were estimated at the dipoles which were located in the vicinity of cortical lesions revealed by neuroimaging studies. These activities were indicated to appear before any change in the scalp EEG. We hope for the clinical application of this method for noninvasive estimation of inconspicuous ictal epileptic activity.The authors thank Prof. Peter K.H. Wong of the Department of Paediatrics, University of British Columbia, Canada, and Prof. Yutaka Tanaka and Mr. Kim Hyun Bin of the Department of Environmental and Mathematical Sciences, Faculty of Environmental Science and Technology, Okayama University, Japan, for their technical suggestions. This study was aided by a grant from the Japan Epilepsy Research Foundation.     

Cumulative lifetime incidence of extracolonic cancers in Lynch syndrome: a report of 121 families with proven mutations

Lynch syndrome or hereditary non-polyposis colorectal cancer is caused by mutations of DNA mismatch repair (MMR) genes. The extracolonic tumour spectrum includes endometrial, ovarian, gastric, small bowel, pancreatic, hepatobiliary, brain, and urothelial neoplasms. Families were referred on the basis of clinical criteria. Tumour immunohistochemistry and microsatellite testing were performed. Appropriate patients underwent sequencing of relevant exons of the MMR genes. Proven and obligate mutation carriers and first-degree relatives (FDRs) with a Lynch syndrome spectrum cancer were considered mutation carriers, as were a proportion of untested, unaffected FDRs based on the proportion of unaffected relatives testing positive in each age group. Kaplan–Meier analysis of risk to 70 years was calculated. One hundred and eighty-four Lynch syndrome spectrum extracolonic cancers in 839 proven, obligate, or assumed mutation carriers were analysed. Cumulative risk for females of an extracolonic tumour is 47.4% (95% CI 43.9–50.8). The risk to males is 26.5% (95% CI 22.6–30.4). There was no reduction in gynaecological malignancies due to gynaecological screening (examination, transvaginal ultrasound scan, hysteroscopy and endometrial biopsy). Males have a higher risk of gastric cancer than females (p = 0.0003). Gastric cancer risk in those born after 1935 does not justify surveillance. These penetrance estimates have been corrected for ascertainment bias and are appropriate for those referred to a high-risk clinic.     

Cancer‐associated somatic DICER1 hotspot mutations cause defective miRNA processing and reverse‐strand expression bias to predominantly mature 3p strands through loss of 5p strand cleavage

Our group recently described recurrent somatic mutations of the miRNA processing gene DICER1 in non‐epithelial ovarian cancer. Mutations appeared to be clustered around each of four critical metal‐binding residues in the RNase IIIB domain of DICER1. This domain is responsible for cleavage of the 3′ end of the 5p miRNA strand of a pre‐mRNA hairpin. To investigate the effects of these cancer‐associated 'hotspot' mutations, we engineered mouse DICER1‐deficient ES cells to express wild‐type and an allelic series of the mutant DICER1 variants. Global miRNA and mRNA profiles from cells carrying the metal‐binding site mutations were compared to each other and to wild‐type DICER1. The miRNA and mRNA profiles generated through the expression of the hotspot mutations were virtually identical, and the DICER1 hotspot mutation‐carrying cells were distinct from both wild‐type and DICER1‐deficient cells. Further, miRNA profiles showed that mutant DICER1 results in a dramatic loss in processing of mature 5p miRNA strands but were still able to create 3p strand miRNAs. Messenger RNA (mRNA) profile changes were consistent with the loss of 5p strand miRNAs and showed enriched expression for predicted targets of the lost 5p‐derived miRNAs. We therefore conclude that cancer‐associated somatic hotspot mutations of DICER1, affecting any one of four metal‐binding residues in the RNase IIIB domain, are functionally equivalent with respect to miRNA processing and are hypomorphic alleles, yielding a global loss in processing of mature 5p strand miRNA. We further propose that this resulting 3p strand bias in mature miRNA expression likely underpins the oncogenic potential of these hotspot mutations. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Thoracic outlet syndrome: evaluation with CT

Diagnosis of the thoracic outlet syndrome is often difficult, particularly in patients without osseous abnormalities on plain radiographs. The radiographic and computed tomographic (CT) findings were reviewed from 27 patients with thoracic outlet syndrome and 21 normal subjects. The plain radiographs and CT scans were assessed by two independent observers without awareness of the clinical history. Fifteen patients with thoracic outlet syndrome had osseous abnormalities (anomalous cervical ribs; abnormally long, drooping C-7 transverse processes) identifiable on plain radiographs. CT did not provide further diagnostic information in the patients with abnormal radiographs. Eight of 12 patients (66%) with normal plain radiographs had abnormal findings on CT scans, consisting of impingement of the C-7 transverse process on the scalene triangle or anteromedial aspect of the middle scalene muscle. Only two of 21 control patients (9.5%) displayed this CT abnormality (P less than .01). CT may be useful in patients with symptoms suggestive of thoracic outlet syndrome and no osseous abnormalities on plain radiographs.