Identification of parasite proteins in a membrane preparation enriched for the surface membrane of erythrocytes infected with Plasmodium knowlesi

A subcellular fraction enriched in erythrocyte membranes has been isolated from rhesus monkey erythrocytes infected with Plasmodium knowlesi. Infected cells were lysed by centrifugation through a zone of hypotonic buffer and membranes isolated by equilibrium density gradient centrifugation in the same tube. The purified membrane fraction was shown to include the erythrocyte surface membrane by several methods: electron microscopy, identification of Coomassie Blue stained erythrocyte membrane proteins, identification of band 3 with a monoclonal antibody, and identification of radioiodinated cell surface proteins. The resulting ghosts were shown to be specifically reactive with monkey sera against the variant surface antigens of P. knowlesi by indirect immunofluorescence and membrane agglutination. No reactivity was seen with a monoclonal antibody (13C11) against the intracellular schizont surface. A number of metabolically labelled parasite proteins were enriched in this membrane function, including peptides of 277, 208, 173, 153, 134, 109, 80, 60 and 48 kDa and the variant surface antigens of variable molecular mass (180-207 kDa). These proteins were distinct from the major parasite proteins of total infected erythrocytes and isolated merozoites. The major glucosamine labelled glycoprotein of the internal schizont (230 kDa) was not found in this fraction. Moreover, no fragment of this parasite glycoprotein was found in this membrane fraction, indicating that no part of this molecule is transported to the erythrocyte surface. In contrast, the variant antigen of P. knowlesi, known to be on the erythrocyte surface, could be readily identified as peptides unique to specific cloned parasite lines. We propose that the other nine parasite proteins found within this membrane fraction represent a starting point for the identification of other parasite proteins transported to the surface membrane of the infected erythrocyte.     

Isolation of exfoliated colonocytes from human stool as a new technique for colonic cytology

Cell exfoliation in the gut is an important cell renewal mechanism. To approach its investigation we applied a novel immunomagnetic technique for isolation of exfoliated cells from human stool. Exfoliated colonocytes were isolated from 168 stool samples. The cells were assessed microscopically using conventional stains and immunohistochemistry. The technique allowed us to obtain well-preserved colonocytes displaying characteristic features of well-differentiated colonic epithelium and positive immunostaining for cytokeratin 5/8. No mucin-producing cells were found. Exfoliated cells did not produce inducible nitric oxide synthase, albeit cultured colon carcinoma cells HT-29 analysed in parallel showed strong immunostaining. Analysis of exfoliated cell numbers in consecutive stool samples from the same subjects revealed considerable interindividual variation. Overall exfoliated colonocyte numbers were relatively low, isolation being unaffected by addition during the procedure of excessive amounts of HT-29 cells. Apoptosis was extremely rare among exfoliated colonocytes. Well-preserved exfoliated colonocytes can be consistently isolated from human faeces using a simple procedure. Our findings suggest that the actual process of cell exfoliation in the human colon may be much less intense than is generally accepted. Exfoliated cell isolation from human stool constitutes a convenient non-invasive approach that can be used for diagnostic and research purposes.     

Primary culture of fetalrat liver bipotential progenitor cells

Summary Techniques are described for the rapid isolation, monolayer culture, and phenotypical characterization of embryonic Day 12 (E12) rat liver epithelial cells. In vitro assays of the differentiation potential of these cells are currently being performed under conditions that make use of-modified Eagle's medium supplemented with fetal bovine serum, insulin, and dexamethasone with or without sodium butyrate. In the absence of sodium butyrate, the cells differentiate along the hepatocytic cell lineage, whereas in its presence they express the phenotype of the biliary ductal epithelial cell lineage.     

Flow-Cytometric Characterization of Hematopoietic Cells in Non-Pregnant Human Endometrium

Lachapelle M-H, Miron P, Hemmings R, Baron C, Roy DC. Flow-cytometric characterization of hematopoietic cells in non-pregnant human endometrium. Am J Reprod Immunol 1996; 35:5–13 cP Munksgaard, Copenhagen PROBLEM: Immunologic evaluation and quantitation of hematopoietic cells in human endometrium has been difficult to perform, particularly in nonpregnant subjects. In this study, we describe a method for the flow-cytometric characterization of hematopoietic cells present in the endometrium of non-pregnant women. METHOD: Endometrial biopsy samples from normal donors were first mechanically disrupted and filtered to generate a single-cell suspension of leukocyte-enriched endometrial cells. Cells were labeled with a panel of monoclonal antibodies, stained with propidium iodide (PI), and one- or two-color flow-cytometric analysis performed on cells excluding PI. RESULTS: The methodology described in this study was highly reproducible in experiments evaluating the interrun and intrarun variability. We then determined the immunophenotypic profile of endometrial leukocytes from 12 normal females. The majority of leukocytes were T cells (CD3: 47%; CD4: 24%; CD8: 28%) with an important contingent of NK cells (CD56: 32%), the majority of which harbored the unusual CD16-CD56 bright phenotype, and a minority of B cells (CD20: 6%) and monocytes (CD14: 7%). CONCLUSIONS: Flow cytometry can be used to assess antigen expression on the surface of endometrial leukocytes from nonpregnant women. In future studies, it will be possible to use this approach to investigate the role of immune cell populations in the endometrium of patient experiencing reproductive failure.     

Neonatal social isolation alters both maternal and pup behaviors in rats

The development of emotional behavior is dependent on the early experiences of the infant and the quality of maternal care. In these experiments, the effects of social isolation during the preweaning period on both pup behavior and maternal responsivity were examined. In the first study, the number of ultrasonic vocalizations (USVs) emitted after brief maternal separation was measured in neonatal rats with differing histories of social isolation. The social isolation procedure consisted of 5 days of daily separation from the dam and littermates for either 3 or 6 hr. At both ages tested, socially isolated pups vocalized significantly less than control pups. In the second study, the effects of prior isolation either daily for 5 previous days (Chronic Isolation) or for 4 hr prior to testing (Acute Isolation) were examined in a T-maze choice test. Pup vocalizations in the presence of the dam and dams' maternal behavior were assessed. When the dam was confined to the start box or during the maternal free access period, both Chronic and Acute Isolates vocalized less than pups that had never left the home nest. Dams spent more time with and licked and groomed more frequently and for a longer time both Chronic and Acute Isolates compared to pups that had always been with dams in the home nest. These results suggest that early isolation experience can alter subsequent responses to separation stress in neonatal rats and that maternal behavior is sensitive to the prior experiences of offspring.     

Rewarding properties of social interactions in adolescent and adult male and female rats: impact of social versus isolate housing of subjects and partners

Social interactions have been shown to be rewarding for adolescent and adult rats; however, there has been little emphasis on comparing the strength of the rewarding value of social stimuli across ontogeny. Since age differences in social interactions may vary with sex or housing circumstances, the present study assessed social conditioned place preference (CPP) in adolescent and adult male and female Sprague-Dawley rats housed either socially or in isolation and conditioned with either group-housed or isolate-housed partners. Isolated animals of both sexes and ages demonstrated social CPP, with the strongest preference emerging in adolescent males. Social CPP was not evident in group-housed adults whereas group-housed adolescents developed a preference for the compartment previously paired with similarly housed partners; however, when socially housed adolescents were conditioned with isolated partners, social CPP did not emerge. Age differences in social CPP may reflect age-related neural alterations in brain systems implicated in regulation of social behavior.     

Isolation of embryonic chick myosatellite and pluripotent stem cells

Summary The current study outlines the isolation and culture of two populations of cells derived from Day 11 embryonic chick leg muscle and associated connective tissues. The two populations consisted of myogenic lineage-committed stem cells (myosatellite stem cells) and lineage-uncommitted stem cells (pluripotent stem cells). After long-term culture the lineage-uncommitted stem cell population displayed differentiated phenotypes suggestive of the following adult tissues, fibroblasts, muscle, fat, cartilage, and bone.     

Isolation and partial characterization of the tegumental outer membrane of schistosomula of Schistosoma mansoni

Separation of the external membranes from freshly converted mechanical schistosomula of Schistosoma mansoni was achieved by osmotic shock under hypertonic conditions, followed by mechanical shearing and ultracentrifugation. Prior to treatment, the schistosomula were surface labeled by introduction of N-DNP-epsilon-aminocaproylphosphatidylethanolamine molecules into their lipid bilayer followed by anti-DNP antibodies and stained with either 125I-protein-A or ferritin labeled secondary anti-DNP antibodies. This label provided a membrane marker by which the purity of the preparation could be assessed at each stage. Fluorescence staining with FITC-conjugated secondary antibodies prior to treatment revealed that the homogeneously stained membrane of the intact schistosomula became swollen and ruptured after the osmotic shock. The isolated membrane pellet was intensely fluorescent. Electron microscopical examination revealed mostly vesicles, some of them with organized multilayer assembly. The vesicles were ferritin labeled, indicating that they originated from the outer surface membrane of the schistosomula. A 100 fold enrichment in the alkaline phosphatase activity and about 300 fold enrichment in acetylcholinesterase activity in the membrane preparations, as compared to the intact schistosomula, was found. The isolated tegument was analyzed by SDS-polyacrylamide gel electrophoresis. The pattern obtained showed three major bands, of molecular weights 69 000, 45 000 and 12 000 alongside with a large number of minor bands. Immunoprecipitation of the isolated 125I-labeled membrane antigens with antisera from chronically infected mice revealed these three major bands together with three other bands of molecular weight 38 000, 23 000 and 16 000.     

中国城乡空巢老人社会隔离状况及其影响因素分析

目的 分析我国城乡空巢老人社会隔离状况及其影响因素差异。方法 社会隔离可被分为主观社会隔离和客观社会隔离,基于2014年中国老年社会追踪调查（CLASS）数据,纳入4 705名空巢老人,运用二分类logistic回归模型分析城乡空巢老人社会隔离的影响因素差异。结果 本研究空巢老人社会隔离发生率为51.3%,主、客观社会隔离同时发生率为11.9%,且农村社会隔离发生率（57.3%）高于城镇（47.4%）（χ2 = 43.871,P＜0.001）。无配偶、自评健康较差（OR = 1.635,95%CI:1.322～2.022;OR = 1.410,95%CI:1.119～1.777）、患有慢性病、低个人年收入、与子女见面（OR = 2.322,95%CI:1.457～3.701;OR = 2.858,95%CI:1.648～4.955）和电话联系频率低是城乡空巢老人出现社会隔离的危险因素,且ADL受损也是城乡空巢老人同时出现主、客观社会隔离的危险因素;教育程度和个人年收入是城镇空巢老人社会隔离发生的特有因素。结论 我国空巢老人社会隔离存在城乡差异,农村空巢老人社会隔离发生率高于城镇,应对城乡空巢老人采取针对性的干预措施。     

Specifics of chronic fatigue syndrome coping strategies identified in a French flash survey during the COVID-19 containment

The COVID-19 pandemic has focused health systems on supporting patients affected by this virus. Meanwhile in the community, many other contained patients could only use self-care strategies, especially in countries that have set up a long and strict containment such as France. The study aimed to compare coping strategies deployed by patients with Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS; a poorly recognised syndrome) to those with better known and referenced chronic conditions. An online flash survey was conducted during the containment period in partnership with French Patients Organizations including ME/CFS national association. Therefore, ‘Brief COPE’ version of Lazarus and Folkman's Ways of Coping Check List has been adapted to the specificity of the containment. The survey was e-distributed in France from 15 April to 11 May 2020. Differences of coping strategies were analyzed using Wilcoxon–Mann–Withney test. Amongst 637 responses, 192 were complete, presenting a wide variety of diseases, including 93 ME/CFS. The latter have significantly different coping strategies than recognised diagnosed diseases patients: similar uses of emotion focused coping but less uses of seek social support and problem-focused copings. In conclusion, coping strategies are different for those who deal with the daily experience of ME/CFS, highly disabling chronic condition with diagnostic ambiguity, low degree of medical and social recognition and without treatment. Better understanding of those strategies is needed to provide the means for health promotion researchers, managers and clinicians, to accompany those patients.