脑血栓形成患者的红细胞变形性测定

采用国产ＢＬ８８－Ｃ型激光衍射红细胞变形仪，对３４例脑血栓形成急性期患者进行红细胞变形性的测定，并与同期住院的３４例上呼吸道感染和周围神经病患者进行对比，结果表明，急性脑血栓形成患者红细胞变形指数（ＤＩ）较对照组明显降低，以低剪切力时尤为明显，说明红细胞变形性降低是脑血栓形成的原因之一，并提示随年龄的增加，急性脑血栓形成的发病率也在增加。     

悬浮介质对红细胞变形性测量的影响

本文研究了悬浮介质粘度对激光衍射仪测量红细胞变形性的影响,所得结果表明:(1)在一定切变率下(800s~(-1),480s~(-1))DI-η曲线的高粘度区与前人(如D.R.Morris等)一致,但低粘度区有一极小值。(2)即使在相同切应力下,DI-η曲线相当复杂,特别是在低切应力水平更是如此,但是当η≥15cP时,DI趋于定值。(3)利用红细胞在切变流场中旋转、取向与变形时间尺度上的明显差异,可以把红细胞旋转取向的贡献从变形中区分出来。实验结果表明,低粘度DI-η复杂行为主要起因于红细胞的旋转和取向。文中对实验观测到的现象进行了讨论。     

Detection of antigen specific rosette formation with the FACS

Donors previously sensitized to conventional antigens PPD and KLH were evaluated for their antigen binding responses, utilizing a rosette forming technique with antigen-conjugated autologous erythrocytes. Reactivity is directly correlated with prior sensitization. Furthermore, antigen specificity is suggested by inhibition of rosette formation by prior incubation with the relevant antigen. The frequency of RFCs detected cytofluorometrically was compared with conventional fluorescent microscopy determinations. RFCs detected in this manner were identified as antibody armed monocytes by cell depletion and histochemical studies. The usefulness of the rosette forming technique for the routine evaluation of donor immunity is discussed.     

In vitro selection of murine antigen-specific T-lymphocytes. I. Description of selection procedure

Enrichment of antigen-responsive murine T-lymphocytes was achieved by two in vitro procedures through the preferential adherence of the antigen-specific cells to the antigen-pulsed macrophages and their consequent multiplication. The first procedure involved the addition of column-purified T-enriched lymph node lymphocytes from immunized mice to a monolayer of antigen-pulsed adherent spleen cells from non-primed syngeneic donors. Lymphocytes which failed to adhere to the antigen-pulsed monolayer were removed after 4 h of incubation. The adherent cells were cultured for a week and the lymphocytes obtained after that period from the selection plate were highly responsive to the antigen for which they were selected and for a T-cell mitogen (Con A). On the other hand, these selected cells demonstrated little or no response to other antigens, to which the original donor of the lymphocytes was immune, and to a B-cell mitogen (LPS). The same preferential response to the selecting antigen and T-cell mitogen was obtained in lymphocytes enriched in the alternate procedures on ‘supernatant cultures’. The enriched population from ‘supernatant culture’ was derived from cells that did not adhere to the antigen-pulsed monolayers during 4 h of incubation. The non-adherent lymphocytes which still contained antigen-specific lymphocytes were transferred to a fresh monolayer of antigen-pulsed adherent spleen cells to be grown for a week in culture. The improvement in the response to the selecting antigen and the decreased reaction to other immunizing antigens show that the cells harvested from either ‘selected’ or ‘supernatant’ cultures were enriched for a given antigen — the selecting antigen. In most individual experiments the enrichment was better in the selected cultures.The enrichment procedures were dependent upon effective antigen presentation to the lymphocytes. Spleen cells from mineral oil-injected mice, which are the most effective antigen-presenting cells, formed the most efficient monolayers for the enrichment of the antigen-specific lymphocytes, both in the ‘selected’ and ‘supernatant’ cultures.     

Opioid Utilization by Pregnant Women with Sickle Cell Disease and the Risk of Neonatal Abstinence Syndrome

Background

Pregnant women with sickle cell disease (SCD) are at increased risk of maternal and fetal complications. There are limited data on the outcome of the treatment of VOCs with opioids in relation to neonatal complications during pregnancy.

A novel serological assay for influenza based on DiD fluorescence dequenching that is free from observer bias and potentially automatable – A proof of concept study

BackgroundSerum hemagglutination inhibition (HAI) and microneutralization (MN) antibodies are often used as a correlate of protection for influenza. However, these manual assays are labor-intensive and difficult to standardize due to variability in biologic reagents used and subjective interpretation of the results.MethodsSera with known HAI and MN titers were used to assess a novel test based on the inhibition of fluorescence ‘dequenching’. Whole influenza virions (A/California/07/2009 (H1N1), A/Hong Kong/4801/2014 (H3N2) and B/Brisbane/60/2008) labelled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindodicarbocyanine perchlorate (DiD) were exposed to serial dilutions of serum and mixed with turkey red blood cells followed by acidification of the media (pH 5.0–5.5). The H1N1 and B/Brisbane strains were high hemagglutinating while the H3N2 strain had low hemagglutinating activity. In some experiments, labelled virions were subjected to repetitive freeze-thaw cycles prior to use in the assay.ResultsIn the absence of detectable HAI/MN antibodies, there were consistent and substantial increases from baseline DiD fluorescence upon acidification. Sera with known high titer HAI/MN antibodies reduced or completely prevented DiD dequenching at low dilutions with progressive increases in fluorescence at higher dilutions, which permitted a reproducible assignment of an antibody ‘titer’ based on baseline and acidified DiD fluorescence values. The ‘titers’ measured by the DiD dequenching assay were highly correlated with HAI/MN results for the H1N1 and B strains (Spearman’s correlation coefficients (r_s) 0.874 to 0.946, p < 10⁻⁷ to 10⁻³⁵). Correlations with HAI/MN titres for the low-hemagglutinating H3N2 strain tested were lower but remained statistically significant (r_s 0.547–0.551, p < 0.004). Freeze-thawing of the DiD pre-stained virus stocks had no significant impact on the results of the assay.ConclusionsThe DiD dequenching assay may be a labour-saving and more objective alternative to the classic serologies. This novel assay could theoretically be standardized across laboratories using pre-stained virions and has the potential to be fully automated.     

Novel method for clearing red blood cell debris from BacT/ALERT blood culture medium for improved microscopic and antimycobacterial drug susceptibility test results

Even though automation in mycobacterial culture has immensely improved the detection of organisms, identification of species and antimycobacterial susceptibility testing from blood culture bottles remain cumbersome and error-prone due to the presence of intact red blood cells (RBCs). The removal or lysis of these RBCs and excessive protein from the blood components could theoretically help improve this process. The present study reports an effective method that uses ammonium chloride (NH(4)Cl) and Triton X-100 to lyse the RBCs in blood culture medium. The method was optimized by preparing various concentrations of NH(4)Cl and Triton X-100, and incubation conditions, leading to eight protocols. The lysis protocol with a concentration of 150 mM of NH(4)Cl, 0.5% Triton X-100, and 1% potassium bicarbonate, pH 7.0, and incubation at 37 degrees C for 15 min was found to be optimal. This method not only made the culture medium clear, the protein concentration decreased from 753.5+/-39.4 to 53.2+/-4.2 mg/mL in the M. tuberculosis-spiked culture medium and in the blood culture medium inoculated with the blood from tuberculosis patients. The method had no adverse effect on mycobacteria, and no depletion of M. tuberculosis colony-forming units was found. The lysate could be used for antimycobacterial susceptibility testing with no difficulty in setting the mycobacterial concentration of inoculum to 0.5 McFarland standards. Furthermore, this method had the added advantage in the microscopy and molecular methods for the speciation of Mycobacterium sp.     

Mammalian acyl-CoA:lysophosphatidylcholine acyltransferase enzymes

The mammalian RBC lacks de novo lipid synthesis but maintains its membrane composition by rapid turnover of acyl moieties at the sn-2 position of phospholipids. Plasma-derived fatty acids are esterified to acyl-CoA by acyl-CoA synthetases and transferred to lysophospholipids by acyl-CoA:lysophospholipid acyltransferases. We report the characterization of three lysophosphatidylcholine (lysoPC) acyltransferases (LPCATs), products of the AYTL1, -2, and -3 genes. These proteins are three members of a LPCAT family, of which all three genes are expressed in an erythroleukemic cell line. Aytl2 mRNA was detected in mouse reticulocytes, and the presence of the product of the human ortholog was confirmed in adult human RBCs. The three murine Aytl proteins generated phosphatidylcholine from long-chain acyl-CoA and lysoPC when expressed in Escherichia coli membranes. Spliced variants of Aytl1, affecting a conserved catalytic motif, were identified. Calcium and magnesium modulated LPCAT activity of both Aytl1 and -2 proteins that exhibit EF-hand motifs at the C terminus. Characterization of the product of the Aytl2 gene as the phosphatidylcholine reacylating enzyme in RBCs represents the identification of a plasma membrane lysophospholipid acyltransferase and establishes the function of a LPCAT protein.