Sezary cell morphology induced in peripheral blood lymphocytes: re- evaluation

In this study we examined the effect of mitogens and epidermal cells in inducing a Sezary cell morphology in normal peripheral blood lymphocytes. Peripheral blood mononuclear cells from six healthy volunteers were stimulated with the mitogens phytohemaglutinin and concanavalin A, and also cocultivated with human epidermal cell cultures. Incubation times with mitogens and epidermal cells were four days and stimulation of the lymphocytes by mitogens was confirmed by standard 3H-thymidine uptake. Standard transmission electron microscopy showed that in the mitogen-driven system 20% to 60% (33 +/- 15%) and in the epidermal cell-driven system 5% to 15% (8 +/- 4%) of the lymphoid cells exhibited mild to moderate indentation of the nuclei with nuclear contour indices (NCI) of 4.6 to 6.5 but no Sezary cells were observed (cells with NCI greater than 6.5 and up to 19.2). In the mitogen- stimulated preparation 2% to 5% (3 +/- 1%) of the lymphoid cells showed nuclear multilobulation resembling the cells seen in adult T cell lymphoma/leukemia. Incubation of mononuclear cells for longer periods of up to 4 weeks with mitogens and exogenous IL-2 resulted in no further morphologic changes. Using an indirect immunogold technique at the electron microscopic level, the cells showing nuclear indentation or lobulation were shown to bear both T helper (CD4) and T suppressor (CD8) cell phenotypes in a similar ratio to the total numbers of T helper and T suppressor cells present. Mitogens and epidermal cells are thus not able to induce a morphologic change to Sezary cells in normal peripheral blood lymphocytes.     

Genetic and phenotypic architecture of metabolic syndrome-associated components in dyslipidemic and normolipidemic subjects: the GEMS Study

Atherogenic dyslipidemia, manifest by low HDL-cholesterol and high TG levels, is an important component of ATP-III defined metabolic syndrome. Here, we dissected the phenotypic and genetic architecture of these traits by assessing their relationships with other metabolically relevant measures, including plasma adipo-cytokines, highly sensitive C-reactive protein (hsCRP) and LDL particle size, in a large family data set (n=2800) and in an independent set of dyslipidemic cases (n=716) and normolipidemic controls (n=1073). We explored the relationships among these phenotypes using variable clustering and then estimated their genetic heritabilities and cross-trait correlations. In families, four clusters explained 61% of the total variance, with one adiposity-related cluster (including hsCRP), one BP-related cluster, and two lipid-related clusters (HDL-C, TG, adiponectin and LDL particle size; apoB and non-HDL-C). A similar structure was observed in dyslipidemic cases and normolipidemic controls. The genetic correlations in the families largely paralleled the phenotype clustering results, suggesting that common genes having pleiotropic effects contributed to the correlations observed. In summary, our analyses support a model of metabolic syndrome with two major components, body fat and lipids, each with two subcomponents, and quantifies their degree of overlap with each other and with metabolic-syndrome related measures (adipokines, LDL particle size and hsCRP).     

Risk Stratification Among Survivors of Cardiac Arrest Considered for Coronary Angiography

BackgroundThe American College of Cardiology Interventional Council published consensus-based recommendations to help identify resuscitated cardiac arrest patients with unfavorable clinical features in whom invasive procedures are unlikely to improve survival.ObjectivesThis study sought to identify how many unfavorable features are required before prognosis is significantly worsened and which features are most impactful in predicting prognosis.MethodsUsing the INTCAR (International Cardiac Arrest Registry), the impact of each proposed “unfavorable feature” on survival to hospital discharge was individually analyzed. Logistic regression was performed to assess the association of such unfavorable features with poor outcomes.ResultsSeven unfavorable features (of 10 total) were captured in 2,508 patients successfully resuscitated after cardiac arrest (ongoing cardiopulmonary resuscitation and noncardiac etiology were exclusion criteria in our registry). Chronic kidney disease was used in lieu of end-stage renal disease. In total, 39% survived to hospital discharge. The odds ratio (OR) of survival to hospital discharge for each unfavorable feature was as follows: age >85 years OR: 0.30 (95% CI: 0.15 to 0.61), time-to-ROSC >30 min OR: 0.30 (95% CI: 0.23 to 0.39), nonshockable rhythm OR: 0.39 (95% CI: 0.29 to 0.54), no bystander cardiopulmonary resuscitation OR: 0.49 (95% CI: 0.38 to 0.64), lactate >7 mmol/l OR: 0.50 (95% CI: 0.40 to 0.63), unwitnessed arrest OR: 0.58 (95% CI: 0.44 to 0.78), pH <7.2 OR: 0.78 (95% CI: 0.63 to 0.98), and chronic kidney disease OR: 0.96 (95% CI: 0.70 to 1.33). The presence of any 3 or more unfavorable features predicted <40% survival. Presence of the 3 strongest risk factors (age >85 years, time-to-ROSC >30 min, and non-ventricular tachycardia/ventricular fibrillation) together or ≥6 unfavorable features predicted a ≤10% chance of survival to discharge.ConclusionsPatients successfully resuscitated from cardiac arrest with 6 or more unfavorable features have a poor long-term prognosis. Delaying or even forgoing invasive procedures in such patients is reasonable.     

Purging of acute myeloid leukemic cells by ether lipids and hyperthermia

Ether lipids (EL) and hyperthermia have been shown to possess a relatively selective cytotoxicity to leukemic cells. In this study, the combined effects of EL (ET-18-OCH3, ET-16-NHCOCH3, or BM 41.440) and hyperthermia on the growth of hematopoietic progenitors, myeloid leukemic cell lines, and leukemic cells obtained from patients with acute myeloid leukemia (AML) were examined to determine if this combination resulted in a greater selective killing of leukemic cells than that achieved by either EL or heat alone. When the cells were treated simultaneously with EL (50 micrograms/mL) and hyperthermia (42 degrees C) for one hour, the killing of leukemic cell line cells was enhanced considerably. Among the three EL, however, the combination of ET-18-OCH3 and heat seemed to be the most cytotoxic to leukemic cell line cells with no effect on the growth of hematopoietic progenitors. An increase in the duration of treatment with ET-18-OCH3 to four hours with heat added during the last hour resulted in a further reduction of leukemic cell line cells while sparing 50% of hematopoietic progenitors after cryopreservation. The combined treatment with ET-18-OCH3 and heat also inhibited the growth of leukemic progenitors obtained from AML patients by 97% to 100%. These data indicate that the combined treatment with EL and hyperthermia might offer an efficient means to eliminate myeloid leukemic cells in vitro.     

Characterization of primitive subpopulations of normal and leukemic cells present in the blood of patients with newly diagnosed as well as established chronic myeloid leukemia

Elevated numbers of primitive Philadelphia chromosome-positive (Ph+) progenitors, including long-term culture-initiating cells (LTC-IC) as well as colony-forming cells (CFC), have been previously described in the blood of patients with chronic myeloid leukemia (CML) in chronic phase with high white blood cell counts. In the present study, which focused primarily on an analysis of circulating progenitors present in such patients at diagnosis, we discovered the frequent and occasionally exclusive presence of circulating normal (Ph-) LTC-IC, often at levels above those seen for LTC-IC in the blood of normal individuals. The presence of detectable numbers of circulating Ph- LTC-IC was independent of the fact that the same peripheral blood samples also contained elevated numbers of predominantly or exclusively Ph+ CFC. Interestingly, both the Ph+ and Ph- LTC-IC in these samples were CD34+CD71- and variably CD38- and Thy-1+, as previously documented for LTC-IC in normal marrow. Thus, neither CD38 nor Thy-1 expression was useful for discriminating between Ph+ and Ph- LTC-IC in mixed populations. Nevertheless, an association of these phenotypes with LTC- IC function did allow highly enriched (> 5% pure) suspensions of either Ph+ or Ph- LTC-IC to be obtained from selected samples of CML blood in which the initial LTC-IC population was either predominantly Ph+ or Ph- , respectively. These findings suggest that the mechanisms causing mobilization of leukemic stem cells in untreated CML patients may affect their normal counterparts. They also indicate a possible new source of autologous cells for the support of intensive therapy of CML patients. Finally, they provide a method for obtaining the most highly purified populations of Ph+ LTC-IC described to date. This method should be useful for further analyses of the molecular activities of these very primitive neoplastic cells.     

Clathrin light chains function in mannose phosphate receptor trafficking via regulation of actin assembly

Clathrin-coated vesicles (CCVs) are major carriers for endocytic cargo and mediate important intracellular trafficking events at the trans-Golgi network (TGN) and endosomes. Whereas clathrin heavy chain provides the structural backbone of the clathrin coat, the role of clathrin light chains (CLCs) is poorly understood. We now demonstrate that CLCs are not required for clathrin-mediated endocytosis but are critical for clathrin-mediated trafficking between the TGN and the endosomal system. Specifically, CLC knockdown (KD) causes the cation-independent mannose-6 phosphate receptor (CI-MPR) to cluster near the TGN leading to a delay in processing of the lysosomal hydrolase cathepsin D. A recently identified binding partner for CLCs is huntingtin-interacting protein 1-related (HIP1R), which is required for productive interactions of CCVs with the actin cytoskeleton. CLC KD causes mislocalization of HIP1R and overassembly of actin, which accumulates in patches around the clustered CI-MPR. A dominant-negative CLC construct that disrupts HIP1R/CLC interactions causes similar alterations in CI-MPR trafficking and actin assembly. Thus, in mammalian cells CLCs function in intracellular membrane trafficking by acting as recruitment proteins for HIP1R, enabling HIP1R to regulate actin assembly on clathrin-coated structures.     

Mitral Valve Repair Using ePTFE Sutures for Ruptured Mitral Chordae Tendineae: A Computational Simulation Study

Mitral valve (MV) repair using expanded polytetrafluoroethylene sutures is an established and preferred interventional method to resolve the complex pathophysiologic problems associated with chordal rupture. We developed a novel computational evaluation protocol to determine the effect of the artificial sutures on restoring MV function following valve repair. A virtual MV was created using three-dimensional echocardiographic data in a patient with ruptured mitral chordae tendineae (RMCT). Virtual repairs were designed by adding artificial sutures between the papillary muscles and the posterior leaflet where the native chordae were ruptured. Dynamic finite element simulations were performed to evaluate pre- and post-repair MV function. Abnormal posterior leaflet prolapse and mitral regurgitation was clearly demonstrated in the MV with ruptured chordae. Following virtual repair to reconstruct ruptured chordae, the severity of the posterior leaflet prolapse decreased and stress concentration was markedly reduced both in the leaflet tissue and the intact native chordae. Complete leaflet coaptation was restored when four or six sutures were utilized. Computational simulations provided quantitative information of functional improvement following MV repair. This novel simulation strategy may provide a powerful tool for evaluation and prediction of interventional treatment for RMCT.     

Biological and Analytical Stability of a Peripheral Blood Gene Expression Score for Obstructive Coronary Artery Disease in the PREDICT and COMPASS Studies

A gene expression score (GES) for obstructive coronary artery disease (CAD) has been validated in two multicenter studies. Receiver-operating characteristics (ROC) analysis of the GES on an expanded Personalized Risk Evaluation and Diagnosis in the Coronary Tree (PREDICT) cohort (NCT no. 00500617) with CAD defined by quantitative coronary angiography (QCA) or clinical reads yielded similar performance (area under the curve (AUC)?=?0.70, N?=?1,502) to the original validation cohort (AUC?=?0.70, N?=?526). Analysis of 138 non-Caucasian and 1,364 Caucasian patients showed very similar performance (AUCs?=?0.72 vs. 0.70). To assess analytic stability, stored samples of the original validation cohort (N?=?526) was re-tested after 5 years, and the mean score changed from 20.3 to 19.8 after 5 years (N?=?501, 95 %). To assess patient scores over time, GES was determined on samples from 173 Coronary Obstruction Detection by Molecular Personalized Gene Expression (COMPASS) study (NCT no. 01117506) patients at approximately 1 year post-enrollment. Mean scores increased slightly from 15.9 to 17.3, corresponding to a 2.5 % increase in obstructive CAD likelihood. Changes in cardiovascular medications did not show a significant change in GES.