Expression of familial Alzheimer disease presenilin 1 gene attenuates vesicle traffic and reduces peptide secretion in cultured astrocytes devoid of pathologic tissue environment

In the brain, astrocytes provide metabolic and trophic support to neurones. Failure in executing astroglial homeostatic functions may contribute to the initiation and propagation of diseases, including Alzheimer disease (AD), characterized by a progressive loss of neurones over years. Here, we examined whether astrocytes from a mice model of AD isolated in the presymptomatic phase of the disease exhibit alterations in vesicle traffic, vesicular peptide release and purinergic calcium signaling. In cultured astrocytes isolated from a newborn wild‐type (wt) and 3xTg‐AD mouse, secretory vesicles and acidic endosomes/lysosomes were labeled by transfection with plasmid encoding atrial natriuretic peptide tagged with mutant green fluorescent protein (ANP.emd) and by LysoTracker, respectively. The intracellular Ca²⁺ concentration ([Ca²⁺]_i) was monitored with Fluo‐2 and visualized by confocal microscopy. In comparison with controls, spontaneous mobility of ANP‐ and LysoTracker‐labeled vesicles was diminished in 3xTg‐AD astrocytes; the track length (TL), maximal displacement (MD) and directionality index (DI) were all reduced in peptidergic vesicles and in endosomes/lysosomes (P < 0.001), as was the ATP‐evoked attenuation of vesicle mobility. Similar impairment of peptidergic vesicle trafficking was observed in wt rat astrocytes transfected to express mutated presenilin 1 (PS1_M146V). The ATP‐evoked ANP discharge from single vesicles was less efficient in 3xTg‐AD and PS1_M146V‐expressing astrocytes than in respective wt controls (P < 0.05). Purinergic stimulation evoked biphasic and oscillatory [Ca²⁺]_i responses; the latter were less frequent (P < 0.001) in 3xTg‐AD astrocytes. Expression of PS1_M146V in astrocytes impairs vesicle dynamics and reduces evoked secretion of the signaling molecule ANP; both may contribute to the development of AD. GLIA 2016;64:317–329     

Systemic response of peripheral blood leukocytes and their phagocytic activity during acute myocardial infarction

OBJECTIVES:

To determine changes in leukocyte counts and phagocytic activity of peripheral blood mononuclear (MN) and polymorphonuclear (PMN) cells as potential cellular markers of systemic immunological events in acute myocardial infarction (AMI).

PATIENTS AND METHODS:

Thirty patients with a first AMI and 30 healthy volunteers were examined. Immunological analyses were performed at admission and repeated at one and seven days after the acute event. MN and PMN cells were obtained from heparinized whole blood after centrifugation and separation on a density gradient, and incubated with a fixed number of heat-inactivated and labelled yeast particles. Total leukocyte counts, leukocyte populations and some parameters of phagocytic activity were determined: percentage phagocytosis, phagocytic index, absolute phagocytic index, count of phagocytes in a fixed volume of peripheral blood (CP) and phagocytic capacity.

RESULTS:

Patients with AMI had increased total leukocyte counts accompanied by increased PMN counts, while there were no significant differences in total MN count and MN populations. Except for the phagocytic index, all phagocytic parameters of MN and PMN cells were increased in patients with AMI at admission and on the first day of disease. On the seventh day after AMI only the CP of MN cells had increased significantly in patients with AMI, while percentage phagocytosis, CP and capacity of phagocytosis of PMN cells increased during the acute phase of AMI.

CONCLUSIONS:

These data suggest that AMI was followed with a strongly systemic inflammatory response to myocardial damage. Furthermore, activated MN and PMN cells may be a significant source of free radicals that may be involved in lipid peroxidation and produce tissue damage in the early postinfarction period.     

The influence of obesity on the oxidative stress status and the concentration of leptin in type 2 diabetes mellitus patients

The aim of this study was to determinate both the oxidative stress/anti-oxidative defense status and the concentration of leptin in obese, overweight and normal weight type 2 diabetes mellitus patients to seek possible association between oxidative stress and hyperleptinemia. Oxidative stress status parameters [thiobarbituric acid-reacting substances (TBARS), superoxide anion (O(2)(-)), superoxide dismutase (SOD) activity and total sulphydryl groups] and the concentration of leptin were measured in 312 subjects (178 patients and in 134 control subjects). Obese patients had a significantly higher concentration of leptin compared to obese subjects in the control population (P<0.001). They also had significantly higher plasma concentrations of TBARS, O(2)(-) and SOD activity in combination with a lower sulphydryl group concentration when compared to control subjects. Obese patients had significantly higher concentrations of both TBARS and O(2)(-) and increased SOD activity compared to normal weight patients. The odds ratio for the degree of association between oxidative stress status parameters and hyperleptinemia was strongest for TBARS [odds ratio 2.66, 95% CI (1.02-6.94), P=0.045]. The observed positive correlation between TBARS and leptin (rho=0.29, P<0.01) in obese patients suggests that increased oxidative stress and hyperleptinemia, both consequences of obesity, may play a role in type 2 diabetes mellitus development.     

Molecular dissection of water and glycerol permeability of the aquaglyceroporin from Plasmodium falciparum by mutational analysis

The selectivity of aquaporins for water and solutes is determined by pore diameter. Paradoxically, the wider pores of glycerol facilitators restrict water passage by an unknown mechanism. Earlier we characterized an aquaglyceroporin from Plasmodium falciparum with high permeability for both glycerol and water. We use point mutations to demonstrate that amino acids directly lining the pore are not responsible for the excellent water permeability of the Plasmodium aquaglyceroporin but affect permeability of pentitols. Within a conserved WET triad in the extracellular C-loop we identified a Plasmodium aquaglyceroporin-specific glutamate (E125) located in proximity to a conserved arginine (R196) at the pore mouth. Mutation of E125 to serine largely abolished water permeability. Concomitantly, the activation energy for water permeation was increased by 4 kcal/mol. Mutation of the adjacent tryptophan to cysteine led to irreversible inhibition of water passage by Hg(2+). This unequivocally proves the proximity of the couple W124/E125 close to the pore mouth. We conclude that in the Plasmodium aquaglyceroporin the electrostatic environment at the extracellular pore entry regulates water permeability.     

Predictors of nurses' experience of verbal abuse by nurse colleagues

Background

Between 45% and 94% of registered nurses (RNs) experience verbal abuse, which is associated with physical and psychological harm. Although several studies examined predictors of RNs' verbal abuse, none examined predictors of RNs' experiences of verbal abuse by RN colleagues.

Leptin-mediated decrease of cyclin A2 and increase of cyclin D1 expression: relevance for the control of prepubertal rat Leydig cell division and differentiation

The number of adult Leydig cells is one of the factors controlling testosterone secretion by sexually mature testis, and it depends on the proliferative capacity of prepubertal Leydig cells. We investigated here whether this capacity is controlled by leptin because this hormone regulates proliferation in other cell types and has a crucial role in male fertility. Our data show that prebupertal Leydig cells express the Ob/Rb form of leptin receptor and are thus direct targets of this hormone. The analysis of G1/S-phase cyclins by quantitative (real-time) RT-PCR and Western blot points to the leptin-induced decrease in cyclin A2 and subsequent increase in cyclin D1 expression that precedes a leptin-triggered decrease in the number of prepubertal Leydig cells. Quantitative assessments of DNA synthesis by bromodeoxyuridine incorporation and of cycling cell population by Ki67 immunocytochemistry indicate that leptin decreases the cell number by inhibiting cell division and increases mRNA levels of Leydig cell differentiation markers such as relaxin-like factor. Immunohistochemistry of cyclin D1 and relaxin-like factor pointed to the parallel increase of their expression coinciding with the onset of Leydig cell differentiation. Moreover, leptin-treated Leydig cells display increased expression of another differentiation marker (3beta-hydroxysteroid dehydrogenase) that is abolished by knocking down cyclin D1 with small interference RNA. Altogether, our data show that leptin inhibits division of prepubertal Leydig cells via a cyclin D-independent mechanism and suggest that cyclin D1 might be involved in leptin-induced differentiation of Leydig cells.     

Activated circulating dendritic cells after hematopoietic stem cell transplantation predict acute graft-versus-host disease

BACKGROUND: Dendritic cells (DC) are central to the development of acute graft-versus-host disease (GVHD) following allogeneic hematopoietic stem cell transplantation (alloHSCT). We hypothesized that DC activation status determines the severity of GVHD and that activated DC may be detected in the circulation prior to clinical presentation of GVHD. METHODS: Following transplant, blood samples were obtained twice weekly from alloHSCT patients. Myeloid (CD11c+) and plasmacytoid (CD123hi) DC were enumerated by flow cytometry, and activated myeloid DC were identified using the CMRF-44 monoclonal antibody. RESULTS: Of 40 alloHSCT patients, 26 developed acute GVHD. Severity of GVHD was associated with low total blood DC counts (P=0.007) and with low myeloid and plasmacytoid DC numbers (P=0.015 and 0.003). The CMRF-44 antigen was expressed on blood CD11c+ DC in all cases prior to GVHD onset, whereas of the 14 patients without GVHD, seven had no CMRF-44+ CD11c DC. Patients with CMRF-44+ CD11c+ DC in more than 20% of samples were more likely to subsequently develop acute GVHD (P=0.001, odds ratio=37.1), while patients who developed grade 2-4 GVHD had prior higher percentages of CMRF-44+ CD11c+ DC compared to grade 0-1 GVHD patients (P=0.001). CMRF-44 expression on >7.9% CD11c+ DC predicted for subsequent development of GVHD with a sensitivity of 87.5% and specificity of 79.2%. CONCLUSIONS: Activation status, as assessed by CMRF-44 antigen expression, of blood CD11c+ DC is highly associated with acute GVHD and these cells may be targets for therapeutic intervention.     

Prospective randomized study comparing sentinel lymph node evaluation with standard pathologic evaluation for the staging of colon carcinoma: results from the United States Military Cancer Institute Clinical Trials Group Study GI-01

BACKGROUND: The principal role of sentinel lymph node (SLN) sampling and ultrastaging in colon cancer is enhanced staging accuracy. The utility of this technique for patients with colon cancer remains controversial. PURPOSE: This multicenter randomized trial was conducted to determine if focused assessment of the SLN with step sectioning and immunohistochemistry (IHC) enhances the ability to stage the regional nodal basin over conventional histopathology in patients with resectable colon cancer. PATIENTS AND METHODS: Between August 2002 and April 2006 we randomly assigned 161 patients with stage I-III colon cancer to standard histopathologic evaluation or SLN mapping (ex vivo, subserosal, peritumoral, 1% isosulfan blue dye) and ultrastaging with pan-cytokeratin IHC in conjunction with standard histopathology. SLN-positive disease was defined as individual tumor cells or cell aggregates identified by hematoxylin and eosin (H&E) and/or IHC. Primary end point was the rate of nodal upstaging. RESULTS: Significant nodal upstaging was identified with SLN ultrastaging (Control vs. SLN: 38.7% vs. 57.3%, P = 0.019). When SLNs with cell aggregates < or =0.2 mm in size were excluded, no statistically significant difference in node-positive rate was apparent between the control and SLN arms (38.7% vs. 39.0%, P = 0.97). However, a 10.7% (6/56) nodal upstaging was identified by evaluation of H&E stained step sections of SLNs among study arm patients who would have otherwise been staged node-negative (N0) by conventional pathologic assessment alone. CONCLUSION: SLN mapping, step sectioning, and immunohistochemistry (IHC) identifies small volume nodal disease and improves staging in patients with resectable colon cancer. A prospective trial is ongoing to determine the clinical significance of colon cancer micrometastasis in sentinel lymph nodes.