Primed for addiction: A critical review of the role of microglia in the neurodevelopmental consequences of adolescent alcohol drinking

Alcohol is one of the most widely used recreational substances worldwide, with drinking frequently initiated during adolescence. The developmental state of the adolescent brain makes it vulnerable to initiating alcohol use, often in high doses, and particularly susceptible to alcohol-induced brain changes. Microglia, the brain parenchymal macrophages, have been implicated in mediating some of these effects, though the role that these cells play in the progression from alcohol drinking to dependence remains unclear. Microglia are uniquely positioned to sense and respond to central nervous system insult, and are now understood to exhibit innate immune memory, or “priming,” altering their future functional responses based on prior exposures. In alcohol use disorders (AUDs), the role of microglia is debated. Whereas microglial activation can be pathogenic, contributing to neuroinflammation, tissue damage, and behavioral changes, or protective, it can also engage protective functions, providing support and mediating the resolution of damage. Understanding the role of microglia in adolescent AUDs is complicated by the fact that microglia are thought to be involved in developmental processes such as synaptic refinement and myelination, which underlie the functional maturation of multiple brain systems in adolescence. Thus, the role microglia play in the impact of alcohol use in adolescence is likely multifaceted. Long-term sequelae may be due to a failure to recover from EtOH-induced tissue damage, altered neurodevelopmental trajectories, and/or persistent changes to microglial responsivity and function. Here, we review critically the literature surrounding the effects of alcohol on microglia in models of adolescent alcohol misuse. We attempt to disentangle what is known about microglia from other neuroimmune effectors, to which we apply recent discoveries on the role of microglia in development and plasticity. Considered altogether, these studies challenge assumptions that proinflammatory microglia drive addiction. Alcohol priming microglia and thereby perturbing their homeostatic roles in neurodevelopment, especially during critical periods of plasticity such as adolescence, may have more serious implications for the neuropathogenesis of AUDs in adolescents.     

Key High‐efficiency Practices of Emergency Department Providers: A Mixed‐methods Study

Objective

The objective of this study was to determine specific provider practices associated with high provider efficiency in community emergency departments (EDs).

Methods

A mixed‐methods study design was utilized to identify key behaviors associated with efficiency. Stage 1 was a convenience sample of 16 participants (ED medical directors, nurses, advanced practice providers, and physicians) identified provider efficiency behaviors during semistructured interviews. Ninety‐nine behaviors were identified and distilled by a group of three ED clinicians into 18 themes. Stage 2 was an observational study of 35 providers was performed in four (30,000‐ to 55,000‐visit) community EDs during two 4‐hour periods and recorded in minute‐by‐minute observation logs. In Stage 3, each behavior or practice from Stage 1 was assigned a score within each observation period. Behaviors were tested for association with provider efficiency (relative value units/hour) using linear univariate generalized estimating equations with an identity link, clustered on ED site.

Results

Five ED provider practices were found to be positively associated with efficiency: average patient load, using name of team member, conversations with health care team, visits to patient rooms, and running the board. Two behaviors, “inefficiency practices,” demonstrated significant negative correlations: non–work‐related tasks and documentation on patients no longer in the ED.

Conclusions

Average patient load, running the board, conversations with team member, and using names of team members are associated with enhanced provider productivity. Identification of behaviors associated with efficiency can be utilized by medical directors, clinicians, and trainees to improve personal efficiency or counsel team members.

     

Cell Labeling with Magneto-Endosymbionts and the Dissection of the Subcellular Location,Fate, and Host Cell Interactions

Purpose

The purposes of this study are to characterize magneto-endosymbiont (ME) labeling of mammalian cells and to discern the subcellular fate of these living contrast agents. MEs are novel magnetic resonance imaging (MRI) contrast agents that are being used for cell tracking studies. Understanding the fate of MEs in host cells is valuable for designing in vivo cell tracking experiments.

Procedures

The ME’s surface epitopes, contrast-producing paramagnetic magnetosomal iron, and genome were studied using immunocytochemistry (ICC), Fe and MRI contrast measurements, and quantitative polymerase chain reaction (qPCR), respectively. These assays, coupled with other common assays, enabled validation of ME cell labeling and dissection of ME subcellular processing.

Results

The assays mentioned above provide qualitative and quantitative assessments of cell labeling, the subcellular localization and the fate of MEs. ICC results, with an ME-specific antibody, qualitatively shows homogenous labeling with MEs. The ferrozine assay shows that MEs have an average of 7 fg Fe/ME, ～30 % of which contributes to MRI contrast and ME-labeled MDA-MB-231 (MDA-231) cells generally have 2.4 pg Fe/cell, implying ～350 MEs/cell. Adjusting the concentration of Fe in the ME growth media reduces the concentration of non-MRI contrast-producing Fe. Results from the qPCR assay, which quantifies ME genomes in labeled cells, shows that processing of MEs begins within 24 h in MDA-231 cells. ICC results suggest this intracellular digestion of MEs occurs by the lysosomal degradation pathway. MEs coated with listeriolysin O (LLO) are able to escape the primary phagosome, but subsequently co-localize with LC3, an autophagy-associated molecule, and are processed for digestion. In embryos, where autophagy is transiently suppressed, MEs show an increased capacity for survival and even replication. Finally, transmission electron microscopy (TEM) of ME-labeled MDA-231 cells confirms that the magnetosomes (the MRI contrast-producing particles) remain intact and enable in vivo cell tracking.

Conclusions

MEs are used to label mammalian cells for the purpose of cell tracking in vivo, with MRI. Various assays described herein (ICC, ferrozine, and qPCR) allow qualitative and quantitative assessments of labeling efficiency and provide a detailed understanding of subcellular processing of MEs. In some cell types, MEs are digested, but the MRI-producing particles remain. Coating with LLO allows MEs to escape the primary phagosome, enhances retention slightly, and confirms that MEs are ultimately processed by autophagy. Numerous intracellular bacteria and all endosymbiotically derived organelles have evolved molecular mechanisms to avoid intracellular clearance, and identification of the specific processes involved in ME clearance provides a framework on which to develop MEs with enhanced retention in mammalian cells.

     

Evaluation of the likelihood of a selective CHK1 inhibitor (LY2603618) to inhibit CYP2D6 with desipramine as a probe substrate in cancer patients

LY2603618 is a selective inhibitor of deoxyribonucleic acid damage checkpoint kinase 1 (CHK1) and has been in development for the enhancement of chemotherapeutic agents. The study described was to assess the potential interaction between LY2603618 and cytochrome P450 isoform 2D6 (CYP2D6) substrate desipramine in patients with cancer. Before clinical investigation, in silico simulations (using Simcyp®) were conducted. An open‐label, two‐period, fixed‐sequence study was planned in 30 patients with advanced or metastatic cancers, in which a 50 mg oral dose of desipramine was administered alone and in combination with 275 mg of LY2603618 (i.v. infusion). An interim analysis was planned after 15 patients completed both periods. Ratios of geometric least squares means (LSMs) of primary pharmacokinetic (PK) parameters and 90% repeated confidence intervals (RCIs) between desipramine plus LY2603618 and desipramine alone were calculated. Lack of an interaction was declared if the 90% RCI fell between 0.8 and 1.25. The LSM ratios (90% RCI) for areas under the plasma concentration–time curve from time zero to t_last (AUC_[0‐tlast]) and to infinity (AUC_[0‐∞]) and maximum plasma concentration (C_max) were 1.14 (1.04, 1.25), 1.09 (0.99, 1.21) and 1.16 (1.05, 1.29). In silico simulations were predictive of clinical results. Single doses of 275 mg LY2603618 administered with 50 mg desipramine were generally well tolerated. In conclusion, no clinically significant interaction was observed between LY2603618 and desipramine in patients with cancer. In silico predictions of clinical results demonstrated that mechanistic and physiologically based PK approaches may inform clinical study design in cancer patients. Copyright © 2014 John Wiley & Sons, Ltd.     

Therapy of metabolic disorders with intravenous (IV) access ports and long term intravenous L-carnitine therapy

With the expansion of newborn screening to include many organic acidurias and fatty acid oxidation defects, effective therapies of these disorders will be needed. Currently severe disorders such as methylmalonic and propionic aciduria. conventional therapy with diet and oral L-camitine often prove ineffective in preventing failure to thrive and recurrent metabolic decompensations. L-carnitine provides a natural pathway for removal of the toxic metabolites in these disorders and is life saving therapy but, with poor oral absorption (25%), it is difficult to supply adequate carnitine to meet the metabolic needs of these patients. Long term intravenous L-carnitine therapy, administered through a subcutaneous venous access port in 5 patients with organic acidurias [propionic aciduria (2), methylmalonic aciduria (2), 3 methylglutaconic aciduria(1)] resulted in improved growth, lower frequency of metabolic decompensations and increased tolerance of natural protein in the diet. An added benefit was the ability to initiate fluid. electrolytes, and antibiotics during metabolic decompensations at home thus averting hospitalizations.     

The epidemiology of cytomegaloviral infection in women attending a sexually transmitted disease clinic

To test the hypothesis that cytomegalovirus (CMV) is sexually transmitted, we examined the association of CMV infection with indices of sexual activity in 347 women attending a sexually transmitted disease (STD) clinic. Stepwise multivariate logistic regression analysis showed that seropositivity to CMV (complement-fixation antibody titer, greater than or equal to 1:8) was most closely associated with number of sex partners in the subjects' lifetime (P less than .0001), young age at first sexual intercourse (P = .0002), and nonwhite race (P = .0007). Among seropositive women, cervical shedding of CMV was most strongly associated with younger age (P = .0001) and the presence of cervical chlamydial infection (P = .016). Among 84 seronegative women followed up for a mean of 18.4 weeks, 11 (13%) developed primary CMV infections, an annual incidence of 37%. Sexual contact seems to be an important mode of acquisition of CMV in some young women.     

Morphologic resemblance of zygomycete spores to Pneumocystis carinii cysts in tissue.

Structures resembling the cyst form of Pneumocystis carinii were noted in the lungs of alloxan-diabetic rabbits to whom spores of several species of fungi belonging to the class zygomycetes had been administered by intranasal instillation. At 24 to 168 hours after the instillation of spores, these rabbits succumbed to diabetes or zygomycete infection, or both, and the possibility of a mixed infection with both fungus and P. carinii could not be ruled out on morphologic criteria alone. To resolve the dilemma, rabbit lung tissue was examined by the fluorescent antibody procedure with labeled P. carinii antibodies. The labeled antibodies did not stain structures that resembled P. carinii but stained P. carinii cells in control rat lung infected with this organism. Investigators should be aware of the difficulty that may arise in differentiating the cyst form of P. carinii from spores of some fungi. The direct fluorescent antibody procedure for P. carinii appears to be very useful for differentiating P. carinii cysts from fungal spores.     

Early changes in phosphatidylcholine metabolism in human acute promyelocytic leukemia cells stimulated to differentiate by phorbol ester

The HL-60 leukemia cell line derived from a human acute promyelocytic leukemia is stimulated to differentiate into macrophages within 24-28 hr after exposure to the phorbol ester, 12-O-tetradecanoylphorbol-13- acetate (TPA). We studied early alterations (within 90 min of exposure to TPA) in phosphatidylcholine metabolism in HL-60 cells and found that phosphatidylcholine synthesis by methylation is phosphatidylethanolamine was inhibited in a dose-dependent fashion. In contrast, synthesis of phosphatidylcholine from endogenous choline was enhanced and correlated inversely with the degree of inhibition of the methylation pathway. Phorbol ester congeners of TPA caused similar alterations in phosphatidylcholine metabolism in direct relationship to their capacity to induce differentiation in HL-60 cells. Perturbation of phosphatidylcholine metabolism is an early membrane even in TPA- induced HL-60 cell differentiation.