Treatment of active tuberculosis in HIV-coinfected patients: a systematic review and meta-analysis.

Abstract:
BACKGROUND Patients with human immunodeficiency virus (HIV) infection and tuberculosis have an increased risk of death, treatment failure, and relapse.
 METHODS A systematic review and meta-analysis of randomized, controlled trials and cohort studies was conducted to evaluate the impact of duration and dosing schedule of rifamycin and use of antiretroviral therapy in the treatment of active tuberculosis in HIV-positive patients. In included studies, the initial tuberculosis diagnosis, failure, and/or relapse were microbiologically confirmed, and patients received standardized rifampin- or rifabutin-containing regimens. Pooled cumulative incidence of treatment failure, death during treatment, and relapse were calculated using random-effects models. Multivariable meta-regression was performed using negative binomial regression.
 RESULTS After screening 5158 citations, 6 randomized trials and 21 cohort studies were included. Relapse was more common with regimens using 2 months rifamycin (adjusted risk ratio, 3.6; 95% confidence interval, 1.1-11.7) than with regimens using rifamycin for at least 8 months. Compared with daily therapy in the initial phase (n=3352 patients from 35 study arms), thrice-weekly therapy (n=211 patients from 5 study arms) was associated with higher rates of failure (adjusted risk ratio, 4.0; 95% confidence interval, 1.5-10.4) and relapse [adjusted risk ratio, 4.8; 95% confidence interval, 1.8-12.8). There were trends toward higher relapse rates if rifamycins were used for only 6 months, compared with > or =8 months, or if antiretroviral therapy was not used.
 CONCLUSIONS This review raises serious concerns regarding current recommendations for treatment of HIV-tuberculosis coinfection. The data suggest that at least 8 months duration of rifamycin therapy, initial daily dosing, and concurrent antiretroviral therapy might be associated with better outcomes, but adequately powered randomized trials are urgently needed to confirm this.


Crystal structure and association behaviour of the GluR2 amino-terminal domain.

Abstract: Fast excitatory neurotransmission is mediated largely by ionotropic glutamate receptors (iGluRs), tetrameric, ligand-gated ion channel proteins comprised of three subfamilies, AMPA, kainate and NMDA receptors, with each subfamily sharing a common, modular-domain architecture. For all receptor subfamilies, active channels are exclusively formed by assemblages of subunits within the same subfamily, a molecular process principally encoded by the amino-terminal domain (ATD). However, the molecular basis by which the ATD guides subfamily-specific receptor assembly is not known. Here we show that AMPA receptor GluR1- and GluR2-ATDs form tightly associated dimers and, by the analysis of crystal structures of the GluR2-ATD, propose mechanisms by which the ATD guides subfamily-specific receptor assembly.


Deficiency in l-serine deaminase results in abnormal growth and cell division of Escherichia coli K-12.

Abstract: The loss of the ability to deaminate l-serine severely impairs growth and cell division in Escherichia coli K-12. A strain from which the three genes (sdaA, sdaB, tdcG) coding for this organism's three l-serine deaminases had been deleted grows well in glucose minimal medium but, on subculture into minimal medium with glucose and casamino acids, it makes very large, abnormally shaped cells, many of which lyse. When inoculated into Luria-Bertani (LB) broth with or without glucose, it makes very long filaments. Provision of S-adenosylmethionine restores cell division in LB broth with glucose, and repairs much of the difficulty in growth in medium with casamino acids. We suggest that replication of E. coli is regulated by methylation, that an unusually high intracellular l-serine concentration, in the presence of other amino acids, starves the cell for S-adenosylmethionine and that it is the absence of S-adenosylmethionine and/or of C1-tetrahydrofolate derivatives that prevents normal cell division.


A key role for Ctf4 in coupling the MCM2-7 helicase to DNA polymerase alpha within the eukaryotic replisome.

Abstract: The eukaryotic replisome is a crucial determinant of genome stability, but its structure is still poorly understood. We found previously that many regulatory proteins assemble around the MCM2-7 helicase at yeast replication forks to form the replisome progression complex (RPC), which might link MCM2-7 to other replisome components. Here, we show that the RPC associates with DNA polymerase alpha that primes each Okazaki fragment during lagging strand synthesis. Our data indicate that a complex of the GINS and Ctf4 components of the RPC is crucial to couple MCM2-7 to DNA polymerase alpha. Others have found recently that the Mrc1 subunit of RPCs binds DNA polymerase epsilon, which synthesises the leading strand at DNA replication forks. We show that cells lacking both Ctf4 and Mrc1 experience chronic activation of the DNA damage checkpoint during chromosome replication and do not complete the cell cycle. These findings indicate that coupling MCM2-7 to replicative polymerases is an important feature of the regulation of chromosome replication in eukaryotes, and highlight a key role for Ctf4 in this process.


Integrating nutrition security with treatment of people living with HIV: lessons from Kenya.

Abstract:
BACKGROUND The increased caloric requirements of HIV-positive individuals, undesirable side effects of treatment that may be worsened by malnutrition (but alleviated by nutritional support), and associated declines in adherence and possible increased drug resistance are all justifications for developing better interventions to strengthen the nutrition security of individuals receiving antiretroviral treatment.
 OBJECTIVE To highlight key benefits and challenges relating to interventions aimed at strengthening the nutrition security of people living with HIV who are receiving antiretroviral treatment.
 METHODS Qualitative research was undertaken on a short-term nutrition intervention linked to the provision of free antiretroviral treatment for people living with HIV in western Kenya in late 2005 and early 2006.
 RESULTS Patients enrolled in the food program while on treatment regimens self-reported greater adherence to their medication, fewer side effects, and a greater ability to satisfy increased appetite. Most clients self-reported weight gain, recovery of physical strength, and the resumption of labor activities while enrolled in dual (food supplementation and treatment) programs. Such improvements were seen to catalyze increased support from family and community.
 CONCLUSIONS These findings provide further empirical support to calls for a more holistic and comprehensive response to the coexistence of AIDS epidemics with chronic nutrition insecurity. Future work is needed to clarify ways of bridging the gap between short-term nutritional support to individuals and longer-term livelihood security programming for communities affected by AIDS. Such interdisciplinary research will need to be matched by intersectoral action on the part of the agriculture and health sectors in such environments.


Acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes

Abstract: Objectives:The orexigenic hormone ghrelin circulates mainly in two forms, acylated and desacyl ghrelin. We evaluated the impact of obesity and obesity-associated type 2 diabetes (T2D) on ghrelin forms and the potential role of acylated and desacyl ghrelin in the control of adipogenesis in humans. Methods:Plasma concentrations of the different ghrelin forms were measured in 80 subjects. The expression of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) was analyzed in omental adipose tissue using western blot and immunohistochemistry, and the effect of acylated ghrelin and desacyl ghrelin (0.1–1000 pmol l−1) on adipogenesis was determined in vitro in omental adipocytes. Results:Circulating concentrations of acylated ghrelin were increased, whereas desacyl ghrelin levels were decreased, in obesity and obesity-associated T2D. Body mass index, waist circumference, insulin and HOMA (homeostasis model assessment) index were positively correlated with acylated ghrelin levels. Obese individuals showed a lower protein expression of GHS-R in omental adipose tissue. In differentiating omental adipocytes, incubation with both acylated and desacyl ghrelin significantly increased PPARγ (peroxisome proliferator-activated receptor γ) and SREBP1 (sterol-regulatory element binding protein-1) mRNA levels, as well as several fat storage-related proteins, including acetyl-CoA carboxylase, fatty acid synthase, lipoprotein lipase and perilipin. Consequently, both the ghrelin forms stimulated intracytoplasmatic lipid accumulation. Conclusions:Both acylated and desacyl ghrelin stimulate lipid accumulation in human visceral adipocytes. Given the lipogenic effect of acylated ghrelin on visceral adipocytes, the herein-reported elevation of its circulating concentrations in obese individuals may play a role in excessive fat accumulation in obesity.


Significant correlation between LKB1 and LGR5 gene expression and the association with poor recurrence-free survival in rectal cancer after preoperative chemoradiotherapy

Abstract: The aim of the present study was to investigate whether the gene expression levels of LKB1 and LGR5 correlated with clinical outcome in patients with locally advanced rectal cancer treated with preoperative chemoradiotherapy (CRT). Residual cancer cells were obtained from 52 patients with locally advanced rectal cancer treated with preoperative CRT. Total RNA was then isolated from formalin-fixed, paraffin-embedded specimens using microdissection. The expression levels of LKB1 and LGR5 genes were measured using real-time reverse-transcription polymerase chain reaction and by immunohistochemistry. In addition, in vitro studies were performed using colon cancer cell lines to study the serial changes of LKB1, LGR5 and PRKAA1 (AMPK) gene expression levels after irradiation. Our data demonstrate that specimens obtained from patients with poor pathological response and tumor recurrence had significantly higher gene expression levels of LKB1 and LGR5 than those without them (P < 0.05), and there was a significant positive correlation between LKB1 and LGR5 gene expression after CRT (Spearman’s ρ: 0.429, P = 0.0023). The patients with high expression levels of both LKB1 and LGR5 had a significantly lower recurrence-free survival compared with the other group (P = 0.0055, 95 % confidence interval: 1.39–11.08). Lastly, in vitro studies demonstrated a similar pattern of serial gene expression among LKB1, LGR5 and PRKAA1 after irradiation. Our results suggest that LKB1 and LGR5 expression may be implicated in resistance to CRT, therefore contributing to tumor relapse in patients with locally advanced rectal cancer treated with preoperative CRT.


Control of signaling-mediated clearance of apoptotic cells by the tumor suppressor p53

Abstract: The inefficient clearance of dying cells can lead to abnormal immune responses, such as unresolved inflammation and autoimmune conditions. We show that tumor suppressor p53 controls signaling-mediated phagocytosis of apoptotic cells through its target, Death Domain1α (DD1α), which suggests that p53 promotes both the proapoptotic pathway and postapoptotic events. DD1α appears to function as an engulfment ligand or receptor that engages in homophilic intermolecular interaction at intercellular junctions of apoptotic cells and macrophages, unlike other typical scavenger receptors that recognize phosphatidylserine on the surface of dead cells. DD1α-deficient mice showed in vivo defects in clearing dying cells, which led to multiple organ damage indicative of immune dysfunction. p53-induced expression of DD1α thus prevents persistence of cell corpses and ensures efficient generation of precise immune responses.


New opportunities: the use of nanotechnologies to manipulate and track stem cells.

Abstract: Nanotechnologies are emerging platforms that could be useful in measuring, understanding, and manipulating stem cells. Examples include magnetic nanoparticles and quantum dots for stem cell labeling and in vivo tracking; nanoparticles, carbon nanotubes, and polyplexes for the intracellular delivery of genes/oligonucleotides and protein/peptides; and engineered nanometer-scale scaffolds for stem cell differentiation and transplantation. This review examines the use of nanotechnologies for stem cell tracking, differentiation, and transplantation. We further discuss their utility and the potential concerns regarding their cytotoxicity.


The emergence of order in the Drosophila pupal retina.

Abstract: During pupation, long-range order is imposed on the autonomously developing ommatidia which compose the Drosophila eye. To accomplish this, eight additional cell types arise: the primary, secondary, and tertiary pigment cells, and the four cells that form the bristle. These cells form an interweaving lattice between ommatidia. The lattice is refined when excess cells are removed to bring neighboring ommatidia into register. Recent evidence suggests that in larval development, local contacts direct cell fate. The same appears to be true during pupal development: the contacts a cell makes predict the cell type it will become. Cells which contact the anterior or posterior cone cells in an ommatidium invariably become primary pigment cells. Cells which contact primary pigment cells from different ommatidia become secondary and tertiary pigment cells. Bristle development is in several ways distinct from ommatidial development. The four cells of each bristle group appear to be immediate descendents of a single founder cell. During their early differentiation, they do not make stereotyped contacts with surrounding ommatidial cells, but do make particular contacts within the bristle group. And unlike the surrounding ommatidia, differentiation of the bristles radiates from the center of the eye to the edges. As cells are removed during two stages of programmed cell death, the bristles are brought into their final position. When all cells in the lattice have achieved their final position, a second stage of retinal development begins as structures specific to each cell type are produced. This paper follows these various stages of pupal development, and suggests how local cell-cell contacts may produce the cells needed for a functional retina.


Autophagy and mTORC1 regulate the stochastic phase of somatic cell reprogramming

Abstract: We describe robust induction of autophagy during the reprogramming of mouse fibroblasts to induced pluripotent stem cells by four reprogramming factors (Sox2, Oct4, Klf4 and c-Myc), henceforth 4F. This process occurs independently of p53 activation, and is mediated by the synergistic downregulation of mechanistic target of rapamycin complex 1 (mTORC1) and the induction of autophagy-related genes. The 4F coordinately repress mTORC1, but bifurcate in their regulation of autophagy-related genes, with Klf4 and c-Myc inducing them but Sox2 and Oct4 inhibiting them. On one hand, inhibition of mTORC1 facilitates reprogramming by promoting cell reshaping (mitochondrial remodelling and cell size reduction). On the other hand, mTORC1 paradoxically impairs reprogramming by triggering autophagy. Autophagy does not participate in cell reshaping in reprogramming but instead degrades p62, whose accumulation in autophagy-deficient cells facilitates reprogramming. Our results thus reveal a complex signalling network involving mTORC1 inhibition and autophagy induction in the early phase of reprogramming, whose delicate balance ultimately determines reprogramming efficiency.


Association of plasma resistin with glomerular filtration rate and albuminuria in hypertensive adults.

Abstract: Resistin, a recently discovered proinflammatory cytokine, has been variably associated with insulin resistance, inflammation, and renal dysfunction. We investigated the association of plasma resistin with estimated glomerular filtration rate and albuminuria in 1575 hypertensive adults without known coronary heart disease or stroke (857 blacks and 718 non-Hispanic whites). Resistin was measured by a solid phase sandwich immunoassay, estimated glomerular filtration rate was estimated from serum creatinine, and albuminuria was expressed as urine albumin:creatinine ratio. After adjustment for coronary heart disease risk factors (age, sex, body mass index, smoking history, systolic blood pressure, diabetes, and total and high-density lipoprotein cholesterol) and use of renin-angiotensin blockers and statins, higher plasma resistin levels were associated with lower estimated glomerular filtration rate in both ethnic groups (each P<0.0001); the association remained significant after further adjustment for a marker of insulin resistance (homeostasis model assessment for insulin resistance) and a marker of inflammation (plasma C-reactive protein) and was seen in subjects with and without diabetes (each P<0.0001) in both ethnic groups. Higher plasma resistin levels were associated with a higher urine albumin:creatinine ratio in black subjects with diabetes (P<0.0001) and non-Hispanic white subjects with diabetes (P=0.032), independent of coronary heart disease risk factors, hypertension medication use, and statin use; the association remained significant after additional adjustment for homeostasis model assessment for insulin resistance and C-reactive protein. In adults with hypertension, higher circulating resistin levels were associated with a lower estimated glomerular filtration rate and with increased urine albumin:creatinine ratio in the presence of concomitant diabetes. This association was independent of coronary heart disease risk factors and markers of insulin resistance and inflammation.


Large-scale candidate gene scan reveals the role of chemoreceptor genes in host plant specialization and speciation in the pea aphid.

Abstract: Understanding the drivers of speciation is critical to interpreting patterns of biodiversity. The identification of the genetic changes underlying adaptation and reproductive isolation is necessary to link barriers to gene flow to the causal origins of divergence. Here, we present a novel approach to the genetics of speciation, which should complement the commonly used approaches of quantitative trait locus mapping and genome-wide scans for selection. We present a large-scale candidate gene approach by means of sequence capture, applied to identifying the genetic changes underlying reproductive isolation in the pea aphid, a model system for the study of ecological speciation. Targeted resequencing enabled us to scale up the candidate gene approach, specifically testing for the role of chemosensory gene families in host plant specialization. Screening for the signature of divergence under selection at 172 candidate and noncandidate loci, we revealed a handful of loci that show high levels of differentiation among host races, which almost all correspond to odorant and gustatory receptor genes. This study offers the first indication that some chemoreceptor genes, often tightly linked together in the genome, could play a key role in local adaptation and reproductive isolation in the pea aphid and potentially other phytophagous insects. Our approach opens a new route toward the functional genomics of ecological speciation.


The anaphase promoting complex/cyclosome: a machine designed to destroy

Abstract: The anaphase promoting complex/cyclosome (APC/C) is a ubiquitin ligase that has essential functions in and outside the eukaryotic cell cycle. It is the most complex molecular machine that is known to catalyse ubiquitylation reactions, and it contains more than a dozen subunits that assemble into a large 1.5-MDa complex. Recent discoveries have revealed an unexpected multitude of mechanisms that control APC/C activity, and have provided a first insight into how this unusual ubiquitin ligase recognizes its substrates.


Declining tuberculosis notification trend associated with strengthened TB and expanded HIV care in Swaziland.

Abstract: This retrospective observational review documents the efforts of the Swaziland National Tuberculosis (TB) Control Programme between 2004 and 2014. The objective is to describe the disparity between actual declines in case notification and increases in estimated incidence. The review of policies and practices shows the most influential factors associated with the decrease in TB case notification to be an increase in access to antiretroviral therapy for co-infected TB patients, the general success of TB and human immunodeficiency virus service integration in the country and improvements in implementation of all components of directly observed treatment, active case finding, and rapid diagnosis using new technologies.


Time from finding abnormality on mass-screening to final diagnosis of lung cancer.

Abstract: Mass-screening for lung cancer is rather a unique system in Japan. This study illustrates time from finding abnormality on mass-screening to final diagnosis of lung cancer. Among the 517 patients with lung cancer who were admitted to our hospital over a 10-year period up to December 2001, 83 (16.1%) were detected by mass-screening. We reviewed medical records of the 83 patients and determined the intervals from the mass-screening to the pathological diagnosis with clinical staging. Time from the mass-screening to the date of hospital visit was <2 months in 62 (74.7%) cases. Five (6.0%) patients visited hospital more than 6 months after the mass-screening. With respect to the interval, there was no statistical difference in gender (p=0.0680) and age (p=0.1532). Among 60 patients who were referred from outside, on average, patients visited our hospital 0.5 month after they first sought medical attention at nearby clinic, and at our hospital 0.5 month was required to make a pathological diagnosis of lung cancer with TNM staging. There was a statistical difference in survival between the patients who were diagnosed <4 months and the patients who were diagnosed >4 months from the screening (p=0.0487). The interval in most cases was acceptable. However, further improvements are still needed to minimize the delay and to maximize the benefits of early cancer detection.


Krüppel-like factor 2 (KLF2) regulates B-cell reactivity, subset differentiation, and trafficking molecule expression.

Abstract: The transcription factor Krüppel-like factor 2 (KLF2) is critical for normal trafficking of T lymphocytes, but its role in B cells is unclear. We report that B cell-specific KLF2 deficiency leads to decreased expression of the trafficking molecules CD62L and β7-integrin, yet expression of sphingosine-1 phosphate receptor 1 (which is a critical target of KLF2 in T cells) was, unexpectedly, minimally altered. Unexpectedly, Klf2 deletion led to a drastic reduction in the B1 B-cell pool and a substantial increase in transitional and marginal zone B-cell numbers. In addition, we observed that KLF2-deficient B cells showed increased apoptosis and impaired proliferation after B-cell receptor cross-linking. Gene expression analysis indicated that KLF2-deficient follicular B cells display numerous characteristics shared by normal marginal zone B cells, including reduced expression of several signaling molecules that may contribute to defective activation of these cells. Hence, our data indicate that KLF2 plays a critical role in dictating normal subset differentiation and functional reactivity of mature B cells.


Thrombopoietin/MPL signaling regulates hematopoietic stem cell quiescence and interaction with the osteoblastic niche.

Abstract: Maintenance of hematopoietic stem cells (HSCs) depends on interaction with their niche. Here we show that the long-term (LT)-HSCs expressing the thrombopoietin (THPO) receptor, MPL, are a quiescent population in adult bone marrow (BM) and are closely associated with THPO-producing osteoblastic cells. THPO/MPL signaling upregulated beta1-integrin and cyclin-dependent kinase inhibitors in HSCs. Furthermore, inhibition and stimulation of THPO/MPL pathway by treatments with anti-MPL neutralizing antibody, AMM2, and with THPO showed reciprocal regulation of quiescence of LT-HSC. AMM2 treatment reduced the number of quiescent LT-HSCs and allowed exogenous HSC engraftment without irradiation. By contrast, exogenous THPO transiently increased quiescent HSC population and subsequently induced HSC proliferation in vivo. Altogether, these observations suggest that THPO/MPL signaling plays a critical role of LT-HSC regulation in the osteoblastic niche.


Interlaboratory reproducibility and proficiency testing within the human papillomavirus cervical cancer screening program in Catalonia, Spain.

Abstract: In Catalonia, a screening protocol for cervical cancer, including human papillomavirus (HPV) DNA testing using the Digene Hybrid Capture 2 (HC2) assay, was implemented in 2006. In order to monitor interlaboratory reproducibility, a proficiency testing (PT) survey of the HPV samples was launched in 2008. The aim of this study was to explore the repeatability of the HC2 assay's performance. Participating laboratories provided 20 samples annually, 5 randomly chosen samples from each of the following relative light unit (RLU) intervals: <0.5, 0.5 to 0.99, 1 to 9.99, and ≥10. Kappa statistics were used to determine the agreement levels between the original and the PT readings. The nature and origin of the discrepant results were calculated by bootstrapping. A total of 946 specimens were retested. The kappa values were 0.91 for positive/negative categorical classification and 0.79 for the four RLU intervals studied. Sample retesting yielded systematically lower RLU values than the original test (P<0.005), independently of the time elapsed between the two determinations (median, 53 days), possibly due to freeze-thaw cycles. The probability for a sample to show clinically discrepant results upon retesting was a function of the RLU value; samples with RLU values in the 0.5 to 5 interval showed 10.80% probability to yield discrepant results (95% confidence interval [CI], 7.86 to 14.33) compared to 0.85% probability for samples outside this interval (95% CI, 0.17 to 1.69). Globally, the HC2 assay shows high interlaboratory concordance. We have identified differential confidence thresholds and suggested the guidelines for interlaboratory PT in the future, as analytical quality assessment of HPV DNA detection remains a central component of the screening program for cervical cancer prevention.


Adhesion disengagement uncouples intrinsic and extrinsic forces to drive cytokinesis in epithelial tissues.

Abstract: Cytokinesis entails cell invagination by a contractile actomyosin ring. In epithelia, E-cadherin-mediated adhesion connects the cortices of contacting cells; thus, it is unclear how invagination occurs, how the new junction forms, and how tissue integrity is preserved. Investigations in Drosophila embryos first show that apicobasal cleavage is polarized: invagination is faster from the basal than from the apical side. Ring contraction but not its polarized constriction is controlled by septin filaments and Anillin. Polarized cleavage is due instead to mechanical anchorage of the ring to E-cadherin complexes. Formation of the new junction requires local adhesion disengagement in the cleavage furrow, followed by new E-cadherin complex formation at the new interface. E-cadherin disengagement depends on the tension exerted by the cytokinetic ring and by neighboring cells. We uncover intrinsic and extrinsic forces necessary for cytokinesis and present a framework for understanding how tissue cohesion is preserved during epithelial division.


The epithelial inward rectifier channel Kir7.1 displays unusual K+ permeation properties.

Abstract: Rat and human cDNAs were isolated that both encoded a 360 amino acid polypeptide with a tertiary structure typical of inwardly rectifying K+ channel (Kir) subunits. The new proteins, termed Kir7.1, were <37% identical to other Kir subunits and showed various unique residues at conserved sites, particularly near the pore region. High levels of Kir7.1 transcripts were detected in rat brain, lung, kidney, and testis. In situ hybridization of rat brain sections demonstrated that Kir7.1 mRNA was absent from neurons and glia but strongly expressed in the secretory epithelial cells of the choroid plexus (as confirmed by in situ patch-clamp measurements). In cRNA-injected Xenopus oocytes Kir7.1 generated macroscopic Kir currents that showed a very shallow dependence on external K+ ([K+]e), which is in marked contrast to all other Kir channels. At a holding potential of -100 mV, the inward current through Kir7.1 averaged -3.8 +/- 1.04 microA with 2 mM [K+]e and -4.82 +/- 1.87 microA with 96 mM [K+]e. Kir7.1 has a methionine at position 125 in the pore region where other Kir channels have an arginine. When this residue was replaced by the conserved arginine in mutant Kir7.1 channels, the pronounced dependence of K+ permeability on [K+]e, characteristic for other Kir channels, was restored and the Ba2+ sensitivity was increased by a factor of approximately 25 (Ki = 27 microM). These findings support the important role of this site in the regulation of K+ permeability in Kir channels by extracellular cations.


Nonoverlapping functions of DNA polymerases mu, lambda, and terminal deoxynucleotidyltransferase during immunoglobulin V(D)J recombination in vivo.

Abstract: DNA polymerases mu (pol mu), lambda (pol lambda), and terminal deoxynucleotidyltransferase (TdT) are enzymes of the pol X family that share homology in sequence and functional domain organization. We showed previously that pol mu participates in light chain but surprisingly not heavy chain gene rearrangement. We show here that immunoglobulin heavy chain junctions from pol lambda-deficient animals have shorter length with normal N-additions, thus indicating that pol lambda is recruited during heavy chain rearrangement at a step that precedes the action of TdT. In contrast to previous in vitro studies, analysis of animals with combined inactivation of these enzymes revealed no overlapping or compensatory activities for V(D)J recombination between pol mu, pol lambda, and TdT. This complex usage of polymerases with distinct catalytic specificities may correspond to the specific function that the third hypervariable region assumes for each immunoglobulin chain, with pol lambda maintaining a large heavy chain junctional heterogeneity and pol mu ensuring a restricted light chain junctional variability.


Moderate regular exercise increases basal production of nitric oxide in elderly women.

Abstract: Vascular endothelial cells produce nitric oxide (NO), which is a potent vasodilator substance and is thought to have antiatherosclerotic properties. Therefore, it has also been proposed that NO may be useful to regulate vascular tonus and prevent progression of atherosclerosis. On the other hand, NO activity reduces with aging. We previously reported that the plasma nitrite/nitrate (NOx: the stable end product of NO) concentration was significantly increased by intense aerobic exercise training in healthy young humans. We hypothesized that lifestyle modification (e.g., even mild regular exercise training) can increase NO production in previously sedentary older humans. We measured the plasma NOx concentration before and after a mild aerobic exercise training regimen (cycling on a leg ergometer at 80% ventilatory threshold for 30 min, 5 days/week) for 3 months in elderly women. In addition, we assessed the plasma concentration of cyclic guanosine monophosphate (cGMP), a second messenger of NO, in the same samples. The individual ventilatory threshold increased significantly after the 3-month exercise training. The blood pressure at rest significantly decreased after exercise training. These results suggest that the 3-month exercise training in the older women produced favorable physiological effects. The plasma concentration of NOx significantly increased by the exercise training, and the plasma concentration of cGMP also increased by the exercise training. The present study suggests that even a mild regular aerobic-endurance exercise increases NO production in previously sedentary older humans, which may have beneficial effects (i.e., antihypertensive and antiatherosclerotic effects by endogenous NO) on the cardiovascular system.


Competition between target sites of regulators shapes post-transcriptional gene regulation

Abstract: Post-transcriptional gene regulation (PTGR) of mRNA turnover, localization and translation is mediated by microRNAs (miRNAs) and RNA-binding proteins (RBPs). These regulators exert their effects by binding to specific sequences within their target mRNAs. Increasing evidence suggests that competition for binding is a fundamental principle of PTGR. Not only can miRNAs be sequestered and neutralized by the targets with which they interact through a process termed 'sponging', but competition between binding sites on different RNAs may also lead to regulatory crosstalk between transcripts. Here, we quantitatively model competition effects under physiological conditions and review the role of endogenous sponges for PTGR in light of the key features that emerge.


Chimeric live, attenuated vaccine against Japanese encephalitis (ChimeriVax-JE): phase 2 clinical trials for safety and immunogenicity, effect of vaccine dose and schedule, and memory response to challenge with inactivated Japanese encephalitis antigen.

Abstract: ChimeriVax-JE is a live, attenuated vaccine against Japanese encephalitis, using yellow fever (YF) 17D vaccine as a vector. In a double-blind phase 2 trial, 99 adults received vaccine, placebo, or YF 17D vaccine (YF-VAX). ChimeriVax-JE was well tolerated, with no differences in adverse events between treatment groups. Viremias resulting from administration of ChimeriVax-JE and YF-VAX were of short duration and low titer; 82 (94%) of 87 subjects administered graded doses (1.8-5.8 log(10)) of ChimeriVax-JE developed neutralizing antibodies. A second dose, administered 30 days later, had no booster effect. Previous inoculation with YF did not interfere with ChimeriVax-JE, but there was a suggestion (not statistically significant) that ChimeriVax-JE interfered with YF-VAX administered 30 days later. A separate study explored immunological memory both in subjects who had received ChimeriVax-JE 9 months before and in ChimeriVax-JE-naive subjects challenged with inactivated mouse-brain vaccine (JE-VAX). Anamnestic responses were observed in preimmune individuals. ChimeriVax-JE appears to be a safe vaccine that provides protective levels of neutralizing antibody after a single dose.


Expression of optineurin, a glaucoma-linked gene, is influenced by elevated intraocular pressure.

Abstract: Optineurin (optic neuropathy inducing protein; OPTN) was recently linked to 16.7% of families with primary open-angle glaucoma. The function of OPTN in the eye is not known, but is present in the trabecular meshwork, which is responsible for maintenance of intraocular pressure (IOP). To gain insight into the role of OPTN in the development of glaucoma we studied its expression in response to factors known to be associated with the disease: elevated IOP, tumor necrosis factor-alpha (TNFalpha), and dexamethasone (DEX). We performed the treatments in human organ cultures under conditions mimicking physiological pressure. We find OPTN significantly upregulated after 2, 4, and 7 days of sustained elevated IOP. OPTN expression is also induced 2.3-fold by TNFalpha and 2.6-fold by prolonged DEX treatment. These results demonstrate that OPTN is part of the transcriptome responding to glaucomatous insults and support the protective role of this protein in the trabecular meshwork.


Activation of AMPKα2 in adipocytes is essential for nicotine-induced insulin resistance in vivo

Abstract: Cigarette smoking promotes body weight reduction in humans while paradoxically also promoting insulin resistance (IR) and hyperinsulinemia. However, the mechanisms behind these effects are unclear. Here we show that nicotine, a major constituent of cigarette smoke, selectively activates AMP-activated protein kinase α2 (AMPKα2) in adipocytes, which in turn phosphorylates MAP kinase phosphatase-1 (MKP1) at serine 334, initiating its proteasome-dependent degradation. The nicotine-dependent reduction of MKP1 induces the aberrant activation of both p38 mitogen-activated protein kinase and c-Jun N-terminal kinase, leading to increased phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307. Phosphorylation of IRS1 leads to its degradation, protein kinase B inhibition, and the loss of insulin-mediated inhibition of lipolysis. Consequently, nicotine increases lipolysis, which results in body weight reduction, but this increase also elevates the levels of circulating free fatty acids and thus causes IR in insulin-sensitive tissues. These results establish AMPKα2 as an essential mediator of nicotine-induced whole-body IR in spite of reductions in adiposity.


Availability of evidence of benefits on overall survival and quality of life of cancer drugs approved by European Medicines Agency: retrospective cohort study of drug approvals 2009-13

Abstract: Objective To determine the availability of data on overall survival and quality of life benefits of cancer drugs approved in Europe. Design Retrospective cohort study. Setting Publicly accessible regulatory and scientific reports on cancer approvals by the European Medicines Agency (EMA) from 2009 to 2013.Main outcome measures Pivotal and postmarketing trials of cancer drugs according to their design features (randomisation, crossover, blinding), comparators, and endpoints. Availability and magnitude of benefit on overall survival or quality of life determined at time of approval and after market entry. Validated European Society for Medical Oncology Magnitude of Clinical Benefit Scale (ESMO-MCBS) used to assess the clinical value of the reported gains in published studies of cancer drugs. Results From 2009 to 2013, the EMA approved the use of 48 cancer drugs for 68 indications. Of these, eight indications (12%) were approved on the basis of a single arm study. At the time of market approval, there was significant prolongation of survival in 24 of the 68 (35%). The magnitude of the benefit on overall survival ranged from 1.0 to 5.8 months (median 2.7 months). At the time of market approval, there was an improvement in quality of life in seven of 68 indications (10%). Out of 44 indications for which there was no evidence of a survival gain at the time of market authorisation, in the subsequent postmarketing period there was evidence for extension of life in three (7%) and reported benefit on quality of life in five (11%). Of the 68 cancer indications with EMA approval, and with a median of 5.4 years' follow-up (minimum 3.3 years, maximum 8.1 years), only 35 (51%) had shown a significant improvement in survival or quality of life, while 33 (49%) remained uncertain. Of 23 indications associated with a survival benefit that could be scored with the ESMO-MCBS tool, the benefit was judged to be clinically meaningful in less than half (11/23, 48%).Conclusions This systematic evaluation of oncology approvals by the EMA in 2009-13 shows that most drugs entered the market without evidence of benefit on survival or quality of life. At a minimum of 3.3 years after market entry, there was still no conclusive evidence that these drugs either extended or improved life for most cancer indications. When there were survival gains over existing treatment options or placebo, they were often marginal.


A cluster randomized, controlled trial of breast and cervix cancer screening in Mumbai, India: methodology and interim results after three rounds of screening.

Abstract: Cervix and Breast cancers are the most common cancers among women worldwide and extract a large toll in developing countries. In May 1998, supported by a grant from the NCI (US), the Tata Memorial Hospital, Mumbai, India, started a cluster-randomized, controlled, screening-trial for cervix and breast cancer using trained primary health workers to provide health-education, visual-inspection of cervix (with 4% acetic acid-VIA) and clinical breast examination (CBE) in the screening arm, and only health education in the control arm. Four rounds of screening at 2-year intervals will be followed by 8 years of monitoring for incidence and mortality from cervix and breast cancers. The methodology and interim results after three rounds of screening are presented here. Good randomization was achieved between the screening (n = 75360) and control arms (n = 76178). In the screening arm we see: High screening participation rates; Low attrition; Good compliance to diagnostic confirmation; Significant downstaging; Excellent treatment completion rate; Improving case fatality ratios. The ever-screened and never-screened participants in the screening arm show significant differences with reference to the variables religion, language, age, education, occupation, income and health-seeking behavior for gynecological and breast-related complaints. During the same period, in the control arm we see excellent participation rate for health education; Low attrition and a good number of symptomatic referrals for both cervix and breast.


Evidence for energy-dependent accumulation of paraquat into rat lung

Abstract: THE herbicide paraquat (N,N′-dimethyl 4,4′-bipyridilium) can produce widespread oedema and fibrosis in the human lung after accidental ingestion1–3. In those cases where death occurs after several weeks, there are no apparent pulmonary changes during the first few days following ingestion. Animal experiments in a variety of species have shown the lung to be the major target organ4–6. After administration of paraquat to animals, the lung has a high initial concentration and retains paraquat7–9. This retention appears to be related to the development of lung damage7 (L. L. Smith and M. S. Rose, unpublished work). The mechanism of retention of paraquat by the lung is at present not understood.


Role of gut commensal microflora in the development of experimental autoimmune encephalomyelitis.

Abstract: Mucosal tolerance has been considered a potentially important pathway for the treatment of autoimmune disease, including human multiple sclerosis and experimental conditions such as experimental autoimmune encephalomyelitis (EAE). There is limited information on the capacity of commensal gut bacteria to induce and maintain peripheral immune tolerance. Inbred SJL and C57BL/6 mice were treated orally with a broad spectrum of antibiotics to reduce gut microflora. Reduction of gut commensal bacteria impaired the development of EAE. Intraperitoneal antibiotic-treated mice showed no significant decline in the gut microflora and developed EAE similar to untreated mice, suggesting that reduction in disease activity was related to alterations in the gut bacterial population. Protection was associated with a reduction of proinflammatory cytokines and increases in IL-10 and IL-13. Adoptive transfer of low numbers of IL-10-producing CD25(+)CD4(+) T cells (>75% FoxP3(+)) purified from cervical lymph nodes of commensal bacteria reduced mice and in vivo neutralization of CD25(+) cells suggested the role of regulatory T cells maintaining peripheral immune homeostasis. Our data demonstrate that antibiotic modification of gut commensal bacteria can modulate peripheral immune tolerance that can protect against EAE. This approach may offer a new therapeutic paradigm in the treatment of multiple sclerosis and perhaps other autoimmune conditions.


Antihypertensive classes, cognitive decline and incidence of dementia: a network meta-analysis.

Abstract:
OBJECTIVES Prevention of cognitive decline and dementia with blood pressure lowering treatments has shown inconsistent results. We compared the effects of different classes of antihypertensive drugs on the incidence of dementia, and on cognitive function.
 METHODS We conducted a systematic review and included 19 randomized trials (18 515 individuals) and 11 studies (831 674 individuals) analysing the effects of antihypertensive treatment on cognition and on the incidence of dementia, respectively, in hypertensive patients without prior cerebrovascular disorders. Network meta-analysis was used for the comparison of antihypertensive classes.
 RESULTS Antihypertensive treatment, regardless of the drug class, had benefits on overall cognition [effect size 0.05, 95% confidence interval (CI) 0.02-0.07] and all cognitive functions except language. Antihypertensive treatment reduced the risk of all-cause dementia by 9%, with reference to the control group (hazard ratio 0.91, 95% CI 0.89-0.94), when randomized trials and observationnal studies were combined (n = 15). Result was not significant with randomized trials alone (n = 4). Angiotensin II receptor blockers (ARBs) had larger benefits than placebo on overall cognition (adjusted effect size 0.60 ± 0.18, P = 0.02). ARBs were more effective than β-blockers (0.67 ± 0.18, P = 0.01), diuretics (0.54 ± 0.19, P = 0.04) and angiotensin-converting enzyme inhibitors (0.47 ± 0.17, P = 0.04) in rank. The mean change in blood pressure did not differ significantly between the different antihypertensive drug classes.
 CONCLUSION Our results support the notion that antihypertensive treatment has beneficial effects on cognitive decline and prevention of dementia, and indicate that these effects may differ between drug classes with ARBs possibly being the most effective.


Cellular adaptation to mechanical stress: role of integrins, Rho, cytoskeletal tension and mechanosensitive ion channels.

Abstract: To understand how cells sense and adapt to mechanical stress, we applied tensional forces to magnetic microbeads bound to cell-surface integrin receptors and measured changes in bead displacement with sub-micrometer resolution using optical microscopy. Cells exhibited four types of mechanical responses: (1) an immediate viscoelastic response; (2) early adaptive behavior characterized by pulse-to-pulse attenuation in response to oscillatory forces; (3) later adaptive cell stiffening with sustained (>15 second) static stresses; and (4) a large-scale repositioning response with prolonged (>1 minute) stress. Importantly, these adaptation responses differed biochemically. The immediate and early responses were affected by chemically dissipating cytoskeletal prestress (isometric tension), whereas the later adaptive response was not. The repositioning response was prevented by inhibiting tension through interference with Rho signaling, similar to the case of the immediate and early responses, but it was also prevented by blocking mechanosensitive ion channels or by inhibiting Src tyrosine kinases. All adaptive responses were suppressed by cooling cells to 4 degrees C to slow biochemical remodeling. Thus, cells use multiple mechanisms to sense and respond to static and dynamic changes in the level of mechanical stress applied to integrins.


Cutting edge: Candida albicans hyphae formation triggers activation of the Nlrp3 inflammasome.

Abstract: The proinflammatory cytokine IL-1beta plays an important role in antifungal immunity; however, the mechanisms by which fungal pathogens trigger IL-1beta secretion are unclear. In this study we show that infection with Candida albicans is sensed by the Nlrp3 inflammasome, resulting in the subsequent release of IL-1beta. The ability of C. albicans to switch from a unicellular yeast form into a filamentous form is essential for activation of the Nlrp3 inflammasome, as C. albicans mutants incapable of forming hyphae were defective in their ability to induce macrophage IL- 1beta secretion. Nlrp3-deficient mice also demonstrated increased susceptibility to infection with C. albicans, which is consistent with a key role for Nlrp3 in innate immune responses to the pathogen C. albicans.


Ghrelin's second life: from appetite stimulator to glucose regulator.

Abstract: Ghrelin, a 28 amino acid peptide hormone produced by the stomach, was the first orexigenic hormone to be discovered from the periphery. The octanoyl modification at Ser³, mediated by ghrelin O-acyltransferase (GOAT), is essential for ghrelin's biological activity. Ghrelin stimulates food intake through binding to its receptor (GRLN-R) on neurons in the arcuate nucleus of the hypothalamus. Ghrelin is widely expressed throughout the body; accordingly, it is implicated in several other physiological functions, which include growth hormone release, gastric emptying, and body weight regulation. Ghrelin and GRLN-R expression are also found in the pancreas, suggesting a local physiological role. Accordingly, several recent studies now point towards an important role for ghrelin and its receptor in the regulation of blood glucose homeostasis, which is the main focus of this review. Several mechanisms of this regulation by ghrelin have been proposed, and one possibility is through the regulation of insulin secretion. Despite some controversy, most studies suggest that ghrelin exerts an inhibitory effect on insulin secretion, resulting in increased circulating glucose levels. Ghrelin may thus be a diabetogenic factor. Obesity-related type 2 diabetes has become an increasingly important health problem, almost reaching epidemic proportions in the world; therefore, antagonists of the ghrelin-GOAT signaling pathway, which will tackle both energy- and glucose homeostasis, may be considered as promising new therapies for this disease.


Integrated genomic profiling of endometrial carcinoma associates aggressive tumors with indicators of PI3 kinase activation.

Abstract: Although 75% of endometrial cancers are treated at an early stage, 15% to 20% of these recur. We performed an integrated analysis of genome-wide expression and copy-number data for primary endometrial carcinomas with extensive clinical and histopathological data to detect features predictive of recurrent disease. Unsupervised analysis of the expression data distinguished 2 major clusters with strikingly different phenotypes, including significant differences in disease-free survival. To identify possible mechanisms for these differences, we performed a global genomic survey of amplifications, deletions, and loss of heterozygosity, which identified 11 significantly amplified and 13 significantly deleted regions. Amplifications of 3q26.32 harboring the oncogene PIK3CA were associated with poor prognosis and segregated with the aggressive transcriptional cluster. Moreover, samples with PIK3CA amplification carried signatures associated with in vitro activation of PI3 kinase (PI3K), a signature that was shared by aggressive tumors without PIK3CA amplification. Tumors with loss of PTEN expression or PIK3CA overexpression that did not have PIK3CA amplification also shared the PI3K activation signature, high protein expression of the PI3K pathway member STMN1, and an aggressive phenotype in test and validation datasets. However, mutations of PTEN or PIK3CA were not associated with the same expression profile or aggressive phenotype. STMN1 expression had independent prognostic value. The results affirm the utility of systematic characterization of the cancer genome in clinically annotated specimens and suggest the particular importance of the PI3K pathway in patients who have aggressive endometrial cancer.


Confusion and memory loss from capsular genu infarction: a thalamocortical disconnection syndrome?

Abstract: We examined six patients with an abrupt change in behavior after infarction involving the inferior genu of the internal capsule. The acute syndrome featured fluctuating alertness, inattention, memory loss, apathy, abulia, and psychomotor retardation, suggesting frontal lobe dysfunction. Contralateral hemiparesis and dysarthria were generally mild, except when the infarct extended into the posterior limb. Neuropsychological testing in five patients with left-sided infarcts revealed severe verbal memory loss. Additional cognitive deficits consistent with dementia occurred in four patients. A right-sided infarct caused transient impairment in visuospatial memory. Functional brain imaging in three patients showed a focal reduction in hemispheric perfusion most prominent in the ipsilateral inferior and medial frontal cortex. We infer that the capsular genu infarct interrupted the inferior and anterior thalamic peduncles, resulting in functional deactivation of the ipsilateral frontal cortex. These observations suggest that one mechanism for cognitive deterioration from a lacunar infarct is thalamocortical disconnection of white-matter tracts, in some instances leading to "strategic-infarct dementia. "


Neoadjuvant androgen ablation before radical prostatectomy in cT2bNxMo prostate cancer: 5-year results.

Abstract:
PURPOSE In the initial report of the Lupron Depot Neoadjuvant Prostate Cancer Study Group patients who received 3 months of androgen deprivation had a significant decrease in the positive margin rate. We monitored these patients for 5 years and to our knowledge present the longest followup of any neoadjuvant trial.    MATERIALS AND METHODS A multi-institutional prospective randomized trial was performed between February 1992 and April 1994 involving patients with stage cT2b prostate cancer, including 138 who received 3 months of leuprolide plus flutamide before radical prostatectomy and 144 who underwent radical prostatectomy only. Patients were followed every 6 months with serum prostate specific antigen (PSA) testing for 5 years. Biochemical recurrence was defined as PSA greater than 0.4 ng./ml.
 RESULTS At 5 years there was no difference in the biochemical recurrence rate. PSA was less than 0.4 ng./ml. in 64.8% of the patients in the neoadjuvant androgen ablation plus prostatectomy and 67.6% in the prostatectomy only group (p = 0.663).
 CONCLUSIONS Although 3 months of androgen deprivation before radical prostatectomy resulted in an apparently significant decrease in positive surgical margins, a 5-year followup does not indicate any difference in the recurrence rate. Until studies document improvement in biochemical or clinical recurrence with longer periods of treatment, induction androgen deprivation before radical prostatectomy is not indicated.


Growth restricted in vitro culture conditions alter the imprinted gene expression patterns of mouse embryonic stem cells.

Abstract: Embryonic stem (ES) cell-derived clones and chimeras are often associated with growth abnormalities during fetal development, leading to the production of over/under-weight offspring that show elevated neonatal mortality and morbidity. Due to the role played by imprinted genes in controlling fetal growth, much of the blame is pointed at improper epigenetic reprogramming of cells used in the procedures. We have analyzed the expression pattern of two growth regulatory imprinted genes, namely insulin like growth factor II (Igf2) and H19, in mouse ES cells cultured under growth restricted conditions and after in vitro aging. Culture of cells with serum-depleted media (starvation) and at high cell density (confluence) increased the expression of both imprinted genes and led to aberrant methylation profiles of differentially methylated regions in key regulatory sites of Igf2 and H19. These findings confirm that growth constrained cultures of ES cells are associated with alterations to methylation of the regulatory domains and the expression patterns of imprinted genes, suggesting a possible role of epigenetic factors in the loss of developmental potential.


When is antipsychotic polypharmacy supported by research evidence? Implications for QI.

Abstract:
BACKGROUND Concurrent use of multiple standing antipsychotics (antipsychotic polypharmacy) is increasingly common among both inpatients and outpatients. Although this has often been cited as a potential quality-of-care problem, reviews of research evidence on antipsychotic polypharmacy have not distinguished between appropriate versus inappropriate use.
 METHODS A MEDLINE search from 1966 to December 2007 was completed to identify studies comparing changes in symptoms, functioning, and/or side effects between patients treated with multiple antipsychotics and patients treated with a single antipsychotic. The studies were reviewed in two groups on the basis of whether prescribing was concordant with guideline recommendations for multiple-antipsychotic use.
 RESULTS A review of the literature, including three randomized controlled trials, found no support for the use of antipsychotic polypharmacy in patients without an established history of treatment resistance to multiple trials of monotherapy. In patients with a history of treatment resistance to multiple monotherapy trials, limited data support antipsychotic polypharmacy, but positive outcomes were primarily found in studies of clozapine augmented with a second-generation antipsychotic.    DISCUSSION Research evidence is consistent with the goal of avoiding antipsychotic polypharmacy in patients who lack guideline-recommended indications for its use. The Joint Commission is implementing a core measure set for Hospital-Based Inpatient Psychiatric Services. Two of the measures address antipsychotic polypharmacy. The first measure assesses the overall rate. The second measure determines whether clinically appropriate justification has been documented supporting the use of more than one antipsychotic medication.


The Liverpool uveal melanoma liver metastases pathway: outcome following liver resection.

Abstract:
AIM To determine the outcome of patients that underwent liver resection for metastases from uveal melanoma.
 METHODS Over a 9-year period, patients referred with uveal melanoma metastases were included. Following treatment of primary uveal melanoma, high-risk patients were offered to be enrolled into a 6-monthly non-contrast liver magnetic resonance imaging (MRI) surveillance. Following detection of liver metastases, patients were staged with a contrast-enhanced (Primovist(®)) liver MRI, computer tomography (CT) of the thorax and staging laparoscopy.
 RESULTS 155 patients were referred with uveal melanoma liver metastases, of which 17 (11.0%) patients had liver resection and one patient was treated with percutaneous radio-frequency ablation. The majority of patients undergoing liver resection were treated with multiple metastectomies (n = 8) and three patients had major liver resections. The overall median survival for patients treated with surgery/ablation was 27 (14-90) months, and this was significantly better compared to patients treated palliatively [median = 8(1-30) months, P < 0.001]. Following surgery, 11 patients had recurrent disease [median = 13(6-36) months]. Patients who had undergone a major liver resection had a significantly poorer disease-free survival (P = 0.037).
 CONCLUSIONS Patients who can undergo surgical resection for metastatic uveal melanoma have a more favorable survival compared to those who do not.


Smooth muscle lineage diversity in the chick embryo. Two types of aortic smooth muscle cell differ in growth and receptor-mediated transcriptional responses to transforming growth factor-beta.

Abstract: Lineage analysis studies in the avian embryo have identified two types of smooth muscle cells (SMCs) in the tunica media of large elastic arteries; one that originates within the cardiac neural crest and is ectoderm in origin (Ect) and another that arises from local mesenchyme of mesodermal origin (Mes). To determine if differences in primary embryonic lineage can give rise to SMCs with stable differences in growth and differentiation properties, we isolated Ect and Mes SMCs from the Day 14 chick embryo aorta. We report that despite different primary embryonic origins, Ect and Mes SMCs express nearly identical levels of seven SMC differentiation markers in vitro, consistent with their common smooth muscle developmental fates in vivo. By contrast, Ect SMCs displayed a greater capacity for growth in serum-free medium than Mes SMCs, but only under conditions permitting short-range cell-cell interactions. Most of the peptide growth factors tested that might account for serum-independent growth (PDGF-AA, PDGF-BB, basic FGF, EGF, or activin) stimulated DNA synthesis to similar extents in Ect and Mes SMCs. However, we found dramatic, lineage-dependent differences in SMC responses to transforming growth factor-beta (TGF-beta). Exposure to TGF-beta 1 (0.4 to 400 pmole/liter) consistently increased DNA synthesis in Ect SMCs, whereas in paired cultures of Mes SMCs, TGF-beta 1 was growth inhibitory. In SMC cultures transfected with p3TP-lux, a luciferase reporter controlled by the TGF-beta 1-response elements of the human PAI-1 promoter, TGF-beta 1 (120 pM) produced 12 +/- 2-fold increases in luciferase activity in Ect SMCs and only 3 +/- 1.5-fold increases in Mes SMCs. Analysis of TGF-beta receptor phenotypes by Northern blot, radioligand binding, and crosslinking assays showed that Ect and Mes SMCs expressed similar levels of types I, II, and III TGF-beta receptors. However, using a polyclonal antibody specific for the chick type II TGF-beta receptor subunit, we demonstrate that Mes SMCs produce a fully glycosylated form of this protein while Ect SMCs elaborate only an unglycosylated type II TGF-beta receptor. These results show that Ect and Mes SMCs exhibit lineage-dependent differences in growth and receptor-mediated transcriptional responses to at least one important class of SMC morphogens and growth modifiers, e.g., the TGF-betas. Our findings suggest that different SMC populations within a common vessel wall may respond in lineage-dependent ways to signals that direct formation of the tunica media in the embryo and to factors involved in the progression of vascular disease later in life.


THE USE OF LEAD CITRATE AT HIGH pH AS AN ELECTRON-OPAQUE STAIN IN ELECTRON MICROSCOPY

Abstract: Aqueous solutions of lead salts (1, 2) and saturated solutions of lead hydroxide (1) have been used as stains to enhance the electron-scattering properties of components of biological materials examined in the electron microscope. Saturated solutions of lead hydroxide (1), while staining more intensely than either lead acetate or monobasic lead acetate (l , 2), form insoluble lead carbonate upon exposure to air. The avoidance of such precipitates which contaminate surfaces of sections during staining has been the stimulus for the development of elaborate procedures for exclusion of air or carbon dioxide (3, 4). Several modifications of Watson's lead hydroxide stain (1) have recently appeared (5-7). All utilize relatively high pH (approximately 12) and one contains small amounts of tartrate (6), a relatively weak complexing agent (8), in addition to lead. These modified lead stains are less liable to contaminate the surface of the section with precipitated stain products. The stain reported here differs from previous alkaline lead stains in that the chelating agent, citrate, is in sufficient excess to sequester all lead present. Lead citrate, soluble in high concentrations in basic solutions, is a chelate compound with an apparent association constant (log Ka) between ligand and lead ion of 6.5 (9). Tissue binding sites, presumably organophosphates, and other anionic species present in biological components following fixation, dehydration, and plastic embedding apparently have a greater affinity for this cation than lead citrate inasmuch as cellular and extracellular structures in the section sequester lead from the staining solution. Alkaline lead citrate solutions are less likely to contaminate sections, as no precipitates form when droplets of fresh staining solution are exposed to air for periods of up to 30 minutes. The resultant staining of the sections is of high intensity in sections of Aralditeor Epon-embedded material. Cytoplasmic membranes, ribosomes, glycogen, and nuclear material are stained (Figs. 1 to 3). STAIN SOLUTION: Lead citrate is prepared by


Amylase, Lipase, and Acute Pancreatitis in People With Type 2 Diabetes Treated With Liraglutide: Results From the LEADER Randomized Trial.

Abstract:
OBJECTIVE To evaluate serum amylase and lipase levels and the rate of acute pancreatitis in patients with type 2 diabetes and high cardiovascular risk randomized to liraglutide or placebo and observed for 3.5-5.0 years.    RESEARCH DESIGN AND METHODS A total of 9,340 patients with type 2 diabetes were randomized to either liraglutide or placebo (median observation time 3.84 years). Fasting serum lipase and amylase were monitored. Acute pancreatitis was adjudicated in a blinded manner.
 RESULTS Compared with the placebo group, liraglutide-treated patients had increases in serum lipase and amylase of 28.0% and 7.0%, respectively. Levels were increased at 6 months and then remained stable. During the study, 18 (0.4% [1.1 events/1,000 patient-years of observation] [PYO]) liraglutide-treated and 23 (0.5% [1.7 events/1,000 PYO]) placebo patients had acute pancreatitis confirmed by adjudication. Most acute pancreatitis cases occurred ≥12 months after randomization. Liraglutide-treated patients with prior history of pancreatitis (n = 147) were not more likely to develop acute pancreatitis than similar patients in the placebo group (n = 120). Elevations of amylase and lipase levels did not predict future risk of acute pancreatitis (positive predictive value <1.0%) in patients treated with liraglutide.
 CONCLUSIONS In a population with type 2 diabetes at high cardiovascular risk, there were numerically fewer events of acute pancreatitis among liraglutide-treated patients (regardless of previous history of pancreatitis) compared with the placebo group. Liraglutide was associated with increases in serum lipase and amylase, which were not predictive of an event of subsequent acute pancreatitis.


Zoledronic acid is unable to induce apoptosis, but slows tumor growth and prolongs survival for non-small-cell lung cancers.

Abstract:
BACKGROUND Although zoledronic acid (ZOL), a third-generation nitrogen-containing bisphosphonate, has been identified as an attractive therapeutic agent against breast cancer, prostate cancer, multiple myeloma as well as small-cell lung cancer (SCLC), as best as we are aware, the anti-tumor effect of ZOL upon non-small-cell lung cancer (NSCLC) remains to be effectively investigated. This study examined the effects of ZOL upon the line-1 tumor cell, using a murine lung adenocarcinoma cell line similar to the behavior of human lung adenocarcinoma.
 METHODS We investigated the anti-tumor effects of ZOL (3-100 microM) on line-1 tumor cells in vitro, including cellular proliferation, by means of an MTT assay, cell-cycle analysis by flow cytometry and by assessing the level of apoptosis by annexin V/propidium iodide (PI) and 4'-6-diamidino-2-phenylindole (DAPI) staining. Further, we evaluated the growth and survival of line-1 tumor cells following ZOL treatment (1 microg/kg/week) using an animal model. We also examined the in vivo cell-cycle pattern using lacZ-expressing line-1 cells (line-1/lacZ).
 RESULTS ZOL significantly slowed the line-1 tumor growth in a dose-dependent manner in vitro. The treated line-1 tumor cells typically arrested at the S/G2/M-phase of the cell-cycle following ZOL exposure, but no apoptotic cells could be detected by either annexin V/PI or DAPI staining. When the ZOL was washed out, the drug-inhibited cells continued to proliferate again and the cell-cycle prolongation elicited earlier by the drug, then disappeared. Within 72-96 h following drug removal, the cell-cycle of the treated cells revealed a similar distribution to that of the untreated controls. In vivo studies demonstrated that ZOL significantly slowed the line-1 tumor growth. Indeed, mice lived significantly longer when they had been ZOL-treated than was the case for untreated mice (p<0.05). Using line-1/lacZ cells, the in vivo cell-cycle distribution of line-1 tumor cells subsequent to ZOL exposure revealed S/G2/M-phase arrest that was identical to the in vitro culture.
 CONCLUSIONS ZOL maintains the potential to reduce tumor burden and prolong survival for murine pulmonary adenocarcinoma. The flow cytometrical analysis of cell-cycle demonstrated that ZOL induces no apoptosis but is able to arrest line-1 tumor cells at the S/G2/M-phase. Although the clinical relevance of these results warrants verification for human lung cancer patients, ZOL combined with chemotherapy and/or radiotherapy appears to be a new therapeutic strategy for the effective treatment of NSCLC.


Contribution of traditional healers to a rural tuberculosis control programme in Hlabisa, South Africa.

Abstract:
SETTING The rural health district of Hlabisa, KwaZulu-Natal, South Africa.   OBJECTIVES To assess the acceptability and effectiveness of traditional healers as supervisors of tuberculosis (TB) treatment in an existing directly observed treatment, short-course (DOTS) programme.
 DESIGN An observational study comparing treatment outcomes among new TB patients in the three intervention sub-districts offered the additional option of traditional healers for directly observed treatment (DOT) supervision with those in the remainder of the district offered the standard range of options for DOT supervision (health facility, community health worker and lay persons). A comparison was also made of treatment outcomes between different options for DOT supervision.
 RESULTS A total of 3461 TB patients were registered in Hlabisa District from April 1999 to December 2000, of whom 2823 were discharged from hospital to the ambulatory DOT programme. Treatment outcomes were known for 1816 patients in Hlabisa District (275 patients in the intervention area and 1541 patients in the control area). There was no significant difference (P < 0.5) in treatment outcome in the intervention and control areas (77% vs. 75%). Among 275 patients with known outcomes in the intervention area, 48 patients were supervised by traditional healers and 227 patients supervised by people other than traditional healers. Treatment completion was not significantly higher among patients supervised by traditional healers than among patients supervised by other categories of DOT supervisor (88% vs. 75%, P = 0.3841). Interviews with 41 of 51 traditional healer patients who had completed treatment revealed high levels of satisfaction with the care received.
 CONCLUSIONS Traditional healers make an effective contribution to TB programme performance in this pilot scheme in Hlabisa district. Further evaluation will be necessary as this approach is scaled up.


Pdgfrβ+ Mural Preadipocytes Contribute to Adipocyte Hyperplasia Induced by High-Fat-Diet Feeding and Prolonged Cold Exposure in Adult Mice.

Abstract: The expansion of white adipose tissue (WAT) in obesity involves de novo differentiation of new adipocytes; however, the cellular origin of these cells remains unclear. Here, we utilize Zfp423(GFP) reporter mice to characterize adipose mural (Pdgfrβ(+)) cells with varying levels of the preadipocyte commitment factor Zfp423. We find that adipose tissue contains distinct mural populations, with levels of Zfp423 distinguishing adipogenic from inflammatory-like mural cells. Using our "MuralChaser" lineage tracking system, we uncover adipose perivascular cells as developmental precursors of adipocytes formed in obesity, with adipogenesis and precursor abundance regulated in a depot-dependent manner. Interestingly, Pdgfrβ(+) cells do not significantly contribute to the initial cold-induced recruitment of beige adipocytes in WAT; it is only after prolonged cold exposure that these cells differentiate into beige adipocytes. These results provide genetic evidence for a mural cell origin of white adipocytes in obesity and suggest that beige adipogenesis may originate from multiple sources.


Regulated nucleo/cytoplasmic exchange of HOG1 MAPK requires the importin beta homologs NMD5 and XPO1.

Abstract: MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran-GSP1, but not the NLS-binding importin alpha/beta heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin beta homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase.


FBW7 regulates endothelial functions by targeting KLF2 for ubiquitination and degradation

Abstract: F-box and WD repeat domain-containing 7 (FBW7), the substrate-binding subunit of E3 ubiquitin ligase SCFFBW7 (a complex of SKP1, cullin-1 and FBW7), plays important roles in various physiological and pathological processes. Although FBW7 is required for vascular development, its function in the endothelium remains to be investigated. In this study, we show that FBW7 is an important regulator of endothelial functions, including angiogenesis, leukocyte adhesion and the endothelial barrier integrity. Using RNA interference, we found that the depletion of FBW7 markedly impairs angiogenesis in vitro and in vivo. We identified the zinc finger transcription factor Krüppel-like factor 2 (KLF2) as a physiological target of FBW7 in endothelial cells. Knockdown of FBW7 expression resulted in the accumulation of endogenous KLF2 protein in endothelial cells. FBW7-mediated KLF2 destruction was shown to depend on the phosphorylation of KLF2 via glycogen synthase kinase-3 (GSK3) at two conserved phosphodegrons. Mutating these phosphodegron motifs abolished the FBW7-mediated degradation and ubiquitination of KLF2. The siRNA-mediated knockdown of FBW7 showed that KLF2 is an essential target of FBW7 in the regulation of endothelial functions. Moreover, FBW7-mediated KLF2 degradation was shown to be critical for angiogenesis in teratomas and in zebrafish development. Taken together, our study suggests a role for FBW7 in the processes of endothelial cell migration, angiogenesis, inflammation and barrier integrity, and provides novel insights into the regulation of KLF2 stability in vivo.


Role of RhoA-specific guanine exchange factors in regulation of endomitosis in megakaryocytes.

Abstract: Polyploidization can precede the development of aneuploidy in cancer. Polyploidization in megakaryocytes (Mks), in contrast, is a highly controlled developmental process critical for efficient platelet production via unknown mechanisms. Using primary cells, we demonstrate that the guanine exchange factors GEF-H1 and ECT2, which are often overexpressed in cancer and are essential for RhoA activation during cytokinesis, must be downregulated for Mk polyploidization. The first (2N-4N) endomitotic cycle requires GEF-H1 downregulation, whereas subsequent cycles (>4N) require ECT2 downregulation. Exogenous expression of both GEF-H1 and ECT2 prevents endomitosis, resulting in proliferation of 2N Mks. Furthermore, we have shown that the mechanism by which polyploidization is prevented in Mks lacking Mkl1, which is mutated in megakaryocytic leukemia, is via elevated GEF-H1 expression; shRNA-mediated GEF-H1 knockdown alone rescues this ploidy defect. These mechanistic insights enhance our understanding of normal versus malignant megakaryocytopoiesis, as well as aberrant mitosis in aneuploid cancers.


Golden Hours in Severe Paraquat Poisoning-The Role of Early Haemoperfusion Therapy.

Abstract:
INTRODUCTION Paraquat is a commonly ingested poison especially in Southern India. There is no antidote for paraquat poison and consumption is often fatal. The usual cause of death is either acute lung injury or multi-organ failure.    AIM To evaluate the role of early haemoperfusion as a therapy in paraquat poisoned patients.    MATERIALS AND METHODS This study was a retrospective analysis of patients admitted to a Tertiary Medical College Hospital between January 2012 and December 2015 with history of paraquat consumption, comparing outcomes in those who received only gastric lavage and symptomatic treatment with those who received haemoperfusion as a therapy. The role of early haemoperfusion (≤ 6 hours) vs late haemoperfusion (> 6 hours) in paraquat poisoned patients was also compared. The data of these patients was extracted and analysed with respect to age, sex, mode of treatment, the outcome in patients who received early and late haemoperfusion.
 RESULTS A total of 101 patients were studied out of which 62 died. Deaths were more in those patients who received only gastric lavage with symptomatic treatment as therapy compared to those who received haemoperfusion i.e., 92.1% vs 42.9% respectively. We also found that, the survival rate was better in patients who received early haemoperfusion.
 CONCLUSION Early haemoperfusion was helpful in the management of severe paraquat poisoning and improved the survival rate in these patients.


Changes in the prevalence of cerebral palsy for children born very prematurely within a population-based program over 30 years.

Abstract:
CONTEXT Although cerebral palsy (CP) among extremely premature infants has been reported as a major morbidity outcome, there are difficulties comparing published CP rates from many sites over various birth years.
 OBJECTIVE To assess the changes in population-based, gestational age-specific prevalence rates of CP among extremely premature infants over 30 years.
 DESIGN Prospective population-based longitudinal outcome study.
 SETTING AND PARTICIPANTS In Northern Alberta, 2318 infants 20 to 27 weeks' gestational age with birth weights of 500 to 1249 g were liveborn from 1974 through 2003. By 2 years of age, 1437 (62%) had died, 23 (1%) were lost to follow-up, and 858 (37%) had received multidisciplinary neurodevelopmental assessment.
 MAIN OUTCOME MEASURE Population-based prevalence rates of CP were determined. Logistic regression with linear spline was used to assess changes in CP prevalence over time.
 RESULTS At age 2 years, 122 (14.2%) of 858 survivors had CP. This diagnosis was confirmed for each child by age 3 years or older. Among those whose gestational age was 20 to 25 weeks, population-based survival increased from 4% to 31% (P<.001), while CP prevalence per 1000 live births increased monotonically from 0 to 110 until the years 1992-1994 (P<.001) and decreased thereafter to 22 in the years 2001-2003 (P<.001). Among those whose gestational age was 26 to 27 weeks, population-based survival increased from 23% to between 75% and 80% (P<.001), while CP prevalence per 1000 live births increased monotonically from 15 to 155 until the years 1992-1994 (P<.001) and then decreased to 16 in the years 2001-2003 (P<.001). For all survivors born in the years 2001-2003, CP prevalence was 19 per 1000 live births.
 CONCLUSION Population-based CP prevalence rates for children whose gestational age was 20 to 27 weeks and whose birth weight ranged from 500 to 1249 g show steady reductions in the last decade with stable or reducing mortality, reversing trends prior to 1992-1994.


Isolation of major pancreatic cell types and long-term culture-initiating cells using novel human surface markers.

Abstract: We have developed a novel panel of cell-surface markers for the isolation and study of all major cell types of the human pancreas. Hybridomas were selected after subtractive immunization of Balb/C mice with intact or dissociated human islets and assessed for cell-type specificity and cell-surface reactivity by immunohistochemistry and flow cytometry. Antibodies were identified by specific binding of surface antigens on islet (panendocrine or alpha-specific) and nonislet pancreatic cell subsets (exocrine and duct). These antibodies were used individually or in combination to isolate populations of alpha, beta, exocrine, or duct cells from primary human pancreas by FACS and to characterize the detailed cell composition of human islet preparations. They were also employed to show that human islet expansion cultures originated from nonendocrine cells and that insulin expression levels could be increased to up to 1% of normal islet cells by subpopulation sorting and overexpression of the transcription factors Pdx-1 and ngn3, an improvement over previous results with this culture system. These methods permit the analysis and isolation of functionally distinct pancreatic cell populations with potential for cell therapy.


Re-engineered CD40 receptor enables potent pharmacological activation of dendritic-cell cancer vaccines in vivo

Abstract: Modest clinical outcomes of dendritic-cell (DC) vaccine trials call for the refinement of DC vaccine design. Although many potential antigens have been identified, development of methods to enhance antigen presentation by DCs has lagged. We have engineered a potent, drug-inducible CD40 (iCD40) receptor that permits temporally controlled, lymphoid-localized, DC-specific activation. iCD40 is comprised of a membrane-localized cytoplasmic domain of CD40 fused to drug-binding domains. This allows it to respond to a lipid-permeable, high-affinity dimerizer drug while circumventing ectodomain-dependent negative-feedback mechanisms. These modifications permit prolonged activation of iCD40-expressing DCs in vivo, resulting in more potent CD8+ T-cell effector responses, including the eradication of previously established solid tumors, relative to activation of DCs ex vivo (P < 0.01), typical of most clinical DC protocols. In addition, iCD40-mediated DC activation exceeded that achieved by stimulating the full-length, endogenous CD40 receptor both in vitro and in vivo. Because iCD40 is insulated from the extracellular environment and can be activated within the context of an immunological synapse, iCD40-expressing DCs have a prolonged lifespan and should lead to more potent vaccines, perhaps even in immune-compromised patients.


Effect of homocysteine lowering on mortality and vascular disease in advanced chronic kidney disease and end-stage renal disease: a randomized controlled trial.

Abstract:
CONTEXT High plasma homocysteine levels are a risk factor for mortality and vascular disease in observational studies of patients with chronic kidney disease. Folic acid and B vitamins decrease homocysteine levels in this population but whether they lower mortality is unknown.
 OBJECTIVE To determine whether high doses of folic acid and B vitamins administered daily reduce mortality in patients with chronic kidney disease.
 DESIGN, SETTING, AND PARTICIPANTS Double-blind randomized controlled trial (2001-2006) in 36 US Department of Veterans Affairs medical centers. Median follow-up was 3.2 years for 2056 participants aged 21 years or older with advanced chronic kidney disease (estimated creatinine clearance < or =30 mL/min) (n = 1305) or end-stage renal disease (n = 751) and high homocysteine levels (> or = 15 micromol/L).
 INTERVENTION Participants received a daily capsule containing 40 mg of folic acid, 100 mg of pyridoxine hydrochloride (vitamin B6), and 2 mg of cyanocobalamin (vitamin B12) or a placebo.
 MAIN OUTCOME MEASURES The primary outcome was all-cause mortality. Secondary outcomes included myocardial infarction (MI), stroke, amputation of all or part of a lower extremity, a composite of these 3 plus all-cause mortality, time to initiation of dialysis, and time to thrombosis of arteriovenous access in hemodialysis patients.
 RESULTS Mean baseline homocysteine level was 24.0 micromol/L in the vitamin group and 24.2 micromol/L in the placebo group. It was lowered 6.3 micromol/L (25.8%; P < .001) in the vitamin group and 0.4 micromol/L (1.7%; P = .14) in the placebo group at 3 months, but there was no significant effect on mortality (448 vitamin group deaths vs 436 placebo group deaths) (hazard ratio [HR], 1.04; 95% CI, 0.91-1.18). No significant effects were demonstrated for secondary outcomes or adverse events: there were 129 MIs in the vitamin group vs 150 for placebo (HR, 0.86; 95% CI, 0.67-1.08), 37 strokes in the vitamin group vs 41 for placebo (HR, 0.90; 95% CI, 0.58-1.40), and 60 amputations in the vitamin group vs 53 for placebo (HR, 1.14; 95% CI, 0.79-1.64). In addition, the composite of MI, stroke, and amputations plus mortality (P = .85), time to dialysis (P = .38), and time to thrombosis in hemodialysis patients (P = .97) did not differ between the vitamin and placebo groups.
 CONCLUSION Treatment with high doses of folic acid and B vitamins did not improve survival or reduce the incidence of vascular disease in patients with advanced chronic kidney disease or end-stage renal disease.
 TRIAL REGISTRATION clinicaltrials.gov Identifier: NCT00032435.


Recipient-type specific CD4+CD25+ regulatory T cells favor immune reconstitution and control graft-versus-host disease while maintaining graft-versus-leukemia.

Abstract: CD4+CD25+ regulatory T cells (Treg's) play a pivotal role in preventing organ-specific autoimmune diseases and in inducing tolerance to allogeneic organ transplants. We and others recently demonstrated that high numbers of Treg's can also modulate graft-versus-host disease (GVHD) if administered in conjunction with allogeneic hematopoietic stem cell transplantation in mice. In a clinical setting, it would be impossible to obtain enough freshly purified Treg's from a single donor to have a therapeutic effect. Thus, we performed regulatory T cell expansion ex vivo by stimulation with allogeneic APCs, which has the additional effect of producing alloantigen-specific regulatory T cells. Here we show that regulatory T cells specific for recipient-type alloantigens control GVHD while favoring immune reconstitution. Irrelevant regulatory T cells only mediate a partial protection from GVHD. Preferential survival of specific regulatory T cells, but not of irrelevant regulatory T cells, was observed in grafted animals. Additionally, the use of specific regulatory T cells was compatible with some form of graft-versus-tumor activity. These data suggest that recipient-type specific Treg's could be preferentially used in the control of GVHD in future clinical trials.


The relationship of the level of cyclic amp to differentiation in primary cultures of chick muscle cells.

Abstract: Abstract The effect of increased levels of cAMP upon the differentiation of primary cultures of chick myo blasts has been investigated. 0.1 mM But 2 cAMP or 1 mM 3-isobutyl-1-methylxanthine was added to the cultures 24 h after plating and maintained throughout the 70 h period of culture examined. Both reagents were found to markedly delay the time of fusion of the myoblasts but had no observable effect upon the increase in activity of creatine phosphokinase. Morphological examination of the cells revealed no difference in the relative numbers of myoblasts and fibroblasts between the control, But 2 cAMP and 3-isobutyl-1-methylxanthine cultures, but the latter reagent appeared to cause some inhibition of cell proliferation.


Intact endothelial autophagy is required to maintain vascular lipid homeostasis.

Abstract: The physiological role of autophagic flux within the vascular endothelial layer remains poorly understood. Here, we show that in primary endothelial cells, oxidized and native LDL stimulates autophagosome formation. Moreover, by both confocal and electron microscopy, excess native or modified LDL appears to be engulfed within autophagic structures. Transient knockdown of the essential autophagy gene ATG7 resulted in higher levels of intracellular (125) I-LDL and oxidized LDL (OxLDL) accumulation, suggesting that in endothelial cells, autophagy may represent an important mechanism to regulate excess, exogenous lipids. The physiological importance of these observations was assessed using mice containing a conditional deletion of ATG7 within the endothelium. Following acute intravenous infusion of fluorescently labeled OxLDL, mice lacking endothelial expression of ATG7 demonstrated prolonged retention of OxLDL within the retinal pigment epithelium (RPE) and choroidal endothelium of the eye. In a chronic model of lipid excess, we analyzed atherosclerotic burden in ApoE(-/-) mice with or without endothelial autophagic flux. The absence of endothelial autophagy markedly increased atherosclerotic burden. Thus, in both an acute and chronic in vivo model, endothelial autophagy appears critically important in limiting lipid accumulation within the vessel wall. As such, strategies that stimulate autophagy, or prevent the age-dependent decline in autophagic flux, might be particularly beneficial in treating atherosclerotic vascular disease.


Induction of circadian gene expression in human subcutaneous adipose-derived stem cells.

Abstract:
OBJECTIVE Genes encoding the circadian transcriptional apparatus exhibit robust oscillatory expression in murine adipose tissues. This study tests the hypothesis that human subcutaneous adipose-derived stem cells (ASCs) provide an in vitro model in which to monitor the activity of the core circadian transcriptional apparatus.    RESEARCH METHODS AND PROCEDURES Primary cultures of undifferentiated or adipocyte-differentiated ASCs were treated with dexamethasone, rosiglitazone, or 30% fetal bovine serum. The response of undifferentiated ASCs to dexamethasone was further evaluated in the presence of lithium chloride. Lithium inhibits glycogen synthase kinase 3, a key component of the circadian apparatus. Total RNA was harvested at 4-hour intervals over 48 hours and examined by real-time reverse transcription polymerase chain reaction (RT-PCR).
 RESULTS Adipocyte-differentiated cells responded more rapidly to treatments than their donor-matched undifferentiated controls; however, the period of the circadian gene oscillation was longer in the adipocyte-differentiated cells. Dexamethasone generated circadian gene expression patterns with mean periods of 25.4 and 26.7 hours in undifferentiated and adipocyte-differentiated ASCs, respectively. Both rosiglitazone and serum shock generated a significantly longer period in adipocyte-differentiated ASCs relative to undifferentiated ASCs. The Bmal1 profile was phase-shifted by approximately 8 to 12 hours relative to Per1, Per3, and Cry2, consistent with their expression in vivo. Lithium chloride inhibited adipogenesis and significantly lengthened the period of Per3 and Rev-erbalpha gene expression profiles by >5 hours in dexamethasone-activated undifferentiated ASCs.    DISCUSSION These results support the initial hypothesis and validate ASCs as an in vitro model for the analysis of circadian biology in human adipose tissue.


BCL9 promotes tumor progression by conferring enhanced proliferative, metastatic, and angiogenic properties to cancer cells.

Abstract: Several components of the Wnt signaling cascade have been shown to function either as tumor suppressor proteins or as oncogenes in multiple human cancers, underscoring the relevance of this pathway in oncogenesis and the need for further investigation of Wnt signaling components as potential targets for cancer therapy. Here, using expression profiling analysis as well as in vitro and in vivo functional studies, we show that the Wnt pathway component BCL9 is a novel oncogene that is aberrantly expressed in human multiple myeloma as well as colon carcinoma. We show that BCL9 enhances beta-catenin-mediated transcriptional activity regardless of the mutational status of the Wnt signaling components and increases cell proliferation, migration, invasion, and the metastatic potential of tumor cells by promoting loss of epithelial and gain of mesenchymal-like phenotype. Most importantly, BCL9 knockdown significantly increased the survival of xenograft mouse models of cancer by reducing tumor load, metastasis, and host angiogenesis through down-regulation of c-Myc, cyclin D1, CD44, and vascular endothelial growth factor expression by tumor cells. Together, these findings suggest that deregulation of BCL9 is an important contributing factor to tumor progression. The pleiotropic roles of BCL9 reported in this study underscore its value as a drug target for therapeutic intervention in several malignancies associated with aberrant Wnt signaling.


The oncogenic microRNA miR-22 targets the TET2 tumor suppressor to promote hematopoietic stem cell self-renewal and transformation.

Abstract: MicroRNAs are frequently deregulated in cancer. Here we show that miR-22 is upregulated in myelodysplastic syndrome (MDS) and leukemia and its aberrant expression correlates with poor survival. To explore its role in hematopoietic stem cell function and malignancy, we generated transgenic mice conditionally expressing miR-22 in the hematopoietic compartment. These mice displayed reduced levels of global 5-hydroxymethylcytosine (5-hmC) and increased hematopoietic stem cell self-renewal accompanied by defective differentiation. Conversely, miR-22 inhibition blocked proliferation in both mouse and human leukemic cells. Over time, miR-22 transgenic mice developed MDS and hematological malignancies. We also identify TET2 as a key target of miR-22 in this context. Ectopic expression of TET2 suppressed the miR-22-induced phenotypes. Downregulation of TET2 protein also correlated with poor clinical outcomes and miR-22 overexpression in MDS patients. Our results therefore identify miR-22 as a potent proto-oncogene and suggest that aberrations in the miR-22/TET2 regulatory network are common in hematopoietic malignancies.


Transmission of atherosclerosis susceptibility with gut microbial transplantation.

Abstract: Recent studies indicate both clinical and mechanistic links between atherosclerotic heart disease and intestinal microbial metabolism of certain dietary nutrients producing trimethylamine N-oxide (TMAO). Here we test the hypothesis that gut microbial transplantation can transmit choline diet-induced TMAO production and atherosclerosis susceptibility. First, a strong association was noted between atherosclerotic plaque and plasma TMAO levels in a mouse diversity panel (n = 22 strains, r = 0.38; p = 0.0001). An atherosclerosis-prone and high TMAO-producing strain, C57BL/6J, and an atherosclerosis-resistant and low TMAO-producing strain, NZW/LacJ, were selected as donors for cecal microbial transplantation into apolipoprotein e null mice in which resident intestinal microbes were first suppressed with antibiotics. Trimethylamine (TMA) and TMAO levels were initially higher in recipients on choline diet that received cecal microbes from C57BL/6J inbred mice; however, durability of choline diet-dependent differences in TMA/TMAO levels was not maintained to the end of the study. Mice receiving C57BL/6J cecal microbes demonstrated choline diet-dependent enhancement in atherosclerotic plaque burden as compared with recipients of NZW/LacJ microbes. Microbial DNA analyses in feces and cecum revealed transplantation of donor microbial community features into recipients with differences in taxa proportions between donor strains that were transmissible to recipients and that tended to show coincident proportions with TMAO levels. Proportions of specific taxa were also identified that correlated with plasma TMAO levels in donors and recipients and with atherosclerotic lesion area in recipients. Atherosclerosis susceptibility may be transmitted via transplantation of gut microbiota. Gut microbes may thus represent a novel therapeutic target for modulating atherosclerosis susceptibility.


Benzodiazepines and injurious falls in community dwelling elders.

Abstract:
BACKGROUND Benzodiazepines are frequently used medications in the elderly, in whom they are associated with an increased risk of falling, with sometimes dire consequences.
 OBJECTIVE To estimate the impact of benzodiazepine-associated injurious falls in a population of elderly persons.
 METHOD A nested case-control study was conducted using data collected during 10 years of follow-up of the French PAQUID (Personnes Agées QUID) community-based cohort. The main outcome measure was the occurrence of an injurious fall, which was defined as a fall resulting in hospitalization, fracture, head trauma or death. Controls (3 : 1) were frequency-matched to cases. Benzodiazepine exposure was the use of benzodiazepines over the previous 2 weeks reported at the follow-up visit preceding the fall.
 RESULTS Benzodiazepine use was significantly associated with the occurrence of injurious falls, with a significant interaction with age. The adjusted odds ratio for injurious falls in subjects exposed to benzodiazepines was 2.2 (95% CI 1.4, 3.4) in subjects aged > or = 80 years and 1.3 (95% CI 0.9, 1.9) in subjects aged <80 years. The population attributable risk for injurious falls in subjects exposed to benzodiazepines was 28.1% (95% CI 16.7, 43.2) for subjects aged > or =80 years. The incidence of injurious falls in subjects aged > or = 80 years exposed to benzodiazepines in the PAQUID cohort was 2.8/100 person-years. Over 9% of these falls were fatal. According to these results and to recent population estimates, benzodiazepine use could be held responsible for almost 20 000 injurious falls in subjects aged > or = 80 years every year in France, and for nearly 1800 deaths.
 CONCLUSION Given the considerable morbidity and mortality associated with benzodiazepine use and the fact that existing good practice guidelines on benzodiazepines have not been effective in preventing their misuse (possibly because they have not been applied), new methods for limiting use of benzodiazepines in the elderly need to be found.