Intrinsic MyD88-Akt1-mTOR Signaling Coordinates Disparate Tc17 and Tc1 Responses during Vaccine Immunity against Fungal Pneumonia

Fungal infections have skyrocketed in immune-compromised patients lacking CD4⁺ T cells, underscoring the need for vaccine prevention. An understanding of the elements that promote vaccine immunity in this setting is essential. We previously demonstrated that vaccine-induced IL-17A⁺ CD8⁺ T cells (Tc17) are required for resistance against lethal fungal pneumonia in CD4⁺ T cell-deficient hosts, whereas the individual type I cytokines IFN-γ, TNF-α and GM-CSF, are dispensable. Here, we report that T cell-intrinsic MyD88 signals are crucial for these Tc17 cell responses and vaccine immunity against lethal fungal pneumonia in mice. In contrast, IFN-γ⁺ CD8⁺ cell (Tc1) responses are largely normal in the absence of intrinsic MyD88 signaling in CD8⁺ T cells. The poor accumulation of MyD88-deficient Tc17 cells was not linked to an early onset of contraction, nor to accelerated cell death or diminished expression of anti-apoptotic molecules Bcl-2 or Bcl-xL. Instead, intrinsic MyD88 was required to sustain the proliferation of Tc17 cells through the activation of mTOR via Akt1. Moreover, intrinsic IL-1R and TLR2, but not IL-18R, were required for MyD88 dependent Tc17 responses. Our data identify unappreciated targets for augmenting adaptive immunity against fungi. Our findings have implications for designing fungal vaccines and immune-based therapies in immune-compromised patients.     

Divergent effects of leptin on luteinizing hormone and insulin secretion are dose dependent

We have shown recently that fasting permits leptin to modulate both luteinizing hormone (LH) and insulin secretion in cows. In rodents, leptin causes divergent effects on LH and insulin release that are dose dependent. To test the hypothesis that leptin effects on LH and insulin secretion in fasted cows are dose related, we examined the effects of various doses of recombinant ovine leptin (oleptin) in mature cows. Twenty ovariectomized beef cows, each bearing an estradiol implant to maintain basal estradiol concentrations, were used. All cows were fasted for 60 hr with free access to water and were assigned randomly to one of four groups (n = 5/group): 1) saline control; 2) leptin, 0.2 microg/kg; 3) leptin, 2.0 microg/kg; and 4) leptin, 20 microg/kg body wt. Blood samples were collected at 10-min intervals for 6 hr on Days 0 and 2, with saline or oleptin injected intravenously immediately after the first intensive sample on Day 2 (54 hr). Leptin caused a dose-related increase (P < 0.001) in mean concentrations of circulating LH. Stimulation of LH release by leptin was significant at the lowest (141% of control) and middle (122% of control) doses used, but no increase was observed for the highest dose. Increased mean concentrations of LH appeared to result from an augmentation of basal secretion, as pulse characteristics were not affected. After 54 hr of fasting, plasma insulin concentrations were lowered (P < 0.01) in all treatment groups compared to Day 0. After leptin injections, plasma insulin concentrations increased (P < 0.01) and reached highest concentrations during the first hour of sampling. However, this increase was sustained for several hours only in the intermediate (2.0 microg/kg) dose group. Collectively, our results show that leptin has potent positive effects on both LH and insulin secretion in fasted cows, but the anterior pituitary and endocrine pancreas appear to become downregulated in the presence of excess ligand.     

Interference of the galactose-dependent binding of lectins by novel pentapeptide ligands

A library of pentapeptides containing the sequence -Y-X-Y- based on rational design was screened with six different lectins. Sequences were identified that modulate galectin binding to its natural carbohydrate ligand. SPR showed inhibition values 2-3 times stronger than galactose and NMR studies suggested real carbohydrate mimicry.     

Heterogeneous Na+ Sensitivity of Na+,K+-ATPase Isoenzymes in Whole Brain Membranes

Abstract: The Na⁺ sensitivity of whole brain membrane Na⁺,K⁺-ATPase isoenzymes was studied using the differential inhibitory effect of ouabain (α1, low affinity for ouabain; α2, high affinity; and α3, very high affinity). At 100 m M Na⁺, we found that the proportion of isoforms with low, high, and very high ouabain affinity was 21, 38, and 41%, respectively. Using two ouabain concentrations (10⁻⁵ and 10⁻⁷ M ), we were able to discriminate Na⁺ sensitivity of Na⁺, K⁺-ATPase isoenzymes using nonlinear regression. The ouabain low-affinity isoform, α1, exhibited high Na⁺ sensitivity [ K _a of 3.88 ± 0.25 m M Na⁺ and a Hill coefficient ( n ) of 1.98 ± 0.13]; the ouabain high-affinity isoform, α2, had two Na⁺ sensitivities, a high ( K _a of 4.98 ± 0.2 m M Na⁺ and n of 1.34 ± 0.10) and a low ( K _a of 28 ± 0.5 m M Na⁺ and an n of 1.92 ± 0.18) Na⁺ sensitivity activated above a thresh old (22 ± 0.3 m M Na⁺); and the ouabain very-high-affinity isoform, α3, was resolved by two processes and appears to have two Na⁺ sensitivities (apparent K _a values of 3.5 and 20 m M Na⁺). We show that Na⁺ dependence in the absence of ouabain is the result of at least of five Na⁺ reactivities. This molecular functional characteristic of isoenzymes in membranes could explain the diversity of physiological roles attributed to isoenzymes.     

Syntenin-1 and Ezrin Proteins Link Activated Leukocyte Cell Adhesion Molecule to the Actin Cytoskeleton

Activated leukocyte cell adhesion molecule (ALCAM) is a type I transmembrane protein member of the immunoglobulin superfamily of cell adhesion molecules. Involved in important pathophysiological processes such as the immune response, cancer metastasis, and neuronal development, ALCAM undergoes both homotypic interactions with other ALCAM molecules and heterotypic interactions with the surface receptor CD6 expressed at the T cell surface. Despite biochemical and biophysical evidence of a dynamic association between ALCAM and the actin cytoskeleton, no detailed information is available about how this association occurs at the molecular level. Here, we exploit a combination of complementary microscopy techniques, including FRET detected by fluorescence lifetime imaging microscopy and single-cell force spectroscopy, and we demonstrate the existence of a preformed ligand-independent supramolecular complex where ALCAM stably interacts with actin by binding to syntenin-1 and ezrin. Interaction with the ligand CD6 further enhances these multiple interactions. Altogether, our results propose a novel biophysical framework to understand the stabilizing role of the ALCAM supramolecular complex engaged to CD6 during dendritic cell-T cell interactions and provide novel information on the molecular players involved in the formation and signaling of the immunological synapse at the dendritic cell side.     

Discovery of 2-(1H-imidazo-2-yl)piperazines as a new class of potent and non-cytotoxic inhibitors of Trypanosoma brucei growth in vitro

The identification of a new series of growth inhibitors of Trypanosoma brucei rhodesiense, causative agent of Human African Trypanosomiasis (HAT), is described. A selection of compounds from our in-house compound collection was screened in vitro against the parasite leading to the identification of compounds with nanomolar inhibition of T. brucei growth. Preliminary SAR on the hit compound led to the identification of compound 34 that shows low nanomolar parasite growth inhibition (T. brucei EC₅₀ 5?nM), is not cytotoxic (HeLa CC₅₀?>?25,000?nM) and is selective over other parasites, such as Trypanosoma cruzi and Plasmodium falciparum (T. cruzi EC₅₀ 8120?nM, P. falciparum EC₅₀ 3624?nM).     

Effect of melatonin on incidence of delirium among patients with hip fracture: a multicentre,double-blind randomized controlled trial

Background:

Disturbance of the sleep–wake cycle is a characteristic of delirium. In addition, changes in melatonin rhythm influence the circadian rhythm and are associated with delirium. We compared the effect of melatonin and placebo on the incidence and duration of delirium.

Methods:

We performed this multicentre, double-blind, randomized controlled trial between November 2008 and May 2012 in 1 academic and 2 nonacademic hospitals. Patients aged 65 years or older who were scheduled for acute hip surgery were eligible for inclusion. Patients received melatonin 3 mg or placebo in the evening for 5 consecutive days, starting within 24 hours after admission. The primary outcome was incidence of delirium within 8 days of admission. We also monitored the duration of delirium.

Results:

A total of 452 patients were randomly assigned to the 2 study groups. We subsequently excluded 74 patients for whom the primary end point could not be measured or who had delirium before the second day of the study. After these postrandomization exclusions, data for 378 patients were included in the main analysis. The overall mean age was 84 years, 238 (63.0%) of the patients lived at home before admission, and 210 (55.6%) had cognitive impairment. We observed no effect of melatonin on the incidence of delirium: 55/186 (29.6%) in the melatonin group v. 49/192 (25.5%) in the placebo group; difference 4.1 (95% confidence interval −0.05 to 13.1) percentage points. There were no between-group differences in mortality or in cognitive or functional outcomes at 3-month follow-up.

Interpretation:

In this older population with hip fracture, treatment with melatonin did not reduce the incidence of delirium. Trial registration: Netherlands Trial Registry, NTR1576: MAPLE (Melatonin Against PLacebo in Elderly patients) study; www.trialregister.nl/trialreg/admin/rctview.asp?TC=1576Delirium in older inpatients is associated with a high risk of dementia and other complications that translate into increased mortality and health care costs.^1,2 The antipsychotic haloperidol has historically been the agent of choice for treating delirium, and it has increasingly been administered as a prophylactic for delirium or to reduce symptoms such as hallucinations and aggressive behaviour.^3,4 However, all antipsychotic treatments may induce serious cerebrovascular adverse effects and greater mortality, particularly among patients with dementia.^5,6 These effects led the US Food and Drug Administration to issue a serious warning against their use.⁷ In addition, benzodiazepines are still frequently used to treat delirium, despite their being known to elicit or aggravate delirium.^8,9Disturbances of the circadian sleep–wake cycle represent one of the core features of delirium,¹⁰ leading to the hypothesis that the neurotransmitter melatonin and changes in its metabolism may be involved in the pathogenesis of delirium.^11,12 Objective measurements have shown that melatonin metabolism is disturbed after abdominal and other types of surgery, insomnia, sleep deprivation and stays in the intensive care unit (ICU), all of which are also known to be factors that contribute to delirium.^13–16 These characteristics suggest an association between melatonin abnormalities and delirium.^17–22 Although proof of a causal relation is still lacking, inpatients might nevertheless benefit from melatonin supplementation therapy through postoperative maintenance or restoration of their sleep–wake cycle.^23–25 Although melatonin depletion is thought to be one of the mechanisms of delirium, few studies have investigated the effects of altering perioperative plasma concentrations of melatonin, in particular, the possible effects on postoperative delirium.The primary objective of this study was to assess the effects of melatonin on the incidence of delirium among elderly patients admitted to hospital as an emergency following hip fracture. Secondary outcomes were duration and severity of delirium, length of hospital stay, total doses of haloperidol and benzodiazepines administered to patients with delirium, mortality during the hospital stay, and functional status, cognitive function and mortality at 3-month follow-up.     

Natural variation in life history strategy of Arabidopsis thaliana determines stress responses to drought and insects of different feeding guilds

Plants are sessile organisms and, consequently, are exposed to a plethora of stresses in their local habitat. As a result, different populations of a species are subject to different selection pressures leading to adaptation to local conditions and intraspecific divergence. The annual brassicaceous plant Arabidopsis thaliana is an attractive model for ecologists and evolutionary biologists due to the availability of a large collection of resequenced natural accessions. Accessions of A. thaliana display one of two different life cycle strategies: summer and winter annuals. We exposed a collection of 308 European Arabidopsis accessions, that have been genotyped for 250K SNPs, to a range of stresses: one abiotic stress (drought), four biotic stresses (Pieris rapae caterpillars, Plutella xylostella caterpillars, Frankliniella occidentalis thrips and Myzus persicae aphids) and two combined stresses (drought plus P. rapae and Botrytis cinerea fungus plus P. rapae). We identified heritable genetic variation for responses to the different stresses, estimated by narrow‐sense heritability. We found that accessions displaying different life cycle strategies differ in their response to stresses. Winter annuals are more resistant to drought, aphids and thrips and summer annuals are more resistant to P. rapae and P. xylostella caterpillars. Summer annuals are also more resistant to the combined stresses of drought plus P. rapae and infection by the fungus Botryris cinerea plus herbivory by P. rapae. Adaptation to drought displayed a longitudinal gradient. Finally, trade‐offs were recorded between the response to drought and responses to herbivory by caterpillars of the specialist herbivore P. rapae.     

Evaluation of the potential of Aspergillus niger species for the bioconversion of L-phenylalanine into 2-phenylethanol

Aspergillus niger was explored, for the first time, for the production of 2-phenylethanol (a rose-like aroma) using L-phenylalanine as precursor. Among the strains screened, A. niger CMICC 298302 was shown to produce, in a culture medium containing 6 g L-phenylalanine l^–1 and 60 g glucose l^–1, 1375 mg 2-phenylethanol l^–1 with a productivity of 153 mg l^–1 day^–1 and a molar yield of 74%. 2-Phenylethanol concentrations of 1 to 2 g l^–1 led to a two-fold and ten-fold decrease, respectively, in the mycelial radial growth rate. However, 2-phenylethanol was synthesized as the sole aromatic product and accumulated in the culture broth.     

High-Dose Benzodiazepine Dependence: A Qualitative Study of Patients’ Perceptions on Initiation,Reasons for Use,and Obtainment

Background

High-dose benzodiazepine (BZD) dependence is associated with a wide variety of negative health consequences. Affected individuals are reported to suffer from severe mental disorders and are often unable to achieve long-term abstinence via recommended discontinuation strategies. Although it is increasingly understood that treatment interventions should take subjective experiences and beliefs into account, the perceptions of this group of individuals remain under-investigated.

Methods

We conducted an exploratory qualitative study with 41 adult subjects meeting criteria for (high-dose) BZD-dependence, as defined by ICD-10. One-on-one in-depth interviews allowed for an exploration of this group’s views on the reasons behind their initial and then continued use of BZDs, as well as their procurement strategies. Mayring’s qualitative content analysis was used to evaluate our data.

Results

In this sample, all participants had developed explanatory models for why they began using BZDs. We identified a multitude of reasons that we grouped into four broad categories, as explaining continued BZD use: (1) to cope with symptoms of psychological distress or mental disorder other than substance use, (2) to manage symptoms of physical or psychological discomfort associated with somatic disorder, (3) to alleviate symptoms of substance-related disorders, and (4) for recreational purposes, that is, sensation-seeking and other social reasons. Subjects often considered BZDs less dangerous than other substances and associated their use more often with harm reduction than as recreational. Specific obtainment strategies varied widely: the majority of participants oscillated between legal and illegal methods, often relying on the black market when faced with treatment termination.

Conclusions

Irrespective of comorbidity, participants expressed a clear preference for medically related explanatory models for their BZD use. We therefore suggest that clinicians consider patients’ motives for long-term, high-dose BZD use when formulating treatment plans for this patient group, especially since it is known that individuals are more compliant with approaches they perceive to be manageable, tolerable, and effective.