Arachidonate metabolite(s) increase the permeability of the plasma membrane of the neutrophils to calcium

The relationship between arachidonate metabolism and stimulated calcium fluxes in rabbit peritoneal neutrophils has been investigated. The addition of arachidonate to the neutrophils was found to cause a rapid and significant increase in the permeability of the plasma membrane to calcium. This effect is specific to calcium, concentration dependent and sensitive to inhibitors of the lipoxygenase mediated metabolic pathway(s). These results strongly suggest that arachidonate metabolites are directly involved in the mechanisms underlying calcium gating in the neutrophils.     

Forced IDO1 expression in dendritic cells restores immunoregulatory signalling in autoimmune diabetes

Indoleamine 2,3‐dioxygenase (IDO1), a tryptophan catabolizing enzyme, is recognized as an authentic regulator of immunity in several physiopathologic conditions. We have recently demonstrated that IDO1 does not merely degrade tryptophan and produce immunoregulatory kynurenines, but it also acts as a signal‐transducing molecule, independently of its enzymic function. IDO1 signalling activity is triggered in plasmacytoid dendritic cells (pDCs) by transforming growth factor‐β (TGF‐β), an event that requires the non‐canonical NF‐κB pathway and induces long‐lasting IDO1 expression and autocrine TGF‐β production in a positive feedback loop, thus sustaining a stably regulatory phenotype in pDCs. IDO1 expression and catalytic function are defective in pDCs from non‐obese diabetic (NOD) mice, a prototypic model of autoimmune diabetes. In the present study, we found that TGF‐β failed to activate IDO1 signalling function as well as up‐regulate IDO1 expression in NOD pDCs. Moreover, TGF‐β‐treated pDCs failed to exert immunosuppressive properties in vivo. Nevertheless, transfection of NOD pDCs with Ido1 prior to TGF‐β treatment resulted in activation of the Ido1 promoter and induction of non‐canonical NF‐κB and TGF‐β, as well as decreased production of the pro‐inflammatory cytokines, interleukin 6 (IL‐6) and tumour necrosis factor‐α (TNF‐α). Overexpression of IDO1 in TGF‐β‐treated NOD pDCs also resulted in pDC ability to suppress the in vivo presentation of a pancreatic β‐cell auto‐antigen. Thus, our data suggest that a correction of IDO1 expression may restore its dual function and thus represent a proper therapeutic manoeuvre in this autoimmune setting.     

The ectopic expression of a pectin methyl esterase inhibitor increases pectin methyl esterification and limits fungal diseases in wheat

Cell wall pectin methyl esterification can influence plant resistance because highly methyl-esterified pectin can be less susceptible to the hydrolysis by pectic enzymes such as fungal endopolygalacturonases (PG). Pectin is secreted into the cell wall in a highly methyl-esterified form and, here, is de-methyl esterified by pectin methyl esterase (PME). The activity of PME is controlled by specific protein inhibitors called PMEI; consequently, an increased inhibition of PME by PMEI might modify the pectin methyl esterification. In order to test the possibility of improving wheat resistance by modifying the methyl esterification of pectin cell wall, we have produced durum wheat transgenic lines expressing the PMEI from Actinidia chinensis (AcPMEI). The expression of AcPMEI endows wheat with a reduced endogenous PME activity, and transgenic lines expressing a high level of the inhibitor showed a significant increase in the degree of methyl esterification. These lines showed a significant reduction of disease symptoms caused by the fungal pathogens Bipolaris sorokiniana or Fusarium graminearum. This increased resistance was related to the impaired ability of these fungal pathogens to grow on methyl-esterified pectin and to a reduced activity of the fungal PG to hydrolyze methyl-esterified pectin. In addition to their importance for wheat improvement, these results highlight the primary role of pectin despite its low content in the wheat cell wall.     

HP2-like protein: a new piece of the facultative heterochromatin puzzle

In Drosophila melanogaster, the two chromosomal proteins HP1 and HP2 colocalize on heterochromatic and euchromatic sites in polytene chromosomes. Mutations in the HP2 gene act as dominant suppressors of position effect variegation, demonstrating a role for HP2 in the formation or maintenance of heterochromatin. In this paper, we investigated whether a putative homolog of the D. melanogaster HP2 is involved in the facultative heterochromatinization process in mealybugs. Using an antibody raised against the Drosophila HP2, we identified in the mealybug Planococcus citri a cross-reactive epitope, which we refer to as HP2-like. We investigated the HP2-like pattern during the male embryo development where the entire paternal haploid chromosome set becomes heterochromatic. The HP2 antibody heavily decorates the chromocenters, where it localizes with HP1, and marks the chromatin before it acquires the full cytological characteristics of the male-specific heterochromatin. In euchromatic chromosomes, HP2-like is mainly concentrated at telomeric sites. The interplay between HP2-like and HP1-like was studied by dsRNA interference experiments. Extinguishing HP1-like expression by RNAi does not prevent the association of HP2-like with facultative heterochromatin, implying that HP2-like binds to chromatin in a HP1-independent manner. Our results confirm and extend the structural and functional conservation of proteins involved in heterochromatin assembly. Silvia Volpi and Silvia Bongiorni contributed equally to the work.     

Reverse signaling through GITR ligand enables dexamethasone to activate IDO in allergy

Glucocorticoid-induced tumor necrosis factor receptor (GITR) on T cells and its natural ligand, GITRL, on accessory cells contribute to the control of immune homeostasis. Here we show that reverse signaling through GITRL after engagement by soluble GITR initiates the immunoregulatory pathway of tryptophan catabolism in mouse plasmacytoid dendritic cells, by means of noncanonical NF-kappaB-dependent induction of indoleamine 2,3-dioxygenase (IDO). The synthetic glucocorticoid dexamethasone administered in vivo activated IDO through the symmetric induction of GITR in CD4(+) T cells and GITRL in plasmacytoid dendritic cells. The drug exerted IDO-dependent protection in a model of allergic airway inflammation. Modulation of tryptophan catabolism via the GITR-GITRL coreceptor system might represent an effective therapeutic target in immune regulation. Induction of IDO could be an important mechanism underlying the anti-inflammatory action of corticosteroids.     

Systematic Review of the Literature and Evidence-Based Recommendations for Antibiotic Prophylaxis in Trauma: Results from an Italian Consensus of Experts

Background

Antibiotic prophylaxis is frequently administered in severe trauma. However, the risk of selecting resistant bacteria, a major issue especially in critical care environments, has not been sufficiently investigated. The aim of the present study was to provide guidelines for antibiotic prophylaxis for four different trauma-related clinical conditions, taking into account the risks of antibiotic-resistant bacteria selection, thus innovating previous guidelines in the field.

Methods

The MEDLINE database was searched for studies comparing antibiotic prophylaxis to controls (placebo or no antibiotic administration) in four clinical traumatic conditions that were selected on the basis of the traumatic event frequency and/or infection severity. The selected studies focused on the prevention of early ventilator associated pneumonia (VAP) in comatose patients with traumatic brain injury, of meningitis in severe basilar skull fractures, of wound infections in long-bone open fractures. Since no placebo-controlled study was available for deep surgical site-infections prevention in abdominal trauma with enteric contamination, we compared 24-hour and 5-day antibiotic prophylaxis policies. A separate specific research focused on the question of antibiotic-resistant bacteria selection caused by antibiotic prophylaxis, an issue not adequately investigated by the selected studies. Randomised trials, reviews, meta-analyses, observational studies were included. Data extraction was carried out by one author according to a predefined protocol, using an electronic form. The strength of evidence was stratified and recommendations were given according to the Grading of Recommendations Assessment, Development and Evaluation (GRADE) criteria.

Results

Uncertain evidence deserving further studies was found for two-dose antibiotic prophylaxis for early VAP prevention in comatose patients. In the other cases the risk of resistant-bacteria selection caused by antibiotic administration for 48 hours or more, outweighed potential benefits.

Conclusions

When accounting for antibiotic-resistant bacteria selection we found no evidence in favour of antibiotic prophylaxis lasting two or more days in the studied clinical conditions.     

Reactive hyperemia is not responsible for stimulating muscle protein synthesis following blood flow restriction exercise

Blood flow restriction (BFR) to contracting skeletal muscle during low-intensity resistance exercise training increases muscle strength and size in humans. However, the mechanism(s) underlying these effects are largely unknown. We have previously shown that mammalian target of rapamycin complex 1 (mTORC1) signaling and muscle protein synthesis (MPS) are stimulated following an acute bout of BFR exercise. The purpose of this study was to test the hypothesis that reactive hyperemia is the mechanism responsible for stimulating mTORC1 signaling and MPS following BFR exercise. Six young men (24 ± 2 yr) were used in a randomized crossover study consisting of two exercise trials: low-intensity resistance exercise with BFR (BFR trial) and low-intensity resistance exercise with sodium nitroprusside (SNP), a pharmacological vasodilator infusion into the femoral artery immediately after exercise to simulate the reactive hyperemia response after BFR exercise (SNP trial). Postexercise mixed-muscle fractional synthetic rate from the vastus lateralis increased by 49% in the BFR trial (P < 0.05) with no change in the SNP trial (P > 0.05). BFR exercise increased the phosphorylation of mTOR, S6 kinase 1, ribosomal protein S6, ERK1/2, and Mnk1-interacting kinase 1 (P < 0.05) with no changes in mTORC1 signaling in the SNP trial (P > 0.05). We conclude that reactive hyperemia is not a primary mechanism for BFR exercise-induced mTORC1 signaling and MPS. Further research is necessary to elucidate the cellular mechanism(s) responsible for the increase in mTOR signaling, MPS, and hypertrophy following acute and chronic BFR exercise.     

Cutting edge: Autocrine TGF-beta sustains default tolerogenesis by IDO-competent dendritic cells

CD8(-) and CD8(+) dendritic cells (DCs) are distinct subsets of mouse splenic accessory cells with opposite but flexible programs of Ag presentation, leading to immunogenic and tolerogenic responses, respectively. In this study, we show that the default tolerogenic function of CD8(+) DCs relies on autocrine TGF-beta, which sustains the activation of IDO in response to environmental stimuli. CD8(-) DCs do not produce TGF-beta, yet externally added TGF-beta induces IDO and turns those cells from immunogenic into tolerogenic cells. The acquisition of a suppressive phenotype by CD8(-) DCs correlates with activation of the PI3K/Akt and noncanonical NF-kappaB pathways. These data are the first to link TGF-beta signaling with IDO in controlling spontaneous tolerogenesis by DCs.     

Sequential muscle biopsies during a 6-h tracer infusion do not affect human mixed muscle protein synthesis and muscle phenylalanine kinetics

Stable isotope tracer experiments of human muscle amino acid and protein kinetics often involve a sequential design, with the same subject studied at baseline and during an intervention. However, prolonged fasting and sequential muscle biopsies from the same area could theoretically affect muscle protein metabolism. The purpose of this study was to determine if sequential muscle biopsies and extended fasting significantly affect parameters of muscle protein and amino acid kinetics in six human subjects. After a 12-h overnight fast, a primed continuous infusion of L-[ring-(2)H(5)]phenylalanine was started. After 120 min, we took the first of a series of five hourly muscle biopsies from the same vastus lateralis to measure mixed muscle protein fractional synthetic rate. Furthermore, between 150-180, 210-240, and 330-360 min, we measured leg phenylalanine kinetics using the two-pool and the three-pool arteriovenous balance models. Tracer enrichments were at steady state, and muscle protein FSR and phenylalanine kinetics did not change throughout the experiment (P=not significant). We conclude that a 6-h tracer infusion during extended fasting (up to 18 h) with five sequential muscle biopsies from the same muscle do not affect basal mixed muscle protein synthesis and muscle phenylalanine kinetics in human subjects. Thus, when using a sequential study design over this period of time, it is unnecessary to include a saline only control group to account for these variables.     

A Cdc7 kinase inhibitor restricts initiation of DNA replication and has antitumor activity

Cdc7 is an essential kinase that promotes DNA replication by activating origins of replication. Here, we characterized the potent Cdc7 inhibitor PHA-767491 (1) in biochemical and cell-based assays, and we tested its antitumor activity in rodents. We found that the compound blocks DNA synthesis and affects the phosphorylation of the replicative DNA helicase at Cdc7-dependent phosphorylation sites. Unlike current DNA synthesis inhibitors, PHA-767491 prevents the activation of replication origins but does not impede replication fork progression, and it does not trigger a sustained DNA damage response. Treatment with PHA-767491 results in apoptotic cell death in multiple cancer cell types and tumor growth inhibition in preclinical cancer models. To our knowledge, PHA-767491 is the first molecule that directly affects the mechanisms controlling initiation as opposed to elongation in DNA replication, and its activities suggest that Cdc7 kinase inhibition could be a new strategy for the development of anticancer therapeutics.