The STING agonist, DMXAA, reduces tumor vessels and enhances mesothelioma tumor antigen presentation yet blunts cytotoxic T cell function in a murine model.

We assessed the murine Stimulator of Interferon Genes (STING) agonist, DMXAA, for anti-mesothelioma potential using the AE17-sOVA model that expresses ovalbumin (OVA) as a neo tumor antigen. Dose response experiments alongside testing different routes of administration identified a safe effective treatment regimen that induced 100% cures in mice with small or large tumors. Three doses of 25mg/kg DMXAA given intra-tumorally every 9 days induced tumor regression and long-term survival (>5 months). Re-challenge experiments showed that tumor-free mice developed protective memory. MTT and propidium-iodide assays showed that DMXAA exerted direct cytotoxic effects at doses >1mg/ml on the murine AE17 and AB1 mesothelioma cell lines. In-vivo studies using a CFSE-based in-vivo proliferation assay showed that DMXAA improved tumor-antigen presentation in tumor-draining lymph nodes, evidenced by OVA-specific OT-1 T cells undergoing more divisions. An in-vivo cytotoxic T lymphocyte (CTL) assay showed that DMXAA blunted the lytic quality of CTLs recognizing the dominant (SIINFEKL) and a subdominant (KVVRFDKL) OVA epitopes. DMXAA reduced tumor vessel size in-vivo and although the proportion of T cells infiltrating tumors reduced, the proportion of tumor-specific T cells increased. These data show careful dosing and treatment protocols reduce mesothelioma cell viability and modulate tumor vessels such that tumor-antigen specific CTLs access the tumor site. However, attempts to enhance DMXAA-induced anti-tumor responses by combination with an agonist anti-CD40 antibody or IL-2 reduced efficacy. These proof-of-concept data suggest that mesothelioma patients could benefit from treatment with a STING agonist, but combination with immunotherapy should be cautiously undertaken.

An international multidisciplinary consensus statement on the prevention of opioid-related harm in adult surgical patients.

This international multidisciplinary consensus statement was developed to provide balanced guidance on the safe peri-operative use of opioids in adults. An international panel of healthcare professionals evaluated the literature relating to postoperative opioid-related harm, including persistent postoperative opioid use; opioid-induced ventilatory impairment; non-medical opioid use; opioid diversion and dependence; and driving under the influence of prescription opioids. Recommended strategies to reduce harm include pre-operative assessment of the risk of persistent postoperative opioid use; use of an assessment of patient function rather than unidimensional pain scores alone to guide adequacy of analgesia; avoidance of long-acting (modified-release and transdermal patches) opioid formulations and combination analgesics; limiting the number of tablets prescribed at discharge; providing deprescribing advice; avoidance of automatic prescription refills; safe disposal of unused medicines; reducing the risk of opioid diversion; and better education of healthcare professionals, patients and carers. This consensus statement provides a framework for better prescribing practices that could help reduce the risk of postoperative opioid-related harm in adults.

Guidelines for Perioperative Care in Elective Colorectal Surgery: Enhanced Recovery After Surgery (ERAS®) Society Recommendations: 2018.

BACKGROUND: This is the fourth updated Enhanced Recovery After Surgery (ERAS®) Society guideline presenting a consensus for optimal perioperative care in colorectal surgery and providing graded recommendations for each ERAS item within the ERAS® protocol. METHODS: A wide database search on English literature publications was performed. Studies on each item within the protocol were selected with particular attention paid to meta-analyses, randomised controlled trials and large prospective cohorts and examined, reviewed and graded according to Grading of Recommendations, Assessment, Development and Evaluation (GRADE) system. RESULTS: All recommendations on ERAS® protocol items are based on best available evidence; good-quality trials; meta-analyses of good-quality trials; or large cohort studies. The level of evidence for the use of each item is presented accordingly. CONCLUSIONS: The evidence base and recommendation for items within the multimodal perioperative care pathway are presented by the ERAS® Society in this comprehensive consensus review.

Elderly dendritic cells respond to LPS/IFN-γ and CD40L stimulation despite incomplete maturation.

There is evidence that dendritic cells (DCs) undergo age-related changes that modulate their function with their key role being priming antigen-specific effector T cells. This occurs once DCs develop into antigen-presenting cells in response to stimuli/danger signals. However, the effects of aging on DC responses to bacterial lipopolysaccharide (LPS), the pro-inflammatory cytokine interferon (IFN)-γ and CD40 ligand (CD40L) have not yet been systematically evaluated. We examined responses of blood myeloid (m)DC1s, mDC2s, plasmacytoid (p)DCs, and monocyte-derived DCs (MoDCs) from young (21-40 years) and elderly (60-84 years) healthy human volunteers to LPS/IFN-γ or CD40L stimulation. All elderly DC subsets demonstrated comparable up-regulation of co-stimulatory molecules (CD40, CD80 and/or CD86), intracellular pro-inflammatory cytokine levels (IFN-γ, tumour necrosis factor (TNF)-α, IL-6 and/or IL-12), and/or secreted cytokine levels (IFN-α, IFN-γ, TNF-α, and IL-12) to their younger counterparts. Furthermore, elderly-derived LPS/IFN-γ or CD40L-activated MoDCs induced similar or increased levels of CD8+ and CD4+ T cell proliferation, and similar T cell functional phenotypes, to their younger counterparts. However, elderly LPS/IFN-γ-activated MoDCs were unreliable in their ability to up-regulate chemokine (IL-8 and monocyte chemoattractant protein (MCP)-1) and IL-6 secretion, implying an inability to dependably induce an inflammatory response. A key age-related difference was that, unlike young-derived MoDCs that completely lost their ability to process antigen, elderly-derived MoDCs maintained their antigen processing ability after LPS/IFN-γ maturation, measured using the DQ-ovalbumin assay; this response implies incomplete maturation that may enable elderly DCs to continuously present antigen. These differences may impact on the efficacy of anti-pathogen and anti-tumour immune responses in the elderly.

Human mesothelioma induces defects in dendritic cell numbers and antigen-processing function which predict survival outcomes.

Mesothelioma is an almost invariably fatal tumor with chemotherapy extending survival by a few months. One immunotherapeutic strategy is to target dendritic cells (DCs), key antigen-presenting cells involved in antigen presentation, to induce antigen-specific T cell responses. However, DC-targeting will only be effective if DCs are fit-for-purpose, and the functional status of DCs in mesothelioma patients was not clear. We found that mesothelioma patients have significantly decreased numbers of circulating myeloid (m)DC1 cells, mDC2 cells and plasmacytoid (p)DCs relative to healthy age and gender-matched controls. Blood monocytes from patients could not differentiate into immature monocyte-derived DCs (MoDCs), indicated by a significantly reduced ability to process antigen and reduced expression of costimulatory (CD40, CD80 and CD86) and MHC (HLA-DR) molecules, relative to controls. Activation of mesothelioma-derived MoDCs with LPS+/-IFNγ generated partially mature MoDCs, evident by limited upregulation of the maturation marker, CD83, and the costimulatory markers. Attempts to rescue mesothelioma-derived DC function using CD40Ligand(L) also failed, indicated by maintenance of antigen-processing capacity and limited upregulation of CD40, CD83, CD86 and HLA-DR. These data suggest that mesothelioma patients have significant numerical and functional DC defects and that their reduced capacity to process antigen and reduced expression of costimulatory molecules could induce anergized/tolerized T cells. Nonetheless, survival analyses revealed that individuals with mesothelioma and higher than median levels of mDC1s and/or whose MoDCs matured in response to LPS, IFNγ or CD40L lived longer, implying their selection for DC-targeting therapy could be promising especially if combined with another treatment modality.

Tissue damage negatively regulates LPS-induced macrophage necroptosis.

Infection is a common clinical complication following tissue damage resulting from surgery and severe trauma. Studies have suggested that cell pre-activation by antecedent trauma/tissue damage profoundly impacts the response of innate immune cells to a secondary infectious stimulus. Cell necroptosis, a form of regulated inflammatory cell death, is one of the mechanisms that control cell release of inflammatory mediators from important innate immune executive cells such as macrophages (Mφ), which critically regulate the progress of inflammation. In this study, we investigated the mechanism and role of trauma/tissue damage in the regulation of LPS-induced Mφ necroptosis using a mouse model simulating long-bone fracture. We demonstrate that LPS acting through Toll-like receptor (TLR) 4 promotes Mφ necroptosis. However, necroptosis is ameliorated by high-mobility group box 1 (HMGB1) release from damaged tissue. We show that HMGB1 acting through cell surface receptor for advanced glycation end products (RAGE) upregulates caveolin-1 expression, which in turn induces caveolae-mediated TLR4 internalization and desensitization to decrease Mφ necroptosis. We further show that RAGE-MyD88 activation of Cdc42 and subsequent activation of transcription factor Sp1 serves as a mechanism underlying caveolin-1 transcriptional upregulation. These results reveal a previous unidentified protective role of damage-associated molecular pattern (DAMP) molecules in restricting inflammation in response to exogenous pathogen-associated molecular pattern molecules.

Enhanced Recovery After Surgery (ERAS) for gastrointestinal surgery, part 2: consensus statement for anaesthesia practice.

BACKGROUND: The present interdisciplinary consensus review proposes clinical considerations and recommendations for anaesthetic practice in patients undergoing gastrointestinal surgery with an Enhanced Recovery after Surgery (ERAS) programme. METHODS: Studies were selected with particular attention being paid to meta-analyses, randomized controlled trials and large prospective cohort studies. For each item of the perioperative treatment pathway, available English-language literature was examined and reviewed. The group reached a consensus recommendation after critical appraisal of the literature. RESULTS: This consensus statement demonstrates that anaesthesiologists control several preoperative, intraoperative and postoperative ERAS elements. Further research is needed to verify the strength of these recommendations. CONCLUSIONS: Based on the evidence available for each element of perioperative care pathways, the Enhanced Recovery After Surgery (ERAS®) Society presents a comprehensive consensus review, clinical considerations and recommendations for anaesthesia care in patients undergoing gastrointestinal surgery within an ERAS programme. This unified protocol facilitates involvement of anaesthesiologists in the implementation of the ERAS programmes and allows for comparison between centres and it eventually might facilitate the design of multi-institutional prospective and adequately powered randomized trials.

Enhanced Recovery After Surgery (ERAS) for gastrointestinal surgery, part 1: pathophysiological considerations.

BACKGROUND: The present article has been written to convey concepts of anaesthetic care within the context of an Enhanced Recovery After Surgery (ERAS) programme, thus aligning the practice of anaesthesia with the care delivered by the surgical team before, during and after surgery. METHODS: The physiological principles supporting the implementation of the ERAS programmes in patients undergoing major abdominal procedures are reviewed using an updated literature search and discussed by a multidisciplinary group composed of anaesthesiologists and surgeons with the aim to improve perioperative care. RESULTS: The pathophysiology of some key perioperative elements disturbing the homoeostatic mechanisms such as insulin resistance, ileus and pain is here discussed. CONCLUSIONS: Evidence-based strategies aimed at controlling the disruption of homoeostasis need to be evaluated in the context of ERAS programmes. Anaesthesiologists could, therefore, play a crucial role in facilitating the recovery process.

Randomized clinical trial investigating the stress response from two different methods of analgesia after laparoscopic colorectal surgery.

BACKGROUND: One of the key elements of managed recovery is thought to be suppression of the neuroendocrine response using regional analgesics. This may be superfluous in laparoscopic colorectal surgery with small wounds. This trial assessed the effects of spinal analgesia versus intravenous patient-controlled analgesia (PCA) on neuroendocrine responses in that setting. METHODS: A randomized clinical trial was conducted with participation of patients undergoing laparoscopic colorectal surgery within a managed recovery programme. Consenting patients were allocated randomly to spinal analgesia or morphine PCA as primary postoperative analgesia. The primary outcome was interleukin (IL) 6 levels; secondary outcomes were levels of cortisol, glucose, insulin and other cytokines, pain scores, morphine use and length of hospital stay. Stress response analysis was conducted before operation, and 3, 6, 12, 24 and 48 h after surgery. RESULTS: Of 143 eligible patients, 133 were randomized and 120 completed the study. Baseline patient characteristics were similar in the two groups. There were no significant differences in median levels of insulin, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, interferon γ, tumour necrosis factor α or vascular endothelial growth factor between the spinal analgesia and PCA groups at any time point. Three hours after surgery (but at no other time point) median (i.q.r.) levels of cortisol (468 (329-678) versus 701 (429-820) nmol/l; P = 0.004) and glucose (6.1 (5.4-7.5) versus 7.0 (6.0-7.7) mmol/l; P = 0.012) were lower in the spinal analgesia group than in the PCA group. Median (i.q.r.) levels of total intravenous morphine were lower in the spinal analgesia group (10.0 (3.3-15.8) versus 45.5 (34.0-60.5) mg; P < 0.001). CONCLUSION: Spinal analgesia reduced early neuroendocrine responses and overall parenteral morphine use. REGISTRATION NUMBER: NCT01128088 (http://www.clinicaltrials.gov).

Functional characterization of calliphorid cell death genes and cellularization gene promoters for controlling gene expression and cell viability in early embryos.

The New World screwworm fly, Cochliomyia hominivorax, and the Australian sheep blow fly, Lucilia cuprina, are major pests of livestock. The sterile insect technique was used to eradicate C. hominivorax from North and Central America. This involved area-wide releases of male and female flies that had been sterilized by radiation. Genetic systems have been developed for making 'male-only' strains that would improve the efficiency of genetic control of insect pests. One system involves induction of female lethality in embryos through activation of a pro-apoptotic gene by the tetracycline-dependent transactivator. Sex-specific expression is achieved using an intron from the transformer gene, which we previously isolated from several calliphorids. In the present study, we report the isolation of the promoters from the C. hominivorax slam and Lucilia sericata bnk cellularization genes and show that these promoters can drive expression of a GFP reporter gene in early embryos of transgenic L. cuprina. Additionally, we report the isolation of the L. sericata pro-apoptotic hid and rpr genes, identify conserved motifs in the encoded proteins and determine the relative expression of these genes at different stages of development. We show that widespread expression of the L. sericata pro-apoptotic genes was lethal in Drosophila melanogaster. The isolated gene promoters and pro-apoptotic genes could potentially be used to build transgenic embryonic sexing strains of calliphorid livestock pests.

Control of the sheep blowfly in Australia and New Zealand--are we there yet?

The last 50 years of research into infections in Australia and New Zealand caused by larvae of the sheep blowfly, Lucilia cuprina, have significantly advanced our understanding of this blowfly and its primary host, the sheep. However, apart from some highly effective drugs it could be argued that no new control methodologies have resulted. This review addresses the major areas of sheep blowfly research over this period describing the significant outcomes and analyses, and what is still required to produce new commercial control technologies. The use of drugs against this fly species has been very successful but resistance has developed to almost all current compounds. Integrated pest management is becoming basic to control, especially in the absence of mulesing, and has clearly benefited from computer-aided technologies. Biological control has more challenges but natural and perhaps transformed biopesticides offer possibilities for the future. Experimental vaccines have been developed but require further analysis of antigens and formulations to boost protection. Genetic technologies may provide potential for long-term control through more rapid indirect selection of sheep less prone to flystrike. Finally in the future, genetic analysis of the fly may allow suppression and perhaps eradication of blowfly populations or identification of new and more viable targets for drug and vaccine intervention. Clearly all these areas of research offer potential new controls but commercial development is perhaps inhibited by the success of current chemical insecticides and certainly requires a significant additional injection of resources.

Macrophage endocytosis of high-mobility group box 1 triggers pyroptosis.

Macrophages can be activated and regulated by high-mobility group box 1 (HMGB1), a highly conserved nuclear protein. Inflammatory functions of HMGB1 are mediated by binding to cell surface receptors, including the receptor for advanced glycation end products (RAGE), Toll-like receptor (TLR)2, TLR4, and TLR9. Pyroptosis is a caspase-1-dependent programmed cell death, which features rapid plasma membrane rupture, DNA fragmentation, and release of proinflammatory intracellular contents. Pyroptosis can be triggered by various stimuli, however, the mechanism underlying pyroptosis remains unclear. In this study, we identify a novel pathway of HMGB1-induced macrophage pyroptosis. We demonstrate that HMGB1, acting through RAGE and dynamin-dependent signaling, initiates HMGB1endocytosis, which in turn induces cell pyroptosis. The endocytosis of HMGB1 triggers a cascade of molecular events, including cathepsin B release from ruptured lysosomes followed by pyroptosome formation and caspase-1 activation. We further confirm that HMGB1-induced macrophage pyroptosis also occurs in vivo during endotoxemia, suggesting a pathophysiological significance for this form of pyroptosis in the development of inflammation. These findings shed light on the regulatory role of ligand-receptor internalization in directing cell fate, which may have an important role in the progress of inflammation following infection and injury.

Sex determination mechanisms in the Calliphoridae (blow flies).

The Calliphoridae or blow flies are a family of insects that occupy diverse habitats and perform important ecological roles, particularly the decomposition of animal remains. Some Calliphoridae species are also important in the forensic sciences, in agriculture (e.g. as livestock pests) and in medicine (e.g. maggot therapy). Calliphoridae provide striking examples in support of the hypothesis that sex determination regulatory gene hierarchies evolve in the reverse order, with the gene at the top being the most recently added. Unlike the model fly Drosophila melanogaster, where sex is determined by the number of X chromosomes, in the Australian sheep blow fly (Lucilia cuprina) sex is determined by a Y-linked male-determining gene (M). A different regulatory system appears to operate in the hairy maggot blow fly (Chrysomya rufifacies) where the maternal genotype determines sex. It is hypothesized that females heterozygous for a dominant female-determining factor (F/f) produce only female offspring and homozygous f/f females produce only sons. The bottom of the regulatory hierarchy appears to be the same in D. melanogaster and L. cuprina, with sex-specific splicing of doublesex transcripts being controlled by the female-specific Transformer (TRA) protein. We discuss a model that has been proposed for how tra transcripts are sex-specifically spliced in calliphorids, which is very different from D. melanogaster.