Human neutrophil clearance of bacterial pathogens triggers anti-microbial gamma delta T cell responses in early infection

Human blood Vc9/Vd2 T cells, monocytes and neutrophils share a responsiveness toward inflammatory chemokines and are rapidly recruited to sites of infection. Studying their interaction in vitro and relating these findings to in vivo observations in patients may therefore provide crucial insight into inflammatory events. Our present data demonstrate that Vc9/Vd2 T cells provide potent survival signals resulting in neutrophil activation and the release of the neutrophil chemoattractant CXCL8 (IL-8). In turn, Vc9/Vd2 T cells readily respond to neutrophils harboring phagocytosed bacteria, as evidenced by expression of CD69, interferon (IFN)-c and tumor necrosis factor (TNF)-a. This response is dependent on the ability of these bacteria to produce the microbial metabolite (E)-4-hydroxy-3-methyl-but-2-enyl pyrophosphate (HMB-PP), requires cell-cell contact of Vc9/Vd2 T cells with accessory monocytes through lymphocyte function-associated antigen-1 (LFA-1), and results in a TNF-a dependent proliferation of Vc9/Vd2 T cells. The antibiotic fosmidomycin, which targets the HMB-PP biosynthesis pathway, not only has a direct antibacterial effect on most HMB-PP producing bacteria but also possesses rapid anti-inflammatory properties by inhibiting cd T cell responses in vitro. Patients with acute peritoneal-dialysis (PD)-associated bacterial peritonitis – characterized by an excessive influx of neutrophils and monocytes into the peritoneal cavity – show a selective activation of local Vc9/Vd2 T cells by HMB-PP producing but not by HMB-PP deficient bacterial pathogens. The cd T celldriven perpetuation of inflammatory responses during acute peritonitis is associated with elevated peritoneal levels of cd T cells and TNF-a and detrimental clinical outcomes in infections caused by HMB-PP positive microorganisms. Taken together, our findings indicate a direct link between invading pathogens, neutrophils, monocytes and microbe-responsive cd T cells in early infection and suggest novel diagnostic and therapeutic approaches.Martin S. Davey, Chan-Yu Lin, Gareth W. Roberts, Sinéad Heuston, Amanda C. Brown, James A. Chess, Mark A. Toleman, Cormac G.M. Gahan, Colin Hill, Tanya Parish, John D. Williams, Simon J. Davies, David W. Johnson, Nicholas Topley, Bernhard Moser and Matthias Eber

Developing evidence-based clinical practice guidelines in hospitals in Australia, Indonesia, Malaysia, the Philippines and Thailand: values, requirements and barriers

<p>Abstract</p> <p>Background</p> <p>Evidence-based clinical practice guidelines support clinical decision-making by making recommendations to guide clinical practice. These recommendations are developed by integrating the expertise of a multidisciplinary group of clinicians with the perspectives of consumers and the best available research evidence. However studies have raised concerns about the quality of guideline development, and particularly the link between research and recommendations. The reasons why guideline developers are not following the established development methods are not clear.</p> <p>We aimed to explore the barriers to developing evidence-based guidelines in eleven hospitals in Australia, Indonesia, Malaysia, the Philippines and Thailand, so as to better understand how evidence-based guideline development could be facilitated in these settings. The research aimed to identify the value clinicians place on guidelines, what clinicians want in guidelines developed in hospital settings and what factors limit rigorous evidence-based guideline development in these settings.</p> <p>Methods</p> <p>Semi-structured, face-to-face interviews were undertaken with senior and junior healthcare providers (nurses, midwives, doctors, allied health) from the maternal and neonatal services of the eleven participating hospitals. Interviews were audio-recorded, transcribed and a thematic analysis undertaken.</p> <p>Results</p> <p>Ninety-three individual, 25 pair and eleven group interviews were conducted. Participants were clear that they want guidelines that are based on evidence and updated regularly. They were also clear that there are major barriers to this. Most of the barriers were shared across countries, and included lack of time, lack of skills in finding, appraising and interpreting evidence, lack of access to relevant evidence and difficulty arranging meetings and achieving consensus.</p> <p>Barriers that were primarily identified in Australian hospitals include cumbersome organisational processes and a feeling that guidelines are being developed for bureaucratic ends. Barriers that were primarily identified in South East Asian hospitals include difficulty accessing evidence due to limited resources available for computers, internet and journal subscriptions and limited skills in computing and English.</p> <p>Conclusions</p> <p>The clinicians in these eleven very different hospitals want evidence-based guidelines. However they are frustrated by guideline development processes that are enormously time, skill and resource intensive. They feel strongly that "there's got to be a better way".</p> <p>The fact that the great majority of the identified barriers were shared across settings may provide an opportunity to develop a more pragmatic way of developing guidelines that can be applied in many contexts.</p

Oral Thromboprophylaxis Following Total Hip or Knee Replacement: Review and Multicentre Experience with Dabigatran Etexilate

The risk of venous thromboembolism (VTE) in patients undergoing total knee or hip replacement surgery is high. As a result, thromboprophylaxis is highly recommended. While current thromboprophylactic agents, such as low molecular weight heparins (LMWH) and vitamin K antagonists, are safe and effective their use can be problematic. Therefore, there is a need for alternative anticoagulants that are as safe and effective as conventional agents, but are more convenient and easier to use. Dabigatran etexilate, a direct thrombin inhibitor, is one such anticoagulant. For VTE prevention following major orthopaedic surgery, dabigatran etexilate shows similar efficacy and safety to the LMWH enoxaparin, and is approved for use in more than 75 countries, including Europe and Canada. Here, we summarize and discuss the experiences of four German clinics that have recently introduced dabigatran etexilate into clinical practice. Overall, dabigatran etexilate was well received by patients, surgeons and nurses, and compared favourably with enoxaparin. Staff appreciated the oral, single-dose administration of dabigatran etexilate. Patient satisfaction was high, especially in those individuals who had previously used LMWHs. In this review, we also address a number of questions that were asked by patients or staff; this will be of relevance to orthopaedic surgeons and nurses. We conclude that, in these four German clinics, dabigatran etexilate offered an effective oral alternative to existing thromboprophylactic agents in patients undergoing major orthopaedic surgery

Nitric oxide production and antioxidant function during viral infection of the coccolithophore Emiliania huxleyi

Emiliania huxleyi is a globally important marine phytoplankton that is routinely infected by viruses. Understanding the controls on the growth and demise of E. huxleyi blooms is essential for predicting the biogeochemical fate of their organic carbon and nutrients. In this study, we show that the production of nitric oxide (NO), a gaseous, membrane-permeable free radical, is a hallmark of early-stage lytic infection in E. huxleyi by Coccolithoviruses, both in culture and in natural populations in the North Atlantic. Enhanced NO production was detected both intra- and extra-cellularly in laboratory cultures, and treatment of cells with an NO scavenger significantly reduced viral production. Pre-treatment of exponentially growing E. huxleyi cultures with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) prior to challenge with hydrogen peroxide (H2O2) led to greater cell survival, suggesting that NO may have a cellular antioxidant function. Indeed, cell lysates generated from cultures treated with SNAP and undergoing infection displayed enhanced ability to detoxify H2O2. Lastly, we show that fluorescent indicators of cellular ROS, NO, and death, in combination with classic DNA- and lipid-based biomarkers of infection, can function as real-time diagnostic tools to identify and contextualize viral infection in natural E. huxleyi blooms

Application of a Mathematical Model to Describe the Effects of Chlorpyrifos on Caenorhabditis elegans Development

The nematode Caenorhabditis elegans is being assessed as an alternative model organism as part of an interagency effort to develop better means to test potentially toxic substances. As part of this effort, assays that use the COPAS Biosort flow sorting technology to record optical measurements (time of flight (TOF) and extinction (EXT)) of individual nematodes under various chemical exposure conditions are being developed. A mathematical model has been created that uses Biosort data to quantitatively and qualitatively describe C. elegans growth, and link changes in growth rates to biological events. Chlorpyrifos, an organophosphate pesticide known to cause developmental delays and malformations in mammals, was used as a model toxicant to test the applicability of the growth model for in vivo toxicological testing.L1 larval nematodes were exposed to a range of sub-lethal chlorpyrifos concentrations (0-75 microM) and measured every 12 h. In the absence of toxicant, C. elegans matured from L1s to gravid adults by 60 h. A mathematical model was used to estimate nematode size distributions at various times. Mathematical modeling of the distributions allowed the number of measured nematodes and log(EXT) and log(TOF) growth rates to be estimated. The model revealed three distinct growth phases. The points at which estimated growth rates changed (change points) were constant across the ten chlorpyrifos concentrations. Concentration response curves with respect to several model-estimated quantities (numbers of measured nematodes, mean log(TOF) and log(EXT), growth rates, and time to reach change points) showed a significant decrease in C. elegans growth with increasing chlorpyrifos concentration.Effects of chlorpyrifos on C. elegans growth and development were mathematically modeled. Statistical tests confirmed a significant concentration effect on several model endpoints. This confirmed that chlorpyrifos affects C. elegans development in a concentration dependent manner. The most noticeable effect on growth occurred during early larval stages: L2 and L3. This study supports the utility of the C. elegans growth assay and mathematical modeling in determining the effects of potentially toxic substances in an alternative model organism using high-throughput technologies

Modulatory effects of heparin and short-length oligosaccharides of heparin on the metastasis and growth of LMD MDA-MB 231 breast cancer cells in vivo

Expression of the chemokine receptor CXCR4 allows breast cancer cells to migrate towards specific metastatic target sites which constitutively express CXCL12. In this study, we determined whether this interaction could be disrupted using short-chain length heparin oligosaccharides. Radioligand competition binding assays were performed using a range of heparin oligosaccharides to compete with polymeric heparin or heparan sulphate binding to I125 CXCL12. Heparin dodecasaccharides were found to be the minimal chain length required to efficiently bind CXCL12 (71% inhibition; P<0.001). These oligosaccharides also significantly inhibited CXCL12-induced migration of CXCR4-expressing LMD MDA-MB 231 breast cancer cells. In addition, heparin dodecasaccharides were found to have less anticoagulant activity than either a smaller quantity of polymeric heparin or a similar amount of the low molecular weight heparin pharmaceutical product, Tinzaparin. When given subcutaneously in a SCID mouse model of human breast cancer, heparin dodecasaccharides had no effect on the number of lung metastases, but did however inhibit (P<0.05) tumour growth (lesion area) compared to control groups. In contrast, polymeric heparin significantly inhibited both the number (P<0.001) and area of metastases, suggesting a differing mechanism for the action of polymeric and heparin-derived oligosaccharides in the inhibition of tumour growth and metastases

Periprosthetic fractures of the femur after total knee arthroplasty

Periprosthetic fracture following total knee arthroplasty is a potentially serious complication. This injury can involve the distal femur, proximal tibia or the patella. This review article analyzes the prevalence, risk factors, classification and treatment options for periprosthetic fractures of the femur

SLI-1 Cbl Inhibits the Engulfment of Apoptotic Cells in C. elegans through a Ligase-Independent Function

The engulfment of apoptotic cells is required for normal metazoan development and tissue remodeling. In Caenorhabditis elegans, two parallel and partially redundant conserved pathways act in cell-corpse engulfment. One pathway, which includes the small GTPase CED-10 Rac and the cytoskeletal regulator ABI-1, acts to rearrange the cytoskeleton of the engulfing cell. The CED-10 Rac pathway is also required for proper migration of the distal tip cells (DTCs) during the development of the C. elegans gonad. The second pathway includes the receptor tyrosine kinase CED-1 and might recruit membranes to extend the surface of the engulfing cell. Cbl, the mammalian homolog of the C. elegans E3 ubiquitin ligase and adaptor protein SLI-1, interacts with Rac and Abi2 and modulates the actin cytoskeleton, suggesting it might act in engulfment. Our genetic studies indicate that SLI-1 inhibits apoptotic cell engulfment and DTC migration independently of the CED-10 Rac and CED-1 pathways. We found that the RING finger domain of SLI-1 is not essential to rescue the effects of SLI-1 deletion on cell migration, suggesting that its role in this process is ubiquitin ligase-independent. We propose that SLI-1 opposes the engulfment of apoptotic cells via a previously unidentified pathway.National Cancer Institute (U.S.) (Award K08CA104890