Design and conduct of Xtreme Everest 2: an observational cohort study of Sherpa and lowlander responses to graduated hypobaric hypoxia

Objective: oxygen availability falls with ascent to altitude and also as a consequence of critical illness. Because cellular sequelae and adaptive processes may be shared in both circumstances, high altitude exposure (‘physiological hypoxia’) assists in the exploration of the response to pathological hypoxia. We therefore studied the response of healthy participants to progressive hypobaric hypoxia at altitude. The primary objective of the study was to identify differences between high altitude inhabitants (Sherpas) and lowland comparators.Methods: we performed an observational cohort study of human responses to progressive hypobaric hypoxia (during ascent) and subsequent normoxia (following descent) comparing Sherpas with lowlanders. Studies were conducted in London (35m), Kathmandu (1300m), Namche Bazaar (3500m) and Everest Base Camp (5300m). Of 180 healthy volunteers departing from Kathmandu, 64 were Sherpas and 116 were lowlanders. Physiological, biochemical, genetic and epigenetic data were collected. Core studies focused on nitric oxide metabolism, microcirculatory blood flow and exercise performance. Additional studies performed in nested subgroups examined mitochondrial and metabolic function, and ventilatory and cardiac variables. Of the 180 healthy participants who left Kathmandu, 178 (99%) completed the planned trek. Overall, more than 90% of planned testing was completed. Forty-four study protocols were successfully completed at altitudes up to and including 5300m. A subgroup of identical twins (all lowlanders) was also studied in detail.Conclusion: this programme of study (Xtreme Everest 2) will provide a rich dataset relating to human adaptation to hypoxia, and the responses seen on re-exposure to normoxia. It is the largest comprehensive high altitude study of Sherpas yet performed. Translational data generated from this study will be of relevance to diseases in which oxygenation is a major facto

Toll-like receptor agonists Porphyromonas gingivalis LPS and CpG differentially regulate IL-10 competency and frequencies of mouse B10 cells

IL-10 expressing regulatory B cells (B10) play a key role in immune system balance by limiting excessive inflammatory responses. Effects of toll-like receptor signaling and co-stimulatory molecules on B10 activity during innate and adaptive immune responses are not fully understood. Objective This study is to determine the effects of P. gingivalis LPS and CpG on B10 cell expansion and IL-10 competency in vitro. Material and Methods Spleen B cells were isolated from C57BL/6J mice with or without formalin-fixed P. gingivalis immunization. B cells were cultured for 48 hours under the following conditions: CD40L, CD40L+LPS, CD40L+CpG, and CD40L+LPS+CpG in the presence or absence of fixed P. gingivalis. Percentages of CD1dhiCD5+ B cells were measured by flow cytometry. IL-10 mRNA expression and secreted IL-10 were measured by real-time quantitative PCR and by ELISA respectively. Results P. gingivalis LPS plus CD40L significantly increased CD1dhiCD5+ B cell percentages and secreted IL-10 levels in both immunized and non-immunized mice B cells in the presence or absence of P. gingivalis, compared with control group. Secreted IL-10 levels were significantly increased in CD40L+LPS treated group compared with CD40L treatment group in the absence of P. gingivalis. CpG plus CD40L significantly decreased CD1dhiCD5+ B cell percentages, but greatly elevated secreted IL-10 levels in immunized and non-immunized mice B cells in the absence of P. gingivalis, compared with CD40L treatment group. Conclusions P. gingivalis LPS and CpG differentially enhance IL-10 secretion and expansion of mouse B10 cells during innate and adaptive immune responses

Monitoring Observations of the Jupiter-Family Comet 17P/Holmes during 2014 Perihelion Passage

We performed a monitoring observation of a Jupiter-Family comet, 17P/Holmes, during its 2014 perihelion passage to investigate its secular change in activity. The comet has drawn the attention of astronomers since its historic outburst in 2007, and this occasion was its first perihelion passage since then. We analyzed the obtained data using aperture photometry package and derived the Afrho parameter, a proxy for the dust production rate. We found that Afrho showed asymmetric properties with respect to the perihelion passage: it increased moderately from 100 cm at the heliocentric distance r_h=2.6-3.1 AU to a maximal value of 185 cm at r_h = 2.2 AU (near the perihelion) during the inbound orbit, while dropping rapidly to 35 cm at r_h = 3.2 AU during the outbound orbit. We applied a model for characterizing dust production rates as a function of r_h and found that the fractional active area of the cometary nucleus had dropped from 20%-40% in 2008-2011 (around the aphelion) to 0.1%-0.3% in 2014-2015 (around the perihelion). This result suggests that a dust mantle would have developed rapidly in only one orbital revolution around the sun. Although a minor eruption was observed on UT 2015 January 26 at r_h = 3.0 AU, the areas excavated by the 2007 outburst would be covered with a layer of dust (<~ 10 cm depth) which would be enough to insulate the subsurface ice and to keep the nucleus in a state of low activity.Comment: 25 pages, 6 figures, 2 tables, ApJ accepted on December 29, 201

Knowledge and exposure to complementary and alternative medicine in paediatric doctors: a questionnaire survey

<p>Abstract</p> <p>Background</p> <p>Complementary and alternative medicines are increasingly used by the general population. A survey was conducted to ascertain the knowledge of Complementary and Alternative Medicines (CAMs) amongst paediatric physicians, and whether seniority increases the likelihood of its use being considered in consultations, or of families discussing it.</p> <p>Methods</p> <p>Anonymous survey of general paediatric doctors in a large inner-city district general hospital (DGH) and tertiary children's centre (TC) using a questionnaire. Statistical analysis was calculated using Minitab.</p> <p>Results</p> <p>43/49 (88%) questionnaires were returned correctly. 13 (30%, CI 17 – 46%) doctors had personally used CAMs. 24 (56%, CI 40 – 71%) of their families had used CAMs. 13 (30%, CI 17 – 46%) had received formal CAMs education. 21 (49%, CI 40 – 71%) could name a total of 5 types of CAMs. Consultants were significantly more likely to ask about CAM use than middle-grades and juniors (p < 0.05, CI 48 – 93%, 35 – 90%, 8 – 33% respectively) and have had a clinical encounter where they felt it was significant. 32 (74%, CI 59 – 86%) of the clinicians had been asked about CAMs. 33 (77%, CI 61 – 88%) of doctors had successful CAM use reported to them, and 20 (47%, CI 31 – 62%) had failure of CAMs reported to them.</p> <p>Conclusion</p> <p>CAM use is relatively common in paediatric doctors and their families. They have received little formal CAMs education. Consultants were more likely than juniors to ask about CAM use and have had a clinical encounter where it played a significant part. Around half of all doctors irrespective of grade have been asked about CAMs in a clinical encounter.</p

Mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine are toll-like receptor 4 antagonists, inhibit NF-κB activation, and decrease TNF-alpha secretion in primary microglia

Toll-like receptor 4 (TLR4) recognizes various endogenous and microbial ligands and is an essential part in the innate immune system. TLR4 signaling initiates transcription factor NF-κB and production of proinflammatory cytokines. TLR4 contributes to the development or progression of various diseases including stroke, neuropathic pain, multiple sclerosis, rheumatoid arthritis and cancer, and better therapeutics are currently sought for these conditions. In this study, a library of 140 000 compounds was virtually screened and a resulting hit-list of 1000 compounds was tested using a cellular reporter system. The topoisomerase II inhibitor mitoxantrone and its analogues pixantrone and mitoxantrone (2-hydroxyethyl)piperazine were identified as inhibitors of TLR4 and NF-κB activation. Mitoxantrone was shown to bind directly to the TLR4, and pixantrone and mitoxantrone (2- hydroxyethyl)piperazine were shown to inhibit the production of proinflammatory cytokines such as tumor necrosis factor alpha (TNFα) in primary microglia. The inhibitory effect on NF-κB activation or on TNFα pro-duction was not mediated through cytotoxity at ≤ 1 μM concentration for pixantrone and mitoxantrone (2- hydroxyethyl)piperazine treated cells, as assessed by ATP counts. This study thus identifies a new mechanism of action for mitoxantrone, pixantrone, and mitoxantrone (2-hydroxyethyl)piperazine through the TLR4.Peer reviewe

Naltrexone inhibits IL-6 and TNFa production in human immune cell subsets following stimulation with ligands for intracellular Toll-like receptors

The opioid antagonist naltrexone hydrochloride has been suggested to be a potential therapy at low dosage for multiple inflammatory conditions and cancers. Little is known about the immune-modulating effects of naltrexone, but an effect on the activity of toll-like receptor 4 (TLR4) has been reported. We analyzed the effects of naltrexone hydrochloride on IL-6 secretion by peripheral blood mononuclear cells (PBMC) in vitro following stimulation with ligands for TLR4 and for the intracellular receptors TLR7, TLR8, and TLR9. Naltrexone did not affect cell viability or induce apoptosis of PBMC. Intracellular staining demonstrated that naltrexone inhibited production of IL-6 and TNFα by monocyte and plasmacytoid dendritic cell subsets within the PBMC population following treatment with ligands for TLR7/8 and TLR9, respectively. No effect of cytokine production by PBMC following stimulation of TLR4 was observed. Additionally, naltrexone inhibited IL-6 production in isolated monocytes and B cells after TLR7/8 and TLR9 stimulation, respectively, but no effect on IL-6 production in isolated monocytes after TLR4 stimulation was observed. These findings indicate that naltrexone has the potential to modulate the secretion of inflammatory cytokines in response to intracellular TLR activity, supporting the hypothesis that it may have potential for use as an immunomodulator

Large Scale Comparison of Innate Responses to Viral and Bacterial Pathogens in Mouse and Macaque

Viral and bacterial infections of the lower respiratory tract are major causes of morbidity and mortality worldwide. Alveolar macrophages line the alveolar spaces and are the first cells of the immune system to respond to invading pathogens. To determine the similarities and differences between the responses of mice and macaques to invading pathogens we profiled alveolar macrophages from these species following infection with two viral (PR8 and Fuj/02 influenza A) and two bacterial (Mycobacterium tuberculosis and Francisella tularensis Schu S4) pathogens. Cells were collected at 6 time points following each infection and expression profiles were compared across and between species. Our analyses identified a core set of genes, activated in both species and across all pathogens that were predominantly part of the interferon response pathway. In addition, we identified similarities across species in the way innate immune cells respond to lethal versus non-lethal pathogens. On the other hand we also found several species and pathogen specific response patterns. These results provide new insights into mechanisms by which the innate immune system responds to, and interacts with, invading pathogens

Ligand engagement of Toll-like receptors regulates their expression in cortical microglia and astrocytes

BACKGROUND: Toll-like receptor (TLR) activation on microglia and astrocytes are key elements in neuroinflammation which accompanies a number of neurological disorders. While TLR activation on glia is well-established to up-regulate pro-inflammatory mediator expression, much less is known about how ligand engagement of one TLR may affect expression of other TLRs on microglia and astrocytes. METHODS: In the present study, we evaluated the effects of agonists for TLR2 (zymosan), TLR3 (polyinosinic-polycytidylic acid (poly(I:C)), a synthetic analogue of double-stranded RNA) and TLR4 (lipopolysaccaride (LPS)) in influencing expression of their cognate receptor as well as that of the other TLRs in cultures of rat cortical purified microglia (>99.5 %) and nominally microglia-free astrocytes. Elimination of residual microglia (a common contaminant of astrocyte cultures) was achieved by incubation with the lysosomotropic agent L-leucyl-L-leucine methyl ester (L-LME). RESULTS: Flow cytometric analysis confirmed the purity (essentially 100 %) of the obtained microglia, and up to 5 % microglia contamination of astrocytes. L-LME treatment effectively removed microglia from the latter (real-time polymerase chain reaction). The three TLR ligands robustly up-regulated gene expression for pro-inflammatory markers (interleukin-1 and interleukin-6, tumor necrosis factor) in microglia and enriched, but not purified, astrocytes, confirming cellular functionality. LPS, zymosan and poly(I:C) all down-regulated TLR4 messenger RNA (mRNA) and up-regulated TLR2 mRNA at 6 and 24 h. In spite of their inability to elaborate pro-inflammatory mediator output, the nominally microglia-free astrocytes (>99 % purity) also showed similar behaviours to those of microglia, as well as changes in TLR3 gene expression. LPS interaction with TLR4 activates downstream mitogen-activated protein kinase and nuclear factor-κB signalling pathways and subsequently causes inflammatory mediator production. The effects of LPS on TLR2 mRNA in both cell populations were antagonized by a nuclear factor-κB inhibitor. CONCLUSIONS: TLR2 and TLR4 activation in particular, in concert with microglia and astrocytes, comprise key elements in the initiation and maintenance of neuropathic pain. The finding that both homologous (zymosan) and heterologous (LPS, poly(I:C)) TLR ligands are capable of regulating TLR2 gene expression, in particular, may have important implications in understanding the relative contributions of different TLRs in neurological disorders associated with neuroinflammation