Total haemoglobin mass, but not haemoglobin concentration, is associated with preoperative cardiopulmonary exercise testing-derived oxygen-consumption variables.

Background: Cardiopulmonary exercise testing (CPET) measures peak exertional oxygen consumption ( V˙O2peak ) and that at the anaerobic threshold ( V˙O2 at AT, i.e. the point at which anaerobic metabolism contributes substantially to overall metabolism). Lower values are associated with excess postoperative morbidity and mortality. A reduced haemoglobin concentration ([Hb]) results from a reduction in total haemoglobin mass (tHb-mass) or an increase in plasma volume. Thus, tHb-mass might be a more useful measure of oxygen-carrying capacity and might correlate better with CPET-derived fitness measures in preoperative patients than does circulating [Hb]. Methods: Before major elective surgery, CPET was performed, and both tHb-mass (optimized carbon monoxide rebreathing method) and circulating [Hb] were determined. Results: In 42 patients (83% male), [Hb] was unrelated to V˙O2 at AT and V˙O2peak ( r =0.02, P =0.89 and r =0.04, P =0.80, respectively) and explained none of the variance in either measure. In contrast, tHb-mass was related to both ( r =0.661, P <0.0001 and r =0.483, P =0.001 for V˙O2 at AT and V˙O2peak , respectively). The tHb-mass explained 44% of variance in V˙O2 at AT ( P <0.0001) and 23% in V˙O2peak ( P =0.001). Conclusions: In contrast to [Hb], tHb-mass is an important determinant of physical fitness before major elective surgery. Further studies should determine whether low tHb-mass is predictive of poor outcome and whether targeted increases in tHb-mass might thus improve outcome.

Transfusion in critical care - a UK regional audit of current practice.

A consistent message within critical care publications has been that a restrictive transfusion strategy is non-inferior, and possibly superior, to a liberal strategy for stable, non-bleeding critically ill patients. Translation into clinical practice has, however, been slow. Here, we describe the degree of adherence to UK best practice guidelines in a regional network of nine intensive care units within Wessex. All transfusions given during a 2-month period were included (n = 444). Those given for active bleeding or within 24 h of major surgery, trauma or gastrointestinal bleeding were excluded (n = 148). The median (IQR [range]) haemoglobin concentration before transfusion was 73 (68-77 [53-106]) g.l-1 , with only 34% of transfusion episodes using a transfusion threshold of < 70 g.l-1 . In a subgroup analysis that did not study patients with a history of cardiac disease (n = 42), haemoglobin concentration before transfusion was 72 (68-77 [50-98]) g.l-1 , with only 36% of transfusion episodes using a threshold of < 70 g.l-1 (see Fig. 3). Most blood transfusions given to critically ill patients who were not bleeding in this audit used a haemoglobin threshold > 70 g.l-1 . The reason why recommendations on transfusion triggers have not translated into clinical practice is unclear. With a clear national drive to decrease usage of blood products and clear evidence that a threshold of 70 g.l-1 is non-inferior, it is surprising that a scarce and potentially dangerous resource is still being overused within critical care. Simple solutions such as electronic patient records that force pause for thought before blood transfusion, or prescriptions that only allow administration of a single unit in non-emergency circumstances may help to reduce the incidence of unnecessary blood transfusions.

Interferon-inducible factor 16 is a novel modulator of glucocorticoid action.

Previously, we used cDNA expression profiling to identify genes associated with glucocorticoid (Gc) sensitivity. We now identify which of these directly influence Gc action. Interferon-inducible protein 16 (IFI16), bone morphogenetic protein receptor type II (BMPRII), and regulator of G-protein signaling 14 (RGS14) increased Gc transactivation, whereas sialyltransferase 4B (SIAT4B) had a negative effect. Amyloid beta (A4) precursor-protein binding, family B, member 1 (APBB1/Fe65) and neural cell expressed developmentally down-regulated 9 (NEDD9) were without effect. Only IFI16 potentiated Gc repression of NF-kappaB. In addition, IFI16 affected basal expression, and Gc induction of endogenous target genes. IFI16 did not affect glucocorticoid receptor (GR) expression, ligand-dependent repression of GR expression, or the ligand-dependent induction of GR phosphorylation on Ser-211 or Ser-203. Coimmunoprecipitation revealed an interaction, suggesting that IFI16 modulation of GR function is mediated by protein crosstalk. Transfection analysis with GR mutants showed that the ligand-binding domain of GR binds IFI16 and is the target domain for IFI16 regulation. Analysis of human lung sections identified colocalization of GR and IFI16, suggesting a physiologically relevant interaction. We demonstrate that IFI16 is a novel modulator of GR function and show the importance of analyzing variation in Gc sensitivity in humans, using appropriate technology, to drive discovery.

Circadian timing in the lung; a specific role for bronchiolar epithelial cells.

In addition to the core circadian oscillator, located within the suprachiasmatic nucleus, numerous peripheral tissues possess self-sustaining circadian timers. In vivo these are entrained and temporally synchronized by signals conveyed from the core oscillator. In the present study, we examine circadian timing in the lung, determine the cellular localization of core clock proteins in both mouse and human lung tissue, and establish the effects of glucocorticoids (widely used in the treatment of asthma) on the pulmonary clock. Using organotypic lung slices prepared from transgenic mPER2::Luc mice, luciferase levels, which report PER2 expression, were measured over a number of days. We demonstrate a robust circadian rhythm in the mouse lung that is responsive to glucocorticoids. Immunohistochemical techniques were used to localize specific expression of core clock proteins, and the glucocorticoid receptor, to the epithelial cells lining the bronchioles in both mouse and human lung. In the mouse, these were established to be Clara cells. Murine Clara cells retained circadian rhythmicity when grown as a pure population in culture. Furthermore, selective ablation of Clara cells resulted in the loss of circadian rhythm in lung slices, demonstrating the importance of this cell type in maintaining overall pulmonary circadian rhythmicity. In summary, we demonstrate that Clara cells are critical for maintaining coherent circadian oscillations in lung tissue. Their coexpression of the glucocorticoid receptor and core clock components establishes them as a likely interface between humoral suprachiasmatic nucleus output and circadian lung physiology.

Combined inhibition of DNA methylation and histone acetylation enhances gene re-expression and drug sensitivity in vivo.

Histone deacetylation and DNA methylation have a central role in the control of gene expression in tumours, including transcriptional repression of tumour suppressor genes and genes involved in sensitivity to chemotherapy. Treatment of cisplatin-resistant cell lines with an inhibitor of DNA methyltransferases, 2-deoxy-5'azacytidine (decitabine), results in partial reversal of DNA methylation, re-expression of epigenetically silenced genes including hMLH1 and sensitisation to cisplatin both in vitro and in vivo. We have investigated whether the combination of decitabine and a clinically relevant inhibitor of histone deacetylase activity (belinostat, PXD101) can further increase the re-expression of genes epigenetically silenced by DNA methylation and enhance chemo-sensitisation in vivo at well-tolerated doses. The cisplatin-resistant human ovarian cell line A2780/cp70 has the hMLH1 gene methylated and is resistant to cisplatin both in vitro and when grown as a xenograft in mice. Treatment of A2780/cp70 with decitabine and belinostat results in a marked increase in expression of epigenetically silenced MLH1 and MAGE-A1 both in vitro and in vivo when compared with decitabine alone. The combination greatly enhanced the effects of decitabine alone on the cisplatin sensitivity of xenografts. As the dose of decitabine that can be given to patients and hence the maximum pharmacodynamic effect as a demethylating agent is limited by toxicity and eventual re-methylation of genes, we suggest that the combination of decitabine and belinostat could have a role in the efficacy of chemotherapy in tumours that have acquired drug resistance due to DNA methylation and gene silencing.

Increased T-regulatory cells within lymphocyte follicles in moderate COPD.

Lymphoid follicles in the lung parenchyma are a characteristic feature of chronic obstructive pulmonary disease (COPD). There are reports of altered CD4 T-regulatory cell numbers in COPD lungs, but the location of these cells within COPD lung tissue specific follicles has not been investigated. The presence of CD4(+)FOXP3(+) T-regulatory cells was assessed in surgically resected lung tissue from 12 COPD patients, 11 smokers with normal lung function and seven nonsmokers by combined immunofluorescence and immunohistochemistry. Organised lymphoid follicles were observed in all three groups of patients, as well as lymphoid clusters lacking organisation. The percentage of CD4 cells that were T-regulatory cells were significantly increased (p = 0.02) within COPD (16%) follicles compared with smokers (10%) and nonsmokers (8%). In contrast, there was no change (p>0.05) in the percentage of T-regulatory cells in clusters or the subepithelium between groups. Lymphoid follicles in COPD patients have increased T-regulatory cells. Therefore, T-regulatory activity may be altered within COPD lymphoid follicles.

Treatment of saline, acidic, metal-contaminated groundwater from the Western Australian Wheatbelt.

Managing acidic, metal-containing saline ground and drainage waters in the Wheatbelt of Western Australia is an environmental and economic challenge. Sulfate-reducing fluidised bed bioreactors are shown to be technically capable of treating high salt, low pH, metal containing waters from the town of Narembeen in the Wheatbelt so as to reduce acidity and to remove most of the undesirable metal contaminants. The hydraulic residence time (HRT) limit for a stable process with groundwater from the region of Narembeen was >16 hours. The maximal rate of sulfate reduction in the laboratory system treating Narembeen groundwater was similar to rates observed in comparable applications of the process at other sites, ca. 3 g sulfate (L-reactor)(-1) day(-1). Salts that are relatively free of metal contaminants can be produced from water that has been treated by the sulfate-reducing fluidised bed bioreactor. It is unlikely that metal precipitates, captured from Wheatbelt waters by the process, would be of economic value. If sulfate-reducing fluidised bed reactors were considered technologically appropriate at larger scale, the decision to use them would be based on the necessity to take action, the comparative effectiveness of competing technologies, and the relative costs of competing technologies.

Cardiopulmonary exercise testing (CPET) in the United Kingdom-a national survey of the structure, conduct, interpretation and funding.

BACKGROUND: Cardiopulmonary exercise testing (CPET) is an exercise stress test with concomitant expired gas analysis that provides an objective, non-invasive measure of functional capacity under stress. CPET-derived variables predict postoperative morbidity and mortality after major abdominal and thoracic surgery. Two previous surveys have reported increasing utilisation of CPET preoperatively in England. We aimed to evaluate current CPET practice in the UK, to identify who performs CPET, how it is performed, how the data generated are used and the funding models. METHODS: All anaesthetic departments in trusts with adult elective surgery in the UK were contacted by telephone to obtain contacts for their pre-assessment and CPET service leads. An online survey was sent to all leads between November 2016 and March 2017. RESULTS: The response rate to the online survey was 73.1% (144/197) with 68.1% (98/144) reporting an established clinical service and 3.5% (5/144) setting up a service. Approximately 30,000 tests are performed a year with 93.0% (80/86) using cycle ergometry. Colorectal surgical patients are the most frequently tested (89.5%, 77/86). The majority of tests are performed and interpreted by anaesthetists. There is variability in the methods of interpretation and reporting of CPET and limited external validation of results. CONCLUSIONS: This survey has identified the continued expansion of perioperative CPET services in the UK which have doubled since 2011. The vast majority of CPET tests are performed and reported by anaesthetists. It has highlighted variation in practice and a lack of standardised reporting implying a need for practice guidelines and standardised training to ensure high-quality data to inform perioperative decision making.

Correction to: Cardiopulmonary exercise testing (CPET) in the United Kingdom-a national survey of the structure, conduct, interpretation and funding.

[This corrects the article DOI: 10.1186/s13741-017-0082-3.].

Effects of the pH dependence of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide-formazan absorption on chemosensitivity determined by a novel tetrazolium-based assay.

The tetrazolium dye, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), is reduced by live but not dead cells, and this reaction is used as the end point in a rapid drug-screening assay. It can also be used for accurate determinations of drug sensitivity but only if a quantitative relationship is established between cell number and MTT-formazan production. We have shown that reduction of MTT to MTT-formazan by cells is dependent on the amount of MTT in the incubation medium. The concentration required to give maximal MTT-formazan production differs widely between cell lines. The absorption spectrum of MTT-formazan varies with cell number and with pH. At a low cell density or a high pH, the absorption maximum is at a wavelength of 560 to 570 nm. However, at a high cell density or a low pH, there are two absorption maxima; one at 510 nm and a second at about 570 nm. Measurements of absorbance at 570 nm underestimate MTT-formazan production and, hence, cell number at high cell densities. This error can result in a 10-fold underestimation of chemosensitivity. Addition of a buffer at pH 10.5 to the solubilized MTT-formazan product can overcome the effects of both cell density and culture medium on the absorption spectrum. Provided that sufficient MTT is used and the pH of the MTT-formazan product is controlled, dye reduction can be used to estimate cell numbers in a simple chemosensitivity assay the results of which agree well with a commonly used clonogenic assay.

Reduction of the growth rate of the Walker 256 tumor in rats by rhodamine 6G together with hypoglycemia.

Previous attempts to use tumor energy metabolism as a target for antineoplastic therapy have used single agents aimed at inhibiting either glycolysis or oxidative phosphorylation. Since most tumor cells use both pathways for energy production, this approach is unlikely to succeed. The aim of this study was to simultaneously manipulate both sources of intracellular ATP to achieve more selective control of tumor growth. Rhodamine 6G (R6G) is a fluorochrome mitochondrial dye which inhibits oxidative phosphorylation. 3-Mercaptopicolinic acid inhibits gluconeogenesis and is a potent hypoglycemic agent in the fasting state. Dose-response relationships were established for R6G and 3-mercaptopicolinic acid, and a nontoxic dose of the compounds was selected for subsequent experiments. Thereafter, groups of rats (n = 7 per group) underwent s.c. implantation of Walker 256 carcinosarcoma. Following a 24-h fast each group received either saline, R6G (0.8 mg/kg), 3-mercaptopicolinic acid (40 mg/kg), or the combination given i.p. Seven days after tumor implantation animals were sacrificed, and tumors were exercised and weighed. Administration of R6G during a period of hypoglycemia significantly reduced the tumor growth rate when compared to control experiments (3.6 +/- 0.3 g cf. 7.1 +/- 0.7 g, mean +/- SE; P less than 0.05). In contrast, neither R6G nor the period of hypoglycemia alone significantly affected tumor growth. These results suggest that simultaneous manipulation of oxidative phosphorylation and glycolysis may be used to selectively inhibit tumor growth in vivo.

Reversal of drug resistance in human tumor xenografts by 2'-deoxy-5-azacytidine-induced demethylation of the hMLH1 gene promoter.

Loss of DNA mismatch repair because of hypermethylation of the hMLH1 gene promoter occurs at a high frequency in a number of human tumors. A role for loss of mismatch repair (MMR) in resistance to a number of clinically important anticancer drugs has been shown. We have investigated whether the demethylating agent 2'-deoxy-5-azacytidine (DAC) can be used in vivo to sensitize MMR-deficient, drug-resistant ovarian (A2780/cp70) and colon (SW48) tumor xenografts that are MLH1 negative because of gene promoter hypermethylation. Treatment of tumor-bearing mice with the demethylating agent DAC at a nontoxic dose induces MLH1 expression. Re-expression of MLH1 is associated with a decrease in hMLH1 gene promoter methylation. DAC treatment alone has no effect on the growth rate of the tumors. However, DAC treatment sensitizes the xenografts to cisplatin, carboplatin, temozolomide, and epirubicin. Sensitization is comparable with that obtained by reintroduction of the hMLH1 gene by chromosome 3 transfer. Consistent with loss of MMR having no effect on sensitivity in vitro to Taxol, DAC treatment has no effect on the Taxol sensitivity of the xenografts. DAC treatment does not sensitize xenografts of HCT116, which lacks MMR because of hMLH1 mutation. Because there is emerging data on the role of loss of MMR in clinical drug resistance, DAC could have a role in increasing the efficacy of chemotherapy for patients whose tumors lack MLH1 expression because of hMLH1 promoter methylation.

In vitro and in vivo reversal of P-glycoprotein-mediated multidrug resistance by a novel potent modulator, XR9576.

The overexpression of P-glycoprotein (P-gp) on the surface of tumor cells causes multidrug resistance (MDR). This protein acts as an energy-dependent drug efflux pump reducing the intracellular concentration of structurally unrelated drugs. Modulators of P-gp function can restore the sensitivity of MDR cells to such drugs. XR9576 is a novel anthranilic acid derivative developed as a potent and specific inhibitor of P-gp, and in this study we evaluate the in vitro and in vivo modulatory activity of this compound. The in vitro activity of XR9576 was evaluated using a panel of human (H69/LX4, 2780AD) and murine (EMT6 AR1.0, MC26) MDR cell lines. XR9576 potentiated the cytotoxicity of several drugs including doxorubicin, paclitaxel, etoposide, and vincristine; complete reversal of resistance was achieved in the presence of 25-80 nM XR9576. Direct comparative studies with other modulators indicated that XR9576 was one of the most potent modulators described to date. Accumulation and efflux studies with the P-gp substrates, [3H]daunorubicin and rhodamine 123, demonstrated that XR9576 inhibited P-gp-mediated drug efflux. The inhibition of P-gp function was reversible, but the effects persisted for >22 h after removal of the modulator from the incubation medium. This is in contrast to P-gp substrates such as cyclosporin A and verapamil, which lose their activity within 60 min, suggesting that XR9576 is not transported by P-gp. Also, XR9576 was a potent inhibitor of photoaffinity labeling of P-gp by [3H]azidopine implying a direct interaction with the protein. In mice bearing the intrinsically resistant MC26 colon tumors, coadministration of XR9576 potentiated the antitumor activity of doxorubicin without a significant increase in toxicity; maximum potentiation was observed at 2.5-4.0 mg/kg dosed either i.v. or p.o. In addition, coadministration of XR9576 (6-12 mg/kg p.o.) fully restored the antitumor activity of paclitaxel, etoposide, and vincristine against two highly resistant MDR human tumor xenografts (2780AD, H69/LX4) in nude mice. Importantly all of the efficacious combination schedules appeared to be well tolerated. Furthermore, i.v. coadministration of XR9576 did not alter the plasma pharmacokinetics of paclitaxel. These results demonstrate that XR9576 is an extremely potent, selective, and effective modulator with a long duration of action. It exhibits potent i.v. and p.o. activity without apparently enhancing the plasma pharmacokinetics of paclitaxel or the toxicity of coadministered drugs. Hence, XR9576 holds great promise for the treatment of P-gp-mediated MDR cancers.

Influence of whole body protein turnover rate on resting energy expenditure in patients with cancer.

Whole body protein turnover and resting energy expenditure are measured simultaneously in weight stable and weight losing patients with lung (n = 22) or colorectal cancer (n = 38). These results were compared with those from weight stable and weight losing non-cancer controls (n = 22). Rates of whole body protein turnover were calculated from the plateau isotopic enrichment of urinary ammonia and urea following a primed, continuous, 24-h infusion of [15N]glycine. Resting energy expenditure was measured by indirect calorimetry. All groups of cancer patients had significantly elevated rates of whole body protein turnover (P less than 0.05) and synthesized, on average, 1.9 g/kg/day more protein compared with weight stable non-cancer controls. In contrast, the resting energy expenditure of cancer patients and controls was similar. Moreover, there was no correlation between individual rates of whole body protein turnover. Thus, although cancer patients had rates of whole body protein turnover which were 50-70% greater than controls, this did not result in a measurable increase in resting energy expenditure. The assumption that elevation of whole body protein turnover or resting energy expenditure causes weight loss in cancer patients must be an oversimplification. An acute phase protein response was observed in the majority of cancer patients. Although the presence of such an inflammatory response did not correlate with the rate of whole body protein turnover, the role of inflammatory mediators in the pathogenesis of disturbed protein metabolism in cancer patients merits further investigation.

ePlantLIBRA: A composition and biological activity database for bioactive compounds in plant food supplements.

The newly developed ePlantLIBRA database is a comprehensive and searchable database, with up-to-date coherent and validated scientific information on plant food supplement (PFS) bioactive compounds, with putative health benefits as well as adverse effects, and contaminants and residues. It is the only web-based database available compiling peer reviewed publications and case studies on PFS. A user-friendly, efficient and flexible interface has been developed for searching, extracting, and exporting the data, including links to the original references. Data from over 570 publications have been quality evaluated and entered covering 70 PFS or their botanical ingredients.

Telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.

Telomerase activation is considered to be a critical step in cancer progression due to its role in cellular immortalization. The prevalence of telomerase expression in human cancers makes it an attractive candidate for new mechanism-based targets for cancer therapy. The selective killing of cancer cells can be achieved by gene-directed enzyme pro-drug therapy (GDEPT). In this study we have tested the feasibility of using the transcriptional regulatory sequences from the hTERT and hTR genes to regulate expression of the bacterial nitroreductase enzyme in combination with the pro-drug CB1954 in a suicide gene therapy strategy. hTERT and hTR promoter activity was compared in a panel of 10 cell lines and showed a wide distribution in activity; low activity was observed in normal cells and telomerase-negative immortal ALT cell lines, with up to 300-fold higher activity observed in telomerase positive cancer lines. Placing the nitroreductase gene under the control of the telomerase gene promoters sensitized cancer cells in tissue culture to the pro-drug CB1954 and promoter activity was predictive of sensitization to the pro-drug (2-20-fold sensitization), with cell death restricted to lines exhibiting high levels of promoter activity. The in vivo relevance of these data was tested using two xenograft models (C33a and GLC4 cells). Significant tumour reduction was seen with both telomerase promoters and the promoter-specific patterns of sensitization observed in tissue culture were retained in xenograft models. Thus, telomerase-specific suicide gene therapy vectors expressing bacterial nitroreductase sensitize human cancer cells to the pro-drug CB1954.