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Flame retardants and legacy contaminants were analyzed in adipose tissue from 11 circumpolar polar bear (Ursus maritimus) subpopulations in 2005–2008 spanning Alaska east to Svalbard. Although 37 polybrominated diphenyl ethers (PBDEs), total-(α)-hexabromocyclododecane (HBCD), 2 polybrominated biphenyls (PBBs), pentabromotoluene, pentabromoethylbenzene, hexabromobenzene, 1,2-bis(2,4,6-tribromophenoxy(ethane) and decabromodiphenyl ethane were screened, only 4 PBDEs, total-(α-)HBCD and BB153 were consistently found. Geometric mean ∑PBDE (4.6–78.4 ng/g lipid weight (lw)) and BB153 (2.5–81.1 ng/g lw) levels were highest in East Greenland (43.2 and 39.2 ng/g lipid weight (lw), respectively), Svalbard (44.4 and 20.9 ng/g lw) and western (38.6 and 30.1 ng/g lw) and southern Hudson Bay (78.4 and 81.1 ng/g lw). Total-(α)-HBCD levels ( more...

An endotoxin-induced form of nitric oxide synthase (EC 1.14.23) was purified to homogeneity from rat liver by sequential anion-exchange chromatography and affinity chromatography using 2',5'-ADP-Sepharose. The enzyme has a subunit molecular mass of 135 kDa as determined by SDS/PAGE, a maximum specific activity of 462 nmol of citrulline formed from arginine per min per mg, and a Km for arginine of 11 microM. The enzyme was strongly stimulated by the addition of calmodulin with an EC50 of 2 nM, but removal of free calcium from the assay medium only reduced activity by 15%. Calmodulin inhibitors significantly reduced the enzyme activity. Tetrahydrobiopterin, FAD, and FMN were all required for full enzyme activity. This form of endotoxin-induced nitric oxide synthase from liver differs from the inducible enzyme found in macrophages and is unusual in that it is stimulated by calmodulin with little dependence on the calcium ion concentration. more...

Recognition of bacterial infection is the first key step to the initiation of an inflammatory response and host defense. Transmembrane proteins of the Toll-like receptor family have long been recognized as key detectors of the extracellular presence of pathogens. Recently, much research has identified a variety of intracellular detectors that also mediate innate immune responses following bacterial infection. These cannot only recognize bacteria that invade the cell cytoplasm, but also a variety of bacterial products that are introduced into cells by specialized secretion systems or are secreted toxins. This article will focus on these intracellular detectors and the bacterial components that they recognize. These detectors are particularly well adapted to recognize the presence of pathogenic bacteria as opposed to commensal organisms. Their growing importance suggests that targeting such intracellular pathways may be important in the future for manipulating the immune response to infection as an aid to augmenting host defense and providing more effective vaccines. more...

BACKGROUND: VCL-CB01, a candidate cytomegalovirus (CMV) DNA vaccine that contains plasmids encoding CMV phosphoprotein 65 (pp65) and glycoprotein B (gB) to induce cellular and humoral immune responses and that is formulated with poloxamer CRL1005 and benzalkonium chloride to enhance immune responses, was evaluated in a phase 1 clinical trial. METHODS: VCL-CB01 was evaluated in 44 healthy adult subjects (22 CMV seronegative and 22 CMV seropositive) 18-43 years old. Thirty-two subjects received 1- or 5-mg doses of vaccine on a 0-, 2-, and 8-week schedule, and 12 subjects received 5-mg doses of vaccine on a 0-, 3-, 7-, and 28-day schedule. RESULTS: Overall, the vaccine was well tolerated, with no serious adverse events. Local reactions included mild to moderate injection site pain and tenderness, induration, and erythema. Systemic reactions included mild to moderate malaise and myalgia. All reactions resolved without sequelae. Through week 16 of the study, immunogenicity, as measured by enzyme-linked immunosorbant assay and/or ex vivo interferon (IFN)-gamma enzyme-linked immunospot assay, was documented in 45.5% of CMV-seronegative subjects and in 25.0% of CMV-seropositive subjects who received the full vaccine series, and 68.1% of CMV-seronegative subjects had memory IFN-gamma T cell responses at week 32. CONCLUSION: The safety and immunogenicity data from this trial support further evaluation of VCL-CB01. more...

The initial step in directed cell movement is lamellipodial protrusion, an action driven by actin polymerization. Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) family proteins are key regulators of this actin polymerization and can control lamellipodial protrusion rate. Ena/VASP proteins are substrates for modification by cyclic-nucleotide-dependent protein kinases at a number of sites. Phosphorylation of Ser239 of VASP in vitro inhibits its anti-capping and filament-bundling activity but the effects of this modification on lamellipodial structure and function are unknown. To examine the functional effects of this modification in living cells, we studied VASP phosphorylation at Ser239 by nitric oxide (NO) stimulation of cGMP-dependent protein kinase. Using live cell imaging of primary cells transfected with GFP-VASP constructs, we found that NO produced rapid retraction of lamellipodia together with cell rounding that was dependent on guanylate cyclase and type II cGMP-dependent protein kinase. In cells expressing a mutant VASP (Ser239Ala) lacking the site preferentially phosphorylated by this kinase, NO had no effect. Phosphorylation of Ser239 of VASP results in loss of lamellipodial protrusions and cell rounding, and is a powerful means of controlling directed actin polymerization within lamellipodia. more...

Purpose To evaluate effects of a 6-month social marketing campaign on awareness of, attitudes toward and use of female as well as male condoms for 15–25 year-old-women. Methods Using a time-space sampling methodology, we conducted a cross-sectional survey of 3407 women at pre-campaign in 12 western U.S. neighborhoods on female and male condom awareness, attitudes, and use. Six of the 12 study neighborhoods were randomly selected to receive the POWER social marketing campaign designed to impact condom knowledge, attitudes, and use. The campaign was followed with another cross-sectional survey of 3,003 women in all 12 study neighborhoods on condom knowledge, attitudes, use and awareness of POWER materials. We compared pre-and post-campaign surveys to determine the efficacy of POWER and conducted post hoc analyses on post-campaign data to determine if exposure to POWER was related to higher levels of positive condom attitudes and norms and condom use. Results We found no differences between neighborhoods with and without the POWER campaign with regard to our primary outcomes. To diagnose reasons for this null effect, we examined outcomes post hoc examining the influence of POWER exposure. Post hoc analyses show some evidence that exposure to POWER was associated with condom use. In the context of the nested trial, this raises concerns that post test only evaluations are limited. Conclusions Establishing the efficacy of a social marketing campaign is challenging. This group randomized trial showed a null effect. Social marketing campaigns may need to have more media channels and saturation before they can show behavioral effects. Using a nested design with randomization at the community level and probability sampling introduces rigor not commonly seen in evaluations of social marketing campaigns. more...

Concentrations of 20 trace elements (V, Cr, Mn, Co, Cu, Zn, Rb, Sr, Mo, Ag, Cd, In, Sn, Sb, Cs, Ba, Hg, Tl, Pb, and Bi) were measured in livers of polar bears (Ursus maritimus) collected from Northern and Western Alaska from 1993 to 2002 to examine differences in the profiles of trace metals between the Beaufort Sea (Northern Alaska) and the Chukchi Sea (Western Alaska) subpopulations in Alaska. Among the trace elements analyzed, concentrations of Cu (50-290 microg/g, dry wt) in polar bear livers were in the higher range of values that have been reported for marine mammals. Concentrations of Hg in polar bears varied widely, from 3.5 to 99 microg/g dry wt, and the mean concentrations in polar bears were comparable to concentrations reported previously for several other species of marine mammals. Mean concentrations of Pb and Cd were 0.67 and 1.0 microg/g dry wt, respectively; these concentrations were lower than levels reported elsewhere for polar bears from Greenland and Canada. Age- and gender-related variations in the concentrations of trace elements in our polar bears were minimal. Concentrations of Hg decreased slowly in samples collected during 1993-2002, whereas Cd and Pb concentrations were found to be stable or slowly increasing, in the livers of Alaskan polar bears. Concentrations of Ag, Bi, Ba, Cu, and Sn were significantly higher in the Chukchi Sea subpopulation than in the Beaufort Sea subpopulation. Concentrations of Hg were significantly higher in the Beaufort Sea subpopulation than in the Chukchi Sea subpopulation. Differences in the profiles and concentrations of Hg, Ag, Bi, Ba, Cu, and Sn suggest that the sources of exposure to these trace elements between Western and Northern Alaskan polar bears are different, in agreement with findings reported earlier for several organic contaminants. more...

Perfluoroalkyl substances are globally distributed anthropogenic contaminants. Their production and use have increased dramatically from the early 1980s. While many recent publications have reported concentrations of perfluorooctane sulfonate (PFOS) and other perfluoroalkyl acids (PFAs) in biotic and abiotic samples, only limited work has addressed temporal trends. In this study we analyzed archived polar bear(Ursus maritimus) livertissue samples from two geographic locations in the North American Arctic, collected from 1972 to 2002. The eastern group, taken from the vicinity of northern Baffin Island, Canada, comprised 31 samples, and the western group, from the vicinity of Barrow, Alaska, comprised 27 samples. Samples were analyzed for perfluorocarboxylic acids (PFCAs) from carbon chain length C8 to C15, perfluorohexane sulfonate, PFOS, the neutral precursor perfluorooctane sulfonamide (PFOSA), as well as 8:2 and 10:2 fluorotelomer acids and their alpha,beta unsaturated acid counterparts. Concentrations of PFOS and PFCAs with carbon chain lengths from C9 to C11 showed an exponential increase between 1972 and 2002 at both locations. Doubling times ranged from 3.6 +/- 0.9 years for perfluorononanoic acid in the eastern group to 13.1 +/- 4.0 years for PFOS in the western group. PFOSA showed decreasing concentrations over time at both locations, while the remaining PFAs showed no significant trends or were not detected in any sample. The doubling time for PFOS was similar to the doubling time of production of perfluoroctylsulfonyl-fluoride-based products during the 1990s. more...

Polybrominated diphenyl ethers (PBDEs) were determined in adipose tissue of adult and subadult female polar bears sampled between 1999 and 2002 from sub-populations in Arctic Canada, eastern Greenland, and Svalbard, and in males and females collected from 1994 to 2002 in northwestern Alaska. Only 4 congeners (BDE47, 99, 100, and 153) were consistently identified in all samples. BDE47 was the major PBDE congener representing from 65% to 82% of the sum (Σ) PBDEs. Age was not a significant co-variate for individual PBDEs or ΣPBDE. Higher proportions of BDE 99, 100, and 153 were generally found in samples from the Canadian Arctic than from Svalbard or the Bering-Chukchi Sea area of Alaska. Geometric mean ΣPBDE concentrations were highest for female polar bear fat samples collected from Svalbard (50 ng/g lipid weight (lw)) and East Greenland (70 ng/g lw). Significantly lower ΣPBDE concentrations were found in fat of bears from Canada and Alaska (means ranging from 7.6 to 22 ng/g lw). For the entire dataset, ΣP... more...

Sepsis and multiple organ failure are common causes of death in patients admitted to intensive care units. The incidence of sepsis and associated mortalities has been steadily increasing over the past 20 years. Sepsis is a complex inflammatory condition, the precise causes of which are still poorly understood. Animal models of sepsis have the potential to cause substantial suffering, and many of them have been poorly representative of the human syndrome. However, a number of non-animal approaches, including in vitro, in silico and clinical studies, show promise for addressing this situation. This report is based on discussions held at an expert workshop convened by Focus on Alternatives and held in 2004 at the Wellcome Trust, London. It provides an overview of some non-animal approaches to sepsis research, including their strengths and weaknesses, and argues that they should be prioritised for further development. more...

Perfluoroalkyl substances were determined in liver tissues and blood of polar bears (Ursus maritimus) from five locations in the North American Arctic and two locations in the European Arctic. Concentrations of perfluorooctane sulfonate (PFOS), perfluorohexane sulfonate, heptadecafluorooctane sulfonamide, and perfluoroalkyl carboxylates with C(8)-C(15) perfluorinated carbon chains were determined using liquid chromatography tandem mass spectrometry. PFOS concentrations were significantly correlated with age at four of seven sampling locations, while gender was not correlated to concentration for any compound measured. Populations in South Hudson Bay (2000-2730 ng/g wet wt), East Greenland (911-2140 ng/g wet wt), and Svalbard (756-1290 ng/g wet wt) had significantly (P < 0.05) higher PFOS concentrations than western populations such as the Chukchi Sea (435-729 ng/g wet wt). Concentrations of perfluorocarboxylic acids (PFCAs) with adjacent chain lengths (i.e., C9:C10 and C10:C11) were significantly correlated (P < 0.05), suggesting PFCAs have a common source within a location, but there were differences in proportions of PFCAs between eastern and western location sources. Concentrations of PFOS in liver tissue at five locations were correlated with concentrations of four polychlorinated biphenyl congeners (180, 153, 138, and 99) in adipose tissue of bears in the same populations, suggesting similar transport pathways and source regions of PFOS or precursors. more...

The physiological role of the duodenal peptide secretin is as a potent stimulant of electrolyte and water movement in pancreatic and biliary epithelium, via activation of G protein-coupled secretin receptors (hSCTR). However, the distribution and potential function of hSCTR in human lung has not previously been addressed. Using real-time quantitative reverse transcriptase-polymerase chain reaction profiling, in situ hybridization, and immunohistochemistry, we demonstrated that the hSCTR is abundantly expressed within the distal regions of human lung (tertiary bronchus and parenchyma), with negligible expression detected in more proximal regions (trachea, primary, and secondary bronchus). Expression was observed predominantly on the basolateral membrane of the bronchial epithelial layer, with some expression also observed in bronchial smooth muscle. In primary cultures of human tertiary bronchial epithelial cells, secretin was demonstrated to potently stimulate channel-mediated Cl- efflux in a concentration-dependent manner. Secretin was also shown to cause concentration-dependent relaxation of human tertiary bronchial smooth muscle. In summary, these data demonstrate that secretin receptors are present in human lung, and that activation of these receptors with human secretin potently stimulates concentration-dependent Cl- efflux from bronchial epithelial cells and bronchorelaxation. more...

Tumor necrosis factor (TNF) is a central cytokine in the pathogenesis of septic shock and other inflammatory states. Assay by immunoassay is convenient, but, because of circulating soluble receptors, does not accurately reflect biological activity of the cytokine. This article describes how to perform a bioassay for TNF, using its cytopathic effect on the murine cell line L929. By suitable manipluation, the assay can determine the two different forms of TNF, alpha and beta. more...

Neutropenia is frequently observed in the practice of oncology, usually resulting from the use of cytotoxic drugs. Less frequently, neutropenia is due to infiltration of the bone marrow by malignant disease. Both of these usually cause some depletion of all haematopoietic linages. True isolated neutropenia is, in contrast, a less frequent phenomenon. We report a case of auto-immune neutropenia occurring in a patient with metastatic melanoma, and discuss the differential diagnosis and implications for patient management. more...

Alaska has two polar bear populations: the Southern Beaufort Sea population, shared with Canada, and the Chukchi/Bering Seas population, shared with Russia. Currently a reliable population estimate for the Chukchi/Bering Seas population does not exist. Land-based aerial and mark-recapture population surveys may not be possible in the Chukchi Sea because variable ice conditions, the limited range of helicopters, extremely large polar bear home ranges, and severe weather conditions may limit access to remote areas. Thus line-transect aerial surveys from icebreakers may be the best available tool to monitor this polar bear stock. In August 2000, a line-transect survey was conducted in the eastern Chukchi Sea and western Beaufort Sea from helicopters based on a U.S. Coast Guard icebreaker under the "Ship of Opportunity" program. The objectives of this pilot study were to estimate polar bear density in the eastern Chukchi and western Beaufort Seas and to assess the logistical feasibility of using ship-based aerial surveys to develop polar bear population estimates. Twenty-nine polar bears in 25 groups were sighted on 94 transects (8257 km). The density of bears was estimated as 1 bear per 147 km² (CV = 38%). Additional aerial surveys in late fall, using dedicated icebreakers, would be required to achieve the number of sightings, survey effort, coverage, and precision needed for more effective monitoring of population trends in the Chukchi Sea. more...

After the publication of the 10-year survival data from Milan on the adjuvant use of the block sequential regimen consisting of four cycles of adriamycin followed by eight cycles of intravenous CMF, many centres adopted this as standard of care for high risk, multiple node-positive breast cancer. For this reason it was identified as the standard arm for the Anglo-Celtic adjuvant high-dose chemotherapy trial. This study reports on the experience of this regimen in 329 women with early breast cancer involving at least four axillary nodes, who were treated outside any adjuvant chemotherapy trial. At a median follow-up of 3 years, the overall 5-year disease-free survival is 61%, and the overall survival is 70%. These data confirm the efficacy of this regimen in non-trial patients, and, for the same high risk subgroup, indicate that this approach offers an outcome at least as good as that seen in the CALGB 9344 AC-Taxol arm, and the NCIC days 1 and 8 CEF. British Journal of Cancer (2002) 87, 1365–1369. doi:10.1038/sj.bjc.6600660 www.bjcancer.com © 2002 Cancer Research UK more...

Polarized tubule epithelial cell functions are dependent on correct delivery of effector proteins to the target apical or basolateral plasma membrane and associated cortical cytoskeleton. PDZ (Postsynaptic density protein 95/Drosophila Disks large/Zona occludens-1) domain-containing proteins have been identified as playing a critical role in membrane trafficking and sorting of ion transporters, receptors and other signalling proteins. These scaffolding proteins coordinate the assembly of functional plasma membrane multiprotein complexes, through PDZ domain binding to a consensus amino acid motif within the carboxyl-terminus of target proteins. The organization of these proteins into submembranous complexes may facilitate downstream signalling. Although several epithelial PDZ proteins that bind to a number of important mammalian proteins have been isolated, in many cases the significance of these interactions is unclear. However, the epithelial PDZ domain-containing Na+/H+ exchanger regulatory factor tethers the Na+/H+ exchanger and cystic fibrosis transmembrane regulator Cl– channel within an apical plasma membrane signalling complex, and has been shown to regulate the activity of these proteins. This article reviews the current evidence that supports a central role for the PDZ protein in the regulation of polarized tubule cell functions, such as vectorial solute transport. more...

MALIGNANT hyperthermia (MH) is a potentially fatal hypermetabolic response to volatile inhalation agents and succinylcholine that may have variable presentations. The authors describe an unusual presentation with late onset of malignant hyperthermia after laparoscopic cholecystectomy, with rhabdomyolysis as the principal sign. more...

Nitric oxide (NO) produced from inducible NO synthase (iNOS) is an important component of host defense against intracellular pathogens. To understand how phagocytes deliver NO to ingested microorganisms while avoiding cytotoxicity, we set out to study the subcellular localization of iNOS within macrophages following phagocytosis. Confocal microscopy of immunostained cells showed that iNOS was located not only diffusely within cytoplasm but also in vesicles, as well as immediately adjacent to the peripheral cell membrane. This peripheral iNOS colocalized with the cortical actin cytoskeleton and was removed by the actin-depolymerizing drug cytochalasin B. Biochemical fractionation of RAW 264 macrophages showed that 32.75% (±5.11%; n = 3) of iNOS was present in a particulate fraction, which cosedimented with low-density cellular vesicles. Following phagocytosis of latex beads, zymosan, immunoglobulin G-coated beads, or complement-coated zymosan, submembranous cortical iNOS was not recruited to phagosomes, nor was there any relocalization of intracellular iNOS. Similarly, following phagocytosis of Salmonella enterica serovar Typhimurium there was no recruitment of iNOS to the Salmonella vacuole at any stage after internalization. NO mediated significant killing of intracellular S. enterica serovar Typhimurium in RAW macrophages treated with lipopolysaccharide and gamma interferon; this was evident 4 h after infection. Although not recruited to phagosomes, iNOS association with the submembranous cortical actin cytoskeleton is ideally suited to deliver NO to microbes in contact with the cell surface and may contribute to early killing of ingested Salmonella. more...

Inoperable cancer of the exocrine pancreas responds poorly to most conventional anti-cancer agents, and new agents are required to palliate this disease. Seocalcitol (EB1089), a vitamin D analogue, can inhibit growth, induce differentiation and induce apoptosis of cancer cell lines in vitro and can also inhibit growth of pancreatic cancer xenografts in vivo. Thirty-six patients with advanced pancreatic cancer received once daily oral treatment with seocalcitol with dose escalation every 2 weeks until hypercalcaemia occurred, following which patients continued with maintenance therapy. The most frequent toxicity was the anticipated dose-dependent hypercalcaemia, with most patients tolerating a dose of 10–15 μg per day in chronic administration. Fourteen patients completed at least 8 weeks of treatment and were evaluable for efficacy, whereas 22 patients were withdrawn prior to completing 8 weeks' treatment and in 20 of these patients withdrawal was due to clinical deterioration as a result of disease progression. No objective responses were observed, with five of 14 patients having stable disease in whom the duration of stable disease was 82–532 days (median=168 days). The time to treatment failure (n=36) ranged from 22 to 847 days, and with a median survival of approximately 100 days. Seocalcitol is well tolerated in pancreatic cancer but has no objective anti-tumour activity in advanced disease. Further studies are necessary to determine if this agent has any cytostatic activity in this malignancy in minimal disease states. British Journal of Cancer (2002) 86, 680–685. DOI: 10.1038/sj/bjc/6600162 www.bjcancer.com © 2002 Cancer Research UK more...

Perfluorooctane sulfonate (PFOS) is a perfluorinated molecule that has recently been identified in the sera of nonindustrially exposed humans. In this study, 247 tissue samples from 15 species of marine mammals collected from Florida, California, and Alaskan coastal waters; the northern Baltic Sea; the Arctic (Spitsbergen); and Sable Island in Canada were analyzed for PFOS. PFOS was detected in liver and blood of marine mammals from most locations including those from Arctic waters. The greatest concentrations of PFOS found in liver and blood were 1520 ng/g wet wt in a bottlenose dolphin from Sarasota Bay, FL, and 475 ng/mL in a ringed seal from the northern Baltic Sea (Bothnian Sea), respectively. No age-dependent increase in PFOS concentrations in marine mammals was observed in the samples analyzed. The occurrence of PFOS in marine mammals from the Arctic waters suggests widespread global distribution of PFOS including remote locations. more...

The Brownlee Centre for Infectious Diseases, Tropical Medicine and Counselling, Gartnaval General Hospital, 1053 Great Western Road, Glasgow G12 3YN, UK; The Centre for Clinical Vaccinology and Tropical Medicine, University of Oxford, Churchill Hospital, Oxford OX3 7LJ, UK; Division of Inflammation, Infection and Immunity, Glasgow Biomedical Research Centre, University of Glasgow, Glasgow G12 8QQ, UK more...

Tumor necrosis factor (TNF) is a central cytokine in the pathogenesis of septic shock and other inflammatory states. Assay by immunoassay is convenient, but, because of circulating soluble receptors, does not accurately reflect biological activity of the cytokine. This article describes how to perform a bioassay for TNF, using its cytopathic effect on the murine cell line L929. By suitable manipluation, the assay can determine the two different forms of TNF, alpha and beta. more...

Part I. Endotoxin. Assay of Endotoxin by Limulus Amebocyte Lysate, Preparation of Endotoxin from Pathogenic Gram-Negative Bacteria, Assay of Anti-Endotoxin Antibodies, Isolation of the Bactericidal/Permeability-Increasing Protein from Polymorphonuclear Leukocytes by Reversible Binding to Target Bacteria, Purification of Lipopolysaccharide Binding Protein, Part II. Other Bacterial Products. Purification of Streptococcal Pyrogenic Exotoxin A, Assay for Superantigens, Shiranee Sriskandan. Part III. Cytokines. Bioassay for Tumor Necrosis Factors-a and b, Assay of Soluble Tumor Necrosis Factor Receptors, Whole-Blood Assays for Cytokine Production, Part IV. Nitric Oxide and Other Reactive Nitrogen Intermediates. Nitric Oxide Synthase Inhibitors, In Situ Detection of Nitric Oxide, Immunochemical Detection of Nitric Oxide Synthase in Human Tissue, Anti-Nitrotyrosine Antibodies for Immunohistochemistry, Detection of Peroxynitrite in Biological Fluids, Part V. Cell Culture Techniques. Myocardial Cells in Culture to Study Effects of Cytokines, Culture of Primary Human Bronchial Epithelial Cells, Primary Culture of Human Proximal Renal Tubular Epithelial Cells, Index more...

Background—Two clinical formulae (CF conference formula and estimation based on 120% of average requirement for energy) have been recommended for the estimation of energy requirements in cystic fibrosis but their accuracy is unknown. Aim—To compare the accuracy of estimates of energy requirement derived from the two formulae. Methods—Energy requirement, defined as total daily energy expenditure, was measured using the doubly labelled water method in 15 patients (six girls, nine boys; mean (SD) age, 10.0 (2.4) years) who were well and clinically stable. The accuracy of the formulae was assessed using calculation of biases and limits of agreement relative to measured energy requirement. Results—Estimates from the CF conference formula were lower than measured values (mean paired diVerence, 0.52 MJ/ day; 95% confidence interval (CI), ˛1.10 to 0.10), but this bias was not significant, and was smaller than that from the alternative formula (mean paired diVerence, 0.77 MJ/day; 95% CI, ˛0.20 to 1.74). Limits of agreement relative to measured total daily energy expenditure were narrower for the CF conference formula (˛2.72 to 1.68 MJ/ day) than for that based on 120% of estimated average requirement (˛2.75 to 4.29 MJ/day), but with both formulae errors in estimation at the individual level were large. Conclusions—The CF conference formula oVers improved prediction of energy requirements, but the accuracy of both formulae at the individual level is not sufficiently good for clinical purposes. (Arch Dis Child 1999;81:120‐124) more...

Acute respiratory exacerbations have been proposed to contribute to the negative energy balance which causes undernutrition in cystic fibrosis. However, no studies have measured their effect on all components of energy balance. The aim of this study was to measure the effect of an acute respiratory exacerbation on energy balance. Fourteen children (six females, eight males, mean+/-SD age 9.9+/-2.4 yrs) were studied when well and during the course of an acute respiratory exacerbation treated with intravenous antimicrobial therapy. The total energy expenditure was measured using the doubly-labelled water method, resting energy expenditure by ventilated hood indirect calorimetry, energy intake by household measures records, and fat malabsorption from measurements of dietary fat intake and faecal fat output. The exacerbation was associated with a significant reduction in energy intake (mean paired difference 47 kJ x kg of body weight(-1) x day(-1), p more...

Abstract —Inflammation appears to have a major role in the development of atherosclerotic lesions affecting native and transplanted coronary arteries. The subsequent risk of plaque rupture and acute ischemic events correlates with the degree of inflammation and may be modified by aspirin, an anti-inflammatory cyclooxygenase inhibitor. Cyclooxygenase-2 (Cox-2) and inducible nitric oxide synthase (iNOS) are involved in the inflammatory response via the rapid and exaggerated production of prostanoids and nitric oxide, both of which may have proatherosclerotic effects. These effects may be mediated by the formation of peroxynitrite in the case of nitric oxide and involve “cross talk” between the two enzyme systems. This study aimed to investigate native and transplant atherosclerosis for the presence and distribution of Cox-2 and iNOS. Immunocytochemical studies were performed on atherosclerotic lesions from patients with native (n=12) and transplant (n=5) coronary disease by using antibodies to Cox-2, iNOS, and nitrotyrosine (an indicator of peroxynitrite production). Control tissue was obtained from unused donor hearts and at the time of autopsy. Cox-2 and iNOS colocalized predominantly in macrophages/foam cells in both types of atherosclerosis. Cox-2 expression was also detected in medial smooth muscle cells and endothelial cells, including those of the vasa vasorum. Nitrotyrosine was found in the same distribution as that of iNOS and was colocalized with Cox-2 in macrophages. Cox-2 and iNOS are coexpressed in native and transplant atherosclerosis, possibly allowing for interaction between the enzymes and suggesting an alternative mechanism for the benefits of aspirin via inhibition of Cox-2 activity. more...

From studies aiming to determine the source of potentially carcinogenic nitrogen compounds within mammals, it was found that cells of the immune system were capable of producing reactive nitrogen intermediates (Green et al. 1981; Stuehr and Marietta 1985). Following the discovery that nitric oxide (NO) was the chemical identity of endothelium-dependent relaxing factor, it was quickly realised that NO was the origin of reactive nitrogen intermediates from activated immune cells. Since that time, it has become clear that a large number of different cells can be stimulated to produce NO, ranging from vascular smooth muscle cells to astrocytes (Nathan and Xie 1994). The stimuli required to produce this NO production are generally pro-inflammatory cytokines and bacterial components such as lipopolysaccharide (LPS). Molecular cloning of the genes responsible for NO production has identified the gene product responsible for cytokine activated NO production, which has been termed inducible NO synthase (iNOS) or type II NOS (Forstermann et al. 1994). This has considerable homology to the constitutive NOS isoforms found in brain and endothelium, as would be expected of enzymes catalysing the same reaction. However, there are important differences. Most notably, iNOS is not ordinarily subject to control by intracellular calcium ion concentrations, so that once induced iNOS produces large and sustained amounts of NO (Vodovotz et al. 1994). more...

But: Etudier la reponse systemique des cytokines a une chirurgie hepatique majeure pour evaluer de nouveaux traitements potentiels. Type d'etude: Ouverte, prospective. Provenance : Hopital universitaire, Royaume-Uni. Patients: Treize patients ayant eu une resection hepatique programmee avec exclusion vasculaire. Methodes: Des prelevements sanguins ont ete realises en preoperatoire, pendant l'intervention et au cours des quatre premiers jours postoperatoires pour mesurer les concentrations d'endotoxine, d'interferon gamma (INF-γ), de facteur de necrose tumoral α (FNT α), d'interleukine-1 (IL-1), et d'interleukine-6 (IL-6). Resultats: Les concentrations d'endotoxine etaient elevees chez 3 patients sur 13 avant l'intervention et chez 6 patients au cours de la periode postoperatoire. Les concentrations de TNF-α etaient nulles. Les doses d'INF-γ et d'IL-1 etaient faibles et leurs variations difficilement interpretables. Les concentrations d'IL-6 etaient augmentees de maniere significative de la sixieme heure au troisieme jour postoperatoire, avec un maximum de 699 (277) pg/ml a la vingtquatrieme heure (p more...

A new style of PGE-Au mineralization has recently been identified in Sudbury Ontario where high grades of PGE-Au are associated with low levels of copper-nickel sulphides (low-sulphide high-PGE-Au mineralization). Low-sulphide high-PGE-Au mineralization has become an increasingly higher profile exploration target in the footwall of the Sudbury Basin and is being explored, evaluated and in some instances mined by a number of companies. The Lonmin-Inco Sudbury PGM Joint Venture was established in January 2005 to explore for low-sulphide high-PGE-Au mineralization on six Inco properties. Geophysical exploration for these targets has focused primarily on using induced polarization methods and, to a lesser degree, electromagnetic methods. Fully integrating all available geologic and geophysical information is essential for directing drilling of target areas. Results from three properties are presented (Denison, Levack North and Wisner) where massive and disseminated sulphide, and low sulphide high PGE-Au mineralization have been intersected during the 2005 exploration program. These properties continue to have significant exploration opportunities for the discovery of low-sulphide high-PGE-Au mineralization and resources. more...

Cork taint in wine is a serious problem which is exacerbated by the difficulty of its assessment. Current analytical procedures are costly, time consuming and require the use of large amounts of solvents. We developed and evaluated a rapid method for the detection of the cork taint compound, 2,4,6-trichloroanisole (TCA), in wine samples. The method employs solid-phase microextraction, a solventless, automated sampling procedure, coupled to GC-MS-selected ion monitoring analysis. Quantification is enabled by a fully deuterated [2H5]TCA analog used as an internal standard. Accuracy (±8%), precision (R.S.D. 5–13%), and limit of quantification (5 ng/l) are comparable to existing methods. more...

A number of different lines of research have revealed the important roles that nitric oxide (NO) plays in mammalian cell biology. Since 1977 it has been known that NO activates soluble guanylate cyclase, producing a rise in intracellular cyclic guanosine monophosphate (cGMP) [1]. This in turn could produce vascular smooth muscle cell relaxation, and accounted for the vasorelaxatory properties of a number of different compounds such as organic nitrates and sodium nitroprusside [2, 3]. The mechanism of action of other vasodilators which did not themselves generate NO remained obscure, but was shown to be dependent on an intact endothelium, leading to the concept of an endothelium-derived relaxing factor (EDRF) [4]. Further work by two groups led to the discovery that the chemical nature of EDRF was in fact NO, or a closely related oxide of nitrogen [5, 6]. Subsequently, NO was found to be of key importance in vasodilatation and macrophage killing of microorganisms and tumor cells, and that NO is important in mediating a diverse number of biological effects, including acting as a neurotransmitter in non-adrenergic, non-cholinergic nerves [7], inhibiting platelet [8], and acting as a negative inotrope and chronotrope [9]. more...

Introduction and Objectives Pseudomonas aeruginosa (PA) is an important respiratory pathogen resulting in damaging neutrophilic responses. The cytokine IL-17 is important in orchestrating such inflammation. Cells producing IL-17, such as Th17 cells, have been shown to be important in host defense in chronic pulmonary PA infection. We set out to determine the origin and role of IL-17 in a model of chronic pulmonary PA infection. Methods Experimental chronic pulmonary infection in mice was produced by intra-tracheal instillation of mucoid PA strains embedded in agar-beads; sterile beads were utilised as controls. Thoracic lymph nodes (LNs), splenocytes and peritoneal B1a cells were restimulated with PA followed by cytokine assay and immunostaining to define responding cell subsets. PA-specific immunoglobulins were measured in sera and culture supernatants. Intrapulmonary B cells were identified via immunohistochemistry. Mice genetically engineered to lack B cells (µMT strain) were utilised to examine the effect on pathogenesis in the absence of B cell responses. Results Chronic PA infection developed in 43% (SD: 25%) of infected animals at 14-days. Following infection, the pulmonary LN B cell compartment expanded, with a large B1 population (B220 + CD19 + CD43 + IgM hi IgD lo and predominantly CD5 + ) that expressed intracellular IL-17A. Infected animals also developed peribronchial B220 + cellular foci. In mediastinal LNs following infection, PA-specific responses were dominated by B220 + CD19 + CD43 + CD23 - B5 + cells expressing and producing IL-17A and IL-22 as well as PA-specific IgM but not IgG. This PA-specific B1 response was not seen in the thoracic lymph nodes of sterile-bead treated animals. In splenocytes, there was a pre-existing B cell response to PA with identical features. Peritoneal B1a cells isolated from untreated controls also produced IL-17A, IL-22 and anti-PA IgM following infection, confirming the existence of pre-existing B1 cells that can respond to PA. In µMT animals, chronic colonisation rates, bacterial burden and neutrophilic inflammation did not differ from WT littermates. However, classical PA-specific Th17 responses dominated following infection in µMT animals, suggesting alternative compensatory IL-17 sources acting in the absence of B cells. Conclusions In chronic pulmonary PA infection, innate-like B1 cells migrate to the site of infection and are a novel source of pro-inflammatory IL-17 cytokines. more...

Introduction and Objectives Pseudomonas aeruginosa (PA) colonisation is a hallmark of cystic fibrosis (CF) resulting in damaging neutrophilic inflammation. Patients with CF produce anti-pseudomonal antibodies but the role of CD4 + T cell responses to PA remains unclear. Novel T helper cell subsets, Th17 and Th22 cells, have important roles in host defense but may also enhance tissue damage. We aimed to define the antigen-specific memory T helper cell responses to Pseudomonas aeruginosa in healthy humans and patients with cystic fibrosis. Methods CD14 + monocytes and memory CD4 + CD45RO + T cells were isolated from peripheral blood of CF patients with PA colonisation (n = 8) and healthy controls (n = 10). Monocyte-derived dendritic cells (DCs) were stimulated with live Pseudomonas strain PA103 and PA isolates derived from CF patients. Autologous T cells were co-cultured with activated DCs. The resultant T helper response was determined by measuring proliferation, immunoassay of cytokine output, and immunostaining of intracellular cytokines. Lavage samples from explanted CF lungs were assayed for IL-22 secretion. Results Healthy individuals andpatients with cystic fibrosis had robust antigen-specific memory CD4 + T cell responses to Pseudomonas aeruginosa that not only contained a Th1 and Th17 component but also Th22 cells. In contrast to previous descriptions of human Th22 cells, these Pseudomonal-specific Th22 cells lacked the skin homing markers CCR4 or CCR10, although they did express the chemokine receptor CCR6 that would direct migration to damaged epithelial surfaces. Furthermore, IL-22 production was evident in the lungs of CF patients colonised with PA. Healthy individuals and patients with cystic fibrosis had similar levels of Th22 and Th1 cells, but the patient group had significantly fewer Th17 cells in peripheral blood. Conclusions MemoryTh22 cells specific to Pseudomonas aeruginosa are induced in both healthy individuals and patients with CF, with IL-22 secretion being demonstrated in the CF lung. These Th22 cells do not express tissue specificity for gut or skin sites and we thus hypothesise may have a role in respiratory defense. Along with Th17 cells, they may play an important role in the pathogenesis of pulmonary infection with this microbe in patients with cystic fibrosis. more...

Although the production of NO within rodent phagocytes is well-characterized, its production and function within human phagocytes are less clear. We show here that neutrophils within human buffy coat preparations stimulated with a mixture of interleukin 1, tumor necrosis factor alpha, and interferon gamma contain inducible NO synthase mRNA and protein, one of the enzymes responsible for NO production. The protein colocalizes with myeloperoxidase within neutrophil primary granules. Using an inhibitor of NO synthase, L-N-monomethyl arginine, we show that activity of this enzyme is required for the formation of nitrotyrosine around phagocytosed bacteria, most likely through the intermediate production of peroxynitrite, a reaction product of NO and superoxide anions. more...

Amongst the many mediators that have been implicated in the pathophysiological changes seen in sepsis and septic shock, nitric oxide (NO) is one of the most recent to be described. Its role in mammalian cell biology is enormous (reviewed in Nathan 1992; Bredt and Snyder 1994), but this chapter focuses on its role in sepsis. While the functions of NO are legion, we concentrate on a few central roles that NO plays in sepsis. In particular, we discuss its hypotensive, cytotoxic, immunomodulatory, cardiovascular, and cerebral effects. In common with other mediators in sepsis there are problems in extending the results obtained in animal models to humans. We therefore discuss the clinical data pertaining to the role of NO in sepsis separately. NO is one example of a potentially toxic oxygen compound, and many others have been identified as possible mediators in sepsis which we do not have space to review. However, it is clear that NO can react with a number of these compounds to generate very toxic molecules, which are described here. more...

s / Pancreatology 13 (2013) e1–e20 e8 University College London, Institute of Liver and Digestive Health, Royal Free Hospital Guy's and St Thomas’ NHS Foundation Trust, London, Department of Gastroenterology King's College London and King's Health Partners, Division of Women's Health University College London, Department of Pathology University College London, HPB & Liver Transplant Unit, Royal Free Hospital Category: Benign and Inflammatory Background and aims: Some 35% of women of reproductive age are obese. Emerging evidence suggests that maternal obesity(MO) may predispose offspring to an increased risk of obesity and its related disorders, including Non-Alcoholic Fatty Pancreas Disease(NAFPD), a newly described disease entity which may link obesity and pancreatic adenocarcinoma. We have investigated the impact of MO on the pathogenesis of NAFPD using a pathophysiologically relevant model and mechanistically explored a role for altered expression of circadian genes(CG). Methods: Female C57BL6 mice were fed standard or obesogenic diet(OD) for 6 weeks prior to and throughout pregnancy and lactation. Litters were standardised to a minimum of 6 pups and offspring then weaned onto either standard or OD to produce 4 groups before sacrifice at 6 months. We assessed biochemical, histological and pro-inflammatory/profibrogenic markers associated with the NAFPD phenotype and the expression of CG in the pancreas. Results and Interpretation: Offspring exposed to MO and OD had increased bodyweights (p more...

s / Pancreatology 13 (2013) e1–e20 e5 Conclusion: Components of the metabolic syndrome, which are associated with insulin resistance, namely elevated diastolic blood pressure, elevated serum HbA1c and decreased serum HDL were positively associated with large increases in the risk of pancreatic cancer. Factors influencing these parameters should be measured in future aetiological work and may suggest mechanisms for carcinogenesis, but support an aetiological role for insulin resistance in pancreatic cancer. Take-home message: Metabolic factors and serum HDL may be predicitve of the risk of developing pancreatic cancer. Abstract previously presented? yes (BSG) Any disclosures? no () more...

The occurrence of viridans streptococcal bacteraemia (VSB) in a London postgraduate teaching hospital was analysed retrospectively. Over a 30-month period, 39 patients with VSB were identified, and the clinical significance of VSB was assessed for each, based upon clinical and laboratory information. Endocarditis accounted for only 13% of the total patients with apparent VSB. A greater proportion of patients with VSB originated from the haematology unit (21%) and in particular, the bonemarrow transplantation unit (15%). VSB also occurred in patients with solid epithelial malignancies and in children aged more...

Nitric oxide (NO) has been proposed as a mediator of hypotension in septic shock. The aim of this study was to determine whether an inhibitor of NO production, NG-monomethyl-L-arginine (L-NMMA), was able to protect against death in two murine models of experimental gram-negative sepsis. L-NMMA (3-300 mg kg-1) did not improve survival in intravenous or intraperitoneal models of sepsis. Seven h after intravenous infection, L-NMMA (100 mg/kg-1) reduced serum nitrite plus nitrate levels (NO breakdown products) from 774 microM in control-treated animals to 282 microM in L-NMMA-treated animals (P < .001). This compared to a level of 103 microM in uninfected mice. L-NMMA produced little change in bacterial load following infection and did not increase hepatic damage, as measured by serum levels of ornithine carbamoyltransferase. Thus, while L-NMMA may reverse the hyporesponsiveness of peripheral circulation in sepsis, it was unable to prevent death in these models of gram-negative septic shock. more...

Nitric oxide is a mediator of a wide range of physiological processes. It is produced by an enzyme family, the nitric-oxide synthases, one form of which is induced in many cells following stimulation with cytokines and lipopolysaccharide. The aim of the experiments reported in this study was to investigate the regulation of mRNA expression for this inducible nitric-oxide synthase in smooth muscle cells and macrophages. Stimulation of these cells with cytokines and lipopolysaccharide results in a marked elevation of nitric-oxide-synthase mRNA levels, which however do not remain elevated, but reach a maximum at 3–6 h after stimulation before returning to baseline levels over the next 20 h. Enzyme activity, however, remained virtually constant for 48 h following stimulation. Inspection of the 3′ untranslated segment of both murine and human inducible nitric-oxide-synthase mRNAs showed the presence of a conserved AU-rich octanucleotide sequence, previously identified in cytokine and oncogene mRNAs and shown to mediate mRNA instability. A particular feature of the breakdown of mRNAs bearing this sequence is that degradation is prevented by protein-synthesis inhibition. We show in this study that the half-life of inducible nitric-oxide-synthase mRNA is 6 h and that in the presence of an inhibitor of protein synthesis this breakdown is prevented. Thus, the mRNA for inducible nitric-oxide synthase shares some features in common with cytokines such as the transient expression and decay of its mRNA which can be prevented by protein-synthesis inhibition. more...

10000 Background: Pts with sarcomas, a family of >70 types of cancer, have limited options once cytotoxic agents fail. Brivanib (B) is an oral once daily selective dual inhibitor of FGF and VEGF signaling currently in phase III. Data from a phase II trial were analyzed to evaluate the potential utility of biomarkers and STS subtypes in predicting response to B. Methods: Pts aged ≥18 y with unresectable STS and no other treatment options received open-label B 800 mg qd for a 12-wk lead-in period and were assessed by CT/MRI. Pts with RECIST response continued on open-label B, those with progressive disease (PD) went off study. Pts with stable disease (SD) were randomized 1:1 to B or placebo (P), stratified by FGF2 expression (IHC + or -), until PD or unacceptable toxicity. Pts with PD on P could crossover to resume open-label B. The primary endpoint was PFS for B vs P in FGF2+ pts; secondary endpoints included ORR, DCR (ORR+SD), and exploratory biomarkers. Results: Of 251 pts (52% with ≥2 prior systemic reg... more...