Toll-like receptor variation in the bottlenecked population of the Seychelles warbler: computer simulations see the 'ghost of selection past' and quantify the 'drift debt'.

Balancing selection can maintain immunogenetic variation within host populations, but detecting its signal in a postbottlenecked population is challenging due to the potentially overriding effects of drift. Toll-like receptor genes (TLRs) play a fundamental role in vertebrate immune defence and are predicted to be under balancing selection. We previously characterized variation at TLR loci in the Seychelles warbler (Acrocephalus sechellensis), an endemic passerine that has undergone a historical bottleneck. Five of seven TLR loci were polymorphic, which is in sharp contrast to the low genomewide variation observed. However, standard population genetic statistical methods failed to detect a contemporary signature of selection at any TLR locus. We examined whether the observed TLR polymorphism could be explained by neutral evolution, simulating the population's demography in the software DIYABC. This showed that the posterior distributions of mutation rates had to be unrealistically high to explain the observed genetic variation. We then conducted simulations with an agent-based model using typical values for the mutation rate, which indicated that weak balancing selection has acted on the three TLR genes. The model was able to detect evidence of past selection elevating TLR polymorphism in the prebottleneck populations, but was unable to discern any effects of balancing selection in the contemporary population. Our results show drift is the overriding evolutionary force that has shaped TLR variation in the contemporary Seychelles warbler population, and the observed TLR polymorphisms might be merely the 'ghost of selection past'. Forecast models predict immunogenetic variation in this species will continue to be eroded in the absence of contemporary balancing selection. Such 'drift debt' occurs when a gene pool has not yet reached its new equilibrium level of polymorphism, and this loss could be an important threat to many recently bottlenecked populations.

Influence of extraction technique on the anti-oxidative potential of hawthorn (Crataegus monogyna) extracts in bovine muscle homogenates.

Six extracts were prepared from hawthorn (Crataegus monogyna) leaves and flowers (HLF) and berries (HB) using solid-liquid [traditional (T) (HLFT, HBT), sonicated (S) (HLFS, HBS)] and supercritical fluid (C) extraction (HLFC, HBC) techniques. The antioxidant activities of HLF and HB extracts were characterised using in vitro antioxidant assays (TPC, DPPH, FRAP) and in 25% bovine muscle (longissimus lumborum) homogenates (lipid oxidation (TBARS), oxymyoglobin (% of total myoglobin)) after 24h storage at 4°C. Hawthorn extracts exhibited varying degrees of antioxidant potency. In vitro and muscle homogenate (TBARS) antioxidant activity followed the order: HLFS>HLFT and HBT>HBS. In supercritical fluid extracts, HLFC>HBC (in vitro antioxidant activity) and HLFC≈HBC (TBARS). All extracts (except HBS) reduced oxymyoglobin oxidation. The HLFS extract had the highest antioxidant activity in all test systems. Supercritical fluid extraction (SFE) exhibited potential as a technique for the manufacture of functional ingredients (antioxidants) from hawthorn for use in muscle foods.

Low-dose acetylsalicylic acid inhibits the secretion of interleukin-6 from white adipose tissue.

BACKGROUND: Chronically elevated interleukin-6 (IL-6) is implicated in obesity-associated pathologies, where a proportion of this cytokine is derived from adipose tissue. Proinflammatory prostaglandins, which regulate this cytokine elsewhere, are also produced by this tissue. OBJECTIVE: To investigate whether constitutively active cyclooxygenase (COX)/prostaglandin (PG) pathway in white adipose tissue (WAT) is responsible for basal IL-6 production. DESIGN: The effect of acetylsalicylic acid (ASA), an inhibitor of COX, on IL-6 was assessed in human subjects and mice. COX, downstream PG synthase (PGS) activity and PG receptor signalling were determined in subcutaneous (SC), gonadal (GN) WAT and adipocytes. METHODS AND RESULTS: In obese humans, low-dose ASA (150 mg day(-1) for 10 days) inhibited systemic IL-6 and reduced IL-6 release from SC WAT ex vivo (0.2 mM). Similarly, in mice, ASA (0.2 and 2.0 mg kg(-1)) suppressed SC WAT 6-keto-PGF(1alpha) (a stable metabolite of prostacyclin) and IL-6 release. Although both COX isoforms are comparably expressed, prostacyclin synthase expression is higher in GN WAT, with levels of activity correlating directly with IL-6. Both ASA (5 mM) and NS-398 (COX-2 selective inhibitor

Genomic comparisons of Salmonella enterica serovar Dublin, Agona, and Typhimurium strains recently isolated from milk filters and bovine samples from Ireland, using a Salmonella microarray.

Salmonella-induced enterocolitis is the leading food-borne illness with a lethal outcome and causes millions of cases of gastroenteritis each year. We examined genomic variation among 12 environmental, veterinary, and clinical Salmonella enterica serovar Dublin, Agona, and Typhimurium strains isolated in Ireland between 2000 and 2003, as well as two clinical isolates from Canada and four archival isolates, which belonged to serovars Dublin and Agona. Using DNA-DNA hybridization to a microarray consisting of most of the predicted protein-encoding sequences of the S. enterica serovar Typhimurium LT2 genome, we identified a number of genomic regions that were absent in one or more serovars. The 34 genomic regions encoded proteins involved in sugar metabolism, transport, fimbrial and phage biogenesis, and transcriptional regulation, as well as inner and outer membrane-associated proteins. Two of the four prophages identified in strain LT2, prophages Gifsy-1 and Gifsy-2, were present in all six serovar Typhimurium strains examined. Prophage Fels-1 was absent from all 18 isolates examined, and Fels-2 was completely absent from the serovar Typhimurium isolates and the Salmonella Reference Collection B serovar Dublin strain Du2. All five Salmonella pathogenicity islands were present in all isolates. Plasmid pSLT was absent from all serovar Agona isolates, and only homologues of the spv genes were present in eight of the nine serovar Dublin strains. Only limited intraserovar diversity was found among the nine serovar Dublin, three serovar Agona, and six serovar Typhimurium isolates examined even though these isolates had extensive geographic, temporal, and source differences.

Deaths from blunt trauma, after arrival at hospital: plus ça change, plus c'est la même chose.

In this retrospective review of 94 deaths from blunt trauma occurring after admission to the hospital, in the region of Northern Ireland, for the year of 2001, data is presented with reference to preventable deaths (10%). Comparison is made with previous audit from the year of 1981 (preventable death rate 16%). Adverse events continue to occur in relation to abdominal haemorrhage, and issues pertaining to multidisciplinary care.

Evaluation of surimi, fat and water content in a low/no added pork sausage formulation using response surface methodology.

Response surface methodology (RSM) was employed for simultaneous analysis of the effects of added surimi (0-40%), fat (5-30%) and water (10-35%), on the physical, textural and sensory characteristics of fresh breakfast pork sausages. Experimental design allowed for evaluation of potential interactive effects between these ingredients. Sausages were evaluated for texture, colour, water holding capacity (WHC) and sensory attributes. Three optimum recipes, R1 (25.3% surimi, 22.2% fat, 12.7% water, 25.3% pork), R2 (12.2% surimi, 5.5% fat, 38.7% water, 33.2% pork) and R3 (25.3% surimi, 6.3% fat, 28.5% water, 25.3% pork), were determined and these were evaluated against a full-fat commercial control (R4). Force values of R1 were not significantly different to R4, however, force values for R2 and R3 were lower (P<0.001). No significant differences were observed between R1, R3 and R4 for visual colour or sensory acceptability scores throughout the study, whereas scores for R2 were lower. Sensory analysis indicated that R2 had lower scores for texture (P<0.01), chewiness (P<0.01), acceptability (P<0.01), flavour (P<0.05) and preference (P<0.01). Results from this study suggest that it is possible to successfully replace pork meat with functional fish proteins in the manufacture of sausage type products.

Possible new role for NF-kappaB in the resolution of inflammation.

Inflammation involves the sequential activation of signaling pathways leading to the production of both pro- and anti-inflammatory mediators. Although much attention has focused on pro-inflammatory pathways that initiate inflammation, relatively little is known about the mechanisms that switch off inflammation and resolve the inflammatory response. The transcription factor NF-kappaB is thought to have a central role in the induction of pro-inflammatory gene expression and has attracted interest as a new target for the treatment of inflammatory disease. We show here that NF-kappaB activation in leukocytes recruited during the onset of inflammation is associated with pro-inflammatory gene expression, whereas such activation during the resolution of inflammation is associated with the expression of anti-inflammatory genes and the induction of apoptosis. Inhibition of NF-kappaB during the resolution of inflammation protracts the inflammatory response and prevents apoptosis. This suggests that NF-kappaB has an anti-inflammatory role in vivo involving the regulation of inflammatory resolution.

Colocalization and interaction of cyclooxygenase-2 with caveolin-1 in human fibroblasts.

Results from our previous study suggest that cyclooxygenase-2 (COX-2) induced by phorbol 12-myristate 13-acetate (PMA) may be localized to caveolae-like structures (Liou, J.-Y., Shyue, S.-K., Tsai, M.-J., Chung, C.-L., Chu, K.-Y., and Wu, K. K. (2000) J. Biol. Chem. 275, 15314-15320). In this study, we determined subcellular localization of COX-2 and caveolin-1 by confocal microscopy. COX-2 in human foreskin fibroblasts stimulated by PMA (100 nm) or interleukin-1beta (1 ng/ml) for 6 h was localized to plasma membrane in addition to endoplasmic reticulum and nuclear envelope. Caveolin-1 was localized to plasma membrane, and image overlay showed colocalization of COX-2 with caveolin-1. This was confirmed by the presence of COX-2 and caveolin-1 in the detergent-insoluble membrane fraction of cells stimulated by PMA. Immunoprecipitation showed complex formation of COX-2 with caveolin-1 in a time-dependent manner. A larger quantity of COX-2 was complexed with caveolin-1 in PMA-treated than in interleukin-1beta-treated cells. Purified COX-2 complexed with glutathione S-transferase-fused caveolin-1, which was not inhibited by the scaffolding domain peptide. Caveolin-1-bound COX-2 was catalytically active, and its activity was not inhibited by the scaffolding domain peptide. These results suggest that COX-2 induced by PMA and interleukin-1beta is colocalized with caveolin-1 in the segregated caveolae compartment. Because caveolae are rich in signaling molecules, this COX-2 compartment may play an important role in diverse pathophysiological processes.

Potential adverse effects of cyclooxygenase-2 inhibition: evidence from animal models of inflammation.

Cyclooxygenase (COX; prostaglandin H synthase, prostaglandin endoperoxidase) is the key enzyme in the synthesis of the prostaglandin and thromboxane families of eicosanoid mediators, and is the target for the nonsteroidal anti-inflammatory drugs (NSAIDs). The identification of an inducible COX isoform, COX-2, and the demonstration of its specific expression at sites of inflammation suggested that it may provide a useful therapeutic target for novel anti-inflammatory drugs. Inhibition of an enzyme that is not expressed in most healthy tissues would potentially avoid most of the adverse effects associated with NSAIDs, which target a constitutively expressed isoform, COX-1. The development of novel 'super aspirins' with high selectivity towards the inhibition of COX-2 showed that this hypothesis was well-founded and that high levels of these drugs could be tolerated without these serious adverse effects. The first two of these new generation NSAIDs, celecoxib and rofecoxib, are now in clinical use. More recently, however, concern has been expressed that COX-2 inhibition may in fact have a number of potential, previously hidden, pitfalls. These have arisen from the demonstration that COX-2 induction is not exclusively associated with the onset of an inflammatory reaction, with expression limited to inflammatory sites. In fact, COX-2 is expressed more chronically, and is also seen during the resolution of inflammation and in areas of wound-healing. The application of COX-2-selective inhibitors during these periods has been shown to be deleterious in that resolution of inflammation is delayed, gastric ulcer healing is delayed and, in some patients, ulcers have been shown to progress further to perforation. The suggestion has now been made that, in these situations, COX-2 may help resolve the pathology, perhaps by generating alternative series of prostaglandins such as the cyclopentenone prostaglandins. The finding that these prostaglandins can affect proteins by direct chemical modifications as well as having their own receptor families has rekindled debate on the deleterious and beneficial effects of prostanoids, and the implications of inhibiting the production of these mediators, in the body. Therefore, in this review we discuss the role of COX-2 in inflammation and the potential adverse effects of its inhibition.

COX-2 expression and cell cycle progression in human fibroblasts.

Cyclooxygenase-2 (COX-2) is continuously expressed in most cancerous cells where it appears to modulate cellular proliferation and apoptosis. However, little is known about the contribution of transient COX-2 induction to cell cycle progression or programmed cell death in primary cells. In this study we determined whether COX-2 regulates proliferation or apoptosis in human fibroblasts. COX-2 mRNA, protein, and prostaglandin E2 (PGE2) were not detected in quiescent cells but were expressed during the G0/G1 phase of the cell cycle induced by serum. Inhibition of COX-2 did not alter G0/G1 to S phase transition or induce apoptosis at concentrations that diminished PGE2. Addition of interleukin-1beta to serum enhanced COX-2 expression and PGE2 synthesis over that by serum alone but had no effect on the progression of these cells into S phase. Furthermore, platelet-derived growth factor drove the G0 fibroblasts into the cell cycle without inducing detectable levels of COX-2 or PGE2. Collectively, these data show that transient COX-2 expression in primary human fibroblasts does not influence cell cycle progression.

Surgical trainees' attitudes to laparoscopic cholecystectomy: a regional survey.

BACKGROUND AND STUDY AIMS: Since the introduction of laparoscopic cholecystectomy (LC), numerous articles have been written emphasising its many advantages over open cholecystectomy (OC). However, reports also highlight increased complications following LC such as bile-duct, vascular and bowel injuries. We aimed to study surgical trainees as a defined population of individuals who, with increasing exposure to cholecystectomy, would become fully aware of LC's advantages and controversies. We wished to test the hypothesis that, with increasing in-depth knowledge, they might opt for OC rather than LC if they themselves required cholecystectomy. MATERIALS AND METHODS: We conducted a postal survey of all 133 Northern Ireland surgical trainees identified as having exposure to LC during their training. Trainees were asked whether they would undergo LC and if so with which preconditions. Similarly, if they stated a preference for open cholecystectomy they were asked to state the reason. A minimum time period of 18 months was considered adequate for trainees to become relatively more experienced in this field compared with their more junior counterparts. RESULTS: A response rate of 80.5% (107/133) was achieved. A total of 51 of 107 trainees had at least 18 months' experience. Of the 107 who replied, 88.8% (95/107) would be willing to undergo LC. A total of 12 of 107 trainees would opt for OC, with twice as many experienced trainees (8 vs. 4) opting for this approach (n.s. [not significant]). Significantly more experienced trainees cited the use of laparoscopic cholangiography as a precondition for LC compared with their inexperienced counterparts (7 vs. 1, p = 0.020). Of 107 trainees, 19 would request use of the open first port (Hasson) technique; 14 of these had at least 18 months' experience (p = 0.009). CONCLUSION: Our survey confirms that the majority of trainees would be willing to undergo LC. However, increased experience of LC may alter an individual's expectations about how LC should ideally be performed.

Selective suppression of CCAAT/enhancer-binding protein beta binding and cyclooxygenase-2 promoter activity by sodium salicylate in quiescent human fibroblasts.

The anti-inflammatory actions of salicylates cannot be explained by inhibition of cyclooxygenase (COX) activity. This study demonstrates that sodium salicylate at a therapeutic concentration suppressed COX-2 gene transcription induced by phorbol 12-myristate 13-acetate and interleukin 1beta by inhibiting the binding of CCAAT/enhancer-binding protein beta to its promoter region of COX-2. By contrast, salicylate did not inhibit nuclear factor kappaB-dependent COX-2 induction by tumor necrosis factor alpha. The inhibitory effect of sodium salicylate was restricted to serum-deprived quiescent cells. These findings indicate that contrary to the current view that salicylate acts via inhibition of nuclear factor kappaB the pharmacological actions of aspirin and salicylates are mediated by inhibiting CCAAT/enhancer-binding protein beta binding and transactivation. These findings have a major impact on the conceptual understanding of the mechanism of action of salicylates and on new drug discovery and design.

Cell cycle-dependent expression of cyclooxygenase-2 in human fibroblasts.

The purpose of this investigation is to determine whether the levels of cyclooxygenase-2 (COX-2) expression are cell cycle dependent. We used a serum-starved human foreskin fibroblast model to determine changes in COX-2 mRNA, protein, and promoter activity in response to stimulation with interleukin-1b (IL-1b) and phorbol 12-myristate 13-acetate (PMA) at G0, G1, S and G2/M phases of the cell cycle. IL-1b (1 ng/ml) and PMA (100 nM) induced robust COX-2 expression in the G0 cells, and the level of COX-2 expression declined progressively after the cells had entered the cell cycle. The COX-2 mRNA level at G1, S and G2/M phases of the cell cycle was 76%, 46%, and 30% of that at G0, respectively. A 5-flanking promoter fragment of COX-2 constructed into a luciferase expression vector was transfected into cells. The promoter activity in response to PMA stimulation was significantly higher in G0 than in S phase cells. These results imply that G0 cells are the key players in inflammation and other COX-2-dependent pathophysiological processes. When the cells are in the proliferative phase, COX-2 inducibility becomes restrained probably by an endogenous control mechanism to avoid COX-2 mediated oxidative DNA damage.

Nitric oxide synthase inhibitors have opposite effects on acute inflammation depending on their route of administration.

The bulk of published data has shown that NO is proinflammatory. However, there also exists the conflicting notion that NO may be protective during an inflammatory insult. In an attempt to resolve this issue, we have compared the effects on inflammation of a range of NO synthase (NOS) inhibitors given either directly to the site of the inflammatory lesion or systemically. It was found that in the carrageenin-induced pleurisy, a single intrapleural injection of the selective inducible NO inhibitors S-(2-aminoethyl) isothiourea (AE-ITU; 3 and 10 mg/kg) and N-(3-(aminomethyl)-benzyl) acetamidine (1400W; 10 mg/kg) or the selective endothelial cell NOS inhibitor L-N(5)(1-iminoethyl)-ornithine (10 mg/kg) not only exacerbated inflammation at the very early stages of the lesion (1-6 h), but also prevented inflammatory resolution. By contrast, administering NOS inhibitors systemically ameliorated the severity of inflammation throughout the reaction. To elucidate the mechanisms by which inhibition of NO synthesis locally worsened inflammation, we found an increase in histamine, cytokine-induced neutrophil chemoattractant, superoxide, and leukotriene B(4) levels at the inflammatory site. In conclusion, this work shows that the local production of NO is protective by virtue of its ability to regulate the release of typical proinflammatory mediators and, importantly, that NOS inhibitors have differential anti-inflammatory effects depending on their route of administration.

Derivation of shellfish harvest reopening criteria following the New Carissa oil spill in Coos Bay, Oregon.

Oil spills in Alaska, California, Maine, and other states have raised concerns regarding potential contamination of fish and shellfish, and have led to temporary closures of seafood harvests while health risks are assessed. Lacking standardized protocols, these assessments are generally ad hoc, site-specific efforts, with significant differences in risk evaluation criteria. This article describes the response of a state health agency to shellfish contamination following an oil spill on the Oregon coast, and discusses some of the factors that can complicate the evaluation of potential health risks from consumption of oil-contaminated shellfish. On 4 February 1999, the Japanese-owned cargo ship M/V New Carissa, carrying an estimated 400,000 gallons of light diesel and heavy fuel oil, ran aground 2 miles north of Coos Bay, Oregon. Damage to the ship's hull from the grounding and pounding surf caused the release of an estimated 25,000 to 70,000 gallons of oil. Concern for potential contamination of local recreational shellfish and commercial oyster beds prompted the Oregon Department of Agriculture (ODA) to close shellfish harvesting in Coos and Douglas counties. ODA requested assistance from the Oregon Health Division in the derivation of risk-based criteria for reopening the shellfish harvest. Criteria were developed for the primary contaminants of concern, polycyclic aromatic hydrocarbons (PAHs) expressed as total benzo[a]-pyrene (BaP) equivalents. "Safe" (<10 microg/kg) and "unsafe" (>45 microg/kg) BaP equivalent levels were derived based on upper end (32.5 g/d) and average (7.5 g/d) estimates of shellfish consumption, respectively. Composite samples of oysters, clams, and mussels (15-20 per composite) were collected from target areas and analyzed for PAHs by gas chromatography/mass spectroscopy (GC/MS). Carcinogenic PAHs were converted to total BaP equivalents (wet weight) and compared with criteria. Two oyster samples, collected from a slough off of Coos Bay, contained 33.9 and 34.5 microg/kg BaP equivalents; all other samples had less than 10 microg/kg BaP equivalents. An evaluation of the PAH profiles in the two higher oyster samples indicated a primary source other than the New Carissa oil. Because shellfish sample BaP equivalents attributable to the New Carissa oil spill did not exceed 10 microg/kg, shellfish harvesting was reopened on 4 March 1999. This study revealed some of the inherent difficulties in attempting to quantify health risks from contaminated shellfish following an oil spill and demonstrated the clear need for standardized protocols for responding to such events.

New insights into the role of COX 2 in inflammation.

Cyclo-oxygenase (COX) is responsible for the synthesis of bioactive prostanoids, the inhibition of which serves as the basis for the mode of action of clinically used nonsteroidal anti-inflammatory drugs. While there were suggestions as early as the 1970s that an inducible isoform of COX exists, it was only in the early 1990s that COX 2 was identified, cloned and sequenced. Not surprisingly, this new isoform was expressed at sites of inflammation and reported to contribute to the inflammatory response. Recently, however, evidence is emerging to suggest that COX 2 also has anti-inflammatory properties. In this review, the two faces of COX 2 are examined, with emphasis on its role in regulating inflammatory resolution, including possible mechanisms of action

Assessing potential health risks from microcystin toxins in blue-green algae dietary supplements.

The presence of blue-green algae (BGA) toxins in surface waters used for drinking water sources and recreation is receiving increasing attention around the world as a public health concern. However, potential risks from exposure to these toxins in contaminated health food products that contain BGA have been largely ignored. BGA products are commonly consumed in the United States, Canada, and Europe for their putative beneficial effects, including increased energy and elevated mood. Many of these products contain Aphanizomenon flos-aquae, a BGA that is harvested from Upper Klamath Lake (UKL) in southern Oregon, where the growth of a toxic BGA, Microcystis aeruginosa, is a regular occurrence. M. aeruginosa produces compounds called microcystins, which are potent hepatotoxins and probable tumor promoters. Because M. aeruginosa coexists with A. flos-aquae, it can be collected inadvertently during the harvesting process, resulting in microcystin contamination of BGA products. In fall 1996, the Oregon Health Division learned that UKL was experiencing an extensive M. aeruginosa bloom, and an advisory was issued recommending against water contact. The advisory prompted calls from consumers of BGA products, who expressed concern about possible contamination of these products with microcystins. In response, the Oregon Health Division and the Oregon Department of Agriculture established a regulatory limit of 1 microg/g for microcystins in BGA-containing products and tested BGA products for the presence of microcystins. Microcystins were detected in 85 of 87 samples tested, with 63 samples (72%) containing concentrations > 1 microg/g. HPLC and ELISA tentatively identified microcystin-LR, the most toxic microcystin variant, as the predominant congener.

Resolution of inflammation.

Acute inflammatory reactions, in contrast to chronic inflammatory reactions, are usually self-limiting and resolve. We have investigated the resolving phase of a number of immune and non-immune inflammatory reactions induced in the pleural cavity of rats. COX-2 is expressed during resolution of these models. Using carrageenan pleurisy, we showed that this enzyme has a proinflammatory role as the reaction develops but an antiinflammatory role as the lesion resolves. This antiinflammatory role is associated with production of cyclopentenone prostaglandins and the absence of PGE2. Dual COX-1/COX-2 inhibitors or COX-2 inhibitors when given at the peak of the inflammatory response delay resolution, an effect reversed by replacing CyPGs into the pleural space. PGF2alpha like the CyPGs appears to have a role in resolving this reaction. Stress proteins are also induced in a variety of acute inflammatory models during resolution. Heme oxygenase-1 (HO-1) is one such protein so too are members of the hsp70 family. An inducer of HO-1 promotes resolution whereas an inhibitor is proinflammatory. In most cases it appears to be the macrophage that is the source of proteins necessary for resolution to occur. Understanding how proinflammatory pathways switch to the antiinflammatory pathways necessary for resolution to take place may eventually allow the exploitation of endogenous antiinflammatory pathways in the treatment of chronic inflammation.