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Importance: Survival probability among individuals critically buried by avalanche is highly time dependent, which was demonstrated 30 years ago. However, it remains unclear whether avalanche survival probability has changed over time., Objective: To assess the avalanche survival rate and probability as well as the rescue probability over the past 4 decades., Design, Setting, and Participants: In this cohort study, avalanche data from Switzerland that were collected by the WSL Institute for Snow and Avalanche Research (SLF) in Davos were analyzed from the winter beginning in 1981 to that beginning in 2020 and compared with data from the period 1981 to 1990. Data were analyzed from January to April 2024., Exposure: Critical avalanche burial (ie, burial involving the head and chest)., Main Outcomes and Measures: Survival rate among individuals critically buried by avalanche, survival probability, and rescue probability in relation to time buried under the avalanche., Results: The study included 1643 individuals critically buried by avalanche (mean [SD] age, 37 [13.7] years; 1090 of 1342 with known sex [81.2%] were male) among 3805 avalanches involving 7059 persons. Compared with the period from 1981 to 1990, the total survival rate over the full study period increased from 43.5% (95% CI, 38.8%-48.3%) to 53.4% (95% CI, 51.0%-55.8%). Survival probability remained high at 91% (95% CI, 80%-100%) for rescue during the first 10 minutes but then decreased to 31% (95% CI, 11%-51%) for rescue between 10 and 30 minutes. The survival rate among those buried long term (>130 minutes) increased from 2.6% (95% CI, 0.7%-6.9%) to 7.3% (95% CI, 4.8%-10.7%). The median rescue time decreased from 45 (IQR, 15-148) minutes to 25 (IQR, 10-85) minutes. Survival rates among individuals rescued from avalanche by organized rescue teams increased from 14.0% (28 of 200) to 22.9% (161 of 704)., Conclusions and Relevance: This cohort study of 1643 individuals critically buried by avalanche found that over the past 4 decades, total survival rates considerably increased and rescue times decreased. Survival rates among those buried long term (>130 minutes) also increased. These findings are likely attributable to collaborative efforts among stakeholders to enhance avalanche search-and-rescue techniques and medical interventions.

Agriculture is the largest source of methane globally, and enteric methane accounts for 32% of methane emissions globally. Dairy-beef is an increasingly important contributor to the beef industry. The objective of this study was to investigate if supplementation with a blend of essential oils (Agolin Ruminant) reduced enteric methane emissions from dairy-bred steers. Methane was measured from thirty-six Holstein Friesian steers (18 control and 18 treatment) in open-circuit respiration chambers, at three time-points relative to the introduction of Agolin Ruminant: (i) -3 (pre-additive introduction co-variate), (ii) 46 days after introduction, and (iii) 116 days after introduction. A significantly lower methane yield was observed in treated animals compared to control animals at both 46 days ( p < 0.05) and 116 days ( p < 0.01) after the introduction of Agolin Ruminant, although there was no difference in methane production (g/day). Control animals appeared to be more affected by isolation in respiration chambers than animals receiving Agolin Ruminant, as indicated by a significant reduction in dry matter intake by control animals in respiration chambers.

Introduction: We aimed to investigate the relationship between the time of the day and the probability of survival of completely buried avalanche victims. We explored the frequency of avalanche burials occurring after sunset, and described victims' characteristics, duration of burial and rescue circumstances compared to daytime avalanches., Methods: In this retrospective, observational study, we analysed avalanche data from the registry of the Swiss Institute for Snow and Avalanche Research, from 1998 to 2020., Results: A total of 3892 avalanche victims were included in the analysis, with 72 of the accidents (1.85%) occurring in the nighttime. Nearly 50% of the victims involved in nighttime avalanche accidents were completely buried, compared to about 25% of victims in daytime avalanches. Completely buried victims were rescued by a companion less often at night than in the daytime (15% vs. 51%, p <.001). The search and rescue of completely buried avalanche victims took longer during the nighttime compared to the daytime (median 89 min vs 20 min, p =.002). The probability of survival decreased as the day progressed; it was highest at around midday (63.0%), but decreased at sunset (40.4%) and was the lowest at midnight (28.7%)., Conclusions: Avalanche accidents at night are a rare event, and probability of survival after complete burial is lower during the nighttime compared to the daytime. The most relevant reason for this is the longer duration of burial, which is explained in part by the lower rate of companion rescue and the lower rate of victim localisation with an avalanche transceiver., (Copyright © 2022 The Author(s). Published by Elsevier B.V. All rights reserved.)

Purpose: To compare the binding and agonistic activity of Acthar ® Gel and synthetic melanocortin receptor (MCR) agonists and examine how the activity of select agonists affects the in vivo production of corticosterone., Materials and Methods: In vitro binding was determined using concentration-dependent displacement of the ligand [ 125 I]Nle 4 , D-Phe 7 -α-melanocyte-stimulating hormone (α-MSH) on cells expressing MC1R, MC3R, MC4R, or MC5R. Functional activity was determined using a time-resolved fluorescence cyclic adenosine monophosphate (cAMP) assay in cells expressing MC1R, MC2R, MC3R, MC4R, or MC5R. In vivo corticosterone analyses were performed by measuring plasma corticosterone levels in Sprague Dawley rats., Results: Acthar Gel and synthetic MCR agonists exhibited the highest binding at MC1R, lowest binding at MC5R, and moderate binding at MC3R and MC4R. Acthar Gel stimulated the production of cAMP in all 5 MCR-expressing cell lines, with MC2R displaying the lowest level of full agonist activity, 3-, 6.6-, and 10-fold lower than MC1R, MC3R, and MC4R, respectively. Acthar Gel was a partial agonist at MC5R. The synthetic MCR agonists induced full activity at all 5 MCRs, with the exception of α-MSH having no activity at MC2R. Acthar Gel treatment had less of an impact on in vivo production of corticosterone compared with synthetic ACTH 1-24 depot., Conclusions: Acthar Gel bound to and activated each MCR tested in this study, with partial agonist activity at MC5R and the lowest level of full agonist activity at MC2R, which distinguished it from synthetic MCR agonists. The minimal activity of Acthar Gel at MC2R corresponded to lower endogenous corticosteroid production.

There is an increasing pressure to identify feed additives which increase productivity or decrease methane emissions. This paper aims to elucidate the effects of supplementing a specific essential oils blend Agolin ® Ruminant on the productivity of dairy cows in comparison to non-treated animals. A total of 23 in vivo studies were identified in which Agolin was supplemented at 1 g/d per cow; then a meta-analysis was performed to determine the response ratio on milk yield, rumen fermentation, methane emissions and health. Results indicated that an adaptation period of at least 4 weeks of treatment is required. Whereas short-term studies showed minor and inconsistent effects of Agolin, long-term studies (>4 weeks of treatment) revealed that Agolin supplementation increases milk yield (+3.6%), fat and protein corrected milk (+4.1%) and feed efficiency (+4.4%) without further changes in milk composition and feed intake. Long-term treatment also decreased methane production per day (-8.8%), per dry matter intake (-12.9%) and per fat and protein corrected milk yield (-9.9%) without changes in rumen fermentation pattern. In conclusion, despite the mode of action is still unclear and the small number of studies considered, these findings show that Agolin represents an encouraging alternative to improve productivity in dairy cows.

Essential oils (EO) are widely used in foods as flavoring and preservative agents. Many of the biological activities of EO have been attributed to major essential oil compounds (EOC) but their direct interaction with colonic epithelial cells and their genotoxic and genoprotective effects are not well established. In this study, the cytotoxicity and genotoxicity of EOC including nerolidol, thymol, geraniol, methylisoeugenol, eugenol, linalool, and a commercial blend (Agolin) were determined. Furthermore, the genoprotective effects of EOC against oxidative and methylating damage were assessed using the comet assay in HT-29 colorectal adenocarcinoma cells. The majority of EOC were cytotoxic to HT-29 cells at or above 250 ppm after 24 hr exposure. At noncytotoxic doses, none of the EOC was genotoxic in the comet assay. Genoprotection against oxidative DNA damage was observed for nerolidol (at 62.5 ppm), thymol (at 12.5 ppm), geraniol, and methylisoeugenol (both at 125 ppm), as well as linalool and Agolin (both at 250 ppm). Thymol was the most protective compound against oxidative DNA damage and geraniol (at 125 ppm) also protected cells against methylating DNA damage. This study highlights the potential of EOC such as thymol to protect the colonic epithelium against oxidative DNA damage and geraniol against methylating DNA damage. Further in vivo studies are needed to confirm these findings for safety and efficacy to exploit their potential pharmaceutical or nutraceutical uses for colonic health., (© 2019 Institute of Food Technologists®.)

Aim: To calculate the first Austrian avalanche survival curve and update a Swiss survival curve to explore survival patterns in the Alps., Methods: Avalanche accidents occurring between 2005/06 and 2012/13 in Austria and Switzerland were collected. Completely buried victims (i.e. burial of the head and chest) in open terrain with known outcome (survived or not survived) were included in the analysis. Extrication and survival curves were calculated using the Turnbull algorithm, as in previous studies., Results: 633 of the 796 completely buried victims were included (Austria n=333, Switzerland n=300). Overall survival was 56% (Austria 59%; Switzerland 52%; p=0.065). Time to extrication was shorter in Austria for victims buried ≤60min (p<0.001). The survival curves were similar and showed a rapid initial drop in survival probability and a second drop to 25-28% survival probability after burial duration of ca. 35min, where an inflection point exists and the curve levels off. In a logistic regression analysis, both duration of burial and burial depth had an independent effect on survival. Victims with an air pocket were more likely to survive, especially if buried >15min., Conclusion: The survival curves resembled those previously published and support the idea that underlying survival patterns are reproducible. The results are in accordance with current recommendations for management of avalanche victims and serve as a reminder that expedient companion rescue within a few minutes is critical for survival. An air pocket was shown to be a positive prognostic factor for survival., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Objective: Avalanches are the primary hazard for winter backcountry recreationists and cause numerous deaths and injuries annually. Although recreationists usually travel in groups, there is little empirical knowledge on group-related risk factors. This study aims to explore the relative risk of avalanche accidents with respect to group size and to discuss underlying reasons for different risk levels., Methods: We compared backcountry usage data in regions in Switzerland and Italy with avalanche accident data in these regions., Results: We found higher avalanche risk for groups of 4 or more people and lower risk for people traveling alone and in groups of 2. The relative risk of group size 4, 5, and 6 was higher compared with the reference group size of 2 in the Swiss and Italian dataset. The relative risk for people traveling alone was not significantly different compared with the reference group size of 2 in the Italian dataset but was lower in the Swiss dataset., Conclusions: These findings are in accordance with avalanche safety recommendations regarding the higher risk of large groups but not regarding lower risks of people traveling alone in avalanche terrain, which is not recommended and requires great caution. Further studies on backcountry usage are necessary to improve our understanding of human behavior and risk factors. New techniques (eg, video monitoring) may be useful for acquiring reliable data on backcountry usage., (Copyright © 2016 Wilderness Medical Society. Published by Elsevier Inc. All rights reserved.)

A static batch culture system inoculated with human faeces was used to determine the influence of essential oil compounds (EOCs) on mixed faecal microbiota. Bacteria were quantified using quantitative PCR of 16S rRNA genes. Incubation for 24 h of diluted faeces from six individuals caused enrichment of Bifidobacterium spp., but proportions of other major groups were unaffected. Thymol and geraniol at 500 p.p.m. suppressed total bacteria, resulting in minimal fermentation. Thymol at 100 p.p.m. had no effect, nor did eugenol or nerolidol at 100 or 500 p.p.m. except for a slight suppression of Eubacterium hallii. Methyl isoeugenol at 100 or 500 p.p.m. suppressed the growth of total bacteria, accompanied by a large fall in the molar proportion of propionate formed. The relative abundance of Faecalibacterium prausnitzii was unaffected except with thymol at 500 p.p.m. The ability of EOCs to control numbers of the pathogen Clostridium difficile was investigated in a separate experiment, in which the faecal suspensions were amended by the addition of pure culture of C. difficile. Numbers of C. difficile were suppressed by thymol and methyl isoeugenol at 500 p.p.m. and to a lesser extent at 100 p.p.m. Eugenol and geraniol gave rather similar suppression of C. difficile numbers at both 100 and 500 p.p.m. Nerolidol had no significant effect. It was concluded from these and previous pure-culture experiments that thymol and geraniol at around 100 p.p.m. could be effective in suppressing pathogens in the small intestine, with no concern for beneficial commensal colonic bacteria in the distal gut., (© 2015 The Authors.)

Aim: Asphyxia is the primary cause of death among avalanche victims. Avalanche airbags can lower mortality by directly reducing grade of burial, the single most important factor for survival. This study aims to provide an updated perspective on the effectiveness of this safety device., Methods: A retrospective analysis of avalanche accidents involving at least one airbag user between 1994 and 2012 in Austria, Canada, France, Norway, Slovakia, Switzerland and the United States. A multivariate analysis was used to calculate adjusted absolute risk reduction and estimate the effectiveness of airbags on grade of burial and mortality. A univariate analysis was used to examine causes of non-deployment., Results: Binomial linear regression models showed main effects for airbag use, avalanche size and injuries on critical burial, and for grade of burial, injuries and avalanche size on mortality. The adjusted risk of critical burial is 47% with non-inflated airbags and 20% with inflated airbags. The adjusted mortality is 44% for critically buried victims and 3% for non-critically buried victims. The adjusted absolute mortality reduction for inflated airbags is -11 percentage points (22% to 11%; 95% confidence interval: -4 to -18 percentage points) and adjusted risk ratio is 0.51 (95% confidence interval: 0.29 to 0.72). Overall non-inflation rate is 20%, 60% of which is attributed to deployment failure by the user., Conclusion: Although the impact on survival is smaller than previously reported, these results confirm the effectiveness of airbags. Non-deployment remains the most considerable limitation to effectiveness. Development of standardized data collection protocols is encouraged to facilitate further research., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

It is known that cyclooxygenase-2 (COX-2) inhibition elicits significant renal hemodynamics alterations when sodium intake is low. However, the mechanisms involved in these renal changes are not well known. Our objective was to evaluate the role of angiotensin II and 5-lipooxygenase-derived metabolites in the renal effects induced by prolonged COX-2 inhibition when sodium intake is low. Conscious dogs were treated during 7 days with a COX-2 inhibitor (1 mg·kg·d, SC75416), and either a vehicle, an AT1 receptor antagonist (0.4 mg · kg · d, candesartan) or a selective 5-lipooxygenase inhibitor (PF-150, 20 and 60 mg · kg · d). The administration of SC75416 alone induced significant changes in renal blood flow (219 ± 14 to 160 ± 10 mL/min), glomerular filtration rate (51 ± 2 to 42 ± 3 mL/min), and plasma potassium (pK) (4.3 ± 0.1 to 4.6 ± 0.1 mEq/L). Similar decrements in renal blood flow (27%) and glomerular filtration rate (20%) and a similar increment in pK (7%) were found when SC75416 was administered in candesartan-pretreated dogs. However, SC75416 administration did not elicit significant changes in renal hemodynamics and pK in dogs pretreated with each dose of PF-150. Our data suggest that leukotrienes but not angiotensin II are involved in the renal effects induced by COX-2 inhibition when sodium intake is low.

Inhibition of mPGES-1, the terminal enzyme in the arachidonic acid/COX pathway to regulate the production of pro-inflammatory prostaglandin PGE2, is considered an attractive new therapeutic target for safe and effective anti-inflammatory drugs. The discovery of a novel series of orally active, selective benzoxazole piperidinecarboxamides as mPGES-1 inhibitors is described. Structure-activity optimization of lead 5 with cyclohexyl carbinols resulted in compound 12, which showed excellent in vitro potency and selectivity against COX-2, and reasonable pharmacokinetic properties. Further SAR studies of the benzoxazole ring substituents lead to a novel series of highly potent compounds with improved PK profile, including 23, 26, and 29, which were effective in a carrageenan-stimulated guinea pig air pouch model of inflammation. Based on its excellent in vitro and in vivo pharmacological, pharmacokinetic and safety profile and ease of synthesis, compound 26 (PF-4693627) was advanced to clinical studies., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The microbiota of the intestinal tract plays an important role in colonic health, mediating many effects of dietary components on colonic health and during enteric infections. In the context of the increasing incidence of antibiotic resistance in gut bacteria, complementary therapies are required for the prevention and treatment of enteric infections. Here we report the potential application of essential oils (EO) and pure EO compounds to improve human gut health. Nerolidol, thymol, eugenol and geraniol inhibited growth of the pathogens Escherichia coli O157 : H7(VT(-)), Clostridium difficile DSM1296, Clostridium perfringens DSM11780, Salmonella typhimurium 3530 and Salmonella enteritidis S1400 at a half-maximal inhibitory concentration (IC(50)) varying from 50 to 500 p.p.m. Most EO showed greater toxicity to pathogens than to commensals. However, the beneficial commensal Faecalibacterium prausnitzii was sensitive to EO at similar or even lower concentrations than the pathogens. The EO showed dose-dependent effects on cell integrity, as measured using propidium iodide, of Gram-positive bacteria. These effects were not strongly correlated with growth inhibition, however, suggesting that cell membrane damage occurred but was not the primary cause of growth inhibition. Growth inhibition of Gram-negative bacteria, in contrast, occurred mostly without cell integrity loss. Principal component analysis showed clustering of responses according to bacterial species rather than to the identity of the EO, with the exception that responses to thymol and nerolidol clustered away from the other EO. In conclusion, the selective effects of some EO might have beneficial effects on gut health if chosen carefully for effectiveness against different species.

Autotaxin is the enzyme responsible for the production of lysophosphatidic acid (LPA) from lysophosphatidyl choline (LPC), and it is up-regulated in many inflammatory conditions, including but not limited to cancer, arthritis, and multiple sclerosis. LPA signaling causes angiogenesis, mitosis, cell proliferation, and cytokine secretion. Inhibition of autotaxin may have anti-inflammatory properties in a variety of diseases; however, this hypothesis has not been tested pharmacologically because of the lack of potent inhibitors. Here, we report the development of a potent autotaxin inhibitor, PF-8380 [6-(3-(piperazin-1-yl)propanoyl)benzo[d]oxazol-2(3H)-one] with an IC(50) of 2.8 nM in isolated enzyme assay and 101 nM in human whole blood. PF-8380 has adequate oral bioavailability and exposures required for in vivo testing of autotaxin inhibition. Autotaxin's role in producing LPA in plasma and at the site of inflammation was tested in a rat air pouch model. The specific inhibitor PF-8380, dosed orally at 30 mg/kg, provided >95% reduction in both plasma and air pouch LPA within 3 h, indicating autotaxin is a major source of LPA during inflammation. At 30 mg/kg PF-8380 reduced inflammatory hyperalgesia with the same efficacy as 30 mg/kg naproxen. Inhibition of plasma autotaxin activity correlated with inhibition of autotaxin at the site of inflammation and in ex vivo whole blood. Furthermore, a close pharmacokinetic/pharmacodynamic relationship was observed, which suggests that LPA is rapidly formed and degraded in vivo. PF-8380 can serve as a tool compound for elucidating LPA's role in inflammation.

Background: Within Europe and North America, the median annual mortality from snow avalanches between 1994 and 2003 was 141. There are two commonly used rescue devices: the avalanche transceiver, which is intended to speed up locating a completely buried person, and the avalanche airbag, which aims to prevent the person from being completely buried., Objective: This retrospective study aimed to evaluate whether these avalanche rescue devices had an effect on mortality., Methods: The study population was 1504 persons who were involved in 752 avalanches either in Switzerland from 1990 to 2000 and from 2002 to 2003 (1296 persons, 86.2%) or in Austria from 1998 to 2004 (208 persons, 13.8%)., Results: Persons equipped with an avalanche airbag had a lower chance of dying (2.9% versus 18.9%; P=0.026, OR 0.09, n=1504). In persons who were completely buried, without visible or audible signs at the surface and who did not rescue themselves (n=317), we found a lower median duration of burial (25min versus 125min; P<0.001) and mortality (55.2% versus 70.6%; P<0.001, OR 0.26) in those using an avalanche transceiver than in those not using the device., Conclusions: Our data showed that both the avalanche airbag and the avalanche transceiver reduce mortality. However, to improve the evaluation of rescue devices in the future, the data collection procedures should be reviewed and prospective trials should be considered, as the reliability of retrospective studies is limited.

Cyclooxygenase-2 (COX-2) overexpression is a widely recognized feature of human breast cancer and inhibitors of the enzyme have antitumor effects in a subset of tumor settings. Previously, we demonstrated that direct overexpression of COX-2 under control of the mammary-specific MMTV promoter/enhancer, was itself oncogenic and lead to the induction of mammary tumors in multiparous, outbred CD1 mice. In the present study, we provide evidence that COX-2 dependent tumor progression can also be studied in FVB/N, an inbred strain widely used for analysis of breast cancer progression. In these mice, the human COX-2 transgene was strongly induced during pregnancy/lactation and mammary tumors developed after multiple pregnancies. However, crossing the COX-2 FVB/N mice with the C57BL6 strain resulted in loss of the mammary tumorigenic phenotype despite the fact that the human COX-2 gene was induced. Treatment of the COX-2 transgenic mice in the FVB/N strain with celecoxib (1600 ppm), a COX-2 selective inhibitor, resulted significant reduction in tumor size and multiplicity when compared to transgenic mice fed with control chow. SC-560 (20 ppm), a COX-1 selective inhibitor did not have significant effect on tumorigenesis. These studies suggest that FVB/N is a susceptible mouse strain well suited to the study of COX-2 mediated tumor progression and may provide a tool for the identification of interacting genes and therapeutic treatments for this clinically important target.

Arachidonic acid is an important polyunsaturated fatty acid involved in cell signaling. It is derived primarily from dietary linoleic acid, and the rate-limiting step in its biosynthesis is the initial desaturation of linoleic acid via Delta-6 desaturase. Evidence suggests that downstream metabolic products of arachidonic acid, e.g. prostaglandins, are involved in colorectal cancer, but involvement of the biosynthetic pathway of arachidonic acid has not been previously investigated. In the present study, we report the effects of a novel selective Delta-6 desaturase inhibitor, SC-26196, on tumorigenesis in two in vivo models of intestinal cancer. SC-26196 treatment resulted in 36-37% fewer tumors in Apc(Min/+) mice and 35% decrease in primary tumor size in nude mice bearing HT-29 human colon cancer cell xenografts (P<0.05). As expected, SC-26196 treatment resulted in significantly higher linoleic acid levels in tissue phospholipids and lower levels of arachidonic acid. The effects on both tissue fatty acid composition and tumorigenesis in Apc(Min/+) mice were abrogated by concomitant treatment with dietary arachidonic acid, indicating that the observed effects were due to interference with the biosynthetic pathway of arachidonic acid.

We provide evidence that cyclooxygenase (COX)-2-derived prostaglandins contribute to tumor growth by inducing newly formed blood vessels (neoangiogenesis) that sustain tumor cell viability and growth. COX-2 is expressed within human tumor neovasculature as well as in neoplastic cells present in human colon, breast, prostate, and lung cancer biopsy tissue. COX-1 is broadly distributed in normal, as well as in neoplastic, tissues. The contribution of COX-2 to human tumor growth was indicated by the ability of celecoxib, an agent that inhibits the COX-2 enzyme, to suppress growth of lung and colon tumors implanted into recipient mice. Mechanistically, celecoxib demonstrated a potent antiangiogenic activity. In a rat model of angiogenesis, we observe that corneal blood vessel formation is suppressed by celecoxib, but not by a COX-1 inhibitor. These and other data indicate that COX-2 and COX-2-derived prostaglandins may play a major role in development of cancer through numerous biochemical mechanisms, including stimulation of tumor cell growth and neovascularization. The ability of celecoxib to block angiogenesis and suppress tumor growth suggests a novel application of this anti-inflammatory drug in the treatment of human cancer.

We investigated the mechanisms by which caffeic acid phenethyl ester (CAPE), a phenolic antioxidant, inhibited the stimulation of prostaglandin (PG) synthesis in cultured human oral epithelial cells and in an animal model of acute inflammation. Treatment of cells with CAPE (2.5 microg/ml) suppressed phorbol ester (12-O-tetradecanoylphorbol-13-acetate; TPA) and calcium ionophore (A23187)-mediated induction of PGE2 synthesis. This relatively low concentration of CAPE did not affect amounts of cyclooxygenase (COX) enzymes. CAPE nonselectively inhibited the activities of baculovirus-expressed hCOX-1 and hCOX-2 enzymes. TPA- and A23187-stimulated release of arachidonic acid from membrane phospholipids was also suppressed by CAPE (4-8 microg/ml). Higher concentrations of CAPE (10-20 microg/ml) suppressed the induction of COX-2 mRNA and protein mediated by TPA. Transient transfections using human COX-2 promoter deletion constructs were performed; the effects of TPA and CAPE were localized to a 124-bp region of the COX-2 promoter. In the rat carrageenan air pouch model of inflammation, CAPE (10-100 mg/kg) caused dose-dependent suppression of PG synthesis. Amounts of COX-2 in the pouch were markedly suppressed by 100 mg/kg CAPE but were unaffected by indomethacin. These data are important for understanding the anticancer and anti-inflammatory properties of CAPE.

A series of sulfonamide-substituted 4,5-diarylthiazoles was prepared via three synthetic routes as selective COX-2 inhibitors. Recently in the synthesis of selective COX-2 inhibitors we have discovered that the sulfonamide moiety is a suitable replacement for the methylsulfonyl moiety yielding compounds with activity both in vitro and in vivo.

The enzymes cyclooxygenase-1 and cyclooxygenase-2 (COX-1 and COX-2) catalyze the conversion of arachidonic acid to prostaglandin (PG) H2, the precursor of PGs and thromboxane. These lipid mediators play important roles in inflammation and pain and in normal physiological functions. While there are abundant data indicating that the inducible isoform, COX-2, is important in inflammation and pain, the constitutively expressed isoform, COX-1, has also been suggested to play a role in inflammatory processes. To address the latter question pharmacologically, we used a highly selective COX-1 inhibitor, SC-560 (COX-1 IC50 = 0.009 microM; COX-2 IC50 = 6.3 microM). SC-560 inhibited COX-1-derived platelet thromboxane B2, gastric PGE2, and dermal PGE2 production, indicating that it was orally active, but did not inhibit COX-2-derived PGs in the lipopolysaccharide-induced rat air pouch. Therapeutic or prophylactic administration of SC-560 in the rat carrageenan footpad model did not affect acute inflammation or hyperalgesia at doses that markedly inhibited in vivo COX-1 activity. By contrast, celecoxib, a selective COX-2 inhibitor, was anti-inflammatory and analgesic in this model. Paradoxically, both SC-560 and celecoxib reduced paw PGs to equivalent levels. Increased levels of PGs were found in the cerebrospinal fluid after carrageenan injection and were markedly reduced by celecoxib, but were not affected by SC-560. These results suggest that, in addition to the role of peripherally produced PGs, there is a critical, centrally mediated neurological component to inflammatory pain that is mediated at least in part by COX-2.

Intraperitoneal administration of urate crystals to mice reduced subsequent macrophage conversion of arachidonic acid (AA) to prostaglandins (PGs) and 12-hydroxyeicosatetraenoic acid for up to 6 h. In contrast, levels of 12-hydroxyheptadecatrienoic acid (12-HHT) were markedly elevated. This metabolic profile was previously observed in vitro when recombinant cyclooxygenase (COX) enzymes were incubated with reduced glutathione (GSH). Analysis of peritoneal GSH levels revealed a fivefold elevation after urate crystal administration. The GSH synthesis inhibitor L-buthionine-[S,R]-sulfoximine partially reversed the urate crystal effect on both GSH elevation and PG synthesis. Moreover, addition of exogenous GSH to isolated peritoneal macrophages shifted AA metabolism from PGs to 12-HHT. Urate crystal administration reduced COX-1, but induced COX-2 expression in peritoneal cells. The reduction of COX-1 may contribute to the attenuation of PG synthesis after 1 and 2 h, but PG synthesis remained inhibited up to 6 h, when COX-2 levels were high. Overall, our results indicate that elevated GSH levels inhibit PG production in this model and provide in vivo evidence for the role of GSH in the regulation of PG biosynthesis.

A novel series of terphenyl methyl sulfones and sulfonamides have been shown to be highly potent and selective cyclooxygenase-2 (COX-2) inhibitors. The sulfonamide analogs 17 and 21 were found to be much more potent COX-2 inhibitors and orally active anti-inflammatory agents than the corresponding methyl sulfone analogs 16 and 20, respectively, albeit with some decrease in COX-2 selectivity. Structure-activity relationship studies have determined that incorporation of two fluorine atoms in the central phenyl group, as in 20 and 21, is extremely advantageous for both in vitro COX-2 potency and selectivity as well as in vivo activity. Several noticeable examples in the 1,2-diaryl-4,5-difluorobenzenesulfonamide series are 21a-c,k,l,n (COX-2, IC50 = 0.002-0.004 microM), in which all have in vitro COX-1/COX-2 selectivity > 1000. In addition, sulfonamides 21a,b,d,g,j,m,n,q were shown to have greatly enhanced oral activity with more than 90% inhibition of prostaglandin E2 production in the air pouch model of inflammation. Furthermore, sulfonamide 21b was found to be very active in the rat adjuvant-induced arthritis model (ED50 = 0.05 mg/kg) and carrageenan-induced hyperalgesia assay (ED50 = 38.7 mg/kg) with no indication of gastrointestinal toxicity in rats at doses as high as 200 mg/kg.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.

ADP-ribose polymers were isolated from living mammalian cells, separated by polyacrylamide gel electrophoresis, visualized in the gel with a novel silver staining agent, and quantified by computer-aided scanning densitometry. This method detects as little as approximately 40 fmol of ADP-ribose polymers of a particular size class and reduces the gel exposure times required for conventionally radiolabeled polymers from 2 months to about 1 h. The method also detects polymers with slow turnover which may be underestimated by techniques requiring metabolic radiolabeling of poly(ADP-ribose).

The molecular mechanisms whereby poly(ADP-ribosyl)ation primes chromatin proteins for an active role in DNA excision repair are not understood. The prevalent view is that the covalent linkage of ADP-ribose polymers is essential for the modification of target protein function. By contrast, we have focused on the possibility that ADP-ribose polymers interact non-covalently with nuclear proteins and thereby modulate their function. The results show that ADP-ribose polymers engage in highly specific and strong non-covalent interactions with a small number of nuclear proteins, predominantly histones, and among these only with specific polypeptide domains. The binding affinities were largely determined by two factors, ie the polymer sizes and the presence of branches. This provides an explanation for the target specificity of the histone shuttle mechanism that was previously reported by our laboratory. Interestingly, the polymer molecules being most effective in protein targeting in vitro, are strictly regulated in mammalian cells during DNA repair in vivo.

Acute inflammatory stimuli elevate both the production of prostaglandins and the synthesis and activity of prostaglandin synthase/cyclooxygenase enzyme (COX) in murine peritoneal macrophages. Adrenalectomy also elevates prostaglandin production, COX synthesis and COX activity in these cells. We have utilized cDNA probes and antisera specific for the products of the prostaglandin synthase/cyclooxygenase-1 (COX-1) and TIS10/prostaglandin synthase-2/cyclooxygenase-2 (COX-2) genes to demonstrate that adrenalectomy causes elevation of mRNA and protein from the COX-2 gene, but not from the COX-1 gene, in peritoneal macrophages. Dexamethasone replacement suppressed the elevation of COX-2 mRNA message, COX-2 protein and the increased COX enzyme activity observed in adrenalectomized animals. In contrast, both COX-1 message and COX-1 protein levels were unaffected either by adrenalectomy or by dexamethasone administration. Thus, under normal physiological conditions, tonic glucocorticoid inhibition appears to play a major role in the in vivo regulation of the COX-2 gene. These data are consistent with COX-1 being the constitutive, housekeeping enzyme in macrophages in normal physiological conditions and with the enhanced prostaglandin synthesis seen after an inflammatory stimulus resulting from the rapid induction and activity of COX-2.

We have examined the role of cyclooxygenase 2 (COX-2) in a model of inflammation in vivo. Carrageenan administration to the subcutaneous rat air pouch induces a rapid inflammatory response characterized by high levels of prostaglandins (PGs) and leukotrienes in the fluid exudate. The time course of the induction of COX-2 mRNA and protein coincided with the production of PGs in the pouch tissue and cellular infiltrate. Carrageenan-induced COX-2 immunoreactivity was localized to macrophages obtained from the fluid exudate as well as to the inner surface layer of cells within the pouch lining. Dexamethasone inhibited both COX-2 expression and PG synthesis in the fluid exudate but failed to inhibit PG synthesis in the stomach. Furthermore, NS-398, a selective COX-2 inhibitor, and indomethacin, a nonselective COX-1/COX-2 inhibitor, blocked proinflammatory PG synthesis in the air pouch. In contrast, only indomethacin blocked gastric PG and, additionally, produced gastric lesions. These results suggest that inhibitors of COX-2 are potent antiinflammatory agents which do not produce the typical side effects (e.g., gastric ulcers) associated with the nonselective, COX-1-directed antiinflammatory drugs.

After Zn2+ finger-mediated binding to a DNA break, poly(ADP-ribose) polymerase becomes automodified with long polymers of ADP-ribose. These nucleic acid-like polymers may facilitate DNA repair by noncovalently interacting with neighboring proteins. Using a novel screening technique, we have identified histones as the predominant poly(ADP-ribose)-binding species in human keratinocytes, rat hepatocytes, frog eggs, and yeast. Polymer binding is confined specifically to the histone domains responsible for DNA condensation, i.e. histone tails. Our results indicate that polymers of ADP-ribose are targeted to sites of DNA strand breaks by poly(ADP-ribose) polymerase and subsequently function to alter chromatin conformation through noncovalent interactions with histone tails.

Polymers of ADP-ribose bind chromatosomal histones in solution and may play a role in chromatin accessibility in vivo. We have enzymatically synthesized a poly(ADP-ribose) affinity resin to further characterize binding of nuclear proteins to ADP-ribose polymers. NAD+- and (ADP-ribose)-derivatized agarose beads were recognized as polymer acceptors by the nuclear enzyme poly(ADP-ribose) polymerase. This polymerase elongated the existing ligands by successive addition of exogenously available ADP-ribose residues to form polymers covalently linked to the agarose beads. Poly(ADP-ribose) formation on the beads was dependent on incubation time and the mode of ligand attachment to the agarose. The resulting poly(ADP-ribose)-derivatized agarose beads possessed polymers which closely resembled those modifying the ADP-ribose polymerase by the automodification reaction. Fractionation of rat liver nuclear lysate over the poly(ADP-ribose) resin revealed a strong affinity of H1 for ADP-ribose polymers, thereby supporting a role for poly(ADP-ribose) in chromatin functions. Poly(ADP-ribose)-agarose beads are extremely stable and will be useful not only for affinity studies, but also for mechanistic studies involving polymer elongation and catabolism.

The effect of endogenous glucocorticoids on the expression of the cyclooxygenase enzyme was studied by contrasting cyclooxygenase expression and prostanoid synthesis in adrenalectomized and sham-adrenalectomized mice with or without the concurrent administration of endotoxin. Peritoneal macrophages obtained from adrenalectomized mice showed a 2- to 3-fold induction in cyclooxygenase synthesis and activity when compared to sham controls. Intravenous injection of a sublethal dose of endotoxin (5 micrograms/kg) further stimulated cyclooxygenase synthesis, resulting in a 4-fold increase in prostaglandin production. Similar cyclooxygenase induction can be achieved in macrophages obtained from normal mice but only after high doses of endotoxin (2.5 mg/kg) that are 100% lethal to adrenalectomized mice. Restoration of glucocorticoids in adrenalectomized animals with dexamethasone completely inhibited the elevated cyclooxygenase and protected these animals from endotoxin-induced death. In contrast, no signs of cyclooxygenase induction were observed in the kidneys of the adrenalectomized mice, even when treated with endotoxin. Dexamethasone did not affect the constitutive cyclooxygenase activity and prostaglandin production present in normal and adrenalectomized kidneys. These data indicate the existence of a constitutive cyclooxygenase that is normally present in most cells and tissues and is unaffected by steroids and of an inducible cyclooxygenase that is expressed only in the context of inflammation by proinflammatory cells, like macrophages, and that is under glucocorticoid regulation. Under normal physiological conditions glucocorticoids maintain tonic inhibition of inducible cyclooxygenase expression. Depletion of glucocorticoids or the presence of an inflammatory stimulus such as endotoxin causes rapid induction of this enzyme, resulting in an exacerbated inflammatory response that is often lethal.

The poly(ADP-ribosyl)ation system of higher eukaryotes produces multiple ADP-ribose polymers of distinct sizes which exhibit different binding affinities for histones. Although precipitation with trichloroacetic acid (TCA) is the standard procedure for isolation of poly(ADP-ribose) from biological material, we show here that poly(ADP-ribose) is not stable under acidic conditions. Storage of poly(ADP-ribose) as TCA pellets results in acid hydrolysis of polymers, the extent of which is dependent on storage time and temperature. The alpha-glycosidic, inter-residue bonds are the preferred sites of attack, thus reducing polymer sizes by integral numbers of ADP-ribose to yield artefactually more and smaller polymers than originally present. Therefore, poly(ADP-ribosyl)ation studies involving TCA precipitation, histone extraction with acids, or acidic incubations of ADP-ribose polymers must account for the impact of acids on resulting polymer populations.

1. Probucol was administered to mature Watanabe heritable hyperlipidaemic (WHHL) rabbits (approximately 9 months old). Groups of WHHL rabbits were randomly selected and treated as follows: Group 1 killed at 9 months (n = 9); Group II placed on sham-treated diet at 9 months and followed for 6 months (n = 8); Group III placed on probucol at 9 months and followed for 6 months (n = 8). Probucol was administered by mixing 1% wt/wt drug with standard laboratory diet. 2. Plasma concentrations of probucol increased to 93 +/- 11 micrograms ml-1 in Group III during the initial 2 weeks and increased further to 149 +/- 24 micrograms ml-1 at the end of the treatment period. 3. Plasma concentrations of total cholesterol, unesterified cholesterol and phospholipids were significantly reduced overall by probucol, while triglycerides were not affected. 4. No statistically significant differences were observed in the presence of oxidized products in low density lipoproteins (LDL) isolated from plasma of controls compared to probucol-treated rabbits. However, LDL from probucol-treated animals was resistant to oxidation in the presence of Cu2+ (3 microM). 5. Group I had aortic atherosclerosis covering 70 +/- 5% of intimal area of thoracic aortae, that increased to 91 +/- 3% in Group II. This was associated with cholesterol contents of aortae increasing from 1.4 +/- 0.2 microgram mg-1 in Group I to 2.7 +/- 0.3 microgram mg-1 in Group II. Probucol administration did not produce a statistically significant reduction of atherosclerotic lesion area (78 +/- 7%). However, probucol treatment reduced cholesterol content to 1.9 + 0.3,ugmg-' (P < 0.01). Collagen content of aortae was not affected by probucol treatment. 6. Thus, while probucol did not promote regression, the drug did retard the continued deposition of cholesterol esters into atherosclerotic lesions of mature WHHL rabbits.

We have studied the effect of glucocorticoids administered in vivo on the activity and synthesis of the cyclooxygenase enzyme (COX) in mice treated with or without concurrent intravenous administration of LPS. Mouse peritoneal macrophages from LPS-treated animals showed a two to three fold increase in COX activity determined by the production of PGE2 and PGI2 after stimulation of the cells with exogenous arachidonate. Dexamethasone injected simultaneously with LPS, 12 h before killing of the animal and removal of the macrophages, completely blocked the LPS-induced increase COX activity in peritoneal macrophages. The regulation observed in COX activity by LPS and dexamethasone are due primarily to changes in COX mass as determined by immunoprecipitation of [35S]methionine endogenously labeled enzyme. In contrast, the COX present in the nonadherent cells and in renal medullary microsomes obtained from the same animals, showed no significant changes between treatments. These results indicate that LPS in vivo stimulates COX synthesis in the peritoneal macrophages but not in the kidney. The effect of dexamethasone to inhibit COX synthesis is selective to the LPS-induced enzyme.

1. Probucol was administered to rabbits fed a cholesterol-enriched (2% wt/wt) diet to determine potential anti-atherogenic effects in a preparation in which the disease process is due to elevated plasma concentrations of cholesterol ester-rich very low density lipoproteins (CER-VLDL). 2. Probucol was supplemented to the diet at 1% wt/wt which resulted in plasma concentrations rising steadily to 53 +/- 8 micrograms ml-1 after 14 days, with no significant changes during continued administration. Dietary consumption and body weight gains were comparable in the drug-treated and control groups during the observation period. 3. Probucol treatment did not significantly affect plasma concentrations of total cholesterol, unesterified cholesterol, triglycerides or phospholipids. 4. The concentration of CER-VLDL in plasma and its physicochemical characteristics were not significantly changed during administration of probucol. CER-VLDL from both control and probucol-treated animals was a potent stimulant of the augmentation of the intracellular incorporation of [3H]-oleate into cholesteryl-[3H]-oleate in cultured macrophages. 5. Despite the lack of effect of probucol on concentrations of plasma lipids and the cell interaction characteristics of CER-VLDL, administration of the drug markedly decreased the extent of intimal aortic surface area covered by grossly discernible atherosclerotic lesions from 55.6 +/- 11.8% to 11.6 +/- 1.9% in thoracic sections, and from 49.1 +/- 10.2% to 7.2 +/- 0.4% in abdominal sections. Furthermore, probucol treatment significantly reduced the deposition of total cholesterol in vascular tissue. 6. Probucol reduced the extent of aortic atherosclerosis produced by diet-induced hypercholesterolemia in rabbits. This reduction occurred in the absence of any significant change in the characteristics of plasma lipoproteins that were determined. These results indicate that either there is a role of oxidation in the disease process of this animal model of atherosclerosis or that probucol is acting via a presently undefined mechanism.

The synthesis and characterization of water-soluble random copolymers containing L-aspartic acid with N5-(4-hydroxybutyl)-l-glutamine, and the thermally induced helix-coil transitions of these copolymers in water and in 0.1 N KCl, are described. The incorporation of L-aspartic acid was found to decrease the helix content of the polymer at both high and low pH, in water and also in 0.1 N KCl. The Zimm-Bragg parameters sigma and s for the helix-coil transition in poly(L-aspartic acid) in water and in 0.1 N KCl were deduced from an analysis of the melting curves of the copolymers in the manner described in earlier papers. Corrections were made for the presence of a small amount of racemized aspartic acid, using data from random copolymers containing D-aspartic acid as the guest residue. The computed values of s indicate that L-aspartic acid destabilizes helical sequences at all temperatures in the range of 0-70 degrees C. Titrations of the copolymers and of N-acetyl-N'-methyl-L-aspartic acid amide in 0.1 N KCl are described.

Atherogenic properties of low density lipoproteins (LDL) in vivo may reflect modification of lipoproteins associated with endothelial translocation and exposure to extracellular matrix and interstitial fluid. To examine whether modifications of LDL occur in vivo, lipoproteins were isolated from plasma and vascular tissue of Watanabe heritable hyperlipidemic (WHHL) rabbits. LDL was extracted from vascular tissue (LDL-V) by homogenization in the presence of a sodium carbonate buffer. Control experiments demonstrated that modifications did not occur under the preparative conditions used to release LDL from tissue. LDL-V contained less esterified cholesterol, but more cholesterol esters, than did LDL from plasma (LDL-P). The diameters of both LDL-V and LDL-P followed gaussian distributions, but LDL-V particles were smaller (20.3 +/- 0.1 and 26.3 +/- 0.1 nm). Mild lipid peroxidation was evident in LDL-V. The sphingomyelin content was increased in LDL-V, with less phosphatidylcholine than in LDL-P. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) indicated that apolipoprotein B was depleted in LDL-V, but Western blot analyses identified lower molecular weight proteins antigenically related to apolipoprotein B. LDL-V markedly stimulated cholesterol esterification in mouse peritoneal macrophages and also in rabbit alveolar macrophages, a cell type that did not respond to acetylated LDL. LDL-V was not recognized by cultured rabbit skin fibroblasts. Thus, LDL isolated from atherosclerotic vascular tissue in vivo was modified in a fashion that could confer atherogenic properties reflected by augmentation of cholesterol esterification in macrophages in vitro.