Cloning and pharmacological characterization of the guinea pig P2X7 receptor orthologue

BACKGROUND AND PURPOSE: The human, rat, and mouse P2X(7) receptors have been previously characterized, and in this study we report the cloning and pharmacological properties of the guinea pig orthologue. EXPERIMENTAL APPROACH: A cDNA encoding for the guinea pig P2X(7) receptor was isolated from a guinea pig brain library. The receptor was expressed in U-2 OS cells using the BacMam viral expression system. A monoclonal antibody was used to confirm high levels of cell surface expression and the functional properties were determined in ethidium bromide accumulation studies. KEY RESULTS: The predicted guinea pig protein is one amino acid shorter than the human and rat orthologues and over 70% identical to the rat and human receptors. In contrast to human and rat P2X(7) receptors, 2'-&3'-O-(4benzoylbenzoyl)ATP (BzATP) was a partial agonist of the guinea pig P2X(7) receptor when compared to ATP and acted as an antagonist in some assays. However, as at other species orthologues, BzATP was more potent than ATP. The guinea pig P2X(7) receptor possessed higher affinity for 1-[N,O-bis(5-isoquinoline sulphonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN62), suramin and Coomassie Brilliant Blue than human or rat P2X(7) receptors suggesting that it is pharmacologically different to other rodent or human P2X(7) receptors. CONCLUSIONS AND IMPLICATIONS: The guinea pig recombinant P2X(7) receptor displays a number of unique properties that differentiate it from the human, rat and mouse orthologues and this structural and functional information should aid in our understanding of the interaction of agonists and antagonist with the P2X(7) receptor.     

Campylobacter enteritis in Portugal: Epidemiological features and biological markers

From 1984 to 1989, stool samples from 2811 gastroenteritis cases were examined for the presence ofCampylobacter jejuni andC. Coli, Salmonella, Shigella andYersinia species. Isolation rates were:Campylobacter jejuni andC. Coli, 5.3%,Salmonella spp., 14.8%,Shigella spp., 4.6% andYersinia enterocolitica, 1.1%. Age group distribution analysis shows a higherCampylobacter isolation rate in children under one year of age. Seasonal distribution revealed a peak incidence in winter as in other Meditteranean countries. Predominant biotypes wereC. jejuni I (51%),C. jejuni II (21.5%) andC. coli I (18.8%). Antimicrobial susceptibility testing did not reveal resistance to erythromycin. Thirty of the strains harboured plasmids with 7 different profiles.     

Neurobehavioral effects of long-term occupational exposure to organic solvents: two comparable studies.

Two comparable cross-sectional studies were carried out employing the same methodology but involving two separate solvent-exposed populations (N = 90, N = 144). In each study, solvent-exposed workers were compared with age-matched controls on tests selected from the Neurobehavioral Evaluation System and on standardized questionnaire measures of symptomatology and psychiatric state. A similar pattern of results was obtained in the two studies indicating a significant effect on cognitive functioning, after controlling for confounding variables, occurring in those with more than 30 years of exposure. A more specific effect on learning processes was observed in those with more than 10 years of exposure. There were no indications in either study of a solvent-related increase in psychiatric symptoms.     

Reply to ‘Indication bias or protopathic bias?’

Aim

The aim of the study was to characterize the extent of indication bias resulting from the excessive use of NSAIDs on the days preceding a spontaneous abortion to relieve pain.

Methods

We used data from a retrospective cohort study assessing the risk for spontaneous abortions following exposure to NSAIDs. Three definitions of exposure for cases of spontaneous abortions were compared, from the first day of pregnancy until the day of spontaneous abortion and until 3 and 2 days before a spontaneous abortion. Statistical analysis was performed using multivariate time programmed Cox regression.

Results

A sharp increase was observed in the dispensation of indomethacin, diclofenac and naproxen, and a milder increase was found in the use of ibuprofen during the week before a spontaneous abortion. Non- selective COX inhibitors in general and specifically diclofenac and indomethacin were found to be associated with spontaneous abortions when the exposure period was defined until the day of spontaneous abortion (hazard ratio (HR) 1.15, 95% confidence interval (CI) 1.04, 1.28; HR 1.31, 95% CI 1.08, 1.59 and HR 3.33, 95% CI 2.09, 5.29, respectively). The effect disappears by excluding exposures occurring on the day before the spontaneous abortion for non-selective COX inhibitors and on the last week before the spontaneous abortion for indomethacin. In general, decreasing HRs were found with the exclusion of exposures occurring on the days immediately before the spontaneous abortion.

Conclusions

The increased use of NSAIDs during the last few days that preceded a spontaneous abortion to relieve pain associated with the miscarriage could bias studies assessing the association between exposure to NSAIDs and spontaneous abortions.     

Effects of serotonin in failing cardiac ventricle: signalling mechanisms and potential therapeutic implications

Previously, cardioexcitation by serotonin (5-hydroxytryptamine, 5-HT) was believed to be confined to atria in mammals including man, and mediated through 5-HT₄ receptors in pig and man, but 5-HT_2A receptors in rat. Recent studies, reviewed here, demonstrate that functional 5-HT₄ receptors can be revealed in porcine and human ventricular myocardium during phosphodiesterase inhibition, and that 5-HT₄ receptor mRNA is increased in human heart failure. In rats, functional 5-HT₄ and 5-HT_2A receptors appear in the cardiac ventricle during heart failure and mediate inotropic responses through different mechanisms. 5-HT_2A receptor signalling resembles that from α₁-adrenoceptors and causes inotropic effects through increased myosin light chain phosphorylation, resulting in Ca²⁺ sensitisation. 5-HT₄ receptor signalling resembles that from β-adrenoceptors and causes inotropic effects through a pathway involving cAMP and PKA-mediated phosphorylation of proteins involved in Ca²⁺ handling, resulting in enhanced contractility through increased Ca²⁺ availability. Cyclic AMP generated through 5-HT₄ receptor stimulation seems more efficiently coupled to increased contractility than cAMP generated through β-adrenoceptor stimulation. Increasing contractility through cAMP is considered less energy efficient than Ca²⁺ sensitisation and this may be one reason why β-adrenoceptor antagonism is beneficial in heart failure patients. Treatment of heart failure rats with the 5-HT₄ antagonist SB207266 (piboserod) resulted in potentially beneficial effects, although small. Further studies are needed to clarify if such treatment will be useful for patients with heart failure.     

5-HT4-elicited positive inotropic response is mediated by cAMP and regulated by PDE3 in failing rat and human cardiac ventricles

Background and purpose:

The left ventricle in failing hearts becomes sensitive to 5-HT parallelled by appearance of functional G_s-coupled 5-HT₄ receptors. Here, we have explored the regulatory functions of phosphodiesterases in the 5-HT₄ receptor-mediated functional effects in ventricular muscle from failing rat and human heart.

Experimental approach:

Extensive myocardial infarctions were induced by coronary artery ligation in Wistar rats. Contractility was measured in left ventricular papillary muscles of rat, 6 weeks after surgery and in left ventricular trabeculae from explanted human hearts. cAMP was quantified by RIA.

Key results:

In papillary muscles from postinfarction rat hearts, 5-HT₄ stimulation exerted positive inotropic and lusitropic effects and increased cAMP. The inotropic effect was increased by non-selective PDE inhibition (IBMX, 10 μM) and selective inhibition of PDE3 (cilostamide, 1 μM), but not of PDE2 (EHNA, 10 μM) or PDE4 (rolipram, 10 μM). Combined PDE3 and PDE4 inhibition enhanced inotropic responses beyond the effect of PDE3 inhibition alone, increased the sensitivity to 5-HT, and also revealed an inotropic response in control (sham-operated) rat ventricle. Lusitropic effects were increased only during combined PDE inhibition. In failing human ventricle, the 5-HT₄ receptor-mediated positive inotropic response was regulated by PDEs in a manner similar to that in postinfarction rat hearts.

Conclusions and implications:

5-HT₄ receptor-mediated positive inotropic responses in failing rat ventricle were cAMP-dependent. PDE3 was the main PDE regulating this response and involvement of PDE4 was disclosed by concomitant inhibition of PDE3 in both postinfarction rat and failing human hearts. 5-HT, PDE3 and PDE4 may have pathophysiological functions in heart failure.     

Physician-based surveillance of occupational disease: developing a methodology

Because of the inadequacies of surveillance data for occupational disease in the United States, this study was undertaken to determine the feasibility of using physicians' and hospital records for occupational disease surveillance. The study was based on Rutstein's sentinel health event (occupational) classification and the use of sentinel physicians. Data were gathered on nonmalignant dermatologic and respiratory diagnoses prospectively at five sites and retrospectively at four of these sites. The combined approaches identified 49 patients whose illnesses were probably work-related. Because the prospective approach took less than 10% of the time for each case identified compared with the time required for the retrospective approach, and prospective data are more timely and easier to access, the prospective approach appears to be better.     

SimSmoke Model Evaluation of the Effect of Tobacco Control Policies in Korea: The Unknown Success Story

Objectives. We evaluated the effect of strict tobacco control policies, implemented beginning in 1995 in the Republic of Korea, on smoking prevalence and deaths.Methods. SimSmoke is a simulation model of the effect of tobacco control policies over time on smoking initiation and cessation. It uses standard attribution methods to estimate lives saved as a result of new policies. After validating the model against smoking prevalence, we used it to determine the Korean policies'' effect on smoking prevalence.Results. The model predicted smoking prevalence accurately between 1995 and 2006. We estimated that 70% of the 24% relative reduction in smoking rates over that period was attributable to tobacco control policies, mainly tax increases and a strong media campaign, and that the policies will prolong 104 812 male lives by the year 2027.Conclusions. Our results document Korea''s success in reducing smoking prevalence and prolonging lives, which may serve as an example for other Asian nations. Further improvements may be possible with higher taxes and more comprehensive smoke-free laws, cessation policies, advertising restrictions, and health warnings.Many Asian nations have smoking prevalence rates among males of at least 50%, leading to a large share of the world''s 5 million deaths attributable to smoking each year.¹ Worldwide, annual tobacco-related mortality is expected to increase to 10 million by 2030,¹ with an increasing share of those deaths in Asia, unless effective tobacco control measures are implemented.Most Asian nations have signed the Framework Convention for Tobacco Control, developed through the World Health Organization. This pact advocates high cigarette taxes, smoke-free indoor air laws, cessation treatment coverage, advertising bans, health warnings, and a well-organized media campaign. Thailand has implemented many of the suggested policies and has shown remarkable success in reducing male and female smoking rates.² Success in other Asian nations has not been documented.As recently as 1995, 67% of males smoked in the Republic of Korea.³ Taxes were increased gradually in the late 1990s, and some of the funds were allocated to tobacco control. The framework was ratified by Korea in May 2005. By the end of 2006, Korea had substantially increased the tax rate on cigarettes, implemented a strong antismoking campaign, strengthened clean air laws and health warnings, and made cessation treatments more accessible.³ No previous study evaluated the effect of these policies.When more than 1 policy is implemented, it is difficult for empirical studies to distinguish each policy''s effects.⁴ Simulation models combine information from diverse sources to examine the effects of different policies over time.^4,5 To determine these effects in Korea, we adopted the SimSmoke tobacco control policy model,^4,6–8 which simultaneously considers a broader array of public policies than do other smoking models.^9–14 The model has accurately explained trends in smoking rates for the United States as a whole and for several states,^7,15–17 as well as for other nations.²We used Korean data to develop a SimSmoke model for that country. We used the model to estimate the effect of individual and combined tobacco control policies implemented between 1995 and 2006 on male smoking prevalence and deaths.     

Exploring Scenarios to Dramatically Reduce Smoking Prevalence: A Simulation Model of the Three-Part Cessation Process

Objectives. We used a simulation model to analyze whether the Healthy People 2010 goal of reducing smoking prevalence from the current 19.8% rate to 12% by 2010 could be accomplished by increasing quit attempts, increasing the use of treatments, or increasing the effectiveness of treatment.Methods. We expanded on previous versions of the tobacco control simulation model SimSmoke to assess the effects of an increase in quit attempts, treatment use, and treatment effectiveness to reduce smoking prevalence. In the model, we considered increases in each of these parameters individually and in combination.Results. Individually, 100% increases in quit attempts, treatment use, and treatment effectiveness reduced the projected 2020 prevalence to 13.9%, 16.7%, and 15.9%, respectively. With a combined 100% increase in all components, the goal of a 12% adult smoking prevalence could be reached by 2012.Conclusions. If we are to come close to reaching Healthy People 2010 goals in the foreseeable future, we must not only induce quit attempts but also increase treatment use and effectiveness. Simulation models provide a useful tool for evaluating the potential to reach public health targets.Since the 1964 Surgeon General''s Report first warned of the hazards of smoking tobacco,¹ enormous strides have been made in reducing adult smoking prevalence. At the peak of US tobacco use in 1965, the adult smoking prevalence was 42.4%²; now, only 19.8% of adults smoke.³ Nevertheless, an estimated 44.4 million American adults continue to smoke, incurring 443 000 premature deaths, with $97 billion in productivity losses and $96 billion in health care expenditures.⁴ In recognition of the problem, Healthy People 2010 set an ambitious goal of reducing smoking prevalence to 12% by 2010.⁵ With that goal now almost certainly unattainable,⁶ new approaches need to be explored.Smoking prevalence can be reduced in 3 ways: (1) by preventing nonsmokers from initiating smoking, (2) by inducing current smokers to quit, and (3) by preventing those who have already quit from relapsing back to smoking. Because prevention strategies apply largely to persons in the 14- to 20-year-old age group,⁷ only a small percentage of the population is affected at any point in time and many years must pass before the strategies lead to large reductions in adult rates.⁸ By contrast, quitting strategies can be targeted at smokers of all ages and can lead to a more immediate drop in adult prevalence. Still, encouraging smokers to quit will only go so far in reducing prevalence unless something is done in tandem to help smokers maintain their abstinence.Each year, fewer than 45% of smokers make a serious quit attempt and quit for even 24 hours.⁹ More than three fourths of smokers making a quit attempt each year do not use efficacious treatment,¹⁰ and only 3% to 5% of those untreated smokers remain abstinent for 12 months.^11,12 Quit success increases 2- to 3-fold when proven treatments are used.¹³ Those at the lowest socioeconomic levels are the most vulnerable to smoking but the least successful at quitting when making a quit attempt.^9,14–18 Thus, much is to be gained by improving treatment effectiveness along with increasing the number of smokers who attempt to quit and who use evidence-based treatment.Simulation models are useful for understanding and predicting how changes in specific inputs (e.g., treatment use) lead to changes in outputs (e.g., quit rates) over time in complex social systems.^19,20 Modeling helps to reveal relationships by organizing the channels of influence and by making assumptions about the relevant relationships more explicit. This process generally proves more robust than relying on intuition alone and is thereby useful for evaluating hypothetical future scenarios.Numerous models of smoking behaviors have been developed to show the effect of tobacco control policies on smoking prevalence and health outcomes.^8,21–28 These models use information from studies of past policies or smoking behaviors to predict future smoking rates. We were motivated by the need to better understand the changes that would be necessary to meet prevalence goals. Because changes in smoking initiation will have minimal impact on smoking prevalence within the next 15 years, we focused on the cessation process. We carried out a series of simulations that focused on the quitting process and were intended to examine hypothetical effects of changes in quitting behaviors on smoking prevalence. Specifically, we generated a model that predicted smoking prevalence by using 3 strategies: (1) by increasing the percentage of current smokers making a quit attempt, (2) by increasing the percentage of current smokers who use an evidence-based treatment, and (3) by improving treatment effectiveness. We explored the magnitude of the various strategies necessary to reach the Healthy People 2010 goal of a 12% smoking prevalence and the time frame in which it could be achieved.