It is widely recognized and accepted that job strain adversely impacts the workforce. Individual responses to stressful situations can vary greatly and it has been shown that certain people are more likely to experience high levels of stress in their job than others. Studies highlighted that there can be age differences in job strain perception.Cross-sectional postal survey of 300 Lithuanian general practitioners. Psychosocial stress was investigated with a questionnaire based on the Reeder scale. Job demands were investigated with the Karasek scale. The analysis included descriptive statistics; logistic regression beta coefficients to find out predictors and interactions between characteristics and predictors.Response rate was 66% (N = 197). Logistic regression as significant predictors for job strain assigned – duration of work in primary care; for job demands- age and duration of working in primary care; for decision latitude- age and patient load.The interactions with regard to job strain showed that GP's age and job strain are negatively associated to a low patient load. Lower decision latitude for older GP age is strongly related to higher patient load. Job demands and GP age are slightly positively related at low patient load.Lithuanian GP's have high patient load and are at risk of stress, they have high job demands and low decision latitude. Older GP's perceive less strain, lower job demands and higher decision latitude in case of low patient load. Young GP's decision latitude has week association to patient load. Regarding to the changes in patient load younger GP's perceive it more sensitively as changes in job demands. The issue of job stress is of utmost important to the public health community and working people because it adversely impacts the workforce. Strain has been considered as an environmental condition, as an appraisal of an environmental condition, as a response to an environmental condition, and as a form of relationship between environmental demands and a person's abilities to meet these demands. Although there are a lot of controversies about the epistemology of job strain, there is an agreement about it as a complex phenomenon related to health. In considering workplace-related stress, it should be recognized that stressors may occur because of individual characteristics of the worker as well as the work environment -5.In general, physicians are at risk of stress. The main experienced pressures at work were uncertainty and insecurity, isolation, poor relationships with other doctors, disillusion with the role of the general practitioner and awareness of changing demands ,7. It haThe importance of job strain understanding as a problem for the general practitioners (GP's) was yielded by Appleton  in a stuThe personal and social conditions have influences on the relationship between age and stress. Continuing problems at work and job strain mostly affects young GP's ,22. On tThe aim of this study was to investigate physician's age, duration of work in primary care and patient load interactions with job strain, decision latitude and perception of job demands.Lithuanian GP's.Cross – sectional study. A mailed survey of random national samples. Computerized random sampling was performed from the registry of Lithuanian physicians. The data collected through the questionnaires filled-in by the GP's.Total number of GP's in Lithuania at the time was 1007 GP's. Sample size was calculated using EpiInfo 2000 Statcalc software which argued the sample size of 192 GP's with the 95% confidence level. From the previous studies the expected response rate was 63%. Therefore, it was decided to send questionnaires to 300 Lithuanian GP's. Our observed response rate was 66%. We collected 197 filled-in questionnaires.Psychosocial stress in this study was investigated by a questionnaire based on the Reeder scale ,27. The A simple inversion of the Coulson scoring system table  was usedWork characteristics were measured by the Karasek's Job Content Questionnaire. This instrument has two scales that measure stressful job character – job decision latitude and psychological workload demands. This model, also known as the "job strain" model -32.Psychological workload demands were defined by questions such as "working very fast," "working very hard," "doing so many things".Job decisions latitude was measured within questions as: "always must learn for new skills", "working a lot".A four point Likert – like scale was used with the coding from 4 to 1 for series, so that the responses were summarised to give a score .Data were computed, coded and analyzed using Statistical Package for the Social Sciences for Windows version 11.0 (SPSS Inc) and Microsoft Excel 2000. The analysis included descriptive statistics; logistic regression beta coefficients were used to assess physician's age, duration of work in primary care and patient load impact on job strain, job demands and decision latitude. Results differences at the p = 0.05 level were considered as statistically significant.Of the 197 respondents, 162 (82.2%) GP's were female, and 35 (17.8%) male. This is very similar to whole GP population in Lithuania. The GP ages ranged from 31 to 66 years . GP's were investigated in 3 age groups: < 44 yr – N = 90 (45.7%); 45–54 yr – N = 85 (43.1%); 55 and > – N = 22 (11.2%).Regarding to our data in general Lithuanian GP's have high patient load and are at risk of stress, they have high job demands and low decision latitude table .The logistic regression beta coefficients showed that job strain development and higher job demands could be predicted by the shorter duration of GP practice. Otherwise older age for GP's can predict lower job demands and higher decision latitude. We found that lower decision latitude can be predicted by high patient load table .In figures the interactions are graphically presented according to the method described by Aiken  and recoThe interactions with regard to job strain fig.  shows thThe age interactions with respect to psychological job demands fig.  shows thRegarding to job decision latitude fig. , the intIn the current social and political climate Lithuanian GPs face many stressors that are peculiar to the medical profession. However there are many stressors that are also attributed to the personality. GPs are the professionals who are at the forefront of helping patients to manage urgent health problems, and as gatekeepers they have to make decisions on patient's health; whether to send them to hospitals. Sometimes it can interfere with personal life that can cause negative feelings about work, frustration, tension and lack of time to make appropriate decisions .Our study has highlighted a matrix of issues contributing to elevated levels of job strain. These issues are rarely attributable to a simple cause and effect formula but there are complex problems with the many linkages. Lithuanian GP's has indicated twofold age interaction with job strain because it depends on patient load. Work related stress development was hardly related to duration of working in primary care. GP's perceive higher job strain and higher job demands when they have shorter duration of GP practice. Older GP's are more vulnerable to job strain, when age interaction compared among low and high patient load groups. This also means different workload and job demands. It seems to be the confirmation of Cox definition of work related stress, where the concept includes an external demand and an internal perception that the response to the demand is uncomfortable: "Work related stress is a person's recognition of his/her inability to cope with demands relating to work, and his/her subsequent experience of discomfort" . We founPeterson's substantial review found that detrimental work environments had social and psychological consequences for all . He mentSeveral weaknesses of the present study have to be mentioned. As main weakness of our study we see its cross-sectional nature, which precludes an evaluation of temporal precedence and causality of the observed associations. Karasek Job Strain model guided our hypothesis about causal relationships between age, patient load and work characteristics, explored causal relations should be interpreted carefully and longitudinal studies should be carried out in the future research.Another limitation is the Karasek's Job Content Questionnaire it self. It was designed to be broadly applicable to a wide range of occupations. However, this generalisability inevitably means that factors that are specific to particular occupations may be overlooked. For example, job demands as it has been conceptualized and operationalised in this survey would not take into account some emotional demands that could be source of stress to general practitioners such as dealing with difficult patients or caring for the dying patients ,36.Third limitation is our exclusive reliance on self-reported rating scales, which raises the issue of systematic positive or negative response tendencies. Furthermore, as no scale is perfectly reliable, the associations between self-reported measures and self-reported workload appear to be weaker than they could be in reality. Several authors have argued that this phenomenon is not a major threat if interactions has been found ,37.On the positive side, our results were obtained among a sample of people working in general practice. Respondents were with similar education level that can be seen as strength of the investigation. The sample was sufficient regarding to sample size calculation and allow exploration of tendencies. The participation rate was acceptable, and the scales we used were previously validated instruments that retained their psychometric properties in our population . OtherwiLithuanian GP's have high patient load and are at risk of stress, they have high job demands and low decision latitude. Job strain development and higher job demands can be influenced by shorter duration of general practice. Older GP's perceive less strain, lower job demands and higher decision latitude in case of low patient load. Young GP's decision latitude has week association to patient load. Regarding to changes in patient load younger GP's perceive it more sensitively as changes in job demands.The author(s) declare that they have no competing interests.GV designed the study, abstracted data, made data analysis, drafted and revised the manuscript.SBA participated in initial study design, participated in data analysis and revised the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Mutual policing is an important mechanism that maintains social harmony in group-living organisms by suppressing the selfish behavior of individuals. In social insects, workers police one another (worker-policing) by preventing individual workers from laying eggs that would otherwise develop into males. Within the framework of Hamilton's rule there are two explanations for worker-policing behavior. First, if worker reproduction is cost-free, worker-policing should occur only where workers are more closely related to queen- than to worker-produced male eggs (relatedness hypothesis). Second, if there are substantial costs to unchecked worker reproduction, worker-policing may occur to counteract these costs and increase colony efficiency (efficiency hypothesis). The first explanation predicts that patterns of the parentage of males  are associated with relatedness, whereas the latter does not. We have investigated how male parentage varies with colony kin structure and colony size in 50 species of ants, bees, and wasps in a phylogenetically controlled comparative analysis. Our survey revealed that queens produced the majority of males in most of the species and that workers produced more than half of the males in less than 10% of species. Moreover, we show that male parentage does not vary with relatedness as predicted by the relatedness hypothesis. This indicates that intra- and interspecific variation in male parentage cannot be accounted for by the relatedness hypothesis alone and that increased colony efficiency is an important factor responsible for the evolution of worker-policing. Our study reveals greater harmony and more complex regulation of reproduction in social insect colonies than that expected from simple theoretical expectations based on relatedness only. Workers of social insects prevent other workers laying eggs to increase colony efficiency and not -- as traditionally thought - purely because workers are more related to the queen of the colony Major evolutionary transitions  require Apis mellifera , and related wasp species, such as Vespa crabro, show patterns of worker reproduction and worker-policing behavior that are consistent with the efficiency hypothesis but not the relatedness hypothesis  was used to make predictions about male parentage based upon colony kin structure. We included colony size in our analyses because it potentially alters expected patterns of male parentage  is dependent upon colony kin structure. Importantly, males should be worker-produced in colonies headed by single, once-mated queens, and queen-produced in colonies headed by multiple related queens, or by multiply mated queens, because worker reproduction is prevented by worker-policing. By contrast, the efficiency hypothesis predicts no association of male parentage or worker-policing with colony kin structure. In this paper we test these predictions by analyzing, using methods that control for phylogenetic dependence, how the proportion of worker-produced males (WPM) varies with both colony kin structure and colony size. The theoretical difference in relatedness of workers to queen- and worker-produced males  and likewise in the number of assignable males , suggesting that there was no systematic bias in our dataset.We found data for 50 species: 16 ants, 20 bees, and 14 wasps . WPM varetection ) that we10WPM, p = 0.016; rdiff, p < 0.001; log10 of colony size [log10nw], p < 0.001) and when within-species variation was included . This confirmed that a comparative approach using an analysis of independent contrasts was warranted , and the mean contrast in log10WPM (–1.70 ± 5.4) was not significantly different from zero when rdiff was coded categorically . Likewise, neither analysis that included within-species variation was significant . The power was high  or when relatedness was controlled for and we included only species with positive rdiff values .The WPM was not significantly correlated with colony kin structure in any of our comparative analyses. Ignoring within-species variation, the slope of the line of regression of contrast in logOur survey revealed that queens produced the majority of males in most of the species, and in less than 10% of the species did workers produce more than half of the males, in line with earlier surveys based largely on behavioral data . Since wrdiff, the analyses of relatedness revealed a tendency for a negative relationship. Importantly, our study included data from 50 species, and our power analyses showed that we had enough power to detect a relationship between male parentage and colony kin structure if it was of moderate or large effect.Our comparative study did not support the view that intra- and interspecific variation in male parentage can be accounted for by the relatedness hypothesis only. First, and most importantly, the proportion of males produced by workers was not significantly associated with colony kin structure. This was true both when within-species variation in colony kin structure was included and when it was ignored. In fact, although the relatedness hypothesis predicts a positive relationship between WPM and A second line of evidence against the relatedness hypothesis came from the finding that workers produce only very few males in a large number of species where, on purely relatedness grounds, they would benefit from producing males. Workers produce less than 10% of males in 30 of the 43 species (70%) in which workers were more related to worker-produced than to queen-produced males.Dolichovespula saxonica  and singly mated stingless bees (tribe Meliponini)  is singly mated, yet males are all queen-produced because workers police one another   and compiponini) . Howeveriponini) . Moreoveiponini) , indicatly mated , whereasly mated . However another , 2002. CTrigona and Plebeia. Finally, we would like to stress that the finding that kin structure alone cannot account for the intra- and interspecific variation in male parentage does not amount to saying that kin structure is unimportant. Rather, it may work in concert with costs as a force influencing patterns of male parentage in social insects. Thus, this study reveals greater harmony and more complex regulation of reproduction in social insect colonies than that expected from simple theoretical expectations based on relatedness alone.In conclusion, our comparative analysis does not support relatedness as the general explanation of patterns of male parentage and occurrence of worker-policing in social Hymenoptera. The concentration of published examples of worker-policing in multiply mated bees and wasps probably reflects the influence of the relatedness hypothesis on the selection of study taxa, rather than relatedness being the ultimate explanation of worker-policing. Moreover, recent studies have revealed worker-policing in species in which the relatedness hypothesis predicts males to be produced by workers . We concFor all analyses, the response variable was WPM see . For almLeptothorax nylanderi c.f. Vespula germanica [Leptothorax unifaciatus and Epimyrma ravouxi , data were for adult or pupal males. For these two exceptional species, we had estimates of WPM at only the egg stage. However, as we found few worker-laid male eggs in both species . For alcies see , our estrdiff, the theoretical difference in relatedness of workers to queen- (rw–qm) and to worker-produced males (rw–wm) /4, where rw–w is the relatedness among adult workers. For species with variation in queen number (polygyny), predictions about worker reproduction are more complicated because both queen number and queen relatedness are important  and among workers (rw–w) as rdiff = (rw–w – rw–q)/2. In our comparative analyses we used rdiff as a continuous explanatory variable, or we coded rdiff categorically as one when rdiff was greater than zero , or as zero when rdiff was less than zero .We made predictions about the parentage of males based on colony kin structure by calculating –wm) see . The relf queens , we calcmportant . For thenw; see 10nw as an explanatory variable.We defined colony size as the number of adult workers per nest nw; see . Where o10WPM, rdiff, and log10nw) by a test for serial independence , as has been suggested for phylogenies containing polytomies (http://www.r-project.org/). All other statistical tests were performed using SPSS (version 11).Analyses using  in CAIC , whereasm PDTREE . We testlytomies , becauserdiff that were mean values for each species. We calculated independent contrasts between log10WPM and rdiff, and with rdiff coded as a categorical variable. In our second set of two analyses, we included within-species variation in colony genetic structure that was present in seven species because of facultative variation in queen number or queen mating frequency , and those where rdiff was negative . We then estimated WPM for each group. We modified the phylogeny by adding an additional bifurcation at the tips corresponding to these seven species , and then compared contrasts in log10WPM with contrasts in log10nw in this subset of the data.We tested the role of colony size in two ways. First, we ignored any effect of relatedness and simply compared contrasts in logrdiff in our dataset. To do that we set WPM to 0% when rdiff was less than zero, to 100% when rdiff was greater than zero, and to 50% when rdiff was equal to zero. An analysis of independent contrasts based on this hypothetical relationship gave a highly significant relationship between WPM and rdiff both when within-species variation was ignored  and included . On the basis of these slopes, we conducted a power analysis by assuming two types of effects. We considered rdiff to have a “large” effect on WPM when β was greater than 4.0, and a “moderate” effect when β was between 2.0 and 4.0. To test the power that our analysis had to detect a large and moderate effect, we used the model y = βx + “resampled residual of y,” where x is the observed standardized contrast in rdiff and “resampled residual of y” is the residual of y estimated by resampling the distribution of residuals from our observed regressions through the origin. From this model, we defined power as the proportion of regressions (forced through the origin) in 1,000 simulated datasets that were significant at α ≤ 0.05 for a given slope β (the effect size). We investigated how power varied with effect size by increasing β incrementally from 1 to 5 in steps of 0.1 .Click here for additional data file.


S. pneumoniae and L. fermentum intranasally inoculated in a mice experimental model. The sensitivity of S. pneumoniae and L. fermentum to antibiotics was evaluated by different "in vitro" techniques. The results showed that both microorganisms have a typical pattern of sensitivity to antibiotics in these assays. The "in vivo" experiments showed that the treatment with Ampicillin increased the number of lactobacilli and neumococci in the groups of mice treated only with one of the microorganisms. In those mice treated with Lactobacillus, challenged later with neumococci and treated with Ampicillin, the pathogen in lung decreased on the 4th day, disappearing completely after on. The histological studies showed that the antibiotic treatment decreased the inflammatory response produced by the pathogen at the lung and trachea levels.Ampicillin was selected to further study the effect of this antibiotic on the colonization capability of  Streptococcus pneumoniae. Extensive antibiotic use for these infections as well as misuse for viral respiratory infections has led to increased penicillin resistance amongst the streptococci [Respiratory tract infections are commonly caused by ptococci -6. Even ptococci -10. The ptococci . There aptococci -14.S. pneumoniae which is a frequent nasopharynx colonizer. In previous papers, the isolation and identification of the microorganisms of the normal microbiota of the respiratory tract of mice was reported by our research group. Also, the evolution from the moment they were born up to two months age was published [Lactobacillus that shared some properties [Lactobacillus fermentum was selected by the beneficial probiotic properties. Later the optimal dose to produce a transitory colonization or permanence [S. pneumoniae [Lactic Acid Bacteria (LAB) has been widely used to restore the ecologic equilibrium of different areas -14, mainublished . In the operties . From thrmanence ,21 and teumoniae  intranasS. pneumoniae and L. fermentum by "in vitro" assays. Also, to study the effect of Ampicillin, orally administered in a mice experimental model, against the intranasal inoculation of S. pneumoniae, L. fermentum or both microorganisms. The "in vivo" assays were complemented with histological and cytological studies to determine if there was some type of general response of the animals to the treatment.The objective of the present paper was to study the effect of specific antimicrobial agents against the growth of L. fermentum was isolated from the respiratory tract (pharynx) of adult BALB/c mice [S. pneumoniae A6 serotype was isolated from human pneumonia-suffering subjects, and identified by standard techniques. The serotypification was performed at the "Servicio de Bacteriología Clínica, Instituto Nacional de Enfermedades Infecciosas-ANLIS "Dr Carlos G. Malbran, Buenos Aires, Argentina by Quellung technique. Pathogenicity in mice was increased by inoculating S. pneumoniae intraperitoneally [B/c mice ,20-22. TB/c mice ,20-22. Sthose antibiotics broadly used for the treatment of Gram Positive microorganisms affecting the respiratory tract were studied by determining the Minimal Inhibitory Concentration: penicillin, ampicillin, ceftriazone, ceftazidine, claritromicine, tetracicline, rifampicin, ciprofloxacin, aztreonam, cloramphenicol and imipenen.Enterococcus faecalis ATCC 29212 and Staphylococcus aureus ATCC 29213.The Minimal Inhibitory Concentration Method (MIC) recommended by the National Committee for Clinical Laboratory Standards (NCCLS) was used, replacing the culture media by MRS agar as basal media , becauseS. pneumoniae ATCC 49619.The behavior of the pathogenic microorganisms with antimicrobial substances was determined by the qualitative and quantitative techniques of diffusion and dilution in agar (MIC), according to the National Committee for Clinical Laboratory Standards, 2001 (NCCLS). The antibiotics assayed for the qualitative method were oxacillin, vancomycin, chloramphenicol, tetracycline, rifampicin, ciprofloxacin, trimetroprim-sulphametoxazol, claritromycin. The antibiotics assayed for the quantitative method were penicillin, ampicillin, ceftriazone and claritromycin. The reference strain used was Each experimental group (or assay) included 24 to 30 animals. Four to six mice were killed in each experimental day. Each one of the "in vivo" experiments was performed twice, and their data used to calculate the media and Standard Deviation. The protocols used were accepted by the Animal Ethics committee of CERELA.L. fermentum strain every 12 h. (50 μl of a suspension containing 1 × 109 CFU/ml). Later, Ampicillin was administered by the oral way in 4 doses of 100 mg/Kg/day, fractionated in two daily doses (1.5 mg/dose/mice) every 12 h. The experimental protocol is schematized in Fig groups of adult (two months old) male BALB/c mice were intranasally inoculated with 4 doses of a RR S. pneumoniae in saline solution (1 × 109 CFU/ml). The antibiotic was administered by the oral way in 4 doses of 100 mg/Kg/day, following the same scheme than before. The experimental protocol is schematized in Fig BALB/c mice were intranasally inoculated with a dose of 50 μl of a suspension of L. fermentum (4 doses of 50 μl of a suspension of 1 × 109 CFU/ml), then challenged with S. pneumoniae A6 by the same way (50 μl of a suspension of 1 × 109 CFU/ml) and later treated with Ampicillin in four doses every 12 hours as before, as shown in Fig Mice were intranasally inoculated with RR All the experimental groups were sacrificed by cervical dislocation on days 2, 4, 7 and 10 post antibiotic administrations. The samples from nasopharynx and pharynx were obtained with a cotton swab. Trachea, bronchia and lung were aseptically removed and homogenized with a Teflon pestle. The number of microorganisms was determined by the successive dilutions method with peptone water, and plated in MRS agar-Rifampicin. The plates were incubated for 24 h. at 37°C in microaerophilic environment. The identification of microorganisms was performed by macroscopic, microscopic characteristics, Gram staining and biochemical tests.the samples for histological assays were obtained from the higher trachea, located at the neck basis, bronchia in the area where the main bronchia are divided, and lung in the terminal bronchiole area and alveolar wall. They were fixed with 10% paraformaldehyde and stained with Hematoxylin-eosin and Ramon and Cajal technique . They weThe left lobule of the lung was used to perform the cytological slices. The organ was first immersed in saline solution, then fixed in methanol for 3 min, and afterwards stained with the Romanovsky method  used routinely in the lab .The numbers in the figures represent the mean and Standard Deviation of the results obtained in the two set of experiments performed for each assay. The Student's t test was applied to determine the differences statistically significant of the data.L. fermentum (ug/ml) are as follows: penicillin, ampicilin, ceftriazone, ceftazidine, rifampicin, ciprofloxacin: ≥100 ug/ml, tetraciclin, imipenem and aztreonam: 10 ug/ml, cloramphenicol 1 ug/ml, claritromicine: 0.1 ug/ml. Comparing these values with those obtained for the MIC of Enterococcus spp and Streptococcus spp, type strains recommended by NCCLS, L. fermentum would be resistant to penicillin, ampicillin, ceftriazone, rifampicin and ciprofloxacin.The MIC results for S. pneumoniae are shown in Table S. pneumoniae is sensitive to all the antibiotics assayed by both, the disc diffusion assay, and the MIC.The results of the antimicrobial sensibility of in vivo" assays at the recommended dose for human beings .from the results obtained in "in vitro" sensitivity assays, that are predictive, the antibiotic Ampicillin was selected to be used in "7–8 CFU/organ on days 1 and 4, and lower values on the following days. The higher number were in nasal and pharynx exudates. In the control mice without antibiotic, Lactobacillus numbers were around 106 CFU on the first days of the assay. On the 7th and 10th days with antibiotic treatment, Lactobacillus were still present in all the organs, except in lung, while in the mice without antibiotics they were only present on nasal and pharynx exudates on day 7 disappearing on day 10  and treated with Ampicillin showed an increased colonization of lactobacilli during all the days studied in nasal and pharynx exudates (p < 0.01), with values of 10neumococci were present a longer time in all the organs of the respiratory tract,  In the case of S. pneumoniae, the previous treatment with Lactobacillus, reduced significantly the number of pathogenic microorganism in the lung, as showed in Fig The colonization of S. pneumoniae and Ampicillin These results are resumed in Table Lactobacillus produces a stimulation of the lung macrophages, and a lymphocytic infiltration at the trachea level, as resumed in Table S. pneumoniae and antibiotic decreased the inflammatory response produced by the pathogen (picture 5b) compared with the pattern produced by the pathogen and antibiotic, represented in Fig The histological studies showed that in all the organs of the respiratory tract there are not significant structural modifications. Only a moderate leukocyte exudation is observed in the alveolar region in the mice treated with Lactobacillus, neumococci, or both. The addition of the antibiotic produces an activation of the non-specific line of defense represented by the alveolar macrophages, as shown in Table The cytological studies showed an increased number of activated macrophages by One of the main goals of our research is to go further into the mechanisms involved in the probiotic effect in the respiratory tract ,27. HaviLactobacilus intranasally increases their colonization in the respiratory tract. These results support the use of antibiotics together with probiotics, which would help in having a higher colonization of the protective microorganisms. A paper published by Dielemen et al [Our results showed that the administration of Ampicillin by the oral route and en et al  demonstrS. pneumoniae strain used was sensitive to Ampicillin in the "in vitro" test, in the experiments performed in mice produces an increase in the number of the pathogenic microorganism that are also present a longer time in the tract. The pathogen produces a lymphocytic infiltration al the tracheal level, together with an increased inflammatory response evidenced in the lung cytological studies.Coincidently with these results, the treatment with Ampicillin, even though the Lactobacillus, there is a broad discussion based on the different results obtained through the application of "in vitro" test, and the differences found in "in vivo" assays. Even though the first one can be used as screening and to predict some characteristics or properties assayed in experimental animals models, one must consider that not always they are coincident [Why the response to the pathogen is different in "in vitro" assays than in "in vivo" experiments?. Referred to the behavior of incident .Lactobacillus, neumococci and Ampicillin, the final results is that the pathogen was cleared faster from the lung supporting the combined use of lactobacilli and antibiotics in the prevention of the infections produced by this pathogen. These results are also demonstrated by the histological slides, (Fig In those experiments performed in mice treated with des, Fig  where thdes, Fig . The hisL. fermentum, S. pneumoniae, or both, indicates the highly active non-specific branch of the immune system, which is an important first line of defense against microbial invasion in the lower airways infection. This activation could also help in the clearance of the neumococci observed on the 4th day of the experiments. These results do not agree with those from one infections produced by Pseudomonas aeruginosa, that does not produce an activation of the Pulmonary Alveolar Macrophages [The activation of lung or alveolar macrophages produced by the administration of rophages . The actrophages -17.More studies must be undertaken trying to elucidate which are the mechanisms involved in the protection exerted by lactobacilli in the respiratory tract, and also which are the reasons of the increased colonization of the pathogen and lactobacilli by the effect of the Ampicillin treatment.Rosa Cangemi de Gutiérrez, carried out the experimental and microbiological assays in animalsViviana Santos, performed the histological and cytological studiesMarta Cecilia, participated in the interpretation of antibiotic assaysClara Silva, carried out the in vitro sensibility testMaría Elena Nader-Macías: conceived the study and participated in its design, evaluation of the results and writing of the manuscript


Oligodendrocytes, neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing complement inhibitor proteins. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis. This vulnerability may be related to a deficiency in their ability to express complement regulatory proteins.This study compared the expression level of complement inhibitor mRNAs by human oligodendrocytes, astrocytes and microglia using semi-quantitative RT-PCR.Semi-quantitative RT-PCR analysis showed that C1 inhibitor (C1-inh) mRNA expression was dramatically lower in oligodendroglial cells compared with astrocytes and microglia. The mRNA expression level of membrane cofactor protein (MCP) by oligodendrocytes was also significantly lower than for other cell types.The lower mRNA expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system. Resident brain cells including oligodendrocytes ,2, astroFor self-protection, resident brain cells also express complement inhibitors, such as membrane cofactor protein (MCP), decay-accelerating factor (DAF), CD59, and C1-esterase inhibitor (C1-inh). The human HOG oligodendroglial cell line produces MCP, DAF, CD59, C1-inh and S-protein, but not complement receptor 1 (CR1)  and DAF,In this study, the expression level of mRNAs for various complement inhibitors by human oligodendrocytes, astrocytes and microglia were compared by semi-quantitative PCR. We show that oligodendrocytes express extremely low levels of mRNA for C1-inh and significantly lower levels of mRNA for MCP compared with astrocytes and microglia. The expression level of mRNAs for CD59 and DAF showed no significant differences between the three cell types.et al. [3) pieces with a sterile scalpel. The fragments were transferred into a 50 ml centrifuge tube containing 10 ml of 0.25% trypsin solution , and incubated at 37°C for 20 min. Subsequently DNase I  was added to reach a final concentration of 50 μg/ml. Tissues were incubated for an additional 10 min at 37°C. The cell suspension was diluted with 10 ml of Dulbecco's modified Eagle's medium (DMEM) and nutrient mixture F12 ham  with 10% fetal bovine serum , and gently triturated by using a 10 ml pipette with a wide mouth. After centrifugation at 275 × g for 10 min, the cell pellet was resuspended in serum containing medium, triturated several times, and passed through a 100 μm nylon cell strainer . The cell suspension was then centrifuged once more (275 × g for 10 min), resuspended into 10 ml of DMEM-F12 with 10% FBS containing gentamicin , and plated onto uncoated 10 cm tissue culture plates (Becton Dickinson). Plates were placed in a humidified 5% CO2, 95% air atmosphere at 37°C for 2 hr in order to achieve adherence of microglial cells. Non-adherent cells with myelin debris were removed from these microglia-enriched cultures and transferred into poly-L-lysine coated 10 cm tissue culture plates in order to achieve adherence of astrocytes. Plates were incubated for 48 hr, after which the culture medium containing myelin debris and non-adherent cells was removed and used to prepare oligodendroglial cell cultures as described below. Both microglial- and astrocyte-enriched cultures were grown for 6 to 7 days before their mRNAs were extracted. Immunostaining with antibodies against CD68  which stains microglia as well as macrophages, and glial fibrillary acidic protein , which is a marker of astrocytes, showed that the microglia-enriched cultures contained 93.5 ± 3.6 % (N = 4) microglial cells, while astrocyte-enriched cultures contained 85.7 ± 3.4 % (N = 4) astrocytes.Human microglial and astrocytic cells were isolated from surgically resected temporal lobe tissues. We thank Dr. J. Maguire, Department of Pathology and Laboratory Medicine, Vancouver General Hospital for providing the surgical specimens. Isolation protocols described by De Groot et al. ,28 were These were prepared as described before . Briefly2) containing 40 U of RNase inhibitor (Pharmacia Biotech) and 1 mM dithiothreitol (DTT), following by incubation at 85°C for 5 min to inactivate the enzyme. Reverse transcription was performed at 42°C for 90 min in 50 μl of the following mixture: 1 × reverse transcriptase buffer containing 2 μg of RNA, 5 mM DTT, 0.2 μg random hexamer primers (Pharmacia Biotech), 1 mM deoxynucleotides , 40 units of RNase inhibitor, and 400 units of SuperScript II reverse transcriptase . At the end of the incubation period, the enzyme was inactivated by heating at 65°C for 10 min [Total RNA from oligodendroglial cells, microglia, and astrocytes were isolated by the acid guanidium thiocyanate-phenol-chloroform method. Two μg of the RNA was then used to prepare cDNA. RNA was treated with 10 U of DNase I  for 60 min at 37°C in 25 μl of 1 × reverse transcriptase buffer , 200 μM dNTPs  and 0.5 μM of each specific primer , 1.25 units AmpliTaq Gold DNA polymerase (Perkin Elmer), 2 mM MgClPrimers were designed to span introns so that cDNA-derived PCR products would be of different sizes to those produced if genomic DNA was amplified  with Fisher's LSD post-hoc test. P < 0.05 was considered to show statistically significant differences.2O2 for 30 min. Blocking was performed for 1 hr at room temperature in 5% skim milk.Oligodendrocytes, astrocytes, and microglia were harvested and air-dried on glass slides. Cells were then fixed with 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100 in phosphate-buffered saline (PBS) for 5 min. For inactivation of endogenous peroxidase, cells were incubated with 0.3% HFor double fluorescence immunostaining, cells were incubated at room temperature overnight with a primary antibody in 1% normal serum. The primary antibody and the dilution used in the first cycle were as follows: O4  for oligodendrocytes, GFAP  for astrocytes, CD68  for microglia. Cells were then treated for 2 hr at room temperature with a biotin conjugated anti-mouse IgM  secondary antibody for O4, a biotin conjugated anti-rabbit IgG  secondary antibody for GFAP and a biotin conjugated anti-mouse IgG  secondary antibody for CD68. Then cells were incubated with Texas Red Avidin DCS (Vector Laboratories) for 1 hr. The primary antibody and the dilution used in the second cycle were as follows: for C1-inh, goat anti-C1-inhbitor ; for CD59, mouse anti-CD59  or rat anti-CD59 . Cells were incubated at 4°C for 3 days with a primary antibody in 1% serum corresponding to the secondary antibody type. Cells were then treated for 2 hr at room temperature with FITC-conjugated anti-mouse IgG , anti-goat IgG , or anti-rat IgG . The glass slides were then rinsed with distilled water, and a drop of Vectashield mounting medium (Vector Laboratories) placed on the slide.RT-PCR was carried out using primers for C1-inh, CD59, DAF and MCP. The housekeeping gene G3PDH was amplified in parallel with each RT-PCR run as an internal standard. Figure To compare the ratio of each of the complement inhibitors to G3PDH, statistical analysis was performed by means of one-way ANOVA with Fisher's LSD post-hoc test Fig. . The oveIn order to establish identity between oligodendroglial cells, astrocytes or microglia and cells expressing the complement inhibitor proteins CD59 or C1-inh, double fluorescence immunostaining was carried out. Oligodendrocytes were detected by O4 staining with a Texas Red tagged secondary antibody Fig.  and 3D iThis report shows that human oligodendrocytes express a much lower level of mRNA for C1-inh than astrocytes and microglia, and a significantly lower level of mRNA for MCP. The mRNA levels of CD59 and DAF were comparable in all the three cell types. Overall our data suggest that oligodendroglial cells, in common with other cell types, can produce complement inhibitors, but at a significantly lower level for C1-inh and MCP.It has already been reported that human neurons and Schwann cells , neuroblActivation of the complement cascade and deposition of activated complement fragments occur in non-infectious diseases such as multiple sclerosis, Pick's disease, Alzheimer's disease and other neurodegenerative conditions ,16,30-34Full activation of the complement cascade requires overcoming a series of endogenous inhibitory factors. Oligodendrocytes are vulnerable to complement attack, which is particularly observed in multiple sclerosis -37 and tSporadic complement attack, in the form of complement activated oligodendroglia (CAO) is also observed in a number of neurodegenerative conditions ,39, inclThese results suggest that the lower expression of C1-inh and MCP by oligodendrocytes could contribute to their vulnerability in several neurodegenerative and inflammatory diseases of the central nervous system, particularly multiple sclerosis.analysis of variance (ANOVA)central nervous system (CNS)complement activated oligodendroglia (CAO)complement receptor 1 (CR1)decay-accelerating factor (DAF)dithiothreitol (DTT)fluorescein isothiocyanate isomer (FITC)glyceraldehyde-3-phosphate dehydrogenase (G3PDH)glial fibrillary acidic protein (GFAP)homologous restriction factor (HRF)membrane cofactor protein (MCP)phosphate-buffered saline (PBS)None declared.MH was responsible for the majority of the experimental studies, and for writing the manuscript. AK contributed to the cell culture and the editing of the manuscript. PLM contributed to the conception, interpretation of results and the writing and editing of the manuscript.


Genetic selections in mammalian cell lines have recently been developed for the isolation of mutant cells that are refractory to infection by retroviruses. These selections have been used to recover lines that block early postentry stages of infection, either before reverse transcription or before nuclear entry. The mechanisms of action of these blocks remain unknown.We have devised a method for the selection of genes from cDNA libraries that suppress the block to virus infection, and so restore virus susceptibility. The protocol involves the transformation of pools of resistant cells by cDNA expression libraries, followed by the selection for rare virus-sensitive cells, using multiple rounds of selection after infection by marked viral vector genomes. The suppressor genes were then recovered from these virus sensitive cells, and their ability to restore virus susceptibility was confirmed by reintroduction of these cDNAs into the resistant line.The identities of these genes provide insights into the mechanism of virus resistance and will help to define new pathways used during retrovirus infection. The methods for gene isolation developed here will also permit the identification of similar suppressors that modify or override other recently identified virus resistance genes. It is becoming increasingly apparent that mammalian cells harbor numerous genes that induce intracellular blocks to retrovirus infection ,2. TheseWe have been involved in the development of screens and selections for virus resistance genes, and have isolated mutant cell lines after chemical mutagenesis that are profoundly resistant to retrovirus infection. Two such lines isolated from a parental fibroblast cell line, Rat2 cells, have been characterized in some detail . Mutant To probe the nature of the blocks in these mutant cell lines, we have sought to identify and characterize suppressor genes that override the restriction exhibited by these cells. To identify such genes, we have developed methodologies that allow for the selection of rare virus-sensitive clones arising after transfer of gene libraries into populations of virus-resistant parents. We here report the isolation of two cDNA constructs that each restore virus sensitivity to the R4-7 mutant cell line. These DNAs constitute valuable tools in the characterization of this line's virus resistance.The R4-7 mutant cell line is approximately 100-fold resistant to transduction by MuLV-based vectors as compared to wild-type Rat2 cells . To idenFour of the pools of transformed cells, with each clone in the pools overexpressing a small number of cDNAs, were sequentially exposed to a series of three genetically marked ecotropic MuLV-based vectors, and the rare successfully infected cells were recovered after each infection by selection for the marker carried by the vector (see Methods). The cells were first exposed to N2 virus, an MuLV vector carrying the neor marker, and infected cells were selected in medium containing G418. These cultures were then expanded and exposed to Eco-TK virus, an MuLV vector carrying the Herpes virus TK gene, and infected cells were selected with HAT medium. These cultures were expanded and finally exposed to Eco-His virus, and infected cells were selected with medium containing histidinol. In all cases, the selecting viral vectors were applied at low multiplicities of infection (MOI) so as not to override the resistance of the parental R4-7 cells, as can happen at high MOI . IndividThe number of colonies of infected cells recovered at each stage of the selection was determined for each of the four pools Table . The numTo determine whether any of these candidate clones had become truly virus sensitive, all 36 colonies were individually picked and expanded into larger cultures. These cultures were then tested by infection with Eco-GFP, a virus vector expressing the green fluorescent protein, and the fraction of the cells expressing the marker was determined by inspection. While all the clones from pools 1 and 2 were as resistant as the parental R4-7 line, a total of 6 clones – 2 from pool 3  and 4 from pool 4  – were fully sensitive to infection. These cloned lines were thus candidates as potentially carrying cDNAs that could restore virus sensitivity to the R4-7 line.To recover the cDNAs present in the virus sensitive cell lines, total genomic DNA was isolated, and polymerase chain reactions were performed to amplify expression cassettes composed of the CMV promoter, the cDNA insert of the library and the poly(A) addition signal. The amplified DNA from each line was directly cloned into the TOPO plasmid DNA and used to transform bacteria. In this way cloned cDNAs were recovered from five of the six lines. Because the lines were expected to each carry a few different cDNAs, and because only one cDNA in each line would be expected to be responsible for the phenotype, a total of 50 bacterial colonies were isolated for each of the five lines. DNAs were prepared from these bacterial colonies and assigned to groups based on the pattern of restriction fragments after produced after digestion with MspI. The number of distinct cDNAs recovered from each of the five lines ranged from 1 to 11, and all together included 29 cDNAs (Table The cDNAs isolated from the virus sensitive lines were then tested directly for their ability to suppress the virus resistance of R4-7 cells. Each cDNA 20 ug) was mixed with pGK-puro DNA (2 ug) and used to transform naive R4-7 cells, and recipients stably expressing the DNAs were selected by growth in puromycin. The resulting puromycin-resistant colonies derived from a given cDNA were pooled and grown into large cultures, and the resulting populations were tested for sensitivity to Eco-neo virus infection. Two of the cDNAs, one from cell line B1 (designated pB1-11) and one from cell line C1 (designated pC1-2), dramatically suppressed the virus resistance of R4-7 cells . These results suggest that the CAPER gene is likely not mutated in the R4-7 line. Nevertheless, to test whether any of these cDNAs could alter virus susceptibility, the cDNA inserts from both R4-7 and Rat2 cells were transferred into the expression vector pcDNA3.1/zeo (see Methods). Overexpression of the various CAPER cDNAs from the Rat2 cells in R4-7 cells did not restore virus susceptibility, and overexpression of the corresponding cDNAs from the R4-7 cells did not induce virus resistance. Thus, of the various CAPER expression constructs, only the original pB1-11 antisense DNA had biological activity.The results here document the development of an effective procedure for the isolation of cDNAs that allow virus infection of virus-resistant cells. The key feature is the repeated infection of the parental resistant line with viral vectors carrying distinct selectable markers, and has become possible only with the development of a multitude of such markers. In this way, rare susceptible cells in the R4-7 population are enriched by as much as 10 to 100 fold in each round of selection. The protocol should allow the recovery of DNAs that confer susceptibility from any large library, if present at an abundance of perhaps at least one in 106 clones. The system can only work if a single DNA is sufficient to enhance virus susceptibility. Once cell lines with restored virus sensitivity were isolated, the recovery of the cDNAs from genomic DNA and the screening for active clones were relatively straightforward.The identities of the two sequences in the active cDNAs isolated here were surprising and the mechanisms of action of the two distinct clones remains mysterious. Both are highly potent, restoring virus susceptibility essentially to wild-type levels figure . The timS. pombe GAR2, a nuclear protein. A later report demonstrated that the gene product interacted with the transcriptional activators AP-1 and the estrogen receptors ERa and ERß, and had potent cotransactivation activity; the gene was renamed CAPER, for coactivator of AP-1 and ER [HCC1.3 and HCC1.4 are two closely related cDNAs first recovered from a patient with hepatocellular carcinoma . The enc1 and ER . The antWhatever the mechanism of action of the pB1-11 cDNA, it is unlikely that the endogenous CAPER gene is the locus of the original virus resistance mutation in the R4-7 line. The levels of the major mRNA are similar in the mutant and the wild-type parent Fig. , and seqgag- and pol-related sequences are often recognizable, nearly all the elements are grossly defective, with multiple frameshift and premature termination mutations interrupting the open reading frames. In addition, the majority of the elements have suffered deletions of various regions relative to the longer family members. Thus, while many of the elements are highly transcribed in rodent cell lines, very few of the transcripts code for protein products of significant length. However, the VL30 RNAs often contain recognition elements for packaging into virion particles encoded by murine leukemia viruses, signals for initiation of DNA synthesis and strong stop DNA translocation, and termini recognized by viral integrase proteins, and thus are competent for transfer by replication-competent viruses acting as helpers.The VL30 elements are a very large family of endogenous virus-like genes found in both mouse and rat genomes -14. The pol gene, specifically the integrase coding region, but is typical of the VL30s in containing no long ORF; furthermore, known cis-acting regions needed for replication are absent. We suppose that the RNA itself may be responsible for the activity, perhaps by binding some cellular protein. The region of the VL30 genome present in pC1-2 – the 3' portion of the pol gene – is not known to contain a binding site for any particular protein. The corresponding region of replication-competent viruses, however, would normally contain the splice acceptor site for the envelope mRNA. Although many VL30 elements do not contain env genes, and although the splice acceptor sites are not readily apparent in the pC1-2 sequence, the transcript might be hypothesized to bind splicing machinery. In this scenario, a possible mechanism of action of the clone is for the overexpressed RNA to bind up a splicing factor or other RNA binding protein, titering out the free protein and removing it from solution. If this factor were responsible for the viral resistance of the R4-7, either directly or indirectly, the binding might relieve that block. The mechanism would be surprising only because the endogenous VL30 RNAs are already so abundant in Rat2 cells, and they are not able to suppress the block to infection. However, the pC1-2 sequence must be an unusual element, in that some distinctive aspect of its sequence must be responsible for its peculiar biological activity. Perhaps identifying proteins that bind to the pC1-2 transcript would be informative.The sequence of the insert in pC1-2 corresponds to a portion of the retroviral The mechanism of resistance exhibited by the R4-7 line remains uncertain. The block is early but likely not at virus entry: it occurs whether ecotropic envelope, amphotropic envelope, or even the VSV G protein is used for entry . FurtherThe power of genetic selections in mammalian cells for alterations in virus susceptibility is increasing rapidly. We believe that selections like the one devised here will be applicable to the isolation of suppressors of other blocks to infection, including the prototypical Fv1 gene , the APOThe Rat2 cell line is a TK-negative fibroblast line that is highly sensitive to MuLV infection. RC-2 is a subclone isolated after mutagen exposure but also sensitive to virus, and was used in many experiments as a wild-type control line. Lines R3-2 and R4-7 are virus-resistant mutants of Rat2 isolated after exposure to ICR-191 . 293T ceA rat kidney cDNA library in the pcDNAI vector  was purcEco-neo or MuLV-N2 virus ; Eco-TK Selections for virus sensitive cells were performed by infecting approximately 105 R4-7 cells per 10-cm dish in each round, with virus titers determined by infection of Rat2 cells. In the first round approximately 104 cfu of N2 virus were applied, and transductants were selected with 800 ug/ml G418. In the second round, approximately 2 × 103 cfu of Eco-TK virus were used, and transductants were selected with HAT medium (Gibco). In the third round, approximately 200 cfu of Eco-His virus were applied, and transductants were selected with medium containing 1 mg/ml histidinol. The multiplicities of all these infections with the selecting viruses were kept low, at less than 0.1 These low MOIs were required because infection of R4-7 cells at high MOI can override the block, perhaps by saturation of a titratable factor. Even at this low MOI, the presence of many siblings of each transformant implied that most cDNAs in the pool were tested for inducing virus susceptibility.cDNA inserts from the expression library were recovered from cell lines by PCR as follows. Genomic DNA was extracted  and subjected to PCR with primers hybridizing upstream from the CMV promoter (sequence 5'-GGGCCAGATATACGCGTT-3') and downstream from the poly(A) addition region (sequence 5'-AATTTGTGATGCTAT-3') of the pcDNAI vector. Conditions for the PCR were: ten cycles of 94°C for 10 sec, 55°C for 30 sec, and 68°C for 3 min, followed by 20 cycles of the same conditions but with an increase in the polymerase reaction time of 5 sec in each cycle. The amplified DNAs were cloned directly into the TOPO vector (Invitrogen) and used to transform DH10b bacteria to ampicillin resistance. DNAs were isolated from approximately fifty bacterial colonies for each original cell line.CAPER cDNAs were prepared from RC-2 mRNA preparations by standard RT-PCR methods using primers spanning the entire ORF (sequences: 5'-ATATAGCTTAAGGCCACCATGGCAGACGATATTGATAT-3' and 5'-ATATAGGCGGCCGCTCATCGTCTACTTGGAAC-3'), and cloned into the pcDNA3.1/zeo expression plasmid using AflII and NotI restriction sites.GG carried out all the experiments and participated in their design. SPG participated in the experimental design and drafted the manuscript. Both authors read and approved the final manuscript.


The scientific community's reliance on active-controlled trials is steadily increasing, as widespread agreement emerges concerning the role of these trials as viable alternatives to placebo trials. These trials present substantial challenges with regard to design and interpretation as their complexity increases, and the potential need for larger sample sizes impacts the cost and time variables of the drug development process. The potential efficacy and safety benefits derived from these trials may never be demonstrated by other methods. Active-controlled trials can develop valuable data to inform both prescribers and patients about the dose- and time-dependent actions of any new drug and can contribute to the management and communication of risks associated with the relevant therapeutic products. In an era of cost containment, the need for rigorous examination of the cost-effectiveness of drugs, as well as their clinical effectiveness, is widely recognized not only by governments but also by the pharmaceutical industry -4. MessaThe latest such example  concernsSince cost-effectiveness is conventionally required for evaluating the efficacy of alternative healthcare interventions, the perspective commonly taken is that of the health services . TherefoCost-effectiveness and clinical effectiveness should be pursued simultaneously to ensure that health care is efficient, ethical, and beneficial to patients. This paper deals with only one aspect of clinical effectiveness: drug treatment benefit and how it may be ascertained from claims of therapeutic equivalence.uncertainty principle, which states that a patient should be enrolled in a randomized controlled trial only when substantial uncertainty exists as to which of the trial treatments would benefit the patient more [The basis for the scientific and ethical underpinnings for the design and conduct of randomized clinical trials is the ent more . From thNevertheless, demonstrating equivalence of the drugs being compared implies starting from the assumption that the new drug is better. In other words, the hypothesis to be tested in equivalence trials  is that one treatment is superior to the other. Altruistic patients are more likely to agree to participate in such a trial, whereas other, less altruistic patients are more likely to decline participation, as their interest lies in treatments with proven efficacy. Obviously, this situation is more patient-favorable than are placebo-controlled trials, in which the individual patient's well-being may be subordinated to the good of others ,14. PlacFor circumstances in which no increased risk for patients is foreseen, use of placebo-controlled trials seems appropriate and ethical, provided the patients are fully informed and that they give their written, informed consent. However, if these patients and their doctors were to find the placebo-controlled studies inappropriate, and if they were to exercise their option in large numbers, these studies would become unfeasible, regardless of the ethical justifications, scientific considerations, views of the trial sponsor, or, ultimately, the expectations of regulatory authorities.Apart from the extreme opinions that challenge the placebo-controlled trials as unethical -24 and tsine qua non for the validity of equivalence claims derived from any active-controlled equivalence/noninferiority study. Methodological flaws affecting one or several of the specific elements inherent in assay sensitivity itself seem to have been more a rule than an exception in many trials carried out during the past decade. Illustrating this point is a systematic review of trials published between 1992 and 1996 that claim equivalence [Defined as "the ability of a study to distinguish between active and inactive treatment," assay sensitivity is the ivalence . In the • 88 papers were evaluated for five equivalence-specific methodological attributes.• Only 45 (51%) of the 88 reports specifically identified demonstration of equivalence as their aim; the others attempted to show superiority or did not state any research aim.• An equivalence boundary was set and confirmed with an appropriate statistical test in 23% of the reports; in 67% of reports, equivalence was declared after a failed test for comparative superiority; in 10%, the claim of equivalence was not evaluated statistically.• Sample sizes were calculated in advance in 33% of reports.• In 25% of reports, sample size was 20 patients per group or fewer.The main concern with such "equivalence claims" is certainly the risk of harm to patients, as poor sensitivity has the potential to cause a type II error  and thereby to thwart satisfaction of public health needs for effective medicines.Just as important as paying careful attention to all aspects of assay sensitivity is acknowledging from the outset that a large number of pharmaceutical products present sensitivity problems. That is, agents otherwise known to be clinically effective are often indistinguishable from placebo in well-designed and well-conducted trials. For this reason, such drugs are useless as comparators in active-controlled trials. A typical example is ondansetron, an antiemetic that, despite its known clinical effectiveness, showed no effect in many placebo-controlled trials . Claims The explanation for this serious problem lies in the great variability of the random placebo effect, which at times may profoundly confound the direction and the magnitude of treatment effects, especially in studies based on small sample sizes . RegardiFurthermore, regarding situations in which multiple trials have demonstrated the efficacy of the active control when compared to placebo, the potential exists for referral bias due to eventual nonreporting of negative results. The risk in such instances is that the smallest clinically relevant effect of the control drug may not be valid .In contrast to the scenarios described above, equivalence and noninferiority trials should be undertaken only when a well-proven standard therapy exists . Investigators should be confident that the efficacy of the control drug was proven to be superior in a previous placebo-controlled trial and that this efficacy will be preserved under the conditions of the current trial . Doubts about the validity of these assumptions mean uncertainty as to whether the two drugs in the current trial, which are allegedly equivalent, really are effective to a similar degree, or are equally ineffective, or cannot be evaluated definitively because the trial design was inadequate to demonstrate the real differences between the two agents.A recent editorial by Alderson  concludeThe primary objective of equivalence/noninferiority trials is to demonstrate that the efficacy of the new treatment matches that of the control treatment. However, "equivalence" should not be interpreted to mean 100% , but that despite some degree of difference, the two agents are clinically indistinguishable. Closer scrutiny should be afforded the secondary objectives of the study, as they might demonstrate some sort of superiority over the control, such as a more favorable safety profile, easier administration, or reduced cost. Alternatively, results might indicate that the new agent would be a reliable second-line treatment.All too often in the past, when trials that were designed to demonstrate the superiority of an agent over its comparator failed to reject the null hypothesis , results were interpreted as proof of the equivalence of the two drugs. A dangerous mismatch of the goals of the superiority and equivalence trials arises when the general reasoning employed in planning and evaluating superiority trials is simply extrapolated to active-controlled trials.The aim of the superiority trial is to rule out the equality of the two agents being compared by rejecting the null hypothesis that the two agents are the same. Failure to reject the null hypothesis does not mean that equivalence can be assumed. Lack of superiority might be consistent with equivalence but does not prove it. In other words, "absence of evidence of a difference is not evidence of absence of a difference" .In equivalence trials, the goal is to rule out all differences of clinical importance between the two agents being compared. This goal is accomplished by rejecting the null hypothesis that the smallest difference of clinical importance exists in favor of the standard-of-care regimen . Therefore, establishing equivalence is contingent upon determining what specifically and precisely constitutes a clinically important difference. This process translates into the need to prove that the two interventions do not differ by more than a certain amount, defined as the "equivalence margin" .To assure the adequacy of the compliance component of assay sensitivity, prescreening of subjects selected to participate in active-controlled trials is necessary, as is reliable assessment of patient compliance with the trial requirements by means of appropriate methodologies ,43. CommThe most compelling example of treatment noncompliance occurs with antihypertensive medications. Noncompliance seems to be the main reason that blood pressure is adequately controlled in fewer than one fourth of patients treated for hypertension, both in the US and in European countries ,48. The Use of co-medication during active-controlled studies, whether the result of self-medication or prescription medication, can distort the study's final results. Co-medication can interfere with response to the tested drug or the control drug, or it can influence the trial endpoints and lead to false-positive conclusions of equivalence. Use of non-trial medication is quite common in clinical trials in general and should be assessed and minimized, particularly in active-controlled studies.A basic assumption is that the active agent in an equivalence/noninferiority study should have retained its known  effect, demonstrated in a previous placebo-controlled comparison. Patients participating in the current study should be as similar as possible to the patients in the placebo-controlled trial with respect to all baseline values and treatment variables that might influence outcome. These variables include symptoms, signs, risk factors, morbidity, compliance with therapy, responsiveness to drug effects, nonuse of prohibited concomitant medication, consistent diagnostic criteria, inclusion and exclusion criteria, unbiased assessment of endpoints, and reasons for dropping out. Failure to achieve this similarity from the outset, failure to ensure high-quality study conduct, or both, can introduce bias into the study and compromise assay sensitivity.randomization, . Further, double blinding is intended to minimize potential biases resulting from differences in management, treatment, or assessment of patients, or differences in interpretation of results that could arise as a result of the subject's or investigator's knowledge of the assigned treatment.The classical method to minimize systematic differences between study groups is The type and frequency of outcome events in the current study are expected to be similar to those in the placebo-controlled comparison. Substantial differences, resulting most often from an imbalance in one or more of the variables mentioned above would render interpretation of differences between the new therapy and the active control very difficult. For example, because of lower baseline blood pressure values and fewer associated risk factors, patients in a hypertension study may display fewer outcome events.Equivalence trials are commonly designed to demonstrate that the test treatment is similar in efficacy to the active control, the assumption being that the control treatment is effective under the conditions of the current trial. In reality, however, most equivalence trials are actually noninferiority trials, attempting to show that the new drug is not less effective than the control by more than the defined amount (margin). Presence of assay sensitivity is essential for interpretation of such a study. In cases with doubtful assay sensitivity, a three-arm study design  might be optimal. Apart from being more complex and requiring a larger sample size, such a trial offers the advantage of measuring the effect of the test drug versus placebo while allowing comparison of the test drug and active control in a setting in which assay sensitivity is established by the active control-versus-placebo comparison. By making the active groups in such trials larger than the placebo groups, it is possible to increase the precision of the active drug comparison while minimizing the chance that patients will be randomly assigned to placebo groups. Furthermore, this design allows distinction between adverse events due to the drug and those due to underlying disease ("background noise") .As mentioned earlier, equivalence/noninferiority trials should not only focus on efficacy, but also should prospectively define an analytical plan for safety assessment as a secondary objective. Accordingly, appropriate statistical power to detect adverse effects is a necessity, as is collection of data on the comparative safety of each treatment. Failure to meet these requirements not only undermines the chance to exploit a potentially favorable safety profile of the test drug versus the control, but also presents the risk of missing dangerous signals with regard to safety. A recent example relates to mibefradil, a calcium inhibitor that appeared to have an excellent safety profile until postmarketing surveillance revealed cases of sudden death in patients at high risk for polymorphic ventricular tachycardia or patients in whom concomitantly administered drugs either inhibited mibefradil metabolism or otherwise amplified its cardiac risk . A more The margin (Δ) itself clearly communicates a judgment as to what is and is not important. The margin defines the largest difference that is clinically acceptable. Setting the margin is critical to the design of both equivalence and noninferiority trials, and it is commonly established for the purpose of excluding a clinically important difference between treatments. However, what constitutes such a difference may vary widely for each patient and clinician and might fall below the margins set by the designer of the trial. For that reason, careful clinical judgment and statistical reasoning should be exercised in selecting a meaningful difference to be ruled out; furthermore, this difference should be specified and justified a priori in all equivalence/noninferiority trials. At the very least, the equivalence margin should be smaller than the lower 95% confidence limit for the absolute risk difference observed between standard therapy and placebo in the relevant superiority trial . That isConfidence interval is the method of choice to interpret equivalence and noninferiority trials. It defines a range for the possible true differences between the test drug and the active control. If every point within this range reflects a difference that is clinically nonrelevant, then the two agents may be considered equivalent. In other words, for an equivalence trial, the two-sided 95% confidence interval – defining the range of possible differences between the test and the control agent – should lie entirely within the interval (-Δ to +Δ) , and the α (type I error) and β (type II error) values should be selected on the basis of the same principles as for comparative trials. The distinction between one-sided and two-sided tests of statistical significance carries over into the confidence interval approach recommended by the Committee for Proprietary Medicine Products (CPMP) guidelines ,57, whicUnlike superiority trials, in which intention-to-treat analysis is the rule, in equivalence/noninferiority trials both intention-to-treat and per-protocol analysis should be run. Both types of analysis would be expected to lead to similar conclusions for a robust interpretation. In a recent article, Gomberg-Maitland et al.  suggesteEach year, major advances in drug discovery generate a seemingly endless supply of new drugs in virtually all therapeutic areas; however, in most cases these new drugs provide only incremental improvement in efficacy, safety, or the overall risk-benefit ratio. In addition, ethics-based restrictions on the use of placebos as comparators have enhanced the viability of equivalence and noninferiority trials as viable alternatives for registration purposes, for risk management once the drug is on the market, and for a marked increase in confidence in the new drug on the part of doctors and patients.Given the complexity of these study designs, careful attention should be paid to the proper use of specific epidemiological features so as to avoid methodological deficiencies that may harm patients if clinically inferior treatments are erroneously deemed equivalent to the standard of care, or if potentially superior therapies are discarded as merely "equivalent."


Although sequences containing regulatory elements located close to protein-coding genes are often only weakly conserved during evolution, comparisons of rodent genomes have implied that these sequences are subject to some selective constraints. Evolutionary conservation is particularly apparent upstream of coding sequences and in first introns, regions that are enriched for regulatory elements. By comparing the human and chimpanzee genomes, we show here that there is almost no evidence for conservation in these regions in hominids. Furthermore, we show that gene expression is diverging more rapidly in hominids than in murids per unit of neutral sequence divergence. By combining data on polymorphism levels in human noncoding DNA and the corresponding human–chimpanzee divergence, we show that the proportion of adaptive substitutions in these regions in hominids is very low. It therefore seems likely that the lack of conservation and increased rate of gene expression divergence are caused by a reduction in the effectiveness of natural selection against deleterious mutations because of the low effective population sizes of hominids. This has resulted in the accumulation of a large number of deleterious mutations in sequences containing gene control elements and hence a widespread degradation of the genome during the evolution of humans and chimpanzees. A comparison of hominid and rodent lineages reveals that the gene control regions of hominids are not conserved and are accumulating mutations, suggesting widespread degradation of the hominid genome Functionally important sequences are expected to evolve more slowly than neutrally evolving sequences. This is because long periods of selection for functional efficiency lead to sequences in which most advantageous mutations have already become fixed. The majority of new mutations in a sequence are then deleterious, because they perturb the highly adapted state. Studies of protein-coding DNA evolution have supported this expectation by showing that rates of amino acid substitution are substantially lower than rates of synonymous substitution in the majority of genes .Recently there has been great interest in using sequence conservation to detect functionally important regions of the genome outside of protein-coding sequences. However, detecting conservation has proven difficult because noncoding DNA sequences often appear to be weakly conserved. In mammals, for example, more than 98.5% of the genome is believed to be intron or intergenic DNA , and at However, the extent to which constraint in noncoding regions varies among species is unclear. In this paper, we investigate sequence conservation in introns and intergenic DNA in interspecific comparisons of mouse and rat (murids) and human and chimpanzee (hominids). To estimate the levels of constraint in segments of non-protein-coding DNA, we compare the observed numbers of substitutions to the number expected from substitution rates at linked sequences assumed to be neutrally evolving. Unexpectedly, we find that selective constraints are essentially absent in hominids in regions upstream of genes and in first introns, in contrast to murids, in which these regions are subject to moderate levels of constraint.We investigated the frequency of deleterious mutations and the pattern of sequence conservation in regions containing gene expression control sequences in hominids by compiling a dataset of 1,000 well-annotated, randomly chosen human genes. The genomic sequences upstream and downstream of each coding sequence and samples of their introns were aligned against the draft chimpanzee sequence. We compared the pattern of evolution in these hominid sequences against that in a previously compiled dataset of murid sequences . NumbersWe calculated levels of selective constraint in blocks of 500 bp, averaged over loci, using the aligned human–chimpanzee and mouse–rat genome sequence datasets. We corrected our constraint estimates for the decline in GC content that appears to be occurring in most mammalian genomes, including hominids and murids , althougSelective constraints in hominid sequences in the regions 6,000 bp upstream and downstream of coding sequences and in the 5′ regions of first introns are clearly far lower than in murid sequences  and 2. IThere are a number of possible explanations for the apparent absence of constraint in hominid 5′ flanking and first intron sequences (and for the lower constraint in the 3′ flanking region), which we examine below.−3. This is expected to reduce constraint by approximately one-tenth since the relationship between the true level of constraint (trueC) and observed level (obsC) is obs = CtrueC/(1 + ɛ/k), and the human–chimp divergence (k) is approximately 1%. If the true levels of constraint in the 5′, 3′, and intron 1 sequences closest to hominid genes were 30%, as we find in murids, the observed constraint in hominids would be 27%. Sequencing errors can therefore explain only a small proportion of the difference in constraint between hominids and murids. Further evidence that our data do not have unusually high rates of sequencing errors is that the divergences  of 1.19 × 10nces see  are veryA second possibility that could explain the differences in constraint between hominids and murids is that our dataset of hominid genes contains large numbers of pseudogenes. However, several lines of evidence argue against this. First, the genes are well annotated and contain no stop codons. Second, the intronic splice donor/acceptor nucleotides are highly conserved. Third, the exons of our gene sample show strong conservation: constraint at second positions of codons estimated in the same manner as for noncoding sequences is 0.750 ± 0.013. And finally, a very substantial proportion of genes would have to be pseudogenes to explain our data. For example, to reduce the constraint by one-half would require 50% of the hominid genes to be pseudogenes.A third possibility is that the lower constraints in hominid 5′ flanking and first intron regions could be a consequence of a reorganization of gene regulation such that murids have a concentration of regulatory sequences in 5′ regions and in first introns and hominids have regulatory sequences concentrated in introns outside intron 1. However, two lines of evidence suggest that this is unlikely. First, there is remarkable conservation of syntenic blocks  and per http://www.niehs.nih.gov/envgenom/home.htm). This dataset was chosen because it represents the most extensive and consistently sampled database of human single nucleotide polymorphisms. The genes were sampled as being “environmentally responsive,” and are therefore not a random sample, but they show average levels of constraint in all regions similar to those of our sample of 1,000 hominid genes. The analysis revealed no reduction in diversity in the 5′, 3′, and intron 1 regions when compared to the levels of diversity in intron sequences outside first introns . Based on polymorphism data and estimates of nucleotide mutation rates (u), estimates of human and chimpanzee eN are typically in the range 10,000–30,000 for nuclear sequences [eN for both species is estimated to be approximately 20,000 [Mus musculus domesticus yielded an estimate of 4euN of 0.0054 [−9 and 2.98 × 10−9 [eN for the house mouse to be between 450,000 and 810,000. The fate of a deleterious mutation depends on the product esN; if e|s|N > 1, then the fixation probability for a deleterious mutation starts to become appreciable. Therefore, deleterious mutations, whose strength of selection falls within the range 1/eN(murids) < |s| < 1/eN(hominids), will tend to be removed by natural selection in murids, but can drift to fixation in hominids. Under the assumption that selection coefficients against deleterious mutations are equivalent in all taxa, the levels of selective constraint in noncoding DNA of murids and hominids imply that approximately 83%, 17%, and 0% of mutations in the first 2,000 bp of 5′ flanking DNA and intron 1 have a strength of selection such that |s| < 1/eN(rodents), 1/eN(rodents) < |s| < 1/eN(hominids) and |s| > 1/eN(hominids), respectively. For the the first 2,000 bp of 3′ flanking DNA we estimate that about 81% of mutations have |s| < 1/eN(rodents), 12% are in the range 1/eN(rodents) < |s| < 1/eN(hominids) and 7% have |s| >1/eN(hominids). In contrast to noncoding DNA, the fraction of slightly deleterious mutations fixed in hominid coding sequences is quite low. Constraint estimates at second codon positions outside CpG-prone sites in hominids and murids are 0.750 ± 0.016 and 0.900 ± 0.0085, respectively. Taking into account sequencing errors in hominids, the predicted “true” constraint value in hominids is 0.84, and this implies that only about 6% of mutations are in the slightly deleterious class.Two lines of evidence suggest that many mutations that affect gene expression may be under only weak purifying selection. First, sequences involved in gene regulation often appear to evolve rapidly . Second,equences ,26,27, ay 20,000 . Unfortuf 0.0054 . Combini8 × 10−9 , we esticis-acting gene control sequences [M. domesticus, and M. spretus. From an analysis of 3,801 orthologous genes across all four species, we found that levels of divergence in expression between human and chimp are very similar to levels of divergence in expression between the two mouse species  and for brain .The lower level of constraint in hominid 5′, 3′, and intron 1 sequences leads to a testable prediction about the evolution of gene expression. Since the flanking regions of genes contain a high concentration of equences , we expeequences , who exa species . At the  species . Thus, wAs demonstrated in an analysis across different primate species, substantial increases in expression distances are still observed when going beyond the evolutionary distances examined here . Thus, oM. musculus on M. spretus samples. Sequence differences between the species will lead to lower hybridization efficiencies in chimpanzee and M. spretus, and will consequently exaggerate expression distances. However, this problem will be more pronounced in the mouse comparisons, since sequence divergence between M. musculus and M. spretus is higher than between human and chimpanzee. Thus, this would bias our results towards higher rates of gene expression evolution in mice, making our test conservative.A potential source of error in this analysis is the use of microarrays designed for humans on chimpanzee samples, and of microarrays designed for eN for hominids, i.e., less than 10−4. It should be noted that it is unlikely that the mutation accumulation is due to a recent relaxation of natural selection in humans due to an improvement in our living conditions [The lack of conservation of regions containing expression control sequences demonstrated above is consistent with the observation that the vast majority of Mendelian genetic disease mutations are located in coding sequences . Our resnditions , since tnditions . FurtherM. musculus and M. spretus it is approximately 1.8 million years [We have also demonstrated that gene expression evolution is significantly accelerated in hominid brain and liver compared to the respective murid tissues. This result has important implications for theories of neutral gene expression evolution ,34. Firson years , so the The importance of effective population size in influencing the organization and complexity of genomes has recently been highlighted . Our finDNA sequences of 1,000 annotated loci were compiled from the reference sequence (build 33) of the human genome. In a preliminary analysis of a smaller dataset, we determined that such a dataset of 1,000 loci would provide standard errors on constraint estimates of less than 2%. We randomly sampled loci by the criterion that each record contained the description of at least one mRNA. We extracted all exons, up to eight introns, including first, second, last, and second last introns, and up to 6 kb of intergenic DNA 5′ and 3′ from the start and stop codon, respectively. Intergenic DNA was extracted up to the midpoint between the sampled coding sequence and the start or stop codon of the following or preceding locus in the genomic contig. We extracted complete introns if they were less than 30 kb in length, otherwise the first and last 10 kb. For more than 80% of the hominid loci sampled, the 6-kb 5′ region includes all annotated untranslated exons and introns.We used reciprocal best-hits BLAST  to identOrthologous genes from 300 well-annotated loci were randomly sampled from the whole-genome assemblies of mouse and rat from GenBank. Loci were chosen for which annotation evidence included at least one complete mRNA sequence in both species. Further details of the sampling are given in . Coding C) for each of the above categories of sites by comparing the observed numbers of substitutions (O) with numbers expected (E) if substitution rates were equal to that of a class of putatively neutral sites. In this analysis, these putatively neutral sites were intronic sites excluding intronic splice control regions  and first introns, since these show evidence of moderate selective constraint [n loci isWe estimated selective constraints  × 4,000 bp (of 5′ and intron 1 sequence) × 0.17 (difference in constraint between murids and hominids) × 0.012/2 (human–chimp divergence/2) + 25,000 (genes) × 2,000 bp (of 3′ sequence) × 0.12 (difference in constraint between murids and hominids) × 0.012/2 = 138,000.http://www.niehs.nih.gov/envgenom/home.htm). Of these, 305 had more than two intron sequences, and were suitable for further analysis. The sequences were aligned against the chimpanzee genome sequence, as described above, and the number of substitutions estimated by counting the number of differences, with no correction made for multiple substitutions . We only considered sites that were not preceded by C or followed by G to maintain consistency with the other analyses reported here. For each gene we calculated the numbers of 5′ (or 3′ or intron 1) substitutions (nD) and polymorphisms (nP), along with the equivalent figures for the other introns, which acted as our neutral standard (respectively iD and iP). To test for adaptive evolution we estimated the proportion of substitutions that were driven by adaptive evolution using the method of Smith and Eyre-Walker [Single nucleotide polymorphism data for 335 genes were compiled from the Environmental Genome Project  and the HG-U95A microarray (used for cortex).Affymetrix oligonucleotide microarray data for brain cortex and liver tissue samples from three individuals each of human, chimpanzee, d et al. . To haved et al. , and thewww.ensembl.org), we obtained a mapping of probe sets to Ensembl gene IDs, and a list of human–mouse orthologs. If more than one probe set matched the same Ensembl gene, we averaged expression levels over all probe sets for this gene. We retained only one-to-one orthologs , further requiring that each sequence cover at least 70% of the other. Expression matrices for the individual experiments were scaled to the same mean. All mouse experiments  were then normalized relative to each other by means of a quantile normalization [We restricted the expression analysis to orthologous genes as follows. From Ensembl  was used to estimate standard errors (standard deviation of bootstrap distances) and confidence intervals (2.5% and 97.5% quantiles of bootstrap distances).M. spretus and M. domesticus, compiled from GenBank. In order to obtain confidence intervals for the ratio of expression divergence d over sequence divergence iK, we used a bootstrap analysis of 1,000 datasets, each combining d values obtained from resampling genes from the expression analyses, and iK values obtained from resampling genes from the divergence analyses.We compared expression differences to intronic nucleotide divergence levels calculated without correction for multiple substitutions, excluding CpG-prone sites as described above. We analyzed the complete 1,000 gene chimp–human intronic dataset described above and a dataset of 39 introns from 24 orthologous loci of