The present study GREECS was conducted in order to evaluate the annual incidence of acute coronary syndromes (ACS) and to delineate the role of clinical, biochemical, lifestyle and behavioral characteristics on the severity of disease. In this work we present the design, methodology of the study and various baseline characteristics of people with ACS.A sample of 6 hospitals located in Greek urban and rural regions was selected. In these hospitals we recorded almost all admissions due to ACS, from October 2003 to September 2004. Socio-demographic, clinical, dietary, psychological and other lifestyle characteristics were recorded. 2172 patients were included in the study (76% were men and 24% women). The crude annual incidence rate was 22.6 per 10,000 people and the highest frequency of events was observed in winter. The in-hospital mortality rate was 4.3%. The most common discharged diagnosis for men was Q-wave MI, while for women it was unstable angina.This study aims to demonstrate current information about the epidemiology of patients who suffer from ACS, in Greece. During the past decades epidemiological investigations have provided a portrait of the potential candidate for acute coronary syndromes, in the US, as well as in many other parts of the world, especially in European countries -9. HowevThe profile of cardiovascular disease patients in Greece has been, mainly, investigated from few large scale, population-based studies, like the Seven Countries Study in the early 1960s , the HelThe aim of the GREEk study of acute Coronary Syndromes (GREECS) is to evaluate the prevalence and annual incidence of acute coronary syndromes (ACS), as well as the characteristics, and management of these patients, in a sample of six Greek urban and rural regions. Secondary goals are to examine the role of adoption to the Mediterranean diet and other lifestyle habits, as well as various clinical, biochemical, psychological and personal characteristics of these patients on the severity and the short (30 days) – long (6, 12 months) – term prognosis. In this work we present the methodology used, and the baseline characteristics of the studied population.Between October 1, 2003 and September 30, 2004 (12 months) we enrolled almost all consecutive patients (participation rate = 98%) that entered in the cardiology clinics or the emergency units of six major General Hospitals, in Greece . By the exception of Athens – where there are several other hospitals -, all the other hospitals cover the whole population of the aforementioned regions, including urban and rural areas. The studied regions were randomly selected from all Greek regions in order to cover a wide range of the county  of them were men and 523 (24%) were women. Power analysis showed that the number of enrolled participants is adequate to evaluate two – sided differences between groups of the study and the investigated parameters greater than 20% (± 5%), achieving statistical power greater than 0.80 at 5% probability level .At entry a 12-lead electrocardiogram was performed and clinical symptoms were evaluated in all patients, by a cardiologist of the Study. Based on the electrocardiographic findings patients were classified as having ST-segment elevations, non-ST segment elevations or other electrocardiographic abnormalities. Moreover, blood tests were performed to detect evidence of myocardial cell death. We measured troponin I levels and the MB fraction of total creatinine posphokinase (CPK). According to the Joint European Society of Cardiology and American College of Cardiology Committee, blood samples were obtained on hospital admission, at 6 to 9 h, and again at 12 to 24 h if earlier samples were negative and the clinical index of suspicion was high . We inclThe study was approved by the Medical Research Ethics Committee of our Institution and was carried out in accordance with the Declaration of Helsinki (1989) of the World Medical Association.In all patients a detailed medical history was recorded, including previous hospitalization for cardiovascular disease (i.e. coronary heart disease, stroke or other cardiovascular disease), presence and management of hypertension, hypercholesterolemia, renal failure and diabetes mellitus. Moreover, we recorded patients' medical family history. In particular, we asked information concerning first-degree relatives  about presence of coronary heart disease, hypertension, dyslipidemias and diabetes. Premature history  of myocardial infarction, sudden death, coronary arteries bypass grafting procedure or percutaneous coronary angioplasty in first-degree relatives classified the participants in the positive family history group for coronary heart disease . PrematuSocio-demographic characteristics included: age, sex, marital status and number of children, years of school, type of occupation and occupational skills, which they were evaluated through a ten-point scale from unskilled – hand workers  to executive – skilled workers  that has been developed for the purposes of the Study; and mean annual income of the family (through self reports) during the last three years. Regarding people in the family who were not working, we used the average family income, while for unemployed individuals we used the basic monthly allowance they take from the Social Service Office.2, while obesity as BMI greater than 29.9 kg / m2.Height and weight was measured, to the nearest 0.5 cm and 100 g respectively. Body mass index (BMI) was then calculated as weight (in kilograms) divided by height (in meters) squared. Based on the World Health Organization , overweiTo evaluate physical activity status of the patients during the past year we used a modified version of a self-reported questionnaire provided by the American College of Sports Medicine . Based oCurrent smokers were defined as those who smoked at least one cigarette per day or have stopped cigarette smoking during the past 12 months. Former smokers were defined as those who had stopped smoking more than one year previously. The rest of them were defined as never smokers or rare smokers. Exposure to environmental cigarette smoke for at least 30 minutes per day and three days per week at workplace, home or other public places was recorded in all patients, too.The evaluation of the nutritional habits was based on a validated semi-quantitative food frequency questionnaire . The conDepressive symptomatology was assessed using a translated and validated version of the Center of Epidemiological Studies Depression Scale (CES-D) . The CESClinical symptomatology of occupational stress was determined by a special self-reported questionnaire based on the survey obtainable by the Job Stress Help Line . By thisIn this, brief, baseline report continuous variables are presented as mean values ± standard deviation. The categorical variables are presented as absolute and relative (%) frequencies. Future analyses will follow with appropriate statistical techniques. Associations between continuous variables and group of patients will be evaluated through the analysis of variance, after controlling for equality of variances (homoscedacity) using the F-test. Due to multiple comparisons we will apply the Bonferroni rule to correct for the inflation of type – I error. Associations between categorical variables will be tested by the use of the chi-squared test, without the correction of continuity. Correlations between continuous variables will be tested by the use of Pearson's correlation coefficient for the normally distributed, and by the use of Sperman's rho coefficient for the ordinal or skewed variables. The association between the investigated socio-demographic, clinical and biochemical characteristics on the short term outcome (i.e. 1 month) will be tested by the development of multiple logistic regression models, while the association of the aforementioned characteristics on the long term outcome (6 and 12 months) will be tested by the use of Cox proportional hazards models. Appropriate tests for goodness-of-fit  will be applied in all models. All statistical calculations will be performed on the SPSS version 12.0 software (SPSS Inc, Texas, U.S.A.).From October 2003 to September 2004, 2172 patients with discharge diagnosis of ACS were enrolled into the study (1649 men, 65 ± 13 years old and 523 women, 62 ± 11 years old, p < 0.001). The men-to-women ratio was 3- to -1. The annual incidence of ACS was 22.6 per 10000 of people (34.0 per 10000 men and 10.9 per 10,000 women). The annual incidence was calculated with the exception of events at Hippokration Hospital in Athens, because it was very difficult to define the referent population since there are many hospitals in the area. The mean number of daily admissions was 6 ± 3 persons per day, averaging 5 ± 4 men and 2 ± 1 women.Table Of the 2172 patients enrolled into the study, 38% had ST segment elevations, 27% had non-ST segment elevations and the rest of them, i.e. 35% patients, had other electrocardiographic findings. According to the discharge diagnosis, 764 (35%) patients were diagnosed as having unstable angina, 699 (32%) patients as having non-Q-wave MI and 709 (33%) patients as having Q-wave MI.Table Table Table th, 75th percentiles) time between the overt of symptoms and the time medical care was sought, was 4 (2, 10) hours. Based on the discharge diagnosis the duration of hospitalization was 7 (5, 8) days for patients with Q-wave MI, 6 (5, 8) days for non-Q-wave patients and 5 (3, 7) days for UA patients. Furthermore, 60% of patients with ST-elevation received trombolytic therapy.The median . The in-hospital mortality rate of patients with ST segment elevation was 74 deaths per 1000 patients, for non-ST segment elevation was 34 deaths per 1000 and for undetermined electrocardiographic findings was 2 deaths per 1000 patients.In this brief report we presented the design, and the aims of an epidemiological study of ACS, that has been conducted in six major general hospitals, in Greece (i.e. the GREECS). We also presented the baseline characteristics of almost all patients who hospitalized in these hospitals for ACS during the study period (i.e. October 2003 to September 2004).The overall incidence rate of ACS observed in our survey was 22.6 events per 10000 of population. Based on this figure it could be speculated that the prevalence of ACS in the investigated Greek areas is 2.6%. In a similar recent study located only in northwestern Greece, which also included sudden cardiac death before hospital admission, the incidence rate of ACS was much higher (i.e. 39 events per 10000 people) . Based oWe also observed that the annual incidence for men was 34 per 10,000 of people, while the incidence for women was significantly lower, i.e. 11 per 10,000 of people. This finding will be tested whether can be attribute to the increased smoking habits observed in women during the past decades, as well as to various other lifestyle habits and work-related conditions, like physical inactivity, unhealthy diet, social insecurity and job stress, observed in female population from late 1960s to present .Furthermore, we observed that the in-hospital mortality rate in was 4.3%, which was similar to the in-hospital mortality rate that has been calculated based on a sample of 25 European countries, i.e. 4.9% [In this work we have also presented various baseline clinical and lifestyle characteristics of the enrolled patients. Future analyses will evaluate the role of the investigated clinical characteristics as well as family history of the common cardiovascular risk factors on the severity and prognosis of the ACS patients. Regarding dietary and other lifestyle related habits, we aim to evaluate whether the adherence to a Mediterranean dietary pattern, the adoption a physically active lifestyle, the abstinence of smoking and the control of psychological factors is associated with less severe disease and a better short and long term outcome. We anticipate that the completion of the follow up and the analysis of the results will provide current, novel and valuable information about the epidemiology of ACS, in Greece, as well as the role of clinical, psychosocial and lifestyle characteristics on the prognosis of cardiac patients.ACS = acute coronary syndromesCPK = creatinine posphokinaseMI = acute myocardial infarctionUA = unstable anginaECG = electrocardiographicCES-D = Center of Epidemiological Studies depressionBMI = body mass indexThe author(s) declare that they have no competing interests.CP, DBP = are the principal investigators of the study, had the concept and the design of the study, and wrote the paper, AA, YK, YM, SZ, PS, CS = contributed to the design of the study, GK = contributed to the data management and analysisThe pre-publication history for this paper can be accessed here:


Locus heterogeneity poses a major difficulty in mapping genes that influence complex genetic traits. A widely used approach to deal with this problem involves modeling linkage data in terms of finite mixture distributions. In its simplest setup, also known as the admixture approach, a single parameter is used to model the probability that the disease-causing gene of a family is linked to a reference marker. This parameter is usually interpreted as the overall proportion of linked families.In this article, we address two issues regarding the admixture approach. First, we tackle the question of whether the single parameter of linked proportion is well defined in general. By formulating the likelihood under a classification scheme based on distributions, we show that such a parameter is meaningful only when a certain well-characterized condition is met. Second, we study a condition given in the literature for validating the admixture approach. A counter example is constructed to illustrate that the condition does not necessarily lead to valid estimates.Estimators from the admixture approach may be inconsistent. This holds even if a condition given in the literature to validate the approach is satisfied. The heterogeneity is modeled by a single parameter (α) that denotes the probability that a family is linked. This parameter is interpreted by many researchers as the overall proportion of linked families.Mapping disease genes influencing complex genetic traits is far more difficult than mapping genes underlying Mendelian traits. One of the difficulties is due to locus heterogeneity, in which the disease in different families  is caused by different loci or non-hereditary factors. Finite mixture distributions have been proposed to model linkage data in the presence of locus heterogeneity , also knα does not even measure the proportion of linked families within the sample, contrary to popular belief. Similar conclusions were obtained in the simulation study by Pal and Greenberg [α. Nevertheless, these studies as well as some other simulation studies  is meaningful when certain assumptions are violated. Janssen et al. [α and θ can be severely biased when the distribution of informativeness in the linked families is not roughly the same as that in the unlinked families, under some measure of informativeness for linkage studies. They used a measure called Effective Number of Informative Meioses (EFNIM) to assess the informativeness of a family. It is based on the Expected LOD (ELOD) score of a family. They argued that, if the Linked and Unlinked Families are nearly Equally Distributed (LUFED) in terms of EFNIM, then the admixture procedure should provide satisfactory, i.e., nearly unbiased, results.Other researchers take a different view, pointing to potentially severe biases of the estimates obtained from the admixture approach e.g., ,9). Whit. Whit9])n et al.  showed, In this article, we consider the same problems discussed in Janssen et al.  and WhitT types such that families in the same type follow the same distribution. That is, families are classified into the same type based on their "structures" that lead to the specification of their distributions. Here we use the term structure in a broad sense that includes not only the pedigree structure but also any other available information about the pedigree (such as knowledge of phase) that contributes to the distribution from which the family is generated. Note that structure does not include any phenotypic or genotypic data of a family. Families with the same structure but different phenotypic and/or genotypic data are simply different realizations from the same distribution and hence are iid, and are classified into the same type. In the most general setup, with g potential linkage scenarios, e.g., linked or unlinked (g = 2) in the simplest setting, the probability distribution, ft*, for a realization (yt) from type t, t = 1,…,T, can be expressed in terms of its component distributions, fti, i = 1,…,g [Suppose that data from independent families in the sample are classified into  = 1,…,g :αti ≤ 1 (i = 1,…,g), , and θ is the linkage parameter vector, including various recombination fractions.where the mixing parameters satisfy 0 ≤ θ or by a locus unlinked to the reference marker. Further, we assume that the genetic (trait) models at the linked and unlinked loci are the same. So, the linkage parameter vector is θ = {θ, 1/2}, and the probability distribution is simplified toTo simplify our presentation without compromising our objectives, we assume that the disease under study is caused either by a locus linked to the reference marker with a recombination fraction ft* = αtft + (1 - αt)ft,αt is the probability that a type t family is linked. Note that the two distributions under the mixture have the same parametric form but differ in their linkage parameter values, being θ or 1/2. Also, note that a single α parameter is used for all families in the same type.where a denote the true proportion of linked families in the entire sample. Let pt, t = 1,…, T, be the true proportion of type t families in the linked group. Thus p = (p1,…, pT) is the distribution of types among the linked families. Recall that all families in a given type have the same distribution, i.e., same likelihood for any value of the recombination fraction. Similarly, define q = (q1,…, qT) to be the distribution of family types among the unlinked group. Then the probability that a family is of type t is st = apt + (1 - a)qt, and the probability that a type t family is linked is thus . Furthermore, let r denote the true common recombination fraction for the linked families. The likelihood contribution for estimating the parameters {αt, θ} by a type t family with data yt isLet Lt(αt, θ | yt) = αtft + (1 - αt)ft.     (1)t can be expressed asThen the expected contribution to the log-likelihood from a family of type r and  are the true underlying quantities, whereas θ and α = (α1,…, αT) are the parameters to be estimated from the data. Combining the expected log-likelihoods, ELLt, t = 1,…, T, we can form the total expected log-likelihood from all family types as follows:Note that α0, r) ≥ ELL(α, θ), for any (α, θ), following Stuart and Ord [α, θ). Note that α is a vector of nuisance parameters; only θ is the true parameter of interest.Since the above formulation of likelihood is correct under the stated assumptions, it can be shown that ELL( and Ord . This imα parameters for families with different structures as well as different phenotypic data. So, families with the same structure have different α parameters if they have different phenotypic data. On the other hand, in (1) families with the same structure (and hence their data coming from the same distribution as elucidated at the beginning of this section) share a common α parameter. Since the issue addressed by Vieland and Logue [We note that the above likelihood, although similar to the one presented in Vieland and Logue , is diffnd Logue  is diffep = q, that is, the distribution of family types among the linked families is the same as that among the unlinked families. In this setup, all αt's are set to be equal to a common parameter, α, the overall proportion of linked families. Hence, the total expected log-likelihood is with respect to the recombination fraction θ and a single proportion parameter α:In this subsection, we show that the two-parameter setup leads to correct likelihood formulation only when a, r) and (α, θ), isThe expected log-likelihood ratio between the two sets of parameter values,  ≥ ELL(α, θ), for any (α, θ), implying that  = a and  = r are the maximum likelihood estimates of α and θ. However, as shown in the examples in the next section, when p ≠ q, ELL(a, r) may be smaller than ELL(α, θ) for some parameter values (α, θ). That is, the maximum likelihood estimates of α and θ may not be consistent because the likelihood model is incorrectly specified (under-parameterized). In order for the two-parameter formulation to be correct, the following constraints should be placed on the makeup of the families:In other words, when p = q given the additional constraints that  and . Notice that p = q is stronger than the LUFED condition. Equal distributions of types in the linked and unlinked families according to the classification scheme based on distributions of the data implies equal distributions of informativeness in the same two groups. Therefore, LUFED is a necessary condition for the admixture approach. We also note that p = q is not necessarily satisfied for every dataset (see Discussion).which implies that α, the relationship between α and αt is as follows:In general, in a parameterization that demands an estimate for the overall proportion of linked families, ut and vt are also unknown parameters (with constraints ∑ut = 1 and ∑vt = 1) that need to be estimated. Note that ut and vt are the parameters to be estimated from the data and are not necessarily the same as pt and qt, respectively, as the latter are the true values of the former. We have shown earlier that the maximum likelihood estimates for αt exist. However, one cannot solve the equations in (3) to obtain a corresponding estimate for α. This is because there are only T + 2 equations (including the two constraints) but 2T + 1 unknown parameters, and thus not all parameters are identifiable when T ≥ 2. Hence, one may not be able to find a meaningful estimate of the overall proportion of linked families, contrary to popular desire for such an estimate. This is consistent with the finding of Whittemore and Halpern [where  Halpern , althougWe have already shown that LUFED is a necessary condition for obtaining consistent estimators using the admixture approach. In this section, we investigate, through a contrived dataset, whether LUFED is also a sufficient condition for achieving satisfactory results, as contended by Janssen et al. . We use p1, and Phase Unknown (PU) families in proportion p2, where p1 + p2 = 1. The unlinked group is also composed of PK and PU families, but in proportions q1 and q2, respectively, where q1 + q2 = 1. We choose to work with the PK and PU families as the PK families were used by Janssen et al. [m children in all PK families and m + 1 children in all PU families. As these two types of families have different distributions [T = 2) under the classification scheme according to distributions.In our contrived dataset, the linked group consists of two types of families: Phase Known (PK) double backcross families in proportion n et al.  to evalum children is:Now, let us consider the ELOD of each family. The ELOD of a PK family with θ is the parameter for recombination fraction while r is the true recombination fraction. Similarly, one can find the ELOD of a PU family with m + 1 children:where, as before, r close to 0, it can be seen that ELODPK(θ) and ELODPU(θ) are both approximately m log10 2, when they are evaluated at θ = r [α parameters.For at θ = r  LUFED com/0, m - 1/1,..., 1/m - 1, 0/m) offspring.It is worth noting that this dataset may serve as an example for understanding classification based on distributions and family realizations that make up a family type. The phase information as well as the number of offspring are part of the family structure, which is the basis of classification according to distributions. However, phenotypes and genotypes do not configure into this classification scheme. For example, the PK data type consists of families with several phenotypes, i.e., various combinations of recombinant/non-recombinant (p = (0.9, 0.1), q = (0.1, 0.9); and (b) p = (0.3, 0.7), q = (0.4, 0.6). For each of these two settings, we take m = 3 children in PK families and m + 1 = 4 children in PU families. In both settings, the overall proportion of linked families, a, is 0.5, and the true  recombination fraction, r, is 0.02. So, as discussed above, the distribution of informativeness among the linked families, under the ELOD measure, is the same as that among the unlinked families. The two pictures in Figure α, θ) where the maximum expected log-likelihood occurs are not the same as their true values. So the estimates are inconsistent. The extent of this inconsistency depends on several factors, including how close p is to q. As seen in Figure p differs greatly from q, the parameter estimates are far away from the true values. On the other hand, in Figure p is close to q, the estimates are not too far from the truth. In fact, the estimates are expected to converge to the true parameter values when the difference between p and q approaches 0.To illustrate our results, we consider two specific settings for the distributions of families in the linked and unlinked groups under the classification scheme according to distributions: (a) = 0.9, 0., q =  in the two groups. Although the problem considered in Vieland and Logue [The first question of interest was addressed in Whittemore and Halpern , where tnd Logue  is diffeno under the ELOD measure for informativeness. The difference between the estimates and the true values of the parameters, even with infinite amount of data, can be large, if one would carry out the analysis under the admixture approach. This should serve as a warning against complacency when the LUFED condition is met. We do realize that, with phase known and phase unknown data, these examples can be quite extreme in human genetic studies, although such data arise frequently in experimental crosses. In situations where data from more general family structures are available, the effect of violation of the admixture approach assumptions may be much smaller.The second question stems from our curiosity on an argument made by Janssen et al.  for the The criteria for classification of families deserve further clarification because it is the heart of the problem. We discuss two criteria for classification, one according to the distributions of the data, and the other according to a measure of informativeness of the data for linkage studies (leading to the LUFED condition). The classification scheme based on the distribution criterion leads to a necessary and sufficient condition for validating the admixture approach. This result is a by-product of our theoretical development for finding an answer to the first question. The classification scheme based on the informativeness criterion, on the other hand, leads to the conclusion that LUFED is only a necessary, but not a sufficient, condition for the admixture approach, contrary to Janssen et al.'s  contentiFinally, we note that although the general formulation of likelihood based on forming groups of families with the same distribution gives consistent estimators, its practical utility seems to be limited. This is mainly due to the difficulty of classifying families according to distributions, in most applications, except for simple situations such as those involving only the PK and PU families. The general likelihood formulation is used here as a vehicle to further the understanding of potential problems in using the two-parameter admixture approach. A practical solution that embeds the correct likelihood formulation in a Bayesian framework is being pursued in a separate study.Both authors were involved in all aspects of this research project. Both authors read and approved the final manuscript.


The rate of total edentulism is said to be increasing in developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries. Several reports have shown that non-disease factors such as attitude, behavior, dental attendance, characteristics of health care systems and socio-demographic factors play important roles in the aetiopathogenesis of edentulism. The aim of this study was to assess the relationship between socio-demographic factors and edentulism.A total of 152 patients made up of 80 (52.6%) males and 72 (47.4%) females who presented in two prosthetic clinics located in an urban and a rural area were included in the study. The relationship between gender, age, socio-economic status and edentulism in this study population was established.rd decade and then started to decline. A significant relationship was found between denture demand and the level of education with a higher demand in lower educational groups (p < 0.001). In addition, the lower socio-economic group had a higher demand more for prostheses than the higher group.No significant relationship between gender and denture demand was noted in the study. The demand for complete dentures increased with age while the demand for removable partial dentures also increased with age until the 3The findings in this study revealed a significant relationship between socio-demographic variables and edentulism with age, educational level and socio-economic status playing vital roles in edentulism and denture demand. Edentulism  is an indicator of the oral health of a population . It may While the rate of total edentulism is decreasing in developed countries, the reverse is the case with developing countries and this had been attributed mainly to the high prevalence of periodontal diseases and caries -7.Previous studies have also shown that several non-disease factors such as attitude, behavior, dental attendance, characteristics of health care system and socio-demographic factors play important roles in the aetiopathogenesis of edentulism .Some studies reported that the incidence of edentulism correlated with educational levels and income status, with those in the lower levels exhibiting higher risks of becoming totally edentulous ,9. In adAlthough, Hoover and McDermount  reportedStudies among Nigerians have linked some of these socio-demographic factors with the prevalence, pattern and rate of dental diseases ,14 but tThe aim of this study therefore was to assess the relationship between socio-demographic variables with types of edentulism.All patients that attended and were treated in the removable prosthetic units of Obafemi Awolowo University Teaching Hospitals Complex (OAUTHC), Ile-Ife  between the months of March and May year 2002 and Lagos University Teaching Hospitals (LUTH), Lagos  between December 2002 and March 2003 were included in the study. Information such as age, gender, occupation and level of education attained were documented. The types of partial denture received following treatment at the clinics were also documentedThere has not been a consensus on various socio-economic classifications in Nigeria because of the unstructured nature of the society. Therefore, for the purpose of this study, a standard occupational classification system designed by Office of population Census and Surveys, London (OPCS 1991)  modifiedClass 1 = Skilled worker e.g professionals and managerial officers and retirees of this cadre.Class 2 = Unskilled workers e.g. Artisans and tradersClass 3 = Dependants. e.g. Retirees of class 2, those not on pensions, house wives of class 2 cadre, students whose parents are unskilled workersData was analysed using SPSS for Windows version 10.0, (SPSS Inc Chicago Illinois, USA). Analysis included frequency, cross tabulations, calculation of means. Association between discrete variables was tested by Chi-Square and the level of significance was set at 5%.One hundred and fifty two patients attended the prosthetic clinics during the study period. Eighty (52.6%) were males while 72 (47.4%) were females Table . Their aThere were no statistically significant age p = 0.312) and gender (p = 0.113) differences between the populations from the two centers. (Tables 2 and gen2 = 7.50 df = 3, P < 0.001) .  patients belonged to class I; 43(28.29%) patients belonged to class II while 81(53.29%) belonged to class III. There was no statistically significant difference in the socio-economic status of patients from the two centers ((χIn both centers, 134 patients (88.2%) received removable partial dentures, 13 patients (8.6%) received complete dentures while 5 patients (3.3%) received either upper or lower complete dentures. There was no significant difference in the demand for different types of dentures between the study locations. (P = 0.315). . Table It was observed that as the age increased, the proportions demanding for complete dentures also increased. In addition, those in age group 21–40 years demanded more for removable partial denture than any other age groups. While those above 61 years asked more for removable complete dentures than removable partial dentures. (Table No significant relationship between gender and pattern of denture demand (p = 0.812) was noted and no statistically significant difference was noted in the pattern of denture demand between the two centers (p = 0.277). Table .The lower educational groups demanded more for complete dentures among those asking for complete denture, while those with higher level of education asked more for removable partial denture. (P < 0.001). Table . More ovAmong the patients that were completely edentulous, there was no significant difference in the demand for complete dentures between those with lower educational status and those with higher educational status. P = 0.276 , hence the likelihood of retaining teeth in the mouth becomes higher as the educational level increases. Although the educational status of the patients from the two centers differ, independent analysis of these centers still showed the same significant effect of educational status on the pattern of denture demand. This is in agreement with the findings of Brodeur et al, where the proportion of completely edentulous adults decreased from 26% in 1980 to 20 % in 1993 due to improved income and educational status [, hence tThe association between edentulism and educational status may be as a result of improved dental health awareness, increased utilization of oral health facilities, proper oral hygiene habits acquired during learning process and peer group influence.Interestingly, about 23.3 % of those in class II who need dentures asked for complete dentures as opposed to 3.6% in class I and 8.6% in class III. The reason for this may be as a result of the fact that they may not be able to afford the exorbitant cost of restorative procedures hence they wait until they have lost their set of teeth to have a complete removable denture which is cheaper.The present study showed that people with low socio-economic status demanded for more dentures than the high socio-economic group. Studies have long established a gradient relationship between socio-economic status and health . In so mThe importance of socio-economic status is further reflected in the urban-rural variance noted in this study population's demand for denture. The Ile-Ife study group, a rural population with a lower socio-economic status, demanded for more dentures than the Lagos study group.However, this study population was an all-inclusive hospital based sample; the result may not be representative of the population at large. Hence, its use can only be limited to the study population. A randomized population based survey may be able to present a better picture among Nigerians.This study observed that edentulism is due to a combination of various factors. Poor education, a risk factor for poverty, has been identified as a major factor in edentulism. So also is the socio-economic status of the patient. These two factors, which are non-disease factors, affect the mortality of teeth arising from disease factors. There is therefore a need for oral health policy formulators to focus on improving the educational and socio-economic status of its citizens (a down stream approach) rather than the present emphasis on disease control (an up stream approach) in oral health care delivery.On the other hand, with increasing level of literacy, and positive social changes in Nigeria, Prosthodontists should brace up to face the challenges that may arise from increased removable partial denture demand and decreased demand for complete dentures. This is because, with increase in level of education and socio-economic status of patients, the demand for removable partial dentures is likely to increase while dentists may be confronted with a significant increase in the number of difficult edentulous mouths requiring treatment.In addition to addressing the non-disease factors, dental education should be targeted at the uneducated populace, the rural dwellers and low-income groups to reduce the rate of total edentulism.No gender relationship with denture demand was noted this study. In addition, the demand for complete dentures increased with age. There was a statistically significant inverse relationship between educational levels and demand for dentures. There was more demand for prostheses among the lower socio economic groups.The pre-publication history for this paper can be accessed here:


To contribute further to the classification of three CFTR amino acid changes (p.I148T, p.R74W and p.D1270N) either as CF or CBAVD-causing mutations or as neutral variations.The CFTR genes from individuals who carried at least one of these changes were extensively scanned by a well established DGGE assay followed by direct sequencing and familial segregation analysis of mutations and polymorphisms.Four CF patients (out of 1238) originally identified as carrying the p.I148T mutation in trans with a CF mutation had a second mutation (c.3199del6 or a novel mutation c.3395insA) on the p.I148T allele. We demonstrate here that the deletion c.3199del6 can also be associated with CF without p.I148T. Three CBAVD patients originally identified with the complex allele p.R74W-p.D1270N were also carrying p.V201M on this allele, by contrast with non CF or asymptomatic individuals including the mother of a CF child, who were carrying p.R74W-p.D1270N alone.These findings question p.I148T or p.R74W-p.D1270N as causing by themselves CF or CBAVD and emphazises the necessity to perform a complete scanning of CFTR genes and to assign the parental alleles when novel missense mutations are identified. Frameshift, splice-site, nonsense, and in-frame but nonfunctional deletions (such as p.F508del) are disease-causing mutations. By contrast, the status of some missense mutations is extremely difficult to assess and functional studies are not available to diagnostic laboratories. Missense mutations may represent, depending on the populations, up to 45% of mutations responsible for CF or CBAVD . Moreover, due to improved scanning strategies, a growing number of complex alleles  are thought to affect the expression of the disease phenotype by modulating the effect of a mutation [Cystic fibrosis (CF) is a common, often fatal disease with a well-defined genetic cause, so that it is now recommended in many countries in Europe and the United States to offer genetic screening for CF mutations to identify carriers among adults with a positive family history of CF, partners of individuals with CF, couples planning a pregnancy, couples seeking prenatal care and, recently, neonatal screening. Because of the mutational heterogeneity and the rarity of many mutations, most clinical DNA laboratories offer tests that aim to detect 75–95 % of CF alleles depending on the ethnic and geographic backgrounds of the population, using available commercial kits motsly including 20 to 31 mutations selected on the basis of their frequency as CF-causing mutations. A few laboratories  have developed the scanning of coding/flanking CFTR sequences to detect unknown mutations. As of February 2004, about 1200 disease-causing mutations have been identified in the cystic fibrosis transmembrane conductance regulator (CFTR) gene mutation  and whiccis with p.I148T in all individuals with a CF phenotype, and that a third missense mutation (p.V201M) was associated in cis with complex allele [p.D1270N;p.R74W] in patients with a CBAVD phenotype in this series.With the start of population screening for CF carriers, new data on the prevalence of some missense mutations have been provided, questioning their involvement as disease-causing mutations. In North American populations, missense mutations p.I148T and p.D1270N were found >100 times and >200 times, respectively, more frequently in carrier screening than in CF patients . Moreovetrans.From 1990 to 2003, we have analysed for CFTR mutations genomic DNA from 437 families with CF and 170 with isolated azoospermia caused by CBAVD, using a combination of mutation screening for known and scanning for unknown mutations. The first step was the search of 31 CF mutations detected by the ABI oligonucleotide ligation assay and 3 common intronic mutations by using restriction analysis. The second step was the scanning of coding/flanking sequences by DGGE (Denaturing Gradient Gel Electrophoresis) using 32 GC-clamped amplimers (which in our experience detected 98% of CFTR mutants), followed by sequencing to resolve abnormal PCR products (BigDye terminator cycle sequencing on ABI 310 automate sequencer). Whenever possible, family members were assayed for the mutations and associated polymorphisms. We detected 160 different mutations in the CF group accounting for 97 % of CF alleles, and 64 different mutations in the CBAVD group accounting for 85% of CBAVD alleles, which represents one of the highest allelic heterogeneity reported so far. Usually, mutation scanning is stopped when two mutations are found to be in trans of c.3195del6 [In this study, we analyzed by DGGE the entire coding and flanking regions of the CFTR gene of individuals who had been previously found to carry p.I148T or the complex allele [p.R74W;p.D1270N] and assayed their relatives for the additional sequence changes identified. We also re-analyzed the CFTR gene of a CF patient who had been originally described with c.394delTT in .Gene variants and mutants are described using DNA and protein designation: intronic changes, deletions, insertions and frameshifts are reported at the cDNA level (c.) and amino acid changes at the protein level (p.), as recommended in the Human Genome Variation Society web page Two out of 437 CF patients analyzed in Montpellier and two out of 801 CF patients analyzed in Lyon were found to carry p.I148T, which was initially thought to be one of the two mutations responsible for CF in these patients. However, thorough re-analysis of the entire CFTR sequence determined that a CF mutation (c.3395insA or c.3199del6) was present on the same gene in both cases  on the same chromosome in three unrelated individuals with CBAVD ; in addition, a completely asymptomatic adult compound heterozygote for p.D1270N and p.I148T has been identified . Althougtrans that are in fact complex alleles.This report further corroborates the recent hypothesis  that p.INone declared.JPA, CG, CT and FC carried out the molecular genetic studies. MDG coordonated the molecular analysis. MC conceived the study and drafted the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress to blindness from retinal detachments or severe retinal scar formation. This is a preliminary report of the effectiveness and safety of a new and original use of topical ketorolac in preterm newborn to prevent the progression of ROP to the more severe forms of this disease.From January 2001 to December 2002, all fifty nine preterm newborns with birthweight less than 1250 grams or gestational age less than 30 weeks of gestational age admitted to neonatal intensive care were eligible for treatment with topical ketorolac (0.25 milligrams every 8 hours in each eye). The historical comparison group included all 53 preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000.Groups were comparable in terms of weight distribution, Apgar score at 5 minutes, incidence of sepsis, intraventricular hemorrhage and necrotizing enterocolitis. The duration of oxygen therapy was significantly longer in the control group. In the ketorolac group, among 43 children that were alive at discharge, one (2.3%) developed threshold ROP and cryotherapy was necessary. In the comparison group 35 children survived, and six child (17%) needed cryotherapy (Relative Risk 0.14, 95%CI 0.00 to 0.80, p = 0.041). Adjusting by duration of oxygen therapy did not significantly change these results. Adverse effects attributable to ketorolac were not detected.This preliminary report suggests that ketorolac in the form of an ophthalmic solution can reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size. Retinopathy of Prematurity (ROP) is a common retinal neovascular disorder of premature infants. It is of variable severity, usually heals with mild or no sequelae, but may progress in some infants to partial vision loss or blindness from retinal detachments or severe retinal scar formation . ROP remThe active disease appears in the premature about 4 to 8 weeks after birth. In this period, the levels of vascular endothelial growth factor (VEGF) increase in the retina, as well as other chemical mediators of inflammation such as platelets activator factor (PAF), prostaglandins (PGs), and eicosanoids which would put again under way the process of vascularization that had stopped in the period of oxidative injury. This vascularization is now degenerated and invasive -17.In models of animal experimentation it was possible to diminish the degree of retinal neovascularization with use of indometacin , dexametKetorolac is a non-steroid anti-inflammatory drug (NSAID) derived from indometacin. Its mechanism of action is developed through the interruption of the synthesis of prostanoids, inhibiting the way of COX 1 and 2 in arachidonic acid metabolism; in this way the tissue levels of prostaglandin F2alfa and thromboxane B2 decrease. Its adverse effects are linked predominantly to their inhibitory action of platelet aggregation. It does not alter either the platelet count, or factors of clotting. High digestive hemorrhage is the principal adverse reaction. Nervous and cardiovascular systems are not generally affected by the use of ketorolac to habitual doses -24.Ketorolac administered as conjunctival topical diminishes prostaglandin E2 concentration in aqueous humor, without modifying the intraocular pressure . In addiKetorolac ophthalmic solution is usually used in older adults with retinal disorders. It is used to diminish the Cystoid Macular Edema that complicates the surgery of cataracts. In this pathology ketorolac has proved to be effective in diminishing the macular edema and improving the visual acuity, providing evidence that their conjunctival instillation produce effects on the most internal layers of the eye. The ophthalmic use of ketorolac only reports occasional episodes of discomfort and ocular burning -28.The use of ketorolac ophthalmic solution in pediatrics is frequent as an analgesic in corneal abrasions, and in allergic and post surgical conjunctivitis. The FDA recognizes its indication for allergic conjunctivitis, ocular pain, post surgical ocular inflammation, ocular pruritus and photophobia ,29.On the basis of experimental evidence and physio-pathogenic rationality, we treated preterm newborns admitted to neonatal intensive care unit (NICU) of our hospital with ketorolac in ophthalmic drops with the aim of decreasing the progression and severity of ROP. This study compares this group of children treated with ketorolac with historic controls to assess the impact of this treatment on the incidence of severe ROP and adverse effects.This is a preliminary report, that compares a cohort of children treated with ketorolac with historic controls that did not received such treatment. From January 2001 to December 2002 all preterm newborns with birthweight less than 1250 g or gestational age less than 30 weeks of gestational age, admitted in the NICU of the University Hospital of Maternity and Neonatology of the city of Córdoba, Argentina, were eligible for treatment with topical ketorolac. The comparison group included all preterm newborns, with the same inclusion criteria, admitted between January 1999 and December 2000. None of these newborns received treatment with ketorolac.There were no differences either in treatment guidelines, equipment or in the number of physicians and nurses that took care of the patients in the two periods. All the children were examined by the same group of ophthalmologists, which have many years of experience in treating patients with ROP. The international classification for ROP  was usedDuring the period in which ketorolac tromethamine was used, when risk signs for ROP were identified   treatmenThe variables considered to assess the comparability among both groups were: birthweight, Apgar score at birth less than 6 at 5 minutes, duration of oxygen therapy, peri-intraventricular hemorrhage (PIVH) equal or greater than 3 degrees, necrotizing enterocolitis (NEC) equal of greater than II degrees, and late sepsis. The analyses included only those children that were alive at discharge, but the number of deaths was reported for both study periods. The presence of undesirable effects of ketorolac such as hemorrhages, oliguresis, local manifestations of intolerance, and conjunctival infection were analyzed.Fischer exact test was used to obtain two tailed p-values. Relative risks (RR) with 95% confidence intervals where also calculated. Adjusted analyses were performed by using Poisson regression with robust estimates . StatistDuring the analyzed period 112 eligible preterm newborns were admitted, 53 between 1999 and 2000 (historic controls) and 59 between 2001 and 2002 (ketorolac group). The number of newborns that died before discharge was similar between groups  than in the control group  is large and in our opinion unlikely to be explained completely by confounding factors, although this possibility cannot be completely ruled out. To our knowledge, no significant change in the standard of care took place between the study periods, but mortality was lower during the period were ketorolac was used, although the difference was not statistically significant. The groups were comparable in terms of their birthweight distribution, Apgar score, and indicators of severe morbidity. A significant difference was found in duration of oxygen therapy, but in our data there was no association between duration of oxygen therapy and severe ROP, and adjusting for oxygen did not change the results. The principal factor involved in the genesis and severity of ROP is the injury due to O2 radicals; there were no substantial changes in the management of O2 supply to the children of our unit in both periods, the equipment used to measured O2 saturation and to administer the oxygen.The prevalence of severe ROP in our maternity was relatively high, considering that for 1998 the Vermont-Oxford Network reported an incidence of 9.5% for severe ROP and of 57.2% for ROP of any degree , but simKetorolac was apparently safe. No patients in the group that received ketorolac presented oliguric, or with biochemical signs of renal failure during treatment. There were no hemorrhagic manifestations that could be attributed to ketorolac; hemorrhages observed in vitreous evolved favorably with collyrium. Neither local intolerance episodes to the drops nor purulent conjunctivitis among the treated cases were observed.The effect on ROP we have observed is compatible with an impact of Ketorolac in the active phase of neovascularization, a fact that is also observed in numerous reports in animal models of ROP with NSAIDs and steroid drugs administered systemically. The hypothesis of an inflammatory component in active ROP has been quite recently recognized by experts, extensive leukocyte adhesion was observed at the leading edge of pathological neovascularization . Recent ROP is a problem that modern medicine has generated due to the survival of very small preterm children and which has been unable to solve so far. About 2% of the children with birthweight less than 1500 grams are blind because of this pathology. Current preventive interventions can prevent blindness in nearly 70% of the cases. Earlier treatment using ablation of the avascular retina in pre-threshold ROP has been proposed , but a pThis preliminary report provides evidence suggesting that ketorolac in the form of an ophthalmic solution can be used to reduce the risk of developing severe ROP in very preterm newborns, without producing significant adverse side effects. These results, although promising, should be interpreted with caution because of the weakness of the study design. We cannot exclude the possibility of bias been responsible of the observed effect. On the other hand, if confirmed, these results can have important public health implications. This is an inexpensive and simple intervention that might ameliorate the progression of a disease with devastating consequences for children and their families. We believe that next logical step would be to assess the effectiveness of this intervention in a randomized controlled trial of adequate sample size to provide a definite answer to this research question.None declared.MAV and RM conceived the study. MAV, MLC, EB and RM design the study. MF carried out the revision of the clinical histories. MS carries out the ophthalmologic exams of all the cases. RM verified the ophthalmologic finds. EB carried out the statistical analysis. MAV and EB drafted the article. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


DMBT1 , a putative tumor suppressor gene, in normal, proliferative, and malignant breast epithelium and its possible relation to cell cycle.We studied the expression of DMBT1 antibody (DMBTh12) and sections from 36 samples, were double-stained also with anti MCM5, one of the 6 pre-replicative complex proteins with cell proliferation-licensing functions. DMBT1 gene expression at mRNA level was assessed by RT-PCR in frozen tissues samples from 39 patients.Sections from 17 benign lesions and 55 carcinomas were immunostained with anti DMBT1 protein expression was down-regulated in the cancerous lesions compared to the normal and/or hyperplastic epithelium adjacent to carcinomas . In 72% of cases RT-PCR confirmed immunohistochemical results. Most of normal and hyperplastic mammary cells positive with DMBTh12 were also MCM5-positive.Normal glands and hyperplastic epithelium in benign lesions displayed a luminal polarized DMBTh12 immunoreactivity. Normal and hyperplastic epithelium adjacent to carcinomas showed a loss of polarization, with immunostaining present in basal and perinuclear cytoplasmic compartments. DMBT1 in normal and hyperplastic tissues flanking malignant tumours and its down-regulation in carcinomas suggests a potential role in breast cancer. Moreover, the concomitant expression of DMTB1 and MCM5 suggests its possible association with the cell-cycle regulation.The redistribution and up-regulation of  DMBT1  at chromosome 10q25.3–26.1, was considered a tumour suppressor gene because frequent deletions and/or lack of expression had been found in malignant tumours of the brain [he brain ,5, and the brain .DMBT1 is not a typical tumour suppressor gene because it is up-regulated in normal or inflamed epithelium adjacent to lung carcinomas as well as in some primary lung cancers and glioblastomas [DMBT1 inactivation in many tumours including breast cancer [However, recent evidence has shown that lastomas . It was t cancer ,10DMBT1 is a multi-functional protein related to the Mac-2 binding protein and mucins that have a protective function and mediate cell-extracellular matrix interactions [GP340(glycoprotein-340) and DMBT1SAG  are DMBT1 variants, present in the respiratory tract and the oral cavity, respectively, that have been linked to innate host defense and epithelial regeneration [GP340is involved in innate host defence by its interaction with the lectins surfactant protein D and A (Sp-D, Sp-A) and by its ability to stimulate alveolar macrophage migration [in vitro and in vivo [DMBT1 expression and localisation observed in other tumor types originating from mono-layered epithelia such as primary esophageal adeno-carcinomas and lung carcinomas [ractions ,11. DMBTneration -16. In tigration ,13,17. A in vivo ,19. Whil in vivo . Remarkarcinomas ,8.DMBT1 [DMBT1 homologue in the mouse, and a novel homologue in the rat (DMBT1 4,7 kb), have been linked to initiation of cell proliferation, differentiation and repair [DMBT1 to the ECM triggers cellular differentiation, whereas a loss of expression of DMBT1 favours tumor cell growth [Hensin, the rabbit homologue of DMBT1, triggers epithelial terminal differentiation by mediating cell-extracellular matrix (ECM) interactions, and this may apply also to DMBT1 ,21-24. Cd repair ,26. It hl growth .DMBT1 in the morphologically normal, hyperplastic, and neoplastic breast epithelium. We also examined in morphologically normal epithelium a possible association of DMBT1 expression with that of an early proliferative marker such as MCM5 (Minichromosome Maintenance protein 5). MCM5 is one of the 6 pre-replicative complex proteins (MCM 2–7) [These findings led us to explore whether similar principles apply to breast epithelium. Here we examined the expression of MCM 2–7)  and its in situ (DCIS) and 45 cases of infiltrating carcinoma (IC) .From the files of the Surgical Pathology at S.Paolo University Hospital, we retrieved paraffin blocks of 72 consecutive breast lesions surgically resected. This material included 16 cases of fibrocystic disease (FD), 1 case of gynecomastia (GM), 10 samples of ductal carcinoma RT-PCR (Reverse-Transcriptase Polymerase Chain Reaction) was performed on 39 samples: i.e. 37 samples out of the 55 carcinomas used for the immunohistochemical studies, 1 sample of normal breast tissue that had been removed with a neoplastic lesion and 1 sample of fibrocystic disease. All 39 samples had been quickly frozen in liquid nitrogen after surgical resection and stored at -80°C. As confirmed by histopathologic examination, in all tumor specimens the amount of tumor cells equalled or exceeded 80% of the overall sample. As controls for each test a frozen sample of the same case positive with immunohistochemical method was used. We also analysed by RT-PCR two breast cancer cell lines (Hs578T and T-47D) obtained from the American Type Culture Collection and maintained as recommended.DMBT1 and anti MCM5 monoclonal antibodies (Novocastra Laboratories Ltd, Newcastle upon Tyne, U.K.) was performed on 17 benign lesions and a subset of 19 in situ and invasive carcinomas where residual normal breast tissue was well represented. On these selected samples a single incubation with anti MCM5 and anti Ki 67 antibody was performed in order to compare the two respective immunoreactivities. Antibodies were diluted as follows: anti-DMBT1h12, 1:500; anti MCM5, 1:40; anti Ki 67 Mib1, 1:100. For anti-DMBT1 and Ki 67 Abs, sections were pre-treated for antigen retrieval in boiling 0.01 M sodium citrate buffer pH 6, in microwave (MW) oven for 12 minutes. DMBT1 and MCM5 double labelling was performed in two steps following a modification of the method of Lan et al. [Sections from blocks containing the predominant lesion were incubated with anti DMBT1h12 monoclonal antibody  as well n et al. , brieflyThree investigators (AM, PB, PGN) evaluated separately DMBTh12 immunopositivity of morphologically normal, hyperplastic, and malignant cells. Immunopositivity was scored in 4 distinct categories according to the percentage of positive cells: 0 = negative, 1 = < 10%, 2 = 10%–50% and 3 = > 50%. MCM5 and Ki67 positivity in morphologically normal epithelium was determined with a semi-quantitative method: percentage of positive glandular structure and number of positive cells observed in each positive gland divided number of glands.Total RNA was extracted from breast specimens using Trizol reagent,  according to the manufacturer's instructions. Total RNA was quantified and checked for purity by UV spectrophotometry.2, 20 units of RNase inhibitor, 50 units of MuLV Reverse Transcriptase, 2.5 μM Random hexamers and 1 μg of total RNA. All reagents were purchased by the supplayer (Applied Biosystems, Foster City, CA, USA). The samples were incubated at 25°C for 10 min, 42°C for 60 min and at 95°C for 5 min.Reverse transcription of RNA was done in a final volume of 20 μl containing 1x buffer (50 mM KCl, 15 mM Tris-HCl, pH 8.3), 500 μM each deoxynucleotide triphosphate, 5 mM MgCl2, 1.25 units of Taq Gold DNA polymerase and each primer at 0.3 μM. The PCR amplification of DMBT1 was carried out in two steps, following a nested procedure. In the first step we used 2 μl of cDNA from the reverse transcription and in the second 2 μl of the first PCR product as template. Primers and thermal condition were used according to Mollenhauer et al [DMBT1) were obtained.Polymerase chain reaction (PCR) of the cDNA was performed in a final volume of 50 μl containing all four dNTPs (each at 200 μM), 1X buffer, 1.5 mM MgClStatistical differences between immunopositive cases/total number of cases were calculated by Fisher's exact test. p value less than 0.05 were considered statistically significant.T-test for independent values was used to evaluate statistically significant differences between immunoreactivity of normal and hyperplastic epithelium in benign, pre-invasive and invasive lesions.Different patterns of immunopositivity were observed in the histologically normal and hyperplastic breast tissue, present in the sections of benign and malignant lesions: a) single supra-nuclear, intra-cytoplasmic dot polarized toward luminal surface Figure ; b) a stDMBT1 at protein level in the mammary gland epithelium. In tumour areas, only 3 out of 55 carcinomas showed variable degrees of immunopositivity with anti-DMBTh12 Ab: in one case there was a widespread and strong decoration of numerous tumour cells (immunopositivity score = 3) with irregularly distributed dots [Figure DMBT1 expression in the cancerous lesions compared to the normal and/or hyperplastic epithelium was statistically significant for both, ductal carcinoma in situ (DCIS)  and invasive carcinoma (IC) .Normal and hyperplastic mammary structures displayed a variable DMBTh12 immunoreactivity: some ducts were decorated while others were negative. Moreover a variable percentage of cells was immunopositive. This heterogeneity suggests that there is no constitutive expression of s Figure ; in the s Figure  and in ts Figure . Down-reDMBT1 expression at protein and mRNA level are summarized in table The results of DMBT1 immunoreactive cells between the normal and the hyperplastic epithelium that flanked benign lesions, in situ and invasive carcinomas  RT-PCR confirmed immunohistochemical results, whereas 11 tumours that were negative with anti-DMBTh12 revealed PCR-products after RT-PCR amplification  and malignant (78%) lesions. Among 55 carcinomas we could detect immunopositivity only in 3 tumours (5%); in one case of infiltrating carcinoma the immunopositivity was widespread with a pattern similar to that seen in hyperplastic lesions near carcinoma; in the other two cases , it was limited to few tumour cells. DMBT1 expression was detected by RT-PCR in 13 of 35 (37%) infiltrating carcinomas and in 2 breast cancer cell lines. Eleven tumours, that were negative with anti-DMBTh12, revealed PCR-products after RT-PCR amplification. This discordance between IHC and RT-PCR results may be due to residual normal epithelial cells present in the sample. Furthermore other possible reasons of this discrepancy could be: 1) the greater sensitivity of molecular technique like RT-PCR in detecting very low expression levels of DMBT1 that were below detection by immunohistochemistry, 2) sampling problems i.e. the presence of few positive tumour cells only in the frozen samples or 3) few immunoreactive cells have been missed by microscopic examination and finally 4) DMBT1 protein could have an altered epitope not recognized by its paratope in the antibody. By either immunohistochemistry or molecular technique we found no correlation between DMBT1 expression and tumor histotype, grade, hormonal receptors status, proliferation index, Her2 Neu expression or lymph-nodal metastases.In the present study we investigated DMBT1 in human breast cancer, but an indirect weak correlation between the degree of differentiation of the breast carcinomas and the extent of the immunoreactivity was found [DMBT1 in breast cancer or to other poorly-defined factors such as race, age or hormonal status of the population under study. Mollenhauer et al. point out that DMBT1 expression may be up-regulated in pathophysiological conditions such as inflammation, liver injury or after carcinogen administration and it is down-regulated in tumors to achieve a growth advantage in a dynamic and complex model. Also according to these authors, the possibility that DMBT1 loss may be a consequence rather than a primary event in carcinogenesis is unlikely (10).In a recent paper on breast carcinogenesis, qualitatively similar data were reported for as found . The disDMBT1 expression by RT-PCR [DMBT1 expression patterns observed in the human respiratory tract and in associated inflammation and carcinomas [DMBT1 expression in the normal mammary gland epithelium may result from the necessity of DMBT1 induction. Indeed, in parallel in vivo studies, it was found that DMBT1 is strongly up-regulated in the mammary gland tissue of the mouse shortly after carcinogen-administration and prior to the onset of histo-pathologically visible symptoms [DMBT1 products [DMBT1 in breast tissue may be part of a protective response as suggested by Kang et al [DMBT1 and Sp-D in pulmonary epithelium.Although the normal mammary gland has been reported to have low y RT-PCR , we founy RT-PCR . Moreovercinomas  and suggsymptoms . It has products . We receproducts ; thus, wng et al  for DMBTDMBT1, the depolarisation of the staining patterns indicates that DMBT1 may also be secreted to other destinations, i.e. the ECM, by the mammary gland epithelial cells. In this regard, the second putative function of DMBT1, i.e. triggering of epithelial differentiation, is noteworthy. It may prevent hyperproliferative epithelial cells from out-of control proliferation and progression to the tumor state. This view is supported by two present findings. The correlation between MCM5 and DMBT1 expression may point to a coupling to cell-cycle processes: basolateral relocation and hyper-expression of DMBT1 at the ECM may provide cells that are licensed to proliferate with protection from unregulated proliferation. On the other hand loss of DMBT1 expression in a significant numbers of DCISs and ICs is consistent with the notion that DMBT1 inactivation may offer growth advantages to neoplastic cells [DMBT1 expression appears to take place early, i.e. already at the time of transition from hyperplastic lesions to carcinoma, as it has been reported in oesophageal squamous cell carcinomas [While this interaction may be attributed to lumenally secreted ic cells . The losDMBT1 inactivation remain unclear. An inactivation by mutation appears not to be a major mechanism for DMBT1 inactivation in tumors [DMBT1 or genes that regulate DMBT1 expression takes place in some tumors [DMBT1 promotor, loss of expression or mutation of DMBT1-regulatory genes might represent alternative candidate mechanisms.The exact mechanisms for n tumors ,31 The se tumors . AccordiDMBT1 expression and cellular location in breast epithelium varies in normal, hyperplastic and neoplastic cells. In normal or hyperplastic tissue flanking carcinomas it is up-regulated and polarized toward glandular lumen or the basolateral membrane while it is down-regulated in carcinomas. These findings closely resemble the patterns observed for its interaction partner SP-A in an earlier study. Additionally we have shown that DMBT1 is co-expressed with MCM5 in normal and hyperplastic tissue. We hypothesize that hyper-expression and basolateral relocation of DMBT1 in these epithelia prevents the out of control growth that characterizes neoplastic epithelium.We have shown that DMBT1: Deleted in Malignant Brain Tumor 1,MCM5: Minichromosome Maintenance protein, 5RT-PCR: Reverse Transcriptase Polymerase Chain ReactionGP 340: Glycoprotein 340SAG: salivary AgglutininSp-A, Sp-D: Surfactant Protein A, DMBP: Mannan Binding ProteinECM: Extracellular MatrixFD: Fibrocystic DiseaseGM: gynecomastiaIn Situ DCIS: Ductal Carcinoma IC: Invasive CarcinomaMW: Micro WaveEDTA Ethylenediaminetetraacetic AcidDAB: DiaminobenzidineNone declared.PB designed the study, participated in the evaluation of immunohistochemical results and drafted the manuscript. PGN participated in the evaluation of immunohistochemical results and performed statistical analysis. JM participated in sequence alignment, criticism and produced DMBTh12 antibody. AP participated in sequence alignment and data evaluation. CP Carried out RT-PCR. AM participated in the evaluation of histologic samples and immunohistochemical results. GB participated in data evaluation and sequence alignment GC participated in study design and data evaluation. SB participated in study design, sequence alignment, criticism and coordinated molecular study. GGP participated in study design, criticism and general revision of paper.The pre-publication history for this paper can be accessed here:


In most parts of the world, microscopy is still the gold standard for diagnosing malaria. An online tool could help to improve your diagnostic skills An online tool allows microscopists to test their knowledge Malaria kills over one million people in Africa each year and contributes 10% of the continent's burden of disease . In thisMalaria affects at least 200–300 million people every year and causes from 1–2 million deaths—mostly children under five and pregnant women in sub-Saharan Africa [Malaria is not limited to Africa—40% of the world's population is at risk of acquiring the disease —and it iDespite the efforts of a global campaign to roll back the disease, the number of deaths from malaria is increasing in Africa . This stThere is another important reason why correct diagnosis matters. Because of rising drug resistance in Africa, the conventional drugs for treating malaria—chloroquine and sulfadoxine-pyrimethamine—are now failing in up to 80% of cases . There iPlasmodium falciparum [We still regard microscopy as the gold standard for the diagnosis and characterisation of malaria infection . Modern lciparum —althoughlciparum ,17—but tSome authors have suggested that these new tests should not be considered a complete substitute for direct microscopic examination of blood smears . We agreP. falciparum or three P. vivax parasites per microlitre of blood [Analysis of DNA by the polymerase chain reaction (PCR) may be a useful tool for diagnosis of malaria when the results of conventional techniques are negative, especially since PCR allows accurate species identification . And wheof blood . Howeverof blood . Even inhttp://www.rph.wa.gov.au/labs/haem/malaria/index.html). The materials are now available in English, French, and Spanish. One of the most important features is a “Test and Teach” section to allow microscopists to develop and sharpen their skills in malaria diagnosis  has been sent on request to institutions and centres in 149 countries, a number that does not include unauthorised (pirated) copies of the CD-ROM. The number of hits to the site (the number of pages or images of the site that are accessed) has risen dramatically, from 1,500 to over 100,000 per week.Graham.Icke@health.wa.gov.au.Through a circulated questionnaire with a 63% response rate, we have received feedback from users in 15 countries. Respondents said they valued the content, presentation, and usefulness of the online information. Who is using the material? A wide range of people—from students through to experienced personnel, and from clinicians to scientific, research, and technical staff. Many tropical medicine institutions have requested copies of the CD-ROM to supplement their own training programs. We have discussed the project at scientific conferences around the world and in scientific journals, advertising the fact that this educational material is freely available. Institutions wishing to obtain a free copy of the CD-ROM should contact E-mail: We also became aware that unauthorised copies of the CD-ROM were being produced. Although there is a real need to guarantee the integrity of the information on the CD-ROMs and ensure proper credit for the authors and patrons, our main aim is to spread the “good word”, and such unauthorised copies are a powerful aid to distribution. Given that there is no need to recoup profit from this project, our only concern is its usefulness. Consequently, we have welcomed the production of these pirated copies, on condition that they were of good quality and were made freely available at no cost.The Internet and associated technologies make it possible for educators with a desire to teach to contact those with a need to learn, regardless of the geographical distances involved. By enlisting assistance from international colleagues, language barriers can be overcome. In addition to the English, French, and Spanish versions, we are aware that our project has been translated into German, Thai, and Vietnamese and has been distributed through small regional health group networks.There can be little doubt that these new technologies have the potential to revolutionise information dissemination, with particularly significant implications for healthcare professionals in developing countries. The malaria educational program could be a model on which future health education programs are based. With rapidly increasing access to these new information technologies, health care professionals from anywhere in the world can now join the global health community.


Waterpipe smoking is becoming a global public health problem, especially in the Eastern Mediterranean region (EMR).We try in this study, which is a cross sectional survey among a representative sample of waterpipe smokers in cafes/restaurants in Aleppo-Syria, to assess the time period for the beginning of this new smoking hype. We recruited 268 waterpipe smokers  30.1 ± 10.2, response rate 95.3%). Participants were divided into 4 birth cohorts (≤ 1960, 1961–1970, 1971–1980, >1980) and year of initiation of waterpipe smoking and daily cigarette smoking were plotted according to these birth cohorts.Data indicate that unlike initiation of cigarette smoking, which shows a clear age-related pattern, the nineties was the starting point for most of waterpipe smoking implicating this time period for the beginning of the waterpipe epidemic in Syria.Maassel), and the proliferation of satellite and electronic media during the nineties may have helped spread the new hype all over the Arab World.The introduction of new flavored and aromatic waterpipe tobacco ( This cee smoker , Recent e smoker . This tre smoker ,7. Althoe smoker -11.Maassel , the media, and social trends [Developing effective intervention strategies to curb this emerging public health problem requires clear understanding of factors influencing the take up of this habit, as well its time course . Accordil trends . UnderstThe current analysis is drawn from a survey conducted in 2003 among a representative sample of waterpipe smokers visiting cafes/restaurants in Aleppo, Syria. The survey details can be found elsewhere , but briThe protocols and informed consent documents for this study were approved by the SCTS' IRB and the University of Memphis' IRB. The questionnaire was anonymous and informed consent was obtained prior to all interviews.First, year of birth was calculated by subtracting current age of participant from the year of survey (2003), while year of initiation of waterpipe smoking and daily cigarette smoking were calculated by adding the age of initiation of smoking to the year of birth. Year of birth was then divided into four decade-long categories , and year of smoking initiation into three decade-long categories (initiation in/before 1990, initiation during 1991–2000, and initiation in/after 2001).p level <0.05 considered significant.The Chi-Square test was used to assess differences between the three smoking initiation time-groups for each birth cohort, with th century, and showing an increasing trend. Based on these results and our previous data regarding factors related to the spread of waterpipe [Maassel was introduced [Figure aterpipe , we prestroduced  simplifytroduced ,15, nicoNone declared.SR designed the study, conducted the analysis and wrote the first draft of the manuscript. KW and TE contributed to the design of survey and revision of the manuscript. WM contributed to the design of survey and wrote the final draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


To examine the associations between vision-targeted health-related quality of life (VT-HRQ) and ocular surface parameters in patients with Sjögren's syndrome, a systemic autoimmune disease characterized by dry eye and dry mouth.Forty-two patients fulfilling European / American diagnostic criteria for Sjögren's syndrome underwent Schirmer testing without anesthesia, ocular surface vital dye staining; and measurement of tear film breakup time (TBUT). Subjects were administered the Ocular Surface Disease Index (OSDI) and the 25-item National Eye Institute Vision Functioning Questionnaire (NEI-VFQ). Main outcome measures included ocular surface parameters, OSDI subscales describing ocular discomfort (OSDI-symptoms), vision-related function (OSDI-function), and environmental triggers, and NEI-VFQ subscales.Participants  all had moderate to severe dry eye. Associations of OSDI subscales with the ocular parameters were modest (Spearman r (ρ) < 0.22) and not statistically significant. Associations of NEI-VFQ subscales with the ocular parameters reached borderline significance for the near vision subscale with TBUT (ρ = 0.32, p = .05) and for the distance vision subscale with van Bijsterveld score (ρ = 0.33, p = .04). The strongest associations of the two questionnaires were for: ocular pain and mental function with OSDI-symptoms (ρ = 0.60 and 0.45, respectively); and general vision, ocular pain, mental function, role function, and driving with OSDI-function (ρ = 0.60, 0.50, 0.61, 0.64, 0.57, and 0.67, respectively).Associations between conventional objective measures of dry eye and VT-HRQ were modest. The generic NEI-VFQ was similar to the disease-specific OSDI in its ability to measure the impact of Sjögren's syndrome-related dry eye on VT-HRQ. Dry eye is a common disorder of the ocular surface and tear film and is estimated to affect from 2% to over 15% of persons in surveyed populations, depending on the definition used . SymptomPatient-reported measurements used to evaluate the specific impact of eye disease and vision on symptoms (discomfort), functioning (the ability to carry out activities in daily life), and perceptions  are referred to as vision-targeted health-related quality of life (VT-HRQ) instruments. Valid and reliable measurements of VT-HRQ have become essential to the assessment of disease status and treatment effectiveness in ocular disease . There a© [The measurement of the impact of dry eye on a patient's daily life, particularly symptoms of discomfort, is a critical aspect of characterizing the disease . Despite©  was deveSjögren's Syndrome is an autoimmune systemic disease characterized by dry mouth and dry eye signs and symptoms ,26. Its Our purpose in this study was to examine VT-HRQ in patients with primary Sjögren's syndrome, using a generic and a dry-eye-disease-specific instrument. We examined the associations of ocular surface parameters with the VT-HRQ scores, hypothesizing that the disease-specific instrument would be more closely related than the generic to the clinical markers of disease. We also examined the association of the generic and disease-specific VT-HRQ scores with each other.The study protocol was approved by the National Eye Institute Internal Review Board. All patients completed an informed consent prior to examination. Consecutive patients with diagnosed primary Sjögren's syndrome were recruited from the NIH Clinical Center, Bethesda, MD. The diagnosis of primary Sjögren's syndrome was based on European-American criteria, which requires at least four of the following six features: signs and symptoms of dry eye and of dry mouth, histopathologic evidence of inflammation on minor salivary gland biopsy, and positive anti-Ro or anti-La antibodies.Before the clinical examination, a trained interviewer administered two questionnaires (described further below) to measure VT-HRQ to each patient. The subsequent clinical examination included a comprehensive anterior segment evaluation, including slit lamp biomicroscopy, evaluation of lid margin thickness and hyperemia, conjunctival erythema, chemosis, tear film debris and mucus, and extent of meibomian gland plugging. Tests of tear function and ocular surface status were performed as described below.The OSDI   ,42 is a Schirmer tests of tear production without and with anesthesia were performed by inserting a Schirmer tear test sterile strip (35 mm, Alcon Laboratories, Inc, Fort Worth, TX) into the inferior fornix, at the junction of the middle and lateral third of the lower eyelid margin, for 5 minutes with the eyes closed. The extent of wetting was measured by referring to the ruler provided by the manufacturer on the envelope containing the strips. Possible scores range from 0 to 35 mm, with lower scores indicating greater abnormality in tear production. This test was repeated after instillation of topical anesthetic, 0.5% proparacaine . A SchirThe assessment of ocular surface damage was performed by a cornea specialist using vital dye staining with 2% unpreserved sodium fluorescein and then 5% lissamine green dye. The corneal, temporal, and nasal regions of the conjunctiva were scored individually from 0–5 (for fluorescein) and 0–5 (for lissamine green) using the Oxford grading scheme . The OxfTear film stability was assessed using fluorescein tear film breakup time (TBUT). Five microliters of 2% sodium fluorescein was instilled into the inferior fornix and the patient was asked to blink several times. Using the cobalt blue filter and slit lamp biomicroscopy, the duration of time required for the first area of tear film breakup after a complete blink was determined. If the TBUT was less than 10 seconds, the test was repeated for a total of 3 values and the average was calculated.For analysis, for each individual, the maximum (worse) score for the two eyes was used for Oxford score and VB, and the minimum (worse) score for the two eyes was used for Schirmer with and without anesthesia and for TBUT. TBUT values greater than or equal to 10 seconds  were codHypotheses of specific associations were formulated based on the areas and domains assessed by the two VT-HRQ instruments. Scatterplots and Spearman's correlation coefficient (ρ)  were useA total of 42 patients, 40 female and 2 male, were included in this study. The average age was 55 years range, 31–81 y). Most (81%) were of European descent. Visual acuity in the better eye was 20/20 or better for 68% of the patients; the remainder had 20/25 or better in the better eye, except for one patient who was 20/32 in both eyes. Ocular examination  indicated moderate problems with symptoms, functioning, and adverse environmental conditions. Mean OSDI-symptoms score was 62.5, mean OSDI-function score was 78.2, and mean OSDI-triggers score was 60.2. However, some patients had no problems with these areas: 12% reported no problems with irritation symptoms, 21% reported no problems with functioning, and 24% had no problems with environmental triggers. Associations of the OSDI subscale and overall scores with ocular surface parameters (Oxford score, VB, TBUT, and Schirmer score with and without anesthesia) are shown in Overall, scores on the NEI-VFQ subscales tended to be high. Average scores for near and distance vision, social and mental functioning, dependency, driving, and peripheral vision were over 80, and a substantial percentage reported no problems at all with any of the items on the subscale: 26% for near vision, 24% for distance vision, 83% for social functioning, 17% for mental functioning, 74% for dependency, 38% for driving, and 79% for peripheral vision. The subscale indicating the most impairment was the ocular pain subscale, with a mean score of 66.7. Associations of the NEI-VFQ subscale and overall scores with ocular surface parameters (Oxford score, VB, TBUT, and Schirmer score with and without anesthesia) are shown in Table In general, stronger associations were observed between subscales of the OSDI and NEI-VFQ Table  than betTable disease-specific instrument (OSDI) with a generic VT-HRQ instrument (NEI-VFQ-25) in patients with a systemic autoimmune disease associated with moderate to severe dry eye. We found that patients with primary Sjögren's syndrome had OSDI scores (mean, 30, before subtraction from 100) similar to those previously published [We compared subscale scores for an ocular surface ublished  for modeublished -20. IndeOne explanation could be that the nature of the items for each of these instruments is quite different. The OSDI queries the frequency of a symptom or difficulty with an activity, over a one week recall period. The NEI-VFQ incorporates questions both the frequency and intensity of symptoms and their impact on activities, with no specified recall period. Perhaps this added element of capturing both the frequency and intensity of a symptom or impact accounts for some of the differences we found. For subscales that are similar, agreement was higher but still moderate, possibly due to differences in the nature of the questions or response options. The OSDI is targeted to assess how much the symptoms of dry eye affect the patient's current status (i.e., in the past week), whereas the NEI-VFQ may be more suited to capturing the overall impact of a chronic ocular disease on VT-HRQ.In this group of primary Sjögren's syndrome patients, associations between subscales of the NEI-VFQ and OSDI were moderate to strong (< 0.70) and in hypothesized directions. Significant associations were seen between OSDI and NEI-VFQ overall scales; OSDI-symptoms and NEI-VFQ ocular pain; and OSDI-function and NEI-VFQ general vision and driving. This suggests that both instruments are capturing important aspects of VT-HRQ. It is not surprising that the highest correlations were observed between subscales with similar domains, which serves to validate the use of alternate methodologies. On the other hand, it is counter-intuitive that the generic and disease-specific instruments appeared similar (or that the generic seemed to do a little better) with respect to their association with objectively measured clinical signs of dry eye, as the NEI-VFQ was designed to capture broader aspects of VT-HRQ. For the NEI-VFQ, we found moderate correlations (greater than 0.3) of distance vision with VB and near vision with TBUT. This was surprising, as one may have expected that subscales measuring ocular discomfort or pain (i.e., more disease-specific for dry eye) would have the strongest correlations with clinical measures of dry eye. Clinical signs of dry eye include measures of tear production, ocular surface staining, and tear film break-up; visual acuity and other aspects of visual function are not generally as widely used. However, some investigators have reported that visual acuity in dry eye patients is correlated with decreased spatial contrast sensitivity  and is fWould we expect the associations to be different in Sjögren's patients? Sjögren's syndrome is an autoimmune exocrinopathy and effects of its systemic nature and chronicity on dry eye may have been more readily captured by the NEI-VFQ's ability to measure both frequency and intensity of problems with VT-HRQ. In contrast, although the OSDI includes items to measure function, responses are limited to the frequency of problems. Because the type of dry eye in Sjögren's syndrome is more likely to be severe, and all patients in our study had Sjögren's-related dry eye, we speculated that somewhat stronger associations between signs and symptoms might be observed. On the other hand, ocular surface inflammation and decreased corneal sensation are features of severe dry eye which might alter a patient's perception of symptoms of ocular irritation and might be the cause of weaker correlations between signs and symptoms ,55. IndeIn addition to clinical signs, it is important to include assessments of VT-HRQ and visual function to fully characterize the impact of dry eye on health status. The correlation between signs and VT-HRQ are modest at best, indicating that VT-HRQ is capturing an additional component of disease that is not captured by the clinical assessment. This does not necessarily mean that the measures of VT-HRQ or the methods of detecting clinical signs are deficient, but rather that VT-HRQ is an additional element of the overall impact of this disease process on affected individuals. Furthermore, in diseases with systemic manifestations, such as Sjögren's syndrome, that may have an influence on quality of life independent of dry eye symptoms, appropriate tests of VT-HRQ are critical to completely characterize quality of life in these patients. It may also be valuable to explore possible differences in associations of clinical signs with VT-HRQ in patient populations with different manifestations or causes of dry eye.VT-HRQ: Vision-targeted health-related quality of life; TBUT: Tearfilm breakup time; OSDI: Ocular Surface Disease Index; NEI-VFQ: National Eye Institute Visual Function Questionnaire; VB: van BijsterveldSV helped to design the study and performed all analyses and took the lead in writing the manuscript. LG performed the patient interviews and assisted with data analyses. GFR provided advice on statistical methods and presentation of the results. JA conceived and helped to design the study and assisted with writing the manuscript.