R) constitute 5–10% of peripheral CD4+ T cells in naive mice and humans, and play an important role in controlling immune responses. Accumulating evidences show that TR cells are involved in some physiological processes and pathologic conditions such as autoimmune diseases, transplantation tolerance and cancer, and might be a promising therapeutic target for these diseases.CD4+CD25+ regulatory T lymphocytes (TR cells in mouse tumor models, CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing BABL/c mice were analyzed by flow cytometry using double staining with CD4 and CD25 antibodies.To evaluate the change of CD4+CD25+ TR in tumor mouse models, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.The proportion of CD4+CD25+/CD4+ in spleen lymphocytes was found to be higher than that in peripheral blood lymphocytes in normal mice. No difference was observed in the proportion in peripheral blood lymphocytes between tumor bearing mice and normal mice, while there was a significant increase in the proportion in spleen lymphocytes in tumor bearing mice as compared with normal mice. Moreover, the proportion increased in accordance with the increase in the tumor sizes. The increase in the proportion was due to the decrease in CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggests the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the T R). TR represents a minor (5–10%) component of peripheral CD4+ T cells but plays an important role in controlling immune responses [R cells possess potent suppressive activity both in vivo and in vitro and are involved in autoimmune diseases, transplantation tolerance and tumor immunity [R cells in total CD4+ T cells was found in peripheral blood of cancer patients [R cells in controlling immune response. The mechanism of how the TR cells control immune response is still unclear. Previous studies show that activated TR cells strongly inhibit proliferative responses of CD4+ or CD8+ T cells in vitro [R cells in immunoregulation makes it be recognized as an attractive therapeutic target for immune-related diseases.Early in 1970s, the concept of suppressor T cells was developed and it was envisioned that this subset of lymphocytes was responsible for the active control, and ultimately the termination, of immune responses . Soon thesponses . Accumulimmunity -5. The timmunity . Similarimmunity , impaireimmunity  and othe minor 5–% componepatients -14 and dpatients ,16. All in vitro ,18, morein vitro , and supin vitro . The impR cells, to our surprise, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood of tumor bearing BALB/c or C57BL/6 mice, this is not in accordance with the increase of the proportion in cancer patients as reported by Wolf et al [R cells in tumor-bearing mice, we analyzed CD4+CD25+ subset in peripheral blood and spleen lymphocytes from normal or C26 colon-carcinoma-bearing mice by flow cytometry.In our animal experiments of antitumor immunotherapy that targeting CD4+CD25+ Tlf et al . In orde5 to 1 × 107 live tumor cells per mouse.6 to 8 weeks BALB/c mice were purchased from the Laboratory Animal Center of Sun Yet-sen University. Mouse C26 colon carcinoma cell line was a gift from Prof. Li-Jian Xian . The C26 Cells were cultured in RPMI 1640 medium (Gibco Invitorogen Corporation) supplemented with 10% fetal calf serum , 100 U/ml of penicillin G and 100 μg/ml of streptomycin, and the medium was renewed every 2 to 3 days. After growing to confluency, the cells were detached with trypsin-EDTA, resuspended in serum-free RPMI 1640 medium and inoculated subcutaneously at right axilla with 1 × 10PE-conjugated anti-mouse CD4, Cychrome-conjugated anti-mouse CD25 antibodies were purchased from eBioscience. Red blood cell lysis buffer is composed of 0.155 M ammonium chloride, 0.01 M potassium bicarbonate, and 0.1 mM EDTA. Fixation solution contains 1% paraformaldehyde in PBS.Mouse peripheral blood was collected from orbital plexus and anticoagulated with 20 U/ml sodium heparin. Single-cell suspensions of splenocytes were prepared by grinding the spleen with the plunger of a disposable syringe, passing the ground spleen through nylon mesh, and suspending the cells in PBS. Mouse peripheral blood or spleen single-cell suspensions were stained with PE-conjugated anti-mouse CD4 and Cychrome-conjugated anti-mouse CD25 antibodies at 4°C for 30 minutes. Then, erythrocytes were lysed by red blood cell lysis buffer. After wash with PBS, the samples were fixed with fixation solution and analyzed on a FACScalibur™ flow cytometer (BD Biosciences) with CELLQuest™ software.t test, statistical significance was accepted at the P < 0.05 level.The data are summarized as the mean ± standard error. Statistical analysis was performed using the Student R cells appear in spleen or peripheral blood in a relative stable percentage manner. The proportion of CD4+CD25+/CD4+ in peripheral blood was 6.19 ± 0.86%, which is in accordance with the results reported by others [P < 0.001). And, the higher level of the proportion in spleen is due to a lower level of the total CD4+ lymphocytes (CD4+CD25+ plus CD4+CD25-) in spleen than that in peripheral blood . The representable figures of peripheral blood and spleen lymphocytes double stained with CD4 and CD25 antibodies were shown in Figure To evaluate the normal proportion of CD4+CD25+/CD4+ in mice, 6 to 8 weeks normal BALB/c mice n = 10) were sacrificed to test the proportion in spleen and peripheral blood lymphocytes by flow cytometry using anti-mouse CD4 and CD25 antibodies. The total CD4+ lymphocytes, CD4+CD25+ subset, and CD4+CD25+/CD4+ in spleen or peripheral blood lymphocytes were shown in Table 0 were say others . Otherwi5 to 1 × 107 live C26 colon carcinoma cells were inoculated subcutaneously at right axilla of BALB/c mice respectively (n = 12). 20 days later, tumor nodules were formed at different sizes from 7 to 40 mm in diameters. The mice were sacrificed and peripheral blood and spleen lymphocytes were prepared for double staining with anti-mouse CD4 and CD25 antibodies. In peripheral blood, we did not find an increase in CD4+CD25+/CD4+ in tumor bearing mice, compared with that in normal mice. Otherwise, an increased proportion of CD4+CD25+/CD4+ in spleen lymphocytes was observed in tumor bearing mice, moreover, the proportion increased in accordance with the increase in tumor sizes, as shown in Figure To investigate the possible changes of the proportion in tumor bearing mice, 1 × 10The proportion of CD4+CD25+/CD4+ was determined by two factors: CD4+CD25+ in lymphocytes (numerator) and total CD4+ in lymphocytes (denominator). The increase of the proportion may be due to the increase of CD4+CD25+ subset or decrease of CD4+ subsets, or both. To investigate the possible reason that the proportion increased in spleen lymphocytes of tumor-bearing mice, we analyzed the CD4+CD25+ and total CD4+ cells in spleen lymphocytes. We found there was no obvious change in CD4+CD25+ in spleen lymphocytes from tumor bearing mice, otherwise, a decrease in total CD4+ lymphocytes was found with the increase of the tumor sizes, and the decrease was mainly due to the decrease of CD4+CD25- subset, as shown in Figure R cells provided a new way to study relationship between tumor development and immune suppression. A higher proportion of CD4+CD25+ TR cells was found in peripheral blood of cancer patients and to be related to poor prognosis of the diseases [R cells using anti-CD25 mAb could promote anti-tumor immunity [R cells maybe an attractive target to restore or improve immune function in cancer treatment.The identification of CD4+CD25+ as the phenotype of regulatory T lymphocytes is one of the highlights of recent immunological progress. These cells are proven to be involved in autoimmune diseases, transplantation tolerance and tumor immunity, etc . The reldiseases ,12. Deplimmunity ,16. All R cells, we analyzed the proportion in peripheral blood and spleen lymphocytes in normal or C26 colon-carcinoma-bearing mice by flow cytometry. In present study, the proportion of CD4+CD25+/CD4+ in peripheral blood of normal mice was about 6.19%, which was compatible with the results reported previously (5–10%). But in spleen lymphocytes from normal mice, we found a higher proportion of CD4+CD25+/CD4+ (around 10%), and the higher proportion is due to a lower level of total CD4+ lymphocytes in spleen, compared with that in peripheral blood, whereas the percentages of the CD4+CD25+ cells are similar.In our animal experiments of antitumor immunotherapy, we did not find an increase of CD4+CD25+/CD4+ in peripheral blood in tumor bearing BALB/c mice, this is not in accordance with the results in cancer patients reported previously . To findIn C26-colon-carcinoma bearing BALB/c mice, we found an increase of CD4+CD25+/CD4+ in spleen but not in peripheral blood, furthermore, the proportion in spleen lymphocytes increased with the increase of tumor sizes. The phenomenon that the increase of the proportion in spleen separates with that in peripheral blood may be due to: 1). Spleen is a professional immune organ, which maybe more sensitive to the changes of immune situation than peripheral blood; 2). In this study, what we used is artificial tumor model, not spontaneous tumor model, and the tumor grew so quickly to cause mice moribund or dead that the increase of the proportion did not appear in peripheral blood. To observe the increase of the proportion in peripheral blood of tumor bearing mice may need a longer observation duration, or had better use spontaneous tumor models. In our experiments, we found the increase of CD4+CD25+/CD4+ is due to the decrease of CD4+ in lymphocytes, which is the result of decreased CD4+CD25- subset in lymphocytes. Our results support the observations reported by Sasada , in whicR cells [The relationship between cancer and immune system has been debated for a long time. Our results provided direct evidence that the tumor might compromise the immune function, since our tumor model was established on BALB/c mice with normal immune function. It is known that tumor cells secrete immunosuppressive cytokines such as IL-10 and TGF-β -27, and R cells ,29. ThesR in tumor mouse models rather than that in peripheral blood lymphocytes, and our results provide some information on strategies of antitumor immunotherapy targeting CD4+CD25+ regulatory T lymphocytes.In normal BALB/c mice, CD4+CD25+/CD4+ proportion in spleen lymphocytes is higher than that in peripheral blood lymphocytes. In C26-colon-carcinoma bearing mice, no difference was found in the proportion in peripheral blood lymphocytes compared with normal mice; Otherwise, the proportion in spleen lymphocytes obviously increased, moreover, the proportion increased in accordance with the increase of tumor sizes. The increase of the proportion is due to the decrease of total CD4+ in lymphocytes, which is resulted from decreased CD4+CD25- subset in lymphocytes. Our observation suggest the CD4+CD25+/CD4+ proportion in spleen lymphocytes might be a sensitive index to evaluate the T


Head injury in England is common. Evidence suggests that socio-economic factors may cause variation in incidence, and this variation may affect planning for services to meet the needs of those who have sustained a head injury.Socio-economic data were obtained from the UK Office for National Statistics and merged with Hospital Episodes Statistics obtained from the Department of Health. All patients admitted for head injury with ICD-10 codes S00.0–S09.9 during 2001–2 and 2002–3 were included and collated at the level of the extant Health Authorities (HA) for 2002, and Primary Care Trust (PCT) for 2003. Incidence was determined, and cluster analysis and multiple regression analysis were used to look at patterns and associations.London, those of the Shire counties, and those of Other Urban authorities. Socio-economic factors were found to account for a high proportion of the variance in incidence for 2001–2. The same pattern emerged for 2002–3 at the PCT level. The use of public transport for travel to work is associated with a decreased incidence and lifestyle indicators, such as the numbers of young unemployed, increase the incidence.112,718 patients were admitted during 2001–2 giving a hospitalised incidence rate for England of 229 per 100,000. This rate varied across the English HA's ranging from 91–419 per 100,000. The rate remained unchanged for 2002–3 with a similar magnitude of variation across PCT's. Three clusters of HA's were identified from the 2001–2 data; those typical of Head injury incidence in England varies by a factor of 4.6 across HA's and PCT's. Planning head injury related services at the local level thus needs to be based on local incidence figures rather than regional or national estimates. Socio-economic factors are shown to be associated with admission, including travel to work patterns and lifestyle indicators, which suggests that incidence is amenable to policy initiatives at the macro level as well as preventive programmes targeted at key groups. It has been estimated that 6.6% of those attending A&E in any given year have a head injury  and overYet, within the UK, evidence upon which to base local service planning is scarce, as recognised in the recent report of the Health Committee of the UK House of Commons . ConsequHospital Episodes Statistics (HES) record all episodes of continuous in-patient care in hospitals in England and can be obtained from the UK Department of Health. For the year 2001–2, statistics are available for the 95 Health Authorities (HA) extant at the time, and for 2002–3 for Primary Care Trusts (PCT's). Concerned with incidence of new cases, data in the current study is based upon the postcode of residence of the patient. 'First episode' was chosen as a filter variable . A second filter identified the relevant primary cause ICD10 codes for head injury (S00.0 to S09.9). There is a long history of debate about the appropriate codes for inclusion in such studies, often surrounding the debate between 'head injury' and 'brain injury' . Much waThe 16–74 year old band was chosen explicitly to match the census key statistics which report on various indicators for the economically active population, deemed to be 16–74 years of age. Census Key Statistics are available from the UK Office of National Statistics and include a range of demographic, social and economic variables that can be presented as percentage indicators. Those chosen for inclusion in the current study are shown in Table In addition, the Townsend Deprivation Index is calculated using the percentage of households with no car, not owner occupied, overcrowded and those economically active who are unemployed . The latThe UK Census key statistics are currently made available at the Primary Care Trust (PCT) level, and thus it was necessary to aggregate these data up to the HA level to match the 2001–2 admission data which was provided at the Health Authority level. The majority of PCT's fit neatly into the 95 Health Authority areas extant in 2002. However, there are some slight variations such that aggregated populations of the HA derived from their respective PCT's match exactly the independent figures (provided separately from the ONS) of 2002 HA populations in 90% of cases, and differ by up to 5% for the rest. For example, a small number of Enumeration Districts (the lowest level census tract) belonging to the Preston PCT, which belongs in North West Lancashire HA, are allocated to East Lancashire. No attempt was made to adjust for these slight variations in boundaries. Key statistic indicators for 2001–2 were thus derived from the aggregated PCT data, based on their aggregated population which, for a few HA's, will differ slightly from the population used to estimate the admission rates for head injury. There was a one-to-one relationship between admission data and census data at the PCT level for the 2002–3 data.Patterns of head injury and socio-economic indicators are determined by Two-Step Cluster Analysis. The procedure is an exploratory tool designed to reveal natural groupings (or clusters) within a data set that would otherwise not be apparent. The association between the various socio-economic factors, and the overall rate for head injury is explored through a multiple regression model using the 2001–2 data at the HA level. This model is validated on the 2002–3 data at the PCT level.The project was funded by the UK Department of Health. The author is independent of the funding body. Ethical approval was not required.A total of 112,718 admissions were recorded for 2001–2. The gives an incidence rate for admission to hospital following a head injury in England in 2001–2, for all ages, of 229.4 per 100,000 Table . The incTaking the overall incidence rate for 2001–2, together with the key statistics in Table The second cluster can be described as the 'Shire' counties, typical of the more rural areas found within England, including Cambridgeshire and Worcestershire. They are characterised by a large proportion of people owning their own home, travelling to work in their own car, and with few of the population born outside of the UK. Although a quarter of the population are without qualifications, the deprivation Index is low at -2.7. The average incidence admission rate for this cluster is 206 per 100,000 all ages.The third cluster can be described as 'Other Urban', and typical of all the Midland and Northern cities of England. They are characterised by a higher proportion of people with limited long standing illness; those of working age who are permanently off sick; of those without qualifications and of those aged 16–24 who are unemployed. The deprivation Index is above average at 1.2, and the admission rate is high at 288 per 100,000 all ages.2 of 0.698, the model explains a large proportion of the variation in admission rates for a head injury . On this occasion the proportion of households without a car was added to the previous set of predictors. Once again, the proportion travelling to work by public transport was the most important variable.The model was then validated upon the 2002–3 data at the PCT level Table . A similHead injury is common; there were 112,718 recorded admissions for English residents during the year April 2001 to March 2002, giving a hospitalised incidence rate of 229.4 per 100,000 all ages. Remaining the same for 2002–3 this incidence is similar to that of stroke although the latter is experienced in a predominately older population . HoweverAdmission rates for England were found to vary by a factor of 4.6 between both health authorities and PCT's. The rates may slightly underestimate the true incidence as those residents of England treated elsewhere are not included. Also, under-reporting may depress incidence to an unknown extent  and, if Half of all health authorities in England, and slightly more PCT's (55%) had an incidence rate which fell below or above the England average by at least 20%. This has important implications for planning local services in response to head injury. Clearly using the overall England incidence rate is unlikely to be helpful. This variation also causes problems in extrapolating the results from research undertaken in a single locality. Estimates of the number of people disabled by head injury, presented as a rate (e.g. 150 per 100000), may have little relation to reality at the local level. The disability rate is conditional upon the local incidence where the study was undertaken. Consequently it is important that research findings should be presented as proportions of the local base incidence if they are to be of any use for planning elsewhere. The extrapolations given above on return to work are based on this approach. Nevertheless such extrapolation assumes equal case mix (e.g. similar mechanisms of injury) which is also unlikely. Case mix will, to a certain extent, be a reflection of local socio-economic factors, particularly associated with sub-populations displaying chronic conditions, including alcohol misuse, shown to have significant impact on outcome ,24,25.The extent of deprivation has also previously been associated with higher incidence of head injury as well as general workload for primary care ,27. The ceteris paribus, we might expect to see the London incidence fall as a consequence of congestion charging and associated increase in the use of public transport. Policies targeted at reducing unemployment amongst the 16–24 year old age group may also be expected to reduce incidence. Also, a continuing emphasis on prevention is clearly needed, both for the young and the old, to reduce their very high incidence rates.Where public transport is not used for journeys to work, incidence is much higher and combined with higher unemployment rates, and other significant indicators associated with 'lifestyle indicators', high incidence rates are observed in the cities and other urban communities in the Midlands and Northern England, including Teeside and Mersyside. As demographic, social and economic factors appear to account for half the variation observed in hospitalised admission rates, one implication is that incidence can be modified at the macro policy level. For example, Some technical limitations are worth mentioning. The likelihood distance measure used in the two-step cluster analysis assumes that variables in the cluster model are independent. Further, each continuous variable is assumed to have a normal (Gaussian) distribution, although in practice the technique is robust to violation of these assumptions. In fact, reassuringly, the results of the linear regression model do suggest independent main effects for several of the key variables and, importantly, the absence of interaction effects. Also it is unknown how, if at all, the slight variation in population denominators used to calculate incidence and the socio-economic indicators for the 2001–2 data could influence these results. However, replication upon the 2002–3 data at the PCT level suggests a robust model. Due to censoring of data of elderly admissions at the PCT level we were not able to confidently include the proportion of females admitted in our validation model, and thus we do not know if variation in male-female ratios at the local level was also a contributing factor to variance in incidence.It is also worth restating that the accuracy of case ascertainment, and the coding of head injuries upon admission to hospital is known to underestimate the true incidence ,22 but hIncidence of head injury in England is high, similar to stroke if just admissions are considered, and the data show considerable variability at the local level. Given that most of the estimates of the potential impact of head injury rely on studies undertaken in a single locality ,28,29 thTo assist in planning for services, data at the local level can now readily be obtained from on-line data sets from the Department of Health. At the national level they can be downloaded on-line at:While we have as yet little understanding of how case mix is related to, or interacts with socio-economic factors to mediate medium or longer term-outcomes, the association of such factors with incidence suggests that economic and social policies, for example in the development of prevention programmes, and in encouraging the use of public transport for journeys to work, may have a significant impact on reducing the incidence of head injury.The author(s) declare that they have no competing interests.The pre-publication history for this paper can be accessed here:Hospitalised Incidence rate for head injury in England in 2001–2. Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and extant Regions and Health Authorities at the time.Click here for fileIncidence of admission to hospital for head injury for England, and each Primary Care Trust (PCT) in England 2002–3; Estimated rate per 100,000; for those aged 0–15; 16–74; and 75 years and over, and in total. Estimates for England, and PCT's.Click here for file


The increasing number of available genotypes for genetic studies in humans requires more advanced techniques of analysis. We previously reported significant univariate associations between gene polymorphisms and antidepressant response in mood disorders. However the combined analysis of multiple gene polymorphisms and clinical variables requires the use of non linear methods.In the present study we tested a neural network strategy for a combined analysis of two gene polymorphisms. A Multi Layer Perceptron model showed the best performance and was therefore selected over the other networks. One hundred and twenty one depressed inpatients treated with fluvoxamine in the context of previously reported pharmacogenetic studies were included. The polymorphism in the transcriptional control region upstream of the 5HTT coding sequence (SERTPR) and in the Tryptophan Hydroxylase (TPH) gene were analysed simultaneously.A multi layer perceptron network composed by 1 hidden layer with 7 nodes was chosen. 77.5 % of responders and 51.2% of non responders were correctly classified . Finally, we performed a comparison with traditional techniques. A discriminant function analysis correctly classified 34.1 % of responders and 68.1 % of non responders (F = 8.16 p = 0.0005).Overall, our findings suggest that neural networks may be a valid technique for the analysis of gene polymorphisms in pharmacogenetic studies. The complex interactions modelled through NN may be eventually applied at the clinical level for the individualized therapy. The increasing number of available genotypes for genetic studies in humans requires more advanced techniques of analysis . MoreoveIn particular, psychiatric disorders are characterized by a non mendelian, multifactorial genetic contribution with a number of susceptibility genes interacting with each other ,8. In thSince our initial study  we invesHowever, both the increasing number of genes associated with response and the limitations of traditional methods of analysis are factors requiring the use of new techniques of analysis that more closely resemble to the underlying biological process, i.e. that allows for non-linear interactions.Neural networks (NN) have been proposed for such studies ,23,24. TNN have been used in other fields of medicine, for example to predict cyclosporine dosage in patients after kidney transplantation , perspecIn fact, neural networks proved to outperform single marker association tests, particularly in the case of a complex mode of inheritance or where multiple mutations result in more than one haplotype associated with the disease ,30,31.In the present paper we have re-analysed our sample where polymorphism in the transcriptional control region upstream of the 5HTT coding sequence (SERTPR) and in the Tryptophan Hydroxylase (TPH) gene were analized , in thatThe sample was already described in the original paper . BrieflyPlasma fluvoxamine levels were determined by high-performance liquid chromatography after 2 weeks of stable 300 mg daily dose . Nine paThis is one of the most popular network architecture in use today, though relatively recent . In MLP Originally developed about 60 years ago by Fisher , in clasIn a radial basis function network the response surface of a single radial unit is a Gaussian (bell-shaped) function, peaked at the center, and descending outwards. RBF networks have advantages and disadvantages over MLPs. First, they can model any non-linear function using a single hidden layer, which removes some design-decisions about numbers of layers. Second, the simple linear transformation in the output layer can be optimized fully using traditional linear modelling techniques, which are fast and do not suffer from problems such as local minima which plague MLP training techniques. However the clumpy approach also implies that RBFs are not inclined to extrapolate beyond known data: the response drops off rapidly towards zero if data points far from the training data are used, therefore they are less reliable for clinical samples such our one. Detailed review of the models are reported elsewhere ,37.An "intent-to-treat" analysis was carried out for all patients who had a baseline assessment and at least 1 assessment after randomization, with the last observation carried forward on the HAM-D. For the current application the inputs to the first layer of the neural network consist of SERTPR and TPH genotypes while the target outputs consist of response status. The network is then trained to attempt to predict response from genotypes. Each node of the input layer of the network is set to a value representing the genotype of each polymorphism. For each polymorphism and for each subject this value is set to genotypes aa, ab or bb. If a marker genotype is missing then the input is assigned a value equal to the average of the values for all subjects in the dataset, however no missing data were present in our sample. The target output for the network is set to 1 or 2 depending on whether the subject is responding or not.The best network was selected on the basis of its discriminating error and performance, positive and negative predictive values were also reported for each model. This last was expressed as area under the Receiving Operator Characteristic (ROC) Curve. The area under a ROC curve ranges from zero to one, with values close to unity indicating better predictive power; an area of 0.5 indicates that the model is not predicting better than a random choice.However, one major problem of NN analyses is to establish if the prediction from genotypes is greater than would be expected by chance. If the whole sample is used for training, the network will to some extent "learn to recognise" particular features of each member of the dataset and can use these to predict response in a way which may not reflect any general association between marker genotypes and disease. Generally, this problem is faced by a set of strategies: dividing the dataset , Jackknife, bootstrapping, cross-validation and so on. However those methods present some disadvantages, in particular if only a part of the data is used to train the network this leads to a loss of power given that subjects in the validating part have different patterns of association between genotypes and drug response.In order to remedy these problems, in the case of MLP, it has been suggested to perform both training and testing on the entire dataset. The statistical significance of any observed association between outputs and affection status can be estimated using a permutation test .Once the network was defined, a statistic, denoted T, is calculated to compare the outputs for responders and non responders in the same way as an unpaired t statistic, although the statistic is not expected to follow a t distribution under the null hypothesis. Instead, in order to estimate statistical significance a permutation procedure is performed. A large number of replicate data sets are generated from the original data and the obtained network model by randomly permuting genotypes with respect to affection status. For each of these replicate data sets we can then train and test the data set as before, each time calculating T. Since each permuted data set will have only random association between genotype and affection status we obtain N values of T which provide a distribution of T under the null hypothesis. We count the number of times any of these values exceeds the value of T we obtained for the real dataset and denote this number R. Then (R + 1)/(N + 1) provides an unbiased estimate of the statistical significance of the association between genotype and affection status in the real dataset.In order to estimate a p-value of alpha, one should carry out approximately 10/alpha replicates. Typically, in order to detect association at a significance of 0.01 one would perform 1000 replicates . In the case of the present paper we performed 10000 replicates.Multiple regression and discriminant function analyses were performed to compare the results obtained with the NN strategy with traditional techniques. Responder status was the dependent variable with SERTPR and TPH as independent variables. Genotypes were scored in the following way according to the hypothesis of codominance .Calculations for the NN selection were performed using STATSOFT (Kernel release 5.5 A). Evaluation was performed using the NNPERM package .MLP showed the best performance and was therefore selected over the other networks , after 5000 epochs we reduced it to 0.01 but after 5000 further epochs we observed no improvement and therefore we finished the selection process and retained the network. Both polymorphisms contributed substantially to the model . This was expected since both markers were individually associated with response. In detail single marker significance, calculated as simple allelic chi-square, was p = 0.00058 for SERTPR and p = 0.025 for TPH. The classification of subjects in responders and non responders was 77.5 % for responders and 51.2% for non responders. Classification may vary depending from the selected threshold, therefore the area under the ROC curve is a better indicator of performance, in this case the area was 0.731. We also evaluated the predictive power of the network with the SERTPR polymorphism only, in this case the area under the ROC curve was 0.698. We may therefore observe that the add of TPH polymorphism increases the predictive power of the system.In order to evaluate the significance of the network we applied a permutation test with 10000 replicates. The t statistic for the network was 4.35, it was achieved in 81 out of 10000 simulations yielding a network p-value = (81+1)/(10000+1) = 0.0082.Finally, we performed a comparison with traditional techniques. A multiple regression analysis showed a significant correlation (p = 0.0004) with a variance explained of 12.5%. The discriminant function analysis correctly classified 34.1 % of responders and 68.1 % of non responders (F = 8.16 p = 0.0005).Following, we tested the possible impact of clinical variables on response. We included in the model the following variables: Age, age at onset, sex, education, diagnosis, presence of delusional features, recurrence index (defined as number of episodes per year), pindolol augmentation and baseline HAM-D. With those variables no satisfactory network was identified. They were therefore not considered as possible confounding factors in the genetic analysis.This paper reports the first attempt to use NN in pharmacogenetic analyses. We applied this technique to short term antidepressant response in mood disorders. Our analyses suggest that MLP network is the most appropriate for this kind of data, in according with previous observations . The groFurther developments of this strategy are the inclusion of more detailed information on the phenotypic side. The classification results we obtained are not sufficient in clinical terms were in particular much higher specificities are needed in order to recognize in advance non responders. To reach this target we should consider that we previously observed that some polymorphism influence only part of the whole depressive symptomatology . FurtherThe relatively small sample we used does not guarantee against a possible overfitting phenomenon, therefore enlargement of the sample is a priority. Moreover we used the same sample for testing and validating our result, this is not a standard technique , this prDifferences in allele frequency for different populations have been reported . HoweverOverall, our findings suggest that neural networks may be a valid technique for the analysis of gene polymorphisms in pharmacogenetic studies. The complex interactions modelled through NN may be eventually applied at the clinical level for the individualized therapy .The author(s) declare that they have no competing interests.AS conceived the study, drafted the manuscript and participated in the design of the study and performed the statistical analysis. ES participated in its design and coordination. All authors read and approved the final manuscriptThe pre-publication history for this paper can be accessed here:


Microarray technologies allow the identification of large numbers of expression differences within and between species. Although environmental and physiological stimuli are clearly responsible for changes in the expression levels of many genes, it is not known whether the majority of changes of gene expression fixed during evolution between species and between various tissues within a species are caused by Darwinian selection or by stochastic processes. We find the following: (1) expression differences between species accumulate approximately linearly with time; (2) gene expression variation among individuals within a species correlates positively with expression divergence between species; (3) rates of expression divergence between species do not differ significantly between intact genes and expressed pseudogenes; (4) expression differences between brain regions within a species have accumulated approximately linearly with time since these regions emerged during evolution. These results suggest that the majority of expression differences observed between species are selectively neutral or nearly neutral and likely to be of little or no functional significance. Therefore, the identification of gene expression differences between species fixed by selection should be based on null hypotheses assuming functional neutrality. Furthermore, it may be possible to apply a molecular clock based on expression differences to infer the evolutionary history of tissues. Analysis of differences in gene expression between primate species suggests that the majority of them are selectively neutral and likely to have little or no functional consequences Advances in microarray technology have made the systematic study of expression levels of thousands of transcripts possible. This has been heralded as a major step forward in understanding the function of genomes, since transcript expression levels are expected to correlate with biological functions. Although this is clearly the case for many genes that change their expression in response to environmental stimuli e.g., , it is nTo test whether this may be the case, we have investigated whether a neutral model can describe transcriptome differences observed among primate and mouse species as well as among various brain regions within a species.(Pan trogodytes), one orangutan (Pongo pygmaea), and one rhesus macaque (Macaca mulatta) using oligonucleotide microarrays. To exclude the influence of DNA sequence differences on the hybridization results, at least between humans and chimpanzees, only oligonucleotide probes that matched perfectly to the chimpanzee DNA sequences were used in the analysis . Due to the greater probe length, these arrays are much less sensitive to DNA sequence differences and therefore can be used to compare gene expression in humans and macaques  as gauged by a permutation test , the vast majority of which are noncoding , although in this case we cannot correct for DNA sequence differences. A correlation between gene expression differences within and between species was recently demonstrated also in teleost fish  in at least one of four brain regions in either the chimpanzees or the humans after masking all probes carrying DNA sequence differences between the species. Of these, 28 contained at least one mutation that leads to a loss of function in both humans and chimpanzees. We therefore assumed that these pseudogenes were nonfunctional in the common ancestor of humans and chimpanzees. Finally, we checked whether these probe sets may crosshybridize with any intact genes by aligning them to the human genome. This left us with 23 expressed pseudogenes.To test this, we considered the expression patterns in four regions of the brain in three humans and three chimpanzees using the Affymetrix U95 array set interrogating approximately 40,000 genes . In order to identify all probe sets on these arrays that interrogate expressed pseudogenes, we aligned the probe sequences, as well as published lists of human pseudogenes, to the human genome . In totap = 0.16 and p = 0.69, respectively)  of the genes had a distribution radically different from that of the pseudogenes. When the data for four brain regions were combined, no visual difference between the two distributions was apparent (ctively) A.Thus, we failed to detect any significant excess of intact genes that diverged faster in expression than pseudogenes. This indicates that the fraction of gene expression differences between the species that are fixed by positive selection is small. Interestingly, there was also no detectable excess of intact genes that diverged slower than pseudogenes. This may seem unexpected, since the expression of many intact genes might be thought to be stabilized by negative selection and therefore to change more slowly than pseudogenes. This may indicate that purifying selection as well is a weak force affecting gene expression. However, it should be noted that the small number of expressed pseudogenes analyzed limits the power to detect positive and negative selection. A targeted effort to study expressed pseudogenes in closely related species would be a worthwhile undertaking.The fact that the overall accumulation of expression differences conforms to a selectively neutral model does not mean, of course, that all expression differences between species are selectively neutral. As for nucleotide changes, some changes in gene expression will have had phenotypic consequences and some of these will have become fixed due to positive selection. To identify such gene expression differences, we propose to use the ratio of divergence between species to diversity within species, akin to the tests suggested for quantitative genetic traits  and in ap = 0.388; Wilcoxon rank sum test, p = 0.134), and both distributions appeared to center around roughly the same values  is very similar to that of the prefrontal cortex in both humans and chimpanzees, although it is known to be involved in speech processing in humans while it must have another function in chimpanzees . This isA noteworthy finding is that the accumulation of expression differences between brain regions within a species is much slower than the accumulation of expression differences within a brain region between species. In fact, the expression differences that have accumulated among the primate species over 20 million years see A are appA possible alternative explanation for the correlation between differences in gene expression and evolutionary divergence time among brain regions could be that differences in gene expression do not correlate with evolutionary divergence time, but instead with divergence time during fetal development. Our observations would then result from the fact that both developmental divergence times and expression differences correlate with evolutionary divergence. A correlation between developmental and evolutionary divergence times has been hypothesized before  according to manufacturer's instructions and purified with Quiagen RNeasy kit  following the “RNA cleanup” protocol. RNAs were of high and comparable quality as gauged by the ratio of 28S to 18S ribosomal RNAs visualized on agarose gels and by the signal ratios between the probes for the 3′ and 5′ ends of the mRNAs of GAPDH and β-actin genes used as quality controls on Affymetrix microarrays .For the primate samples, approximately 200 mg of gray matter was collected from post mortem brain samples from prefrontal cortex region corresponding to Brodmann's area 9 in the left hemisphere from six male humans who were 45, 45, 63, 65, 70, and 70 years old; five male chimpanzees that were 7, 12, 12, 12, and approximately 40 years old; one 16-year-old male orangutan; five approximately 10-year-old male rhesus macaques; and five approximately 15-year-old male crab-eating macaques. All individuals had no history of brain-related diseases and suffered sudden deaths without associated brain damage. For the mouse samples, approximately 50 mg of gray matter was collected from the frontal cortex regions of six For Affymetrix microarrays, labeling of 5 μg of the RNA, hybridization, staining, washing steps, and array scanning were carried out following Affymetrix protocols. Expression data were collected using Affymetrix HG U95Av2 arrays for the primate samples and Affymetrix MG U74Av2 arrays for the mice samples. The Affymetrix CEL files containing expression data for the different regions of the mouse brain, including amygdala, cerebral cortex, hippocampus, hypothalamus, cerebellum, olfactory bulb, and two regions of spinal cord were provided by John Hogenesch.http://www.ebi.ac.uk/arrayexpress/).Arrays containing 51,000 cDNAs corresponding to approximately 40,000 UniGene clusters were manufactured in the laboratory of W.A. as described elsewhere (Anonymous 2003). Labeling, hybridization, staining, washing, and array scanning were carried out as described by http://www.affymetrix.com/analysis/index.affx) first to the human genome (build 33) and then to a draft version of the chimpanzee genome (the assembly was given courtesy of David Jaffe in June 2003). Using BLAT  in gene expression between humans and chimpanzees if the real divergence is zero. Then using this distribution and the observed distribution of differences (Z), we generated a distribution (Y) that-added to values from X-would give Z. In order to underestimate the divergence, we generated Y assuming that the correlation of X and Y is one. We then generated random samples in the following way: For each gene (g), we chose a random difference of expression (d) from our generated distribution. We then drew six samples from a normal distribution whose mean is zero and whose variance is the diversity in humans for gene g, and three samples from a normal distribution whose mean is d and whose variance is the diversity of chimps for gene g. For these expression values we then calculated the correlation between diversity and divergence. We repeated the whole procedure 1,000 times. None of these randomizations generated a correlation that is as strong as the observed one. To make sure that the whole test is conservative, we generated 100 datasets of three types, all of which had a similar diversity, but had a “real” divergence distribution of (1) zero, (2) the underestimated divergence, or (3) the measured divergence, and had uncorrelated diversity and divergence. We then performed the whole test described above, doing just one randomization test. If the test was not conservative, one would expect the correlation in the dataset to be higher than the correlation after randomization in 50% of the cases. Instead, the correlation after randomization was higher in 98, 98, and 99 cases respectively-showing that our test is indeed conservative.http://vega.sanger.ac.uk). These sequences, as well as the Affymetrix target sequences, were mapped to the human genome (build 34) using BLAT (http://genome.ucsc.edu). Thus, we identified 889 probe sets that overlapped with a pseudogene, but not with a known gene. Combined with gene expression data collected in four brain regions  in three humans and three chimpanzees, 33 of these probe sets had detectable expression levels in at least one brain region in either three chimpanzees or three humans. For these probe sets, we checked whether at least one of the identified interruptions of the human pseudogene was also present in the chimpanzee, indicating that the pseudogene was already nonfunctional at the time of the chimpanzee–human divergence. This left us with 28 probe sets that were checked for crosshybridization with other genes by aligning oligonucleotide probes from these probe sets to the human genome. Finally, we were left with 23 expressed pseudogenes that did not match perfectly to any other gene by more than seven out of 16 probes in the probe set.We retrieved sequences of all pseudogenes as determined by ing BLAT , and the


Malnutrition in children can be a consequence of unfavourable socioeconomic conditions. However, some families maintain adequate nutritional status in their children despite living in poverty. The aim of this study was to ascertain whether family-related factors are determinants of stunting in young Mexican children living in extreme poverty, and whether these factors differ between rural or urban contexts.A case-control study was conducted in one rural and one urban extreme poverty level areas in Mexico. Cases comprised stunted children aged between 6 and 23 months. Controls were well-nourished children. Independent variables were defined in five dimensions: family characteristics; family income; household allocation of resources and family organisation; social networks; and child health care. Information was collected from 108 cases and 139 controls in the rural area and from 198 cases and 211 controls in the urban area. Statistical analysis was carried out separately for each area; unconditional multiple logistic regression analyses were performed to obtain the best explanatory model for stunting.In the rural area, a greater risk of stunting was associated with father's occupation as farmer and the presence of family networks for child care. The greatest protective effect was found in children cared for exclusively by their mothers. In the urban area, risk factors for stunting were father with unstable job, presence of small social networks, low rate of attendance to the Well Child Program activities, breast-feeding longer than six months, and two variables within the family characteristics dimension .This study suggests the influence of the family on the nutritional status of children under two years of age living in extreme poverty areas. Factors associated with stunting were different in rural and urban communities.Therefore, developing and implementing health programs to tackle malnutrition should take into account such differences that are consequence of the social, economic, and cultural contexts in which the family lives. Despite improvements in the health of children under five years of age, malnutrition remains as an important public health problem in Mexico  and in oPrevious reports have stated that malnutrition occurs mainly in rural areas and worsens under conditions of extreme poverty ,14,15. IPoverty is not confined to rural areas, nor is malnutrition; they are also present in urban environments . UncontrMalnutrition is an outcome of various factors resulting from unfavourable socioeconomic circumstances such as difficulties in obtaining food, unemployment -which determines an irregular income for the family's breadwinner-, limited access to education and health services, or illness caused by unsanitary conditions ,14,20-24A case-control study was conducted from August through December 1998. Two extreme poverty level areas  were selected. Each area was analysed separately.The study took place in the south-western State of Guerrero, one of the poorest in Mexico, with a population of nearly three million.The rural area, named Alto Balsas, is located next to the Balsas River; the urban area, named El Sinai, is a poor neighbourhood on the outskirts of the port of Acapulco, which has half a million inhabitants. For the rural area, four villages with a combined population of 4,638 were included in the study. The closest city is approximately 45 miles away. The principal language spoken in these villages is Náhuatl, and agriculture is the main economic activity. Because of seasonal conditions there is a high rate of cyclic migration each year. This migration occurs during harvest season, where farmers migrate from the State of Guerrero to another states. There is one medical doctor and one primary care centre per approximately 2300 people.El Sinai is located in the outskirts of Acapulco and has 6,860 inhabitants, most people speak Spanish and they work in the local industry as unskilled manual workers. In this area, there is one medical doctor and one clinic per 3400 people. In both study areas, sanitation and public services are deficient. Table The units of study were children between 6 and 23 months of age. Children older than 6 months were included on the assumption that this is the minimum amount of time the child is exposed to family-related factors; also, this is the average age in which children are weaning (this is defined as the time when mothers begin to introduce food other than milk into the child's diet). Thus infants become more exposed to environmental causes of under-nutrition. The family was defined as next of kin, or persons sharing the same household and food expenses . Only onCases were stunted children. This was ascertained by using the height-for-age indicator . The criControls were children without stunting: equal to or above -2.00 SDs below the median in families that had no stunted children.The sample size was calculated by using the case-control study formula in accordance with the following assumptions  a number of conceptually grounded dimensions that could be associated with stunting.As mentioned earlier, rural and urban environments are different. Regarding the results of the rural area, within the family income dimension, the variable father's occupation (farming) was found to be a risk factor for child's stunting. In this area, availability of food depends upon local production, which in turn is related to father's occupation. Maize is the staple, and there is lack of food variability or inability to produce sufficient food for the family's nutritional requirements .per capita family income, which was statistically significant in the bivariate analysis, did not show significance in the multivariate analysis. This finding could be interpreted as the ability of the family to cope with a difficult environment. Analysis of microeconomic variables shows the need of further studies to confirm plausibility of our findings and its association with malnutrition.In the urban area the instability of the father's employment (working in the same place for less than 2 years) was found to be a risk factor. Lack of stable employment is a common problem among unskilled workers; their income is low and irregular, thus affecting their capacity to purchase goods and food, which in turn affects child nutrition . NeverthThe dimension of family structure, which included socio-demographic characteristics of the parents, migration, and literacy attainment, has been repeatedly associated with the nutritional status of children. ,23,34,35An interesting finding in this study was the relationship between social networks and children's nutritional status. It is often assumed that extended families have extensive social networks, and that this can be advantageous for the care of young children. However, we found quite the opposite in the rural area: the presence of family networks was associated with stunting, while exclusive provision of child-care by the mother showed a protective effect. These findings stress the role of the mother as primary caregiver, at least for young children; such findings also suggest that when a mother is present to care for the child, some of the effects of living in a poor community can be ameliorated.In the urban area, conditions seem to be different, and the sizes of social networks influenced the nutritional status of the children positively. Urban families living in a more aggressive environment might be more likely to obtain health benefits if they have greater family or social support.Use of health services, based on preventive measures from the Well Child Program, was also analyzed. Few visits to the Well Child Program activities were associated with stunting in the urban area. Restricted access to health care services in deprived areas is an important shortcoming with regard to monitoring child's growth and health . Based oMigration was also explored. In the final explanatory model addressing the urban area, parents' migration from rural to urban area was found to be a risk factor for stunting. Migration from rural to urban settings is frequent among young people looking for improving their living conditions. However, families' adaptation to the new situation is a long process, given that such families live in a hostile environment and they have limited access to information regarding how to care for the child. In contrast, migration of one member of the family from one urban setting to another did not show a relationship with nutritional status of children. Adult male family members, mainly the heads of household, migrate to other states in Mexico or to the U.S. in search of better income. Perhaps the influence of this variable could be identified by a qualitative approach. Therefore, further research on this aspect is necessary.The study has some limitations. Firstly, the study groups were of unequal size; this is a consequence of including all children aged from 6 to 23 months of age identified by the census. Additionally, since children of the control group were significantly younger than cases, some of them could have become stunted by the time they were of comparable age to the cases. To solve this limitation, the covariate age was adjusted in the multivariate analysis. However, it is possible that an age-matched cases-control design would have been preferable.Secondly, the fact that almost 25% of eligible children in the rural area were not included due to migration of their families could have led to selection bias.Another limitation is the lack of precise information regarding food practices among participating families. The interview allowed identifying general aspects such as duration of breast-feeding and age of weaning. Breast-feeding beyond 6 months of age entailed a risk of stunting, but only in the urban area. This issue has been controversial; some authors suggest that the relationship between prolonged breast-feeding and malnutrition may indicate a maternal decision to continue breast-feeding to a nutritionally disadvantaged child -39, rathThis study suggests the influence of the family on the nutritional status of children under two years of age living in extreme poverty areas. Factors associated with stunting were different in rural and urban communities.Therefore, developing and implementing health programs to tackle malnutrition should take into account such differences that are consequence of the social, economic, and cultural contexts in which the family lives.The author(s) declare that they have no competing interests.HR and AS conceived, designed and co-ordinated the study. RPC participated in the design of the study, statistical analysis and interpretation of data, and drafted the manuscript. RC co-ordinated the acquisition of data and participated in the statistical analysis. SD collaborated in the data analysis and drafted the manuscript. JIS and GG participated in the conception and design of the study, interpretation of data and critical revision for important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Individual patient meta-analysis to determine the analgesic efficacy and adverse effects of single-dose rofecoxib in primary dysmenorrhoea.Individual patient information was available from three randomised, double blind, placebo and active controlled trials of rofecoxib. Data were combined through meta-analysis. Number-needed-to-treat (NNT) for at least 50% pain relief and the proportion of patients who had taken rescue medication over 12 hours were calculated. Information was collected on adverse effects.For single-dose rofecoxib 50 mg compared with placebo, the NNTs (with 95% CI) for at least 50% pain relief were 3.2 (2.4 to 4.5) at six, 3.1 (2.4 to 9.0) at eight, and 3.7 (2.8 to 5.6) at 12 hours. For naproxen sodium 550 mg they were 3.1 (2.4 to 4.4) at six, 3.0 (2.3 to 4.2) at eight, and 3.8 (2.7 to 6.1) at 12 hours. The proportion of patients who needed rescue medication within 12 hours was 27% with rofecoxib 50 mg, 29% with naproxen sodium 550 mg, and 50% with placebo. In the single-dose trial, the proportion of patients reporting any adverse effect was 8% (4/49) with rofecoxib 50 mg, 12% (6/49) with ibuprofen 400 mg, and 6% (3/49) with placebo. In the other two multiple dose trials, the proportion of patients reporting any adverse effect was 23% (42/179) with rofecoxib 50 mg, 24% (45/181) with naproxen sodium 550 mg, and 18% (33/178) with placebo.Single dose rofecoxib 50 mg provided similar pain relief to naproxen sodium 550 mg over 12 hours. The duration of analgesia with rofecoxib 50 mg was similar to that of naproxen sodium 550 mg. Adverse effects were uncommon suggesting safety in short-term use of rofecoxib and naproxen sodium. Future research should include restriction on daily life and absence from work or school as outcomes. Dysmenorrhoea is associated with painful cramping of the lower abdominal or back muscles, with or without other symptoms such as nausea, vomiting, and diarrhoea. Onset of dysmenorrhoea is common during adolescence, and up to 50% of women of reproductive age may be affected , and 10%Raised concentrations of uterine prostaglandins are thought to cause the pain and cramping associated with dysmenorrhoea -6. NonstSystematic reviews have shown NSAIDs to be effective in the treatment of primary dysmenorrhoea ,8 While At least moderate pain relief over a cycle was reported in 14 comparisons between NSAID  and placebo in 599 women, with an NNT of 2.1 (1.9 to 2.5). The most studied NSAID was naproxen, with 287 women in seven trials, with an NNT of 2.5 (2.0 to 3.3) for this outcome. An earlier review [Cox-2 selective inhibitors (coxibs) provide an alternative to conventional NSAIDs, with a potential advantage of once daily dosing -11. ThisQUOROM guidelines  for qualMerck Research Laboratories, Rahway, New Jersey provided individual patient data from three Phase III trials of rofecoxib in dysmenorrhoea , with the guarantee that all relevant studies completed by July 2002 had been made available. One of the trials has been published in full . SearchiOur intention was to use two pain outcomes  with the outcome being that closest to at least half pain relief. Over the first dose, this might be a measure of total pain relief (TOTPAR), while over a whole cycle it may be a patient global evaluation of good or excellent, rather than mild, fair, no improvement, or worse. Analyses for comparator treatments were based on information available only from the trials of rofecoxib in this report. Outcome data were pooled in an intention to treat (number of patients randomised) analysis. Neither heterogeneity tests nor funnel plots were used ,17. InstRelative benefit (or risk) was calculated using a fixed effects model  with no The mean age of women in the trials was 31 years, and at baseline pain was moderate in 66% of women and severe in 34%. All trials were randomised, double blind, and compared oral doses of rofecoxib with an active control and placebo in women with moderate to severe pain due to dysmenorrhoea. One trial was a single dose crossover (study 38), and two were multiple dose crossovers (studies 55 and 56), where the crossover was between single doses in different menstrual cycles. Single dose efficacy data were available for the first 12 hours of treatment in all trials, but not summary estimates over a cycle. Study designs and quality and validity scores are shown in Table The single dose trial (study 38) was conducted over three cycles. Each of 49 women received three of four treatments . For the two multiple dose studies, one (study 55) compared a single dose of rofecoxib 50 mg followed by 25 mg daily as required, naproxen sodium 550 mg every 12 hours, or placebo in 60 women over three menstrual cycles. The other (study 56) compared rofecoxib 50 mg as required, rofecoxib 50 mg followed by 25 mg daily as required, naproxen sodium 550 mg every 12 hours, or placebo in 122 women over four menstrual cycles. Both trials reported multiple dose adverse effects. In the multiple dose trials, all women received each treatment regimen for one cycle.Pain intensity and pain relief were measured using the standard 4-point categorical pain intensity scale  and a 5-point point pain relief scale . Pain measurements were collected using patient diaries. Patients were assessed at baseline, then at least hourly for eight hours, and again at 12 hours for single dose efficacy data. The exact time at which a patient requested remedication , if required, was recorded. Adverse effects were recorded as the number of patients with any adverse effect(s), or particular adverse effects.Full efficacy results over six, eight and 12 hours are shown in Table Rofecoxib 25 mg was tested in a single trial, rofecoxib 50 mg in three, ibuprofen 400 mg in one and naproxen sodium 550 mg in two. For all the active analgesics the proportion of patients with at least 50% pain relief was about 60% at all time points, and with placebo it was about 30% at all time points , ibuprofen 400 mg , or naproxen sodium 550 mg, or between NNTs of rofecoxib 50 mg and ibuprofen 400 mg , or naproxen sodium 550 mg .The proportion of patients who remedicated at different time points over 12 hours is shown in Figure Few adverse effects of a particular type were reported, and none were serious in any trial. The most commonly reported adverse effects were nausea and somnolence, but these occurred infrequently. A single dose in one trial (study 38) gave the proportion of patients reporting any adverse effect(s) as 10% (5/49 patients) with rofecoxib 25 mg, 8% (4/49) with rofecoxib 50 mg, 12% (6/49) with ibuprofen 400 mg, and 6% (3/49) with placebo. With multiple doses over a cycle, the proportion of patients reporting any adverse effect(s) was 23% (42/179 patients) with rofecoxib 50 mg, 24% (45/181) with naproxen sodium 550 mg, and 18% (33/178) with placebo.Pain with dysmenorrhoea usually lasts for about three days, though with considerable individual variation. Trials of analgesics can have various forms. The simplest might be to give the same analgesic for the whole of the painful cycle, and ask a global question concerning efficacy at the end. Women might then be crossed over to a different treatment at the next cycle. A variation would be to use the same basic structure, but make more detailed evaluations of pain or pain relief over a limited time during the first day, though a global question could always be added. A more complicated design would use a cross-over within a single cycle.The three trials described here used a cross-over between cycles, with detailed pain measurements over 12–24 hours in the first painful day. Twelve-hour and 24-hour outcomes have also been reported in two other recent studies of coxibs in dysmenorrhoea ,11. WithAll three trials were of the highest reporting quality, and had high validity scores, indicating that known sources of bias are unlikely to occur ,8. We knIndividual patient information from three trials of high quality showed that for the outcome of at least half pain relief over 12 hours, rofecoxib, naproxen sodium and ibuprofen were similarly effective in the treatment of pain associated with dysmenorrhoea. This confirms what was known from previous meta-analysis , in whicRofecoxib 50 mg was statistically indistinguishable from naproxen sodium 550 mg Table  at all tPain relief and duration of analgesia are not the only issues of importance in dysmenorrhoea. The impact of dysmenorrhoea on activities of daily living, disability or function, and absence from work or school are additional factors to be considered. These outcomes were not addressed by the trials for rofecoxib, which were conducted for regulatory purposes. This limits the utility of the information, but trials of other coxibs  have also concentrated on pain relief and duration of analgesia ,11.Future trials should examine a range of short-term analgesia and longer outcomes like interference with daily living or absence from work or school. The analysis by Zhang and colleagues  did examFuture individual patient analysis of trials in dysmenorrhoea would have the potential to examine issues around the efficacy of analgesics in women with heavy menstrual loss, or who use combined oral contraceptive pills. In this analysis information was not available for these analyses, and in any event any sub-groups would probably have been too small for any definitive answer.In the trials for rofecoxib, information on adverse effects was collected using diaries. Few adverse effects were reported to have occurred and none were serious. The most common adverse effects were nausea and somnolence. These and headache have been frequently reported with other coxibs ,11 and NBased on information from three trials, a single dose of rofecoxib 50 mg is as effective as a single dose of naproxen sodium 500 mg in controlling the pain associated with dysmenorrhoea, and causes relatively few adverse effects.RAM has been a consultant for Merck, Sharpe and Dohme Ltd, UK. RAM, JE and HJM have received lecture fees from pharmaceutical companies. The authors have received research support from charities and government sources at various times, but no such support was received for this work. Neither author has any direct stock holding in any pharmaceutical company. The terms of the financial support from MSD included freedom for authors to reach their own conclusions, and an absolute right to publish the results of their research, irrespective of any conclusions reached. MSD did have the right to view the final manuscript before publication, and did so.JE conducted the analyses, which were checked by RAM. All authors contributed equally to the design, writing and reviewing of the paper.The pre-publication history for this paper can be accessed here:


Epidemiological studies have, so far, identified factors associated with increased risk for incident or progressive OA, such as age, sex, heredity, obesity, and joint injury. There is, however, a paucity of long-term data that provide information on the nature of disease progression on either group or individual levels. Such information is needed for identification of study cohorts and planning of clinical trials. The aim of the study was, thus, to assess the variation in pain and function on group and individual level over 2 years in previously meniscectomized individuals with and without radiographic knee osteoarthritis (OA).143 individuals  were assessed twice; approximately 14 and 16 years after isolated meniscectomy, with a median interval of 2.3 years (range 2.3–3.0). Radiographic OA  was present in the operated knee in 40%, and an additional 19% had a single osteophyte grade 1 in one or both of the tibiofemoral compartments. Subjects completed the self-administered and disease-specific Knee injury and Osteoarthritis Outcome Score (KOOS).There were no significant changes in the group mean KOOS subscale scores over the 2-year period. However, a great variability over time was seen within individual subjects. Out of 143 subjects, 16% improved and 12% deteriorated in the subscale Pain, and 13% improved and 14% deteriorated in the subscale ADL ≥ 10 points . Similar results were seen for remaining subscales.Group mean scores for this study cohort enriched in incipient and early-stage knee OA were similar over 2 years, but pain, function and quality of life changed considerably in individuals. These results may be valid also for other at risk groups with knee OA, and motivate further careful examination of the natural history of OA, as well as properties of the OA outcome instruments used. Longitudinal outcome data in OA studies need to be analyzed both on an individual and a group level. Drugs that may slow or halt the breakdown of cartilage and other joint tissues in osteoarthritis (OA) and possibly improve symptoms and function are now being developed in the pharmaceutical industry. The potential availability of disease modifying OA drugs has focused attention on our relative lack of information on the 'natural disease history' of OA with regard to changes in symptoms, functional limitations, joint structure and other markers of disease change .Epidemiological studies have identified factors associated with increased risk for incident or progressive OA, such as age, sex, heredity, obesity, and joint injury, pain, alignment, or laxity. There is, however, a paucity of long-term data that document the rate and nature of natural OA disease progression on either group or individual levels. Such information is needed for identification of study cohorts and planning of clinical trials of disease modifying OA drugs. Even more importantly, knowledge of natural disease progression in different patient groups will be needed to select those future groups that may benefit from such drugs.Only a few of the previously published studies have presented information on longitudinal variation in pain and function in the natural history of knee OA. The "Bristol 500 OA study" noted, that although pain changed little on a group level over a 3-year follow-up period, it varied greatly in individuals, with some subjects reporting marked improvements. Similarly, a minority improved functionally -4.Yet another report suggested that most patients with OA attending rheumatology clinics do not deteriorate radiographically or symptomatically over an 11-year period . A more However, outcome is usually heterogeneous: study subjects may report improvement or deterioration while they do not change radiographically over the time period assessed. It may also be that a few individuals alone generate much of any change detected at group level -11. A fuThe objective of this investigation was to assess both group and individual variation in knee pain, function and quality of life over two years in a study group enriched in incipient and early-stage radiographic knee OA.Approval was obtained from the Research Ethics Committee of the Medical Faculty of Lund University, Sweden. All patients who underwent meniscectomy between 1983 and 1985 were identified by searching the surgical records at the Department of Orthopedics, Lund University Hospital. In this period 552 meniscectomies were performed. Inclusion and exclusion criteria  who returned the questionnaires, 6 were excluded because they matched one of the exclusion criteria. At 2 years after the first assessment 5 subjects had died, but the remaining 200 individuals were asked to provide a second evaluation using an identical questionnaire. Replies were received from 143 (72%). Of these 143 participants, 102 were meniscectomized by open surgery, and 41 by arthroscopy. Nineteen underwent an additional meniscus operation in the index knee.All re-operations were performed within 3 years after the original meniscectomy. Twenty-three participants were treated with subsequent meniscectomy of the contralateral knee. One of them underwent high tibial osteotomy and 1, because of OA, received a knee prosthesis in the contralateral knee. Data concerning subsequent surgeries were based on the medical records of Lund University Hospital and on self-reported information.At the time of the participants' second evaluation with questionnaires, standing anteroposterior (AP) radiographs of both knees were taken in 15 degrees of flexion using a CGR Phasix 60 generator at 70 kV, 16 mA, film-focus distance 1.5 m . Ten out of the 143 participants (7%) declined the radiographic examination. All AP radiographs of the tibiofemoral joints from the follow-up were assessed for joint space narrowing (JSN) and osteophytes according to the atlas from Osteoarthritis Research Society International (OARSI) .The presence of these features was graded on a 4-point scale . We considered radiographic knee OA to be present if any of the following criteria was achieved in any of the 2 tibiofemoral compartments: JSN ≥ grade 2 or the sum of the 2 marginal osteophyte grades from the same compartment ≥ 2, or JSN grade 1 in combination with an osteophyte grade 1 in the same compartment ,14. ThisThe Knee injury and Osteoarthritis Outcome Score  is a 42-item self-administered knee-specific questionnaire based on the WOMAC Osteoarthritis Index ,17. KOOS. The KOOS is valid, reliable and responsive in follow-up of meniscectomy [The questionnaire and scoring manual can be downloaded from scectomy , anterioscectomy  and totascectomy . The parThe minimal perceptible clinical improvement (MPCI) represents the difference on the measurement scale associated with the smallest change in the health status detectable by the individual. Since the KOOS questionnaire contains the full and original version of the WOMAC LK 3.0 index, we used the MPCI as described for WOMAC . Thus, aP-values for categoric data were calculated with Fisher's exact test. All tests were 2-tailed and a P-value of ≤ 0.05 was considered statistically significant .If questions were left unanswered in any part of the questionnaire, we returned the questionnaire to be completed. The questionnaires were then completed fully. The Mann-Whitney U-test was used to determine differences between the groups. The study group comprised 143 individuals, of whom 23 (16%) were women. The participants' mean age at the first follow-up was 51 (range 27–83) years. The assessment was carried out twice: at approximately 14 and 16 years after the surgery, with a median interval of 2.3 (range 2.3 to 3.0) years. Fifty-three (40%) of the 133 individuals who had undergone radiographic examination had radiographic tibiofemoral OA in their index (operated) knee  and 80 were classified as non having OA . An additional 25 (19%) (not classified as radiographic OA) had a single osteophyte grade 1 in either one or both tibiofemoral compartments.P = 0.004), other Symptoms Δ = 9 points (P = 0.013), ADL Δ = 10 points (P = 0.003), Sport/Rec Δ = 17 points (P = 0.005), and QOL Δ = 16 points (P = 0.003) assessed in 2000, and in the dimensions Sport/Rec Δ = 14 points (P = 0.020) and QOL Δ = 12 points (P = 0.041) evaluated in 1998.Mean scores for the KOOS subscales at the first assessment did not change significantly over the 2-year study period Table . MoreoveWe analyzed separately those subjects (N = 57) that did not participate in the second assessment. Their mean KOOS scores at the first examination did not differ significantly from the remainder of the study cohort, indicating little or no inclusion bias for the second follow-up (data not shown). The scores in the 5 patients that underwent additional surgery  did not differ significantly from the rest of the group.In spite of the lack of change on a group level, we found substantial intra-individual variability in the questionnaire subscale scores measured 2 years apart. Out of the total 143 study subjects, 40 had either improved or deteriorated (n = 23 (16%) and n = 17 (12%), respectively) 10 points or more for the KOOS subscale Pain. Of the 23 subjects who had improved in their pain score by these criteria, 14 had also improved in the subscale Symptoms, 17 in ADL, 16 in Sports/Rec, and 17 in QOL.P = 0.24, Table Only 1 of these subjects deteriorated in Symptoms, and 2 in Sports/Rec, none in the other subscales. Of the 17 subjects who deteriorated in Pain, 13 similarly deteriorated in Symptoms, 12 in ADL, 10 in Sports/Rec, and 10 in QOL. When evaluating those who had undergone radiographic examination, there were no significant differences in variability detected whether the subject had radiographic tibiofemoral OA or not  obtained for the WOMAC; since KOOS contains the WOMAC items and is similar in format. The KOOS subscale ADL is equivalent to the WOMAC subscale Function, while new items have been added to the KOOS subscales Pain and Symptoms. The dimensions assessed by the KOOS subscales Sport and Recreation Function and knee-related Quality of Life are not assessed by the WOMAC. The MPCI for the WOMAC is in the range of 8 to 12 points on a 0–100 scale .This threshold coincides with the change observed in KOOS scores between 3 and 6 months postoperatively when assessing rehabilitation following reconstruction of the anterior cruciate ligament and concurs with the OARSI definition of moderate improvement in the knee pain assessment for clinical trials in OA ,22. HoweIt may be argued that the subject-related changes observed in this study represent inherent instrument instability. However, validation studies of KOOS support the reproducibility and stability of the KOOS instrument -19. TestThe proportion of subjects changing over 5 days was approximately half of that changing over 2 years in the present study, in further support that the variation in the present study cannot be explained solely by instrument noise (data not shown). A 'frame shift' in the priorities of the individual patient may occur during long term studies. However, we suggest that a significant frame shift is unlikely to have occurred over this 2 year study period of a cohort with a mean age of 50 years.Significant change of KOOS scores over time were noted in 1/3 of the cohort studied. About half of those who changed clinically improved. This was true in particular for patients with lower (worse) baseline scores. It is thus possible that the lower proportion of 'changers' among those with better baseline scores may have been, at least in part, due to a ceiling effect.We conclude that despite unchanged group mean scores over 2 years, pain, function and quality of life change considerably over time in individuals, in this study cohort enriched in incipient and early-stage knee OA. These findings may be applicable also to other at risk patient groups in different phases of OA development, and motivate further careful examination of the natural history of OA, as well as properties of the OA outcome instrument used. We suggest that longitudinal OA study data should be analyzed both on the individual and group level.Our findings may have further relevance to clinical trials of OA that seek to document long term benefit in the form of symptom improvement and structural improvement. It is clear that much additional effort will need to be spent on selection of groups at high risk of progression of symptoms and structural joint change, and the identification of predictors for deterioration. Our results also suggest that the use of responder criteria may be an important aspect of analyzing the outcome of such trials ,33.EMR and LSL planned study and collected the data. PTP performed the statistical analysis and drafted the manuscript. ME formed the database of patients and participated in the statistical analysis. ME, EMR, LSL corrected the manuscript. All authors read and approved the manuscript


Obesity is a growing international health problem that has already reached epidemic proportions, particularly within the United States where a majority of the population is overweight or obese. Effective methods of treatment are needed, and should be taught to physicians by efficient means. There exists a disconnect between the rising obesity prevalence with its high toll on medical resources, and the lack of obesity education provided to practitioners in the course of their training. One particular shortfall is the lack of representation of obesity on standardized medical examinations. Physician attitudes toward obesity are influenced by their lack of familiarity with the management of the disease. This may include dietary restriction, increasing physical activity, behavior modification, pharmacotherapy, and surgical interventions. Thus, curricular changes in the medical education of obesity could help reduce morbidity and mortality associated with this disease. Wegeners's granulomatosis, errlichiosis, and tetrology of Fallot are examples of some of the rare diseases that I have learned to identify and look for in patients for over seven years as a medical student and resident in Internal Medicine. As a physician in training, I was frequently challenged to consider broad differentials and to watch out for the zebra that masquerades as a common disease throughout my clinical encounters. I have had my antennas up in search of these diseases for quite a while now. I have not, however, encountered any patients with these diseases so far. What I have usually encountered instead are common diseases, like type 2 diabetes, hypertension, and arthritis. More than anything, I have been struck by the incidence of obesity in our population. I find it alarming that many clinicians fail to actively address this growing epidemic in a formal manner with their patients, but rather regard obesity as a lifestyle choice that is the patient's sole responsibility.My personal experience with obesity, having trained at a quaternary referral center in the relatively thin state of California, is that over one third of the patients I cared for were obese (not just overweight). Consistent with national trends, patients in my region have been becoming more obese and accordingly have been suffering increasingly from the complications of the disease, such as hypertension, insulin resistance, fatty liver disease, dyslipidemia, coronary heart disease, osteoarthritis, obesity-related cancer, and premature death . I have Many physicians cite their unfamiliarity with obesity as an impediment to their ability to adequately address the problem with their patients . In one I recalled at least five examination questions addressing Wegener's granulomatosis, but none directly on obesity. The differences in prevalence are remarkable. In the United States, approximately 61% (110 million) of adults (age 20–74 years) are overweight or obese. . EducatoDiabetes, coronary heart disease, dyslipidemias, hypertension, and cancer were appropriately well represented in this ABIM exam and previous standardized examinations I have taken. Obesity should be right up there with these "big players". There is a lot to understand within this field. It is a lot more complex than "eat less, make better nutritional choices, and exercise more". The complexities of nutritional decision making are very important to the patient, and they want to discuss these choices with physicians as well as dieticians. Pharmacotherapy, although limited in terms of number of options, does have a role. Bariatric surgery is an evolving field and there are complexities associated with the use of the procedure that surgeons and internists alike must understand. Also, such psychosocial aspects of obesity as diminished quality-of-life, self-esteem, and work performance must be stressed and understood. Fogelman reported that in one survey of family practitioners, 72% believed that they had limited efficacy in treating obesity and considered themselves not well prepared by medical school to treat overweight patients . Some 60Primary prevention is the way to go in obesity research expenditure. In my opinion, the greatest healthcare cost-savings' "bang-for-the-buck" comes from preventive care and education. Consider how many fewer cases of diabetes, hypertension, CHD, fatty liver disease, dyslipidemia, thromboembolic disease, obesity-hypoventilation, obstructive sleep apnea, gout, osteoarthritis, cancer, GERD, reproductive and urinary tract abnormalities, cataracts, psychosocial abnormalities we would have to battle if the inciting factor (obesity) was aggressively treated at the onset. Is this not one prudent management step toward curtailing an overtaxed health care system?The war on obesity seems winnable. We just need the tools and awareness to address the disease. Obesity is not a silent disease to be tolerated: it is an aggressive yet insidious disease (much like type 2 diabetes) that must be arrested before its negative health consequences become irreversible. The first step is to teach practitioners what to look out for, and in turn, to teach patients what to look out for. Meanwhile, I will have my antennas up in search of Wegener's granulomatosis.I hereby declare that I do not have a financial association or other conflict of interest with the subjects mentioned in this manuscript.