This scarcity of evidence is observed especially among children with the virus. Therefore, this study aimed to assess the characteristics and outcome profile of children with COVID-19 admitted to Millennium COVID-19 Care Center in Ethiopia.rd to September 17th, 2020. Data were summarized using frequency tables, mean ± standard deviation or median with inter quartile range values. A chi-square test/Fischer´s exact test was used to compare disease severity between groups.a prospective cohort study was conducted among 90 children with COVID-19 who were admitted from June 23the median age of the participants was 15 years and 57 were females. The most common reported route of disease transmission was through close contact with a diagnosed person (41/90). Only three had a history of pre-existing comorbid illness. One-third (31/90) had one or more symptoms at diagnosis, the most common being cough (20/90). Among the 90 patients, 59 were asymptomatic, 14 had mild disease and the rest 17 had moderate disease. Based on the chi-square/Fischer´s exact test result, no statistically significant difference was observed between the age groups and sex.pediatric patients seemed to have a milder disease presentation and a favorable outcome compared to other countries report and also the adult pattern observed in our country. The majBoth symptomatic and asymptomatic cases are reported, with a number of studies including systematic review report showing that a higher proportion of children had one or more symptom at presentation. The commonly reported symptoms are fever and cough, with fever being the predominant symptom in many countries. Nonspecific symptoms like fatigue and myalgia are also reported -9. This A study conducted in South Africa among children younger than 13 years showed that among hospitalized children, majority were symptomatic, one-fifth had pre-existing medical condition and respiratory support was required in half of the children . LaboratDue to scarcity of study among children with COVID-19 in Africa, understanding the disease pattern and its outcome is crucial to guide decision making. Therefore, in this study we aimed to assess the characteristics and outcome profile of children with RT-PCR confirmed COVID-19 and admitted to Millennium COVID-19 Care Center in Ethiopia.Study setting, design and population: an institution based prospective cohort study design was conducted at Millennium COVID-19 Care Center (MCCC), a makeshift hospital in Addis Ababa, Ethiopia. The center is remodeled from the previous Millennium Hall which was a recreational center. The center started accepting patients on June 2nd, 2020 with a capacity of 1000 beds, 40 intensive care unit (ICU) beds and 12 mechanical ventilators. At that time the national admission criteria was having positive real time polymerase chain reaction (RT-PCR) test result irrespective of the presence of symptoms or severity of the disease including cases identified through contact tracing. Therefore, the center was used as both quarantine and treatment center in order to halt the transmission of the disease. The center was modelled to admit adult and none pregnant patients only but since the other centers were not ready for COVID-19 case admission, all age groups and pregnant women were also being admitted for few months. The center accepts patients from referral centers  only with a positive RT-PCR result and the service is provided for free.rd to September 17th, 2020 [The source population was all consecutively admitted children with COVID-19 admitted to MCCC with a confirmed diagnosis of COVID-19 using RT-PCR, as reported by a laboratory given mandate to test such patients by the Ethiopian Federal Ministry of Health and who were on follow up and/or treatment from June 23th, 2020 . All eliSample size determination and sampling technique: all consecutively admitted children with COVID-19 during the three months follow up period were included in the study. During this interval a total of 115 children with COVID-19 were admitted to the center. Finally, 90 children who fulfilled the eligibility criteria were included in the final analysis.Eligibility criteria: all children 18 years and younger with RT-PCR confirmed COVID-19 who were on follow up and/or treatment at the center from June 23rd to September 17th, 2020 and with complete follow up data were included in the study.Asymptomatic patient: any patient who tested positive for COVID-19 but does not have any symptoms. These patients are detected after isolation and contact tracing as done by the Ethiopian Public Health Institute (EPHI).COVID-19 severity score: was determined based on the WHO classification as follows: 1) Mild disease: characterized by fever, malaise, cough, upper respiratory symptoms, and/or less common features of COVID-19 ; 2) moderate disease: patients with lower respiratory symptom/s. They may have infiltrates on chest X-ray. These patients are able to maintain oxygenation on room air.Data collection and quality assurance: a pre-tested interviewer administered questionnaire was used to collect data from patients and their medical charts to capture their socio-demographic, clinical and disease outcome profile. The data were collected by trained data collectors and all the necessary precautions in order to prevent the acquisition and disseminate the virus were taken during the data collection process. To improve data quality, data cleaning through checking for inconsistencies, numerical errors and missing parameters was done. Where discrepancies are observed, data entered was verified with the primary data source.Statistical analysis: once the collected data were coded and cleaned, it was exported and stored into SPSS version 23 for analysis. Categorical covariates were summarized using frequencies and percentages and numerical variables were summarized with a mean ± standard deviation (SD) or median with inter quartile range (IQR) values. A chi-square test (or Fischer´s exact test for those variables which do not meet the chi-square assumptions) were used to determine the presence of a significant difference between the age groups and sex based on COVID-19 disease severity. A statistically significant difference was detected for variables with a P-value of ≤ 0.05.Ethics: the study was conducted after obtaining ethical clearance from St. Paul´s Hospital Millennium Medical College Institutional Review Board. Written informed consent was obtained from the participants and/or their care givers. The study had no risk/negative consequence on those who participated in the study. Medical record numbers were used for data collection and personal identifiers were not used in the research report. Access to the collected information was limited to the principal investigator and confidentiality was maintained throughout the project.Socio-demographic, co-morbid illness and presenting symptom characteristics: the median age of the participants was 15  years. The majority (33/90) of the participants were 15 to 18 years. Almost two third (57/90) of the participants were females. The majority (80/90) were from Addis Ababa, the capital city of Ethiopia. The most common reported route of disease transmission was through close contact with a diagnosed person (41/90). Only three had a history of pre-existing comorbid illness, two had bronchial asthma and one had a history of valvular heart disease. One-third (31/90) of the patients had one or more symptom at admission and rest 59 were asymptomatic. The most common symptom was cough (20/90), followed by sore throat and headache (each constitutes 9 cases), fever, runny nose, chest pain and fatigue (each constitutes 5 cases) and then nausea and vomiting (4 cases) (4 cases) .Disease severity and outcome: among the 90 patients, 59 were asymptomatic, 14 had mild disease and the rest 17 had moderate disease. From the 90 children, 67 were discharged improved and the rest 23 were transferred to another hospital. Among the 23 transferred cases, 17 were asymptomatic, 4 had mild disease and 2 had moderate disease. One child with valvular heart disease was transferred to a center where there is a pediatrician for better management and follow up as MCCC is setup for an adult care. Eight were transferred to another center with family request for convenience of visit. The rest 14 were transferred to another center as an adult family member with whom the children were admitted with were transferred due to different reasons.Management of the admitted children: mild cases were managed with analgesic and antipyretic to relieve symptoms and the moderate cases received antibiotics treatment in addition. Since there was no severe case during the observation period, none of the children required an oxygen therapy or intensive care unit admission.Characteristics of the children with moderate COVID-19: seventeen of the children had moderate COVID-19 at admission. The profile of the 17 patients showed that, 11/17 were 15-18 years old and 12/17 were females. Only one child had valvular heart disease, the rest didn´t have any known pre-existing co-morbid illness. Fifteen out of seventeed were discharged improved and 2/17 were transferred to another hospital . The complete blood count, renal function and liver enzyme laboratory report of the moderate cases showed that all values were with in normal range  years. A similar finding was observed in the North American study [In this study we have assessed the characteristics and outcome profile of 90 children with RT-PCR confirmed COVID-19 who were admitted to Millennium COVID-19 Care Center in Ethiopia from June 23an study . Two thiAlmost two third of the participants were females unlike the pediatric studies conducted in other countries where the majority of infected children are males ,4. In adOnly three had a history of pre-existing comorbid illness, two had bronchial asthma and one had a history of valvular heart disease. This seems to be in contrast to other studies where 42% up to 83% of admitted children had a history of one or more pre-existing co-morbid illness ,8. The mAmong the entire pediatric admission during the study period in this largest COVID-19 Care Center in the country, the majority (59/90) of the children were asymptomatic, 14 had mild disease and the rest 17 had moderate disease. There was no severe or critical case. On the contrary, studies from other countries revealed that a considerable proportion of children were diagnosed with severe and critical illness with a need of Intensive care unit admission and invasive ventilation -6,8. In The variation in report among studies could be attributed to the small sample sizes included in most studies or due to the difference in underlying population characteristics and difference in admission criteria between countries. Therefore, though the finding from this study can be used as a baseline, further study with large sample size is necessary to have a better understanding of the pandemic in pediatric group as the small sample size population included in this study might not generalize the actual pattern and outcome of the disease at the national level.The majority of the identified cases were asymptomatic and there was no death during the observation period. The pediatric patients seemed to have a milder disease presentation and favorable outcome compared to other countries report and also the adult pattern observed in our country. Further multicenter study with a large sample size is recommended to gain a better perspective of the disease in children.COVID-19 among children is reported to have a diverse outcome among different countries with majority reporting a favorable disease course and outcome than adults;There is scarcity of research among children with COVID-19 compared to the adults, especially in the African setup.This study assessed children with COVID-19, it is the first of few studies in Africa;Pediatric patients seemed to have a milder disease presentation and a favorable outcome compared to the adult pattern observed in our country and other setups;The finding can be used as a baseline for further large scale study and can be used to guide the practice in similar the mean time.


The total amount of the tax increase in the past 3 years was 3 yen per stick or 60 yen (about 0.5 United States dollar [USD]) per pack.3 The same price  may be considered cheap and affordable in higher-income countries but expensive and less affordable in lower-income countries. There are several measures of tobacco affordability.3 Among them, the “relative income price” is readily available for cross-national comparisons including Japan. This measure is defined as the proportion of gross domestic product (GDP) per capita required to purchase 100 packs of cigarettes, with a smaller proportion indicating higher affordability.For cross-national comparisons of tobacco prices, affordability should be considered.4 As of September 2021, the latest available year was 2018. To identify the level of Japan’s tobacco affordability, we compared the relative income price  across 38 countries belonging to the Organisation for Economic Co-operation and Development (OECD) and examined its relationship with GDP per capita to investigate whether tobacco affordability is proportional. Data on GDP per capita in 2018 were obtained from the OECD.5We obtained data on the relative income price from the World Health Organization, which compiles tobacco tax and price information from Member States every 2 years.The proportion of GDP per capita required to purchase 100 packs of the most-sold brand of cigarettes among OECD countries in 2018 is illustrated in Figure Figure 6,7 but such estimates are not available for cross-national comparisons. In summary, tobacco prices in Japan appear to be low relative to the wealth of the country. The government should use tobacco price increases as an effective method of achieving a tobacco-free society.8–10We acknowledge that tobacco affordability presented herein is based on the price of the most-sold brand cigarettes and GDP per capita, which reflects neither the income distribution in the population nor the income level in the smoking population. Tobacco affordability can be more accurately estimated with individual rather than aggregate data,


To evaluate the safety and preliminary efficacy of topical rVA576, a dual inhibitor of complement component 5 (C5) and leukotriene B4 (LTB4), in patients with recalcitrant atopic keratoconjunctivitis (AKC) in the open label phase 1 TRACKER clinical trial.Three patients diagnosed with moderate or severe AKC who had been on maximal topical treatment (antihistamines and ciclosporin) for at least three months prior to entry, and showed persistent symptoms and signs of inflammation, were recruited into the trial. Patients received rVA576 eye drops twice a day for 8 weeks. Patients were seen at baseline and weeks 1, 2, 4, 6 and 8. Safety data was recorded and a composite sum score of symptoms and signs was obtained. This score comprised symptoms such as itching, mucous discharge and photophobia, and conjunctival and corneal signs such as hyperemia, tarsal papillae, punctate keratitis and corneal neovascularization, all rated individually from 0 to 3 for a maximum score of 33.Two of the three patients completed the initial open label phase of the trial. The third patient was unable to attend appointments and terminated the study early at day 14. Topical rVA576 was well tolerated with no serious adverse events reported. There was an average improvement in overall clinical score of 53%, composed of an improvement in symptoms of 65% [63.64–66.67%] and signs of 40% [40–40.12%] by day 56.In this open label phase 1 TRACKER trial, rVA576 eye drops were well tolerated and showed a response across signs and symptoms of active inflammation. This study is exploratory but supports topical rVA576 safety and shows promising efficacy for recalcitrant AKC. A phase 2 randomised control trial is currently underway.The online version contains supplementary material available at 10.1186/s13023-021-01890-6. The recombinant protein rVA576  is derived from a protein discovered in the saliva of the Ornithodoros moubata tick, also known as OMCI , where iAtopic keratoconjunctivitis (AKC) is a severe chronic allergic eye disease that primarily affects the adult population. Compared to other forms of allergy, AKC is characterised by a more chronic and insidious course, with recurrent episodes of inflammation that can lead to corneal scarring and neovascularisation, and ultimately vision loss. AKC involves mast cell activation secondary to the predominance of inflammatory mediators such as eosinophils and Th2-type cytokines . ManagemThis study was an open label phase 1 clinical trial of topical rVA576 eye drops in patients with moderate to severe AKC with a treatment period of 8 weeks and a further follow-up of 4 weeks beyond rVA576 therapy. It is to be followed up by a randomized, double blinded, placebo-controlled phase 2 clinical trial (ongoing at present).Patients included were aged 18 and above who had been on maximal topical therapy for at least three months without improvement but were not receiving systemic immunotherapy. A full list of inclusion and exclusion criteria has been provided in Table The primary trial objective was to demonstrate the safety and tolerability of rVA576 when given by topical ocular administration to patients with AKC. Tolerability of the study drug was assessed by patient diary cards. The diary cards measured the comfort profile by asking the patient to describe in one word how comfortable or not they felt immediately after the instillation of the study drug and after 1, 2, 3 and 5 min. Safety data was assessed by review of adverse event reporting.Safety data was recorded and a composite sum score of signs and symptoms was obtained. This scoring system is described in Table The secondary objective was to evaluate efficacy. Efficacy was assessed in the eye judged to be worst affected by the patient, or if equally affected, in the right eye.Three patients were enrolled in the study. Two of them completed 56 days (8 weeks) of treatment and one completed 14 days and then withdrew for reasons unrelated to the study treatment. The two patients who completed the study continued a further 4-week follow-up after cessation of rVA576 use. The drops were found to be comfortable and well-tolerated throughout the trial for all three patients. Post-instillation comfort was reported by patients as excellent with high levels of acceptance of eye drops, which were described as comfortable. Tolerability in ocular surface inflammatory disease is important to ensure long-term compliance of therapy. There were no serious adverse events (SAE) reported in this trial.All patients saw a reduction in average sign and symptom scores by day seven of treatment Figs. . By day TRACKER  is the first clinical trial to evaluate rVA576 as a treatment for any ocular disease (first-in-eye study). It was administered topically to patients with recalcitrant atopic keratoconjunctivitis. Systemic rVA576 has already been studied for the treatment of non-ocular disease. Paroxysmal nocturnal haemoglobinuria (PNH) is one such disease where subcutaneous injections of rVA576 have been used with success [The data from this study suggests rVA576 is safe and well tolerated as an eye drop medication. In terms of efficacy, albeit in a small number of patients, the results of this first-in-eye open label clinical trial of topical rVA576 showed clinically relevant reduction across signs and symptoms of active inflammation in a group of patients with recalcitrant disease. It is therefore a promising new topical treatment for atopic keratoconjunctivitis filling a stepladder space of clinical need. Limitations of this trial include a small number of participants and lack of placebo comparison. However, part 2 of the TRACKER clinical trial is underway with a control arm to the study.Overall the results of this first-in-eye open label trial are novel and important, showing a potentially relevant role for rVA576 eye drops in the treatment of atopic keratoconjunctivitis. This work also highlights the need for further investigation of the role of complement in inflammatory ocular surface disease.Additional file 1. Supplementary table with complete patient scores.


The prevalence of self-reported preventive behaviors in response to an anticipated local human-to-human H5N1 transmission outbreak and factors associated with such behaviors have not been examined.A random, anonymous, cross-sectional telephone survey of 503 Hong Kong Chinese adults.The public in Hong Kong is likely to adopt self-protective behaviors , increasing the frequency of handwashing (86.7%)) and behaviors that protect others , immediately seeking medical consultation (94.2%), making declarations when crossing the border with ILI (87.1%), complying to quarantine policies (88.3%)). Multivariate analyses indicated that factors related to age, full-time employment, perceived susceptibility, perceived efficacy of preventive measures, perceived higher fatality as compared to SARS, perceived chance of a major local outbreak, and being worried about self/family members contracting the virus were significantly associated with the inclination to adopt self-protective measures. Similar analyses showed that education level, variables related to perceived efficacy, perceived major local outbreak and such were significantly associated with various behaviors directed towards protecting others.In the event of a human-to-human H5N1 outbreak, the public in Hong Kong is likely to adopt preventive measures that may help contain the spread of the virus in the community. As of December 7, 2006, bird-to-bird transmission of the H5N1 virus have been reported in over 50 countries ,4, experDuring the SARS epidemic, preventive measures were commonly practiced  and a nuThis study investigated the prevalence of self-reported preventive behaviors in response to a hypothetical local outbreak of human-to-human H5N1 transmission. These behaviors included both self-protective measures as well and those protecting others from contracting the virus. Factors associated with these behaviors were investigated.The study population was comprised of male and female Hong Kong Chinese adults aged 18–60 years. An anonymous cross-sectional telephone survey using a structured questionnaire was conducted in November 2005 (n = 503). Table Respondents were asked how likely they would be to adopt the following preventive behaviors if a local human-to-human H5N1 outbreak (defined as "if 2–3 new human-to-human transmission of H5N1 cases were to be reported in Hong Kong") were to occur: face mask use in public venues, increased frequency of handwashing, avoidance of eating poultry, declaration of influenza-like illness (ILI) symptoms at border health checkpoints, the seeking of medical consultation immediately with the onset of a fever, face mask use in public venues when having ILI symptoms and compliance with any quarantine policies. Responses were recoded into 2 categories (yes or no) from a 5-point Likert scale.Respondents were asked about perceptions related to human-to-human H5N1 transmission, including perceived modes of transmission , perceived susceptibility to H5N1 in different groups of people , perceived chance of having a major outbreak in Hong Kong in the next 12 months and perceived efficacy of various prevention measures . Respondents were also asked their perceptions of the current availability of effective treatments, whether they believed health outcomes would be worse than those of SARS  and the degree of concern they had about oneself or one's family contracting the virus if 2–3 new human-to-human H5N1 were to be reported in Hong Kong.Univariate odds ratios of the associations between the studied perception variables and individual studied preventive behaviors were derived. Variables that were significant in the univariate analyses were further analyzed using multivariate logistic regression analyses. Statistical significance was set at p = 0.05 and SPSS software version 12.0.0  was used for data analyses.The majority would adopt self-protecting behaviors such as wearing face mask in public venues (73.8%), increasing frequency of handwashing (86.7%), avoiding eating poultry (63.8%), wearing face mask in public venues when having ILI symptoms 92.4%), seeing a doctor immediately when having a fever (94.2%), making declarations at border health checkpoints when traveling with ILI (87.1%), and full compliance with any quarantine policies (88.3%). Gender and educational differences were, in general, non-significant, whereas some significant age differences were noted. Overall, 41.6% of the respondents would practice all 7 types of protective behaviors  believed that effective drugs are now unavailable, fewer (12.9%) thought that the efficacy of treatment for this disease would be worse that that of SARS . An even higher frequency of handwashing is expected if the anticipated outbreak occurs. This is consistent with the local government campaigns for promoting handwashing . HandwasDuring the SARS epidemic, the reported prevalence of face mask use when having ILI while traveling abroad was not very high, indicating that a substantial proportion of the general population was not practicing behaviors directed towards protecting others . This isQuarantine was an effective means contributing to the control of SARS ,21 but aFrom 2003 until the present, the reported fatality rate of avian influenza in human has been over 50% , which iWith full population compliance with quarantine policies, the critical battle front of the avian influenza epidemic would shift to effective hospital infection control. It is unlikely that health care workers will be able to comply to all the stringent occupational guidelines in the event of nosocomial human H5N1 outbreaks. High fatality rates may occur in these health settings if panic or widespread non-adherence to safety measures occur. Research, counseling and emergency plans are essential to ensure that front-line health care workers are psychologically prepared and that the operations of health systems will not be disrupted.The public regarded older people, children, health care workers and food vendors as particularly at risk of contracting the virus. During the SARS epidemic, it was reported that discriminatory attitudes have been expressed toward some at-risk groups, such as health workers ,25. It iApproximately one-quarter of the respondents believed that they or their family members would likely/very likely be affected by an H5N1 outbreak whereas approximately one-third of the respondents believed this to be true for the general population. It was reported that during the SARS epidemic, the general public worried about themselves or their family members' contracting the virus . This isAlthough prevalence of health-seeking behaviors are usually higher among females than males, gender was not associated with any of the studied behaviors in this study. The health threat in this case may have overridden the aforementioned gender differentials. Education level was not associated with self-directed preventive behaviors but was associated with one of the others-directed preventive behaviors. It is possible that altruism is associated with education level. The reverse was true with age. Another study indicated that higher age was associated with the more use of preventive measures .In general, factors related to perceived susceptibility, perceived clinical severity of outcomes  were associated with anticipated preventive behaviors. Such variables are the key factors prescribed by the Health Belief Model (HBM), which stated that adoption of health behaviors is a function of an individual's attitudes and beliefs about the health issue/behavior of concern . These vThe study has a number of limitations. First, the study was conducted using telephone surveys and some households may not have been included. In Hong Kong, however, almost all households have telephones  and a laWhile the results of this study may have meaningful regional policy implications, caution should be given when generalizing the results of this study to other countries. It is less clear whether populations which were relatively unaffected by SARS and populations that demonstrated less frequent face mask use and other relevant public health measures would exhibit the same magnitude of intended behavioral responses as the population of Hong Kong. Cultural and perceptions factors (such as perceived efficacy of prevention means) would also result in different prevalence of behavioral responses. International comparisons are therefore also greatly warranted.In the event of a human-to-human H5N1 outbreak, the Hong Kong public is very likely to adopt strong preventive measures in order to protect themselves and others. The magnitude of these behavioral responses may be even greater than those witnessed during the SARS epidemic and would be likely to increase if a high fatality rate or high infectivity rate were reported. These preventive behaviors may be an effective firewall to the continued spread of the virus in the community. Surveillance of public responses is an integral part of the government's H5N1 preparedness plan. It should address issues related to the potentially underestimated fatality associated with human H5N1 cases. Surveillance should also closely monitor prevalence of important public health behaviors such as quarantine compliance and cross-border preventive measures. Up-to-date surveillance information is necessary for the government to implement and make rapid adjustments these public health measures.The author(s) declare that they have no competing interests.JL conceptualized, oversaw the project, drafted and made final revisions of the manuscript. JHK helped to draft the manuscript and assisted in the statistical analysis. HYT participated in the design and coordination of the study and performed the statistical analysis. SG was involved in the conceptualization of the study andediting of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


There is, however, clinical need to forecast the likelihood of future occurrence of VTE, at least qualitatively, to support decisions about intensity of diagnostic or preventive measures.Community-based cohort studies are not available that evaluated the predictive power of both clinical A 10-year observation period of the Bavarian Thromboembolic Risk (BATER) study, a cohort study of 4337 women (18–55 years), was used to develop a predictive model of VTE based on clinical and genetic variables at baseline (1993). The objective was to prepare a probabilistic scheme that discriminates women with virtually no VTE risk from those at higher levels of absolute VTE risk in the foreseeable future. A multivariate analysis determined which variables at baseline were the best predictors of a future VTE event, provided a ranking according to the predictive power, and permitted to design a simple graphic scheme to assess the individual VTE risk using five predictor variables.4 WYs for no, moderate, high, and very high risk, respectively.Thirty-four new confirmed VTEs occurred during the observation period of over 32,000 women-years (WYs). A model was developed mainly based on clinical information  and one composite genetic risk markers (combining Factor V Leiden and Prothrombin G20210A Mutation). Four levels of increasing VTE risk were arbitrarily defined to map the prevalence in the study population: No/low risk of VTE (61.3%), moderate risk (21.1%), high risk (6.0%), very high risk of future VTE (0.9%). In 10.6% of the population the risk assessment was not possible due to lacking VTE cases. The average incidence rates for VTE in these four levels were: 4.1, 12.3, 47.2, and 170.5 per 10Our prognostic tool – containing clinical information  – seems to be worthwhile testing in medical practice in order to confirm or refute the positive findings of this study. Our cohort study will be continued to include more VTE cases and to increase predictive value of the model. Internationally there are several models available to assess the long-term risk of cardiovascular disease and they are broadly used in clinical practice and research -3. A 10-A prediction of the absolute risk of venous thromboembolism can only be developed on the basis of a specifically designed long-term cohort study. The study reported here is the first long-term, community-based cohorts study that observed genetic and clinical thromboembolic risk factors and their multivariate impact on VTE incidence to address this issue in young women.BAvarian ThromboEmbolic Risk study (BATER), focused on women in the reproductive age [A population-based thromboembolic risk factor study started in the mid-1990s in Bavaria, the tive age -6. CliniThis study provides a probability scheme that enables identification of women at high risk for VTE compared to women with virtually no risk for a theoretical period of maximal 10 years (defined by the model used). These findings could contribute to weigh the prognostic importance of clinical and laboratory data for medical decisions and better counseling of patients.Material and methods of this long-term cohort study has been described in detail in earlier publications -6. In brWe collected data from demographics, reproductive life, lifestyle pattern, conditions/diseases, and particularly potential risk factors for VTE through a questionnaire for self-administration. Whenever possible, time-related information was documented. Using this method we were able to set up the common starting point for the cohort as 1993. Telephone enquiries were made to supplement, clarify and verify the data in the questionnaires.The primary source for the data on VTE was the follow-up questionnaire . This information was completed by telephone interviews with the woman and with the treating physician. All available information about diagnostic and therapeutic measures taken was recorded. Clinical data and/or invasive or non-invasive diagnostic procedures were assigned to one of the following categories of likelihood of a VTE:Unequivocal positive finding in at least one imaging test, e.g., phlebography or duplex sonography for deep venous thrombosis (DVT); pulmonary angiogram, VQ scan, spiral computed tomography  for pulmonary embolism (PE).and subsequent specific therapy over a longer period (low-dose heparin or other anticoagulants).Typical clinical symptoms for VTE without unequivocal imaging test but positive findings in other diagnostic tests (e.g. Doppler US or plethysmography) and no subsequent specific therapy .Typical clinical symptoms for VTE and unknown or equivocal result on imaging, only suspicion of VTE suggested by a non-imaging diagnostic tests  Typical clinical symptoms for VTE without further diagnostic tests or negative results or diagnostics unknown. Unspecific therapy – but nonetheless the treating physician maintained the diagnosis VTE based on clinical findings.All possible and potential "VTE cases" were excluded from the analyses in this paper because of diagnostic uncertainty, i.e. lacking information whether they should be better classified as VTE cases or non-cases.Women with a history of cancer or with known antiphospolipid syndrome were not in our follow-up study.After having given informed consent the women included in this study gave a blood sample at one time point during the observational period (1996/97). An independent ethics committee approved all study related activities.Whole blood samples were obtained from resting subjects. Blood was put into tubes with trisodium citrate. Plasma was prepared soon after venipuncture by centrifugation for 15 minutes with 3000 to 4000 / min at room temperature and stored at -20°C.Protein C and antithrombin activities in plasma were measured by chromogenic substrate assays . For "antithrombin" the activity against factor IIa was determined, Protein C activity was measured after activation of the proenzyme by snake venom. Plasma activities are given as percentage (units/dl) of pooled human normal plasma.® DNA Blood Kit (Qiagen) according to the manufacturer's instructions. The genetic polymorphisms Factor V R506Q (G1691A), the prothrombin promoter G2010A and the 5-, 10-methylenetetrahydrofolate reductase (MTHFR) A223V (C677T) were determined using a multiplex PCR with allele-specific primers slightly modifying a previously described method [Genomic DNA was isolated by mean of QIAmp A223V C67T were dAll blood tests were performed blinded, i.e. the investigators had no clinical information, and had no access to the clinical database.Due to the importance of the temporal relationship the database was structured to accommodate both concurrent as well as time-dependent variables. Concurrent variables are variables, which describe the woman's status at the time of questionnaire response, whereas the outcome variable (VTE) is time-dependent. While concurrent variables were held in a fixed data set, a periodic data set containing information on lifetime exposures and the occurrence of VTE events along a time axis was created for the time-dependent variables of each participant, using months as a unit of measurement. The exposures of interest in this publication, such as VTE risk factors including genetic markers, refer to the baseline time point.Some of the variables in the database  were continuous. These variables were dichotomized in order to define a categorical exposure status (exposed – non-exposed) for the analyses based on incidence or logistic regression. We arbitrarily separated the continuum in two roughly equal intervals such as age under/over 30 or BMI under/over 25 in order to have sufficient case numbers for analyses with further stratification. For protein C and AT we used the 5th percentile (lower 5% of the distribution in non-cases) as cut off point. This limit was considered as usual definition for deficiency and clinically relevant .All analyses concerning the occurrence of VTE events over time were performed by adding up individual observation time (1993 until the last contact) for different exposure-cohorts and in total. The incidence rate of VTE was calculated per 10,000 women years of observation (WY).The predictive model was developed using the discriminant function analysis technique . This teWe multivariately ranked the predictive power of the variables with suspected effect on occurrence of VTE . Other available information in the database or information not known at baseline  were not included into the model since they cannot contribute to a predictive model (i.e. the setting for the application of the results of this study is women consulted by a physician independent of an acute VTE event). Technically, we used stepwise discriminant analysis with forward inclusion or backward elimination of variables. The p-value for entry into the model was 0.49 and for removal 0.50.The p-value of the parameter provided by the discriminant analysis at the last step determines its rank. Variables with the smallest p-value get the highest rank. This permits the comparison of predictive power of potential risk factors – documented at baseline (1993) – for later occurring VTEs.This technique permits to classify persons by the discriminant function value (separated by the case status). The true incidence of new VTE cases was determined in strata of persons with different pattern of risk profile to characterize groups with lower or higher absolute VTE risk according their risk profile at entry. We used for this analysis those variables that depicted the highest 5 ranks in the stepwise discriminant analysis.All analyses were performed with the statistical packages SPSS 10.2, SAS 8.2 or STATA 8.2.The overall cohort encompasses 4337 women with sufficient information in 1993 and one follow-up at minimum. The observational period for our current analysis was 32,656 WYs since 1993.The follow-up was continued until 2003 at most, or was otherwise terminated at the time when the last contact was possible to get information about new conditions that may have had occurred. 2076 women could be followed up until 2002/3 (47.9 %), 595 (13.7%) women dropped out between 1999 and 2001, and the largest proportion of women dropped out before 1999 (38.4%). Thus, the follow-up period was censored some time before 2002/3 for approximately half of the cohort members.Thirty-four new cases of VTE occurred in the observational period. These cases were finally confirmed and categorized according to diagnostic certainty by an independent medical reviewer as definite (n = 31) or probable (n = 3). Cases with possible/potential VTE (n = 17) were excluded from further analyses because of low diagnostic certainty, i.e. it was not clear whether to classify them in the group "cases" or "non-cases".Out of the 34 definite or probable VTE cases 18 cases (= 52.9%) were associated with "clinical causes for VTE" and 16 (= 47.1%) were so-called "idiopathic" VTEs. The following previously described "acute clinical causes" for VTE the following were observed: 4 with previous VTE, 3 with pregnancy/delivery, 4 after accident, 2 after surgery, 3 with immobilization, and 2 after long travel.Table The mean age was 26 ± 8.6 years, however, for the dichotomized age variable we used as cut-off point 30 years resulting in strata that contained VTE cases in both age groups. The frequency of other conditions, family history of potentially relevant diseases, lifestyle pattern and genetic tests is provided in the table The stepwise discriminant analysis was used to rank relevant variables at baseline according to their power to predict the case and non-case status many years later. We initially used 7 clinically available, potential risk factors for VTE and 5 laboratory parameters. Later, we combined genetic markers (Factor V Leiden and Prothrombin mutation G20210A) together in one composite variable due to low prevalence in the cases: FVL (n = 4) and PTM (n = 2). We used only categorical variables in the model, i.e., dichotomized continuous variables.st degree relatives), age at baseline 1993, body mass index (BMI), factor V Leiden (FVL) or prothrombin mutation (PTM), family history of varicose veins, protein C, AT level, hormone ever use, MTHFR carrier status, and OC ever use. We considered only the 5 highest-ranking variables for the development of our predictive model for practical use  in declining order: Medical history of VTE, family history of VTE , 12.3 (moderate), 47.2 (high), and 170.5 (very high).The majority of the study population 61.3%) had a small VTE risk  and usually based on clinical observations or case-control studies or studies in administrative databases, and also a few cohort studies . OneOther studies with focus on markers for hereditary thrombophilia were performed in patients (e.g. in anticoagulation clinics), in relatives of carriers of genetic mutations but not in the "normal female population" -18. In a4 WYs in OC non-users and 2 to 10 per 104 WYs in OC users [4 person-years [Our BATER cohort study covers more than 4,000 cohort members with a fairly long observation period (1993 – 2003), translating into over 32,000 WYs. Thirty-four VTE cases, classified as definite or probable, occurred within this period. This is equivalent to an incidence of about 10 per 10,000 WYs. It is important to realize that we put great effort on the detection of potential cases and – even more important – we included all definite and probable cases, whereas most reported incidence rates refer only to definite and so-called "idiopathic VTE", i.e. most reported rates excluded all cases that occurred in temporal relationship to possible "acute" causes such as pregnancy/delivery, surgery, and immobilization. Idiopathic VTEs, however, reflect only a part of all confirmed VTE cases . We founOC users  came to on-years .The objective of this study was to provide a simple algorithm for medical practice to predict the future VTE risk with a simple scheme based on usually available information, i.e. to discriminate women with virtually no VTE risk in the foreseeable future from those at a high absolute risk to suffer from VTE. Incidence rates associated with different clinical and genetic factors will be published separately .It is a limitation of this long-term cohort study, however, that the number of confirmed (definitive and probable), incident VTE cases was still too small in absolute numbers (n = 34). In other words, some sub-cohorts with certain combinations of risk factors did not contain one single new VTE case. The consequence was that the number of subgroups at risk was minimized to the extent possible to make it a feasible tool for the practice. In so far, the results and conclusions should be considered as rough but the best we can possibly do at this stage, i.e. future analyses will benefit from an improved point of departure . Another limitation is that we did not have the chance yet to test the validity of the model in another, independent cohort. This is a task for the future. Therefore we focused this paper on a simple scheme with a rough classification of the future VTE risk.The interested (or worried) women and her treating physician might like to know (or to get confirmation) whether the future VTE risk is higher than "normal" (no/low risk). This information could have an impact on further medical surveillance, especial counseling, proposals as how to reduce of changeable risk factors or on suggestions for preventive measures under certain circumstances and – of course- with respect to the compliance regarding preventive measures.Using stepwise discriminant analysis the rank order of 12 (11) clinical or laboratory data at baseline (1993) was multivariately determined concerning the power to predict future VTEs. This was the information needed to select a minimal set of parameter combinations to build a "VTE prediction model". Finally we ended up with a model covering the five variables with highest ranking (impact) regarding predictive importance for future VTE only: history of previous VTE, family history of VTE, higher age, higher body mass index, and carrier of FVL or PTM. The decision to form the composite genetic marker "FVL or PTM" was guided by the small numbers of cases who were carrier of this two mutations and the low predictive power of all other lab parameters we had in the data set (see table No/low risk (4 per 104 WY), moderate (12/104 WY), high risk (47/104 WY), or very high (171/104 WY). The low-risk group was chosen to reflect an assumed VTE risk of the normal population (see above), the group with "moderate risk" because VTE risk over 10/104 is indicative for an increased risk, and the "high and very high risk" groups are clearly out of the normal range. One should also consider in this context, that these cut-off points reflect an average risk with an assumed variation within these groups – as can be seen in the scheme of a decision tree  depicts a low risk of a future VTE. Only a minority of 6% and 0.9% is facing a high or very high VTE risk. The women who fall into the two highest risk categories have a previous own history of VTE or a positive VTE family history, have a higher BMI or a genetic mutation (FVL or PTM). Even though, the contribution of genetic appears to be limited. Using an analysis based only on clinically available data, i.e., without use of the information about lab parameters, we arrived at very similar three risk categories with almost identical absolute VTE risk  and are also labelled as risk factor in drugs containing sexual hormones .We assume that physicians will appreciate these results as a possibility to double-check if their own decision are supported by evidence coming from this large cohort study or may even alter their decision. At least the proposed model contains a reassuring element. It should be underlined that these algorithms do not obviate the need of weighing individual risks and benefits.We conclude from our observation that the prediction of future VTEs can well be done on clinical data alone – at least until better genetic markers are established. In other words, well-established genetic parameters alone are relatively weak long-term risk factors, the occurrence of VTE requires interaction of both inherited and acquired risk factors . ResultsForecast of VTE risk cannot be based on genetic characteristics alone but only in combination with important clinical data (acquired risk information). Genetic markers play obviously a limited role in the long-term prediction of VTE – at least in the age group under 50. Genetic markers together with these "personal characteristics" constitute the disposition. Family history of cardiovascular events, specifically venous events have to be taken into account. As described before and confirmed by our data the probability whether the disposition translates into an event is obviously more influenced by "personal characteristics" such as higher age, or higher BMI. However, there are obviously other important, more acutely affecting environmental factors such as immobilization, surgery, accidents, and treatment with drugs that influence coagulation. The latter factors can be used to reduce the risk as estimated by the model .4 per year and therefore very low for women who were using oral contraceptives as the average population. We like to stress that our study was rigorous in documenting the VTE diagnosis. We conclude that the data can be generalized for the female population in the fertile age range. In analogy, the incidence might be compared with results of a prospective, community-based cohort study [4 person-years .Another issue for discussion is the validity of our calculated incidence rates: The lowest VTE incidence level observed in this model was 4 per 10,000 WYs. Due to an active search for findings compatible with the diagnosis of VTE, the inclusion of definite and probable diagnosis as well as of so-called "non-idiopathic VTEs", the incidence figures were expected to be higher than in normal "medical statistics" or administrative databases as discussed above. The equivalent incidence rate for only definite and idiopathic VTE could be expected to be approximately 2 per 10rt study  that fouThe influence of other, more acutely acting risk modifiers – such as immobilization, surgery, long-haul flights, and use of drugs (e.g. OCs and other hormones) was intentionally excluded from this model. Only parameters that were available at baseline and likely to affect the long-term development were eligible for this prognostic model. We saw no possibility to introduce parameters in the model that may or may not operate later, shorter or longer during the observational period. In other words, only long-term characteristics at baseline  were initially included into the modeling. Other influential risk factors or preventive measures have to be considered when discussing activities to reduce a predicted increased risk in the medical practice. It was not the aim of the study and data are neither available to test the effect of preventive measures nor the effect of additional risk factors in the immediate period before the event occurred. This would require another study design and a separate study with sufficient power for such questions.The variables selected for the model fit the expectations of the skilled clinician. The model was developed to assist physicians- we hope for feedback from medical practice.This "prognostic model" seems to be worthwhile to be tested in clinical practice. There is a minority of women that would need additional genetic testing, intense counseling, suggestions for risk reduction (if possible), and efforts to prevent avoidable risk situations (e.g. treatment with OCs or hormones) or to take other appropriate preventive measures in situation of an acute risk , e.g. compression stockings/ heparin. Several other options to reduce the VTE risk profile are principally available, but sometimes not easy to achieve (e.g. reduction of BMI). Prevention of VTE in medical practice can be improved if the main risk factors are known , but also knowledge of their relative importance in the risk-network as well as of interactions with environmental factors. The predictive models discussed in this paper may assist doctors to pay particular attention to labeling prior to prescription  and to use convincing evidence-based data when counseling women of a predicted higher risk.We abstained – at the current stage – from the temptation to use a complex equation to calculate an apparently exact risk for the individual person  because it suggests inadequate accuracy and we rather prefer to provide a very simple scheme that can be handled during routine work. Moreover, we are planning a validation of this model in an independent cohort study, as the first step the part related to clinical risk factors.Our prognostic tool – containing clinical information  – seems to be worthwhile testing in medical practice in order to confirm or refute the positive findings of this study. Our cohort study will be continued to include more VTE cases and to increase predictive value of the model.The five authors from research institutes  and the two authors from industry  see no conflict of interest.LAJH: designed together with WS the cohort study and both are the principal investigators, LAJH planned all analyses, wrote a first draft of the manuscript. DMT: developed and maintained the database, performed the majority of analysis, and contributed to the manuscript. AA: responsible for running all field work, performing quality control and designing the validation of diagnoses, contributed to the manuscript. WS: one of the PIs, contributed to the manuscript. RS: responsible together with JH for interpretation of the findings, major contributions to the manuscript. JH see RS. MS: responsible for the haemostasiological lab work during the entire study period, contributed to the manuscript.


The will to eat is a decision associated with conditioned responses and with unconditioned body sensations that reflect changes in metabolic biomarkers. Here, we investigate whether this decision can be delayed until blood glucose is allowed to fall to low levels, when presumably feeding behavior is mostly unconditioned. Following such an eating pattern might avoid some of the metabolic risk factors that are associated with high glycemia.In this 7-week study, patients were trained to estimate their blood glucose at meal times by associating feelings of hunger with glycemic levels determined by standard blood glucose monitors and to eat only when glycemia was < 85 mg/dL. At the end of the 7-week training period, estimated and measured glycemic values were found to be linearly correlated in the trained group  but not in the control (untrained) group . Fewer subjects in the trained group were hungry than those in the control group (p = 0.001). The 18 hungry subjects of the trained group had significantly lower glucose levels (80.1 ± 6.3 mg/dL) than the 42 hungry control subjects . Moreover, the trained hungry subjects estimated their glycemia  more accurately than the control hungry subjects . Also the estimation error of the entire trained group (4.7 ± 3.6%) was significantly lower than that of the control group . A value of glycemia at initial feelings of hunger was provisionally identified as 87 mg/dL. Below this level, estimation showed lower error in both trained (p = 0.04) and control subjects (p = 0.001).Subjects could be trained to accurately estimate their blood glucose and to recognize their sensations of initial hunger at low glucose concentrations. These results suggest that it is possible to make a behavioral distinction between unconditioned and conditioned hunger, and to achieve a cognitive will to eat by training. The will to eat is a decision associated with conditioned responses and with body feelings reflecting changes in metabolic biomarkers. The body feelings are often described as hunger, but have components that are strongly conditioned by time, social, and metabolic factors, for which there are salient unconditioned physiologic correlates. Blood glucose has long been considered a biomarker of hunger . In exteBlood glucose has a central role in the regulation of energy metabolism. It provides energy to the brain, has limited and exhaustible storage, is regulated by the availability of other fuels, and its blood levels correlate with the time interval between spontaneously requested meals ,8-10. OuPrevious investigations have reported the use of hunger feelings with ,13 or wiThe investigation was carried out in patients with functional disorders of the bowel such as dyspepsia, abdominal pain, and diarrhea . Data frth experimental week.In this 7-week pilot study, 158 adults suffering from diarrhea, abdominal pain, and dyspepsia were recruited and randomized to experimental  and control  groups Figure . After 7Subjects in training measured capillary blood by glucometer  in the quarter-of-an-hour before intended meal consumption. Accuracy of measurements by the glucometer was validated at periodic laboratory visits with measurements by autoanalyzer on blood samples from the same subjects. In contrast, control subjects did not have their glycemia measured until the final laboratory session.On the first training day, subjects were told to ignore previous meal times and to pay attention to their feelings of hunger or discomfort. At the earliest feelings of hunger or discomfort, the subjects measured glucose concentrations with the portable instrument. This first event of hunger appeared after the training session with a time interval that varied widely from 0 up to 48 hours (average 2 h) and was often far from the usual meal times during the next 3 training days. This suggests that the recorded behavioral responses were largely spontaneous (unconditioned). Measurements obtained less than 1 h after a small amount of food consumption, intense physical activity, or changes in environmental temperature were excluded from the analysis. When glycemia was higher than 85 mg/dL, patients were instructed to delay or skip the meal, to engage in some activity as a distraction from food, and to wait for the spontaneous development of novel hunger feelings for at least one hour before making further blood glucose measurements. When glycemia was under 85 mg/dL, patients were instructed to remember their feelings and to proceed to meal consumption. The glycemic level of 85 mg/dL was chosen based on the hypothesis, suggested by previous studies, that it represents the upper limit of homeostatic control of feeding ,11-14. TAt the final investigative session, the subjects returned to the laboratory, stated whether they were hungry or not hungry, and estimated their glucose concentrations before blood sampling and before breakfast. Control subjects had ignored the relation between glycemia (referred to the subjects as "nutrient levels") and feelings of hunger up to the final session. They were asked to estimate their glucose levels referring to a range of values that could vary between the extremes of 60 mg/dL during intense hunger and 110 mg/dL after a satiating meal. Blood was sampled, centrifuged immediately, and analyzed in duplicate. The subjects were then free to eat food that they brought from home or from the hospital cafeteria under the observation of an investigator.t-test on difference and analyses of simple, linear correlation (r = correlation coefficient in linear regression), agreement limits and estimation error between the estimated and measured values of glucose. The estimation of error was calculated as the mean ± SD of the absolute values of differences from the reference measurement. The significance of difference and correlation was analyzed by two-tailed t-test analysis and Yates test, and was set at p < 0.05 when one difference was analyzed between two groups and at p < 0.025 when two differences were analyzed between the same two subject groups, e.g. measured glycemia and estimation error . Subjects showed an estimation error lower than ± 4 mg/dL after less than a week of training (n = 8) or within the first two weeks of training (n = 47). The remaining 9 subjects either reached an estimation error lower than ± 4 mg/dL in > 2 weeks or still showed an estimation error higher than ± 4 mg/dL at the end of the 7-week-training.At the final session, the number of trained subjects stating that they were hungry (18 of 64) was significantly lower than that of hungry control subjects  in the hungry trained group  was significantly lower than that in the hungry control group  described the subtle feelings they employed to estimate glycemic concentrations. Thus, compared to controls – who did not report equivalents of hunger (n = 30) – a significantly higher proportion of the 46 not-hungry trained subject (p = 0.001) was able to report feelings other than gastric hunger, which were useful in estimating their glycemic levels, and this ability prevailed below 87 mg/dL. In their reports, these 15 subjects described physical (3 subjects) or mental (10) weakness or abdominal changes in tension or movement (2). Another 6 of the 46 not-hungry trained subjects, but none of the control subjects, had felt gastric hunger before entering the hospital for the final session; however the feeling faded while waiting for the laboratory session.In the not-hungry subjects' reports, the feelings of mental weakness consisted of difficulty in sustained mental concentration, impatience, irritability, drowsiness, gnawing feeling, loss of enthusiasm and effectiveness at mental work, or poor mood at their jobs. The mental feelings emerged alone or in addition to gastric or other feelings and ceased with the meal. Sensing impairment during physical activity was associated with heavy physical exercise outdoors and often accompanied a change from a sedentary life style. This feeling was used regularly to indicate meal signal with an increased requirement of high-energy-dense food for the next meal(s). The prevalence of these 'hunger equivalents' ranged from an occasional occurrence to less than 15% of the meals in the phone reports. Two subjects reported that they never felt (gastric) hunger, but estimated glycemic concentrations within 6% estimation error always by assessing mental or muscular weakness during training or during the final investigative session. In their reports, these subjects consumed meals at glycemic estimation of 78 to 85 mg/dL.At the final laboratory session, the 64 trained subjects showed a decrease of 43.1% in reporting hunger events before breakfast compared to the reported events of the previous week (Table red symbols). In contrast, control (untrained) subjects were unable to recognize their glycemic levels at meal times  and expressed the will to eat at a wide range of glycemic values.The main result of this study is that adult individuals can be trained to accurately estimate their glucose levels at meal times. This cognition was achieved by conditioning the subjects to associate feelings of hunger with low glucose concentrations  that food consumption at high glycemic concentrations in control subjects may lead to higher energy intake than in trained subjects -14, and a levels ,14, and a levels ,22.The collected evidence supports the interpretation that trained subjects learned to recognize the unconditioned feelings of hunger. The training in this study was intended to cut off excess food consumption, i.e. caloric intake occurring at high > 85 mg/dL) glycemic concentrations, through conscious exposure of the subjects to the initial sensations of hunger arising when glycemia declined below 85 mg/dL. A 7-week period with association of estimated and measured glycemic values repeated 3 times per day was sufficient to train the subjects to accurately recognize their glycemia  and unexpectedly during working or entertaining activities, and persisted for at least 1 h. Third, at the final laboratory session, the number of trained subjects that recognized the appearance of sensations of initial hunger similar to those experienced during the training at low glycemic concentrations was significantly lower than in the control group /square height (m)r: linear correlation coefficientThe author(s) declare that they have no competing interests.


Our goal was to identify subgroups of sib pairs from the Framingham Heart Study data set that provided higher evidence of linkage to particular candidate regions for cardiovascular disease traits. The focus of this method is not to claim identification of significant linkage to a particular locus but to show that tree models can be used to identify subgroups for use in selected sib-pair sampling schemes.We report results using a novel recursive partitioning procedure to identify subgroups of sib pairs with increased evidence of linkage to systolic blood pressure and other cardiovascular disease-related quantitative traits, using the Framingham Heart Study data set provided by the Genetic Analysis Workshop 13. This procedure uses a splitting rule based on Haseman-Elston regression that recursively partitions sib-pair data into homogeneous subgroups.Using this procedure, we identified a subgroup definition for use as a selected sib-pair sampling scheme. Using the characteristics that define the subgroup with higher evidence for linkage, we have identified an area of focus for further study. Determining the underlying genetic basis in a complex disease challenges researchers due to the presence of multiple genes with modest effects, known and unknown environmental factors, and possible gene × gene and gene × environment interactions. In contrast, a homogeneous data set provides an optimal situation for identifying genes involved in a particular disease's etiology because confounding factors, either genetic or environmental, do not mask the power to detect linkage. Recursive partitioning or tree-based models  have Cardiovascular disease (CVD) is a prime example of a complex disorder with numerous disease-related traits, determined by multiple genes and environmental factors, in which recursive partitioning can potentially aid in the detection and localization of the underlying genes. In this paper, we apply a recursive partitioning splitting rule developed by Shannon et al.  to assesThe Framingham Heart Study data set consists of two longitudinal cohorts with information relating individuals across both sets. In the interest of brevity, we refer the reader to published descriptions of the data in this issue and in the literature ,6. Previibd) for all loci on chromosomes 17 and 20 using the program ibdn (a modified version of ERPA . For the categorical variables we created sib-pair variates that count the number of sibs in the pair that fit a given condition: number of males (psex), number with high blood pressure (phbp), and number on hypertensive medication (phrx).The recursive partitioning method developed by Shannon et al.   has been programmed as an extension to the RPART  function in S-PLUS [Shannon et al.  describen S-PLUS . Simulatn S-PLUS  show tha2 pruning rule for association tests in which the χ2 statistic at each node is compared to a χ2 critical value from a preset alpha level. If the χ2 statistic at the node and subsequent nodes is not significant, the node is pruned. We propose an analogous pruning rule based on the H-E sib-pair linkage test to prune the trees we obtain using the previously described methodology. Since the H-E method is a simple linear regression with the null hypothesis that the regression slope equals zero versus an alternative hypothesis that the slope is negative, a t-statistic can be calculated for the regression coefficient at each node and compared to a predetermined one-sided significance level. If the t-value at the node and subsequent nodes is greater than the critical value then we prune the branch. We call this the minT pruning rule.Tree-based models follow the same rule as all modeling procedures: a balance between parsimony and good fit. Therefore, large trees need to be pruned in order to make better interpretations. Traditional pruning rules  prune ofsqsbp or sqtrig onto ibd at each marker on chromosomes 17 and 20 and based splits on the constructed sib pair covariates. We utilized the Shannon method splitting rule and defined a set of parameters for the splits: 1) a node must have at least 150 sib pairs to be considered for a split, 2) at least 100 sib pairs were required for each terminal node, and 3) surrogate splits were permitted to allow for missingness. We set the complexity parameter equal to zero to allow each tree to grow to its full size and then pruned the tree back using our proposed minT pruning procedure to remove branches containing nodes with a t-value greater than -1.645 .We constructed regression trees, one per marker on chromosomes 17 and 20, using the data from the FHS with 10-fold cross-validation. We used H-E regression as our response, in which we regressed We conducted tree-based analysis on regions around candidate loci located on chromosomes 17 and 20. Figure sqsbp onto ibd for the 11 markers on chromosome 20 (trees not shown). Sib pairs in which neither sib had high blood pressure showed higher evidence for linkage across the chromosome. For example, at marker D20S482 located at 12 cM, the node with increased evidence of linkage identified a subgroup of same-sex sib pairs for which neither sib had high blood pressure, average age of the sibs was less than 32 years, and on average they smoked less than 4 cigarettes per day and had average triglycerides less than 75. Xu et al. [We further constructed trees for the H-E regression of u et al.  reportedu et al.  reportedsqtrig onto ibd for markers D20S604 (33 cM) and D20S470 (39 cM) on chromosome 20. Subgroups of sib pairs with average cholesterol greater than 196 and less than 140 were both identified from the trees. For marker D20S604, sib pairs who had average cholesterol greater than 196, where each sib drank less than one drink per day on average and who had an average HDL around 50 displayed highest linkage. A similar subgroup was identified in the tree for marker D20S604, except the average age of the sibs were greater than 43 years. The subgroup with the highest evidence for linkage for marker D20S604 contained sib pairs that had average cholesterol less than 140, each sib drank less than two drinks per day on average, and the average age of the sibs was greater than 46.We subsequently constructed trees for the H-E regression of Data mining methodologies have gained popularity in the field of genetic epidemiology. We applied the tree-based methods described above to identify subgroups in the FHS data set that provide more evidence for linkage. We do not claim identification of significant linkage to this region on chromosome 17 in FHS. Rather, we suggest that identification of pairs in subsequent studies with similar characteristics would enhance the power to detect linkage. Furthermore, Shannon et al.  proposedIn this investigation, we constructed linkage trees at each marker on chromosomes 17 and 20. A more optimal approach would have been to construct multipoint regression trees. However, at the time of this investigation, the methodology had not been developed to handle this type of analysis. Therefore, we chose to complete our investigation one marker at a time. One might be concerned that investigating a tree for every marker represents a risk of multiple testing issues. Multiple testing is regarded as a gray area in genetic analysis, and also for tree modeling. However, our goal was not to determine true linkage to a trait, but to show that tree modeling can be used to refine selected sib-pair sampling. Subdividing sib pairs for linkage analysis is valuable because different subgroups of sib pairs are useful under different genetic models . We alsoWe are in the process of completing secondary analyses using SOLAR  to asses


Loads acting on scoliotic spines are thought to be asymmetric and involved in progression of the scoliotic deformity; abnormal loading patterns lead to changes in bone and disc cell activity and hence to vertebral body and disc wedging. At present however there are no direct measurements of intradiscal stresses or pressures in scoliotic spines. The aim of this study was to obtain quantitative measurements of the intradiscal stress environment in scoliotic intervertebral discs and to determine if loads acting across the scoliotic spine are asymmetric. We performed in vivo measurements of stresses across the intervertebral disc in patients with scoliosis, both parallel  and perpendicular  to the end plate, using a side mounted pressure transducer (stress profilometry)Stress profilometry was used to measure horizontal and vertical stresses at 5 mm intervals across 25 intervertebral discs of 7 scoliotic patients during anterior reconstructive surgery. A state of hydrostatic pressure was defined by identical horizontal and vertical stresses for at least two consecutive readings. Results were compared with similar stress profiles measured during surgery across 10 discs of 4 spines with no lateral curvature and with data from the literature.Profiles across scoliotic discs were very different from those of normal, young, healthy discs of equivalent age previously presented in the literature. Hydrostatic pressure regions were only seen in 14/25 discs, extended only over a short distance. Non-scoliotic discs of equivalent age would be expected to show large centrally placed hydrostatic nuclear regions in all discs. Mean pressures were significantly greater (0.25 MPa) than those measured in other anaesthetised patients (<0.07 MPa). A stress peak was seen in the concave annulus in 13/25 discs. Stresses in the concave annulus were greater than in the convex annulus indicating asymmetric loading in these anaesthetised, recumbent patients.Intradiscal pressures and stresses in scoliotic discs are abnormal, asymmetrical and high in magnitude even in the absence of significant applied muscle loading. The origin of these abnormal stresses is unclear. The aim of this study was to obtain measurements of pressures and stresses in the intervertebral discs of scoliotic patients in order to try to prove the hypothesis that asymmetrical loading was present in the scoliotic spine and hence could realistically be involved in curve progression. We were also very interested in the stress environment of the intervertebral disc in scoliosis in general. We considered that recording from discs prior to excision during anterior scoliosis reconstructive surgery involved minimal additional patient risk. However, in anaesthetised patients with additional muscle relaxation  no loading due to muscle activity is present. We therefore were unsure if our approach would show any meaningful results.Previous authors have theorised about the biomechanical role of asymmetrical loading in progression of the scoliotic deformity -3. Non-oThe cellular mechanisms by which abnormal loading may generate deformity has been shown previously in cell culture and in animal models. Cells of bone and disc are very responsive to mechanical stress,15, withMost of the information on loads acting on the scoliotic spine is derived from measurements of changes in spinal morphology and from modelling,24. TherPatients having anterior surgery for thoraco-lumbar scoliosis were considered for inclusion in this study. The control group was patients without scoliosis having anterior surgery involving total disc excision. These were patients undergoing kyphosis surgery or anterior lumbar surgery for back pain. They were chosen as a comparison group to the scoliotics because of the lack of lateral curvature at the disc levels operated on. Ethical approval was obtained from the Oxford Regional Ethics Committee. Written and verbal information about the study was given to patients 4–6 weeks before surgery. In those patients under the age of 16 years, the parents' consent was obtained as well. Details of the patients and controls tested are given in tables The pressure transducer used in the study was the same type as that used previously for pressure profile measurements -28. It hThe transducer was calibrated up to 1 MPa using a pressure vessel(a tube of circular cross section) with a known cross sectional area filled with water to which known loads were applied by a servocontrolled hydraulic materials testing machine . The rate of application of load during calibration was approximately 0.2 MPa/s. Over the range 0–1 MPa the voltage recorded was found to be linear with respect to pressure applied. Re-calibrations of the transducer showed that it was very stable and showed negligible drift over time. Before surgical use, the pressure transducer was sterilised using Perisafe  according to standard hospital protocols.In all the scoliosis and kyphosis cases the spine was exposed by a conventional transpleural and retroperitoneal approach, dividing the diaphragm close to its peripheral attachment. In the back pain patients, a retroperitoneal approach to the low lumbar spine was performed. Our normal practice is to insert spinal needles into exposed discs and to check the anatomical level by taking an x-ray. In these scoliosis and control patients, one of the needles was substituted by the pressure transducer. It was inserted through the convex annulus and the transducer pushed across the disc to the concave annulus Figure , the patThe initial position of the transducer/electrode tip and of the transducer membrane in each disc was assessed from the intra-operative X-ray film. These were scaled using the known diameter and length of the pressure transducer. The disc dimensions were obtained from the same X-ray. Results are given as the position of the transducer diaphragm relative to the concave border of the disc.Chart software  was used to convert the voltages obtained into stresses/pressures in accordance with the measured calibration coefficient. The data were then exported to Microsoft Excel for further analysis.Where appropriate, measurements are presented as means and standard deviations in n discs where n is the number of discs tested.In this study, pressure profiles were measured in 25 scoliotic and 10 non-scoliotic discs from 7 scoliotic and 4 non-scoliotic patients over a period of one year (July 2001 – July 2002). Details of the scoliotic patients in the study are given in Table The stress profiles generally showed asymmetries between the concave and convex sides of the curve for both the vertical and horizontal stresses.A peak in the vertical stress recorded in the concave annulus (far left side of the profile) was a common feature of the profiles in the scoliotic discs in this study, a typical example being shown in figure Previous pressure profiles measured in non-scoliotic, non-degenerate discs of similar age to the scoliotic patients show large hydrostatic central areas ,26 In maIn 5 cases, peaks in the convex annulus also occurred. On only 2 occasions was this higher than the maximum vertical stress in the concave annulus in the same disc.We analysed the pressures recorded in those scoliotic discs that showed hydrostatic regions. The hydrostatic pressure values varied between 0.1 and 0.43 MPa; the mean value was 0.25 ± 0.10 MPa (SD). The values for each patient in relation to the apical disc are shown in figure Hydrostatic disc pressures in the apical disc were not obviously higher overall or higher than at adjacent levels and the apical discs had more hydrostatic nuclei 6/7 (86%) compared with 10/18 non-apical discs (56%).We found a slight trend towards higher mean pressure progressing caudally for absolute disc level, shown in figure We analysed the maximum value of the stress in the concave annulus. No readings were obtained in the concave annulus of the T10/11 disc of patient 4 hence this disc is not represented. Stress levels were very variable, ranging from 0.1–1.15 MPa. For all discs where a recording was made in the concave annulus, the mean stress was 0.55 ± 0.28 MPa (SD), The magnitude of the peak vertical stresses recorded in the concave annulus for each of the scoliotic patients relative to apical disc level is shown in Figure The stress change across the disc was calculated from the first reading shown to be taken inside the anatomical disc minus the last reading taken before the transducer exited on the convex side. On some occasions, the last reading was probably out of the disc . In 18/24 discs, this value was positive indicating that higher stresses were found on the concave side of the curve. In only 2/7 patients was the stress positive and greater in the apical disc than adjacent discs. In 7/25 discs, stress was higher in the concave annulus than the convex without a concave stress peak being present.The stress change across each of the scoliotic study discs is shown in Figure Due to the low numbers of patients who both were having anterior surgery for non-scoliotic conditions and who consented to having pressure measurements performed during the time period of the study, we only have pressure profiles from 10 discs for comparison.Table Profiles showed significant variability in this group of patients.Patient P10 who was 26 years old showed a pressure profile with higher stresses towards the posterior annulus, a hydrostatic region and a trough of horizontal pressure in the anterior annulus. In both discs of patient 11, stresses were essentially zero throughout apart from an increase in vertical stress in both and a horizontal stress peak in the anterior annulus of the L5/S1 disc with no hydrostatic regions.In the kyphotic patients, patient 8 who had quite a flexible curve clinically showed profiles with pressure peaks mainly in the anterior annulus with an under pressurised central region. The T11/12 and L1/2 levels showed hydrostatic nuclei. Patient 9 who was a kyphotic with a curve that was noted to be very stiff intra-operatively had very narrow discs and made introduction of the pressure probe difficult. Only a few recordings per disc (mean of 2.75) were made and so profile shape could not be accurately determined.5/10 (50%) non-scoliotic discs showed no regions of hydrostatic pressure.For the patients with back pain, hydrostatic regions were seen in the L4/5 disc of patient 10 (0.214 MPa). The central regions of both discs in patient 11 showed negative pressures very close to zero level  and the horizontal and vertical pressures were very close in value over these regions. In the kyphotic patients, only the T11/12 and L1/2 discs of patient 8 showed hydrostatic regions with a mean of 0.030 ± 0.022 MPa. For patient 9, there were no hydrostatic regions however in order to indicate the general size of the stresses present, the mean vertical stress was 0.327 ± 0.33 MPa and horizontal was 0.627 ± 0.46 MPa over all 4 recorded discs.5/6 (83%) non-scoliotic discs showed stress peaks in either the anterior or posterior annulus (data from Patient 9 was disregarded due to too few data points per disc) with the T11/12 disc of patient 8 showing both anterior and posterior peaks. Of these stress peaks, 5/7 (71%) were anterior.All the non-scoliotic discs showed some stress difference across the disc. It was very variable in magnitude ranging from .057 to 1.33 MPa with a mean of 0.35 ± 0.39 MPa  we have also compared our results with intradiscal pressures measured in Japanese subjects with back pain prior to laser nucleotomy. In thisIn our study, the mean value of hydrostatic pressure for the scoliotic patients was 0.25 ± 0.10 MPa, > 3 fold higher than that measured in Yonezawa's patients.Figure We present new data on intra-disc pressure profiles of patients with thoraco-lumbar scoliosis. The overall hypothesis we were attempting to test in this study was that abnormal, asymmetrical loading was present in the scoliotic spine and contributed to the progression of the scoliotic deformity. However, due to the surgical positioning and anaesthetised state of the scoliotic subjects we were unsure whether any physiologically significant pressure readings would be obtained since the majority of loading across the motion segment is due to body weight and muscle activity -32. We wRather surprisingly, the readings obtained in the scoliotic discs were much higher in magnitude than we expected. The mean pressure reading in those discs with a hydrostatic nucleus (14/25) was 0.25 ± 0.10 MPa which was on average >3 fold greater than in the available control data Figure .Despite the lack of muscle activity, our results also showed evidence of asymmetrical loading in the scoliotic spine. Stresses were higher in the concave than in the convex annulus in 18/25 discs Figure . We did A number of scoliotic and non-scoliotic discs in this study had non-hydrostatic regions, 44% of scoliotic and 50% of non-scoliotic. For the nuclear matrix to behave in a non-hydrostatic manner it must be abnormal in composition or relatively dehydrated. This would be most likely due to glycosaminoglycan loss or potentially, mechanical loss of fluid due to chronic high loading. With regards to the non-scoliotic discs, the back pain patients would be expected to have degenerate discs with glycosaminoglycan loss. Patient 11 was known to have severe degeneration on MRI imaging and hence showed disc pressures close to zero and non-hydrostatic discs. Patient 10 was younger and had less severe degeneration consistent with higher pressures and a hydrostatic disc. For the kyphotic patients, patient 8 was a severely disabled, paraplegic from T4 distally due to invasion of the spinal cord by tumour. She had flexible kyphosis clinically and the T12/L1 level was non-hydrostatic presumably due to secondary degenerative change at that level. Patient 9 had a very stiff curve and almost complete loss of disc height at all levels and severely dehydrated discs. With regards to the scoliotic discs however, non-hydrostatic discs are less easily explainable. Glycosaminoglycan loss in not know to be severe in the scoliotic disc and althThe pressures we measured in discs with hydrostatic regions in the scoliotic patients were considerably higher than those measured in patients with kyphosis and with chronic low back pain, which were on average low and of similar magnitude to that reported by Yonezawa. In addiThe finding of high pressures and stresses in recumbent, anaesthetised scoliotic patients with minimal loading due to muscle activity was unexpected and its origins are unclear.The internal mechanical environment of the intervertebral disc is complex. The factors influencing disc pressure/stress at any point in the disc will arise from both intrinsic factors, viz. disc swelling pressure and matrix organisation and extriWith regard to externally imposed forces, body weight and muscle forces, though altered in scoliotic patients, should Ligamentous tethering or changes in annulus organisationcould be As well as differences in pressure levels, there were also profound qualitative differences between the pressure profiles measured in the study scoliotic discs during surgery Figure  and 5 anThe origin of disc stress peaks has been discussed previously in the literature,27 in noIn 18/24 discs, higher stresses were found on the concave side of the curve compared to the convex indicating asymmetrical loading. This is obviously not explainable by asymmetrical muscle loading since the spinal muscles were relaxed in these patients. The simple presence of concave annular stress peaks, due to the possible previously mentioned factors, could lead to a stress gradient across the disc. However, even in some discs without a defined stress peak, asymmetrical loading was present.c. 1.0 MPa, Figure This study has found that stresses in scoliotic discs are abnormal. Scoliotic discs in recumbent anaesthetised, muscle relaxed patients have higher nuclear hydrostatic pressures than those measured in non-scoliotic discs in this study and reported in the literature. In 18/24 discs from patients, a stress gradient from concave to convex sides of the disc was measured, with some very high differential stresses (Without the presence of muscle activity, these findings are very surprising and intriguing. Previous studies have implied that asymmetrical muscle loading is the origin of asymmetrical loading which via changes in cellular activity and hence asymmetrical growth causes a "vicous cycle" of progression in an otherwise physiologically normal spine,58. The AM carried out the pressure measurements in vivo, analysed the data and drafted the manuscript. DJ and MS were responsible for setting up and validating the pressure measurements in vitro and providing technical help. JF provided the clinical overview and expertise. JU and JF conceived the study, were involved in the study design and co-ordination and helped draft the manuscript. All authors read and approved the final manuscript.