Cigarette smoking takes a staggering toll on human health and attracts considerable public health attention, yet real solutions seem distant. The 2004 Family Smoking Prevention and Tobacco Control Act (US Senate bill S2461) would have given the US Food and Drug Administration limited authority to regulate cigarettes to "protect the public health." However, such legislation is unlikely to substantially reduce smoking or related deaths.The past 500 years of tobacco control efforts demonstrate that nicotine prohibition is a practical impossibility for numerous reasons, state revenue being one of the most ominous. The FDA already has regulatory authority over pharmaceutical grade nicotine products, and requires pharmacists to dispense the most addictive of these only with prescriptions. Meanwhile, every corner store can sell far more addictive and dangerous cigarettes to any adult. The FDA could immediately increase competition between cigarettes and clean nicotine products by approving available nicotine products for over-the-counter sales to adults. Similarly permissive regulation of cigarettes and addictive nicotine products will reduce tobacco use and improve smokers' health, but increase nicotine use in the population. Fortunately, restricted youth access and accurate labeling of nicotine's absolute risks will dissuade many non-smokers from experimenting with it, while accurate depiction of its risks relative to cigarette smoking will encourage many smokers to switch. The FDA could take a series of small steps that might ultimately replace a large proportion of cigarette smoking with equally addictive nicotine products, without risking serious public health setbacks. Vaccine, methadone, and injury prevention policies establish relevant public health precedents.Cigarettes, or an equally addictive alternative, will be a permanent and common product in most societies. Regulations restricting only the safest addictive nicotine products are hard to justify. Addictive nicotine compliments other tobacco control strategies. Modern tobacco control policies are applicable to addictive nicotine. Controlled trials and test market studies are urgently needed to evaluate addictive nicotine as an alternative to smoking. Meanwhile, legislators should preserve the Food and Drug Administration's option to permit non-prescription sales of addictive nicotine. Cigarette smoking is a source of worldwide misery ,2 and reThe recently defeated  2004 FamA complimentary strategy called "harm reduction" proposes that smokers could improve their health by frequently substituting less hazardous tobacco products, such as 'smokeless cigarettes,' chewing tobacco, and newer smokeless tobacco products for cigarettes -14. 'SmoSome tobacco control advocates suggest a third strategy: national policies encouraging competition between safer nicotine products and cigarettes ,23-34. TThere are different ways to level this playing field. At one extreme, the FDA could increase regulation of cigarettes to match that of nicotine products. This is politically challenging, as the FDA discovered in the 1990's , The FamAt another extreme, the FDA could regulate other nicotine products more permissively, as cigarettes have been regulated. The FDA has that authority, but would face criticism for facilitating a common addiction. For instance, the FDA could approve non-prescription nicotine nasal sprays and slow acting inhalers for indefinite use by adults. These nicotine replacement therapies have excellent safety records and low potential for addicting non-smokers, although a few ex-smokers become addicted. The widespread misconception that nicotine prohibition is practical and desirable makes it politically difficult for the FDA to pursue this path.A recent Lancet editorial called for the criminalization of tobacco products . HoweverThe first problem is that historically, smokers accept personal and public hazards that make the dangers we associate with tobacco look quaint. After Christopher Columbus failed to control his crew's tobacco use, monarchs from England to China tried to contain the weed by execution, disfigurement, exile, and onerous taxation . TobaccoThe second problem is that risk-tolerant smokers are no small group. Smoking persists where Mormon and Islamic prohibitions discourage it ,49. If 7The third problem is a common genetic predisposition to nicotine addiction . The modThe fourth problem is that so much money is involved. When state and local taxes raised New York City cigarette prices to US$7 per pack, tax revenues and black marketing both increased . During The fifth problem is that government taxation compromises tobacco control efforts. Historically, States willingly trade citizens' health for wealth. Seventeenth century monarchs who opposed smoking relented as tobacco tax revenue accumulated. The States demonstrated the same perverse values with petitions to protect their MSA payments when an Illinois court threatened Altria with a US$12 billion bond . The StaThe sixth problem is the lack of a popular mandate for prohibition. Tobacco control advocates in the United States are rightfully pleased with smoking's declining prevalence, reduced teen smoking, rising cigarette prices, spreading restrictions on public smoking, and fire-safe cigarette initiatives. However, none of this demonstrates popular support for prohibition. Citizens will balk at limiting the supposedly personal choice to smoke in private. Midwesterners have already rejected modest cigarette tax hikes . FurtherThe seventh problem is that nicotine use could have a favorable risk-benefit profile for some informed users. Nicotine causes a mild euphoria without intoxication, in contrast to more tightly regulated drugs: it does not destroy relationships as intoxicating drugs routinely do. Many smokers may use nicotine to treat various problems -60 incluNicotine accounts for very few of the long-term hazards of smoking. A smokeless tobacco proponent has likened nicotine's risks to the risks of consuming caffeine . Fetal eIn summary, however desirable tobacco prohibition may be, it is hopelessly impractical – unless smokers, governments, and producers have an equally satisfying alternative.Legal and regulatory pressures have prevented the development of meaningful alternatives to cigarettes. Directed to approve drugs as "safe and effective" for specific indications, and to ignore tobacco, the FDA has had little reason to approve chronic, addictive nicotine. Although the FDA approves more dangerous drugs for specified indications, without an indication there is no benefit to weigh against any nicotine risk. Consequently the FDA requires prescriptions for slow acting inhalers and nasal spray because of a small risk of inconsequential addiction in ex-smokers. Unfortunately, seemingly risk averse regulation of nicotine forces public health policies to rely on difficult smoking prevention, cessation, use restrictions, and treatment. The net effect is a risky public health policy, and very slow development of new nicotine delivery systems . AnotherTort threats have also delayed product improvement efforts within the industry. In 1963 an industry lawyer, anticipating condemnation in the 1964 Surgeon General's report, suggested competing on safe nicotine delivery, but was overruled . ProductIn the mid 1960's, motor vehicle accidents were also taking a terrific health toll in the United States. While alternatives to driving existed, no one expected private automobiles to disappear. Instead, systematic efforts transformed traffic safety. Haddon's matrix illustrated how complimentary strategies could work together to reduce the morbidity and mortality of driving . Table 1italicized) [For tobacco control, smoking replaces accidents as the focal event (Table licized) . As withlicized) ,97. but licized) . AvailabFor the reasons outlined above, the health benefits of addictive pharmaceutical grade nicotine products would likely outweigh the harms. If so, the FDA could improve public health by regulating nicotine much as the government has regulated cigarettes.A permissive nicotine regulatory policy would allow sales of pharmaceutical grade nicotine delivery systems to adults without a prescription. The FDA could suggest warnings appropriate for classes of products and delivery system constituents. Manufacturers would accurately label contents, and would be legally liable for undisclosed harms caused by the delivery system and constituents other than nicotine, as with any pharmaceutical product. The FDA could prohibit inherently risky delivery systems, and would undertake a full, traditional review of nicotine systems that give users a faster or higher peak arterial level of nicotine than cigarettes. The FDA would also review additives intended to provide antidepressant or other effects familiar to smokers ,100. YouStates could tax cigarettes and nicotine at different rates, in theory recovering expenses related to each product. This tax policy would discourage cigarette use, encourage switching to nicotine, and maintain some State revenue as cigarette sales decline. A satisfying, safe, legal, and affordable alternative to cigarettes would discourage black markets. The FDA and Federal Trade Commission could permit advertising of nicotine as an alternative to smoking, and monitor relative harm claims. Nicotine manufacturers would pay for monitoring of adverse health effects from their products until remaining health questions are answered.The FDA could implement a permissive policy in a series of simple, informative steps. First, the FDA could immediately approve non-prescription sales of the existing nicotine spray and inhaler, and similar competitors, with restraints consistent with the Synar amendment, the MSA, and the FCTC. In particular, it should require disclosure of known nicotine risks, such as addiction, invite comparisons to smoking risks, and forbid marketing to minors.Data collected during this first step will inform subsequent steps. Economists have shown that the price of nicotine gum and patches affects demand for cigarettes . This meIf experience with the current nicotine spray and inhaler is reassuring, the FDA can take a second step, to approve clearly addictive nicotine inhalers for non-prescription use. These fast acting inhalers will deliver nicotine in a powder  or aerosSeveral trends could develop that encourage smokers to switch to addictive nicotine inhalers, while limiting recruitment of non-smokers. Smokers will appreciate the healthier alternative to cigarettes, especially as they develop smoking related illnesses, while warning labels will discourage casual experimentation by non-smokers. Smoke-free workplace regulations would spread with less controversy, as fast acting inhalers give smokers a reasonable alternative. Employers might voluntarily forbid smoking to reduce health care and workman's compensation costs; reduce workplace fires; and increase productivity by eliminating both smoking breaks and withdrawal symptoms. Physicians might strongly encourage smokers to switch for their own health, primarily, but also for the health of family members. The government might permit more aggressive advertising of nicotine inhalers to further undermine cigarette smoking. Governments will probably find nicotine taxation irresistible, especially if their tobacco revenue declines. Nicotine taxation will raise the cost of experimenting with inhalers beyond some teenager's means. Communities might press smokers to switch so that fire departments, health care providers, and research funding agencies could shift resources to many other pressing problems. Ultimately, palatable inhaled nicotine products could finally allow governments to ban tobacco cigarettes.The public health benefits of these policy shifts could be substantial. We can estimate the long term relative public health burdens of different policies as the product of the risks posed by a delivery system, relative to cigarettes, and the fraction of the adult population using it. Using this formula, the current burden is more than 20% use × 100% risk = 0.2. Healthy People 2010 calls for 12% use × 100% risk = 0.12, about half of the current burden. If nicotine accounted for a surprising 10% of smoking risks, eliminating smoking by quintupling the prevalence of inhaled nicotine use – universal addiction – would cause 100% use × 10% risk = 0.1, a slight improvement over HP2010. A slightly less pessimistic scenario would be that all past and present smokers become inhaled nicotine addicts, but their risk is only twice that of smokeless tobacco users. This scenario is no more than 70% use × 4% risk = 0.028, about a quarter of the HP2010 burden. The most likely scenario is that nicotine risks are about 2% of smoking risks, and that policy changes will slowly shift the probabilities that smokers will attempt and succeed in quitting, that smokers will switch to nicotine indefinitely or use both nicotine and cigarettes, that ex-smokers relapse to nicotine use, and that non-smokers begin using nicotine. If the risks of inhaled nicotine are this low and if at least one smoker can switch to addictive inhaled nicotine for every 50 ex-smokers and non-smokers who start using nicotine, then public health will benefit from permissive nicotine regulation. One public health risk is that more people might begin or continue smoking cigarettes in the belief that inhaled nicotine will provide an easy escape path, but suffer irreparable harm before switching. We should quantify these probabilities through surveillance of the public's behavior and health consequences following each step toward more permissive nicotine regulation.Although S2461 was defeated, the continuing carnage will motivate similar legislative proposals. Tobacco policies must preserve the possibility of permissive nicotine regulation. Bad legislation could solidify the position of cigarettes, delay safer products, prohibit disclosure of relative harms, or otherwise interfere with market forces that ought to benefit nicotine addicts . The FDANicotine use will remain common indefinitely. Pharmaceutical grade nicotine is the safest known substance that could replace cigarettes, but inherently addictive products are required to compliment traditional tobacco control policies effectively. The FDA could apply restrictions, similar to those on cigarettes, to an increasingly addictive portfolio of nicotine products, requiring honest portrayals of absolute and relative risks, and expect a significant reduction in cigarette smoking and related illness with modest recruitment of new nicotine addicts at each step. A brief and focused research effort could solidify the already substantial justification for such permissive nicotine regulation. Meanwhile, federal legislation must not obstruct free market ideals of competitive innovation and informed consumption.The author(s) declares that he has no competing interests.The pre-publication history for this paper can be accessed here:


Surgeons seem to love publishing case series, which are of limited usefulness. How can we encourage them to do randomized clinical trials? Lancet, chastised much of current surgical research and, in particular, questioned the usefulness of the case series as a predominant form of communication among surgeons [In 1996, Richard Horton, editor of the Randomized controlled trials (RCTs) have become the pillar of clinical research. Such trials attempt to obtain an unbiased randomization of patients with respect to known and unknown baseline conditions and to assess the effects of an intervention. However, only a minority of surgical studies involve a valid randomization scheme. The case series remains a favored method of clinical investigation in surgery.Case series are easy to perform, require less resources in terms of personnel and funds, can be performed at a single center, and, for many surgeons, represent a means to illustrate their surgical method and skills. In many instances, case series also serve as valuable intellectual background for future clinical or scientific work. For example, consider Dennis Burkitt's report on jaw tumors in African children, Alfred Blalock's initial efforts in cardiac surgery, or, more recently, Starzl and colleagues' observations, in a small collection of patients, of donor leukocyte chimerism, whereby recipients acquire tolerance to foreign donor cells. In all three cases, the authors' work led to powerful shifts in our understanding of the biology and treatment of disease ,3,4. AllThere are many reasons why RCTs in surgical patients may be more difficult to perform than those in non-surgical patients. One of the most important—though least understood—is that the complexities of human disease in surgical patients makes them a more difficult group to study. Surgical patients are often heterogeneous in many more ways than non-surgical patients. So it would be inherently easier, for example, to study a new medication for generally healthy young adults with essential hypertension than a surgical technique for older patients with hepatic failure needing transplantation.In addition, while there may be value in studying patients from multiple centers, there may be important differences in the skill levels of different surgeons, either between centers or across the country. For example, the skill levels of surgeons in trials of carotid endarterectomy may be greater than those across the surgical community as a whole. This makes the applicability of some surgical RCTs to the wider community less certain than trials of medical therapies.So when it comes to surgical research, for both researchers and funding agencies, it is easier to grapple with a difficult, but ultimately soluble, basic science question than to face the uncertainty of clinical research. Investigators understand these implicit issues and trim their sails accordingly.Nonetheless, too much surgical work is conducted in the less rigorous format of the case series. What can and should be done to improve the rigor of surgical investigation? It would seem that improvements are required from within and beyond the surgical world.First, as Horner observed, and several eminent surgeons have since agreed, reforms must begin within the field itself ,5,6,7. BSecond, surgeons must establish firm and friendly relations with biostatisticians so that the latter may play a strong role in helping to develop adequately powered studies that can answer critical questions raised by new therapies and techniques. This is an especially acute need in an accelerating age of targeted therapies and disease biomarkers.Third, surgeons must re-engage in the clinical research enterprise and resume leadership roles in local and national clinical trials that involve surgical patients. In the United States, for example, an important step in this regard has been the establishment of the American College of Surgeons Oncology Group, which invites surgeons from all sectors, including private practice, to become active participants in well-designed, multi-institutional trials . SimilarFinally, similar to the pressures faced by their colleagues elsewhere in academia, surgeon clinician-investigators must be nurtured, protected, and valued by their colleagues and medical administrators. The financial health of academic medical centers relies heavily on the generation of clinical revenue, which in many centers falls disproportionately on the shoulders of surgeons. New paradigms for revenue generation and funding of clinical research are needed.Beyond the walls of the academic medical center, there also needs to be greater recognition of the value of scientifically sound surgical research and clinical investigation. However, the National Institutes of Health (NIH), the major source of biomedical funding in the United States, continues to convey a less welcoming attitude toward surgical research than toward other types of clinical or basic science,9.At the NIH, the principal instrument for performing peer review and making grant funding decisions is the study section, composed of about 10–20 members with expertise in a given field. There are few study sections devoted to surgically oriented clinical research and only two study sections (from among more than 100) in which surgeons make up even a reasonable minority of the committee members . In compFunding agencies need to recognize the importance of the surgical endeavor to modern medicine.Surgical research is also impeded by processes affecting other types of research as well. The number of researchers under 35 years of age receiving a first RO1 grant, the main NIH mechanism for external funding, in any field, is below 4%. The average age of initial funding for US physicians is about 44 years, and shows a trend toward advancing age that has progressed significantly in the past two decades. Thus, the NIH appears to reward experience and proven results very heavily, which may stifle innovation and likely serves as an innate barrier for younger physician-investigators contemplating research careers .To help correct for this worrisome trend, the NIH created the “K” award system—career development grants designed to help starting researchers gain the experience needed to compete for RO1 grants. However, nearly 40% of the clinicians who receive KO8 awards never apply for RO1 funding , which sFinally, outside the US, surgeons face similar, if not greater problems. This bodes poorly for countries where the cost of evaluating new therapies and technologies may be an unaffordable luxury. These challenges to the surgical research enterprise are therefore global issues and should merit the attention of surgeons, medical institutions, and funding agencies in all countries.http://nihroadmap.nih.gov) designed to provide “new pathways to discovery.” Clear, careful, scientific surgical investigation must be part of this roadmap, although it is not specifically mentioned. Outreach efforts to include surgeons in a variety of study sections should be made to ensure that important insights into the pathophysiology and treatment of disease, with which surgeons are concerned on a daily basis, are not overlooked. Additional efforts are needed to improve funding for clinical research, both for individuals at early stages of their careers and for multi-disciplinary clinical research and clinical trials. Locally, and individually, surgeons must join efforts to improve the clinical research enterprise by including training in clinical investigation at an early stage in medical school and during surgical residency training, fostering the careers of young surgeon-investigators through committed, protected time, participating in local and national clinical research groups, and recognizing that development as a clinical researcher takes time—many years in fact.What can be done? On the national and international level, funding agencies need to recognize the importance of the surgical endeavor to modern medicine. Recently, in the US the NIH unveiled a “roadmap” (These efforts may help ensure that surgical research is a vital part of the future of medicine and that it leads to the kind of high-quality work that shapes and remodels the face of medicine. To foster these efforts, surgeons must change and adapt to the currents of modern medical research. If this is successful, the case series will become the occasional rather than the common form of surgical communication. And surgeons, other clinicians, and, most importantly, basic scientists will be better able to take advantage of the new avenues of biomedical science opening before us.But the case series will always represent one important tool for early studies or uncommon conditions. It remains true that while the method one uses influences the answer one receives, it can be just as important to ask the right questions, which can be asked even in a series of one patient . And sur


The assumption that lifestyles formed early in life track into adulthood has been used to justify the targeting of health promotion programmes towards children and adolescents. The aim of the current study was to use data from the Northern Ireland Young Hearts Project to ascertain the extent of tracking, between adolescence and young adulthood, of physical activity, aerobic fitness, selected anthropometric variables, and diet.n 245) and females (n 231) were assessed at age 15 y, and again in young adulthood [mean (SD) age 22 (1.6) y]. At both timepoints, height, weight and skinfold thicknesses were measured, and physical activity and diet were assessed by questionnaire and diet history method respectively. At 15y, fitness was assessed using the 20 metre shuttle run, while at young adulthood, the PWC170 cycle ergometer test was used. For each measurement made at 15y, subjects were ranked into 'low' , 'medium'  or 'high'  categories. At young adulthood, similar categories  were created. The extent of tracking of each variable over time was calculated using 3 × 3 matrices constructed using these two sets of categories, and summarised using kappa (κ) statistics.Males (Tracking of diet and fitness was poor (κ ≤ 0.20) in both sexes, indicating substantial drift of subjects between the low, medium and high categories over time. The tracking of physical activity in males was fair (κ 0.202), but was poor in females (κ 0.021). In contrast, anthropometric variables such as weight, body mass index and sum of skinfolds tracked more strongly in females  than in males .The poor tracking of fitness and diet in both sexes, and physical activity in females, suggests that these aspects of adolescent lifestyle are unlikely to be predictive of behaviours in young adulthood. In contrast, the fair to moderate tracking of anthropometric variables, particularly in females, suggests that attempts to reduce the ever increasing incidence of overweight and obesity in adults, should probably begin in earlier life. Numerous epidemiological studies in adults have identified environmental and physiological risk factors that are associated with increased risk for cardiovascular disease (CVD). Among the many that have been identified , the majAlthough the clinically relevant effects of CVD are often not manifest until middle age or later, it is now generally well accepted that the disease is likely to have its antecedents in childhood -4. In adHowever, if health promotion interventions in younger life are to have any hope of success, it must be assumed that physiological and behavioural risk factors exhibited in early life track into adulthood. Tracking has been defined as the maintenance of relative position in rank of behaviour over time, such that subjects who rank highly for unfavourable risk profiles at a young age are likely to maintain their ranks through into adulthood ,11.The aim of the current study was to use data from the Northern Ireland Young Hearts Project to ascertain the extent of tracking, between adolescence and young adulthood, of selected modifiable risk factors for CVD. Specifically, we investigated the tracking of physical activity, aerobic fitness, selected anthropometric variables, and diet.n 251;12-year old girls, n 258; 15-year old boys, n 252; and 15-year old girls, n 254) randomly selected from post-primary schools. At that time, the resulting cohort represented a 2% sample of each of the two age populations in Northern Ireland. In 1992/93, a follow up study (YH2) was undertaken, in which subjects from the original 12 year old cohort were reassessed using procedures identical to those used in YH1. The response rate in the follow-up study was 90%. Between October 1997 and October 1999, all YH1 subjects were invited to participate in the third, hospital-based screening phase (YH3), and a 48.2% response rate was achieved. Reasons for the low response rate in YH3 have been described in full elsewhere . Ethical approval for each phase of the study was obtained from the Medical Research Ethical Committee of The Queen's University of Belfast, and written informed consent was obtained from all subjects prior to participation.The analyses reported in the current paper are restricted to males . Two measurements were taken at each site and the average was recorded. The sum of the four skinfolds thicknesses was then calculated for each subject.Each subject's height, weight and skinfold thicknesses were measured at all study timepoints. Standing height was measured to the nearest millimetre using a Harpenden portable stadiometer , and body weight was measured to the nearest 0.1 kg using an electronic balance . For both measurements, subjects wore light indoor clothing and no shoes. Body mass index (BMI) was then calculated as weight (kg)/ [height (m)]At all study timepoints, dietary data were obtained using the diet history method . This coAt age 15y, habitual physical activity was assessed by self-report questionnaire, and scored according to the method of . This meAs the school-based questionnaire was not relevant to the young adult subjects, a modification  of the B2max (ml/kg/min), the number of laps completed by each subject in this maximal endurance test was entered into a sex-specific regression equation, based on data obtained in the Northern Ireland Fitness Survey [Aerobic fitness at age 15y was assessed by the 20 metre shuttle test (20MST). In order to estimate maximal aerobic capacity, or VOs Survey .2max was assessed using the Physical Work Capacity at a heart rate of 170 beats per minute (PWC170) cycle ergometer test [2 in ml/kg/min), and this was used to estimate VO2max at the age-adjusted maximum heart rate [As it was not feasible to conduct the 20MST at young adulthood , VOter test . PWC170 art rate .All data were analysed using SPSS version 11.0.1 . Means and standard deviations were used to summarise the data for physical characteristics, aerobic fitness, physical activity scores and energy and macronutrient intakes of males and females at age 15y and young adulthood.Tracking of each of these variables over time was assessed by determining the extent to which subjects who were placed into low, medium and high categories at age 15y, maintained their ranking in young adulthood. Owing to the fact that different techniques were used to measure physical activity and aerobic fitness at each timepoint, a method based on ranks, rather than actual measurements, was employed for assessing the tracking of these variables. Tracking of the other variables was also assessed using the rank based method because of its relative simplicity, and its ability to show the numbers of subjects making the transition between low, medium and high categories . For exa2max assessed at young adulthood was significantly lower than at age 15y. At age 15y, males and females were 4.8% and 8.4% heavier than the British age-specific reference population, while at young adulthood, they were 7.9% and 9.9% heavier respectively. Details of the reference populations are described in Annex 1 of the 'Dietary Reference Values for Food Energy and Nutrients for the United Kingdom' [The physical characteristics, aerobic fitness and physical activity levels of the Northern Ireland Young Hearts cohort at age 15y and at follow-up (young adulthood) are summarised in Table Kingdom' .P 0.04), total fat  and total carbohydrate  than at age 15y. In contrast, intakes of protein  reported by males at young adulthood were significantly greater than at age 15y. Similar patterns were observed for females, with the exception that % energy derived from total carbohydrate did not change significantly between age 15y and young adulthood.The energy and macronutrient intakes reported by the Young Hearts cohort at age 15y, and at follow-up, are presented in Table 2max) was poor, but was greater than the κ value obtained for the females (κ 0.150 vs κ 0.076). A similar pattern was observed for physical activity scores (κ 0.202 vs κ 0.021).Table The extent of tracking of energy and macronutrient intakes reported at age 15y and at young adulthood is presented in Table The current paper describes the extent of tracking for a range of behavioural and biological risk factors for CVD, between adolescence (15y) and young adulthood (22y) in 245 males and 231 females from Northern Ireland.In relation to nutrient intakes, the poor tracking between 15 and 22 years revealed in this study reflects previous findings in this cohort between 12 and 15 years . This suWhile adolescence is widely regarded to be a time of transition, it could also be argued that young adulthood is an equally important time of change, especially with regard to dietary habits. This is a time when people in this age group are likely to move out of home, go to university, start a family or to encounter other environmental or psychosocial factors that influence food intakes. Thus it is possible that the poor maintenance of ranks that we have observed in this study has arisen simply because we attempted to assess tracking between two very unstable periods of time in the life-cycle.It is also possible that at least part of the explanation for poor dietary tracking in a cohort of this nature lies in the unsuitability of the diet history method for this purpose. Although the diet history method has shown good validity at the group level in adolescents, it is prone to significant problems of precision at the individual level . MoreoveBoth physical fitness and physical activity are now accepted as independent risk factors for several chronic diseases. The identification of low levels of fitness and/or activity at an early stage in the lifecourse might, therefore, enable early remedial strategies, provided that a degree of tracking for these risk factors is demonstrable. The results of the present study fail to provide such evidence, with poor tracking being demonstrated for fitness in both sexes, and poor tracking in females and only moderate levels of tracking in males for activity. By and large, these results are in keeping with the handful of other studies which have examined tracking of fitness and activity in this age group ,27. WhilIn contrast to diet and physical activity/fitness, anthropometric variables relating to body weight and adiposity showed stronger and consistent tracking, particularly in females. Good tracking of BMI from adolescence to young adulthood has been noted in previous studies ,24,33, wThe authors declare that they have no competing interests.CB conceived of the study, participated in its design and co-ordination and co-drafted the manuscript. PR supervised the collection of dietary data and co-drafted the manuscript. AG and GC carried out the statistical analysis. LM participated in the design and co-ordination of the project. JS conceived of the study and participated in its design and co-ordination. All authors read and approved the final manuscript.


In view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in gastroesophageal reflux disease, (GERD), we investigated the hypothesis that these changes result from a disordered myoelectric activity of the esophagus.The electric activity of the esophagus  was studied in 27 patients with GERD  and 10 healthy volunteers as controls . According to the Feussner scoring system, 7 patients had a mild (score 1), 10 a moderate (score 2) and 10 a severe (score 3) stage of the disease. One electrode was applied to the upper third and a second to the lower third of the esophagus, and the electric activity was recorded. The test was repeated after the upper electrode had been moved to the mid-esophagus.The EEG of the healthy volunteers showed slow waves and exhibited the same frequency, amplitude and conduction velocity from the 2 electrodes of the individual subject, regardless of their location in the upper, middle or lower esophagus. Action potentials occurred randomly. In GERD patients, score 1 exhibited electric waves' variables similar to those of the healthy volunteers. In score 2, the waves recorded irregular rhythm and lower variables than the controls. Score 3 showed a "silent" EEG without waves.The electric activity in GERD exhibited 3 different patterns depending on the stages of GERD. Score 1 exhibited a normal EEG which apparently denotes normal esophageal motility. Score 2 recorded irregular electric waves variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" EEG was recorded with probably no acid clearance. It is postulated that the interstitial cells of Cajal which are the electric activity generators, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The EEG seems to have the potential to act as an investigative tool in the diagnosis of GERD stages. Reflux esophagitis is a multifactorial disease that entails the reflux of gastric contents into the esophageal lumen . EsophagAbnormal esophageal motility results in an increased exposure of the esophageal mucosa to gastric contents. Progressing severity of the reflux disease is associated with failing primary peristalsis -9. The fPrevious studies have shown that the esophagus possesses electric activity presenting as slow waves (SWs) followed or superimposed by fast activity spikes or action potentials (APs) ,13. ActiIn view of the disturbed esophageal peristaltic activity and abnormal esophageal motility in GERD, we hypothesized that these findings result from a disordered myoelectric activity of the esophagus. This hypothesis was investigated in the current study.Twenty seven patients with GERD  were enrolled in the study. The diagnosis was confirmed by 24-hour pH monitoring, endoscopy and esophageal motility test. According to Feussner's scoring system , seven pThe study also included 10 healthy volunteers  who had no reflux esophagitis. They had no gastrointestinal complaints in the past or at the time of enrollment in the study.Physical examination of both the patients and healthy volunteers had normal findings. The results of laboratory work comprising blood count, renal and hepatic function tests as well as electrocardiogram were unremarkable. The studied subjects gave an informed consent after having been fully informed about the nature of the study, the tests to be done and their role in the study. The study was approved by the Review Board and Ethics Committee of the Cairo University Faculty of Medicine.The electric activity of the esophagus was recorded in the patients with GERD and the healthy volunteers. We used a monopolar silver-silver chloride electrodes of 0.8 mm diameter  introduced through a 6F catheter  with the electrode protruding by 1 cm from the catheter tip. The catheter was attached to the esophageal mucosa by negative pressure suction of 50 to 100 mmHg which was maintained during the test.Two electrodes were introduced into the esophagus by means of an endoscope and fixed to the esophageal mucosa by suction; one electrode was applied to the upper third, and the second to the lower third of the esophagus, and the electric activity was recorded. The upper electrode was then transferred to the middle third of the esophagus and recordings of the electric activity from the middle and lower third electrodes were performed. Signals from the electrodes were fed into an AC amplifier with a frequency response within ± 3 dB from 0.016 Hz to 1 kHz; they were displayed on a recorder at a sensitivity of 1 mV/cm. A metal disc applied to the abdominal skin served as the indifferent electrode. A strain gauge respiration transducer was attached to the thoracic wall for respiratory artefacts. We allowed the esophagus a 30 minute period to adapt to the electrodes applied to its wall, before we started a 120-min recording session for each subject.The results were analyzed statistically using the Student's t test. Differences assumed significance at p < 0.05 and values were given as the mean ± standard deviation (SD).No adverse side effects were encountered during or after the performance of the tests and all the subjects were evaluated. There was no difficulty in applying the electrodes to the esophageal mucosa. We found that a suction pressure of 50–60 mmHg was sufficient to keep the electrode fixed to the esophageal mucosa during the testing period in most of the subjects; in few cases we had to increase the suction pressure to 100 mm to keep the electrodes in position. Applying the aforementioned pressures, we encountered neither migration nor detachment of the electrodes during the entire test. We met no mucosal bleeding, tears or ulcers during application or after removal of the electrodes either.Monophasic negatively deflected SWs were recorded from the 2 electrodes of each subject of all the studied individuals fig. . They haIn score 1 GERD, the electric waves' variables were similar to those of the healthy volunteers in all the subjects (p > 0.05, fig Score 2 GERD patients exhibited a different electromyographic pattern. The SWs had an irregular rhythm with varying but lower frequency, amplitude and conduction velocity compared to the normal controls fig. . The APsThe current study has demonstrated that the esophagus possesses an electric activity in the form of regular SWs and APs. The waves in the healthy volunteers were reproducible with identical frequency, amplitude and conduction velocity in the same subject. Previous studies have shown that the APs were coupled with increased esophageal pressure, while the SWs were not ,13, thesEffective esophageal motility is a critical determinant for esophageal clearance of refluxed gastric contents . A singlThe relationship between esophageal peristalsis and gastroesophageal reflux has been studied using stationary and ambulatory prolonged esophageal manometry. Various motor-disorders have been detected in GERD including a significantly high rate of incomplete primary peristalsis, changes in esophageal motility and an increased number of non-transmitted contractions ,19. ContPatients with abnormal GER but mild esophagitis, or none, had normal amplitude of contractions with increased prevalence of simultaneous contractions ,21. MeanThe electric activity in GERD exhibited different patterns depending on the stage of the GERD. In score 1 GERD, an electroesophagram similar to that of healthy volunteers was recorded. This apparently denotes that the motile activity of the esophagus in score 1 is normal and that the esophageal peristaltic activity can probably clear the esophagus of the refluxed gastric contents. The irregular and diminished esophageal electric wave variables displayed in score 2 GERD are presumably indicative of diminished motile activity and peristalsis of the esophagus with a resulting inhibited reflux clearance rate. The failure of adequate esophageal clearance is probably responsible for the clinical manifestations and investigative results encountered in score 2 GERD. With progress of the condition to score 3, there is probably no motor or peristaltic esophageal activity as evidenced by the absence of the esophageal electric waves. In such case, we presume that there is no esophageal clearance.We do not know the cause of the diminished esophageal electric activity in GERD. Is it due to the refluxed acid material or to the resulting esophageal inflammation? It may be argued that the refluxed acid content into the esophagus inhibits its motile and peristaltic activity. However, the current and earlier studies have demonstrated normal peristalsis in score 1 GERD in which acid reflux was manifest . FurtherWhat then could be the cause of the deranged electric activity and peristaltic movement in the more advanced stages of GERD?The electric waves seem to be generated from the interstitial cells of Cajal that are located at the level of the myenteric plexus and in the circular muscle layer of the esophageal wall -24. TheyIn the advanced stages of GERD it may be assumed that the inflammatory changes in the esophageal wall have involved the interstitial cells of Cajal. Destruction of these cells appears to occur in grades that are in accordance with the GERD socres. It seems that in score 1 GERD, the mucosal inflammatory changes of the esophagus, if present, have not involved the Cajal cells yet. With the more advanced stages of the disease as in scores 2 and 3, the Cajal cells are presumingly being gradually destroyed by the advancing esophageal inflammatory process. The diminished SW variables encountered in score 2 GERD seem to be due to partial involvement of Cajal cells in the inflammatory process; the cells are not yet completely destroyed and are still mediating electric and peristaltic activity. However the Cajal cells in score 3 GERD are believed to be extremely injured so that they cannot generate electric activity.There are various methods for the diagnosis of GERD. They include pressure measurements using water-perfused manometry catheters, external transducers or intraluminal transducers, pH-metry and others ,29. HoweThe electric activity in GERD expressed 3 different patterns depending on the stage of GERD. In score 1 GERD, a normal electroesophagogram was recorded which would denote normal esophageal motile activity and acid clearance. Score 2 GERD exhibited irregular and diminished esophageal electric waves' variables which are presumably indicative of decreased esophageal motility and reflux clearance. In score 3, a "silent" electroesophagogram was recorded with probably no acid clearance. A new theory of the pathogenesis of diminished electric activity in GERD is put forward. It is postulated that the interstitial cells of Cajal which are considered as the generators of the pacemaker activity and electric waves, are involved in the inflammatory process of GERD. Destruction of these cells appears to occur in grades that are in accordance with GERD scores. The electroesophagogram may serve as an investigative tool in diagnosing the various GERD stages, especially if they can be recorded percutaneously.gastroesophageal reflux disease = GERD(electroesophagogram = EEGslow waves = SWsaction potentials = APsstandard deviation = SDAS: Study design/ planningOES: Data collection/entry, Data analysis/statistics, Literature analysis/searchIS: Data collection/entry, Data analysis/statistics, Literature analysis/searchAS: Data collection/entry, Preparation manuscriptThe pre-publication history for this paper can be accessed here:


Serologic tests for HIV infection in infants less than 18 months do not differentiate exposure and infection since maternally acquired IgG antibodies may be detected in infants. Thus, the gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA or reverse transcriptase polymerase chain reaction. There is an urgent need to evaluate alternative and cost effective laboratory methods for early diagnosis of infant HIV-1 infection as well as identifying infected infants who may benefit from cotrimoxazole prophylaxis and/or initiation of highly active antiretroviral therapy.Whole blood was collected in EDTA from 137 infants aged 0 to 18 months. DNA polymerase chain reaction was used as the reference standard for diagnosis of HIV-1 infection. T-cell subset profiles were determined by flow cytometry.Seventy-six infants were DNA PCR positive while 61 were negative. The median CD4 counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001. The median CD4/CD8 ratio and the %CD4 of the PCR positive infants were both significantly lower than those of the negative infants, p < 0.001. The CD4/CD8 ratio had a >98% sensitivity for diagnosis of HIV-1 infection and a specificity of >98%.The CD4/CD8 ratio appears useful in identifying HIV-infected infants. The development of lower cost and more robust flow cytometric methods that provide both CD4/CD8 ratio and %CD4 may be cost-effective for HIV-1 diagnosis and identification of infants for cotrimoxazole prophylaxis and/or highly active antiretroviral therapy. Endemic HIV infection, in sub-Saharan Africa, where in many countries more than 20% of pregnant women are HIV seropositive leads to a diagnostic problem in the evaluation of their infants. Without intervention, more than 25% of infants born to seropositive women will acquire HIV infection in the first year of life. HIV testing with enzyme immunoassay-based rapid tests have expanded capacity to identify seropositive women and provide interventions, but even with single dose Nevirapine and other antiretrovirals, infection of infants still exceeds 10% in the first year of life. Serologic tests for HIV do not accurately identify those infants who have acquired infection within the first 18 months of life because of transplacentally acquired maternal IgG antibodies. As antimicrobial and antiviral interventions are developed to reduce morbidity and mortality, among infants born to seropositive women, the early diagnosis of infection is increasingly important. The gold standard for diagnosis of HIV-1 infection in infants under the age of 2 years is DNA polymerase chain reaction (PCR) or reverse transcriptase (RT)-PCR . With thThe World Health Organization (WHO) recommends initiation of highly active antiretroviral therapy (HAART) in HIV-seropositive infants under the age of 18 months, who have WHO Pediatric Stage III disease and CD4 percentage (%CD4) <20% in resource-poor countries where %CD4 are available but virologic tests  for confirmation of HIV infection are not available . Thus, i+ cells, a transient increase in CD8+ cells, total lymphocytes and inversion of the CD4/CD8 ratio [+ cells decline, while the CD8+ cells which may remain at high levels for long periods, eventually decrease but not to baseline levels. Since in healthy children the CD4+ and CD8+ cells account for 60% and 30% of the T lymphocytes respectively, a normal CD4/CD8 ratio should always be >1.0. Thus, in HIV-1 infection where there is a decrease in CD4+ cells and an increase in CD8+ cells, the reversal of the CD4/CD8 to <1.0 should in theory be useful for diagnosis of HIV-1 infection.Immunological changes in HIV-1 infection include a decrease in CD4D8 ratio ,7. As HI+ cell counts, their percentages as well as the CD4/CD8 ratio. The use of the ratio in combination with %CD4, may lead to a timely identification of infected infants, who meet the WHO criteria for initiation of CTX prophylaxis and/or ARV therapy. While the infrastructure needed to conduct flow-cytometric analyses of HIV infection are still largely confined to a few centers, an increasing number of point of care diagnostic testing systems, inexpensive methods to measure CD4 cells are currently in development. These include the development and evaluation of simplified volumetric flow cytometric methods using a low cost flow cytometer that can be powered from a car battery or by solar panels  by Cassens and colleagues [Certain flow cytometers when used in tandem with a haematological analyzer can provide absolute CD4lleagues  and modilleagues . Furtherlleagues ,11.The main objective of the current study was to evaluate the CD4/CD8 ratio for diagnosis of subtype C HIV-1 infection in infants under the age of 2 years among infants where DNA PCR was performed to diagnose HIV infection.The infant specimens used in this study were obtained from two independent prospective studies; the short course zidovudine (AZT) and Pediatric AIDS Clinical definition (PACD). The Medical Research Council and the Institutional Ethics Committees approved both studies and present study. The infants included in the study were under the age of 2 years. All the infants were breastfeeding at the time of specimen collection.Infants who were followed in the short course AZT study aimed at preventing MTCT were enrolled between May 2001 and June 2002. In the AZT study, mothers received short course AZT starting at 36 weeks gestation and throughout labor. Their infants received AZT for 7 days.In the PACD study, hospitalized children aged between 2 months and 18 months were prospectively enrolled into the study, between July 2002 and July 2003, following informed consent from their mothers. The mothers of these children did not receive antiretroviral therapy for prevention of MTCT nor for HIV disease. The PACD study is a hospital based analytical cross-sectional survey. Laboratory specimens were obtained once from each subject. Children presenting in the moribund state, requiring immediate resuscitation or those with known HIV status or those whose mothers/guardians refused to sign the informed consent were excluded from the study. The objective of the PACD study is to identify clinical symptoms, associated with HIV-1 infection in infants under the age of 18 months, which may be used in the absence of laboratory tests, for HIV-1 diagnosis.A total volume of 2 ml whole blood was collected in ethylenediamine tetraacetic acid from each of the 137 infants in the short course AZT and the PACD studies. The whole blood was then aliquoted into two tubes (500 microlitres in each) for determination of T cell subset profiles, and the second for DNA PCR and the rest centrifuged at 200 g to obtain plasma, which was stored at -80°C.The laboratory tests described below were conducted in a blind fashion.+ and CD8+ cell counts, CD4/CD8 ratios, as well as the %CD4 and %CD8 values among lymphocytes were automatically calculated.T cell subset profiles were determined by flow cytometry using a Coulter Epics XL equipped with System II software  within 4 hours of blood collection. This flow cytometer was run, in a double platform setting where the absolute counts for both white blood cells and lymphocytes were obtained on a Celldyn 3500R haematological analyzer . Then from the combined results, the absolute CD4DNA PCR Roche amplification assay version 1.5  was employed as the reference standard for diagnosis of infant HIV-1 infection status. DNA extraction, amplification and detection were performed and results interpreted following the manufacturer's instructions  as we previously described ,13.Reference ranges of T cell subset profiles for infants and children are usually stratified by age as <12 months, 1 to 5 years and 6–12 years. In the current study, in addition to overall evaluation of samples obtained from all the infants aged 0 to 18 months, the evaluated parameters were also compared based on infant age groups 0–11, and 12–18 months.Diagnostic tests , negative predictive value (NPV) and test efficiency (TE) with 95% confidence interval) were used to assess the assay under evaluation with DNA PCR as the reference standard. A MTCT prevalence rate of 30.7% (based on the current rate in Zimbabwe) was used in the calculations of PPV and NPV. Sensitivity was defined as the percentage of reference standard HIV positive samples found reactive by assay under evaluation. Specificity was defined as the percentage of reference standard HIV-negative samples that were negative by the assay under evaluation. TE refers to the overall ability of a test to correctly identify all positives and negatives . It is a combination of the sensitivity and the specificity of an assay and gives an idea of the total effectiveness of the assay. PPV was defined as the probability that a specimen had CD4:CD8 ratio less than 1 when the test was DNA PCR positive. NPV was defined as the probability that a specimen does not have a CD4:CD8 ratio ≥ 1 when the test was DNA PCR negative.P values less than 0.05 were considered statistically significant.Comparisons of T cells between infected and uninfected infants were done using non-parametic equivalent of the T-test . in utero transmission as determined by serial DNA PCR of birth and subsequent samples tested in longitudinal short course AZT MTCT studies .Of the 137 infant specimens tested using DNA PCR, 76 were HIV-1 positive and 61 were HIV-1 negative. The 76 PCR positive infants included 9 infants who had evidence of p = 0.08). As expected, the median CD4+ cell counts of PCR negative infants were significantly higher than those of the PCR positive infants, p < 0.001  and 8.0 months (IQR: 4–14) for HIV infected and uninfected respectively (Seventy-five infants of the 76 PCR positive group (98.7%) had CD4/CD8 ratio <1 while 60 infants of the 61 who were PCR negative had CD4/CD8 ratio ≥ 1. All the 9 infants infected in utero had a CD4:CD8 ratio less than 1. Their median CD4/CD8 ratio was similar to that of the rest of the PCR positive babies , albeit the numbers are too small for statistical significance considerations. The infant (aged 11 months at specimen collection) who was DNA PCR positive with a CD4/CD8 ratio of 1.1 was from the PACD study. The PCR negative infant (aged 4 months at specimen collection) with a CD4/CD8 ratio of 0.3 was also from the PACD cohort.The overall sensitivity and specificity of the CD4/CD8 ratio were 98.7% : 95.1–100) respectively with PPV and NPV of 96.3% and 99.4% respectively and a test efficiency of 98.5% (95% CI: 96.5–100) Table . CompariIn resource-poor countries, the major constraint in the use of DNA PCR for diagnosis of HIV-1 infection in infants under the age of 18 months is the cost of the equipment and the reagents. In addition, highly trained laboratory personnel and stringent quality assurance measures are needed to run this assay for routine diagnosis of HIV infection in infants.In Zimbabwe, enzyme linked immunosorbent assay (ELISA) is routinely performed in both public and private laboratories for diagnosis of various infections including HIV-1. However, because of the transplacental transfer of maternal IgG antibodies, which may persist in infants for up to 18 months, ELISA is not suitable for diagnosis of HIV-1 infection in these infants. Therefore alternative methods are needed for this purpose. In addition, both public and private laboratories have flow cytometers or FACSCount machines for enumeration of T cell subset profile and automated haematological analysers for routine full blood counts with differential. With this equipment available in the country we evaluated the CD4/CD8 ratio as an alternative diagnostic test for infant HIV-1 infection.+ cell counts and both %CD4 and %CD8 values among lymphocytes. The %CD4 value among lymphocytes is the recommended parameter for analyzing pediatric samples, as absolute counts for infants are age sensitive and variable. A simpler single platform system such as the FACScount , is not fully suited for pediatric work as it provides CD4+, CD8+, CD3+ T lymphocyte counts and CD4/CD8 ratio but %CD4 are not available. Of note, %CD4 expressed among CD3+ T lymphocytes, is a different parameter from the customary %CD4 expressed among lymphocytes. The %CD4 expressed among lymphocytes and not the %CD4 expressed among CD3+ T lymphocytes is recommended for decision making to initiate ARV therapy in children under the age of 18 months [In our investigation we used Epics XL Coulter flow cytometer equipped with System II software  for the following reasons. With this instrument, CD4/CD8 ratios can be conveniently obtained. In addition, when used in double platform setting tandem with a haematological analyzer, the results also show absolute CD48 months .+ cell count, is less expensive than PCR and may provide additional information of value to the clinician with respect to prognosis, and the need for prophylaxis and treatment. Optimal flow cytometry for determination of T cell subset profile, offers the added advantage that CD4/CD8 ratio will determine the infection status of the infant while the % CD4 will provide information on decision-making for commencement of HAART.In Zimbabwe, CD4Overall, the CD4/CD8 ratio had a ≥ 98% sensitivity for diagnosis of infant HIV-1 infection and a specificity ≥ 98%. Both sensitivity and specificity were 100% for infants in the 12–18 months age group. Interestingly, in parallel studies performed in 250 HIV-1 seropositive adults, 249 had a CD4/CD8 ratio of <1. The CD4/CD8 ratio of the one patient was 1 at enrollment and has remained so for over one year.When interpreting our data, it is important to note that normal T cell subset values among African children differ from those of other populations -16. A st+ cell count rather than a CD4+ cell count was a more specific prognostic marker of disease progression in HIV infected children.A very few studies in Africa have compared T cell subset profiles between HIV-1 infected and uninfected infants under the age of 2 years ,17. MoodEmbree and colleagues, in Kenya, also reported that HIV-1 infected children had lower %CD4 and higher %CD8 by 3 months when compared to uninfected children . The autThe two African studies mentioned above, have documented the clinical value of %CD4, CD4/CD8 ratio and CD8 counts in HIV-1 infection in infants.In summary, the CD4/CD8 ratio may be useful in identifying infected infants while %CD4 will identify infants who may benefit from cotrimoxazole prophylaxis and/or initiation of HAART, and for management of HIV-infected infants in developing countries in general. We thus propose use of flow cytometry, where available, as a point of care diagnostic tool for ill infants admitted to hospitals with clinical symptoms suggestive of HIV infection and/or AIDS.The author(s) declare that they have no competing interests.LSZ designed the study, analyzed the data and drafted the manuscript. DAK, KJN, AB and GJ participated in the design of the study and preparation of the manuscript. SR conducted the statistical analysis and participated in the preparation of the manuscript. OT performed DNA PCR and analysed the data. AB and CG conducted flow cytometry and analysed the data, with the guidance of GJ. MN and PM participated in the collection of infant specimens and demographic data as well as preparation of the manuscript. All authors read and approved the final manuscript.Table 3. Gender-based comparison of T cell subset profiles of HIV infected and uninfected babies.Click here for file


The callipyge mutation is located within an imprinted gene cluster on ovine chromosome 18. The callipyge trait exhibits polar overdominant inheritance due to the fact that only heterozygotes inheriting a mutant paternal allele  have a phenotype of muscle hypertrophy, reduced fat and a more compact skeleton. The mutation is a single A to G transition in an intergenic region that results in the increased expression of several genes within the imprinted cluster without changing their parent-of-origin allele-specific expression.DLK1, PEG11, and MEG8 in the muscles of lambs with the callipyge allele. DLK1 and PEG11 transcript levels were elevated in the hypertrophied muscles of paternal heterozygous animals relative to animals of the other three genotypes. The PEG11 locus produces a single 6.5 kb transcript and two smaller antisense strand transcripts, referred to as PEG11AS, in skeletal muscle. PEG11AS transcripts were detectable over a 5.5 kb region beginning 1.2 kb upstream of the PEG11 start codon and spanning the entire open reading frame. Analysis of PEG11 expression by quantitative PCR shows a 200-fold induction in the hypertrophied muscles of paternal heterozygous animals and a 13-fold induction in homozygous callipyge animals. PEG11 transcripts were 14-fold more abundant than PEG11AS transcripts in the gluteus medius of paternal heterozygous animals. PEG11AS transcripts were expressed at higher levels than PEG11 transcripts in the gluteus medius of animals of the other three genotypes.There was a significant effect of genotype (p < 0.0001) on the transcript abundance of DLK1, GTL2, PEG11 and MEG8 in the hypertrophied skeletal muscles. Transcript abundance of DLK1 and PEG11 was highest in paternal heterozygous animals and exhibited polar overdominant gene expression patterns; therefore, both genes are candidates for causing skeletal muscle hypertrophy. There was unique relationship of PEG11 and PEG11AS transcript abundance in the paternal heterozygous animals that suggests a RNA interference mechanism may have a role in PEG11 gene regulation and polar overdominance in callipyge sheep.The effect of the callipyge mutation has been to alter the expression of  CLPGPat) exhibit the callipyge phenotype. Maternal heterozygotes (CLPGMat/+Pat) and callipyge allele homozygotes (CLPGMat/CLPGPat) have muscling and carcass compositions that are similar to normal sheep .The mutation responsible for the callipyge trait is located within an imprinted gene cluster on the distal end of ovine chromosome 18 . The calDelta-like 1 (DLK1), DLK associated transcript (DAT), gene-trap locus 2 (GTL2), paternal expressed gene 11 (PEG11 / PEG11AS) and maternal expressed gene 8 (MEG8). The same conserved gene order was also found as an imprinted domain on human chromosome 14 and mouse chromosome 12 -dATP . Unincorporated nucleotides were removed by spin column chromatography . The probes were hybridized to the membranes without denaturation using Ultrahyb™ (Ambion Inc.) at 42°C overnight. After hybridization, the membranes were washed in 2X SSC /0.5% SDS followed by 3 washes in 1X SSC/ 0.1% SDS at 65°C for 30 min and a final high stringency wash in 0.1X SSC/ 0.1%SDS at 65°C for 30 min. The northern blots were exposed to Kodak XAR autoradiography film for 18 to 72 h at -80°C. After autoradiography, the probe was degraded and removed from the membranes using Strip-EZ™ reagents (Ambion Inc.).Northern blots were prepared using denaturing formaldehyde gel electrophoresis  of 10 μg of total RNA and transferred to positively charged nylon membranes using standard methods . Primer et al. , and the32P]-UTP (Amersham-Pharmacia). The RPA were conducted using RPA III™ reagents (Ambion Inc.) and standard urea/acrylamide gel electrophoresis methods [Templates for synthesizing strand specific RNA probes for the ribonuclease protection assay (RPA) were generated from PCR products Table  by ligat methods . Dried g8 to 102 molecules. The standards were used to calculate a regression of threshold cycle on molecule copy number to determine a log value of starting abundance for each of the cDNA samples based on their threshold cycle. The PCR reactions were run for 40 cycles in an iCycler Real-Time PCR Detection System (Bio-Rad Inc.). The log value of starting abundance for each gene was analyzed by analysis of variance using the PROC MIXED procedure of SAS [et al. [Complementary DNA was synthesized from 3.2 μg of total RNA using random hexamer priming and MMLV reverse transcriptase reagents  with RNase inhibitor supplementation . The cDNA samples were diluted with water and aliquoted so that the quantification was based on 213 ng of total RNA for analysis of glyceraldehyde-3-phosphate dehydrogenase, DLK1 and MEG8 transcripts. PEG11 and PEG11AS transcripts were measured using gene-specific priming of cDNA synthesis from 1.6 μg of total RNA. The cDNA was diluted with water and aliquoted for quantification based on 400 ng of total RNA. Each cDNA sample was amplified in triplicate using the SYBR Green Jump Start™ system . Quantification standards were composed of aliquots of plasmids containing target PCR products in 10-fold serial dilutions ranging from 10e of SAS . The anaCB participated in planning the study and developing the experimental design, conducted the northern blot analysis and ribonuclease protection assays, and wrote the drafts of the manuscript. LK and AP isolated RNA and performed the quantitative PCR assays. TH collected the muscle samples and genotyped the animals used in this study. DM participated in experimental design and performed the statistical analysis. NC participated in planning the study, set up a series of matings to generate all four genotypes and provided the experimental animals. All authors read and approved the final manuscript.


The growing amount of structural, pharmacological and physiological data for these receptors indicates the need for a dedicated and accurate database to provide a means to access this information in a coherent manner.Nicotinic acetylcholine receptors (nAChRs) are pentameric proteins that are important drug targets for a variety of diseases including Alzheimer's, schizophrenia and various forms of epilepsy. One of the most intensively studied nAChR subunits in recent years has been α7. This subunit can form functional homomeric pentamers (α7) is a new relational database of manually curated experimental physiological data associated with the α7 nAChR. It aims to store as much of the pharmacology, physiology and structural data pertaining to the α7 nAChR. The data is accessed via web interface that allows a user to search the data in multiple ways: 1) a simple text query 2) an incremental query builder 3) an interactive query builder and 4) a file-based uploadable query. It currently holds more than 460 separately reported experiments on over 85 mutations.A7DB A7DB will be a useful tool to molecular biologists and bioinformaticians not only working on the α7 receptor family of proteins but also in the more general context of nicotinic receptor modelling. Furthermore it sets a precedent for expansion with the inclusion of all nicotinic receptor families and eventually all cys-loop receptor families. Distinceceptors . All areeceptors ,5. Mutateceptors ,7 and seeceptors . There ird level normalization, the principal information prototype stored in the database is referred to as an "experiment event". An experiment event is a collection of simultaneous measurements and their associated measurement conditions. For example, two drugs tested against the wild type α7 and a mutant under the same experimental conditions would be described by 4 (2 drugs × 2 sequences) experiment events. If the identical measurements were repeated in, say, calcium-free saline, this would yield 8 experiment events. Using this prototype a unique set of experimental conditions is associated with a single set of data. Therefore, the database tables  the positions of these data can be highlighted on a homology model based on the acetylcholine binding protein AChBP . The modFor any database, data integrity will ultimately determine its usefulness. The initial population of the database has been carried out by the authors, but it is desirable and practical in the long term to have a procedure whereby any laboratory can submit data. To help ensure reliable deposition procedure, each depositor will have a unique identifier (thus making the data accountable). A depositor will submit their data which is then tabulated and presented as a form which the depositor is then asked to confirm as being correct. Only when this confirmation is received is the data committed to the database. Although this will reduce some entry-based mistakes, the database will nonetheless need to be curated to maintain integrity.The database does not attempt to duplicate the functionality of other databases. For example we store a local alignment of α7 subunits taken directly from pfam , but we Access to the database is through various routes. The first route is by a very simple search string (e.g. citation = Smith*). The user is then presented with a page informing him of the number of hits and asking what information should be displayed from those hits. The second is a simple incremental search in which the user applies criteria sequentially until he chooses to examine the results. As the query is built, the number of hits returned by the query in its current state is shown. The third search method is also incremental but via the use of a set of tabbed pages which divide the information into intuitive categories such as pharmacology and physiology. Additionally, the user can make choices about alignments used and even upload their own. As the query is built, summary information of its status is also displayed. The fourth method to access the data is provided by a 'fast lane' route which provides more experienced users with a quicker and more direct route to the data of interest. The user formulates and constructs a query offline and then uploads it as a simple ASCII file. A sample query form and details of the format are presented in the supplementary information.The result of a query is presented in tabular form. Only the information requested is actually presented with the option for further or refined searches. In addition to this, the results of any position matches can be displayed via the homologous 3-dimensional structure of the AChBP. This is automatically available if the user selects the alignments available from the server. If the user uploads their own alignment the model will only be produced if AChBP was included. The server streams a Rasmol script or spawns a Chime window depending on browser configuration. In addition to these query routes, the user is also able to browse the contents of the database.The current database has been designed such that it could easily be extended to include other nAChR subunits and then on to other members of the cys-loop ligand-gated ion channel family. We are currently pursuing this aspect. Such expansion will allow trends that link structure to function in this receptor family to be more readily identified.The database is very complementary to some existing resources, in particular the LGICdb ,15. ThisTorpedo marmorata [We have shown here how the collation and careful storage of experimental data pertaining to one sub-family (the α7 nAChR sub-family) of the ligand-gated ion channels can be assembled into a useful resource. However, the real power of the database will be when it is combined with machine-learning technologies to explore complex relationships between sequence, structure and function . We beliarmorata . Its devThe database is freely available at: The project was conceived and technical aspects managed by PCB. The coding and design was done by SB. MSPS and DBS provided critical evaluation of the design and construction process. DBS, LP, AKJ and LB populated the database. All authors have approved the manuscript.Supplementary information. This doc file provides a complete description of the table entries and also provides a description of the uploadable query file. Further information about the online help is also provided.Click here for file


Haemophilus influenzae (HI). OM often occurs to young children, and is mostly treated with antibiotics. Due to concerns over bacterial resistance toward antibiotics, reliable prophylactic treatments such as administrating anti-adhesion agents are now viewed as viable alternatives.One of the primary causes of otitis media (OM), an inflammation of the middle ear, is the bacterium 111- indium-oxine, pre-incubated with various individual phosphoinositides for 15 minutes at 37°C, and incubated with a monolayer of human pharynx carcinoma (DT 562) cells for 20 minutes at 37°C. The result showed that at 0.1 mg/mL dipalmitoylphosphatidylinositol-3,4-diphosphate  had the highest anti-adhesion activity, followed by phosphatidylinositol-3-phosphate (PI-3-P) and phosphatidylinositol-4-phosphate (PI-4-P). The anti-adhesion activity of PI-3,4-PP was dose-dependent ranging from 0.006 to 0.1 mg/mL. In addition, results from an in vivo study demonstrated that pre-incubation of HI cells with PI-3,4-PP at 1 mg/mL suppressed the growth of HI in nasopharynx of neonatal rats.The present study tested the feasibilty of using phosphoinositides as anti-adhesion agents against HI cells. Cells of non-typeable HI were radiolabeled with These findings suggest that PI-3-P and PI-4-P and more so PI-3,4-PP may serve as prophylactic agents against HI adhesion and colonization. Haemophilus influenzae (HI). Complications of OM include permanent hearing loss and perforation of the tympanic membrane.Otitis media (OM) is an inflammation of the middle ear, often seen in children younger than six year of age. OM is caused by infection of nasopharyngeal cells by the bacterium Generally, OM is treated with antibiotics such as penicillin derivatives. However, in spite of the effectiveness of antibiotic prophylaxis, the increasing bacterial resistance to antibiotics has caused some concerns. This has prompted the development of anti-adhesive agents against HI infection . Today, in vitro effect of various phosphoinositides on the attachment of HI to nasopharyx was evaluated. The activity was defined as the percent of inhibition of HI adhesion to human pharynx carcinoma cells as compared to the control. Results in Table The attachment (adhesion) of bacteria to a mammal's nasopharynx area is believed to be the first stage of the bacterial infection, which can lead to OM and other disorders and diseases caused by HI. For this study, 10 [cfu/mL]) of HI recovered for a total of 10 rat pups for each treatment. In trial 1, at the inoculum dose of approximately 100 cfu/pup, the treated group  containing 1 mg/mL of PI-3,4-PP showed an 80-fold (1.9 logs) reduction in the number of bacteria recovered 24 hours post-inoculation as compared to the control group (HBSS). In trial 2, the rat pups were exposed to much higher doses (approximately 700 cfu/pup) of bacteria. The treated group , which was protected by the same level of PI-3,4-PP, showed a 4-fold (0.6 log) reduction in the number of bacteria recovered. The result demonstrates that PI-3,4-PP can inhibit the attachment and thus the growth of nontypeable HI in the nasopharynx of neonatal rats.The inhibitory activity of PI-3,4-PP against the attachment of nontypeable HI was further demonstrated in two trials using a neonatal rat model. Figure myo-inositol hexaphosphate), myo-inositol penta-and tetra-phosphates (data not shown). Taken together, these results suggests that the presence of both a phosphatidyl group and an additional phosphate group is essential for the high anti-adhesion activity of PI-3,4-PP as compared to PI-3-P and PI-4-P.The exact mechanism for the anti-adhesion activity of the phosphoinositides is not known at this time. However, the anti-adhesion activity may be associated with the structures of the phosphoinositides. Molecules containing no phosphatidyl group such as Ins-1,2,6-P, Ins-1,3,4-P and GPI, exerted no effects on adhesion. PI, which contains a phosphatidyl group, also had no activity. In addition to the presence of a phosphatidyl group, phosphorylation of inositol seems to be required for the bioactivity. Since PI-3,4-PP had a higher anti-adhesion activity than PI-3-P and PI-4-P, it is possible that the presence of extra phosphate groups at the positions 3 and 4 of the inositol moiety may exert an even greater effect. Results from a preliminary study  showed the presence of multi-phosphate groups in a molecule  were inhibitory to HI adhesion. On the other hand, results from our study showed that PI-3,4-PP was more active than phytate  and 1-D-myo-inositol-1,3,4-triphosphate sodium salt  , class III L-α-phosphatidylinositol ammonium salt (PI), class IV dipalmitoylphosphatidyl-inositol-3-phosphate ammonium salt (PI-3-P), and L-α-phosphatidylinositol-4-monophosphate sodium salt (PI-4-P), and class V dipalmitoylphosphatidylinositol-3,4-diphosphate ammonium salt   of phosphoinositides were used for this study Figure . Class I) Figure . Class I) Figure . Ins-1,22 at 37°C until reaching at least 90% confluency. Plates were washed three times with 20 mL of Hanks Balanced Saline Solution (HBSS)  to remove serum proteins.The Detroit 562 human pharynx carcinoma cell line (DT 562) was obtained from the American Culture Type Collection. The DT 562 cells were seeded into Costar 96-well plates  at a density of 20,000 to 25,000 cells per well, and cultured in Dulbeco's modified Eagle Medium  supplemented with 10% fetal bovine serum (FBS) . The plates were incubated in a humidified atmosphere of 95% air: 5% CO2 for 18 hours. Bacteria were then harvested in phosphate buffered solution (PBS) supplemented with 0.05% bovine serum albumin (BSA) . After centrifugation, the cell pellets were resuspended in a volume of PBS/BSA yielding an optical density of 2.4 at a wavelength of 660 nm. The bacteria were then radiolabelled with 111-Indium-oxine (111- In), a high energy, short-lived tracer. Fifty μCi of 111-In solution was added to 2.5 mL of the bacterial suspension and incubated for 20 minutes at 37°C. The radiolabeled bacteria were then washed two times with 10 mL HBSS and unbound 111- In were removed by centrifugation. The bacteria pellets were then resuspended in 5 mL HBSS supplemented with 30 mM 2-hydroxyethyl-piperazine-N'-2-ethane sulfonic acid buffer .For the adhesion study, an HI nontypeable bacterial strain was used. The HI isolated from the middle ear of an infected child was a gift from Dr. Lauren Bakaletz of The Ohio State University, Columbus, Ohio. HI was streaked onto Chocolate agar plates  from frozen aliquots of a low passage number. The plates were then incubated at 37°C in a humidified atmosphere of 95% air: 5% CO111- In-labeled bacterial suspension were pre-incubated with 25 μL of the test chemical (containing various phosphoinositides) in a Costar polypropylene 96-well plate for 15 minutes at 37°C to allow binding of the test agent to the HI. For adhesion quantitation, aliquots (25 μL) of the pre-incubated mixture were pipetted into the wells of an assay plate containing the DT 562 human pharynx carcinoma cells. The assay plate was incubated for about 15 to 20 minutes at 37°C to allow adhesion of the bacteria to the cell monolayer. Nonadherent bacteria were removed by washing the plate three times with HBSS. The cell monolayer and the adhering of the HI cells were disrupted by the addition of 100 μL of 0.05 N sodium hydroxide. The contents of each well were placed in Cobra polypropylene tubes and the radioactivity counted on a Cobra Gamma Counter . After calibration of the background, the average radiation count of four replicates (per sample) was calculated. The percents of inhibition of bacterial adhesion, as compared to bacterial attachment in control wells containing no test chemical were then calculated.Prior to adhesion test, aliquots (25 μL) of the in vivo activity of PI-3,4-PP against nontypeable HI. Prior to the test, overnight cultures of nontypeable HI were prepared, washed twice and diluted with HBSS to obtain a bacterial suspension of less than 105 colony-forming-units (cfu) per mL. Three different test chemicals were prepared for this test: (i) HI + PI-3,4-PP, an aliquot (0.5 mL) of PI-3,4-PP (2 mg/mL in HBSS) mixed with 0.5 mL of the diluted bacterial suspension (5 × 104 cfu/mL) and incubated for one hour at 37°C; (ii) HI + HBSS, This bacterial control prepared by mixing and incubating 0.5 mL HBSS with 0.5 mL of the diluted bacterial suspension; and (iii) HBSS, the solvent blank prepared by incubating 1 mL of HBSS under the same conditions. A 10 μL of the test material was used to inoculate 24-hour-old or younger Sprague Dawley rats  by intranasal administration. Twenty-four hours after the inoculation, samples of nasopharyngeal fluid were collected by the slow instillation of 25 μL of HBSS into the left naris, and the initial 10-μL discharge from the right naris was collected for plate count. This procedure insured that the fluid had passed through the nasopharynx. The nasal wash was then spread (or diluted and then spread), onto Chocolate agar plates. The plates were incubated at 37°C overnight and counted for the number of cfu's, an indicator of the number of viable bacteria.A neonatal rat model ,15 was mYSH conceived of the study. JWL screened and identified the phosphoinositides, and drafted the manuscript. SNA carried out cell adhesion assay. JM carried out assay for in vivo activity. SMH and PM participated in experiment coordination.


Deregulation of the Wnt/ β-catenin signal transduction pathway has been implicated in the pathogenesis of tumours in the mammary gland, colon and other tissues. Mutations in components of this pathway result in β-catenin stabilization and accumulation, and the aberrant modulation of β-catenin/TCF target genes. Such alterations in the cellular transcriptional profile are believed to underlie the pathogenesis of these cancers. We have sought to identify novel target genes of this pathway in mouse mammary epithelial cells.Gene expression microarray analysis of mouse mammary epithelial cells inducibly expressing a constitutively active mutant of β-catenin was used to identify target genes of this pathway.Hig2, previously shown to be induced by both hypoxia and glucose deprivation in human cervical carcinoma cells, was strongly repressed upon ΔNβ-catenin induction. The predicted N-terminus of Hig2 contains a putative signal peptide suggesting it might be secreted. Consistent with this, a Hig2-EGFP fusion protein was able to enter the secretory pathway and was detected in conditioned medium. Mutation of critical residues in the putative signal sequence abolished its secretion. The expression of human HIG2 was examined in a panel of human tumours and was found to be significantly downregulated in kidney tumours compared to normal adjacent tissue.The differential expression in response to ΔNβ-catenin for five putative target genes, Autotaxin, Extracellular Matrix Protein 1 (Ecm1), CD14, Hypoxia-inducible gene 2 (Hig2) and Receptor Activity Modifying Protein 3 (RAMP3), was independently validated by northern blotting. Each of these genes encodes either a receptor or a secreted protein, modulation of which may underlie the interactions between Wnt/β-catenin tumour cells and between the tumour and its microenvironment. One of these genes, HIG2 represents a novel non-cell autonomous target of the Wnt pathway which is potentially involved in human cancer. The Wnt/β-catenin signal transduction pathway plays a central role in metazoan development, controlling such diverse processes as cell growth, proliferation and organogenesis . Wnt-1 iAPC was identified as the tumour suppressor gene mutated in the hereditary colorectal cancer syndrome, Familial Adenomatous Polyposis [Axin and β-catenin have also been detected in tumours of the colon and other tissues [Overexpression of Wnt-1 in the mammary glands of transgenic mice leads to extensive hyperplasia and tumorigenesis . APC wasolyposis ,11. Muta tissues . DeregulMany of the Wnt targets thus far identified are cell-cycle regulators ,14 and t2 at 37°C in RPMI 1640, supplemented with 10% Foetal Bovine Serum, 2 mM L-glutamine, 2.5 μg/ml insulin, 5 ng/ml epidermal growth factor and 50 μg/ml gentamycin [lacZ and HC11-ΔNβ-catenin cells were routinely cultured in 2 μg/ml tetracycline to repress transcription of the tetracycline-regulated transgene. HEK293 and MDCK cells were grown in DMEM supplemented with 10% Foetal Bovine Serum.All reagents were purchased from Sigma unless otherwise noted. HC11 mouse mammary epithelial cells were cultured in 5% COntamycin . HC11-lalacZ and HC11-ΔNβ-catenin cell lines were generated by infecting the cells with an ecotropic retrovirus (TRE-tTA) in which the tTA cDNA is under the control of a tetracycline responsive promoter. Consequently, tTA expression is minimal in the presence of tetracycline and, upon tetracycline withdrawal, tTA activates its own transcription in an autoregulatory manner [The HC11-y manner . HC11 ceBosc23 cells were used to produce ecotropic retroviruses . Cells wCell monolayers were washed twice in ice-cold Phosphate Buffered Saline and lysed by addition of Trizol (Invitrogen). Total RNA was isolated according to the manufacturer's instructions. PolyA+ RNA was purified from total RNA using Oligotex (Qiagen) according to the manufacturer's instructions.Autotaxin – 533819; CD14 – 2936787; Ecm1 717050; Hig2 – 367488; Ramp3 – 615797, HIG2 4366895). Following overnight hybridisation with the labelled probe, the membranes were washed twice in 1X SSC, 0.1% (w/v) SDS at room temperature for 20 mins, and twice in 0.2X SSC, 0.1% (w/v) SDS at 68°C for 10 mins and exposed to film at -80°C for 48 hours. Bound probe was quantitated using a phosphorimager (Molecular Dynamics).10 μg of total RNA from each cell line was fractionated on a denaturing formaldehyde agarose gel and transferred to a positively charged nylon membrane  in 10x SSC. Membranes were prehybridised for four hours in 50% (v/v) formamide, 5X SSPE, 2X Denhardt's reagent, 0.1% (w/v) SDS and 100 μg/ml denatured herring sperm DNA. Radiolabelled probes were prepared from PCR-amplified cDNA clones using the Rediprime II kit (Amersham Pharmacia Biotech) according to the manufacturer's instructions. EST sequences corresponding to the coding sequence of the genes-of-interest were identified by BLAST  and obtaCell monolayers were rinsed twice with ice-cold Phosphate Buffered Saline and total cell lysates were prepared by scraping cells into a minimal volume of 50 mM Tris. HCl pH 7.5, 150 mM NaCl, 0.5% NP40 and Complete protease inhibitor cocktail (Roche). Aliquots containing 80 μg protein from each sample were analysed by SDS-PAGE , and tralacZ cDNA was excised from the CMV-lacZ construct  and sub-cloned into BamHI digested RevTRE to make RevTRE-lacZ. A plasmid containing a myc-tagged ΔNβ-catenin was obtained from Hans Clevers. The myc-tagged ΔNβ-catenin was excised with KpnI and NotI and the ends were blunted, and subcloned into HpaI digested RevTRE to make RevTRE-ΔNβ-catenin. The mouse Hig2 open reading frame was amplified by PCR from I.M.A.G.E. cDNA clone 367488 using the primers TTTACTAGTAGGAGCTGGGCACCGTCGCC and TTTTACCGGTGCCTGCACTCCTCGGGATGGATGG. The PCR product was digested with AgeI and SpeI and subcloned into the AgeI and NheI sites in pEGFP-C1 (Clontech) to make the Hig2-EGFP fusion gene. Site directed mutagenesis was carried out by the method of Sawano and Miyawaki (2000) [A BgIII fragment containing the i (2000) . The pri5 cells were plated in 3.5 cm wells on the day prior to transfection. Each well was transfected with a total of 0.9 μg DNA under serum-free conditions for six hours, after which the cells were washed and incubated for a further 48 hours before assaying expression.Transient transfections were performed using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Briefly, 1.5 × 10lacZ cells for each condition, were cultured in triplicate in 96 well plates for 72 hours, and beta-galactosidase activity was determined as previously described [For the tetracycline dose response curve, 5000 HC11-escribed .Stable cell lines were generated in which either ΔNβ-catenin or β-galactosidase was expressed in a tetracycline dependent manner. These cell lines were established using a novel autoregulatory system in which the expression level of the tetracycline transactivator (tTA) protein is minimised during routine culture and is induced upon withdrawal of tetracycline with concomitant upregulation of the transgene-of-interest . This stlacZ cell line is shown in Figure A dose-response analysis for the HC11-lacZ and HC11-ΔNβ-catenin cells by withdrawal of tetracycline for 72 hours. Total RNA was isolated, from which mRNA was purified. cDNAs were labelled and hybridized to an 8962 element Incyte mouse GEM1 cDNA microarray . These data are provided as supplementary material , Autotaxin, Receptor Activity Modifying Protein 3 (Ramp3), Cd14 and Hypoxia Inducible Gene 2 (Hig2). Each putative target gene was initially subjected to a secondary screen by Northern blotting to confirm the differential expression in response to ΔNβ-catenin  cells by transient transfection   Figure . The levcDNA microarray analysis of the transcriptional changes resulting from overexpression of a constitutively active β-catenin revealed a panel of putative target genes of the Wnt/β-catenin pathway in mouse mammary epithelial cells. This differential expression was confirmed by Northern blotting in five cases.Autotaxin [in vivo [Autotaxin was originally identified as a secreted enzyme with potent motility stimulating activity  and has utotaxin , and pur[in vivo . Autotax[in vivo . Autotax[in vivo ,41.In situ hybridisation showed that Ecm1 was strongly expressed in most newly formed blood vessels and experiments using purified recombinant Ecm1 showed that it could increase the proliferation rate of vascular endothelial cells in vitro and also stimulate angiogenesis in vivo. The ability to induce de novo angiogenesis is an absolute requirement for tumours to grow beyond a size which can be readily perfused by oxygen and nutrients from the interstitial fluid. ECM1 is overexpressed in many epithelial tumours including 73% of breast tumours analyzed [Autotaxin and Ecm1 as genes upregulated by activation of this pathway, together with VEGF [Extracellular Matrix Protein 1 was first identified as a novel 85 KDa protein secreted by a mouse osteogenic stromal cell line . In situanalyzed . Homozyganalyzed . The ideith VEGF  suggestsCD14, which can function as both a receptor and a secreted protein, was downregulated upon ΔNβ-catenin expression. CD14 is a glycosyl-phosphatidylinositol-linked cell surface protein, preferentially expressed in monocytes, where it acts as a receptor for Lipopolysaccharide Binding Protein:Lipopolysaccharide complexes [in vitro and has been detected in human milk where it is postulated to play a role in neonatal immunity [in vivo at the onset of involution [omplexes . Solubleimmunity , and is volution .Receptor Activity Modifying Protein 3 (RAMP3) was downregulated upon ΔNβ-catenin induction and is one of three members of the RAMP family. These proteins are involved in mediating the cellular response to the neuropeptides calcitonin, calcitonin gene related peptide, amylin and adrenomedullin. The RAMP family members function as chaperones for the seven transmembrane domain G-protein coupled receptors for these neuropeptides, shuttling the receptor to the cell surface and altering receptor glycosylation. The ligand binding phenotype of the receptor is dependent on the RAMP family member with which it is associated [sociated . RAMP3-Csociated , and RAMsociated .in vivo [Expression of both CR and CRLR was detected in HC11 by RT-PCR (data not shown) suggesting that functional receptor-RAMP complexes are present in this cell line. Adrenomedullin, the ligand for the CRLR/RAMP3 receptor dimer, functions as a growth factor in several human tumour cell lines , in addiin vivo  via CRLRin vivo ,55.Hypoxia-inducible gene 2 (Hig2) was one of several genes identified in a representational difference analysis screen for genes regulated by hypoxia in a human cervical epithelial cell line. The human gene encodes a 63 amino acid polypeptide of unknown function [Hig2 was downregulated in HC11 cells overexpressing ΔNβ-catenin. The identification of a group of mammalian orthologues revealed a well conserved hydrophobic region in the N-terminus, reminiscent of a signal peptide. A Hig2-EGFP fusion protein entered the secretory pathway and was detected in conditioned medium of transfected cells. The introduction of a pair of charged amino acids into the hydrophobic region abolished secretion, lending support to the hypothesis that this region contains a functional signal peptide. The nature of the large vesicular structures observed in Hig2-EGFP overexpressing cells is as yet unclear. Mammary epithelial cells are known to contain membrane-enclosed lipid droplets, as well as a variety of vesicular compartments involved in the secretion of casein, citrate, lactose and calcium [function . Express calcium , howeverHIG2 using a matched Tumour/Normal tissue cDNA array showed that HIG2 is widely expressed. In most cases, the levels of HIG2 in the tumours and the associated normal tissue controls were similar. HIG2 was, however, strongly and consistently downregulated in the majority of the kidney and stomach tumours analysed. This represents an significant validation of our in vitro findings in human tumours, and suggests that HIG2 may exert a tumour suppressive effect in vivo. Human HIG2 is located on 7q32.2, a commonly deleted region in several tumour types, most prominently leukaemias and lymphomas [Analysis of the expression of ymphomas . Deletioymphomas .The identification of this panel of candidate target genes for this clinically important signal transduction pathway adds to those identified by other workers in a variety of model systems and suggests that, as well as promoting tumour cell proliferation and survival in a cell autonomous manner, this activation of this pathway is likely to have a series of non-cell autonomous effects. Here we have focussed on the identification of Wnt/β-catenin target genes that are either secreted signalling molecules or receptors. It is likely that such targets are involved in mediating autocrine proliferation, promotion of angiogenesis and the mediation of reciprocal communication between Wnt/β-catenin tumours and their microenvironmental milieu.The authors declare that they have no competing interests.PK carried out all of the experimental procedures and drafted the manuscript. PK, TE and AA contributed to the design of the study. All authors read and approved the final version of this manuscript.The pre-publication history for this paper can be accessed here:cDNA microarray dataset: HC11 ΔNβ-catenin v. HC11 lacZ. This file contains the dataset from the Incyte GEM1 cDNA microarray comparison between HC11 cells overexpressing ΔNβ-catenin (Probe 1) and β-galactosidase (Probe 2). This file may be easily imported into MS Excel, Genespring or other microarray analysis software to facilitate further analysis.Click here for file