Pulp necrosis is one of the main complications of dental trauma. When it happens on an immature tooth, pulp necrosis implies a lack of root maturation and apical closure. A therapy called apexification is required to induce the formation of a calcified apical barrier allowing a permanent and hermetic root filling. The aim of this prospective randomized clinical trial is to compare Mineral Trioxide Aggregate(MTA)with Calcium Hydroxide(CH)as materials used to induce root-end closure in necrotic permanent immature incisors.This study, promoted by AP-HP, was approved by the ethics committee(CPP Paris Ile de France IV). 34 children aged from 6 to 18 years and presenting a non-vital permanent incisor are selected. Prior to treatment, an appropriate written consent has to be obtained from both parents and from children. Patients are then randomly assigned to either the MTAor CH(control)groups. Recalls are performed after 3, 6 and 12 months to determine the presence or absence of a calcified apical barrier through the use of clinical and radiographic exams. Additional criteria such as clinical symptoms, apical radiolucencies, periapical index(PAI)are also noted.NCT00472173 ClinicalTrials.gov no.  Dental injuries are very common in children between six and nine years old. A serious complication of these traumas is the pulp necrosis whose prevalence varies with the type of traumatism from 1-6% for crown fractures to nearly 100% for intrusions . Many st- The treatment involves numerous visits over a prolonged period and the patients may therefore fail to attend their appointments. It may also lead to a loss of temporary dressings and a re-infection of teeth. It is moreover impossible to perform a permanent restoration .- In retrospective studies, cervical root fractures were said to occur on teeth during or following treatment with calcium hydroxide due to thin dentinal walls in immature teeth as well as to weakened dental structure induced by calcium hydroxide -7.® MTA, Dentsply Maillefer France)is a medical device benefiting of EC marking. It is a powder mixed with sterile water to obtain a dental filling cement, used routinely for different clinical protocols in odontology. All clinical cases reported in the literature show, after a period of about six to twelve months, an absence of pathological clinical signs, the healing of a possible periapical lesion and the development of an apical closure [To avoid these inconveniences, the use of new materials potentially inducing mineralisation such as Mineral Trioxyde Aggregate(MTA)was suggested . A revie closure -40. Howe- The main objective of this study is to test the ability of MTA, used as a root-end filling material, to induce a hard calcific barrier to seal hermetically non vital immature anterior permanent teeth with open apices over a 6 month's period.MTA is a filling material allowing the creation of an artificial apical barrier-the MTA apical plug-but it is not good enough. The advantage of MTA is to induce a very close formation of mineralized tissue which is going to coat gradually the entire apex adhering to the MTA and the root walls. The interest of MTA is not only to create an immediate apical sealingto induce a successful healing but also to promote the root-end growth or apexification, and finally the "bio-integration" of the non-vital immature permanent tooth.- Secondary objectives:• To assess the presence or absence of periapical radioluciency indicating the emergence or persistence of an apical periodontitis• To compare the time required to obtain the clinical healing and the disappearance of clinical symptoms with the two materials: MTA and Calcium Hydroxide, when symptoms such as pain, swelling, sinus tract, abscess or abnormal mobility are present at the beginning of the study.In children with pulp necrosis of a permanent immature incisor, MTA is not better than calcium hydroxide in the rates of a calcified apical barrier, but MTA is more valuable to achieve a biologic periapical barrier before 6 months.Randomized controlled trial(RCT), monocentric, open-labeled, superiority trial® 6.01. For ethical reasons, the sample size was limited while remaining compatible with the ability to obtain a significant difference between the two materials. The case reports published in the literature show a periapical repair within the first 6 months whereas the apical bridging induced with calcium hydroxide is more often obtained after a period of 6 to 18 months. That is why we hypothesized that after a period of 6 months, at least half of the MTA cases would be a success for the main objective as opposed to a minimal percentage for the calcium hydroxide group.The studied sample is composed of 34 children and teenagers, aged from 6 to 18 years presenting a pulp necrosis of a permanent immature incisor. The root maturation is normally completed 3 or 4 years after the tooth eruption, that is to say around the age of 9 or 10 years as far as incisors are concerned. However some teenagers present teeth damaged by a trauma before their maturation was achieved. That is the reason why such patients are included in the study when those traumas were not or unsuccessfully dealt with. The number of patients needed for this study was calculated prior to investigation with the hypothesis of a higher successful apexification rate in the MTA group compared to the control CH group. The anticipated success rates at 6 months were 5% in the CH group and 50% in the MTA group; Given these hypotheses, it was necessary to include 15 patients in each group that is to say a total of 30 patients to have a 80% statistical power(with a α risk of 5%), to compare the proportion of apical closure at 6 months with a Chi-2 test(two sided). Taking into account an anticipated dropout of 5%, it was decided to include 34 patients. Sample size calculations were computed with nQuery AdvisorI1-Patient presenting a non-vital permanent immature incisor for which an apexification treatment is indicatedI2-Aged 6-18 yearsI3-Written informed consent obtained from each parent and from the childI4-Medical examE1-General pathologyE1.1-History of uncontrolled diabetesE1.2-ImmunosuppressionE1.3-Severe asthmaE1.4-Chronic systemic disease if a treatment is requiredE1.5-Eating disorderE2-Oral pathology: Periodontal diseaseE3-Corticosteroid treatment during the last 3 monthsE4-Non-affiliation to a social security schemeS1-Poor complianceS2-New trauma or new complicationS3-Intercurrent illness requiring discontinuation of the trialWhen patients are lost sight of, observations must be noted down in the CRF until the last effected visit. The investigators will do their best to contact the patient and find out why the patient ceased being in the trial.Participants are assigned in a treatment group by an independent statistician. Random numbers were randomly determined via a block randomization with a 1:1 ratio and blocks size of 4. The block size was concealed from all researchers. The randomization of the trial is centralized and conducted by an external authority: the Unity of Clinical Research(UCR). After the informed consent form is obtained, the investigator sends a randomization request fax to the UCR that sends back the random number according to the random sequence list. The latter fax is also sent to the hospital pharmacy that delivers the products necessary to the study.For each patient the protocol is composed of 7 sessionsTable . The fol1. Day 0-14/Day 0: Inclusion and selection visitDiagnosis of pulp necrosis, clinical and radiographic examsWritten informed consent is obtained from both parents and from the child.2. Day 0: Calcium hydroxide canal conditioning regardless of the treatment groupThis treatment is performed under local anesthesia with Articaïne with adrenalin 1/200000, after placement of a rubber dam. After preparing an access cavity and establishing the working length by taking a radiograph with a file inserted into the root canal within 1 mm of the radiographic apex, the canal is cleaned by irrigation with 3% sodium hypochloriteand the use of manual files. The cleaning and shaping are realized with files with a very light parietal action to avoid the canal widening and the weakening of the root walls. Above all, it consists in removing the pulp remnants. The most important thing is to observe the vacuity and cleanliness of the canal using operative microscopy. Considering the root immaturity, it is actually a cleaning and desinfection without shaping. Then the canal is dried with paper point and can be filled with calcium hydroxide. Calcium hydroxide paste is prepared by mixing the calcium hydroxide powderwith an anaesthetic solution. A plug of calcium hydroxide is deposed in the canal and condensed to the apical end of the root with a plugger. Other applications of calcium hydroxide are realized, until complete canal filling. The excess moisture is dried between each input with sterile paper points. The intracanal dressing quality is checked with a radiograph. The access cavity is temporarily sealed with a resin modified glass ionomer cement. This calcium hydroxide canal conditioning is performed for all patients to allow the complete disinfection already obtained thanks to irrigation with sodium hypochlorite and the use of manual files, to control acute symptoms and to allow further treatments.3. Day0+15: MTA apical filling for the testing group/calcium hydroxide renewal for the control groupThis session starts with a local anesthesia, the placement of a rubber dam and the removal of all the calcium hydroxide. For this procedure, the canal has to be cleaned by irrigation and the use of ultrasonic files to remove all the calcium hydroxide. The use of EDTA allows a more complete elimination but due to these teeth immaturity the EDTA is not recommended. Then, the treatment differs among the treatment group:For the MTA group, a MTA plug is placed into the canal with a root canal messing gun and condensed to the apical end of the root with a plugger to create a 4 mm apical plug of MTA. The pipe of the MTA gun is adjusted for the piston to deliver a 3 mm plug. Coarse paper points are used upside down to perfect the MTA adaptation to the apical walls. A radiographic control allows the practitioner to verify the good placement of MTA. A paper point moistened with sterile water is placed in the canal to produce an ambient humidity for the MTA to achieve its solidification. The access cavity is filled with conventional glass ionomer cement.- For the CH group, the complete filling with calcium hydroxide is obtained as the first treatment session.4. Day 0+21: Control for the CH group/complete filling with gutta-percha for the MTA group. The hardness of the MTA is checked with an endodontic file and the root canal filling with gutta-percha can be realized in contact with MTA. This filling is realized with the Schilder's technique: warm gutta-percha compacted with the Touch'n Heat. The access cavity is then sealed with a composite resin.5. 3 months: clinical and radiographic control for all patients, and calcium hydroxide renewal for CH group patients.6. 6 months: clinical and radiographic control for all patients and for CH group patients, calcium hydroxide renewal or complete root filling with gutta-percha if an apical barrier is present.7. 12 months: clinical and radiographic controlFor the teeth of CH group not filled with gutta-percha, the following treatment is realized outside study.All the treatment sessions are realized in the same dental office, under local anesthesia, after placement of a rubber dam and with optical aids, by the same two operators, one assistant and the other alternately.The X-Rays of each patient are standardized so that they can be compared. When each patient has his first X-Ray taken, the patient is asked to bite into a piece of silicone which enables the operator to put the film holder in the same position at each sessionFigure . Thus thThe assessment criteria are both clinical and radiographicTable .First, the evaluation is realized at each session by the investigators-operators, who assess the presence or absence of clinical symptoms such as pain, abnormal mobility, swelling, sinus tract, abcess and percussion tenderness. Radiographic exams allow the evaluation of the main success criterion, the presence or absence of a calcified apical barrier as well as the presence of a radioluscent image and the form and thickness of the apical barrier. These investigators can compare all the patient's radiographs.All these data are immediately entered in the CRF.Two independent investigators will study all the anonymized X-Rays. This assessment will be done without the investigators having any information about the treated patients, their treatment or the nature of the × rays. If the conclusions of the two investigators diverge, a third one will settle the matter. These anonymous data will be sent to the UCR that will then remove the anonymity before sending the service statistics for analysis.The dental root canal can be hermetically filled immediately after the MTA hardens thus reducing the number of sessions and the length of the treatment.in Vitro and In Vivo studies on MTA biocompatibility have been realized [Many realized ,25,26,41realized . Souza erealized . This prAny adverse event observed during the study or the follow-up has to be reported in the CRF. Some information including occurrence time, duration, severity and treatment of this undesirable event should be noted as well. The relationship between adverse event and the apexification treatment has to be assessed.Monitor should regularly visit the dental office to control the process of study and the CRF, check storage of investigational dental products and record of data.This study has been promoted by AP-HPand approved by the ethics committee(CPP Paris Ile de France IV)and other French institutions:- CNIL: French data protection watchdog- AFSSAPS: the French Medecine and healthcare products regulatory agency, equivalent of the Food and Drug Administration in the United States.Informed consent form(ICF)has been reviewed and approved by the ethics committee before study. Information about the protocol should be given to children and their parents both orally and in writing in an easily understandable language. The ICF must be dated and signed by the child and the parents(or representatives), and 3 copies must be made to be kept by the participants, the investigators and the UCR(15 years).All information about the participants must be kept confidential.All the analyses followed an intention-to-treat principle and will be conducted to compare the efficacy of MTA with CH to induce root-end closure in necrotic permanent immature incisors at 6 months. Secondary efficacy endpoints are apical radiolucencies and PeriApical IndexPrimary endpoints, ie, proportions of patients with an apical barrier close at 6 months will be compared between treatment groups with the Fisher's exact test, such as with the secondary endpoints.To handle missing data on the primary endpoint, several procedures will be used where the missing values will be either dropped-out or replaced according to 3 simple imputation methods as follows:- Observed data: patients with missing values are dropped out.- Worst-case analysis: missing values are considered as a failurein MTA group and as a success in CH group.- Best-case analysis: missing values are considered as a successin MTA group and as a failure in CH group.- Last Observation Carried Forward(LOCF):previous diagnostic is used to replace missing values.The assessment of concordance between the two independent investigators about the closure of apical barrier at baseline, months 3, months 6 and months 12 will be given with the Kappa coefficient and the 95% confidence interval.Analyses will be performed by two statisticians of Biostatistics and Epidemiology unit of Cochin Hospital(Paris)using SAS 9.1; tests will be 2-sided, and 0.05 will be used as the threshold for statistical significance.AFSSAPS: Agence Française de Sécurité Sanitaire des aliments et produits de santé; AP-HP: Assistance Publique-Hôpitaux de Paris; CH: Calcium Hydroxide; CNIL: Commission Nationale Informatique et Liberté; CRF: Case Report Form; EDTA: Ethylenediaminetetraacetic acid; ICF: Informed Consent Form; MTA: Mineral Trioxide Aggregate; PAI: Periapical Index; UCR: Unity of Clinical ResearchThe authors declare that they have no competing interests.ABN, RS, JJL: participated in the design of the study. RS: is in charge of the trial management. FA is in charge of monitoring. BB manage the dental materials. ABN, JJL: do treatment and data collection. ABN, EB, JJL: are analyzing X-rays data. LQ, SG: are responsible of the statistical analysis. ABN particularly developed this publication. JJL: chair.All authors read and approved the final manuscript.Aurélie Beslot-Neveu: DDS, Hôpital Bretonneau, Assistance Publique-Hôpitaux de Paris(AP-HP), Department of Pediatric Dentistry, Paris Descartes University, Unité de Recherche Biomatériaux Innovants et Interfaces EA 4462.Eric Bonte: DDS, Associate Professor, Paris Descartes University, AP-HP, Hôpital Bretonneau, Department of conservative dentistry and endodontics, ParisBruno Baune: Hospital Pharmacist, AP-HP Hôpital Bretonneau, ParisRaphaël Serreau: MD, PhD, pharmacologist, Project Manager, Unity of Clinical Research CIC-URC Necker Cochin ParisFawzia Aissat: Monitor, Unity of Clinical Research CIC-URC Necker Cochin ParisLaurent Quinquis: Statistician, Unité de Biostatistique et d'Epidémiologie Hôpital Cochin, ParisSophie Grabar: MD, PhD, Paris Descartes University, AP-HP, Hôpital Cochin, Unité de Biostatistique et d'Epidémiologie, ParisJean-Jacques Lasfargues: DDS, Pr PhD, PD, Paris Descartes University, AP-HP, Hopital Bretonneau, Department of conservative dentistry and endodontics, Paris


Physiological respiratory motion of tumors growing in the lung can be corrected with respiratory gating when treated with radiotherapy (RT). The optimal respiratory phase for beam-on may be assessed with a respiratory phase optimizer (RPO), a 4D image processing software developed with this purpose.Fourteen patients with lung cancer were included in the study. Every patient underwent a 4D-CT providing ten datasets of ten phases of the respiratory cycle (0-100% of the cycle). We defined two morphological parameters for comparison of 4D-CT images in different respiratory phases: tumor-volume to lung-volume ratio and tumor-to-spinal cord distance. The RPO automatized the calculations (200 per patient) of these parameters for each phase of the respiratory cycle allowing to determine the optimal interval for RT.Lower lobe lung tumors not attached to the diaphragm presented with the largest motion with breathing. Maximum inspiration was considered the optimal phase for treatment in 4 patients (28.6%). In 7 patients (50%), however, the RPO showed a most favorable volumetric and spatial configuration in phases other than maximum inspiration. In 2 cases (14.4%) the RPO showed no benefit from gating. This tool was not conclusive in only one case.The RPO software presented in this study can help to determine the optimal respiratory phase for gated RT based on a few simple morphological parameters. Easy to apply in daily routine, it may be a useful tool for selecting patients who might benefit from breathing adapted RT. Lung cancer is the first cause of cancer death in the world with an overall 5 year survival rate inferior to 15%. It has been shown that local control after radiotherapy (RT) is dose-dependent with a better overall-survival for patients with the disease locally controlled . NeverthThere are presently several approaches under investigation aiming to correct for tumor motion potentially leading to a better conformality of RT: tumor tracking, synchronizing the beam-on/beam-off time with respiratory motion (gating), or using 4D-CT to determine the average tumor motion during a respiratory cycle in order to define an internal target volume . A 4D-CTAlthough there are techniques compensating for respiratory motion during RT and delivering RT during one specific moment of the respiratory cycle, the optimal moment for delivering RT remains unknown and controversial. Irradiation during deep inspiratory breath hold (DIBH) is considered by some to have dosimetric advantages in terms of lung sparing through the inspiratory expansion of the healthy lung tissue ,13. HoweIn this report we present a respiratory phase optimizer (RPO) for breathing adapted RT (BART) in order to determine the optimal irradiation phase based on a few simple morphological parameters.Fourteen patients with a primary or recurrent lung cancer were retrospectively studied. 4D-CTs were acquired during 4 to 6 respiratory cycles for every patient in the study. Patient data sets were provided by the Geneva University Hospital (6 cases), the CRLC Val d'Aurelle (6 cases), and by the General Electric Corporation (2 cases).et al. originally called "maximum activity projection", for nuclear medicine use [Ten 4D-CT axial images corresponding to ten time bins (phases) of the respiratory cycle  were reconstructed, using a maximum intensity projection (MIP) system , ideally as small as possible and b) the tumor to spinal cord distance (T-Cdist), sought to be as large as possible. A low T/L ratiovol may obviously result in optimal target coverage with a simultaneous reduced lung irradiation. DIBH has shown the potential for a reduced lung V20  [dist is the largest is based on the fact that dose constraints to the spinal cord have the highest priority in ongoing trials [®", developed by the Intrasense Company, Montpellier, France) was used for delineation and volume determination of the tumor and OARs : the GTV, the right lung, the left lung and, the spinal cord.As shown in Figure g 20 Gy) . Choosing trials . An imag®" is not able in the current version to calculate T/L ratiovol and T-Cdist, all these data are then transferred to the RPO, where their calculations and graphical presentation are automatized for each respiratory phase. In this process, "Myrian®" is unable to perform an automatic propagation of TV and OAR delineated from one phase to the other nine. So, this sequence is repeated for each of the ten phases of the respiratory cycle and with the CT data acquired in maximal inspiration (the reference). Once the data are collected, the RPO is able to display a bar graph for both comparison parameters: the T/L ratiovol and the T-Cdist. A graph displays, in addition, the absolute ipsilateral lung volume measured at each respiratory phase. A synoptic summary of the two graphs is presented to the user who may then proceed to the assessment of the optimal respiratory phase.Because "MyrianFor the present study the percentual difference between the optimal respiratory phase and maximal inspiration (the reference) was assessed. If both were coincident, we computed, in addition, the percentual difference between the optimal respiratory phase and the least optimal one. We considered that there was no gain if the difference was ≤ 20%.vol) and (T-Cdist) for the 14 patients according to the ten sequential respiratory phases chosen in our study.Patients and tumor characteristics are presented in Table vol, the optimal respiratory phase coincided with maximal inspiration in only 6 cases. The mean difference between the optimal respiratory phase and maximal inspiration (the reference) was 15% (SD ± 19) ranging from 0  to 67%. Compared to the worst phase of the respiratory cycle, the mean difference between the optimal phase and the less optimal one was 34% (SD ± 18) ranging from 12 to 79%.As also shown in Table dist, the optimal phase coincided with maximal inspiration in only 3 cases  and differing from maximal inspiration in 7 (50%). In only one patient (7%) (Patient #4) maximal inspiration was optimal for the T/L ratiovol, but was suboptimal for the T-Cdist Figure With a cut-off of 20% only 2 cases showed no benefit in either of both parameters (patients #7 and #10). In 11 patients, however, a substantial gain was observed for the T/L ratioet al., observed large diaphragm displacements in the cranio-caudal direction during free breathing with an average range of 34 mm and a maximum of 67 mm between inspiration and expiration. Reduced motion, however, has been reported for tumors in the lung apices with an average of 8 mm displacement in the cranio-caudal direction between inspiration and expiration [Physiological respiratory motion is a major challenge for lung cancer RT. The range of motion can reach an average up to 12 ± 6 mm for tumors in the lower lung lobes . Giraud piration . A patiepiration . As a copiration . The mospiration ,23. It iet al., in his analysis of intrathoracic organ motion during breathing [vol or T-Cdist., translating in a potential benefit from respiratory gating techniques. Giraud et al., observed also that the smallest displacements were in the apices and near the tracheal carina. This is in agreement with our observation that centrally located tumors may benefit less from gating based on the present algorithm, especially when fibrous attachments to the mediastinum restrict their mobility . Five tumors growing in the superior lung regions  presented less, though not negligible, changes in the chosen comparative parameters. For patients with tumors growing in the posterior mediastinum, close to the spinal cord, the RPO helped to find the optimal respiratory phase other than maximal inspiration . Maximum inspiration, the reference, was optimal in only 28.6% of cases  benefitted the most from gating [Although, gating techniques are reasonably time consuming, and they may not be needed for every patient. A threshold of tumor motion or tumor volume needs to be defined above which gating can be recommended. Starkschall m gating . TherefoEasy to apply in daily routine, fast in getting the optimization result, and no special hardware needed are the main practical advantages of the RPO worth to be highlighted. It is important, however, to plan on a 4D-CT to be able to acquire synchronized image sets. Data analysis represents about 2000 calculations  for every patient.Variability in target volume delineation is a major source of error in 4D-CT treatment planning. Because all the contours were defined by the same author, inter-observer variability was unavailable in our study in response to the need of technique novelties claimed in some recent literature in the 4D-CT era . In a neAn evident limitation of our study is the reduced number of patients studied so far and the restricted morphological parameters of the comparison not including dose-volume parameters in the analysis. Nevertheless, it seems reasonable to assume a dosimetric gain when treating patients in the optimal respiratory phase selected by the RPO.®" may also be used to assess the treatment response after treatment.Further development of the presented software is planned in order to adapt it for tumor locations in the upper abdomen as treatment reproducibility may also be conditioned by respiratory motion. In addition, the density histograms obtained with "MyrianThe RPO software presented in this study can help to determine the optimal respiratory phase for gated RT based on a few simple morphological parameters. Easy to apply in daily routine, it may be a useful tool for selecting patients who might benefit from BART.The authors declare that they have no competing interests.NP conceived the RPO software, provided and cared for study patients, performed all target volume and OAR delineation, contributed to data acquisition and drafted the manuscript. JV contributed to the study design, provided and cared for study patients, contributed to data acquisition and revised the manuscript critically. VVH contributed to the presentation of our results and revised the manuscript critically. PF contributed to the study design , and provided Montpellier patient data. DA contributed to the study design  and provided cooperation with CLRC Val d'Aurelle. HZ conceived and introduced the use of low dose 4DCT in Geneva and contributed to data acquisition. MW provided collaboration with the Nuclear Medicine department in Geneva and provided and cared for study patients in the Nuclear Medicine department. OR provided collaboration with the Nuclear Medicine department in Geneva and assumed the overall responsibility from the Nuclear Medicine department. RM permitted to NP to develop this study in the Radiation Oncology department in Geneva, revised the manuscript critically and assumed the overall responsibility for the study. All authors read and approved the final manuscript.


Monoclonal antibodies are widely used both in infants and in adults for several indications. Humanized monoclonal antibodies  have been used for many years for the prevention of respiratory syncytial virus infection in pediatric populations  at high risk of severe and potentially lethal course of the infection. This drug was reported to be safe, well tolerated and effective to decrease the hospitalization rate and mortality in these groups of infants by several clinical trials. In the present paper we report the development and the current use of monoclonal antibodies for prophylaxis against respiratory syncytial virus. Pneumovirus genus and Paramyxoviridae family. It has an RNA genome which is enveloped, negative-sense single-stranded, and nonsegmented; it is composed of 10 genes encoding for 11 proteins. The envelope is formed by 4 proteins in a lipid bilayer: the two glycosylated surface G and F (fusion) proteins, the M (matrix) protein, and the SH  protein.Respiratory syncytial virus (RSV) was discovered in 1956 and it was classified as a member of The G and F proteins play a key role in the pathogenesis of the infection since the G protein determines the adhesion to the cells of the respiratory epithelium, while the F protein is responsible for the entry of the virus in the cells and determines the insertion of viral RNA in the cell which is responsible for the formation of syncytia . Two subtypes of RSV, A and B, are different for the G protein structure; RSV A and B coexist during every RSV epidemic season and the subtype A seems to be associated with more severe infections , 3. NeutRSV is responsible for respiratory tract infections that could lead to severe respiratory failure and death in infants, especially in those born extremely preterm or affected by some chronic conditions. RSV is a worldwide infection whose specific antibodies are detected in 87% of 18-month-old infants  and virtRSV is one of the major causes of lower respiratory tract infections (LRTI) during infancy with high rates of hospitalization and mortality during the first years of life , 6. The widespread diffusion of the virus and the high interhuman diffusion lead to large epidemics in infants younger than 5 years with a strong economic impact due to the increase of pediatric visits, emergency room accesses, and hospitalizations .Moreover, it was observed that RSV-associated mortality during the first year of life was ninefold higher than influenza-associated mortality .The number of worldwide episodes of RSV infections  in childThe incidence of RSV-related hospitalization in the USA increased from 22.2% in 1980 to 47% in 1996 during the first year of life and from 5.4% to 16.4% at any age . The estPediatric patients at high risk of RSV morbidity and mortality include preterm infants, especially those with CLD, infants with CHD, neuromuscular diseases, cystic fibrosis, and congenital or acquired immunodeficiency , 13.Considering the great impact of RSV infections on children health, several studies were conducted to identify risk factors and to develop an effective tool for specific prophylaxis in high-risk infants.Prematurity is one of most important risk factors for severe RSV infections in young infants because of their immature immune response and their incomplete development of the lungs and the airways. Low GA is one of the most relevant risk factors for RSV-related hospitalization during the first months of life: the yearly hospitalization rate is lower in term infants (4.4%) than in infants born before 28 weeks' GA (9.4%) .Multicenter studies were conducted to analyze RSV epidemiology in large cohorts of term and preterm infants; a higher risk for RSV infections was reported among preterm infants, including also late preterm infants (33–35 weeks GA); these studies also evaluated the risk factors for RSV-related infection and hospitalization in preterm infants –22. The A multicenter study conducted in Italy  reportedRisk factors for severe RSV infections in late preterm were also investigated in the PICNIC study (Pediatric Investigators Collaborative Network on Infections in Canada) by Wang et al.  (1995) aMoreover, a multicenter cohort Italian study started in November 2009 enrolled newborns born at GA ≥ 33 weeks: preliminary data showed that infants of the lower GA group (33 weeks + 0 days–34 weeks + 6 days) were at a slight higher risk of hospitalization for LRTI during the first year of life .Risk factors for RSV hospitalization reported in these three studies are summarized in CLD is a chronic pulmonary disease which may affect premature infants characterized by oxygen requirement after 28 days of age . The patThe RSV-related hospitalization rate in infants younger than 6 months with CLD is 56.2/100 children/year [In our experience , infantsOther congenital malformations are associated to a more severe course of RSV LRTI; in a birth cohort study conducted in Colorado from 1997 to 2004, the risk for RSV-related hospitalization was higher in infants <2 years with spina bifida, agenesis, hypoplasia or dysplasia of the lung, cleft palate alone, and biliary atresia .Congenital or acquired immunodeficiency may also increase the risk of RSV related hospitalization.Moreover, Luján-Zilbermann et al.  reportedNeuromuscular diseases are also associated with severe respiratory infections, especially in the presence of technology dependence and respiratory support ; in a prThe prevention of RSV infections in infants is extremely important to decrease the great amount of complications and hospitalization in young infants. For this reason, pharmacological research during the last 20 years aimed at the development of a safe, well-tolerated, and effective drug and led to the current use of monoclonal antibodies.The development of a safe, effective, and well-tolerated drug for RSV prophylaxis in high-risk infants has been studied for many years through clinical trials. Many authors reviewed the “history” of the development of the drug used for specific prophylaxis –32.Passive prophylaxis was at first introduced in the 1990s with standard intravenous immunoglobulin (IGIV) after studies developed in cotton rats ; neutralThe effectiveness of RSV-IGIV to prevent RSV infections requiring hospitalization in children with cardiovascular disease was analyzed in a randomized controlled trial enrolling 416 children younger than 4 years with CHD or cardiomyopathy . MonthlyBeyond the environmental prevention used to limit the virus diffusion especially in hospitalized patients, nowadays the prophylaxis of RSV-related hospital admissions is based on the administration of specific monoclonal antibodies to the infants at high risk.At first, a murine monoclonal anti-RSV antibody (mAb) was developed; it was an IgA intended for topical nasal administration . HoweverThe mAbs neutralizing G surface glycoprotein are not enough effective because of the variability of this protein between the two viral subtypes A and B. F protein has less heterogeneity, and it is stable in different seasons and in different geographic areas ; thus itSB 209763 is an IgG1 specific antibody against the C epitope of the F protein. Its effectiveness is unclear because a large, multicenter, placebo-controlled clinical trial did not report a significant reduction in the number of RSV-related hospital admission after the monthly 10 mg/kg dose administered to 800 European and American children .Palivizumab is an IgG1 antibody specific for a different epitope (A) of F protein that was introduced in the United States in 1998; it is currently the only approved monoclonal antibody used for RSV prophylaxis , 45. It The mAbs have the same properties of a human IgG1, with a long half-life (28 days). It is free from the risk of transmission of blood-borne pathogens and it can be produced in large batch lots that provide sufficient supply. Beeler and Van Wyke Coelingh in 1989  evaluatePalivizumab reduces RSV replication  through μg/mL. This serum concentration was considered to be a protective value for human receiving palivizumab to prevent RSV infections.The biological characteristics of palivizumab were analyzed in cotton rat models . The ratSeveral trials were also conducted to determine if a noninhibitory concentration of antibodies could promote viral replication or virus-mediated respiratory disorders; this hypothesis was derived from previous studies conducted in the 1960s . It was The possibility of failure of palivizumab efficacy due to genetic variation of the A epitope of the F protein was excluded by surveillance studies , 50.The pharmacokinetic characteristics of palivizumab after a single intramuscular (IM) 15 mg/kg injection were analyzed with the aim of defining the prophylaxis schedule , 52; theThe effect of the drug on RSV detection in the airways was investigated in hospitalized children with severe RSV disease after an intravenous (IV) dose . The RSVμg/mL after the first dose, 57 ± 2.4 μg/mL after the second dose, 68  ±  2.9 μg/mL after the third dose, and 72 ± 1.7 μg/mL after the fourth dose. Serum antibody concentration in infant with CHD was not different after the first and the fourth doses [A study conducted to investigate pharmacokinetic properties of palivizumab in infants <2 years without CHD receiving 15 mg/kg of palivizumab IM monthly reported a half-life of approximately 20 days and a mean serum antibody titer  of 37 ± 1.2 th doses . μg/mL after the first administration and 86.2 ± 4.2 μg/mL after the fourth administration in the second season of prophylaxis [Children who received palivizumab in the previous epidemic season had mean concentrations 60.7 ± 2.4 phylaxis .μg/mL before cardiac bypass and of 41 ± 33 μg/mL after bypass; this 58% decrease had no defined clinical consequences [Another study administered palivizumab with the same schedule to 139 infants younger than 2 years with haemodynamically significant CHD and reported a serum concentration of 98 ± 52 equences . Pharmacequences . A phaseSubsequently a phase II trial was conducted enrolling 31 Japanese children (19 preterm and 13 with CLD) with the previous prophylaxis schedule; mean serum titer through levels and AUC was not different in both Japanese and overseas infants . No adveThese pharmacokinetics trials guided the development of current indications for palivizumab use.The monthly schedule of palivizumab administration should be continued also if an RSV infection occurs; the prophylaxis should start before the beginning of the RSV season (from November to April in the northern hemisphere) . Since the antibody serum titer is lower after cardiac bypass, patients undergoing this procedure should receive an injection of palivizumab as soon as possible after surgery.The monthly dose should be injected by aseptic technique in the anterolateral area of the thigh, avoiding the gluteal area because of the risk of injury of the sciatic nerve. Volumes over 1 mL should be administered in divided doses. The efficacy and safety of palivizumab were investigated in two randomized, double-blind, placebo-controlled trials: the Impact-RSV trial  and anotThe Impact trial was performed during one RSV season in 1998 and enrolled a cohort of 1502 infants younger than 2 years with CLD or infants born before 36 weeks' GA and aged less than 6 months. The cohort was divided as follows: 1002 randomized children received 15 mg/kg palivizumab at monthly intervals, and 500 received placebo during the RSV season; all children were followedup for 150 days after the enrollment. Primary endpoints were an RSV-positive respiratory illness requiring hospital admission or a moderate respiratory illness in infants who had had a positive RSV test during a previous hospital stay; the number of RSV-related hospitalizations decreased by 55% in the overall palivizumab group, with a reduction in days of hospitalization; the number of days requiring mechanical ventilation, days of hospitalization for other causes, and the incidence of otitis media did not differ between the two groups. This reduction was greater in preterm infants  than in infants with CLD , who often require hospitalization also for mild respiratory disease. The reported adverse events were not different between the two groups and included injection site reactions , rash (0.9% versus 0.2%), fever (2.8% versus 3%), and nervousness (2.6% versus 2.5%). The mortality rate was 0.4% in the palivizumab group and 1% in the placebo group; none of the deaths was related to palivizumab administration.The mortality during RSV-related hospitalization was 6.7% in studies that included deaths for causes not related to the infection , 20.The efficacy and safety of palivizumab in two seasons were analyzed in 88 infants of the Impact study . The preA randomized, double-blind, placebo-controlled multicenter trial enrolled 1287 children aged ≤2 years with haemodynamically significant CHD  who receThe incidence of adverse events  was not different in the palivizumab group and in the placebo group and never led to drug discontinuation. Mortality was not different in the two groups and no deaths were related to palivizumab; moreover, the drug did not affect the management of the CHD.A recent review  stated tPalivizumab was also reported to reduce by 80% the risk of recurrent wheezing in children aged 2–5 years and born prematurely, with a 68% decrease in infants without family history of asthma and 80% in infants without atopic background, but this protective effect was not observed in infants with an atopic family . This reMoreover, a prospective Italian study comparing 154 palivizumab recipients to 71 palivizumab nonrecipients  observedThe treatment of bronchiolitis is essentially based on respiratory support, adequate fluids and nutrition supply, and therapy of respiratory symptoms, since an etiologic therapy does not exist. The safety and effectiveness of palivizumab in children hospitalized for acute RSV infections were investigated in a phase I/II, randomized, multicenter, placebo-controlled, double-blind trial that reported the absence of differences in clinical outcomes between palivizumab and placebo group; no adverse events requiring trial interruption were observed . Another drug of the group of monoclonal antibodies  is a molecule that differs from palivizumab in 13 amino acid residuals; both mAbs are specific for the site A of the F protein of respiratory syncytial virus. The neutralizing effect was measured both in vitro and in animal models and found to be higher for motavizumab –66.Large trials examined clinical effects of this drug for RSV prophylaxis. A double-blind, randomized, phase 2 trial  comparedA multicenter phase 3 trial involving more than 300 centers  analyzedSafety, immunogenicity, and pharmacokinetics of motavizumab were determined in a randomized, open-label phase I-II study enrolling 217 high-risk children; motavizumab characteristics were comparable to palivizumab .In January 2008 MedImmune submitted to the Food and Drug Administration the request of a biologic license application for motavizumab, but the advisory committee refused to endorse the request for the use of motavizumab in the prophylaxis of RSV infections in the high-risk pediatric population because it had the same safety, efficacy, and tolerability of palivizumab, which has been in use since the 1990s.Since palivizumab is a safe, well-tolerated, effective but expensive drug, its use is regulated by national and international guidelines based on pharmacoeconomic studies.The use of the prophylaxis also in late preterm infants has been a matter of concern for many years , 71, sinThe prophylaxis was observed to be useful especially in late preterm infants with >1 risk factors for RSV LRTI  . A prospective, multicenter Italian study  includinClinical trials are ongoing in order to establish the cost-effectiveness of prophylaxis in this population and to improve the existing guidelines for prophylaxis schedule with the support of epidemiological data. A cost-utility analysis was made in 4 cohorts of preterm infants ; palivizThe positive impact of prophylaxis with palivizumab on healthcare expense was also confirmed by two retrospective analyses made in Austria  and in SA pharmacoeconomic study could also improve the guidelines in use for the prophylaxis of RSV infections in infants with other risk factors than prematurity . Palivizumab cost-effectiveness was confirmed in infants with neuromuscular diseases, congenital diaphragmatic hernia, but it is still controversial in infants with other diseases .RSV has a worldwide diffusion and it is responsible for a large amount of hospital admissions in young infants, with a subsequent strong impact on infants' health and healthcare costs. RSV infections may lead to respiratory failure and death, especially in infants with chronic pathological conditions. An etiologic treatment for RSV bronchiolitis does not exist at the present, while the use of palivizumab for prophylaxis of RSV infections in high-risk infants is recommended since it is well tolerated and effective in the reduction of RSV-related hospitalizations. Palivizumab is the only monoclonal antibody currently used for anti-infectious purpose.The extension of prophylaxis with palivizumab to late preterm infants (GA 33–35 weeks) is still a matter of concern since this drug is too expensive to be used for the entire population of late preterm infants. The cost-effectiveness of the use of palivizumab in the late preterm has been analyzed by several studies to identify environmental or individual risk factors for severe RSV infection. The use of risk scores derived from this study is helpful to detect the subjects for whom the administration of palivizumab could be effective to reduce RSV-related mortality and morbidity.These positive results subsequent to the use of palivizumab to prevent an infectious disease could encourage the use of monoclonal antibodies for prevention and treatment of other infectious diseases in infants and adults.


Drosophila development, prior to the establishment of MSL-mediated dosage compensation, suggesting the existence of a novel mechanism.Mmany genes from the X chromosome are expressed at the same level in female and male embryos during early  Drosophila melanogaster embryos initiate zygotic transcription around mitotic cycle 10, the dose-sensitive expression of specialized genes on the X chromosome triggers a sex-determination cascade that, among other things, compensates for differences in sex chromosome dose by hypertranscribing the single X chromosome in males. However, there is an approximately 1 hour delay between the onset of zygotic transcription and the establishment of canonical dosage compensation near the end of mitotic cycle 14. During this time, zygotic transcription drives segmentation, cellularization, and other important developmental events. Since many of the genes involved in these processes are on the X chromosome, we wondered whether they are transcribed at higher levels in females and whether this might lead to sex-specific early embryonic patterning. To investigate this possibility, we developed methods to precisely stage, sex, and characterize the transcriptomes of individual embryos. We measured genome-wide mRNA abundance in male and female embryos at eight timepoints, spanning mitotic cycle 10 through late cycle 14, using polymorphisms between parental lines to distinguish maternal and zygotic transcription. We found limited sex-specific zygotic transcription, with a weak tendency for genes on the X to be expressed at higher levels in females. However, transcripts derived from the single X chromosome in males were more abundant that those derived from either X chromosome in females, demonstrating that there is widespread dosage compensation prior to the activation of the canonical MSL-mediated dosage compensation system. Crucially, this new system of early zygotic dosage compensation results in nearly identical transcript levels for key X-linked developmental regulators, including giant (gt), brinker (brk), buttonhead (btd), and short gastrulation (sog), in male and female embryos.When  Drosophila compensate for the difference in X chromosome dosage  with a mechanism that allows for more transcription of the single X chromosome in males. But this mechanism is not established until over an hour after the embryo begins transcription, during which time a number of important events in development occur such as cellularization and segmentation. Here we use an mRNA sequencing method to characterize gene expression in individual female and male embryos before the onset of the previously characterized dosage compensation system. While we find more transcripts from X chromosomal genes in females, we also find many genes with equal transcript levels in males and females. These results indicate that there is an alternate mechanism to compensate for dosage acting earlier in development, prior to the onset of the previously characterized dosage compensation system.Variation in gene dose can have profound effects on animal development. Yet every generation, animals must cope with differences in sex chromosome numbers.  Drosophila melanogster, the MZT occurs amidst the 14 rapid and synchronous mitotic divisions that mark the first several hours of development, with zygotic transcripts appearing as early as mitotic cycle 8 The earliest stages of animal development are under maternal control until mRNAs deposited prior to fertilization degrade and zygotic transcription is initiated during a period known as the maternal to zygotic transition (MZT). In Sex lethal (Sxl) rox1 and rox2) that bind to the X chromosome, hyperacetylate histone H4K16, and induce hypertranscription of the male X chromosome As zygotic transcription begins, the different numbers of X chromosomes  results in different transcript levels for a small number of genes on the X chromosome , which lead to female-specific expression of the master sex control gene giant (gt), buttonhead (btd), orthodenticle (otd) and runt (run), D–V factors brinker (brk), short gastrulation (sog) and neijire (nej), and the cellularization factor nullo.However, there is a lag between the onset of zygotic transcription and the establishment of MSL-mediated dosage compensation: the complex is not localized on DNA, and H4K16 acetylation is not detectable, until after the blastoderm stage We were intrigued by the possibility that the absence of MSL-mediated dosage compensation during the MZT might lead to higher levels of mRNAs derived from genes on the X chromosome in females, and sex-specific differences in patterning or cellularization that have not been detected because systematic studies of early developmental transcription have never differentiated male and female embryos.A variety of approaches have been used to profile zygotic transcription during the MZT, including genome-wide expression profiling with microarrays D. melanogaster embryos, which we combined with methods to precisely stage and sex single embryos to generate sex-specific time courses of maternal and zygotic transcript abundance spanning the first wave of early zygotic transcription through the MZT to the end of the blastoderm stage when MSL-mediated dosage compensation is thought to begin To address these limitations, we developed methods to characterize, by sequencing, the mRNA content of individual In order to create a precise time series of zygotic transcription in male and female embryos during embryonic development, we needed methods to demarcate small differences in developmental time, to determine the sex of embryos, and to measure the entire pool of transcripts in these embryos in a way that distinguished mRNAs of maternal and zygotic origin.We chose to focus on the period of development bounded by cycle 10 (when early zygotic transcription is detectable) and the completion of cellularization in mitotic cycle 14 .To determine developmental stage, we took advantage of two characteristics of early embryos: the tightly controlled synchronous mitotic cycles and the process of cellularization as the embryo transitions from a syncytium to a cellular blastoderm . We examWe selected at least four embryos each for cycles 10, 11, 12, 13, 14A, 14B, 14C, and 14D, and extracted DNA and RNA from each embryo independently. We carried out whole-genome amplification on the DNA from each embryo and genotyped it for Y chromosomal markers to determine the sex of the embryo, and selected at least one male and female embryo from every stage for transcriptome analysis. We obtained 75 to 100 ng of total RNA from each embryo. As this was less starting material than required for standard mRNA sequencing protocols, we modified the Illumina mRNA-Seq protocol to obtain reliable data from such small quantities of input mRNA without amplification by performing all purification and size selection steps using magnetic beads, and reducing the volume of some reactions (a complete protocol is available in D. melanogaster reference sequence (version 5.23) using Bowtie We sequenced a total of 24 mRNA samples on an Illumina GAIIx Genome Analyzer. We aligned reads to the The single embryo mRNA-Seq method was highly reproducible and has a wide dynamic range . TranscrD. melanogaster lines: a w1 derived maternal line (which contained the His2Av-RFP marker) and a Canton-S (CaS) paternal line. We sequenced both lines to roughly 35× coverage . This is consistent with these strains representing independent samples drawn from resident populations in the United States. Although both lines have been in laboratories for decades, we found that each harbored a significant amount of residual polymorphism, especially CaS. We therefore restricted our subsequent analyses to a set of 122,672 SNPs that were fixed between strains.The vast majority of these differences were biallelic single-nucleotide polymorphisms (SNPs) known from resequencing projects to be polymorphic in the North American w1 or CaS  reads must have been the result of zygotic transcription. We were thus able to partition the overall expression of any mRNA containing w1-CaS differences into its maternal and zygotic component  contained at least one fixed polymorphism, allowing us to assign RNA-Seq reads spanning the polymorphism to either 1 or CaS . Since momponent . As expeomponent . We obseomponent .w1-CaS polymorphism into 20 groups based on similarity of their inferred abundance of maternally and paternally derived transcripts. We classified each cluster as maternal (only w1 mRNAs detected with levels declining over time), zygotic , or maternal and zygotic . Because of the absence of paternal alleles for genes on the X chromosome in males, all assignments were based on data from females only. We classified genes lacking polymorphisms distinguishing the strains by comparing their mRNA abundances from the eight female samples to the average patterns from each of the previously assigned groups. We assigned genes to the group with which their expression pattern was best correlated (if the correlation coefficient was greater than 0.8). Overall, 5,598 genes were classified as maternal, 2,210 as zygotic, and 1,195 and maternal+zygotic  the 5,226 genes with at least 10 reads spanning a sisA, sisB , and run are expressed at higher levels in females  the X chromosome. Total expression of zygotic genes from the paternal and maternal X chromosomes of females was very similar , our data speak only indirectly to the mechanism by which it occurs. Assuming that, in an uncompensated system, we would expect transcription to produce twice as many zygotically derived copies of X chromosomal genes in females than in males, the generally lower levels we observe in females must arise through sex and X-chromosome-specific transcriptional or post-transcriptional regulation.The simplest explanation is that the MSL-based dosage compensation system is active before and during cycle 14, leading to hypertranscription of the male X. However, several imaging studies of the male-specific localization of MSL proteins to, and the subsequent acetylation of histones on, the male X chromosome describe an at least 1 h lag between the onset of zygotic transcription and these hallmarks of MSL-mediated dosage compensation run hypomorphic alleles, Gergen demonstrated that the X-linked gene run, which is involved in both sex-determination and segmentation, is functionally dosage compensated run is expressed throughout embryogenesis, the effects on larval cuticle patterns these studies examined arise during the blastoderm stage and are thus an example of EZDC. We also find that run is dosage compensated during cycle 14. Gergen run is MSL independent but requires the early female-specific form of Sxl.Through an analysis of larval cuticle patterns of male and female embryos carrying various combinations of run might result from direct SXL-mediated reduction of the translation or stability of run in females run mRNA contains several matches to the SXL consensus sequence run dosage compensation has not been confirmed.Since SXL is an RNA-binding protein known to modulate splicing and translation, it was proposed that dosage compensation of msl-2 mRNA, which it regulates by translational repression, and its own mRNA, which it regulates by controlling how it is spliced. However, a total of 88 genes (including run) have transcripts whose 3′UTRs contain three or more SXL target sites (AUUUUUUU or UUUUUUUU). And of these an astonishing 76 are on the X chromosome. This striking enrichment, originally noted by Kelley et al. The two best-characterized targets of SXL are gt, for example, is not a predicted target) and many predicted SXL targets are not or are poorly dosage compensated  Canton-S males, and eggs were collected from many 3–6-d-old females, thus minimizing chances that multiple embryos sampled would come from the same mother. After collection, eggs were dechorionated, placed on a slide in halocarbon oil, and visualized using a Nikon Eclipse 80i microscope, with a Nikon DS-UI camera, and the NIS Elements F 2.20 software. Embryos were photographed both for fluorescence with an RFP filter to visualize nuclei and under white light with a DIC filter to visualize the extent of cellularization in mitotic cycle 14 embryos , and a region on chromosome 2R  as a control.RNA and DNA extraction from single embryos was done with TRIzol (Invitrogen) reagent according to the manufacturer's protocol, except with a higher volume of reagent relative to the amount of material . Extracted DNA was amplified using the Illustra GenomiPhi V2 DNA Amplification Kit , and embryos were sexed by detecting the presence of a Y chromosome, using PCR with primers to a region of the male fertility factor kl5 on the Y chromosome  using maq (maq.sourceforge.net). We found that consensus base and SNP calling algorithm was adversely affected by the high level of polymorphism, especially in the CaS sample, so we exported the base-by-base pileup from maq and developed our own SNP calling algorithm. We designated a position as a CaS-w1 SNP if there were at least 13 reads covering the base in each strain, if the frequency of the most common base in each strain was at least 95%, and if these most frequent bases differed. We also generated a w1-CaS consensus sequence consisting of the reference D. melanogaster bases, except where the sequences of the two strains agreed but differed from the reference. We identified all RNA reads expected to differ between the strains, counted their frequencies in each sample, and partitioned the RPKM values for individual genes into their w1 and CaS components in proportion to the fraction of reads in that sample that mapped to the maternal or paternal chromosome. Upon examination of the data, we became concerned that absence of reads from the paternal X chromosome and the low levels of Sxl in embryo F13 arose from a genotyping error. So for graphs showing single genes, we use an average between F12 and F14A for this time point.Reads from each RNA-Seq sample were mapped to the reference w1-CaS polymorphism into 20 groups based on similarity of their inferred abundance of maternally and paternally derived transcripts using Cluster 3.0 w1 mRNAs detected with levels declining over time), zygotic , or maternal and zygotic . Because of the absence of paternal alleles for genes on the X chromosome, all assignments were based on data from females only. We classified genes lacking polymorphisms distinguishing the strains by comparing their mRNA abundances from the eight female samples to the average patterns from each of the previously assigned groups. We assigned genes to the group with which their expression pattern was best correlated (if the correlation coefficient was greater than 0.8).We used the strain-specific time series to classify genes as maternal, zygotic, or maternal and zygotic. We clustered (k-medians) the 5,226 genes with at least 10 reads spanning a All reads have been deposited in the NCBI GEO under the accession number GSE25180 and will be made available at the time of publication. The processed data are available at the journal website  and at eFigure S1Extent of dosage compensation for zygotic genes on the X chromosome. Each gene was assigned a female to male (F∶M) ratio score equal to the slope of the line fit (by least squares) to the male and female transcript levels for that gene over all time points. (A) Proportion of genes with F∶M ratios between 1.0  and 2.0 . (B) Transcript abundance time series for zygotic genes on the X chromosome, in female and male embryos, over all time points. Genes sorted by F∶M ratio.(2.11 MB PDF)Click here for additional data file.Dataset S1Normalized read counts per gene for each individual embryo.(2.89 MB TXT)Click here for additional data file.Protocol S1Small sample RNA-Seq protocol.(0.14 MB PDF)Click here for additional data file.Protocol S2Low concentration sequencing library loading protocol.(0.08 MB PDF)Click here for additional data file.


The aim of our study was to compare the intake of nutrients and nutritional status of cow’s milk allergic (CMA) children supported by a dietician and children with no dietary limitations (NDL).We observed 54 children  with CMA which had at least one dietetic evaluation and received dietary support to adequately integrate their diet, and 54 children  with no dietary restrictions and no dietary intervention. The assessment of nutrient intakes  was based on a 7-day food diary. All values were compared to the Italian Recommended Daily Allowance (RDA). Children were divided by age groups: 1-5years (1), 6-10years (2), 11-18years (3). Nutritional status was assessed by measuring weight and height related to the standard for age and sex.The mean weight and height were within the norm range in both populations. 32% of NDL children and 23% of CMA had a mean weight-for-age percentile >75. In all age groups, CMA patients had a medium energy intake (EI) higher than the controls. The mean protein intakes of both populations at different ages were higher than the RDA. Mean carbohydrates (CHO) intakes were lower in healthy children than in children with CMA and there was no difference in mean fat intakes. The mean intake of calcium, iron and fibre were below the RDA in both populations Table .Our data show that children with CMA on an elimination diet, who received dietetic support, have a nutrient intake similar to children with no dietary limitations. The low calcium quantity in CMA children is a critical issue that should be monitored in other to supplement children, especially in the older groups (group 2 and group 3) with a higher RDA. These preliminary data confirm the importance of a dietetic support in allergic children in all age groups. The nutrient intake suggests that, also in children with no dietary restrictions, more attention should be paid to the nutritional aspects by paediatricians.None declared.


Genome-wide association studies have been successful at identifying common disease variants associated with complex diseases, but the common variants identified have small effect sizes and account for only a small fraction of the estimated heritability for common diseases. Theoretical and empirical studies suggest that rare variants, which are much less frequent in populations and are poorly captured by single-nucleotide polymorphism chips, could play a significant role in complex diseases. Several new statistical methods have been developed for the analysis of rare variants, for example, the combined multivariate and collapsing method, the weighted-sum method and a replication-based method. Here, we apply and compare these methods to the simulated data sets of Genetic Analysis Workshop 17 and thereby explore the contribution of rare variants to disease risk. In addition, we investigate the usefulness of extreme phenotypes in identifying rare risk variants when dealing with quantitative traits. Finally, we perform a pathway analysis and show the importance of the vascular endothelial growth factor pathway in explaining different phenotypes. In disease association studies, the common disease/common variants (CDCV) model states that common diseases are caused by common variants with minor allele frequencies (MAFs) in the range of 1–5%. Recent studies suggest that although many more common disease susceptibility variants may still exist, they will likely have even smaller effect sizes and thus will be unlikely to explain most of the missing heritability for many of the traits ,3. ThereMany statistical methods have been developed to test association between common SNPs and disease traits, but these methods have low power for identifying rare variants in complex diseases because of the low frequencies and large number of such variants ,6. SeverIn this paper, we apply these methods to the simulated data sets provided by Genetic Analysis Workshop 17 (GAW17) to analyze the effect of rare variants on various phenotypes and evaluate the performance of each method by comparing the results with the true disease model. We also study the effect of trait dichotomization when dealing with quantitative traits. In addition, we investigate the importance of the vascular endothelial growth factor (VEGF) pathway to different phenotypes in the GAW17 data set.The simulated data set from GAW17 consists of 3,205 autosomal genes with 24,487 SNPs genotyped on 697 subjects. Three quantitative phenotypes  and the Affected Status phenotype are generated for each of the unrelated individuals, and 200 simulated replicates are available to us. In particular, each simulation contains 209 case subjects and 488 control subjects for the Affected Status phenotype and three normally distributed quantitative traits . In the disease model, Q1 is caused by SNPs in the VEGF pathway and Q2 is caused by SNPs related to cardiovascular risk and inflammation; the trait Affected Status is affected by SNPs from both biological processes. See Blangero et al.  for moreWe implemented three recently developed strategies for association testing with rare variants: the gene-based combined multivariate and collapsing (CMC) method of Li and Leal , the weiBecause the methods mentioned are designed for case-control studies, we make some adjustments in order to apply them to quantitative traits. First, we dichotomize the quantitative traits by treating the individuals in the upper quartile as case subjects and those in the lower quartile as control subjects and then apply the CMC, WS, and RB methods to these pseudo-case-control data. Another strategy that uses all the available data is to use the modified weighted-sum test suggested by Price et al.  in whichhttp://www.genome.jp/kegg/pathway.html) and in the NCI-pathway interaction database (http://pid.nci.nih.gov/) and found that 53 genes in GAW17 data sets participate in the VEGF pathway. We grouped the 344 rare SNPs  from these 53 genes and evaluated their effects on Affected Status and Q1.The true disease model suggests that Q1 and Affected Status are affected by genes from the VEGF pathway ; therefoA preliminary analysis indicates that the distribution of SNP frequencies is heavily skewed toward low frequencies. In particular, about 87% of SNPs have a MAF less than 0.05, and about 74% have a MAF less than 0.01. Motivated by the large proportion of rare variants, we decided to investigate their effects on different phenotypes even without prior knowledge of the true disease model.p-value and the relative rank of the gene among all genes in each method, using only the first simulated data set. We also obtained the power (or replicability) for each gene, calculated as the percentage of times the resulting gene-based p-value was less than 0.05 across the 200 replicates. After the true disease model was reviewed in GAW17, our results were compared with the true model. In Table We applied the gene-based CMC, WS, and RB methods for the case-control design and dichotomized quantitative traits; we used Price’s method for quantitative traits only. For each gene we computed the In our analyses, we set two different thresholds to define rare variants: MAF < 0.01 (T1) or MAF < 0.05 (T5). We note here that the results from T1 and T5 are consistent but using T5 tends to give higher power compared to T1 because the sample size (697) is relatively small and the disease model involves variants with MAF greater than 0.01. Therefore, for the sake of clarity, we report here and in Table p-values and their ranks from the WS, CMC, and RB methods highly agree with each other; this makes us confident about the true effect if we observe strong signals across different methods. Our analyses included either all the genes  harboring rare variants or only those genes  containing nonsynonymous rare variants. Among these genes, FLT1 is the easiest to identify. For Q1, FLT1 is statistically significant with p-values less than the Bonferroni-corrected significance level (~1.67 × 10−5) and ranks first among all genes in the first simulation. For Affected Status, FLT1 has a very low p-value, close to the Bonferroni-corrected threshold, especially when only nonsynonymous rare mutations are used. For Q2, disease gene VNN3 is top-ranked with all three methods.We observe that for case-control or dichotomized quantitative trait designs, FLT1 and KDR for Q1 and VNN3 for Q2, which may suggest that they are true positives.For quantitative traits, the two strategies (dichotomization or not) result in different risk genes being detected; in particular, dichotomization identifies more genes with higher power compared with Price’s method. This is partly because dichotomization likely eliminates rare disease variants contained only in individuals with modest phenotype values but it amplifies the signal for rare variants with strong effect that show up in extreme phenotypes. Genes identified by dichotomization tend to have high power because the statistical signal can be consistently detected after removing unidentifiable noise. Therefore selecting extreme phenotypes can sometimes be an alternative strategy in dealing with quantitative traits. On the other hand, these two strategies both detect the same disease genes: Overall, our results are encouraging on these simulated data. In particular, we successfully detected six true disease genes for Q1, five for Q2, and three for Affected Status in the top 50 most significant genes using only the first simulated data set.GOLGA1 for Affected Status, PPP1R14BP1 for Q1, and MAP3K8 for Q2, all have power greater than 50% (data not shown). This can happen for various reasons. Both sequencing errors and population stratification can create these replicable false signals, especially in these data sets, where the genotype data are fixed across simulations. Another reason might be long-range correlation between markers. Our investigation showed that certain consistent false positives are highly correlated with true causal SNPs and that it is difficult to eliminate this artificial linkage effect because of the fixed-genotype design across all simulations.Many of the disease genes are highly replicable in 200 simulations with power greater than 50% Table , indicatWe compared the results using only nonsynonymous rare variants with those using all rare variants and observed that nonsynonymous rare variants improve the rank of true disease genes and the power for most disease genes, especially for trait Q2 Table . This isp-value than using all variants does; this is consistent with previous gene-based analyses and emphasizes again that filtering nonsynonymous mutations is important for identifying influential rare SNPs.We collapsed the rare SNPs (MAF < 0.05) in the VEGF pathway and evaluated their effects on traits Affected Status and Q1 using different statistical strategies Table . The WS In this study, we applied three statistical methods to detect rare variants and successfully pinpointed several true disease genes containing rare SNPs in the GAW17 data sets. We started with the first data set and then made use of the remaining 199 replicates to evaluate the replicability of the discoveries in the first data set. This strategy agrees with the natural procedure when dealing with real data; namely, a first data set is used for discovery purposes, and additional independent data sets are used for replication purposes. We showed that using 5% as the MAF cutoff is better than using 1% in the GAW17 data set because the current sample size is small and disease variants occur at higher frequencies as well. Also, including only nonsynonymous mutations can substantially increase the signal. We also showed that selection of extreme phenotypic individuals can be a useful strategy for rare variant analysis with quantitative traits. Our analyses encountered the difficulty of numerous false positives, some probably resulting from sequencing errors or population stratification. Nevertheless, in real life without knowledge of the true model, the methods we investigated here can help us to detect rare disease SNPs. The consistency of results from different methods can be an indicator of true signal. The three methods performed similarly for the disease genes in these simulations. However, we did observe that the replication-based approach was the most powerful approach for large genetic regions such as pathways, followed by the weighted-sum and collapsing approaches.The authors declare that they have no competing interests.II and RF designed the study. II, RF, SHL and TZ performed the research. II, RF and CHH analyzed data. II and RF wrote the paper. All authors read and approved the final manuscript.


Substantial increases in height have occurred concurrently with economic development in most populations during the last century. In high-income countries, environmental exposures that can limit genetic growth potential appear to have lessened, and variation in height by socioeconomic position may have diminished. The objective of this study is to investigate inequalities in height in a cohort of children born in the early 1990s in England, and to evaluate which factors might explain any identified inequalities.12,830 children from The Avon Longitudinal Study of Parents and Children (ALSPAC), a population based cohort from birth to about 11.5 years of age, were used in this analysis. Gender- and age-specific z-scores of height at different ages were used as outcome variables. Multilevel models were used to take into account the repeated measures of height and to analyze gender- and age-specific relative changes in height from birth to 11.5 years. Maternal education was the main exposure variable used to examine socioeconomic inequalities. The roles of parental and family characteristics in explaining any observed differences between maternal education and child height were investigated.Children whose mothers had the highest education compared to those with none or a basic level of education, were 0.39 cm longer at birth (95% CI: 0.30 to 0.48). These differences persisted and at 11.5 years the height difference was 1.4 cm (95% CI: 1.07 to 1.74). Several other factors were related to offspring height, but few changed the relationship with maternal education. The one exception was mid-parental height, which fully accounted for the maternal educational differences in offspring height.In a cohort of children born in the 1990s, mothers with higher education gave birth to taller boys and girls. Although height differences were small they persisted throughout childhood. Maternal and paternal height fully explained these differences. Height is a highly heritable trait Attained adult height is determined by the potential of a child's genotype and the restrictions that the environment places on this Thus, the aim of this study is to better understand what drives socioeconomic differentials in height from birth to childhood in a contemporary population of UK children born in the early 1990s.http://www.alspac.bris.ac.uk). Briefly, pregnant women living in three health districts in Bristol, England, who had an expected date of delivery between 1 April 1991 and 31 December 1992, were eligible. The recruited ALSPAC sample consists of 14,541 pregnancies that resulted in 14,676 known foetuses.The Avon Longitudinal Study of Parents and Children (ALSPAC) is a population-based study investigating environmental and genetic factors that affect health and development of children. The study methods are described in detail elsewhere Detailed data about her socioeconomic background, health, welfare and lifestyle characteristics were obtained from the mother using four self-reported questionnaires throughout the pregnancy. Data on delivery and birth measurements were obtained by ALSPAC staff or were otherwise extracted from medical records. Since delivery, regular questionnaires have been completed by the child's main caregiver (most commonly their mother) and as they became older, the children themselves.Ethical approval for the study was obtained from the ALSPAC Law and Ethics Committee and the Local Research Ethics Committees. Written informed consent was obtained from all participants involved in the study.st, 2nd and 3rd trimesters was measured. A variable categorized as “Never” or “Ever” smoking during pregnancy and a variable indicating the number of trimesters the mother smoked were created. Finally, the number of previous pregnancies (both live births and stillbirths) and number of living children were reported by the mother.Maternal education was ascertained from the antenatal 32-week questionnaire. Education was coded using an ascending mutually exclusive five point scale of highest educational achievement: “None/Certificate of Secondary Education (CSE)”, 2: “Vocational”, 3: “Ordinary- (O-) level )”, 4: “Advanced- (A-) level  of educational qualifications most commonly attained at 16 years of age ; level 4 refers to educational qualifications gained at 18 years of age. Mothers with no educational qualifications most often left the question unanswered which was recoded to none, and those who responded ‘not known’ were left as missing. A previous report on this cohort found similar socioeconomic differentials in birth length and childhood growth irrespective of whether maternal education, head of household occupational social class or father's education was used as the measure of socioeconomic position (SEP) The height, weight, and smoking habit during pregnancy of the mother's partner were obtained from a partner's self-completed questionnaire that was passed to them via the mother. For 95.5% of the children the mother's partner was the biological father (by mother's self-report). Mid-parental height was calculated using both parents' height adapting Galton's formula Data on delivery and birth measures (crown-heel length and head circumference) were obtained by trained staff of the ALSPAC team for babies born in the two major maternity hospitals in the region and from medical records for the other participants. Gestational age was estimated using the mother's last menstrual period in most cases and through obstetric assessment for the rest. Whether the mother breastfed, and duration of breastfeeding, were ascertained at 6 months and categorized into a composite variable, as “Never or up to one month” versus “more than one month”.th week , 8th month , 18th month  and at the pre-school child visit at 3.5 years . Thereafter, the whole cohort of children was invited to attend clinical examinations. The first ALSPAC direct measurement of height occurred at an average of 7.5 years (range: 6.8 to 9.2 years) and four subsequent yearly examinations were held at ages 8.5 years (range: 7.5 to 10.5 years), 9.5 years (range: 8.7 to 11.7 years), 10.5 years (range: 9.8 to 11.3 years) and 11.5 years (range: 10.4 to 13.6 years). There are in total a maximum of 10 measurements of height per child. In the clinics (from age 7.5 years) height was measured by trained technicians to the last complete millimetre using the Harpenden stadiometer (Holtain Ltd). As far as possible, all children were measured in their underclothes with their shoes removed. For all measurements taken, the tester recorded any problems that may have affected accuracy. In a previous study we have shown that heights assessed from birth to pre-school by health visitors were accurate, by comparing these with research clinic measurements completed on a random 10% sub-sample of the ALSPAC cohort Height after birth was measured by health visitors and general practitioners as part of standard childcare in the UK. The examinations take place at around the 8st week and 6 months, child's age in 1 week intervals; and, iii) heights beyond 6 months, child's age in 1 month intervals. Time intervals with too few observations for appropriate calculation of a z-score were combined with the earlier interval. An alternative method to standardise height using a locally weighted smoother was evaluated but produced similar standardised values (results available from the authors). Z-scores were preferred as they are more easily interpreted and translated to the original scale.Gender- and age-specific z-scores for length/height were calculated. Z-scores control for the association of age and gender on height and its change, and standardise for the increasing variance of the measurements with increasing age. As there was considerable variation in the ages at which the children had their measurements taken, z-scores were calculated within the following time intervals, irrespective of the visit when they were obtained: i) for birth length, gestational age in 1 week intervals for those born from 37 to 43 weeks; ii) for length/height between the 1Exploratory cross-sectional analyses were carried out at each visit to evaluate the association of mother's education with child's height at each age. Multilevel modelling was carried out to model height (in z-scores) change with age and to evaluate the role of the mother's educational level on the child's height z-score trajectory. There was strong statistical evidence in both boys and girls, that a random intercept and random slope model provided a better fit to the data than a model that included a random intercept only . The association with maternal education was evaluated as a categorical and ordinal variable and gender differences in the educational effect on height were tested with an interaction term.2), number of children, gestational age (weeks) maternal smoking during gestation, child's early nutrition measured with breastfeeding and maternal food frequency questionnaire at 32-week pregnancy, paternal height (cm), BMI and smoking habit during gestation. These variables were added in the multilevel model as fixed effects using restricted maximum likelihood. Wald tests were used to evaluate the effect of adding each fixed term. All continuous variables were centred on their mean value. Models were also adjusted for a dummy variable indicating the visit at which the measurement took place to adjust for potential differences occurring between measurements.The following explanatory variables were evaluated to test mechanisms that could explain differences in child's height according to mother's education. These variables were chosen based on previous reports of the literature of determinants of child's height. Maternal age at delivery (years), maternal height (cm) and body mass index  to test the robustness of the results and the assumptions of the models and improve the normality distribution of the outcome variable. All analyses were carried out using STATA (version 10.1 for Windows).The analyses were restricted to singletons as in-utero conditions may differ for multiple pregnancies (390 multiple pregnancies were excluded) and to pregnancies that resulted in a child alive at 27 days after birth  and were term births (≥37 weeks) (693 observations excluded). Children who had no height measure available throughout the entire follow-up were excluded (n = 115). Finally, measurements of birth length that were obtained later than one week after delivery were not considered (180 measurements), as these may no longer reflect birth length, although all subsequent measures of these children were used in the analyses. The final sample included 12,830 children : 4 to 9); 6,251 girls with a median of 7 measurements (IQR: 4 to 9).Mother's educational level was available for 89.4% of the children . Almost 20%  had either no education or education to CSE level, 9.8% had vocational education, 34.8% had O-levels, 22.5% had A-levels and 12.9% of the mothers had a university degree. The median number of height measurements was greater with higher maternal educational level: 4 measurements (IQR: 3 to 5) for mothers with no education or CSE level, 6 measurements (IQR: 4 to 9) for those with vocational training, 7 measurements (IQR: 5 to 9) for mothers with O-levels, and 8 measurements (IQR: 5 to 9) for mothers with A-level or university degrees. Mean height increased with age similarly among boys and girls. Boys tended to be slightly taller than girls up until the age of 7–8 years. Birth length and height were consistently higher with increasing levels of mother's education although the magnitude of these differences across educational groups was relatively small .When considering growth until 10 years of age only in order to assess whether pubertal changes already occurring in some children could have influenced the results, educational inequalities in child's growth remained similar . When all analyses were repeated excluding observations with standardised height values above 2 or below −2 the effect of maternal education on standardised height growth remained although the magnitude of the effect was slightly reduced . Maternal and partner's height remained as the main explanatory variables of the educational differences in child's height growth , whereas adjustment for all other characteristics, but not mid-parental height, resulted in attenuation but some association remained . Finally, educational inequalities in child's growth were slightly greater after removing those with only one or all ten height measures  and the role of the explanatory variables remained the same .Among children born in the UK in the early 1990s, those born to mothers with higher educational levels were taller than those born to mothers of lower educational levels. These height inequalities were present at birth and persisted over time . Mid-parental height fully explained the differentials in child's height growth across maternal educational levels. Although most of the other explanatory variables investigated were associated with the child's height and were socially patterned, they accounted for little of the maternal educational inequalities in the child's height.The improvements in pre-natal and maternal care and child nutrition, along with fewer childhood infections would suggest that inequalities in height due to environmental exposures should have decreased or disappeared in high income countries The Boyd Orr study, a cohort of 4999 children surveyed between 1937–39 in the UK, showed a general pattern of greater stature and body proportions  with better childhood socioeconomic and housing circumstances as well as diet We found a similar pattern of association of potentially modifiable exposures with height growth in ALSPAC with those reported in the literature However, although these factors were associated with child's height they explained little of the inequalities, whereas educational inequalities in childhood height were no longer present when comparing children whose parents had the same mid-parental height. Li et al compared the effect of parental height in the two generations and found parental height had a stronger association in the offspring than the parents' generation Parental height is often used as proxy for the genetic component of height. Indeed, the fact that the combination of both parents' height was necessary to account for the height differences, rather than maternal height alone, does seem to point to a strong genetic contribution. However, parental height partly reflects the embodiment of a range of social and environmental characteristics shared within a family that relate to the parents' stature and will also influence offspring height . These could be transmitted to subsequent generations through several mechanisms. Assortative mating by social class can result in parents of higher education, who are taller, transmitting their genetic, social and environmental characteristics to their children who then also grow to be taller. In the ALSPAC cohort, maternal and partner's height increased with increasing education and the educational level of the mother's partner tended to be similar to her own (65% of the mothers with no or basic education had partner with no or basic education whereas 75% of mothers with a degree had a partner with a degree). On the other hand, a gene-by-environment correlation with respect to genetic variants related to height could also generate this pattern. Mid-parental height might also incorporate effects due to exposures that are not measured accurately, e.g. maternal smoking during pregnancy. Smoking has a negative effect on height The next step in understanding the associations of height and height inequalities in high income countries requires understanding the determinants of parental height that are transmitted across generations, and disentangling the different aspects, genetic and environmental, that are captured by this variable. On the one hand, we need more knowledge on the genetic variants related to height, as up until now these can only explain about 45% of the height variance Some methodological limitations need to be considered in interpreting results from this study. Birth measures were available for about 60% of the total sample. Height measures from the first child visit were available for more than 80% of the cohort but this decreased to about 50% by age 11.5 years. There were fewer height measurements for children of mothers with lower levels of education. This loss to follow-up will only bias the results if the direction of the association in those who did not participate or were lost to follow-up was in a different direction to the one reported here. A previous report from this cohort analysed participants with at least 9 height measures and found similar results as to when children with 1 or more measures were included As some heights were standardised over a wider age range  this resulted in a positive correlation of height z-scores with age, and therefore all models were additionally adjusted for an age z-score to fully account for differential ages at measurement. The effect of education on height did not differ between the models that included this additional adjustment and those that didn't.In conclusion, inequalities in child's growth, although relatively small in magnitude, persist in England. These were fully explained by maternal and paternal reported height. Disentangling the genetic and environmental factors that this variable captures will help understanding the preventable factors that underlie height inequalities in rich income countries.Appendix S1Calculation of mid-parental height.(DOCX)Click here for additional data file.