Acinetobacter genus are present mainly in the external environment  and in animals, while in humans they may comprise physiological flora. The main pathogenic species is Acinetobacter baumannii complex, which constitutes a common cause of nosocomial infections, particularly in patients with underlying diseases and risk factors ; however, infections of the eye caused by strains of Acinetobacter spp. are very rare. We report a unique case of community-acquired corneal ulcer caused by Acinetobacter non-baumannii (possibly A. junii), in a patient with no risk factors identified. The case highlights the need for obtaining a sample from the cornea for bacteriological culture in the case of suspected ophthalmic infection as identification of the pathogen, and assessment of its susceptibility profile enables proper antibiotic therapy, improves the outcome and may constitute an eyesight-saving management.Rods of the  Acinetobacter genus are Gram-negative coccobacilli. They are present mainly in the external environment  and in animals, while in humans they may comprise physiological flora, particularly in hot and humid climate countries [Acinetobacter baumannii complex, which constitutes a common cause of nosocomial infections, particularly in patients with underlying diseases and risk factors  [Acinetobacter spp. are uncommon. We present a unique case of community-acquired Acinetobacter non-baumannii (possibly A. junii) corneal ulcer in a patient without recognised risk factors.Rods of the ountries . The maiilation) , 2. HoweA. junii in a 51-year-old woman with no significant past medical history. She was admitted to the E.R. with a diagnosis of corneal ulcer (ulcerative keratitis) and anterior uveitis of the right eye. For the previous 2 days she complained of flu-like symptoms and pain of the affected eyeball. She was generally in good health, with no history of ophthalmological diseases or treatment. Eight years earlier she was diagnosed with unexplained anaemia, but results of the recent blood tests were within the normal range.We report a community-acquired corneal ulcer caused by On examination the eyelid and conjunctiva were swollen, with deep and superficial conjunctival injection. There was a large , white and deep inflammatory infiltrate. A 2-mm hypopyon (accumulation of pus in the anterior chamber) was noticed. Best corrected visual acuity (BCVA) was 0.2.A. junii was isolated in pure culture and identified susceptible to all tested antimicrobials: aminoglycosides , tetracycline, chloramphenicol, norfloxacin and sulphonamides. The species identification and susceptibility testing was performed in a clinical microbiology laboratory using an automated system VITEK® 2 Compact (bioMerieux). It should be noted, however, that according to the present data the identification of A. junii by this automated system is uncertain, in contrast to A. baumannii complex [Corneal scrapings were taken at presentation for microbiological examination. A strain of  complex .Empiric antibiotic therapy was started on admission and comprised systemic ciprofloxacin (0.2 g b.d.) and topical chloramphenicol ointment (q.i.d.). After obtaining the culture result and an antibiogram of the isolate, this treatment was continued as the strain was susceptible to the antibiotics used in empiric therapy. In the meantime (whilst awaiting the culture results) there was a clinical improvement of the condition of the patient’s eye.Four days later the corneal ulcer continued to diminish in size and depth, with gradual epithelisation noted in the periphery of the affected area. Descemet’s membrane folds decreased substantially and hypopyon virtually resolved, while fine dust-like deposits were detected on the corneal endothelium. The infection resolved within 2 weeks, with only slight opacity, thinning and single pigment granules still present in the lower nasal quadrant of the cornea. The patient was discharged with BCVA 0.7.Acinetobacter species are opportunistic pathogens causing nosocomial infections [Acinetobacter spp. are community acquired, reported primarily from countries with tropical or subtropical climate, and mainly affected patients with some form of comorbidity or associated with heavy smoking and excess alcohol consumption [Acinetobacter infections include mainly the respiratory tract, bloodstream infections, peritoneum, urinary tract infection, surgical wounds, meningitis, skin and soft tissue infections, while eye infections are rare [fections . Relativsumption , 5. Clinare rare .Acinetobacter spp. comprises acute or chronic conjunctivitis, corneal ulcers and dacryocystitis [Acinetobacter [Acinetobacter have been implicated in the aetiology of endophthalmitis and preseptal cellulitis usually preceded by bacteraemia [Acinetobacter spp. is rare and may result from trauma, contact lens wear or ocular surgery, e.g. penetrating keratoplasty—PKP or cataract surgery [Acinetobacter spp. comprised 2 % of strains cultured from clinical specimens, being second most common pathogen (after Pseudomonas) among Gram-negative bacteria [Acinetobacter may precede its appearance in sputum or blood cultures 1–2 days later [Acinetobacter keratitis has also been reported in a patient with chronic lymphocytic leukaemia [The spectrum of ocular infections caused by cystitis , 7. Expotobacter . Rods ofteraemia , 8–10. K surgery –13. In abacteria . Interesys later . Acinetoeukaemia .Acinetobacter spp. may be particularly important in sedated intensive care unit patients [Ocular surface colonisation by patients . Incomplpatients .Acinetobacter spp. are endogenous, without any obvious predisposing factors [A. baumannii endophthalmitis has been reported, which resulted from intravitreal ranibizumab injection [A. baumannii as well as other species such as A. anitratus or A. lwoffii [Acinetobacter species, e.g. A. gyllenbergii-like isolate, have been implicated in ocular infections as well [In some cases infections caused by  factors , 9. Clin factors , 13. Recnjection . Eye inf lwoffii , 8, 11.  as well .Acinetobacter junii rarely causes infections in humans [A. junii constitute 3.6–4.8 % of all Acinetobacter spp. isolates—12/331 and 9/186, respectively [A. junii infections 80 % were hospital-acquired; four patients died (11.4 %) despite adequate antibiotic therapy in three cases [Acinetobacter junii has been associated mainly with bacteremia and sepsis in neonates and paediatric oncologic patients [A. junii in an adult oncologic patient [n humans . In largectively , 21. It ectively . Out of ee cases . Only onpatients . Linde e patient .A. junii-johnsonii. Corneal perforation due to A. junii has been reported very rarely [Henao-Martinez et al.  describey rarely , 24.A. non-baumannii species, including A. junii, may cause a community-acquired corneal ulceration in an otherwise healthy patient. Ophthalmologists should obtain a sample from the cornea for bacteriological culture in the case of suspected infection as identification of the unique causative pathogen, and its susceptibility profile enables administration of proper antibiotic therapy, improves the outcome and may constitute an eyesight-saving management.In conclusion it should be noted that


The second issue of Journal of Neonatal Surgery has been successfully published. It is a matter of great pride and celebration for the editorial team. Every editor has contributed to the best of the capacity to bring forth the issue within the specified time. Professor Yogesh Kumar Sarin led us from the front; his meticulous efforts are worth a mention.The primary scope of the journal is provision of a platform for publication of evidence-based neonatal surgery. It is the only available journal dedicated to the specialty. Neonatal surgery is often performed by pediatric surgeons in many parts of the world. Surgical neonates, the targeted proportion of the population, are entirely different with other age groups. Their body composition, requirements, immunity, pathophysiology, and response to various diseases are specific to this age group [1]. Management of surgical neonates often starts while in-utero, especially in those with congenital malformations. The decision to continue or terminate pregnancy in a case of congenital malformation is generally based on the type and severity of the malformation, parents’ will, country’s law, or religious recommendations etc. Similarly, there are other issues like management of surgical neonates in surgical neonatal intensive care unit (NICU) or medical NICU; should they be operated in operation theatre or in surgical NICU etc. Here, the role of a specialty journal as a forum for policy making and recommending cannot be ignored. In this issue, a debate on “where should the surgical neonates be nursed?” is published to help find the answer.Often the surgical practice in neonatal population is minimally evidenced. Hall et al reviewed the literature and found that most of the surgical practice in neonatal surgery is not supported by randomized controlled trials (RCTs) [2]. The current practice is largely based on the retrospective reviews and individual preferences [2]. The better scope of the journal is to publish highly evidenced neonatal surgery as RCTs. Various categories have been developed for different kind of publications each with its own level of evidence and significance. Case reports are also published to support clinical evidence, though they are considered to represent lowest level of clinical evidence.Electronic publishing has recently evolved as a preferred mode of publication. Print journals have also started publishing online for their continued existence [3]. This mode of publishing has given us the opportunity and freedom of space, as not the case with print journals. A great deal of manuscripts can be published at a time, without delaying the delivery of latest research to the readers. Since the launch [4], a few original articles have been published; of which majority are descriptive analyses. We are committed to publish RCTs in future; nevertheless, a limited span of the age is related to the specialty which may be the reason of more retrospective analyses than proper RCTs. The same is true for more case reports that are being published in the age group. This problem can be handled by publishing Meta-analyses of these descriptive series that might infer more concrete evidence.The parallel scope of the journal is Continuing Professional Development (CPD) which states that everyone in any field or discipline must have up-to-date knowledge-base and skills [5]. The clinical data on various diseases and their management are dynamic and optimum therapies are continually being evolved. Therefore, it is our endeavor to publish recent literature on neonatal surgery help support the CPD. The journal also provides an opportunity to the junior surgeons and residents to learn the basic research and publication. Research papers are welcome from the junior lot. A complete section under the heading of “Face the Examiner” is especially devoted to the residents to check their basic knowledge about important neonatal surgical disorders. Similarly, “Athena’s Pages” will keep the readers abreast of latest research in the field.The journal is in the beginning. It has been indexed with few journal-databases. Indexing with PubMed Central, PubMed, and acquisition of Impact Factor are the goals that would be achieved once the journal qualifies the recommended number of publications. Every manuscript that has been published since the launch would be indexed with all known and credulous indexing agencies. Moreover, neonatal/pediatric surgery associations and groups are invited to become our partners. We also invite conference officials to use this journal as a platform for their publications.Source of Support: NilConflict of Interest: None declared


Acinetobacter sp clinical isolates frequency and its antibiotic susceptibility pattern by comparing results obtained from the Intensive Care Units (ICUs) to that of other units at the Mohammed V Military Teaching Hospital in Rabat.This study aims to determine the Acinetobacter sp obtained from samples for infection diagnosis performed on hospitalized patients between 2012 to 2014.This is a retrospective study over a 2-years period where we collected all clinical isolates of Acinetobacter sp were collected representing 6.94% of all bacterial clinical isolates (n = 6352) and 9.6% of Gram negative rods (n = 4569). More than a half of the isolates were from the ICUs and were obtained from 293 infected patients of which 65, 2% (191 cases) were males (sex ratio = 1.9) and the median age was 56 years (interquartile range: 42-68 years). Acinetobacter clinical isolates were obtained from respiratory samples (44.67%) followed by blood cultures (14.51%). The resistance to ciprofloxacin, ceftazidime, piperacillin / tazobactam, imipenem, amikacin, tobramycin, netilmicin, rifampicin and colistin was respectively 87%, 86%, 79%, 76%; 52%, 43%, 33% 32% and 1.7%. The difference in resistance between the ICUs and the other units was statistically significant (p <0.05) except for colistin, tetracycline and rifampicin.During the study period, 441 clinical and non-repetitive isolates of Acinetobacter infection which represents a therapeutic impasse, requires the control of the hospital environment and optimizing hands hygiene and antibiotics use in the hospital.This paper shows that solving the problem of prevalence and high rate of multidrug resistant  Acinetobacter is a non-fermenting Gram negative coccobacillus with a high capacity to colonize the human body and the environmental reservoirs [Acinetobacter infection prevalence is variable depending on the geographical localization and the patient's socio-economic status [Acinetobacter infections rate was 19.2% in Asia; 17.1% in Eastern Europe; 14.8% in Africa; 13.8% in Central and South America; 5.6% in Western Europe; 4.4% in Oceania and 3.7% in North America [Acinetobacter prevalence have been performed. Acinetobacter is an opportunistic pathogen known for its intrinsic resistance to antibiotics and greater ability to rapidly acquire resistance genes as mobile genetic elements  [Acinetobacter baumannii is becoming a global threat with a therapeutic impasse increasingly described in literature [Acinetobacter strains develop resistance to colistin [Acinetobacter's antibiotic resistance rates were of 50.3 to 68.7% for ceftazidime, 23.8 to 42.6% for the imipenem, 17 to 77.5% for aminoglycosides, 65 to 68% for ciprofloxacin and no clinical isolates were resistant to colistin [Acinetobacter by comparing data from the intensive care units versus other units of the Mohammed V Military Teaching Hospital (HIMMV).servoirs . It has servoirs –5. In Moservoirs  from 200servoirs  and in aservoirs , it reprc status –10. In aquences) –13. Multterature . Indeed colistin –21. Resicolistin . In Morocolistin  however This study was conducted in HMIMV, a teaching hospital with 700-bed located in Rabat, Kingdom of Morocco. The hospital has different departments mainly 2 intensive care units  with 10 beds each, a center for burns treatment, surgical and medical units, as well as laboratory and imagery departments.st, 2012 to April, 1stst, 2014.This retrospective study was conducted by the laboratory of medical microbiology over a 2-years period. Clinical isolates were collected from diagnosis samples performed on patients who were hospitalized in different units of the HMIMV from April, 1Acinetobacter was performed on blood agar and bromo-cresol purple lactose agar and identification of clinical isolates was performed by classical bacteriological techniques  and biochemical characters using API20NE galleries . The study of antibiotic susceptibility was performed by the disc diffusion method on Mueller-Hinton agar plates, and interpreted as recommended by the antibiogram committee of the French Society of Microbiology in their 2014 recommendations. The antibiotic discs tested were: ticarcillin, ticarcillin-clavulanate, piperacillin, piperacillin-tazobactam, cefepime, ceftazidime, imipenem, amikacin, gentamicin, tobramycin, netilmicin, ciprofloxacin, sulfamethoxazole -trimethoprime and colistin. The reading of the antibiograms was performed using the OSIRIS expert system. The resistance to colistin was confirmed by the determination of the Minimum Inhibitory Concentrations (MICs) using the E-test method  according to the manufacturer′s recommendations. All isolates of Acinetobacter resistant to three or more classes of antibiotics represented by piperacillin / tazobactam, ceftazidime, imipenem, ciprofloxacin, aminoglycosides and colistin were considered as MDR [Isolation of d as MDR , 21, 23.Acinetobacter infection prevalence, resistance rates and MDR between ICUs and the other units. The p values less than 0.05 were considered statistically significant.The statistical analysis was performed using the SPSS Statistics 17.0. The Chi 2 test was used to compare the percentages of Acinetobacter were collected, representing 6.94% of all bacterial isolates (n = 6352) and 9.6% of all Gram-negative bacilli (n = 4569) throughout the hospital. These isolates were obtained from 293 Acinetobacter infected patients of which 65.2% (191cases) were males, so a sex ratio M/F of 1.9. These patients represented 8, 2% of all patients having a bacterial infection (n = 3565). The median age of Acinetobacter infected patients was 56 years (interquartile range: 42-68 years) and the distribution by age showed that 64.9% of the isolates came from patients aged between 18-64 years; 31.3% aged 65-101 years and 3.8% of patients ≤ 17 years. Site sampling analysis of the Acinetobacter's isolates showed that the proportion of broncho-pulmonary samples was 44.67%, followed by the blood cultures (14.51%), the deep pus (12.47%), the urine (12%), the superficial pus (9%), the catheters (3.85%), the tissue (1.81%) and the puncture liquid (1.59%)  were Acinetobacter baumannii, and 4 (1%) were Acinetobacter lwoffii. The breakdown by department analysis showed that 54.9% of clinical isolates were obtained from the ICUs, 36.7% and 8.4% from the medical and surgical units respectively. In the ICUs, the isolates of collected Acinetobacter strains (n = 242) represented 24.85% of all isolates (n = 974) and 31.5% of all Gram-negative bacilli (n = 769). They were collected from 156 patients infected with Acinetobacter. For these patients 61.53% were male with a sex ratio of 1.6 and represented a 49.2% of patients with bacterial infection (n = 317). In the other units, 199 isolates of Acinetobacter were collected, representing 3.7% of all isolates (n = 5378) and 5.23% of Gram-negative rods (n = 3800) obtained in these units. These isolates were obtained from 137 infected patients. Among these patients, 69.3% were male, so a sex ratio equal to 2.3 and represented a rate of 4.2% of whole patients with bacterial infection (n = 3248). The difference in the prevalence of Acinetobacter related-infections between the ICUs and the other units was statistically significant (p <0.001).During the study period, 441clinical isolates of  (1.59%) . IsolateAcinetobacter sp with the resistance rate to colistin of 1.7%. The resistance rates difference between the ICUs and the other units was statistically significant (p< 0.05) except for colistin, tetracycline and rifampicin . The isolation rate of Acinetobacter in the various samples was 6.94%. These results are higher compared to those from the study conducted by Mushtaq and al. (2013) [Acinetobacter species was 4.2% [Acinetobacter (8.8%) was significantly lower than that of the ICUs (24.85%) in our hospital. These clinical isolates represented 9.6% of all gram-negative bacilli in the hospital and 31.5% in the ICUs. On the other hand, a study carried out in an Indian hospital reported a comparable rate as A. baumannii constituted 9.4% of all Gram-negative rods throughout the hospital and 22.6% in the ICUs [Acinetobacter isolates are increasing in our hospital. Indeed, in 2001, 147 clinical isolates of A. baumannii were isolated from all patients hospitalized in our institution and checked for bacterial infection [st, 2012 to April 1st, 2014. This high prevalence observed in our study is probably related to non-compliance with the recommendations for mastery the hospital environment [Acinetobacter [The present study shows that the infection . 2013)  in Pakiswas 4.2% . In an iwas 4.2% , the iso13  in Pathe ICUs .85% in oironment . Some stironment . Since hthe ICUs .85% in othe ICUs .85% in oAcinetobacter has been verified in other studies but the reason is not justified [A. baumannii infection [Acinetobacter strains in broncho-pulmonary samples [Acinetobacter spp infections are generally involved in anatomical sites with a high fluid content manifested by pneumonia, bacteremia, urinary tract infection, meningitis and wound infection [A. baumannii pneumonia is associated with mechanical ventilation [A. baumannii (87.1%). The research by Chuang and al. (2011) demonstrated that among Acinetobacter species, A. baumannii is the main cause of Acinetobacter infections with the antibiotic resistance rate being very high causing more serious infections than other species of Acinetobacter [Acinetobacter isolates are known for their resistance to various antibiotics despite their weak virulence limiting the control and infections treatment due to these microorganisms [Acinetobacter infection such as immunocompromised persons, longer duration of stay in hospitals, invasive devices use on patients, the broad spectrum antibiotics therapy, possible and frequent contaminations and cross transmission of this bacteria through environmental reservoirs and hands of healthcare workers [Acinetobacter clinical isolates to this class of antibiotics  has been described in the literature [In our study, 65.2% of affected patients were male. The predominance of male patients infected with ustified , 5, 11. ustified , 5, 11.  samples , 20, 27.tilation , 28 resutilation , 28. Thetobacter . In generganisms , 20, 30. workers . For the workers , the higterature . In our terature . In a Literature . The resterature . It rangterature .A. baumannii is often due to the expression of OXA carbapenemase types, Metallo-beta-lactamases (MBL) carbapenemase and the impermeability associated with mutations altering the expression of porins and efflux pumps [Acinetobacter strains showed the phenotype having resistance to beta-lactam antibiotics associated with the expression of carbapenemase or alteration of porins . These results are not in accordance with those found in another study conducted in the same hospital where the penicillinase phenotype and high level cephalosporinase production were predominant with a rate of 33% [A. baumannii trains had the penicillinase phenotype in 26.3% of cases [Acinetobacter spp. involves the production of aminoglycosides modifying enzymes and genes encoding these enzymes can be acquired through plasmids, transposons or integrons [Acinetobacter infections is rare because of the high resistance to this antibiotic in our institution. Rifampicin was very effective (but less than colistin) with a resistance rate of 32.11%, but the use of this drug in the treatment of Acinetobacter infections is limited because Morocco is a country of endemic tuberculosis. Colistin was the most active antibiotic against Acinetobacter. In this study, the resistance to colistin was 1.7%.Carbapenems  remain one of the most important therapeutic options for these infections despite the fact that carbapenem-resistant strains are increasing . In our ux pumps , 30. In e of 33% . In a Tuof cases . This emof cases . The amintegrons . The ratntegrons , 17–20. Acinetobacter was resistant to colistin [Acinetobacter infections in cases where this bacterium is strongly suspected to be resistant to other antibiotics [Some studies have reported that no clinical isolate of colistin , 22 but colistin , 33. Sevibiotics , 19. Theibiotics , 35. Synibiotics . There aibiotics . The comAcinetobacter was 77.4% throughout the hospital and extents 92.6% in ICUs and 75.3% in the other units with a rate difference that was statistically significant between the ICUs and the other units (p <0.001). Depending on the nature of each sampling site, the highest MDR Acinetobacter percentage (94.1%) was observed in vascular catheters followed by broncho-pulmonary samples (89.4%) and the lowest MDR Acinetobacter percentage (52.83%) was found in urine samples. These results are comparable to conclusions of a Lebanese study where rates of MDR Acinetobacter varied between 73.4% and 77.7% [The current study demonstrated that the percentage of MDR nd 77.7%  but highnd 77.7% .Acinetobacter infection are high and could pose a real problem and a management impasse. A strict control of the hospital environment, hand hygiene and optimizing the use of antibiotics is recommended in order to reduce the MDR frequency.In this study, we showed that, in our hospital, the frequency and rates of MDR Acinetobacter is a non-fermenting Gram negative coccobacillus with a high capacity to colonize the human body and the environmental reservoirs and it is associated with a high morbidity and a high mortality rate especially in immunocompromised patients.Acinetobacter is an opportunistic pathogen known for its intrinsic resistance to antibiotics and greater ability to rapidly acquire resistance genes.Acinetobacter baumannii is becoming a global threat with a therapeutic impasse increasingly described in literature.It generally has resistance to several antibiotics and MDR Acinetobacter by comparing data from the intensive care units versus other units in our hospital and in our region.This is the first study which shows the prevalence rate of infections and antibiotic resistance level in clinical isolates of Acinetobacter isolates are increasing in our hospital.This study shows that the frequency and resistance of Acinetobacter. The infection Acinetobacter's prevalence and the MDR Acinetobacter rate were statically higher in the intensive care units compared to other units.This is the first Moroccan study that reports the rate of MDR


The reporting of serious adverse events (SAEs) in clinical trials is crucial to assess the balance between benefits and risks. For trials with serious adverse events posted at ClinicalTrials.gov, we assessed the consistency between SAEs posted at ClinicalTrials.gov and those published in corresponding journal articles.All records from ClinicalTrials.gov up to February 2014 were automatically exported in XML format. Among these, we identified all phase III or IV randomized controlled trials with at least one SAE posted. For a random sample of 300 of these trials, we searched for corresponding publications using MEDLINE via PubMed and extracted safety results from the articles.Among the sample of 300 trials with SAEs posted at ClinicalTrials.gov, 78 (26 %) did not have a corresponding publication, and 20 (7 %) had a publication that did not match the ClinicalTrials.gov record. For the 202 remaining trials, 26 published articles (13 %) did not mention SAEs, 4 (2 %) reported no SAEs, and 33 (16 %) did not report the total number of SAEs per treatment group. Among the remaining 139 trials, for 44 (32 %), the number of SAEs per group published did not match those posted at ClinicalTrials.gov. For 31 trials, the number of SAEs was greater at ClinicalTrials.gov than in the published article, with a difference ≥30 % for at least one group for 21. Only 33 trials (11 %) had a publication reporting matching numbers of SAE and describing the type of SAE.Many trials with SAEs posted at ClinicalTrials.gov are not yet published, omit the reporting of these SAEs in corresponding publications, or report a discrepant number of SAEs as compared with ClinicalTrials.gov. These results underline the need to consult ClinicalTrials.gov for more information on serious harms. Randomized controlled trials (RCTs) are considered the gold standard for assessing the effects of health care interventions and form the basis for treatment decision-making. Thus, all results from clinical trials should be transparent and accessible to all –3. ResulTo increase transparency, the 2007 US Food and Drug Administration Amendments Act (FDAAA 801) required that, after September 2008, results from clinical trials conducted in the United States be made publicly available at ClinicalTrials.gov within 1 year of the completion of the trial –15. In aIn this study, we examined the consistency between SAEs posted at ClinicalTrials.gov and those published in journals. We identified a random sample of trials with SAEs posted at ClinicalTrials.gov to assess whether these safety results were published and to compare the timing of availability of SAEs between ClinicalTrials.gov and publications as well as the number and type of SAEs posted at ClinicalTrials.gov and those published in corresponding journal articles.On February 2, 2014, we exported all records from ClinicalTrials.gov and, using R 3.1.1  with theIn June 2014, one of the authors (ET) searched for corresponding publications in journals using the link to publications provided at ClinicalTrials.gov whenever possible. Also, MEDLINE was systematically searched via PubMed by using the ClinicalTrials.gov identification number (NCT number). If no publication was identified, MEDLINE was searched again by using keywords for drug names and condition studied. The articles identified by the search had to match the corresponding trial in terms of the information registered at ClinicalTrials.gov . If there were several publications for a trial, all publications that matched the time-frame indicated at ClinicalTrials.gov were selected. Trials for which the published article reported a different time-frame or number of groups were excluded.A second reviewer (CR) checked the matching between ClinicalTrials.gov and the corresponding published articles for all trials. All disagreements were resolved by discussion between the two reviewers with the help of a third reviewer (AD) if needed.General characteristics of the trial: lead sponsor, condition, and countries where the trial was conducted; primary completion date  and the date when results were first posted. This date was extracted from the archive record and differs from the date on which results were first received, which is available under Study Results at ClinicalTrials.gov. The difference between these two dates relates to ClinicalTrials.gov production and the vetting of the results by the US National Institutes of Health.Design of the trial: phase III or IV trial and parallel or cross-over trial.Interventions: details concerning the interventions for the experimental and control groups.SAEs: total number of SAEs, number of SAEs per group, types of SAEs, number of SAEs per type per group, and number of participants at risk per group. We recorded the date when SAEs were first posted from the archive record.For the random sample of 300 trials with SAEs posted at ClinicalTrials.gov, we extracted the following characteristics from the records exported from ClinicalTrials.gov by using R 3.1.1 :General General characteristics of the publication: journal of publication, first author, date of online publication, type of journal , and whether ClinicalTrials.gov NCT number was reported in the published article.SAEs: whether SAEs were reported and whether they were reported by number per group. If the total number of SAEs per group was not reported, we noted whether the number of the most common SAEs (those observed above a certain frequency or threshold rate), number of SAEs related to treatment, or toxicity-graded events were reported instead. We also extracted the types of SAEs reported and noted whether the number per type and per group was reported. Again, if all types were not reported, we noted whether the types reported were common event types, those observed above a certain frequency or threshold rate, those related to treatment, or toxicity-graded events. We also collected the number of participants at risk for each group.For all trials with corresponding publications, the following information was collected from published articles, including online supplements:If several publications were identified for the same trial, we extracted safety results from all corresponding publications having the same time-frame as reported at ClinicalTrials.gov. For trials with multiple phases  reported at both ClinicalTrials.gov and in published articles, we extracted SAE data only for the double-blind randomized treatment period. For published articles reporting pooled results from multiple trials, we considered the data to be missing if we were unable to extract SAE data that corresponded to the specific trial.All data were extracted from the published article independently by two reviewers , independently of data collected from ClinicalTrials.gov. One of the reviewers (CR) was blinded to the hypothesis. All disagreements were resolved by discussion to reach a consensus, including intervention by a third reviewer (AD) in case of discrepancies.Descriptive data are reported with numbers (percentages) and median (quartile 1–3 [Q1–3]). We compared time between primary completion date as reported at ClinicalTrials.gov and date of the SAEs first publicly posted at ClinicalTrials.gov or the date of the first online publication in journals reporting the number of SAEs per group by the Kaplan–Meier method. Trials for which the number of SAEs per group were not reported in a published article on June 2014 were censored at this date for the estimation of time between the primary completion date and online publication of SAEs. For trials with both results posted and published, we compared the number of SAEs reported at ClinicalTrials.gov and in the published article. To simplify, for three-group trials with two groups representing different doses of the same treatment, we combined these two groups. For trials comparing three different treatments, we selected the main comparison according to the ClinicalTrials.gov posting and without knowledge of the results. Analyses involved the use of R 3.1.1 .Figure The difference between the median time to the availability of SAEs at ClinicalTrials.gov and in published articles was 50 months  with the median time between primary completion of trials and SAEs publicly posted at ClinicalTrials.gov of 22 months  and median time between primary completion date and availability of SAEs in published articles of 72 months ; 26 (13 %) published articles did not mention SAEs at all and 4 (2 %) reported no SAEs. Of these 30 articles, for 12 trials, the number of SAEs posted at ClinicalTrials.gov was >10 and for 6, >100. For 33 trials, the reporting of SAEs was incomplete in the published article: 10 reports (5 %) described only drug-related SAEs or SAEs of interest and 9 (4 %) reported adverse events with grade ≥3 instead. In 8 articles, the results were pooled from several trials and we were not able to extract the number of SAEs for each individual trial. Three published articles (1 %) did not report the number of SAEs per group, and 3 (1 %) reported only SAEs for the experimental group , the number of SAEs per group did not match that posted at ClinicalTrials.gov; for 32 of these, the number of SAEs was different for both groups and, for 12, only one group. For the 32 trials with number of SAEs different for both groups, 22 (69 %) had more SAEs reported at ClinicalTrials.gov than in the published articles. For 15 of these trials (68 %), the difference between the number posted at ClinicalTrials.gov and the published article was ≥30 % for at least one group. For the 12 trials where number of SAEs was different for one group only, 9 trials had more SAEs reported at ClinicalTrials.gov than in the published article. For 6 of these trials, the difference between the number posted at ClinicalTrials.gov and the published article was ≥30 %.Of the 95 trials with number of SAEs matching that at ClinicalTrials.gov, all types of SAEs that occurred were reported for 33 (35 %) in the corresponding published articles. In 34 published articles (36 %), the type of SAE was not reported at all. Other publications reported the types of SAEs for only drug-related SAEs (n = 12), the most common SAEs (n = 10), the SAEs of interest (n = 5), or SAEs leading to withdrawal (n = 1).Overall, only 33 trials (11 %) out of the random sample of 300 trials with SAEs posted at ClinicalTrials.gov had a publication reporting matching SAE numbers and describing the type of SAE  did not mention SAEs or reported no SAEs in the corresponding publications. The number of SAEs per group was frequently not reported in the published articles and when it was reported, discrepancies with the numbers posted at ClinicalTrials.gov were common, with frequently more SAEs reported at ClinicalTrials.gov than in the published article.Restricted space in articles is a frequently cited reason for incomplete reporting of harms , 18. HowP <0.0001) [In a previous article focusing on completeness of reporting, we found that SAEs were significantly more completely reported at ClinicalTrials.gov than in the published articles  . This reOur results identified some trials not mentioning SAEs or reporting no SAEs in the published article, despite these being reported at ClinicalTrials.gov. Furthermore, when SAEs were reported in published articles, discrepancies with the number posted at ClinicalTrials.gov were common, with frequently more SAEs reported at ClinicalTrials.gov than in the published article. Although we do not know which the ‘true’ results are, we believe that these discrepancies clearly outline problems in the reporting of SAEs. Two studies comparing results posted at ClinicalTrials.gov and in peer-reviewed publications also showed discrepancies in the number of SAEs , 25, 26.Our results have important implications. Our results highlight that ClinicalTrials.gov provide more information on serious harms, whereas these events are frequently underreported in published articles. For systematic reviewers, they outline the interest of using ClinicalTrials.gov to find safety results not yet published in journals and for trials with both SAEs posted and published, to compare the rate of SAEs. In case of discrepancies, we recommend systematically contacting authors for clarification and performing sensitivity analyses in case of non-response to assess to what extent these discrepancies may affect the meta-analysis result. For journals, they question the peer-review process, in that the assessment of data recorded in registries including results and harms when available should be part of the process to assess if there are any discrepancies that could bias the results. In case of discrepancies, investigators should be contacted for clarification. They also raise questions about how reporting guidelines, especially the CONSORT harms, are implemented by journals, with a need for more active endorsement. Templates with mandatory reporting of critical elements, such as that used at ClinicalTrials.gov , could iWe may not have identified all published articles because we searched only MEDLINE for publications. Further, for trials without publications, the results could be published at a future date because publication in journals may take time due to multiple submissions. Some trials may have multiple publications with different results reported. In this case, we did not include all reports resulting from the trial but only the reports that included safety data and matched the time frame reported at ClinicalTrials.gov. Finally, this study focused on trials assessing pharmacological treatments, but non-pharmacological treatments can also incur SAEs.Our results reveal that many trials with SAEs posted at ClinicalTrials.gov are not yet published, omit the reporting of these SAE in corresponding publications, or report a discrepant number of SAEs as compared with ClinicalTrials.gov. Consulting safety results posted at ClinicalTrials.gov, when available, is crucial for more information on serious harms.


Ahlström and Elisabet WentzRegarding the paper titled: Difficulties in everyday life: “Young persons with attention-deficit/hyperactivity disorder and autism spectrum disorders perspectives. A chat-log analysis” International Journal of Qualitative Studies on Health and Well-being, 28 May 2014. Citation: Int J Qualitative Stud Health Well-being 2014, 9: 23376 - http://dx.doi.org/10.3402/qhw.v9.23376Published in Internet-based support and coaching”), the last sentence currently reads: “The coaches chatted with the participants from their work places or homes.”On page 3, first paragraph .”This sentence should read: “The coaches chatted with the participants from their work places or homes European Child and Adolescent Psychiatry, 21(11), 611–622. doi: 10.1007/s00787-012-0297-2.”The references have been supplemented with: “Wentz, E., Nydén., A. & Krevers, B. (2012). Development of an internet-based support and coaching model for adolescents and young adults with ADHD and autism spectrum disorders: A pilot study.


We recently identified T cell epitopes associated with human allergic responses. In a majority of cases, responses focused on a few immunodominant epitopes which can be predicted on the basis of MHC binding characteristics. Several observations from our studies challenged the assumption that T cell epitopes are derived from the same allergen proteins that bind IgE. Transcriptomic and proteomics analysis identified pollen proteins, not bound by IgE. These novel Timothy Grass proteins elicited vigorous Th2 responses, suggesting that unlinked T cell help is operational in pollen-specific responses. Thus, the repertoire of antigens recognized by T cells is much broader than IgE-binding allergens. Additionally, we evaluated the use of epitopes from these novel antigens to assess immunological changes associated with Specific Immunotherapy (SIT). We found that a marked decrease in IL5 production is associated with clinically efficacious SIT, suggesting that these novel antigens are potential immunomarkers for SIT efficacy.The online version of this article (doi:10.1186/1939-4551-7-26) contains supplementary material, which is available to authorized users. Herein we will briefly summarize data generated in our laboratory pertaining to the identification of T cell epitopes associated with allergic responses in humans. We will further discuss this data in the context of the potential use of allergen-derived T cell epitopes as biomarkers of specific immunotherapy (SIT).These studies were prompted by the observation that T cells contribute to pathology associated with allergy both directly and indirectly through modulation of immune responses . The invOur approach and results will be articulated in three main steps. First, we will review the development of techniques and general approaches for the identification of allergen-derived epitopes and their application to some of the most common allergen sources. Second, we will describe a series of studies that questioned the assumption that T cell epitopes are derived from the same allergen proteins that bind IgE. The final series of experiments described here will relate to the use of epitopes to assess immunological changes associated with SIT maintenance phase.A number of different methods are available for the identification and characterization of peptide epitopes recognized by human class II restricted T cells. They include generation of stable T cell lines and clones, isolation of naturally processed ligands, screening of libraries of overlapping peptides, and generation of mimotope libraries and prediction of HLA class II binding capacity by bioinformatics analysis.Our group in particular has been interested in the use of bioinformatics predictions combined with experimental tests of predicted binders using lymphocytes derived from allergic patients –10. One To address this challenge, we put forth the hypothesis that while the large number of HLA class II variants would likely be reflected in a large degree of heterogeneity of responses, a large fraction of the response might be accounted for by a limited number of more dominant epitopes. We further hypothesized that T cell epitopes capable of cross-binding multiple HLA class II molecules  would likely be amongst such dominant epitopes. Hence, we suggest that predicting promiscuous HLA class II epitopes might offer a practical means to identify a rather significant fraction of T cell allergen epitope specificities.in vitro restimulation with TG extract. Positive pools were subsequently deconvoluted to identify the individual 15-mer(s) eliciting allergen-specific responses.The potential merit of the hypothesis formulated above was tested experimentally in the Timothy Grass (TG) system . For theUpon completion of the screen that included 10 non-allergic donors and 33 allergic donors, 8 of which were in maintenance phase of SIT, we noted, as expected, a large degree of heterogeneity of responses. At least 43 different epitopes/antigenic regions were defined. However, we also noted that a few regions accounted for a disproportionately large fraction of the responses. In fact, just the top nine regions accounted for over half of the total response. Subsequent analysis verified that these epitopes were indeed promiscuous . Further analysis also revealed that binding predictions could identify the most promiscuous epitopes accounting for a relatively large fraction of the responses.Based on these results, we developed a predictive strategy to identify specifically those peptides binding to multiple HLA. We applied this strategy to a larger scale effort targeting epitope identification for an additional set of 28 allergen sources , 9, 15. In conclusion, our studies have found that in a majority of cases allergen-specific responses are directed against relatively few immunodominant epitopes. Further, these epitopes can be identified and/or predicted on the basis of MHC binding characteristics.It was generally assumed that T cell epitopes are derived from the same allergens that bind and elicit IgE responses. However, in the course of the studies described above, we made two observations that led us to question the validity of this assumption.First, in both the TG and cockroach allergy systems, we observed that T cell responses and IgE responses did not correlate at the level of individual patients , 10. MorSecond, in several patients, no T cell response could be detected against any of the known IgE-binding allergens, despite vigorous T cell responses to the TG extract. We hypothesized that these observations might be explained by T cell responses targeting additional pollen proteins other than the known allergens. The absence of an exact correlation of T and B cell responses on the antigen level is not really surprising. In fact, this notion, called “antigen bridge” –21, whilBased on these data, we hypothesized that Th2 responses in allergic patients might recognize antigens contained in the pollen particles but not necessarily limited to the ones bound by IgE. Testing this hypothesis required a multidisciplinary approach Figure , includiIdentification of novel proteins was accomplished by isolating TG pollen mRNA and analyzing high-throughput sequencing from an Illumina Genome Analyzer. A total of 1,016,285 unique, putative transcripts  were identified. Next, we used gel separation and MS analysis to identify which of the newly identified transcripts encoded proteins detectable in TG pollen. More specifically, pollen extract was separated on 2D gels and specific spots were picked for further analysis based on antibody or protein staining. These spots were then cut out from the gel and analyzed by mass spectrometry. As a result, 83 new proteins derived from the 2D gel and an additional ten proteins derived from whole extract MS analysis were chosen for further studies [To identify T cell epitopes on the basis of HLA class II binding predictions, we predicted the capacity of peptides derived from the novel proteins to bind a panel of 25 common HLA class II DR, DP, and DQ molecules selected to represent the most prevalent alleles in the general world-wide population, inclusive of all major ethnicities . A set oin vitro expansion, and showing that responses to both conventional and novel TG antigens can be detected directly ex vivo.When compared to known allergens, the results clearly indicated that a majority of Th2 responses in TG allergic individuals target epitopes derived from novel antigens, some of which are not targeted by IgE responses. Further experiments confirmed this discovery by showing that memory T cells are the source of IL5 T cell responses after In conclusion, the results summarized in this section indicate that novel TG proteins elicited Th2 responses, explaining the reactivity gap between known allergens and whole extract. IL5 production was directed to several antigens not targeted by IgE or IgG, suggesting that unlinked T-cell help is operational in pollen-specific responses. In the end, the universe of T cell inducing antigens was found to be much broader than that delineated by IgE-binding allergens.Despite unquestionable evidence demonstrating that SIT is effective in treating allergic disease –25, seveWith these considerations in mind, we initiated a comparison of cytokine production in allergic and SIT donors . In thesWhen epitopes from known and novel antigens were compared, the decrease in IL5 production associated with SIT was similar, and, if anything, more pronounced in the case of the novel antigens and epitopes. Taken together, these data have suggested several potential immunomarkers for SIT efficacy as well as possible approaches towards defining a mechanism of action.Based on these results, we selected a subset of novel Timothy Grass antigens (NTGAs) and epitopes for which the IL-5 response was found to be particularly down-modulated in SIT and used it for additional studies in new donors . An impoIn the course of recruitment of the SIT donors utilized in these experiments, each donor provided a subjective evaluation as to whether or not the treatment had helped to alleviate their symptoms. When the data pertaining to the modulation of the IL5 response was subsequently analyzed, it was found that modulation of IL5 responses were much more pronounced in the donors self-reporting benefit from treatment . That isThese data raise the possibility that T cell responses to certain modulated novel antigen (MNA) epitopes can be used as biomarkers of SIT efficacy. However, additional studies are clearly required, including those addressing larger patient cohorts as well as longitudinal studies  and those incorporating more objective evaluations of SIT clinical efficacy. These subsequent studies should also evaluate whether modulation of Th2 responses to specific epitopes follows or precedes the clinical benefit, and determine how long the modulation lasts. These studies should also correlate changes in epitope specific responses with other immunological changes that have been proposed as biomarkers of SIT efficacy, such as increases in IgG4 titers.We utilized a bioinformatics-based approach to characterize T cell responses to common allergens. Peptides from novel, non-IgE-binding proteins elicited Th2 responses, suggesting that unlinked T-cell help is operational in pollen-specific responses. Thus, the repertoire of T cell allergens is much broader than that defined by IgE-binding. IL5 responses to these novel antigens from SIT donors that subjectively reported to have benefitted from treatment were significantly lower than those of individuals that did not benefit from SIT. These observations raise the possibility that allergen specific epitopes can be used as biomarkers of SIT efficacy.


Despite significant improvement in pancreas allograft survival, rejection of the pancreas remains a major clinical problem. In addition to cellular rejection of the pancreas, antibody-mediated rejection of the pancreas is now a well-described entity. The 2011 Banff update established comprehensive guidelines for the diagnosis of acute and chronic AMR. The pancreas biopsy is critical in order to accurately diagnose and treat pancreas rejection. Other modes of monitoring pancreas rejection we feel are neither sensitive nor specific enough. In this review, we examine recent advances in the diagnosis and treatment of pancreas rejection as well as describe practical diagnostic and treatment algorithms. Solid organ pancreas transplant outcomes continue to improve , with thClinically, pancreas allograft function is commonly monitored by a number of parameters including hyperglycemia, serum amylase, serum lipase, C-peptide level, hemoglobin A1C, or, if bladder drained, urinary amylase. However, in order to accurately identify and define rejection, these clinical tools are insufficient, because they are too non-specific. There are multiple potential causes for hyperamylasemia, hyperlipasemia, and hyperglycemia. Even stable kidney function, or a normal serum creatinine in SPK transplants, is not a consistently reliable indicator that pancreatic graft immunological damage is not occurring . Indeed,Elevated pancreatic enzymes is the most common presentation of pancreatic allograft rejection, and most patients are asymptomatic or have only mild graft tenderness upon presentation. If a patient is asymptomatic, the source of the elevated enzymes is more likely to be from the transplanted pancreas as the graft itself is insensate. In contrast, symptomatic increases in pancreatic enzymes can point to either transplant or native gland disease. Lipase is considered to be more specific than amylase and has greater sensitivity in the author’s experience. Naturally, these signs and symptoms can arise from multiple causes in the setting of an enterically drained graft. Hence, it is useful to consider the differential diagnosis and the timing of presentation when evaluating these patients Fig. , Table 2An algorithm for the diagnosis and management of patients with increased pancreatic enzymes is shown in Fig. Medication non-compliance, recent intolerance of oral medications, alcohol consumption, recent abdominal trauma, constipation, presence of a ventral hernia, abdominal pain, nausea, vomiting, and obstructive symptoms are important points to elicit from the patient. Duodenal or parenchymal enzyme leak, pseudocyst, mycotic aneurysm, SBO, intrinsic pancreatic abnormalities such as duct strictures, and tumors are included in the differential diagnosis of elevated enzymes and are usually diagnosed with imaging studies.In our experience, transplant pancreatitis that is not attributable to an anatomic or immunological cause is very uncommon. Instead, transplant pancreatitis, putatively defined as increased pancreatic enzymes without evidence of other anatomic abnormality on CT scan other than peri-pancreatic transplant inflammation and graft swelling, can also be indicative of acute or chronic rejection or CMV pancreatitis. In our large series of for-cause pancreas allograft biopsies, non-immunological pancreatitis was only diagnosed on biopsy in 4 % 16/422) of biopsies, a frequency similar to that reported previously [6/422 of While most patients who have acute rejection present with elevated pancreatic enzymes, it is not uncommon to see patients develop chronic rejection and graft failure without significant increases in their serum enzymes. Why chronic rejection does not always coincide with serological evidence of acinar injury is not presently known, but may be related to progressive loss of acinar/parenchymal tissue resulting in less release of enzymes and/or reduced lab frequency resulting in missed detection.In order to diagnose pancreas rejection accurately, a biopsy of the pancreas allograft is necessary. It is currently the only available means of grading the severity of rejection and distinguishing antibody-mediated rejection (AMR) from acute cellular rejection (ACR). The most commonly performed approach to allograft biopsy is percutaneous ultrasound-guided biopsy of the pancreas parenchyma, which can be performed in either bladder-drained or enterically drained allografts safely and effectively , 6. AlteIn our practice, we prefer ultrasound-guided biopsy with an 18-gauge automatic biopsy device. Our trajectory for biopsy is ideally towards the tail, avoiding the splenic artery and vein. Because of the nest of vessels and larger duct in the head region, we prefer to biopsy the body/tail of the graft aiming either transversely or preferably longitudinally with respect to the axis of the pancreas. We have performed 406 percutaneous biopsies since 1992, with a very low complication rate and no graft losses. Two patients required reoperation: one for bleeding and one for evacuation of pancreatic ascites, which ultimately resolved, and both patients currently still retain excellent graft function. If we are unsuccessful, or there is no suitable safe window free of overlying bowel, then we proceed with CT-guided biopsy using a posterior approach, or alternatively an open or laparoscopic biopsy, but this is required rarely. The majority of pancreas biopsies were diagnostic, only 6 % (24/422) were non-diagnostic , race mismatch , and incThe deleterious effects of both pre-formed donor-specific antibodies (DSA) and de novo DSA are well established in the kidney transplant literature . Few stuMittal et al. demonstrated in a large cohort of pancreas transplant patients that de novo DSA was also an independent risk factor for graft loss •. In thiIn our practice, the presence of pretransplant DSA aids our assessment of the posttransplant immunologic risk. For patients with pretransplant DSA and a negative flow crossmatch, we tend to favor using a depleting antibody for induction therapy. We monitor all our patients with DSA measurements postoperatively with the frequency of monitoring determined by a preoperative risk stratification. However, it is currently unknown what to do with de novo DSA in the setting of normal pancreas allograft function. One hypothesis is that it indicates under immunosuppression, and increasing immunosuppression maybe warranted. Elevated DSA in the setting of normal graft function could also be a harbinger of eventual graft dysfunction or simply could be of no clinical consequence. Certainly more studies are needed. If the emergence or rise in DSA does in fact accompany abnormal graft function, then a biopsy is warranted to rule out rejection.A normal fasting C-peptide and HbA1c confirms a functioning pancreas allograft prior to the initiation of anti-rejection therapy. Consideration of pancreas graft functional reserve prior to initiating any anti-rejection therapy is paramount. Hyperglycemia, as evidenced by significantly elevated fasting blood glucose or HbA1c, or low fasting C-peptide level should be taken into account when weighing the potential benefits and risks of pursuing therapy. Additionally, a significant chronic component to the patient’s rejection status, as evidenced by fibrotic changes on biopsy, should factor into the potential benefit of therapy. Treatment of chronic or acute rejection if significant hyperglycemia is present prior to initiation of steroid therapy, or if the patient is on insulin, is controversial, but traditionally, hyperglycemia had been thought to be a marker of poor salvagability in a pancreas allograft . If the H&E staining can diagnose and grade ACR, and we treat accordingly with steroids +/− anti-thymocyte globulin (ATG) as illustrated in Fig. If DSA is positive with histologic findings consistent with AMR, even in the absence of C4d, we proceed with treatment consisting of IVIG and plasmapheresis as we would in the setting of significant C4d staining. In cases of a mixed rejection, we generally add ATG. If the amylase/lipase decline, we continue treatment until they have normalized. If their descent stalls or if they start to rise again, we usually repeat the biopsy. If the re-biopsy demonstrates persistent AMR, additional anti-B cell therapies, such as bortezomib, rituximab, or eculizumab, are considered. Admittedly, treatment of pancreas AMR is currently pursued without clear data and the aforementioned approach is merely proposed as a guide. Even in the kidney AMR literature, there is a paucity of randomized clinical trails. Despite this, there have been advances in the treatment of AMR resulting in improved outcomes. The main treatment goal in AMR is for the reduction of DSA and elimination of the B cell and plasma cell population responsible for the production of DSA. Hopefully, carefully protocolized treatments and the examination of outcomes will be prospectively conducted.Overall, key points of our treatment algorithm Fig.  are as fIn our practice of evaluating each patient for the possibility of pancreas graft rejection, we gather serologic and histologic data in the form of DSA and biopsy, and if hyperglycemic, then fasting C-peptide and HbA1c. Once a diagnosis of rejection is made, we treat accordingly as described in Fig.


An important diagnostic criterion for social communication deficits in autism spectrum disorders (ASD) are difficulties in adjusting behavior to suit different social contexts. While the BTBR T+tf/J (BTBR) inbred strain of mice is one of the most commonly used mouse models for ASD, little is known about whether BTBR mice display deficits in detecting changes in social context and their ability to adjust to them. Here, it was tested therefore whether the emission of isolation-induced ultrasonic vocalizations (USV) in BTBR mouse pups is affected by the social odor context, in comparison to the standard control strain with high sociability, C57BL/6J (B6). It is known that the presence of odors from mothers and littermates leads to a calming of the isolated mouse pup, and hence to a reduction in isolation-induced USV emission. In accordance with their behavioral phenotypes with relevance to all diagnostic core symptoms of ASD, it was predicted that BTBR mouse pups would not display a calming response when tested under soiled bedding conditions with home cage bedding material containing maternal odors, and that similar isolation-induced USV emission rates would be seen in BTBR mice tested under clean and soiled bedding conditions. Unexpectedly, however, the present findings show that BTBR mouse pups display such a calming response and emit fewer isolation-induced USV when tested under soiled as compared to clean bedding conditions, similar to B6 mouse pups. Yet, in contrast to B6 mouse pups, which emitted isolation-induced USV with shorter call durations and lower levels of frequency modulation under soiled bedding conditions, social odor context had no effect on acoustic call features in BTBR mouse pups. This indicates that the BTBR mouse model for ASD does not display deficits in detecting changes in social context, but has a limited ability and/or reduced motivation to adjust to them. The BTBR T+tf/J (BTBR) inbred strain of mice is one of the most commonly used mouse models for autism spectrum disorders (ASD). BTBR mice display behavioral phenotypes with relevance to all diagnostic core symptoms of ASD, namely persistent deficits in reciprocal social interaction and communication across multiple contexts, together with restricted, repetitive patterns of behavior, activities, and interests DSM-5, , as compSpecifically, BTBR mice display reduced reciprocal social interaction behavior as juveniles  and N = 30 C57BL/6J (B6) mice. Breeding pairs were purchased from The Jackson Laboratory  and bred at the National Institute of Mental Health in Bethesda, MD, USA. About 2 weeks after pairing for breeding, females were individually housed and subsequently inspected daily for pregnancy and delivery. The day of birth was considered as postnatal day (PND) 0. All mice were housed in polycarbonate Makrolon cages . Bedding, paper strips, a nestlet square, and a cardboard tube were provided in each cage. Standard rodent chow and water were available ad libitum. The colony room was maintained on a 12:12 light/dark cycle with lights on at 06:00 h, at 20°C temperature and 55% humidity. All procedures were conducted in strict compliance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and approved by the National Institute of Mental Health Animal Care and Use Committee.Subject mice were N = 15 per strain and social odor context. To this aim, mouse pups from four different litters per strain  were tested on PND8, using either clean bedding or soiled bedding from the home cage. Random group assignment was used, with approximately 50% of pups per sex from a given litter being tested under clean bedding or soiled bedding conditions, respectively. Pups were tested only once to avoid carry over effects. Home cages used to obtain soiled bedding material were not cleaned for at least 2 days prior testing in order to expose mouse pups to sufficiently distinct odor stimuli.An experimental design with two independent factors was used in order to study the effects of social odor context on isolation-induced pup USV in a strain-dependent manner, namely strain (BTBR vs. B6) and social odor context (clean bedding vs. soiled bedding), with Pups were isolated from their mother and littermates on PND8 for 5 min under room temperature . Pups were removed individually from the nest at random and gently placed into an isolation container made of glass , containing either clean bedding or soiled bedding depending on experimental group. The isolation container was surrounded by a sound attenuating box (18 × 18 × 18 cm) made of Styrofoam . USV emission was monitored by an UltraSoundGate Condenser Microphone CM16  placed in the roof of the sound attenuating box, 10 cm above the floor. The microphone was connected via an UltraSoundGate 116 USB audio device (Avisoft Bioacoustics) to a personal computer, where acoustic data were recorded with a sampling rate of 250,000 Hz in 16 bit format by Avisoft RECORDER . The microphone was sensitive to frequencies of 15–180 kHz with a flat frequency response (±6 dB) between 25 and 140 kHz. After the 5 min isolation period, body weight and body temperature were determined. Body weight was measured using a palmscale . For body temperature determination a DiGiSense Thermistor Thermometer  was used. Body temperature was measured by gentle application of the thermal probe onto the stomach of the mouse pup for 20 s. Isolation occurred between 8:00 and 12:00 h during the light phase of the 12:12 h light/dark cycle. Prior to each test, behavioral equipment was cleaned using a 70% ethanol solution, followed by water, and dried with paper towels.For acoustical analysis, recordings were transferred to Avisoft SASLab Pro  and a fast Fourier transform was conducted , resulting in spectrograms with 488 Hz of frequency resolution and 0.512 ms of time resolution. Detection of isolation-induced USV was provided by an automatic threshold-based algorithm (amplitude threshold: −40 dB) and a hold-time mechanism (hold time: 10 ms). Since no USV were detected below 30 kHz, a high-pass filter of 30 kHz was used to reduce background noise outside the relevant frequency band to 0 dB. The accuracy of USV detection by the software was verified manually by an experienced user. When necessary, missed USV were marked by hand to be included in the automatic parameter analysis. Total number of isolation-induced USV was calculated for the entire 5 min recording session. Based on previous studies on isolation-induced USV in mouse pups , the ones two before (N-2), and the ones three before (N-3) in mouse pups that emitted >3 isolation-induced USV. Paired t-tests were used to compare correlation coefficients against chance level. Sex had no effect on the emission of isolation-induced USV . A p-value of < 0.050 was considered statistically significant.For statistical comparisons, Two-Way ANOVAs with the between-subject factors strain (BTBR vs. B6) and social odor context (clean bedding vs. soiled bedding) were calculated, followed by unpaired F = 15.646, p < 0.001; Figure F = 124.807, p < 0.001; interaction social odor context × strain: F = 0.001, p = 0.983; Figure t(28) = 2.939, p = 0.007 and t(28) = 2.664, p = 0.013; respectively]. In BTBR mouse pups, a significant reduction in isolation-induced USV was seen in the first 2 min of testing , and in line with the results obtained under clean bedding conditions, BTBR mouse pups also emitted significantly more isolation-induced USV than B6 mouse pups under soiled bedding conditions .Social odor context significantly affected the emission of isolation-induced USV , despite total calling times being significantly higher in BTBR than in B6 mouse pups under clean and soiled bedding conditions . The reductions in isolation-induced USV emission rates and total calling times seen in mouse pups tested in a soiled odor context were not due to longer latencies to start calling , yet latencies to start calling differed significantly between strains . Specifically, the latency to start calling was significantly reduced in BTBR mouse pups as compared to B6 mouse pups under both, clean and soiled bedding conditions , consistent with an overall higher level of isolation-induced USV emission in BTBR mouse pups.Consistently, total calling time was significantly lower in mouse pups tested in soiled bedding than in littermates tested in clean bedding  and frequency modulation . In contrast to BTBR mouse pups, mean call duration was affected by social odor context in B6 mouse pups, which emitted significantly shorter isolation-induced USV when tested in soiled bedding than littermates tested in clean bedding . Furthermore, frequency modulation was affected by social odor context, with B6 mouse pups tested in soiled bedding emitting significantly less frequency-modulated isolation-induced USV than littermates tested in clean bedding , again in contrast to BTBR mouse pups. Under both, clean and soiled bedding conditions, isolation-induced USV emitted by BTBR mouse pups were significantly longer  and higher in frequency modulation  when compared to isolation-induced USV emitted by B6 mouse pups.Importantly, two out of four acoustic call features determined were also significantly affected by social odor context, namely mean call duration , but higher in peak amplitude  when compared to isolation-induced USV emitted by B6 mouse pups.Finally, however, the two other acoustic call features determined were not significantly affected by social odor context, namely peak frequency , yet significant differences between strains were detected for both measures . Specifically, as expected, body weight was significantly higher in BTBR than in B6 mouse pups under clean and soiled bedding conditions . Yet, when comparing strains tested either in clean or soiled bedding, no significant differences in body temperature were detected, but BTBR tended to have higher body temperatures than B6 .Of note, body weight and temperature did not differ significantly between the two social odor contexts , by the ones two before , and by the ones three before [N–3; clean bedding: t(14) = 6.353, p < 0.001; soiled bedding: t(14) = 7.313, p < 0.001; Figure t(13) = 6.255, p < 0.001; soiled bedding: t(12) = 5.647, p < 0.001; N–2; clean bedding: t(13) = 3.970, p = 0.002; soiled bedding: t(12) = 3.187, p = 0.008; N–3; clean bedding: t(13) = 3.992, p = 0.002; soiled bedding: t(12) = 1.479, p = 0.165; Figure p > 0.100).An additional sequential analysis of the durations of subsequent isolation-induced USV finally indicated that the call emission pattern is not random in BTBR mouse pups tested under both, clean and soiled, bedding conditions, since the durations of given isolation-induced USV could be predicted by the durations of the previous ones [N–1; clean bedding: An important diagnostic criterion for social communication deficits in ASD are difficulties in adjusting behavior to suit different social contexts DSM-5, . In expeConsidering the diagnostic criteria for ASD and the experimental findings obtained in human ASD studies, surprisingly little is known about difficulties in adjusting behavior to suit different social contexts in mouse models for ASD (Wöhr and Scattoni, This is in contrast to what was expected, considering that BTBR mice display behavioral phenotypes with relevance to all diagnostic core symptoms of ASD (for review see: Blanchard et al., The fact that emission of isolation-induced USV is affected by social context in BTBR mouse pups might be viewed as a challenge for the BTBR inbred strain of mice as a mouse model for ASD. However, it has to be emphasized that very little evidence is available up to now supporting the notion that the inhibition of isolation-induced USV caused by the presence of odors from mothers and littermates allows the reliable assessment of ASD-relevant behavioral alterations in mouse pups. Probably the strongest finding in support of such a notion was reported by Moles et al. . They shIt has further to be emphasized that also little is known about the general mechanisms underlying the inhibition of isolation-induced USV caused by the presence of odors from mothers and littermates in mouse pups. In a pioneering study by Branchi et al. , compariWhile BTBR mouse pups unexpectedly displayed a calming response and emitted fewer isolation-induced USV when tested under soiled as compared to clean bedding conditions, social odor context had no effect on acoustic call features, such as call duration, peak frequency, peak amplitude, and frequency modulation, in BTBR mouse pups. This is in stark contrast to what was seen in B6 mouse pups, which emitted isolation-induced USV with shorter call durations and lower levels of frequency modulation under soiled bedding conditions as compared to clean bedding conditions.At least three possible mechanisms for the reduced adjustment to different social contexts in BTBR mouse pups can be considered. Firstly, the fact that social odor context had no effect on acoustic call features in BTBR mouse pups could be due to deficits in detecting changes in the social context caused by olfactory impairments. However, BTBR mice displayed normal olfactory abilities, both in non-social test paradigms, such as the buried food task (Moy et al., Secondly, a limited ability to adjust to different social contexts could be the reason for the fact that social odor context had no effect on acoustic call features in BTBR mouse pups. The unusual repertoire of USV categories seen in BTBR mouse pups, including high levels of harmonics, two-syllable, and composite calls (Scattoni et al., Thirdly, a reduced motivation to adjust to different social contexts could also be the reason and on the basis of the data available it is currently not possible to differentiate between the two possible mechanisms. In support of the latter mechanism it was shown that BTBR mice are characterized by a reduction in social motivation (Pearson et al., In line with the findings obtained by Scattoni et al.  and SchwIt is further in line with the findings obtained by Scattoni et al.  that theThe present study further identified clusters of isolation-induced USV emitted by BTBR and B6 mouse pups under clean and soiled bedding conditions by means of density plots. In BTBR mouse pups, four prominent call clusters were detected, virtually independent from social odor context, further highlighting their limited ability and/or reduced motivation to adjust to different social contexts. One cluster was characterized by short call durations and low levels of frequency modulation. The other three clusters were characterized by long call durations, with varying levels of frequency modulation, namely low, moderate, and high. Therefore, by means of the quantitative approach applied here, no clear evidence for the existence of 10 distinct USV subtypes as reported by Scattoni et al.  was obtaCall clustering markedly differed between BTBR and B6 mouse pups. While in BTBR four prominent call clusters were detected, only two prominent call clusters were detected in B6 mouse pups. One cluster was characterized by short call durations and low levels of frequency modulation, whereas the second one was characterized by comparably long call durations and moderate levels of frequency modulation. Interestingly, in B6 mouse pups, social odor context affected call clustering, with the second call cluster characterized by comparably long call durations and moderate levels of frequency modulation being less prominent and coherent, in line with the overall reduced mean call duration and frequency modulation. Shorter call durations in mouse pups tested in soiled bedding were reported before (Marchlewska-Koj et al., Shank1 deficient mouse (Wöhr, Shank1 deficient mice display a variety of behavioral alterations with relevance to ASD (Hung et al., Shank2 deficient mice see: Ey et al., Finally, an additional sequential analysis of the durations of subsequent isolation-induced USV indicated that the USV emission pattern is not random in BTBR mouse pups tested under both, clean and soiled bedding conditions, and that the temporal pattern does not differ significantly from the one obtained in B6 mouse pups. Specifically, in both, BTBR and B6 mouse pups, the durations of given isolation-induced USV could be predicted by the durations of the previous ones. Considering the unusual pattern of USV categories displayed by BTBR mouse pups (Scattoni et al., se Wöhr, . Shank1 In accordance with their behavioral phenotypes with relevance to all diagnostic core symptoms of ASD, it was predicted that BTBR mouse pups would not display a calming response when tested under soiled bedding conditions with home cage bedding material containing maternal odors, and that similar isolation-induced USV emission rates would be seen in BTBR mice tested under clean and soiled bedding conditions. Unexpectedly, however, the present findings show that BTBR mouse pups display such a calming response and emit fewer isolation-induced USV when tested under soiled as compared to clean bedding conditions, similar to B6 mouse pups. Yet, in contrast to B6 mouse pups, which emitted isolation-induced USV with shorter call durations and lower levels of frequency modulation under soiled bedding conditions, social odor context had no effect on acoustic call features in BTBR mouse pups. This indicates that the BTBR mouse model for ASD does not display deficits in detecting changes in social context, but has a limited ability and/or reduced motivation to adjust to them.The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


We here discuss these techniques in relation to important structural features and modifications of starches such as: starch phosphorylation, starch hydrolysis, chain transfer/branching and novel concepts of hybrid starch-based polysaccharides. In planta starch bioengineering is generally challenged by yield penalties and inefficient production of the desired product. However, in some situations, GM crops for starch bioengineering without deleterious effects have been achieved.Plant starches regularly require extensive modification to permit subsequent applications. Such processing is usually done by the use of chemical and/or physical treatments. The use of recombinant enzymes produced by large-scale fermentation of GM microorganisms is increasingly used in starch processing and modification, sometimes as an alternative to chemical or physical treatments. However, as a means to impart the modifications as early as possible in the starch production chain, similar recombinant enzymes may also be expressed  Raw starch is usually modified by industrial chemical and/or physical treatments. The most frequent chemical modifications are esterification, etherification or oxidation of hydroxyl groups. Physical treatments usually involve shaping and/or disruption by mechanical force or thermal treatments or a combination of both  recently concluded that an equivalent level of hazard can be associated with cis-genesis as for conventional plant breeding, which should favor the cis-genic strategy as a non-GM technology (3) or by high-energy radiation. When combined with high-throughput molecular DNA screening techniques, targeted mutations in genes of interest can be identified  and rice , which hydrolyze α-1,4 linkages, producing maltodextrins, and β-amylase (EC 3.2.1.2) acting specifically on α-1,4 linkages from the non-reducing ends to produce β-maltose. Glucoamylases (EC 3.2.1.3) can be used to further hydrolyse β-limit dextrins, maltodextrins and maltose to glucose. Finally, hydrolases may be used in combination with isomerases to convert between isomeric forms of saccharides, such as in the case of glucose isomerase (EC 5.3.1.5) used to convert glucose into fructose . Such hyBranching and de-branching enzymes represent two types of starch hydrolases that affect the number of α-1,6 linkages, and thereby the branching structure of starch. De-branching enzymes, such as pullulanase/limit dextrinase (EC 3.2.1.41) and isoamylase (EC 3.2.1.68), specifically hydrolyse α-1,6 linkages. Specific homologs of debranching enzymes are naturally involved in the biosynthesis of starch in plants. Their suppression results in production of so-called phytoglycogen , which iKlebsiella has been expressed in potato tubers to generate cyclodextrins. However, the amount of cyclodextrin was ≤0.01% of the starch fraction (E. coli has been used to increase the branching in potato tuber starch granules (amylose-extender (ae) maize starch types contain above 70% amylose, which is caused by loss-of-function mutations in the SBEIIb gene   . Natural starch) , and the starch) . Both en starch)  demonstr starch) . A similin vitro . Howeverin vitro . The usein wheat  and cornin wheat  leading ct yield . To thishreefold .in vitro by the action of different types of enzymes and their respective substrates, a technique which has been referred to as “enzymatic polymerization to unnatural HP”   are defined as having covalent bonds between two or more different natural (poly)-saccharide systems. It is possible to engineer such HP by enzymatic polymerization ural HP” . Hybrid  g–1 FW) . A simil dextran . Expressrization  and thato starch . Expresso starch .in planta starch modification in cereal grains can jeopardize germination efficiency and seedling establishment (Sorghum bicolor allelic variation of a gene encoding the starch debranching enzyme pullulanase was shown to be a potential target for breeding toward higher starch digestibility without deleterious pleiotropic effects (Plants generally demonstrate a tendency to display negative pleiotropic effects in response to transgenic events, which changes their starch biosynthesis . Strateglishment . In othelishment . In Sorg effects .in planta starch bioengineering by expression of starch modifying enzymes directly in crop storage organs faces a number of challenges that need to be addressed further. In particular, starch bioengineering may sometimes be associated with significant yield loss, e.g., by pleiotropic effects of the transgenic enzyme or due to effects of the modified starch structure on the ability of the starch storage organ to re-mobilize the energy that is stored in the starch. Only a few studies have been carried through to agronomic field trials. The physiological conditions in amyloplasts of crop starch organs may not be optimal for starch modifying enzymes of non-plant origin, and in several studies only very small amounts of the desired product is formed. However, the method looks promising for situations where the transgenic enzymes remain inactive during crop development, so that the above mentioned deleterious effects are avoided. For example crops expressing thermophilic hydrolytic enzymes, which are activated by heat, have been shown to reduce production costs and energy and water usage of grain processing. Other methods of “post-harvest” activation of transgenic enzymes in crops could be explored. In other situations there may not be a biotechnological alternative to transgenic enzyme expression directly in developing crop organs. For example starch kinases have been used to increase starch phosphate content in cereal grains and in potatoes, whereas there are currently no reports that a similar modification can be made during post-harvest starch processing by adding enzymes produced by GM microorganism.In this review we have compared the use of starch modifying enzymes produced by GM microorganisms with the expression of these enzymes directly in crops. In summary we find that The Editor Giuseppe Dionisio declares that, despite being affiliated to the same institution as the authors Kim H. Hebelstrup and Domenico Sagnelli, the review process was handled objectively and no conflict of interest exists. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.