Influenza Vaccines: Successes and Continuing Challenges.

Influenza vaccines have been available for over 80 years. They have contributed to significant reductions in influenza morbidity and mortality. However, there have been limitations in their effectiveness, in part due to the continuous antigenic evolution of seasonal influenza viruses, but also due to the predominant use of embryonated chicken eggs for their production. The latter furthermore limits their worldwide production timelines and scale. Therefore today, alternative approaches for their design and production are increasingly pursued, with already licensed quadrivalent seasonal influenza vaccines produced in cell cultures, including based on a baculovirus expression system. Next-generation influenza vaccines aim at inducing broader and longer-lasting immune responses to overcome seasonal influenza virus antigenic drift and to timely address the emergence of a new pandemic influenza virus. Tailored approaches target mechanisms to improve vaccine-induced immune responses in individuals with a weakened immune system, in particular older adults.

Aging and Options to Halt Declining Immunity to Virus Infections.

Immunosenescence is a process associated with aging that leads to dysregulation of cells of innate and adaptive immunity, which may become dysfunctional. Consequently, older adults show increased severity of viral and bacterial infections and impaired responses to vaccinations. A better understanding of the process of immunosenescence will aid the development of novel strategies to boost the immune system in older adults. In this review, we focus on major alterations of the immune system triggered by aging, and address the effect of chronic viral infections, effectiveness of vaccination of older adults and strategies to improve immune function in this vulnerable age group.

The Human Cytomegalovirus pUL145 Isoforms Act as Viral DDB1-Cullin-Associated Factors to Instruct Host Protein Degradation to Impede Innate Immunity.

Human cytomegalovirus (HCMV) causes diseases in individuals with immature or compromised immunity. To evade immune control, HCMV evolved numerous antagonists targeting the interferon system at multiple levels. By comparative analysis of naturally arising variants of the most widely studied HCMV strain, AD169, and a panel of targeted mutants, we uncover the UL145 gene as indispensable for STAT2 downregulation. Ribosome profiling confirms the translation of the canonical pUL145 protein (pUL145-Long) and newly identifies a shorter isoform (pUL145-Short). Both isoforms recruit DDB1-containing ubiquitin ligases to induce proteasomal degradation of STAT2. An alanine-scanning mutagenesis discloses the DDB1 interaction motif of pUL145 that resembles the DDB1-binding interface of cellular substrate receptors of DDB1-containing ubiquitin ligases. Thus, pUL145 constitutes a viral DDB1-cullin-associated factor (vDCAF), which mimics cellular DCAFs to exploit the ubiquitin-proteasome system to impede antiviral immunity. Notably, the viral exploitation of the cullins can be targeted to restore the efficacy of the host immune response.

Cellular Cullin RING Ubiquitin Ligases: Druggable Host Dependency Factors of Cytomegaloviruses.

Human cytomegalovirus (HCMV) is a ubiquitous betaherpesvirus that frequently causes morbidity and mortality in individuals with insufficient immunity, such as transplant recipients, AIDS patients, and congenitally infected newborns. Several antiviral drugs are approved to treat HCMV infections. However, resistant HCMV mutants can arise in patients receiving long-term therapy. Additionally, side effects and the risk to cause birth defects limit the use of currently approved antivirals against HCMV. Therefore, the identification of new drug targets is of clinical relevance. Recent work identified DNA-damage binding protein 1 (DDB1) and the family of the cellular cullin (Cul) RING ubiquitin (Ub) ligases (CRLs) as host-derived factors that are relevant for the replication of human and mouse cytomegaloviruses. The first-in-class CRL inhibitory compound Pevonedistat (also called MLN4924) is currently under investigation as an anti-tumor drug in several clinical trials. Cytomegaloviruses exploit CRLs to regulate the abundance of viral proteins, and to induce the proteasomal degradation of host restriction factors involved in innate and intrinsic immunity. Accordingly, pharmacological blockade of CRL activity diminishes viral replication in cell culture. In this review, we summarize the current knowledge concerning the relevance of DDB1 and CRLs during cytomegalovirus replication and discuss chances and drawbacks of CRL inhibitory drugs as potential antiviral treatment against HCMV.

Nonpalpable testes: Ultrasound and contralateral testicular hypertrophy predict the surgical access, avoiding unnecessary laparoscopy.

INTRODUCTION: In up to 20% of patients presenting with undescended testes, one or both are non-palpable. Whereas the most reliable means to exclude an abdominal testis is laparoscopy, there has been a lot of debate about the role of inguinal ultrasound (US) in detecting non-palpable inguinal testis. While we do not aim to add another paper claiming the benefits of US, we wanted to determine the excess capability of US to determine the correct surgical approach - inguinal or laparoscopy. In the light of avoiding unnecessary diagnostic laparoscopies, even the cost-effectiveness raised in many current papers might be called into question. PATIENTS AND METHODS: Of a total of 684 boys who underwent surgery for undescended testes at our department between 2011 and 2014, in 58 (8.5%), one or both testes were neither palpable preoperatively nor under general anesthesia. These boys were examined by two experienced pediatric urologists clinically as well as by US. Besides the size of the contralateral testis, the presence of a testis in the inguinal channel was investigated. The additional impact of US over clinical exam and consideration of the size of the contralateral testis was assessed by means of intra-individual comparisons using Cochran-Q as well as McNemar tests. RESULTS: Clinical exam without considering the size of the contralateral testis had a sensitivity of 9% (95% CI 2-24%) and a specificity of 100% (95% CI 86-100%) to accurately predict the surgical approach deemed appropriate postoperatively. The consideration of the size of the contralateral testis - taken as an isolated factor - accurately predicted the surgical approach with a sensitivity of 21% (95% CI 9-38%) and a specificity of 88% (95% CI 68-97%). Ultrasound accounted for a sensitivity of 53% (95% CI 35-70%) and a specificity of 100% (95% CI 86-100%). The addition of US increased the sensitivity to correctly predict an inguinal incision from 29% to 71% and specificity slightly increased from 88% to 92%. This difference is significant (p = 0.008) in the bilateral McNemar test (Figure). CONCLUSION: Inguinal US of non-palpable testes and measurement of the contralateral testis are synergistic in predicting the surgical approach. The addition of ultrasound to a clinical exam, performed also under general anesthesia and by an experienced pediatric urologist significantly increases the prediction of the correct surgical approach. Our results translate into five boys needing an US of the NPT to prevent one laparoscopy. Whereas cost-effectiveness of US might be debatable in regard to different healthcare systems, it is proven to be an effective, non-harmful tool to avoid unnecessary diagnostic laparoscopies.

Evaluation of Mathisen's technique for ureteral reimplantation in children with primary vesicoureteral reflux.

INTRODUCTION: Although cross-trigonal ureteral reimplantation (Cohen) is a commonly used technique in children, it represents a non-physiological transfer of the ureteral orifices and may prove challenging with regard to endoscopic ureteral operations in later life. In 1964, Mathisen described an alternative method of ureteral reimplantation with lateralization of the neohiatus, creating an orthotopic course of the submucosal ureter. We have evaluated success and complication rates of both techniques that were applied sequentially at our departments. METHODS: Forty-eight consecutive patients (83 ureters, 24 males/24 females) following Mathisen reimplantation were compared with 53 consecutive patients (98 ureters, 30 males/23 females) following Cohen reimplantation. Inclusion criteria were primary vesicoureteral reflux (VUR) and no previous intervention. Reflux grades (Mathisen 58 ureters/69.9% VUR ≥ III; Cohen 66 ureters/66.7% VUR ≥ III) and the occurence of other complicating factors (ureteroceles, megaureters, posterior urethral valves) in both groups were comparable. RESULTS: After Cohen's reimplantation there were no immediate complications requiring intervention; during follow-up (mean 28.2 months) three patients (5.6%) suffered febrile urinary tract infections (UTIs), of which one (1.8%) was diagnosed with a persisting VUR. Persistent hydronephroses (≥II SFU) were recorded in six patients (13.2%). After reimplantation using Mathisen's technique, two patients (4.1%) suffered significant intravesical bleeding; during follow-up (mean 23.06 months) four patients (8.3%) suffered febrile UTIs, and seven patients (14.5%) were diagnosed with persisting VUR after a mean follow-up of 10.8 months. The patients with persistent VUR had more commonly high-grade (IV and V) VUR initially, compared to the whole group. Two patients (4.1%) had persistent hydronephroses (≥II SFU). Mathisen's technique for ureteral reimplantation yielded a significantly (p = 0.0256 patients, p = 0.006 ureterorenal units) lower success rate (85.5% patients, 89.2% ureterorenal units) in comparison with Cohen's technique (98.2% patients, 99% ureterorenal units). Although there was no intervention for obstruction, persistent hydronephrosis was more common in the Cohen group (13.2% vs. 4.1%, n.s.). CONCLUSIONS: Despite the advantages of an orthotopic ureteral orifice close to the bladder neck, as achieved by Mathisen's reimplantation, cross-trigonal ureteral reimplantation proved more reliable for VUR correction. As regards optimizing the results, patient selection for either technique could prove essential. Nevertheless, as regards the difficulties with ectopic ureteral orifices in the Cohen technique in the long-term follow-up, the concept of anatomic, orthotopic ureteral reimplantation should be pursued and the technique should be further refined.

Penile appearance after hypospadias correction from a parent's point of view: Comparison of the hypospadias objective penile evaluation score and parents penile perception score.

INTRODUCTION: As there is only scarce information on the parents' view of the cosmetic outcome after hypospadias surgery we aimed to evaluate whether the results of the hypospadias objective penile evaluation (HOPE) score are transferable to parents satisfaction as measured by the pediatric penile perception score (PPPS). PATIENTS: 42 patients after hypospadias correction were included (2 (6.9%) glandular, 20 (68.9%) coronal, 6 (20.6%) penile and 1 (3.4%) scrotal hypospadias, median age 15.0 months). Two surgeons independently assessed HOPE score; the PPPS score as well as 4 questions specifically designed by a psychologist were completed by fathers and mothers. 29 (69.9%) full datasets were available for evaluation. RESULTS: Parents' assessment of the cosmetic results was worse than surgeons' assessment (81.13% [PPPS] vs. 92.81% [HOPE] of the respectively possible highest score, P < 0.0001). All 58 parents (100%) were convinced that surgery led to a better cosmetic aspect of their sons' genitalia although both, mothers and fathers, perceived the operation as a major encumbrance (fathers 3.62 vs. mothers 3.97 on a scale from 0-6, P = 0.22). CONCLUSION: Parents can be encouraged preoperatively that a hypospadias operation, seen from their point of view will be a major amendment to the cosmetic appearance of their sons' genitalia even if the operation itself is perceived as a major psychological burden. In direct comparison of the highest possible score of either tool (HOPE or PPPS), the cosmetic results were judged significantly more optimistic by surgeons as compared to parents using validated tools. HOPE score results therefore may not be transferred uncritically to the parents view on the cosmetic results.

Evaluation of the Charité Jet Lag Scale: Further Assessment of Jet Lag Using a Method-Comparison Approach.

The first German Charité Jet Lag Scale (CJS), based on the only validated English questionnaire, was introduced in a former study. In addition to providing further jet lag investigations, this second method-comparison study aims to evaluate the CJS. Consistent interviewing methods are essential for future research in identifying possible cross-cultural tendencies, advancing jet lag definition, and establishing potential alleviation methods. Jet lag in 17 German chorus members (5 male, 12 female, mean age 42.35 ± 13.06 years) was monitored on their 2½-week trip from Germany to Argentina and back by use of actigraphy, sleep diaries, and the CJS. Cronbach's alpha, Pearson and Lin correlations, and Bland-Altman diagrams were applied to assess CJS reliability and validity between all measurement methods. CJS study analysis results reconfirmed the prevalence of jet lag at approximately 60%. Moreover, the study assessed high Cronbach's alpha values and significant positive correlations for several corresponding and related sleep parameters and CJS items such as total sleep time (TST) and sleep onset latency (SOL). The confidence limits in the Bland-Altman diagrams remained within the limits of earlier studies (TST ± 1 h, SOL ± 30 min). Overall, jet lag measures in this study confirmed previous jet lag studies. Evidence indicates positive reliability and validity of the CJS, with lower specificity in some parameters. We conclude that the CJS is applicable for complex jet lag studies. The findings provide support for further development of the scale and its capability as a consistent jet lag interviewing method.

Persistence and recurrence of vesicoureteric reflux in children after endoscopic therapy - implications of a risk-adapted follow-up.

INTRODUCTION: There is no well-defined follow-up scheme available to reliably detect persistent or recurrent vesicoureteric reflux (VUR) after endoscopic therapy (ET), but also to reduce postoperative invasive diagnostics in these children. Our aim was the evaluation of possible predictors of persistence and recurrence of VUR, in order to elaborate and test a risk-adapted follow-up regimen. MATERIAL AND METHODS: 92 patients (85/92%f, 7/8%m, age 2.99y) underwent direct isotope cystography (DIC) three months after ET. Persistent or recurrent VUR, scarring on dimercaptosuccinic acid (DMSA) scans and further fUTIs after therapy (follow-up 24.6 m) were documented and analysed. RESULTS: VUR persistence 3 months after ET was found in 11 (11.9%) patients; recurrent VUR in 4 (4.3%) patients. Scarring on preoperative DMSA and dilating VUR (°III and °IV) were significantly associated with recurrent VUR. If only children with preoperative positive DMSA scan or dilating VUR would have undergone DIC, only 58/92 DICs (64%) would have been necessary. Only 45.5% of otherwise detected VURs would have been identified using this risk-adapted strategy. CONCLUSIONS: Limiting invasive follow-up diagnostics (VCUG) and, therewith, the radiation burden in a predefined group of patients at risk for persistence or recurrence of VUR is not recommended, due to the significant chance of missing persistent or new onset contralateral VUR. Therefore, we recommend a routine follow-up VCUG after ET. Further prospective scientific efforts to evaluate new, alternative factors influencing persistence and recurrence of VUR, in order to establish an effective follow-up strategy, are warranted.

Lower radiation burden in state of the art fluoroscopic cystography compared to direct isotope cystography in children.

INTRODUCTION: Both, fluoroscopic voiding cystourethrography (fVCUG) and direct isotope cystography (DIC) are diagnostic tools commonly used in pediatric urology. Both methods can detect vesicoureteral reflux (VUR) with a high sensitivity. Whilst the possibility to depict anatomical details and important structures as for instance the urethra in boys or the detailed calyceal anatomy are advantages of fVCUG, a lower radiation burden is thought to be the main advantage of DIC. In the last decade, however, a rapid technical evolution has occurred in fluoroscopy by implementing digital grid-controlled, variable rate, pulsed acquisition technique. As documented in literature this led to a substantial decrease in radiation burden conferred during fVCUGs. OBJECTIVE: To question the common belief that direct isotope cystography confers less radiation burden compared to state of the art fluoroscopic voiding cystography. STUDY DESIGN: Radiation burden of direct isotope cystography in 92 children and in additional 7 children after an adaption of protocol was compared to radiation burden of fluoroscopic voiding cystourethrography in 51. The examinations were performed according to institutional protocols. For calculation of mean effective radiation dose [mSv] for either method published physical models correcting for age and sex were used. For DIC the model published by Stabin et al., 1998 was applied, for fVCUG two different physical models were used (Schultz et al., 1999, Lee et al., 2009). RESULTS: The radiation burden conferred by direct isotope cystography was significantly higher as for fluoroscopic voiding cystourethrography. The mean effective radiation dose for direct isotope cystography accounted to 0.23 mSv (± 0.34 m, median 0.085 mSv) compared to 0.015 mSv (± 0.013, median 0.008 mSv, model by Schultz et al.) - 0.024 mSv (± 0.018, median 0.018 mSv, model by Lee et al.) for fluoroscopic voiding cystourethrography. After a protocol adaption to correct for a longer examination time in DIC that was caused by filling until calculated bladder capacity, mean radiation burden accounted to .07 mSv (median 0.07 mSv) and the values were less scattered. DISCUSSION: As it had to be expected from literature, radiation dose from fVCUG, if modern image acquisition techniques are used, is even less than from DIC. In our protocol, according to nuclear medicine standards, bladders were filled until calculated capacity. This resulted in a longer examination time for the patients with a higher functional capacity, resulting in relatively higher radiation burden. However, also if the protocol is changed or only the patients with relatively fast bladder emptying are considered, radiation burden conferred by DIC is higher (at least × 2.9, comparing the "worst" case for fVCUG with the "best" case for DIC). Absolute radiation burden conferred by either exam is extremely low compared to other medical radiation exposures as well as to environmental radiation. Consequently it is most probably not relevant for the individual childs future risk for cancer or other radiation damage. However, because of repeated investigations with correspondingly higher radiation burden in this patient group the ALARA (as low as reasonably achievable) principle should lead to a optimized use of fVCUG rather than an uncritical use of DIC, given that modern acquisition standards are available and radiation measurement is performed. Also, fVCUG provides more information concerning anatomical details compared to DIC. CONCLUSION: Contrary to common beliefs, effective radiation dose conferred during fluoroscopic voiding cystourethrography is significantly lower than during direct isotope cystography. The prerequisite for our findings, however, is the use of modern image acquisition tools and an optimized protocol. Both exams confer low radiation doses probably only relevant to children undergoing repeated radiation exposure. Nevertheless, this findings should be considered in indication for either exam in order to reduce the radiation burden to a minimum whilst optimizing the information yield.

A new German Charité Jet Lag Scale for jet lag symptoms and application.

Travelling across multiple time zones provokes adaptation of endogenous circadian rhythm to the new time zone. Within the context of previous studies, an English-language state-of-health questionnaire, the Columbia Jet Lag Scale, is the only sufficiently validated scale for jet lag and its symptoms. This study presents a new state-of-health questionnaire in German, one intended to achieve standardisation of surveys on jet lag. The questionnaire was applied to define the baseline for the prevalence of jet lag symptoms based on a reference group (n = 36). The jet lag score ascertained was subsequently applied to determine the frequency of jet lag in a group of 53 subjects. Systematic investigation of the frequency of jet lag symptoms had not been previously presented. Among the group of 53 travelling test subjects, 60% demonstrated moderate jet lag symptoms. Practitioner Summary: This introduction of the Charité Jet Lag Scale, the first German jet lag questionnaire, calls attention to this topic for the first time since a 2000 publication in Ergonomics. Our systematic investigation of jet lag frequency, with the new scale, determined moderate jet lag symptoms among 60% of subjects.

Gender-related fetal development of the internal urethral sphincter.

OBJECTIVE: To investigate the fetal development of the internal urethral sphincter and the gender-related morphologic differences of the bladder outlet. MATERIALS AND METHODS: Thirty-seven (14 female, 23 male) fetal bladder neck specimens (mean gestational age, 19.4 weeks) with the smooth muscle complex of the internal sphincter were investigated histologically. After immunostaining serial sections in 3 reference planes (sagittal, frontal, and horizontal) of the bladder neck, the internal sphincter volumes and bladder outlet diameters were measured and correlated with gender and age of gestation. RESULTS: Between the 18th and 40th week of gestation, an exponential growth of the internal sphincter muscle with significant higher volumes could be observed in male fetuses compared with female fetuses (internal sphincter volumes, P = .006; radius of the sphincter complex, P = .001). As a result of this gender difference, the bladder outlet was significantly (P = .001) narrower in male than in female fetuses. Moreover, we found a significant positive correlation between age and all measured parameters in both male and female specimens. CONCLUSION: The present study indicates a significant closer bladder outlet in male fetuses compared than in females. It thereby provides evidence of a gender-related functional obstruction in addition to a suppositious transient infravesical obstruction in male human fetuses.

Prevalence of the human papillomavirus (HPV) expression of the inner prepuce in asymptomatic boys and men.

OBJECTIVE: To investigate the prevalence of high-risk (HR) and low-risk (LR) human papillomavirus (HPV) in prepuces of boys and men without any HPV related lesions. PATIENTS AND METHODS: Between 2009 and 2011, a total collective of 250 boys and men were investigated in this prospective study. The samples were subdivided into 3 groups regarding their age, consisting of 125 (50%) children (0-10 years), 38 (15.2%) adolescents (11-20 years) and 87 (34.8%) adults (>20 years). In situ hybridization (ISH) was performed to detect HR and LR virus types within the epithelium, followed by microscopic interpretation and determination between episomal and integrative signal pattern. RESULTS: Our results revealed high levels of HPV concentration (HR and LR) in all age groups: HR versus LR positivity was seen in 45.5 versus 35% (children), 60.6 versus 63.6% (adolescents) and 58.3 versus 48.6% (adults). The topmost rate of HR (59.8%)- and LR (50.6%)-positive probes was found in the group with high estimated sexual activity (>14 years). CONCLUSION: Concerning the high prevalence of male HPV infection even in children, indicating non-sexual transmission pathways, inclusion of boys in the vaccination program seems to be required reducing their burden of HPV related disease.

Positioning irrigation of contrast cystography for diagnosis of occult vesicoureteric reflux: association with technetium-99m dimercaptosuccinic acid scans.

OBJECTIVE: Positioning irrigation of contrast (PIC) cystography identifies occult or PIC vesicoureteral reflux (PIC-VUR) in children with recurrent febrile urinary tract infections (UTI) but no vesicoureteric reflux (VUR) on standard voiding cystourethrogram (VCUG). We sought to identify the relationship between PIC-VUR and renal scarring in technetium-99m dimercaptosuccinic acid (DMSA) scans. PATIENTS AND METHODS: We retrospectively analysed PIC cystograms and DMSA scans for 154 kidneys in 81 children (65 girls; 16 boys; median age, 4.7 years; range, 0.9-15.2). Renal scarring was graded on a scale of 0-3. DMSA scans were pathologic in 66 patients (81%). Children had experienced mean 3.8 febrile UTI (range 1-25). Forty-seven (58%) children had a history of reflux, including 15 (19%) with previous anti-reflux operations. Indications for PIC cystography were recurrence of febrile UTI after either bilateral negative VCUG (66 children) or unilateral VUR (15 children) with contralateral/bilateral scarring or reflux that had changed sides in subsequent VCUGs. RESULTS: PIC-VUR was bilateral in 63, unilateral in 12, and absent in 6 children. Statistically significant associations between PIC-VUR grade and severity of renal scarring were identified in inter-individual (n = 77, p = 0.017) and intra-individual (refluxing vs. nonrefluxing kidney; n = 12, p = 0.008) analyses. After excluding patients with history of VUR, statistical significance was maintained in inter-individual analysis (n = 49; p = 0.018). CONCLUSION: The data suggest an association between PIC-VUR and severity of renal scarring, and legitimise the use of PIC cystography in children with renal scarring due to recurrent febrile UTI but negative findings on VCUG.

An orphan sensor kinase controls quinolone signal production via MexT in Pseudomonas aeruginosa.

Pseudomonas aeruginosa employs both N-acylhomoserine lactone and 2-alkyl-4(1H)-quinolone (AQ)-mediated interbacterial signalling for the orchestration of a genome-wide gene regulatory network. Despite the many advances that have been made in understanding the target genes of quorum sensing regulation, little is known on how quorum sensing systems are influenced by environmental cues. In this study, we show that AQ production is modulated by an orphan P. aeruginosa sensor kinase. Transcriptional studies of the sensor kinase (MxtR) mutant demonstrated that an induced expression of MexT, a LysR-type transcriptional regulator, largely determined the global transcriptional profile. Thereby, overexpression of the MexT-regulated MexEF-OprN efflux pump led to a delayed expression of the AQ biosynthetic genes and of AQ-dependent virulence factors. Furthermore, we demonstrated that autophosphorylation of MxtR was inhibited by ubiquinone, the central electron carrier of respiration in in vitro experiments. Our results elucidate on a mechanism by which P. aeruginosa senses environmental conditions and adapts by controlling the production of interbacterial AQ signal molecules. A regulatory function of a sensor kinase may indicate that there is a pre-emptive role of adaptation mechanisms that are turned on under distinct environmental conditions and that are important for efficient colonization and pathogenesis.

Long-term follow-up after botulinum toxin A (BTX-A) injection into the detrusor for treatment of neurogenic detrusor hyperactivity in children.

PURPOSE: To prove the long-term efficacy of BTX-A injection in the management of children with neurogenic detrusor hyperactivity. MATERIALS AND METHODS: 28 out of 145 children with neurogenic bladder (15 male and 13 female, mean age 10.7 years) who were treated between 2002 and 2010 and became non-responders to conservative treatment were included into the retrospective study. We injected 10-12 U/kg of BTX-A (Botox(®)) into the detrusor at 20-30 sites, sparing the trigone. The mean follow-up was 48 months (range 6-84 months). RESULTS: Group 1. 14 patients had a single injection of BTX-A. Five of them were successful. Mean bladder reflex volume increased (from 62.9 to 117.5 ml), maximum detrusor pressure decreased (from 59 to 37.5 cm H2O), detrusor compliance increased (from 4.8 to 9.5 ml/cm H2O), and leak-point-pressure decreased (from 46.5 to 24.2 cm H2O). Four patients did not respond and were treated by ileocystoplasty. Another five were lost to follow-up. Group 2. 14 patients had repeated (mean 2.5) injections of BTX-A with a mean interval of 13.7 months. In thirteen patients, urodynamic parameters of the first and last injection were similar to those obtained in Group 1, showing a good response. One patient received an ileocystoplasty. CONCLUSION: BTX-A is a safe alternative in the treatment of detrusor hyperactivity in children with myelomeningocele (MMC). The efficacy lasted a mean of 12 months and urodynamic response was unchanged even after several injections. In our series, 21.7% of children with severe low-compliance bladders were non-responders.

The Pseudomonas aeruginosa chemotaxis methyltransferase CheR1 impacts on bacterial surface sampling.

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa.

Global genotype-phenotype correlations in Pseudomonas aeruginosa.

Once the genome sequence of an organism is obtained, attention turns from identifying genes to understanding their function, their organization and control of metabolic pathways and networks that determine its physiology. Recent technical advances in acquiring genome-wide data have led to substantial progress in identifying gene functions. However, we still do not know the function of a large number of genes and, even when a gene product has been assigned to a functional class, we cannot normally predict its contribution to the phenotypic behaviour of the cell or organism--the phenome. In this study, we assessed bacterial growth parameters of 4030 non-redundant PA14 transposon mutants in the pathogenic bacterium Pseudomonas aeruginosa. The genome-wide simultaneous analysis of 119 distinct growth-related phenotypes uncovered a comprehensive phenome and provided evidence that most genotypes are not phenotypically isolated but rather define specific complex phenotypic clusters of genotypes. Since phenotypic overlap was demonstrated to reflect the relatedness of genotypes on a global scale, knowledge of an organism's phenome might significantly contribute to the advancement of functional genomics.

Genomewide identification of genetic determinants of antimicrobial drug resistance in Pseudomonas aeruginosa.

The emergence of antimicrobial drug resistance is of enormous public concern due to the increased risk of delayed treatment of infections, the increased length of hospital stays, the substantial increase in the cost of care, and the high risk of fatal outcomes. A prerequisite for the development of effective therapy alternatives is a detailed understanding of the diversity of bacterial mechanisms that underlie drug resistance, especially for problematic gram-negative bacteria such as Pseudomonas aeruginosa. This pathogen has impressive chromosomally encoded mechanisms of intrinsic resistance, as well as the potential to mutate, gaining resistance to current antibiotics. In this study we have screened the comprehensive nonredundant Harvard PA14 library for P. aeruginosa mutants that exhibited either increased or decreased resistance against 19 antibiotics commonly used in the clinic. This approach identified several genes whose inactivation sensitized the bacteria to a broad spectrum of different antimicrobials and uncovered novel genetic determinants of resistance to various classes of antibiotics. Knowledge of the enhancement of bacterial susceptibility to existing antibiotics and of novel resistance markers or modifiers of resistance expression may lay the foundation for effective therapy alternatives and will be the basis for the development of new strategies in the control of problematic multiresistant gram-negative bacteria.

The Pho regulon influences biofilm formation and type three secretion in Pseudomonas aeruginosa.

Research into the molecular mechanisms of the switch from highly motile to biofilm forming Pseudomonas fluorescens bacteria recently uncovered a role of inorganic phosphate as an important environmental regulatory factor to control c-di-GMP levels in the cell. In this study we present evidence that in the opportunistic pathogen P. aeruginosa the Pho regulon inhibits biofilm formation and is required for the repression of the type three secretion system. We furthermore identified an EAL domain protein as a downstream effector of the Pho regulon, which at least partially mediated the observed inhibition. Interestingly, inhibition of the P. aeruginosa virulence phenotype was Pho regulon-dependent in both a PA14 and a PAO1 strain background; however, in PA14 this inhibition was independent on the availability of inorganic phosphate, whereas in PAO1 phosphate enhanced biofilm formation independently of the inhibitory activity of the Pho regulon. These results clearly show that the Pho regulon contributes to the expression of the virulence phenotype in P. aeruginosa and add even more complexity to the strain-specific regulation of bacterial behaviour by environmental cues.