Comparative analysis in continuous expansion of bovine and human primary nucleus pulposus cells for tissue repair applications.

Autologous NP cell implantation is a potential therapeutic avenue for intervertebral disc (IVD) degeneration. However, monolayer expansion of cells isolated from surgical samples may negatively impact matrix production by way of dedifferentiation. Previously, we have used a continuous expansion culture system to successfully preserve a chondrocyte phenotype. In this work, we hypothesised that continuous expansion culture could also preserve nucleus pulposus (NP) phenotype. We confirmed that serial passaging drove NP dedifferentiation by significantly decreasing collagen type II, aggrecan and chondroadherin (CHAD) gene expression, compared to freshly isolated cells. Proliferation, gene expression profile and matrix production in both culture conditions were compared using primary bovine NP cells. Both standard culture and continuous culture produced clinically relevant cell populations. However, continuous culture cells maintained significantly higher collagen type II, aggrecan and CHAD transcript expression levels. Also, continuous expansion cells generated greater amounts of proteoglycan, collagen type II and aggrecan protein deposition in pellet cultures. To our surprise, continuous expansion of human intervertebral disc cells - isolated from acute herniation tissue - produced less collagen type II, aggrecan and CHAD genes and proteins, compared to standard culture. Also, continuous culture of cells isolated from young non-degenerate tissue did not preserve gene and protein expression, compared to standard culture. These data indicated that primary bovine and human NP cells responded differently to continuous culture, where the positive effects observed for bovine cells did not translate to human cells. Therefore, caution must be exercised when choosing animal models and cell sources for pre-clinical studies.

Imidazolium and benzimidazolium-containing compounds: from simple toxic salts to highly bioactive drugs.

The toxicity of simple imidazolium and benzimidazolium salts started to be more and more investigated in the last few years and was taken in consideration in the context of microorganisms, plants and more evolved organisms' exposure. However, the toxicity of these salts can be exploited in the development of different biological applications by incorporating them in the structure of compounds that specifically target microorganisms and cancer cells. We highlight in this minireview the way researchers became aware of the inherent problem of the stability and bioaccumulation of imidazolium and benzimidazolium salts and how they found inspiration to exploit their toxicity by incorporating them into new highly potent drugs.

Alphaherpesvirus-associated disease in greater bilbies (Macrotis lagotis).

CASE REPORT: A captive breeding colony of 9 greater bilbies (Macrotis lagotis) exhibited mild upper respiratory signs and sudden deaths with 100% mortality over a 2-week period. Histologically, acute necrotising and erosive epithelial lesions throughout the upper respiratory system and bronchi were associated with eosinophilic intranuclear inclusion bodies. Inclusions were also present in hepatocytes and adrenocortical cells, but were not always associated with necrosis. Transmission electron microscopy of lung sections revealed nucleocapsids forming arrays within some nuclei. A pan-herpesvirus PCR yielded a 440-bp product, with sequencing confirming homology with the alphaherpesviruses. Viral culture in a marsupial cell line resulted in cytopathic effect consistent with an alphaherpesvirus. CONCLUSION: This is the first report of a herpesvirus-associated disease in greater bilbies.

Associations between exposure to viruses and bovine respiratory disease in Australian feedlot cattle.

Bovine respiratory disease (BRD) is the most important cause of clinical disease and death in feedlot cattle. Respiratory viral infections are key components in predisposing cattle to the development of this disease. To quantify the contribution of four viruses commonly associated with BRD, a case-control study was conducted nested within the National Bovine Respiratory Disease Initiative project population in Australian feedlot cattle. Effects of exposure to Bovine viral diarrhoea virus 1 (BVDV-1), Bovine herpesvirus 1 (BoHV-1), Bovine respiratory syncytial virus (BRSV) and Bovine parainfluenza virus 3 (BPIV-3), and to combinations of these viruses, were investigated. Based on weighted seroprevalences at induction (when animals were enrolled and initial samples collected), the percentages of the project population estimated to be seropositive were 24% for BoHV-1, 69% for BVDV-1, 89% for BRSV and 91% for BPIV-3. For each of the four viruses, seropositivity at induction was associated with reduced risk of BRD (OR: 0.6-0.9), and seroincrease from induction to second blood sampling (35-60 days after induction) was associated with increased risk of BRD (OR: 1.3-1.5). Compared to animals that were seropositive for all four viruses at induction, animals were at progressively increased risk with increasing number of viruses for which they were seronegative; those seronegative for all four viruses were at greatest risk (OR: 2.4). Animals that seroincreased for one or more viruses from induction to second blood sampling were at increased risk (OR: 1.4-2.1) of BRD compared to animals that did not seroincrease for any viruses. Collectively these results confirm that prior exposure to these viruses is protective while exposure at or after feedlot entry increases the risk of development of BRD in feedlots. However, the modest increases in risk associated with seroincrease for each virus separately, and the progressive increases in risk with multiple viral exposures highlights the importance of concurrent infections in the aetiology of the BRD complex. These findings indicate that, while efficacious vaccines could aid in the control of BRD, vaccination against one of these viruses would not have large effects on population BRD incidence but vaccination against multiple viruses would be expected to result in greater reductions in incidence. The findings also confirm the multifactorial nature of BRD development, and indicate that multifaceted approaches in addition to efficacious vaccines against viruses will be required for substantial reductions in BRD incidence.

Effects of exposure to Bovine viral diarrhoea virus 1 on risk of bovine respiratory disease in Australian feedlot cattle.

Viruses play a key role in the complex aetiology of bovine respiratory disease (BRD). Bovine viral diarrhoea virus 1 (BVDV-1) is widespread in Australia and has been shown to contribute to BRD occurrence. As part of a prospective longitudinal study on BRD, effects of exposure to BVDV-1 on risk of BRD in Australian feedlot cattle were investigated. A total of 35,160 animals were enrolled at induction (when animals were identified and characteristics recorded), held in feedlot pens with other cattle (cohorts) and monitored for occurrence of BRD over the first 50days following induction. Biological samples collected from all animals were tested to determine which animals were persistently infected (PI) with BVDV-1. Data obtained from the Australian National Livestock Identification System database were used to determine which groups of animals that were together at the farm of origin and at 28days prior to induction (and were enrolled in the study) contained a PI animal and hence to identify animals that had probably been exposed to a PI animal prior to induction. Multi-level Bayesian logistic regression models were fitted to estimate the effects of exposure to BVDV-1 on the risk of occurrence of BRD. Although only a total of 85 study animals (0.24%) were identified as being PI with BVDV-1, BVDV-1 was detected on quantitative polymerase chain reaction in 59% of cohorts. The PI animals were at moderately increased risk of BRD (OR 1.9; 95% credible interval 1.0-3.2). Exposure to BVDV-1 in the cohort was also associated with a moderately increased risk of BRD (OR 1.7; 95% credible interval 1.1-2.5) regardless of whether or not a PI animal was identified within the cohort. Additional analyses indicated that a single quantitative real-time PCR test is useful for distinguishing PI animals from transiently infected animals. The results of the study suggest that removal of PI animals and/or vaccination, both before feedlot entry, would reduce the impact of BVDV-1 on BRD risk in cattle in Australian feedlots. Economic assessment of these strategies under Australian conditions is required.

Determinants Of Oral corticosteroid Responsiveness in Wheezing Asthmatic Youth (DOORWAY): protocol for a prospective multicentre cohort study of children with acute moderate-to-severe asthma exacerbations.

INTRODUCTION: Oral corticosteroids are the cornerstone of acute asthma management in the emergency department. Recent evidence has raised doubts about the efficacy of this treatment in preschool-aged children with viral-induced wheezing and in smoking adults. The aims of the study were to: (1) document the magnitude of response to oral corticosteroids in children presenting to the emergency department with moderate or severe asthma; (2) quantify potential determinants of response to corticosteroids and (3) explore the role of gene polymorphisms associated with the responsiveness to corticosteroids. METHODS AND ANALYSIS: The design is a prospective cohort study of 1008 children aged 1-17 years meeting a strict definition of asthma and presenting with a clinical score of ≥4 on the validated Pediatric Respiratory Assessment Measure. All children will receive standardised severity-specific treatment with prednisone/prednisolone and cointerventions (salbutamol with/without ipratropium bromide). Determinants, namely viral aetiology, environmental tobacco smoke and single nucleotide polymorphism, will be objectively documented. The primary efficacy endpoint is the failure of emergency department (ED) management within 72 h of the ED visit. Secondary endpoints include other measures of asthma severity and time to recovery within 7 days of the index visit. The study has 80% power for detecting a risk difference of 7.5% associated with each determinant from a baseline risk of 21%, at an α of 0.05. ETHICS AND DISSEMINATION: Ethical approval has been obtained from all participating institutions. An impaired response to systemic steroids in certain subgroups will challenge the current standard of practice and call for the immediate search for better approaches. A potential host-environment interaction will broaden our understanding of corticosteroid responsiveness in children. Documentation of similar effectiveness of corticosteroids across determinants will provide the needed reassurance regarding current treatment recommendations. RESULTS: Results will be disseminated at international conferences and manuscripts targeted at emergency physicians, paediatricians, geneticists and respirologists. TRIAL REGISTRATION NUMBER: This study is registered at Clinicaltrials.gov (NCT02013076).

Use of a dual-labelled oligonucleotide as a DNA dosemeter for radiological exposure detection.

A reporter molecule consisting of a synthetic oligonucleotide is being characterised for a novel damage detection scenario for its potential use as a field-deployable, personal deoxyribonucleic acid (DNA) dosemeter for radiation detection. This dosemeter is devoid of any biological properties other than being naked DNA and therefore has no DNA repair capabilities. It supports biodosimetry techniques, which require lengthy analysis of cells from irradiated individuals, and improves upon inorganic dosimetry, thereby providing for a more relevant means of measuring the accumulated dose from a potentially mixed-radiation field. Radiation-induced single strand breaks (SSBs) within the DNA result in a quantifiable fluorescent signal. Proof of concept has been achieved over 250 mGy-10 Gy dose range in radiation fields from 6°Co, with similar results seen using a linear accelerator X-ray source. Further refinements to both the molecule and the exposure/detection platform are expected to lead to enhanced levels of detection for mixed-field radiological events.

[Multicenter validation of the clinical dehydration scale for children]. / Validation multicentrique du score clinique de déshydratation pédiatrique.

INTRODUCTION: Dehydration is an important complication for sick children. The Clinical Dehydration Scale for children (CDS) measures dehydration based on 4 clinical signs: general appearance, eyes, saliva, and tears. OBJECTIVE: To validate the association between the CDS and markers of dehydration in children aged 1 month to 5 years visiting emergency departments (EDs) for vomiting and/or diarrhea. METHOD: An international prospective cohort study conducted in 3 university-affiliated EDs in 2009. Participants were a convenience sample of children aged 1-60 months presenting to the ED for acute vomiting and/or diarrhea. Following triage, a research nurse obtained informed consent and evaluated dehydration using the CDS. A few days after recovery, another research assistant weighed participants at home. The primary outcome was the percentage of dehydration calculated by the difference in weight at first evaluation and after recovery. Secondary outcomes included proportion of blood test measurements, intravenous use, hospitalization, and inter-rater agreement. RESULTS: During the study period, 264 children were recruited and data regarding weight and dehydration scores were complete for 219 (83%). According to the CDS, 88 had no dehydration, 159 some dehydration, and 15 moderate or severe dehydration. A Chi-square test showed a statistical association between CDS and weight gain, the occurrence of blood tests, intravenous rehydration, hospitalization, and abnormal plasmatic bicarbonate. Good inter-rater correlation was found among participants (linear weighted Kappa score of 0.65; (95% CI, 0.43-0.87). CONCLUSION: CDS categories correlate with markers of dehydration for young children complaining of vomiting and/or diarrhea in the ED.

Respiratory rhythms generated in the lamprey rhombencephalon.

Brainstem networks generating the respiratory rhythm in lampreys are still not fully characterized. In this study, we described the patterns of respiratory activities and we identified the general location of underlying neural networks. In a semi-intact preparation including the brain and gills, rhythmic discharges were recorded bilaterally with surface electrodes placed over the vagal motoneurons. The main respiratory output driving rhythmic gill movements consisted of short bursts (40.9+/-15.6 ms) of discharge occurring at a frequency of 1.0+/-0.3 Hz. This fast pattern was interrupted by long bursts (506.3+/-174.6 ms) recurring with an average period of 37.4+/-24.9 s. After isolating the brainstem by cutting all cranial nerves, the frequency of the short respiratory bursts did not change significantly, but the slow pattern was less frequent. Local injections of a glutamate agonist (AMPA) and antagonists (6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) or D,L-amino-5-phosphonopentanoic acid (AP5)) were made over different brainstem regions to influence respiratory output. The results were similar in the semi-intact and isolated-brainstem preparations. Unilateral injection of AP5 or CNQX over a rostral rhombencephalic region, lateral to the rostral pole of the trigeminal motor nucleus, decreased the frequency of the fast respiratory rhythm bilaterally or stopped it altogether. Injection of AMPA at the same site increased the rate of the fast respiratory rhythm and decreased the frequency of the slow pattern. The activity recorded in this area was synchronous with that recorded over the vagal motoneurons. After a complete transverse lesion of the brainstem caudal to the trigeminal motor nucleus, the fast rhythm was confined to the rostral area, while only the slow activity persisted in the vagal motoneurons. Our results support the hypothesis that normal breathing depends on the activity of neurons located in the rostral rhombencephalon in lampreys, whereas the caudal rhombencephalon generates the slow pattern.

Modulation of respiratory activity by locomotion in lampreys.

In vertebrates, locomotion is associated with changes in respiratory activity, but the neural mechanisms by which this occurs remain unknown. We began examining this in lampreys using a semi-intact preparation of young adult Petromyzon marinus, in which respiratory and locomotor behaviors can be recorded simultaneously with the activity of the underlying neural control systems. Spontaneous fictive respiration was recorded with suction electrodes positioned over the glossopharyngeal or the rostral vagal motor nucleus. In this preparation, locomotor activity, characterized by symmetrical tail movements (electromyogram recordings), was evoked by mechanical stimulation of the skin. During locomotion, the mean respiratory frequency and the mean area of the motor bursts were significantly increased (81.6+/-28.6% and 62.8+/-25.4%, respectively; P<0.05). The frequency returned to normal 92+/-51 s after the end of locomotion. There were fluctuations in the instantaneous respiratory and locomotor frequencies that were rhythmical but antiphasic for the two rhythmic activities. The changes in respiratory activity were also examined during bouts of locomotion occurring spontaneously, and it was found that a modification in respiratory activity preceded the onset of spontaneous locomotion by 3.5+/-2.6 s. This suggests that the early respiratory changes are anticipatory and are not caused by feedback generated by locomotion. The increase in respiratory frequency during locomotion induced by sensory stimulation persisted after removal of the mesencephalon. When both the mesencephalon and spinal cord were removed, resulting in the isolation of the rhombencephalon, changes in the respiratory activity were also present following skin stimulations that would have normally induced locomotion. Altogether, the results suggest that respiratory changes are programmed to adjust ventilation prior to motor activity, and that a central rhombencephalic mechanism is involved.

Experiment and simulations on the energy reservoir effect in femtosecond light filaments.

We report the results of an experiment and numerical simulations that demonstrate the large spatial extent and the effect of the so-called energy reservoir during the filamentation of femtosecond laser pulses in air. By inserting pinholes of different sizes in the filament path we observe different stages of development ranging from the termination of the filament, through its partial survival, to undisturbed propagation. A background containing up to 50% of the pulse energy is found to be necessary to maintain the filament formation, including a first refocusing.

Sensing of halocarbons using femtosecond laser-induced fluorescence.

A femtosecond laser-induced clean fluorescence technique was explored as a means to monitor halogenated alkanes in the atmosphere. Characteristic difluorocarbene radical (CF2) fluorescence in the UV-vis can be generated inside a femtosecond laser-induced filament for different halocarbons. We show that, due to different dissociation and excitation kinetics leading to fluorescence emission, it is possible to temporally resolve the characteristic fluorescence of CF2-containing halocarbons from that of background species, therefore enhancing the signal-to-noise ratio. Laboratory-scale experiments demonstrate the potential use of femtosecond laser-induced clean fluorescence for the remote sensing of halocarbons in the atmosphere. The combination of this detection strategy with LIDAR could allow the long-range monitoring of several atmospheric species with a single laser source, eventually leading to a better understanding of chemical and dynamic processes affecting global warming, ozone loss, tropospheric pollution, and weather prediction.

A preliminary account of phonological and morphophonological perception in young children with and without otitis media.

This investigation examined the effects of otitis media with effusion (OME) and its associated fluctuating conductive hearing loss on the perception of phonological and morphophonological /s/ and /z/ in young children. We predicted that children free of OME (OME-) would perform better than children with histories of OME (OME+). We also predicted that for the OME+ group morphological perception would be harder than phonological perception, because the former category carries an additional linguistic load (i.e., plurality). Sixteen children, ages 26 to 28 months (M = 26.5, SD = 0.6) were divided into two groups, the OME- (n = 8) and OME+ (n = 8) based on OME history during the first year of life. Subjects in the OME- group were free of the disease for 4/5 visits and pure tone average (PTA) was 12.6 dB HL (SD = 4.8). Subjects in the OME+ group had the disease on 3/5 visits and PTA was 23 dB HL (SD = 2.7). Experimental stimuli were six monosyllabic novel word-pairs. Members of each word-pair differed only in the presence of final voiced or voiceless fricative, marking the targets phonologically (e.g., [g [symbol: see text]]/[g [symbol: see text] s] as in 'law', 'loss') or morphophonologically (e.g., [daep]/[daeps] as in 'map' 'maps'). Subjects were taught the unfamiliar word pairs using a fast mapping procedure. Perception was tested with the bimodal preferential looking paradigm. Children in the OME- group performed significantly better than their OME+ counterparts. Individual word-pair analyses showed that OME+ group performed more poorly than the OME- group on one phonological and on two morphological targets, all ending with [s]. For the OME+ group, targets with final [s] posed greater difficulty than those with final [z], especially on morphophonological plural-(s) targets. The results suggested that the fluctuating hearing loss associated with OME might have a negative impact on speech perception.

Practice parameter: screening and diagnosis of autism: report of the Quality Standards Subcommittee of the American Academy of Neurology and the Child Neurology Society.

Autism is a common disorder of childhood, affecting 1 in 500 children. Yet, it often remains unrecognized and undiagnosed until or after late preschool age because appropriate tools for routine developmental screening and screening specifically for autism have not been available. Early identification of children with autism and intensive, early intervention during the toddler and preschool years improves outcome for most young children with autism. This practice parameter reviews the available empirical evidence and gives specific recommendations for the identification of children with autism. This approach requires a dual process: 1) routine developmental surveillance and screening specifically for autism to be performed on all children to first identify those at risk for any type of atypical development, and to identify those specifically at risk for autism; and 2) to diagnose and evaluate autism, to differentiate autism from other developmental disorders.

Effects of otitis media with effusion on hearing in the first 3 years of life.

Hearing sensitivity was examined prospectively in young children as a function of otitis media with effusion (OME) status in Years 1, 2, and 3. Hearing and OME status were sampled bimonthly from 5 to 36 months of age. Behavioral thresholds were obtained at 4 test frequencies (500, 1000, 2000, and 4000 Hz) using visual reinforcement audiometry and conditioned play audiometry techniques. The majority of children's audiograms were obtained using a computer-controlled test procedure. Thresholds for the test frequencies were averaged for each visit and then averaged across all visits in each year. Reference values were developed for infants and children in Years 1, 2, and 3 who were OME free. Results reveal that children who were classified as bilaterally OME positive in Years 1, 2, and 3 had significantly poorer hearing than children classified as bilaterally OME free in each of these time periods. There was no difference in hearing as a function of gender, socioeconomic status, or birth-risk status.

Effect of neonatal test environment on recording transient-evoked otoacoustic emissions.

OBJECTIVE: To examine the effect of test environment on recording transient-evoked otoacoustic emissions (TEOAE) in neonates. METHODS: Thirty-two infants from the newborn nursery (NBN) who passed a screening auditory brainstem response (SABR) test and were at least 34 weeks' postconceptional age were studied. One ear of each newborn was tested using TEOAE in 5 different test environments: open bassinet in the NBN (E1), working isolet located in the NBN (E2), nonfunctioning isolet in the NBN (E3), nonfunctioning isolet in a quiet room off the NBN (E4), and open bassinet in a quiet room (E5). The number of high noise samples (HNS), the test duration (in seconds), the signal-to-noise ratio (SNR; in dB) measured at bandwidths centered at 1.6, 2.4, 3.2, and 4.0 kHz, and the percentage of neonates with a fail screening outcome based on a common pass-fail screening criteria were compared in the 5 test environments. RESULTS: There were statistically significant differences in the number of HNS accumulated in the 5 test environments (F = 6.79). The use of a nonfunctioning isolet in both the NBN and within a room off the NBN (E3 and E4, respectively) resulted in significantly fewer HNS than when TEOAEs were recorded in the other 3 test environments (E1, E2, and E5). Mean test duration was significantly different among the 5 locations (F = 6. 53). Posthoc analyses revealed that test time in E3 and E4 was significantly shorter than in E1 and E2. The percentage of newborns with a fail (

Assessing growth of loudness in children by cross-modality matching.

This study examined the clinical feasibility, validity, and reliability of loudness growth assessment using cross-modality matching (CMM) between line length and loudness in 16 children 4 to 12 years old with normal hearing or bilateral sensorineural hearing losses ranging from moderate to severe in degree. Eight adult listeners with normal hearing were used as a comparison group. Loudness growth functions and real-ear measures were obtained for 500-Hz and 2,000-Hz narrowband noise stimuli for each individual. No significant differences were found between the loudness slope values for the adults and children with normal hearing. Loudness growth functions of the children with sensorineural hearing loss were significantly steeper (larger) than the slopes obtained from children with normal hearing. The numeric slope value of the loudness growth function became larger and more variable as children's hearing threshold increased and differed for children with similar thresholds. The loudness functions obtained for retested participants at two different test sessions were highly correlated. Real-ear measurements revealed that for equivalent input stimulus levels, significantly higher stimulus levels were present in the ear canals of children versus adults. Although adults and children with normal hearing had similar overall rates of loudness growth, discrete points along the loudness growth function were judged to be louder by the children. This preliminary study suggests that measures of loudness growth using CMM between line length and loudness are feasible, valid, and reliable in children with normal hearing or sensorineural hearing loss. The individual variability noted in slope values for children with hearing loss attests to the importance of subjective assessments of loudness. The protocol used in this study may have potential as a clinical tool for selecting and fitting amplification technology for children with hearing loss as young as 6 years.

New York State universal newborn hearing screening demonstration project: inpatient outcome measures.

OBJECTIVE: To evaluate the feasibility of universal newborn hearing screening by examining inpatient outcome measures from 8 hospitals located in geographically diverse areas of New York State over a 3-yr period. DESIGN: Funding was provided by the New York State Department of Health to implement predischarge hearing screening programs in the neonatal intensive care units (NICUs) and well-baby nurseries (WBNs) of eight hospitals. Various screening protocols including transient evoked otoacoustic emissions alone or in combination with conventional auditory brain stem response or screening auditory brain stem response were implemented by each site. Measured outcomes included rate of misses, refusals, and fails. Results were analyzed as a function of year of operation, nursery type, and geographic location. RESULTS: Six out of eight hospitals successfully implemented universal hearing screening during the first year, and the remaining 2 hospitals implemented programs during the second year of the project. Over a period of 3 yr, 69,761 newborns were screened at the eight hospitals representing 96.9% of all live births. The overall fail rate (4.04%) combined with the miss rate (2.61%) resulted in 6.63% of infants referred for outpatient follow-up. Mean data indicated that inpatient outcome measures improved with year of operation, with most individual hospitals also showing improvements. Both fail and miss rates were higher in the NICU than in the WBN and for hospitals located in New York City than in other regions of the state. CONCLUSIONS: Inpatient outcome measures of a universal newborn hearing screening project, which involved multiple centers across geographically diverse regions of New York State, were acceptable in terms of successfully screening a high percentage of live births and attaining low refer rates for outpatient screening. This study adds to the growing body of literature supporting the feasibility of screening all newborns before hospital discharge.