Anti-NMDAR Encephalitis After Neonatal HSV-1 Infection in a Child With Low TLR-3 Function.

Neonatal herpes simplex virus encephalitis (HSVE) often results in long-lasting neuro-disability in affected children. In addition to primary HSVE and HSVE relapses, children with herpes simplex virus are at increased risk of developing anti-N-methyl-d-aspartate receptor encephalitis (NMDARe), an autoimmune encephalitis. In this study, we describe a patient with neonatal disseminated herpes infection, who developed HSVE after discontinuation of 2 years of acyclovir suppressive therapy. After resolution of HSVE, the patient rapidly deteriorated with significant behavioral and neurologic changes including emotional outbursts, fearfulness, involuntary movements, and focal seizures. The patient was diagnosed with anti-NMDARe and was later found to have low toll-like receptor-3 function. In this study, we review published pediatric cases of anti-NMDARe after HSVE as well as previous literature and primary data examining the presentation, predisposing risk factors, predictive outcomes, future directions, and the role of immunodeficiency in HSVE-mediated anti-NMDARe. The neonatal immune system and developing brain are disproportionately vulnerable to early viral exposure; therefore, it is important to recognize the value of early immunodeficiency screening in patients with neonatal herpes simplex virus. By understanding the immune landscape within this patient population, we can mitigate long-term neurologic disability and improve the quality of life of affected children.

Themes in Superhero-Based Television Shows: An Opportunity for the Development of Children and Adolescents Through Co-Viewing and Active Mediation.

The depiction of superheroes and comic book characters in television shows has become incredibly popular. The objective of this study was to determine the positive and negative themes depicted in a select number of superhero-based television shows. A total of 49 episodes from 10 superhero-based television shows were analyzed by four independent reviewers. The average number of positive and negative themes was 18.8 and 36.9 mean events per hour, respectively, for all included episodes. The most common positive themes in our sample were associated with service, teamwork, and encouragement, and the most common negative themes were associated with violence, bullying, and alcohol use. Although exposure to positive themes depicted in superhero-based television shows may be beneficial to the development of children and adolescents, pediatric health care providers should counsel families in an attempt to limit their exposure to violence and other negative themes depicted in this genre of television shows.

Cryptococcal Meningitis in an Apparent Immunocompetent Patient.

Cryptococcal meningitis is an uncommon and severe infection that tends to affect immunocompromised hosts worldwide and in the United States. Annually it is estimated that there are 200 000 cases of cryptococcal meningitis, with the most recent estimate of 3400 cases per year in the United States alone. However, despite the low incidence, 1-year mortality is estimated at 20% to 30% even with long-term consolidation antifungal therapy. A 37-year-old man presented to the emergency department with headaches, dysarthria, hallucinations, and acute worsening of altered mental status, and he was found to have increased intracranial pressure, cerebrospinal fluid leukocytosis, and few encapsulated yeasts consistent with Cryptococcus neoformans meningitis in addition to radiologic evidence consistent with a cryptococcoma of the lungs. This report highlights the occurrence of Cryptococcus neoformans meningitis in a presumed immunocompetent host. The clinician should be aware of sources of minor immunosuppression, as they may contribute to development of Cryptococcus neoformans meningitis. Mortality in this condition remains high due to subacute presentations and delayed diagnosis in non-immunocompromised patients.

Characterization of mucoid and serous middle ear effusions from patients with chronic otitis media: implication of different biological mechanisms?

BACKGROUND: Chronic otitis media with effusion (COME) is characterized by persistent middle ear effusions that are in most cases highly viscous, but some patients present with serous fluid. This study aimed at comprehensively characterizing the macromolecular composition of mucoid vs. serous middle ear effusions (MEEs). METHODS: MEEs from patients with COME were analyzed for proteins by mass spectrometry (MS) and western blot techniques, total DNA quantity, bacterial DNA (16S sequencing), and cytokine content. Proteomics datasets were studied in Ingenuity Pathway Analysis (IPA). RESULTS: Mucoid samples showed a global tendency of increased pro-inflammatory mediators. Interleukin-1ß (IL-1ß) and IL-10 were significantly more abundant in serous samples (p < 0.01). Mucoid samples had higher DNA quantity (p = 0.04), more likely to be positive in MUC5B protein (p = 0.008) and higher peptide counts (12,786 vs. 2225), as well as an overall larger number of identified proteins (331 vs. 177), compared to serous. IPA found the mucoid sample dataset to be related to immune cell function and epithelial remodeling, whereas the serous sample dataset showed acute responses and blood-related proteins. Interestingly, serous samples showed more bacterial DNA than mucoid ones, with less bacterial genera variability. CONCLUSION: This study demonstrates divergent immune responses in children with COME by effusion quality.

Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

Nontypeable Haemophilus influenzae (NTHi), one of the most common acute otitis media (OM) pathogens, is postulated to promote middle-ear epithelial remodeling in the progression of OM from acute to chronic. The goal of this study was to examine early quantitative proteomic secretome effects of NTHi lysate exposure in a human middle-ear epithelial cell (HMEEC) line. NTHi lysates were used to stimulate HMEEC, and conditional quantitative stable isotope labeling with amino acids in cell culture of cell secretions was performed. Mass spectrometry analysis identified 766 proteins across samples. Of interest, several heterogeneous nuclear ribonucleoproteins (hnRNPs) were regulated by NTHi lysate treatment, especially hnRNP A2B1 and hnRNP Q, known to be implicated in microRNA (miRNA) packaging in exosomes. After purification, the presence of exosomes in HMEEC secretions was characterized by dynamic light scattering (<100 nm), transmission electron microscopy, and CD63/heat shock protein 70 positivity. hnRNP A2B1 and hnRNP Q were confirmed to be found in exosomes by Western blot and proteomic analysis. Finally, exosomal miRNA content comprised 110 unique miRNAs, with 5 found to be statistically induced by NTHi lysate (miR-378a-3p + miR-378i, miR-200a-3p, miR-378g, miR30d-5p, and miR-222-3p), all known to target innate immunity genes. This study demonstrates that NTHi lysates promote release of miRNA-laden exosomes from middle-ear epithelium in vitro. -Val, S., Krueger, A., Poley, M., Cohen, A., Brown, K., Panigrahi, A., Preciado, D. Nontypeable Haemophilus influenzae lysates increase heterogeneous nuclear ribonucleoprotein secretion and exosome release in human middle-ear epithelial cells.

Purification and characterization of microRNAs within middle ear fluid exosomes: implication in otitis media pathophysiology.

BACKGROUND: Otitis media (OM) is characterized by acute infection progressing to chronic middle ear effusion (MEE). Extracellular secretion of microRNAs (miRNAs) in exosomes is a newly discovered mechanism for cells exerting distant cell genetic regulation. Whether MEE contains exosomes with specific miRNAs is unknown. This study aimed to purify and characterize the exosomal and miRNA content of MEE. METHOD: MEEs were subjected to Exoquick exosomal purification and EXOCET exosomal quantification. Extracted vesicles were analyzed by dynamic light scattering (DLS), transmission electron microscopy (TEM), and immunoblotting of HSP-70. NanoString hybridization was performed to profile miRNAs. Exosomal protein content was profiled by Liquid chromatography tandem mass spectrometry (LC-MS/MS). RESULTS: EXOCET assays showed presence of exosomes (0-0.5 × 107/ml) in MEEs. DLS confirmed exosomal size between 10 and 200 nm. Western blot analysis showed presence of HSP-70. Twenty-nine miRNAs were found to be unique to MEEs. The most abundant miRNA was miR-223, a miRNA typically secreted by neutrophils. Proteomics demonstrated typical neutrophil markers as well as common innate immune molecules. CONCLUSION: To our knowledge, this the first report demonstrating the presence of exosomes transporting miRNAs in MEEs. These findings open a broad and novel area of research in OM pathophysiology as driven by miRNA cell communication.

Relationship of the Middle Ear Effusion Microbiome to Secretory Mucin Production in Pediatric Patients With Chronic Otitis Media.

BACKGROUND: Acute otitis media, an infection of the middle ear, can become chronic after multiple episodes. Microbial influence on chronic otitis media remains unclear. It has been reported that mucin glycoproteins are required for middle ear immune defense against pathogens. We aim to characterize the middle ear effusion (MEE) microbiome using high-throughput sequencing and assess potential associations in microbiome diversity with the presence of the secretory mucins MUC5B and MUC5AC. We hypothesize that MEEs containing MUC5B will exhibit a microbiome largely devoid of typical acute otitis media bacteria. METHODS: Fifty-five MEEs from children undergoing myringotomy at Children's National Health System were recovered. Mucin was semiquantitatively determined through Western blot analysis. DNA was subjected to 16S rRNA amplicon sequencing using the Illumina MiSeq platform. Raw data were processed in mothur (SILVA reference database). Alpha- and beta-diversity metrics were determined. Abundance differences between sample groups were estimated. RESULTS: MUC5B was present in 94.5% and MUC5AC in 65.5% of MEEs. Sequencing revealed 39 genera with a relative abundance ≥0.1%. Haemophilus (22.54%), Moraxella (11.11%) and Turicella (7.84%) were the most abundant. Turicella and Pseudomonas proportions were greater in patients older than 24 months of age. In patients with hearing loss, Haemophilus was more abundant, while Turicella and Actinobacteria were less abundant. Haemophilus was also more abundant in samples containing both secretory mucins. CONCLUSIONS: The microbiome of MEEs from children with chronic otitis media differs according to specific clinical features, such as mucin content, age and presence of hearing loss. These associations provide novel pathophysiologic insights across the spectrum of otitis media progression.

Proteomic Characterization of Middle Ear Fluid Confirms Neutrophil Extracellular Traps as a Predominant Innate Immune Response in Chronic Otitis Media.

BACKGROUND: Chronic Otitis Media (COM) is characterized by middle ear effusion (MEE) and conductive hearing loss. MEE reflect mucus hypersecretion, but global proteomic profiling of the mucosal components are limited. OBJECTIVE: This study aimed at characterizing the proteome of MEEs from children with COM with the goal of elucidating important innate immune responses. METHOD: MEEs were collected from children (n = 49) with COM undergoing myringotomy. Mass spectrometry was employed for proteomic profiling in nine samples. Independent samples were further analyzed by cytokine multiplex assay, immunoblotting, neutrophil elastase activity, next generation DNA sequencing, and/or immunofluorescence analysis. RESULTS: 109 unique and common proteins were identified by MS. A majority were innate immune molecules, along with typically intracellular proteins such as histones and actin. 19.5% percent of all mapped peptide counts were from proteins known to be released by neutrophils. Immunofluorescence and immunoblotting demonstrated the presence of neutrophil extracellular traps (NETs) in every MEE, along with MUC5B colocalization. DNA found in effusions revealed unfragmented DNA of human origin. CONCLUSION: Proteomic analysis of MEEs revealed a predominantly neutrophilic innate mucosal response in which MUC5B is associated with NET DNA. NETs are a primary macromolecular constituent of human COM middle ear effusions.

A proteomic characterization of NTHi lysates.

BACKGROUND: Non-typeable Haemophilus influenzae (NTHi) is a ubiquitous bacterial pathogen which accounts for a majority of human upper respiratory tract infections. Laboratory lysate preparations from this bacterium are commonly utilized to investigate the promulgation of inflammatory responses in respiratory and middle ear epithelium both in vivo and in vitro. We undertook an unbiased proteomics based analysis of NTHi lysate preps to: (a) identify abundant bacterial proteins present in these lysates that could play a role in NTHi biological effects and (b) determine the protein content variability in different lysate prep batches from the same NTHI strain. STUDY DESIGN: Proteomic analysis of laboratory NTHi lysate preparations from clinical strain 12. METHODS: NTHi lysates were denatured, gel-fractionated, digested by trypsin and the generated peptides were identified using a liquid chromatography tandem mass spectrometry (LC-MS/MS). Western blot analyses for the important proinflammatory enhancer, outer membrane protein 6 (OMP6), was performed to validate the MS findings. Luciferase assays for NF-kB activation were used to measure the pro-inflammatory biologic effects from each NTHi lysate preparation. RESULTS: The MS identified 793 unique NTHi proteins. Most common and abundant proteins found have been described to either contribute to biofilm formation, elude the innate immune system, or activate epithelial pro-inflammatory pathways such as Toll Like Receptor 2 (TLR-2) signaling and NF-kB transcription factor. Strong positive signal for OMP6 was found in each of the NTHi lysate preparations. Significant NF-kB promoter response activation as expected with NTHi stimulation over control was also noted for each NTHi lysate preparation. CONCLUSIONS: Proteomics was a successful technique to broadly define the protein content of NTHi lysates. This is the first report of the proteome of NTHi lysates widely used in laboratories to study the biological effect of NTHi. Despite the variability of the protein composition from different preps, all the batches of NTHi lysates induced similar NFκB activation. LEVEL OF EVIDENCE: NA.

Trends in U.S. Pediatric Otolaryngology Fellowship Training.

IMPORTANCE: Interest in pediatric otolaryngology fellowship training is growing. The workforce implications of this growing interest are unclear and understudied. OBJECTIVES: To analyze trends in pediatric otolaryngology training, determine where fellows who graduated over the past 10 years are currently practicing, and test the hypothesis that graduates from Accreditation Council for Graduate Medical Education (ACGME)­accredited programs were more likely to have academic tertiary positions with faculty appointments. DESIGN, SETTING, AND PARTICIPANTS: We conducted a web-based analysis of pediatric otolaryngology fellowship graduates. The names of all 274 applicants who were matched to pediatric otolaryngology fellowships from May 31, 2003, to May 31, 2014, were obtained from the SF Match website. Accreditation status of each program for each match year was obtained from the ACGME website. We then performed an Internet search for the current practice location of each matched applicant. Analysis was conducted from January 1, 2015, to May 1, 2015. MAIN OUTCOMES AND MEASURES: Practice setting per year of fellowship match and accreditation status of program. RESULTS: For the 2003 to the 2014 match years, there was an increase from 5 to 22 accredited pediatric otolaryngology fellowship programs overall; simultaneously, the number of yearly matched applicants increased from 14 to 35. More graduates with ACGME accreditation practice at academic settings compared with graduates without ACGME accreditation although the difference was not statistically significant (67.1% vs. 50.7%; P = .15). Graduates from accredited programs, however, were significantly more likely to practice at a hospital-based setting compared with those from nonaccredited programs (81.7% vs. 65.5%; P = .003). Fellows trained in the last 10 years are relatively well distributed across the country. CONCLUSIONS AND RELEVANCE: The number of pediatric otolaryngology fellowship applicants as well as total number of matched applicants and ACGME-accredited positions has risen in the last 10 years. It appears that a higher proportion of fellows trained in accredited programs work in academic positions in hospital-based practices. The long-term effect on the pediatric otolaryngology workforce of training more fellows in accredited fellowships remains to be seen.

Does pediatric cochlear implant insertion technique affect intraoperative neural response telemetry thresholds?

OBJECTIVE: Recent reports of mostly adult patients suggest round window insertion is less traumatic than cochleostomy for cochlear implantation (CI), while other reports have indicated that curved electrode arrays lower the neural response telemetry (NRT) threshold and consume less power. We aimed to compare the intraoperative neural response telemetry (NRT) thresholds in children receiving cochlear implants through a cochleostomy (COCH) vs. a round window (RW) approach, as well as patients receiving a curved array vs. a straight one. DESIGN: A direct case-cohort comparison of NRT in pediatric CI recipients at two large tertiary pediatric hospitals from 2008 through 2014 was done. Univariate Mann-Whitney analyses and one-wayANOVA were performed to compare average NRT in RW vs. COCH insertion, and curved vs. straight electrodes. Multivariate regression was performed to control for age and pre- vs. postlingual patient status. RESULTS: Overall, 236 children were included, between January 2008 and October 2014 at two large tertiary referral medical centers. A total of 52 patients received a RW insertion and 184 received a cochleostomy. There was no statistically significant difference between RW insertion (187.9±18.7) and COCH (183.4±17.1) (p=0.125). The patients were divided into four categories: RW insertion with curved electrode (175.0±11.2), RW with straight electrode (192.1±18.8), COCH with curved electrode (182.2±16.7), and COCH with straight electrode (193.0±20.8). The lowest NRT current thresholds were achieved with curved electrode array insertions through the RW (p=0.001). Multivariate regression analysis revealed the following parameters were not independently associated with differences in NRT responses: RWI vs. COCH (p=0.12) and pre- vs. postlingual (p=0.18). The difference in NRT levels between curved electrode arrays and straight was shown to be statistically significant (p=0.00075). CONCLUSIONS: When controlling for insertion technique or pre- vs. postlingual hearing loss, the curved electrode array correlates with lower NRT thresholds. Although studies to examine functional language performance of these recipients are pending, initial results of this multi-institutional trial suggest that curved electrodes indeed result in lower NRT levels, particularly when inserted through the RW.