Management of Pediatric Persistent Asymptomatic Cervical Lymphadenopathy.

OBJECTIVE: Persistent asymptomatic cervical lymphadenopathy (PACL) is a common outpatient referral diagnosis for pediatric otolaryngologists. Historically, excisional biopsy under general anesthesia has been the gold standard for diagnosis but is associated with some risks. Current literature provides little guidance on less invasive monitoring. Our hypothesis is that the majority of children who present with PACL can be safely monitored with ultrasound and avoid the risks of excisional biopsy. STUDY DESIGN: A retrospective review was performed of patients <18 years of age, referred to a tertiary care children's hospital for PACL who also underwent at least 1 neck ultrasound from 2007 to 2021. Patients with acute neck infections, congenital masses, or known rheumatologic, immunologic, or malignant conditions were excluded. A multivariate logistic regression model was used to determine patient and nodal factors associated with the decision for operative management. SETTING: University of California, San Francisco Pediatric Otolaryngology Department. RESULTS: Among the 197 patients meeting inclusion criteria, 30 (15.2%) underwent surgical biopsy. Overall, 26% underwent repeat ultrasound with a mean interval of 6.6 months, and a mean decrease in nodal size of 0.34 cm. Of the 30 surgical cases, 27 (90%) patients had benign pathology. Multivariate regression analysis revealed pain (p = .04), firmness (p < .001), and lack of a normal fatty hilum on ultrasound (p = .04) as statistically significantly correlated with decisions for surgical management. CONCLUSION: The majority of pediatric PACL is benign and does not require an excisional biopsy to rule out lymphoma. Serial clinical follow-up with neck ultrasound can be used to safely monitor patients.

Inactivation of HIV-1 in Polarized Infant Tonsil Epithelial Cells by Human Beta-Defensins 2 and 3 Tagged with the Protein Transduction Domain of HIV-1 Tat.

Mother-to-child transmission (MTCT) of HIV-1 may occur during pregnancy, labor, and breastfeeding; however, the molecular mechanism of MTCT of virus remains poorly understood. Infant tonsil mucosal epithelium may sequester HIV-1, serving as a transient reservoir, and may play a critical role in MTCT. Innate immune proteins human beta-defensins 2 (hBD-2) and -3 may inactivate intravesicular virions. To establish delivery of hBD-2 and -3 into vesicles containing HIV-1, we tagged hBDs with the protein transduction domain (PTD) of HIV-1 Tat, which facilitates an efficient translocation of proteins across cell membranes. Our new findings showed that hBD-2 and -3 proteins tagged with PTD efficiently penetrated polarized tonsil epithelial cells by endocytosis and direct penetration. PTD-initiated internalization of hBD-2 and -3 proteins into epithelial cells led to their subsequent penetration of multivesicular bodies (MVB) and vacuoles containing HIV-1. Furthermore, PTD played a role in the fusion of vesicles containing HIV-1 with lysosomes, where virus was inactivated. PTD-initiated internalization of hBD-2 and -3 proteins into ex vivo tonsil tissue explants reduced the spread of virus from epithelial cells to CD4+ T lymphocytes, CD68+ macrophages, and CD1c+ dendritic cells, suggesting that this approach may serve as an antiviral strategy for inactivating intraepithelial HIV-1 and reducing viral MTCT.

Human Immunodeficiency Virus (HIV) and Human Cytomegalovirus (HCMV) Coinfection of Infant Tonsil Epithelium May Synergistically Promote both HIV-1 and HCMV Spread and Infection.

Mother-to-child transmission (MTCT) of human immunodeficiency virus type 1 (HIV-1) and human cytomegalovirus (HCMV) may occur during pregnancy, labor, or breastfeeding. These viruses from amniotic fluid, cervicovaginal secretions, and breast milk may simultaneously interact with oropharyngeal and tonsil epithelia; however, the molecular mechanism of HIV-1 and HCMV cotransmission through the oral mucosa and its role in MTCT are poorly understood. To study the molecular mechanism of HIV-1 and HCMV MTCT via oral epithelium, we established polarized infant tonsil epithelial cells and polarized-oriented ex vivo tonsil tissue explants. Using these models, we showed that cell-free HIV-1 and its proteins gp120 and tat induce the disruption of tonsil epithelial tight junctions and increase paracellular permeability, which facilitates HCMV spread within the tonsil mucosa. Inhibition of HIV-1 gp120-induced upregulation of mitogen-activated protein kinase (MAPK) and NF-κB signaling in tonsil epithelial cells, reduces HCMV infection, indicating that HIV-1-activated MAPK and NF-κB signaling may play a critical role in HCMV infection of tonsil epithelium. HCMV infection of tonsil epithelial cells also leads to the disruption of tight junctions and increases paracellular permeability, facilitating HIV-1 paracellular spread into tonsil mucosa. HCMV-promoted paracellular spread of HIV-1 increases its accessibility to tonsil CD4 T lymphocytes, macrophages, and dendritic cells. HIV-1-enhanced HCMV paracellular spread and infection of epithelial cells subsequently leads to the spread of HCMV to tonsil macrophages and dendritic cells. Our findings revealed that HIV-1- and HCMV-induced disruption of infant tonsil epithelial tight junctions promotes MTCT of these viruses through tonsil mucosal epithelium, and therapeutic intervention for both HIV-1 and HCMV infection may substantially reduce their MTCT. IMPORTANCE Most HIV-1 and HCMV MTCT occurs in infancy, and the cotransmission of these viruses may occur via infant oropharyngeal and tonsil epithelia, which are the first biological barriers for viral pathogens. We have shown that HIV-1 and HCMV disrupt epithelial junctions, reducing the barrier functions of epithelia and thus allowing paracellular penetration of both viruses via mucosal epithelia. Subsequently, HCMV infects epithelial cells, macrophages, and dendritic cells, and HIV-1 infects CD4+ lymphocytes, macrophages, and dendritic cells. Infection of these cells in HCMV- and HIV-1-coinfected tonsil tissues is much higher than that by HCMV or HIV-1 infection alone, promoting their MTCT at its initial stages via infant oropharyngeal and tonsil epithelia.

Airway Hemangiomas in PHACE Syndrome: A Multicenter Experience.

OBJECTIVE: To describe the prevalence and clinical characteristics of airway findings in a multi-institutional cohort of PHACE patients. STUDY DESIGN: Multicenter retrospective case series. SETTING: Multidisciplinary vascular anomalies clinics at 2 institutions. METHODS: Data were collected from the electronic medical record, including clinical presentation, airway findings, treatment, and outcomes. RESULTS: Of 55 PHACE patients, 22 (40%) had airway hemangiomas. Patients with airway involvement were more commonly female (P = .034, odds ratio [OR] 23, 95% confidence interval [CI] 1.3-410) and of Caucasian ethnicity (P = .020, OR 5.3, 95% CI 1.3-21). Anatomically, patients with bilateral S3 involvement had higher rates of airway disease (P = .0012, OR 15, 95% CI 2.9-77). Most patients with airway hemangiomas had stridor (68%). Of the patients managed in the propranolol era (2008 or later, n = 35), 14 had airway involvement. All 14 were treated with propranolol, whereas 13 (62%) of 21 nonairway patients were treated with propranolol. The average treatment duration was longer in the airway patients (22.1 vs 16.7 months). All patients who underwent tracheostomy (n = 4) did so before 2008. CONCLUSION: Risk factors for airway involvement in PHACE include female gender, Caucasian ethnicity, and stridor. Since the widespread use of propranolol, fewer patients have required surgical management of their airway disease. Given the high prevalence of airway involvement even in patients without stridor, assessment of the airway is a crucial component of a comprehensive PHACE workup.

Advanced practice providers and children's hospital-based pediatric otolarynology practices.

INTRODUCTION: Advanced practice providers (APPs), including nurse practitioners and physician assistants, have been deployed in children's hospital-based academic pediatric otolaryngology practices for many years. However, this relationship in terms of prevalence, roles, financial consequences and satisfaction has not been examined. The objective of this study is to explore how APPs impact healthcare delivery in this setting. METHODS: Pediatric otolaryngology chiefs of all academic children's hospitals in the US were electronically surveyed about the ways APPs intersected clinically and financially in their respective practice. RESULTS: A total of 29 of 36 children's hospital-based pediatric otolaryngology practices completed the survey, of which 26 practices (90%) utilized APP. There were large variances within the APP practice cohort in faculty size (mean/median/range = 9.4/8.5/3-29); annual patient visits (mean/median = 18,373/17,600); number of practice site (mean/median/range = 4.3/4/2-9) and number of outpatient APP (mean/median/range = 6.3/5/1-30). No factors (faculty size, annual visits and number of practice sites) differentiated between the APP and non-APP practices. Among APP practices, significant correlation (p<.00001) was observed between size of APP cohort to faculty size and annual visits. 69% of the practices did not differentiate job functions of nurse practitioners and physician assistants. 85% of the practices utilized APPs in all practice sites and 19% utilized APPs in the operating room. 77% of APPs billed independently and 46% had on-site supervision. The most prevalent APP salary bracket based on 0-5, 6-10 and > 11 years of tenure were $76-100K (65%), $100-150K (77%) and $100-150K (86%), respectively. In 46% of the practices, APPs were able to generate enough revenue to cover more than 75% of their salary and 23% of practices generated a profit. 81% of the chiefs ranked the effectiveness of APPs as high (4 and 5) on a 5-point Likert scale. DISCUSSION: The majority of academic pediatric otolaryngology practices employed APPs. Despite the diversity seen in practice complexity, APP functionality and financial impact, most found the APP model to be beneficial in improving patient care, patient access and faculty productivity.

HIV-1 proteins gp120 and tat induce the epithelial-mesenchymal transition in oral and genital mucosal epithelial cells.

The oral, cervical, and genital mucosa, covered by stratified squamous epithelia with polarized organization and strong tight and adherens junctions, play a critical role in preventing transmission of viral pathogens, including human immunodeficiency virus (HIV). HIV-1 interaction with mucosal epithelial cells may depolarize epithelia and disrupt their tight and adherens junctions; however, the molecular mechanism of HIV-induced epithelial disruption has not been completely understood. We showed that prolonged interaction of cell-free HIV-1 virions, and viral envelope and transactivator proteins gp120 and tat, respectively, with tonsil, cervical, and foreskin epithelial cells induces an epithelial-mesenchymal transition (EMT). EMT is an epigenetic process leading to the disruption of mucosal epithelia and allowing the paracellular spread of viral and other pathogens. Interaction of cell-free virions and gp120 and tat proteins with epithelial cells substantially reduced E-cadherin expression and activated vimentin and N-cadherin expression, which are well-known mesenchymal markers. HIV gp120- and tat-induced EMT was mediated by SMAD2 phosphorylation and activation of transcription factors Slug, Snail, Twist1 and ZEB1. Activation of TGF-ß and MAPK signaling by gp120, tat, and cell-free HIV virions revealed the critical roles of these signaling pathways in EMT induction. gp120- and tat-induced EMT cells were highly migratory via collagen-coated membranes, which is one of the main features of mesenchymal cells. Inhibitors of TGF-ß1 and MAPK signaling reduced HIV-induced EMT, suggesting that inactivation of these signaling pathways may restore the normal barrier function of mucosal epithelia.

Cryoprobe retrieval of an airway foreign body: A case report and literature review.

We report a case of a 7-year-old boy with clinical and radiographic evidence of foreign body (FB) aspiration with a 2-week delay in diagnosis. The retrieval of the pushpin with traditional bronchoscopic instrumentation was made difficult by granulation tissue formation. A cryoprobe through a flexible bronchoscope was used to successfully remove the FB. To our knowledge, this is the first report of interventional bronchoscopy with a cryoprobe to remove a pushpin in a child under suspension laryngoscopy and spontaneous ventilation. A high index of suspicion is crucial for identifying FBs early and minimizing granulation tissue development which complicates FB removal.

Hearing loss in PHACE syndrome: clinical and radiologic findings.

PURPOSE: To characterize the types of hearing loss, auditory-related imaging findings, and hemangioma characteristics in patients with Posterior fossa malformations, Hemangiomas, Arterial anomalies, Cardiac defects, and abnormalities of the Eye (PHACE) syndrome. METHODS: Retrospective medical records, audiologic data, and imaging review of all patients presenting to a tertiary care children's hospital with a proven diagnosis of PHACE syndrome from 2005 to 2016. RESULTS: Twelve patients were identified with hearing and imaging data. 5/12 had hearing loss, 1 had unilateral severe sensorineural loss with minor conductive component, 1 had unilateral moderate sensorineural loss with minor conductive component, 1 had mild bilateral conductive loss, 1 had bilateral hearing loss (left severe mixed and right severe sensorineural), and 1 had moderate bilateral conductive loss. All patients passed their newborn hearing screening. Of the 5 patients with hearing loss, 3 had IAC hemangiomas (1 bilateral), 3 had enlarged IACs with prominent posterior petrous bones (1 bilateral), 2 had dysgenesis of the cerebellar vermis and hemispheres, there was 1 patient each with a deformed pinna and middle ear and mastoid effusions, and 1 patient had no abnormal auditory-related imaging findings. Patients with hearing loss were more likely to have more areas of cutaneous hemangioma involvement (mean 6.4 vs 3.1, p = .05). Laterality of hearing impairment correlated with the side of cutaneous hemangioma in all patients with hearing loss. Treatment with systemic propranolol did not improve hearing. CONCLUSIONS: Patients with PHACE are at risk for hearing loss and may demonstrate radiologic abnormalities within the ear structures, although the type of hearing loss, imaging findings, and their respective correlation vary. While our results are limited by our small sample size, comprehensive audiology evaluations (as opposed to newborn screening testing only) should be considered for PHACE patients who have extensive cutaneous hemangioma or auditory-related imaging abnormalities, such as internal auditory canal hemangiomas.

Genetic engineering in primary human B cells with CRISPR-Cas9 ribonucleoproteins.

Genome editing in human cells with targeted nucleases now enables diverse experimental and therapeutic genome engineering applications, but extension to primary human B cells remains limited. Here we report a method for targeted genetic engineering in primary human B cells, utilizing electroporation of CRISPR-Cas9 ribonucleoproteins (RNPs) to introduce gene knockout mutations at protein-coding loci with high efficiencies that in some cases exceeded 80%. Further, we demonstrate knock-in editing of targeted nucleotides with efficiency exceeding 10% through co-delivery of oligonucleotide templates for homology directed repair. We delivered Cas9 RNPs in two distinct in vitro culture systems to achieve editing in both undifferentiated B cells and activated B cells undergoing differentiation, reflecting utility in diverse experimental conditions. In summary, we demonstrate a powerful and scalable research tool for functional genetic studies of human B cell biology that may have further applications in engineered B cell therapeutics.

Sirolimus for management of complex vascular anomalies - A proposed dosing regimen for very young infants.

Neonates with vascular anomalies causing airway compromise and other complications require early initiation of medical therapy. Sirolimus has emerged as a safe and effective treatment, but standard recommendations for dosing start at seven months. Guidelines are needed for dosing in very young infants, who have reduced hepatic metabolism of sirolimus. We present our experience treating six neonates (mean age 14.8 days) with complicated vascular anomalies. Standard dosing caused supratherapeutic levels in this population. Our modified dosing regimen has resulted in safe therapeutic concentrations. Properly dosed, sirolimus is a viable and potentially lifesaving option for neonates with severe morbidity from vascular anomalies.

HIV internalization into oral and genital epithelial cells by endocytosis and macropinocytosis leads to viral sequestration in the vesicles.

Recently, we showed that HIV-1 is sequestered, i.e., trapped, in the intracellular vesicles of oral and genital epithelial cells. Here, we investigated the mechanisms of HIV-1 sequestration in vesicles of polarized tonsil, foreskin and cervical epithelial cells. HIV-1 internalization into epithelial cells is initiated by multiple entry pathways, including clathrin-, caveolin/lipid raft-associated endocytosis and macropinocytosis. Inhibition of HIV-1 attachment to galactosylceramide and heparan sulfate proteoglycans, and virus endocytosis and macropinocytosis reduced HIV-1 sequestration by 30-40%. T-cell immunoglobulin and mucin domain 1 (TIM-1) were expressed on the apical surface of polarized tonsil, cervical and foreskin epithelial cells. However, TIM-1-associated HIV-1 macropinocytosis and sequestration were detected mostly in tonsil epithelial cells. Sequestered HIV-1 was resistant to trypsin, pronase, and soluble CD4, indicating that the sequestered virus was intracellular. Inhibition of HIV-1 intraepithelial sequestration and elimination of vesicles containing virus in the mucosal epithelium may help in the prevention of HIV-1 mucosal transmission.

Release of HIV-1 sequestered in the vesicles of oral and genital mucosal epithelial cells by epithelial-lymphocyte interaction.

Oropharyngeal mucosal epithelia of fetuses/neonates/infants and the genital epithelia of adults play a critical role in HIV-1 mother-to-child transmission and sexual transmission of virus, respectively. To study the mechanisms of HIV-1 transmission through mucosal epithelium, we established polarized tonsil, cervical and foreskin epithelial cells. Analysis of HIV-1 transmission through epithelial cells showed that approximately 0.05% of initially inoculated virions transmigrated via epithelium. More than 90% of internalized virions were sequestered in the endosomes of epithelial cells, including multivesicular bodies (MVBs) and vacuoles. Intraepithelial HIV-1 remained infectious for 9 days without viral release. Release of sequestered intraepithelial HIV-1 was induced by the calcium ionophore ionomycin and by cytochalasin D, which increase intracellular calcium and disrupt the cortical actin of epithelial cells, respectively. Cocultivation of epithelial cells containing HIV-1 with activated peripheral blood mononuclear cells and CD4+ T lymphocytes led to the disruption of epithelial cortical actin and spread of virus from epithelial cells to lymphocytes. Treatment of epithelial cells with proinflammatory cytokines tumor necrosis factor-alpha and interferon gamma also induced reorganization of cortical actin and release of virus. Inhibition of MVB formation by small interfering RNA (siRNA)-mediated silencing of its critical protein hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) expression reduced viral sequestration in epithelial cells and its transmission from epithelial cells to lymphocytes by ~60-70%. Furthermore, inhibition of vacuole formation of epithelial cells by siRNA-inactivated rabankyrin-5 expression also significantly reduced HIV-1 sequestration in epithelial cells and spread of virus from epithelial cells to lymphocytes. Interaction of the intercellular adhesion molecule-1 of epithelial cells with the function-associated antigen-1 of lymphocytes was important for inducing the release of sequestered HIV-1 from epithelial cells and facilitating cell-to-cell spread of virus from epithelial cells to lymphocytes. This mechanism may serve as a pathway of HIV-1 mucosal transmission.

Sleep-disordered breathing in pediatric head and neck vascular malformations.

OBJECTIVES: To determine the prevalence of sleep-disordered breathing (SDB) symptoms among children with head and neck vascular malformations and to compare obstructive sleep apnea (OSA)-18 scores between children with head and neck vascular malformations and children with non-head and neck vascular malformations. STUDY DESIGN: Retrospective cohort and prospective cross-sectional studies METHODS: Forty-three pediatric subjects with head and neck vascular malformations evaluated at a tertiary-care multidisciplinary vascular anomalies center were included in a retrospective cohort study. Eighty-three consecutive pediatric subjects with vascular malformations evaluated at the same center were included in the prospective cross-sectional study. RESULTS: In the retrospective cohort study, 20 (47%) subjects with head and neck malformations had documented SDB symptoms. Of those with SDB symptoms, five (25%) required long-term tracheotomy. The children with SDB symptoms had greater vascular malformation size, more extensive pharyngeal involvement, greater vascular malformation mass effect on airway, and closer proximity of malformation to airway when compared to children without SDB symptoms. For the prospective cross-sectional study, 23% of pediatric subjects had malformations of the head and neck. Those with head and neck malformations had a higher OSA-18 score and a lower overall quality of life (QOL) score when compared to subjects with non-head and neck malformations. CONCLUSION: Nearly half of children with head and neck vascular malformations have SDB symptoms. Children with head and neck vascular malformations have a higher OSA-18 score and lower overall QOL score when compared to children with non-head and neck vascular malformations. LEVEL OF EVIDENCE: 4. Laryngoscope, 127:2159-2164, 2017.

Conversion of traditional osseointegrated bone-anchored hearing aids to the Baha® attract in four pediatric patients.

Bone-anchored hearing aids are external devices attached to the skull via a titanium implant, and can be used for multiple types of hearing loss. Traditionally, osseointegrated implants have been coupled to the external processor with a percutaneous abutment, but more recently, a fully implanted, transcutaneous magnet-based system has become available. Skin reactions from the percutaneous portion are a common complication that can prevent use of the device during critical windows of language development and learning in children. We describe our experience replacing the Baha® abutment system with the Baha® Attract in four pediatric patients. Specific operative considerations for incision placement, and magnet and implant coverage are discussed. All patients maintained osseointegration, had excellent long-term wound healing without post-operative infection, and were able to wear their devices more consistently.

Pediatric sialendoscopy indications and outcomes.

PURPOSE OF REVIEW: Chronic sialadenitis can affect patients of all age ranges and typically presents as recurrent or chronic painful swelling of the salivary glands. In children, the most common cause of sialadenitis is juvenile recurrent parotitis. Salivary stones, or sialolithiasis, are a much less common cause. Historically, for patients with chronic sialadenitis who failed conservative management, salivary gland removal was the standard treatment option. Recently, however, sialendoscopy has emerged as an effective gland-preserving procedure for sialadenitis evaluation and treatment in adults and children. The aim of this review is to discuss pediatric sialadenitis evaluation and treatment, including sialendoscopy indications, technique, and outcomes. RECENT FINDINGS: Sialendoscopy is a well tolerated and effective treatment for sialadenitis Sialendoscopy and salivary duct irrigation have been shown to improve frequency and severity of sialadenitis episodes in patients with juvenile recurrent parotitis. Salivary stones are managed successfully with endoscopic and combined endoscopic-assisted open approaches. Minimally invasive approaches with sialendoscopy have improved the ability to preserve salivary glands in patients with recurrent sialadenitis. SUMMARY: Sialendoscopy is a well tolerated and effective procedure for recurrent sialadenitis in children.

Pneumothorax after tracheostomy closure with successful nonsurgical management.

A 3-year-old girl presented for routine closure of her tracheostomy site. She was intubated easily for the procedure, and the wound was closed with a drain in place. In recovery, the mother noticed fullness in the patient's submandibular region, and on examination, the girl had subcutaneous emphysema in the neck bilaterally. She returned to the operating room for exploration, and air was released from the surgical site. The wound was again closed with a drain in place, and the patient was extubated uneventfully. After arriving to the pediatric intensive care unit for monitoring, the patient acutely developed respiratory distress and was found to have pneumomediastinum and pneumothorax and was emergently intubated. She was observed closely, and the following day, the pneumothorax improved, and she successfully extubated without further complication.

Human beta-defensins 2 and -3 cointernalize with human immunodeficiency virus via heparan sulfate proteoglycans and reduce infectivity of intracellular virions in tonsil epithelial cells.

We previously showed that expression of the anti-HIV innate proteins human beta-defensin 2 (hBD2) and hBD3 in adult oral epithelial cells reduces HIV transepithelial transmission by inactivation of virus. However, fetal/infant oral epithelia lack beta-defensin expression, leading to transmission of HIV. The mechanisms of hBD2- and hBD3-mediated HIV inactivation in adult oral epithelial cells are poorly understood. Here we found that heparan sulfate proteoglycans (HSPGs) on the apical surfaces of epithelial cells facilitate simultaneous binding of hBDs and HIV gp120 to the cell surface. HSPG-facilitated binding of hBDs and HIV gp120 to the cell surface did not affect viral attachment. HBD2 or -3 cointernalized with virions in endosomes, formed oligomers, and reduced infectivity of HIV. The anti-HIV effect of combining hBD2 and hBD3 was substantially higher than that of the individual peptides. These findings advance our understanding of the mechanisms of anti-HIV resistance in adult oral epithelium.