17ß-estradiol Attenuates the Middle Ear Inflammatory Response to Nontypeable Haemophilus influenzae.

OBJECTIVES: 17ß-estradiol (E2) is a steroidal hormone with immunomodulatory functions that play a role in infectious and inflammatory diseases. E2 was recently identified as the leading upstream regulator of differentially expressed genes in a comparative RNA sequencing study of pediatric patients with otitis media (OM) versus OM-free counterparts and may therefore play a role in the inflammatory response to bacterial otopathogens during pediatric OM. This study examined the effect of E2 on bacterial-induced inflammatory cytokine expression in an in vitro pediatric OM model. METHODS: An immortalized middle ear (ME) epithelial cell line, ROM-SV40, was developed from a pediatric recurrent OM patient. The culture was exposed to E2 at physiological levels for 1-48 h prior to 6 h-stimulation with nontypeable Haemophilus influenzae (NTHi) whole cell lysate. TNFA, IL1B, IL6, and IL8 were assayed by qPCR and ELISA. RESULTS: E2 pretreatment (24 h) abrogated NTHi induction of IL6; a longer pretreatment (1-10 nM, 48 h) abrogated IL1B induction (p < 0.05). E2 pretreatment (5 nM, 48 h) abrogated NTHi-induced IL8 secretion (p = 0.017). CONCLUSION: E2 pretreatment partially rescued NTHi-induced cytokine production by ME epithelia. These data support a role for E2 in moderating the excessive inflammatory response to middle ear infection that contributes to OM pathophysiology. LEVELS OF EVIDENCE: NA Laryngoscope, 134:3815-3819, 2024.

Amprenavir inhibits pepsin-mediated laryngeal epithelial disruption and E-cadherin cleavage in vitro.

Background: Laryngopharyngeal reflux (LPR) causes chronic cough, throat clearing, hoarseness, and dysphagia and can promote laryngeal carcinogenesis. More than 20% of the US population suffers from LPR and there is no effective medical therapy. Pepsin is a predominant source of damage during LPR which disrupts laryngeal barrier function potentially via E-cadherin cleavage proteolysis and downstream matrix metalloproteinase (MMP) dysregulation. Fosamprenavir (FDA-approved HIV therapeutic and prodrug of amprenavir) is a pepsin-inhibiting LPR therapeutic candidate shown to rescue damage in an LPR mouse model. This study aimed to examine amprenavir protection against laryngeal monolayer disruption and related E-cadherin proteolysis and MMP dysregulation in vitro. Methods: Laryngeal (TVC HPV) cells were exposed to buffered saline, pH 7.4 or pH 4 ± 1 mg/mL pepsin ± amprenavir (10-60 min). Analysis was performed by microscopy, Western blot, and real time polymerase chain reaction (qPCR). Results: Amprenavir (1 µM) rescued pepsin acid-mediated cell dissociation (p < .05). Pepsin acid caused E-cadherin cleavage indicative of regulated intramembrane proteolysis (RIP) and increased MMP-1,3,7,9,14 24-h postexposure (p < .05). Acid alone did not cause cell dissociation or E-cadherin cleavage. Amprenavir (10 µM) protected against E-cadherin cleavage and MMP-1,9,14 induction (p < .05). Conclusions: Amprenavir, at serum concentrations achievable provided the manufacturer's recommended dose of fosamprenavir for HIV, protects against pepsin-mediated cell dissociation, E-cadherin cleavage, and MMP dysregulation thought to contribute to barrier dysfunction and related symptoms during LPR. Fosamprenavir to amprenavir conversion by laryngeal epithelia, serum and saliva, and relative drug efficacies in an LPR mouse model are under investigation to inform development of inhaled formulations for LPR.

Topical Alginate Protection against Pepsin-Mediated Esophageal Damage: E-Cadherin Proteolysis and Matrix Metalloproteinase Induction.

Epithelial barrier dysfunction is a hallmark of gastroesophageal reflux disease (GERD) related to symptom origination, inflammatory remodeling and carcinogenesis. Alginate-based antireflux medications were previously shown to topically protect against peptic barrier disruption, yet the molecular mechanisms of injury and protection were unclear. Herein, Barrett's esophageal (BAR-T) cells were pretreated with buffered saline (HBSS; control), dilute alginate medications (Gaviscon Advance or Gaviscon Double Action, Reckitt Benckiser), a viscosity-matched placebo, or ADAM10 and matrix metalloproteinase (MMP) inhibitors before exposure to HBSS pH7.4 or pH4 ± 1 mg/mL pepsin for 10-60 min. Cell viability was assessed by ATP assay; mediators of epithelial integrity, E-cadherin, ADAM10, and MMPs were examined by Western blot and qPCR. Alginate rescued peptic reduction of cell viability (p < 0.0001). Pepsin-pH4 yielded E-cadherin fragments indicative of regulated intramembrane proteolysis (RIP) which was not rescued by inhibitors of known E-cadherin sheddases. Transcriptional targets of E-cadherin RIP fragments were elevated at 24 h (MMP-1,2,9,14; p < 0.01). Alginate rescued E-cadherin cleavage, ADAM10 maturation, and MMP induction (p < 0.01). Results support RIP as a novel mechanism of peptic injury during GERD. Alginate residue after wash-out to mimic physiologic esophageal clearance conferred lasting protection against pepsin-induced molecular mechanisms that may exacerbate GERD severity and promote carcinogenesis in the context of weakly acidic reflux.

Cancer Risk in Barrett's Esophagus: A Clinical Review.

Esophageal adenocarcinoma (EAC) is rapidly increasing in incidence and is associated with a poor prognosis. Barrett's esophagus (BE) is a known precursor of esophageal adenocarcinoma. This review aims to explore Barrett's esophagus, esophageal adenocarcinoma, and the progression from the former to the latter. An overview of the definition, diagnosis, epidemiology, and risk factors for both entities are presented, with special attention being given to the areas of debate in the literature. The progression from Barrett's esophagus to esophageal adenocarcinoma is reviewed and the relevant molecular pathways are discussed. The definition of Barrett's esophagus remains debated and without international consensus. This, alongside other factors, has made establishing the true prevalence of Barrett's esophagus challenging. The degree of dysplasia can be a histological challenge, but is necessary to guide clinical management. The progression of BE to EAC is likely driven by inflammatory pathways, pepsin exposure, upregulation of growth factor pathways, and mitochondrial changes. Surveillance is maintained through serial endoscopic evaluation, with shorter intervals recommended for high-risk features.

The Protease Inhibitor Amprenavir Protects against Pepsin-Induced Esophageal Epithelial Barrier Disruption and Cancer-Associated Changes.

Gastroesophageal reflux disease (GERD) significantly impacts patient quality of life and is a major risk factor for the development of Barrett's esophagus (BE) and esophageal adenocarcinoma (EAC). Proton pump inhibitors (PPIs) are the standard-of-care for GERD and are among the most prescribed drugs in the world, but do not protect against nonacid components of reflux such as pepsin, or prevent reflux-associated carcinogenesis. We recently identified an HIV protease inhibitor amprenavir that inhibits pepsin and demonstrated the antireflux therapeutic potential of its prodrug fosamprenavir in a mouse model of laryngopharyngeal reflux. In this study, we assessed the capacity of amprenavir to protect against esophageal epithelial barrier disruption in vitro and related molecular events, E-cadherin cleavage, and matrix metalloproteinase induction, which are associated with GERD severity and esophageal cancer. Herein, weakly acidified pepsin (though not acid alone) caused cell dissociation accompanied by regulated intramembrane proteolysis of E-cadherin. Soluble E-cadherin responsive matrix metalloproteinases (MMPs) were transcriptionally upregulated 24 h post-treatment. Amprenavir, at serum concentrations achievable given the manufacturer-recommended dose of fosamprenavir, protected against pepsin-induced cell dissociation, E-cadherin cleavage, and MMP induction. These results support a potential therapeutic role for amprenavir in GERD recalcitrant to PPI therapy and for preventing GERD-associated neoplastic changes.

Association of e-Cigarette Exposure with Pediatric Otitis Media Recurrence.

OBJECTIVE: Otitis media (OM) is a common inflammatory disease spectrum in children and a leading cause of pediatric physician visits, antibiotic prescriptions and surgery. Tobacco exposure is associated with increased risk of OM recurrence, chronicity and surgeries. Tobacco products have changed dramatically in recent years with the advent of electronic cigarettes (e-cigarettes). While users frequently perceive vape as less harmful than traditional cigarettes, burgeoning evidence supports its contribution to respiratory pathologies. The consequences of secondhand exposure, particularly among children, are understudied. The aim of this study was to examine the association of e-cigarette emissions (EE) with OM recurrence and surgeries in the US. METHODS: Questionnaire data regarding ear infections and tobacco exposure was gathered for all pediatric respondents of the National Health and Nutrition Examination Survey (NHANES) 2017 to 2018. Weighted analyzes and logistic regression models were used to assess associations. RESULTS: Data was available for 2022 participants (aged 6-17); all were included for analyzes. Tobacco exposure was observed in 42%; 9% were exposed to EE. EE contributed to risk of ≥3 ear infections (OR = 1.61, 95% CI 1.01-2.58, P = .047). After adjustment for significant covariates (race and asthma), the association fell below significance (P = .081). No other significant associations were observed between ear infections, or tympanostomy tube insertion and exposure variables (EE, gestational or other household exposure). CONCLUSIONS: Exposure to EE may confer greater risk of pediatric OM than previously identified factors such as household smoke, or gestational exposure. Further investigation of EE and its health implications in children is warranted. LEVEL OF EVIDENCE: IV.

Pepsinogen/Proton Pump Co-Expression in Barrett's Esophageal Cells Induces Cancer-Associated Changes.

EDUCATIONAL OBJECTIVE: At the conclusion of this presentation, participants should better understand the carcinogenic potential of pepsin and proton pump expression in Barrett's esophagus. OBJECTIVE: Barrett's esophagus (BE) is a well-known risk factor for esophageal adenocarcinoma (EAC). Gastric H+ /K+ ATPase proton pump and pepsin expression has been demonstrated in some cases of BE; however, the contribution of local pepsin and proton pump expression to carcinogenesis is unknown. In this study, RNA sequencing was used to examine global transcriptomic changes in a BE cell line ectopically expressing pepsinogen and/or gastric H+ /K+ ATPase proton pumps. STUDY DESIGN: In vitro translational. METHODS: BAR-T, a human BE cell line devoid of expression of pepsinogen or proton pumps, was transduced by lentivirus-encoding pepsinogen (PGA5) and/or gastric proton pump subunits (ATP4A, ATP4B). Changes relative to the parental line were assessed by RNA sequencing. RESULTS: Top canonical pathways associated with protein-coding genes differentially expressed in pepsinogen and/or proton pump expressing BAR-T cells included those involved in the tumor microenvironment and epithelial-mesenchymal transition. Top upstream regulators of coding transcripts included TGFB1 and ERBB2, which are associated with the pathogenesis and prognosis of BE and EAC. Top upstream regulators of noncoding transcripts included p300-CBP, I-BET-151, and CD93, which have previously described associations with EAC or carcinogenesis. The top associated disease of both coding and noncoding transcripts was cancer. CONCLUSIONS: These data support the carcinogenic potential of pepsin and proton pump expression in BE and reveal molecular pathways affected by their expression. Further study is warranted to investigate the role of these pathways in carcinogenesis associated with BE. LEVEL OF EVIDENCE: NA Laryngoscope, 133:59-69, 2023.

Context and mechanisms that enable implementation of specialist palliative care Needs Rounds in care homes: results from a qualitative interview study.

BACKGROUND: Improving quality of palliative and end of life care in older people's care homes is essential. Increasing numbers of people die in these settings, yet access to high quality palliative care is not routinely provided. While evidence for models of care are growing, there remains little insight regarding how to translate evidence-based models into practice. Palliative Care Needs Rounds (hereafter Needs Rounds) have a robust evidence base, for providing palliative care in care homes, reducing resident hospitalisations, improving residents' quality of death, and increasing staff confidence in caring for dying residents. This study aimed to identify and describe the context and mechanisms of change that facilitate implementation of Needs Rounds in care homes, and enable other services to reap the benefits of the Needs Rounds approach to care provision. METHODS: Qualitative interviews, embedded within a large randomised control trial, were conducted with a purposive sample of 21 staff from 11 care homes using Needs Rounds. The sample included managers, nurses, and care assistants. Staff participated in individual or dyadic semi-structured interviews. Implementation science frameworks and thematic analysis were used to interpret and analyse the data. RESULTS: Contextual factors affecting implementation included facility preparedness for change, leadership, staff knowledge and skills, and organisational policies. Mechanisms of change that facilitated implementation included staff as facilitators, identifying and triaging residents, strategizing knowledge exchange, and changing clinical approaches to care. Care home staff also identified planning and documentation, and shifts in communication. The outcomes reported by staff suggest reductions in hospitalisations and problematic symptoms for residents, improved staff skills and confidence in caring for residents in their last months, weeks and days of life. CONCLUSIONS: The significance of this paper is in offering care homes detailed insights into service contexts and mechanisms of change that will enable them to reap the benefits of Needs Rounds in their own services. The paper thus will support the implementation of an approach to care that has a robust evidence base, for a population under-served by specialist palliative care. TRIAL REGISTRATION: ACTRN12617000080325 .

RNA Sequencing and Pathways Analyses of Middle Ear Epithelia From Patients With Otitis Media.

OBJECTIVES: Otitis media (OM) is the most common pediatric diagnosis in the United States. However, our understanding of the molecular pathogenesis of OM remains relatively poor. Investigation of molecular pathways involved in OM may improve the understanding of this disease process and elucidate novel therapeutic targets. In this study, RNA sequencing (RNA-Seq) was used to discern cellular changes associated with OME compared to healthy middle ear epithelium (MEE). STUDY DESIGN: Ex vivo case-control translational. METHODS: Middle ear epithelia was collected from five pediatric patients diagnosed with OME undergoing tympanostomy tube placement and five otherwise healthy pediatric patients undergoing cochlear implantation. Specimens underwent RNA-Seq and pathways analyses. RESULTS: A total of 1,292 genes exhibited differential expression in MEE from OME patients compared to controls including genes involved in inflammation, immune response to bacterial OM pathogens, mucociliary clearance, regulation of proliferation and transformation, and auditory cell differentiation. Top networks identified in OME were organismal injury and abnormalities, cell morphology, and auditory disease. Top Ingenuity canonical pathways identified were axonal guidance signaling, which contains genes associated with auditory development and disease and nicotine degradation II and III pathways. Associated upstream regulators included ß-estradiol, dexamethasone, and G-protein-coupled estrogen receptor-1 (GPER1), which are associated with otoprotection or inflammation during insult. CONCLUSIONS: RNA-Seq demonstrates differential gene expression in MEE from patients with OME compared to healthy controls with important implications for infection susceptibility, hearing loss, and a role for tobacco exposure in the development and/or severity of OME in pediatric patients. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:2590-2597, 2021.

RNA Sequencing Reveals Cancer-Associated Changes in Laryngeal Cells Exposed to Non-Acid Pepsin.

OBJECTIVE: Laryngopharyngeal reflux (LPR) is a common affliction that contributes to laryngeal inflammation, symptoms that impact quality of life, and life-threatening illnesses such as cancer. Effective treatment strategies for LPR are lacking. Pepsin is a proinflammatory and carcinogenic element of refluxate. Investigation of molecular pathways involved in pepsin-mediated damage may lead to identification of novel biomarkers and therapeutic targets for LPR. In this study, RNA sequencing was used to examine changes in human laryngeal epithelial cells following brief pepsin insult. Cells were immortalized to generate a model to aid future study of laryngeal injury and therapeutics. STUDY DESIGN: In vitro translational. METHODS: Laryngeal epithelial cells were cultured from a patient without signs or symptoms of LPR or laryngeal cancer. Cells were treated with 0.1 mg/ml pepsin for 1 hour or normal growth media (control) prior to RNA sequencing. Cells were immortalized via HPV E6/7 and characterized by microscopy, immunohistochemistry, G-banding, and soft agar assay. RESULTS: Three hundred ninety-seven genes exhibited differences in expression with pepsin treatment (P < .05). Pathway analysis revealed association with cancer and related signaling processes including dysregulation of cancer-associated molecules, Metastasis-Associated Lung Adenocarcinoma Transcript 1 and KRT82, and the long-noncoding RNA, lipoprotein receptor-related protein 1 (LRP1)-AS, which regulates the putative pepsin receptor LRP1. CONCLUSIONS: A single, brief exposure to pepsin activated cancer-associated signaling pathways in laryngeal cells in vitro, revealing novel mechanisms by which chronic reflux may contribute to carcinogenesis. The cell line developed herein represents a novel tool in which to investigate pepsin-dysregulated pathways identified by RNA sequencing and disparities of tumor proneness of laryngeal subsites. LEVEL OF EVIDENCE: N/A Laryngoscope, 131:121-129, 2021.

Analysis of Inflammatory Signaling in Human Middle Ear Cell Culture Models of Pediatric Otitis Media.

OBJECTIVES/HYPOTHESIS: Cell culture models are valuable tools for investigation of the molecular pathogenesis of diseases including otitis media (OM). Previous study indicates that age-, sex-, and race-associated differences in molecular signaling may impact disease pathophysiology. Currently, a singular immortalized middle ear epithelial (MEE) cell line exists, HMEEC-1, derived from an adult without known middle ear disease. In this study, HMEEC-1 and primary MEE cultures from pediatric patients with and without OM were stimulated with inflammatory cytokines or OM-pathogenic bacterial lysates to examine differences in the response of molecules associated with OM pathogenesis. STUDY DESIGN: Case-control series. METHODS: MEE cultures were established from patients aged <6 years: two with recurrent OM (ROM), two with OM with effusion (OME), and one patient without OM who was undergoing cochlear implant surgery control undergoing cochlear implantation (Peds CI). Primary MEE cultures and HMEEC-1 cells were stimulated with tumor necrosis factor-α, interleukin (IL)-1ß, or nontypeable Haemophilus influenzae lysate. TNFA, IL1B, IL6, IL8, IL10, and MUC5B were assayed via quantitative polymerase chain reaction. IL-8 was assayed by enzyme-linked immunosorbent assay. RESULTS: Gene/protein target expressions were frequently higher in pediatric OM lines than in HMEEC-1 and Peds CI. HMEEC-1 cells were frequently less responsive to stimuli than all pediatric lines. OME lines were often more responsive than ROM lines. CONCLUSIONS: OM may be associated with specific molecular phenotypes that are retained in primary cell culture. Adult-derived HMEEC-1 cells differ significantly in baseline expression and response of OM-associated molecules relative to pediatric MEE cells. Work is underway to immortalize pediatric OM MEE cultures as improved tools for the OM research community. LEVEL OF EVIDENCE: 4 Laryngoscope, 131:410-416, 2021.

H+/K+ATPase Expression in the Larynx of Laryngopharyngeal Reflux and Laryngeal Cancer Patients.

OBJECTIVES: The gastric H+/K+ ATPase proton pump has previously been shown to be expressed in the human larynx, however its contribution to laryngopharyngeal reflux (LPR) signs, symptoms and associated diseases such as laryngeal cancer is unknown. Proton pump expression in the larynx of patients with LPR and laryngeal cancer was investigated herein. A human hypopharyngeal cell line expressing the proton pump was generated to investigate its effects. STUDY DESIGN: In-vitro translational. METHODS: Laryngeal biopsies were obtained from three LPR and eight LSCC patients. ATP4A, ATP4B and HRPT1 were assayed via qPCR. Human hypopharyngeal FaDu cell lines stably expressing proton pump were created using lentiviral transduction and examined via transmission electron microscopy and qPCR for genes associated with inflammation or laryngeal cancer. RESULTS: Expression of ATP4A and ATP4B was detected in 3/3 LPR, 4/8 LSCC-tumor and 3/8 LSCC-adjacent specimens. Expression of ATP4A and ATP4B in FaDu elicited mitochondrial damage and expression of IL1B, PTGS2, and TNFA (P < .0001); expression of ATP4B alone did not. CONCLUSIONS: Gastric proton pump subunits are expressed in the larynx of LPR and LSCC patients. Mitochondrial damage and changes in gene expression observed in cells expressing the full proton pump, absent in those expressing a single subunit, suggest that acid secretion by functional proton pumps expressed in upper airway mucosa may elicit local cell and molecular changes associated with inflammation and cancer. LEVEL OF EVIDENCE: NA Laryngoscope, 131:130-135, 2021.

Pepsin in gastroesophageal and extraesophageal reflux: molecular pathophysiology and diagnostic utility.

PURPOSE OF REVIEW: Gastroesophageal and extraesophageal reflux are prevalent and costly diseases. Recognition of the pathogenicity of nonacid reflux has stimulated interest in alternatives to acid-targeting diagnostics and therapeutics. Pepsin is the most deleterious enzyme in refluxate, eliciting inflammatory and carcinogenic effects irrespective of acid. Its presence in all refluxate and detection in saliva have situated pepsin as the most widely researched biomarker for reflux today. This review summarizes emerging findings regarding pepsin-mediated damage during reflux and developments in pepsin-targeting diagnostics. RECENT FINDINGS: New evidence supports a role for pepsin in epithelial--mesenchymal transition, an important process in carcinogenesis and fibrosis. The first global transcriptomic analysis of pepsin-exposed laryngeal cells was described, yielding evidence of a putative airway pepsin receptor. Evaluation of pepsin diagnostics highlighted the need for rigorous validation in which pepsin concentrations are corroborated by a secondary quantitative assay, and reflux is confirmed or excluded by multichannel intraluminal impedance pH testing. Standards for sample collection and storage, and normative and pathological values are lacking. SUMMARY: Progress continues to be made in our understanding of pepsin-mediated damage with implications for novel therapeutic strategies. Salivary pepsin diagnostics continue to garner interest; however, further work appears necessary to improve their accuracy and reproducibility.

The impact of introducing Palliative Care Needs Rounds into rural residential aged care: A quasi-experimental study.

OBJECTIVE: This study examined the impact of introducing Palliative Care Needs Rounds (hereafter Needs Rounds) into residential aged care on hospitalisations (emergency department presentations, admissions and length of stay) and documentation of advance care plans. DESIGN: A quasi-experimental study. SETTING: Two residential aged care facilities in one rural town in the Snowy Monaro region of New South Wales, Australia. PARTICIPANTS: The intervention group consisted of all residents who died during the study period (April 2018-March 2019), and included a subgroup of decedents who were discussed in a Needs Round. The control cohort included all residents who died in the three-year period prior to introducing Needs Rounds (2015-2017). INTERVENTION: Needs Rounds are monthly onsite triage/risk stratification meetings where case-based education and staff support help to identify residents most at risk of dying without an adequate plan in place. Needs Rounds were attended by residential aged care staff and led by a palliative medicine physician. MAIN OUTCOME MEASURES: Decedents' hospitalisations (emergency department presentations, admissions and length of stay) in the last three months of life, place of death and documentation of advance care plans. RESULTS: Eleven Needs Rounds were conducted between April and September 2018. The number of documented advance care plans increased (P < .01). There were no statistically significant changes in hospitalisations or in-hospital deaths. CONCLUSION: Needs Rounds are an effective approach to increase the documentation of advance care plans within rural residential aged care. Further studies are required to explore the rural influence on outcomes including hospital transfers and preferred place of death.

Reducing time in acute hospitals: A stepped-wedge randomised control trial of a specialist palliative care intervention in residential care homes.

BACKGROUND: Care home residents are frequently transferred to hospital, rather than provided with appropriate and timely specialist care in the care home. AIM: To determine whether a model of care providing specialist palliative care in care homes, called Specialist Palliative Care Needs Rounds, could reduce length of stay in hospital. DESIGN: Stepped-wedge randomised control trial. The primary outcome was length of stay in acute care (over 24-h duration), with secondary outcomes being the number and cost of hospitalisations. Care homes were randomly assigned to cross over from control to intervention using a random number generator; masking was not possible due to the nature of the intervention. Analyses were by intention to treat. The trial was registered with ANZCTR: ACTRN12617000080325. Data were collected between 1 February 2017 and 30 June 2018. SETTING/PARTICIPANTS: 1700 residents in 12 Australian care homes for older people. RESULTS: Specialist Palliative Care Needs Rounds led to reduced length of stay in hospital (unadjusted difference: 0.5 days; adjusted difference: 0.22 days with 95% confidence interval: -0.44, -0.01 and p = 0.038). The intervention also provided a clinically significant reduction in the number of hospitalisations by 23%, from 5.6 to 4.3 per facility-month. A conservative estimate of annual net cost-saving from reduced admissions was A$1,759,011 (US$1.3 m; UK£0.98 m). CONCLUSION: The model of care significantly reduces hospitalisations through provision of outreach by specialist palliative care clinicians. The data offer substantial evidence for Specialist Palliative Care Needs Rounds to reduce hospitalisations in older people approaching end of life, living in care homes.

Detection of Pepsin in Oral Secretions of Infants with and without Laryngomalacia.

OBJECTIVES: Laryngomalacia is a common cause of stridor in infants and is associated with laryngopharyngeal reflux (LPR). Although pepsin in operative supraglottic lavage specimens is associated with severe laryngomalacia, detection of pepsin in oral secretions has not been demonstrated in an outpatient setting. METHODS: Children <2 years old with laryngomalacia diagnosed by flexible laryngoscopy and children without stridor were selected. Oral secretion samples were obtained in clinic from all subjects. Pepsin, IL-1ß, and IL-8 enzyme-linked immunosorbent assays were performed to determine presence of LPR. RESULTS: Sixteen laryngomalacia and sixteen controls were enrolled. Pepsin was detected more frequently in oral secretions of patients with laryngomalacia (13/16) than in controls (2/16; P < .001). Four patients with laryngomalacia developed symptoms requiring supraglottoplasty. Presence and level of salivary pepsin was not significantly associated with need for surgical management, nor were the levels or presence of IL-1ß or IL-8 significantly associated with presence or level of pepsin, diagnosis of laryngomalacia, or need for operative management. CONCLUSION: Pepsin in saliva appears to be associated with laryngomalacia, suggesting a role for salivary pepsin as a noninvasive marker of LPR in patients with laryngomalacia. Future studies will determine the utility of this test in laryngomalacia.

Defining 'specialist palliative care': findings from a Delphi study of clinicians.

Objective This study aimed to achieve consensus regarding what distinguishes specialist from non-specialist palliative care to inform service organisation and delivery to patients with life-limiting conditions. Methods A three-phase Delphi study was undertaken, involving qualitative interviews and two questionnaire cycles. Thirty-one clinicians (nurses, doctors and social workers) working with a wide range of patients participated in interviews, of whom 27 completed two questionnaire cycles. Results Consensus was gained on 75 items that define specialist palliative care and distinguish it from non-specialist palliative care. Consensus was gained that specialist palliative care clinicians have advanced knowledge of identifying dying, skills to assess and manage complex symptoms to improve quality of life, have advanced communication skills and perform distinct clinical practices (e.g. working with the whole family as the unit of care and providing support in complex bereavement). Non-specialist palliative care involves discussions around futile or burdensome treatments, and care for people who are dying. Conclusions Areas of connection were identified: clinicians from disease-specific specialties should be more involved in leading discussions on futile or burdensome treatment and providing care to people in their last months and days of life, in collaboration with specialists in palliative care when required. What is known about the topic? At present there is no evidence-based definition or agreement about what constitutes specialist palliative care (as opposed to palliative care delivered by non-specialists) in the Australian Capital Territory. An agreed definition is needed to effectively determine the workforce required and its clinical skill mix, and to clarify roles and expectations to mitigate risks in not adequately providing services to patients with life-limiting conditions. What does this paper add? This paper offers, for the first time, an evidence-based definition that distinguishes specialist palliative care from non-specialist palliative care. End of life care and bereavement support are not just the remit of specialist palliative care clinicians. Clinicians from beyond specialist palliative care should lead discussions about futile or burdensome treatment. What are the implications for practitioners? The findings of this study can facilitate implementation of palliative care strategies by enabling practitioners and patients to distinguish who should be delivering what care.

The role of pepsin in epithelia-mesenchymal transition in idiopathic subglottic stenosis.

OBJECTIVES: Idiopathic subglottic stenosis (iSGS) is commonly characterized by laryngeal fibrosis thought to arise by epithelia-mesenchymal transition (EMT) induced by chronic inflammation. Pepsin is a potent inducer of inflammation in the airways during chronic laryngopharyngeal reflux and has been observed in the subglottic mucosa of patients with iSGS, absent in normal mucosa. The aim of this study was to examine the effect of pepsin on mechanisms of EMT in laryngeal cells with implications for iSGS. STUDY DESIGN: In vitro translational research study. METHODS: Human laryngeal epithelial cell cultures were exposed to 0.1 mg/mL or 1.0 mg/mL pepsin at pH7 for 24 and 48 hours, or media pH5 ± 0.1 mg/mL pepsin for 10 minutes and harvested after 24 and 48 hours. EMT marker expression was measured by qPCR and enzyme-linked immunosorbent assays. Wound-healing scratch assay was performed on immortalized human vocal fold fibroblasts pretreated with media pH5 ± 0.1 mg/mL pepsin (10 minutes) or continuously treated with media pH7 ± 0.1 to 1 mg/mL pepsin for 24 hours. RESULTS: Pepsin yielded no effect on MMP1, MMP9, FN1, COL1A1, HAS2, or CDH1 gene expression or matrix metalloproteinase-9 or fibronectin protein expression, either alone or in the presence of weak acid. Pepsin and/or acid produced no effect on fibroblast migration. CONCLUSION: Whereas pepsin has been shown to be present in the subglottic mucosa of patients with iSGS, this in vitro acute exposure investigation does not provide evidence of a direct causal role for development of fibrosis in subglottic epithelial cell cultures. LEVELS OF EVIDENCE: NA. Laryngoscope, 130:154-158, 2020.

Improved Quality of Death and Dying in Care Homes: A Palliative Care Stepped Wedge Randomized Control Trial in Australia.

OBJECTIVES: Mortality in care homes is high, but care of dying residents is often suboptimal, and many services do not have easy access to specialist palliative care. This study examined the impact of providing specialist palliative care on residents' quality of death and dying. DESIGN: Using a stepped wedge randomized control trial, care homes were randomly assigned to crossover from control to intervention using a random number generator. Analysis used a generalized linear and latent mixed model. The trial was registered with ANZCTR: ACTRN12617000080325. SETTING: Twelve Australian care homes in Canberra, Australia. PARTICIPANTS: A total of 1700 non-respite residents were reviewed from the 12 participating care homes. Of these residents, 537 died and 471 had complete data for analysis. The trial ran between February 2017 and June 2018. INTERVENTION: Palliative Care Needs Rounds (hereafter Needs Rounds) are monthly hour-long staff-only triage meetings to discuss residents at risk of dying without a plan in place. They are chaired by a specialist palliative care clinician and attended by care home staff. A checklist is followed to guide discussions and outcomes, focused on anticipatory planning. MEASUREMENTS: This article reports secondary outcomes of staff perceptions of residents' quality of death and dying, care home staff confidence, and completion of advance care planning documentation. We assessed (1) quality of death and dying, and (2) staff capability of adopting a palliative approach, completion of advance care plans, and medical power of attorney. RESULTS: Needs Rounds are associated with staff perceptions that residents had a better quality of death and dying (P < .01; 95% confidence interval [CI] = 1.83-12.21), particularly in the 10 facilities that complied with the intervention protocol (P < .01; 95% CI = 6.37-13.32). Staff self-reported perceptions of capability increased (P < .01; 95% CI = 2.73-6.72). CONCLUSION: The data offer evidence for monthly triage meetings to transform the lives, deaths, and care of older people residing in care homes. J Am Geriatr Soc 68:305-312, 2020.

Esophageal pepsin and proton pump synthesis in barrett's esophagus and esophageal adenocarcinoma.

OBJECTIVES/HYPOTHESIS: Gastroesophageal reflux disease and associated metaplasia of the esophagus (Barrett's esophagus [BE]) are primary risk factors for esophageal adenocarcinoma (EAC). Widespread use of acid suppression medications has failed to stem the rise of EAC, suggesting that nonacid reflux may underlie its pathophysiology. Pepsin is a tumor promoter in the larynx and has been implicated in esophageal carcinogenesis. Herein, specimens from the esophageal cancer spectrum were tested for pepsin presence. Pepsin-induced carcinogenic changes were assayed in an esophageal cell culture model. STUDY DESIGN: Laboratory analysis. METHODS: Pepsin was assayed in reflux and cancer free esophagi, BE, EAC, and esophageal cancer lacking association with reflux (squamous cell carcinoma [SCC]). Refluxed or locally synthesized pepsin was assayed by Western blot. Local synthesis of pepsin and proton pumps was assayed via reverse transcription-polymerase chain reaction. The effect of pepsin on BE and EAC markers was investigated via enzyme-linked immunosorbent assay and quantitative polymerase chain reaction in human esophageal epithelial cells treated with pepsin or control diluent. RESULTS: Pepsinogen and proton pump mRNA were observed in BE (3/5) and EAC (4/4) samples, but not in normal adjacent specimens, SCC (0/2), or reflux and cancer-free esophagi. Chronic pepsin treatment (0.1-1 mg/mL, 4 weeks) of human esophageal cells in vitro induced BE and EAC markers interleukin 8 and KRT8 and depleted normal esophageal marker KRT10 (P < .05) expression. CONCLUSIONS: Local synthesis of pepsin and proton pumps in BE and EAC is not uncommon. Absence of these molecules in normal (noncancer) esophagi, SCC, and in vitro data support a role for pepsin in reflux-attributed carcinogenic changes in the esophagus. LEVEL OF EVIDENCE: NA Laryngoscope, 129:2687-2695, 2019.