Characterization of an engineered mucus microenvironment for in vitro modeling of host-microbe interactions.

The human mucus layer plays a vital role in maintaining health by providing a physical barrier to pathogens. This biological hydrogel also provides the microenvironment for commensal bacteria. Common models used to study host-microbe interactions include gnotobiotic animals or mammalian-microbial co-culture platforms. Many of the current in vitro models lack a sufficient mucus layer to host these interactions. In this study, we engineered a mucus-like hydrogel Consisting of a mixed alginate-mucin (ALG-MUC) hydrogel network by using low concentration calcium chloride (CaCl2) as crosslinker. We demonstrated that the incorporation of ALG-MUC hydrogels into an aqueous two-phase system (ATPS) co-culture platform can support the growth of a mammalian monolayer and pathogenic bacteria. The ALG-MUC hydrogels displayed selective diffusivity against macromolecules and stability with ATPS microbial patterning. Additionally, we showed that the presence of mucin within hydrogels contributed to an increase in antimicrobial resistance in ATPS patterned microbial colonies. By using common laboratory chemicals to generate a mammalian-microbial co-culture system containing a representative mucus microenvironment, this model can be readily adopted by typical life science laboratories to study host-microbe interaction and drug discovery.

Development of a physiological model of human middle ear epithelium.

INTRODUCTION: Otitis media is an umbrella term for middle ear inflammation; ranging from acute infection to chronic mucosal disease. It is a leading cause of antimicrobial therapy prescriptions and surgery in children. Despite this, treatments have changed little in over 50 years. Research has been limited by the lack of physiological models of middle ear epithelium. METHODS: We develop a novel human middle ear epithelial culture using an air-liquid interface (ALI) system; akin to the healthy ventilated middle ear in vivo. We validate this using immunohistochemistry, immunofluorescence, scanning and transmission electron microscopy, and membrane conductance studies. We also utilize this model to perform a pilot challenge of middle ear epithelial cells with SARS-CoV-2. RESULTS: We demonstrate that human middle ear epithelial cells cultured at an ALI undergo mucociliary differentiation to produce diverse epithelial subtypes including basal (p63+), goblet (MUC5AC+, MUC5B+), and ciliated (FOXJ1+) cells. Mature ciliagenesis is visualized and tight junction formation is shown with electron microscopy, and confirmed by membrane conductance. Together, these demonstrate this model reflects the complex epithelial cell types which exist in vivo. Following SARS-CoV-2 challenge, human middle ear epithelium shows positive viral uptake, as measured by polymerase chain reaction and immunohistochemistry. CONCLUSION: We describe a novel physiological system to study the human middle ear. This can be utilized for translational research into middle ear diseases. We also demonstrate, for the first time under controlled conditions, that human middle ear epithelium is susceptible to SARS-CoV-2 infection, which has important clinical implications for safe otological surgery. LEVEL OF EVIDENCE: NA.

Transmastoid Occlusion Surgery for Superior Semicircular Canal Dehiscence Syndrome Improves Patient-Reported Quality-of-Life Measures and corrects cVEMP Thresholds and Amplitudes.

OBJECTIVE: To determine the pre- and postoperative clinical, audiological, vestibular, and patient-reported measures in patients undergoing transmastoid occlusion surgery for superior canal dehiscence syndrome (SCDS). STUDY DESIGN: Retrospective case review. SETTING: Tertiary referral centre, UK. PATIENTS: All primary transmastoid occlusion surgeries for SCDS were included (tertiary centre, single-surgeon), January 2008 to July 2019. INTERVENTIONS: Transmastoid superior canal occlusion surgery for SCDS. MAIN OUTCOME MEASURES: We collated audiological (pure tone audiogram), vestibular (cervical vestibular evoked myogenic potentials [cVEMPs]), and patient-reported outcome measures (Dizziness Handicap Inventory and subjective symptom grading). RESULTS: Fifty-two patients (55 ears) met the inclusion criteria. Thirty-one (56%) were female. Mean age was 47 years (range 29-63) and mean follow-up of 11.2 months. Six patients had bilateral disease, four of whom underwent sequential, bilateral surgery.Autophony was the most frequent presenting symptom, improving in 92%.Significant improvements were self-reported in patients' autophony (p < 0.0001), pressure- and noise-induced dizziness (p < 0.0001 and p < 0.0001), aural fullness (p = 0.0159), pulsatile tinnitus (p < 0.0001), perceived hearing loss (p = 0.0058), and imbalance (p = 0.0303).Overall Dizziness Handicap Inventory scores reduced from 45.9 to 27.4 (p < 0.0001), and across all subgroups of functional (p = 0.0003), emotional (p < 0.0001), and physical handicap (p = 0.0005).A 6.4-dB HL improvement in the air-bone gap (500-1000 Hz) occurred (95% confidence intervals 3.3-9.4 dB HL, p < 0.0001). There were no dead ears. cVEMP thresholds, when recordable, normalized in all except two ears. CONCLUSIONS: Transmastoid occlusion is effective at improving patient-reported outcomes and normalizing cVEMP thresholds, though some symptoms, notably disequilibrium, persist to a variable degree.

The Power of First Impressions: Can Influenza Imprinting during Infancy Inform Vaccine Design?

Influenza virus infection causes severe respiratory illness in people worldwide, disproportionately affecting infants. The immature respiratory tract coupled with the developing immune system, and lack of previous exposure to the virus is thought to synergistically play a role in the increased disease severity in younger age groups. No influenza vaccines are available for those under six months, although maternal influenza immunization is recommended. In children aged six months to two years, vaccine immunogenicity is dampened compared to older children and adults. Unlike older children and adults, the infant immune system has fewer antigen-presenting cells and soluble immune factors. Paradoxically, we know that a person's first infection with the influenza virus during infancy or childhood leads to the establishment of life-long immunity toward that particular virus strain. This is called influenza imprinting. We contend that by understanding the influenza imprinting event in the context of the infant immune system, we will be able to design more effective influenza vaccines for both infants and adults. Working through the lens of imprinting, using infant influenza animal models such as mice and ferrets which have proven useful for infant immunity studies, we will gain a better understanding of imprinting and its implications regarding vaccine design. This review examines literature regarding infant immune and respiratory development, current vaccine strategies, and highlights the importance of research into the imprinting event in infant animal models to develop more effective and protective vaccines for all including young children.

2019-nCoV (Wuhan virus), a novel Coronavirus: human-to-human transmission, travel-related cases, and vaccine readiness.

On 31 December 2019 the Wuhan Health Commission reported a cluster of atypical pneumonia cases that was linked to a wet market in the city of Wuhan, China. The first patients began experiencing symptoms of illness in mid-December 2019. Clinical isolates were found to contain a novel coronavirus with similarity to bat coronaviruses. As of 28 January 2020, there are in excess of 4,500 laboratory-confirmed cases, with > 100 known deaths. As with the SARS-CoV, infections in children appear to be rare. Travel-related cases have been confirmed in multiple countries and regions outside mainland China including Germany, France, Thailand, Japan, South Korea, Vietnam, Canada, and the United States, as well as Hong Kong and Taiwan. Domestically in China, the virus has also been noted in several cities and provinces with cases in all but one provinence. While zoonotic transmission appears to be the original source of infections, the most alarming development is that human-to-human transmission is now prevelant. Of particular concern is that many healthcare workers have been infected in the current epidemic. There are several critical clinical questions that need to be resolved, including how efficient is human-to-human transmission? What is the animal reservoir? Is there an intermediate animal reservoir? Do the vaccines generated to the SARS-CoV or MERS-CoV or their proteins offer protection against 2019-nCoV? We offer a research perspective on the next steps for the generation of vaccines. We also present data on the use of in silico docking in gaining insight into 2019-nCoV Spike-receptor binding to aid in therapeutic development. Diagnostic PCR protocols can be found at https://www.who.int/health-topics/coronavirus/laboratory-diagnostics-for-novel-coronavirus.

Comparing Ocular Responses to Caloric Irrigation and Electrical Vestibular Stimulation in Vestibular Schwannoma.

Electrical Vestibular Stimulation (EVS) is a non-invasive technique for activating the vestibular-ocular reflex, evoking mainly a torsional eye movement response. We have previously demonstrated that this response can be used to detect vestibular asymmetry in patients with vestibular schwannoma (VS). Here we perform a direct comparison of EVS with caloric irrigation in this patient group. We studied 30 patients with unilateral VS, alongside an equal number of aged-matched healthy control subjects. EVS current was delivered to the mastoid process in a monaural configuration using a sinusoidal stimulus (2 Hz; ± 2 mA; 10 s), with an electrode placed over the spinous C7 process. Evoked eye movements were recorded from the right eye in darkness using an infra-red sensitive camera while the subject sat relaxed with their head on a chinrest. Ocular torsion was subsequently tracked off-line using iris striations. Each subject separately underwent water caloric irrigation, in accordance with the British Society of Audiology guidelines. For the caloric test, eye movement was recorded in the yaw axis using electro-oculography. For both EVS and calorics, inter-aural response asymmetry was calculated to determine the extent of canal paresis. Both tests revealed impaired vestibular function in the ipsilesional ear of VS patients, with a mean asymmetry ratio of 15 ± 17% and 18 ± 16% for EVS and calorics, respectively. Overall, the caloric test results discriminated controls from patients slightly more effectively than EVS (Cohen's D effect size = 1.44 vs. 1.19). Importantly, there was a significant moderate correlation between the AR values produced by EVS and calorics (r = 0.53, p < 0.01), and no significant difference between mean AR estimates. When questioned, ≥85% of participants subjectively preferred the EVS experience, in terms of comfort. Moreover, it took ~15 min to complete, vs. ~1 h for caloric. These results confirm that the results of the EVS test broadly agree with those of caloric irrigation, in terms of detecting vestibular asymmetry. Furthermore, they suggest a higher degree of convenience and patient comfort.

Historical H1N1 Influenza Virus Imprinting Increases Vaccine Protection by Influencing the Activity and Sustained Production of Antibodies Elicited at Vaccination in Ferrets.

Influenza virus imprinting is now understood to significantly influence the immune responses and clinical outcome of influenza virus infections that occur later in life. Due to the yearly cycling of influenza viruses, humans are imprinted with the circulating virus of their birth year and subsequently build a complex influenza virus immune history. Despite this knowledge, little is known about how the imprinting strain influences vaccine responses. To investigate the immune responses of the imprinted host to split-virion vaccination, we imprinted ferrets with a sublethal dose of the historical seasonal H1N1 strain A/USSR/90/1977. After a +60-day recovery period to build immune memory, ferrets were immunized and then challenged on Day 123. Antibody specificity and recall were investigated throughout the time course. At challenge, the imprinted vaccinated ferrets did not experience significant disease, while naïve-vaccinated ferrets had significant weight loss. Haemagglutination inhibition assays showed that imprinted ferrets had a more robust antibody response post vaccination and increased virus neutralization activity. Imprinted-vaccinated animals had increased virus-specific IgG antibodies compared to the other experimental groups, suggesting B-cell maturity and plasticity at vaccination. These results should be considered when designing the next generation of influenza vaccines.

Swallowing after transoral surgery for oropharyngeal cancer: comparison with primary chemoradiotherapy outcomes.

PURPOSE OF REVIEW: Survival equipoise is recognized between the contemporary surgical and oncological approaches to oropharyngeal squamous cell carcinoma treatment. Primary transoral surgery (TOS) options have emerged that utilize either laser or robotic techniques. Our review presents an overview of the evidence available for swallowing outcomes following TOS approaches and compares these with outcomes following primary oncological management. RECENT FINDINGS: Meta-analysis of swallow outcomes following TOS or (chemo)radiotherapy is not possible given the heterogeneity of the available data. There are suggestions of less swallowing impairment following primary TOS, but the favourable selection of patients to these case series must be considered. SUMMARY: Minimizing swallowing impairment following oropharyngeal squamous cell carcinoma treatment, while ensuring oncological efficacy, should be a priority for head and neck healthcare providers. Primary TOS may offer an advantage to patients, but only through a team approach that considers how adjuvant oncological therapy could be tailored to individuals. High-quality clinical trials are in progress that will inform future practice.

Merkel cell carcinoma of the head and neck: challenges in implementing best practice.

OBJECTIVE: Merkel cell carcinoma (MCC) is a rare, aggressive skin tumor. Controversies regarding optimal management persist due to inadequate data and knowledge regarding tumor biology. Head and neck MCC increases both oncological and reconstructive challenges, compounded by predominantly elderly patients. We review our practice and outcomes, review evidence, and discuss the difficulties in delivering best practice management. METHODS: All patients with primary head and neck MCC, managed by a single multidisciplinary team between January 2001 and December 2010, were identified through retrospective analysis of a pathology coding database. A literature review was performed. RESULTS: Twenty patients, with a mean age of 83.5 years (40-99 years) and presenting with mean symptom duration of 5 months, had primary tumors involving the nose (n = 2), periorbital region (n = 5), cheek (n = 6), and the temple and scalp (n = 7). Mean tumor size was 2.1 cm (range, 0.5-7.5 cm). Reconstructive techniques were direct closure (n = 8), skin grafting (n = 7), local flaps (n = 4), and free anterolateral thigh flap (n = 1). Two (10%) patients presented with nodal disease. Eight (40%) patients re-presented with nodal recurrence at a mean of 7 months with 6 undergoing salvage neck dissections. Adjuvant radiotherapy was completed in 5 cases, and chemotherapy used for palliation in 1 case. Most of the patients declined radiotherapy due to adverse effects or frailty. CONCLUSIONS: Our series demonstrates the profound challenges in managing head and neck MCC, including tailoring definitive primary treatment and current consensus management to elderly patients. Regional nodal assessment and management remains crucial to achieving this goal.

The temporal and spatial expression patterns of ABCG2 in the developing human heart.

BACKGROUND: The discovery that the adult heart is not a terminally differentiated organ and contains stem/progenitor cells has important implications for the development of cellular therapeutics to treat heart disease. Moreover the discovery of cardiac stem cells might be important in furthering our understanding of both normal and abnormal cardiac development and yet little is known about these cell populations in the developing human heart, which we have focused on in this study. METHODS: The presence of ABCG2 and islet-1 expressing cells in human heart was determined using immunohistochemistry and RT-PCR (and western blotting for ABCG2). Cardiac SP cells were isolated using FACS. Co-localisation immunohistochemistry was used to determine if ABCG2 positive cells expressed other known stem/progenitor cell, endothelial markers or cardiac markers. RESULTS: We observed that ABCG2 expressing cells show a difference in both their temporal and spatial patterns of expression from Islet-1 expressing cardiac progenitors. We identified rare cells that expressed both ABCG2 and markers of other cell lineages including CD31, CD34 and alpha-actinin. We also noted the presence of cells that only expressed ABCG2. We isolated cardiac SP cells and confirmed the SP cell phenotype. CONCLUSIONS: Our results suggest that the developing human heart contains at least two distinct cardiac stem/progenitor cell populations one of which, the ABCG2 positive cells, can be readily isolated, suggesting that this tissue could be a useful source of cardiac stem cells.

Pneumatosis intestinalis presenting with a pneumoperitoneum in a patient with chronic bronchiectasis: a delayed diagnosis of superior mesenteric artery ischaemia.

We describe a case of pneumatosis intestinalis (PI) in a 69-year-old woman presenting to the surgical department with non-specific abdominal pain and a pneumoperitoneum. PI complicated by pneumoperitoneum was diagnosed on the basis of the characteristic clinical and abdominal CT findings, demonstrating a cystic gas pattern within the small bowel wall. In the context of resolving clinical findings, non-peritonitic abdomen and with no evidence of portal venous gas or perforation the patient was managed conservatively. The findings were considered benign and attributed to her chronic acquired bronchiectasis. Following recurrence of symptoms after several months, further imaging confirmed chronic mesenteric ischaemia (CMI) involving the superior mesenteric artery. Angioplasty was performed and symptoms resolved. PI is one of the few imaging signs to occur in CMI. PI presenting as a pneumoperitoneum is rare and may often lead to avoidable surgical intervention. However, no clear algorithm exists to guide surgical management.