SARS-CoV-2 is the viral pathogen responsible for COVID-19. Although morbidity and mortality frequently occur as a result of lung disease, the gastrointestinal (GI) tract is recognized as a primary location for SARS-CoV-2. Connections and interactions between the microbiome of the gut and respiratory system have been linked with viral infections via what has been referred to as the ‘gut-lung axis’ with potential aerodigestive communication in health and disease. This research explored the relationship between the microbiomes of the upper respiratory and GI tracts in patients with COVID-19 and examined Extraesophageal reflux (EOR), a mechanism which could contribute to dysregulated communication between the GI and respiratory tract (as identified in COVID-19). 97 patients with a laboratory diagnosis of COVID-19 infection, and 50 age-matched controls were recruited and stool, saliva and sputum were obtained from each participant. ELISA Pepsin tests and Reflux Symptom Index scores (RSI) were conducted for EOR assessment. DNA sequencing of the V4 region of the 16 S rRNA gene was performed for microbiome analysis. No differences were observed between the fecal microbiome’s alpha and Shannon diversity indices; however, a distinct microbial composition was observed in COVID-19 patients (when compared to the controls). The respiratory microbiota from individuals with COVID-19 demonstrated a statistically significant reduction in Shannon diversity and bacterial richness alongside an overall reduction in the prevalence of organisms from a typical healthy respiratory microbiome. Furthermore, the bacterial richness of the stool and sputum samples was significantly lower among COVID-19 patients admitted to ICU. A significantly higher RSI score and salivary pepsin level were detected among those with COVID-19. The data indicates that COVID-19 is associated with a dysregulation of both the gut and lung microbiome with a more marked perturbation in the lung, particularly among COVID-19 patients who had been admitted to the ICU. The presence of increased RSI scores, combined with elevated levels of Pepsin, suggests that increased micro-aspiration may occur, which is consistent with of under-recognized interactions between the GI and lung microbiomes in COVID-19 patients and requires additional study. Such studies would benefit from the insights provided by biological samples which reflect the continuum of the aerodigestive tract.

MUC2 mucin, the primary gel-forming component of intestinal mucus, is well researched and a model of polymerisation and post-secretory organisation has been published previously. Recently, several significant developments have been made which either introduce new ideas or challenge previous theories. New ideas include an overhaul of the MUC2 C-terminal globular structure which is proposed to harbour several previously unobserved domains, and include a site for an extra intermolecular disulphide bridge dimer between the cysteine 4379 of adjacent MUC2 C-termini. MUC2 polymers are also now thought to be secreted attached to the epithelial surface of goblet cells in the small intestine and removed following secretion via a metalloprotease meprin β-mediated cleavage of the von Willebrand D2 domain of the N-terminus. It remains unclear whether MUC2 forms intermolecular dimers, trimers, or both, at the N-termini during polymerisation, with several articles supporting either trimer or dimer formation. The presence of a firm inner mucus layer in the small intestine is similarly unclear. Considering this recent research, this review proposes an update to the previous model of MUC2 polymerisation and secretion, considers conflicting theories and data, and highlights the importance of this research to the understanding of MUC2 mucus layers in health and disease.

"The Potential Role of Gastric Microbiology in Respiratory Disease." American Journal of Respiratory and Critical Care Medicine, 0(ja), pp.

When initiated the human microbiome project did not include the lungs and airways in its sampling sites, indicating an under appreciation of the role of the human lung microbiome in health and disease. This paradigm has recently changed through the use of culture independent methods to characterise the human lung microbiome. The original thinking, that the normal lung was essentially sterile, had previously been challenged by findings of microaspiration in normal volunteers and in patients with decreased levels of consciousness. The sterile lung was also questioned by findings of clinically occult infection markers in lung allograft recipients. What is arguably a “rediscovery” of the importance of the human lung microbiome may still underappreciate physiological and patho-physiological inter-relationships between organ systems, studied in separate research disciplines. In particular, microaspiration may be an important, direct mechanism through which the lung microbiome is modulated. As well as aspiration related to gastro-oesophageal reflux and microaspiration the authors feel that the importance of dysphagia in chronic lung disease, will be increasingly recognised in frailty related microbiome exchange between the oropharynx into the lung. This review therefore discusses interconnections in the human microbiome, with a focus on the potential for aerodigestive pathophysiology and microaspiration. Potential connections with human lung disease are discussed and contextualised within a developing literature. This review therefore highlights much needed new targets for translational intervention in lung pathophysiology and underlies the importance of a mixed disciplinary team approach for the future.

The high antibiotic resistance of Pseudomonas aeruginosa (PA) makes it critical to develop alternative antimicrobial agents that are effective and affordable. One of the many applications of silver nanoparticles (Ag NPs) is their use as an antimicrobial agent against bacteria resistant to common antibiotics. The key purpose of this research was to assess the antibacterial and antibiofilm effectiveness of biosynthesized Ag NPs against six biofilm-forming clinically isolated strains of PA and one reference strain (ATCC 27853). Ag NPs were biosynthesized using a seed extract of Peganum harmala as a reducing agent. Ag NPs were characterized by Ultraviolet–visible (UV–Vis) spectroscopy and scanning transmission electron microscopy (STEM). The effect of Ag NPs on biofilm formation and eradication was examined through micro-titer plate assays, and the minimal inhibitory (MIC) and minimum bactericidal (MBC) concentrations determined. In addition, real-time polymerase chain reactions (RT-PCR) were performed to examine the effects of Ag NPs on the expression of seven PA biofilm-encoding genes (LasR, LasI, LssB, rhIR, rhII, pqsA and pqsR). The biosynthesized Ag NPs were spherically-shaped with a mean diameter of 11 nm. The MIC for each PA strain was 15.6 µg/ml, while the MBC was 31.25 µg/ml. All PA strains exposed to Ag NPs at sub-inhibitory concentrations (0.22–7.5 µg/ml) showed significant inhibitory effects on growth and biofilm formation. Biomass and biofilm metabolism were reduced dependent on Ag NP concentration. The expression of the quorum-sensing genes of all strains were significantly reduced at an Ag NP concentration of 7.5 µg/ml. The results demonstrate the extensive in-vitro antibacterial and antibiofilm performance of Ag NPs and their potential in the treatment of PA infection. It is recommended that future studies examine the possible synergy between Ag NPs and antibiotics.

Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) lung infection has represented a global challenge. Intriguingly, it has been shown that the alveolar lung epithelium expresses little Angiotensin Converting Enzyme receptor protein (ACE2), the entry receptor for SARS-CoV-2. Upper airway establishment of infection and translocation to the lung is well documented but other anatomical niches may be relevant to potentially serious lung infection. ACE2 is heavily expressed in the gastrointestinal tract and gastrointestinal symptoms support a clinical diagnosis of Coronavirus disease 2019 (COVID-19). This suggests a research question and the need to gather patient data exploring potential aerodigestive links in SARS-CoV-2 tranlocation and infection which may be relevant in the peripheral lung. This recognizes anatomical proximity and concepts of bi-directional movement between the Gastrointestinal and lung systems in normal physiology and disease. We have therefore explored the potential for gastro oesophageal reflux disease (GORD) micro aspiration and aeorodigestive pathophysiology in a novel prospective investigation of patients hospitalized with COVID-19. Methods This is a prospective descriptive cohort study of 210 patients who were hospitalized with a confirmed diagnosis of COVID-19. The cohort was divided into three groups of patients based on symptom severity and radiological results. The Reflux Symptom Index (RSI) was used to evaluate the presence and severity of GOR. An RSI greater than 13 is considered to be abnormal. Patients’ saliva samples were tested using enzyme-linked immunosorbent assay (ELISA) to determine the level of salivary pepsin among the cohort of patients. Results A total of 210 patients with COVID-19 were enrolled in the study with 55.2% (116/210) classified as mildly ill, 31.9% (67/210) moderately ill and 12.9% (27/210) as severely ill. 34% (72/210) of the patients had an RSI score of over 13 and a median salivary pepsin value of 54 ± 29 ng/ml which suggested an incidence of extraesophageal reflux (EOR) in around a third of patients. The presence of respiratory comorbid conditions, an RSI score of over 13 and a salivary pepsin level of > 76ng/ml increased the risk of developing a more severe COVID-19 infection. Conclusion The study showed a high prevalence of EOR among the study cohort and provide the first prospective evidence suggesting the potential for aerodigestive pathophysiology including microaspiration in COVID-19 disease. We believe that the results of our study support the need for more extensive research.

There is a limitation in the range of effectual antibiotics due to the Pseudomonas aeruginosa (PA) infection due to its innate antimicrobial resistance. Researchers have therefore been concentrating their efforts to discover advanced and cost effective antibacterial agents among the ever-increasing PA bacterial resistance strains. It has been discovered that various nanoparticles can be employed as antimicrobial agents. Here, we evaluated the antibacterial properties of the Zinc Oxide nanoparticles (ZnO NPs), which was biosynthesized, being examined on six hospital strains of PA alongside a reference strain (ATCC 27853). A chemical approach was applied to biosynthesize the ZnO NPs from Olea europaea was performed, and confirmed by using X-ray diffraction and Scanning Electron Microscopes. The nanoparticles then applied their antibacterial properties to examine them against six clinically isolated PA strains alongside the reference strain. This process tested for the results of the minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC). The Growth, biofilm formation and eradication were analyzed. The influence of the differentiating degrees ZnO NPs in regard to Quorom sensing gene expression were further examined. The ZnO NPs exhibited a crystalline size and diameter (Dc) of 40–60 nm and both the MIC and MBC tests revealed positive outcomes of concentrations of 3 and 6 mg/ml for each PA strain, respectively. At sub inhibitory concentration, The ZnO NPs were found to significantly inhibit the growth and biofilm formation of all PA strains and decreases in the biomass and metabolic behavior of PA established biofilms; these decreases varied depending on the dosage. At ZnO NPs concentrations of 900 µg/ml, the expression of majority of quorum sensing genes of all strains were significantly reduced, at ZnO NPs concentrations of 300 µg/ml, few genes were significantly impacted. In conclusion, the treatment of PA and could be other antibiotic resistant bacteria can therefore be approached by using ZnO NPs as it has been uncovered that they withhold advanced antibacterial properties.

Abstract White kidney bean extract (WKBE) is a nutraceutical often advocated as an anti-obesity agent. The main proposed mechanism for these effects is alpha-amylase inhibition, thereby slowing carbohydrate digestion and absorption. Thus, it is possible that WKBE could impact the gut microbiota and modulate gut health. We investigated the effects of supplementing 20 healthy adults with WKBE for 1 week in a randomised, placebo-controlled crossover trial on the composition of the gut microbiota, gastrointestinal (GI) inflammation (faecal calprotectin), GI symptoms, and stool habits. We conducted in vitro experiments and used a gut model system to explore potential inhibition of alpha-amylase. We gained qualitative insight into participant experiences of using WKBE via focus groups. WKBE supplementation decreased the relative abundance of Bacteroidetes and increased that of Firmicutes, however, there were no significant differences in post-intervention gut microbiota measurements between the WKBE and control. There were no significant effects on GI inflammation or symptoms related to constipation, or stool consistency or frequency. Our in vitro and gut model system analyses showed no effects of WKBE on alpha-amylase activity. Our findings suggest that WKBE may modulate the gut microbiota in healthy adults, however, the underlying mechanism is unlikely due to active site inhibition of alpha-amylase.

Primary epithelial cell models to provide insights into subglottic stenosis. We read with interest the recent study by Sharif et al. Using computational analyses of a previous scRNA-seq from seven idiopathic subglottic stenosis (iSGS) patients and three matched controls, the authors described a central role for a dysregulated epithelium in the pathophysiology of SGS. This study of genetic risk in SGS showed that TGFβ response pathways and epithelial–mesenchymal transition (EMT) were prominently implicated. The findings compliment and extend previous work from the group, and the North American Airway Collaborative (NoAAC), to provide collaborative new translational research into SGS. We thank Sharif et al. for referencing our work investigating the role of EMT in lung allograft dysfunction and collaborative research with Australian investigators in smoking-related pathophysiology, which provided context for their work in SGS. We agree that EMT-related airways remodeling in SGS might be a key area to research and have previously developed primary and cell-line-based models to study pathways of epithelial injury in SGS. We think our results may be complimentary to the computational data presented by Sharif et al. Gastroesophageal reflux disease has been implicated in patients with iSGS and we found that bile acids were a potent stimulus for EMT in primary epithelial cells grown from patient subglottic brushings. Related work indicated that having a role in airways remodeling EMT is an important mechanism in malignancy and may be a mechanistic link for the increased risk associated with airflow obstruction per se, over and above risk associated with smoking. We suggest that primary airway culture systems may have a key role in further research. Patientderived cell models are amenable to studying differences in disease pathophysiology related to age and sex, for example, and allow the screening of potential new treatment approaches. We suggest that this has the potential to compliment the elegant computational methods of Sharif et al. and work of others implicating EMT in iSGS. ADIL ALDHAHRANI, PhD Department of Clinical Laboratory Sciences, Turabah University College, Taif University, Taif, Saudi Arabia

Background A “balanced, adequate, and varied diet” is recommended as the basis of nutritionally sound diet by the World Health Organisation and national public health agencies. Huel is a proprietary, on-the-go, powdered, plant based food, providing all 26 essential vitamins and minerals, protein, essential fats, carbohydrate, fibre, and phytonutrients. Objectives Assessing the effect of solely consuming Huel on micronutrient status, dietary intake and markers of health was achieved through a 4-week intervention of solely Huel powder. Methods Habitual energy intake was assessed through a one-week lead in period with healthy adult participants (aged 18 or over) logging their food intake, after which only Huel was consumed for 4 weeks. Blood samples and body composition was assessed before and after the lead in week as well the end of the intervention. Thirty participants were recruited with 20 (11 females, median age 31, range 22–44) completing the study, 19 sets of blood samples were collected. 22 blood markers were analysed along with weight, BMI, waist circumference, visceral adipose tissue (VAT), and body composition. All blood micronutrients, except for Thyroid Stimulating Hormone and choline were sent to Royal Victoria Infirmary NHS, Newcastle Laboratory (Newcastle upon Tyne, United Kingdom) for analysis. Results Fourteen of the parameters significantly changed over the course of the study with circulating haemoglobin, iron, vitamins B12 and D as well as selenium significantly increasing (p < 0.05). HbA1c, total and non-HDL cholesterol, vitamins A and E, potassium, BMI, VAT, and waist circumference all significantly decreased (p < 0.05) post intervention. Conclusion Although energy intake decreased during the intervention period, the adherence to recommended micronutrient intake, as quantified by the dietary Total Adherence Score, significantly increased which tallies with the preservation or improvement of micronutrient status. This study potentially demonstrates that consuming only Huel for 4 weeks does not negatively affect micronutrient status.

Studies of microbiota reveal inter-relationships between the microbiomes of the gut and lungs. This relationship may influence the progression of lung disease, particularly in patients with cystic fibrosis (CF), who often experience extraoesophageal reflux (EOR). Despite identifying this relationship, it is not well characterised. Our hypothesis is that the gastric and lung microbiomes in CF are related, with the potential for aerodigestive pathophysiology. We evaluated gastric and sputum bacterial communities by culture and 16S rRNA gene sequencing in 13 CF patients. Impacts of varying levels of bile acids, pepsin and pH on patient isolates of Pseudomonas aeruginosa (Pa) were evaluated. Clonally related strains of Pa and NTM were identified in gastric and sputum samples from patients with symptoms of EOR. Bacterial diversity was more pronounced in sputa compared to gastric juice. Gastric and lung bile and pepsin levels were associated with Pa biofilm formation. Analysis of the aerodigestive microbiomes of CF patients with negative sputa indicates that the gut can be a reservoir of Pa and NTM. This combined with the CF patient’s symptoms of reflux and potential aspiration, highlights the possibility of communication between microorganisms of the gut and the lungs. This phenomenon merits further research.

Background: Satiety in the stomach is often caused by distension and the satiating feeling is triggered through afferent vagal signals. Increasing or prolonging the distension of the stomach with a low-calorie agent would be beneficial in reducing the energy intake and potentially aid in the management of weight. The aim of this work was to quantify the rheological properties of an alginate formulation to induce satiety (AFIS) as it passes through a physiologically relevant model of human digestive tract. Methods: A physiologically relevant model of oral, gastric and small intestinal digestion was used to simulate in vivo conditions, including digestive capacity and physical forces. Samples were taken from the model and the rheological properties and viscosity of them assessed. This was repeated in the presence of a mixed meal. Results: The addition of the AFIS gelled strongly in the gastric phase of the model gut system and reformed the gel after shear stress disrupted the gel network. The inclusion of the formulation to induce satiety with a mixed meal to the model gut system increased the viscosity in the gastric phase to a greater extent than just the formulation alone. Conclusions: The forces generated by the stomach in vivo would be sufficient to eventually overcome the gelled formulation and with the repeated breakdown and additional gastric secretions would eventually allow passage into the small intestine. The synergistic increase in viscosity seen with the mixed meal and the formulation indicated an interaction between the formulation and the meal. The AFIS would potentially increase the retention time of gastric contents as well as gelling strongly. However, the forces generated by the stomach in vivo would eventually be sufficient to breakdown the formulation, and with the additional gastric secretion, allow it to pass into the small intestine, avoiding indefinite retention.

This review discusses the utility and limitations of model gut systems in accurately modelling the mucosa of the digestive tract from both an anatomical and functional perspective, with a particular focus on the oesophagus and the upper digestive tract, and what this means for effective in vitro modelling of oesophageal pathology. Disorders of the oesophagus include heartburn, dysphagia, eosinophilic oesophagitis, achalasia, oesophageal spasm and gastroesophageal reflux disease. 3D in vitro models of the oesophagus, such as organotypic 3D culture and spheroid culture, have been shown to be effective tools for investigating oesophageal pathology. However, these models are not integrated with modelling of the upper digestive tract—presenting an opportunity for future development. Reflux of upper gastrointestinal contents is a major contributor to oesophageal pathologies like gastroesophageal reflux disease and Barratt’s oesophagus, and in vitro models are essential for understanding their mechanisms and developing solutions. The limitations of current model gut systems in modelling the mucosa is not only limited to the oesophagus. Integration of modelling of the mucus covered epithelia of the stomach and small intestine in to upper digestive tract models is limited and often not considered at all. In this paper we discuss mucus structure and function and current approaches to modelling of the mucus layer in isolation, and in integrated systems with cell culture systems and digestive models. We identify a need for relevant modelling of the viscoelastic properties of mucus and its protective function to allow complete integration in modelling. Addressing limitations of current in vitro models and integrating upper gastrointestinal models with those of the oesophagus presents an opportunity for better understanding oesophageal physiology and pathophysiology where reflux of digestive fluids is involved.

Two seaweeds; Ascophyllum nodosum and Fucus vesiculosus, were incorporated into bread at 0.5 and 2% and their effect on blood glucose in vivo and carbohydrate digestion in vitro were studied. In the five way randomised placebo controlled double blind pilot trial (n = 10) each volunteer consumed 100 g of available carbohydrate (from bread) and their blood glucose was measured over two hours. The breads were tested in a human digestion model and compared against control bread and control bread with the equivalent amount of seaweed. In the pilot human study the enriched breads did not cause any significant reductions in iAUC of blood glucose with average reductions of 0.1 ± 44.4%, 8.2 ± 19.3%, 1.0 ± 54.3% and 2.7 ± 31.9% for 0.5% F.vesiculosus, 0.5% A.nodosum, 2% F.vesiculosus, and 2% A.nodosum respectively. However, seaweed added alongside the control bread in vitro significantly reduced the level of carbohydrate digestion compared to the control bread. F.vesiculosus or A.nodosum can reduce carbohydrate digestion, however baking into bread reduces the effect.

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The inter-relationship between chronic respiratory disease and reflux disease in the airway reflux paradigm is extremely complex and remains poorly characterised. Reflux disease is reported to cause or contribute to the severity of a number of respiratory tract diseases including laryngeal disorders, sinusitis, chronic cough, asthma, COPD, idiopathic pulmonary fibrosis, cystic fibrosis, bronchiectasis and bronchiolitis obliterans post lung transplant. It is now appreciated that reflux disease is not simply caused by liquid acid reflux but rather by a variety of chemical refluxates originating from the stomach and duodenum due to a number of different mechanisms. Reflux disease can be challenging to diagnose, particularly proving its role in the causation of direct respiratory epithelial damage. Significant advances in oesophageal assessment and gastric biomarkers have emerged in recent years as our understanding increases. There are a number of treatments available for reflux disease, both medical and surgical, but there is a paucity of large randomised trials to evaluate their efficacy in the setting of chronic respiratory disease. Everyday clinical practice, however, informs us that treatment failure in reflux disease is common. This clinical review summarises associations between reflux disease in the setting of chronic respiratory diseases and examines available evidence regarding potential therapeutic strategies.

Searchable abstracts of presentations at key conferences in obesity ISSN 2632-9808 (online)

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)