A Monoclonal Human Alveolar Epithelial Cell Line ("Arlo") with Pronounced Barrier Function for Studying Drug Permeability and Viral Infections.

In the development of orally inhaled drug products preclinical animal models regularly fail to predict pharmacological as well as toxicological responses in humans. Models based on human cells and tissues are potential alternatives to animal experimentation allowing for the isolation of essential processes of human biology and making them accessible in vitro. Here, the generation of a novel monoclonal cell line "Arlo," derived from the polyclonal human alveolar epithelium lentivirus immortalized cell line hAELVi via single-cell printing, and its characterization as a model for the human alveolar epithelium as well as a building block for future complex in vitro models is described. "Arlo" is systematically compared in vitro to primary human alveolar epithelial cells (hAEpCs) as well as to the polyclonal hAELVi cell line. "Arlo" cells show enhanced barrier properties with high transepithelial electrical resistance (TEER) of ≈3000 Ω cm2 and a potential difference (PD) of ≈30 mV under air-liquid interface (ALI) conditions, that can be modulated. The cells grow in a polarized monolayer and express genes relevant to barrier integrity as well as homeostasis as is observed in hAEpCs. Successful productive infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in a proof-of-principle study offers an additional, attractive application of "Arlo" beyond biopharmaceutical experimentation.

Zonulin, a marker of gut permeability, is associated with mortality in a cohort of hospitalised peruvian COVID-19 patients.

Zonulin has previously been related to intestinal permeability in various inflammatory diseases, and more recently to the physiopathology of severe COVID-19 infections. We analysed serum samples from a previous study of a Peruvian cohort of hospitalised COVID-19 patients, for the quantification of zonulin by sandwich ELISA. Comparisons with clinical data, haematological and biochemical parameters and cytokine/chemokine levels were made. We found higher baseline zonulin levels in deceased patients, and zonulin was associated with fatal outcome in multivariable analyses, even after adjustment for age, gender, and obesity. There were also positive correlations between zonulin, creatinine, D-dimer values and prothrombin time, while inverse correlations were found for Sa/FiO2 ratio and CCL5 (RANTES). Further longitudinal studies are recommended to analyse the variation of zonulin levels over time as well as their relationship with long-COVID.

'Poly phenolic phytoceutical loaded nano-bilosomes for enhanced caco-2 cell permeability and SARS-CoV 2 antiviral activity': in-vitro and insilico studies.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) predisposed to the emergence of worldwide catastrophe that impels the evolution of safe and effective therapeutic system. Polyphenols as resveratrol (RSV) exhibit a well evidenced antiviral activity. Unfortunately, like most phenolic nutraceuticals, RSV suffers from restrained solubility and massive degradation in GIT and liver which in turn prohibit its clinical use. Herein, PEGylated bilosomes (PBs) contain PEGylated edge activator along with the traditional components as (Span 60, cholesterol and bile salts) were proposed to boost both permeability and bioavailability of RSV. The investigation of the prominent effect of the diverse variables on the characteristics of the vesicles and picking of the optimum formula were conducted via construction of 23 factorial experiment. The appraisal of the formulae was conducted on the basis of entrapment efficiency percent (EE%), particle size (PS) and zeta potential (ZP). In addition, the spherical shaped optimal formula (F5) exhibited EE% of 86.1 ± 2.9%, PS of 228.9 ± 8.5 nm, and ZP of -39.8 ± 1.3 mV. The sorted optimum formula (F5) exhibited superior dissolution behaviors, and boosted Caco-2 cells cellular uptake by a round 4.7 folds relative to RSV dispersion. In addition, F5 demonstrated a complete in vitro suppression of SARS-CoV-2 at a concentration 0.48 µg/ml with 6.6 times enhancement in antiviral activity relative to RSV dispersion. The accomplished molecular modeling heavily provided proof for the possible interactions of resveratrol with the key residues of the SARS-CoV2 Mpro enzyme. Finally, F5 could be proposed as a promising oral panel of RSV for curation from SARS-CoV-2 infection.

IL-6, IL-10, sFas, granulysin and indicators of intestinal permeability as early biomarkers for a fatal outcome in COVID-19.

The clinical presentation of coronavirus disease 2019 (COVID-19), which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), ranges between mild respiratory symptoms and a severe disease that shares many of the features of sepsis. Sepsis is a deregulated response to infection that causes life-threatening organ failure. During sepsis, the intestinal epithelial cells are affected, causing an increase in intestinal permeability and allowing microbial translocation from the intestine to the circulation, which exacerbates the inflammatory response. Here we studied patients with moderate, severe and critical COVID-19 by measuring a panel of molecules representative of the innate and adaptive immune responses to SARS-CoV-2, which also reflect the presence of systemic inflammation and the state of the intestinal barrier. We found that non-surviving COVID-19 patients had higher levels of low-affinity anti-RBD IgA antibodies than surviving patients, which may be a response to increased microbial translocation. We identified sFas and granulysin, in addition to IL-6 and IL-10, as possible early biomarkers with high sensitivity (>73 %) and specificity (>51 %) to discriminate between surviving and non-surviving COVID-19 patients. Finally, we found that the microbial metabolite d-lactate and the tight junction regulator zonulin were increased in the serum of patients with severe COVID-19 and in COVID-19 patients with secondary infections, suggesting that increased intestinal permeability may be a source of secondary infections in these patients. COVID-19 patients with secondary infections had higher disease severity and mortality than patients without these infections, indicating that intestinal permeability markers could provide complementary information to the serum cytokines for the early identification of COVID-19 patients with a high risk of a fatal outcome.

In Silico Prediction of Intestinal Permeability by Hierarchical Support Vector Regression.

The vast majority of marketed drugs are orally administrated. As such, drug absorption is one of the important drug metabolism and pharmacokinetics parameters that should be assessed in the process of drug discovery and development. A nonlinear quantitative structure-activity relationship (QSAR) model was constructed in this investigation using the novel machine learning-based hierarchical support vector regression (HSVR) scheme to render the extremely complicated relationships between descriptors and intestinal permeability that can take place through various passive diffusion and carrier-mediated active transport routes. The predictions by HSVR were found to be in good agreement with the observed values for the molecules in the training set (n = 53, r2 = 0.93, q CV 2 = 0.84, RMSE = 0.17, s = 0.08), test set (n = 13, q2 = 0.75-0.89, RMSE = 0.26, s = 0.14), and even outlier set (n = 8, q2 = 0.78-0.92, RMSE = 0.19, s = 0.09). The built HSVR model consistently met the most stringent criteria when subjected to various statistical assessments. A mock test also assured the predictivity of HSVR. Consequently, this HSVR model can be adopted to facilitate drug discovery and development.

Ultralight and Ultrathin Electrospun Membranes with Enhanced Air Permeability for Chemical and Biological Protection.

With the growing interest in chemical and biological warfare agents (CWAs/BWAs), the focus has shifted toward aerosol protection using protective clothing. However, compared to air-permeable membranes, those with water vapor permeability have been investigated more extensively. Filtering membranes without air permeability have limited practical usage in personal protective suits and masks. In this study, polyacrylonitrile membranes with tightly attached activated carbon and doped copper(II) oxide were prepared via electrospinning. The nanofibers with uniformly controlled diameters and smooth morphologies enable water/air breathability and protection against aerosol (100 nm polystyrene nanobeads similar to SARS-CoV-2) penetration. The uniformly distributed and tightly attached activated carbon and doped copper(II) oxide particles enhance the sorptive performance of the membranes by blocking gaseous CWAs, including soman, nerve chemical agents, and BWAs. Such dual-purpose membranes can be implemented in protective equipment owing to their high performance and easy processing.

Evaluation of the Microbiological Effectiveness of Three Accessible Mask Decontamination Methods and Their Impact on Filtration, Air Permeability and Physicochemical Properties.

The need to secure public health and mitigate the environmental impact associated with the massified use of respiratory protective devices (RPD) has been raising awareness for the safe reuse of decontaminated masks by individuals and organizations. Among the decontamination treatments proposed, in this work, three methods with the potential to be adopted by households and organizations of different sizes were analysed: contact with nebulized hydrogen peroxide (H2O2); immersion in commercial bleach (NaClO) (sodium hypochlorite, 0.1% p/v); and contact with steam in microwave steam-sanitizing bags (steam bag). Their decontamination effectiveness was assessed using reference microorganisms following international standards (issued by ISO and FDA). Furthermore, the impact on filtration efficiency, air permeability and several physicochemical and structural characteristics of the masks, were evaluated for untreated masks and after 1, 5 and 10 cycles of treatment. Three types of RPD were analysed: surgical, KN95, and cloth masks. Results demonstrated that the H2O2 protocol sterilized KN95 and surgical masks (reduction of >6 log10 CFUs) and disinfected cloth masks (reduction of >3 log10 CFUs). The NaClO protocol sterilized surgical masks, and disinfected KN95 and cloth masks. Steam bags sterilized KN95 and disinfected surgical and cloth masks. No relevant impact was observed on filtration efficiency.

Safety and preliminary efficacy of sequential multiple ascending doses of solnatide to treat pulmonary permeability edema in patients with moderate to severe ARDS in a randomized, placebo-controlled, double-blind trial: preliminary evaluation of safety and feasibility in light of the COVID-19 pandemic.

BACKGROUND: In May 2018, the first patient was enrolled in the phase-IIb clinical trial "Safety and Preliminary Efficacy of Sequential Multiple Ascending Doses of Solnatide to Treat Pulmonary Permeability Edema in Patients with Moderate to Severe ARDS." With the onset of the COVID-19 pandemic in early 2020, the continuation and successful execution of this clinical study was in danger. Therefore, before the Data Safety Monitoring Board (DSMB) allowed proceeding with the study and enrollment of further COVID-19 ARDS patients into it, additional assessment on possible study bias was considered mandatory. METHODS: We conducted an ad hoc interim analysis of 16 patients (5 COVID-19- ARDS patients and 11 with ARDS from different causes) from the phase-IIB clinical trial. We assessed possible differences in clinical characteristics of the ARDS patients and the impact of the pandemic on study execution. RESULTS: COVID-19 patients seemed to be less sick at baseline, which also showed in higher survival rates over the 28-day observation period. Trial specific outcomes regarding pulmonary edema and ventilation parameters did not differ between the groups, nor did more general indicators of (pulmonary) sepsis like oxygenation ratio and required noradrenaline doses. CONCLUSION: The DSMB and the investigators did not find any evidence that patients suffering from ARDS due to SARS-CoV-2 may be at higher (or generally altered) risk when included in the trial, nor were there indications that those patients might influence the integrity of the study data altogether. For this reason, a continuation of the phase IIB clinical study activities can be justified. Researchers continuing clinical trials during the pandemic should always be aware that the exceptional circumstances may alter study results and therefore adaptations of the study design might be necessary.

Exploring the permeability of covid-19 drugs within the cellular membrane: a molecular dynamics simulation study.

The diffusion of drugs into the cellular membrane is an important step in the drug delivery systems. Furthermore, predicting the interaction and permeability of drugs across the cellular membrane could help scientists to design bioavailable and high-efficient drugs. Discovering the COVID-19 drugs has recently drawn remarkable attention to tackle its outbreak. Due to the rapid replication of the coronavirus in the human body, searching for highly permeable drugs into the cellular membrane is vital. Herein, we performed the molecular dynamics (MD) simulation and density functional (DFT) calculations to investigate the permeability of keto and enol tautomers of the favipiravir (FAV) as well as hydroxychloroquine (HCQ) COVID-19 drugs into the cellular membrane. Our results reveal that though both keto and enol tautomers of the FAV are feasible to transfer through the cellular membrane, the keto form moves faster and diffuses deeper; however, the HCQ molecules aggregate in the water phase and remain near the cellular membrane. It is worth pointing out that the obtained results are consistent with the reactivity trends projected by the calculated reactivity descriptors of the considered drugs. Despite the pair correlation function and H-bond analyses revealing the interactions between the membrane and HCQ, the aggregation of the HCQ molecules resists their passage through the cellular membrane. Besides, the lower free energy barrier of FAV confirms its higher permeability than HCQ. These findings suggest that due to the deeper permeability of the FAV drug, its effectiveness can be more than that of HCQ. These molecular insights might help with a better understanding of the interactions between COVID-19 drugs and cellular membranes. Moreover, these theoretical findings could help experimental researchers find high-efficient strategies for COVID-19 therapy.

A stability indicating RP-HPLC method for determination of the COVID-19 drug molnupiravir applied using nanoformulations in permeability studies.

Antiviral drugs have gained much more attention in recent years due to severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and many drug candidates are currently under investigation in order to end pandemic. Molnupiravir, a prodrug of the synthetic nucleoside derivative N4-hydroxycytidine, is one of the promising candidates for SARS-CoV-2 treatment. In this study, a RP-HPLC method was developed for the determination of Molnupiravir and applied for in vitro permeability studies of self-emulsifying drug delivery system (SEDDS) formulations using Caco-2 cell line. Discovery® HS C18 Column (75 ×4.6 mm, 3 µm) was used at 30 °C. Isocratic elution was performed with ACN:water (20:80 v/v) mixture. The flow rate was 0.5 mL/min and UV detection was at 240 nm. Molnupiravir eluted within 5 min. Molnupiravir was exposed to thermal, photolytic, hydrolytic, and oxidative stress conditions. Peak homogeneity data of Molnupiravir in the stressed samples peak obtained using photodiode array detector, in the stressed sample chromatograms, demonstrated the specificity of the method for their estimation in presence of degradants. The developed method was validated according to the International Council for Harmonisation (ICH) guidelines and found to be linear within the range 0.1-60.0 µg/mL. The method was simple, rapid, selective, sensitive, accurate, precise, robust and rugged. Thus, it was applied successfully for permeability quantitation of Molnupiravir in nanoformulations. The apparent permeability of Molnupiravir in SEDDS formulations, which have droplet size under 350 nm, was calculated as 3.20 ± 0.44 × 10-6 cm/s.

Safety and preliminary efficacy of sequential multiple ascending doses of solnatide to treat pulmonary permeability edema in patients with moderate-to-severe ARDS-a randomized, placebo-controlled, double-blind trial.

BACKGROUND: Acute respiratory distress syndrome (ARDS) is a complex clinical diagnosis with various possible etiologies. One common feature, however, is pulmonary permeability edema, which leads to an increased alveolar diffusion pathway and, subsequently, impaired oxygenation and decarboxylation. A novel inhaled peptide agent (AP301, solnatide) was shown to markedly reduce pulmonary edema in animal models of ARDS and to be safe to administer to healthy humans in a Phase I clinical trial. Here, we present the protocol for a Phase IIB clinical trial investigating the safety and possible future efficacy endpoints in ARDS patients. METHODS: This is a randomized, placebo-controlled, double-blind intervention study. Patients with moderate to severe ARDS in need of mechanical ventilation will be randomized to parallel groups receiving escalating doses of solnatide or placebo, respectively. Before advancing to a higher dose, a data safety monitoring board will investigate the data from previous patients for any indication of patient safety violations. The intervention (application of the investigational drug) takes places twice daily over the course of 7 days, ensued by a follow-up period of another 21 days. DISCUSSION: The patients to be included in this trial will be severely sick and in need of mechanical ventilation. The amount of data to be collected upon screening and during the course of the intervention phase is substantial and the potential timeframe for inclusion of any given patient is short. However, when prepared properly, adherence to this protocol will make for the acquisition of reliable data. Particular diligence needs to be exercised with respect to informed consent, because eligible patients will most likely be comatose and/or deeply sedated at the time of inclusion. TRIAL REGISTRATION: This trial was prospectively registered with the EU Clinical trials register (clinicaltrialsregister.eu). EudraCT Number: 2017-003855-47 .

Effectiveness of Common Fabrics to Block Aqueous Aerosols of Virus-like Nanoparticles.

Layered systems of commonly available fabric materials can be used by the public and healthcare providers in face masks to reduce the risk of inhaling viruses with protection that is about equivalent to or better than the filtration and adsorption offered by 5-layer N95 respirators. Over 70 different common fabric combinations and masks were evaluated under steady-state, forced convection air flux with pulsed aerosols that simulate forceful respiration. The aerosols contain fluorescent virus-like nanoparticles to track transmission through materials that greatly assist the accuracy of detection, thus avoiding artifacts including pore flooding and the loss of aerosol due to evaporation and droplet breakup. Effective materials comprise both absorbent, hydrophilic layers and barrier, hydrophobic layers. Although the hydrophobic layers can adhere virus-like nanoparticles, they may also repel droplets from adjacent absorbent layers and prevent wicking transport across the fabric system. Effective designs are noted with absorbent layers comprising terry cloth towel, quilting cotton, and flannel. Effective designs are noted with barrier layers comprising nonwoven polypropylene, polyester, and polyaramid.

Multisystem inflammatory syndrome in children is driven by zonulin-dependent loss of gut mucosal barrier.

BACKGROUNDWeeks after SARS-CoV-2 infection or exposure, some children develop a severe, life-threatening illness called multisystem inflammatory syndrome in children (MIS-C). Gastrointestinal (GI) symptoms are common in patients with MIS-C, and a severe hyperinflammatory response ensues with potential for cardiac complications. The cause of MIS-C has not been identified to date.METHODSHere, we analyzed biospecimens from 100 children: 19 with MIS-C, 26 with acute COVID-19, and 55 controls. Stools were assessed for SARS-CoV-2 by reverse transcription PCR (RT-PCR), and plasma was examined for markers of breakdown of mucosal barrier integrity, including zonulin. Ultrasensitive antigen detection was used to probe for SARS-CoV-2 antigenemia in plasma, and immune responses were characterized. As a proof of concept, we treated a patient with MIS-C with larazotide, a zonulin antagonist, and monitored the effect on antigenemia and the patient's clinical response.RESULTSWe showed that in children with MIS-C, a prolonged presence of SARS-CoV-2 in the GI tract led to the release of zonulin, a biomarker of intestinal permeability, with subsequent trafficking of SARS-CoV-2 antigens into the bloodstream, leading to hyperinflammation. The patient with MIS-C treated with larazotide had a coinciding decrease in plasma SARS-CoV-2 spike antigen levels and inflammatory markers and a resultant clinical improvement above that achieved with currently available treatments.CONCLUSIONThese mechanistic data on MIS-C pathogenesis provide insight into targets for diagnosing, treating, and preventing MIS-C, which are urgently needed for this increasingly common severe COVID-19-related disease in children.

The role of the envelope protein in the stability of a coronavirus model membrane against an ethanolic disinfectant.

Ethanol is highly effective against various enveloped viruses and can disable the virus by disintegrating the protective envelope surrounding it. The interactions between the coronavirus envelope (E) protein and its membrane environment play key roles in the stability and function of the viral envelope. By using molecular dynamics simulation, we explore the underlying mechanism of ethanol-induced disruption of a model coronavirus membrane and, in detail, interactions of the E-protein and lipids. We model the membrane bilayer as N-palmitoyl-sphingomyelin and 1-palmitoyl-2-oleoylphosphatidylcholine lipids and the coronavirus E-protein. The study reveals that ethanol causes an increase in the lateral area of the bilayer along with thinning of the bilayer membrane and orientational disordering of lipid tails. Ethanol resides at the head-tail region of the membrane and enhances bilayer permeability. We found an envelope-protein-mediated increase in the ordering of lipid tails. Our simulations also provide important insights into the orientation of the envelope protein in a model membrane environment. At ∼25 mol. % of ethanol in the surrounding ethanol-water phase, we observe disintegration of the lipid bilayer and dislocation of the E-protein from the membrane environment.

Plasma Markers of Disrupted Gut Permeability in Severe COVID-19 Patients.

A disruption of the crosstalk between the gut and the lung has been implicated as a driver of severity during respiratory-related diseases. Lung injury causes systemic inflammation, which disrupts gut barrier integrity, increasing the permeability to gut microbes and their products. This exacerbates inflammation, resulting in positive feedback. We aimed to test whether severe Coronavirus disease 2019 (COVID-19) is associated with markers of disrupted gut permeability. We applied a multi-omic systems biology approach to analyze plasma samples from COVID-19 patients with varying disease severity and SARS-CoV-2 negative controls. We investigated the potential links between plasma markers of gut barrier integrity, microbial translocation, systemic inflammation, metabolome, lipidome, and glycome, and COVID-19 severity. We found that severe COVID-19 is associated with high levels of markers of tight junction permeability and translocation of bacterial and fungal products into the blood. These markers of disrupted intestinal barrier integrity and microbial translocation correlate strongly with higher levels of markers of systemic inflammation and immune activation, lower levels of markers of intestinal function, disrupted plasma metabolome and glycome, and higher mortality rate. Our study highlights an underappreciated factor with significant clinical implications, disruption in gut functions, as a potential force that may contribute to COVID-19 severity.

SARS-CoV-2 uses major endothelial integrin αvß3 to cause vascular dysregulation in-vitro during COVID-19.

The unprecedented global COVID-19 pandemic has prompted a desperate international effort to accelerate the development of anti-viral candidates. For unknown reasons, COVID-19 infections are associated with adverse cardiovascular complications, implicating that vascular endothelial cells are essential in viral propagation. The etiological pathogen, SARS-CoV-2, has a higher reproductive number and infection rate than its predecessors, indicating it possesses novel characteristics that infers enhanced transmissibility. A unique K403R spike protein substitution encodes an Arg-Gly-Asp (RGD) motif, introducing a potential role for RGD-binding host integrins. Integrin αVß3 is widely expressed across the host, particularly in the endothelium, which acts as the final barrier before microbial entry into the bloodstream. This mutagenesis creates an additional binding site, which may be sufficient to increase SARS-CoV-2 pathogenicity. Here, we investigate how SARS-CoV-2 passes from the epithelium to endothelium, the effects of αVß3 antagonist, Cilengitide, on viral adhesion, vasculature permeability and leakage, and also report on a simulated interaction between the viral and host protein in-silico.

Potential use of serum-derived bovine immunoglobulin/protein isolate for the management of COVID-19.

COVID-19 manifests as a mild disease in most people but can progress to severe disease in nearly 20% of individuals. Disease progression is likely driven by a cytokine storm, either directly stimulated by SARS-CoV-2 or by increased systemic inflammation in which the gut might play an integral role. SARS-CoV-2 replication in the gut may cause increased intestinal permeability, alterations to the fecal microbiome, and increased inflammatory cytokines. Each effect may lead to increased systemic inflammation and the transport of cytokines and inflammatory antigens from the gut to the lung. Few interventions are being studied to treat people with mild disease and prevent the cytokine storm. Serumderived bovine immunoglobulin/protein isolate (SBI) may prevent progression by (1) binding and neutralizing inflammatory antigens, (2) decreasing gut permeability, (3) interfering with ACE2 binding by viral proteins, and (4) improving the fecal microbiome. SBI is therefore a promising intervention to prevent disease progression in COVID-19 patients.

Is the Intestine a Portal of Entry for the Serious COVID-19 Complications of Endotoxemia and Thrombosis?

Severe acute respiratory syndrome coronavirus 2 infection has been associated with both endotoxemia and thrombosis of small and large vessels, but the relationship between these 2 phenomena has not been pursued. Oliva et al. in this issue of Clinical and Translational Gastroenterology demonstrate an association between the 2 findings and suggest that increased intestinal permeability is a possible mechanism to explain the endotoxemia. Although the evidence to support this hypothesis is only suggestive, the role of the small intestine in the illness produced by the virus needs to be further explored.

Low-Grade Endotoxemia and Thrombosis in COVID-19.

INTRODUCTION: Patients with community-acquired pneumonia display enhanced levels of lipopolysaccharides (LPS) compared with controls, suggesting that low-grade endotoxemia may be implicated in vascular disturbances. It is unknown whether this occurs in patients with coronavirus 2019 (COVID-19) and its impact on thrombotic complications. METHODS: We measured serum levels of zonulin, a marker of gut permeability, LPS, and D-dimer in 81 patients with COVID-19 and 81 healthy subjects; the occurrence of thrombotic events in COVID-19 during the intrahospital stay was registered. RESULTS: Serum LPS and zonulin were higher in patients with COVID-19 than in control subjects and, in COVID-19, significantly correlated (R = 0.513; P < 0.001). Among the 81 patients with COVID-19, 11 (14%) experienced thrombotic events in the arterial (n = 5) and venous circulation (n = 6) during a median follow-up of 18 days (interquartile range 11-27 days). A logistic regression analysis showed that LPS (P = 0.024) and D-dimer (P = 0.041) independently predicted thrombotic events. DISCUSSION: The study reports that low-grade endotoxemia is detectable in patients with COVID-19 and is associated with thrombotic events. The coexistence of low-grade endotoxemia with enhanced levels of zonulin may suggest enhanced gut permeability as an underlying mechanism.