Urothelium removal does not impact mucosal activity in response to muscarinic or adrenergic receptor stimulation.

The inner lining of the urinary bladder (urothelium and lamina propria, or bladder mucosa) has an important role as a tissue barrier between stored urine and the underlying smooth muscle, as well as in the modulation and regulation of bladder contractility. However, the individual influence of the apical urothelial layer on the contractile activity of this tissue is uncertain. The aim of this experiment was to identify the contractile activity of the lamina propria after removal of the urothelium. Several methods were used to mechanically disrupt the urothelium, including dabbing the tissue with a paper towel, longitudinal swipes with a cotton bud, or a longitudinal scrape with the edge of a scalpel. Hematoxylin-eosin staining was utilized to determine the level of removal of the apical urothelial cells. Spontaneous contractile activity was measured in organ baths, and responses to the agonists carbachol and isoprenaline were obtained. Three longitudinal swipes with a cotton bud was found to be the optimal method to remove the majority of the urothelium without damaging the lamina propria. Upon removal of the urothelium, the spontaneous activity of the tissue was unaltered. Similarly, responses to carbachol (1 µM) and isoprenaline (1 µM) were not affected after removal of the urothelium. The urothelium can be effectively removed without damaging the lamina propria. This apical tissue layer is not responsible for mediating the increases to spontaneous phasic activity or tonic contractions of the bladder mucosa (urothelium with lamina propria) when muscarinic or adrenergic receptors are stimulated. This research presents the lamina propria as the important cell layer mediating the overall contractile activity of the bladder wall.

Calcium-dependent ESCRT recruitment and lysosome exocytosis maintain epithelial integrity during Candida albicans invasion.

Candida albicans is both a commensal and an opportunistic fungal pathogen. Invading hyphae of C. albicans secrete candidalysin, a pore-forming peptide toxin. To prevent cell death, epithelial cells must protect themselves from direct damage induced by candidalysin and by the mechanical forces exerted by expanding hyphae. We identify two key Ca2+-dependent repair mechanisms employed by epithelial cells to withstand candidalysin-producing hyphae. Using camelid nanobodies, we demonstrate candidalysin secretion directly into the invasion pockets induced by elongating C. albicans hyphae. The toxin induces oscillatory increases in cytosolic [Ca2+], which cause hydrolysis of PtdIns(4,5)P2 and loss of cortical actin. Epithelial cells dispose of damaged membrane regions containing candidalysin by an Alg-2/Alix/ESCRT-III-dependent blebbing process. At later stages, plasmalemmal tears induced mechanically by invading hyphae are repaired by exocytic insertion of lysosomal membranes. These two repair mechanisms maintain epithelial integrity and prevent mucosal damage during both commensal growth and infection by C. albicans.

Physiological properties, composition and structural profiling of porcine gastrointestinal mucus.

The gastrointestinal mucus is a hydrogel that lines the luminal side of the gastrointestinal epithelium, offering barrier protection from pathogens and lubrication of the intraluminal contents. These barrier properties likewise affect nutrients and drugs that need to penetrate the mucus to reach the epithelium prior to absorption. In order to assess the potential impact of the mucus on drug absorption, we need information about the nature of the gastrointestinal mucus. Today, most of the relevant available literature is mainly derived from rodent studies. In this work, we used a larger animal species, the pig model, to characterize the mucus throughout the length of the gastrointestinal tract. This is the first report of the physiological properties (physical appearance, pH and water content), composition (protein, lipid and metabolite content) and structural profiling (rheology and gel network) of the porcine gastrointestinal mucus. These findings allow for direct comparisons between the characteristics of mucus from various segments and can be further utilized to improve our understanding of the role of the mucus on region dependent drug absorption. Additionally, the present work is expected to contribute to the assessment of the porcine model as a preclinical species in the drug development process.

Influence of Nutrition and Maternal Bonding on Postnatal Lung Development in the Newborn Pig.

Background: Microbial colonization and immune cell maturation coincide at mucosal sites and are decisive for postnatal lung development. How external factors influence neonatal pulmonary immune development is poorly understood. Objective: To elucidate the impact of key determinants in early life, nutrition, and maternal bonding, on postnatal lung maturation in a human-relevant animal model. To investigate the underlying immunological changes of impaired lung maturation and study the mechanisms of conversion. Methods: Newborn piglets were kept with or without isolation from their mothers and fed bovine milk-based infant formula or received milk of sow. Lung growth, histomorphology, respiratory immune responses, and lung microbiota were analyzed. Mother- and sow-milk-deprived piglets received maternal material or were reintroduced to the maternal environment at varying intervals to study options for reversal. Results: Formula feeding combined with isolation of newborn piglets resulted in disturbed postnatal lung maturation. Reduced lung growth correlated with dampened IL-33 expression, impaired lung myeloid cell activation, and decreased Th1 differentiation, along with diminished richness and diversity of the lung microbiota. Transfer of bacteria-enriched maternal material reversed the negative effects on pulmonary immune maturation. Early (within 3 days) but not late (within 7 days) reintroduction to the mother allowed restoration of normal lung development. Conclusion: Our findings reveal that lung growth, respiratory immunity, and microbial lung colonization in newborns depend on postnatal diet and maternal contact, and targeting these key regulators could promote lung development during this critical life stage. Summary: Disturbances in natural diet and reduced maternal contact during the neonatal period impair postnatal lung maturation. In pediatrics, timely breast milk feeding and intensive maternal bonding represent valuable intervention measures to promote early postnatal lung development.

A Comparative Study of Early Mucosal Healing Following Hot Polypectomy and Cold Polypectomy.

BACKGROUND Cold polypectomy (CP) and hot polypectomy (HP) are both accepted methods for polypectomy. In recent years, the use of CP has increased for reasons of safety. However, there have been few investigations of conditions at follow-up early after resection. This prospective study from a single center aimed to compare colonic mucosal healing at 1 week following HP vs CP of benign colonic polyps <10 mm in diameter. MATERIAL AND METHODS Six patients with a total of 52 lesions under 10 mm in size were randomized to either the HP group (n=25) or CP group (n=27) using information in opaque envelopes. One week after endoscopic treatment, the site of treatment was evaluated using colonoscopy. We assessed the mean tumor size, ulcer diameter, exposed blood vessels, residual lesion, and complications. RESULTS Mean tumor size did not differ between the 2 groups (CP vs HP: 5.41 mm vs 5.68 mm). The CP group had a smaller ulcer base diameter (2.70 mm vs 4.84 mm; P<0.05) and fewer exposed blood vessels than the HP group (3.7% vs 36.0%; P<0.05). One residual lesion was found in the CP group. No patients experienced delayed perforation or post-polypectomy bleeding. CONCLUSIONS Our study findings showed that at 1-week follow-up, cold polypectomy resulted in improved colonic mucosal healing, with a smaller ulcer diameter and fewer blood vessels, when compared with hot polypectomy.

Roles of Lipids in the Permeability Barriers of Skin and Oral Mucosa.

PubMed searches reveal much literature regarding lipids in barrier function of skin and less literature on lipids in barrier function of the oral mucosa. In terrestrial mammals, birds, and reptiles, the skin's permeability barrier is provided by ceramides, fatty acids, and cholesterol in the outermost layers of the epidermis, the stratum corneum. This layer consists of about 10-20 layers of cornified cells embedded in a lipid matrix. It effectively prevents loss of water and electrolytes from the underlying tissue, and it limits the penetration of potentially harmful substances from the environment. In the oral cavity, the regions of the gingiva and hard palate are covered by keratinized epithelia that much resemble the epidermis. The oral stratum corneum contains a lipid mixture similar to that in the epidermal stratum corneum but in lower amounts and is accordingly more permeable. The superficial regions of the nonkeratinized oral epithelia also provide a permeability barrier. These epithelial regions do contain ceramides, cholesterol, and free fatty acids, which may underlie barrier function. The oral epithelial permeability barriers primarily protect the underlying tissue by preventing the penetration of potentially toxic substances, including microbial products. Transdermal drug delivery, buccal absorption, and lipid-related disease are discussed.

Evolution and function of the epithelial cell-specific ER stress sensor IRE1ß.

Barrier epithelial cells lining the mucosal surfaces of the gastrointestinal and respiratory tracts interface directly with the environment. As such, these tissues are continuously challenged to maintain a healthy equilibrium between immunity and tolerance against environmental toxins, food components, and microbes. An extracellular mucus barrier, produced and secreted by the underlying epithelium plays a central role in this host defense response. Several dedicated molecules with a unique tissue-specific expression in mucosal epithelia govern mucosal homeostasis. Here, we review the biology of Inositol-requiring enzyme 1ß (IRE1ß), an ER-resident endonuclease and paralogue of the most evolutionarily conserved ER stress sensor IRE1α. IRE1ß arose through gene duplication in early vertebrates and adopted functions unique from IRE1α which appear to underlie the basic development and physiology of mucosal tissues.

Gastrointestinal biofilms in health and disease.

Microorganisms colonize various ecological niches in the human habitat, as they do in nature. Predominant forms of multicellular communities called biofilms colonize human tissue surfaces. The gastrointestinal tract is home to a profusion of microorganisms with intertwined, but not identical, lifestyles: as isolated planktonic cells, as biofilms and in biofilm-dispersed form. It is therefore of major importance in understanding homeostatic and altered host-microorganism interactions to consider not only the planktonic lifestyle, but also biofilms and biofilm-dispersed forms. In this Review, we discuss the natural organization of microorganisms at gastrointestinal surfaces, stratification of microbiota taxonomy, biogeographical localization and trans-kingdom interactions occurring within the biofilm habitat. We also discuss existing models used to study biofilms. We assess the contribution of the host-mucosa biofilm relationship to gut homeostasis and to diseases. In addition, we describe how host factors can shape the organization, structure and composition of mucosal biofilms, and how biofilms themselves are implicated in a variety of homeostatic and pathological processes in the gut. Future studies characterizing biofilm nature, physical properties, composition and intrinsic communication could shed new light on gut physiology and lead to potential novel therapeutic options for gastrointestinal diseases.

Conjunctival mucous membrane colour as an indicator for the targeted selective treatment of haemonchosis and of the general health status of peri-urban smallholder goats in southern Malawi.

The world's growing population is becoming increasingly centred around large cities, affording opportunities for peri-urban food production. Goats are well-suited to conversion of resources that are available in peri-urban settings into meat and occasionally milk. Haemonchus contortus has been described as "the nemesis of small ruminant production systems in tropical and subtropical regions"; hence control of haemonchosis through planned animal health management affords a pragmatic first step in improving the production efficiency of peri-urban goats. This study of peri-urban goat production investigated the potential value of targeted selective treatment of haemonchosis. 452 peri-urban goat keepers in southern Malawi were visited during three seasonal periods with relevance to the epidemiology of haemonchosis. 622, 599 and 455 individually identified goats were clinically examined during the dry season, the rainy season, and shortly after the end of the rainy season, respectively. Data were recorded for sex, age, weight, conjunctival mucous membrane colour score (FAMACHA©), body condition score (BCS) and faecal worm egg count (FEC); and where possible for pregnancy and lactation status. Animals with pale ocular mucous membranes were treated with 10 mg/kg albendazole, then re-examined 14 days later. Animals with pink mucous membranes, but FECs ≥250 eggs per gram were also re-examined and treated 14 days later. The results show high variability in growth rates deduced from the ages and bodyweights of each of 999 goats at the time of their enrolment. FAMACHA© scores alone were a poor index for the targeted selective treatment of haemonchosis, because they failed to identify too many animals that would have required treatment at different times of year and using different FAMACHA© and FEC cut-offs. Combining the indices of FAMACHA© scores ≥4, body condition scores ≥2, and age >18 months was more reliable in identifying those animals requiring treatment when different epidemiologically-relevant FEC thresholds for different seasons were taken into account. Inclusion of late pregnancy or early lactation status would have resulted in very few animals requiring treatment being missed. The use of conjunctival mucous membrane colour scoring in this way provided a valuable insight of the general health status of the peri-urban goats, to create opportunities for planned animal health management to improve productivity. The efficacy of albendazole treatment was poor, putatively due to drug resistance, or poor drug bioavailability in goats. In summary, our study shows opportunities for better production efficiency in peri-urban goats, and demonstrates the value of simple clinical diagnostic indices as decision support tools in planned animal health management.

γδ T cells in tissue physiology and surveillance.

γδ T cells are a unique T cell subpopulation that are rare in secondary lymphoid organs but enriched in many peripheral tissues, such as the skin, intestines and lungs. By rapidly producing large amounts of cytokines, γδ T cells make key contributions to immune responses in these tissues. In addition to their immune surveillance activities, recent reports have unravelled exciting new roles for γδ T cells in steady-state tissue physiology, with functions ranging from the regulation of thermogenesis in adipose tissue to the control of neuronal synaptic plasticity in the central nervous system. Here, we review the roles of γδ T cells in tissue homeostasis and in surveillance of infection, aiming to illustrate their major impact on tissue integrity, tissue repair and immune protection.

Mucosal Immunity and Liver Metabolism in the Complex Condition of Lactation Insufficiency.

Lactation insufficiency is variously defined and includes the inability to produce milk, not producing enough milk to exclusively meet infant growth requirements, and pathological interruption of lactation (e.g., mastitis). Of women with intent-to-breastfeed, lactation insufficiency has been estimated to affect 38%-44% of newly postpartum women, likely contributing to the nearly 60% of infants that are not breastfed according to the World Health Organization's guidelines. To date, research and clinical practice aimed at improving feeding outcomes have focused on hospital lactation support and education, with laudable results. However, researchers' reports of recent rodent studies concerning fundamental lactation biology have suggested that the underlying pathologies of lactation insufficiency may be more nuanced than is currently appreciated. In this article, we identify mucosal biology of the breast and lactation-specific liver biology as two under-researched aspects of lactation physiology. Specifically, we argue that further scientific inquiry into reproductive state-dependent regulation of immunity in the human breast will reveal insights into novel immune based requirements for healthy lactation. Additionally, our synthesis of the literature supports the hypothesis that the liver is an essential player in lactation-highlighting the potential that pathologies of the liver may also be associated with lactation insufficiency. More research into these biologic underpinnings of lactation is anticipated to provide new avenues to understand and treat lactation insufficiency.

Trefoil Factor Family (TFF) Peptides and Their Diverse Molecular Functions in Mucus Barrier Protection and More: Changing the Paradigm.

Trefoil factor family peptides (TFF1, TFF2, TFF3) are typically co-secreted together with mucins. Tff1 represents a gastric tumor suppressor gene in mice. TFFs are also synthesized in minute amounts in the immune and central nervous systems. In mucous epithelia, they support rapid repair by enhancing cell migration ("restitution") via their weak chemotactic and anti-apoptotic effects. For a long time, as a paradigm, this was considered as their major biological function. Within recent years, the formation of disulfide-linked heterodimers was documented for TFF1 and TFF3, e.g., with gastrokine-2 and IgG Fc binding protein (FCGBP). Furthermore, lectin activities were recognized as enabling binding to a lipopolysaccharide of Helicobacter pylori (TFF1, TFF3) or to a carbohydrate moiety of the mucin MUC6 (TFF2). Only recently, gastric TFF1 was demonstrated to occur predominantly in monomeric forms with an unusual free thiol group. Thus, a new picture emerged, pointing to diverse molecular functions for TFFs. Monomeric TFF1 might protect the gastric mucosa as a scavenger for extracellular reactive oxygen/nitrogen species. Whereas, the TFF2/MUC6 complex stabilizes the inner layer of the gastric mucus. In contrast, the TFF3-FCGBP heterodimer (and also TFF1-FCGBP) are likely part of the innate immune defense of mucous epithelia, preventing the infiltration of microorganisms.

Research Note: Effect of age on the distribution of lymphocytes in the oviduct in Turkey breeder hens.

Considering the lack of research on the local immunity of the reproductive tract in other poultry species than chickens, the aim of this study was to determine the contribution of T and B cell subpopulations in different parts of breeder turkey oviduct mucous membrane with the use of flow cytometry. In addition, the study aimed to establish an impact of bird age and different stages of the egg production cycle on the systemic and local oviduct-related immune system structure. Our study results demonstrated a lower percentage of T lymphocytes in 32-wk turkey hens followed by a successively increasing population of these cells up to the 38th week of bird's life. The results of our study have also shown a similar dependency between birds' age and number of B lymphocytes. In addition, we demonstrated a decrease in the number of immune system cells in the oviduct, blood, and spleen of turkey hens in the late and end laying period. The differences reported in the number of lymphocyte subpopulations in the reproductive system of laying turkey hens at various stages of the production cycle may, to some extent, explain the frequency and periods of increased predilection to the incidence of infectious diseases in birds under field conditions.

Development of human ectocervical tissue models with physiologic endocrine and paracrine signaling†.

There is a shortage of research models that adequately represent the unique mucosal environment of human ectocervix, limiting development of new therapies for treating infertility, infection, or cancer. We developed three microphysiologic human ectocervix models to study hormone action during homeostasis. First, we reconstructed ectocervix using decellularized extracellular matrix scaffolds, which supported cell integration and could be clinically useful. Secondly, we generated organotypic systems consisting of ectocervical explants co-cultured with murine ovaries or cycling exogenous hormones, which mimicked human menstrual cycles. Finally, we engineered ectocervix tissue consisting of tissue-specific stromal-equivalents and fully-differentiated epithelium that mimicked in vivo physiology, including squamous maturation, hormone response, and mucin production, and remained viable for 28 days in vitro. The localization of differentiation-dependent mucins in native and engineered tissue was identified for the first time, which will allow increased efficiency in mucin targeting for drug delivery. In summary, we developed and characterized three microphysiologic human ectocervical tissue models that will be useful for a variety of research applications, including preventative and therapeutic treatments, drug and toxicology studies, and fundamental research on hormone action in a historically understudied tissue that is critical for women's health.

The emerging roles of eosinophils in mucosal homeostasis.

Eosinophils are granulocytes, typically implicated as end-stage effector cells in type-II immune responses. They are capable of producing a wide array of pre-formed molecules which render them with vast potential to influence a wide variety of processes. Nonetheless, eosinophil research has traditionally focused on their role in anti-helminthic responses and pathophysiological processes in type-II immune disorders, such as allergy and asthma, where eosinophilia is a hallmark phenotype. However, a number of key studies over the past decade have placed this restricted view of eosinophil function into question, presenting additional evidence for eosinophils as critical regulators of various homeostatic processes including immune maintenance, organ development, and tissue regeneration.

Clostridioides difficile-Associated Antibiotics Alter Human Mucosal Barrier Functions by Microbiome-Independent Mechanisms.

A clinically relevant risk factor for Clostridioides difficile-associated disease (CDAD) is recent antibiotic treatment. Although broad-spectrum antibiotics have been shown to disrupt the structure of the gut microbiota, some antibiotics appear to increase CDAD risk without being highly active against intestinal anaerobes, suggesting direct nonantimicrobial effects. We examined cell biological effects of antibiotic exposure that may be involved in bacterial pathogenesis using an in vitro germfree human colon epithelial culture model. We found a marked loss of mucosal barrier and immune function with exposure to the CDAD-associated antibiotics clindamycin and ciprofloxacin, distinct from the results of pretreatment with an antibiotic unassociated with CDAD, tigecycline, which did not reduce innate immune or mucosal barrier functions. Importantly, pretreatment with CDAD-associated antibiotics sensitized mucosal barriers to C. difficile toxin activity in primary cell-derived enteroid monolayers. These data implicate commensal-independent gut mucosal barrier changes in the increased risk of CDAD with specific antibiotics and warrant further studies in in vivo systems. We anticipate this work to suggest potential avenues of research for host-directed treatment and preventive therapies for CDAD.

The 1st international symposium on mucosal health in aquaculture - MHA2019.

The 1st International Symposium on Mucosal Health and Aquaculture (MHA2019) was held on 11-13 September 2019 in Oslo, Norway. This was the first platform of its kind that gathered people from academia, R&D institutes, and industry to discuss the state-of-the-art and future directions of mucosal health research in aquaculture. The symposium was divided into four scientific sessions: Session 1, Mucosal structures and functions; Session 2, Mucosal health and nutrition; Session 3, Mucosal health and microbiome; and Session 4, Mucosal health and the changing environment. A plenary talk from a prominent authority in the field opened each session. The papers presented at MHA2019 exemplified the dynamic evolution of the field of mucosal health in aquaculture - from an area solely explored from an immunological aspect about 20 years ago, to the multidisciplinary field it is today. Moreover, papers presented highlighted the complementary application of both classical (e.g., histology) and modern platforms (e.g., omics, artificial intelligence) to characterize mucosal health. The interactive discussion during the meeting underscored the importance of mucosal health research in modern aquaculture and collectively emphasized the role of both fundamental and applied approaches in advancing this timely and highly relevant field. The symposium was organized by Nofima, The Norwegian Institute of Food, Fisheries, and Aquaculture Research, with support from the Research Council of Norway.

Contractile elements and their sympathetic regulations in the pig urinary bladder: a species and regional comparative study.

Contractile behaviour of the urinary bladder and its sympathetic inhibition during storage phases are not well understood. Here, we explore muscularis mucosae (MM) as a predominant mucosal contractile element and the capability of sympathetic nerves to relax detrusor smooth muscle (DSM) or MM. Distribution of α-smooth muscle actin (α-SMA)-immunoreactive cells was compared in pig, human, guinea pig, rat and mouse bladders by immunohistochemistry, while contractility of the bladder mucosa was compared in these species by isometric tension recordings. In pig, human and guinea pig bladders, DSM and MM located in the lamina propria expressed α-SMA immunoreactivity, while both rat and mouse bladders lacked a MM. Consistent with this presence or absence of MM, bladder mucosa of pig, human and guinea pig but not rat and mouse developed spontaneous phasic contractions (SPCs). Distribution of tyrosine hydroxylase (TH)-immunoreactive sympathetic nerve fibres was compared in pig DSM, MM, trigone and urethra, as were their sympathetic nerve-evoked contractile/relaxing responses examined. In pig DSM or MM, where TH-immunoreactive sympathetic fibres exclusively projected to the vasculature, sympathetic relaxations were difficult to demonstrate. In contrast, sympathetic contractions were invariably evoked in pig trigone and urethra where the smooth muscle cells receive TH-immunoreactive sympathetic innervations. Thus, SPCs of bladder mucosa appear to predominantly arise from the MM displaying species differences. Despite the currently accepted concept of sympathetic nerve-mediated DSM relaxation during the storage phase, it is unlikely that neurally released noradrenaline acts on ß-adrenoceptors to relax either DSM or MM due to the anatomical lack of sympathetic innervation.

Engineering drug delivery systems to overcome mucosal barriers for immunotherapy and vaccination.

Mucosal surfaces protect our bodies from pathogens and external irritants using a system of biological barriers. Overcoming these barriers is a significant drug delivery challenge, particularly for immunotherapies that aim to modulate the local immune response. Reaching local lymphoid tissues and draining lymph nodes (LNs) requires crossing the mucus mesh, mucosal epithelium, and either targeting M cells covering lymphoid tissues or utilizing lymphatic transport that shuttles molecules and particulates from the periphery to the LN. We first highlight the barrier properties of mucus and mucosal epithelium, and the function of the mucosal immune system. We then dive into existing drug delivery technologies that have been engineered to overcome each of these barriers. We particularly focus on novel strategies for targeting lymphoid tissues, which has been shown to enhance immunotherapies and vaccinations, via directly targeting LNs, lymphatic vessels, and M cells that transport samples of mucosal content to the lymphoid tissues.

Personal and Clinical Vaginal Lubricants: Impact on Local Vaginal Microenvironment and Implications for Epithelial Cell Host Response and Barrier Function.

BACKGROUND: A majority of US women report past use of vaginal lubricants to enhance the ease and comfort of intimate sexual activities. Lubricants are also administered frequently in clinical practice. We sought to investigate if hyperosmolar lubricants are toxic to the vaginal mucosal epithelia. METHODS: We tested a panel of commercially available lubricants across a range of osmolalities in human monolayer vaginal epithelial cell (VEC) culture and a robust 3-dimensional (3-D) VEC model. The impact of each lubricant on cellular morphology, cytotoxicity, barrier targets, and the induction of inflammatory mediators was examined. Conceptrol, containing nonoxynol-9, was used as a cytotoxicity control. RESULTS: We observed a loss of intercellular connections, and condensation of chromatin, with increasing lubricant osmolality. EZ Jelly, K-Y Jelly, Astroglide, and Conceptrol induced cytotoxicity in both models at 24 hours. There was a strong positive correlation (r = 0.7326) between lubricant osmolality and cytotoxicity in monolayer VECs, and cell viability was reduced in VECs exposed to all the lubricants tested for 24 hours, except McKesson. Notably, select lubricants altered cell viability, barrier targets, and inflammatory mediators in 3-D VECs. CONCLUSIONS: These findings indicate that hyperosmolar lubricants alter VEC morphology and are selectively cytotoxic, inflammatory, and barrier disrupting in the 3-D VEC model.