Growth factors and growth factor gene therapies for treating chronic wounds.

Chronic wounds are an unmet clinical need affecting millions of patients globally, and current standards of care fail to consistently promote complete wound closure and prevent recurrence. Disruptions in growth factor signaling, a hallmark of chronic wounds, have led researchers to pursue growth factor therapies as potential supplements to standards of care. Initial studies delivering growth factors in protein form showed promise, with a few formulations reaching clinical trials and one obtaining clinical approval. However, protein-form growth factors are limited by instability and off-target effects. Gene therapy offers an alternative approach to deliver growth factors to the chronic wound environment, but safety concerns surrounding gene therapy as well as efficacy challenges in the gene delivery process have prevented clinical translation. Current growth factor delivery and gene therapy approaches have primarily used single growth factor formulations, but recent efforts have aimed to develop multi-growth factor approaches that are better suited to address growth factor insufficiencies in the chronic wound environment, and these strategies have demonstrated improved efficacy in preclinical studies. This review provides an overview of chronic wound healing, emphasizing the need and potential for growth factor therapies. It includes a summary of current standards of care, recent advances in growth factor, cell-based, and gene therapy approaches, and future perspectives for multi-growth factor therapeutics.

Micronutrients at Supplemental Levels, Tight Junctions and Epithelial Barrier Function: A Narrative Review.

Disease modifiers, whether from cancer, sepsis, systemic inflammation, or microbial pathogens, all appear to induce epithelial barrier leak, with induced changes of the Tight Junctional (TJ) complex being pivotal to the process. This leak-and the ensuant breakdown of compartmentation-plays a central role in disease morbidity on many levels. Accumulation of lung water in the luminal compartment of airways was a major driver of morbidity and mortality in COVID-19 and is an excellent example of the phenomenon. Increasing awareness of the ability of micronutrients to improve basal barrier function and reduce barrier compromise in pathophysiology may prove to be a low-cost, safe, and easily administered prophylactic and/or therapeutic option amenable to large populations. The growing appreciation of the clinical utility of supplemental doses of Vitamin D in COVID-19 is but one example. This narrative review is intended to propose a general theory on how and why micronutrients-at levels above normal dietary intake-successfully remodel TJs and improve barrier function. It discusses the key difference between dietary/Recommended Daily Allowance (RDA) levels of micronutrients versus supplemental levels, and why the latter are needed in disease situations. It advances a hypothesis for why signal transduction regulation of barrier function may require these higher supplemental doses to achieve the TJ remodeling and other barrier element changes that are clinically beneficial.

Calcitriol modifies tight junctions, improves barrier function, and reduces TNF-α-induced barrier leak in the human lung-derived epithelial cell culture model, 16HBE 14o.

Using the 16HBE 14o- human airway epithelial cell culture model, calcitriol (Vitamin D) was shown to improve barrier function by two independent metrics - increased transepithelial electrical resistance (TER) and reduced transepithelial diffusion of 14 C-D-mannitol (Jm ). Both effects were concentration dependent and active out to 168 h post-treatment. Barrier improvement associated with changes in the abundance of specific tight junctional (TJ) proteins in detergent-soluble fractions, most notably decreased claudin-2. TNF-α-induced compromise of barrier function could be attenuated by calcitriol with a concentration dependence similar to that observed for improvement of control barrier function. TNF-α-induced increases in claudin-2 were partially reversed by calcitriol. The ERK 1,2 inhibitor, U0126, itself improved 16HBE barrier function indicating MAPK pathway regulation of 16HBE barrier function. Calcitriol's action was additive to the effect of U0126 in reducing TNF- α -induced barrier compromise, suggesting that calcitriol may be acting through a non-ERK pathway in its blunting of TNF- α - induced barrier compromise. This was supported by calcitriol being without effect on pERK levels elevated by the action of TNF-α. Lack of effect of TNF- α on the death marker, caspase-3, and the inability of calcitriol to decrease the elevated LC3B II level caused by TNF-α, suggest that calcitriol's barrier improvement does not involve a cell death pathway. Calcitriol's improvement of control barrier function was not additive to barrier improvement induced by retinoic acid (Vitamin A). Calcitriol improvement and protection of airway barrier function could in part explain Vitamin D's reported clinical efficacy in COVID-19 and other airway diseases.

The multiphasic TNF-α-induced compromise of Calu-3 airway epithelial barrier function.

Purpose: Airway epithelial barrier leak and the involvement of proinflammatory cytokines play a key role in a variety of diseases. This study evaluates barrier compromise by the inflammatory mediator Tumor Necrosis Factor-α (TNF-α) in the human airway epithelial Calu-3 model. Methods: We examined the effects of TNF-α on barrier function in Calu-3 cell layers using Transepithelial Electrical Resistance (TER) and transepithelial diffusion of radiolabeled probe molecules. Western immunoblot analyses of tight junctional (TJ) proteins in detergent soluble fractions were performed. Results: TNF-α dramatically reduced TER and increased paracellular permeability of both 14C-D-mannitol and the larger 5 kDa probe, 14C-inulin. A time course of the effects shows two separate actions on barrier function. An initial compromise of barrier function occurs 2-4 hours after TNF-α exposure, followed by complete recovery of barrier function by 24 hrs. Beginning 48 hrs. post-exposure, a second more sustained barrier compromise ensues, in which leakiness persists through 144 hrs. There were no changes in TJ proteins observed at 3 hrs. post exposure, but significant increases in claudins-2, -3, -4, and -5, as well as a decrease in occludin were seen at 72 hrs. post TNF-α exposure. Both the 2-4 hr. and the 72 hr. TNF-α induced leaks are shown to be mediated by the ERK signaling pathway. Conclusion: TNF-α induced a multiphasic transepithelial leak in Calu-3 cell layers that was shown to be ERK mediated, as well as involve changes in the TJ complex. The micronutrients, retinoic acid and calcitriol, were effective at reducing this barrier compromise caused by TNF-α. The significance of these results for airway disease and for COVID-19 specifically are discussed.

Exploring the failing right ventricle in pulmonary hypertension by cardiac magnetic resonance: An in vivo study utilizing Macitentan.

Cardiac magnetic resonance (CMR) imaging is used to assess the right ventricle (RV) of pulmonary hypertensive (PH) patients and more recently to track changes in response to therapy. We wished to investigate if repeat CMRs could be used to assess ventricular changes in the Sugen 5416 hypoxic (Su/Hx) rat model of PH treated with the dual endothelin receptor antagonist Macitentan. Male Sprague Dawley Su/Hx rats were dosed for 3 weeks with either vehicle or Macitentan (30 mg/kg) daily, control rats received only vehicle. All rats underwent three CMR scans; before treatment, 2 weeks into treatment, and end of the study. A separate group of Su/Hx and control rats, treated as above, underwent terminal hemodynamic measurements. Using terminal and CMR measurements, Macitentan was found to lower RV systolic pressure pulmonary artery remodeling and increase RV ejection fraction but not change RV hypertrophy (RVH). Repeat CMRs determined that Su/Hx rats treated with Macitentan had significantly reversed RVH via reducing RV mass as well as reducing elevated left ventricular eccentricity index; reductions in RV mass were also observed in Su/Hx vehicle rats exposed to normoxic conditions. We have demonstrated that repeat CMRs can be used to assess the volume and structural changes in the ventricles of the Su/Hx rat model. Using repeat CMRs has allowed us to build a more complete picture of the response of the RV and the left ventricle to treatment. It is unknown if these effects are a consequence of direct action on the RV or secondary to improvements in the lung vasculature.

Micronutrient Improvement of Epithelial Barrier Function in Various Disease States: A Case for Adjuvant Therapy.

The published literature makes a very strong case that a wide range of disease morbidity associates with and may in part be due to epithelial barrier leak. An equally large body of published literature substantiates that a diverse group of micronutrients can reduce barrier leak across a wide array of epithelial tissue types, stemming from both cell culture as well as animal and human tissue models. Conversely, micronutrient deficiencies can exacerbate both barrier leak and morbidity. Focusing on zinc, Vitamin A and Vitamin D, this review shows that at concentrations above RDA levels but well below toxicity limits, these micronutrients can induce cell- and tissue-specific molecular-level changes in tight junctional complexes (and by other mechanisms) that reduce barrier leak. An opportunity now exists in critical care-but also medical prophylactic and therapeutic care in general-to consider implementation of select micronutrients at elevated dosages as adjuvant therapeutics in a variety of disease management. This consideration is particularly pointed amidst the COVID-19 pandemic.

SARS-CoV-2 Envelope (E) protein interacts with PDZ-domain-2 of host tight junction protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2-induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway and/or gastrointestinal barrier damage and mitigate virus spread.

Best Practices in the Development and Validation of Physiologically Based Biopharmaceutics Modeling. A Workshop Summary Report.

This workshop report summarizes the proceedings of Day 2 of a three-day workshop on "Current State and Future Expectations of Translational Modeling Strategies toSupportDrug Product Development, Manufacturing Changes and Controls". From a drug product quality perspective, physiologically based biopharmaceutics modeling (PBBM) is a tool to link variations in the drug product quality attributes to in vivo outcomes enabling the establishment of clinically relevant drug product specifications (CRDPS). Day 2 of the workshop focused on best practices in developing, verifying and validating PBBM. This manuscript gives an overview of podium presentations and summarizes breakout (BO) session discussions related to (1) challenges and opportunities for using PBBM to assess the clinical impact of formulation and manufacturing changes on the in vivo performance of a drug product, (2) best practices to account for parameter uncertainty and variability during model development, (3) best practices in the development, verification and validation of PBBM and (4) opportunities and knowledge gaps related to leveraging PBBM for virtual bioequivalence simulations.

Retinoic acid improves baseline barrier function and attenuates TNF-α-induced barrier leak in human bronchial epithelial cell culture model, 16HBE 14o.

Retinoic acid (RA) has been shown to improve epithelial and endothelial barrier function and development and even suppress damage inflicted by inflammation on these barriers through regulating immune cell activity. This paper thus sought to determine whether RA could improve baseline barrier function and attenuate TNF-α-induced barrier leak in the human bronchial epithelial cell culture model, 16HBE14o- (16HBE). We show for the first time that RA increases baseline barrier function of these cell layers indicated by an 89% increase in transepithelial electrical resistance (TER) and 22% decrease in 14C-mannitol flux. A simultaneous, RA-induced 70% increase in claudin-4 attests to RA affecting the tight junctional (TJ) complex itself. RA was also effective in alleviating TNF-α-induced 16HBE barrier leak, attenuating 60% of the TNF-α-induced leak to 14C-mannitol and 80% of the leak to 14C-inulin. Interleukin-6-induced barrier leak was also reduced by RA. Treatment of 16HBE cell layers with TNF-α resulted in dramatic decrease in immunostaining for occludin and claudin-4, as well as a downward "band-shift" in occludin Western immunoblots. The presence of RA partially reversed TNF-α's effects on these select TJ proteins. Lastly, RA completely abrogated the TNF-α-induced increase in ERK-1,2 phosphorylation without significantly decreasing the TNF-driven increase in total ERK-1,2. This study suggests RA could be effective as a prophylactic agent in minimizing airway barrier leak and as a therapeutic in preventing leak triggered by inflammatory cascades. Given the growing literature suggesting a "cytokine storm" may be related to COVID-19 morbidity, RA may be a useful adjuvant for use with anti-viral therapies.

Epithelial barrier function properties of the 16HBE14o- human bronchial epithelial cell culture model.

The human bronchial epithelial cell line, 16HBE14o- (16HBE), is widely used as a model for respiratory epithelial diseases and barrier function. During differentiation, transepithelial electrical resistance (TER) increased to approximately 800 Ohms × cm2, while 14C-d-mannitol flux rates (Jm) simultaneously decreased. Tight junctions (TJs) were shown by diffusion potential studies to be anion-selective with PC1/PNa = 1.9. Transepithelial leakiness could be induced by the phorbol ester, protein kinase C (PKC) activator, 12-O-tetradecanoylphorbol-13-acetate (TPA), and the proinflammatory cytokine, tumor necrosis factor-α (TNF-α). Basal barrier function could not be improved by the micronutrients, zinc, or quercetin. Of methodological significance, TER was observed to be more variable and to spontaneously, significantly decrease after initial barrier formation, whereas Jm did not significantly fluctuate or increase. Unlike the strong inverse relationship between TER and Jm during differentiation, differentiated cell layers manifested no relationship between TER and Jm. There was also much greater variability for TER values compared with Jm. Investigating the dependence of 16HBE TER on transcellular ion conductance, inhibition of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) chloride channel with GlyH-101 produced a large decrease in short-circuit current (Isc) and a slight increase in TER, but no significant change in Jm. A strong temperature dependence was observed not only for Isc, but also for TER. In summary, research utilizing 16HBE as a model in airway barrier function studies needs to be aware of the complexity of TER as a parameter of barrier function given the influence of CFTR-dependent transcellular conductance on TER.

Current status and future opportunities for incorporation of dissolution data in PBPK modeling for pharmaceutical development and regulatory applications: OrBiTo consortium commentary.

In vitro dissolution experiments are used to qualitatively assess the impact of formulation composition and process changes on the drug dosage form performance. However, the use of dissolution data to quantitatively predict changes in the absorption profile remains limited. Physiologically-based Pharmacokinetic(s) (PBPK) models facilitate incorporation of in vitro dissolution experiments into mechanistic oral absorption models to predict in vivo oral formulation performance, and verify if the drug product dissolution method is biopredictive or clinically relevant. Nevertheless, a standardized approach for using dissolution data within PBPK models does not yet exist and the introduction of dissolution data in PBPK relies on a case by case approach which accommodates from differences in release mechanism and limitations to drug absorption. As part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project a cross-work package was set up to gather a realistic understanding of various approaches used and their areas of applications. This paper presents the approaches shared by academic and industrial scientists through the OrBiTo project to integrate dissolution data within PBPK software to improve the prediction accuracy of oral formulations in vivo. Some general recommendations regarding current use and future improvements are also provided.

IMI - Oral biopharmaceutics tools project - Evaluation of bottom-up PBPK prediction success part 4: Prediction accuracy and software comparisons with improved data and modelling strategies.

Oral drug absorption is a complex process depending on many factors, including the physicochemical properties of the drug, formulation characteristics and their interplay with gastrointestinal physiology and biology. Physiological-based pharmacokinetic (PBPK) models integrate all available information on gastro-intestinal system with drug and formulation data to predict oral drug absorption. The latter together with in vitro-in vivo extrapolation and other preclinical data on drug disposition can be used to predict plasma concentration-time profiles in silico. Despite recent successes of PBPK in many areas of drug development, an improvement in their utility for evaluating oral absorption is much needed. Current status of predictive performance, within the confinement of commonly available in vitro data on drugs and formulations alongside systems information, were tested using 3 PBPK software packages (GI-Sim (ver.4.1), Simcyp® Simulator (ver.15.0.86.0), and GastroPlus™ (ver.9.0.00xx)). This was part of the Innovative Medicines Initiative (IMI) Oral Biopharmaceutics Tools (OrBiTo) project. Fifty eight active pharmaceutical ingredients (APIs) were qualified from the OrBiTo database to be part of the investigation based on a priori set criteria on availability of minimum necessary information to allow modelling exercise. The set entailed over 200 human clinical studies with over 700 study arms. These were simulated using input parameters which had been harmonised by a panel of experts across different software packages prior to conduct of any simulation. Overall prediction performance and software packages comparison were evaluated based on performance indicators (Fold error (FE), Average fold error (AFE) and absolute average fold error (AAFE)) of pharmacokinetic (PK) parameters. On average, PK parameters (Area Under the Concentration-time curve (AUC0-tlast), Maximal concentration (Cmax), half-life (t1/2)) were predicted with AFE values between 1.11 and 1.97. Variability in FEs of these PK parameters was relatively high with AAFE values ranging from 2.08 to 2.74. Around half of the simulations were within the 2-fold error for AUC0-tlast and around 90% of the simulations were within 10-fold error for AUC0-tlast. Oral bioavailability (Foral) predictions, which were limited to 19 APIs having intravenous (i.v.) human data, showed AFE and AAFE of values 1.37 and 1.75 respectively. Across different APIs, AFE of AUC0-tlast predictions were between 0.22 and 22.76 with 70% of the APIs showing an AFE > 1. When compared across different formulations and routes of administration, AUC0-tlast for oral controlled release and i.v. administration were better predicted than that for oral immediate release formulations. Average predictive performance did not clearly differ between software packages but some APIs showed a high level of variability in predictive performance across different software packages. This variability could be related to several factors such as compound specific properties, the quality and availability of information, and errors in scaling from in vitro and preclinical in vivo data to human in vivo behaviour which will be explored further. Results were compared with previous similar exercise when the input data selection was carried by the modeller rather than a panel of experts on each in vitro test. Overall, average predictive performance was increased as reflected in smaller AAFE value of 2.8 as compared to AAFE value of 3.8 in case of previous exercise.

Understanding longitudinal biventricular structural and functional changes in a pulmonary hypertension Sugen-hypoxia rat model by cardiac magnetic resonance imaging.

Cardiac magnetic resonance-derived ventricular variables are predictive of mortality in pulmonary arterial hypertension. Rodent models which emphasize ventricular function, allowing serial monitoring, are needed to identify pathophysiological features and novel therapies for pulmonary arterial hypertension. We investigated longitudinal changes in the Sugen-hypoxia model during disease progression. Sprague Dawley rats (n = 32) were divided into two groups. (1) Sugen-hypoxia: a dose of subcutaneous Sugen-5416 and placed in hypobaric hypoxia for two weeks followed by normoxia for three weeks. (2) Normoxia: maintained at normal pressure for five weeks. Rats were examined at five or eight weeks with right-heart catheter, cardiac magnetic resonance, and autopsy. Compared to normoxic controls (23.9 ± 4.1 mmHg), right ventricular systolic pressure was elevated in Sugen-hypoxia rats at five and eight weeks (40.9 ± 15.5 mmHg, p = 0.026; 48.9 ± 9.6 mmHg, p = 0.002). Right ventricular end-systolic volume index was increased in eight weeks Sugen-hypoxia (0.28 ± 0.04 µlcm-2, p = 0.003) compared to normoxic controls (0.18 ±0.03 mlcm-2). There was progressive dilatation of the right ventricular at eight weeks Sugen-hypoxia compared to normoxic controls (0.75 ± 0.13 µlcm-2 vs 0.56 ± 0.1 µlcm-2 p = 0.02). Ventricle mass index by cardiac magnetic resonance at five weeks (0.34 ± 0.06, p = 0.003) and eight weeks Sugen-hypoxia (0.34 ± 0.06, p = 0.002) were higher than normoxic controls (0.21 ± 0.04). Stroke volume, right ventricular ejection fraction, and left ventricular variables were preserved in Sugen-hypoxia. Ventricular changes during the course of illness in a pulmonary arterial hypertension rodent model can be examined by cardiac magnetic resonance. These changes including right ventricular hypertrophy and subsequent dilatation are similar to those seen in pulmonary arterial hypertension patients. Despite the persisting pulmonary hypertension, there are features of adaptive cardiac remodeling through the study duration.

SARS-CoV-2 Envelope (E) Protein Interacts with PDZ-Domain-2 of Host Tight Junction Protein ZO1.

Newly emerged SARS-CoV-2 is the cause of an ongoing global pandemic leading to severe respiratory disease in humans. SARS-CoV-2 targets epithelial cells in the respiratory tract and lungs, which can lead to amplified chloride secretion and increased leak across epithelial barriers, contributing to severe pneumonia and consolidation of the lungs as seen in many COVID-19 patients. There is an urgent need for a better understanding of the molecular aspects that contribute to SARS-CoV-2 induced pathogenesis and for the development of approaches to mitigate these damaging pathologies. The multifunctional SARS-CoV-2 Envelope (E) protein contributes to virus assembly/egress, and as a membrane protein, also possesses viroporin channel properties that may contribute to epithelial barrier damage, pathogenesis, and disease severity. The extreme C-terminal (ECT) sequence of E also contains a putative PDZ-domain binding motif (PBM), similar to that identified in the E protein of SARS-CoV-1. Here, we screened an array of GST-PDZ domain fusion proteins using either a biotin-labeled WT or mutant ECT peptide from the SARS-CoV-2 E protein. Notably, we identified a singular specific interaction between the WT E peptide and the second PDZ domain of human Zona Occludens-1 (ZO1), one of the key regulators of TJ formation/integrity in all epithelial tissues. We used homogenous time resolve fluorescence (HTRF) as a second complementary approach to further validate this novel modular E-ZO1 interaction. We postulate that SARS-CoV-2 E interacts with ZO1 in infected epithelial cells, and this interaction may contribute, in part, to tight junction damage and epithelial barrier compromise in these cell layers leading to enhanced virus spread and severe respiratory dysfunction that leads to morbidity. Prophylactic/therapeutic intervention targeting this virus-host interaction may effectively reduce airway barrier damage and mitigate virus spread.

Improving Transient Transfection Efficiency in a Differentiated, Polar Epithelial Cell Layer.

Polar, differentiated epithelial cell culture models (especially at confluence) are difficult to transfect compared with the higher transfection efficiencies that one obtains with relatively less differentiated, nonpolar cell culture models. Here, we sought to develop a strategy to enhance the efficiency of transfecting polar, differentiated epithelial cells. We found that chemically abrading the differentiated CACO-2 human intestinal epithelial cell layer by a trypsin and EDTA pretreatment (before the use of detergent-like transfection reagents) dramatically improved transfection efficiency in this polar, differentiated model. Although this treatment did improve the transfection efficiency, it also induced leakiness in the epithelial barrier by both opening tight junctional complexes and by creating holes in the cell layer because of low-level cell death and detachment. Thus, this approach to enhance the transfection efficiency of polar, differentiated cells will be useful for assessment of the effect of the transfected/expressed protein on (re)formation of an epithelial barrier rather than on a functional barrier itself.

Intestinal barrier tightening by a cell-penetrating antibody to Bin1, a candidate target for immunotherapy of ulcerative colitis.

Patients afflicted with ulcerative colitis (UC) are at increased risk of colorectal cancer. While its causes are not fully understood, UC is associated with defects in colonic epithelial barriers that sustain inflammation of the colon mucosa caused by recruitment of lymphocytes and neutrophils into the lamina propria. Based on genetic evidence that attenuation of the bridging integrator 1 (Bin1) gene can limit UC pathogenicity in animals, we have explored Bin1 targeting as a therapeutic option. Early feasibility studies in the dextran sodium sulfate mouse model of experimental colitis showed that administration of a cell-penetrating Bin1 monoclonal antibody (Bin1 mAb 99D) could prevent lesion formation in the colon mucosa in part by preventing rupture of lymphoid follicles. In vivo administration of Bin1 mAb altered tight junction protein expression and cecal barrier function. Strikingly, electrophysiology studies in organ cultures showed that Bin1 mAb could elevate resistance and lower 14 C-mannitol leakage across the cecal mucosa, consistent with a direct strengthening of colonic barrier function. Transcriptomic analyses of colitis tissues highlighted altered expression of genes involved in circadian rhythm, lipid metabolism, and inflammation, with a correction of the alterations by Bin1 mAb treatment to patterns characteristic of normal tissues. Overall, our results suggest that Bin1 mAb protects against UC by directly improving colonic epithelial barrier function to limit gene expression and cytokine programs associated with colonic inflammation.

Accuracy of phase-contrast velocimetry in systems with skewed intravoxel velocity distributions.

Phase contrast velocimetry (PCV) has been widely used to investigate flow properties in numerous systems. Several authors have reported errors in velocity measurements and have speculated on the sources, which have ranged from eddy current effects to acceleration artefacts. An often overlooked assumption in the theory of PCV, which may not be met in complex or unsteady flows, is that the intravoxel displacement distributions (propagators) are symmetric. Here, the effect of the higher moments of the displacement distribution (variance, skewness and kurtosis) on the accuracy of PCV is investigated experimentally and theoretically. Phase and propagator measurements are performed on tailored intravoxel distributions, achieved using a simple phantom combined with a single large voxel. Asymmetric distributions (Skewness ≠ 0) are shown to generate important phase measurement errors that lead to significant velocimetry errors. Simulations of the phase of the spin vector sum, based on experimentally measured propagators, are shown to quantitatively reproduce the relationship between measured phase and experimental parameters. These allow relating the observed velocimetry errors to a discrepancy between the average phase of intravoxel spins considered in PCV theory and the vector phase actually measured by a PFG experiment. A theoretical expression is derived for PCV velocimetry errors as a function of the moments of the displacement distribution. Positively skewed distributions result in an underestimation of the true mean velocity, while negatively skewed distributions result in an overestimation. The magnitude of these errors is shown to increase with the variance and decrease with the kurtosis of the intravoxel displacement distribution.

Spontaneous and cytokine-induced hole formation in epithelial cell layers: Implications for barrier function studies with the gingival cell culture, Gie-3B11, and other epithelial models.

Epithelial barrier function studies often attribute alterations in barrier function to induced changes in tight junctional (TJ) complexes. The occurrence of spontaneous and cytokine-induced, focal cell detachment in cell layers of the human gingival epithelial cell line, Gie-3B11, highlights the danger of this assumption without confirmatory experimentation. Gie-3B11 cell layers manifest morphological polarity, TJ complexes and barrier function after confluence but fail to then maintain a stable epithelial barrier. Transepithelial electrical resistance rises to over 100 ohms x cm2 a few days after seeding cell layers at a confluent density, but then spontaneously declines, with simultaneous, inverse changes in transepithelial 14C-D-mannitol diffusion rates. This barrier decline correlates with the appearance of focal cell detachment/hole formation in cell layers. Both barrier compromise (decreased electrical resistance; increased 14C-D-mannitol leak) and hole formation are accelerated and exaggerated by exposing cell layers to proinflammatory cytokines. Both are inhibited by increasing the basal-lateral medium compartment volume, suggesting that cell layers are secreting factor(s) across their basal-lateral surfaces that are causal to hole formation. The molecular mechanism of cell death/detachment here is not as significant as the implications of hole formation for the correct interpretation of barrier function studies. Barrier changes in any epithelial model should be attributed to induced changes in TJ complexes only after thorough investigation.

Improvement of Human-Oral-Epithelial-Barrier Function and of Tight Junctions by Micronutrients.

The oral epithelium represents a major interface between an organism and its external environment. Improving this barrier at the molecular level can provide an organism added protection from microbial-based diseases. Barrier function of the Gie-3B11-human-gingival-epithelial-cell-culture model is enhanced by the micronutrients zinc, quercetin, retinoic acid, and acetyl-11-keto-ß-boswellic acid, as observed by a concentration-dependent increase in transepithelial electrical resistance and a decrease in transepithelial 14C-d-mannitol permeability. With this improvement of tight-junction (TJ)-barrier function (reduced leak) comes a pattern of micronutrient-induced changes in TJ claudin abundance that is specific to each individual micronutrient, along with changes in claudin subcellular localization. These micronutrients were effective not only when administered to both cell surfaces simultaneously but also when administered to the apical surface alone, the surface to which the micronutrients would be presented in routine clinical use. The biomedical implications of micronutrient enhancement of the oral-epithelial barrier are discussed.

The Host Microbiome Regulates and Maintains Human Health: A Primer and Perspective for Non-Microbiologists.

Humans consider themselves discrete autonomous organisms, but recent research is rapidly strengthening the appreciation that associated microorganisms make essential contributions to human health and well being. Each person is inhabited and also surrounded by his/her own signature microbial cloud. A low diversity of microorganisms is associated with a plethora of diseases, including allergy, diabetes, obesity, arthritis, inflammatory bowel diseases, and even neuropsychiatric disorders. Thus, an interaction of microorganisms with the host immune system is required for a healthy body. Exposure to microorganisms from the moment we are born and appropriate microbiome assembly during childhood are essential for establishing an active immune system necessary to prevent disease later in life. Exposure to microorganisms educates the immune system, induces adaptive immunity, and initiates memory B and T cells that are essential to combat various pathogens. The correct microbial-based education of immune cells may be critical in preventing the development of autoimmune diseases and cancer. This review provides a broad overview of the importance of the host microbiome and accumulating knowledge of how it regulates and maintains a healthy human system. Cancer Res; 77(8); 1783-812. ©2017 AACR.