Epithelial Uptake Research Articles

Microvesicle-Mediated Communication Within the Alveolar Space: Mechanisms of Uptake by Epithelial Cells and Alveolar Macrophages.

Intra-alveolar microvesicles (MVs) are important mediators of inter-cellular communication within the alveolar space, and are key components in the pathophysiology of lung inflammation such as acute respiratory distress syndrome (ARDS). Despite the abundance of data detailing the pro-inflammatory effects of MVs, it remains unclear how MVs interact or signal with target cells in the alveolus. Using both in vivo and in vitro alveolar models, we analyzed the dynamics of MV uptake by resident alveolar cells: alveolar macrophages and epithelial cells. Under resting conditions, the overwhelming majority of MVs were taken up by alveolar macrophages. However, following lipopolysaccharide (LPS)-mediated inflammation, epithelial cells internalized significantly more MVs (p<0.01) whilst alveolar macrophage internalization was significantly reduced (p<0.01). We found that alveolar macrophages adopted a pro-inflammatory phenotype after internalizing MVs under resting conditions, but reduction of MV uptake following LPS pre-treatment was associated with loss of inflammatory phenotype. Instead, MVs induced significant epithelial cell inflammation following LPS pre-treatment, when MV internalization was most significant. Using pharmacological inhibitors, we interrogated the mechanisms of MV internalization to identify which endocytic pathways and cell surface receptors are involved. We demonstrated that epithelial cells are exclusively dependent on the clathrin and caveolin dependent endocytotic pathway, whereas alveolar macrophage uptake may involve a significant phagocytic component. Furthermore, alveolar macrophages predominantly engulf MVs via scavenger receptors whilst, epithelial cells internalize MVs via a phosphatidylserine/integrin receptor mediated pathway (specifically alpha V beta III), which can be inhibited with phosphatidylserine-binding protein (i.e. annexin V). In summary, we have undertaken a comprehensive evaluation of MV internalization within the alveolar space. Our results demonstrate that different environmental conditions can modulate MV internalization, with inflammatory stimuli strongly enhancing epithelial cell uptake of MVs and inducing epithelial cell activation. Our data reveal the unique mechanisms by which alveolar macrophages and epithelial cells internalize MVs thereby elucidating how MVs exert their pathophysiological effect during lung inflammation and injury. As MVs are potential novel therapeutic targets in conditions such as ARDS, these data provide crucial insights into the dynamics of MV-target cell interactions and highlight potential avenues for researchers to modulate and inhibit their pro-inflammatory actions within the alveolar space.

Protease inhibitor Camostat Mesyalte blocks wild type SARS-CoV-2 and D614G viral entry in human engineered miniature lungs

The catastrophic global effects of the SARS-CoV-2 pandemic highlight the need to develop novel therapeutics strategies to prevent and treat viral infections of the respiratory tract. To enable this work, we need scalable, affordable, and physiologically relevant models of the human lung, the primary organ involved in the pathogenesis of COVID-19. To date, most COVID-19 in vitro models rely on platforms such as cell lines and organoids. While 2D and 3D models have provided important insights, human distal lung models that can model epithelial viral uptake have yet to be established. We hypothesized that by leveraging techniques of whole organ engineering and directed differentiation of induced pluripotent stem cells (iPSC) we could model human distal lung epithelium, examine viral infection at the tissue level in real time, and establish a platform for COVID-19 related research ex vivo. In the present study, we used type 2 alveolar epithelial cells (AT2) derived from human iPSCs to repopulate whole rat lung acellular scaffolds and maintained them in extended biomimetic organ culture for 30 days to induce the maturation of distal lung epithelium. We observed emergence of a mixed type 1 and type 2 alveolar epithelial phenotype during tissue formation. When exposing our system to a pseudotyped lentivirus containing the spike of wildtype SARS-CoV-2 and the more virulent D614G, we observed progression of the infection in real time. We then found that the protease inhibitor Camostat Mesyalte significantly reduced viral transfection in distal lung epithelium. In summary, our data show that a mature human distal lung epithelium can serve as a novel moderate throughput research platform to examine viral infection and to evaluate novel therapeutics ex vivo.

Human albumin enhances the pathogenic potential of Candida glabrata on vaginal epithelial cells.

The opportunistic pathogen Candida glabrata is the second most frequent causative agent of vulvovaginal candidiasis (VVC), a disease that affects 70-75% of women at least once during their life. However, C. glabrata is almost avirulent in mice and normally incapable of inflicting damage to vaginal epithelial cells in vitro. We thus proposed that host factors present in vivo may influence C. glabrata pathogenicity. We, therefore, analyzed the impact of albumin, one of the most abundant proteins of the vaginal fluid. The presence of human, but not murine, albumin dramatically increased the potential of C. glabrata to damage vaginal epithelial cells. This effect depended on macropinocytosis-mediated epithelial uptake of albumin and subsequent proteolytic processing. The enhanced pathogenicity of C. glabrata can be explained by a combination of beneficial effects for the fungus, which includes an increased access to iron, accelerated growth, and increased adhesion. Screening of C. glabrata deletion mutants revealed that Hap5, a key regulator of iron homeostasis, is essential for the albumin-augmented damage potential. The albumin-augmented pathogenicity was reversed by the addition of iron chelators and a similar increase in pathogenicity was shown by increasing the iron availability, confirming a key role of iron. Accelerated growth not only led to higher cell numbers, but also to increased fungal metabolic activity and oxidative stress resistance. Finally, the albumin-driven enhanced damage potential was associated with the expression of distinct C. glabrata virulence genes. Transcriptional responses of the epithelial cells suggested an unfolded protein response (UPR) and ER-stress responses combined with glucose starvation induced by fast growing C. glabrata cells as potential mechanisms by which cytotoxicity is mediated.Collectively, we demonstrate that albumin augments the pathogenic potential of C. glabrata during interaction with vaginal epithelial cells. This suggests a role for albumin as a key player in the pathogenesis of VVC.

Microbial Transglutaminase Is a Very Frequently Used Food Additive and Is a Potential Inducer of Autoimmune/Neurodegenerative Diseases.

Microbial transglutaminase (mTG) is a heavily used food additive and its industrial transamidated complexes usage is rising rapidly. It was classified as a processing aid and was granted the GRAS (generally recognized as safe) definition, thus escaping full and thorough toxic and safety evaluations. Despite the manufacturers claims, mTG or its cross-linked compounds are immunogenic, pathogenic, proinflammatory, allergenic and toxic, and pose a risk to public health. The enzyme is a member of the transglutaminase family and imitates the posttranslational modification of gluten, by the tissue transglutaminase, which is the autoantigen of celiac disease. The deamidated and transamidated gliadin peptides lose their tolerance and induce the gluten enteropathy. Microbial transglutaminase and its complexes increase intestinal permeability, suppresses enteric protective pathways, enhances microbial growth and gliadin peptide’s epithelial uptake and can transcytose intra-enterocytically to face the sub-epithelial immune cells. The present review updates on the potentially detrimental side effects of mTG, aiming to interest the scientific community, induce food regulatory authorities’ debates on its safety, and protect the public from the mTG unwanted effects.

Oral Core-Shell Nanoparticles Embedded in Hydrogel Microspheres for the Efficient Site-Specific Delivery of Magnolol and Enhanced Antiulcerative Colitis Therapy.

Although magnolol (Mag), an anti-inflammatory natural compound, has been demonstrated to play protective effects on ulcerative colitis (UC), its application as an alternative therapeutic reagent for UC treatment is still greatly impeded due to its poor stability in the gastrointestinal tract and insufficient accumulation in the inflamed colon lesion. Nano-/microsized drug delivery systems can potentially overcome some challenges regarding the oral administration of phytochemicals, which still confront premature early drug release, degradation of NPs, or the sustained drug release of MPs. In this study, we primarily loaded Mag into the core-shell zein-based nanoparticles with chondroitin sulfate coating (Mag@CS-Zein NPs) with an average size of 142.27 ± 5.11 nm, showing significant macrophage-targeting and enhanced colon epithelial cellular uptake capacity. Then, we embedded Mag@CS-Zein NPs into hydrogel microspheres via an electrospraying technology. The Mag@CS-Zein NPsinMPs presented a uniform-sized sphere with an average size of 164.36 ± 6.29 μm and sustained drug-release profiles. Compared to CS-Zein NPs, the developed CS-Zein NPsinMPs exhibited prolonged colon retention on the inflammatory surface, as seen from ex vivo and in vivo imaging fluorescence adhesion experiments. Based on the advantage of the combination of hybrid nanoparticles-in-microparticles, oral administration of Mag@CS-Zein NPsinMPs significantly alleviated colitis symptoms in DSS-treated mice by regulating the expression levels of proinflammatory cytokines (TNF-α, IL-6, and IL-1β) and anti-inflammatory cytokines (IL-10) and factor accelerated colonic mucosal barrier repair via upregulating the expression of ZO-1 and occludin. This study provides great insights into the oral drug delivery of natural compounds for UC therapy.

Tubular epithelial cells derived-exosomes containing CD26 protects mice against renal ischemia/reperfusion injury by maintaining proliferation and dissipating inflammation

Ischemia-reperfusion injury (IR) is the leading cause of acute kidney injury (AKI). No effective drugs to treat IR-related AKI are currently available. Recent pre-clinical trials have evaluated the therapeutic potential of extracellular vesicles-exosomes to chronic kidney disease. Here, we found exosomes derived from the tubular epithelial cell in IR condition (ExoIR) enriched CD26, compared with control (ExoNormal). Tracking exosomes in vivo certified tubular epithelial cell uptake exosomes. We have isolated exosomes with overexpression of CD26 (ExoCD26+) from culture media from tubular epithelial cell line transferred by adenovirus vectors. After administration of exosomes (100 mg) or bovine serum albumin (BSA, equivalent protein control) in IR or sham operation mice after 72 h via tail vein injection, the renal function impairment and histology injury were relived in mice receiving ExoCD26+. Immunofluorescence staining with proliferating cell nuclear antigen revealed ExoCD26+ recovered proliferation of cells partly after IR injury. Cell cycle modulator, p53 and p21 were upregulated in IR mice receiving BSA control, ExoNormal, and ExoIR. ExoCD26+ significantly blunt this protein upregulation. Inflammatory cell infiltration and chemokine receptor (CXCR4) were dissipated in IR mice receiving ExoCD26+. Downstream chemokine of CXCR4, stromal derived factor-1 (SDF1) also decreased after administration of ExoCD26+ in IR mice. Finally, ExoCD26+ suppressed inundant collagenⅠ expression in IR kidney. In conclusion, Tubular epithelial cells derived-exosomes containing CD26 might be one of the therapy modes for IR-AKI by maintaining proliferation and dissipating inflammation.

Gastrointestinal lipolysis and trans-epithelial transport of SMEDDS via oral route

Self-microemulsifying drug delivery systems (SMEDDSs) have recently returned to the limelight of academia and industry due to their enormous potential in oral delivery of biomacromolecules. However, information on gastrointestinal lipolysis and trans-epithelial transport of SMEDDS is rare. Aggregation-caused quenching (ACQ) fluorescent probes are utilized to visualize the in vivo behaviors of SMEDDSs, because the released probes during lipolysis are quenched upon contacting water. Two SMEDDSs composed of medium chain triglyceride and different ratios of Tween-80 and PEG-400 are set as models, meanwhile Neoral® was used as a control. The SMEDDS droplets reside in the digestive tract for as long as 24 h and obey first order kinetic law of lipolysis. The increased chain length of the triglyceride decreases the lipolysis of the SMEDDSs. Ex vivo imaging of main tissues and histological examination confirm the trans-epithelial transportation of the SMEDDS droplets. Approximately 2%–4% of the given SMEDDSs are transported via the lymph route following epithelial uptake, while liver is the main termination. Caco-2 cell lines confirm the cellular uptake and trans-epithelial transport. In conclusion, a fraction of SMEDDSs can survive the lipolysis in the gastrointestinal tract, permeate across the epithelia, translocate via the lymph, and accumulate mainly in the liver.

Disparities between Uptake of Germline BRCA1/2 Gene Tests and Implementation of Post-test Management Strategies in Epithelial Ovarian, Fallopian Tube, or Primary Peritoneal Cancer Patients.

BackgroundTo assess the rate of germline BRCA gene tests in epithelial ovarian cancer (EOC) patients and uptake of post-test risk management strategies in BRCA1/2-mutated patients.MethodsInstitutional databases were searched to identify patients who were diagnosed with epithelial ovarian, fallopian tube, or primary peritoneal cancer (EOC) between 2009 and 2019 in two academic hospitals. Retrospective review on medical records was performed to collect clinico-pathologic variables, including performance of germline BRCA gene test and its results, as well as conduct of breast cancer screening tests and cascade testing. If annual mammography +/− breast ultrasonography was performed, it was considered that regular breast cancer surveillance was done.ResultsA total of 840 women with EOC were identified during the study period. Of these, 454 patients (54.0%) received BRCA gene testing and 106 patients (106/454, 23.3%) were positive for BRCA1/2 mutations. The rate of BRCA tests has markedly increased from 25.8% in 2009-2012 to 62.7% in 2017-2019. Among the 93 patients with BRCA1/2 mutation without previous personal breast cancer history, 20 patients (21.5%) received annual mammography with or without breast ultrasonography for regular surveillance. Among the 106 BRCA1/2-mutated EOC patients, cascade testing on family members was performed only in 13 patients (12.3%).ConclusionAlthough BRCA1/2 gene tests have been substantially expanded, the uptake of post-test risk management strategies, including breast cancer screening for BRCA1/2-mutated patients and cascade testing for family members, has remained low. Strategies to increase its uptake and education about the importance of post-test risk managements are needed.

PEGylation of recombinant human deoxyribonuclease I decreases its transport across lung epithelial cells and uptake by macrophages.

Conjugation to high molecular weight (MW ≥ 20 kDa) polyethylene glycol (PEG) was previously shown to largely prolong the lung residence time of recombinant human deoxyribonuclease I (rhDNase) and improve its therapeutic efficacy following pulmonary delivery in mice. In this paper, we investigated the mechanisms promoting the extended lung retention of PEG-rhDNase conjugates using cell culture models and lung biological media. Uptake by alveolar macrophages was also assessed in vivo. Transport experiments showed that PEGylation reduced the uptake and transport of rhDNase across monolayers of Calu-3 cells cultured at an air-liquid interface. PEGylation also decreased the uptake of rhDNase by macrophages in vitro whatever the PEG size as well as in vivo 4h following intratracheal instillation in mice. However, the reverse was observed in vivo at 24h due to the higher availability of PEGylated rhDNase in lung airways at 24 h compared with rhDNase, which is cleared faster. The uptake of rhDNase by macrophages was dependent on energy, time, and concentration and occurred at rates indicative of adsorptive endocytosis. The diffusion of PEGylated rhDNase in porcine tracheal mucus and cystic fibrosis sputa was slower compared with that of rhDNase. Nevertheless, no significant binding of PEGylated rhDNase to both media was observed. In conclusion, decreased transport across lung epithelial cells and uptake by macrophages appear to contribute to the longer retention of PEGylated rhDNase in the lungs.

Bigger or Smaller? Size and Loading Effects on Nanoparticle Uptake Efficiency in the Nasal Mucosa.

PLGA nanoparticles hold great promise for nasal administration, but only with careful design will efficient, effective, and safe delivery systems be developed. To better understand the size dependence of nasal epithelial uptake, PLGA nanoparticles (60nm or 125nm) loaded with Nile Red were prepared, and their uptake into excised sections of bovine nasal respiratory or olfactory mucosa was measured for 30 or 60min. The epithelial layer and the submucosal tissues were separated, and the amount of Nile Red was used to calculate the number of nanoparticles in each tissue region. Both particle sizes were able to be internalized into the nasal tissues in as little as 30min, but their total uptake represented less than 5% of the nanoparticles available. Nanoparticles were present both in the epithelial cells and in the submucosal tissues, and greater numbers of the 60-nm particles were present in the submucosa than the epithelium, while greater numbers of the 125-nm particles remained in the epithelial cell layer. The amount of Nile Red recovered from the mucosal tissues after exposure to 125-nm nanoparticles was at least 2-fold greater than from the 60-nm nanoparticles, however, due to the higher (~ 9-fold) loadingcapacity of the larger particles. The greater mass transfer of the Nile Red from the larger particles suggests that it may not be necessary to develop small nanoparticulate delivery systems for efficient drug delivery via the nasal mucosa. Well-designed nanoparticles with diameters > 100nm show good uptake into the nasal epithelium and are capable of transfer to the submucosal tissues, near the location of significant populations of blood and lymphatic vessels. Graphical abstract.

Highly bioavailable curcumin preparation with a co-grinding and solvent-free process.

Curcumin (Cur.) is a natural product isolated from the rhizome of Curcuma longa, with a variety of biological and pharmacological activities in food and pharmaceutical products. However, curcumin's poor solubility in water greatly limits its bioavailability and clinical applications. In this study, co‐grinding curcumin with food additives produced a mixture, which was evaluated for the solubility in water, dissolution, material morphology, in vivo bioavailability, cell uptake and entry mechanism. We tested 9 food additives in total and found that poloxamers performed the best. The 2 co‐grinding mixtures Cur./Kolliphor® P407 and Cur./Kolliphor® P188 with high drug loading at 65.5% significantly improved the curcumin aqueous solubility, subsequently increased its intestinal epithelial cell uptake and oral bioavailability. The relative bioavailabilities for the 2 co‐grinding mixtures were 309% and 163%, respectively, compared with curcumin API. Co‐grinding process has a broad application prospect and is suitable for industrial production.

A Comparison of Chitosan, Mesoporous Silica and Poly(lactic-co-glycolic) Acid Nanocarriers for Optimising Intestinal Uptake of Oral Protein Therapeutics

Efficacious oral delivery of therapeutic proteins remains challenging and nanoparticulate approaches are gaining interest for enhancing their permeability. In this study, we explore the ability for three comparably sized nanocarriers, with diverse physicochemical properties [i.e., chitosan (CSNP), mesoporous silica nanoparticles (MSNP) and poly(lactic-co-glycolic) acid (PLGA-NP)], to successfully facilitate epithelial uptake of a model protein, ovalbumin (OVA). We report the effect of nanoparticle surface chemistry and nanostructure on protein release, cell toxicity and the uptake mechanism in a Madin Darby Canine Kidney (MDCK) cell model of the intestinal epithelium. All nanocarriers exhibited bi-phasic OVA release kinetics with sustained and incomplete release after 4 days, and more pronounced release from MSNP than either polymeric nanocarriers. CSNP and MSNP displayed the highest cellular uptake, however CSNP was prone to significant dose-dependent toxicity attributed to the cationic surface charge. Approximately 25% of MSNP uptake was governed by a clathrin-independent endocytic mechanism, while CSNP and PLGA-NP uptake was not controlled via any endocytic mechanisms investigated herein. Furthermore, endosomal localisation was observed for CSNP and MSNP, but not for PLGA-NP. These findings may assist in the optimal choice and engineering of nanocarriers for specific intestinal permeation enhancement for oral protein delivery.

Ultrafine Bubble Water Usefulness in Fecal Microbiota Transplantation: Recognition of Transplanted Microbiota in Intestinal Epithelial Cells

Background: Indications for various diseases such as diabetes mellitus and metabolic syndrome, etc. of Fecal Microbiota Transplantation (FMT) have been investigated. For the establishment of the FMT therapy, the examination of the solvent has not been carried out, though the use of the physiological saline is fixed, at present. We have produced the Mr. Shimizu made ultrafine bubble water (UFB) that produces a larger number of bubbles than existing UFB.Objective: To verify the usefulness of UFB, we prepared a conventional Saline and UFB-prepared microbial preparation (Bio3); (Bio3/Saline) and (Bio3/UFB). The bacterial preparations, Bio3, contain glycated bacteria, lactic acid bacteria, and butyric acid bacteria. FMT was carried out to the diabetic model mouse using these microbial preparations, and whether the disease state was improved was examined. Cultured intestinal epithelial cells (CaCO-2) were also used to test for differentiation by UFB and Saline from variations in several receptors that recognize microbiota-mRNA. In addition, glucose uptake into cells was measured.Methods: FMT with (Bio3/UFB) and (Bio3/Saline) was performed in streptozotocin-induced diabetic mice (STZ-mice) in duplicate at 0, 7 days. Blood glucose levels (7, 14 days) and blood insulin levels (14 days) were measured. Cultured intestinal epithelial cells were also used to test for differentiation by UFB and Saline from change in several receptors that recognize microbiota-mRNA (Toll-like receptors, NOD-like receptors, and RIG-I-like receptors). In addition, glucose uptake into cells was measured using fluorescently labeled glucose analog (NBDG).Results: UFB could reduce the blood glucose level of STZ-induced diabetic model mice. However, no such effects were observed in Saline. Stimulation of serially diluted Bio3 with UFB-suspension were showed significant alteration in TLR4 and IL-1B-uPNA. The amount of glucose uptake in the (Bio3/UFB) group was significantly different at 30 min, inhibited or delayed. Conclusion: It is concluded that UFB-mediated cross-talk between intestinal bacteria and intestinal epithelial cells and inhibition or delay of intestinal epithelial glucose uptake may have been associated with the reduction of blood glucose levels in diabetic model mice. The superiority of UFB as a suspension used for the transfer of bacteria has been suggested.

Ecdysone steroid hormone remote controls intestinal stem cell fate decisions via the PPARγ-homolog Eip75B in Drosophila.

Developmental studies revealed fundamental principles on how organ size and function is achieved, but less is known about organ adaptation to new physiological demands. In fruit flies, juvenile hormone (JH) induces intestinal stem cell (ISC) driven absorptive epithelial expansion balancing energy uptake with increased energy demands of pregnancy. Here, we show 20-Hydroxy-Ecdysone (20HE)-signaling controlling organ homeostasis with physiological and pathological implications. Upon mating, 20HE titer in ovaries and hemolymph are increased and act on nearby midgut progenitors inducing Ecdysone-induced-protein-75B (Eip75B). Strikingly, the PPARγ-homologue Eip75B drives ISC daughter cells towards absorptive enterocyte lineage ensuring epithelial growth. To our knowledge, this is the first time a systemic hormone is shown to direct local stem cell fate decisions. Given the protective, but mechanistically unclear role of steroid hormones in female colorectal cancer patients, our findings suggest a tumor-suppressive role for steroidal signaling by promoting postmitotic fate when local signaling is deteriorated.

Investigating the Effect of Esterification on Retinal Pigment Epithelial Uptake Using Rhodamine B Derivatives.

PurposeThis study investigated the effects of esterification and increased lipophilicity on cellular penetration, accumulation and retention in ARPE-19-nic cells using ester functionalized rhodamine B dyes.MethodsRhodamine B was esterified to generate four dyes with increasing lipophilicity. Cellular uptake, retention and mitochondrial localization were investigated in vitro using ARPE-19-nic cells using direct intracellular and extracellular and mitochondrial fluorescence quantitation, confocal and high-resolution live cell imaging and co-localization with Mito-GFP.ResultsCellular penetrance, mitochondrial accumulation, and retention of the esterified dyes were increased in ARPE-19-nic cells compared with the nonesterified parent dye by direct fluorescence quantitation. Imaging demonstrated intracellular accumulation was confined to mitochondria as confirmed by colocalization with Mito-GFP.ConclusionsEsterification is an effective way to increase lipophilicity of a dye to improve cellular penetration of chemical entities. These observations may be key to improving retinal drug delivery for retinal pigment epithelium–based diseases.Translational RelevanceUnderstanding the intracellular distribution of drugs into retinal pigment epithelium cells is a critical component for identifying potential therapies for retinal pigment epithelium–based diseases.

Proximal Tubule Epithelial Cell Reabsorption of the Drug Delivery Protein Elastin‐like Polypeptide

Elastin like polypeptide (ELP) is a protein composed of a pentapeptide repeat domain found in human tropoelastin. ELP is used for drug delivery by altering the ELP coding sequence to attach drugs, therapeutic peptides, or therapeutic proteins. One of the focuses of our group has been development of ELP – delivered therapeutics for treatment of kidney disease. Thus, understanding how ELP is filtered, reabsorbed, and distributed within the kidney is of key importance. In vivo studies in our lab showed that ELPs accumulated preferentially in the kidney after systemic administration. Renal deposition and intra‐renal distribution was dependent on ELP size, with smaller ELPs accumulating exclusively in the renal cortex and larger ELPs having a more mixed cortical and medullary distribution. All ELPs were present at high levels in tubular epithelial cells. However, whether the tubular distribution is the result of ELP reabsorption after filtration and whether the amount or rate of reabsorption is dependent on ELP size is unknown.To address this issue, uptake and intracellular distribution of fluorescein‐5‐malemide labeled ELPs of varying sizes were determined in primary human proximal tubule epithelial cells (HRPteC). HRPteC were seeded in a 6 well plate (4x104/well) and incubated with fluorescein labeled ELP (10μM) for various times (24hr, 6hr, 4hr, 2hr and 1hr). HRPteC were collected, and mean cellular fluorescence intensity was measured using flow cytometry. Percentage of ELP internalization was determined by quenching the extracellular fluorescein label with trypan blue (0.13% v/v), then repeating the flow cytometric analysis to measure only intracellular fluorescence. To obtain a qualitative representation of ELP interaction with kidney cells, HRPteC were seeded in an 8‐chamber glass slide (2x103 cells/chamber) and incubated with fluorescein labeled ELP (10μM) for various times as above. Following fixation and staining with 4′,6‐diamidino‐2‐phenylindole (DAPI), cells were imaged by laser scanning confocal microscopy. Our data demonstrate that a longer incubation time led to a greater accumulation of ELP in the proximal tubule epithelial cells. Analysis of internalization rate revealed that all ELPs entered the cells slowly over the course of a 24‐hour time frame. This finding was supported by confocal microscopy. Between 1 and 12 hours, localization of ELP occurred at the cellular membrane of the proximal tubule cells, but at time points between 12 and 24 hours, ELP was visible in punctate cytoplasmic structures likely to be endocytic vesicles. A smaller ELP‐63 (~25 kDa) accumulated in cells at higher levels than did a larger ELP‐159 (~62 kDa) after 24 hours of incubation (390.7 ±5.0 versus 651.6 ±42.7 RFU; p&lt;0.05). These data suggest that there may be a common mechanism for tubular epithelial cell uptake of ELPs of various sizes, but that smaller ELPs may be internalized to a greater extent than larger ELPs. The results of these and future studies to determine the exact renal handling of ELPs of various sizes will be crucial to the design of ELPs for delivery of therapeutics to specific renal compartments or cell types.

Systemic delivery of peptides by the oral route: Formulation and medicinal chemistry approaches.

In its 33 years, ADDR has published regularly on the po5tential of oral delivery of biologics especially peptides and proteins. In the intervening period, analysis of the preclinical and clinical trial failures of many purported platform technologies has led to reflection on the true status of the field and reigning in of expectations. Oral formulations of semaglutide, octreotide, and salmon calcitonin have completed Phase III trials, with oral semaglutide being approved by the FDA in 2019. The progress made with oral peptide formulations based on traditional permeation enhancers is against a background of low and variable oral bioavailability values of ~1%, leading to a current perception that only potent peptides with a viable cost of synthesis can be realistically considered. Desirable features of candidates should include a large therapeutic index, some stability in the GI tract, a long elimination half-life, and a relatively low clearance rate. Administration in nanoparticle formats have largely disappointed, with few prototypes reaching clinical trials: insufficient particle loading, lack of controlled release, low epithelial particle uptake, and lack of scalable synthesis being the main reasons for discontinuation. Disruptive technologies based on engineered devices promise improvements, but scale-up and toxicology aspects are issues to address. In parallel, medicinal chemists are synthesizing stable hydrophobic macrocyclic candidate peptides of lower molecular weight and with potential for greater oral bioavailability than linear peptides, but perhaps without the same requirement for elaborate drug delivery systems. In summary, while there have been advances in understanding the limitations of peptides for oral delivery, low membrane permeability, metabolism, and high clearance rates continue to hamper progress.

Influenza A Virus-Infected Lung Epithelial Cell Co-Culture with Human Peripheral Blood Mononuclear Cells.

Sensing of influenza A virus (IAV) infection by pattern recognition receptors can occur by either direct infection of lung epithelial cells or uptake of virus-infected cells by innate cells such as dendritic cells/monocytes. This triggers a series of downstream events including activation of the inflammasome, the production of cytokines, chemokines, and the upregulation of stress-induced ligands that can lead to the activation of innate cells. These cells include innate lymphocytes such as MAIT, NKT, NK, and γδ T cells. Here we describe a method used to allow activation of human innate lymphocytes in co-culture with an IAV-infected human lung epithelial cell line (A549) to measure ex vivo effector functions (TNF and IFNγ) in a mixed culture environment. We describe (1) infection of the human lung epithelial cell line, (2) co-culture with PBMC, and (3) measurement of activation using intracellular cytokine staining.

Identification of the amino acid residue responsible for the myricetin sensitivity of human proton-coupled folate transporter

Human proton-coupled folate transporter (hPCFT/SLC46A1) has recently been found to be inhibited by myricetin by a sustained mechanism, raising a concern that the inhibition might lead to malabsorption of folates in the intestine, where hPCFT works for their epithelial uptake. However, rat PCFT (rPCFT) has more recently been found not to be inhibited by myricetin. Prompted by this finding, we attempted to determine the amino acid residue involved in that by analyses comparing between hPCFT and rPCFT. In the initial analysis, chimeric constructs prepared from hPCFT and rPCFT were examined for myricetin sensitivity to determine the hPCFT segment involved in the sensitivity. Focusing on the thereby determined segment from 83rd to 186th amino acid residue, hPCFT mutants having a designated amino acid residue replaced with its counterpart in rPCFT were prepared for the subsequent analysis. Among them, only G158N-substituted hPCFT was found to be transformed to be insensitive to myricetin and, accordingly, oppositely N158G-substituted rPCFT was transformed to be sensitive to myricetin. These results indicate the critical role of Gly158 in the myricetin sensitivity of hPCFT. This finding would help advance the elucidation of the mechanism of the myricetin-induced inhibition of hPCFT and manage the potential risk arising from that.

Cathelicidin-WA Facilitated Intestinal Fatty Acid Absorption Through Enhancing PPAR-γ Dependent Barrier Function.

The molecular mechanisms underlying the cellular uptake of long-chain fatty acids and the regulation of this process have been debated in recent decades. Here, we established an intestinal barrier dysfunction model in mice and Caco2 cell line by Lipopolysaccharide (LPS), and evaluated the fatty acid uptake capacity of the intestine. We found that LPS stimulation restricted the absorption of long chain fatty acid (LCFA), while Cathelicidin-WA (CWA) pretreatment facilitated this physiological process. At the molecular level, our results demonstrated that the stimulatory effects of CWA on intestinal lipid absorption were dependent on cluster determinant 36 and fatty acid transport protein 4, but not fatty acid–binding protein. Further, an enhanced intestinal barrier was observed in vivo and in vitro when CWA alleviated the fatty acid absorption disorder induced by LPS stimulation. Mechanistically, peroxisome proliferator-activated receptor (PPAR-γ) signaling was considered as a key pathway for CWA to enhance LCFA absorption and barrier function. Treatment with a PPAR-γ inhibitor led to impaired intestinal barrier function and suppressed LCFA uptake. Moreover, once PPAR-γ signaling was blocked, CWA pretreatment could not maintain the stability of the intestinal epithelial cell barrier or LCFA uptake after LPS stimulation. Collectively, these findings suggested that PPAR-γ may serve as a target for specific therapies aimed at alleviating fatty acid uptake disorder, and CWA showed considerable potential as a new PPAR-γ agonist to strengthen intestinal barrier function against fatty acid malabsorption.