Porcine Intestinal Tissue Research Articles

Evaluation of Southern African wild edible plants for potential herb-drug interactions through ex vivo p-glycoprotein and in vitro cytochrome P450 3A4 inhibitory effects

BackgroundWild edible plants (WEPs) including herbs provide staple foods as well as income for local communities on the African continent. However, these commonly used plant materials interact with orthodox or conventional drugs through both p-glycoprotein (P-gp) and cytochrome P450 enzymes inhibition. Hence, it is vital to explore the possibility of herb-drug interactions when concomitantly taking conventional drug dosage forms with some of the WEPs. P-gp and CYP3A4 show analogous substrate specificities and work together to establish an intestinal absorption barrier against xenobiotics. This study investigated the ex vivo p-glycoprotein inhibition and the in vitro inhibition of cytochrome P450 3A4 (CYP3A4) isoezyme by selected wild edible plants to identify potential food/herb-drug interactions. Methods: Ethanolic extracts of 30 wild edible plants sourced from Lesotho, Eswatini and the Limpopo province of South Africa were used for the study. Drug transport studies using propranolol, a P-glycoprotein substrate, across porcine intestinal tissue were conducted using intestinal sacs with test solutions in Krebs-Ringer bicarbonate buffer (pH 7.4) under a 95 % O2/5 % CO2 atmosphere. For the in vitro luminescent-based CYP3A4 inhibition assay, both porcine liver and intestinal microsomes were extracted from their respective tissues by homogenization, followed by high-speed centrifugation and standardization before use. ResultsSisymbrium thelungii O. E. Schulz significantly (p < 0.05) inhibited P-gp with an effective apparent permeability coefficient (Papp eff) of (7.53±0.33) × 10–6 cm/s and effective flux (Jeff) of 1.38±0.01 µg/cm2/min which was similar to sodium orthovanadate (positive control), indicating potential for herb/food-drug interaction possibly by absorption enhancement of poorly permeable P-gp substrates by this plant. Additionally, both Rubus cuneifolius Pursh and Hypoxis hirsuta (L.) Coville had significant (p < 0.05) p-glycoprotein inhibitory effects with Papp eff of (6.60±0.52) × 10–6 cm/s and Jeff of 1.11±0.08 µg/cm2/min, and Papp eff of 6.58±0.67 × 10–6 cm/s and Jeff of 1.19±0.12 µg/cm2/min, respectively. Significantly (p < 0.05), Rubus cuneifolius at a concentration of 0.2 mg/mL exhibited the strongest CYP3A4 inhibition in liver microsomes suggesting its potential as a bioavailability enhancer of CYP3A4 substrates. Meanwhile, Wahlengergia androsacea A.DC exhibited the most significant (p < 0.05) CYP3A4 inhibitory effect at the same concentration in intestinal microsomes. ConclusionThe observed inhibitory effects of certain wild edible plants on P-gp-mediated drug efflux and CYP3A4 drug metabolism indicate potential food/herb-drug interactions challenges for clinical practice. The herb/food-drug interactions also present opportunities for discovery of new functional excipients for enhancing the permeability of poorly absorbed orally administered drugs that are substrates of P-gp and CYP3A4.

Effect of Tannic Acid Concentrations on Temperature-Sensitive Sol-Gel Transition and Stability of Tannic Acid/Pluronic F127 Composite Hydrogels.

Recently, interest in polyphenol-containing composite adhesives for various biomedical applications has been growing. Tannic acid (TA) is a polyphenolic compound with advantageous properties, including antioxidant and antimicrobial properties. Additionally, TA contains multiple hydroxyl groups that exhibit biological activity by forming hydrogen bonds with proteins and biomacromolecules. Furthermore, TA-containing polymer composites exhibit excellent tissue adhesion properties. In this study, the gelation behavior and adhesion forces of TA/Pluronic F127 (TA/PluF) composite hydrogels were investigated by varying the TA and PluF concentrations. PluF (above 16 wt%) alone showed temperature-responsive gelation behavior because of the closely packed micelle aggregates. After the addition of a small amount of TA, the TA/PluF hydrogels showed thermosensitive behavior similar to that of PluF hydrogels. However, the TA/PluF hydrogels containing more than 10 wt% TA completely suppressed the thermo-responsive gelation kinetics of PluF, which may have been due to the hydrogen bonds between TA and PluF. In addition, TA/PluF hydrogels with 40 wt% TA showed excellent tissue adhesion properties and bursting pressure in porcine intestinal tissues. These results are expected to aid in understanding the use of mixtures of TA and thermosensitive block copolymers to fabricate adhesive hydrogels for versatile biomedical applications.

Activation of the NLRP1 Inflammasome and Its Role in Transmissible Gastroenteritis Coronavirus Infection.

The inflammasome pathway is a critical early response mechanism of the host that detects pathogens, initiates the production of inflammatory cytokines, and recruits effector cells to the infection site. Nonetheless, the mechanism of inflammasome activation in coronavirus infection and its biological functions in host defense remain unclear. Transmissible gastroenteritis virus (TGEV), a member of the genus Alphacoronavirus, is a significant pathogen that mainly infects piglets and causes intestinal inflammation and inflammatory cell infiltration. Here, we investigated the mechanism of inflammasome activation in intestinal epithelial cells (IECs) infected with TGEV. We observed a substantial increase in interleukin 1β (IL-1β) and IL-18 levels in both IECs and TGEV-infected porcine intestinal tissues. Furthermore, TGEV infection resulted in increased activation of caspase-1 and the NLRP1 (NOD-like receptor [NLR]-containing pyrin domain [PYD]) inflammasome. Our findings revealed that TGEV infection impeded the interaction between porcine NLRP1 (pNLRP1) and porcine dipeptidyl peptidases 9 (pDPP9), yet it did not reduce the expression of pDPP9. Importantly, the ZU5 domain, not the function-to-find domain (FIIND) reported in human NLRP1, was identified as the minimal domain of pNLRP1 for pDPP9 binding. In addition, the robust type I IFN expression induced by TGEV infection also upregulated pNLRP1 expression and pNLRP1 itself acts as an interferon-stimulated gene to counteract TGEV infection. Our data demonstrate that pNLRP1 has antiviral capabilities against coronavirus infection, which highlights its potential as a novel therapeutic target for coronavirus antiviral therapy. IMPORTANCE Coronavirus primarily targets the epithelial cells of the respiratory and gastrointestinal tracts, leading to damage in both humans and animals. NLRP1 is a direct sensor for RNA virus infection which is highly expressed in epithelial barrier tissues. However, until recently, the precise molecular mechanisms underlying its activation in coronavirus infection and subsequent downstream events remained unclear. In this study, we demonstrate that the alphacoronavirus TGEV induces the production of IL-1β and IL-18 and upregulates the expression of pNLRP1. Furthermore, we found that pNLRP1 can serve as an interferon-stimulated gene (ISG) to inhibit the infection of enterovirus TGEV. Our research highlights the crucial role of NLRP1 as a regulator of innate immunity in TGEV infection and shows that it may serve as a potential therapeutic target for the treatment of coronavirus infection.

Investigation of the effect of colloidal structures formed during lipolysis of lipid-based formulation on exemestane permeability using the in vitro lipolysis-permeation model

This study aimed to examine the effect of colloidal structures forming and changing with dispersion and digestion of lipid-based formulation (LBF) on the permeability of exemestane (EXE), a poorly water-soluble drug. Self-nanoemulsifying drug delivery system (SNEDDS), one of the LBF, was developed using Imwitor® 988, Kolliphor® RH 40, and Labrasol® according to the solubility study and optimized using Box-Behnken Design. The EXE-loaded SNEDDS formulation has a 14.46 ± 0.23 nm droplet size with a 0.119 ± 0.053 polydispersity index. EXE was loaded into SNEDDS at 15 mg/g. In vitro digestion was conducted to investigate the EXE permeability at the digestion medium and the flux of EXE through a dialysis membrane and porcine intestinal tissue was assessed with the permeability step. In vitro digestion study revealed that the SNEDDS formulation retained the solubilization of EXE and showed moderate supersaturation. The permeation of EXE through the dialysis membrane was observed that the EXE amount on the acceptor side was similar during different digestion phases, but the permeation of EXE through porcine intestinal tissue was slightly different during different digestion phases and EXE permeability was higher at 60 min of digestion although statistically not significant. However, EXE precipitation was observed since the solvation capacity of colloidal structures was slightly decreased at 60 min of digestion. It can be explained by the fact that EXE re-dissolved and increased the free fraction, increasing permeability. This study showed that the drug precipitation in the digestion study does not always result in low permeation and minor differences could be observed more evident in the porcine intestine tissue.

Isolation and immortalization of macrophages derived from fetal porcine small intestine and their susceptibility to porcine viral pathogen infections.

Macrophages are a heterogeneous population of cells that are present in all vertebrate tissues. They play a key role in the innate immune system, and thus, in vitro cultures of macrophages provide a valuable model for exploring their tissue-specific functions and interactions with pathogens. Porcine macrophage cultures are often used for the identification and characterization of porcine viral pathogens. Recently, we have developed a simple and efficient method for isolating primary macrophages from the kidneys and livers of swine. Here, we applied this protocol to fetal porcine intestinal tissues and demonstrated that porcine intestinal macrophages (PIM) can be isolated from mixed primary cultures of porcine small intestine-derived cells. Since the proliferative capacity of primary PIM is limited, we attempted to immortalize them by transferring the SV40 large T antigen and porcine telomerase reverse transcriptase genes using lentiviral vectors. Consequently, immortalized PIM (IPIM) were successfully generated and confirmed to retain various features of primary PIM. We further revealed that IPIM are susceptible to infection by the African swine fever virus and the porcine reproductive and respiratory syndrome virus and support their replication. These findings suggest that the IPIM cell line is a useful tool for developing in vitro models that mimic the intestinal mucosal microenvironments of swine, and for studying the interactions between porcine pathogens and host immune cells.

Open-source force analyzer with broad sensing range based on an optical pickup unit

In the pharmaceutical field, oral drug delivery devices continue to shrink down to the micrometer scale, driving a trending demand to investigate ex vivo mucoadhesive force down to the micro-Newton scale. However, owing to the limitation of measuring sensitivity, conventional methods (e.g., a texture analyzer) lack reliability while measuring forces in this range. Herein, we report on an open-source force analyzer that utilizes an optical-pickup-unit (from a DVD player) to detect cantilever-based force transducers and thereby, achieves a wide force-sensing range from 1.1 N to 0.99 nN. The cantilever force transducers can easily be adjusted to fit different force ranges by adjusting the steel shim, magnets, and 3D printed components. To validate the analyzer, we conducted a preliminary study to investigate the effect of time and humidity of mucoadhesion of porcine intestinal tissues. Besides, we measured the mucoadhesive force of a single oral drug delivery microdevice with an average force of 93.7 µN on the top sides of the device. This analyzer offers the possibility of measuring e.g. mucoadhesion of individual microdevices in the micro-Newton range. Hence, the analyzer can assist in the development of miniaturized oral drug delivery devices but has a much wider field of potential force sensing applications.

Intestinal explant barrier chip: long-term intestinal absorption screening in a novel microphysiological system using tissue explants

The majority of intestinal in vitro screening models use cell lines that do not reflect the complexity of the human intestinal tract and hence often fail to accurately predict intestinal drug absorption. Tissue explants have intact intestinal architecture and cell type diversity, but show short viability in static conditions. Here, we present a medium throughput microphysiological system, Intestinal Explant Barrier Chip (IEBC), that creates a dynamic microfluidic microenvironment and prolongs tissue viability. Using a snap fit mechanism, we successfully incorporated human and porcine colon tissue explants and studied tissue functionality, integrity and viability for 24 hours. With a proper distinction of transcellular over paracellular transport (ratio >2), tissue functionality was good at early and late timepoints. Low leakage of FITC-dextran and preserved intracellular lactate dehydrogenase levels indicate maintained tissue integrity and viability, respectively. From a selection of low to high permeability drugs, 6 out of 7 properly ranked according to their fraction absorbed. In conclusion, the IEBC is a novel screening platform benefitting from the complexity of tissue explants and the flow in microfluidic chips.

A retrospective evaluation of heparin product reactions in patients with alpha-gal allergies

BackgroundAlpha-gal allergy, also known as red meat allergy or alpha-gal syndrome, can present after bites of certain tick species that contain galactose-alpha-1,3-galactose (alpha-gal) carbohydrate. Following this exposure, patients may develop an allergic reaction after mammalian meat consumption. Some heparin products are derived from porcine intestinal tissue, and it is therefore possible that administering these medications to a patient with an alpha-gal allergy may trigger a reaction. ObjectiveThe purpose of this study was to evaluate the incidence of reactions to porcine heparin products in patients with an alpha-gal allergy. MethodsA retrospective case series was conducted by review of electronic medical record data. Patients included were between the ages of 18 and 89 years, with a documented alpha-gal or red meat allergy and an admission to a hospital in the Sentara Healthcare system. The primary outcome was the incidence of allergic reactions upon exposure to heparin products in patients with a documented alpha-gal allergy. ResultsPatients with a documented alpha-gal allergy received a heparin product in 57 of 158 hospital visits (36.1%). Heparin products were tolerated in 56 of the 57 visits (98.3%). The incidence of an alpha-gal reaction to unfractionated heparin was 2.6% (1/39) while the incidence of an alpha-gal reaction to enoxaparin was 0% (0/22). Conclusion and RelevanceHeparin products were associated with a low incidence of alpha-gal reactions among patients with documented alpha-gal allergy. It is possible that enoxaparin poses less of a risk for reaction in these patients compared to unfractionated heparin.

Design and Testing of Efficient Mucus-Penetrating Nanogels-Pitfalls of Preclinical Testing and Lessons Learned.

Mucosal surfaces pose a challenging environment for efficient drug delivery. Various delivery strategies such as nanoparticles have been employed so far; yet, still yielding limited success. To address the need of efficient transmucosal drug delivery, this report presents the synthesis of novel disulfide-containing dendritic polyglycerol (dPG)-based nanogels and their preclinical testing. A bifunctional disulfide-containing linker is coupled to dPG to act as a macromolecular crosslinker for poly-N-isopropylacrylamide (PNIPAM) and poly-N-isopropylmethacrylamide (PNIPMAM) in a precipitation polymerization process. A systematic analysis of the polymerization reveals the importance of a careful polymer choice to yield mucus-degradable nanogels with diameters between 100 and 200nm, low polydispersity, and intact disulfide linkers. Absorption studies in porcine intestinal tissue and human bronchial epithelial models demonstrate that disulfide-containing nanogels are highly efficient in overcoming mucosal barriers. The nanogels efficiently degrade and deliver the anti-inflammatory biomacromolecule etanercept into epithelial tissues yielding local anti-inflammatory effects. Over the course of this work, several problems are encountered due to a limited availability of valid test systems for mucosal drug-delivery systems. Hence, this study also emphasizes how critical a combined and multifaceted approach is for the preclinical testing of mucosal drug-delivery systems, discusses potential pitfalls, and provides suggestions for solutions.

Different responses of the blockade of the P2Y1 receptor with BPTU in human and porcine intestinal tissues and in cell cultures.

Gastrointestinal smooth muscle relaxation is accomplished by activation of P2Y1 receptors, therefore this receptor plays an important role in regulation of gut motility. Recently, BPTU was developed as a negative allosteric modulator of the P2Y1 receptor. Accordingly, the aim of this study was to assess the effect of BPTU on purinergic neurotransmission in pig and human gastrointestinal tissues. Ca2+ imaging in tSA201 cells that express the human P2Y1 receptor, organ bath and microelectrodes in tissues were used to evaluate the effects of BPTU on purinergic responses. BPTU concentration dependently (0.1 and 1µmolL-1 ) inhibited the rise in intracellular Ca2+ evoked by ADP in tSA201 cells. In the pig small intestine, 30µmolL-1 BPTU reduced the fast inhibitory junction potential by 80%. Smooth muscle relaxations induced by electrical field stimulation were reduced both in pig ileum (EC50 =6µmolL-1 ) and colon (EC50 =35µmolL-1 ), but high concentrations of BPTU (up to 100µmolL-1 ) had no effect on human colonic muscle. MRS2500 (1µmolL-1 ) abolished all responses. Finally, 10µmolL-1 ADPβS inhibited spontaneous motility and this was partially reversed by 30µmolL-1 BPTU in pig, but not human colonic tissue and abolished by MRS2500 (1µmolL-1 ). BPTU blocks purinergic responses elicited via P2Y1 receptors in cell cultures and in pig gastrointestinal tissue. However, the concentrations needed are higher in pig tissue compared to cell cultures and BPTU was ineffective in human colonic tissue.

IDISCO Allows Complete Visualization and Analysis of Postnatal Enteric Nervous System Development in a Comparative Pig Model

The enteric nervous system consists of a dense, complex network of neurons and glia which are instrumental in the maturation of normal intestinal physiology after birth. Our lab uses a comparative pig model to study the postnatal development of the enteric glial network and its role in regulating intestinal barrier functions in neonates. Immunolabeling‐enabled three‐dimensional (3D) imaging of solvent‐cleared organs (iDISCO) is a method of preparing tissue samples for volume imaging with a light sheet microscope. iDISCO has been optimized for use in primarily mouse organs and embryos for the study of early development and offers a more complete picture of the tissue than traditional histological analysis. Our objective was to optimize the iDISCO protocol for use in porcine intestinal tissue to allow complete qualitative and quantitative analysis of postnatal development of the enteric glial network in our comparative pig model. Antibodies against glial cell markers S100b, Sox10, and glial fibrillary acidic protein (GFAP) were used to triple‐stain fixed full‐thickness 3mm by 5mm samples of porcine jejunum using the iDISCO protocol. Samples were imaged with a light‐sheet microscope (Ultra‐II, LaVision BioTec®) using three different fluorescent channels and datasets were visualized and analyzed in 3D with Imaris software (Oxford Instruments®). The percent volume of GFAP+ glial cells was quantified by manually masking individual intestinal villi and optimizing a surface algorithm to identify glial network structures within those villi. Antibodies against all three markers tested produced the predicted staining pattern with minimal non‐specific staining. Percent of jejunal villus volume occupied by GFAP+ glia is higher in 6‐week‐old versus 2‐week‐old pig (0.49% versus 0.23%, *P≤0.05). Ongoing work will optimize quantification techniques for S100b and Sox10, and assess co‐localization patterns of these glial markers in the jejunum at discrete timepoints postnatally. iDISCO is a powerful imaging modality which will allow our lab to directly assess the expansion, complexity, and localization of glial cell subtypes by marker co‐expression analysis at discrete postnatal timepoints, and will be utilized in future studies to explore effects of disease and external interventions on the enteric glial network.Support or Funding InformationNIH P30 DK034987 UNC CGIBD Pilot Feasibility; NIH T32 5T32DK007737‐22 UNC Basic Science GI Research Training Fellowship; USDA National Institute of Food and Agriculture, Animal Health Projects 1007263 and 07985; 2017 NC State CMI TPP Seed Grant

In Vitro, Ex Vivo and In Vivo Evaluation of Microcontainers for Oral Delivery of Insulin.

Enhancing the oral bioavailability of peptides has received a lot of attention for decades but remains challenging, partly due to low intestinal membrane permeability. Combining a permeation enhancer (PE) with unidirectionally releasing microcontainers (MCs) has previously been shown to increase insulin permeation across Caco-2 cell monolayers. In the present work, this setup was further employed to compare three common PEs—sodium caprate (C10), sodium dodecyl sulfate (SDS), and lauroyl carnitine. The concept was also studied using porcine intestinal tissue with the inclusion of 70 kDa fluorescein isothiocyanate-dextran (FD70) as a pathogen marker. Moreover, a combined proteolysis and Caco-2 cell permeation setup was developed to investigate the effect of soybean trypsin inhibitor (STI) in the MCs. Lastly, in vivo performance of the MCs was tested in an oral gavage study in rats by monitoring blood glucose and insulin absorption. SDS proved to be the most potent PE without increasing the ex vivo uptake of FD70, while the implementation of STI further improved insulin permeation in the combined proteolysis Caco-2 cell setup. However, no insulin absorption in rats was observed upon oral gavage of MCs loaded with insulin, PE and STI. Post-mortem microscopic examination of their gastrointestinal tract indicated lack of intestinal retention and optimal orientation by the MCs, possibly precluding the potential advantage of unidirectional release.

Investigation of Mucoadhesion and Degradation of PCL and PLGA Microcontainers for Oral Drug Delivery.

Microfabricated devices have been introduced as a promising approach to overcome some of the challenges related to oral administration of drugs and, thereby, improve their oral bioavailability. In this study, we fabricate biodegradable microcontainers with different polymers, namely poly-ɛ-caprolactone (PCL), poly(lactic-co-glycolic acid) (PLGA) 50:50 and PLGA 75:25 by hot punching. The mucoadhesion of the microcontainers is assessed with an ex vivo retention model on porcine intestinal tissue. Finally, in vitro degradation studies of the biodegradable microcontainers are completed for six weeks in simulated intestinal medium with the addition of pancreatic enzymes. Through SEM inspection, the PLGA 50:50 microcontainers show the first signs of degradation already after two weeks and complete degradation within four weeks, while the other polymers slowly degrade in the medium over several weeks.

A higher throughput and physiologically relevant two-compartmental human ex vivo intestinal tissue system for studying gastrointestinal processes

A majority of the preclinical intestinal screening models do not properly reflect the complex physiology of the human intestinal tract, resulting in low translational value to the clinical situation. The often used cell lines such as Caco-2 or HT-29 are not well suited to investigate the different processes that predict oral bioavailability in real life, or processes involved in general gut health aspects. Therefore, highly realistic models resembling the human in vivo situation are needed; application of ex vivo intestinal tissue is an interesting and feasible alternative. After previously using porcine intestinal tissue as a predictive model for human intestinal absorption, we now have successfully applied human intestinal tissue into a newly developed InTESTine™ two-compartmental disposable device suitable for standard 6- or 24-well plate format. With this set-up we demonstrated (regional differences in) drug absorption, by using a subset of compounds with known varying Fa (fraction absorbed) values. A rank-order relationship of R2 = 0.85 could be established between the Fa and Papp of these commercially available drugs. Additionally, comparison between the InTESTine system and the established Ussing chamber technology showed a correlation of R2 = 0.94 (10 drugs) with respect to Papp values, indicating good comparison of both models. Besides absorption, intestinal wall metabolism of testosterone (CYP3A4) was determined by showing a linear formation (R2 = 0.99; up to 165 min) of the main metabolites androstenedione and 6Beta-hydroxytestosterone, indicating no loss of metabolic capacity of the intestinal tissue within the system. Enteroendocrine responses were assessed of the satiety hormones GLP-1 and PYY after stimulation with rebaudioside A and casein, resulting in significantly increased secretion to the luminal side as well as to the basolateral side. Incubation with the probiotic strain LGG showed to enhance the viability of the tissue by showing to decrease the LDH secretion compared to blank intestinal tissue. In conclusion, we show that human ex vivo intestinal tissue mounted in the higher throughput InTESTine 6- 24-transwell plate system is easy to handle and a suitable system to study diverse functional GI processes.

Fully replicable and automated retention measurement setup for characterization of bio-adhesion

<h2>Abstract</h2> The retention model by Rao and Buri is often used to characterize microparticles and other drug delivery systems for their bio-adhesive properties. Currently, these experiments are performed on customized setups, reducing reproducibility of results obtained in different labs. As a solution, we propose a fully replicable retention model, which can be constructed by parts mostly made by 3D printing and laser cutting as well as a limited amount of other easy to source and commercially available parts. In addition of being fully replicable, the setup features integration of a climate-controlled chamber, a peristaltic pump and an autosampler, thereby enabling fully automated but customized control of the experiments. Using the presented retention model setup and an automated experimental sequence, the setup has been proven capable of investigating mucoadhesion of differently shaped particles to porcine intestinal tissue.

Polymeric Lids for Microcontainers for Oral Protein Delivery.

Oral delivery of proteins and peptides is one of the main challenges in pharmaceutical drug development. Microdevices have the possibility to protect the therapeutics until release is desired, avoiding losses by degradation. One type of microdevice is polymeric microcontainers. In this study, lysozyme is chosen as model protein and loaded into microcontainers with the permeation enhancer sodium decanoate (C10). The loaded microcontainers are sealed and functionalized by applying polymeric lids onto the cavity of the devices. The first lid is poly(lactic-co-glycolic) acid (PLGA) and on top of this either polyethylene glycol (PEG) or chitosan is applied (PLGA+PEG or PLGA+chitosan, respectively). The functionalization is evaluated in vitro for morphology, drug release, and mucoadhesive properties. These are coupled with in vitro and ex vivo studies using Caco-2 cells, Caco-2/HT29-MTX-E12 co-cultures, and porcine intestinal tissue. PLGA+chitosan shows slower release compared to PLGA+PEG or only PLGA in buffer and the transport of lysozyme across cell cultures is not enhanced compared to the bulk powder. Microcontainers coated with chitosan or PEG demonstrate a three times stronger adhesion during ex vivo mucoadhesion studies compared to samples without coatings. Altogether, functionalized microcontainers with mucoadhesive properties and tunable release for oral protein delivery are developed and characterized.

Porcine Response to a Multidrug-Resistant Salmonella enterica serovar I 4,[5],12:i:- Outbreak Isolate.

Salmonella enterica serovar I 4,[5],12:i:- has emerged as a common nontyphoidal Salmonella serovar to cause human foodborne illness. An interesting trait of serovar I 4,[5],12:i:- is that it only expresses the fliC gene for bacterial motility (i.e., monophasic), while most Salmonella strains alternately express two flagellin genes (fliC and fljB). The goal of this study was to characterize the porcine response following inoculation with a multidrug-resistant (MDR) serovar I 4,[5],12:i:- isolate associated with a multistate pork outbreak to determine if the increased prevalence of serovar I 4,[5],12:i:- in swine is due to enhanced pathogenicity. Pigs were inoculated and subsequently evaluated for the ability of the isolate to colonize intestinal tissues, cause clinical symptoms, induce an immune response, and alter the fecal microbiota over a 7-day period. Pigs exhibited a significant increase in rectal temperature (fever) (p < 0.01) and fecal moisture content (diarrhea) (p < 0.05) at 2 days postinoculation (d.p.i.) compared with preinoculation (day 0). Serum analyses revealed significantly increased interferon-gamma (IFN-γ) levels at 2 (p ≤ 0.0001) and 3 (p < 0.01) d.p.i. compared with day 0, and antibodies against Salmonella lipopolysaccharide (LPS) were present in all pigs by 7 d.p.i. Serovar I 4,[5],12:i:- colonized porcine intestinal tissues and was shed in the feces throughout the 7-day study. Analysis of the 16S rRNA gene sequences demonstrated that the fecal microbiota was significantly altered following MDR serovar I 4,[5],12:i:- inoculation, with the largest shift observed between 0 and 7 d.p.i. Our data indicate that the pork outbreak-associated MDR serovar I 4,[5],12:i:- isolate induced transient clinical disease in swine and perturbed the gastrointestinal microbial community. The porcine response to MDR serovar I 4,[5],12:i:- is similar to previous studies with virulent biphasic Salmonella enterica serovar Typhimurium, suggesting that the absence of fljB does not substantially alter acute colonization or pathogenesis in pigs.

Storage stability study of porcine hepatic and intestinal cytochrome P450 isoenzymes by use of a newly developed and fully validated highly sensitive HPLC-MS/MS method.

Microsomes are an ideal medium to investigate cytochrome P450 (CYP450) enzyme-mediated drug metabolism. However, before microsomes are prepared, tissues can be stored for a long time. Studies about the stability of these enzymes in porcine hepatic and intestinal tissues upon storage are lacking. To be able to investigate CYP450 stability in microsomes prepared from these tissues, a highly sensitive and rapid HPLC-MS/MS method for the simultaneous determination of six CYP450 metabolites in incubation medium was developed and validated. The metabolites, paracetamol (CYP1A), 7-hydroxy-coumarin (CYP2A), 1-hydroxy-midazolam (CYP3A), 4-hydroxy-tolbutamide (CYP2C), dextrorphan (CYP2D), and 6-hydroxy-chlorzoxazone (CYP2E) were extracted with ethyl acetate at pH1.0, followed by evaporation and separation on an Agilent Zorbax Eclipse Plus C18 column. The method was fully validated in a GLP-compliant laboratory according to European guidelines and was highly sensitive (LOQ = 0.25-2.5ng/mL), selective, had good precision (RSD-within, 1.0-9.1%; RSD-between, 1.0-18.4%) and accuracy (within-run, 83.3-102%; between-run, 78.5-102%), and showed no relative signal suppression and enhancement. Consequently, this method was applied to study the stability of porcine hepatic and intestinal CYP450 isoenzymes when tissues were stored at - 80°C. The results indicate that porcine CYP450 isoenzymes are stable in tissues at least up to 4months when snap frozen and stored at - 80°C. Moreover, the results indicate differences in porcine CYP450 stability compared to rat, rabbit, and fish CYP450, as observed by other research groups, hence stressing the importance to investigate the CYP450 stability of a specific species.

Degree of SGLT1 phosphorylation is associated with but does not determine segment-specific glucose transport features in the porcine small intestines.

Glucose‐induced electrogenic ion transport is higher in the porcine ileum compared with the jejunum despite equal apical abundance of SGLT1. The objective of this study was a detailed determination of SGLT1 and GLUT2 expressions at mRNA and protein levels along the porcine small intestinal axis. Phosphorylation of SGLT1 at serine 418 was assessed as a potential modulator of activity. Porcine intestinal tissues taken along the intestinal axis 1 h or 3 h after feeding were analyzed for relative mRNA (RT‐PCR) and protein levels (immunoblot) of SGLT1, pSGLT1, GLUT2, (p)AMPK, β2‐receptor, and PKA substrates. Functional studies on electrogenic glucose transport were done (Ussing chambers: short circuit currents (I sc)). Additionally, effects of epinephrine (Epi) administration on segment‐specific glucose transport and pSGLT1 content were examined. SGLT1 and GLUT2 expression was similar throughout the small intestines but lower in the duodenum and distal ileum. pSGLT1 abundance was significantly lower in the ileum compared with the jejunum associated with significantly higher glucose‐induced I sc. SGLT1 phosphorylation was not inducible by Epi. Epi treatment decreased glucose‐induced I sc and glucose flux rates in the jejunum but increased basal I sc in the ileum. Epi‐induced PKA activation was detectable in jejunal tissue. These results may indicate that SGLT1 phosphorylation at Ser418 represents a structural change to compensate for certain conditions that may decrease glucose transport (unfavorable driving forces/changed apical membrane potential) rather than being the cause for the overall differences in glucose transport characteristics between the jejunum and ileum.

Porcine intestinal lymphoid tissues synthesize estradiol

Estradiol (17β-estradiol) is synthesized primarily in the gonads of both sexes and regulates the development and function of reproductive organs. Recently, we reported that intestinal lymphocyte homeostasis is regulated by estradiol synthesized de novo in the endothelial cells of the high endothelial venules (HEVs) of mesenteric lymph nodes and Peyer's patches in mice. This observation prompted us to hypothesize that HEVs of intestinal lymphoid tissues are sites of estradiol synthesis across species. In this study, we examined whether estradiol is synthesized in the intestinal lymphoid tissues of adolescent piglets. Comparisons of estradiol levels in blood and tissue showed that estradiol concentrations in mesenteric lymph nodes and Peyer's patches were significantly higher than the level in serum. Reverse transcription polymerase chain reaction showed that porcine intestinal lymphoid tissues express mRNAs for steroidogenic enzymes (StAR, 17β-Hsd, 3β-Hsd, Cyp17a1, and Cyp19a1), and immunohistochemical results in ilial tissue showed expression of aromatase (CYP19) in Peyer's patch-localized endothelial cells of HEVs. When mesenteric lymph node and Peyer's patch tissues were cultured in vitro, they produced estradiol. Taken together, the results indicate that mesenteric lymph nodes and Peyer's patches are sites of estradiol synthesis in adolescent piglets.