Application of a 3D bioprinter: jet technology for 'biopatch' development using cells on hydrogel supports.

Additive manufacturing (3D printing) has been deployed across multiple platforms to fabricate bioengineered tissues. We demonstrate the use of a Thermal Inkjet Pipette System (TIPS) for targeted delivery of cells onto manufactured substrates to design bio-bandages. Two cell lines - HEK 293 (kidney) and K7M2 wt (bone) - were applied using TIPS. We demonstrate a novel means for targeted cell delivery to a hydrogel support structure. These cell/support constructs (bio-bandages) had a high viability for survival and growth over extended periods. Combining a flexible biosupport with application of cells via TIPS printing now for the first time allows for custom cell substrate constructs with various densities to be deployed for regenerative medicine applications.

Three-Dimensional Bioprinting Applications for Bone Tissue Engineering.

The skeletal system is a key support structure within the body. Bones have unique abilities to grow and regenerate after injury. Some injuries or degeneration of the tissues cannot rebound and must be repaired by the implantation of foreign objects following injury or disease. This process is invasive and does not always improve the quality of life of the patient. New techniques have arisen that can improve bone replacement or repair. 3D bioprinting employs a printer capable of printing biological materials in multiple directions. 3D bioprinting potentially requires multiple steps and additional support structures, which may include the use of hydrogels for scaffolding. In this review, we discuss normal bone physiology and pathophysiology and how bioprinting can be adapted to further the field of bone tissue engineering.

3D Bioprinting Using Hydrogels: Cell Inks and Tissue Engineering Applications.

3D bioprinting is transforming tissue engineering in medicine by providing novel methods that are precise and highly customizable to create biological tissues. The selection of a "cell ink", a printable formulation, is an integral part of adapting 3D bioprinting processes to allow for process optimization and customization related to the target tissue. Bioprinting hydrogels allows for tailorable material, physical, chemical, and biological properties of the cell ink and is suited for biomedical applications. Hydrogel-based cell ink formulations are a promising option for the variety of techniques with which bioprinting can be achieved. In this review, we will examine some of the current hydrogel-based cell inks used in bioprinting, as well as their use in current and proposed future bioprinting methods. We will highlight some of the biological applications and discuss the development of new hydrogels and methods that can incorporate the completed print into the tissue or organ of interest.

Colonic Fluid and Electrolyte Transport 2022: An Update.

Colonic epithelial cells are responsible for maintaining a delicate balance between luminal secretion and the absorption of fluids and ions. This review aims to discuss and update the model of colonic electrolyte secretion and absorption via the cystic fibrosis transmembrane regulator (CFTR), epithelial sodium channel (ENaC), Na-K-Cl cotransporters (NKCC1 and 2), Na-H exchangers (NHE1-4), colonic H,KATPase, and several other key components involved in multi-level transepithelial ion transport. Developments in our understanding of the activity, regulation, localization, and relationships of these ion transporters and their interactions have helped forge a more robust understanding of colonic ion movement that accounts for the colonic epithelium's role in mucosal pH modulation, the setting of osmotic gradients pivotal for fluid retention and secretion, and cell death regulation. Deviations from homeostatic ion transport cause diarrhea, constipation, and epithelial cell death and contribute to cystic fibrosis, irritable bowel syndrome (IBS), ulcerative colitis, and cancer pathologies. Signal transduction pathways that regulate electrolyte movement and the regulatory relationships between various sensors and transporters (CFTR as a target of CaSR regulation and as a regulator of ENaC and DRA, for example) are imperative aspects of a dynamic and comprehensive model of colonic ion homeostasis.

3D Bioprinting of Vascularized Tissues for in vitro and in vivo Applications.

With a limited supply of organ donors and available organs for transplantation, the aim of tissue engineering with three-dimensional (3D) bioprinting technology is to construct fully functional and viable tissue and organ replacements for various clinical applications. 3D bioprinting allows for the customization of complex tissue architecture with numerous combinations of materials and printing methods to build different tissue types, and eventually fully functional replacement organs. The main challenge of maintaining 3D printed tissue viability is the inclusion of complex vascular networks for nutrient transport and waste disposal. Rapid development and discoveries in recent years have taken huge strides toward perfecting the incorporation of vascular networks in 3D printed tissue and organs. In this review, we will discuss the latest advancements in fabricating vascularized tissue and organs including novel strategies and materials, and their applications. Our discussion will begin with the exploration of printing vasculature, progress through the current statuses of bioprinting tissue/organoids from bone to muscles to organs, and conclude with relevant applications for in vitro models and drug testing. We will also explore and discuss the current limitations of vascularized tissue engineering and some of the promising future directions this technology may bring.

Bile Microbiota in Liver Transplantation: Proof of Concept Using Gene Amplification in a Heterogeneous Clinical Scenario.

Objective: Historically, bile in the biliary tract has been considered sterile. Most of the series are based on patients with biliary tract diseases or the bile has been obtained with procedures susceptible to contamination. Methods: We evaluated the bile in a heterogeneous cohort of liver donors and recipient patients, with samples obtained in a sterile way, directly from the gallbladder and the common bile duct. Results: We assessed the bile microbiota in six liver donors and in six liver recipients after whole or split liver procedures in adult or pediatric recipients. Bile samples were studied using PCR sequencing of the 16S ribosomal RNA gene amplification (rDNA). Conclusions: We demonstrated that the bile is sterile, thereby ruling this out as a source of contamination following transplant.

Hydrogel-based 3D bioprints repair rat small intestine injuries and integrate into native intestinal tissue.

3D Printing has become a mainstay of industry, with several applications in the medical field. One area that could benefit from 3D printing is intestinal failure due to injury or genetic malformations. We bioprinted cylindrical tubes from rat vascular cells that were sized to form biopatches. 2 mm enterotomies were made in the small intestine of male Sprague-Dawley rats, and sealed with biopatches. These intestinal segments were connected to an ex vivo perfusion device that provided independent extraluminal and intraluminal perfusion. The fluorescence signal of fluorescein isothiocyanate (FITC)-inulin in the intraluminal perfusate, a non-absorbable fluorescent marker of intestinal integrity, was measured every 15 min over 90 min, and used to assess the integrity of the segments under both continuous perfusion and alternate-flow perfusion. Enterotomies were made an inch away from the ileocecal junction in male Wistar rats and sealed with biopatches. The animals were monitored daily and euthanized at post-operative days 7, 14, 21, and 30. Blinded histopathological analysis was conducted to compare the patch segments to native intestine. Biopatch-sealed intestinal segments withstood both continuous and pulsatile flow rates without leakage of FITC-inulin above the control baseline. 21 of 26 animals survived with normal activity, weight gain, and stool output. Histopathology of the explanted segments showed progressive villi and crypt formation over the enterotomies, with complete restoration of the epithelium by 30 days. This study presents a novel application of 3D bioprinting to develop a universal repair patch that can seal lesions in vivo, and fully integrate into the native intestine.

Penicillin G Induces H+, K+-ATPase via a Nitric Oxide-Dependent Mechanism in the Rat Colonic Crypt.

BACKGROUND/AIMS: The colonic H+, K+ ATPase (HKA2) is a heterodimeric membrane protein that exchanges luminal K+ for intracellular H+ and is involved in maintaining potassium homeostasis. Under homeostatic conditions, the colonic HKA2 remains inactive, since most of the potassium is absorbed by the small intestine. In diarrheal states, potassium is secreted and compensatory potassium absorption becomes necessary. This study proposes a novel mechanism whereby the addition of penicillin G sodium salt (penG) to colonic crypts stimulates potassium uptake in the presence of intracellular nitric oxide (NO), under sodium-free (0-Na+) conditions. METHODS: Sprague Dawley rat colonic crypts were isolated and pHi changes were monitored through the ammonium prepulse technique. Increased proton extrusion in 0-Na+ conditions reflected heightened H+, K+ ATPase activity. Colonic crypts were exposed to penG, L-arginine (a NO precursor), and N-nitro l-arginine methyl ester (L-NAME, a NO synthase inhibitor). RESULTS: Isolated administration of penG significantly increased H+, K+ ATPase activity from baseline, p 0.0067. Co-administration of arginine and penG in 0-Na+ conditions further upregulated H+, K+ ATPase activity, p <0.0001. Crypt perfusion with L-NAME and penG demonstrated a significant reduction in H+, K+ ATPase activity, p 0.0058. CONCLUSION: Overall, acute exposure of colonic crypts to penG activates the H+, K+ ATPase in the presence of NO. This study provides new insights into colonic potassium homeostasis.

Human Small Intestine Transplantation: Segmental Susceptibility to Ischemia Using Different Preservation Solutions and Conditions.

BACKGROUND AND AIMS: Among all transplanted abdominal organs, the small intestine is one of the most ischemia sensitive. Appropriate graft selection, procurement, and preservation are crucial for optimum graft and patient survival. We evaluated ischemic damage in human small intestine grafts under different hypothermic preservation conditions (cold static and continuous perfusion) and solutions: histidine-tryptophan-ketoglutarate (HTK) and University of Wisconsin (UW). METHODS: Fourteen small intestinal grafts were procured from deceased donors. HTK and UW were used for the vascular perfusion at the cross clamp, and UW, HTK, or Ringer Lactate were used for the luminal flush at the back table. Therefore, part of the same harvested intestine was stored in cold static storage and in continuous perfusion preservation (with intestinal perfusion unit) simultaneously. Histological samples were collected from the jejunum and ileum at different time points and different preservation conditions. The samples were collected before the initiation of cold storage (T0), after 8 hours of cold static (ST8), or after 8 hours of continuous perfusion preservation (PT8) (n = 161 samples). Blinded histological evaluation was conducted and ischemic damage was determined using the Park/Chiu scale. RESULTS: The ileum had less ischemic damage than the jejunum, regardless of using static or continuous perfusion preservation. There was no significantly ischemic damage difference between intestinal grafts flushed and perfused with UW or HTK. CONCLUSION: The jejunum is more susceptible to ischemic injury than the ileum. UW and HTK are equivalent to preserve intestinal graft. This suggests that selective transplantation of ileum could reduce ischemia-related postoperative complications.

Induction of Secretagogue Independent Gastric Acid Secretion via a Novel Aspirin-Activated Pathway.

Aspirin has been widely recommended for acute and chronic conditions for over 2,000 years. Either single or repetitive doses are commonly used for analgesic and antipyretic reasons and to prevent heart attacks, stroke, and blood clot formation. Recent studies show that it can also be used chronically to dramatically reduce the risk of a variety of cancers. However, prolonged usage of aspirin can cause severe damage to the mucosal barrier, increasing the risk of ulcer formation and GI-bleeding events. In the present study, we show the effects of acute low-dose aspirin exposure as an active secretagogue-inducing gastric acid secretion. Studies were carried out with isolated gastric glands using the pH-sensitive dye BCECF-AM to assess acid secretion. The non-selective NOS inhibitor L-NAME (30 µM), or the specific inhibitor ODQ (1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one) was applied while monitoring intracellular pH. The effects of basolateral exposure to aspirin (acetylsalicylic acid, ASA) caused activation of gastric acid secretion via the H+, K+-ATPase. Our data suggest that aspirin increases nitric oxide (NO) production, which in turn activates acid secretion. Exposure of gastric glands to either the non-selective NOS inhibitor L-NAME, and the highly selective, soluble guanylyl cyclase inhibitor 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) effectively inhibited aspirin-dependent gastric acid secretion. Aspirin can be considered as a novel secretagogue, in the way that it activates the H+, K+-ATPase. With increased daily aspirin consumption, our findings have important implications for all individuals consuming aspirin even in low doses and the potential risks for increased acid secretion.

The calcium-sensing receptor: A novel target for treatment and prophylaxis of neratinib-induced diarrhea.

Diarrhea is one of the most commonly reported adverse effect of hemotherapy and targeted cancer therapies, such as tyrosine kinase inhibitors (TKI), which often significantly impact patient quality of life, morbidity, and mortality. Neratinib is an oral, irreversible pan-HER tyrosine kinase inhibitor, which is clinically active in HER2-positive breast cancer. Diarrhea is the most common side effect of this potent anticancer drug and the reasons for this adverse effect are still largely unclear. We have recently shown that activation of the calcium-sensing Receptor (CaSR) can inhibit secretagogue-induced diarrhea in the colon, therefore we hypothesized that CaSR activation may also mitigate neratinib-induced diarrhea. Using an established ex vivo model of isolated intestinal segments, we investigated neratinib-induced fluid secretion and the ability of CaSR activation to abate the secretion. In our study, individual segments of the rat intestine (proximal, middle, distal small intestine, and colon) were procured and perfused intraluminally with various concentrations of neratinib (10, 50, 100 nmol L-1). In a second set of comparison experiments, intraluminal calcium concentration was modulated (from 1.0 to 5.0 or 7.0 mmol L-1), both pre- and during neratinib exposure. In a separate series of experiments R-568, a known calcimimetic was used CaSR activation and effect was compared to elevated Ca2+ concentration (5.0 and 7.0 mmol L-1). As a result, CaSR activation with elevated Ca2+ concentration (5.0 and 7.0 mmol L-1) or R-568 markedly reduced neratinib-induced fluid secretion in a dose-dependent manner. Pre-exposure to elevated luminal calcium solutions (5.0 and 7.0 mmol L-1) also prevented neratinib-induced fluid secretion. In conclusion, exposure to luminal neratinib resulted in a pronounced elevation in fluid secretion in the rat intestine. Increasing luminal calcium inhibits the neratinib-associated fluid secretion in a dose-dependent manner. These results suggest that CaSR activation may be a potent therapeutic target to reduce chemotherapy-associated diarrhea.

Activation of the Calcium Sensing Receptor Decreases Secretagogue-Induced Fluid Secretion in the Rat Small Intestine.

BACKGROUND: The calcium-sensing receptor (CaSR) has been localized and characterized in numerous tissues throughout the body. In the mammalian gastrointestinal tract, the CaSR is known to act as a nutrient sensor and has recently been found to play a role in intestinal fluid and electrolyte balance. This study aims to demonstrate the functionality of the CaSR as a modulator of fluid secretion and absorption along the small intestine. METHODS: Small intestine regions (proximal, middle, and distal) were isolated from Sprague Dawley rats and loaded into an ex vivo intestinal perfusion device that provides independent intraluminal and extraluminal (serosa/basolateral) perfusion. The regions were perfused with 5 and 7 mM of Ca2+, both in the presence and absence of forskolin (FSK), a potent secretagogue. Control experiments were conducted with intraluminal perfusate containing standard Ringer-HEPES buffer with a physiological concentration of Ca2+ (1 mM). A second set of comparison experiments was performed with intraluminal perfusates containing AC-265347, a CaSR activator and agonist, in the presence of FSK. In all experimental conditions, the intraluminal perfusate contained fluorescein isothiocyanate (FITC)-inulin, a nonabsorbable fluorescent marker of secretion and/or absorption. Intraluminal fluorescence signal was utilized as a measure of water movement at the start of the experiment and every 15 min for 90 min. RESULTS: Under physiological conditions, increasing the concentration of Ca2+ in the luminal perfusate reduced intestinal fluid secretion in all regions. At a Ca2+ concentration of 7 mM, net fluid absorption was observed in all regions. In the presence of FSK, 5 mM Ca2+ significantly decreased fluid secretion and 7 mM Ca2+ abolished FSK-induced fluid secretion. Intraluminal perfusion with 5 mM Ca2+ was as effective as AC-265347, in reducing secretagogue-induced fluid hypersecretion in the proximal and middle regions. CONCLUSION: This study concludes that apical CaSR is active along the small intestine. Its activation by Ca2+ and/or calcimimetics reduces fluid secretion in a dose-dependent manner, with higher Ca2+ concentrations, or application of a calcimimetic, leading to fluid absorption. We furthermore show that, in the presence of FSK, receptor activation abates FSK secretagogue-induced fluid secretion. This presents a new therapeutic target to address secretory diarrheal illnesses.

Design and implementation of novel nutraceuticals and derivatives for treating intestinal disorders.

Gastrointestinal illnesses pose a significant worldwide disease burden and are associated with an array of medicinal and surgical therapies. Standard pharmaceutical options have adverse effects, prompting the rise of nutraceutical or food-derivative therapies. Here, we present an overview of the current nutraceutical therapies in gastrointestinal disease. We then introduce the calcium-sensing receptor (CaSR) as a novel therapeutic target. A G-protein-coupled receptor found in apical and basal intestinal cells, the CaSR modulates intestinal fluid secretion and mucosal integrity. Applying nutraceuticals that upregulate the CaSR may alleviate symptoms seen across a spectrum of illnesses. At last, we discuss how nanoparticle technology can be implemented to effectively deliver nutraceuticals to diseased regions of the intestine, thereby minimizing systemic side effects.

In Pursuit of the Parietal Cell - An Evolution of Scientific Methodology and Techniques.

The stomach has unique embryologic and anatomic properties, making the study of the parietal cell technically challenging. Numerous individuals have devoted decades of research to unraveling the pathophysiological basis of this cell type. Here, we perform a scoping review of novel in vitro and in vivo methodology pertaining to the parietal cell. First, we evaluate early in vitro methods of parietal cell analysis. This section focuses on three major techniques: gastric gland isolation, parietal cell isolation, and parietal cell culture. We also discuss parietal cell physiology and pathophysiology. Second, we discuss more contemporary efforts involving confocal microscopy and gastric organoids, a new technique that holds much promise in unveiling the temporal-spatial dynamics of the cell. Finally, we will discuss findings from our laboratory where we identified an active gastric vacuolar H+-ATPase as a putative mechanism for refractory GERD. Overall, this review aims to highlight the major milestones in understanding an elusive yet important cell. Though in no way comprehensive, we hope to provide a birds-eye view to the study of this unique cell type in the gastrointestinal tract.

Modulations in extracellular calcium lead to H+-ATPase-dependent acid secretion: a clarification of PPI failure.

The H+,K+-ATPase was identified as the primary proton secretory pathway in the gastric parietal cell and is the pharmacological target of agents suppressing acid secretion. Recently, we identified a second acid secretory protein expressed in the parietal cell, the vacuolar H+-ATPase (V-type ATPase). The aim of the present study was to further characterize H+-ATPase activation by modulations in extracellular calcium via the calcium sensing receptor (CaSR). Isolated gastric glands were loaded with the pH indicator dye BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester] to measure intracellular pH. Experiments were conducted in the absence of sodium and potassium to monitor H+-ATPase-specific transport activity. CaSR was activated with the calcimimetic R568 (400 nM) and/or by modulations in extracellular Ca2+. Elevation in calcium concentrations increased proton extrusion from the gastric parietal cell. Allosteric modification of the CaSR via R568 and calcium increased vacuolar H+-ATPase activity significantly (ΔpH/minlowCa2+(0.1mM) = 0.001 ± 0.001, ΔpH/minnormalCa2+(1.0mM) = 0.033 ± 0.004, ΔpH/minhighCa2+(5.0mM) = 0.051 ± 0.005). Carbachol significantly suppressed calcium-induced gastric acid secretion via the H+-ATPase under sodium- and potassium-free conditions. We conclude that the V-type H+-ATPase is tightly linked to CaSR activation. We observed that proton pump inhibitor (PPI) exposure does not modulate H+-ATPase activity. This elevated blood calcium activation of the H+-ATPase could provide an explanation for recurrent reflux symptoms while taking a PPI therapy. NEW & NOTEWORTHY This study emphasizes the role of the H+-ATPase in acid secretion. We further demonstrate the modification of this proton excretion pathway by extracellular calcium and the activation of the calcium sensing receptor CaSR. The novelty of this paper is based on the modulation of the H+-ATPase via both extracellular Ca (activation) and the classical secretagogues histamine and carbachol (inactivation). Both activation and inactivation of this proton pump are independent of PPI modulation.

Generating vascular conduits: from tissue engineering to three-dimensional bioprinting.

Vascular disease - including coronary artery disease, carotid artery disease, and peripheral vascular disease - is a leading cause of morbidity and mortality worldwide. The standard of care for restoring patency or bypassing occluded vessels involves using autologous grafts, typically the saphenous veins or internal mammary arteries. Yet, many patients who need life- or limb-saving procedures have poor outcomes, and a third of patients who need vascular intervention have multivessel disease and therefore lack appropriate vasculature to harvest autologous grafts from. Given the steady increase in the prevalence of vascular disease, there is great need for grafts with the biological and mechanical properties of native vessels that can be used as vascular conduits. In this review, we present an overview of methods that have been employed to generate suitable vascular conduits, focusing on the advances in tissue engineering methods and current three-dimensional (3D) bioprinting methods. Tissue-engineered vascular grafts have been fabricated using a variety of approaches such as using preexisting scaffolds and acellular organic compounds. We also give an extensive overview of the novel use of 3D bioprinting as means of generating new vascular conduits. Different strategies have been employed in bioprinting, and the use of cell-based inks to create de novo structures offers a promising solution to bridge the gap of paucity of optimal donor grafts. Lastly, we provide a glimpse of our work to create scaffold-free, bioreactor-free, 3D bioprinted vessels from a combination of rat vascular smooth muscle cells and fibroblasts that remain patent and retain the tensile and mechanical strength of native vessels.

Activation of Secretagogue Independent Gastric Acid Secretion via Endothelial Nitric Oxide Synthase Stimulation in Rats.

BACKGROUND/AIMS: L-arginine is an important mediator of cell division, wound healing, and immune function. It can be transformed by the nitric oxide synthase (NOS) to nitric oxide (NO), an important cell signaling molecule. Recent studies from our laboratory demonstrate specific effects of L-arginine (10mM) exposure on gastric acid secretion in rat parietal cells. METHODS: Studies were performed with isolated gastric glands and the pH sensitive dye BCECF-AM +/- L-arginine to examine its effects on acid secretion. The direct NO-donor diethylamine NONOate sodium salt hydrate, was also used while monitoring intracellular pH. The specific inhibitor of the intracellular NO signal cascade ODQ was also used. RESULTS: We found that gastric proton extrusion was activated with application of L-arginine (10mM), in a separate series when L-arginine (10mM) + L-NAME (30µM) were added there was no acid secretion. Addition of the NO-donor diethylamine NONOate sodium salt hydrate (10µM) also induced acid secretion. When the selective sGC-inhibitor ODQ was added with NONOate we did not observe acid secretion. CONCLUSION: We conclude that L-arginine is a novel secretagogue, which can mediate gastric acid secretion. Furthermore, the intake of L-arginine causes direct activation of the H+, K+ ATPase and increased proton extrusion from parietal cells resulting in the increased risk for acid-related diseases. The NO/sGC/cGMP pathway has never been described as a possible intracellular mechanism for H+, K+ ATPase activation before and presents a completely new scientific finding. Moreover, our studies demonstrate a novel role for L-NAME to effectively eliminate NOS induced acid secretion and thereby reducing the risk for L-arginine inducible ulcer disease.

Stomach and Bone.

The relation between gastrointestinal organs and bone metabolism has become clearer during the last decades. Of paramount importance is the tight and intertwined regulation of gastric acid secretion and bone metabolism in regard of diseases caused by dysfunction of any of these or intermediary organs or mediators. The importance of the functions of the endocrine modulators 1,25(OH)2 vitamin D (calcitriol), PTH, and calcitonin becomes clear when seeing misbalances and its impact on the skeleton. Another important player in the gut-bone signaling axis is calcium, which is operating through the calcium-sensing receptor (CaSR). The CaSR is located on diverse tissues of the human body, such as the parathyroid glands, stomach, intestine, and kidney. The strict regulation of calcium homeostasis is of high importance and any disturbances have immense consequences for the body. Mechanisms and therapeutic implications, as well as diseases caused by imbalances on the stomach-bone signaling axis, are highlighted in the following chapter.

Acute Effects of Vitamin C Exposure On Colonic Crypts: Direct Modulation of pH Regulation.

BACKGROUND/AIM: Colorectal cancer is still considered a leading cause of death in the United States and worldwide. One potential way to improve survival besides detection is to look to new therapeutic agents that can be taken prophylactically to reduce the risk of tumor formation. For cancer cells to grow and invade, a higher (more alkaline) intracellular pH must occur. We chose to examine a specific nutraceutical agent, which is Vitamin C. The acute effect of Vitamin C exposure on normal colonic crypts has been studied, providing some insight into how Vitamin C achieve its effect. METHODS: Distal colon was excised from rats. Following enzymatic digestion single colonic crypts were isolated. Colonic crypts were loaded with pH sensitive dye to measure the intracellular pH changes. Crypts were exposed to solutions +/- Vitamin C. RESULTS: 10 mM Vitamin C decreased Na+-dependent intracellular pH recovery. Vitamin C modulates SVCT leading to changes in proton extrusion. Vitamin C entry occurs via either SVCT2 on the basolateral membrane or by transcellular passive diffusion through tight junctions to the apical membrane and then active transport via SVCT1. CONCLUSION: Acute addition of Vitamin C to the basolateral membrane maintains low intracellular pH for a longer period which could halt and/or prevent tumor formation.

Modulation of Intestinal Microbiome Prevents Intestinal Ischemic Injury.

Background: Butyrate protects against ischemic injury to the small intestine by reducing inflammation and maintaining the structure of the intestinal barrier, but is expensive, short-lived, and cannot be administered easily due to its odor. Lactate, both economical and more palatable, can be converted into butyrate by the intestinal microbiome. This study aimed to assess in a rat model whether lactate perfusion can also protect against intestinal ischemia. Materials and Methods: Rat intestinal segments were loaded in an in vitro bowel perfusion device, and water absorption or secretion was assessed based on fluorescence of FITC-inulin, a fluorescent marker bound to a biologically inert sugar. Change in FITC concentration was used as a measure of ischemic injury, given the tendency of ischemic cells to retain water. Hematoxylin and eosin-stained sections at light level microscopy were examined to evaluate intestinal epithelium morphology. Comparisons between the data sets were paired Student t-tests or ANOVA with p < 0.05 performed on GraphPad. Results: Lactate administration resulted in a protective effect against intestinal ischemia of similar magnitude to that observed with butyrate. Both exhibited approximately 1.5 times the secretion exhibited by control sections (p = 0.03). Perfusion with lactate and methoxyacetate, a specific inhibitor of lactate-butyrate conversion, abolished this effect (p = 0.09). Antibiotic treatment also eliminated this effect, rendering lactate-perfused sections similar to control sections (p = 0.72). Perfusion with butyrate and methoxyacetate did not eliminate the observed increased secretion, which indicates that ischemic protection was mediated by microbial conversion of lactate to butyrate (p = 0.71). Conclusions: Lactate's protective effect against intestinal ischemia due to microbial conversion to butyrate suggests possible applications in the transplant setting for reducing ischemic injury and ameliorating intestinal preservation during transport.