Gut Organoid as a New Platform to Study Alginate and Chitosan Mediated PLGA Nanoparticles for Drug Delivery

Intestinal organoids can be used as an ex vivo epithelial model to study different drug delivery effects on epithelial cells’ luminal surface. In this study, the impact of surface charge on the delivery of 5-ASA loaded PLGA nanoparticles into the lumen of organoids was investigated. Alginate and chitosan were used to coat the nanoparticles and provide negative and positive charges on the particles, respectively. The organoid growth and viability were not affected by the presence of either alginate- or chitosan-coated nanoparticles. It was shown that nanoparticles could be transported from the serosal side of the organoids to the lumen as the dye gradually accumulated in the lumen by day 2–3 after adding the nanoparticles to the Matrigel. By day 5, the dye was eliminated from the lumen of the organoids. It was concluded that the positively charged nanoparticles were more readily transported across the epithelium into the lumen. It may be attributed to the affinity of epithelial cells to the positive charge. Thus, the organoid can be utilized as an appropriate model to mimic the functions of the intestinal epithelium and can be used as a model to evaluate the benefits of nanoparticle-based drug delivery.

Adenomyosis: Mechanisms and Pathogenesis

Adenomyosis is a common disorder of the uterus, and is associated with an enlarged uterus, heavy menstrual bleeding (HMB), pelvic pain, and infertility. It is characterized by endometrial epithelial cells and stromal fibroblasts abnormally found in the myometrium where they elicit hyperplasia and hypertrophy of surrounding smooth muscle cells. While both the mechanistic processes and the pathogenesis of adenomyosis are uncertain, several theories have been put forward addressing how this disease develops. These include intrinsic or induced (1) microtrauma of the endometrial–myometrial interface; (2) enhanced invasion of endometrium into myometrium; (3) metaplasia of stem cells in myometrium; (4) infiltration of endometrial cells in retrograde menstrual effluent into the uterine wall from the serosal side; (5) induction of adenomyotic lesions by aberrant local steroid and pituitary hormones; and (6) abnormal uterine development in response to genetic and epigenetic modifications. Dysmenorrhea, HMB, and infertility are likely results of inflammation, neurogenesis, angiogenesis, and contractile abnormalities in the endometrial and myometrial components. Elucidating mechanisms underlying the pathogenesis of adenomyosis raise possibilities to develop targeted therapies to ameliorate symptoms beyond the current agents that are largely ineffective. Herein, we address these possible etiologies and data that support underlying mechanisms.

Collision Tumor of Adenocarcinoma and Gastrointestinal Stromal Tumor in the Small Bowel

We report a very rare case of collision tumor composed of primary adenocarcinoma of the jejunum and gastrointestinal stromal tumor (GIST). The patient was a 63-year-old man who visited our hospital for epigastralgia and vomiting. Abdominal computed tomography revealed a mass in the upper jejunum, with gastric and duodenal dilatation. Endoscopy of the small bowel showed a circumferential tumor in the upper jejunum, which was diagnosed as primary adenocarcinoma by tissue biopsy. Thereafter, partial resection of the small bowel from the third part of the duodenum over the upper jejunum was performed. A tumor colliding with the primary adenocarcinoma was identified on the serosal side of the jejunum in the excised specimen and was histologically diagnosed as GIST. The annual incidence of primary adenocarcinoma of the small bowel (i.e., jejunum and ileum excluding the duodenum) has been reported to be 7 in 1 million people, and only 6 cases of collision tumor of the small bowel (i.e., duodenum: 5, ileum: 1) have been reported thus far. Although esophageal, gastric, and large intestinal collision tumors composed of primary cancer and GIST have been reported, to our knowledge, the present patient is the first case of the small bowel. The cause of or correlation between 2 tumors forming a collision tumor remains unclear. In the present patient, there was no pathological finding of infiltration between the 2 tumors. Although the collision of the 2 tumors was unclear, the findings indicate their independent development in closely located regions consistent with collision tumors.

Abstract 17268: ApoA-I Mimetic Peptide 4F Suppresses the Elevation of Pro-Inflammatory Lipid Mediators in Mouse Models of Inflammatory Bowel Disease

Introduction: Inflammatory bowel disease (IBD) has been linked to an increased prevalence of early stage vascular disease. ApoA-I mimetic peptides including 4F are potential therapeutic agents for the treatment of inflammatory diseases including atherosclerosis, and their mechanism of action appears localized to the intestine. We have reported that 4F protects against the development of disease in both the piroxicam-accelerated IL10-/- and myeloid COX2-/- mouse models of IBD. Hypothesis: We previously reported that plasma and lesion levels of oxidized products of linoleic and arachidonic acid correlate with disease in mouse models of atherosclerosis, and that 4F protects against disease in these models while inhibiting accumulation of these pro-inflammatory mediators. We thus sought to determine the complete lipid pro-inflammatory mediator profiles of both the COX2- and IL10-dependent models of IBD, while also determining the effect of 4F on the pro-inflammatory lipid profiles. Methods: We developed and validated a LC-ESI-MS/MS method for determining the levels of 40 lipid inflammatory mediators in both intestinal tissue and plasma, and we analyzed the effects of both disease and 4F upon these mediators in both IBD models. We also employed Ussing chambers to investigate ex vivo the direct effect of 4F on the clearance of pro-inflammatory lipid mediators from intestinal explants and serosal-side lipoproteins. Results: Disease in both models correlated with significantly elevated tissue and plasma levels of multiple lipid pro-inflammatory mediators, while the protective effects of 4F correlated with the significant suppression of most of these mediators. Of interest, 4F inhibited the disease dependent increase of 15HETE, 12HETE, 5HETE, 13HODE, LTB4, 6ketoPGF1α, PGF2α, and TXB2 in the COX2-/- model; and 15HETE, 12HETE, 13HODE, LTB4, and LTE4 in the IL10-/- model. Ex vivo, we showed that 4F could directly clear the pro-inflammatory mediators from inflamed intestinal explants, while also mediating their trans-intestinal efflux from serosal-side lipoproteins. Conclusions: 4F appears to protect against IBD in part by inhibiting the accumulation of pro-inflammatory lipid mediators, through a mechanism that involves the intestinal clearance of these mediators from tissue and plasma.

Absorption and metabolism of volatile fatty acids by rumen and omasum

Volatile fatty acids (VFA) absorption and metabolic capacity of rumen and omasum were compared, in vitro. Fragments of rumen wall and omasum laminae were taken from eight adult crossbred bovines. An isolated fragment of the mucosa was fitted in a tissue diffusion chamber. Valeric acid and CrEDTA were added to ruminal fluid and placed on the mucosal side and buffer solution was placed on the serosal side. Fractional absorption rates were measured by exponential VFA:Cr ratio decay over time. Metabolism rate was determined as the difference between VFA absorbed and VFA which appeared on the serosal side over time. Mitotic index was higher in omasum (0.52%) than in rumen epithelium (0.28%). VFA fractional absorption rate was higher in omasum (4.6%/h.cm²) than in rumen (0.4%/h.cm²). Acetate, propionate, butyrate, and valerate showed similar fractional absorption rates in both fragments. Percentage of metabolized acetate and propionate was lower than butyrate and valerate in both stomach compartments. In the rumen, individual VFA metabolism rates were similar (mean of 7.7 , but in the omasum, valerate (90.0 was more metabolized than butyrate (59.6 propionate (69.8 and acetate (51.7 . Correlation between VFA metabolism and mitotic index was positive in the rumen and in the omasum. In conclusion, VFA metabolism and absorption potential per surface of the omasum is higher than that of the rumen. Variations on rumen and omasum absorption capacities occur in the same way, and there are indications that factors capable of stimulating rumen wall proliferation are similarly capable of stimulating omasum walls.

The relationships between potassium intakes, transmural potential difference of the rumen epithelium and magnesium absorption in wethers

In vitro studies with isolated sheep rumen epithelium have shown that an increase in the lumen K concentration induces an increase in the transmural potential difference across the rumen epithelium (serosal side: positive), which is associated with a decrease in Mg transport. However, at lumen K concentrations >80 mmol/l, Mg transport across the epithelium became independent of the lumen K concentration. The present study was carried out to determine whether this observation also occurs in vivo. Four ruminally fistulated wethers were fed four rations supplemented with KHCO3 (15·7, 37·6, 59·4 or 77·4 g K/kg DM) in a 4×4 Latin square design. Increased K intakes significantly increased the rumen K concentration. For all data combined, Mg absorption expressed as % intake was negatively correlated with the rumen K concentration. However, apparent Mg absorption either expressed in absolute terms (g/d) or as % intake was not significantly affected when the dietary K concentration was increased from 59·4 to 77·4 g/kg DM. Rumen K concentration was inversely correlated with the transmural potential difference (blood side: positive) (Pearson's r −0·709; R2adj 0·468, P=0·002, n 16). It is concluded that in wethers apparent Mg absorption becomes independent of the dietary K concentration when the K concentration is >60 g/kg DM or equivalent to a postprandial rumen K concentration of about 125 mmol/l.

Amino acids and carbohydrates absorption by Na+-dependent transporters in the pyloric ceca of Hoplias malabaricus (Erythrinidae)

Information about amino acids and carbohydrate absorption in fish is important to formulate an adequate diet to obtain optimal growth. Therefore, the objective of this study was to investigate if Na+-dependent transporters are involved on the absorption of glycine, L-glutamine, L-leucine, L-lysine, L-proline, L-alanine, and the carbohydrates fructose and glucose in the pyloric ceca of Hoplias malabaricus. The pyloric ceca were mounted in a system of continuous perfusion "in vitro". Amino acids and carbohydrates were placed on the mucosal side at concentrations of 10, 20, and 40mM. The serosal side of the pyloric ceca was positive in relation to the mucosal side. The addition of glycine, L-glutamine, L-leucine, L-lysine, L-proline (all tested concentrations), and glucose (at concentrations of 20 and 40mM) increased the positivity of the serosal side, indicating the presence of Na+-dependent transporters in the absorption of these substances. L-alanine and fructose did not change the positivity of the serosal side. The pyloric ceca seem to be the main site of nutrient absorption in the digestive tract of H. malabaricus.

BAFILOMYCIN A1 AT NANOMOLAR CONCENTRATIONS SATURABLY INHIBITS A PORTION OF TURTLE BLADDER ACIDIFICATION CURRENT

SUMMARY An earlier report indicated that acid secretion in turtle urinary bladder is driven by an unusual vacuolar H+-ATPase and that the ATPase accounts for essentially all acid secreted. These results, however, are difficult to reconcile with the acid transporters currently ascribed to the renal collecting duct. Here, we re-examine the effect of bafilomycin A1, an inhibitor of vacuolar (V-type) H+-ATPases, on acid secretion by intact isolated bladders from Pseudemys scriptaturtles. Serosal-side bafilomycin had no effect on the transepithelial acidification current (AC). In the mucosal solution, bafilomycin inhibited the AC, with inhibition developing over the range 0.1-10 nmol l-1, with a sigmoidal dose—response curve, and an IC50 of 0.47 nmol l-1. At saturation, approximately 70 % of H+ secretion was inhibited. The remaining 30 % could be abolished by 30 μmol l-1 Sch-28080, which is a level that in other systems is known to inhibit H+/K+-ATPase transport activity specifically and essentially completely. When the order of addition was reversed (Sch-28080 first), there was no change in the magnitude of the effect produced by either inhibitor, and the two together again eliminated the AC. The data indicate that baseline acid secretion in intact bladders is due (i) in part to a highly bafilomycin-sensitive process, with sensitivity typical of vacuolar H+ ATPases; and (ii) in part to a more bafilomycin-resistant process that is sensitive to Sch-28080.

Putrescine as a source of instant energy in the small intestine of the rat

Background and aims—It has been suggested that putrescine acts as a growth factor in the gut, but its exact function in some aspects of cellular metabolism is still in question. The aim of the present work was to identify some functions of putrescine in small bowel metabolism.Animals—Rats (about 80 g), in groups of five, were given either phytohaemagglutinin- or lactalbumin-containing diets, fed ad libitum or were fasted for 48 hours and re-fed for six or twelve hours before being killed.Methods—Uptake of intraperitoneally or intragastrically administered [14C]putrescine and its conversion to succinate by the rat small bowel mucosa was measured. Tissue polyamine and succinate contents were measured by high performance liquid chromatography and amino acid analysis respectively.Results—Uptake of putrescine by the small bowel mucosa from the systemic circulation and conversion of about 30% of this to succinate occurs in the epithelium of the healthy small bowel. Compared with rats given food ad libitum, putrescine uptake was doubled in fasted animals and more than 70% of it was converted to succinate. All these changes returned to control values on refeeding. Using phyto- haemagglutinin induced gut growth as a model, the uptake of putrescine from the systemic circulation by the serosal side of the small intestinal epithelium was increased immediately after growth was stimulated. During phytohaemagglutinin induced growth of the gut, putrescine was converted to succinate in the same proportion as in the healthy small bowel.Conclusions—The experiments identified a novel function for putrescine in gut metabolism: it can be used as an instant energy source when required.