Fibrogenesis in Human Mucosa and Muscularis Precision-Cut Intestinal Slices.

In Crohn's Disease (CD), intestinal fibrosis is a prevalent yet unresolved complication arising from chronic and transmural inflammation. The histological assessment of CD intestines shows changes in tissue morphology in all the layers, including the mucosa and muscularis. This study aimed to determine the differences in fibrogenesis between mucosa and muscularis. Human precision-cut intestinal slices (hPCIS) were prepared from human intestine mucosa and muscularis and treated with TGF-ß1 and/or PDGF-BB for 72 h. Gene and protein expression and matrix metalloproteinase (MMP) activity were determined. The basal gene expression of various fibrosis markers was higher in muscularis compared to mucosa hPCIS. During incubation, Pro-Collagen-1A1 secretion increased in muscularis but not in mucosa hPCIS. MMP gene expression increased during incubation in mucosa and muscularis hPCIS, except for MMP9, MMP12, and MMP13 in muscularis hPCIS. Incubation with TGF-ß1 caused increased COL1A1 expression in the mucosa but not in muscularis hPCIS. In muscularis hPCIS, TGF-ß1 treatment caused a decrease in MMP1 and CTSK expression, while MMP13 was increased. In the presence of TGF-ß1, protease inhibitor expression was stable, except for SERPINE1, which was increased in muscularis hPCIS. We conclude that fibrogenesis is more pronounced in muscularis hPCIS compared to mucosa hPCIS, especially when stimulated with TGF-ß1.

Matrix metalloproteinases in intestinal fibrosis.

Intestinal fibrosis is a common complication in patients with inflammatory bowel disease [IBD], in particular Crohn's disease [CD]. Unfortunately, at present intestinal fibrosis is not yet preventable, and cannot be treated by interventions other than surgical removal. Intestinal fibrosis is characterized by excessive accumulation of extracellular matrix [ECM], which is caused by activated fibroblasts and smooth muscle cells. Accumulation of ECM results from an imbalanced production and degradation of ECM. ECM degradation is mainly performed by matrix metalloproteinases [MMPs], enzymes that are counteracted by tissue inhibitors of MMPs [TIMPs]. In IBD patients, MMP activity [together with other protease activities] is increased. At the same time, CD patients have a generally lower MMP activity compared to ulcerative colitis patients, who usually do not develop intestinal strictures or fibrosis. The exact regulation and role[s] of these MMPs in fibrosis are far from understood. Here, we review the current literature about ECM remodelling by MMPs in intestinal fibrosis and their potential role as biomarkers for disease progression or druggable targets.

Rifampicin Induces Gene, Protein, and Activity of P-Glycoprotein (ABCB1) in Human Precision-Cut Intestinal Slices.

P-glycoprotein (ABCB1), an ATP-binding cassette efflux transporter, limits intestinal absorption of its substrates and is a common site of drug-drug interactions. Drug-mediated induction of intestinal ABCB1 is a clinically relevant phenomenon associated with significantly decreased drug bioavailability. Currently, there are no well-established human models for evaluating its induction, so drug regulatory authorities provide no recommendations for in vitro/ex vivo testing drugs' ABCB1-inducing activity. Human precision-cut intestinal slices (hPCISs) contain cells in their natural environment and express physiological levels of nuclear factors required for ABCB1 induction. We found that hPCISs incubated in William's Medium E for 48 h maintained intact morphology, ATP content, and ABCB1 efflux activity. Here, we asked whether rifampicin (a model ligand of pregnane X receptor, PXR), at 30 µM, induces functional expression of ABCB1 in hPCISs over 24- and 48-h incubation (the time to allow complete induction to occur). Rifampicin significantly increased gene expression, protein levels, and efflux activity of ABCB1. Moreover, we described dynamic changes in ABCB1 transcript levels in hPCISs over 48 h incubation. We also observed that peaks of induction are achieved among donors at different times, and the extent of ABCB1 gene induction is proportional to PXR mRNA levels in the intestine. In conclusion, we showed that hPCISs incubated in conditions comparable to those used for inhibition studies can be used to evaluate drugs' ABCB1-inducing potency in the human intestine. Thus, hPCISs may be valuable experimental tools that can be prospectively used in complex experimental evaluation of drug-drug interactions.

Survival and cellular heterogeneity of epithelium in cultured mouse and rat precision-cut intestinal slices.

Precision-cut intestinal slices (PCIS) are used to study intestinal (patho)physiology, drug efficacy, toxicity, transport and metabolism ex vivo. One of the factors that limit the use of PCIS is a relatively short life-span. Moreover, culture-induced changes in cellular composition of PCIS remain largely uncharacterized. In this study, we demonstrated the epithelial cell heterogeneity in mouse and rat PCIS and its alterations during culture. In addition, we evaluated whether the presence of niche growth factors impacts the survival of PCIS epithelial cells. We showed that freshly prepared PCIS retained the main epithelial cell types, namely absorptive enterocytes, goblet cells, enteroendocrine cells, stem cells, transit-amplifying cells and Paneth cells. Once placed in culture, PCIS displayed progressive epithelial damage, and loss of these epithelial cell types. Cells comprising the intestinal stem cell niche were especially sensitive to the damage, and the addition of niche growth factors beneficially affected the survival of stem cells and transit-amplifying cells in PCIS during culture. In conclusion, this study provides new insights into the dynamic changes in cellular composition of epithelium in cultured PCIS, paving the way to future toxicological and pharmacological studies in an informed and reliable ex vivo setting.

Predictive Value of Precision-Cut Kidney Slices as an Ex Vivo Screening Platform for Therapeutics in Human Renal Fibrosis.

Animal models are a valuable tool in preclinical research. However, limited predictivity of human biological responses in the conventional models has stimulated the search for reliable preclinical tools that show translational robustness. Here, we used precision-cut kidney slices (PCKS) as a model of renal fibrosis and investigated its predictive capacity for screening the effects of anti-fibrotics. Murine and human PCKS were exposed to TGFß or PDGF pathway inhibitors with established anti-fibrotic efficacy. For each treatment modality, we evaluated whether it affected: (1) culture-induced collagen type I gene expression and interstitial accumulation; (2) expression of markers of TGFß and PDGF signaling; and (3) expression of inflammatory markers. We summarized the outcomes of published in vivo animal and human studies testing the three inhibitors in renal fibrosis, and drew a parallel to the PCKS data. We showed that the responses of murine PCKS to anti-fibrotics highly corresponded with the known in vivo responses observed in various animal models of renal fibrosis. Moreover, our results suggested that human PCKS can be used to predict drug efficacy in clinical trials. In conclusion, our study demonstrated that the PCKS model is a powerful predictive tool for ex vivo screening of putative drugs for renal fibrosis.

Decellularized Human Gut as a Natural 3D Platform for Research in Intestinal Fibrosis.

BACKGROUND: The current methodologies for the identification of therapeutic targets for inflammatory bowel disease (IBD) are limited to conventional 2-dimensional (2D) cell cultures and animal models. The use of 3D decellularized human intestinal scaffolds obtained from surgically resected intestine and engineered with human intestinal cells may provide a major advancement in the development of innovative intestinal disease models. The aim of the present study was to design and validate a decellularization protocol for the production of acellular 3D extracellular matrix (ECM) scaffolds from the human duodenum. METHODS: Scaffolds were characterized by verifying the preservation of the ECM protein composition and 3D architecture of the native intestine and were employed for tissue engineering with primary human intestinal myofibroblasts for up to 14 days. RESULTS: Engrafted cells showed the ability to grow and remodel the surrounding ECM. mRNA expression of key genes involved in ECM turnover was significantly different when comparing primary human intestinal myofibroblasts cultured in 3D scaffolds with those cultured in standard 2D cultures on plastic dishes. Moreover, incubation with key profibrogenic growth factors such as TGFß1 and PDGF-BB resulted in markedly different effects in standard 2D vs 3D cultures, further emphasizing the importance of using 3D cell cultures. CONCLUSIONS: These results confirm the feasibility of 3D culture of human intestinal myofibroblasts in intestinal ECM scaffolds as an innovative platform for disease modeling, biomarker discovery, and drug testing in intestinal fibrosis.

Growth factors of stem cell niche extend the life-span of precision-cut intestinal slices in culture: A proof-of-concept study.

Precision-cut intestinal slices (PCIS) is an ex vivo culture technique that found its applications in toxicology, drug transport and drug metabolism testing, as well as in fibrosis research. The main limiting factor of PCIS as experimental model is the relatively short viability of tissue slices. Here, we describe a strategy for extending the life-span of PCIS during culture using medium that is routinely used for growing intestinal organoids. Mouse and rat PCIS cultured in standard medium progressively showed low ATP/protein content and severe tissue degradation, indicating loss of tissue viability. In turn, organoid medium, containing epithelial growth factor (EGF), Noggin and R-spondin, maintained significantly higher ATP/protein levels and better preserved intestinal architecture of mouse PCIS at 96 h. In contrast, organoid medium that additionally contained Wnt, had a clear positive effect on the ATP content of rat PCIS during 24 h of culture, but not on slice histomorphology. Our proof-of-concept study provides early evidence that employing organoid medium for PCIS culture improved tissue viability during extended incubation. Enabling lasting PCIS cultures will greatly widen their range of applications in predicting long-term intestinal toxicity of xenobiotics and elucidating their mechanism of action, among others.

Passive inhalation of dry powder influenza vaccine formulations completely protects chickens against H5N1 lethal viral challenge.

Bird to human transmission of high pathogenicity avian influenza virus (HPAIV) poses a significant risk of triggering a flu pandemic in the human population. Therefore, vaccination of susceptible poultry during an HPAIV outbreak might be the best remedy to prevent such transmissions. To this end, suitable formulations and an effective mass vaccination method that can be translated to field settings needs to be developed. Our previous study in chickens has shown that inhalation of a non-adjuvanted dry powder influenza vaccine formulation during normal breathing results in partial protection against lethal influenza challenge. The aim of the present study was to improve the effectiveness of pulmonary vaccination by increasing the vaccine dose deposited in the lungs and by the use of suitable adjuvants. Two adjuvants, namely, Bacterium-like Particles (BLP) and Advax, were spray freeze dried with influenza vaccine into dry powder formulations. Delivery of dry formulations directly at the syrinx revealed that BLP and Advax had the potential to boost either systemic or mucosal immune responses or both. Upon passive inhalation of dry influenza vaccine formulations in an optimized set-up, BLP and Advax/BLP adjuvanted formulations induced significantly higher systemic immune responses than the non-adjuvanted formulation. Remarkably, all vaccinated animals not only survived a lethal influenza challenge, but also did not show any shedding of challenge virus except for two out of six animals in the Advax group. Overall, our results indicate that passive inhalation is feasible, effective and suitable for mass vaccination of chickens if it can be adapted to field settings.