Assessment of the effect of ethanol extracts from Cinnamomum camphora seed kernel on intestinal inflammation using simulated gastrointestinal digestion and a Caco-2/RAW264.7 co-culture system.

Cinnamomum camphora seeds have multiple bioactivities. There were few studies on the effect of C. camphora seeds on intestinal inflammation in vitro and in vivo. The study aimed to investigate the effects of ethanol extracts from C. camphora seed kernel on intestinal inflammation using simulated gastrointestinal digestion and a Caco-2/RAW264.7 co-culture system. Results showed that the digested ethanol extracts (dEE) were rich in polyphenols, and a total of 17 compounds were tentatively identified using UPLC-LTQ-Orbitrap-MS/MS. dEE increased cell viability, while decreasing the production of reactive oxygen species, and the secretion and gene expression of inflammatory markers (NO, PGE2, TNF-α, IL-1ß and IL-6). dEE also down-regulated NF-κB/MAPK pathway activities by suppressing the phosphorylation of relevant signaling molecules (p65, IκBα, ERK and p38), as well as the expression of TLR4 receptor protein. Furthermore, dEE may improve intestinal barrier function by increasing the TEER value, and the expression of tight junction proteins (ZO-1, claudin-1 and occludin). The results suggest the ethanol extracts from C. camphora seed kernel may have strong anti-inflammatory activities, and a potential application in the prevention or treatment of intestinal inflammation and enhancement of intestinal barrier function in organisms.

Functional Assessment of Intestinal Permeability and Neutrophil Transepithelial Migration in Mice using a Standardized Intestinal Loop Model.

The intestinal mucosa is lined by a single layer of epithelial cells that forms a dynamic barrier allowing paracellular transport of nutrients and water while preventing passage of luminal bacteria and exogenous substances. A breach of this layer results in increased permeability to luminal contents and recruitment of immune cells, both of which are hallmarks of pathologic states in the gut including inflammatory bowel disease (IBD). Mechanisms regulating epithelial barrier function and transepithelial migration (TEpM) of polymorphonuclear neutrophils (PMN) are incompletely understood due to the lack of experimental in vivo methods allowing quantitative analyses. Here, we describe a robust murine experimental model that employs an exteriorized intestinal segment of either ileum or proximal colon. The exteriorized intestinal loop (iLoop) is fully vascularized and offers physiological advantages over ex vivo chamber-based approaches commonly used to study permeability and PMN migration across epithelial cell monolayers. We demonstrate two applications of this model in detail: (1) quantitative measurement of intestinal permeability through detection of fluorescence-labeled dextrans in serum after intraluminal injection, (2) quantitative assessment of migrated PMN across the intestinal epithelium into the gut lumen after intraluminal introduction of chemoattractants. We demonstrate feasibility of this model and provide results utilizing the iLoop in mice lacking the epithelial tight junction-associated protein JAM-A compared to controls. JAM-A has been shown to regulate epithelial barrier function as well as PMN TEpM during inflammatory responses. Our results using the iLoop confirm previous studies and highlight the importance of JAM-A in regulation of intestinal permeability and PMN TEpM in vivo during homeostasis and disease. The iLoop model provides a highly standardized method for reproducible in vivo studies of intestinal homeostasis and inflammation and will significantly enhance understanding of intestinal barrier function and mucosal inflammation in diseases such as IBD.

Accuracy of real-time shear wave elastography in the assessment of normal small intestine mucosa in dogs.

The aim of the study was to assess the physiological stiffness of the normal canine jejunal mucosa based on shear wave elastography. The study was carried out on 60 dogs. In all the animals studied, the abdominal ultrasound was carried out using the SuperSonic Imagine Aixplorer system. The site of the jejunal elastography was determined using standard ultrasonography and all the measurements were carried out thrice. The stiffness of the area examined was determined during each measurement. Mean values were calculated based on the results obtained. The normal stiffness of the jejunal mucosa ranged from 1.305 kPa to 9.319 kPa (mean 5.31 ± 2.04 kPa). Based on our findings, we determined the range of normal values of the jejunal mucosal stiffness in healthy dogs. In addition, shear wave elastography was found to be safe and easy to perform. Moreover, it did not require anaesthesia or patient immobilisation for long periods.

Assessment of Crohn's Disease Activity: Magnetic Resonance Enterography in Comparison with Clinical and Endoscopic Evaluations.

Crohn's disease (CD) is a chronic inflammatory condition characterized by continuous mucosal damage and ongoing wound healing of the intestines. The fibrinolytic system is involved in early parts of the wound healing process. Fibrin is a key mediator of primary blood clot formation and is formed by cross-linking of fibrinogen. To gain insights into the dynamics of wound healing in CD patients we investigated the conversion of fibrinogen into fibrin by the pro-peptide FPA, the amount of factor XIII cross-linked fibrin and total fibrin clot. METHODS: Serum samples of 35 CD patients, 15 non-inflammatory bowel disease (non-IBD) patients and 39 age-matched healthy controls were analyzed for three novel neo-epitope markers: D-fragment and D-dimer, reflecting the degradation of total fibrin clot and factor XIII cross-linked fibrin, as well as FPA, reflecting synthesis of fibrin. RESULTS: Crohn's disease patients had a significantly lower D-dimer level (p=0.0001) compared to healthy controls. Crohn's disease and non-IBD patients had a significantly higher level of FPA (p<0.0001) and D-fragment/D-dimer ratio (p<0.0001 and p=0.02). FPA, D-dimer and D-fragment/D-dimer ratio could distinguish CD patients from healthy controls with area under the curve of 0.92 (95% CI 0.83-0.97), 0.78 (95% CI 0.67-0.87) and 0.85 (95% CI 0.75-0.93), respectively. CONCLUSION: Wound healing parameters were clearly changed in CD patients. FPA levels were higher in CD patients as compared to healthy controls, indicating more ongoing wound healing. D-dimer levels were lower in CD patients than in healthy controls, indicating impaired wound healing due to poor quality of factor XIII cross-linked fibrin and clot resolution.

Serological Assessment of the Quality of Wound Healing Processes in Crohn's Disease.

BACKGROUND AND AIMS: Crohn's disease (CD) is a chronic inflammatory condition characterized by continuous mucosal damage and ongoing wound healing of the intestines. The fibrinolytic system is involved in early parts of the wound healing process. Fibrin is a key mediator of primary blood clot formation and is formed by cross-linking of fibrinogen. To gain insights into the dynamics of wound healing in CD patients we investigated the conversion of fibrinogen into fibrin by the pro-peptide FPA, the amount of factor XIII cross-linked fibrin and total fibrin clot. METHODS: Serum samples of 35 CD patients, 15 non-inflammatory bowel disease (non-IBD) patients and 39 age-matched healthy controls were analyzed for three novel neo-epitope markers: D-fragment and D-dimer, reflecting the degradation of total fibrin clot and factor XIII cross-linked fibrin, as well as FPA, reflecting synthesis of fibrin. RESULTS: Crohn's disease patients had a significantly lower D-dimer level (p=0.0001) compared to healthy controls. Crohn's disease and non-IBD patients had a significantly higher level of FPA (p<0.0001) and D-fragment/D-dimer ratio (p<0.0001 and p=0.02). FPA, D-dimer and D-fragment/D-dimer ratio could distinguish CD patients from healthy controls with area under the curve of 0.92 (95% CI 0.83-0.97), 0.78 (95% CI 0.67-0.87) and 0.85 (95% CI 0.75-0.93), respectively. CONCLUSION: Wound healing parameters were clearly changed in CD patients. FPA levels were higher in CD patients as compared to healthy controls, indicating more ongoing wound healing. D-dimer levels were lower in CD patients than in healthy controls, indicating impaired wound healing due to poor quality of factor XIII cross-linked fibrin and clot resolution.

Assessment of small bowel mucosal healing by video capsule endoscopy for the prediction of short-term and long-term risk of Crohn's disease flare: a prospective cohort study.

BACKGROUND: The optimal monitoring strategy for predicting disease course in Crohn's disease remains undefined. We aimed to evaluate the accuracy, safety, and tolerability of an intensive monitoring strategy designed to predict the future course of Crohn's disease in patients with quiescent disease. METHODS: In a prospective observational cohort study, we recruited patients older than 18 years with quiescent (for 3-24 months) Crohn's disease involving the small bowel with confirmed small bowel patency from three tertiary medical centres in Israel. Enrolled patients underwent baseline magnetic resonance enterography (MRE) and patency capsule, clinical or biomarker assessment every 3 months, and video capsule endoscopy (VCE) at baseline and every 6 months for 2 years or until a clinical flare (the primary outcome, defined as an increase in the Crohn's disease activity index score by 70 points or more) or disease worsening necessitating treatment intensification. We assessed the ability of the different Crohn's disease monitoring methods used to predict the occurrence of a flare during the 24-month follow-up period. FINDINGS: Of 90 screened patients, 29 were excluded (17 because of non-patent small bowel). Of the 61 patients enrolled between July 3, 2013, and Feb 1, 2015, 17 (28%) had a flare during the 24-month follow-up. No clinicodemographic parameter predicted future flare. A baseline VCE Lewis score of 350 or more identified patients with future flare (area under the curve [AUC] 0·79, 95% CI 0·66-0·88; p<0·0001; hazard ratio 10·7, 3·8-30·3). C-reactive protein at baseline had an AUC of 0·73 (0·6-0·84; p=0·0013) for predicting flare. The AUC of baseline faecal calprotectin for the prediction of flare occurring within 2 years was 0·62 (0·49-0·74; p=0·17), but progressively improved for shorter timespans and reached an AUC of 0·81 (0·76-0·85) for the prediction of flare occurring within 3 months. Of four MRE-based indices, only MRE global score correlated with 2-year flare risk (AUC 0·71, 0·58-0·82; p=0·024). During follow-up, a Lewis score increase of 383 points or more from baseline predicted imminent disease exacerbation within 6 months (AUC 0·79, 0·65-0·89; p=0·011). The safety and tolerability of the 231 VCEs ingested was excellent, with none being retained. INTERPRETATION: In patients with quiescent Crohn's disease involving the small bowel, faecal calprotectin predicts short-term flare risk, whereas VCE predicts both short-term and long-term risk of disease exacerbation. If corroborated by additional studies, protocols incorporating VCE could expand the scope of available methods for monitoring disease activity and predicting outcomes in small bowel Crohn's disease. FUNDING: The Leona M & Harry B Helmsley Charitable Trust.

Advanced endoscopic techniques in the assessment of inflammatory bowel disease: new technology, new era.

Endoscopic assessment of inflammation and mucosal healing is crucial for appropriate management in IBD. Current definition of endoscopic mucosal healing has been derived using previous generation of standard white light endoscopes. New endoscopy technologies widely available provide much more detailed images of mucosal and vascular patterns. Novel endoscopic techniques with high definition image, optical and digital enhancement have enhanced the quality and fine details of vascular and mucosal pattern so that endoscopic images have started to reflect histological changes for lesions and inflammation/healing. These technologies can now define subtle inflammatory changes and increase detection and characterisation of colonic lesions in patients with IBD. The best endoscopic technique to detect dysplasia in IBD is still debated. Dye chromoendoscopy with targeted biopsies is considered by Surveillance for Colorectal Endoscopic Neoplasia Detection and Management in inflammatory Bowel Disease Patients: International Consensus Recommendations (SCENIC consensus the standard of care and recommended for adoption by gastroenterologists in practice. In future, it is possible that well-trained colonoscopists using high definition equipment with image enhancements may be able to obtain equivalent yield without pan-colonic dye spraying and characterise lesions. Finally, SCENIC introduced endoscopic resectability of some dysplastic colonic lesions-new techniques may now better characterise endoscopic resectability and limit the number of colectomies. In this review, we will provide a state-of-the-art opinion on the direction of technological advances in the assessment of IBD and how new concepts will refine clinical practice.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions.NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.

Mechanically evoked cortical potentials: A physiological approach to assessment of anorectal sensory pathways.

BACKGROUND: Normal defaecation involves activation of anorectal mechanoreceptors responsive to pressure and stretch. The aim of this study was to develop selective anal and rectal mucosal light-touch stimulation suitable for measurement of cortical evoked potentials (EPs) in order to explore the sensory arm of these pathways. NEW METHOD: A novel device was manufactured to deliver selective rectal and/or anal light-touch stimulation using a shielded inter-dental brush mounted on a rotating stepper motor (1Hz, 1ms, 15° rotation). Resultant somatosensory EPs recorded with a 32-channel cortical multi-electrode array were compared to those elicited by electrical anorectal stimulation (2mm anal plug electrode [1Hz, 1ms, 10V]). RESULTS: Eighteen anaesthetized female Wistar rats (body mass 180-250g) were studied. Electrical and mechanical stimulation provoked similar maximal response amplitudes (electrical anorectal 39.0µV[SEM 5.5], mechanical anal 42.2µV[8.1], mechanical rectal 45.8µV[9.0]). Response latency was longer following mechanical stimulation (electrical anorectal 8.8ms[0.5], mechanical anal 16.4ms[1.1], mechanical rectal 18.3ms[2.5]). The extent of activated sensory cortex was smaller for mechanical stimulation. Sensory inferior rectal nerve activity was greater during anal compared to rectal mechanical in a subgroup of 4 rats. Evoked potentials were reproducible over 40min in a subgroup of 9 rats. COMPARISON WITH EXISTING METHODS: Cortical EPs are typically recorded in response to non-physiological electrical stimuli. The use of a mechanical stimulus may provide a more localized physiological method of assessment. CONCLUSIONS: To the authors' knowledge these are the first selective brush-elicited anal and rectal EPs recorded in animals and provide a physiological approach to testing of anorectal afferent pathways.

Confocal laser endomicroscopy: a new gold standard for the assessment of mucosal healing in ulcerative colitis.

BACKGROUND AND AIM: Endoscopic assessment of mucosal healing in ulcerative colitis (UC) is increasingly accepted as a measure of disease activity, therapeutic goal, and the key prognostic indicator. While regular endoscopy evaluates appearance of the mucosal surface, confocal laser endomicroscopy (CLE) enables in vivo visualization of subepithelial mucosa at 1000× magnification during ongoing endoscopy. Our aims were to determine using CLE whether endoscopically normal appearing colonic mucosa in patients with UC in remission (UC-IR) has fully regenerated mucosal structures, resolved inflammation, and to identify the mechanisms. METHODS: Twelve patients (six controls and six with UC-IR) underwent colonoscopy using CLE and intravenous fluorescein infusion. During colonoscopy, CLE images of colonic mucosa and conventional mucosal biopsies were obtained and evaluated using image-analysis systems. We quantified; (i) regeneration of colonic crypts and blood microvessels; (ii) cyclooxygenase 2 (COX2) expression; (iii) mitochondrial DNA (mtDNA) mutations; (iv) inflammatory infiltration; and (v) vascular permeability (VP). RESULTS: In control subjects, CLE demonstrated normal colonic crypts and microvasculature. COX2 expression was minimal, and < 7% crypts showed mtDNA mutations. Colonic mucosa of UC-IR patients had impaired and distorted crypt regeneration, increased COX2, 69% crypts with mtDNA mutations, persistent inflammation, and abnormal vascular architecture with increased VP (all P < 0.001 vs normal mucosa). CONCLUSIONS: (i) Endoscopically normal appearing colonic mucosa of patients with UC-IR remains abnormal: CLE demonstrates impaired crypt regeneration, persistent inflammation, distinct abnormalities in angioarchitecture and increased vascular permeability; molecular imaging showed increased COX2 and mtDNA mutations; (ii) CLE may serve as a new gold standard for the assessment of mucosal healing in UC.

In vitro assessment of epithelial electrical resistance in human esophageal and jejunal mucosae and in Caco-2 cell layers.

OBJECTIVE: There is a need for a technique allowing studies of human mucosal specimens collected during different clinical conditions. This study elucidates if square wave pulse analysis discriminates between epithelial and transmural electrical resistance and if there is an association with transepithelial permeability of molecular probes. METHODS: Mucosae from esophagus (surgical resections: n = 14; endoscopic biopsies: n = 15) and jejunum (n = 12) and Caco-2 cell monolayers were investigated in Ussing chambers. Transmural and epithelial electrical resistance were recorded by the use of standardized current pulses. Permeability was assessed using two fluorescein-labeled probes (weight 376 and 4000 Da). RESULTS: Baseline epithelial electrical resistance was higher in esophageal mucosa (~280 Ω*cm(2)), than in jejunal (~10 Ω*cm(2)) and Caco-2 cells (~140 Ω*cm(2)). The subepithelial contribution to the transmural resistance was higher in jejunal preparations (+88%) and Caco-2 cells (+75%), than in esophageal (+30%). During hypoxia the subepithelial resistance was unchanged, whereas the epithelial resistance decreased significantly in jejunal mucosa and Caco-2 cells. These findings coincided with increased transepithelial probe permeability and signs of disturbed morphology. Esophageal epithelia were resistant to hypoxia. However, exposure to deoxycholic acid and trypsin abolished the esophageal epithelial resistance and increased probe permeability. Endoscopic esophageal biopsies from patients with erosive reflux disease exhibited significantly lower epithelial resistance and higher current than healthy subjects. CONCLUSION: Square wave pulse analysis in Ussing chambers is suitable for assessment of epithelial electrical resistance that can reflect transepithelial permeability of molecular probes with known size. Moreover, the technique discriminated between healthy and reflux-diseased esophageal mucosal biopsies.

[Dietary treatment in acute pancreatitis]. / Leczenie iywieniowe w ostrym zapaleniu trzustki.

Proper nourishment is one of the basic elements in treatment patients suffering from acute pancreatitis and that's why it should be introduced in early phase of the disease. Patients suffering from light pancreatitis don't need dietary treatment because regular nourishment being.introduced a few days after the disease has developed itself. The proper supply of nourishing elements is crucial to patients with acute or chronic pancreatities. In this group of patient intravenous feeding or enteral nutrition methods are being used. They work as self sufficient and independent methods or they complete one another. The most recommended is the oligomeric diet with glutamine. Despite constant controversy over nourishment, early enternal nutrition is said to be better than intravenous feeding. Due to protection of intestinal barrier the enternal nutrition decrease the translocation of bacteria and endotoxin and as a result decrease the possibility of pancreaties parenchyma. The analysis of randomized clinical studies shows the improvement of clinical treatment and the improvement of prognosis in the group of patients using enternal nutrition. The number of complications and mortality rate has also decreased in this group. It allows also to shorten the hospitalization and cut the treatment costs. The patients, which do not tolerate internal nutrition or which cannot put up with intestinal entrance are to be fed with intravenous feeding. Presented above positive results of acute panctreatitis treatment, which were achieved after using internal nutrition, are the best basis for introducing this method in clinical practice.

PASSCLAIM--gut health and immunity.

BACKGROUND: The gut and immune system form a complex integrated structure that has evolved to provide effective digestion and defence against ingested toxins and pathogenic bacteria. However, great variation exists in what is considered normal healthy gut and immune function. Thus, whilst it is possible to measure many aspects of digestion and immunity, it is more difficult to interpret the benefits to individuals of variation within what is considered to be a normal range. Nevertheless, it is important to set standards for optimal function for use both by the consumer, industry and those concerned with the public health. The digestive tract is most frequently the object of functional and health claims and a large market already exists for gut-functional foods worldwide. AIM: To define normal function of the gut and immune system and describe available methods of measuring it. RESULTS: We have defined normal bowel habit and transit time, identified their role as risk factors for disease and how they may be measured. Similarly, we have tried to define what is a healthy gut flora in terms of the dominant genera and their metabolism and listed the many, varied and novel methods for determining these parameters. It has proved less easy to provide boundaries for what constitutes optimal or improved gastric emptying, gut motility, nutrient and water absorption and the function of organs such as the liver, gallbladder and pancreas. The many tests of these functions are described. We have discussed gastrointestinal well being. Sensations arising from the gut can be both pleasant and unpleasant. However, the characteristics of well being are ill defined and merge imperceptibly from acceptable to unacceptable, a state that is subjective. Nevertheless, we feel this is an important area for future work and method development. The immune system is even more difficult to make quantitative judgements about. When it is defective, then clinical problems ensure, but this is an uncommon state. The innate and adaptive immune systems work synergistically together and comprise many cellular and humoral factors. The adaptive system is extremely sophisticated and between the two arms of immunity there is great redundancy, which provides robust defences. New aspects of immune function are discovered regularly. It is not clear whether immune function can be "improved". Measuring aspects of immune function is possible but there is no one test that will define either the status or functional capacity of the immune system. Human studies are often limited by the ability to sample only blood or secretions such as saliva but it should be remembered that only 2% of lymphocytes circulate at any given time, which limits interpretation of data. We recommend assessing the functional capacity of the immune system by: measuring specific cell functions ex vivo. measuring in vivo responses to challenge, e. g. change in antibody in blood or response to antigens. determining the incidence and severity of infection in target populations during naturally occurring episodes or in response to attenuated pathogens.

Intestinal permeability assessment before and after ileal pouch-anal anastomosis.

AIM: Intestinal permeability is considered an index of anatomic and functional integrity of the small intestine mucosa. Altered intestinal permeability has been suggested to be a possible cause of pouchitis. Aim of this paper was to assess variations in intestinal permeability during the first year of a pouch reconstruction. METHODS: Intestinal permeability (IP) was investigated in 8 ulcerative colitis patients before and after total proctocolectomy, with ileal pouch-anal anastomosis (IPAA), by means of the cellobiose/mannitol test. To each patient a basal test (before surgery) and 3 more tests during a 1 year follow-up were administered. RESULTS: Individual data were altered despite clinical findings in 9 of 30 IP measured values. An overall pattern of unaffected permeability was however shown and none of our patients, during the first year follow-up, has developed pouchitis. CONCLUSIONS: Six of the 8 investigated patients presented at least 1 altered IP value. A longer follow-up aimed to further investigate patients beyond the first year after IPAA confection as to the occurrence of pouchitis and its possible correlation with a previous permeability alteration of the pouch mucosa is in progress.

Experimental assessment of small intestinal submucosa as a small bowel graft in a rat model.

BACKGROUND/PURPOSE: Small intestinal submucosa (SIS) is an extracellular matrix used in tissue engineering. The purpose of this study is to evaluate the feasibility of using SIS as a scafford for small bowel regeneration in a rat model. METHODS: A 2-cm length tubular SIS graft from donor Sprague Dawley rats was interposed with bilateral anastomosis in the median tract of an isolated ileal loop of Lewis rats used to construct an ileostomy. The grafts were harvested and analyzed at each of the time-points ranging from 2 weeks to 24 weeks after operation using histology and immunohistochemistry. RESULTS: Macroscopic examination found no adhesion in the surrounding area of neointestine by 24 weeks, and no stenosis was visible. The shrinkage of neointestine was indicated from 20% to 40%. Histologic and immunohistochemical evaluation showed that SIS grafts were colonized by numerous inflammation cells by 2 weeks. Neovascularization was evident, but the luminal surface was not epithelized. By 4 weeks, transitional mucosal epithelial layer began to line the luminal surface of the graft, and nearly 70% luminal surface of the graft had been covered by mucosal epithelium at 8 weeks. By 12 weeks, the luminal surface was covered completely by a mucosal layer with distinct bundles of smooth muscle cells in the neointestine. At 24 weeks, the neointestine wall showed 3 layers of mucosa, smooth muscle, and serosa. CONCLUSIONS: The preliminary study suggested that SIS allow rapid regeneration of mucosa and smooth muscle and might be a viable material for the creation of neointestine.

In vitro biocompatibility assessment of naturally derived and synthetic biomaterials using normal human urothelial cells.

The reconstruction of urinary tissues often employs various types of biomaterials, and adequate material biocompatibility is essential for the successful reconstruction of urinary tissues. In this study we utilized a primary normal human urothelial cell culture system to evaluate the in vitro biocompatibility of a number of naturally derived biomaterials [i.e., bladder submucosa, small intestinal submucosa, collagen, and alginate] and polymeric biomaterials [i.e., poly(glycolic acid), poly(L-lactic acid), poly(lactic-co-glycolic acid), and silicone] that are either experimentally or clinically used in urinary reconstructive surgery. To determine the cytotoxic and bioactive effects of these biomaterials, the cell viability, metabolic activity, apoptotic properties, and DNA-synthesis activity were measured with four types of assays [Neutral Red, 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyl tetrazolium bromide, apoptotic activity, and tritiated thymidine incorporation assays] using extract and direct contact methods. Most of the biomaterials tested did not induce significant cytotoxic effects and exhibited normal metabolic function and cell growth in vitro. This normal primary human urothelial cell culture model is suitable for in vitro biocompatibility assessments and is able to provide information on the cell-biomaterial interactions and the ability of biomaterials to support bioactive cell functions.

In vivo assessment of villous microcirculation in the rat small intestine in indomethacin-induced inflammation: role of mast-cell stabilizer Ketotifen.

Indomethacin induces an inflammatory reaction in the small intestine in several rat strains. This animal model is widely used to study the implications of intestinal inflammation. Macroscopically, there is evidence that Indomethacin induces a hyperaemic inflammatory reaction in the mucosa, and in our previous studies, we found a significant increase in villous perfusion in the acutely inflamed small intestine. Mast-cell activation was found to take part in inflammatory reactions in several organ systems in various species. However, no data are available about the effect of mast-cell degranulation on the perfusion of the mucosa in Indomethacin-induced intestinal inflammation. Therefore, we used the mast-cell stabilizer Ketotifen to identify if mast-cell activation may contribute to changes in the perfusion of single villi in the rat ileum. We found that Ketotifen significantly reduced the Indomethacin induced increased blood flow in the main arteriole 60 min after i.v. application, indicating that mast-cell degranulation effects microcirculatory disturbances in the mucosa in Indomethacin-induced intestinal inflammation.

Analysis of differential gene expression in human colorectal tumor tissues by RNA arbitrarily primed-PCR: a technical assessment.

RNA arbitrarily primed (RAP)-PCR is a powerful tool for studying differential gene expression in cancer cells. Systematic analysis of human tumor samples may provide a list of markers with potential application to the diagnosis, prognostic assessment, and treatment of the disease. Nevertheless, because of characteristics inherent to the samples and technique, artifactual results are likely. We have assessed the effects of several factors on RAP-PCR performance to determine the sensitivity and reproducibility of the technique, as well as the accuracy of its results, under different conditions in human cell lines and in a series of 129 paired human normal colonic mucosa-colorectal carcinoma samples. Our results show that RAP-PCR provides reliable fingerprints in a relatively wide spectrum of circumstances, including variations in RNA concentration and contamination by DNA. Densitometric analysis indicated that relative band-intensity variations more than 20% were reproducible in 95% of the cases. Serial analysis of paired normal-tumor cases yielded a number of bands that were recurrently either underexpressed or overexpressed in tumor relative to normal mucosa. These differentially expressed bands are prime targets of research because they represent candidate tumor-specific up- or down-regulated genes with a relevant role in carcinogenesis.