In the porcine colon, the submucous plexus is divided into an inner submucous plexus (ISP) on the epithelial side and an outer submucous plexus (OSP) on the circular muscle side. Although both plexuses are probably involved in the regulation of epithelial functions, they might differ in function and neurochemical coding according to their localization. Therefore, we examined expression and co-localization of different neurotransmitters and neuronal markers in both plexuses as well as in neuronal fibres. Immunohistochemical staining was performed on wholemount preparations of ISP and OSP and on cryostat sections. Antibodies against choline acetyltransferase (ChAT), substance P (SP), somatostatin (SOM), neuropeptide Y (NPY), vasoactive intestinal peptide (VIP), neuronal nitric oxide synthase (nNOS) and the pan-neuronal markers Hu C/D and neuron specific enolase (NSE) were used. The ISP contained 1,380 ± 131 ganglia per cm2 and 122 ± 12 neurons per ganglion. In contrast, the OSP showed a wider meshwork (215 ± 33 ganglia per cm2) and smaller ganglia (57 ± 3 neurons per ganglion). In the ISP, 42% of all neurons expressed ChAT. About 66% of ChAT-positive neurons co-localized SP. A small number of ISP neurons expressed SOM. Chemical coding in the OSP was more complex. Besides the ChAT/±SP subpopulation (32% of all neurons), a nNOS-immunoreactive population (31%) was detected. Most nitrergic neurons were only immunoreactive for nNOS; 10% co-localized with VIP. A small subpopulation of OSP neurons was immunoreactive for ChAT/nNOS/±VIP. All types of neurotransmitters found in the ISP or OSP were also detected in neuronal fibres within the mucosa. We suppose that the cholinergic population in the ISP is involved in the control of epithelial functions. Regarding neurochemical coding, the OSP shares some similarities with the myenteric plexus. Because of its location and neurochemical characteristics, the OSP may be involved in controlling both the mucosa and circular muscle.

The expression of different coagulation factors suggests that RPE cells provide a procoagulant surface for the formation of thrombin from prothrombin via the extrinsic coagulation pathway. Thrombin stimulates the secretion of VEGF-A, the expression of FLT1, and the chemotaxis of RPE cells via transactivation of TGF-beta and PDGF receptors, respectively.

Background: Primary cultures of epithelial cells are suitable models for studying epithelial function and, in particular, the regulation of epithelial tightness in vitro. The aim of our study was to develop a protocol for the isolation and culture of porcine colonic epithelial cells and to establish transepithelial electrical resistance (TEER) as a functional parameter for epithelial tightness. Methods: Epithelial cells were obtained from the proximal colon of piglets by enzymatic dispase digestion. Cells were cultured on collagen-coated membrane supports for 21 days. The epithelial origin of the cells was shown by immunohistochemical detection of cytokeratin and zonula occludens protein 1 (ZO-1). Scanning electron microscopy, transmission electron microscopy and confocal microscopy were used for further morphological characterization. The integrity and tightness of the artificial epithelium were determined by measuring TEER. Results: The cultured epithelial cells were immunoreactive for cytokeratin and ZO-1. They showed dense microvilli on their apical membranes and expression of Na⁺/K⁺-ATPase on their basolateral membranes. Adjacent cells were connected by tight junctions. We observed TEER to continuously increase up to 870 ± 38 Ω·cm² during the culture period. TEER correlated with the amount of epithelial cells expressing ZO-1. Conclusions: The properties of primary cultured epithelial cells resemble the structural properties of polarized colonic epithelium in vivo. Measurement of TEER seems to be suitable for studying epithelial tightness in vitro. We suggest that these primary epithelial cultures be used to investigate the regulation of the epithelial barrier function.

Background: Within the gut, acetylcholine (ACh) is synthesised by enteric neurons, as well as by ‘non-neuronal' epithelial cells. In studies of non-intestinal epithelia, ACh was involved in the generation of an intact epithelial barrier. In the present study, primary cultured porcine colonocytes were used to determine whether treatment with exogenous ACh or expression of endogenous epithelium-derived ACh may modulate epithelial tightness in the gastrointestinal tract. Methods: Piglet colonocytes were cultured on filter membranes for 8 days. The tightness of the growing epithelial cell layer was evaluated by measuring transepithelial electrical resistance (TEER). To determine whether ACh modulates the tightness of the cell layer, cells were treated with cholinergic, muscarinic and/or nicotinic agonists and antagonists. Choline acetyltransferase (ChAT), cholinergic receptors and ACh were determined by immunohistochemistry, RT-PCR and HPLC, respectively. Results: Application of the cholinergic agonist carbachol (10 µm) and the muscarinic agonist oxotremorine (10 µM) resulted in significantly higher TEER values compared to controls. The effect was completely inhibited by the muscarinic antagonist atropine. Application of atropine alone (without any agonist) led to significantly lower TEER values compared to controls. Synthesis of ACh by epithelial cells was proven by detection of muscarinic and nicotinic receptor mRNAs, immunohistochemical detection of ChAT and detection of ACh by HPLC. Conclusion: ACh is strongly involved in the regulation of epithelial tightness in the proximal colon of pigs via muscarinic pathways. Non-neuronal ACh seems to be of particular importance for epithelial cells forming a tight barrier.

Chitosan (CS) and its derivatives show antimicrobial properties. This is of interest in preventing and treating denture stomatitis, which can be caused by fungi. Therefore, the aim of this study was the development of a novel antifungal denture base material by modifying polymethyl methacrylate (PMMA) with CS-salt and characterizing its antifungal and surface properties in vitro. For this purpose, the antifungal effect of chitosan-hydrochloride (CS-HCl) or chitosan-glutamate (CS-G) as solutions in different concentrations was determined. To obtain modified PMMA resin specimens, the CS-salts were added to the PMMA before polymerization. The roughness of these specimens was measured by contact profilometry. For the evaluation of the antifungal properties of the CS-salt modified resins, a C. albicans biofilm assay on the specimens was performed. As solutions, both the CS-G and CS-HCl-salt had an antifungal effect and inhibited C. albicans growth in a dose-dependent manner. In contrast, CS-salt modified PMMA resins showed no significant reduced C. albicans biofilm formation. Furthermore, the addition of CS-salts to PMMA significantly increased the surface roughness of the specimens. This study shows that despite the antifungal effect of CS-salts in solution, a modification of PMMA resin with these CS-salts does not improve the antifungal properties of PMMA denture base material.

Recent advances have been made in the understanding of Fanconi anemia (FA), a hereditary disease that increases the risk for head and neck squamous cell carcinomas (HNSCC) by 500- to 700-fold. FA patients harbour germline mutations in genes of cellular DNA repair pathways that are assumed to facilitate the accumulation of mutations during HNSCC development. Mutations in these FA genes may also contribute to HNSCC in general. In the present study, we analysed three FA genes; FANCF, FANCG and BRIP1, that are involved in the repair of DNA inter strand cross-links, in HNSCC and their potential role for patient survival. We measured loss of heterozygosity (LOH) mutations at eight microsatellite loci flanking three FA genes in 54 HNSCC of the oral cavity and corresponding blood samples. Survival analyses were carried out using mutational data and clinical variables. LOH was present in 17% (FANCF region), 41% (FANCG region) and 11% (BRIP1 region) of the patients. Kaplan-Meier survival curves and log-rank tests indicated strong clinical predictors (lymph node stages with decreased survival: p=2.69e-12; surgery with improved survival: p=0.0005). LOH in the FANCF region showed a weaker association with decreased overall survival (p=0.006), which however, did not hold in multivariate analyses. LOH may predominantly indicate copy number gains in FANCF and losses in FANCG and BRIP1. Integration of copy number data and gene expression proved difficult as the available sample sets did not overlap. In conclusion, LOH in FA genes appears to be a common feature of HNSCC development seen here in 57% of patients and other mutation types may increase this mutation frequency. We suggest larger patient cohorts would be needed to test the observed association of LOH in FANCF and patient survival comprehensively.

Bone drill chips that are collected during implant site preparation can be reused as autologous bone-grafting material for alveolar ridge augmentation. This study characterized five market-leading implant drill sets regarding their geometric properties and ability to produce vital bone chips. The drill geometry of each tool of five commercial implant drill sets was characterized while using optical profile projector devices and SEM. Bone chips were collected during the in vitro preparation of porcine jaw bone with the various drill sets. Produced bone chip masses were measured. The bone chips were cultured in vitro and the number of outgrown cells was determined and measurand for vitality. Furthermore, the thrust force and cutting torque were recorded to examine the mechanical loads of the manual drilling process. The tool geometry and set configuration of one out of five implant drill sets appears to be superior regarding chip mass, vitality, and thrust force. It could be proven that there is a correlation between vitality and thrust force. The thrust force is influenced by the cutting behavior of the tool, which in turn depends on the geometry of the tool. The tool geometry has an influence on the vitality of the augmentation material due to this relationship.

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