Semipermeable Hollow Fiber Membranes in Hepatocyte Bioreactors: A Prerequisite for a Successful Bioartificial Liver?

Abstract: Recent studies have shown that liver support systems based on viable hepatocytes can prolong life in animal models of acute liver failure. Now the time has come to elucidate the design characteristics that are essential to construct an efficient bioreactor. The gold standard remains the intact liver. Despite the very high cell density in this organ, individual cell perfusion is guaranteed resulting in low diffusional gradients which are essential for optimal mass transfer. These conditions are not met in bioreactors based on hollow fiber membranes. Moreover, the semipermeable membranes can foul and act as a diffusional barrier between the hepatocytes and the blood or plasma of the recipient. We devised a novel bioreactor for use as a bioartificial liver that does not include hollow fiber membranes for blood or plasma perfusion. The device is based on an integral oxygenator and a nonwoven polyester matrix material for hepatocyte culture as small aggregates. The efficacy of this original design was tested in rats with liver ischemia. Preliminary results show statistically significantly improved survival; life was prolonged 100% compared to the control experiments.     

Self‐perceived mouthfeel and physico‐chemical surface effects after chewing gums containing sorbitol and Magnolia bark extract

The European Food Safety Authority recognizes the contribution of sugar‐free chewing gum to oral health through increased salivation, clearance of food debris, and neutralization of biofilm pH. Magnolia bark extract is a gum additive shown to reduce the prevalence of bad‐breath bacteria but its effects on self‐perceived mouthfeel are unknown. This paper aims to relate the effects of sorbitol‐containing chewing gum, with and without Magnolia bark extract, on tooth‐surface hydrophobicity and salivary‐film composition with self‐perceived mouthfeel. In a crossover clinical trial, volunteers chewed sorbitol‐containing gum, with or without Magnolia bark extract added, three times daily during a 4‐wk time period. A subset of volunteers also chewed Parafilm as a mastication control. Oral moistness and tooth smoothness were assessed using questionnaires, and intra‐oral water‐contact angles were measured before, immediately after, and 60 min after, chewing. Simultaneously, saliva samples were collected, placed on glass slides, and the compositions of the adsorbed film were measured using X‐ray photoelectron spectroscopy. Chewing of gum, regardless of whether or not it contained Magnolia bark extract, improved self‐perceived mouthfeel up to 60 min, concurrent with a more hydrophilic tooth surface and an increased amount of O_1s electrons bound at 532.6 eV in salivary films. Chewing of Parafilm affected neither tooth‐surface hydrophobicity nor salivary‐film composition. Accordingly, adsorption of sorbitol, rather than the presence of Magnolia bark extract or increased salivation, is responsible for improved self‐perceived mouthfeel.     

Effects of Dietary Plant Sterols and Stanol Esters with Low- and High-Fat Diets in Chronic and Acute Models for Experimental Colitis

In this study, we evaluated the effects of dietary plant sterols and stanols as their fatty acid esters on the development of experimental colitis. The effects were studied both in high- and low-fat diet conditions in two models, one acute and another chronic model of experimental colitis that resembles gene expression in human inflammatory bowel disease (IBD). In the first experiments in the high fat diet (HFD), we did not observe a beneficial effect of the addition of plant sterols and stanols on the development of acute dextran sulphate sodium (DSS) colitis. In the chronic CD4CD45RB T cell transfer colitis model, we mainly observed an effect of the presence of high fat on the development of colitis. In this HFD condition, the presence of plant sterol or stanol did not result in any additional effect. In the second experiments with low fat, we could clearly observe a beneficial effect of the addition of plant sterols on colitis parameters in the T cell transfer model, but not in the DSS model. This positive effect was related to the gender of the mice and on Treg presence in the colon. This suggests that especially dietary plant sterol esters may improve intestinal inflammation in a T cell dependent manner.     

ATG16L1 and NOD2 polymorphisms enhance phagocytosis in monocytes of Crohn’s disease patients

AIM:To investigate if the presence of relevant genetic polymorphisms has effect on the effectual clearance of bacteria by monocytes and granulocytes in patients with Crohn’s disease(CD).METHODS:In this study,we assessed the differential responses in phagocytosis by measuring the phagocytic activity and the percentage of active phagocytic monocytes and granulocytes in inflammatory bowel disease patients as well as healthy controls.As both autophagy related like 1(ATG16L1)and immunityrelated guanosine triphosphatase gene are autophagy genes associated with CD and more recently nucleo-tide-binding ligomerization domain-containing protein2(NOD2)has been identified as a potent inducer of autophagy we genotyped the patients for these variants and correlated this to the phagocytic reaction.The genotyping was done with restriction fragment length polymorphisms analysis and the phagocytosis was determined with the pHrodo?Escherichia coli Bioparticles Phagocytosis kit for flowcytometry.RESULTS:In this study,we demonstrate that analysis of the monocyte and granulocyte populations of patients with CD and ulcerative colitis showed a comparable phagocytic activity(ratio of mean fluorescence intensity)between the patient groups and the healthy controls.CD patients show a significantly higher phagocytic capacity(ratio mean percentage of phagocytic cells)compared to healthy controls(51.91%±2.85%vs 37.67%±7.06%,P=0.05).The extend of disease was not of influence.However,variants of ATG16L1(WT:2.03±0.19 vs homozygoot variant:4.38±0.37,P<0.009)as well as NOD2(C-ins)(heterozygous variant:42.08±2.94 vs homozygous variant:75.58±4.34(P=0.05)are associated with the phagocytic activity in patients with CD.CONCLUSION:Monocytes of CD patients show enhanced phagocytosis associated with the presence of ATG16L1 and NOD2 variants.This could be part of the pathophysiological mechanism resulting in the disease.     

MRI characteristics of proctitis in Crohn’s disease on perianal MRI

Purpose

Multiple features have been described for assessing inflammation in Crohn’s disease (CD) in MR enterography, but have not been validated in perianal magnetic resonance imaging (MRI). Retrospectively, we studied which MRI features are valuable in assessing proctitis.

Materials and methods

CD patients (≥18 years) who underwent colonoscopy (reference standard) and perianal fistula MRI within 8 weeks were included. Seventeen MRI features were blindly scored by three observers and correlated to endoscopy (regression analysis). Reproducibility (multirater kappa, intraclass correlation coefficient) was determined for all three observer pairs. MRI features were considered relevant when significantly correlated to endoscopy for ≥2 observers, and reproducibility was ≥0.40 for ≥2 observer pairs.

Results

Perianal MRI of 58 CD patients were included. Wall thickness, rectal mural fat, creeping fat, and size of mesorectal lymph nodes showed a significant correlation with endoscopy for ≥2 observers (p = 0.000–0.023, p = 0.011–0.172, p = 0.007–0.011 and p = 0.000–0.005, respectively) with a kappa/intraclass correlation coefficient of ≥0.60 for ≥2 observer pairs. Perimural T2 signal and perimural enhancement significantly correlated to endoscopy (all p values ≤0.05) for all three observers and the reproducibility was ≥0.40 for ≥2 observer pairs. Mural T2 signal and degree and pattern of T1 enhancement showed significant correlation to endoscopy for two observers, but with poor to moderate reproducibility.

Conclusion

Wall thickness, mural fat, and mesorectal features (perimural T2 signal, perimural enhancement, creeping fat, and size of mesorectal lymph nodes) had significant correlation to endoscopy and were reproducible in diagnosing proctitis. Some established luminal features in MRE were considered not useful.

     

Quantitative structure-activity relationship analysis of the cation permeability of the P2X2 channel

The membrane-embedded, ligand-gated P2X glycoprotein receptor is a monovalent-bivalent cation channel that is activated by physiological concentrations of extracellular ATP. A quantitative structure-activity relationship (QSAR) analysis was developed to model the cation permeability of the P2X2 channel and its mutants. As chemical properties, the helix-coil equilibrium constants and the distribution coefficients of the system octanol/water at pH 7.4 were applied and modified (sliding windows) according to Eroshkin et al. (Comput. Appl. Biosci., 1995, 11, 49-44). The results were visualized by a dimeric P2X2 channel construct. The results support the hypothesis that residues which put into the cavity and contribute to hydrogen bonding forces are involved to a control of the transport of hydrated cations through the P2X2 channel. The model may be useful to develop P2X2 receptor antagonists.     

Arginine deficiency affects early B cell maturation and lymphoid organ development in transgenic mice

Apart from its role in the synthesis of protein and nitric oxide (NO), and in ammonia detoxification, the amino acid arginine exerts an immunosupportive function. We have studied the role of arginine in immune defense mechanisms in the developing postnatal immune system. In suckling mice, arginine is produced in the small intestine. In F/A-2(+/+) transgenic mice, which overexpress arginase in their enterocytes, circulating and tissue arginine concentrations are reduced to 30-35% of controls. In these mice, the development and composition of the T cell compartment did not reveal abnormalities. However, in peripheral lymphoid organs and the small intestine, B cell cellularity and the number and size of Peyer's patches were drastically reduced, and serum IgM levels were significantly decreased. These phenotypes could be traced to an impaired transition from the pro- to pre-B cell stage in the bone marrow. Cytokine receptor levels in the bone marrow were normal. The development of the few peripheral B cells and their proliferative response after in vitro stimulation was normal. The disturbance in B cell maturation was dependent on decreased arginine levels, as this phenotype disappeared upon arginine supplementation and was not seen in NO synthase- or ornithine transcarbamoylase-deficient mice. We conclude that arginine deficiency impairs early B cell maturation.     

Generation of regulatory gut-homing human T lymphocytes using ex vivo interleukin 10 gene transfer

BACKGROUND & AIMS: Systemic treatment of Crohn's disease patients using recombinant interleukin (rIL)-10 has not resulted in significant therapeutic benefit presumably because of limited bioavailability and unexpected proinflammatory effects of high-dose rIL-10. Ex vivo gene transfer of the interleukin (IL)-10 gene to gut-homing CD4(+) cells may lead to improved long-term management. METHODS: Peripheral blood mononuclear cells (PBMCs) were transduced with a retroviral vector containing the IL-10 and green fluorescent protein (GFP) gene or a control vector containing GFP only. Transduced CD4(+) cells were sorted and maintained in culture for phenotypic and functional analysis. RESULTS: Stimulated IL-10-GFP CD4(+) cells produced significantly higher levels of IL-10 than control cells for at least 4 months. The IL-10 transgene was biologically active and decreased proliferation of IL-10-GFP CD4(+) cells as well as expression of major histocompatibility class (MHC) class II, proliferation of autologous responder cells, and IL-12 production by dendritic cells (DCs). The majority of transduced CD4(+) cells had a gut-homing potential because they expressed the mucosal integrin alpha4beta7, and displayed efficient binding to MAdCAM-1-expressing cells in vitro. CONCLUSIONS: Transduction of peripheral blood CD4(+) lymphocytes with IL-10 results in a regulatory phenotype. The use of regulatory gut-homing human CD4(+) cells may provide a novel approach to local delivery of immunomodulatory signals to the intestine in Crohn's disease.     

Targeting regulatory factors to intranuclear replication sites

Plenty of evidence exists that mammalian nuclei are highly organized. Complex biochemical processes like DNA replication take place at specialized subnuclear sites and proteins directly or indirectly involved are concentrated at these sites. DNA replication is being used as a paradigm to study this functional organization of the nucleus, its underlying principles, and its potential regulatory consequences. In this review we discuss which factors were shown to be localized at nuclear replication sites, how they get there, and what role this might play in the precise, genome-wide regulation and coordination of complex biochemical processes.     

The role of interferon-gamma in murine pneumococcal pneumonia.

To determine the role of interferon (IFN)-gamma in pneumonia, IFN-gamma receptor-deficient (IFN-gamma R(-/-)) and 129/Sv (wild-type [wt]) mice were inoculated intranasally with Streptococcus pneumoniae. Although mortality did not differ between the groups 48 h after inoculation, IFN-gamma R(-/-) mice had significantly fewer pneumococci in their lungs than the wt mice. Similarly, IFN-gamma(-/-) mice had fewer colony-forming units in lungs than wt mice. The relatively increased resistance of IFN-gamma R(-/-) mice was not related to favorable effects on defense mechanisms known to contribute to antibacterial immunity-that is, the neutrophilic influx was reduced and the cytokine and nitric oxide levels were similar or lower in IFN-gamma R(-/-) mice. In contrast, mice treated with anti-IFN-gamma did not demonstrate a consistently altered bacterial outgrowth, compared with mice treated with a control antibody. These data suggest that endogenous IFN-gamma, despite its protective role in defense against intracellular pathogens, does not serve a protective role during pneumococcal pneumonia.