Ida Schoultz

Combined Polymorphisms in Genes Encoding the Inflammasome Components NALP3 and CARD8 Confer Susceptibility to Crohn's Disease in Swedish Men

OBJECTIVES:Crohns disease (CD) is characterized by overproduction of proinflammatory cytokines like interleukin (IL)-1β. Production of mature IL-1β is dependent on a caspase-1-activatingprotein complex called the NALP3 inflammasome, composed of NALP3, ASC, and CARD8. NALP3 shares structural similarities with Nod2, and both of these proteins are required forbacteria-induced IL-1β secretion. The combination of the polymorphisms CARD8 (C10X)and NALP3 (Q705K) was recently shown to be associated with rheumatoid arthritis.Our aim was to investigate whether these combined polymorphisms play a role in the susceptibility to CD.METHODS:The study included 498 CD patients in two cohorts from different regions and 742 control individuals from a Swedish population. DNA was isolated from whole blood. Polymorphisms of (Q705K) NALP3 and (C10X) CARD8, as well as the Nod2 variants, R702W and G908R, were genotyped using the Taqman single nucleotide polymorphism assay. The Nod2 frameshift mutation, L1007fs, was detected by Megabace SNuPe genotyping.RESULTS:Our results show that men who have both the C10X and Q705K alleles in CARD8 and NALP3, and who express wild-type alleles of Nod2 are at an increased risk of developing CD (odds ratio, OR: 3.40 range: 1.32–8.76); P=0.011). No association with these polymorphisms was found in women (OR: 0.89 (range: 0.44–1.77); P=0.74).CONCLUSIONS:We suggest a role for combined polymorphisms in CARD8 and NALP3 in the development of CD in men, with obvious sex differences in the genetic susceptibility pattern. These findings give further support to the importance of innate immune responses in CD.

TRIM27 Negatively Regulates NOD2 by Ubiquitination and Proteasomal Degradation

NOD2, the nucleotide-binding domain and leucine-rich repeat containing gene family (NLR) member 2 is involved in mediating antimicrobial responses. Dysfunctional NOD2 activity can lead to severe inflammatory disorders, but the regulation of NOD2 is still poorly understood. Recently, proteins of the tripartite motif (TRIM) protein family have emerged as regulators of innate immune responses by acting as E3 ubiquitin ligases. We identified TRIM27 as a new specific binding partner for NOD2. We show that NOD2 physically interacts with TRIM27 via the nucleotide-binding domain, and that NOD2 activation enhances this interaction. Dependent on functional TRIM27, ectopically expressed NOD2 is ubiquitinated with K48-linked ubiquitin chains followed by proteasomal degradation. Accordingly, TRIM27 affects NOD2-mediated pro-inflammatory responses. NOD2 mutations are linked to susceptibility to Crohns disease. We found that TRIM27 expression is increased in Crohns disease patients, underscoring a physiological role of TRIM27 in regulating NOD2 signaling. In HeLa cells, TRIM27 is partially localized in the nucleus. We revealed that ectopically expressed NOD2 can shuttle to the nucleus in a Walker A dependent manner, suggesting that NOD2 and TRIM27 might functionally cooperate in the nucleus. We conclude that TRIM27 negatively regulates NOD2-mediated signaling by degradation of NOD2 and suggest that TRIM27 could be a new target for therapeutic intervention in NOD2-associated diseases.

Yersinia pseudotuberculosis induces transcytosis of nanoparticles across human intestinal villus epithelium via invasin-dependent macropinocytosis

Crohns disease is characterized by a defect in intestinal barrier function, where bacteria are considered the most important inflammation-driving factor. Enteric bacteria, including E. coli and Yersinia spp, affect tight junctions in enterocytes, but little is known about bacterial effects on the transcellular pathway. Our objective was to study the short-term effects of Y. pseudotuberculosis on uptake of nanoparticles across human villus epithelium. Monolayers of human colon epithelium-derived Caco-2 cells and biopsies of normal human ileum were studied after 2 h exposure to Y. pseudotuberculosis expressing (inv+) or lacking (inv−) the bacterial adhesion molecule, invasin. Transepithelial transport of fluorescent nanoparticles (markers of transcytosis) was quantified by flow cytometry, and mechanisms explored by using inhibitors of endocytosis. Epithelial expressions of β1-integrin and particle uptake pathways were studied by confocal microscopy. The paracellular pathway was assessed by electrical resistance (TER), mannitol flux, and expression of tight junction proteins occludin and caludin-4 by confocal microscopy. Inv+ Y. pseudotuberculosis adhered to the apical surface of epithelial cells and induced transcytosis of exogenous nanoparticles across Caco-2 monolayers (30-fold increase, P<0.01) and ileal mucosa (268±47% of control; P<0.01), whereas inv− bacteria had no effect on transcytosis. The transcytosis was concentration-, particle size- and temperature-dependent, and possibly mediated via macropinocytosis. Y. pseudotuberculosis also induced apical expression of β1-integrin on epithelial cells. A slight drop in TER was seen after exposure to inv+ Y. pseudotuberculosis, whereas mannitol flux and tight junction protein expression was unchanged. In summary, Y. pseudotuberculosis induced apical expression of β1-integrin and stimulated uptake of nanoparticles via invasin-dependent transcytosis in human intestinal epithelium. Our findings suggest that bacterial factors may initiate transcytosis of luminal exogenous particles across human ileal mucosa, thus presenting a novel mechanism of intestinal barrier dysfunction.

Combinatorial diversity of fission yeast SCF ubiquitin ligases by homo- and heterooligomeric assemblies of the F-box proteins Pop1p and Pop2p

BackgroundSCF ubiquitin ligases share the core subunits cullin 1, SKP1, and HRT1/RBX1/ROC1, which associate with different F-box proteins. F-box proteins bind substrates following their phosphorylation upon stimulation of various signaling pathways. Ubiquitin-mediated destruction of the fission yeast cyclin-dependent kinase inhibitor Rum1p depends on two heterooligomerizing F-box proteins, Pop1p and Pop2p. Both proteins interact with the cullin Pcu1p when overexpressed, but it is unknown whether this reflects their co-assembly into bona fide SCF complexes.ResultsWe have identified Psh1p and Pip1p, the fission yeast homologues of human SKP1 and HRT1/RBX1/ROC1, and show that both associate with Pop1p, Pop2p, and Pcu1p into a ~500 kDa SCFPop1p-Pop2p complex, which supports polyubiquitylation of Rum1p. Only the F-box of Pop1p is required for SCFPop1p-Pop2p function, while Pop2p seems to be attracted into the complex through binding to Pop1p. Since all SCFPop1p-Pop2p subunits, except for Pop1p, which is exclusively nuclear, localize to both the nucleus and the cytoplasm, the F-box of Pop2p may be critical for the assembly of cytoplasmic SCFPop2p complexes. In support of this notion, we demonstrate individual SCFPop1p and SCFPop2p complexes bearing ubiquitin ligase activity.ConclusionOur data suggest that distinct homo- and heterooligomeric assemblies of Pop1p and Pop2p generate combinatorial diversity of SCFPop function in fission yeast. Whereas a heterooligomeric SCFPop1p-Pop2p complex mediates polyubiquitylation of Rum1p, homooligomeric SCFPop1p and SCFPop2p complexes may target unknown nuclear and cytoplasmic substrates.

Is metabolic stress a common denominator in inflammatory bowel disease

&NA; The enteric epithelium represents the major boundary between the outside world and the body, and in the colon it is the interface between the host and a vast and diverse microbiota. A common feature of inflammatory bowel disease (IBD) is decreased epithelial barrier function, and while a cause‐and‐effect relationship can be debated, prolonged loss of epithelial barrier function (whether this means the ability to sense bacteria or exclude them) would contribute to inflammation. While there are undoubtedly individual nuances in IBD, we review data in support of metabolic stress—that is, perturbed mitochondrial function—in the enterocyte as a contributing factor to the initiation of inflammation and relapses in IBD. The postulate is presented that metabolic stress, which can arise as a consequence of a variety of stimuli (e.g., infection, bacterial dysbiosis, and inflammation also), will reduce epithelial barrier function and perturb the enterocyte‐commensal flora relationship and suggest that means to negate enterocytic metabolic stress should be considered as a prophylactic or adjuvant therapy in IBD. (Inflamm Bowel Dis 2010;)

Indomethacin-induced translocation of bacteria across enteric epithelia is reactive oxygen species-dependent and reduced by vitamin C

The enteric epithelium must absorb nutrients and water and act as a barrier to the entry of luminal material into the body; this barrier function is a key component of innate immunity. Nonsteroidal anti-inflammatory drug (NSAID)-induced enteropathy occurs via inhibition of prostaglandin synthesis and perturbed epithelial mitochondrial activity. Here, the direct effect of NSAIDs [indomethacin, piroxicam (cyclooxygenase 1 and 2 inhibitors), and SC-560 (a cyclooxygenase 1 inhibitor)] on the barrier function of human T84 epithelial cell line monolayers was assessed by transepithelial electrical resistance (TER) and internalization and translocation of a commensal Escherichia coli. Exposure to E. coli in the presence and absence of drugs for 16 h reduced TER; however, monolayers cotreated with E. coli and indomethacin, but not piroxicam or SC-560, displayed significant increases in internalization and translocation of the bacteria. This was accompanied by increased reactive oxygen species (ROS) production, which was also increased in epithelia treated with E. coli only. Colocalization revealed upregulation of superoxide synthesis by mitochondria in epithelia treated with E. coli + indomethacin. Addition of antioxidants (vitamin C or a green tea polyphenol, epigallocathechin gallate) quenched the ROS and prevented the increase in E. coli internalization and translocation evoked by indomethacin, but not the drop in TER. Evidence of increased apoptosis was not observed in this model. The data implicate epithelial-derived ROS in indomethacin-induced barrier dysfunction and show that a portion of the bacteria likely cross the epithelium via a transcellular pathway. We speculate that addition of antioxidants as dietary supplements to NSAID treatment regimens would reduce the magnitude of decreased barrier function, specifically the transepithelial passage of bacteria.

Exploring the concept of optimal functionality in old age

Background Aging is characterized by loss of function and represents a perspective that puts the focus on the negative aspects of aging. Thus, it is fundamental to shift the focus from loss of function to maintaining good health and personal satisfaction through life; in other words, to promote optimal functionality at a level appropriate for older adults. However, it is not yet known what constitutes optimal functionality from the older adult’s own perspective. Objective To explore the concept of optimal functionality in old age from the older adult’s perspective (ie, people over 65 years of age) in industrialized Western countries. Methods We undertook a scoping review and searched two electronic databases (PubMed and the Cumulative Index to Nursing and Allied Health Literature [CINAHL]) from January 2002 to July 2013 for scientific studies, using the key search term personal satisfaction. In total, 25 scientific studies were analyzed. Results Only six of the included articles applied a qualitative methodology. By analyzing the results of these articles, three major themes were identified as cornerstones in the concept of optimal functionality at old age: 1) self-related factors (eg, mental well-being); 2) body-related factors (eg, physical well-being); and 3) external factors equal to demographic and environmental factors. Conclusion There is a lack of qualitative studies in the current literature, and hence of what constitutes optimal functionality from the older adult’s perspective. The results outlined in this review identify three cornerstones (self-related factors, body-related factors, and external factors) of what constitutes optimal functionality at old age. However, it is vital that these findings are taken further and are evaluated through qualitative studies to reflect older adults’ opinions.

A β-Glucan-Based Dietary Fiber Reduces Mast Cell-Induced Hyperpermeability in Ileum From Patients With Crohn’s Disease and Control Subjects

Abstract Background Administration of β-glucan has shown immune-enhancing effects. Our aim was to investigate whether β-glucan could attenuate mast cell (MC)-induced hyperpermeability in follicle-associated epithelium (FAE) and villus epithelium (VE) of patients with Crohn’s disease (CD) and in noninflammatory bowel disease (IBD)-controls. Further, we studied mechanisms of β-glucan uptake and effects on MCs in vitro. Methods Segments of FAE and VE from 8 CD patients and 9 controls were mounted in Ussing chambers. Effects of the MC-degranulator compound 48/80 (C48/80) and yeast-derived β-1,3/1,6 glucan on hyperpermeability were investigated. Translocation of β-glucan and colocalization with immune cells were studied by immunofluorescence. Caco-2-cl1- and FAE-cultures were used to investigate β-glucan-uptake using endocytosis inhibitors and HMC-1.1 to study effects on MCs. Results β-glucan significantly attenuated MC-induced paracellular hyperpermeability in CD and controls. Transcellular hyperpermeability was only significantly attenuated in VE. Baseline paracellular permeability was higher in FAE than VE in both groups, P<0.05, and exhibited a more pronounced effect by C48/80 and β-glucan P<0.05. No difference was observed between CD and controls. In vitro studies showed increased passage, P<0.05, of β-glucan through FAE-culture compared to Caco-2-cl1. Passage was mildly attenuated by the inhibitor methyl-β-cyclodextrin. HMC-1.1 experiments showed a trend to decreasing MC-degranulation and levels of TNF-α but not IL-6 by β-glucan. Immunofluorescence revealed more β-glucan-uptake and higher percentage of macrophages and dendritic cells close to β-glucan in VE of CD compared to controls. Conclusions We demonstrated beneficial effects of β-glucan on intestinal barrier function and increased β-glucan-passage through FAE model. Our results provide important and novel knowledge on possible applications of β-glucan in health disorders and diseases characterized by intestinal barrier dysfunction.

Increasing the qualitative understanding of optimal functionality in older adults: a focus group based study

BackgroundDecreased independence and loss of functional ability are issues regarded as inevitably connected to old age. This ageism may have negative influences on older adults’ beliefs about aging, making it difficult for them to focus on their current ability to maintain a good health. It is therefore important to change focus towards promoting Optimal Functionality (OF). OF is a concept putting the older adult’s perspective on health and function in focus, however, the concept is still under development. Hence, the aim was to extend the concept of optimal functionality in various groups of older adults.MethodsA qualitative study was conducted based on focus group discussions (FGD). In total 6 FGDs were performed, including 37 older adults from three different groups: group 1) senior athletes, group 2) free living older adults, group 3) older adults living in senior living homes. All data was transcribed verbatim and analyzed following the process of deductive content analysis.ResultsThe principal outcome of the analysis was “to function as optimally as you possibly can”, which was perceived as the core of the concept. Further, the concept of OF was described as multifactorial and several new factors could be added to the original model of OF. Additionally the findings of the study support that all three cornerstones comprising OF have to occur simultaneously in order for the older adult to function as optimal as possible.ConclusionsOF is a multifaceted and subjective concept, which should be individually defined by the older adult. This study further makes evident that older adults as a group are heterogeneous in terms of their preferences and views on health and should thus be approached as such in the health care setting. Therefore it is important to promote an individualized approach as a base when caring for older adults.

Are self-reported gastrointestinal symptoms among older adults associated with increased intestinal permeability and psychological distress?

BackgroundDespite the substantial number of older adults suffering from gastrointestinal (GI) symptoms little is known regarding the character of these complaints and whether they are associated with an altered intestinal barrier function and psychological distress. Our aim was to explore the relationship between self-reported gut health, intestinal permeability and psychological distress among older adults.MethodsThree study populations were included: 1) older adults with GI symptoms (n = 24), 2) a group of older adults representing the general elderly population in Sweden (n = 22) and 3) senior orienteering athletes as a potential model of healthy ageing (n = 27). Questionnaire data on gut-health, psychological distress and level of physical activity were collected. Intestinal permeability was measured by quantifying zonulin in plasma. The level of systemic and local inflammation was monitored by measuring C-reactive protein (CRP), hydrogen peroxide in plasma and calprotectin in stool samples. The relationship between biomarkers and questionnaire data in the different study populations was illustrated using a Principal Component Analysis (PCA).ResultsOlder adults with GI symptoms displayed significantly higher levels of both zonulin and psychological distress than both general older adults and senior orienteering athletes. The PCA analysis revealed a separation between senior orienteering athletes and older adults with GI symptoms and showed an association between GI symptoms, psychological distress and zonulin.ConclusionsOlder adults with GI symptoms express increased plasma levels of zonulin, which might reflect an augmented intestinal permeability. In addition, this group suffer from higher psychological distress compared to general older adults and senior orienteering athletes. This relationship was further confirmed by a PCA plot, which illustrated an association between GI symptoms, psychological distress and intestinal permeability.