Bacteroides thetaiotaomicron-derived outer membrane vesicles promote regulatory dendritic cell responses in health but not in inflammatory bowel disease

Background: Bacteroides thetaiotaomicron (Bt) is a prominent member of the human intestinal microbiota that, like all Gram-negative bacteria, naturally generates nanosized outer membrane vesicles (OMVs) which bud off from the cell surface. Importantly, OMVs can cross the intestinal epithelial barrier to mediate microbe-host cell crosstalk involving both epithelial and immune cells to help maintain intestinal homeostasis. Here we have examined the interaction between Bt OMVs and blood or colonic mucosa-derived dendritic cells (DC) from healthy individuals and patients with Crohn’s disease (CD) or ulcerative colitis (UC). Results: In healthy individuals, Bt OMVs stimulated significant (p<0.05) IL-10 expression by colonic DC, whereas in peripheral blood-derived DC they also stimulated significant (p<0.001 and p<0.01, respectively) expression of IL-6 and the activation marker CD80. Conversely, in UC Bt OMVs were unable to elicit IL-10 expression by colonic DC. There were also reduced numbers of CD103 + DC in the colon of both UC and CD patients compared to controls, supporting a loss of regulatory DC in both diseases. Furthermore, in CD and UC, Bt OMVs elicited a significantly lower proportion of DC which expressed IL-10 (p<0.01 and p<0.001, respectively) in blood compared to controls. These alterations in DC responses to Bt OMVs were seen in patients with inactive disease, and thus are indicative of intrinsic defects in immune responses to this commensal in inflammatory bowel disease (IBD). Conclusions: Overall, our findings suggest a key role for OMVs generated by the commensal gut bacterium Bt in directing a balanced immune response to constituents of the microbiota locally and systemically during health which is altered in IBD patients

Characterization of Polyphosphoesters by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry

FT-ICR mass spectrometry, together with collision-induced dissociation and electron capture dissociation, has been used to characterize the polyphosphoester poly[1,4-bis(hydroxyethyl)terephthalate-alt-ethyloxyphosphate] and its degradation products. Three degradation pathways were elucidated: hydrolysis of the phosphate–[1,4-bis(hydroxyethyl)terephthalate]bonds; hydrolysis of the phosphate–ethoxy bonds; and hydrolysis of the ethyl–terephthalate bonds. The dominant degradation reactions were those that involved the phosphate groups. This work constitutes the first application of mass spectrometry to the characterization of polyphosphoesters and demonstrates the suitability of high mass accuracy FT-ICR mass spectrometry, with CID and ECD, for the structural analysis of polyphosphoesters and their degradation products

A Study of the PDGF Signaling Pathway with PRISM

In this paper, we apply the probabilistic model checker PRISM to the analysis of a biological system -- the Platelet-Derived Growth Factor (PDGF) signaling pathway, demonstrating in detail how this pathway can be analyzed in PRISM. We show that quantitative verification can yield a better understanding of the PDGF signaling pathway.Comment: In Proceedings CompMod 2011, arXiv:1109.104

Mitochondrial contact site and cristae organizing system (MICOS) machinery supports heme biosynthesis by enabling optimal performance of ferrochelatase

Heme is an essential cofactor required for a plethora of cellular processes in eukaryotes. In metazoans the heme biosynthetic pathway is typically partitioned between the cytosol and mitochondria, with the first and final steps taking place in the mitochondrion. The pathway has been extensively studied and its biosynthetic enzymes structurally characterized to varying extents. Nevertheless, understanding of the regulation of heme synthesis and factors that influence this process in metazoans remains incomplete. Therefore, we investigated the molecular organization as well as the physical and genetic interactions of the terminal pathway enzyme, ferrochelatase (Hem15), in the yeast Saccharomyces cerevisiae. Biochemical and genetic analyses revealed dynamic association of Hem15 with Mic60, a core component of the mitochondrial contact site and cristae organizing system (MICOS). Loss of MICOS negatively impacts Hem15 activity, affects the size of the Hem15 high-mass complex, and results in accumulation of reactive and potentially toxic tetrapyrrole precursors that may cause oxidative damage. Restoring intermembrane connectivity in MICOS-deficient cells mitigates these cytotoxic effects. These data provide new insights into how heme biosynthetic machinery is organized and regulated, linking mitochondrial architecture-organizing factors to heme homeostasis

Under pressure: Response urgency modulates striatal and insula activity during decision-making under risk

When deciding whether to bet in situations that involve potential monetary loss or gain (mixed gambles), a subjective sense of pressure can influence the evaluation of the expected utility associated with each choice option. Here, we explored how gambling decisions, their psychophysiological and neural counterparts are modulated by an induced sense of urgency to respond. Urgency influenced decision times and evoked heart rate responses, interacting with the expected value of each gamble. Using functional MRI, we observed that this interaction was associated with changes in the activity of the striatum, a critical region for both reward and choice selection, and within the insula, a region implicated as the substrate of affective feelings arising from interoceptive signals which influence motivational behavior. Our findings bridge current psychophysiological and neurobiological models of value representation and action-programming, identifying the striatum and insular cortex as the key substrates of decision-making under risk and urgency

Deficient resident memory T-cell and Cd8 T-cell response to commensals in inflammatory bowel disease

Background Aims: The intestinal microbiota is closely associated with resident memory lymphocytes in mucosal tissue. We sought to understand how acquired cellular and humoral immunity to the microbiota differ in health versus inflammatory bowel disease (IBD). Methods Resident memory T-cells (Trm) in colonic biopsies and local antibody responses to intraepithelial microbes were analyzed. Systemic antigen-specific immune T- and B-cell memory to a panel of commensal microbes was assessed. Results Systemically, healthy blood showed CD4 and occasional CD8 memory T-cell responses to selected intestinal bacteria but few memory B-cell responses. In IBD, CD8 memory T-cell responses decreased although B-cell responses and circulating plasmablasts increased. Possibly secondary to loss of systemic CD8 T-cell responses in IBD, dramatically reduced numbers of mucosal CD8+ Trm and γδ T-cells were observed. IgA responses to intraepithelial bacteria were increased. Colonic Trm expressed CD39 and CD73 ectonucleotidases, characteristic of regulatory T-cells. Cytokines/factors required for Trm differentiation were identified, and in vitro-generated Trm expressed regulatory T-cell function via CD39. Cognate interaction between T-cells and dendritic cells induced T-bet expression in dendritic cells, a key mechanism in regulating cell-mediated mucosal responses. Conclusions A previously unrecognized imbalance exists between cellular and humoral immunity to the microbiota in IBD, with loss of mucosal T-cell-mediated barrier immunity and uncontrolled antibody responses. Regulatory function of Trm may explain their association with intestinal health. Promoting Trm and their interaction with dendritic cells rather than immunosuppression may reinforce tissue immunity, improve barrier function and prevent B-cell dysfunction in microbiota-associated disease and IBD etiology

Role of Porphyromonas gingivalis gingipains in multi-species biofilm formation

BackgroundPeriodontal diseases are polymicrobial diseases that cause the inflammatory destruction of the tooth-supporting (periodontal) tissues. Their initiation is attributed to the formation of subgingival biofilms that stimulate a cascade of chronic inflammatory reactions by the affected tissue. The Gram-negative anaerobes Porphyromonas gingivalis, Tannerella forsythia and Treponema denticola are commonly found as part of the microbiota of subgingival biofilms, and they are associated with the occurrence and severity of the disease. P. gingivalis expresses several virulence factors that may support its survival, regulate its communication with other species in the biofilm, or modulate the inflammatory response of the colonized host tissue. The most prominent of these virulence factors are the gingipains, which are a set of cysteine proteinases (either Arg-specific or Lys-specific). The role of gingipains in the biofilm-forming capacity of P. gingivalis is barely investigated. Hence, this in vitro study employed a biofilm model consisting of 10 ¿subgingival¿ bacterial species, incorporating either a wild-type P. gingivalis strain or its derivative Lys-gingipain and Arg-gingipan isogenic mutants, in order to evaluate quantitative and qualitative changes in biofilm composition.ResultsFollowing 64 h of biofilm growth, the levels of all 10 species were quantified by fluorescence in situ hybridization or immunofluorescence. The wild-type and the two gingipain-deficient P. gingivalis strains exhibited similar growth in their corresponding biofilms. Among the remaining nine species, only the numbers of T. forsythia were significantly reduced, and only when the Lys-gingipain mutant was present in the biofilm. When evaluating the structure of the biofilm by confocal laser scanning microscopy, the most prominent observation was a shift in the spatial arrangement of T. denticola, in the presence of P. gingivalis Arg-gingipain mutant.ConclusionsThe gingipains of P. gingivalis may qualitatively and quantitatively affect composition of polymicrobial biofilms. The present experimental model reveals interdependency between the gingipains of P. gingivalis and T. forsythia or T. denticola

Low temperature method for the production of calcium phosphate fillers

BACKGROUND: Calcium phosphate manufactured samples, prepared with hydroxyapatite, are used as either spacers or fillers in orthopedic surgery, but these implants have never been used under conditions of mechanical stress. Similar conditions also apply with cements. Many authors have postulated that cements are a useful substitute material when implanted in vivo. The aim of this research is to develop a low cristalline material similar to bone in porosity and cristallinity. METHODS: Commercial hydroxyapatite (HAp) and monetite (M) powders are mixed with water and compacted to produce cylindrical samples. The material is processed at a temperature of 37–120 degrees C in saturated steam to obtain samples that are osteoconductive. The samples are studied by X-ray powder diffraction (XRD), Vickers hardness test (HV), scanning electron microscopy (SEM), and porosity evaluation. RESULTS: The X-ray diffractions of powders from the samples show patterns typical of HAp and M powders. After thermal treatment, no new crystal phase is formed and no increase of the relative intensity of the peaks is obtained. Vicker hardness data do not show any relationship with treatment temperature. The total porosity decreases by 50–60% according to the specific thermal treatment. Scanning electron microscopy of the surfaces of the samples with either HAp 80%-M 20% (c) or Hap 50%-M 50% (f), show cohesion of the powder grains. CONCLUSIONS: The dissolution-reprecipitation process is more intesive in manufactured samples (c) and (f), according to Vickers hardness data. The process occurs in a steam saturated environment between 37 degrees and 120 degrees C. (c) (f) manufactured samples show pore dimension distributions useful to cellular repopulation in living tissues