BACKGROUND AND AIMS:Inflammatory bowel diseases (IBD) result in chronic inflammation of the gastrointestinal tract. Genetic studies have shown that the GPR65 gene, as well as its missense coding variant, GPR65*Ile231Leu, is associated with IBD. We aimed to define the signalling and biological pathways downstream of GPR65 activation and evaluate the impact of GPR65*231Leu on these. METHODS:We used HEK 293 cells stably expressing GPR65 and deficient for either Gαs, Gαq/11 or Gα12/13, to define GPR65 signalling pathways, IBD patient biopsies and a panel of human tissues, primary immune cells and cell lines to determine biologic context, and genetic modulation of human THP-1-derived macrophages to examine the impact of GPR65 in bacterial phagocytosis and NLRP3 inflammasome activation. RESULTS:We confirmed that GPR65 signals via the Gαs pathway, leading to cAMP accumulation. GPR65 can also signal via the Gα12/13 pathway leading to formation of stress fibers, actin remodeling and RhoA activation; all impaired by the IBD-associated GPR65*231Leu allele. Gene expression profiling revealed greater expression of GPR65 in biopsies from inflamed compared to non-inflamed tissues from IBD patients or control individuals, potentially explained by infiltration of inflammatory immune cells. Decreased GPR65 expression in THP-1-derived macrophages leads to impaired bacterial phagocytosis, increased NLRP3 inflammasome activation and IL-1β secretion in response to an inflammatory stimulus. CONCLUSIONS:We demonstrate that GPR65 exerts its effects through Gαs- and Gα12/13-mediated pathways, that the IBD-associated GPR65*231Leu allele has compromised interactions with Gα12/13 and that KD of GPR65 leads to impaired bacterial phagocytosis and increased inflammatory signalling via the NLRP3 inflammasome. This work identifies a target for development of small molecule therapies.

BACKGROUND:Genome wide association studies (GWAS) have identified and validated more than 200 genomic loci associated with the inflammatory bowel disease (IBD), although for most the causal gene remains unknown. Given the importance of myeloid cells in IBD pathogenesis, the current study aimed to uncover the role of genes within IBD genetic loci that are endogenously expressed in this cell lineage. METHODS:The open reading frames (ORF) of 42 genes from IBD-associated loci were expressed via lentiviral transfer in the THP-1 model of human monocytes and the impact of each of these on the cell's transcriptome was analyzed using a RNA sequencing-based approach. We used a combination of genetic and pharmacologic approaches to validate our findings in the THP-1 line with further validation in human induced pluripotent stem cell (hiPSC)-derived-monocytes. RESULTS:This functional genomics screen provided evidence that genes in four IBD GWAS loci (PTGIR, ZBTB40, SLC39A11 and NFKB1) are involved in controlling S100A8 and S100A9 gene expression, which encode the two subunits of calprotectin (CP). We demonstrated that increasing PTGIR expression and/or stimulating PTGIR signaling resulted in increased CP expression in THP-1. This was further validated in hiPSC-derived monocytes. Conversely, knocking-down PTGIR endogenous expression and/or inhibiting PTGIR signaling led to decreased CP expression. These analyses were extended to the known IBD gene PTGER4, whereby its specific agonist also led to increased CP expression. Furthermore, we demonstrated that the PTGIR and PTGER4 mediated control of CP expression was dependent on signaling via adenylate cyclase and STAT3. Finally, we demonstrated that LPS-mediated increases in CP expression could be potentiated by agonists of PTGIR and PTGER4, and diminished by their antagonists. CONCLUSION:Our results support a causal role for the PTGIR, PTGER4, ZBTB40, SLC39A11 and NFKB1 genes in IBD, with all five genes regulating the expression of CP in myeloid cells, as well as potential roles for the prostacyclin/prostaglandin biogenesis and signaling pathways in IBD susceptibility and pathogenesis.

Intestinal permeability is increased in unaffected 1st degree relatives of patients with inflammatory bowel disease (IBD), and is considered a risk factor for the development of IBD, likely increasing the interactions between intestinal microorganisms and the immune system. We recently reported that C1orf106, a gene located within a genomic region associated with IBD, regulates epithelial permeability. We further demonstrated that a rare coding variant within C1orf106 (p.Y333F) decreases protein stability and that lower levels of C1orf106 protein leads altered stability of adherens junctions (AJ) and to an increase in epithelial permeability. In addition to altering AJ, we believe that C1orf106 is also involved in the regulation of tight junction (TJ) formation, which also impacts epithelial permeability. The objectives of the project are to (a) validate the impact of C1orf106 on tight junctions and (b) verify the impact of C1orf106 IBD-associated variants on intestinal barrier integrity. We observed that knocking down the expression of C1orf106 in Caco-2 cells leads to a number of phenotypes in human epithelial monolayer (2D) and spheroid (3D) cultures that are associated with alterations in TJs. Specifically, when studying the dynamic reformation of TJ in 2D cultures after transient withdrawal of calcium, which is required for TJ stability, we observed that lower levels of C1orf106 resulted in (1) decreased recovery of barrier function as measured by transepithelial electrical resistance (TEER); (2) an alteration of tight junction protein localization; and (3) thickening of the circumferential actin belt. Moreover, in 3D cultures, we observed an altered spheroid formation associated with impaired epithelial polarization. In addition, our preliminary studies of human induced pluripotent stem cell (hiPSC)-derived epithelial cultures support that Y333F heterozygotes also have altered structure and function of their tight junctions. Our observations indicate an important role of C1orf106 in apical junctional complex (AJC) formation likely mediated by a regulation of the circumferential actin belt. This can affect other functions of AJC, like the establishment of cell polarity. AJC formation is important for epithelial repair after an injury and its dysregulation impairs the formation of an impermeable epithelial barrier, which likely facilitates the passage of microorganisms and the induction and maintenance of intestinal inflammation.

Background Genetic studies have been tremendously successful in identifying genomic regions associated with a wide variety of phenotypes, although the success of these studies in identifying causal genes, their variants, and their functional impacts has been more limited. Methods We identified 145 genes from IBD-associated genomic loci having endogenous expression within the intestinal epithelial cell compartment. We evaluated the impact of lentiviral transfer of the open reading frame (ORF) of these IBD genes into the HT-29 intestinal epithelial cell line via transcriptomic analyses. By comparing the genes in which expression was modulated by each ORF, as well as the functions enriched within these gene lists, we identified ORFs with shared impacts and their putative disease-relevant biological functions . Results Analysis of the transcriptomic data for cell lines expressing the ORFs for known causal genes such as HNF4a, IFIH1, and SMAD3 identified functions consistent with what is already known for these genes. These analyses also identified two major clusters of genes: Cluster 1 contained the known IBD causal genes IFIH1, SBNO2, NFKB1, and NOD2, as well as genes from other IBD loci (ZFP36L1, IRF1, GIGYF1, OTUD3, AIRE and PITX1), whereas Cluster 2 contained the known causal gene KSR1 and implicated DUSP16 from another IBD locus. Our analyses highlight how multiple IBD gene candidates can impact on epithelial structure and function, including the protection of the mucosa from intestinal microbiota, and demonstrate that DUSP16 acts a regulator of MAPK activity and contributes to mucosal defense, in part via its regulation of the polymeric immunoglobulin receptor, involved in the protection of the intestinal mucosa from enteric microbiota. Conclusions This functional screen, based on expressing IBD genes within an appropriate cellular context, in this instance intestinal epithelial cells, resulted in changes to the cell’s transcriptome that are relevant to their endogenous biological function(s). This not only helped in identifying likely causal genes within genetic loci but also provided insight into their biological functions. Furthermore, this work has highlighted the central role of intestinal epithelial cells in IBD pathophysiology, providing a scientific rationale for a drug development strategy that targets epithelial functions in addition to the current therapies targeting immune functions.

Regulatory T cell (Treg) therapy is a potential curative approach for a variety of immune-mediated conditions, including autoimmunity and transplantation, in which there is pathological tissue damage. In mice, IL-33R (ST2)–expressing Tregs mediate tissue repair by producing the growth factor amphiregulin, but whether similar tissue-reparative Tregs exist in humans remains unclear. We show that human Tregs in blood and multiple tissue types produced amphiregulin, but this was neither a unique feature of Tregs nor selectively upregulated in tissues. Human Tregs in blood, tonsil, synovial fluid, colon, and lung tissues did not express ST2, so ST2+ Tregs were engineered via lentiviral-mediated overexpression, and their therapeutic potential for cell therapy was examined. Engineered ST2+ Tregs exhibited TCR-independent, IL-33–stimulated amphiregulin expression and a heightened ability to induce M2-like macrophages. The finding that amphiregulin-producing Tregs have a noneffector phenotype and are progressively lost upon TCR-induced proliferation and differentiation suggests that the tissue repair capacity of human Tregs may be an innate function that operates independently from their classical suppressive function.

Leigh syndrome French Canadian type (LSFC) is a mitochondrial disease caused by mutations in the leucine-rich pentatricopeptide repeat-containing (LRPPRC) gene leading to a reduction of cytochrome-c oxidase (COX) expression reaching 50% in skin fibroblasts. We have shown that under basal conditions, LSFC and control cells display similar ATP levels. We hypothesized that this occurs through upregulation of mechanistic target of rapamycin (mTOR)-mediated metabolic reprogramming. Our results showed that compared with controls, LSFC cells exhibited an upregulation of the mTOR complex 1 (mTORC1)/p70 ribosomal S6 kinase pathway and higher levels of hypoxia-inducible factor 1α (HIF-1α) and its downstream target pyruvate dehydrogenase kinase 1 (PDHK1), a regulator of mitochondrial pyruvate dehydrogenase 1 (PDH1). Consistent with these signaling alterations, LSFC cells displayed a 40-61% increase in [U-13C6]glucose contribution to pyruvate, lactate, and alanine formation, as well as higher levels of the phosphorylated and inactive form of PDH1-α. Interestingly, inhibition of mTOR with rapamycin did not alter HIF-1α or PDHK1 protein levels in LSFC fibroblasts. However, this treatment increased PDH1-α phosphorylation in control and LSFC cells and reduced ATP levels in control cells. Rapamycin also decreased LRPPRC expression by 41 and 11% in LSFC and control cells, respectively, and selectively reduced COX subunit IV expression in LSFC fibroblasts. Taken together, our data demonstrate the importance of mTORC1, independent of the HIF-1α/PDHK1 axis, in maintaining LRPPRC and COX expression in LSFC cells.

Polymorphisms in C1orf106 are associated with increased risk of inflammatory bowel disease (IBD). However, the function of C1orf106 and the consequences of disease-associated polymorphisms are unknown. Here we demonstrate that C1orf106 regulates adherens junction stability by regulating the degradation of cytohesin-1, a guanine nucleotide exchange factor that controls activation of ARF6. By limiting cytohesin-1-dependent ARF6 activation, C1orf106 stabilizes adherens junctions. Consistent with this model, C1orf106(-/-) mice exhibit defects in the intestinal epithelial cell barrier, a phenotype observed in IBD patients that confers increased susceptibility to intestinal pathogens. Furthermore, the IBD risk variant increases C1orf106 ubiquitination and turnover with consequent functional impairments. These findings delineate a mechanism by which a genetic polymorphism fine-tunes intestinal epithelial barrier integrity and elucidate a fundamental mechanism of cellular junctional control.

Regulatory T cell (Treg)-based therapy is a promising curative approach for allograft rejection. Beyond their effects on immune cells, emerging evidence suggests that Tregs have direct effects on tissue repair. Specifically, Tregs in mice promote tissue repair after infection or injury by secreting the EGF family member amphiregulin (AREG) under the control of alarmin IL-33 and its receptor ST2. We investigated the potential of human blood Tregs to mediate tissue repair via the IL-33/ST2 axis and AREG production. AREG expression was measured by flow cytometry in blood Tregs (flow-sorted as CD4+CD25+CD127-) stimulated with PMA and ionomycin. Human Tregs could produce AREG ex vivo, upregulated by TCR activation, but at a lower proportion than their Tconv counterparts (flow-sorted as CD4+CD25-CD127+). AREG expression was enriched in non-effector Tregs (CD39-, CCR4-, TIGIT-), a phenotype maintained after TCR activation. Moreover, AREG production potential was lost upon Treg proliferation and differentiation, suggesting that AREG production may comprise a distinct modality of human Tregs. In contrast to reports from mouse Tregs, IL-33 did not affect human blood Treg production of AREG. However, ST2 was undetectable in blood Tregs ex vivo and after activation. To more accurately measure human ST2 expression, we used phage display to generate a series of anti-ST2 antibodies. Experiments in transfected and endogenous ST2+ cells revealed several candidate antibodies that were superior to commercially available options for flow cytometric detection of human ST2. Investigations to define the tissue localization and biology of human ST2+ Tregs are in progress. Meanwhile, because of the importance of IL-33 signalling in promoting both the function and maintenance of mouse ST2+ Tregs in tissues, we sought to generate a plentiful source of human ST2+ Tregs to evaluate their potential as a cell therapy. Human blood naïve Tregs (flow-sorted as CD4+CD25+CD127-CD45RA+) were engineered to overexpress ST2 and expanded for 12 days with artificial antigen-presenting cells, anti-CD3, and IL-2. ST2 overexpression conferred IL-33 responsiveness, as determined by signal transduction and increased proliferation. IL-33 upregulated AREG in a TCR-independent manner on ST2-engineered Tregs, suggesting that the tissue repair capacity of human Tregs may be controlled innately. Overall, human Treg expression of AREG is associated with an innate-like program, potentially regulated by IL-33 and uncoupled from classical TCR-dependent Treg effector functions. Knowledge of the mechanisms by which human Tregs mediate tissue repair and the signals controlling this process will help delineate their therapeutic potential when used as a cell therapy. Thus, future investigations will focus on the tissue repair capacity of human Treg-derived AREG in vitro, as well as the in vivo functions of ST2-engineered Tregs in a humanized allotransplant model. CIHR Doctoral Research Award. CIHR Foundation Grant.

The French-Canadian variant of Leigh Syndrome (LSFC) is an autosomal recessive oxidative phosphorylation (OXPHOS) disorder caused by a mutation in LRPPRC, coding for a protein involved in the stability of mitochondrially-encoded mRNAs. Low levels of LRPPRC are present in all patient tissues, but result in a disproportionately severe OXPHOS defect in the brain and liver, leading to unpredictable subacute metabolic crises. To investigate the impact of the OXPHOS defect in the liver, we analyzed the mitochondrial phenotype in mice harboring an hepatocyte-specific inactivation of Lrpprc. Loss of LRPPRC in the liver caused a generalized growth delay, and typical histological features of mitochondrial hepatopathy. At the molecular level, LRPPRC deficiency caused destabilization of polyadenylated mitochondrial mRNAs, altered mitochondrial ultrastructure, and a severe complex IV (CIV) and ATP synthase (CV) assembly defect. The impact of LRPPRC deficiency was not limited to OXPHOS, but also included impairment of long-chain fatty acid oxidation, a striking dysregulation of the mitochondrial permeability transition pore, and an unsuspected alteration of trans-membrane H2O2 diffusion, which was traced to the ATP synthase assembly defect, and to changes in the lipid composition of mitochondrial membranes. This study underscores the value of mitochondria phenotyping to uncover complex and unexpected mechanisms contributing to the pathophysiology of mitochondrial disorders.

Purpose: The current standard reconstruction algorithm for computed tomography (CT) scans is filtered back projection. Alternative algorithms using iterative reconstruction (IR)-in our case, "sinogram affirmed iterative reconstruction"-have been increasingly implemented in diagnostic CT imaging. We studied its potential in improving radiation therapy planning images.Methods and materials: Raw planning CT data sets of patients from varied disease sites were reconstructed using filtered back projection and IR levels 1, 3, and 5 with equal radiation dose. For each site, 2-7 patient scans were selected; 2-3 physicians blindly evaluated the 4 3-dimensional image sets. Using a visual analogue scale, they rated the sharpness, noise, perceived ease in delineating gross tumor/clinical target volume and organs at risk, and overall appreciation of the images. Interobserver correlation was calculated with the Spearman correlation coefficient (rho). Generalized estimating equations assessed the differences in the mean score for each criterion between reconstructions. When significant differences existed, pairwise comparisons compared the least-squares means. The preference for each reconstruction was rank ordered for each observer. The rank occurrences were computed; generalized estimating equations and pairwise comparisons were again calculated.Results: Sixteen physicians reviewed 548 image sets. The sharpness had a rho = -0.22-0.53, noise. = -0.34-0.38, delineating gross tumor/;clinical target volume rho = -0.28-0.53, delineating organs rho at risk rho = -0.47-0.42, and overall appreciation rho = -0.17-0.38, suggesting a low level of agreement among observers. IR 3 and 5 had consistently higher scores and ranks than filtered back projection (P =.02 and P =.015, respectively). Paradoxically, IR 5 scored both highest and worst the most frequently. IR 3 was more consistently well-ranked for all criteria.Conclusions: This report is the first to clinically evaluate IRin radiation therapy planning. When used to reduce noise in current CT simulation protocols, IR images were generally preferred. Although highly processed images polarized observers, the use of moderate IR was appreciated for most disease sites. (C) 2015 American Society for Radiation Oncology. Published by Elsevier Inc. All rights reserved.

IL-23 is part of the IL-12 family of cytokines and is composed of the p19 subunit specific to IL-23 and the p40 subunit shared with IL-12. IL-23 specifically contributes to the inflammatory process of multiple chronic inflammatory autoimmune disorders, including psoriasis, multiple sclerosis, inflammatory bowel disease, and rheumatoid arthritis. So far, one antibody targeting the shared p40 subunit of IL-12 and IL-23, Ustekinumab, is approved clinically to treat psoriasis. However, there are no treatments inhibiting specifically the IL-23 proinflammatory response. We have developed small IL-23R-specific antagonists by designing all D-peptides arising from flexible regions of IL-23R. Of these peptides, we selected 2305 (teeeqqly), since in addition to its soluble properties, it inhibited IL-23-induced STAT3 phosphorylation in spleen cells. Peptide 2305 specifically binds to IL-23R/IL-12Rβ1-expressing HEK-293 cells and not to cells devoid of the receptor. Peptide 2305 showed functional selectivity by modulating IL-23-induced gene expression in IL-23R/IL-12Rβ1-expressing cells and in Jurkat cells; 2305 does not inhibit IL-12-induced cytokine expression in IL-12Rβ-IL-12Rβ2-HEK-293 cells. Finally, compared with anti-p40 treatment, 2305 effectively and selectively inhibits IL-23-induced inflammation in three in vivo mouse models: IL-23-induced ear inflammation, anti-CD40-induced systemic inflammatory response, and collagen-induced arthritis. We, hereby, describe the discovery and characterization of a potent IL-23R small-peptide modulator, 2305 (teeeqqly), that is effective in vivo. 2305 may be more convenient, less cumbersome, less costly, and most importantly, more specific than current biologics for the treatment of inflammatory conditions, and conceivably complement the actual therapies for these chronic and debilitating inflammatory diseases.

IL-12 and IL-23 cytokines respectively drive Th1 and Th17 type responses. Yet, little is known regarding the biology of these receptors. As the IL-12 and IL-23 receptors share a common subunit, it has been assumed that these receptors are co-expressed. Surprisingly, we find that the expression of each of these receptors is restricted to specific cell types, in both mouse and human. Indeed, although IL-12Rβ2 is expressed by NK cells and a subset of γδ T cells, the expression of IL-23R is restricted to specific T cell subsets, a small number of B cells and innate lymphoid cells. By exploiting an IL-12- and IL-23-dependent mouse model of innate inflammation, we demonstrate an intricate interplay between IL-12Rβ2 NK cells and IL-23R innate lymphoid cells with respectively dominant roles in the regulation of systemic versus local inflammatory responses. Together, these findings support an unforeseen lineage-specific dichotomy in the in vivo role of both the IL-12 and IL-23 pathways in pathological inflammatory states, which may allow more accurate dissection of the roles of these receptors in chronic inflammatory diseases in humans.

Purpose/Objective(s)The current standard reconstruction algorithm for CT scans is filtered back projection (FBP). Recently, an alternative algorithm of iterative reconstruction (IR) has been increasingly implemented in the realm of diagnostic CT imaging, allowing decoupling of spatial resolution and image noise while reducing radiation doses. We studied a variant of IR, known as "Sinogram affirmed iterative reconstruction" (SAFIRE) and assessed its potential to improve the quality of radiation therapy (RT) planning images.Materials/MethodsRaw CT data sets of patients planned for brain, spine, head and neck, lung, breast, gastrointestinal, liver, gynecological, genitourinary and limb tumors were included. A total of 50 scans were acquired using our standard imaging protocols and reconstructed using FBP and SAFIRE levels 1, 3 and 5 (higher levels referring to more noise correction). For each disease site, two to seven scans were selected. For each site, 2 to 3 specialized radiation oncologists evaluated the 3D image sets in a blind fashion. Using a visual analogue scale, they assessed the image sharpness, noise, perceived ease in delineating gross tumor/clinical target volume (GTV/CTV) and organs at risk (OAR) and overall appreciation of the planning images. Inter-observer correlation was calculated with the Spearman correlation coefficient (ρ). Generalized estimating equations (GEA) assessed the differences in the mean score for each criteria, between reconstructions, adjusted for observer status. When there were significant differences, pairwise comparisons (PC) were done to compare the least-squares means. The preference for each scan was rank ordered for each observer. The mean rank across all observers and the ranks occurrences were computed. GEA was calculated again for the mean ranks; PC were done when differences existed.ResultsThe sharpness of borders had ρ = -0.22-0.53, the ease of GTV/CTV delineation ρ = -0.28-0.53, the ease of OAR delineation ρ = -0.47-0.42, the image noise ρ = -0.34-0.38 and the overall appreciation ρ = -0.17-0.38. Although there were discrepancies between physicians, SAFIRE levels 3 and 5 had consistently higher scores than FBP scans and were the highest rated scans for all criteria and for all disease sites (p = 0.02 and p = 0.015, respectively). Paradoxically, although SAFIRE level 5 scored well on average, it was ranked as worst the most often. SAFIRE level 3 was consistently well ranked for all criteria.ConclusionsThis report is the first to report the potential benefit of IR in RT planning scans. Although highly processed images polarized observers, the use of IR is globally preferred over standard FBP for planning CT scans. The preference for IR is seen for all disease sites, for all facets of images. This work will lead to clinical implementation of intermediate IR processing and further study investigating quantitatively IR's impact on contouring. Purpose/Objective(s)The current standard reconstruction algorithm for CT scans is filtered back projection (FBP). Recently, an alternative algorithm of iterative reconstruction (IR) has been increasingly implemented in the realm of diagnostic CT imaging, allowing decoupling of spatial resolution and image noise while reducing radiation doses. We studied a variant of IR, known as "Sinogram affirmed iterative reconstruction" (SAFIRE) and assessed its potential to improve the quality of radiation therapy (RT) planning images. The current standard reconstruction algorithm for CT scans is filtered back projection (FBP). Recently, an alternative algorithm of iterative reconstruction (IR) has been increasingly implemented in the realm of diagnostic CT imaging, allowing decoupling of spatial resolution and image noise while reducing radiation doses. We studied a variant of IR, known as "Sinogram affirmed iterative reconstruction" (SAFIRE) and assessed its potential to improve the quality of radiation therapy (RT) planning images. Materials/MethodsRaw CT data sets of patients planned for brain, spine, head and neck, lung, breast, gastrointestinal, liver, gynecological, genitourinary and limb tumors were included. A total of 50 scans were acquired using our standard imaging protocols and reconstructed using FBP and SAFIRE levels 1, 3 and 5 (higher levels referring to more noise correction). For each disease site, two to seven scans were selected. For each site, 2 to 3 specialized radiation oncologists evaluated the 3D image sets in a blind fashion. Using a visual analogue scale, they assessed the image sharpness, noise, perceived ease in delineating gross tumor/clinical target volume (GTV/CTV) and organs at risk (OAR) and overall appreciation of the planning images. Inter-observer correlation was calculated with the Spearman correlation coefficient (ρ). Generalized estimating equations (GEA) assessed the differences in the mean score for each criteria, between reconstructions, adjusted for observer status. When there were significant differences, pairwise comparisons (PC) were done to compare the least-squares means. The preference for each scan was rank ordered for each observer. The mean rank across all observers and the ranks occurrences were computed. GEA was calculated again for the mean ranks; PC were done when differences existed. Raw CT data sets of patients planned for brain, spine, head and neck, lung, breast, gastrointestinal, liver, gynecological, genitourinary and limb tumors were included. A total of 50 scans were acquired using our standard imaging protocols and reconstructed using FBP and SAFIRE levels 1, 3 and 5 (higher levels referring to more noise correction). For each disease site, two to seven scans were selected. For each site, 2 to 3 specialized radiation oncologists evaluated the 3D image sets in a blind fashion. Using a visual analogue scale, they assessed the image sharpness, noise, perceived ease in delineating gross tumor/clinical target volume (GTV/CTV) and organs at risk (OAR) and overall appreciation of the planning images. Inter-observer correlation was calculated with the Spearman correlation coefficient (ρ). Generalized estimating equations (GEA) assessed the differences in the mean score for each criteria, between reconstructions, adjusted for observer status. When there were significant differences, pairwise comparisons (PC) were done to compare the least-squares means. The preference for each scan was rank ordered for each observer. The mean rank across all observers and the ranks occurrences were computed. GEA was calculated again for the mean ranks; PC were done when differences existed. ResultsThe sharpness of borders had ρ = -0.22-0.53, the ease of GTV/CTV delineation ρ = -0.28-0.53, the ease of OAR delineation ρ = -0.47-0.42, the image noise ρ = -0.34-0.38 and the overall appreciation ρ = -0.17-0.38. Although there were discrepancies between physicians, SAFIRE levels 3 and 5 had consistently higher scores than FBP scans and were the highest rated scans for all criteria and for all disease sites (p = 0.02 and p = 0.015, respectively). Paradoxically, although SAFIRE level 5 scored well on average, it was ranked as worst the most often. SAFIRE level 3 was consistently well ranked for all criteria. The sharpness of borders had ρ = -0.22-0.53, the ease of GTV/CTV delineation ρ = -0.28-0.53, the ease of OAR delineation ρ = -0.47-0.42, the image noise ρ = -0.34-0.38 and the overall appreciation ρ = -0.17-0.38. Although there were discrepancies between physicians, SAFIRE levels 3 and 5 had consistently higher scores than FBP scans and were the highest rated scans for all criteria and for all disease sites (p = 0.02 and p = 0.015, respectively). Paradoxically, although SAFIRE level 5 scored well on average, it was ranked as worst the most often. SAFIRE level 3 was consistently well ranked for all criteria. ConclusionsThis report is the first to report the potential benefit of IR in RT planning scans. Although highly processed images polarized observers, the use of IR is globally preferred over standard FBP for planning CT scans. The preference for IR is seen for all disease sites, for all facets of images. This work will lead to clinical implementation of intermediate IR processing and further study investigating quantitatively IR's impact on contouring. This report is the first to report the potential benefit of IR in RT planning scans. Although highly processed images polarized observers, the use of IR is globally preferred over standard FBP for planning CT scans. The preference for IR is seen for all disease sites, for all facets of images. This work will lead to clinical implementation of intermediate IR processing and further study investigating quantitatively IR's impact on contouring.

Patients with very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency frequently present cardiomyopathy and heartbeat disorders. However, the underlying factors, which may be of cardiac or extra cardiac origins, remain to be elucidated. In this study, we tested for metabolic and functional alterations in the heart from 3- and 7-mo-old VLCAD null mice and their littermate counterparts, using validated experimental paradigms, namely, 1) ex vivo perfusion in working mode, with concomitant evaluation of myocardial contractility and metabolic fluxes using13C-labeled substrates under various conditions; as well as 2) in vivo targeted lipidomics, gene expression analysis as well as electrocardiogram monitoring by telemetry in mice fed various diets. Unexpectedly, when perfused ex vivo, working VLCAD null mouse hearts maintained values similar to those of the controls for functional parameters and for the contribution of exogenous palmitate to β-oxidation (energy production), even at high palmitate concentration (1 mM) and increased energy demand (with 1 μM epinephrine) or after fasting. However, in vivo, these hearts displayed a prolonged rate-corrected QT (QTc) interval under all conditions examined, as well as the following lipid alterations: 1) age- and condition-dependent accumulation of triglycerides, and 2) 20% lower docosahexaenoic acid (an omega-3 polyunsaturated fatty acid) in membrane phospholipids. The latter was independent of liver but affected by feeding a diet enriched in saturated fat (exacerbated) or fish oil (attenuated). Our finding of a longer QTc interval in VLCAD null mice appears to be most relevant given that such condition increases the risk of sudden cardiac death.

Purpose of the study: Recent studies have shown that the incidence of certain cancers would be due to ionising radiation received during diagnostic radiological explorations. It is thus important to optimise dosimetry. In this context, slot scanners have demonstrated potential for generating images with a quality comparable with conventional systems but with a considerable reduction in dose. We wanted to verify this proposition. Material and method: Radiographs were obtained in 50 scoliosis patients (posteroanterior and lateral incidences) using the slot scanner (EOS, Biospace) and with a conventional machine (FCR-7501S, Fuji). A dosimeter was placed on the patient after each exam. Phantoms were used to adjust radiographic parameters for each system in order to obtain comparable quality images. Patient images were then acquired ad the dose calculated at several entry points. These measures were used to compare skin radiation and to initialise a Monte-Carlo simulation calculating the effective dose. Two orthopaedic surgeons and two radiologists then evaluated the visibility of the structures of interest using a standard check list. They read the images in random order and were blinded to all information concerning the patient and the system used to acquire the images. Visibility was noted on a non-parametric scale with 4 levels. Wilcoxon’s test was used to compare the visibility scores. Results: Mean radiation of the skin in the thoracoab-dominal region varied from 0.11 to 0.30 mGy (effective dose 0.057 mSv) for the EOS and 0.73 to 2.47 mGy (effective dose 0.460 mSv) for the FCR-7501S. EOS provided significantly superior visibility for all structures (frontal view, p Discussion: Using the slot scanner, the patients received 6 to 9 times less radiation to the skin for the thoracoab-dominal region and an 8-fold reduction in effective dose than with the conventional system. In addition, the doses presented in the literature for the same exam are much higher than reported for EOS. Conclusion: The EOS slot scanner offers image quality which is globally superior to conventional systems while considerably reducing radiation dose.

CD36, a multifunctional protein, is involved in cardiac long chain fatty acid (LCFA) metabolism and in the etiology of heart diseases, yet the functional impact of Cd36 gene variants remains unclear. In 7-week-old spontaneously hypertensive rats (SHR), which, like humans, carry numerous mutations in Cd36, we tested the hypothesis that their restricted cardiac LCFA utilization occurs prior to hypertrophy due to defective CD36 post-translational modifications (PTM), as assessed by ex vivo perfusion of (13)C-labeled substrates and biochemical techniques. Compared to their controls, SHR hearts displayed a lower (i) contribution of LCFA to β-oxidation (-40%) and triglycerides (+2.8 folds), which was not explained by transcriptional changes or malonyl-CoA level, a recognized β-oxidation inhibitor, and (ii) membrane-associated CD36 protein level, but unchanged distribution. Other results demonstrate alterations in CD36 PTM in SHR hearts, specifically by N-glycosylation, and the importance of O-linked-β-N-acetylglucosamine for its membrane recruitment and role in LCFA use in the heart.

BACKGROUND:Crohn's disease (CD) and ulcerative colitis (UC) are inflammatory bowel diseases (IBDs) presumably caused by dysregulated immune responses to the gut microbiota. Genetic association studies have implicated dozens of chromosomal regions or loci in IBD susceptibility. The next challenge is to explain the individual role of each of these modest effect loci in the disease state. We have previously identified MAST3 as an IBD susceptibility gene through genetic fine-mapping of the 19p linkage region. Testing MAST3 in a reporter assay provided preliminary evidence that MAST3 modulates the activity of inflammation-related transcription factor nuclear factor kappa B. METHODS:Here we characterized the function of MAST3 through an examination of the influence of the modulation of MAST3 expression on endogenous genome-wide expression patterns. More specifically, we looked at differential gene expression resulting from overexpression and knockdown of the MAST3 gene in epithelial and macrophage cell lines. From we highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-jB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. RESULTS:We highlight a group of genes whose expression is modulated by MAST3 and correlate their expression with NF-κB activity. Their expression was found to be enriched in inflamed mucosal tissue of UC patients, confirming the importance of these genes in IBD. These MAST3-regulated genes are central to mucosal immune responses. Among them are proinflammatory cytokines (e.g., CCL20, IL8), regulators of NF-κB (e.g., TNFAIP3, LY96, NFKBIA), genes involved in interferon-induced defense against pathogen invasion (e.g., IFIT1, ISG15), and genes involved in cell adhesion and/or migration (e.g., CD44, TMOD1). CONCLUSIONS:Taken together, these results confirm MAST3 as a modulator of the inflammatory response through regulation of immune gene expression in the gut of IBD patients.