DSS Models Research Articles

P169 Circulating eNAMPT predicts anti-TNF response in IBD patients: possible place in therapy of anti-eNAMPT antibody

Abstract Background extracellular Nicotinamide phosphoribosyltrasferase (eNAMPT) is a cytokine with paracrine and autocrine effects on different cell types. Importantly, eNAMPT levels are increased in patients suffering of Inflammatory Bowel Diseases (IBD). Biologic drugs have been found effective in many IBD patients; however, a large proportion of patients with severe disease fail to achieve remission due to lack of drug response, loss of response, drug intolerance, or severe side effects that require cessation of therapy. Therefore, there is a clinical need for predictive response biomarkers as well as for new therapeutic strategies. Methods First, we investigated the expression of NAMPT in biopsies, in stools and the secretion of eNAMPT in serum in four cohorts of IBD patients. Second, we investigated if circulating eNAMPT levels correlate with the clinical response to biologics (infliximab, adalimumab, ustekinumab, vedolizumab). Clinical response is defined as a reduction of >2 points in HBI (for CD) and in pMAYO (for UC) from baseline. Third, we have developed a monoclonal anti-eNAMPT antibody and we have evaluated its preclinical efficacy in acute and chronic preclinical models of IBD. Results We have determined the levels of circulating eNAMPT in three cohorts of patients that were not controlled by DMARDs and were treated with infliximab (IFX, cohort 1 and 3) or adalimumab (ADA, cohort 2). Notably, we confirmed a pronounced variability through the cohorts, identifying a group of patients with eNAMPT serum levels comparable with healthy adult populations and a group that showed elevated levels of eNAMPT. Performing a ROC curve analysis, a cutoff of 4.5 ng/ml can be extrapolated to discriminate these two populations. Noteworthy, 100% patients with levels of eNAMPT below 4.5 ng/ml were responsive to infliximab/adalimumab. In contrast, anti-TNF therapy failed either at 14 or 22 weeks in some patients with high circulating levels of eNAMPT, indicating that high systemic eNAMPT might be associated with an increased risk of resistance to anti-TNF therapy. Notably, we found also that eNAMPT levels in stools of IBD patients are elevated compared to healthy subjects. Then, we have developed and validated a candidate monoclonal antibody (called C269) which bind to eNAMPT, block is cytokine activity and reduces IBD symptoms, immune infiltrate and fibrosis in DSS and DNBS models. Conclusion Our data demonstrate that eNAMPT serum levels correlate with the clinical response to anti-TNF therapies suggesting that eNAMPT is not a simple by- stander of IBD, and that local and serum eNAMPT could be define as a biomarker to define the responsiveness to biologics. Notably, its neutralization might be a pharmacological strategy worth investigating.

P117 Transglutaminase 2 is elevated in Crohn’s disease associated strictures and exerts profibrotic activities in myofibroblasts and experimental intestinal fibrosis

Abstract Background Intestinal fibrosis is a significant clinical problem in Inflammatory bowel disease (IBD). This may lead to intestinal strictures induced by excessive accumulation of extracellular matrix (ECM) produced by human intestinal myofibroblasts (HIMF). Transglutaminase (TG)2 covalently cross-links ECM-associated proteins, increases ECM stiffness and has direct pro-fibrotic effects. We investigated the role of TG2 in intestinal fibrosis in human tissues, HIMF and transgenic mouse models. Methods TG2 was detected in human tissues from Crohn’s disease (CD) strictures (CDs; n=10), CD non-strictured (CDns; n=20), ulcerative colitis (UC; n=10) and non-IBD controls (NL; n=14) by immunofluorescence (IF), qPCR, in-situ hybridization (ISH), and biotinylated cadaverin incorporation (BCI). Relative contribution of TG2 was measured by BCI using a TG2-selective blocking antibody (TAb). The cellular source of TG2 was evaluated by IF. HIMF ECM production was measured using a novel ECM deposition assay and TG2-inhibitors. HIMF were exposed to anti-TG2 antibody or isotype control and bulk RNA-sequencing (RNAseq) analysis was performed. Tamoxifen-inducible global Cre/TG2 floxed conditional KO (cKO) mice were used in the DSS and TNBS fibrosis models. Results Total TG activity was increased in CDs and UC vs. NL in the submucosa (SM) and muscularis propria (MP; P≤ 0.04). Strikingly 90-99% of TG activity were attributable to TG2 in all groups. TG2 activity correlated with fibrosis scores (P≤0.02), but not inflammation scores. TG2 amount was elevated in CDs patients vs. NL in qPCR and ISH (P≤0.05). IF revealed HIMF as the major source of TG2 in the intestine as indicated by colocalization with alpha-SMA. HIMF, invitro, produced 82–97% of active TG2. Inhibition of TG2 by cell-permeable inhibitor or Tab reduced fibronectin (FN) and collagen (COL)1 and 3 deposition by HIMF. RNAseq showed TAb-treated cells expressed 432 downregulated and 402 upregulated genes. Functional enrichment analysis showed pathways associated with fibroblast activation/proliferation, ECM, and fibrosis-associated signaling (e.g. AKT) pathways to be downregulated in TAb-treated HIMF. Associated genes included nuclear protein (NUPR1) and sphingolipid signaling pathway proteins (SMPD1 and CTSD), which may be potential targets for TG2-mediated activity in HIMF. Deletion of TGM2 prior to inducing murine DSS or TNBS experimental fibrosis did not lead to any difference in inflammation, but decreased fibrosis (picrosirius red intensity, COL1/FN expression, wall thickness) in TG2 cKO mice vs. wild type (P≤0.05). Conclusion TG2 is involved in inflammation-independent progression of intestinal fibrosis and may represent a future anti-fibrotic target.

A numerical investigation of an indoor dry storage facility on heat transfer

This study conducted a numerical simulation of the dry storage system and indoor facility heat transfer problem. Four standard DSS models have been constructed to investigate the thermal performance. The results show a high-temperature difference found on the surfaces between the sealing canister and the cask, indicating a high thermal resistance in the radial direction. It is also found that the boundary conditions of DSS only affect the indoor facility ambient parameters instead of the inside components. The adjacent pitch distance between DSSs does not significantly change the indoor temperature but affects the view factor. However, convection accounts for 90% of the heat transfer, while radiation only contributes less than 4%. Thus, it is not essential to emphasize the indoor DSS arrangement optimization. This is the first time indoor facility DSS simulation offers suggestions and physical insights into DSS heat transfer, which is beneficial to nuclear energy science and technology.

Preventive role of Dietary Phytochemical Lupeol in Preclinical Ulcerative Colitis Models

Background: The primary cause of intestinal and colon inflammation is inflammatory bowel disease (IBD).Preclinical ulcerative colitis is poorly definedin this article we providea scientific validation in preclinical systemic inflammation in ulcerative colitis by the means of phytochemical lupeol as a source of triterpenoid rich food. Aim: The aim of the present study was to evaluate the bioactive phytochemical, lupeol for the management of IBD through two different experimental models. Method: The intestinal anti-inflammatory models for rat colitis were performed by using TNBS and DSS to access the use of lupeol (25μg/ml and 50μg/ml) in ulcerative colitis. Change in body weight, stool consistency, colon weight/length and histological examination of colon were performed. Results: Lupeol showed significant anti-inflammatory impact in the colon at both the doses in TNBS and DSS models. The results of lupeol showed marked evidence both by histologically and also by the tissue recovery seen in various parameters like, Change in body weight, stool consistency and colon weight/length. Conclusion: The findings of this study provide the full proof rationale for additional research using lupeol in the successful management of ulcerative colitis.

IL-20 Activates ERK1/2 and Suppresses Splicing of X-Box Protein-1 in Intestinal Epithelial Cells but Does Not Improve Pathology in Acute or Chronic Models of Colitis.

The cytokine Interleukin (IL)-20 belongs to the IL-10 superfamily. IL-20 levels are reported to increase in the intestines of Ulcerative Colitis (UC) patients, however not much is known about its effects on intestinal epithelial cells. Here, we investigated the influence of IL-20 on intestinal epithelial cell lines and primary intestinal organoid cultures. By using chemical-induced (dextran sodium sulphate; DSS) colitis and a spontaneous model of colitis (Winnie mice), we assess whether recombinant IL-20 treatment is beneficial in reducing/improving pathology. Following stimulation with IL-20, intestinal primary organoids from wild-type and Winnie mice increased the expression of ERK1/2. However, this was lost when cells were differentiated into secretory goblet cells. Importantly, IL-20 treatment significantly reduced endoplasmic reticulum (ER) stress, as measured by spliced-XBP1 in epithelial cells, and this effect was lost in the goblet cells. IL-20 treatment in vivo in the DSS and Winnie models had minimal effects on pathology, but a decrease in macrophage activation was noted. Taken together, these data suggest a possible, but subtle role of IL-20 on epithelial cells in vivo. The therapeutic potential of IL-20 could be harnessed by the development of a targeted therapy or combination therapy to improve the healing of the mucosal barrier.

Therapeutic role of ursodeoxycholic acid in colitis-associated cancer via gut microbiota modulation

Inflammatory bowel disease (IBD) is a predisposing factor for colitis-associated cancer (CAC). The association between bile acids and the gut microbiota has been demonstrated in colon neoplasia; however, the effect of ursodeoxycholic acid (UDCA) on gut microbiota alteration in development of colitis and CAC is unknown. Our analysis of publicly available datasets demonstrated the association of UDCA treatment and accumulation of Akkermansia. UDCA-mediated alleviation of DSS-induced colitis was microbially dependent. UDCA treatment significantly upregulated Akkermansia colonization in a mouse model. Colonization of Akkermansia was associated with enhancement of the mucus layer upon UDCA treatment as well as activation of bile acid receptors in macrophages. UDCA played a role in CAC prevention and treatment in the AOM-DSS and ApcMin/+-DSS models through downregulation of inflammation and accumulation of Akkermansia. This study suggests that UDCA intervention could reshape intestinal gut homeostasis, facilitating colonization of Akkermansia and preventing and treating colitis and CAC.

INCREASE IN MK2 ACTIVITY IN CROHN’S DISEASE: ROLE IN THE INFLAMMATORY ACTIVATION OF FIBROBLASTS

Abstract INTRODUCTION AND HYPOTHESIS Mesenchymal cells known as myo-/fibroblasts (MFs) are critical immunosuppressors under gut mucosal homeostasis. Expression of immune checkpoint PD-L1 by MFs plays a key role in the control of T cell inflammatory responses. In Crohn’s disease (CD), MFs switch their activity from immunosuppressive to pro-inflammatory, where they are also known as Inflammatory Fibroblasts. However, the mechanisms responsible for these pathological changes in MF activity are unknown. Map-kinase-activated protein kinase 2 (MK2) is a major regulator of inflammation in the gut. MK2 is downstream of p38 signaling, it evokes a sub-pathway that directly regulates the production of key inflammatory cytokines implicated in CD (such as TNF-α, IL-1, and IL-6). Thus, we hypothesized that activation of MK2 signaling is critical to the pathological changes in MFs during the immunopathogenesis of CD. METHODS Human normal and CD tissues and derived MFs, as well as animal models relevant to CD, were used in this study. MF signaling/activity was analyzed using RNAseq, qRT-PCR, western blot (WB), cytokine/chemokine multiplex arrays, and confocal microscopy. RESULTS In situ analysis demonstrated an increase in MK2 activity within the inflamed compared to the non-inflamed CD and healthy control intestinal tissues, which was confirmed by WB and multiplex signaling array analysis. In situ increase in MK2 activity in CD intestinal mucosa was greatly associated with mesenchymal stromal cells that bear a “myofibroblast” phenotype (positive for α-SMA expression). An increase in MK2 activity was also observed in primary MF cultures isolated from CD (CD-MFs) when compared to normal (N-) MFs. MK2 activity within CD-MFs was also associated with a significant decrease in the expression of the immunosuppressive checkpoint PD-L1 and an increase in the expression of inflammatory CCL2 and IL-6. Inhibition of MK2 activity within CD-MFs through using the MK2-specific inhibitor PF-3644022 (10 μM) reversed the inflammatory activity of MFs. Remarkably, we observed a differential role of p38 and MK2 in the regulation of PD-L1 expression in MFs: while p38 was required for basal expression of PD-L1, activation of MK2 downregulates PD-L1 expression. These data indicate a unique role of MK2 activation in pathological reprogramming of MFs in CD. Use of MK2 inhibitor in a therapeutic modality in chronic DSS and IL-10 KO murine models of CD also significantly reduce MF-linked inflammatory responses in vivo. CONCLUSION Our data suggest that an increase in MK2 activity in CD is critical to the reprogramming of the MF from immunosuppressive toward pathological Inflammatory fibroblasts. Targeting MK2 activity within MFs could be a desirable strategy for improving the efficacy of current IBD therapeutic approaches.

MTADV 5-MER PEPTIDE SUPPRESSES IBD PATHOLOGY AND UNVEILS A NEW POTENTIAL TARGET-SERUM AMYLOID A

Abstract We reported in the previous Crohn’s and Colitis Congress and published in Refs 1 and 2 ,that a segment of 5 aa of a CD44 variant -Methionine, Threonine, Alanine, Aspartic Acid, Valine (MTADV), called 5-MER peptide (5MP), inhibits the inflammation in DSS and TNBS mouse models of IBD, when subcutaneously injected or orally delivered. Similarly, 5MP inhibited the inflammation in mouse models of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA; Ref 2). As IBD, MS and RA share up-regulation of pathological Serum Amyloid A (SAA) (which fuels chronic inflammations), we predicted and then showed that 5-MP targets and hampers the pathological SAA, thus ameliorating SAA-associated inflammatory diseases by inhibiting the release of pro-inflammatory cytokines from SAA-activated innate-immunity cells. Now we focus on deciphering 5MP mechanism of action. To this end, we show that 1. Rats with Collagen-Induced Arthritis (CIA; the animal model of RA), in contrast to mice or man, do not produce under inflammatory conditions SAA. Therefore, 5MP, in the absence of the SAA target, cannot inhibits the rat CIA, that is almost identical to mouse CIA. This fact supports our prediction that SAA is a 5MP target. 2. SAA binds 5MP, as indicated by mass spectrometry. 3. Polymerized and aggregated forms of SAA are the pathological forms of SAA, generating inflammation and amyloidosis, the last could be a severe complication of Crohn’s (Ref 3). Co-Incubation with 5MP interferes with SAA polymerization and aggregation (Figure 1), providing mechanistic explanation for 5MP therapeutic effect in IBD. 4. Human Fibroblasts, monocytes and Peripheral Blood Mononuclear Cells (PBMCs), stimulated with human SAA, release the pro-inflammatory cytokines IL-6, IL-1β and TNFα. Co-incubation of SAA with 5MP (but not with scrambled peptide TMVAD) suppresses the cytokines release from the cells, providing evidence on the 5-MP potency to interfere with SAA-cytokine destructive cross-talk. In support, mice with IBD reduce inflammation and alleviate IL-6 and IL-1β expression in colonic tissue after 5-MP injection (Figure 2). 5. Normal immune responses, such as DTH or antibody response to foreign antigen, are not inhibited by 5MP. 6. 5-MP is stable in room temperature for at least 5 months. 7. Dogs can tolerate (without signs of harmful effects) 5MP doses, which are much higher than the 5MP therapeutic dose, and finally, a finding which merits a special attention 8. Gene Set Enrichment Analysis reveals that 5-MP activates genes, inducing resistance to IBD, e.g., Batf2, Fgl2, Ifitm3, Pyrin (Nefv), TNFR2 and Tnfrsf9. 1. Nedvetzki, S., and Naor, D. J. Clin. Invest. 111, 1211-1220, 2003. 2. Hemed-Shaked M. and Naor D. J Autoimmun. Aug 12;124:102713, 2021. 3. Denis MA. Eur J Clin Invest. 2013;43:292-301

MTADV 5-MER PEPTIDE SUPPRESSES IBD PATHOLOGY AND UNVEILS A NEW POTENTIAL TARGET-SERUM AMYLOID A

We reported in the previous Crohn’s and Colitis Congress and published in Refs 1 and 2 ,that a segment of 5 aa of a CD44 variant -Methionine, Threonine, Alanine, Aspartic Acid, Valine (MTADV), called 5-MER peptide (5MP), inhibits the inflammation in DSS and TNBS mouse models of IBD, when subcutaneously injected or orally delivered. Similarly, 5MP inhibited the inflammation in mouse models of Multiple Sclerosis (MS) and Rheumatoid Arthritis (RA; Ref 2). As IBD, MS and RA share up-regulation of pathological Serum Amyloid A (SAA) (which fuels chronic inflammations), we predicted and then showed that 5-MP targets and hampers the pathological SAA, thus ameliorating SAA-associated inflammatory diseases by inhibiting the release of pro-inflammatory cytokines from SAA-activated innate-immunity cells.

P072 The involvement of extracellular Nicotinamide Phosphoribosyltransferase (eNAMPT) and Nicotinate Phosphoribosyltransferase (eNAPRT) in inflammatory bowel disease

Abstract Background Nicotinamide phosphoribosyltrasferase (NAMPT) is a pleiotropic enzyme which catalyses the first and rate-limiting step in the biosynthesis of NAD. It is present in two different forms: an intracellular form, called iNAMPT, (Chiarugi et al., 2012), and an extracellular form, eNAMPT. eNAMPT is considered an important factor for granulocyte-colony stimulating factor-(G-CSF)-induced myeloid differentiation, with paracrine and autocrine effects on different cell types (i.e. immune and cancer cells), binding TLR4. NAMPT is structurally and functionally related to the enzyme nicotinate phosphoribosyltransferase (iNAPRT), which is rate-limiting in the NAD salvage pathway that starts from nicotinic acid. The NAD biosynthetic pathways controlled by NAMPT and NAPRT are closely interconnected and can compensate for each other. Also, NAPRT is identified as an extracellular ligand (eNAMPRT) for TLR4 and a mediator of inflammation (Managò et al., 2020). Importantly, iNAMPT and eNAMPT levels are increased in several pathologies, included inflammatory bowel disease (IBD). It has been reported that serum eNAMPT levels correlate with the stage of the pathology: in an active state of the disease the levels of NAMPT are very high, however its levels are partially reduced in a remission stage (Moschen et al., 2007). Methods First, we investigated the role of eNAMPT and eNAPRT in murine IBD models (especially in DNBS and DSS model). We took into account phenotypic effect as weight loss and colon shortening, but also the reduction of mRNA of inflammatory genes with RT-PCR, tissue damage with H&E and IHC analysis and systemic and local production through colon explant. Secondly, we determined serum eNAMPT and eNAPRT levels in a cohort of adult IBD patients. Results Both eNAMPT and eNAPRT have been found elevated in 180 IBD patients, as proinflammatory marker of the pathologies. These levels are also elevated in serum and colonic explant of DSS and DNBS preclinical models, associated to an active state of the disease, as a pro-inflammatory response developed locally and systemically. Moreover, we performed ELISA analysis on sera of 100 IBD patients, eligible for anti-TNF treatment, both pediatric and adults. Serum eNAMPT levels are increased before the treatment, responsive patients verified a reduction of these levels, while no-responsive ones verified higher levels. Conclusion eNAMPT and eNAPRT could be considered pro-inflammatory markers of IBD and possible druggable targets.

ABIN3 Negatively Regulates Necroptosis-induced Intestinal Inflammation Through Recruiting A20 and Restricting the Ubiquitination of RIPK3 in Inflammatory Bowel Disease.

There is evidence for a disturbed necroptosis function in many inflammatory diseases, but its role in inflammatory bowel diseases [IBD] and the underlying mechanisms are unclear. Here, we studied the functional significance and molecular mechanisms of ABIN3, a ubiquitin-binding protein, in regulating the ubiquitination and activation of necroptosis in IBD. The expression of necroptosis hallmarks and ABIN3 were assessed in inflamed samples of IBD patients, dextran sodium sulphate [DSS]-induced colitis models, and azoxymethane [AOM]/DSS models in mice. ABIN3 was overexpressed and silenced to explore its function in regulating necroptosis, inflammation, and intestinal barrier function. Immuoprecipitiation [IP] and co-IP assays were performed to investigate the cross-talk between ABIN3 and deubiquitinating enzyme A20, and the mechanisms of coordinating ubiquitination modification to regulate necroptosis. Excessive necroptosis is an important contributory factor towards the uncontrolled inflammation and intestinal barrier defects in IBD and experimental colitis. Blocking necroptosis by Nec-1s or GSK'872 significantly prevented cell death and alleviated DSS-induced colitis in vivo, whereas in the AOM/DSS model, necroptosis inhibitors aggravated the severity of colitis-associated colon carcinogenesis [CAC]. Mechanistically, ABIN3 is rapidly recruited to the TNF-RSC complex, which interacts and coordinates with deubiquitinating enzyme A20 to control the K63 deubiquitination modification and subsequent activation of the critical necroptosis kinase, RIPK3, to suppress necroptosis. ABIN3 regulates inflammatory response and intestinal barrier function by interacting with A20 and coordinating the K63 deubiquitination modification of necroptosis in IBD.

DSS Model for the Adipura Trophy

Every year the Indonesian government presents challenges to the regional and city governments to obtain the adiputra trophy. The Adipura Cup is given to the municipal and regional governments in Indonesia who meet several requirements such as cleanliness, beauty, peace and several other criteria. During this time the criteria assessment process is done manually and this research aims to create a Decision Support System to help provide an assessment of the Indonesian government to determine the winner of The Adipura trophy. The research method uses Analytical Hierarchical Processing (AHP) The results of this research are DSS models for The Adipura trophies. The conclusion of this research is that the resulting model facilitates the process of selecting the The Adipura cup.

Abstract 2601: HMGA1 is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis

Abstract Cancer cells undergo chromatin remodeling and epigenetic reprogramming to hijack stem cell transcriptional networks and drive tumorigenesis, although the molecular underpinnings remain unclear. High Mobility Group A (HMGA1) chromatin remodeling proteins are architectural transcription factors that bind AT-rich sequences where they “open” chromatin and recruit transcriptional complexes to modulate gene expression. The HMGA1 gene is highly expressed during embryogenesis, but silenced postnatally in differentiated tissues. HMGA1 is re-expressed in aggressive cancers where high levels portend adverse clinical outcomes. In colorectal cancer, HMGA1 is among the genes most overexpressed compared to normal colon epithelium. We previously found that HMGA1 induces genes involved in EMT and drives tumor progression in colon cancer models. Hmga1 transgenic mice develop aberrant proliferation and polyposis in the small intestine and colon. In intestinal stem cells, Hmga1 amplifies Wnt signals to enhance self-renewal. Surprisingly, Hmga1 also induces Sox-9 to drive Paneth cell differentiation and “build” a stem cell niche. To determine how HMGA1 functions in the colon, we examined mouse models with varied levels of Hmga1. Hmga1 overexpression in colon stem cells leads to expansion of the colon stem cell pool. Both goblet cells and colonic mucous are increased. In DSS models of inflammatory bowel disease, Hmga1 enhances repair and tissue regeneration, while Hmga1 overexpression promotes aberrant proliferation and polyposis with advancing age. To determine how HMGA1 is induced during carcinogenesis, we examined mice harboring a heterozygous Apc mutation (Min+/-) following colonization with the human symbiotic bacteria, enterotoxigenic Bacterioides fragilis (ETBF). ETBF triggers increased Wnt signaling by inducing E-cadherin cleavage with release of membrane bound β-catenin, leading to enhanced distal colon tumorigenesis in mice. Further, ETBF colonization in humans is linked to colon cancer. ETBF colonization in mice induces Hmga1 and increases stem cell self-renewal in preliminary studies. Inflammatory cytokine genes are also induced. Bacteroides fragilis toxin (BFT) induces Hmga1 in wildtype and Min+/- mouse colon organoids. HMGA1 also increases modestly in human HT29/C1 cells after exposure to BFT. Studies are underway to identify transcriptional networks and epigenetic alterations governed by HMGA1 in this setting. This work not only provides new insights into the role of HMGA1 in colon epithelial homeostasis by maintaining both the stem cell pool and promoting regeneration following injury, but also suggests that HMGA1 can be aberrantly induced by signals from the microbiome to promote tumorigenesis. Our results also highlight the interactions between the microbiome and HMGA1 as a potential therapeutic opportunity in colon carcinogenesis. Citation Format: Linda M S Resar, Lingling Xian, Jawara Allen, Shaoguang Wu, Lionel Chia, Cynthia Sears. HMGA1 is induced by procarcinogenic bacteria within the microbiome where it drives expansion in the colon stem cell pool and tumorigenesis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2601.

P137 LOCAL CRAC CHANNEL BLOCKADE TREATS COLITIS IN MICE

The Ca2+ release activated Ca2+ (CRAC) channel is an exciting new pharmacological target for the treatment of Inflammatory Bowel Disease (IBD) because it is upstream in the inflammatory cascade and absolutely necessary for immune responses. Calcium-selective CRAC channels become activated when T cells recognize antigen and allow calcium influx to the cytoplasm leading to increased NF-κB and NFAT-mediated gene expression driving inflammation. We have developed VV2003 which is a small molecule that blocks the CRAC channel and can be delivered orally. It has limited epithelial permeability allowing VV2003 to function locally at the site of inflammation with little risk for systemic complications. Following oral administration in healthy and colitic mice, VV2003 demonstrates low systemic absorption while maintaining therapeutic levels in the colon tissue for greater than 12 hours. In rat pharmacokinetic studies, VV2003 demonstrates low percent bioavailability and rapid clearance. The drug candidate diffuses into the intestinal mucosa and submucosa where it ameliorates inflammation without systemic activity. We have tested VV2003 in the DSS and Adoptive Transfer mouse models for colitis and have seen improvements in multiple endpoints including: disease activity index, fecal calprotectin and histologic scores. Notably, it is non-inferior to several systemically-active treatments which are currently the standard of care. We find that the CRAC channel is upregulated in biopsy samples from patients with IBD, and VV2003 suppresses inflammatory cytokines from PBMCs in-vitro. Importantly, there have been no signs of toxicity in any of our experiments. Overall, these data demonstrate that VV2003 has potential to become a novel next-generation oral therapy for colitis because it effectively limits leukocyte function in-vitro and in-vivo and is expected to have local delivery to the sites of pathology with minimal off target effects. In the near future, we plan to begin clinical trials to determine if VV2003 will be effective in treating patients with IBD. This project is supported by: VA CDA 5IK2BX003518, NIH NCATS TR001415; NIH NIAID R43AI134205.

P079 NEONATAL SUPPLEMENTATION OF A LACTOBACILLUS RHAMNOSUS GG (LGG)-DERIVED PROTEIN, P40, PROMOTES INTESTINAL DEVELOPMENT AND PREVENTS COLITIS IN ADULTHOOD

p40 transactivates EGF receptor in intestinal epithelial cells, leading to protection of intestinal epithelial cells under the inflammatory condition. This study aimed to determine the effects of neonatal supplementation of p40 on intestinal development and its long-term outcomes. We generated pectin/zein hydrogels for specifically delivering p40 to the small intestine and the colon. Wt, Egfrfl/fl-Vil-Cre with constitutive EGF receptor deletion in the intestinal epithelial cells, and Egfr fl/fl (littermate control) pups were gavaged with p40-containing hydrogels at 0.25, 0.5, and 0.75 μg/day during postnatal days 2–6, 7–13, and 14–21, respectively. Six- to seven-week old mice were treated with DSS and TNBS to induce colitis. The intestinal tissues were prepared for immunostaining and real-time PCR analysis. The fecal IgA level was examined using ELISA. Compared to wt mice receiving hydrogels without p40, treatment with p40-containing hydrogels significantly enhanced bodyweight gain and functional maturation of the intestine, including epithelial cell proliferation and differentiation examined by increasing numbers and gene expression levels of Ki67, and Muc2 and sucrose-isomaltase, respectively, tight junction formation by ZO-1 apical localization and claudin 3 expression prior to weaning, and increased persistent IgA production from early life to adulthood. These p40-induced effects were observed in Egfrfl/fl, but abolished in Egfrfl/fl-Vil-Cre mice. Neonatal p40 treatment decreased inflammatory score and production of proinflammatory cytokines (TNF and IFN-γ in TNBS and TNF, KC, IL-6 in DSS models) in adult mice. These findings reveal novel roles of neonatal supplementation of probiotic-derived factors in promoting EGF receptor-mediated maturation of intestinal functions and innate immunity, which likely promote long-term beneficial outcomes for reducing the susceptibility to intestinal injury and colitis in adulthood.

P079 NEONATAL SUPPLEMENTATION OF A LACTOBACILLUS RHAMNOSUS GG (LGG)-DERIVED PROTEIN, P40, PROMOTES INTESTINAL DEVELOPMENT AND PREVENTS COLITIS IN ADULTHOOD

p40 transactivates EGF receptor in intestinal epithelial cells, leading to protection of intestinal epithelial cells under the inflammatory condition. This study aimed to determine the effects of neonatal supplementation of p40 on intestinal development and its long-term outcomes. We generated pectin/zein hydrogels for specifically delivering p40 to the small intestine and the colon. Wt, Egfrfl/fl-Vil-Cre with constitutive EGF receptor deletion in the intestinal epithelial cells, and Egfr fl/fl (littermate control) pups were gavaged with p40-containing hydrogels at 0.25, 0.5, and 0.75 μg/day during postnatal days 2-6, 7-13, and 14-21, respectively. Six- to seven-week old mice were treated with DSS and TNBS to induce colitis. The intestinal tissues were prepared for immunostaining and real-time PCR analysis. The fecal IgA level was examined using ELISA. Compared to wt mice receiving hydrogels without p40, treatment with p40-containing hydrogels significantly enhanced bodyweight gain and functional maturation of the intestine, including epithelial cell proliferation and differentiation examined by increasing numbers and gene expression levels of Ki67, and Muc2 and sucrose-isomaltase, respectively, tight junction formation by ZO-1 apical localization and claudin 3 expression prior to weaning, and increased persistent IgA production from early life to adulthood. These p40-induced effects were observed in Egfrfl/fl, but abolished in Egfrfl/fl-Vil-Cre mice. Neonatal p40 treatment decreased inflammatory score and production of proinflammatory cytokines (TNF and IFN-γ in TNBS and TNF, KC, IL-6 in DSS models) in adult mice. These findings reveal novel roles of neonatal supplementation of probiotic-derived factors in promoting EGF receptor-mediated maturation of intestinal functions and innate immunity, which likely promote long-term beneficial outcomes for reducing the susceptibility to intestinal injury and colitis in adulthood.

Of mice and men: a novel dietary supplement for the treatment of ulcerative colitis.

Background:Curcumin, green tea polyphenols and selenium possess anti-inflammatory and anti-oxidant properties. Individually they have demonstrated some efficacy in animal models and human subjects with inflammatory bowel disease (IBD). To evaluate the efficacy and safety of Coltect [Curcumin (500 mg), green tea (250 mg) and selenium (100 µg)] in vivo and in patients with ulcerative colitis (UC).Methods:Each component was compared to placebo in a DSS mice colitis model. The efficacy was validated in a 2,4,6-trinitrobenzenesulfonic acid (TNBS) rat colitis model. Twenty patients with mild-to-moderate UC received two Coltect tablets twice daily for 8 weeks. Enrollees underwent sigmoidoscopy at study entrance and closure, and physical and laboratory evaluation at baseline, 4 and 8 weeks.Results:Coltect showed a synergistic therapeutic effect in the DSS and TNBS models. Disease activity was significantly higher in the placebo versus the treated group (p < 0.05). Selenium was the more active component. The contribution of green tea was minor. In the TNBS model, the Wallace scores for macroscopic lesions were 4.8 ± 1.5 (treatment) and 8.2 ± 0.5 (placebo) (p = 0.01). In humans, Coltect was well tolerated and effective. Fourteen subjects (70%) improved: nine (45%) went into complete remission, four (20%) experienced marked improvement and one (5%) experienced moderate improvement at the end of the trial. Clinical activity index decreased significantly at 4 and 8 weeks (p < 0.001). Two patients had no change in their symptoms, and one withdrew after 4 weeks. Flare-up in four subjects caused three to withdraw from the study after less than 4 weeks. Endoscopic improvement was observed in 11 (69%) patients, and four patients (25%) achieved complete remission.Conclusions:Coltect may serve as a first-line or add-on therapy in patients with mild-to-moderate UC.

Metabolic Variability of a Multispecies Probiotic Preparation Impacts on the Anti-inflammatory Activity.

Background: In addition to strain taxonomy, the ability of probiotics to confer beneficial effects on the host rely on a number of additional factors including epigenetic modulation of bacterial genes leading to metabolic variability and might impact on probiotic functionality.Aims: To investigate metabolism and functionality of two different batches of a probiotic blend commercialized under the same name in Europe in models of intestinal inflammation.Methods: Boxes of VSL#3, a probiotic mixture used in the treatment of pouchitis, were obtained from pharmacies in UK subjected to metabolomic analysis and their functionality tested in mice rendered colitis by treatment with DSS or TNBS.Results: VSL#3-A (lot DM538), but not VSL#3-B (lot 507132), attenuated “clinical” signs of colitis in the DSS and TNBS models. In both models, VSL#3-A, but not VSL#3-B, reduced macroscopic scores, intestinal permeability, and expression of TNFα, IL-1β, and IL-6 mRNAs, while increased the expression of TGFβ and IL-10, occludin, and zonula occludens-1 (ZO-1) mRNAs and shifted colonic macrophages from a M1 to M2 phenotype (P < 0.05 vs. TNBS). In contrast, VSL#3-B failed to reduce inflammation, and worsened intestinal permeability in the DSS model (P < 0.001 vs. VSL#3-A). A metabolomic analysis of the two formulations allowed the identification of two specific patterns, with at least three-folds enrichment in the concentrations of four metabolites, including 1–3 dihydroxyacetone (DHA), an intermediate in the fructose metabolism, in VSL#3-B supernatants. Feeding mice with DHA, increased intestinal permeability.Conclusions: Two batches of a commercially available probiotic show divergent metabolic activities. DHA, a product of probiotic metabolism, increases intestinal permeability, highlighting the complex interactions between food, microbiota, probiotics, and intestinal inflammation.

Association of colorectal cancer with pathogenicEscherichia coli: Focus on mechanisms using optical imaging

To investigate the molecular or cellular mechanisms related to the infection of epithelial colonic mucosa by pks-positive Escherichia coli (E. coli) using optical imaging. We choose to evaluate the tumor metabolic activity using a fluorodeoxyglucose analogue as 2-deoxyglucosone fluorescent probes and to correlate it with tumoral volume (mm(3)). Inflammation measuring myeloperoxidase (MPO) activity and reactive oxygen species production was monitored by a bioluminescent (BLI) inflammation probe and related to histological examination and MPO levels by enzyme-linked immunosorbent assay (ELISA) on tumor specimens. The detection and quantitation of these two signals were validated on a xenograft model of human colon adenocarcinoma epithelial cells (HCT116) in nude mice infected with a pks-positive E. coli. The inflammatory BLI signal was validated intra-digestively in the colitis-CEABAC10 DSS models, which mimicked Crohn's disease. Using a 2-deoxyglucosone fluorescent probe, we observed a high and specific HCT116 tumor uptake in correlation with tumoral volume (P = 0.0036). Using the inflammation probe targeting MPO, we detected a rapid systemic elimination and a significant increase of the BLI signal in the pks-positive E. coli-infected HCT116 xenograft group (P < 0.005). ELISA confirmed that MPO levels were significantly higher (1556 ± 313.6 vs 234.6 ± 121.6 ng/mL P = 0.001) in xenografts infected with the pathogenic E. coli strain. Moreover, histological examination of tumor samples confirmed massive infiltration of pks-positive E. coli-infected HCT116 tumors by inflammatory cells compared to the uninfected group. These data showed that infection with the pathogenic E. coli strain enhanced inflammation and ROS production in tumors before tumor growth. Moreover, we demonstrated that the intra-digestive monitoring of inflammation is feasible in a reference colitis murine model (CEABAC10/DSS). Using BLI and fluorescence optical imaging, we provided tools to better understand host-pathogen interactions at the early stage of disease, such as inflammatory bowel disease and colorectal cancer.

Implementation Web Decision Support Model for Predicting Performance of Field Machinery Operation (DWDSS)

Farm machinery planning, design and operation are complicated undertaking due to time and cost constraint and due to prevalence of complicated interacting and overlapping field operations involving capacity constraints and cooperating units. The classical DSS models that applied in the past to machinery planning and policy analysis as well as to performance assessment and simulation of machinery demand, and supplies are criticized by limitations in programming and the difficulty in manipulation and storing the bulky data usually encountered in machinery records. In contrast by application of a web-based decision support system (DWDSS) the user can enjoy the facility to store the data in the server. (DWDSS), is a user-friendly interactive program which permits the user to interact by entering the required input records. The model estimates machinery performance of various farm machines. It consists of one model, which helps the farm manager to take the correct optimum selection of his agricultural machinery. DWDSS predicts field efficiency, field capacity, draft power required to operate machines and PTO power. The DWDSS was successfully validated statistically in comparison to the published data from the ASAE (2009). The comparison indicated that there were no significant differences (probability = 0.05) between them in the calculations that were executed. The DWDSS model was applied to real case conditions in Wad Salma and Rahad irrigated schemes in the central clay plains under similar treatments. The DWDSS results of field efficiency, theoretical field capacity, working rate and draw bar power was found fairly identical to the actual Wad Salma and Rahad data. The results indicated that, generally, the actual field efficiencies of the studied machines were found to be lower by 7% than ASAE published data and t-test comparison between Wad Salma and Rahad schemes in working rate of the three tillage implement, indicated no significant difference between the two means at probability level =0.05. In general, the results indicated that the DWDSS could be applied to any real-life case successfully and with confidence. This is reached by helping the decision maker in planning and operation of a farm fleet by deciding size of farm power.