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Anterior gradient 2 (AGR2) is often overexpressed in several types of cancer. AGR2 is cytoplasmic or secreted as an extracellular signal. Intracellular AGR2 properties and role in cancer have been well studied, but its extracellular function is largely unclear. It has been shown that extracellular AGR2 activates endothelial cells and fibroblasts in culture, but the mechanism of AGR2 signaling is not well elucidated. Here, we report that tumor secreted or externally added AGR2 translocates into cytoplasm by endocytosis, binds to β-catenin and further co-translocates to the nucleus in NIH3T3 fibroblasts. Externally added AGR2 also increased β-catenin expression, stability, and accumulation in the nucleus in both fibroblasts and cancer cells. External AGR2 rescued the expression of β-catenin, which was suppressed by EGFR inhibitor AG1478 indicating an alternative pathway to regulate β-catenin independent of EGFR signal. These effects were abolished when a monoclonal antibody against AGR2 was added to the experiments, confirming the effects are caused by AGR2 only. Putting together, our results show that extracellular AGR2 signaling pathway involves endocytosis mediated cellular translocation, direct binding and regulating β-catenin nuclear accumulation. It is also a target against tumor initiated AGR2 signaling to form and maintain tumor microenvironment.

Endoplasmic reticulum (ER) stress is associated with Crohn's disease (CD), but its impact on host-microbe interaction in disease pathogenesis is not well defined. Functional deficiency in the protein disulfide isomerase anterior gradient 2 (AGR2) has been linked with CD and leads to epithelial cell ER stress and ileocolitis in mice and humans. Here, we show that ileal expression of AGR2 correlates with mucosal Enterobactericeae abundance in human inflammatory bowel disease (IBD) and that Agr2 deletion leads to ER-stress-dependent expansion of mucosal-associated adherent-invasive Escherichia coli (AIEC), which drives Th17 cell ileocolitis in mice. Mechanistically, our data reveal that AIEC-induced epithelial cell ER stress triggers CD103

Primary sclerosing cholangitis (PSC) is a progressive fibrosing cholestatic liver disease that is strongly associated with inflammatory bowel disease (IBD). PSC-associated IBD (PSC-IBD) displays a unique phenotype characterized by right-side predominant colon inflammation and increased risk of colorectal cancer compared to non-PSC-IBD. The frequent association and unique phenotype of PSC-IBD suggest distinctive underlying disease mechanisms from other chronic liver diseases or IBD alone. Multidrug resistance protein 2 knockout (Mdr2−/−) mice develop spontaneous cholestatic liver injury and fibrosis mirroring human PSC. As a novel model of PSC-IBD, we treated Mdr2−/− mice with dextran sulfate sodium (DSS) to chemically induce colitis (Mdr2−/−/DSS). Mdr2−/− mice demonstrate alterations in fecal bile acid composition and enhanced colitis susceptibility with increased colonic adhesion molecule expression, particularly mucosal addressin cell adhesion molecule 1 (MAdCAM-1). In vitro, ursodeoxycholic acid (UDCA) co-treatment resulted in a dose dependent attenuation of TNF-α-induced endothelial MAdCAM-1 expression. In the combined Mdr2−/−/DSS model, UDCA supplementation attenuated colitis severity and down-regulated intestinal MAdCAM-1 expression. These findings suggest a potential mechanistic role for alterations in bile acid signaling in modulating MAdCAM-1 expression and colitis susceptibility in cholestasis-associated colitis. Together, our findings provide a novel model and new insight into the pathogenesis and potential treatment of PSC-IBD.

Hepatocellular carcinoma (HCC) is the fifth most common malignant tumor in the world. It ranks third among cancer-induced deaths worldwide and has the characteristics of high metastasis and high recurrence rate. Long non-coding RNA (LncRNA) LINC00460 is significantly up-regulated in multiple types of cancers and is closely related to the progression of tumors. However, effects of LINC00460 and corresponding regulatory path in HCC are still poorly investigated. In our study, we found that expression of LINC00460 was up-regulated in HCC tissues and cell lines compared with the control. Then we revealed that knockdown of LINC00460 suppressed cell proliferation and cell mobility and induced cell apoptosis in HCC cells. Further study demonstrated that knockdown of LINC00460 suppressed the progression of HCC by elevating the expression of microRNA (miRNA, miR)-342-3p. Besides that, metastasis marker, Anterior gradient homolog 2 (AGR2) was found to be a target of miR-342-3p and overexpression of AGR2 promoted the progression of HCC. Finally, the in vivo experiments further verified the anti-tumor effects of LINC00460 / miR-342-3p / AGR2 axis in HCC. The LINC00460 / miR-342-3p / AGR2 axis exerts anti-tumor effect in HCC in vitro and in vivo, consolidating and expanding the research about targeted gene therapy for early diagnosis and treatment of HCC.

Aberrant glycosylation resulting from altered expression of sialyltransferases, such as ST3 β-galactoside α2-3-sialyltransferase 6, plays an important role in disease progression in multiple myeloma (MM). Hypersialylation can lead to increased immune evasion, drug resistance, tumor invasiveness, and disseminated disease. In this study, we explore the in vitro and in vivo effects of global sialyltransferase inhibition on myeloma cells using the pan-sialyltransferase inhibitor 3Fax-Neu5Ac delivered as a per-acetylated methyl ester pro-drug. Specifically, we show in vivo that 3Fax-Neu5Ac improves survival by enhancing bortezomib sensitivity in an aggressive mouse model of MM. However, 3Fax-Neu5Ac treatment of MM cells in vitro did not reverse bortezomib resistance conferred by bone marrow (BM) stromal cells. Instead, 3Fax-Neu5Ac significantly reduced interactions of myeloma cells with E-selectin, MADCAM1 and VCAM1, suggesting that reduced sialylation impairs extravasation and retention of myeloma cells in the BM. Finally, we showed that 3Fax-Neu5Ac alters the post-translational modification of the α4 integrin, which may explain the reduced affinity of α4β1/α4β7 integrins for their counter-receptors. We propose that inhibiting sialylation may represent a valuable strategy to restrict myeloma cells from entering the protective BM microenvironment, a niche in which they are normally protected from chemotherapeutic agents such as bortezomib. Thus, our work demonstrates that targeting sialylation to increase the ratio of circulating to BM-resident MM cells represents a new avenue that could increase the efficacy of other anti-myeloma therapies and holds great promise for future clinical applications.

The anterior gradient-2 (AGR2) is an endoplasmic reticulum (ER)-resident protein belonging to the protein disulfide isomerase family that mediates the formation of disulfide bonds and assists the protein quality control in the ER. In addition to its role in proteostasis, extracellular AGR2 is responsible for various cellular effects in many types of cancer, including cell proliferation, survival, and metastasis. Various OMICs approaches have been used to identify AGR2 binding partners and to investigate the functions of AGR2 in the ER and outside the cell. Emerging data showed that AGR2 exists not only as monomer, but it can also form homodimeric structure and thus interact with different partners, yielding different biological outcomes. In this review, we summarize the AGR2 “interactome” and discuss the pathological and physiological role of such AGR2 interactions.

BACKGROUND: Mounting evidence summarizes that circRNA is closely implicated in the development of numerous cancers. Our study aimed to investigate the role of circ_0119412 whose function was not explored in cervical cancer. METHODS: RT‐qPCR analysis was utilized for the expression analysis of circ_0119412, miR‐217, and anterior gradient 2 (AGR2). CCK‐8 assay, transwell assay, and MTT assay were employed to assess cell proliferation, migration, and adhesion, respectively. Animal study was performed to check the role of circ_0119412 in vivo. Bioinformatics analysis was applied to predict the downstream targets of circ_0119412. RIP assay was utilized to examine miRNAs potentially bound by circ_0119412. The interplays between miR‐217 and circ_0119412 or AGR2 were validated by dual‐luciferase reporter assay. RESULTS: circ_0119412 expression was highly enhanced in cervical tumor tissues and cancer cells. circ_0119412 overexpression aggravated cervical cancer cell proliferation, migration, and adhesion, and its overexpression was also conducive to tumor formation and growth in animal models. AGR2 was upregulated in cervical cancer by the public bioinformatics data. circ_0119412 bound to miR‐217, and miR‐217 bound to AGR 3’UTR. The promoting effects of circ_0119412 overexpression on cancer cell malignant phenotypes were reversed by miR‐217 enrichment. In addition, increased expression of miR‐217 suppressed AGR2 expression, thus weakening the functional effects of AGR2. CONCLUSION: circ_0119412 functioned as an oncogenic driver to promote the malignant development of cervical cancer by targeting the miR‐217/AGR2 pathway.

Anterior gradient protein 2 homolog (AGR2), an endoplasmic reticulum protein, is secreted in the tumor microenvironment. AGR2 is a member of the disulfide isomerase family, is highly expressed in multiple cancers, and promotes cancer metastasis. In this study, we found that etravirine, which is a non-nucleoside reverse transcriptase inhibitor, could induce AGR2 degradation via autophagy. Moreover, etravirine diminished proliferation, migration, and invasion in vitro. Moreover, in an orthotopic xenograft mouse model, the combination of etravirine and paclitaxel significantly suppressed cancer progression and metastasis. This drug may be a promising therapeutic agent for the treatment of ovarian cancer.

Primary biliary cholangitis (PBC) is characterised by ductopenia, ductular reaction, impairment of anion exchanger 2 (AE2) and the 'bicarbonate umbrella'. Ductulo-canalicular junction (DCJ) derangement is hypothesised to promote PBC progression. The secretin (Sct)/secretin receptor (SR) axis regulates cystic fibrosis transmembrane receptor (CFTR) and AE2, thus promoting choleresis. We evaluated the role of Sct/SR signalling on biliary secretory processes and subsequent injury in a late-stage PBC mouse model and human samples.At 32 weeks of age, female and male wild-type and dominant-negative transforming growth factor beta receptor II (late-stage PBC model) mice were treated with Sct for 1 or 8 weeks. Bulk RNA-sequencing was performed in isolated cholangiocytes from mouse models.Biliary Sct/SR/CFTR/AE2 expression and bile bicarbonate levels were reduced in late-stage PBC mouse models and human samples. Sct treatment decreased bile duct loss, ductular reaction, inflammation, and fibrosis in late-stage PBC models. Sct reduced hepatic bile acid levels, modified bile acid composition, and restored the DCJ and 'bicarbonate umbrella'. RNA-sequencing identified that Sct promoted mature epithelial marker expression, specifically anterior grade protein 2 (Agr2). Late-stage PBC models and human samples exhibited reduced biliary mucin 1 levels, which were enhanced by Sct treatment.Loss of Sct/SR signalling in late-stage PBC results in a faulty 'bicarbonate umbrella' and reduced Agr2-mediated mucin production. Sct restores cholangiocyte secretory processes and DCJ formation through enhanced mature cholangiocyte phenotypes and bile duct growth. Sct treatment may be beneficial for individuals with late-stage PBC.Secretin (Sct) regulates biliary proliferation and bicarbonate secretion in cholangiocytes via its receptor, SR, and in mouse models and human samples of late-stage primary biliary cholangitis (PBC), the Sct/SR axis is blunted along with loss of the protective 'bicarbonate umbrella'. We found that both short- and long-term Sct treatment ameliorated ductular reaction, immune cell influx, and liver fibrosis in late-stage PBC mouse models. Importantly, Sct treatment promoted bicarbonate and mucin secretion and hepatic bile acid efflux, thus reducing cholestatic and toxic bile acid-associated injury in late-stage PBC mouse models. Our work perpetuates the hypothesis that PBC pathogenesis hinges on secretory defects, and restoration of secretory processes that promote the 'bicarbonate umbrella' may be important for amelioration of PBC-associated damage.

Asthma is a complex airway disease that affects more than 350 million humans worldwide. Allergic asthma symptoms are induced by Th2 immune response with the release of cytokines and allegro-inflammatory mediators that amplify the inflammatory response, airway hyper-responsiveness (AHR) and hyperproduction of mucus. Higenamine, as a chemical compound, is a β2 adrenoreceptor agonist and can be used as bronchodilator in allergic asthma. BALB/c mice were allocated in four groups and then allergic asthma was induced in three groups. One of the asthmatic groups was treated with albuterol and other one was treated with higenamine. At least, methacholine challenge to determine the AHR, measurement of cytokines, total immunoglobulin E (IgE), LTB4 and LTC4 levels, evaluation of gene expression of Muc5ac, Muc5b, Agr2 and Arg1, and histopathological study were done. Higenamine treatment reduced AHR, interleukin (IL)-4, IL-13 levels, mRNA expression of MUC5ac, MUC5b, Arg1 and Agr2, goblet cell hyperplasia and mucus hypersecretion. Higenamine had no significant effect on IL-5, interferon-γ (INF-γ), IgE, LTB4, LTC4 levels and eosinophilic inflammation in lung tissue. Higenamine treatment controls asthma acute attack and breathlessness and can be used as asthma treatment with control of AHR and decrease of airflow obstruction and mucus hypersecretion and had allegro-immune-regulatory effect. But higenamine treatment had no notable effect on the inflammation and inflammatory factors.

Previous studies have explored the association between protein-coding genes and microRNAs (miRNAs) in lung adenocarcinoma (LUAD). However, the influence of the miR-199a-3p/anterior gradient 2 (AGR2) axis in LUAD has not yet been fully explored. Therefore, this study aimed to examine the underlying roles of AGR2 and miR-199a-3p in the development of LUAD. The expression levels of miR-199a-3p and AGR2 in LUAD tissues and cells were detected via quantitative reverse transcription-polymerase chain reaction (qRT-PCR). A luciferase assay was also performed to identify the interaction between AGR2 and miR-199a-3p. Moreover, the cell counting kit 8 (CCK-8), 5ʹ-bromo-2ʹ-deoxyuridine (BrdU), and adhesion assays were used along with flow cytometry to verify the malignancy of LUAD in vitro, while a xenograft tumor assay was performed to confirm the tumor growth in vitro. The findings showed a decrease in the expression of miR-199a-3p in LUAD. Additionally, miR-199a-3p overexpression inhibited the growth of LUAD cells in vitro and in vivo, while elevating the apoptosis rate of the cells. AGR2 knockdown had the same effect in the cells as that of miR-199a-3p overexpression. It was also found that miR-199a-3p directly targeted AGR2 in LUAD cells to suppress tumorigenesis. In conclusion, this study suggests that miR-199a-3p plays an anti-tumorigenic role in LUAD by targeting AGR2. Moreover, our study provides insights into the development of novel therapeutic targets for the treatment of LUAD.

Lymphocyte "homing", the physiologic trafficking of lymphocytes from the circulation to secondary lymphoid organs, is regulated by sequential adhesive interactions between lymphocytes and endothelial cells that constitute high endothelial venules (HEVs). Initial lymphocyte "rolling" is mediated by relatively weak, transient adhesive interactions between L-selectin expressed on lymphocytes and sulfated mucin-type O-glycans expressed on HEVs. Keratan sulfate galactose (Gal)-6-O-sulfotransferase (KSGal6ST) catalyzes 6-O-sulfation of Gal in keratan sulfate glycosaminoglycan chains but also transfers sulfate to Gal in much shorter glycan chains, such as sialylated N-acetyllactosamine (LacNAc)-capped O-glycans. In mice, KSGal6ST is reportedly expressed in HEVs and functions in synthesizing 6-sulfo Gal-containing O-glycans on HEVs. However, in humans, the presence of 6-sulfo Gal-containing O-glycans on HEVs is not reported. Employing the newly developed monoclonal antibody 297-11A, which recognizes non-sialylated terminal 6'-sulfo LacNAc, we demonstrate that sialyl 6'-sulfo (and/or 6,6'-disulfo) LacNAc-capped O-glycans are preferentially displayed on HEVs in human peripheral lymph nodes (PLNs) and to a lesser extent in mesenteric LNs (MLNs) but not in Peyer's patches (PPs). We also found that the scaffold protein mucosal addressin cell adhesion molecule 1 (MAdCAM-1), which is expressed on HEVs in PPs and MLNs but not PLNs, was modified by 297-11A-positive sulfated glycans less efficiently than was CD34. Moreover, 297-11A-positive sulfated glycans were also displayed on HEV-like vessels induced in tumor-infiltrating lymphocyte (TIL) aggregates formed in various cancers. These findings collectively indicate that 297-11A-positive sulfated glycans potentially play a role in physiologic lymphocyte homing as well as in lymphocyte recruitment under pathologic conditions.

The most prominent cancer-associated fibroblasts (CAFs) in tumor stroma is known to form a protective structure to support tumor growth. Anterior gradient-2 (AGR2), a tumor secretory protein is believed to play a pivotal role during tumor microenvironment (TME) development. Here, we report that extracellular AGR2 enhances fibroblasts elongation and migration significantly. The early stimulation of RhoA showed the association of AGR2 by upregulation of G1-S phase-regulatory protein cyclin D1 and FAK phosphorylation through fibroblasts growth factor receptor (FGFR) and vascular endothelial growth factor receptor (VEGFR). Our finding indicates that secretory AGR2 alters fibroblasts elongation, migration, and organization suggesting the secretory AGR2 as a potential molecular target that might be responsible to alter fibroblasts infiltration to support tumor growth.

Human anterior gradient homolog 2 (AGR2) reportedly acts as an oncogene in multiple types of cancers. As a secreted protein, the oncogenic roles of extracellular AGR2 have been the focus of the increasing number of studies. In contrast, the oncological functions of intracellular AGR2 (iAGR2) remain elusive. Here, we report that intracellular AGR2 (iAGR2) is sufficient to promote CRC metastasis. iAGR2 binds to KDEL receptors (KDELRs) via its KTEL motif to activate downstream Gs-PKA signaling. Activated PKA upregulates the expression of NF-κB subunit c-Rel (REL) and acetylates histone H3 at lysine 9 (H3K9ac) to promote the transcription of SNAIL and SLUG. AGR2 can be upregulated by prostaglandin E2 (PGE2) via EP4-PI3K-AKT pathway and is indispensable for PGE2-induced CRC metastasis. AGR2 knockdown enhances therapeutic effects of a COX-2 inhibitor, celecoxib, in CRC metastasis. Collectively, our study reveals a promoting role and molecular mechanisms of iAGR2 in CRC metastasis and uncovers a new tumor microenvironment signal regulating AGR2 expression, which may provide new targets for treating metastatic CRC.

Background & Aims Promoted by pancreatitis, oncogenic KrasG12D triggers acinar cells' neoplastic transformation through acinar-to-ductal metaplasia (ADM) and pancreatic intraepithelial neoplasia. Anterior gradient 2 (Agr2), a known inhibitor of p53, is detected at early stage of pancreatic ductal adenocarcinoma (PDAC) development. RNA polymerase II (RNAPII) is a key nuclear enzyme; regulation of its nuclear localization in mammalian cells represents a potential therapeutic target. Methods A mouse model of inflammation-accelerated KrasG12D-driven ADM and pancreatic intraepithelial neoplasia development was used. Pancreas-specific Agr2 ablation was performed to access its role in pancreatic carcinogenesis. Hydrophobic hexapeptides loaded in liposomes were developed to disrupt Agr2–RNAPII complex. Results We found that Agr2 is up-regulated in ADM-to-pancreatic intraepithelial neoplasia transition in inflammation and KrasG12D-driven early pancreatic carcinogenesis. Genetic ablation of Agr2 specifically blocks this metaplastic-to-neoplastic process. Mechanistically, Agr2 directs the nuclear import of RNAPII via its C-terminal nuclear localization signal, undermining the ATR-dependent p53 activation in ADM lesions. Because Agr2 binds to the largest subunit of RNAPII in a peptide motif-dependent manner, we developed a hexapeptide to interfere with the nuclear import of RNAPII by competitively disrupting the Agr2-RNAPII complex. This novel hexapeptide leads to dysfunction of RNAPII with concomitant activation of DNA damage response in early neoplastic lesions; hence, it dramatically compromises PDAC initiation in vivo. Moreover, the hexapeptide sensitizes PDAC cells and patient-derived organoids harboring wild-type p53 to RNAPII inhibitors and first-line chemotherapeutic agents in vivo. Of note, this therapeutic effect is efficient across various cancer types. Conclusions Agr2 is identified as a novel adaptor protein for nuclear import of RNAPII in mammalian cells. Also, we provide genetic evidence defining Agr2-dependent nuclear import of RNAPII as a pharmaceutically accessible target for prevention and treatment in PDAC in the context of wild-type p53.

Background & Aims The gastrointestinal epithelium plays a crucial role in maintaining homeostasis with the gut microbiome. Mucins are essential for intestinal barrier function and serve as a scaffold for antimicrobial factors. Mucin 2 (MUC2) is the major intestinal gel-forming mucin produced predominantly by goblet cells. Goblet cells express anterior gradient 2 (AGR2), a protein disulfide isomerase that is crucial for proper processing of gel-forming mucins. Here, we investigated 2 siblings who presented with severe infantile-onset inflammatory bowel disease. Methods We performed whole-genome sequencing to identify candidate variants. We quantified goblet cell numbers using H&E histology and investigated the expression of gel-forming mucins, stress markers, and goblet cell markers using immunohistochemistry. AGR2-MUC2 binding was evaluated using co-immunoprecipitation. Endoplasmic reticulum (ER) stress regulatory function of mutant AGR2 was examined by expression studies in Human Embryonic Kidney 293T (HEK293T) using tunicamycin to induce ER stress. Results Both affected siblings were homozygous for a missense variant in AGR2. Patient biopsy specimens showed reduced goblet cells; depletion of MUC2, MUC5AC, and MUC6; up-regulation of AGR2; and increased ER stress. The mutant AGR2 showed reduced capacity to bind MUC2 and alleviate tunicamycin-induced ER stress. Conclusions Phenotype–genotype segregation, functional experiments, and the striking similarity of the human phenotype to AGR2-/- mouse models suggest that the AGR2 missense variant is pathogenic. The Mendelian deficiency of AGR2, termed “Enteropathy caused by AGR2 deficiency, Goblet cell Loss, and ER Stress” (EAGLES), results in a mucus barrier defect, the inability to mitigate ER stress, and causes infantile-onset inflammatory bowel disease., Graphical abstract

Anterior gradient 2 (AGR2), a protein disulfide isomerase (PDI), is a well-established oncogene. Here, we found that Agr2-/- mice had a decreased fat mass and hepatic and serum lipid levels compared with their wild-type littermates after fasting, and exhibited reduced high-fat diet (HFD)-induced fat accumulation. Transgenic mice overexpressing AGR2 (Agr2/Tg) readily gained fat weight on a HFD but not a normal diet. Proteomic analysis of hepatic samples from Agr2-/- mice revealed that depletion of AGR2 impaired long-chain fatty acid uptake and activation but did not affect de novo hepatic lipogenesis. Further investigations led to the identification of several effector substrates, particularly fatty acid binding protein-1 (FABP1) as essential for the AGR2-mediated effects. AGR2 was coexpressed with FABP1, and knockdown of AGR2 resulted in a reduction in FABP1 stability. Physical interactions of AGR2 and FABP1 depended on the PDI motif in AGR2 and the formation of a disulfide bond between these two proteins. Overexpression of AGR2 but not a mutant AGR2 protein lacking PDI activity suppressed lipid accumulation in cells lacking FABP1. Moreover, AGR2 deficiency significantly reduced fatty acid absorption in the intestine, which might be resulted from decreased fatty acid transporter CD36 in mice. These findings demonstrated a novel role of AGR2 in fatty-acid uptake and activation in both the liver and intestine, which contributed to the AGR2-mediated lipid accumulation, suggesting that AGR2 is an important regulator of whole-body lipid metabolism and down-regulation of AGR2 may antagonize the development of obesity.

Marta Truffi,1 Marta Sevieri,2 Lucia Morelli,3 Matteo Monieri,2 Serena Mazzucchelli,2 Luca Sorrentino,2 Raffaele Allevi,2 Arianna Bonizzi,2 Pietro Zerbi,2 Beatrice Marchini,2 Erika Longhi,2 Gianluca Matteo Sampietro,4 Francesco Colombo,4 Davide Prosperi,3 Miriam Colombo,3 Fabio Corsi2,5 1Nanomedicine and Molecular Imaging Laboratory, Istituti Clinici Scientifici Maugeri IRCCS, Pavia 27100, Italy; 2Department of Biomedical and Clinical Sciences “Luigi Sacco, Università degli studi di Milano, Milano, 20157, Italy; 3NanoBioLab, Department of Biotechnology and Biosciences, Università degli studi di Milano-Bicocca, Milano, 20126, Italy; 4IBD Surgery Unit, ASST Fatebenefratelli Sacco - Ospedale “Luigi Sacco” Polo Universitario, Milano, 20157, Italy; 5Surgery Department, Breast Unit, Istituti Clinici Scientifici Maugeri IRCCS, Pavia, 27100, ItalyCorrespondence: Fabio CorsiSurgery Department, Breast Unit, Istituti Clinici Scientifici Maugeri IRCCS, via Maugeri 4, Pavia 27100, ItalyTel +39 02 5031 9850Fax +39 02 5031 9846Email fabio.corsi@icsmaugeri.itPurpose: Assessment of inflammatory bowel disease (IBD) currently relies on aspecific clinical signs of bowel inflammation. Specific imaging of the diseased bowel regions is still lacking. Here, we investigate mucosal addressin cell adhesion molecule 1 (MAdCAM-1) as a reliable and specific endothelial target for engineered nanoparticles delivering imaging agents to obtain an exact mapping of diseased bowel foci.Materials and Methods: We generated a nanodevice composed of PLGA-PEG coupled with anti-MAdCAM-1 antibody half-chains and loaded with quantum dots (P@QD-MdC NPs). Bowel localization and systemic biodistribution of the nanoconjugate were analyzed upon injection in a murine model of chronic IBD obtained through repeated administration of dextran sulfate sodium salt. Specificity for diseased bowel regions was also assessed ex vivo in human specimens from patients with IBD. Potential for development as contrast agent in magnetic resonance imaging was assessed by preliminary study on animal model.Results: Synthesized nanoparticles revealed good stability and monodispersity. Molecular targeting properties were analyzed in vitro in a cell culture model. Upon intravenous injection, P@QD-MdC NPs were localized in the bowel of colitic mice, with enhanced accumulation at 24 h post-injection compared to untargeted nanoparticles (p< 0.05). Nanoparticles injection did not induce histologic lesions in non-target organs. Ex vivo exposure of human bowel specimens to P@QD-MdC NPs revealed specific recognition of the diseased regions vs uninvolved tracts (p< 0.0001). After loading with appropriate contrast agent, the nanoparticles enabled localized contrast enhancement of bowel mucosa in the rectum of treated mice.Conclusion: P@QD-MdC NPs efficiently detected bowel inflammation foci, accurately following the expression pattern of MAdCAM-1. Fine-tuning of this nanoconjugate with appropriate imaging agents offers a promising non-invasive tool for specific IBD diagnosis.Keywords: polymeric nanoparticles, inflamed bowel, adhesion molecule, targeted imaging

Background & Aims The heterodimeric integrin receptor α4β7 regulates CD4 T cell recruitment to inflamed tissues, but its role in the pathogenesis of non-alcoholic steatohepatitis (NASH) is unknown. Herein, we examined the role of α4β7-mediated recruitment of CD4 T cells to the intestine and liver in NASH. Methods Male littermate F11r+/+ (control) and junctional adhesion molecule A knockout F11r−/− mice were fed a normal diet or a western diet (WD) for 8 weeks. Liver and intestinal tissues were analyzed by histology, quantitative reverse transcription PCR (qRT-PCR), 16s rRNA sequencing and flow cytometry. Colonic mucosa-associated microbiota were analyzed using 16s rRNA sequencing. Liver biopsies from patients with NASH were analyzed by confocal imaging and qRT-PCR. Results WD-fed knockout mice developed NASH and had increased hepatic and intestinal α4β7+ CD4 T cells relative to control mice who developed mild hepatic steatosis. The increase in α4β7+ CD4 T cells was associated with markedly higher expression of the α4β7 ligand mucosal addressin cell adhesion molecule 1 (MAdCAM-1) in the colonic mucosa and livers of WD-fed knockout mice. Elevated MAdCAM-1 expression correlated with increased mucosa-associated Proteobacteria in the WD-fed knockout mice. Antibiotics reduced MAdCAM-1 expression indicating that the diet-altered microbiota promoted colonic and hepatic MAdCAM-1 expression. α4β7 blockade in WD-fed knockout mice significantly decreased α4β7+ CD4 T cell recruitment to the intestine and liver, attenuated hepatic inflammation and fibrosis, and improved metabolic indices. MAdCAM-1 blockade also reduced hepatic inflammation and fibrosis in WD-fed knockout mice. Hepatic MAdCAM-1 expression was elevated in patients with NASH and correlated with higher expression of α4 and β7 integrins. Conclusions These findings establish α4β7/MAdCAM-1 as a critical axis regulating NASH development through colonic and hepatic CD4 T cell recruitment. Lay summary Non-alcoholic steatohepatitis (NASH) is an advanced and progressive form of non-alcoholic fatty liver disease (NAFLD), and despite its growing incidence no therapies currently exist to halt NAFLD progression. Herein, we show that blocking integrin receptor α4β7-mediated recruitment of CD4 T cells to the intestine and liver not only attenuates hepatic inflammation and fibrosis, but also improves metabolic derangements associated with NASH. These findings provide evidence for the potential therapeutic application of α4β7 antibody in the treatment of human NASH.

Pancreatic cancer remains among the most lethal cancers worldwide, with poor early detection rates and poor survival rates. Patient-derived xenograft (PDX) models have increasingly been used in preclinical and clinical research of solid cancers to fulfil unmet need. Fresh tumour samples from human pancreatic adenocarcinoma patients were implanted in severe combined immunodeficiency (SCID) mice. Samples from 78% of treatment-na&iuml, ve pancreatic ductal adenocarcinoma patients grew as PDX tumours and were confirmed by histopathology. Frozen samples from F1 PDX tumours could be later successfully passaged in SCID mice to F2 PDX tumours. The human origin of the PDX was confirmed using human-specific antibodies, however, the stromal component was replaced by murine cells. Cell lines were successfully developed from three PDX tumours. RNA was extracted from eight PDX tumours and where possible, corresponding primary tumour (T) and adjacent normal tissues (N). mRNA profiles of tumour vs. F1 PDX and normal vs. tumour were compared by Affymetrix microarray analysis. Differential gene expression showed over 5000 genes changed across the N vs. T and T vs. PDX samples. Gene ontology analysis of a subset of genes demonstrated genes upregulated in normal vs. tumour vs. PDX were linked with cell cycle, cycles cell process and mitotic cell cycle. Amongst the mRNA candidates elevated in the PDX and tumour vs. normal were SERPINB5, FERMT1, AGR2, SLC6A14 and TOP2A. These genes have been associated with growth, proliferation, invasion and metastasis in pancreatic cancer previously. Cumulatively, this demonstrates the applicability of PDX models and transcriptomic array to identify genes associated with growth and proliferation of pancreatic cancer.

The enhanced recruitment of leukocytes to the inflamed colon is a key feature of ulcerative colitis (UC). The gut-specific adhesion molecules involved in leukocyte recruitment have emerged as recent therapeutic targets. Nicotine absorbed from smoking has been reported to work protectively in UC patients. Our hypothesis is that nicotine may suppress the aberrant leukocyte recruitment and colonic inflammation via the suppression of the overexpressed gut-specific adhesion molecules in the inflamed colon. To test this hypothesis, the severity of colitis and the degree of leukocyte recruitment induced by gut-specific adhesion molecules were assessed in dextran sulfate sodium (DSS) colitis mice (C57BL/6J mice treated with 3% DSS) with or without nicotine treatment. We also studied the in vitro changes in the expression of adhesion molecules by using a vascular endothelial cell line. DSS-induced colitis was accompanied by increases in disease activity index (DAI), histological score, recruitment of leukocytes, and the expression of adhesion molecules, mucosal vascular addressin cell adhesion molecule-1 (MAdCAM-1) and VCAM-1. Nicotine treatment significantly attenuated MAdCAM-1 expression, leukocyte recruitment, DAI, and histological score. The expression of β7-integrin, the ligand for MAdCAM-1, on leukocytes was not affected by nicotine treatment. In vitro study, the TNF-α-enhanced mRNA expression of MAdCAM-1 was reduced by the coadministration of nicotine in a dose-dependent manner, possibly via nicotinic receptor activation. These results supported our hypothesis that nicotine treatment ameliorated colitis through the suppression of MAdCAM-1 expression on the microvessels in the inflamed colon. Further investigation is warranted on the role of nicotine in the treatment of UC. Nicotine ameliorated the over-expressed MAdCAM-1 and inhibited leukocyte recruitment in murine colitis.

Chorioamnionitis, mechanical ventilation, oxygen therapy, and postnatal infection promote inflammation in the newborn lung. The long-term consequences of pulmonary inflammation during infancy have not been well characterized. The aim of this study was to examine the impact of inflammation during the late saccular to alveolar stages of lung development on lung structure and function in adulthood. To induce IL-1β expression in the pulmonary epithelium of mice with a tetracycline-inducible human IL-1β transgene, doxycycline was administered via intraperitoneal injections to bitransgenic pups and their littermate controls on postnatal days (PN) 0, 0.5, and 1. Lung structure, inflammation, and airway reactivity were studied in adulthood. IL-1β production in early life resulted in increased numbers of macrophages and neutrophils on PN21, but inflammation subsided by PN42. Permanent changes in alveolar structure, i.e., larger alveoli and thicker alveolar walls, were present from PN21 to PN84. Lack of alveolar septation thus persisted after IL-1β production and inflammation had ceased. Early IL-1β production caused goblet cell hyperplasia, enhanced calcium-activated chloride channel 3 (CLCA3) protein expression, and increased airway reactivity in response to methacholine on PN42. Lymphoid follicles were present adjacent to small airways in the lungs of adult bitransgenic mice, and levels of the B cell chemoattractant CXC-motif ligand (CXCL) 13 were elevated in the lungs of bitransgenic mice compared with controls. In conclusion, IL-1β-induced pulmonary inflammation in early life causes a chronic lung disease in adulthood.

Integrins are cell surface receptors with bidirectional signalling capabilities that can bind to adhesion molecules in order to mediate homing of leukocytes to peripheral tissues. Gut-selective leukocyte homing is facilitated by interactions between α4β7 and its ligand, mucosal addressin cellular adhesion molecule-1 [MAdCAM-1], while retention of lymphocytes in mucosal tissues is mediated by αEβ7 binding to its ligand E-cadherin. Therapies targeting gut-selective trafficking have shown efficacy in inflammatory bowel disease [IBD], confirming the importance of leukocyte trafficking in disease pathobiology. This review will provide an overview of integrin structure, function and signalling, and highlight the role that these molecules play in leukocyte homing and retention. Anti-integrin therapeutics, including gut-selective antibodies against the β7 integrin subunit [etrolizumab] and the α4β7 integrin heterodimer [vedolizumab and abrilumab], and the non-gut selective anti-α4 integrin [natalizumab], will be discussed, as well as novel targeting approaches using small molecules.

Bacterial biofilms are highly resistant to the action of antibiotics. Presence of persisters, phenotypically resistant populations of bacterial cells, is thought to contribute toward recalcitrance of biofilms. The phage-derived lysins, by virtue of their ability to cleave the peptidoglycan of bacterial cells in an enzymatic manner, have the unique ability to kill dormant cells. Several lysins have shown potent antibiofilm activity in vitro. The fact that lysins have shown better efficacy than conventional drugs in animal models of endocarditis and other infections involving biofilms suggests that the lysins can potentially be developed against difficult-to-treat bacterial infections.

Protein evolution is driven by the sum of different physiochemical and genetic processes that usually results in strong purifying selection to maintain biochemical functions. However, proteins that are part of systems under arms race dynamics often evolve at unparalleled rates that can produce atypical biochemical properties. In the marine mollusk abalone, lysin and VERL are a pair of rapidly coevolving proteins that are essential for species-specific interactions between sperm and egg. Despite extensive biochemical characterization of lysin, including crystal structures of multiple orthologs, it was unclear how sites under positive selection may facilitate recognition of VERL. Using a combination of targeted mutagenesis and multidimensional NMR, we present a high-definition solution structure of sperm lysin from red abalone (Haliotis rufescens). Unapparent from the crystallography data, multiple NMR-based analyses conducted in solution reveal clustering of the N-and C-termini to form a nexus of 13 positively selected sites that constitute a VERL binding interface. Evolutionary rate was found to be a significant predictor of backbone flexibility, which may be critical for lysin bioactivity and / or accelerated evolution. These flexible, rapidly evolving segments that constitute the VERL binding interface were also the most distorted regions of the crystal structure relative to what was observed in solution. While lysin has been the subject of extensive biochemical and evolutionary analyses for more than 30 years, this study highlights the enhanced insights gained from applying NMR approaches to rapidly evolving proteins.SignificanceThe fertilization of eggs by sperm is a critical biological process for nearly all sexually reproducing organisms to propagate their genetic information. Despite the importance of fertilization, the molecules that mediate egg-sperm interactions have been characterized for only a few species, and the biochemical mechanisms underlying these interactions are even less well understood. In the marine mollusk abalone, sperm lysin interacts with egg VERL in a species-specific manner to facilitate fertilization. In this report, we characterized the solution structure and molecular evolution of sperm lysin from red abalone (Haliotis rufescens), and identified the VERL binding interface as well as important lysin dimer interactions that have emerged as part of an incessant sexual arms race.

AGR2 is a well-studied secreted protein that is involved in multiple biological processes including cell proliferation and migration. The mechanism by which AGR2 increases the growth and migration of non-small cell lung cancer cells (NSCLC) is still unknown. In this study, we report that AGR2 is directly targeted by miR-342-3p. Functional studies suggest that overexpression of miR-342-3p inhibits the proliferation and migration of non-small cell lung cancer cells. Overexpression of AGR2 counteracts the phenotypes induced by miR-342-3p. Moreover, AGR2 expression is up-regulated and negatively correlated with miR-342-3p levels in NSCLC cells and tissues. A meta-analysis of survival data indicates that NSCLC patients with high levels of AGR2 in their tumors have a worse prognosis. Collectively, the identification of miR-342-3p and AGR2 might facilitate the development of biomarkers and therapeutic targets for this devastating disease.

Background & Aims Oral therapies targeting the integrin α4β7 may offer unique advantages for the treatment of inflammatory bowel disease. We characterized the oral α4β7 antagonist peptide PTG-100 in preclinical models and established safety, pharmacokinetic/pharmacodynamic relationships, and efficacy in a phase 2a trial in patients with ulcerative colitis (UC). Methods In vitro studies measured binding properties of PTG-100. Mouse studies measured biomarkers and drug concentrations in blood and tissues. The phase 1 study involved healthy volunteers. In phase 2a, patients with moderate to severe active UC were randomized to receive PTG-100 (150, 300, or 900 mg) or placebo once daily for 12-weeks. Results PTG-100 potently and selectively blocks α4β7. Oral dosing of PTG-100 in mice showed high levels of target engagement and exposure in gut-associated lymphoid tissues. In healthy volunteers, PTG-100 showed dose-dependent increases in plasma exposure and blood target engagement. Although this phase 2a study initially did not meet the primary endpoint, a blinded reread of the endoscopy videos by a third party indicated clinical efficacy in conjunction with histologic remission at doses correlating with less than 100% receptor occupancy in peripheral blood. Conclusions PTG-100 showed local gastrointestinal tissue target engagement and inhibition of memory T-cell trafficking in mice. It was safe and well tolerated in phase 1 and 2 studies. Phase 2a data are consistent with biological and clinical response and showed a dose response reflecting similar activities in preclinical models and healthy individuals. These data suggest that local gut activity of an oral α4β7 integrin antagonist, distinct from full target engagement in blood, are important for efficacy and the treatment of UC. (ClinicalTrials.gov, Number NCT02895100; EudraCT, Number 2016-003452-75)

Objective This study investigates the differential expression and the mechanism of long intergenic non-protein coding RNA (LINC) 01857 in hepatocellular carcinoma (HCC) proliferation and apoptosis. Methods LINC01857 expression in HCC tissues and cells was evaluated. In addition, gain-of and loss-of functions were carried out to assess HCC cell proliferation and apoptosis. After that, LINC01857 subcellular localization was predicted and verified. Additionally, the binding relations between LINC01857 and microRNA (miRNA)-197-3p and between miR-197-3p and anterior GRadient 2 (AGR2) were detected and confirmed. Besides, HCC cell proliferation and apoptosis were assessed after silencing LINC01857 or overexpressing AGR2. Next, levels of key factors in the AKT and ERK pathways were measured. Additionally, xenograft transplantation was also conducted to confirm the effect of LINC01857 in HCC. Results LINC01857 was overexpressed in HCC. Silencing LINC01857 leads to a blockage in HCC cell proliferation but improved apoptosis. LINC01857 could competitively bind to miR-197-3p and thus upregulate AGR2. miR-197-3p was poorly expressed in HCC, while AGR2 was overexpressed. Mechanistically, downregulated miR-197-3p or overexpressed AGR2 were observed to attenuate the effect of the LINC01857 knockdown on suppressing cell proliferation and enhancing apoptosis. Moreover, LINC01857 activated the AKT and ERK pathways through the manipulation of the miR-197-3p/AGR2 axis in HCC. Conclusion The results of this study indicated that LINC01857 was highly expressed in HCC, and it could improve HCC cell proliferation and reduce apoptosis via competitively binding to miR-197-3p, promoting AGR2 and upregulating the AKT and ERK pathways.

Asthma tolerance can be induced by breast-feeding or oral feeding with ovalbumin (OVA). Anergy or deletion of specific T cells and generation of T regulatory cells might contribute to this process. However, whether respiratory syncytial virus (RSV) infection would affect asthma tolerance is not very clear. Here, we first established asthma and oral tolerance mouse models and then analyzed airway hypersensitivity and asthma-related genes in the lung, CCR6-expressing IL-17A+ cells in the lungs, hilar or mesenteric lymph nodes (HLN or MLN) among control, asthmatic, tolerized, RSV infection, and RSV-infected asthmatic and tolerized groups. We also administrated CCL20 or IL-17A neutralizing antibody to RSV-infected tolerized mice to test whether RSV infection would mobilize CCR6-expressing IL-17A+ cells from MLN to the infected lungs. We found that tolerized mice infected with RSV developed asthma-like responses manifested by increasing airway hypersensitivity, exacerbating peribronchial inflammation, elevating lung asthma-related genes (Il17a, Mu5ac, and Gob5), accumulating CCR6-expressing IL-17A+ cells in the lungs and HLN with a reduction of this cell population in MLN. CCL20-CCR6 co-expression in RSV-infected tolerized MLN was reduced. Neutralization of CCL20 reduced CD3+CD4+CCR6+ cells in the RSV-infected tolerized HLN. Neutralization of IL-17A mitigated the compromising effects of RSV infection on asthma tolerance. Taken together, RSV infection impairs asthma tolerance by recruiting IL-17A-producing cells via CCR6-CCL20 signaling. The findings provide novel insight into exacerbation and therapeutic strategy of asthma under RSV infection.

Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Despite recent identification of metabolic alterations in this lethal malignancy, the metabolic dependencies of obesity-associated PDA remain unknown. Here we show that obesity-driven PDA exhibits accelerated growth and a striking transcriptional enrichment for pathways regulating nitrogen metabolism. We find that the mitochondrial form of arginase (ARG2), which hydrolyzes arginine into ornithine and urea, is induced upon obesity, and silencing or loss of ARG2 markedly suppresses PDA. In vivo infusion of 15N-glutamine in obese mouse models of PDA demonstrates enhanced nitrogen flux into the urea cycle and infusion of 15N-arginine shows that Arg2 loss causes significant ammonia accumulation that results from the shunting of arginine catabolism into alternative nitrogen repositories. Furthermore, analysis of PDA patient tumors indicates that ARG2 levels correlate with body mass index (BMI). The specific dependency of PDA on ARG2 rather than the principal hepatic enzyme ARG1 opens a therapeutic window for obesity-associated pancreatic cancer., Obesity is an established risk factor for pancreatic ductal adenocarcinoma (PDA). Here the authors show that obesity induces the expression of the mitochondrial form of arginase ARG2 in PDA and that ARG2 silencing or loss results in ammonia accumulation and suppression of obesity-driven PDA tumor growth.

The importance of the tumor microenvironment in targeted anticancer therapies has been well recognized. Various protein factors participate in the cross-talk between tumor cells and non-malignant cells. Anterior gradient-2 (AGR2) is overexpressed in diverse human adenocarcinomas and it exists in both intracellular and extracellular spaces. Although intracellular AGR2 has been intensively investigated, the function of secreted AGR2, especially its exact mechanism of action is still poorly understood. Here we report that the secreted AGR2 promotes the angiogenesis and the invasion of vascular endothelial cells and fibroblasts by enhancing the activities of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2). Further study indicated that AGR2 directly binds to these extracellular signaling molecules, and enhances their homodimerization. The extracellular AGR2 activity can be blocked to reduce angiogenesis and inhibit tumor growth in vitro and in vivo by a monoclonal antibody targeting the AGR2 self-dimerization region, and combined treatment with bevacizumab produced maximum inhibition effect. In conclusion, our investigation reveals a mechanism that directly links the secreted AGR2 with extracellular signaling networks, and we propose that the secreted AGR2 is a blockable molecular target, which acts as a chaperon-like enhancer to VEGF and FGF2.

AIM To study mucosal addressin cellular adhesion molecule-1 (MAdCAM-1) and vascular endothelial growth factor (VEGF)-targeted contrast enhanced ultrasound (CEUS) for the assessment of murine colitis and carcinogenesis. METHODS C57BL/6 mice were challenged with 3% dextran sodium-sulfate (DSS) for three, six or nine days to study the development of acute colitis. Ultrasound was performed with and without the addition of unspecific contrast agents. MAdCAM-1-targeted contrast agent was used to detect and quantify MAdCAM-1 expression. Inflammatory driven colorectal azoxymethane (AOM)/DSS-induced carcinogenesis was examined on day 42 and 84 using VEGF-targeted contrast agent. Highly specific tissue echogenicity was quantified using specialized software. Sonographic findings were correlated to tissue staining, western blot analysis and immunohistochemistry to quantify the degree of inflammation and stage of carcinogenesis. RESULTS Native ultrasound detected increased general bowel wall thickening that correlated with more progressed and more severe DSS-colitis (healthy mice: 0.3 mm ± 0.03 vs six days DSS: 0.5 mm ± 0.2 vs nine days DSS: 0.6 mm ± 0.2, P < 0.05). Moreover, these sonographic findings correlated well with clinical parameters such as weight loss (r2 = 0.74) and histological damage (r2 = 0.86) (P < 0.01). In acute DSS-induced murine colitis, CEUS targeted against MAdCAM-1 detected and differentiated stages of mild, moderate and severe colitis via calculation of mean pixel contrast intensity in decibel (9.6 dB ± 1.6 vs 12.9 dB ± 1.4 vs 18 dB ± 3.33, P < 0.05). Employing the AOM/DSS-induced carcinogenesis model, tumor development was monitored by CEUS targeted against VEGF and detected a significantly increased echogenicity in tumors as compared to adjacent healthy mucosa (healthy mucosa, 1.6 dB ± 1.4 vs 42 d, 18.2 dB ± 3.3 vs 84 d, 18.6 dB ± 4.9, P < 0.01). Tissue echogenicity strongly correlated with histological analysis and immunohistochemistry findings (VEGF-positive cells in 10 high power fields of healthy mucosa: 1 ± 1.2 vs 42 d after DSS start: 2.4 ± 1.6 vs 84 d after DSS start: 3.5 ± 1.3, P < 0.01). CONCLUSION Molecularly targeted CEUS is a highly specific and non-invasive imaging modality, which characterizes murine intestinal inflammation and carcinogenesis in vivo.

Sexual selection can explain the rapid evolution of fertilization proteins, yet sperm proteins evolve rapidly even if not directly involved in fertilization. In the marine mollusk abalone, sperm secrete enormous quantities of two rapidly evolving proteins, lysin and sp18, that are stored at nearly molar concentrations. We demonstrate that this extraordinary packaging is achieved by associating into Fuzzy Interacting Transient Zwitterion (FITZ) complexes upon binding the intrinsically disordered FITZ Anionic Partner (FITZAP). FITZ complexes form at intracellular ionic strengths and, upon exocytosis into seawater, lysin and sp18 are dispersed to drive fertilization. NMR analyses revealed that lysin uses a common molecular interface to bind both FITZAP and its egg receptor VERL. As sexual selection alters the lysin-VERL interface, FITZAP coevolves rapidly to maintain lysin binding. FITZAP-lysin interactions exhibit a similar species-specificity as lysin-VERL interactions. Thus, tethered molecular arms races driven by sexual selection can generally explain rapid sperm protein evolution.

Aims Hepatocellular carcinoma (HCC) is a leading cause of cancer mortality worldwide. Decrease in NKG2D ligand levels and exhaustion of NK cells in HCC patients are major causes of immune escape, high recurrence, poor prognosis, and low overall survival. Enhancing the susceptibility of HCC to NK cells by upregulating NKG2DLs on tumor cells is an effective treatment strategy. This study aimed to identify the effect of the Anterior gradient 2 (AGR2)-derived peptide P1, which was reported to bind to HLA-A*0201 as an epitope, on both the expression of major histocompatibility complex class I-related chains A/B (MICA/B) on HCC cells and the cytotoxicity of NK cells. Main methods The effect of P1 on MICA/B expression on HCC cells was determined by qRT-PCR, western blotting, and flow cytometry analysis. HCC cells were pre-treated with various pathway inhibitors to identify the molecular pathways associated with P1 treatment. The cytotoxicity of NK cells toward HCC was investigated by LDH cytotoxicity assay. The tumor-suppression effect of P1 was determined in vivo using a NOD/SCID mice HCC model. Key findings P1 significantly increased MICA/B expression on HCC cells, thereby enhancing their susceptibility to the cytotoxicity of NK cells in vitro and in vivo. Further, p38 MAPK cell signaling pathway inhibitor SB203580 significantly attenuated the effects of P1 in vivo and in vitro. Significance P1 upregulates MICA and MICB expression on HCC cells, thereby promoting their recognition and elimination by NK cells, which makes P1 an attractive novel immunotherapy agent.

ObjectivesMyositis is a heterogeneous family of diseases that includes dermatomyositis (DM), antisynthetase syndrome (AS), immune-mediated necrotising myopathy (IMNM), inclusion body myositis (IBM), polymyositis and overlap myositis. Additional subtypes of myositis can be defined by the presence of myositis-specific autoantibodies (MSAs). The purpose of this study was to define unique gene expression profiles in muscle biopsies from patients with MSA-positive DM, AS and IMNM as well as IBM.MethodsRNA-seq was performed on muscle biopsies from 119 myositis patients with IBM or defined MSAs and 20 controls. Machine learning algorithms were trained on transcriptomic data and recursive feature elimination was used to determine which genes were most useful for classifying muscle biopsies into each type and MSA-defined subtype of myositis.ResultsThe support vector machine learning algorithm classified the muscle biopsies with >90% accuracy. Recursive feature elimination identified genes that are most useful to the machine learning algorithm and that are only overexpressed in one type of myositis. For example, CAMK1G (calcium/calmodulin-dependent protein kinase IG), EGR4 (early growth response protein 4) and CXCL8 (interleukin 8) are highly expressed in AS but not in DM or other types of myositis. Using the same computational approach, we also identified genes that are uniquely overexpressed in different MSA-defined subtypes. These included apolipoprotein A4 (APOA4), which is only expressed in anti-3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) myopathy, and MADCAM1 (mucosal vascular addressin cell adhesion molecule 1), which is only expressed in anti-Mi2-positive DM.ConclusionsUnique gene expression profiles in muscle biopsies from patients with MSA-defined subtypes of myositis and IBM suggest that different pathological mechanisms underly muscle damage in each of these diseases.

Mucosal tissues play critical roles in health and disease as the primary barrier between the external world and the inner body, lining the digestive, respiratory, urinary, mammary, and reproductive tracts. Clinical evaluation of mucosal tissues is currently performed using endoscopy, such as ileocolonoscopy for the intestinal mucosa, which causes substantial patient discomfort and can lead to organ damage. Here, we developed a contrast agent for molecular magnetic resonance imaging (MRI) that is targeted to mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1), an adhesion molecule overexpressed by inflamed mucosal tissues. We investigated the diagnostic performance of molecular MRI of MAdCAM-1 to detect mucosal inflammation in several models of acute and chronic intestinal inflammation in mice. We demonstrated that molecular MRI of MAdCAM-1 reveals disease activity and can evaluate the response to inflammatory treatments along the whole intestinal mucosa in clinically relevant models of inflammatory bowel diseases. We also provide evidence that this technique can detect low, subclinical mucosal inflammation. Molecular MRI of MAdCAM-1 has potential applications in early diagnosis, longitudinal follow-up, and therapeutic response monitoring in diseases affecting mucosal tissues, such as inflammatory bowel diseases.

Extra-intestinal manifestations (EIMs) of the eyes are found in IBD patients, but the underlying pathogenesis remains unknown. To investigate the pathogenesis of IBD-associated retinal dysfunction, chronic colitis was induced in mice by oral administration of dextran sodium sulfate (DSS). Electroretinography (ERG) was performed to evaluate retinal function. Retinal neuron degeneration was analyzed by immunohistochemistry. Colitic mice displayed aberrant amplitudes of ERG a-, b-wave and oscillatory potentials (OP). Importantly, we observed severe degeneration of bipolar and ganglion cells. In contrast, outer retinal neurons (mainly photoreceptor cells) are mildly affected by colitis. Moreover, retinal inflammatory responses were significantly upregulated during colitis, including microglia activation, lymphocyte infiltration and cytokine/chemokine production. Notably, mucosal addressin cell adhesion molecule 1 (MAdCAM-1) was upregulated in retinal microvessels, especially the superficial and deep plexuses, and recruited gut-homing CD4

International audience; Anterior gradient 2 (AGR2) is a dimeric protein disulfide isomerase family member involved in the regulation of protein quality control in the endoplasmic reticulum (ER). Mouse AGR2 deletion increases intestinal inflammation and promotes the development of inflammatory bowel disease (IBD). Although these biological effects are well established, the underlying molecular mechanisms of AGR2 function toward inflammation remain poorly defined. Here, using a protein-protein interaction screen to identify cellular regulators of AGR2 dimerization, we unveiled specific enhancers, including TMED2, and inhibitors of AGR2 dimerization, that control AGR2 functions. We demonstrate that modulation of AGR2 dimer formation, whether enhancing or inhibiting the process, yields pro-inflammatory phenotypes, through either autophagy-dependent processes or secretion of AGR2, respectively. We also demonstrate that in IBD and specifically in Crohn's disease, the levels of AGR2 dimerization modulators are selectively deregulated, and this correlates with severity of disease. Our study demonstrates that AGR2 dimers act as sensors of ER homeostasis which are disrupted upon ER stress and promote the secretion of AGR2 monomers. The latter might represent systemic alarm signals for pro-inflammatory responses.

Frontiers in immunology 10, 366 (2019). doi:10.3389/fimmu.2019.00366, Published by Frontiers Media, Lausanne

Frontiers in immunology 10, 903 (2019). doi:10.3389/fimmu.2019.00903, Published by Frontiers Media, Lausanne

Pancreatic cancer is the fourth leading cause of cancer death in the United States. Better understanding of pancreatic cancer biology may help identify new oncotargets towards more effective therapies. This study investigated the mechanistic actions of microRNA-1291 (miR-1291) in the suppression of pancreatic tumorigenesis. Our data showed that miR-1291 was downregulated in a set of clinical pancreatic carcinoma specimens and human pancreatic cancer cell lines. Restoration of miR-1291 expression inhibited pancreatic cancer cell proliferation, which was associated with cell cycle arrest and enhanced apoptosis. Furthermore, miR-1291 sharply suppressed the tumorigenicity of PANC-1 cells in mouse models. A proteomic profiling study revealed 32 proteins altered over 2-fold in miR-1291-expressing PANC-1 cells that could be assembled into multiple critical pathways for cancer. Among them anterior gradient 2 (AGR2) was reduced to the greatest degree. Through computational and experimental studies we further identified that forkhead box protein A2 (FOXA2), a transcription factor governing AGR2 expression, was a direct target of miR-1291. These results connect miR-1291 to the FOXA2-AGR2 regulatory pathway in the suppression of pancreatic cancer cell proliferation and tumorigenesis, providing new insight into the development of miRNA-based therapy to combat pancreatic cancer.

The current study was designed to determine the effects of antibody blockade of mucosal addressin cell adhesion molecule-1 (MAdCAM-1) on the proinflammatory activity of mesenteric lymph after hemorrhagic shock and resuscitation (HS/R).Rats were subjected to HS/R with or without treatment with MAdCAM-1 polyclonal antibody. MAdCAM-1 expression and lymphocyte infiltration in rats were examined. Post-shock mesenteric lymph was collected, filtered to remove lymphocytes, and transfused into recipient mice to induce systemic inflammation and intestinal injury. The proinflammatory activity of post-shock lymph in mice was determined by examining intestinal permeability, enterocyte apoptosis, intestinal lactate levels, lung myeloperoxidase (MPO) activity, and serum cytokine levels. Survival of recipient mice was determined over a 1-week time period.Rats subjected to HS/R had increased MAdCAM-1 expression and lymphocyte infiltration in the intestine. Antibody blockade of MAdCAM-1 attenuated the increased lymphocyte infiltration after HS/R (P.05). Post-shock mesenteric lymph transfusion significantly increased mortality accompanied by increases in gut permeability, enterocyte apoptosis, intestinal lactate levels, lung MPO activity, and serum levels of interleukin (IL)-1β, IL-6, tumor necrosis factor (TNF)-α, IL-10, and transforming growth factor-β (all P.05). Antibody blockade of MAdCAM-1 in rats subjected to HS/R attenuated the proinflammatory activity of post-shock mesenteric lymph, with abrogation of lymph transfusion-induced increases in mortality, gut permeability, epithelial cell apoptosis, intestinal lactate levels, lung MPO activity, and serum levels of IL-1β, IL-6, and TNF-α (all P.05).These findings demonstrate that antibody blockade of MAdCAM-1 attenuates the proinflammatory activity of mesenteric lymph after HS/R.

Staphylococcus aureus (S. aureus) is an important food-borne pathogen in dairy products contaminated through raw ingredients or improper food handling. Rapid detection of S. aureus with high sensitivity is of significance for food quality and safety. In this study, a new method was developed for detecting S. aureus in milk by coupling immunomagnetic separation with enzyme linked cell wall binding domain (CBD) of lysin plyV12, which can bind to S. aureus with high affinity. There are millions of binding sites present on the cell surface of S. aureus for the CBD attachment, which greatly improves the detection sensitivity. The method has the overall testing time of only 1.5h with the detection limit of 4 × 10(3)CFU/mL in spiked milk. Because it is simple, rapid and sensitive, this method could be used for the detection of S. aureus in various food samples.

This study aimed to examine eugenic acid (EA) as an alternative therapeutic approach against pancreatic cancer. The pancreatic cancer xenograft mouse model was employed to determine the impacts of treatment with EA on the growth of tumors. Expressions of NF-κB subunit RelA as well as Anterior gradient 2 (AGR2) were quantified in pancreatic cells treated with EA. Chromatin immunoprecipitation and luciferase report assay were performed to examine the regulation of AGR2 by RelA. The function of AGR2 as a downstream effector EA treatment was further assessed through overexpression of AGR2 in pancreatic cells. EA suppressed the growth of xenograft pancreatic tumor, and promoted the overall survival of animals with xenograft tumors. Furthermore, EA downregulated the expression of AGR2 in pancreatic cancer cells via the RelA binding site. Ectopic AGR2 overexpression attenuated the EA-elicited inhibition on the growth of xenograft pancreatic tumor, and negated the EA-induced enhancement of mouse survival. EA ameliorates pancreatic cancer through suppression of AGR2 expression, and future studies in clinical settings are needed to further assess the anti-cancer efficacy of EA.

Background & Aims Aberrant lymphocyte homing could potentially link inflammatory processes in the intestine and the liver, as distinct hepatobiliary diseases frequently develop as extra-intestinal manifestations in inflammatory bowel disease. In this study, we examined the role of the gut-tropic leukocyte adhesion molecule β7 integrin and its endothelial ligand mucosal addressin cell-adhesion molecule-1 (MAdCAM-1) in immune-mediated hepatitis in mice. Methods Wild-type (WT) mice, MAdCAM-1-deficient mice, β7 integrin-deficient mice, RAG-2–deficient mice, RAG-2/MAdCAM-1 double-deficient mice, and RAG-2/β7 integrin double-deficient mice were subjected to concanavalin A (ConA)-induced hepatitis. The degree of hepatitis was evaluated by histology, flow cytometry, and expression analysis of inflammatory mediators. The motility of lymphocytes in progressive liver damage was assessed by intravital laser scanning multiphoton microscopy. Results Ablation of MAdCAM-1 or β7 integrin ameliorated ConA-induced hepatitis in mice. β7 integrin-deficient lymphocytes caused less liver damage than WT lymphocytes in ConA-treated RAG-2–deficient mice. Moreover, WT lymphocytes caused less liver damage in ConA-treated RAG-2/β7 integrin double-deficient mice than in similarly treated RAG-2–deficient mice, indicating that β7 integrin expression contributes significantly to the liver damage mediated by innate immune cells. MAdCAM-1 expression was dependent on β7 integrin expression on adaptive and innate immune cells. Most importantly, lymphocytes in ConA-treated MAdCAM-1-deficient mice displayed more motility and less adhesion in the liver sinusoids in vivo, than lymphocytes in similarly treated WT mice. Conclusions These data suggest that β7 integrin expression on lymphocytes and innate immune cells contributes to MAdCAM-1 upregulation and liver damage in acute immune-mediated hepatitis, most likely by facilitating lymphocyte/sinusoidal endothelial cell interactions., Graphical abstract