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Multidrug resistance (MDR) mediated by ATP binding cassette subfamily B member 1 (ABCB1) is significantly hindering effective cancer chemotherapy. However, currently, no ABCB1-inhibitory drugs have been approved to treat MDR cancer clinically, mainly due to the inhibitor specificity, toxicity, and drug interactions. Here, we reported that three polyoxypregnanes (POPs) as the most abundant constituents of Marsdenia tenacissima (M. tenacissima) were novel ABCB1-modulatory pro-drugs, which underwent intestinal microbiota-mediated biotransformation in vivo to generate active metabolites. The metabolites at non-toxic concentrations restored chemosensitivity in ABCB1-overexpressing cancer cells via inhibiting ABCB1 efflux activity without changing ABCB1 protein expression, which were further identified as specific non-competitive inhibitors of ABCB1 showing multiple binding sites within ABCB1 drug cavity. These POPs did not exhibit ABCB1/drug metabolizing enzymes interplay, and their repeated administration generated predictable pharmacokinetic interaction with paclitaxel without obvious toxicity in vivo. We further showed that these POPs enhanced the accumulation of paclitaxel in tumors and overcame ABCB1-mediated chemoresistance. The results suggested that these POPs had the potential to be developed as safe, potent, and specific pro-drugs to reverse ABCB1-mediated MDR. Our work also provided scientific evidence for the use of M. tenacissima in combinational chemotherapy., Graphical abstract Type I polyoxypregnanes (POPs) as prodrugs can be biotransformed by gut microbiota to form active metabolites (type II POPs) that are specific, safe, and potent non-competitive inhibitors of ABCB1, showing unique mechanisms to reverse ABCB1-mediated cancer MDR.Image 1

Cancer therapy is a strategic measure in inhibiting breast cancer stem cell (BCSC) pathways. Naringenin, a citrus flavonoid, was found to increase breast cancer cells’ sensitivity to chemotherapeutic agents. Bioinformatics study and 3D tumorsphere in vitro modeling in breast cancer (mammosphere) were used in this study, which aims to explore the potential therapeutic targets of naringenin (PTTNs) in inhibiting BCSCs. Bioinformatic analyses identified direct target proteins (DTPs), indirect target proteins (ITPs), naringenin-mediated proteins (NMPs), BCSC regulatory genes, and PTTNs. The PTTNs were further analyzed for gene ontology, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment, protein–protein interaction (PPI) networks, and hub protein selection. Mammospheres were cultured in serum-free media. The effects of naringenin were measured by MTT-based cytotoxicity, mammosphere forming potential (MFP), colony formation, scratch wound-healing assay, and flow cytometry-based cell cycle analyses and apoptosis assays. Gene expression analysis was performed using real-time quantitative polymerase chain reaction (q-RT PCR). Bioinformatics analysis revealed p53 and estrogen receptor alpha (ERα) as PTTNs, and KEGG pathway enrichment analysis revealed that TGF-ß and Wnt/ß-catenin pathways are regulated by PTTNs. Naringenin demonstrated cytotoxicity and inhibited mammosphere and colony formation, migration, and epithelial to mesenchymal transition in the mammosphere. The mRNA of tumor suppressors P53 and ERα were downregulated in the mammosphere, but were significantly upregulated upon naringenin treatment. By modulating the P53 and ERα mRNA, naringenin has the potential of inhibiting BCSCs. Further studies on the molecular mechanism and formulation of naringenin in BCSCs would be beneficial for its development as a BCSC-targeting drug.

Background & Aims Despite recent advances in antiviral therapy for hepatitis C virus (HCV), a proportion of patients with genotype 3 (G3) HCV infection do not respond to current all oral treatment regimens. Genomic analyses have identified key polymorphisms correlating with increased resistance to direct-acting antivirals. We previously reported that amino acid polymorphisms (A150V and K206E) in the polymerase (NS5B) of G3 HCV reduce response to sofosbuvir. We now demonstrate that these polymorphisms alter the response to interferon alpha. Methods Quantitative polymerase chain reaction, immunofluorescence, luciferase activity assay, immunoblotting, and flow cytometry were used to study the antiviral effect of interferon (IFN) on DBN G3 HCV-infected cells and G3 HCV replicons. Results We show the presence of the A150V polymorphism markedly reduces the response to IFN alpha (IC50 of S52_WT = 1.162 IU/mL and IC50 of S52_A150V = 14.45 IU/mL, 12.4-fold difference). The induction of IFN-stimulated genes in A150V replicon cells is unaffected, but nuclear localization of active protein kinase R (PKR) is reduced. Blockade of PKR activity reduced the antiviral effect of IFN on wild-type replicons, whereas augmented PKR activation promoted the antiviral effect of IFN on A150V replicons. Furthermore, we show that impaired activation of PKR in A150V replicon cells diminishes cellular apoptosis. Conclusions These results demonstrate that polymorphisms reducing response rates to direct-acting antivirals may function beyond conferring drug resistance by modulating the intrinsic cellular antiviral response.

Graphical abstract, HER2-positive breast cancer is one of its most challenging subtypes, forming around 15–25% of the total cases. It is characterized by aggressive behavior and treatment resistance. On the other hand, poly (amidoamine) (PAMAM) dendrimers are widely used in drug delivery systems and gene transfection as carriers. PAMAMs can modulate gene expression and interfere with transactivation of the human epidermal growth factor receptor family members (HER1-4). Nevertheless, the outcome of PAMAMs on HER2-positive breast cancer remains unknown. Thus, in this study, we investigated the anti-cancer effects of different generations of PAMAM dendrimers (G4 and G6) and the outcome of their surface chemistries (cationic, neutral, and anionic) on HER2-positive breast cancer cell lines, SKBR3 and ZR75. Our data showed that PAMAM dendrimers, mainly cationic types, significantly reduce cell viability in a dose-dependent manner. More significantly, PAMAMs induce substantial cell apoptosis, accompanied by the up-regulation of apoptotic markers (Bax, Caspases-3, 8 and 9) in addition to down-regulation of Bcl-2. Moreover, our data pointed out that cationic PAMAMs inhibit colony formation compared to controls and other types of PAMAMs. The molecular pathway analysis of PAMAM exposed cells revealed that PAMAMs enhance JNK1/2/3 expression while blocking ERK1/2, in addition to EGFR1 (HER1) and HER2 activities, which could be the major molecular pathway behind these events. These observed effects were comparable to lapatinib treatment, a clinically used inhibitor of HER1 and 2 receptors phosphorylation. Our findings implicate that PAMAMs may possess important therapeutic effects against HER2-positive breast cancer via JNK1/2/3, ERK1/2, and HER1/2 signalling pathways.

Background & Aims Rapid gastric epithelial progenitor cell (EPC) proliferation and inflammatory response inhibition play key roles in promoting the repair of gastric mucosal damage. However, specific targets inducing these effects are unknown. In this study, we explored the effects of a potential target, Ankyrin repeat domain 22 (ANKRD22). Methods An acute gastric mucosal injury model was established with Ankrd22-/- and Ankrd22+/+ mice by intragastric administration of acidified ethanol. Organoid culture and flow cytometry were performed to evaluate the effects of ANKRD22 on leucine-rich repeat-containing G-protein–coupled receptor 5–positive (Lgr5+) gastric EPC proliferation. The mechanisms by which ANKRD22 affects gastric EPC proliferation and inflammatory responses were explored by mitochondrial Ca2+ influx and immunoblotting. Candidate ANKRD22 inhibitors then were screened virtually and validated in vitro and in vivo. Results After acute gastric mucosal injury, the number of Lgr5+ gastric EPCs was increased significantly in Ankrd22-/- mice compared with that in Ankrd22+/+ mice. Moreover, Ankrd22 knockout attenuated inflammatory cell infiltration into damaged gastric tissues. ANKRD22 deletion also reduced mitochondrial Ca2+ influx and cytoplasmic nuclear factor of activated T cells in gastric epithelial cells and macrophages, which further induced Lgr5+ gastric EPC proliferation and decreased macrophage release of tumor necrosis factor-α and interleukin 1α. In addition, a small molecule, AV023, was found to show similar effects to those produced by ANKRD22 deletion in vitro. Intraperitoneal injection of AV023 into the mouse model promoted the repair of gastric mucosal damage, with increased proliferation of Lgr5+ gastric EPCs and visible relief of inflammation. Conclusions ANKRD22 inhibition is a potential target-based therapeutic approach for promoting the repair of gastric mucosal damage., Graphical abstract

Background & Aims How benign liver steatosis progresses to nonalcoholic steatohepatitis (NASH), fibrosis, and hepatocellular carcinoma (HCC) remains elusive. NASH progression entails diverse pathogenic mechanisms and relies on complex cross-talk between multiple tissues such as the gut, adipose tissues, liver, and the brain. Using a hyperphagic mouse fed with a Western diet (WD), we aimed to elucidate the cross-talk and kinetics of hepatic and extrahepatic alterations during NASH–HCC progression, as well as regression. Methods Hyperphagic mice lacking a functional Alms1 gene (Foz/Foz) and wild-type littermates were fed WD or standard chow for 12 weeks for NASH/fibrosis and for 24 weeks for HCC development. NASH regression was modeled by switching back to normal chow after NASH development. Results Foz+WD mice were steatotic within 1 to 2 weeks, developed NASH by 4 weeks, and grade 3 fibrosis by 12 weeks, accompanied by chronic kidney injury. Foz+WD mice that continued on WD progressed to cirrhosis and HCC within 24 weeks and had reduced survival as a result of cardiac dysfunction. However, NASH mice that were switched to normal chow showed NASH regression, improved survival, and did not develop HCC. Transcriptomic and histologic analyses of Foz/Foz NASH liver showed strong concordance with human NASH. NASH was preceded by an early disruption of gut barrier, microbial dysbiosis, lipopolysaccharide leakage, and intestinal inflammation. This led to acute-phase liver inflammation in Foz+WD mice, characterized by neutrophil infiltration and increased levels of several chemokines/cytokines. The liver cytokine/chemokine profile evolved as NASH progressed, with subsequent predominance by monocyte recruitment. Conclusions The Foz+WD model closely mimics the pathobiology and gene signature of human NASH with fibrosis and subsequent HCC. Foz+WD mice provide a robust and relevant preclinical model of NASH, NASH-associated HCC, chronic kidney injury, and heart failure., Supplemental Graphical Summary

Background & Aims Crohn’s disease (CD) likely represents decreased immune tolerance to intestinal bacterial antigens. Most CD patients have high titers of antibodies to intestinal commensal proteins, including the outer membrane porin C (OmpC) of Escherichia coli. Methods By using major histocompatibility complex II tetramers, we identified an HLA-DRB1∗15:01-restricted peptide epitope of OmpC recognized by CD4+ T cells in peripheral blood mononuclear cells from HLA-DRB1∗15:01+ healthy control (HC) and CD patients. Results The precursor frequency of these cells in CD correlated with anti-OmpC IgA titers, but did not differ from that of HCs. In both cohorts, they showed a CD161+, integrin α4β7+ phenotype ex vivo by flow cytometry, distinct from the C-X-C Motif Chemokine Receptor 3 phenotype of autologous influenza hemagglutinin (Flu) peptide-specific T cells. The T-cell receptor α and β chains of in vitro–expanded OmpC-specific T-cell clones often contained public amino acid sequences that were identical in cells from different patients. Expanded T-cell clones from CD subjects produced significantly less interleukin (IL)10 (P < .0001) than those from HCs, and a trend toward decreased production of the T helper 2 cell–associated IL4, IL5, and IL13 by CD clones also was seen. Conclusions Both HCs and CD patients have detectable OmpC-specific T cells in circulation, with similar immunophenotypes and often identical T-cell–receptor sequences. However, expanded clones from patients with CD produce less of the immunoregulatory cytokine IL10, showing a selective defect in the regulatory function of intestinal microbial antigen-specific T cells in patients with CD., Graphical abstract

Middle East respiratory syndrome (MERS) is a threat to public health worldwide. A vaccine against the causative agent of MERS, MERS-coronavirus (MERS-CoV), is urgently needed. We previously identified a peptide ligand, Co4B, which can enhance antigen (Ag) delivery to the nasal mucosa and promote Ag-specific mucosal and systemic immune responses following intranasal immunization. MERS-CoV infects via the respiratory route; thus, we conjugated the Co4B ligand to the MERS-CoV spike protein receptor-binding domain (S-RBD), and used this to intranasally immunize C57BL/6 and human dipeptidyl peptidase 4-transgenic (hDPP4-Tg) mice. Ag-specific mucosal immunoglobulin (Ig) A and systemic IgG, together with virus-neutralizing activities, were highly induced in mice immunized with Co4B-conjugated S-RBD (S-RBD-Co4B) compared to those immunized with unconjugated S-RBD. Ag-specific T cell-mediated immunity was also induced in the spleen and lungs of mice intranasally immunized with S-RBD-Co4B. Intranasal immunization of hDPP4-Tg mice with S-RBD-Co4B reduced immune cell infiltration into the tissues of virus-challenged mice. Finally, S-RBD-Co4B-immunized mice exhibited were better protected against infection, more likely to survive, and exhibited less body weight loss. Collectively, our results suggest that S-RBD-Co4B could be used as an intranasal vaccine candidate against MERS-CoV infection.

Highlights • PCC and LG-134 had direct cytotoxicity in murine melanoma cell lines (IC50 values ranged from 0.19–1.8 µM). • PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. • PCC treatment induced G0-G1 cell cycle arrest of melanoma cells. • PCC treatment caused complete regression of established melanoma tumors in all mice. • 17β-hydroxywithanolides have the potential to improve melanoma therapeutic outcome., Melanoma is an aggressive skin cancer that metastasizes to other organs. While immune checkpoint blockade with anti-PD-1 has transformed the treatment of advanced melanoma, many melanoma patients fail to respond to anti-PD-1 therapy or develop acquired resistance. Thus, effective treatment of melanoma still represents an unmet clinical need. Our prior studies support the anti-cancer activity of the 17β-hydroxywithanolide class of natural products, including physachenolide C (PCC). As single agents, PCC and its semi-synthetic analog demonstrated direct cytotoxicity in a panel of murine melanoma cell lines, which share common driver mutations with human melanoma; the IC50 values ranged from 0.19–1.8 µM. PCC treatment induced apoptosis of tumor cells both in vitro and in vivo. In vivo treatment with PCC alone caused the complete regression of established melanoma tumors in all mice, with a durable response in 33% of mice after discontinuation of treatment. T cell-mediated immunity did not contribute to the therapeutic efficacy of PCC or prevent tumor recurrence in YUMM2.1 melanoma model. In addition to apoptosis, PCC treatment induced G0-G1 cell cycle arrest of melanoma cells, which upon removal of PCC, re-entered the cell cycle. PCC-induced cycle cell arrest likely contributed to the in vivo tumor recurrence in a portion of mice after discontinuation of treatment. Thus, 17β-hydroxywithanolides have the potential to improve the therapeutic outcome for patients with advanced melanoma.

Leukemogenic potential of MLL fusion with the coiled-coil domain-containing partner genes and the downstream target genes of this type of MLL fusion have not been clearly investigated. In this study, we demonstrated that the coiled-coil–four-helix bundle structure of EB1 that participated in the MLL/EB1 was required for immortalizing mouse bone marrow (BM) cells and producing myeloid, but not lymphoid, cell lines. Compared to MLL/AF10, MLL/EB1 had low leukemogenic ability. The MLL/EB1 cells grew more slowly owing to increased apoptosis in vitro and induced acute monocytic leukemia with an incomplete penetrance and longer survival in vivo. A comparative analysis of transcriptome profiling between MLL/EB1 and MLL/AF10 cell lines revealed that there was an at least two-fold difference in the induction of 318 genes; overall, 51.3% (163/318) of the genes were known to be bound by MLL, while 15.4% (49/318) were bound by both MLL and MLL/AF9. Analysis of the 318 genes using Gene Ontology–PANTHER overrepresentation test revealed significant differences in several biological processes, including cell differentiation, proliferation/programmed cell death, and cell homing/recruitment. The Ets1 gene, bound by MLL and MLL/AF9, was involved in several biological processes. We demonstrated that Ets1 was selectively upregulated by MLL/EB1. Short hairpin RNA knockdown of Ets1 in MLL/EB1 cells reduced the expression of CD115, apoptosis rate, competitive engraftment to BM and spleen, and incidence of leukemia and prolonged the survival of the diseased mice. Our results demonstrated that MLL/EB1 upregulated Ets1, which controlled the balance of leukemia cells between apoptosis and BM engraftment/clonal expansion. Novelty and impact of this study The leukemogenic potential of MLL fusion with cytoplasmic proteins containing coiled-coil dimerization domains and the downstream target genes of this type of MLL fusion remain largely unknown. Using a retroviral transduction/transplantation mouse model, we demonstrated that MLL fusion with the coiled-coil–four-helix bundle structure of EB1 has low leukemogenic ability; Ets1, which is upregulated by MLL/EB1, plays a critical role in leukemic transformation by balance between apoptosis and BM engraftment/clonal expansion.

Background Sialic acids are widely distributed in nature and have biological relevance owing to their varied structural and functional roles. Immobilized neuraminidase can selectively remove terminal N-acetyl neuraminic acid from glycoproteins without altering the protein backbone while it can be easily removed from the reaction mixture avoiding sample contamination. This enables the evaluation of changes in glycoprotein performance upon desialylation. Methods Neuraminidase was immobilized onto agarose activated with cyanate ester groups and further used for desialylation of model glycoproteins, a lysate from tumour cells and tumour cells. Desialylation process was analysed by lectin binding assay, determination of sialyl-Tn or flow cytometry. Results Clostridium perfringens neuraminidase was immobilized with 91 % yield and expressed activity yield was of 41%. It was effective in the desialylation of bovine fetal serum fetuin, bovine lactoferrin and ovine submaxilar mucin. A decrease in sialic-specific SNA lectin recognition of 83% and 53 % was observed for fetuin and lactoferrin with a concomitant increase in galactose specific ECA and PNA lectin recognition. Likewise, a decrease in the recognition of a specific antibody (82%) upon mucin desialylation was observed. Moreover, desialylation of a protein lysate from the sialic acid-rich cell line TA3/Ha was also possible leading to a decrease in 47 % in SNA recognition. Immobilized neuraminidase kept 100% of its initial activity upon five desialylation cycles. Conclusions Immobilized neuraminidase is an interesting as well as a robust biotechnological tool for enzymatic desialylation purposes. General significance Immobilized neuraminidase would contribute to understand the role of sialic acid in biological processes., Graphical abstract Image 1, Highlights • Neuraminidase immobilization was successfully achieved. • Immobilized neuraminidase was effective in the desialylation of several model glycoproteins. • TA3/Ha tumour cell lysates were desialylated with immobilized neuraminidase. • Immobilized neuraminidase was successfully re-used.

Monoclonal antibodies can acquire the property of engagement of a second antigen via fusion methods or modification of their CDR loops, but also by modification of their constant domains, such as in the mAb2 format where a set of mutated amino acid residues in the CH3 domains enables a high-affinity specific interaction with the second antigen. We tested the possibility of introducing multiple binding sites for the second antigen by replacing the Fab CH1/CL domain pair with a pair of antigen-binding CH3 domains in a model scaffold with trastuzumab variable domains and VEGF-binding CH3 domains. Such bispecific molecules were produced in a “Fab-like” format and in a full-length antibody format. Novel constructs were of expected molecular composition using mass spectrometry. They were expressed at a high level in standard laboratory conditions, purified as monomers with Protein A and gel filtration and were of high thermostability. Their high-affinity binding to both target antigens was retained. Finally, the Her2/VEGF binding domain-exchanged bispecific antibody was able to mediate a potentiated surface Her2-internalization effect on the Her2-overexpressing cell line SK-BR-3 due to improved level of cross-linking with the endogenously secreted cytokine. To conclude, bispecific antibodies with Fabs featuring exchanged antigen-binding CH3 domains offer an alternative solution in positioning and valency of antigen binding sites., Graphical abstract Image 1, Highlights • Fab constant domains can be efficiently exchanged for antigen-binding CH3 domains. • Such mutagenesis results in bispecific antibodies with correct chain pairing. • Domain-exchanged bispecific Fab- and IgG-like formats are of favorable biophysical properties. • Resulting bispecific antibodies show high-affinity binding to both target antigens.

Numerous observations indicate that red blood cells (RBCs) affect T-cell activation and proliferation. We have studied effects of packed RBCs (PRBCs) on T-cell receptor (TCR) signaling and the molecular mechanisms whereby (P)RBCs modulate T-cell activation. In line with previous reports, PRBCs attenuated the expression of T-cell activation markers CD25 and CD69 upon costimulation via CD3/CD28. In addition, T-cell proliferation and cytokine expression were markedly reduced when T-cells were stimulated in the presence of PRBCs. Inhibitory activity of PRBCs required direct cell–cell contact and intact PRBCs. The production of activation-induced cellular reactive oxygen species, which act as second messengers in T-cells, was completely abrogated to levels of unstimulated T-cells in the presence of PRBCs. Phosphorylation of the TCR-related zeta chain and thus proximal TCR signal transduction was unaffected by PRBCs, ruling out mechanisms based on secreted factors and steric interaction restrictions. In large part, downstream signaling events requiring reactive oxygen species for full functionality were affected, as confirmed by an untargeted MS-based phosphoproteomics approach. PRBCs inhibited T-cell activation more efficiently than treatment with 1 mM of the antioxidant N-acetyl cysteine. Taken together, our data imply that inflammation-related radical reactions are modulated by PRBCs. These immunomodulating effects may be responsible for clinical observations associated with transfusion of PRBCs.

Recombinant vesicular stomatitis virus (VSV)-fusion and hemagglutinin (FH) was developed by substituting the promiscuous VSV-G glycoprotein (G) gene in the backbone of VSV with genes encoding for the measles virus envelope proteins F and H. Hybrid VSV-FH exhibited a multifaceted mechanism of cancer-cell killing, and improved neurotolerability over parental VSV in preclinical studies. In this study, we evaluated VSV-FH in vitro and in vivo in models of hepatobiliary and pancreatic cancers. Our results indicate that high intrahepatic doses of VSV-FH did not result in any significant toxicity and were well tolerated by transgenic mice expressing the measles virus receptor CD46. Furthermore, single intratumoral treatments with VSV-FH yielded improved survival and complete tumor regressions in a proportion of mice in the Hep3B hepatocellular carcinoma model, but not in mice xenografted with BxPC3 pancreatic cancer cells. Our preliminary findings indicate that VSV-FH can induce potent oncolysis in hepatocellular and pancreatic cancer cell lines with concordant results in vivo in hepatocellular cancer and discordant in pancreatic cancer, without the VSV-mediated toxic effects previously observed in laboratory animals. Further study of VSV-FH as an oncolytic virotherapy is warranted in hepatocellular carcinoma and pancreatic cancer, to understand broader applicability and mechanisms of sensitivity and resistance., Graphical Abstract

Background & Aims Psoriasis and inflammatory bowel disease (IBD) are both chronic inflammatory diseases occurring in the skin and gut, respectively. It is well established that psoriasis and IBD have high concordance rates, and similar changes in immune cells and microbiome composition have been reported in both conditions. To study this connection, we used a combination murine model of psoriatic dermatitis and colitis in which mice were treated topically with the Toll-like receptor 7 agonist imiquimod (IMQ) and fed dextran sulfate sodium (DSS). Methods We applied IMQ topically to B6 mice (IMQ mice) and subsequently fed them 2% DSS in their drinking water. Disease activity and immune cell phenotypes were analyzed, and the microbial composition of fecal samples was investigated using 16S ribosomal RNA sequencing. We transplanted feces from IMQ mice to germ-free IQI/Jic (IQI) mice and fed them DSS to assess the effect of the gut microbiome on disease. Results We first confirmed that IMQ mice showed accelerated DSS colitis. IMQ mice had decreased numbers of IgD+ and IgM+ B cells and increased numbers of non–cytokine-producing macrophages in the gut. Moreover, the gut microbiomes of IMQ mice were perturbed, with significant reductions of Lactobacillus johnsonii and Lactobacillus reuteri populations. Germ-free mice transplanted with feces from IMQ mice, but not with feces from untreated mice, also developed exacerbated DSS colitis. Conclusions These results suggest that skin inflammation may contribute to pathogenic conditions in the gut via immunologic and microbiological changes. Our finding of a novel potential skin–gut interaction provides new insights into the coincidence of psoriasis and IBD., Graphical abstract

Background & Aims GPBAR1, also known as TGR5, is a G protein–coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis. Material and methods Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s. Results In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1–/– mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model. Conclusion Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity., Graphical abstract

Obtaining T cells by reprogramming is one of the major goals in regenerative medicine. Here, we describe a protocol for generating functional T cells from Hoxb5-expressing pro/pre-B cells in vivo. This protocol includes the construction of Hoxb5 recombinant plasmids, retroviral packaging, isolation and viral transduction of pro/pre-B cells, cell transplantation, and phenotypic analysis of induced T cells. The procedure is reproducible and straightforward, providing an approach for generating induced T cells for translational research. Keywords: Hoxb5, Retrovirus, Pro/pre-B cells, T cells

Objective We previously demonstrated that diabetes exacerbates stroke-induced brain injury, and that this correlates with brain methylglyoxal (MG)-to-glutathione (GSH) status. Cerebral injury was reversed by N-acetylcysteine (NAC). Here we tested if the pro-thrombotic phenotype seen in the systemic circulation and brain during diabetes was associated with increased MG-glycation of proteins, and if NAC could reverse this. Methods The streptozotocin (STZ)-induced mouse model of type 1 diabetes was used. Thrombus formation in venules and arterioles (pial circulation) was determined by intravital videomicroscopy using the light-dye method. Circulating blood platelet-leukocyte aggregates (PLAs) were analyzed by flow cytometry 1 wk before other measurements. GSH and MG levels in platelets were measured by HPLC. MG-modified proteins, glutathione peroxidase-1 (GPx-1), and superoxide dismutase-1 (SOD1) levels were detected in platelets by western blot at 20 weeks. Proteins involved in coagulation were quantified by ELISA. NAC (2 mM) was given in drinking water for 3 weeks before the terminal experiment. Results Thrombus development was accelerated by diabetes in a time-dependent manner. % PLAs were significantly elevated by diabetes. Plasma activated plasminogen activator inhibitor type 1 levels were progressively increased with diabetes duration, with tail bleeding time reduced by 20 wks diabetes. Diabetes lowered platelet GSH levels, GPx-1 and SOD-1 expression. This was associated with higher MG levels, and increased MG-adduct formation in platelets. NAC treatment partly or completely reversed the effects of diabetes. Conclusion Collectively, these results show that the diabetic blood and brain become progressively more susceptible to platelet activation and thrombosis. NAC, given after the establishment of diabetes, may offer protection against the risk for stroke by altering both systemic and vascular prothrombotic responses via enhancing platelet GSH, and GSH-dependent MG elimination, as well as correcting levels of antioxidants such as SOD1 and GPx-1., Graphical abstract fx1, Highlights • Diabetes elevates dicarbonyl stress leading to enhanced thrombosis in the brain. • Glutathione levels decrease leading to impaired elimination of methylglyoxal in platelets during diabetes. • Platelet proteins are glycated and platelets form aggregates with leukocytes in diabetes. • Diabetes increases circulating levels of plasminogen activator inhibitor-1. • NAC, via GSH synthesis, reverses the platelet activation, protein glycation and pro-coagulation responses & protects against thrombosis in the diabetic brain.

Background & Aims Chronic inflammation is a predisposing condition for colorectal cancer. Many studies to date have focused on proinflammatory signaling pathways in the colon. Understanding the mechanisms that suppress inflammation, particularly in epithelial cells, is critical for developing therapeutic interventions. Here, we explored the roles of transforming growth factor β (TGFβ) family signaling through SMAD4 in colonic epithelial cells. Methods The Smad4 gene was deleted specifically in adult murine intestinal epithelium. Colitis was induced by 3 rounds of dextran sodium sulfate in drinking water, after which mice were observed for up to 3 months. Nontransformed mouse colonocyte cell lines and colonoid cultures and human colorectal cancer cell lines were analyzed for responses to TGFβ1 and bone morphogenetic protein 2. Results Dextran sodium sulfate treatment was sufficient to drive carcinogenesis in mice lacking colonic Smad4 expression, with resulting tumors bearing striking resemblance to human colitis–associated carcinoma. Loss of SMAD4 protein was observed in 48% of human colitis–associated carcinoma samples as compared with 19% of sporadic colorectal carcinomas. Loss of Smad4 increased the expression of inflammatory mediators within nontransformed mouse colon epithelial cells in vivo. In vitro analysis of mouse and human colonic epithelial cell lines and organoids indicated that much of this regulation was cell autonomous. Furthermore, TGFβ signaling inhibited the epithelial inflammatory response to proinflammatory cytokines. Conclusions TGFβ suppresses the expression of proinflammatory genes in the colon epithelium, and loss of its downstream mediator, SMAD4, is sufficient to initiate inflammation-driven colon cancer. Transcript profiling: GSE100082., Graphical abstract

Background & Aims Virus-specific T cell dysfunction is a common feature of HBV-related hepatocellular carcinoma (HBV-HCC). Conventional T (ConT) cells can be redirected towards viral antigens in HBV-HCC when they express an HBV-specific receptor; however, their efficacy can be impaired by liver-specific physical and metabolic features. Mucosal-associated invariant T (MAIT) cells are the most abundant innate-like T cells in the liver and can elicit potent intrahepatic effector functions. Here, we engineered ConT and MAIT cells to kill HBV expressing hepatoma cells and compared their functional properties. Methods Donor-matched ConT and MAIT cells were engineered to express an HBV-specific T cell receptor (TCR). Cytotoxicity and hepatocyte homing potential were investigated using flow cytometry, real-time killing assays, and confocal microscopy in 2D and 3D HBV-HCC cell models. Major histocompatibility complex (MHC) class I-related molecule (MR1)-dependent and MR1-independent activation was evaluated in an Escherichia coli THP-1 cell model and by IL-12/IL-18 stimulation, respectively. Results HBV TCR-MAIT cells demonstrated polyfunctional properties (CD107a, interferon [IFN] γ, tumour necrosis factor [TNF], and IL-17A) with strong HBV target sensitivity and liver-homing chemokine receptor expression when compared with HBV TCR-ConT cells. TCR-mediated lysis of hepatoma cells was comparable between the cell types and augmented in the presence of inflammation. Coculturing with HBV+ target cells in a 3D microdevice mimicking aspects of the liver microenvironment demonstrated that TCR-MAIT cells migrate readily towards hepatoma targets. Expression of an ectopic TCR did not affect the ability of the MAIT cells to be activated via MR1-presented bacterial antigens or IL-12/IL-18 stimulation. Conclusions HBV TCR-MAIT cells demonstrate anti-HBV functions without losing their endogenous antimicrobial mechanisms or hepatotropic features. Our results support future exploitations of MAIT cells for liver-directed immunotherapies. Lay summary Chronic HBV infection is a leading cause of liver cancer. T cell receptor (TCR)-engineered T cells are patients’ immune cells that have been modified to recognise virus-infected and/or cancer cells. Herein, we evaluated whether mucosal-associated invariant T cells, a large population of unconventional T cells in the liver, could recognise and kill HBV infected hepatocytes when engineered with an HBV-specific TCR. We show that their effector functions may exceed those of conventional T cells currently used in the clinic, including antimicrobial properties and chemokine receptor profiles better suited for targeting liver tumours., Graphical abstract, Highlights • Mucosal-associated invariant T (MAIT) cells expanded in vitro can be engineered to kill HBV antigen-presenting hepatoma cells. • MAIT cells produce IL-17A upon encountering their HBV targets, a functional property not observed in conventional T cells currently used in immunotherapy. • HBV-specific MAIT cells migrate readily towards liver targets in a 3D microfluidics system. • Co-expression of an HBV-specific TCR does not affect the ability of MAIT cells to respond to their physiological stimuli.

CD4+ CD25+ regulatory T cells (Treg), an essential subset for preventing autoimmune diseases, is implicated as a negative regulator in anti-tumor immunity. We found that metformin (Met) reduced tumor-infiltrating Treg (Ti-Treg), particularly the terminally-differentiated CD103+ KLRG1+ population, and also decreased effector molecules such as CTLA4 and IL-10. Met inhibits the differentiation of naïve CD4+ T cells into inducible Treg (iTreg) by reducing forkhead box P3 (Foxp3) protein, caused by mTORC1 activation that was determined by the elevation of phosphorylated S6 (pS6), a downstream molecule of mTORC1. Rapamycin and compound C, an inhibitor of AMP-activated protein kinase (AMPK) restored the iTreg generation, further indicating the involvement of mTORC1 and AMPK. The metabolic profile of iTreg, increased Glut1-expression, and reduced mitochondrial membrane-potential and ROS production of Ti-Treg aided in identifying enhanced glycolysis upon Met-treatment. The negative impact of Met on Ti-Treg may help generation of the sustained antitumor immunity., Highlights • Metformin downregulates CD4+ CD25+ regulatory T cells (Treg) in tumors but not in peripheral lymphoid tissues. • Metformin administration results in activation of mTORC1 in Treg in tumors. • Metformin administration results in elevation of glycolysis, while suppressing oxidative phosphorylation in Treg in tumors. CD4+ CD25+ regulatory T cells (Treg) is a negative regulator that inhibits T cell mediated anti-tumor immunity. Therefore, targeting Treg is one of the important therapeutic intervention in cancers. We found that metformin reduces Treg in the number and the function in tumors. Metabolism of Treg is usually dependent on oxidative phosphorylation through fatty acid oxidation (FAO). However, metformin treatment causes metabolic reprogramming of Treg toward the glycolysis, resulting in the failure in survival in tumors. Metformin as a metabolic modifier for Treg may contribute to generation of sustained anti-tumor immunity, combined with currently emerging cancer immunotherapy.

Objectives The goal of this study was to determine whether ticagrelor reduces high-sensitivity troponin I concentrations in patients with established coronary artery disease and high-risk coronary plaque. Background High-risk coronary atherosclerotic plaque is associated with higher plasma troponin concentrations suggesting ongoing myocardial injury that may be a target for dual antiplatelet therapy. Methods In a randomized, double-blind, placebo-controlled trial, patients with multivessel coronary artery disease underwent coronary 18F-fluoride positron emission tomography/coronary computed tomography scanning and measurement of high-sensitivity cardiac troponin I. Patients were randomized (1:1) to receive ticagrelor 90 mg twice daily or matched placebo. The primary endpoint was troponin I concentration at 30 days in patients with increased coronary 18F-fluoride uptake. Results In total, 202 patients were randomized to treatment, and 191 met the pre-specified criteria for inclusion in the primary analysis. In patients with increased coronary 18F-fluoride uptake (120 of 191), there was no evidence that ticagrelor had an effect on plasma troponin concentrations at 30 days (ratio of geometric means for ticagrelor vs. placebo: 1.11; 95% confidence interval: 0.90 to 1.36; p = 0.32). Over 1 year, ticagrelor had no effect on troponin concentrations in patients with increased coronary 18F-fluoride uptake (ratio of geometric means: 0.86; 95% confidence interval: 0.63 to 1.17; p = 0.33). Conclusions Dual antiplatelet therapy with ticagrelor did not reduce plasma troponin concentrations in patients with high-risk coronary plaque, suggesting that subclinical plaque thrombosis does not contribute to ongoing myocardial injury in this setting. (Dual Antiplatelet Therapy to Reduce Myocardial Injury [DIAMOND]; NCT02110303), Central Illustration

Basophils, which are the rarest granulocytes, play crucial roles in protective immunity against parasites and development of allergic disorders. Although immunoglobulin (Ig)E-dependent responses via receptor for IgE (FcεRI) in basophils have been extensively studied, little is known about cell surface molecules that are selectively expressed on this cell subset to utilize the elimination in vivo through treatment with monoclonal antibody (mAb). Since CD200 receptor 3 (CD200R3) was exclusively expressed on basophils and mast cells (MCs) using a microarray screening, we have generated anti-CD200R3 mAb recognizing CD200R3A. In this study we examined the expression pattern of CD200R3A on leukocytes, and the influence of the elimination of basophils by anti-CD200R3A mAb on allergic responses. Flow cytometric analysis showed that CD200R3A was primarily expressed on basophils and MCs, but not on other leukocytes. Administration with anti-CD200R3A mAb led to the prominent specific depletion of tissue-resident and circulating basophils, but not MCs. Furthermore, in vivo depletion of basophils ameliorated IgE-mediated systemic and local anaphylaxis. Taken together, these findings suggest that CD200R3A is reliable cell surface marker for basophils in vivo, and targeting this unique molecule with mAb for the elimination of basophils may serve as a novel therapeutic strategy in ameliorating the allergic diseases., Highlights • CD200R3A was primarily expressed on basophils and mast cells. • Administration with anti-CD200R3A mAb depleted basophils, but not MCs. • Depletion of basophils by anti-CD200R3A mAb ameliorated IgE-mediated systemic and local anaphylaxis.