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Background Nalfurafine is the first clinically approved kappa-opioid receptor (KOP-r) agonist as an antipruritus drug with few side effects in humans (e.g., sedation, depression, and dysphoria). No study, however, has been done using nalfurafine on alcohol drinking in rodents or humans. Methods We investigated whether nalfurafine alone or in combination with mu-opioid receptor (MOP-r) antagonist naltrexone changed excessive alcohol drinking in male and female C57BL/6J (B6) mice subjected to a chronic intermittent-access drinking paradigm (2-bottle choice, 24-hour access every other day) for 3 weeks. Neuronal proopiomelanocortin enhancer (nPE) knockout mice with brain-specific deficiency of beta-endorphin (endogenous ligand of MOP-r) were used as a genetic control for the naltrexone effects. Results Single administration of nalfurafine decreased alcohol intake and preference in both male and female B6 mice in a dose-dependent manner. Pretreatment with nor-BNI (a selective KOP-r antagonist) blocked the nalfurafine effect on alcohol drinking, indicating a KOP-r-mediated mechanism. Pharmacological effects of a 5-dosing nalfurafine regimen were further evaluated: The repeated nalfurafine administrations decreased alcohol consumption without showing any blunted effects, suggesting nalfurafine did not develop a tolerance after the multidosing regimen tested. Nalfurafine did not produce any sedation (spontaneous locomotor activity), anhedonia-like (sucrose preference test), anxiety-like (elevated plus maze test), or dysphoria-like (conditioned place aversion test) behaviors, suggesting that nalfurafine had few side effects. Investigating synergistic effects between low-dose naltrexone and nalfurafine, we found that single combinations of nalfurafine and naltrexone, at doses lower than individual effective dose, profoundly decreased excessive alcohol intake in both sexes. The effect of nalfurafine on decreasing alcohol consumption was confirmed in nPE-/- mice, suggesting independent mechanisms by which nalfurafine and naltrexone reduced alcohol drinking. Conclusion The clinically utilized KOP-r agonist nalfurafine in combination with low-dose naltrexone has potential in alcoholism treatment.

Eukaryotic ribosome biogenesis is initiated with the transcription of pre-ribosomal RNA at the 5' external transcribed spacer, which directs the early association of assembly factors but is absent from the mature ribosome. The subsequent co-transcriptional association of ribosome assembly factors with pre-ribosomal RNA results in the formation of the small subunit processome. Here we show that stable rRNA domains of the small ribosomal subunit can independently recruit their own biogenesis factors in vivo. The final assembly and compaction of the small subunit processome requires the presence of the 5' external transcribed spacer RNA and all ribosomal RNA domains. Additionally, our cryo-electron microscopy structure of the earliest nucleolar preribosomal assembly - the 5' external transcribed spacer ribonucleoprotein - provides a mechanism for how conformational changes in multi-protein complexes can be employed to regulate the accessibility of binding sites and therefore define the chronology of maturation events during early stages of ribosome assembly.

Background and Aims Inflammatory bowel disease [IBD] is accompanied by lesions in the epithelial barrier, which allow translocation of bacterial products from the gut lumen to the host's circulation. IMM-124E is a colostrum-based product containing high levels of anti-E.coli-LPS IgG, and might limit exposure to bacterial endotoxins. Here, we investigated whether IMM-124E can ameliorate intestinal inflammation. Methods Acute colitis was induced in WT C57Bl/6J mice by administration of 2.5% dextran sodium sulphate [DSS] for 7 days. T cell transfer colitis was induced via transfer of 0.5 x 10(6) naive T cells into RAG2(-/-) C57Bl/6J mice. IMM-124E was administered daily by oral gavage, either preventively or therapeutically. Results Treatment with IMM-124E significantly ameliorated colitis in acute DSS colitis and in T cell transfer colitis. Maximum anti-inflammatory effects were detected at an IMM-124E concentration of 100 mg/kg body weight, whereas 25 mg/kg and 500 mg/kg were less effective. Histology revealed reduced levels of infiltrating immune cells and less pronounced mucosal damage. Flow cytometry revealed reduced numbers of effector T helper cells in the intestine, whereas levels of regulatory T cells were enhanced. IMM-124E treatment reduced the DSS-induced increase of serum levels of lipopolysaccharide [LPS]-binding protein, indicating reduced systemic LPS exposure. Conclusions Our results demonstrate that oral treatment with IMM-124E significantly reduces intestinal inflammation, via decreasing the accumulation of pathogenic T cells and concomitantly increasing the induction of regulatory T cells. Our study confirms the therapeutic efficacy of IMM-124E in acute colitis and suggests that administration of IMM-124E might represent a novel therapeutic strategy to induce or maintain remission in chronic colitis.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) loci in prokaryotes are composed of 30-40-base-pair repeats separated by equally short sequences of plasmid and bacteriophage origin known as spacers(1-3). These loci are transcribed and processed into short CRISPR RNAs (crRNAs) that are used as guides by CRISPR-associated (Cas) nucleases to recognize and destroy complementary sequences (known as protospacers) in foreign nucleic acids(4,5). In contrast to most Cas nucleases, which destroy invader DNA(4-7), the type VI effector nuclease Cas13 uses RNA guides to locate complementary transcripts and catalyse both sequence-specific cis-and non-specific trans-RNA cleavage(8). Although it has been hypothesized that Cas13 naturally defends against RNA phages(8), type VI spacer sequences have exclusively been found to match the genomes of double-stranded DNA phages(9,10), suggesting that Cas13 can provide immunity against these invaders. However, whether and how Cas13 uses its cis- and/or trans-RNA cleavage activities to defend against double-stranded DNA phages is not understood. Here we show that trans-cleavage of transcripts halts the growth of the host cell and is sufficient to abort the infectious cycle. This depletes the phage population and provides herd immunity to uninfected bacteria. Phages that harbour target mutations, which easily evade DNA-targeting CRISPR systems(11-13), are also neutralized when Cas13 is activated by wild-type phages. Thus, by acting on the host rather than directly targeting the virus, type VI CRISPR systems not only provide robust defence against DNA phages but also prevent outbreaks of CRISPR-resistant phage.

One successful class of cancer immunotherapies, immune checkpoint inhibitory antibodies, disrupts key pathways that regulate immune checkpoints, such as cytotoxic T lymphocyte-associated antigen-4 (CTLA-4). These agents unleash the potency of antigen-experienced T cells that have already been induced as a consequence of the existing tumor. But only 20% of cancers naturally induce T cells. For most cancers, vaccines are require to induce and mobilize T effector cells (T-effs) to traffick into tumors. We evaluated the effects of anti-CTLA-4 given in combination with an antigen-specific dendritic cell vaccine on intratumoral T-effs in a murine pancreatic cancer model. The dendritic cell-targeted tumor antigen plus anti-CTLA-4 significantly increased the number of vaccine-induced CD4(+) T-effs within the tumor. This increase was accompanied by a reduction in the size of the peripheral CD4(+) T-eff pool. We also found that IL-3 production by activated CD4(+) T cells was significantly increased with this combination. Importantly, the CD4(+) T-eff response was attenuated in Il3(-/-) mice, suggesting mediation of the effect by IL-3. Finally, the induced T cell infiltration was associated with activation of the tumor endothelium by T cell-derived IL-3. Our findings collectively provide a new insight into the mechanism driving T-eff infiltration and vascular activation in a murine pancreatic cancer model, specifically identifying a new role for IL-3 in the anticancer immune response.

Mutational profiling has demonstrated utility in predicting the likelihood of disease progression in patients with myelofibrosis (MF). However, there is limited data regarding the prognostic utility of genetic profiling in MF patients undergoing allogeneic hematopoietic stem cell transplantation (allo-HCT). We performed high-throughput sequencing of 585 genes on pre-transplant samples from 101 patients with MF who underwent allo-HCT and evaluated the association of mutations and clinical variables with transplantation outcomes. Overall survival (OS) at 5 years post-transplantation was 52%, and relapse-free survival (RFS) was 51.1 % for this cohort. Nonrelapse mortality (NRM) accounted for most deaths. Patient's age, donor's age, donor type, and Dynamic International Prognostic Scoring System score at diagnosis did not predict for outcomes. Mutations known to be associated with increased risk of disease progression, such as ASXL1, SRSF2, IDH1/2, EZH2, and TP53, did not impact OS or RFS. The presence of U2AFI (P=.007) or DNMT3A (P=.034) mutations was associated with worse OS. A Mutation-Enhanced International Prognostic Scoring System 70 score was available for 80 patients (79%), and there were no differences in outcomes between patients with high risk scores and those with intermediate and low risk scores. Collectively, these data identify mutational predictors of outcome in MF patients undergoing allo-HCT. These genetic biomarkers in conjunction with clinical variables may have important utility in guiding transplantation decision making. (C) 2019 Published by Elsevier Inc. on behalf of American Society for Blood and Marrow Transplantation.