BACKGROUND: Atrial fibrillation (AF) is the most common cardiac arrhythmia. Although treatment options for AF exist, many patients cannot be maintained in normal sinus rhythm. Amiodarone is an effective medication for AF but has limited clinical utility because of off-target tissue toxicity. METHODS: Here, we use a pig model of AF to test the efficacy of an amiodarone-containing polyethylene glycol-based hydrogel. The gel is placed directly on the atrial epicardium through the pericardial space in a minimally invasive procedure using a specially designed catheter. RESULTS: Implantation of amiodarone-containing gel significantly reduced the duration of sustained AF at 21 and 28 days; inducibility of AF was reduced 14 and 21 days post-delivery. Off-target organ drug levels in the liver, lungs, thyroid, and fat were significantly reduced in animals treated with epicardial amiodarone gel compared with systemic controls in small-animal distribution studies. CONCLUSIONS: The pericardium is an underutilized therapeutic site and may be a new treatment strategy for AF and other cardiovascular diseases.

Background & Aims: The intestinal epithelium must be resilient to physiochemical stress to uphold the physiological barrier separating the systemic compartment from the microbial and antigenic components of the gut lumen. Identifying proteins that mediate protection and enhancing their expression is therefore a clear approach to promote intestinal health. We previously reported that oral ingestion of the probiotic Lactobacillus rhamnosus GG not only induced the expression of several recognized cytoprotective factors in the murine colon, but also many genes with no previously described function, including the gene encoding proline-rich acidic protein 1 (PRAP1). PRAP1 is a highly expressed protein in the epithelium of the gastrointestinal tract and we sought to define its function in this tissue. Methods: Purified preparations of recombinant PRAP1 were analyzed biochemically and PRAP1 antisera were used to visualize localization in tissues. Prap1-/- mice were characterized at baseline and challenged with total body irradiation, then enteroids were generated to recapitulate the irradiation challenge ex vivo. Results: PRAP1 is a 17-kilodalton intrinsically disordered protein with no recognizable sequence homology. PRAP1 expression levels were high in the epithelia of the small intestine. Although Prap1-/- mice presented only mild phenotypes at baseline, they were highly susceptible to intestinal injury upon challenge. After irradiation, the Prap1-/- mice showed accelerated death with a significant increase in apoptosis and p21 expression in the small intestinal epithelium. Conclusions: PRAP1 is an intrinsically disordered protein highly expressed by the gastrointestinal epithelium and functions at exposed surfaces to protect the barrier from oxidative insult.

Among responders to microbial invasion, neutrophils represent one of the earliest and perhaps most important factors that contribute to initial host defense. Effective neutrophil immunity requires their rapid mobilization to the site of infection, which requires efficient extravasation, activation, chemotaxis, phagocytosis, and eventual killing of potential microbial pathogens. Following pathogen elimination, neutrophils must be eliminated to prevent additional host injury and subsequent exacerbation of the inflammatory response. Galectins, expressed in nearly every tissue and regulated by unique sensitivity to oxidative and proteolytic inactivation, appear to influence nearly every aspect of neutrophil function. In this review, we will examine the impact of galectins on neutrophils, with a particular focus on the unique biochemical traits that allow galectin family members to spatially and temporally regulate neutrophil function.

The use of beneficial bacteria to promote health is widely practiced. However, experimental evidence corroborating the efficacy of bacteria promoted with such claims remains limited. We address this gap by identifying a beneficial bacterium that protects against tissue damage and injury-induced inflammation in the gut. We first employed the Drosophila animal model to screen for the capacity of candidate beneficial bacteria to protect the fly gut against injury. From this screen, we identified Lactococcus lactis subsp. cremoris as a bacterium that elicited potent cytoprotective activity. Then, in a murine model, we demonstrated that the same strain confers powerful cytoprotective influences against radiological damage, as well as anti-inflammatory activity in a gut colitis model. In summary, we demonstrate the positive salutary effects of a beneficial bacterium, namely, L. lactis subsp. cremoris on intestinal tissue and propose the use of this strain as a therapeutic to promote intestinal health.

Background & Aims: Ileal bile acid absorption is mediated by uptake via the apical sodium-dependent bile acid transporter (ASBT), and export via the basolateral heteromeric organic solute transporter α-β (OSTα-OSTβ). In this study, we investigated the cytotoxic effects of enterocyte bile acid stasis in Ostα-/-mice, including the temporal relationship between intestinal injury and initiation of the enterohepatic circulation of bile acids. Methods: Ileal tissue morphometry, histology, markers of cell proliferation, gene, and protein expression were analyzed in male and female wild-type and Ostα-/-mice at postnatal days 5, 10, 15, 20, and 30. Ostα-/-Asbt-/-mice were generated and analyzed. Bile acid activation of intestinal Nrf2-activated pathways was investigated in Drosophila. Results: As early as day 5, Ostα-/-mice showed significantly increased ileal weight per length, decreased villus height, and increased epithelial cell proliferation. This correlated with premature expression of the Asbt and induction of bile acid–activated farnesoid X receptor target genes in neonatal Ostα-/-mice. Expression of reduced nicotinamide adenine dinucleotide phosphate oxidase-1 and Nrf2–anti-oxidant responsive genes were increased significantly in neonatal Ostα-/-mice at these postnatal time points. Bile acids also activated Nrf2 in Drosophila enterocytes and enterocyte-specific knockdown of Nrf2 increased sensitivity of flies to bile acid–induced toxicity. Inactivation of the Asbt prevented the changes in ileal morphology and induction of anti-oxidant response genes in Ostα-/-mice. Conclusions: Early in postnatal development, loss of Ostα leads to bile acid accumulation, oxidative stress, and a restitution response in ileum. In addition to its essential role in maintaining bile acid homeostasis, Ostα-Ostβ functions to protect the ileal epithelium against bile acid–induced injury. NCBI Gene Expression Omnibus: GSE99579.

BACKGROUND: While very low birth weight (VLBW) infants often require multiple red blood cell transfusions, efforts to minimize transfusion-associated risks have resulted in more restrictive neonatal transfusion practices. However, whether restrictive transfusion strategies limit transfusions without increasing morbidity and mortality in this population remains unclear. Recent epidemiologic studies suggest that severe anemia may be an important risk factor for the development of necrotizing enterocolitis (NEC). However, the mechanism whereby anemia may lead to NEC remains unknown. STUDY DESIGN AND METHODS: The potential impact of anemia on neonatal inflammation and intestinal barrier disruption, two well-characterized predisposing features of NEC, was defined by correlation of hemoglobin values to cytokine levels in premature infants and by direct evaluation of intestinal hypoxia, inflammation and gut barrier disruption using a pre-clinical neonatal murine model of phlebotomy-induced anemia (PIA). RESULTS: Increasing severity of anemia in the preterm infant correlated with the level of IFN-gamma, a key pro-inflammatory cytokine that may predispose an infant to NEC. Gradual induction of PIA in a pre-clinical model resulted in significant hypoxia throughout the intestinal mucosa, including areas where intestinal macrophages reside. PIA-induced hypoxia significantly increased macrophage pro-inflammatory cytokine levels, while reducing tight junction protein ZO-1 expression and increasing intestinal barrier permeability. Macrophage depletion reversed the impact of anemia on intestinal ZO-1 expression and barrier function. CONCLUSIONS: Taken together, these results suggest that anemia can increase intestinal inflammation and barrier disruption likely through altered macrophage function, leading to the type of predisposing intestinal injury that may increase the risk for NEC.

Intestinal homeostasis is regulated in-part by reactive oxygen species (ROS) that are generated in the colonic mucosa following contact with certain lactobacilli. Mechanistically, ROS can modulate protein function through the oxidation of cysteine residues within proteins. Recent advances in cysteine labeling by the Isotope Coded Affinity Tags (ICATs) technique has facilitated the identification of cysteine thiol modifications in response to stimuli. Here, we used ICATs to map the redox protein network oxidized upon initial contact of the colonic mucosa with Lactobacillus rhamnosus GG (LGG). We detected significant LGG-specific redox changes in over 450 proteins, many of which are implicated to function in cellular processes such as endosomal trafficking, epithelial cell junctions, barrier integrity, and cytoskeleton maintenance and formation. We particularly noted the LGG-specific oxidation of Rac1, which is a pleiotropic regulator of many cellular processes. Together, these data reveal new insights into lactobacilli-induced and redox-dependent networks involved in intestinal homeostasis.

This study aimed to enhance antitumor immune responses to pancreatic cancer via Ab-based blockade of IL-6 and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4). Mice bearing s.c. or orthotopic pancreatic tumors were treated with blocking Abs to IL‑6 and/or CTLA-4. In both tumor models, dual IL-6 and CTLA-4 blockade significantly inhibited tumor growth. Additional investigations revealed that dual therapy induced an overwhelming infiltration of T cells into the tumor as well as changes in CD4+ T cell subsets. Dual blockade therapy elicited CD4+ T cells to secrete increased IFN-γ in vitro. Likewise, in vitro stimulation of pancreatic tumor cells with IFN-γ profoundly increased tumor cell production of CXCR3-specific chemokines, even in the presence of IL-6. In vivo blockade of CXCR3 prevented orthotopic tumor regression in the presence of the combination treatment, demonstrating a dependence on the CXCR3 axis for antitumor efficacy. Both CD4+ and CD8+ T cells were required for the antitumor activity of this combination therapy, as their in vivo depletion via Abs impaired outcomes. These data represent the first report to our knowledge of IL-6 and CTLA‑4 blockade as a means to regress pancreatic tumors with defined operative mechanisms of efficacy.

Acetaminophen is the most common cause of acute drug-induced liver injury in the United States. However, research into the mechanisms of acetaminophen toxicity and the development of novel therapeutics is hampered by the lack of robust, reproducible, and cost-effective model systems. Herein, we characterize a novel Drosophila-based model of acetaminophen toxicity. We demonstrate that acetaminophen treatment of Drosophila results in similar pathophysiologic alterations as those observed in mammalian systems, including a robust production of reactive oxygen species, depletion of glutathione, and dose-dependent mortality. Moreover, these effects are concentrated in the Drosophila fat body, an organ analogous to the mammalian liver. Utilizing this system, we interrogated the influence of environmental factors on acetaminophen toxicity which has proven difficult in vertebrate models due to cost and inter-individual variability. We find that both increasing age and microbial depletion sensitize Drosophila to acetaminophen toxicity. These environmental influences both alter oxidative stress response pathways in metazoans. Indeed, genetic and pharmacologic manipulations of the antioxidant response modify acetaminophen toxicity in our model. Taken together, these data demonstrate the feasibility of Drosophila for the study of acetaminophen toxicity, bringing with it an ease of genetic and microbiome manipulation, high-throughput screening, and availability of transgenic animals.

A paucity of effector T cells within tumors renders pancreatic ductal adenocarcinoma (PDAC) resistant to immune checkpoint therapies. While several under-development approaches target immune-suppressive cells in the tumor microenvironment, there is less focus on improving T cell function. Here we show that inhibiting vasoactive intestinal peptide receptor (VIP-R) signaling enhances anti-tumor immunity in murine PDAC models. In silico data mining and immunohistochemistry analysis of primary tumors indicate overexpression of the neuropeptide vasoactive intestinal peptide (VIP) in human PDAC tumors. Elevated VIP levels are also present in PDAC patient plasma and supernatants of cultured PDAC cells. Furthermore, T cells up-regulate VIP receptors after activation, identifying the VIP signaling pathway as a potential target to enhance T cell function. In mouse PDAC models, VIP-R antagonist peptides synergize with anti-PD-1 antibody treatment in improving T cell recruitment into the tumors, activation of tumor-antigen-specific T cells, and inhibition of T cell exhaustion. In contrast to the limited single-agent activity of anti-PD1 antibodies or VIP-R antagonist peptides, combining both therapies eliminate tumors in up to 40% of animals. Furthermore, tumor-free mice resist tumor re-challenge, indicating anti-cancer immunological memory generation. VIP-R signaling thus represents a tumor-protective immune-modulatory pathway that is targetable in PDAC.