Commensal bacteria influence host physiology, without invading host tissues. We show that proteins from segmented filamentous bacteria (SFB) are transferred into intestinal epithelial cells (IECs) ...through adhesion-directed endocytosis that is distinct from the clathrin-dependent endocytosis of invasive pathogens. This process transfers microbial cell wall-associated proteins, including an antigen that stimulates mucosal T helper 17 (T
17) cell differentiation, into the cytosol of IECs in a cell division control protein 42 homolog (CDC42)-dependent manner. Removal of CDC42 activity in vivo led to disruption of endocytosis induced by SFB and decreased epithelial antigen acquisition, with consequent loss of mucosal T
17 cells. Our findings demonstrate direct communication between a resident gut microbe and the host and show that under physiological conditions, IECs acquire antigens from commensal bacteria for generation of T cell responses to the resident microbiota.
Abstract
Nonoperative management for hepatic injuries requires observation and supportive care in the case of hemodynamically stable patients. If there is active bleeding on presentation, hepatic ...artery embolization is an option to achieve hemostasis in the acute setting. Although interventional radiology procedures are well documented in adults, there is limited literature regarding these procedures in the pediatric population. In this report, we present a case of a pediatric patient who sustained blunt abdominal trauma, resulting in a grade IV liver injury. Treatment involved fluoroscopically guided right hepatic segmental arterial gel-foam embolization.
Abstract only
Lactobacillus rhamnosus GG
(LGG) is a Gram positive facultative anaerobic lactic acid bacterium that has been associated with the treatment and prevention of intestinal inflammatory ...disorders. Proposed mechanisms, including increased barrier function, exclusion of pathogenic species, and modulation of cytokine production, were obtained mainly from experimental approaches relying on
in vivo
LGG incorporation into feed and drinking water where LGG effects may be confounded by trillions of resident bacteria as well as on
in vitro
LGG incubation of isolated cells and immortalized cell lines that may not fully replicate
in vivo
LGG‐mucosa interactions. Thus, we perfused LGG at 37°C into the chyme‐free intestinal lumen of anesthetized nongerm‐free mice, to determine acute mucosal responses when LGG are the sole bacteria challenging the gut.
Commercial grade lyophilized LGG was reconstituted and determined to be viable via confocal scanning laser microscopy and LIVE/DEAD BacLight viability staining, as well as growth curve analysis. OD
600
absorbance values were obtained and used to calculate a generation time of 2.27 hours, further showing the viability of LGG prior to perfusion. Mice were placed under general anesthesia and surgery was performed to directly perfuse LGG into the proximal small intestine via catheters (15 mL/h over 4 h). The inflowing LGG perfusate had 3.3×10^8 CFU/mL in Krebs Ringer Buffer with inulin (7 mg/mL), well above the recommended 10^7 CFU/mL probiotic concentration shown to induce mucosal responses. Control perfusate contained only inulin.
Mucosal mRNA expression of cytokines
IL6
and
IL2
as well as mucin
Muc 2
increased with LGG perfusion. Multiplex ELISA analysis of whole tissue proteins showed LGG‐induced increases in cytokines IFN‐γ, IL10, IL2, IL4, IL5, and IL6 (
P
<0.05). RNA from mucosal scrapes were then submitted for Next Generation Sequencing to determine LGG‐induced changes in the mucosal transcriptome. Analysis of sequencing data determined that LGG perfusion (
n
= 4) altered the mRNA levels of 441 genes (fold change>1.5
, P
<0.01).
Chodl
(endocytosis of bacteria)
, Cyp1a1
(expression depends on TLR2)
,
and
Tmprss15
(serine protease in host defense) expression increased by > 7‐fold while
Defa3
(a defensin)
, Plb1 and Pla2g2
(phospholipases)
, CCR6
(dendritic and T cells migration and recruitment) and
Lyz
(lysozyme) were each downregulated by > 5‐fold. Ingenuity Pathway Analysis (IPA) was performed to identify canonical pathways affected by LGG. The most significant were the LPS/IL‐1 Mediated Inhibition of RXR Function, Glutathione‐mediated Detoxification, and Aryl Hydrocarbon Receptor Signaling (
P
<0.01). IPA also revealed upstream regulators
Cftr, mir‐21, Tp53, Tcf3, CDKN1A, POR and TGFb1
to have the greatest predicted activation (z‐score >2.7,
P
<0.01), and upstream regulators
Rabl6, Foxm1, Myc, S100a6 and NFkb1
to have the greatest predicted inhibition (z‐score <‐2.4,
P
<0.01). Most of these LGG‐affected genes, pathways and transcription factors are involved in bacterial recognition, host defense, host metabolism and inflammation.
Our initial findings indicate strong modulation of the gut mucosa of mice perfused solely with LGG, and parallel those of previous probiotic research relying on feeding and
in vitro
techniques. Thus, intestinal perfusion is an excellent method for use in studies seeking to understand acute, singular effects of probiotic and prebiotic supplements on the intestinal mucosa.
Support or Funding Information
Supported by NSF Grant No. IOS‐1121049 and 1456673 (RPF), NIH Grant No. R01‐DK‐102934 (NG)
Background & Aims: Lacticaseibacillus rhamnosus GG (LGG) is the world’s most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions ...with an essential source of signaling metabolites, dietary tryptophan (trp), are unclear. Methods: Untargeted metabolomic and transcriptomic analyses were performed in LGG monocolonized germ-free mice fed trp-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific tight junction proteins and enterocyte-promoting factors with gain and loss of function approaches. Results: LGG, strictly with dietary trp, promotes the enterocyte program and expression of tight junction genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in patients with inflammatory bowel disease. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in dextran sulfate sodium colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or vitamin B3 absorption abolishes barrier recovery in vivo. Conclusions: Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.
Lacticaseibacillus rhamnosus GG (LGG) is the world’s most consumed probiotic but its mechanism of action on intestinal permeability and differentiation along with its interactions with an essential ...source of signaling metabolites, dietary tryptophan (trp), are unclear.
Untargeted metabolomic and transcriptomic analyses were performed in LGG monocolonized germ-free mice fed trp-free or -sufficient diets. LGG-derived metabolites were profiled in vitro under anaerobic and aerobic conditions. Multiomic correlations using a newly developed algorithm discovered novel metabolites tightly linked to tight junction and cell differentiation genes whose abundances were regulated by LGG and dietary trp. Barrier-modulation by these metabolites were functionally tested in Caco2 cells, mouse enteroids, and dextran sulfate sodium experimental colitis. The contribution of these metabolites to barrier protection is delineated at specific tight junction proteins and enterocyte-promoting factors with gain and loss of function approaches.
LGG, strictly with dietary trp, promotes the enterocyte program and expression of tight junction genes, particularly Ocln. Functional evaluations of fecal and serum metabolites synergistically stimulated by LGG and trp revealed a novel vitamin B3 metabolism pathway, with methylnicotinamide (MNA) unexpectedly being the most robust barrier-protective metabolite in vitro and in vivo. Reduced serum MNA is significantly associated with increased disease activity in patients with inflammatory bowel disease. Exogenous MNA enhances gut barrier in homeostasis and robustly promotes colonic healing in dextran sulfate sodium colitis. MNA is sufficient to promote intestinal epithelial Ocln and RNF43, a master inhibitor of Wnt. Blocking trp or vitamin B3 absorption abolishes barrier recovery in vivo.
Our study uncovers a novel LGG-regulated dietary trp-dependent production of MNA that protects the gut barrier against colitis.
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Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic processes, including autophagy, and aberrant activation of such receptors is a common ...feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated VPS34 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors and restores autophagy in non-small-cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.
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•EGFR negatively regulates autophagy by binding to Beclin 1•Active EGFR phosphorylates Beclin 1 and alters its interactome•EGFR suppression of Beclin 1 may contribute to tumor progression in lung cancer•Lung cancer responses to EGFR inhibitors may involve activation of Beclin 1
The oncogenic receptor tyrosine kinase EGFR regulates the core autophagy machinery by phosphorylating the autophagy protein Beclin 1. This regulation may contribute to the progression and chemoresistance of non-small-cell lung carcinoma with active EGFR mutations.
Despite the established efficacy of statins, many patients do not achieve recommended LDL cholesterol (LDL-C) goals. Contributing factors may be inadequate dosing, increased risk for adverse effects ...with high-dose monotherapy, and increased potential for intolerance and adverse effects with combinations of available agents.
In a double-blind study, 628 patients with baseline LDL-C 145 to 250 mg/dL and triglycerides < or =350 mg/dL were randomly assigned to receive 1 of the following for 12 weeks: ezetimibe (10 mg/d); atorvastatin (10, 20, 40, or 80 mg/d); ezetimibe (10 mg) plus atorvastatin (10, 20, 40, or 80 mg/d); or placebo. The primary efficacy end point was percentage reduction in LDL-C for pooled ezetimibe plus atorvastatin versus pooled atorvastatin treatment groups. Ezetimibe plus atorvastatin significantly improved LDL-C, HDL cholesterol (HDL-C), triglycerides, total cholesterol:HDL-C, and high-sensitivity C-reactive protein (hs-CRP) compared with atorvastatin alone (P<0.01). Coadministration of ezetimibe provided a significant additional 12% LDL-C reduction, 3% HDL-C increase, 8% triglyceride reduction, and 10% hs-CRP reduction versus atorvastatin alone. Ezetimibe plus atorvastatin provided LDL-C reductions of 50% to 60%, triglyceride reductions of 30% to 40%, and HDL-C increases of 5% to 9%, depending on atorvastatin dose. LDL-C reductions with ezetimibe plus 10 mg atorvastatin (50%) and 80 mg atorvastatin alone (51%) were similar.
Ezetimibe plus atorvastatin was well tolerated, with a safety profile similar to atorvastatin alone and to placebo. When coadministered with atorvastatin, ezetimibe provided significant incremental reductions in LDL-C and triglycerides and increases in HDL-C. Coadministration of ezetimibe and atorvastatin offers a well-tolerated and highly efficacious new treatment option for patients with hypercholesterolemia.
Intrinsically disordered proteins (IDPs) that lack a unique 3D structure and comprise a large fraction of the human proteome play important roles in numerous cellular functions. Prostate-Associated ...Gene 4 (PAGE4) is an IDP that acts as a potentiator of the Activator Protein-1 (AP-1) transcription factor. Homeodomain-Interacting Protein Kinase 1 (HIPK1) phosphorylates PAGE4 at S9 and T51, but only T51 is critical for its activity. Here, we identify a second kinase, CDC-Like Kinase 2 (CLK2), which acts on PAGE4 and hyperphosphorylates it at multiple S/T residues, including S9 and T51. We demonstrate that HIPK1 is expressed in both androgen-dependent and androgen-independent prostate cancer (PCa) cells, whereas CLK2 and PAGE4 are expressed only in androgen-dependent cells. Cell-based studies indicate that PAGE4 interaction with the two kinases leads to opposing functions. HIPK1-phosphorylated PAGE4 (HIPK1-PAGE4) potentiates c-Jun, whereas CLK2-phosphorylated PAGE4 (CLK2-PAGE4) attenuates c-Jun activity. Consistent with the cellular data, biophysical measurements (small-angle X-ray scattering, single-molecule fluorescence resonance energy transfer, and NMR) indicate that HIPK1-PAGE4 exhibits a relatively compact conformational ensemble that binds AP-1, whereas CLK2-PAGE4 is more expanded and resembles a random coil with diminished affinity for AP-1. Taken together, the results suggest that the phosphorylation-induced conformational dynamics of PAGE4 may play a role in modulating changes between PCa cell phenotypes. A mathematical model based on our experimental data demonstrates how differential phosphorylation of PAGE4 can lead to transitions between androgen-dependent and androgen-independent phenotypes by altering the AP-1/androgen receptor regulatory circuit in PCa cells.
Background Reduction in low-density lipoprotein cholesterol (LDL-C) improves clinical outcomes in patients with chronic coronary artery disease and acute coronary syndromes (ACSs). The combination of ...ezetimibe/simvastatin produces greater reductions in LDL-C compared to simvastatin monotherapy. The IMProved Reduction of Outcomes: Vytorin Efficacy International Trial (IMPROVE-IT) is a multicenter, randomized, double-blind, active-control trial designed to test the hypothesis that the addition of ezetimibe to statin therapy, using ezetimibe/simvastatin, will translate into increased clinical benefit on cardiovascular outcomes relative to simvastatin monotherapy in patients with ACS. Study Design The study will recruit up to 18,000 moderate- to high-risk patients stabilized after ACS. Patients are randomized in a 1:1 ratio to once-daily doses of either ezetimibe/simvastatin 10/40 mg or simvastatin monotherapy 40 mg. Follow-up visits are at 1 and 4 months, and every 4 months thereafter. If consecutive measures of LDL-C are >79 mg/dL at follow-up visits, the simvastatin dose will be increased to 80 mg in a double-blind manner. The primary end point is the first occurrence of cardiovascular death, nonfatal myocardial infarction, rehospitalization for unstable angina, coronary revascularization (occurring at least 30 days after randomization), or stroke. Patients will be followed for a minimum of 2.5 years and until at least 5,250 patients experience a primary end point. Summary IMPROVE-IT will determine whether the addition of ezetimibe to statin therapy, using ezetimibe/simvastatin, improves cardiovascular outcomes compared with simvastatin monotherapy in patients after ACS. In addition, the difference in achieved LDL-C levels between the groups will provide data on whether the target for LDL-C lowering should be reduced further.
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