Dietary intervention with serum-derived bovine immunoglobulins protects barrier function in a mouse model of colitis

2015 ◽  
Vol 308 (12) ◽  
pp. G1012-G1018 ◽  
Author(s):  
Anna Pérez-Bosque ◽  
Lluïsa Miró ◽  
Mònica Maijó ◽  
Javier Polo ◽  
Joy Campbell ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Mouse Model ◽  
Inducible Nitric Oxide Synthase ◽  
Inflammatory Markers ◽  
Dietary Intervention ◽  
Dietary Supplementation ◽  
Tnf Α ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Dietary supplementation with immunoglobulins from animal plasma has anti-inflammatory effects on intestinal and lung models of acute inflammation. Here, we aimed to establish whether dietary intervention with serum-derived bovine immunoglobulin (SBI) can prevent alterations in intestinal barrier function in a mouse model with a genetic predisposition to inflammatory bowel disease (IBD). Wild-type (WT) mice and mice lacking the mdr1a gene (KO) were fed diets supplemented with either SBI (2% wt/wt) or milk proteins (control diet), from day 21 (weaning) until day 56. The epithelial permeability of distal colon crypts was measured by confocal microscopy using a fluorescent marker. The expression of junctional epithelial E-cadherin and β-catenin proteins were determined by Western blot and zonula occludens-1 (ZO-1) by immunofluorescence. Mucins (MUC1, MUC2, MUC4), TFF3, cytokines (TNF-α, IFN-γ), and inducible nitric oxide synthase RNA expression were quantified by real-time PCR. SBI blocked the increase in colon crypt permeability and partially prevented the reduction in E-cadherin and ZO-1 expression that characterize the KO mouse model (both P < 0.05). SBI inclusion also reduced the mucosal expression of the inflammatory markers TNF-α, IFN-γ, and inducible nitric oxide synthase (all P < 0.005). The number of goblet cells in the colon of KO mice was low and correlated well with MUC2 and TFF3 expression ( P < 0.001), whereas dietary supplementation with SBI attenuated these effects (all P < 0.05). In short, dietary SBI ameliorated colonic barrier alterations and reduced the expression of mucosal inflammatory markers in a genetic model of IBD.

scholarly journals Enhanced Immunohistochemical Detection of Autonomic Nerve Fibers, Cytokines and Inducible Nitric Oxide Synthase by Light and Fluorescent Microscopy in Rat Spleen

1997 ◽  
Vol 45 (4) ◽  
pp. 599-610 ◽  
Author(s):  
Jon C. Meltzer ◽  
Paul C. Grimm ◽  
Arnold H. Greenberg ◽  
Dwight M. Nance
Keyword(s):  
Nitric Oxide ◽  
Inducible Nitric Oxide Synthase ◽  
Fluorescent Microscopy ◽  
Nerve Fibers ◽  
Autonomic Nerve ◽  
Detection Systems ◽  
Tnf Α ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

We have developed enhanced immunohistochemical protocols for detecting autonomic nerve fibers and splenocyte-associated proteins in rat spleen. This includes norepinephrine-synthesizing enzymes (dopamine-βeta hydroxylase (DBH) and tyrosine hydroxylase (TH)), neuropeptide Y (NPY), tumor necrosis factor -α (TNF-α), interferon-γ (IFN-γ), c-fos protein, inducible nitric oxide synthase (iNOS), and the macrophage cell marker ED1. Animals were divided into sham-operated and splenic nerve-sectioned groups for detection of DBH, TH, and NPY. For immunodetection of TNF-α, iNOS, IFN-γ and c-fos, animals were injected IV with saline or 100 μg of lipopolysaccharide (LPS) and were sacrificed at various time intervals post injection. Rats were perfused with 4% paraformaldehyde, spleens removed and cryoprotected, and 50-μm floating sections were cut on a freezing microtome. Immunodetection was performed with various detection systems and substrate/chromogen solutions, and in some cases using pretreatment with proteinase K (PK) for antigen unmasking. PK pretreatment increased immunostaining for DBH, TH, NPY, IFN-γ, iNOS, and ED1, and the improvement was concentration-dependent. Using NPY immunostaining to index the signal-to-noise ratio for various substrates and detection systems, we found that an alkaline phosphatase detection system with NBT/BCIP as a substrate was the best procedure for light microscopy, whereas the CY3-labeled secondary antibody technique proved optimal for fluorescent microscopy. Surgical transection of the splenic nerve eliminated all nerve fiber staining for DBH, TH, and NPY. TNF-α, IFN-γ, c-fos, and iNOS proteins were observed in the spleen in a time-dependent manner after LPS stimulation. Fluorescent double labeling, visualized with fluorescent confocal scanning laser microscopy, revealed many NPY fibers distributed among the ED1-labeled macrophages. These results demonstrate that immunohistochemistry can be used to index the activational effects of an immune challenge on splenocytes in situ and verifies that splenic immune cells are innervated by the sympathetic nervous system.

Extracellular superoxide dismutase is upregulated with inducible nitric oxide synthase after NF-κB activation

1997 ◽  
Vol 273 (5) ◽  
pp. L1002-L1006 ◽  
Author(s):  
Todd C. Brady ◽  
Ling-Yi Chang ◽  
Brian J. Day ◽  
James D. Crapo
Keyword(s):  
Nitric Oxide ◽  
Superoxide Dismutase ◽  
Nitric Oxide Synthase ◽  
Inducible Nitric Oxide Synthase ◽  
Dermal Fibroblasts ◽  
Extracellular Superoxide Dismutase ◽  
Tnf Α ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Inflammatory cytokines have been shown to upregulate secretion of the antioxidant enzyme extracellular superoxide dismutase (EC-SOD) in dermal fibroblasts and, in other cells, to stimulate production of nitric oxide (⋅ NO). Because superoxide rapidly scavenges ⋅ NO, forming the injurious peroxynitrite anion (OONO−), we hypothesize that stimulated cells upregulate EC-SOD expression concurrently with ⋅ NO release. To test for coregulation of EC-SOD and ⋅ NO within the same cell, the timing of inducible nitric oxide synthase (iNOS) and EC-SOD transcription was measured after exposure of a rat type II pneumocyte analog, the L2 cell line, to a combination of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α). Upregulation of iNOS and EC-SOD transcription occurred after 6 h of exposure, and transcription of both genes was linked by activation of the transcription factor nuclear factor-κB. Both EC-SOD and iNOS were elevated in rat lung homogenates 24 h after intratracheal instillation with IFN-γ and TNF-α. The observation that EC-SOD and iNOS are temporally coregulated after cytokine exposure suggests the possibility of a critical mechanism by which cells might protect ⋅ NO and avoid the formation of OONO−during inflammation.

scholarly journals Effects of Immunomodulatory Substances on Phagocytosis of A by Human Microglia

2010 ◽  
Vol 2010 ◽  
pp. 1-18 ◽  
Author(s):  
Erik Hjorth ◽  
Dan Frenkel ◽  
Howard Weiner ◽  
Marianne Schultzberg
Keyword(s):  
Nitric Oxide ◽  
Membrane Protein ◽  
Inducible Nitric Oxide Synthase ◽  
Inflammatory Markers ◽  
Interleukin 1 ◽  
Type I ◽  
Human Microglia ◽  
Amyloid A ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

Glial activation and increased inflammation characterize neuropathology in Alzheimer's disease (AD). The aim was to develop a model for studying phagocytosis of -amyloid (A) peptide by human microglia and to test effects thereupon by immunomodulatory substances. Human CHME3 microglia showed intracellular A colocalized with lysosome-associated membrane protein-2, indicating phagocytosis. This was increased by interferon-, and to a lesser degree with Protollin, a proteosome-based adjuvant. Secretion of brain-derived neurotrophic factor (BDNF) was decreased by A and by interferon- and interleukin-1. These cytokines, but not A, stimulated interleukin-6 release. Microglia which phagocytosed A exhibited a higher degree of expression of interleukin-1 receptor type I and inducible nitric oxide synthase. In conclusion, we show that human microglia are able to phagocytose A and that this is associated with expression of inflammatory markers. A and interferon- decreased BDNF secretion suggesting a new neuropathological role for A and the inflammation accompanying AD.

scholarly journals Both Inducible Nitric Oxide Synthase and NADPH Oxidase Contribute to the Control of Virulent Phase I Coxiella burnetii Infections

2004 ◽  
Vol 72 (11) ◽  
pp. 6666-6675 ◽  
Author(s):  
Robert E. Brennan ◽  
Kasi Russell ◽  
Guoquan Zhang ◽  
James E. Samuel
Keyword(s):  
Nitric Oxide ◽  
Nitric Oxide Synthase ◽  
Cell Lines ◽  
Inducible Nitric Oxide Synthase ◽  
Coxiella Burnetii ◽  
Wild Type ◽  
Primary Mouse ◽  
Ifn Γ ◽  
Oxide Synthase ◽  
Inducible Nitric Oxide

ABSTRACT Host control of Coxiella burnetii infections is believed to be mediated primarily by activated monocytes/macrophages. The activation of macrophages by cytokines leads to the production of reactive oxygen intermediates (ROI) and reactive nitrogen intermediates (RNI) that have potent antimicrobial activities. The contributions of ROI and RNI to the inhibition of C. burnetii replication were examined in vitro by the use of murine macrophage-like cell lines and primary mouse macrophages. A gamma interferon (IFN-γ) treatment of infected cell lines and primary macrophages resulted in an increased production of nitric oxide (NO) and hydrogen peroxide (H2O2) and a significant inhibition of C. burnetii replication. The inhibition of replication was reversed in the murine cell line J774.16 upon the addition of either the inducible nitric oxide synthase (iNOS) inhibitor NG-monomethyl-l-arginine (NGMMLA) or the H2O2 scavenger catalase. IFN-γ-treated primary macrophages from iNOS−/− and p47phox−/− mice significantly inhibited replication but were less efficient at controlling infection than IFN-γ-treated wild-type macrophages. To investigate the contributions of ROI and RNI to resistance to infection, we performed in vivo studies, using C57BL/6 wild-type mice and knockout mice lacking iNOS or p47phox. Both iNOS−/− and p47phox−/− mice were attenuated in the ability to control C. burnetii infection compared to wild-type mice. Together, these results strongly support a role for both RNI and ROI in the host control of C. burnetii infection.

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