Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown

Background

Intestinal barrier failure may lead to systemic inflammation and distant organ injury in patients following severe injury. Enteric glia cells (EGCs) have been shown to play an important role in maintaining gut barrier integrity through secretion of S-Nitrosoglutathione (GSNO). We have recently shown than Vagal Nerve Stimulation (VNS) increases EGC activation, which was associated with improved gut barrier integrity. Thus, we sought to further study the mechanism by which EGCs prevent intestinal barrier breakdown utilizing an in vitro model. We postulated that EGCs, through the secretion of GSNO, would improve intestinal barrier function through improved expression and localization of intestinal tight junction proteins.

Methods

Epithelial cells were co-cultured with EGCs or incubated with GSNO and exposed to Cytomix (TNF-α, INF-γ, IL-1β) for 24 hours. Barrier function was assessed by permeability to 4kDa FITC-Dextran. Changes in tight junction proteins ZO-1, occludin, and phospho-MLC (P-MLC) were assessed by immunohistochemistry and immunoblot.

Key Results

Co-culture of Cytomix-stimulated epithelial monolayers with EGCs prevented increases in permeability and improved expression and localization of occludin, ZO-1, and P-MLC. Further, treatment of epithelial monolayers with GSNO also prevented Cytomix-induced increases in permeability and exhibited a similar improvement in expression and localization of occludin, ZO-1, and P-MLC.

Conclusions & Inferences

The addition of EGCs, or their secreted mediator GSNO, prevents epithelial barrier failure after injury and improved expression of tight junction proteins. Thus, therapies that increase EGC activation, such as VNS, may be a novel strategy to limit barrier failure in patients following severe injury.     

Impact of microbial tolerance in persistent secondary Klebsiella pneumoniae peritonitis

Purpose and methodsMicrobial tolerance represents a diminished pro-inflammatory response following repeated stimulation by a host of pathogen-associated molecular patterns (PAMPs) of varying origins. Toll-like receptors (TLRs) have been centrally implicated in the development of tolerance. The purpose of this study was to investigate the impact of tolerance in a previously described murine model.C57BL/6 mice were pretreated intraperitoneally with phosphate buffered saline (PBS), heat-killed Klebsiella 2 × 10⁸ CFU (hkKlebsiella), LPS 10 mg/kg (LPS 10), or BLP 10 mg/kg (BLP 10). Following pretreatment, peritonitis was induced 24 h later using 10³ intraperitoneal Klebsiella CFU. Peritoneal concentrations of TNF-α, IL-10 and nitric oxide (NO), as well as characteristic cell patterns, were determined. Long-term consequences of microbial tolerance were assessed by measuring survival and weight-loss.ResultsFollowing in vitro stimulation with Klebsiella 10⁵ and 10³ CFU, TNF-α and IL-10 secretion were diminished in macrophages harvested from mice pretreated with hkKlebsiella, LPS 10 and BLP 10. Pretreated animals had significantly lower bacterial counts. Conversely, local NO levels were elevated. Survival was not different between the groups.ConclusionPretreatment with TLR ligands induced microbial tolerance, with reduced peritoneal cytokine concentrations and enhanced early bacterial clearance. However, this did not translate into improved survival.     

Vacuolar reticulum in oomycete hyphal tips: An additional component of the Ca2+Regulatory system?

Cultures of Achlya sp., Phytophthora cinnamomi, Saprolegnia diclina, S. ferax, and S. parasitica, treated with 6-carboxyfluorescein diacetate solution, accumulate 6-carboxyfluorescein in a reticulate system of fine tubules. The network shows longitudinal polarity within the hyphae, tubules being finest toward the hyphal tips. In more mature subapical regions the network is connected with large vacuoles that also accumulate 6-carboxyfluorescein. A morphologically similar system has also been identified in freeze-substituted hyphae of S. ferax. The network is considered to be vacuolar, but differs from the tubular vacuole system of true fungi in that tubules are less motile, more frequently branched, and do not alternate with clusters of spherical vacuoles. The appearance of the network resembles patterns of calcium-sensitive dye staining and it is suggested that the vacuolar reticulum in the tip region of oomycete hyphae may act as a Ca2+ sink. The tubular reticulum in oomycetes is very fragile and can be shown with 6-carboxyfluorescein in only those hyphal tips with a motility and organelle distribution characteristic of growing hyphae with normal morphology. Diverse abnormal hyphae show a range of other fluorochrome localizations. These include large irregular compartments filled with fluorochrome, and fluorescent cytoplasm with organelles and vacuoles standing out in negative contrast. These localizations in abnormal hyphae are correlated with other structural changes indicative of damage. Special care is required in experiments with oomycetes to avoid such artefacts of localization. Copyright 1997 Academic Press. Copyright 1997 Academic Press     

VESSELS IN JUNCALES: II. CENTROLEPIDACEAE AND RESTIONACEAE

The occurrence and specialization of vessels in 6 genera and 14 species of Centrolepidaceae and 25 and 72, respectively, of Restionaceae are described. Those in Anarthriaceae (1 and 3) and Ecdeiocoleaceae (1 and 1), newly separated from Restionaceae, were also examined. Vessels in Ecdeiocolea are highly specialized; those in Centrolepidaceae are relatively primitive. Vessels in Restionaceae and Anarthriaceae are intermediate. Vessels in Cyperaceae and Gramineae are compared with those in Juncales in a review of vessels in Glumiflorae. Thurniaceae has most primitive vessels on the average, Juncales has more primitive vessels in leaves and inflorescence axes than does Cyperales; the opposite is true for roots, rhizomes and stems. Vessels in Graminales on the average are much more specialized than those in Cyperales and Juncales.     

Gene Expression Profile of Herpesvirus-Infected T Cells Obtained Using Immunomicroarrays: Induction of Proinflammatory Mechanisms

Herpesvirus infections can frequently lead to acute inflammation, yet the mechanisms regulating this event remain poorly understood. In order to determine some of the immunological mechanisms regulated by human herpesvirus infections, we studied the gene expression profile of lymphocytes infected with human herpesvirus 6 (HHV-6) by using a novel immunomicroarray. Our nylon-based immunomicroarray contained more than 1,150 immune response-related genes and was highly consistent between experiments. Experimentally, we found that independently of the HHV-6 strain used to infect T cells, multiple proinflammatory genes were increased and anti-inflammatory genes were decreased at the mRNA and protein levels. HHV-6 strains A and B increased expression of the genes for interleukin-18 (IL-18), the IL-2 receptor, members of the tumor necrosis factor alpha superfamily receptors, mitogen-activated protein kinase, and Janus kinase signaling proteins. As reported previously, CD4 protein levels were also increased significantly. Specific type 2 cytokines, including IL-10, its receptor, and IL-14, were downregulated by HHV-6 infection and, interestingly, amyloid precursor proteins and type 1 and 2 presenilins. Thus, T cells respond to HHV-6 infection by inducing a type 1 immune response that may play a significant role in the development and progression of diseases associated with HHV-6, including pediatric, hematologic, transplant, and neurologic disorders.     

PubMatrix: a tool for multiplex literature mining

Background  

Molecular experiments using multiplex strategies such as cDNA microarrays or proteomic approaches generate large datasets requiring biological interpretation. Text based data mining tools have recently been developed to query large biological datasets of this type of data. PubMatrix is a web-based tool that allows simple text based mining of the NCBI literature search service PubMed using any two lists of keywords terms, resulting in a frequency matrix of term co-occurrence.     

Genetic background influences natural killer cell activation during bacterial peritonitis in mice, and is interleukin 12 and interleukin 18 independent

Some mouse strains produce strong pro-inflammatory, T-helper (Th)1 responses (e.g. C57BL/6), or strong anti-inflammatory, Th2 responses (e.g. BALB/c). The exact mechanisms for development of distinct immune responses to infection are not completely understood, although cytokines such as interleukin (IL)-12, IL-18 and IL-4 are known to play roles. Natural killer T (NKT)/natural killer (NK) cells are important regulators of immune responses in infection and non-infection models, and NKT/NK activation is also regulated by IL-12 and IL-18 in many models. We investigated the role of IL-12/IL-18 in NKT/NK activation in murine bacterial peritonitis, as well as differential NKT and NK cell activation in C57BL/6 and BALB/c mice. No differences in NKT or NK cell activation or intracellular interferon (IFN)-gamma were determined between mice given control, anti-IL-12 or anti-IL-18 antibodies or in NKT/NK cell activation in STAT4-/- mice (deficient in IL-12 signaling) or wild type controls. However, there were significant differences in the activation of NKT and NK cells between C57BL/6 mice and BALB/c mice, with NKT/NK cytokine production following Th1 or Th2 lines dependent on strain. This suggests a role for NKT and NK cell activation in the development of Th1 and Th2 responses during bacterial infection independently of IL-12 or IL-18.