Endophytic bacterial diversity in roots of Typha angustifolia L. in the constructed Beijing Cuihu Wetland (China)

We investigated the community structure of endophytic bacteria in narrowleaf cattail (Typha angustifolia L.) roots growing in the Beijing Cuihu Wetland, China, using the 16S rDNA library technique. In total, 184 individual sequences were used to assess the diversity of endophytic bacteria. Phylogenetic analysis revealed that 161 clones (87.5%) were affiliated with Proteobacteria, other clones grouped into Cytophaga/Flexibacter/Bacteroids (3.3%), Fusobacteria (3.8%), and nearly 5% were uncultured bacteria. In Proteobacteria, the beta and gamma subgroups were the most abundant, accounting for approximately 46% and 36.6% of all Proteobacteria, respectively. The dominant genera included Rhodoferax, Pelomonas, Uliginosibacterium, Pseudomonas, Aeromonas, Rhizobium, Sulfurospirillum, Ilyobacter and Bacteroides. While some of these endophytic bacteria are capable of fixing nitrogen and can therefore improve plant growth, other endophytes may play important biological roles by removing nitrogen, phosphorus and/or organic matter from the water body and thus have the potential to enhance the phytoremediation of eutrophic water bodies. These bacteria have the potential to degrade xenobiota such as methane, methanol, methylated amines, catechol, oxochlorate, urea, cyanide, and 2,4-dichlorophenol. Hence, the use of certain endophytic bacteria in the process of phytoremediation could be a powerful approach for the restoration of eutrophic systems.     

Deficient innervation characterizes intestinal strictures in a rat model of colitis

Intestinal strictures are a common complication of Crohn's disease leading to serious consequences. With unknown etiology and cellular composition, strictures can be neither prevented nor reversed by current therapeutic strategies, and research has been limited by the lack of a well-developed animal model. We observed the sporadic occurrence of intestinal strictures at Day 35 in the TNBS rat model of colitis, which persisted beyond Day 90. Strictured tissue showed fusion, thickening, and disorganization of the smooth muscle layers. Immunocytochemistry revealed that all strictures were characterized by deficient innervation with a complete loss of intrinsic neurons, and a 92% loss of total axons per area. The number of alpha-smooth muscle actin-positive smooth muscle cells (SMC) increased in strictures, but immunolabeling showed phenotypic modulation of these cells, with the SMC phenotype (desmin-positive, vimentin-negative) entirely replaced by a myofibroblast phenotype (desmin-negative, vimentin-positive). Although cellular structure still predominated in the strictured regions, histochemistry showed increased extracellular matrix collagen, from 6 +/- 0.9% to 22 +/- 4% of total area. With previous evidence for neural loss in colitis, and in vitro studies showing neural regulation of smooth muscle cell (SMC) growth, we conclude that the regional loss of innervation may initiate tissue re-modeling that is characteristic of stricture formation.     

Caffeic acid phenethyl ester (CAPE) reverses fibrosis caused by chronic colon inflammation in murine model of colitis

Fibrosis is known to be the hallmarks of chronic inflammation of the bowel. Epithelial damage due to inflammation compromises the barrier function of the gastrointestinal tract. This barrier dysfunction leads to further spread of inflammation resulting in a chronic state of inflammation. This chronic inflammation leads to development of fibrosis, which has very limited therapeutic options and usually requires surgical removal of the affected tissue. Our previous work has shown that Caffeic acid phenethyl ester (CAPE) is a naturally occurring anti-inflammatory agent, found in propolis, has been found to be protective in experimental colitis via enhancement of epithelial barrier function. However, the impact of CAPE on resolution of fibrosis in the long-term is unknown. The aim of this follow up study was to investigate the effect of CAPE on colon fibrosis in a chronic model of Dextran sulphate sodium induced colitis in mice. Dextran sulphate sodium (DSS) 2.5% w/v was administered in drinking water to induce colitis in C57/BL6 mice for 5 days on the 6th day DSS was stopped and test group mice were treated with intraperitoneal administration of CAPE (30?mg?kg^?1 day^?1) for a further 7 days. Disease activity index (DAI) score, colon length and tissue histology and level of tissue fibrosis was observed. CAPE-treated mice had significantly lower levels of DAI, tissue inflammation scores and fibrosis as compared with control group. Our results show that CAPE is effective in resolving colon fibrosis in chronic inflammation. Thus, we can conclude CAPE could be a potential therapeutic agent for further clinical investigations for treatment of fibrosis in inflammatory bowel diseases in humans.     

In vivo treatment with the herbal phenylethanoid acteoside ameliorates intestinal inflammation in dextran sulphate sodium-induced colitis

Recently we demonstrated that in inflammatory bowel disease (IBD) macrophage-oxidative burst activity is increased and NADPH oxidase mRNA is induced. The herbal phenylethanoid acteoside isolated from Plantago lanceolata L. was shown to exhibit anti-oxidative potential. Using the dextran sulphate sodium (DSS)-induced colitis model, in this study we have assessed whether systemic application of acteoside affects colitis. Colitis was induced by DSS in Balb/c mice. Treatment with acteoside (120, 600 microg/mouse/day) was performed intraperitoneally. The colon lengths were determined. Colonic tissue was scored histologically (max. score 8) by a blinded investigator. T cells isolated from mesenteric lymph nodes (MLN) were stimulated with anti-CD3 antibody in the presence of interleukin (IL)-2 (final concentration 10 U/ml). After incubation for 24 h, IL-1beta, IL-6, IL-12 tumour necrosis factor (TNF)-alpha and interferon (IFN)-gamma levels in supernatants were analysed by the beadlyte cytokine detection system. Histological scoring of colonic tissue revealed that application of acteoside was followed by a significantly improved histological score. In acute colitis the histological score was 3.2 with acteoside versus 5.2 with phosphate-buffered saline (PBS) (P < 0.02). In chronic colitis both 120 microg (3.3 versus 5.2) or 600 microg acteoside (3.0 versus 5.2) significantly ameliorated colitis (both P < 0.02). Stimulated MLN from mice with chronic DSS-induced colitis treated with acteoside showed a significant down-regulation of IFN-gamma secretion (195 pg/ml with 600 microg acteoside versus 612 pg/ml with PBS, P < 0.02). Inhibition of oxidative burst activity with acteoside reduced mucosal tissue damage in DSS colitis and could be a therapeutic alternative for IBD treatment. Further studies of this agent are warranted.     

Saccharomyces boulardii decreases inflammation and intestinal colonization by Candida albicans in a mouse model of chemically-induced colitis.

The present study was designed to investigate the effects of Saccharomyces boulardii on inflammation and intestinal colonization by Candida albicans in a BALB/c mouse model of colitis that had been induced by dextran-sulfate-sodium (DSS). Colonization with C. albicans was established by oral gavage with a 200 microL suspension of 10(7) yeast cells. A 1.5% solution of DSS was administered in drinking water 1 h after C. albicans oral challenge, while 10(7) cells of S. boulardii was inoculated daily by oral gavage for 1 week. Faeces were collected daily for 2 weeks. Seven groups of mice consisting of those that were administered either C. albicans or S. boulardii or both were sacrificed after 14 days and samples of the colon were taken for histological scoring and real-time PCR (RT-PCR) analysis of inflammatory cytokines and toll-like receptors (TLRs). Compared to control animals that did not receive DSS, the number of C. albicans colonies recovered from faeces was significantly greater in mice receiving DSS. In contrast, the colony forming units (CFUs) of C. albicans were greatly reduced in mice receiving S. boulardii. The administration of this yeast decreased the severity of DSS-induced clinical scores and histological inflammation. At the mRNA expression level, an increase in TLR2 and TLR4 resulting from the presence of S. boulardii was associated with a reduction in the inflammatory cytokines TNFalpha and INFgamma. In mice receiving DSS and C. albicans, TLR4 was over-expressed by stimulation with both yeasts, but TLR2 and TNFalpha, which were increased by the administration of C. albicans alone, were decreased in the presence of S. boulardii. These results indicate that S. boulardii decreased inflammation and C. albicans colonization in this BALB/c mouse model of colitis.     

Nod1 and Nod2 regulation of inflammation in the Salmonella colitis model

The pattern recognition molecules Nod1 and Nod2 play important roles in intestinal homeostasis; however, how these proteins impact on the development of inflammation during bacterial colitis has not been examined. In the streptomycin-treated mouse model of Salmonella colitis, we found that mice deficient for both Nod1 and Nod2 had attenuated inflammatory pathology, reduced levels of inflammatory cytokines, and increased colonization of the mucosal tissue. Nod1 and Nod2 from both hematopoietic and nonhematopoietic sources contributed to the pathology, and all phenotypes were recapitulated in mice deficient for the signaling adaptor protein Rip2. However, the influence of Rip2 was strictly dependent on infection conditions that favored expression of the Salmonella pathogenicity island 2 (SPI-2) type III secretion system (TTSS), as Rip2 was dispensable for inflammation when mice were infected with bacteria grown under conditions that promoted expression of the SPI-1 TTSS. Thus, Nod1 and Nod2 can modulate inflammation and mediate efficient clearance of bacteria from the mucosal tissue during Salmonella colitis, but their role is dependent on the expression of the SPI-2 TTSS.     

Structure and function of the intestinal epithelial cells in the perch (Perca fluviatillis L.)

An ultrastructural study of the intestinal absorptive epithelium in perch (Perca fluviatilis) has shown that the perch intestine can be divided into three segments: the proximal segment, the middle segment and the distal segment. The enterocytes of the proximal segment are found to be concerned with lipid absorption. The adsorbed fat gives rise to the presence of two forms of inclusions: lipid particles and lipid droplets. Enterocytes of the middle segment exhibit the typical ultrastructural features of pinocytosis; these consist of extensive invaginations of the luminal surface membrane and acculation of vacuoles in the apical cytoplasm. Exogenous proteins are ingested by absorptive cells from the intestinal lumen by a process similar to that described in neonatal mammals. In the distal segment the absorptive cells have few, short microvilli. Besides the absorptive epithelial cells, goblet cells, endocrine cells, pear-shaped cells, and plasma cells are occasionally found.     

Flufenamic acid prevents behavioral manifestations of salicylate-induced tinnitus in the rat

Introduction

Tinnitus is defined as a phantom auditory sensation, the perception of sound in the absence of external acoustic stimulation. Given that flufenamic acid (FFA) blocks TRPM2 cation channels, resulting in reduced neuronal excitability, we aimed to investigate whether FFA suppresses the behavioral manifestation of sodium salicylate (SSA)-induced tinnitus in rats.

Material and methods

Tinnitus was evaluated using a conditioned lick suppression model of behavioral testing. Thirty-one Wistar rats, randomly divided into four treatment groups, were trained and tested in the behavioral experiment: (1) control group: DMSO + saline (n = 6), (2) SSA group: DMSO + SSA (n = 6), (3) FFA group: FFA (66 mg/kg bw) + saline (n = 9), (4) FFA + SSA group: FFA (66 mg/kg bw) + SSA (400 mg/kg bw) (n = 10). Localization of TRPM2 to the plasma membrane of cochlear nucleus neurons was demonstrated by confocal microscopy.

Results

Pavlovian training resulted in strong suppression of licking, having a mean value of 0.05 ±0.03 on extinction day 1, which is below the suppression training criterion level of 0.20 in control tinnitus animals. The suppression rate for rats having both FFA (66 mg/kg bw) and SSA (400 mg/kg bw) injections was significantly lower than that for the rats having SSA injections (p < 0.01).

Conclusions

We suggest that SSA-induced tinnitus could possibly be prevented by administration of a TRPM2 ion channel antagonist, FFA at 66 mg/kg bw.     

The schistosome glutathione S-transferase P28GST,a unique helminth protein,prevents intestinal inflammation in experimental colitis through a Th2-type response with mucosal eosinophils

Intestinal helminth parasites are potent inducers of T helper type 2 (Th2) response and have a regulatory role, notably on intestinal inflammation. As infection with schistosomes is unlikely to provide a reliable treatment of inflammatory bowel diseases, we have investigated the beneficial effect of a schistosome enzymatic protein, the 28-kDa glutathione S-transferase (P28GST), on the modulation of disease activity and immune responses in experimental colitis. Our results showed that immunization with recombinant P28GST is at least as efficient as established schistosome infection to reduce colitis lesions and expression of pro-inflammatory cytokines. Considering underlying mechanisms, the decrease of inflammatory parameters was associated with the polarization of the immune system toward a Th2 profile, with local and systemic increases of interleukin (IL)-13 and IL-5. Dense eosinophil infiltration was observed in the colons of P28GST-immunized rats and mice. Depletion of eosinophils by treatment with an anti-Siglec-F monoclonal antibody and use of IL-5-deficient mice led to the loss of therapeutic effect, suggesting the crucial role for eosinophils in colitis prevention by P28GST. These findings reveal that immunization with P28GST, a unique recombinant schistosome enzyme, ameliorates intestinal inflammation through eosinophil-dependent modulation of harmful type 1 responses, representing a new immuno-regulatory strategy against inflammatory bowel diseases.     

Crucial role of plasmacytoid dendritic cells in the development of acute colitis through the regulation of intestinal inflammation

Disruption of intestinal homeostasis can lead to inflammatory bowel diseases endowed susceptibility genes and environmental factors affecting intestinal accumulation and activation of colitogenic phagocytes. Plasmacytoid dendritic cells (pDCs) are immune cells that had been proposed to control innate and adaptive immunity through the massive secretion of type I interferon (IFN-I). However, the contribution of pDCs to the progression of intestinal inflammation remains unclear. Here we show a critical role of pDCs in the initiation of acute colonic inflammation using T-cell-independent acute colitis model with a selective ablation of pDCs. Although pDCs accumulated in the inflamed colon upon mucosal injury, deficiency of pDCs attenuated the development of acute colitis independent of IFN-I signaling, accompanied by the diminished colonic production of proinflammatory cytokines. Furthermore, deficiency of pDCs impaired the mobilization of colitogenic phagocytes into the inflamed colon possibly mediated by the abrogated mucosal production of C–C chemokine receptor 2 ligand. Thus, our findings highlight a critical role of pDCs in the induction of the colonic inflammation that regulates the colonic accumulation of inflammatory phagocytes leading to the initiation and exacerbation of acute colitis, and they may serve a key role in controlling gut mucosal immune homeostasis.     

Helicobacter species in the intestinal mucosa of patients with ulcerative colitis

In a search for Helicobacter species in the intestinal mucosae of 42 patients with ulcerative colitis (UC) and 74 without UC, only H. pylori was found. Although the bacterium was detected in UC patients by culture (7.1%) and nested PCR (19.0%), its presence was not associated with the disease (P = 0.13).     

Tumor necrosis factor alpha is a key mediator of gut inflammation seen in amebic colitis in human intestine in the SCID mouse-human intestinal xenograft model of disease

We used Entamoeba histolytica infection in human intestinal xenografts to study the roles interleukin-1 (IL-1) and tumor necrosis factor alpha (TNF-alpha) in the pathogenesis of amebic colitis. We found that blockade of TNF-alpha reduced inflammation and intestinal damage in amebic infection, while inhibition of IL-1 reduced cytokine production but had less marked effects on inflammation and disease.     

Blockade of macrophage migration inhibitory factor (MIF) prevents the antigen-induced response in a murine model of allergic airway inflammation

Objective and Design: The role of macrophage migration inhibitory factor (MIF), a proinflammatory cytokine, was tested using a mouse asthma model. Materials: One hundred and four male BALB/c mice were used in this study. Treatment: Mice were actively sensitized with an intraperitoneal injection of ovalbumin (OVA) and challenged with repeated nebulization of 1 w/v% OVA. Polyclonal anti-MIF antibody was intraperitoneally injected at 10 mg/kg during the antigen challenge period. Methods: Bronchoalveolar lavage (BAL) was performed 8 h after the last challenge. Airway hyperresponsiveness to inhaled methacholine was measured 24 h after the last challenge. Results: Antigen challenge to immunized mice induced increase in inflammatory cells and concentration of Th2 cytokines in BAL fluid (BALF), and caused the development of airway hyperresponsiveness. Anti-MIF antibody significantly decreased the numbers of inflammatory cells including macrophages, eosinophils, lymphocytes and neutrophils in BALF from OVA-challenged mice. Prednisolone decreased the numbers of eosinophils, lymphocytes and neutrophils but not macrophages. Anti-MIF antibody reduced airway hyperresponsiveness. Anti-MIF antibody affected neither the cytokine levels in BALF nor the IgE levels in serum. Conclusion: MIF was involved in the antigen-induced inflammatory cell accumulation in the lung and airway hyperresponsiveness without affecting immune responses. Received 17 November 2005; returned for revision 14 June 2006; accepted by M. Katori 25 July 2006     

Antiinflammatory effects of various drugs on acetic acid induced colitis in the rat

The efficacy of various drugs used to treat ulcerative colitis, (sulfasalazine, 5-aminosalicylate, hydrocortisone) was investigated in a model of acetic acid-induced colitis in the rat. Subsequently, we tested the ability of antioxidant/5-lipoxygenase inhibitors (gossypol and nordihydroguiaretic acid [NDGA]) and a cyclooxygenase inhibitor (indomethacin) to attenuate the macroscopic colonic damage and/or neutrophil influx (myeloperoxidase activity [MPO]) associated with this model of colitis. Oral pretreatment with either sulfasalazine, gossypol, or NDGA significantly decreased colonic MPO activity induced by acetic acid. Intrarectal administration of such drugs resulted in an even larger reduction of the colonic inflammation, with gossypol being the most potent compound. Oral or intrarectal administration of corticosteroids (dexamethasone, hydrocortisone) also attenuated the parameters of acetic acid induced colitis. In contrast, pretreatment with indomethacin was ineffective, or when administered daily after colitis induction, indomethacin actually increased colonic neutrophil influx significantly. Our data suggest that both the route of drug administration and dosing regimen employed affect the antiinflammatory potency and/or efficacy of compounds on colitis induced by acetic acid in the rat. Drugs which were effective against this colitis may act by scavenging of oxygen derived free radicals.     

Fibronectin in chronic inflammation: studies using the rat air pouch model of chronic allergic inflammation

Fibronectin is a large glycoprotein of plasma, tissue fluids and tissues. The rat air pouch model of mesenchymal inflammation was used to examine changes in fibronectin levels during inflammation within a mesenchymal cavity. Rat plasma fibronectin levels showed a rapid and significant rise in relation to the induction of an air pouch. In contrast pouch fluid fibronectin levels were initially low and gradually increased with chronicity. They were unrelated to plasma levels. Pouch fluid fibronectin showed no relationship to cell content of the fluid, its volume, nor the weight of granulation tissue. Two-dimensional immuno-electrophoresis showed pouch fluid fibronectin was partially complexed but plasma fibronectin was not. These results show plasma and tissue fluid fibronectin have different patterns of response to inflammation. In rats plasma fibronectin is an acute-phase reactant, although this is not the case in humans.     

Helicobacter hepaticus-induced colitis in interleukin-10-deficient mice: cytokine requirements for the induction and maintenance of intestinal inflammation

We have previously shown that specific-pathogen-free interleukin-10 (IL-10)-deficient (IL-10 KO) mice reconstituted with Helicobacter hepaticus develop severe colitis associated with a Th1-type cytokine response. In the present study, we formally demonstrate that IL-12 is crucial for disease induction, because mice deficient for both IL-10 and IL-12 p40 show no intestinal pathology following H. hepaticus infection. By using monoclonal antibodies (MAbs) to IL-12, gamma interferon (IFN-gamma), and tumor necrosis factor alpha (TNF-alpha), we have further analyzed the role of these cytokines in the maintenance of the Th1 response and inflammation in IL-10 KO mice with established H. hepaticus-induced colitis. Treatment of infected colitic IL-10 KO mice with anti-IL-12 p40 resulted in markedly reduced intestinal inflammation, colonic IFN-gamma, TNF-alpha, and inducible nitric oxide synthase (iNOS) mRNA levels, and H. hepaticus-specific IFN-gamma secretion by mesenteric lymph node (MLN) cells compared to the findings in control MAb-treated mice. Moreover, the diminished pathology was associated with decreased numbers of colonic CD3(+) T cells and significantly reduced frequencies of Helicobacter-reactive CD4(+) Th1 cells in MLN. In contrast, anti-IFN-gamma and/or anti-TNF-alpha had no effect on intestinal inflammation in IL-10 KO mice with established colitis. Using IL-10/IFN-gamma double-deficient mice, we further show that IFN-gamma is not required for the development of colitis following H. hepaticus infection. MLN cells from infected IL-10/IFN-gamma KO animals secreted elevated amounts of IL-12 and TNF-alpha following bacterial antigen stimulation, indicating alternative pathways of disease induction. Taken together, our results demonstrate a crucial role for IL-12 in both inducing and sustaining intestinal inflammation through recruitment and maintenance of a pool of pathogenic Th1 cells.     

A model of persistent antigen-induced chronic inflammation in the rat air pouch

Continuing antigen-induced inflammation was established in a subcutaneous air pouch in rats by recurrent local challenge. The animals were sensitized using bovine serum albumin in Freund's complete adjuvant and were challenged 14 days later by injection of the antigen in a solution containing sodium carboxymethylcellulose into the air pouch to produce allergic inflammation. A single antigenic challenge induced acute inflammation with a predominantly polymorph infiltration in the first 48 h. Later samples showed a low-grade mononuclear response which persisted for 4-5 days. Repeated challenge produced chronic inflammation with an accentuated mononuclear response. Connective tissue activation involving fibronectin and collagen was seen as the inflammation progressed, and this was associated with production of ferritin by mononuclear cells. Discontinuation of challenge injections resulted in resolution of the granuloma. We suggest this model can be used to investigate the mechanisms involved in chronic inflammatory diseases with an immunological component and to evaluate the effects of therapeutic intervention upon chronic allergic inflammation.