Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 12 de 12
Filter
Add more filters










Publication year range
1.
PLoS One ; 13(2): e0193003, 2018.
Article in English | MEDLINE | ID: mdl-29447283

ABSTRACT

Gp96 is an endoplasmic reticulum chaperone for multiple protein substrates. Its lack in intestinal macrophages of Crohn's disease (CD) patients is correlated with loss of tolerance against the host gut flora. Gp96 has been stablished to be an essential chaperone for Toll-like receptors (TLRs). We studied the impact of gp96-knockdown on TLR-function in macrophages. TLR2 and TLR4 expression was only decreased but not abolished when gp96 was knocked-down in cell lines, whereas in a monocyte/macrophage specific knock-out mouse model (LysMCre) TLR4 was abolished, while TLR2 was still present. Lipopolysaccharide (LPS)-induced NF-κB activation was still observed in the absence of gp96, and gp96-deficient macrophages were able to up-regulate surface TLR4 upon LPS treatment, suggesting that there is another chaperone involved in the folding of TLR4 upon stress responses. Moreover, LPS-dependent pro-inflammatory cytokines were still expressed, although to a lesser extent in the absence of gp96, which reinforces the fact that gp96 is involved in regulating signaling cascades downstream of TLR4 are impaired upon loss of gp96. In addition, we have also found a reduced phosphorylation of ERK and p38 kinases and an impaired response upon CSF1R activation in gp96 deficient macrophages. Our findings indicate that the loss of gp96 not only impairs TLR4 signaling, but is also associated with a diminished phosphorylation of ERK and mitogen-activated stress kinases resulting in an impaired signalling through several receptors, including CSF1R.


Subject(s)
Extracellular Signal-Regulated MAP Kinases/metabolism , Membrane Glycoproteins/deficiency , Toll-Like Receptor 4/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Animals , Cell Line , HEK293 Cells , Humans , Interleukin-8/metabolism , Membrane Glycoproteins/genetics , Mice, Inbred C57BL , Mice, Transgenic , NF-KappaB Inhibitor alpha/metabolism , NF-kappa B/metabolism , Phosphorylation/physiology , Toll-Like Receptor 2/genetics , Toll-Like Receptor 2/metabolism , Toll-Like Receptor 4/genetics
2.
J Crohns Colitis ; 12(3): 355-368, 2018 Feb 28.
Article in English | MEDLINE | ID: mdl-29136128

ABSTRACT

BACKGROUND AND AIMS: During active inflammation, intraluminal intestinal pH is decreased in patients with inflammatory bowel disease [IBD]. Acidic pH may play a role in IBD pathophysiology. Recently, proton-sensing G-protein coupled receptors were identified, including GPR4, OGR1 [GPR68], and TDAG8 [GPR65]. We investigated whether GPR4 is involved in intestinal inflammation. METHODS: The role of GPR4 was assessed in murine colitis models by chronic dextran sulphate sodium [DSS] administration and by cross-breeding into an IL-10 deficient background for development of spontaneous colitis. Colitis severity was assessed by body weight, colonoscopy, colon length, histological score, cytokine mRNA expression, and myeloperoxidase [MPO] activity. In the spontaneous Il-10-/- colitis model, the incidence of rectal prolapse and characteristics of lamina propria leukocytes [LPLs] were analysed. RESULTS: Gpr4-/- mice showed reduced body weight loss and histology score after induction of chronic DSS colitis. In Gpr4-/-/Il-10-/- double knock-outs, the onset and progression of rectal prolapse were significantly delayed and mitigated compared with Gpr4+/+/Il-10-/- mice. Double knock-out mice showed lower histology scores, MPO activity, CD4+ T helper cell infiltration, IFN-γ, iNOS, MCP-1 [CCL2], CXCL1, and CXCL2 expression compared with controls. In colon, GPR4 mRNA was detected in endothelial cells, some smooth muscle cells, and some macrophages. CONCLUSIONS: Absence of GPR4 ameliorates colitis in IBD animal models, indicating an important regulatory role in mucosal inflammation, thus providing a new link between tissue pH and the immune system. Therapeutic inhibition of GPR4 may be beneficial for the treatment of IBD.


Subject(s)
Colitis/metabolism , Receptors, G-Protein-Coupled/genetics , Receptors, G-Protein-Coupled/metabolism , Rectal Prolapse/etiology , Animals , Chemokine CCL2/metabolism , Chemokine CXCL1/metabolism , Chemokine CXCL2/metabolism , Colitis/chemically induced , Colitis/complications , Colitis/pathology , Dextran Sulfate , Endothelial Cells/metabolism , Female , Hydrogen-Ion Concentration , Interferon-gamma/metabolism , Interleukin-10/genetics , Intestinal Mucosa/pathology , Macrophages/metabolism , Male , Mice , Mice, Knockout , Myocytes, Smooth Muscle/metabolism , Nitric Oxide Synthase Type II/metabolism , Peroxidase/metabolism , Protons , RNA, Messenger/metabolism , Rectal Prolapse/genetics , T-Lymphocytes, Helper-Inducer/pathology
3.
PLoS One ; 8(10): e76350, 2013.
Article in English | MEDLINE | ID: mdl-24146856

ABSTRACT

The chaperone function of the ER-residing heat shock protein gp96 plays an important role in protein physiology and has additionally important immunological functions due to its peptide-binding capacity. Low amounts of gp96 stimulate immunity; high quantities induce tolerance by mechanisms not fully understood. A lack of gp96 protein in intestinal macrophages (IMACs) from Crohn`s disease (CD) patients correlates with loss of tolerance against the host gut flora, leading to chronic inflammation. Since gp96 shows dose-dependent direction of immunological reactions, we studied primary IMACs and developed cell models to understand the regulation of gp96 expression. Induction of gp96-expression was higher in in vitro differentiated dendritic cells (i.v.DCs) than in in vitro differentiated macrophages (i.v.MACs), whereas monocytes (MOs) expressed only low gp96 levels. The highest levels of expression were found in IMACs. Lipopolysaccharide (LPS), muramyl dipeptide (MDP), tumour necrosis factor (TNF), and Interleukin (IL)-4 induced gp96-expression, while IL12, IL-17, IL-23 and interferon (IFN)-γ were not effective indicating that Th1 and Th17 cells are probably not involved in the induction of gp96. Furthermore, gp96 was able to induce its own expression. The ER-stress inducer tunicamycin increased gp96-expression in a concentration- and time-dependent manner. Both ulcerative colitis (UC) and CD patients showed significantly elevated gp96 mRNA levels in intestinal biopsies which correlated positively with the degree of inflammation of the tissue. Since gp96 is highly expressed on the one hand upon stress induction as during inflammation and on the other hand possibly mediating tolerance, these results will help to understand the whether gp96 plays a role in the pathophysiology of inflammatory bowel disease (IBD).


Subject(s)
Dendritic Cells/metabolism , Gene Expression Regulation , Macrophages/metabolism , Membrane Glycoproteins/genetics , Acetylmuramyl-Alanyl-Isoglutamine/pharmacology , Biopsy , Case-Control Studies , Cell Differentiation/drug effects , Cell Differentiation/genetics , Cell Wall/drug effects , Cell Wall/metabolism , Cells, Cultured , Dendritic Cells/drug effects , Dendritic Cells/pathology , Endoplasmic Reticulum Stress/drug effects , Endoplasmic Reticulum Stress/genetics , Gene Expression Regulation/drug effects , Humans , Ileum/drug effects , Ileum/metabolism , Ileum/pathology , Immunity, Innate/drug effects , Immunity, Innate/genetics , Inflammatory Bowel Diseases/pathology , Lipopolysaccharides/pharmacology , Macrophages/drug effects , Macrophages/pathology , Membrane Glycoproteins/metabolism , Monocytes/drug effects , Monocytes/metabolism , Monocytes/pathology , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Up-Regulation/drug effects , Up-Regulation/genetics
4.
Toxicol In Vitro ; 27(5): 1525-32, 2013 Aug.
Article in English | MEDLINE | ID: mdl-23562973

ABSTRACT

Retinoid treatment is suggested to promote development of inflammatory bowel disease, although preclinical studies are not supportive. We evaluated the effect of retinoids on cytokine response in in vitro-differentiated human dendritic cells (ivDCs) and macrophages (ivMACs) derived from healthy human donors and in cultured human THP-1 cells. Effect on human intestinal epithelial cell integrity was also assessed. Each cell type was incubated (±lipopolysaccharide [LPS]) with all-trans retinoic acid (ATRA), 13-cis-RA (isotretinoin) and 4-oxo-13-cis-RA. Cytokine analysis was performed by array analysis. Cultured human endothelial colorectal adenocarcinoma (Caco-2) cells were incubated with these retinoids and media analyzed for leakage by spectrofluorometric analysis. ATRA consistently and significantly inhibited LPS-induced release of the pro-inflammatory cytokines tumor necrosis factor, interleukin (IL)-6, macrophage inflammatory protein (MIP)-1α and MIP-1ß. All retinoids tested stimulated release of the anti-inflammatory cytokines granulocyte-macrophage colony-stimulating factor and IL-10, and also monocyte chemotactic protein-1, vascular endothelial growth factor and eotaxin-1. Incubation with retinoids did not significantly alter the permeability of Caco-2 monolayers. Pre-treatment of each cell type with retinoids promoted an anti-inflammatory cytokine profile with only minimal effect on intestinal epithelial cell permeability; consistent with in vivo studies.


Subject(s)
Dendritic Cells/drug effects , Macrophages/drug effects , Retinoids/pharmacology , Adult , Caco-2 Cells , Cell Line , Cells, Cultured , Cytokines/metabolism , Dendritic Cells/metabolism , Female , Humans , Immunity, Innate/drug effects , Intestinal Mucosa/metabolism , Lipopolysaccharides , Macrophages/metabolism , Male , Permeability
5.
J Med Microbiol ; 61(Pt 1): 23-30, 2012 Jan.
Article in English | MEDLINE | ID: mdl-21903829

ABSTRACT

Recently, dupA was reported as a new virulence factor in Helicobacter pylori, but its association with gastroduodenal disorders and its mode of action are still unclear. Here, an association of the dupA status with different disease groups was determined and a biological explanation for the observed associations was tested. In total, 216 H. pylori isolates were obtained from 232 presumed H. pylori-infected patients. A positive association was observed between the occurrence of duodenal ulcer (DU) and the presence of dupA [odds ratio (OR) 24.2; 95 % confidence interval (CI) 10.6-54.8]. In addition, an inverse association between the occurrence of gastric cancer (GC) [OR 0.16; 95 % CI 0.05-0.47] and gastric ulcer (GU) [OR 0.34; 95 % CI 0.16-0.68] with the presence of dupA was observed. A putative explanation for the observed associations might be a more corpus-located infection (pan-gastritis) by the dupA-positive strains due to their increased acid resistance. Indeed, a strong association between dupA-positive H. pylori isolated from gastritis patients and in vitro acid resistance was observed (P<0.05). The observed higher acid resistance of the dupA-positive strains suggests that these strains are adapted to a stomach with high gastric acid output. This may in part explain the observed associations, as an increased gastric acid output is thought to be typical for an antrum-predominant H. pylori infection and, whilst this is associated with an increased risk of DU formation, it also decreases the risk for the genesis of GUs and GC.


Subject(s)
Gene Deletion , Helicobacter Infections/microbiology , Helicobacter pylori/pathogenicity , Virulence Factors/genetics , Adolescent , Adult , Aged , Culture Media , Duodenal Ulcer/genetics , Female , Helicobacter Infections/complications , Helicobacter pylori/genetics , Helicobacter pylori/isolation & purification , Helicobacter pylori/physiology , Humans , Hydrogen-Ion Concentration , Male , Middle Aged , Stomach Neoplasms/genetics , Stomach Ulcer/genetics , Virulence , Virulence Factors/metabolism , Young Adult
7.
Am J Physiol Gastrointest Liver Physiol ; 300(4): G598-607, 2011 Apr.
Article in English | MEDLINE | ID: mdl-21233279

ABSTRACT

Transketolase-like (TKTL) 1 indirectly replenishes NADPH preventing damage induced by reactive oxygen species (ROS) formed upon intestinal inflammation. We investigated the function of TKTL1 during murine colitis and ROS detoxification for prevention of tissue damage. Mucosal damage in TKTL1(-/-) and wild-type (WT) mice was assessed by miniendoscopy and histology during dextran sodium sulfate (DSS) colitis. mRNA levels of interferon (IFN)-γ, inducible nitric oxide synthase (iNOS), interleukin (IL)-6, tumor necrosis factor (TNF), transketolase (TKT), and TKTL2 were determined by PCR and/or Western blotting. To assess oxidative and nitrosative stress nitrosylation, carbonylation and antioxidative enzymes catalase (Cat), superoxide dismutase 1 and 2, as well as glutathione (GSH) were determined. Myeloperoxidase (MPO) was determined for assessment of tissue neutrophils. TKTL1 knockout or DSS treatment did not influence TKT and TKTL2 mRNA or protein expression. Mucosal damage was significantly increased in TKTL1(-/-) mice indicated by miniendoscopy as well as a significantly shorter colon and more severe histological scores compared with WT mice during DSS colitis. This was associated with higher mRNA levels of IFN-γ, iNOS, IL-6, and TNF. In addition, iNOS protein expression was significantly enhanced in TKTL1(-/-) mice as well as MPO activity. Protein modification by nitric oxide (nitrotyrosine) was induced in TKTL1(-/-) mice. However, introduction of carbonyl groups by ROS was not induced in these mice. The expression of SOD1, SOD2, Cat, as well as GSH content was not significantly changed in TKTL1(-/-) mice. We conclude that induced colitis in TKTL1(-/-) mice was more severe compared with WT. This indicates a role of TKTL1 during mucosal repair and restoration.


Subject(s)
Colitis/metabolism , Colon/metabolism , Intestinal Mucosa/metabolism , Transketolase/metabolism , Animals , Blotting, Western , Colitis/chemically induced , Colitis/genetics , Colitis/pathology , Colon/pathology , Interleukin-6/genetics , Interleukin-6/metabolism , Intestinal Mucosa/pathology , Mice , Mice, Knockout , Nitric Oxide Synthase Type II/genetics , Nitric Oxide Synthase Type II/metabolism , Reactive Oxygen Species/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Transketolase/genetics , Tumor Necrosis Factor-alpha/genetics , Tumor Necrosis Factor-alpha/metabolism
8.
Helicobacter ; 15(6): 505-9, 2010 Dec.
Article in English | MEDLINE | ID: mdl-21073606

ABSTRACT

BACKGROUND: The aim of this study was to investigate the prevalence of resistances in Helicobacter pylori against commonly used antibiotics including metronidazole, clarithromycin, amoxicillin, and tetracycline in Iranian patients. METHODS: H. pylori isolates were collected from gastric biopsies from patients referred for upper gastrointestinal endoscopy at Tooba Medical Center, Sari, Iran, from 2007 to 2010. None of them had been using antibiotics for at least 8 months. H. pylori was identified based on morphological shape and positive biochemical tests for catalase, oxidase, and urease activity. Antibiotic resistance for metronidazole, clarithromycin, amoxicillin, and tetracycline was investigated by using epsilometer test. Resistance was defined by minimal inhibitory concentration (MIC) > 0.5 mg/L for amoxicillin (AMX), >4 mg/L for tetracycline (TET), >8 mg/L for metronidazole (MTZ), and >1 mg/L for clarithromycin (CLR). RESULTS: Strains were collected from 132 patients, mean age 45.8 years, 52 (39%) were women. Patients had diverse diagnoses: gastritis 42 (31.8%), duodenal ulcer 45 (34%), gastric cancer 15 (11.3%), or gastric ulcer 30 (22.7%). The prevalences of resistance of H. pylori strains isolated from the patients were 73.4% for metronidazole, 30% for clarithromycin, 6.8% for amoxicillin, and 9% for tetracycline. Twenty-eight (21.2%) were double resistant to MTZ-CLR, 16 (12.1%) showed triple resistance to MTZ-CLR-AMX, and 8 (6%) were resistant to all four tested antibiotics (MTZ-CLR-AMX-TET). No associations were detected between multiple resistant strains and clinical manifestations (p > .05). CONCLUSIONS: The prevalence of H. pylori antibiotic resistance to metronidazole and clarithromycin was high in Iran consistent with the reported low success rates for H. pylori treatment in this country.


Subject(s)
Drug Resistance, Multiple, Bacterial , Helicobacter Infections/microbiology , Helicobacter pylori/drug effects , Adolescent , Adult , Aged , Amoxicillin/pharmacology , Clarithromycin/pharmacology , Female , Helicobacter Infections/drug therapy , Helicobacter pylori/isolation & purification , Humans , Iran , Male , Metronidazole/pharmacology , Microbial Sensitivity Tests , Middle Aged , Tetracycline/pharmacology , Young Adult
9.
Helicobacter ; 14(4): 264-70, 2009 Aug.
Article in English | MEDLINE | ID: mdl-19674130

ABSTRACT

BACKGROUND: Nickel-dependent urease activity and nickel supply are essential for successful colonization of Helicobacter pylori in the acidic environment of the human stomach. A comparison of media effects on these two activities have never been carried out. Additionally to H. pylori we cultivated an Escherichia coli strain expressing the urease and the nickel transporter NixA of H. pylori on the same four media and measured in all cases urease and nickel uptake activity. AIM: To compare nickel uptake and urease activity on an inter- and intraspecies level. RESULTS: In H. pylori nickel uptake (four to 200 times) and urease activities (400 to 30,000 times) were found to be much higher in comparison to the tested E. coli strain after growth on all media. These differences could not be explained by reduced protein amounts in the heterologous host E. coli. On which media the two bacteria extracted most of the nickel were organism-dependent: E. coli on Brucella Broth, H. pylori on Trypticase Soy Broth, and Minimal Media. CONCLUSION: H. pylori took nickel much more efficiently up than E. coli. The observed differences in urease activity are most likely due to additional protein components absent in the recombinant E. coli strain. The observed variety in nickel uptake and urease activities on the different media in the same organism depended on the intrinsic nickel content and chelating capacities of media components. Different culture conditions may lead to varying results; generalizations should be concluded only after excluding their media dependence.


Subject(s)
Bacterial Proteins/metabolism , Culture Media/metabolism , Escherichia coli/metabolism , Helicobacter pylori/metabolism , Nickel/metabolism , Urease/metabolism , Bacterial Proteins/genetics , Biological Transport , Escherichia coli/enzymology , Escherichia coli/genetics , Helicobacter pylori/enzymology , Helicobacter pylori/genetics , Urease/genetics
10.
J Bacteriol ; 188(4): 1245-50, 2006 Feb.
Article in English | MEDLINE | ID: mdl-16452405

ABSTRACT

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. NixA was identified as a specific nickel uptake system in this organism. Addition of small amounts of nickel to media strongly stimulates urea hydrolysis. On the other hand, high nickel concentrations are deleterious to cell growth. As a possible protective reaction, nickel uptake seems to be reduced in H. pylori grown in nickel-rich media. These observations led to investigations of regulation of the expression of the nickel permease NixA. We found that increasing the nickel concentration in media reduced the amount of NixA. In order to address the question of whether this phenomenon was subject to transcriptional or translational regulation, we quantified nixA mRNA from H. pylori by real-time PCR. The amount of nixA mRNA was gradually reduced five- to sevenfold in a time- and concentration-dependent manner. Repression could be measured as soon as 5 min after nickel addition, and the maximum repression occurred after 20 to 30 min. The maximum repression was obtained with an external nickel concentration of 100 microM. The observed nickel repression of NixA was dependent on nikR encoding the nickel-responsive regulatory protein NikR. In conclusion, we demonstrated that synthesis of the NixA nickel permease of H. pylori shows nickel-responsive regulation mediated by NikR to maintain the balance between effective nickel acquisition and a toxic overload.


Subject(s)
Bacterial Proteins/metabolism , Cation Transport Proteins/metabolism , Gene Expression Regulation, Bacterial , Helicobacter pylori/genetics , Nickel/metabolism , Bacterial Proteins/biosynthesis , Biological Transport , Cation Transport Proteins/biosynthesis , Dose-Response Relationship, Drug , Down-Regulation , Escherichia coli Proteins/metabolism , Helicobacter pylori/growth & development , Helicobacter pylori/metabolism , Nickel/pharmacology , Polymerase Chain Reaction , RNA, Bacterial/genetics , RNA, Messenger/genetics , Repressor Proteins/metabolism , Time Factors
11.
J Bacteriol ; 184(5): 1438-43, 2002 Mar.
Article in English | MEDLINE | ID: mdl-11844775

ABSTRACT

Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79-both part of the characteristic sequence motif-and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.


Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Cation Transport Proteins/chemistry , Cation Transport Proteins/metabolism , Helicobacter pylori/enzymology , Nickel/metabolism , Amino Acid Motifs , Bacterial Proteins/genetics , Cation Transport Proteins/genetics , Cell Membrane/metabolism , Conserved Sequence , Molecular Sequence Data , Mutagenesis, Site-Directed , Urease/metabolism
12.
J Ethnopharmacol ; 79(1): 43-52, 2002 Jan.
Article in English | MEDLINE | ID: mdl-11744294

ABSTRACT

As part of an ethnopharmacological field study 48 medicinal plants were evaluated using several biological assays with the goal to obtain information on the pharmacological effects of these plants, which may be of direct relevance to the indigenous uses. Three species used to treat gastrointestinal disorders showed remarkable activity against Helicobacter pylori. One of them showed activity against Giardia duodenalis. Cytotoxic effects against KB cells were found for six species. In the group of plants used for dermatological conditions several species were active against gram-positive bacteria and Candida albicans. Two plant species of this group were found to be active in an Nuclear Factor-kappaB (NF-kappaB) assay measuring inhibition of this pro-inflammatory transcription factor. A species of the Solanaceae, applied in cases of pain and fever, showed a weak activity against Plasmodium falciparum. One species traditionally used for diabetes exhibited antihyperglycemic activity. None of the six species from the group of 'women's medicine' showed relevant affinity to the D(2) dopamine receptor. Based on this evaluation, plants with strong activities should be further investigated phytochemically and pharmacologically to identify active fractions and compounds.


Subject(s)
Drug Evaluation/methods , Ethnopharmacology , Medicine, Traditional , Plant Extracts/therapeutic use , Animals , Anti-Bacterial Agents/therapeutic use , Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Humans , Mexico , Microbial Sensitivity Tests , Plants, Medicinal
SELECTION OF CITATIONS
SEARCH DETAIL
...