Nazanin Navabi

Human gastric mucins differently regulate Helicobacter pylori proliferation, gene expression and interactions with host cells.

Helicobacter pylori colonizes the mucus niche of the gastric mucosa and is a risk factor for gastritis, ulcers and cancer. The main components of the mucus layer are heavily glycosylated mucins, to which H. pylori can adhere. Mucin glycosylation differs between individuals and changes during disease. Here we have examined the H. pylori response to purified mucins from a range of tumor and normal human gastric tissue samples. Our results demonstrate that mucins from different individuals differ in how they modulate both proliferation and gene expression of H. pylori. The mucin effect on proliferation varied significantly between samples, and ranged from stimulatory to inhibitory, depending on the type of mucins and the ability of the mucins to bind to H. pylori. Tumor-derived mucins and mucins from the surface mucosa had potential to stimulate proliferation, while gland-derived mucins tended to inhibit proliferation and mucins from healthy uninfected individuals showed little effect. Artificial glycoconjugates containing H. pylori ligands also modulated H. pylori proliferation, albeit to a lesser degree than human mucins. Expression of genes important for the pathogenicity of H. pylori (babA, sabA, cagA, flaA and ureA) appeared co-regulated in response to mucins. The addition of mucins to co-cultures of H. pylori and gastric epithelial cells protected the viability of the cells and modulated the cytokine production in a manner that differed between individuals, was partially dependent of adhesion of H. pylori to the gastric cells, but also revealed that other mucin factors in addition to adhesion are important for H. pylori-induced host signaling. The combined data reveal host-specific effects on proliferation, gene expression and virulence of H. pylori due to the gastric mucin environment, demonstrating a dynamic interplay between the bacterium and its host.

Gastrointestinal cell lines form polarized epithelia with an adherent mucus layer when cultured in semi-wet interfaces with mechanical stimulation.

Mucin glycoproteins are secreted in large quantities by mucosal epithelia and cell surface mucins are a prominent feature of the glycocalyx of all mucosal epithelia. Currently, studies investigating the gastrointestinal mucosal barrier use either animal experiments or non-in vivo like cell cultures. Many pathogens cause different pathology in mice compared to humans and the in vitro cell cultures used are suboptimal because they are very different from an in vivo mucosal surface, are often not polarized, lack important components of the glycocalyx, and often lack the mucus layer. Although gastrointestinal cell lines exist that produce mucins or polarize, human cell line models that reproducibly create the combination of a polarized epithelial cell layer, functional tight junctions and an adherent mucus layer have been missing until now. We trialed a range of treatments to induce polarization, 3D-organization, tight junctions, mucin production, mucus secretion, and formation of an adherent mucus layer that can be carried out using standard equipment. These treatments were tested on cell lines of intestinal (Caco-2, LS513, HT29, T84, LS174T, HT29 MTX-P8 and HT29 MTX-E12) and gastric (MKN7, MKN45, AGS, NCI-N87 and its hTERT Clone5 and Clone6) origins using Ussing chamber methodology and (immuno)histology. Semi-wet interface culture in combination with mechanical stimulation and DAPT caused HT29 MTX-P8, HT29 MTX-E12 and LS513 cells to polarize, form functional tight junctions, a three-dimensional architecture resembling colonic crypts, and produce an adherent mucus layer. Caco-2 and T84 cells also polarized, formed functional tight junctions and produced a thin adherent mucus layer after this treatment, but with less consistency. In conclusion, culture methods affect cell lines differently, and testing a matrix of methods vs. cell lines may be important to develop better in vitro models. The methods developed herein create in vitro mucosal surfaces suitable for studies of host-pathogen interactions at the mucosal surface.

Dynamic changes in mucus thickness and ion secretion during Citrobacter rodentium infection and clearance.

Citrobacter rodentium is an attaching and effacing pathogen used as a murine model for enteropathogenic Escherichia coli. The mucus layers are a complex matrix of molecules, and mucus swelling, hydration and permeability are affected by many factors, including ion composition. Here, we used the C. rodentium model to investigate mucus dynamics during infection. By measuring the mucus layer thickness in tissue explants during infection, we demonstrated that the thickness changes dynamically during the course of infection and that its thickest stage coincides with the start of a decrease of bacterial density at day 14 after infection. Although quantitative PCR analysis demonstrated that mucin mRNA increases during early infection, the increased mucus layer thickness late in infection was not explained by increased mRNA levels. Proteomic analysis of mucus did not demonstrate the appearance of additional mucins, but revealed an increased number of proteins involved in defense responses. Ussing chamber-based electrical measurements demonstrated that ion secretion was dynamically altered during the infection phases. Furthermore, the bicarbonate ion channel Bestrophin-2 mRNA nominally increased, whereas the Cftr mRNA decreased during the late infection clearance phase. Microscopy of Muc2 immunostained tissues suggested that the inner striated mucus layer present in the healthy colon was scarce during the time point of most severe infection (10 days post infection), but then expanded, albeit with a less structured appearance, during the expulsion phase. Together with previously published literature, the data implies a model for clearance where a change in secretion allows reformation of the mucus layer, displacing the pathogen to the outer mucus layer, where it is then outcompeted by the returning commensal flora. In conclusion, mucus and ion secretion are dynamically altered during the C. rodentium infection cycle.

Local cytokine and inflammatory responses to candidate vaginal adjuvants in mice

The current study was undertaken to explore the correlation of adjuvanticity and local inflammatory response elicited in the murine vagina and the draining lymph nodes following local administration of two candidate vaginal adjuvants, Toll like receptor (TLR) 9 agonist CpG ODN, and a non-TLR targeting molecule alpha-galactosylceramide (alpha-GalCer). Using real-time PCR array analysis, we could show that a group of 13 common cytokine genes are activated in the vagina within 24h after vaginal administration of these adjuvants, including Ccl2, Ccl7, Ccl12, Ccl19, Ccl20, Ccl22, Cxcl1, Cxcl5, Il10 and the Th1-inducing molecules Ifng, Cxcl9, Cxcl10 and Cxcl11. A high degree of inflammation in and damage to the epithelium was exclusively observed in the vagina of the CpG ODN treated mice, which was reversed within 48h. These results indicate that there is a group of common genes that correlate with the adjuvanticity of CpG ODN and alpha-GalCer in the vagina, and that alpha-GalCer induces less of local inflammatory reactions in the murine vagina compared to CpG ODN.

IL-4 Protects the Mitochondria Against TNFα and IFNγ Induced Insult During Clearance of Infection with Citrobacter rodentium and Escherichia coli

Citrobacter rodentium is a murine pathogen that serves as a model for enteropathogenic Escherichia coli. C. rodentium infection reduced the quantity and activity of mitochondrial respiratory complexes I and IV, as well as phosphorylation capacity, mitochondrial transmembrane potential and ATP generation at day 10, 14 and 19 post infection. Cytokine mRNA quantification showed increased levels of IFNγ, TNFα, IL-4, IL-6, and IL-12 during infection. The effects of adding these cytokines, C. rodentium and E. coli were hence elucidated using an in vitro colonic mucosa. Both infection and TNFα, individually and combined with IFNγ, decreased complex I and IV enzyme levels and mitochondrial function. However, IL-4 reversed these effects, and IL-6 protected against loss of complex IV. Both in vivo and in vitro, the dysfunction appeared caused by nitric oxide-generation, and was alleviated by an antioxidant targeting mitochondria. IFNγ −/− mice, containing a similar pathogen burden but higher IL-4 and IL-6, displayed no loss of any of the four complexes. Thus, the cytokine environment appears to be a more important determinant of mitochondrial function than direct actions of the pathogen. As IFNγ and TNFα levels increase during clearance of infection, the concomitant increase in IL-4 and IL-6 protects mitochondrial function.

Neutrophil Elastase and Interleukin 17 Expressed in the Pig Colon during Brachyspira hyodysenteriae Infection Synergistically with the Pathogen Induce Increased Mucus Transport Speed and Production via Mitogen-Activated Protein Kinase 3

ABSTRACT Brachyspira hyodysenteriae colonizes the pig colon, resulting in mucoid hemorrhagic diarrhea and mucus layer changes. These changes are characterized by a disorganized mucus structure and massive mucus induction with de novo expression of MUC5AC and increased production of MUC2. To investigate the mechanisms behind this altered mucin environment, we quantified the mRNA levels of mucin pathway genes and factors from the immune system in the colons of infected and control pigs and observed upregulation of neutrophil elastase, SPDEF, FOXA3, MAPK3/ERK1, IL-17A, IL-1β, IL-6, and IL-8 expression. In vitro, colonic mucus-producing mucosal surfaces were treated with these factors along with B. hyodysenteriae infection and analyzed for their effect on mucin production. Neutrophil elastase and infection synergistically induced mucus production and transport speed, and interleukin 17A (IL-17A) also had similar effects, in both the presence and absence of infection. A mitogen-activated protein kinase 3 (MAPK3)/extracellular signal-regulated kinase 1 (ERK1) inhibitor suppressed these effects. Therefore, we suggest that the SPDEF, FOXA3, and MAPK3/ERK1 signaling pathways are behind the transcriptional program regulating mucin biosynthesis in the colon during B. hyodysenteriae infection. In addition to furthering the knowledge on this economically important disease, this mechanism may be useful for the development of therapies aimed at conditions where enhancing mucus production may be beneficial, such as chronic inflammatory disorders of the colon.

Colonic levels of vasoactive intestinal peptide decrease during infection and exogenous VIP protects epithelial mitochondria against the negative effects of IFNγ and TNFα induced during Citrobacter rodentium infection

Citrobacter rodentium infection is a model for infection with attaching and effacing pathogens, such as enteropathogenic Escherichia coli. The vasoactive intestinal peptide (VIP) has emerged as an anti-inflammatory agent, documented to inhibit Th1 immune responses and successfully treat animal models of inflammation. VIP is also a mucus secretagogue. Here, we found that colonic levels of VIP decrease during murine C. rodentium infection with a similar time dependency as measurements reflecting mitochondrial function and epithelial integrity. The decrease in VIP appears mainly driven by changes in the cytokine environment, as no changes in VIP levels were detected in infected mice lacking interferon gamma (IFNγ). VIP supplementation alleviated the reduction of activity and levels of mitochondrial respiratory complexes I and IV, mitochondrial phosphorylation capacity, transmembrane potential and ATP generation caused by IFNγ, TNFα and C. rodentium infection, in an in vitro mucosal surface. Similarly, VIP treatment regimens that included the day 5–10 post infection period alleviated decreases in enzyme complexes I and IV, phosphorylation capacity, mitochondrial transmembrane potential and ATP generation as well as increased apoptosis levels during murine infection with C. rodentium. However, VIP treatment failed to alleviate colitis, although there was a tendency to decreased pathogen density in contact with the epithelium and in the spleen. Both in vivo and in vitro, NO generation increased during C. rodentium infection, which was alleviated by VIP. Thus, therapeutic VIP administration to restore the decreased levels during infection had beneficial effects on epithelial cells and their mitochondria, but not on the overall infection outcome.

Characterization of new human gastric epithelial cell lines

no.: WS1.1 HELICOBACTER PYLORI INFECTION AND MARKERS OF GASTRIC CANCER RISK IN ALASKA NATIVE PEOPLE J. Keck,* K. Miernyk,* L. Bulkow,* J. Kelly, B. McMahon, F. Sacco, T. Hennessy* and M. G. Bruce* *Centers for Disease Control and Prevention, Anchorage, AK, USA; Alaska Native Tribal Health Consortium, Anchorage, AK, USA Background: Alaska Native gastric cancer incidence and mortality rates are 3 to 4-times higher than general US population rates. We evaluated pepsinogen I, pepsinogen I/II ratio, anti-H. pylori and CagA antibodies, and blood group to determine their association with gastric cancer development in Alaska Native people. Methods: We conducted a retrospective case-control study that matched gastric cancers reported to the Alaska Native Tumor Registry from 1969–2008 to three controls on known demographic risk factors for H. pylori infection, using previously collected sera from the Alaska Area Specimen Bank. Conditional logistic regression evaluated the associations between serum markers and gastric cancer. Results: We included 122 gastric cancer cases with sera predating cancer diagnosis (mean = 13 years) and 346 matched controls. One hundred and twelve cases (91.8%) and 285 controls (82.4%) had evidence of previous or ongoing H. pylori infection as measured by anti-H. pylori antibodies. Gastric cancer cases had 2.63-fold increased odds of positive anti-H. pylori antibodies compared with their matched controls (p = .01). In a multivariate model, non-cardia gastric cancer (n = 94) was associated with anti-H. pylori antibodies (adjusted OR 3.92, p = .004) and low pepsinogen I (aOR 6.04, p = .04). We found no association between gastric cancer and blood group, anti-CagA antibodies, or pepsinogen I/II ratio. Conclusions: Alaska Native people with gastric cancer had increased odds of previous H. pylori infection. Low pepsinogen I might function as a pre-cancer marker for non-cardia cancer. Impact: Future research to identify Alaska Native individuals with increased gastric cancer risk includes H. pylori genotype and host characteristic studies. Abstract no.: WS1.2 CLUSTERING OF HELICOBACTER PYLORI STRAINS FROM GASTRIC CANCER C. Wang, S. Zhan and Q. Dong Qingdao Municipal Hospital, Qingdao City, Chinano.: WS1.2 CLUSTERING OF HELICOBACTER PYLORI STRAINS FROM GASTRIC CANCER C. Wang, S. Zhan and Q. Dong Qingdao Municipal Hospital, Qingdao City, China Genetic differences between strains play an important role in the determination of clinical outcomes of Helicobacter pylori infection. This study aimed to determine the sequencing types of H. pylori strains from gastric cancer. Materials and Methods: Twenty-two strains of H. pylori were enrolled, including 12 strains from patients with gastric cancer. MLST was used to determine the sequencing type. Results: The seven genetic loci of H. pylori were PCR amplified and sequenced. Those sequences of the seven genes were concatenated, and aligned with the sequences of strains from Europe (5), Africa (5), Asia (5) and other parts of China (16) extracted from the MLST database. A neighbour-joining tree with a kimura 2-parameter model was subsequently constructed. The results showed that all 22 strains, as well as Asia strains from database fell into the HpEastAsia haplogroup which could divided into two groups, groups I and II. Group I consisted of seven cancer strains but only one non-cancer strain of H. pylori, in addition to five strains form database. Fisher’s exact test revealed a statistically significant difference (p = .027). Discussion and Conclusion: The clustering of cancer strains of H. pylori is consistent with a recent report showing that the phylogeopraphic origin of H. pylori is a determinant of gastric cancer risk. This may reflect the consequence of long-term interaction of the bacterium with individual hosts of different genetic ground. The results suggested that the sequencing types could possibly be used to predict the clinical outcomes of H. pylori infection. Abstract no.: WS1.3 LACK OF ASSOCIATION BETWEEN GENE POLYMORPHISMS OF ANGIOTENSIN CONVERTING ENZYME, NOD-LIKE RECEPTOR 1, TOLL-LIKE RECEPTOR 4 AND FAS/FASL WITH THE PRESENCE OF HELICOBACTER PYLORI-INDUCED PREMALIGNANT GASTRIC LESIONS AND GASTRIC CANCER IN CAUCASIANS J. Kupcinskas,* T. Wex, J. Bornschein, M. Selgrad, M. Leja, L. Jonaitis* and P. Malfertheiner *Department of Gastroenterology, Lithuanian University of Health Sciences, Kaunas, Lithuania; Clinic of Gastroenterology, Hepatology and Infectious Diseases, Otto von Guericke University, Magdeburg, Germany; Faculty of Medicine, University of Latvia, Digestive Diseases Center, Hospital Lizeners, Riga, Latvia Background: Several polymorphisms of genes involved in the immunological recognition of Helicobacter pylori and regulating apoptosis and proliferation have been linked to gastric carcinogenesis, however reported data are partially conflicting. The aim of our study was to evaluate potential associations between the presence of gastric cancer (GC) and high risk atrophic gastritis (HRAG) and polymorphisms of genes encoding Angiotensin converting enzyme (ACE), Nod-like receptor 1 (NOD1), Toll-like receptor 4 (TLR4) and FAS/FASL. Methods: Gene polymorphisms were analyzed in 574 subjects (GC: n = 114; HRAG: n = 222, controls: n = 238) of Caucasian origin. ACE I/D (rs4646994), NOD1 796G>A (rs5743336), TLR4 3725G>C (rs11536889), FAS 1377G>A (rs2234767), FAS 670A>G (rs1800682) and FASL 844T>C (rs763110) were genotyped by different PCR approaches and RFLP analysis. Results: Frequencies of genotypes in our study are similar to the data reported on subjects of Caucasian ethnicity. There was a tendency for NOD1 796G/G genotype to be associated with increased risk of HRAG (62.4% vs 54.5% in controls, p = .082). FAS 670G/G genotype was more frequent in HRAG when compared to controls, 23.9% and 17.2% respectively, however it failed to reach significance level (p = .077). We did not find any significant associations for all examined polymorphism in relation to GC or HRAG. NOD1 796G>A and TLR4 3725G>C gene polymorphisms were also not linked with Helicobacter pylori seropositivity status. Conclusions: ACE, NOD1, TRL4 and FAS/FASL gene polymorphisms are not linked with gastric carcinogenesis in Caucasians, and therefore they should not be considered as potential biomarkers for identifying individuals with higher risk for GC. Abstract no.: WS1.4 ATROPHIC GASTRITIS BY THE OLGA STAGES AND HELICOBACTER CAGA SEROPOSITIVITY IN GASTRIC CANCER T. Vorobjova,* K. Kull,* H. Saar,* R. Labotkin,* A. Zimmermann and H. Maaroos* *University of Tartu, Tartu, Estonia; University of Bern, Bern, Switzerlandno.: WS1.4 ATROPHIC GASTRITIS BY THE OLGA STAGES AND HELICOBACTER CAGA SEROPOSITIVITY IN GASTRIC CANCER T. Vorobjova,* K. Kull,* H. Saar,* R. Labotkin,* A. Zimmermann and H. Maaroos* *University of Tartu, Tartu, Estonia; University of Bern, Bern, Switzerland Introduction: Operative Link on Gastritis Assessment (OLGA) express extent of gastric atrophy in terms of gastritis staging, which severity should be related to gastric cancer. Aim: To study how the OLGA stages of atrophic gastritis are associated with the morphological type, and Helicobacter pylori CagA positivity in gastric cancer. Patients: Twenty two gastric carcinoma patients (8 male, 14 female; mean age 64 ± 12) were operated on. The intestinal type of carcinoma was diagnosed in 12, diffuse in 8, mixed and indeterminate type in two cases (according to Lauren). Methods: Gastric mucosa samples (altogether up to 15) from the each operation specimen were stained with haematoxylin and eosin. Tissue material was received from the primary tumour and the tumour surrounding antral and corpus mucosa. The stage of atrophy by OLGA was established by combining the extent of histologically scored atrophy with the topography of atrophy. IgG antibodies to H. pylori cell surface proteins and CagA were evaluated using ELISA. Results: Of the 12 patients with intestinal type of gastric cancer eight had OLGA stage III or IV, four had OLGA stage II and nobody had OLGA stage I (p < .05). Five patients with diffuse cancer had OLGA stage I and II, two had III stage and one had IV stage. There was no association of OLGA stage or cancer type with CagA positivity. Conclusion: Gastric cancer patients represented all stages of gastric atrophy from OLGA stage I to OLGA stage IV which was not associated with cancer type and CagA seropositivity. a 2011 Blackwell Publishing Ltd, Helicobacter 16 (Suppl. 1): 77–143 79 WS1 Gastric Cancer

P02-11. Correlate of local adjuvanticity and inflammation for experimental vaginal adjuvants in mice

Background Development of mucosal adjuvants to elicit immunity in the female genital tract may have important implications for the development of vaccines to counter sexually transmitted infections. We have recently documented the vaginal adjuvant effect of the TLR9 agonist CpG-ODN, and a non-TLR targeting molecule α galactosylceramide (α-GalCer), an invariant natural killer T cell ligand, for induction of protective immunity in the murine female genital tract.