Richard L Ferrero

Publications of Richard L Ferrero

• The use of AlbuMAX II(®) as a blood or serum alternative for the culture of Helicobacter pylori.

  Authors: Melanie L Hutton, Maria Kaparakis-Liaskos, Richard L Ferrero

  Helicobacter. 02/2012; 17(1):68-76.

  Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch-to-batch variation,Growth of Helicobacter pyloriin vitro depends on supplementation of the medium with blood or serum. However, these supplements often require frozen storage and can show batch-to-batch variation, resulting in differences in bacterial growth. In this study, we introduce the use of a commercially available, lipid-rich supplement called AlbuMAX II(®) (Gibco BRL, Grand Island, NY, USA) for use as a serum/blood replacement for H. pylori culture. The growth of H. pylori on solid and liquid media was examined by comparing growth after supplementation with horse blood, fetal calf serum, β-cyclodextrin or AlbuMAX II(®) (Gibco BRL). Human gastric adenocarcinoma (AGS) cellular responses to H. pylori were measured by NF-κB luciferase assays and IL-8 ELISA. We show that the growth of H. pylori on both solid and liquid media containing AlbuMAX II(®) (Gibco BRL) were comparable to levels obtained on blood agar or liquid media supplemented with serum. Growth was consistently higher in media supplemented with AlbuMAX II(®) (Gibco BRL) than media containing β-cyclodextrin. Furthermore, bacteria grown in AlbuMAX II(®) (Gibco BRL) induced proinflammatory responses in AGS cells. AlbuMAX II(®) (Gibco BRL) can be used as a serum/blood replacement for the cultivation of H. pylori in solid and liquid media. This medium could be useful for an improved understanding of H. pylori metabolism or for antigen production. Furthermore, AlbuMAX II(®) (Gibco BRL) may be suitable for use in remote locations, particularly in areas where frozen storage of serum may be a problem.

• The molecular pathogenesis of STAT3-driven gastric tumourigenesis in mice is independent of IL-17.

  Authors: Catherine L Kennedy, Meri Najdovska, Gareth W Jones, Louise McLeod, Norman R Hughes, Cody Allison, Chia Huey Ooi, Patrick Tan, Richard L Ferrero, Simon A Jones, Anouk Dev, William Sievert, Prithi S Bhathal, Brendan J Jenkins

  The Journal of pathology. 05/2011; 225(2):255-64.

  Chronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by theirChronic activation of the gastric mucosal adaptive immune response is a characteristic trait of gastric cancer. It has recently emerged that a new class of T helper (Th) cells, defined by their ability to produce interleukin (IL)-17A (Th17), is associated with a host of inflammatory responses, including gastritis. However, the role of these Th17 cells in the pathogenesis of gastric cancer is less clear. To formally address this, we employed gp130(F/F) mice, which spontaneously develop gastric inflammation-associated tumours akin to human intestinal-type gastric cancer. At the molecular level, these tumours demonstrate hyper-activation of the latent transcription factor signal transducer and activator of transcription (STAT)3 via the IL-6 cytokine family member, IL-11. In gp130(F/F) mice, the generation of Th17 cells, as well as the gastric expression of IL-17a and other Th17-related factors (Rorγt, IL-23), were augmented compared to wild-type gp130(+/+) mice. Consistent with a role for IL-6 and STAT3 in regulating IL-17A, increased Th17 generation and gastric expression of Th17-related factors in gp130(F/F) mice were reduced to wild-type levels in gp130(F/F) :Stat3(-/+) mice displaying normalized STAT3 activity, and also in gp130(F/F) :IL-6(-/-) mice. Importantly, genetic ablation of IL-17A in gp130(F/F) :IL-17a(-/-) mice did not suppress the initiation and growth of gastric tumours. Furthermore, IL-17A and RORC gene expression was strongly increased in human gastric biopsies from patients with gastritis, but not gastric cancer. Collectively, our data suggest that increased expression of Th17-related factors does not correlate with the molecular pathogenesis of gastric tumourigenesis.

• Helicobacter pylori exploits cholesterol-rich microdomains for induction of NF-kappaB-dependent responses and peptidoglycan delivery in epithelial cells.

  Authors: Melanie L Hutton, Maria Kaparakis-Liaskos, Lorinda Turner, Ana Cardona, Terry Kwok, Richard L Ferrero

  Infection and immunity. 11/2010; 78(11):4523-31.

  Infection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes aInfection with Helicobacter pylori cag pathogenicity island (cagPAI)-positive strains is associated with more destructive tissue damage and an increased risk of severe disease. The cagPAI encodes a type IV secretion system (TFSS) that delivers the bacterial effector molecules CagA and peptidoglycan into the host cell cytoplasm, thereby inducing responses in host cells. It was previously shown that interactions between CagL, present on the TFSS pilus, and host α(5)β(1) integrin molecules were critical for CagA translocation and the induction of cytoskeletal rearrangements in epithelial cells. As the α(5)β(1) integrin is found in cholesterol-rich microdomains (known as lipid rafts), we hypothesized that these domains may also be involved in the induction of proinflammatory responses mediated by NOD1 recognition of H. pylori peptidoglycan. Indeed, not only did methyl-β-cyclodextrin depletion of cholesterol from cultured epithelial cells have a significant effect on the levels of NF-κB and interleukin-8 (IL-8) responses induced by H. pylori bacteria with an intact TFSS (P < 0.05), but it also interfered with TFSS-mediated peptidoglycan delivery to cells. Both of these effects could be restored by cholesterol replenishment of the cells. Furthermore, we demonstrated for the first time the involvement of α(5)β(1) integrin in the induction of proinflammatory responses by H. pylori. Taking the results together, we propose that α(5)β(1) integrin, which is associated with cholesterol-rich microdomains at the host cell surface, is required for NOD1 recognition of peptidoglycan and subsequent induction of NF-κB-dependent responses to H. pylori. These data implicate cholesterol-rich microdomains as a novel platform for TFSS-dependent delivery of bacterial products to cytosolic pathogen recognition molecules.

• Structural and solution studies of phenylbismuth(III) sulfonate complexes and their activity against Helicobacter pylori.

  Authors: Philip C Andrews, Madleen Busse, Glen B Deacon, Richard L Ferrero, Peter C Junk, Katie K Huynh, Ish Kumar, Jonathan G Maclellan

  Dalton transactions (Cambridge, England : 2003). 10/2010; 39(40):9633-41.

  Three bis-phenylbismuth sulfonates [Ph(2)Bi(O(3)SR)](∞) (R = p-tolyl 1, mesityl 2 or S-(+)-10-camphoryl 3) have been synthesised and characterised. Their tendency for ligand redistribution inThree bis-phenylbismuth sulfonates [Ph(2)Bi(O(3)SR)](∞) (R = p-tolyl 1, mesityl 2 or S-(+)-10-camphoryl 3) have been synthesised and characterised. Their tendency for ligand redistribution in solution, and activity against the bacterium Helicobacter pylori have been investigated. The structures of 2 and 3 have been authenticated by X-ray diffraction crystallography. They are structurally very similar with polymeric helical chain structures composed of four coordinate Bi atoms which bridge between two sulfonate O atoms with near linear O-Bi-O bond angles. The two phenyl rings are cis to one another and trans to the stereochemically active lone pair. Upon dissolution of the pure compounds 1, 2 and 3, a ligand redistribution reaction occurs in which the mono-phenylbismuth bis-sulfonates, the bismuth tris-sulfonates and triphenylbismuth are formed. Two further complexes of general formula [PhBi(O(3)SR)(2)](∞) (where R = p-tolyl 4, and mesityl 5) were thus obtained and their crystal structures determined. The presence of the single sulfonato ligand in compounds 1, 2 and 3 resulted in a dramatic increase in bacteriocidial activity towards H. pylori (MIC values of ≥6.25 μg mL(-1)) relative to BiPh(3) (>64 μg mL(-1)) and the sulfonic acids, which were essentially inactive.

• Both the p33 and p55 subunits of the Helicobacter pylori VacA toxin are targeted to mammalian mitochondria.

  Authors: Jung Hock Foo, Janetta G Culvenor, Richard L Ferrero, Terry Kwok, Trevor Lithgow, Kipros Gabriel

  Journal of molecular biology. 09/2010; 401(5):792-8.

  Helicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells asHelicobacter pylori infection causes peptic ulcers and gastric cancer. A major toxin secreted by H. pylori is the bipartite vacuolating cytotoxin A, VacA. The toxin is believed to enter host cells as two subunits: the p55 subunit (55 kDa) and the p33 subunit (33 kDa). At the biochemical level, it has been shown that VacA forms through the assembly of large multimeric pores composed of both the p33 subunit and the p55 subunit in biological membranes. One of the major target organelles of VacA is the mitochondria. Since only the p33 subunit has been reported to be translocated into mitochondria and the p55 subunit is not imported, it has been contentious as to whether VacA assembles into pores in a mitochondrial membrane. Here we show the p55 protein is imported into the mitochondria along with the p33 protein subunit. The p33 subunit integrally associates with the mitochondrial inner membrane, and both the p33 subunit and the p55 subunit are exposed to the mitochondrial intermembrane space. Their colocalization suggests that they could reassemble and form a pore in the inner mitochondrial membrane.

• Genetic modulation of TLR8 response following bacterial phagocytosis.

  Authors: Michael P Gantier, Aaron T Irving, Maria Kaparakis-Liaskos, Dakang Xu, Vanessa A Evans, Paul U Cameron, James A Bourne, Richard L Ferrero, Matthias John, Mark A Behlke, Bryan R G Williams

  Human mutation. 09/2010; 31(9):1069-79.

  Human Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 inHuman Toll-like receptors (TLRs) TLR7, TLR8, and TLR9 are important immune sensors of foreign nucleic acids encountered by phagocytes. Although there is growing evidence implicating TLR7 and TLR9 in the detection of intracellular pathogenic bacteria, characterization of such a role for TLR8 is currently lacking. A recent genetic study has correlated the presence of a TLR8 single nucleotide polymorphism (SNP) (rs3764880:A>G; p.Met1Val) with the development of active tuberculosis, suggesting a role for TLR8 in the detection of phagosomal bacteria. Here we provide the first direct evidence that TLR8 sensing is activated in human monocytic cells following Helicobacter pylori phagocytosis. In addition, we show that rs3764880 fine tunes translation of the two TLR8 main isoforms, without affecting protein function. Although we show that TLR8 variant 2 (TLR8v2) is the prevalent form of TLR8 contributing to TLR8 function, we also uncover a role for the TLR8 long isoform (TLR8v1) in the positive regulation of TLR8 function in CD16(+)CD14(+) differentiated monocytes. Thus, TLR8 sensing can be activated following bacterial phagocytosis, and rs3764880 may play a role in the modulation of TLR8-dependent microbicidal response of infected macrophages.

• Role of virulence factors and host cell signaling in the recognition of Helicobacter pylori and the generation of immune responses.

  Authors: Cody C Allison, Richard L Ferrero

  Future microbiology. 08/2010; 5(8):1233-55.

  Helicobacter pylori colonizes a large proportion of the world's population, with infection invariably leading to chronic, lifelong gastritis. While the infection often persists undiagnosed andHelicobacter pylori colonizes a large proportion of the world's population, with infection invariably leading to chronic, lifelong gastritis. While the infection often persists undiagnosed and without causing severe pathology, there are a number of host, bacterial and environmental factors that can influence whether infection provokes a mild inflammatory response or results in significant morbidity. Intriguingly, the most virulent H. pylori strains appear to deliberately induce the epithelial signaling cascades responsible for activating the innate immune system. While the reason for this remains unclear, the resulting adaptive immune responses are largely ineffective in clearing the bacterium once infection has become established and, as a result, inflammation likely causes more damage to the host itself.

• The innate immune molecule, NOD1, regulates direct killing of Helicobacter pylori by antimicrobial peptides.

  Authors: Alexandra Grubman, Maria Kaparakis, Jérôme Viala, Cody Allison, Luminita Badea, Abdulgader Karrar, Ivo G Boneca, Lionel Le Bourhis, Shane Reeve, Ian A Smith, Elizabeth L Hartland, Dana J Philpott, Richard L Ferrero

  Cellular microbiology. 05/2010; 12(5):626-39.

  The cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in hostThe cytosolic innate immune molecule, NOD1, recognizes peptidoglycan (PG) delivered to epithelial cells via the Helicobacter pylori cag pathogenicity island (cagPAI), and has been implicated in host defence against cagPAI(+)H. pylori bacteria. To further clarify the role of NOD1 in host defence, we investigated NOD1-dependent regulation of human beta-defensins (DEFBs) in two epithelial cell lines. Our findings identify that NOD1 activation, via either cagPAI(+) bacteria or internalized PG, was required for DEFB4 and DEFB103 expression in HEK293 cells. To investigate cell type-specific induction of DEFB4 and DEFB103, we generated stable NOD1'knockdown' (KD) and control AGS cells. Reporter gene assay and RT-PCR analyses revealed that only DEFB4 was induced in an NOD1-/cagPAI-dependent fashion in AGS cells. Moreover, culture supernatants from AGS control, but not AGS NOD1 KD cells, stimulated with cagPAI(+)H. pylori, significantly reduced H. pylori bacterial numbers. siRNA studies confirmed that human beta-defensin 2 (hBD-2), but not hBD-3, contributes to the antimicrobial activity of AGS cell supernatants against H. pylori. This study demonstrates, for the first time, the involvement of NOD1 and hBD-2 in direct killing of H. pylori bacteria by epithelial cells and confirms the importance of NOD1 in host defence mechanisms against cagPAI(+)H. pylori infection.

• Bismuth(III) complexes derived from non-steroidal anti-inflammatory drugs and their activity against Helicobacter pylori.

  Authors: Philip C Andrews, Richard L Ferrero, Peter C Junk, Ish Kumar, Quynh Luu, Kim Nguyen, James W Taylor

  Dalton transactions (Cambridge, England : 2003). 03/2010; 39(11):2861-8.

  The formation of bismuth(III) complexes of carboxylates and benzoates derived from the 1 : 3 reaction of BiPh(3) with the common non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen, naproxen,The formation of bismuth(III) complexes of carboxylates and benzoates derived from the 1 : 3 reaction of BiPh(3) with the common non-steroidal anti-inflammatory drugs (NSAIDs) ketoprofen, naproxen, ibuprofen, mefenamic acid, diflunisal, 5-chlorosalicylic acid, fenbufen, sulindac, tolfenamic acid and flufenamic acid, has been achieved using both solvent-free and solvent-mediated methods. The thermochemical profiles of the solvent-free reactions were studied using DSC-TGA. All reactions produced the tris-substituted complexes of general formula [BiL(3)](n), with the complexes derived from ketoprofen and sulindac having an additional single bismuth bound H(2)O molecule in the inner coordination sphere. The complexes are stable in air over a period of six months, do not undergo significant decomposition when suspended overnight in water, but decompose in 1 M HCl solution to release the free acid form of the NSAID. All ten complexes show excellent in vitro activity against Helicobacter pylori with MIC values of > or = 6.25 microg mL(-1).

• Vitamin B6 is required for full motility and virulence in Helicobacter pylori.

  Authors: Alexandra Grubman, Alexandra Phillips, Marie Thibonnier, Maria Kaparakis-Liaskos, Chad Johnson, Jean-Michel Thiberge, Fiona J Radcliff, Chantal Ecobichon, Agnès Labigne, Hilde de Reuse, George L Mendz, Richard L Ferrero

  mBio. 01/2010; 1(3).

  Despite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. ToDespite recent advances in our understanding of how Helicobacter pylori causes disease, the factors that allow this pathogen to persist in the stomach have not yet been fully characterized. To identify new virulence factors in H. pylori, we generated low-infectivity variants of a mouse-colonizing H. pylori strain using the classical technique of in vitro attenuation. The resulting variants and their highly infectious progenitor bacteria were then analyzed by global gene expression profiling. The gene expression levels of five open reading frames (ORFs) were significantly reduced in low-infectivity variants, with the most significant changes observed for ORFs HP1583 and HP1582. These ORFs were annotated as encoding homologs of the Escherichia coli vitamin B(6) biosynthesis enzymes PdxA and PdxJ. Functional complementation studies with E. coli confirmed H. pylori PdxA and PdxJ to be bona fide homologs of vitamin B(6) biosynthesis enzymes. Importantly, H. pylori PdxA was required for optimal growth in vitro and was shown to be essential for chronic colonization in mice. In addition to having a well-known metabolic role, vitamin B(6) is necessary for the synthesis of glycosylated flagella and for flagellum-based motility in H. pylori. Thus, for the first time, we identify vitamin B(6) biosynthesis enzymes as novel virulence factors in bacteria. Interestingly, pdxA and pdxJ orthologs are present in a number of human pathogens, but not in mammalian cells. We therefore propose that PdxA/J enzymes may represent ideal candidates for therapeutic targets against bacterial pathogens.

• Helicobacter pylori-induced histone modification, associated gene expression in gastric epithelial cells, and its implication in pathogenesis.

  Authors: Song-Ze Ding, Wolfgang Fischer, Maria Kaparakis-Liaskos, George Liechti, D Scott Merrell, Patrick A Grant, Richard L Ferrero, Sheila E Crowe, Rainer Haas, Masanori Hatakeyama, Joanna B Goldberg

  PloS one. 01/2010; 5(4):e9875.

  Histone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the majorHistone modifications are critical in regulating gene expression, cell cycle, cell proliferation, and development. Relatively few studies have investigated whether Helicobacter pylori, the major cause of human gastric diseases, affects histone modification. We therefore investigated the effects of H. pylori infection on histone modifications in a global and promoter-specific manner in gastric epithelial cells. Infection of gastric epithelial cells by wild-type H. pylori induced time- and dose-dependent dephosphorylation of histone H3 at serine 10 (H3 Ser10) and decreased acetylation of H3 lysine 23, but had no effects on seven other specific modifications. Different cag pathogenicity island (PAI)-containing-clinical isolates showed similar abilities to induce H3 Ser10 dephosphorylation. Mutation of cagA, vacA, nonphosphorylateable CagA mutant cagA(EPISA), or disruption of the flagella showed no effects, while deletion of the entire cagPAI restored the H3 Ser10 phosphorylation to control levels. Analysis of 27 cagPAI mutants indicated that the genes that caused H3 Ser10 dephosphorylation were similar to those that were previously found to induce interleukin-8, irrespective of CagA translocation. This effect was independent of ERK or p38 pathways and type I interferon signaling. Additionally, c-Jun and hsp70 gene expression was associated with this histone modification. These results demonstrate that H. pylori alters histone modification and host response via a cagA-, vacA-independent, but cagPAI-dependent mechanisms, which contribute to its persistent infection and pathogenesis.

• Helicobacter pylori Induces MAPK Phosphorylation and AP-1 Activation via a NOD1-Dependent Mechanism.

  Authors: Cody C Allison, Thomas A Kufer, Elisabeth Kremmer, Maria Kaparakis, Richard L Ferrero

  Journal of immunology (Baltimore, Md. : 1950). 12/2009; 183(12):8099-109.

  Helicobacter pylori rapidly activates MAPKs and transcription factors, NF-kappaB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largelyHelicobacter pylori rapidly activates MAPKs and transcription factors, NF-kappaB and AP-1, in gastric epithelial cells following host attachment. Activation of these signal transducers is largely dependent on the cag pathogenicity island (cagPAI)-encoded Type IV Secretion System. H. pylori was shown to translocate peptidoglycan through the Type IV Secretion System, which is recognized by the pathogen recognition molecule, NOD1, thus resulting in NF-kappaB activation. The mechanisms of H. pylori-induced MAPK and AP-1 activation, however, are less well defined and therefore, we assessed the contribution of NOD1 to their activation. For this, we used gastric epithelial cell lines, stably expressing siRNA to either NOD1 or a control gene. In siNOD1-expressing cells stimulated with cagPAI(+) H. pylori, we observed significant reductions in p38 and ERK phosphorylation (p < 0.05), whereas the levels of Jnk phosphorylation remained unchanged. Consistent with a previous report, however, we were able to demonstrate NOD1-dependent Jnk phosphorylation by the invasive pathogen Shigella flexneri, highlighting pathogen-specific host responses to infection. We also show that NOD1 was essential for H. pylori induction of not only NF-kappaB, but also AP-1 activation, implying that NOD1 induces robust proinflammatory responses, in an attempt to rapidly control infection. Pharmacological inhibition of p38 and ERK activity significantly reduced IL-8 production in response to H. pylori, further emphasizing the importance of MAPKs in innate immune responses to the pathogen. Thus, for the first time we have shown the important role for NOD1 in MAPK and AP-1 activation in response to cagPAI(+) H. pylori.

• Bacterial membrane vesicles deliver peptidoglycan to NOD1 in epithelial cells.

  Authors: Maria Kaparakis, Lynne Turnbull, Leticia Ccarneiro, Stephen Firth, Harold A Coleman, Helena C Parkington, Lionel Le Bourhis, Abdulgader Karrar, Jérôme Viala, Johnson Mak, Melanie L Hutton, John K Davies, Peter J Crack, Paul J Hertzog, Dana J Philpott, Stephen E Girardin, Cynthia B Whitchurch, Richard L Ferrero

  Cellular microbiology. 11/2009;

  Summary Gram negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells areSummary Gram negative bacterial peptidoglycan is specifically recognized by the host intracellular sensor NOD1, resulting in the generation of innate immune responses. Although epithelial cells are normally refractory to external stimulation with peptidoglycan, these cells have been shown to respond in a NOD1 dependent manner to Gram negative pathogens that can either invade or secrete factors into host cells. In the present work, we report that Gram negative bacteria can deliver peptidoglycan to cytosolic NOD1 in host cells via a novel mechanism involving outer membrane vesicles (OMVs). We purified OMVs from the Gram negative mucosal pathogens: Helicobacter pylori, Pseudomonasaeruginosa and Neisseria gonorrhoea and demonstrated that these peptidoglycan containing OMVs upregulated NF-kappaB and NOD1 dependent responses in vitro. These OMVs entered epithelial cells through lipid rafts thereby inducing NOD1 dependent responses in vitro. Moreover, OMVs delivered intragastrically to mice induced innate and adaptive immune responses via a NOD1 dependent but TLR independent mechanism. Collectively, our findings identify OMVs as a generalized mechanism whereby Gram-negative bacteria deliver peptidoglycan to cytosolic NOD1. We propose that OMVs released by bacteria in vivo may promote inflammation and pathology in infected hosts.

• Protease Activated Receptor-1 down-regulates the murine inflammatory and humoral response to Helicobacter pylori.

  Authors: Janet L K Wee, Yok-Teng Chionh, Garrett Z Ng, Stacey N Harbour, Cody Allison, Charles N Pagel, Eleanor J Mackie, Hazel M Mitchell, Richard L Ferrero, Philip Sutton

  Gastroenterology. 09/2009;

  BACKGROUND AND AIMS: Helicobacter pylori infection results in a diversity of pathologies, from asymptomatic gastritis to adenocarcinoma. The reason for these diverse outcomes is multifactorial, andBACKGROUND AND AIMS: Helicobacter pylori infection results in a diversity of pathologies, from asymptomatic gastritis to adenocarcinoma. The reason for these diverse outcomes is multifactorial, and includes host factors that regulate severity of Helicobacter-induced gastritis. Protease-Activated Receptors (PAR) are environmental sensors that can detect tissue damage and pathogens. While PAR-2 has proinflammatory activity and PAR-1 can protect the gastric mucosa against chemical damage, neither have previously been examined for their potential roles in regulating Helicobacter pathogenesis. METHODS: PAR-1(-/-), PAR-2(-/-) and wild-type mice were infected with H. pylori for up to two months, then colonization levels determined by colony-forming assay, gastritis by histology and serum antibody levels by ELISA. Responsiveness of primary epithelial cells to PAR-1 activation was assessed by calcium mobilization assay. Primary epithelial cells, macrophages and dendritic cells were co-cultured with H. pylori and NF-kappaB and cytokine secretion determined by ELISA. RESULTS: Two months post-infection, H. pylori levels were significantly reduced in PAR-1(-/-) and increased in PAR-2(-/-) mice. This effect on colonization was inversely correlated with inflammation severity. Infection of PAR-1(-/-) mice induced an increased serum antibody response. Primary epithelial cells were activated by a PAR-1 activating peptide. H. pylori stimulation of primary epithelial cells, but not macrophages or dendritic cells, from PAR-1(-/-) mice induced increased levels of NF-kappaB and the proinflammatory cytokine, MIP-2. PAR-1 also downregulated MIP-2 secretion in response to cag pathogenicity island activity. DISCUSSION: PAR-1 protects the host against severe Helicobacter-induced gastritis. This may be mediated by suppressing the production of proinflammatory cytokines such as MIP-2.

• Bismuth(iii) 5-sulfosalicylate complexes: structure, solubility and activity against Helicobacter pylori.

  Authors: Philip C Andrews, Glen B Deacon, Richard L Ferrero, Peter C Junk, Abdulgader Karrar, Ish Kumar, Jonathan G Maclellan

  Dalton transactions (Cambridge, England : 2003). 08/2009;

  Treatment of 5-sulfosalicylic acid (H(3)Ssal) with BiPh(3) results in the formation of the first dianionic carboxylate-sulfonate bismuth complex, [PhBi(HSsal)H(2)O](infinity), and its ethanolTreatment of 5-sulfosalicylic acid (H(3)Ssal) with BiPh(3) results in the formation of the first dianionic carboxylate-sulfonate bismuth complex, [PhBi(HSsal)H(2)O](infinity), and its ethanol analogue [PhBi(HSsal)EtOH](infinity) (space group P2(1)/c), while Bi(OAc)(3) gives the mixed monoanionic and dianionic complex, {[Bi(HSsal)(H(2)Ssal)(H(2)O)(3)](2).2H(2)O}(infinity) (space group P1[combining macron]). The three complexes are all polymeric in the solid state as determined by single crystal X-ray diffraction, with extended frameworks constructed from dimeric [Bi(HSsal)](2), and , or from [Bi(HSsal)(H(2)Ssal)](2) units, . The heteroleptic bismuth complexes and display remarkable aqueous solubility, 10 and 2.5 mg ml(-1) respectively, resulting in a clear solution of pH 1.5. In contrast, is essentially insoluble in aqueous environments. All three complexes show significant activity against the bacterium Helicobacter pylori of <6.25 mug ml(-1).

• A commensal Helicobacter sp. of the rodent intestinal flora activates TLR2 and NOD1 responses in epithelial cells.

  Authors: Nadia Chaouche-Drider, Maria Kaparakis, Abdulgader Karrar, Maria-Isabel Fernandez, Letitia A M Carneiro, Jérôme Viala, Ivo Gomperts Boneca, Anthony P Moran, Dana J Philpott, Richard L Ferrero

  PLoS ONE. 02/2009; 4(4):e5396.

  Helicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induceHelicobacter spp. represent a proportionately small but significant component of the normal intestinal microflora of animal hosts. Several of these intestinal Helicobacter spp. are known to induce colitis in mouse models, yet the mechanisms by which these bacteria induce intestinal inflammation are poorly understood. To address this question, we performed in vitro co-culture experiments with mouse and human epithelial cell lines stimulated with a selection of Helicobacter spp., including known pathogenic species as well as ones for which the pathogenic potential is less clear. Strikingly, a member of the normal microflora of rodents, Helicobacter muridarum, was found to be a particularly strong inducer of CXC chemokine (Cxcl1/KC, Cxcl2/MIP-2) responses in a murine intestinal epithelial cell line. Time-course studies revealed a biphasic pattern of chemokine responses in these cells, with H. muridarum lipopolysaccharide (LPS) mediating early (24-48 h) responses and live bacteria seeming to provoke later (48-72 h) responses. H. muridarum LPS per se was shown to induce CXC chemokine production in HEK293 cells stably expressing Toll-like receptor 2 (TLR2), but not in those expressing TLR4. In contrast, live H. muridarum bacteria were able to induce NF-kappaB reporter activity and CXC chemokine responses in TLR2-deficient HEK293 and in AGS epithelial cells. These responses were attenuated by transient transfection with a dominant negative construct to NOD1, and by stable expression of NOD1 siRNA, respectively. Thus, the data suggest that both TLR2 and NOD1 may be involved in innate immune sensing of H. muridarum by epithelial cells. This work identifies H. muridarum as a commensal bacterium with pathogenic potential and underscores the potential roles of ill-defined members of the normal flora in the initiation of inflammation in animal hosts. We suggest that H. muridarum may act as a confounding factor in colitis model studies in rodents.

• The beta1 integrin activates JNK independent of CagA, and JNK activation is required for Helicobacter pylori CagA+-induced motility of gastric cancer cells.

  Authors: Jared L Snider, Cody Allison, Bryan H Bellaire, Richard L Ferrero, James A Cardelli

  The Journal of biological chemistry. 06/2008; 283(20):13952-63.

  The Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associatedThe Helicobacter pylori CagA protein is translocated into gastric epithelial cells through a type IV secretion system (TFSS), and published studies suggest CagA is critical for H. pylori-associated carcinogenesis. CagA is thought to be necessary and sufficient to induce the motogenic response observed in response to CagA+ strains, as CagA interacts with proteins involved in adhesion and motility. We report that H. pylori strain 60190 stimulated AGS cell motility through a CagA- and TFSS-dependent mechanism, because strains 60190DeltacagA or 60190DeltacagE (TFSS-defective) did not increase motility. The JNK pathway is critical for H. pylori-dependent cell motility, as inhibition using SP600125 (JNK1/2/3 inhibitor) or a JNK2/3-specific inhibitor blocked motility. JNK mediates H. pylori-induced cell motility by activating paxillin, because JNK inhibition blocked paxillinTyr-118 phosphorylation, and paxillin expression knockdown completely abrogated bacteria-induced motility. Furthermore, JNK and paxillinTyr-118 were activated by 60190DeltacagA but not 60190DeltacagE, demonstrating CagA-independent signaling critical for cell motility. A beta1 integrin-blocking antibody significantly inhibited JNK and paxillinTyr-118 phosphorylation and cell scattering, demonstrating that CagA-independent signaling required for cell motility occurs through beta1. The requirement of both Src and focal adhesion kinase for signaling and motility further suggests the importance of integrin signaling in H. pylori-induced cell motility. Finally, we show that JNK activation occurs independent of known upstream kinases and signaling molecules, including Nod1, Cdc42, Rac1, MKK4, and MKK7, which demonstrates novel signaling leading to JNK activation. We report for the first time that H. pylori mediates CagA-independent signaling that promotes cell motility through the beta1 integrin pathway.

• NF-kappaB activation during acute Helicobacter pylori infection in mice.

  Authors: Richard L Ferrero, Patrick Avé, Delphine Ndiaye, Jean-Christophe Bambou, Michel R Huerre, Dana J Philpott, Sylvie Mémet

  Infection and immunity. 03/2008; 76(2):551-61.

  Nuclear factor kappaB (NF-kappaB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pyloriNuclear factor kappaB (NF-kappaB) plays a key regulatory role in host cell responses to Helicobacter pylori infection in humans. Although mice are routinely used as a model to study H. pylori pathogenesis, the role of NF-kappaB in murine cell responses to helicobacters has not been studied in detail. We thus investigated the abilities of different Helicobacter isolates to induce NF-kappaB-dependent responses in murine gastric epithelial cells (GECs) and in transgenic mice harboring an NF-kappaB-responsive lacZ reporter gene. H. pylori and Helicobacter felis strains up-regulated the synthesis in mouse GECs of the NF-kappaB-dependent chemokines KC (CXCL1) and MIP-2 (CXCL2). These responses were cag pathogenicity island (cagPAI) independent and could be abolished by pretreatment with a pharmacological inhibitor of NF-kappaB. Consistent with the in vitro data, experimental Helicobacter infection of transgenic mice resulted in increased numbers of GECs with nuclear beta-galactosidase activity, which is indicative of specific NF-kappaB activation. The numbers of beta-galactosidase-positive cells in mice were significantly increased at day 1 postinoculation with wild-type H. pylori strains harboring or not harboring a functional cagPAI, compared to naive animals (P = 0.007 and P = 0.04, respectively). Strikingly, however, no differences were observed in the levels of gastric NF-kappaB activation at day 1 postinoculation with H. felis or at day 30 or 135 postinoculation with H. pylori. This work demonstrates for the first time the induction of NF-kappaB activation within gastric mucosal cells during acute H. pylori infection. Furthermore, the data suggest that helicobacters may be able to regulate NF-kappaB signaling during chronic infection.

• Nod1-mediated innate immune recognition of peptidoglycan contributes to the onset of adaptive immunity.

  Authors: Jörg H Fritz, Lionel Le Bourhis, Gernot Sellge, Joao Gamelas Magalhaes, Hafida Fsihi, Thomas A Kufer, Cathy Collins, Jérôme Viala, Richard L Ferrero, Stephen E Girardin, Dana J Philpott

  Immunity. 05/2007; 26(4):445-59.

  Recent evidence has suggested that signals other than those from Toll-like receptors (TLRs) could contribute to the elicitation of antigen-specific immunity. Therefore, we examined the role of theRecent evidence has suggested that signals other than those from Toll-like receptors (TLRs) could contribute to the elicitation of antigen-specific immunity. Therefore, we examined the role of the Nod-like receptor (NLR) family member, Nod1, in the generation of adaptive immune responses. Our findings show that innate immune sensing of peptidoglycan by Nod1 is key for priming antigen-specific T cell immunity and subsequent antibody responses in vivo. Nod1 stimulation alone was sufficient to drive antigen-specific immunity with a predominant Th2 polarization profile. In conjunction with TLR stimulation, however, Nod1 triggering was required to instruct the onset of Th1 and Th2 as well as Th17 immune pathways. Cells outside of the hematopoietic lineage provided the early signals necessary to orchestrate the development of Nod1-dependent immune responses. These findings highlight Nod1 as a key innate immune trigger in the local tissue microenvironment that drives the development of adaptive immunity.

• Nod-like proteins in immunity, inflammation and disease.

  Authors: Jörg H Fritz, Richard L Ferrero, Dana J Philpott, Stephen E Girardin

  Nature immunology. 01/2007; 7(12):1250-7.

  The intracellular Nod-like proteins or receptors are a family of sensors of intracellularly encountered microbial motifs and 'danger signals' that have emerged as being critical components of theThe intracellular Nod-like proteins or receptors are a family of sensors of intracellularly encountered microbial motifs and 'danger signals' that have emerged as being critical components of the innate immune responses and of inflammation in mammals. Several Nod-like receptors, including Nod1, Nod2, NALP3, Ipaf and Naip, are strongly associated with host responses to intracellular invasion by bacteria or the intracellular presence of specific bacterial products. An additional key function of Nod-like receptors is in inflammatory conditions, which has been emphasized by the identification of several different mutations in the genes encoding Nod1, Nod2 and NALP3 that are associated with susceptibility to inflammatory disorders. Those and other issues related to the Nod-like receptor family are discussed here.