scholarly journals Development of an Interleukin-12-Deficient Mouse Model That Is Permissive for Colonization by a Motile KE26695 Strain of Helicobacter pylori

2003 ◽  
Vol 71 (5) ◽  
pp. 2534-2541 ◽  
Author(s):  
Paul S. Hoffman ◽  
Neeraj Vats ◽  
Donna Hutchison ◽  
Jared Butler ◽  
Kenneth Chisholm ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Immune Modulation ◽  
Limit Of Detection ◽  
Genomic Research ◽  
Interleukin 12 ◽  
Deficient Mouse ◽  
Loss Of Function ◽  
Double Knockout ◽  
H Pylori ◽  
Deficient Mice

ABSTRACT The identification of genes associated with colonization and persistence of Helicobacter pylori in the gastric mucosa has been limited by the lack of robust animal models that support infection by strains whose genomes have been completely sequenced. Here we report that an interleukin-12 (IL-12)-deficient mouse (IL-12−/− p40 subunit knockout in C57BL/6 mouse) is permissive for infection by a motile variant (KE88-3887) of The Institute For Genomic Research-sequenced strain (KE26695) of H. pylori. The IL-12-deficient mouse was also more permissive for colonization by the mouse-colonizing Sydney 1 strain of H. pylori than were wild-type C57BL/6 mice. Differences in colonization efficiency were demonstrated by mouse challenge with SS1 strains containing loss-of-function mutations in two genes (hspR and hrcA), whose products negatively regulate several heat shock genes. At 5 weeks postinfection, double-knockout mutants (SS1 hspR hrcA) efficiently colonized IL-12-deficient mice (5 of 5 animals compared to 4 of 10 for C57BL6 mice) and bacterial counts were higher in stomachs of IL-12-deficient mice (106 versus 105 CFU/g of stomach, respectively). IL-12-deficient mice were efficiently colonized by KE88-3887 (29 of 30), but not by nonmotile KE26695, and bacterial numbers (104 to 105 CFU/g of stomach) were unchanged over an 8-week period postinfection. In contrast, C57BL/6 mice were inefficiently colonized by KE88-3887 (8 of 20 animals with bacterial loads at the limit of detection, ∼103 CFU/g), and infection did not persist much beyond 5 weeks. Cytokine responses (tumor necrosis factor alpha and gamma interferon), pathology, and antral-predominant infection were indistinguishable between IL-12-deficient and C57BL/6 mice. The increased permissiveness of the IL-12-deficient mouse for infection with H. pylori should facilitate whole-genome-based strategies to study genes associated with virulence and immune modulation.

scholarly journals A Helicobacter pylori TolC Efflux Pump Confers Resistance to Metronidazole

2005 ◽  
Vol 49 (4) ◽  
pp. 1477-1482 ◽  
Author(s):  
Karin van Amsterdam ◽  
Aldert Bart ◽  
Arie van der Ende
Keyword(s):  
Helicobacter Pylori ◽  
Efflux Pump ◽  
Sodium Deoxycholate ◽  
Loss Of Function ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Active Efflux ◽  
Knockout Mutants ◽  
H Pylori ◽  
Susceptibility Profiles

ABSTRACT In Helicobacter pylori, the contribution of efflux proteins to antibiotic resistance is not well established. As translocases that act in parallel may have overlapping substrate specificities, the loss of function of one such translocase may be compensated for by that of another translocase with no effect on susceptibilities to antibiotics. The genome of H. pylori 26695 was assessed for the presence of putative translocases and outer membrane efflux or TolC-like proteins which could interact to form efflux systems involved in drug resistance. Twenty-seven translocases were identified, of which HP1184 was the sole representative of the multidrug and toxic compound extrusion family of translocases and which could thus have a unique substrate specificity. In addition, four TolC-like proteins (HP0605, HP0971, HP1327, and HP1489) were identified. Thus, it is feasible that inactivation of a TolC-like protein would affect the functions of multiple translocases. We aimed to determine whether efflux systems contribute to antimicrobial susceptibility by evaluation of the susceptibility profiles of an HP1184-knockout mutant, four mutants in which one of the four TolC homologs was inactivated, as well as a mutant in which both HP0605 and HP0971 were inactivated. The HP1184- and HP1489-knockout mutants both showed increased susceptibilities to ethidium bromide, while the HP0605-knockout mutant exhibited increased susceptibilities to novobiocin and sodium deoxycholate. The HP0605 and HP0971 double-knockout mutant was also more susceptible to metronidazole, in addition to being susceptible to novobiocin and sodium deoxycholate. Thus, active efflux is an eminent means of resistance to antimicrobials in H. pylori and resembles the situation in other bacteria.

scholarly journals Vaccine-Induced Protection against Helicobacter pylori in Mice Lacking Both Antibodies and Interleukin-4

2003 ◽  
Vol 71 (6) ◽  
pp. 3628-3633 ◽  
Author(s):  
Christine A. Garhart ◽  
John G. Nedrud ◽  
Frederick P. Heinzel ◽  
Norma E. Sigmund ◽  
Steven J. Czinn
Keyword(s):  
Helicobacter Pylori ◽  
B Cell ◽  
Knockout Mice ◽  
Cellular Responses ◽  
Interleukin 4 ◽  
Wild Type ◽  
Double Knockout ◽  
Th2 Response ◽  
H Pylori ◽  
Deficient Mice

ABSTRACT To test the hypothesis that a Th2 response to Helicobacter pylori is necessary for protection and to address the possibility that humoral and Th2 cellular responses may compensate for each other, we generated mice deficient in both interleukin-4 (IL-4) and antibodies. The immunized double-knockout mice were protected from H. pylori challenge, as were the parental strains and wild-type C57BL/6 mice. Neutralization of IL-4 in B-cell-deficient mice did not prevent protection. Immunized IL-5-deficient mice were also protected. Thus, IL-4 and IL-5 are not essential for protection.

scholarly journals Systems Modeling of the Role of Interleukin-21 in the Maintenance of Effector CD4+ T Cell Responses during Chronic Helicobacter pylori Infection

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Adria Carbo ◽  
Danyvid Olivares-Villagómez ◽  
Raquel Hontecillas ◽  
Josep Bassaganya-Riera ◽  
Rupesh Chaturvedi ◽  
...  
Keyword(s):  
Gastric Cancer ◽  
Helicobacter Pylori ◽  
T Cell ◽  
Wild Type ◽  
Content Type ◽  
Interleukin 21 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACTThe development of gastritis duringHelicobacter pyloriinfection is dependent on an activated adaptive immune response orchestrated by T helper (Th) cells. However, the relative contributions of the Th1 and Th17 subsets to gastritis and control of infection are still under investigation. To investigate the role of interleukin-21 (IL-21) in the gastric mucosa duringH. pyloriinfection, we combined mathematical modeling of CD4+T cell differentiation within vivomechanistic studies. We infected IL-21-deficient and wild-type mice withH. pyloristrain SS1 and assessed colonization, gastric inflammation, cellular infiltration, and cytokine profiles. ChronicallyH. pylori-infected IL-21-deficient mice had higherH. pyloricolonization, significantly less gastritis, and reduced expression of proinflammatory cytokines and chemokines compared to these parameters in infected wild-type littermates. Thesein vivodata were used to calibrate anH. pyloriinfection-dependent, CD4+T cell-specific computational model, which then described the mechanism by which IL-21 activates the production of interferon gamma (IFN-γ) and IL-17 during chronicH. pyloriinfection. The model predicted activated expression of T-bet and RORγt and the phosphorylation of STAT3 and STAT1 and suggested a potential role of IL-21 in the modulation of IL-10. Driven by our modeling-derived predictions, we found reduced levels of CD4+splenocyte-specifictbx21androrcexpression, reduced phosphorylation of STAT1 and STAT3, and an increase in CD4+T cell-specific IL-10 expression inH. pylori-infected IL-21-deficient mice. Our results indicate that IL-21 regulates Th1 and Th17 effector responses during chronicH. pyloriinfection in a STAT1- and STAT3-dependent manner, therefore playing a major role controllingH. pyloriinfection and gastritis.IMPORTANCEHelicobacter pyloriis the dominant member of the gastric microbiota in more than 50% of the world’s population.H. pyloricolonization has been implicated in gastritis and gastric cancer, as infection withH. pyloriis the single most common risk factor for gastric cancer. Current data suggest that, in addition to bacterial virulence factors, the magnitude and types of immune responses influence the outcome of colonization and chronic infection. This study uses a combined computational and experimental approach to investigate how IL-21, a proinflammatory T cell-derived cytokine, maintains the chronic proinflammatory T cell immune response driving chronic gastritis duringH. pyloriinfection. This research will also provide insight into a myriad of other infectious and immune disorders in which IL-21 is increasingly recognized to play a central role. The use of IL-21-related therapies may provide treatment options for individuals chronically colonized withH. pylorias an alternative to aggressive antibiotics.

scholarly journals Deficiencies of Myeloid Differentiation Factor 88, Toll-Like Receptor 2 (TLR2), or TLR4 Produce Specific Defects in Macrophage Cytokine Secretion Induced by Helicobacter pylori

2007 ◽  
Vol 75 (5) ◽  
pp. 2408-2414 ◽  
Author(s):  
Marygorret Obonyo ◽  
Mojgan Sabet ◽  
Sheri P. Cole ◽  
Joerg Ebmeyer ◽  
Satoshi Uematsu ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Myeloid Differentiation ◽  
Interleukin 12 ◽  
Proinflammatory Response ◽  
Differentiation Factor ◽  
Myeloid Differentiation Factor 88 ◽  
H Pylori ◽  
Molecular Patterns ◽  
Myd88 Signaling ◽  
Myeloid Differentiation Factor

ABSTRACT Helicobacter pylori is a gram-negative microaerophilic bacterium that colonizes the gastric mucosa, leading to disease conditions ranging from gastritis to cancer. Toll-like receptors (TLRs) play a central role in innate immunity by their recognition of conserved molecular patterns on bacteria, fungi, and viruses. Upon recognition of microbial components, these TLRs associate with several adaptor molecules, including myeloid differentiation factor 88 (MyD88). To investigate the contribution of the innate immune system to H. pylori infection, bone marrow-derived macrophages from mice deficient in TLR2, TLR4, TLR9, and MyD88 were infected with H. pylori SS1 and SD4 for 24 or 48 h. We demonstrate that MyD88 was essential for H. pylori induction of all cytokines investigated except alpha interferon (IFN-α). The secretion of IFN-α was substantially increased from cells deficient in MyD88. H. pylori induced interleukin-12 (IL-12) and IL-10 through TLR4/MyD88 signaling. In addition, H. pylori induced less IL-6 and IL-1β in TLR2-deleted macrophages, suggesting that the MyD88 pathway activated by TLR2 stimulation is responsible for H. pylori induction of the host proinflammatory response (IL-6 and IL-1β). These observations are important in light of a recent report on IL-6 and IL-1β playing a role in the development of H. pylori-related gastric cancer. In conclusion, our study demonstrates that H. pylori activates TLR2 and TLR4, leading to the secretion of distinct cytokines by macrophages.

scholarly journals Microarray-Based Detection and Clinical Evaluation for Helicobacter pylori Resistance to Clarithromycin or Levofloxacin and the Genotype of CYP2C19 in 1083 Patients

2018 ◽  
Vol 2018 ◽  
pp. 1-12 ◽  
Author(s):  
Yi Song ◽  
Fengna Dou ◽  
Zhe Zhou ◽  
Ningmin Yang ◽  
Jing Zhong ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Antibiotic Resistance ◽  
Limit Of Detection ◽  
Eradication Therapy ◽  
Gastric Biopsy ◽  
Individual Therapy ◽  
23S Rrna ◽  
Antibiotic Administration ◽  
Cyp2c19 Polymorphism ◽  
H Pylori

Background. Helicobacter pylori (H. pylori) is one of the most frequent and persistent bacterial infections that affect nearly half of the world’s population. Antibiotic resistance is a constantly evolving process and local surveillance of antibiotic resistance is warranted to guide clinicians in their choice of therapy. The aim of this study was to establish a microarray-based detection to identify H. pylori infection, clarithromycin and levofloxacin susceptibility, and CYP2C19 genetic polymorphism and guide to potential choice of proton pump inhibitor (PPI), antibiotic administration for tailored H. pylori eradication therapy. Methods. By analyzing the sequence of human genomic CYP2C19⁎2 and CYP2C19⁎3 and mutations within the 23S rRNA and gyrA gene regions conferring clarithromycin and levofloxacin resistance, respectively, we developed a microarray for individual therapy detection of H. pylori infection. Plasmids were established as positive or limit of detection (LOD) reference materials. The specificity and sensitivity of the microarray had been performed. And a total of 1083 gastric biopsy samples were tested and the Kappa value had been calculated between the array and Sanger sequencing. We also analyzed the resistance to clarithromycin and levofloxacin in China, as well as the CYP2C19 polymorphisms. Results. The LOD of detecting H. pylori was 103 CFU/mL and human genome DNA was 2 ng/μL. The detection results of 1083 gastric biopsy samples showed that 691 (63.80%) were H. pylori positive, of which 266 (38.49%) were resistant to clarithromycin, 192 (27.79%) were resistant to levofloxacin, and 61 (8.83%) were resistant to both of them. For the type of CYP2C19 polymorphism, 412 (38.04%) were homozygous fast type (HomEM), 574 (53%) were heterozygous EM (HetEM), and 97 (8.96%) were poor metabolizer (PM). Conclusions. The proposed microarray-based detection has high specificity, sensitivity, and reproducibility for detecting the resistance of clarithromycin or levofloxacin as well as CYP2C19 polymorphism, which may help to improve the clinical eradication rate of H. pylori.

scholarly journals Enzymes Associated with Reductive Activation and Action of Nitazoxanide, Nitrofurans, and Metronidazole in Helicobacter pylori

2002 ◽  
Vol 46 (7) ◽  
pp. 2116-2123 ◽  
Author(s):  
Gary Sisson ◽  
Avery Goodwin ◽  
Ausra Raudonikiene ◽  
Nicky J. Hughes ◽  
Asish K. Mukhopadhyay ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Reductase Activity ◽  
Loss Of Function ◽  
Reductive Activation ◽  
E Coli ◽  
Redox Active ◽  
Bactericidal Effects ◽  
Pyruvate Oxidoreductase ◽  
Essential Enzyme ◽  
H Pylori

ABSTRACT Nitazoxanide (NTZ) is a redox-active nitrothiazolyl-salicylamide prodrug that kills Helicobacter pylori and also many anaerobic bacterial, protozoan, and helminthic species. Here we describe development and use of a spectrophotometric assay, based on nitroreduction of NTZ at 412 nm, to identify H. pylori enzymes responsible for its activation and mode of action. Three enzymes that reduce NTZ were identified: two related NADPH nitroreductases, which also mediate susceptibility to metronidazole (MTZ) (RdxA and FrxA), and pyruvate oxidoreductase (POR). Recombinant His-tagged RdxA, FrxA, and POR, overexpressed in nitroreductase-deficient Escherichia coli, each rapidly reduced NTZ, whereas only FrxA and to a lesser extent POR reduced nitrofuran substrates (furazolidone, nitrofurantoin, and nitrofurazone). POR exhibited no MTZ reductase activity either in extracts of H. pylori or following overexpression in E. coli; RdxA exhibited no nitrofuran reductase activity, and FrxA exhibited no MTZ reductase activity. Analysis of mutation to rifampin resistance (Rifr) indicated that NTZ was not mutagenic and that nitrofurans were only weakly mutagenic. Alkaline gel DNA electrophoresis indicated that none of these prodrugs caused DNA breakage. In contrast, MTZ caused DNA damage and was strongly mutagenic. We conclude that POR, an essential enzyme, is responsible for most or all of the bactericidal effects of NTZ against H. pylori. While loss-of-function mutations in rdxA and frxA produce a Mtzr phenotype, they do not contribute much to the innate susceptibility of H. pylori to NTZ or nitrofurans.

scholarly journals Galectin-3 Plays an Important Role in Innate Immunity to Gastric Infection by Helicobacter pylori

2016 ◽  
Vol 84 (4) ◽  
pp. 1184-1193 ◽  
Author(s):  
Ah-Mee Park ◽  
Satoru Hagiwara ◽  
Daniel K. Hsu ◽  
Fu-Tong Liu ◽  
Osamu Yoshie
Keyword(s):  
Innate Immunity ◽  
Helicobacter Pylori ◽  
Bacterial Cells ◽  
Mucus Layer ◽  
Iodide Uptake ◽  
Gastric Glands ◽  
Content Type ◽  
Galectin 3 ◽  
H Pylori ◽  
Deficient Mice

We studied the role of galectin-3 (Gal3) in gastric infection byHelicobacter pylori. We first demonstrated that Gal3 was selectively expressed by gastric surface epithelial cells and abundantly secreted into the surface mucus layer. We next inoculatedH. pyloriSydney strain 1 into wild-type (WT) and Gal3-deficient mice using a stomach tube. At 2 weeks postinoculation, the bacterial cells were mostly trapped within the surface mucus layer in WT mice. In sharp contrast, they infiltrated deep into the gastric glands in Gal3-deficient mice. Bacterial loads in the gastric tissues were also much higher in Gal3-deficient mice than in WT mice. At 6 months postinoculation,H. pylorihad successfully colonized within the gastric glands of both WT and Gal3-deficient mice, although the bacterial loads were still higher in the latter. Furthermore, large lymphoid clusters mostly consisting of B cells were frequently observed in the gastric submucosa of Gal3-deficient mice.In vitro, peritoneal macrophages from Gal3-deficient mice were inefficient in killing engulfedH. pylori. Furthermore, recombinant Gal3 not only induced rapid aggregation ofH. pyloribut also exerted a potent bactericidal effect onH. pylorias revealed by propidium iodide uptake and a morphological shift from spiral to coccoid form. However, a minor fraction of bacterial cells, probably transient phase variants of Gal3-binding sugar moieties, escaped killing by Gal3. Collectively, our data demonstrate that Gal3 plays an important role in innate immunity to infection and colonization ofH. pylori.

scholarly journals Possible Correlates of Long-Term Protection against Helicobacter pylori following Systemic or Combinations of Mucosal and Systemic Immunizations

2007 ◽  
Vol 75 (7) ◽  
pp. 3462-3469 ◽  
Author(s):  
Jennifer M. Taylor ◽  
Melanie E. Ziman ◽  
Julie Fong ◽  
Jay V. Solnick ◽  
Michael Vajdy
Keyword(s):  
Helicobacter Pylori ◽  
Protective Immunity ◽  
Interleukin 12 ◽  
Effective Vaccine ◽  
Correlates Of Protection ◽  
Oral Group ◽  
Efficient Route ◽  
H Pylori

ABSTRACT The ability to induce long-term immunity to Helicobacter pylori is necessary for an effective vaccine. This study was designed to establish the most efficient route(s) (systemic, mucosal, or a combination) of immunization for induction of long-term immunity and to define correlates of protection. Mice were immunized orally alone (oral group), intramuscularly (i.m.) alone (i.m. group), orally followed by i.m. (oral/i.m. group), or i.m. followed by orally (i.m./oral group). Long-term protective immunity to oral H. pylori challenge was observed 3 months after immunization through the i.m. or oral/i.m. route. Protection correlated with an increase in H. pylori-specific interleukin-12 and both immunoglobulin G1 (IgG1) and IgG2a serum titers following challenge. Mice that were not protected (oral or i.m./oral) had increased levels of IgA in both sera and Peyer's patches. This study demonstrates the ability to induce long-term immunity against H. pylori, provides correlates of protection, and illustrates the crucial role of the immunization route(s).

scholarly journals Intact Gram-Negative Helicobacter pylori, Helicobacter felis, and Helicobacter hepaticus Bacteria Activate Innate Immunity via Toll-Like Receptor 2 but Not Toll-Like Receptor 4

2004 ◽  
Vol 72 (11) ◽  
pp. 6446-6454 ◽  
Author(s):  
Leisa Mandell ◽  
Anthony P. Moran ◽  
Andrew Cocchiarella ◽  
JeanMarie Houghton ◽  
Nancy Taylor ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Cytokine Response ◽  
Innate Immune ◽  
Cytokine Secretion ◽  
Helicobacter Hepaticus ◽  
Toll Like Receptor ◽  
Helicobacter Felis ◽  
Toll Like Receptor 2 ◽  
H Pylori ◽  
Deficient Mice

ABSTRACT Molecular and genetic studies have demonstrated that members of the Toll-like receptor (TLR) family are critical innate immune receptors. TLRs are recognition receptors for a diverse group of microbial ligands including bacteria, fungi, and viruses. This study demonstrates that distinct TLRs are responsible for the recognition of Helicobacter lipopolysaccharide (LPS) versus intact Helicobacter bacteria. We show that the cytokine-inducing activity of Helicobacter LPS was mediated by TLR4; i.e., TLR4-deficient macrophages were unresponsive to Helicobacter pylori LPS. Surprisingly, the cytokine response to whole Helicobacter bacteria (H. pylori, H. hepaticus, and H. felis) was mediated not by TLR4 but rather by TLR2. Studies of HEK293 transfectants revealed that expression of human TLR2 was sufficient to confer responsiveness to intact Helicobacter bacteria, but TLR4 transfection was not sufficient. Our studies further suggest that cag pathogenicity island genes may modulate the TLR2 agonist activity of H. pylori as cagA + bacteria were more active on a per-cell basis compared to cagA mutant bacteria for interleukin-8 (IL-8) cytokine secretion. Consistent with the transfection studies, analysis of knockout mice demonstrated that TLR2 was required for the cytokine response to intact Helicobacter bacteria. Macrophages from both wild-type and TLR4-deficient mice produced a robust cytokine secretion response (IL-6 and MCP-1) when stimulated with intact Helicobacter bacteria. In contrast, macrophages from TLR2-deficient mice were profoundly unresponsive to intact Helicobacter stimulation, failing to secrete cytokines even at high (100:1) bacterium-to-macrophage ratios. Our studies suggest that TLR2 may be the dominant innate immune receptor for recognition of gastrointestinal Helicobacter species.

scholarly journals GroEL of Lactobacillus johnsonii La1 (NCC 533) Is Cell Surface Associated: Potential Role in Interactions with the Host and the Gastric Pathogen Helicobacter pylori

2006 ◽  
Vol 74 (1) ◽  
pp. 425-434 ◽  
Author(s):  
Gabriela E. Bergonzelli ◽  
Dominique Granato ◽  
Raymond D. Pridmore ◽  
Laure F. Marvin-Guy ◽  
Dominique Donnicola ◽  
...  
Keyword(s):  
Helicobacter Pylori ◽  
Epithelial Cells ◽  
Immune Modulation ◽  
Enzyme Linked Immunosorbent Assay ◽  
Lactobacillus Johnsonii ◽  
Gram Positive ◽  
Groel Protein ◽  
Intestinal Pathogens ◽  
Gastric Pathogen ◽  
H Pylori

ABSTRACT Heat shock proteins of the GroEL or Hsp60 class are highly conserved proteins essential to all living organisms. Even though GroEL proteins are classically considered intracellular proteins, they have been found at the surface of several mucosal pathogens and have been implicated in cell attachment and immune modulation. The purpose of the present study was to investigate the GroEL protein of a gram-positive probiotic bacterium, Lactobacillus johnsonii La1 (NCC 533). Its presence at the bacterial surface was demonstrated using a whole-cell enzyme-linked immunosorbent assay and could be detected in bacterial spent culture medium by immunoblotting. To assess binding of La1 GroEL to mucins and intestinal epithelial cells, the La1 GroEL protein was expressed in Escherichia coli. We report here that La1 recombinant GroEL (rGroEL) binds to mucins and epithelial cells and that this binding is pH dependent. Immunomodulation studies showed that La1 rGroEL stimulates interleukin-8 secretion in macrophages and HT29 cells in a CD14-dependent mechanism. This property is common to rGroEL from other gram-positive bacteria but not to the rGroEL of the gastric pathogen Helicobacter pylori. In addition, La1 rGroEL mediates the aggregation of H. pylori but not that of other intestinal pathogens. Our in vitro results suggest that GroEL proteins from La1 and other lactic acid bacteria might play a role in gastrointestinal homeostasis due to their ability to bind to components of the gastrointestinal mucosa and to aggregate H. pylori.

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