This Month in Gastroenterology

Abstract

IL-23 Receptor Variations and Susceptibility for Inflammatory Bowel DiseaseStudies of genetic association may help to unravel the pathogenesis of multifactorial disorders with putative genetic predisposition, such as inflammatory bowel disease (IBD). Previous studies have already identified a number of candidate IBD-associated gene polymorphisms. However, the currently identified IBD susceptibility genes explain only a fraction of the genetic predisposition, and larger and confirmatory studies are needed for several candidate genes. A genome-wide scan study comparing North American Crohn’s disease patients with controls, published in Science last year, reported an association with the interleukin-23 receptor (IL23R) gene. Moreover, the paper included an internal replication studies that confirmed IL23R associations with both Crohn’s disease and ulcerative colitis.In this issue of Gastroenterology, Tremelling et al report the results of a study that investigated the association between IL23R and IBD. DNA samples of a total of 2877 IBD patients (two thirds Crohn’s disease and one third ulcerative colitis) in the United Kingdom were compared with 1345 control samples. Genotyping for 8 IL23R markers was performed, and the association with CARD15 mutations was also analyzed.Clear statistically significant associations were found between variants in the IL23R gene and IBD (Figure 1). The strongest association was found between Crohn’s disease and the IL23R coding variant Arg381Gln, with a significantly lower odds ratio of this protective allele in Crohn’s disease (odds ratio 0.38; 95% confidence interval 0.29–0.50). Analysis accounting for Arg381Gln demonstrated that other loci within IL23R also influenced Crohn’s disease susceptibility. No association of IL23R and CARD15 mutations were found. Similarly, several single nucleotide polymorphisms of the IL23R gene were significantly associated with ulcerative colitis, but the overall susceptibility impact was more modest in ulcerative colitis than in Crohn’s disease. The odds ratio for ulcerative colitis associated with Arg381Gln was 0.73, with a 95% confidence interval of 0.55–0.96. The protective Arg381Gln allele is relatively uncommon, with a low prevalence of 6.2% in the control population, and 2.5% and 4.6% in Crohn’s disease and ulcerative colitis, respectively; the more common G allele is a disease risk allele. Both for Crohn’s disease and ulcerative colitis, IL23R polymorphisms were not significantly linked to disease phenotype subgroups (eg, ileal versus colonic involvement in Crohn’s disease) was found.This study confirms the association between IL23R and IBD, which is also found outside of North America. With an association with both ulcerative colitis and Crohn’s disease, and not with disease phenotypes, it seems that IL23R variants may exert a generic effect on susceptibility to chronic intestinal inflammation, although the effect size is biggest for Crohn’s disease. The findings further support an important role for IL-23, the ligand of IL23R , as a key player in the immune system and in chronic IBD. IL-23 or its receptor may become important targets for the development of novel therapeutic strategies for IBD.See page 1657.Monoclonal Antibodies to α4-Integrin in the Treatment of Active Crohn’s DiseaseThe development of biological agents allows to target crucial steps in the inflammatory cascade. The anti–tumor necrosis factor agent, infliximab, has profoundly altered the management of Crohn’s disease. Despite this progress, major unmet needs persist in the medical treatment of inflammatory bowel disease, and the success of this agent has stimulated the exploration of several other anti-inflammatory targets. Natalizumab, a recombinant humanized immunoglobulin G4 monoclonal antibody that binds to α4-integrin, is a member of a group of molecules known as selective adhesion molecule inhibitors. The α4-integrins are glycoproteins expressed on the surface of leukocytes, which interact with endothelial vascular cell adhesion molecule-1, expressed on the vascular endothelium, to allow leukocyte adhesion and subsequent migration into surrounding tissue. In Crohn’s disease, recruitment of leukocytes into inflamed areas of the gut is a key event, and blocking the adhesion molecules that allow this process to happen is expected to decrease chronic intestinal inflammation.Previously, the ENACT-1 and -2 studies evaluated the use of natalizumab in the induction and maintenance of remission in patients with moderately to severely active Crohn’s disease. Although the ENACT-1 trial did not meet the primary endpoint of higher response rates after 3 infusions of natalizumab, efficacy of natalizumab as maintenance therapy for up to 56 weeks in responders to induction therapy was demonstrated in the ENACT-2 trial.In this issue of Gastroenterology, Targan et al report the results of the Efficacy of Natalizumab in Crohn’s disease Response and Remission (ENCORE) trial, which was designed to confirm the hypothesis that natalizumab is effective as induction therapy in patients with active Crohn’s disease. In this multicenter, randomized, double-blind, placebo-controlled, parallel-group study, a total of 509 patients with moderate to severely active Crohn’s disease (Crohn’s Disease Activity Index [CDA] scores ≥220 to ≤450) and active inflammation (C-reactive protein [CRP] >2.87 mg/L) were recruited from 114 centers. Patients were randomized to receive 3 intravenous infusions of placebo or natalizumab 300 mg at weeks 0, 4, and 8, and they were followed up through week 12 for safety and efficacy assessments.Response rate, defined as a reduction of ≥70 points in the CDAI score at week 12 from week 0, occurred in 60% of natalizumab-treated patients compared with 44% of placebo-treated patients (P < .001). Remission, defined as a CDAI score <150 points, was obtained in 38% of patients after natalizumab and 25% after placebo (P = .001). Significant differences in response rates occurred as early as week 4 and were sustained throughout the 12-week evaluation period (Figure 2A and B).Figure 2(A) Proportion of patients with a clinical response at various time points. (B) Proportion of patients in remission at various time points.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The beneficial effects of natalizumab on the CDAI were associated with significant greater decreases in CRP plasma levels and significantly greater improvements in quality of life scores. These results confirm that natalizumab effectively induces response and remission in patients with moderately to severely active Crohn’s disease and objective evidence of inflammation. Tolerance of natalizumab in the present study was good, with adverse events and serious adverse events occurring equally frequently in both study arms. Rates of antibody formation and of infusion reactions were low. Antibodies to natalizumab occurred in <10% of patients and were not associated with decreased response rates, but with a higher incidence of acute infusion reactions.These observations suggest that natalizumab is effective as an induction therapy for Crohn’s disease. The agent is attractive as it derives its anti-inflammatory activity from a mode of action that differs from that of other available therapies. Although no serious events occurred in this trial, in previous studies the use of natalizumab has been associated with development of progressive multifocal leukoencephalopathy and other opportunistic infections. Clinicians and patients therefore need to carefully consider the risk/benefit ratio of this agent and available alternative therapies that can be used in the treatment of active Crohn’s disease.See page 1672.Mechanism for Helicobacter pylori-induced T-cell Suppression in Persistent InfectionHelicobacter pylori is a common colonizer of human gastric mucosa, and how it evades immune surveillance is not clear. The H pylori virulence factors cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) have been suggested as mediators to inhibit lymphocyte proliferation, but this is inconsistent in the literature, and may induce inflammation rather than reduce it. An unknown, low-molecular-weight secreted product from H pylori, separate from CagA and VacA factors, was recently shown to inhibit T-cell proliferation and induce a G1 cell-cycle phase arrest. The identity of the secreted product was not known until the current study was performed.In the study by Schmees et al, low-molecular-weight fractions from the H pylori G27 strain was purified by size exclusion chromatography and SDS-PAGE, and the secreted protein γ-glutamyl transpeptidase (GGT) was identified from supernatant fractions that inhibited lymphocyte proliferation, and had activity in a photometric GGT activity assay. An isogenic GGT knock-out strain of H pylori G27 that had no effect on survival or growth of the bacteria revealed complete abrogation of GGT activity and loss of the ability to inhibit lymphocyte proliferations (Figure 3), and both activities appear to be mediated by serine residues 451 and 452 of the H pylori GGT as determined by site-directed mutagenesis. Additionally, purified recombinant H pylori GGT demonstrated high GGT activity and efficiently inhibited lymphocyte proliferation in a dose-dependent manner, and the GGT inhibitor acivicin reversed these effects. Equine GGT, although possessing GGT enzymatic activity, failed to inhibit lymphocyte proliferation, presumably owing to possessing only 22% homology with H pylori GGT. Wild-type or recombinant H pylori GGT, but not mutant strains, stimulated a G1 cell-cycle phase arrest, but there was no effect on IL-2 or γ-IFN secretion by lymphocytes, nor any change in apoptosis. In assessing signaling mechanisms that could reduce lymphocyte proliferation, both wild-type and recombinant H pylori GGT, but not mutant strains, reduced the downstream mediators of Ras-dependent signaling, c-Raf and c-Myc, and increased levels of the Cdk-inhibitor p27, while preserving PI3 kinase signaling mediators.Figure 3GGT-deficient H pylori mutants fail to inhibit lymphocyte proliferation. (A) Cell proliferation of stimulated peripheral blood mononuclear cells in the presence or absence of indicated H pylori supernatants was determined by 3H-thymidine incorporation assay. (B) GGT phenotype of constructed knock-out strains was confirmed by enzyme activity assay and immunoblotting using a polyclonal antibody raised against the large GGT subunit. For immunoblotting, 30 μg protein of H pylori supernatants were used. Immunoblotting with anti-VacA antibody served as a loading control. Data represent mean ± SD of 3 independent experiments. *P < .001 as determined by Student t test were considered significant. WT, wild type.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The study indicates that secreted H pylori GGT is a lymphocyte proliferation-inhibiting factor that may allow persistent colonization of this bacteria in the human stomach.See page 1820.Enterochromaffin Cells Pass the “Sniff” Test: Newly Found Gustatory Function Triggers Serotonin Release in the IntestineThe sparsely located enterochromaffin cells reside in intestinal crypts and release substances, such as serotonin, in response to local mechanical stimulation or in response to certain nutrients in the lumen of the intestine. The secreted serotonin can then stimulate sensory components of the enteric nervous system, ultimately controlling gut peristalsis as well as water and chloride transport by enterocytes. It has been hypothesized that enterochromaffin cells may be able to sense other substances that cause serotonin release. Microarray gene chip data suggested that enterochromaffin cells might express olfactory G-protein–coupled receptors that are typically found in the oral cavity and nose, most of which have overlapping oderant ligand profiles. This possibility was examined in the current study.In the study by Braun et al, BON carcinoid cancer cells, human intestinal specimens, and laser capture-microdissected human intestinal enterochromaffin cells were examined by reverse transcriptase polymerase chain reaction (RT-PCR) for expression of olfactory receptors, and BON cells were further used to study oderant stimulatory responses. Both BON cells and human enterochromaffin cells expressed the same 4 (of 339 potential) olfactory receptor genes by RT-PCR: OR73, hOR17-7/11, OR1G1, and hOR17-210. Specific oderant ligands, most based on the organic structure of phenol, were tested for activity in BON cells. Thymol, which binds to OR1G1, is a component of thyme spice. Thymol triggers a transient rise in intracellular calcium and a dose-dependent increase in serotonin release in BON cells, whereas phenol did not (Figure 4). Other oderant ligands showed similar responses: eugenol and isoeugenol (binds OR73), methylsalicylate (receptor unknown), geraniol (binds hOR17-7/11 and OR1G1), bourgeonal (binds hOR17-7/11 and hOR17-4), and helional (binds hOR17-7/11 and hOR17-40) increased intracellular calcium, and eugenol, bourgeonal, and helional all stimulated serotonin release by exocytosis from BON cells. Both the calcium rise and serotonin release was reversibly blocked by the L-type calcium channel blocker nifedipine and was unaffected by thapsigargin, indicating that extracellular calcium was required for ligand–olfactory receptor activity. Indeed, removing extracellular calcium or the use of a specific phospholipase C inhibitor or inositol-1,4,5-triphosphate receptor blocker diminished the oderant ligand activity.Figure 4Oderants stimulate serotonin release by enterochromaffin BON cells. BON cells were incubated for 15 minutes in serum-free medium with oderant. Serotonin release was measured in the culture supernatant by a serotonin enzyme immunoassay kit (ELISA). Concentration-dependent serotonin release by BON cells was triggered by thymol, but not phenol. P values correspond to Student–Newman–Keuls test.View Large Image Figure ViewerDownload Hi-res image Download (PPT)This study indicated that enterochromaffin cells express 4 olfactory G-protein–coupled receptors that may be stimulated by oderant ligands in the intestinal lumen to release serotonin via influx of intracellular calcium. Although the authors did not show that inhibition of olfactory receptor mRNA reversed the observations, the study suggests that luminal oderants may influence gut motility and secretion.See page 1890. IL-23 Receptor Variations and Susceptibility for Inflammatory Bowel DiseaseStudies of genetic association may help to unravel the pathogenesis of multifactorial disorders with putative genetic predisposition, such as inflammatory bowel disease (IBD). Previous studies have already identified a number of candidate IBD-associated gene polymorphisms. However, the currently identified IBD susceptibility genes explain only a fraction of the genetic predisposition, and larger and confirmatory studies are needed for several candidate genes. A genome-wide scan study comparing North American Crohn’s disease patients with controls, published in Science last year, reported an association with the interleukin-23 receptor (IL23R) gene. Moreover, the paper included an internal replication studies that confirmed IL23R associations with both Crohn’s disease and ulcerative colitis.In this issue of Gastroenterology, Tremelling et al report the results of a study that investigated the association between IL23R and IBD. DNA samples of a total of 2877 IBD patients (two thirds Crohn’s disease and one third ulcerative colitis) in the United Kingdom were compared with 1345 control samples. Genotyping for 8 IL23R markers was performed, and the association with CARD15 mutations was also analyzed.Clear statistically significant associations were found between variants in the IL23R gene and IBD (Figure 1). The strongest association was found between Crohn’s disease and the IL23R coding variant Arg381Gln, with a significantly lower odds ratio of this protective allele in Crohn’s disease (odds ratio 0.38; 95% confidence interval 0.29–0.50). Analysis accounting for Arg381Gln demonstrated that other loci within IL23R also influenced Crohn’s disease susceptibility. No association of IL23R and CARD15 mutations were found. Similarly, several single nucleotide polymorphisms of the IL23R gene were significantly associated with ulcerative colitis, but the overall susceptibility impact was more modest in ulcerative colitis than in Crohn’s disease. The odds ratio for ulcerative colitis associated with Arg381Gln was 0.73, with a 95% confidence interval of 0.55–0.96. The protective Arg381Gln allele is relatively uncommon, with a low prevalence of 6.2% in the control population, and 2.5% and 4.6% in Crohn’s disease and ulcerative colitis, respectively; the more common G allele is a disease risk allele. Both for Crohn’s disease and ulcerative colitis, IL23R polymorphisms were not significantly linked to disease phenotype subgroups (eg, ileal versus colonic involvement in Crohn’s disease) was found.This study confirms the association between IL23R and IBD, which is also found outside of North America. With an association with both ulcerative colitis and Crohn’s disease, and not with disease phenotypes, it seems that IL23R variants may exert a generic effect on susceptibility to chronic intestinal inflammation, although the effect size is biggest for Crohn’s disease. The findings further support an important role for IL-23, the ligand of IL23R , as a key player in the immune system and in chronic IBD. IL-23 or its receptor may become important targets for the development of novel therapeutic strategies for IBD.See page 1657. Studies of genetic association may help to unravel the pathogenesis of multifactorial disorders with putative genetic predisposition, such as inflammatory bowel disease (IBD). Previous studies have already identified a number of candidate IBD-associated gene polymorphisms. However, the currently identified IBD susceptibility genes explain only a fraction of the genetic predisposition, and larger and confirmatory studies are needed for several candidate genes. A genome-wide scan study comparing North American Crohn’s disease patients with controls, published in Science last year, reported an association with the interleukin-23 receptor (IL23R) gene. Moreover, the paper included an internal replication studies that confirmed IL23R associations with both Crohn’s disease and ulcerative colitis. In this issue of Gastroenterology, Tremelling et al report the results of a study that investigated the association between IL23R and IBD. DNA samples of a total of 2877 IBD patients (two thirds Crohn’s disease and one third ulcerative colitis) in the United Kingdom were compared with 1345 control samples. Genotyping for 8 IL23R markers was performed, and the association with CARD15 mutations was also analyzed. Clear statistically significant associations were found between variants in the IL23R gene and IBD (Figure 1). The strongest association was found between Crohn’s disease and the IL23R coding variant Arg381Gln, with a significantly lower odds ratio of this protective allele in Crohn’s disease (odds ratio 0.38; 95% confidence interval 0.29–0.50). Analysis accounting for Arg381Gln demonstrated that other loci within IL23R also influenced Crohn’s disease susceptibility. No association of IL23R and CARD15 mutations were found. Similarly, several single nucleotide polymorphisms of the IL23R gene were significantly associated with ulcerative colitis, but the overall susceptibility impact was more modest in ulcerative colitis than in Crohn’s disease. The odds ratio for ulcerative colitis associated with Arg381Gln was 0.73, with a 95% confidence interval of 0.55–0.96. The protective Arg381Gln allele is relatively uncommon, with a low prevalence of 6.2% in the control population, and 2.5% and 4.6% in Crohn’s disease and ulcerative colitis, respectively; the more common G allele is a disease risk allele. Both for Crohn’s disease and ulcerative colitis, IL23R polymorphisms were not significantly linked to disease phenotype subgroups (eg, ileal versus colonic involvement in Crohn’s disease) was found. This study confirms the association between IL23R and IBD, which is also found outside of North America. With an association with both ulcerative colitis and Crohn’s disease, and not with disease phenotypes, it seems that IL23R variants may exert a generic effect on susceptibility to chronic intestinal inflammation, although the effect size is biggest for Crohn’s disease. The findings further support an important role for IL-23, the ligand of IL23R , as a key player in the immune system and in chronic IBD. IL-23 or its receptor may become important targets for the development of novel therapeutic strategies for IBD. See page 1657. Monoclonal Antibodies to α4-Integrin in the Treatment of Active Crohn’s DiseaseThe development of biological agents allows to target crucial steps in the inflammatory cascade. The anti–tumor necrosis factor agent, infliximab, has profoundly altered the management of Crohn’s disease. Despite this progress, major unmet needs persist in the medical treatment of inflammatory bowel disease, and the success of this agent has stimulated the exploration of several other anti-inflammatory targets. Natalizumab, a recombinant humanized immunoglobulin G4 monoclonal antibody that binds to α4-integrin, is a member of a group of molecules known as selective adhesion molecule inhibitors. The α4-integrins are glycoproteins expressed on the surface of leukocytes, which interact with endothelial vascular cell adhesion molecule-1, expressed on the vascular endothelium, to allow leukocyte adhesion and subsequent migration into surrounding tissue. In Crohn’s disease, recruitment of leukocytes into inflamed areas of the gut is a key event, and blocking the adhesion molecules that allow this process to happen is expected to decrease chronic intestinal inflammation.Previously, the ENACT-1 and -2 studies evaluated the use of natalizumab in the induction and maintenance of remission in patients with moderately to severely active Crohn’s disease. Although the ENACT-1 trial did not meet the primary endpoint of higher response rates after 3 infusions of natalizumab, efficacy of natalizumab as maintenance therapy for up to 56 weeks in responders to induction therapy was demonstrated in the ENACT-2 trial.In this issue of Gastroenterology, Targan et al report the results of the Efficacy of Natalizumab in Crohn’s disease Response and Remission (ENCORE) trial, which was designed to confirm the hypothesis that natalizumab is effective as induction therapy in patients with active Crohn’s disease. In this multicenter, randomized, double-blind, placebo-controlled, parallel-group study, a total of 509 patients with moderate to severely active Crohn’s disease (Crohn’s Disease Activity Index [CDA] scores ≥220 to ≤450) and active inflammation (C-reactive protein [CRP] >2.87 mg/L) were recruited from 114 centers. Patients were randomized to receive 3 intravenous infusions of placebo or natalizumab 300 mg at weeks 0, 4, and 8, and they were followed up through week 12 for safety and efficacy assessments.Response rate, defined as a reduction of ≥70 points in the CDAI score at week 12 from week 0, occurred in 60% of natalizumab-treated patients compared with 44% of placebo-treated patients (P < .001). Remission, defined as a CDAI score <150 points, was obtained in 38% of patients after natalizumab and 25% after placebo (P = .001). Significant differences in response rates occurred as early as week 4 and were sustained throughout the 12-week evaluation period (Figure 2A and B).The beneficial effects of natalizumab on the CDAI were associated with significant greater decreases in CRP plasma levels and significantly greater improvements in quality of life scores. These results confirm that natalizumab effectively induces response and remission in patients with moderately to severely active Crohn’s disease and objective evidence of inflammation. Tolerance of natalizumab in the present study was good, with adverse events and serious adverse events occurring equally frequently in both study arms. Rates of antibody formation and of infusion reactions were low. Antibodies to natalizumab occurred in <10% of patients and were not associated with decreased response rates, but with a higher incidence of acute infusion reactions.These observations suggest that natalizumab is effective as an induction therapy for Crohn’s disease. The agent is attractive as it derives its anti-inflammatory activity from a mode of action that differs from that of other available therapies. Although no serious events occurred in this trial, in previous studies the use of natalizumab has been associated with development of progressive multifocal leukoencephalopathy and other opportunistic infections. Clinicians and patients therefore need to carefully consider the risk/benefit ratio of this agent and available alternative therapies that can be used in the treatment of active Crohn’s disease.See page 1672. The development of biological agents allows to target crucial steps in the inflammatory cascade. The anti–tumor necrosis factor agent, infliximab, has profoundly altered the management of Crohn’s disease. Despite this progress, major unmet needs persist in the medical treatment of inflammatory bowel disease, and the success of this agent has stimulated the exploration of several other anti-inflammatory targets. Natalizumab, a recombinant humanized immunoglobulin G4 monoclonal antibody that binds to α4-integrin, is a member of a group of molecules known as selective adhesion molecule inhibitors. The α4-integrins are glycoproteins expressed on the surface of leukocytes, which interact with endothelial vascular cell adhesion molecule-1, expressed on the vascular endothelium, to allow leukocyte adhesion and subsequent migration into surrounding tissue. In Crohn’s disease, recruitment of leukocytes into inflamed areas of the gut is a key event, and blocking the adhesion molecules that allow this process to happen is expected to decrease chronic intestinal inflammation. Previously, the ENACT-1 and -2 studies evaluated the use of natalizumab in the induction and maintenance of remission in patients with moderately to severely active Crohn’s disease. Although the ENACT-1 trial did not meet the primary endpoint of higher response rates after 3 infusions of natalizumab, efficacy of natalizumab as maintenance therapy for up to 56 weeks in responders to induction therapy was demonstrated in the ENACT-2 trial. In this issue of Gastroenterology, Targan et al report the results of the Efficacy of Natalizumab in Crohn’s disease Response and Remission (ENCORE) trial, which was designed to confirm the hypothesis that natalizumab is effective as induction therapy in patients with active Crohn’s disease. In this multicenter, randomized, double-blind, placebo-controlled, parallel-group study, a total of 509 patients with moderate to severely active Crohn’s disease (Crohn’s Disease Activity Index [CDA] scores ≥220 to ≤450) and active inflammation (C-reactive protein [CRP] >2.87 mg/L) were recruited from 114 centers. Patients were randomized to receive 3 intravenous infusions of placebo or natalizumab 300 mg at weeks 0, 4, and 8, and they were followed up through week 12 for safety and efficacy assessments. Response rate, defined as a reduction of ≥70 points in the CDAI score at week 12 from week 0, occurred in 60% of natalizumab-treated patients compared with 44% of placebo-treated patients (P < .001). Remission, defined as a CDAI score <150 points, was obtained in 38% of patients after natalizumab and 25% after placebo (P = .001). Significant differences in response rates occurred as early as week 4 and were sustained throughout the 12-week evaluation period (Figure 2A and B). The beneficial effects of natalizumab on the CDAI were associated with significant greater decreases in CRP plasma levels and significantly greater improvements in quality of life scores. These results confirm that natalizumab effectively induces response and remission in patients with moderately to severely active Crohn’s disease and objective evidence of inflammation. Tolerance of natalizumab in the present study was good, with adverse events and serious adverse events occurring equally frequently in both study arms. Rates of antibody formation and of infusion reactions were low. Antibodies to natalizumab occurred in <10% of patients and were not associated with decreased response rates, but with a higher incidence of acute infusion reactions. These observations suggest that natalizumab is effective as an induction therapy for Crohn’s disease. The agent is attractive as it derives its anti-inflammatory activity from a mode of action that differs from that of other available therapies. Although no serious events occurred in this trial, in previous studies the use of natalizumab has been associated with development of progressive multifocal leukoencephalopathy and other opportunistic infections. Clinicians and patients therefore need to carefully consider the risk/benefit ratio of this agent and available alternative therapies that can be used in the treatment of active Crohn’s disease. See page 1672. Mechanism for Helicobacter pylori-induced T-cell Suppression in Persistent InfectionHelicobacter pylori is a common colonizer of human gastric mucosa, and how it evades immune surveillance is not clear. The H pylori virulence factors cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) have been suggested as mediators to inhibit lymphocyte proliferation, but this is inconsistent in the literature, and may induce inflammation rather than reduce it. An unknown, low-molecular-weight secreted product from H pylori, separate from CagA and VacA factors, was recently shown to inhibit T-cell proliferation and induce a G1 cell-cycle phase arrest. The identity of the secreted product was not known until the current study was performed.In the study by Schmees et al, low-molecular-weight fractions from the H pylori G27 strain was purified by size exclusion chromatography and SDS-PAGE, and the secreted protein γ-glutamyl transpeptidase (GGT) was identified from supernatant fractions that inhibited lymphocyte proliferation, and had activity in a photometric GGT activity assay. An isogenic GGT knock-out strain of H pylori G27 that had no effect on survival or growth of the bacteria revealed complete abrogation of GGT activity and loss of the ability to inhibit lymphocyte proliferations (Figure 3), and both activities appear to be mediated by serine residues 451 and 452 of the H pylori GGT as determined by site-directed mutagenesis. Additionally, purified recombinant H pylori GGT demonstrated high GGT activity and efficiently inhibited lymphocyte proliferation in a dose-dependent manner, and the GGT inhibitor acivicin reversed these effects. Equine GGT, although possessing GGT enzymatic activity, failed to inhibit lymphocyte proliferation, presumably owing to possessing only 22% homology with H pylori GGT. Wild-type or recombinant H pylori GGT, but not mutant strains, stimulated a G1 cell-cycle phase arrest, but there was no effect on IL-2 or γ-IFN secretion by lymphocytes, nor any change in apoptosis. In assessing signaling mechanisms that could reduce lymphocyte proliferation, both wild-type and recombinant H pylori GGT, but not mutant strains, reduced the downstream mediators of Ras-dependent signaling, c-Raf and c-Myc, and increased levels of the Cdk-inhibitor p27, while preserving PI3 kinase signaling mediators.The study indicates that secreted H pylori GGT is a lymphocyte proliferation-inhibiting factor that may allow persistent colonization of this bacteria in the human stomach.See page 1820. Helicobacter pylori is a common colonizer of human gastric mucosa, and how it evades immune surveillance is not clear. The H pylori virulence factors cytotoxin-associated gene A (CagA) and vacuolating cytotoxin A (VacA) have been suggested as mediators to inhibit lymphocyte proliferation, but this is inconsistent in the literature, and may induce inflammation rather than reduce it. An unknown, low-molecular-weight secreted product from H pylori, separate from CagA and VacA factors, was recently shown to inhibit T-cell proliferation and induce a G1 cell-cycle phase arrest. The identity of the secreted product was not known until the current study was performed. In the study by Schmees et al, low-molecular-weight fractions from the H pylori G27 strain was purified by size exclusion chromatography and SDS-PAGE, and the secreted protein γ-glutamyl transpeptidase (GGT) was identified from supernatant fractions that inhibited lymphocyte proliferation, and had activity in a photometric GGT activity assay. An isogenic GGT knock-out strain of H pylori G27 that had no effect on survival or growth of the bacteria revealed complete abrogation of GGT activity and loss of the ability to inhibit lymphocyte proliferations (Figure 3), and both activities appear to be mediated by serine residues 451 and 452 of the H pylori GGT as determined by site-directed mutagenesis. Additionally, purified recombinant H pylori GGT demonstrated high GGT activity and efficiently inhibited lymphocyte proliferation in a dose-dependent manner, and the GGT inhibitor acivicin reversed these effects. Equine GGT, although possessing GGT enzymatic activity, failed to inhibit lymphocyte proliferation, presumably owing to possessing only 22% homology with H pylori GGT. Wild-type or recombinant H pylori GGT, but not mutant strains, stimulated a G1 cell-cycle phase arrest, but there was no effect on IL-2 or γ-IFN secretion by lymphocytes, nor any change in apoptosis. In assessing signaling mechanisms that could reduce lymphocyte proliferation, both wild-type and recombinant H pylori GGT, but not mutant strains, reduced the downstream mediators of Ras-dependent signaling, c-Raf and c-Myc, and increased levels of the Cdk-inhibitor p27, while preserving PI3 kinase signaling mediators. The study indicates that secreted H pylori GGT is a lymphocyte proliferation-inhibiting factor that may allow persistent colonization of this bacteria in the human stomach. See page 1820. Enterochromaffin Cells Pass the “Sniff” Test: Newly Found Gustatory Function Triggers Serotonin Release in the IntestineThe sparsely located enterochromaffin cells reside in intestinal crypts and release substances, such as serotonin, in response to local mechanical stimulation or in response to certain nutrients in the lumen of the intestine. The secreted serotonin can then stimulate sensory components of the enteric nervous system, ultimately controlling gut peristalsis as well as water and chloride transport by enterocytes. It has been hypothesized that enterochromaffin cells may be able to sense other substances that cause serotonin release. Microarray gene chip data suggested that enterochromaffin cells might express olfactory G-protein–coupled receptors that are typically found in the oral cavity and nose, most of which have overlapping oderant ligand profiles. This possibility was examined in the current study.In the study by Braun et al, BON carcinoid cancer cells, human intestinal specimens, and laser capture-microdissected human intestinal enterochromaffin cells were examined by reverse transcriptase polymerase chain reaction (RT-PCR) for expression of olfactory receptors, and BON cells were further used to study oderant stimulatory responses. Both BON cells and human enterochromaffin cells expressed the same 4 (of 339 potential) olfactory receptor genes by RT-PCR: OR73, hOR17-7/11, OR1G1, and hOR17-210. Specific oderant ligands, most based on the organic structure of phenol, were tested for activity in BON cells. Thymol, which binds to OR1G1, is a component of thyme spice. Thymol triggers a transient rise in intracellular calcium and a dose-dependent increase in serotonin release in BON cells, whereas phenol did not (Figure 4). Other oderant ligands showed similar responses: eugenol and isoeugenol (binds OR73), methylsalicylate (receptor unknown), geraniol (binds hOR17-7/11 and OR1G1), bourgeonal (binds hOR17-7/11 and hOR17-4), and helional (binds hOR17-7/11 and hOR17-40) increased intracellular calcium, and eugenol, bourgeonal, and helional all stimulated serotonin release by exocytosis from BON cells. Both the calcium rise and serotonin release was reversibly blocked by the L-type calcium channel blocker nifedipine and was unaffected by thapsigargin, indicating that extracellular calcium was required for ligand–olfactory receptor activity. Indeed, removing extracellular calcium or the use of a specific phospholipase C inhibitor or inositol-1,4,5-triphosphate receptor blocker diminished the oderant ligand activity.This study indicated that enterochromaffin cells express 4 olfactory G-protein–coupled receptors that may be stimulated by oderant ligands in the intestinal lumen to release serotonin via influx of intracellular calcium. Although the authors did not show that inhibition of olfactory receptor mRNA reversed the observations, the study suggests that luminal oderants may influence gut motility and secretion.See page 1890. The sparsely located enterochromaffin cells reside in intestinal crypts and release substances, such as serotonin, in response to local mechanical stimulation or in response to certain nutrients in the lumen of the intestine. The secreted serotonin can then stimulate sensory components of the enteric nervous system, ultimately controlling gut peristalsis as well as water and chloride transport by enterocytes. It has been hypothesized that enterochromaffin cells may be able to sense other substances that cause serotonin release. Microarray gene chip data suggested that enterochromaffin cells might express olfactory G-protein–coupled receptors that are typically found in the oral cavity and nose, most of which have overlapping oderant ligand profiles. This possibility was examined in the current study. In the study by Braun et al, BON carcinoid cancer cells, human intestinal specimens, and laser capture-microdissected human intestinal enterochromaffin cells were examined by reverse transcriptase polymerase chain reaction (RT-PCR) for expression of olfactory receptors, and BON cells were further used to study oderant stimulatory responses. Both BON cells and human enterochromaffin cells expressed the same 4 (of 339 potential) olfactory receptor genes by RT-PCR: OR73, hOR17-7/11, OR1G1, and hOR17-210. Specific oderant ligands, most based on the organic structure of phenol, were tested for activity in BON cells. Thymol, which binds to OR1G1, is a component of thyme spice. Thymol triggers a transient rise in intracellular calcium and a dose-dependent increase in serotonin release in BON cells, whereas phenol did not (Figure 4). Other oderant ligands showed similar responses: eugenol and isoeugenol (binds OR73), methylsalicylate (receptor unknown), geraniol (binds hOR17-7/11 and OR1G1), bourgeonal (binds hOR17-7/11 and hOR17-4), and helional (binds hOR17-7/11 and hOR17-40) increased intracellular calcium, and eugenol, bourgeonal, and helional all stimulated serotonin release by exocytosis from BON cells. Both the calcium rise and serotonin release was reversibly blocked by the L-type calcium channel blocker nifedipine and was unaffected by thapsigargin, indicating that extracellular calcium was required for ligand–olfactory receptor activity. Indeed, removing extracellular calcium or the use of a specific phospholipase C inhibitor or inositol-1,4,5-triphosphate receptor blocker diminished the oderant ligand activity. This study indicated that enterochromaffin cells express 4 olfactory G-protein–coupled receptors that may be stimulated by oderant ligands in the intestinal lumen to release serotonin via influx of intracellular calcium. Although the authors did not show that inhibition of olfactory receptor mRNA reversed the observations, the study suggests that luminal oderants may influence gut motility and secretion. See page 1890. IL23R Variation Determines Susceptibility But Not Disease Phenotype in Inflammatory Bowel DiseaseGastroenterologyVol. 132Issue 5PreviewBackground & Aims: Identification of inflammatory bowel disease (IBD) susceptibility genes is key to understanding pathogenic mechanisms. Recently, the North American IBD Genetics Consortium provided compelling evidence for an association between ileal Crohn’s disease (CD) and the IL23R gene using genome-wide association scanning. External replication is a priority, both to confirm this finding in other populations and to validate this new technique. We tested for association between IL23R and IBD in a large independent UK panel to determine the size of the effect and explore subphenotype correlation and interaction with CARD15. Full-Text PDF Open AccessNatalizumab for the Treatment of Active Crohn’s Disease: Results of the ENCORE TrialGastroenterologyVol. 132Issue 5PreviewBackground & Aims: A randomized placebo-controlled trial evaluated the efficacy of natalizumab induction therapy in patients with Crohn’s disease. Methods: Patients (N = 509) with moderately to severely active Crohn’s disease and active inflammation characterized by elevated C-reactive protein concentrations were randomized (1:1) to receive natalizumab 300 mg or placebo intravenously at Weeks 0, 4, and 8. The primary end point was induction of response (≥70-point decrease from baseline in the Crohn’s Disease Activity Index score at Week 8 sustained through Week 12). Full-Text PDF Inhibition of T-Cell Proliferation by Helicobacter pylori γ-Glutamyl TranspeptidaseGastroenterologyVol. 132Issue 5PreviewBackground & Aims: Helicobacter pylori colonizes the human gastric mucosa of >50% of the world’s population. Most of the patients have no overt clinical symptoms. However, the infection is invariably associated with the development of active chronic gastritis, leading in some cases to the development of peptic ulcer disease, distal gastric adenocarcinoma, and mucosa-associated lymphoid tissue lymphoma. In contrast to most other pathogens, infection with H pylori persists lifelong, but reasons for the persistence remain obscure. Full-Text PDF Enterochromaffin Cells of the Human Gut: Sensors for Spices and OdorantsGastroenterologyVol. 132Issue 5PreviewBackground & Aims: Release of serotonin from mucosal enterochromaffin cells triggered by luminal substances is the key event in the regulation of gut motility and secretion. We were interested to know whether nasal olfactory receptors are also expressed in the human gut mucosa by enterochromaffin cells and whether their ligands and odorants present in spices, fragrances, detergents, and cosmetics cause serotonin release. Methods: Receptor expression was studied by the reverse-transcription polymerase chain reaction method in human mucosal enterochromaffin cells isolated by laser microdissection and in a cell line derived from human enterochromaffin cells. Full-Text PDF