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Immunological Tolerance to Helicobacter Pylori Infection Conferred by Adaptive Gene Loss in Humans
41
IMMUNOLOGICAL TOLERANCE TO HELICOBACTER PYLORI INFECTION CONFERRED BY ADAPTIVE GENE LOSS IN HUMANS Garrett Z. Ng, Louise O'Connor, Michelle Scurr, Lincon A. Stamp, Louise M. Judd, Andrew S. Giraud, Philip Sutton, Trevelyan R. Menheniott
EFFECTS OF AGE AND BODY MASS INDEX ON PELVIC FLOOR STRUCTURE AND FUNCTION IN 129 HEALTHY WOMEN AS DETERMINED BY MAGNETIC RESONANCE DEFECOGRAPHY David O. Prichard, Pratyusha Tirumanisetty, Kelly Feuerhak, Joel G. Fletcher, Adil E. Bharucha
BACKGROUND & AIMS: Most individuals infected with the gastric bacterium, Helicobacter (H.) pylori, remain asymptomatically colonised throughout life, with only a minority developing disease sequelae. Mechanisms that sustain this host-microbe dynamic are unclear. Gastrokine (GKN)3 belongs to a family of gastric mucosal proteins that putatively regulate the host response to H. pylori. In mice, GKN3 is expressed in the mucous neck, antral gland base and metaplastic lesions, however its physiological role remains undefined. While functional in most mammals, GKN3 has been inactivated in all humans, suggesting a process of adaptive gene loss during recent evolution. Defining the role of GKN3 would help elucidate selective pressures underlying its non-functionality in humans and illuminate a potential link with H. pylori. Here, we generated Gkn3-/- (knockout) mice to model functional effects of ‘GKN3 loss' on the host response to H. pylori infection. METHODS: Gkn3-/- mice were created by conventional gene targeting. Gastric mucosal differentiation was assessed by histopathology and immunofluorescence. Impact of GKN3 loss on the host response to H. pylori was investigated in Gkn3-/- mice infected with mouse-adapted H. pylori SS1 for 2 and 6 months. Infection-related immune profiles were assessed by quantitative (Q)-RTPCR analysis of gastric cytokine and chemokine expression, and by flow cytometric analysis of gastric immune cell subsets. H. pylori colonisation levels were determined by Q-PCR. Serum anti-H. pylori antibody levels were determined by enzyme immunoassay. RESULTS: Gkn3-/mice had normal gastric mucosal differentiation at baseline and were not differentially responsive to H. pylori at 2 months post-infection. However, at 6 months post-infection, Gkn3-/- mice displayed significantly decreased susceptibility to H. pylori-related immunopathology, relative to WT mice, as evidenced by reduced gastric mucosal inflammation, glandular atrophy and metaplasia. Immune profiling revealed an attenuated induction of T-helper 1 (Th1) type cytokines and chemokines in stomachs of 6 month infected Gkn3-/- compared to WT mice, with concomitantly decreased serum levels of Th1-related anti-H. pylori IgG2c antibodies. 6 month infected Gkn3-/- mice also had significantly higher H. pylori colonisation levels compared to WT mice, possibly consistent with increased immune tolerance. Finally, we found that decreased immune responsiveness of Gkn3-/- mice to H. pylori was associated with an increased mucosal prevalence of tolerogenic CD4+CD25+FOXP3+ regulatory T cells. CONCLUSIONS: Our data implicate loss of GKN3 in humans as a functionally important adaptation favouring tolerance that helps minimise the severity of H. pylori-induced gastric immunopathology in order to protect against disease, with the likely consequence of sustained H. pylori colonisation.
Background: The American College of Gastroenterology and Rome criteria recommend the use of defecography for diagnosing defecatory disorders (DD) in selected patients. However, most studies that evaluated the utility of MR defecography for diagnosing DD have been uncontrolled. Indeed, there is a paucity of normal values for MR defecography and endoanal sphincter imaging in asymptomatic women. Furthermore, the effects of age and BMI on anorectal motion are unclear. Our aims were to assess normal values and the effects of age and BMI on pelvic floor structure and motion in asymptomatic women without functional bowel disorders. Methods: Using established approaches, anal sphincter appearance and pelvic floor motion were assessed with endoanal MRI and dynamic MR defecography respectively in 129 women (age, 52 years [36-63]; BMI, 25.7 Kg/m2 [22.8-30.1], median [IQ range]) without a functional bowel disorder. Mann-Whitney U, Spearman Rho and Chi Square tests were used to evaluate the effects of age and BMI on anorectal motion. Results: At rest, the location of the anorectal junction (ρ=0.35, P<0.001) but not the anorectal angle was correlated with age (Table). Older women had a greater reduction in the anorectal angle (ρ=0.24, P=0.007) but less perineal ascent (ρ=-0.23, P=0.009) during squeeze. During defecation, the anorectal angle was greater (ρ=0.27, P=0.002) in older women. Rectal evacuation was not associated with age. BMI was only correlated with one parameter, i.e., anorectal junction location during simulated evacuation (ρ=0.24, P=0.006). A rectocele ≥3cm was identified in 23 women (18%); these women had greater opening of their anorectal angle (29° [15-37] vs. 16° [6-31], P=0.03), greater perineal descent (4.1cm [3.1-5.1] vs. 2.7cm [1.9-3.9], P=0.002) and greater rectal emptying (76% [46-94] vs. 51% [10-81], P=0.01) during evacuation. A patulous anal canal, rectal intussusception, rectal prolapse, or other prolapse (peritoneocele, sigmoidocele, enterocele) was identified in 5%, 13%, 3% and 5% respectively. There was no association between age or BMI and pelvic organ prolapse. The anal sphincters were normal in most asymptomatic women (Table). Conclusion: In asymptomatic women, age is associated with perineal laxity at rest. However, the reduction in anorectal angle during voluntary contraction was preserved, indeed greater, in older women, which may function to maintain fecal continence. By comparison, age was associated with minimal effects on anorectal motion during evacuation and did not significantly affect evacuation per se. Among asymptomatic women BMI was not associated with significant effects on anorectal or pelvic floor motion. Approximately 1 in 5 asymptomatic women have a rectocele 3 cm or larger; these are associated with greater perineal descent and rectal evacuation. Normal Reference Values for Pelvic Floor Anatomy and Motion, and Their Relationships to Age and BMI, as Determined during Magnetic Resonance Defecography in 129 Healthy Women.
40 IMPLANTATION OF AUTOLOGOUS BIOSPHINCTERS IN A NON-HUMAN PRIMATE (NHP) MODEL OF FECAL INCONTINENCE Khalil N. Bitar, Elie Zakhem, Jaime L. Bohl, Riccardo Tamburrini, Prabhash Dadhich, Christie L. Scott, Dylan T. Knutson, John H. Gilliam Introduction: The Internal Anal Sphincter (IAS) is primarily responsible for maintaining anorectal resting pressure and fecal continence. IAS dysfunction can lead to passive fecal incontinence (FI). We previously developed a rabbit model of fecal incontinence and successfully treated it with implantation of autologous-engineered BioSphincters. Objective: The objective of this study was (1) to develop a pre-clinical NHP model of fecal incontinence, (2) to scale up the engineering process of the BioSphincter, and (3) to implant the BioSphincters and reinstate fecal continence in the FI NHP. Methods: FI was induced in male cynomolgus (7-10Kg) as previously described. A partial IAS hemi-sphincterectomy was performed in all NHPs. Smooth muscle cells were isolated from the excised IAS tissue. Neural progenitor cells were isolated from a jejunal biopsy obtained through laparotomy. Autologous BioSphincters with a 12mm internal diameter were engineered using both types of cells. Quality control was performed on engineered BioSphincters prior to implantation. Anorectal manometry was performed on all NHPs at baseline, 3 weeks following sphincterectomy and 1 month following implantation of sphincters. Results: (1) Anorectal manometry performed following sphincterectomy revealed 15-20% reduction in basal tone and 30-40% reduction in rectoanal inhibitory reflex (RAIR) compared to baseline. (2) BioSphincter constructs with an internal diameter of 12mm were successfully engineered using autologous cells (3) Constructs were tested for physiological functionality: (i) KCl induced a rapid and robust contraction that averaged 696 ± 9 µN. (ii) Acetylcholine induced an average contraction of 430 ± 11 µN which was partially inhibited (40-50%) in the presence of tetrodotoxin. (iii) Neural functionality was tested using electrical field stimulation (EFS) which caused smooth muscle relaxation of an average of -367 ± 20 µN. Relaxation was attenuated by 60% in the presence of nitric oxide synthase inhibitor, indicating functional nitrergic contribution to the response. (4) Constructs were successfully implanted in the NHPs. (5) Anorectal manometry following 1 month of implantation revealed reinstatement of basal tone and RAIR back to baseline. Conclusions: Fecal incontinence was induced in a pre-clinical large animal model. This study demonstrated the feasibility of scaling up the engineering process of the BioSphincters from a rabbit model to an NHP model with maintenance of functionality. Implantation of the BioSphincters reinstated in vivo physiological functionality of the anorectum similar to normal continent NHPs. This is a strong proof of concept that the scaled-up BioSphincters provide a potential therapeutic approach to treat a scaled-up model of fecal incontinence. Funding: Brooks Medical Research Fellowship, Sons of Confederate Veterans.
a
Perpendicular distance from pubococcygeal line; positive and negative values are below and above the line respectively. Data are median (10th and 90th centile) or percentages. For Chi Square analyses, sphincter injuries were compared with age greater/lesser than the median age (52 years) and with the BMI groups <24.9, 25-29.9, 30-34.9 and >35 Kg/m2.
S-15
AGA Abstracts
AGA Abstracts
39
IMMUNOLOGICAL TOLERANCE TO HELICOBACTER PYLORI INFECTION CONFERRED BY ADAPTIVE GENE LOSS IN HUMANS Garrett Z. Ng, Louise O'Connor, Michelle Scurr, Lincon A. Stamp, Louise M. Judd, Andrew S. Giraud, Philip Sutton, Trevelyan R. Menheniott
EFFECTS OF AGE AND BODY MASS INDEX ON PELVIC FLOOR STRUCTURE AND FUNCTION IN 129 HEALTHY WOMEN AS DETERMINED BY MAGNETIC RESONANCE DEFECOGRAPHY David O. Prichard, Pratyusha Tirumanisetty, Kelly Feuerhak, Joel G. Fletcher, Adil E. Bharucha
BACKGROUND & AIMS: Most individuals infected with the gastric bacterium, Helicobacter (H.) pylori, remain asymptomatically colonised throughout life, with only a minority developing disease sequelae. Mechanisms that sustain this host-microbe dynamic are unclear. Gastrokine (GKN)3 belongs to a family of gastric mucosal proteins that putatively regulate the host response to H. pylori. In mice, GKN3 is expressed in the mucous neck, antral gland base and metaplastic lesions, however its physiological role remains undefined. While functional in most mammals, GKN3 has been inactivated in all humans, suggesting a process of adaptive gene loss during recent evolution. Defining the role of GKN3 would help elucidate selective pressures underlying its non-functionality in humans and illuminate a potential link with H. pylori. Here, we generated Gkn3-/- (knockout) mice to model functional effects of ‘GKN3 loss' on the host response to H. pylori infection. METHODS: Gkn3-/- mice were created by conventional gene targeting. Gastric mucosal differentiation was assessed by histopathology and immunofluorescence. Impact of GKN3 loss on the host response to H. pylori was investigated in Gkn3-/- mice infected with mouse-adapted H. pylori SS1 for 2 and 6 months. Infection-related immune profiles were assessed by quantitative (Q)-RTPCR analysis of gastric cytokine and chemokine expression, and by flow cytometric analysis of gastric immune cell subsets. H. pylori colonisation levels were determined by Q-PCR. Serum anti-H. pylori antibody levels were determined by enzyme immunoassay. RESULTS: Gkn3-/mice had normal gastric mucosal differentiation at baseline and were not differentially responsive to H. pylori at 2 months post-infection. However, at 6 months post-infection, Gkn3-/- mice displayed significantly decreased susceptibility to H. pylori-related immunopathology, relative to WT mice, as evidenced by reduced gastric mucosal inflammation, glandular atrophy and metaplasia. Immune profiling revealed an attenuated induction of T-helper 1 (Th1) type cytokines and chemokines in stomachs of 6 month infected Gkn3-/- compared to WT mice, with concomitantly decreased serum levels of Th1-related anti-H. pylori IgG2c antibodies. 6 month infected Gkn3-/- mice also had significantly higher H. pylori colonisation levels compared to WT mice, possibly consistent with increased immune tolerance. Finally, we found that decreased immune responsiveness of Gkn3-/- mice to H. pylori was associated with an increased mucosal prevalence of tolerogenic CD4+CD25+FOXP3+ regulatory T cells. CONCLUSIONS: Our data implicate loss of GKN3 in humans as a functionally important adaptation favouring tolerance that helps minimise the severity of H. pylori-induced gastric immunopathology in order to protect against disease, with the likely consequence of sustained H. pylori colonisation.
Background: The American College of Gastroenterology and Rome criteria recommend the use of defecography for diagnosing defecatory disorders (DD) in selected patients. However, most studies that evaluated the utility of MR defecography for diagnosing DD have been uncontrolled. Indeed, there is a paucity of normal values for MR defecography and endoanal sphincter imaging in asymptomatic women. Furthermore, the effects of age and BMI on anorectal motion are unclear. Our aims were to assess normal values and the effects of age and BMI on pelvic floor structure and motion in asymptomatic women without functional bowel disorders. Methods: Using established approaches, anal sphincter appearance and pelvic floor motion were assessed with endoanal MRI and dynamic MR defecography respectively in 129 women (age, 52 years [36-63]; BMI, 25.7 Kg/m2 [22.8-30.1], median [IQ range]) without a functional bowel disorder. Mann-Whitney U, Spearman Rho and Chi Square tests were used to evaluate the effects of age and BMI on anorectal motion. Results: At rest, the location of the anorectal junction (ρ=0.35, P<0.001) but not the anorectal angle was correlated with age (Table). Older women had a greater reduction in the anorectal angle (ρ=0.24, P=0.007) but less perineal ascent (ρ=-0.23, P=0.009) during squeeze. During defecation, the anorectal angle was greater (ρ=0.27, P=0.002) in older women. Rectal evacuation was not associated with age. BMI was only correlated with one parameter, i.e., anorectal junction location during simulated evacuation (ρ=0.24, P=0.006). A rectocele ≥3cm was identified in 23 women (18%); these women had greater opening of their anorectal angle (29° [15-37] vs. 16° [6-31], P=0.03), greater perineal descent (4.1cm [3.1-5.1] vs. 2.7cm [1.9-3.9], P=0.002) and greater rectal emptying (76% [46-94] vs. 51% [10-81], P=0.01) during evacuation. A patulous anal canal, rectal intussusception, rectal prolapse, or other prolapse (peritoneocele, sigmoidocele, enterocele) was identified in 5%, 13%, 3% and 5% respectively. There was no association between age or BMI and pelvic organ prolapse. The anal sphincters were normal in most asymptomatic women (Table). Conclusion: In asymptomatic women, age is associated with perineal laxity at rest. However, the reduction in anorectal angle during voluntary contraction was preserved, indeed greater, in older women, which may function to maintain fecal continence. By comparison, age was associated with minimal effects on anorectal motion during evacuation and did not significantly affect evacuation per se. Among asymptomatic women BMI was not associated with significant effects on anorectal or pelvic floor motion. Approximately 1 in 5 asymptomatic women have a rectocele 3 cm or larger; these are associated with greater perineal descent and rectal evacuation. Normal Reference Values for Pelvic Floor Anatomy and Motion, and Their Relationships to Age and BMI, as Determined during Magnetic Resonance Defecography in 129 Healthy Women.
40 IMPLANTATION OF AUTOLOGOUS BIOSPHINCTERS IN A NON-HUMAN PRIMATE (NHP) MODEL OF FECAL INCONTINENCE Khalil N. Bitar, Elie Zakhem, Jaime L. Bohl, Riccardo Tamburrini, Prabhash Dadhich, Christie L. Scott, Dylan T. Knutson, John H. Gilliam Introduction: The Internal Anal Sphincter (IAS) is primarily responsible for maintaining anorectal resting pressure and fecal continence. IAS dysfunction can lead to passive fecal incontinence (FI). We previously developed a rabbit model of fecal incontinence and successfully treated it with implantation of autologous-engineered BioSphincters. Objective: The objective of this study was (1) to develop a pre-clinical NHP model of fecal incontinence, (2) to scale up the engineering process of the BioSphincter, and (3) to implant the BioSphincters and reinstate fecal continence in the FI NHP. Methods: FI was induced in male cynomolgus (7-10Kg) as previously described. A partial IAS hemi-sphincterectomy was performed in all NHPs. Smooth muscle cells were isolated from the excised IAS tissue. Neural progenitor cells were isolated from a jejunal biopsy obtained through laparotomy. Autologous BioSphincters with a 12mm internal diameter were engineered using both types of cells. Quality control was performed on engineered BioSphincters prior to implantation. Anorectal manometry was performed on all NHPs at baseline, 3 weeks following sphincterectomy and 1 month following implantation of sphincters. Results: (1) Anorectal manometry performed following sphincterectomy revealed 15-20% reduction in basal tone and 30-40% reduction in rectoanal inhibitory reflex (RAIR) compared to baseline. (2) BioSphincter constructs with an internal diameter of 12mm were successfully engineered using autologous cells (3) Constructs were tested for physiological functionality: (i) KCl induced a rapid and robust contraction that averaged 696 ± 9 µN. (ii) Acetylcholine induced an average contraction of 430 ± 11 µN which was partially inhibited (40-50%) in the presence of tetrodotoxin. (iii) Neural functionality was tested using electrical field stimulation (EFS) which caused smooth muscle relaxation of an average of -367 ± 20 µN. Relaxation was attenuated by 60% in the presence of nitric oxide synthase inhibitor, indicating functional nitrergic contribution to the response. (4) Constructs were successfully implanted in the NHPs. (5) Anorectal manometry following 1 month of implantation revealed reinstatement of basal tone and RAIR back to baseline. Conclusions: Fecal incontinence was induced in a pre-clinical large animal model. This study demonstrated the feasibility of scaling up the engineering process of the BioSphincters from a rabbit model to an NHP model with maintenance of functionality. Implantation of the BioSphincters reinstated in vivo physiological functionality of the anorectum similar to normal continent NHPs. This is a strong proof of concept that the scaled-up BioSphincters provide a potential therapeutic approach to treat a scaled-up model of fecal incontinence. Funding: Brooks Medical Research Fellowship, Sons of Confederate Veterans.
a
Perpendicular distance from pubococcygeal line; positive and negative values are below and above the line respectively. Data are median (10th and 90th centile) or percentages. For Chi Square analyses, sphincter injuries were compared with age greater/lesser than the median age (52 years) and with the BMI groups <24.9, 25-29.9, 30-34.9 and >35 Kg/m2.
S-15
AGA Abstracts
AGA Abstracts
39