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OC.05.2: PATHOGEN AND PROBIOTIC BACTERIA DIFFERENTIALLY STIMULATE NITRIC OXIDE PRODUCTION AND S100B PROTEIN EXPRESSION IN HUMAN ENTEROGLIAL CELLS
S128
Abstracts of the XVII National Congress of Digestive Diseases / Digestive and Liver Disease 43S (2011) S115–S264
Material and methods: We extracted information from a prospectively maintained data-base on characteristics of CD patients diagnosed in our CD Clinic. Information included total cholesterol (TC), triglycerides, HDL cholesterol, LDL cholesterol (Friedewal formula), homocysteinemia, GGT level and BMI measured in patients studied twice, before and after at least 12 months GFD. Patients with associated diseases affecting lipid profile at baseline were excluded. Results are expressed as mean ± SD; paired and unpaired t-tests have been used as appropriate for statistical analysis. Results: A cohort of 765 CD patients (72% F) was identified. At baseline, BMI was = 25 in 74/572 (13%), homocysteinemia was >15 μmol/L in 24/68 (35%), and TC was below 200 mg/dL in 525/650 (81%). During GFD, TC increased from 171±38 to 182±36 mg/dL (p<0.0001), tryglicerides decreased from 89±48 to 80±42 (p<0.0001), GGT increased form 17±15 to 20±20 U/L (p<0.0001) and BMI increased form 21.3±3.4 to 22.6±3.5 (p<0.0001). More detailed information was available on a subgroup of 38 patients HDL increased from 47±2 mg/dL at baseline to 53±1 mg/dL (p=0.002) during GFD. LDL remained unchanged and LDL/HDL ratio decreased from 2.5±1.0 at baseline to 2.3±0.9 (p=0.05) during GFD. Homocysteinemia remained unchanged during GFD at 15.2±9.8 μmol/L Vs 17.4±8.9 at baseline (NS). Conclusions: Our study indicates that risk and protective factors for cardiovascular diseases coexist in CD both at baseline and during GFD. Hyperhomocysteinemia accompanied by increased GGT level and by increased proportion of overweight patients predicts a pro-atherogenic effect of GFD, but reduced tryglicerides and increased HDL point to the opposite effect. Although these results are in keeping with the uncertainty on prevalence of vascular outcomes in CD, they suggest that in order to be healthy GFD has to go beyond gluten exclusion to include control of body weight and of quality of nutrients.
OC.05.1 COLONIC MUCOSAL NEUROPLASTICITY AND ITS INVOLVEMENT IN SYMPTOM GENERATION IN PATIENTS WITH IRRITABLE BOWEL SYNDROME C. Cremon ∗ , V. Vasina, G. Dothel, L. Gargano, L. Zecchi, G. Carini, L. Bellacosa, R. De Giorgio, V. Stanghellini, F. De Ponti, R. Corinaldesi, G. Barbara University Of Bologna, Bologna, Italy Background and aim: Growing evidence suggests that neuro-immune crosstalk contributes to symptom generation in irritable bowel syndrome (IBS). The aims of the present study were to evaluate in the colonic mucosa of IBS patients in comparison with healthy controls (HC): 1) phenotypic changes in colonic nerves (including growth-associated protein 43 [GAP-43]), and the expression of nerve growth factor (NGF) and tirosine kinase receptors A (Trk-A); 2) the impact of colonic mediators on cultured human neuronal cells differentiation and sprouting; 3) correlation between phenotypic changes and gastrointestinal symptoms. Material and methods: Mucosal biopsies were obtained from the descending colon of 42 patients with IBS and 21 HC. The expression of neuronal specific enolase (NSE), GAP-43, NGF and Trk-A was assessed by quantitative immunohistochemistry. The impact of mucosal supernatants and its NGF content on nerve sprouting and differentiation was assessed on cultured neuronal cell lines (SH-Sy5Y) by means of morphometric analysis. IBS symptoms severity and frequency were graded and their correlation with biological parameters was evaluated by non parametric Spearman rank test. Results: Patients with IBS displayed a significant increase in NSE+ and GAP43+ fibers compared with HC (P=0.036 and P=0.047; respectively). In addition, NGF and Trk-A immunoreactivity was significantly enhanced in patients with IBS (P<0.05). The majority of NGF+ reactivity was localized in tryptase + mast cells. Mucosal supernatants from IBS patients induced a 60% neuronal sprouting on cultured neuronal cell lines. This was comparable to that obtained with retinoic acid (68%) and significantly higher than that obtained with HC (41%) (P<0.05). Neuronal sprouting was partly NGF-dependent, since it was significantly reverted by NGF immunoblocking. There was no significant correlation between these biological parameters and the severity or frequency of abdominal pain/discomfort and bloating in patients with IBS. Conclusions: Nerve fibers, sprouting, NGF and Trk-A expression are significantly enhanced in patients with IBS. Mucosal mediators, mainly mast
cell-derived NGF, participate to neuronal sprouting. These biological parameters are not correlated with classical symptom patterns.
OC.05.2 PATHOGEN AND PROBIOTIC BACTERIA DIFFERENTIALLY STIMULATE NITRIC OXIDE PRODUCTION AND S100B PROTEIN EXPRESSION IN HUMAN ENTEROGLIAL CELLS F. Turco ∗ , G. Sarnelli, C. Cirillo, A. Mango, A. D’Alessandro, I. Palumbo, R. Cuomo Università “Federico II”, Dipartimento di Medicina Clinica e Sperimentale, Napoli, Italy Background and aim: Enteric glial cells (EGC) are involved in intestinal homeostasis and may contribute to regulate host-bacteria interaction. Astrocytes, the equivalent of enteroglial cells (EGC) in Central Nervous System respond to bacteria releasing nitric oxide (NO), whether this does occur in bacterial-EGC interaction is not known. We aimed to investigate whether human EGC generate NO in response to pathogen and probiotic bacteria and whether this is associated with S100B overexpression. Material and methods: Human EGC were obtained according to a method previously described by our group. Briefly, myenteric plexus preparations were isolated from ileum of patients undergoing surgery and enzimatically dissociated. Ganglia were plated and cell cultures were grown to subconfluence. After 21 days, EGC were purified by incubation with the anti-Thy-1.1 ab-coated magnetic beads and separated using a Dynal Magnet® . EGC were incubated for 24 hours with the probiotic Lactobacillus Paracasei F19 (LP F19) and the pathogen Enteroinvasive Escherichia Coli (EIEC). 2 different bacteria/cells ratios were used (0.1/1 and 10/1, respectively). Nitrite assay and Western Blot analysis were respectively used to evaluate NO release and S100B expression in stimulated cells compared to unstimulated cells that served as controls. Data are expressed as mean±SD of 3 independent experiments. Results: Glial derived S100B protein expression was significantly higher in response to EIEC than to LP F19 (+2.9±0.2 and +0.9±0.3 fold increase vs control; p<0.05). EIEC induced a significantly higher NO release than LP F19 both at a 0.1/1 (17.7±0.7 vs 4.0±0.1 nmol x 10ˆ6 cells; p<0.001) and at 10/1 ratio (20.7±2.1 vs 9.0±0.1 nmol x 10ˆ6 cells; p<0.001). Compared to control conditions (3.7±0.1 nmol x 10ˆ6 cells), EIEC and high concentration of LP F19 induced a significant increase of NO release (all p<0.001). Conclusions: We show that EGC are able to release nitric oxide when challenged with bacteria and that this is likely dependent on the different expression of S100B protein. As EGC-released NO was different between pathogen and probiotic bacteria we suggest that human EGCs likely participate to host-bacteria interaction via a different NO release.
OC.05.3 UTILITY OF THE BALLOON-EVACUATION TEST FOR IDENTIFYING PATIENTS WITH DYSSYNERGIC DEFECATION G. Chiarioni ∗ ,1 , O. Pieramico 2 , I. Vantini 1 , S. Heymen 3 , W.E. Whitehead 3 1 Divisione di Gastroenterologia Universitaria, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 2 Servizio di Endoscopia Digestiva, Ospedale Generale, Merano, Italy; 3 Center for Functional Gi Motility Disorders, University of North Carolina, Chapel Hill, United States
Background and aim: Some have suggested the balloon evacuation test (BET) could substitute for anorectal manometry (ARM) in identifying patients with dyssynergic defecation (DD) and save cost. Aims: (1) Assess test-retest reliability of the BET. (2) Determine its optimal duration. (3) Determine sensitivity and specificity of BET for detecting DD when DD is defined by ARM. (4) Explore reasons for lack of agreement between BET and ARM. Material and methods: 110 consecutive constipated patients were invited into a one-month study; 4 declined. All patients completed symptom questionnaires, underwent BET and increased iber intake up to 30 g per day, while keeping a symptom diary for 30 days. The BET was repeated after 30 days.
Abstracts of the XVII National Congress of Digestive Diseases / Digestive and Liver Disease 43S (2011) S115–S264
Material and methods: We extracted information from a prospectively maintained data-base on characteristics of CD patients diagnosed in our CD Clinic. Information included total cholesterol (TC), triglycerides, HDL cholesterol, LDL cholesterol (Friedewal formula), homocysteinemia, GGT level and BMI measured in patients studied twice, before and after at least 12 months GFD. Patients with associated diseases affecting lipid profile at baseline were excluded. Results are expressed as mean ± SD; paired and unpaired t-tests have been used as appropriate for statistical analysis. Results: A cohort of 765 CD patients (72% F) was identified. At baseline, BMI was = 25 in 74/572 (13%), homocysteinemia was >15 μmol/L in 24/68 (35%), and TC was below 200 mg/dL in 525/650 (81%). During GFD, TC increased from 171±38 to 182±36 mg/dL (p<0.0001), tryglicerides decreased from 89±48 to 80±42 (p<0.0001), GGT increased form 17±15 to 20±20 U/L (p<0.0001) and BMI increased form 21.3±3.4 to 22.6±3.5 (p<0.0001). More detailed information was available on a subgroup of 38 patients HDL increased from 47±2 mg/dL at baseline to 53±1 mg/dL (p=0.002) during GFD. LDL remained unchanged and LDL/HDL ratio decreased from 2.5±1.0 at baseline to 2.3±0.9 (p=0.05) during GFD. Homocysteinemia remained unchanged during GFD at 15.2±9.8 μmol/L Vs 17.4±8.9 at baseline (NS). Conclusions: Our study indicates that risk and protective factors for cardiovascular diseases coexist in CD both at baseline and during GFD. Hyperhomocysteinemia accompanied by increased GGT level and by increased proportion of overweight patients predicts a pro-atherogenic effect of GFD, but reduced tryglicerides and increased HDL point to the opposite effect. Although these results are in keeping with the uncertainty on prevalence of vascular outcomes in CD, they suggest that in order to be healthy GFD has to go beyond gluten exclusion to include control of body weight and of quality of nutrients.
OC.05.1 COLONIC MUCOSAL NEUROPLASTICITY AND ITS INVOLVEMENT IN SYMPTOM GENERATION IN PATIENTS WITH IRRITABLE BOWEL SYNDROME C. Cremon ∗ , V. Vasina, G. Dothel, L. Gargano, L. Zecchi, G. Carini, L. Bellacosa, R. De Giorgio, V. Stanghellini, F. De Ponti, R. Corinaldesi, G. Barbara University Of Bologna, Bologna, Italy Background and aim: Growing evidence suggests that neuro-immune crosstalk contributes to symptom generation in irritable bowel syndrome (IBS). The aims of the present study were to evaluate in the colonic mucosa of IBS patients in comparison with healthy controls (HC): 1) phenotypic changes in colonic nerves (including growth-associated protein 43 [GAP-43]), and the expression of nerve growth factor (NGF) and tirosine kinase receptors A (Trk-A); 2) the impact of colonic mediators on cultured human neuronal cells differentiation and sprouting; 3) correlation between phenotypic changes and gastrointestinal symptoms. Material and methods: Mucosal biopsies were obtained from the descending colon of 42 patients with IBS and 21 HC. The expression of neuronal specific enolase (NSE), GAP-43, NGF and Trk-A was assessed by quantitative immunohistochemistry. The impact of mucosal supernatants and its NGF content on nerve sprouting and differentiation was assessed on cultured neuronal cell lines (SH-Sy5Y) by means of morphometric analysis. IBS symptoms severity and frequency were graded and their correlation with biological parameters was evaluated by non parametric Spearman rank test. Results: Patients with IBS displayed a significant increase in NSE+ and GAP43+ fibers compared with HC (P=0.036 and P=0.047; respectively). In addition, NGF and Trk-A immunoreactivity was significantly enhanced in patients with IBS (P<0.05). The majority of NGF+ reactivity was localized in tryptase + mast cells. Mucosal supernatants from IBS patients induced a 60% neuronal sprouting on cultured neuronal cell lines. This was comparable to that obtained with retinoic acid (68%) and significantly higher than that obtained with HC (41%) (P<0.05). Neuronal sprouting was partly NGF-dependent, since it was significantly reverted by NGF immunoblocking. There was no significant correlation between these biological parameters and the severity or frequency of abdominal pain/discomfort and bloating in patients with IBS. Conclusions: Nerve fibers, sprouting, NGF and Trk-A expression are significantly enhanced in patients with IBS. Mucosal mediators, mainly mast
cell-derived NGF, participate to neuronal sprouting. These biological parameters are not correlated with classical symptom patterns.
OC.05.2 PATHOGEN AND PROBIOTIC BACTERIA DIFFERENTIALLY STIMULATE NITRIC OXIDE PRODUCTION AND S100B PROTEIN EXPRESSION IN HUMAN ENTEROGLIAL CELLS F. Turco ∗ , G. Sarnelli, C. Cirillo, A. Mango, A. D’Alessandro, I. Palumbo, R. Cuomo Università “Federico II”, Dipartimento di Medicina Clinica e Sperimentale, Napoli, Italy Background and aim: Enteric glial cells (EGC) are involved in intestinal homeostasis and may contribute to regulate host-bacteria interaction. Astrocytes, the equivalent of enteroglial cells (EGC) in Central Nervous System respond to bacteria releasing nitric oxide (NO), whether this does occur in bacterial-EGC interaction is not known. We aimed to investigate whether human EGC generate NO in response to pathogen and probiotic bacteria and whether this is associated with S100B overexpression. Material and methods: Human EGC were obtained according to a method previously described by our group. Briefly, myenteric plexus preparations were isolated from ileum of patients undergoing surgery and enzimatically dissociated. Ganglia were plated and cell cultures were grown to subconfluence. After 21 days, EGC were purified by incubation with the anti-Thy-1.1 ab-coated magnetic beads and separated using a Dynal Magnet® . EGC were incubated for 24 hours with the probiotic Lactobacillus Paracasei F19 (LP F19) and the pathogen Enteroinvasive Escherichia Coli (EIEC). 2 different bacteria/cells ratios were used (0.1/1 and 10/1, respectively). Nitrite assay and Western Blot analysis were respectively used to evaluate NO release and S100B expression in stimulated cells compared to unstimulated cells that served as controls. Data are expressed as mean±SD of 3 independent experiments. Results: Glial derived S100B protein expression was significantly higher in response to EIEC than to LP F19 (+2.9±0.2 and +0.9±0.3 fold increase vs control; p<0.05). EIEC induced a significantly higher NO release than LP F19 both at a 0.1/1 (17.7±0.7 vs 4.0±0.1 nmol x 10ˆ6 cells; p<0.001) and at 10/1 ratio (20.7±2.1 vs 9.0±0.1 nmol x 10ˆ6 cells; p<0.001). Compared to control conditions (3.7±0.1 nmol x 10ˆ6 cells), EIEC and high concentration of LP F19 induced a significant increase of NO release (all p<0.001). Conclusions: We show that EGC are able to release nitric oxide when challenged with bacteria and that this is likely dependent on the different expression of S100B protein. As EGC-released NO was different between pathogen and probiotic bacteria we suggest that human EGCs likely participate to host-bacteria interaction via a different NO release.
OC.05.3 UTILITY OF THE BALLOON-EVACUATION TEST FOR IDENTIFYING PATIENTS WITH DYSSYNERGIC DEFECATION G. Chiarioni ∗ ,1 , O. Pieramico 2 , I. Vantini 1 , S. Heymen 3 , W.E. Whitehead 3 1 Divisione di Gastroenterologia Universitaria, Azienda Ospedaliera Universitaria Integrata, Verona, Italy; 2 Servizio di Endoscopia Digestiva, Ospedale Generale, Merano, Italy; 3 Center for Functional Gi Motility Disorders, University of North Carolina, Chapel Hill, United States
Background and aim: Some have suggested the balloon evacuation test (BET) could substitute for anorectal manometry (ARM) in identifying patients with dyssynergic defecation (DD) and save cost. Aims: (1) Assess test-retest reliability of the BET. (2) Determine its optimal duration. (3) Determine sensitivity and specificity of BET for detecting DD when DD is defined by ARM. (4) Explore reasons for lack of agreement between BET and ARM. Material and methods: 110 consecutive constipated patients were invited into a one-month study; 4 declined. All patients completed symptom questionnaires, underwent BET and increased iber intake up to 30 g per day, while keeping a symptom diary for 30 days. The BET was repeated after 30 days.