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OC3.05.6 DOES ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) REINDUCE IMMUNE TOLERANCE TO GLUTEN IN COELIAC DISEASE?
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Abstracts / Digestive and Liver Disease 40S (2008), S1–S195
aberrant, clonal T-cells. It is supposed that high production of proinflammatory cytokines, and chronic antigenic stimulation, due to gluten ingestion, plays a key role in inducing inflammation, resistance to apoptosis and the emergence of these T-clones. Regulatory T-cells (Treg) maintain immunological self-tolerance by active suppression of auto aggressive T-cells. Among them, Tr1 subset plays a role in the suppression of naïve and memory T-cells through IL-10 production. The role of Tr1 in human diseases is not well understood, even because there are no specific markers able to identify these cells, however recently, Galectin-10 has been proposed as a marker for functional Tr1 To better understand pathogenetic mechanisms associated with CD and clonal T-cell proliferations we investigated galectin-10 expression from gut epithelium by 2D-DIGE approaches. Material and methods: Patients were selected and grouped for histological inflammatory degree and for T pattern defined by TCR genescan analysis. Groups consisted of 7 individuals with Marsh-0 (4/7 oligoclonal), 3 with a Marsh-1 or -2 (3/3 polyclonal) and 5 with Marsh-3 (2/5 clonal T). Control consisted of 4 individuals with excluded CD. Results: We found, a parallel increase in galectin-10 levels and Marsh index in individuals with polyclonal T cells (p=0.0092), while reduced levels were evidenced from patients with clonal T cells and Marsh-3 (p=0.017). Conclusions: Data demonstrates up-production of galectin-10 in relation with inflammatory degree. From the critical role of Tr1 in immune regulation, we assume that galectin-10 up-expression is induce to an attempt to extinguish inflammation. By converse, down-regulation of galectin-10, found in the samples with clonal T and Marsh-3, suggests a reduction in Tr1 function allowing to clonal proliferation. If these overexpanded clones are those more susceptible to a malignant progression reserve further exploration. # C. Small bowel diseases 1. Celiac disease
OC3.05.5 INVOLVEMENT OF THE CD40/CD40 LIGAND PATHWAY IN COELIAC DISEASE A. Di Sabatino ∗ ,1 , L. Rovedatti 1 , L. Cantoro 1 , S. Vetrano 3 , P. Biancheri 1 , P. Cazzola 1 , E. Strada 2 , F. Broglia 2 , S. Danese 3 , G.R. Corazza 1 1 First
Department of Medicine and Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura della Malattia Celiaca, University of Pavia, Pavia; 2 Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Pavia; 3 Division of Gastroenterology, IRCCS Istituto Clinico Humanitas, Milano Background and aim: The CD40/CD40 ligand (CD40L) system is crucially invoved in immunity. Cognate interaction between CD40, expressed on immune (monocytes, macrophages, dendritic cells) and non-immune cells (fibroblasts, epithelial and endothelial cells), and CD40L, mainly expressed by activated T cells, results in up-regulation of a number of cell surface and soluble molecules which ultimately induce inflammation. To determine if CD40/CD40L pathway is involved in the pathogenesis of coeliac disease (CD), we explored the effect of CD40L blockade on T helper cell type 1 (Th1) cytokine production and expression of T-bet, a T-box transcription factor required for Th1 differentiation, in treated CD biopsies grown ex vivo. Material and methods: Multiple perendoscopic duodenal biopsies were collected from 11 treated CD patients (mean age 34.6 yrs, range 21-57). Biopsies were placed on iron grids in the central well of an organ culture dish in a tight container with 95% O2/5% CO2 at 37°C, and cultured for 24h with or without 1mg/ml peptic-tryptic digest of gliadin (PT-gliadin) in the presence or absence of 10mcg/ml antiCD40L neutralising antibody or its isotype matched control (mouse IgG). After culture, interferon (IFN)-gamma and interleukin (IL)-17 were measured in the organ culture supernatants by ELISA. T-bet was
determined in mucosal homogenates by immunoblotting and normalised for β-actin. Results: Supernatants of coeliac biopsies cultured with PT-gliadin showed significantly higher levels of IFN-gamma (mean 183.6±68.6 pg/ml; p<0.001) and IL-17 (mean 54.8±21.4 pg/ml; p<0.005) in comparison to biopsies treated with control IgG (mean 102.4±36.2 and 16.2 ± 5.7 pg/ml, respectively). The addition of the anti-CD40L neutralising antibody significantly (p<0.001) inhibited the PT-gliadininduced up-regulation of both IFN-gamma (mean 98.3±29.1) and IL-17 (18.5±8.1 pg/ml). Mucosal T-bet expression was significantly (p<0.001) down-regulated when coeliac biopsies were treated with anti-CD40L antibody. Conclusions: Our results indicate that CD40/CD40L pathway plays a key pathogenic role in CD. Disruption of CD40/CD40L interactions through monoclonal antibodies against CD40 or CD40L may offer a therapeutic alternative to gluten-free diet in CD. # C. Small bowel diseases 1. Celiac disease
OC3.05.6 DOES ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) REINDUCE IMMUNE TOLERANCE TO GLUTEN IN COELIAC DISEASE? R. Ciccocioppo ∗ ,1 , M.E. Bernardo 2 , R. Maccario 2 , A.M. Cometa 2 , F. Biagi 2 , P.I. Bianchi 1 , O. Luinetti 2 , M. Valli 1 , G. Parigi 2 , F. Locatelli 2 , G.R. Corazza 2 1 University of Pavia, Pavia; 2 IRCCS Policlinico San Matteo Foundation, Pavia
Background and aim: It is well known that allogeneic HSCT is an efficacious therapy for many non-malignant diseases such as inherited defects of haematopoiesis, immune deficiencies, metabolic diseases and autoimmune hematological cytopenias. Several case reports have also demonstrated that HSCT can cure autoimmune disorders occurring in patients with concomitant haematological malignancies. We report the case of a 5-year-old boy with thalassaemia major and coeliac disease (CD) who underwent allogeneic HSCT from an unrelated donor. Material and methods: CD was diagnosed on the basis of commonly accepted histological, serological and genetical criteria at the age of 2 years and 6 months; a second duodenal biopsy, taken after a course of 16 month gluten-free diet, showed a significant improvement of the mucosal lesions. The patient received a successful allogeneic HSCT from a fully HLA-matched unrelated volunteer after a myeloablative preparative regimen at the age of 3 years. The peripheral blood dendritic and lymphocyte subpopulations, including FoxP3+ regulatory T cells, were evaluated by flow cytometry at the time of CD diagnosis and after HSCT during both gluten-free and gluten-containing diet. Results: After HSCT a complete donor chimerism was observed. After cyclosporine discontinuation (i.e. 12 months after the allograft), the parents were instructed to reintroduce gluten in the diet being the child blind about this procedure. The antiendomysial antibodies were monitored every three months. A duodenal biopsy, taken after further 12 months, showed no modification in comparison to the second one and antiendomysial antibodies resulted always negative after HSCT. The patient growth is just above the 85th percentile for both height and weight. The cytometric characterization showed a recover in the number of both circulating dendritic cells (54 cells/ul after- vs 22 cells/ul before-HSCT) and regulatory T cells (35% of CD4+ after- vs 21% before-HSCT). Conclusions: Our data suggest that the substitution of the patient immune system with the donor one, possibly together with phenomenon of recapitulation of immune system ontogeny, may result into induction of immune tolerance towards gluten. The increase of peripheral blood dendritic cells and FoxP3+ regulatory T cells may play a major role in silencing the pathological effect of autoreactive T cells in CD. # C. Small bowel diseases 1. Celiac disease
Abstracts / Digestive and Liver Disease 40S (2008), S1–S195
aberrant, clonal T-cells. It is supposed that high production of proinflammatory cytokines, and chronic antigenic stimulation, due to gluten ingestion, plays a key role in inducing inflammation, resistance to apoptosis and the emergence of these T-clones. Regulatory T-cells (Treg) maintain immunological self-tolerance by active suppression of auto aggressive T-cells. Among them, Tr1 subset plays a role in the suppression of naïve and memory T-cells through IL-10 production. The role of Tr1 in human diseases is not well understood, even because there are no specific markers able to identify these cells, however recently, Galectin-10 has been proposed as a marker for functional Tr1 To better understand pathogenetic mechanisms associated with CD and clonal T-cell proliferations we investigated galectin-10 expression from gut epithelium by 2D-DIGE approaches. Material and methods: Patients were selected and grouped for histological inflammatory degree and for T pattern defined by TCR genescan analysis. Groups consisted of 7 individuals with Marsh-0 (4/7 oligoclonal), 3 with a Marsh-1 or -2 (3/3 polyclonal) and 5 with Marsh-3 (2/5 clonal T). Control consisted of 4 individuals with excluded CD. Results: We found, a parallel increase in galectin-10 levels and Marsh index in individuals with polyclonal T cells (p=0.0092), while reduced levels were evidenced from patients with clonal T cells and Marsh-3 (p=0.017). Conclusions: Data demonstrates up-production of galectin-10 in relation with inflammatory degree. From the critical role of Tr1 in immune regulation, we assume that galectin-10 up-expression is induce to an attempt to extinguish inflammation. By converse, down-regulation of galectin-10, found in the samples with clonal T and Marsh-3, suggests a reduction in Tr1 function allowing to clonal proliferation. If these overexpanded clones are those more susceptible to a malignant progression reserve further exploration. # C. Small bowel diseases 1. Celiac disease
OC3.05.5 INVOLVEMENT OF THE CD40/CD40 LIGAND PATHWAY IN COELIAC DISEASE A. Di Sabatino ∗ ,1 , L. Rovedatti 1 , L. Cantoro 1 , S. Vetrano 3 , P. Biancheri 1 , P. Cazzola 1 , E. Strada 2 , F. Broglia 2 , S. Danese 3 , G.R. Corazza 1 1 First
Department of Medicine and Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Centro per lo Studio e la Cura della Malattia Celiaca, University of Pavia, Pavia; 2 Endoscopy Unit, Fondazione IRCCS Policlinico S. Matteo, Pavia; 3 Division of Gastroenterology, IRCCS Istituto Clinico Humanitas, Milano Background and aim: The CD40/CD40 ligand (CD40L) system is crucially invoved in immunity. Cognate interaction between CD40, expressed on immune (monocytes, macrophages, dendritic cells) and non-immune cells (fibroblasts, epithelial and endothelial cells), and CD40L, mainly expressed by activated T cells, results in up-regulation of a number of cell surface and soluble molecules which ultimately induce inflammation. To determine if CD40/CD40L pathway is involved in the pathogenesis of coeliac disease (CD), we explored the effect of CD40L blockade on T helper cell type 1 (Th1) cytokine production and expression of T-bet, a T-box transcription factor required for Th1 differentiation, in treated CD biopsies grown ex vivo. Material and methods: Multiple perendoscopic duodenal biopsies were collected from 11 treated CD patients (mean age 34.6 yrs, range 21-57). Biopsies were placed on iron grids in the central well of an organ culture dish in a tight container with 95% O2/5% CO2 at 37°C, and cultured for 24h with or without 1mg/ml peptic-tryptic digest of gliadin (PT-gliadin) in the presence or absence of 10mcg/ml antiCD40L neutralising antibody or its isotype matched control (mouse IgG). After culture, interferon (IFN)-gamma and interleukin (IL)-17 were measured in the organ culture supernatants by ELISA. T-bet was
determined in mucosal homogenates by immunoblotting and normalised for β-actin. Results: Supernatants of coeliac biopsies cultured with PT-gliadin showed significantly higher levels of IFN-gamma (mean 183.6±68.6 pg/ml; p<0.001) and IL-17 (mean 54.8±21.4 pg/ml; p<0.005) in comparison to biopsies treated with control IgG (mean 102.4±36.2 and 16.2 ± 5.7 pg/ml, respectively). The addition of the anti-CD40L neutralising antibody significantly (p<0.001) inhibited the PT-gliadininduced up-regulation of both IFN-gamma (mean 98.3±29.1) and IL-17 (18.5±8.1 pg/ml). Mucosal T-bet expression was significantly (p<0.001) down-regulated when coeliac biopsies were treated with anti-CD40L antibody. Conclusions: Our results indicate that CD40/CD40L pathway plays a key pathogenic role in CD. Disruption of CD40/CD40L interactions through monoclonal antibodies against CD40 or CD40L may offer a therapeutic alternative to gluten-free diet in CD. # C. Small bowel diseases 1. Celiac disease
OC3.05.6 DOES ALLOGENEIC HAEMATOPOIETIC STEM CELL TRANSPLANTATION (HSCT) REINDUCE IMMUNE TOLERANCE TO GLUTEN IN COELIAC DISEASE? R. Ciccocioppo ∗ ,1 , M.E. Bernardo 2 , R. Maccario 2 , A.M. Cometa 2 , F. Biagi 2 , P.I. Bianchi 1 , O. Luinetti 2 , M. Valli 1 , G. Parigi 2 , F. Locatelli 2 , G.R. Corazza 2 1 University of Pavia, Pavia; 2 IRCCS Policlinico San Matteo Foundation, Pavia
Background and aim: It is well known that allogeneic HSCT is an efficacious therapy for many non-malignant diseases such as inherited defects of haematopoiesis, immune deficiencies, metabolic diseases and autoimmune hematological cytopenias. Several case reports have also demonstrated that HSCT can cure autoimmune disorders occurring in patients with concomitant haematological malignancies. We report the case of a 5-year-old boy with thalassaemia major and coeliac disease (CD) who underwent allogeneic HSCT from an unrelated donor. Material and methods: CD was diagnosed on the basis of commonly accepted histological, serological and genetical criteria at the age of 2 years and 6 months; a second duodenal biopsy, taken after a course of 16 month gluten-free diet, showed a significant improvement of the mucosal lesions. The patient received a successful allogeneic HSCT from a fully HLA-matched unrelated volunteer after a myeloablative preparative regimen at the age of 3 years. The peripheral blood dendritic and lymphocyte subpopulations, including FoxP3+ regulatory T cells, were evaluated by flow cytometry at the time of CD diagnosis and after HSCT during both gluten-free and gluten-containing diet. Results: After HSCT a complete donor chimerism was observed. After cyclosporine discontinuation (i.e. 12 months after the allograft), the parents were instructed to reintroduce gluten in the diet being the child blind about this procedure. The antiendomysial antibodies were monitored every three months. A duodenal biopsy, taken after further 12 months, showed no modification in comparison to the second one and antiendomysial antibodies resulted always negative after HSCT. The patient growth is just above the 85th percentile for both height and weight. The cytometric characterization showed a recover in the number of both circulating dendritic cells (54 cells/ul after- vs 22 cells/ul before-HSCT) and regulatory T cells (35% of CD4+ after- vs 21% before-HSCT). Conclusions: Our data suggest that the substitution of the patient immune system with the donor one, possibly together with phenomenon of recapitulation of immune system ontogeny, may result into induction of immune tolerance towards gluten. The increase of peripheral blood dendritic cells and FoxP3+ regulatory T cells may play a major role in silencing the pathological effect of autoreactive T cells in CD. # C. Small bowel diseases 1. Celiac disease