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Modulation of a-1-proteinase inhibitor secretion in epithelial Caco-2 cells by different cytokines
A1340 AGA ABSTRACTS
GASTROENTEROLOGY Vol. 118, No.4
6103
6105
PRO·INFLAMMATORY CYTOKINE EXPRESSION IN SURGI· CAL WOUNDS. Brian Endlich, David Armstrong, Jason A. Brodsky, Michael Novotny, Fredrick J. Brody, Thomas A. Hamilton, Jeffrey L. Ponsky, Cleveland Clin Fdn, Cleveland, OH.
MODULATION OF A·I·PROTEINASE INHIBITOR SECRETION IN EPITHELIAL CACO·2 CELLS BY DIFFERENT CYTOKINES. Dominik Faust, Katrin Raschke, Julia Schlichting, Wolfgang F. Caspary, Juergen Stein, 2nd Dept of Internal Medicine and Gastroenterology, Frankfurt/Main, Germany; 2nd Dept of Medicine and Gastroenterology, Frankfurt/Main, Germany. Introduction: o-l-proteinase inhibitor (a-I-PI) is the main serine proteinase inhibitor in human plasma. Although it is predominantly liver-derived, recent studies showed that a-I-PI is also synthesized and secreted locally by human intestinal epithelial cell lines. Furthermore we have shown that a-I-PI secretion is enhanced by coculture with macrophages (M-derived cytokines. Methods: Confluent enterocyte-like Caco-2 cells(l4-21 days of culture) were treated with I or 10 Units/ml (1 or IOng/ml) of each cytokine. After twenty-four hours supernatants were tested for a-I-PI concentrations (ELISA), and the cells were prepared for a-I-PI Western blot analysis. Results: Both, Interleukin (IL)-2 and 8 cause a significant and dose dependent increase of a-I-PI secretion, whereas Interferon (INF)-r decreases a-I-PI secretion. IL-I {3 also decreases a-I-PI secretion but not in a dose dependent manner. However, IL-6 had no effect on a-I-PI secretion. All results obtained by ELISA were confirmed by the Western blot analysis performed with lysed Caco-2 cells (data not shown). Conclusion: These data imply that in vitro a-I-PI secretion of enterocytelike Caco-2 cells is highly regulated by the cytokines IL-I {3, IL-2, IL-8, and INF--y. IL-I {3, IL-6 and IL-8 are released by M<1>. IL-I {3 causes a decrease and IL-8 an increase of a-I-PI secretion. Although IL-6 is known to enhance synthesis of acute phase proteins and hepatic a-I-PI release, it failed to modulate a-I-PI secretion in Caco-2 cells. IL-2, which increases, and INF-r which decreases, a-I-PI secretion are both synthesized by CD4 + T lymphocytes. These results may allow the hypothesis that in humans, Iymphocyte- and M-derived cytokines are involved in the local regulation of a-I-PI secretion by enterocytes.
Surgical wounds provoke an inflammatory cascade which includes the temporally ordered production of cytokines and chemokines. This sequence of secretory events orchestrates the sequential infiltration of inflammatory leukocytes and regulates their function. The objective of this study is to define the cause and effect relationships between cytokine expression patterns, leukocyte infiltration, and normal and pathogenic wound healing outcomes. To examine the expression of cytokines and chemokines in surgical wounds a 20 mm abdominal incision though the dermis and peritoneum was made in anesthetized C57BL6 mice. The incisions were closed and at specified times skin, peritoneum, and serum were collected. Cytokine mRNA expression was evaluated by Northern hybridization and RNAse protection assays. Cytokine protein levels in tissue and serum were determined by ELISA. Neutrophil and monocyte infiltration was monitored using immunohistochemistry. Cytokine/chemokine mRNA expression was detected in 3 temporal waves. Within 2 hours IL-I{3, MIP-Ia, MIP-I{3, MIP-2, KC, Eotaxin and RANTES were detected; while some cytokine/chemokine mRNAs were sustained (IL-I (3, MIP-la, MIP-2) others were transient (KC, Eotaxin, RANTES). No TNFaexpression was detected. An intermediate wave of chemokine expression, represented by MCP-I followed the early wave by approximately 2-4 hr. A later wave of cytokine expression was also observed which included IL-6 and TGF{3. In general, cytokine/chemokine protein levels detected in tissue reflected the levels of the corresponding mRNA. Serum cytokines were detectable within I hr and peaked between 2 and 4 hrs post wounding. Neutrophil infiltration was detected within 3-4 hrs while monocytes were first seen between 8 and 16 hrs. The current data suggest that surgical trauma induces the expression and secretion of a wide range of pro-inflammatory cytokines and chemokines. The early events are suggestive of a critical role for IL-I as the inducer of chemokines which are, in turn, responsible for the sequential inflammatory leukocyte infiltration. Ongoing experiments will provide more precise cause and effect relationships through selective manipulation (addition or deletion) of specific cytokine/chemokine participants.
ELISA Results (~g/I a-1-PI) Cytokine IL-1Jj IL-2 IL·6 IL·a INF-y
6104 RAPID AND PROLONGED CLOSURE OF CROHN'S DISEASE FISTULAS UNDER INTENSIFIED IMMUNOSUPPRESSIVE THERAPY. Eva Engelhardt, A. Bantosz Rozhowski, Bertram Wiedenmann, Axel U. Dignass, Charite Med Sch, Berlin, Germany. Objective: The management of fistulas complicating Crohn's disease remains a difficult clinical challenge. Relapse rates following medical and surgical treatment are high and medical treatment is often limited by side effects. Infliximab and intravenous administration of cyclosporine A (CyA) have shown dramatic beneficial effects in patients with refractory fistulizing Crohn's disease, however, the long-term benefit after discontinuation of this drugs has been disappointing. The aim of this study was to assess the outcome of an intensified immunosuppressive therapy on patients with refractory fistulizing Crohn s disease. Patients: Six patients with Crohn's disease complicated by chronic draining perianal fistulas unresponsive to previous surgery, steroids and antibiotics were enrolled in this open study. All patients were initially treated with continuous intravenous CyA (4mg/ kg/day) for 7 to 12 days. Azathioprine (2mg/kg/d), prednisolone and aminosalicylates in varying doses were also given. Intravenous CyA was converted to oral CyA (6-8mg/kg/d) after 7 to 12 days and steroids were tapered. Effectiveness of this treatment was evaluated clinically and side effects were recorded. Results: All six patients responded to this immunosuppressive regimen with decreased drainage and with improvement in perianal inflammation, perianal pain and patient comfort. Complete resolution of drainage was observed in 12 from 15 fistulas. No deterioration was observed, when intravenous CyA was converted to oral CyA. Steroids were tapered and discontinued in five patients. Oral CyA was discontinued after ten to fourteen weeks. In two patients a prolonged improvement of fistulas was observed. Three patients deteriorated slightly with intermittent drainage of at least one fistula without inflammation and pain and one patient had a relapse. No serious side effects were observed. Side effects included intermittent paresthesia of hands and feet, minor increase of creatinine and were completely reversible after dose reduction of CyA and did not necessitate discontinuation of medical treatment. Conclusions: An intensified immunosuppressive regimen with cyclosporine A, azathioprine and steroids leads to marked and rapid improvement of perianal fistulas in Crohn's disease that was maintained at least partially in the majority of patients. These findings warrant further studies with more patients and long-term follow-up of patients to assess the long-term benefit of this therapeutic approach.
Control
1ng(U)/ml
10ng(Uyml
P
857±177 1009±87 1191±77 140±1 332±24
953±93 1225±155 1145±160 160±8 305+13
421±25 1574±116 1123±37 166±5 214+25
0.0091 0.0012 not significant 0.0424 0.0018
6106 A SIX-MONTH OPEN LABEL ASSESSMENT OF STEROID SPARING PROPERTIES OF CDP571, A HUMANIZED ANTIBODY TO TNFA IN STEROID DEPENDENT PATIENTS WITH CROHN'S DISEASE. Brian G. Feagan, Stephen B. Hanauer, F. Cominelli, Lloyd R. Sutherland, C. D. Elson, J. P. Baker, G. R. Lichtenstein, A. Archambault, C. Bernstein, Patricia K. Heath, William J. Sandborn, Univ of Western Ontario, London, ON, Canada; Univ of Chicago, Chicago, IL; Univ of Virginia, Charlottesville, VA; Univ of Calgary, Calgary, AB, Canada; Univ of Alabama at Birmingham, Birmingham, AL; Univ of Toronto, Toronto, ON, Canada; Univ of Pennsylvania, Philadelphia, PA; Univ of Montreal, Montreal, PQ, Canada; Univ of Manitoba, Winnipeg, MB, Canada; Celltech Therapeutics Inc, Slough, United Kingdom; Mayo Clin, Rochester, MN. Background. The humanized anti-TNFa antibody, CDP571, has been shown in a 16 week, double-blind, placebo controlled study to be steroid sparing in patients who are dependent upon steroids to maintain remission of Crohn's disease (CDAI score <150) (Trial 010). Aims. To evaluate the long-term steroid sparing (off steroids and without flare) potential of CDP57 I over 10 months. To evaluate the safety and immunogenicity of CDP57 I in steroid dependent patients, following multiple intravenous infusions for up to one year. Methods. Patients who had entered Trial 010 were eligible to enter the open label extension phase if they had either (a)flared during steroid withdrawal or (b)had successfully completed (withdrawn steroids without flaring) the full four months of blind treatment (CDP571 vs placebo, every 8 weeks). CDP57 I (lOmg/kg) was dosed every 8 weeks by slow i.v. infusion. Crohn's Disease Activity Index, Inflammatory Bowel Disease Questionnaire, C reactive protein and sedimentation rate (ESR) were assessed throughout the 24 weeks of open-label study. Adverse events, anti-idiotype response and auto-antibody formation were continuously monitored. Steroid and immunosuppressant use was monitored. Results. 17/39 CDP571 treated patients successfully completed blinded phase of Trial 01O(versus 7/32 placebo treated patients, p=0.049). Fifty-five of the initial 71 patients who entered the blinded phase entered the open label phase, 32 had originally received CDP571 and 23 had received placebo. Data will be presented on the number of patients who received CDP571 and remained off steroids without flare for this additional 24-week open phase. Data will also be presented on treatment response to open-label CDP571 in patients who failed to respond to placebo in the blinded phase. Safety and immunogenicity of CDP57 I following multiple doses of CDP571 over I year in steroid dependent patients will be presented.
GASTROENTEROLOGY Vol. 118, No.4
6103
6105
PRO·INFLAMMATORY CYTOKINE EXPRESSION IN SURGI· CAL WOUNDS. Brian Endlich, David Armstrong, Jason A. Brodsky, Michael Novotny, Fredrick J. Brody, Thomas A. Hamilton, Jeffrey L. Ponsky, Cleveland Clin Fdn, Cleveland, OH.
MODULATION OF A·I·PROTEINASE INHIBITOR SECRETION IN EPITHELIAL CACO·2 CELLS BY DIFFERENT CYTOKINES. Dominik Faust, Katrin Raschke, Julia Schlichting, Wolfgang F. Caspary, Juergen Stein, 2nd Dept of Internal Medicine and Gastroenterology, Frankfurt/Main, Germany; 2nd Dept of Medicine and Gastroenterology, Frankfurt/Main, Germany. Introduction: o-l-proteinase inhibitor (a-I-PI) is the main serine proteinase inhibitor in human plasma. Although it is predominantly liver-derived, recent studies showed that a-I-PI is also synthesized and secreted locally by human intestinal epithelial cell lines. Furthermore we have shown that a-I-PI secretion is enhanced by coculture with macrophages (M
Surgical wounds provoke an inflammatory cascade which includes the temporally ordered production of cytokines and chemokines. This sequence of secretory events orchestrates the sequential infiltration of inflammatory leukocytes and regulates their function. The objective of this study is to define the cause and effect relationships between cytokine expression patterns, leukocyte infiltration, and normal and pathogenic wound healing outcomes. To examine the expression of cytokines and chemokines in surgical wounds a 20 mm abdominal incision though the dermis and peritoneum was made in anesthetized C57BL6 mice. The incisions were closed and at specified times skin, peritoneum, and serum were collected. Cytokine mRNA expression was evaluated by Northern hybridization and RNAse protection assays. Cytokine protein levels in tissue and serum were determined by ELISA. Neutrophil and monocyte infiltration was monitored using immunohistochemistry. Cytokine/chemokine mRNA expression was detected in 3 temporal waves. Within 2 hours IL-I{3, MIP-Ia, MIP-I{3, MIP-2, KC, Eotaxin and RANTES were detected; while some cytokine/chemokine mRNAs were sustained (IL-I (3, MIP-la, MIP-2) others were transient (KC, Eotaxin, RANTES). No TNFaexpression was detected. An intermediate wave of chemokine expression, represented by MCP-I followed the early wave by approximately 2-4 hr. A later wave of cytokine expression was also observed which included IL-6 and TGF{3. In general, cytokine/chemokine protein levels detected in tissue reflected the levels of the corresponding mRNA. Serum cytokines were detectable within I hr and peaked between 2 and 4 hrs post wounding. Neutrophil infiltration was detected within 3-4 hrs while monocytes were first seen between 8 and 16 hrs. The current data suggest that surgical trauma induces the expression and secretion of a wide range of pro-inflammatory cytokines and chemokines. The early events are suggestive of a critical role for IL-I as the inducer of chemokines which are, in turn, responsible for the sequential inflammatory leukocyte infiltration. Ongoing experiments will provide more precise cause and effect relationships through selective manipulation (addition or deletion) of specific cytokine/chemokine participants.
ELISA Results (~g/I a-1-PI) Cytokine IL-1Jj IL-2 IL·6 IL·a INF-y
6104 RAPID AND PROLONGED CLOSURE OF CROHN'S DISEASE FISTULAS UNDER INTENSIFIED IMMUNOSUPPRESSIVE THERAPY. Eva Engelhardt, A. Bantosz Rozhowski, Bertram Wiedenmann, Axel U. Dignass, Charite Med Sch, Berlin, Germany. Objective: The management of fistulas complicating Crohn's disease remains a difficult clinical challenge. Relapse rates following medical and surgical treatment are high and medical treatment is often limited by side effects. Infliximab and intravenous administration of cyclosporine A (CyA) have shown dramatic beneficial effects in patients with refractory fistulizing Crohn's disease, however, the long-term benefit after discontinuation of this drugs has been disappointing. The aim of this study was to assess the outcome of an intensified immunosuppressive therapy on patients with refractory fistulizing Crohn s disease. Patients: Six patients with Crohn's disease complicated by chronic draining perianal fistulas unresponsive to previous surgery, steroids and antibiotics were enrolled in this open study. All patients were initially treated with continuous intravenous CyA (4mg/ kg/day) for 7 to 12 days. Azathioprine (2mg/kg/d), prednisolone and aminosalicylates in varying doses were also given. Intravenous CyA was converted to oral CyA (6-8mg/kg/d) after 7 to 12 days and steroids were tapered. Effectiveness of this treatment was evaluated clinically and side effects were recorded. Results: All six patients responded to this immunosuppressive regimen with decreased drainage and with improvement in perianal inflammation, perianal pain and patient comfort. Complete resolution of drainage was observed in 12 from 15 fistulas. No deterioration was observed, when intravenous CyA was converted to oral CyA. Steroids were tapered and discontinued in five patients. Oral CyA was discontinued after ten to fourteen weeks. In two patients a prolonged improvement of fistulas was observed. Three patients deteriorated slightly with intermittent drainage of at least one fistula without inflammation and pain and one patient had a relapse. No serious side effects were observed. Side effects included intermittent paresthesia of hands and feet, minor increase of creatinine and were completely reversible after dose reduction of CyA and did not necessitate discontinuation of medical treatment. Conclusions: An intensified immunosuppressive regimen with cyclosporine A, azathioprine and steroids leads to marked and rapid improvement of perianal fistulas in Crohn's disease that was maintained at least partially in the majority of patients. These findings warrant further studies with more patients and long-term follow-up of patients to assess the long-term benefit of this therapeutic approach.
Control
1ng(U)/ml
10ng(Uyml
P
857±177 1009±87 1191±77 140±1 332±24
953±93 1225±155 1145±160 160±8 305+13
421±25 1574±116 1123±37 166±5 214+25
0.0091 0.0012 not significant 0.0424 0.0018
6106 A SIX-MONTH OPEN LABEL ASSESSMENT OF STEROID SPARING PROPERTIES OF CDP571, A HUMANIZED ANTIBODY TO TNFA IN STEROID DEPENDENT PATIENTS WITH CROHN'S DISEASE. Brian G. Feagan, Stephen B. Hanauer, F. Cominelli, Lloyd R. Sutherland, C. D. Elson, J. P. Baker, G. R. Lichtenstein, A. Archambault, C. Bernstein, Patricia K. Heath, William J. Sandborn, Univ of Western Ontario, London, ON, Canada; Univ of Chicago, Chicago, IL; Univ of Virginia, Charlottesville, VA; Univ of Calgary, Calgary, AB, Canada; Univ of Alabama at Birmingham, Birmingham, AL; Univ of Toronto, Toronto, ON, Canada; Univ of Pennsylvania, Philadelphia, PA; Univ of Montreal, Montreal, PQ, Canada; Univ of Manitoba, Winnipeg, MB, Canada; Celltech Therapeutics Inc, Slough, United Kingdom; Mayo Clin, Rochester, MN. Background. The humanized anti-TNFa antibody, CDP571, has been shown in a 16 week, double-blind, placebo controlled study to be steroid sparing in patients who are dependent upon steroids to maintain remission of Crohn's disease (CDAI score <150) (Trial 010). Aims. To evaluate the long-term steroid sparing (off steroids and without flare) potential of CDP57 I over 10 months. To evaluate the safety and immunogenicity of CDP57 I in steroid dependent patients, following multiple intravenous infusions for up to one year. Methods. Patients who had entered Trial 010 were eligible to enter the open label extension phase if they had either (a)flared during steroid withdrawal or (b)had successfully completed (withdrawn steroids without flaring) the full four months of blind treatment (CDP571 vs placebo, every 8 weeks). CDP57 I (lOmg/kg) was dosed every 8 weeks by slow i.v. infusion. Crohn's Disease Activity Index, Inflammatory Bowel Disease Questionnaire, C reactive protein and sedimentation rate (ESR) were assessed throughout the 24 weeks of open-label study. Adverse events, anti-idiotype response and auto-antibody formation were continuously monitored. Steroid and immunosuppressant use was monitored. Results. 17/39 CDP571 treated patients successfully completed blinded phase of Trial 01O(versus 7/32 placebo treated patients, p=0.049). Fifty-five of the initial 71 patients who entered the blinded phase entered the open label phase, 32 had originally received CDP571 and 23 had received placebo. Data will be presented on the number of patients who received CDP571 and remained off steroids without flare for this additional 24-week open phase. Data will also be presented on treatment response to open-label CDP571 in patients who failed to respond to placebo in the blinded phase. Safety and immunogenicity of CDP57 I following multiple doses of CDP571 over I year in steroid dependent patients will be presented.