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Azathioprine downregulates trail expression in a T cell line and in normal human peripheral blood T cells
S256
Abstracts
AJG – Vol. 98, No. 9, Suppl., 2003
Results: Twelve patients, 9 men and 3 women (mean age: 46.5 years) with active, refractory UC received infliximab as single (2/12) or multiple (10/12) infusions. All patients were steroid-dependent at the time of initial infusion (mean dose 45 mg/day) and all but one were receiving at least 1 immunomodulator, or had failed therapy. Six patients had pancolitis and six with left-sided disease. Demographic, clinical information and response to infliximab are given in tables 1 and 2. The overall clinical response rate was 83.3% (10/12 patients); the remaining 2 patients who failed therapy underwent total colectomy. The mean response time was 5.2 days and the mean duration of response was 10 weeks. Patients with left-sided colitis required fewer infusions (mean 1.6) than those with pancolitis (mean 3.6). There was 1 serious complication of therapy: Herpes Zoster. Clinical Data in Left-side Colitis Patients Disease Response F/U after 1st Duration # Infusions Time Total # Infusion Age Sex (years) to Remission (days) Infusions (months) Outcome 38 70 36 35 75 61
M M M F M M
15 7 1 2 3 8
2 2 2 1 1 NA
2 2 14 3 2 NA
3 3 5 1 1 2
10 19 6 1 6 11
Remission Remission Remission Remission Remission Surgery
Clinical Data in Pancolitis Patients Duration Response F/u after 1st disease # infusions time Total # infusion Age Sex (years) to remission (days) infusions (months) Outcome 34 60 39 18 49 43
M F F M M M
2 19 21 2 34 2
6 Lost to f/u 7 2 1 NA
2 7 3 14 3 NA
11 2 14 4 10 2
34 months 2 months 29 months 5 months 17 months 12
Remission Lost to f/u Remission Remission Remission Surgery
Conclusions: Our experience suggests that infiximab is effective in refractory UC, inducing remission in the majority of these patients. Patients with left-sided colitis may require fewer infusions than those with pancolitis to induce remission.
771 AZATHIOPRINE DOWNREGULATES TRAIL EXPRESSION IN A T CELL LINE AND IN NORMAL HUMAN PERIPHERAL BLOOD T CELLS Carlton W. Thomas, M.D., Laurence J. Egan, M.D., Gennett M. Myhre, Renee C. Tschumper, Deborah L. Rasmussen, Sreekumar Raghavakaimal, Ph.D., Michael R. Charlton, M.D., Diane F. Jelinek, Ph.D., William J. Sandborn, M.D.*. Mayo Clinic, Rochester, MN. Purpose: Azathioprine (AZA) is a useful drug in the treatment of inflammatory bowel disease but its mechanism of action is poorly understood. We sought to investigate changes in expression of inflammatory cytokines at the gene and protein level in a T cell line and in normal human T lymphocytes in the presence of AZA. Methods: Jurkat T cells stably expressing the interleukin-1 receptor were used for these studies. Log growth phase cells were cultured with standard medium, or medium supplemented with AZA 1mol/L. After 48 hours, cells were left untreated or were stimulated with 5g/ml cross-linked OKT3 plus 0.25 ng/ml interleukin-1␣. After 12 hours, RNA was harvested and pooled from 3 independent experiments. Isolated total RNA was amplified, labeled with fluorescent probe, fragmented and hybridized to the Affymetrix U133A gene chip containing about 15,000 genes. A cutoff of a two-fold change in gene expression was used for statistical significance. Real time PCR (RTPCR) was used to verify gene chip results in an AZA dose-response fashion. Changes in protein expression were confirmed with
TRAIL ELISA (Biosource). Human T cells were extracted from whole blood using ficoll centrifugation and T cell rosetting. Results: Gene chip studies revealed a 2.5 fold reduction in TRAIL expression in AZA treated stimulated Jurkat cells compared to untreated cells. RTPCR confirmed AZA-related down-regulation of TRAIL expression with maximal effect at 10mol/L. TRAIL ELISA demonstrated a dramatic reduction in TRAIL expression from 22ng/g protein in stimulated untreated cells to undetectable levels in 10mol/L AZA treated stimulated cells. An AZA dose-dependent reduction in TRAIL expression was seen in cells with maximal inhibition at 10mol/L. A time course study showed maximal inhibition of TRAIL expression at 48 hours of AZA exposure. Dose response studies for TRAIL expression with 6-mercaptopurine (6MP) and 6-thioguanine (6TG) showed a similar dose response compared to AZA. Stimulated human T lymphocytes demonstrated a dramatic reduction in TRAIL expression in the presence of AZA from 42 ng/g protein to 17 ng/g protein. Conclusions: TRAIL expression is down-regulated in the presence of AZA in studies using a T cell line and in normal human T cells in cell culture. The effect appears to be 6-thioguanine nucleotide related as similar effects were seen with 6TG and 6MP. 772 CONVENTIONAL CROHN’S DISEASE ACTIVITY INDEX DEFINITION OF REMISSION DOSE NOT PREDICT PATIENT’S PERCEPTION OF DISEASE ACTIVITY WITH HIGH SPECIFICITY Chinyu Su, M.D., Gary R. Lichtenstein, M.D., Julius Deren, M.D., James D. Lewis, M.D.*. University of Pennsylvania, Philadelphia, PA. Purpose: Crohn’s Disease Activity Index (CDAI) is the most commonly used instrument for evaluating disease activity in patients with Crohn’s disease (CD). It incorporates 8 weighted variables that reflect physicians’ overall rating of patients’ disease state. A cutoff value of 150 was previously determined to identify most patients with a physician’s rating of remission, and has been widely accepted as the definition of remission in clinical trials of CD. No study has examined how well this instrument predicts patient’s perception of disease activity. The aim of this study is to determine the ability of CDAI to predict patients’ perception of disease activity. Methods: CDAI was prospectively calculated for patients with CD. Patients and physicians also rated disease activity on a 4-point Likert scale ranging from remission to severely active. Two-sample t test was used to compare total and subcomponents of CDAI and IBDQ among patients with CDAI⬍150. Results: There were 261 visits from 171 patients. The mean CDAI was 146.8 (STD 110.9). Most patients rated their disease activity as remission (28%) or mildly active (45%). The sensitivity and specificity of the conventional definition of remission (CDAI⬍150) for predicting patient’s perception of remission were 86% and 51%, respectively. These values were 75% and 72%, respectively, for CDAI⬍100. The table summarizes the sensitivity and specificity of different cut-points of CDAI at 25-point intervals for predicting remission. Among patients with CDAI⬍150, those who rated their disease as mildly active had significantly lower mean IBDQ, higher mean CDAI, and higher values for the following components of CDAI than those who rated their disease as in remission: number of loose stools, abdominal pain, presence of extraintestinal manifestation (p⬍0.05 for all comparisons). Patient’s perception of remission Cut-off for CDAI ⬍100 ⬍125 ⬍150 ⬍175
Physician’s assessment of remission
Sensitivity (%)
Specificity (%)
Sensitivity (%)
Specificity (%)
75 81 86 90
72 60 51 44
74 86 90 93
84 75 64 56
Abstracts
AJG – Vol. 98, No. 9, Suppl., 2003
Results: Twelve patients, 9 men and 3 women (mean age: 46.5 years) with active, refractory UC received infliximab as single (2/12) or multiple (10/12) infusions. All patients were steroid-dependent at the time of initial infusion (mean dose 45 mg/day) and all but one were receiving at least 1 immunomodulator, or had failed therapy. Six patients had pancolitis and six with left-sided disease. Demographic, clinical information and response to infliximab are given in tables 1 and 2. The overall clinical response rate was 83.3% (10/12 patients); the remaining 2 patients who failed therapy underwent total colectomy. The mean response time was 5.2 days and the mean duration of response was 10 weeks. Patients with left-sided colitis required fewer infusions (mean 1.6) than those with pancolitis (mean 3.6). There was 1 serious complication of therapy: Herpes Zoster. Clinical Data in Left-side Colitis Patients Disease Response F/U after 1st Duration # Infusions Time Total # Infusion Age Sex (years) to Remission (days) Infusions (months) Outcome 38 70 36 35 75 61
M M M F M M
15 7 1 2 3 8
2 2 2 1 1 NA
2 2 14 3 2 NA
3 3 5 1 1 2
10 19 6 1 6 11
Remission Remission Remission Remission Remission Surgery
Clinical Data in Pancolitis Patients Duration Response F/u after 1st disease # infusions time Total # infusion Age Sex (years) to remission (days) infusions (months) Outcome 34 60 39 18 49 43
M F F M M M
2 19 21 2 34 2
6 Lost to f/u 7 2 1 NA
2 7 3 14 3 NA
11 2 14 4 10 2
34 months 2 months 29 months 5 months 17 months 12
Remission Lost to f/u Remission Remission Remission Surgery
Conclusions: Our experience suggests that infiximab is effective in refractory UC, inducing remission in the majority of these patients. Patients with left-sided colitis may require fewer infusions than those with pancolitis to induce remission.
771 AZATHIOPRINE DOWNREGULATES TRAIL EXPRESSION IN A T CELL LINE AND IN NORMAL HUMAN PERIPHERAL BLOOD T CELLS Carlton W. Thomas, M.D., Laurence J. Egan, M.D., Gennett M. Myhre, Renee C. Tschumper, Deborah L. Rasmussen, Sreekumar Raghavakaimal, Ph.D., Michael R. Charlton, M.D., Diane F. Jelinek, Ph.D., William J. Sandborn, M.D.*. Mayo Clinic, Rochester, MN. Purpose: Azathioprine (AZA) is a useful drug in the treatment of inflammatory bowel disease but its mechanism of action is poorly understood. We sought to investigate changes in expression of inflammatory cytokines at the gene and protein level in a T cell line and in normal human T lymphocytes in the presence of AZA. Methods: Jurkat T cells stably expressing the interleukin-1 receptor were used for these studies. Log growth phase cells were cultured with standard medium, or medium supplemented with AZA 1mol/L. After 48 hours, cells were left untreated or were stimulated with 5g/ml cross-linked OKT3 plus 0.25 ng/ml interleukin-1␣. After 12 hours, RNA was harvested and pooled from 3 independent experiments. Isolated total RNA was amplified, labeled with fluorescent probe, fragmented and hybridized to the Affymetrix U133A gene chip containing about 15,000 genes. A cutoff of a two-fold change in gene expression was used for statistical significance. Real time PCR (RTPCR) was used to verify gene chip results in an AZA dose-response fashion. Changes in protein expression were confirmed with
TRAIL ELISA (Biosource). Human T cells were extracted from whole blood using ficoll centrifugation and T cell rosetting. Results: Gene chip studies revealed a 2.5 fold reduction in TRAIL expression in AZA treated stimulated Jurkat cells compared to untreated cells. RTPCR confirmed AZA-related down-regulation of TRAIL expression with maximal effect at 10mol/L. TRAIL ELISA demonstrated a dramatic reduction in TRAIL expression from 22ng/g protein in stimulated untreated cells to undetectable levels in 10mol/L AZA treated stimulated cells. An AZA dose-dependent reduction in TRAIL expression was seen in cells with maximal inhibition at 10mol/L. A time course study showed maximal inhibition of TRAIL expression at 48 hours of AZA exposure. Dose response studies for TRAIL expression with 6-mercaptopurine (6MP) and 6-thioguanine (6TG) showed a similar dose response compared to AZA. Stimulated human T lymphocytes demonstrated a dramatic reduction in TRAIL expression in the presence of AZA from 42 ng/g protein to 17 ng/g protein. Conclusions: TRAIL expression is down-regulated in the presence of AZA in studies using a T cell line and in normal human T cells in cell culture. The effect appears to be 6-thioguanine nucleotide related as similar effects were seen with 6TG and 6MP. 772 CONVENTIONAL CROHN’S DISEASE ACTIVITY INDEX DEFINITION OF REMISSION DOSE NOT PREDICT PATIENT’S PERCEPTION OF DISEASE ACTIVITY WITH HIGH SPECIFICITY Chinyu Su, M.D., Gary R. Lichtenstein, M.D., Julius Deren, M.D., James D. Lewis, M.D.*. University of Pennsylvania, Philadelphia, PA. Purpose: Crohn’s Disease Activity Index (CDAI) is the most commonly used instrument for evaluating disease activity in patients with Crohn’s disease (CD). It incorporates 8 weighted variables that reflect physicians’ overall rating of patients’ disease state. A cutoff value of 150 was previously determined to identify most patients with a physician’s rating of remission, and has been widely accepted as the definition of remission in clinical trials of CD. No study has examined how well this instrument predicts patient’s perception of disease activity. The aim of this study is to determine the ability of CDAI to predict patients’ perception of disease activity. Methods: CDAI was prospectively calculated for patients with CD. Patients and physicians also rated disease activity on a 4-point Likert scale ranging from remission to severely active. Two-sample t test was used to compare total and subcomponents of CDAI and IBDQ among patients with CDAI⬍150. Results: There were 261 visits from 171 patients. The mean CDAI was 146.8 (STD 110.9). Most patients rated their disease activity as remission (28%) or mildly active (45%). The sensitivity and specificity of the conventional definition of remission (CDAI⬍150) for predicting patient’s perception of remission were 86% and 51%, respectively. These values were 75% and 72%, respectively, for CDAI⬍100. The table summarizes the sensitivity and specificity of different cut-points of CDAI at 25-point intervals for predicting remission. Among patients with CDAI⬍150, those who rated their disease as mildly active had significantly lower mean IBDQ, higher mean CDAI, and higher values for the following components of CDAI than those who rated their disease as in remission: number of loose stools, abdominal pain, presence of extraintestinal manifestation (p⬍0.05 for all comparisons). Patient’s perception of remission Cut-off for CDAI ⬍100 ⬍125 ⬍150 ⬍175
Physician’s assessment of remission
Sensitivity (%)
Specificity (%)
Sensitivity (%)
Specificity (%)
75 81 86 90
72 60 51 44
74 86 90 93
84 75 64 56