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The effect of adenosine on intestinal wound healing in an invitro model
April 1995
THE EFFECT OF ADENOSINE ON INTESTINAL WOUND HEALING IN AN INVITRO MODEL. T. Smith, R. Emerson, V. Wang, B. Purtic, and P. Harmatz. Children's Hospital Oakland Research Institute, Oakland, CA. The intestinal epithelial barrier acts to limit uptake of foreign proteins, toxins, and microorganisms from the intestinal lumen. Failure of this barrier occurs in ulcerative colitis with probable uptake of chemotaedc bacterial products from the gut lumen and accumulation of neutrophils in the intestine. Adenosine is a potent inhibitor of neutrophil adhesion to endothelium and accumulation at sites of inflammation. The aim of this study was to determine whether adenosine might influence development of inflammatory process in the intestine by enhancing intestinal wound healing in an invitro model. Methods: IEC-6 cells were grown to confluence in 5% FBS/DMEM. After 24 hrs culture in 0.1% FBS, monolayers wounded with a sterile razor blade and treated with adenosine at concentrations 0.001 mM, 0.01 mM, 0.1 raM, 1.0 raM, and 10 raM. DMEM/5% FBS and DMEM/0.1% FBS were used as positive and negative controls, respectively. Following 24 hours of incubation at 37 C in 10% CO2, wound healing was assessed by quantitating the number of cells crossing a 0.5 cm length of wound edge. Treatment effects were compared by ANOVA. Results: Adenosine significantly enhanced wound healing compared to DMEM/0A% controls. This effect was greatest at 0.01 mM adenosine (M+SEM: 250+12 vs 182+15, p <0.05). Conclusions: Our results suggest that adenosine promotes intestinal wound healing in an in vitro model. We speculate that adenosine may play an important role in the treatment of ulcerative colitis by enhancing reconstitution of the epithelial barrier, limiting uptake of inflammatory agents from the gut, and reducing migration of neutrophils into the colonic epithelium.
• SENSITIVE MARKERS OF RENAL DYSFUNCTION ARE ELEVATED IN CHRONIC ULCERATIVE COLITIS (CUC). Asacol ® Study Group: C. Sninsky, S. Hanauer, B. Powers, M. Robinson, J. Mayle, C. Elson, M. DeMicco, J. Butt, R. Pruitt, J. McHattie, J. Bozdech, M. Safdi, M. Gurney, A. Fixelle, D. Wolf, A. Levin, J. Smoots (with sites located in FL, IL, CO, OK, MI, AL, CA, MO, TN, Canada, OH, OR, GA, WA). Most clinical studies have demonstrated the safety of mesalamine preparations in man (NEJM 317:1625,1987 and Ann.Int.Med 115:350,1991), however dose-related nephrotoxicity occurs in animals. Besides commonly used renal function tests [serum blood urea nitrogen (BUN), serum creatinine (Cr), and creatinine clearance (CrC1)], we evaluated three sensitive markers of renal function [urinary B2 microglobulin (B2M), alanine aminopeptidase (AaP), and N-acetyl-B-Dglueosaminidase (NAG)] during a multicenter randomized 6-month trial comparing mesalamine 0.8 g/d, mesalamine 1.6 g/d or placebo in the maintenance of remissions in 264 patients. Appropriate samples were obtained at entry and at regular intervals. Results: BUN, Cr, CrC1, and B2M values remained within the normal range in all groups. Although the overall mean(+SEM) AaP and NaG values were normal at baseline(10.0+0.5 (nl AaP range 2.0-11.0 U/L) and 5.1+0.3 (nl NaG range 0.0-5.9 U/L), respectively) a substantial st/bset of subjects had an elevated AaP or NaG value. These values did not change in any group during the 6-month period. AaP and NaG values were significantly(p<0.003) higher in males than females, 11.85.-0.7 vs. 7.9+0.6 and 6.0+0.6 vs. 4.1+0.3, respectively, but no correlation was observed with the dose or extent and duration of disease. Entry mean values for AaP and NaG were similar whether the patienfwas maintained prestudy on sulfasalazine(SASP) or Asacol. At entrY, 40% of SASPtreated and 33% of Asacol-treated patients had an elevated AaP Value. An elevated NaG value was noted in 26% of SASP-treated and 28% of Asacol-lreated patients at entry. In conclusion, Aap and NaG'values are elevated in the absence of clinically significant renal dysfunction in a substantial subgroup of patients maintained on mesalamine-containing formulations(including SASP). Further studies will be required to determine 1) the clinieal significance of these findings, 2) if there is an intrinsic renal defeet or 3) a drug-related abnormality in CUC patients. (This work was supported by Procter&Gamble Pharmaceuticals.)
Immunology, Microbiology, and Inflammatory Disorders A919
I N C R E A S E D EXPRESSION OF CARCINOEMBRYONIC ANTIGEN (CEA) IN THE NORMAL PROXIMAL COLON AND UPREGULATION IN ACTIVE ULCERATIVE COLITIS. JE Smithson, BF Warren, S Young*, R Pigott** and DP Jewelt. Radcliffe Infirmary and **British Biotechnology Ltd, Oxford and *ICRF Applied Development Laboratory, St Bartholomew's Hospital, London, UK. Aims To investigate the relative expression of epithelial molecules in different parts of the normal colon and in ulcerative colitis (UC) using novel monoclonal antibodies (MAbs). Methods Mice were immunized using homogenized biopsies of normal proximal and distal colon. Tolerization with cyclophosphamide (Gastro 106 A776) was used in order to generate MAbs with site-specific reactivity in the normal colon. MAbs produced from 5 fusions were screened on frozen sections by immunoperoxidase staining. Results MAb 2E8, a colon-specific antibody reactive with apical epithelial membranes, displayed increased staining with normal proximal colon compared to the rectum and enhanced reactivity with inflamed UC colon. Average semiquantitafive scores for MAb 2E8 assigned by two blinded observers are shown. Scale: 0-3 (none to dense staining). Scores for paired sections of normal proximal colon (NP) and normal rectum (NR) are connected by lines. Other groups: quiescent UC proximal colon (UCPQ), quiescent UC rectum (UCQR), inflamed UC proximal colon (UCPA) and inflamed UC rectum (UCRA). 0 DDO
Score
1%. g:o
,,.
"'"
C~Q
U~RQ UCPA UCRA NP NR U Statistics (2-tailed): NR v NP, p
• DIETHYLHOMOSPERMINE (DEHSPM) REDUCES REFRACTORY AIDS-RELATED DIARRHEA(PHASE I STUDY). C.A. Sninsky, R.R. Streiff, R.J. Bergemn Dept. Medicine & Medicinal Chemistry, VA Medical Center and University of Florida, Gainesville, FL. Refractory diarrhea is common in the later stages of AIDS and is generally due to an untreatable or unresponsive opportunistic infection or AIDS enteropathy. We previously showed that DEHSPM is a potent inhibitor of castor oil-induced diarrhea, cholera toxin-induced intestinal secretion, and inhibited gastrointestinal motility in rats. We postulated that DEHSPM would reduce AiDS-related diarrhea and embarked on a FDAapproved(IND 43,746) single center, open label pilot study to treat five patients (Phase I study). Patients were admitted to the Clinical Research Center at the Un. FL for a 10 d admission consisting of an initial hydration period (24 hr) followed by a 3 d baseline; 3 d DEHSPM infusion at 0,25 mg/kg Lv, t.i.d.; and a 3 d washout period. We studied patients with diarrhea in whom aggressive medical management had either become exhausted or provided suboptimal symptomatic relief. To be eligible, patients had to have >500 gm of stool/d for >4wks. A "complete response" was defined as a reduction in stool volume <250 gm/d and a "partial response" was defined as a reduction >50% on the last day of DEHSPM. Baseline diarrheal parameters were calculated from the mean of the initial three day observational period regarding daily stool weight, bowel movements per day and stool consistency(1-4;formed to liquid). Figure 1,2 and 3 summarizes the results= for each patient (data expressed mean +SEM).
DEHSPM significantly reduced stool weight from 1,218+490 to 422+_.253 gm/day; stool frequency decreased from 9.6+3.2 to 5.8.+.2.8 and stool consistency improved from 3.3i~0.5 to 1.8i~0.7. A "complete response" was observed in two patients and two patients were considered a "partial response". Two of the patients had cryptosporidiosis and one had CMV. DEHSPM infusions were well tolerated and no significant changes were noted in the CBC, serum electrolytes, liver function tests, Urinanalysis, EKG, Or mean arterial blood pressure. Further trials with DEHSPM for refractory AIDS-related diarrhea are strongly indicated.: (funded predominantly by the CRC at the Un. F1 and partially by SunPharm Corp.)
THE EFFECT OF ADENOSINE ON INTESTINAL WOUND HEALING IN AN INVITRO MODEL. T. Smith, R. Emerson, V. Wang, B. Purtic, and P. Harmatz. Children's Hospital Oakland Research Institute, Oakland, CA. The intestinal epithelial barrier acts to limit uptake of foreign proteins, toxins, and microorganisms from the intestinal lumen. Failure of this barrier occurs in ulcerative colitis with probable uptake of chemotaedc bacterial products from the gut lumen and accumulation of neutrophils in the intestine. Adenosine is a potent inhibitor of neutrophil adhesion to endothelium and accumulation at sites of inflammation. The aim of this study was to determine whether adenosine might influence development of inflammatory process in the intestine by enhancing intestinal wound healing in an invitro model. Methods: IEC-6 cells were grown to confluence in 5% FBS/DMEM. After 24 hrs culture in 0.1% FBS, monolayers wounded with a sterile razor blade and treated with adenosine at concentrations 0.001 mM, 0.01 mM, 0.1 raM, 1.0 raM, and 10 raM. DMEM/5% FBS and DMEM/0.1% FBS were used as positive and negative controls, respectively. Following 24 hours of incubation at 37 C in 10% CO2, wound healing was assessed by quantitating the number of cells crossing a 0.5 cm length of wound edge. Treatment effects were compared by ANOVA. Results: Adenosine significantly enhanced wound healing compared to DMEM/0A% controls. This effect was greatest at 0.01 mM adenosine (M+SEM: 250+12 vs 182+15, p <0.05). Conclusions: Our results suggest that adenosine promotes intestinal wound healing in an in vitro model. We speculate that adenosine may play an important role in the treatment of ulcerative colitis by enhancing reconstitution of the epithelial barrier, limiting uptake of inflammatory agents from the gut, and reducing migration of neutrophils into the colonic epithelium.
• SENSITIVE MARKERS OF RENAL DYSFUNCTION ARE ELEVATED IN CHRONIC ULCERATIVE COLITIS (CUC). Asacol ® Study Group: C. Sninsky, S. Hanauer, B. Powers, M. Robinson, J. Mayle, C. Elson, M. DeMicco, J. Butt, R. Pruitt, J. McHattie, J. Bozdech, M. Safdi, M. Gurney, A. Fixelle, D. Wolf, A. Levin, J. Smoots (with sites located in FL, IL, CO, OK, MI, AL, CA, MO, TN, Canada, OH, OR, GA, WA). Most clinical studies have demonstrated the safety of mesalamine preparations in man (NEJM 317:1625,1987 and Ann.Int.Med 115:350,1991), however dose-related nephrotoxicity occurs in animals. Besides commonly used renal function tests [serum blood urea nitrogen (BUN), serum creatinine (Cr), and creatinine clearance (CrC1)], we evaluated three sensitive markers of renal function [urinary B2 microglobulin (B2M), alanine aminopeptidase (AaP), and N-acetyl-B-Dglueosaminidase (NAG)] during a multicenter randomized 6-month trial comparing mesalamine 0.8 g/d, mesalamine 1.6 g/d or placebo in the maintenance of remissions in 264 patients. Appropriate samples were obtained at entry and at regular intervals. Results: BUN, Cr, CrC1, and B2M values remained within the normal range in all groups. Although the overall mean(+SEM) AaP and NaG values were normal at baseline(10.0+0.5 (nl AaP range 2.0-11.0 U/L) and 5.1+0.3 (nl NaG range 0.0-5.9 U/L), respectively) a substantial st/bset of subjects had an elevated AaP or NaG value. These values did not change in any group during the 6-month period. AaP and NaG values were significantly(p<0.003) higher in males than females, 11.85.-0.7 vs. 7.9+0.6 and 6.0+0.6 vs. 4.1+0.3, respectively, but no correlation was observed with the dose or extent and duration of disease. Entry mean values for AaP and NaG were similar whether the patienfwas maintained prestudy on sulfasalazine(SASP) or Asacol. At entrY, 40% of SASPtreated and 33% of Asacol-treated patients had an elevated AaP Value. An elevated NaG value was noted in 26% of SASP-treated and 28% of Asacol-lreated patients at entry. In conclusion, Aap and NaG'values are elevated in the absence of clinically significant renal dysfunction in a substantial subgroup of patients maintained on mesalamine-containing formulations(including SASP). Further studies will be required to determine 1) the clinieal significance of these findings, 2) if there is an intrinsic renal defeet or 3) a drug-related abnormality in CUC patients. (This work was supported by Procter&Gamble Pharmaceuticals.)
Immunology, Microbiology, and Inflammatory Disorders A919
I N C R E A S E D EXPRESSION OF CARCINOEMBRYONIC ANTIGEN (CEA) IN THE NORMAL PROXIMAL COLON AND UPREGULATION IN ACTIVE ULCERATIVE COLITIS. JE Smithson, BF Warren, S Young*, R Pigott** and DP Jewelt. Radcliffe Infirmary and **British Biotechnology Ltd, Oxford and *ICRF Applied Development Laboratory, St Bartholomew's Hospital, London, UK. Aims To investigate the relative expression of epithelial molecules in different parts of the normal colon and in ulcerative colitis (UC) using novel monoclonal antibodies (MAbs). Methods Mice were immunized using homogenized biopsies of normal proximal and distal colon. Tolerization with cyclophosphamide (Gastro 106 A776) was used in order to generate MAbs with site-specific reactivity in the normal colon. MAbs produced from 5 fusions were screened on frozen sections by immunoperoxidase staining. Results MAb 2E8, a colon-specific antibody reactive with apical epithelial membranes, displayed increased staining with normal proximal colon compared to the rectum and enhanced reactivity with inflamed UC colon. Average semiquantitafive scores for MAb 2E8 assigned by two blinded observers are shown. Scale: 0-3 (none to dense staining). Scores for paired sections of normal proximal colon (NP) and normal rectum (NR) are connected by lines. Other groups: quiescent UC proximal colon (UCPQ), quiescent UC rectum (UCQR), inflamed UC proximal colon (UCPA) and inflamed UC rectum (UCRA). 0 DDO
Score
1%. g:o
,,.
"'"
C~Q
U~RQ UCPA UCRA NP NR U Statistics (2-tailed): NR v NP, p
• DIETHYLHOMOSPERMINE (DEHSPM) REDUCES REFRACTORY AIDS-RELATED DIARRHEA(PHASE I STUDY). C.A. Sninsky, R.R. Streiff, R.J. Bergemn Dept. Medicine & Medicinal Chemistry, VA Medical Center and University of Florida, Gainesville, FL. Refractory diarrhea is common in the later stages of AIDS and is generally due to an untreatable or unresponsive opportunistic infection or AIDS enteropathy. We previously showed that DEHSPM is a potent inhibitor of castor oil-induced diarrhea, cholera toxin-induced intestinal secretion, and inhibited gastrointestinal motility in rats. We postulated that DEHSPM would reduce AiDS-related diarrhea and embarked on a FDAapproved(IND 43,746) single center, open label pilot study to treat five patients (Phase I study). Patients were admitted to the Clinical Research Center at the Un. FL for a 10 d admission consisting of an initial hydration period (24 hr) followed by a 3 d baseline; 3 d DEHSPM infusion at 0,25 mg/kg Lv, t.i.d.; and a 3 d washout period. We studied patients with diarrhea in whom aggressive medical management had either become exhausted or provided suboptimal symptomatic relief. To be eligible, patients had to have >500 gm of stool/d for >4wks. A "complete response" was defined as a reduction in stool volume <250 gm/d and a "partial response" was defined as a reduction >50% on the last day of DEHSPM. Baseline diarrheal parameters were calculated from the mean of the initial three day observational period regarding daily stool weight, bowel movements per day and stool consistency(1-4;formed to liquid). Figure 1,2 and 3 summarizes the results= for each patient (data expressed mean +SEM).
DEHSPM significantly reduced stool weight from 1,218+490 to 422+_.253 gm/day; stool frequency decreased from 9.6+3.2 to 5.8.+.2.8 and stool consistency improved from 3.3i~0.5 to 1.8i~0.7. A "complete response" was observed in two patients and two patients were considered a "partial response". Two of the patients had cryptosporidiosis and one had CMV. DEHSPM infusions were well tolerated and no significant changes were noted in the CBC, serum electrolytes, liver function tests, Urinanalysis, EKG, Or mean arterial blood pressure. Further trials with DEHSPM for refractory AIDS-related diarrhea are strongly indicated.: (funded predominantly by the CRC at the Un. F1 and partially by SunPharm Corp.)