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UBC Theses and Dissertations

Changes in the O-acetyl substituted sialic acids in carrageenan induced large bowel ulceration in rabbits Al-Suhail, A. Amir A. Aziz 1981

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CHANGES IN W O-ACETYL SUBSTITUTED SIALIC ACIDS IN CARRAGEENAN INDUCED LARGE BOWEL ULCERATION IN RABBITS BY A. AMIR A. AZIZ AL-SUHAIL BVM & S U n i v e r s i t y of Baghdad, 1970 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n THE FACULTY OF GRADUATE STUDIES (Department of Pathology) We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA October 1981 A. Amir A. A z i z A l - S u h a i l , 1981 I n p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f t h e r e q u i r e m e n t s f o r a n a d v a n c e d d e g r e e a t t h e U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e t h a t t h e L i b r a r y s h a l l m a k e i t f r e e l y a v a i l a b l e f o r r e f e r e n c e a n d s t u d y . I f u r t h e r a g r e e t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p u r p o s e s m a y b e g r a n t e d b y t h e h e a d o f m y d e p a r t m e n t o r b y h i s o r h e r r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t b e a l l o w e d w i t h o u t m y w r i t t e n p e r m i s s i o n . D e p a r t m e n t o f T h e U n i v e r s i t y o f B r i t i s h C o l u m b i a 2 0 7 5 W e s b r o o k P l a c e V a n c o u v e r , C a n a d a V 6 T 1W5 D E - 6 ( 2 / 7 9 ) ABSTRACT Mucus is a glycoprotein containing viscous f l u i d secreted continuously, by goblet c e l l s and by mucous glands, into the lumen of the g a s t r o i n t e s t i n a l t r a c t . Mucous forms a continuous coating over the underlying epithelium and presumably both provides protection to the sensitive mucosa and lubricates the fe c a l stream. Its protective properties have been at t r i b u t e d , i n part, to the r e l a t i v e resistance of i t s glycoproteins to digestion with p r o t e o l y t i c enzymes. This resistance has been ascribed to the presence of terminal s i a l i c acids residues. It is generally agreed that s i a l i c acids bearing an O-acetyl substituents at the C^ p o s i t i o n are more r e s i s t a n t to hydrolysis with V i b r i o  cholera neuraminidase, and therefore i t has been suggested that the presence of such substituents may protect s i a l i c acid residues from digestion by enzymes in the fecal stream. Alt e r a t i o n s i n the large i n t e s t i n a l glycoproteins have been shown to be associated with u l c e r a t i v e c o l i t i s , but the s i g n i f i c a n c e of these findings i s unclear because i t is not known whether the observed changes are a cause or a consequence of the disease. Furthermore, most chemical studies are d i f f i c u l t to interpret because the s t a r t i n g material could have been contaminated with connective tissue elements or products of the f e c a l stream. Therefore, a r a t i o n a l approach is to investigate changes i n the glycoproteins under controlled laboratory conditions. In the present study, the carrageenan model of ulcerations has been used to investigate the e f f e c t s of the large bowel ulcerations on the e p i t h e l i a l glycoproteins of the lower digestive t r a c t of the rabbit. The r e s u l t s of t h i s study showed t h a t : a) There are s i g n i f i c a n t r e g i o n a l d i f f e r e n c e s i n the percentage of s i a l i c acids bearing 0-acetyl s u b s t i t u e n t s on the polyhydroxy side chain i n normal r a b b i t s . Upper colon e p i t h e l i a l g l y c o p r o t e i n s have the smallest percentage of 0-acetyl s u b s t i t u t e d s i a l i c a c i d s . b) There i s a s i g n i f i c a n t reduction i n the percentage of side chain 0-acetyl s u b s t i t u t e d s i a l i c acids and a s i g n i f i c a n t increase i n the percentage of s i a l i c acids l a b i l e to d i g e s t i o n with V i b r i o cholera neuraminidase i n the e p i t h e l i a l g l y c o p r o t e i n s of the c e c i of the carrageenan treated r a b b i t s , . These changes were shown to be progressive and preceded both mucosal u l c e r a t i o n and the presence of s i g n i f i c a n t inflammatory response. c) Removal of carrageenan from the d i e t r e s u l t e d i n an apparent i n i t i a l p rogressive recovery c h a r a c t e r i z e d by a progressive increase i n the percentage of 0-acetyl s u b s t i t u t e d s i a l i c a c i d and a reduction i n the percentage of s i a l i c acids l a b i l e to d i g e s t i o n w i t h V i b r i o cholera neuraminidase together with gross and micro anatomical evidence of h e a l i n g . These observations were p a r t i c u l a r l y evident i n the group of animals 12 days a f t e r removal of carrageenan from the d i e t . A f t e r 20 days, however, there was an exacerbation of the disease c h a r a c t e r i z e d by inflammation and u l c e r a t i o n together w i t h reduction i n the percentage of the 0-acetyl s u b s t i t u t e d s i a l i c a c i d s . I t was concluded that i n t h i s model mucosal u l c e r a t i o n i s associated w i t h a reduction i n the percentage of 0-acetyl s u b s t i t u t e d s i a l i c acids of the e p i t h e l i a l g l y c o p r o t e i n s . - i v -TABLE OF CONTENTS Page Abs t r a c t i i Table of Contents i v L i s t of Tables v i i i L i s t of Figures x i L i s t of Appendix x i v Acknowledgment xv INTRODUCTION 1 I . Objective 1 I I . Chemistry of the i n t e s t i n a l g l y c o p r o t e i n s 1 A. D e f i n i t i o n 1 B. Stru c t u r e of the i n t e s t i n a l g l y c o p r o t e i n s 4 a) Histochemical studies 4 b) Chemical studies 12 1) General s t r u c t u r a l features 12 2) Rabbit i n t e s t i n a l g l y c o p r o t e i n s 19 I I I . Function of g a s t r o i n t e s t i n a l g l y c o p r o t e i n s 20 IV. U l c e r a t i v e c o l i t i s 21 A. General 21 B. Changes i n e p i t h e l i a l g l y c o p r o t e i n s a s s o c i a t e d with 23 u l c e r a t i v e c o l i t i s C. Histochemical studies 23 D. Chemical studies 23 V. Animal model of lower d i g e s t i v e t r a c t u l c e r a t i o n 27 A. Spontaneous c o l i t i s 27 B. Vascular impairment 29 C. Immune c o l i t i s 29 - v -Page D. As a secondary l e s i o n 30 E. Carrageenan c o l i t i s 30 VI. Rationale 35 MATERIALS AND METHODS 36 I. Materials 36 I I . General experimental design 36 III . Methods 38 A. Induction of large bowel u l c e r a t i o n 38 B. I s o l a t i o n of the e p i t h e l i a l c e l l s 40 C. Extraction of the e p i t h e l i a l glycoproteins (105,000 x g) 42 D. Frac t i o n a t i o n of the e p i t h e l i a l glycoproteins 42 E. H i s t o l o g i c a l and histochemical studies 45 F. Chemical studies 47 a) estimation of the percentage of s i a l i c acids substituted 47 at p o s i t i o n CJ/CQ b) V i b r i o cholera neuraminidase digestion studies 50 c) estimation of the percentage of s i a l i c acids substituted 52 at p o s i t i o n Cg/Cg G. S t a t i s t i c s 52 RESULTS 54 I. Preliminary studies 54 I I . Experiment #1 61 A. Experimental design 61 B. C l i n i c a l observations 61 C. Post-mortem observations 63 D. H i s t o l o g i c a l and histochemical studies 71 a) histology 71 b) histochemistry 77 - v i -Page E. Chemical studies 84 a) percentage s i a l i c acids substituted at Cy/Cg 84 b) digestion with V i b r i o cholera neuraminidase 84 F. Summary 93 I I I . Experiment #2 94 A. Experimental design 94 B. C l i n i c a l observation 94 C. Post-mortem observations 96 D. H i s t o l o g i c a l and histochemical observations 97 a) histology 97 b) histochemistry 101 E. Chemical studies 105 a) percentage s i a l i c acids substituted at Cy/Cg 105 b) digestion studies 109 F. Summary 111 IV. Experiment #3 114 A. Experimental design 114 B. C l i n i c a l observation 115 C. Post-mortem findings 115 D. H i s t o l o g i c a l and histochemical observations 115 a) histology 115 b) histochemistry 125 E. Chemical studies 133 a) percentage s i a l i c acids substituted at Cj/Cg 133 b) digestion studies 133 V. Experiment #4 139 A. Experimental design 139 B. C l i n i c a l observations 139 - v i i -Page C. Post-mortem examination 141 D. H i s t o l o g i c a l and his t o c h e m i c a l observations 141 a) h i s t o l o g y 142 b) h i s t o c h e m i s t r y 149 E. Chemical studies 156 DISCUSSION 161 PROPOSED FUTURE INVESTIGATION 189 LITERATURE CITED 192 APPENDIX 212 - v i i i -LIST OF TABLES Table Page 1. Properties and d i s t r i b u t i o n of mucusubstance i n normal gastro- 5 i n t e s t i n a l t r a c t : histochemical studies A. Stomach 5 B. Small int e s t i n e 7 C. Large i n t e s t i n e 9 2. G a s t r o i n t e s t i n a l glycoproteins: chemical studies 13 3. Ulcerative c o l i t i s , a l t e r a t i o n s in mucusubstance: histochemical 24 studies 4. Large i n t e s t i n a l glycoproteins and u l c e r a t i v e c o l i t i s : chemical 25 studies 5. Inflammatory bowel disease: animal studies 28 6. Carrageenan induced c o l i t i s 30 7. Comparison between u l c e r a t i v e c o l i t i s and carrageenan induced 34 c o l i t i s 8. E f f e c t s of various doses of carrageenan fed to guinea pigs, 55 rabbits and rats 9. Preliminary studies: i s o l a t i o n of e p i t h e l i a l glycoproteins from 56 rabbits and guinea pigs large i n t e s t i n e 10. Experiment #1: c l i n i c a l observations 62 11. Experiment #1: h i s t o l o g i c a l and histochemical observations 72 12. Percentage of C 7 / C 3 substituted s i a l i c acids in the 105,000 85 x g supernatants obtained from the e p i t h e l i a l c e l l s of the large i n t e s t i n e of the controls and carrageenan treated rabbits 13. Analysis of the s i a l i c acids i n the 105,000 x g supernatants 86 obtained from the e p i t h e l i a l c e l l s of d i f f e r e n t regions of large i n t e s t i n e of controls and degraded carrageenan treated r a b b i t s . The data presented is pooled data from both males and females 14. Percentage of s i a l i c acids in the saponified 105,000 x g super- 87 natants obtained from the e p i t h e l i a l c e l l s of the large bowel of the controls and from the carrageenan treated rabbits released a f t e r digestion with V i b r i o cholera neuraminidase - ix -Table Page 15. Percentage of the s i a l i c acids in the 105,000 x g supernatants 88 obtained from the e p i t h e l i a l c e l l s of the large bowel of the controls and from the carrageenan treated rabbits released a f t e r digestion with V i b r i o cholera neuraminidase. 16. Analysis of the s i a l i c acids i n the glycoproteins i s o l a t e d from 90 the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . 17. Experiment #2: c l i n i c a l observations 95 18. Experiment #2: h i s t o l o g i c a l and histochemical observations 19. Percentage of s i a l i c acids substituted at positions Cj/Cg and 106 C 4 and the percentage released with V i b r i o cholera neuraminidase in the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . 20. Percentage of s i a l i c acids substituted at positions Cy/Cg and 107 C 4 and the percentage of s i a l i c acids released by digestion with V i b r i o cholera neuraminidase i n the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s of the upper colon of the controls and from the degraded carrageenan treated rabbits. 21. Percentage of s i a l i c acids substituted at positions Cy/Cg and 108 C 4 and the percentage s i a l i c acids released with V i b r i o cholera neuraminidase i n the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s of the lower colon of the controls and from the degraded carrageenan treated rabbits 22. Analysis of the pooled 105,000 x g and of the pure glycoproteins 112 isolated from the cecal e p i t h e l i a l c e l l s . 23. Experiment #3: c l i n i c a l observations 116 24. Experiment #3: gross anatomical observations 117 25. Experiment #3: h i s t o l o g i c a l and histochemical observations 120 26. Percentage of s i a l i c acids substituted at positions Cf/Cg and 134 C4 and the percentage released with V i b r i o cholera neuraminidase with and without sap o n i f i c a t i o n i n the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of controls and of degraded carrageenan treated rabbits. 27. Percentage s i a l i c acids substituted at positions Cy/Cg, 137 CgCg, C 4 and percentage s i a l i c acids released from the 105,000 x g supernatants and the p u r i f i e d glycoproteins from cecal epithelium of controls and carrageenan treated r a b b i t s . - x -Table Page 28. Experiment #4: c l i n i c a l observations 140 29. Experiment #4: h i s t o l o g i c a l and histochemical observations 144 30. Percentage of s i a l i c acids substituted at positions Cj/Cg 157 and at C 4 and percentage released by digestion with V i b r i o cholera neuraminidase i n the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of control and carrageenan treated rabbits. 31. Percentage s i a l i c acids substituted at positions C 7/Cg, 160 Cg/Cg, and percentage s i a l i c acids released a f t e r digestion with V i b r i o cholera neuraminidase i n the p u r i f i e d glycoproteins and i n the pooled 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . 32. Expected colour reaction of the 0-acetyl substituted s i a l i c 169 acids of e p i t h e l i a l glycoproteins a f t e r staining with the PBT/KOH/PAs and PAT/KOH/PAS staining procedures. 33. Experiment #2: percentage of CQ, C 7/Cg, Cg/C g 0-acetyl 177 substituted s i a l i c acids i n the glycoproteins isolated from pools of the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s from controls and degraded carrageenan treated rabbits. 34. Experiment #3: percentage of CQ, C 7 , C 7/Cg, Cg/C 9 178 0-acetyl substituted s i a l i c acids of the glycoproteins isolated from pools i n the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s from controls and degraded carrageenan treated rabbits. 35. Experiment #4: percentage of CQ, C 7, C 7/C g, Cg/C 9 179 0-acetyl substituted s i a l i c acids of the e p i t h e l i a l glycoproteins i s o l a t e d from pools of 105,000 x g supernatants obtained from cecal e p i t h e l i a l c e l l s from controls and degraded carrageenan treated r a b b i t s . 36. Comparison of percentage CQ substituted s i a l i c acids i n the 180 glycoproteins isolated from pools of the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s of controls and 5 days degraded carrageenan treated rabbits from experiments number 2, 3 and 4. - x i -LIST OF FIGURES Figure Page 1. Structure of s i a l i c acids 2 2. Structure of O-acetyl substituted s i a l i c acids 3 3. Schematic diagram of the experimental design 37 4. Diagram of the d i v i s i o n of the rabbit large i n t e s t i n e into 39 regions according to anatomic markers 5. Flow chart of the procedures used for the i s o l a t i o n of lower GI 41 tract e p i t h e l i a l c e l l s from rabbits and guinea pigs 6. Flow chart of the procedures used for the preparation of 105,000 43 x g supernatants from isolated e p i t h e l i a l c e l l s 7. Flow chart of the f r a c t i o n a t i o n procedures used to i s o l a t e the 44 e p i t h e l i a l glycoproteins from the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s of the large bowel of the controls and the carrageenan treated rabbits 8. Flow chart of the h i s t o l o g i c a l and histochemical procedures 46 used to investigate the anatomical and the chemical nature of the e p i t h e l i a l glycoproteins of the large bowel of the rabbit 9. Flow diagram of the method used for the estimation of t o t a l 48 s i a l i c acid and percentage side chain s u b s t i t u t i o n at positions Cj/Cg 10. Flow diagram i l l u s t r a t i n g the method used for the estimation 51 of the percentage of s i a l i c acids released by digestion with V i b r i o cholera neuraminidase 11. Flow diagram i l l u s t r a t i n g the method used for the estimation 53 of the percentage of s i a l i c acids substituted at positions CgCq 12. Typical f r a c t i o n a t i o n of the 105,000 x g supernatant obtained 57 from rabbits cecal e p i t h e l i a l c e l l s by gel chromatography on Biogel A15M 13. Diagram of the c e l l u l o s e acetate electrophoresis performed on 58 1" x 6" c e l l u l o s e polyacetate s t r i p s i n t r i s barbital-sodium b a r b i t a l buffer pH 8.8 14. Typical f r a c t i o n a t i o n of the carbohydrate r i c h f r a c t i o n with 60 DEAE-cellulose ion exchange column chromatography 15. Ventral view of the cadaver of 1% carrageenan treated r a b b i t : 65 note evidence of diarrhea; the dark brown f e c a l material indicates the presence of v i s i b l e f e c a l blood - x i i -Figure Page 16. Comparison between the gross anatomical features of the everted 67 cecum and upper colon of control and carrageenan treated rabbits 17. Gross anatomical comparison between the everted lower colon of 69 controls and of degraded carrageenan treated rabbits 18. Photomicrographs of H & E stained sections that compare the 73 cecal mucosa of control rabbits with the mucosa of degraded carrageenan treated rabbits 19. Photomicrographs of H & E sections to demonstrate the differences 75 between the upper colons of control degraded carrageenan treated rabbits 20. Photomicrographs of sections of control and carrageenan treated 80 rabbit cecum stained by PAT/KOH/PAS and PBT/KOH/PAS techniques to demonstrate the 0-acetyl substituted s i a l i c acids i n the e p i t h e l i a l mucins 21. The PAT/KOH/PAS and PBT/KOH/PAS techniques were used to 82 investigate the nature and d i s t r i b u t i o n of the 0-acetyl substituted s i a l i c acids i n the upper colon of con t r o l and carrageenan treated rabbits 22. Analysis of the O-acetyl substitution pattern and neuraminidase 91 s u s c e p t i b i l i t y of the s i a l i c acids i n the 105,000 x g supernatants prepared from e p i t h e l i a l c e l l s of large bowel of control rabbits 23. Analysis of the 0-acetyl s u b s t i t u t i o n pattern and neuraminidase 92 s u s c e p t i b i l i t y of the s i a l i c acids i n the 105,000 x g supernatants prepared from cecal e p i t h e l i a l c e l l s of controls and carrageenan treated rabbits 24. Photomicrograph of H & E stained sections demonstrating the 99 progressive nature of the pathological process i n the cecum of the carrageenan treated rabbits 25. Photomicrographs of sections of rabbit cecum stained by the 102 PAT/KOH/PAS technique that show the t y p i c a l e f f e c t s of carrageenan on the e p i t h e l i a l mucin 26. Percentage of s i a l i c acids substituted at positions Cy/Cg 110 and C4 and the percentage released a f t e r digestion with V i b r i o  cholera neuraminidase i n the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated rabbits. The data c i t e d for the % of s i a l i c acid substituted at Cg+Cg was obtained from the p u r i f i e d glycoproteins - x i i i -F i g " r e Page 27. Photomicrographs of H & E stained sections that show the 121 progressive nature of the carrageenan induced c o l i t i s i n the ceci of carrageenan treated rabbits 28. Photomicrographs of sections of rabbit c e c i stained with the 129 PAT/KOH/PAS and PBT/KOH/PAS procedures to demonstrate the pro-gressive nature of the changes i n the staining of the e p i t h e l i a l mucins upon treatment of the rabbits with 1% degraded carrageenan 29. Percentage of s i a l i c acids substituted at positions C-j/Cg 135 and and percentage released with V i b r i o cholera neuraminidase in the 105,000 x g supernatants obtained from cecal e p i t h e l i a l c e l l s of controls and degraded carrageenan treated r a b b i t s . The data c i t e d for the percentage of s i a l i c acid substituted at Cg/Cg was obtained from the isolated glycoproteins 30. Photomicrographs of H & E stained sections that show the 145 anatomical changes i n the cecal mucosa a f t e r removing degraded carrageenan from the diet of the treated rabbits 31. Photomicrographs of sections of rabbit cecum stained with the 150 PAT/KOH/PAS and the PBT/KOH/PAS procedures that show the changes i n the e p i t h e l i a l mucins upon removing the carrageenan from the di e t 32. Percentage of s i a l i c acids substituted at positions Cj/Cg, 158 Cg/Cg, C 4 and the percentage released with V i b r i o cholera neuraminidase i n the 105,000 x g supernatants prepared from the cecal epitheliam c e l l s of controls and carrageenan treated r a b b i t s . The data c i t e d for the percentage of s i a l i c acid substituted at Cg/Cg was obtained from p u r i f i e d glycoproteins 33. Comparison of 0-acetyl substituted s i a l i c acids i n the 105,000 184 x g supernatants prepared from cecal e p i t h e l i a l c e l l s of controls used i n experiments 1, 2, 3 and 4. The data c i t e d for the percentage of s i a l i c acid substituted at Cg/Cg was obtained from the p u r i f i e d glycoproteins - x i v -Appendix S i a l i c acids contents (ug/ml), percentages of 0-acetyl s u b s t i t u t i o n p o s i t i o n s Cj/Cg, CgCg and percentage of s i a l i c acids released by d i g e s t i o n w i t h V i b r i o cholera neuraminidase both with and without s a p o n i f i c a t i o n i n bovine s a l i v a r y mucin used as q u a l i t y c o n t r o l . - XV -Acknowledgment My many thanks and gratitude to my supervisor Dr. P.E. Reid. Indeed, this work was made possible only through his encouragement, endless support, patience and above a l l his good sense of humour. A s p e c i a l thanks and deep appreciation are extended to the members of my research committee, Professor C.F.A. C u l l i n g , Dr. D.J. Campbell, Dr. W.L. Dunn, and Dr. M.G. Clay, for t h e i r support and the many valuable suggestions and discussions. I am also g r a t e f u l to Dr. R.H. Pearce, the advisor of graduate studies i n the Department of Pathology, for h i s concern and support. His o f f i c e is always an open drop-in place to a worthy discussion. A s p e c i a l thanks to Mr. C. Ramey for providing technical help. I am going to miss his friendship, his rea d i l y available solutions to the many problems and h i s neatness i n running an overcrowded lab. My thanks are extended to the many s k i l f u l people in the histology lab who were involved i n preparing the h i s t o l o g i c a l and histochemical materials used i n th i s study. To Janet Reid and a l l the members of the Reid family, my family and I express our thanks for your h o s p i t a l i t y and caring which meant a l o t to us. We are going to remember your friendship. Thanks to Jake and Eugene. You both work hard to keep my rabbits happy. Thanks also to Sandra Sturgeon for the expert typing of this thesis. I would l i k e to express my gratitude to the Iraqi government and to the Minist r y of Higher Education and S c i e n t i f i c Research for granting me the scholarship which made my dreams a r e a l i t y . Thanks are extended to the a Awards O f f i c e i n the University of B r i t i s h Columbia for providing f i n a n c i a l support when i t was needed. - xv i -Sp e c i a l thanks to my f a m i l y , i n p a r t i c u l a r to my wif e Samira. She t o l e r a t e d me for those so many hard years and su f f e r e d to keep me happy. At l a s t , but not l e a s t , my thanks to the many people i n the Department Pathology f o r the wonderful years I spent among you, I r e a l l y f e l t at home. Thanks to everybody. - x v i i -r U - 1 -INTRODUCTION I. Objective The object of the i n v e s t i g a t i o n described i n t h i s t h e s i s was to study the changes, i f any, i n the e p i t h e l i a l g l y c o p r o t e i n s during the process of e x p e r i -mentally induced large bowel u l c e r a t i o n i n the r a b b i t . The sequence and the s i g n i f i c a n c e of these changes, and the p o s s i b l e c o r r e l a t i o n w i t h and t h e i r a p p l i c a t i o n to the disease process i n man w i l l be discussed. I I . Chemistry of the I n t e s t i n a l Glycoproteins A) D e f i n i t i o n The chemistry of the g l y c o p r o t e i n s has been e x t e n s i v e l y reviewed by Gottschalk ( 1 ) , Terho ( 2 ) , Kent (3,4), Clamp ( 5 ) , Sharon ( 6 ) , Horowitz and Pigman ( 7 ) , Horowitz (8,9), F i l i p e (10) and i n a number of papers i n a recent volume of the B r i t i s h Medical B u l l e t i n (11). "Glycoproteins can be best defined as conjugated p r o t e i n s c o n t a i n i n g as p r o s t h e t i c group(s) one or more he t e r o s a c c h a r i d e ( s ) , the l a t t e r are u s u a l l y branched, with a r e l a t i v e l y low number of sugar r e s i d u e s , l a c k i n g repeating u n i t s , and bound c o v a l e n t l y to the peptide c h a i n . " ( 1 ) . While the i n t e s t i n a l g l y c o p r o t e i n s have no unique amino a c i d composition they do c o n t a i n a c h a r a c t e r i s t i c group of sugars that includes D-galactose, L-fucose, N-acetylglucosamine, N-acetylgalactosamine, and various d e r i v a t i v e s of neuraminic a c i d - the s i a l i c acids ( 4 ) . The s i a l i c a c i d s , the N-acetyl or N - g l y c o l y l d e r i v a t i v e s of the parent D-neuraminic a c i d ( F i g . 1), are the t e r m i n a l l y located non-reducing residue of the carbohydrate p r o s t h e t i c groups of numerous i n t e s t i n a l g l y c o p r o t e i n s (1,3,6,8,38,42). Some of these s i a l i c acids are known to c a r r y 0-acetyl s u b s t i t u e n t s located at C^ and/or at various p o s i t i o n s of the polyhydroxy side chain (38,42) ( F i g . 2 ) . - 2 -Figure 1. S t r u c t u r e of the s i a l i c a c i d s . H (CgH17OgN) D-Neuramlnic acid H N-Acetyl Neuraminic Acid (Cj^H^OgN) H OH ft N-glycolylNeuraminic Acid (C..H Q0.nN) - 3 -Figure 2. S t r u c t u r e of O-acetyl S u b s t i t u t e d S i a l i c Acids Crt = u n s u b s t i t u t e d s i a l i c a c i d s - 4 -B) Structure of the I n t e s t i n a l Glycoproteins Information concerning the s t r u c t u r e of the i n t e s t i n a l g l y c o p r o t e i n s has been obtained as a r e s u l t of both h i s t o c h e m i c a l and chemical i n v e s t i g a t i o n s . a) Histochemical studies Histochemical i n v e s t i g a t i o n s of the g a s t r o i n t e s t i n a l g l y c o p r o t e i n s (10, 12-65) i n both man and animals have shown that there are v a r i a t i o n s i n the nature of these g l y c o p r o t e i n s associated with the d i f f e r e n t regions o f the g a s t r o i n t e s t i n a l t r a c t and with d i f f e r e n t l e v e l s of the e p i t h e l i a l c r y p t s . These f i n d i n g s are summarised i n Table 1. In the stomach, the mucus s e c r e t i n g c e l l s l i n i n g the surface e p i t h e l i u m , p i t , p y l o r i c and cardiac glands and the mucus neck c e l l s c o n t a i n predominantly n e u t r a l g l y c o p r o t e i n s . A c i d mucins, both s i a l y l a t e d and sulphated, have a l s o been demonstrated at the base of the f^veolae and i n the mucus neck c e l l s of the fundic region (10,11). In the f e t u s , the surface e p i t h e l i u m secretes a neuraminidase r e s i s t a n t a c i d mucin (18,19). In the small i n t e s t i n e (duodenum, jejunum, and the ileum), both n e u t r a l fucomucins and a c i d non-sulphated mucins are present (10,35,29); goblet c e l l s c l o s e to the i l e o c e c a l valwe a l s o c o n t a i n sulphated a c i d mucin (29). The sialomucins are l a r g e l y neuraminidase r e s i s t a n t . Glycoproteins c o n t a i n i n g s i a l i c acids with 0-acetyl s u b s t i t u e n t s i n t h e i r side chains have been found i n man (31), p a r t i c u l a r l y i n the ter m i n a l ileum (33,35), and i n the Brunner's glands of r a b b i t s (30,31,39,40). In the human fetus the small i n t e s t i n e contains predominantly n e u t r a l g l y c o p r o t e i n s (18,65). In the colon, the goblet c e l l s predominantly cont a i n a c i d sulphated and a c i d non-sulphated g l y c o p r o t e i n s together w i t h minimal q u a n t i t i e s of n e u t r a l mucins (10). In the l e f t c olon, sulphated g l y c o p r o t e i n s are found Table 1A. Histochemical distribution of the mucus substances of the normal stomach Investigator Species Observations Neutral mucins Sialic acid containing mucins Sulphates containing mucins Lev and Spicer (12) human surface epithelium not detected not detected Lev (13) human fundic surface and foveolar epithelium. Antrum surface and crypt cells deep fovealae, mucus neck cells, antrum crypt. Cells sialidase susceptible sulphated and uronic acid - probably mesenchymal Goldman & Ming (14) human surface epithelium fundic mucosa, deep foveolae, some neck cells occasionally (antrum) Gad (15) human surface epithelium, foveolae, cardiac and antrum glands surface epithelium foveolae (few cells); most mucus neck cells deep foveolae cells and mucus neck cells (occasional) Sippon, P. (16) Lida et al (17) human throughout surface epithelium NR NR Filipe (10) human surface epithelium mucus neck cells base of the foveolae trace Lev (18) H. foetus throughout lining epithelium Lev (19) H. foetus abundant throughout lining epithelium adult surface epithelium cardiac and pyloric glands crypt cells (small amount) NR Sheahan & Jervis (20) 11 species (all species) Throughout surface epithelium & pits Also present in deep cardiac glands, mucus neck cells of corpus and antral glands. a) Man throughout lining epithelium foveolar & mucus neck cells (scant) not detected b) Mouse cardiac surface and deep glandular epithelium (trace) antral glands c) Rat antral glands - not detected surface & foveolar epithelium of cardiac & corpus, deep cardiac glands and mucus neck cells upper mucus neck cells & antrum glands d) Hamster not seen in card La or corpus foveolae and antrum glands - 6 -Investigator Species Observations Neutral mucins Sialic acid containing mucins Sulphates containing mucins e) Gerbil NR cardiac glands, deeper antrum foveolae and glands f) Guinea pig chief cell8 cardiac and arum glands. Surface -epithelium of corpus (trace), chief cells antrum glands g) Cat surface epithelium, cardia and antrum surface epithelium, lower mucus neck cells of the corpus, antrum glands and chief cells h) Dog surface epithelium, antrum (sialo-mucin only) predominant throughout surface epithelium, traces chief cells, cardiac and antrum glands i) R. monkey parietal cells antrum epithelium, surface epithelium surface epithelium j) Baboon cardiac glands, antrum epithelium cardiac glands, mucus neck cells, antrum epithelium k) Rabbit cardiac glands cardiac glands Lev (19) Dog surface epithelium abundant in surface epithelium, abundant in the surface small amounts crypt cells Spicer (21) Dog mucus neck cells, pyloric glands abundant in surface epithelium, superfacial convolutions abundant walls surface epithelium, foveolar Spicer et a l . (22) rat super facial surface epithelium & mucus neck cells foveolar surface epithelium, deeper stratum and pits foveolar the pits cells, particularly deep in Yamada & Mitsuoukali (24) rat germ free rat surface and foveolar cells less intense reaction as compared present to conventional rats present Katsuyama & Spicer (24) rat surface epithelium orifice of gastric pits foveolar epithelium, pyloric glands Spicer (24) HID/EM, no staining rat _ .._ - - • •' •-. ; . . f - 7 -Table IB: Histochemical distribution of mucosubstance of the normal small intestine Investigator Species Observations Remarks Lev & Spicer (12) human intensely PAS reactive neutral & sialidase sensitive mucins Lev (18) H. foetus intensely PAS reactive/basophilia (AB 2-5) neutral & sialomucins, more sialidase susceptible than adult Goldman & Ming (14) human PAS reactive/basophilia 'AB 2.5) acid nonsulphated mucins Gad (15,26) human goblet cells AB/PAS reactive only acid nonsulphated partially sialidase resistant Lev (19) human PAS reactive & strong basophilia weakly reactive for sulphate fucose, siali c acid containing mucins Subba8wammy (27) human AB/PAS (2.5), v i l l i goblet cells pink base of the crypt purple neutral mucin neutral & sialomucins Pizzolato & Berger (28) human Brunner's glands, PAS reactive neutral mucins Filipe & Fenger (29) human Brunner's gland, PAS reactive goblet cells goblet cells - AB + PAS (2.5) reactive ileocaecal valve neutral neutral & sialomucins, more sialated at top of the v i l l i & the crypts traces of sulphated Filipe (10) human goblet cells Brunner's glands neutral & sialomucin (largely sialidase resistant), sulphated, may present at ileocaecal valve neutral mucins Yamada & Mitsuoukali (23) rat germ free rat PAS-reactive and basophilia more intense reaction neutral & sialomucin, with or without traces of sulphate with increased sialomucins Katsuyama & Spicer (24) rat brush borders goblet cells neutral & sialomucins sialomucin and/or sulphomucins \ Sheahan & Jervis (20) 11 species including man al l species a) brush border 2 layers b) goblet cells outer edge, acid mucin, sulphated predominant (in man & duodenum of rat sialomucin), inner layer, neutral mucins neutral mucins are present, acid mucins (sulphated and nonsulphated) considerably variable from specie to specie c) Brunner's glands, rat, cat, dog & man only neutral mucin Investigator Special Observation* lamarka rabbit •) goblet c e l l a aulphated mucins, aialoaucin predominant only i n few c e l l a b) Brunner's glaod*| aeroua acinic neutral mucin aucua tubea neutral, a i a l o and eulphated aucin C u l l i n g et a l . human goblet c e l l a - PAS reactive (30-38) rat basophilia (AB 2.S) Said et a l . mouse saponification guinea pig rabbit aialoaucin, a i a l i c acid bearing 0-acetyl aubstitutions increaaed PAS r e a c t i v i t y (30,31,39) at ileocaecal valve - a l l species, Brunner's gland - rabbit Table 1C. Hietochemical d i i t r i b u t i o n of the mucus substance of th« normal Urge intestine Investigator(s) Special Region Obaervatione Remarks Neutral mucin Sialomucin Sulphated mucin Lev » Spicer (12) human colon present moderate predoainant KOH s i g n i f i c a n t l y increaaed PAS r e a c t i v i t y Greco et a l . (47) human crypta Intermediate a bottom aurface epithelium a necka of epithelium crypta crypts fundus & intermediate part sigmoid free of neutral mucin Gad (15,26) human colon 6 rectus aurface G.C. (mainly) brush border lower part of tha crypts, bruah border sulphomucin vary greatly i n d i f -ferent segments of the colon r i l i p e (48) human aurface epithelium upper crypta moderate surface epithelium aurface epithelium (predominant), lower 4 upper portion of crypta Johnaon a Kay (49) human rectus present r e l a t i v e l y n'daae resistant present preaent mixture of acid 4 neutral mucins Lev (18,19) human a) adult b) foetua mainly predominant preaent Goldaan 4 Ming (14) human goblet c e l l s goblet c e l l a Sabbusvaamy (27) present preaent G.C. aurface epithelium and appendix - predominantly aialomucine - 10 -Histochemical distribution of the mucus substance of the normal large intestine Investigator(8) Species Region Observations Remarks Neutral mucin Sialomucin Sulphated mucin Filipe & Dawson (50) human rectum small amount, upper 1/3 of crypts upper 1/3 of crypts predominant, lower 2/3 of crypts lower 2/3 of crypts, contain no neutral mucins Dawson (51) human surface & upper 1/3 of crypts predominant upper 1/3 of crypta lower 1/3-whole crypt Filipe (52) human rectum predominant (goblet cells) Korhanen et a l . (53) human present present present sialomucin relatively resistant to to sialidase Makela et a l . (54) human colon a) brush border b) G.C. ++ largely n'dase resistant predominant ++ poximal colon more PAS reactive than distal colon, n'dase resistance due to N-acetyl-O-diacetyl sialic ac id Filipe (55,58) human upper half of crypts predominant; lower H of crypts upper H of crypts brush border 35 35 S brush border, more S in the upper than the lower *i of the epithelium, crypts Filipe & Branfoot (56) human large intestine left colon brush border brush border upper crypts & surface epithelium brush border (predominant), upper acid sulphated and nonsulphated may be present in some c e l l or in different cells right colon fetus -early l i f e lower 1/3 of crypts (predominant) predominant crypts & surface epithelium upper 2/3 of crypts (predominant) sulphated (later) Penelope et a l . (57) human colon upper crypts surface epithelium (may present) predominant throughout crypts Filipe & Branfoot (59) human colon upper crypts surface coat & lower crypts (predominant) upper crypts c Lev & Orlic (60) human colon a) adult (intense) transverse colon. Right colon -moderate. Left colon - least G.C. sulphomucin vary with location in crypts, age and with region of large intestine Histochemical d i s t r i b u t i o n of the micus substance of the normal large intestine Inveatigator(a) Spec ies Region Observations Remarks Neutral mucin Sialomucin Sulphated mucin b) 10 wk fetus up to 20 months predominant n'dase resistant, reversed after KOH sialomucins, carry H-acetyl-0-diacetyl s i a l i c acid Fenger & F i l i p e (61) human anal glands predominant n'daae resistant due to 0-acetyl substituted s i a l i c acid Subbuswamwy (62) human a) adult superfacial 4 upper crypts deep crypta (predominant) b) fetua 8 month-up aacending colon +++ considerable amounts before 3 months of age Riddle « Levin (63) human upper part of crypts (almost e n t i r e l y ) predominant lower 2/3 of crypts Isaacson 6 Ah wood (64) human recto-sigmoid present at aurface predominant F i l i p e (10) human • sialomucins bearing 0-acetyl substitution, regional 4 crypta l e v e l variations Sheahan 4 Jer v i s (20) U species including a) brush • border ( a l l species) species 4 regional v a r i a t i o n man rabbit b) goblet • c e l l ( a l l species) lower crypts proximal colon 4 lower crypts appendix •+ predominant i n d i a t a l colon, upper crypts (exclusively) spec lea 4 regional variations predominant i n d i s t a l • Caecum 4 d i s t a l colon only sulphomucln Culli n g et a l . Raid et a l . (39-467 human guinea pig rat rabbit colon eaponification (30,31,33,45,46,39,40) PAS reaction due to 0-acetyl a i a l i c acid at the aide chain s i a l i c acids are largely resistant to digestion with V. cholera neuraminidase (44) rat cytoplssmic and brush border mucins showed sim i l a r antigenic determinants (108) man - throughout the crypta rat - crypta baae guinea pig - variable rabbit - upper portion of the crypta (31) and Brunner'a gland - 1 2 -predominantly in the lower half of the epithelial crypts. In the upper half of the crypts, both sialo and sulpho mucins are present. In contrast, in the right colon s i a l i c acid-containing glycoproteins occupy the lower half of the crypts. In both the right and left colon the uppermost portion of the crypts and the surface epithelium contain neutral glycoproteins (12,32,56). The s i a l i c acids are predominantly neuraminidase resistant and contain O-acetyl substituents in the polyhydroxy side chain (30-46). In addition to the histochemical variations discussed above, variation among the individuals of the same strain, in different strains of the same species and amongst individuals of different species has been reported (20). b) Chemical studies i) General structural features The many chemical studies of the glycoproteins of the stomach (1-11,66-68, 70,120,121), small intestine (1-11,69-85,111,117,121,161-165) and large intestine (1-11,69,79,84-107,111-121,160,166-174) of both man and animals are summarized in Table 2. It is well established that the GI glycoproteins are made up of a protein core to which groups of oligosaccharides are attached (1) but the molecular weight, the molar ratio of the component sugars and the presence or absence of certain sugar residues (e.g. mannose) have been the subject of a great deal of controversy. This confusion and the incomplete knowledge of chemistry of these glycoproteins could be the result of a number of factors: (a) Many of the studies carried out in this f i e l d were incomplete and, in most cases, only analytical data of limited scope were reported. (b) Crude procedures such as mincing of the whole organ, irrigation of the mucosa and collection of the mucosal secretion, mucosal scraping, heating of - 13 -Table 2. Gastrointestinal glycoproteins: chemical investigations Investigator(s) Species GI region Methodology and Observations Remarks Gottschalk (1) glycoproteins and glycosaminoglycans - extensive review Tehro (2) human GI hexosamine containing molecules in GI homogenates and in mucosal scrapings rat 1. glycosaminoglycans - low in mucosal scrapings 2' 4"* a . H M W T glycopeptide (sulfated and sialylated), a major traction in human and in rat colon, and rat small intestine (b) neutral glycopeptide, human & rat stomach (c) LMWT glycopeptide (free of sialic acid & sulfate), contain ribose and mannose colonic glycopeptide containing 15 x more sulphate than the small intestine Clamp (5) mucus glycoproteins in health & disease Horowitz & Pigman (7) glycoconjugates Sharon (6) complex carbohydrate British Medical Journal (11)- mucus Horowitz (8) human GI tract mucopolysaccharides & glycoproteins of GI tract Horowitz (9) mucus glycoproteins - chemistry, methodology Kent (4) human GI tract Different GI tract regions produce chemically different muco-proteins. NANA is a characteristic constituent of mucus glycoproteins. Stomach and above rich in blood group active substance -Kent (3) GI tract a review - structure & function of mucus glycoproteins 1 1 Tiba (66) human gastric juice 3 fractions (ppt with alcohol), f? (non antiagglutinable carbohydrate) -in water, y (antiagglutinative carbohydrate) - inhibits agglutination agglutination or agglutination inhibition activities) blood group active, non soluble ' - specifically, o (with no Stanley & Edward (67) human s tomach gastric juice - fasting. ABO secretor: 8.8% fucose, 0.79% sialic % acids, 14.8% hexosamine, 30.2% galactose, 6.6% nitrogen, 29.7% protein. Non sec tutors has 50% less fucose dry weight \ - 14 -Investigator(s) Species GI region Methodology and Observations Remarks Masuda et a l . (68)„. human stomach mucosal scraping + proteolytic digestion, ppt with TCA zone electrophoresis, (a) hyaluronic acid, (b) heparin (c) chondroitin sulfate preparative sulfate Azuuimi et a l . rat (120) stomach extracted - triton x 100, fractionated with Biogel A1.5 (a) macromolecules - neutral & acidic glycoprotein with or with out without sulfate, (b) protein bulk - with or without carbohydrate. (c) Low mwt - hexose containing fraction fraction (a) diminished after aspirin administration Kawasaki et a l . human (70) stomach small intestine lmmunofluorescent staining, mucus glycoproteins 80% carbohydrate (hexosamine, galactose, fucose, s i a l i c acid & sulfate) and protein (principal amino acids, threonine, serine & proline) SI glycoproteins contain more sialic acid than the stomach glycoproteins Andere et a l . (71) human duodenum analysis by GLC (sugars), N-acetylglucose (3.00), N-acetyl galactose (1.31), fucose (2.5), galactose (3.45), mannose (0.47), glucose (1.65), sulfate (2.06) - molar ratio Deluca et a l . (72) rat SI Forsner (73) rat SI Coffey et a l . (74) — rat SI glucose, mannose, from serum but could be from goblet cells or from structural glycoproteins mucosal scrapiung + proteolytic digestion, chemical studies: 9.5% fucose, 27% galactose, 29.0% galactosamine, 6.0% sialic acid, 4.0% uronic acid (% weight) [l-1^C]-gluco'samine lumen contents brush border cytoplasm (membrane free) Radioactive glycoproteins isolated from: hexose hexosamine sialic acid fucose 2-82 1.0 07l8 0.22 1-75 1.0 0.08 0.15 1-22 1.0 0.20 0.23 (molar ratios to hexosamine) [1- C]-fucose IP 1 hour, mucosal scrapings, radioactivity in (40,000 mwt) fraction, 20.5% protein, 44.5% carbohydrate (fucose, hexosamine, siali c acid, sulfate and traces of phosphorous) cytoplasmic glycoproteins had least hexose content Be Ilia & Kim (75) Forstner et a l . rat SI SI Kleinman & Wolf (77) SI mucosal scrapings - water soluble extract fractionated with sephadex DEAE• Hmwt glycoprotein (major fraction), contains fucose, galactose, hexosamine, ?"d s u l f a t e " threonine, serine, proline; major amino acids, acid hydrolysis removed, si a l i c acid, fucose, galactose galactosamine mucosal scrapings, fractionated with DEAE sephadex and sepharose 4B; viscosity rose with decrease 3 fractions, negatively charged Hmwt (2x10°) glycoprotein made up concentration - flhnlf!hlHK sluT^lx 2 2 ' 4 % h e X ° 8 a * i n e ' 1 0 2 «•«• '-co., and ^ £ ' S S i c * homogenate - fucose 1, galactose 2.75, hexosamine 4.60, sialic acid 0.32 (molar ratio to fucose), 15% protein (threonine & serine major amino acids) vitamin A deficiency hexosamine decreased - 15 -Investigator(s) Species .GI region Methodology and Observations Remarks Jabbal et a l . (78) rat SI mucosal scrapings, hexose 1.26, hexosamine 0.97, s i a l i c acid 0.60, fucose 0.38 (ug/ug protein) Wold et al (79) germ free rat SI, large l^C-glucose, radioactive water soluble extract contained intestine galactose, mannose, fucose, galactosamine, glucosamine faecal contents & mucosal scrapings SI similar to large intestine but contain more protein & less sulfate Wold et a l . (80) germ free rat faecal extract & mucosal scrapings glycoproteins chemically similar, 23% galactose, 8.6% fucose, 18.6% N-acetylgalactosamine, 13.5% siali c acid, 1.3% sulfate groups fucose & siali c acids are terminally located blood group activity present Jabbal et al (81) human SI mucosal scrapings fractionated with sepharose 4B and 2B. chemically, antigenic feature and polyacrylamide gel electrophoresis, human glycoproteins similar to rat small intestine glycoproteins but hexosamine, fucose and hexosamine si a l i c acid ratio being higher in human glycoprotein Bouhours et_ a_l. guinea SI, brush fucose 2.04, galactose 3.45, glucosamine 0.49, mannose 1.34, (82) pig border galactose 0.44, glucosamine 6.04, siali c acid 0.36 and sulfate 0.50 (molar ratio to protein) Munahata & Yosizawa (83) rabbit SI f a s t i n g — * increased fucose, siali c acid and sulfate, and decreased protein mucosal scraping, Hmwt: 40% protein, 52.7% carbohydrate and 1.6% sulfate. Sugars: galactose, glucosamine, galactosamine and sialic acid, fucose, glucose and mannose present in small quantities, glutaminic acid, threonine aspartic acid, alanine, serine, lucine, proline, glycine, valine and histadine are major amino acids Keranen (84) human GI tracts gangliosides neuraminic acid 9 gangliosides were obtained from stomach 0.16 umol/g, SI 0.07 umol/g, Large I 0.11 umol/g (dry weight).each part of the GI tracts Qureshi et_ al_. human GI tract goblet cell mucin - high rawt glycoprotein contains siali c acid: (69) fucose:N-acetyl glucosamine:N-acetylgalactosamine (0.23:0.94:1.23: 0.95:1.0, molar ratio to galactosamine). 13% (w/w) protein, serine, threonine and proline major amino acids. Antigenetically rectum, SI and colonic glycoproteins are similar. Small intestine glycoproteins with DEAE Biogel fAl r run fractions (a) fucose containing glycoprotein (b) siali c acid containing glycoproteins Warner (117) Pig SI mucosal scraping + proteolytic digestion (a) fucomucin - 50% carbohydrate 50% proteins (b) sialomucins - 16 -Investigator(s) Species GI region Kupchella & dog Steggarda (85) Skypeck et a l . (86) human Korhonen & Make la (88) human Methodology and Observations Remarks GI tract mucosal scraping - homogenized ppt with cpc, fractionated with Dowex 1 x 2 (a) heparin; (b) hyaluronic acid - highest in colon; (c) chondroitin sulfate colon Rolfs et a l . (87) human rectum colon Kim et a l . (89) human colon Fraser and Clamp muconium (90) SI contained least acid muco-polysaccharides minces & papain digestion; (a) glycopeptides; (b) glycosaminoglycans 0.09M-NaCl-Lavaged-hydrolysed (100°C) acid medium. GLC fucose, mannosamine. galactosamine, glucosamine, N-acetyl-hexosamine and siali c acid The only study to report mannosamine mucosa - removed at level of muscularis mucosi - homogenized, pronase digested;15% protein and carbohydrate sugars hexose 14%, sialic acid 9%, hexosamine 6%, fucose 3%, uronic acid 3% normal appearing (away from carcinoma) mucosal scrapings homogenate. 1.18 fucose, 1.0 mannose, 1.96 galactose, i N 7 a c e t y 1 glucosamine, 1.61 N-acetylgalactosamine, 1.50 siali c acid (molar ratio to mannose) fractionated with Sepharose 2B a) mucous type glycoprotein b) mannose containing glycoproteins Gold & Miller (91) human colon mucosal scraping - phenol H20 extraction, Hmwt mucoprotein containing N-glycolyl neuraminic «rlH blc. 0d egro 8; l asub°s S:;„c 8e a l a C t 0 8 a , D i n e' « « ^ labLTall Z ^ l ^ with Gold & Miller (94) colon tissue homogenate, fucose, galactose, glucosamine, galactos and siali c acid (mostly N-glycolyl) Filipe & Cooke (95) Rogers et a l . (96) human colon mucosal scrapings, minced surgical specimens - papain digestion, V. cholerae n dase digestion released 60% of.sialic acids sialic acids might be an 0-acetyl form human colon mucosal scrapings, fractionated with Biorad AG50 and AG 2x8 ion exchanger. Acid hydrolysis followed by TLC: N-acetylneuraminic acids acids with five 0-acetyl variants - 17 -L Investigator's) Species GI region Methodology and Observations Draper & Kent (118) colon mucosal scraping - ultracentrif high mwt 4.6% ester lo in6T ~: --ugation; fraction 9.58S (major) (2xl0&) contained 25% peptide, 72% carbohydrate, sulfate ' sialic acid 8.6% N-glycolyl, 5.1% N-acetyl Kent & Marsden sheen (119) F colon Inoue & Yosizawa pie (116) Marshall & Allen pie (97) colon mucosal scrapings - proteolytic digestion, ultracentrifugation -a heavy fraction (4.96S) and a light fraction (1.73S), fraction 4.96S made up of 3.8% nitrogen, 2.4% hydrolyzable sulfate, 6.8% si a l i c acid, 11.5% neutral sugars, 20.6% aminosugars (12.4% glucosamine and 5.4% galactosamine). Threonine is the major amino acid. ^SO^ a n ( j J-^_14Q] glucose were incorporated in the heavy fraction mucosal scraping + pronase digestion, a major sulfated s i a l i c acid containing glycopeptides (mwt 2-3x10^), a minor uronic aicd containing fraction mucosal scrapings + extracted with NaN3, the water soluble extract contained 72% protein, 12% nucleic acids and 6% glycoprotein (dry weight), the glycoprotein (15x10^ mwt) and contained 10.4% fucose, 23.9% glucosamine, 8.3% galactosamine, 9.9% si a l i c acid, 20.8% galactose, 3% sulfate, 13.3% protein and was positive for blood group activity galactose and fucose were present in small quantities Winsnes et al. caecal contents f r ! " i ° n " ! - 2 W 4 i 5 h n o e p n a 2 e x 9-200 and DEAE Sephadex A50, mwt 3.2x105-2.4x100, a n d contained 81.3% carbohydrate, 0.8% sulfate, 16.1% protein, blood group activity present Murty et al (100) ~~ Reid et a l . human (102-104,106,107) Culling et al human & (105) r a t rat LI (42 106) colon 104) colon 105) (102 (103 epithelial cells were isolated by shaking with EDTA (102-105) or homogenized surgical specimens (103,106,42), isolated intact epithelial glycoproteins (fractionated with agarose A15m and then with DEAE cellulose A22). Detailed study of the si a l i c acid showed they could be (a) with no 0-acetyl substitution (polyhydroxy side chain); (b) mono, di or t r i 0-acetyl substituted (poly-hydryxy side chain); (c) the s i a l i c acid are largely resistant to digestion with vibrio cholera neuraminidase; (d) upper half of the rat colon glycoprotein contained larget percentage of 0-acetyl substituted sial i c acids than the lower half (103) Investigstor(s) Specie* CI region Methodology and Ob*erv*tion* Bemarks Nemo to k rabbit SI t large mucoaal acraping • pronaae digeation, fractionated with Sephadex SI contained more uronic acid than Yoaiaawa (101) i o t e i t i n e DBAS 25 and GSO, 3 fr a c t i o n * resolved, f r a c t i o n * (0.35 and 0.9S) colon were aulfated and a i a l i c acid containing glycopeptide* andsuronic acid, highly sulfated fraction, major amino acids include threonine, proline and s e r i n * - 19 -the crude e x t r a c t and d i g e s t i o n with p r o t e o l y t i c enzymes such as papain have of t e n been u t i l i z e d i n the preparation of the s t a r t i n g m a t e r i a l . The use of such methodology makes i t d i f f i c u l t , i f not impossible, to r e l a t e any of the s t r u c t u r a l data obtained to any s p e c i f i c g l y c o p r o t e i n because of p o s s i b l e contamination with e i t h e r connective t i s s u e components (glycosaminoglycan) and/or the f e c a l contents and p o s s i b l e degradation of the g l y c o p r o t e i n by d i g e s t i o n with p r o t e o l y t i c enzymes. However t h i s problem has been recognized and m i l d e r methods have been employed, e.g. i n our l a b o r a t o r y i s o l a t e d e p i t h e l i a l c e l l s have been used as s t a r t i n g m a t e r i a l and m i l d f r a c t i o n a t i o n and i s o l a t i o n methods used (102). (c) Species v a r i a t i o n s have been shown to occur i n the chemistry of the g a s t r o i n t e s t i n a l g l y c o p r o t e i n s (20,31). In a d d i t i o n there are d i f f e r e n c e s between the regions of the alimentary t r a c t and a l s o i n the nature of the g l y c o p r o t e i n s at various l e v e l s i n the e p i t h e l i a l c r y p t s (10,31,56). I t i s apparent that these problems cannot be resolved without e s t a b l i s h i n g a w e l l standardized i n v e s t i g a t i o n procedure. i i ) Rabbit I n t e s t i n a l Glycoproteins There have only been two studies of r a b b i t small and large i n t e s t i n a l g l y c o p r o t e i n s (83,101). In general they appear to be t y p i c a l mucin-type g l y c o p r o t e i n s : r i c h i n carbohydrate, c o n t a i n i n g s i a l i c a c i d s , fucose, g a l a c t o s e , glucosamine and galactosamine and poor i n p r o t e i n w i t h s e r i n e and threonine being the predominant amino a c i d s . The carbohydrate p r o s t h e t i c groups of the c o l o n i c g l y c o p r o t e i n s contain O-sulphate e s t e r s and appear to be l i n k e d , O - g l y c o s i d i c a l l y , to threonine and s e r i n e r e s i d u e s . Nemoto and Yosizawa (101) have i s o l a t e d glycopeptides from p r o t e o l y t i c d i g e s t s of mucosal scraping of both r a b b i t colon and small i n t e s t i n e . - 20 -Di g e s t i o n with pronase and p r e c i p i t a t i o n w i t h t r i c h l o r a c e t i c a c i d (to pre-c i p i t a t e p r o t e i n s ) were used i n preparing the s t a r t i n g m a t e r i a l s . On f r a c t i o n a t i o n of the supernatant w i t h DEAE Sephadex A 25, three f r a c t i o n s were obtained from both the small i n t e s t i n e and the colon. The most a c i d i c f r a c t i o n was r i c h i n sulphate and contained uronic a c i d , hexosamine, fucose and peptide. No s i a l i c a c i d was detected. The other two f r a c t i o n s were sulphated and con-tained s i a l i c a c i d . Threonine and serine were the major amino a c i d s i n a l l these f r a c t i o n s . The n a t i v e g l y c o p r o t e i n s of r a b b i t small i n t e s t i n e mucin were studied by Munakata and Yosizawa (83). The g l y c o p r o t e i n s were ext r a c t e d from s l i c e d small i n t e s t i n e with normal s a l i n e and then p r e c i p i t a t e d w i t h c e t y l p y r i d i n i u m c h l o r i d e . F r a c t i o n a t i o n w i t h DEAE Sephadex A25 y i e l d e d two f r a c t i o n s ; a carbohydrate r i c h f r a c t i o n and a p r o t e i n r i c h f r a c t i o n . On c e l l u l o s e acetate and zone e l e c t r o p h o r e s i s , the carbohydrate r i c h f r a c t i o n was separated i n t o two components; a slow moving f r a c t i o n ( A l ) s t a i n a b l e with a l c i a n b l u e , t o l u i d i n e blue and with the PAS, and a f a s t moving f r a c t i o n (A2) s t a i n a b l e w i t h a l c i a n blue. F r a c t i o n (A2) could be removed with hyaluronidase and was thought to represent a connective t i s s u e contaminant. The slow moving f r a c t i o n ( A l ) was shown to be a high molecular weight, e s t e r sulphate c o n t a i n i n g g l y c o p r o t e i n . The p r i n c i p a l sugars were ga l a c t o s e , glucosamine, galactosamine, and s i a l i c a c i d . Small amounts of mannose, fucose, and glucose were a l s o present. The major amino acids were glutamic a c i d , threonine, a s p a r t i c a c i d and s e r i n e . I I I . Function of G a s t r o i n t e s t i n a l Glycoproteins Mucous g l y c o p r o t e i n s are secreted continuously by the goblet c e l l s and mucus s e c r e t i n g glands of the g a s t r o i n t e s t i n a l t r a c t i n the form of a h i g h l y - 21 -hydrated g e l . They form a continuous thin layer over the underlying epithelium and, presumably, provide protection to the s e n s i t i v e mucosa and l u b r i c a t i o n to the f e c a l stream (1,3,4,7,11,123-127). Their protective properties have been attributed to the r e l a t i v e resistance of these glycoproteins to digestion with p r o t e o l y t i c enzymes (1,79,128-133), a property thought to be a function of the presence of terminal s i a l i c acid residue (129-133). The b i o l o g i c a l s i g n i f i c a n c e of the presence of 0-acetyl substituents i n the s i a l i c acid is not known (134,135). It is generally agreed that s i a l i c acids bearing an 0-acetyl substituent at the p o s i t i o n are more r e s i s t a n t to hydrolysis with V i b r i o cholera neuraminidase (1,136-137) than the unsubstituted s i a l i c acids. Therefore, i t was suggested by Reid et a l . (101,104), and C u l l i n g et a l . (30,33,35), that the presence of s i a l i c acids with 0-acetyl substituents at C^ (and possibly on the side chain) may protect s i a l i c acid residues from digestion by enzymes in the fecal stream. IV. Ulcerative C o l i t i s A. General Ulcerative c o l i t i s is a disease of unknown etiology (51,143-156) which is defined as "a d i f f u s e , non-specific inflammatory condition i t primarily a f f e c t s the mucosa of the large i n t e s t i n e , the inflammation is of exudative and vascular type and is generally non-productive of granulation tissue or f i b r o s i s " (151). Although, u l c e r a t i v e c o l i t i s can be acquired at any age, the age-disease rel a t i o n s h i p studies indicate that u l c e r a t i v e c o l i t i s has a bimodal d i s t r i -bution with a large population at r i s k between the ages of 15-20 and a small peak between the ages of 55-60 (146,151). In adults u l c e r a t i v e c o l i t i s is - 22 -l e s s common among men than women (2:3) where i t may be as s o c i a t e d w i t h preg-nancy. I t i s more common amongst whites, urban dwellers and c e r t a i n e t h n i c groups (Jewish) where the presence of f a m i l i a l f a c t o r s i s p o s s i b l e (151). There have been numerous attempts to e l u c i d a t e the f a c t o r ( s ) which lead to the establishment of u l c e r a t i v e c o l i t i s . The f o l l o w i n g have been suggested to be involved (151): (a) i n f e c t i o n , (b) mucolytic enzymes, (c) food a l l e r g y and h y p e r s e n s i t i v i t y , (d) p s y c h o l o g i c a l f a c t o r s , and (e) autoimmunity)but i n f e c t i o n and autoimmunity have been most e x t e n s i v e l y i n v e s t i g a t e d . The hypothesis that u l c e r a t i v e c o l i t i s i s caused by an i n f e c t i v e agent(s) has been accompanied by claims that many organisms, i n c l u d i n g Diplococus, Bacterium necrophorus, Shigella, f l e x i n e r i , E s h e r i c h i a c o l i , Entomobia h i s t o -l y t i c a might be f a c t o r s i n the e t i o l o g y o f u l c e r a t i v e c o l i t i s . The presence of lymphotoxic antibodies i n the sera of u l c e r a t i v e c o l i t i s p a t i e n t s and i n the blood of t h e i r r e l a t i v e s , together w i t h the p o s s i b l e i n d u c t i o n of c o l i t i s -l i k e l e s i o n s i n r a b b i t s i n j e c t e d w i t h f i l t e r e d colon homogenates suggests the p o s s i b i l i t y that a f i l t e r a b l e agent ( i . e . a v i r u s ) might be i n v o l v e d . However, no known i n f e c t i v e agent has been shown to be c o n s i s t e n t l y present i n abnormal q u a n t i t i e s i n p a t i e n t s w i t h u l c e r a t i v e c o l i t i s and, f u r t h e r , no organism has been shown to transmit the disease i n human beings (156). There i s (157) considerable i n t e r e s t i n the suggestion that u l c e r a t i v e c o l i t i s i s an autoimmune disease but the hypothesis i s s t i l l q u estionable. Antibodies have been demonstrated i n 90% of c h i l d r e n and i n 40% of a d u l t s w i t h u l c e r a t i v e c o l i t i s (158). Although these antibodies react w i t h the cytoplasm of c o l o n i c e p i t h e l i a l c e l l s (154-159), they a l s o cross react w i t h the b a c t e r i a l f l o r a of the gut p a r t i c u l a r l y with E. c o l i . The r e a c t i v i t y of g l y c o p r o t e i n antigens i s o l a t e d from the mucosa of r a t colon was s i g n i f i c a n t l y reduced when - 23 -subjected to mild acid hydrolysis (160), conditions known to remove s i a l i c a cids. This strongly implies that the s i a l i c acids are important for the i n t e g r i t y of the i s o l a t e d antigens. B. Changes in e p i t h e l i a l glycoproteins associated with u l c e r a t i v e c o l i t i s a) Histochemical studies Changes in the normal histochemical pattern of the colonic glycoproteins, summarised i n Table 3, have been shown to occur i n a s s o c i a t i o n with u l c e r a t i v e c o l i t i s (19,26,35,38,47,48,50,56,107,109-115,138-142) but the s i g n i f i c a n c e of these changes i s unknown. The most consistent findings have been a s i g n i f i c a n t reduction in the quantity of mucos substance i n the goblet c e l l s (19,26,47,48, 50,109,110,140). Of s p e c i a l i n t e r e s t to t h i s project are the findings of C u l l i n g e_t a_l. (35,38) who showed that u l c e r a t i v e c o l i t i s was associated with a reduction i n the degree of side chain s u b s t i t u t i o n of the 0-acetyl s i a l i c acids of the e p i t h e l i a l glycoproteins. b) Chemical studies There have been only a few chemical studies of colonic e p i t h e l i a l glyco-proteins in u l c e r a t i v e c o l i t i s . There is no general agreement as to the changes that occur and some of the methodology used i s open to c r i t i c i s m (Table 4). Sorgel and Ingelfinger (112) obtained r e c t a l mucus, from 11 normals and from 8 patients with u l c e r a t i v e c o l i t i s , by i r r i g a t i n g the rectum with a hypertonic phosphate solution. Chemical analysis showed no differences i n the composition of the carbohydrate from i r r i g a t e s of the two groups. However, the nitrogen content was 50% higher i n the r e c t a l i r r i g a t e s obtained from patients with u l c e r a t i v e c o l i t i s . Immunological studies showed that the glycoproteins of the patients with u l c e r a t i v e c o l i t i s were d e f i c i e n t i n some Table 3. Histochemical a l t e r a t i o n of the colonic mucosubstance i n u l c e r a t i v e c o l i t i s I n v e s t i g a t o r ( s ) Severity Neutral Mucins Observations Sialomucins Sulphomucins Remarks Hardaky et a l . (109) mucus substance e i t h e r decreased or absent Greco et a_l. (47) mild severe present (goblet of sigmoid) absent c e l l present absent decreased absent F i l i p e (48) absent trace decreased corre l a t e with the sev e r i t y of the disease Gad (26) moderate severe moderately increased decreased increased decreased Lev (19) ea r l y stage moderate reduced increased reduced reduced adjacent to u l c e r a t i o n F i l i p e & Dawson (50) moderate reduction moderate reduction cor-r e l a t e with the s e v e r i t y of the inflammatory response i n lamina propria Hellstrom & Fisher (110) mucosubstance e i t h e r moderately decreased or p a r t i a l l y preserved C u l l i n g et a l . (35,38) reduction i n the 0-acetyl substituted s i a l i c acid most evident at the u l c e r margins Table 4. Large i n t e s t i n a l glycoproteins and u l c e r a t i v e c o l i t i s : chemical investigations I n v e s t i g a t o r ( s ) Region of Methodology and Observations large i n t e s t i n e Remarks Sorgel & I n g e l -f i n g e r (112) r e c t a l i r r i g a t i o n with hypertonic phosphate s o l u t i o n ; (a) nitrogen contents 50Z higher i n u l c e r a t i v e c o l i t i s ; (b) immunological studies -u l c e r a t i v e c o l i t i s glycoproteins d e f i c i e n t i n a and 8 globulins Teague et a l . (113) colon scrapings - mucus biopsies - fractionated with Sephadex G50 and with Sepharose 2B; two fractions (a) included (b) excluded; There was more mannose i n the included f r a c t i o n and the amount of mannose was s i g n i f i c a n t l y increased i n u l c e r a t i v e c o l i t i s Fraser et a l . ( I l l ) colon mucosal scrapings; (a) increased mannose, (b) s i g n i f i c a n t l y smaller q u a n t i t i e s of threonine and serine, therefore u l c e r a t i v e c o l i t i s associated with changes i n glycoprotein composition involving a fewer number of carbohydrate prosthetic groups Ettwood et a l . (114) r e c t a l epithelium c e l l s t i s s u e culture - u l c e r a t i v e c o l i t i s - e p i t h e l i a l c e l l s - have an increased turnover and migration rate MacDennott et a l . (115) r e c t a l e p i t h e l i a l c e l l s organ c u l t u r e , u l c e r a t i v e c o l i t i s - e p i t h e l i a l c e l l s - e x h i b i t an increased glycoprotein synthesis and secretion Reid et a l . formaline t i s s u e specimens from u l c e r a t i v e c o l i t i s patients - is o l a t e d glycoproteins; (107) f i x e d (a) s i a l i c acids - s i g n i f i c a n t l y reduced 0-acetyl substitute (at C4 and at colon polyhydroxy side chain); (b) a l t e r a t i o n s with f u c o s e / s i a l i c a c i d , galactose/fucose and glucosamine/galactosamine molar r a t i o s - 26 -serum p r o t e i n s , p a r t i c u l a r l y a and 3 g l o b u l i n s . The authors p o s t u l a t e d that these serum g l o b u l i n s might form an i n t e g r a l part of normal r e c t a l mucus and that the h i g h l y viscous o t l - g l y c o p r o t e i n might c o n t r i b u t e to the p h y s i c a l and the chemical p r o p e r t i e s of the mucus. Teague et a l . (113) studied c o l o n i c mucus i s o l a t e d from mucosal b i o p s i e s obtained from normal i n d i v i d u a l s and from i n d i v i d u a l s w i t h i n t e s t i n a l d i s -orders i n c l u d i n g u l c e r a t i v e c o l i t i s . A f t e r f r a c t i o n a t i o n w i t h Sephadex G50 f u r t h e r f r a c t i o n a t i o n with Sepharose 2B y i e l d e d two f r a c t i o n s , one included i n t o and one excluded from the g e l . Both f r a c t i o n s contained fucose, galactose, mannose, N-acetyl glucosamine, N-acetyl galactosamine and s i a l i c a c i d . There was more mannose i n the included f r a c t i o n , and the amount of mannose i n t h i s f r a c t i o n was s i g n i f i c a n t l y increased i n u l c e r a t i v e c o l i t i s . The o r i g i n of the "high mannose g l y c o p r o t e i n " was unknown but the authors claimed that i t d i d not o r i g i n a t e from the plasma p r o t e i n s . Fraser e_t a_l. ( I l l ) i n v e s t i g a t e d the g l y c o p r o t e i n s i s o l a t e d from mucosal scrapings of both normal colons and colons from u l c e r a t i v e c o l i t i s p a t i e n t s . In u l c e r a t i v e c o l i t i s , the mucous g l y c o p r o t e i n s were found to c o n t a i n increased q u a n t i t i e s of mannose and s i g n i f i c a n t l y smaller q u a n t i t i e s of the amino acids threonine and s e r i n e . They concluded that i n u l c e r a t i v e c o l i t i s , there are changes i n the g l y c o p r o t e i n composition i n v o l v i n g fewer carbohydrate p r o s t h e t i c groups. Reid et a_l. (107) found that the e p i t h e l i a l g l y c o p r o t e i n s i s o l a t e d from fo r m a l i n f i x e d specimens of colon resected f o r u l c e r a t i v e c o l i t i s p a t i e n t s contained s i a l i c acids with s i g n i f i c a n t l y l e s s 0-acetyl s u b s t i t u e n t s at and i n the polyhydroxy side chains. In a d d i t i o n there were changes i n the molar r a t i o of f u c o s e / s i a l i c a c i d , galactose/fucose and glucosamine/ galactosamine. - 27 -Eastwood et al_. (114), using t i s s u e c u l t u r e s , studied the renewal rate of r e c t a l e p i t h e l i a l c e l l s from both normal i n d i v i d u a l s and pa t i e n t s with u l c e r a t i v e c o l i t i s . E p i t h e l i a l c e l l s from u l c e r a t i v e c o l i t i s p a t i e n t s were found to have an increased turnover and mi g r a t i o n r a t e . MacDermott e^ t a l . (115), i n organ c u l t u r e s t u d i e s , showed that r e c t a l e p i t h e l i a l c e l l s from u l c e r a t i v e c o l i t i s p a t i e n t s have increased g l y c o p r o t e i n synthesis and s e c r e t i o n . V. Animal models of lower d i g e s t i v e t r a c t u l c e r a t i o n An animal model for u l c e r a t i v e c o l i t i s would be very h e l p f u l i n the e l u c i d a t i o n of the disease process and might a i d i n r e s o l v i n g the e t i o l o g i c a l f a c t o r s and the pathogenesis of the disease, thus p o t e n t i a l l y c o n t r i b u t i n g to a b e t t e r treatment. Many methods have been t r i e d i n attempts to induce u l c e r -a t i v e c o l i t i s i n animals but with l i t t l e success. The f o l l o w i n g b r i e f review emphasizes carrageenan induced c o l i t i s ; f o r d e t a i l e d l i t e r a t u r e reviews see Tables 5 and 6. A. Spontaneous c o l i t i s has been reported to occur i n r a t s (175), mice (176), pigs (177-180), horses (181) and dogs (182-185). Of p a r t i c u l a r i n t e r e s t i s the c o l i t i s reported i n boxer dogs (184,185). C l i n i c a l l y i t resembles u l c e r -a t i v e c o l i t i s i n man, having c h a r a c t e r i s t i c a l l y loose feces, d i a r r h e a , followed by blood and excess mucus i n the feces. I t i s more common i n females than i n males (2:1), may be associated with pregnancy, or changes i n food and environment and has a h i s t o r y of remission and r e l a p s e . I n i t i a l l y i t s t a r t s i n the rectum and then progresses to the colon. However, i n the lamina p r o p r i a there are large pale macrophages co n t a i n i n g PAS p o s i t i v e m a t e r i a l and the disease may lack crypt abscesses. -28-Table 5. Inflammatory bowel disease: animals studies Investigator Animal Cause/Agent Observations: clinical and/or microscopic Stewart & Jones (175) rats spontaneous periarteritis, end arteries obliteration, lymphangetis, healing - intensive scar formation Edgier et a l . (176) mic spontaneous (outbreak) variably enlarged colon and rectal prolapse, citrobacter freudii were isolated Embso (177) swine spontaneous terminal ileum, grossly and histologically comparable to human regional enteritis Gross & Kohler (178) Glika (179) Moon (180) young pigs unknown similar to human u.c. - contaminated with E. coli Stand et a l . (182) Van Kraingen (183) dogs unknown similar, i f not identical, to human regional enteritis Kennedy & Cello (184) Lawson et a l . (185) boxer dogs unknown involved colon and rectum, prevalence male to female 1:2, associated with pregnancy, remission and relapse, micro-scopically different from human U.C; lamina propria and submucosa infiltrated with macrophages with PAS positive material, crypts abscesses and pseudopolyps were not feature of the disease stage examined Smith & Jones (186) Hartson et a l . (187) dogs dogs vascular bloody diarrhea, pathological appearance depends upon the impairment degree of ischemia induced mechanical removing the insult—»recovery orcholenergic drugs MacPherson & Pfieffer (190,191) rats cats acetic acid effective (100% ulcerated), diffuse ulceration, nonspecific inflammatory response, healing after 60 days Berger & Liutn (188) dogs ganglioectomy (celiac & sup-erior mesentric ganglia) tenesmous, diarrhea with blood after 3 days, inflammation and ulceration. Spasm^o blocked venous flow Halpern & Zweibaum (192) Zweibaum et a l . (193) rats immunization with E. coli dead live produced colitis were similar to human U.C. can be prevented by oral administration of E. coli ulceration resulted from immunological breakdown of colonic bacterial balance Kirsner et a l . (194) Kirsner & Gold-graber (195) Kirsner (196) rabbits sensitization with egg a l -bumin followed by formalin (rectum) & moderate to severe inflammation, erosions and ulceration -distal colon, multiple egg albumin challenge were needed to maintain the disease, Ab-Ag complexes were found localized at the site of the lesion (197) Kraft et a l . (197) challenged with egg albumin Ip or IV -28a-Investigator Animal Cause/Agent Observations: clinical and/or microscopic Shean et a l . (199) dogs passive & massive rectal bleeding - death, haemmorhagic co l i t i s and active immun- jejunitis ization (anti-dog colon antibodies) Hodgson et a l . (210) rabbits pre-formed Ag-Ab complexes HSA-Anti HSA-IV-rabbits followed by local irritation with formalin (rectum) severe c o l i t i s , ulceration and crypts abscessation Mee et al. (201) rabbi ts a) immunized chronic ulcerative colitis-like lesion, inflammation s t i l l with entobac- present after 6 months terial antigen of Kunn b) IV Ag-Ab complexes c) rectal irritation with formation Mitschke & Kracht (202) rats rabbits auto immunized (colonic mucosa rat colonic mucosa chronic ulceration ) no lesion Rosenberg & Fischer (204) Rosenberg & Hoeberlin (205) Bick et al. (206) Bick & Rosenberg (207) guinea Pig + many species including man sensitization edema, mucosal necrosis and prevascular cuffing, severity of with DNCB f o l - the inflammation depends upon frequency of DNCB application lowed by chal-lenge per rectum Rabine & Susan (208) Rabine (209) rabbits DNCB sensiti- 3 day after challenge - ulceration, crypts Absesses and zation (skin) inflammatory response - compatible with human U.C. Healing -followed by 5 weeks, loss of goblet cells & submucosal fibrosis rectal challenge Leveen et a l . (198) dogs anticolon, bloody diarrhea (1-2 day after IV injection), mucosal ulcer-antibodies ations, remission and exacerbation. Failed to induce chronic prepared in lesion rabbits or ducks Bicks & Walker (200) dogs immunized with anticolon rabbit serum acute immune reaction primarily limited to the colon and evidence of systemic involvement Palou et a l . (203) rat immunized with colonic mucosa disease produced was strictly confined to the colon, clinically resemble human U.C. Gross anatomical and histologically different Veteri et a l . (213) Barlet et a l . (215) Katz et a l . (216) Refkin et a l . (217) Humphrey & Contey (218,219) Alio et a l . (220) human hamsters rabbits lincomycin clindemycin mild watery diarrhea (human). Severe: bloody diarrhea, ulceration, pseudomembranous and pseudopolyps Vancomycin protective, bacterial involved in etiology Caused by C. defficile toxins, can be prevented by C. sordellii antitoxin (220) - 29 -B. Vascular impairment. In many animal species i n c l u d i n g dogs (186-188), pigs (189), r a t (190-192) and cats (191), c o l o n i c l e s i o n s have been induced by d i r e c t blood v e s s e l impairment e i t h e r by l i g a t i o n or by the a c t i o n of c h o l i n e r g i c drugs. The p a t h o l o g i c a l appearance of t h i s type of c o l i t i s i s dependent upon the degree of the induced ischemia. In severe cases, there i s edema, hemorrhage and deep u l c e r a t i o n , more s i m i l a r to ischemic c o l i t i s than to u l c e r a t i v e c o l i t i s . C. Immune c o l i t i s . The p o s s i b i l i t y of inducing a c o l i t i s - l i k e l e s i o n i n labo r a t o r y animals by means of immunological methods has been e x t e n s i v e l y i n v e s t i g a t e d (194-210). K i r s n e r et^ al. (194-196) and K r a f t et a l . (197) produced c o l i t i s i n r a b b i t s s e n s i t i z e d w i t h egg albumin. In the acute phase of the disease they noted edema and hemorrhage, along with a mononuclear i n f i l t r a t e i n the lamina p r o p r i a and the submucosa. On subsequent challenge there were s u p e r f i c i a l mucosal u l c e r a t i o n s , together w i t h c r y p t i t i s and f i b r o s i s . Leveen et_ a l . (198) and Shean et^ a l . (199) produced c o l i t i s i n dogs by i n j e c t i o n o f a n t i c o l o n a n t i b o d i e s prepared i n r a b b i t s (or i n ducks) or by immunization with a n t i c o l o n r a b b i t serum (200). The disease was colon s p e c i f i c and c h a r a c t e r i s e d by d i a r r h e a followed by blood i n the feces. However, continuous challenge was needed to prevent remission. Microscopy demonstrated a c t i v e c o l o n i c u l c e r a t i o n , crypt abscesses, edema, and congestion along with a mononuclear i n f i l t r a t e . S e n s i t i z a t i o n w i t h b a c t e r i a l antigens (201), c o l o n i c mucous (202,203) and w i t h dinitrochlorobenzene (204-209) followed by subsequent l o c a l challenge per rectum with or without i r r i t a t i o n w ith f o r m a l i n were reported to cause c o l i t i s - l i k e l e s i o n s i n r a t s (202,203), guinea pigs (204), and r a b b i t s (201,202,205-210). - 30 -D. C o l i t i s and c o l o n i c u l c e r a t i o n s were noted as secondary l e s i o n s i n experimental i r r a d i a t i o n of r a b b i t s (211) and r a t s (212) and a l s o during the course of treatment with drugs, i n p a r t i c u l a r w i t h clindomycin (213-220). A l i o ejt a l . (220) reported that clindomycin c o l i t i s can be prevented by i n j e c t i n g C l o s t r i d i u m s o r d e l l i i a n t i t o x i n s . E. Carrageenan c o l i t i s Carrageenans are obtained by the aqueous e x t r a c t i o n of many species of the Rhodophyceae where they occur i n the c e l l s and the c e l l w a l l s (221). They are sulphated p o l y s a c c h a r i d e s , p r i m a r i l y polymers of galactose and 3,6-anhydro-D-galactose. The exact chemical nature and the degree of s u l p h a t i o n of carrageenan v a r i e s from species to species. Native carrageenan has a molecular weight of 1-9 x 10^ and e x i s t s i n two forms; kappa carrageenan and lambda carrageenan. The former has 88% anhydro-galactose repeated u n i t s , and i s l e s s sulphated and l e s s s o l u b l e i n potassium c h l o r i d e . The l a t t e r c o n s i s t s of a 50:50 mixture of 1-4 and 1-3 l i n k e d galactose u n i t s , and i s more sulphated and more s o l u b l e i n KCl (221). Native carrageenan forms a t h i c k viscous s o l u t i o n even at low concen-t r a t i o n s (146,221,222). Carrageenans are e x t e n s i v e l y used i n the food i n d u s t r y as thickeners and s t a b i l i z e r s p a r t i c u l a r l y i n m i l k and d a i r y products. The l i s t of foods c o n t a i n i n g carrageenan includes i c e cream, chocolate m i l k , s o l i d chocolates, cottage cheese, frozen custards, salad d r e s s i n g s , sauces, s o f t d r i n k s , bread, yogurt, syrups and a r t i f i c i a l l y sweetened jams and j e l l i e s (222). Carrageenan can be degraded with p e r i o d i c a c i d or w i t h m i l d a c i d . A c i d degraded carrageenan has a smaller molecular weight ( 25,000), i s more s o l u b l e w i t h a greater d i s s o l u t i o n r a t e , and forms l e s s v i scous s o l u t i o n than n a t i v e - 31 -carrageenan but i t retains i t s polyanionic properties and sulphate content (221,223). Mild acid degraded carrageenan i s more stable than periodic acid treated carrageenan. Native carrageenan, l i k e some other b i o l o g i c a l sulphated polysaccharides has anticoagulant and anticomplement e f f e c t s and i n t e r f e r e s with both humoral and c e l l mediated immunity (224-226). Acid degraded carrageenan when given o r a l l y to guinea pigs and rabbits has been shown to be deposited i n the Kupfer c e l l s of the l i v e r and i n the lymph nodes and to be excreted in the urine. Subcutaneous i n j e c t i o n of degraded carrageenan results i n granuloma formation followed by necrosis and, eventually, replacement with adipose tissue (222). Native carrageenan, administered intravenously, i s more toxic than degraded carrageenan (146,221). Since carrageenans are polyanions, they in t e r a c t with polycations such as proteins at a pH below t h e i r i s o e l e c t r i c points. In Europe, degraded carrageenans are widely used medically, as antipeptic agents (158,227). The proposed mechanism i s that the carrageenan in t e r a c t s with g a s t r i c mucus forming a gel l i k e protective layer impermeable to pepsin and further decreases pepsin a c t i v i t y by rendering the substrate unavailable to pepsin (228-233). Watt and Marcus (227,234-243) and other investigators (244-254) (Table 6) have shown that carrageenan, both native and degraded, causes large bowel ul c e r a t i o n i n laboratory animals. In 1969 Marcus and Watt (234) reported that guinea pigs fed 5% degraded carrageenan or 1% native carrageenan i n the drinking water for 30 days developed mucosal u l c e r a t i o n i n the cecum and the colon. From that time there have been numerous reports on both the t o x i c i t y of carrageenan and i t s c a p a b i l i t y to induce large bowel u l c e r a t i o n i n various species of laboratory animals. Carrageenan causes large bowel u l c e r a t i o n i n guinea pigs (234,235,238-240, 244-248,251,252), and r a b b i t s (227,235,237,239,241,244,251,252). The e f f e c t of carrageenan i n mice and r a t s , however, i s c o n t r o v e r s i a l . Marcus and Watt (235,236) reported that a d m i n i s t r a t i o n of 5% degraded carrageenan i n the d r i n k i n g water over a period of 6 months caused large bowel u l c e r a t i o n s i n r a t s (236). In c o n t r a s t , Sharrat et a l . (244) and other i n v e s t i g a t o r s (250-252) have reported negative carrageenan e f f e c t s i n r a t s . As w e l l squamous metaplasia i n the r e c t a l mucosa (250) and carcinoma of the rectum and colon have been reported i n r a t s kept on more than 5 g/kg bodyweight carrageenan while h y p e r p l a s i a i n the colon of r a b b i t has been induced by feeding 1%-0.1% degraded carrageenan for a period of 2 years (227). Carrageenan (2% degraded or 5% undegraded) apparently has no e f f e c t s on f e r r e t s , s q u i r r e l monkeys, mice, or r a t s ; and 2% degraded carrageenan i n milk (252) had no a f f e c t on guinea p i g s . In general, carrageenan induced, dose dependent large bowel u l c e r a t i o n s seem w e l l documented i n guinea pigs and r a b b i t s (234,206). The v a l i d i t y of carrageenan c o l i t i s as an experimental model for human u l c e r a t i v e c o l i t i s was discussed by Marcus and Watt (238,239). The hypothesis that the two diseases were s i m i l a r was supported by Mottet (255) but was disputed by Sharrat et a l . (244,253). M o r p h o l o g i c a l l y , carrageenan c o l i t i s occurs i n i t i a l l y i n the cecum and the l e s i o n progresses d i s t a l l y to i n v o l v e the c e c a l part of the colon, the colon proper and the rectum. H i s t o l o g i c a l s t u d i e s showed mucosal u l c e r a t i o n s which i n severe cases or i n lengthy carrageenan treatment involved the muscularis mucosa and could be accompanied by f i b r o s i s . In the lamina p r o p r i a and the submucosa there was congestion and edema and an inflammatory i n f i l t r a t e composed mostly of vacuolated macro-phages, lymphocytes, plasma c e l l s and polymorphonuclear leukocytes. There Table 6. Carrageenan induced colitis studies Investigator Animal Cause/Agent Observations: clinical and/or microscopic Watt & Marcus (227,234-243) guinea undegraded pigs carrageenan rabbits degraded rats carrageenan a) 1Z solution - undegraded carrageenen caused colonic ulcer-ation only in guinea pigs and rabbits over a period of 5 and 3 months respectively b) degraded carrageenan produced the above effects as well as lesion in the GI tracts of rats and mice. Rabbits + 5Z solution _»acute fulminating colitis and death within 10-14 days. 1Z - less tendency for diarrhea, bleeding occurred within 3 weeks. 0.1Z - colonic ulcerations after 3 months only in SOZ of the rabbits. Caecum is primary site of ulceration, on longer treatment the colon and the rectum will be involved The carrageenan model of ulceration (235) was successfully produced only in the rabbits and guinea pigs (244-249, 250-253). Watt and Marcus (239), Mottet (255), Anwer and Cohen (247); carrageenan colitis is comparable in many aspects to human co l i t i s , it is a valuable model. CTJ Shorratt et a l . (244) (253) (a) carrageenan induced colitis is a species specific (guinea pigs and rabbits) carrageenan effects (b) i t is primarily in the caecum (c) ulceration is due to ability of carrageenan to cross the mucosa of the caecum in rabbits and guinea pigs where in lamina propria will be phago-cytosed by the macrophages, death of the carrageenan laden macrophages and the subsequent release of the macrophage lysosomal enzymes will result in the mucosal ulceration Abraham et a l . (251) I Grasso et a l . (252) Abraham et a l . (251) 2Z degraded carrageenan in milk fa i l to induce ulceration in guinea pigs Fabian et a_l. (250) rats taken more than 5 g/kg bwt degraded carrageenan after .6 months developed squamous metaplasia of the rectum. Mucosal hyperplasia had been reported to occur in rabbits kept on 0.1Z degraded carrageenan (242) Grasso et a l . (252) Neomycin (diet) + carrageenan treated rabbits - no effect on the mucosal ulceration, reduction - polymorph.es population Van der Waaij (248), Onderdonk and Barlett (254) antimocrobial drugs active against obligate anaerobes prevented mucosal ulceration in the carrageenan treated guinea pigs. - 33 -was necrosis i n v o l v i n g the e n t i r e c r y p t s , a few crypt abscesses and e p i t h e l i a l h y p e r p l a s i a at the margin of the u l c e r . Increased a c i d phosphatase a c t i v i t y was reported i n the macrophages and i n the e p i t h e l i a l c e l l s (251). E l e c t r o n microscopic studies showed an increase i n macrophage lysosomes along with m i t o c h o n d r i a l degeneration. A comparison of carrageenan and human c o l i t i s i s shown i n Table 7. Grasso et a l . (252) studied the sequence of c e c a l u l c e r a t i o n i n guinea pigs kept on 2% degraded carrageenan i n the d r i n k i n g water and on 5% native carrageenan i n the d i e t . They showed that the e a r l i e s t h i s t o l o g i c a l change was a heavy aggregation o f carrageenan loaded macrophages. Subsequently there was the appearance of polymorphs and lymphocytes which was accompanied by mucosal u l c e r a t i o n s . A d d i t i o n of neomycin reduced the polymorph po p u l a t i o n but d i d not a f f e c t the s e v e r i t y of the mucosal u l c e r a t i o n s . They proposed that the process of u l c e r a t i o n was the r e s u l t of the a c t i v a t i o n of macrophage lysosomes by the phagocytosed carrageenan leading to the death of the macro-phages and the l i b e r a t i o n of lysosomal hydrolases which, i n t u r n , caused the mucosal u l c e r a t i o n s . Van Der Waaij e_t al_. (248) showed that s e l e c t i v e e l i m i n a t i o n of aerobic gram negative i n t e s t i n a l f l o r a w i t h 0.25 mg/ml Trimethoprim with sulpha-methozazole given o r a l l y along with 2% degraded carrageenan to guinea pigs r e s u l t e d i n a granulomatous type response and no u l c e r a t i o n . These authors postulated that mucosal u l c e r a t i o n could be the product of b a c t e r i a l a c t i o n on the weakened mucosa. These f i n d i n g s were confirmed by the study of Onderdonk et a l . (254) who i n a d d i t i o n found an increase i n the gram negative organisms i n guinea pigs treated w i t h carrageenan f o r periods i n excess of 6 days. - 34 -Table 7. Comparison between u l c e r a t i v e Ulcerative C o l i t i s (Robbins 256) Unknown etiology, 65-75% colon, 10-20% terminal ileum involved I n i t i a t e d at rectosigmoid Diffuse with pseudopolyps Microabscesses at the depth of the crypts Mucosa and submucosa affected Inflammatory metaplasia (ulcer margins, dysplasia of the glands) In l a t e r stage, some evidence of f i b r o s i s and scarring Polymorphs, lymphocytes, plasma c e l l s c o l i t i s and carrageenan induced c o l i t i s Carrageenan C o l i t i s Caused by carrageenan, mostly cecum, cecal end of the colon, rectum may be involved I n i t i a t e d i n the cecum Pinpoint (2x2 mm), l i n e a r on transverse folds Necrosis of the entire crypts, occasionally with microabscesses Mucosa and submucosa, muscularis mucosae i n severe and chronic cases Inflammatory metaplasia and u l c e r -ation, no dysplasia Present Granuloma, vacuolated macrophages, lymphocytes, plasma c e l l s with eosinophils i n l a t e r stage - 35 -VI. Rationale Changes i n the e p i t h e l i a l g l y c o p r o t e i n have been shown to occur i n u l c e r a t i v e c o l i t i s . Since these r e s u l t s were obtained by a study of s u r g i c a l specimens,they represent changes at an advanced stage of the disease process and therefore t h e i r s i g n i f i c a n c e i s d i f f i c u l t to evaluate. Carrageenan induced c o l i t i s provides a simple model f o r i n v e s t i g a t i n g u l c e r a t i v e disease of the colon (255). As f a r as we are aware there has been no systematic study of the r o l e of the e p i t h e l i a l g l y c o p r o t e i n s i n e i t h e r l a rge bowel u l c e r a t i o n i n man or i n experimental models of large i n t e s t i n a l u l c e r a t i o n . The object of t h i s t h e s i s therefore was to study the changes, i f any, i n the e p i t h e l i a l g l y c o p r o t e i n s during experimentally induced large bowel u l c e r a t i o n i n laboratory animals under c o n t r o l l e d l a b o r a t o r y c o n d i t i o n s . - 36 -MATERIALS AND METHODS I. Materials Young adult white New Zealand rabb i t s , guinea pigs and rats were purchased from the animal unit University of B r i t i s h Columbia. Degraded carrageenan ( l o t #18206P010L) was obtained from Glaxo Laboratories, Paris, V i b r i o cholera neuraminidase (aldolase, protease, le c i t h i n a s e - c - f r e e ) 1.0 IU/ml from Behring-werke, W. Germany, and N-acetyl neuraminic acid A grade from C a l i f o r n i a Biochemicals. Bovine submaxillary mucin (BSM) was purchased from Boehringer Mannheim Corp. Human serum mucoid ( s i a l i c acid concentration 54.5 yg/ml H 2 O ) was prepared from the 0.6M perchloric acid soluble f r a c t i o n of human serum by Mr. C.W. Ramey, Department of Pathology, U.B.C. IN sulphuric acid, 2 N hydrochloric acid, sodium borohydride, acetic acid, hydrogen peroxide, sodium arsenite, t-butanol and N-butanol were purchased from Fisher S c i e n t i f i c Co.; IN potassium hydroxide was obtained from the J.T. Baker Chemical Co., P h i l l i p s b u r g , N.J. orthotolidine was kindly donated by Dr. R.H. Pearce, Department of Pathology, U.B.C. A l l the chemicals used were of a n a l y t i c a l grade or better. • I I . General Experimental Design This is i l l u s t r a t e d in figure 3 and consisted of the following steps: A. Induction of large bowel u l c e r a t i o n and c l i n i c a l observations B. S a c r i f i c e of the animals and gross anatomic observations C. H i s t o l o g i c a l and histochemical observation D. Is o l a t i o n of e p i t h e l i a l c e l l s E. Preparation and analysis of 105,000 x g supernatants prepared from e p i t h e l i a l c e l l homogenates F. I s o l a t i o n and analysis of the e p i t h e l i a l glycoproteins - 37 -Figure 3. Schematic diagram of the experimental design Induction of Lower GI Tract U l c e r a t i o n s c l i n i c a l observations post-mortem .observations H i s t o l o g y & h i s t o c h e m i s t r y I s o l a t i o n of e p i t h e l i a l c e l l s cecum upper colon lower colon P r e p a r a t i o n of 105,000 x g supernatants I s o l a t i o n of the e p i t h e l i a l g l y c o p r o t e i n s 1 Chemical studies - 38 -III . Methods A. Induction of large bowel u l c e r a t i o n Prior to the administration of the carrageenan a l l the rabbits were i d e n t i -f i e d with tattoo numbers in the l e f t ear and were housed in separate cages. The animals were kept on a standard laboratory d i e t (Purina rabbit chow) and were provided with water ad l i b . Feces were col l e c t e d d a i l y and examined for con- sistency, colour and the presence of occult blood. Samples were also examined microscopically for the presence of Co c c i d i o s i s , infected animals being removed and replaced with uninfected specimens. During this period the normal, base l i n e food and water intake was established. On the f i f t h day the rabbits were weighed and were then divided into two groups: (a) Carrageenan treated group fed 1% degraded Carrageenan (prepared fresh daily i n tap water) i n the drinking water and (b) Control group fed water ad l i b . Both groups were fed the standard laboratory d i e t but were given no green food supplement. The animals were monitored d a i l y as follows: (a) the food and carrageenan intake were estimated by weighing the drinking bottles and the feeders, (b) feca l samples were inspected for apparent physical changes and tested for the presence of occult blood with the orthotolidine method (257) and (c) the general state of health of the animals was assessed. Body weight changes were estimated weekly. At the conclusion of the experiments, a l l the l i v i n g rabbits were s a c r i -ficed with chloroform anesthesia, the abdominal and chest c a v i t i e s were opened and the digestive t r a c t was inspected in s i t u . The lower digestive t r a c t was resected and was divided, according to the anatomic markers shown in Figure 4, into cecum, upper colon, lower colon and appendix (the appendix was not - 40 -examined a f t e r the preliminary work). Each region of the large i n t e s t i n e was emptied of the f e c a l content, everted, washed with ice cold saline (8 g sodium chloride/1 d i s t i l l e d water) and examined v i s u a l l y or with the aid of a dissecting microscope. Tissue specimens (approximately l x l cm) were taken from each region of the large i n t e s t i n e and fixed i n 10% buffered formalin for further h i s t o l o g i c a l and histochemical studies. The remaining tissue from the lower digestive tract was processed for the removal of e p i t h e l i a l c e l l s (see below). In experiments 3 and 4, e p i t h e l i a l c e l l s were removed only from the c e c i , the upper and lower colon being fixed i n 10% buffered formalin). The remainder of the digestive t r a c t (small i n t e s t i n e and stomach), the l i v e r , mesenteric lymph nodes and the respiratory system were examined v i s u a l l y and preserved i n 10% buffered formalin. B. I s o l a t i o n of the e p i t h e l i a l c e l l s The general procedures used for i s o l a t i o n of e p i t h e l i a l c e l l s are outlined i n Figure 5. Each washed everted organ was i n f l a t e d with normal saline and a l t e r n a t e l y shaken for 5 minutes i n and incubated for 10 minutes at 37°C with phosphate buffered s a l i n e (PBS), pH 7.0, containing 0.02% EDTA (8.2 g sodium chloride, 0.2 g potassium chloride, 1.15 g disodium hydrogen phosphate, 0.2 g potassium dihydrogen phosphate and 0.2 g EDTA sodium s a l t i n 1 l i t r e of d i s t i l l e d water ( C u l l i n g et a_l., 37). This process was repeated four times, the incubating medium (PBS-EDTA) being changed each time. The c e l l s were recovered by ce n t r i f u g a t i o n at 2,000 rpm for 10 minutes at 4°C, washed with PBS and stored at -20°C for further processing. The extent of c e l l removal was checked by microscopic examination of sections of formalin fixed tissue specimen taken af t e r the f i n a l treatment. - 41 -Figure 5. Flow chart of the procedures used for the i s o l a t i o n of lower GI tr a c t e p i t h e l i a l c e l l s from guinea pigs and r a b b i t s . Guinea pig or rabbit k i l l e d by anesthesia cecum upper colon lower colon tissue specimens main portion 1. everted 2. i n f l a t e d with physio-l o g i c a l s a l i n e 3. shaken with 0.02% EDTA PAS tissue specimens c e l l s suspension centrifuge 2,500 rpm 15 minutes e p i t h e l i a l c e l l s 1. wash with PBS 2. centrifuge 2,500 rpm for 10 mins iso l a t e d e p i t h e l i a l c e l l s - 42 -C. Extraction of the e p i t h e l i a l glycoproteins (105,000 x g) The procedures employed are outlined in figure 6. The i s o l a t e d e p i t h e l i a l c e l l s were suspended i n 1M NaCl and sonicated at 0°C for 6x60 second periods at a setting of 2 and 80 with a Biosonic Sonicator (Bronwill S c i e n t i f i c ) . Microscopic examination of smears of the sonicates was used to determine the degree of membrane rupture. The suspension was centrifuged at 105,000 x g for one hour at 4°C and the supernatant was dialysed overnight against d i s t i l l e d water. The contents of the d i a l y s i s bag were centrifuged at 1,000 x g for 10 minutes and the supernatant was concentrated by means of an Amicon u l t r a f i l t e r (Amicon Corp., Lexington, MA) f i t t e d with an XM50 membrane (retention l i m i t 50,000 mwt). The concentrated dialysed e p i t h e l i a l c e l l extract (105,000 x g supernatant) was stored frozen at -20°C. D. Fractionation of the e p i t h e l i a l glycoproteins The f r a c t i o n a t i o n procedure employed i s shown in F i g . 7. The 105,000 x g supernatants were dialysed against 1M NaCl, and concentrated to a volume of less than 10 ml. They were then applied to a 2.5 x 90 cm column (Pharmacia K25) packed with Biogel A15M (100-200 mesh) (Biorad Laboratories) and eluted with 1M NaCl containing 0.02% sodium azide. The carbohydrate-rich f r a c t i o n was concentrated, and was then dialysed successively against d i s t i l l e d water and 0.02M pyridine-HCl buffer pH 5.5. The s o l u t i o n obtained was c l a r i f i e d by centrifugation at 1,000 x g for 10 minutes and then applied to a 2.5 x 25 cm, Pharmacia K25 column packed with DEAE c e l l u l o s e (DE22-fibrous Whatman). The column was eluted with 0.02M pyridine-HCl buffer, pH 5.5, containing a convex gradient 0-2M NaCl, Fractions (10 ml) were c o l l e c t e d with a LKB 3400 B Radirac (LKB, Produktr. AB, Sweden) f r a c t i o n c o l l e c t o r f i t t e d with a 10 ml - 43 -Figure 6. Flow chart of the procedures used for the preparation of 105,000 x g supernatant from is o l a t e d e p i t h e l i a l c e l l s . i solated e p i t h e l i a l c e l l s 1. suspended i n 1M NaCl - 0°C 2. sonicated 6 minutes 3. centrifuge 2,500 rpm 10 mins 4. supernatants c o l l e c t e d , residues resuspended i n 1M NaCl repeat (2-4) pooled supernatant residues centrifuge at 105,000 x g for 1 hr residue supernatant 1. concentrate 2. d i a l y s e , d i s t i l l e d water or 1M NaCl concentrated 105,000 x g - 44 -Figure 7. Flow chart of the f r a c t i o n a t i o n procedures used to i s o l a t e the e p i t h e l i a l glycoproteins from the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s of the large bowel of the controls and the carrageenan treated r a b b i t s . 105,000 x g Agarose (A15M) gel chromatography i n 1M NaCL protein r i c h f r a c t i o n carbohydrate r i c h f r a c t i o n 1. concentrate by amicon u l t r a f i l t r a t i o n x M 50 2. dialyse against 0.02M pyridine-HCl pH 5.5 3. fractionate DEAE-cellulose with 0-2M NaCl gradient DNA glycoprotein I 1. dialyse against d i s t i l l e d water 2. concentrate and/or freeze dry glycoproteins - 45 -siphon (LKB 3408B-1012). The s i a l i c acid containing fractions were concentrated, dialysed against d i s t i l l e d water, l y o p h i l i z e d and weighed. The fr a c t i o n a t i o n procedures were monitored for proteins by the Lowry procedure (258), for s i a l i c acids by the periodate r e s o r c i n o l procedure of Jourdian e_t a l . (259), and/or for hexoses by the phenol-sulphuric acid procedure of Dubois et a l . (260), and for DNA (deoxyribonucleic acid) by the Indole procedure of Keck (261). Fractions were examined by electrophoresis performed on (1" x 6") ce l l u l o s e polyacetate s t r i p s (Sepraphore I I I , Gelman Inst. Co.), in T r i s -barbital-sodium b a r b i t a l buffer (high r e s o l u t i o n buffer, Gelman), pH. 8.8, for 30 minutes at 300 V. The s t r i p s were stained with A l c i a n blue pH 3.0 (262). E. H i s t o l o g i c a l and histochemical studies Tissue specimens (approximately l x l cm) from each region of the large i n t e s t i n e (cecum, upper and lower colons) were fixed i n 10% formol calcium for a period i n excess of 24 hours, processed through alcohols and chloroform, and embedded in Paraplast ( Laftcer, Sherwood Medical, U.S.A). Sections cut at a thickness of 5 u were stained with (A) hematoxylin and eosin (H and E) - for h i s t o l o g i c a l studies; and (B) with two histochemical staining procedures (a) the PBT/KOH/PAS - Reid et a l . (39) and (b) the PAT/KOH/PAS - C u l l i n g et a l . (263) known to demonstrate the 0-acetyl side chain substituted s i a l i c acids (see F i g . 8). Tissue sections from the carrageenan treated rabbits were compared with the co n t r o l s . The anatomical a l t e r a t i o n s , severity and the nature of the c e l l u l a r inflammatory f i l t r a t e , the severity and the extent of tissue damage and the severity of the inflammatory process were assessed. Figure 8. Flow chart of the h i s t o l o g i c a l and the histochemical procedures used to investigate the anatomical and the chemical nature of the e p i t h e l i a l glycoproteins of the large bowel of the rabbit. Cecum chemistry histology 1. f i x i n 10% formal calcium buffer 2. p a r a f f i n processed 3. sections were, cut 5 microns thick st a i n for PBT/KOH/PAS PAT/KOH/PAS H and E demonstration of the side chain s u b s t i t u t i o n of s i a l i c acid H & E = hematoxylin and eosin PAT/KOH/PAS = periodic acid thionine/saponification/periodic acid s c h i f f , C u l l i n g et a l . (263) PBT/KOH/PAS = periodic acid borohydride technique/saponification/periodic acid s c h i f f , , Reid et a l . (39). - 47 -The degree of histochemical change was assessed by estimating: (a) a l t e r a t i o n s i n the d i s t r i b u t i o n of the s t a i n , (b) a l t e r a t i o n s i n staining i n t e n s i t y and (c) any changes i n colour of the staining reaction of the e p i t h e l i a l mucins. The PBT/KOH/PAS staining was graded as l i g h t to dark while sections stained with the PAT/KOH/PAS were graded as red, red purple, purple, blue purple and blue. F. Chemical studies A l l analyses were carried out i n duplicate. Bovine submaxillary mucin (BSM) (1 mg/ml in d i s t i l l e d water) was used as q u a l i t y c o n t r o l throughout t h i s study. Saponification (removal of the 0-acetyl substituents) was accomplished by treating the glycoproteins solutions (9 volumes) with (1 volume) of IN KOH for 30 minutes at room temperature. The solutions were then neutralized with the equivalent quantity of IN ^ SO^. a) Estimation of the percentage of s i a l i c acids substituted at positions c 7 / c 8 This was performed by the method of Reid et a l . (107). As i l l u s t r a t e d i n F i g . 9, duplicate saponified (A) and unsaponified (B) samples (50 pi ) of the glycoproteins (105,000 x g or pure glycoproteins) were transferred (by use of an adjustable 0-200 p i , pipettor Pipetman, Gilson, France) to 10 ml screw capped tubes (100 x 10 mm pyrex). They were then oxidized with 0.04M periodic acid (10 pi) for 35 minutes at 0°C, reduced with 1.4M sodium borohydride (15 pi) at room temperature for 20 minutes, re-saponified with IN KOH (8 pi) for - 48 -Figure 9 . Flow diagram of the method used f o r the es t i m a t i o n of t o t a l s i a l i c a c i d and % side chain s u b s t i t u t i o n at p o s i t i o n C 7 KOH/PRT (A) standard KOH add 1-4 (premixed) e p i t h e l i a l g l y c o p r o t e i n s PRT (C) KOH PRS(B) NKOH 1 . oxidase 0.04M H I O 5 2 . reduce 1 . 4 M N A B H 4 3. saponify IN KOH 4 . n e u t r a l i s e 2 N H C 1 1 . o x i d i z e 0.04M H I O 5 2 . 400 u l r e s o r c i n o l reagent 3. heat b o i l i n g water bath 8 minutes 4. 400 y l t-butanol mix 5. heat 37°C 5 minute - mix w e l l read at 630 nm KOH = s a p o n i f i e d NKOH = unsaponified C-B 1-/B % C 7/C 8 = L-(A) T o t a l s i a l i c a c i d (B) PRS (C) Non-saponified PRT - X 100 / - 49 -30 minutes at room temperature and then neutralized with 2N HC1 (18 j j l ) . To further duplicate saponified samples (C) and to the samples used to construct the standard curve (human serum mucoid 54.5 ug/ml) was added 50 y l of a premixed solution containing 10 y l periodic acid, 15 y l sodium borohydride, 8 y l KOH, and 18 y l HC1. A l l the samples were then oxidised with 20 y l of 0.04M periodic acid for 35 minutes at 0°C. Resorcinol reagent (200 y l ) was added and the solutions were heated in a b o i l i n g water bath for 8 minutes, cooled, mixed with (200 y l ) of t-butanol and incubated for 3-5 minutes at 37°C. The solutions were mixed and the absorbance read at 630 nm and the percentage sub s t i t u t i o n at + Cg was calculated according to the following equation (Reid et a l . , 103). PRT - PRS PRS % C 7/Cg = 1 - KOH PRT x 100 (KOH PRT) PRS = sample A PRT = sample B KOH PRT = sample C - 50 -b) V i b r i o cholera neuraminidase digestion studies These were carried out as i l l u s t r a t e d in F i g . 10. Aliquots (81 y l ) of saponified and unsaponified glycoprotein (63 y l glycoprotein + 8 y l IN KOH + 8 -2 y l IN E^SO^) were incubated with V i b r i o cholera neuraminidase (10 I.U. i n 0.05M sodium acetate buffer pH 5.5 containing 0.02% sodium azide and 0.1% calcium c h l o r i d e ) , at 37°C. Af t e r 24 hours, a further 10~ 2I.U. of the enzyme was added and the incubation was continued for a further 24 hours. The incubate was then saponified with IN KOH (12 yl) for 30 minutes at room temperature and then neutralized with IN E^SO^ (12 y l ) . Duplicate aliquots (25 y l ) were analysed for free and k e t o s i d i c a l l y bound s i a l i c acids by the method of C u l l i n g et^ a_l. (105), human serum mucoid (54.5 yg/ml) and N-acetyl neuraminic acid (100 yg/ml) were used as standards i n the bound and free assays r e s p e c t i v e l y . The percentage of s i a l i c acids released from the saponified and unsaponified glycoproteins was used to cal c u l a t e the percentage s i a l i c acid substituted at position C,. 4 % s i a l i c acid substituted at C 4 = K0H% released - NKOH % released x 100 Total s i a l i c acid % released s i a l i c acid = NKOH TB/A (released) x 100 Total s i a l i c acid KOH - % released = % s i a l i c acid released by the enzyme from the saponified glycoproteins NKOH - % released = % s i a l i c acids released by the enzymes from the unsaponified glycoprotein Total s i a l i c acid = t o t a l s i a l i c acid contents (released + bound) - 51 -Figure 10. Flow diagram i l l u s t r a t i n g the method used for the estimation of the percentage of s i a l i c acids released by digestion with V i b r i o cholera neuraminidase. e p i t h e l i a l glycoprotein KOHGP NKOHGP (+)K0H (IN) 30 mins (+)H2S04 (IN) — rm. temp. + 0.5M K 2 S O 4 + acetate buffer (pH 5.5) + V i b r i o cholera neuraminidase 1. incubate 37°C, 24 hrs 2. V i b r i o cholera neuraminidase 3. incubate 37°C 24 hrs 4. treated with IN KOH then n e u t r a l i z e with IN H 2 S O 4 Analyse Bound SA PRB/A released SA TB/A KOH = saponified NKOH = unsaponified TBA = t h i o b a r b i t u r i c acid assay PRB/A = modified r e s o r c i n o l assay for bound s i a l i c acid (105) GP = glycoproteins SA = s i a l i c acids (+) = add - 52 -c) Estimation of the percentage of s i a l i c acids substituted at pos i t i o n C^/Cg This analysis was car r i e d out only on p u r i f i e d glycoprotein samples. As i l l u s t r a t e d i n F i g . 11, t r i p l i c a t e (70 y l ) saponified and unsaponified samples of glycoprotein were oxidized with 0.04M periodic acid (15 y l ) for 35 minutes at 0°C and then a 1:1 (v/v) mixture of 0.5 M potassium iodide and 0.5M sodium thiosulphate (15 y l ) was added. Aliquots (30 y l ) of a premixed periodic acid-potassium iodide-sodium thiosulphate s o l u t i o n were added to unoxidised saponified and unsaponified samples to give the unoxidized sample blank. Formaldehyde was then estimated by the procedure of Nash (222) and read at 412 nm. Standard curves were constructed using N-acetyl neuraminic acid (NANA) and the percentage su b s t i t u t i o n at po s i t i o n Cg/Cg was calculated by the equation below. %C 8/C 9 = NKOH - NKOH blank x 1 0 Q KOH - KOH blank NKOH = non saponified formaldehyde NKOH blank = no oxidized non saponified sample KOH = saponified formaldehyde KOH blank = saponified non oxidized sample Student t-test was calculated according to the following equation: x - y - D - 53 -Figure 11. Flow diagram i l l u s t r a t i n g the method used for the estimation of the percentage of s i a l i c acids substituted at positions Cg / C 9 , PURE GLYCOPROTEIN Saponified Non-saponified (+)0.0AM periodic acid 35 min 0°C (+)0.5M KI 0.5M Na thiosulphate + premixed periodic acid (0.04M) 0.5M KI - 0.5M Na thiosulphate, add formaldehyde reagent (acetyl acetone*) heat at 60°C - 8 mins read at 412 nm Cal c u l a t i o n = % Cg/Co = D ~ c v 100 A - B *Nash (264) - 54 -RESULTS I. Preliminary Studies Preliminary experiments were ca r r i e d out to determine optimum conditions for a study of lower GI tract u l c e r a t i o n . Tables 8 and 9 and figures 4 and 12-14 show the r e s u l t s of preliminary experiments designed to ascertain: (1) the f e a s i b i l i t y of applying published techniques (37,102) for the i s o l a t i o n of large i n t e s t i n a l glycoproteins to the large i n t e s t i n e of rabbits and guinea pigs, (2) which of the animal species, rabbit, guinea pig and rat was the most suitable for the study of lower g a s t r o i n t e s t i n a l t r a c t u l c e r a t i o n , and (3) the dose of degraded carrageenan required to induce such u l c e r a t i o n . As w i l l be seen: (1) the rabbit yielded more large i n t e s t i n a l e p i t h e l i a l glycoproteins than the guinea pigs (Table 8). (2) degraded carrageenan, 1% i n tap water, was a suitable dose (Table 9) for the induction of lower digestive tract u l c e r a t i o n i n both rabbits and guinea pigs; induction of s i m i l a r u l c e r a t i o n did not occur i n rats even at a much higher (5%) dose of degraded carrageenan. (3) Anatomically ( F i g . 4, p. 39), i t was possible to divide the large i n t e s t i n e of the rabbits and the guinea pigs into four d i s t i n c t i v e regions. In the rabbits the length of these regions were; (a) cecum - 25-30 cm. (b) appendix - 5-10 cm. (c) upper colon - 25-30 cm. (d) lower colon - 60-70 cm. Table 8. E f f e c t s of various doses of degraded carrageenan fed ad l i b i n tap water to guinea pigs, rabbits and r a t s . Animal species % Carrageenan (w/v) Occult blood Treatment duration (wks) Ulceration Guinea pigs 1 + 7 days 7 + 2 + 4-5 days 5 + 5 + 1 day 2 + Rabbits 1 + 6 days 6 + 2 + 4 days 6 + Rats 1 - 5 -2 - 5 -5 + 4 weeks 5 -+ = present - = absent - 56 -Table 9. The results of preliminary studies of the i s o l a t i o n of e p i t h e l i a l glycoproteins from rabbit and guinea pig large i n t e s t i n e . % s i a l i c acids Animal mg glycoprotein ug SA/mg glyco- substituted at Species obtained protein obtained positions C-j/Cg Rabbits Cecum 1.75 Upper colon 4.21 Lower colon 2.5 Guinea Pigs 109.0 40.42 95.0 65.41 118.0 69.40 Cecum Upper colon Lower colon 0.5 0.84 3.75 29.0 55.0 160.0 28.55 50.44 87.59 - 57 -Figure 12. Typical fractionation of the 105,000 x g supernatants obtained from rabbit cecal epithelial cells by gel chromatography on Biogel A15M (100-200 mesh). The gel columns were eluted with 1M NaCl containing 0.02% sodium azide. A carbohydrate rich fraction (A) and protein rich fraction (B) were resolved. s i a l i c acid rich fraction protein rich fraction - 58 -Figure 13. Diagram of the cellulose acetate electrophoresis of the products of the fractionation procedures shown in Figs. 12 and 13. Column chromato-grapy fractionation were examined by electrophoresis performed on 1" x 6" cellulose polyacetate strips (Sepraphore III, Gelman Inst. Co.), in t r i s -barbital-sodium barbital buffer (high resolution buffer, Gelman) pH 8.8 for 30 minutes at 300V. The strips were stained with alcian blue at pH 3.0' a b c d e (a) Heparin standard (b) 105,000 x g supernatant (c) Biogel A15M column chromatography, Fraction A (Carbohydrate rich fraction), see Fig. 12. DEAE-cellulose chromatography of fraction A (see Fig. 14). (d) Homogenous sialic acid rich fraction (A) (e) Fast moving DNA rich fraction (B) - 59 -Figure 14. T y p i c a l f r a c t i o n a t i o n on DEAE-cellulose of the carbohydrate r i c h f r a c t i o n obtained by the Agarose A15M g e l chromatography of r a b b i t c e c a l e p i t h e l i a l c e l l s . loo 2oo Soo 400 5oo VOLUME OF ELUENT (ML) • s i a l i c a c i d r i c h f r a c t i o n » DNA r i c h f r a c t i o n - NaCl gradient The carbohydrate rich fraction (fraction A) was dialysed against 0.02M pyridine-HCl buffer pH 5.5 and was further fractionated with a 2.5 x 25 cm of DEAE cellulose (DE22-fibrous Whatman) column, eluted with 0.0M pyridine-HCl buffer, pH 5.5 containing a convex gradient 0.15M NaCl. Two fractions were resolved, a major sialic acid fraction (A) and a DNA containing fraction (B). - 60 -(4) H i s t o l o g i c a l and histochemical studies i n rabbits showed that there are d i s t i n c t i v e regional v a r i a t i o n s between the cecum and the upper and the lower colons; these w i l l be discussed i n d e t a i l l a t e r i n the r e s u l t s . (5) Complete removal of the cecal e p i t h e l i a l c e l l s was accomplished within three treatments. It was more d i f f i c u l t , however, to remove completely the e p i t h e l i a l c e l l s from the upper and lower colons; those located at the base of the crypts being more d i f f i c u l t to remove than those i n the upper parts of the crypts. (6) As described below i t was possible to i s o l a t e p u r i f i e d e p i t h e l i a l glycoproteins. Figure 12 i l l u s t r a t e s the r e s u l t s of gel chromatography on Agarose A15M, of the 105,000 x g supernatant prepared from cecal e p i t h e l i a l c e l l s of r a b b i t s . As can be seen, the 105,000 x g supernatant yielded two f r a c t i o n s , a carbohydrate r i c h f r a c t i o n (A), and a protein r i c h f r a c t i o n (B). E l e c t r o -phoresis of the carbohydrate r i c h f r a c t i o n showed that i t consisted of two subfractions ( F i g . 13), f r a c t i o n 1 and f r a c t i o n 2; f r a c t i o n 2 having approxi-mately twice the mobility of f r a c t i o n 1. DEAE c e l l u l o s e chromatography of the carbohydrate r i c h f r a c t i o n yielded two e l e c t r o p h o r e t i c a l l y homogeneous fr a c t i o n s , the slower moving being s i a l i c acid r i c h , while the other was r i c h i n DNA ( F i g . 14). On the basis of the findings i n these preliminary studies r a b b i t s , fed a dose of 1% degraded carrageenan i n tap water, were chosen for this i n v e s t i g a t i o n . - 61 -Experiment #1 This experiment was designed to test whether or not changes i n e p i t h e l i a l glycoproteins were associated with u l c e r a t i o n of the lower dig e s t i v e t r a c t . A. Experimental design 30 young adult rabbits (2.960 + 0.248 kg), 15 males and 15 females were used. Ten rabbits (5 males and 5 females) fed water ad l i b served as c o n t r o l s . The remaining twenty rabbits (10 males and 10 females) which were given 1% degraded carrageenan in t h e i r drinking water ad l i b for a maximum period of nine weeks served as the test group. Three degraded carrageenan treated animals and one animal from the control group died before the completion of the experiment and were not included i n the study. B. C l i n i c a l observations The observations are summarized in Table 10. As can be seen, a l l the carrageenan treated rabbits developed diarrhea and showed occult and v i s i b l e f e c a l blood by the f i n i s h of the experiment. A l l animals, except #16, 20 and 30, l o s t body weight. Treatment with degraded carrageenan apparently resulted i n acute disease. Fecal occult blood was present i n some animals as early as 24 hours a f t e r the commencement of the carrageenan treatment. In a l l animals this was r a p i d l y followed by soft feces and then diarrhea with v i s i b l e blood. At t h i s time there was a sharp drop i n the carrageenan and food intake accompanied by a rapid loss of body weight and a progressive d e t e r i o r a t i o n i n health. The animals were weak, emaciated and anemic to a va r i a b l e extent. During t h i s Table 10. C l i n i c a l observations made upon the carrageenan treated and the controls grOup i n experiment #1. Group Average Food Intake gm/day Average Carrageenan (or H 20) ml/day Occult Blood V i s i b l e Blood Average Bodyweight Change (kg) Controls 170+25 440 +41 ( a ) Normal Feces ( + H.310 +0.657 Carrageenan treated 85+34 250+90 + + Soft/diarrhea (-)*0.647 +0.038 *Three carrageenan treated rabbits (#16, 20 and 30) gained body weight (0.271+0.271 kg) + = present - = not detected (+) = gained body weight (-) = l o s t body weight (a) = water intake - 63 -stage of the carrageenan treatment (1-2 weeks) three animals died and f i v e others had to be s a c r i f i c e d . The animals which survived this period became progressively weaker and emaciated. They showed p o s i t i v e f e c a l occult blood continuously. Diarrhea and v i s i b l e f e c a l blood were intermittent and were always preceded by the intake of substantial amounts of carrageenan. Animals #16, 20 and 30 consistently showed p o s i t i v e f e c a l occult blood and had mild diarrhea frequently accompanied by v i s i b l e blood. The average food intake of the experimental animals was 85 +_ 35 g/day, and the average carrageenan intake was 250 + 90 ml/day (0.99 +_ 1 g/kg BWt); compared with a food intake of 170 g/day and water intake of 440 + 41 ml/day for the controls. A l l the control animals were healthy (except #25 which died of an abdominal hernia) and gained body weight (+ 1.310 +_ 0.065 kg). The carrageenan treated animals a l l l o s t body weight 0.647 + 0.038 kg with the exception of #16, 20 and 30 which gained body weight 0.271 + 0.271 kg. No f e c a l occult blood or change i n the physical properties of the feces of the controls was observed throughout the course of the study. C. Post-morten observations A l l carrageenan treated animals (except #16, 20 and 30) were emaciated, the blood was l i g h t in colour and the body fat was v a r i a b l y depleted ( i t was severely depleted in animal #19 which showed ascites 500 ml, straw-coloured f l u i d ) and i n addition white-grey deposits were observed on the serosal surface of the cecum, and the upper and lower colons and on the capsule of the l i v e r . In the rabbits s a c r i f i c e d early, the cecum was f i l l e d with l i q u i d dark brown coloured feces with an offensive smell. The cecum was thin and floppy and the mucosa was congested and covered with pinpoint hemorrhagic spots - 64 -which, under the d i s s e c t i n g microscope were found to be mucosal u l c e r s . The transverse f o l d s of the cecum were edematous, severely congested, hemorrhagic and u l c e r a t e d . The small i n t e s t i n e s were inflammed and there were mucosal erosions i n the stomach of many animals at t h i s stage of the carrageenan treatment. In r a b b i t s t r e a t e d for longer periods, s i m i l a r but more severe p a t h o l o g i c a l changes were noted. In p a r t i c u l a r , i n the cecum there was frank mucosal u l c e r a t i o n c o n s i s t i n g of m u l t i p l e u l c e r s of v a r i a b l e s i z e and shape throughout the l i n i n g e p i t h e l i u m and on the transverse f o l d s . In a d d i t i o n i n t h i s group the spleen and the mesenteric lymph nodes were enlarged, the l i v e r was swollen and g r a y i s h yellow i n colour and showed white-grey deposits on the capsule. I t was i n t e r e s t i n g to note that at t h i s stage of the experiment the upper d i g e s t i v e t r a c t s were less a f f e c t e d . Topographically the p a t h o l o g i c a l changes were most prominent i n the cecum, infrequent i n the lower colon and l e a s t n o t i c e a b l e i n the upper co l o n . In the cecum the mucosal changes were d i f f u s e u l c e r a t i o n being more common on the transverse f o l d s . In the lower colon, however, p a t h o l o g i c a l changes, when present, were l o c a l i s e d and l i m i t e d to the r e c t a l end of the organ, u s u a l l y i n a s s o c i a t i o n with severe u l c e r a t i o n . Only m i l d changes (congestion and very few p e t i c h e a l hemorrhages) were observed i n the upper colon and these were l i m i t e d to the c e c a l end of the organ. The s e v e r i t y and extent of the p a t h o l o g i c a l changes were c o r r e l a t e d w i t h the d u r a t i o n of the carrageenan treatment, i . e . the longer the treatment, the more e s t a b l i s h e d the disease and the more evident the mucosal u l c e r a t i o n . None of the preceding observations were noticed i n the c o n t r o l animals. The gross anatomical observations f o r the carrageenan t r e a t e d and c o n t r o l r a b b i t s are i l l u s t r a t e d i n f i g u r e s 15-17. - 65 -Figure 15. Ventral view of the cadaver of a 1% carrageenan treated rabbit note evidence of diarrhea; the dark brown fec a l material indicates the presence of v i s i b l e f e c a l blood. - 67 -Figure 16. Comparison between the gross anatomical feature of the everted cecum and upper colon of controls and carrageenan treated r a b b i t s . (a) Normal cecum. (b) Cecum from rabbit treated with 1% degraded carrageenan. Note the congestion and mucosal ulcerations (arrow) which are more common on and around transverse f o l d s . (c) Normal upper colon. (d) Upper colon from degraded carrageenan treated r a b b i t . - 69 -Figure 17. Gross anatomical comparison between the everted lower colon of controls and of degraded carrageenan treated rabbits. (a) Normal lower colon (b) Lower colon from carrageenan treated rabbit, the arrow indicates mucosal u l c e r a t i o n at the r e c t a l end of the lower colon. - 71 -D. H i s t o l o g i c a l and histochemical studies The r e s u l t s of these studies are summarised i n Table 11 and discussed below; a) H i s t o l o g i c a l observations  Controls Normal rabbit cecum ( F i g . 18) i s lined with a si n g l e layer of columnar epithelium which form crypts. The crypts contain a large number of goblet c e l l s , are short and extend deeply into the t h i n lamina propria. The lamina propria i s made up of a loose connective tissue together with blood vessels and is i n f i l t r a t e d with some macrophages, plasma c e l l s and a very few eosino-p h i l s . Underneath the lamina propia i s a t h i n muscular layer (muscularis mucosae) which covers the muscular layers proper. These are covered on the peritoneal surface by smooth, thin serosa. Carrageenan treated animals Sections from the carrageenan treated rabbits stained with H & E showed a variable extent of h i s t o l o g i c a l change which was well correlated with both the gross anatomical findings and with the duration of the carrageenan treatment. Sections prepared from the ce c i of rabbits s a c r i f i c e d early (#14, 15 and 19), showed mild h i s t o l o g i c a l changes as compared to the sections taken from rabbits treated for longer periods. In the c e c i of the animals which died or were s a c r i f i c e d i n the early stage of the carrageenan treatment, there was congestion, edema, submucosal hemorrhage and mucosal u l c e r a t i o n . The crypts were necrotic and reduced i n both length and number. In the lamina propria, there was an increased inflammatory c e l l u l a r i n f i l t r a t i o n composed mainly of macrophages, plasma Table 1 1 . The r e s u l t s of the h i s t o l o g i c a l and histochemical observations of the ceci of the controls and of the carrageenan treated r a b b i t s . Histochemistry  Group Histology H & E PAT/KOH/PAS PBT/KOH/PAS Single layer columnar e p i -thelium. E p i t h e l i a l crypts loosely placed i n the cecum. Lamina propria contains few plasma c e l l s , lympocytes, macrophages and with very few eosinophils (occasional) Entire crypt stained. Over-a l l s t a i n i n g red purple, upper portion of the crypts more red, lower portion more blue, upper and lower colons clear d i s t i n c t i o n between upper and lower halves of the crypts . red along the entire length of the crypts, l i g h t red at the base. Upper & lower colons, upper half of the crypts red, lower h a l f l i g h t red or unstained II Edema and congestion, hemor-rhages i n the submucosa, mucosal u l c e r a t i o n , e p i t h e l i a l crypts were reduced i n number and height, d i s t o r t e d and ne c r o t i c . In the lamina propria severe i n f i l t r a t i o n of macrophages, plasma c e l l s , lymphocytes, massive aggre-gation of eosinophils i n and around the ulcerations Reduction i n the stained material. Stained f a i n t i n color, o v e r a l l color was blue, i t was s o l i d blue at the ulcer border with some red away from the ulce r a t i o n , loss of staining pattern. In the upper & lower colons the entire crypts stained blue or purple - no d i s t i n c t i between the upper and lower halves Overall decrease i n staining a f f i n i t y (stained f a i n t ) , loss of pattern, not stained or very l i g h t red at the ulcer border. Upper & lower colon stained very l i g h t red i n the upper h a l f . No staining i n the lower h a l f of the crypts Group I = controls Group II = degraded carrageenan treated - 73 -Figure 18. Photomicrographs of H & E stained sections that compare the cecal mucosa of control rabbits with the mucosa of degraded carrageenan treated rabb i t s . (a) Controls (x 6.3) (b) Degraded carrageenan treated rabbit (b x 6.3, c x 2.5); Note mucosal u l c e r a t i o n and inflammatory reaction. - 75 -Figure 19. Photomicrographs of H & E section to demonstrate the d i f f e r e n c e between the upper colons of control (a x 6.3) and degraded carrageenan treated rabbits (b, c, x 2.5). In the majority of the carrageenan treated rabbits (75%) there was a degeneration of the e p i t h e l i a l crypts together with an inflammatory exudate (b x 2.5). The rest of the carrageenan treated rabbits (25%) showed mucosal ulcerations i n the upper colon (c x 2.5). c e l l s and eosinophils. In the l a t e r stage animals s a c r i f i c e d at the termin-ation of the experiment there were very well established mucosal u l c e r a t i o n s . In the ulcerated areas, a mass o f necrotic tissue replaced e p i t h e l i a l crypts or surface epithelium. The lamina propria was thick and f i l l e d with inflam-matory c e l l s composed of plasma c e l l s , macrophages and a massive aggregation of eosinophils with a p r o l i f e r a t i o n of scar tissue at the depth of the u l c e r . At the ulcer marginsthere was an increased number of goblet c e l l s while at a distance from the margins, there were frequently active, twisted, long, d i s -torted e p i t h e l i a l crypts ( F i g . 18). In the upper and the lower colon the anatomical outlines of the tis s u e were comparable to that of the co n t r o l s . Detailed study indicated that the e p i t h e l i a l c e l l s on the luminal surface were degenerated and, i n some sections, n e c r o t i c . The c e l l s l i n i n g the crypts were vacuolated and there were signs of an increase i n the goblet c e l l population. The lamina propria was congested, with s l i g h t edema, and early inflammatory changes. In some sections from animals treated with carrageenan for long periods there was a massive i n f i l t r a t i o n of eosinophils. Typical l o c a l i s e d mucosal u l c e r a t i o n was noted i n sections of both the upper and lower colons from animals #'s 1, 4, 16, 26 ( F i g . 19). b) Histochemical studies 1. PBT/KOH/PAS In the normal cecum stained with the PBT/KOH/PAS procedure ( F i g . 20), the red stai n i n g occurred throughout the ent i r e length of the crypts; the i n t e n s i t y being greater towards the top of the crypts. In the neck of the crypt the staining was intense and the stained c e l l s were vacuolated. On the surface epithelium only a few c e l l s were stained. - 78 -In the normal upper and the lower colons, p a r t i c u l a r l y i n the upper colon, the upper ha l f of the crypt was stained more intensely than the lower h a l f of the crypts with the most basal part being unstained ( F i g . 21). In the lower colon, a s i m i l a r but less d i s t i n c t pattern was seen, the major part of the crypts being red with less staining i n the basal portion. In the c e c i of the carrageenan treated rabbits (except #12, 14 and 15 which showed an increase i n the red s t a i n i n g ) , there was an o v e r a l l decrease i n the stained area, not a l l crypts were stained and there were fewer stained c e l l s per crypt. In addition, there was a decrease i n the i n t e n s i t y of the red stai n i n g along with a loss of pattern. The e p i t h e l i a l crypts i n and around the ulcerated mucosa were eit h e r unstained or the staining was very f a i n t . The st a i n i n g became more intense away from the margins of the ulcers ( F i g . 20). 2. PAT/KOH/PAS Figure 20 shows sections from normal control cecum, upper colon, and lower colon stained by the PAT/KOH/PAS procedure. In the cecum, a l l the e p i t h e l i a l crypts were stained with an o v e r a l l red-red purple colour. In the i n d i v i d u a l crypts, however, a blend of the blue and red colours was observed. In the base of the e p i t h e l i a l crypts the mucus stained blue, and i n the middle purple s t a i n i n g predominated. The neck stained a red-red purple, while i n the surface epithelium where the c e l l s tended to be vacuolated the s t a i n i n g was red. In the upper and the lower colon, two d e f i n i t e regions could be recognized i n the crypts. The upper h a l f stained red-red purple, the lower h a l f stained blue purple with blue at the base of the crypts. In the cecum of the carrageenan treated rabbits ( F i g . 20), there were many unstained c e l l s within the i n d i v i d u a l crypts and many unstained crypts. There was loss of pattern together with a decrease i n red s t a i n i n g , blue s t a i n i n g predominating throughout the sections. It was of int e r e s t that while the e p i t h e l i a l c e l l s at the ulcers margins stained a pure blue, those away from the area of u l c e r a t i o n stained blue purple. Overall the s t a i n i n g reaction was f a i n t and the colour was blue to blue purple i n d i c a t i n g that the cecal e p i t h e l i a l c e l l s of the ulcerated rabbits contained less glycoprotein with less substituted s i a l i c acids. In the upper and the lower colon ( F i g . 21), there was complete loss of the normal stainin g pattern, i . e . a loss of the d i s t i n c t i o n between the upper and the lower halves of the crypts. There was decrease i n the red colour and enti r e crypts were stained blue (with blue plus some l i g h t reddish colour close to the lumen). Overall the colour was blue to blue purple. Compared to controls animals #'s 12, 14 and 15 showed a s l i g h t increase i n the red colour ( F i g . 21). Animals #'s 1, 4, 16, and 26 had ulcers which showed histochemical changes s i m i l a r to those observed i n the ulcerated c e c i , i n that at the ulcer margins there was a d e f i n i t i v e decrease i n the 0-acetyl substituted s i a l i c acid, the colour of the PAT/KOH/PAS being pure blue; i t became redder away from the margin, F i g . 20. In summary, i n the controls, the upper portion of the e p i t h e l i a l crypts contained more side chain 0-acetyl substituted s i a l i c acids than the basal portion. In the carrageenan treated animals, there was a decrease i n the 0-acetyl substituted s i a l i c acids; these changes were most prominent i n the cecum and occurred p a r t i c u l a r l y at the margins of the u l c e r s . - 82 -: t t ? ( i Figure 21. The PAT/KOH/PAS and PBT/KOH/PAS techniques were used to investigate the nature and d i s t r i b u t i o n of the 0-acetyl substituted s i a l i c acids i n the upper colon of control and carrageenan treated rabbits. With the PAT/KOH/PAS procedure, two d i s t i n c t i v e regions can be recognized i n the e p i t h e l i a l crypts of normal upper colon (a x 10); the lower h a l f , stained more blue, and the upper ha l f stained with more red. This corresponds with l i g h t red at the lower h a l f and intense red i n the upper h a l f of the crypts stained with the PBT/KOH/PAS procedure (b x 10). In the degraded carrageenan rabbits there is an a l t e r a t i o n of the normal staining pattern, the difference between the upper and the lower halves of the e p i t h e l i a l crypts i s no longer present, the entire crypt stained l i g h t blue with the PAT/KOH/PAS (c x 6.3) and f a i n t red with the PBT/KOH/PAS (d x 6.3) procedures. - 84 -D. Chemical studies a) Percentage of s i a l i c acids substituted at C^/Co The percentage of the s i a l i c acids substituted at positions C^/Cg present i n the 105,000 x g supernatants prepared from e p i t h e l i a l c e l l s i s o l a t e d from the large intestines of both the control and the degraded carrageenan treated rabbits are shown in Table 12, 13 and Figures 22, 23. Preliminary analysis of this data indicated that i n both the control and treated groups of animals there were no s t a t i s t i c a l differences between the males and the females. Table 13 shows the res u l t s obtained from pooling the data. Reference to Table 13 shows that there were s i g n i f i c a n t differences between the d i f f e r e n t regions of the lower GI tract of the normal animals with regard to the percentage of s i a l i c acids substituted at C,/C0. The 7 o percentage s u b s t i t u t i o n at C 7/Cg in the cecum and lower colon was s i m i l a r and greater than that found i n the upper colon. In the cecum and the lower colon of the carrageenan treated animals there were, as compared to the controls, decreases i n the percentage of the s i a l i c acids substituted at C^ and/or Cg. The decrease was, however, s t a t i s t i c a l l y s i g n i f i c a n t only i n the cecum. No differences were found i n the upper colon. b) Digestion with V i b r i o cholera neuraminidase Preliminary analysis of the V i b r i o cholera neuraminidase digestion studies showed that both i n controls and in the carrageenan treated group there were no differences between the males and the females (Tables 14 and 15). The re s u l t s obtained from pooling the data from the males and the females are shown in Table 13. Table 14 shows that: - 85 -Table 12. Percentage of C 7 / C 8 substituted s i a l i c acids i n the 105,000 x g supernatant obtained from the e p i t h e l i a l c e l l s of the large i n t e s t i n e of the controls and the carrageenan treated r a b b i t s . Mean % S i a l i c Acids Substituted at C 7 / C 8 Male Female Region of Large Intestine Controls Carrageenan treated Controls Carrageenan treated Cecum 64.6 + 4.8 42.8 + 15.3 73.2 + 8.6 37.6 + 12.0 Upper colon 43.2 + 10.0 46.7 + 9.9 46.9 + 10.7 43.0 + 9.9 Lower colon 65.7 + 5.9 52.4 + 12.6 61.5 + 10.8 54.6 + 17.3 Controls; Males vs. females t - t e s t DF = 6 Cecum = 1. 76 Upper colon = 0.16 Lower colon = 0.81 Controls vs. carrageenan treated Females vs. females t = Df = 10 Cecum =5.25 Upper colon = 0.64 Lower colon = 0.72 Carrageenan treated: t-test DF = 15 Cecum = 0.46 Upper colon = 0.66 Lower colon = 0.31 males vs males t = DF = 11 Cecum =2.67 Upper colon =0.17 Lower colon = 2.10 Males vs. females - 86 -Table 13. Analysis of the s i a l i c acids i n the 105,000 x g supernatants i s o l a t e d from the e p i t h e l i a l c e l l s of d i f f e r e n t regions of the large intestines of control and degraded carrageenan treated r a b b i t s . The data presented is pooled data from both males and females. (a) Cecum %Cy/C8 % released n'dase % released % C 4 KOH n'dase Controls (a>68.9 + 7.9 < b )22.7 + 6.9 (c>42.1 + 8.0 64.8 + 10.0 C. treated *40.4 + 13.7 *33.9 + 13.1 *58.9 + 10.9 41.6 + 21.9 (b) Upper colon Controls 47.4 + 8.2 29.5 + 15.3 75.9 + 3.6 61.1 + 19.6 C. treated 45.0 + 11.5 25.6 + 14.6 75.3 + 5.1 66.0 + 17.7 (c) Lower colon Controls 64.0 + 8.6 17.8 + 8.0 74.3 + 8.7 76.0 + 9.0 C. treated 53.4 + 14.6 22.6 + 11.6 73.9 + 7.8 69.4 + 17.3 * S t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the controls t = DF = 24 Controls %C7/C8 DF = 7 t = (a) = 5.45 Cecum vs upper colon = 5.35 (b) = =2.71 Cecum vs lower colon = 1.18 (c) = -3.33 Lower colon vs upper colon = -3.96 - 87 -Table 14. Percentage of s i a l i c acids i n the saponified 105,000 x g supernatant obtained from the e p i t h e l i a l c e l l s of the large bowel of the controls and from the carrageenan treated rabbits released a f t e r d i g e s t i o n with V i b r i o cholera neuraminidase. Region of Mean % s i a l i c acid released with V.C. n'dase large in t e s t i n e Male Female Controls Treated Controls Treated C e c u m 42.1 + 8.0 57.1 + 11.4 46.6 + 7.9 59.0 + 11.0 Upper colon 76.0+ 4.8 73.5+ 5.5 75.9+2.1 75.3+ 5.1 Lower colon 72.9 + 10.8 73.9 + 7.8 76.0 + 6.5 75.4 + 10.4 Controls = males vs. females DF = 7 Cecum t = -0.8 Upper colon = 0.05 Lower colon = -0.5 Controls vs. treated (a) males vs. females DF = 12 Cecum t = -2.53 Upper colon = 0.86 Lower colon = 0.07 Treated = males vs. females DF = 15 Cecum t = -0.35 Upper colon = -1.89 Lower colon = - 0.71 Controls vs. treated (b) females vs. males DF = 10 Cecum t = -1.99 Upper colon = -1.36 Lower colon = 0.11 - 88 -Table 15. Percentage of the s i a l i c acids i n the 105,000 x g supernatants obtained from the e p i t h e l i a l c e l l s of the large bowel of the controls and from the carrageenan treated rabbits released a f t e r digestion with V i b r i o cholera neuraminidase. Region of large intestine Mean % s i a l i c acid released with V.C. n'dase Males Females Controls Treated Controls Treated Cecum 23.7 + 8.6 37.1 + 10.9 21.4 + 4.9 31.5 + 13.1 Upper colon 32.4 + 15.0 21.9 + 14 .8 26.5 + 16.8 30.5 + 13.7 Lower colon 17.7 + 8.6 22.1 + 11 .6 18.0 + 8.6 23.1 + 12.3 Controls males vs. females Treated males vs. females t = DF = 6 Cecum = 0.47 t = DF Cecum =0.98 = 15 Upper colon = 0.51 Lower colon = -0.05 Controls vs. treated (a) females vs. females t = DF = 10 Cecum = -1.45 Upper colon = -0.43 Lower colon = -0.74 Upper colon = -1.19 Lower colon = - 0.17 Controls vs. treated (b) males vs. males t = DF = 12 Cecum = -2.37 Upper colon = 1.21 Lower colon = -0.74 - 89 -(a) i n a l l regions of the large i n t e s t i n e of both the controls and carrageenan treated animals, sapo n i f i c a t i o n resulted i n an increased neuraminidase s u s c e p t i b i l i t y . Such increases have been a t t r i b u t e d to the presence of s i a l i c acids bearing 0-acetyl substituents at and/or at (127). The f a i l u r e to l i b e r a t e a l l s i a l i c acids upon digestion of the saponified supernatants i s presumably due to some unrecognized s t r u c t u r a l feature i n the glycoproteins. (b) The s i a l i c acids of the 105,000 x g supernatant obtained from the c e c i of the carrageenan treated animals were more susceptible to neuraminidase than those i n supernatants obtained from the controls. No such differences were noted between the supernatants obtained from the upper or the lower colons, see Table 15. It was necessary to demonstrate that the differences between the s i a l i c acids i n the 105,000 x g supernatants isolated from the control and carrageenan treated animals were r e f l e c t e d in the p u r i f i e d e p i t h e l i a l glycoproteins isolated from them. Therefore, pools were made of s i m i l a r volumes of the concentrated supernatants from the c e c i upper colon and lower colon of the controls and of the carrageenan treated animals. Each pool was fractionated by agarose gel chromatography followed by f r a c t i o n a t i o n with DEAE c e l l u l o s e chromatography. Analysis of the pooled supernatants and of the isolated glycoproteins (Table 16) confirmed the findings obtained by analysis of the i n d i v i d u a l 105,000 x g supernatants and showed that, i n the carrageenan treated ra b b i t s , there was a large reduction i n the percentage of the 0-acetyl substituted s i a l i c acids ( C 7 / C g and C^) accompanied by a large increase i n the percentage of the s i a l i c acids susceptible to dig e s t i o n with V i b r i o  cholera neuraminidase. - 90 -Table 16. Analysis of the s i a l i c acids i n the glycoproteins isolated from the cecal e p i t h e l i a l c e l l s of control and carrageenan treated r a b b i t s . Analysis Controls Treated % c 7 / c 8 (A) 69.2 32.1 (B) 73.0 48.3 % c 4 (A) 61.5 30.8 (B) 80.9 39.5 KOH % (A) 43.1 59.7 released (B) 58.0 57.2 % released (A) 16.1 41.3 (B) 11.1 34.6 A - pooled 105,000 x g supernatants B - is o l a t e d glycoproteins ( p u r i f i e d ) (KOH-released) % s i a l i c acids released with V i b r i o cholera neuraminidase a f t e r saponi f i c a t i o n - 91 -Figure 22. Analysis of the 0-acetyl substitution pattern and neuraminidase susceptibility of the sialic acids in the 105,000 x g supernatants prepared from epithelial cells of large bowel of control rabbits. Figure 23. Analysis of the 0-acetyl substitution pattern and neuraminidase susceptibility of the sialic acids in 105,000 x g supernatants prepared from cecal epithelial cells of controls and carrageenan treated rabbits. 100 THICK LINE = CONTROL THIN LINE = TREATED - 9 3 -E. Summary Chemical inv e s t i g a t i o n of the e p i t h e l i a l glycoproteins i s o l a t e d from the lower dig e s t i v e tracts of degraded carrageenan treated and control rabbits showed that: 1. In the controls there were s i g n i f i c a n t differences between the anatomic regions of the large i n t e s t i n e i n the percentage s i a l i c acids substituted at po s i t i o n C^/Cgj the cecum and the lower colon had more 0-acetyl side chain substituted s i a l i c acids, the upper colon had least substituted s i a l i c acids. (2) There were no s i g n i f i c a n t sexual differences i n the percentage of the substituted s i a l i c acids. ( 3 ) In the c e c i of the carrageenan treated rabbits there was a s t a t i s t i c a l l y s i g n i f i c a n t decrease i n the percentage of the s i a l i c acids substituted at pos i t i o n C^/Cg, and a s i g n i f i c a n t increase i n the percentage s i a l i c acids l a b i l e to digestion with V i b r i o cholera neuraminidase ( i . e . there was decrease i n the percentage s i a l i c acids substituted at C^ or (C^). (4) In the cecum, there was a general agreement between the chemical and the histochemical studies. - 94 -Experiment 2 A. Experimental Design On the basis of the findings i n the previous study, this experiment was designed to investigate the time course of the lower dig e s t i v e t r a c t u l c e r a t i o n . Forty-two young adult male rabbits (body weight 2.630 + 0.270 Kg) were divided into two groups: (a) 8 animals fed water ad l i b , which served as controls and (b) 34 animals fed 1% w/v degraded carrageenan i n t h e i r drinking water. A l l animals were monitored for the c l i n i c a l parameters described previously (see page 38) and the carrageenan treated animals were s a c r i f i c e d i n four sub-groups according to c l i n i c a l markers of the disease as follows: group 1 - 4 8 hours treatment (no occult blood), 12 rabbits group II - occult blood, no v i s i b l e blood (5 days), 12 rabbits group III - v i s i b l e blood (9 days), 11 rabbits group IV - ulcerated (17 days), 4 rabbits. Three carrageenan treated rabbits died and were not included in the experiment. The large i n t e s t i n e of one rabbit from group IV, one rabbit from the controls, and 3 rabbits from each of groups I, II and III were fixed i n 10% formol calcium for future studies. B. C l i n i c a l observations and post mortem examination The re s u l t s are summarised i n Table 17. C l i n i c a l observations made for this experiment were s i m i l a r to the c l i n i c a l observation i n experiment 1. The treated animals i n groups I and II showed a s l i g h t gain i n body weight. The remainder of the treated groups however showed a progressive loss i n body - 95 -Table 17. C l i n i c a l observations, Experiment #2 Group Average Food intake gm/day Average carrageenan or H 20 ml /day Occult blood V. blood Average BWT Change (Kg) Controls 165 + 30 420 + 35* (+)0.310 + 0.110 I-(48H) 150 + 40 340 + 70 (+)0.024 + 0.015 II-(5 days) 120 + 35 210 + 31 Soft Feces (+)0.040 + 0.010 III-(9days) 90 + 20 170 + 15 IV-(17days) 110 + 20 205 + 60 + + Soft-very soft/diarrhea (-)0.30 + 0.019 (-)0.080 +0.040 + = present - = not detected (+) = gained body weight (-) = lost body weight * = average water intake - 96 -weight. Following 24 hours of treatment, a l l the treated rabbits showed a progressive decrease i n food and carrageenan intake accompanied by det e r i o r a t i o n i n health. A l l animals i n group II showed p o s i t i v e f e c a l occult blood. The feces of the animals i n Group III were soft with a va r i a b l e severity of v i s i b l e blood while those of group IV were soft to very soft with v i s i b l e blood. A l l the control animals were c l i n i c a l l y normal and showed no evidence of f e c a l occult blood. C. Post mortem observations group I - The lower digestive tracts of the treated rabbits were comparable to the controls except for s l i g h t congestion, edema and a few petecheal hemorrhages. groups II -The c e c i were congested, edematous and showed a greater number of petecheal hemorrhages. The cec i were f i l l e d with soft dark brown feces, group III - This group showed gross pathological changes s i m i l a r to the changes described i n experiment 1. There was severe congestion, edema, petecheal hemorrhages and mucosal u l c e r a t i o n . The cecum was f i l l e d with black-brown sof t , or watery feces which had an offensive smell, group IV - At this stage there was more advanced mucosal u l c e r a t i o n , congestion and edema. In the upper and lower colon i n group IV s l i g h t congestion and edema was sometimes noted. Groups I-III however had apparently normal upper and lower colons. - 97 -D. H i s t o l o g i c a l and histochemical observations The r e s u l t s of these studies are summarized i n Table 18, and i l l u s t r a t e d i n figure 24. a) Histology Controls; The c e c i of the controls i n this study were anatomically s i m i l a r to the previous observations for the controls i n experiment I. Therefore, for a detai l e d d e s c r i p t i o n r e f e r to page 71, and figure 18. The re s u l t s of the h i s t o l o g i c a l examination and the anatomical changes i n the c e c i of the carrageenan treated rabbits are i l l u s t r a t e d i n figure 24, and discussed below: Group I - In this group, there were early signs of an inflammatory reaction. There was s l i g h t congestion of the blood vessels, a mild inflammatory response and edema. In the lamina propria, there was a s l i g h t increase i n the inflammatory c e l l aggregate. The l i n i n g epithelium of the crypts showed mucosal degeneration and necrosis. C e l l s at the neck of the crypts were vacuolated while c e l l s at the base were large with large darkly stained n u c l e i . Group II - At this stage the pathological observations were s i m i l a r to but more severe than those observed i n the previous stage of the disease. In the submucosa there was severe congestion of the blood vessels, tissue edema and hemorrhage. There was an increase i n the number of mononuclear inflammatory c e l l s i n the lamina propria together with a few eosinophils. E p i t h e l i a l necrosis and mucosal ulcerations were r e a d i l y seen at t h i s stage. Table 18. Histology and histochemical observations, Experiment #2 Group Histology H & E Histochemistry Controls See Experiment I Table 11 PAT/KOH/PAS PBT/KOH/PAS red purple, blue purple at the base of the crypts r e d - l i g h t e r red at the base of the crypts I-(48H) Edema and congestion, s l i g h t increase treatment i n the inflammatory c e l l s i n the lamina pr o p r i a , degeneration of the e p i t h e l i a l c e l l s e s p e c i a l l y of the c r y p t s , early avidence of microulcerations Unstained areas, s i n g l e stained c e l l s i n the crypts, s t a i n i n g f a i n t , stained c e l l s red-purple Stained c e l l s , stained red, pattern l o s s , less stained material I I - ( 5 days) Severe congestion, edema and submucosal Occult hemorrhage, some inflammatory i n f i l t r a t e blood i n the lamina propria along with eosino-p h i l s , degeneration & necrosis of the l i n i n g epithelium, microulcerations Pattern l o s s , decrease i n the red colour e s p e c i a l l y around u l c e r a t i o n . Over-a l l blue-blue purple More pattern l o s s , fewer stained c e l l s , decrease i n the red colour I I I - More advanced pathological changes com-(9 days) pared with the previous stage, large mucosal u l c e r a t i o n s . In lamina propria there was massive aggregation of eosino-p h i l s . Crypts i n unulcerated areas were dist o r t e d ) twisted, large; l i n e d with acti v e c e l l s , formed fi n g e r l i k e pro-j e c t i o n s i n t o the lumen more pattern l o s s , larger very f a i n t s t a i n i n g , unstained areas, fewer single stained c e l l s com-pared to previous stage the colour i s blue with some red away from u l c e r a t i o n pattern l o s s , o v e r a l l f a i n t red and only few stained c e l l s IV-C17 S i m i l a r to previous stage, with more severe u l c e r a t i o n , ulcers are larger and deeper, involved the submucosa & also i n t o the muscularia mucosa, at the base of the ulcers there were evidence of f i b r o s i s , massive eosinophil i n f i l t r a t i o n . In the non ulcerated areas signs of e p i t h e l i a l p r o l i f e r a t i o n large unstained areas. s i m i l a r to above, c e l l s In the ulcerated areas i n the stained areas, the colour was f a i n t blue, i . e . the p r o l i f e r a t e d i n the p r o l i f e r a t e d c e l l s mucosa finger l i k e (away from ulceration) projections tend to have stained blue-purple more red colour - 99 -Figure 24. Photomicrographs of H and E stained sections demonstrating the progressive nature of the pathological process i n the cecum of the carrageenan treated r a b b i t s . As compared to normal ( f i g . 18) there was an increase i n the mucous production within 48 hours of the s t a r t of the carrageenan treatment (a x 10) followed by an inflammatory exudate together with d i s t o r t i o n and reduction in the e p i t h e l i a l crypts within 5 days (b x 10). In the rabbits given carrageenan for a period of 9 days ( v i s i b l e blood stage) there was mucosal u l c e r a t i o n (c x 2.5), arrow. - 101 -Group III - Sections from animals i n th i s group exhibited large mucosal ul c e r a t i o n s . The lamina propria was heavily i n f i l t r a t e d with macrophages, plasma c e l l s , lymphocytes and i n , and near, the ulcerated areas large aggregates of eosinophils. The e p i t h e l i a l crypts i n the areas which showed no mucosal u l c e r a t i o n were enlarged, twisted and d i s t o r t e d and the l i n i n g epithelium formed projections of active c e l l s into the lumen. Group IV - At this stage tissue a l t e r a t i o n s were more severe. There were well established mucosal u l c e r a t i o n and an increase i n the number of inflammatory c e l l s i n f i l t r a t i n g the lamina propria which on occasion extended deep into the muscularis mucosi. At the base of the ulcers there was a p r o l i f e r a t i o n of fibrous t i s s u e . H i s t o l o g i c a l changes i n the upper and lower colon were comparable to the findings i n experiment I. b) Histochemistry The histochemical properties of the e p i t h e l i a l mucins of the c e c i of the control and carrageenan treated groups are shown i n Table 18 and Figure 25. In the controls, the staining c h a r a c t e r i s t i c s observed with the PAT/KOH/PAS and PBT/KOH/PAS procedures were s i m i l a r to the previous finding (see the controls, page 77-78). In the carrageenan treated groups, there was a progressive reduction i n the tissue a f f i n i t y toward both stains, i . e . the stained sections were f a i n t e r than the controls and there were fewer stained c e l l s . The histochemical observations for each group are given below: - 102 -F i g . 25. Photomicrographs of sections of rabbit cecum stained by the 1 t PAT/KOH/PAS technique that show the t y p i c a l e f f e c t s of carrageenan on the !. . . . ' e p i t h e l i a l mucin. As compared to control ( f i g . 20) within 48[hours (a x 6.3) there i s a s i g n i f i c a n t reduction i n the amount of glycoproteins a v a i l a b l e for staining together with a reduction i n the red color and loss of the normal staining pattern, b x 6.3 and c x 2.5 (5 days and 9 days) show that the above changes i n the e p i t h e l i a l glycoproteins are progressive. ; I It - 104 -1. PAT/KOH/PAS Group I (48 hours treatment) - At this stage there was some pattern loss; some crypts had no staining, i n others few c e l l s stained while i n others the entire crypt stained. The stained portions appeared large, and were stained prominently red. Overa l l , the colour was purple-red but there were also unstained areas. Group II (occult blood) - There were an increase i n the number and s i z e of the unstained areas and a decrease i n staining i n t e n s i t y . The colour of the st a i n was more blue than red and there was a greater loss of pattern. Overall the c e l l s stained blue-blue purple. Group III - There were large unstained areas, an even greater pattern loss and a further decrease i n staining i n t e n s i t y . The borders of the ulcers stained blue; there was an increase i n the red stainin g i n areas distant from the u l c e r . Group IV - The histochemical changes i n this group were s i m i l a r to but more severe than i n the previous stage. Away from the ulcerated areas there were signs of tissue p r o l i f e r a t i o n shown by f i n g e r - l i k e projections of e p i t h e l i a l c e l l s i n which the c e l l s were large, active and had a reddish purple colour. 2. PBT/KOH/PAS The observations with this s t a i n i n g procedure were well correlated with the previous staining procedure. - 105 -Group I - There was some pattern loss, and a reduction i n the number of stained c e l l s . The stained c e l l s appeared large, and stained s i m i l a r l y to or sometimes even more deeply than the controls. Group II - There was an increase i n the unstained areas, a greater loss of pattern, and fewer stained c e l l s . The staining was f a i n t and the ulcer margins were either unstained or only very f a i n t l y stained. Group III - The d i s t r i b u t i o n of staining was s i m i l a r to that i n the previous group but the i n t e n s i t y of the staining was decreased. Group IV - Overall the mucins were very f a i n t l y stained and there were large unstained areas. Away from the ulcerated areas and i n the p r o l i f e r a t i n g c e l l s the staining was a moderate red. E. Chemical studies a. Percentage s u b s t i t u t i o n at p o s i t i o n C^/Cg Tables 19-21 show the percentage of s i a l i c acids substituted at p o s i t i o n Cy/Cg in the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s i solated from the large i n t e s t i n e of the control and the carrageenan treated r a b b i t s . S t a t i s t i c a l analysis of the data showed that s i g n i f i c a n t differences between the controls and the carrageenan treated rabbits occurred only i n the cecum. The percentage of side chain substituted s i a l i c acids i n the 105,000 x g supernatants i s o l a t e d from the c e c i of the control rabbits was s u r p r i s i n g l y low as compared to the r e s u l t s from both experiment I (see p. 81, Table 13) - 106 -Table 19. Percentage of s i a l i c acids substituted at positions C 7 / C g and C 4 and the percentage released with V i b r i o cholera neuraminidase in the 105,000 x g supernatant prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . Analys is Controls 2 days (I) 5 days (II) 9 days ( i l l ) 17 days (IV) % Cy/Cg 46.0+ 4.8 55.2+11.0 39.3+10.6 *a23.0+10.8 38.9+ 4. 7 % c 4 38.7+19.5 37.1+15.0 * b13.4+ 9.5 * c 11.3+12.1 26.7+19. 1 KOH % released 59.4+13.2 63.2+ 8.4 53.1+ 7.5 55.7+ 9.6 54.1+ 7. 2 % released 35.6+11.7 39.5+ 9.7 *d45.7+ 6.0 * e50.1+ 6.3 38.4+ 4. 9 * S t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the controls t = DF = 13 a = 4.82 b = 3.39 c = 3.31 d = _2.2 e = -2.98 KOH = s a p o n i f i c a t i o n (IN KOH) % released = % s i a l i c acid released with V i b r i o cholera neuraminidase digestion ( ) = Group - 107 -Table 20. Percentage of s i a l i c acids substituted at positions C7/C0 and C 4 and the percentage s i a l i c acids released by digestion with V i b r i o cholera neuraminidase i n the 105,000 x g supernatant prepared from the e p i t h e l i a l c e l l s of the upper colon of the controls and from the degraded carrageenan treated rabbits. Analys is Controls 48 hours (I) 5 days (II) 9 days ( i l l ) 17 days (IV) % c 7 / c 8 41.2+16.1 47.4+10.6 41.4+10 .6 50.9+12.3 41.6+5.4 % c 4 56.7+ 6.8 58.9+ 7.3 50.5+16 .4 *a66.2+ 8.7 53.6+13.8 KOH Z released 73.2+ 6.8 73.8+ 4.1 72.1+ 5 .7 *b65.7+ 4.9 74.6+ 3.9 % released 31.9+ 7.4 30.4+ 5.8 35.8+12 .9 *c22.4+ 6.6 34.4+ 9.0 * S t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the controls t = DF = 11 a = -2.08 t> = 2.32 c = 2.44 KOH = sa p o n i f i c a t i o n (IN KOH) % released = % s i a l i c acid released with V i b r i o cholera neuraminidase digestion ( ) = group - 108 -Table 21. Percentage of s i a l i c acids substituted at positions Cy/Cg and C 4 and the percentage s i a l i c acids released with V i b r i o cholera neuraminidase i n the 105,000 x g supernatant prepared from the e p i t h e l i a l c e l l s of the lower colon of the controls and from the degraded carrageenan treated rabbits. Analys is Controls 48 hours ( i ) 5 days (II) 9 day; s (III) 17 days (IV) % Cj/Cg 52.4+ 7.1 49.7+ 9.5 41.1+ 3.2 51.4+ 9.5 41.1+21.2 % c 4 56.8+18.6 55.8+14.9 54.2+16.7 66.3+ 9.5 66.1+ 9.5 KOH % released 76.8+ 7.2 76.1+ 6.9 79.6+ 5.7 81.2+ 7.9 79.2+ 6.7 % released 33.5+15.0 33.3+10.6 36.9+13.9 27.5+ 8.6 27.2+ 9.1 % released - % s i a l i c acid released with v i b r i o cholera neuraminidase digestion KOH = sa p o n i f i c a t i o n (IN KOH) ( ) = Group - 109 -IS and experiments 3 and 4 (see p. 134, 157, Tables 26, 30). As i l l u s t r a t e d i n Table 19 i n the carrageenan treated groups there was apparently an increase in side chain s u b s t i t u t i o n i n group I (48 hours) followed by a decrease at the next two stages, Groups II and I I I . However, group IV displays an apparent return of s u b s t i t u t i o n towards the co n t r o l value. S t a t i s t i c a l l y s i g n i f i c a n t differences were shown between the controls and group I I I . The increase observed in Group I was not s t a t i s t i c a l l y s i g n i f i c a n t . As w i l l be seen i n Table 19 there were no s i g n i f i c a n t differences between the control and carrageenan treated animals i n groups I, II and IV. b. Digestion studies Tables 19-21 show the percentage of the s i a l i c acids released, by diges-tion with V i b r i o cholera neuraminidase, from the saponified and unsaponified 105,000 x g supernatants prepared from e p i t h e l i a l c e l l s i s o l a t e d from the large i n t e s t i n e of controls and the degraded carrageenan treated r a b b i t s . As w i l l be seen, i n the cecum, there was a progressive increase in the percentage of neuraminidase l a b i l e s i a l i c acids reaching a maximum with group I I I . Group IV however contained more neuraminidase r e s i s t a n t s i a l i c acids than group I I I . There was a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e between the controls and carrageenan treated animals in groups II and I I I , betwween group I (carrageenan treated) and group II, and also between group III and IV. The difference between percentage of s i a l i c acids released from the saponified supernatants prepared from groups I and II was also s t a t i s t i c a l l y s i g n i f i c a n t . The r e s u l t s obtained from a c a l c u l a t i o n of the percentage of s i a l i c acids substituted at p o s i t i o n are shown i n Tables 19-21. As w i l l be seen there was a progressive decrease in the % of C, substituted s i a l i c acids i n groups - 110 -Figure 26. Percentage of s i a l i c acids substituted at positions C-,/CQ and / o and the percentage released a f t e r digestion with V i b r i o cholera neuraminidase i n the 105,000 x g supernatant prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . The data c i t e d for the % of s i a l i c acid substituted at cg^Cg was obtained from the p u r i f i e d glycoproteins. 40 CO => »« 40 1 1 JL 1 C 1 2 3 4 x c 7 / c 8 I A 1 J . C 1 2 3 4 X RELEASED C 1 2 3 4 C 8 / C 9 C 1 2 3 4 C - CONTROL 1 - 2 DAYS 2 * 5 DAYS 3 - 9 DAYS 4 - 17 DAYS - I l l -I, II and I I I . In group IV, however, an increase was noted, as compared to group I I I . S t a t i s t i c a l l y s i g n i f i c a n t differences were found between (a) the controls and group II and III and (b) group I and groups II and I I I . Table 22 shows the r e s u l t s obtained from the analysis of the p u r i f i e d glycoproteins isolated from the pooled 105,000 x g supernatants of the cecal e p i t h e l i a l c e l l s . It i s c l e a r that there was a large progressive difference between the carrageenan treated group and the c o n t r o l s . E. Summary This study confirmed the disease related observation of experiment I, and demonstrated that 1. Although the controls were c l i n i c a l l y , h i s t o l o g i c a l l y and histochemically s i m i l a r to the controls in the previous study, chemical analysis showed that the cecal e p i t h e l i a l glycoproteins contained a lower percentage of O-acety substituted (C^/Cg) s i a l i c acids than the controls from experiments 1, 3 and 4. 2. Group I of the carrageenan treated animals (48 hours treatment) showed an apparent increase i n the percentage of C^/Cg substituted s i a l i c acids. On further treatment there was a progressive decrease in the percentage of the side chain substituted s i a l i c acids; group III had the largest differences, i . e . the e p i t h e l i a l glycoproteins of this group (stage of v i s i b l e blood) had the smallest percentage of the substituted s i a l i c acids. 3. These changes were associated with an increase in the s u s c e p t i b i l i t y of the s i a l i c acid to digestion with V i b r i o cholera neuraminidase. 4. A l l these changes were observed in the isolated glycoproteins. - 112 -Table 22. Analysis of the pooled 105,000 x g and of the pure e p i t h e l i a l glycoprotein i s o l a t e d from the cecal e p i t h e l i a l c e l l s i n Experiment #2. Analysis Controls 2 days 5 days 9 days 17 days c 7 / c 8 (a) 52.4 53.9 36.0 27.0 36.0 (b) 55.2 58.2 29.0 32.7 38.5 Cg/Cg (a) _ — _ — -(b) 31.4 51.2 17.6 32.6 c 4 (a) 40.8 27.3 34.8 16.5 29.7 (b) 47.3 35.1 24.1 9.6 40.0 KOH (a) 74.0 69.6 72.9 80.4 73.8 N1 dase (b) 71.1 68.3 73.2 78.4 76.6 NKOH N'dase (a) 43.8 50.6 47.5 67.1 51.9 (b) 37.5 44.3 55.6 70.9 46.0 (a) = 105,000 x g pooled (b) = pure glycoproteins KOH N'dase = % of s i a l i c acids released from saponified samples NKOH N'dase = % s i a l i c acid released from the unsaponified samples - 113 -5. This study showed that there were no s i g n i f i c a n t changes i n the upper and lower colons of the carrageenan treated animals. 6. There was a general agreement between the chemical, histochemical, the pathological and the histopathological observation. A l l these parameters showed that the disease, the pathological changes and the chemical changes were progressive. - 114 -IV. Experiment 3 In the previous study (experiment 2), i t was shown that the carrageenan induced decreases i n the 0-acetyl side chain s u b s t i t u t i o n of the s i a l i c acids of the c e c a l e p i t h e l i a l g l y c o p r o t e i n s were progressive with a maximum decreast at the v i s i b l e blood stage ( c l i n i c a l u l c e r a t i o n ) . In t h i s study we attempted both to confirm our previous f i n d i n g s and to i n v e s t i g a t e the sequence of changes p r i o r to the stage of c l i n i c a l u l c e r a t i o n . Since most of the carrageenan tr e a t e d r a b b i t s showed v i s i b l e blood w i t h i n 5 days of the treatment, the f o l l o w i n g experiment was designed to proceed f o r 5 days. A. Experimental design 50 young adult r a b b i t s (2.710 _+ 0.232 kg) were d i v i d e d i n t o two groups: (a) c o n t r o l s (10 r a b b i t s ) and (b) the carrageenan treated group which was f u r t h e r subdivided i n t o 5 experimental groups each c o n s i s t i n g of 8 r a b b i t s as f o l l o w s : 1. group I were s a c r i f i c e d at e x a c t l y 24 hours from the s t a r t of the carrageenan treatment 2. group I I were s a c r i f i c e d a f t e r 48 hours of treatment 3. group I I I were s a c r i f i c e d a f t e r 3 days of the treatment 4. group IV were s a c r i f i c e d a f t e r 4 days of the treatment 5. group V were s a c r i f i c e d a f t e r 5 days o f treatment 6. A l l the c o n t r o l s were s a c r i f i c e d on the 6th day o f the experiment A l l the animals were housed, fed and monitored as described p r e v i o u s l y . - 115 -B. C l i n i c a l observations A summary of the c l i n i c a l observations i s shown i n Table 23. A l l the animals gained body weight, i n variable amounts, although there was a s l i g h t drop i n the food and the carrageenan intake. P o s i t i v e f e c a l occult blood (traces) were noted as early as 48 hours a f t e r the star t of the carrageenan treatment. The animals in group V showed variable degrees of v i s i b l e blood along with soft feces; diarrhea was sometimes present i n t h i s group. C. Post mortem findings The r e s u l t s of the gross anatomical examination (see Table 24) indicated the presence of progressive pathological changes. In group I, a l l the rabbits (except #6 which showed s l i g h t congestion) had c e c i of normal appearance. A l l the animals i n group II showed a s l i g h t to moderate congestion of the cecum with s l i g h t edema and sometimes s l i g h t petecheal hemorrhages. Rabbits i n group III showed congestion, petecheal hemorrhages and edema of the cecum. The small i n t e s t i n e and the stomach were congested i n t h i s group. The c e c i of the animals i n group IV showed severe congestion, edema and submucosal petecheal hemorrhages. Animals i n group V, i n addition to findings s i m i l a r to those of group IV, showed mucosal u l c e r a t i o n ( 2 x 2 mm). The mesenteric lymph nodes and the spleen were enlarged; the l i v e r was swollen. The small i n t e s t i n e and the stomach were inflammed. The cecum was f i l l e d with blood. D. H i s t o l o g i c a l and histochemical studies a. Histology The r e s u l t s of the h i s t o l o g i c a l examination of the cecal tissue obtained from both control and carrageenan treated rabbits are i l l u s t r a t e d i n Figure 27 and summarised i n Table 25. There appeared to be a ser i e s of progressive - 116 -Table 23. C l i n i c a l observations, Experiment #3. Group Average Food Intake gm/day Average Carrageenan intake (or H 20) (ml/day) Occult blood V i s i b l e b lood Average BWT change (kg) Controls 171+23 *415+35 - (+)0.261+0.082 I-C24H) 149+30 390+90 - (+)0.045+0.017 II-C48H) 175+85 356+25 + - (+)0.118+0.030 III-C72H) 197+72 211+50 + - (+)0.007+0.015 IV-(96H) 177+35 374+85 + + soft feces (+)0.140+0.090 V-(120H) 157+46 348+93 + + soft-very soft (+)0.202+0.086 + = pos i t i v e + = trace - = absent (+) = gained body weight * = average water intake - 117 -Table 24. Gross anatomical observation of the controls and the carrageenan treated groups in Experiment #3 Group Congestion Oedema Hemorrhage Ulcerations Controls Group I Group II Group III Group IV Group V +* ++ + severe + severe Normal looking + severe (petechael) + ++ petechael small i n t e s t i n e congested +++ + severe bleeding u l c e r a t i o n i n the cecum + = trace + = present ++ = moderate +++ = marked * = one animal - 118 -changes from day one to day 5. These changes were mild from day to day but the longer the period of treatment, the more extensive were the changes noted. There were no clear h i s t o l o g i c a l differences between the controls and the carrageenan treated rabbits at day 1 but there were clear h i s t o l o g i c a l differences between the controls and the 5 day treated group (see Table 25 and figures 27). The general h i s t o l o g i c a l changes were as follows: Controls - The h i s t o l o g i c a l observations were similar to the previous findings for detailed description see page 71 and figures 18. Carrageenan treated 24 hour treatment (group I) This group d i f f e r e d l i t t l e from the controls. Most of the differences were of a degenerative type being mainly limited to the goblet c e l l s . There was a variable degree of congestion and a s l i g h t inflammatory edema. The goblet c e l l s appeared empty and there were i n t e r c e l l u l a r spaces, most l i k e l y due to the edema. In some sections the l i n i n g epithelium was severely interrupted by degenerative f o c i . In the lamina propria there were enlarged active macrophages with vacuolated cytoplasm . 48 hours (group I i ) In this group there was, compared with group I, an increase i n congestion and edema, and the goblet c e l l s i n the crypts were more vacuolated. There was c e l l u l a r necrosis i n the crypts and also i n the surface epithelium, together with submucosal hemorrhages. The lamina propria was i n f i l t r a t e d with vacuo-lated macrophages. Mucosal p r o l i f e r a t i o n i n form of polyps and d i s t o r t e d e p i t h e l i a l crypts was noted i n some sections. - 119 -72 hours (group III) At this time of the treatment the h i s t o l o g i c a l changes varied from an apparently normal, to an extremely degenerated mucosa. The crypts were eit h e r p r o l i f e r a t i v e , forming mucosal f i n g e r - l i k e projections into the lumen, or were degenerated, necrotic and fewer in number. Overall the changes represented an advanced stage of degeneration and a well established stage of e p i t h e l i a l necrosis. There was severe congestion, submucosal hemorrhages, edema and well established micro-ulcers. In the lamina propria there were macrophages, plasma c e l l s and a moderate aggregate of eosinophils. 96 hours (group IV) The major change noted in this group was the tendency to form a more degenerated mucosa and the presence of more inflammatory c e l l s i n the submucosa. In most of the animals (with the exception of #25 which showed mucosal polyps), there was edema and hemorrhage, the mucosa tended to be degenerated, with fewer and dis t o r t e d necrotic crypts. The lamina propria tended to be severely crowded with inflammatory c e l l s . There were micro-ulcers and an increased number of eosinophils. In some other s i t e s the mucosa tended to be a simple layer of columnar epithelium with few or no e p i t h e l i a l crypts and the lamina propria was i n f i l t r a t e d with very few inflammatory c e l l s . 120 hours (group V) At this stage, there was more severe mucosal degeneration and necrosis. Congestion, hemorrhage and u l c e r a t i o n were prominent with extreme u l c e r a t i o n i n the majority of animals. Overall there was more hemorrhage, a simpler Group Histology Histochemistry Controls see controls table H PAT/KOH/PAS PBT/KOH/PAS red purple, base blue-purple r e d»lighter at the base I s l i g h t edema & congestion. Goblet 24 hours c e l l s , large vacuolated, some are empty, lamina propria had increased vacuolated macrophages s l i g h t decrease in s t a i n - s l i g h t reduction in red ing a f f i n i t y , some colour along with some pattern loss, o v e r a l l pattern loss colour b l u i s h purple II edema & congestion with submucosal hem-48 hours orrhage. E p i t h e l i a l c e l l s p a r t i c u l a r l y of the crypts were degenerated and n e c r o t i c , some crypts were hyperplastic, polyps l i k e p r o j e c t i o n into the lumen, early changes of microulceration, more inflammatory c e l l s i n the lamina propria pattern loss, singly stained c e l l s i n the crypts, o v e r a l l staining is blue, p r o l i f e r a t e d c e l l s stained purple-purple red pattern l o s s , l i g h t red. In the p r o l i f e r a t e d c e l l s the colour was red III 72 hours Variable - normal appearing to extremely more pattern loss, fewer fewer c e l l s , unstained degenerated mucosa, polyps, severe con-gestion, edema and hemorrhages, micro-u l c e r s , less and shorter crypts i n -creased inflammatory c e l l s stained c e l l s , c e l l s i f stained looked active, large and stained purple o v e r a l l stained c e l l s looked red IV more edema, congestion and submucosal 96 hours hemorrhage, mucosal u l c e r a t i o n , mucosa looked simple i n structure with fewer, shorter crypts, lamina propria i n f i l -trated with vacuolated c e l l s large unstained areas, l i g h t ( i . e . obvious faint staining, few decrease in stainable stained c e l l s , complete material) pattern loss, stained c e l l s were blue-blue purple V More advanced compared to previous stage very f a i n t l y stained, 120 large ulcerated areas. In lamina dominant color blue, hours propria there were increased eosinophils s p e c i a l l y at ulcer margin very few stained c e l l s , staining very f a i n t red - 121 -Figure 27. Photomicrographs of H and E stained sections that show the progressive nature of the carrageenan induced c o l i t i s i n the c e c i of carrageenan treated rabbits. a - control (x 10) b - 24 hours treatment (x 10) c - 48 hours treatment (x 10) ( i n s e r t x 6.3) d - 72 hours (x 10) e - 4 days treatment (x 6.2), (e x 10) f - 5 days treatment, note d i s t o r t i o n , reduction of e p i t h e l i a l crypts, inflammatory exudate and mucosal u l c e r a t i o n (x 10) -122a-Figure 27. (c) 48 hours treatment with % degraded carrageenan (x 10) (i n s e r t x 6.3) (d) a f t e r 72 hours treatment with degraded carrageenan (x 10) -123a-Figure 27 (e) 4 days treatment (e x 6.3, e x 10) ( f ) 5 days treatment, note d i s t o r t i o n , reduction of e p i t h e l i a l crypts, inflammatory exudate and mucosal u l c e r a t i o n (x 10 ) . - 125 -mucosa, fewer and more degenerative or even necrotic crypts, and more eosinophils i n the lamina propria. The muscularis mucosae was involved i n most of the sections studied. (b) Histochemical observation The r e s u l t s of these studies are i l l u s t r a t e d i n figure 28 and summarized i n Table 25. Controls As shown i n figure 28, tissue sections from the cecum stained with the PAT/KOH/PAS and the PBT/KOH/PAS procedures were s i m i l a r to those i n experiments 1 (see page 77). Carrageenan treated Results of examining tissue sections from the cec i of the carrageenan treated rabbits are shown i n figure 28 . 1. PAT/KOH/PAS  Group I (24 hours) The s t a i n i n g pattern was more or less s i m i l a r to that of the con t r o l s . Overall however there was a variable reduction i n the i n t e n s i t y of the red colour of the s t a i n and an increase i n the blue colour; o v e r a l l the st a i n i n g was purplish-bluish purple. In animal #1, however, the red component of the st a i n predominated and the o v e r a l l colour was red purple. The i n t e n s i t y of the staining reaction was less intense than that of the controls and, i n the main, a l l the section was stained except for sections #4, 3 and 1 where the colour of the s t a i n was f a i n t . - 126 -Group II (48 hours) At this stage, the staining pattern was distorted and was l a r g e l y limited to singly scattered c e l l s i n the crypts. There were many unstained crypts. The staini n g i n t e n s i t y varied from dark to f a i n t , stained c e l l s , i f present, appearing more active, being large and darkly stained. The mucins stained more blue than the previous stage. Ove r a l l , the s t a i n i n g was blue to blue purple. Group III (72 hours) In this group the staini n g was l i t t l e changed from the previous stage. There was a lack of the normal staining pattern with more unstained areas; the colour of the stained areas, mostly single c e l l s , was v a r i a b l e from section to section. In #'s 18 and 19 i t was red purple, while i n #'s 23, 24 i t was blue purple. Overall the colour was blue purple. Group IV (96 hours) In this stage there was a marked lack of stained tissue components. The majority of the sections showed nothing more than a f a i n t b l u i s h or sometimes reddish background with a few scattered f a i n t blue or blue purple stained c e l l s . Group V (120 hours) In general, sections from t h i s group stained s i m i l a r l y to those i n group IV. In the areas of the micro-ulcerations, s t a i n i n g , i f i t was present, tended to be a f a i n t blue. At points removed from the ulcerated areas the stainin g had a redder hue. - 127 -2. PBT/KOH/PAS Generally, a l l the alt e r a t i o n s observed with the previous PAT/KOH/PAS were noted with the PBT/KOH/PAS technique and were comparable, i . e . there was a progressive loss of pattern, a decrease i n tissue a f f i n i t y toward the s t a i n , and increasingly unstained areas i n the sections. A l l these observations are 'illustrated i n figure 28 and summarized i n Table 25. Group I (24 hours) At this stage a l l sections, except #7 which showed no apparent change, showed a decrease i n the red staining i n t e n s i t y but the normal pattern of d i s t r i b u t i o n of the staining was to some extent preserved. Group II (48 hours) In a l l the sections there were large unstained areas with the exception of one section #9 i n which a l l areas were f a i n t l y stained. Stained c e l l s were scattered singly a l l over the d i f f e r e n t parts of the tissue section. These c e l l s look active and large and stained either f a i n t l y or moderately. Overall the staini n g reaction was a fa i n t red. Group III (72 hours) In this group there was a greater loss of staining pattern, i . e . stained c e l l s were more scattered and were fewer than i n the previous stage. Stained c e l l s i n most of the sections i f stained (except #17 and 23, where the staining was f a i n t red) tended to be stained well with a moderate red colour; o v e r a l l there were very few c e l l s stained a moderate red. - 128 -Group IV (96 hours) At this stage there was a marked reduction in the s t a i n i n g reaction of the tissue with an increase i n the number of unstained areas. The stained c e l l s were more scattered and had no pattern. The staini n g i n t e n s i t y of the c e l l s was variable being from no colour to l i g h t red. Group V (120 hours) There was a sharp decrease in the number of stained c e l l s together with an increase i n the unstained parts of the sections and a complete loss of the s t a i n i n g pattern. There was no s t a i n i n g i n and around the ulcerated areas. The i n t e n s i t y of the stained c e l l s was v a r i a b l e ; some c e l l s stained a very l i g h t red, while others, p a r t i c u l a r l y i n the areas away from u l c e r a t i o n s , were stained red. Summary - histology and histochemistry Although i t was not easy to pinpoint the pathological and the histochemical a l t e r a t i o n s during the progressive stages i n the carrageenan treated animals, there were evident differences between controls and the carrageenan treated groups and between the early and the late stages of the carrageenan treated rab b i t s . H i s t o l o g i c a l l y there was a progressive increase in the inflammatory response. Twenty-four hours of treatment resulted i n degenerative changes which progressed as treatment continued to give, on day 5, an extensive inflammatory process accompanied by severe tissue damage i n the form of mucosal u l c e r a t i o n and a c e l l u l a r inflammatory response. The histochemical staining procedures were well correlated with each other and also with the - 129 -Figure 28. Photomicrographs of sections of rabbit c e c i stained with the PAT/KOH/PAS (a,b,d,f,h,j,l) and PBT/KOH/PAS (c,e,g,i,k,m) procedures to i demonstrate the progressive nature of the changes i n the staining of the <i e p i t h e l i a l mucin upon treatment of the rabbits with 1% degraded carrageenan. a,b,c = normal control, a and c x 10, b x 16 ' d and e = 24 hours - note there was an immediate reduction i n the quantity of stained mucin together with a loss of the normal pattern of staining x 6.3 f and g = 48 hours x 10 } h and i = 72 hours x 10 j and k = 96 hours x 10 \ 1 and m = 120 hours - note the almost complete absence o f : s t a i n i n g for e p i t h e l i a l glycoprotein x 10 r -130a-Figure 28. 48hours treatment with 1% degraded carrageenan :' ] (f)PAT/KOH/PAS (x 10),(g)PBT/KOH/PAS (x 10) (h) PAT/KOH/PAS (x 10) , ( i ) PBT/KOH/PAS (X 10)f .treated for 72 hours . -131a-Fagure 28. J (PAT/KOH/PAS, x 10) and K (PBT/KOH/PAS, x 10); 96 hours Icarrageenan t treatment. j I (PAT/KOH/PAS, x 10) and M (PBT/KOH/PAS, x 10); 120 hoursi treatment; note the almost complete absence of staining reaction. j i. i t ' I I j i i - 133 -progressive nature of the disease. In the diseased animals there was a progressive decrease i n the stainable tissue components (glycoproteins) associated with a progressive decrease i n the degree of side chain s u b s t i -t u t i o n of the s i a l i c a c ids. In terms of colour, there were progressive increases i n the blue component of the PAT/KOH/PAS and progressive decreases i n the red colour of the PBT/KOH/PAS procedures. These findings were more prominent i n and around the areas of u l c e r a t i o n . It i s of interest to note that, i n this experiment, histochemical changes i n the e p i t h e l i a l glycoprotein apparently took place before the presence of an evident inflammatory response. E. Chemical observations a. Percentage of s i a l i c acids substituted at positions C,/CQ • / o Tables 26 shows the percentage of s i a l i c acids substituted at C 7 /c_ / 8 i n the 105,000 x g supernatant obtained from the e p i t h e l i a l c e l l s of the ceci of the control and carrageenan treated r a b b i t s . As w i l l be seen, there was a progressive decrease i n the percentage of s i a l i c acids substituted at positions C^/Cg. S t a t i s t i c a l analysis showed the presence of s i g n i f i c a n t differences between the controls and degraded carrageenan treated groups I I I , IV and V. There were no such differences between the controls and eit h e r group I or group I I . b. Digestion Studies Table 26 show the re s u l t s obtained from an i n v e s t i g a t i o n of the neuramin-idase s u s c e p t i b i l i t y of the s i a l i c acids i n the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of the control and degraded - 134 -Table 26. Percentage of s i a l i c acids substituted at positions C 7/Cg and C 4 and the percentage released with V i b r i o cholera neuraminidase i n the 105,000 x g supernatant prepared from the cecal e p i t h e l i a l c e l l s of controls and of degraded carrageenan treated r a b b i t s . Analysis Controls 24 hours 48 hours 72 hours 96 hours 120 hours 62.8+10.3 54.8+ 9.6 57.9+ 7.5 *a 53.2+ 7.0 *b 45.9+12.1 *c 46.9+14.6 % c 4 33.7+16.1 *d 9.0+ 9.2 *e 17.5+14.7 * f 14.7+19.3 *g 14.8+13.3 *h 11.9+ 6.5 KOH % released 57.2+ 9.9 57.2+ 9.6 56.1+ 4.4 55.5+ 8.7 55.1+ 7.7 57.3+ 6.2 % released 38.2+14.0 * i 52.4+12.0 47.6+11.6 47.3+11.1 49.3+12.9 * j 52.1+ 8.3 • S t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the controls t = DF = 16 a = 2.25 f = 2.28 b = 3.22 g = 2.67 c = 2.71 h = 3 > 6 o d = 3.86 i = 2.27 e = 2.22 j = 2.47 KOH = sap o n i f i c a t i o n (IN KOH) % released = % s i a l i c a c i d released with V i b r i o cholera neuraminidase digestion - 135 -Figure 29. Percentage of s i a l i c acids substituted at positions C-JCQ and C4 and percentage released with V i b r i o cholera neuraminidase in the 105,000 x g superntants obtained from cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . The data c i t e d for the percentage of s i a l i c ac substituted at Cg/Cg was obtained from the is o l a t e d glycoproteins. C - CONTROL 1 • 24 HOURS TREATMENT 2 - 4 8 HOURS TREATMENT 3 - 7 2 HOURS TREATMENT 4 « 96 HOURS TREATMENT 5 « 120 HOURS TREATMENT - 136 -carrageenan treated r a b b i t s . As w i l l be seen, carrageenan treatment resulted in an increased s u s c e p t i b i l i t y to V i b r i o cholera neuraminidase r e f l e c t e d in a decrease in the percentage s u b s t i t u t i o n at C^. S t a t i s t i c a l analysis of the data indicated that the s i a l i c acids of group I and V were s i g n i f i c a n t l y more susceptible to digestion than those of the controls while a l l groups d i f f e r e d s i g n i f i c a n t l y from the controls with respect to s u b s t i t u t i o n at p o s i t i o n C^. There were no s t a t i s t i c a l differences between the s u s c e p t i b i l i t y to digestion with V i b r i o cholera neuraminidase of saponified glycoproteins. On adding the t h i o b a r b i t u r i c acid reagents to the neuraminidase digest of the 105,000 x g supernatants i n experiment 3 and 4 a pinkish colour was developed i n some of the specimens p r i o r to the heating step. The cause of this a r t i f a c t is not known. Since i t seemed to a f f e c t the t o t a l amounts of the recovered s i a l i c acids, the data obtained from the digestion studies of the 105,000 x g supernatants (Experiment 3, Table 26 and Experiment 4, Table 30) was considered to be unreliable and the emphasis was directed toward the data obtained from digestion of the isolated glycoproteins presented i n Table 27. As w i l l be seen t h i s data confirmed that presented i n Table 26 with respect to side chain s u b s t i t u t i o n . In addition i t showed that the c a r r a -geenan treatment had resulted in an immediate and consistent reduction in the percentage of the s i a l i c acid substituted at positions C-,/CQ, CoC n and along with an immediate increase in the percentage of s i a l i c acids susceptible to digestion with V i b r i o cholera neuraminidase. A r t i f a c t s i m i l a r to that noted upon addition of t h i o b a r b i t u r i c acid reagent was noted i n some of the pooled 105,000 x g supernatants and therefore the percentage of s i a l i c acids released by V i b r i o cholera neuraminidase was - 137 -Table 27. Percentage s i a l i c acids substituted at positions C-j/Cg, C 8Cg, and percentage s i a l i c acids released from the 105,000 x g supernatant and the p u r i f i e d glycoproteins prepared from cecal epithelium of controls and carrageenan treated r a b b i t s . Analysis Controls 24 hrs 48 hrs 72 hrs 96 hrs 120 hrs c 7 / c 8 (a) 59.9 55.0 60.4 55.7 49.3 53.1 (b) 64.5 56.4 61.7 56.8 46.6 50.9 c 8 / c 9 (a) - - - - - -(b) 53.6 21.3 29.7 41.3 29.5 30.4 c 4 (a) 58.5 23.3 41.5 46.2 32.9 38.3 (b) 66.8 40.8 50.7 53.6 54.7 53.3 % released (a) 67.2 58.0 62.6 63.4 63.0 61.1 KOH N'dase (b) 66.8 40.8 50.7 53.6 54.7 53.3 % released (a) 27.9 44.5 36.6 34.1 42.3 37.7 N'dase (b) 21.7 35.0 31.0 28.8 29.1 29.1 (a) = 105, 000 x g pooled (b) = pure glycoproteins KOH = sa p o n i f i c a t i o n % released = percentage s i a l i c acids released by digestion with V.C. N'dase - 138 -calculated from the t o t a l s i a l i c acid content before and a f t e r the digestion as follows: % released s i a l i c acid = t o t a l s i a l i c acid before digestion-bound s i a l i c acid after digestion t o t a l s i a l i c acid before digestion - 139 -Experiment 4 This experiment was planned to confirm our previous observations and to study the changes i n the 0-acetyl substituted s i a l i c acids i n the healing stage when the carrageenan treatment was discontinued. A. Experimental design Six groups (8 rabbits each) were kept on 1% degraded carrageenan, 12 rabbits were given water and kept as controls. Each carrageenan treated group was predetermined, they were s a c r i f i c e d as follows: 1. group I plus 2 controls were s a c r i f i c e d 48 hours from the st a r t of the experiment 2. group II plus 2 controls were s a c r i f i c e d at the stage of v i s i b l e blood i n the feces (5 days) and termed the ulcerated group. At th i s time the carrageenan treatment was discontinued and a l l treated animals were given water ad l i b with no added carrageenan. Groups I I I , IV, V and VI and 2 controls for each group were s a c r i f i c e d on days 3, 6, 12 and 20 a f t e r removing the degraded carrageenan from the d i e t . B. C l i n i c a l observations The re s u l t s of these studies are summarised i n Table 28. One rabbit from each of groups I I , I I I , IV, V and 2 rabbits from group VI died p r i o r to the completion of the treatment; they were not included i n the study. The c l i n i c a l observations for groups I and II were s i m i l a r to those reported i n experiment 3. At day 5 of the carrageenan treatment, a l l the treated animals showed v i s i b l e blood i n t h e i r feces. Carrageenan treated rabbits i n group I II were l i t t l e improved from group I I . A l l animals were weak, emaciated and - 140 -Table 28. C l i n i c a l observations made for control and degraded carrageenan treated ra b b i t s , Experiment #4. Group Average Average Food Intake Carrageenan gm/day intake (or H 20) (ml/day) Average Occult blood V i s i b l e BWT change Blood (kg) Controls I- (48H) I I - (5 days) 109+30 180+30 °440+50 150+35 320+70 III-(3 days)* 80+25 IV-(6 day)* 140+60 V-(12 day)* 150+70 VI-(20 day)* 155+85 195+56 60+15 295+80 320+80 410+60 (+)0.420+0.186 + - (+)0.025+0.020 + + (+)0.089+0.026 soft-very soft feces + + (+)0.060+0.025 s o f t - s o l i d feces s o l i d - s o f t s o l i d feces s o l i d feces (+)0.040+0.025 (+)0.080+0.023 (+)0.105+0.020 = on this day the carrageenan was removed from the d i e t , a l l rabbits were given water * a l l data calculated from the day on which carrageenan was removed from the di e t - = absent + = present + = trace (+) = body weight o = water intake ml/day - 141 -anemic and had soft formed feces with traces of v i s i b l e blood and strongly p o s i t i v e f e c a l occult blood. Rabbits i n group IV showed a s l i g h t l y increased food and water intake and appeared to be i n better health. They had s o l i d feces with p o s i t i v e f e c a l occult blood and were s t i l l weak, emaciated, and anemic. C. Post mortem examination Animals i n group I showed, to a variable extent, congestion, edema, and petecheal hemorrhages. In group II and I I I , there was severe congestion, edema and mucosal u l c e r a t i o n s ; i n groups II the cecum was f i l l e d with dark brown feces with an offensive smell. Groups IV and V showed less edema and congestion. Group VI showed severe edema, congestion and petecheal hemorrhages. Groups I I I , IV, V and VI (respectively, 3, 6, 12, 20 days o f f carrageenan treatment) had mucosal u l c e r a t i o n and this was p a r t i c u l a r l y evident in Group VI. The ulcers i n group VI were large, i r r e g u l a r , l o c a l i z e d and deep. The l i n i n g mucosa was rough, patchy and corrugated. The spleen and the mesentric lymph nodes were enlarged. The l i v e r was swollen and d u l l in colour. The small int e s t i n e and the rest of the large i n t e s t i n e (upper and lower colon) were apparently normal. A l l the rabbits at this stage of the treatment were apparently healthy, although s l i g h t l y anemic. D. H i s t o l o g i c a l and histochemical observations The r e s u l t s of these studies are i l l u s t r a t e d in Figure 30 and summarised in Table 29. - 142 -a. Histology  Controls The h i s t o l o g i c a l findings were s i m i l a r to those i n previous experiments (see p. 71). Carrageenan treated Group I (48 hours treatment) At this stage there were advanced degenerative changes, congestion, sub-mucosal hemorrhage and edema. The e p i t h e l i a l c e l l s of the crypts were vacuo-lated with i n t e r c e l l u l a r spaces. Some c e l l s had undergone necrosis. The sections were f a i n t l y stained, the blue component of the haeraatoxylin s t a i n being prominent. The c e l l s showed large vacuolated n u c l e i . In the lamina propria there were a few inflammatory c e l l s made up of vacuolated macro-phages and polymorphs. Group II (ulcerated) The mucosa was ulcerated and there were fewer crypts with more necrotic c e l l s . There was congestion and edema along with submucosal hemorrhages and early involvement of the muscularis mucosa. Some e p i t h e l i a l crypts were active and p r o l i f e r a t i v e , being large and di s t o r t e d with d i l a t e d lumina. There were e p i t h e l i a l f i n g e r - l i k e projections (polyps) into the lumen. The lamina propria was i n f i l t r a t e d with macrophages, plasma c e l l s and an increased eosinophil aggregation. Generally, the mucosa appeared simple i n type. - 143 -Group III (3 days o f f treatment) A l l sections at this stage showed var i a b l e degrees of regeneration and r e - e p i t h e l i z a t i o n . There was congestion, edema, submucosal hemorrhages and c e l l u l a r i n f i l t r a t i o n i n the lamina propria with the inflammatory c e l l s con-s i s t i n g mostly of macrophages, plasma c e l l s and eosinophils. The e p i t h e l i a l crypts appeared regenerative and p r o l i f e r a t i v e being l i n e d with active e p i t h e l i a l c e l l s and an increased number of goblet c e l l s . The upper part of the crypts formed f i n g e r - l i k e projections f l o a t i n g f r e e l y into the lumen. In places the epithelium appeared to be made up of a single layer of columnar epithelium which might indicate the i n i t i a t i o n of r e - e p i t h e l i z a t i o n . There were mucosal u l c e r a t i o n s . Group IV (6 day o f f treatment) A l l the tissue changes mentioned above were seen at this stage. The prominent difference from the previous stage was that the crypts appeared more active and were longer, twisted and contained an increasing number of goblet c e l l s . Group V (12 day o f f treatment) There was apparently an advanced stage of healing, characterized by an extremely active, p r o l i f e r a t i v e mucosa with many goblet c e l l s i n the bottom and shoulders of the crypts. However, the lamina propria was s t i l l i n f i l t r a t e d with inflammatory c e l l s (macrophages, plasma c e l l s & eo s i n o p h i l s ) . A few micro-ulcers were observed i n most of the sections studied. Table 29. Histological and histochemical observation of the control and carrageenan treated rabbits Group Histology H & E Histochemistry Controls See eeneral controls (table 11 ) PAT/KOH/PAS PBT/KOH/PAS red purple, blue purple at the base of the crypts red, lighter at base of the crypts the I 48H see table 25 treatment group II, 48 hours reduction in red colour lighter red II 5 days see table 25 ulcerated group V, 5 days, overall blue, specially at the ulcer border unstained at ulcer border III 3 overall it was with less severe inflam- comparable with previous poorly stained, very light days off matory reaction than the previous stage stage, pattern loss, red in few cells treat. and showed signs of regeneration, ulcer- overall colour blue, few ation, congestion, edema & hemorrhage, cells purple epithelial crypts lined with active, regenerating cells, finger-like projections IV 6 more regeneration (group III), crypts compared to III, more stained cells (few) days off were large, long twisted, lined with stained areas, light blue, light red treat. large number of goblet cells. Epithelial purple away from ulcer-cells, active, large, darkly stained ation V 12 advanced stage of healing, active improved staining affin- stained cells with more days off mucosa, with polyps, crypts had large ity, mostly stained intense red compared with treat. number of goblet cells blue, the colour, with stage IV more stainable mucous was dark i.e. more stainable contents VI 20 variable tissue reaction i.e. an ad- very poorly stained, poorly stained, stained days off vanced healing, proliferative mucosa, mostly blue background cells (few) looked active treat. large crypts with large number of goblet large unstained areas, and stained dark red cells, on the other hand some areas reactive cells stained showed deep chronic mucosal ulceration, dark blue-purple (only severe inflammatory infiltrate mostly few cells) plasma cells with large aggregate of eosinophils - 145 -Figure 30. Photomicrographs of H and E sections that show the antomical changes i n the cecal mucosa a f t e r removing degraded carrageenan from the di e t of the carrageenan treated rabbits. Note the inflammatory exudate, absence of e p i t h e l i a l crypts and mucosal u l c e r a t i o n induced by feeding 1% degraded carrageenan for a period of 5 days ( a ) . Removing carrageenan from the d i e t resulted i n progressive healing characterized by active regenerating e p i t h e l i a l crypts within 3 days (b). Re-ep i t h e l i z a t i o n together with increased goblet c e l l s population (c) and normal looking mucosa within 12 days (d). After 20 days (e) there was an active inflammatory process together with mucosal u l c e r a t i o n . The inflammatory exudate consisted of massive aggregation of e o s i n o p h i l i c c e l l s (star) and large aggregates of lymphocytes and plasma c e l l s at the ulcer margins and away from the ulcerations ( i n s e r t b). a-d x 10 e x 6.3 in s e r t , star x 16 insert b x 40 1 -146a-Figure 30 (c) 6 days af t e r removing degraded carrageenan from the d i e t , (et) 12 days a f t e r removing carrageenan from the d i e t . -147a-Figure 30 (e) 20 days af t e r removing carrageenan from the diet (x 10), insert b; lymphocytes at u l c e r margins (x 40) and star ( i n s e r t ) shows eosinophils i n and around ulcers (x 16). - 149 -Group VI (20 day o f f treatment) At this stage, the h i s t o l o g i c a l findings are complicated c o n s i s t i n g of an advanced stage of healing with an extremely active p r o l i f e r a t i v e mucosa together with very well established mucosal u l c e r a t i o n , congestion, edema, hemorrhage and a massive e o s i n o p h i l i a . Other sections showed a tendency to form simple mucosa (see ulcerated stage, 5 day, experiment 3 and ulcerated stage, 0.0 time of treatment, experiment 4). The lamina propria and occasionally the muscularis mucosi were severely i n f i l t r a t e d with plasma c e l l s , eosinophils and macrophages. b. Histochemistry  Controls As shown i n figure 31, tissue sections obtained from the c e c i of the controls stained with the PAT/KOH/and the PBT/KOH/PAS procedures follow the normal staini n g properties mentioned i n experiment I (p. 77). With the exception of the sections taken from controls #'s 9, 29 and 60 (stained f a i n t purple) and from control #59 (stained predominantly b l u i s h with PAT/KOH/PAS), a l l the remaining controls were stained red-red purple with the PAT/KOH/PAS and red with the PBT/KOH/PAS. Carrageenan treated groups 1) PAT/KOH/PAS  Group I (48 hours treatment) There was a loss of normal s t a i n i n g pattern characterized by large un-stained areas, the c e l l s which stained were scattered throughout the e p i t h e l i a l crypts and stained less intensely than the controls. The colour of the PAT/ - 150 -i Figure 31. Photomicrographs of section of rabbit cecum stained with the PAT/KOH/PAS (a,b,c,e,g) and the PBT/KOH/PAS (d,f) procedures. a - 5 days carrageenan treated, note the almost complete absence of st a i n i n g . Compare with the H and •# stained section. Note the progressive increase i n both mucin and the red component of the staining following removal of carrageenan for 3 days (b); 6 days (c) and 12 days (e and f ) . This was followed by a reduction i n both mucin and the red component of the s t a i n at 20 days (g). a-c x 10 d-g x 6.3 : -151a-Figure 31 (d) PBT/KOH/PAS; 6 days a f t e r removing the carrageenan from tlie d i e t . I (e) PAT/KOH/PAS and ( f ) PBT/KOH/PAS; 12 days af t e r removing degraded carrageenan from the d i e t . • •• (i i f -152a-3 •i : Figure 31 (g) PAT/KOH/PAS; 20 days after removing carrageenan from the d i e t . J f i I i - / 1 T J . - 154 -KOH/PAS was variable from c e l l to c e l l . In some areas, the c e l l s stained purple while other c e l l s were s o l i d blue. Overall there was a reduction i n staining i n t e n s i t y and the blue staining predominated. Group II (ulcerated) In sections from this group only a very few randomly scattered c e l l s were stained with a f a i n t blue colour. The blue staining tended to become redder away from ulcer margins. Group III (3 days o f f treatment) Tissue sections at this stage showed a very poor s t a i n i n g reaction v^ith loss of the pattern of d i s t r i b u t i o n . In most sections the staining consisted of nothing more than a blue background (except #26, 27 which showed dark blue stained c e l l s ) . Group IV (6 days off treatment) There was an increase i n the mucin present as compared to the previous stage, the colour being more intense. The staining was a mixture of blue and red being more red i n some areas (away from ulcerated, affected areas) and vice versa. Overall the staining was purple-red purple. Group V (12 days off treatment) At this stage the colour of the s t a i n was more intense but more v a r i a b l e than i n the previous group. No pattern could be observed and there were many unstained areas. The blue colour was more prominent than the red p a r t i c u l a r l y close to the ulcerated areas. - 155 -Group VI (20 days o f f treatment) The tissue sections were f a i n t l y stained, blue being the prominent colour (with the exception of section #52 purple). There were large unstained areas and a complete d i s t o r t i o n of the pattern of the s t a i n d i s t r i b u t i o n of the crypts. 2. PBT/KOH/PAS Group I (48 hours treatment) A l l sections i n th i s group of treated animals showed a very f a i n t s t a i n i n g reaction. In most cases i t was nothing more than a red background, 0-2 red. Group II (ulcerated) No stainin g reaction other than f a i n t pinkish background was seen at th i s stage. Group III (3 day o f f treatment) No stainin g reaction was seen i n the section taken for th i s stage of treatment. Group IV (6 days o ff treatment) The majority of the sections were unstained. Some i n d i v i d u a l crypts or single c e l l s stained a f a i n t red. Group V (12 day o f f treatment) Most of the tissue sections were unstained, where s t a i n i n g occurred i t varied from f a i n t red to some darkly stained active c e l l s , i . e . there was no stai n i n g pattern, and a severe decrease i n the st a i n i n g r e a c t i o n . - 156 -Group VI (20 days o f f treatment) Generally the stainin g i n t e n s i t y was low, e s p e c i a l l y i n and around the ulcerated areas; p r o l i f e r a t e d crypts with an increased number of goblet c e l l s stained dark red. E. Chemical observations These are shown i n Table 30. Analysis of the 105,000 x g supernatants prepared from the e p i t h e l i a l c e l l s removed from the c e c i of the carrageenan treated groups and from the controls showed that i n a l l of the carrageenan treated groups there was a s i g n i f i c a n t decrease i n the percentage of s i a l i c acids substituted at p o s i t i o n C 7/Cg. The reduction i n the percentage of the C^/Cg substituted s i a l i c acid was observed immediately (group I) and occurred throughout the experimental groups. A study of the trend of this reduction showed that i n groups IV and V there was s l i g h t r i s e in percentage of the C^/Cg substituted s i a l i c acids. This was followed by a further reduction i n the percentage of 0-acetyl substituted s i a l i c acids in group VI. S t a t i s t i c a l analysis of this data showed s i g n i f i c a n t d i f f e r e n c e s to be present only between a l l the experimental groups and the controls. No s i g n i f i c a n t differences were noted between the group i n th'e disease induction and recovery studies. The V i b r i o cholera neuraminidase digestion studies of the 105,000 x  g supernatants were considered to be unreliable (see page 36). However, analysis of the data obtained showed that treatment with degraded carrageenan produced an immediate increase in the percentage of V i b r i o cholera neur-aminidase l a b i l e s i a l i c acids i n groups I to I I I . Group II had the highest percentage of s i a l i c acids released, Table 30. The rest of the recovery stage (group IV-V) were s i m i l a r to the co n t r o l s . E p i t h e l i a l glycoproteins (105,000 - 157 -Table 30. Percentage of s i a l i c acids substituted at positions C-J/CQ and at C 4 and percentage released by digestion with V i b r i o cholera neuraminidase in the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of cont r o l and carrageenan treated rabbits Analysis Control 48 hours 5 days ••3 days ••6 days ••12 days *+20 days XC7/C0. 73.8 (o)50.3 (o)55.l (o)52.0 (o)61.1 (o)58.7 (o)54.8 •9.1 • 13.5 •13.7 • 11.7 •10.0 •8.3 •4.8 XC4 31 . 9 28.6 (o)9.0 20.4 31.5 22.5 19.1 •16.4 •15.5 •8.5 •13.5 •15.7 •15.2 • 12.9 X KOH- 63.8 71.1 70 .9 64.6 59.8 57.1 (o)40.9 released • 9 . 9 •5.5 •9.4 •9.1 •6.5 •13.5 •3.0 Z 43.4 50.7 (o)65.7 52.2 40.7 43.0 (o)32.9 released • 11.2 •11.5 •10.0 •15.5 •7.9 •7.6 •4.3 •healing stage (carrageenan treatment discontinued) KOH " s a p o n i f i c a t i o n (IN KOB) Z released " Z s i a l i c acids released by digestion with v i b r i o cholera neuraminidase (o) * s t a t i s t i c a l l y s i g n i f i c a n t l y d i f f e r e n t from the controls (a) C7 • Cg t - DF Controls vs 48 hours • 4.68 18 5 days - 3.59 17 (•)3 days - 4.53 (•)6 days - 2.83 (•)12 days - 3.39 16 (•)20 days - 5.07 17 (b) V.C. A"date d i g e s t i o n Z released Controls vs 5 days - 4.35 17 vs (*)20 days - 2.37 48 hours vs 5 days " 2.5 13 (•)20 days - 3.86 5 days vs (+)6 days - 5.19 12 (•)12 days - 4.52 11 (•)20 days - 7.97 12 (•)3 days vs (*)20 days - 3.19 12 (•)6 days vs ( + )20 days - 22.98 (•)12 days vs (*)20 days - 3.03 11 (c) Z released (KOH N'dase) t - DF Controls vs (•)20 days • 5.62 17 48 hours vs ( + )6 days - 3.63 13 (•)12 days - 2.43 12 (•)20 days - 12.9 13 5 days vs (*)6 days - 2.56 12 (•)12 days - 2.43 11 (•)20 days - 8.04 12 (•)3 days vs (+)20 days - 6.59 (•)6 days vs (*)20 days - 6.99 (•)12 days vs (+)20 days • 2.95 11 (d) Z C 4 Controls vs 5 days • 3.4 17 48 hours vs 5 days - 2.41 13 S days vs (*)6 days - 3.34 12 (•)12 days - 2.02 11 - 158 -Figure 32. Percentage of s i a l i c acids substituted at C^/Cg, Cg/Cg and C^ and the percentage released with V i b r i o cholera neuraminidase i n the 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of controls and carrageenan treated r a b b i t s . The data c i t e d for the percentage of s i a l i c acid substituted at Cg/Cg was obtained from p u r i f i e d glycoproteins. I 1 2 3 4 5 6 i c7/c8 1 2 3 4 5 6 ( RELEASED C 1 2 3 4 5 6 C • CONTROL 1 - 48 HOURS TREATMENT 2 - ULCERATED (S DAYS) 3 - 3 DAYS OFF TREATMENT 4 * 6 DAYS OFF TREATMENT 5 - 1 2 DAYS OFF TREATMENT 6 • 20 DAYS OFF TREATMENT - 159 -x g) obtained from group VI contained s i a l i c acids with an increased resistance to V i b r i o cholera neuraminidase. S t a t i s t i c a l analysis of the data showed that only group II and VI were s i g n i f i c a n t l y d i f f e r e n t from the c o n t r o l s . In the non-saponified neuraminidase digest, there were s t a t i s t i c a l s i g n i f i c a n t differences between the controls and the carrageenan treated groups II and VI, between groups I and VI, between groups II and V, between groups III and VI and between groups V and VI. In the KOH pretreated digest, there were s i g n i f i c a n t differences between the controls and group VI, between groups I and groups IV, V, VI, between groups II and IV, V and VI, between groups III and VI, between groups IV and V and group VI. S t a t i s t i c a l analysis of s u b s t i t u t i o n showed there were s i g n i f i c a n t differences between the controls and group I I , between groups I and I I , and between groups II and IV and V. Analysis of the is o l a t e d glycoprotein (Table 31) confirmed the above data and showed that i n e p i t h e l i a l glycoproteins i s o l a t e d from the carrageenan treated groups there was an immediate reduction i n the percentage of the s i a l i c acids substituted at positions C 7/Cg, Cg/C g and followed by apparent increase i n the s u b s t i t u t i o n and then by a further reduction i n the percentage of the 0-acetyl substituted s i a l i c acids (C^/Cg, Cg/Cg and C^) i n group VI. This was accompanied by a s i m i l a r trend of changes i n the percentage of s i a l i c acid l a b i l e to digestion with V i b r i o cholera neuraminidase. - 160 -Table 31. Percentage s i a l i c acids substituted at positions Cy/Cg, Cg/Cg, C 4 and percentage s i a l i c acid released a f t e r digestion with V i b r i o cholera neuraminidase i n the p u r i f i e d glycoproteins and i n pooled 105,000 x g supernatants prepared from the cecal e p i t h e l i a l c e l l s of control and carrageenan treated rabbits Analysis Control 48 hours 5 days *+3 days *+6 days *+12 days *+20 days % c 7 / c 8 (a) 72.1 56.1 58.4 50.2 65.6 63.3 43.7 (b) 74.8 54.1 52.5 55.3 60.0 61.5 44.8 %Cg+Cg (a) _ (b) 56.8 37.4 42.2 44.5 47.0 51.6 36.5 % c 4 (a) 56.5 43.2 43.2 53.6 53.6 51.1 54.2 (b) 63.4 45.2 39.3 53.3 52.8 52.3 33.9 % KOH- (a) 75.1 60.0 63.0 57.5 62.8 68.7 72.2 re leased (b) 67.7 59.7 52.4 56.1 63.5 59.1 66.3 % (a) 32.7 34.1 25.9 26.7 29.1 33.4 33.0 released (b) 24.8 32.4 31.8 26.2 30.0 28.2 43.8 * - healing stage (carrageenan treatment discontinued) (a) = pooled 105,000 x g supernatant (b) = iso l a t e d glycoproteins from pooled 105,000 x g supernatants KOH = s a p o n i f i c a t i o n (IN KOH) % released = % s i a l i c acids released by digestion with V i b r i o cholera neuraminidase - 161 -DISCUSSION Ga s t r o i n t e s t i n a l mucus i s a thick viscous glycoprotein containing f l u i d secreted continuously into the lumen of the i n t e s t i n e by the goblet c e l l s and by the mucus producing glands, eg. Brunners glands. The mucus forms a coating over the epithelium, where i t i s assumed to function to lubricate the f e c a l stream and to provide protection to the se n s i t i v e underlying mucosal e p i t h e l i a l c e l l s . A l t e r a t i o n s i n the large i n t e s t i n a l glycoproteins have been shown to be associated with u l c e r a t i v e c o l i t i s (19,26,35,38,47,48,50,56,107,109-115,148-152) but the s i g n i f i c a n c e of these findings i s unclear because i t i s not known whether the observed changes are a cause or a consequence of the disease process. Furthermore, most chemical studies (111-113) are d i f f i c u l t to interpret because the s t a r t i n g materials could have been contaminated with connective tissue elements and/or products of the f e c a l stream. The degraded carrageenan model of large bowel u l c e r a t i o n , described by Marcus and Watt (237), was used to study u l c e r a t i o n under controlled laboratory conditions. Although carrageenan induced c o l i t i s i s not an exact model of u l c e r a t i v e c o l i t i s , the model provides an excellent opportunity to study chemical changes i n the e p i t h e l i a l glycoproteins associated with the induction, recovery and healing stages of u l c e r a t i v e disease and to co r r e l a t e such changes with c l i n i c a l , gross anatomical, h i s t o p a t h o l o g i c a l and h i s t o -chemical findings. P o t e n t i a l l y i t should provide basic b i o l o g i c a l information about the processes occurring during large i n t e s t i n a l u l c e r a t i o n and hence may help i n the understanding of human disease. Marcus and Watt (239) induced large bowel u l c e r a t i o n i n both rabbits and guinea pigs by feeding them various doses of degraded carrageenan i n t h e i r - 162 -drinking water. Preliminary studies established that i t was possible under our conditions to induce u l c e r a t i o n i n both guinea pigs and r a b b i t s . Rabbits were selected as the experimental model because (a) they yielded more e p i t h e l i a l glycoprotein than guinea pigs, (b) they contracted the disease at a moderate dose of carrageenan (1% w/v) and (c) p u r i f i e d e p i t h e l i a l glycoproteins could r e a d i l y be prepared. Feeding 1% (1 g/kg body weight) degraded carrageenan ad l i b to New Zealand white rabbits resulted i n the rapid onset of acute disease. Fecal occult blood was detected as early as 48 hours a f t e r the commencement of treatment while v i s i b l e blood was present i n the feces within 5 days. There was variable diarrhea ( s o f t , watery feces), a sharp drop i n the food and carrageenan intake accompanied by rapid loss of body weight, and a deter-i o r a t i o n i n health. Rabbits treated for longer periods became progressively weaker and emaciated. In contrast, Marcus and Watt (239) found that while 5% degraded carrageenan fed ad l i b to C a l i f o r n i a s t r a i n rabbits resulted i n acute disease and death within 10-14 days the course with 1% degraded carrageenan was less severe, i n t e s t i n a l bleeding occurring within 3 weeks of the st a r t of the treatment. S i m i l a r l y Grasso et a_l. (252) i n a study of New Zealand white rabbits found that a higher carrageenan intake (1.5 g/fcg/day) was required to produce a c l i n i c a l l y comparable disease to that observed i n the present i n v e s t i g a t i o n . The acute nature of the disease i n the current study and the lower d a i l y carrageenan intake required to induce u l c e r a t i o n i s presumably due either to d i f f e r e n t environmental conditions (eg. the exclusion of green food supplement) or to genetic f a c t o r s . Evidence obtained i n th i s study indicates that the carrageenan induced c o l i t i s was a progressive disease, the longer the treatment the more severe - 163 -and more evident the mucosal u l c e r a t i o n . In the cecum, the u l c e r a t i o n was widespread but was p a r t i c u l a r l y common on and around the transverse f o l d s . The ulcers were shallow and of variable size and shape. In contrast, i n the upper and the lower colon, i f ulcers were present they tended to be deep, large and l o c a l i z e d . Further, they were usually limited to the r e c t a l portion of the lower colon and to the cecal end of the upper colon. Watt and Marcus (239) reported that i n rabbits u l c e r a t i o n occurred primarily i n the cecum, then extended to the upper colon and f i n a l l y involved the r e c t a l portion of the lower colon. Sharrat et a l . (244) and M a i l l e t et^ al_. (245) found that u l c e r a t i o n i n the rabbits and guinea pigs was primarily i n the cecum and that on longer treatment i t might extend to the upper colon. This d i s t r i b u t i o n was att r i b u t e d to the a b i l i t y of carrageenan to cross the cecal mucosa i n these animal species (251,252). In some of the animals which died or were s a c r i f i c e d a f t e r a short period of carrageenan treatment, the small i n t e s t i n e was severely congested and on occasions was hemorrhagic. Furthermore i n the stomach there were black spots, presumably due to bleeding. Such observations are consistent with the micro-scopic findings of Fabian e_t a K (250) who demonstrated that i n r a t s , i n addition to metaplasia i n the r e c t a l mucosa, there was a v a r i a b l e inflammatory response i n a l l regions of the digestive t r a c t . H i s t o l o g i c a l l y the inflam-matory response observed i n our study was s i m i l a r to that described by Marcus and Watt (237,239,241), Sharrat et a l . (244) and Abraham et a l . (251). Ulceration was normally limited to the mucosa and the inflammatory response to the lamina propia. In severely ulcerated i n d i v i d u a l s , however, where the muscularis mucosa was destroyed, the inflammatory response extended deeply into the submucosa and occasionally into the muscle layer proper. The nature - 164 -and severity of the inflammatory reaction correlated with the period of carrageenan treatment. In the early stages macrophages predominated, i n the l a t e r stages the predominant c e l l s were plasma c e l l s and massive aggregations of eosinophils. There were few, i f any, true crypt abscesses. These findings confirm those of Sharatt £t a_l. (244,253) but are not i n t o t a l agreement with those of Marcus and Watt (239) who found crypt abscesses. The find i n g of massive numbers of eosinophils has only been reported very recently i n the carrageenen model (254). The inflammatory response observed i n our studies of the carrageenan model show some differences from and some s i m i l a r i t i e s to that found i n human ulc e r a t i v e c o l i t i s . In the la t e r stages of the carrageenan c o l i t i s the presence of plasma c e l l s and eosinophils i s sim i l a r to that reported for chronic u l c e r a t i v e c o l i t i s i n man (155). However the model d i f f e r s i n that i t lacks severe congestion and hemorrhage and there are few i f any crypt abscesses. In the early stages the major s i m i l a r i t y i s the acute hemorrhage and congestion but the inflammatory c e l l s d i f f e r i n that macrophages predominate whereas i n ul c e r a t i v e c o l i t i s the predominant c e l l s are polymorphs and plasma c e l l s . The presence of tissue e o s i n o p h i l i a has been suggested to represent an immunological response. The current hypothesis i s that the responding eosinophils serve to contain and terminate h y p e r s e n s i t i v i t y reactions, e s p e c i a l l y those of the immediate type (265). E o s i n o p h i l i a i n the colon has been noted i n association with long standing active c o l i t i s (266) where i t i s thought to be associated with mast c e l l s and basophil degradation (267-268), the l a t t e r being common i n active c o l i t i s i n man (269). Therefore, the presence of massive tissue e o s i n o p h i l i a associated with plasma c e l l i n f i l -t r a t i o n , i n the carrageenan treated rabbits could represent an immunological - 165 -process, humoral and/or c e l l u l a r , occurring i n the tissue of the affected organ. The e o s i n o p h i l i a was correlated with the se v e r i t y of the disease i n that i t was observed i n the c h r o n i c a l l y ulcerated rabbits but not i n the non-ulcerated r a b b i t s . Therefore i t i s assumed that these aggregates are assoc-iated with a phenomenon i n the tissue which occurs a f t e r the establishment of the u l c e r a t i o n . It might represent a h y p e r s e n s i t i v i t y reaction i n i t i a t e d by s e n s i t i s i n g the host tissue towards (a) i t s e l f ( a u t o - s e n s i t i s a t i o n ) , (b) the carrageenan, (c) the b a c t e r i a l f l o r a , or (d) some combination of these f a c t o r s . The primary gross anatomic and histopathologic observations i n experiments 1, 2 and 3 indicated that the carrageenan induced c o l i t i s was a progressive disease the early stages of which were mucosal degeneration followed by an inflammatory response and u l c e r a t i o n . Experiment 4, i n contrast, was designed to investigate the healing process attendant on removal of the carrageenan. The r e s u l t s showed that 20 days after removal of the degraded carrageenan from the d i e t , ulcers were s t i l l present and were associated with an inflammatory response, consisting of plasma c e l l s , polymorphs, massive aggregates of eosinophils, severe blood vessel congestion and hemorrhages i n the lamina properia. These findings were shown to be associated with a s i g n i f i c a n t reduction i n the percentage of the glycoprotein 0-acetyl substituted s i a l i c acids. Groups of animals s a c r i f i c e d p r i o r to this point, however, showed evidence of a progressive healing process i n that there was a reduction i n the inflammatory response and a regeneration of the l i n i n g epithelium. Apparently, therefore, there was an exacerbation of the disease between days 12 and 20 associated with renewed mucosal u l c e r a t i o n . There have been few experimental studies of the healing of the large i n t e s t i n a l mucosa. Melnik et a l . - 166 -(272,273), and Braucher and Kirsner (271), investigated healing a f t e r mucosal biopsy i n man and dogs, and i n addition after a mucosal burn i n dogs, and found that complete healing required at least 3 months (271). In contrast, Grasso et a l . (252) found that i n guinea pigs treated with 1% carrageenan there were no cecal ulcerations i n animals s a c r i f i c e d 1 or 4 weeks af t e r cessation of the carrageenan treatment. Further the cec i and upper colons of the animals k i l l e d a f t e r 4 weeks were h i s t o l o g i c a l l y i ndistinguishable from the co n t r o l s . Large bowel u l c e r a t i o n can occur spontaneously, or can be induced i n many species of laboratory animals by ph y s i c a l , chemical or b i o l o g i c a l means (146,270). The major problem encountered i n such studies was to maintain the disease long enough to simulate the c l i n i c a l course of the human disease. Analysis of the information obtained from the recovery stage of experiment 4, p a r t i c u l a r l y from group VI (20 day-off carrageenan treatment), showed that a severe inflammatory process and mucosal ulcerations were present a f t e r removing the carrageenan from the d i e t . These were associated with an evident a l t e r a t i o n i n the nature of the inflammatory response i n that plasma c e l l s and massive aggregates of eosinophils replaced the macrophages which were the major inflammatory c e l l u l a r response i n the induction stage of the carrageenan induced c o l i t i s (251,252). These observations, therefore, were suggestive of the p o s s i b i l i t y that an e t i o l o g i c f a c t o r ( s ) other than the carrageenan was involved i n the mucosal u l c e r a t i o n at t h i s stage. Ulcerative c o l i t i s i s a disease characterized by remission and relapse (151,152,155). Therefore the observations i n experiment 4 might present a c l i n i c a l s i m i l a r i t y to the disease i n man. These observations should be investigated further therefore to e s t a b l i s h whether they are due to the short period of recovery used i n this experiment or are a consequence of a - 167 -pathological process i n i t i a t e d by the carrageenan treatment and maintained by other f a c t o r ( s ) such as b a c t e r i a l i n f e c t i o n or immunological phenomena. Such factors are thought to play a major part i n large bowel u l c e r a t i o n i n man (146, 157) and also b a c t e r i a are considered to be involved i n the carrageenan model of u l c e r a t i o n (248,254). One p o s s i b i l i t y would be to reproduce experiment 4 and extend the recovery stage for a period i n excess of the 3 months period found by Braucher and Kirsner to be necessary for complete recovery (271). Simultaneously an immunological study could be performed to determine whether or not the sera of the ulcerated rabbits contains anticolon antibodies. The aim of this thesis was to investigate the changes i n the e p i t h e l i a l glycoproteins associated with experimentally induced large bowel u l c e r a t i o n i n the r a b b i t . Hence i t was necessary to compare the properties of the glyco-proteins i n both normal and diseased r a b b i t s . This comparison depended upon histochemical and chemical assessments of the e p i t h e l i a l glycoproteins. It was chosen because previous and current studies (35,38,107) i n our laboratory have shown that changes i n 0-acetyl s i a l i c acids are associated with u l c e r a t i v e c o l i t i s . Histochemical studies were performed with two s t a i n i n g techniques, the PBT/KOH/PAS (39) and the PAT/KOH/PAS (263). In the PBT/KOH/PAS procedure, pre-existing v i c i n a l d i o l s are oxidised to aldehydes with periodic acid and then converted to S c h i f f unreactive primary alcohols by reduction with sodium borohydride. On subsequent treatment with potassium hydroxide, any 0-acetyl esters blocking a v i c i n a l d i o l are removed and stained red with the standard PAS procedure. Thus s i a l i c acids which are substituted at C o r a t cQ or / o which are d i ( C 7 c 8 , c 7 C 9 , C 8 C 9 ) or t r i (C 7CgC 9) substituted w i l l s t a i n red, while those which have no side chain s u b s t i t u t i o n or which are substituted at C g d o n o t 8 t a i n . - 168 -In the PAT/KOH/PAS procedure, pre-existing tissue d i o l s were oxidised with periodic acid and stained blue with t h i o n i n S c h i f f reagent. Following s a p o n i f i c a t i o n v i c i n a l d i o l s , generated by the removal of 0-acetyl e s t e r s , were stained red with the standard PAS procedure. In this technique, s i a l i c acids substituted at C g (or which are d i or t r i substituted) s t a i n red, a l l other s i a l i c acids s t a i n blue. Mixtures of these classes of s i a l i c acids s t a i n i n various shades of purple. Using both these stain i n g procedures, i t i s possible to d i f f e r e n t i a t e between s i a l i c acids substituted at p o s i t i o n and s i a l i c acids substituted at carbon Cg. The results expected from the st a i n i n g procedures are i l l u s t r a t e d i n Table 32. Chemical analyses were performed with procedures developed i n t h i s labor-atory for the measurement of s i a l i c acids substituted at positions C^/Cg and Cg/Cg i n the polyhydroxy side chain. The theory of these methods has been discussed extensively (102,104). In addition the neuraminidase suscept-i b i l i t y of the s i a l i c acid was measured and the data obtained used to measure the percentage of s i a l i c acids substituted at C^ (104). The comparison between the r e s u l t s of chemical and histochemical methods for the assessment of side chain s u b s t i t u t i o n has several inherent d i f f i c u l t i e s (42). "The major problem of making v i s u a l assessments of histochemical reactions i s that a reading of 0 means that there i s an i n s u f f i c i e n t l o c a l concentration of dye binding reactive groups for the s t a i n i n g to be v i s i b l e , not that such groups are absent, and that i n most instances t h i s lower l i m i t i s unknown. At the opposite end of the scale, there i s probably a maximum concentration above which changes cannot be recognized. It may also be assumed that, as i n colorimetric analyses, there w i l l be a concentration above - 169 -Table 32. Expected colour reaction of the 0-acetyl substituted s i a l i c acids of the e p i t h e l i a l glycoproteins after s t a i n i n g with the PBT/KOH/PAS and PAT/KOH/PAS staining procedures 0-acetyl s u b s t i t u t i o n * PBT/KOH/PAS colour - red PAT/KOH/PAS colour - red/blue c 0 unstained blue C7 red blue red red c 9 unstained blue d i or t r i substituted red red *mixture of these classes of s i a l i c acids s t a i n i n with the PAT/KOH/PAS procedure various shades of purple CQ = s i a l i c acid with no 0-acetyl s u b s t i t u t i o n at the polyhydroxy side chain - 170 -which there w i l l be a non l i n e a r r e l a t i o n s h i p between depth of colour and concentration." Further the quantitative r e l a t i o n s h i p between the h i s t o -chemical staining reaction and the degree of side chain is not accurately known although i n previous studies a good c o r r e l a t i o n was obtained (42). The chemical analysis measures the percentage of the t o t a l s i a l i c acids present which contain side chain substituents. Consequently the value w i l l be the same regardless of the number of s i a l i c acid residues present/molecule. Therefore a reduction i n the t o t a l number of s i a l i c acid end groups could a f f e c t the colour of staining i n the PAT/KOH/PAS, or the i n t e n s i t y of st a i n i n g i n the PBT/KOH/PAS, without decreasing the percentage of s i a l i c acid with side chain substituents as determined by chemical assessment. A further complication i s introduced by the fact that chemical analysis represents an average of the changes over a r e l a t i v e l y large area whereas histochemical change can be observed over a very small area. For example histochemically i t is easy to observe a change i n the c e l l s of a single crypt among completely normal crypts whereas such changes would probably not be observed by chemical analysis. As a consequence c o r r e l a t i o n between chemical and histochemical data can be affected by both the extent and the nature of the changes. These factors probably explain why (a) i n the cecum we often observed histochemically a greater reduction i n side chain s u b s t i t u t i o n than would be accounted for by chemical analysis and (b) i n the upper and lower colons reductions i n side chain s u b s t i t u t i o n observed histochemically could not be detected by chemical a n a l y s i s . Therefore, although i n th i s study there was a general agreement between the chemical and histochemical r e s u l t s , t h i s agreement should not be interpreted i n absolute terms. - 171 -Histochemical Studies Histochemical studies of the large i n t e s t i n a l e p i t h e l i a l mucins of the normal rabbit indicate that they are composed of s i a l o and sulpho glycoproteins (20,31). Previous investigations carried out i n t h i s laboratory indicated that these mucins also contain side chain O-acylated s i a l i c acids (31) and that such mucins predominate i n the upper halves of the crypts. The current work confirmed and amplified these l a t t e r observations, and i n addition, demonstrated the presence of such glycoproteins i n the cecal epithelium. In the upper and lower colon there was an abundance of mucin and a c l e a r d i s t i n c t i o n between the upper and lower halves of the crypts; O-acylated s i a l i c acids c l e a r l y predominating i n the upper halves. The s i g n i f i c a n c e of this d i s t r i b u t i o n i s unknown but i t d i f f e r s from that i n r a t s , where O-acylated s i a l i c acids predominate i n the base of the crypts, and from guinea pigs and man where such s i a l i c acids are found a l l along the length of the crypts (31). In the cecum there was both less mucin o v e r a l l and a less c l e a r d i s t i n c t i o n between the upper and lower halves of the crypts. The most consistent histochemical finding i n the c e c i of the carrageenan treated rabbits was a decrease i n both the quantity of mucin and i n the degree of side chain s u b s t i t u t i o n of the O-acylated s i a l i c acids r e s u l t i n g i n the loss of the normal staining pattern of the e p i t h e l i a l crypt. The reduction i n O-acylated s i a l i c acids was r e l a t i v e l y greater i n the epithelium at the margins of the u l c e r s . Although i n experiment #1 u l c e r a t i o n occurred i n either or both the upper and lower colon, i n only 25% of the animals were the h i s t o -chemical findings s i m i l a r to those found i n the cecum. In those animals i n which there was no h i s t o l o g i c a l evidence of colonic u l c e r a t i o n (experiments 1 and 2) the most consistent f i n d i n g was an apparent increase i n mucin - 172 -production. In many of these rabbits there was i n addition a decrease i n 0-acetylated s i a l i c acids presenting as a loss of the normal st a i n i n g pattern. These data imply, but do not prove, that changes i n the e p i t h e l i a l glyco-proteins occur prior to the production of frank u l c e r a t i o n and may indic a t e that the quantity of mucin produced could be an important factor i n the process of u l c e r a t i o n . In human ul c e r a t i v e c o l i t i s a reduction i n mucin i s a consistent finding (19,26,47,48,50,109,110) and a reduction i n the degree of side chain a c e t y l a t i o n of s i a l i c acids, correlated with the " s e v e r i t y " of the disease, has been reported by C u l l i n g et^ al_. (35,38). Experiments 2, 3 and groups I and II of experiment 4 showed that the decrease i n the degree of side chain 0-acetyl s u b s t i t u t i o n was progressive and was correlated with severity of the disease i n that the longer the treatment the more the decrease i n the s u b s t i t u t i o n . In p a r t i c u l a r the data from group 1, 24 hours treatment, of experiment 3 showed that these changes i n the e p i t h e l i a l glycoprotein s i a l i c acids were not associated with the inflammatory response. These findings imply that the decrease i n the percentage of the substituted s i a l i c acid might be a primary tissue response to the i n s u l t rather than an e f f e c t of the inflammatory process. Hence i t i s considered that the changes i n the e p i t h e l i a l glycoproteins are not a l i k e l y consequence of the release of lysosomal enzymes from degenerating macrophages considered by Sharrat et_ a_l. to be the cause of the mucosal u l c e r a t i o n (244,251,252,253). Healing, as established by h i s t o l o g i c c r i t e r i a , was associated with a progressive increase i n the degree of side chain s u b s t i t u t i o n of the s i a l i c a c i d as compared to ulcerated animals. However, i n the f i n a l group of animals (experiment #4, group VI) i n which there was gross anatomic and h i s t o l o g i c evidence for active u l c e r a t i o n along with advanced regeneration there was a - 173 -reduction i n the degree of side chain s u b s t i t u t i o n of the s i a l i c acids. Regardless therefore of the actual course of events there is a c l e a r as s o c i a t i o n between u l c e r a t i o n and a reduction i n side chain s u b s t i t u t i o n . Chemical Studies P r i o r to the present study apparently there has been no i n v e s t i g a t i o n of the chemistry of native rabbit large i n t e s t i n a l glycoproteins of known e p i t h e l i a l o r i g i n . The chemical studies performed i n the present work confirm the histochemical conclusions that these glycoproteins contain side chain O-acylated s i a l i c acids. In addition they show that the s i a l i c acids are, i n the main, r e s i s t a n t to digestion with V i b r i o cholera neuraminidase. This resistance was p a r t i a l l y reversed by p r i o r s a p o n i f i c a t i o n of the glycoproteins i n d i c a t i n g the presence of s i a l i c acids with O-acyl substituent at p o s i t i o n C^ (or C^) (102-105). The f a i l u r e to remove a l l the s i a l i c acids a f t e r such treatment is presumably due to some other s t e r i c factor since the c o n t r o l samples, analysed simultaneously, always released a higher percentage of s i a l i c acids. These phenomena have been detected previously i n colonic e p i t h e l i a l glycoproteins i s o l a t e d from both man and rat (103,104). The data from experiment #1 indicated that there were s i g n i f i c a n t d i f -ferences between the O-acetyl s u b s t i t u t i o n patterns of the s i a l i c acids of the glycoproteins i s o l a t e d from the cecum, and upper colon and from the upper and lower colon. The values obtained for these cecal glycoproteins were s i m i l a r to those obtained i n experiments #3 and 4 but those obtained i n experiment #2 were s i g n i f i c a n t l y lower.' The reason for this finding is unknown. Since a l l the controls i n experiment #2 were apparently normal by c l i n i c a l , gross anatomical and h i s t o p a t h o l o g i c a l c r i t e r i a i t is u n l i k e l y that they had an extraneous large bowel disease. Further, since s i m i l a r technical procedures - 174 -and reagents were used throughout t h i s work and s a t i s f a c t o r y control values were obtained, the values cannot be attributed to technical errors. Examin-ation of the chemical data for the i n d i v i d u a l control animals i n experiments 1, 3 and 4 showed that on occasion there were apparently normal individuals with lower than expected values. Since the genetic homogeneity of the animals used i s unknown, i t i s possible that the low values i n experiment #2 resulted from uncontrolled factors such as genetic c h a r a c t e r i s t i c s and housing and/or feeding p r i o r to the study. Differences between the percentage of s i a l i c acids with O—acetyl substituents at and/or on the polyhydroxy side chain have been demonstrated i n the e p i t h e l i a l glycoproteins i s o l a t e d from the upper and lower halves of rat colon (103) but no such difference has been detected in the large i n t e s t i n e of man (104). Chemical analysis of the 105,000 x g supernatants i s o l a t e d in experiment #1 from the cecal e p i t h e l i a l c e l l s of the carrageenan treated rabbits indicated, i n confirmation of the histochemical data, that there was a s t a t i s t i c a l l y s i g n i f i c a n t reduction i n the percentage of s i a l i c acids with side chain 0-acyl substituents at positions C^/CQ. In addition the s i a l i c acids were more susceptible to digestion with V i b r i o cholera neuraminidase. As found with the normal rabbit glycoproteins a l l the s i a l i c acids were not susceptible to d i g e s t i o n with neuraminidase even a f t e r s a p o n i f i c a t i o n to remove ester substituents. The increased s u s c e p t i b i l i t y , p r i o r to s a p o n i f i -cation, was r e f l e c t e d i n a decrease i n the calculated percentage of C^ (or C^) substituted s i a l i c acids. The association between changes i n s i a l i c acid 0-acylation pattern and u l c e r a t i o n was confirmed i n experiments 2, 3 and - 175 -To prove that the changes noted i n the 105,000 x g supernatants were associated with the e p i t h e l i a l glycoproteins the l a t e r were i s o l a t e d and analysed. Previous studies (102) had demonstrated that, i n normal r a t s , analysis of the 105,000 x g supernatants and glycoproteins i s o l a t e d from them gave si m i l a r r e s u l t s with regard to s u b s t i t u t i o n of the s i a l i c acids at positions C^/Cg. However this had not been demonstrated for either normal or carrageenan treated rabbits and furthermore had not been demonstrated with respect to the neuraminidase s u s c e p t i b i l i t y of the s i a l i c acids. The 105,000 x g supernatants were obtained from sonicates of washed e p i t h e l i a l c e l l s and therefore represent a crude extract presumably uncontaminated with extra-c e l l u l a r and membrane components. Ideall y the e p i t h e l i a l glycoproteins should have been i s o l a t e d from each i n d i v i d u a l supernatant but due to the r e l a t i v e l y small amounts of material and the very large number of specimens to be processed i t was decided to pool the supernatants. The close correspondence between the mean re s u l t s obtained from the i n d i v i d u a l 105,000 x g supernatants and those obtained from the pooled supernatants demonstrates the v a l i d i t y of the method used to prepare the pools. The correspondence between these r e s u l t s and those obtained from the is o l a t e d glycoproteins shows that the changes observed with the crude extracts were a c t u a l l y associated with the e p i t h e l i a l glycoproteins and were not a consequence of other s i a l i c acid containing molecules. I s o l a t i o n of the p u r i f i e d glycoproteins also enabled an estimate to be made of the percentage of s i a l i c acids substituted at Cg/C^ which had proved to be impossible with the 105,000 x g supernatants. These analyses showed that i n addition to a decrease i n the percentage of s i a l i c substituted a t C^/Cg there was also a decrease i n su b s t i t u t i o n at Cg/Cg. - 176 -Calculations were made (104) using t h i s data and assuming that the glyco-proteins either contain no 9-0-acyl s i a l i c acid or that i f such acids are present they are not oxidised by periodate. These showed that, i n comparison to the controls, the glycoproteins i s o l a t e d from the ulcerated groups of animals (5 days carrageenan treatment, experiments 2, 3 and 4) contained a greater percentage of s i a l i c acids without side chain substituents. Apparently, therefore, the process of u l c e r a t i o n i s accompanied by the production of such unsubstituted s i a l i c acids (Tables 33-36). In the upper and lower colons of a number of the carrageenan treated animals i n experiments 1 and 2 the histochemical data suggested a reduction i n the degree of side chain s u b s t i t u t i o n of the e p i t h e l i a l glycoprotein s i a l i c a cids. However these differences could not be demonstrated upon chemical analysis of the 105,000 x g supernatants (experiments 1 and 2) or the i s o l a t e d glycoproteins (experiment 1). Furthermore examination of the chemical data obtained from those i n d i v i d u a l s (20%) which showed clear h i s t o l o g i c a l evidence of u l c e r a t i o n f a i l e d to indicate any clear difference from normal. This apparent discrepancy probably arises from the fact that the lesions were l o c a l i s e d . Therefore, although h i s t o l o g i c a l l y i t i s possible to observe changes within such an area, such changes w i l l probably not be observed by chemical analysis since the chemical data represents an average over the ent i r e specimen of colon which includes both normal and affected t i s s u e s . A l t e r n a t i v e l y the histochemical changes could be a consequence of a l t e r a t i o n s i n residues other than s i a l i c a cid. For example an increase i n the number of fucose residues at the expense of s i a l i c acids would be expected to r e s u l t i n an increase i n the blue component of the PAT/KOH/PAS s t a i n . I f at the same time there was no change i n the percentage s i a l i c acid side chain s u b s t i t u t i o n - 177 -Table 33. Percentage of C Q, c 7 , C 7/C 8, C 8/C 9 0-acetyl substituted s i a l i c acids i n the glycoproteins i s o l a t e d from pools of the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s from controls and degraded carrageenan treated rabbits, Experiment #2. % s u b s t i t u t i o n at p o s i t i o n Controls 48 hours 5 days 9 days 17 days % C 7 / C g 55.2 58.2 29.0 32.7 38.7 % C8 / C 9 31.4 51.2 17.6 - 32.6 * c 7 23.8 7.0 11.4 - 5.9 % C 45.8 41.8 71.0 - 61.5 ^ CQ = s i a l i c acid with no 0-acetyl substituent at the polyhydroxy side chain - 178 -Table 34. Percentage of C 0, C 7, C 7/Cg, C 3 / C 9 0-acetyl substituted s i a l i c acids i n the glycoproteins i s o l a t e d from pools of the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s from control and degraded carrageenan treated rabbits, Experiment #3. % s u b s t i t u t i o n at positions i Controls 24 hours 48 hours 3 days 4 days 5 days % C 7/Cg 64.5 56.4 61.7 56.8 46.6 50.9 % Cg/Cg 53.6 21.3 29.7 41.3 29.5 30.4 % c 7 10.9 35.1 32.0 15.5 17.1 19.5 % c 0 35.5 43.6 38.3 43.2 53.4 49.1 % Cg = s i a l i c chain acid bearing no 0-acetyl substituents at the polyhydroxy side - 179 -Table 35. Percentage of CQ, C 7 , Cy/Cg, Cg/C Y 0-acetyl substituted s i a l i c acids i n the glycoproteins i s o l a t e d from pools of the 105,000 x g supernatants obtained from the cecal e p i t h e l i a l c e l l s from control and degraded carrageenan treated rabbits, Experiment #4. % s u b s t i t u t i o n at positions Controls 48 hours 5 days *+3 days *+6 days *+12 days *+20 days 74.8 54.1 52.5 55.3 60.0 61.5 44.8 %Cg/Cg 56.8 37.4 42.2 44.5 47.0 51.6 36.3 %c7 18.0 16.7 10.3 10.8 13.0 9.9 8.3 %c0 25.2 45.9 47.5 44.7 40.0 38.5 55.2 •healing stage (carrageenan treatment discontinued) %CQ = s i a l i c acids bearing no 0-acetyl s u b s t i t u t i o n at the polyhydroxy side chain - 180 -Table 36. Comparison of percentage CQ- substituted s i a l i c acid i n the glycoproteins i s o l a t e d from pools of the 105,000 x g supernatant obtained from the cecal e p i t h e l i a l c e l l s of controls and 5 days degraded carrageenan treated rabbits from experiments number 2, 3 and 4. %CQ = % s i a l i c acids bearing no 0-acetyl substituents at the polyhydroxy side chain Experiment # Controls Carrageenan treated 2 45.8 71.0 3 35.5 49.1 4 25.2 47.5 - 181 -no differences would be detected with the chemical procedures employed. In view of the f a i l u r e to detect chemical differences i n the upper and lower colons these organs were not examined i n experiments #3 and #4 although the tissues were preserved i n formalin for any future studies. Analysis of the 105,000 x g supernatants obtained i n experiment 3 indicated that carrageenan treatments resulted i n an immediate reduction i n the percentage of s i a l i c acids substituted at C 7/Cg. This immediate e f f e c t was followed by a trend towards a further reduction i n s u b s t i t u t i o n . This data was i n general q u a l i t a t i v e agreement with the histochemical observations but the chemical changes were not s t a t i s t i c a l l y s i g n i f i c a n t u n t i l 72 hours at which time there was h i s t o l o g i c evidence of u l c e r a t i o n . In experiment #4 however the reduction was s t a t i s t i c a l l y s i g n i f i c a n t at 48 hours where again, h i s t o l o g i c a l l y , there were early signs of u l c e r a t i o n . It i s clear therefore that u l c e r a t i o n is accompanied by a reduction i n side chain a c e t y l a t i o n but, although suggestive, the chemical analyses cannot be used to confirm the histochemical observation that reductions i n side chain a c e t y l a t i o n occur at a time when ulcers are absent and the inflammatory response minimal. As discussed above, t h i s apparent discrepancy could be a function of the difference between chemical and histochemical methodology. The i n t e r p r e t a t i o n of the r e s u l t s of the neuraminidase digests i n experiments 3 and 4 are complicated by the observation that i n some of the 105,000 x g supernatants a pink colour developed p r i o r to the heating step of the t h i o b a r b i t u r i c acid assay. The reason for t h i s i s unknown but the i n t e n s i t y was small as compared to that developed a f t e r heating. However the t o t a l s i a l i c a c i d , as measured i n the digests by the sum of the r e s o r c i n o l and t h i o b a r b i t u r i c acid assays, was not s i m i l a r to that obtained by an - 182 -independent periodate r e s o r c i n o l assay. This phenomenon was not observed i n d i g e s t s of the p u r i f i e d g l y c o p r o t e i n s and the r e s u l t s obtained showed the same trends as those obtained with the 105,000 x g supernatants although the a c t u a l r e s u l t s d i f f e r . In view of t h i s , r e l i a n c e can only be placed upon the r e s u l t s obtained wi t h the p u r i f i e d g l y c o p r o t e i n s and these data are that r e f e r r e d to i n the d i s c u s s i o n below. Carrageenan treatment in"experiment #3 r e s u l t e d i n an immediate increase i n s u s c e p t i b i l i t y to d i g e s t i o n w i t h V i b r i o cholera neuraminidase which was then l i t t l e changed through the course of the experiment. S i m i l a r l y , i n experiment 4 there was an increased s u s c e p t i b i l i t y a f t e r 48 hours and at 5 days. Therefore an increased s u s c e p t i b i l i t y to neuraminidase accompanies h i s t o l o g i c a l evidence of u l c e r a t i o n and the data suggests but does not prove that such an increase occurs p r i o r to both u l c e r a t i o n and a s i g n i f i c a n t inflammatory response. In experiments 3 and 4 carrageenan treatment f o r up to 5 days r e s u l t e d i n no change i n the s u s c e p t i b i l i t y of the s i a l i c acids of the s a p o n i f i e d g l y c o -p r o t e i n s to d i g e s t i o n with V i b r i o cholera neuraminidase. When, however, the r e s u l t s were used to c a l c u l a t e the percentage of s i a l i c acids s u b s t i t u t e d at 0^, there was r e d u c t i o n at a l l the time periods i n experiment 3 and at both 48 hours and at day 5 i n experiment 4. Therefore u l c e r a t i o n i s accompanied by a reduction i n the percentage of s i a l i c acids s u b s t i t u t e d at C^ and p o s s i b l y t h i s reduction occurs p r i o r to the u l c e r a t i o n . During the f i r s t 12 days a f t e r the removal of carrageenan from the d i e t of the animals i n experiment 4 there was no s t a t i s t i c a l l y s i g n i f i c a n t increase i n the percentage of s i a l i c a c i d s s u b s t i t u t e d at C^/Cg, Cg/Cg although there was h i s t o c h e m i c a l evidence of such an increase and h i s t o l o g i c a l evidence - 183 -of healing. In contrast, during this time period, there was a decrease i n the s u s c e p t i b i l i t y of the s i a l i c acids to digestion with V i b r i o cholera neur-aminidase which was r e f l e c t e d i n an increase i n the percentage of s i a l i c acids substituted at p o s i t i o n C^. This implies that healing i s accompanied by an increased resistance to neuraminidase at a time when s u b s t i t u t i o n at C 7 + Cg is not affected. Analyses of the glycoproteins obtained from the group of animals s a c r i f i c e d at 20 days, however, indicated an increased suscept-i b i l i t y to neuraminidase and a decrease in s u b s t i t u t i o n i n the side chain and at C^. These findings suggest that the loss of 0-acetyl group accompanies exacerbation of the disease as observed with h i s t o l o g i c a l procedures. Interpretation of the data obtained i n experiment #2 i s complicated by the design chosen i n which the groups of animals were selected on the basis of c l i n i c a l observations rather than being predetermined. Thus the animals s a c r i f i c e d at 48 hours were selected because they showed no evidence of occult blood at a time although some of the animals i n the remaining group did show occult blood. S i m i l a r l y on day 5 only the animals with occult blood were selected although some animals showed v i s i b l e blood. The la s t groups (17 days) consisted of those animals which had v i s i b l e blood but were c l i n i c a l l y healthy enough to withstand a prolongation of the treatment. Therefore i t is possible that the data obtained was influenced by a process which selected animals on the basis of their s u s c e p t i b i l i t y to the disease. Regardless of these problems, this experiment demonstrated that the disease process was accompanied by a reduction i n side chain s u b s t i t u t i o n and an increased s u s c e p t i b i l i t y to digestion with neuraminidase presumably as a r e s u l t i n the reduction of s u b s t i t u t i o n at p o s i t i o n C^. i n the f i n a l group of animals - 184 -Figure 33. Comparison of 0-acetyl substituted sialic acids in the 105,000 x g supernatants prepared from cecal epithelial cells of controls used in experiments 1, 2, 3 and 4. The data cited for the percentage of sialic acid substituted at C R / c 0 was obtained from the purified glycoproteins. 100 1 • EXPERIMENT 11 2 • EXPERIMENT 12 3 • EXPERIMENT #3 4 • EXPERIMENT #4 - 185 -there was apparently an increase in both side chain and s u b s t i t u t i o n . The s i g n i f i c a n c e of t h i s finding i s unknown because the number of animals i n the group was small (3 rabbits) and as discussed they could have been selected as being less susceptible. SUMMARY The results of t h i s i n v e s t i g a t i o n indicate that degraded carrageenan induced u l c e r a t i o n is accompanied by a reduction in the percentage of the s i a l i c acids of the e p i t h e l i a l glycoproteins with substituents i n the side chain and at p o s i t i o n C^. This change i s progressive and apparently precedes the mucosal u l c e r a t i o n or a s i g n i f i c a n t inflammatory response. On cessation of carrageenan feeding healing occurs accompanied by an increase in 0-acetyl s i a l i c acids, but following this phase exacerbation of the disease process was accompanied by a reduction in O-acetylated s i a l i c acids. The pathogenesis of carrageenan induced c o l i t i s is not f u l l y understood. Carrageenan laden macrophages have been shown to be a major inflammatory c e l l u l a r i n f i l t r a t e i n the lamina properia of carrageenan treated animals (239). Grasso e_t a_l. (252) showed that the pre-ulcerative changes consisted p r i n c i p a l l y of an accumulation of carrageenan laden macrophages i n the lamina propria. Abraham e_t a_l. (251), a f t e r detailed studies of the carrageenan induced c o l i t i s i n guinea pigs, claimed that the mucosal ulcerations were i n i t i a t e d by an uptake of carrageenan by the macrophages i n the lamina propria. This caused death of the macrophages r e s u l t i n g i n the release of the lysosomal enzymes and subsequent tissue damage and u l c e r a t i o n . Van Der Waaij et a l . (248) showed that i n guinea pigs given 2% degraded carrageenan along with trimethoprim with sulfamethoxazole to eliminate the anaerobic - 186 -gram-negative m i c r o f l o r a , granulomas occurred rather than u l c e r a t i o n . They concluded therefore that gram negative bacteria were necessary to produce mucosal u l c e r a t i o n i n guinea pigs treated with carrageenan. In an independent, systematic study Onderdonk e_t al_. (254) confirmed these findings and showed that the feeding of guinea pigs with carrageenan plus clindomycin or metro-nidazole f a i l e d to cause mucosal u l c e r a t i o n s . In addition i n guinea pigs with carrageenan induced caecal u l c e r a t i o n they showed that there was a s i g n i f i c a n t increase i n the gram negative b a c t e r i a l count accompanying the u l c e r a t i o n s . It i s i n t e r e s t i n g to note that clindomycin has been shown to cause pseudo-membranous l i k e c o l i t i s i n man, rabbits and hamsters (213-220). Grasso et a l . (252) however reported that feeding neomycin along with carrageenan to guinea pigs resulted i n a reduction i n the polymorphs population but f a i l e d to a f f e c t the mucosal u l c e r a t i o n s . The functions of the digestive t r a c t mucins are assumed to include l u b r i -cation and protection of the epithelium against damage from the various constituents of the f e c a l stream (1,274). The colonic mucins form a con-tinuous layer over the l i n i n g epithelium and are known to be degraded by the colonic f l o r a (7). Under normal conditions, i t has been suggested that there is a steady state between mucus production and mucus loss due to degradation and mechanical loss i n the f e c a l stream (275). Since s i a l i c acids bearing an 0-acetyl substituent at carbon 4 are known to be r e s i s t a n t to neuraminidase digestion (137), i t has been suggested that such s i a l i c acids might p a r t i a l l y protect the colonic glycoproteins against b a c t e r i a l degradation (30,44). Observations i n t h i s laboratory have shown that b a c t e r i a l free f i l t r a t e s from rat colonic contents and rat feces remove a high percentage of the s i a l i c acids of rat e p i t h e l i a l glycoproteins due, i t i s thought, to the presence of a - 187 -de-O-acetylase i n addition to a neuraminidase (unpublished data). However, the rate of removal of O-acetylated s i a l i c acids i s slower than that of s i a l i c acids without ester substituents. The findings i n t h i s work suggest that the glycoproteins produced i n carrageenan c o l i t i s are less r e s i s t a n t than normal to b a c t e r i a l neuraminidase and therefore more prone to b a c t e r i a l degradation. In these circumstances there could be an eventual f a i l u r e of the mucous b a r r i e r leading to a di r e c t exposure of the l i n i n g epithelium to the b a c t e r i a l f l o r e , b a c t e r i a l toxins or other hazardous agents i n the f e c a l stream. This could lead to tissue damage and i n i t i a t e an inflammatory response. U l c e r a t i o n could r e s u l t from the action of tissue (or macrophage) lysosomes and/or the di r e c t action of the b a c t e r i a l f l o r a . As a res u l t there could be an increase i n the b a c t e r i a l population and an aggravation of the condition. The reason for the a l t e r a t i o n i n e p i t h e l i a l glycoproteins i n carrageenan c o l i t i s i s unknown but there are a number of p o s s i b i l i t i e s discussed below; i ) Eastwood and T r i e r (114) have shown that, i n tissue culture, e p i t h e l i a l c e l l s from u l c e r a t i v e c o l i t i s patients have a faster turnover and migration rate than normal. MacDermott et a l . (115) i n organ culture studies showed that r e c t a l e p i t h e l i a l c e l l s from u l c e r a t i v e c o l i t i s patients had an increased glycoprotein synthesis and secretion. Similar observations have been made with e p i t h e l i a l c e l l s from patients with peptic ulcer (276,277). I f these phenomena occur i n carrageenan induced c o l i t i s they might lead to an increased mucus production and possibly the secretion of glycoproteins containing a smaller percentage of O-acetylated s i a l i c acids. L i t t l e i s known about the biosynthesis of O-acetylated s i a l i c acids i n the large i n t e s t i n e . Schauer (135), Schauer et a l . (136), Schauer and Wember (278), and C o r f i e l d et a l . (134) have demonstrated the presence of enzymes, 0-acetyl transferases, i n - 188 -bovine, equine and porcine submandibular gland responsible for the synthesis of 0-acetylated s i a l i c acids. Such enzymes have not been demonstrated i n rabbit large i n t e s t i n e but recent work i n our laboratory has demonstrated them i n rat colonic e p i t h e l i a l c e l l s (unpublished data). If dietary carrageenan modifies the a c t i v i t y of such an enzyme system the glycoprotein synthesised could be d e f i c i e n t i n 0-acetyl substitued s i a l i c acids, i i ) Smyth et^ al_. (279) have shown that carrageenan changes the physical properties of g a s t r i c mucin. Kim _et a l . (280) have demonstrated that the binding of amylopectin sulphate (a sulphated polysaccharide s i m i l a r to carrageenan) to g a s t r i c mucin i s pH dependent; about 35-40% being bound at pH 7.0. I f such binding occurs with large i n t e s t i n a l mucin i t might a l t e r the properties of the mucus and a l t e r the steady state to cause an increased mucus production. I f t h i s r e s u l t s i n an exhaustion of the mucous producing c e l l s , i t could r e s u l t either i n the f a i l u r e to produce any mucin or the production of an "incomplete" (lacking 0-acetyl s i a l i c acids) mucin. Depletion of mucus i s a common find i n g i n human u l c e r a t i v e c o l i t i s (10). - 189 -Proposed Future Investigation 1. To investigate the possible r o l e of the b a c t e r i a l f l o r a i n the process of u l c e r a t i o n (248,254) i t i s suggested that the carrageenan model of u l c e r a t i o n and any changes i n the 0-acetyl side chain s u b s t i t u t i o n of the s i a l i c acids of the e p i t h e l i a l glycoproteins be studied i n the presence and absence of the b a c t e r i a l f l o r a through the use of a n t i b i o t i c s administered with the carrageenan. 2. To investigate the presence and nature of 0-acetyl transferases i n the rabbit large i n t e s t i n e and the a l t e r a t i o n s ( i f any) i n association experimentally induced large bowel u l c e r a t i o n s . 3. To investigate the e f f e c t and the importance of the 0-acetyl substituted s i a l i c acids on the p r o t e o l y t i c digestion, and the rheology of the glycoproteins. 4. The carrageenan model of u l c e r a t i o n i s successful only i n rabbits and guinea pigs (245,252). Since there are differences i n the digestive tract and i n the feeding habits of man and rabbits and guinea pigs, i t would be useful to search for a procedure to induce large bowel u l c e r a t i o n i n an experimental animal with a closer s i m i l a r i t y to man such as the dogs, cats or r a t s . 5. In a l l the i n v e s t i g a t i o n of u l c e r a t i v e c o l i t i s , the chemical data were presented i n terms of a l t e r a t i o n s of the molar r a t i o of the component sugars or amino acids i n the glycoprotein. I f we assume that there are quantitative changes i n the amount of mucus produced p a r t i c u l a r l y i n the early stage with no a l t e r a t i o n s i n the molar r a t i o of the component sugars, such treatment of the data might f a i l to reveal a s i g n i f i c a n t f i n d i n g . I t i s suggested there-fore that experiment 1 be repeated and that the r e s u l t s be expressed i n terms of the quantity of the glycoproteins. This may require the development of a - 190 -radioimmunoassay. This should be possible since such an assay has been developed for human i n t e s t i n a l goblet c e l l mucin (69) and antibodies can be prepared against large i n t e s t i n e e p i t h e l i a l glycoproteins of other species (108). 6. Our study showed that carrageenan was apparently capable of inducing pathological a l t e r a t i o n s i n the upper digestive t r a c t i n rabbits contrary to the claim (244,253) that only the large bowel was affected. Short carrageenan treatment of patients with u l c e r a t i v e c o l i t i s f a i l e d to aggravate the disease (252). Since carrageenan i s widely used i n the food industry (222) such findingswould not exclude the p o s s i b i l i t y of long term pathological e f f e c t s of carrageenan. In t h i s respect i t i s suggested that the model be further investigated to re-evaluate the ef f e c t s of carrageenan upon the upper D g a s t r o i n t e s t i n a l t r a c t . - 1 9 1 -Append ix S i a l i c acids contents (ug/ml), percentages of 0 - a c e t y l s u b s t i t u t i o n at pos i t i o n C 7 / C 3 , CgCg and percentage of s i a l i c acids released by digestion with v i b r i o cholera neuraminidase both with and without saponification in bovine s a l i v a r y mucin (BSM) used as q u a l i t y c o n t r o l . S i a l i c acid % Cj/Cg % CgCtj % released % released yg/ml KOH-N'dase N'dase 99.3 + 3.9 61.4 + 3.9 56.1 + 4.3 83.7 + 9.2 48.5 + 3.4 - 192 -REFERENCES 1. Gottschalk, A. The glycoproteins. BBA Library, V ol. 5A, 5B, 2nd ed., E l s e v i e r (1972). 2. Terho, T. Studies on hexosamine containing macromolecules. Ann. Acad. S c i . Fennicae A l l , Chemica 175, 4-83 (1974). 3. Kent, P.W. 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