COLONIC EPITHELIAL CELL APOPTOSIS IN INFLAMMATORY BOWEL DISEASE

BY

Bradley M. Hann

A thesis submitted to the Department of Microbiology and Irnmunology in conformity with the requirements for

the degree of Master of Science

Queen's University Kingston, Ontario, Canada

May, 1999

copyright O Bradley M. Ham

National Library Bibiiot hèque nationale du Canada

Acquisitions and Acquisitions et Bibliographie Services seMces bibliographiques 395 Wellingtori Street 395, nie Weliigton OnamObi K 1 A W OrtawaON K 1 A W Canade canada

The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microforni, vendre des copies de cette thèse sous paper or electronic formats. la fonne de microfiche/film, de

reproduction sur papier ou sur format électronique.

The author retains ownership of the L'auteur conserve la propiété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts frorn it Ni la thèse ni des extraits substantiels may be p ~ t e d or othenvise de celle-ci ne doivent être imprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation.

ABSTRACT

Bradley M. Hano: Colonic Epithelial Cell Apoptosis in Inflammatory Bowel Disease

In this study apoptosis was studied in the colonic mucosa of patients with Crohn's

Disease (CD) and ulcerative colitis (UC). CD and UC are both relapsing and remitting

inflarnrnatory disorders of the gastrointestinal tract known as inflarnrnatory bowel disease

(IBD) which are identified and diagnosed according to clinical, endoscopic, and histologie

features. One of the typical features of chronic CD and UC is a reduction in the size of

the colonic epithelial regenerative compartments (crypts). The factors responsible for this

reduction are unknown. Since epithelial regeneration is an important response to injury.

impaired regecoration may contribute to disease activity and progression. It is

hypothesized that increased epithelial ce11 apoptosis is an important factor in the reduction

of crypt size described in colonic mucosa of patients with IBD. The present research was

carried out to define the role of apoptosis in the crypt epithelium in colonic mucosal

biopsies from patients with CD and UC. Biopsies were taken from both invoivrd and

uninvolved bowel regions. Biopsies taken from otherwise healthy patients with non-

inflammatory bowel disease (non-IBD) served as disease-related controls. Biopsies taken

fiom subjects undergoing colonic surveillance for cancer served as normal controls.

Apoptosis was detected in colonic mucosal biopsies from patients with IBD, non-

IBD, and normal controls using the following techniques: (a) Lieht Microscop~ of HPS

Stained Tissue to detect clusters of apoptotic bodies, morphological evidence of

apoptosis; (b) TUNEL Method to detect cells containing fragmented DNA, a biochernical

hallmark of apoptosis; (c) Electron Microscopy to locate ultrastructural features typical of

apoptotic cells; (d) Gel Electrophoresis of DNA extracted from tissue biopsies (a ladder

pattern is evidence of the presence of apoptotic cells containing fiagmented DNA); (e)

Immunohistochemistq of Apoptotic Related Proteins to locate the presence of Fas, p52.

CPP-32, Ich-IL, TIAR, Bcl-x, and BAD in mucosal biopsies.

Study results indicated that mucosal epithelial cell apoptosis detected wiih the

TUNEL technique was reduced in biopsies affected by active disease for both CD and UC.

In contrat. colonic crypt cell apoptosis was dramatically more frequent in normal and

non-IBD disease related controls, and in uninvolved IBD biopsies relative to the inflarned

state. Immunohistochemistry of apoptotic regulatory proteins (CPP-32, [ch-1 L, TIAR,

and BAD) confirmed the results obtained with the TUNEL staining pattern. These results

suggest that apoptosis plays a role as an intrinsic mechanism for normal homeostasis of

epithelial cells in healthy and IBD uninvolved intestinal mucosa, located mainly in the

crypts, and this regulatory process is inhibited in the crypts of inflamed mucosa of patients

with ulcerative colitis and Crohn's disease, perhaps in response to an increased demand for

surface epithelial ce11 repopulation.

ACKNOWLEDGMENT

First and foremost 1 would like to extend my deepest thanks and appreciation to

my supervisor Dr. Myron Szewcnik. His inexhaustible reserve of patience,

undentanding, and support facilitated completion of this very interesting and exciting

Master's thesis project.

Also, thanks to my CO-supervisors Dr. David Hurlbut and Dr. William Depew

without whom this work would not have been possible. The suggestions and advice that 1

received from Drs. Szewczuk, Hurlbut, and Depew made this educational experience most

fulfilling. Invariably, they were always willing to take time out of thcir busy schedules to

answer any questions that 1 may have had. For this, they deserve my deepest gratitude.

1 would also like to recognize two former project students of Dr. Szewczuk. Jane

Shearer researched many of the apoptosis regulatory proteins and some of her data is

included in this thesis. Similarly, a portion of the TUNEL measurements was contnbuted

by Catherine Irwin. To both of them 1 am grateful.

To the members of the Gastrointestinal Diseases Research Unit (GIDRU) at Hotel

Dieu Hospital 1 thank you for providing a learning environment that was second to none.

The Fnendly atmosphere of the Department of Microbiology and Immunology

made studying enjoyable and to al1 of you 1 offer my sincere thanks.

Finally, to my parents Anne and James Ham for their undying support of me and

my goals. Your belief and prayers gave me strength. 1 am truly grateful.

The financial support fiom the Department of Microbiology and Imrnunology, and

School of Graduate Studies and Research is duly acknowledged.

TABLE OF CONTENTS

ABSTRACT ...................................................................................................................... i ... ACKNOWLEDGMENT ............................................................................................... 111

TABLE OF CONTENTS ............................................................................................ iv LIST OF FIGURES .................................................................................................... vii LIST OF ABBREVIATIONS ............................................................... ix

INTRODUCTION .......................................................................................................... 1 lnflarnmatory Bowel Disease ................................................................................. 1

..................................... Clinicopathologic Characteristics of Crohn's Disease ... 2 ......................................... Clinicopathologic Characteristics of Ulcerative Colitis 2

.................................................... Pathogenesis of Inflammatory Bowel Disease 3 ............................................................................................................... Apoptosis 4

Apoptotic Related Proteins . Intracellular Mediators of the Cell Death Pathway ................................................................................ 13 (1 ) Bcl-2 Family of Proteins .................................................................... 13 (2) p53 ........................................................................*............................. 15 (3) Fas ...................................................................................................... 17 (4) CPP-32 and Ich-1 ............................................................................. 18

(a) CPP-32 ................................................................................... 19 (b) Ich- 1: ................................................................................... 21

37 ( 5 ) TIAR ................................................................................................... ,, Apoptosis and In flarnmatory Bowel Disease (IBD):

1s there a co~ect ion? ............................................................................ 28 ............................................................................ Cytokines, IBD, and Apoptosis 31

................................ Reactive Oxygen Metabolites (ROM), IBD, and Apoptosis 33 Conclusion ........................................................................................................... 37

MATEMALS AND METHODS ............................................................................. 39 Patient Selection .................................................................................................. 39 Histologic Assessrnent of IBD ............................................................................ 40 Procedures ............................................................................................................ 43

.................................... .................................... (1) Morphology Studies ... 43 ...................... (a) Hematoxylin/Phloxine/Saffion (HPS) Staining 43

(b) Electron Microscopy ........................................................... 44 ............................................................. (2) Studies on Fragmented DNA 45

.................................. (a) Gel Electrophoresis of Extracted DNA 45 .......................................................... (i) DNA Extraction 46

...................................... (ii) Agarose Gel Electrophoresis 46 (b) The TUNEL Method - In Situ Terminal

Deoxy nucleotidy 1 Transferase 3' ............................................ Hydroxy Nick End Labeling 47

(3) TUNEL Controls ................................................................................ 5 1 (a) Positive Control .................................................................. 51

.................................................................... (b) Negative Control 51 (c) Non-mucosal Tissue Controls ................................................ 52

.................................................... (4) Quantification of TUNEL Staining 52 ..................... (a) "Northem Exposw" Image Analysis Software 52

@)The Tissue Unit ..................................................................... 54 .......... (c) Procedure for Measurement of TUNEL Positive Cells 55

........................................... (d) Data Analysis-Statistical Method 57 (5) Immunohistochemical Analysis of Apoptotic Related

.......................................................... Proteins in Mucosal Tissue 57 (6) Immunohistochemistry Controls ....................................................... 59

...................................................................... (a) Positive Control 59 .................................................................... (b) Negative Control 60

RESULTS ...................................................................................................................... 61 Study Population .................................................................................................. 61

(1) Crohn's Disease .................................................................................. 61 ................................................................................ (2) Ulcerative Colitis 62

..................................................... (3) Non-lnflammatory Bowel Disease 64 (4) Normal .............................................................................................. 64

..................................................................................... Assessrnent of Apoptosis 65 (1) Observations of Hematoxylin/Phloxine/Saffron (HPS)

........................................................................... Stained Sections 65 (2) In Silu Detection of DNA Strand Breaks by the

........................................................................... TUNEL Method 67 (a) Controls .......................... .. .............................................. 67

..................... (i) Positive and Negative TUNEL Controls 67 ............................................................ (ii) Other Controls 67

..................... (b) TUNEL Analysis of IBD and Normal Controls 70 (i) TUNEL Analysis of Uninvolved and

Involved IBD Tissue ........................................... 70 CD ....................................................................... 70 UC ........................................................................ 73

.............. (ii) TUNEL Analysis of Normal Control Tissue 75 (c) TUNEL Analysis and Association

with Therapy States .......................................................... 75

(d) TUNEL Analysis and the Effect of ........................................................... Anatomic Location 78

CD ................................................................................ 78 UC .................................................................................... 81

............................................................................. Normal 81

LIST OF FIGURES

Fipre 1 . Fipre 2 . Figure 3 . Figure 4 .

Figure 5 . Figure 6 . Figure 7 . Figure 8 . Figure 9 . Figure 10 . Figure 11 . Figure 12 . Fipre 13 . Figure 14 . Figure 15 . Figure 16 . Figure 17 . Figure 18 . Figure 19 .

.......................................................... Overview of the Apoptosis Cascade 6

Diagram to Illustrate the Morphological Features of Apoptosis ................ 9

Histologie Assessrnent of IBD ............................................................. 42

Mechansim of TUNEL Analysis According to the Oncor Apoptag Detection Kit .............................................................. 49

Analysis of Hematoxylid Phloxinel Safion (HPS) Stained Tissue ........ 66

TUNEL Controls-1 ............................................................................... 68

TUNEL Controls-II ............................................................................. 69

TUNEL Analysis of Uninvolved CD Mucosa ........................................ 71

TUNEL Analysis of lnvolved CD Mucosa .............................................. 72

TUNEL Analysis of Uninvolved UC Mucosa ......................................... 74

TüNEL Analysis of Involved UC Mucosa .............................................. 76

TUNEL Analysis of Normal Mucosa .................................................... 77

TUNEL Analysis and Association with Therapy States .......................... 79

........................................ Anatomical Analysis of TUNEL in CD Bowel 80

Anatomical Analysis of TUNEL in UC Bowel ........................................ 82

.................... Anatomical Analysis of TUNEL in Normal Control Bowel 83

Anatomical Analysis of TUNEL of Non-IBD Bowel Specimens ............ 85

Correlation of Two Measurements: Manual vs . Cornputer Counts ......... 87

Percent TUNEL Positive Cells in the Involved and Uninvolved IBD Mucosa ............................................................................................. 89

vii

Fipre 20 . Figure 21 . Figure 22 . Figure 23 . Figure 24 . Fipre 25 . Figure 26 . Figure 27 . Figure 28 .

Electron Microscopy of UC Bowel Tissue ............................... ... . . . 9 1

DNA Gel Electrophoresis of Extracted DNA .......................................... 92

Expression and Localization of Fas Receptor .......................................... 94

Expression and Localization of pS3 ......................................................... 95

Expression and Localization of CPP-32 Protease (Caspase-3) ................ 97

.................................................... Expression and Localization of Ich-I L 98

Expression and Localization of TIAR Binding Protein ..................... .... 100

.................................................. Expression and Localization of B ~ 1 - x ~ 101

Expression and Localization of BAD .................................................... 103

viii

ADP AICD AIDS ALD Bad Bag BAF Bak Bax Bcl-2 B ~ 1 - x ~ BcI-x, bp C. eleguns c-myc Ca" CD CD4' CD36 Ced-3 Ced-4 Ced-9 CPP-32 CTL DAB DDW DISC DNA DNA-ESB dUTP EDTA EM FADD Fas

FasL FAST FLICE g GDP

adenosine diphosphate activation induced cell death acquired immunodeficiency syndrome autoimmwe lymphoproliferative disease bcl-2 homologous protein; promotes ce11 death by binding to bcl-x, bcl-2-associated athanogene bocasparty l (Orne)-fluoromethy lketone bcl-2 homologous antagonistkiller bcl-2-associated protein X b-ce11 leukemia\ lymphoma 2 bcl-2 related gene; larger mRNA splice; inhibitor of apoptosis bcl-2 related gene; shorter mRNA splice; promoter of apoptosis base pair Caenorhabditis elegans cellular proto-oncogene calcium ion crohn's disease cluster of differentiation 4 88 kD glycoprotein IV ce11 death defective 3 ce11 death defective 4 cell death defective 9 apoptosis related cysteine protease; caspase-3 cytotoxic T lymphocyte 3 ,Y-diaminobenzidine 4-HCI double distilled water death initiating signalling complex deoxyribonucleic acid DNA electrophoretic sampling buffer deoxyuridine triphosphate ethylene diamine tetra-acetic acid electron microscopy fas-associating protein with death domain tumor necrosis factor receptor family memberlcan transmit cell death signal fas ligand fas activated serine threonine kinase FADD like ICE gr- guanosine diphosphate

GI gld H,O H202 HIV HPS hTIA- 1 hTIAR 1-KB IBD ICAM ICE k h - 1 Ich- 1, Ich- 1, IFN-y IgG IL- 1 IL-1p IL-2 IL-6 IL-8 IL- 1 O IL-12 iNOS kDa kpb lpr lvi Mg" ml mM mg min mo mRNA mTIA- 1 mTIAR NAD' NADPH nedd-2 NGF NF-KB

gastrointestinal general ized 1 y mphoproli ferative disease water hydrogen peroxide human imrnunodeficiency virus hematoxy lin/ phloxinel samon human TIA-1 human TIAR inhibitor of NF-KB inflarnmatory bowel disease intracellular adhesion molecule- l interleukin I p converting enzyme ICE/ced3 homolog- 1 ICEIced3 homolog- 1 long ICE/crd3 homolog- 1 short interferon gamma immunoglobulin G interleukin 1 interleukin 1 beta interleukin 2 interleukin 6 interleukin 8 interleukin 10 interleukin 12 nitric oxide synthase kilodalton ki lobase pair lymphoproli feration molar magnesium ion millilitre millimolar mi lligrarn minute month messenger RNA murine TIA- 1 murine TIAR nicotinarnide adenine dinucleotide, oxidized nicotinarnide-adenine dinucleotide phosphate, reduced mouse gene homologous to hurnan Ich-1 nerve growth factor nuclear factor kappa B

NK rlm NO non-IBD NUC 18 02- Ocl- OH PZ 1 W"'C'P'

P53 PARP PBS PCD PK PMN RAIDD ras RNA ROM rPm RT TCR TdT TGF-P Th0 Th1 Th2 TIA- 1 TlAR TMB rnF TNF-a TNF-R TRPE TUNEL UC P 1 v VCAlM- 1 Zn2+

natural killer nanometre nitric oxide non in flammatory bowel disease calcium dependent endonuclease, 18 kDa superoxide radical hypochlorite ion hydroxyl radical 2 1 kDa protein of WAF 1 gene turnor suppressor protein poly (ADP) ribose polymerase phosphate buffered saline programmed cell death proteinase K pol y morphonuclear ceIl RIP-associated Ich- 1 Ked-3 homologous protein with a death domain oncogene protein regulating ceIl proliferation ribonucleic acid reactive oxygen metabolites revolutions per minute room temperature T cell receptor terminal deoxynucleotidyl transferase transforming growth factor beta T helper lymphocyte type O T helper lymphocyte type 1 T helper lymphocyte type 2 T-ceIl intracellular antigen- 1 ma binding protein; may be an effector of apoptotic cell death 3,3',5,5'-tetramethlybenzidine turnor necrosis factor turnor necrosis factor alpha turnor necrosis factor receptor monoclonal antibody terminal deoxynucleotidyl transferase 3' hydroxy nick end labelling ulcerative colitis microlitre volt vascular cell adhesion molecule- 1 zinc ion

INTRODUCTION

Inflammatory Bowel Disease

Inflammatoy bowel disease (IBD) is a general term used to encompass two

debilitating chronic inflarnmatory disorders of the gastrointestinal tract, narnely Crohn's

disease (CD) and ulcerative colitis (UC) as identified and diagnosed by the appearance of

characteristic sets of clinical, endoscopic, and pathologic features (Stenson, 1995). The

incidence and prevalence of Crohn's disease and ulcerative colitis Vary according to

geographic location, as well as arnong ethnic and racial groups within those geographic

areas (Stenson, 1 995).

One of the first documented descriptions of UC was made by Wilks and Moxon in

1859 (Kirsner, 1985). However distinguishing the disease as a distinct entity was

probably postponed due to difficulties in differentiating it from many of the infectious

dysenteries (Kirsner, 1985). In 19 13, nine patients with an illness now known as CD were

reportcd by Kennedy Dalziel (Dalziel, 19 13). In 1932, Crohn, Ginzburg, and

Oppenheimer, recorded 14 cases seen at Mount Sinai Hospital in New York (Crohn et al..

1932). UC was the predominant inflarnmatory bowel disease in the 1940's. but by the late

1970's and early 1980's CD was being reported more frequently than UC in western

patient populations (Kirsner, 1985). The eventual classification of CD as a clinical entity

separate from UC was established throughout the 1950's starting with Wells in 1952 and

finishing with Morson and Lockhart-Mumrnery in 1959 (Farmer, 1977).

Clinico pathologie C harocteristics of Cro hn's Disease

Crohn's disease is a relapsing and remitting inflarnmatory disorder which can affect

any portion of the gastrointestinal tract fiom the mouth to the anus (Stenson, 1995).

Histologically, the inflammatory ce11 infiltrate consisting of lymphocytes, with plasma cells,

polymorphonuclear leukocytes, and eosinophils (in smaller numbers) can extend

transmurally from the luminal aspect of the mucosa through the subrnucosa and muscularis

propria to the serosal surface (Stenson. 1995). Granulomas composed of lymphocytes

and epithelioid histiocytes can be found in the mucosa or submucosa in 60% of patients

(Kirsner and Shorter, 1982). Macroscopically , there is thickening of al1 layers of the bowel

wall with subsequent narrowing of the intestinal lumen. It is a segmenta1 disease with

intlamed areas of the colon and small bowel interrupted by apparently normal mucosa.

The inflamed segments of the GI tract rnay display deep linear and transverse ulcers with

intervening edematous mucosa resulting in a "cobblestone" appearance (Stenson, 1995).

The predominant symptoms of CD include diarrhea, abdominal pain, and weight loss.

Anal and perianai lesions includicg pendulous skin tags, abscesses, and fistular are

characteristic of this disorder (Stenson, 1995). Th2 c!inical presentation and prognosis

depend on the site and extent of bowel involved.

Clinicopathologic Cbaracteristics of Ulcerative Colitis

Ulcerative colitis is also a relapsing and remitting disease which is confined

exclusively to the colon (Stenson, 1995). Histologically, the inflarnmatory ce11 infiltrate

can occupy the mucosa and adjacent submucosa. Active disease is characterized by an

intense neutrophilic infiltration with crypt abscesses, cellular mucin depletion, and rnucosal

edema. Crypt (epithelial regenerative cornpartment) shortening and branching is typical of

UC (Stenson, 1995). Chronic disease is characterized by the presence of lymphoid

aggregates, plasma cells, mast cells, and eosinophils in the lamina propria (Stenson, 1995).

Colonoscopy has s h o w that the rectum of patients with active UC is always inflamed

with visible inflammation then extending proximally and continuously throughout the

bowel to a point at which the mucosal pathology dissipates and the appearance becomes

normal (Stenson, 1995). The predominant symptom of UC is diarrhea which is often

bloody. Other symptoms such as weight loss, malaise, fever, and tachycardia may be

present if al1 or most of the colon is involved (Stenson, 1995). According to Truelove and

Witts, the severity of UC cm be classified as mild, moderately severe, and severe based

upon clinical criteria (Truelove and Witts, 1955). Utilizing empirical clinical correlations.

Edwards and Truelove discovered that 54% of initial attacks were of mild severity, 27%

were moderately severe, and 19% were diagnosed as having severe disease (Edwards and

Truelove, 1963). The prognosis of an initial attacck of ulcerativc colitis cm be predicted

based upon the extent of the disease and the severity of symptorns (Stenson, 1995).

Pnthogeoesis of Inflammatory Bowel Disease

The onset of inflarnmatory bowel disease is believed to be due to an exogenous

sensitization to luminal antigens (dietary or bacterial) promoted by unknown genetic

factors. This sensitization process leads to an abnormally active immune response in the

mucosa of patients with CD and UC. T cells bind these luminal antigens that are

presented on macrophages, become activated, and secrete IL-2. This results in clona1

expansion of cytotoxic and helper T cells. B cells subsequently synthesize and secrete

increased quantities of antibody. Monokines and T ce11 lymphokines fùrther activate

neutrophils and macrophages. Epithelial ce11 damage and loss within the rnucosa is

characteristic of patients witb inflammatory bowel disease. The factors responsible for this

damage are unknown, but rnay result fiom the combined effects of cytotoxic T cells,

activated macrophages, and proteases and free radicals that are released from activated

neutrophils. The results of such epithelial ce11 loss allows more antigen exposure and

inflammation and may be a kry step in the perpetuation of IBD. A focus on how epithelial

cells die during the progression of IBD should provide insight into the impact that

components of the inflammatory milieu have on intestinal epithelium. Apoptosis rnay be

activated by inflarnmatory celis and mediators. An understanding of the role of apoptosis

in the loss of epithelial ceils during active IBD would provide insight into factors

responsible for architectural distortions within the mucosa including superficial ulceration

and the shortrning and branching of crypis (opitl~rlial rrgemrative conipartments of

intestinal mucosa).

Apoptosis

Apoptosis was coined in 1972 by Kerr et al. (Kerr et al., 1972) to describe a

particular form of programmed cell death. It is derived fiom the Greek 'apo ', meaning

'off and 'ptosis ' meaning ' falling ' and is used to describe the "dropping off 'or "falling

off' of petals from flowers, or leaves from trees (Que and Gores, 1996). Although the

relative importance of apoptosis as a mechanism in ce11 biology has only been realized in

the past decade. the study of cell death has been in progress for well over one hundred

years. More than one hundred publications from the nineteenth century deal with naturally

occurring cell death beginning soon after the establishment of the ce11 theory by Schleiden

and Schwann (Kerr et al., 1972). A distinction must be made at this point between

'programmed cell death' and 'apoptosis' because of the common yet misguided

interchangeability of these terms. According to Jacobson et al. programmed ceIl death

(PCD) now generally refers to any ceIl death that is mediated by the intracellular death

prograrn, no matter what triggers it and whether or not it displays al1 of the characteristic

features of apoptosis (Jacobson et al., 1997). The intracellular death program is a

cascade of interdependent enzyme-substrate interactions controlled by a set of genes

which the ce11 can activate to cause its own dernise (Figure 1). The overall apoptosis

mechanism involves an interaction between factors signaling for cell death and regulatory

proteins controlling the susceptibility of the ce11 to apoptosis (Wyllie, 1997). Apoptosis

susceptible celis then proceed through typical structural changes mediated by the cascade

of proteases culminating in ceIl degradation known as the terminal effector events (Wyllie.

1997). Apoptosis can be triggered by both physiological stimuli or injury to various parts

of the ceIl (Wy llie, 1997). Furthemore, both transcriptional and non-transcriptional

apoptotic pathways exist.

Physiologically, cytokine receptors such as the tumour necrosis factor receptor

(TNFR) and Fas can bind tumour necrosis factor (TNF) and Fas ligand (FasL),

respectively. These receptors then recruit a series of proteins known as the death initiating

INJURY 1 1 PHYSIOLOCICAL STIhlULl

TSF f4

mitochondria

B

Figure 1. Scheme of cellular events in apoptosis (Taken €rom Wyllie AH., 1997)

signaling complex (DISC) to their cytoplasmic domains and through a non-transcriptional

pathway can activate the cascade of proteases which leads to the typical structural

apoptotic changes and death of the ce11 (Nagata, 1997) (Figure 1). The R I F signaling

pathway also involves nuclear factor kappa B (NF-KB) activation which c m initiate

transcription of survival factors (Nagata, 1 997). Other transcriptional pathway s have been

implicated in positive or negative regulation of apoptosis by proteins including ras, rho. c-

myc, and bax (Wyllie. 1997) (Figure l). Collectively, these proteins al1 contribute to the

susceptibility of a cell to apoptosis.

Cells that have incurred injury such as DNA damage (Evan et al., 1995), loss of

plasma membrane integrity (Wyllie, 1997). mitochondrial alterations (Wallach et al..

1997), or injury as a result of cytotoxic T lymphocyte granzyme B assault (Qum et al..

1996) undergo apoptosis via the sarne transcriptional and non-transcriptional pathways

utilized under normal physiological conditions (Figure 1).

Although a large variety of potential death triggering stimuli exist, the pathways

iniriated by these stimuli converge to a few or aven a single final pathway that directs a ce11

to its demise (Wallach et al., 1997). This final pathway is in part regulated by Bcl-2, an

anti-apoptotic protein that can block ce11 death in response to multiple, various stimuli

(Karsan et al., 1996; Armstrong et al., 1996; Borner, 1996) (Figure 1). Bcl-2 defines a

farnily of structurally related proteins that have both antiapoptotic and pro-apoptotic

capacities.

An uninhibited apoptotic stimulus results in the activation of a cascade o f

proteases that coordinates the structural alterations associated with apoptotic ceIl death.

The cascade is an irreversible step that drives the final stages of the ce11 death process and

completes the apoptotic cycle. The above description provides only a brief outline of the

steps involved in apoptosis and a much more detailed analysis of apoptosis and the

proteins coordinating this process are provided below.

Apoptosis was originally characterized by Kerr et al. on morphological grounds

which presently remains the gold standard for ce11 death identification. According to Kerr,

the structural changes occurred in two distinct steps. First, there was nuclear (involving

DNA degradation) and cytoplasmic condensation followed by budding of the ceIl into

apoptotic bodies which may or may not contain nuclear material. Secondly, the apoptotic

bodies were either sloughed off fiom epithelial linings or more likely were phagocytosed

by neighboring cells. There was a breakdown of the apoptotic bodies resembling autolysis

within the phagosomes previous to lysosome fusion and digestion into electron dense

bodies (Kerr et al.. 1972; Searle et al., 1982) (Figure 2). The phagocytosis of apoptotic

bodies occurs rapidly, within a few hours of body formation and without the release of

cellular contents into the surroundiny environment. Uiilikz necrosis. apoptotic col1 dsath

does not incite an inflammatory response (Searle et al., 1982; Que and Gores, 1996).

Apoptosis also has distinct biochemical features. One of the biochemical hallmarks

of apoptosis is the cleavage of DNA into 300 andor 50 kbp segments followed by further

intemucleosomal DNA fragmentation into traditional 180-200 bp multiples of DNA (Que

and Gores, 1996; Martin et al., 1994; Hale et al., 1 996).

Figure 2. Diagram to illustrate the morpholoyical features of apoptosis (Taken from Kerr et ai., 1973)

In 25 years. the rnorphological and biochemical distinctions of apoptosis have

remained constant. New studies however have contributed a greater understanding of the

molecular biology underlying the structural changes observed during apoptosis. Firstly,

the mechanisms controlling nuclear envelope breakdown and chromatin condensation are

largely unknown but may be a result of proteolysis of lamin B. Larnin B binds to specific

DNA sequences which mediate the at tachent of chromatin to the nuclear matrix and

envelope. It has been proposed that the destruction of lamin B would result in the

formation of large fragments of DNA thereby providing access to endogenous nucleases

responsible for DNA fragmentation. It is now accepted that the nuclease active during

apoptosis is Ca2'- and Mg2*- dependent and is inhibited by Zn". In 1993, Peitsch et al.

isolated a nuclease from rat thymocyte and lymph node with Ca2'- and ~g"-dependence

(Peitsch et al., 1993). Transfection experirnents and further immunohistochemical staining

contirmed the isolated nuclease as DNase 1 (Peitsch et al.. 1 993). At the same time, a

nuclease dependent on acidic conditions was purified from Chinese hamster ovary which

could mediate DNA degradation identical to that observed during apoptosis when added

to isolated nuclei. Barry et al. concluded that this nuclease was DNase II (Barry and

Eastman, 1993). A third potential nuclease was purified and characterized by Gaido and

Cidlowski (Gaido and Cidlowski, 1991). Apoptosis can be stimulated in rat thymocytes

by the application of glucocorticoids, and by using this system along with a modified

nuclease assay using [ P 3 ' ] ~ N ~ as substrate, a ca2'-dependent, 1 8kDa nuclease labeled

NUC 18 was isolated (Gaido and Cidlowski, 199 1). Presently, Dnase 1, Dnase II, and

NUC 18 remain potential effector candidates for the DNA degradation observed during

apoptosis, however further research will determine the exact role, if any, that the above

nucleases play, or whether the DNA fragmentation is due to an as yet unidentified

nuclease. Secondly, and characteristically seen during the final stages of apoptosis is the

formation of apoptotic bodies. In order for this to occur, there must be a dismption of the

microfilament network. In their review, Hale et al. state that rnicrotubule-disrupting

agents such as colchicine, vinblastine, and nocodazole al1 induce apoptosis. suggesting that

disruption of the microtubule network initiates events which lead to apoptosis (Hale et al..

1996).

Tissue transglutaminases are cytoplasmic proteins which depend on calcium to

catalyze acyl transfer reactions resulting in the assembly of highly cross-linked protein

scaffolds. These enzymes have been implicated in the cytoplasmic changes which take

place during apoptosis. With respect to apoptosis. the construction of the protein

scaffolds prevents the leakage of the intracellular contents from the dying cells and any

subsequent inflammatory reaction (Cummings, 1996; Hale et al., 1996). In three epithelial

models of apoptosis. including castration-induced prostatic atrophy, mild ischaemia in the

liver, and hydronephrosis due to ureteric ligation, tissue transglutaminase protein was

consistently expressed with the attendant ce11 death (Cummings, 1996).

Inflammation is also avoided by the swift phagocytosis of the dying cells and

apoptotic bodies. In tissues, the phagocytic cells are not always "professional" in nature

(i.e. macrophages). Resident tissue cells such as epithelial cells surrounding an apoptotic

event can also perform the phagocytic function if required. Ce11 membrane alterations

which precede the actual engulhent of apoptotic bodies allow recognition of the dying

ce11 by macrophages and other phagocytic cells. In one such mechanism, anionic

phosphatidyl serine which is nonally located on the imer plasma membrane is

translocated to the outer plasma membrane eliciting the phagocytic response (Hale et al..

1996). The a$, vitronectin receptor, the 88 kD glycoprotein IV (CD36), and

thrombospondin are mammalian phagocytic ce11 membrane components vital to the

recognition and engulfment of apoptotic entities (Hale et al., 1996). Savill et al. were

able to demonstrate that acquisition of the vitronectin receptor by monocyte-derived

macrophages "anned" these phagocytic cells with the ability to recognize apoptotic

neutrophils, human lymphocytes, and eosinophils (Savill, 1997). Recognition is followed

by ingestion which is negotiated by thrombospondin, a glycoprotein secreted by

macrophages. Thrombospondin binds the vitronectin receptor to an as yet unidentified

moiety on the apoptotic ce11 (Hale et al., 1996). The exact role of CD36 is uncertain, but

is believed to provide enhanced phagocytic function by cooperating with a$, to bind

thrornbospondin (Hale et al., 1996). Evidence also exists supporting a role for other

receptors in the identification of apoptotic bodies and cells, but further study is required in

order to clarifj the macromolecular environment of this end stage of apoptosis.

Studies in C. elegans have identified seven genes that are involved in the

engulfment of dying cells. Perhaps other phagocytic pathways are present in mammalian

cells and these may act in parallel or simultaneously. The nematode C. elegans mode1 will

undoubtedly provide fùrther insight into questions surrounding this and other stages of

apoptosis.

Apoptotic Related Proteins - Intracel1ular Mediators of the Ce11 Death Pathway

Current research in apoptosis has focused on elucidating the molecular biology of

the signaling, activation, execution, and regulatory components of the programmed ce11

death pathways controlling the demise of affected cells. To catalogue in detail the array of

molecules confirmed or postulated to play roles in apoptotic ce11 death is beyond the scope

of this review; however pertinent to the present study are the following apoptotic related

proteins: (1) Bcl-2, and family members B~1-x~. BAD (2) p53, (3) Fas, (4) ICE proteases

CPP-32 and Ich- 1. and (5) TIAR.

(1) Bcl-2 Family of Proteins

The Bcl-2 family of proteins are well established mediators of apoptosis. The

farnily contains members with both anti-apoptotic and pro-apoptotic activity. Bcl-2, the

defining member of this class, was discovered as a result of a chromosomal translocation

(t[14: 181) which led to inappropriate expression of the bcl-2 gene in neoplastic 6-cells

characteristic of follicular non-Hodgkin's B-ce11 lymphoma (Kemohan and Cox, 1996).

Bcl-2 is able to block apoptosis (Karsan et al., 1996; Armstrong et al., 1996; Borner.

1996) in a number of systems in response to a variety of stimuli (Figure 1). For example,

bcl-2 and bcl-x, (anti apoptotic) expression in erythroid progenitors blocked cell death in

responsc to erythropoietin withdrawal (Silva et al., 1996). Similady, an in vitro mode1 of

apoptotic ce11 death due to hypoxia was inhibited by the overexpression of bcl-2 and bcl-

x, (Shimizu et al., 1996). Cytokines, can control the persistence or elimination of

activated T cells by inducing bel-2 and bcl-x, expression (Akbar et al., 1996). B ce11

lineage development is also dependent on bel-2 expression as the pro-B ce11 line C 1.92

synthesized Bcl-2 in the presence of the stromal ce11 line S 10. Removal of stromal ce11

support generated an upregulation of bax (pro-apoptotic) expression. correlating direct1 y

with initiation of apoptosis (Gibson et al., 1996). Many other lines of evidence implicate

Bcl-2 and its family mernbers in other biological environrnents including cancer (Frankfurt

et al., 1996; Baretton et al.. 1996). Regulation of the bel-2 gene family is an influential

factor in the intracellular decision between survival and apoptosis following activation of

the ce11 death pathways controlled by the bcl-2 family of genes. Constituents of the Bcl-2

family of proteins including anti-apoptotic Bcl-2 and B ~ 1 - x ~ and pro-apoptotic Bax, BAD.

Bak, Bag, and Bcl-x, c m form both homodimers and heterodimers thereby rnodulating the

action of the monomers and providing a mechanism controlling opposing apoptotic forces

(Kemohan and Cox, 1996). For example, displacement of Bax fiom B ~ 1 - x ~ by BAD

promotes apoptosis (Kemohan and Cox, 1996). Furthemore, varying the expression of

bcl-2 and its homologous proteins defines a second mode of cellular regulation of Bcl-2

activity (Kernohan and Cox, 1996). The tumor suppressor protein p53 (see below) is

upregulated during DNA damage and induces apoptosis if the darnage is irreparable. p53

can downregulate Bcl-2 protein levels while simultaneously transactivating the bax gene.

By shifting the balance in favor of pro-apoptotic activities. p53 can drive a ce11 to

apoptosis thereby eliminating the potential of passing on mutated or darnaged DNA to

friture generations of cells. In general, the ability of Bcl-2 to inhibit apoptosis is dependent

on the repulation of expression of the bcl-2 gene, the regulation of expression of other

members of the bcl-2 gene family, and finally the species of the dimers then formed

(Kemohan and Cox, 1996). The mechanisms by which Bcl-2 exerts its anti-apoptotic

activity are unclear at this point, but research has provided some interesting possibilities

including inhibition of ceramide-induced poly-ADP ribose polymerase (PARP) cleavage

(Smyth et al., 1 W6), prevention of mitochondrial membrane permeabi lity transitions

(Zarnzarni et al., 1996; Shimizu et al.. 1996), an antioxidant function in maintaining a

cellular redox balance in a reducing state (Hedley and McCulloch. 1996), and

maintenance of intracytoplasmic and intranuclear Ca2+ levels (Marin et al., 1996).

(2) ~ 5 3

p53 is a tumor suppressor gene whose wild type expression yields a 393-amino

acid, 53-kDa nuclear phosphoprotein (Krishna et al.. 1995). This nuclear phosphoprotein

causes G , ceIl cycle arrest in response to DNA darnage (Martin et al., 1994). If the DNA

is irreparable, p53 will direct the cell into an apoptotic pathway to ensure that damaged

DNA is not replicated (Evan et al., 1995). Cell type, the nature and severity of the insult.

cytokine status, and arnbient milieu are al1 factors which determine whether a cell's fate

will be growth arrest or death by apoptosis. p53 acts as a transcription factor (Hale et al..

1996; Martin et al., 1994; Evan et al., 1995) by binding to DNA which c m modulate

target genes to coordinate ce11 cycle arrest. One such target gene, p21wufl "lp' i s

upregulated following accumulation of p53, and potently inhibits cyclin-dependent kinases

resulting in growth cessation at the G,/S border (Hale et al., 1996; Evan et al., 1995).

The rnechanism utilized by p53 to regulate apoptosis is less clearly understood, but may

involve transactivation of the bax gene (Evan et al., 1995). pS3 is also the gene most

fiequently dysfunctional in human cancer attesting to the vital role that p53 plays in

orchestrating cellular responses to DNA darnage. Clarke et al. were able to demonstrate

an increased mutation fiequency at the Dlb-l locus within intestinal epithelial cells of mice

with partiaily or totally defective p53 -mediated apoptotic responses (Clarke et al., 1 997).

Unrepaired and unchecked DNA darnage is fertile ground for cellular transformation. For

example, 15 of 40 DNA samples extracted from laryngeal squamous ce11 cancer tissue

were discovered to have mutations in the p.53 gene (Golusinski et al., 1997). Similarly,

mutated p53 has been correlated with heightened radiation resistance and tumor survival

in squamous ceIl carcinoma of the head and neck (Chang et al., 1997). p53-dependent

pathways have also been implicated in neuronal ce11 loss associated with Alzheimer's

(Kitamura et al., 1 997) and Parkinson's (Blum et al ., 1 997) diseases, during B-ce1 l

maturation in the bone marrow (Shick et al., 1997). and also in apoptosis of T-cells that

have incurred pathological DNA damage (Malcomson et al., 1997). Pertinent to the

present study are irnmunohistochemical experiments conducted by Krishna et al.

documenting an increased expression ofp53 in the nuclei of ciypt epithelial cells in the

acutely inflarned intestinal mucosa of patients with UC and CD (Krishna et al., 1995).

The association between inflammation. ce11 stress, and p53 is not well established and

fùrther study is necessary to clarify the impact inflammation has on affected tissues and

cells and what, if any, is the involvement of p53 in the mucosal destruction and epithelial

ce11 changes associated with infiammatory bowel disease.

(3) Fas

Fas is a member of the tumor necrosis factor receptor family of surface proteins

which when cross-linked either through binding of its substrate Fas ligand (FasL) or anti-

Fas monoclonal antibodies transduces a death signal to the interior of the ce11 (Nagata and

Suda, 1995). Fas is located on a variety of ce11 types throughout the body whereas FasL,

a protein of the tumor necrosis factor family, is associated particularly with cells of known

cytolytic and killer fùnction along with other specialized cells such as B lymphocytes

(Hahne et al., 1996). The Fas/FasL systern has been implicated in a number of

physiological and pathological processes ranging fiom T-ce11 development and

irnmunoregulation to cancer, AIDS and autoimmunity. Lymphoproliferation (Ipr) and

generalized lymphoproliferative disease (gld) are two mouse strains with similar

phenotypes characterized by lymphadenopathy and splenomegaly. The mutations in the

Ipr and gld mice were localized to genes encoding Fas and FasL, respectively (Nagata

and Suda, 1995). The cells which accumulated in these mice were of T-ceil lineage and

led scientists to suspect that the FasIFasL control of apoptosis occurred during T ce11

maturation. Specifically, it was discovered that FadFasL system mediated clonal deletion

of autoreactive T-cells in the periphery (Nagata and Suda, 1995). Persistence of

autoreactive T-cells can lead to autoimmune diseases as depicted by Dianzani et al.

(Dianzani et al., 1997). Six of seven patients with autoirnmune/lymphoproliferative

disease (ALD) were relatively resistant to PCD induced by monoclonal antibodies to Fas.

The defect was not related to the expression of Fas, but to sites downstream of ceramide

(intracellular messenger of apoptosis) action (Dianzani et al., 1997). The FaslFasL

system also controls the activation induced ceIl death (AICD) of T cells (Wang et al.,

1997; Varadhachary et al., 1997). A study of T ce11 clones by Varadhachary et al., found

Th1 type T cells sensitive to AICD while AlCD resistant Th2 and Th0 clones were a result

of signals generated from ligation of the CD3lTCR complex (Varadhachary et al., 1997).

Cytotoxic T lymphocytes (CTLs) also utilize the FadFasL system to initiate ce11 death in a

variety of turnor targets (Komada et al.. 1397). Increased neutrophil survival, necessary

during an inflammatory response. has been s h o w by Watson et al. to be a result of

blockage of the FadFasL signaling pathway by increased iniracellular glutathione in

response to costirnulatory signals delivered through P2 integrins or activation by

lipopolysaccharide (Watson et al., 1997). Pathologically, decreased T ceIl counts dunng

the progression of HIV/AIDS (Di Marzio et al., 1997), the apoptosis associated with

progressing and regressing tumors (Meterissian et al., 1997; Wang et al., 1997) and

defects leading to autoimmunity (Ito et ai., 1997; Dianzani et al., 1997) are disease States

in which alterations to the FadFasL signaling pathway contributes in whole or in part to

the pathogenesis. The Fas/FasL system provides a target for therapeutic intervention in a

number of disorders.

(4) CPP-32 and Ich-1

CPP-32 and Ich-1 are two members of the interleukin 1 -P converting enzyme

(ICE) family of cysteine proteases. It is generally considered that this family of proteases

and CPP-32 in particular play a major role in the effector ann of the apoptotic pathway.

ICE is the structural standard for the family and was first discovered in the cytosol of

monocytes and monocyte-like ce11 lines (Howard et al., 199 1 ). ICE has significant

sequence similarity to Ced-3, the pro-apoptotic protein necessary for al1 ce11 death in C.

elegans (Hale et al., 1996). As a result, ICE or an ICE-like protease is hypothesized to

control apoptotic pathways in vertebrates. ICE cleaves the cytokine interleukin 1-P (IL-

1 p) from its inactive 3 1 -kDa Tom to its active 17.5-kDa form. Activated IL- I P can

rnediate a variety of both physiological phenornena including inflammation, septic shock,

wound healing, hematopoiesis and pathologicai processes such as the growth of certain

leukemias (Hale et al., 1996). However. IL-1 p does not seem to be involved in any way

in the apoptotic process; therefore an as yet undiscovered substrate of ICE probably exists

which controls apoptosis in certain circumstances. Such circumstances include Fas- and

tumor necrosis factor receptor (RJF-R)-mediated apoptosis. For example. apoptosis of

normal thymocytes via Fas stimulation with anti-Fas antibodies can be blocked by a

tetrapeptide ICE inhibitor (Hale et al.. 1996). Since the discovery of ICE, a farnily of

proteins with sequence similarity to ICE has been identified.

(a) CPP-32

CPP-32 is described by Nicholson et al. as a precursor which is cleaved to fom an

active enzyme called apopain. Apopain is composed of two subunits of relative molecular

mass 17K and 12K and is responsible for cleavage of poly-(ADP-ribose) polymerase

(PARP), and is also considered to be necessary for the majonty, if not al1 foms of

apoptotic ce11 death (Nicholson et al., 1995). PARP is an enzyme which transfers ADP-

ribose from NAD' to nuclear proteins following DNA damage. Recent data suggests that

CPP-32 may effect the apoptotic process by inactivating DNA repair enzymes in general.

McConnell et al., using in vivo Fas-mediated apoptosis and in vitro cell-free systems were

able to demonstrate that DNA-PKc, a serine-threonine kinase that repairs double stranded

DNA breaks (present during fragmentation of DNA during apoptosis) is cleaved by CPP-

32 (McConnell et al.. 1997). The 70 kDa protein component of the U 1 -ribonucleoprotein

(involved in DNA repair) is also inactivated by CPP-32 (Casciola-Rosen et al., 1996).

Other substrates of CPP-32 consist of actin (Mashima et al., 1997) and D4-GDI, the

hematopoietic ce11 GDP dissociation inhibitor for the Ras-related Rho family of GTPases

(Na et al., 1996). CPP-32 has been documented as playing an active role in many

diseases. A decrease in C PP-32 activi ty and Ca2'-dependent endonucleases has been

linked to increased metastatic potential of both murine and human cancer ce11 lines

(Glinsky et al.. 1997). Goldberg et al. demonstrated that CPP-32 was able to cleave the

protein huntingtin, a product of the Huntington's disease gene, which has led to

speculation that inappropriate apoptosis may explain the underlying pathogenic mechanism

of this disorder (Goldberg et al., 1996). Cytotoxic T lymphocytes (CTLs), those cells of

the immune system that specialize in ridding the body of viral infected and tumor cells

secrete granzyme B, a senne protease and effector of apoptotic ceIl death in susceptible

targets. Not surprisingly, granzyme B can activate CPP-32 leading to PARP cleavage

( Q u m et al., 1996). CPP-32 is a member of the ICE family of cysteine proteases that acts

as a major effector of apoptosis in response to a variety of stimuli. Because CPP-32 is

required for practically al1 foms of cellular suicide, this protease provides a key focus for

further scientific study which will provide insight into both the basic mechanism of

apoptosis and its clinical implications.

(b) Ich-1:

Ich-l is the human homologue of a murine gene nedd-2 which was first recognized

as a genetic element highly expressed during the embryonic developrnent of mouse brain.

but subsequently down-regulated in the adult murine brain. Overexpression of nedd-2 in

mouse fibroblast and neuroblastoma cell lines induced apoptosis (Hale et al.. 1996).

Alternative splicing of Ich-1 mRNA produces two protein products with opposing

apoptotic effector functions. Ich- 1 L is a 435-arnino-acid protein with a pro-apoptotic

capacity, where as Ich-1 S, is a 3 12-amino-acid protein with anti-apoptotic ability (Hale et

al., 1996). Initial experiments have documented a role for Ich-1 L during neuronal

apoptosis. Deshmukh et al. have suggested that Ich- 1 L is activated during neuronal

apoptosis (Deshmukh et al.. 1996). This is based on bocaspartyl (Orne)-

fluoromethylketone (BAF) inhibition of rat sympathetic neuron apoptosis in response to

nerve growth factor withdrawal. Cleavage of PARP, pro-ICE, and Ich-1 was shown to be

inhibitable by BAF (Deshmukh et al.. 1996). Alpha-spectrin, a non-erythroid cytoskeletal

protein is broken down in neuronal cells undergoing apoptosis. Ich- 1 L has shown an in

vitro ability to produce alpha-spectrin breakdown products sirnilar to those produced by

calpain dunng neuronal ce11 necrosis (Nath et al., 1996). According to Harvey et al., Ich-

1 activation occurs early following an apoptotic stimuli (Harvey et al., 1997), similar to

the early upregulation of Ich-l L in apoptotic THP. 1 cells observed by MacFarlane's group

(MacFarlane et al., 1997). It seems therefore, that Ich- 1 L is an upstrearn protease which

may be involved in the early activation of the cascade of proteases leading to CPP-32

mobilization and PARP cleavage. In support of this, is the description of an adaptor

rnolecule RAIDD that can bind via protein-protein interactions to Ich-1, thereby linking

the surface signaling molecules to the effector ami (proteases) of the ce11 death pathway

(Duan and Dixit, 1997). Additional research is needed to unravel the sequencing order of

this protease cascade which ultimately leads to the irreversible cornmitment of the ceIl to

death.

(5) TIAR

TIAR is a member of a subset of RNA binding proteins which also includes TIA-1 .

RNA binding proteins (Mondino and Jenkins, 1999, and in particular TIAR and TIA- 1

have been implicated in the mechanism of apoptotic cell death (Chang. 1995; Tian et al..

1995; Dember et al., 1996), however their mode of action is completely unknown.

Scientists have observed that application of TIAR and TIA- I to permeabilized thymocytes

has resulted in DNA fragmentation and apoptosis, respectively (Lowin et al.. 1996;

Taupin et al., 1 995). A study by Beck et al., focused on characterizing TIAR and TIA- 1,

concluded that murine TIAR (mTIAR) and murine TIA- 1 (mTIA-1) protein were 80%

similar to each other, and 99 and 96% similar to hTIAR and hTIA- 1, respectively (Beck

et al., 1996). mTIAR and mTIA- 1 were localized predominantly to brain, testis and

spleen. A complementary study by Lowin et al. determined that during murine

embryogenesis, abundant TIA-1 mRNA was detected in the neuronal cells of the brain and

retina. TL44 transcripts were also discovered in the lung, kidney, and thymus. Adult

mice expressed TM-1 mainly in T cells and NK cells (Lowin et al., 1996). Specifically.

TIA-1 is a component of cytotoxic T cell (CTL) granules along with perforin (Akbar et

al., 1994) and may be responsible for the induction of apoptosis in target cells during CTL

attack (Lowin et al., 1996). TIAR and TIA- 1 have also been documented as active

members of the apoptotic pathway triggered by Fas ligation. TIAR, known to be

concentrated in the nucleus of hematopoietic and non-hematopoietic cells is translocated

to the cytoplasm 30 minutes following Fas ligation (Taupin et al., 1995). On the other

hand, TIA- 1 is rapidly phosphorylated by Fas-activated serineheonine kinase (FAST)

which is dephosphorylated and activated following Fas ligation in Jurkat cells (Tian et al..

1995). TIAR may be a general feature of the apoptotic program whereas TIA- I may play

a role in signaling downstream events during the course of ce11 death. Presently, the

notion that TIAR and TIA-1 regulate in some manner the process of apoptosis remains

more of an hypothesis than actual fact. TIAR and TIA- 1 are newly discovered proteins

and as a result have received the least attention. Preliminary data have shown physiologic

changes associated with TIAR and TIA-1 during apoptosis, particularly that triggered by

Fas ligation. As a result, these two RNA binding proteins deserve fùrther study in order

to elucidate specifically the roles and the importance of those roles that TIAR and TIA-1

play in the grand scheme of apoptosis.

Apoptosis is a genetically controlled process of ce11 deletion that c m be triggered

by nurnerous extemal and intemal, physiological and pathological stimuli. These death

signals are received by molecules of the control and execution stage upon which an

intracellular decision is made regarding the activation of proteases and the completion of

the cell death cycle. Regulation of the various Bcl-2 family members will have a large

impact on whether or not a ce11 dies or swives. Death of apoptosis susceptible cells is

promoted in the absence of adequate Bcl-2 protection. Subsequentl y, proteases suc h as

Ich-IL and CPP-32 among many others are activated leading to cleavage of their

respective substrates. DNA cleavage by activated nucleases guarantees irreversible

progress to cellular fragmentation and the formation of apoptotic bodies. Phagocytosis

clears the surrounding tissue of any cellular fragments and completes the death cycle.

Apoptosis has been studied intensely in the past couple of years. The reason is

because of the far reaching implications that this type of ce11 death has in both the normal

physiology and pathophysiology of living organisrns including humans. Conceptually. it

has long been realized that cells must be lost continuously from many normal tissues in

order to balance the cell mitosis which is readily evident (Kerr et al.. 1972). Accordingly.

apoptosis can act as a homeostatic mechanism to control the balance between stem ce11

production and maturation and loss of those cells which are functionally inactive or

terminally differentiated (McKenna and Cotter, 1997). Apoptosis may also operate to

eliminate potentially pathological cells from the organisrn. For example, the extensive

apoptosis of marrow-derived lymphocytes which recognize self antigens inhibits

autoimmune reactions (McKenna and Cotter, 1997; Fleisher, 1997). In addition,

cytotoxic T lymphocytes via granzyme B assault induce apoptosis in targets such as virus-

infected cells or turnor cells. Finally, the d o m regulation of a beneficial immune response

is achieved by apoptosis of activated T cells (Akbar and Salmon, 1997). Apoptosis has

also found roles in organogenesis during ernbryological development (Lovschall and

Mosekilde, 1997), in the development of the mammalian brain (Weller et al., 1997) and

central and peripheral nervous systems (Narayanan, 1997), in proper gastrointestinal

system functioning (Potten et al.. 1997), and in ovarian follicle function to control the

number of embryos which c m successfblly complete pregnancy (Amsterdam et al., 1997).

Conversely, dysregulated apoptosis is suspected to be an underlying factor in many

pathological states. Excessive apoptosis has been shown to be responsible for the massive

decrease in CD4' T ceIl nurnbers associated with HIV infection (McKenna and Cotter.

1997). It is possible that diseases such as cancer, autoimmune (egsystemic lupus

erythematosus), neurodegenerative (eg.Alzheimers, Parkinsons, and Huntingtons diseases)

and neurodevelopment disorders, and inflammatory conditions may al1 have inappropriate

apoptosis as an underlying pathogenic factor. Elucidation of the pathways controlling this

form of programmed cell death should clariQ the mechanisms of ceIl death in both

physiological and disease states. Detailed knowledge of such pathways miiy ultimately

provide new approaches to the treatment of many common pathologies.

Much of our knowledge conceminp apoptosis and in particular the genetic

regulation of the process has corne from studies of the nematode Caenorhabditis elegans

(C. elegans). Thirteen genes have been identified whose products are mem bers of the C.

elegans ceil death machinery (Yuan, 1996). Ced-3, ced-4, and ced-9 are three key genes

of the programmed ce11 death pathway in C. elegans. The gene products of ced-3 and

ced-4 are both promoters of cell death whereas the Ced-9 protein inhibits apoptosis

(James et al., 1997; Hengartner, 1996). Research has aiso revealed that Ced-3 and Ced-4

have structural homologies to human proteins. In particular. Ced-3 has a 35% amino acid

identity and a 58% similarity with CPP-32 or caspase-3, a member of the interleukin-1 P

converting enzyme (ICE) fmily of cysteine proteases which is absolutely necessary for

apoptosis in marnrnaiian cells (Momey et al., 1996; Xue et al., 1996; Miura, 1996). The

mammalian homolog of Ced-9 is Bcl-2, a well established inhibitor of apoptosis with an as

yet undiscovered mechanism of suppression (Monney et al., 1996; Driscoll, 19%;

Shaharn'ànd Horvitz, 1996). No marnmalian homolog to Ced-4 has been discovered.

however several lines of evidence have shed light on its possible role in the rnarnmalian

apoptotic pathway. By inducing wild type Cedd expression in Schizosaccharomyces

pombe (a metazoa with no identified cellular suicide machinery), James et al. were able to

demonstrate rapid focal chromatin condensation and lethality, establishing a possible role

for Ced-4 in chromatin condensation (James et al., 1997). Altematively, Bauer et al.

using genetic techniques discovered that the N-terminal region of Ced-4 contained amino

acid identity (14 out of a possible 71 amino acids) with the dcath effector domain of PEA-

15 (Bauer et al., 1997). The death effector domain has previously been demostrated to act

as an important protein interaction motif (Bauer et al., 1997). Therefore. Crd-4 may

function as an adaptor protein in death signaling pathways similar to marnmalian FADD

and FLlCE (Bauer et al.. 1997). Complementary to the research directed at elucidating

the function of the aforernentioned Ced proteins were studies focused on their position in

the death pathway and how these proteins exerted pro-apoptotic and anti-apoptotic

effecis. Shaham et al. have over expressed ced-3, ced-4, and ced-9 in C. elegans neurons

that normally live. Their results concluded that both pro-apoptotic (Ced-3, Ced-4) and

anti-apoptotic (Ced-9) actions are simultaneously present in C. elegans neurons and other

cells, and it is the balance between the protective and killer activities which will determine

whether a ce11 will live or die (Shaham and Horvitz, 1996). A number of lines of evidence

have s h o w that Ced-9 may exert an anti-apoptotic effect by direct physical association

with Ced-4 (James et al.. 1997; Spector et al., 1997; Chi~aiyan et al., 1997; Wu et al.,

1997). In addition, Chinnaiyan et al., utilizing genetic studies determined that Ced-4 can

simultaneously interact with Ced-3 and its mammalian counterparts interleukin- 1 beta-

converting enzyme (ICE) and FLlCE (Chi~aiyan et al., 1997). Moreover, Wu et al.

showed that in mammalian cells Ced-9 which is localized primari ly to intracellular

membranes and the perinuclear region could target Ced-4 from the cytosol by direct

physical association, relocating Ced-4 to cellular positions occupied by Ced-9 (Wu et al..

1997). Interactions between Ced-3, Ced-4, and Ced-9 could in essence define a

regulatory mechanism for apoptosis in the worm C. elegans . Apoptosis in mammalian

cells is inevitably more complex than that which is found in C. elegans; however. it is

obvious that apoptosis at a "grass roots" level has been evolutionarily conserved from

nematode to man, and that C. elegans will remain a vital study mode1 for further insight

into apoptosis in humans.

Apoptosis is a fundamental hiological phenomenon whose scientific and clinical

relevance has only become to be appreciated in the past decade. The above discussion

provides an overview of the process along with a depiction of its role in both normal and

pathophysiological States. Genetic control of ce11 death by apoptosis presently remains a

focus of many labomtories around the world. Fas, p53, CPP-32, Ich-1 L, the Bcl-2 farnily

of proteins, and TIAR comprise a small portion of the genetic elements controlling the

many pathways leading to this controlled ce11 deletion known as apoptosis. Despite

extensive research, apoptosis remains an incomplete puule. Future in vivo and in vitro

studies will be necessary to elucidate the underlying molecular biology regulating

apoptotic ce11 death. Such an understanding holds the hopes of developing new

therapeutic interventions currentl y unavailable in the fight against many hurnan diseases

including cancer, autoimmune disorders, AIDS, and neurodevelopment and

neurodegenerative dysfùnction.

Apoptosis and Inflammatoty Bowel Disease (IBD). 1s there a connection?

A variety of experiments have been undertaken to define what are the "normal"

stages in the life of small or large intestinal epithelial cells. These investigations will

provide an introduction to data relevant to apoptosis and IBD. For exarnple. "the mouse

intestinal epithelium expresses a sequence of 'developmental events'- proliferation. lineage

allocation, migration, differentiation and death- throughout life (Henniston and Gordon.

1995)." Death is a programmed event as intestinal epithelial cells reaching the luminal

surface die by apoptosis and are then exfoliated or eliminated via phagocytosis. The same

is certainly true for cells of the hurnan small and large intestine. Enterocytes. mucus-

producing goblet cells, enteroendocrine cells, and Paneth cells are the four ce11 lineages

which constitute the crypt regenerative compartments of the small and large bowels. They

al1 aise from multipotent, and genotypically identical cells temed stem celis. Control of

ce11 production by the stem ce11 population is by a process termed by Merritt et al. as

spontaneous apoptosis (Merritt et ai., 1995). A murine mode1 (BDF,) was used to

estimate crypt epithelial cell apoptosis. TUNEL analysis identified apoptotic cells in the

small intestine focused at position 4, also considered to be the position harboring the stem

cells. In the large intestine, TUNEL positive epithelial cells were attenuated and found

dispersed throughout the proliferative compartment, not confined to the area containing

the stem cells (position 1 and 2) (Memtt et al., 1995). More recently, Potten reiterated

many of the findings issued by M e d t et al. including the differences in bci-2 expression

associated with the small and large intestine. Bcf-2 is a gene whose product has anti-

apoptotic capabilities. It is not expressed in the murine or human small intestine. but is

expressed in the stem ce11 compartment of the large bowel crypts (Potten, 1997).

According to Potten, this fact explains why the level of apoptosis in the large bowel

epithelium is reduced relative to the srnall bowel and may also explain the higher incidence

of large bowel cancers (Potten, 1997; Merritt et al., 1995). In general, spontaneous

apoptosis in the normal small intestine is tightly regulated with cellular homeostasis being

maintained by elimination of stem cells. Diminished apoptosis of large bowel enterocytes

is a result of the survival advantage conferred upon these cells by the expression of bcl-2.

Apoptosis in the large bowel is loosely regulated and apoptotic cells are observed at

various sites dong the length of the crypts. Morphologically observable apoptotic cells.

usually identified by the presence of apoptotic bodies are rare. A study by Lee

demonstrates that even an intense inflammatory reaction such as that seen in untreated

IBD only marginally increases the crypt apoptotic count over the n o m (Lee, 1993).

Little is known about the role apoptosis might play, if any, in the mucosal

inflarnrnatory changes in either Crohn's disease (CD) or ulcerative colitis (UC). Presently.

apoptosis is considered the process responsible for the crypt epithelial ce11 loss which

equates to a gradua1 reduction in the size of the crypts in IBD (Iwamoto et al., 1996).

Iwamoto et al. (Iwarnoto et al., 1996) found that apoptosis, detected by TLJNEL, in

either involved or uninvolved biopsies from patients with UC was located in both crypt

and luminal epithelium while in normal control subjects apoptosis was restricted to the

luminal epithelium. TUNEL is a technique originally developed by Gavrieli et al. that

enables specific in situ labelling of the exposed 3' OH terrnini of cleaved DNA fragments,

thereby identifjing cells destined to die by apoptosis (Gavrieli et al., 1992).

Immunohistochemical staining implicated FasFasL- two components of the apoptosis

death program - in the mechanism of observed ce11 death (Iwarnoto et al., 1996).

Historically, necrotic ce11 death has k e n associated with inflammation and tissue

darnage and a change in scientific thought towards other modes of cell death, specifically

apoptosis, may be necessary to make advances in understanding the etiology and/or

pathogenesis of inflammatory bowel disease. The study of apoptosis as it relates to IBD

remains in its infancy and the role that apoptosis rnight play in IBD is not clearly defined.

The present study was designed to investigate the hypothesis that increased apoptosis is an

important factor in the reduction of crypt size described in the colonic mucosa of

inflamrnatory bowel disease (CD and UC). The results fiom this will provide additional

information conceming the mechanisms responsible for ce11 death by apoptosis in

inflammatory bowel disease.

Cytokines, IBD, and Apoptosis

Cytokines are low molecular weight glycoproteins that have been widely accepted

as major mediators in the development of the mucosal lesions observed in CD and UC.

Cytokines are capable of directing a plethora of cellular process including in some cases,

ceIl death by apoptosis.

Mucosal inflammation associated with active IBD is in part characterized by an

upregulation of a number of proinflarnmatory cytokines. Interleukin- 1 (IL- 1 ) (Shortman

and Scollay. 1994), IL-6 (Funakoshi et al., 1995; Murata et al., 1995), IL-8 (Funakoshi et

al., 1 995; Murata et al., 1999, interferon-gamma (INF-y ) (Parronchi et al., 1 997), and

tumor necrosis factor-alpha (MF-a) (Murata et al., 1995) have al1 been observed to be

upregulated (mRNA and/or protein) in the mucosa of patients with IBD. Furthemore.

many of these interleukins (IL-1 P, IL-6, IFN-y, RIF-a) have been shown to prolong the

survival of polymorphonuclear leukocytes (PMN) in culture (Colotta et al., 1992; Biffl et

al., 1996). It is quite conceivable that extended survival of PMN in vivo at an

inflamrnatory site could contribute to mucosal damage in patients with IBD.

Proinflammatory cytokines also have a significant impact on intestinal epithelial

cells. For example, a fûnctional consequence of augmented IFN-y production in the gut is

high antibody dependent cellular cytotoxicity against colonic epithelial cells (Unno et al.,

1995; Hibi et al., 1993). IFN-y has been observed to increase permeability when cultured

with monolayers of intestinal epithelial cells (UMO et al., 1995). An in vivo increase in the

permeability of an intestinal epithelial barrier could allow for the breakdown of tolerance

to intestinal flora leading to mucosal inflammation and cytokine production. Jung et al.

infected monolayers of human colon epithelial ce11 lines with invasive strains of bacteria

which resulted in the upregulation of IL-8, monocyte chemotactic protein-1, granulocyte

macrophage-colony stimulating factor, and TNF-a (Jung et al., 1 995). Epithelium

hyperpermeability and subsequent inflammatory reactions in the adjacent mucosa could

theoretically result in damage and premature death of surface and crypt enterocytes.

The mucosal injury caused by proinflammatory cytokines may be intesified by the

fact that the inflammatory reaction involved in IBD may be self-perpetuating. Human

intestinal epithelial cells express intercellular adhesion molecule- 1 (ICAM- 1 ) during active

inflammation which may favor the interaction between epithelial cells and circulating

leukocytes that express the natural ligand for ICAM- 1. A continuous human intestinal

epithelial ce11 line cultured with TNF-a ancilor IFN-y upregulated ICAM- 1 expression

(Paolieri et al., 1997). If this is the case in vivo, then proinflarnmatory cytokines

generated during active IBD could indirect] y recrui t and activate further leukocytes by

upregulating ICAM-1 on gut epithelial cells. This would in tum generate increased

quantities of proinflammatory cytokines thereby perpetuating chronic inflammation

t h x p h initiation and maintenance of a local T ce11 mediated immune response

(Romagnani et al., 1997).

Cytokines are undoubtedly involved in either the initiation of IBD or its

perpetuation. The difliculty in assessing the effects of cytokines is that these protein

hormones form very cornplex pathways and cascades with members having pleiotropic

effects. It becomes imperative then, to decipher what exactly defines a cytokine profile

associated with diseases such as IBD. Only then will therapeutic interventions based on

cytokines be feasible. IL- I O which acts as a natural damper of the abnormal and intense

inflammation associated with IBD has been tested in clinical trials on patients with CD.

IL- l O administration to patients with CD attentuates the clinical expression of disease

activity and there is endoscopic correlation with healing (Dr. Depew, personal

communication). Further research might consider investigation of IL- 12 antagonists

which would block the differentiation of naive T cells into IFN-y producing Th1 cells

(Strober et al., 1997).

Reactive Oxygen Metabolites (ROM), IBD, and Apoptosis

ROM refers to reactive molecules that are centered around the oxygen atom.

Hydrogen peroxide (H202). superoxide anion (O2'), hypochlonte (OCI'), and the very

reactive hydroxyl radical (OH.) are ROM prototypes.

ROM are generated at physiological levels during normal metabolism. Similarly.

during inflammation ROM are generated at enhanced levels, however both enzymatic and

non-enzyrnatic antioxidants exist to scavenge ROM thereby limiting their deleterious

effects to the immediate surrounding tissue. During a state of chronic inflammation such

as that which characterizes IBD however, the inherent antioxidant defenses may become

ovenvhelmed leading to oxidative tissue darnage (Grisham, 1994). Indeed, mucosal

biopsies from patients wîth CD and UC have harboured increased quantities of ROM, iron

(transition metal responsible for Fenton chemistry), DNA oxidation products (CD), and

decreased superoxide dismutase (scavenger of O,) (CD) (Lih-Brody et al., 1996; Sedghi

et al., 1993). McKenzie et al. observed decreased [14-Cl-iodoacetamide labeling of a

nurnber of proteins indicative of oxidation of thiol groups in colon epithelial crypt cells

from patients with active IBD (McKenzie et al., 1996). Moreover, biologically relevant

oxidants such as H,02, OCI-, NO, and a mode1 chlorarnine T molecule applied to colon

epithelial crypt cells in vitro were capable of duplicating the in vivo redox status of the

isolated colon epithelial crypt cells (McKenzie et al., 1996).

The source of ROM is suspected to be activated neutrophils and macrophages that

infiltrate the mucosa during active flares of IBD. Proinflammatory compounds such as

leukotriene B,, platelet-activating factor, immune complexes, complement components. or

bacterial products bind to specific receptors on the phagocyte plasma membrane,

activating NADPH oxidase which results in the liberation of an excess amount of O,',

H202, as well as the myeloperoxidase-derived oxidants hypochlorous acid and N-

chloramines (Grisharn, 1994; Zimmerman and Jewell, 1996). OH (generated by the

superoxide driven Fenton reaction) and hypochlorous acid are extremely reactive species

that can attack virtually al1 biomolecules (Zimmerman and Jewell, 1 996). The ce11 death

pathway mediated by ROM i s poorly understood, but it is speculated that cell death is

imminent following alterations to cellular components such as proteins, carbohydrates.

lipids and DNA ( C h i et al., 1995; Sirnrnonds and Rarnpton, 1993). Activated

neutrophils also secrete proteases into the extracellular space including elastase,

collagenase, and gelatinase (Zimrnerman and Jewell, 1996). Neutrophil-derived ROM

may alter the protease-antiprotease balance that normally exists in the intestinal

interstitium leading to mucosal interstitial matrix and epithelial ce11 degradation by these

proteases (Zirnmerman and Jewell, 1996). The potential pathogenicity of ROM however.

may lie in their ability to mobilize the expression of genes controlling many other aspects

of the inflammatory, immune, and acute phase response, by activation of the transcription

factor NF-KB (Jourd'heuil et al., 1997). Inactive NF-KB is normally sequestered in the

cytoplasm bound to its inhibitor protein I-KB . Activation of NF-KB involves degradation

of 1-KB by the proteasorne followed by translocation of NF-KB into the nucleus. Once in

the nucleus, NF-KB binds to its consensus sequence on the promoter-enhancer region of

different genes, where it upregulates the expression of a variety of proinflammatory

cytokines (IL- 1, IL-2, IL-6. IL-& TNF-a), adhesion molecules (ICAM- 1, E-selectin,

VCAM-1), and enzymes (iNOS) (Jourd'heuil et al., 1997). The upregulation of these

compounds contribute to an environment favourable to mucosal, including epithelial ce11

darnage while the acute and chronic inflammatory processes are sustained by iûrther

recruitment of cells to inflammatory sites by enhanced expression of adhesion molecules.

Jourd'heuil et al. have proposed that inhibition of NF-KB activation will lead to significant

anti-inflarnmatory activity which may be mediated by the inhibition of certain

proinflammaiory mediators and adhesion moleciiles (Jourd'heuil et al.? 1997: Zimrneman

and Jewell, 1996).

ROM are hypothesized to play an important role in the apoptotic pathway, but the

exact nature of the involvement is unclear. Studies by Satoh et al. demonstrated that

apoptosis of PC12 cells and rat cortical neurons upon senun deprivation was associated

with an increase in ROM (Satoh et al., 1996). Antioxidants (Le. superoxide dismutase)

have also shown an ability to block staurosporine induced neurotoxicity (Prehn et al.,

1997) and death of cultured sympathetic neurons deprived of NGF (Greenlund et al.,

1995). Bcl-2 can block apoptosis due to increases in ROM, but how Bcl-2 accomplishes

this is unclear. Bcl-2 is localized to endoplasmic reticulum, nuclear membranes, and

mitochondria, which are also sites of ROM generation. This led researchers to speculate

that the anti-apoptotic capacity of Bcl-2 stemmed from an ability to act as an anti-oxidant

(i.e. as a fiee radical scavenger). Expression of Bcl-2 has been observed to decrease the

net cellular generation of ROM (Sarafian and Bredesen, 1994). Conversely, Steinman has

proposed that Bcl-2 acts as a prooxidant generating an intracellular oxidative stress which,

in tum, results in the expression of certain antioxidants, such as catalase (Steinman,

1995). However, it is generally accepted that instead of scavenging radicals or creating an

oxidative stress within cells, Bcl-2 functions to prevent oxidative darnage to cellular

constituents (Korsmeyer et al., 1993; Korsrneyer et al., 1995). Regardless of the

mechanism by which Bcl-2 mediates its activity, these results suggest that ROM may

represent important cellular messengers involved in the control of cellular apoptosis.

A study by Reinshagen et al. demonstated that during inflammation in a rat mode1

of colitis. Bcl-2 expression was downregulated and the pro-apoptotic Bax protein was

upregulated (Gerlach et al., 1996). If an equivalent mechanism exists in hurnans with

active IBD, then it is conceivable that epithelial and lamina propria ce11 damage and death

could result from the altered expression of Bcl-2 farnily members. Further research

investigating these important apoptosis regulatory proteins and their relationship to ROM

under both physiological and pathophysiological conditions may consequently provide

new insights into the regulation of inflammation and apoptosis in the gut.

Conclusion

Crohn's disease and ulcerative colitis are chronic inflamrnatory diseases of the

intestines and despite extensive study the etiology of the inflarnmatory processes remains a

mystery. It is believed that a genetic predisposition. endogenous anomalies. and

exogenous tnggers lead to the onset of the disease. Proinflarnmatory cytokines IL- 1, IL-

6, IL-8, IFN-y, and TNF-a are upregulated in mucosal biopsies from inflamed sections of

small or large bowel (Murata et al.. 1995) or stimulated whole blood ce11 cultures isolated

from patients aMicted with CD or UC (Elsasser-Beile et al., 1994). Similady, reactive

oxygen metabolites which are produced as vital components of many physiological

operations are synthesized and released in excess during chronic inflammation ty pical of

IBD (Sedghi et al.. 1993). Proinflammatory cytokines and ROM together contribute to a

mucosal environment with the potential for mucosal interstitial rnatrix and epithelial cell

damage. A loss of epithelial cells leading to crypt shortening and branching is a typical

histologic observation of chronic Crohn's disease and ulcerative colitis. Enhanced

apoptosis has recent ly been postuiated as responsi ble for this loss of epi theliurn (Iwamoto

et al., 1996). Apoptosis is a term used to describe a type of genetically controlled ce11

deletion with characteristic morphology including condensation of the cytoplasm and

nucleus, chromatin compaction, DNA fragmentation, and apoptotic body formation (Kerr

et al., 1972). Apoptosis acts as a normal homeostatic mechanism to control ce11 numbers

in self-renen-ing tissues, but is also believed to underly many pathological conditions. The

molecular biology of the apoptotic ce11 death pathway includes the Bcl-2 family of

proteins, p53, and the ICE farnily of proteases. The complete picture of the "inner-

workings" of apoptosis is far fiom being deciphered, but remains under the intense

scrutiny of many molecular biologists.

The role of apoptosis is as yet not clearly defined in IBD and the present study

was designed to support the hypothesis that it does play an important role, particularly in

the crypt reduction described in the colonic mucosa of inflammatory bowel disease.

MATERIALS AND METHODS

Patient Selection

The research study received previous approval from the Ethics Review Board of

the Faculty of Health Sciences at Queen's University (Kingston, ON).

IBD patients for this study had been previously diagnosed and had longstanding

disease ranging from 2 to 32 years. The initial diagnosis of IBD in these patients was

made based on clinical evaluation, endoscopy and/or radiography, and histology of biopsy

specimens. The clinical evaluation is guided by the history of symptoms (eg. severity and

frequency of diarrhea, systemic symptoms) and physical examination of the patient.

Endoxopy and radiography are the two major diagnostic tools used by physicians to

establish a diagnosis of IBD. Endoscopy is usually required following initial presentation

of symptoms to help establish a diagnosis and define the extent of mucosal disease.

Biopsies can be obtained during endoscopy and subsequently processed for

histopathological examination. CD and UC each have characteristic histopathological

appearances. Patients with IBD were followed periodically at a university hospital (Hotel

Dieu Hospital, Kingston, ON).

A schedule of IBD patients undergoing colonoscopic follow-up (for flares of IBD,

cancer surveillance in longstanding UC patients, etc.) was examined at the begiming of

each week prior io the actual procedw dates. Medical charts of patients undergoing

colonoscopic follow-up for that week were exarnined and patients with previously

diagnosed IBD and normal patients undergoing colonic surveillance for cancer were

identified. It was anticipated that biopsies fiom these patients would be obtained. The

colonoscopic procedures of the prospective patients as previously identified were attended

by the author. Pnor to the colonoscopy, the attending gastroenterologist informed the

patient of the nature of the research study and the attendant risks of colonoscopy and

biopsy. Informed patients willing to participate in the study were required to sign a

consent form (Appendix C).

During the actual colonoscopy, extra biopsies fiom various locations throughout

the bowel from ileum and/ or cecum to rectum were obtained for the purposes of the

study. Biopsies were immediately fixed in 10% neutral buffered formalin. The site of

biopsy and any macroscopic mucosal abnormalities were recorded. Patient information

including medical treatrnent was also recorded. Following fixation, colonic rnucosal

biopsies were embedded in paraffin wax and microtome (Sorvall M2MT-8) sectioned at 5

pm in thickness and adhered to glass slides. A total of 49 patients were used in these

studies. This group included 1 1 CD (patient # 1 - 1 O,49), 1 1 UC (patient # 1 1-20,48), 19

non-IBD (patient # 21 -39), and 8 normal (patient # 40-47) patients (see Appendix 8- table

1). Examination of the histopathology was done by a pathologist and was used to

determine areas of bowel that were involved or uninvolved microscopically.

Histologie Assessmen t of IBD

The histologic assessrnent of IBD was made by completing a microscopie analysis

of HPS stained tissue fiom patients with previously diagnosed IBD.

Mucosa afTected by longstanding IBD shows features of chronic colitis. Chronic

colitis or chronic injury is characterized on histologic (HPS) specimens by a

plasmolymphocytic infiltrate in the lamina propria (Figure 3A and 38). This

plasmolymphocytic inflammation including lymphoid aggregates, plasma cells, mast cells.

and eosinophils has varying degrees of intensity from mild to severe. The histopathology

of chronic colitis also includes crypt architectural distortions. Crypts become shortened

and branched during chronic IBD (Figure 3A).

The histopathology of active coli tis is characterized by epithelial inj ury .

Neutrophils infiltrate the crypt epithelium (cryptitis) and crypt lumens (crypt abscesses)

leading to destruction of the affected crypts (Figure 38). The sxtent of epithelial darnage

will Vary according to the degree of the inflarnmatory process. Surface epithelium erosion

and mucosal ulceration indicate more severe acute inflammation. Actively inflarned

mucosa will also demonstrate evidence of epithelial regeneration. This is indicated on

histologic specimens by increased mitotic activity of epithelial cells and goblet ce11

depletion. The histopathology of actively infiarned bowel also exhibits extensive

neutrophil infiltration in the lamina propria (Figure 3).

In the present study, histopathologic exarnination of HPS stained tissue fiom

patients with IBD was completed by a pathologist. Mucosal tissue displayed

histopathologic characteristics of either inactive chronic colitis or active chronic colitis.

The inflammation in tissues with active chronic colitis was relatively mild (Figure 3B).

Histopathologic analysis was also cornpleted of HPS stained tissue fiom normal

control and non-IBD patients. Healthy mucosa contains a normal physiologic population

of chronic infiammatory cells. The mucosal architecture is normal with regularly sized

Figure 3 Histoloeic Assessrnent of IBD. HPS stained rnucosal tissues fiom IBD patients

were analyzed rnicroscopically to determine the activity and severity of the colitis.

Histopathologic examination determined that mucosal biopsies from IBD patients had

either inactive chronic colitis (A) or active chronic colitis (B). In both (A) and (B) there is

an increase in mononuclear cells in the lamina propria. Both specimens are fiom patients

with longstanding IBD and the chronic nature of the disease is evident by the crypt

architectural distortion. Crypts are shortened and branched and irregularly spaced. The

epitheliurn in (A) is normal with no cryptitis or crypt abscesses. Neutrophil infiltration

into crypt epithelium (cryptitis) is characteristic of actively inflamed bowel (B). The arrow

in (B) indicates an area of cryptitis and partial destruction of crypt epithelium. The degree

of active inflammation in (B) is relatively mild. There is no rvidence of crypt abscesses,

ulceration, or extensive crypt injury. The inflammation in the majority of cases of active

chronic IBD was determined to be relatively mild. Histologie assessrneni was also

completed of HPS stained mucosal tissues from normal and non-IBD patients. Original

Magnification: (.4)100X, (B) 200X.

crypts that have uniform spacing.

Procedures

(1) Morphology Studies

(a) HematoxylinlPhloxinelSaffron (HPS) Staining

HPS stained mucosal sections were examined using light rnicroscopy for

morphological evidence of cellular apoptosis. Evidence of an apoptotic event is indicated

by the presence of apoptotic bodies. Apoptotic bodies are membrane bound remnants of a

previously intact ce11 that has undergone apoptosis. The apoptotic bodies may or may not

contain nuclear material. Only the larger rnembers of a cluster of apoptotic bodies can be

discerned using a light microscope (Kerr et al., 1972). Clusters of apoptotic bodies were

examined in involved and uninvolved colonic rnucosal sections of bowel using

photomicroscopy. Since light microscopy can only detect end stage apoptosis (i.e.

apoptotic bodies) the actual arnount of apoptosis in the tissue sections is generally

undçrestirnated.

The Pathology Department at Hotel Dieu Hospital in Kingston, ON perfoned the

HPS staining for this study using routine standard protocols (see Appendix A). The

results of HPS staining of mucosa tissues indicate a blue nucleus, pink cytoplasm and

smooth muscle and yellow connective tissue. HPS stained sectons of al1 colonic mucosal

biopsies fiom al1 49 patients (see Appendix B - table 1) were examined. HPS stained

biopsies h m patients 8,9. 16, 18, and 44 were randomly selected for apoptosis

morphology analysis using light photomicroscopy.

(b) Electron Microscopy

Electron microscopy was used to study the ultrastructure of colonic epithelial cells

from mucosal biopsies isolated fiom involved or uninvolved areas of a patient with UC.

Evidence of apoptotic cells was exarnined for qualitative indication of the arnount of cell

death that may be ongoing in the gut and whether the amount of ce11 death is changed

during active IBD. Gross evidence of apoptotic cells using EM include cytoplasmic and

nuclear condensation, compactness of cytoplasmic organelles, the appearance of

protuberances on the ce11 surface. and condensation of chromatin that relocates to the

nuclear membrane (Gavrieli et al., 1992).

Colonic mucosal biopsies were isolated from involved and uninvolved colon from a

patient with UC (patient # 48-Appendix B-table 1). Mr. John DaCosta in the department

of Pathology (Kingston General Hospital, Kingston, ON) prepared the biopsy for EM

analysis. Bnefly, the biopsies were fixed in a solution of 2 % paraformaldehyde and 0.5 %

glutaraldehyde in 0.2 M sodium cacodylate buffer. The tissues were then processed using

a Lynx EL autocatalytic processor. The piocessed tissues were then rinsed in 2 changes

of 0.2 M sodium cacodylate buffer to remove salts. Osmium tetroxide was then applied to

post fix the tissues followed by 3 rinses in 0.2 M sodium cacodylate buffer. The tissues

were then dehydrated by applying a graded ethanol series. Two changes in each of 70%,

85%, and 95% ethanol were followed by 3 changes in 1 00% ethanol. Plastic was

gradually introduced into the tissues by applying 3 changes of a solution 1 part propylene

oxide : 1 part epon (plastic), followed by 2 changes of a solution of 1 part propylene oxide

: 2 parts epon, and finally embedding of the tissue in 100% epon. Thick tiss~le sections at

1 pm were cut using a glass knife mounted on a Sorvall M2MT-B microtome. These

thick sections were stained using a solution of toluene blue/borax/H20. Stained tissues

were mounted on slides using glass coverslips and then viewed using light microscopy for

specific areas of interest that would be cut for viewing using electron microscopy. Ultra-

thin sections for electron microscopy and photography were cut using a diarnond knife.

Sections were cut at a pale gold colour or approximately 90 qm and then picked up on

100 mesh copper grids. Next, the ultra-thin tissue sections were stained with uranyl

acetate and lead citrate. Photographs were taken with an H500 transmission electron

microscope.

(2) Studios on Fragmented DNA

(a) Gel Electrophoresis of Extracted DNA

One of the biochemical hallmarks of apoptosis is the activation of an endonuclease

that mediates the cleavage of DNA at intemucleosomal sites generating 180-200 bp

fngments of DNA or multiples thereof. These pieces of DNA cm be extracted from

apoptotic cells and can be separated using gel electrophoresis. A characteristic ladder

pattern observed after electrophoresis is an indication of cellular apoptotic activity in the

cells. Therefore, DNA gel electrophoresis was applied in this study to DNA extracted

from involved or uninvolved colonic mucoral biopsies from a patient with CD (patient 49-

Appendix B-Table 1). According to Iwamoto et al., a potential limitation to this technique

is that the arnount of fiagmented DNA in the test samples must be relatively large in order

to obtain a ladder pattern (Iwarnoto et al., 1996). The absence of a ladder does not

necessarily eliminate the possibility of apoptotic activity .

(i) DNA Extraction

The extraction procedure was developed from two published protocols (Gerlach et

al., 1996; Wyllie et al., 1984). The biopsies initially tixed in 70% ethanol were each taken

in turn and homogenized in fresh 70% ethanol using a dounce homogenizer. The

homogenates were then transferred using a pipet to 1.5 ml cryovials (Simport Itd., Beloeil,

Quebec) followed by centrifugation (IEC Centra-7R, Damon/ IEC Division, Needham

Hts., MA) at 700g for 5 mins at room temperature (RT). The ethanol was decanted off

leaving behind the cell pellets. The cells were lysed by the addition of 50 pl of a hypotonic

buffer consisting of 5 m M Tris pH 7.5,5 mM ethylene diamine tetra-acetic acid (EDTA)

and 0.5% Triton X-100 to each cryovial. The viais were agitated by vortexing for 5 min

followed by centrifugation at -800g for another 5 min at RT. The supematants were

transferred to new cryovials. To remove any RNA from the reaction mixture, 3 pl of

0.25% non-idet NP-40 and 6 pl of DNase free RNase A (Amersharn Pharmacia Biotech

Inc., Baie d' Urfe, Quebec) was added to each supernatant and incubated for 30 min at

37°C in a hurnid chamber. 3 pl of 1 mdml proteinase K (Life Technologies Inc..

Gaithersburg, MD) was then added to each of the reaction mixtures followed by

incubation ai 37°C in a hurnid chamber for an additional 30 min. The samples of extracted

DNA were stored ovemight at 4"C.

(ii) Agarose Gel Electrophoresis

A solution of 0.4g of agarose in 49 ml of distilled water and 1 ml of SOX TAE

(2.OM tr is acetate, 0.5M EDTA, 1 .OM glacial acetic acid) was made for casting a 0.8%

agarose gel. 20 pl of ethidiurn bromide was added to the solution. The mixture was

heated in a rnicrowave until a clear solution and then cooled to 37°C in a water bath. A

volume of 2 pl of DNA electrophoretic sampling buffer (DNA-ESB) (48% sucrose,

0.075% bromphenol blue, 10 m M EDTA, p H 4 . 3 ) was added to each via1 containing 15

pl of the extracted DNA samples. After the 0.8% agarose gel had solidified, it was

removed From the casting box and re-settled in the electrophoretic charnber which was

filled with 1X TAE. 10 pl of each DNA sample in DNA-ESB buffer as well as the

molecular weight markers (Boehringer Mannheim Canada, Laval, Quebec) were pipetted

into the respective loading chambers. A Biorad Mode1 500/200 power supply (Biorad.

Mississauga, ON) was used to apply 50V to the charnber and electrophoresis was carried

out for 60 min. DNA was visualized by fluorescence in ultraviolet light (260 nm) and

photographed.

(b) The TUNEL Method - In Situ Terminal Deoxynucleotidyl Transferase 3'

Hydroxy Nick End Labeling

The TUNEL procedure was originally developed by Gavrieli et al. in order to

visualize programmed cell death (PCD) in situ at the single ce11 level while preserving

tissue architecture (Gavrieli et al., 1992). Previous to this, identification of PCD was

inferred mainly from gel electrophoresis of pooled DNA extracts as PCD was s h o w to be

associated with DNA fragmentation. The TUNEL technique can identiS, DNA strand

breaks due to endogenous nuclease activated during apoptosis. As a result, cells destined

to die can be identified earlier and more accurately cornpared to morphological

recognition of apoptosis which relies on end stage apoptosis (i.e. apoptotic bodies). The

extent of tissue-PCD revealed by the TUNEL method is considerably greater than

apoptosis detected by nuclear morphology (Gavrieli et al., 1992). The TUNEL technique

as applied in this study not only gave an indication of the arnount of apoptosis in the

mucosa, but because labeling occurs in situ, the location of the dying cells could also be

identified. Since the original TUNEL technique was published a number of variations on

the procedure have been developed, including the one applied in the present study.

The in situ labeling of fiagmented DNA was can-ied out using the Apop Tag

(Oncor, Gaithersberg, MD) In Situ Apoptosis Detection Kit (Figure 4). The feature of

this kit is that it applies digoxigenin-dUTP as the molecules used for DNA end extension.

The DNA end extension reaction is controlled by Tdt enzyme. The natural source of

digoxigenin is the digitalis plant. Therefore, background staining due to

imrnunclchemically similar ligands is generally insignificant. Furthemore. the Fc portion

of the anti-digoxigenin antibody has been enzymatically removed in order to eliminate any

nonspecific adsorption to cellular Fc receptors. The digoxigeninhti-digoxigenin system

has been f o n d to be equally sensitive to avididbiotin systems (Gavrieli et al., 1997).

The following protocol was used to examine the extent of TUNEL positive cells in

mucosal biopsies taken from normal, non-IBD and IBD tissue samples. Microtome

sections of mucosal biopsies on microscope slides were deparafinized by heating followed

by two changes in xylene for 5 min each. Hydration of specimens was carried out by two

changes in 100% ethanol for 5 min each proceeded by 3 min each in 95% and 70%

Figure 4 Mechanism of TUNEL Anal~sis Accordine to the Oncor A D O D ~ ~ P Detection Kit.

This TUNEL technique is a variation of the TUNEL protocol initially developed by

Gavrieli et al. (Gavrieli et al., 1992). The sirnplified cartoon indicates DNA fragmentation

as a result of an endogenous nuclease activated during apoptotic ceIl death. The ends of

the DNA contain 3' hydroxy moieties that cm be labelled with digoxigenin-dUTP via

terminal transferase enzymatic action. Following DNA end extension, anti-digoxigenin

labelled with peroxidase antibody is applied and bound to attached digoxigenin-dUTP

molecules. Finally, sites of cellular DNA fragmentation (characteristic of apoptotic ce11

death) c m be visualized following staining with peroxidase substrate (in this case 3,3',5,5'-

tetrarnethylbenzidine) using a light microscope.

End result of apoptosis: nucleosome sized DNA fragments.

ApopTaga Step 1 : Tail with Digoxigenin-dUTP.

ApopTag" Step 2: Bind Antibody Peroxidase Conjugate.

ApopTaga Step 3: Stain with Substrate.

ethanol. The specimens were then washed in phosphate buffered saline (PBS) (50mM

KH2P0,, 2OOmM NaCI, pH 7.4) for 5 min. Nuclei of tissue sections were stripped of

proteins by incubation in 20 pg/ml proteinase K (PK) (Life Technologies Inc.,

Gaithersburg, MD) for 15 min at RT followed by washing in double distilled water

(DDW) twice for 2 min each. A hydrophobie pen ("PAP pen") (Research Products

International Corp., Mt. Prospect, IL) was then used to circle each tissue specimen on the

glass slides to ensure that the reagents remained concentrated on the tissue since

covealips were not used for this purpose. Endogenous peroxidase in the tissue was

inactivated by covenng the biopsy sections with 2%H201 in PBS for 5 min at RT. The

tissues were washed twice in PBS for 5 min. 13 pl of equilibration buffer (#S7 100- 1 ) was

then added to each tissue section and incubation for 2 min at RT. AHer removing the

equilibration buffer, the DNA 3'OH end extension was initiated by immediately covering

the tissue section with a working strength of TdT enzyme (76 pl reaction buffer [#S7 100-

2]+ 32 pl TdT enzyme [#S7100-31) for I hr in a 37°C humid chamber. The reaction was

stoppcd by preheated (37°C) stop/~!ash buffer (#S7100-4) for 10 min. The tissues were

then washed 3 times for 5 min each in PBS at RT. Each tissue section was then covered

with anti-digoxigenin antibody (#S7 100-5) for 30 min in a humid chamber at RT.

Following the 30 min, the tissues were washed 3 times for 5 min each in PBS.

Localization of DNA fragmentation was accomplished by adding the chromogenic

substrate 3,3',5,5'-tetramelhylbenzidine (TMB) in HzOz ("Ttue Blue" peroxidase

substrate) (Kirkegaard and Perry Laboratories, Gaithersburg, MD). "True Blue"

peroxidase substrate was added to each tissue for 10 min at RT. The tissues were then

washed 3 times in DDW for 1 min each followed by once in DDW for 5 min. The tissues

were dehydrated through a graded ethanol series of 3 min in each of 20% 40%, 80%, and

100% ethanol, and allowed to dry completely before being mounted under a glas

coverslip using an organic mounting media (Permount, Fisher Scientific, Fair Lawn. NJ).

A TUNEL positive signal is characterized by a dark blue nuclear staining.

(3) TUNEL Controls

Each experiment included a positive and negative control. This was to ensure that

the reagents were working (positive control) and that an excess of background staining

was not observed (negative control).

(a) Positive Control

A positive TUNEL control involved treating a designated tissue sarnple (normal.

CD, or UC colonic mucosa) with DNase. After quenching the endogenous peroxidase

with 2% H,O,IPBS and washing, positive control tissue was treated with DNase buffer

(1 OmM Tris-HCl, l OmM NaCl, 5mM MgCI,O, 25mM HCl, 0.1 m M CaCl,, pH 7.4) for I O

min in a 37°C hurnid charnber. DNase ( l p g h l ) (Pharrnacia Biotech, Ste-Anne-de-

Bellevue, Quebec) was applied to the tissue section. and incubated at 37°C in a humid

chamber for 20 min. The tissue was washed and then processed normally along with the

test samples.

(b) Negative Control

A negative control tissue was treated according to the TUNEL procedure except

the Tdt enzyme was replaced with DDW in the working strength Tdt solution. This

control indicates whether endogenous peroxidase was present or had been adequately

inactivated by the use of the quenching reaction (i.e. H20,/PBS treatment).

(c) Non-mucosal Tissue Controis

Other tissues were utilized as TUNEL controls to ensure that the staining pattern

that was being observed in the normal, CD, UC, and non-IBD test tissues was not a

general phenornena of the staining protocol. Tissues taken fiom the appendix and tonsil

were treatrd according to the TUNEL procedure as described above.

(4) Quantification of TUNEL Staining

Qualitative observations of the colonic rnucosal TüNEL staining pattem were

made by photomicroscopy. Quantification of TUNEL staining was accomplished by

capturing the stained image by a CDD video camera mounted on the microscope and

using the "Northern Exposure" (Empix Imaging Inc., Mississauga, ON) image analysis

software. Image analysis of stained tissue sections from patients 6,9, 10, 16, 1 7, 19,20,

and 4 1 (Appendix B-table 1 ) was completed.

(a) Worthern Exposure" Image Analysis Software

Stained tissues were viewed using an Olympus BX60 light microscope (Carsen

Group Inc., Markham, Ontario) and a CDD video camera. AAer areas of stained tissue

were located, the live video output From the colour camera was converted to a digitized

image using the freeze tool of the software. The program assigns a gray value from 0-255

(O=black, 255=white) to each primary color (Red, Green, and Blue) of the image. A

digital picture allows for image analysis, because given gray values can be isolated by

binarization and measured. Once a digitized image has been synthesized. objects of

interest are measured by thresholding.

Thresholding is the essential measuring aspect of image analysis allowing

distinctions to be made between interesting and uninteresting parts of an image.

Thresholding creates a new binary image, defined by objects in an image based on gray or

color intensity values. In this study, cells with dark stained nuclei (TUNEL positive) were

the quantity to be thresheld and counted. In other words, al1 darker pixels (TUNEL

positive cells) or gray values would be of interest and al1 lighter background pixels would

be excluded. Using "Northem Exposure"soHware, the newl y created binary image was

stored in the red colour plane so that selected objects appeared red while image integrity

undemeath the red binary colour was still visible. The soflware program has two distinct

mechanisms for thresholding, monochrome and colour. It was determined that, after

experimenting with both thresholding systems, converting the colour digitized image to

black and white (monochrome) using the colour correction tool of the software allowed

for easier and more accurate thresholding of TCINEL positive cells. After the digitized

image was colour corrected to monochrome, the threshold range of gray values was set.

The range of gray values determines what objects are measured based upon the intensity

of gray colour on the black and white digitized image. It becarne necessary to determine

what would be the standard for positive staining against which al1 other positive cells

would be measured. The DNase positive control served as the standard for positivity

since al1 cells in a DNase positive control contained nuclei with abundant DNA

fragmentation. Before measurements were taken of the test tissues, a DNase positive

control tissue was digitized for that experiment, and converted to monochrome and

thresheld. By adjusting the threshold slide bars on the computer screen it was possible to

view what was being thresheld and what was not. Thresheld objects appeared red on a

blue background. The threshold slide bars were adjusted until al1 nuclei of the DNase

positive control were red and al1 background was Mue. The staining of DNase positive

controls varied from experiment to experiment, and therefore it was necessary to adjust

the threshold range to each DNase positive control for each TUNEL experiment. Once

the threshold was set, it was applied consistently to mesure al1 test slides for that

respective experiment. Threshold gray range values for measuring positive cells for this

study invariably began at O (black) but the upper setting varied from as low as 105 to as

high as 140.

(b)The Tissue Unit

The TUNEL positive ce11 was the single entity being counted, but it was necessary

to define in what areas (i.e. surface andor crypt epithelium, lamina propria) of the mucosa

these measurements were going to be made. A "tissue unit" was implemented in which

TUNEL positive ce11 measurements were calculated. The length of mucosa at 20X

magnification captured and digitized as viewed on the computer was defined as the tissue

unit. On a microscopic level, the tissue unit was equivalent to approximately 500 microns

(pm) of mucosa. Along the length of the tissue unit, TUNEL positive ceIl counts could be

made within the surface and crypt epitheliurn and lamina propria (at 20X magnification.

the entire thickness of the mucosa was in the field of view). The standard unit of length

defined by the tissue unit also accommodated changes in ce11 number and volume in the

lamina propna due to the inflammatory process. The tissue unit standardized the

measwing procedure so that valid cornparisons between involved and uninvolved IBD and

normal bowel could be made.

(c) Procedure for Measurement of TUNEL Positive Cells

A TUNEL stained tissue section of involved or uninvolved IBD or normal bowel

from a specific bowel location was examined under an Olympus BX60 light microscope

(Carsen Group Inc., Markham. Ontario). An appropriate (Le. full thickness mucosa with

crypts and surface epithelium) area of tissue was captured as a digitized image. That

digitized length of mucosa also defined the tissue unit. The digitized image was converted

to black and white using the colour correction tool and then thresheld. The threshold

range values were obtained from thresholding the DNase positive control for that

expenment. The tracing selection tool of the software was then applied to outline specific

areas (surface or crypt epithelium, lamina propria) of mucosa within the tissue unit. The

tracing selection tool enabled outlining of thresheld images. Once a trace was completed

the cornputer automatically enumerated al1 thresheld objects within the outlined area. For

simpli Qing measurements of complicated digi tized images the mask editor of the software

was applied. Using the mask editor it was possible to trace specific areas of interest on a

digitized, but non-thresheld image. The outline was then saved as a mask (.msk) file. The

original image was thresheld and then using the mask tool the stored measurement mask

was recalled and the thresheld objects were autornatically counted by the image analysis

software. Measurements were made using either the trace selection tool or mask editod

measure using mask tool.

Three separate tissue units within a tissue section from each bowel location were

measured. Within each tissue unit, TUNEL positive cells in the surface epithelium, the

crypt epithelium. and the lamina propria were counted. Using the procedure stated above

the total ce11 numbers were determined from the HPS staining. This was possible because

the location of tissue in which TUNEL positive cells were measured could be matched

with relative accuracy to the same location in a section of tissue sliced from the same

tissue block stained with HPS. The nuclei of al1 cells of an HPS stained tissue are labeled.

thresheld and measured using the image software analysis.

The percentages of TUNEL positive cells for each area of the tissue unit

(surfacekrypt epithelium. lamina propria) were calculated for al1 three tissue units

measured witliin each tissue section from each bowel location for each patient. Three

tissue units for each bowel location were measured so that an average of TUNEL positive

cells for each mucosal area (Le. surfacelcrypt epithelium, lamina propria) would be

calculated. Percent TUNEL positive ce11 calculations was decided to be the only

meaningful measurement hecause the bowel tissues were not well oriented. The averages

of percent TUNEL positive cells for each mucosal location were then used for

comparative purposes to illustrate differences in the quantity of apoptotic cells (as

measured by TUNEL) between normal, uninvolved. and involved bowel.

The "Nonhem Exposure" image analysis software was applied with relative

success to the measurernent of apoptosis in the mucosa of patients with IBD or normal

subjects undergoing colonoscopy for surveillance of cancer. The quantification of

TUNEL positive cells was necessary to validate the qualitative observations. One

potential test limitation was in matching the tissue unit location of the TUNEL stained

sections to the exact same location in the HPS tissue. This caused measurement of cells

within HPS stained tissue that were not included as part of the tissue unit of the TUNEL

stained bowel mucosa. However, tissue units of TUNEL stained bowel were generally

matched with enough accuracy to HPS stained bowel to obtain meaningful data without

being influenced by minor changes in tissue location.

(d) Data Analysis - Statistical Method

A linear regression analysis was applied to assess the best fit line correlating

computer counts versus manual counts for TUNEL positive stained cells. The linear

regression fits a mode1 relating the manual counts (dependent variable) to the computer

counts (independent variable) by minimizing the sum of the squares of the residuals for the

fitted line. The linear regression analysis displays the estimated intercept and dope of the

line, a standard error for each estimate, and a t-statistic (at 95 % confidence) usefùl for

testing whether the true value of the coefficient is equal to zero. The statistical

computations were completed using Statistica 6.0 software progrm.

(5) Immunobistochemistry Analysis of Apoptotic Related Proteins in Mucosal Tissue

Immunohistochemical detection of the apoptotic regulatory proteins Fas, p53,

CPP-32, Ich-lL, TIAR, Bcl-x,, and BAD in tissue sections isolated from patients 8, 1 1.

13, 15, 18,40, and 44 (Appendix B-table 1) was exarnined initially to verifi the TUNEL

results and to possibly derive some insight into the molecular biology controlling ce11 death

in the gut. The antibodies necessary for this task were obtained fiom an "Apoptosis

Sarnpler" kit purchased from Transduction Laboratories (Lexington, KT). Antibody

characteristics are summarized in Table 2 (Appendix B).

The respective formalin-fixed paraffin embedded sections were deparaffinized by

heating until the wax was clear. Melted parafin was removed by consecutive immersions

in xylene, twice for 5 min each at RT. Hydration was camed out by two changes in 100%

ethanol for 5 min each, proceeded by 3 min in each of 95% and 70% ethanol. Tissues

were then rinsed in DDW at UT. Antigenic determinants masked by formalin-fixation and

parafin-embedding were retrieved by pressure cooking . Tissue sections on slides were

submerged in 200 ml of 1 M sodium citrate buffer, pH 6.0 and placed in a pressure cooker.

The specimens were pressure treated for 10 min. The tissues were then cooled to RT by

bathing in two changes of sodium citrate buffer followed by changes in DDW for 1 O sec

and 1 min respectively . The tissue specimens were then treated with 20 pglml proteinase

K (PK) (Life Technologies Inc., Gaithersburg, MD) for 15 min in order to permeabilize

the cells. A hydrophobie pen ("PAP pen") (Research Products International Corp., Mt.

Prospect, IL) was then used to circle dl the tissue specimens to ensure that the reagents

remained concentrated on the tissue since coverslips were not used for this purpose.

Endogenous peroxidase was inactivated by covering the sections with 2%H20JPBS for 5

min at RT. The tissues were then washed in PBS two times for 5 min each. In order to

suppress non-specific binding of immunoglobulin, 10% goat serurn (blocking serum) in

PBS was added to each tissue specimen and incubation was carried out at 4°C in a humid

chamber for 90 min. The tissues were then rinsed twice in PBS for 5 min each. The

prirnary antibody (Appendix B-table 2) was pipetted ont0 the tissue followed by

incubation ovemight at 4°C. The next day, the primary antibody was removed and the

specimens were washed three times for 5 min each in PBS. Biotinylated secondary

antibody (1 :30 dilution in 5% Tris buffer) was then applied to each section for 30 min.

After 3 washes in PBS for 5 min each at RT. with constant agitation, tissues were then

each exposed to ABC Immunoperoxidase Reagent (i.e. 1 :30: 1 solution of avidin-PBS-

biotin) and then incubated for 30 min at RT. The tissues were washed 3 times in PBS for

5 min each. Colour development was achieved via incubation in 3,3'-diaminobenzidine 4-

HCI @AB) (Vector Laboratories, Burlingarne, CA) and H20, for 5 min at RT. The

tissues were then washed 3 times in DDW for 1 min each followed by once in DDW for 5

min, after which the tissues were dehydrated through a graded ethanol series of 2 min in

each of 20%, 40%, 80%. and 100% ethanol. Finally , the tissues were allowed to dry

completely before being mounted under a g l a s coverslip using an organic mounting media

(Permount, Fisher Scientific, Fair Law, NJ). A positive result was observed as a dark

brown cellular staining. Positive cellular staining of the apoptotic related proteins BAD,

Bcl-x,, CPP-32, and Ich-1L will be localized ptimtrily to the cytoplasm, Fas to the cell

surface, TIAR to the nucleus, and p53 to both the cytoplasm and nucleus.

(6) Immunohistochemistry Controls

(a) Positive Control

A breast carcinoma slide was used as a positive control for staining of the p53

protein. Breast carcinoma and most cancer tissues in general upregulate wild-type or

mutated versions of the p53 gene. Hence these types of tissues can serve as positive

controls for p53. The tissue was processed exactly as described above for the test

samples. Positive controls for the remaining apoptotic regulatory proteins were not

available.

(b) Negative Control

Negative controls were run with every experiment in order to confirm a minimal

quantity of non-specific background staining. A negative control was run by substituting

the primary anti-apoptotic regulatory protein antibody with 20 pL of TRPE total IgG in a

1 :20 dilution with Tris buffer. The negative control was processed as described above for

the test samples.

RESULTS

Study Population

The study population consisted of 49 patients (29 males and 20 females) including

1 1 CD, 1 1 UC, 19 non-IBD, and 8 normal patients (Appendix B- table 1).

(1) Crohn's Disease

All CD patients had been previously diagnosed. The patients had longstanding CD

with duration of disease ranging from 2 y 10 mo to 20 y. The mean disease duration was

10 y 3 mo (median 9 y 6 mo). The disease duration for one CD case was unavailable.

There were 5 males and 6 females with ages from 16 to 76 y. 9 CD patients were

receiving treatment and 1 was not on any medication at the time of colonoscopy.

Treatment information for 1 CD patient was unavailable. Colonoscopic examination and

analysis of the histopathology revealed that the majority (7 of 1 1) of CD patients had

sections of bowel that displayed evidence of both acute and chronic inflammation (i.e

involved bowel). lnvolved bowel in CD has characteristic colonoscopic and histologie

features. The earliest manifestation of CD observed using colonoscopy is the aphrhous

ulcer. Apthous ulcers can grow to form large stellate or linear ulcers as the disease

progresses. Furthemore, intersecting longitudinal and transverse ulcers give involved

mucosa a "cobblestone" appearance. In CD, areas of involved bowel are typically

interspersed with normal mucosa or "skip areas." Histologic analysis of CD biopsy

specimens fiom involved bowel with active inflammation reveal an intense trammural

infiltrate of neutrophils. Cryptitis and crypt abscesses are observed in more severel y

inflamed CD mucosa. Along with active inflammation are usually signs of chronicity

within the involved mucosa including lymphoid aggregates, plasma cells, mast cells, and

eosinophils in the lamina propria. The presence of granulomas in histologie preparations

are indicative of CD. Inactive or quiescent CD mucosa has less inflammatory infiltrate and

the mucosal architecture is distorted. The number of crypts are reduced and remaining

crypts may be shortened and branched.

The study population also included patients with multiple sections of involved

bowel (6 of I l ) and patients in which inflammation was localized to the ileum only (4 of

1 1-inflammation in 2 of 4 of these patients was non-specific). One patient (1 of 1 1 ) had

acute inflammation only in the sigmoid colon that resulted in complete ulceration of the

mucosa. The study population included CD cases with either active chronic colitis or

inactive chronic colitis. The inflammation in patients with active disease (8 of 1 1) was

relatively mild with only one case ( 1 of I l ) having inflammation classified as moderate to

severe (with ulcers observed on histopathologic specimens). A complete description of al1

CD patients c m be found in Table 1 (see patients 1 - 10.49).

(2) Ulcerative Colitis

Al1 UC patients had been previously diagnosed. The patients had longstanding

UC with duration of disease ranging fiom 2 y 6 mo to 32 y. The rnean disease duration

was 13 y 1 1 mo (median 13 y). The disease duration for two UC cases was unavailable.

There were 9 males and 2 females with ages from 29 to 73 y. 6 UC cases were receiving

treatment and 3 were not on any medication at the time of colonoscopy. Treatment

information for 2 UC patients was unavailable. Colonoscopic examination and analysis of

the histopathology revealed that the majority (9 of 1 1) of UC patients had sections of

bowel that displayed evidence of both acute and chronic inflammation (i.e. involved

bowel). The earliest manifestation of UC observed using colonoscopy is the loss of fine

vascular pattern in affected mucosa. The mucosa becomes diffisely erythematous and

edematous. As UC becomes more severe, blood vessels cannot be seen at d l . The

mucosa bleeds spontaneously and small ulcerations begin to appear. Macroscopic

inflammation is contiguous begiming at the rectum and extending proximally to a variable

point at which overt pathology disappears and normal mucosa appears. The histologie

characteristics of acute and chronic inflammation in UC are similar to those observed in

CD (see above). Actively inflarned bowel is defined by an intense neutrophilic infiltration

in the mucosa and submucosa. Cryptitis is common with crypt abscesses present during

more severe inflarnmatory activity. Chronic inflammation is associated with lymphoid

aggregates, plasma cells, mast cells, and eosinophils in the lamina propria. Inactive UC

'2

mucosa has a distorted architecture with reduced nurnbers of crypts. The remaining crypts

arc usually shortencd and branched. Crypt drophy, poly rnorphonuclem leukocy tes in the

mucosal epithelium, and surface erosions are al1 more prevalent in UC than in CD.

The study population also included patients with multiple sections of involved

bowel(8 of 1 1) and patients in which inflammation was localized to a single bowel area (2

of 1 1-inflammation in 1 of 2 of these cases was non-specific). Histologie analysis of

biopsies obtained throughout the entire bowel of a single UC patient (1 of 1 1 ) with acute

and chronic inflammation confirmed pancolitis. The bowel of another patient (1 of 1 1)

displayed no specific abnomality (i.e. no acute or chronic inflammation suggestive of

UC). The inflammation in patients with active disease (9 of 1 1) was classified as mild (5

of 9), moderate to severe (3 of 9), and mild to moderate to severe (1 of 9). A complete

description of al1 UC patients can be found in Table 1 (see patients 1 1-20,48).

(3) Non-Inflammatory Bowel Disease

Patients diagnosed as having non-IBD presented with a variety of GI complaints.

Disease duration for the non-IBD group ranged from 13 d to 15 y. Disease durations for

3 cases of non-IBD were unavailable. There were 10 males and 9 females with ages from

25 to 83 y. 9 non-IBD cases were receiving treatrnent and 8 were not on any medication

at the timc of colonoscopy. Treatment information for 2 non-IBD patients was

unavailable. A complete description of al1 non-IBD patients can be found in Table 1 (see

patients 2 1 -39).

(4) Normal

All normal patients undenvent colonoscopy as part of a surveillance program for

colon cancer. There were 5 males and 3 females with ages from 28 to 7 1 y. 7 normal

cases were receiving some type of medication and 1 patient was not on any medication at

the time of colonoscopy. A complete description of al1 normal patients can be found in

Table 1 (see patients 40-47).

Assessrnent of Apoptosis

(N.B. - the term u-crypts is used to refer to crypts in tissue specimens that were

unoriented)

(1) Observations of HematoxylinlPhloxine/Saffroa (HPS) Stained Sections

Microscopie observation of HPS stained tissue revealed very little morphological

evidence of apoptosis (i.e. apoptotic bodies). Although apoptotic events were not

quantitated it was observed that the fiequency of apoptosis was much less than one

apoptotic event per crypt. This frequency did not appear to vary drarnatically across

diseases (i.e. CD, UC, non-IBD, normals), between involved or uninvolved IBD bowel. or

along the length of bowel from cecum to rectum. Apoptotic bodies were located primarily

within the crypt epitheiium and to a lesser extent in the surface epithelium. The great

majority of cells within the whole of the mucosa remained morphologically normal. Figure

5A depicts a section of bowel isolated from the inflamed cecum of a UC patient (patient

16). The crypt outlined by the box contains apoptotic bodies, illustrated at higher

magnification in Figure 5B. The arrow in Figure 58 is pointing to a cluster of dmk ovoid

structures of varying size which are classic apoptotic bodies, most likely the result of an

apoptotic enterocyte. In general, there was minimal evidence for apoptosis by light

microscopy in the large bowel mucosa of CD, UC, and non-IBD patients and normal

control subjects.

Figure 5 Analvsis of Hematox~lid Phloxine/ Saffkon IHPS) Stained Tissue. Light

microscopic analysis of HPS stained tissue revealed very little evidence of apoptotic

events. (A) HPS stained tissue fiom the inflamed cecum of a UC patient (patient 16).

Inflammation is indicated by the increase in cells of the lamina propria. The crypt outlined

by the black box in (A) contains apoptotic bodies which are illustrated at a higher

magnification in (B). In (B) the arrow points to a dark ovoid structure which is typical of

an apoptotic body. There are numerous other smaller apopt~tic bodies beneath the one

indicated by the arrow. These apoptotic bodies were most likely the result of an

enterocyte that has undergone apoptosis. Detection of apoptosis in HPS stained tissue did

not vary according to disease, disease activity, or bowel location. The large majority of

cells maintained a normal morphology as shown in (A) and (B). Original magnification:

(A) 60X, phase 1, (B) 1 OOX, phase 2.

(2) In Situ Detection of DNA Strand Breaks by the TUNEL Method

(a) Controls

(i) Positive and Negative TUNEL Controls

Al1 nuclei of all cells of a positive control tissue were stained by the TUNEL

procedure (Figure 6A). A positive ce11 stains darkly relative to background (light staining

or absent staining). In Figure 6A the staining appears black against a grey background.

The TUNEL procedure labels fragmented DNA and hence the positive staining observed

was localized to the nucleus.

Negative controls showed no tissue staining (Figure 68).

(ii) Otber Controls

Initial TUNEL staining of IBD, non-IBD, and normal tissues indicated fragmented

DNA in a large majority of cells in the mucosa. Intial concems were that the TUNEL

procedure was flawed and staining al1 cells without regard for the presence of DNA

Fragmentation. In order to confirm the qualitative accuracy of the TUNEL staining results

the procedure p as applied to non-mucosal tissues. TUNEL analysis of tonsillar (Figure

7A) and appendix (Figure 7B) tissues localized positive staining to small clusters of cells

or to single cells within what could be considered "normal limits" for apoptotic events

during normal tissue physiology. TUNEL staining of tonsillar tissue indicated positive

cells in srnall clusters scattered throughout the tissue section (Figure 7A). TUNEL

positive cells were darkly stained and distributed singly throughout the appendix tissue

sample (Figure 7B). The remaining cells of both the tonsil and appendix tissue were either

very lightly stained or unstained (Le. TUNEL negative). TUNEL staining of non-mucosal

Figure 6 TUNEL Controls-1. (A) DNase positive control. A DNase positive control was

completed with every expriment and was a tissue sample that had been treated with

DNase in order to create abundant quantities of 3' OH ends (moiety to which TUNEL

procedure is directed). In (A), al1 cells of a DNase positive control stain darkly ensuring

that the reagents are working. The DNase positive control dso defi ned a TUNEL positive

cell. A TUNEL positive ce11 was a ce11 that was darkly stained relative to background as

seen in (A). The exact localization of staining within the cell was dificult to discem as

cells appeared to be stained completely. (B) Negative TüNEL control. A negative

control was achieved by replacing the TdT enzyme with water in the working strength

TdT solution during the TUNEL protocol. There is no tissue staining seen in (B). The

negative TUNEL control ensured a minimal amount of background staining. Original

magnification: (A) 40X, (B) 40X.

Figure 7 TUNEL Controls-II. Others TüNEL controls were implemented to confirm that

TUNEL staining of IBD test tissues was authentic and not simply a general phenornena of

the TUNEL staining procedure. (A) Tonsil tissue stained by TLTNEL. Positive ce11

staining was localized to single cells or small clusten of cells. The vast percentage of cells

within the tond tissue were TUNEL negative. (B) Appendix tissue stained by TUNEL.

Single scattered cells were stained intensely. TLJNEL positive cells in the tonsil and

appendix tissues were localized to small clusten of cells or to single cells. Staining was

specific to certain cells and not to al1 cells thereby ensuring the validity of IBD test tissues

stained by the TüNEL protocol. Original magnification: (A) IOX. phase 2, (B) IOX,

phase 2.

tissues was localized to specific cells and not to al1 cells. Therefore, the pattern of

TUNEL staining observed in IBD, non-IBD, and normal tissues was concluded to be

accurate. The TUNEL method was properly labelling cells with fiagmented DNA.

(b) TUNEL Analysis of IBD and Normal Controls

(i) TUNEL Analysis of Uninvolved and lnvolved IBD Tissue

The TUNEL positive staining was in general reduced dramatically in the crypts of

involved IBD tissue relative to the uninvolved IBD and normal control mucosal

specimens.

CD

Figure 8 is a series of 3 photomicrographs at increasing magnifications of TUNEL

staining in the crypt epithelium of an uninvolved CD bowel specirnen (patient 8). The full

thickness mucosa (Figure 8A) showed no inflammatory activity in the lamina propria.

Cells in the bottom half of oriented crypts were darkly stained with cells in the top half of

oriented crypts and surface epithelium stained very lightly (TUNEL negative). All cells of

the u-crypts exhibited intense TLrNEL staining (Figure 8A and B). A small percentage of

lamina propria cells, particularly those underlying the surface epithelium were positively

stained (Figure 8A). High magnification of the basal crypt (Figure 8C) illustrated the high

level of DNA fragmentation found in the cells in the bottom portions of oriented crypts.

The exact location of positive TUNEL staining within the cells was dificult to discern

even at higher magnifications and remains more of an overall cellular staining pattern.

Figure 9 is another series of 3 photomicrographs at increasing magnifications of

TUNEL staining in the crypt epithelium of an inflamed CD bowel specimen (patient 9).

Figure 8 TUNEL Analvsis of Uninvolved CD Mucosa. Figure 8 illustrates at increasing

magnifications the TUNEL staining of an uninvolved rectal specimen fiom a CD patient

(patient 8). The full thickness mucosa in (A) shows TUNEL positive cells within the

bottom half of oriented crypts. The surface epithelium and superficial crypt sections

illustrate negative TUNEL staining. The vast majority of cells within the crypts are

stained dark as seen in (A) and (B). High magnification of a basal crypt in (C) indicates

intense TUNEL staining of epithelial cells containing abundant DNA fragmentation. The

lamina propria is not inflamed and contains TUNEL positive cells located pnmady

beneath the surface epithelium (A). Staining within TUNEL positive cells was difficult to

localize even at higher magnifications. It was not possible to pinpoint only nuclear or

cytoplasmic staining. Original magnification: (A) 20X, (B) 40X, (C) 1 OOX.

Figure 9 TUNEL Analvsis of Involved CD Mucosa. TUNEL Analysis of inflarned tissue

from the mid-transverse splenic colon of a CD patient (patient 9) resulted in reduced

TUNEL staining of epithelial cells particularly in the bottom half of oriented crypts (see

outlined box in [A]). Figure (B) is a higher magnification of the mucosa outlined in (A).

Only a very limited number of cells in the extreme basal portion of the crypt are TUNEL

positive. Cells along the sides of the crypt exhibit very light staining (B). TUNEL

staining within cells of the u-crypts was decreased drarnatically relative to the uninvolved

mucosa with only a very limited number of u-crypt enterocytes darkly stained. Figure (C)

focuses at higher magnification on the mucosa outlined in (B). Enterocytes in the extreme

boaom portion of oriented crypts are only lightly stained, in contnst to observations made

of crypts in the uninvolved mucosa. The lamina propria is inflamed showing an increase in

TUNEL positive cells (A). These cells are located beneath the surface epithelium and

sunounding the bottom portion of the oriented crypt in figure (A). The staining pattern of

single TüNEL positive cells was difficult to discem even at higher magnifications and was

observed to bc dark staining of the cntire cell. Original magnification: (A) 30X, (B) l O X ,

(C) 100X.

There were increased numbers of inflarnmatory cells within the lamina propria (Figure

9A). Crypt epithelial cells were lightly stained throughout the length of the crypts with an

especially noticeable reduction in staining intensity in the bottom half of the crypts (Figure

9B). Cells of the u-crypts were also lightly stained with only an extremely limited number

of cells indicated as TUNEL positive (Figure 9A and B). There was an increase in

TUNEL positive cells within the lamina propria due primarily to the elevated inflammatocy

ce11 numbers in this mucosal cornpartment. High magnification of the basal crypt cells

(Figure 9C) showed a decrease in staining intensity relative to crypts of the uninvolved CD

bowel.

UC

The TUNEL analysis of crypts in the bowel of UC patients was very similar to that

observed for CD. Figure 10 is representative of the T W E L staining pattern in the

uninvolved bowel of UC patients (patient 20). Figure 10A is a low magnification

photomicrograph showing dark TUNEL staining in cells in the bottom third of the

oriented crypt (see box in Figure 10.4). The top two thirds of the crypt and the surface

epitheliurn show no positive staining. There is no inflarnmatory activity in the lamina

propria. Only a random few cells in the lamina propria are darkly stained. Figures 1OB

and 1 OC are higher magnifications focused on the crypt outlined in Figure 1 OA. Figure

1 OB illustrates more clearly the basal localization and staining intensity of TUNEL positive

cells in the crypts. High magnification of the bottom third of the crypt (Figure 10C)

depicts dark cellular staining in the majority of cells in this area. TUNEL positive cells at

high magnification (Figure 1 OC) showed dark staining that was Iocalized primarily to the

Figure 10 TUNEL Analvsis of Uninvolved UC Mucosa. TUNEL positive staining of

uninvolved mucosa fiom the hepatic flexure of a UC patient (patient 20) localized to the

bottom and middle sections of oriented crypts (A), (B). The full thickness mucosa and

crypt outlined by the box in (A) is represented at higher magnification in (B). In both (A)

and (B) the cells of the bottom third of the oriented crypt are stained darkly. A few cells

extending into the middle section of the crypt are also TUNEL positive. There is no

TUNEL positive staining in the surface epithelium or upper half of the otienied crypt

(surface epithelial cells that appear to be TUNEL positive are most likely debtis fiom the

staining procedure). A very limited number of cells scattered throughout the lamina

propna are stained darkly. The basal section of the oriented crypt isolated by the outlined

box in (B) is illustrated at higher magnification in (C). Positive TUNEL staining of the

basal crypt enterocytes is dark. but not localized specifically to the nucleus or cytoplasm.

Original magnification: (A) 20X, (B) 40X, ( C ) 100X.

nucleus. TUNEL positive cells were absent in the crypts of UC involved bowel (patient

20) (Figure 1 1). In Figure 1 1A the mucosa is inflamed indicated by the increase in cells in

the lamina propria. There was an elevation in TUNEL positive cells in the lamina propria

compared to the uninvolved bowel (Figures 1 1A and Figure 10A) and this is likely a result

of the inflammatory process. The TüNEL positive cells were scattered throughout the

lamina propria. The bottom third of the crypts contained zero TUNEL positive cells (see

outlined boxes in Figure 1 1 A and 1 1 B). U-crypts were also free of TUNEL positive

staining. Figure 1 1C is a high magnification photomicrograph of the extreme bottorn

section of a crypt, clearly illustrating the complete absence of dark staining in this area.

(ii) TUNEL Analysis of Normal Control Tissue

Figure 12 is the TUNEL staining pattern typical of normal undiseased colonic

mucosa (patient 45). Normal bowel at Iow magnifications contained intense TUNEL

staining in cells of the bottom one half of oriented crypts (Figure 12A and 12B). U-crypts

were a mixture of TUNEL positive and negative cells (Figure 12A and 12B). A small

percentrige of cells were darklp stained and scattered throughout the lamina proprin

(Figure 12A). The surface epithelium was devoid of TUNEL positive cells. Figure 12C is

a high magnification photomicrograph of the outlined box in Figure 128. The basal

sections of normal oriented crypts contained intense staining of cells with extensive DNA

fragmentation.

(c) TUNEL Analysis and Association with Therapy States

The TUNEL technique was applied to bowel tissue removed fiom patients that

were receiving therapy at the time of colonoscopy. These TUNEL staining results were

Figure 11 TUNEL Analysis of Involved UC Mucosa. Figure (A) is a TUNEL stained

specimen from the involved rectum of a UC patient (patient 20). The lamina propria

contains an increase in cells due to the inflarnmatory process. The lamina propria also

contains an increase in TUNEL positive cells compared to UC uninvolved mucosa.

Darkly stained cells are scattered throughout the entire lamina propria. The small section

of surface epithelium in (A) contains cells with little or no TUNEL staining. The focus of

the outlined box in (A) is the lack of dark staining in the basal crypt. Figure (B) illustrates

the mucosa outlined in (A) at a higher magnification. Basal crypt enterocytes display little

or no staining with no crypt cells indicated as TUNEL positive. Some lamina propria cells

surrounding the crypt are TWEL positive. The outlined box in (B), magnified and

displayed in (C) illustrates enterocytes at the extreme base of a crypt with very light

staining. The majority of crypt enterocytes contain no staining. Original magnification:

(A) 20X, (B) 40X, (C) 100X.

Figure 12 TUNEL Analvsis of Normal Mucosa. TUNEL analysis of sigmoid tissue from

a normal control patient (patient 45) submitted for surveillance for colon cancer is shown.

In (A), intense TUNEL staining of cells is localized to the bottom half of the oriented

crypt outlined by the box. The surface epithelium has very little positive staining and the

top half of the oriented crypt contains a few TUNEL positive cells. U-crypts are a

mixture of TUNEL positive and negative cells. In (B), the mucosa outlined in (A) is

magnified and illustrates the basal localization of TUNEL positive cells in the crypt. It is

possible to decipher a nuclear staining pattern particularly within TUNEL positive cells

localized to the middle section of the oriented crypt and also within some TIMEL positive

cells of the lamina propria. The mucosa outlined in (B) is displayed in (C) at a higher

magnification. Al1 cells in this portion of the crypt are stained intensely. There is a

nuclear localization of staining seen in some of the cells. The remaining cells are also

stained intensely, but a specific cellular staining panem is not obvious. TUNEL positive

cells are almost exclusively confined to the basal crypt as s h o w in (C). Original

magnification: (A) 70X, (B) 40X, (C) 100X.

then compared to TUNEL stained tissue fiom patients receiving no prior therapy at the

time of colonoscopy. Although the overall study design was not controlled specifically to

determine the effects of therapy on TUNEL staining in mucosal tissues, preliminary

observations indicated that TUNEL staining was not affected by therapy regiments.

Figure 13A and 13B are tissues isolated fiom the inflarned transverse colon of one patient

on no medication (patient 13) and one patient receiving salofalk (4 g/d) and budesonide

enemas daily (patient 1 1 ), respectively. In both circumstances, TüNEL analysis revealed

dark staining of cells in the lamina propria and light or absent staining of the crypt and

surface enterocytes, consistent with inflarned bowel tissue (see above).

(d) TUNEL Analysis and the Effect of Anatomic Location

CD

TUNEL analysis of mucosa showed consistent results throughout the small and

large bowel of a CD patient (patient 8) (Figure 14). An inflarned ileal tissue (Figure 14B)

presented elevated numbers of TUNEL positive cells within the lamina propria. The u-

crypts of the ileal specimen contained a small fraction of cells with fiagrnented DN.4 with

the majority of cells only very lightly stained. The large bowel specimens (Figure 14C-

14H) were macroscopically normal. The surface epithelium of al1 colonic sections

contained very little or absent staining as did the top half oriented crypts. Dark staining of

cells was localized to the bottom half of oriented crypts, particularly in the tissue from the

hepatic flexure (Figure 14C), mid transverse colon (Figure 14D), and splenic flexure

(Figure 14E). The u-crypts were a mixture of TUNEL positive and negative cells. Many

of the lamina propna cells underlying the surface epitheliurn were darkly stained (Figure

Figure 13 TUNEL Anal~sis and Association with T h e r a ~ ~ States. Figure (A) and (B) are

results fiom TUNEL analysis completed on involved bowel tissue isolated frorn a UC

patient (patient 13) receiving no medication. and a UC patient (patient I 1) on a therapy

regiment for IBD at the time of colonoscopy, respectively. The inflammation in both

sections is indicated by the large increase in cells in the lamina propria. TUNEL positive

staining of tissue fiom both patients is localized primarily to cells of the lamina propria.

The TUNEL positive cells in this mucosal compartment are numerous, stained darkly, and

scattered throughout the entire thickness of the mucosa. Only a very small fraction of

crypt enterocytes are stained deeply with varying locations along the length of the crypt.

The large majority of crypt epithelial cells are TUNEL negative. The surface epithelium in

(A) has -6 cells that are stained darkly and indicated as TUNEL positive with remaining

surface epitheliurn containing no TLJNEL positive cells. The staining in both (A) and (B)

is very similar and resembles the staining of involved UC tissue regardless of the drug

therapy . Original rnagnification: (A) 1 OX, phase 2, (B) 1 OX, phase 2.

Figure 14 Anatomical Anal~sis of TUNEL in CD Bowel. TUNEL was performed on a

series of biopsy specimens isolated from different anatomical locations fiom the bowel of a

CD patient (patient 8). (A) DNase positive control. (B) neoterminal ileum, (C) hepatic

flexure, (D) mid-transverse colon, (E) splenic flexure, (F) descending colon, (G) sigmoid

colon, (H) rectum. The neoteminal ileum (B) shows active inflammation while the

remainder of the bowel (C)-(H) is microscopically notmal. The lamina propria of the

neoteminal ileum contains an increase in cells due to inflammation. There are numerous

darkly stained cells scattered throughout the lamina propria. The u-crypts in (B) display a

small fraction of cells with fragmented DNA. TUNEL staining in the colon (C-H) is

consistent with macroscopically normal CD bowel. The surface epithelium contains very

little TUNEL staining. T W E L positive enterocytes are localized to the bottom half of

oriented crypts (C,D,E) and mixed with TUNEL negative cells in the u-crypts (F). There

is no inflammatory activity in the lamina propria of the large bowel. Lamina propria cells

directly undemeath the luminal epithelium exhibit dark staining. Original magnification:

(A)-(H) 20X

14C- 14F). TUNEL staining of tissue from the sigrnoid colon (Figure 14G) and rectum

(Figure 14H) appeared light to moderate, however relative to the DNase control (Figure

14A) many of the enterocytes in the bottom half of oriented crypts were positive as were

some of the lamina propria cells undemeath the surface epithelium (Figure 14G and 14H).

UC

TUNEL analysis of mucosa showed consistent results throughout the large bowel

of a UC patient (patient 1 9) (Figure 1 5). Mucosal tissue from the ascending colon that

showed no indication of inflammation displayed positive staining of cells within the lamina

propria (Figure 158). Numerous u-crypt enterocytes were also TUNEL positive (Figure

15B). Specimens from the descending colon (Figure 15C), sigmoid colon (Figure 1 5D

and 1 SE), and rectum (Figure 15F) had drastically reduced quantities of TUNEL staining

particularly within the crypts. Oriented crypts had no positive staining along their entire

length (Figure 15D). Surface epithelium was devoid of TUNEL positive cells. The

inflarned tissue similarly had reduced numbers of darkly stained cells in the lamina propria

(Figure 15C- 15E).

Normal

TUNEL analysis of rnucosa showed consistent results throughout the large bowel

of a normal control subject (patient 44) (Figure 16). The surface epithelium contained no

TUNEL positive cells in any of the large bowel locations (Figure 16B- 16H). The

specimens nom the ascending colon (Figure MC), hepatic flexure (Figure 16D), and

transverse colon (Figure 16E) illustrated dark staining of enterocytes extending from the

base of the crypt upwards and into the middle sections of the crypts. TUNEL positive

Figure 15 Anatomical Analvsis of TUNEL in UC Bowel. TUNEL was performed on a

series of biopsy specimens isolated from different anatomical locations from the bowel of a

UC patient (patient 19). (A) DNase positive control, (B) ascending colon, (C) descending

colon, (D) sigmoid colon, (E) sigmoid colon, (F) rectum. Mucosa fiom the ascending

colon is microscopically normal while the remainder of the large bowel (C-F) displays

active inflammation. Dark staining of cells in the ascending colon mucosa is localized to a

large number of cells distributed along the bottom and middle sections of the lamina

propria (B). The u-crypts also contain a large percentage of TWEL positive cells that

are admixed with enterocytes showing liale or no TUNEL staining. TUNEL staining in

general then is decreased in al1 mucosal specimens throughout the remainder of the bowel

consistent with an involved IBD large intestine. The u-crypts in (C) present dark TUNEL

staining in a very limited number of enterocytes and the lamina propria has drastically

reduced numbers of TUNEL positive cells relative to the uninvolved ascending colon

specimen. The inflamed sigmoid colon (D,E) displays very little TUNEL staining in either

the crypts or the lamina propria. Staining of the rectal specimcn (F) appears to increasc

fiom the sigmoid colon, however, relative to the DNase positive control, this intenrity of

staining should be considered negative staining (DNase positive control defines positive

staining for al1 tissues for that respective experiment). Original magnification: (A)-(F)

20X.

Figure 16 Anatomical Analysis of TUNEL in Normal Control Bowel. TUNEL was

perfonned on a series of biopsy specimens isolated from different anatomical locations

fiom the bowel of a normal control patient subrnitted for surveillance for colon cancer

(patient 44). (A) DNase positive control, (B) cecum, (C) ascending colon, (D) hepatic

flexure, (E) transverse colon, (F) splenic flexure. (G) descending colon, (H) sigmoid

colon. TüNEL analysis throughout the large bowel revealed staining results consistent

with previous observations of staining of normal control tissue. In general, TUNEL

positive enterocytes were contained in the bottom half of oriented crypts (C,D,E). The

lamina propria of the cecum (B) has an increase in cells relative to the rest of the bowel,

but this is not unusual activity for this area of the bowel. The lamina propria also presents

numerous TüNEL positive cells scattered throughout the thickness of the mucosa (B).

Lamina propria staining decreases frorn the hepatic flexure (D) to the descending colon

(G). TUNEL positive cells, if present, are located directly beneath the surface epithelium

(D,E). There is no dark TUNEL staining in the surface epithelium in any bowel section

(B-F). A large portion of enterocytcs within u-crypts are TUNEL positive (B,H). The

TUNEL staining in figure G is light with no cells stained darkly. Original rnagnification:

(A)-(H) 20X.

cells were scattered extensively thoughout the lamina propria of the cecum (Figure 16B)

and ascending colon (Figure 16C). The amount of lamina propria staining is reduced

extending fiom the hepatic flexure (Figure 16D) to the descending colon (Figure 16G).

The u-crypts were also extensively stained (Figure 168 and 16H). The TUNEL staining

in Figure 16G is light with no cells darkly stained. This should be considered an artifact

and not a true indication of the cellular DNA fragmentation that may be ongoing in this

large bowel location.

Non-IBD

Anatomical analysis of TüNEL for the non-IBD group included specimens from

six different patients with GL complaints including diarrhea (patients 22,23.26,32)

collagenous colitis (patient 25), and campylobacter colitis (patient 28) (Figure 17). Five

of the specimens were inflamed (Figure 1 7B- 17F) with one being microscopically normal

(Figure 17A). TUNEL staining within the diarrhea group (inflarned) (Figure 17C- 17E)

was consistent. There was extensive and dark staining of lamina propria cells in the

rectum (Figure 1 7C) and sigmoid colon (Figure 17D and 17E) tissue. A large fraction of

cells of u-crypts in the sigmoid colon were TUNEL positive (Figure 17D and 17E).

TUNEL staining in uîrypts of the rectum specimen (diarrhea, inflamed) was light with

only a very few number of cells containing DNA fragmentation. The microscopically

normal tissue fiom a patient with diarrhea exhibited typical staining patterns (Figure 17A).

Crypt enterocytes in the bottom half of oriented crypts exhibited positive staining. The

surface epithelium and superficial crypt epithelial cells showed very light staining. A small

Fraction of cells scattered throughout the lamina propria were also stained darkly. A

Figure 17 Anatomical Analvsis of TUNEL of Non-IBD Bowel Swcimens. TUNEL was

performed on a series of biopsy specimens isolated from different anatomical locations

fiom patients with a variety of GI cornplaints including diarrhea, collagenous colitis, and

carnpylobacter colitis. (A) descending colon fiom patient with diarrhea (B) rectum from

patient with collagenous colitis, (C) rectum from patient with chronic diarrhea, (D)

sigmoid colon from patient with chronic diarrhea, (E) sigmoid colon from patient with

recurrent diarrhea, (F) sigmoid colon fiom patient with campylobacter colitis. Five of the

specirnens were inflamed (B-F) with one being microscopically normal (A). TUNEL

positive staining in the normal descending colon (A) is localized to cells in the bottom half

of oriented crypts and to a limited nurnber of lamina propria cells surrounding the base of

ciypts and beneath the surface epithelium. The inflamed sigmoid colon from patients with

chronic (D) and recurrent (E) diarrhea has dark and extensive staining of cells in the u-

crypts and lamina propria. The inflamed rectum specimen of a patient with chronic

diarrhea (C) also shows dark staining of approximately half of lamina propria cells.

Staining in the u-crypts is rcduccd compared to the sigmoid specimens ( D E ) with only a

scattered few crypt enterocytes indicated as TUNEL positive. The rectal specimen from

the coilagenous colitis patient (B) shows dark staining of a large fraction of cells in the u-

crypts and lamina propria. Similady, dark TUNEL staining is found throughout the lamina

propria and u-crypts of the sigmoid colon of the patient with campylobacter colitis (F).

Original magnification: (A)-(F) 20X.

rectum specimen fiom the patient with collagenous colitis displayed dark staining of cells

in both the lamina propria and u-crypts (Figure 178). Inflarned sigmoid colon tissue from

the carnpylobacter colitis patient was stained extensively (Figure 17F). Al1 cells within the

u-crypts were stained darkly as were cells of the lamina propria.

(3) Quantification of TUNEL Positive Cells in tbe IBD and Normal Mucosa

(a) Manual versus Automated Cell Counts

Data were collected to ensure that the cornputer counts of thresheld objects (cells)

were indeed giving an accurate picture of the relative nurnber of cells containing

fiagmented DNA (Le. TUNEL positive). Therefore, TUNEL positive cells were

enumerated manually for a series of increasingly larger stained tissue sections and

compared to computer totals for the exact sarne tissue locations. Manual (actual) counts

were consistently higher than automated tallies and this was a result of the thresholding

mechanism. At the magnification used (20X) to obtain data, many thresheld cells would

be touching cach other and as a result, two or more cells would be counted by the imaging

prograrn as one cell. Figure 18 is a linear regression of computer versus manual counts at

95% confidence based on data collected to answer the question as to whether the

computer totals correlated significantly with the manual (actual) counts. The manual and

computer variables were determined to be highly correlated with r=0.97798 @=0.00).

Hence, computer counts of TUNEL positive cells were accurate relative to manual

(actual) counts. The counting capacity of the software was therefore concluded to be

applicable to generating data in this study.

Figure 18 Correlation of Two Measurements: Manual vs Com~uter Counts. A linear

regression at 95% confidence of TUNEL positive cells enumerated manually versus

automatically (using "Northem Exposure" Image Analysis software) produced a

correlation coefficient of r=0.97798 at p < 0.05000. Such a high correlation at 95%

confidence verified the applicability of the counting capacity of the Northern Exposure

computer program.

X Regression 95% confid.

20 60 100 140 180

COMPUTER IMAGING COUNTS

(b) Percentage of TUNEL Positive Cells in the IBD and Normal Mucosa

TUNEL stained slides fiom four patients with UC, three with CD, and one normal

patient under colonic surveillance were digitized and subsequently analyzed quantitatively

using the "Northem Exposure" (Empix Imaging, Inc., Mississauga, ON) image analysis

software. Involved and uninvolved specimens existed within each disease classification

(UC, CD. normal) and these were consequently pooled and averaged with respect to

surface and crypt epithelium and lamina propria. Speci ficall y, 8 involved (n=8) and 1 3

uninvolved (n= 13) , 5 involved (n=5) and 9 uninvolved (n=9), and 2 undiseased biopsy

specimens defined the disease activity base for UC, CD, and normal, respectively. The

raw data collected were transformed into percent TUNEL positive cells and then graphed

as shown in Figures 19A and 198. The numbers support the qualitative observations

docurnented above. The percentage of TUNEL positive cells was greater in the

macroscopically uninvolved crypts of CD when compared to CD crypt epithelium that was

involved (2 1.79% vs. 4.37%). Such was also the case for UC (14.57% vs. 8.46%). The

surface epitheliurn displayed a similar trend with CD and UC uninvolved surfacc mucosa

harbouring a greater percentage of TüNEL positive cells when maiched against involved

lwninal epithelium (1 1.32% vs. 4.34% for CD, 15.73% vs. 3.96% for UC). Normal

specimens from patients under colonic surveillance had percentages of TUNEL positive

cells for surface and crypt epithelium of 16.28% and 20.36%, respectively. These values

were also significantly above those calculated for CD and UC inflamed bowel lending

m e r support to the notion that apoptosis is somehow downregulated in the actively

diseased srnall and large intestine of IBD patients.

Figure 19 Percent TLMEL Positive Cells in the Involved and Uninvolved IBD Mucosa.

(A) Bar graph of % TUNEL positive cells/tissue unit vs tissue diagnosis for UC. (B) Bar

graph of % TUNEL positive cells/tissue unit vs tissue diagnosis for CD. Solid, clear. and

hatched bars represent crypt epitheli~m~ surface epithelium, and lamina propria,

respectively. The percentage of TUNEL positive cells/tissue unit in the crypt epthelium

was greater in the uninvolved CD and UC mucosa compared to crypt epithelium of

involved IBD mucosa (2 1.79% vs. 4.37% for CD, 14.57% vs. 8.46% for UC). This was

also the trend for the percentage of TUNEL positive cells/tissue unit in the surface

epithelium (1 1.32% vs. 4.34% for CD, 15.73% vs. 3.96% for UC). The bowel of normal

control subjects had percentages of TUNEL positive cellshissue unit for surface and crypt

epithelium of 16.28% and 20.36%. respectively. These values were also greater than the

percentages calculated for CD and UC inflamed bowel. N values represent the number of

image analysis measurements averaged for each tissue diagnosis. The bars represent the

standard error of the mean for each group of tissue analyses. The percentage of TUNEL

positive cells/tissue unit values were not significant.

(A) Ulcerative colitis crypt epitheliurn

O surface epithelium lamina propria

- crypt epithelium

O surface epithelium ESl lamina propria

(4) Other Studies of Apoptosis

(a) Electron Microscopy

Electron rnicroscopic analysis of tissue sections isoiated from inflamed portions

and macroscopically normal sections of colonic mucosa fiom a UC patient (patient 48)

revealed that the fiequency of apoptotic features were similar to that documented for the

HPS stained sections. That is, regardless of disease activity, histologic and/or

colonoscopic examination, prominent morphologie features of apoptosis were extremely

iirnited (Figure 20A). Figure 20A depicts an electron micrograph from an inflamed section

of UC bowel. Ultrastructural features of crypt epithelial cells attached to the basement

membrane appeared normal. Rare ultrastructural evidence of apoptotic cells did however

exist within the epithelium (Figure 208) and lamina propria. For example, in Figure 208.

a compacted and fiagmented nucleus surrounded by condensed cytoplasm of a ce11 that

has broken contact fiom the neightboring cells most likely indicates an enterocyte

proceeding through ce11 death by apoptosis.

(b) DNA Gel Elcctrophoresis

DNA was extracted from mucosal biopsies taken from involved and uninvolved

segments of bowel fiom a CD patient (patient 49) with active disease and then run on a

gel (Figure 21). DNA from both involved (lanes 1,4,5,6) and uninvolved (lanes 2,3)

biopsy specimens separated as smears consisting of multiple sized fragments of DNA. The

bright bands at the top of lanes 2,3, and 6 indicated full length, uncut, genomic DNA. The

mono- and oligonucleosomose sized fragments of DNA generated during apoptosis

separate as a ladder of DNA on gel electrophoresis and this phenornena was not readiiy

Figure 20 Electron Microsco~v of UC Bowel Tissue. Electron microscopie analysis was

performed on biopsies isolated from a UC patient (patient 48-table 1) with active

inflammation. (A) and (B) are electron photomicrographs of UC bowel that was actively

and chronically inflamed. In (A), enterocytes with normal ultrastructural morphology are

shown attached to the basement membrane. The nuclei of these enterocytes also have a

normal ultrastructural appearance. These enterocytes display no characteristic features of

apoptosis. Although the great majority of cells within the mucosa showed no evidence of

apoptosis, rare instances of apoptotic activity did exist. For example, the ce11 illustrated in

(B) most likely resembles a ceIl proceeding through apoptosis. The compacted cell has

detached fiom the surrounding enterocytes. The cytoplasm has condensed and the

nucleus has fragmented. Intact organelles are observed within the cytoplasm. Cellular

protuberances and plasma membranes alterations are not present. Magnification: (A)

3500X, (B) 5000X.

Figure 21 DNA Gel Electrophoresis of Extracted DNA. DNA extracted from mucosal

biopsies of involved and uninvolved areas of colon of a CD patient (patient 49) was nin on

a 0.8% agarose gel. Lane 1 : Ileocecal region of bowel. Lane 2: ileocecal region of bowel,

Lane 3: transverse colon, Lane 4: sigmoid colon, Lane 5: rectum, Lane 6: rectum.

Biopsies fiom the ileocecal region (lane 1), sigmoid colon (lane 4), and rectum (lane 5 and

6) were from inflamed sections of bowel. Biopsies from the ileocecal region (iane 2) and

transverse colon (lane 3) were from normal sections of bowei. Smears of DNA in al1 six

lanes indicates variable numbers and sizes of nucleic acids with no observable ladder

characteristic of apoptosis. The bright bands at the top of lanes 2. 3, and 6 represent

uncut full lenght genomic DNA. Lane M is a 100 bp molecular weight DNA ladder.

observable in any of the gel lanes (Figure 2 1).

(5) Immunohistochemistry of Apoptotic Regdatory Proteins

(a) Fas Receptor (inducer of apoptosis)

In the normal colon (Figure 22A) there was virtually no staining of cells indicating

the absence of Fas. A similar situation existed for the the uninvolved UC or CD bowel.

Figure 22B and 22C illustrate the staining or lack thereof in the inflamed transverse and

sigmoid colon, respectively. The few Fas positive cells observed were isolated in the

lamina propria (Figure 228) with the crypt epithelium being totally devoid of Fas staining.

Individual Fas positive cells within the lamina propria had dark brown staining along the

surface of the cells. The exact type of the Fas positive lamina propria cells was not

determined. The immunohistochemistry studies for al1 the apoptotic related proteins did

not characterize the exact nature of positive cells. Labelled cells could only be identified

as epithelial (surface or crypt) or lamina propria cells.

(b) p53 (inducer of apoptosis)

There was no observable pS3 staining in either normal or actively inflamed 1BD

mucosa (Figure 23). Figure 23A is a photomicrograph of breast carcinoma tissue

dernonstrating strong p5 3 reactivity . In many cancers including breast carcinoma, wild-

type or mutated p53 is upregulated and therefore such tissue can serve as a positive

control for p53. The positive staining in breast cancer ensured that the staining method

was working properly. The normal ileal tissue shown in Figure 23C shows very linle p53

staining, apart fiom occasional cells of the lamina propria (see arrows).

ression and Localization of Fas Rece~tor. (A) Normal colon (patient 40).

(B) UC inflamed transverse colon (patient 13). (C) UC inflamed sigmoid colon (patient

15). Black arrows indicate u-crypts in (A), (B), and (C). There is cornpiete absence of

Fas staining in the crypts of normal (A) and both involved (B,C) and uninvolved (not

shown) UC bowel. Isolated positivity indicating cells with surface Fas c m be seen rarely

in the lamina propria (Figure (B)). Individual Fas positive cells were stained darkly with

no specific staining pattern. Staining was not observed to be localized to either the ce11

surface. cytoplasm, or nucleus. Although not shown, a similar staining pattern was

observed in CD bowel tissue. Original magnification: (A)-(C) 20X.

Figure 23 Ex~ression and Localization of ~ 5 3 . (A) Breast carcinoma positive control,

(B) Negative control, (C) Normal ileum (patient 40). (D) UC inflamed sigmoid (patient

1 3 , (E) UC inflamed transverse (patient 13). The dark staining in (A) as indicated by the

black arrow e n s w s that the reagents are working and the absence of staining observed in

(B) demonstrates a minimal amount of background staining. The ileal tissue in (C) shows

a lack of staining in the crypt indicated by the upper black arrow with perhaps a few

scattered cells within the lamina propria marked as positive for p53 indicated by the lower

black arrow. The u-crypts (black arrows) in (D) and (E) show very little p53 staining.

The lamina propria in (E) contains a very small number of darkly stained cells. The

staining within individual p53 positive cells did not display a specific staining pattern, only

a dark staining throughout the entire cell. The uninvolved state, although not shown did

not indicate the presence of p53. CD staining (not shown) was very similar to that

docurnented above for UC. Original magnification: (A)-(E) 20X.

The pattern of positive staining within individual cells was dificult to discem and was

observed as dark staining throughout the entire cell. P53 staining was absent in the lamina

propria or epithelium of the inflamed sigmoid and transverse colon as seen in Figures 23D

and 23E, respectively.

(c) CPP-32 (easpase-3) (inducer of a poptosis)

lmmunohistochemical experirnents have localized CPP-32 positive cells in the

crypt and lurninal epithelium of a normal cecurn specimen (Figure 24A) as well as in the

crypt epithelium of a non-inflarned transverse colon from a Crohn's patient (Figure 248).

n i e darkest CPP-32 staining was concentrated in the basal sections of oriented crypts.

CPP-32 staining was reduced dramatically in the crypt and luminal epithelium of mucosa

from an inflamed CD small bowel specimen (Figure 24). CPP-32 positive ceils were

increased and scattered throughout inflarned lamina propria (Figure 24). The pattern of

CPP-32 staining in positive cells was dark and covers the entire cell.

(d) Ich-1L (inducer of apoptosis)

Anti-lch- 1 L antibodies detected the presence of ?ch- I L protein

concentrated in the crypt and lurninal epithelium of specimens biopsied from normal

cecum (Figure 25A) and UC uninvolved transverse colon (Figure 25B). Ich-1 L positive

cells in crypts were located primarily in bottom half of oriented crypts and throughout the

u-crypts (Figure 25A and 258) . Furthemore, it was observed that Ich- 1 L positive cells

were also located in cells of the lamina propria, particularly in the normal colon (Figure

25A). These Ich-1 L positive cells in the lamina propria were scattered thoughtout the

entire thickness of the normal mucosa. Staining for I c h 1 L protein in the involved

ression and Localization of CPP-32 Protease (Cas~ase-3). (A) Normal

colon cecum (patient 44). (B) CD uninvolved transverse colon (patient 8). (C) CD

inflamed ileum (patient 8). CPP-32 positive staining is localized (black arrows) to the

crypts and luminal epithelium of nomal bowel (A). In (B) oriented crypts contain CPP-32

positive cells in the basal compartments (black arrows) with little or no staining in the

lamina propria. Epithelial cells (surface and crypt) are stained darkly. The staining was

observed throughout the surface of each individual cell. A portion of the cells that have

infiltrated the lamina propria during inflammation contain CPP-32 (C-bottom black

arrow). nie staining of these positive lamina propria cells was dark and covers the

surface of the cells. Crypts of inflamed mucosa contain undetectable levels of CPP-32 (C-

upper black arrow). Original rnagnitication: (A)-(C) 20X.

Figure 25 Ex~ression and Localization of Ich-1 L. (A) Normal colon cecum (patient 44).

(B) UC uninvolved transverse colon (patient 1 8). (C) UC inflamed cecum (patient 18).

Anti-Ich- I L antibodies detected extensive arnounts of Ich- 1 L protein in the crypts and

luminal epithelium as well as the lamina propria of normal undiseased bowel as shown by

the black arrows (A). Positive Ich-IL staining was observed primarily in the crypts of

uninvolved IBD bowel as shown in (B). Dark Ich- 1L staining was concentrated in the

bottom half of oriented crypts and throughout the u-crypts (A-B). Ich-1 L positive lamina

propria cells were scattered throughout the thickness of the mucosa (A). Staining in the

UC inflamed cecum appears light with only a few single cells of the lamina propria

indicated as containing Ich-1 L (C). The staining pattern of individual Ich-1 L positive cells

was dark staining over the entire surface of the cells. Biack arrows in ali figures indicate

utrypts. Original magnification: (A)-(C) 20X.

colon is s h o w in Figure 25C. The staining appeared light (negative staining) with only a

few scattered lamina propria cells indicaied as positive for Ich- 1 L. The u-crypts and

surface epithelium in the involved colon contained no Ich-1 L positive cells.

(e) TIAR (inducer of apoptosis)

TIAR positively stained crypt epithelial cells were identified in the normal cecum

(Figure 26A) and Crohn's uninvolved transverse colon (Figure 268). Specifically, cells

containing TIAR protein extended uniformly from the very bottom of crypts along the

sides of the regenerative compartments and even into parts of the luminal epithelium

(Figure 26A). Conversely, specimens taken from uninvolved IBD colon showed TIAR

positive cells only at very basal locales within the crypts (Figure 268). The positive

staining in the epithelial cells was intense and marks the entire surface of the cells. The

superficial lamina propria in the normal colon (Figure 26A) showed a rnoderate arnount of

TIAR positive cells. The lamina propria in the CD uninvolved colon showed no positive

staining. The inflamed ileum in Figure 26C has a decreased quantity and intensity of

staining. The lamina propria showed no TlAR positive cells and the crypt epithelium

exhibited only very light staining in the bottom sections of crypts.

( f ) B&xL (inhibitor of apoptosis)

Figure 27A, 27B, and 27C illustrate tissue fiom a normal cecurn, Crohn's

uninvolved transverse colon, and Crohn's inflamed ileum stained with anti-Bcl-x,.

Positive cells were absent in the crypt and luminal epithelium of boih normal (Figure 27A)

and CD uninvolved colon (Figure 27B). Lamina propria was similarly without B ~ 1 - x ~

positive staining (A,B). U-crypts of inflamed ileum showed an absence of

Figure 26 Expression and Localization of TIAR Bindina Protein. (A) normal colon

cecum (patient 44). (B) CD uninvolved ûansverse colon (patient 8). (C) CD inflarned

ileum (patient 8). Crypts of normal cecum contain cells positive for TIAR protein that

extend fiom the very bottom of the crypts along the walls of the glands and even into the

luminal epithelium (A-upper black arrow). Conversely, crypts of uninvolved bowel show

TIAR positive cells in only very basal locales (B-black arrow). TIAR positive crypt and

luminal epithelial cells were stained intensely. The staining covers the entire ce11 without

an obvious specific staining pattern. In (A), a limited number of lamina propria cells are

darkly stained. These TIAR positive cells were located adjacent to the crypt walls (A-

bottom black arrow). In (C), tissue from CD inflamed ileum depict no dark staining of

cells for TIAR. The crypts (black arrow) have very light (negative) staining and the

lamina propria cells are negative for TIAR. Original magnification: (A)-(B) 40X, (C)

20X.

Figure 27 Expression and Localization of Bcl-x,. (A) Normal colon cecum (patient 44).

(B) CD uninvolved transverse colon (patient 8). (C) CD inflamed ileum (patient 8).

Positive ceils for B ~ 1 - x ~ were limited to the lamina propria of inflamed mucosal tissue (C-

upper arrow). lndividual B ~ 1 - x ~ positive cells in this mucosal cornpartment showed dark

staining at the periphery of the cells (C). The cells were scattered throughout the inflamed

lamina propria (C). Crypt epithelium of normal undiseased bowel (A-arrow) and

uninvolved (B-lower arrow) and involved IBD mucosa (C-lower arrow) was devoid of

B ~ 1 - x ~ positive cells. The surface epithelium in any of the mucosal specimens did not

shown any dark B ~ 1 - x ~ staining. Original magnification: (A)-(C) 20X.

Bcl-x, positive cells (C). Scattered cells positive for B ~ 1 - x ~ were located in the lamina

propna of the inflamed CD ileal mucosa (C). The staining appeared to be localized to the

periphery of individual B ~ 1 - x ~ positive cells.

(g) BAD (inducer of apoptosis)

In Figure 28B, immun~histochemistry has revealed intense BAD staining in cells of

the uîrypt, lamina propria, and smaller sections of the luminal epithelium of a CD

uninvolved tranverse colon specimen. The BAD positive cells were scattered throughout

the thickness of the lamina propria, the entire u-crypt and a large portion of the luminal

epithelium (Figure 28B). Intense BAD staining was located in the basal sections of

oriented crypts and u-crypts in normal bowel (Figure 28A). There were a very limited

nurnber of BAD positive lamina propria ceils and the surface epithelium did not show any

positive BAD staining (Figure 28A). The inflamed small (Figure 28C) and large bowel of

CD did not show any positive BAD staining in the crypt epithelium. BAD positive cells

were located in moderate arnounts in the lamina propria (Figure 28C).

Figure 28 Exmession and Locdization of BAD. Normal colon cecum (patient 44). (B)

CD uninvolved transverse colon (patient 8). (C) CD inflamed ileum (patient 8). The

uninvolved CD bowel was characterized by deep anti-BAD staining in a large majority of

the cells of the u-crypt (B-upper arrow), lamina propria (B-lower arrow), and smaller

sections of the luminal epithelium (B). The BAD positive lamina propria cells were

scattered throughout the thickness of the mucosa. The BAD staining pattern of individual

cells was dark staining over the entire surface of positive cells. The staining of normal

undiseased bowel was similar to the uninvolved IBD. Dark BAD staining was located in

the basal sections of oriented crypts and u-crypts (A-arrows). The lamina propria

contained very limited nwnbers of BAD positive cells (A). Crypt epithelium of inflamed

CD mucosa showed no dark BAD staining (C). Individual BAD positive cells were

located at moderate levels throughout the inflamed lamina propria (C). Original

magnitïcation: (A)-(C) 20X.

DISCUSSION

Crohn's Disease and ulcerative colitis are inflarnmatory disorders of the

gastrointestinal tract of unknown etiopathogenesis. The onset of the disease is believed to

be due to a combination of genetic predisposition, exogenous triggers, and endogenous

anomalies. The chronic inflammation characteristic of CD and UC is associated with

histological changes within the intestinal rnucosa. One of these changes is the gradua1

reduction in the size of the crypts in affected tissue throughout the progression of the

disease. The factors responsible for this reduction are unknown. Dysregulated apoptosis

may contribute to the loss of epitheliurn and crypt size reduction in the rnucosa of patients

with CD and UC. The present study was initiated to investigate the role of apoptosis in

the loss of crypt epithelium during the pathogenesis of chronic IBD.

Colonic mucosal biopsies were obtained during routine colonoscopy from patients

with CD, UC, non-IBD, and normal patients undergoing colonic surveillance for cancer.

Biopsies from each patient were obtained from involved and uninvolved areas along the

length of the bowel from ileum a d :or cccum to rectum. To invcstigate apoptosis in the

crypt epithelium of patients with IBD, a series of standard techniques were applied to the

colonic mucosal biopsy tissue. TUNEL, light microscopy of HPS stained tissue, electron

microscopy, gel electrophoresis of extracted DNA, and immunohistochemistry of

apoptotic related proteins were used to detect and localize apoptotic cells in the mucosa of

patients with IBD, non-IBD, and normal controls. Quantification of TUNEL staining was

completed using an image analysis software program.

(1) TUNEL

In the present study. apoptotic cells with fiagmented DNA were identified by

TUNEL primarily in the bottom half of oriented crypts in uninvolved colonic mucosa of

IBD patients, non-IBD mucosa, and normal control tissue. TUNEL staining results in

normal control mucosa were consistent with TUNEL staining reported by others (Potten,

1997; Strater et al., 1995). Crypts of inflamed mucosa had significantly reduced quantities

of epithelial cells with DNA fragmentation. TUNEL positive cells in the inflarned tissue

were observed at increased levels in the lamina propria. The increase in TUNEL positive

cells in this area is most likely due to the inflammatory process and the infiltration of

massive numbers of inflammatory cells into the lamina propria.

Iwarnoto et al. reported TUNEL staining in the normal, uninvolved and involved

colon of untreated UC patients contrary to the results of the present study (Iwamoto et al..

1996). According to Iwamoto et al., cells labelled with biotin after TUNEL were

localized to the luminal epithelium of normal colonic mucosa (Iwamoto et al., 1996). In

addition to surface epithelial cells, TUNEL positive cells were sczttered dong the walls of

crypts in uninvolved and involved UC mucosa (Iwamoto et al., 1996). The staining

reported in the Iwamoto study however displayed inconsistencies. Some crypts of

uninvolved and involved UC mucosa contained TUNEL positive cells in the bottom two

thirds of oriented crypts whereas other crypts displayed no TUNEL positive staining. In

the present study, crypts of inflarned bowel consistently showed very rare cells labelled by

TUNEL, and positive TüNEL staining of normal and uninvolved mucosa was observed

primarily in the bottom half of oriented crypts. Furthemore, uninvolved biopsies were

obtained by the Iwamoto group only centimetres fiom involved areas towards the

descending colon. Although the mucosa may appear uninvolved, active inflammation in

such close proximity may still have similar effects on the mucosa compared to

histologically inflamed bowel. This fact may explain the parallel staining results reported

between uninvolved and involved crypts (Iwamoto et al., 1 996). It is possible that

TUNEL staining is localized to a specific area of bowel within or near involved or

uninvolved mucosa and it is presently unknown whether this staining will Vary according

to differences in anatomical bowel location. In order to study changes in TUNEL staining

throughout the colon, biopsies were taken from cecum and along the length of the large

bowel to rectum and stained using TUNEL. The expression and localization of TUNEL

positive staining did not vary with changes in bowel location. The differences in TUNEL

staining were attributed only to changes in the intensity of inflammation (Le. active vs

inactive). TUNEL staining was consistent within each of involved, uninvolved, and

normal areas of mucosa.

Many patients within the case population were receiving rnedical therapy to

control recurrence of inflammation and it was possible that TLTNEL results could be

influenced by therapy regiments. A previous study by Lee (1993) investigated the

possibility that the incidence of apoptotic bodies in the crypt epitheliurn might help to

identify colonic lesions due to drugs (Lee, 1993). A drug effect was observed as

apoptotic bodies were increased in crypt epithelium when there was a partial response to

dmg treatment (Lee, 1993). Although the present study was not controlled to study the

effect of h g treatments on TUNEL staining, preliminary observations demonstrate no

drug effect. The intensity of staining and locaiization of TUNEL positive cells was

consistent in intestinal mucosal tissue fiom patients with or without medical therapy for

IBD. The difference in results between the two studies is most likely related to the stage

of apoptosis being evaluated. How dmg therapy affects DNA fragmentation, apoptotic

body formation. or apoptosis in general is unknown.

Results of the TUNEL technique were not altered by different anatomical bowel

locations or by dnig therapy. However, it was unknown whether the relatively large

quantity of TUNEL positive staining observed in intestinal rnucosal tissue (normal, non-

IBD, uninvolved IBD) was specific to the large bowel or was a general result of the

TUNEL procedure. Application of the TUNEL technique to tissues distinct from

intestinal mucosa including appendix and tonsil revealed that staining of intestinal mucosa

was indeed specific to the colon. Only single cells or clusters of cells in appendix and

tonsil tissue were TUNEL positive. These control tissues confirmed that the TUNEL

procedure was not staining al1 cells unspecifically, but only cells with signi ficant DNA

fragmentation were being labelled. The authenticity of TCTNEL staining results was

further ensured by positive and negative experiment controls. Positive and negative

controls were included with every TUNEL experiment. Positive controls were intestine

mucosal tissues treated with DNase in order cleave DNA in al1 cells. Experiment reagents

were working properly if al1 cells in a positive control were darkly stained following

TUNEL. False positive staining due to factors other than DNA fragmentation was

monitored by the negative control.

Controls are vital to the reliability of any experimental procedure. Many

researchers either do not utilize proper experimental controls or do not accurately report

the use of controls. In the present study, positive and negative controls were included in

every TUNEL expriment. Appendix and tonsil tissue were used to confirm the specificity

of TUNEL staining in the large bowel. Moreover, TLMEL testing was conducted to

observe variations in staining based on anatomical location throughout the large bowel or

due to medical therapy. TUNEL experiments were tightly controlled and staining results

are reliable and reproducible.

In order to substantiate the qualitative TUNEL staining results, a senes of

measurements of TüNEL positive cells were taken of selected IBD and normal tissue. An

image analysis sobare prograrn called "Northem Exposure" was used to quanti&

TUNEL positive staining. Previous studies have evaluated apoptotic phenornena

(apoptotic bodies, DNA fragmentation) in situ in the large colon that have focused

specifically on crypt epithelium (Iwarnoto et al., 1996; Lee, 1993). Measurements in these

studies are made on crypts that are focused at a relatively high magnification so that cells

(positive and negative) can be enurnerated manually. Furthermore, the crypts are oriented

and hence measurements can be made on a series of similar oriented crypts in one stained

tissue specimen (Iwamoto et al., 1996; Lee. 1993). The "Northem Exposure" image

analysis software program was utilized to improve the efficiency of completing

measurements as opposed to manual evaluation. Measurements of TUNEL positive

staining would be made on full thickness mucosa including surface and crypt epithelium

and lamina propna. Measurements of TUNEL positive cells were made within a fixed

length of tissue (5OOpm on a microscopie level) called a "tissue unit". A tissue unit was

implemented to account for the increase in ce11 numbers in the lamina propna of involved

IBD bowel due to inflammation. Within a tissue unit, TUNEL positive cells and total cells

were counted separately for the surface epitheliurn. the crypt epithelium, and the lamina

propria. These numbers were used to calculate the percentage of TUNEL positive cells

for crypt epithelium, surface epithelium, and lamina propria.

The percentage of TUNEL positive cells was significantly higher in crypt

epithelium of normal and uninvolved IBD bowel compared io inflamed IBD mucosa. The

surface epithelium demonstrated a similar trend. The quantitative anaiysis of selected IBD

and normal control cases supported the expression and localization of TUNEL positive

cells in normal, uninvolved and involved IBD intestinal rnucosa. Moreover, the "Northern

Exposure" image analysis software program was an efficient. accurate, and usefùl method

of rneasuring TUNEL staining in al1 mucosal compartments of the large bowel (surface

and crypt epithelium. lamina propria).

TUNEL analysis localized cells with DNA Fragmentation primarily in the bonom

half of oriented crypts in normal and uninvolved IBD mucosa. TUNEL positive cells were

also observed in the surface epithelium. TCMEL positive staining was drasticall y reduced

in epithelial cells of inflamed IBD mucosa, but increased in the lamina propria. These

observations were supported by quantitative analysis. A series of standard techniques

were then applied to investigate the expression and pattern of apoptosis presented by the

TUNEL shidy.

(2) Morphology Studies

Apoptosis was originally characterized by Kerr et al. based on morphological

criteria (Kerr et al., i 972). Morphology presently remains the "gold standard" for

identification of apoptosis (Ken et al., 1972). According to Kerr et al., morphology

changes of cells undergoing apoptosis occurred in two distinct steps. There was nuclear

and cytoplasmic condensation and budding of the ce11 into apoptotic bodies followed by

phagocytosis of apoptotic bodies by neighboring cells or macrophages. Morphology

studies were completed using light microscopy of standard HPS stained mucosal tissue

and electron microscopy. Light microscopy can identiS, apoptotic bodies containing

fragmented DNA and electron microscopy can detect ultrastructural morphology changes

such as compaction of cytoplasm and nucleus, condensed DNA, plasma membrane

alterations, and apoptotic bodies.

Results of the morphology studies did not support the quantity of apoptosis as

detected by TUNEL. Light and electron microscopy located rare cells in various stages of

apoptosis, but the large majority of cells in the mucosa were morphologically normal. The

apoptotic bodies and ultrastructural apoptotic changes that were found were located

primarily in the crypt epitheliurn. The quantity and location of apoptosis based on

morphology did not Vary significantly in inflamed mucosa relative to uninvolved IBD, non

IBD, and nona l control mucosa.

A study by Lee et al. also reported rare apoptotic bodies in the crypts as detected

by light microscopy and noted that an intense inflammatory reaction characteristic of IBD

only marginally increased the apoptotic count over the nom (Lee, 1993). Apoptotic

bodies form only momentarily in the pathway of apoptosis and may explain the lack of

cells with apoptotic morphology according to light microscopy of HPS stained tissue.

Phagocytosis of apoptotic bodies is enhanceci due to the translocation of anionic

phosphatidyl serine to the outer plasma membrane leaflet during apoptosis (Hale et al.,

1996). Furthemore, only a fraction of cells undergoing apoptosis may be recognized by

the presence of apoptotic bodies (Iwamoto et al., 1996).

In addition to apoptotic bodies. electron microscopy can detect various and earlier

changes in morphology of cells undergoing apoptosis. Contrary to the present results,

Iwamoto et al. reported cells that were frequently found at various stages of apoptosis in

the luminal epithelium of normal colon and in the crypts of uninvolved and involved UC

mucosa (Iwamoto et al., 1996). The difference may be accounted for by medical therapy .

Biopsies for electron microscopy in the present study were obtained from a patient

receiving treatment for ulcerative colitis whereas al1 patients in the Iwamoto study had

untreated active UC. Both studies also focused on differences in apoptosis markers

between involved, uninvolved and normal mucosa without regard for specific disease

activities. The severity of inflammation in the tissues used for electron microscopy may

also explain the discrepancy between the morphology results of the two studies.

The pathway of apoptosis from signaling death to removal of the dying ce11 lasts

from 1 to 3 hours (Gavrieli et al., 1992). The ptocess is efficient and therefore it seems

intuitively, that the number of cells with apoptotic morphology as detected by light or

electron microscopy should be low and relatively the same between both procedures. The

abundance of TUNEL staining in the normal, uninvolved and non-IBD mucosa was not

substantiated by the morphology studies. Although only rare cells were detected by

morphology, this does not mean that large scale cell death was not ongoing (Jacobson et

al., 1997).

(3) DNA Gel Electrophoresis

One of the structural changes observed during the final stages of apoptosis is the

fragmentation of DNA. Endogenous nucleases activated during apoptosis cleave DNA

into mono- and/or oligonucleosomose sized fragments (Hale et al., 1996; Martin et al..

1994). These pieces of DNA typically form a ladder upon separation via DNA gel

electrophoresis. Hence, this technique has found widespread use in the field of apoptosis

research particularly as a means of confirmation of previously detected ce11 death events

(eg. TUNEL).

DNA gel electrophoresis was applied in this study to confimi the TUNEL results.

DNA was extracted from biopsies from involved and uninvolved sections of large bowel

fiom a single patient with LTC. -4 ladder was not observed following electrophoresis of

any of the DNA samples. Only smears of DNA indicating variable numbers and sizes of

DNA were observed for both involved and uninvolved bowel.

Results fiom the TUNEL experiments certainly imply the existence of a ladder as

has been previously reported (Iwamoto et al., 1996). In order to apply gel

electrophoresis, DNA must be extracted from tissues that contain a significant quantity of

apoptotic cells at approximately the same stage in the ce11 death process (Potten, 1997).

I Since DNA was extracted from total mucous membrane, the amount of DNA obtained

fiom normal healthy non-apoptotic cells was likely in much larger quantities relative to

DNA removed fiom apoptotic cells. Therefore, smears of DNA could resemble

separation of large arnounts of normal non-apoptotic DNA along with much smaller

quantities of apoptotic fiagmented DNA. The ladder may exist, however it was covered

by the separation pattern of normal unfiagmented DNA. Indeed, a ladder can be

recognized in DNA from normal control tissue as faint bands when the micrographs are

image enhanced (Iwarnoto et al., 1996). Moreover, DNA gel electrophoresis in this study

was conducted on DNA from biopsies isolated fiom a single UC patient with mildly active

disease. Obtaining biopsies from a larger population of IBD patients (Iwarnoto et al.,

1996)and normal controls and pooling the DNA extracts (Le. involved, uninvolved,

normal) could increase the relative quantity of fragmented DNA. This would increase the

probability of separating the characteristic ladder on gel electrophoresis.

(4) Apoptotic Regulatory Proteins

In order to further examine the role of apoptosis in the ciypt reduction and loss of

epithelial cells during chronic IBD, immunohistochemistry of a variety of proteins was

completed. Expression and localization of apoptotic regulatory proteins Fas, p53, CPP-32.

Ich-1 L, TIAR, Bcl-xL, and BAD were investigated as part of the present study. Staining

results of apoptosis inducer proteins CPP-32, Ich-1 L, TIAR, and BAD confirmed the

TUNEL analysis.

Fas is a cell surface receptor that c m transmit an apoptotic signal to the interior of

a target ce11 following binding of its natural substrate FasL (Nagata and Suda, 1995). In

this study, no significant Fas staining was observed in any tissues using

imrnunohistochemistry. This suggests that factors other than Fas signal apoptosis of

colonic surface epithelium or in crypts. it is generally believed that FasL is associated

particularly with cells of known cytolytic and killer fhction (Hahne et al., 1996; Nagata

and Suda, 1995) and not with surface or crypt enterocytes (Ma, 1997; Strater et al., 1997)

as was reported by Iwamoto (Iwamoto et al., 1996). Moreover, the lack of epithelial

disturbances in lpr/lpr (Fas-deficient) or gldfgld (FasL-deficient) mice combined with the

absence of FasL on normal enterocytes suggest that apoptosis signals other than Fas

regulate the homeostatic tumover of colonic epithelial cells under normal conditions (Ma.

1997). During active IBD, the number of lymphocytes in the lamina propria bearing FasL

is increased (Strater et al., 1997). A previous study (Strater et al., 1997) has suggested

that focal association of activated lymphocytes bearing FasL in the lamina propria with

crypt epithelial cells during active IBD results in apoptosis of the affected epithelial cells

and microlesions in the crypts. It is presently unclear exactly what extemal trigger(s) are

responsible for the tumover of epithelial cells in intestinal mucosa during nonal gut

physiology and IBD.

P53 protein is a transcription factor that is upreguiated following ceIl injury such as

DNA damage. P53 will arrest injured cells in the G 1 stage of the ce11 cycle to repair

DNA, or if the DNA is irreparable, will push the ce11 into apoptosis. I t has been

speculated that ce11 stress due to inflammation can cause an intracellular upregulation of

p53 (Krishna et al., 1995). Earlier work has shown upregulated quantities of p53 positive

tells in the crypt and surface epithelium of acutely inflamed IBD mucosa compared to

regenerated and normal mucosa (Krishna et al.. 1995). In the present study,

immunohistochemistry of actively inflamed bowel of IBD patients did not reveal any cells

with detectable levels of p53. Similar staining results were observed for normal and

uninvolved bowel. The inflammation characteristic of IBD may indeed stress cells and the

degree to which cells are affected (i.e. p53 levels) rnay also depend on the severity of

inflammation, which may account for the p53 staining differences between the two studies.

Presently there is no established comection between the severity of inflammation, ce11

stress, p53 levels, and apoptosis. Currently, p53 positive cells were not detected in

normal, uninvolved, or involved IBD mucosa suggesting that p53 does not play an

important role in controlling apoptosis in the gut.

CPP-32 (caspase 3) is an enzyme that cleaves a DNA repair enzyme known as

PARP during the execution stage of the apoptotic pathway. CPP-32 is also considered to

be necessary for the majority, if not al1 foms of apoptotic ce11 death (Nicholson et al.,

1995). It was expected that CPP-32 would also contribute to executing apoptosis in the

small and large bowels. Positive staining of cells containing CPP-32 w r e localized

primarily to crypt epithelium of normal control and uninvolved IBD mucosa. These

results implicated CPP-32 as an active member of the cascade of proteases controlling the

execution of apoptosis in normal and quiescent IBD mucosa. More importantly, results

fiom the TUNEL analysis were confirmed by the mucosal staining pattern of CPP-32.

Presumably, apoptotic ciypt enterocytes would upregulate CPP-32 in response to signals

generated following DNA fragmentation. Enzymes activated to repair the damaged DNA

would be inactivated by CPP-32, thereby pushing the ce11 into the final stages of the

apoptotic pathway. Therefore, it seems logical that the pattern of TUNEL positive cells in

normal and quiescent IBD mucosa was paralled by the pattern of CPP-32 positive cells in

the same mucosa.

Normal and uninvolved IBD bowel tissue also contained upregulated quantities of

another pro-apoptotic enzyme Ich-1 L (caspase 2). Cells containing Ich- 1 L were located

principally along the walls of the crypts and to a minor degree in the surface epithelium.

Again, TUNEL results were supported by this staining pattern of Ich-1 L positive cells.

This is the first study that has characterized the expression and localization of Ich-1 L in

intestinal mucosa. Ich- 1 L detected at elevated arnounts in normal and quiescent IBD

mucosa suggests that this pro-apoptotic enzyme also plays a role in apoptosis in the

intestines. Initial studies in other systems have suggested that Ich- 1 L is an early enzyme

(Harvey et al., 1997) in the cascade of proteases. whose activation would eventually lead

to mobilization of late proteases such as CPP-32 (Duan and Dixit, 1997). Ich- 1 L is

suspected to play a similar role in apoptosis in the gut.

TlAR is an RNA binding protein which has been implicated in apoptotic pathways.

TIAR has not been studied extensively and no studies have characterized the expression

and localization of TIAR in intestinal mucosa. In the present study, TIAR was detected in

the crypt and luminal epithelium of normal mucosa and in basal sections of crypts in

uninvolved IBD mucosa. This suggests that TIAR may have a function in apoptotic

processes in normal and quiescent IBD mucosa. The nature of that function however is

completely unknown. In a preliminary study, DNA fragmentation was observed to be the

result of application of TIAR to permeabilized thymocytes (Lowin et al., 1996). Perhaps,

TIAR is in part responsible for fragmentation of DNA in apoptotic enterocytes. In

general, TIAR can be considered an inducer of apoptosis. Results of TIAR staining in the

colon have supported staining patterns of DNA fragmentation by TUNEL.

The Bcl-2 family of proteins are well established mediators of apoptosis. B ~ 1 - x ~

(anti-apoptotic) and BAD (pro-apoptotic) are two rnembers of this family that were

selected for detailed study using immunohistochemistry.

Dark staining of cells containing B ~ 1 - x ~ were essentially absent in the crypt and

surface epithelium of al1 tissues that were examined. The expression of Bcl-x, in the small

and large bowels has not been previously studied. However, Bcl-2 (anti-apoptotic) is

expressed in the stem ce11 region of large bowel crypts in mice and prelirninary results

suggest a simiiar scenario in colonic crypts of hurnans (Potten et al., 1997). Therefore. ii

is probable that Bcl-2. instead of BcI-x,, is prirnarily responsible for mediating survival of

epithelial cells in large bowel crypts.

intense BAD staining was localized in basal sections of oriented crypts of normal

control md uninvolved IBD mucosa. Smaller sections of the lamina propria and surface

epitheliurn were also stained. These results support the T W E L analysis and moreover

suggest a role for BAD in apoptotic pathways in normal and quiescent IBD intestinal

mucosa. BAD has not been otherwise investigated in colonic mucosa and how BAD

influences apoptotic pathways dong with other Bcl-2 family mernbers rernains unclear.

Rie immunohistochemistry of apoptosis inducer proteins CPP-32. Ich-1 L, TIAR.

and BAD support the staining results from the TUNEL analysis. The staining results of

the apoptotic regulatory proteins also suggest that CPP-32, Ich- 1 L, TIAR, and BAD may

play a role in controlling apoptosis in the gut, although the exact nature of that role in

most cases remains unclear.

(5) Cytokines

The TUNEL analysis and supporting irnrnunohistochemistry studies demonstrate a

drastic reduction of apoptotic cells in the crypt epitheliurn of inflarned IBD bowel.

In the normal undiseased bowel the mucosa contains a normal population of

chronic inflamrnatory cells. However, durhg an active flue of IBD, the intensity of

inflammation becomes so great as to overwhelm anti-inflarnmatory TGF-P mechanisms in

the Peyer's patches as has been suggested using a TNBS mouse mode1 of colitis (Strober

et al., 1997). It is hypothesized that this intense inflarnmatory process of active CD and

UC may be responsible in downregulating epithelial ceIl apoptosis in crypts of inflarned

IBD mucosa. The mechanism by which inflammation downregulates apoptosis is

unknown, but the components of an infiammatory milieu such as proinflammatory

cytokines may provide an answer. Proinflammatory cytokines are major mediators of

inflammation in IBD that are found at increased levels both systemically in the blood

(Elsasser-Beile et al., 1994) and locally within the inflamed IBD mucosa (Murata et al..

1995). Proidammatory cytokines have been shown to extend the life span of neutrophils.

Neutrophils are the predominant ce11 type infiltrating the lamina propria during acute flues

of CD and UC. For example, IL-1 P (Colotta et al., l992), IL-6 (Biffl et al., 1996), IFN-y

(Colotta et al., 1992), and TNF-a (Colotta et al., 1992), are sorne of a group of cytokines

that can al1 extend the life span of polyrnorphonuclear leukocytes (PMNs) following in

vitro culture. Similarly, IL4 O, an anti-inflarnmatory cytokine, has been shown to block

apoptosis of human umbilical vein endothelial cells (HUVECS) (Lindner et al., 1997).

More recently, Estaquier et al. demonstrated that Th1 -type cytokines (IBD is associated

with a skewed Th- 1 type cytokine profile) reduced apoptosis of cultured monocytes in a

number of experimental systems (Estaquier and Ameisen, 1997). Furthemore. IL-1 2

enhanced survival in long-term (10 days) culture of adherent monocytes (Estaquier and

Ameisen, 1997). Kiener et al. also reported that addition of proinflammatory cytokines to

cultured monocytes significantly reduced spontaneous apoptotic events (Kiener et al..

1997). Monocytes and monocyte derived macrophages along with PMNs, constitute a

large portion of the ce11 population in the mucosal lamina propria during an active flare of

IBD. A number of other experiments have shown similar trends (Lotem and Sachs. 1997;

Lin and Benchimol, 1997; Yousefi et al., 1997). It is possible then, that the sarne

proinflarnmatory cytokines that downregulate apoptosis of PMN and monocytes during

active IBD, c m also mediate a reduction in the capacity of intestinal enterocytes or crypt

epithelial cells to undergo ce11 death and this may indeed explain why the TLrNEL malysis

of actively inflarned IBD tissue demonstrated a greatly reduced quantity of staining

relative to the normal, non-IBD, and uninvolved conditions.

(6) Summary

The role of apoptosis in the loss of epithelial cells and reduction of crypt size

during chronic IBD was investigated. The hypothesis of the study was that increased

apoptosis would play a role in the reduction of crypt size and loss of epithelial cells during

the pathogenesis of chronic IBD, as had been reported by others (Iwamoto et al., 1996).

Apoptosis in colonoscopie biopsies fiom patients with IBD and normal controls was

investigated using a variety of standard techniques including TWEL, light microscopy of

HPS stained tissue, electron microscopy, DNA gel electrophoresis, and

immunohistochemistry of apoptotic related proteins. Unexpectedly, TUNEL positive cells

were localized primarily to the bottom half of crypts in normal control and uninvolved

IBD mucosa. Epithelial ce11 apoptosis in crypts of inflamed bowel was significantly

reduced. These results were confimed by quantitative analysis and the expression and

staining patterns of apoptosis inducer proteins CPP-32, Ich- 1 L, TIAR, and BAD.

The initial research hypothesis was shown to be incorrect. Apoptosis does not

play a role in crypt reduction and loss of epithelium during chronic IBD. It is proposed

that during the intense inflarnmatory reaction of active CD and UC, the downregulation of

apoptosis in the crypts is a "homeostatic response" designed to increase crypt ce11

production and aid in epithelial restitution. This downregulation of the apoptotic process

in the colonic mucosa is most likely due to the effects of components of the inHammatory

microenvironment such as proinfiammatory cytokines. Furthemore, that apoptotic ce11

death in the crypts of normal and uninvolved IBD bowel acts to eliminate stem cells in

excess of tissue needs.

(7) Future Studies

Future studies would focus on what was causing the reduction of crypt epithelial

ce11 apoptosis in the inflamed bowel of IBD patients. A study of proliferation markers on

stem cells would determine whether apoptosis in the inflamed bowel was reduced due to

increased proliferation of stem cells or whether stem ce11 proliferation was decreased; with

other factors in the inflarnmatory microenvironment possibly blocking apoptosis of crypt

enterocytes. For exarnple, certain proinflammatory cytokines have been observed to

extend the lifespan of neutrophils and monocytes (Colotta et al., 1992; Biffl et al., 1996;

Estaquier and Ameisen, 1997; Kiener et al., 1997). In vitro culture systems of crypt

epithelial cells with the sarne proinflammatory cytokines upregulated in IBD could

demonstrate an increase in the lifespan of crypt enterocytes similar to neutrophils and

monocytes. Further Iines of study would address in more detail the effect of IBD drug

treatments on apoptosis in the mucosa. The relationship between specific disease

activities and mucosal apoptosis would clarify how the intensity of inflammation correlates

with the quantity of ce11 death in the gut. Future studies of apoptosis in intestinal rnucosa

would be completed using properly oriented biopsies so that important information

regarding topographical location of apoptotic crypt epithelial cells could be obtained.

REFERENCES

Akbar A N , Borthwick N.J., Wickremasinghe R.G., Panayoitidis P., Pilling D., Bofill M., Krajewski S., Reed J.C. and Salmon M. (1996) Interleukin-2 receptor common gamma-chain signaling cytokines regulate activated T ceIl apoptosis in response to growth factor withdrawal: selective induction of anti- apoptotic (bcl-2. bcl-xL) but not pro-apoptotic (bax, bcl-xS) gene expression. European Journal o/lmmunology 26,294-299.

Akbar AsN. and Salmon M. (1997) Cellular environrnents and apoptosis: tissue microenvironments control activated T-ce11 death. [Review] [43 refs]. Immunology Today 18,72-76.

Akbar A.N., Savill J., Gombert W., Bofill M., Borthwick N.J., Whitelaw F., Grundy J., Janossy G. and Salmon M. (1994) The specific recognition by macrophages of CD8+,CD45RO+ T cells undergoing apoptosis: a mechanism for T ce11 clearance during resol ution of viral infections. Journal of Experimental Medicine 180, 194301947,

Amsterdam A., Dantes A., Selvaraj Ns and Aharoni D. ( 1 997) Apoptosis in steroidogenic cells: structure-function analysis. [Review] [33 refs]. Strroids 62. 207-2 1 1.

Armstrong R.C., Aja T., Xiang J., Gaur S., Krebs J.F., Hoang K., Bai X., Korsmeyer S.J., Karanewsky D.S., Fritz L.C. and Tomaselli K.J. (1996) Fas- induced activation of the ce11 death-related protease CPP32 1s inhibited by Bcl-2 and by ICE family protease inhibitors. Journal of Biologieal Chemistry 271, 16850-16855.

Baretton C.B., Diebold J., Christoforis G., Vogt M., Muller C., Dopfer K., Schneiderbanger K., Schmidt M. and Lohrs U. (1996) Apoptosis and immunohistochemical bcl-2 expression in colorectal adenomas and carcinomas. Aspects of carcinogenesis and prognostic significance. Cancer 77, 255-264.

Barry M.A. and Eastman A. (1993) Identification of deoxyribonuclease ii as an endonuclease involved in apoptosis. Arch. Biochem. Biophys. 300,440-450.

Bauer MsK.9 Wesselborg S. and Schulze-Osthoff K. (1997) The Caenorhabditis elegans death protein Ced-4 contains a motif with similarity to the mammalian 'death effector domain'. FEBS Letiers 402,256-258.

Beck A.R., Medley Q.G., O'Brien S., Anderson P. and Streuli M. (1996) Structure. tissue distribution and genornic organization of the murine RRM-type RNA binding proteins TI A- l and TI AR. Nucleic Acids Research 24,3 829-3 835.

Bifn W.L., Moore E.E., Moore F.A., Barnett C.C., Jr., Carl V.S. and Peterson V.N. (1 996) Interleukin-6 delays neutrophil apoptosis. Archives of Surgery 131, 24-29.

Blum Do, Wu Y., Nissou M.F., Arnaud S., Alim-Louis-Benabid and Verna J.M. (1 997) p53 and Bax activation in 6-hydroxydopamine-induced apoptosis in PC 12 cells. Brain Res. Mol. Brain Res. 751, 1 39- 1 42.

Borner C. ( 1996) Diminished ce11 proliferation associated with the death- protective activity of Bcl-2. Journal of Biologieal Chemistry 271, 1269542698.

Camhi S.L., Lee P. and Choi A.M. (1995) The oxidative stress response. [Review] [ l 14 refs]. New Horiz. 3, 1 70- 1 82.

Casciola-Rosen Le, Nicholson D.W., Chong Te, Rowan K.R, Thornberry N.A., Miller D.K. and Rosen A. (1 996) ApopaidCPP32 cleaves proteins that are essential for cellular repair: a fundamental principle of apoptotic death [see comments]. Journal cf Experimental Medicine 183, 1 95 7- 1 964.

Chang E.H., Jang Y.J., Hao Z., Murphy G., Rait A., Fee W.E., Jr., Sussman H.H., Ryan P., Chiang Y. and Pirollo K.F. (1997) Restoration of the G1 checkpoint and the apoptotic pathway mediated by wild-type p53 sensitizes squamous ce11 carcinoma of the head and neck to radiotherapy. Arch. Otolaryngoi. Head. Neck Surg. 123,507-5 12.

Chang N.S. ( 1 995) Hyaluronidase induces murine L929 fibrosarcoma cells resistant to tunior necrosis factor and Fas cytotoxicity in the prrsence of actinomycin D. C d Biochem. Biophys. 27, 10% 1 32.

Chinnaiyan A.M., O'Rourke K., Lane B.R. and Dixit V.M. (1997) Interaction of CED-4 with CED-3 and CED-9: a molecular framework for ce11 death [see comrnents]. Science 275, 1 122-1 126.

Clarke A.R, Howard L.A., Harrison D.J. and Winton D.J. (1997) p53, mutation fiequency and apoptosis in the murine small intestine. Oncogene 14,20 15-201 8.

Colotta F., Re F., Polentarutti N., Sovani S. and Mantovani A. (1992) Modulation of granulocyte survival and programmed ce11 death by cytokines and bacterial products. Blood 80,20 1 2-2020.

Crohn B.B., Ginsburg Le and Oppenheim GD. (1932) Regional ileitis. A pathologie and clinical entity. J A M 99, 1323- 1329.

Cummings M. (1 996) Apoptosis of epithelial cells in vivo involves tissue transglutaminase upregulation. Journal of Puthology 179,288-293.

Dahiel T.K. (19 13) Chronic interstitial enteritis. Br. MedJ ii, 1068- 1070.

Dember L.M., Kim N.D., Liu K.Q. and Anderson P. (1996) Individual RNA recognition motifs of TIA-1 and TIAR have different RNA binding specificities. Journal ofBiological Chemistry 271,2783-2788.

Desbmukh M., Vasilakos J., Decherth TL., Lampe P.A., Shivers B.D., Johnson and EM Jr. (1 996) Genetic and metabolic status of NGF-deprived sympathetic neurons saved by an inhibitor of ICE family proteases. Journal of Ce11 Biology 135, 1341-1354.

Di Manio Lm, Alesse E., Roncaioli P., Muzi P., Moretti S., MatceIlini S., Amicosante Ga, De Simone Ca and Cifone M.G. (1 997) Influence of L-camitine on CD95 cross-lining-induced apoptosis and ceramide generation in human cell lines: correlation with its effects on purified acidic and neutral sphingomyelinases in vitro. Proc. Assoc. Am. Physicians. 109, 1 54- 1 63.

Dianzani U., Bragardo M., DiFranco D., Alliaudi C., Scagni P., Buonfiglio, D, Redoglia V., Bonissoni S., Correra A., Dianzani I. and Ramenghi U. (1 997) Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lyrnphoproliferation. Biood 89,287 1-2879.

Driscoll M. (1996) Ce11 death in C. elegans: molecular insights into mechanisms conserved between nematodes and mammals. [Review] [107 refs]. Bruin Pathol. 6,4 1 1-425.

Duan Hm and Dixit V.M. (1997) RAlDD is a new 'death' adaptor molecule. Nurure 385. 86-89.

Edwards E.C. and Truclove S.C. ( 1 963) The course and prognosis of ulcerative colitis. Gut 4,299

Elsasser-Beile U., von Kleist S., Gerlach S., GaIIati H. and Montiag J.S. (1994) Cytokine production in whole blood ce11 cultures of patients with Crohn's disease and ulcerative colitis. J. Clin. Lab. Anal. 8,447-45 1 .

Estaquier J. and Ameisen J.C. (1997) A role for t-helper type-1 and type-2 cytokines in the regulation of human monocyte apoptosis. BIood 90, 1 6 1 8- 1625.

Evan G.I., Brown L., Wbyte M. and Harrington E. (1995) Apoptosis and the celi cycle. [review] [84 refs]. Curr. Opin. Ce11 Biol. 7,825-834.

Farmer R.G. (1977) Clinical features of Crohn's disease and ulcerative colitis. in Infammatory Bowel Diseuse (Anonymous), p. 4. Pharmacia,

Fleisber T.A. (1 997) Apoptosis. [Review] [28 refs]. Ann-Allergv Asthma hrnunol. 78, 245-9; quiz 249-50.

Frankfurt O.S., Robb J.A., Sugarbaker E.V. and Villa La (1996) Apoptosis in human breast and gastrointestinal carcinomas. Detection in histological sections with monoclonal antibody to single-stranded DNA. Anticancer Res. 16, 1979- 1988.

Funakoshi K., Sugimura K., Sasakawa T., Bannai Ha, Anezaki K., Ishizuka K., Yoshida K., Narisawa R. and Asakura H. (1 995) Study of cytokines in ulcerative colitis. J. Gastroenterol. 30 Suppl8,6 1 -63.

Gaido M.L. and Cidlowski L A . ( 199 1 ) Identification, purification, and characterization of a calcium- dependent endonuclease (nuc 18) fiom apoptotic rat thymocytes. nuc 18 is not histone h2b. Journal of Biological Chemistry 266, 1 8580- 1 8585.

Gavrieli Y., Sherman Y. and Ben-Saswn S.A. (1992) Identification of programmed cell death in situ via specific labeling of nuclear DNA fragmentation. Journal of Cell Biology 1 19.493 -50 1.

Gerlach M., Riederer P. and Youdim M.B. (1996) Molecular mechanisms for neurodegeneration. Synergism between reactive oxygen species, calcium, and excitotoxic arnino acids. [Review] [Il9 refs]. Adv.Neuro1. 69, 177- 194.

Gibson L.F., Piktel D., Narayanan R a , Nunez G. and Landreth K.S. (1 996) Stroma1 cells regulate bcl-2 and bax expression in pro-B cells. Experirnental Hernatology 24 ,628-637.

Glinsky G.V., Glinsky V.V., Ivanova A.B. and Hueser C.J. ( 1 997) Apoptosis and metastasis: increased apoptosis resistance of metastatic cancer cells is associated with the profound deficiency of apoptosis execution mechanisms. Cancer Letters 115, 185-193.

Goldberg Y.P., Nicholson D.W., Rasper D.M., Kalcbman M.A., Koide H.B., Graham, RK, Bromrn M., Kuemi-Esfarjani P., Thornberry N.A., Vaillancourt J.P. and Hayden M.R. (1 996) Cleavage of huntingtin by apopain, a proapoptotic cysteine protease, is modulated by the polyglutarnine tract [see comments]. Nat.Genet. 13,4420449.

Golusinski W., Olofsson J., Szmeja Z., Szyfter K., Szyfter W., Biczysko W. aod Hemminki K. (1 997) Alteration of p53 gene structure and fûnction in laryngeal squamous ceil cancer. Eur. Arch. Olorhinolaryngol. Suppl. 1 , S 133-7.

Greenluad L.J., Deckwerth T.L. and Johnson E.M., Jr. (1995) Superoxide dismutase delays neuronal apoptosis: a role for reactive oxygen species in programmed neuronal death. Neuron 14,303-3 15.

Grisham M.B. (1 994) Oxidants and fiee radicals in inf'larnmatory bowel disease. [Review]. Lancer 344, 859-86 1 .

Hahne M., Remo Ta, Schroeter M., lrmler M., French Le, Bornard T., MacDonald H.R. and Tschopp J. (1 996) Activated B cells express functional Fas ligand. European Journal of Intmunologv 26,72 1-724.

Hale AJ., Smith C.A., Sutherland L.C., Stoneman V.E., Longthorne V.L., Culhane, AC and Williams G.T. (1996) Apoptosis: molecular regulation of ce11 death. [Review]. EurJ Biochem. 236, 1-26.

Harvey N.L., Butt A.J. and Kumar S. (1 997) Functional activation of NeddZnCH- 1 (caspase-2) is an early process in apoptosis. Journal ojBiofogical Chemistry 272 , 13134-13139.

Hedley D.W. and McCulloeh E.A. (1 996) Generation of reactive oxygen intermediates after treatment of blasts of acute myeloblastic leukemia with cytosine arabinoside: role of bcl-2. Leukemia 10, 1 143-1 149.

Hengartner M.O. (1 996) Programrned ce11 death in invertebraies. [Review] [37 refs]. Curr. Opin.. Genet. Dev. 6,34-38.

Hermiston M.L. and Gordon J.I. (1995) Inflammatory bowel disease and adenornas in rnice expressing a dominant negative N-cadherin. Science 270, 1 203- 1 207.

Hibi T., Ohara M., Watanabe M., Kanai T., Takaisbi H., Hayashi A., Hosoda, Y, Ogata H., Iwao Y., Aiso S. and et al. (1993) Interleukin 2 and interferon-gamma augment anticolon antibody dependent cellular cytotoxicity in ulcerative colitis. Gur 34,788-793.

Howard A.D., Chartrain N., Ding C.F., Kostura MJ., Limjuco G., Schmidt J.A. and Tocci M.J. (1991) Probing the role of interleukin-1 beta convertase in interleukin- 1 beta secretion. Agents & Actions - Supplements 35, 77-83.

Ito M.R., Terasaki S., Itob J., Katoh H., Yonehara S. and Nose M. (1997) Rheumatic diseases in an MRL strain of mice with a deficit in the functional Fas ligand. Arthritis & Rheumatism 40, 1 054- 1 063.

Iwamoto M., Koji Ta, Makiyama K., Kobayashi N. and Nakane P.K. (1996) Apoptosis of crypt epithelial cells in ulcerative colitis. Journal of Pathology 180, 152- 1 59.

Jaeobson M.D., Weil M. and Raff M.C. (1997) Programmed cell death in animal development. [review] [7 1 refs]. Cell88,347-54:6.

James C., Cschmeissaer S., Fraser A. and Evaa G.I. (1997) CED-4 induces chromatin condensation in Schizosaccharomyces pombe and is inhibited by direct physical association with CED-9. Curr. Biol. 7,246-252.

Jourd'heuil D., Morise Z., Conner E.M., Kurose 1. and Grisham M.B. (1997) Oxidant-regulation of gene expression in the chronically inflamed intestine. [Review] [58 refs]. Kei0.J. Med. 46,1001 5 .

Jung H.C., Eckmann L., Yang S.K., Panja A., Fierer J., Monycka-Wroblewska E. and Kagnoff M.F. (1995) A distinct array of proinflammatory cytokines is expressed in human colon epithelial cells in response to bacterial invasion. Journal of Clinical Investigution 95, 55-65.

Kanan A., Yee E., Kaufihansky K. and Harlan J.M. (1996) Cloning of human Bcl-2 homologue: inflammatory cytokines induce human Al in cultured endothelial cells. Blood 87,3089-3096.

Kernohan N.M. and Con L.S. (1 996) Regulation of apoptosis by bcl-2 and its related proteins: irnmunochemical challenges and therapeutic implications [editorial]. Journal of Pathology 179. 1 -3 : 7 .

Kerr J.F., Wyllie A.H. and Currie A.R. (1972) Apoptosis: a basic biological phenornenon with wide-ranging implications in tissue kinetics. [review] [68 refs]. British Journal of Cancer 26,239-57.

Kiener P.A., Davis P.M., Starling G.C., Mehlin C., Klebanoff S.J., Ledbetter, JA and Liles W.C. ( 1997) Differential induction of apoptosis by Fas-Fas ligand interactions in human monocytes and macrophages. Journal of Experimental Medicine 185, 15 1 1 - 15 16.

Kinner J.B. (1985) Inflammatory bowel disease at the university of chicago-the first 50 years: some persona1 reflections. American Journal of Gastroenterology 80,2 1 9- 26: 18.

Kinner J.B. and Shorter R.G. (1982) Recent developments in nonspecific inflammatory bowel disease (second of two parts). [Review] [ 1 97 refs]. New England Journal ofMedicine 306,837-848.

Kitamura Y., Shimohama S., Kamoshima W., Matsuoka Y., Nomura Y., Tanigucbi and T. (1997) Changes of p53 in the brains of patients with Alzheimer's disease. Biochernical & Biophysical Research Communications 232,418-42 1.

Komada Y., Zhou Y.W., Zhang X.L., Cben T.X., Tanaka S., Azuma E. and Sakurai M. (1 997) Fas/APO-1 (CD95)-mediated cytotoxicity is responsible for the apoptotic ceIl death of leukaemic cells induced by interleukin-2- activated T cells. British Journal of Haernatology 96, 147-157.

Korsmeyer S.J., Shutter JmRm, Veis D.J., Merry DmE. and Oltvai Z.N. ( 1 993) Bcl- 2/Bax: a rheostat that regulates an anti-oxidant pathway and ce11 death. [Review]. Semin. Cancer Bio l. 4 , 3 27-3 32.

Korsmeyer S.J., Yin X.M., Olhtai ZN., Veis-Novack D.J. and Linette GmPm (1995) Reactive oxygen species and the regulation of ce11 death by the Bcl-2 gene family. [Review]. Biochimica et Biophysica Acta 1271,63066.

Krishna M., Woda B., Savas L., Baker S. and Banner B. (1 995) Expression of p53 antigen in inflamed and regenerated mucosa in ulceraiive colitis and crohn's disease. Mod. Pathol. 8,654-657.

Lee F.D. (1993) Importance of apoptosis in the histopathology of dmg related lesions in the large intestine. Journal of Cfinical Puthologv 46, 1 18-122.

Lih-Brody Lm, Powell S.R., Collier K.P., Reddy G.M., Cerchia R., Kahn E., Weissman G.S., Katz S., Floyd RA., McKinley MM, Fisher S.E. and Mullin C.E. (1 996) Increased oxidative stress and decreased antioxidant defenses in mucosa of inflammatory bowel disease. Digestive Diseases & Sciences 41,2078- 2086.

Lin Y. and Benchimol S. (1 997) P53-mediated ceIl cycle arrest and apoptosis. Leukemia 11, Suppl3 3324-6

Lindner Hm, Holler E., Ertl B., Multhoff G., Schregimann M., Klauke I., Schultx- Hector S. and Eissner G. (1997) Penpheral blood mononuclear cells induce programmed cell death in human endotheliai cells and may prevent repair: role of cytokines. Blood 89, 193 1 - 1938.

Lotem J. and Sachs Lm (1997) Cytokine suppression of protease activation in wild-type p53- dependent and p53-independent apoptosis. Proceedings of the National Academy of Sciences of the United States of America 94,9349-9353.

Lovschall H. and Mosekilde L. (1 997) [Apoptosis: cellular and clinical aspects]. [Review] [15 refs] [Danish]. Nord. Med. 112, 133- 137.

Lowin B., French Lm, Martinou J.C. and Tschopp J. (1996) Expression of the CTL- associated protein TIA- I during murine embryogenesis. Journal of lmmunology 157, 1448- 1454.

Ma A. ( 1 997) Fas and the imrnunologically underprivileged intestine [editorial; comment]. Gastrcenterology 1 13,345-8.

MacFarlane M., Cain K., Sun X.M., Alnemri E.S. and Cohen C.M. (1997) Processing/activation of at least four interleukin-1 beta converting enzyme-like proteases occurs during the execution phase of apoptosis in human monocytic tumor cells. Journal ofCell Biology 137,469-479.

Malcornson R.D., Clarke A.R., Peter A., Coutts S.B., Howie S.E. and Harrison D.J. (1997) Apoptosis induced by gamma-irradiation, but not CD4 ligation, of peripheral T lymphocytes in vivo is p53-dependent. Journal of Pathology 181. 166-171.

Marin M.C., Fernandez A., Bick R.J., Brisbay S., Buja L.M., Snuggs M., McConkey D.J., von Eschenbach A C , Keating M.J. and McDonnell T.J. (1 996) Apoptosis suppression by bcl-2 is correlated with the regulation of nuclear and cytosolic C d + . Oncogene 12,2259-2266.

Martin S.J., Green D.R. and Cotter T.C. ( 1 994) Dicing with death: dissecting the components of the apoptosis machinery. [review] [45 refs]. Trends. Biochem.Sci. 19,2630.

Mashima Tm, Noito M., Noguchi K., Miller D.K., Nicholson D.W. and Tsuruo T. ( 1 997) Actin cleavage by CPP-32lapopain during the development of apoptosis. Oncogene 14, 1007-1012.

McConnell K.R., Dynan W.S. and Hardin J.A. (1997) The DNA-dependent protein kinase catalytic subunit (p460) is cleaved during Fas-mediated apoptosis in Jurkat cells. Journal of Immunology 158,2083-2089.

McKenna S.L. and Cotter T.G. (1 997) Functional aspects of apoptosis in hematopoiesis and consequences of failure. [Review] [284 refs] . Adv. Cancer Rer 71, 12 1 - 164.

McKenzie S.J., Baker M.S., Bufiinton C.D. and Doe W.F. (1996) Evidence of oxidant-induced injury to epithelial cells during inflammatory bowel disease. Journal of Clinical Investigation 98, 1 3 6- 1 4 1 .

Merritt A.J., Potten C.S., Watson A.J., Loh D.Y., Nakayama K. and Hickman J.A. (1 995) Differential expression of bcl-2 in intestinal epithelia. correlation with attenuation of apoptosis in colonic crypts and the incidence of colonic neoplasia. Journal of Cell Science 108,226 1 -227 1.

Meterissian S.H., Kontogiaanea M., Po L, Jensen G. and Ferdinand B. (1997) Apoptosis induced in human colorectal carcinoma by anti-Fas antibody . Ann. Surg. Oncol. 4 , 169- 175.

Miura M. ( 1 996) [IceIcedJ farnily gene and apoptosis]. [Review] [23 refs] [Japanese]. Nippon. Rinsho. 54, 1860- 1 868.

Mondino A. and Jenkins M.K. (1995) Accumulation of sequence-specific RNA-binding proteins in the cytosol of activated T cells undergoing RNA degradation and apoptosis. Journal of Biological Chemistry 270,26593-2660 1 .

Monney Le, Otter I., Olivier R, Rava U., Minasaleb Ha, Fdlay I., Poirier G.G. and Borner C. (1996) Bcl-2 overexpression blocks activation of the death protease CPP32Namaiapopain. Biochemical & Biophysical Research Communications 22 1,340-345.

Murata Y., Ishiguro Y., Itoh J., Munakata A. and Yoshida Y. ( 1 995) The role of proinflammatory and immunoregulatory cytokines in the pathogenesis of ulcerative colitis. J. Gastroenterol. 30 Suppl8, 56-60.

Na S., Chuang T.H., Cunningham A., Turi T.G., Hanke J.H., Bokoch G.M., Danley and DE. (1996) D4-GDI, a substrate of CPP32, is proteolyzed dunng Fas-induced apoptosis. Journal of Biological Chemistry 271, 1 1 209- 1 12 13.

Nagata S. (1997) Apoptosis by death factor. [review] [85 refs]. Ce11 88,355-65: 12.

Nagata S. and Suda Tm (1995) Fas and fas ligand: Ipr and gld mutations. [review] [46 refs]. Immunologv Today 16, 39-43.

Narayanan V. (1997) Apoptosis in development and disease of the nervous system: 1. Naturally occurring cell death in the developing nervous system. [Review] [48 refs]. Pediatr. Neurol. 16,9- 1 3.

Nath Ra, Raser K.J., Stafford De, Hajimohrrnmadrcza l., Posner A., Allen H., Talanian R.V., Yuen P., Gübertsen R.B. and Wang K.K. (1996) Non- erythroid alpha-spectrin breakdown by calpain and interleukin 1 beta-converting- enzyme-like protease(s) in apoptotic cells: contributory roles of both protease families in neuronal apoptosis. Biochemicul Journal 319,683-690.

Nicholson D.W., Ali A., Thornberry N.A., Vaillancourt J.P., Ding C.K., Gallant, M, Gareau Y., Grinin P.R., Labelle M., Lazebnik Y.A. and et a1 (1995) Identification and inhibition of the ICEICED-3 protease necessary for marnmalian apoptosis [see comments]. Nature 376,37943.

Paolieri Fa, Battifora M., Riccio A.M., Pace Ga, Canoaica G.W. and Bapasco M. (1 997) Intercellular adhesion molecule- 1 on cultured human epithelial ce11 lines: influence of proinflammatory cytokines. Allergy 52,52 1-53 1 .

Parronchi P., Romagnani P., Annuoziato Fa, Sampognaro S., Becchio A., Giannarioi

La, Maggi E., Pupilli Ca, Tonelli Fa and Romagnani S. (1997) Type 1 T-helper ce11 predominance and interleukin-12 expression in the gui of patients with Crohn's disease. American Journal of Pathology 150,823-832.

Peitsch M.C., Pobar B., Stephan H., Cromptoa Ta, MacDonald H.R., Mannhen H.G. and Tschopp J. (1 993) Characterization of the endogenous deoxyribonuclease involved in nuclear dna degradation during apoptosis (prograrnmed ce11 death). EMBO J. 12 . 37 1 -3 77.

Potten C.S. (1 997) Epithelial ce11 growth and di fferentiation. ii. intestinal apoptosis. [review] [16 refs]. American Journal of Physiology 273, P t 1):G253-7 2

Potten CS., Wilson J.W. and Booth Cm (1997) Regulation and significance of apoptosis in the stem cells of the gastrointestinal epithelium. [Review] [96 refs]. Stem.Cells 15, 82-93.

Ptehn J.H., Jordan J., Ghadge G.D., Preis E., Galindo M.F., Roos R.P., Krieglstein J. and Milbr R.J. (1997) Ca2+ and reactive oxygen species in staurosporine- induced neuronal apoptosis. Journal of Neurochemisfry 68, 1679- 1685.

Quan L.T., Tewari M., O'Rourke K., Dixit Va, Snipas S.J., Poirier G.G., Ray, C, Pickup D.J. and Salvesen G.S. (1 996) Proteolytic activation of the ce11 death protease Y arnaKPP3 2 by granzyme B. Proceedings of the National A cademy of Sciences of the United States ojAmerica 93, 1972- 1 976.

Que F.G. and Gores GJ. (1996) Cell death by apoptosis: basic concepts and disease relevance for the gastroentemlogist [see comments] . [Review]. Gastroenferology 110, 1238-1243.

Romagnani P., Annunziato Fa, Baccari M.C. and Parronchi P. (1997) T cells and cytokines in Crohn's disease. [Review] [67 refs]. Current Opinion in lmmunology 9,793-799.

Saralian T.A. and Bredesen D.E. (1994) 1s apoptosis mediated by reactive oxygen species?. [Review]. Free Radic. Res. 2 1 , 1 -8.

Satob Ta, Sakai N., Enokido Y., Uchiyama Y. and Hataoaka Ha (1996) Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells. Brain Res. Mol. Brain Res. 733,9- 14.

Savill J. (1 997) Apoptosis in resolution of inflammation. [Review] [58 refs]. Journal of Leukocyte Biology 61,375-380.

Searle J., Kerr J.F. and Bishop C.J. (1982) Necrosis and apoptosis: distinct modes of ce11 death with fùndamentally different significance. Pathologv Annuul 17, Pt

Sedgbi S., Fields J.Z., Klamut M., Urban C., Durkin MW, Winship Dm, Fretland Dm, Olyaee M. and Keshavanian A. (1993) Increased production of luminol enhanced cherniluminescence by the inflarned colonic mucosa in patients with ulcerative colitis. Gut 34, 1 1 9 1 - 1 197.

Shaham S. and Horvib H.R. (1 996) An altematively spliced C. elegans ced-4 RNA encodes a novel cell death inhibitor. CelZ 86,20 1-208.

Shaham S. and Horvib H.R. (1996) Developing caenorhabditis elegans neurons may contain both cell- death protective and killer activities. Genes Dev. 10. 578-59 1.

Shick Lw, Carman J.H., Choi JeK., Somasundaram K., Burrell M., Hill D.E., Zeng Y.X., Wang Y., Wiman K.GO, Salhany K., Kadesch T.R., Monroe J.G., Donehower L.A. and ECDeiry W.S. (1997) Decreased immunoglobulin deposition in tumors and increased immature B cells in p53-nul1 mice. Cell Growth Dlffer. 8, 121-13 1.

Shimizu S., Eguchi Y., Kamiike W., ltoh Y., Hasegawa J., Yamabe K., Otsuki, Y, Matsuda H. and Tsujirnoto Y. (1 996) Induction of apoptosis as well as necrosis by hypoxia and predominant prevention of apoptosis by Bcl-2 and Bcl-XL. Cancer Aesearch 56,2 1 6 1 -2 1 66.

Shimizu S., Eguchi Y., Kamiike W., Waguri S., Uchiyama Y., Matsudv Hw and Tsujimoto Y. (1996) Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhi bitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 13,2 1 -29.

Shortman K. and Scollay R. (1994) Immunoiogy. Death in the thymus [news; comment]. Nature 372,44-45.

Silva M., Grillot Dm, Benito A., Richard C., Nunez G. and Fernandez-Luna J.L. ( 1996) Erythropoietin c m promote erythroid progenitor survival by repressing apoptosis through Bcl-XL and Bcl-2. Blood 88, 1576-1 582.

Simmoads NJ. and Rampton DwSm (1993) Inflamrnatory bowel diseaseœ-a radical view. Gut 34,865-868.

Smyth MJ.9 Perry D.K., Zhang Jw, Poirier G.C., Hannun Y.A. and Obeid L.M. (1996) prICE: a downstream target for ceramide-induced apoptosis and for the inhibitory action of Bcl-2. Biochemical Journal 316,25-28.

Spector M.S., Desnoyers S., Hoeppner D.J. and Hengartner M.O. (1 997) Interaction between the C. elegans cell-death regulators CED-9 and CED-4. Nature 385,

Steinman H.M. (1995) The Bcl-2 oncoprotein functions as a pro-oxidant. Journal of Biological Chemistry 270,3487-3490.

Stenson W.F. (1995) lnflammatory Bowel Disease. In Textbook ofGastroenterology (Edited by Yamada T.), p. 1748. J.B. Lippincott Co., Philadelphia.

Strater J., Koreb K., Cunthert A.R. and Moller P. (1995) In situ detection of enterocytic apoptosis in normal colonic mucosa and in familial adenomatous polyposis. Gut 37,8 1 9-825.

Strater J., Wellisch I., Riedl S., Walczak H., Koretz K., Tandara A., Krammer P.H. and Moller P. (1997) Cd95 (apo- I/fas)-mediated apoptosis in colon epithelial cells: a possible role in ulcerative colitis [see comments]. Gastroenterologv 113, 160- 167.

Strober W., Kelsall B., Fuss I., Marth Tm, Ludviksson B., Ehrhardt R. and Neurath M. (1997) Reciprocal ifn-gamma and tgf-beta responses regulate the occurrence of mucosal inflammation. [review] [ I l refs]. Imrnunology Today 18, 6 1-64.

Taupin J.L., Tian Q., Kedersha N., Robertson M. and Anderson P. (1995) The RNA- binding protein TIAR is translocated from the nucleus to the cytoplasm during Fas-mediated apoptotic cell death. Proceedings of the National Academy of Sciences of the United States of A rnerica 92, 1629- 1 63 3.

Tian Q., Taupin J., Elledp S., Robertson M. and Anderson P. (1995) Fas-activated serine/threonine kinase (FAST) phosphorylates TIA- 1 during Fas-mediated apoptosis. Journal of Experimental Medicine 182,865-874.

'huelove S.C. and Witts L.J. (1955) Cortisone in ulcerative colitis. Final report on a clinical trial. Brit. Med Journal. 2, 104 1 - 1048.

Unno Na, Menconi M.J., Smith M. and Fink M.P. (1995) Nitric oxide mediates interferon-gamma-induced hyperpermeability in cultured human intestinal epithelial rnonolayers. Critical Cure Medicine 23, 1 170- 1 1 76.

Varadhachary AS., Perdow S.N., Hu C., Ramanarayanan M. and Salgame P. (1997) Differential ability of T ce11 subsets to undergo activation- induced ce11 death. Proceedings of the Narional Academy of Sciences of the United States of America 94,5778-5783.

Wallach Da, Boldin M., Varfolomeev E., Beyaert R, Vandenabeele P. and Fiers W. (1997) Ce11 death induction by receptors of the tnf family: towards a molecular understanding. [review] [155 refs]. F E N Letters 410,96- 106: 17.

Wang D., Freeman G.J., Levine H., Ritz J. and Robertson M.J. (1997) Role of the CD40 and CD95 (APO- 1 /Fm) antigens in the apoptosis of human B-ceIl malignancies. British Journal of Haematoloty 97,409-4 1 7 .

Watson RW., Rotstein O.D., Jimenez M., Parodo J. and Marshall J.C. (1997) Augmented intracellular glutathione inhibits Fas-triggered apoptosis of activated human neutrophils. Blood 89,4 1 75-4 1 8 1.

Weller M., Schulz J.B., Wullner U., Loschmaan P.A., Klockgether T., Dichgans and S. (1 997) Developrnental and genetic regulation of programmed neuronal death. [Review] [29 re fs] . J. Neural Transm. Suppl. 50, 1 1 5- 1 23.

Wu D., Wallen H.D. and Nunez C. ( 1 997) Interaction and regulation of subcellular localization of CED-4 by CED-9 [see comments]. Science 275, 1 126- 1 129.

Wyllie A.H. (1997) Apoptosis: an overview. [Review] [5 1 refs]. British Medical Bulletin 53,45 1-465.

Wyllie A.H., Morris R.G., Smith A.L. and Dunlop D. (1 984) Chromatin cleavage in apoptosis: association with condensed chromatin morphology and dependence on macromolecular synthesis. < None Specifieb

Xue Dm, Shaham S. and Horvib H.R. (1 996) The Caenorhabditis elegans cell-death protein CED-3 is a cysteine protease with substrate specificities similar to those of the human CPP32 protease. Genes Dev. 10. 1073- 1083.

Yousefi S., Blaser K. and Simon H.U. (1997) Activation of signaling pathways and prevention of apoptosis by cytokines in eosinophils. [Review] [29 refs]. [nt. Arch. Allergy Immunol. 1 12,9- 12.

Yuan J. (1 996) Evolutionary conservation of a genetic pathway of programmed ce11 death. [Review] [53 refs]. Journal of Cellular Biochemistry 60,441.

Zamzami N., Susin S.A., Marchetti P., Hirsch T., Cornez-Monterrey I., Castedo M. and Guido Kroemer. (1 996) Mitochondrial control of nuclear apoptosis [see comments]. Journal of Expcrimental Medicine 183, 1533- 1544.

Zimmerman M.J. and Jewell D.P. (1 996) Cytokines and mechanisms of action of glucocorticoids and aminosalicylates in the treatrnent of ulcerative colitis and C rohn's disease. [Review] [56 re fs]. Alimentary Pharmacology & Therapeutics 10, Suppl-8

APPENDIX A

Standard Hematoxylin/Phloxine/Saffron (HPS) Staining Protocol

Mucosal biopsies removed fiom small or large bowel were immediately fixed in

10% formaiin, ernbedded in paraffin wax. sectioned at 5 pm in thickness, and then adhered

to glass slides. The sections are then deparaffinized by applying two changes of xylene for

5 min each. Hydration of tissues was carried out by bringing the sections twice to 100%

ethanol for 5 min each and then to 95% and 70% ethanol for 3 min each. The tissues were

then stained in hematoxylin solution for 3-5 min. The slides were then washed in

lukewarm tap water. The nuclei were differentiated by dipping the slides 3-5 times in acid

alcohol ( 1% HCl in 70% alcohol) and then the sections were rinsed in tap water. AAer

rinsing, ammonium water (approximately 2 ml of ammonium hydroxide in 4 litres of

water) was applied to blue the tissues. The sarnples were then washed well using running

tap water. In order to ensure proper nuclear staining, the tissues were observed under a

microscope. If the nuclei were too dark then the nuclear differentiation procedure (acid

alcohoVrinse/ammonium water) was conducted again and the slides were rechecked. If

the nuclear staining was too light then the siides were restained in hematoxylin and

differentiated again. Confirmation of appropriate nuclear staining was followed by rinsing

the slides in distilled water and then staining the tissue sections in phloxine for 1-2 min.

The sections were rinsed in tap water. The smooth muscle and cytoplasm were

differentiated fiom the connective tissue by treating the sections in 70% alcohol. The

tissues were then dehydrated in 3 changes of absolute alcohol for 1 min each. The slides

were then stained in safion (time varies with age of stain fiom 10 seconds to 3 minutes).

Excess siain was rernoved by passing the tissues through 4 changes of absolute alcohol ( 1

dip for each change of alcohol). The sections were cleared in toluol and mounted in a

synthetic resin medium.

APPENDIX B

Table 1: Study Population

pentasa- 1 g p.0.q.i.d.

ileum, cecum, rectum

cecum, sigmoid 1 losec-20 mg 1 ld

random colonic sites 1 none

ileum, cecum 1 pentasa-l g q.i.d. - -

sigmoid imuran- 125 mg 0.d. prednisone-40 mg 0.d.

al1 sites normai 1 salofalk-l g t.i.d.

ileum, transverse, splenic budesonide- 1 5 mg oral/d flexure omeprazole-20 mg oral/d

ileum-anastomosis prednisone-25 mg pentasa-1 g twice/d ranitidine- 1 50 mg iwiceld

ileurn, cecum budesonide-enemadd salofal k-4 g/d

cecum to sigmoid colon prednisone- 10 mg 0.d. asacol-4 tabletdd

cecum, ascending, hepatic 1 NA

cecum, transverse, descending

salazopyrin-intermittent

sigmoid NA

ileum-cecum-hepatic salazopyrin- 1 g t . i .d.

dl-sites-normal colace/laxatives/aspirin

cecurn

cecurn, splenic, descending, sigmoid, rectum

sigmoid, rectum

metamucil sigmoid, rectum

sigmoid lanoxin-0.125 mg 0.d. trental-400 mg 0.d. ventolin puffershitropaste

sigmoid

sigmoid, rectum

M 1 6-7 rno 50- 1 Ocm-multiple biopsies

asa- l tablet t.i.d. enalaprii- 1 tablet 0.d. trental-400 mg t.i.d. moduret- l tablet t.i.d. nitropaste and spray

rectum

1 5cm-non-speci fic changes imodium, premarin, cardizem, lasix, dicetal

sigmoid-no pathological diagnosis

sigmoid-no pathological diagnosis

triphasil

ileum-lymphoid aggregate rec turn-normal

Güodenurn/descending- increase in eosinophils

sigmoid-multi ple tubular adenornas

I ileum-nodular lymphoid hyperplasia

years 1 ileum-normal 1 prodium, buscopan

i lem-normal transverse-focal acute inflammation

rectum-hyperplastic polyp nadolol-40 mg 0.d. aspirin- 1 Id metamuci l

S Y 1 duodenurn-normal 1 none

I ileum-normal hydrochlorothiazide-pm transverse-no abnormal i ties

i leudascending-normal imodium descending-involved rectum-possible early polyp

1 i ieurn-rectum-normal 1 ranitidine- 150 mg ad.

1 ileum-sigrnoid-normal

cecum-sigmoid-normal vasotecd mg/d rectum-hyperplastic polyp eltroxin-0.1 mg/d

diabeta-10 mg b.i.d. vitamin E anti-histamine

surveillance cecum-sigrnoid-normal losec-20 mg/d estrogen, amitri pty line

surveillance cecum-rectum-normal pi11 for psoriasis

3 Y ileum-rectum-normal imovane, ty lenol plain . . -

surveillance I cecurn-rectum-normal 1 none

13 Y transverse-rectum prednisone-55 mg asacol-3.2 g

49 32 M 16y sigmoid, rectum, distal ciprofloxacin-5OOmg b.i.d. ileum, gastroduodenum prevacid-30 mg-

altemating days

Table 2: Description of Immunohistochemistry Antibodies

amino acids 38- 198 of mouse BAD

amino acids 1 8- 233 of human Bcl-X,

amino acids 1 - 219 of human CPP-32

amino acids 1 - 163 of human Fas

amino acids 225- 401 of human Ich- 1 L

amino acids 195- 393 of monkey ~ 5 3

arnino acids 16 1 - 365 of human TIAR

APPENDIX C

PATIENT CONSENT FORM

A STUDY OF THE SIGNIFICANCE OF APOPTOSIS (PROGRAMMED CELL DEATH) IN CHRONIC INFLAMMATORY BOWEL DISEASE: BLOOD and BIOPSY SAMPLES

You are being asked to participate in a research project which will study the significance of apoptosis (prograrnmed cell death) in chronic inflammatory bowel disease. The study is being conducted by Drs. M.R. Szewczuk and W.T. Depew of the Departments of Microbiology & lmmunology and Medicine, respectively. The research is designed to explore the possibility that certain stressproteins, someofwhich rnay reside in the intestine, rnay be important ineither initiating or perpetuating the disease in the lining of the intestine.

Procedures, Techniques To Be Used: 1 understand chat 1 suffer from chronic inflarnmatory bowel disease. My physician has

explained the nature of this disease to me in detail and 1 understand the implications of the diagnosis in terrns of my health and the requirements for treatment. My physician has indicated to me that the exact cause of these conditions remains unknown. Because of this there is no specific cure for these diseases. The research study will involve the donation of approximately 2 oz. (60 ml) of blood so that the blood serum and the cells in the blood can be analyzed to determine if there is any sign of a reaction to the stress proteins which are the focus of this research. The blood sample will be drawn from a vein in my a m by venipuncture with a standard needle.

1 understand that it rnay be necessary to examine my intestine directly with a colonoscope/sigmoidoscope to determine the extent and severity of my inflammatory bowel disease. Such exam inations are used when decisions about treatment are king considered. The requirement for such an examination in my case will be detemined by my doctor based on my disease progression. If such a colonoscopy/sigmoidoscopy is performed. the donation of a biopsy of normal and affected tissue from the colon will be requested. This will be a one time donation. The cells in the colon can be analyzed to determine if there is any sign of a reaction to the stress proteins. I understand that the biopsy sample will be taken during the clinically nccessary colonoscop~, and that such a biopsy donation wiII not alter the risks of the procedure. The extra biopsies will prolong the procedure by 20 to 40 seconds.

Benefitsl Risks: The risks associated with the venipuncture procedure are minimal but rnay include transient

discornfort at the needle puncture site and minor local bruising. Normally, only a singlevenipuncture. an additional 5 minutes of time, will be required. If subsequent samples of blood are needed, a su bsequent separate consent wil l be obtained.

The risks associated with the donation of biopsy procedure are minimal, and have already been explained to me by the doctor who organized the colonoscopy/sigmoidoscopy to evaluate my disease. Perforation due to biopsy is very rare (less than 1/100,000). Bleeding due to biopsy is also rare (less than 1 / 10,000).

The results of these research investigations rnay or rnay not be of direct benefit to me. Future patients might be helped through results derived from this project.

Voluntary Participation: 1 undentand that participation in this study is voluntary. 1 may withdraw my consent to

participate in this study at any point intime without jeopardizing my ongoing medical care at present or in the future. If 1 refuse to participate, my medical care will not be compromised in any way.

Confidentiality: All information obtained during this study is confidential. The information obtained will be

stored in a locked cabinet and availableonly to the principal investigator, Drs. W.P. Depew and M.R. Szewczuk. My identity will not be disclosed in any published findings of the study.

Copy for the Subject: 1 may retain a copy of this consent/information form for my records.

Contact People: I may discuss any aspects of the trial at any time with the CO-investigator Dr. W.T. Depew

(544-33 1 O ext. 2495); the principal investigator Dr. Myron Szewczuk (545-2457); or the Heads ofthe Department of Medicine. Dr. P.W. Munt (545-6327); and Microbiology and lmmunology - Dr. W.P. Aston (545-2450).

Signatures: By signing this consent form, 1 agree to participate in the above named research project.

Signature of Participant Date

The information within this consent has been explained to the participant, and to the best of my knowledge the subject understands the nature of the study and the risks and benefits involved.

Signature of 1 nvestigator or Designate Date

CONTROL CONSENT FORM

A STUDY OF THE SIGNIFICANCE OF APOPTOSIS (PROGRAMMED CELL DEATH) IN CHRONK INFLAMMATORY BOWEL DISEASE: BLOOD and BIOPSY SAMPLES

You are king asked to participate in a research project which will study the significance of apoptosis (prograrnmed cell death) in chronic inflarnmatory bowel disease. The study is being conducted by Drs. M.R. Szewczuk and W.T. Depew of the Departments of Microbiology & Irnmunology and Medicine, respectively. The research is designed to explore the possibility that certain stress proteins, someofwhich rnay reside in the intestine, rnay be important in either initiating or perpetuating the disease in the lining of the intestine.

Proceâures, Techniques To Be Used: 1 understand that 1 am being asked to participate in a research study designed to determine the

importance of immunity to stress proteins. 1 further understand that 1 do not suffer from chronic inflarnmatory bowel disease, and that rny participation is specifically sought as a control subject because the lining of my bowel is normal. The research study will involve the donation of approxirnately 2 oz. (60 ml) of blood so that the blood serum and the cells in the blood can be analyzed to determine if there is any sign of a reaction to the stress proteins. The blood sample will be drawn from a vein in my a m by venipuncture with a standard needle.

1 understand that it rnay be necessary to examine my intestine directly with a colonoscope/sigmoidoscope to determ ine the extent and severity of my disease. Such exam inations are used when decisions about treatment are being considered. The requirernent for such an examinat ion in my case will be determined by my doctor based on my disease progression. if such a colonoscopyhigmoidoscopy is perfoned, the donation of a biopsy of normal and affected tissue from the colon will be requested. This will be a one time donation. The cells in the colon can be analyzed to determine if there is any sign of a reaction to the stress proteins. 1 understand that the biopsy sample will be taken during the clinically necessary colonoscopy, and that such a biopsy donation will not alter the risks of the procedure. The extra biopsies will prolong the procedure by 20 to 40 seconds.

Benefits/ Risks: The risks associated with the venipuncture procedure are minimal but rnay include minor

discornfort at the needle puncture site and minor local bruising. Normally, only a single venipuncture, an additional 5 minutes of time, will be required. If subsequent samples of blood are needed, a subsequent separate consent will be obtained.

The risks associated with the donation of biopsy procedure are minimal, and have already been explained to me by the doctor who organized the colonoscopy/sigmoidoscopy to evaluate my disease. Perforation due to biopsy is very rare (less than Il 100,000). Bleeding due to biopsy is also rare (less than 1 / 10,000).

The results of these research investigations rnay or rnay not be of direct benefit to me. Future patients might be helped through results derived from this project.

Voluatary Participation: 1 understand that participation in this study is voluntary. 1 rnay withdraw rny consent to

participate in this study at any point in time without jeopardizing my ongoing rnedical care at present

or in the future. If 1 refuse to participate, my medical care will not be compromised in any way.

Confidentiality: All information obtained during this study is confidential. The information obtained will be

stored in a locked cabinet and available only to the principal investigator, Drs. W.T. Depew and M.R. Szewczuk. My identity will not be disclosed in any published findings of the study.

Copy for the Subject: 1 may retain a copy of this consent/information form for my records.

Contact People: 1 may discuss any aspects of the trial at any time with the CO-investigator Dr. W.T. Depew

(544-33 10 ext. 2495); the principal investigator Dr. Myron Szewczuk (545-2457); or the Heads ofthe Department of Medicine, Dr. P.W. Munt(545-6327); and Microbiology and lmmunology - Dr. W.P. Aston (545-2450).

Signatures: By signing this consent form. 1 agree to participate in the above named research project.

- -

Signature of Participant Date

The information within this consent has been explained to the participant. and to the best of my knowledge the subject understands the nature of the study and the risks and benefits involved.

Signature of lnvestigator or Designate Date