COLONIC EPITHELIAL CELL APOPTOSIS INFLAMMATORY BOWEL...
Transcript of COLONIC EPITHELIAL CELL APOPTOSIS INFLAMMATORY BOWEL...
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
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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.
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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