-
Gut 1996; 38: 886-893
Mucosal enzyme activity for butyrate oxidation;no defect in
patients with ulcerative colitis
E S Allan, S Winter, A M Light, A Allan
AbstractBackground-Butyrate is an importantenergy source for the
colon and its metab-olism has been reported to be defective
inulcerative colitis. One mechanism fordefective butyrate
metabolism in patientswith ulcerative colitis could be an
enzymedeficiency in the P-oxidation pathway ofbutyrate.Aims-This
study was undertaken tomeasure the activity of each enzymeinvolved
in the 3-oxidation pathway ofbutyrate in colonic
epithelium.Patients-Patients with ulcerative colitis(n=33), Crohn's
colitis (n= 10), and controlsubjects with colorectal cancer or
diver-ticular disease (n=73) were studied.Methods--Analysis was
carried out usingfluorometric and spectrophotometric tech-niques on
homogenised epithelial biopsyspecimens.Results-Significantly
increased butyrylCoA dehydrogenase activity was found inmucosa from
patients with ulcerativecolitis (33.2 (28.3, 38.1) pumolIg wet
weightlmin:mean (95% CI)) compared withactivity in mucosa from
control patients(24.3 (20.9, 27.7) gmollg wet weightlmin:mean
(950/o CI)) p
-
Mucosal enzyme activity for butyrate oxidation
Butyric acid CH3.CH2.CH2.COOH
ATP
AMP
Butyryl CoA
Crotonyl CoA
Hydroxybutyryl Cl
NA
NAI
Acetoacetyl CoA
Acetyl CoA
CH3.CH2.CH2.CO-S.CoA
H2
CH3.CH.CH.CO-S.CoA
H20 -
oA CH3.CHOH.CH2.CO-S.CoA
D+
DH
CH3.CO.CH2.CO-S.CoA
Butyryl.CoA.synthetase
EC: 6.2.1.2.
Butyryl.CoA.dehydrogenase
EC: 1.3.99.2.
Crotonase
EC: 4.2.1.17.
Hydroxybutyryl.CoA.dehydrogenase
EC: 1.1. 1.35.
Acetoacetyl.CoA.thiolase
EC: 2.3.1.9.
CH3.CO-S.CoA + CH3.CO-S.CoA
Figure 1: Pathway of /3-oxidation of butyrate.
mucosal specimen. Therefore as manyenzymes as possible were
assayed dependingon the amount of mucosa available
forhomogenisation from each patient. The effectof age, sex, or site
of mucosa along the colon,or method of harvesting was assessed
usingnormal mucosa.
Sample preparationSamples of colorectal mucosa were
transportedto the laboratory in ice cold saline (0. 15 MNaCl).
Samples were examined and excessmoisture removed by blotting.
Samples not forimmediate analysis were snap frozen and storedat
-40°C. Lamina propria lymphocytes wereisolated and stored according
to the method ofSachdev et al. l 1 Lymphocytes were thawedslowly at
room temperature and enzymesassessed similarly to whole tissue
samples.Mucosa was removed from the bowel wall by
scraping with the edge of a clean microscopeslide and then
weighed. Biopsy specimens wereweighed and disrupted in entirety.
Previouslyisolated lamina propria lymphocytes wereweighed and then
homogenised. All cells werehomogenised in enzyme extraction buffers
at aratio of 100 mg/ml (wet weight/volume) byusing an Omni 5000
hand held homogeniser at40C.
SubjectsMucosal specimens were taken from 73 subjectsundergoing
investigation or treatment for colo-rectal cancer (n= 69) or
diverticular disease(n=4). The median age of these patients was
67
(range 28-9 1) years and 39 were male. Allmucosal specimens in
these control patientswere taken from a site at least 10 cm from
anymacroscopic abnormality and adjacent mucosalsamples were
histologically normal. Sixty ofthese mucosal specimens were taken
at laparo-tomy and 13 at rigid sigmoidoscopy.
Mucosal specimens were taken from 33subjects with ulcerative
proctocolitis, medianage 46 (range 18-83) years, 18 were
men.Adjacent mucosal specimens were taken in eachcase for
histological examination and assess-ment of severity of ulcerative
colitis. Mucosalspecimens were taken from 13 patients atlaparotomy
and 20 at rigid sigmoidoscopy.
Mucosal specimens were taken from 10subjects with Crohn's
colitis, median age 34(21-67) years, two were men. All of
thesemucosal specimens were taken at laparotomy.All of these
patients had coexisting small bowelCrohn's disease. The diagnosis
of ulcerative orCrohn's colitis was established using conven-tional
clinical, radiological, endoscopic, andhistological criteria.
Enzyme extractionButyryl-CoA dehydrogenase and hydroxybu-
tyryl-CoA dehydrogenase - mucosal homogenateswere extracted into
50 mM phosphate buffer(pH=7.0) containing 0.3 mM EDTA, 200
uMphenylmethylsulphonyl fluoride, and 2 mMdithiothreitol. Crude
homogenate was reservedfor hydroxybutyryl-CoA dehydrogenase
assay.Homogenate for butyryl-CoA dehydrogenaseassay was
fractionated by addition of solidammonium sulphate to 40% w/v with
mixingfor 30 minutes on ice and then centrifugation at5000xg at 4°C
for 15 minutes. The supernatantwas assayed.
Crotonase, acetoacetyl-CoA thiolase, andbutyryl-CoA synthetase
were extracted into 10mM TRIS buffer (pH= 7.4) containing 1 mMEDTA,
3 mM magnesium chloride, and 300mM sucrose.
Cytological assessment of cell suspensionsBefore complete
disruption by homogenisa-tion, aliquots of mucosal cell suspensions
fromthe normal colon of three patients were spreadon glass slides
and sprayed with polyethyleneglycol, ethyl alcohol, and glacial
acetic acidfixative and stained with Papanicolaou stain.The
proportions of lymphocytes and epithelialcells in these
preparations were then estimatedmicroscopically for 10 high power
(X400)fields.
Classification of mucosal samples from patientswith ulcerative
proctocolitisRoutine haematoxylin and eosin stained paraf-fin wax
sections were prepared from samples ofmucosa adjacent to those used
for assay.Inflammatory changes were classified usingthe criteria of
Truelove and Richards12(Appendix). Epithelial cell density
wasassessed using the grading system described byGoodman et al.
13
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Allan, Winter, Light, Allan
TABLE I Enzyme activity in normal biopsy specimens.Enzyme is
expressed in ,umol/g wet weight/minute. Numbersofsamples and 95%
confidence limits are also shown
Enzyme Men Women
Butyryl-CoA synthetaseMean 0-058 0.049Number 4 995% CI -0-062 to
0-178 0-019 to 0 079
Butyryl-CoA dehydrogenaseMean 21-97 25-54Number 6 1295% CI 14-77
to 29-2 20-7 to 30-38
CrotonaseMean 9-26 9-25Number 16 1995% CI 7-2 to 11-3 6-52 to
11-98
Hydroxybutyryl-CoA dehydrogenaseMean 4-24 4.59Number 34 3095% CI
3.79 to 4-69 4-1 to 5-08
ThiolaseMean 5-96 5-42Number 34 3195% CI 5-27 to 6-65 4-4 to
6-44
Enzyme assaysAll chemicals and enzymes were purchasedfrom Sigma
Chemical Company UK. Assayswere performed either
spectrophotometricallyusing a Philips PU8720 spectrophotometer
orfluorimetically using a Shimadzu RF-5001PCfluorimeter.
All enzyme assays were optimised for humancolonic epithelial
cells.
Butyryl-CoA synthetase (BuCoAs) wasassayed according to the
method of Lageweget al.'4 This enzyme catalyses the activation
ofbutyrate to its CoA ester. The reaction wasassessed by coupling
the reaction to acyl-CoAoxidase and peroxidase. The rate of
peroxideformation was assessed fluorimetrically. Theintra-assay
coefficient of variation was 9-6%.Buyryl-CoA dehydrogenase
(BuCoADH) was
assayed according to the method of Engel.'5BuCoADH catalyses the
conversion ofbutyryl-CoA to crotonyl-CoA. The reaction
Butyryl-CoA Synthetase
TABLE ii Enzyme activity in ,umol/wet weight/min forcontrol
subjects in samples collectedfrom rectal biopsy andsurgical
specimens from rectum, left colon, and right colon
Biopsy Surgical specimenspecimen
Enzyme (rectum) Rectum Left colon Right colon
Synthetase 0-0783 0.0375 0-0251BuCoA DH 23-41 32-24
21-50Crotonase 8-58 7.90 8-45 13-26HoBuCoADH 4-19 4-53 4-16
4-71Thiolase 4.13* 6-14 5.44 6-75Protein 3-71 4-22 3-62 3-53
Statistical analysis of loglo transformed data shows no
significantdifferences from synthetase, butyryl-CoA
dehydrogenase,crotonase and hydroxybutyryl-CoA dehydrogenase.
Thiolaseactivity, however, showed significant differences
(*p=0.046)between rectal biopsy samples and surgical samples.
Allsubsequent statistical analyses of thiolase activity
wereperforned on like samples.
was assessed by coupling this reaction
todichlorophenolindophenol and phenazinemethosulphate. The rate of
decreasingabsorbance was assessed spectrophotometri-cally at 600
nM.
Crotonase was assayed according to themethod of Fong and
Schultz.16 Crotonasecatalyses the conversion of crotonyl-CoA
tohydroxybutyryl-CoA. The formation of themagnesium complex was
assessed spectro-photometrically at 280 nm. The intra-assayand
inter-assay coefficients of variation were4% and 13%
respectively.
Hydroxybutyryl-CoA dehydrogenase (Hobu-CoADH) activity was
measured accordingto the method of Fong and Schulz.'7HobuCoADH
catalyses the conversion ofhydroxybutyryl-CoA to
acetoacetyl-CoA.However, in vitro the forward reaction
rapidlyequilibrates thus the reaction was assayed inthe reverse
direction. The rate ofreaction result-ing from nicotinamide adenine
dinucleotide(NAD) formation was assessed spectrophoto-metrically at
340 nm. The intra-assay and inter-
Butyryl-CoA dehydrogenase Crotonase130.20 r
0.15 e-
60
40 F-
0-10 |-
30 r
20 F-
30K
* 80
* O
Hydroxybutyryl-CoAdehydrogenase
20 H
10
_4h O13
0
a 4+* |
12
10 e-
o
-I
8
itiII
Thiolase
18 r15
a
£1
12
9
6
3
0
+-e-c
C,C
Figure 2: Comparison ofenzyme activities between control
subjects and ulcerative colitis patients classified as Truelove
andRichards class 1. Control values are shown as O and uleerative
colitis samples as *. The mean enzyme activity is shownby
horizontal line.
Ea)CD
3._
0)
3.10E
0
a)ENcwL
0.05
0.00
1211109876543210
888
.
a12
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Mucosal enzyme activity for butyrate oxidation
TABLE III Variation ofenzyme activity with age for control and
ukerative colitis samples
30-50 51-70 71-90
Enzyme Control UC Control UC Control UC
Synthetase 0.03 0-032 0-052 0-034 0-067 0-021BuCoA DH 29-31
28-84 27-25 24-31 18-91 25-45Crotonase 6-63 8-95 10-76 11-37 7.97
7-63HoBuCoA DH 4-51 3-85 4-33 3-13 4-66 2-96Thiolase 3-43 3-69 5-74
2-37 6-12 5-84
assay coefficients of variation were 8.2% and16.7%
respectively.Acetoacetyl-CoA thiolase (Thiolase) activity
was assayed according to the method ofWilliamson et al.18
Thiolase catalyses theconversion of acetoacetyl-CoA to
twoacetyl-CoA units. The rate of reaction due toacetyl-CoA
formation was assessed spectro-photometrically at 303 nm. The
intra-assaycoefficient of variation was 4%.
Protein concentration was determined usingthe method of
Bradford.'9 Protein concen-trations in control samples, ulcerative
colitis,and Crohn's samples were compared. Nodifferences were seen
between normal controlsamples and samples from patients with
ulcer-ative colitis. Therefore no adjustment forprotein
concentration was made. In the caseof Crohn's samples differences
in protein
100°
10
a)
zC.).)0)
0)
a)
E
0.1 [_
0.011 2 3
Truelove and Richards classFigure 3: Comparison ofenzyme
activity between Truelove and Richards classes. Butyryl-CoA
synthetase (M), butyryl-CoA dehydrogenase (Ol), crotonase (M),
hydroxybutyryl-CoA dehydrogenase (E), thiolase (u).
concentration with the control samples wereobserved and
therefore all results wereexpressed per gram of protein.
Statistical methodsComparisons in enzyme activity betweengroups
of patients were assessed using anunpaired t test with two tail p
values on logl0transformations of the enzyme data. Otherstatistical
tests are as indicated in the text.
EthicsApproval for the study was given by theNorth Birmingham
Research and EthicsCommittees.
Results
Enzyme activity in histologically normal mucosaTable I
summarises enzyme activity in normalbiopsy specimens. No
differences in enzymeactivity were found between men and womenfor
any of the enzymes tested (two samplet test). One way analysis of
variance showedno site related difference in enzyme activitybetween
samples from the rectum, left or rightcolon. There was increased
thiolase activitywith age when patients were subdivided intothree
groups: those below 50 years, thosebetween 50 and 70 years, and
those over 70years of age. Statistical analysis (one wayanalysis of
variance) on logl0 transformed datashowed significant differences
in enzymeactivity between groups (F=4-83: p=0 01).No other enzyme
activity varied significantlywith age. Thiolase activity also
varied signifi-cantly between rectal biopsy and laparotomysamples
with the biopsy specimens givinglower values (two sample t test p=0
008).Table II gives these results. All furthercomparisons were
stratified into biopsy andrectal excision samples. No other
enzymeactivity varied with the mode of collection.
ProteinFifty four specimens were assayed for proteincontent. The
mean (950/O CI) protein concen-tration was 3.79 (3.52, 4.06) g/g
wet weighttissue. No differences were seen across the agerange of
patients tested (45-91 years).Comparative analysis by site of
origin ofsample showed no differences between rectum,left colon or
right colon (Table II).
Enzyme activity in ulcerative colitis mucosaButyryl-CoA
synthetase - 18 patients donated
biopsy specimens, eight were male and 10 werefemale. The median
age was 55 years and theage range spanned 20 to 80.Buyl-CoA
dehydrogenase - 24 patients
donated biopsy samples, 15 were male andnine were female. The
median age was 39years and the age range spanned 18 to 83.
Crotonase - 20 patients donated biopsyspecimens, 13 were male
and seven female.
889
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Allan, Winter, Light, Allan
The median age was 38 years and the age rangespanned from 18 to
83.
Hydroxybutyryl-CoA dehydrogenase - 24patients donated biopsy
specimens, 13 weremale and 11 female. The median age was 39years
and the age range spanned 18 to 83.
Thiolase - 20 patients donated biopsysamples, 13 were male and
seven were female.The median age was 38 years and the age
rangespanned 18 to 72.
Comparison ofenzyme activity in ulcerativecolitic mucosa with
histologically normal mucosaFor each enzyme studied a comparison
wasmade between the enzyme activity in controlbiopsy specimens and
the enzyme in Trueloveand Richards (T and R) class 1 biopsy
speci-mens (two sample t test on loglo transformeddata). The mean
(95% CI) butyryl CoAdehydrogenase activity (pumol/g wet weight!min)
in normal mucosa was 24.3 (20.9, 27.7)n=18 compared with 33.2
(28.3, 38.1) n=10in biopsy specimens of T and R class 1(p=002). The
mean thiolase activity was 5-69(5.09, 6.29) n=65 in normal
mucosacompared with 3.21 (2.61, 3.81) n=9 insamples ofT and R class
1 (p=0.001). Therewas no significant difference in butyryl-CoA
100 r
synthetase, crotonase, or hydroxybutyryl-CoAdehydrogenase
activity between control and Tand R class 1 biopsy specimens (Fig
2).The mean thiolase activity in T and R class 1
biopsy specimens was significantly lower thanthe mean thiolase
activity in control samples.Within theT and R class 1 biopsy
specimens themedian age of the donor patients was 37 (range28-62)
years compared with a median age of 67(range 28-85) years in
patients with normalmucosa. In view of this the youngest
ninecontrol patients: median age 49 (range 28-52)years were
selected from the control group andthiolase activity in these
biopsy specimens wascompared with the thiolase activity in the T
andR class 1 samples. No significant difference wasfound (two
sample t test on loglo transformeddata p=06). Table III shows these
data.
Differences in enzyme activity in Truelove andRichard
classesEnzyme activity was compared between eachof the three
Truelove and Richards classes(one way analysis of variance on loglo
trans-formed data). Butyryl-CoA dehydrogenaseactivity was
significantly different between Tand R classes. Hydroxybutyryl-CoA
dehydro-genase activity was also significantly differentbetween T
and R classes with enzyme activityfalling with increasing
inflammation in theadjacent mucosal samples (Fig 3).
Correlation of enzyme activity with epithelial celldensityBiopsy
specimens from patients with ulcerativecolitis were classified
according to their epithe-lial cell density. There was a trend
towardsdecreasing enzyme activity with decreasingcell density, but
between the grades 1, 2, 3there was no significant difference (Fig
4). Nospecimens were classified as class 4.
Correlation of enzyme activity with drugs takenby patientThe
mean enzyme activity in ulcerative colitispatients taking either
oral or intravenous corti-costeroids (n= 8) was compared with
theenzyme activity in those patients taking neithertopical nor
systemic corticosteroids (n=9). Anunpaired t test on the log1o
transformed datashowed no significant difference in any
enzymeactivity.A similar analysis comparing the enzyme
activity in patients taking any 5-aminosalicyliccontaining drug
orally or rectally (n= 17) withthe enzyme activity in those
patients takingno 5-aminosalicylic containing drug eitherorally or
rectally (n= 5) showed no significantdifferences.
Enzyme activity in the mucosa ofpatients with3 colonic Crohn 's
disease
Butyyl-CoA dehydrogenase - samples fromIcell nine patients were
assayed for butyryl-CoAcse ( dehydrogenase activity. The mean
activity was
1-33 (0-83, 1.83) p.mol/g protein/min.
10 i
nccC.)
._
0)
0
c
0E=L
0.1 r-
0.01 IH
I I0 2
Epithelial cell densityFigure 4: Comparison ofenzyme activity in
tissue classified according to epithelialdensity. ButyIyl-CoA
synthetase (E), butyryl-CoA dehydrogenase (Ol),
crotonahydroxybutyryl-CoA dehydrogenase (s), thiolase (m).
H=hypertrophied crypt.
F=EL-iF==]EL-3==1a__AF=:=1
890
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Mucosal enzyme activity for butyrate oxidation
1.4r
1.2 F-
'E 0.8c
0
0.
0 0.6
_-
0.4 _
0.2
0 WOWOI0%1A I,,,,5Y% Q,/,%ABucoA DH *Crotonase *HoBucoA DH
Thiolase
Figure 5: Comparison ofenzyme activity in Crohn 's and control
tissue. The enzymeactivity is expressed in 1ttmollg protein/minute.
Crohn 's disease (E), control (W).*p
-
Allan, Winter, Light, Allan
E
._
0
0
E
4
2
BucoA DH Crotonase HoBucoA DH Thiolase
Figure 6: Enzyme activities in lamina propria lymphocytes from
control (E), ulcerativecolitis ( D), and Crohn 's disease (0)
tissues. Enzyme activity is expressed as ,umol/g wetweight
tissue/min.
certainly due to the increased age of the controlgroup, with
correspondingly increased enzymeactivity, compared with the younger
age of thecolitic group. Furthermore thiolase activity isrequired
to catalyse the final step in the oxida-tion of butyrate and it is
probable that butyryl-CoA synthetase controls the rate limiting
stepof the oxidation pathway.
This study therefore shows a rise in enzymeactivity in minimally
inflamed mucosa frompatients with ulcerative colitis compared
withnormal mucosa.There was a tendency for all enzyme activity
studied to fall in parallel with falling epithelialcell density
in the mucosa of patients withulcerative colitis. This type of
relation waspreviously described as a property of thecolonic
epithelial cell enzyme glucosaminesynthetase.13 This is probably a
generalisedresponse to epithelial cell depletion and may beshared
by many colonic epithelial cell enzymesin patients with ulcerative
colitis.
Previous studies show that 5-aminosalicylicacid has no effect on
butyrate metabolism incolonic epithelial cells from normal
controls.This study complements this finding bysuggesting that
neither corticosteroid nor5-aminosalicylic acid treatment have
anyeffect on the enzyme activity of the oxidationpathway of
butyrate.
Enzyme activity in colonic mucosa frompatients with Crohn's
colitis was raised com-pared with that from the mucosa of
controlsubjects. This rise in activity was significant forthe
enzymes butyryl-CoA dehydrogenase andthiolase. This rise in
activity mirrors theincreased activity seen in Truelove andRichards
class 1 mucosa from patients withulcerative colitis. It probably
occurred becauseof selection of the least inflamed tissue in
thecolon for assay. Previous studies of the enzymeglucosamine
synthetase13 report a rise in theactivity of this enzyme in the
mucosa ofpatients with Crohn's colitis even at sites dis-tant from
the macroscopically evident disease.The rise in enzyme activity
found in mucosafrom patients with Crohn's colitis is probablynot
caused by an increased cell turnover in theCrohn's tissue because
cell proliferation in rec-tal biopsy specimens from patients with
rectalCrohn's disease is normal irrespective of thedegree of
inflammation in the rectum.2' Itseems more probable that the
disease processtriggers a generalised reactive increase in
theactivity of many intracellular enzymes. Thisfinding suggests
that the observed rise inglucosamine synthetase activity in
patientswith Crohn's colitis is part of a more wide-spread response
to inflammation in the colonicepithelial cell.
This study used biopsy specimens and notepithelial cell
preparations to study colonicepithelial cell enzyme activity and
thereforeconsideration must be given to sources ofenzyme activity
within the samples but notarising from the colonic epithelial cells
them-selves. Although colonic wall muscle does notmetabolise
butyrate3 the mucosal homogenateused for enzyme assay contained a
mixtureof both colonic epithelial cells and laminaproprial
lymphocytes. Cytological assessmentof these cell mixtures suggested
that laminalproprial lymphocytes accounted for only 10%of the cells
in the mixture. Further studies onisolated laminal proprial
lymphocytes showeda small but detectable enzyme activity fromthese
cells. This activity was similar irrespec-tive of whether the cells
were from controlsubjects or patients with inflammatory
boweldisease. They are therefore likely to have madea minimal but
relatively constant contributionto the total enzyme activity in the
homogenatesirrespective of the source of the specimens.
In conclusion, this study suggests that ifabnormalities of
butyrate metabolism occur inthe colonic epithelium of patients with
ulcera-tive colitis then the mechanism of this abnor-mality does
not entail decreased enzymeactivity in the 3-oxidation pathway
forbutyrate. None the less an abnormality ofbutyrate metabolism in
patients with ulcera-tive colitis remains possible. It seems
unlikelythat supply of butyrate to colonic epithelialcells is ever
in danger because studies ofcolonic luminal bacterial flora in
patients withulcerative colitis show few changes.22 In addi-tion
absorption of butyrate in the remissionphase of the disease is no
different fromabsorption in control subjects.23 However,other
abnormalities of intracellular butyrate
892
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Mucosal enzyme activity for butyrate oxidation 893
metabolism may occur such as abnormalintracellular transport of
butyric acid, alter-ation of mitochondrial oxidation or
abnormalincorporation of butyrate products into thecitric acid
cycle for energy production.This study was supported by a grant
from the West MidlandsRegional Health Authority and the Sutton
Municipal Charities.The isolated lamina proprial lymphocytes were a
generous giftfrom Dr D P Jewell, The Radcliffe Infirmary, Oxford.
Dr P JHanson, Department of Pharmaceutical Sciences, University
ofAston gave invaluable advice on aspects of the enzymes assays.Mrs
J H Plant typed the manuscript.
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Appendix Summary of Truelove and Richardsclassification for
histological severity of ulcerative colitis
No significant inflammationInflammation NilCrypt abscesses
AbsentEpithelium IntactGland architecture Glands reduced in
number
Stunted and atrophicMild to moderate inflammationInflammation
Increased chronic inflammatory cell
Mild increase in neutrophilsVariable intensity
Crypt abscesses OccasionalEpithelium Increased proliferation
Intact (apart from crypt abscess)Gland architecture Irregular
shape and densitySevere inflammationInflammation Chronic
inflammatory cell infiltrateCrypt abscesses Plentiful+erosions of
crypt epithelium
Inflammation extending into laminapropria
Epithelium Small breaches and erosionsIrregular and often cuboid
cells
adjacent to erosionsGland architecture Markedly irregular in
density and
shape with gland destruction
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