Research in Veterinary Science 2000, 68, 189Ð196

doi:10.1053/rvsc.1999.0359, available online at http://www.idealibrary.com on

Cell proliferation and apoptosis in the pathogenesis of

oesophagogastric lesions in pigs
R. PREZIOSI, G. SARLI, P. S. MARCATO

Dipartimento di Sanitˆ Pubblica Veterinaria e Patologia Animale, Sezione di Patologia Generale ed Anatomia
Patologica, Universitˆ di Bologna, via Tolara di Sopra 50, Ozzano Emilia, 40064, Italy

SUMMARY

Recent studies have stressed the importance of epithelial hyperproliferation in the pathogenesis of early lesions (parakeratotic
hyperkeratosis) of the porcine gastric pars oesophagea (PO). In this study, immunohistochemical staining with Ki67 (clone MIB1)
and AgNOR proteins silver staining were used to evaluate, by means of image analysis, cell proliferation in normal and parakera-
totic (parakeratotic hyperkeratosis) epithelia of the PO. Apoptotic activity was also assessed with the TUNEL assay and compared
with cell proliferative parameters. Early lesions of the PO were characterised by a significant increase in epithelial proliferative
activity while there was no difference in the apoptotic activity between normal and parakeratotic epithelia.
Our data confirm the hyperproliferative nature of epithelial changes preceding degeneration and erosion/ulcer of the PO and
suggest that an underlying feature of gastric ulcers in pigs is an imbalance between cell proliferation and programmed cell death.
© 2000 Harcourt Publishers Ltd

THE occurrence of hyperplastic lesions (parakeratotic
hyperkeratosis), erosions and ulcers of the pars
oesophagea (PO) is a common finding in slaughtered pigs
and is attributed to different dietary and environmental
stresses, associated with intensive pig husbandry (O’Brien
1992). Physical and/or chemical composition of feed is
often considered the most important aetiological factor
(Embaye et al 1990) and recent studies emphasised the role
of microbial production of undissociated short chain fatty
acids (SCFA) in the mechanism of epithelial injury
(Argenzio and Eisemann 1996). Gross pathological and
microscopical findings observed in the naturally occurring
disease, have been reported in different studies
(Muggenburg et al 1964, Asdrubali 1966, Kowalczyk
1969, Macari et al 1976, Embaye et al 1990).
In spite of the numerous reports which focus on the aeti-
ology and description of these lesions, their pathogenesis
remains largely unknown. Recently, Roels et al (1997), sug-
gested an important role for epithelial proliferation (paraker-
atotic hyperplasia) in the pathogenesis of early lesions of
this porcine gastric zone. However, programmed cell death
(apoptosis) has recently gained scientific recognition as an
active regulatory mechanism, complementary but function-
ally opposite to proliferation, with an important role in
maintaining homeostasis of continually renewing tissues,
especially epithelia, and in the pathogenesis of different
diseases (Haake and Polakowska 1993). In this study, we
assessed cell proliferation and apoptosis in areas of PO
exhibiting gross lesions and compared the results with the
Correspondence to: Rosario Preziosi, Dipartimento di Sanità Pubblica
Veterinaria e Patologia Animale, Sezione di Patologia Generale ed
Anatomia Patologica, Università di Bologna, via Tolara di Sopra 50,
Ozzano Emilia, 40064, Italia
degree of severity in tissue injury. Cell proliferation was
evaluated using Ki67 monoclonal antibody (clone MIB1)
that recognises a nuclear protein expressed by actively
cycling cells but not by resting G0 cells (Gerdes et al 1992),
and AgNOR proteins silver staining. AgNOR proteins are a
group of argyrophilic nucleolar proteins associated with
nucleolar organiser regions in which the amount is strictly
related to cell duplication rate (Trerè et al 1989).
Apoptotic activity was evaluated in normal and parakera-
totic stomachs, using the terminal deoxynucleotidyl trans-
ferase (TdT)-mediated digoxigenin dUTP end labelling
(TUNEL) (Thiry 1992), the aim in which is to test the hypoth-
esis that in fundamental to parakeratotic lesions there may
be an imbalance between cell proliferation and cell death.
MATERIALS AND METHODS
Source of stomach tissues
Stomachs from a total of 1131 pigs (160 kilos 1iveweight.)
belonging to seven different herds but on the same
fattening ration [3/4 whey + 1/4 dry part (60 per cent maize
silage, 10 per cent barley, 15 percent soybeans, 9 per cent
bran, 6 per cent feed supplement)]. Stomachs were examined
on line in the same slaughter plant and dissected so as to
leave undamaged the pars oesophagea and the contents were
discarded. The stomachs were washed in tap water and the
pars oesophagea (PO) was examined and assessed for the
presence of macroscopic lesions. Then the PO was classified
according to its gross appearance into normal (N), present-
ing a white, smooth, and glistening surface; early or mild
parakeratosis (P1) and more severe parakeratosis (P2),
presenting a PO mucosal surface less (P1) or more (P2)
irregularly thickened and yellow or yellow green; severe

0034-5288/00/020189 + 8 $35.00/0 © 2000 Harcourt Publishers Ltd

190
TABLE 1 Mean values (± standard deviation) of the epithelial proliferative and apoptotic indices in gastric pars oesophagea of pigs
assessed by AgNOR proteins quantification, MIB1 index and apoptotic index: N = normal pars oesophagea; P1 = mild parakeratotic
lesions; P2 = heavy parakeratotic lesions; P3 = parakeratosis + erosions; ulcers (U), (ANOVA for MIB1 index and AgNOR area between
N vs P1, P2, P3 vs U, P < 0·05; ANOVA for apoptotic index N vs P1 or P2, P = 0·71).
Mean values (± SD) of epithelial proliferative activity and apoptosis
Parameter (N) (P1)
AgNOR area 4·39 ± 0·67 4·90 ± 0·44
(µm2)
MIB1 index 10 ± 0·03 15 ± 0·09
(%)
Apoptotic index 18 ± 11·58 19 ± 8·58
(%)
// = No available data
parakeratosis with macroscopic erosions (linear superficial
clefts in the parakeratotic surface) (P3) and ulceration (U)
(round, oval or linear deep lesions devoid of epithelium and
presenting the base with granular appearance and with a
blood or brown-tinged exudate). Routine histological exam-
ination was also performed on selected samples (18 for each
of the seven scores) to confirm, microscopically, this gross
classification. P1 and P2 were histologically discriminated
according to the extent of parakeratotic changes, while ero-
sions and ulcer were defined according to the involvement
or not of the lamina propria in the erosive process. For the
histochemical and immunohistochemical studies, 46 sam-
ples were collected from pigs with lesions belonging to each
of the four groups (P1 = 15; P2 = 17; P3 = 4; U = 10) and 18
specimens from pigs with macroscopically normal PO were
also included. Sampling of the visible ulcerative areas was
performed taking care to always obtain a wide margin con-
taining the gastric epithelia beside the lesion. The specimens
were formalin-fixed and paraffin wax-embedded. From each
sample two consecutive sections, 4-µm thick, were cut for
AgNOR proteins silver staining and immunohistochemical
analysis with Ki67 Mab. A third section from normal, P1
and P2 samples was cut for TdT-mediated digoxigenin
dUTP end labelling method (TUNEL) staining.
Ki 67 immunohistochemistry
The sections were dewaxed in toluene and rehydrated in a
graded acetone series. Endogenous peroxidase was blocked
by means of 3 per cent hydrogen peroxide for 30 minutes.
Sections were then rinsed in Tris buffer, immersed in citrate
buffer (2·1 g citric acid monohydrate/l distilled water), pH
6·0 and incubated for four periods of 5 minutes each in a
microwave oven at 750 W. After microwave irradiation, sec-
tions were allowed to cool down to room temperature (RT)
(approximately 20 minutes).
Ki 67 immunohistochemistry was performed with the
clone MIB1 (Immunotech, Int, Marseilles, France) using a
highly sensitive streptavidin-biotin-peroxidase technique
with a commercial kit (BIO SPA, Milan, Italy). In control
sections, the primary antibody was substituted by PBS buffer.
AgNOR silver staining
The sections were stained with a specific silver staining
method (Öfner et al 1994). The dewaxed sections were
immersed in citrate buffer at pH 6·0 and underwent the same
R. Preziosi et al
(P2) (P3) (U)
4·94 ± 0·66 4·60 ± 0·89 7·26 ± 1·46
20 ± 0·07 22 ± 0·03 26 ± 0·03
21 ± 7·10 // //
microwave treatment as mentioned above for MIB1
immunohistochemistry; they were then allowed to cool
down to RT, washed in double distilled water and immersed
in a fresh solution of one volume of gelatin (2 per cent) in
1 per cent aqueous formic acid and two volumes of 50 per cent
silver nitrate, at 37°C for 14 minutes in the dark. The slides
were washed in distilled water, dehydrated and mounted.
Detection of apoptosis in tissues
The sections were stained with the Apoptag Kit (Oncor,
Resnova, Rome, Italy). The kit utilises reagents for non-iso-
topic DNA end-extension in situ (digoxigenin-11-dUTP), and
other reagents for immunohistochemical staining (anti-
digoxigenin-peroxidate antibody) of the extended DNA.
Residues of digoxigenin-nucleotide are catalytically added
to the DNA by terminal deoxynucleotidyl transferase (TdT)
which generates, to the 3′-OH ends of double or single
stranded DNA, tails of digoxigenin d-UTP. The latter was
revealed immunohistochemically by means of the anti-
digoxigenin antibody. In control slides, TdT-enzyme solu-
tion was substituted by distilled water.
Scoring method
Quantification of cell proliferation and apoptosis was per-
formed with the image cytometer Cytometrica (Byk Gulden,
Milan, Italy).
AgNOR Quantitative evaluation of interphase AgNORs
was performed by measuring the area (square micrometres)
occupied by AgNOR proteins in the nucleus. Every image
was obtained with a 40x objective. One hundred random
nuclei of basal and suprabasal epithelial cells were evalu-
ated in four different fields of each section and twenty-five
nuclei were assessed in each field. AgNOR quantity was
expressed as mean AgNORarea (MNORA).
MIB1 index For each case, 10 fields with the highest posi-
tivity to MIB1 were selected using a 25x objective. In each
field a first image of the total nuclear area was mapped with
a green (575/10 nm) bandpass filter; a second area consist-
ing of all the MIB1 positive nuclei, was mapped with a blue
(490/10 nm) bandpass filter. In each field inflammatory or
stromal cells were masked. MIB1 index was calculated
as the percentage of labelled nuclei compared with
total nuclear area (MIB1 index = total nuclear area of the
 Cell turnover in porcine gastric ulcers 191

positively stained nuclei in ten fields/total nuclear area in USA) to compare the proliferative activity and apoptotic
ten fields per 100). expression among the groups.

Apoptotic index The apoptotic fraction was quantitated

choosing, in each case, 10 fields in the gastric epithelial lay-
ers (superficial and midzonal layers) where apoptosis was
recognisable. All images were obtained using a 40x objec-
tive. In each field, the number of apoptotic nuclei, the total
nuclear area and the mean area of a nucleus were evaluated.
Apoptosis has been expressed as the number of positive
nuclei per 100 cells, named apoptotic index and calculated
as follows:
Total nuclear area of the fields
=N
mean area of a nucleus
N = number of cells present in the total nuclear area of the
chosen fields.
Apoptotic index = Total number of apoptosis × 100
N (Fig 1a, 1b), whereas in parakeratotic lesions numerous Ki
Statistical analysis
Analysis of variance (ANOVA) was performed with the
Complete Statistical System (CSS Statistic, Statsoft, Tulsa,
RESULTS
Histological changes of different degrees were observed
in all PO with macroscopic epithelial alterations (P1, P2, P3),
including increased epithelial thickness, elongation of rete
pegs, more or less severe parakeratosis and ballooning
degeneration. In P2, the parakeratotic epithelium showed
microvesicular changes and microabscessation which con-
tributed to weakening and erosions. In P3 the erosions
reached the papillae of the lamina propria.
In MIB1 immunostained sections, Ki 67 positivity was
expressed by granular nuclear staining and often most
prominent nucleolar positivity. In the squamous epithelium
of normal PO the scattered labelled nuclei were mostly con-
fined to the basal cell layer adjacent to basal membrane
67 positive nuclei were present in the stratum basale and
extended up to four or five cell layers (Fig 2a, 2b).
Lymphoid follicles in the lamina propria showed strong pro-
liferative activity in the germinal centres whilst scattered

FIG 1: MIB1 immunostaining in normal squamous epithelium of porcine pars oesophagea (a) x67; the scattered labelled nuclei were mostly confined to the
basal cell layer adjacent to basal membrane (b) x178.
192
b
FIG 2: MIB1 immunostaining in parakeratotic squamous epithelium of porcine pars oesophagea (a) x67; numerous MIB1 positive nuclei were present in the
stratum basale and extended up to 4 or 5 cell layers (b) x178
lymphoid cells in the epithelium and outside germinal cen-
tres were Ki 67 positive. In ulcerative lesions labelled prolif-
erating cells were immediately adjacent to migrating
epithelial cells at the edges of the ulcer (Fig 3), while the
granulation tissue in the lamina propria showed Ki 67 posi-
tivity in fibroblasts, endothelial and inflammatory cells.
AgNOR proteins appeared in light microscopy as black
dots, irregular in size and shape, scattered in the light
nuclear background (Fig 4a, 4b).
The mean values of the kinetic parameters, MIB1 index
and mean AgNOR area (MNORA) are summarised in Table 1.
Both the proliferative indices trended to increase progres-
sively from normal pars oesophagea (N) to P1, P2, P3
lesions to ulcerative (U) lesions.
Analysis of variance revealed a significant difference, for
both parameters used to assess proliferative activity between
normal PO (N) versus P1, P2, P3 lesions vs. ulcerative (U)
lesions (P < 0·05) (Figs 5, 6). No significant difference was
apparent, on the contrary, between P1 to P2 to P3 lesions
(P > 0·05). The apoptotic signal was localised in the nucleus
of epithelial cells and belonged, mostly, to the superficial
layers of the normal mucosa (i.e. stratum corneum) (Fig 7).
If apoptotic cells were identified, in some cases of parakera-
totic epithelium, they were located both in the superficial
R. Preziosi et al
and in the midzonal layers (i.e. the first viable layers
beneath the stratum corneum) (Fig 8).
Out of the 50 cases selected for the detection of apoptosis,
only 21 were positive to TUNEL staining (N = 7, P1 = 6,
P2 = 8). The negative staining of the 29 specimens dis-
carded could probably be related to the loss of the superfi-
cial layers during the steps of TUNEL assay.
By means of the TUNEL method, nuclei in apoptosis
appeared brown. Various morphological phases of apoptosis
were present: chromatin margination, that is the earliest
morphological appearance of the phenomenon, nuclear con-
densation of uniformly stained nuclei and the terminal stage
of apoptotic bodies. Among these phases, other nuclei did
not appear condensed but stained uniformly, representing an
earlier phase than chromatin margination and revealed
because of the amplification of DNA fragmentation signal
due to the method (Sarli et al 1998).
No significant difference in the apoptotic activity was
revealed with ANOVA comparing normal and mild (P1) or
heavy (P2) parakeratotic epithelia of PO (P = 0·71), while a
significant difference was present between N versus P1 or
P2 lesions, for both proliferative parameters, including the
cases selected for the apoptotic activity evaluation (P = 0·02
for MIB1 index; P = 0·03 for AgNOR).
 Cell turnover in porcine gastric ulcers 193

GT

FIG 3: MIB1 immunostaining in oesophagogastric ulcerative lesion; labelled proliferating cells are present both in the granulation tissue (GT) in the submucosa
(arrow) and in the epithelium (E) adjacent to the edges of the ulcer (arrow-head). x170.

a b

FIG 4: AgNOR proteins silver staining in normal (a) and parakeratotic squamous epithelium of porcine pars oesophagea (b) x266.
194 R. Preziosi et al

32 9

28 8
24
7
MIB1 index

AgNOR area
20
6
16
5
12
4
8
±Std. Dev.
±Std. Dev. ±Std. Err.
4 ±Std. Err. 3
N P1 P2 P3 U Mean
N P1 P2 P3 U Mean
FIG 6: Epithelial proliferative activity (mean values ± standard deviation ±
FIG 5: Epithelial proliferative activity (mean values ± standard deviation ±
standard error) expressed by AgNOR proteins quantification (µm2) in normal
standard error) expressed by MIB1 index (%) in normal pars oesophagea (N),
pars oesophagea (N), mild parakeratotic lesions (P1), heavy parakeratotic
mild parakeratotic lesions (P1), heavy parakeratotic lesions (P2), parakerato-
lesions (P2), parakeratosis + erosions (P3), ulcers (U) (Analysis of variance:
sis + erosions (P3), ulcers (U) – (Analysis of variance: P < 0·05).
P < 0·05).

SL

FIG 7: TUNEL staining of normal oesophagogastric epithelium; nuclei in apoptosis are evidenced by labelled chromatin margination in the most superficial
layers (SL) of epithelium. x672.

DISCUSSION AND CONCLUSIONS
Microscopical examination of the early stages of the
ulcerative process in porcine gastric PO confirmed O’Brien’s
(1992) findings of different epithelial changes including
parakeratosis, presence of ‘balloon’ cells in the mid-zonal
layers of the stratified squamous mucosa, increased depth of
the rete pegs in the lamina propria and basal zone thickness.
Moreover, we observed mild focal infiltrations of leuko-
cytes in the lamina propria and epithelium (microabscesses),
which may contribute to erosions and/or may represent a
secondary inflammatory reaction. A recent study by Roels et
al (1997) stressed the hyperproliferative nature of the early
parakeratotic lesions in the PO. These authors demonstrated
this by an immunohistochemical reactivity with the mono-
clonal antibodies LL020 and LHK6, which are specific for
K6 keratin, a prominent marker of epithelial hyperprolifera-
tion (Tyner and Fuchs 1986, Kopan and Fuchs 1989), which
was present in the suprabasal layers of the epithelium of the
parakeratotic PO but not in the normal epithelium. This sup-
ports the use of K6 immunohistochemistry as a useful
marker to study the early pathogenesis of gastric ulcers in
pigs.
In our study, attempting to outline better the features of
this proliferative behaviour, we employed two different
methods to evaluate cell proliferative activity: MIB1
 Cell turnover in porcine gastric ulcers 195

sl

ml

FIG 8: TUNEL staining of parakeratotic oesophagogastric epithelium; different phases of apoptosis (chromatin margination, nuclear condensation, uniformly
stained nuclei and apoptotic bodies) are evident in the midzonal (ml) and superficial layers (sl) of the mucosa. x435.

immunohistochemistry that allowed us to assess the pool of
proliferating basal cells or growth fraction (number of
cycling cells/number of total cells of the tissue) (Woosley
1991) and AgNOR proteins quantification that measures cell
duplication rate (Trerè et al 1989). Both parameters demon-
strated an increased proliferative activity of the epithelial
germinative compartment in the parakeratotic lesions in
respect to normal PO and analysis of these data supported the
hyperproliferative nature of these lesions observed by Roels
et al (1997).
Until recently, proliferation was thought to be the primary
point of control in the regulation of normal tissue kinetic
homeostasis and, as such, has been the major focus in both
understanding the aetiology of different diseases and devel-
oping therapeutic strategies. However, in recent years,
several reports investigated the role of programmed cell
death (apoptosis) in the turnover of continually renewing
tissues (especially epithelia like those of the gastrointestinal
tract and the skin) mainly in those diseases where the patho-
genetic importance of the balance between cell proliferation
and apoptosis was demonstrated (Haake and Polakowska
1993, Leoncini et al 1993, Jones et al 1997, Wrone-Smith et
al 1997).
Our data suggested that the apoptotic pattern in the nor-
mal and parakeratotic squamous epithelium of porcine pars
gastrooesophagea is quite similar to that of the skin, where
keratinocytes in the superficial layers of the epithelium
undergo apoptosis and thereby regulate or balance the pro-
liferation in the basal cell layer (Wrone-Smith et al 1997).
Our results show that apoptotic cells are also present in the
midzonal layers of the PO stratified squamous mucosa where
early morphological changes consisting of cellular balloon-
ing degeneration (hydropic degenerative change affecting
stratified squamous epithelia) are often reported both in
macroscopically normal and affected stomachs (Embaye et
al 1990). It is quite difficult, in this case, to explain the
simultaneous evidence of a physiological event like apopto-
sis and a pathological process such as ballooning degenera-
tion; it is only possible to hypothesise that in these cells,
affected by degenerative changes but still viable, a program
for fragmentation of DNA has been initiated and progresses
even if ballooning degeneration could again an advantage
over the activated mechanisms of programmed cell death,
transforming a controlled action into a pathological process
often leading to cell swelling and necrosis (Argenzio and
Eisemann 1996).
196 R. Preziosi et al
Regarding the lack of difference in apoptotic activity
between normal and affected squamous epithelium of PO, it
can be considered that, together with the higher proliferative
activity in parakeratotic stomachs compared to normal ones,
this is an important factor in determining the increased
epithelial thickness in these lesions. In fact, apoptosis,
together with cell proliferation, has an important role in
shaping and maintaining tissue size. Therefore, an imbal-
ance between cell proliferation and programmed cell death,
as demonstrated in our study, is characterised by a higher
pool of proliferative basal cells not balanced by an equal cell
elimination rate, leading to an increase of cells with an
altered differentiation pattern (parakeratosis) in the superfi-
cial layers of the epithelial tissue.
The pattern of cell proliferation in gross ulcerative lesions
had quite similar features to those normally found in wound
skin healing and repair (Schaffer and Nanney 1996). Both
lack of MIB1 positivity in the migratory cell population at
the ulcer edge and high growth fraction of the immediately
adjacent basal epithelium are consistent with the reepithe-
lialisation of the tissue, a complex process that involves the
detachment, reattachment, proliferation and differentiation
of epithelial cells. Moreover, proliferating fibroblasts,
endothelial cells and local macrophages are basilar compo-
nents of an immature granulation tissue (Schaffer and
Nanney 1996).
AKNOWLEDGEMENTS
The authors are grateful to John Mucera for his assistance
in English translation.
REFERENCES
ARGENZIO, R.A. & EISEMANN, J. (1996) Mechanisms of acid injury in porcine
gastrooesophageal mucosa. American Journal of Veterinary Research 57,
564–573.
ASDRUBALI, G. (1966) Osservazioni sulla incidenza delle ulcere gastriche nel
suino. Archivio Veterinario Italiano 17, 455–463.
EMBAYE, H., THOMLINSON, J.R. & LAWRENCE, T.L.J. (1990) Histopathology of
oesophagogastric lesions in pigs. Journal of Comparative Pathology 103, 253–264.
GERDES, J., BECKER, M.H.G., KEY, G. & CATTORETTI, G. (1992)
Immunohistological detection of tumour growth fraction (Ki67 antigen) in forma-
lin-fixed and routinely processed tissues. Journal of Pathology 168, 85–87.
HAAKE, R.A. & POLAKOWSKA, R.R. (1993) Cell death by apoptosis in epidermal
biology. Journal of Investigative Dermatology 101, 107–112.
JONES, L.N., SHANNON, P.T., CUTZ, E., YEGER, H. & SHERMAN, P.M. (1997)
Increase in proliferation and apoptosis of gastric epithelial cells early in the natural
history of helicobacter pylori infection. American Journal of Pathology 151,
1695–1703.
KOPAN, R. & FUCHS, S.E. (1989) The use of retinoic acid to probe the relation
between hyperproliferation-associated keratins and cell proliferation in normal
and malignant epithelial cells. Journal of Cell Biology 109, 295–307.
KOWALCZYK, T. (1969) Etiologic factors of gastric ulcers in swine. American
Journal of Veterinary Research 30, 393–400.
LEONCINI, L., DEL VECCHIO, M.T., MEGHA, T., BARBINI, P., GALIENI, P.,
PILERI, S., SABATTINI, E., GHERLINZONI, F., TOSI, P., KRAFT, R. & COT-
TIER H. (1993) Correlations between apoptotic and proliferative indices in
malignant non-Hodgkin’s lymphomas. American Journal of Pathology 142,
755–763.
MACARI, I., BRIUZOIU, M., BUNEA, M. & SEICIU, L. (1976) Aspecte morfopato-
logice ale ulcerului gastroesofagiau la porc. Lucrari Stiintifice I.A.M.B., Series C
18Ð19, 77–82.
MUGGENBURG, B.A., MCNUTT, S.H. & KOWALCZYK, T. (1964) Pathology of
gastric ulcers in swine. American Journal of Veterinary Research 25, 1354–1365.
O’BRIEN, J.J. (1992) Gastric ulcers. In: Diseases of Swine. 7th edition. A.D. Leman
et al, Eds. Wolfe Publishing Ltd, London, 680–691.
ÖFNER, D., BAUKFALVI, A., RIENEMANN, K., BOCKER, W. & SCHMID, K.W.
(1994) Standardized AgNOR stain method for formalin-fixed and paraffin-
embedded material. Pathologie 15, 226–230.
ROELS, S., DUCATELLE, R. & BROEKAERT D. (1997) Keratin pattern in hyper-
keratotic and ulcerated gastric pars oesophagea in pigs. Research in Veterinary
Science 62, 165–169.
SARLI, G., DELLA SALDA, L., ZACCARO, L., BENDINELLI, M, PIEDIMONTE,
G. & MARCATO P.S. (1998) Apoptotic fraction in lymphoid tissue of FIV-
infected SPF cats. Veterinary Immunology and Immunopathology 64, 33–44.
SCHAFFER, J.C. & NANNEY, L.B. (1996) Cell biology of wound healing. In:
International Review of Cytology, 169, 151–181.
THIRY, M. (1992) Highly sensitive immunodetection of DNA on sections with exoge-
nous terminal deoxynucleotidyltransferase and non isotopic nucleotide analogs.
Journal of Histochemistry and Cytochemistry 40, 411–419.
TRERÈ D., PESSION, A. & DERENZINI, M. (1989) The silver-stained proteins of
interphasic nucleolar organizer regions as a parameter of cell duplication rate.
Experimental Cell Research 184, 131–137.
TYNER, A.L. & FUCHS, S.E. (1986) Evidence for post-transcriptional regulation of
the keratins expressed during hyperproliferation and malignant transformation in
human epidermis. Journal of Cell Biology 103, 1945–1955.
WOOSLEY, J.T. (1991) Measuring cell proliferation. Archives of Pathology and
Laboratory Medicine 115, 555–557.
WRONE-SMITH, T., MITRA, R.S., THOMPSON, C.B., JASTY, R., CASTLE, V.P.
& NICKOLOFF B.J. (1997) Keratinocytes derived from psoriatic plaques are
resistant to apoptosis compared with normal skin. American Journal of Pathology
151, 1321–1329.
Accepted November 20, 1999