Int. J. Exp. Path.

(2005), 86, 19–24

ORIGINAL ARTICLE

Apoptosis in chronic viral hepatitis parallels histological activity:

An immunohistochemical investigation using anti-activated
caspase-3 and M30 Cytodeath antibody

Jo L. McPartland*, Muna Ali Guzail†, Charles H. Kendall* and James Howard Pringle†
*
Department of Histopathology, Sandringham Building, Leicester Royal Infirmary, Leicester, UK, and †Department of Pathology,
University of Leicester, Robert Kilpatrick Clinical Sciences Building, Leicester Royal Infirmary, Leicester, UK

I N T E R N AT I O N A L
JOUR NAL OF
E X P E R I M E N TA L
PAT H O L O G Y
Received for publication:
16 June 2004
Accepted for publication:
14 October 2004
Correspondence:
Charles H. Kendall
Department of Histopathology
Sandringham Building
Leicester Royal Infirmary
Leicester LE1 5WW, UK
Tel.: +44 116 2586582
Fax: +44 116 2586585
E-mail: charles.kendall@uhl-tr.nhs.uk
Summary
Apoptosis is implicated as a major pathogenic mechanism in chronic hepatitis B and C.
Previous studies of the relationship between apoptotic rates and histological necroinflamma-
tory activity have produced conflicting results. Hepatocyte apoptosis was assessed in liver
tissue from 32 cases of chronic viral hepatitis, seven cases of hepatocellular carcinoma (HCC)
and six cases of steatohepatitis as non-viral disease controls and eight cases of control liver.
Apoptotic rates were measured using H&E morphological assessment and immunohisto-
chemical staining with antibodies to activated caspase-3 and M30. Histological necroinflam-
matory activity of viral hepatitis cases was scored using the Knodell scoring system, and the
cases were divided according to their score into group 1 (mean 2.43 – 0.48) and group 2 (mean
7.80 – 0.49). Apoptotic indices were significantly higher in group 2 than group 1 using H&E
(11.53 – 2.70 vs. 0 – 0, P 5 0.015) and activated caspase-3 (22.01 – 5.27 vs. 1.79 – 1.79,
P 5 0.03) methods but were not significantly higher with M30 (3.80 – 1.74 vs. 0 – 0,
P 5 0.207). Apoptotic scores using an antibody to activated caspase-3 are significantly higher
in cases of chronic viral hepatitis with greater histological necroinflammatory scores, support-
ing a central role for apoptosis in disease pathogenesis. This method offers an alternative to
routine histological assessment for measuring disease activity.
Keywords
apoptosis, caspase-3, hepatitis B, hepatitis C, Knodell score, M30

The hepatitis viruses B and C are important causes of morbid-
ity and mortality and can lead to chronic viral hepatitis, cir-
rhosis and hepatocellular carcinoma (HCC). The exact
mechanisms of hepatocyte damage remain to be elucidated,
but both immune-mediated reactions and direct cytopathic
effects are likely to be involved. Much evidence suggests that
apoptosis plays a major role in the pathogenesis of chronic
viral hepatitis. In both hepatitis B and C, cytotoxic T lympho-
cytes are involved in the immune clearance of virally infected
hepatocytes (Chisari 1997). The Fas/Fas ligand system plays a
major role; Fas ligand expressed on cytotoxic T lymphocytes
binds to Fas antigen expressed on hepatocytes, inducing apop-
tosis (Galle et al. 1995; Hayashi & Mita 1999).
Apoptosis is a genetically programmed form of cell death
that plays a major role in development and tissue homeostasis
in addition to pathological processes (Wyllie et al. 1980).
Most of the morphological changes of nuclear and cytoplas-
mic condensation, membrane blebbing and cell shrinkage

 2005 Blackwell Publishing Ltd 19

20 J. L. McPartland et al.
observed in apoptotic cells (Kerr et al. 1972) are caused by
caspases, a group of evolutionarily conserved cysteine pro-
teases that all cleave substrates after aspartic acid residues
(Cohen 1997). At least 14 mammalian caspases have been
identified (Creagh & Martin 2001). The caspases are secreted
as inactive zymogens and are activated in sequence, some such
as caspase-8 and -9 being initiator caspases which trigger acti-
vation of downstream effector caspases including caspase-3, -6
or -7. In vitro studies have elucidated two main apoptotic path-
ways, both which converge at the level of caspase-3 activation,
triggering a cascade of enzymatic events that culminate in cell
death (Hengartner 2000). Caspase-3 activation is required to
produce apoptotic chromatin condensation and DNA fragment-
ation; these features are absent in apoptotic cells of caspase-3-
defective mice and MCF-7 breast carcinoma cells in which the
caspase-3 gene is functionally deleted (Jänicke et al. 1998; Woo
et al. 1998). Its importance in liver-cell apoptosis was con-
firmed by studies in caspase-3 knockout mice which show
resistance to Fas-mediated liver damage (Woo et al. 1999). As
well as caspase-3 itself, its substrates such as PARP, poly (ADP-
ribose) polymerase (Stroh & Schulze-Osthoff 1998) and M30
can also be used as markers of apoptosis. M30 recognizes a neo-
epitope of cytokeratin 18 that becomes available after cleavage
by caspase-3, before nick-end labelling identifies the cell as
apoptotic (Leers et al. 1999).
Previous studies of apoptosis in chronic viral hepatitis have
used a variety of different methods, including using antibodies
to activated caspase-3 and -7 and PARP (Bantel et al. 2001), the
TUNEL assay (Rodrigues et al. 2000; Papakyriakou et al. 2002)
and Fas expression as a surrogate marker of apoptosis
(Hiramatsu et al. 1994; Mochizuki et al. 1996). While most of
these studies show higher apoptotic rates or Fas expression in cases
of chronic viral hepatitis with more severe histological necroin-
flammatory activity (Hiramatsu et al. 1994; Mochizuki et al.
1996; Bantel et al. 2001; Papakyriakou et al. 2002), one study
found the opposite in cases of hepatitis C (Rodrigues et al. 2000).
The use of different methods of scoring apoptosis and classi-
fying histological activity makes comparison between these stu-
dies difficult. Also, none of these studies compared their
methods of measuring apoptosis with counting of morphologi-
cally apoptotic cells, which must remain the gold standard while
assessing relatively novel antibody techniques (Galle 1997).
The aims of this study are to resolve these issues by quanti-
fying apoptosis in chronic viral hepatitis by three different
methods and scoring histological necroinflammatory activity
using the internationally recognized Knodell scoring system
(Knodell et al. 1981). We examined 32 cases of chronic viral
hepatitis where patients were either hepatitis C or B positive, or
both. In addition to control samples of normal liver, we also
examined cases of steatohepatitis and HCC as non-viral disease
 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 19–24
controls. This gave a larger pool of cases for comparison of
different methods of apoptosis quantification. Apoptotic rates
were assessed by using H&E morphology and immunohistochem-
istry for activated caspase-3 and the monoclonal antibody M30.
Materials and methods
Case material
This is a retrospective study using archival formalin-fixed and
paraffin-embedded tissue. Liver biopsies and resections were
retrieved from the archive and anonymized according to local
Ethical Committee guidelines. There were 32 cases of chronic
viral hepatitis, including 26 from patients with hepatitis C
virus infection, four from patients with hepatitis B virus infec-
tion and two from patients with both hepatitis B and C virus
infection. Seven cases of HCC and six of steatohepatitis were
used as non-viral disease controls. In addition, blocks of back-
ground normal liver from eight liver resections for metastatic
adenocarcinoma were selected as control material.
Immunohistochemical procedures
Formalin-fixed, paraffin-embedded sections were cut to 4 mm
thickness, dewaxed in xylene and rehydrated through graded
alcohol to distilled water. The sections were subjected to
microwave antigen retrieval for 14 min in 10 mM citrate buf-
fer (pH 6.0). The indirect alkaline phosphatase method was
used for activated caspase-3 detection. The primary antibody
(Affinity-purified Rabbit Anti-human/mouse Caspase-3 Active,
R&D systems, Minneapolis, MN, USA) was applied at a dilu-
tion of 1 : 1000 after normal goat serum, incubated overnight at
4  C, then treated with goat anti-mouse/rabbit alkaline phos-
phatase conjugate (N series ready to use, Dako Ltd, Ely,
Cambridgeshire, UK) for 15 min. The alkaline phosphatase
anti-alkaline phosphatase (APAAP) method was used for M30
detection. The primary antibody (M30 Cytodeath mouse
monoclonal antibody, Roche, Basel, Switzerland) was applied
at a dilution of 1 : 50 after normal rabbit serum, incubated
overnight at 4  C, then treated with rabbit anti-mouse immuno-
globulin (Dako P314) at a dilution of 1 : 50 and then APAAP at
a dilution of 1 : 100 (Dako D0651). The slides were immersed
in naphthol phosphate/fast red substrate for 35 min to demon-
strate alkaline phosphatase activity. A negative control reaction
with no primary antibody was carried out for every case.
Quantification of apoptosis
Sections were examined by light microscopy using a high-
power objective, and apoptotic cells in a parenchymal location
 Apoptosis in chronic viral hepatitis 21

were counted. On H&E-stained sections, apoptotic cells were Table 1 Apoptotic indices (mean – SEM) for all cases and subsets
identified by their characteristic features of nuclear and cyto- of conditions
plasmic condensation. For sections stained with antibodies to
H&E Activated
activated caspase-3 and M30, whole-cell cytoplasmic staining
apoptotic caspase-3 M30
was counted; any weak background granular staining was index apoptotic index apoptotic index
ignored. For core biopsies, all of the tissue was examined,
and the number of high-power fields (field diameter All cases (n 5 53) 9.33 – 1.89 31.87 – 9.74 13.4 – 8.33
0.55 mm) of hepatic parenchyma on each slide was counted. Control (n 5 8) 2.5 – 0.95 0.63 – 0.63 0–0
For large blocks from liver resections, 20 high-power fields Hepatitis B or 9.01 – 2.27 17.58 – 4.38 2.97 – 1.38
C (n 5 32)
were selected randomly and the same area of the block was
Hepatocellular 23.16 – 8.05 158.2 – 51.5 87.9 – 58.7
examined for H&E, activated caspase-3 and M30 sections.
carcinoma (n 5 7)
For each case, an apoptotic index was calculated as the num- Steatohepatitis (n 5 6) 3.99 – 1.42 2.4 – 1.12 0–0
ber of apoptotic cells · 100/number of high-power fields.

Apoptotic cells were distributed in both periportal and

Grading of histological activity
For the cases of chronic viral hepatitis, an experienced liver
histopathologist (Charles H. Kendall) scored the grade of histo-
logical activity according to the Knodell system, incorporating
scores for periportal inflammation and bridging necrosis (scored
from 0 to 10), intralobular degeneration and focal necrosis
(scored from 0 to 4) and portal inflammation (scored from 0
to 4) (Knodell et al. 1981). This was performed independently of
apoptotic scoring. Fibrosis scoring was not included.
Statistical analysis
Data are expressed as mean – SEM. Spearman’s rank correla-
tions and Mann–Whitney U-tests were used to compare sets of
data. All statistical analyses were performed using MINITAB
version 13 or SPSS version 11.01 software.
Results
Taking all cases together, the apoptotic indices were highest
lobular locations. The activated caspase-3 and M30 stains
commonly highlighted cells that were not overtly apoptotic
morphologically. This was especially the case with large HCC
cells, a possible explanation for the large difference between the
H&E apoptotic index and the activated caspase-3 and M30
apoptotic indices for these cases (Figure 3). The use of these
antibodies therefore enhances detection of apoptotic cells com-
pared with the use of H&E morphology alone, marking cells
before definitive morphological features develop.
None of the cases of steatohepatitis showed positive staining
with M30, although apoptotic cells were identified on H&E
slides and with activated caspase-3. Figure 4 illustrates control
tissue showing no apoptosis.
Histological activity of chronic viral hepatitis cases
The 32 cases were placed into two groups according to their
Knodell necroinflammatory activity score, the sum of scores for
220

using activated caspase-3 (31.87 – 9.74), followed by M30 200 H&E apoptotic index
CP3 apoptotic index
(13.4 – 8.33), with the H&E apoptotic index being lowest 180 M30 apoptotic index

(9.33 – 1.89). However, the M30 apoptotic index was the 160
lowest of the three in all individual groups of conditions
Apoptotic index

140
except HCCs (Table 1) (Figure 1). 120
Cases of HCC showed the highest apoptotic indices; those of 100
chronic viral hepatitis and steatohepatitis were intermediate, 80
and very low apoptotic indices were observed in the control 60
tissue from liver resections. Although of different magnitudes, 40
the three different apoptotic indices of all cases were signifi- 20
cantly correlated with each other (H&E index vs. activated 0
caspase-3 index r 5 0.481, P < 0.001; H&E index vs. M30 All cases Control Hepatitis C/B HCC Steatohepatitis

index r 5 0.365, P 5 0.007; activated caspase-3 index vs.

Figure 1 H&E, activated caspase-3 (CP3) and M30 apoptotic
M30 r 5 0.553, P < 0.001). Figure 2 illustrates apoptotic cells indices (mean – SEM) displayed for all cases and individual groups
with H&E, activated caspase-3 and M30 staining. of conditions.

 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 19–24
22 J. L. McPartland et al.

Figure 2 (a) An apoptotic cell in a case of steatohepatitis. Note

chromatin condensation and cell shrinkage (H&E, ·400); whole-cell
cytoplasmic staining with antibodies to activated caspase-3 in a case
of hepatitis B and C (b) and M30 in a case of hepatitis C (c) (·400).
Figure 4 Control liver showing no apoptosis. (a) H&E,
(b) activated caspase-3 and (c) M30 (·400).
categories (1) periportal inflammation and necrosis, (2) lobular
inflammation and (3) portal inflammation. Seven cases were in
group 1, activity score 0–4 (mean 2.43 – 0.48) and 25 cases
were in group 2 with activity scores of 5 and above, ranging
from 5 to 14 (mean 7.80 – 0.49). An activity score cut-off of 4
excludes cases with bridging necrosis from group 1.
All three apoptotic indices were higher in group 2 than in group
1, with no apoptotic cells identified with H&E or M30 staining in
any of the cases in group 1 (Table 2) (Figure 5). The H&E and
activated caspase-3 apoptotic indices of the two groups were
significantly different, P 5 0.015 and P 5 0.030, respectively

Table 2 Apoptotic indices (mean – SEM) for cases of chronic viral

hepatitis divided into two groups based on histological necroin-
flammatory activity

H&E Activated M30

apoptotic caspase-3 apoptotic
index index index

Figure 3 (a) Cells positively staining with antibodies to activated Knodell 0–0 1.79 – 1.79 0–0
caspase-3 in a case of hepatocellular carcinoma. Note that cell activity
labelled with closed arrow shows nuclear pyknosis, but cells grade 0–4 (n 5 7)
labelled with open arrows show nuclear features not sufficiently Knodell 11.53 – 2.70 22.01 – 5.27 3.80 – 1.74
different from surrounding tumour cells to allow identification as activity
apoptotic by morphology alone (·400). (b) M30-positive cells grade 5–14 (n 5 25)
in the same case show similar features (·400).

 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 19–24

30
Knodell activity grade 0–4
Knodell activity grade 5–14
25
P = 0.030
20
Apoptotic index
P = 0.015
15
10
5
0 et al. (2001) found no correlation overall between fibrosis
H&E apoptotic index CP3 apoptotic index
Figure 5 Apoptotic indices (mean – SEM) for chronic viral
hepatitis cases divided into two groups according to Knodell
activity grade. Note that H&E and M30 apoptotic indices are
zero for group 1. The difference between the two groups is
significant for the H&E (P 5 0.015) and activated caspase-3
(P 5 0.030) indices but not significant for the M30 index
(P 5 0.207) (Mann–Whitney U-test).
(Mann–Whitney U-test). The difference between the groups was
not significant for the M30 apoptotic index (P 5 0.207), as many
cases in group 2 had much lower apoptotic indices with M30 than
with H&E or activated caspase-3, as was seen generally with
chronic viral hepatitis, steatohepatitis and control cases.
Discussion
Two important conclusions can be drawn from this study.
Firstly, we have shown that the use of antibodies to activated
caspase-3 and M30 enables the identification of apoptotic hepa-
tocytes more frequently than by the use of H&E morphological
features alone. Activated caspase-3 identified more cells than
M30 in all of the different disease groups tested in this study.
This may reflect the fact that activated caspase-3 identifies an
earlier stage of apoptosis, as caspase-3 must be activated to cleave
cytokeratin 18 and reveal the M30 epitope (Leers et al. 1999).
The other major finding is that, in chronic viral hepatitis due
to hepatitis C and B, apoptotic rates are higher in cases show-
ing higher necroinflammatory scores. This is in accord with
the findings of a study using activated caspase-3 and -7 and
PARP in cases of chronic hepatitis C (Bantel et al. 2001). The
differing results found between studies using the TUNEL assay
(Rodrigues et al. 2000; Papakyriakou et al. 2002) may be due
to technical artefacts (Labat-Moleur et al. 1998) and the fact
that this assay is not specific for apoptotic cells (Grasl-Kraupp
et al. 1995). Papakyriakou et al. 2002 found increased apop-
totic rates in cases of severe hepatitis B and C compared with
mild cases, concordant with our findings. However, Rodrigues
 2005 Blackwell Publishing Ltd, International Journal of Experimental Pathology, 86, 19–24
Apoptosis in chronic viral hepatitis 23
et al. (2000) found higher apoptotic rates in cases of hepatitis
C with a total Knodell histological activity index of 5 and
below compared with those above 5. Their study included
the fibrosis score, which could explain this discrepancy.
Expression of bcl-2, an antiapoptotic protein, has been
shown to be low in liver biopsies with hepatitis C infection
but high in cirrhotic livers (Frommel et al. 1999). If apoptosis
is suppressed by bcl-2 as fibrosis progresses towards cirrhosis,
P = 0.207 then cases with a higher total Knodell histological activity
index incorporating significant fibrosis scores might be
expected to show lower apoptotic rates. However, Bantel
M30 apoptotic index
and caspase-3 and -7 expression.
Our findings in non-viral disease controls have suggested
interesting avenues of further research. We found high rates of
apoptosis in HCC compared with chronic viral hepatitis, steato-
hepatitis and control liver tissue. An increased frequency of
apoptosis has been reported in preneoplastic nodules and
HCCs in rat hepatocarcinogenesis accompanied by very high
replicative rates (Columbano et al. 1984; Bursch et al. 1994). A
recent study of caspase-3 expression in HCCs found overexpres-
sion of caspase-3 in hepatoma cell lines and a subset of human
HCCs by Western blot and immunohistochemistry (Persad et al.
2004). Although the antibody used in this study recognised the
activated form of caspase-3, surprisingly, immunoblot for an
active subunit of caspase-3 revealed no caspase activation. This
is in contrast to our finding of a high rate of caspase-3 activation
in all of our HCC cases. Future work in this area is required to
resolve these differences.
We also found higher rates of apoptosis in cases of steatohe-
patitis compared with control liver tissue. The aetiology of these
cases is not known to us, but alcohol is a major cause of steato-
hepatitis (Burt et al. 1998); hepatocyte apoptosis has been shown
to be significantly increased in cases of alcoholic hepatitis using
the TUNEL assay and antibodies to activated caspase-3 (Natori
et al. 2001). A larger study of steatohepatitis cases including
correlation with the underlying aetiology may further elucidate
the mechanism of apoptosis in this group.
We have demonstrated that, in chronic viral hepatitis, apop-
totic rates scored using a commercial antibody to activated
caspase-3 are significantly higher with greater histological
necroinflammatory scores. In the United Kingdom’s National
Health Service, interferon-a and ribavirin treatment for
chronic hepatitis C is only recommended for patients who
show significant histological fibrosis or necroinflammation
(National Institute for Clinical Excellence 2000). Interobser-
ver variation in Knodell scoring has been shown to be only fair
to moderate (Grønbæk et al. 2002); immunohistochemistry
for activated caspase-3 could therefore provide a more
reproducible method of measuring disease activity, making
24 J. L. McPartland et al.
such treatment decisions more robust and the monitoring of
treatment effects more accurate.
Acknowledgements
We thank Angela Gillies and Linda Potter for technical
assistance and Dr Angus McGregor for helpful comments on
the manuscript.
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