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Battle
of the New, Non-invasive Measures of Fibrosis: FibroScan versus FibroTest
By Ronald Baker, PhD
Liver fibrosis
is the principal feature of the injury caused by chronic liver disease and
determines the major clinical events that lead to
liver-related deaths. For this reason alone, an accurate
assessment of fibrosis is vital to the management of patients with liver
disease.
Measuring the extent of liver disease is also
a significant factor when considering whether to use
treatment,
to assess the response to therapy and to make other important decisions
related to progression of fibrosis, such as screening for
hepatocellular carcinoma and
varices.
For 60 years,
liver biopsy has been regarded as the
gold standard diagnostic
for assessing the
progression of fibrosis in chronic hepatitis C patients.
However, despite its longstanding utility, liver biopsy has some
significantly negative features. First, patients often resist undergoing
liver biopsy due to the discomfort resulting from its invasiveness. There is
also some risk to the patient of experiencing an adverse event from liver
biopsy. In addition to these negative features, there is a sampling error of
at least 24% due to inadequate liver specimen length or fragmentation.
Finally, there are inconsistencies in the interpretation of liver biopsy
results because of errors on the part of one or more observers of the
specimens.
As a result of these drawbacks to liver
biopsy, interest continues to grow in new, non-invasive methods of assessing
fibrosis, including biochemical markers, biomarkers, and new imaging
techniques.
FibroScan and FibroTest
One such advance in the field is
FibroScan, a type of ultrasound machine that uses
transient elastography to measure liver stiffness. The device reports a
value that is measured in kilopascals (kPa). This value can be extrapolated
to a fibrosis score.
FibroTest (aka
FibroTest-ActiTest)
is another non-invasive diagnostic for
assessing fibrosis. Available through BioPredictive (www.biopredictive.com),
FibroTest uses an algorithm to combine the results of serum tests of beta
2-macroglobulin, haptoglobulin, apolipoprotien A1, total bilirubin, gamma
glutamyltranspeptidase (GGT), and
alanine
aminotransferase (ALT) to assess the level of fibrosis and
necroinflammatory activity.
Comparison of FibroScan and FibroTest to
Detect Fibrosis Progression among HCV Carriers with
Normal Aminotransferases
An article published in the October 2005 issue
of Hepatology by Colletta et al compared the value of FibroScan and
FibroTest in HCV carriers with normal ALT levels [1]. The study
evaluated 40 untreated HCV RNA positive subjects who had two liver biopsies,
with a median interval of 78.5 months, during which ALT levels never
exceeded 1.2 times the upper normal limit.
The study authors
concluded that FibroScan yielded results that showed perfect
agreement between FibroScan and liver biopsy [emphasis
added—Ed]. In addition, the study concludes that the diagnostic
accuracy of FibroScan was 100%. Further, the authors write, “FibroScan is
superior to the FibroTest in the noninvasive identification of fibrosis, for
which excess
alcohol consumption in the past and high viral load represent
risk factors” [2].
The glowing review by Colletta et al of the
diagnostic superiority of FibroScan compared to FibroTest has now been
strongly challenged by other experts in the field, specifically L Castera et
al and T Poynard et al. Their contrasting opinions appear in the
“Correspondence” section of the February 2006 issue of Hepatology
[3,4]. A reply by Colletta et al to Castera et al and to Poynard et al
also appears in the February 2006 issue of Hepatology. The major
arguments of each of the three teams of experts concerning the original
study by Colletta et al are summarized here.
FibroScan and FibroTest to
Assess Liver Fibrosis in HCV with
Normal Aminotransferases (L Castera and
others)
Although Castera et al agree with the opinion
of Colletta et al that these non invasive diagnostics could “someday become
an alternative” to liver biopsy in patients with
persistently normal ALT levels (PNAL), the authors state they
feel compelled to voice several methodological concerns about the study by
Colletta et al.
Castera et al raise three major issues about the study:
1. The stated capability of FibroScan to identify the entire spectrum of
fibrosis stage in individuals with PNAL contracts sharply with the results
noted in all other published studies. Due in part to the small number of
patients in their study, Colletta et al need to interpret their data more
cautiously concerning the “perfect agreement” between FibroScan and liver
biopsy, write Castera et al.
The performance of FibroScan in this study shows a surprising diagnostic
accuracy of 100%. A critical issue in assessing accuracy is the cutoff value
for identifying patients with significant fibrosis. Colletta et al give no
justification for the choice of their cutoff point of O.31. Using the cutoff
proposed by Castera et al, the results by Colletta et al most likely would
be quite different than what they report, with lower diagnostic accuracy:
sensitivity 100%, specificity 46%, diagnostic accuracy 46%, positive
predictive value 50%, negative predictive value 100%).
2. Second, Colletta
et al offer no information on the proportion of patients in whom liver
elasticity measurements could not be obtained.
3.
Third, Colletta et al should be
cautious in asserting that FibroScan has much better correlation with liver
biopsy than FibroTest. In their study, FibroTest yields a surprisingly high
false positive rate, which contrasts with the results of prior published
studies that show high specificity for FibroTest.
Castera et al propose avoiding the limitations
of both FibroScan and FibroTest by employing an algorithm that combines both
tests. When doing so, the authors found that in 97 of 100 consecutive HCV
patients with normal ALT levels, concordance between FibroScan and FibroTest
for significant fibrosis was 67%.
In conclusion, Castera et al write, “We
believe combining FibroScan and FibroTest as a first-line noninvasive
assessment of liver fibrosis might prove particularly useful in the setting
of HCV carriers with normal ALT levels and should be further evaluated.”
Service d'Hépato-Gastroenterologie,
Hôpital Haut Léveque, C.H.U.Bordeaux, Pessac, France
Diagnostic Value of
FibroTest with
Normal Serum Aminotransferases (T Poynard
et al)
Colletta et al have
concluded that FibroScan is superior to
FibroTest. Their study yielded “perfect” diagnostic measurements for
FibroScan, but for FibroTest (at the 0.31 cutoff) only 64% sensitivity and
31% specificity for the diagnosis of advanced fibrosis.
In the observations of Poynard et al, such
“poor” results have not been found for FibroTest, nor are they reflected in
the results of other published studies using the services of BioPredictive,
“the sole company allowed to market FibroTest.”
In a FibroTest analysis of 537 patients with
chronic hepatitis C (129 with normal ALT and 408 with elevated ALT), there
were no differences in area under the ROC curves between patients with
normal or elevated ALT. Furthermore, the AUROC was 0.76, higher than the 43%
accuracy observed by Colletta et al.
The Poynard group also analyzed prospectively
the specificity of FibroTest in 954 blood donors without liver biopsy, 917
with normal ALT. Excluding the 25 patients with high risk of false
positive/negative, 877 of the remaining 892 patients (98.2%) have normal
FibroTest and 15 (1.8%) had FibroTest between 0.31 and 0.48 and none above.
“These figures are very different than the 69% false positive of Colletta et
al,” according to Poynard at al.
Poynard et al propose four possible
explanations for these discrepancies:
1. The small number of patients in the study
by Colletta et al.
2.An error in the calculation of FibroTest if
the professional website (www.BioPredictive.com)
was not used.
3.
The non exclusion of patients with high risk profile of false
positive/negative. (These patients would have been identified by the
security algorithms on the BioPredictive website).
4.
Use of the FibroTest threshold at 0.31 for F2F3F4 detection instead
of 0.48 which is the recommended threshold.
Finally, in evaluating the concordance between
the two tests in 70 consecutive subjects with baseline normal ALT: 60
patients (44 HCV, 5 hepatitis B, 11 others), and 10 apparently healthy
volunteers, FibroScan was not applicable in 3 subjects (abdominal fat) and
FibroTest in 5 (high risk profile). In the remaining 62 subjects the
concordance was fair with 78% (48/62) of concordance for stage F2F3F4 when
using the 0.31 threshold, and even better (82% (51/62) at the recommended
threshold (0.48). These concordance rates were similar to the 77% observed
by Castera et al.
In conclusion, Poynard et al write, “We think
the comparisons between noninvasive markers should be performed according to
professional recommendations, respecting applicability reports to exclude
high risk of false negative or false positive results and in populations
with sufficient sample size.”
Service d Hepato-Gastroenterologie,
GH Pitie-Salpetriere, Pans,
France; BioPredictive,
Paris,
France
Colletta et al Reply to Poynard et al and
Castera at al
Colletta et al dismiss two of the explanations
for the discrepancies between their study and the study by Poynard et al by
stating that all FibroTest values were calculated using the BioPredictive
website. Further, the five serum markers used in this fibrosis index were
measured following the strict requirements specified by BioPredictive, say
Colletta et al.
Concerning the use of the 0.31 cutoff,
Colletta et al maintain that this is the most sensitive cutoff to exclude
significant fibrosis. Further, if the data are re-analyzed using the 0.48
cutoff, the diagnostic value of FibroTest in the studied patients would be:
sensitivity, 21%, specificity, 81%, positive predictive value, 38%, negative
predictive value, 66%.
While acknowledging that future, larger
studies might reach different conclusions than theirs, Colletta et al
emphasize that the selection of patients for these future studies will
require the use of very strict criteria, such as those used by them (no
value >1.2 times the upper normal limit in a minimum of 17 determinations,
along more than 5 years), and which Poynard et al did not follow. This
included the using patients with PNAL who have ALT within the normal limits
on the day a liver biopsy was performed.
Regarding the objections of Castera et al
related to the cutoff chosen for liver elasticity (8.7%kPa), using a cutoff
that reduces the overall value of a test is not justifiable, say Colletta et
al.
With regard to the second issue raised by
Castera et al., the proportion of patients in whom liver elasticity
measurements could not be obtained, Colletta et al reply, “Unfortunately,
Castera et al neither specify why their patients could not have liver
elasticity measured, nor define the HCV carriers with normal ALT they
studied in terms of length of follow-up and number of ALT determinations.”
Finally, the authors question the
appropriateness of Castera et al to equate the specificity of FibroTest in
blood donors without hepatitis C (and no liver biopsy) to that in HCV
carriers with normal ALT, “since these two populations should not be
considered equivalent.”
It would appear that FibroScan has certain
advantages over other diagnostic indices or predictive models based on
laboratory tests in that it is completely noninvasive, provides a more
direct measure of fibrosis, should not be affected by other disease states,
and should theoretically be applicable to all chronic liver diseases,
explain Ghany and Doo in an editorial that accompanies the study by Colletta
et al [7].
Conclusion
What will be the future of the liver biopsy
and the newer, non-invasive diagnostics such as FibroScan and FibroTest?
If as expected, new antivirals become
available for the treatment of chronic hepatitis C, there will be an
increasing need to assess fibrosis as a method of monitoring the effect of
these treatments. Liver biopsy no doubt will continue to be employed in the
diagnosis, grading and assessment of chronic liver disease. Yet despite a
continuing role for liver biopsy, non invasive methods likely will soon
become the diagnostic of choice for assessing liver fibrosis.
02/07/06
Sources
C Colletta C and others. Value of two
noninvasive methods to detect progression of fibrosis among HCV carriers
with normal aminotransferases. Hepatology 42(4): 838-845. October
2005.
L Castera, J Foucher,
J Bertet, P Couzigou, and V de Ledinghen.
FibroScan and
FibroTest to assess liver fibrosis in HCV with normal aminotransferases.
Hepatology 43(2): 373-374. February 2006.
T Poynard, M Munteanu, Y Ngo, M Torres, Y Benhamou, D Thabut, and V Ratziu.
Diagnostic
value of FibroTest with normal serum aminotransferases.
Hepatology 43(2): 374-375. February 2006.
References
1.
C Colletta
and others.
Value of two noninvasive methods to detect progression of fibrosis among HCV
carriers with normal aminotransferases. Hepatology 42(4): 838-845.
October 2005.
2.
Ibid.
3.
L Castera, J Foucher, J Bertet, P Couzigou, and V de Ledinghen.
FibroScan and FibroTest to assess liver fibrosis in HCV with normal
aminotransferases. Hepatology 43(2): 373-374. February 2006.
4.T Poynard, M Munteanu, Y Ngo, M Torres, Y
Benhamou, D Thabut, and V Ratziu. Diagnostic value of FibroTest with normal
serum aminotransferases. Hepatology 43(2): 374-375. February
2006.
5.C Colletta and
others. Reply to L Castera et al and T Poynard et al. Hepatology
43(2): 375-376. February 2006.
6.M G Ghany and
E Doo. Assessment of liver fibrosis:
Palpate, poke or pulse? (editorial). Hepatology 42(4): 759-761.
February 2006.
http://www.hivandhepatitis.com/hep_c/news/2006/020706_a.html
Losartan May Reduce
Liver Fibrosis in Hepatitis C Patients
By Matas A. Loewy BUENOS AIRES (Reuters Health) Feb
10 - Antihypertensive drug losartan may reduce liver fibrosis in patients
with chronic hepatitis C infection who do not respond to the standard
antiviral therapy, the results of a pilot study suggest.
Dr. Silvia Sookoian, and colleagues at the University of Buenos Aires
Medical Research Institute Alejandro Lanari, enrolled 14 hepatitis C
patients with biopsy-proven fibrosis (median age= 49. 6 years old) who did
not respond to interferon plus ribavirin therapy or who did not comply
with treatment. The subjects received 50 mg/day of losartan.
After 6 months, "a decrease in fibrosis stage was observed in 7/14
(treated) patients vs 1/9 control patients" (p< 0,04), authors reported in
the current issue of the World Journal of Gastroenterology.
The drug was well tolerated and only one treated patient had a single
episode of mild orthostatic hypotension.
"Our findings should be confirmed in a randomized, controlled trial
with a larger number of patients. But losartan appears to be a safe drug
and it also diminishes portal hypertension in a high proportion of
patients," Dr. Sookoian told Reuters Health.
Losartan and other drugs with antifibrotic activity might become a
feasible option for the 50% to 70% of patients who do not respond to the
current hepatitis C treatment.
Losartan, an angiotensin II type 1 receptor blocker, appears to prevent
liver fibrosis and portal hypertension by blocking circulating angiotensin
II-mediated activation of hepatic stellate cells.
"Much evidence suggests that hepatic stellate cells play important
roles in the pathogenesis of liver fibrosis, since they were shown to
undergo (an activation) during the (chronic) injury," researchers add.
World J Gastroenterol 2005;11:7560-7563.
http://www.medscape.com/viewarticle/523426
Liver Fibrosis in HIV+ Patients
With Hepatitis C Virus: Role of Persistently Normal Alanine
Aminotransferase Levels
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JAIDS Journal of Acquired Immune Deficiency Syndromes:
Volume 41(1) 1 January 2006 pp 63-67
"......The follow-up of our PNAL (persistently normal ALT) patients
showed a heterogeneous progression of HCV-related liver disease, ranging
from high stable clinical status for 5 years after liver biopsy to fast
progression and even lethal evolution during the same interval. In
conclusion, these findings highlights the threat hidden by persistently
normal ALT levels, which are too often considered a sign of
well-balanced liver status in HIV-positive subjects with chronic HCV
infection...."
Uberti-Foppa, Caterina MD; De Bona, Anna MD; Galli, Laura MSc; Sitia,
Giovanni MD; Gallotta, Giulia MD; Sagnelli, Caterina MD; Paties, Carlo
MD; Lazzarin, Adriano MD
From Clinic of Infectious Diseases, Vita-Salute University, San Raffaele
Scientific Institute, Milan, Italy.
Abstract
Background: Liver fibrosis requiring treatment in HIV/hepatitis C virus
(HCV)-coinfected patients with persistently normal alanine
aminotransferase (ALT) values (PNAL) is currently not well defined; in
this study clinical and histologic features of PNAL were compared with
those of subjects with elevated ALT (EAL).
Methods: A total of 326 liver biopsies of HIV/HCV-coinfected patients,
performed from 1997-2003, were retrospectively identified. Subjects with
at least 3 consecutive normal ALT determinations during a prebiopsy
follow-up of 12 months were grouped as PNAL (24 patients) and compared
with EAL subjects (302 patients). Liver biopsy was classified with the
modified Ishak score.
Results: Age, HCV viral load, and genotype, CD4 T-cell count, and
antiretroviral drugs did not show a statistical difference between the 2
groups. Statistical significance was found when comparing mean grading
(1.4 ± 1.8 vs. 7.2 ± 2.6, P < 0.0001) and staging (1.4 ± 1.79 vs. 2.5 ±
1.7, P < 0.0003) between PNAL and EAL subjects. The proportion of PNAL
patients fulfilling histologic criteria for anti-HCV treatment (25% with
stage 2-6) was also significantly different from EAL subjects (69%; P =
0.0001). At multivariate analysis, only age, CD4 count (>500 vs. ≦500
cells/mL), and patient's group (EAL vs. PNAL) were found to be
independently associated with a fibrosis score of ≥2.
Conclusion: Liver fibrosis requiring treatment was found in 25%
of HIV/HCV-coinfected subjects with PNAL values.
DISCUSSION
Published data on HIV/HCV-coinfected subjects pertain mostly to patients
with EAL values, and little is known about the subpopulation showing
PNAL values. In our retrospective study, 2 groups of HIV/HCV-coinfected
patients similar in terms of gender, age, and risk factors presenting
persistently normal or elevated values of ALT for 12 months before liver
biopsy were compared to assess how ALT values are related to severity of
histopathologic liver damage. The rationale was to select a population
that is presently considered not eligible for liver biopsy, as also
indirectly shown by the relatively small number of PNAL patients
referred by primary HIV care providers. A bias of the present study may
be the unreported true duration of HCV disease, which may have
influenced either the ALT values or histology.
In the absence of HIV and HCV seroconversion dates for each patient, we
decided to address this point analyzing the relationship between staging
and age in each group or staging and IVDU initiation date. The grouping
of the median age in the same 5-year range of time suggests a comparable
duration of HCV disease as also shown by the similar mean duration of
coinfection in the 2 subgroups of previous or active IVDU.
Analysis of liver biopsies showed that 70% of PNAL patients presented
some degree of liver fibrosis (1-6) and that 1 of 4 required adequate
treatment (2-6). Although overall histologic abnormalities found in
patients with PNAL levels were milder than those found in EAL patients,
incomplete and frank cirrhosis (staging ≥5) were found in PNAL subjects
(12.5%) as also described for HIV-seronegative HCV-infected patients.5,6
Our proportions of significant fibrosis and also of histologic cirrhosis
differ from those recently described by Fonquernie et al.4 These authors
did not observe cirrhosis in the coinfected PNAL subjects. This
discordance may be explained by the different definition used to select
the PNAL population in the French study compared with ours (3 years of
prebiopsy follow-up vs. our 1 year of follow-up). It is possible that
longer observation periods would allow a more strict selection of PNAL
patients. However, our findings clearly show the threat hidden by PNAL
in patients who have had 1 year of follow-up. Although mean CD4 T-cell
counts were similar in both groups of patients, this parameter was shown
to be inversely related to the patient's staging, as also observed in
other cohorts.8 Notably, older age and CD4+ T-cell count <500 cell/mL
were independently associated with a stage of liver fibrosis fulfilling
histologic criteria for anti-HCV treatment in patients with PNAL. These
parameters may help to select difficult-to-treat PNAL patients eligible
for liver biopsy. In HCV-monoinfected PNAL subjects, 18%-50% of patients
developed intermittent or persistent elevation of ALT levels during a
6-month to 7-years follow-up, a percentage comparable to that observed
in our study (37.5% during 5 years of follow-up).9,10 The follow-up of
our PNAL patients showed a heterogeneous progression of HCV-related
liver disease, ranging from high stable clinical status for 5 years
after liver biopsy to fast progression and even lethal evolution during
the same interval. In conclusion, these findings highlights the threat
hidden by persistently normal ALT levels, which are too often considered
a sign of well-balanced liver status in HIV-positive subjects with
chronic HCV infection.
RESULTS
Comparison of Demographic and Immunovirologic Status of HIV/HCV-Coinfected
Patients With Persistently Normal ALT and Elevated ALT
PNAL and EAL subjects, comprising 326 HIV/HCV-coinfected patients, did
not present statistically significant differences in term of gender,
age, and behavioral risk factors as summarized in Table 1. Median age
was in the majority of each group distribution in the same 5-year range
of time (EAL = 41 years, interquartile range 39-44; PNAL = 40 years,
interquartile range 39-42). The average interval since diagnosis of HIV
infection was >10 years in both groups; the duration from diagnosis of
HCV infection was shorter probably owing to a delay in managing HCV
infection. For previous or active IVDU, the estimated mean duration of
coinfection, using the date of IVDU initiation, was similar in the 2
groups (14 years). HCV genotype 3 was the most judgement genotype in the
overall sample (44%), followed by genotype 1 (40%), genotype 4 (13%),
and genotype 2 (3%). Genotype distribution analysis showed a higher
proportion, not statistically significant, of genotype 1 in PNAL (61%,
11 cases) compared with EAL (38%, 97 cases; P = 0.08), followed by
genotype 3 (22%, 4 cases vs. 46%, 116 cases) and genotype 4 (17%, 3
cases vs. 13%, 32 cases). No genotype 2 was found in the PNAL group vs.
3% of EAL (9 cases). HCV RNA levels in plasma did not significantly
differ in PNAL and EAL subjects; high-level HCV viremia (HCV RNA ≥2 X
106 copies/mL) generally associated with a reduced response to treatment
was also homogeneously distributed. The mean CD4+ T-cell count did not
differ significantly. Among patients receiving antiretroviral therapy,
46% were on protease inhibitor-based highly active antiretroviral
therapy (HAART) and 17% were receiving nonnucleoside reverse
transcriptase inhibitor (NNRTI)-based HAART. Three nucleoside reverse
transcriptase inhibitors (NRTIs) were administered in 10% of subjects
and 2 NRTIs in 27%. Statistical analysis did not show any difference in
the antiretroviral therapy distribution between PNAL and EAL patients
(data not shown).
Comparison of Liver Histologic Features in PNAL and EAL Patients:
Correlation With Immunovirologic Status and Antiretroviral Therapy
One third of all patients presented an absent or minimal liver fibrosis;
in PNAL and EAL subjects stage 0-1 was shown in 75% and 31% of cases,
respectively. Mild to moderate fibrosis was more frequent in PNAL with
respect to EAL patients, despite the presence of some cases of advanced
fibrosis in the first group (1 patient in stage 5 precirrhosis and 2
patients in stage 6 cirrhosis). Stage 0 was described in 31 subjects
(10%) (PNAL 7 patients, EAL 24); stage 1 in 81 subjects (25%) (PNAL 11
patients, EAL 70); stage 2 in 81 subjects (25%) (PNAL 2 patients, EAL
79); stage 3 in 53 subjects (16%) (PNAL 1 patient, EAL 52); stage 4 in
26 subjects (8%) (PNAL 0 patients; EAL 26); stage 5 in 21 subjects (6%)
(PNAL 1 patient, EAL 20); and stage 6 in 33 subjects (10%) (PNAL 2
patients, EAL 31). The necroinflammatory status was found to be minimal
or moderate (≦7) in more than half of patients. The analysis of CD4
count and liver fibrosis showed a significant inverse association (P =
0.0036) between immune status and staging: staging 0-1 was detected in
46 subjects (42%) and 63 (58%), respectively, for CD4 count <500 cells/mL
and ≥500 cells/mL, whereas higher stage ≥2-6 was evaluated in 127 (61%)
and 81 (39%) of the same CD4 categories with available CD4 count at the
time of biopsy. A multivariate analysis was done, aimed at evaluating
factors possibly related to liver fibrosis ≥2. Three variables were
found, independently associated with mild to severe liver fibrosis:
higher age (for 1-unit increment: odds ratio [OR] = 1.09, 95% CI: 1.03
to 1.18, P = 0.01); CD4+ T-cell count at the time of liver biopsy (<500
vs. ≥500: OR = 1.98, 95% CI: 1.07 to 3.7; P = 0.03); and patient's group
(EAL vs. PNAL: OR = 3.83, 95% CI: 1.23 to 14.28; P = 0.02). Neither the
HCV genotype (1 vs. other than 1), nor high HCV RNA (<2,000,000 vs.
≥2,000,000 copies/mL), log HIV RNA (for 1-unit increment), and
antiretroviral therapy (no vs. yes) were found to be associated to
staging score ≥2.
Clinical Follow-Up of PNAL Patients
Because HIV has a deleterious impact on chronic HCV infection and no
data are available on the course of liver disease in HIV/HCV-coinfected
patients with PNAL, we performed a post-biopsy clinical follow-up of 2-8
years (mean duration: 5.13 years ± 1.9 SD) as summarized in Figure 1.
More than half the patients in the PNAL group (n = 14, 58%) had a
follow-up duration of ≥5 years, 3 of whom (12%) were followed for 9
years. Considering HIV-related clinical follow-up, a consistent number
of patients (n = 22, 91%) had stable clinical conditions at the end of
follow-up (December 2003; 8 patients of this number, accounting for >1/3
of the whole PNAL group, remained naive for antiretroviral therapy
during the whole period). Conversely, HCV-related clinical follow-up
showed that progression rate of liver disease in this population was
heterogeneous (Fig. 1). The patients displayed a range of HCV-related
conditions from clinical and biochemical steady state of liver function,
with PNAL for 14 subjects with different fibrosis stage at baseline
(58%), to 10 with progressive disease who presented elevated ALT at last
observation (42%). Of the 10 with progressive disease, 1 died from
cirrhosis (5 years after stage 0 at biopsy; liver specimen was 1.5 cm
long, 1.4 mm wide, and contained 8 portal tracts) and 1 developed
clinical decompensated cirrhosis (3 years after stage 6 at liver
histology).We emphasize that at biopsy no clinical or biochemical signs
of advanced liver disease (spider angiomata, palmar erythema,
splenomegaly, ascites, platelets lower than the upper limit of normal,
abnormal PT, aspartate > alanine aminotransferase) were present in
patients 1, 7, and 12 (Fig. 1). All of the 6 patients treated for
chronic active hepatitis remained clinically stable for HCV-related
conditions independently from the therapy outcome.
PATIENTS AND METHODS
Eligibility and Study Design
All outpatients with HIV/HCV coinfection, presented to the Clinic of
Infectious Diseases, Vita-Salute University, San Raffaele Scientific
Institute of Milano, between 1997-2003 were retrospectively identified.
Patients who had a liver biopsy performed were eligible in the study.
Exclusion criteria were other possible causes of chronic liver disease,
including hepatitis B virus infection, autoimmune liver disease,
hemochromatosis, and heavy alcohol consumption (defined as average daily
consumption >50 grams/d for ≥2 years. No patients had received prior
antiviral treatment of HCV infection. Data for this study were available
for 326 subjects. The patient cohort was divided into 2 groups,
according to ALT values: the PNAL group comprised subjects presenting 3
consecutive PNAL values (≦55 IU/L for male and ≦50 for female according
to our laboratory normal values) during the 12-month follow-up prior to
liver biopsies; and the EAL group comprised subjects showing elevated
ALT values during the same period of observation. Because in most
patients, the date of HCV acute infection was unknown, we analyzed the
duration of this disease addressing the distribution of staging in
relation to age and to the date of initiation of intravenous drug use (IVDU)
(drug users are likely to acquire HCV during the 1st year of drug use).
Signed consent for the study was obtained from all eligible patients.
Laboratory Tests of HCV and HIV Diseases
Immunologic and virologic profiles related to HCV and HIV infections
were evaluated at the time of liver biopsy. For PNAL patients, some
clinical and laboratory tests were also monitored at the end of 2003.
HCV antibody status was assessed by second- or third-generation enzyme
immunoassay (Ortho Diagnostics, Raritan, NJ) and HCV viral load by the
use of the Amplicor Monitor Diagnostic System 2.0 (Hoffman-La Roche,
Basel, Switzerland). HCV genotyping was performed using the InnoLIPA HCV
II assay (Innogenetics, Gent, Belgium). ALT was measured using an
ultraviolet spectrophotometric method on a Hitachi 911 analyzer (Roche
Diagnostics, Lewes, UK). Ultrasound-guided liver biopsy was performed
using a modified Menghini needle. Formalin-fixed, paraffin-embedded
specimens were stained with hematoxylin-eosin, Masson trichrome, and the
Perl test for iron and evaluated by one local hepatopathologist blinded
to clinical and biochemical markers, other than HCV antibody status. All
the specimens examined were ≥1.5 cm long (range: 1.5-3 cm, average: 1.8
cm) and 1.4 mm wide. The slides were reevaluated in a blinded fashion.
The degree of inflammation was graded and the amount of fibrosis was
staged by using the Ishak modified hepatic activity index.7 Any
necroinflammatory score ≦7 was defined as minimal to mild and any score
>7 was defined as moderate to severe liver inflammation. Due to the
small numbers in the PNAL group, patients displaying fibrosis staging
0-1 (absent or minimal) vs. ≥2 (from mild fibrosis to overt cirrhosis)
were compared. This subgroup analysis finds its rationale in the fact
that efficacy of treatment in patients with normal ALT levels should be
assessed in the context of study protocols or might be initiated after a
liver biopsy has proved the presence of clinically significant fibrosis
(ie, staging ≥2).
Statistical Analysis
Analyses were performed by use of SAS software (version 8.2; SAS
Institute, Cary, NC). All tests of significance were 2-sided, and P <
0.05 was considered to be statistically significant. Some continuous
parameters were stratified according to their median values and then
used and compared as categorical variables. Univariate analysis on mean
independent values of continuous variables were carried out using the
Mann-Whitney rank sum test for nonparametric data. Associations between
discrete variables were tested by ƒÔ2 or Fisher exact test, as
appropriate. Multiple logistic regression was carried out to assess the
independent contribution on staging (0+1 vs. >1) of some potential risk
factors: patient's groups (PNAL vs. EAL), genotype (1 vs. others than
1), HCV RNA, log HIV RNA, antiretroviral therapy (no vs. yes), CD4 count
(<500 vs. ≥500), and age.
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Antifibrotic therapy in
chronic liver disease
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Clinical Gastroenterology and Hepatology (AGA)
Pages 95-107 (February 2005)
Don C. Rockey
Departments of Cell Biology, Duke University Medical Center, Durham,
North CarolinaUSA and Medicine, Duke University Medical Center, Durham,
North Carolina, USA
Supported by the National Institutes of Health (grants R01 DK 50574 and
R01 DK 57830) and the Burroughs Welcome Fund. D.C.R. is the recipient of
a Burroughs Welcome Fund Translational Scientist Award.
The response to injury is one of wound healing and, subsequently,
fibrosis. This response is generalized, occurring in diverse organ
systems. Injury and wounding in the liver ultimately lead to cirrhosis
in many patients (although not all patients), and are the result of many
different diseases. The fact that various diseases result in cirrhosis
suggests a common pathogenesis. Study over the past 2 decades has shed
considerable light on the pathogenesis of fibrosis and cirrhosis. A
growing body of literature indicates that the hepatic stellate cell is a
central component in the fibrogenic process. Stellate cells undergo a
transformation during injury that has been termed activation. Activation
is complex and multifaceted, but one of its most prominent features is
the synthesis of large amounts of extracellular matrix, resulting in
deposition of scar or fibrous tissue. The fibrogenic process is dynamic;
it is noteworthy that even advanced fibrosis (or cirrhosis) is
reversible. The best antifibrotic therapy is treatment of the underlying
disease. For example, eradication of hepatitis B or C virus can lead
to the reversal of fibrosis. In situations in which treating the
underlying process is not possible, specific antifibrotic therapy is
desirable. A number of specific antifibrotic therapies have been tried,
but have been met with poor or mediocre success. However, elucidation of
the mechanisms responsible for fibrogenesis, with particular emphasis on
stellate cell biology, has highlighted many putative novel therapies.
This article emphasizes mechanisms underlying fibrogenesis, and reviews
current antifibrotic therapies as well as potential future approaches.
Chronic injury results in a wound-healing response and, subsequently,
fibrosis. The response is a generalized one, with features common among
multiple organ systems. In the liver, a variety of different types of
injury lead to fibrogenesis-implying a common pathogenesis. Although a
number of specific therapies for patients with different liver diseases
have been developed, including antiviral therapies for patients with
hepatitis B and hepatitis C virus infection, specific and effective
antifibrotic therapy remains elusive.
Over the past 2 decades, great advances in the understanding of fibrosis
have been made and multiple mechanisms underlying hepatic fibrogenesis
have been uncovered. Elucidation of these mechanisms has been of
fundamental importance in highlighting novel potential therapies.
Indeed, preclinical studies have pointed to a number of putative
therapies to abrogate fibrogenesis. This article emphasizes mechanisms
underlying fibrogenesis, and reviews the current status of the field
with regard to available and future therapeutics.
ARTICLE FROM LA TIMES
Traditional herb may help liver disease
Elena Conis
LA Times
December 19, 2005
The spindly, yellow-flowered Bupleurum chinense and some closely related
species are key herbs in traditional Chinese medicine prescribed for
mood swings and gastrointestinal conditions. The root of the plant is
one of the main ingredients in an herbal formula widely known by its
Japanese name, Sho-saiko-to - in Chinese it's known as xiao chai hu tang
- that contains ginseng, licorice, ginger and a handful of other herbs
in addition to Bupleurum. Sho-saiko-to has recently gained scientific
attention for its potential in managing chronic liver disease.
Uses: In traditional Asian medicine, Bupleurum root has been used to
treat bloating, colds, fever, malaria and liver diseases, including
hepatitis. In the U.S., Bupleurum supplements are commonly marketed for
liver health.
Dose: Traditional Chinese herbalists generally recommend 1 to 5 grams of
dried Bupleurum root a day, or 5 to 7 grams of Sho-saiko-to. Bupleurum
is widely available in health food stores; Sho-saiko-to should be
available from some traditional Chinese herbalists and Asian herbal
shops.
Precautions: Large doses may cause nausea and vomiting. Sho-saiko-to
appears to pose a tiny but measurable risk of pneumonitis (lung
inflammation), particularly among patients also taking the drug
interferon.
Research: In test tube studies, Bupleurum has displayed antiviral and
anti-inflammatory capabilities. In animals, the root has been shown to
act like an antihistamine, curbing asthma and other allergy symptoms.
It's also shielded the liver from damage and expedited healing in livers
already injured. On its own, Bupleurum hasn't been well-studied in
humans. Clinical trials on Sho-saiko-to, mostly in Japan, reported
reduced symptoms in people with hepatitis B. Perhaps most promising is a
decade-old trial showing that in cirrhosis patients, Sho-saiko-to helped
prevent liver cancer. But a review published last year in the journal
Clinical Gastroenterology and Hepatology concluded that there was still
not enough evidence to recommend the concoction for chronic liver
disease.
Dietary supplement makers are not required by the U.S. government to
demonstrate that their products are safe or effective. Ask your
healthcare provider for advice on selecting a brand.
Fibrogenesis and pathophysiology
The fibrogenic process
The response to recurrent injury, in the liver and in other organs, is
one of wound healing. The wounding process (in multiple tissues,
including the liver) is complicated, but characterized by a typical
constellation of features such as increased production of extracellular
matrix, secretion of various cytokines and biologically active peptides,
and proliferation of a unique population of cells known as
myofibroblasts. Inflammation is a common theme in most forms of chronic
wound healing. This is true in particular in liver disease, in which
inflammation is often a prominent component, and frequently drives the
fibrogenic response (it is noteworthy that in some diseases,
inflammation may not be as important as in others). Further, it appears
that temporal and functional relationships are important with regard to
inflammation. For example, chronicity of inflammation is typical and
often important in many types of liver disease. Further, the type of
inflammation (ie, Th2 vs Th1) and the interplay of inflammation with
environmental/metabolic factors and genetic factors appear to be
important in fibrogenesis.
Many different types of injury (ie, chronic hepatitis, ethanol, biliary
tract disease, iron overload, copper overload, and so forth) lead to
hepatocellular injury and ultimately to hepatic fibrosis and cirrhosis.
The point that the response to recurrent injury in the liver is similar
in multiple different types of liver disease underscores the value of
identifying common regulatory components of the fibrotic response
because such components theoretically could be targeted without respect
to cause of disease. However, it is important to emphasize that, at
least in theory, different pathologic patterns of fibrosis (biliary,
perisinusoidal, pericentral) can occur and thus may merit different
types of therapies.
One of the most remarkable aspects of the wounding response in the liver
(as in other tissues) is enhanced extracellular matrix production, or
fibrogenesis. Regardless of the specific cause of liver injury (in both
experimental models and human cirrhosis), increased content of
extracellular matrix constituents occurs after injury. Specific changes
in matrix composition are highly similar in all forms of liver injury
and hepatic fibrogenesis. Hepatic fibrogenesis is characterized by
increases in multiple matrix components, including the interstitial
collagens, basement membrane collagens, proteoglycans, and matrix
glycoproteins such as laminin, and fibronectin, including its EDA (or
cellular fibronectin) isoforms.1 Many-fold increases in collagens (type
I > III > IV) are typical, but increases in the other matrix proteins
also are prominent. The wounding process is complex and integrated, and
moreover is a dynamic one that involves aspects of matrix synthesis and
deposition as well as degradation.2 Thus, there are a number of
well-defined situations in which fibrosis clearly is reversible.3-6 In
addition, in some instances, advanced cirrhosis may be reversible.7,8
Hepatic stellate cells and their activation in fibrogenesis
Although multiple liver cell types, including periportal and pericentral
fibroblasts, myofibroblasts, and even bile duct epithelial cells and
endothelial cells, play a role in fibrogenesis, stellate cells (also
known as lipocytes, Ito cells, and perisinusoidal cells) have attracted
great attention. Abundant evidence points mechanistically to a critical
role for perisinusoidal stellate cells in the pathogenesis of hepatic
fibrosis. Stellate cells, which are distributed throughout the hepatic
lobule, serve as the principal storage site for retinoids (vitamin A
metabolites) and are well known for their vitamin A handling capacity.9
It is important to emphasize that the identification and isolation of
this cell type represents a major advance in understanding the
pathogenesis of hepatic fibrogenesis because it has allowed careful
characterization of its biology.10
A central feature of the fibrogenic response is the transformation of
stellate cells (lipocytes, Ito, and perisinusoidal cells) from quiescent
(normal) to an activated (injured liver) state (Figure 1).11 Although
simple in concept, the activation process is remarkably complex and
consists of many important cellular changes. Characteristic features of
this transition include loss of vitamin A, acquisition of stress
bundles, and development of prominent rough endoplasmic reticulum. Among
the more prominent features of activation is a striking increase in
secretion of extracellular matrix proteins, including types I, III, and
IV collagens, fibronectin, laminin, and proteoglycans, some of which are
increased by greater than 50-fold, consistent with the position that
stellate cells are the cellular source of the enhanced extracellular
matrix production at the whole organ level.12 A further critical feature
of activation is de novo expression of smooth muscle-specific proteins,
such as smooth muscle a actin.13 This feature identifies stellate cells
as liver-specific myofibroblasts, and has important implications for
their contractile properties.
Although the most prominent feature of activation is enhanced
extracellular matrix production, activation also is associated with
other important cellular phenotypes including proliferation,
contractility, release of proinflammatory cytokines, and release of
matrix degrading enzymes and their inhibitors.2,11,14 It is important to
emphasize that each of these features of activation (and fibrogenesis)
represent a potential target for therapy. Key pathogenic events in
stellate cell activation are related intricately and are interdependent
(ie, in fibrogenesis, proliferation, contractility, and so forth);
several important components of the activation process are highlighted
later.
Stellate cell fibrogenesis and activation: regulatory factors
Multiple factors play a key pathogenic role in stellate cell
fibrogenesis. Prominent among these factors are cytokines, small
peptides, and the extracellular matrix itself. Transforming growth
factor-B-1 (TGF-B1) appears to be the most profibrogenic cytokine
present in the liver.15-17 TGF-B1 is produced in a paracrine manner by
Kupffer cells, sinusoidal endothelial cells, bile duct epithelial cells,
hepatocytes, or by stellate cells themselves.11,18 TGF-B1 production by
stellate cells is important, and thus points to this cytokine as a
classic autocrine factor.11,18 When overexpressed in the liver, it leads
to fibrosis,15 and when inhibited during experimental liver injury,
fibrosis is decreased.19 TGF-B1 appears to act via direct (and to a
lesser extent, indirect) stimulation of extracellular matrix production
in stellate cells. A number of other cytokines and peptides appear to
exhibit profibrogenic properties toward stellate cells (Table 1);
however, none is as potent as TGF-B1. Finally, cytokines and growth
factors that drive stellate cell proliferation are important in the
fibrogenic cascade because they help expand the total number of
fibrogenic (stellate) cells. Included in this group are platelet-derived
growth factor (PDGF), monocyte chemotactic factor, insulin-like growth
factors-1 and -2, interleukin-6, and, possibly, hepatocyte growth
factor.11
A body of literature indicates that vasoactive peptides including
endothelin-1 and angiotensin II, each of which have pleiotrophic cell
biologic and molecular effects, are important in hepatic
fibrogenesis.20-22 Additionally, the inheritance of single nucleotide
polymorphism for angiotensinogen correlated with fibrosis progression in
patients with chronic hepatitis C,23 raising the possibility of a
genetic role for angiotensin II in fibrogenesis. Because these compounds
also have vasoactive properties including presumably in portal
hypertension, the data have opened an entirely new therapeutic area (ie,
targeting both fibrogenesis and portal hypertension). Other biologically
active peptides (including unidentified compounds) also are important in
mediating hepatic fibrogenesis. Included in this group are compounds
involved in adrenergic signaling (ie, norepinephrine), which appear to
be profibrogenic.24,25 For example, the exposure of rats undergoing
liver injury to 6-hydroxydopamine, a toxin that destroys noradrenergic
fibers, significantly decreased fibrosis.24 Additionally, dopamine B-hydroxylase-deficient
mice, which cannot make norepinephrine, are resistant to fibrogenesis.25
A number of cytokines and peptides appear to have anti-activation or
antifibrogenic properties toward stellate cells. Included in this group
are interferon r,26 interferon a,27 stellate cell activation-associated
protein,28 and, possibly, adiponectin29 and hepatocyte growth factor.30
Finally, although cytokines, growth factors, and other soluble
substances are important components of fibrogenesis, it is clear that
the matrix itself modulates stellate cell activation. For example,
culture of stellate cells on a basement membrane mimicking the normal
basement membrane inhibits stellate cell activation and matrix
synthesis,31 whereas culture of stellate cells on abnormal substrates
such as the EDA isoform of fibronectin leads to increased activation of
stellate cells.32 Recent data suggest that stellate cells sense their
surrounding environment and can respond to cell-matrix tension.33
Finally, integrins, which link the extracellular matrix to stellate (and
other cells), also may play an important role in transmitting fibrogenic
signals.34
A prominent feature of liver fibrosis is extracellular matrix turnover,
including not only its synthesis, but also its degradation.14 During
fibrosis progression there is increased expression of matrix
metalloproteinases (MMPs) and in particular their tissue inhibitors (TIMPs).
Available data indicate that increases in expression of MMP-2 and
membrane type 1 MMP as well as TIMP-1 and TIMP-2 are prominent during
fibrogenesis,14,35-37 and that the overexpression of the TIMPs in
particular contributes to the profibrogenic phenotype.14 Interestingly,
overexpression of MMP8 led to partial reversal of fibrosis, providing
proof of concept for a therapeutic role for overexpression of MMPs.38
Nonfibrogenic features of stellate activation
An increase in stellate cell number is typical after both experimental
and human liver injury.11 Indeed, proliferation is an important
component of the activation cascade because it amplifies the stellate
cell-mediated response to injury. A number of mitogens appear to be
important in stimulation of stellate cell proliferation and include PDGF,
epidermal growth factor, fibroblast growth factor, endothelin-1,
angiotensin II, insulin-like growth factor, thrombin, peroxisomal
proliferator activated receptor agonists, and TGF-a to name several.11
The major mitogen driving cellular proliferation appears to be PDGF, a
cytokine that also plays a key role in cellular proliferation during
other forms of injury and wounding. Studies have shown further that
increased responsiveness to PDGF accompanies stellate cell activation
through up-regulation of its receptors.39 Thus, neutralization of PDGF
activity, by either competitive antagonists or receptor blockade, is an
important putative therapeutic approach.
Importantly, not only is stellate cell proliferation important in liver
fibrosis, but it recently has been shown that during spontaneous
recovery of experimental liver fibrosis, stellate cell apoptosis (ie,
programmed cell death) is prominent.3 These data suggest that apoptosis
of activated stellate cells may play a role in resolution of fibrosis,
and imply that a balance between cell proliferation and death is
important in determining the dynamics of the total overall stellate cell
population in the liver. Indeed, based on these data, stimulation of
stellate cell apoptosis could be an attractive therapeutic approach.40
Although apoptosis of activated stellate cells may well be critical in
fibrosis resolution, other work suggests that activated stellate cell
apoptosis may stimulate activation, and thus could be detrimental with
regard to fibrogenesis.41
Activation of stellate cells is accompanied by a marked increase in
proteins that are characteristic of contractile cells (ie, such as
smooth muscle a actin and smooth muscle myosins13,42). Stellate cell
contraction is important in the injured liver because it may contribute
to the collapse and shrunken state of cirrhotic livers, and because it
also appears to play a role in portal hypertension.22 Thus, stellate
cell contractility, although not directly related to fibrosis, is an
important physiologic target.
Approach to therapy for fibrosis
Although obvious in principle, it is important to emphasize that the
most effective antifibrotic therapies are likely to be those that target
or remove the underlying stimulus to fibrogenesis (Table 2). For
example, eradication or inhibition of hepatitis B virus4,5 or hepatitis
C virus (HCV)6 leads to reversion of fibrosis, even in some patients
with histologic cirrhosis. Additionally, fibrosis (and cirrhosis) in
patients with autoimmune hepatitis who respond to medical treatment
(prednisone or the equivalent) is reversible.8 Fibrosis may improve in
patients with alcohol-induced liver disease who respond to
anti-inflammatory therapy such as corticosteroids.43,44 Fibrosis reverts
in patients with hemochromatosis during iron depletion45,46 and after
relief of bile duct obstruction.47 Finally, in a preliminary report in
patients with non-alcohol-induced steatohepatitis treated with the
peroxisomal proliferator active receptor-r agonist, rosiglitazone
decreased both steatosis and fibrosis.48
Preclinical studies have highlighted a number of therapies that
specifically could abrogate fibrogenesis. Such therapies have been
targeted at inhibition of collagen synthesis, matrix deposition,
modulation of stellate cell activation, stimulation of matrix
degradation, or stimulation of stellate cell death. A number of these
preclinical approaches have been transitioned to clinical trials in
humans, which are highlighted later and in Table 3. The summary
presented later indicates that as of the current writing, a specific
antifibrotic that fits the profile of an ideal agent-one that is potent,
safe, orally bioavailable, and inexpensive-is not yet available.
Specific antifibrotic therapies
Colchicine
Colchicine, a plant alkaloid, inhibits polymerization of microtubules, a
process that is believed to be required for collagen secretion and thus
has been touted as an antifibrotic compound. Further, abundant evidence
supports the antifibrotic properties of colchicine in experimental
animal models,49 and thus this compound has been studied in a number of
clinical trials,50-53 including in primary biliary cirrhosis,
alcohol-induced cirrhosis, as well as in miscellaneous other liver
diseases.51 In a double-blind, randomized, controlled trial examining
colchicine in primary biliary cirrhosis, improvements were noted in a
number of biochemical markers, but colchicine failed to decrease
fibrosis.50 In a double-blind, randomized, controlled trial of
colchicine vs. placebo in patients with various liver diseases,
colchicine led to improved fibrosis as well as a dramatic improvement in
survival.51 However, this study has been criticized because of
methodologic concerns because many patients were lost to follow-up
evaluation and because there was substantial unexplained excess
mortality in the control group from causes unrelated to liver disease. A
meta-analysis including 1138 subjects found that colchicine had no
effect on fibrosis or mortality.53 In a recent multicenter study
involving 549 patients comparing colchicine (0.6 mg orally twice daily)
with placebo in patients with alcohol-induced liver disease, there was
no effect of active treatment on survival (histologic data were not
obtained).52 The aggregate data led to the conclusion that colchicine is
safe and may lead to improvement in markers of liver disease and even
mortality from liver disease. However, the failure of colchicine to
clearly decrease hepatic fibrosis makes recommendation of this drug as
an antifibrotic problematic.
Polyenylphosphatidylcholine
Polyenylphosphatidylcholine, a mixture of polyunsaturated
phosphatidylcholines, is extracted from soybeans. This compound has
gained interest as an antifibrotic agent, particularly in
alcohol-induced liver injury, because this disease often is associated
with oxidative stress. Oxidative stress in turn leads to lipid
peroxidation, cellular injury, inflammation, and a wounding response.
Thus, it has been proposed that because phosphatidylcholine is a
prominent component of cell membranes, that supplementation of it should
protect cell membranes and might lead to decreased cellular injury and
fibrogenesis. Experimental data support this notion.54 Given the
available experimental data and the apparent safety of
polyenylphosphatidylcholine, a Veterans Affairs cooperative clinical
trial examining its effect in patients with alcohol-induced hepatitis
was performed.55 This multicenter, prospective, randomized,
double-blind, placebo-controlled trial study examined 789 alcoholic
subjects (average alcohol intake of 16 drinks/day). Study subjects were
randomized to either polyenylphosphatidylcholine or placebo for 2 years.
Although the majority of subjects substantially decreased their ethanol
consumption during the trial (which was felt to result in improvement in
fibrosis in the control group), polyenylphosphatidylcholine failed to
lead to a significant improvement in fibrosis.
Interleukin-10
Interleukin-10 has both anti-inflammatory and immunomodulatory effects.
Interleukin-10 can down-regulate production of proinflammatory
cytokines, such as tumor necrosis factor-a, interleukin-1, interferon r,
and interleukin-2 from T cells. Endogenous interleukin-10 appears to
decrease the intrahepatic inflammatory response and decrease fibrosis in
several models of liver injury.56 Notably, a direct antifibrotic effect
for interleukin-10 has not been established. Nonetheless, it has been
postulated that in vivo administration of interleukin-10 to patients
with HCV infection may shift the intrahepatic immunologic balance away
from Th1 cytokine predominance, and thus have an anti-inflammatory and
subsequent antifibrotic effect.57 Thirty patients with advanced fibrosis
who had failed typical current antiviral therapy were enrolled in a
12-month treatment trial of subcutaneous interleukin-10 given daily or 3
times a week. In these patients, the hepatic inflammation score
decreased by at least 2 points (Ishak) in 13 of 28 patients, and 11 of
28 patients had a decrease in fibrosis score (mean change from 5.0 ± 0.2
to 4.5 ± 0.3, P < .05). However, serum HCV-RNA levels increased during
therapy (mean HCV-RNA level at day 0: 12.3 ± 3.0 mEq/mL; at 12 months:
38 mEq/mL; P < .05), and returned to baseline at the end of the
follow-up period. The changes in liver histology and HCV-RNA levels were
accompanied by an apparent shift in lymphocyte response toward a
Th2-predominant phenotype. Thus, long-term therapy with interleukin-10
decreased hepatic inflammatory activity and fibrosis, but led to
increased HCV viral levels by virtue of interleukin-10-induced
immunologic modifications. Thus, although potentially antifibrotic,
interleukin-10 may have detrimental effects on human HCV biology, and
thus has not been pursued.
Interferon r
The interferons consist of a family of 3 major isoforms including a, B,
and r. Each of these isoforms is unique, not only in terms of protein
structure, but also with regard to their biologic actions. There are
many different interferon a subtypes, whereas there appear to be only
single interferon B and interferon r species. Interferon a and B bind to
the same receptor and therefore share many common signaling properties.
Interferon a has much more potent antiviral effects than does interferon
r. However, interferon r has been shown specifically to inhibit
extracellular matrix synthesis in fibroblasts.58
Several studies have indicated that interferon r has potent effects on
stellate cells, inhibiting multiple aspects of stellate cell
activation.26,59 With regard to use of interferon r in patients with
hepatic fibrogenesis, there has been concern about its use because its
overexpression in the liver leads to chronic hepatitis,60 and because of
potential long-term side effects related to its profound
immunomodulatory effects. However, a recent report in patients with
chronic hepatitis C infection and fibrosis suggested that the compound
is safe and well tolerated, and that a subgroup of patients may have an
antifibrotic response.61 Although this pilot study provides a foundation
for the potential use of interferon r in patients, larger studies will
be required to document its therapeutic potential.
Silymarin
Silymarin extract, derived from the milk thistle Silybum marianum (the
major active component of which is silybinin), decreases lipid
peroxidation and inhibits fibrogenesis in animal models,62,63 including
in baboons.64 It has been tested in several carefully performed trials,
although fibrosis was not used as an outcome. The compound has been
found to be safe, but had mixed effects.65,66 In one study,65 a benefit
on mortality was shown specifically in the subgroup of alcoholic
patients. Those with early stages of cirrhosis also appeared to benefit.
However, in another study focused solely on alcoholic patients, no
survival benefit could be identified.66 Thus, although silymarin is
safe, data supporting its use are lacking.
Ursodeoxycholic acid
Ursodeoxycholic acid binds to hepatocyte membranes and presumably is
cytoprotective, thereby reducing inflammation and thus fibrogenesis.67
Although neither experimental data nor human studies indicate a primary
antifibrotic effect of ursodeoxycholic acid in the liver, a number of
studies have examined its overall effects.68-76 Ursodeoxycholic acid has
been studied in patients with cystic fibrosis, primary biliary injury
(primary biliary cirrhosis, primary sclerosing cholangitis, and
progressive familial intrahepatic cholestasis), and miscellaneous liver
diseases. The data regarding the use of ursodeoxycholic acid in these
conditions are controversial. Both symptomatic and biochemical
improvement have been observed in these diseases, but data on histologic
improvement (and survival) are mixed. For example, in one study,
survival was improved in ursodeoxycholic acid-treated patients, but
fibrogenesis was no different than in controls.73 In a randomized
controlled trial of ursodeoxycholic acid in primary biliary cirrhosis,
active treatment led to decreased fibrosis in those with mild liver
disease, but had no effect on those with severe disease.69 Further, in a
histopathologic study of 54 patients with primary biliary cirrhosis and
paired liver biopsy examinations, 4 years of ursodeoxycholic acid
therapy was associated with a significant decrease in the prevalence of
florid interlobular bile duct lesions, lobular inflammation, and
necrosis. Worsening of fibrosis was observed in 14 patients (the
majority had only a 1-grade progression in fibrosis score), whereas
stabilization was noted in the 40 remaining patients.74 Although the
results of meta-analyses have been mixed, and largely have reported that
ursodeoxycholic acid is not effective in primary biliary cirrhosis,72 a
recent combined analysis of the histologic effect of ursodeoxycholic
acid on paired liver biopsy examinations (a total of 367 patients-200
ursodeoxycholic acid and 167 placebo), revealed that when patients with
initial stages I-II were considered, ursodeoxycholic acid significantly
delayed histologic stage progression (P < .03).75 The aggregate data
suggest that ursodeoxycholic acid may impede progression of fibrosis in
primary biliary cirrhosis via effects on bile ductal inflammation,
particularly if given early in the disease course. Ursodeoxycholic acid
is safe, and although expensive, it is this author's belief that the
available data justify its use in patients with primary biliary
cirrhosis as an antifibrotic.
A beneficial effect of ursodeoxycholic acid on fibrogenesis was shown in
a small number of children with progressive familial intrahepatic
cholestasis.70 Further, a case series indicated that 7 of 10 patients
with cystic fibrosis treated with ursodeoxycholic acid had a decrease in
liver fibrosis.71 It should be emphasized that although these effects
are promising, the number of patients studied has been small. Finally,
in a large, randomized, controlled trial of the effect of a 2-year
course of ursodeoxycholic acid in patients with nonalcoholic
steatohepatitis, including 107 subjects who had paired biopsy data,
there was no improvement in fibrosis.76
Herbal medicines
A number of herbal medicines have been shown to have antifibrotic
properties in animal models, and in some, specific mechanisms have been
identified.77-79 Herbal medicines with putative antiviral,
anti-inflammatory, and antifibrotic effects are being used extensively
in the far East in patients with a variety of liver diseases.80
Medications containing herbs of the Salvia genus have been popular in
particular as antifibrotics.80 Although human trials have suggested
effectiveness of specific herbal medicines in some studies,80 data in
peer-reviewed Western journals remain lacking. Because it is well
appreciated that such herbal medicines may have significant toxicity,
including hepatotoxicity,81 these medications should be used with
caution.
Miscellaneous
Given the role of oxidative stress in injury and in stellate cell
activation and stimulation of extracellular matrix production,
antioxidants have received considerable attention as antifibrotics.
Vitamin E has been examined in animal models82 as well as in
humans.83-86 The vitamin E precursor, d-a-tocopherol (1200 IU/day for 8
weeks) was tested in 6 patients with HCV infection who failed to respond
to interferon therapy,83 resulting in inhibition of parameters of
stellate cell activation. However, it did not affect fibrosis. A
randomized controlled trial examined vitamin E in patients with mild to
moderate alcohol-induced hepatitis and found that vitamin E decreased
serum hyaluronic acid levels, but did not lead to a change in type III
collagen.85 Antioxidant therapy, including vitamin E, in patients with
severe alcohol-induced hepatitis, had no effect on outcome, although
fibrosis was not addressed specifically.86
Malotilate appears to be cytoprotective, perhaps via inhibition of
cytochrome P450 2E1, and additionally may have anti-inflammatory
effects. Unfortunately, in patients with primary biliary cirrhosis,
although it was found to decrease plasma cell and lymphocytic infiltrate
and piece-meal necrosis, it had no significant effect on fibrogenesis.87
The heavy metal chelating properties of penicillamine have been proposed
to contribute to its anti-inflammatory and antifibrogenic effects88;
however, this compound was ineffective in mitigating fibrogenesis in
patients with primary biliary cirrhosis.89,90
Methotrexate also is considered to be an anti-inflammatory compound, and
typically has been considered to be profibrogenic,91 although it is
noteworthy that the risk for fibrosis progression may be less prominent
than classically believed.91,92 Nonetheless, because of its
anti-inflammatory properties, it has been assessed as a potential
therapeutic agent in patients with primary biliary cirrhosis; although
improvement in disease and fibrosis have been reported, including
reversion of fibrosis,93 the majority of the data on methotrexate are
either negative94,95 or show that its effects are marginal, either
alone,94 or in combination with colchicine.96 If methotrexate is used to
treat patients with primary biliary cirrhosis, an experienced
hepatologist should manage its use (with caution).
S-adenosylmethionine is important in the synthesis of the antioxidant,
glutathione. Since it was proposed to have antioxidant properties in the
liver, and decreased expression of the enzyme responsible for its
synthesis (methionine adenosyltransferase) has been found after liver
injury,97S-adenosylmethionine has been tested in a large randomized
trial in patients with alcohol-induced cirrhosis. Histologic assessment
of fibrosis was not measured specifically as an outcome, although there
was an improvement in overall mortality/need for liver transplantation
in the treatment arm, especially in patients with Child's A/B cirrhosis,
raising the possibility fibrosis may have improved.98
Propylthiouracil, an antithyroid drug that reacts with some of the
oxidizing species derived from the respiratory burst, may be protective
in alcohol-induced liver disease, a disease in which an increase in
hepatic oxygen consumption may predispose the liver to ischemic injury.
Thus, propylthiouracil has been tested in randomized clinical trials in
patients with alcohol-induced liver disease. Unfortunately, a systematic
review and meta-analysis found that propylthiouracil led to no benefit
in fibrosis (or other outcome variables).99
Anabolic-androgenic steroids such as oxandrolone have been tested in
patients with alcohol-induced liver disease, but have not been found to
have significant effects on fibrosis (or other outcomes).100
Several recent pilot studies have examined the effect of anti-tumor
necrosis factor-a compounds in patients with alcohol-induced liver
disease.101-104 The rationale for these therapies is that tumor necrosis
factor-a is up-regulated in the liver injured by alcohol, and thus these
compounds should decrease inflammation, and is the stimulus for
fibrosis. Although there are little data on the effect of these
interventions on hepatic fibrosis, preliminary analyses suggest an
improvement in inflammation and acute injury (which presumably precedes
fibrosis in this disease).103 However, the use of these compounds will
require great caution because they may increase the risk for serious
infection.105
It is important to emphasize that for many human studies (ie, involving
S-adenosylmethionine, propylthiouracil, androgenic steroids, and
anti-tumor necrosis factor-a, and so forth) subjects with
alcohol-induced hepatitis and liver injury were examined (see Table 3),
and in these studies fibrosis typically was not measured as a specific
outcome. Thus, it is not entirely appropriate to consider these agents
as primary antifibrotics, but rather as compounds that could have
secondary effects on fibrogenesis owing to other properties.
Ones to watch
There is scientific rationale for the use of a number of other
compounds, many of which have been studied in experimental models and
have been shown to have antifibrotic effects (Table 4). For example, TGF-B
plays a central role in the fibrogenic cascade and therefore is an
important therapeutic target. Several approaches to inhibit the action
of TGF-B have been proposed and include the use of molecules such as
decorin, the protein core component of proteoglycan, which binds and
inactivates TGF-B,106 antibodies directed against TGF-B1, and soluble
receptors that typically encode for sequences that bind active TGF-B and
prevent it from binding to cognate receptors.19,107 The concept has been
well established experimentally; indeed, the effect of inhibition of TGF-B
in animal models of liver injury and fibrogenesis has been
striking.19,107 Additionally, stellate cells express angiotensin II and
endothelin receptors and stimulation of these receptors with their
cognate ligands leads to prominent stellate cell effects.22 Inhibition
of endothelin signaling leads to decreased fibrogenesis21; likewise, the
blockade of angiotensin II function (ie, with angiotensin enzyme
inhibitors, angiotensin 2 receptor antagonists) in vivo also inhibited
stellate cell activation and fibrosis.108 Thus, inhibition of their
binding in human liver disease may be beneficial clinically. Among
others, compounds such as pirfenidone,109 peroxisomal proliferator-activated
receptor-r ligands,110 pentoxifylline,111 halofuginone,112 and
5'-lipoxygenase inhibitors113 appear to have direct effects on stellate
cells and/or in vivo effects in hepatic fibrogenesis. A recent report
suggests that the adipocytokine, adiponectin, inhibits PDGF-induced
proliferation and attenuates the effect of TGF-B1 in stellate cells, and
thereby leads to inhibition of fibrogenesis.29
Table 4.Potential Antifibrotic Therapies
HGF, hepatocyte growth factor; STAP, stellate cell
activation-associated protein; PAR, protease activated receptor; PPAR,
peroxisomal proliferator activated receptor. a-Also angiotensin
converting enzyme inhibitors.
Diagnosis and monitoring of hepatic fibrosis and cirrhosis
Liver biopsy examination is considered to be the gold standard for
determining the extent of fibrosis. Liver biopsy examination also is
used to assess fibrosis progression. Connective tissue stains, including
reticulin, Masson's trichrome, and sirius red, readily identify
extracellular matrix within tissue sections. A quantitative measure of
collagen content can be made by colorimetric assay of sirius red in
liver tissue or by image analytic quantitation of collagen-containing
tissue.21 Additionally, scoring systems have been developed114-116 to
quantitate fibrosis and to help standardize the interpretation of biopsy
examinations among different centers; such systems are most useful for
standardization and comparison of fibrosis in studies. For individual
patients, direct comparison of biopsy specimens over time is most
useful. It is important to emphasize that although histology is helpful
for judging the presence and degree of fibrosis, clinical tools such as
Child-Pugh classification are most useful for assessing overall disease
severity.
Liver biopsy examination, although considered the gold standard tool to
assess fibrosis, is inexact. Not only is liver biopsy examination
subject to interobserver variability, but sampling error may be
important, as evidenced by studies examining liver samples from
different regions of the liver.117 Additionally, liver biopsy
examination also is associated with significant potential morbidity,
including a significant risk for death.118 Thus, noninvasive measures
that can monitor fibrogenesis would be ideal. Noninvasive tools used to
assess fibrosis include radiographic tests,119 combinations of routine
laboratory tests,120,121 and specific serum markers.122,123 In
particular, serum marker panels, including several that use mathematic
algorithms,120,121 have recently been emphasized. However, as of this
writing, they have proven to be of limited clinical use.
Summary and future directions
Understanding of the basis of hepatic fibrogenesis has advanced
significantly in the past 2 decades, and with it, a field dedicated to
therapeutic antifibrotics has emerged. The central event in fibrogenesis
appears to be activation of hepatic stellate cells. Stellate cell
activation is characterized by a number of important features including
enhanced matrix synthesis and a prominent contractile
phenotype-processes that each undoubtedly contribute to physical
distortion and dysfunction of the liver in advanced disease. It is
important to emphasize that factors controlling activation are
multifactorial and complex, and thus multiple potential therapeutic
interventions are possible. A further critical concept is that the
fibrogenic lesion, in particular, the extracellular matrix, is dynamic
and reversible. Even advanced fibrosis may be reversible under the
appropriate conditions. Currently, effective therapy for hepatic
fibrogenesis exists for several diseases-in the form of removal of the
underlying disease process. As for specific therapy directed only at the
fibrotic lesion, the most effective therapies most likely will be
directed at the stellate cell. Additionally, approaches that address
matrix remodeling (ie, by enhancing matrix degradation or inhibiting
factors that prevent matrix breakdown) will be attractive. Thus,
although specific, effective, safe, and inexpensive antifibrotic
therapies do not yet exist, multiple potential targets have been
identified, and it is highly likely that candidates will emerge.
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