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 dHepato-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

	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.

http://www.natap.org/

Antifibrotic therapy in chronic liver disease

	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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Reviewed Feb 21 2006