ACKNOWLEDGEMENTS Authors wish to thank Swedish Institute for the financial support rendered through the New Visby progra

1. Invasive and Non-invasive Monitoring of Hepatitis C Virus-induced Liver Fibrosis, Alternatives or Complements?Znoiko Olga O.*(1, 2) Yuschuk Nikolai D. (1)

2. INTRODUCTIONChronic hepatitis C is estimated to affect 170 million persons worldwide, and is now the single major reason for liver transplantation. Natural history studies suggest that 5%–20% of persons develop cirrhosis during the 20 years of HCV infection, while other patients remain asymptomatic, without significant liver disease for many decades, if not for life [1, 2]. Detection and characterization of hepatic fibrosis by liver histology and by non-invasive monitoring is a primary instrument to assess chronic hepatitis C natural history and prognosis. This topic review will focus on the hepatic fibrosis diagnosis.

4. However, in doing so liver function mayultimately become impaired. With recurrent bouts of inflammation, the liver's normal architecture can be replaced by fibrous scar tissue, ultimately resulting in the advanced liver disease known as cirrhosis [1, 3]. Typically, injury is present for months to years before significant scar accumulates, although the time course may be accelerated in congenital liver disease. Being a hallmark of hepatic cirrhosis, the fibrosis worsening is probably the best surrogate marker for the progression of chronic liver disease.

5. Stellate cells undergo a process known as activation, in response to any insult (Fig. (2)). Upon activation, HSC is transformed from a quiescent cell filled with retinoids to anactivated cell characterized by a loss of retinoids, expression of new receptors, cellular proliferation, expression of new genes such as a-smooth muscle actin, and synthesis of extracellular matrix (ECM) proteins (Fig. (2)) [Brenner DA.The role of hepatitis C in hepatic fibrosis; http://www.postgrado.com/5htm)].

6. ECM is a dynamic regulator of cell function. Early, subendothelial matrix accumulation leading to "capillarization"of the subendothelial space of Disse is a key event, and may be more important than overall increases in matrix content (Fig. 1); [7]. Normally, this space contains the components of a basement membrane. Replacement of the normally low-density matrix of basement membrane by high-density interstitial matrix directly perturbs hepatocyte function. Activated HSCs produce fibrogenic environment through combination of ECM overproduction, diminished activation of metallproteinases responsible for degradation of ECM proteins and inhibition of this degradation by metalloproteinase tissue inhibitors.

7. FIBROSIS STAGING AND GRADINGThe risk of developing cirrhosis depends on the stage (degree of fibrosis) and the grade (degree of inflammation and necrosis) observed in the initial liver biopsy (Figs. (3, 4); for more images, see also http://janis7hepc.com/learning_about_liver_fibrosis.htm#Predicting). The degrees of fibrosis, portal, then peripheral, then bridging ending at cirrhosis) are illustrated by Figure 3.

8. It is widely excepted that the fibrosis intensity grade correlates with the activity of inflammatory changes in the liver (Fig. (4)); inflammation grades from I to IV as illustrated at the web-site http://www.ikp.unibe.ch/lab2/hepC). This, however, is not absolutely true. According tothe liver biopsy data, in 36% of patients with chronic hepatitis C the severity of hepatitis did not correspond to the grade of fibrosis. If the recommendations to treat were based on the degree of hepatitis intensity, 56% of patients would not undergo medical treatment despite the severe fibrosis [9].

9. FIBROSIS DEVELOPMENTFibrosis RateThe speed of fibrosis development in the case of hepatitis C is of top interest for the clinicians. The speed of fibrosis development is determined as a ratio of the difference in fibrosis stages expressed in METAVIR and the interval between biopsies expressed in years [9]. This method may only be deemed significant if one knows exactly theinfection duration and if the speed of fibrosis development is constant. However, the rate of fibrosis development is not a constant value.

10. There are three excepted options of fibrosis progress: quick (10 and less years), average (about 30 years) and slow (more than 50 years) [1, 9]. For the untreated patients, the average number of life years from the on-set of the infection to the development of cirrhosis amounts to 30, with 33% of the patients having the expected time of cirrhosis development of less than 20 years, while for 31% of patients the expected time of cirrhosis development exceeds 50 or more years. Different patients progress to cirrhosis at different rates. The overall rate of fibrosis progression suggests that the average patient would require 49 years to develop cirrhosis.

11. Factors Effecting Fibrosis DevelopmentLiver fibrosis in chronic hepatitis C is related to sex, age at infection, duration of infection, and alcohol consumption. A link between age at HCV infection and the presence of hepatic fibrosis is supported by the strong and consistent trend of increasing risk with age at infection, and the independence of effect. This relationship has been seen in several previous cross-sectional studies (Fig. (5)) [17, 23, 24].Recent longitudinal studies had found very low rates of progression to advanced liver disease among young adults infected through injecting drug use and contaminated anti-D immunoglobulin injections [25-27] and among children infected through blood transfusion [28].

12. The progression of liver fibrosis seems to be influenced by polymorphisms in the genes encoding immunoregulatory proteins, proinflammatory cytokines, and fibrogenic factors [29]. These genetic factors could explain the broad spectrum of responses to HCV found in patients with chronic liver diseases. For example, analysis of patients with end stage liver cirrhosis and blood donors demonstrated a reduced risk for HCV-induced end stage liver disease in the presence ofDRB1*11 and DQB1*03 [30]. Large-scale, well-designed studies are required to clarify the actual role of different factors and genetic variants in liver fibrosis development.

13. ReversibilityDense cirrhosis, the end-stage consequence of fibrosis, with nodule formation, portal hypertension, and early liver failure is generally considered irreversible. The exact moment at which fibrosis becomes irreversible is unknown, either in terms of a histologic marker or a specific change in the matrix composition or content [7]; www.uptodate.com/patient_info/topicpages/topics/Cirrhosi/9999.asp # 13).Less advanced lesions can show remarkable reversibility when the underlying cause of the liver injury is controlled.

14. FIBROSIS DIAGNOSIS BY LIVER PUNCTURE BIOPSY AND ITS RISKSLiver biopsy serves as a “gold standard” for the diagnosis of fibrosis with chronic hepatitis C being responsible for over 50% liver biopsy cases all over the world [19, 33]. A morphological study of the punctate determines the grade of hepatitis activity and fibrosis stage, eliminates the alternative diagnoses and helps to detect additional pathologies, as well as to evaluate the efficiency of antiviral therapy.

15. However, the biopsy is an invasive technique withpossible complications and even the lethal outcomes. The number of lethal cases due to biopsy varies from 0 to 3,3 out of 1000 [9, 34, 35]. The study by Gadranel et al. Contains data analysis of 2,084 liver biopsies carried out in 89 medical centers in France during 1997 with 54% of cases due to HCV infection [33]. A percutaneous biopsy was carried in 91%, and transvenous, in 9% of cases. In 20% of cases,moderate pains were registered after the procedure, while 3% of patients had considerable pains that required intravenous analgesia with further hospitalization (in cases when the biopsy was carried out in the outpatient setting).

16. LIVER BIOPSY LIMITATIONSSome clinicians believe that since liver biopsies sample only 1/50,000 of the entire liver mass, sampling errors are bound to occur. There is a probability of “spotting error” when the biopsy needle hits the area of tissue where thechanges are less or on the contrary more intense than on the whole in the liver. For example, liver biopsy samples taken from the right and left hepatic lobes differed in histological grading and staging in a large proportion of hepatitis Cpatients [37].

17. NON-INVASIVE BIOCHEMICAL SCALING OF FIBROSISThe above-mentioned limitations of the liver biopsy plus considerable biopsy costs substantiate the necessity todevelop simple, inexpensive, and reliable non-invasive tests to assess disease severity in patients with chronic hepatitis C in which peripheral blood is to be used [38-41].

18. Three-Marker Biochemical Fibrosis IndexOne of the biochemical approaches of non-invasivefibrosis monitoring makes use of three parameters: the number of thrombocytes, the prothrombin time and the ALT/AST ratio with evaluation range from 0 to 11 points [45]. The scale shows a specificity of 98% and a sensitivity of 46% [45]. The average score for the patients having histologically detected fibrosis of 0-2 amounts to 4.3±2.0, while the score for the patients having stage 3 and 4 fibrosis is considerably higher (7.9±1.4; p< 0.0001). Thus, without recurring to liver biopsy, one may suggest that patients with the number of scores equal to 7 and more are more likely to have fibrosis stage 3 to 4.

19. Thrombocytopenia is commonly seen in patients with cirrhosis and in patients with acute liver failure [50]. Both splenomegaly and inadequate thrombopoietin (TPO) production by the cirrhotic liver are responsible for thrombocytopenia. In addition, thrombocytopenia is frequently observed in chronic hepatitis patients who received interferon therapy, and may even lead to the discontinuation of treatment. The serum TPO response to interferon-induced thrombocytopenia is interferon-dose dependent, and together with the changesin platelet counts may serve as a marker for the degree of liver fibrosis [51].

20. Five-Marker Biochemical Fibrosis IndexThe newest fibrosis index devised included a wide panel of clinical and laboratory markers combined with age and sex. The most informative markers were: alpha2 macroglobulin, alpha2 globulin (or haptoglobin), gamma globulin, apolipoprotein A1, gamma glutamyltranspeptidase (GGT), and total bilirubin [52, 53]. Five-markers index accurately predicts significant fibrosis in patients with HIV/HCV co-infection, and reduce the necessity for liver biopsy by 55% while maintaining an accuracy of 89% [53].

21. IronIn CHC patients, hepatic iron concentration correlates with liver fibrosis [21]. In CHC patients, hepatic iron could play a role in the activation of hepatic stellate cells and in the progression of fibrosis. In CHC patients with low grading score (< 3), hepatic iron concentration correlates positively with the number of stellate cells and proliferating hepatocytes (72 CHC patients) [56]. Ferritin serum level correlated significantly with hepatic iron concentration (r = 0.59, P < 0.001), with grading (r = 0.47, P < 0.001) and staging (r = 0.51, P < 0.001) scores for chronic hepatitis in the whole group of patients [56].

22. Enzymatic and Nonenzymatic AntioxidantsFree radicals play a role in CHC liver damage.Antioxidants (AO) (enzymatic and nonenzymatic) scavenge free radicals and prevent tissue injury. The levels of malondialdehyde (MDA) and AO, namely retinol, alpha- and gamma-tocopherol, lutein, beta-cryptoxanthin, lycopene, andalpha- and beta-carotene levels, were estimated by highpressure liquid chromatography in the pretreatment serum and liver biopsy specimens [58].

23. Alpha-FetoproteinSerum levels of alpha-fetoprotein (AFP) were analysed at the time of diagnosis in 77 patients with CHC fibrosis and HCC [59]. AFP levels were elevated in 78% of cases, but only 34% reached >500 ng/ml, a value considered to besignificant for the diagnosis of HCC. AFP levels correlated with the stage of fibrosis. The sensitivity of AFP serum levels as a tumour marker is poor but might help to detect atleast a minority of cases.

24. Conflicting Biochemical Fibrosis MarkersThe relationship between serum transaminase levels and risk of liver disease progression remains controversial.Although risk of disease progression is considerably higher among people with abnormal serum transaminase levels compared with those with consistently normal levels [60].There is conflicting evidence on the association between degree of ALT abnormality and extent of histological fibrosis (see, for example, Fig. (6)).

25. A reasonable explanation for this discrepancy is that ALT/AST levels vary during the course of this disease, and the values found at the time of a single liver biopsy may not accurately reflect values that have occurred in previous years.These findings indicate that regular determination of aminotransferase values is a helpful and reliable means of monitoring disease, judging prognosis, and recommending therapy and support the recommendation that patients with normal aminotransferase levels and mild liver histology can safely defer treatment [2].

26. EXTRACELLULAR MATRIX TESTS OF FIBROSIS SCALINGAnother route of non-invasive fibrosis scaling that has recently received much attention is the evaluation of serumlevels of extracellular matrix break down products. The rationale is that since there is extensive deposition of fibrous tissue, serum levels of the constituents of fibrous tissue will increase as a result of remodeling and recurrent scarring.

27. The biochemical tests of fibrosis severity are based on the detection of molecular junctions that activate fibrosis, or participate in the pathophysiology of the extracellular matrix generation process. The most extensively studied are: hyaluronic acid (HA); metalloproteinases (MMP) and inhibitors of metalloproteinases (TIMP); type IV collagen (IV-C) and N-terminal propeptide of type III procollagen(PIIIP) [21, 40, 68, 69].

28. Hyaluronic Acid (HA)The particularly intense discussion arises over thediagnostic significance of the high content of HA. HA is a glycosaminoglycan, polysaccharide with a heavy molecular weight, widely present in extracellular space. Some of the produced HA is supplied by the lymphatic system to the venous blood and may be identified in the blood by enzymeimmune detection. In liver, HA is synthesized by stellate cells and is considered to be a factor contributing to fibrogenesis. The increase in HA production may reflect the induction of stellate cell proliferation and the synthesis of extracellular matrix due to the inflammation.

29. According to Ueno et al., HA levels reflected the degree of fibrosis in HCV-RNA positive non-responders to antiviral therapy [73]. Changes in the levels of HA were compared among the patients who showed a sustained normalization of serum ALT levels with and without eradication of serum HCV RNA (complete responders and biochemical responders) and nonresponders. Hyaluronate levels were significantly decreased 24 weeks after the end of the treatment, as compared with those prior to the treatment [76]. In nonresponders, IFNalpha therapy was unable to decrease the HA levels and did not improve the histological outcome. It was reflected by unchanged HA levels during the therapy and the follow up [77].

30. Propeptide of Procollagen Type III (PIIIP) and Type IV Collagen (IV-C)Collagens type III and V and fibronectin accumulate in early, and collagens type I, III, and IV, indulin, elastan and laminin in late injury [80]. Serum pro-collagen III peptide levels (PIIIP) are related to lobular necrosis in untreated patients with chronic hepatitis C [71]. Serum N-terminal procollagen III peptide (PIIINP) displayed a significant and persistent decrease in sustainedresponders but not in non-responders (14 sustainedresponders and 17 non-responders [81, 82], PIIIP normalized when fibrosis improved (P< 0.01).

31. CHC patients showed a mean level of serum type IV collagen (IV-C) (133.6+/-93.3 ng/ml) significantly (P < 0.01) higher than controls (100.2+/-10.5 ng/ml). A good relationship between the degree of liver fibrosis and IV-C serum level was found (r = 0.68; P < 0.005). In both responders and non-responders IV-C levels decreased during interferon therapy. During the follow-up, in responders IV-C remained unchanged, while in non-responders/relapsers it returned rapidly to the pretreatment levels (139.1+/-100.7 ng/ml). Thus, in CHC type IV collagen appears to be a useful tool for evaluating fibrogenic activity, and can be used to monitor the effects of antiviral treatment [85].

32. Matrix MetalloproteinasesRecovery from fibrosis involve remodelling and breakdown of multiple ECM components with degradation of predominant component collagen I. The matrix metalloproteinases (MMP), a family of zink dependent endoporteinases, have a capacity to degrade these various ECM components. MMPs are expressed particlularly by HSCs and Kupffer cells [88]. Matrix metalloproteinases 1, 2, and 7 (MMP-1, -2, -7) and tissue inhibitor of metalloproteinase-1 (TIMP-1) are closely involved in the fibroproliferativeprocess in the liver during chronic hepatitis C [88-91].

33. In another study, serum levels of MMP-1, MMP-2, TIMP-1 and TIMP-2 before and after interferon-alpha (IFNalpha) treatment demonstrated a significant increase in the MMP-1/TIMP-1 ratio in the responder group, while in the non-responder group the MMP-1/TIMP-1 ratio tended towards a decrease [93]. Moreover, there was not only a significant increase in TIMP-2 levels but also a tendency towards an increase in TIMP-1 levels. The results of Ninomiya et al. suggested that an elevated MMP-1/TIMP-1 ratio may ameliorate liver fibrosis by interferon in CHC, whereas elevated levels of TIMP-1 and TIMP-2 might impede improvement [93].

34. Cell Adhesion MoleculesLymphocyte adhesion to endothelium, extravasation, and adhesion to hepatocytes are mediated by adhesion molecules and constitute important steps in the liver inflammation dueto CHC. A number of publciations has described increased levels of soluble adhesion molecules in the serum of patients with hepatitis C, and their correlation with cytokine concentrations and liver inflammation and fibrosis. Measurement ofsoluble adhesion molecules may be a perspective way to follow liver inflammation and fibrosis during CHC [94].

35. CYTOKINES: TRANSFORMING GROWTH FACTOR BETA 1Host genetic factors influencing fibrogenesis may account for some of the variability in progression of this disease. Progressive fibrosis of other organs, particularly heart and kidney, involve overproduction of the profibrogenic cytokine transforming growth factor beta1 (TGF-beta1) that may be enhanced by angiotensin II, the principal effector molecule of the renin-angiotensin system. Powell et al. examined the inheritance of polymorphisms in TGF-beta1, interleukin 10 (IL-10), tumor necrosis factor alpha (TNF-alpha), and genes of the renin-angiotensin system in 128 CHC patients [96].

36. TGF-beta 1 is an important cytokine involved in thepathobiology of tissue fibrosis through its stimulation of the production of, and inhibition of the degradation of, extracellular matrix proteins. TGF-beta 1 impairment of extracellular matrix breakdown is related to stimulation oftissue inhibitor of metalloproteinases-1 (TIMP-1) gene. There is a close correlation between TGF-beta serum levels and the rate of fibrosis progression. Plasma TGF-beta 1 levels are significantly elevated in patients with mild and moderate chronic hepatitis, but not in those with minimal chronic hepatitis, compared with the levels in the controls.

37. CONCLUSIONThere has been considerable effort to identify serum markers as noninvasive measures of hepatic fibrosis [67]. However, noninvasive markers have several limitations:1. They typically reflect the rate of matrix turnover, not deposition, and thus tend to be more elevated when there is high inflammatory activity. In contrast, extensive matrix deposition can go undetected if there is minimal inflammation.2. None of the molecules are liver-specific, so that concurrent sites of inflammation may contribute to serum levels.

38. ACKNOWLEDGEMENTSAuthors wish to thank Swedish Institute for the financial support rendered through the New Visby program.REFERENCES