The clinical and the prognostic value of Insulin, Growth Hormone, TNF-R (P55) and IL-1 receptor antagonist (IL-1ra) in Chronic Hepatitis due to HCV

Genotype 4 before and after combination therapy with Pegylated Interferon α-2a and Ribavirin.

Nihal M.El Assaly 1 , Naema El Ashri1, Omnia El Bendary1, Shendy M.Shendy2 , M. Ali Saber 3 and Ehab El Dabaa3 1. Clinical Chemistry Department. Theodor Bilharz Research Institute (TBRI), 2. Gastroenterology and Hepatology Department (TBRI). 3. Biochemistry Department (TBRI).

Abstract

Combined therapy using Interferon alfa (IFN) and Ribavirin (RIB) represents the standard treatment in patients with chronic hepatitis C. However, the percentage of responders to this regimen is still low, while its cost and side effects are elevated. Therefore, the possibility to predict patient's response to the above treatment is of paramount importance. Aim of this work is to estimate the clinical and prognostic role of IL-1ra and TNFR (P55) which are receptors related to inflammatory cytokines and, GH and Insulin hormones metabolized in the liver in HCV infection, cirrhotic and non-cirrhotic. Also to find their significance as a noninvasive biochemical markers that may correlate with HCV infection on predicting the outcome of interferon alpha 2a therapy in patients with chronic HCV infection. Methods 54 patients infected by HCV genotype 4 were enrolled in this study. They were classified into two groups according to the liver histology. Group A of 42 chronic compensated HCV patients with no cirrhosis, Group B of 12 chronic HCV patients with established cirrhosis and 12 healthy controls. Patients were treated by Pegylated INF α-2a (180 µg for group A and 130 µg for group B) once weekly & 1200 mg Ribavirin/ day in two doses. Tested parameters have been done by ELISA method before and after treatment for group A, group B and control group. Results: end of treatment response (ETR) and sustained virological response (SVR) were 73.817% and 61.91% for group A, and 58.33% and 33.33% for group B respectively. Serum IL-1ra was increased after treatment but this increase was not significant (P<0.25). There was a significant increase of serum insulin (P<0.01) of group A after treatment compared to group A before treatment, group B and control group. On the other hand, serum TNF-R P55 showed significant decrease (p <0.05) in group A after treatment compared to group A before treatment, group B and control group. TNF-R P55 showed positive correlation with sALT and sAST. Also, serum GH level decreased in group A after treatment compared to the other studied groups; but, this decrease was not statistically significant Conclusion Pegylated INF α-2a and Ribavirin are effective combination in treatment of chronic HCV genotype 4. Insulin and TNFR (P55) correlate with HCV infection and could be used as a marker of peg - IFN α-2a and Ribavirin response while IL-1ra and GH are of no value.

INTRODUCTION:

Chronic Hepatitis C ( HCV ) is a major public health problem in Egypt. It is caused by genotype 4 in

more than 90 % of the patients (Zekry et al., 2001). Early treatment of HCV will markedly reduce the

progression to cirrhosis, decompensated disease and hepatocellular carcinoma (Attia, 1998).

Combined therapy using Interferon alfa (IFN) and Ribavirin (RIB) represents the standard treatment

in patients with chronic hepatitis C. However, the percentage of responders to this regimen is still low,

while its cost and side effects are elevated. Therefore, the possibility to predict patient's response to the

above treatment is of paramount importance. The viral genotype, the degree of inflammation, fibrosis and

the viral load prior to treatment are considered the strongest predictors of response to antiviral therapy

(Maiellaro et al., 2004).

Until recently interferon alfa (INF) and ribavirin were the combination of choice in treatment of

HCV. Response of Egyptian patients with chronic hepatitis C to standard therapy of interferon and ribavirin

combination is unsatisfactory and only less than 40 % of Egyptian patients had sustained virologic response

(SVR) (Mc Hutchinson et al., 1998 and Poynard et al., 1998). Factors that influence response rate to

interferon therapy are numerous and include both host and viral factors (El-Zayadi et al., 1999).

Pegylated INF has longer half life than standard INF and can be administered once weekly. So, its

combination with ribavirin resulted in SVR in 82 % of patients with genotype 2 and 3 and 42% of patients

with genotype 1 (Mannervik et al., 1992). Good response of other genotypes (2 & 3) to Pegylated INF

therapy pushed us to study its efficacy on genotype 4 (Maiellaro et al., 2004).

The number of patients with genotype 4 who were enrolled in European studies was too small to be

included in statistical analysis. Also, the response of this genotype 4 to pegylated INF with ribavirin was

not properly studied in the Middle East and north Africa (Zekry et al., 2001).

Interleukin-1 (IL-1) is a cytokine that plays an important role in initiating the cascade of events of

immuno-inflammatory responses through co-stimulation of T lymphocytes, B-cell proliferation and

induction of adhesion molecules and stimulation of the production of other inflammatory cytokines. The

role of IL-1 in immuno-inflammatory responses is highlighted by the presence of endogenous regulators

(IL-1 receptor antagonist, soluble receptors type 1 and II, human IL-1 accessory protein) that, when

secreted into the blood stream may serve as endogenous regulators of IL-1 action (Libra et al., 2002).

Hepatitis C virus (HCV) infection is resistant to interferon alpha (IFN-Alpha) in some patients. The

mechanism of this resistance is unknown (Mannervik et al., 1992). It was proved that Interleukin-1 receptor

antagonist (ILI-Ra) is induced by IFN-alpha and is a good indicator of IFN activity, and this increase

indicates that IFN receptors are functioning in patients with IFN-resistant hepatitis C and that the lack of

response is related to other virologic or immunologic factors (Cotler et al., 2002).

Cirrhosis is characterized by high growth hormone (GH) levels which fail to decrease and often

paradoxically increase after administration of glucose or insulin (Riley and McCann, 1981 and S hanker et

al., 1986). The cause of this high GH level remains uncertain. It may be a decrease in its metabolic

clearance rate or diminish in liver growth hormone receptors (Shmueli et al., 1994). Insulin resistance is

present in nearly all patients with liver cirrhosis, but its etiology remains unclear. Recent studies have

shown that tumor necrosis factor-a (TNF-) system is involved in the insulin resistance of liver cirrhosis,

as serum concentrations of TNF-, and soluble TNF receptors (sTNF-RI and sTNF-RII) are increased in

cirrhotic patients (S.Y. Lin et al., 2004). Itoh et al (1999), proved that the serum levels of sTNFRs

increased in proportion to the severity of liver disease; and, that the levels of sTNFRs revealed significant

correlations with the serum levels of alanine aminotransferase and aspartate aminotransferase (Itoh et al,

1999).

It is presumed that resolution of hepatitis C, as evidenced by normalization of liver function tests and

disappearance of hepatitis C virus (HCV) RNA from serum reflects virus eradication (Pham et al., 2004).

In this study our aim is to evaluate the role of IL-1ra, and TNFR (P55) which are receptors related to

inflammatory response and, GH and Insulin which are hormones metabolized in the liver in HCV infection,

whether cirrhotic or non-cirrhotic. Another aim is to find their significance as non invasive biochemical

markers that may predict the outcome of interferon alpha 2a therapy in patients with chronic HCV infection

genotype 4.

Patients and methods:

This study was conducted on 12 healthy control persons and 54 chronic hepatitis patients with HCV of

genotype 4 who did not receive antiviral therapy for HCV before (naïve patients). They were collected

according to the following exclusion criteria: HBsAg positivity (by ELISA), diabetes, disturbed thyroid

function, Hb < 11g/dl, platelets count < 100.000/cumm, and WBCs < 1500/cumm, and their inclusion

criteria: Genotype 4, HCV-RNA +ve, ALT was more than double fold the upper limit of normal and

negative anti-ANA, anti-AMA, anti-thyroid globulin and anti LKM antibodiesd. Chronic hepatitis C

patients were subdivided into two groups:

Group A: 42 patients with chronic hepatitis but no evidences of cirrhosis (by liver biopsy), and who

were highly selected from a group of HCV patients receiving a schedule of treatment consisting of 180 µg

pegylated interferon / week (once weekly), and ribavirin 1200 mg/day for 1 year treatment.

Group B: 12 patients who had the same inclusion and exclusion criteria but with cirrhosis and

received 135 µg Pegylated INF α-2a once weekly & 1200 mg Ribavirin/ day in two doses .

All patients were subjected to thorough clinical examination and routine laboratory investigations

which include CBC, liver function tests (ALT, AST, serum bilirubin, serum albumin and prothrombin time

and concentration), fasting & post-prandial blood sugar and renal function tests. Abdominal

ultrasonography was done to all patients. HCV RNA was done by RT- PCR amplicor molecular system (F

Hoffmann - La roche Basel Switzerland) using patient serum. Genotyping was done by the Inno Lippa

HCV II assay (innogenetics inc., GA, USA). HBsAg was negative in all patients (done by ELISA). All

patients were Egyptian.

The study was done over a period of 30 months from March 2003. The initial response (IR) was

defined as the clearance of the virus or reduction of the viral load by two logs after treatment for 12 weeks.

The end of treatment response (ETR) was defined as clearance of the virus at 48 weeks of treatment.

Reassessment of ALT and HCV RNA were done 6 months after stopping therapy. Patients who had absent

HCV after these 6 months were defined to have sustained virological response SVR.

The dose of Pegylated INF α-2a was reduced to 135 µg in two patients from group A when the WBCs

count dropped below 1500/cmm and rose again in one of them after improvement of the count. The dose of

Ribavirin was reduced to 600 mg / day in 8 patients when the Hb level dropped below 10 mg/dl.

Strategy of treatment and timing of blood collection:

CBC and ALT were examined every two weeks. After 12 weeks serum from all patients was collected

again and HCV-RNA was re-assayed to diagnose the initial response to treatment. Patients who achieved

initial response continued treatment for 48 weeks to prevent relapse. Patients who failed to achieve IR

discontinued treatment and not included in the study.

Those patients who responded to pegylated IFN alfa as proved by negative PCR and normal ALT after

6 months from stopping the treatment were considered to have sustained viral response (SVR). Sera of

these patients obtained before the start (before treatment group) and 6 months after completion of the

treatment (SVR group) were assayed for our studied parameters.

I. Clinical examination:

- A detailed history and clinical examination was performed for all the patients with special emphasis

on the possible duration of the HCV infection, age and sex.

- Ultrasonography was done by an ultrasound machine Hitachi EUB 515 A , using a convex linear

transducer 3.5 MHz for complete evaluation and to exclude cirrhotic patients.

- Biopsy was done by a Hepafix needle 14 mm in diameter and the biopsy was done according to the

Menghini technique to confirm the none cirrhotic diagnosis. All biopsy specimens were immersed in 10%

formol and sent for histopathological analysis.

II. Laboratory tests:

Blood samples were collected under aseptic conditions before, after 12 weeks and after 30 months of

the combined treatment. Samples were divided into two groups: before treatment group and SVR group.

All subjects were fasting overnight before sampling. Serum was separated from the other contents and

stored at –70 C until assayed.

The following investigations had been done:-

- Liver function tests (Serum albumin, direct bilirubin, AST and ALT) were done using standard

laboratory methods.

- Fasting blood sugar was done using standard laboratory method to exclude diabetes.

- HCV-RNA by (PCR).

- ANA, AMA, Anti-thyroid globulin Ab, anti LKM by indirect immunofluorescence antibody test

(the Binding site LTD, Birmingham, England).

- Serum Insulin by ELISA using (Biosource Europe S.A.)

- Serum growth hormone level by enzyme immune assay method (IBL, Hamburg, Germany, and

quorum Diagnostics Inc., Vancover, British Colombia, Canada respictevely).

- Serum Interleukin I- receptor antagonist by ELISA (Biosource IL-ra Cytoscreen Kit, Europe S.A.

Rue de L' Industrie, 8 B-1400 Nivelles Belgium.)

- Serum TNF RI by EASIA using Biosource Kit

Statistical analysis:

Medians were compared using the median test, continuous variables, expressed as mean SD,

were compared by using student’s t-test or correlated by using simple regression done by Excell program.

Differences were considered significant if P< 0.01.

RESULTS:

This study was conducted on 54 HCV patients, their ages range (20 - 70) all were Egyptian; 41 males

and 13 females. Results were presented as mean ±SD. Table (1) showing the characteristics of the studied

group and the main positive findings of the studied parameters after the combined treatment of pegylated

interferon α 2a and ribavirin. P55 showed a significant decrease after treatment for group A and B (P <

0.001 and P> 0.05 respectively).

Table (1): shows characteristics of the 54 genotype 4 patients

Gender: Count %

Male 41 76.00%

Female 13 24.00%

Total 54 100.00%

Age: Total 54 100.00%

From 20 to 39 15 27.78%

From 40 to 54 34 62.96%

From 55 to 70 5 9.26%

Non- cirrhotic 42 77.7%

Cirrhotic 12 22.3%

Table 2: Clinical data of Chronic HCV patients:

Clinical Data Non-cirrhotic (n = 42) Cirrhotic (n = 12)

Presenting symptoms: Fatigue:

Dyspepsia

Upper abdominal pain

History of Jaundice

Bleeding tendency

Non (accidental)

31

23

19

8

5

5

9

7

8

6

5

0

Signs:

jaundice

Hepatomegaly

15

28

6

3

Splenomegaly

Oedema

Ascites

Foetor hepaticus/ palmar erythema

Spider Naevi/ flabbing tremors

13

0

0

0/0

0/0

7

3

0

0/3

2/0

Table 3: Endoscopic and ultrasonographic findings in patients with chronic HCV infection:

Clinical Data Non-cirrhotic (n = 42) Cirrhotic (n = 12)

Endoscopic findings:

Oesophageal varices:

1. Grade: 1/2

2. Grade: 3/4

Gastric varices

Congestive gastropathy

1. mild

2. severe

Peptic ulcer

Chronic gastritis

2/1

0/0

0

4

0

2

15

4/3

1/0

2

7

2

1

7

Ultrasonographic findings:

Liver size:

a. Average size

b. Mildly enlarged

c. Markedly enlarged

d. Shrunken

Echopattern:

Long axis of spleen

Collaterals:

Gall stones

23

11

7

1

diffuse

13+/- 1.65

3

5

3

4

0

5

coarse cirrhotic

16 +/- 3.27

7

4

Table 4: Liver function tests and viral load in HCV patients before and after treatment.

Clinical Data Non-cirrhotic Group A (n =

42)

Cirrhotic group B (n = 12)

ALT before/after treatment U/L 101.5 8.5 / 39.4 4.93 76.3 5.92 / 45.43 4.54

AST before/after treatment U/L 81 7.21 / 40.17 4.37 93 4.62 / 49.94 6.73

Bilirubin before/after treatment

(mg/dl)1.8 0.61 / 1.1 0.23 2.67 0.83 / 1.52 0.63

Serum albumin before/after treatment 3.9 0.23 / 4.05 0.31 3.1 0.41/3.31 0.47

Prothr. time before/after treatment 12.16 1.04/ 11.74 1.0113.54 1.48 / 12.62

1.36

HCV RNA load > 2 X106 copies/ml 11/7 3/2

HCV RNA load < 2 X106 copies/ml 31/9 9/6

Table 5: Histological findings in patients with chronic HCV infection:

Clinical Data Non-cirrhotic Group A (n = 42) Cirrhotic group B (n = 12)

Liver biopsy findings:

Activity:

1. Mild

2. Moderate

3. Severe

Grade:

1. Grade 1

2. Grade 2

3. Grade 3

4. Grade 4

5. Grade 5

(cirrhosis)

27

11

4

32

5

4

1

0

3

7

2

0

0

0

0

12

Patients with initial response (IR) in both cirrhotic and non Cirrhotic groups showed decreased end of

treatment (ETR) response that became 73.817% (31/42 patients) in non cirrhotic and 58.33% (7/12

patients) of the initial responders in cirrhotic. Sustained virological response (SVR) was 26/42 (61.91%)

and 4/12 (33.33%) in both groups. The biochemical response and the studied parameters were also initially

high then decreased in both groups. The difference was significant between both groups concerning

virological response and the biochemical response (P > 0.01).

Table 6: Virological response (disappearance of HCV RNA in serum)in both groups of chronic HCV:

Initial response (IR) End of treatment

Response (ETR)

Sustained Virological

response (SVR)

Group A 42/42 31/42 (73.817%) 26/42 (61.91%)

Group B 12/12 7/12 (58.33%) 4/12 (33.33%)

Table 7: showing the difference of the studied parameters at the two HCV patient groups (before and after

treatment) and the reference group.

HCV patients without cirrhosisGroup A

HCV patients with cirrhosis group B

Reference group

Before ttt After ttt Before ttt After ttt

P55mean ± SD

3.91±1.98** 1.85 ±0.55* 7.4 ±2.98** 6.53 ±2.79* 3.23 ± 0.28

range 1.4 - 9.5 1.3 - 3.9 2.9 - 11.2 2.8 - 11.2 2.9 - 3.7

IL -1 raMean± SD 193.4±42.07 214.7±44.04* 167.08±33.8 166.25±44.42* 133.5

range 145 - 256 145 - 276 135 - 215 115 - 225 71 - 184

GHMean±SD

7.56±1.33** 4.93±1.45* 23.15±10.97** 22.34±10.93 4.57±1.93

range 2.5 - 8.0 2.5 - 7 5.5 - 30.5 5.5 - 30.5 2.0 - 7.0

InsulinMean±SD

26.72±8.27** 48.88±16.07* 51.12±16.82** 53±19.62 14.49±5.51

Range 14.5 - 36.5 28 - 70 14.5 - 85 32 - 80 8.5 - 28

*Significant statistically compared to their level before.**Significant statistically compared to the reference group.

Table 8: Success Rate According to Degree of Severity:Cured Uncured

Count Row %Count Row % Count Row %

Non-cirrhotic 31 73% 11 27% 42 100.00%

Cirrhotic 4 33.3% 8 66.6% 12 100.00%

Total 35 64.8% 19 35.2% 54 100.00%

Table 9: showing the p value of the studied parameters in all groups including group A and B (before and after treatment) and the reference group:

HCV without cirrhosisGroup A

HCV with cirrhosis group B Reference group

Before ttt After ttt (31/42) Before ttt After ttt (4/12)

P55 P** > 0.01P**** >0.01

IL-1 ra P* >0.01

GH P** >0.01P**** >0.01

Insulin

P* compared to the reference group.P** compared to the non cirrhotic group, before treatmentP*** compared to the non cirrhotic group after treatmentP**** compared to the cirrhotic group before treatment

The two groups showed significantly higher P55 before ttt compared to the reference group p> 0.001 and a

significantly lower P55 in group A and B after treatment compared to their level before treatment and to the

cirrhotic group. There were higher levels of IL-ra of the two treatment groups before treatment compared to

the reference group but of no significant values and a significantly lower levels of IL-1ra in group A after

treatment compared to their level before treatment. There were higher levels of GH of the two treatment

groups before treatment compared to the reference group but of no significant values and a significant

decrease of GH at group A after treatment compared to their level before treatment and compared to the

cirrhotic group before treatment.

Table 10: showing the p value of the studied parameters at the two groups A and B (before treatment and after ) at SVR state and the reference group.:

HCV without cirrhosisBefore ttt

HCV without cirrhosisafter ttt R31/42

Cirrhosis Before ttt

cirrhosis after ttt

R 4/12

Reference group

P55 P**> 0.005P****>0.005

P**>0.01P****0.005

IL-! raP*>0.01 P*>0.01

GH P**>0.01P****>0.01

Insulin P**>0.01P****>0.01

P**>0.01P ****>0.01

P* compared to the reference groupP** compared to the non cirrhotic group, before tttP*** compared to the non cirrhotic group after tttP**** compared to the cirrhotic group Before ttt

The two groups at SVR show a significant increase of P55 before treatment compared to the reference

group p> 0.005and a significant decrease in P55 in group A and B after treatment compared to their level

before treatment and to the cirrhotic group.

Increase of IL-ra of the two groups at R before treatment compared to the reference group but of no

significant values and a significant decrease of IL-1ra at group A and B after treatment compared to their

level before treatment.

Increase of GH of the two groups at R before treatment compared to the reference group but of no

significant values and a significant decrease of GH at group A after treatment compared to their level

before treatment and compared to the cirrhotic group before treatment.

Discussion:

Serum ALT and HCV-RNA by PCR are the standard markers to assess liver disease and to monitor

response to therapy in patients with chronic HCV infection. Other factors as viral load, genotype and grade

of fibrosis are also, used to predict the treatment outcome of such patients (Libra et al., 2002). In 1995

Martinot et al., proved that viral genotype is a major predictor of SVR and patients with genotype 4 were

considered as “difficult to treat” by the standard interferon (Cotler et al., 2002)).

Also our study confirmed that modification of the pharmacokinetic profile of interferon-α (IFN-

α) through pegylation (addition of a polyethylene glycol molecule) with ribavirin for 48 weeks has

resulted in a marked improvement of drug efficacy in the treatment of infection due to hepatitis C virus in

Egyptian patients with genotype 4.

In this study we tried to estimate the role of IL-1ra, TNF-α R (P55) as a receptors related to

inflammatory response and, GH and Insuline as a hormones metabolized in the liver in HCV infection,

cirrhotic and non-cirrhotic. Also to find their significance as non invasive biochemical markers that may

correlate with HCV infection and predict the outcome of interferon-α 2a therapy in patients with chronic

HCV infection genotype 4.

In our study the growth hormone levels were high in the two studied groups before treatment

compared to the reference group while after treatment its level decreased only in group A responders and in

group A and B in SVR group. This was agreed by others who prove that cirrhosis is characterized by high

growth hormone (GH) levels (Riley and McCann, 1981 and S hanker et al., 1986). The cause of this high

(GH) level remains uncertain. It may be a decrease in its metabolic clearance rate or diminish in liver

growth hormone receptors (Shmueli et al., 1994). But in our study we proved that after treatment it

decreased significantly where p> 0.01 in SVR and in after treatment in those with no cirrhosis.

We also revealed a significant increase in serum insulin level in (SVR) group compared to (before

treatment) group P< 0.01 and compared to the reference group as all the selected patients were non diabetic

with normal serum glucose level and that insulin secretion is increased in non-diabetic patients with HCV

infection treated by peg-interferon2a with ribavirin and is associated with an amelioration of glucose

tolerance in non-diabetic HCV-infected patients. This was previously reported by others (Mc Hutchinson et

al., 1998)

Our study revealed a high level of IL-1ra in both groups before and after treatment and in SVR

group compared to the reference group but of low statistical value were p> 0.1. In 1999, Gramantieri et., al

proved that the increased level of IL-1Ra may contribute to the pathogenesis and the activity of chronic

active hepatitis C (Gramantieri et al., 1999). In 2002 Cotler et al proved that Serum IL-1Ra levels

increased rapidly in all patients with hepatitis C after IFN-alpha administration, irrespective of their

virologic response (Cotler et al., 2002). IL-1Ra levels remained elevated at 1 week but were similar to

baseline by week 2 of treatment in patients receiving continuous therapy. The increase in IL-1Ra indicates

that IFN receptors are functioning in patients with IFN-resistant hepatitis C and that the lack of response is

related to other virologic or immunologic factors (Gramantieri et al., 1999).

Our study revealed a significantly high level of P55 in the HCV patients before treatment

compared to the reference group which decreases significantly in the non cirrhotic group after treatment

and SVR group of both groups p> 0.01 and 0.005 respectively with positive correlation between serum

levels of sTNF-R p55 and the severity of infection. In 1999, Itoh et al proved that in the sustained

responder group, the levels of sTNF-R p55 showed a significant decrease (p < 0.0002.). He also proved that

the TNF alpha-R- mediated pathway, is involved in the hepatic inflammation-fibrosis process in chronic

hepatitis C.

In conclusion: pegylated INF-α combined with ribavirin therapy is the regimen of choice in treating

chronic HCV infection of genotype 4 especially in non cirrhotic patients for at least 48 weeks. The tested

parameters specially P55 can be used successfully as a liver function test denoting response of these

patients to this treatment. Thereby, P55 could be considered a sensitive, non-invasive and cheap parameter

than HCV RNA denoting regression of the viremia and hepatic inflammatory affection of the liver. It could

be also used in selecting the patients with a high percentage of showing sustained response.

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