The Effect of Stem Cell Transplantation Therapy for Post Viral Chronic Liver Cell Failure on Associated Type II Diabetes Mellitus: A Pilot Study.

BACKGROUND: It was observed that type II diabetes mellitus associated with chronic liver failure improved after stem cell transplantation. However, there were no adequate studies regarding this issue. The aim of this study was to evaluate the effect of stem cell transplantation on associated type II diabetes mellitus and on the liver function tests. METHODS: This pilot study included 30 patients of post-hepatitis chronic liver failure who were classified into two groups: Group I included patients with chronic liver cell failure associated with type 2 diabetes. Group II included patients without type II diabetes. Autologous CD34+ and CD133+ stem cells were percutaneously infused into the portal vein. Responders (regarding the improvement of diabetes as well as improvement of liver condition) and non-responders were determined. Patients were followed up for one, three and six months after the intervention evaluating their three-hour glucose tolerance test, C- peptide (Fasting and postprandial), Child-Pugh score and performance score one month, three months, and six months after stem cell therapy. RESULTS: Both synthetic and excretory functions of the liver were improved in 10 patients (66.66 %) of group I and in 12 patients (80 %) of group II. Significant improvement in the Oral Glucose Tolerance Test in the responders of both the groups was well defined from the 3rd month and this was comparable to changes in liver function tests and Child-Pugh score. CONCLUSION: Successful stem cell therapy in chronic liver cell failure patients can improve but not cure the associating type 2 diabetes by improving insulin resistance.

Role of relevant immune-modulators and cytokines in hepatocellular carcinoma and premalignant hepatic lesions.

AIM: To assess the levels of different immune modulators in patients with hepatocellular carcinoma (HCC), in relation to other hepatic diseases. METHODS: Eighty-eight patients were included in the current study and represented patients with HCC (20), liver cirrhosis (28) and chronic hepatitis (CH; 25), and normal controls (NC; 15). Peripheral blood was isolated for immunophenotyping of active myeloid dendritic cells (mDCs; CD1c and CD40), mature inactive myeloid cells (CD1c and HLA), active plasmacytoid cells (pDCs; CD303 and CD40), mature inactive pDCs (CD30 and HLA), active natural killer (NK) cells (CD56 and CD161), active NK cells (CD56 and CD314) and inactive NK cells (CD56 and CD158) was done by flow cytometry. Serum levels of interleukin (IL)-2, IL-10, IL-12, IL-1ß, interferon (IFN)-α, IFN-γ and tumor necrosis factor (TNF)-αR2 were assessed by ELISA. RESULTS: Active mDCs (CD1C+/CD40+) and inactive mDCs (CD1c+/HLA+) were significantly decreased in HCC patients in relation to NC (P < 0.001). CD40+ expression on active pDCs was decreased in HCC patients (P < 0.001), and its level was not significantly changed among other groups. Inactive pDCs (CD303+/HLA+), inactive NKs (CD56+/CD158+) and active NKs (CD56+/CD161+) were not statistically changed among the four groups studied; however, the latter was increased in CH (P < 0.05). NKG2D was statistically decreased in HCC, CH and cirrhosis (P < 0.001), and it was not expressed in 63% (12/20) of HCC patients. There was significant decrease of IL-2, IFN-α and IFN-γ (P < 0.001), and a significant increase in IL-10, IL-1ß, and TNF-αR2 (P <0.01, P < 0.001 and P < 0.001; respectively) in HCC patients. There was inverted correlation between IL-12 and IL-1ß in HCC (r = -0.565, P < 0.01), with a strong correlation between pDCs (CD303+/CD40+) and NKs (CD56+/CD161+; r = 0.512, P < 0.05) as well as inactive mDCs (CD1c+/HLA+) and inactive NK cells (CD56+/CD158+; r = 0.945, P < 0.001). CONCLUSION: NKG2D, CD40, IL-2 and IL-10 are important modulators in the development and progression of HCC.

Potential Diagnostic and Prognostic Value of Lymphocytic Mitochondrial DNA Deletion in Relation to Folic Acid Status in HCV-Related Hepatocellular Carcinoma

Objective: We assessed the possibility of using mitochondrial (mt) DNA deletion as a molecular biomarker for disease progression in HCV-related hepatocellular carcinoma (HCC) and to identify its association with folic acid status. Methods: Serum folic acid and lymphocytic mtDNA deletions were assessed in 90 patients; 50 with HCC, 20 with liver cirrhosis (LC), and 20 with chronic hepatitis C (CHC) compared to 10 healthy control subjects. The diagnostic accuracy of mtDNA deletions frequency was evaluated using receiver-operating characteristic (ROC) curve analysis Survival analysis was performed using the Kaplan-Meier method. Differences in the survival rates were compared using log-rank test. Result: Our data revealed a significant elevation of mtDNA deletions frequency in the HCC group compared to the other groups (P-value <0.01). Also, our data showed a significant correlation between folate deficiency and high frequency of mtDNA deletions in patients with HCV-related HCC when compared to the other groups (r= -0.094 and P-value <0.05). Moreover, the size of the hepatic focal lesion in the HCC patients was positively correlated with mtDNA deletions (r= 0.09 and P-value <0.01). The median survival time for the HCC patients with high frequency of mtDNA deletions (ΔCt ≥3.9; 5.7+ 0.6 months) was significantly shorter than those with low mtDNA deletions frequency (ΔCt < 3.9; 11.9+ 0.04 months, P-value <0.01). Conclusion: Our data provided an evidence that lymphocytic mtDNA deletion could be used as non-invasive biomarker for disease progression and patients' survival in HCV-related HCC. Also, our findings implied a causal relationship between the folate deficiency and the high mtDNA deletions frequency among Egyptian patients with HCV related HCC.

Arabinoxylan rice bran (Biobran) suppresses the viremia level in patients with chronic HCV infection: A randomized trial.

Current treatments for Hepatitis C virus (HCV) have severe side effects and are very expensive. There is a need to explore effective natural therapies against HCV that are less toxic and more cost-effective. In the current study, 37 chronic HCV patients were randomized into two groups and treated with either pegylated interferon (PEG IFN) plus ribavirin (n = 21) or Biobran, an arabinoxylan from rice bran (1 g/day) (n = 16). We examined viremia, liver enzymes, interferon-γ (IFN-γ) levels in serum, and toxicity before and three months after treatment. Both groups showed a significant and similar reduction in viral load after three months of treatment relative to the baseline viral load (P <0.05). In addition, treatment with Biobran resulted in a significant increase in the level of IFN-γ (P <0.001). Patients in the PEG IFN plus ribavirin group showed fever, anemia, thrombocytopenia, and easy fatigue. Patients in the Biobran group showed no side effects and reported good health. We conclude that Biobran is a potential novel therapeutic regimen that has a similar effect to PEG IFN plus ribavirin and is safe and cost-effective in the treatment of chronic HCV. Our finding of Biobran's efficacy against HCV infection warrants further investigation in multiple clinical trials (Clinical Trials Registration: NCT02690103).

Three Gene Signature for Predicting the Development of Hepatocellular Carcinoma in Chronically Infected Hepatitis C Virus Patients.

Hepatitis C virus (HCV) is the leading cause of liver fibrosis and hepatocellular carcinoma (HCC). At present, there is no predictive biomarker for the patients at high risk of developing HCC. In this study, we examined the association between single-nucleotide polymorphisms (SNPs) in 3 innate immunity genes [2'-5'oligoadenylate synthetase 1 (OAS1) rs10774671, interleukin 28B (IL28B) rs12979860, and low molecular mass polypeptide 7 (LMP-7) at codon 49] besides cytomegalovirus (CMV) coinfection and susceptibility to HCC in genotype 4 (GT4) chronically infected Egyptian patients. SNPs were determined using restriction fragment length polymorphism analysis in DNA from HCC patients (n = 34) and compared with either controls (n = 70) or patients with early grades of liver fibrosis (n = 49). Our results demonstrated that patients bearing the genetic combination consisting of LMP-7 CA/AA [OR 4.75, 95% confidence interval (CI) 1.443-15.631, P = 0.007] and IL28B rs12979860 CT/TT (OR 6.00, 95% CI 1.603-22.455, P = 0.004) and positive for CMV viremia (OR 3.11, 95% CI 1.151-8.412, P = 0.02) were more likely to have HCC. However, OAS1 rs10774671 does not seem to contribute to the development of HCC. Binary regression analysis indicated that HCC risk significantly increases with the presence of each unfavorable genotype (LMP-7 CA/AA, IL28B rs12979860 CT/TT), when accompanied by the existence of CMV coinfection (probability of HCC risk is 0.8 for combined factors versus 0.14, 0.07, and 0.07 for individual factor IL28B, LMP-7, and CMV; respectively). These data suggest that the 2 SNPs and the coinfection in concert have potential in predicting the risk of HCC development in patients infected with HCV GT4.

The impact of repeated autologous infusion of haematopoietic stem cells in patients with liver insufficiency.

INTRODUCTION: The worldwide shortage of donor livers has prompted the search for alternative cell therapies. Previous data from our laboratory proved a supportive role for stem cell therapy in the treatment of end-stage liver disease patients. Therefore; this study was conducted to assess the clinical and biochemical effects of repeated stem cell infusion. METHODS: Ninety patients with liver cirrhosis were randomized to receive either one session treatment (G-I) or two sessions 4 months apart (G-II) of autologous haematopoietic stem cells (HSCs) transplantation and a control group (G-III) who received regular liver treatment. G-CSF was administered to transplanted patients before infusion; HSCs were isolated from 400 cc bone marrow (BM) aspirate. CD34+/CD133+ cells were purified: 50 % of the cells were infused locally in the portal vein on the same day and the other 50 % were differentiated to MSC and infused systemically in a peripheral vein (one session treatment G-I). In G-II, the same process was repeated after 4 months from the first treatment (two session's treatment G-II). Liver function was monitored for 12 months after stem cell therapy (SCT). RESULTS: Statistically significant improvement was reported in the transplanted patients (G-1) as regards the mean serum albumin, bilirubin and INR levels which started to improve after 2 weeks of treatment and continued to improve till the 6(th) month in the single infusion group. The two sessions infused group (G-II) showed sustained response which continued throughout the all follow-up period (12 month). By the end of the study, 36.7 % of the patients in G-I and 66.7 % in G-II showed improvement in the degree of ascites compared to the control group (G-III). We also reported an improvement in the hepatic functional reserve as assessed by the Child-Pugh and MELD score. Safety of the procedure was evidenced by the low incidence of complications encountered. CONCLUSION: In patients with end-stage liver disease, the repeated infusion with combined routes portal and peripheral veins has a beneficial effect on liver functions with minimal adverse events and more lasting clinical efficacy after repeated HSCs infusion.

Serum Soluble CD14 in Egyptian Patients with Chronic Hepatitis C: Its Relationship to Disease Progression and Response to Treatment.

Hepatitis C virus (HCV) is a major public health problem. Soluble CD14 (sCD14) level was shown to be associated with HCV infection. In this study, we aimed to investigate the relationship between sCD14 concentration and disease progression, as well as the response to pegylated interferon/ribavirin (peg-IFN/RBV) therapy in Egyptian patients with chronic hepatitis C (CHC). The ELISA technique was used to test 80 patients with CHC and 20 healthy control persons for serum levels of sCD14 (pretreatment and after 12 weeks of treatment). CHC patients were 65 males and 15 females. Normal healthy controls included 20 age- and sex-matched volunteers. The mean age of the CHC patients was 39.94 years, while that of the controls was 39.2 years The serum sCD14 level was significantly higher in chronic HCV-infected patients (3.6±0.18 µg/mL) compared to healthy control subjects (3.1±0.18 µg/mL). The serum sCD14 level was significantly directly correlated with the hepatic fibrosis score (r=0.24, P=0.03), histological activity index (r=0.26, P=0.02), and serum aminotransferases [r=0.28, P=0.005 for alanine aminotransferase (ALT) and r=0.30, P=0.003 for aspartate aminotransferase (AST)]. The pretreatment sCD14 level was not significantly correlated to the treatment response, but it increased after 12 weeks of peg-IFN/RBV therapy and values were significantly higher in nonresponders (P=0.02). The pretreatment sCD14 level cannot predict the treatment response in chronic HCV patients receiving peg-IFN/RBV therapy. However, the serum sCD14 level after 12 weeks of treatment can serve as a negative predictor of treatment response.

Ambovex(®) as a novel immunological modulator drug for the treatment of hepatocellular carcinoma (HCC) in the liver: a Phase II clinical trial.

Hepatocellular carcinoma (HCC) is a global public health problem, based on it being the fifth most common cancer and third leading cause of cancer-related mortality worldwide. The approved conventional treatment methods for HCC have shown life-threatening side effects with limited or negligible success, especially in multifocal HCC. As a consequence, new therapeutic approaches are being explored, including immunoregulatory molecules that may have the potential to treat or delay the progression of HCC. A novel pharmaceutical botanical drug - Ambovex(®), an immune-modulator molecule - was tested to treat or delay the progress of HCC. We conducted a 6-month randomized clinical trial with an additional 3-month washing period (no treatment) to evaluate the safety and efficacy of low-dose Ambovex oral spray in treating patients with HCC. The clinical study involved a total of 40 patients, with 33 in the treatment group and seven in the control group. The α-fetoprotein (AFP) levels were measured every month and ultrasound scans were performed at time zero and every 2 months thereafter. Computed tomography (CT) scans were performed for patients in the treatment group. Ambovex proved to be safe, as there were no significant side effects although some patients found that the drug has unpleasant taste. AFP analysis showed a significant decrease in its level (α=0.05; 95% confidence interval) in the treatment group when compared to the control group at 3 months (P=0.0031) and at 6 months (P=0.007). The ultrasound results showed improvement in the treated group, as evidenced by a significant decrease in the lesion numbers and sizes. The lesions in 38% of treated patients decreased from multiple to single with major improvements; 35% of patients exhibited a decrease from multiple lesions to multiple lesions with minor improvements, whereas 27% had stabilized lesions. CT scans in the treated group showed significant improvement, as there was complete disappearance of the lesions after 6 months of treatment with Ambovex in two patients. This clinical study showed the effective and promising results of Ambovex as an immunological modulator in treating HCC. Further exploration of Ambovex is recommended.

IL28B rs12979860 gene polymorphism in Egyptian patients with chronic liver disease infected with HCV.

BACKGROUND: Egypt has one of the highest prevalences of hepatitis C virus (HCV) infection worldwide. Although the IL28B gene polymorphism has been shown to modify the course of chronic HCV infection, this has not been properly assessed in the Egyptian population. MATERIALS AND METHODS: The IL28B rs12979860 single nucleotide polymorphism (SNP) was therefore examined in 256 HCV-infected Egyptian patients (group II) at different stages of disease progression and in 48 healthy volunteers (group I). Group II was subdivided into GII-A (chronic hepatitis patients, n=119), GII-B (post hepatitis cirrhosis, n=66) and GII-C (HCC on top of cirrhosis, n=71). RESULTS: The C/T genotype was the commonest in all groups. It was more frequent in GI (52%) than in GII (48%). There was no significant difference in the frequency of C/T and C/C or T/T genotypes between groups and subgroups (p=0.82). Within the subgroups; the C/C genotype was more common in GII-B while C/T and T/T genotypes were more common in GII-C, though with no significant difference (p=0.59 and p=0.80). There was no significant association between IL28B rs12979860 SNP and viral load, ALT, AFP level, METAVIR scores for necro-inflammation and fibrosis, and Child-Pugh classification. CONCLUSIONS: 1) IL28Brs12979860 C/T genotype is the commonest genotype in HCV-associated CH and HCC in Egypt. 2) IL28Brs12979860 polymorphisms are not associated with disease progression or aggression (histological staging, severity of fibrosis in CH or the incidence of post-HCV HCC). 3) Differences in IL28Brs12979860 genotypes could be a consequence of environmental or ethnic variation.

Peripheral vein infusion of autologous mesenchymal stem cells in Egyptian HCV-positive patients with end-stage liver disease.

INTRODUCTION: We have assessed the utility of autologous mesenchymal stem cell (MSC) peripheral vein infusion as a possible therapeutic modality for patients with end-stage liver diseases. METHODS: Forty patients with post-hepatitis C virus (HCV) end-stage liver disease were randomized into two groups: Group 1 (GI): 20 patients who received granulocyte colony-stimulating factor (G-CSF) for 5 days followed by autologous MSCs peripheral-vein infusion and group 2 (GII): 20 patients who received regular liver-supportive treatment only (control group). RESULTS: In MSC-infused patients (GI), 54% showed near normalization of liver enzymes and improvement in liver synthetic function. Significant changes were reported in albumin (P = 0.000), bilirubin (P = 0.002), increased international normalized ratio (INR) (P = 0.017), prothrombin concentration (P = 0.029) and alanine transaminase (ALT) levels (P = 0.029), with stabilization of clinical and biochemical status in 13% of cases. None of the patients in GII showed any significant improvement. Hepatic fibrosis was assessed in GI by detection of procollagen IIIC peptide level (PIIICP) and procollagen III N peptide level (PIIINP). The pretreatment values of s-PIIICP and s-PIIINP were 9.4 ± 4.2 and 440 ± 189, respectively, with a decrease to 8.1 ± 2.6 and 388 ± 102, respectively, 3 months after MSC therapy. However, the difference was statistically nonsignificant (P = 0.7). A significant correlation coefficient was reported after 3 months between the s-PIIINP and prothrombin concentration (P = -0.5) and between s-PIIICP and ascites (P = 0.550). CONCLUSIONS: First, autologous MSC infusion into a peripheral vein is as effective as the previously reported intrahepatic infusion. Second, MSCs have a supportive role in the treatment of end-stage liver disease, with satisfactory tolerability and beneficial effects on liver synthetic functions and hepatic fibrosis. Third, IV infusion of MSCs after G-CSF mobilization improves s-albumin within the first 2 weeks and prothrombin concentration and alanine Taransaminase after 1 month. According to the data from this current study and those previously reported by our group, we recommend further studies on patients' infusion with pure CD133 and CD34 followed by IV infusion of in vitro-differentiated MSCs within 1 week and another infusion after 3 months. TRIAL REGISTRATION: ClinicalTrials.gov NCT01729221. Registered 17 November 2012.

Assessment of health-related quality of life in patients receiving stem cell therapy for end-stage liver disease: an Egyptian study.

INTRODUCTION: This prospective cohort study aimed to assess the influence of stem cell therapy (SCT) on health-related quality of life (HRQOL) by using the SF-36 v2 and to elucidate the influence of objective clinical variables on subjective HRQOL. METHODS: The study included 100 chronic liver disease patients (50 received SCT, and 50 received supportive medical treatment (SMT)). Both groups completed a modified SF-36 v2 form before therapy and at 1-, 3-, 6-, and 12-month intervals. Fifty healthy Egyptian volunteers were enrolled in the study and completed the SF-36 v2 form once. RESULTS: Both SCT and SMT groups showed significantly lower pretherapy SF 36 v2 scores compared with healthy volunteers. In SCT-treated patients, limited complications were encountered (SF-36 v2 scores showed significant improvement in all domains throughout the follow-up period) compared with the deterioration shown by SMT patients after therapy. A significant association was detected between SF-36 v2 scores and laboratory data in SCT patients during the first month after therapy. The grade of ascites improved during the follow-up in SCT compared with SMT patients. The mean survival time was 277.56 days (95% CI, 246.217 to 308.903) for SMT and 359.300 days (95% CI, 353.022 to 365.578) for SCT patients (log rank, 0.00). Stem cell-treated patients showed no malignancies. CONCLUSIONS: SCT positively affects health-related quality of life in cirrhosis patients. The survival rate was significantly improved after SCT.

Autologous CD34+ and CD133+ stem cells transplantation in patients with end stage liver disease.

AIM: To assess the utility of an autologous CD34(+) and CD133(+) stem cells infusion as a possible therapeutic modality in patients with end-stage liver diseases. METHODS: One hundred and forty patients with end-stage liver diseases were randomized into two groups. Group 1, comprising 90 patients, received granulocyte colony stimulating factor for five days followed by autologous CD34(+) and CD133(+) stem cell infusion in the portal vein. Group 2, comprising 50 patients, received regular liver treatment only and served as a control group. RESULTS: Near normalization of liver enzymes and improvement in synthetic function were observed in 54.5% of the group 1 patients; 13.6% of the patients showed stable states in the infused group. None of the patients in the control group showed improvement. No adverse effects were noted. CONCLUSION: Our data showed that a CD34(+) and CD133(+) stem cells infusion can be used as supportive treatment for end-stage liver disease with satisfactory tolerability.

Detection of aberrant p16INK4A methylation in sera of patients with HCV-related liver diseases: An Egyptian study.

BACKGROUND: The present study was performed to estimate the frequency of methylated p16INK4A in the sera of patients with hepatitis C virus (HCV)-related chronic active hepatitis (CAH), liver cirrhosis (LC), and hepatocellular carcinoma (HCC) and to evaluate the role of p16INK4A as a tumor marker of HCC. MATERIAL/METHODS: The sera of 17 CAH, 20 LC, and 25 HCC patients were examined in this study. The methylation status of p16INK4A was evaluated by methylation-specific PCR of the serum samples. RESULTS: Methylated p16INK4A was detected in 47.1% (8/17) of the CAH patients, 5% (4/20) of the LC patients, and in 92% (23/25) of the HCC patients. HBV markers were detected in (4/25) of HCC patients; all had methylated p16INK4A. No association was demonstrated between p16INK4A methylation and serum AFP level in the HCC group. CONCLUSIONS: The results of this study indicate that aberrant DNA methylation contributes to hepatocarcinogenesis and it may be an early event during hepatocarcinogenesis. As the status of p16INK4A methylation was not associated with serum AFP level, it may have a complementary role with AFP as a tumor marker of HCC.

Autologous hematopoietic stem cell transplantation in 48 patients with end-stage chronic liver diseases.

The only presently viable treatment for end-stage liver disease is whole organ transplantation. However, there are insufficient livers available. The aim of the present study is to provide autologous bone marrow-derived stem cells as a potential therapeutic for patients with end-stage cirrhosis. This is a retrospective chart review of autologous stem cell treatment in 48 patients, 36 with chronic end-stage hepatitis C-induced liver disease and 12 with end-stage autoimmune liver disease. For all patients, granulocyte colony-stimulating factor was administered to mobilize their hematopoietic stem cells. Following leukapheresis, CD34(+) stem cells were isolated, amplified, and partially differentiated in culture, then reinjected into each subject via their hepatic artery or portal vein. Treatment was generally well tolerated with the expected moderate but transient bone pain from G-CSF in less than half of the patients. Three patients had serious treatment-related complications, and only 20.8% of these end-stage liver disease patients died during 12 months of follow up. For all patients there was a statistically significant decrease in ascites. There was clinical and biochemical improvement in a large percentage of patients who received the transplantation. In the viral group, there were marked changes in albumin (p = 0.0003), bilirubin (p = 0.04), INR (p = 0.0003), and ALT levels (p = 0.02). In the autoimmune group, values also improved significantly for albumin (p = 0.001), bilirubin (p = 0.002), INR (p = .0005), and ALT levels (p = 0.003). These results suggest that autologous CD34(+) stem cell transplantation may be safely administered and appears to offer some therapeutic benefit to patients with both viral and autoimmune-induced end-stage liver disease.

Consensus siRNA for inhibition of HCV genotype-4 replication.

BACKGROUND: HCV is circulating as a heterogeneous group of quasispecies. It has been addressed that siRNA can inhibit HCV replication in-vitro using HCV clone and/or replicon which have only one genotype. The current study was conducted to assess whether siRNA can inhibit different HCV genotypes with many quasispecies and to assess whether consensus siRNA have the same effect as regular siRNA. METHODS: We generated two chemically synthesized consensus siRNAs (Z3 and Z5) which cover most known HCV genotype sequences and quasispecies using Ambium system. Highly positive HCV patient's serum with nine quasispecies was transfected in-vitro to Huh-7 cell line which supports HCV genotype-4 replication. siRNA (Z3&Z5) were transfected according to Qiagen Porta-lipid technique and subsequently cultured for eight days. HCV replication was monitored by RT-PCR for detection of plus and minus strands. Real-time PCR was used for quantification of HCV, whereas detection of the viral core protein was performed by western blot. RESULTS: HCV RNA levels decreased 18-fold (P = 0.001) and 25-fold (P = 0.0005) in cells transfected with Z3 and Z5, respectively, on Day 2 post transfection and continued for Day 3 by Z3 and Day 7 by Z5. Reduction of core protein expression was reported at Day 2 post Z3 siRNA transfection and at Day 1 post Z5 siRNA, which was persistent for Day 4 for the former and for Day 6 for the latter. CONCLUSION: Consensus siRNA could be used as a new molecular target therapy to effectively inhibit HCV replication in the presence of more than one HCV quasispecies.

Non-invasive fibrosis seromarkers as a predictor of liver fibrosis in chronic hepatitis C and/or non-alcoholic steatohepatitis.

BACKGROUND AND STUDY AIMS: In patients with chronic hepatitis C, the precise stage of hepatic fibrosis is the most important predictor of disease progression and it determines the need for antiviral therapy. Although liver biopsy is acknowledged as the gold standard for evaluating fibrosis, it is occasionally prone to sampling error and complications. We aimed to correlate an index of biochemical markers with histological features of fibrosis to predict hepatic fibrosis in patients with chronic hepatitis C virus, patients with combined hepatitis C virus and non-alcoholic steatohepatitis and those with non-alcoholic steatohepatitis, aiming to reduce the use of the liver biopsy. PATIENTS AND METHODS: Out of those attending our out patient clinic for clinical, haematological, biochemical, virological, histological and ultrasonographic assessment prior to interferon therapy for hepatitis C virus, we enrolled 41 patients and grouped them according to histopathological examination of their liver biopsies into: Group I: 21 chronic hepatitis C virus patients as defined by positive 3rd generation ELISA; Group II: 20 patients with combined hepatitis C virus and NASH. We added a third group (Group III) of 15 patients having non alcoholic steatohepatitis as defined clinically, biochemically and through diagnostic percutanous liver biopsy. There were 33 male 23 female patients; 35 (62.5%) of them were from rural areas and 21 (37.5%) were from urban areas; the mean ages were 40.5±9, 46.6±7.7 and 42.13±11.06 in Group I, II and III respectively. Twenty apparently healthy individuals served as the control group. All the patients and the control group were submitted to full clinical history and examination, abdominal ultrasonography, CBC, liver biochemical profile and fibrosis biomarkers (apolipoprotein A1, haptoglobin, α2 marcoglobulin, GGT). Liver biopsy was done for suitable patients after taking a consent and the results of fibrosis seromarkers were compared with the results of liver biopsy using the Metavir scoring system. We also estimated patients' body mass index, fasting and post prandial blood glucose. We excluded patients with other causes of chronic liver disease and co-morbidities that could confound the results of the non-invasive markers adopted, including schistosomiasis which was excluded by serological test. RESULTS: 43% of Group I and 40% of Group II had advanced fibrosis. None of Group III had advanced fibrosis; mild fibrosis was detected in 80% of them. γ-GT was found positively correlated to the degree of hepatic fibrosis in Groups I, II and III (r=0.667, 0.656 and 0.121, respectively) with P values of 0.001, 0.002, 0.668, respectively. α2 macroglobulin was found to be a reliable predictor of fibrosis (r=0.30, P=0.02) with ROC curve (area under the curve=0.70) best cutoff value 2.55g/L with sensitivity of 0.80 and specificity of 0.50. The results of haptoglobin were negatively related to the degree of hepatic fibrosis in Group I and II with ROC curve area under the curve of 0.33 and P value of 0.04. Significant direct correlation was seen in Group III (r=0.55, P=0.03), so by regression analysis, haptoglobin can be used as a good predictor for fibrosis in Group III (r=0.54, P=0.04). Apolipoprotein A1 has negative correlation to the stage of fibrosis in Groups I and II although the results were statistically insignificant. APRI index was found significantly directly correlated to the fibrosis stage and the grade of inflammation of all studied groups (r=0.57, P<0.01 and r=0.36, P<0.01, respectively) with a best cutoff value of 0.62, with sensitivity of 0.86 and specificity of 0.57. In patients with advanced fibrosis the best cutoff value was found to be 0.72 with sensitivity of 0.94 and specificity of 0.67. Modified APRI test showed AUC of 0.79 (P<0.01) with a best cutoff value of 0.067 at which sensitivity and specificity were 0.82 and 0.61, respectively. CONCLUSION: α2 macroglobulin, haptoglobin, apolipoprotein A1, APRI index and a modified APRI index, were found to be significant predictors of hepatic fibrosis and were reprocessed by stepwise logistic regression.

Interferon-alpha gene therapy prevents aflatoxin and carbon tetrachloride promoted hepatic carcinogenesis in rats.

Retrovirus-mediated interferon alpha (IFN-alpha) gene transfer was evaluated with regard to its possible protective effects against aflatoxin B1 (AFB1)-initiated and carbon tetrachloride (CCl4)-promoted hepatic carcinogenesis in rats. To our knowledge, this is the first time an experimental in vivo gene therapy trial was conducted in Egypt. Two genes were examined in liver tissue by RT-PCR: the first was glutathione-S-transferase placental (GST-P) isoenzyme, as an early marker to detect hepatic malignancy; the second was IFN-alpha gene expression to detect the efficiency of gene uptake and its persistence after transduction. Forty male rats, divided equally into 4 groups, were included in the study: the first group was the control; the second group received CCl4 0.2 ml subcutaneously twice weekly for 12 weeks and AFB1 0.25 mg/kg body wt intraperitoneally twice weekly for 6 weeks; the third group received IFN-alpha (10(8) pfu) intravenously in the tail vein prior to the start of CCl4 and AFB1 injections; and the fourth group received IFN-alpha (10(8) pfu) by intrahepatic injection under ultrasonography guide after termination of the CCl4 and AFB1 injection schedule. The results showed that IFN-alpha has a marked and significant protective effect against hepatic fibrogenesis as well as hepatic carcinogenesis. Pathological examination of liver tissue proved that IFN-alpha minimized both fibrotic and cirrhotic processes. The amount of fibrosis was less in both groups receiving IFN-alpha, with more protection in the group that received IFN-alpha intravenously prior to CCl4 and AFB1. The results of RT-PCR showed that the IFN-alpha gene was significantly expressed in both groups receiving IFN-alpha, with a more intense expression in the group that received IFN-alpha by intrahepatic injection after termination of CCl4 and AFB1 injections. The IFN-alpha gene was detected after three months of gene transduction in rats receiving IFN-alpha intravenously prior to CCl4 and AFB1 and after one month of gene transduction in the post CCl4 and AFB1 rats. IFN-alpha gene was not expressed in the two groups that did not undergo gene transfer. Histopathological signs of premalignant macronodules were evident in the group receiving CCl4 and AFB1, but not IFN-alpha as well as in the group that received IFN-alpha at the end of the experiment. GST-P gene expression was also detected in these two groups, confirming early malignant transformation. In conclusion, IFN-alpha exerts significant protective effects, but more so when the gene is administered before fibrogenic and carcinogenic induction in hepatic tissues. IFN-alpha gene therapy may be justified in clinical trials for high-risk candidates with hepatic carcinogenesis.

Clinical trial of E1B-deleted adenovirus (dl1520) gene therapy for hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide. The prognosis of HCC is poor and current therapies are largely ineffective. Genetic abnormalities are commonly seen in HCC tumors particularly with inactivation of the p53 tumor suppressor. Gene therapy with E1B-deleted (dl1520) adenovirus could be of therapeutic value as it offers the potential of tumor growth control in patients with p53 mutation. Ten patients with posthepatitis cirrhosis and histologically proven HCC were enrolled into an open label, randomized prospective study. Randomization was to receive either percutaneous ethanol injection (control group) or dl1520. Toxicity and complications in the ethanol group were pain and fever, whereas in the gene therapy group complications were minimal. Grade I-II toxicity fever, stable performance status, and no significant rise in liver enzymes were observed in patients treated with dl1520. Analysis of patients' response to treatment in the gene therapy group showed one patient with a partial response and four patients with progressive disease. In the ethanol-treated group two patients had stable disease and three patients showed disease progression. In conclusion, this study showed that the adenovirus was well tolerated, but did not seem to offer significant tumor control. Although only a small number of patients were treated here it appears that more effective vectors are needed to achieve a useful clinical impact.