Abdelfattah M. Attallah

Immunodetection of Collagen Types I, II, III, and IV for Differentiation of Liver Fibrosis Stages in Patients with Chronic HCV

Abstract The current study is aimed at evaluating serum collagens and other serum biochemical markers as useful, non‐invasive markers of hepatic fibrosis associated with chronic hepatitis C virus (HCV). Collagen types I, II, III, and IV were detected in serum using ELISA and Western blot techniques. The ELISA levels of collagen I, II, III, and IV increased significantly with the progression of fibrosis staging. Based on receiver‐operating characteristic (ROC) curve analysis, the collagen type III (70 kDa) and type IV (200 kDa) were more useful than other serum bio‐markers for differentiating severe fibrosis from mild fibrosis. Multivariate discriminant analysis (MDA) selected a fibrosis discriminant score (FDS)=[2.345+Collagen III (µg/mL) × 1.923+Collagen IV (µg/mL) × 1.544+ALT (U/mL) × 0.005] ‐ [albumin(g/L) × 0.046]. The FDS correctly classified 87% of the severe fibrosis patients at a cut‐off score=2.2 (i.e., more than 2.2 indicated severe fibrotic liver and less than 2.2 indicated mild fibrotic liver) with specificity of 97%. In a validation study, the FDS was applied to the second cohort of patients and the results were reproduced without significant difference. In conclusion, the developed four‐parameter based FDS is useful for identifying severe liver fibrosis in patients with chronic HCV infection.

Evaluation of serum procollagen aminoterminal propeptide III, laminin, and hydroxyproline as predictors of severe fibrosis in patients with chronic hepatitis C.

Abstract In an attempt to identify biochemical analytes that could enhance the discrimination between the patients with severe liver fibrosis (F3‐F4) and mild fibrosis (F1‐F2) based on absolute values of biochemical markers, we measured 12 analytes, including procollagen III aminoterminal propeptide (PIIINP), laminin, proline, hydroxylproline, glycine, AST, ALT, alkaline phosphatase, albumin, total bilirubin, total protein, and prothrombin time in 252 individuals with chronic hepatitis C infection (CHC). PIIINP and laminin were determined by radio‐immunoassay; the degraded amino acids were determined using high performance liquid chromatography. Statistical analyses were performed by logistic regression, and receiver operating characteristic (ROC) curves. The best linear combination of blood markers was selected by multivariate discriminant analysis (MDA) for construction of the fibrosis discriminant score (FDS). FDS, an index of five markers (PIIINP, laminin, hydroxyproline, prothrombin activity, and AST/ALT) correctly classified 82% of the patients with severe liver fibrosis at a discriminant cut‐off score=−0.5 (i.e., less than −0.5 indicated severe liver fibrosis and greater than −0.5 indicated mild liver fibrosis with sensitivity (76%) and specificity (89%). This result was reproduced in a validation study with no significant difference. In conclusion, FDS is useful for identifying severe liver fibrosis in patients with CHC.

Detection of serum p53 protein in patients with different gastrointestinal cancers.

Overexpression of p53 has been found in many types of human malignancy. The present study aimed to detect preoperative serum p53 among 158 patients with different gastrointestinal cancers using ELISA technique based on mouse anti-p53 DO-7 monoclonal antibody and anti-p53 rabbit polyclonal antibody. A single band of 53kDa was detected in nuclear protein tissue extracts of selected cancer patients and in 96% of the corresponding sera using Western blot assay. The ELISA technique revealed that the serum p53 was detected in 100% of patients with cholangiocarcinoma, 76% of pancreatic carcinoma, 75% of hepatocellular carcinoma, 70% of colon cancer, 60% of esophagus carcinoma, and 35% of gastric carcinoma. The serum p53 concentrations of the positive patients were highly elevated (P<0.001) compared with healthy individuals. These results suggest that immunodetection of serum p53 could be valuable for post-operative monitoring during follow up in preoperatively positive patients with gastrointestinal cancers.

DNA ploidy of liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma: a flow cytometric analysis

Flow cytometric DNA analysis was used to assess cellular kinetics of needle liver biopsies from patients with liver cirrhosis and hepatocellular carcinoma (HCC). An abnormal DNA content was shown in 44.5% of liver cirrhosis cases and in 78.6% of tumor sites. The number of proliferating cells (S + G2M%) was significantly increased in cirrhotic liver (P < 0.05). Dysplasia was found in 66% of cirrhotic specimens. All negative dysplasia specimens showed a diploid pattern while 69% of positive dysplastic specimens were aneuploid (P < 0.001). In conclusion, cell proliferation, aneuploidy and liver cell dysplasia are important indicators in liver cirrhosis for the development of HCC.

A novel antigen detection immunoassay for field diagnosis of hepatitis C virus infection.

Abstract The limitations of dominant methods-based on the detection of anti-HCV antibodies or HCV viremia currently used for the diagnosis of HCV infection enhance efforts to have a rapid, simple, sensitive, and specific alternative diagnostic approach to detect viral antigens. A highly reactive IgG antibody was raised to HCV-NS4 recombinant antigen. The produced antibody showed no cross-reactivity with the other HCV structural and nonstructural recombinant antigens (C1 + 2, C3 + 4, E2/NS1, NS3, NS5). The well established ELISA technique was adapted to detect the new target HCV-NS4 antigen in serum samples. Extremely high agreement was found between the results of ELISA and qualitative detection of HCV-RNA, using a RT-PCR test as a gold standard for the diagnosis of HCV infection. Based on these encouraging results, a novel enzyme immunoassay; dot-ELISA was developed for rapid (˜5 min) and simple qualitative detection of the target HCV antigen in serum. The developed method detected the HCV target antigen in 95% of serum samples from HCV infected individuals, with a specificity of 97% using sera of noninfected individuals in comparison with PCR test. The antigen detection method showed high predictive values of positive (99%) and negative (90%). Moreover, the dot-ELISA could detect the HCV target antigen in sera negative for anti-HCV Abs, but positive for HCV-RNA, and in sera of HCV infected individuals with low viremia, as well as those with high viremia, using quantitative RT-PCR. Accordingly, the developed highly sensitive and specific HCV antigen detection method could be applied for mass screening of HCV infection.

Expression of p53 protein in liver and sera of patients with liver fibrosis, liver cirrhosis or hepatocellular carcinoma associated with chronic HCV infection

OBJECTIVES Hepatitis C virus (HCV) is a major aetiological agent of chronic hepatitis and it leads to the development of liver cirrhosis and hepatocellular carcinoma (HCC). The significances of p53 protein and anti-p53 antibodies levels in HCV genotype IV infected patients with different liver pathology were evaluated. DESIGN AND METHODS Immunostaining and western blot based on monospecific anti-p53 antibody were used for the identification of p53 protein in liver tissues and serum samples. The serum levels of p53 protein and anti-p53 IgG antibodies were evaluated using enzyme linked immunosorbent assay (ELISA). RESULTS Mild and diffuse p53 cytoplasmic immunostaining was found in liver tissues of patients with liver fibrosis [F1-F3] and liver cirrhosis [F4] in comparison with strong and diffuse p53 cytoplasmic immunostaining in patients with HCC. The target p53 protein was identified in sera of patients with liver fibrosis, liver cirrhosis and HCC at 53-kDa. The detection rate of serum p53 protein increases significantly (p<0.05) with the progression of the liver pathology. However, a significant difference (p<0.05) was only shown between serum p53 protein level of HCC patients and those of other liver pathology. In contrast, anti-p53 IgG antibodies positive rates showed only a significant decrease (p<0.05) in HCC in comparison with liver cirrhosis. CONCLUSIONS The serum and cytoplasmic p53 protein expressions were more pronounced in patients with HCC more than liver cirrhosis, and in liver cirrhosis more than liver fibrosis. These results suggest that HCV genotype IV and p53 protein levels may have a role in the development of HCC among Egyptian patients.

Efficacy of passive immunization with IgY antibodies to a 58-kDa H. pylori antigen on severe gastritis in BALB/c mouse model.

Abstract Consecutive triple doses of 1 × 108 CFU/mL of a pathogenic H. pylori strain isolated from stomach of Egyptian patients with severe gastritis were used to establish infection in BALB/c mice model. White Leghorn hens were immunized with H. pylori whole cell lysate (HpLysate) antigen and with a highly reactive 58-kDa H. pylori (Hp58) antigen. Two months later, IgY antibodies (IgY-HpLysate & IgY-Hp58) were purified from egg yolk and its efficacy was evaluated in the adopted model. Microbiological culture and immunohistochemical staining revealed that H. pylori infection was inhibited 1 week after oral passive immunization in 70% of infected BALB/c mice with a significant decrease (p < 0.05) in the degrees of gastritis. In conclusion, we have adapted BALB/c mice model for human H. pylori pathogenic strain and oral passive immunization with specific IgY antibodies to the 58-kDa antigen inhibited active H. pylori infection and decreased gastritis.

Rapid and simple detection of a Mycobacterium tuberculosis circulating antigen in serum using dot-ELISA for field diagnosis of pulmonary tuberculosis.

Abstract Tuberculosis (TB) has re-emerged as a major health problem worldwide. Developing an easy, inexpensive immunodiagnostic test is extremely important for TB diagnosis, especially in developing countries. A target mycobacterial circulating antigen of 55-kDa molecular weight was identified in sera from confirmed Mycobacterium tuberculosis infected individuals by using Western blotting based on a specific mouse IgG anti-M. tuberculosis monoclonal antibody (TB-55 mAb). No bands were identified in sera of healthy individuals. The target TB antigen was isolated and characterized as a protein. It consists of 15 amino acids; 24.6% of the amino acids are hydrophobic and 46.4% are hydrophilic. A dot-ELISA format, based on TB-55 mAb, was developed for the direct demonstration of the 55-kDa TB antigen in serum samples of pulmonary TB patients. The technical aspects of the developed dot-ELISA are simple, rapid (5 min), and reproducible, as well as sensitive (87%) and specific (93%). Using the more sensitive immunoassay; Western blot, the 55-kDa TB antigen was detected in all (100%) sera that have been shown false negative by dot-ELISA, as well as in true positive sera. In conclusion, we have developed a simple and rapid immunoassay for the direct detection of a circulating mycobacterial antigen in sera of TB infected individuals and, therefore, the developed assay can be applied for laboratory and field diagnosis of TB infection in developing countries.

Development of a novel score for liver fibrosis staging and comparison with eight simple laboratory scores in large numbers of HCV-monoinfected patients.

BACKGROUND This study aimed to develop and evaluate a predictive score named Fibrosis Routine Test (FRT) for liver fibrosis staging and to compare FRT with APRI, Lok, GUCI, FI, FibroQ, FCI, FIB-4 and 4RLB scores in large numbers of untreated HCV-monoinfected patients. METHODS Large numbers of estimation (N=2045) and validation patients (N=3212) were included in this study. Stepwise linear discriminant analysis and area under receiver-operating characteristic curves (AUCs) were used to create a predictive score comprising age, AFP, APRI and albumin. RESULTS In the estimation study, FRT produced AUCs 0.84, 0.85 and 0.86 for significant (F2-F4), advanced fibrosis (F3-F4) and cirrhosis (F4), respectively. FRT > 4 was 83% specific and 73% sensitive for F2-F4, FRT > 5 was 83% specific and 71% sensitive for F3-F4 and FRT > 5.5 was 81% specific and 73% sensitive for F4. In the validation study, FRT produced AUCs 0.81, 0.89 and 0.95 for F2-F4, F3-F4 and F4, respectively. The above eight scores demonstrated lower AUCs than FRT. CONCLUSION While liver biopsy is invasive, costly and associated with complications, Fibrosis Routine Test (FRT) is a non-invasive, inexpensive, simple and may reduce the need of liver biopsy.

Evaluation of cytokeratin-1 in the diagnosis of hepatocellular carcinoma

BACKGROUND This study was undertaken to investigate whether serum cytokeratin-1 (CK1) could complement alpha-fetoprotein (AFP) to improve the diagnosis of hepatocellular carcinoma (HCC). METHODS CK1 was identified using western blot and ELISA in serum samples from 250 Egyptian patients including 150 with HCC, 100 with liver cirrhosis (LC) and 50 healthy controls. Multivariate discriminant analysis (MDA) and ROC curve analyses were used to create a predictive model including CK1 in addition to a panel of routine blood markers. RESULTS CK1 was identified at 67 kDa and quantified in sera of HCC patients using western blot and ELISA. MDA selected a score for the prediction of HCC from LC patients based on levels of CK1, albumin and AFP. An area under the ROC curves (AUC) of the score was 0.87. The score showed a sensitivity of 87% vs 39% sensitivity of AFP at cutoff value of 200 IU/ml for prediction HCC. Absolute specificity (100%) was obtained to discriminate HCC from healthy individuals. CONCLUSIONS This study suggests that the use of a combination of score including CK1, AFP and albumin in clinical practice provides a non invasive and simple test that could increase significantly the sensitivity of HCC diagnosis.