Abstract Type 2 diabetes (T2DM) and its complications represent a main worldwide public health challenge, with high-rate morbidity and mortality. Non-alcoholic fatty liver disease (NAFLD) has a great focus worldwide, as it is expected to affect 75% of patients with type2 diabetes mellitus. In this study, we aimed to assess reduced glutathione plasma level then evaluate its correlation with fasting blood glucose levels, HBA1c and lipid profile in type 2 diabetic patients with fatty liver. This study included 40 participants divided into 20 type2 diabetics with fatty liver group and 20 healthy control group. Estimation of fasting blood glucose levels, HBA1c, lipid profile and liver function enzymes were done. reduced glutathione plasma levels were measured by ELISA. The results revealed that plasm levels of reduced glutathione were lower in type 2 diabetics with fatty liver compared to control subjects (P < 0.01). These low plasma GSH levels in diabetics was negatively correlated with FBG and HBA1C% (r= −0.641, −.0547 respectively; P < 0.01) indicating that chronic hyperglycemia and its resulted oxidative stress have negative impact on GSH concentrations in the body. This low GSH levels in type 2 diabetics with fatty liver can be an important prognostic factor as, GSH reduction causing increase of oxidative stress and may worsen the liver status that may progress to NASH.

Abstract Background Diabetes mellitus is considered the ninth major cause of death, biomarkers have been investigated in DM research field to understand the pathophysiology of DM, help in diagnosis and find new therapeutics. This study evaluated the role of Galectin 1, LncRNA MEG3, miR22 in T2DM. Aim of the work we aimed to evaluate the role of galectin 1 expression in type 2 DM and to explore the epigenetic regulatory effect of miRNA22 and LncRNA, MEG3 on gelatin 1 expression in type 2 DM Materials and methods The study included two groups: Fifteen type 2 diabetes mellitus patients and fifteen normal healthy individuals. Blood samples were collected from every participant and serum was separated. Expressions of Gal-1, LncRNA MEG3, miR22 were assessed in each serum sample by quantitative real time polymerase chain reaction (qRT-PCR). Results The study revealed higher levels of gal-1, miR22 and lower levels of MEG3 in serum samples of T2DM Patients compared to normal healthy individuals with statistical significance difference (p < 0.05). Conclusion Gal-1, MEG3, miR22 may play a role in the pathophysiology of T2DM. Studies with larger sample sizes are required to further confirm their role in early detection and diagnosis of T2DM.

Aim The aim of this study was to explore the expression of exosomal non-coding RNAs (ncRNAs) in the sera of patients with HCC versus control. Methods Firstly, Bioinformatics analysis was conducted to retrieve ncRNAs specific to HCC (hsa-miRNA-1298 and lncRNA-RP11-583F2.2). Afterwards, extraction and characterization of exosomes were performed. We measured the expression of the chosen exosomal RNAs by reverse transcriptase quantitative real-time PCR in sera of 60 patients with HCC, 42 patients with chronic hepatitis C (CHC) infection and 18 healthy normal volunteers. Results The exosomal ncRNAs [hsa-miRNA-1298, lncRNA-RP11-583F2.2] had better sensitivity and specificity than alpha-fetoprotein (AFP) in HCC diagnosis. Conclusion The exosomal hsa-miRNA-1298, lncRNA-RP11-583F2.2 can be potential biomarkers for HCC diagnosis.

Autism spectrum disorder (ASD) refers to a group of neurodevelopmental disorders characterized by impairments in communication and stereotypic patterns of behavior. Epigenetics is a rapidly evolving field of genetic study implicated in pathogenesis of ASD. Changes in biomarkers theme are thought to be early events in ASD development. We aimed to investigate the expression of serum RNA-based biomarkers exploring their potential utility as diagnostic or prognostic biomarkers for ASD. Based on bioinformatics analysis, the current work has been designed to study the expression of miR-106a and ADARB1 as a novel circulating biomarkers that might improve the specificity of diagnosing ASD. This study was conducted on 32 children, 16 children with ASD as patient group (A) and 16 age, sex matched healthy children as control group (B). Quantitative PCR was carried out to evaluate the expression pattern of selected biomarkers in serum of autistic cases. The expression level of circulating miR-106a and ADARB1 total mRNA were significantly up-regulated in ASD children than in control group (p = 0.000). Notably there was a highly positive significant correlation between both studied biomarkers and with autism severity assessed by CARS. This study may open a new gate for the use of different potential circulating biomarkers, side by side with routine parental questionnaires to improve detection of ASD.

Worldwide, hepatocellular carcinoma (HCC) is the major subtype of primary liver cancers. HCC is typically diagnosed late in its course. With respect to cancer, the genomic actions of vitamin D are mediated through binding to the Vitamin D Receptor (VDR), which allows it to modulate the expression of genes in a cell-and tissue-specific manner. Epigenetics is a rapidly evolving field of genetic study applicable to HCC. Changes in DNA methylation patterns are thought to be early events in hepatocarcinogenesis. Curcumin has great potential as an epigenetic agent. Accordingly, the current study has been designed to study the methylation status of VDR gene promoter for the first time in HCC aiming to find its clinical significance and potential screening role in chronic Liver Disease (CLD). Additionally, we aimed to investigate, the effect of Curcumin on HCC cell line, aiming to discover new therapeutic targets through epigenetics. This study was conducted on 45 formalin-fixed, paraffin-embedded liver tissue blocks including 15 HCC samples (group A), 15 CLD samples (group B) and 15 apparently normal tissue taken from around benign lesions (group C). Methylation Specific Restriction Digestion and qPCR were done on all samples after DNA extraction. The percentage of VDR gene promoter methylation was significantly higher in the HCC group compared to both CLD and control groups (p < 0.01). VDR promoter methylation by (MS-qPCR) was decreased and the relative expression of VDR by (qRT-PCR) was markedly increased in a dose-dependent fashion in cells grown in Curcumin-adequate medium. In conclusion, this study may open a new gate for the use of VDR promoter methylation as a potential biomarker in HCC.

Background: Nowadays, microRNAs (miRNAs) have proved their significant role in the diagnosis and prognosis of various types of malignancy. Accordingly, this study aimed at evaluating the role of miRNA-155 in diagnosis as well as prognosis of lung cancer. Methods: In this prospective case-control study, exhaled breath condensate (EBC) samples were collected from a group of 15 pathologically-confirmed, chemotherapy/radiotherapy-naive lung cancer patients, as well as from another control group comprised of 15 patients at high risk for lung cancer development. The expression of miRNA-155 (RQ value) in EBC samples was measured using Quantitative real time Polymerase Chain Reaction (PCR). Results: There was significant statistical difference between the lung cancer group and the control group as regards RQ value (p <= .01) being higher among the lung cancer group. RQ value among the lung cancer group showed significant statistical difference as regards tumor extension (p = .004), and smoking index (p = .004). RQ levels correlated significantly with tumor extension (p = .001), smoking status (p = .000), smoking index (p = .000), and family history of lung cancer (p = .007), whereas tumor location, histopathological type of the tumor, and sex did not correlate significantly (p > .05). The best cut-off value for EBC RQ level was >= 3.338 with an overall sensitivity 100%, specificity 100%, positive predictive value 100%, negative predictive value 100%, and overall accuracy 100%. Conclusion: The expression of miRNA-155 in EBC can be considered as an accurate, non-invasive, promising biomarker for early lung cancer diagnosis and prognosis. (C) 2017 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V.

Acute lymphoblastic leukemia (ALL) is the most common pediatric hematologic tumor. MiR-181a was expected to have a role in the development of hematological malignancies; it might act as tumor suppressor or oncogene. Smad7 was selected as miR-181a target pair. It is a negative regulator for the TGF-β1 signaling pathway. In this study, relative expression levels of miR-181a by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), both Smad 7 and TGF-β1 proteins levels by enzyme linked immunosorbent assay (ELISA) were all measured in serum of 60 child, 30 with ALL and 30 age and sex matched healthy child as control group. MiR-181a expression showed highly significant decrease; plus a significant increase and decrease of Smad7 and TGF-β1 protein levels respectively, in serum samples of ALL as compared to control group. MiR-181a expression achieved a highly significant positive and a significant negative correlation with TGF-β1 and Smad7 respectively. Furthermore, the levels of Smad7 and TGF-β1 were negatively correlated with each other (p<0.05). Although, positivity rate of both Smad7 and TGF-β1 in ALL group increased with presence of hepatosplenomegaly, still there was no statistical significance. In conclusion, miR-181a could act as a tumor suppressor in pediatric ALL with over expression of its target pair, Smad7. Smad7 regulates TGF-β1 signaling via a negative feedback loop and mediates the interaction between TGF-β1 and other signaling pathways; suggesting that Smad7 over expression may have therapeutic potential in ALL.

Rationale: The global burden of lung cancer is attributed to its poor outcome as it is usually discovered in an advanced stage therefore the constant search for screening protocols among the high risk groups like smokers and chronic obstructive pulmonary disease (COPD). Biomarker testing in exhaled breath condensate (EBC) samples is a simple inexpensive non invasive method. Many previous researches linked the dys-Regulation of microRNAs to the development of lung carcinogenesis. Consequently the aim of this study is to investigate the role microRNA7 as a potential biomarker for early detection of lung cancer among smokers and COPD patients using EBC samples. Methods: The expression level of microRNA7 was evaluated in EBC samples of 15 COPD patients (7 GOLD II and 8 GOLD III) who were currently smokers and 5 healthy never smokers volunteers by quantitative real time PCR analysis using the miScript II RT kit, Qiagen, Germany (catalog number 218161). Results: The levels of microRNA 7 in EBC samples collected from COPD patients (GOLD stage II and III) who were currently smokers were significantly down regulated (p < 0.007) compared with the nonsmoking control samples. In addition, among the COPD patients, the levels of microRNA 7 in EBC samples were significantly associated to the degree of airflow limitation and the severity of the disease represented by FEV1 (% predicted) (p < 0.024). There was also a negative correlation between the levels of microRNA 7 and smoking index. ROC curve analysis was plotted displaying an AUC of 0.909 for EBC samples of microRNA 7. Conclusion: MicroRNA 7 levels should be considered as a potential biomarker for the detection of early lung carcinogenesis as part of microRNA microarrays. (C) 2017 The Egyptian Society of Chest Diseases and Tuberculosis. Production and hosting by Elsevier B.V.

There is an obvious need to diagnose hepatocellular carcinoma using novel non-invasive and sensitive biomarkers. In this regard, the aim of this study was to evaluate and correlate both relative quantification of microRNA-7 using quantitative real time polymerase chain reaction and quantitative analysis of selenoprotein P using enzyme-linked immunosorbent assay in sera of hepatocellular carcinoma patients, chronic liver disease patients, as well as normal healthy subjects in order to establish a new diagnostic biomarker with a valid non-invasive technique. In addition, this study aimed to investigate whether changes in selenium supply affect microRNA-7 expression and selenoprotein P levels in human hepatocarcinoma cell line (HepG2). The results showed a highly significant decrease in serum microRNA-7 relative quantification values and selenoprotein P levels in malignant group in comparison with benign and control groups. The best cutoff for serum microRNA-7 and selenoprotein P to discriminate hepatocellular carcinoma group from benign and control groups was 0.06 and 4.30 mg/L, respectively. Furthermore, this study showed that changes in selenium supply to HepG2 cell line can alter the microRNA-7 profile and are paralleled by changes in the concentration of its target protein (selenoprotein P). Hence, serum microRNA-7 and selenoprotein P appear to be potential non-invasive diagnostic markers for hepatocellular carcinoma. Moreover, the results suggest that selenium could be used as an anticancer therapy for hepatocellular carcinoma by affecting both microRNA-7 and selenoprotein P.

Background/Purpose: There have been many conflicting reports on the effects of type 2 diabetes mellitus (DM) and the level of vitamin B12 on bone mineral density (BMD) in elderly patients. Moreover, conflicting data exists regarding the prevalence of vitamin B12 deficiency among elderly diabetics. The aim of this study was to investigate the link between vitamin B12 levels, type 2 DM, and BMD in elderly patients.Methods: A caseecontrol study was conducted on 61 participants, >= 60 years of age, divided into 31 cases of patients with diabetes and 30 age-matched healthy controls. Patients receiving vitamin B12 supplements were excluded. The relationship between BMD and serum levels of vitamin B12 was examined.Results: Borderline/deficient serum B12 status was more common in the control group; it was found in 53.33% of the controls and 25.80% of diabetic patients. The mean serum vitamin B12 concentration was 820.65 +/- 544.77 pg/mL in patients with diabetes and 677.80 +/- 619.89 pg/mL in healthy control participants (p = 0.34). Serum vitamin B12 concentration showed no significant difference between osteoporotic patients, osteopenic patients, and normal patients among the diabetic group.Conclusion: The prevalence of vitamin B12 deficiency was higher in the control group than the diabetic group who did not receive oral B12 supplementation. Low serum vitamin B12 is commonly overlooked in the elderly, with or without diabetes. The presence of diabetes mellitus did not affect BMD in the elderly. Furthermore, there is no significant relationship between serum vitamin B12 levels and BMD among diabetics. Copyright (C) 2015, Asia Pacific League of Clinical Gerontology & Geriatrics. Published by Elsevier Taiwan LLC

PURPOSE:There is an obvious need to diagnose lung cancer using novel noninvasive and sensitive biomarkers. In this regard, the aim of the present study was to evaluate and compare sputum matrix metalloproteinase 2 (MMP-2) in relation to serum MMP-2 of lung cancer patients and other nonmalignant lung diseases in order to establish a new diagnostic and prognostic biomarker with a valid noninvasive technique.EXPERIMENTAL DESIGN:Group 1 included 32 newly diagnosed lung cancer patients and group 2 included 20 patients with benign pulmonary diseases. In addition, 38 healthy subjects served as control group. MMP-2 activity levels were evaluated in serum and sputum samples of the studied groups using ELISA and zymography techniques.RESULTS:There was a highly significant increase in serum and sputum MMP-2 levels in malignant group in comparison with benign and control groups. In addition, there was a significant difference in the levels of serum and sputum MMP-2 as regards the different histopathological types of lung cancer and advanced stages of lung cancer. Gelatin zymography was used to confirm the enzymatic activity of MMP-2. A higher MMP-2 activity was detected in lung cancer group in comparison with benign and control groups.CONCLUSIONS AND CLINICAL RELEVANCE:Serum and to a larger extent sputum MMP-2 appear to be potential noninvasive markers for detecting lung cancer.

There is an intimate relation between disturbance in survivin and SMAC/DIABLO expressions and cancer formation in many tissues; however, this was not confirmed in the primary breast cancer. The aim of this study was to evaluate the relationship between survivin and SMAC/DIABLO mRNA expressions in primary breast cancer using RT-PCR and their relationship to some of the risk factors (age, family history, breast-feeding, use of contraceptive pills and hormonal receptors). Breast cancer tissues were studied for the detection of the mRNA expression of survivin and SMAC/DIABLO. This was done by qualitative reverse transcription-polymerase chain reaction (RT-PCR). SMAC/DIABLO mRNA was detected by RT-PCR only in 1 sample in breast cancer tissues (6.25%). However, it was expressed in 16 out of 25 (64%) of the benign tumor tissues (P = 0.00). In contrast, survivin mRNA was highly expressed in breast cancer tissues, 36 out of 48 (75%) (P = 0.00), while its expression was very low in benign tumor tissues, only 1 out of 37 (2.7%) (P = 0.00). Expressions of SMAC/DIABLO and survivin were significantly reciprocal in breast cancer and benign tumor tissues. SMAC/DIABLO inhibits apoptosis of breast cancer cells by suppression of survivin. These two genes probably form an important link in the signaling pathway of formation of breast cancer cells, which may be chosen as therapeutic targets in the primary breast cancer.

Results Cirrhotic patients with HCV had higher serum NO level and MPO activity while lower AE and PON1 activities than the chronic. Significant inverse correlation was observed between MDA and PON1 activity in patients with HCV. The most significant HCV biomarker was MDA, AE, NO and PON1. The best combined ones for sensitivity, specificity were MDA + albumin, PON1 + AST, and PON1 + albumin. Conclusions The use of the MDA, MPO, AE, NO and PON1 as biomarkers might be useful tools, helping in the monitoring of patients with HCV. Keywords Oxidative stress Malondialdehyde (MDA) Nitric oxide (NO) Myloperoxidase (MPO) Arylesterase (AE) Paraxonase (PON1) Hepatitis C virus (HCV) Introduction Hepatitis C virus (HCV) infection is a major worldwide health problem that has an increasing prevalence and mortality, with more than 170 million people infected. Approximately 70–80% of infected individuals develop chronic HCV infection. The hallmarks of chronic HCV infection are inflammation and liver fibrosis and 20–30% of patients develop cirrhosis with a risk of hepatocellular carcinoma [1] . Egypt has the highest prevalence HCV genotype-4 with more than 19% of the population infected and chronic HCV representing one of the top five leading causes of death [2] . Hepatitis C virus has the capacity to generate substantial oxidative stress within hepatocytes. Subsequently, oxidative stress has been identified as a significant mechanistic pathway culminating in the development of hepatic cirrhosis, liver failure and liver cancer [3] . Reactive oxygen species (ROS) mediated liver injury may be triggered by lipid peroxidation. It leads to oxidative stress and contributes to the initiation and progress of liver damage [4] . Cytotoxic products of lipid peroxidation, such as malondialdehyde (MDA), may impair cellular functions including nucleotide and protein synthesis, which may play a role in hepatic fibrogenesis [5] . Nitric oxide (NO), synthesized from l -arginine by nitric oxide synthase (NOS), is a relaxing factor for the vascular endothelium that may mediate hepatic injury from ROS and lipid peroxidation products. Nitric oxide and superoxide radical interact to form peroxinitrite, which is an important mediator of free radical toxicity [6] . Nitric oxide is an important signaling molecule that acts in many tissues to regulate a wide range of physiological processes. Consequently, abnormal regulation and control of NO synthesis affect a number of important biological processes and are involved in a variety of diseases. The NO system disturbances appear to play a key role in the pathogenesis of chronic liver diseases [7,8] . The host immune response to HCV infected hepatocytes is responsible for liver injury and subsequent development of hepatic fibrosis. The cellular elements comprising the host immune response include neutrophils, macrophages (Kupffer cells) and lymphocytes. Neutrophils and Kupffer cells contain myeloperoxidase (MPO). Consequently, the infiltration of neutrophils into the liver is evaluated by hepatic MPO activity. Thus, measuring the MPO level is also helpful for the monitoring oxidative stress [9,10] . Arylesterase (AE) and paraoxonase-1 (PON1) are synthesized mainly in the liver, which hydrolyze organophosphates like pesticides, neurotoxins and arylesters [11,12, and 13] . PON1 level is tightly linked with HDL in the serum and contributes to the protection conferred by HDL against LDL oxidation. Human PON1, an HDL-associated enzyme carried on apolipoprotein A-I, is believed to protect lipoproteins against oxidative modification. Both AE and PON1 prevent LDL oxidation. These enzymes are recognized as antioxidant enzymes because they hydrolyze lipid peroxides and oxidized lipoproteins [14] . The role of PON1 and AE activities may be particularly meaningful as an index of liver function and useful for monitoring the disease of chronic hepatitis [15] . ROC plots are constructed by calculating the true positive fraction (sensitivity percent) and false positive fraction (100-specificity) of the different investigated markers at several cut off points, then the best cutoff value which differentiates between normal and HCV patients is calculated as the best value that maximizes the sum of sensitivity and specificity at which the highest predictive values are reached. Area under the curve approaching 1 indicates a perfect discriminator of the positive cases from the negative ones. Thus, the ROC plot itself and ROC analysis provide information useful to the clinical laboratorian in making practical decisions about laboratory operation and clinical application [16] . The goal of the present study was to investigate the diagnostic and prognostic efficiencies of some biomarkers of liver dysfunction in Egyptian patients with chronic and cirrhotic HCV infection. These markers included MDA and NO levels, and MPO activity as oxidant biomarkers, and AE and PON1 activities as antioxidants biomarkers. The measurement of these biomarkers may provide a better predicting sensitivity and specificity than those obtained from the traditional standard liver biochemical tests. Moreover, these markers may be valuable for the assessment of the extent of liver damage in HCV patients, thus, assisting physicians in fast diagnosis and early treatment of chronic and cirrhotic liver disease associated with HCV infection. Materials and methods Chemicals Thiobarbituric acid; p -nitrophenyl acetate; paraoxone and o -dianisidine dihydrochloride were purchased from Sigma Chemical Co. (St. Louis, MO, USA). All other chemicals were of analytical grade and purchased from Sigma or Merck (Darmstadt, Germany). Subjects This study was carried out on 44 patients with HCV (mean age: 47 ± 11 years; 31 males and 13 females) and 21 age-and-gender matched healthy subjects (mean age: 48 ± 8 years; 17 males and 4 females). Patients were admitted to outpatient's clinics of the Tropical Disease Department of Ain Shams University Hospitals. The Control group included healthy volunteers, mostly, relatives of the patients who came with them to the clinic. They shared the same socio-economic status of the patients group. HCV infection was diagnosed by the positivity of anti-HCV Ab and HCV-RNA. All patients with HCV were confirmed to be chronic (23 patients) or cirrhotic disease (21 patients) by clinical and biochemical evaluation. The healthy subjects didn't have any clinical or laboratory renal insufficiency, liver damage, neoplasia or neurological disorders. Samples Informed consent was obtained from patients and healthy relative volunteers prior to the study. The study was approved by Ethics Committees of the Ain Shams University Hospitals. All blood samples were collected in the morning after an overnight fast. Serum samples were separated and stored at − 70 °C until assay. Methods Fasting blood samples were obtained from all subjects. Qualitative determination of antibodies to hepatitis C virus (anti-HCV) was performed to confirm diagnosis by using anti-HCV. Serum alanine aminotransferase (ALT); aspartate aminotransferase (AST); alkaline phosphatase activities and serum albumin; total and direct bilirubin; cholesterol; HDL cholesterol and LDL cholesterol levels were assayed on an automated analyzer (HITACHI 7170A, Hitachi Koki Co., Ltd, Hitachinaka City, Japan). The lipid peroxides as thiobarbituric acid reactive substance (TBARS) was assayed according to the method of Esterbauer and Cheeseman [17] . Briefly, serum was precipitated by 10% trichloroacetic acid (TCA). Thiobarbituric acid reacted with products of lipid peroxidation, mainly malondialdehyde (MDA), producing a pink color that could be measured at 535 nm. MDA was calculated by using extinction coefficient 153,000 M − 1 cm − 1 . Nitric oxide was determined as described previously by Vodovotz [18] . The concentration of NO was measured as nitrite and nitrate. Briefly, serum nitrate was reduced to nitrite by cadmium and assayed by Greiss reaction. Myeloperoxidase was quantified spectrophotometrically by the method described by Buchmann et al. [19] . Myeloperoxidase catalyzes the oxidation of hydrogen peroxide in the presence of o -dianisidine dihydrochloride as a substrate of oxidizing agent. 100 μl of 1 mM hydrogen peroxide and 100 μl deionized water was added to 0.5 ml 0.2 M citric acid buffer (pH 5.5) containing 0.125% Triton-X-100, then 50 μl serum was added. After that 50 μl of 16 mM o -dianisidine was added and the mixture was incubated at 37 °C for 7 min. After incubation; 200 μl 0.2 M of glycine/NaOH (pH 10.4) was added to the mixture. The change in absorbance at 405 nm was recorded. The change in absorbance was converted to micromoles of oxidized o -dianisidine using the extinction coefficient ( ɛ = 133,000 M − 1 cm − 1 ). One unit of myeloperoxidase activity is defined as 1 nmol of oxidized o -dianisidine formed per minute under the above assay conditions. Arylesterase was measured using p -nitrophenyl acetate as substrate as described by Kao et al. [20] . The reaction mixture consisted of 50 mM phosphate buffer, pH 7.5, 5 μl of serum and 0.5 μmol p -nitrophenyl acetate (acetone 0.3%) in a total volume of 1 ml. The mixture was incubated at 37 °C and followed by 2 min, and the change in absorbance at 405 nm was recorded. The change in absorbance was converted to micromoles using the extinction coefficient ( ɛ = 17,000 M − 1 cm − 1 ) for p- nitrophenol. One unit of arylesterase activity is defined as 1 nmol of p- nitrophenol formed per minute under the above assay conditions. Paraoxonase was quantified spectrophotometrically by the method described by Gil et al. [21] using 0.1 M Glycine-NaOH buffer pH 10, calcium chloride 1 mM, sodium chloride 0.3 M, paraoxone 2 mM in 1 ml of the reaction mixture containing 50 μl of serum. Reaction was followed for 5 min at 37 °C by monitoring the appearance of p -nitrophenol at 405 nm. One unit of paraoxonase activity is defined as 1 nmol of p -nitrophenol formed per minute under the above assay conditions. Statistical analysis All statistical analysis was performed using the Statistical Package for Social Science (SPSS) (version 12.0, USA). Results were expressed as mean ± standard deviation (SD). Comparison between two groups using Student's t -test for continuous variable and p values were performed. Correlation between two parameters was determined by Pearson's correlation coefficient ( r ). Sensitivity, specificity, positive predictive value (PPV) and negative predictive value were determined. p values were considered significant values at p < 0.05. Results Serum ALT, AST, ALP activities, total and direct bilirubin levels were higher, whereas serum albumin levels were lower in the cirrhotic and chronic patients with HCV than the control group. Serum ALT activity in cirrhotic patients was 2 fold higher than those in chronic patients. Serum HDL cholesterol were lower whereas LDL cholesterol were higher in cirrhotic patients than controls ( Table 1 ). Serum MDA and NO levels and MPO activity were highly significantly elevated in chronic and cirrhotic patients with HCV as compared to the control subjects. In addition, serum NO level and MPO activity were higher in the cirrhotic patients than the chronic patients. On the other hand, serum AE and PON activities were significantly lower in the chronic and cirrhotic patients than in the controls. Moreover, serum PON activity was significantly reduced in the cirrhotic patients as compared to the chronic patients ( Table 2 ). A statistically positive correlation was obtained between serum MDA levels and with serum ALT, AST, ALP activities; total and direct bilirubin levels in patients with HCV. On the other hand, a negative correlation had been found between serum MDA levels and serum PON1 activities in patients with HCV ( r = − 0.458, p = 0.002) ( Fig. 1a ). Negative correlations were observed between serum MPO activity and AST/ALT ratio ( r = − 0.385, p = 0.01) in patients with HCV ( Fig. 1b ). Serum AE activity was negatively correlated with serum ALT and AST activities but positive correlated with serum PON1 activity in HCV patients. Moreover serum PON1 activity showed a negative correlation to serum ALT, AST, ALP activities and total and direct bilirubin levels in patients with HCV. The best cutoff values that maximize the sum of sensitivity and specificity of serum MDA, NO, MPO, AE and PON1 ( Figs. 2 and 3 ) were 7.25 nmol/ml, 33.48 μmol/l, 6.1 nmol/min/ml, 764 nmol/min/ml and 482 nmol/min/ml respectively. AE was more sensitive than MDA, MPO, PON1 and NO, as a biomarker for hepatitis C where its sensitivity was 86% but for MDA, MPO, NO and PON1 the sensitivities were 68%, 75%, 66% and 79.5%, respectively. MDA and PON1 were more specific than AE, MPO and NO where their specificities were 100% but for AE, MPO and NO the specificities were 90%, 90% and 95%, respectively. The best combined ones for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were MDA + albumin, PON1 + albumin and PON1 + AST, as their values were 100%. Discussion Hepatocytes are continuously exposed to ROS, but protected from oxidative injury by a range of antioxidant pathways. The defenses against free radical-mediated injury include enzymatic deactivation and direct reaction with free radicals [22] . Although free radicals are normally produced by many reactions essential for cell metabolism and energy production, they are implicated also in the pathogenesis of several different human diseases. In patients with viral or alcoholic liver disease, the consequent alternation of cellular redox state is potentiated by a correlated decrease in antioxidant enzymes and increase of free radical-mediated damage and apoptosis of liver cells [23] . Several investigations suggested that free radical generation and oxidative stress play an important role in the mechanisms developed by HCV to survive and progress in the infected host [24–26] . Lipid peroxidation is caused by free radicals leading to oxidative destruction of polyunsaturated fatty acids constitutive of cellular membranes. The destruction of polyunsaturated fatty acids leads to the production of toxic and reactive aldehyde metabolites such as MDA [27] . MDA content usually reflects the level of lipid peroxidation and indirectly reflects the extent of hepatocellular injury in vivo [22] . In the present study, we found that serum MDA levels were higher in patients with HCV than the healthy ones. This result considers the consequence of lipid peroxidation in the pathophysiology of patients with HCV. Serum MDA concentration in patients with chronic active hepatitis C and as well as transaminase activities and bilirubin level were also found to be increased in many other studies [22,27,28] . MDA was also elevated in the liver and the blood of patients with hepatitis C as reported by De Maria et al. [29] . Moreover, Boya et al. [30] reported that, the peripheral blood mononuclear cells separated from chronic hepatitis C patient had increased MDA concentrations. Serum MDA levels were reported to be correlated with the severity of chronic hepatitis [22] . In the present study, although the levels of serum MDA were higher in cirrhotic than chronic HCV patients, the difference between both groups was not significant, thus its value as a prognostic marker is unsure. Considering bilirubin, its function as an antioxidant was previously reported, it can scavenge the chain-carrying peroxyl radical by donating a hydrogen atom attached to the C-10 bridge of the tetrapyrrole molecule to form a carbon-centered radical bilirubin. Therefore, both unconjugated bilirubin and conjugated bilirubin can serve as antioxidants, protecting human LDL from lipid peroxidation in condition of impairement hepatic bilirubin excretion [ 31,32 ]. Nevertheless, serum MDA levels in the present study showed a positive correlation to total bilirubin and direct bilirubin levels in patients with HCV as well as ALT, AST and ALP activities. The area under the ROC curve of serum MDA level was 0.901, and the best cutoff value of serum MDA was 7.25 nmol/ml. Applying this cutoff value, the maximum sensitivity, specificity, positive predictive and negative predictive values of MDA were 68%, 100%, 100% and 60% respectively. Hepatocytes and macrophages produce NO in response to several inflammatory stimuli [8] . Inducible nitric oxide synthase (NOS) is expressed in hepatocytes and macrophages and produces high amounts of NO in response to cytokines e.g. interferon-γ. The role of NO in the pathogens is though to be the inactivation of enzymes requiring iron and sulfur prosthetic groups for their catalytic activity by forming nitrosyl-iron sulfur complexes [7] . In our study, serum NO levels had increased in the HCV patients than normal controls. Furthermore, NO levels had increased with the severity of liver disease, as its mean level was significantly higher in cirrhotic than chronic patients, this overproduction in liver cirrhosis might be via increased inducible NO synthase (iNOS) [7] . Therefore, the data presented in this study indicated an association between NO levels and the development and progression of liver cirrhosis, but no correlation was found between serum NO level and serum MDA. The best cutoff of NO in this work was 33.48 μmol/l and the area under the roc curve was 0.867. Applying this cutoff value, the sensitivity of NO was 66% and its specificity was 95%. Zhu and Fung [33] found that nitric oxide protects against liver injury by scavenging lipid radicals and inhibiting the lipid peroxidation. On the other hand, Sass et al. [34] reported that inducible NOS-derived NO regulates pro-inflammatory genes in vivo, contributing to inflammatory liver injury. Previous studies also showed that patients with viral cirrhosis, revealed an important increase in serum NO, and that HCV infection induced high synthesis of NO compared with HBV infection [34,35] . However, in the presence of massive injury (high level of inducers and elevated oxidative stress), greatly increased NO production might induce the hepatocytes to progress to irreversible channel necrosis and cell death [8] . Myeloperoxidase (MPO) is an oxidative enzyme present in phagocytes and atherosclerotic lesions [11] . MPO generated oxidants function as antimicrobial agents, yet can also inadvertently damage stander cells, such that MPO has been implicated as a contributory agent in a number of inflammatory or autoimmune diseases [36] . It has been involved in the pathogenesis of several diseases through excessive production of reactive oxygen species as well as through its genetic polymorphism [37] . In the present study, serum myeloperoxidase activity was significantly elevated in chronic and cirrhotic hepatitis compared to the control group. Also there was a highly significant elevation in MPO activity in the patients with cirrhotic HCV compared to the patients with chronic HCV, indicating its usefulness as both diagnostic and prognostic hepatic biomarker in hepatitis C infection. MPO activity was also estimated in plasma HCV patients by Nakamuta et al. [38] they found that the mean concentration of plasma myeloperoxidase in cirrhotic patients was markedly higher than that in chronic hepatitis patients and normal control groups. One possible mechanism is that invading neutrophils or monocyte-macrophages release MPO at inflammatory sites within the liver, and MPO generated hypochlorous acid and other oxidants damage by stander hepatic cells, eventually leading to fibrosis. Also hepatic clearance of MPO may be decreased because of liver dysfunction [38] . The best cutoff of MPO in this work was 6.1 nmol/min/ml and the area under the curve was equal to 0.913. Applying this cutoff value, the sensitivity of MPO was 75% and its specificity was 90%. Increased plasma MPO has a pathogenic role in the progression of liver dysfunction through hepatic endothelial cell damage. The respiratory burst of Kupffer cells adequately explains the oxidative damage that is observed in chronic liver disease. Kupffer cells express MPO that may be an important source of oxidative damage during chronic liver injury [39] . Oxidative free radicals play a role in liver cell damage and apoptosis through impairment of antioxidant enzymatic defense system [22] . Serum AE and PON1 originate from the liver and the role of their antioxidant activities may be particularly meaningful as an index of liver function status. In the present study it was found that, serum AE and PON1 activities have decreased in the chronic and cirrhotic hepatitis C patients compared to the control group. Some recent studies are in agreement with the present investigation such as Gangadharan et al. [40] who found a decreasing in serum AE and PON1 activities in chronic and cirrhotic HCV patients compared with healthy controls. Serum AE and PON1 carried in circulation bounded to HDL particles protect LDL from peroxidation [12] . Thus, there are two possible explanations for decreased PON1 and AE activities in chronic hepatitis. Firstly, the decrease in PON1 and AE enzymatic activities or gene expression could be the consequence of the hepatic dysfunction. The second is that serum PON and AE activities could be decreased as a consequence of an altered synthesis and/or secretion of HDL. PON1 and AE act as antioxidants that protect LDL from oxidative modifications and can reduce oxidized lipid in oxidized lipoproteins [12,15] . The other salient observation in this study was that the activities of both enzymes have significantly decreased in cirrhotic more than in chronic HCV patients, indicating that the level of decrease in AE and PON1 activity in the serum of patients with chronic liver diseases is probably a consequence of liver dysfunction. That could emphasize the prognostic value of AE and PON1 in HCV infected patients and give clinicians a signal for deterioration of the liver function with the progression of the disease. The results also demonstrated that serum AE was negatively correlated with serum ALT and AST activities; whereas serum PON1 was negatively correlated with serum ALT, AST and ALP activities and serum total bilirubin and direct bilirubin levels in HCV patients group. Kilic et al. [15] is in agreement with our observation that serum AE and PON1 activities were also negative correlated with serum ALT and AST activities and albumin and bilirubin concentrations as well as between serum PON1 and ALP activity. In the current study, a negative significant correlation was found between PON1 and MDA in HCV patients. Ferre et al. [41] reported that, increased serum PON1 concentrations and raised hepatic PON1 expression were associated with higher peroxidation in serum and liver biopsies. On the other hand, Bafikol et al. [42] found that no statistically significant correlations between hepatic PON1 activity and serum lipid levels or serum MDA levels in patients with non-alcoholic steatohepatitis. Moreover, PON1 has been found to influence the antiapoptotic ability of the HDL molecule, because it has the capacity to protect the lipoprotein against oxidation. PON1 has an active role in the regulation of oxidative stress, fibrosis and hepatic cell apoptosis in chronic liver diseases [41] . Marsillach et al. [43] reported that, PON1 activity measurement would be very useful in discriminating between alcoholic patients and healthy subjects. On studying the ROC curves of both AE and PON1, it was found that the areas under the curves were 0.968 and 0.966 respectively, and best cutoff values of serum AE and PON1 were 764 and 482 nmol/min/ml respectively. Applying these cutoff values, the maximum sensitivity, specificity, positive predictive and negative predictive values of (AE and PON1) were (86% and 79.5%), (90% and 100%), (95% and 100%) and (76% and 70%) respectively. Marsillach et al. [43] reported that, PON1 activity measurement would be very useful in discriminating between alcoholic patients and healthy subjects; on studying the ROC curve of PON1 activity, the area under the curve of PON1 activity was ≥ 0.90, the increased sensitivity up to 83.5–96.6% and specificity up to 95.8–99.6%. In the present study, AE was the hepatitis C biomarker marker which showed the highest sensitivity, when measured as a single marker without any combination with others, while MDA or PON1 was the marker which gave the highest specificity, when each of them was measured alone without combination. But in combination, the best combined ones for sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) were MDA + albumin, PON1 + albumin and PON1 + AST. In conclusion, the over release of the oxidative free radicals [MDA, NO and MPO] and the decrement of the antioxidants [PON1 and AE] might play a role in the pathogenesis of viral hepatitis. 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Purpose: Angiogenesis is tightly regulated by a large number of pro-angiogenic factors, including vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor and angiogenin. We adapted and evaluated the measurement of these factors using enzyme-linked immunosorbent assay and compared the results with Western blot and voided urine cytology.Materials and Methods: This study included 240 patients diagnosed with bladder carcinoma, 108 with benign bladder lesions and 110 healthy individuals who served as controls. All participants underwent serological schistosomiasis antibody assay in serum, urine cytology and estimation of angiogenic factors in voided urine.Results: Intra-assay and interassay CVs of the investigated markers were 10.3 to 12.3 and 10 to 13.7, respectively. The recovery rate of the added angiogenic factor to the urine pool was 98% to 103%, 97% to 103%, 98% to 104% and 97% to 100% for vascular endothelial growth factor, basic fibroblast growth factor, angiogenin and hepatocyte growth factor, respectively. The concordance rate with Western blot was 97.5%. The levels and positive rates of urinary angiogenic markers and urine cytology were significantly higher in the malignant group than in the benign and healthy groups. Basic fibroblast growth factor increased significantly in bladder squamous cell carcinoma cases. Moreover, basic fibroblast growth factor and hepatocyte growth factor significantly correlated with tumor grade. Angiogenic markers showed significant association with clinical stage.Conclusions: Quantitative measurement of urinary angiogenic factors in voided urine samples by enzyme-linked immunosorbent assay was reliable. The sensitivity of basic fibroblast growth factor and hepatocyte growth factor was superior to that of the other investigated markers and of cytology in low grade and early stage cases, suggesting their convenience as sensitive, noninvasive diagnostic and screening tools for bladder cancer.

Purpose: New, noninvasive methods are needed for the diagnosis, followup and screening of patients with bladder cancer. Three methods of detecting telomerase were evaluated in this aspect.Materials and Methods: This study included 200 patients diagnosed with bladder carcinoma, 85 with benign bladder lesions and 30 healthy individuals who served as the control group. All underwent serological. schistosomiasis antibody assay in serum, urine cytology and estimation of relative telomerase activity by telomeric repeat amplification protocol, human telomerase RNA by reverse transcriptase-polymerase chain reaction and human telomerase reverse transcriptase by realtime reverse transcriptase-polymerase chain reaction in urothelial cells from voided urine.Results: The concordance between the positive rates of telomerase detected by the 3 methods was high (90% to 95%). Results were significantly higher in the malignant group than in the benign and control groups. There was a significant difference among the results of the 3 methods in relation to different clinicopathological factors. Overall the sensitivity of human telomerase reverse transcriptase for detecting bladder cancer was the highest compared to that of human telomerase RNA, relative telomerase activity and urine cytology (96%, 92%, 75% and 75%, respectively). Combinations of telomerase results with urine cytology were not useful except in cases of relative telomerase activity.Conclusions: Detection of human telomerase reverse transcriptase in urine by real-time polymerase chain reaction, followed by human telomerase RNA by reverse transcriptase-polymerase chain reaction, improves sensitivity and specificity for the diagnosis of bladder cancer. However, regarding cost-effectiveness, human telomerase RNA is superior.

Objectives: TIMPs control the activity of MMPs, one of the key molecules for tumor invasion and metastasis. The aim of this study was to assess the usefulness of MMP-2 and MMP-9 in relation to their inhibitor (TIMP2) as noninvasive diagnostic tests for bilharzial bladder cancer.Material and methods: Voided urine samples were provided from 244 subjects (154 bladder cancer [136 bilharzial]; 60 benign urologic disorders; 30 healthy volunteers). Urine sediment was used for cytology, and the supernatant for estimation of MMPs and TIMP-2 by ELISA and gelatin zymography.Results: The best cut-off values for the investigated markers were determined by ROC curve. Positivity rates and median levels for MMP-2, MMP-9, TIMP-2, MMP-2/TIMP-2, and MMP-9/TIMP-2 showed significant difference among the three investigated groups (p < 0.001). MMP-9 and MMP-2/TIMP-2 were related to pathologic type, MMP-2/TIMP-2 was inversely related to the grade, and MMP-9/TIMP-2 was related to bilharziasis (p < 0.05). MMP zymography results were comparable to those from ELISA.Conclusion: The sensitivity and specificity of MMP zymography, MMP-9/ TIMP-2 ratio, and MMP-2/TIMP2 ratio were superior among all investigated parameters; furthermore, combined testing of cytology with them improves the sensitivity even in superficial and low-grade tumors. (c) 2007 European Association of Urology. Published by Elsevier B.V. All rights reserved.

BACKGROUND. A new, sensitive, noninvasive method for the detection of urothelial carcinomas of the urinary bladder would open new possibilities in both the diagnosis and followup of patients.METHODS. This study included 228 patients diagnosed with bladder carcinoma, 68 patients with benign bladder lesions, and 44 healthy persons served as the control group. All were subjected to: serologic schistosomiasis antibody assay in serum, urine cytology, estimation of urine hyaluronic acid (HA) by enzyme-linked immunosorbent assay, and detection of CK-20 and hyaluronidase (HAase) by reverse transcription polymerase chain reaction (RT-PCR) in urothelial cells from voided urine.RESULTS. HA mean rank was higher in benign and malignant groups than in the healthy group (P < 0.0001) and was significantly related to tumor grade (P = 0.021). HA best-cutoff, determined using receiver operating characteristic curve to discriminate between malignant and nonmalignant groups, was 58.5 units/mg protein at 85.8% sensitivity and 60.7% specificity. HAase RNA showed superior sensitivity (90.8%) over cytology (68.9%) and CK-20 (78.1%) with specificity of 93.4%, 98.1% and 80.2%, respectively. The sensitivity reached 94.7% at a specificity of 91.5% when combined with CK-20. All 4 of the investigated markers were related to grade at P < 0.05. Whereas only HAase and CK-20 were significantly related to stage (P < 0.05). As to schistosomiasis, only HAase RNA positivity was significantly associated (P = 0.038).CONCLUSIONS. HAase RNA is a promising noninvasive test with high sensitivity and specificity in bladder carcinoma detection. (c) 2005 American Cancer Society.

The invasive potential of epithelial ovarian cancer is the main factor determining its biological behavior. Ets-1 transcription factor is involved in the activation of several proteases participating in tumor invasion and metastasis. The balance of matrix metalloproteinase-2 and its inhibitor is important in the regulation of tumor invasion. This study included 31 tumors from patients with epithelial ovarian cancer in different stages, and 20 tissue samples from benign ovarian lesions as a control group. Ets-1 was assessed using immunohistochemistry. Matrix metalloproteinase-2 (MMP-2) and the MMP-2 inhibitor (TIMP-2) were measured in the cytosolic fractions using enzyme immunoassay. MMP-2 results were confirmed by gelatin zymography. ETS-1 was expressed only in the malignant group. MMP-2 and the MMP-2:TIMP-2 ratio were significantly higher in the malignant group (p=0.045 and 0.026, respectively) with significant correlation to stage and poor survival above the specified cut-off values (p=0.006 and 0.002, respectively). Log rank of Kaplan-Meier survival analysis was significant for FIGO stage, MMP-2 and the MMP-2:TIMP-2 ratio (p<0.05). Multivariate analysis demonstrated that the MMP-2:TIMP-2 ratio is an independent prognostic parameter. Ets-1 could be a future target for therapeutic strategies. Moreover, the MMP-2:TIMP-2 ratio might serve as a new independent prognostic indicator of poor prognosis in epithelial ovarian cancer patients.

Objectives: This study was undertaken to evaluate the diagnostic efficacy of telomerase in urine, and bladder wash and also the matrix metalloproteinase-9 (MMP-9) in urine, compared with voided urine cytology (VUC) and bladder wash cytology (BWC) for the detection of bladder cancer cells.Material and Methods: A total of 110 subjects provided a single preoperative voided morning urine sample for telomerase, matrix metalloproteinase-9 (MMP-9) and cytology. Bladder wash samples were obtained for telomerase and cytology. Cystoscopy was done for all patients as the reference standard for the identification of bladder cancer. Biopsy of any suspicious lesion was performed for histopathological examination. Of 110 cases 73 were histologically diagnosed as bladder cancer, whereas the remaining 16 had benign urological disorders. A group of 21 healthy volunteers were also enrolled in this study.Results: The optimal threshold values for telomerase activity in urine, bladder wash and MMP-9 were calculated by receiver-operator characteristics (ROC) curves as 0.05, 0.088 and 0.51 (ng/ml), respectively. The levels and the positivity rates of the 2 parameters were significantly higher in the malignant group compared to either the benign group or normal controls. Of the entire group, telomerase activity in urine, bladder wash, and MMP-9 were positive in 92%, 87% and 61%, respectively in bladder cancer patients with positive cytology. Moreover, these positive rates for them were significantly higher in bilharzial bladder cancer cases (88%, 89%, 69%, respectively) compared to non-bilharzial cases (50%, 62.5%, 50%). The overall sensitivity and specificity were 83% and 88.6%, 86.3% and 78.3% for telomerase activity in urine, and in bladder wash, respectively; 66.6% and 80% for MMP-9 and 58.5% and 100% for voided urine cytology and 64.4% and 100% for bladder wash cytology. Combined sensitivity of VUC with the 2 biomarkers together was higher than either combined sensitivity of VUC with one of the biomarkes or than that of the biomarker alone.Conclusions: Our data indicate that urinary telomerase and MMP-9 had superior sensitivities over VUC. The combined use of markers increased the sensitivity of cytology from 58.46% to 95%. The higher sensitivities of markers in bilharzial bladder cancer than non-bilharzial type highlight their clinical utility in screening patients with urinary bilharziasis. (C) 2003 Elsevier B.V. All rights reserved.

The role of urokinase plasminogen activator receptor (uPAR) and c-erbB-2 in breast and ovarian cancer was investigated. Eighty patients of breast and ovarian cancer and benign lesions, as well as twenty normal controls were evaluated for the expression of c-erbB-2 by Western blotting and uPAR levels by ELISA. The c-erbB-2 and uPAR showed a significant increase in both types of cancer investigated compared to normal control and benign lesions. The frequency of c-erbB-2 was significantly higher in breast cancer lesions (p < 0.01). Levels of CA15.3 in breast cancer and CA125 in ovarian cancer were significantly higher in cases expressing c-erbB-2 (p < 0.01) than in negative c-erbB-2 cases. The uPAR showed a significant positive correlation with advanced stages of breast cancer (r = 0.7971) and ovarian cancer (r = 0.83662), while significant correlations were found for CA15.3 in breast cancer (r = 0.64967) and CA125 in ovarian cancer (r = 0.83996). Taken together, our data suggest that the c-erbB-2 and uPAR in the sera of ovarian and breast cancer act as valuable markers for the evaluation of the patients preoperatively.