Ahmed Barakat

Human cytomegalovirus infection inhibits response of chronic hepatitis-C-virus-infected patients to interferon-based therapy

Background and Aim:  Cytomegalovirus (CMV) is a ubiquitous pathogen that infects the majority of humans. Co‐infection of CMV and hepatitis C virus (HCV) may deteriorate the prognosis of HCV‐infected patients. This study was conducted to examine the role of CMV reactivation in determining the response rate to treatment with interferon and ribavirin therapy in chronic HCV patients.

Disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma is associated with increasing DNA promoter methylation

BACKGROUND Changes in DNA methylation patterns are believed to be early events in hepatocarcinogenesis. A better understanding of methylation states and how they correlate with disease progression will aid in finding potential strategies for early detection of HCC. The aim of our study was to analyze the methylation frequency of tumor suppressor genes, P14, P15, and P73, and a mismatch repair gene (O6MGMT) in HCV related chronic liver disease and HCC to identify candidate epigenetic biomarkers for HCC prediction. MATERIALS AND METHODS 516 Egyptian patients with HCV-related liver disease were recruited from Kasr Alaini multidisciplinary HCC clinic from April 2010 to January 2012. Subjects were divided into 4 different clinically defined groups - HCC group (n=208), liver cirrhosis group (n=108), chronic hepatitis C group (n=100), and control group (n=100) - to analyze the methylation status of the target genes in patient plasma using EpiTect Methyl qPCR Array technology. Methylation was considered to be hypermethylated if >10% and/or intermediately methylated if >60%. RESULTS In our series, a significant difference in the hypermethylation status of all studied genes was noted within the different stages of chronic liver disease and ultimately HCC. Hypermethylation of the P14 gene was detected in 100/208 (48.1%), 52/108 (48.1%), 16/100 (16%) and 8/100 (8%) among HCC, liver cirrhosis, chronic hepatitis and control groups, respectively, with a statistically significant difference between the studied groups (p-value 0.008). We also detected P15 hypermethylation in 92/208 (44.2%), 36/108 (33.3%), 20/100 (20%) and 4/100 (4%) , respectively (p-value 0.006). In addition, hypermethylation of P73 was detected in 136/208 (65.4%), 72/108 (66.7%), 32/100 (32%) and 4/100 (4%) (p-value <0.001). Also, we detected O6MGMT hypermethylation in 84/208 (40.4%), 60/108 (55.3%), 20/100 (20%) and 4/100 (4%), respectively (p value <0.001. CONCLUSIONS The epigenetic changes observed in this study indicate that HCC tumors exhibit specific DNA methylation signatures with potential clinical applications in diagnosis and prognosis. In addition, methylation frequency could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC. We can conclude that methylation processes are not just early events in hepatocarcinogenesis but accumulate with progression to cancer.

Antigenic diversity and cross-reactivity of avian influenza H5N1 viruses in Egypt between 2006 and 2011

Influenza epidemics are a major health concern worldwide. Highly pathogenic avian influenza (HPAI) H5N1 viruses in Egypt have been subject to rapid genetic and antigenic changes since the first outbreak in February 2006 and have been endemic in poultry in Egypt since 2008. In this study, 33 H5N1 viruses isolated from avian hosts were antigenically analysed by using a panel of eight mAbs raised against the A/Viet Nam/1203/04 (H5N1; clade 1) and A/bar-headed goose/Qinghai-lake/1A/05 (H5N1; clade 2.2) influenza viruses. Rats were immunized with inactivated whole-virus vaccine produced by reverse genetics with the haemagglutinin and neuraminidase genes of eight antigenically different HPAI H5N1 virus isolates and six internal genes from A/Puerto Rico/8/1934 (PR8) to produce polyclonal antibodies. Cross-reactivity between the obtained polyclonal antibodies and the isolated viruses was assayed. Antigenic cartography of the isolated viruses showed that three antigenic clusters were defined based on haemagglutination inhibition (HI) analysis using mAbs and the majority of viruses isolated in 2010 and 2011 fell into two of these clusters. An antigenic map based on polyclonal rat antisera showed that all virus isolates fell within one extended cluster. Accordingly, continuous surveillance and antigenic characterization will help us determine which virus isolate(s) should be used in poultry vaccine preparation.

Generation of a reassortant avian influenza virus H5N2 vaccine strain capable of protecting chickens against infection with Egyptian H5N1 and H9N2 viruses

BACKGROUND Avian influenza H5N1 viruses have been enzootic in Egyptian poultry since 2006. Avian influenza H9N2 viruses which have been circulating in Egyptian poultry since 2011 showed high replication rates in embryonated chicken eggs and mammalian cells. METHODS To investigate which gene segment was responsible for increasing replication, we constructed reassortant influenza viruses using the low pathogenic H1N1 PR8 virus as backbone and included individual genes from A/chicken/Egypt/S4456B/2011(H9N2) virus. Then, we invested this finding to improve a PR8-derived H5N1 influenza vaccine strain by incorporation of the NA segment of H9N2 virus instead of the NA of H5N1. The growth properties of this virus and several other forms of reassortant H5 viruses were compared. Finally, we tested the efficacy of this reassortant vaccine strain in chickens. RESULTS We observed an increase in replication for a reassortant virus expressing the neuraminidase gene (N2) of H9N2 virus relative to that of either parental viruses or reassortant PR8 viruses expressing other genes. Then, we generated an H5N2 vaccine strain based on the H5 from an Egyptian H5N1 virus and the N2 from an Egyptian H9N2 virus on a PR8 backbone. This strain had better replication rates than an H5N2 reassortant strain on an H9N2 backbone and an H5N1 reassortant on a PR8 backbone. This virus was then used to develop a killed, oil-emulsion vaccine and tested for efficacy against H5N1 and H9N2 viruses in chickens. Results showed that this vaccine was immunogenic and reduced mortality and shedding. DISCUSSION Our findings suggest that an inactivated PR8-derived H5N2 influenza vaccine is efficacious in poultry against H5N1 and H9N2 viruses and the vaccine seed replicates at a high rate thus improving vaccine production.

Cyclooxygenase-2 expression is associated with elevated aspartate aminotransferase level in hepatocellular carcinoma

BACKGROUND Cyclooxygenase-2 (COX-2), the inducible rate-limiting enzyme of prostaglandins biosynthesis, is involved in the pathogenesis of many chronic inflammation-related human malignancies including hepatocellular carcinoma (HCC). However, its clinical significance in HCC remains obscure. The aim of our study was to evaluate COX-2 expression in HCC and correlate its expression to both clinicopathological parameters and patients survival. MATERIALS AND METHODS The present study was conducted on 17 HCC and 21 adjacent nontumor liver tissues obtained from 22 HCC patients underwent hepatectomy. Eight normal liver tissues taken from normal donors and HepG2 cells were used as controls. Total RNA was extracted and COX-2 mRNA was detected by reverse transcription polymerase chain reaction and correlated to the clinicopathological criteria and to patients survival. RESULTS COX-2 mRNA was detected in 58.8% of the HCC tissues and in 28.6% of the adjacent nontumor liver tissues. COX-2 expression was significantly associated with elevated levels of serum aspartate aminotransferase (AST) with high specificity for disease detection. There was no significance between COX-2 expression and any of the histopathological criteria. CONCLUSIONS COX-2 expression may be involved in HCC carcinogenesis with high specificity for disease detection. COX-2 expression is significantly associated with elevated AST levels indicating a mechanism that may correlate both markers. However COX-2 expression seems to be an independent factor with no correlation to any of the histopathological data or patients survival.

Transcriptional Dysregulation of Upstream Signaling of IFN Pathway in Chronic HCV Type 4 Induced Liver Fibrosis.

IFN orchestrates the expression of various genes to halt hepatitis C virus (HCV) replication with the possibility of either reduced or increased liver fibrosis; due to controlled viral replication or overproduction of inflammatory mediators, repectively. In this study, we examined the transcriptional profiling of type I IFN related genes in HCV-chronically infected patients with varying degrees of liver fibrosis. PCR array was used to examine the expression of 84 type I IFN related genes in peripheral blood mononuclear cells (PBMCs) RNA from 12 treatment-naïve chronic HCV patients (5 F0-F1 and 7 F2-F4) and 5 healthy subjects. We further validated our results by quantitative real time PCR (qRT-PCR) in 103 treatment-naïve chronic HCV patients (43 F0-F1 and 60 F2-F4) and 15 controls. PCR array data revealed dysregulation in TLR7 pathway. The expression of TLR7 was decreased by 4 folds and MyD88 was increased by 3 folds in PBMCs of F2-F4 patients when compared to the healthy volunteers (p = 0.03 and 0.002, respectively). In addition, IRF7 and TLR7 showed dramatic downregulation (6 and 8 folds, respectively) in F2-F4 patients when compared to F0-F1 ones. qRT-PCR confirmed the altered expression patterns of TLR7 and MyD88 in F2-F4 patients when compared to either controls or F0-F1 patients. However, by qRT-PCR, IRF7 and NF-κB1 (TLR7 pathway transcription factors) exhibited similar mRNA abundance among F2-F4 and F0-F1 patients. These results suggest that TLR7 and MyD88 are possible candidates as biomarkers for the progression of HCV-induced liver fibrosis and/ or targets for therapeutic intervention.

Molecular cloning and immunogenicity evaluation of rotavirus structural proteins as candidate vaccine.

The middle capsid protein of rotavirus, VP6, constitutes approximately 51% of the virion by weight. The high degree of identity (>87-99%) in the primary amino acid sequences of VP6 proteins from mammalian rotaviruses suggests VP6-based vaccines could potentially provide heterotypic protection. For this reason, significant effort has been directed toward producing recombinant rotavirus VP6 vaccines. We have cloned and expressed 155bp of the VP6 most frequent in Egyptian rotavirus sewage isolates, encoding the VP6 protein in the pET30b vector having an apparent molecular mass of 12.5kDa. The recombinant VP6 expressed by the transformants was detected by SDS-PAGE analysis. Rabbits immunized with the recombinant rotavirus expressed VP6 elicited VP6-specific IgG antibodies that provided significant reductions in the infectious units of the Egyptian rotavirus sewage isolate and human reference rotavirus Wa strain in vitro assay.

Gene expression profiling of circulating CD133+ cells of hepatocellular carcinoma patients associated with HCV infection

AIM Identifying the genetic expression profile of CD133+ cells from HCC patients compared to CD133+ cells from healthy volunteers that may contribute in hepatocarcinogenesis process. METHOD Circulating CD133+ cells were sorted from the peripheral blood of HCC patients as well as from healthy volunteers using magnetic activated cell sorting. The differential expression profile of stem cell related genes was performed using the Stem Cell PCR profiling assay. RESULTS Data analysis of stem cells related genes in CD133+ cells of the HCC group compared to the control group showed that; CCND2, COL1A1, CTNNA1, DLL3, JAG1, KRT15, MYC, NOTCH2, T and TERT were up-regulated (fold change=80, 68.6, 6.67, 7.22, 3.8, 15.2, 14.5, 105.6, 26.6 and 99 respectively while only CD3D was down-regulated (fold change=0.055) in HCC patients. However, after application of Beferroni correction to adjust P-value; KRT15 was the only gene that was significantly over expressed in CD133+ cells of HCC compared to control group (P-value=0.012). CONCLUSION KRT15 can be used to differentiate between circulating CD133+ cells from HCC group and control group. However, further study may be needed to confirm on the protein level.

Dysregulation of fibrosis related genes in HCV induced liver disease

BACKGROUND Liver fibrosis results from a wound healing response to chronic injury, which leads to excessive matrix deposition. Genome wide association studies have showen transcriptional dysregulation in mild and severe liver fibrosis. Recent studies suggested that genetic markers may be able to define the exact stage of liver fibrosis. AIM To define genes or genetic pathways that could serve as markers for staging or as therapeutic targets to halt progression of liver fibrosis. METHODS The study was performed on 105 treatment naïve HCV genotype 4 infected patients [F0-F2, n = 56; F3-F4, n = 49] and 16 healthy subjects. The study included PCR array on 84 fibrosis related genes followed by customization of a smaller array consisting of 11 genes that were designed on the bases of results obtained from the larger array. Genes that displayed significant dysregulation at mRNA levels were validated at protein levels. RESULTS AND DISCUSSION Two major pathways exhibited high dysregulation in early fibrosis as compared with controls or when compared with late fibrosis, these were the TGFβ - related pathway genes and Matrix - deposition associated genes. Hepatic stellate cell (HSC) activators i.e. TGFβ pathway genes [TGFβ1, 2 and 3, their receptors TGFβR1 and 2, signaling molecules SMAD genes and PDGF growth factors] were considerably over-expressed at transcriptional levels as early as F0, whereas expression of their inhibitor TGIF1 was simultaneously down regulated. Matrix proteins including collagen and MMPs were upregulated in early fibrosis whereas tissue inhibitors TIMPs 1 and 2 began over expression in late fibrosis. Expression at protein levels was concordant with RNA data excluding dysregulation at post transcriptional levels. CONCLUSION Since these 2 gene sets are closely interrelated regarding HSC activation and proliferation, we assume that the current findings suggest that they are favorable targets to further search for stage specific markers.

Operational Risk and Reputation in Financial Institutions: Does Media Tone Make a Difference?

Operational risk announcements are unexpected adverse media news that potentially harm the reputation of financial institutions. This paper examines the equity-based and debt-based reputational effects of financial sentiment tones in operational risk announcements and shows how such reputational effects are moderated by alternative sources of public information. Our analysis reveals that the net negative tone and litigious tone have adverse reputational effects, and the uncertainty tone mitigates the adverse reputational impact. Additionally, alternative, simultaneous sources of information neutralize the reputational effects of textual tones. First, third-party information about the event (i.e. regulatory announcements and final settlements) dissolves the favorable (adverse) reputational impact of the uncertainty tone (litigious tone). Second, loss amount disclosure and firm recognition substitute the reputational effects of the net negative tone and uncertainty tone only in Anglo-Saxon countries and market-based economies. Overall, our findings indicate that the reputational effects of the media materialize most when there is lack of certain, quantifiable and regulated public information about the operational risk event.