Shiu-Wan Chan

Detection of three types of hepatitis C virus in blood donors : investigation of type-specific differences in serologic reactivity and rate of alanine aminotransferase abnormalities

The serologic reactivity and epidemiology associated with different hepatitis C virus (HCV) variants were investigated in a cohort of 113 anti‐HCV‐positive donors. In Scotland, HCV type 1 accounted for one‐ half of all infections; 40 percent of subjects were infected with HCV type 3, and the remainder were infected with type 2. Reactivity with the NS‐4‐encoded antigens in the first‐generation anti‐c100 assay was absent in 68 percent of donors infected with types 2 and 3, as compared with 10 percent for those infected with type 1. Even when combined with surrogate marker testing, first‐generation tests would have failed to detect 12 percent of HCV‐infected blood donors. The age distribution, incidence of past infection with hepatitis B virus, and reported risk factors were similar in donors infected with types 1 and 3 (mean ages were 31.9 and 29.9; 18 and 17.5% were positive for antibody to hepatitis B core antigen; and 47 and 48% had past intravenous drug abuse). However, the distributions of alanine aminotransferase levels were significantly different in those infected with type 3 (abnormally raised in 83%) and those infected with type 1 (55% abnormal alanine aminotransferase; p < 0.05) or type 2 (60%; p < 0.01) and those who were nonviremic (8%; p < 0.0001). These data suggest that HCV type 1 is the most common HCV infection in blood donors and that infection with HCV type 3 may be associated with more severe liver disease, because of more recent infection or because of a greater inherent pathogenicity of type 3 variants.

Cooperative Interactions Between RB and p53 Regulate Cell Proliferation, Cell Senescence, and Apoptosis in Human Vascular Smooth Muscle Cells From Atherosclerotic Plaques

Compared with vascular smooth muscle cells (VSMCs) from normal vessels, VSMCs from human atherosclerotic plaques proliferate more slowly, undergo earlier senescence, and demonstrate higher levels of apoptosis in culture. The tumor suppressor genes p105RB (retinoblastoma, acting through the E2F transcription factor family) and p53 regulate cell proliferation, cell senescence, and apoptosis in many cell types. We have therefore determined whether these stable growth properties of plaque VSMCs reflect altered activity of RB and/or p53. VSMCs were derived from coronary atherectomies or from normal coronary arteries from transplant recipients. Compared with normal VSMCs, plaque VSMCs showed a higher ratio of the active (hypophosphorylated) to the inactive (phosphorylated) form of RB and a lower level of E2F transcriptional activity. Cells were stably transfected with retrovirus constructs that inhibited RB or p53 alone or in combination. Suppression of RB alone increased rates of cell proliferation and apoptosis and inhibited cell senescence in normal VSMCs. Suppression of p53 and RB together had similar effects but, additionally, resulted in immortalization of normal VSMC cultures. In contrast, inhibition of RB binding to E2F or ectopic expression of E2F-1 in plaque VSMCs induced massive apoptosis, which required suppression of p53 to rescue cells. Suppression of RB and p53 together increased cell proliferation and delayed senescence but failed to immortalize plaque VSMCs. Inhibition of p53 alone had minimal effects on plaque VSMCs but increased the lifespan of normal VSMCs. We conclude that human plaque VSMCs have slower rates of cell proliferation and earlier senescence than do cells from normal vessels because of a defect in phosphorylation of RB. Furthermore, both disruption of RB/E2F and inhibition of p53 are required for plaque VSMCs to proliferate without apoptosis. This observation may explain the relatively low level of cell proliferation and high level of apoptosis seen in VSMCs in human atherosclerotic plaques.

Hepatitis C virus envelope proteins regulate CHOP via induction of the unfolded protein response

Unfolded protein response (UPR) is a cellular adaptive response that functions to reduce stress caused by malfolded proteins in the endoplasmic reticulum (ER). UPR can be induced under physiological or pathological conditions and is responsible for the pathogenesis of many human diseases. Hepatitis C virus (HCV) is a single‐stranded, positive‐sense RNA virus causing chronic diseases. Its genome encodes two envelope proteins E1 and E2, which mature in the ER to form a noncovalently bound, native complex and disulfide aggregates and have previously been shown to induce expression of the molecular chaperone immunoglobulin heavy chain binding protein. In this study, we show that HCV envelope protein expression regulates another stress indicator CCAAT/enhancer‐binding protein‐homologous protein (CHOP). The ER‐stress element and the activating transcription factor 4 element in the CHOP promoter were activated to a similar extent by HCV envelope protein expression. Using mouse embryonic fibroblasts deficient in the ER stress kinase RNA‐activated protein kinase‐like ER‐resident kinase (PERK), we showed that PERK was necessary and sufficient for activating the CHOP promoter. Expression of HCV E1 and/or E2 also induced splicing of X‐box binding protein 1 and transactivation of the unfolded protein response element, leading to the speculation that HCV E1 and E2 not only regulate the UPR but also ER‐associated degradation.

Serological responses to infection with three different types of hepatitis C virus

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Sensitivity to Fas-Mediated Apoptosis Is Determined Below Receptor Level in Human Vascular Smooth Muscle Cells

Despite Fas expression, many cells resist Fas-induced apoptosis. Although differences in surface Fas expression can explain Fas resistance, multiple proteins below receptor level also inhibit Fas-induced apoptosis. To examine the mechanism of Fas resistance, we studied Fas-induced apoptosis in human medial vascular smooth muscle cells (VSMCs) from healthy coronary arteries. VSMCs showed marked heterogeneity to Fas-induced apoptosis, exhibiting both Fas-resistant (98.1+/-2.3% viable, n = 4, P = NS) and Fas-sensitive (31.3+/-2.6% viable, n = 3, P<0.01) cells. Fas-resistant VSMCs expressed surface Fas and could recruit RIP, indicating that functional receptor complexes were formed. However, Fas-resistant cells showed reduced expression of FADD, Fas ligand, and caspases 3, 7, and 8 and increased expression of FLIP and c-IAP-1. Fas-induced apoptosis was associated with cleavage of caspase 3 and blocked by inhibitors of caspase 3 or 8 but not caspase 1, 6, or 7. Selective inhibition of caspase 3 or 8 by antisense transfection inhibited Fas-induced apoptosis, but their reexpression could not rescue the Fas-resistant phenotype. In vivo, medial VSMCs showed marked heterogeneity of expression of caspase 3. We conclude that Fas sensitivity is determined not only by expression of surface Fas but by differential expression of Fas-signaling proteins below receptor level. Subpopulations of cells within the same tissue have different sensitivities to apoptosis, determined by expression of specific death-signaling proteins.

Pyrimethamine resistant mutations in Plasmodium falciparum

Three mutations in Plasmodium falciparum yielding increased resistance to pyrimethamine were obtained following treatment with chemical mutagens and selection in presence of pyrimethamine. From parasite clone TM4/8.2 a mutant, TM4/8.2/4.1, was produced which raised pyrimethamine resistance about 500 times and was found to involve an amino acid change in the DHFR-TS enzyme molecule from Ser108 to Asn108. A clone of another isolate, T9/94, yielded a mutant, T9/94/300.300, raising pyrimethamine resistance about 10 times and involving an amino acid change from Ile164 to Met164. However, another mutant from T9/94, T9/94/M1-1(b3), although it raised the pyrimethamine resistance 100 times, did not involve any changes in the coding sequence of the DHFR-TS gene, but resulted in the production of about twice as much DHFR-TS enzyme as the original clone T9/94. No amplification of the DHFR-TS gene was detected. It is concluded that changes in pyrimethamine resistance of malaria parasites may arise in at least 2 ways: (1) by structural changes in the DHFR domain of the DHFR-TS gene (as previously found by other workers); (2) by other changes, possibly affecting the expression of the DHFR-TS gene. The relative importance of these 2 mechanisms in causing resistance in wild populations of P. falciparum is discussed.

HUMAN RECOMBINANT ANTIBODIES SPECIFIC FOR HEPATITIS C VIRUS CORE AND ENVELOPE E2 PEPTIDES FROM AN IMMUNE PHAGE DISPLAY LIBRARY

Hepatitis C virus (HCV) is the aetiological agent responsible for most cases of non-A non-B hepatitis. Hepatitis C is a disease of clinical importance because of its high infection rate in blood donors and its persistence as chronic infections which may lead to cirrhosis and hepatocellular carcinoma in the long term. The variability of the HCV genome has posed difficulties in serological detection and vaccine design. The recent advance in phage technology offers a means of cloning human anti-HCV antibodies of a defined specificity that may have potential therapeutic use. We now report the generation of a phage display library using the V(H) genes of a HCV-infected patient and the V(L) genes of two non-immune individuals. From this library we were able to obtain specific IgG single-chain Fvs (scFvs) that recognize viral core and envelope proteins by selection on synthetic peptides derived from the core sequence PKARRPEGRTWAQPG and the envelope E2 sequence RPIDDFDQGWGPITY. The specificity of the scFvs was demonstrated by their specific reactions with homologous peptides in ELISA and the specific blocking of scFv binding by homologous peptides, in a dose-dependent manner, in inhibition ELISA. The binding of the anticore 4c2 to homologous peptide was blocked by HCV-positive human sera in an antibody-concentration-dependent manner, suggesting that the scFv recognizes a similar if not identical epitope to those of one or more of the polyclonal antibodies present in the sera.

Unfolded protein response in hepatitis C virus infection

Hepatitis C virus (HCV) is a single-stranded, positive-sense RNA virus of clinical importance. The virus establishes a chronic infection and can progress from chronic hepatitis, steatosis to fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). The mechanisms of viral persistence and pathogenesis are poorly understood. Recently the unfolded protein response (UPR), a cellular homeostatic response to endoplasmic reticulum (ER) stress, has emerged to be a major contributing factor in many human diseases. It is also evident that viruses interact with the host UPR in many different ways and the outcome could be pro-viral, anti-viral or pathogenic, depending on the particular type of infection. Here we present evidence for the elicitation of chronic ER stress in HCV infection. We analyze the UPR signaling pathways involved in HCV infection, the various levels of UPR regulation by different viral proteins and finally, we propose several mechanisms by which the virus provokes the UPR.

Effects of hepatitis C virus envelope glycoprotein unfolded protein response activation on translation and transcription.

The hepatitis C virus (HCV) envelope glycoproteins have been shown to cause ER stress and induce the unfolded protein response (UPR). Using a bicistronic reporter, we show that the envelope glycoproteins repressed both cap-dependent and HCV IRES-mediated translation in HeLa cells but displayed a differential repression of cap-dependent translation in Huh-7 cells. In contrast, the envelope glycoproteins repressed E2F transcriptional activity in both HeLa and Huh-7 cells and caused increased accumulation of the underphosphorylated retinoblastoma protein. Expression of the envelope glycoproteins induced eIF2α phosphorylation, suggesting a role of the UPR in regulating translation and E2F transcriptional activity. The envelope glycoproteins also enhanced transcriptional activity from the COX-2 promoter and endogenous COX-2 expression in HeLa cells, but not in Huh-7 cells. Together, these results suggest that the envelope glycoproteins may assume more functional roles in viral replication and host cell interactions.

Human recombinant single‐chain antibody fragments, specific for the hypervariable region 1 of hepatitis C virus, from immune phage‐display libraries

The hypervariable region 1 (HVR1) of hepatitis C virus (HCV) may contain a potential neutralization site and the generation of human single‐chain antibody fragments (scFv) to HVR1 may therefore provide a useful tool for the study of HCV. In this report, we have isolated and characterized three anti‐HVR1 scFv clones from two patient‐derived phage‐displayed libraries and HCV HVR1 peptides. scFv S52/20 and S53/6 were selected with serologically cross‐reactive HVR1 peptides. scFv p3f10 was obtained by screening the library from patient MH with an autologous HVR1 peptide. Nucleotide sequencing showed that the VH chains and Vκ chains of all three scFv antibodies were derived from VH3 and Vκ1 family germline V‐genes, respectively. The specificity and affinity of the recombinant scFv antibodies were examined by enzyme‐linked immunosorbent assay (ELISA) and an affinity biosensor, using HVR1 peptides. S52/20 scFv binding to S52 HVR1 peptide was blocked by preincubation with soluble peptide S52 and was partially competed by one of three HCV‐infected patient sera. In addition, scFv S52/20 blocked the binding of HCV‐susceptible Molt‐4 cells to immobilized S52 peptide. This study demonstrates that recombinant human scFv antibodies to HCV HVR1 can be produced in vitro and directly confirms that HVR1 of HCV elicits highly specific antibodies. The very high specificity of these antibodies to HVR1 may limit their potential use in passive immunization therapy against HCV, and further engineering of the scFvs needs to be performed to generate broad‐spectrum blocking scFvs.