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In situ distribution of hepatitis C virus replicative-intermediate RNA in hepatic tissue and its correlation with liver disease 下载免费PDF全文
Chang M Marquardt AP Wood BL Williams O Cotler SJ Taylor SL Carithers RL Gretch DR 《Journal of virology》2000,74(2):944-955
Liver failure from chronic hepatitis C is the leading indication for liver transplantation in the United States. However, the pathogenesis of liver injury resulting from chronic hepatitis C virus (HCV) infection is not well understood. To examine the relationship between HCV replication in liver tissue and hepatocellular injury, a strand-specific in situ hybridization procedure was developed. The sensitivity and specificity of digoxigenin-labeled riboprobes were optimized by analyzing Northern blots and cell lines expressing HCV RNAs. For the current study, both genomic (sense) and replicative-intermediate (antisense) HCV RNAs were detected and quantified in 8 of 8 liver tissue specimens from infected patients versus 0 of 11 liver tissue specimens from noninfected controls. The distribution pattern for HCV replicative-intermediate RNA in liver was different from that for HCV genomic RNA. HCV genomic RNA was variably distributed throughout infected livers and was located primarily in the cytoplasm of hepatocytes, with some signal in fibroblasts and/or macrophages in the surrounding fibroconnective tissue. However, HCV replicative-intermediate RNA showed a more focal pattern of distribution and was exclusively localized in the cytoplasm of hepatocytes. There was no significant relationship between the distribution pattern for HCV genomic RNA and any indices of hepatocellular injury. However, a highly significant correlation was observed between the percentage of cells staining positive for replicative-intermediate RNA and the degree of hepatic inflammatory activity (P, < 0.0001). Furthermore, the ratio of cells staining positive for HCV replicative-intermediate versus genomic RNA correlated with the histological severity of liver injury (P, 0. 0065), supporting the hypothesis that active replication of HCV in liver tissue may be a significant determinant of hepatocellular injury. 相似文献
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Regina Helena Saramago Peralta Jorge Néstor Velásquez Flavia de Souza Cunha María Laura Pantano Fernando Campos Sodré Sidnei da Silva Osvaldo Germán Astudillo José Mauro Peralta Silvana Carnevale 《Memórias do Instituto Oswaldo Cruz》2016,111(1):30-36
The identification and characterisation of Cryptosporidiumgenotypes
and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding
in prevention and control strategies. The objective was to determine the genetic
diversity ofCryptosporidium in samples obtained from hospitals of
Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by
microscopy and TaqMan polymerase chain reaction (PCR) assays
forCryptosporidium detection, genotyped by nested-PCR-restriction
fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA
sequencing of the gp60 gene. Among the 89 samples from Rio de
Janeiro, Cryptosporidium spp were detected in 26 by
microscopy/TaqMan PCR. In samples from Buenos Aires,Cryptosporidium
was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the
nested-PCR-RFLP detected Cryptosporidium parvum,
Cryptosporidium hominis, and co-infections of both species. In
Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found
in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed
subtypes of Ia and IIa families were detected in the co-infections. C.
hominis was the species more frequently detected, and subtype family Ib
was reported in both countries. Subtype diversity was higher in Buenos Aires than in
Rio de Janeiro and two new subtypes were described for the first time. 相似文献
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Thaís Augusto Marinho Carmen Luci Rodrigues Lopes Sheila Araújo Teles Nádia Rúbia Silva Reis Megmar Aparecida dos Santos Carneiro Andreia Alves de Andrade Regina Maria Bringel Martins 《Memórias do Instituto Oswaldo Cruz》2013,108(4):519-522
The prevalence of hepatitis C virus (HCV) in a population of
recyclable waste collectors (n = 431) was assessed using a cross-sectional
survey in all 15 cooperatives in the city of Goiânia, Central-West Brazil. The
HCV prevalence was 1.6% (95% confidence interval: 0.6-3.6) and a history of
sexually transmitted infections was independently associated with this
infection. HCV RNA (corresponding to genotype 1; subtypes 1a and 1b) was
detected in five/seven anti-HCV-positive samples. Although the study population
reported a high rate (47.3%) of sharps and needle accidents, HCV infection was
not more frequent in recyclable waste collectors than in the general Brazilian
population. 相似文献
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Samantha Therezinha Almeida Pereira Leite Marilene Borges da Silva Marco Andrey Pepato Francisco José Dutra Souto Raquel Alves dos Santos Carmen Lucia Bassi-Branco 《Memórias do Instituto Oswaldo Cruz》2014,109(1):15-20
In this study, we analysed the frequency of micronuclei (MN), nucleoplasmic bridges
(NPBs) and nuclear buds (NBUDs) and evaluated mutagen-induced sensitivity in the
lymphocytes of patients chronically infected with hepatitis B virus (HBV) or
hepatitis C virus (HCV). In total, 49 patients with chronic viral hepatitis (28
HBV-infected and 21 HCV-infected patients) and 33 healthy, non-infected blood donor
controls were investigated. The frequencies (‰) of MN, NPBs and NBUDs in the controls
were 4.41 ± 2.15, 1.15 ± 0.97 and 2.98 ± 1.31, respectively. The frequencies of MN
and NPBs were significantly increased (p < 0.0001) in the patient group (7.01 ±
3.23 and 2.76 ± 2.08, respectively) compared with the control group. When considered
separately, the HBV-infected patients (7.18 ± 3.57) and HCV-infected patients (3.27 ±
2.40) each had greater numbers of MN than did the controls (p < 0.0001). The
HCV-infected patients displayed high numbers of NPBs (2.09 ± 1.33) and NBUDs (4.38 ±
3.28), but only the HBV-infected patients exhibited a significant difference (NPBs =
3.27 ± 2.40, p < 0.0001 and NBUDs = 4.71 ± 2.79, p = 0.03) in comparison with the
controls. Similar results were obtained for males, but not for females, when all
patients or the HBV-infected group was compared with the controls. The lymphocytes of
the infected patients did not exhibit sensitivity to mutagen in comparison with the
lymphocytes of the controls (p = 0.06). These results showed that the lymphocytes of
patients who were chronically infected with HBV or HCV presented greater chromosomal
instability. 相似文献
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From 2006 to 2010, hospitals in Hanoi treated 10 human patients for dirofilariasis. The worms were collected from parasitic places, and identification of the species was completed by morphology and molecular methods. Ten parasites were recovered either from the conjunctiva (n=9) or subcutaneous tissue (n=1). The parasites were 4.0-12.5 cm in length and 0.5-0.6 mm in width. Morphological observations suggested all parasites as Dirofilaria repens. Three of the 10 parasites (1 from subcutaneous tissue and 2 from eyes) were used for molecular confirmation of the species identification. A portion of the mitochondrial cox1 (461 bp) was amplified and sequenced. Nucleotide and amino acid homologies were 95% and 99-100%, respectively, when compared with D. repens (Italian origin, GenBank AJ271614; DQ358814). This is the first report of eye dirofilariasis and the second report of subcutaneous tissue dirofilariasis due to D. repens in Vietnam. 相似文献
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Agha S Al-Gendy M El-Fiky A El-Emshaty W 《Microbes and infection / Institut Pasteur》1999,1(13):1091-1094
The quantity of circulating hepatitis C virus (HCV) RNA, aminotransferases and the degree of liver cell injury in relation to HCV serotype have not been fully studied. In this work, we estimated the HCV RNA titre in serum and correlated the findings with levels of aminotransferases, gamma glutamyltransferase (GGT), and liver histopathological changes and with HCV serotype. HCV RNA was found in 22 out of 30 HCV-seropositive cases included in this study (73. 3%) and serotype 4 represented 90.9% (20/22). Levels of aminotransferases and GGT correlated with the levels of serum HCV RNA. Noticeably, GGT showed the highest positive correlation with the level of HCV RNA. Liver histopathological findings of 15 patients showed that eight had hepatocellular carcinoma and seven had cirrhosis. There was no significant difference between these two groups regarding levels of enzymes or serum HCV RNA titre. 相似文献
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Roles of the AX(4)GKS and arginine-rich motifs of hepatitis C virus RNA helicase in ATP- and viral RNA-binding activity 下载免费PDF全文
The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) possesses protease, nucleoside triphosphatase, and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase are not well-characterized. In this study, NS3 proteins with point mutations in the conserved helicase motifs were expressed in Escherichia coli, purified, and analyzed for their effects on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV cross-linking experiments indicate that the lysine residue in the AX(4)GKS motif is directly involved in ATP binding, whereas the NS3(GR1490DT) mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutation at Arg1488 (R1488A) and was also affected by the K1236E substitution in the AX(4)GKS motif and the R1490A and GR1490DT mutations in the arginine-rich motif. Previously, Arg1490 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the gamma-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding. 相似文献
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PAOLA A. FILOMENO KYUNG-PHIL KIM NARA YOON IRAN RASHEDI VICTOR DAYAN RITA A. KANDEL XING-HUA WANG TANIA C. FELIZARDO ELLIOT BERINSTEIN SALOMEH JELVEH ANDREA FILOMENO JEFFREY A. MEDIN PETER C. FERGUSON ARMAND KEATING 《Cytotherapy》2018,20(8):1001-1012
Background. Mesenchymal stromal cells (MSCs) promote wound healing, including after radiotherapy (RT) and surgery. The use of MSCs in regenerative medicine in the context of malignancy, such as to enhance wound healing post-RT/surgery in patients with soft tissue sarcomas (STSs), requires safety validation. The aim of this study was to determine the effects of human MSCs on STS growth in vitro and local recurrence and metastasis in vivo. Methods. Human primary STS and HT-1080 fibrosarcoma lines were transduced to express luciferase/eGFP (enhanced green fluorescent protein). Sarcoma cells were co-cultured or co-injected with bone marrow–derived MSCs for growth studies. Xenograft tumor models were established with STS lines in NOD/SCID/γcnull mice. To emulate a clinical scenario, subcutaneous tumors were treated with RT/surgery prior to MSC injection into the tumor bed. Local and distant tumor recurrence was studied using histology and bioluminescence imaging. Results. MSCs did not promote STS proliferation upon co-culture in vitro, which was consistent among MSCs from different donors. Co-injection of MSCs with sarcoma cells in mice exhibited no significant tumor-stimulating effect, compared with control mice injected with sarcoma cells alone. MSC administration after RT/surgery had no effect on local recurrence or metastasis of STS. Discussion. These studies are important for the establishment of a safety profile for MSC administration in patients with STS. Our data suggest that MSCs are safe in STS management after standard of care RT/surgery, which can be further investigated in early-phase clinical trials to also determine the efficacy of MSCs in reducing morbidity and to mitigate wound complications in these patients. 相似文献
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Miriam YH Ueda Paulo G Alvarenga Juliana M Real Eloisa de Sá Moreira Aripuan? Watanabe Ana Maria Passos-Castilho Matheus Vescovi Yana Novis Vanderson Rocha Adriana Seber Jose SR Oliveira Celso A Rodrigues Celso FH Granato 《Memórias do Instituto Oswaldo Cruz》2015,110(4):461-467
Human herpesvirus 6 (HHV-6) may cause severe complications after haematopoietic stem
cell transplantation (HSCT). Monitoring this virus and providing precise, rapid and
early diagnosis of related clinical diseases, constitute essential measures to
improve outcomes. A prospective survey on the incidence and clinical features of
HHV-6 infections after HSCT has not yet been conducted in Brazilian patients and the
impact of this infection on HSCT outcome remains unclear. A rapid test based on
real-time quantitative polymerase chain reaction (qPCR) has been optimised to screen
and quantify clinical samples for HHV-6. The detection step was based on reaction
with TaqMan® hydrolysis probes. A set of previously described primers and
probes have been tested to evaluate efficiency, sensitivity and reproducibility. The
target efficiency range was 91.4% with linearity ranging from 10-106
copies/reaction and a limit of detection of five copies/reaction or 250 copies/mL of
plasma. The qPCR assay developed in the present study was simple, rapid and
sensitive, allowing the detection of a wide range of HHV-6 loads. In conclusion, this
test may be useful as a practical tool to help elucidate the clinical relevance of
HHV-6 infection and reactivation in different scenarios and to determine the need for
surveillance. 相似文献
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《Bioorganic & medicinal chemistry letters》2014,24(17):4276-4280
Using our recently developed assay system for full-genome-length hepatitis C virus (HCV) RNA replication in human hepatoma-derived Li23 cells (ORL8), we identified 4-(1,1,1,3,3,3-hexafluoro-2-hydroxy-2-propyl)aniline analog 1a as a novel HCV inhibitor. Structural modifications of 1a provided a series of sulfonamides 7 with much more potent HCV RNA replication-inhibitory activity than ribavirin. Compound 7a showed an additive anti-HCV effect in combination with standard anti-HCV therapy (IFN-α plus ribavirin). Since 7a generated reactive oxygen species (ROS) in the ORL8 system and its anti-HCV activity was blocked by vitamin E, its anti-HCV activity may be mediated at least in part by ROS. 相似文献
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Klumpp K Lévêque V Le Pogam S Ma H Jiang WR Kang H Granycome C Singer M Laxton C Hang JQ Sarma K Smith DB Heindl D Hobbs CJ Merrett JH Symons J Cammack N Martin JA Devos R Nájera I 《The Journal of biological chemistry》2006,281(7):3793-3799
Hepatitis C virus (HCV) polymerase activity is essential for HCV replication. Targeted screening of nucleoside analogs identified R1479 (4'-azidocytidine) as a specific inhibitor of HCV replication in the HCV subgenomic replicon system (IC(50) = 1.28 microM) with similar potency compared with 2'-C-methylcytidine (IC(50) = 1.13 microM). R1479 showed no effect on cell viability or proliferation of HCV replicon or Huh-7 cells at concentrations up to 2 mM. HCV replicon RNA could be fully cleared from replicon cells after prolonged incubation with R1479. The corresponding 5'-triphosphate derivative (R1479-TP) is a potent inhibitor of native HCV replicase isolated from replicon cells and of recombinant HCV polymerase (NS5B)-mediated RNA synthesis activity. R1479-TP inhibited RNA synthesis as a CTP-competitive inhibitor with a K(i) of 40 nM. On an HCV RNA-derived template substrate (complementary internal ribosome entry site), R1479-TP showed similar potency of NS5B inhibition compared with 3'-dCTP. R1479-TP was incorporated into nascent RNA by HCV polymerase and reduced further elongation with similar efficiency compared with 3'-dCTP under the reaction conditions. The S282T point mutation in the coding sequence of NS5B confers resistance to inhibition by 2'-C-MeATP and other 2'-methyl-nucleotides. In contrast, the S282T mutation did not confer cross-resistance to R1479. 相似文献
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Lettuce‐produced hepatitis C virus E1E2 heterodimer triggers immune responses in mice and antibody production after oral vaccination 下载免费PDF全文
Jihong Liu Clarke Lisa Paruch Mihaela‐Olivia Dobrica Iuliana Caras Catalin Tucureanu Adrian Onu Sonya Ciulean Crina Stavaru Andre Eerde Yanliang Wang Hege Steen Sissel Haugslien Catalina Petrareanu Catalin Lazar Costin‐Ioan Popescu Ralph Bock Jean Dubuisson Norica Branza‐Nichita 《Plant biotechnology journal》2017,15(12):1611-1621
The hepatitis C virus (HCV) is a major etiologic agent for severe liver diseases (e.g. cirrhosis, fibrosis and hepatocellular carcinoma). Approximately 140 million people have chronic HCV infections and about 500 000 die yearly from HCV‐related liver pathologies. To date, there is no licensed vaccine available to prevent HCV infection and production of a HCV vaccine remains a major challenge. Here, we report the successful production of the HCV E1E2 heterodimer, an important vaccine candidate, in an edible crop (lettuce, Lactuca sativa) using Agrobacterium‐mediated transient expression technology. The wild‐type dimer (E1E2) and a variant without an N‐glycosylation site in the E2 polypeptide (E1E2?N6) were expressed, and appropriate N‐glycosylation pattern and functionality of the E1E2 dimers were demonstrated. The humoral immune response induced by the HCV proteins was investigated in mice following oral administration of lettuce antigens with or without previous intramuscular prime with the mammalian HEK293T cell‐expressed HCV dimer. Immunization by oral feeding only resulted in development of weak serum levels of anti‐HCV IgM for both antigens; however, the E1E2?N6 proteins produced higher amounts of secretory IgA, suggesting improved immunogenic properties of the N‐glycosylation mutant. The mice group receiving the intramuscular injection followed by two oral boosts with the lettuce E1E2 dimer developed a systemic but also a mucosal immune response, as demonstrated by the presence of anti‐HCV secretory IgA in faeces extracts. In summary, our study demonstrates the feasibility of producing complex viral antigens in lettuce, using plant transient expression technology, with great potential for future low‐cost oral vaccine development. 相似文献