Decreased level of intracellular cholesterol in peripheral blood mononuclear cells is associated with chronic hepatitis C virus infection

Peripheral blood mononuclear cells (PBMCs) constitute the main extrahepatic place of, hepatitis C virus (HCV) replication. We aimed to determine the impact of CHC infection and microRNA-, 122 expression on cholesterol expression in PBMCs. HCV RNA strand, intracellular cholesterol, HMGCoA, reductase and miR-122 expression in PBMC were determined in 54 CHC patients. The study shows that significant decrease of intracellular cholesterol level in PBMC (p = 0.000000), accompanied by serum hypocholesterolemia is the characteristic feature of chronic hepatitis C infection. Although, microRNA-122 expression was detectable in PBMCs of CHC patients (52.5%), the alteration of intracellular cholesterol level was independent of miR-122 expression.     

The impact of chronic hepatitis C infection on cholesterol metabolism in PBMCs is associated with microRNA-146a expression

Chronic hepatitis C (CHC) infection is known to induce important changes in host cholesterol metabolism. MicroRNAs (miRNAs) regulate the expression of many genes and, in consequence, control various processes, including human metabolism and response to viral infection. Recently, the alteration of the immune-associated miR-146a, which is abundantly present in peripheral blood mononuclear cells (PBMCs), was found in some viral infections. The study aimed to analyse the influence of hepatitis C virus (HCV) infection on miR-146a expression in PBMCs in vivo and in vitro, as well as to assess the possible impact of miR-146a alteration on the intracellular cholesterol level in PBMCs. Blood samples collected from 42 healthy donors and 72 CHC patients were the source of materials. HCV RNA, intracellular cholesterol level and miR-146a expression were determined in PBMCs, as well as HCV genotype and interferon (IFN)α concentration in sera. The influence of miR-146a inhibition on cholesterol expression in PBMCs was analysed in vitro after transient cell transfections with mirVana? anti-miR-146a Inhibitor. Our data demonstrated an alteration of miR-146a and intracellular cholesterol expression in PBMCs and of IFNα concentration in sera of genotype 1, HCV-infected patients compared to the healthy donors. Also, in cultured PBMCs, miR-146a expression and intracellular cholesterol level were significantly decreased in CHC patients compared to the healthy donors. In vitro blockage of miR-146a expression in PBMCs of CHC patients greatly impaired intracellular cholesterol expression. In these conditions, miR-146a expression was positively correlated with the intracellular cholesterol level. These results suggest that genotype 1 HCV infection may alter miR-146a expression in PBMCs and, consequently, contribute to the observed dysregulation of cholesterol synthesis.     

MicroRNA‐155 and microRNA‐196b: promising biomarkers in hepatitis C virus infection?

MicroRNAs (miRNAs) are a class of small, evolutionarily conserved, noncoding RNA that regulate several important cellular processes. The versatility of these molecules allowed the accurate predictions that they would also affect the replication and life cycle of HCV. In this review, emphasis has been given to two selected miRNAs: miR‐155 and miR‐196b. Recent data indicate that miR‐155 is overexpressed in HCV‐infected patients, inducing an inflammatory state, and promoting virus replication and persistence even after the completion of antiviral treatment. It is also associated with the increased proliferation and inhibited apoptosis of hepatocytes, which promotes the growth of liver tumors. In contrast, miR‐196b is reported as a factor inhibiting HCV replication with cytoprotective, anti‐inflammatory, and antioxidant properties. Growing evidence suggests that these molecules could be used as potential prognostic and predictive factors and their antagonists or mimics as a promising therapeutic approach in HCV‐infected patients. Copyright © 2014 John Wiley & Sons, Ltd.     

Lack of BIC and microRNA miR-155 expression in primary cases of Burkitt lymphoma

We previously demonstrated high expression of primary-microRNA BIC (pri-miR-155) in Hodgkin lymphoma (HL) and lack of expression in most non-Hodgkin lymphoma subtypes including some Burkitt lymphoma (BL) cases. Recently, high expression of BIC was reported in BL in comparison to pediatric leukemia and normal peripheral-blood samples. In this study, we extended our series of BL cases and cell lines to examine expression of BIC using RNA in situ hybridization (ISH) and quantitative RT-PCR (qRT-PCR) and of miR-155 using Northern blotting. Both BIC RNA ISH and qRT-PCR revealed no or low levels of BIC in 25 BL tissue samples [including 7 Epstein-Barr virus (EBV)-positive cases] compared to HL and normal controls. In agreement with these findings, no miR-155 was observed in BL tissues. EBV-negative and EBV latency type I BL cell lines also showed very low BIC and miR-155 expression levels as compared to HL cell lines. Higher levels of BIC and miR-155 were detected in in vitro transformed lymphoblastoid EBV latency type III BL cell lines. An association of latency type III infection and induction of BIC was supported by consistent expression of BIC in 11 and miR-155 in 2 posttransplantation lymphoproliferative disorder (PTLD) cases. In summary, we demonstrated that expression of BIC and miR-155 is not a common finding in BL. Expression of BIC and miR-155 in 3 latency type III EBV-positive BL cell lines and in all primary PTLD cases suggests a possible role for EBV latency type III specific proteins in the induction of BIC expression.     

Expression of CD81, SR‐BI and LDLR in lymphocytes and monocytes from patients with classic and occult hepatitis C virus infection

CD81, the scavenger receptor‐BI (SR‐BI) and the low‐density lipoprotein receptor (LDLR) are involved in peripheral blood mononuclear cells (PBMCs) hepatitis C virus (HCV) entry. To investigate if these molecules are altered by HCV, 20 controls and 66 patients: 37 untreated and 29 sustained virological responders, were studied. CD81 and LDLR expression, measured the percentage of cells expressing the HCV‐receptors and their mean fluorescence intensity (MFI), was analyzed on lymphocytes and monocytes, as well as SR‐BI on monocytes by flow cytometry. RNA was extracted from PBMCs and detection of the HCV‐RNA positive and negative strands was performed by strand‐specific RT‐PCR. A statistically significant increase of CD81 expression was observed on lymphocytes, a higher percentage of LDLR on lymphocytes and monocytes, as well as SR‐BI on monocytes was found in the patients as compared to the controls (P < 0.05 in all cases). Untreated patients showed a higher percentage of LDLR⁺ lymphocytes than sustained virological responders (P = 0.025). Nineteen sustained virological responders bore the HCV‐RNA positive strand in PBMCs; nine of them the negative strand too. Sustained virological responders with occult infection and viral replication, showed a higher expression of LDLR on lymphocytes (P < 0.05) and a higher LDLR MFI on monocytes (P = 0.011) than those without viral replication. In conclusion, HCV exposure modifies expression levels of the receptors studied, being LDLR related with HCV replication, not only in the classic but also in the occult infection. J. Med. Virol. 84:1727–1736, 2012. © 2012 Wiley Periodicals, Inc.     

Quantitation of replication of the HCV genome in human livers with end‐stage cirrhosis by strand‐specific real‐time RT‐PCR assays: Methods and clinical relevance

HCV replicates in liver via an intermediate negative strand RNA. To study the relevance of HCV genome replication, quantitative strand‐specific HCV real‐time RT‐PCR assays were developed and applied to livers explanted because of end‐stage cirrhosis. The assays have broad ranges of determination and a high reproducibility and accuracy. Analysis of five different samples showed an even distribution of HCV genomes in four livers. Hepatic concentrations of positive (PS)‐ and negative (NS)‐strand RNA did correlate with each other, with PS/NS ratios ranging between 3 and 340. Hepatic concentrations of HCV‐PS or ‐NS RNA did not correlate with serum HCV‐RNA levels or with genotypes. A high HCV envelope‐2 protein expression correlated with a low NS concentration. HCV‐PS and ‐NS levels, E2 protein expression and genotype did not correlate with biochemical tests or with histological changes in the explanted liver, but the ratio NS/PS, a marker of viral replication, correlated with the severity of the recurrent post‐transplant hepatitis caused by HCV. This suggests the existence of an extra‐hepatic location of HCV with comparable viral replication rate being responsible for the infection of the newly transplanted liver. J. Med. Virol. 81:1569–1575, 2009. © 2009 Wiley‐Liss, Inc.     

The role of microRNAs in hepatitis C virus RNA replication

Replication of hepatitis C virus (HCV) RNA is influenced by a variety of microRNAs, with the main player being the liver-specific microRNA-122 (miR-122). Binding of miR-122 to two binding sites near the 5′ end of the 5′ untranslated region (UTR) of the HCV genomic RNA results in at least two different effects. On the one hand, binding of miR-122 and the resulting recruitment of protein complexes containing Argonaute (Ago) proteins appears to mask the viral RNA′s 5′ end and stabilizes the viral RNA against nucleolytic degradation. On the other hand, this interaction of miR-122 with the 5′-UTR also stimulates HCV RNA translation directed by the internal ribosome entry site (IRES) located downstream of the miR-122 binding sites. However, it is suspected that additional, yet undefined roles of miR-122 in HCV replication may also contribute to HCV propagation. Accordingly, miR-122 is considered to contribute to the liver tropism of the virus. Besides miR-122, let-7b, miR-196, miR-199a* and miR-448 have also been reported to interact directly with the HCV RNA. However, the latter microRNAs inhibit HCV replication, and it has been speculated that miR-199a* contributes indirectly to HCV tissue tropism, since it is mostly expressed in cells other than hepatocytes. Other microRNAs influence HCV replication indirectly. Some of those are advantageous for HCV propagation, while others suppress HCV replication. Consequently, HCV up-regulates or down-regulates, respectively, the expression of most of these miRNAs.     

Application of circular consensus sequencing and network analysis to characterize the bovine IgG repertoire

Background

Co-infection with human immunodeficiency virus-1 (HIV-1) and hepatitis C virus (HCV) is associated with faster progression of liver disease and an increase in HCV persistence. However, the mechanism by which HIV-1 accelerates the progression of HCV liver disease remains unknown.

Results

HIV-1/HCV co-infection is associated with increased expression of interferon gamma-induced protein-10 (IP-10) mRNA in peripheral blood mononuclear cells (PBMCs). HCV RNA levels were higher in PBMCs of patients with HIV-1/HCV co-infection than in patients with HCV mono-infection. HIV-1 Tat and IP-10 activated HCV replication in a time-dependent manner, and HIV-1 Tat induced IP-10 production. In addition, the effect of HIV-1 Tat on HCV replication was blocked by anti-IP-10 monoclonal antibody, demonstrating that the effect of HIV-1 Tat on HCV replication depends on IP-10. Taken together, these results suggest that HIV-1 Tat protein activates HCV replication by upregulating IP-10 production.

Conclusions

HIV-1/HCV co-infection is associated with increased expression of IP-10 mRNA and replication of HCV RNA. Furthermore, both HIV-1 Tat and IP-10 activate HCV replication. HIV-1 Tat activates HCV replication by upregulating IP-10 production. These results expand our understanding of HIV-1 in HCV replication and the mechanism involved in the regulation of HCV replication mediated by HIV-1 during co-infection.     

丙型肝炎患者外周血单个核细胞HCV感染的电镜研究

目的 以常规电镜和免疫电镜技术,发现和证实慢性丙型肝炎患者外周血单个核细胞９ＰＢＭＣｓ）内丙型肝炎病毒（ＨＣＶ）颗粒。试图在病毒形态学和形态发生学上证实ＰＢＭＣｓ的ＨＣＶ感染和复制。方法 以逆转录多聚酶链反应（ＲＴ－ＰＣＲ）和免疫组织化学方法,分别检测２８例患者ＰＢＭＣｓ内ＨＣＶ ＲＮＡ和ＨＣＶＡｇ,对其中阳性标本重点进行电镜研究。结果 ＨＣＶ ＲＮＡ和ＨＣＶ Ａｇ阳性检出率分别为７７．２７％（１     

Detection of hepatitis C virus RNA in serum and peripheral blood mononuclear cells in patients with chronic hepatitis C treated with interferon alpha

PCR was used to detect hepatitis C virus (HCV) RNA in serum and peripheral blood mononuclear cells (PBMCs) for evaluation of a six-month course of Interferon therapy in 18 patients with histologically confirmed chronic hepatitis C. At follow-up six months after the end of therapy positive-stranded (genomic) and negative-stranded (anti-genomic, presumptive replicative intermediate) HCV RNA could be detected in PBMCs of all ten patients who either did not respond to therapy or suffered a relapse; genomic strand RNA was detected in five patients who responded but then relapsed. The study confirms that interferon therapy leads to inhibition of HCV replication but not eradication of the virus. Persistence of the virus at extrahepatic sites may explain its reactivation after cessation of interferon therapy.     

Identification and visualization of virus-like particles in peripheral blood mononuclear cells]

OBJECTIVE: PBMCs from patients with chronic hepatitis C were examined by electron microscopy (EM), immune EM (IEM) combined with immunohistochemistry to trace the infection and morphology of HCV in the PBMCs. METHODS: The PBMCs from 28 patients with chronic hepatitis C were analyzed firstly for HCV RNA and HCV antigens by RT-PCR and immunohistochemistry respectively. The PBMCs with positive HCV RNA and HCV antigens were then observed by electron microscopy. RESULTS: The positive rate of HCV RNA and HCV antigens were 77.27% (17/22) and 75.00% (21/28), respectively. Two types of polymorphic HCV-like particles with diameter of approximately 65 nm and 110 nm were observed in cytoplasmic vesicles of the PBMCs from HCV Ag positive patients. 10 patients with HCV Ag over expression were studied by electron microscopy and immune election microscopy. The budding phenomenon of the particles could be also visualized in the cytoplasmic vesicles of the PBMCs. Some ultrastructural changes showed an increased ratio of cytoplasm to nucleus and pyknosis of nucleus was also suggestive of the virus infection. CONCLUSIONS: The EM study demonstrated the infection and replication of HCV in the PBMCs of chronic hepatitis C patients by morphology and morphogenesis.     

Fibroblast-derived exosomal miRNA-133 promotes cardiomyocyte-like differentiation

ObjectiveTo investigate the role of exosomal miRNA-133 secreted by cardiac fibroblasts (CFs) in promoting cardiomyocyte differentiation.MethodsNeonatal rat CFs were cultured in vitro, and the cultured CFs were divided into three groups as follows: induction, miRNA-133 high expression, and miRNA-133 inhibition. miRNA-133 was transfected into CFs with lentivirus as a vector. CFs were transfected with the miRNA-133 inhibitor, and the markers of cardiomyocyte were detected through immunofluorescence staining, Western blotting, and real-time quantitative polymerase chain reaction (qRT-PCR) at 3, 8, and 14 days, respectively. The expression levels of cardiac troponin T (cTnT) and cardiac actin (α-actin) were determined, and qRT-PCR was used to detect the expression of miRNA-133 in the fibroblast exosomes.ResultsCFs subjected to immunofluorescence staining expressed vimentin and discoid domain receptor 2. The exosomes secreted by CFs were observed as small vesicles of 30–100 nm via transmission electron microscopy, and Western blotting was used to detect exosome-specific protein CD63 and CD9 expression. The expression levels of cTnT, α-actin, and exosomal miRNA-133 secreted into the supernatant of the miRNA-133 high-expression group increased gradually at different time points and reached the highest level at 14 days. The expression levels of cTnT, α-actin, and exosome miRNA-133 in the miRNA-133 inhibition group were the lowest.ConclusionThe exosomal miRNA-133, which is derived from CFs, can promote the differentiation of fibroblasts into cardiomyocyte-like cells.     

HCV in peripheral blood mononuclear cells are predominantly carried on the surface of cells in HIV/HCV co-infected individuals

HCV replication in extra-hepatic reservoirs has been suggested to occur in many tissues including PBMCs. A recent study showed evidence for compartmentalization and evolution of HCV in PBMCs. However, the cells that support HCV replication in PBMCs have not been identified. In this study we have fractionated the PBMC from HIV/HCV co-infected patients into T, monocytes, B and NK cells, and most of the HCV was located in CD3-cell fractions. Protease treatment of PBMCs to remove cell surface receptors resulted in the loss of HCV RNA suggesting that most of the HCV is present on the cell surface. PBMCs were treated by freeze-thaw nuclease method that would protect the HCV RNA in the virus but not the intracellular viral RNA. Data from this analysis support the conclusion that most of HCV is present on the cell surface. Even though the presence of minus strand RNA in PBMCs suggests that a low level HCV replication takes place within the PBMCs of HIV/HCV co-infected individuals, HCV in PBMC is present mainly on the surface of non-T cells, mostly on NK, monocytes and B cells. These results suggest a unique pathogenic role of NK, monocyte and B cells as carriers of HCV.     

Increased HDAC3 and decreased miRNA-130a expression in PBMCs through recruitment HDAC3 in patients with spinal cord injuries

The study was performed to investigate the molecular mechanism for SCI patients. The interaction between miRNA-130a and HDAC was demonstrated in PBMCs from SCI patients. Increased HDAC3 and decreased miRNA-130a were observed in PBMCs from AS patients. Next, HDAC3 loss-of-function or HAAC3 inhibition promoted the expression of miRNA-130a, and HDAC3 could be recruited to the promoter region of the gene, miRNA-130a, in PBMCs. In addition, linear regression analysis indicated that mRNA expression results were highly negative correlated between HDAC3 and miRNA-130a in PBMCs from SCI patients. Furthermore, miRNA-130a down expression increased the expression of HDAC3 in PBMCs. Loss-of-function of miRNA-130a promoted PBMCs apoptosis, but HDAC3 loss-of-function had no significant effect on the apoptotic cell. In addition, miR-130a overexpression decreased, whereas miR-130a inhibition increased, the expression of TNF-α in PBMCs. Furthermore, HDAC3 loss-of-function or HAAC3 inhibition associated with simultaneous up-regulation the expression of miR-130a and down-regulation the expression of TNF-α in PBMCs. In conclusion, HDAC3 regulated a distinct underlying molecular and pathogenic mechanism of SCI by forming a negative feedback loop with miR-130a and enhanced TNF-1α expression.     

A novel strand-specific RT-PCR for detection of hepatitis C virus negative-strand RNA (replicative intermediate): evidence of absence or very low level of HCV replication in peripheral blood mononuclear cells

Hepatitis C virus (HCV) is reported to be lymphotropic under certain circumstances. In order to evaluate viral replication in peripheral blood mononuclear cells (PBMCs), a novel strand-specific RT-PCR was developed for the determination of HCV negative-strand RNA. The detection limit of this strand-specific RT-PCR was 100 copies of HCV negative-strand RNA in the presence of 1 μg liver RNA and 10⁷–10⁸ copies of positive-strand RNA. False positive PCR signals occurred only when HCV positive-strand RNA exceeded 10⁹ copies. With this RT-PCR, the replicative-intermediates could be detected specifically in eight of ten liver tissues, but not in any samples of serum or plasma (0/65) of patients with chronic hepatitis C. When examining the PBMCs of 46 hepatitis C patients, including 12 patients who had undergone orthotopic liver transplantation, HCV negative-strand RNA was detected in only one patient (1/46). In addition, HCV replicative intermediates were not detected in PBMCs of patients using immunosuppressive agents. It is concluded that the replication of HCV in PBMCs is very unusual.