MYC-Targeting Inhibitors Generated from a Stereodiversified Bicyclic Peptide Library.

Here, we present the second generation of our bicyclic peptide library (NTB), featuring a stereodiversified structure and a simplified construction strategy. We utilized a tandem ring-opening metathesis and ring-closing metathesis reaction (ROM-RCM) to cyclize the linear peptide library in a single step, representing the first reported instance of this reaction being applied to the preparation of macrocyclic peptides. Moreover, the resulting bicyclic peptide can be easily linearized for MS/MS sequencing with a one-step deallylation process. We employed this library to screen against the E363-R378 epitope of MYC and identified several MYC-targeting bicyclic peptides. Subsequent in vitro cell studies demonstrated that one candidate, NT-B2R, effectively suppressed MYC transcription activities and cell proliferation.

[Effect of Polygonati Rhizoma in improving pyroptosis injury of diabetic macroangiopathy via NLRP3/caspase-1/GSDMD pathway].

This study aims to explore the influence of Polygonati Rhizoma on the pyroptosis in the rat model of diabetic macroangiopathy via the NOD-like receptor thermal protein domain associated protein 3(NLRP3)/cysteinyl aspartate specific proteinase-1(caspase-1)/gasdermin D(GSDMD) pathway. The rat model of diabetes was established by intraperitoneal injection of streptozotocin(STZ) combined with a high-fat, high-sugar diet. The blood glucose meter, fully automated biochemical analyzer, hematoxylin-eosin(HE) staining, enzyme-linked immunosorbent assay, immunofluorescence, immunohistochemistry, and Western blot were employed to measure blood glucose levels, lipid levels, vascular thickness, inflammatory cytokine levels, and expression levels of pyroptosis-related proteins. The mechanism of pharmacological interventions against the injury in the context of diabetes was thus explored. The results demonstrated the successful establishment of the model of diabetes. Compared with the control group, the model group showed elevated levels of fasting blood glucose, total cholesterol(TC), triglycerides(TG) and low-density lipoprotein cholesterol(LDL-c), lowered level of high-density lipoprotein cholesterol(HDL-c), thickened vascular intima, and elevated serum and aorta levels of tumor necrosis factor-α(TNF-α), interleukin-1ß(IL-1ß) and interleukin-18(IL-18). Moreover, the model group showed increased NLRP3 inflammasomes and up-regulated levels of caspase-1 and GSDMD in aortic vascular cells. Polygonati Rhizoma intervention reduced blood glucose and lipid levels, inhibited vascular thickening, lowered the levels of TNF-α, IL-1ß, IL-18 in the serum and aorta, attenuated NLRP3 inflammasome expression, and down-regulated the expression levels of caspase-1 and GSDMD, compared with the model group. In summary, Polygonati Rhizoma can slow down the progression of diabetic macroangiopathy by inhibiting pyroptosis and alleviating local vascular inflammation.

Clinical characteristics, risk factors and antiviral treatments of influenza in immunosuppressed inpatients in Beijing during the 2015-2020 influenza seasons.

BACKGROUND: Compared with immunocompetent patients, immunosuppressed patients have higher morbidity and mortality, a longer duration of viral shedding, more frequent complications, and more antiviral resistance during influenza infections. However, few data on this population in China have been reported. We analysed the clinical characteristics, effects of antiviral therapy, and risk factors for admission to the intensive care unit (ICU) and death in this population after influenza infections and explored the influenza vaccination situation for this population. METHODS: We analysed 111 immunosuppressed inpatients who were infected with influenza virus during the 2015-2020 influenza seasons. Medical data were collected through the electronic medical record system and analysed. Univariate analysis and multivariate logistics analysis were used to identify risk factors. RESULTS: The most common cause of immunosuppression was malignancies being treated with chemotherapy (64.0%, 71/111), followed by haematopoietic stem cell transplantation (HSCT) (23.4%, 26/111). The most common presenting symptoms were fever and cough. Dyspnoea, gastrointestinal symptoms and altered mental status were more common in HSCT patients than in patients with immunosuppression due to other causes. Approximately 14.4% (16/111) of patients were admitted to the ICU, and 9.9% (11/111) of patients died. Combined and double doses of neuraminidase inhibitors did not significantly reduce the risk of admission to the ICU or death. Risk factors for admission to the ICU were dyspnoea, coinfection with other pathogens and no antiviral treatment within 48 h. The presence of dyspnoea and altered mental status were independently associated with death. Only 2.7% (3/111) of patients less than 12 months old had received a seasonal influenza vaccine. CONCLUSION: Fever and other classic symptoms of influenza may be absent in immunosuppressed recipients, especially in HSCT patients. Conducting influenza virus detection at the first presentation seems to be a good choice for early diagnosis. Clinicians should pay extra attention to immunosuppressed patients with dyspnoea, altered mental status, coinfection with other pathogens and no antiviral treatment within 48 h because these patients have a high risk of severe illness. Inactivated influenza vaccines are recommended for immunosuppressed patients.

Design and Development of Virtual Medical System Interface Based on VR-AR Hybrid Technology.

With the continuous development of information technology and digital medicine, computer-assisted virtual medicine has become the development trend of a new generation of clinical surgery, which aims to improve the accuracy of surgery, reduce the risk of surgery, and achieve precise and minimally invasive treatment. The interface design in the computer-aided virtual medical system is a medium for transmitting and exchanging information between humans and machines. This article uses virtual reality technology and augmented reality technology to develop a virtual medical system interface, which aims to solve the interaction problem between users and virtual medical systems and satisfy users. The multidemand psychology is an effective way of interaction. It provides users with a multichannel and comprehensive communication method, which truly meets the design goals that meet the user's psychological needs. It also expands applications for virtual reality technology and augmented reality technology.

Correlation analysis of hepatitis C virus core antigen and low viral loads: Can core antigen replace nucleic acid test?

Value of hepatitis C virus (HCV) core antigen (cAg) test has been controversy in patients with low HCV loads for its lower sensitivity. We assessed correlation between HCV-cAg and HCV RNA in serum samples with low viral loads and analyzed the performance of HCV-cAg assay in determining diagnosis and treatment outcomes in chronic hepatitis C patients. Both HCV RNA and HCV-cAg were detected for 2298 serum samples. Correlation analysis was performed between the two tests. Receiver operating characteristics (ROC) curve was used to assess value of HCV-cAg test in determining diagnosis and response outcomes at the different HCV RNA thresholds. The two tests were correlated very well, and moreover, correlation in the low viral load group was higher than that in the high viral load group (r value: 0.901 and 0.517). Positive agreement of HCV-cAg ≥ 3 fmol/L was as high as 97.0% for HCV RNA ≥ 1000 IU/mL, and its negative agreement for HCV RNA < 15 IU/mL was up to 98.9% in all samples. Area under ROCs ranged from 0.939 to 0.992, regardless of HCV RNA thresholds. When lower limit of detection of HCV RNA was 15, 100 or 1000 IU/mL, positive predictive value of HCV-cAg was 96.8%, 98.8% or 92.4%, and its negative predictive value was 87.0%, 89.9% or 98.3%, respectively, on the basis of different cutoff values. High-sensitivity HCV-cAg detection may likely replace HCV RNA to confirm the existence of HCV and to guide the treatment of chronic HCV infection.

Increased regulatory T cells and eosinophils characterize atopic dermatitis-like graft-versus-host disease compared with lichen planus-like graft-versus-host disease.

BACKGROUND: Graft-versus-host disease (GVHD) has various cutaneous manifestations. Little is known about the mechanisms of cutaneous GVHD with different clinical features. OBJECTIVE: To characterize the immunologic features and skin barrier functions of cutaneous GVHD. METHODS: The study included 19 patients with atopic dermatitis (AD)-like GVHD, 8 with lichen planus (LP)-like GVHD, 24 with AD, and 15 healthy controls. The subpopulation of T cells in peripheral blood and skin lesions was measured by flow cytometry and immunofluorescence, respectively. Filaggrin expression in skin lesions was measured by Western blot and immunohistochemistry. Transepidermal water loss was also measured using Tewameter TM 300 (Courage & Khazaka Electronic GmbH, Köln, Germany). RESULTS: The number of peripheral blood eosinophils in AD-like GVHD was significantly higher than that in LP-like GVHD. Type 2 helper T cells in peripheral blood and skin lesions were increased in AD-like GVHD and LP-like GVHD. Regulatory T cells in peripheral blood and skin lesions were increased in AD-like GVHD. Filaggrin expression and transepidermal water loss were increased in skin lesions of AD-like GVHD and LP-like GVHD. LIMITATIONS: The number of patients is limited. CONCLUSION: Although AD-like GVHD and LP-like GVHD both had elevated type 2 helper T cells and impaired skin barrier, increased eosinophils and regulatory T cells were found only in AD-like GVHD.

Regulatory factor X5 promotes hepatocellular carcinoma progression by transactivating tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta and suppressing apoptosis.

BACKGROUND: Our previous studies have shown that regulatory factor X5 (RFX5), a classical transcription regulator of MHCII genes, was obviously overexpressed in hepatocellular carcinoma (HCC) tumors. However, the role of RFX5 in the carcinogenesis and progress of HCC remains unknown. This study aimed to reveal its biological significance and the underlying mechanism in HCC. METHODS: RFX5 mRNA expression level and copy number variation in HCC tumors and cell lines were determined by analyzing deposited data sets in the Cancer Genome Atlas and Gene Expression Omnibus database. The biological significance of RFX5 in HCC was investigated by monitoring the colony formation and subcutaneous tumor growth capacity when RFX5 was silenced with lentiviral short hairpin RNA and CRISPR/Cas9 system in HCC cell lines. The downstream gene transcriptionally activated by RFX5 in HCC cells was determined by chromatin immunoprecipitation and luciferase reporter assay. The involvement of tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein theta (YWHAQ) in HCC development was further determined by performing colony formation rescue assay and subcutaneous tumor growth rescue experiment. The association of YWHAQ with recurrence-free survival of patients with HCC was assessed by Kaplan-Meier analysis. Moreover, apoptosis level and the protein level of p53 pathway were determined to reveal the mechanism of RFX5 in driving HCC development. RESULTS: RFX5 was amplified and highly overexpressed in HCC tumor tissues compared with the corresponding non-tumor tissues. The mRNA expression level of RFX5 was significantly correlated with its DNA copy number (r = 0.4, P < 0.001). Functional study demonstrated that RFX5 was required for both clonogenic forming in vitro and subcutaneous tumor growth in vivo of HCC cells. Further study identified YWHAQ, namely 14-3-3 tau, as a key downstream transcriptional target gene of RFX5, which was tightly regulated by RFX5 in HCC. Moreover, overexpression of YWHAQ largely rescued the clonogenic growth of HCC cells that was suppressed by RFX5 knockdown. In addition, overexpression of YWHAQ in primary tumor was linked to poor prognosis of patients with HCC. These results demonstrated that YWHAQ was a downstream effector of RFX5 in HCC. Notably, RFX5-YWHAQ pathway could protect cells from apoptosis by suppressing the p53 and Bax in HCC. CONCLUSION: RFX5 is a putative HCC driver gene that plays an important role in the development and progression of HCC by transactivating YWHAQ and suppressing apoptosis.

PPPDE1 promotes hepatocellular carcinoma development by negatively regulate p53 and apoptosis.

We have previously identified that PPPDE1 is a deubiquitinase (DUB) belonging to a cysteine isopeptidase family. Here we sought to explore the biological significance of PPPDE1 in hepatocellular carcinoma and its underlying molecular mechanism. In the present study, we found that amplification and overexpression of PPPDE1 were associated with poor prognosis in hepatocellular carcinoma (HCC). We also demonstrated that knocking down of PPPDE1 could significantly block the clonal growth and tumorigenicity of human HCC cells, which revealed a critical role for PPPDE1 in HCC development. Furthermore, we proved that PPPDE1 is a key modulator of p53 protein level and its down stream apoptosis pathway. Taken together, these results suggested that PPPDE1 is a putative HCC driver gene and extensive studies should be conducted in the future to investigate the role of PPPDE1 in HCC and other tumors.

Effect of Upregulated DNA Replication and Sister Chromatid Cohesion 1 Expression on Proliferation and Prognosis in Hepatocellular Carcinoma.

BACKGROUND: DNA replication and sister chromatid cohesion 1 (DSCC1) (also called DCC1) is a component of an alternative replication factor C complex that loads proliferating cell nuclear antigen onto DNA during S phase of the cell cycle. It is located at 8q24 and frequently amplified in hepatocellular carcinoma (HCC). However, the role of DSCC1 in the carcinogenesis and progress of HCC has not been fully investigated. Here, we aimed to assert the importance of DSCC1 in the HCC. METHODS: In this study, copy number variation data and RNA sequencing data were used to calculate the DNA copy number and mRNA expression of DSCC1 in HCC. Quantitative polymerase chain reaction, Western blotting, and immunohistochemistry analysis were used to determine the mRNA and protein level of DSCC1 in HCC. The Kaplan-Meier analysis and univariate and multivariate Cox regression analysis were used to assess the association of DSCC1 with the overall survival (OS) of HCC patients. Moreover, lentiviral shRNA was used to knockdown DSCC1, and then, colony-forming assay, cell cycle assay, and cell proliferation assay were performed to evaluate the impact of DSCC1 silencing on HCC cell lines. RESULTS: We found that DSCC1 was amplified and highly expressed in HCC tumor tissues than in nontumor tissues. We then found that the overexpression of both mRNA and protein of DSCC1 was linked to the bad prognosis of HCC patients. Astonishingly, the protein level of DSCC1 was an independent prognostic factor for OS (hazard ratio, 1.79; 95% confidence interval, 1.17-2.74; P = 0.007). Furthermore, the clonogenic capacity of DSCC1-amplified HCC cell lines (MHCC-97H, MHCC-97L, and Hep3B) was significantly inhibited by transduction of a lentiviral shRNA that targets DSCC1. We also showed that knockdown of DSCC1 induced G0-G1 cell cycle arrest (increased from 60% to more than 80%) and greatly inhibited the proliferation of HCC cell lines. CONCLUSION: These results suggest that DSCC1 is a putative HCC driver gene that promotes proliferation and is associated with poor prognosis in HCC.

HBsAg stimulates NKG2D receptor expression on natural killer cells and inhibits hepatitis C virus replication.

BACKGROUND: Higher hepatitis B surface antigen (HBsAg) facilitates hepatitis C virus (HCV) clearance in patients with hepatitis B virus (HBV)/HCV co-infection. We investigated the effect of exogenous HBsAg on the inhibition of HCV replication mediated by natural killer (NK) cells. METHODS: After isolated from peripheral blood of 42 chronic hepatitis B (CHB) patients and 16 healthy individuals, NK cells were co-cultured with HCV-infected Huh7 cells, respectively, with or without HBsAg. Three days later, the co-cultured supernatants were collected and HCV RNA levels were measured by real-time quantitative PCR. NKG2D, NKp46 and NKG2A expression levels were measured by flow cytometry. NKG2D on NK cells from CHB responsive subgroup was blocked and HCV RNA levels were examined again. RESULTS: HCV RNA levels in the co-cultured system were significantly reduced by NK cells isolated from healthy donors (P < 0.01) but not from CHB patients. However, HCV RNA levels in CHB cultures were significantly decreased following HBsAg addition (P < 0.05), whereas no such effect was seen in control cultures. No significant difference was observed in basic NKG2D expression between the CHB patients and healthy donors. On NK cells from CHB patients, the expression of NKG2D was increased significantly by HBsAg stimulation (P < 0.01), and higher than that from healthy controls (P < 0.05). HCV RNA levels were increased significantly after the blockage of NKG2D on NK cells from responsive CHB patients in the co-cultured system (P < 0.05). CONCLUSION: Exogenous HBsAg stimulated NKG2D expression on NK cells from CHB patients which inhibit HCV replication, suggesting that HBsAg may facilitate the clearance of HCV in patients with HBV/HCV co-infection.

MYL6B, a myosin light chain, promotes MDM2-mediated p53 degradation and drives HCC development.

BACKGROUND: Identification of novel MDM2 or p53 binding proteins may reveal undefined oncogenes, tumor suppressors, signaling pathways and possible treatment targets. METHODS: By means of immunoprecipitation and Mass Spectrometry analysis, we aimed to identify novel regulators of the MDM2-p53 pathway. We further clarified the impact of MYL6B on the p53 protein level and on the process of apoptosis. We also investigated the role of MYL6B in hepatocellular carcinoma by clone formation assay and by determining the correlation between its expression and prognosis of HCC patients. RESULTS: We identified a novel MDM2 and p53 binding protein, MYL6B. It is a myosin light chain that could bind myosin II heavy chains to form non-muscle myosin II holoenzymes (NMII). We found that MYL6B could facilitate the binding of MDM2 to p53, which consequently promotes the ubiquitination and degradation of p53 protein. We further proved that MYL6B exerts the suppression effect on p53 as part of NMII holoenzymes because inhibiting the ATPase activity of myosin II heavy chain largely blocked this effect. We also discovered that MYL6B is overexpressed in HCC tissues and linked to the bad prognosis of HCC patients. Knocking out of MYL6B dramatically suppressed the clonogenic ability and increased the apoptosis level of HCC cell lines. CONCLUSIONS: To summary, our results demonstrate that MYL6B is a putative tumor driver gene in HCC which could promote the degradation of p53 by enhancing its' MDM2-mediated ubiquitination.

PPPDE1 is a novel deubiquitinase belonging to a cysteine isopeptidase family.

Ubiquitinlation of proteins is prevalent and important in both normal and pathological cellular processes. Deubiquitinating enzymes (DUBs) can remove the ubiquitin tags on substrate proteins and dynamically regulate the ubiquitination process. The PPPDE family proteins were predicted to be a novel class of deubiquitinating peptidase, but this has not yet been experimentally proved. Here we validated the deubiquitinating activity of PPPDE1 and revealed its isopeptidase activity against ubiquitin conjugated through Lys 48 and Lys 63. We also identified ribosomal protein S7, RPS7, as a substrate protein of PPPDE1. Moreover, PPPDE1 could mediate the ubiquitin chain editing of RPS7, deubiquitinating Lys 48-linked ubiquitination, and finally stabilize RPS7 proteins. Taken together, we report that PPPDE1 is a novel deubiquitinase that belongs to a cysteine isopeptidase family.

Role of intrahepatic B cells in non-alcoholic fatty liver disease by secreting pro-inflammatory cytokines and regulating intrahepatic T cells.

OBJECTIVE: To investigate the alterations and functions of intrahepatic B (IHB) cells in non-alcoholic fatty liver disease (NAFLD) model induced by high-fat diet (HFD). METHODS: C57BL/6 J mice were fed with HFD for 16 weeks to induce NAFLD. Flow cytometry was used to analyze lymphocytes from liver, spleen and peripheral blood mononuclear cells (PBMC). Real-time polymerase chain reaction and immunofluorescence stain were applied to investigate cytokine expression in the intrahepatic lymphocytes and IHB cells. CD4(+) intrahepatic T (IHT) cells and IHB cells were enriched by a magnetic-activated cell sorting method and cultured in vitro. The cytokines and immunoglobulin (Ig) levels in the plasma, cultural supernatants and liver homogenates were monitored with cytometric bead arrays or multiplex immunoassays. RESULTS: The percentage of IHB cells in CD45(+) cells was significantly higher in the NAFLD group than in the control group (P < 0.05). IHB cells expressed higher levels of interleukin (IL)-6 and tumor necrosis factor (TNF)-α in the NAFLD group, and produced higher levels of IL-6 and TNF-α under the stimulation of lipopolysaccharide (LPS) than the control group. IgG2a levels were higher in the plasma, liver homogenates and the culture supernatants of IHB cells after stimulated by LPS and anti-CD40/IgM in the NAFLD group than in the control group. Moreover, IHB cells enhanced the activation of CD4(+) IHT cells and promoted the differentiation into T helper (Th) 1 cells in the NAFLD group. CONCLUSION: IHB cells might be involved in NAFLD both by inducing the secretion of IL-6, TNF-α and IgG2a and by enhancing the activation of CD4(+) IHT cells and their differentiation into Th1 cells.

CD4+CXCR5+ T cells activate CD27+IgG+ B cells via IL-21 in patients with hepatitis C virus infection.

BACKGROUND: Chronic hepatitis C virus (HCV) infection causes the skewing and activation of B cell subsets, but the characteristics of IgG+ B cells in patients with chronic hepatitis C (CHC) infection have not been thoroughly elucidated. CD4+CXCR5+ follicular helper T (Tfh) cells, via interleukin (IL)-21 secretion, activate B cells. However, the role of CD4+CXCR5+ T cells in the activation of IgG+ B cells in CHC patients is not clear. METHODS: The frequency of IgG+ B cells, including CD27-IgG+ B and CD27+IgG+ B cells, the expression of the activation markers (CD86 and CD95) in IgG+ B cells, and the percentage of circulating CD4+CXCR5+ T cells were detected by flow cytometry in CHC patients (n=70) and healthy controls (n=25). The concentrations of serum IL-21 were analyzed using ELISA. The role of CD4+CXCR5+ T cells in the activation of IgG+ B cells was investigated using a co-culture system. RESULTS: A significantly lower proportion of CD27+IgG+ B cells with increased expression of CD86 and CD95 was observed in CHC patients. The expression of CD95 was negatively correlated with the percentage of CD27+IgG+ B cells, and it contributed to CD27+IgG+ B cell apoptosis. Circulating CD4+CXCR5+ T cells and serum IL-21 were significantly increased in CHC patients. Moreover, circulating CD4+CXCR5+ T cells from CHC patients induced higher expressions of CD86 and CD95 in CD27+IgG+ B cells in a co-culture system; the blockade of the IL-21 decreased the expression levels of CD86 and CD95 in CD27+IgG+ B cells. CONCLUSIONS: HCV infection increased the frequency of CD4+CXCR5+ T cells and decreased the frequency of CD27+IgG+ B cells. CD4+CXCR5+ T cells activated CD27+IgG+ B cells via the secretion of IL-21.

Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients

Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expen- sive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen) had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%), and hepatitis C virus core antigen decrease greater than 1 log10 IU/mL (early virological response based on hepatitis C virus core antigen) had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.Z%). Analysis on the validation group demonstrated a positive predictivevalue of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to hepatitis C virus RNA in predicting sustained virologic response of chronic genotype 1 hepatitis C virus infected patients, and can be used to guide anti-hepatitis C virus treatment, especially in resource-limited areas.

Early predictive efficacy of core antigen on antiviral outcomes in genotype 1 hepatitis C virus infected patients.

Response-guided therapy is of limited use in developing countries because hepatitis C virus RNA detection by sensitive molecular methods is time- and labor-consuming and expensive. We evaluated early predictive efficacy of serum hepatitis C virus core antigen kinetics on sustained virologic response in patients with genotype 1 hepatitis C virus during pegylated interferon plus ribavirin treatment. For 478 patients recruited, hepatitis C virus RNAs were detected at baseline, and at weeks 4, 12, 24, 48, and 72 using Cobas TaqMan. Architect hepatitis C virus core antigen was performed at baseline, and weeks 4 and 12. Predictive values of hepatitis C virus core antigen on sustained virologic response were compared to hepatitis C virus RNA. In the first 12 weeks after treatment initiation the dynamic patterns of serum hepatitis C virus core antigen and hepatitis C virus RNA levels were similar in sustained virologic response, relapse, and null response patients groups. Although areas under the receiver operating characteristics curves of hepatitis C virus core antigen were lower than those of hepatitis C virus RNA at the same time points, modeling analysis showed that undetectable hepatitis C virus core antigen (rapid virological response based on hepatitis C virus core antigen) had similar positive predictive value on sustained virologic response to hepatitis C virus RNA at week 4 (90.4% vs 93.3%), and hepatitis C virus core antigen decrease greater than 1log10IU/mL (early virological response based on hepatitis C virus core antigen) had similar negative predictive value to hepatitis C virus RNA at week 12 (94.1% vs 95.2%). Analysis on the validation group demonstrated a positive predictive value of 97.5% in rapid virological response based on hepatitis C virus core antigen and a negative predictive value of 100% in early virological response based on hepatitis C virus core antigen. In conclusion, hepatitis C virus core antigen is comparable to hepatitis C virus RNA in predicting sustained virologic response of chronic genotype 1 hepatitis C virus infected patients, and can be used to guide anti-hepatitis C virus treatment, especially in resource-limited areas.

Construction, expression, and function of 6B11ScFv-mIL-12, a fusion protein that attacks human ovarian carcinoma.

We previously produced an anti-idiotypic monoclonal antibody, 6B11, which mimics ovarian cancer antigen CA166-9 and induces cellular and humoral immunity. Here, to enhance the immunogenicity of 6B11, we constructed the 6B11ScFv-mIL-12 fusion protein (FP), by fusing single-chain fragment of 6B11 variable region (6B11ScFv) with mouse interleukin-12 (mIL-12), which was expressed in eukaryotic 293EBNA cells transfected with pSBI vectors. A binding activity assay showed 6B11ScFv-mIL-12 to have activities of both 6B11 and mIL-12-it specifically bound both ovarian monoclonal antibody COC166-9 and rabbit anti-mouse IL-12 antibody. The immune activity assay showed 6B11ScFv-mIL-12 to promote proliferation of lymphocytes stimulated by phytohemagglutinin, increase the absolute numbers and percentages of CD3(-)/CD56(+) natural killer cells and CD3(+)/CD56(+) natural killer T cells among peripheral lymphocytes, and increase interferon-γ. The FP was specifically cytotoxic to the CA166-9(+) ovarian cancer cell lines HOC1A and SKOV3 and inhibited growth of ID8 subcutaneous tumors in C57BL/6J mice. This study provides an experimental basis for clinical use of 6B11ScFv-mIL-12 in ovarian cancer therapy. To our knowledge, this is the first report of a fusion protein from an anti-idiotypic antibody and IL-12.

Hepatitis C virus core antigen, an earlier and stronger predictor on sustained virological response in patients with genotype 1 HCV infection.

BACKGROUND: Earlier kinetics of serum HCV core antigen (HCVcAg) and its predictive value on sustained virological response (SVR) were investigated in patients with genotype 1 HCV infection during antiviral treatment. METHODS: In a multi-centered, randomized and positive drug-controlled phase IIb clinical trial on type Y peginterferon α-2b ( NCT01140997), forty-eight CHC patients who participated in pharmacokinetics were randomly divided into 4 cohorts and treated with PegIFNα (type Y peginterferon α-2b 90 µg, 135 µg, 180 µg and PegIFNα-2a 180 µg, respectively, once a week) and ribavirin (< 75 kg, 1000 mg daily and ≥ 75 kg, 1200 mg daily) for 48 weeks, and then followed up for 24 weeks. 32 patients infected with genotype 1 HCV and completed the whole process were included in this study. HCV RNAs were detected at baseline, and weeks 4, 12, 24, 48 and 72 using Cobas TaqMan. ARCHITECT HCVcAg was performed at 24, 48, 72, 96, 120 and 144 h in addition to the above time points. The receiver operating curves (ROCs) were performed to study the predictive values of HCVcAg decline on SVR. RESULTS: Following antiviral treatment, serum HCVcAg levels rapidly declined within the first week and correlated well with corresponding HCV RNA at baseline, weeks 4, 12, 24, 48 and 72 (rs = 0.969, 0.928, 0.999, 0.983, 0.985 and 0.946, respectively, P < 0.001). All of the areas under the receiver operating curves (AUROCs) were more than 0.80 and showed good predictive power on SVR at 24, 48, 72, 96, 120 and 144 h. The144 h was the best predictive time point of HCVcAg decline on SVR because of its largest AUROC (more than 0.90). CONCLUSIONS: Early kinetics of serum HCVcAg predicts SVR very well in genotype 1 CHC patients during antiviral treatment, and its reduction value at 144 h is an earlier and stronger predictor on SVR than rapid virological response and early virological response. (TRN: NCT01140997).

In vitro interactions between rat bone marrow-derived endothelial progenitor cells and hepatic stellate cells: interaction between EPCs and HSCs.

Transplantation of bone marrow (BM)-derived endothelial progenitor cells (EPCs) has been reported to improve liver fibrosis, but there is no direct evidence for the mechanism of improvement. We investigated the mechanism in vitro by coculturing BM-derived EPCs with activated hepatic stellate cells (HSCs) to mimic the hepatic environment. EPCs and HSCs were cultured alone and indirectly cocultured at a 1:1 ratio in a Transwell system. The characteristics of HSCs and EPCs were examined at different time points. An invasion assay showed the time-dependent effect on degradation of the extracellular matrix (ECM) layer in EPCs cultured alone. Real-time PCR and enzyme-linked immunosorbent assay analysis revealed that EPCs served as a source of matrix metalloproteinase-9 (MMP-9), and MMP-9 expression levels significantly increased during the 2 d of coculture. CFSE labeling showed that EPCs inhibited proliferation of HSCs. Annexin-V/PI staining, erminal deoxynucleotidyl transferase X-dUTP nick end labeling analysis, and (cleaved) caspase-3 activity revealed that EPCs promoted HSC apoptosis. However, the proliferation and apoptosis of EPCs were unaffected by cocultured HSCs. Coculturing increased the expression of inducible nitric oxide synthase, vascular endothelial growth factor, and hepatocyte growth factor (HGF) in EPCs, promoted differentiation of EPCs, and reduced the expression of types I and III collagens and transforming growth factor beta 1. Knockdown of HGF expression attenuated EPC-induced activation of HSC apoptosis and profibrotic ability. These findings demonstrated that BM-derived EPCs could degrade ECM, promoting activated HSC apoptosis, suppressing proliferation and profibrotic ability of activated HSCs. HGF secretion by EPCs plays a key role in inducing activated HSC apoptosis and HSC profibrotic ability.

BST2/tetherin inhibits dengue virus release from human hepatoma cells.

UNLABELLED: Type I interferons (IFN) have been shown to play an important role for inhibiting Dengue virus (DENV) infection. Identifying IFN-induced cellular proteins are essential for understanding its mechanisms against DENV. Here we established stable Huh7-derived cell lines expressing the IFN-induced cell membrane protein BST2 (Huh7-BST2) or its variant bearing a V5 tag at the C-terminal (Huh7-BST5CV5). These cell lines were infected with DENV to determine proteins modulating their anti-DENV response. We found that expression of BST2 did not affect the efficiency of DENV infection and intracellular replication. Rather, it significantly reduced the virion yield of the infected cells, particularly at low MOI infection. In addition, BST2 also decreased the foci formation and the size of infectious foci in cultured Huh7 monolayers with media containing methocellulose. The addition of the V5 tag at C-terminal inhibited the GPI modification of BST2 and blocked its shift from endoplasm to cytoplastic membrane. BST2CV5 did not affect DENV infection and foci formation in Huh7 cells but reduced virion yield by 1 log at low MOI infection. Interestingly, intracellular BST2CV5 expression was reduced by high level of DENV production. CONCLUSION: Our results imply that BST2 is a functional mediator of the IFN response against DENV infection. BST2 inhibits the release of DENV virions from Huh7 cells and limits viral cell-to-cell transmission. BST2CV5 variant is unable to inhibit DENV release but impairs viral infection in cells.