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Osteoblast differentiation is a key process in bone homeostasis. Mutations in plastin 3 have been reported to be responsible for X-linked osteoporosis. Plastin 3 and plastin 2 act synergistically to regulate osteoblast differentiation. However, the bone-related function of plastin 1, another family member of plastins, has not been assessed. In this study, we addressed the functional importance of plastin 1 in osteoblasts. We characterized the expression patterns of plastin 1 during osteoblast differentiation and revealed its important role in this process. In both HEK 293T and hFOB1.19 cells, plastin 1 was demonstrated to regulate intracellular Ca2+. Accordingly, we revealed that higher Ca2+ concentration promotes osteoblast differentiation. Finally, we found that plastin 1 may play a compensatory role in osteoporosis patients with plastin 3 deficiency. Together, our results indicate that plastin 1 promotes osteoblast differentiation by regulating intracellular Ca2+. Our work sheds new light on the role played by plastins in bone homeostasis.

Unconventional T cells (UTCs) are a heterogenous group of T cells that typically exhibit rapid responses towards specific antigens from pathogens. Chronic hepatitis C virus (HCV) infection causes dysfunction of several subsets of UTCs. This altered phenotype and function of UTCs can persist over time even after direct acting antiviral (DAA) mediated clearance of chronic HCV. However, it is less clear if and how UTCs respond in acute, symptomatic HCV infection, a rare clinical condition, and if rapid DAA treatment of such patients reverses the caused perturbations within UTCs. Here, we comprehensively analyzed the phenotype and reinvigoration capacity of three major UTC populations, mucosal associated invariant T (MAIT) cells, γδ T cells, and CD4 and CD8 double-negative αβ T cells (DNT cells) before, during, and after DAA-mediated clearance of acute symptomatic HCV infection. Furthermore, MAIT cell functionality was systematically studied. We observed a reduced frequency of MAIT cells. However, remaining cells presented with a near-to-normal phenotype in acute infection, which contrasted with a significant dysfunction upon stimulation that was not restored after viral clearance. Notably, DNT and γδ T cells displayed a strong activation ex-vivo in acute HCV infection, which subsequently normalized during the course of treatment. In addition, DNT cell activation was specifically associated with liver inflammation and inflammatory cytokines. Altogether, these data provide evidence that UTCs respond in a cell type-specific manner during symptomatic HCV infection. However, even if early treatment is initiated, long-lasting imprints within UTCs remain over time. This article is protected by copyright. All rights reserved.

Soluble inflammatory mediators (SIM) can be predictive of treatment outcome in antiviral treatment of chronic hepatitis C. Recently, it was shown that a subgroup of patients can be cured with four weeks of therapy. We here profiled patients for 70 SIM before and during treatment of hepatitis C with glecaprevir/pibrentasvir (GLE/PIB) +/− ribavirin. Proximity extension assay was performed in a total of 32 patients. Pre-treatment SIM profiles did not distinguish patients achieving an SVR (n = 21) from patients experiencing antiviral relapse (n = 11). However, after 4 weeks of therapy, eight markers were identified that could distinguish patients with SVR from the relapsed group, namely MMP-10, CCL20, CXCL11, FGF-23, TNF, MCP-2, IL-18R1 and CXCL10. Thus, this study shows that a distinct on-treatment immune profile is associated with cure of HCV infection after ultrashort treatment. in press

Background Treatment with direct-acting antivirals (DAAs) in patients with chronic hepatitis C infection leads to partial restoration of soluble inflammatory mediators (SIMs). In contrast, we hypothesized that early DAA treatment of acute hepatitis C virus (HCV) with DAAs may normalize most SIMs. Methods In this study, we made use of a unique cohort of acute symptomatic hepatitis C patients who cleared HCV with a 6-week course of ledipasvir/sofosbuvir. Plasma samples were used for proximity extension assay measuring 92 proteins. Results Profound SIM alterations were observed in acute HCV patients, with marked upregulation of interleukin (IL)-6 and CXCL-10, whereas certain mediators were downregulated (eg, monocyte chemoattractant protein-4, IL-7). During treatment and follow-up, the majority of SIMs decreased but not all normalized (eg, CDCP1, IL-18). Of note, SIMs that were downregulated before DAA treatment remained suppressed, whereas others that were initially unchanged declined to lower values during treatment and follow-up (eg, CD244). Conclusions Acute hepatitis C was associated with marked changes in the soluble inflammatory milieu compared with both chronic hepatitis patients and healthy controls. Whereas early DAA treatment partly normalized this altered signature, long-lasting imprints of HCV remained.

The mechanism of exosomes derived from activated hepatic stellate cells (HSCs) involved in liver fibrosis is poorly understood. We previously reported that hypoxia-inducible factor 1 (Hif-1) regulated HSC activation, and, therefore, we investigated in current work whether Hif-1 regulates exosome secretion and the metabolic switch of HSCs, thus affecting the metabolism of liver nonparenchymal cells. In this study, the characteristics of exosomes from HSCs were assessed via electron microscopy, Western blot analysis, and acetylcholinesterase activity. Confocal microscopy was used to measure the uptake of exosomes by quiescent HSCs, Kupffer cells (KCs), and liver sinusoidal endothelial cells (LSECs). Hif-1α was inhibited via 2-ME or specific small interfering RNAs to investigate its role in exosomes derived from HSCs. It was determined that glucose transporter 1 and pyruvate kinase M2 were increasingly expressed in fibrotic liver samples, cell lysates, and exosomes derived from activated HSCs. Exosomes released from HSCs were associated with activation and glucose uptake of HSCs. Delivery of exosomes from activated HSCs induced glycolysis of quiescent HSCs, KCs, and LSECs. Disruption of Hif-1 expression suppressed the glycolysis effect delivered by exosomes. Conclusively, our results demonstrated that exosomes secreted by activated HSCs affect the metabolic switch of liver nonparenchymal cells via delivery of glycolysis-related proteins. These findings represent a novel mechanism that contributes to liver fibrosis and has significant implications for new diagnosis and treatment of liver diseases.-Wan, L., Xia, T., Du, Y., Liu, J., Xie, Y., Zhang, Y., Guan, F., Wu, J., Wang, X., Shi, C. Exosomes from activated hepatic stellate cells contain GLUT1 and PKM2: a role for exosomes in metabolic switch of liver nonparenchymal cells.

Background & Aims: During chronic hepatitis B virus (HBV) infection, suppressed functionality of natural killer (NK) cells might contribute to HBV persistence but the underlying mechanisms remain elusive. A peculiar feature of HBV is the secretion of large amount of hepatitis B surface antigen (HBsAg). However, the effect of HBsAg quantities on NK cells is unclear. The aim was to determine the effects of HBsAg quantities on NK cell functionality in patients with chronic hepatitis B (CHB). Methods: Eighty CHB patients were included and categorized into four groups based on their HBsAg levels. As a control, 30 healthy donors were enrolled. NK cell frequency, phenotype and function were assessed using flow cytometry and correlated with HBsAg levels and liver enzymes. Results: Compared to the healthy controls, a reshaping of NK cell pool towards more CD56brⁱght NK cells was observed during CHB infection. Importantly, NK cells in patients with low HBsAg levels (

Functional maturation of liver sinusoidal endothelial cells (LSECs) plays an important role in intrahepatic T‐cell activation and control of viral infections. Natural killer (NK) cells have been reported to prompt the maturation of antigen‐presenting cells (APCs), especially for dendritic cells (DCs), but the interaction between NK cells and LSECs is elusive. Here, we investigated whether and how NK cells are involved in regulating LSEC maturation and if this has a role in controlling hepatitis B virus (HBV) infection in a mouse model. A chronic HBV replication mouse model was established by hydrodynamic injection (HI) of 6 µg adeno‐associated virus plasmid (pAAV)/HBV 1.2. The nucleotide‐binding oligomerization domain‐containing protein 1 (NOD1) ligand diaminopemelic acid (DAP) was imported into liver by HI at day 14 after plasmid injection. We found that HI of DAP recruited conventional NK cells (cNK) into the liver and promoted tumor necrosis factor alpha (TNF‐α) and interferon‐gamma (IFN‐γ) production of NK cells in a chemokine (C‐X‐C motif) receptor 3 (CXCR3)‐dependent manner. Importantly, the maturation of LSECs and the anti‐HBV effects of DAP were impaired in CXCR3−/− mice; this possibly was associated with the decreased number of intrahepatic cNK cells. Consistently, depleting cNK cells but not liver‐resident NK cells also impaired the maturation and antigen‐presenting function of LSECs, which reduced intrahepatic HBV‐specific T‐cell responses and thus inhibited HBV clearance both in wild‐type and in Rag1−/− mice. Moreover, TNF‐α or IFN‐γ stimulation as well as coculture with intrahepatic NK cells partly promoted LSEC phenotypic and functional maturation in vitro. Conclusion: NOD1‐triggered NK cell activation may lead to the enhancement of intrahepatic T‐cell responses by promoting maturation of LSECs through soluble cytokines and cell–cell contact, thereby controlling HBV replication and expression., NK cells promote intrahepatic T cell responses by regulating LSEC maturation. The potential mechanisms by which NK cells regulate the maturation of LSECs may involve soluble cytokines as well as cell‐cell contact. The recruitment and activation of conventional NK cells (cNK) cells after NOD1 activation depend on CXCR3 axis.

It remains controversial how interferon (IFN) response contributes to hepatitis B virus (HBV) control and pathogenesis. A previous study identified that hydrodynamic injection (HI) of type I IFN (IFN-I) inducer polyinosinic-polycytidylic acid (poly(I:C)) leads to HBV clearance in a chronic HBV mouse model. However, recent studies have suggested that premature IFN-I activation in the liver may facilitate HBV persistence. In the present study, we investigated how the early IFN-I response induces an immunosuppressive signaling cascade and thus causes HBV persistence. We performed HI of the plasmid adeno-associated virus (pAAV)/HBV 1.2 into adult BALB/c mice to establish an adult acute HBV replication model. Activation of the IFN-I signaling pathway following poly(I:C) stimulation or murine cytomegalovirus (MCMV) infection resulted in subsequent HBV persistence. HI of poly(I:C) with the pAAV/HBV 1.2 plasmid resulted in not only the production of IFN-I and the anti-inflammatory cytokine interleukin (IL)-10 but also the expansion of intrahepatic regulatory T cells (Tregs), Kupffer cells (KCs) and myeloid-derived suppressor cells (MDSCs), all of which impaired the T cell response. However, when poly(I:C) was injected at day 14 after the HBV plasmid injection, it significantly enhanced HBV specific T cell responses. In addition, interferon-alpha/beta receptor (IFNAR) blockade rescued T cell response by downregulating of IL-10 expression and decreasing Treg and KC expansion. Consistently, Treg depletion or IL-10 blockade also controlled HBV replication. Importance: IFN-I plays a double-edged sword role during chronic HBV infection. Here, we identified that application of IFN-I at different time points causes contrast outcome. Activation of the IFN-I pathway before HBV replication induces an immunosuppressive signaling cascade in the liver, and consequently caused HBV persistence while IFN-I activation post HBV infection enhances HBV-specific T cell responses and thus promote HBV clearance. This result provided an important clue to the mechanism of HBV persistence in adult individuals.

Hepatitis B virus (HBV) is a global health issue, but currently available anti-HBV drugs have limited success. Previously, introduction of the Toll-like receptor (TLR)-3 ligand poly(I:C) to the liver via hydrodynamic injection (HI) was shown to effectively suppress HBV replication in a chronic HBV replication mouse model. However, this method cannot be applied in human beings. To improve the liver targeting of poly(I:C) via intravenous injection, calcium phosphate nanoparticles (CPNs) carrying poly(I:C) with or without antibodies were constructed, and their anti-HBV effects were investigated. We found that significantly more anti-F4/80-conjugated and IgG2α-conjugated nanoparticles were taken up in liver cells both in vivo and in vitro. In addition, these nanoparticles produced pronounced immunostimulatory effects in vitro in primary liver cells. Importantly, treatment with nanoparticles carrying poly(I:C) increased the production of intrahepatic cytokines and chemokines and enhanced T cell responses, significantly reducing HBsAg, HBeAg and HBV DNA levels in the mice. Compared to nonconjugated and isotype-antibody-conjugated nanoparticles, the anti-F4/80-conjugated nanoparticles demonstrated the strongest anti-HBV effects. In summary, nanoparticles carrying poly(I:C) conjugated with an F4/80 antibody promoted liver targeting, and they may represent a suitable alternative to HI for future anti-HBV treatment. Statement of significance HBV chronically infects approximately 250 million individuals worldwide but current anti-HBV drugs have limited success. Introduction of toll-like receptor 3 ligand poly(I:C) into liver by hydrodynamic injection has been proven to promote HBV clearance in mouse model. However, this technique is not clinically suitable for human patients. We have constructed calcium phosphate nanoparticles carrying poly(I:C) with specific antibody targeting liver nonparenchymal cells. The uptake into relevant liver cells and the anti-HBV effects were studied. After intravenous injection into mice, the uptake rate of anti-F4/80-conjugated nanoparticels was enhanced in liver, and these nanoparticles exert effective anti-HBV effects in vivo. This may provide important insight into future HBV immunotherapy based on nanoparticle-mediated drug delivery.

Liver sinusoidal endothelial cells (LSECs) are organ resident APCs capable of antigen presentation and subsequent tolerization of T cells under physiological conditions. In this study, we investigated whether LSEC pretreatment with NOD-like receptor (NLR) agonists can switch the cells from a tolerogenic to an immunogenic state and promote the development of T cell immunity. LSECs constitutively express NOD1, NOD2 and RIPK2. Stimulation of LSECs with DAP induced the activation of NF-κB and MAP kinases and upregulated the expression of chemokines (CXCL2/9, CCL2/7/8) and cytokines (IFN-γ, TNF-α and IL-2). Pretreatment of LSECs with DAP induced significantly increased IFN-γ and IL-2-production by HBV-stimulated CD8+ T cells primed by DAP-treated LSECs. Consistently, a significant reduction in the HBV DNA and HBsAg level occurred in mice receiving T cells primed by DAP-treated LSECs. MDP stimulation had no impact on LSECs or HBV-stimulated CD8+ T cells primed with MDP-treated LSECs except for the upregulation of PD-L1. DAP stimulation in vitro could promote LSEC maturation and activate HBV-specific T cell responses. These results are of particular relevance for the regulation of the local innate immune response against HBV infections.

Functional maturation of liver sinusoidal endothelial cells (LSECs) induced by a NOD1 ligand (diaminopimelic acid [DAP]) during viral infection has not been well defined. Thus, we investigated the role of DAP-stimulated LSEC maturation during hepatitis B virus (HBV) infection and its potential mechanism in a hydrodynamic injection (HI) mouse model. Primary LSECs were isolated from wild-type C57BL/6 mice and stimulated with DAP in vitro and in vivo and assessed for the expression of surface markers as well as for their ability to promote T cell responses via flow cytometry. The effects of LSEC maturation on HBV replication and expression and the role of LSECs in the regulation of other immune cells were also investigated. Pretreatment of LSECs with DAP induced T cell activation in vitro. HI-administered DAP induced LSEC maturation and subsequently enhanced T cell responses, which was accompanied by an increased production of intrahepatic cytokines, chemokines, and T cell markers in the liver. The HI of DAP significantly reduced the HBsAg and HBV DNA levels in the mice. Importantly, the DAP-induced anti-HBV effect was impaired in the LSEC-depleted mice, which indicated that LSEC activation and T cell recruitment into the liver were essential for the antiviral function mediated by DAP application. Taken together, the results showed that the Ag-presenting ability of LSECs was enhanced by DAP application, which resulted in enhanced T cell responses and inhibited HBV replication in a mouse model.

The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action was discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. A modular system where the immunoactive toll-like-receptor ligand 3 (TLR-3) poly(I:C) was incorporated into calcium phosphate nanoparticles was developed. The nanoparticles had a hydrodynamic diameter of 275 nm and a zeta potential of +20 mV, measured by dynamic light scattering. The diameter of the solid core was 120 nm by scanning electron microscopy. In vitro, the nanoparticle uptake was investigated after 1 and 24 h of incubation of THP-1 cells (macrophages) with nanoparticles by fluorescence microscopy. After intravenous injection into BALB/c and C57BL/6J mice, respectively, the in vivo uptake was especially prominent in lung and liver, 1 and 3 h after the injection. Pronounced immunostimulatory effects of the nanoparticles were found in vitro with primary liver cells, i.e. Kupffer cells (KC) and liver sinusoidal endothelial cells (LSEC) from wild-type C57BL/6J mice. Thus, they represent a suitable alternative to hydrodynamic injection treatments for future vaccination concepts. Statement of Significance The selective activation of the immune system is a concurrent problem in the treatment of persistent diseases like viral infections (e.g. hepatitis). For the delivery of the toll-like receptor ligand poly(I:C), an immunostimulatory action has been discovered earlier by hydrodynamic injection. However, this technique is not clinically transferable to human patients. We have developed a modular system where poly(I:C) was incorporated into calcium phosphate nanoparticles. The uptake into relevant liver cells was studied both in vitro and in vivo. After intravenous injection into mice, the in vivo uptake was especially prominent in lung and liver, 1 and 3 h after the injection. The corresponding strong immune reaction proves their high potential to turn up the immune system, e.g. against viral infections, without adverse side reactions.

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Fetal electrocardiogram (FECG) is an objective index of the activities of fetal cardiac electrophysiology. The acquired FECG is interfered by maternal electrocardiogram (MECG). How to extract the fetus ECG quickly and effectively has become an important research topic. During the non-invasive FECG extraction algorithms, independent component analysis(ICA) algorithm is considered as the best method, but the existing algorithms of obtaining the decomposition of the convergence properties of the matrix do not work effectively. Quantum particle swarm optimization (QPSO) is an intelligent optimization algorithm converging in the global. In order to extract the FECG signal effectively and quickly, we propose a method combining ICA and QPSO. The results show that this approach can extract the useful signal more clearly and accurately than other non-invasive methods.

The first thing of doing laparoscopic surgery is to puncture the abdominal wall, it depends on the clinical experience. However, there will be differences in individual patients, so experience alone will cause puncture failure or puncture too deep to injuries internal organs. This paper presents an automatically protective method of puncture abdominal. Implementation method is: measure the air pressure of puncture carbon dioxide channel, judge the puncture whether penetrate abdominal or not by the diversification of the air pressure, open the electromagnetic lock immediately when detected puncture through the abdominal cavity, make the needle core retracted to puncture tube under the pressure of the spring. We choose MPX5050 which made by Motorola as the pressure sensor, selected MSP430F135 as microcontroller in this article. Studies show that the proposed automatic protection of abdominal puncture technique is simple, safe, has great practical value.