Diagnostic Efficacy of a Novel Rotating Brush for Endoscopic Sampling of Malignant Biliary Strictures: A Multicenter Prospective Study.

INTRODUCTION: Although cytologic examination of biliary stricture brushings obtained by endoscopic retrograde cholangiopancreatography is commonly used for diagnosing malignant biliary strictures (MBSs), it has low sensitivity. Several new brushes have capabilities that are still being debated. We have developed a novel brush working from conventional back-and-forth movement to rotation in situ (RIS) that may be more efficient for MBS sampling. We aimed to compare the MBS detection sensitivity of our RIS brush with that of the conventional brush. METHODS: In this multicenter prospective study, we enrolled patients who underwent endoscopic retrograde cholangiopancreatography for suspected MBSs involving biliary stricture brushings obtained using our RIS brush. The historical control group consisted of the 30-brushing arm of our previous randomized trial (patient inclusion, 2018-2020) that used the study design in the same centers and with the same endoscopists as were used in this study. The primary outcome was to compare the sensitivity and specificity of detecting MBSs by cytologic evaluation of biliary stricture brushings between the 2 groups. RESULTS: We enrolled 155 patients in the intent-to-treat analysis. Using the same number of brushing cycles, the RIS brush showed a higher sensitivity than the conventional brush (0.73 vs 0.56, P = 0.003). In per-protocol population, the sensitivity was also higher in the RIS brush group than in the conventional brush group (0.75 vs 0.57, P = 0.002). Multivariate analysis revealed that the RIS brush was the only predictive factor for MBS detection. No significant differences were observed in procedure-related complications between the 2 groups. DISCUSSION: The RIS brush was a promising tool for effective and safe MBS sampling and diagnosis. Further randomized studies are warranted to confirm our results (Chictr.org.cn, identifier: ChiCTR2100047270).

Yes-associated protein promotes the proliferation and differentiation of liver progenitor cells during liver fibrosis.

Liver fibrosis is closely related to the proliferation and differentiation of liver progenitor cells (LPCs). Yes-associated protein (YAP) is a key effector molecule of the Hippo signaling pathway and plays an important role in regulating cell proliferation and liver homeostasis. However, its role in LPCs proliferation and differentiation during liver fibrosis are not well understood. Using immunohistochemistry, immunofluorescence staining, quantitative PCR and Western blotting, we discovered that LPCs expansion and enhanced YAP expression in LPCs in either choline-deficient, ethionine-supplemented (CDE) diet or 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) diet-induced fibrotic mice, as well as in patients with liver fibrosis. By injecting adeno-associated virus vectors under the transcriptional control of Lgr5 promoter, we found that targeted knockdown of YAP in LPCs attenuated the CDE/DDC diet-induced ductular reaction and liver fibrosis. Using EdU incorporation and Cell Counting Kit-8 assays, we demonstrated that YAP can modulate LPCs proliferation. Importantly, spleen transplantation of YAP-overexpressing LPCs improved their ability to differentiate into hepatocytes and alleviated carbon tetrachloride-induced liver fibrosis. Collectively, our findings indicate that LPCs expansion and differentiation during liver fibrosis could be modulated by YAP, further suggesting the possibility of manipulating YAP expression in LPCs as a potential treatment for chronic liver diseases.

YAP affects the efficacy of liver progenitor cells transplantation in CCl4-induced acute liver injury.

The liver is a highly regenerative organ. During acute liver injury, the remaining hepatocytes rapidly proliferate to restore liver parenchyma and liver function. However, hepatocytes-driven regeneration is compromised in severe liver injury; instead, liver progenitor cells (LPCs) proliferate and differentiate into hepatocytes or cholangiocytes to restore mass and function of liver. The Hippo signaling pathway is of vital importance in liver regeneration, and Yes-associated protein (YAP) is the key component of the Hippo pathway. The therapeutic role of YAP has been well studied in hepatocytes-driven liver regeneration. However, the role of LPCs transplantation in acute liver injury has not been defined. Here, we investigated the therapeutic effect of splenic-transplantation of LPCs in CCl4-induced acute liver injury and explored the role of YAP during the procedure. LPCs isolated from choline-deficient, ethionine-supplemented diet (CDE) model were infected with GFP-YAP cDNA lentiviral vector, GFP-YAP shRNA lentiviral vector, and GFP lentiviral vector as control, respectively. At 48 h after CCl4 injection, PBS (control group), GFP lentiviral vector-infected LPCs (GFP-LPC group), GFP-YAP cDNA lentiviral vector-infected LPCs (YAP-LPC group) and GFP-YAP shRNA lentiviral vector-infected LPCs (sh-YAP-LPC group) were injected into spleens in CCl4-treated mice. Histological and serological analyses were performed to evaluate pathology and liver function. The effect of LPCs on the proliferation of hepatocytes and inflammation was investigated. We demonstrated that intra-splenic transplantation of LPCs alleviates CCl4-induced acute liver injury and YAP signaling acts a key role during the procedure. Further studies suggested that LPCs alleviate acute liver injury by promoting pre-existing hepatocytes proliferation rather than differentiating into hepatocytes. Furthermore, intra-splenic transplantation of LPCs attenuates inflammation, which facilitates tissue repair in acute liver injury. In conclusion, LPCs transplantation is a potential treatment for acute liver injury and YAP is a prospective therapeutic target in acute liver injury.

More Endoscopy-Based Brushing Passes Improve the Detection of Malignant Biliary Strictures: A Multicenter Randomized Controlled Trial.

INTRODUCTION: Endoscopic biliary brushing is the most common method used for sampling in patients with malignant biliary strictures (MBSs); however, its sensitivity is relatively low. There is still no consensus on endoscopy-based biliary brushing, although brushing 10 times in 1 specimen is routinely performed. This study was designed to compare the sensitivity of brush cytology for 10, 20, or 30 brushing times of a pass in 1 specimen in patients with MBSs. METHODS: In this multicenter, prospective, randomized controlled study, patients who underwent endoscopic retrograde cholangiopancreatography for suspected MBSs were enrolled. Patients were randomly assigned to receive 10, 20, and 30 brushing times of a pass. The primary outcome was to compare the sensitivity of brush cytology among the 3 groups. Patients were prospectively followed up for 6 months after endoscopic brushing for malignancy diagnosis. RESULTS: A total of 443 patients were enrolled for intention-to-treat analysis (147, 148, and 148 patients in the 10-times, 20-times, and 30-time groups, respectively). The 3 groups were similar in baseline characteristics. The sensitivity of brush cytology was 38%, 47%, and 57% in the 10-times, 20-times, and 30-times groups, respectively, and the 30-times group showed significantly higher sensitivity than the 10-times group (P = 0.001). The multivariate analysis revealed that stricture length and the number of brushing passes were significant factors for the detection of biliary malignancy. No significant differences were observed in procedure-related complications among the 3 groups. DISCUSSION: Brushing 30 times could increase the diagnostic sensitivity without increasing complications and seems to be preferred for the endoscopic sampling and diagnosis of MBSs (chictr.org.cn, identifier: ChiCTR1800015978).

Identification of LBH and SPP1 involved in hepatic stellate cell activation during liver fibrogenesis.

Liver fibrosis is a pathological response driven by the activation of hepatic stellate cell (HSC). However, the mechanisms of liver fibrosis and HSC activation are complicated and far from being fully understood. We aimed to explore the candidate genes involved in HSC activation during liver fibrogenesis. Five genes (LBH, LGALS3, LOXL1, S100A6 and SPP1) were recurrent in the DEGs derived from the seven datasets. The expression of these genes gradually increased as liver fibrosis staging advanced, suggesting they might be candidate genes involved in HSC activation during hepatic fibrosis. These candidate genes were predicted to be coregulated by miRNAs such as hsa-miR-125a-5p and has-miR-125b, or by transcription factors including JUN, USF1, TP53 and TFAP2C. PPI analysis showed that LGALS3, LOXL1, S100A6 and SPP1 might interact with each other indirectly, but no interaction was found between them and LBH. The candidate genes and their interaction partners were enriched in focal adhesion, extracellular matrix organization and binding. Upregulation of LBH, S100A6 and SPP1 were further validated in TGF-ß-treated LX-2 as well as in DDC or CCL4-treated mice models. Decreased LBH and SPP1 expression reduces the expression of HSC activation-related markers in TGF-ß-treated LX-2. Our results indicated that LBH, LGALS3, LOXL1, S100A6 and SPP1 were candidate genes which may participate in the HSC activation during liver fibrosis.

Expansion of macrophage and liver sinusoidal endothelial cell subpopulations during non-alcoholic steatohepatitis progression.

Liver non-parenchymal cells (NPCs) play a critical role in the progression of non-alcoholic steatohepatitis (NASH). We aimed to explore the heterogeneity of NPCs and identify NASH-specific subpopulations contributing to NASH progression. Through single-cell RNA sequencing, we uncovered a proinflammatory subpopulation of Itgadhi/Fcrl5hi macrophages with potential function of modulating macrophage accumulation and promoting NASH development. We also identified subpopulations of Egr1hi and Ly6ahi liver sinusoidal endothelial cells (LSECs), which might participate in pathological angiogenesis and inflammation regulation. The Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs, and Ly6ahi LSECs emerged in the early stage and expanded significantly along with pathological progression of liver injury during NASH. Cell-cell interactions between hepatic stellate cells (HSCs) and Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs or Ly6ahi LSECs were enhanced in NASH liver. Our results revealed that expansion of Itgadhi/Fcrl5hi macrophages, Egr1hi LSECs or Ly6ahi LSECs was strongly associated with NASH severity, suggesting these subpopulations might be involved in NASH progression.

FABP4 in LSECs promotes CXCL10-mediated macrophage recruitment and M1 polarization during NAFLD progression.

BACKGROUND AND AIMS: Non-alcoholic liver disease (NAFLD) is emerging as the leading cause of end-stage liver disease with a serious threat to global health burden. Fatty acid-binding protein 4 (FABP4) is closely associated with metabolic syndromes. We aimed to explore the potential mechanisms of FABP4 in NAFLD progression. MATERIALS AND METHODS: For NAFLD mice, animals were fed with high fat diet (HFD) for 20 weeks. The assays of hematoxylin and eosin, Sirius Red, oil red O staining and immunohistology were performed to evaluate hepatic pathology. Flow cytometric analysis was used to distinguish macrophage subtypes. RESULTS: Serum FABP4 level was positively correlate with the severity of hepatic steatosis in NAFLD patients. FABP4 expression was mainly distributed in liver sinusoidal endothelial cells (LSECs), which was significantly increased in HFD mice. The level of CXCL10 was positively correlated with FABP4 at mRNA and serum level. FABP4 inhibition resulted in decreased expression of CXCL10. The percentage of M1 macrophage and CXCR3+ cells in infiltrated macrophage was increased in liver of HFD mice. Inhibition of FABP4 ameliorated HFD-induced M1 macrophage polarization as well as CXCR3+ macrophages recruitment. Recombinant CXCL10 and co-culturing with TMNK-1 stimulated macrophage toward M1 polarization, which could be reversed by CXCR3 inhibitor. Palmitic acid treatment resulted in increased nuclear P65 expression, which could be reversed by inhibiting FABP4. Cxcl10 expression was dramatically suppressed by NF-κB inhibitor. CONCLUSIONS: FABP4 in LSECs may play a pathogenic role in NAFLD course by promoting CXCL10-mediated macrophage M1 polarization and CXCR3+ macrophage infiltration via activating NF-κB/p65 signaling.

Kuhuang alleviates liver fibrosis by modulating gut microbiota-mediated hepatic IFN signaling and bile acid synthesis.

Background: Liver fibrosis is a common outcome of the pathological progression of chronic liver disease; however, no specific and effective therapeutic agent has been approved for its treatment. We investigated the effects of Kuhuang on liver fibrosis and the underlying mechanisms of action. Materials and methods: To induce hepatic fibrosis, either 3,5-diethoxycarbonyl-1,4-dihydro-collidine (DDC) diet was administered, or bile duct ligation (BDL) surgery was performed on C57BL/6 mice. Kuhuang was orally administered to mice for 7 days before and after bile duct ligation or 4 weeks with a DDC diet. Hematoxylin and eosin, Sirius red staining, and immunohistochemical analyses were performed to evaluate hepatic pathology. Hepatic interferon-ß (IFN-ß) levels were measured using an enzyme-linked immunosorbent assay. RNA sequencing was performed to examine the gene expression profiles of liver tissues. The mRNA expression of inflammatory, profibrotic, and bile acid (BA)-related genes was further validated by qRT-PCR. A targeted metabolomics assay revealed the alteration of the hepatic bile acid (BA) composition. The composition of the gut microbiota was determined via 16S rRNA sequencing. Results: Treatment with Kuhuang attenuated liver fibrosis and reduced the inflammatory response in bile duct ligation and DDC mouse models. In addition, the hepatic IFN signaling pathway was activated following Kuhuang treatment. Kuhuang treatment also significantly decreased hepatic levels of both primary and secondary BAs. In addition, Kuhuang treatment altered gut microbiota composition, with an increased abundance of interferon-inducing Akkermansia and decreased abundance of bile salt hydrolase-producing Lactobacillus, Clostridium, and Bifidobacterium. Furthermore, the abundance of Akkermansia was positively correlated with the hepatic mRNA expression levels of Ifna4, Ifnb, and Isg15, whereas that of Lactobacillus, Clostridium - sensu - stricto - 1, and Bifidobacterium was positively correlated with levels of bile acid synthesis-related genes. Conclusion: Our results suggest that Kuhuang plays a protective role during the progression of liver fibrosis, potentially by altering the composition of the gut microbiota, which consequently activates interferon signaling and inhibits bile acid synthesis in the liver.

Dissecting the single-cell transcriptomeunderlying chronic liver injury.

Chronic liver disease (CLD) is currently a major health problem worldwide, which is accompanied by chronic liver injury and lack of clinically effective treatment; however, systematic characterization of chronic liver injury procedures at single-cell resolution is lacking. In the present study, we established chronic liver injury mouse models and conducted single-cell RNA sequencing (scRNA-seq), including choline-deficient, ethionine-supplemented (CDE) and 3,5-diethoxycarbonyl 1,4-dihydrocollidinen (DDC) mouse models. We captured in total 16,389 high-quality cells and identified 12 main cell types in scRNA-seq data. Macrophages and endothelial cells are the largest cell populations in our dataset. Transcriptional trajectory analysis revealed different expression patterns of cells between CDE and DDC models and identified potential liver injury markers, such as Ets1, Gda, Itgam, and Sparc. Differential analysis identified 25 and 152 differentially expressed genes in CDE and DDC macrophages, respectively. In addition, 413 genes were detected to exclusively express in specific pseudotime states of macrophages. These genes were found to participate in immune-related biological processes. Further cell-cell communication analysis found extensive receding of cell-cell interactions between different cell types in the liver injury process, especially in the DDC model. Our study characterized the single-cell transcriptional landscape in the process of chronic liver injury, promoting the understanding of the underlying molecular mechanisms and providing candidate clinical strategy for effective intervention of chronic liver diseases.

The immunogenicity and safety of a Hib-MenAC vaccine: a non-inferiority randomized, observer-blind trial in infants aged 3-5 months.

BACKGROUND: The objective of this study was to evaluate the immunogenicity and safety of the novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroup A and C-tetanus toxoid conjugate vaccine (Hib-MenAC). METHODS: We conducted a non-inferiority, randomized, observer-blind, positive control clinical trial in 900 healthy infants aged between 3-5 months in Funing County, Jiangsu Province, China. Participants were randomly allocated, in a ratio of 2:1 (block = 6), to receive experimental combined Hib-MenAC vaccines co-administrated with placebo or the co-administration of licensed Hib vaccine and MenAC vaccine, according to a three-dose immunization schedule. The seroconversion of antibody titer against meningococcal serogroups A, C and Hib was the primary endpoint. RESULTS: The experimental vaccines was non-inferior to the licensed two control vaccines. Participants receiving experimental Hib-MenAC vaccines showed a seroconversion rate of 99.0%, 96.1% and 97.7% for rSBA-MenA, rSBA-MenC and anti-PRP antibodies, respectively. The Hib-MenAC vaccine did not result in an increase in adverse reaction, and no serious adverse event was judged to be related to the vaccination. CONCLUSIONS: The novel combined Hib-MenAC conjugate vaccine was safe and highly immunogenic in infants aged between 3 to 5 months.

Safety and immunogenocity of a novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine in healthy Chinese children aged 6 months to 5 years old.

A novel combined Haemophilus influenzae type b-Neisseria meningitidis serogroups A and C-tetanus-toxoid conjugate vaccine (Hib-MenAC vaccine) has been developed to protect children against diseases caused by Hib, MenA, and MenC. This study investigated the safety and immunogenicity of the Hib-MenAC vaccine administered in 2-dose series to children aged 6-23 months and in a single dose to children aged 2-5 y. A randomized, positive-controlled, non-inferiority clinical trial was conducted for 1200 healthy participants in each age group. Within each age group, participants were randomly allocated to the Hib-MenAC group or the control group at a ratio of 1:1. Adverse reactions were recorded within 28 d after each dose. Blood samples were obtained to assess immunogenicity on day 0 and at 28 d after a complete vaccination course. For the investigational vaccine, the incidence of total adverse reactions in vaccinees aged 6-23 months was 46.8% and that in vaccinees aged 2-5 y was 29.8%. Most adverse reactions were mild or moderate. One non-fatal serious adverse event occurred in the Hib-MenAC group, but was unrelated to vaccination. The seroconversion rate to the 3 components reached 94.0%, and the proportion of vaccinees with rSBA titers ≥ 1:8 and PRP ≥ 0.15 g/mL reached 97.0% in both age groups. The safety and immunogenicity of the Hib-MenAC vaccine were non-inferior when compared to the licensed vaccines. It was concluded that the novel vaccine would be expected to protect children against all of the targeted diseases.

Retrospective study of the incidence of HFMD and seroepidemiology of antibodies against EV71 and CoxA16 in prenatal women and their infants.

BACKGROUND: Hand, foot, and mouth disease (HFMD) has been emerging as an important public problem over the past few decades, especially in Asian and Pacific regions. A national program on EV71 vaccine development against HFMD was initiated in China, in 2008, which called for a need for seroepidemiological study for the target population. METHODOLOGY/PRINCIPAL FINDINGS: This was a retrospective study conducted in Jiangsu Province, in October, 2010. We measured the neutralizing antibodies against EV71 and CoxA16 in a cohort of infants aged of 2, 7, 12, and 27-38 months and their mothers just before delivery. Series sera samples from 975 infants and 555 mothers were collected and analyzed. Questionnaires on the history of HFMD were completed in the survey. A total of 143 HFMD cases were collected, but only 11.2% were reported to the National Infectious Disease Information Management System. The level of maternal antibody titers decreased dramatically during the first 7 month and remained at a relatively low level thereafter. But it increased significantly from month 12 to months 27-38. The accumulate incidence density of HFMD demonstrated a significant increase after 14 months of age, resulting in a accumulate incidence density of 50.8/1000 person-years in survey period. Seropositivity of EV71 antibody in infants at the age of 2 months seems to demonstrate a protective effect against HFMD. CONCLUSIONS AND SIGNIFICANCE: High seropositive rate of EV71 and CoxA16 antibody was found in prenatal women in mainland China, and there is a need to enhance the HFMD case management and the current surveillance system. We suggest that infants aged between 6 to 14 months should have the first priority to receive EV71 vaccine.

A decoupled circular-polarized volume head coil pair for studying two interacting human brains with MRI.

One of the major functions of the human brain is to mediate interactions with other people. Until recently, studying brain social interactions has not been possible due to the lack of measurable methods to observe two interacting minds simultaneously. We have developed a novel dual-head MRI coil that can scan two subjects' brains simultaneously while the subjects are socially interacting in one MRI scanner. Meanwhile, a novel scheme for decoupling two quadrature coils (not surface coils) is introduced and validated.