Ultrasonic microbubbles promote mesenchymal stem cell homing to the fibrotic liver via upregulation of CXCR4 expression.

OBJECTIVE: To investigate the mechanism of ultrasound microbubbles (UTMB) promoting stem cells homing to fibrotic liver. METHODS: Bone marrow derived mesenchymal stem cells (BMSCs) were divided into 5 groups with or without ultrasound microbubbles and continuously irradiated with ultrasound conditions of frequency 1 MHZ and output power 0.6 W/cm2 for different times, and then injected into a mouse model of liver fibrosis through the tail vein with or without ultrasound microbubbles, with sound intensity. The effect of ultrasound microbubbles on MSC expression of CXC chemokine receptor 4 (CXCR4) and homing fibrotic liver was evaluated by flow cytometry (FCM), western blot (WB) and immunohistochemistry (IHC) analysis. RESULTS: The level of CXCR4 expression was significantly higher in the ultrasound microbubble group than in the non-intervention group (P < 0.05), and the number of MSC and the rate of CXCR4 receptor positivity in the ultrasound microbubble-treated liver tissues were significantly higher than in the non-intervention group (P < 0.01). CONCLUSION: Ultrasonic microbubbles can promote the expression of CXCR4 on the surface of MSCs, thus improving the homing rate of MSCs in fibrotic liver.

IL-2/GM-CSF enhances CXCR3 expression in CAR-T cells via the PI3K/AKT and ERK1/2 pathways.

OBJECTIVE: To investigate the effects of cytokines IL-2 and GM-CSF on CXCR3 expression and chemotaxis of CAR-T cells. BACKGROUND: High lymphocyte infiltration within the tumor is a basic requirement for good results in tumor immunotherapy; C-X-C motif chemokine receptor 3 (CXCR3) is an important factor for the chemotaxis of lymphocytes to tumor tissues. The tumor microenvironment can exhibit diverse cytokine suppression or promote antitumor immunity. Both interleukin (IL)-2 and granulocyte macrophage colony-stimulating factor (GM-CSF) contribute to the regulation of immunosuppression in the tumor microenvironment. However, the effects of IL-2 and GM-CSF on CXCR3 expression on the T cell surface and its mechanisms are not well understood. Here, we explored the effects of polycytokines on CXCR3 expression in chimeric antigen receptor T cells (CAR-T cells) and on HuH-7 in situ hepatocellular carcinoma. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated, followed by purifying using CD3 immunomagnetic beads. Cells were divided into three groups. After 24h of activation using CD3/CD28 antibody, T cells were transfected using lentiviral vector, pGC-SV40-EGFP-GPC3-CAR. Three culture methods were used to amplify the transfected T cells. Method 'A' was to incubate T cells with CD3/CD28 antibody; method 'B' was with CD3/CD28 antibody and IL-2 at a final concentration of 1000 U/ml; method 'C' was with method B in addition of GM-CSF at a final concentration of 1000 U/ml. The phosphorylation of MAPK and PI3K/AKT was determined by western blot. The chemotaxis effect of CAR-T cells on Huh-7 HCCIA in situ was assayed by immunofluorescence and immunohistochemistry. RESULTS: The CD3/CD28/IL-2/GM-CSF combination is the most potent for stimulating activated CAR-T cell proliferation and CXCR3 expression in vitro; CD3/CD28/IL-2 induces CAR-T cell expression of CXCR3 through the activation of the PI3K/APK pathway and GM-CSF induces CXCR3 expression in CAR-T cells through the activation of ERK1/2 rather than the p38 MAPK signaling pathway. CAR-GPC3-T cells with high CXCR3 expression showed increased chemotaxis ability to HuH in situ hepatocellular carcinoma, and considerably inhibited the growth of in situ tumors in nude mouse livers. CONCLUSION: A multi-factorial amplification protocol can effectively improve CXCR3 expression on the surface of activated CAR-T cells in vitro, as well as enhance the chemotaxis ability of CAR-T cells in vivo, which significantly inhibit the growth of liver cancer.

Low Serum Soluble Transferrin Receptor Levels Are Associated with Poor Prognosis in Patients with Hepatitis B Virus-Related Acute-on-Chronic Liver Failure.

Iron metabolism disorder is closely related to acute-on-chronic liver failure (ACLF). This study was conducted to analyze the serum levels of soluble transferrin receptor (sTfR) in hepatitis B virus (HBV)-related ACLF and to evaluate the predictive value of sTfR for the short-term prognosis of HBV-ACLF. A total of 359 patients, including 139 with HBV-ACLF, 103 with chronic hepatitis B (CHB), and 117 healthy controls (HCs), participated in this study. We measured serum levels of ferritin, transferrin, and sTfR using nephelometry and performed data analysis using SPSS software. Ferritin levels were significantly higher in HBV-ACLF patients (both P < 0.001), while transferrin and sTfR were significantly lower (all P < 0.001) than in patients with CHB and HCs. Spearman correlation analysis demonstrated that serum sTfR significantly correlated with the alanine aminotransferase (ALT) (r = -0.366, P < 0.001), aspartate aminotransferase (AST) (r = -0.322, P < 0.001), total bilirubin (TBIL) (r = -0.222, P = 0.009), alpha fetoprotein (AFP) (r = 0.329, P < 0.001), prothrombin time-international normalization ratio (PT-INR) (r = -0.428, P < 0.001), and model for end-stage liver disease (MELD) (r = -0.459, P < 0.001). Nonsurviving HBV-ACLF patients who died within 30 days had much lower serum sTfR levels than surviving patients (P < 0.001). Logistic regression analysis showed that decreased serum sTfR levels were independently associated with 30-day mortality in patients with HBV-ACLF (P = 0.003). Receiver operating characteristic (ROC) curve analysis for predicting 30-day mortality showed that the area under the curve (AUC) for serum sTfR was 0.813 (95% CI: 0.738-0.874, P < 0.001). This was similar to that of the MELD score (AUC = 0.812, 95% CI: 0.737-0.873, P < 0.001). Serum sTfR combined with MELD score significantly improved the predictive capacity for 30-day mortality in patients with HBV-ACLF (AUC = 0.871, 95% CI: 0.803-0.922, P < 0.001). Kaplan-Meier analysis revealed that the overall cumulative 30-day mortality rate was significantly higher in patients with serum sTfR levels ≤ 0.55 mg/L compared to those with serum sTfR levels > 0.55 mg/L (P < 0.001).

Serum sCD14 as a Biomarker for Significant Liver Inflammation in Chronic Hepatitis B Patients with Normal or Mildly Elevated ALT.

BACKGROUND: CD14 is a pattern recognition receptor constitutively expressed in different types of immune cells, either in a membrane-anchored (mCD14) or in a soluble (sCD14) form. This study investigated whether hepatic CD14 expression levels were correlated with the grades of liver inflammation as well as the potential usefulness of serum sCD14 as a biomarker for predicting liver inflammation in chronic hepatitis B (CHB) patients with normal or mildly elevated ALT. METHODS: A total of 216 treatment-naive CHB patients with normal or mildly elevated ALT who underwent liver biopsy were recruited. Hepatic expression level of CD14 was measured using immunohistochemistry and real-time PCR. Serum sCD14 concentrations were determined with an enzyme-linked immunosorbent assay. Correlations between hepatic CD14, serum sCD14, and liver inflammation grade were analyzed. Univariate and multivariate analysis were performed to identify significant liver inflammation-associated factors. The receiver operating characteristic curve was used to assess the discriminating power of serum sCD14 to significant liver inflammation in CHB patients with normal or mildly elevated ALT. RESULTS: Both hepatic expression levels of CD14 and serum sCD14 concentrations significantly increased with the aggravation of liver inflammation. Moreover, hepatic expression levels of CD14, serum sCD14 concentrations, and liver inflammation grades were positively correlated with each other. Three parameters including alkaline phosphatase (ALP), neutrophil, and sCD14 were identified as independent predictors of significant liver inflammation. Subsequently, a diagnostic equation named model-sCD14 was developed incorporating sCD14 and other variables (ALP and neutrophils) with p < 0.05 in multivariate logistic analysis. The area under receiver operating characteristic curve (AUC) of sCD14 for predicting significant liver inflammation was 0.788 and the optimal cutoff was 27.14 ng/mL, with a sensitivity of 66.67%, a specificity of 81.70%, positive predictive value of 60.01%, and negative predictive value of 85.62%. When sCD14 was replaced by model-sCD14, the AUC value increased from 0.788 to 0.843 (z = 2.311, p = 0.021), with sensitivity of 77.78%, specificity of 77.12%, positive predictive value of 58.33%, and negative predictive value of 89.39%. CONCLUSIONS: Serum sCD4 has the potential to discriminate significant liver inflammation from CHB patients with normal or mildly increased ALT levels.