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Abstract Background Excessive consumption of alcohol contributes to alcoholic liver disease. Fatty liver is the early stage of alcohol-related liver disease. The aim of this study was to search for specific serological biomarkers of alcoholic fatty liver (AFL) compared to healthy controls, non-alcoholic fatty liver (NAFL) and liver fibrosis in a rodent model. Methods Serum samples derived from animals with AFL, NAFL, or liver fibrosis were characterized and compared using two-dimensional differential gel electrophoresis. A matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer in conjunction with mascot software was used for protein identification. Subsequently, Western blotting and flexible multi-analyte profiling were used to measure the expressions of the putative biomarkers present in the serum of animals and clinical patients. Results Eight differential putative biomarkers were identified, and the two most differentiated proteins, including upregulated C-reactive protein (CRP) and downregulated haptoglobin (Hp), were further investigated. Western blotting validated that CRP was dramatically higher in the serum of AFL compared to healthy controls and other animals with liver disease of NAFL or liver fibrosis (p < 0.05). Moreover, we found that CRP and Hp were both lower in liver fibrosis of TAA-induced rats and clinical hepatitis C virus-infected patients. Conclusion The results suggest that increased levels of CRP are an early sign of AFL in rats. The abnormally elevated CRP induced by ethanol can be used as a biomarker to distinguish AFL from normal or otherwise diseased livers.

Abstract Background The gold standard of assessing liver fibrosis is liver biopsy, which is invasive and not without risk. Therefore, searching for noninvasive serologic biomarkers for liver fibrosis is an importantly clinical issue. Methods A total of 16 healthy volunteers and 45 patients with chronic hepatitis C virus (HCV) were enrolled (F0: n = 16, F1: n = 7, F2: n = 17, F3: n = 8 and F4: n = 13, according to the METAVIR classification). Three serum samples of each fibrotic stage were analyzed by two-dimension difference gel electrophoresis (2D-DIGE). The differential proteins were identified by the cooperation of MALDI-TOF/TOF and MASCOT; then western blotting and Bio-Plex Suspension Array were used to quantify the protein levels. Results Three prominent candidate biomarkers were identified: alpha 2 macroglobulin (A2M) is up regulated; vitamin D binding protein (VDBP) and apolipoprotein AI (ApoAI) are down regulated. The serum concentration of A2M was significantly different among normal, mild (F1/F2) and advanced fibrosis (F3/F4) (p < 0.01). The protein levels of VDBP and ApoAI were significantly higher in normal/mild fibrosis, when compared to those in advanced fibrosis (both p < 0.01). Conclusions This study not only reveals three putative biomarkers of liver fibrosis (A2M, VDBP and ApoAI) but also proves the differential expressions of those markers in different stages of fibrosis. We expect that combination of these novel biomarkers could be applied clinically to predict the stage of liver fibrosis without the need of liver biopsy.

Radiotherapy (RT) not only damages tumors but also induces interferon (IFN) expression in tumors. IFNs mediate PD-L1 to exhaust CD8+ T cells, but which also directly impact tumor cells and potentially activate anti-tumor immune surveillance. Little is known about the contradictory mechanism of IFNs in regulating CD8+ T-mediated anti-tumor activity in lung cancer. This study found that RT induced IFNs and CXCL9/10 expression in the RT-treated lung cancer cells. Specifically, RT- and IFNγ-pretreated A549 significantly activated CD8+ T cells, resulting in significant inhibition of A549 colony formation. RNAseq and consequent qPCR results revealed that IFNγ induced PD-L1, CXCL10, and ICAM-1, whereas PD-L1 knockdown activated CD8+ T cells, but ICAM-1 knockdown diminished CD8+ T cell activation. We further demonstrated that CXCR3 and CXCL10 decreased in the CD8+ T cells and nonCD8+ PBMCs, respectively, in the patients with lung cancer that expressed lower reactivation as co-cultured with A549 cells. In addition, inhibitors targeting CXCR3 and LFA-1 in CD8+ T cells significantly diminished CD8+ T cell activation and splenocytes-mediated anti-LL/2shPdl1. In conclusion, we validated that RT suppressed lung cancer and overexpress PD-L1, CXCL10, and ICAM-1, which exhibited different roles in regulating CD8+ T cell activity. We propose that CXCR3highCD8+ T cells stimulated by CXCL10 exhibit anti-tumor immunity, possibly by enhancing T cells-tumor cells adhesion through CXCL10/CXCR3-activated LFA-1-ICAM-1 interaction, but CXCR3lowCD8+ T cells with low CXCL10 in patients with lung cancer were exhausted by PD-L1 dominantly. Therefore, RT potentially activates CD8+ T cells by inducing IFNs-mediated CXCL10 and ICAM-1 expression in tumors to enhance CD8+ T-tumor adhesion and recognition. This study clarified the possible mechanisms of RT and IFNs in regulating CD8+ T cell activation in lung cancer. [ABSTRACT FROM AUTHOR]

Irradiation-broken DNA fragments increase type I interferon and chemokines secretion in tumor cells. Since radiotherapy may augment tumor immunotherapy, we hypothesize that the chemokines increased by irradiation could recruit CD8+ T cells to suppress tumor proliferation. This study intended to unveil the secreted factors activating and recruiting CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive A549 was selected and treated by X-irradiation (IR) to identify the overexpression of chemokines associated to CD8+ T cell cytotoxicity and recruitment. A transwell assay with Alexa 488-labeled CD8+ T cells was used to evaluate CD8+ T cell motility in vitro. A nuclear imaging platform by In111-labeled nivolumab was used to track CD8+ T cells homing to tumors in vivo. The activation markers GZMB, PRF-1, and IFNγ, migration marker CD183 (CXCR3), and inhibitory marker CD274 (PD-1), were measured and compared in CD8+ T cells with A549 co-cultured, chemokines treated, and patients with late-stage lung cancer. We found that IR not only suppressed A549 proliferation but also induced IFNα and CXCL9 expression (p < 0.05). IFNα majorly increased IFNγ levels in CD8+ T cells (p < 0.05) and synergistically with CXCL9 enhanced CD8+ T cell migration in vitro (p < 0.05). We found that CXCR3 and PD-1 were down-regulated and up-regulated, respectively, in the peripheral blood CD8+ T cells in patients with lung cancer (n = 4 vs. healthy n = 3, both p < 0.05), which exhibited reduction of cell motility (p < 0.05). The in vivo nuclear imaging data indicated highly CD8+ T cells migrated to A549-induced tumors. In addition, we demonstrated that healthy PBMCs significantly suppressed the parallel tumor growth (p < 0.05) and the radioresistant tumor growth in the tumor xenograft mice (p < 0.05), but PBMCs from patients with lung cancer had lost the anti-tumor capacity. We demonstrated that IR induced IFNα and CXCL9 expression in A549 cells, leading to CD8+ T cell migration. This study unveiled a potential mechanism for radiotherapy to activate and recruit CD8+ T cells to suppress lung tumors. [ABSTRACT FROM AUTHOR]

Tumor cells express immune checkpoints to exhaust CD8+ T cells. Irradiation damages tumor cells and augments tumor immunotherapy in clinical applications. However, the radiotherapy-mediated molecular mechanism affecting CD8+ T cell activity remains elusive. We aimed to uncover the mechanism of radiotherapy augmenting cytotoxic CD8+ T cells in non-small-cell lung cancer (NSCLC). EGFR-positive NSCLC cell lines were co-cultured with CD8+ T cells from healthy volunteers. Tumor cell viability and apoptosis were consequently measured. IFNγ was identified secreted by CD8+ T cells and PBMCs. Therefore, RNAseq was used to screen the IFNγ-mediated gene expression in A549 cells. The irradiation effect to IFNγ-mediated gene expression was investigated using qPCR and western blots. We found that the co-culture of tumor cells stimulated the increase of granzyme B and IFNγ in CD8+ T, but A549 exhibited resistance against CD8+ T cytotoxicity compared to HCC827. Irradiation inhibited A549 proliferation and enhanced apoptosis, augmenting PBMCs-mediated cytotoxicity against A549. We found that IFNγ simultaneously increased phosphorylation on STAT1 and STAT3 in EGFR-positive lung cancer, resulting in overexpression of PD-L1 (p < 0.05). In RNAseq analysis, MCL1 was identified and increased by the IFNγ-STAT3 axis (p < 0.05). We demonstrated that irradiation specifically inhibited phosphorylation on STAT1 and STAT3 in IFNγ-treated A549, resulting in reductions of PD-L1 and MCL1 (both p < 0.05). Moreover, knockdowns of STAT3 and MCL1 increased the PBMCs-mediated anti-A549 effect. This study demonstrated that A549 expressed MCL1 to resist CD8+ T cell-mediated tumor apoptosis. In addition, we found that irradiation suppressed IFNγ-mediated STAT3 phosphorylation and PD-L1 and MCL1 expression, revealing a potential mechanism of radiotherapy augmenting immune surveillance. [ABSTRACT FROM AUTHOR]