Microbiota transfer following liver surgery involves microbial extracellular vesicle migration that affects liver immunity.

BACKGROUND: Short-term perioperative administration of probiotics was shown to alleviate postoperative complications and promote liver recovery among patients undergoing resection for liver malignancy. The mechanisms by which probiotic bacteria effectively influence the gut microbiome composition during the perioperative time are controversial. Here, we aim to elucidate the short-term direct biological effect of probiotic microbiota-derived vesicles on host liver cells during the perioperative period. METHODS: Probiotic-derived vesicles (pbMVs) were administered postoperatively. pbMVs were isolated and characterized from probiotics, mainly from the bacteria genus Lactobacillus, Bifidobacterium, and Lactococcus. Mice underwent bile duct ligation, sham laparotomy (SHAM), or 70% partial hepatectomy (70%PH). pbMVs were tracked in vivo, and intrahepatic cellular and molecular aspects were analyzed by flow cytometry and qRT-PCR techniques. Liver sinusoidal endothelial cells (LSECs) analysis for Vascular Cell Adhesion Molecule-1(VCAM-1) expression following pbMV stimulation of cultured liver non-parenchymal cells which had been activated by LPS. RESULTS: The administered pbMV rapidly translocated to the liver after surgery. pbMV administrations following surgeries enhanced neutrophil clearance; there was a dramatic decline in the liver neutrophil-to-lymphocyte ratio Ly6G+/CD3+ and an increase in IL6 levels. pbMVs reduced intrahepatic VCAM1 and ICAM2 expression compared with control following SHAM and decrease in IL10 levels following 70%PH. The administration of pbMV improved liver regeneration 72 hours following surgical liver resection with a significant decrease in IL17 expression. pbMVs modulated VCAM-1 on liver sinusoidal endothelial cells in liver cell culture. CONCLUSIONS: Our study findings provide mechanistic insights into the liver-gut axis following surgery and illustrate how probiotic vesicles can reduce adhesion molecule expression and affect immune cell invasion and liver immunity, resulting in improved liver recovery following hepatic surgery.

Novel Proteome Extraction Method Illustrates a Conserved Immunological Signature of MSI-H Colorectal Tumors.

Using a simple, environment friendly proteome extraction (TOP), we were able to optimize the analysis of clinical samples. Using our TOP method we analyzed a clinical cohort of microsatellite stable (MSS) and unstable (MSI-H) colorectal carcinoma (CRC). We identified a tumor cell specific, STAT1-centered, immune signature expressed by the MSI-H tumor cells. We then showed that long, but not short, exposure to Interferon-γ induces a similar signature in vitro We identified 10 different temporal protein expression patterns, classifying the Interferon-γ protein temporal regulation in CRC. Our data sheds light on the changes that tumor cells undergo under long-term immunological pressure in vivo, the importance of STAT proteins in specific biological scenarios. The data generated could help find novel clinical biomarkers and therapeutic approaches.

Micro-RNA Expression Patterns Predict Metastatic Spread in Solid Pseudopapillary Neoplasms of the Pancreas.

Solid pseudopapillary neoplasm (SPN) of pancreas is a rare pancreatic neoplasm with a low metastatic potential. Up to 10% of patients with localized disease at presentation will develop systemic metastases, usually in the peritoneum or the liver. Due to the rarity of SPNs and the overall excellent prognosis, reliable prognostic factors to predict malignant biological behavior remain undetermined. Therefore, we aimed to define clinical, histological, and microRNA patterns that are associated with metastatic disease. We conducted a retrospective single center study on all patients operated for SPN of pancreas between 1995 and 2018. Clinical and pathological data were collected, and expression patterns of 2,578 human microRNAs were analyzed using microRNA array (Affimetrix 4.1) in normal pancreases (NPs), localized tumors (LTs), and metastatic tumors (MTs). The diagnosis of SPN was confirmed in 35 patients who included 28 females and 3 males, with a mean age of 33.8 ± 13.9 years. The only clinical factor associated with metastases was tumor size (mean tumor size 5.20 ± 3.78 in LT vs. 8.13± 1.03 in MT, p < 0.012). Microscopic features of malignancy were not associated with metastases, nor were immunohistochemical stains, including the proliferative index KI67. Higher expressions of miR-184, miR-10a, and miR-887, and lower expressions of miR-375, miR-217, and miR-200c were observed in metastatic tissues on microarray, and validated by real-time polymerase chain reaction. Hierarchal clustering demonstrated that the microRNA expression pattern of MTs was significantly different from that of LTs. The only clinical factor associated with metastases of SPN of pancreas was tumor size. Histological features and immunohistological staining were not predictive of metastases. A panel of six microRNAs was differentially expressed in MTs, and these findings could potentially be used to predict tumor behavior. Validation of these results is needed in larger series.

A Harmonization Study for the Use of 22C3 PD-L1 Immunohistochemical Staining on Ventana's Platform.

INTRODUCTION: Immunotherapy is a novel treatment for lung cancer. Pembrolizumab (Merck Sharp and Dohme, Kenilworth, NJ) is a monoclonal antibody against programmed cell death 1 that has been approved for use with NSCLC together with a companion diagnostic by Dako (Carpinteria, CA). Ventana's BenchMark XT (Ventana Medical Systems, Tucson, AZ) is a widely used immunohistochemical (IHC) platform. However, data on its reliability and reproducibility with the 22C3 antibody are scant. METHODS: We performed a comprehensive calibration of 22C3 programmed cell death ligand 1 (PD-L1) staining on the BenchMark XT platform using Dako's prediluted 22C3 anti-PD-L1 primary antibody with two of Ventana's detection systems. Forty-one random cases of NSCLC were then independently evaluated by two pathologists. Each case was scored using Dako- or Ventana-stained slides. The scores obtained with the two 22C3 Ventana assays were compared with those obtained using the Dako 22C3 IHC platform. RESULTS: The Dako IHC platform stratified eight, seven, and 26 cases as being strongly positive, weakly positive, and negative for PD-L1, respectively, whereas 36 of 41 cases (87.8%) had the same results with Ventana's UltraView 22C3 protocol (Pearson's correlation score 0.91, p < 0.0001). Moreover, 35 of 41 cases (85.3%) had the same results with Ventana's OptiView 22C3 protocol (Pearson's correlation score 0.89, p < 0.0001). CONCLUSIONS: The results of this study demonstrate that the same PD-L1 IHC algorithm can be reliably applied to Ventana's BenchMark XT platform. Furthermore, we were able to detect all of the strongly positive cases with high interobserver and intraobserver agreement by using the Ventana platform. These findings suggest that the Ventana platform can be used to stratify patients for pembrolizumab-based immunotherapy.

PF-4708671 activates AMPK independently of p70S6K1 inhibition.

The P70 ribosomal protein S6 kinase 1 (P70S6K1) is activated by the mammalian target of rapamycin (mTORC1) and regulates proliferation, growth, and metabolism. PF-4708671 is a novel, cell-permeable, has been proposed to be a highly specific inhibitor of p70S6K1. It is used in micromolar concentration range to dissect signaling pathways downstream of mTORC1 and to study the function of p70S6K1. Here we show that PF-4708671 induces AMP-activated protein kinase (AMPK) phosphorylation and activation in immortalized mouse embryonic fibroblasts (MEF) independently of p70S6K1, due to specific inhibition of mitochondrial respiratory chain Complex I.