Development of an immune-related prognostic biomarker for triple-negative breast cancer.

Purpose: Oncology studies employing digital dissection methodologies have provided some insight on the biological features of tumor microenvironment of Triple-negative breast cancer (TNBC), but molecular diagnostics rarely have therapeutic impact. We aimed to identify a novel prognostic biomarker to investigate immune characteristics of TNBC using transcriptomic features.Patients and Methods: We extracted whole transcriptome from breast cancer tissue of 30 TNBC patients and then used bioinformatics approaches to characterize the different immune cell contents in tumor tissue and para-cancerous tissue. We extract 2 indicators to describe the major differences in immune infiltration in the microenvironment between tumor tissue and para-cancerous tissue. We then combined the 2 indicators that represent the levels of increased and decreased infiltration in each sample to obtain the Immune Infiltration Score (IIS). Then we compared the tumor-infiltrating immune cell contents and immune infiltrating status in TNBC samples with CIBERSORT and ESTIMATE score to validate the IIS. Finally, 132 TNBC patients from the Cancer Genome Atlas program (TCGA) dataset was used to validate the predictive power of IIS.Results: 4 types of upregulated and 4 types of downregulated immune cells were identified in the tumor tissue samples of the TNBC patients. Then we developed a novel biomarker, IIS. Results showed that IIS score can clearly separate cancer and para-cancerous tissue. Using the same cutoff value of 0 in the TNBC-TCGA cohort, we show that those patients with a higher IIS had significantly higher PD-L1 expression and shorter progression-free survival time than those with a lower IIS value, indicating IIS score can be generalized to other TNBC datasets.Conclusion: we explored the immune infiltration landscape in 30 TNBC patients and provided IIS as a novel and reliable biomarker to evaluate the progression-free survival and prognosis of the TNBC patients.

Ketamine promotes inflammation through increasing TLR4 expression in RAW264.7 cells.

SUMMERY: Ketamine (KTM), a N-methyl-D-aspartate (NMDA) receptor antagonist, was found to has an anti-inflammatory effect, but some patients suffered from exacerbated pro-inflammatory reactions after anesthesia with KTM. The present study was aimed to examine the underlying mechanism of pro-inflammatory effects of KTM. In this study, RAW264.7 cells were exposed to KTM and NMDA alone or combined for 30 min before lipopolysaccharide (LPS) stimulation. The expression levels of IL-6 and TNF-α were detected by RT-PCR and ELISA, and those of NMDA receptors by RT-PCR in RAW264.7 cells. Additionally, the TLR4 expression was determined by RT-PCR and flow cytometry, respectively. The results showed that in RAW264.7 cells, KTM alone promoted the TLR4 expression, but did not increase the expression of IL-6 or TNF-α. In the presence of LPS, KTM caused a significantly higher expression of IL-6 and TNF-α than LPS alone. NMDA could neither alter the IL-6 and TNF-α mRNA expression, nor reverse the enhanced expression of IL-6 and TNF-α mRNA by KTM in LPS-challenged cells. After TLR4-siRNA transfection, RAW264.7 cells pretreated with KTM no longer promoted the IL-6 and TNF-α expression in the presence of LPS. In conclusion, KTM accelerated LPS-induced inflammation in RAW264.7 cells by promoting TLR4 expression, independent of NMDA receptor.

Volatile anesthetics inhibit the activity of calmodulin by interacting with its hydrophobic site.

BACKGROUND: Volatile anesthetics (VAs) may affect varied and complex physiology processes by manipulating Ca(2+)-calmodulin (CaM). However, the detailed mechanism about the action of VAs on CaM has not been elucidated. This study was undertaken to examine the effects of VAs on the conformational change, hydrophobic site, and downstream signaling pathway of CaM, to explore the possible mechanism of anesthetic action of VAs. METHODS: Real-time second-harmonic generation (SHG) was performed to monitor the conformational change of CaM in the presence of VAs, each plus 100 µmol/L Ca(2+). A hydrophobic fluorescence indicator, 8-anilinonaphthalene-1-sulfonate (ANS), was utilized to define whether the VAs would interact with CaM at the hydrophobic site or not. High-performance liquid chromatography (HPLC) was carried out to analyze the activity of CaM-dependent phosphodiesterase (PDE1) in the presence of VAs. The VAs studied were ether, enflurane, isoflurane, and sevoflurane, with their aqueous concentrations 7.6, 9.5, 11.4 mmol/L; 0.42, 0.52, 0.62 mmol/L; 0.25, 0.31, 0.37 mmol/L and 0.47, 0.59, 0.71 mmol/L respectively, each were equivalent to their 0.8, 1.0 and 1.2 concentration for 50% of maximal effect (EC50) for general anesthesia. RESULTS: The second-harmonic radiation of CaM in the presence of Ca(2+) was largely inhibited by the VAs. The fluorescence intensity of ANS, generated by binding of Ca(2+) to CaM, was reversed by the VAs. HPLC results also showed that AMP, the product of the hydrolysis of cAMP by CaM-dependent PDE1, was reduced by the VAs. CONCLUSIONS: Our findings demonstrate that the above VAs interact with the hydrophobic core of Ca(2+)-CaM and the interaction results in the inhibition of the conformational change and activity of CaM. This in vitro study may provide us insight into the possible mechanism of anesthetic action of VAs in vivo.

Adenovirus-delivered angiopoietin 1 accelerates the resolution of inflammation of acute endotoxic lung injury in mice.

BACKGROUND: The immune system plays a key role in protecting the organism from infection. Timely resolution of the inflammatory response to infection plays a vital role in returning homeostasis and maintaining normal organ function. Angiopoietin1 prevents endothelial activation, part of the inflammatory response to a pathogen, and has an anti-inflammatory effect in acute lung injury. We designed this study to investigate whether increasing serum production of angiopoietin1 by IV administration of adenoviral-delivered angiopoietin1 could accelerate the resolution of inflammation in endotoxin-induced acute lung injury in mice. METHODS: Lipopolysaccharide was intratracheally instilled to induce acute lung injury in animals pretreated for 24 hours with adenoviral-GFP vector or adenoviral-GFP-angiopoietin1, respectively. An additional 6 mice in each pretreatment group were killed before lipopolysaccharide instillation to serve as controls. Indices of resolution of inflammation were analyzed. Apoptotic polymorphonuclear leukocytes and their phagocytosis by macrophages were determined by fluorescent activated cell sorter. The expression of angiopoietin1 in tissues and granulocyte macrophage colony-stimulating factor in the bronchoalveolar lavage fluid were measured. RESULTS: Lipopolysaccharide induced leukocyte infiltration into air spaces, with maximal infiltration 48 hours after lipopolysaccharide instillation. Pretreatment with adenovirus-GFP-angiopoietin1 markedly increased angiopoietin1 expression, reduced leukocyte, and neutrophil infiltration and shortened the duration of inflammation. Adenovirus-GFP-angiopoietin1 pretreatment augmented the magnitude without altering the time course of granulocyte macrophage colony-stimulating factor. CONCLUSIONS: Our results suggest that angiopoietin1 pretreatment promotes resolution of inflammation in endotoxin-induced acute lung injury in mice by accelerating the apoptosis of neutrophils and their phagocytosis by macrophages.