Dissection of DLBCL microenvironment provides a gene expression-based predictor of survival applicable to formalin-fixed paraffin-embedded tissue.

Background: Gene expression profiling (GEP) studies recognized a prognostic role for tumor microenvironment (TME) in diffuse large B-cell lymphoma (DLBCL), but the routinely adoption of prognostic stromal signatures remains limited. Patients and methods: Here, we applied the computational method CIBERSORT to generate a 1028-gene matrix incorporating signatures of 17 immune and stromal cytotypes. Then, we carried out a deconvolution on publicly available GEP data of 482 untreated DLBCLs to reveal associations between clinical outcomes and proportions of putative tumor-infiltrating cell types. Forty-five genes related to peculiar prognostic cytotypes were selected and their expression digitally quantified by NanoString technology on a validation set of 175 formalin-fixed, paraffin-embedded DLBCLs from two randomized trials. Data from an unsupervised clustering analysis were used to build a model of clustering assignment, whose prognostic value was also assessed on an independent cohort of 40 cases. All tissue samples consisted of pretreatment biopsies of advanced-stage DLBCLs treated by comparable R-CHOP/R-CHOP-like regimens. Results: In silico analysis demonstrated that higher proportion of myofibroblasts (MFs), dendritic cells, and CD4+ T cells correlated with better outcomes and the expression of genes in our panel is associated with a risk of overall and progression-free survival. In a multivariate Cox model, the microenvironment genes retained high prognostic performance independently of the cell-of-origin (COO), and integration of the two prognosticators (COO + TME) improved survival prediction in both validation set and independent cohort. Moreover, the major contribution of MF-related genes to the panel and Gene Set Enrichment Analysis suggested a strong influence of extracellular matrix determinants in DLBCL biology. Conclusions: Our study identified new prognostic categories of DLBCL, providing an easy-to-apply gene panel that powerfully predicts patients' survival. Moreover, owing to its relationship with specific stromal and immune components, the panel may acquire a predictive relevance in clinical trials exploring new drugs with known impact on TME.

Cytokine expression profile and blood parameter evaluation of patients undergoing cardiac surgery.

Cardiac surgery is accompanied by an important immune response that is poorly understood. This inflammatory response is caused by several stimuli: surgical trauma, cardiopulmonary bypass apparatus, aortic-cross clamping, reperfusion injury and hypothermia. The aim of the present study is to investigate the cytokine level profile involved in the inflammatory pathway of patients undergoing cardiac surgery. One hundred and two patients undergoing elective cardiac surgery utilizing cardiopulmonary bypass (CPB) apparatus were enrolled in the study. In the hematological and biochemical profiles investigated, we observed a significant increase of WBC and blood glucose concentration and a strong decrease of RBC, HB, HCT and PLT 24 h post-surgery compared to baseline and immediately after surgery groups. Furthermore, we found a modulation of cytokine levels mostly for IL-10 and an increase of IL-6, detected at 6 h post-surgery, IL-8 at 6 and 24 h, and TNFα only at 24 h post-surgery. In conclusion, these findings evidence a time course profile on cytokine levels and a balance between pro- and anti-inflammatory cytokine activation during and after cardiac surgery. In fact, IL-6 and IL-10, a pro- and an anti-inflammatory cytokine, respectively, increased immediately after surgery. The plasma level of TNF-α could be inhibited by the high concentration of IL-10 up to 6 h post-surgery. An IL-10 reduction at baseline level, after 24 h post-surgery, could explain a rise of TNF-α plasma concentration. On the other hand, considering the dual role of IL-6 on inflammation acting both as an activator of inflammatory cascade or an anti-inflammatory agent, the increased IL-6 levels 24 h after surgery could be related to the negative feedback action on TNFα activity.

Cytokine modulation in patients with idiopathic pulmonary fibrosis undergoing treatment with steroids, immunosuppressants, and IFN-γ 1b.

Idiopathic pulmonary fibrosis (IPF) is a chronic lung disease of unknown etiology and pathogenic mechanisms. From an etiopathogenic point of view, alveolar macrophages play a key role in accumulation of fibroblasts and deposition of collagen and extracellular matrix by releasing specific cytokines and inflammatory mediators. IPF seems to be also associated with circulating fibrocytes, which might be involved with an abnormal pulmonary vascular repair and remodeling. Based on its hypothesized pathologic mechanisms, anti-inflammatory, anti-fibrotic and immunosuppressive therapies are often used. For these reasons, Interferon-g (IFN-g) has been used to exploit its activity on macrophages and fibroblasts. The aim of this study was to investigate the response to corticosteroids and/or IFN-g 1b treatments based on pulmonary function tests and on inflammatory cytokine patterns of expression on bronchoalveolar lavage (BAL), at baseline and during and after the therapies. Unlike previous studies, we analyzed a period of therapy longer than 1 year. Our results demonstrated the effectiveness of IFN-γ in a group of IPF patients in whom the treatment was prolonged for over a year. These data suggest a positive role of IFN-γ; treatment in patients in the initial stage of the disease.

Effect of low energy light irradiation by light emitting diode on U937 cells.

Photobiomodulation (PBM) can induce a set of different biological modulators either in vitro or in vivo. Experimental evidence has highlighted the role of light effects on the mechanisms related to inflammation, apoptosis and autophagy. The goal of this project was the evaluation of PBM on U937, an established cell line of histiocytic lymphoma origin. Several aspects of modulation of proinflammatory pathways were analyzed and autophagic and proapoptotic mechanisms related to low laser light exposure of cells were studied. As a source of low energy light emission, we used an NIR-LED device, characterized by an 880 nm-wavelength as light source. Flow cytometry analysis was performed on supernatants of controls and treated U937 cells to detect inflammatory cytokine levels. In order to evaluate NF-kB and caspase3 expressions, Western blot analysis was performed according to standard procedures. In this report, we show the effect of PBM on a monocyte/macrophage established tumor cell line (U-937). We demonstrate that LED exposure, in the presence or absence of lipopolysaccharide (LPS), activates cell degranulation, increased expression of Interleukin-8 (IL-8) and modulation of beta galactosidase activity. Evidence shows that the well-known pro-inflammatory nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and the apoptotic marker (caspase3/cleaved-caspase3 ratio) are up-regulated in response to a proinflammatory biochemical pathway.