URINARY PROTEOMIC MARKERS OF IGA NEPHROPATHY, LUPUS NEPHRITIS AND MEMBRANOUS NEPHROPATHY

INTRODUCTION: Chronic kidney disease (CKD) is a worldwide public health problem, related to increased morbidity and mortality. Glomerulopathies represent major causes of CKD and require complicated diagnostics. Standard of care includes kidney biopsy in order to confirm the type of nephropathy. However, biopsy brings specific risks. Therefore, non-invasive diagnostic and prognostic methods are sought. Urinary proteomics emerged as safe and promising tool, but still requires development and improvements. Our previous studies which are part of European Patent Application from 10th June 2016 (WO/2017/212463), identified urinary markers of IgA nephropathy. They included among others: alpha-1B-glycoprotein (A1BG), alpha-l-acid glycoprotein 1 (ORM-1), ferritin light chain (FTL) and serotransferrin (TF). The aim of this study was to evaluate them in comparison to patients with glomerulopathies of different etiologies, such as lupus nephritis (LN) and membranous nephropathy (MN). METHODS: This proteomic study included patients with CKD (41 IgAN, 33 LN, 26 MN, 6 with erytrocyturia of unknown etiology) and 19 healthy controls. Urine samples were obtained from a midstream of the: first-morning (FM) and second- or third-morning (SPOT) sample. The SPOT samples were processed up to 2 h and FM samples up to 4 h after collection, by agitating and gently inverting 4-6 times, portioned into 2-ml aliquots and stored at -80°C for further measurements. Western Blotting was used for analysis of the SPOT and FM samples, ELISA and mass spectrometry for SPOT urine only. The results were related to demographic data, standard laboratory tests and GFR estimated with use of Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) equation. RESULTS: The urinary concentrations of A1BG, ORM-1, FTL and TF were found to be higher in CKD patients than in healthy controls. Moreover, these proteins varied depending on the disease. According to ELISA measurements, patients with IgAN, erytrocyturia and LN had significantly more A1BG and ORM-1 (p < 0.05), whereas TF was more elevated in LN and MN individuals comparing to healthy controls. The western blot analysis revealed significantly elevated level of A1BG, ORM-1 and FTL in IgAN, LN and MN, comparing to healthy control. Additionally, it revealed fragmentation of A1BG in several patients and the bottom range bands tended to be most prominently elevated in IgAN patients. Mass spectrometry confirmed differences between the diseases according to the specific amino acids fragments of each tested protein. Figure 1. Western blot scans for urinary A1BG, ORM-1 and FTL in CKD patients (2-4) and healthy controls (1). CONCLUSIONS: The urinary concentrations of A1BG, ORM-1, FTL and TF are elevated in CKD patients and vary depending on the type of nephropathy. This observation suggests their differential roles in the pathophysiology of the given diseases, and we believe their evaluation may help distinguishing between nephropathies. Further studies are desired to establish the role of these urinary proteins in non-invasive disease differentiation

Dimeric peroxiredoxins are druggable targets in human Burkitt lymphoma

Burkitt lymphoma is a fast-growing tumor derived from germinal center B cells. It is mainly treated with aggressive chemotherapy, therefore novel therapeutic approaches are needed due to treatment toxicity and developing resistance. Disturbance of red-ox homeostasis has recently emerged as an efficient antitumor strategy. Peroxiredoxins (PRDXs) are thioredoxin-family antioxidant enzymes that scavenge cellular peroxides and contribute to red-ox homeostasis. PRDXs are robustly expressed in various malignancies and critically involved in cell proliferation, differentiation and apoptosis. To elucidate potential role of PRDXs in lymphoma, we studied their expression level in B cell-derived primary lymphoma cells as well as in cell lines. We found that PRDX1 and PRDX2 are upregulated in tumor B cells as compared with normal counterparts. Concomitant knockdown of PRDX1 and PRDX2 significantly attenuated the growth rate of lymphoma cells. Furthermore, in human Burkitt lymphoma cell lines, we isolated dimeric 2-cysteine peroxiredoxins as targets for SK053, a novel thiol-specific small-molecule peptidomimetic with antitumor activity. We observed that treatment of lymphoma cells with SK053 triggers formation of covalent PRDX dimers, accumulation of intracellular reactive oxygen species, phosphorylation of ERK1/2 and AKT and leads to cell cycle arrest and apoptosis. Based on site-directed mutagenesis and modeling studies, we propose a mechanism of SK053-mediated PRDX crosslinking, involving double thioalkylation of active site cysteine residues. Altogether, our results suggest that peroxiredoxins are novel therapeutic targets in Burkitt lymphoma and provide the basis for new approaches to the treatment of this disease

Immune Evasion as the Main Challenge for Immunotherapy of Cancer

Immune evasion is currently considered one of the most prominent hallmarks of cancer [...

Bioinformatic Analysis Reveals Central Role for Tumor-Infiltrating Immune Cells in Uveal Melanoma Progression

Tumor-infiltrating immune cells are capable of effective cancer surveillance, and their abundance is linked to better prognosis in numerous tumor types. However, in uveal melanoma (UM), extensive immune infiltrate is associated with poor survival. This study aims to decipher the role of different tumor-infiltrating cell subsets in UM in order to identify potential targets for future immunotherapeutic treatment. We have chosen the TCGA-UVM cohort as a training dataset and GSE22138 as a testing dataset by mining publicly available databases. The abundance of 22 immune cell types was estimated using CIBERSORTx. Then, to determine the significance of tumor-infiltrating cell subsets in UM, we built a multicell type prognostic signature, which was validated in the testing cohort. The created signature was built upon the negative prognostic role of CD8+ T cells and M0 macrophages and the positive role of neutrophils. Based on the created signature score, we divided the patients into low- and high-risk groups. Kaplan-Meier, Cox, and ROC analyses demonstrated superior performance of our risk score compared to either clinical or pathologic characteristics of both cohorts. Further, we found the molecular pathways associated with cancer immunoevasion and metastasis to be enriched in the high-risk group, explaining both the lack of adequate immune surveillance despite increased infiltration of CD8+ T cells as well as the higher metastatic potential. Genes associated with tryptophan metabolism (IDO1 and KYNU) and metalloproteinases were among the most differentially expressed between the high- and low-risk groups. Our correlation analyses interpreted in context of published in vitro data strongly suggest the central role of CD8+ T cells in shifting the UM tumor microenvironment towards suppressive and metastasis-promoting. Therefore, we propose further investigations of IDO1 and metalloproteinases as novel targets for immunotherapy in lymphocyte-rich metastatic UM patients

Vadadustat, a HIF Prolyl Hydroxylase Inhibitor, Improves Immunomodulatory Properties of Human Mesenchymal Stromal Cells

The therapeutic potential of mesenchymal stromal cells (MSCs) is largely attributed to their immunomodulatory properties, which can be further improved by hypoxia priming. In this study, we investigated the immunomodulatory properties of MSCs preconditioned with hypoxia-mimetic Vadadustat (AKB-6548, Akebia). Gene expression analysis of immunomodulatory factors was performed by real-time polymerase chain reaction (real-time PCR) on RNA isolated from six human bone-marrow derived MSCs populations preconditioned for 6 h with 40 &mu;M Vadadustat compared to control MSCs. The effect of Vadadustat preconditioning on MSCs secretome was determined using Proteome Profiler and Luminex, while their immunomodulatory activity was assessed by mixed lymphocyte reaction (MLR) and Culturex transwell migration assays. Real-time PCR revealed that Vadadustat downregulated genes related to immune system: IL24, IL1B, CXCL8, PDCD1LG1, PDCD1LG2, HIF1A, CCL2 and IL6, and upregulated IL17RD, CCL28 and LEP. Vadadustat caused a marked decrease in the secretion of IL6 (by 51%), HGF (by 47%), CCL7 (MCP3) (by 42%) and CXCL8 (by 40%). Vadadustat potentiated the inhibitory effect of MSCs on the proliferation of alloactivated human peripheral blood mononuclear cells (PBMCs), and reduced monocytes-enriched PBMCs chemotaxis towards the MSCs secretome. Preconditioning with Vadadustat may constitute a valuable approach to improve the therapeutic properties of MSCs

Inhibition of autophagy sensitizes cancer cells to Photofrin-based photodynamic therapy

Abstract Background Accumulating evidence suggest that autophagy plays a pivotal role in various anticancer therapies, including photodynamic therapy (PDT), acting as a pro-death or pro-survival mechanism in a context-dependent manner. Therefore, we aimed to determine the role of autophagy in Photofrin-based PDT. Methods In vitro cytotoxic/cytostatic effects of PDT were evaluated with crystal violet cell viability assay. Autophagy induction was analyzed by immunoblotting and immunofluorescence using anti-LC3 antibody. Autophagy was inhibited by shRNA-mediated ATG5 knockdown or CRISPR/Cas9-mediated ATG5 knockout. Apoptosis was assessed by flow cytometry analysis of propidium iodide and anexin V-positive cells as well as by detection of cleaved PARP and caspase 3 proteins using immunoblotting. Protein carbonylation was evaluated by the 2,4-dinitrophenylhydrazine (DNPH) method. Results Photofrin-PDT leads to robust autophagy induction in two cancer cell lines, Hela and MCF-7. shRNA-mediated knockdown of ATG5 only partially blocks autophagic response and only marginally affects the sensitivity of Hela and MCF-7 cells to PDT. ATG5 knockout in HeLa cell line utilizing CRISPR/Cas9 genome editing results in increased PDT-mediated cytotoxicity, which is accompanied by an enhanced apoptotic response and increased accumulation of carbonylated proteins. Conclusions Altogether, these observations imply that autophagy contributes to Photofrin-PDT resistance by enabling clearance of carbonylated and other damaged proteins. Therefore, autophagy inhibition may serve as a strategy to improve PDT efficacy

Gene Expression Profile of Human Mesenchymal Stromal Cells Exposed to Hypoxic and Pseudohypoxic Preconditioning—An Analysis by RNA Sequencing

Mesenchymal stromal cell (MSC) therapy is making its way into clinical practice, accompanied by research into strategies improving their therapeutic potential. Preconditioning MSCs with hypoxia-inducible factors-α (HIFα) stabilizers is an alternative to hypoxic priming, but there remains insufficient data evaluating its transcriptomic effect. Herein, we determined the gene expression profile of 6 human bone marrow-derived MSCs preconditioned for 6 h in 2% O2 (hypoxia) or with 40 μM Vadadustat, compared to control cells and each other. RNA-Sequencing was performed using the Illumina platform, quality control with FastQC and adapter-trimming with BBDUK2. Transcripts were mapped to the Homo_sapiens. GRCh37 genome and converted to relative expression using Salmon. Differentially expressed genes (DEGs) were generated using DESeq2 while functional enrichment was performed in GSEA and g:Profiler. Comparison of hypoxia versus control resulted in 250 DEGs, Vadadustat versus control 1071, and Vadadustat versus hypoxia 1770. The terms enriched in both phenotypes referred mainly to metabolism, in Vadadustat additionally to vesicular transport, chromatin modifications and interaction with extracellular matrix. Compared with hypoxia, Vadadustat upregulated autophagic, phospholipid metabolism, and TLR cascade genes, downregulated those of cytoskeleton and GG-NER pathway and regulated 74 secretory factor genes. Our results provide valuable insight into the transcriptomic effects of these two methods of MSCs preconditioning