ATPergic signaling disruption in human sepsis as a potential source of biomarkers for clinical use.

Sepsis is a life-threatening organ dysfunction caused by a dysregulated inflammatory response to infection. To date, there is no specific treatment established for sepsis. In the extracellular compartment, purines such as adenosine triphosphate (ATP) and adenosine play essential roles in the immune/inflammatory responses during sepsis and septic shock. The balance of extracellular levels among ATP and adenosine is intimately involved in the signals related to immune stimulation/immunosuppression balance. Specialized enzymes, including CD39, CD73, and adenosine deaminase (ADA), are responsible to metabolize ATP to adenosine which will further sensitize the P2 and P1 purinoceptors, respectively. Disruption of the purinergic pathway had been described in the sepsis pathophysiology. Although purinergic signaling has been suggested as a potential target for sepsis treatment, the majority of data available were obtained using pre-clinical approaches. We hypothesized that, as a reflection of deregulation on purinergic signaling, septic patients exhibit differential measurements of serum, neutrophils and monocytes purinergic pathway markers when compared to two types of controls (healthy and ward). It was observed that ATP and ADP serum levels were increased in septic patients, as well as the A2a mRNA expression in neutrophils and monocytes. Both ATPase/ADPase activities were increased during sepsis. Serum ATP and ADP levels, and both ATPase and ADPase activities were associated with the diagnosis of sepsis, representing potential biomarkers candidates. In conclusion, our results advance the translation of purinergic signaling from pre-clinical models into the clinical setting opening opportunities for so much needed new strategies for sepsis and septic shock diagnostics and treatment.

TLR4 expression and functionality are downregulated in glioblastoma cells and in tumor-associated macrophages: A new mechanism of immune evasion?

Glioblastoma (GB) is the most common and aggressive form of primary brain tumor, in which the presence of an inflammatory environment, composed mainly by tumor-associated macrophages (TAMs), is related to its progression and development of chemoresistance. Toll-Like Receptors (TLRs) are key components of the innate immune system and their expression in both tumor and immune-associated cells may impact the cell communication in the tumor microenvironment (TME), further modeling cancer growth and response to therapy. Here, we investigated the participation of TLR4-mediated signaling as a mechanism of induced-immune escape in GB. Initially, bioinformatics analysis of public datasets revealed that TLR4 expression is lower in GB tumors when compared to astrocytomas (AST), and in a subset of TAMs. Further, we confirmed that TLR4 expression is downregulated in chemoresistant GB, as well as in macrophages co-cultured with GB cells. Additionally, TLR4 function is impaired in those cells even following stimulation with LPS, an agonist of TLR4. Finally, experiments performed in a cohort of clinical primary and metastatic brain tumors indicated that the immunostaining of TLR4 and CD45 are inversely proportional, and confirmed the low TLR4 expression in GBs. Interestingly, the cytoplasmic/nuclear pattern of TLR4 staining in cancer tissues suggests additional roles of this receptor in carcinogenesis. Overall, our data suggest the downregulation of TLR4 expression and activity as a strategy for GB-associated immune escape. Additional studies are necessary to better understand TLR4 signaling in TME in order to improve the benefits of immunotherapy based on TLR signaling.

Immortalization of Mesenchymal Stromal Cells by TERT Affects Adenosine Metabolism and Impairs their Immunosuppressive Capacity.

Mesenchymal stromal cells (MSCs) are promising candidates for cell-based therapies, mainly due to their unique biological properties such as multipotency, self-renewal and trophic/immunomodulatory effects. However, clinical use has proven complex due to limitations such as high variability of MSCs preparations and high number of cells required for therapies. These challenges could be circumvented with cell immortalization through genetic manipulation, and although many studies show that such approaches are safe, little is known about changes in other biological properties and functions of MSCs. In this study, we evaluated the impact of MSCs immortalization with the TERT gene on the purinergic system, which has emerged as a key modulator in a wide variety of pathophysiological conditions. After cell immortalization, MSCs-TERT displayed similar immunophenotypic profile and differentiation potential to primary MSCs. However, analysis of gene and protein expression exposed important alterations in the purinergic signaling of in vitro cultured MSCs-TERT. Immortalized cells upregulated the CD39/NTPDase1 enzyme and downregulated CD73/NT5E and adenosine deaminase (ADA), which had a direct impact on their nucleotide/nucleoside metabolism profile. Despite these alterations, adenosine did not accumulate in the extracellular space, due to increased uptake. MSCs-TERT cells presented an impaired in vitro immunosuppressive potential, as observed in an assay of co-culture with lymphocytes. Therefore, our data suggest that MSCs-TERT have altered expression of key enzymes of the extracellular nucleotides/nucleoside control, which altered key characteristics of these cells and can potentially change their therapeutic effects in tissue engineering in regenerative medicine.

Identification of valid endogenous control genes for determining gene expression in C6 glioma cell line treated with conditioned medium from adipose-derived stem cell.

INTRODUCTION: There is growing evidence that mesenchymal stem cells (MSCs) can be important players in the tumor microenvironment. They can affect the glioma progression through the modulation of different genes. This modulation can be evaluated through a very useful model, treating the tumor cells with MSC-conditioned medium. However, for an accurate and reliable gene expression analysis, normalization of gene expression data against reference genes is a prerequisite. METHODS: We performed a systematic review in an attempt to find a reference gene to use when analyzing gene expression in C6 glioma cells lines. Considering that we were not able to find a reference gene originated by an appropriate validation, in this study we evaluated candidate genes to be used as reference gene in C6 cells under different treatments with adipose-derived stem cells conditioned medium (CM-ADSCs). ß-actin (ACTB); glyceraldehyde-3-phosphate dehydrogenase (GAPDH); hypoxanthine-guanine phosphoribosyltransferase I (HPRT-1); TATA box binding protein (TBP) and beta-2-microglobulin (B2M) were evaluated by real-time reverse transcription PCR (RT-qPCR). The mean Cq, the maximum fold change (MFC) and NormFinder software were used for reference gene evaluation and selection. RESULTS: The GAPDH and ACTB genes have been the most widely used reference genes to normalize among the different investigated genes in our review, however, controversially these genes underwent a substantial variability among the genes evaluated in the present work. Individually, TBP gene was more stable when compared with other genes analyzed and the combination of TBP and HPRT-1 was even more stable. CONCLUSION: These results evidence the importance of appropriate validation of reference genes before performing qPCR experiments. Besides, our data will contribute with researchers that work analyzing the role of ADSCs in glioma microenvironment through gene expression.