Tumor-neutrophil crosstalk promotes in vitro and in vivo glioblastoma progression.

Introduction: The tumor microenvironment (TME) of glioblastoma (GB) is characterized by an increased infiltration of immunosuppressive cells that attenuate the antitumor immune response. The participation of neutrophils in tumor progression is still controversial and a dual role in the TME has been proposed. In this study, we show that neutrophils are reprogrammed by the tumor to ultimately promote GB progression. Methods: Using in vitro and in vivo assays, we demonstrate the existence of bidirectional GB and neutrophil communication, directly promoting an immunosuppressive TME. Results and discussion: Neutrophils have shown to play an important role in tumor malignancy especially in advanced 3D tumor model and Balb/c nude mice experiments, implying a time- and neutrophil concentration-dependent modulation. Studying the tumor energetic metabolism indicated a mitochondria mismatch shaping the TME secretome. The given data suggests a cytokine milieu in patients with GB that favors the recruitment of neutrophils, sustaining an anti-inflammatory profile which is associated with poor prognosis. Besides, glioma-neutrophil crosstalk has sustained a tumor prolonged activation via NETs formation, indicating the role of NFκB signaling in tumor progression. Moreover, clinical samples have indicated that neutrophil-lymphocyte ratio (NLR), IL-1ß, and IL-10 are associated with poor outcomes in patients with GB. Conclusion: These results are relevant for understanding how tumor progression occurs and how immune cells can help in this process.

The Impact of Mesenchymal Stromal Cells on Neutrophils: A Concise Review.

This review describes the current state of knowledge concerning interactions between mesenchymal stromal cells (MSCs) and neutrophils. MSCs are known as somatic multipotent cells with regenerative and anti-inflammatory abilities and immunomodulatory effects over other immune cells. Several studies reported that MSCs could affect the function and viability of neutrophils in their recruitment, activation, activity, survival, production of reactive oxygen species, phagocytosis capacity, and apoptosis. Moreover, neutrophils could be involved in the pro-metastatic effects of MSCs. Inversally, only a few studies pointed to the possibility of the opposite effect of neutrophils on MSCs. Understanding the interactions between MSCs and neutrophils could help promote therapeutic strategies using stromal cell-based therapeutic approaches, especially for hyper-immune pathologies, immunodeficiencies, and infectious diseases. However, further in vitro and in vivo studies are essential to determine the complete mechanisms of MSCs and neutrophils interaction.

Cholinergic immunomodulation in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders characterized by dysregulated immune responses to resident microbiota in genetically susceptible hosts. The activation of the cholinergic system has been proposed for the treatment of IBD patients according to its potential anti-inflammatory effect in vivo. The α-7-nicotinic-acetylcholine receptor (α7nAChR) is involved in the inhibition of inflammatory processes, modulating the production of cytokines, suppressing dendritic cells and macrophage activity, leading to the suppression of T cells. In this review, we address the most recent studies and clinical trials concerning cholinergic signaling and its therapeutic potential for inflammatory bowel diseases.

Mesenchymal Stem Cells From Bone Marrow, Adipose Tissue, and Lung Tissue Differentially Mitigate Lung and Distal Organ Damage in Experimental Acute Respiratory Distress Syndrome.

OBJECTIVES: Mesenchymal stem cells-based therapies have shown promising effects in experimental acute respiratory distress syndrome. Different mesenchymal stem cells sources may result in diverse effects in respiratory diseases; however, there is no information regarding the best source of mesenchymal stem cells to treat pulmonary acute respiratory distress syndrome. We tested the hypothesis that mesenchymal stem cells derived from bone marrow, adipose tissue, and lung tissue would lead to different beneficial effects on lung and distal organ damage in experimental pulmonary acute respiratory distress syndrome. DESIGN: Animal study and primary cell culture. SETTING: Laboratory investigation. SUBJECTS: Seventy-five Wistar rats. INTERVENTIONS: Wistar rats received saline (control) or Escherichia coli lipopolysaccharide (acute respiratory distress syndrome) intratracheally. On day 2, acute respiratory distress syndrome animals were further randomized to receive saline or bone marrow, adipose tissue, or lung tissue mesenchymal stem cells (1 × 10 cells) IV. Lung mechanics, histology, and protein levels of inflammatory mediators and growth factors were analyzed 5 days after mesenchymal stem cells administration. RAW 264.7 cells (a macrophage cell line) were incubated with lipopolysaccharide followed by coculture or not with bone marrow, adipose tissue, and lung tissue mesenchymal stem cells (10 cells/mL medium). MEASUREMENTS AND MAIN RESULTS: Regardless of mesenchymal stem cells source, cells administration improved lung function and reduced alveolar collapse, tissue cellularity, collagen, and elastic fiber content in lung tissue, as well as decreased apoptotic cell counts in liver. Bone marrow and adipose tissue mesenchymal stem cells administration also reduced levels of tumor necrosis factor-α, interleukin-1ß, keratinocyte-derived chemokine, transforming growth factor-ß, and vascular endothelial growth factor, as well as apoptotic cell counts in lung and kidney, while increasing expression of keratinocyte growth factor in lung tissue. Additionally, mesenchymal stem cells differently modulated the secretion of biomarkers by macrophages depending on their source. CONCLUSIONS: Mesenchymal stem cells from different sources led to variable responses in lungs and distal organs. Bone marrow and adipose tissue mesenchymal stem cells yielded greater beneficial effects than lung tissue mesenchymal stem cells. These findings may be regarded as promising in clinical trials.

Membrane particles generated from mesenchymal stromal cells modulate immune responses by selective targeting of pro-inflammatory monocytes.

Mesenchymal stromal cells (MSC) are a promising therapy for immunological disorders. However, culture expanded MSC are large and get trapped in the capillary networks of the lungs after intravenous infusion, where they have a short survival time. Hypothetically, living cells are a risk for tumor formation. To reduce risks associated with MSC infusion and improve the distribution in the body, we generated membrane particles (MP) of MSC and MSC stimulated with IFN-γ (MPγ). Tracking analysis and electron microscopy indicated that the average size of MP was 120 nm, and they showed a round shape. MP exhibited ATPase, nucleotidase and esterase activity, indicating they are enzymatically active. MP and MPγ did not physically interact with T cells and had no effect on CD4+ and CD8+ T cells proliferation. However, MP and MPγ selectively bound to monocytes and decreased the frequency of pro-inflammatory CD14+CD16+ monocytes by induction of selective apoptosis. MP and MPγ increased the percentage of CD90 positive monocytes, and MPγ but not MP increased the percentage of anti-inflammatory PD-L1 monocytes. MPγ increased mRNA expression of PD-L1 in monocytes. These data demonstrate that MP have immunomodulatory properties and have potential as a novel cell-free therapy for treatment of immunological disorders.

Assisted reproduction: what factors interfere in the professional's decisions? Are single women an issue?

BACKGROUND: With the development of medical technology, many countries around the world have been implementing ethical guidelines and laws regarding Medically Assisted Reproduction (MAR). A physician's reproductive decisions are not solely based on technical criteria but are also influenced by society values. Therefore, the aim of this study was to analyze the factors prioritized by MAR professionals when deciding on whether to accept to perform assisted reproduction and to show any existing cultural differences. METHODS: Cross-sectional study involving 224 healthcare professionals working with assisted reproduction in Brazil, Italy, Germany and Greece. Instrument used for data collection: a questionnaire, followed by the description of four special MAR cases (a single woman, a lesbian couple, an HIV discordant couple and gender selection) which included case-specific questions regarding the professionals' decision on whether to perform the requested procedure as well as the following factors: socio-demographic variables, moral and legal values as well as the technical aspects which influence decision-making. RESULTS: Only the case involving a single woman who wishes to have a child (without the intention of having a partner in the future) demonstrated significant differences. Therefore, the study was driven towards the results of this case specifically. The analyses we performed demonstrated that professionals holding a Master's Degree, those younger in age, female professionals, those having worked for less time in reproduction, those in private clinics and Brazilian health professionals all had a greater tendency to perform the procedure in that case. A multivariate analysis demonstrated that the reasons for the professional's decision to perform the procedure were the woman's right to gestate and the duty of MAR professionals to help her. The professionals who decided not to perform the procedure identified the woman's marital status and the child's right to a father as the reason to withhold treatment. CONCLUSION: The study indicates differences among countries in the evaluation of the single woman case. It also discloses the undervaluation of bioethics committees and the need for a greater participation of healthcare professionals in debates on assisted reproduction laws.

Age-dependent availability and functionality of bone marrow stem cells in an experimental model of acute and chronic myocardial infarction.

The aim of this study was to investigate the effect of aging and timing of left ventricular ischemic injury on the availability and functionality of stem cells. We studied young and aged male inbred Lewis rats that were used as donors of bone marrow mononuclear cells (BM-MNCs), divided in four experimental groups: controls, sham operated, 48 h post-myocardial infarction (MI), and 28 days post-MI. In vitro studies included flow cytometry analysis, hematopoietic colony-forming capacity, and invasion assays of migration capacity. BM-MNCs from these groups were transplanted in female rats after MI induction. Late engraftment was evaluated by real-time PCR of the SRY chromosome. Percentage of CD34+/CD45+(low) cells was similar among different experimental groups in young rats, but was significantly higher in aged animals (p < 0.001), particularly 28 days post-MI. KDR+/CD34+ cells were increased 48 h after MI and decreased 28 days post-MI in young animals, while they were profoundly reduced in the aged group (p < 0.001). Triple staining for CD44+/CD29+/CD71+ cells was similar in different groups of aged rats, but we observed an intense increase 48 h post-MI in young animals. Colony-forming units and cytokine-induced migration were significantly attenuated 28 days after the MI. Late engraftment in infarcted transplanted female hearts was present, but considerably heterogeneous. Finally, recovery of left ventricular systolic function in transplanted female recipients was significantly influenced by donors' BM-MNCs groups (p < 0.01). We have demonstrated that aging and timing of myocardial injury are factors that may act synergistically in determining stem cell availability and function. Such interaction should be considered when planning new cell therapy strategies for acute and chronic ischemic heart disease in the clinical arena.

Betacellulin overexpression in mesenchymal stem cells induces insulin secretion in vitro and ameliorates streptozotocin-induced hyperglycemia in rats.

Betacellulin (BTC), a ligand of the epidermal growth factor receptor, has been shown to promote growth and differentiation of pancreatic ß-cells and to improve glucose metabolism in experimental diabetic rodent models. Mesenchymal stem cells (MSCs) have been already proved to be multipotent. Recent work has attributed to rat and human MSCs the potential to differentiate into insulin-secreting cells. Our goal was to transfect rat MSCs with a plasmid containing BTC cDNA to guide MSC differentiation into insulin-producing cells. Prior to induction of cell MSC transfection, MSCs were characterized by flow cytometry and the ability to in vitro differentiate into mesoderm cell types was evaluated. After rat MSC characterization, these cells were electroporated with a plasmid containing BTC cDNA. Transfected cells were cultivated in Dulbecco's modified Eagle medium high glucose (H-DMEM) with 10 mM nicotinamide. Then, the capability of MSC-BTC to produce insulin in vitro and in vivo was evaluated. It was possible to demonstrate by radioimmunoassay analysis that 10(4) MSC-BTC cells produced up to 0.4 ng/mL of insulin, whereas MSCs transfected with the empty vector (negative control) produced no detectable insulin levels. Moreover, MSC-BTC were positive for insulin in immunohistochemistry assay. In parallel, the expression of pancreatic marker genes was demonstrated by molecular analysis of MSC-BTC. Further, when MSC-BTC were transplanted to streptozotocin diabetic rats, BTC-transfected cells ameliorated hyperglycemia from over 500 to about 200 mg/dL at 35 days post-cell transplantation. In this way, our results clearly demonstrate that BTC overabundance enhances glucose-induced insulin secretion in MSCs in vitro as well as in vivo.

Germinative testicular cells and bone marrow mononuclear cells transplanted to a rat model of testicular degeneration.

The aim of this study was to evaluate the ability of rat mononuclear bone marrow cells to recover testis cell associations and multiplication in busulfan-treated rats, and to compare these data to germinative testicular cell transplant. The germinative testicular cells were obtained by the trypsin digestion method, and bone marrow cells were harvested from femurs and tibias, and purified using by Ficoll gradient. Cell transplantation was performed by the injection of cells through the efferent ducts into the rete testis in busulfan-treated animals. Fifteen days after transplantation, the recipient rats were sacrificed and the testes were collected and analyzed by histology (hematoxilin-eosin and DAPI staining). Results demonstrated that germ cells transplantation promoted cellular reorganization of seminiferous epithelium 15 days later. On the other hand, no improvement in spermatogenesis regeneration was found after heterologous mononuclear bone marrow cell transplantation.