GFAP as Astrocyte-Derived Extracellular Vesicle Cargo in Acute Ischemic Stroke Patients-A Pilot Study.

The utility of serum glial fibrillary acidic protein (GFAP) in acute ischemic stroke (AIS) has been extensively studied in recent years. Here, we aimed to assess its potential role as a cargo protein of extracellular vesicles (EVs) secreted by astrocytes (ADEVs) in response to brain ischemia. Plasma samples from eighteen AIS patients at 24 h (D1), 7 days (D7), and one month (M1) post-symptoms onset, and nine age, sex, and cardiovascular risk factor-matched healthy controls were obtained to isolate EVs using the Exoquick ULTRA EV kit. Subsets of presumed ADEVs were identified further by the expression of the glutamate aspartate transporter (GLAST) as a specific marker of astrocytes with the Basic Exo-Flow Capture kit. Western blotting has tested the presence of GFAP in ADEV cargo. Post-stroke ADEV GFAP levels were elevated at D1 and D7 but not M1 compared to controls (p = 0.007, p = 0.019, and p = 0.344, respectively). Significant differences were highlighted in ADEV GFAP content at the three time points studied (n = 12, p = 0.027) and between D1 and M1 (z = 2.65, p = 0.023). A positive correlation was observed between the modified Rankin Scale (mRS) at D7 and ADEV GFAP at D1 (r = 0.58, p = 0.010) and D7 (r = 0.57, p = 0.013), respectively. ADEV GFAP may dynamically reflect changes during the first month post-ischemia. Profiling ADEVs from peripheral blood could provide a new way to assess the central nervous system pathology.

Astrocyte Involvement in Blood-Brain Barrier Function: A Critical Update Highlighting Novel, Complex, Neurovascular Interactions.

Neurological disorders have been linked to a defective blood-brain barrier (BBB), with dysfunctions triggered by stage-specific disease mechanisms, some of these being generated through interactions in the neurovascular unit (NVU). Advanced knowledge of molecular and signaling mechanisms in the NVU and the emergence of improved experimental models allow BBB permeability prediction and the development of new brain-targeted therapies. As NVU constituents, astrocytes are the most numerous glial cells, characterized by a heterogeneity that occurs as a result of developmental and context-based gene expression profiles and the differential expression of non-coding ribonucleic acids (RNAs). Due to their heterogeneity and dynamic responses to different signals, astrocytes may have a beneficial or detrimental role in the BBB's barrier function, with deep effects on the pathophysiology of (and on the progression of) central nervous system diseases. The implication of astrocytic-derived extracellular vesicles in pathological mechanisms, due to their ability to pass the BBB, must also be considered. The molecular mechanisms of astrocytes' interaction with endothelial cells at the BBB level are considered promising therapeutic targets in different neurological conditions. Nevertheless, a personalized and well-founded approach must be addressed, due to the temporal and spatial heterogeneity of reactive astrogliosis states during disease.

Primary MSCs for Personalized Medicine: Ethical Challenges, Isolation and Biocompatibility Evaluation of 3D Electrospun and Printed Scaffolds.

Autologous cell therapy uses patients' own cells to deliver precise and ideal treatment through a personalized medicine approach. Isolation of patients' cells from residual tissue extracted during surgery involves specific planning and lab steps. In the present manuscript, a path from isolation to in vitro research with human mesenchymal stem cells (MSCs) obtained from residual bone tissues is described as performed by a medical unit in collaboration with a research center. Ethical issues have been addressed by formulating appropriate harvesting protocols according to European regulations. Samples were collected from 19 patients; 10 of them were viable and after processing resulted in MSCs. MSCs were further differentiated in osteoblasts to investigate the biocompatibility of several 3D scaffolds produced by electrospinning and 3D printing technologies; traditional orthopedic titanium and nanostructured titanium substrates were also tested. 3D printed scaffolds proved superior compared to other substrates, enabling significantly improved response in osteoblast cells, indicating that their biomimetic structure and properties make them suitable for synthetic tissue engineering. The present research is a proof of concept that describes the process of primary stem cells isolation for in vitro research and opens avenues for the development of personalized cell platforms in the case of patients with orthopedic trauma. The demonstration model has promising perspectives in personalized medicine practices.

Effects of Laser Application on Alveolar Bone Mesenchymal Stem Cells and Osteoblasts: An In Vitro Study.

Mesenchymal stem cells isolated from the bone marrow have a great differentiation potential, being able to produce many cell lines, including osteoblasts. Osteoblasts have an important role in bone remodeling by actively participating in the maturation and mineralization of the extracellular matrix. The aim of this study was to determine the effect of laser application on the viability and proliferation of osteoblasts. METHODS: Alveolar bone was harvested from 8 patients and placed into a culture medium to induce proliferation of mesenchymal stem cells. These were differentiated into osteoblasts in special conditions. The cells from each patient were split into two groups, one was treated using a 980 nm laser (1W output power, pulsed mode, 20 s, 50 mm distance) (laser "+") and the other one did not receive laser stimulation (laser "-"). RESULTS: Using the confocal microscope, we determined that the cells from the laser "+" group were more active when compared to the laser "-" group. The number of cells in the laser "+" group was significantly greater compared to the laser "-" group as the ImageJ-NIH software showed (p = 0.0072). CONCLUSIONS: Laser application increases the proliferation rate of osteoblasts and intensifies their cellular activity.

Treatment With Cladribine Selects IFNγ+IL17+ T Cells in RRMS Patients - An In Vitro Study.

Background: Multiple sclerosis (MS) is an incurable autoimmune disease mediated by a heterogeneous T cell population (CD3+CD161+CXCR3-CCR6+IFNγ-IL17+, CD3+CXCR3+CCR6+IFNγ+IL17+, and CD3+CXCR3+IFNγ+IL17- phenotypes) that infiltrates the central nervous system, eliciting local inflammation, demyelination and neurodegeneration. Cladribine is a lymphocyte-depleting deoxyadenosine analogue recently introduced for MS therapy as a Disease Modifying Drug (DMD). Our aim was to establish a method for the early identification and prediction of cladribine responsiveness among MS patients. Methods: An experimental model was designed to study the cytotoxic and immunomodulatory effect of cladribine. T cell subsets of naïve relapsing-remitting MS (RRMS) patients were analyzed ex vivo and in vitro comparatively to healthy controls (HC). Surviving cells were stimulated with rh-interleukin-2 for up to 14days. Cell proliferation and immunophenotype changes were analyzed after maximal (phorbol myristate acetate/ionomycin/monensin) and physiological T-cell receptor (CD3/CD28) activation, using multiparametric flow cytometry and xMAP technology. Results: Ex vivo CD161+Th17 cells were increased in RRMS patients. Ex vivo to in vitro phenotype shifts included: decreased CD3+CCR6+ and CD3+CD161+ in all subjects and increased CD3+CXCR3+ in RRMS patients only; Th17.1 showed increased proliferation vs Th17 in all subjects; CD3+IL17+ and CD3+IFNγ+IL17+ continued to proliferate till day 14, CD3+IFNγ+ only till day 7. Regarding cladribine exposure: RRMS CD3+ cells were more resistant compared to HC; treated CD3+ cells proliferated continuously for up to 14 days, while untreated cells only up to 7 days; both HC/RRMS CD3+CXCR3+ populations increased from baseline till day 14; in RRMS patients vs HC, IL17 secretion from cladribine-treated cells increased significantly, in line with the observed proliferation of CD3+IL17+ and CD3+IFNγ+IL17+ cells; in both HC/RRMS, cladribine led to a significant increase in CD3+IFNγ+ cells at day 7 only, having no further effect at day14. IFNγ and IL17 secreted in culture media decreased significantly from ex vivo to in vitro. Conclusions: CD3+ subtypes showed different responsiveness due to selectivity of cladribine action, in most patients leading to in vitro survival/proliferation of lymphocyte subsets known as pathogenic in MS. This in vitro experimental model is a promising tool for the prediction of individual responsiveness of MS patients to cladribine and other DMDs.