Articulation recovery in ALS patients after lineage-negative adjuvant cell therapy - preliminary report.

Background: Amyotrophic lateral sclerosis (ALS) is one of the most frequently occurring neurodegenerative diseases affecting speech and swallowing. This preliminary study aimed to investigate whether an autologous lineage-negative stem/progenitor cell therapy applied to ALS patients affects the level of selected trophic and proinflammatory factors, and subsequently improves the articulation. Methods: We enrolled 12 patients with sporadic ALS, who underwent autologous bone marrow-derived lineage negative (LIN-) cells administration into cerebrospinal fluid (CSF). We evaluated patients' articulation using the Frenchay Dysarthria Assessment on days 0 and 28 following the LIN- cells administration. Concentrations of various factors (BDNF, NGF, ANGP-2, VEGF, PDGF-AA, PEDF, COMP-FH, CRP, C3, C4) in CSF were quantified by multiplex fluorescent bead-based immunoassays in the samples collected on the day of LIN- cells administration and 28 days later. On top of this, we assessed levels of BDNF and NGF in the patients' plasma on the day of the injection, three, seven days and three months after the treatment. Results: Of the 12 patients who received the LIN- cell therapy 8 showed short-termed improvement in articulatory functions (group I), which was particularly noticeable in better phonation time, lips and soft palate performance, swallowing reflex and voice loudness. Four patients (group II) did not show substantial improvement. CSF concentrations of BDNF, ANGP-2 and PDGF-AA in group I decreased significantly 28 days after LIN- cells administration. The highest concentration levels of BDNF in group II and NGF in both groups in blood plasma were observed on day 3 following the injection. Conclusions: The outcomes of the LIN- cell application in ALS treatment of articulatory organs are promising. The procedure proved to be safe and feasible. A short-lasting trophic effect of autologous LIN- administration could encourage repeated cell's application in order to sustain their beneficial effects, however this approach needs further investigation.

The Interplay Between Systemic Inflammatory Factors and MicroRNAs in Age-Related Macular Degeneration.

We aimed to explore the expression of systemic inflammatory factors and selected intracellular miRNAs that regulate inflammatory signaling pathways potentially involved in age-related macular degeneration (AMD) pathogenesis. A total of 179 patients with wet AMD, 175 with dry AMD and 121 controls were enrolled in the study. Soluble inflammatory factors were analyzed in plasma samples using Luminex technology. Expression of selected miRNAs was analyzed in isolated nucleated peripheral blood cells (PBNCs) using real-time qPCR. Wet AMD was an independent factor associated with higher concentrations of IL-6 (ß = +0.24, p = 0.0004), GM-CSF (ß = +0.31, p < 0.001), IFN-γ (ß = +0.58, p < 0.001), higher expression of miRNA-23a-3p (ß = +0.60, p < 0.0001), miRNA-30b (ß = +0.32, p < 0.0001), miRNA-191-5p (ß = +0.28, p < 0.0001) and lower concentration of IL-1ß (ß = -0.25, p = 0.0003), IL-5 (ß = -0.45, p < 0.001), IL-10 (ß = -0.45, p < 0.001), IL-12 (ß = -0.35, p < 0.001), lower expression of miRNA-16-5p (ß = -0.31, p < 0.0001), miRNA-17-3p (ß = -0.18, p = 0.01), miRNA-150-5p (ß = -0.18, p = 0.01) and miRNA-155-5p (ß = -0.47, p < 0.0001). Multivariate analysis revealed that dry AMD was an independent factor associated with higher concentration of GM-CSF (ß = +0.34, p < 0.001), IL-6 (ß = +0.13, p = 0.05), higher expression of miRNA-23a-3p (ß = +0.60, p < 0.0001), miRNA-126-3p (ß = +0.23, p = 0.0005), miRNA-126-5p (ß = +0.16, p = 0.01), miRNA 146a (ß = +0.14, p = 0.03), and mRNA191-5p (ß = +0.15, p = 0.03) and lower concentrations of TNF-α (ß = +0.24, p = 0.0004), IL-1ß (ß = -0.39, p < 0.001), IL-2 (ß = -0.20, p = 0.003), IL-5 (ß = -0.54, p < 0.001), IL-10 (ß = -0.56, p < 0.001), IL-12 (ß = -0.51, p < 0.001), lower expression of miRNA-16-5p (ß = -0.23, p = 0.0004), miRNA-17-3p (ß = -0.20, p = 0.003) and miRNA-17-5p (ß = -0.19, p = 0.004). Negative correlations between visual acuity and WBC, lymphocyte count, TNF-α, IL-1 ß, IL-2, IL-4, IL-6, IL-10 concentrations and miRNA-191-5p, as well as positive correlations between visual acuity and miRNA-126-3p, -126-5p, and -155-5p PBNCs expression were found in AMD patients. No such correlations were found in the control group. Our results may suggest the role of both intra- and extracellular mechanisms implicated in inflammatory response regulation in multifactorial AMD pathogenesis.

Electrostatic complex of neurotrophin 4 with dendrimer nanoparticles: controlled release of protein in vitro and in vivo.

Background: NT4 has been regarded as a promising therapeutic protein for treatment of damaged retinal pigment epithelium cells. Purpose: Here, we studied physicochemical parameters of an NT4-polyamidoamine (PAMAM) electrostatic complex, which can provide a sustained concentration of protein in intraocular space over an extended period after delivery. Adsorption/desorption of NT4 molecules to/from positively charged PAMAM dendrimers were precisely determined to control the concentration of bounded/unbounded protein molecules, diffusion coefficient, and size of a protein-laden dendrimer structure. We determined kinetics of NT4 desorption in PBS, vitreous, and damaged retina. Methods: Initially, adsorption of NT4 molecules on PAMAM dendrimers was studied in PBS using dynamic light scattering, electrophoresis, solution depletion, ELISA, and atomic force microscopy. This allowed us precisely to determine desorption of NT4 from nanoparticles under in situ conditions. The maximum coverage of irreversibly adsorbed NT4 determined by ELISA allowed us to devise a robust procedure for preparing stable and well-controlled coverage of NT4 on PAMAM nanoparticles. Thereafter, we studied diffusion of nanospheres containing NT4 molecules by injecting them into vitreous cavities of mice exposed to intravenous injections of sodium iodate and evaluated their intraocular desorption kinetics from drug carriers in vivo. Results: Our measurements revealed NT4-dendrimer nanoparticles can be used for continuous neurotrophic factor delivery, enhancing its distribution into mouse vitreous, as well as damaged retina over 28 days of postinjury observation. Conclusion: Understanding of polyvalent neurotrophin interactions with dendrimer nanoparticles might be useful to obtain well-ordered protein layers, targeting future development of drug-delivery systems, especially for neuroprotection of damaged retinal neurons.

Extracellular vesicles in hematological malignancies.

Extracellular vesicles (EVs) act as transporters that carry regulatory molecules between cells in physiologic and pathologic states; therefore, they play a crucial role in thrombosis, inflammation, angiogenesis, vascular dysfunction and other processes that affect the course of hematologic diseases. Within the tumor microenvironment, for example the leukemic bone marrow, EVs-mediated signaling may direct the activities of surrounding cells and act as a positive feedback loop that contributes to cancer progression. The importance of EVs in hematological malignancies is also attributed to their involvement in development of multidrug resistance and the hypercoagulable state related to hematologic disorders, which may be partially influenced by an increase in the total number of EVs. In this review, we focused on the role of EVs in hematologic malignancies and in particular on their influence on the BM microenvironment, their role in angiogenesis and the possible use of EVs as biomarkers of disease progression and drug resistance.

Expression of selected angiogenesis-related small microRNAs in patients with abnormally increased secretion of glucocorticoids.

INTRODUCTION: Higher cortisol levels are associated with cardiovascular morbidity and mortality in the elderly, partially resulting from biologic effects of glucocorticoids (GCs) on endothelial cells observed in an experimental setting. These features are replicated in patients with endogenous GC excess (Cushing's syndrome) or with exogenous hypercortisolism due to excessive pharmacological application of GCs. Both groups present also an increased cardiovascular disease event rate. GCs may also adversely influence recovery after myocardial infarction. Recently it was proposed that microRNAs (miRNAs) - small noncoding RNAs functioning as antisense regulators of gene expression by targeting mRNA - may have a central role in regulating endothelial function through multiple mechanisms. Thus, the purpose of this study was to evaluate the effects of chronic GC excess on the expression of selected endothelium-controlling miRNAs expressed in nucleated cells circulating in peripheral blood (PBNCs) of patients with endogenous hypercortisolism either due to corticotrophin-independent or corticotrophin-dependent Cushing's syndrome (CS). MATERIAL AND METHODS: Peripheral blood nuclear cells were collected from 35 healthy subjects and 31 patients with endogenous hypercortisolism as a source of miRNAs. A self-validated individual quantitative RT-PCR study was then performed to evaluate the expression levels of selected miRNAs in PBNCs. Additionally, endothelin-1 (ET-1) expression in peripheral blood was assessed with respect to endothelial dysfunction using Western blotting. RESULTS: The ET-1 expression levels in CS were higher than in controls, confirming endothelial dysfunction in the CS group. Furthermore, miRNA analysis revealed a significantly decreased intracellular expression of selected endothelium-related miRNAs in patients with endogenous hypercortisolism, including miRNA-17-5p, miRNA-126-3p, and miRNA-126-5p, compared to controls. In contrast, two other angiogenic miRNAs, miRNA-150-5p and miRNA-223-3p, were significantly upregulated compared to controls. CONCLUSIONS: Cardiovascular events related to hypercortisolism remain a challenging problem in medical practice. This study has demonstrated that the chronic excess of GCs in endogenous CS might induce significant dysregulation of selected miRNAs involved in the control of endothelium biology. However, the lack of knowledge about specific miRNA expression postpones the full understanding of the biological roles of such miRNAs in hypercortisolism. Moreover, dysregulated miRNAs seem to be promising targets for further research, especially to search for potential therapies for several GC-induced cardiovascular complications.

Safety and Feasibility of Lin- Cells Administration to ALS Patients: A Novel View on Humoral Factors and miRNA Profiles.

Therapeutic options for amyotrophic lateral sclerosis (ALS) are still limited. Great hopes, however, are placed in growth factors that show neuroprotective abilities (e.g., nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and vascular endothelial growth factor (VEGF)) and in the immune modulating features, in particular, the anti-inflammatory effects. In our study we aimed to investigate whether a bone marrow-derived lineage-negative (Lin-) cells population, after autologous application into cerebrospinal fluid (CSF), is able to produce noticeable concentrations of trophic factors and inflammatory-related proteins and thus influence the clinical course of ALS. To our knowledge, the evaluation of Lin- cells transplantation for ALS treatment has not been previously reported. Early hematopoietic Lin- cells were isolated from twelve ALS patients’ bone marrow, and later, the suspension of cells was administered into the subarachnoid space by lumbar puncture. Concentrations of selected proteins in the CSF and plasma were quantified by multiplex fluorescent bead-based immunoassays at different timepoints post-transplantation. We also chose microRNAs (miRNAs) related to muscle biology (miRNA-1, miRNA-133a, and miRNA-206) and angiogenesis and inflammation (miRNA-155 and miRNA-378) and tested, for the first time, their expression profiles in the CSF and plasma of ALS patients after Lin- cells transplantation. The injection of bone marrow cells resulted in decreased concentration of selected inflammatory proteins (C3) after Lin- cells injection, particularly in patients who had a better clinical outcome. Moreover, several analyzed miRNAs have changed expression levels in the CSF and plasma of ALS patients subsequent to Lin- cells administration. Interestingly, the expression of miR-206 increased in ALS patients, while miR-378 decreased both in the CSF and plasma one month after the cells’ injection. We propose that autologous lineage-negative early hematopoietic cells injected intrathecally may be a safe and feasible source of material for transplantations to the central nervous system (CNS) environment aimed at anti-inflammatory support provision for ALS adjuvant treatment strategies. Further research is needed to evaluate whether the observed effects could significantly influence the ALS progression.

miRNAs in chronic myeloid leukemia: small molecules, essential function.

Chronic myeloid leukemia (CML) is a myeloproliferative disorder associated with clonal expansion of cancerous bone marrow stem cells. Tyrosine kinase inhibitors (TKIs) targeting Bcr-Abl oncoprotein are the first-line therapy for most CML patients, however, some are unresponsive to it or develop resistance. Recently, microRNAs (miRNAs) have been implicated in the progression of CML and the development of TKI resistance based on their important regulatory function in cell homeostasis. MicroRNAs are small noncoding RNAs that post-transcriptionally regulate gene expression. Since microRNAs can function either as oncogenes or tumor suppressor genes in leukemogenesis, the potential of using them as therapeutic targets by inhibiting or amplifying their activity, opens up new opportunities for leukemia therapy. In this review, we focus on recent studies on the important roles of microRNAs in the pathogenesis of CML and their relevance as biomarkers for diagnosis, monitoring disease progression, and treatment response.