Investigation of Bone Marrow Hematopoietic Stem Cell Migration during Inflammation in BALB/c Mice

The aim of dissertation work to investigate the anti-inflammatory effect of murine bone marrow hematopoietic stem cells and their migration in the BALB/c mouse contact hypersensitivity model in vivo. It was found that isolated hematopoietic cell populations had a significant anti-inflammatory effect and inhibited the edema. The studies showed that the most efficient (up to 66%) inhibition of foot edema was obtained when using the HSC population. For the first time the HSC population migration kinetics in the BALB/c mouse contact hypersensitivity model in vivo was investigated. We determined that cells of this population can be found in mice paw edema and liver after just one hour. A little bit later they are detected in the spleen. We did the HSC population quantitative migration kinetic studies and found that in case of foot inflammation there is a secondary migration of the transplanted HSC migrating from the bone marrow to the spleen hematopoietic niche. We have shown that these cells selectively migrate into the inflammation areas of the foot edema. Transplanted cells quantity in the samples of foot edema, as compared with the untreated foot, was more than 1000 times higher. A transplanted hematopoietic stem cell migration research during inflammation, carried out in this work, contributes to clarification of stem cell migration patterns in case of pathological processes. This is particularly important in ensuring a safe and effective stem cell application in practice

Skin extracellular matrix components accelerate the regenerative potential of Lin-cells

Due to their unique properties, bone marrow-derived Lin− cells can be used to regenerate damaged tissues, including skin. The objective of our study was to determine the influence of the skin tissue-specific microenvironment on mouse Lin− cell proliferation and migration in vitro. Cells were analyzed for the expression of stem/progenitor surface markers by flow cytometry. Proliferation of MACS-purified cells in 3D cultures was investigated by WST-8 assay. Lin− cell migration was evaluated by in vitro scratch assay. The results obtained show that basement membrane matrix is more effective for Lin− cell proliferation in vitro. However, type I collagen matrix better enhances the re-epithelization process, that depends on the cell migratory properties. These studies are important for preparing cells to be used in transplantation

Quantitative microlaryngoscopic measurements of vocal fold polyps, glottal gap and their relation to vocal function

Objectives. The purpose of this study was to quantify the size of vocal fold polyps and to investigate the relationship between the glottal gap and parameters of acoustic voice analysis and phonetography. Material and methods. Eighty-one microlaryngoscopic images and digital recordings of voices (acoustic analysis and phonetogram) acquired from the patients with vocal fold polyps (VFPs) were employed in this study. Vocal fold (VF) images were collected during routine direct microlaryngoscopy using Moller-Wedel Universa 300 surgical microscope, 3-CCD Elmo 768×576- pixel color video camera and a 300 W Xenon light source. Acoustic voice analysis and phonetography were established using Dr. Speech (Tiger Electronics Inc.) software. Microlaryngoscopic images were processed by original software created by ELINTA and displayed on a monitor. The relative lengths and widths of vocal fold polyps as well as percentage area of VFP were calculated. The Pearson’s correlation was applied to reveal the correlation between VFP dimensions and acoustic voice parameters. Results. There were no statistically significant differences between the dimensions of left and right vocal folds and VFPs. Statistically significant slight to mild correlations between measured dimensions of VFP acoustic and phonetogram parameters were revealed, with HNR and phonetogram area showing the strongest correlation to the size of VFPs. Conclusion. The results of our study confirm that quantitative microlaryngoscopic measurements of vocal fold polyp and glottal gap dimensions may be a useful tool for objective assessment of glottic incompetence and voice impairment

High-Frequency Nanosecond Bleomycin Electrochemotherapy and its Effects on Changes in the Immune System and Survival

In this work, a time-dependent and time-independent study on bleomycin-based high-frequency nsECT (3.5 kV/cm × 200 pulses) for the elimination of LLC1 tumours in C57BL/6J mice is performed. We show the efficiency of nsECT (200 ns and 700 ns delivered at 1 kHz and 1 MHz) for the elimination of tumours in mice and increase of their survival. The dynamics of the immunomodulatory effects were observed after electrochemotherapy by investigating immune cell populations and antitumour antibodies at different timepoints after the treatment. ECT treatment resulted in an increased percentage of CD4+ T, splenic memory B and tumour-associated dendritic cell subsets. Moreover, increased levels of antitumour IgG antibodies after ECT treatment were detected. Based on the time-dependent study results, nsECT treatment upregulated PD 1 expression on splenic CD4+ Tr1 cells, increased the expansion of splenic CD8+ T, CD4+CD8+ T, plasma cells and the proportion of tumour-associated pro inflammatory macrophages. The Lin− population of immune cells that was increased in the spleens and tumour after nsECT was identified. It was shown that nsECT prolonged survival of the treated mice and induced significant changes in the immune system, which shows a promising alliance of nanosecond electrochemotherapy and immunotherapy

Extracellular Vesicles from Human Teeth Stem Cells Trigger ATP Release and Promote Migration of Human Microglia through P2X4 Receptor/MFG-E8-Dependent Mechanisms

Extracellular vesicles (EVs) effectively suppress neuroinflammation and induce neuroprotective effects in different disease models. However, the mechanisms by which EVs regulate the neuroinflammatory response of microglia remains largely unexplored. Here, we addressed this issue by testing the action of EVs derived from human exfoliated deciduous teeth stem cells (SHEDs) on immortalized human microglial cells. We found that EVs induced a rapid increase in intracellular Ca2+ and promoted significant ATP release in microglial cells after 20 min of treatment. Boyden chamber assays revealed that EVs promoted microglial migration by 20%. Pharmacological inhibition of different subtypes of purinergic receptors demonstrated that EVs activated microglial migration preferentially through the P2X4 receptor (P2X4R) pathway. Proximity ligation and co-immunoprecipitation assays revealed that EVs promote association between milk fat globule-epidermal growth factor-factor VIII (MFG-E8) and P2X4R proteins. Furthermore, pharmacological inhibition of αVβ3/αVβ5 integrin suppressed EV-induced cell migration and formation of lipid rafts in microglia. These results demonstrate that EVs promote microglial motility through P2X4R/MFG-E8-dependent mechanisms. Our findings provide novel insights into the molecular mechanisms through which EVs target human microglia that may be exploited for the development of new therapeutic strategies targeting disease-associated neuroinflammation

Improving NonViral Gene Delivery Using MHz Bursts of Nanosecond Pulses and Gold Nanoparticles for Electric Field Amplification

Gene delivery by the pulsed electric field is a promising alternative technology for nonviral transfection; however, the application of short pulses (i.e., nanosecond) is extremely limited. In this work, we aimed to show the capability to improve gene delivery using MHz frequency bursts of nanosecond pulses and characterize the potential use of gold nanoparticles (AuNPs: 9, 13, 14, and 22 nm) in this context. We have used bursts of MHz pulses 3/5/7 kV/cm × 300 ns × 100 and compared the efficacy of the parametric protocols to conventional microsecond protocols (100 µs × 8, 1 Hz) separately and in combination with nanoparticles. Furthermore, the effects of pulses and AuNPs on the generation of reactive oxygen species (ROS) were analyzed. It was shown that gene delivery using microsecond protocols could be significantly improved with AuNPs; however, the efficacy is strongly dependent on the surface charge of AuNPs and their size. The capability of local field amplification using AuNPs was also confirmed by finite element method simulation. Finally, it was shown that AuNPs are not effective with nanosecond protocols. However, MHz protocols are still competitive in the context of gene delivery, resulting in low ROS generation, preserved viability, and easier procedure to trigger comparable efficacy