Elevated Fibronectin Levels in Profibrotic CD14+ Monocytes and CD14+ Macrophages in Systemic Sclerosis.

Background: Systemic sclerosis (SSc) is an autoimmune disease characterized by overproduction of extracellular matrix (ECM) and multiorgan fibrosis. Animal studies pointed to bone marrow-derived cells as a potential source of pathological ECM-producing cells in immunofibrotic disorders. So far, involvement of monocytes and macrophages in the fibrogenesis of SSc remains poorly understood. Methods and Results: Immunohistochemistry analysis showed accumulation of CD14+ monocytes in the collagen-rich areas, as well as increased amount of alpha smooth muscle actin (αSMA)-positive fibroblasts, CD68+ and mannose-R+ macrophages in the heart and lungs of SSc patients. The full genome transcriptomics analyses of CD14+ blood monocytes revealed dysregulation in cytoskeleton rearrangement, ECM remodeling, including elevated FN1 (gene encoding fibronectin) expression and TGF-ß signalling pathway in SSc patients. In addition, single cell RNA sequencing analysis of tissue-resident CD14+ pulmonary macrophages demonstrated activated profibrotic signature with the elevated FN1 expression in SSc patients with interstitial lung disease. Peripheral blood CD14+ monocytes obtained from either healthy subjects or SSc patients exposed to profibrotic treatment with profibrotic cytokines TGF-ß, IL-4, IL-10, and IL-13 increased production of type I collagen, fibronectin, and αSMA. In addition, CD14+ monocytes co-cultured with dermal fibroblasts obtained from SSc patients or healthy individuals acquired a spindle shape and further enhanced production of profibrotic markers. Pharmacological blockade of the TGF-ß signalling pathway with SD208 (TGF-ß receptor type I inhibitor), SIS3 (Smad3 inhibitor) or (5Z)-7-oxozeaenol (TGF-ß-activated kinase 1 inhibitor) ameliorated fibronectin levels and type I collagen secretion. Conclusions: Our findings identified activated profibrotic signature with elevated production of profibrotic fibronectin in CD14+ monocytes and CD14+ pulmonary macrophages in SSc and highlighted the capability of CD14+ monocytes to acquire a profibrotic phenotype. Taking together, tissue-infiltrating CD14+ monocytes/macrophages can be considered as ECM producers in SSc pathogenesis.

Photobiomodulation with Red and Near-Infrared Light Improves Viability and Modulates Expression of Mesenchymal and Apoptotic-Related Markers in Human Gingival Fibroblasts.

Photobiomodulation (PBM), also called low-level laser treatment (LLLT), has been considered a promising tool in periodontal treatment due to its anti-inflammatory and wound healing properties. However, photobiomodulation's effectiveness depends on a combination of parameters, such as energy density, the duration and frequency of the irradiation sessions, and wavelength, which has been shown to play a key role in laser-tissue interaction. The objective of the study was to compare the in vitro effects of two different wavelengths-635 nm and 808 nm-on the human primary gingival fibroblasts in terms of viability, oxidative stress, inflammation markers, and specific gene expression during the four treatment sessions at power and energy density widely used in dental practice (100 mW, 4 J/cm2). PBM with both 635 and 808 nm at 4 J/cm2 increased the cell number, modulated extracellular oxidative stress and inflammation markers and decreased the susceptibility of human primary gingival fibroblasts to apoptosis through the downregulation of apoptotic-related genes (P53, CASP9, BAX). Moreover, modulation of mesenchymal markers expression (CD90, CD105) can reflect the possible changes in the differentiation status of irradiated fibroblasts. The most pronounced results were observed following the third irradiation session. They should be considered for the possible optimization of existing low-level laser irradiation protocols used in periodontal therapies.

CRISPR/Cas9 in Cancer Immunotherapy: Animal Models and Human Clinical Trials.

Even though chemotherapy and immunotherapy emerged to limit continual and unregulated proliferation of cancer cells, currently available therapeutic agents are associated with high toxicity levels and low success rates. Additionally, ongoing multi-targeted therapies are limited only for few carcinogenesis pathways, due to continually emerging and evolving mutations of proto-oncogenes and tumor-suppressive genes. CRISPR/Cas9, as a specific gene-editing tool, is used to correct causative mutations with minimal toxicity, but is also employed as an adjuvant to immunotherapy to achieve a more robust immunological response. Some of the most critical limitations of the CRISPR/Cas9 technology include off-target mutations, resulting in nonspecific restrictions of DNA upstream of the Protospacer Adjacent Motifs (PAM), ethical agreements, and the lack of a scientific consensus aiming at risk evaluation. Currently, CRISPR/Cas9 is tested on animal models to enhance genome editing specificity and induce a stronger anti-tumor response. Moreover, ongoing clinical trials use the CRISPR/Cas9 system in immune cells to modify genomes in a target-specific manner. Recently, error-free in vitro systems have been engineered to overcome limitations of this gene-editing system. The aim of the article is to present the knowledge concerning the use of CRISPR Cas9 technique in targeting treatment-resistant cancers. Additionally, the use of CRISPR/Cas9 is aided as an emerging supplementation of immunotherapy, currently used in experimental oncology. Demonstrating further, applications and advances of the CRISPR/Cas9 technique are presented in animal models and human clinical trials. Concluding, an overview of the limitations of the gene-editing tool is proffered.

Cortical Granule Distribution and Expression Pattern of Genes Regulating Cellular Component Size, Morphogenesis, and Potential to Differentiation are Related to Oocyte Developmental Competence and Maturational Capacity In Vivo and In Vitro.

Polyspermia is an adverse phenomenon during mammalian fertilization when more than one sperm fuses with a single oocyte. The egg cell is prepared to prevent polyspermia by, among other ways, producing cortical granules (CGs), which are specialized intracellular structures containing enzymes that aim to harden the zona pellucida and block the fusion of subsequent sperm. This work focused on exploring the expression profile of genes that may be associated with cortical reactions, and evaluated the distribution of CGs in immature oocytes and the peripheral density of CGs in mature oocytes. Oocytes were isolated and then processed for in vitro maturation (IVM). Transcriptomic analysis of genes belonging to five ontological groups has been conducted. Six genes showed increased expression after IVM (ARHGEF2, MAP1B, CXCL12, FN1, DAB2, and SOX9), while the majority of genes decreased expression after IVM. Using CG distribution analysis in immature oocytes, movement towards the cortical zone of the oocyte during meiotic competence acquisition was observed. CGs peripheral density decreased with the rise in meiotic competence during the IVM process. The current results reveal important new insights into the in vitro maturation of oocytes. Our results may serve as a basis for further studies to investigate the cortical reaction of oocytes.

Photobiomodulation-Underlying Mechanism and Clinical Applications.

The purpose of this study is to explore the possibilities for the application of laser therapy in medicine and dentistry by analyzing lasers' underlying mechanism of action on different cells, with a special focus on stem cells and mechanisms of repair. The interest in the application of laser therapy in medicine and dentistry has remarkably increased in the last decade. There are different types of lasers available and their usage is well defined by different parameters, such as: wavelength, energy density, power output, and duration of radiation. Laser irradiation can induce a photobiomodulatory (PBM) effect on cells and tissues, contributing to a directed modulation of cell behaviors, enhancing the processes of tissue repair. Photobiomodulation (PBM), also known as low-level laser therapy (LLLT), can induce cell proliferation and enhance stem cell differentiation. Laser therapy is a non-invasive method that contributes to pain relief and reduces inflammation, parallel to the enhanced healing and tissue repair processes. The application of these properties was employed and observed in the treatment of various diseases and conditions, such as diabetes, brain injury, spinal cord damage, dermatological conditions, oral irritation, and in different areas of dentistry.

The Proliferation and Differentiation of Adipose-Derived Stem Cells in Neovascularization and Angiogenesis.

Neovascularization and angiogenesis are vital processes in the repair of damaged tissue, creating new blood vessel networks and increasing oxygen and nutrient supply for regeneration. The importance of Adipose-derived Mesenchymal Stem Cells (ASCs) contained in the adipose tissue surrounding blood vessel networks to these processes remains unknown and the exact mechanisms responsible for directing adipogenic cell fate remain to be discovered. As adipose tissue contains a heterogenous population of partially differentiated cells of adipocyte lineage; tissue repair, angiogenesis and neovascularization may be closely linked to the function of ASCs in a complex relationship. This review aims to investigate the link between ASCs and angiogenesis/neovascularization, with references to current studies. The molecular mechanisms of these processes, as well as ASC differentiation and proliferation are described in detail. ASCs may differentiate into endothelial cells during neovascularization; however, recent clinical trials have suggested that ASCs may also stimulate angiogenesis and neovascularization indirectly through the release of paracrine factors.

Genes regulating hormone stimulus and response to protein signaling revealed differential expression pattern during porcine oocyte in vitro maturation, confirmed by lipid concentration.

Genes influencing oocyte maturation may be valuable for predicting their developmental potential, as well as discerning the mechanistic pathways regulating oocyte development. In the presented research microarray gene expression analysis of immature and in vitro matured porcine oocytes was performed. Two groups of oocytes were compared in the study: before (3 × n = 50) and after in vitro maturation (3 × n = 50). The selection of viable oocytes was performed using the brilliant cresyl blue (BCB) test. Furthermore, microarrays and RT-qPCR was used to analyze the transcriptome of the oocytes before and after IVM. The study focused on the genes undergoing differential expression in two gene-ontology groups: "Cellular response to hormone stimulus" and "Cellular response to unfolded protein", which contain genes that may directly or indirectly be involved in signal transduction during oocyte maturation. Examination of all the genes of interest showed a lower level of their expression after IVM. From the total number of genes in these gene ontologies ten of the highest change in expression were identified: FOS, ID2, BTG2, CYR61, ESR1, AR, TACR3, CCND2, EGR2 and TGFBR3. The successful maturation of the oocytes was additionally confirmed with the use of lipid droplet assay. The genes were briefly described and related to the literature sources, to investigate their potential roles in the process of oocyte maturation. The results of the study may serve as a basic molecular reference for further research aimed at improving the methods of oocyte in vitro maturation, which plays an important role in the procedures of assisted reproduction.

Inclusion Biogenesis, Methods of Isolation and Clinical Application of Human Cellular Exosomes.

Exosomes are a heterogenous subpopulation of extracellular vesicles 30-150 nm in range and of endosome-derived origin. We explored the exosome formation through different systems, including the endosomal sorting complex required for transport (ESCRT) and ESCRT-independent system, looking at the mechanisms of release. Different isolation techniques and specificities of exosomes from different tissues and cells are also discussed. Despite more than 30 years of research that followed their definition and indicated their important role in cellular physiology, the exosome biology is still in its infancy with rapidly growing interest. The reasons for the rapid increase in interest with respect to exosome biology is because they provide means of intercellular communication and transmission of macromolecules between cells, with a potential role in the development of diseases. Moreover, they have been investigated as prognostic biomarkers, with a potential for further development as diagnostic tools for neurodegenerative diseases and cancer. The interest grows further with the fact that exosomes were reported as useful vectors for drugs.

New markers for regulation of transcription and macromolecule metabolic process in porcine oocytes during in vitro maturation.

Oocyte maturation is essential for proper fertilization, embryo implantation and early development. While the physiological conditions of these processes are relatively well­known, its exact molecular mechanisms remain widely undiscovered. Oocyte growth, differentiation and maturation are therefore the subject of scientific debate. Precious literature has indicated that the oocyte itself serves a regulatory role in the mechanisms underlying these processes. Hence, the present study performed expression microarrays to analyze the complete transcriptome of porcine oocytes during their in vitro maturation (IVM). Pig material was used for experimentation, as it possesses similarities to the reproductive processes and general genetic proximities of Sus scrofa to human. Oocytes, isolated from the ovaries of slaughtered animals were assessed via the Brilliant Cresyl Blue test and directed to IVM. A number of oocytes were left to be analyzed as the 'before IVM' group. Oocyte mRNA was isolated and used for microarray analysis, which was subsequently validated via RT­qPCR. The current study particularly focused on genes belonging to 'positive regulation of transcription, DNA­dependent', 'positive regulation of gene expression', 'positive regulation of macromolecule metabolic process' and 'positive regulation of transcription from RNA polymerase II promoter' ontologies. FOS, VEGFA, ESR1, AR, CCND2, EGR2, ENDRA, GJA1, INHBA, IHH, INSR, APP, WWTR1, SMARCA1, NFAT5, SMAD4, MAP3K1, EGR1, RORA, ECE1, NR5A1, KIT, IKZF2, MEF2C, SH3D19, MITF and PSMB4 were all determined to be significantly altered (fold change, >|2|; P<0.05) among these groups, with their downregulation being observed after IVM. Genes with the most altered expressions were analyzed and considered to be potential markers of maturation associated with transcription regulation and macromolecule metabolism process.

Human Umbilical Vein Endothelial Cells (HUVECs) Co-Culture with Osteogenic Cells: From Molecular Communication to Engineering Prevascularised Bone Grafts.

The repair of bone defects caused by trauma, infection or tumor resection is a major clinical orthopedic challenge. The application of bone grafts in orthopedic procedures is associated with a problem of inadequate vascularization in the initial phase after implantation. Meanwhile, the survival of cells within the implanted graft and its integration with the host tissue is strongly dependent on nutrient and gaseous exchange, as well as waste product removal, which are effectuated by blood microcirculation. In the bone tissue, the vasculature also delivers the calcium and phosphate indispensable for the mineralization process. The critical role of vascularization for bone healing and function, led the researchers to the idea of generating a capillary-like network within the bone graft in vitro, which could allow increasing the cell survival and graft integration with a host tissue. New strategies for engineering pre-vascularized bone grafts, that apply the co-culture of endothelial and bone-forming cells, have recently gained interest. However, engineering of metabolically active graft, containing two types of cells requires deep understanding of the underlying mechanisms of interaction between these cells. The present review focuses on the best-characterized endothelial cells-human umbilical vein endothelial cells (HUVECs)-attempting to estimate whether the co-culture approach, using these cells, could bring us closer to development and possible clinical application of prevascularized bone grafts.

The Unique Mechanisms of Cellular Proliferation, Migration and Apoptosis are Regulated through Oocyte Maturational Development-A Complete Transcriptomic and Histochemical Study.

The growth and development of oocyte affect the functional activities of the surrounding somatic cells. These cells are regulated by various types of hormones, proteins, metabolites, and regulatory molecules through gap communication, ultimately leading to the development and maturation of oocytes. The close association between somatic cells and oocytes, which together form the cumulus-oocyte complexes (COCs), and their bi-directional communication are crucial for the acquisition of developmental competences by the oocyte. In this study, oocytes were extracted from the ovaries obtained from crossbred landrace gilts and subjected to in vitro maturation. RNA isolated from those oocytes was used for the subsequent microarray analysis. The data obtained shows, for the first time, variable levels of gene expression (fold changes higher than |2| and adjusted p-value < 0.05) belonging to four ontological groups: regulation of cell proliferation (GO:0042127), regulation of cell migration (GO:0030334), and regulation of programmed cell death (GO:0043067) that can be used together as proliferation, migration or apoptosis markers. We have identified several genes of porcine oocytes (ID2, VEGFA, BTG2, ESR1, CCND2, EDNRA, ANGPTL4, TGFBR3, GJA1, LAMA2, KIT, TPM1, VCP, GRID2, MEF2C, RPS3A, PLD1, BTG3, CD47, MITF), whose expression after in vitro maturation (IVM) is downregulated with different degrees. Our results may be helpful in further elucidating the molecular basis and functional significance of a number of gene markers associated with the processes of migration, proliferation and angiogenesis occurring in COCs.

The effects of chosen plant extracts and compounds on mesenchymal stem cells-a bridge between molecular nutrition and regenerative medicine- concise review.

Mesenchymal stem cells (MSC) stand as a promising tool in regenerative medicine because of their high therapeutic potential in treatment of degenerative, metabolic and other types of diseases. The cellular therapies involving MSCs include their isolation mainly from the bone marrow, adipose tissue or umbilical cord and in vitro expansion for further autologous or allogeneic transplantation. Recent studies revealed, that bioactive compounds, naturally occurring in seaweeds, herbs, fruits and vegetables, possess the ability to modulate self-renewal and differentiation potential of adult stem cells, targeting a broad range of intracellular signal transduction pathways. Number of ongoing trials aim to find a herbal extract that may become less toxic and affordable natural therapeutic. Mesenchymal stem cells are treated with crude extracts or individual compounds to investigate its effects and mechanism on stem cells proliferation and differentiation. Deeply investigated, herbal extract which increases tissue regeneration and promotes stem cell growth may be successfully applied in the field of biomaterials. Promoting the endogenous stem cell multipotency and their differentiation potential may additionally support the regenerative processes after MSCs transplantation. The review focuses on the beneficial effects of chosen plant derived substances on MSCs proliferative activity and their osteogenic differentiation potential. Copyright © 2017 John Wiley & Sons, Ltd.

The Cladophora glomerata Enriched by Biosorption Process in Cr(III) Improves Viability, and Reduces Oxidative Stress and Apoptosis in Equine Metabolic Syndrome Derived Adipose Mesenchymal Stromal Stem Cells (ASCs) and Their Extracellular Vesicles (MV's).

This study investigated in vitro effects of freshwater alga Cladophora glomerata water extract enriched during a biosorption process in Cr(III) trivalent chromium and chromium picolinate on adipose-derived mesenchymal stromal stem cells (ASCs) and extracellular microvesicles (MVs) in equine metabolic syndrome-affected horses. Chemical characterisation of natural Cladophora glomerata was performed with special emphasis on: vitamin C, vitamin E, total phenols, fatty acids, free and protein-bound amino acids as well as measured Cr in algal biomass. To examine the influence of Cladophora glomerata water extracts, in vitro viability, oxidative stress factor accumulation, apoptosis, inflammatory response, biogenesis of mitochondria, autophagy in ASCs of EMS and secretory activity manifested by MV release were investigated. For this purpose, various methods of molecular biology and microscopic observations (i.e., immunofluorescence staining, SEM, TEM, FIB observations, mRNA and microRNA expression by RT-qPCR) were applied. The extract of Cladophora glomerata enriched with Cr(III) ions reduced apoptosis and inflammation in ASCs of EMS horses through improvement of mitochondrial dynamics, decreasing of PDK4 expression and reduction of endoplastic reticulum stress. Moreover, it was found, that Cladophora glomerata and Cr(III) induce antioxidative protection coming from enhanced SOD activity Therefore, Cladophora glomerata enriched with Cr(III) ions might become an interesting future therapeutic agent in the pharmacological treatment of EMS horses.