Current methods for the microglia isolation: Overview and comparative analysis of approaches.

Microglia represent a distinct population of neuroglia, constituting ~ 10% of all CNS cells and exhibit high plasticity. Proper functioning of microglia is critical in the event of CNS damage due to the rapid modulation of their functions. Microglia are not only the first stage of immune defense against injury and infection, contributing to both the innate and adaptive local immune response, but also play a vital role in maintaining homeostasis of the brain and spinal cord. For this reason, microglia deserve special attention in the study of neuropathological responses. Studying microglia behavior in various in vivo models of neuropathologies is certainly a priority, as it allows us to evaluate the behavior in the context of the changing microenvironment of nervous tissue. However, sometimes there are some technological problems that hinder the identification of the features of intercellular interactions, ensured cooperation between microglia and other cell types. In this regard, the use of in vitro models remains relevant today, contributing to a more in-depth understanding of the mechanisms of microglial involvement in neuropathology. The methods considered in this review for obtaining an isolated culture of microglia, along with their advantages and disadvantages, can help researchers in selecting the appropriate source and method for obtaining these cells, thereby opening up opportunities for gaining new neurobiological knowledge.

In Vivo DYSF Gene Viral Delivery Provides a Histoprotective Effect in Skeletal Muscle Tissue in Dysferlin-Deficient Mice.

We studied the effects of a dual-vector DYSF gene delivery system based on adeno-associated virus serotype 9 capsids on pathological manifestations of dysferlinopathy in skeletal muscles of Bla/J mice lacking DYSF expression. The mice received intravenous injection of 3×1013 genomic copies of the virus containing the dual-vector system. M. gastrocnemius, m. psoas major, m. vastus lateralis, and m. gluteus superficialis were isolated for histological examination in 3, 6, and 12 weeks after treatment. Healthy wild-type (C57BL/6) mice served as positive control and were sacrificed 3 weeks after injection of 150 µl of 0.9% NaCl into the caudal vein. To detect dysferlin in muscle cryosections, immunohistochemical analysis with diagnostic antibodies was performed; paraffin sections were stained with hematoxylin and eosin for morphometric analysis. After administration of gene-therapeutic constructs, muscle fibers with membrane or cytoplasmic dysferlin location were detected in all examined muscles. The proportion of necrotic muscle fibers decreased, the number of muscle fibers with central location of the nucleus increased, and the mean cross-section area of the muscle fibers decreased.

[COVID-19: is transmission through eye contact possible?] / COVID-19: vozmozhen li put' peredachi cherez glaza?

The article reviews international and Russian scientific papers concerning the possibility of transmitting coronavirus infections, particularly the COVID-19, through eye surface. According to the studied literature, the incidence of ocular symptoms in COVID-19 is around 0.8-31.6%, with conjunctivitis being the most frequent manifestation. The review summarizes data on virus detection in conjunctival discharge of COVID-19 patients. Across six studies, the total number of patients is 252, among which were 8 cases (3.17%) of virus detection in the conjunctival cavity. The review discusses the reasons for infrequent detection of the virus in the lacrimal fluid. The analyzed data shows that COVID-19 associated conjunctivitis can be the first symptom, the primary manifestation, or sometimes be detected in the lacrimal fluid of patients without any concomitant signs of eye surface inflammation. The article also presents two clinical cases of patients with keratoconjunctivitis and conjunctivitis associated with COVID-19, as well as the results of experimental transconjunctival and respiratory exposure of Rhesus macaques to SARS-CoV-2 with conclusion of possibility of this type of transmission. Additionally, the review contains the opinion of researchers concerning the influence of several factors on the possibility of virus detection in the lacrimal fluid. The conclusion was made that there is possibility of COVID-19 transmission through the eye surface. While it is not being considered a major transmission route, it should not be ignored. Conjunctival cavity of COVID-19 patients can be the source of infection. Eye protection measures should be undertaken when working with potentially infected patients.

Stem-like tumor cells and proinflammatory cytokines in the ascitic fluid of ovarian cancer patients.

Ovarian cancer (OC) is able to develop implantation metastases in the abdominal cavity. Ascites is potentially useful for evaluating cancer features. The aim of the study was to assess the content of stem-like tumor cells and inflammatory mediators in ascites of OC. The prospective study included 11 patients with primary OC having ascites, 8 patients with benign ovarian tumors having ascites and 22 healthy women. In ascitic fluid obtained by laparocentesis, the populations of tumor stem-like cells were determined on a Cytoflex S` flow cytometer (Beckman Coulter, USA) and CytExpert Software using monoclonal antibodies to CD45, CD44 and CD133. The cytokine profiles of ascitic fluid and blood serum (IL-1ß, IL-18, IL-4, IL-10 and VEGF) were assessed by ELISA. Stem-like cells were found in all samples. 5 cell populations were evaluated. The number of cells expressing both markers: CD44 + and CD133+, was the lowest. The highest, about 32%, was the number of CD44+ cells. The number of cells CD45-CD44+CD133- in ascites strongly positively correlated with the content of IL-10 in ascites, and the numbers of CD45-CD133+ and CD45-CD44-CD133+ - with the level of VEGF in blood serum. No correlations were found between the numbers of stem-like cells and the disease stage or the level of CA125 in blood. The combination of IL-4 and IL-10 in ascites had the greatest significance in predicting the disease stage. These results suggest a relationship between the levels of VEGF, IL-10, and cancer stem cells in the OC ascites. Stem-like cells in OC ascites are heterogeneous and are present even at an early stage of the disease. It seems promising to study cell populations and cytokine profile of ascites together, to assess the biomarker potential of their combination.

Potential therapeutic application of mesenchymal stem cells in ophthalmology.

At present a wide variety of methods have been proposed to treat eye disorders, drug therapies are most commonly used. It should be noted that effective treatment modalities especially for degeneration of the retina and optic nerve are lacking. In the last few years stem cell transplantation has been proposed as an alternative method. The opportunities that stem cells provide within clinical use are almost unlimited. These cells are presently applied to treat various traumatic and degenerative disorders due to their unique biologic properties. Stem cells have high proliferative capabilities and are a self-maintained population of cells capable of differentiating into different cell types. Thus, they are represent a very primary stage of a cell lineage. Their ability to differentiate into different pathways provides animals with great plasticity in the renewal of somatic cells in postnatal ontogenesis. Pre-clinical and clinical ophthalmology studies where mesenchymal stem cells are applied and various methods of their administration are discussed herein. In addition the safety and efficacy of using bone marrow- and adipose tissue-derived mesenchymal stem cells have been discussed.

PTEN expression in astrocytic processes after spinal cord injury.

The role of the Rho/ROCK/PTEN signaling pathway in the regulation of astrocyte function for consolidation/stabilization of the synapse has not been thoroughly studied. In this study, the expression of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in GFAP-positive astrocytic processes in the ventral horns (VH) of the rat spinal cord has been evaluated in the normal condition and in a delayed period (30 days) after dosed contusion spinal cord injury (SCI) in caudal thoracic segments. In intact rats and at 30 days post-injury (dpi), semi-quantitative immunohistochemical analysis showed that there is approximately 2 folds less synaptophysin reactivity in the motoneuron perikarya than outside the perikarya, i.e., on dendritic spines, in the VH area. At 30 dpi, the square occupied by synaptophysin reactivity on the motoneuron perikarya and dendritic spines decreased ~2.4 and ~2.1 folds, respectively. Western blotting of the postsynaptic density protein 95 (PSD95) showed a decreased amount in the area of injury of ~3 folds at 30 dpi. Expression of GFAP in the astrocytic processes around the synaptophysin spots (APAS) was less than in the astrocytic processes that were located at distance from the synapses (APFS) both in the intact and SCI groups. In the APAS, the expression level of PTEN increased significantly after SCI. In these astrocytic processes, the PTEN expression level was significantly higher than in the APFS for both the intact and SCI rats. In the intact spinal cord, different PTEN expression levels were detected both in APAS and APFS. This may be due to the varying degree of integration of PTEN in the membrane compartment of astrocyte stem processes and possibly the increased delivery of PTEN from the GFAP-positive stem into fine GFAP-negative peripheral processes. The observed shifts after SCI reflect the imbalance in the mechanisms of synaptic plasticity after injury. Thus, strategies that have been developed for the deletion or knockdown of the PTEN gene are quite promising.

Electrophysiological, Morphological, and Ultrastructural Features of the Injured Spinal Cord Tissue after Transplantation of Human Umbilical Cord Blood Mononuclear Cells Genetically Modified with the VEGF and GDNF Genes.

In this study, we examined the efficacy of human umbilical cord blood mononuclear cells (hUCB-MCs), genetically modified with the VEGF and GDNF genes using adenoviral vectors, on posttraumatic regeneration after transplantation into the site of spinal cord injury (SCI) in rats. Thirty days after SCI, followed by transplantation of nontransduced hUCB-MCs, we observed an improvement in H (latency period, LP) and M(Amax) waves, compared to the group without therapy after SCI. For genetically modified hUCB-MCs, there was improvement in Amax of M wave and LP of both the M and H waves. The ratio between Amax of the H and M waves (Hmax/Mmax) demonstrated that transplantation into the area of SCI of genetically modified hUCB-MCs was more effective than nontransduced hUCB-MCs. Spared tissue and myelinated fibers were increased at day 30 after SCI and transplantation of hUCB-MCs in the lateral and ventral funiculi 2.5 mm from the lesion epicenter. Transplantation of hUCB-MCs genetically modified with the VEGF and GNDF genes significantly increased the number of spared myelinated fibers (22-fold, P > 0.01) in the main corticospinal tract compared to the nontransduced ones. HNA+ cells with the morphology of phagocytes and microglia-like cells were found as compact clusters or cell bridges within the traumatic cavities that were lined by GFAP+ host astrocytes. Our results show that hUCB-MCs transplanted into the site of SCI improved regeneration and that hUCB-MCs genetically modified with the VEGF and GNDF genes were more effective than nontransduced hUCB-MCs.

Epidemiological dynamics of nephropathia epidemica in the Republic of Tatarstan, Russia, during the period of 1997-2013.

This report summarizes epidemiological data on nephropathia epidemica (NE) in the Republic of Tatarstan, Russia. NE cases identified in the period 1997-2013 were investigated in parallel with the hantavirus antigen prevalence in small rodents in the study area. A total of 13 930 NE cases were documented in all but one district of Tatarstan, with most cases located in the central and southeastern districts. The NE annual incidence rate exhibited a cyclical pattern, with the highest numbers of cases being registered once in every 3-5 years. The numbers of NE cases rose gradually from July to November, with the highest morbidity in adult males. The highest annual disease incidence rate, 64·4 cases/100 000 population, was observed in 1997, with a total of 2431 NE cases registered. NE cases were mostly associated with visiting forests and agricultural activities. The analysis revealed that the bank vole Myodes glareolus not only comprises the majority of the small rodent communities in the region, but also consistently displays the highest hantavirus prevalence compared to other small rodent species.

Reflective Schmidt-Cassegrain system for large-aperture telescopes.

A reflective modification of the Schmidt-Cassegrain system was built and tested. Ultraviolet (UV) and soft x-ray applications are discussed. The system consists of a planoid mirror with an aspheric profile and prime concave and secondary convex spherical mirrors. Spherical aberration in a wide field of view and astigmatism are compensated by the aspheric profile of the planoid. The main parameters of the scheme are as follows: an entrance aperture of 180 mm, a focal ratio F/3.2, an angular resolution better than 3'' (corresponding to a pixel size of a back-side illuminated CCD), a field of view of ±1.5° (2ω=3°) and a flat image field with a diameter of 30.4 mm. Due to the absence of chromatic aberrations and wide field of view, the scheme is of considerable interest for hyperspectral instruments. In particular, the operating range of the instruments can be expanded into vacuum UV and UV regions.

Construction of recombinant adenovirus containing picorna-viral 2A-peptide sequence for the co-expression of neuro-protective growth factors in human umbilical cord blood cells.

STUDY DESIGN: Experimental study. OBJECTIVE: Several neuro-degenerative disorders such as Alzheimer's dementia, Parkinson's disease and amyotrophic lateral sclerosis (ALS) are associated with genetic mutations, and replacing or disrupting defective sequences might offer therapeutic benefits. Single gene delivery has so far failed to achieve significant clinical improvements in humans, leading to the advent of co-expression of multiple therapeutic genes. Co-transfection using two or more individual constructs might inadvertently result in disproportionate delivery of the products into the cells. To prevent this, and in order to rule out interference among the many promoters with varying strength, expressing multiple proteins in equimolar amounts can be achieved by linking open reading frames under the control of only one promoter. SETTING: Kazan, Russian Federation. METHODS: Here we describe a strategy for adeno-viral co-expression of vascular endothelial growth factor (VEGF) and fibroblast growth factor 2 (FGF2) interconnected through picorna-viral 2A-amino-acid sequence in transfected human umbilical cord blood mono-nuclear cells (hUCB-MCs). RESULTS: Presence of both growth factors, as well as absence of immune response to 2A-antigen, was demonstrated after 28-52 days. Following injection of hUCB-MCs into ALS transgenic mice, co-expression of VEGF and FGF2, as well as viable xeno-transplanted cells, were observed in the spinal cord after 1 month. CONCLUSION: These results suggest that recombinant adeno-virus containing 2A-sequences could serve as a promising alternative in regenerative medicine for the delivery of therapeutic molecules to treat neurodegenerative diseases, such as ALS.

Effects of intranasal administration of the peptide antagonist of type I vaniloid receptor (TRPV1) in the rodent central nervous system.

Intranasal administration of the polypeptide APHC3, an antagonist of the TRPV1 receptor, had acute anxiolytic and antidepressant effects, as well as an ability to modify the microglial response to proinflammatory stress and cytokine profile of the hippocampus. However, the acute antidepressant effect of the polypeptide was not related to the attenuation of neuroiflammation and probably had a different mechanism. The use of intranasal administration of the APHC3 peptide as a therapeutic approach aimed at decreasing depression symptoms needs additional studies in order to find the mechanism of action of this polypeptide in the central nervous system (CNS).

Synaptosome-associated protein 25 (SNAP25) synthesis in terminal buttons of mouse motor neuron.

Previously, we formulated the hypothesis of compartmentalized protein synthesis in axons of motor neurons. In the axon hillock, along the entire length of the axon and in its ending, specific proteins are locally synthesized, which ensure the function of each compartment. In support of this hypothesis, in this work we studied the local protein synthesis in mouse motor nerve ending.

Upregulation of IFN-γ and IL-12 is associated with a milder form of hantavirus hemorrhagic fever with renal syndrome.

Hantavirus hemorrhagic fever with renal syndrome (HFRS) is a zoonotic disease characterized by acute onset, fever, malaise, and back pain. As the disease progresses, hemorrhagic disturbances and kidney dysfunctions predominate. The examination of tissue collected postmortem supports the premise that virus replication is not responsible for this pathology; therefore, it is widely believed that virus-induced immune responses lead to the clinical manifestations associated with HFRS. The overproduction of inflammatory cytokines is commonly reported in subjects with HFRS and has given rise to the hypothesis that a so-called "cytokine storm" may play a pivotal role in the pathogenesis of this disease. Currently, supportive care remains the only effective treatment for HFRS. Our data show that serum levels of interferon (IFN)-γ, interleukin (IL)-10, CCL2, and IL-12 are upregulated in HFRS cases when compared to healthy controls and the level of upregulation is dependent on the phase and severity of the disease. Furthermore, we observed an association between the mild form of the disease and elevated serum levels of IFN-γ and IL-12. Collectively, these observations suggest that the administration of exogenous IFN-γ and IL-12 may provide antiviral benefits for the treatment of HFRS and, thus, warrants further investigations.

Usage of plasmid vector carrying vegf and fgf2 genes after spinal cord injury in rats.

Using rat model of spinal cord contusion injury at TVIII, we compared the effectiveness of immediate single transplantation of human mononuclear umbilical cord blood cells transfected with pBud-VEGF-FGF2 plasmid and immediate direct injection of the same plasmid into the lesion area. The results suggest that the delivery of therapeutic genes vegf and fgf2 in cells is more effective than direct injection of plasmid DNA with the same genes (judging from the number of myelinated fibers). Better tissue preservation and motor function recovery in experiments with direct injection of plasmid pBud-VEGF-FGF2 suggest that direct gene therapy seems to be an effective additional procedure to the method of gene delivery with transfected stem and progenitor cells.

Analysis of the efficiency of gene-cell therapy in transgenic mice with amyotrophic lateral sclerosis phenotype.

Amyotrophic lateral sclerosis is a neurodegenerative disease characterized by progressive death of cerebral and spinal motorneurons. Using behavioral tests we studied the efficiency of gene-cell therapy in SOD1 G93A transgenic mice receiving xenotransplantation of human umbilical cord blood mononuclear cells genetically modified with adenoviral vectors encoding vascular endothelial growth factor (VEGF) and reporter green fluorescent protein (EGFP) genes. The cells were transplanted to mice on week 27 of life (preclinical stage of the disease). Behavioral tests (open field, grip strength test) showed that transplantation of umbilical cord blood mononuclear cells expressing VEGF significantly improved the parameters of motor and explorative activity, grip strength, and animal survival. Thus, gene-cell therapy based on genetically modified mononuclear cells expressing VEGF can be efficient for the treatment of amyotrophic lateral sclerosis.

[Effects of transplantation of human umbilical cord blood mononuclear cells, expressing VEGF and FGF2 genes, into the area of spinal cord traumatic lesion].

Effects of immediate single transplantation of human umbilical cord blood mononuclear cells (UCB-MC) transfected with recombinant vascular endothelial growth factor (VEGF) and fibroblast growth factor-2 (FGF2) genes into the area of injury were studied on the model of rat spinal cord dosed contusion at TVIII level. UCB-MC transfected with EGFP-N2 plasmid were transplanted into the rats of the control group under similar conditions. The presence of EGFP- labeled cells were traced in white matter during 21 days after transplantation at a distance no less than 10 mm in rostral and caudal directions from the nearest point of the injection. By 30 days after the transplantation of UCB-MC transfected with pBud-VEGF-FGF2 plasmid, the cross-sectional area of sparing grey matter increased by more than 60% at a distance of 3 mm from the epicenter of injury. By that time, in the animals of this group, the number of perivascular cells expressing beta receptor of platelet-derived growth factor (PDGFbetaR) was increased by an average of 30% in the outer zones of white matter 1.5 cm from the injury epicenter. Delivery of the therapeutic genes VEGF and FGF2 to the damaged region and their expression in cell carriers stimulates vascularization and post-traumatic spinal cord regeneration.

Mechanisms of ERK phosphorylation triggered via mouse formyl peptide receptor 2.

Formyl peptide receptors (FPRs) are expressed in the cells of the innate immune system and provide binding with pathogen and damage-associated molecular patterns with subsequent activation of the phagocytes for defense reactions such as chemotaxis, secretory degranulation and ROS generation. Probably, FPR2 is one of the unique receptors in the organism; it is able to recognize numerous ligands of different chemical structure, and moreover, these ligands can trigger opposite phagocyte responses promoting either pro- or anti-inflammatory reactions. Therefore, FPR2 and its signaling pathways are of intense research interest. We found only slight activation of ERK1/2 in the response to peptide ligand WKYMVM in the accelerating phase of ROS generation and more intense ERK1/2 phosphorylation in the declining phase of it in mouse bone marrow granulocytes. Lipid agonist BML-111 did not induce significant ERK phosphorylation when applied for 10-1800 s. To some extent co-localization of ERK1/2 and NADPH oxidase subunits was observed even in the intact cells and didn't change under FPR2 stimulation by WKYMVM, while direct PKC activation by PMA resulted to more efficient interaction between ERK1/2 and p47phox/p67phox and their translocation to plasma membrane. We have shown that phosphorylation and activation of ERK1/2 in bone marrow granulocytes depended on FPR2-triggered activity of PI3K and PKC, phosphatase DUSP6, and, the most but not the least, on ROS generation. Since blocking of ROS generation led to a slowdown of ERK activation indicating a significant contribution of ROS to the secondary regulation of ERK activity.

[Posttraumatic changes of rat spinal cord after transplantation of human umbilical cord blood mononuclear cells transfected with VEGF and FGF2 genes].

Using the model of the rat spinal cord dosed contusion injury at T8 level, cross sectional area of the pathological cavities was measured and the number of myelinated nerve fibers was calculated in the outer zones of white matter after immediate single injection in the damaged area of human umbilical cord blood mononuclear cells (UCB-MC) transfected with plasmid with vegf and fgf2 genes. UCB-MC transfected with pEGFP-N2 plasmid with egfp gene of enhanced green fluorescent protein were injected into the rats of control group under similar conditions. By Day 30 after the injection of UCB-MC transfected with vegf and fgf2 genes, total cross-sectional area of the cavities in outer zones of white matter at a distance of 3 mm caudally from the epicenter of the injury was reduced more than twice as compared with that found in control group. Number of myelinated nerve fibers in the same zones of white matter at the same distance from the epicentre in rostral and caudal directions, was increased by 20% on the average as compared with control, and at a distance of 5 mm in rostral direction--by 40 to 70%. Thus, the delivery to the injury region of the therapeutic genes vegf and fgf2 reduced cavitation, restrained the processes of secondary degeneration and supported the number of myelinated fibers in the injured spinal cord.

Pathogenesis, Diagnosis, and Treatment of Hemostatic Disorders in COVID-19 Patients.

The novel coronavirus infection named COVID-19 was first detected in Wuhan, China, in December 2019, and it has been responsible for significant morbidity and mortality in scores of countries. At the time this article was being written, the number of infected and deceased patients continued to grow worldwide. Most patients with severe forms of the disease suffer from pneumonia and pulmonary insufficiency; in many cases, the disease is generalized and causes multiple organ failures and a dysfunction of physiological systems. One of the most serious and prognostically ominous complications from COVID-19 is coagulopathy, in particular, decompensated hypercoagulability with the risk of developing disseminated intravascular coagulation. In most cases, local and diffuse macro- and microthromboses are present, a condition which causes multiple-organ failure and thromboembolic complications. The causes and pathogenic mechanisms of coagulopathy in COVID-19 remain largely unclear, but they are associated with systemic inflammation, including the so-called cytokine storm. Despite the relatively short period of the ongoing pandemic, laboratory signs of serious hemostatic disorders have been identified and measures for specific prevention and correction of thrombosis have been developed. This review discusses the causes of COVID-19 coagulopathies and the associated complications, as well as possible approaches to their early diagnosis, prevention, and treatment.

Isolation and characterization of stem cells derived from human third molar tooth germs of young adults: implications in neo-vascularization, osteo-, adipo- and neurogenesis.

A number of studies have reported in the last decade that human tooth germs contain multipotent cells that give rise to dental and peri-odontal structures. The dental pulp, third molars in particular, have been shown to be a significant stem cell source. In this study, we isolated and characterized human tooth germ stem cells (hTGSCs) from third molars and assessed the expression of developmentally important transcription factors, such as oct4, sox2, klf4, nanog and c-myc, to determine their pluri-potency. Flow-cytometry analysis revealed that hTGSCs were positive for CD73, CD90, CD105 and CD166, but negative for CD34, CD45 and CD133, suggesting that these cells are mesenchymal-like stem cells. Under specific culture conditions, hTGSCs differentiated into osteogenic, adipogenic and neurogenic cells, as well as formed tube-like structures in Matrigel assay. hTGSCs showed significant levels of expression of sox2 and c-myc messenger RNA (mRNA), and a very high level of expression of klf4 mRNA when compared with human embryonic stem cells. This study reports for the first time that hTGSCs express developmentally important transcription factors that could render hTGSCs an attractive candidate for future somatic cell re-programming studies to differentiate germs into various tissue types, such as neurons and vascular structures. In addition, these multipotential hTGSCs could be important stem cell sources for autologous transplantation.