Gene Therapy Using Plasmid DNA Encoding VEGF164 and FGF2 Genes: A Novel Treatment of Naturally Occurring Tendinitis and Desmitis in Horses

This clinical study describes the intralesional application of the plasmid DNA encoding two therapeutic species-specific growth factors: vascular endothelial growth factor (VEGF164) and fibroblast growth factor 2 (FGF2) in seven horses to restore naturally occurring injuries of the superficial digital flexor tendon (SDFT) (tendinitis) and in three horses with suspensory ligament branch desmitis. Following application all horses were able to commence a more rapid exercise program in comparison to standardized exercise programs. Clinical observation and ultrasonic imaging was used to evaluate the regeneration rate of the tendon and ligament injury recovery and to confirm the safety of this gene therapy in horses, throughout a 12 month period. Follow-up data of the horses revealed a positive outcome including significant ultrasonographic and clinical improvements in 8 out of 10 horses with SDFT and suspensory ligament branch lesions, with return to their pre-injury level of performance by 2–6 months after the completion of treatment. The ninth horse initially presenting with severe suspensory ligament branch desmopathy, showed no significant ultrasonographic improvements in the first 2 months after treatment, however, it improved clinically and became less lame. The final horse, presenting with severe tendinitis of the SDFT returned to their pre-injury level of performance, but experienced re-injury 6 months after treatment. This data is highly promising, however, further research in experimental models, with the histopathological, immunohistochemical and gene expression evaluation of the equine tendon/ligament after gene therapy application is required in order to fully understand the mechanisms of action. This treatment and the significant clinical impacts observed represents an important advancement in the field of medicine

Spatial patterns and cell surface clusters in perineuronal nets

© 2016 Elsevier B.V.Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29 µm2 in mouse and 1.44 µm2 in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh – with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity

Peripheral blood mono-nuclear cells derived from Alzheimer's disease patients show elevated baseline levels of secreted cytokines but resist stimulation with β-amyloid peptide

Objectives: Among several other factors, the neuro-toxic β-amyloid peptide (βAP)-induced inflammatory mechanisms have also been implicated in the pathogenesis of Alzheimer's dementia (AD). Cytokines have recently emerged as prime candidates underlying this immune reaction. The purpose of this study was to evaluate the inflammatory response of peripheral blood mono-nuclear cells (PBMC) in AD. Design: Cross-sectional (observational) study. Setting: Behavioral and cognitive neurology clinic of the Universidade Federal de Minas Gerais in Belo Horizonte, Brazil. Participants: AD patients (n = 19), healthy elderly (n = 19) and young (n = 14) individuals. Measurements: Cytokine levels were assessed by enzyme-linked immuno-sorbent assay (ELISA) after exposing cells to a broad range of βAP concentrations (10 -4-10 -10M) as a stimulus. AD samples were weighed against leukocytes harvested from non-demented young and elderly subjects. Results: Cytokine production of PBMCs in the youth was characterized by low baseline levels when compared to cells from the older generation. In the aging population, AD cells were distinguished from the healthy elderly sub-group by an even higher basal cytokine secretion. The low resting concentration in young individuals was markedly increased after treatment with βAP, however cells from the elderly, irrespective of their disease status, showed unchanged cytokine release following βAP administration. Non-specific activation of PBMCs with anti-CD3/CD28 antibodies resulted in elevated interleukin (IL)-1β concentrations in AD. Conclusions: These results demonstrate a general over-production of cytokines and resistance to βAP in the old comparison group, with a more pronounced disruption/boosted pattern in AD. Our findings are in line with the hypothesis of "inflammaging", i.e. an enhanced inflammatory profile with normal aging and a further perturbed environment in AD. The observed cytokine profiles may serve as diagnostic biomarkers in dementia. © 2011 Elsevier Inc

Proangiogenic Effect of 2A-Peptide Based Multicistronic Recombinant Constructs Encoding VEGF and FGF2 Growth Factors

Coronary artery disease remains one of the primary healthcare problems due to the high cost of treatment, increased number of patients, poor clinical outcomes, and lack of effective therapy. Though pharmacological and surgical treatments positively affect symptoms and arrest the disease progression, they generally exhibit a limited effect on the disease outcome. The development of alternative therapeutic approaches towards ischemic disease treatment, especially of decompensated forms, is therefore relevant. Therapeutic angiogenesis, stimulated by various cytokines, chemokines, and growth factors, provides the possibility of restoring functional blood flow in ischemic tissues, thereby ensuring the regeneration of the damaged area. In the current study, based on the clinically approved plasmid vector pVax1, multigenic constructs were developed encoding vascular endothelial growth factor (VEGF), fibroblast growth factors (FGF2), and the DsRed fluorescent protein, integrated via picornaviruses’ furin-2A peptide sequences. In vitro experiments demonstrated that genetically modified cells with engineered plasmid constructs expressed the target proteins. Overexpression of VEGF and FGF2 resulted in increased levels of the recombinant proteins. Concomitantly, these did not lead to a significant shift in the general secretory profile of modified HEK293T cells. Simultaneously, the secretome of genetically modified cells showed significant stimulating effects on the formation of capillary-like structures by HUVEC (endothelial cells) in vitro. Our results revealed that when the multicistronic multigene vectors encoding 2A peptide sequences are created, transient transgene co-expression is ensured. The results obtained indicated the mutual synergistic effects of the growth factors VEGF and FGF2 on the proliferation of endothelial cells in vitro. Thus, recombinant multicistronic multigenic constructs might serve as a promising approach for establishing safe and effective systems to treat ischemic diseases

Онлайн-курс по первой помощи при критических состояниях на международной платформе ˝Coursera˝

The pandemic of the novel coronavirus infection (COVID-19) has had an enormous impact on the education system. Higher educational institutions were forced to adapt to remote interaction with students and urgently switch to online learning. Currently, there are many educational platforms, among which one of the leading is the Californian Coursera service. In 2020, Kazan Federal University developed and published the first Coursera course on first aid — “Staying Alive! First Aid in Emergency”. The purpose of the current study was to demonstrate the course results in one calendar year. We analyzed the general indicators of Coursera — the global online learning platform. We reviewed the course indicators from December 21, 2020, to December 21, 2021. The research assesses the advantages and disadvantages of using online courses in the medical education system. It was concluded that massive open online courses effectively teach theoretical disciplines. In case of implementation of applied courses, it is necessary to search and introduce new approaches for practical skill development.Пандемия новой коронавирусной инфекции COVID-19 оказала чрезвычайное влияние на систему образования. Высшие учебные заведения были вынуждены адаптироваться к удаленному взаимодействию с обучающимися и в ургентном порядке перейти на систему онлайн-обучения. В настоящее время функционирует множество образовательных платформ, среди которых одним из ведущих является калифорнийский сервис ˝Сoursera˝. В 2020 году Казанский федеральный университет разработал и опубликовал первый на ˝Сoursera˝ курс на тему оказания первой помощи пострадавшим — ˝Staying Alive! First Aid in Emergency˝ (Остаться в живых! Первая помощь в неотложной ситуации). Целью данного исследования явилась демонстрация результатов реализации курса за один календарный год. Проанализированы общие показатели международной платформы онлайн-образования ˝Сoursera˝. Проведен аналитический обзор показателей курса за период с 21 декабря 2020 по 21 декабря 2021 года. Дана оценка преимуществам и недостаткам использования онлайн-курсов в системе медицинского образования. Сделан вывод о том, что массовые открытые онлайн-курсы достаточно эффективны при преподавании теоретических дисциплин. В случае реализации прикладных курсов необходим поиск и внедрение новых подходов для освоения обучающимися практических навыков

Intestinal mucosal lymphocytes in neonatal sepsis

© 2019 National Academy of Pediatric Science and Innovation. All rights reserved. We studied the autopsy material obtained from 7 children who died in the neonatal period in order to evaluate the composition of lymphocytes of the intestinal mucosa against the background of morphological changes in the tissues of the gastrointestinal tract in newborns with sepsis. The main group consisted of 4 children with neonatal sepsis, the control group – of 3 newborns who died from other causes. The research material included the specimen of the small and large intestine. Results. Small intestine: it was found that there were less CD4 + lymphocytes in the small intestinal mucosa in the group of children who died from neonatal sepsis in 75% of cases than in the control group, but this difference was not statistically significant (p=0.1). There were no differences in the number of CD8 + and CD20 + cells in the studied groups. Large intestine: the number of CD4 + lymphocytes of the mucous membrane of the colon was greater in the main group of children than in the control group (p=0.03). An increase in the number of CD4 + cells was registered in 3 of 4 cases of neonatal sepsis. The number of CD8+ and CD20+ lymphocytes in the studied groups was the same (р>0.05). Conclusion. The increase in T-lymphocytes CD4+ in the mucous membrane of the large intestine is probably connected with the antigenic stimulation of opportunistic intestinal bacteria. We found no morphological signs of the suppression of the cells of adaptive immunity associated with the intestinal mucosa

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

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. 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). 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. 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

Intestinal mucosal lymphocytes in neonatal sepsis

We studied the autopsy material obtained from 7 children who died in the neonatal period in order to evaluate the composition of lymphocytes of the intestinal mucosa against the background of morphological changes in the tissues of the gastrointestinal tract in newborns with sepsis. The main group consisted of 4 children with neonatal sepsis, the control group – of 3 newborns who died from other causes. The research material included the specimen of the small and large intestine.Results. Small intestine: it was found that there were less CD4 + lymphocytes in the small intestinal mucosa in the group of children who died from neonatal sepsis in 75% of cases than in the control group, but this difference was not statistically significant (p=0.1). There were no differences in the number of CD8 + and CD20 + cells in the studied groups. Large intestine: the number of CD4 + lymphocytes of the mucous membrane of the colon was greater in the main group of children than in the control group (p=0.03). An increase in the number of CD4 + cells was registered in 3 of 4 cases of neonatal sepsis. The number of CD8+ and CD20+ lymphocytes in the studied groups was the same (р>0.05).Conclusion. The increase in T-lymphocytes CD4+ in the mucous membrane of the large intestine is probably connected with the antigenic stimulation of opportunistic intestinal bacteria. We found no morphological signs of the suppression of the cells of adaptive immunity associated with the intestinal mucosa

Intestinal mucosal lymphocytes in neonatal sepsis

© 2019 National Academy of Pediatric Science and Innovation. All rights reserved. We studied the autopsy material obtained from 7 children who died in the neonatal period in order to evaluate the composition of lymphocytes of the intestinal mucosa against the background of morphological changes in the tissues of the gastrointestinal tract in newborns with sepsis. The main group consisted of 4 children with neonatal sepsis, the control group – of 3 newborns who died from other causes. The research material included the specimen of the small and large intestine. Results. Small intestine: it was found that there were less CD4 + lymphocytes in the small intestinal mucosa in the group of children who died from neonatal sepsis in 75% of cases than in the control group, but this difference was not statistically significant (p=0.1). There were no differences in the number of CD8 + and CD20 + cells in the studied groups. Large intestine: the number of CD4 + lymphocytes of the mucous membrane of the colon was greater in the main group of children than in the control group (p=0.03). An increase in the number of CD4 + cells was registered in 3 of 4 cases of neonatal sepsis. The number of CD8+ and CD20+ lymphocytes in the studied groups was the same (р>0.05). Conclusion. The increase in T-lymphocytes CD4+ in the mucous membrane of the large intestine is probably connected with the antigenic stimulation of opportunistic intestinal bacteria. We found no morphological signs of the suppression of the cells of adaptive immunity associated with the intestinal mucosa

Spatial patterns and cell surface clusters in perineuronal nets

© 2016 Elsevier B.V.Perineuronal nets (PNN) ensheath GABAergic and glutamatergic synapses on neuronal cell surface in the central nervous system (CNS), have neuroprotective effect in animal models of Alzheimer disease and regulate synaptic plasticity during development and regeneration. Crucial insights were obtained recently concerning molecular composition and physiological importance of PNN but the microstructure of the network remains largely unstudied. Here we used histochemistry, fluorescent microscopy and quantitative image analysis to study the PNN structure in adult mouse and rat neurons from layers IV and VI of the somatosensory cortex. Vast majority of meshes have quadrangle, pentagon or hexagon shape with mean mesh area of 1.29 µm2 in mouse and 1.44 µm2 in rat neurons. We demonstrate two distinct patterns of chondroitin sulfate distribution within a single mesh – with uniform (nonpolar) and node-enriched (polar) distribution of the Wisteria floribunda agglutinin-positive signal. Vertices of the node-enriched pattern match better with local maxima of chondroitin sulfate density as compared to the uniform pattern. PNN is organized into clusters of meshes with distinct morphologies on the neuronal cell surface. Our findings suggest the role for the PNN microstructure in the synaptic transduction and plasticity