Сytotoxic effect of CAR-T cells against modified MCF-7 breast cancer cell line.

The most often diagnosed and fatal malignancy in women is breast cancer. The International Agency for Research on Cancer (IARC) estimates that there are 2.26 million new cases of cancer in 2020. Adoptive cell therapy using T cells with chimeric antigen receptor shows potential for the treatment of solid tumors, such as breast cancer. In this work the effectiveness of CAR-T cells against monolayer and three-dimensional bioprinted tumor-like structures made of modified MCF-7 breast cancer cells was assessed. The cytokine profile of supernatants after co-cultivation of MCF-7 tumor cell models with CAR-T cells was also measured to reveal the inflammatory background associated with this interaction.

Severity- and Time-Dependent Activation of Microglia in Spinal Cord Injury.

A spinal cord injury (SCI) initiates a number of cascades of biochemical reactions and intercellular interactions, the outcome of which determines the regenerative potential of the nervous tissue and opens up capacities for preserving its functions. The key elements of the above-mentioned processes are microglia. Many assumptions have been put forward, and the first evidence has been obtained, suggesting that, depending on the severity of SCI and the post-traumatic period, microglia behave differently. In this regard, we conducted a study to assess the microglia behavior in the model of mild, moderate and severe SCI in vitro for various post-traumatic periods. We reported for the first time that microglia make a significant contribution to both anti- and pro-inflammatory patterns for a prolonged period after severe SCI (60 dpi), while reduced severities of SCI do not lead to prolonged activation of microglia. The study also revealed the following trend: the greater the severity of the SCI, the lower the proliferative and phagocytic activity of microglia, which is true for all post-traumatic periods of SCI.

Induction of Angiogenesis by Genetically Modified Human Umbilical Cord Blood Mononuclear Cells.

Stimulating the process of angiogenesis in treating ischemia-related diseases is an urgent task for modern medicine, which can be achieved through the use of different cell types. Umbilical cord blood (UCB) continues to be one of the attractive cell sources for transplantation. The goal of this study was to investigate the role and therapeutic potential of gene-engineered umbilical cord blood mononuclear cells (UCB-MC) as a forward-looking strategy for the activation of angiogenesis. Adenovirus constructs Ad-VEGF, Ad-FGF2, Ad-SDF1α, and Ad-EGFP were synthesized and used for cell modification. UCB-MCs were isolated from UCB and transduced with adenoviral vectors. As part of our in vitro experiments, we evaluated the efficiency of transfection, the expression of recombinant genes, and the secretome profile. Later, we applied an in vivo Matrigel plug assay to assess engineered UCB-MC's angiogenic potential. We conclude that hUCB-MCs can be efficiently modified simultaneously with several adenoviral vectors. Modified UCB-MCs overexpress recombinant genes and proteins. Genetic modification of cells with recombinant adenoviruses does not affect the profile of secreted pro- and anti-inflammatory cytokines, chemokines, and growth factors, except for an increase in the synthesis of recombinant proteins. hUCB-MCs genetically modified with therapeutic genes induced the formation of new vessels. An increase in the expression of endothelial cells marker (CD31) was revealed, which correlated with the data of visual examination and histological analysis. The present study demonstrates that gene-engineered UCB-MC can be used to stimulate angiogenesis and possibly treat cardiovascular disease and diabetic cardiomyopathy.

Evaluation of CAR-T Cells' Cytotoxicity against Modified Solid Tumor Cell Lines.

In recent years, adoptive cell therapy has gained a new perspective of application due to the development of technologies and the successful clinical use of CAR-T cells for the treatment of patients with malignant B-cell neoplasms. However, the efficacy of CAR-T therapy against solid tumor remains a major scientific and clinical challenge. In this work, we evaluated the cytotoxicity of 2nd generation CAR-T cells against modified solid tumors cell lines-lung adenocarcinoma cell line H522, prostate carcinoma PC-3M, breast carcinoma MDA-MB-231, and epidermoid carcinoma A431 cell lines transduced with lentiviruses encoding red fluorescent protein Katushka2S and the CD19 antigen. A correlation was demonstrated between an increase in the secretion of proinflammatory cytokines and a decrease in the confluence of tumor cells' monolayer. The proposed approach can potentially be applied to preliminarily assess CAR-T cell efficacy for the treatment of solid tumors and estimate the risks of developing cytokine release syndrome.

Characteristics and Resistance to Cisplatin of Human Neuroblastoma Cells Co-Cultivated with Immune and Stromal Cells.

We investigated the features of the morphology and cytokine profiles of neuroblastoma SH-SY5Y cells, bone marrow-derived mesenchymal stromal/stem cells (BM-MSCs), and peripheral blood mononuclear cells (PBMCs) in double (BM-MSCs + SH-SY5Y cells) and triple (BM-MSCs + SH-SY5Y cells + PBMCs) co-cultures incubated on plastic and Matrigel. Cells in the co-cultures communicated by vesicular transport and by exchanging membrane and cytoplasmic components. The cytokine profile of double and triple co-cultures incubated on Matrigel and plastic had differences and showed the highest concentration of a number of chemokines/cytokines, such as CXCL8/IL-8, I-TAC/CXCL11, IP10/CXCL10, MDC/CCL22, MIP-1α/CCL3, IL-1ß, ENA-78/CXCL5, Gro-α/CXCL1, MCP-1/CCL2, TERC/CCL25, CXCL8/IL-8, and IL-6. High concentrations of inflammatory chemokines/cytokines in the conditioned medium of triple co-culture form a chronic inflammation, which brings the presented co-cultivation system closer to a natural tumor. Triple co-cultures were more resistant to cisplatin (CDDP) than the double- and monoculture of SH-SY5Y. The mRNA levels of BCL2, BCL2L1, RAC1, CAV1, CASP3, and BAX genes were changed in cells after co-culturing and CDDP treatment in double and triple co-cultures. The expression of the BCL2, BAX, CAV1, and CASP3 proteins in SH-SY5Y cells after the triple co-culture and CAV1 and BAX protein expression in SH-SY5Y cells after the double co-culture were determined. This study demonstrated the nature of the cellular interactions between components of tumor niche and the intercellular influence on chemoresistance observed in our tumor model, which should enable the development of novel test systems for anti-tumor agents.

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.

Rab GTPase Mediating Regulation of NALP3 in Colorectal Cancer.

The NALP3 inflammasome signaling contributes to inflammation within tumor tissues. This inflammation may be promoted by the vesicle trafficking of inflammasome components and cytokines. Rab5, Rab7 and Rab11 regulate vesicle trafficking. However, the role of these proteins in the regulation of inflammasomes remains largely unknown. To elucidate the role of these Rab proteins in inflammasome regulation, HCT-116, a colorectal cancer (CRC) cell line expressing pDsRed-Rab5 wild type (WT), pDsRed-Rab5 dominant-negative (DN), pDsRed-Rab7 WT, pDsRed-Rab7 DN, pDsRed-Rab11 WT and pDsRed-Rab11 DN were treated with lipopolysaccharide (LPS)/nigericin. Inflammasome activation was analyzed by measuring the mRNA expression of NLRP3, Pro-CASP1, RAB39A and Pro-IL-1ß, conducting immunofluorescence imaging and western blotting of caspase-1 and analysing the secretion levels of IL-1ß using enzyme-linked immunosorbent assay (ELISA). The effects of Rabs on cytokine release were evaluated using MILLIPLEX MAP Human Cytokine/Chemokine Magnetic Bead Panel-Premixed 41 Plex. The findings showed that LPS/nigericin-treated cells expressing Rab5-WT indicated increased NALP3 expression and secretion of the IL-1ß as compared to Rab5-DN cells. Caspase-1 was localized in the nucleus and cytosol of Rab5-WT cells but was localized in the cytosol in Rab5-DN cells. There were no any effects of Rab7 and Rab11 expression on the regulation of inflammasomes. Our results suggest that Rab5 may be a potential target for the regulation of NALP3 in the treatment of the CRC inflammation.

Improving Culture Conditions, Proliferation, and Migration of Porcine Mesenchymal Stem Cells on Spinal Cord Contusion Injury Model in vitro.

Adipose tissue-derived mesenchymal stem cells (AD-MSCs) are promising for cell therapy in spinal cord injury (SCI). The pig is one of the most approximate models of many human diseases, including SCI. In our study, we selected the optimal conditions for the culture of porcine AD-MSCs and developed an in vitro SCI model based on the culture of cells in injured spinal cord extracts (SCE) 3 days and 6 weeks after SCI. We show that Dulbecco's Modified Eagle Medium (DMEM) with 20% serum content, supplemented with a combination of 5 mM L-ascorbate-2-phosphate and nonessential amino acids, stimulated a typical fibroblast-like morphology and high proliferation of porcine AD-MSCs. SCE caused a higher proliferation of porcine AD-MSCs compared with extracts from an intact spinal cord. The optimal proliferating effect was achieved using rostral 3 days SCE, and proliferation was lower in caudal and central SCE. Porcine AD-MSCs migration to the 3 days and 6 weeks SCE was higher than to an intact one and preferred the rostral SCE, avoiding central and caudal SCE. We also studied 13 cytokines contained in SCE but did not observe any definite relationship between some analyte concentrations and a change in the behavior of AD-MSCs.

Cytokine Storm Combined with Humoral Immune Response Defect in Fatal Hemorrhagic Fever with Renal Syndrome Case, Tatarstan, Russia.

Hemorrhagic fever with renal syndrome (HFRS) is endemic in Tatarstan, where thousands of cases are registered annually. Puumalaorthohantavirus is commonly detected in human case samples as well as in captured bank voles, the rodent hosts. The pathogenesis of HFRS is still not well described, although the cytokine storm hypothesis is largely accepted. In this study, we present a comprehensive analysis of a fatal HFRS case compared with twenty four non-fatal cases where activation of the humoral and cellular immune responses, pro-inflammatory cytokines and disturbed blood coagulation were detected using immunological, histological, genetic and clinical approaches. Multiple organ failure combined with disseminated intravascular coagulation syndrome and acute renal failure was the cause of death. Decreased Interleukin (IL)-7 and increased IL-18, chemokine (C-C motif) ligand (CCL)-5, stem cell growth factor (SCGF)-b and tumor necrosis factor-beta (TNF-ß) serum levels were found, supporting the cytokine storm hypothesis of hantavirus pathogenesis.

Use of Biologically Active 3D Matrix for Extensive Skin Defect Treatment in Veterinary Practice: Case Report.

Objectives: Large full-thickness skin defects represent a serious veterinary problem. Methods: We have developed novel bioactive 3D-matrixes based on fibrin glue Tissucol (Baxter), containing the combination of the adenoviral constructs with genes vascular endothelial growth factor 165 (VEGF165) and fibroblast growth factor two (FGF2; construct Ad5-VEGF165 + Ad5-FGF2) or multipotent mesenchymal stem cells, genetically modified with these constructs. Results: In vitro studies confirmed the biosynthesis of VEGF165 and FGF2 mRNA in the transduced cells. Ad5-VEGF165 + Ad5-FGF2- transduced multipotent mesenchymal stem cells showed an enhanced capacity to form capillary-like tubes in vitro. Bioactive 3D-matrix application enhanced granulation tissue formation and epithelialization of non-healing, large bite wounds in a dog. Successful wound healing was observed with a positive clinical outcome for the canine patient. This research and application of regenerative gene therapy alongside a novel bioactive 3D-matrix shows promising clinical applications for the future in both dogs and other mammals including humans.

Adipose-Derived Mesenchymal Stem Cells Applied in Fibrin Glue Stimulate Peripheral Nerve Regeneration.

Mesenchymal stem cells (MSCs) hold a great promise for cell therapy. To date, they represent one of the best choices for the treatment of post-traumatic injuries of the peripheral nervous system. Although autologous can be easily transplanted in the injured area, clinical advances in this filed have been impaired by lack of preservation of graft cells into the injury area after transplantation. Indeed, cell viability is not retained after injection into the blood stream, and cells injected directly into the area of injury either are washed off or inhibit regeneration through scar formation and neuroma development. This study proposes a new way of MSCs delivery to the area of traumatic injury by using fibrin glue, which not only fixes cells at the site of application but also provides extracellular matrix support. Using a sciatic nerve injury model, MSC derived from adipose tissue embedded in fibrin glue were able to enter the nerve and migrate mainly retrogradely after transplantation. They also demonstrated a neuroprotective effect on DRG L5 sensory neurons and stimulated axon growth and myelination. Post-traumatic changes of the sensory neuron phenotype were also improved. Importantly, MSCs stimulated nerve angiogenesis and motor function recovery. Therefore, our data suggest that MSC therapy using fibrin glue is a safe and efficient method of cell transplantation in cases of sciatic nerve injury, and that this method of delivery of regeneration stimulants could be beneficial for the successful treatment of other central and peripheral nervous system conditions.

Angiogenic Activity of Cytochalasin B-Induced Membrane Vesicles of Human Mesenchymal Stem Cells.

: The cytochalasin B-induced membrane vesicles (CIMVs) are suggested to be used as a vehicle for the delivery of therapeutics. However, the angiogenic activity and therapeutic potential of human mesenchymal stem/stromal cells (MSCs) derived CIMVs (CIMVs-MSCs) remains unknown. OBJECTIVES: The objectives of this study were to analyze the morphology, size distribution, molecular composition, and angiogenic properties of CIMVs-MSCs. METHODS: The morphology of CIMVs-MSC was analyzed by scanning electron microscopy. The proteomic analysis, multiplex analysis, and immunostaining were used to characterize the molecular composition of the CIMVs-MSCs. The transfer of surface proteins from a donor to a recipient cell mediated by CIMVs-MSCs was demonstrated using immunostaining and confocal microscopy. The angiogenic potential of CIMVs-MSCs was evaluated using an in vivo approach of subcutaneous implantation of CIMVs-MSCs in mixture with Matrigel matrix. RESULTS: Human CIMVs-MSCs retain parental MSCs content, such as growth factors, cytokines, and chemokines: EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, GM-CSF, Fractalkine, IFNα2, IFN-γ, GRO, IL-10, MCP-3, IL-12p40, MDC, IL-12p70, IL-15, sCD40L, IL-17A, IL-1RA, IL-1a, IL-9, IL-1b, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP_1a, MIP-1b, TNF-α, TNF-ß, VEGF. CIMVs-MSCs also have the expression of surface receptors similar to those in parental human MSCs (CD90+, CD29+, CD44+, CD73+). Additionally, CIMVs-MSCs could transfer membrane receptors to the surfaces of target cells in vitro. Finally, CIMVs-MSCs can induce angiogenesis in vivo after subcutaneous injection into adult rats. CONCLUSIONS: Human CIMVs-MSCs have similar content, immunophenotype, and angiogenic activity to those of the parental MSCs. Therefore, we believe that human CIMVs-MSCs could be used for cell free therapy of degenerative diseases.

In vitro assessment of electrospun polyamide-6 scaffolds for esophageal tissue engineering.

Artificial tissue-engineered grafts offer a potential alternative to autologous tissue grafts for patients, which can be traumatic. After decellularizing Papio hamadryas esophagus and studying the morphology and physical properties of the extracellular matrix (ECM), we generated electrospun polyamide-6 based scaffolds to mimic it. The scaffolds supported a greater mechanical load than the native ECM and demonstrated similar 3D microstructure, with randomly aligned fibers, 90% porosity, 29 µm maximal pore size, and average fiber diameter of 2.87 ± 0.95 µm. Biocompatibility studies showed that human adipose- and bone marrow-derived mesenchymal stromal cells (AD-MSC and BMD-MSC) adhered to the scaffold surface and showed some proliferation: scaffold cell coverage was 25% after 72 h of incubation when seeded with 1000 cells/mm2 ; cells elongated processes along the polyamide-6, although they flattened 1.67-4 times less than on cell culture plastic. Human umbilical vein endothelial cells, however, showed poor adherence and proliferation. We thus provide in vitro evidence that polyamide-6 scaffolds approximating the esophageal biomechanics and 3D topography of nonhuman primates may provide a biocompatible substrate for both AD-MSC and BMD-MSCs, supporting their adhesion and survival to some degree. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 253-268, 2019.

Transplantation of Microglia in the Area of Spinal Cord Injury in an Acute Period Increases Tissue Sparing, but Not Functional Recovery.

Microglial cells are known as important mediators of inflammation and immune response in the central nervous system (CNS). However, a neuroprotective role of these cells in post-traumatic processes should not be overlooked. Microglial cells are the first to respond to CNS injury and are further involved in all critical events of pathogenesis. When activated microglia clear the cellular debris and release anti- and proinflammatory cytokines and chemokines, nitric oxide, neurotrophins, and antioxidants capable of producing both neurotoxic and neuroprotective effects. The aim of this study was to determine to what extent the phagocytic activity of microglia in an acute period of spinal cord injury (SCI) in rats can effect the post-traumatic processes. For this purpose we implanted genetically modified Ad5-EGFP or Ad5-GDNF microglial cells into the area of acute SCI. Our experiments demonstrate that the area of intact tissue was lower in the group transplanted with Ad5-GDNF-transduced microglial cells with reduced phagocytic activity than that in the group of animals transplanted with Ad5-EGFP-transduced microglia cells which did not affect the cell activity. At the same time, there was no significant difference in the functional recovery index between these groups. Thus, the increased number of microglia cells with good phagocytic activity in the area of acute SCI may contribute to the improved nervous tissue integrity without a significant effect on the functional recovery within 30 days after injury.

Cytochalasin B-induced membrane vesicles convey angiogenic activity of parental cells.

Naturally occurring extracellular vesicles (EVs) play essential roles in intracellular communication and delivery of bioactive molecules. Therefore it has been suggested that EVs could be used for delivery of therapeutics. However, to date the therapeutic application of EVs has been limited by number of factors, including limited yield and full understanding of their biological activities. To address these issues, we analyzed the morphology, molecular composition, fusion capacity and biological activity of Cytochalasin B-induced membrane vesicles (CIMVs). The size of these vesicles was comparable to that of naturally occurring EVs. In addition, we have shown that CIMVs from human SH-SY5Y cells contain elevated levels of VEGF as compared to the parental cells, and stimulate angiogenesis in vitro and in vivo.

Human adipose-derived stem cells stimulate neuroregeneration.

Traumatic brain injuries and degenerative neurological disorders such as Alzheimer's dementia, Parkinson's disease, amyotrophic lateral sclerosis and many others are characterized by loss of brain cells and supporting structures. Restoring microanatomy and function using stem cells is a promising therapeutic approach. Among the many various sources, adipose-derived stem cells (ADSCs) are one of the most easily harvested alternatives, they multiply rapidly, and they demonstrate low immunogenicity with an ability to differentiate into several cell types. The objective of this study was to evaluate the effect of xenotransplanted human ADSCs on post-traumatic regeneration of rat sciatic nerve. Peripheral reconstruction following complete sciatic transection and autonerve grafting was complemented by intra-operative injection of hADSCs into the proximal and distal stumps. The injury caused gliosis and apoptosis of sensory neurons in the lumbar 5 (L5) ganglia in the control rodents; however, animals treated with hADSCs demonstrated a smaller amount of cellular loss. Formation of amputation neuroma, which hinders axonal repair, was less prominent in the experimental group, and immunohistochemical analysis of myelin basic protein showed good myelination 65 days after surgery. At this point, control groups still exhibited high levels of microglia/macrophage-specific marker Iba-1 and proliferating cell nuclear antigen, the mark of an ongoing inflammation and incomplete axonal growth 2 months after the injury. This report demonstrates that hADSCs promote neuronal survival in the spinal ganglion, fuel axonal repair and stimulate the regeneration of peripheral nerves.