[High Heterogeneity of Virus-Neutralizing and RBD-Binding Activities of COVID-19 Convalescent Sera].

The parameters of the humoral response are an important immunological characteristic of donors who recovered from COVID-19 and vaccinated individuals. Analysis of the level of virus-binding antibodies has become widespread. The most accurate predictor of effective immune protection against symptomatic SARS-CoV-2 infection is the activity of virus-neutralizing antibodies. We determined virus-neutralizing activities in plasma samples of individuals (n = 111) who had COVID-19 from April to September 2020. Three independent methods were used: conventional with live virus, with virus-like particles pseudotyped with spike protein, and a surrogate virus-neutralization test (cVNT, pVNT and sVNT, respectively). For comparison, the levels of IgG, IgA and IgM antibodies against the receptor-binding domain of the SARS-CoV-2 spike protein were also evaluated. The levels of virus-binding as well as virus-neutralizing antibodies in cVNT and pVNT showed high heterogeneity. A comparison of cVNT and pVNT results showed a high correlation, sVNT results also correlated well with both cVNt and pVNT. To the greatest extent, the level of IgG antibodies correlated with the results of cVNT, pVNT and sVNT. These results can be used in the selection of plasmas that are best suited for transfusion and treatment of acute COVID-19. In addition, data on the virus-neutralizing activity of plasma are important for the selection of potential donors, for the isolation of SARS-CoV-2-specific B-lymphocytes, in order to further generate monoclonal virus-neutralizing antibodies.

[Enhanced Natural Killers with CISH and B2M Gene Knockouts Reveal Increased Cytotoxicity in Glioblastoma Primary Cultures].

In an experimental study using the CRISPR/Cas9 system, "enhanced" NK cell lines with knockout of CISH, the gene for the CIS protein (a negative regulator of NK cytotoxicity), as well as two lines with a knocked-out ß2-microglobulin gene, which provides membrane exposure of MHC class I, were obtained from two parental lines of human natural killers (YT wild type and YT-VAV1^(+) overexpressing the VAV1 cytotoxicity enhancing protein). The knockout efficiency was determined by real-time PCR as well as by flow cytometry with specific antibodies. The resulting CISH^(-/-) or B2M^(-/-) knockout lines were tested for cytotoxicity in primary monolayer cultures of human glioblastoma multiforme. The cytotoxicity of the lines was assessed using a cell analyzer that records the cell index based on cell impedance. YT-CISH^(-/-) has been shown to be significantly more effective than wild-type YT in eliminating primary glioblastoma cells in an in vitro cell monolayer experiment. The cytotoxicity of the YT-VAV1^(+)-CISH^(-/-) and YT-VAV1^(+)B2M^(-/-) lines against glioblastoma cells was the highest, but overall, it did not significantly differ from the initially increased cytotoxicity of the YT-VAV1^(+) line. The lines of NK-like cells obtained may serve as a prototype for the creation of "enhanced" allogeneic and autologous NK- and CAR-NK cells for the immunotherapy of glioblastoma multiforme.

[Transcription Factors of Direct Neuronal Reprogramming in Ontogenesis and ex vivo].

Direct reprogramming technology allows several specific types of cells, including specialized neurons, to be obtained from readily available autologous somatic cells. It presents unique opportunities for the development of personalized medicine, from in vitro models of hereditary and degenerative neurological diseases to novel neuroregenerative technologies. Over the past decade, a plethora of protocols for primary reprogramming has been published, yet reproducible generation of homogeneous populations of neuronally reprogrammed cells still remains a challenge. All existing protocols, however, use transcription factors that are involved in embryonic neurogenesis. This is presumably be the key issue for obtaining highly efficient and reproducible protocols for ex vivo neurogenesis. Analysis of the functional features of transcription factors in embryonic and adult neurogenesis may not only lead to the improvement of reprogramming protocols, but also, via cell marker analysis, can exactly determine the stage of neurogenesis that a particular protocol will reach. The purpose of this review is to characterize the general factors that play key roles in neurogenesis for the embryonic and adult periods, as well as in cellular reprogramming, and to assess correspondence of cell forms obtained as a result of cellular reprogramming to the ontogenetic series of the nervous system, from pluripotent stem cells to specialized neurons.

Cell Reprogramming Preserving Epigenetic Age: Advantages and Limitations.

Our understanding of cell aging advanced significantly since the discovery of this phenomenon by Hayflick and Moorhead in 1961. In addition to the well-known shortening of telomeric regions of chromosomes, cell aging is closely associated with changes of the DNA methylation profile. Establishing, maintaining, or reversing epigenetic age of a cell is central to the technology of cell reprogramming. Two distinct approaches - iPSC- and transdifferentiation-based cell reprogramming - affect differently epigenetic age of the cells. The iPSC-based reprogramming protocols are generally believed to result in the reversion of DNA methylation profiles towards less differentiated states, while the original methylation profiles are preserved in the direct trans-differentiation protocols. Clearly, in order to develop adequate model of CNS pathologies, one has to have thorough understanding of the biological roles of DNA methylation in the development, maintenance of functional activity, tissue and cell diversity, restructuring of neural networks during learning, as well as in aging-associated neuronal decline. Direct cell reprogramming is an excellent alternative and a valuable supplement to the iPSC-based technologies both as a source of mature cells for modeling of neurodegenerative diseases, and as a novel powerful strategy for in vivo cell replacement therapy. Further advancement of the regenerative and personalized medicine will strongly depend on optimization of the production of patient-specific autologous cells involving alternative approaches of direct and indirect cell reprogramming that take into account epigenetic age of the starting cell material.

Chemical Reprogramming of Somatic Cells in Neural Direction: Myth or Reality?

In in vitro experiments on cultures of human multipotent stem cells from the human bonearrow and dental pulp, we studied direct reprogramming towards neuro-glial lineage cells using a cocktail of small molecules. Reprogramming by the previously published protocol (with a cocktail containing ß-mercaptoethanol, LIF, VPA, CHIR99021, and RepSox) and by the optimized protocol (VPA, RG108, А83-01, dorsomorphin, thiazovivin, CHIR99021, forskolin, and Isx9) allows obtaining cells with immunophenotypic and genetic signs of neural stem cells. However, neither the former, nor the optimized protocols allowed preparing neural progenitors capable of adequate terminal differentiation from both bone marrow-derived mesenchymal stem cells and nestin-positive neural crest-derived mesenchymal stem cells. Real-time PCR demonstrated the expression of some neurogenesis markers, but neural stem cell-specific expression pattern was not observed. The findings lead us to a conclusion that reprogramming with small molecules without additional factors modifying gene expression does not allow reproducible production of human neural stem cell-like progenitors that can be used as the source of neural tissue for the regenerative therapy.

Internalization of Vectorized Liposomes Loaded with Plasmid DNA in C6 Glioma Cells.

We studied internalization of vector nanocarriers loaded with plasmid DNA into C6 glioma cells. For improving selectivity of plasmid delivery, the liposomes were conjugated with monoclonal antibodies to VEGF and its receptor VEGFR2. Flow cytofluorometry and laser scanning confocal microscopy showed more intensive (more than 2-fold) internalization and accumulation of antibody-vectorized liposomes in C6 glioma cells in comparison with the control (liposomes conjugated with non-specific antibodies and non-vectorized liposomes). Using quantitative analysis of fluorescent signal, we showed that cationic immunoliposomes significantly more effective delivered pCop-Green-N plasmid DNA and ensured effective transfection of C6 glioma cells.

Bioluminescent Study of the Distribution of High-Molecular-Weight Protein Fraction of Cellex Daily Preparation in the Brain after Intranasal Administation.

Permeability of the blood-brain barrier for protein fractions 50-100 kDa (PF50-100) of Cellex Daily preparation labeled with fluorescent tracer FITC and non-conjugated FITC were compared after intranasal administration of the preparations to healthy rats. Fluorimetrical analysis of the serum and cerebrospinal fluid samples showed that Cellex Daily PF50-100-FITC administered intranasally penetrated into the blood and cerebrospinal fluid with maximum accumulation in 2 h after administration and persists in the circulation for 24 h probably due to binding with plasma proteins. The differences in the kinetic profile of PF50-100-FITC and free FITC indirectly suggest that the major part of the preparation is not degraded within 24 h and FITC is probably not cleaved from the protein components of the preparation. In vivo fluorescence analysis showed significant fluorescent signal in the olfactory bulbs in 6 h after intranasal administration; hence, the preparation administered via this route can bypass the blood-brain barrier. Scanning laser confocal microscopy of rat brain sections confirmed penetration of the high-molecular weight protein fraction PF50-100-FITC into CNS structures. The most pronounced accumulation of the labeled drug was observed in the olfactory bulb in 6 and 12 h after administration. In contrast to free FITC administered in the control group, significant accumulation of PF50-100-FITC in the olfactory cortex and frontal cortex neurons with functionally active nuclei was observed in 6, 12 and 24 h after intranasal administration.

Sensitivity of C6 Glioma Cells Carrying the Human Poliovirus Receptor to Oncolytic Polioviruses.

A humanized line of rat C6 glioma cells expressing human poliovirus receptor was obtained and tested for the sensitivity to oncolytic effects of vaccine strains of type 1, 2, and 3 polioviruses. Presentation of the poliovirus receptor on the surface of C6 glioma cells was shown to be a necessary condition for the interaction of cells with polioviruses, but insufficient for complete poliovirus oncolysis.

Antitumor Activity of Rat Mesenchymal Stem Cells during Direct or Indirect Co-Culturing with C6 Glioma Cells.

The tumor-suppressive effect of rat mesenchymal stem cells against low-differentiated rat C6 glioma cells during their direct and indirect co-culturing and during culturing of C6 glioma cells in the medium conditioned by mesenchymal stem cells was studied in an in vitro experiment. The most pronounced antitumor activity of mesenchymal stem cells was observed during direct co-culturing with C6 glioma cells. The number of live C6 glioma cells during indirect co-culturing and during culturing in conditioned medium was slightly higher than during direct co-culturing, but significantly differed from the control (C6 glioma cells cultured in medium conditioned by C6 glioma cells). The cytotoxic effect of medium conditioned by mesenchymal stem cells was not related to medium depletion by glioma cells during their growth. The medium conditioned by other "non-stem" cells (rat astrocytes and fibroblasts) produced no tumor-suppressive effect. Rat mesenchymal stem cells, similar to rat C6 glioma cells express connexin 43, the main astroglial gap junction protein. During co-culturing, mesenchymal stem cells and glioma C6 cells formed functionally active gap junctions. Gap junction blockade with connexon inhibitor carbenoxolone attenuated the antitumor effect observed during direct co-culturing of C6 glioma cells and mesenchymal stem cells to the level produced by conditioned medium. Cell-cell signaling mediated by gap junctions can be a mechanism of the tumor-suppressive effect of mesenchymal stem cells against C6 glioma cells. This phenomenon can be used for the development of new methods of cell therapy for high-grade malignant gliomas.

Mono- and Combined Therapy of Metastasizing Breast Carcinoma 4T1 with Zoledronic Acid and Doxorubicin.

The efficiency of monotherapy with zoledronic acid (Resorba), doxorubicin, and their combination was studied on the model of metastasizing breast carcinoma in BALB/c mice. Doxorubicin monotherapy was accompanied by a significant increase in median survival up to 57 days (vs. 34 and 35 days in control groups); 27% animals survived for 90 days (duration of the study). Bioluminescence area of the primary tumor significantly decreased on days 21 and 28; the total number of visceral metastases also decreased according to magnetic-resonance imaging data. Resorba monotherapy produced no general toxic effect, the median survival increased to 64 days, and 90-day survival was 33%. Imaging techniques (magnetic-resonance imaging, microtomography, bioluminescent analysis) showed that Resorba delayed the development of the primary tumor (regression of luminescence area on days 21 and 28, regression of standardized bioluminescence intensity on day 28) and significantly reduced the number of visceral metastases in comparison with the control. Combination therapy was less effective than monotherapy with the same medications. Median survival was 55 days, 90-day survival was 13%, but magnetic-resonance imaging and bioluminescence analysis after combination therapy also showed delayed growth of the primary tumor and reduced number of visceral metastases. Microtomography revealed bone metastases in ~30% animals of the control group; in experimental groups, no bone metastases were found. The experiment with periosteal (distal epiphysis of the femur) injection of 4T1-Luc2 tumor cells demonstrated pronounced selective effectiveness of Resorba in relation to bone metastases. Monotherapy with Resorba can prevent the development of not only bone, but also visceral metastases of breast cancer.

Internalization of Vectored Liposomes in a Culture of Poorly Differentiated Tumor Cells.

Internalization of liposomal nanocontainers conjugated with monoclonal antibodies to VEGF, VEGFR2 (KDR), and proteins overproduced in the tumor tissue was studied in vitro on cultures of poorly differentiated tumor cells. Comparative analysis of accumulation of vectored liposomes in the tumor cells was performed by evaluating co-localization of labeled containers and cell organelles by laser scanning confocal microscopy. We observed nearly 2 times more active penetration and accumulation of liposomes vectored with antibodies in the tumor cells in comparison with non-vectored liposomes. Selective clathrin-dependent penetration of vectored liposomes into tumor cells was demonstrated by using pharmacological agents inhibiting endocytosis.

Functionally Active Gap Junctions between Connexin 43-Positive Mesenchymal Stem Cells and Glioma Cells.

The formation of functional gap junctions between mesenchymal stem cells and cells of low-grade rat glioma C6 cells was studied in in vitro experiments. Immunocytochemical analysis with antibodies to connexin 43 extracellular loop 2 showed that mesenchymal stem cells as well as C6 glioma cells express the main astroglial gap junction protein connexin 43. Analysis of migration activity showed that mesenchymal stem cells actively migrate towards C6 glioma cells. During co-culturing, mesenchymal stem cells and glioma C6 form functionally active gap junctions mediating the transport of cytoplasmic dye from glioma cells to mesenchymal stem cells in the opposite direction. Fluorometry showed that the intensity of transport of low-molecular substances through heterologous gap junctions between mesenchymal stem cells and glioma cells is similar to that through homologous gap junctions between glioma cells. This phenomenon can be used for the development of new methods of cell therapy of high-grade gliomas.

Modeling and integral X-ray, optical, and MRI visualization of multiorgan metastases of orthotopic 4T1 breast carcinoma in BALB/c mice.

A model of highly metastasizing orthotopic allogeneic breast carcinoma was reproduced and standardized in experiments on BALB/c mice. 4T1 cells characterized by high metastatic activity were transfected with red fluorescent protein (RFP) gene or firefly luciferase (Luc2) gene. Unmodified 4T1 cells and modified 4T1-RFP and 4T1-Luc2 cells were subcutaneously injected to mature female mice into the second mammary fat pads. Quantitative evaluation of the primary node and visceral metastases was performed using magnetic-resonance imaging, X-ray and optical tomography. Modification of 4T1 cells with RFP gene considerably reduced their invasive and metastatic potential and led to spontaneous regression of the primary tumor in 20% cases. Modification of 4T1 cells with Luc2 gene had practically no effect on proliferative, invasive, and metastatic characteristics of the tumor and provided the possibility of quantitative analysis of the primary tumor dynamics by the luminescence intensity. The survival median in mice receiving unmodified 4T1 cells and transfected 4T1-RFP and 4Т1-Luc2 cells was 32, 42, and 38 days, respectively. Neither primary node nor tumor metastases accumulated gadolinium-containing contrast agent and Alasens fluorescent tracer. After implantation of 4T1 and 4Т1-Luc2 cells, multiple metastases were more often detected in the lungs, liver, spleen, spine, and regional lymph nodes and less frequently in the brain, which corresponded to metastasizing profile of human breast cancer. The developed model of orthotopic breast carcinoma 4T1 in BALB/c mice with complex detection of multiple organ metastases using X-ray microCT, optical, and MRI can be recommended for preclinical studies of new antitumor preparations.

Site-directed delivery of VEGF-targeted liposomes into intracranial C6 glioma.

The efficiency of conventional chemotherapy for aggressive tumors in the CNS remains low and new strategies for the targeted delivery of anti-tumor substances are now actively developed. Pegylated liposomes covalently conjugated with monoclonal antibodies to VEGF synthesized by us are nanoparticle characterized by narrow size distribution and high dispersion stability. Immunochemical activity of antibodies after conjugation was 70% of initial level. The anti-VEGF liposomes developed by us were highly specific for VEGF(+) tumor cells (in vitro and in vivo). Intravenous injection of VEGF-liposomes to rats with intracranial C6 glioma was followed by their specific accumulation in the malignant tissues and engulfment by glioma cells, which attested to target delivery and selective accumulation of anti-VEGF-liposomes in the brain tumor. Thus, the use of targeting molecules can significantly increase the distribution and efficiency of delivery of nanocontainers to a tumor characterized by hyperexpression of the target proteins.

[Blood-brain barrier permeability in healthy rats and rats with experimental C6 glioma after fractionated radiotherapy of the brain].

OBJECTIVE: To evaluate the effect of fractionated radiotherapy on permeability of the blood-brain barrier in healthy rats and rats with C6 glioma in vivo. MATERIAL AND METHODS: An increase in BBB permeability in C6 glioma was assessed by dynamic MRI monitoring (glioma size before and after radiation therapy in combination with immunotherapy, n=30) and confocal microscopy (fluorescence imaging of tumor invasion boundaries in a dose-dependent experiment for the amount of injected antibodies). In healthy rats, BBB permeability to macromolecular substances (MMS) was assessed by ELISA (n=23, 192 plasma samples) and confocal microscopy (n=7). RESULTS: It was shown that BBB permeability to biological macromolecules in blood-brain and brain-blood directions was increased after fractionated radiotherapy. CONCLUSION: Drug delivery to the brain can be improved using therapeutic doses of radiation treatment that affects the BBB and minimizes the risk of serious side effects that are often associated with the drug dose.

Generation of recombinant extracellular fragment of vascular endothelial growth factor receptor 2 and specific monoclonal antibodies to this receptor.

cDNA extracellular Ig-like domains I-III of vascular endothelial growth factor receptor 2 (VEGFR2) was cloned in an expressing vector pET_32a. Western blotting showed immunochemical identity of recombinant VEGFR2I-III produced by prokaryotic expression system to the native receptor. BALB/c mice were immunized with VEGFR2I-III for obtaining specific antibodies to VEGFR2. Hybridomas producing monoclonal antibodies were selected by ELISA, Western blotting, and immunocytochemical assay. Thus, we obtained hybridoma producing monoclonal antibodies to VEGFR2 that selectively interact with both recombinant and native extracellular fragment of the receptor.

Treatment of poorly differentiated glioma using a combination of monoclonal antibodies to extracellular connexin-43 fragment, temozolomide, and radiotherapy.

Antitumor efficiencies of monoclonal antibodies to connexin-43 second extracellular loop (MAbE2Cx43), temozolomide, and fractionated γ-irradiation in the monotherapy mode and in several optimized combinations were studied in Wistar rats with induced C6 glioma. The survival of animals with glioma and the dynamics of intracerebral tumor development were evaluated by MRT. Temozolomide monotherapy (200 mg/m(2)) and isolated radiotherapy in a total dose of 36 Gy shifted the survival median from 28 days (no therapy) to 34 and 38 days, respectively; 100% animals died under conditions of temozolomide monotherapy and radiotherapy. Monotherapy with MAbE2Cx43 in a dose of 5 mg/kg led to significant regression of the tumor (according to MRT data), cure of 19.23% animals with glioma, and prolongation of the survival median to 39.5 days after tumor implantation. Combined therapy with MAbE2Cx43 and temozolomide completely abolished the antitumor effect (survival median 29 days). Treatment with MAbE2Cx43 in combination with radiotherapy was associated with mutual boosting of the therapeutic efficiencies, leading to a significant inhibition of tumor development and prolongation of the survival median to 60 days. The mechanism of tumorsuppressive activity of the antibodies could be due to connexon blockade in Cx43-positive glioma cells in the peritumor invasion zone. Higher efficiency of combined therapy was presumably due to the increase in blood-brain barrier permeability for antibodies after irradiation of the brain and to additional inhibitory effect of antibodies towards radioresistant migrating glioma cells. The results suggested that MAbE2Cx43 could be effective as the first-line drug in combined therapy for poorly differentiated gliomas.

Targeted delivery of cisplatin by сonnexin 43 vector nanogels to the focus of experimental glioma C6.

The aim of this study was to create a nanocontainer conjugated with monoclonal antibodies to connexin 43 (Cx43) that is actively expressed at the periphery of C6 glioma and in the astroglia roll zone. Stable vector nanogels with high (up to 35%) cisplatin load were synthesized. The antitumor effects of Cx43-modified cisplatin-loaded nanogels, free cisplatin, and nonspecific drugs were carried out on C6 glioma model. Vector nanogels reduced systemic toxicity of cisplatin, effectively inhibited tumor growth, and significantly prolonged the lifespan of animals with experimental tumors.

[Modern molecular approaches to diagnosis and treatment of high-grade brain gliomas].

The review analyzes the current state of the problem of diagnosis and therapy of high-grade gliomas on the basis of the most promising present-day approaches. The diagnostic and treatment perspectives of the molecular genetic analysis of glioblastoma markers located on the tumor cell surface are considered. Gene therapy and the use of dendritic cells and oncolytic viruses are considered as the most interesting approaches to therapy of high-grade gliomas.

[Connexin-43 antibodies in intraoperative diagnosis of experimental poorly differentiated gliomas].

Fluorescent diagnosis was first proposed in the early XX century and has been used in neurosurgery for about 15 years. The method relies on selective accumulation of strongly fluorescent protoporphyrin IX in tumor cells. Over the past years, the method of intraoperative fluorescence diagnosis has occupied its niche in many neurosurgical clinics around the world and is now used for fast intraoperative diagnosis in brain tumor surgery. However, the efficiency of fluorescent intraoperative diagnosis using 5-aminolevulinic acid is 80-90% and 58.8% for surgery of Grade III-IV and I-II gliomas, respectively. One of the methods to improve the efficiency of fluorescent diagnosis is to use vector systems for delivering fluorescent drugs into the tumor. This paper reports the results of an experimental study of systems for delivering fluorescent agents (protoporphyrin IX, Alexa 488, Alexa 660) using connexin-43 antibodies in rats with transplanted C6 glioma.