Barnase-barstar Specific Interaction Regulates Car-T Cells Cytotoxic Activity toward Malignancy.

The development of CAR-T specific therapy made a revolution in modern oncology. Despite the pronounced therapeutic effects, this novel approach displayed several crucial limitations caused by the complications in pharmacokinetics and pharmacodynamics controls. The presence of the several severe medical complications of CAR-T therapy initiated a set of attempts aimed to regulate their activity in vivo. We propose to apply the barnase-barstar system to control the cytotoxic antitumor activity of CAR-T cells. To menage the regulation targeting effect of the system we propose to use barstar-modified CAR-T cells together with barnase-based molecules. Barnase was fused with designed ankyrin repeat proteins (DARPins) specific to tumor antigens HER2 (human epidermal growth factor receptor 2) The application of the system demonstrates the pronounced regulatory effects of CAR-T targeting.

A new mouse unilateral model of diffuse alveolar damage of the lung.

OBJECTIVE AND DESIGN: The existing biological models of diffuse alveolar damage (DAD) in mice have many shortcomings. To offset these shortcomings, we have proposed a simple, nonsurgical, and reproducible method of unilateral total damage of the left lung in ICR mice. This model is based on the intrabronchial administration of a mixture of bacterial lipopolysaccharide (LPS) from the cell wall of S. enterica and α-galactosylceramide (inducing substances) to the left lung. METHODS: Using computer tomography of the lungs with endobronchial administration of contrast material, we have been able to perform an operative intravital verification of the targeted delivery of the inducer. The model presented is characterized by more serious and homogeneous damage of the affected lung compared to the existing models of focal pneumonia; at the same time, our model is characterized by longer animal survival since the right lung remains intact. RESULTS: The model is also characterized by diffuse alveolar damage of the left lung, animal survival of 100%, abrupt increases in plasma levels of TNFa, INFg, and IL-6, and significant myocardial overload in the right heart. It can be used to assess the efficacy of innovative drugs for the treatment of DAD and ARDS as the clinical manifestations that are developed in patients infected with SARS-CoV-2. Morphological patterns of lungs in the noninfectious ("sterile") model of DAD induced by LPS simultaneously with α-galactosylceramide (presented here) and in the infectious model of DAD induced by SARS-CoV-2 have been compared. CONCLUSION: The DAD model we have proposed can be widely used for studying the efficacy of candidate molecules for the treatment of infectious respiratory diseases, such as viral pneumonias of different etiology, including SARS-CoV-2.

Influence of electromagnetic waves, with maxima in the green or red range, on the morphofunctional properties of multipotent stem cells.

This paper examines the effect of electromagnetic waves, with maxima in the green or red regions of the spectrum, on the morphofunctional state of multipotent mesenchymal stromal cells. The illumination regimes used in our experiments did not lead to any substantial heating of the samples; the physical parameters of the lighting were carefully monitored. When the samples were illuminated with a green light, no significant photostimulatory effect was observed. Red light, on the other hand, had an evident photostimulatory effect. It is shown that photostimulation with a red light decreases the enzymatic activities of mitochondrial dehydrogenases and enhances the viability of cells, their proliferative activity, and their ability to form bone tissue. It is also established that red light stimulates cell proliferation, while not activating the genes that increase the risk of the subsequent malignant transformation of cells or their death. This paper discusses the possible role of hydrogen peroxide in the processes examined.

Analysis of Immunogenicity of Intracellular CTAR Fragments of Epstein-Barr Virus Latent Phase Protein LMP1.

Intracellular fragments of latent phase protein LMP1 of Epstein-Barr virus, denoted as CTAR1/2/3, can trigger a variety of cell cascades and contribute to the transforming potential of the virus. Generation of recombinant proteins CTAR1/2/3 is expected to yield more ample data on functional and immunogenic characteristics of LMP1. We created genetic constructs for prokaryotic expression of LMP1 CTAR fragments and selected optimal conditions for their production and purification. Using a new library of LMP1 CTAR fragments, we carried out epitope mapping of a diagnostic anti-LMP1 antibody S12. Analysis of polyclonal serum antibodies from mice immunized with full-length LMP1 confirmed immunogenicity of CTAR elements comparable with that of full-length protein.

Analysis of the Effect of ß-Endorphin-Like Peptide Pentarphin on Intracellular Ca²âº Concentration in Early Mouse Embryos Using Fluorescent Probes.

At the early stages of mammalian embryo development, Ca(2+) influences the processes of cleavage and differentiation. An important role in these processes is played by peptide growth factors. One of them, pentarphin, is a peptide growth factor similar to the central part of ß-endorphin molecule and a fragment 369-373 of IgG heavy chain. We used fluorescent Ca(2+) indicators Fluo-3 AM and Fura Red AM. Pentarphin added to embryos in a concentration of 0.1 µM 1.5-2 fold increased intercellular Ca(2+) level in comparison with the initial level.

DTI-MRI at 2 days after spinal cord injury accurately predicts long-term locomotor function recovery in rats.

OBJECTIVE: Spinal cord injury (SCI) damages an individual's sensory, motor, and autonomic functions and represents a social emergency, mostly in developed countries. Accurate and timely diagnosis of the severity of SCI must be carried out as quickly as possible to allow time for drug and therapy testing in the early stages after injury. MATERIALS AND METHODS: Male Dark Agouti (DA) rats underwent spinal cord cryoinjury at the T13 level of the spine. Under typical conditions, in vivo magnetic resonance imaging (MRI) T2 and echo-planar imaging - diffusion tensor imaging (EPI-DTI) examinations were conducted. This involved the reconstruction of nerve tracts and the measurement of the fractional anisotropy (FA) index, as well as measurements of the ratio of Hyper/Hypo intensive areas and spinal cord injury severity scores. RESULTS: Our study shows that, after cryoinjury, the FA significantly decreased in all animals. An increase in FA level, derived from EPI-DTI within 2 days after SCI, accurately predicts long-term locomotor function recovery. In rats with higher FA, recorded on day 2 after injury, complete restoration of locomotor function was observed, while at low FA values, the animals maintained stable monoplegia. CONCLUSIONS: Our results, though validating the T2 10-grade MRI scale for SCI, indicate that FA would represent the MRI technical instrument, which would better monitor the evolution of SCI and, accordingly, better objectively evaluate the impact of potentially therapeutic protocols for spinal cord traumatic injury. Despite the results achieved, significant difficulties must be overcome on the way to successful clinical implementation of the findings in humans.

CSF - injected contrast medium enhances post-traumatic spinal cord cysts. An experimental study in rats.

OBJECTIVE: Spinal cord injury (SCI) is still one of the most challenging problems in neurosurgical practice. One of the major obstacles to neural regeneration following trauma is the formation of glial scarring and post-traumatic cysts which acts against proper growth of axons through the site of injury. Cerebrospinal fluid (CSF) delivery of bioactive agents into cystic cavities could represent a promising therapeutic strategy. In the present study, we investigated specifically the dynamics of intradural delivery of contrast medium and its relocation into post-traumatic cysts in an experimental model of spinal cord cryoinjury in rats. MATERIALS AND METHODS: 32 male Sprague Dawley SPF rats were submitted to injury as previously described. Omnipaque-240 was injected either into the cisterna magna or at the level of the cauda equina. Subsequently, cerebral CT scan examinations were performed in order to check the CSF dynamics of the contrast medium. RESULTS: There was a steady accumulation of contrast medium into post-traumatic cysts as early as five minutes after injection. A dosage of 65 mg of iodine per kilogram ensured an adequate feeling of the cysts at an average of 30 minutes. CONCLUSIONS: Our data indicate that intraspinal injection of bioactive agents can easily reach the site of injury and fill post-traumatic cysts. This could represent an interesting potential therapeutic protocol for SCI.

[Investigation of beta-endorphin nonopioid reception in preimplantation development of mouse embryo in vitro].

The effect of beta-endorphin on 2-, 4- and 8-cell embryo development in vitro was studied. It is shown, that hormone has no effect on 2-cell embryos development, but it has enhanced viability of 4- and 8-cell mouse embryos. The number ofblastocyst formation increases in presence of 0.1 microM beta-endorphin in embryo cultured medium but the number of blastocyst with abnormal structure decreases. The effect of hormone on the change of intracellular concentration of Ca2+ ion in 2-, 4- and 8-cell mouse embryo has been studied with the help of fluorescent microscopy. The effect of adenylate cyclase, and phospholipase activity blockers and opioid blocker naloxone on the change of intracellular concentration of Ca2+ ion in early mouse embryo in the presence of beta-endorphin have been also studied. It is shown that 2-cell embryo has opioid and nonopioid beta-endorphin receptors, whereas 4- and 8-cell mouse embryos have only nonopoioid beta-endorphin receptors. It is also shown that the effect of beta-endorphin in the early mouse embryo through a nonopioid receptors occurs with the participation of intracellular Ca2+ and adenylate cyclase signaling system.

[Alimemazine in treatment of agitation in schizophrenia]. / Primenenie Alimemazina pri kupirovanii vozbuzhdeniya u bol'nykh shizofreniei.

OBJECTIVE: An analysis of the efficacy and safety of additional therapy of excitement with the injectable form of alimemazine during exacerbations of schizophrenia with psychomotor agitation, impulsivity, including dangerous behavior, irritability, conflict, hostility, aggressiveness, anxiety, sleep disturbances (insomnia). MATERIAL AND METHODS: Thirty patients, aged 18 to 65 years, with a diagnosis of «paranoid schizophrenia¼, established in accordance with the ICD-10 criteria, were studied. The patients received treatment with a second-generation antipsychotic and alimemazine (intramuscular injection solution) in daily dose from 25 mg to 150 mg during no more than 9 days. The patients were assessed with psychometric scales (PANSS, ABS, HARS and VAS) four times during the observation period. RESULTS AND CONCLUSION: During therapy with a combination of second-generation antipsychotics and alimemazine solution for intramuscular injection, a reliable (p<0.001) reduction in the severity of psychotic symptoms assessed with PANSS was achieved by 8-9 days (the average total score decreased by 30% relative to the initial level) that indicated the improvement in all manifestations of schizophrenia exacerbation. A decrease of 34.8% (p=0.007) in the risk of aggression (PANSS points S1-S3) was established. The level of excitation on the agitation scale (ABS) decreased by 3.6% (p<0.001). In 50% of patients, manifestations of anxiety disappeared, and the average HARS score decreased by 2.2 times compared with the initial level (p<0.001). Almost half of the patients noted the normalization of sleep, and the average value of sleep disturbance on a visual analogue scale decreased threefold compared with the initial level (p<0.001). The observed adverse events were moderate or mild. Alimemazine shows the highest efficacy in the treatment of anxiety arousal in patients with schizophrenia with affective-delusional attacks.

Cytokine Profile As a Marker of Cell Damage and Immune Dysfunction after Spinal Cord Injury.

This study reviews the findings of recent experiments designed to investigate the cytokine profile after a spinal cord injury. The role played by key cytokines in eliciting the cellular response to trauma was assessed. The results of the specific immunopathogenetic interaction between the nervous and immune systems in the immediate and chronic post-traumatic periods are summarized. It was demonstrated that it is reasonable to use the step-by-step approach to the assessment of the cytokine profile after a spinal cord injury and take into account the combination of the pathogenetic and protective components in implementing the regulatory effects of individual cytokines and their integration into the regenerative processes in the injured spinal cord. This allows one to rationally organize treatment and develop novel drugs.

[Allelic polymorphism of the IL-2and IL-4 genes in herpesvirus infection].

Herpesvirus infection is accompanied by imbalance of peripheral mononuclear production of cytokines, by polarizing an immune response by the Th2 pathway. The homozygous TT genotype of T330G polymorphism in the IL-2 gene is an immunogenetic factor that has a protective activity against susceptibility to herpesvirus infection. The risk of progression, the recurrent course of the infection and its poor outcome is positively associated with the heterozygous TG genotype in the IL-2 gene T-330G promoter region and with the homozygous CC type and C allele in the IL-4 gene C-590T promoter region.

Surgical Simulation of a Posttraumatic Spinal Cord Glial Scar in Rats.

We developed and verified an original, minimally invasive method for surgical simulation of a posttraumatic spinal cord glial scar in rats. The model is intended for use as a biological platform for testing the stimulation of regenerative processes in the central nervous system. Unification of the model enables one to achieve versatility both for implantation techniques and for the development of system-action approaches. Faced with a standard structural defect of the spinal cord, researchers will have the unique opportunity to test in vivo promising methods for spinal function recovery in the posttraumatic period. We developed anesthetic support, surgical tactics, and a set of rehabilitation measures for the chronic postoperative period. Experimental exposure effects were preliminarily assessed in vivo using a standard technique for recording the motor activity of rats in the postoperative period of spinal cord injury. Our final conclusions were drawn based on an analysis of histological sections of the rat spinal cord glial scar in three mutually perpendicular planes.

Influence of wideband visible light with an padding red component on the functional state of mice embryos and embryonic stem cells.

It is known that visible light, including sunlight and laboratory lighting, adversely affect the development of embryos in vitro. In with article we present a technology for the synthesis of composite screens, capable to photoconvert UV and a part of the blue spectrum into red light with the maximum ~630 nm. It is established that the application of such transformed light with an evident red component raises the chances of embryos to survive and protects embryonic stem cells. To create photoconversion screens, the CdZn/Se quantum dots were obtained, the average size being about 7 nm. When the quantum dots are excited by electromagnetic waves of the UV and blue spectral range, photoluminescence is observed. The average photon energy for photoluminescence is of the order of 2 eV. On the basis of CdZn/Se quantum dots and methylphenylsiloxane polymer, light-transforming composite screens were made. In case of the light-transforming composite screen, the UV component disappeared from the energy spectrum, and the intensity of the blue region of the spectrum was reduced. On the contrary, in the red region (λmax = 630 nm) one can see a little more than two-fold increase of intensity. It is shown that when exposed to 2-cell embryos by transformed light, the proportion of normally developing embryos increases by 20%, the number of dead embryos decreases twice, and number of dead and apoptotic cells was lower in blastocysts, what's decreased by 70%, as compared to the control group. When blastocysts are transferred to the feeder substrate, colonies of embryonic stem cells are formed. Cells obtained from blastocysts irradiated with transformed visible light are in a normal state in 90% of cases and did not change expression levels, biochemistry and morphology for at least 20 passages. It is assumed that the data obtained can be used for the design of systems of efficient cultivation of embryonic cells for tissue engineering and cell therapy.

Experimental Models of Spinal Cord Injury in Laboratory Rats.

Pathologies associated with spinal cord injury are some of the leading diseases in the world. The search for new therapeutic agents and 3D biodegradable materials for the recovery of spinal cord functions is a topical issue. In this review, we have summarized the literature data on the most common experimental models of spinal cord injury in laboratory rats and analyzed the experience of using 3D biodegradable materials (scaffolds) in experimental studies of spinal trauma. The advantages and disadvantages of the described models are systematically analyzed in this review.

Studying the Specific Activity of the Amide Form of HLDF-6 Peptide using the Transgenic Model of Alzheimer's Disease.

The neuroprotective and nootropic activities of the amide form (AF) of the HLDF-6 peptide (TGENHR-NH2) were studied in transgenic mice of the B6C3-Tg(APPswe,PSEN1de9)85Dbo (Tg+) line (the animal model of familial Alzheimer's disease (AD)). The study was performed in 4 mouse groups: group 1 (study group): Tg+ mice intranasally injected with the peptide at a dose of 250 µg/kg; group 2 (active control): Tg+ mice intranasally injected with normal saline; group 3 (control 1): Tg- mice; and group 4 (control 2): C57Bl/6 mice. The cognitive functions were evaluated using three tests: the novel object recognition test, the conditioned passive avoidance task, and the Morris water maze. The results testify to the fact that the pharmaceutical substance (PhS) based on the AF of HLDF-6 peptide at a dose of 250 µg/kg administered intranasally efficiently restores the disturbed cognitive functions in transgenic mice. These results are fully consistent with the data obtained in animal models of Alzheimer's disease induced by the injection of the beta-amyloid (ßA) fragment 25-35 into the giant-cell nucleus basalis of Meynert or by co-injection of the ßA fragment 25-35 and ibotenic acid into the hippocampus, and the model of ischemia stroke (chronic bilateral occlusion of carotids, 2VO). According to the overall results, PhS based on AF HLDF-6 was chosen as an object for further investigation; the dose of 250 µg/kg was used as an effective therapeutic dose. Intranasal administration was the route for delivery.

[The surface architectonics, functional characteristics, and metabolism in peripheral blood lymphocytes in acute herpes virus infection]. / Poverkhnostnaia arkhitektonika, funktsional'nye osobennosti i metabolizm limfotsitov perifericheskoi krovi pri khronicheskoi gerpesvirusnoi infektsii.

Twenty-five patients (males--11, females--14; aged 18 to 45) with chronic herpes virus infection relapsing at the time of clinical tests were examined, including investigations of the surface architectonics, the ability to blast-transformation and the metabolic status of peripheral-blood lymphocytes. Chronic herpes virus infection was found to be concurrent with changes of the percentage ratio of lymphocytes with the surface micro-patterns of different types, with a more intense activity of the immunity T-chain and with a changed nature of the intracellular metabolism of lymphocyte cells.