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.

COVID-19 in Russia: Clinical and Immunological Features of the First-Wave Patients.

The coronavirus disease outbreak in 2019 (COVID-19) has now achieved the level of a global pandemic and affected more than 100 million people on all five continents and caused over 2 million deaths. Russia is, needless to say, among the countries affected by SARS-CoV-2, and its health authorities have mobilized significant efforts and resources to fight the disease. The paper presents the result of a functional analysis of 155 patients in the Moscow Region who were examined at the Central Clinical Hospital of the Russian Academy of Sciences during the first wave of the pandemic (February-July, 2020). The inclusion criteria were a positive PCR test and typical, computed tomographic findings of viral pneumonia in the form of ground-glass opacities. A clinical correlation analysis was performed in four groups of patients: (1) those who were not on mechanical ventilation, (2) those who were on mechanical ventilation, and (3) those who subsequently recovered or (4) died. The correlation analysis also considered confounding comorbidities (diabetes, metabolic syndrome, hypertension, etc.). The immunological status of the patients was examined (levels of immunoglobulins of the M, A, G classes and their subclasses, as well as the total immunoglobulin level) using an original SARS-CoV-2 antibody ELISA kit. The ELISA kit was developed using linear S-protein RBD-SD1 and NTD fragments, as well as the N-protein, as antigens. These antigens were produced in the prokaryotic E. coli system. Recombinant RBD produced in the eukaryotic CHO system (RBD CHO) was used as an antigen representing conformational RBD epitopes. The immunoglobulin A level was found to be the earliest serological criterion for the development of a SARS-CoV-2 infection and it yielded the best sensitivity and diagnostic significance of ELISA compared to that of class M immunoglobulin. We demonstrated that the seroconversion rate of "early" N-protein-specific IgM and IgA antibodies is comparable to that of antibodies specific to RBD conformational epitopes. At the same time, seroconversion of SARS-CoV-2 N-protein-specific class G immunoglobulins was significantly faster compared to that of other specific antibodies. Our findings suggest that the strong immunogenicity of the RBD fragment is for the most part associated with its conformational epitopes, while the linear RBD and NTD epitopes have the least immunogenicity. An analysis of the occurrence rate of SARS-CoV-2-specific immunoglobulins of different classes revealed that RBD- and N-specific antibodies should be evaluated in parallel to improve the sensitivity of ELISA. An analysis of the immunoglobulin subclass distribution in sera of seropositive patients revealed uniform induction of N-protein-specific IgG subclasses G1-G4 and IgA subclasses A1-A2 in groups of patients with varying severity of COVID-19. In the case of the S-protein, G1, G3, and A1 were the main subclasses of antibodies involved in the immune response.

Production of Recombinant Human Transferrin in Eukaryotic Pichia pastoris Expression System.

The development and manufacturing of serum-free culture media allowing reducing the costs of preparations and standardizing the biotechnological process are important trends in biotechnology. Substitution of protein compounds in the serum-free media with recombinant analogues reduces the risk of contamination with various infectious agents. Human transferrin is a protein component of serum-free media responsible for the transport of Fe3+ ions into cells. We generated a producing strain P. pastoris secreting human transferrin to the culture medium. The use of constitutive GAP promoter and maintenance of medium pH at 6.5 allows attaining maximum level of transferrin expression (20 mg/liter).

Loading Rate of Exogenous and Autoantigenic Determinants on Major Histocompatibility Complex Class II Mediates Resistance to Multiple Sclerosis.

Genetic analysis of thousands of patients with multiple sclerosis (MS) and healthy Russian donors showed that the carriage of groups of HLA-DRB1*15 and HLA-DRB1*03 alleles is associated with the risk of MS, whereas the carriage of groups of HLA-DRB1*01 and HLA-DRB1*11 alleles is protective. Recombinant HLA-DRB1*01:01 with a high affinity can recognize the fragments of myelin basic protein (MBP), one of the autoantigens in MS. However, the comparison of the kinetic parameters of the load of MBP and viral HA peptides on HLA-DRB1*01:01, which is catalyzed by HLA-DM, showed a significantly lower rate of exchange of CLIP for MBP peptides. We assume that the observed protective properties of the group of HLA-DRB1*01 alleles may be directly associated with the ability of HLA-DRB1*01:01 to kinetically distinguish peptides of exogenous and endogenous nature.

"Shielding" of Cytokine Induction by the Periodontal Microbiome in Patients with Periodontitis Associated with Type 2 Diabetes Mellitus.

Periodontal diseases, especially those with polymicrobial etiology, are often associated with type 2 diabetes mellitus, proceeding more severely and affecting the course of diabetes mellitus. Recently, this feature has been associated with the ability of periodontopathogen microflora to cause not only a local infectious process in the oral cavity, but also to interact with the human immune system and induce various systemic effects. We investigated changes in the salivary cytokine profile of patients with chronic periodontitis, associated and not associated with type 2 diabetes mellitus. We observed a statistically significant decrease of MCP-1/CCL2, GM-CSF, IL-5, IL-6, and IFN-γ in the saliva of patients with chronic periodontitis associated with type 2 diabetes mellitus in comparison with patients with chronic periodontitis only. All of these cytokines are associated with macrophage activation. These data are an important contribution to the elucidation of the mechanism of periodontopathogens involvement in the manifestation of the systemic effects of type 2 diabetes.

Genetic Engineering of Native Chain Combinations of B-Cell Repertoires on the Surface of Methylotrophic Yeasts Pichia pastoris.

We designed genetic constructs for exposing Fab-fragment library of natively paired single cell B-cell receptors on the surface of Pichia pastoris yeast cells. We have previously obtained the A17 antibody in our laboratory [6]. In this study we showed that the newly designed genetic constructs provide a compatible level of A17 antibody Fab fragment on the surface of yeast cells as well as in the case of vectors containing DNA fragments corresponding to each chain of the antibody. The data suggest that the developed approach for constructing immunoglobulin gene libraries is adequate and fully convenient for studying properties of the real human B-lymphocyte repertoire.

Three-dimensional structures of noncovalent complexes of Citrobacter freundii methionine γ-lyase with substrates.

Crystal structures of Citrobacter freundii methionine γ-lyase complexes with the substrates of γ- (L-1-amino-3-methylthiopropylphosphinic acid) and ß- (S-ethyl-L-cysteine) elimination reactions and the competitive inhibitor L-norleucine have been determined at 1.45, 1.8, and 1.63 Å resolution, respectively. All three amino acids occupy the active site of the enzyme but do not form a covalent bond with pyridoxal 5'-phosphate. Hydrophobic interactions between the active site residues and the side groups of the substrates and the inhibitor are supposed to cause noncovalent binding. Arg374 and Ser339 are involved in the binding of carboxyl groups of the substrates and the inhibitor. The hydroxyl of Tyr113 is a potential acceptor of a proton from the amino groups of the amino acids.

[Vegetative disbalance in pathogenesis of metabolic syndrome].

The aim of the work was to elucidate peculiarities of neurovegetative mechanisms underlying cardiovascular regulation in patients with metabolic syndrome (MS) compared with healthy subjects and patients with arterial hypertension (AH). 46 patients presented with AH without obesity and metabolic disturbances, 42 with MS. 24-hour AD and ECG monitoring was performed and cardiac rhythm variability indices (CVI) were calculated. AH patients showed lowered CVI in association with decreased high-frequency vagal effects compared with healthy subjects. MS patients had smaller CVI than healthy and AH ones due to much poorer involvement of both parasympathetic and sympathetic divisions of vegetative nervous system. Vegetative balance index remained normal. It is concluded that the use of LF/HF ratio alone is insufficient for the evaluation of sympatovagal index in MS patients due to markedly decreased CVI; a complex analysis of all CVI temporal and spectral characteristics is needed.

[Influence of nebivolol on left ventricular remodeling in patients with arterial hypertension without chronic heart failure].

In the recent years the problem of heart remodeling under drugs in patients with arterial hypertension (AH) have been extensively discussed. The beta-blocker of the third generation nebivolol is known as a good antihypertensive agent. The aim of our study was to evaluate effects of nebivolol on structural and functional parameters of cardiac remodeling in patients with AH without heart failure. We examined 28 patients with AH aged 49.99 +/- 5.26 years, with average arterial pressure 154.42 +/- 94.37 mm Hg and ejection fraction (EF) > 60%. All of them underwent routine clinical examination and echocardiography (EchoCG) with calculation of additional indexes, and than were given nebivolol 5 mg. After 3 months and 1 year of treatment with nebivolol we assessed antihypertensive effect and the state of cardiac hemodynamics using EchoCG with calculation of remodeling indexes. After 3 months of therapy myocardial stresses (systolic - MSs and diastolic - MSd) were reduced (from 154.28 +/- 11.65 to 132.77 +/- 11.37 U, p < 0.02, and from 184.17 +/- 14.1 to 159.87 +/- 13.34 U, p < 0.02, respectively), and diastolic function improved. After 1 year of antihypertensive treatment LV diastolic function remained normal, while thickness of LV wall decreased (from 0.41 +/- 0.03 to 0.37 +/- 0.02 U, p=0.01). MS continued to decrease with high degree of significance. At the end of the first year we revealed decrease of MSs/ESVI from 9.66 +/- 1.77 at the start of treatment to 6.1 +/- 0.69 (p < 0.005) and MSd/EDVI from 3.21 +/- 0.4 to 2.57 +/- 0.38 (p < 0,005). Increase of EF/MSs (from 0.47 +/- 0.05 to 0.61 +/- 0.07, p < 0.02) was also revealed after 1 year treatment with nebivolol. There were 32% individuals in the studied group, who had concentric type of LV remodeling at the beginning, after 3 months this number decreased to 16%, and at the end of first year no patients had this type of remodeling. Thus, it was shown that nebivolol is able to interfere with the processes of structural and functional rearrangement of the myocardium, preventing development of systolic and diastolic dysfunction of the heart.

[Mono Mac 50D treatment results in patients with chronic coronary heart disease]. / Opyt primeneniia Mono Maka 50D u bol'nykh s khronicheskimi formami IBS.

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Interaction of tyrosine phenol-lyase with phosphoroorganic analogues of substrate amino acids.

The phosphinic analogues of tyrosine and pyruvate were first demonstrated to be substrates in the reactions of elimination and synthesis catalyzed by tyrosine phenol-lyase. Kinetic parameters of the enzymatic process were determined, and the first enzymic synthesis of an aminophosphinic acid was carried out. Replacement of the planar HOOC-group by the tetrahedral (HO)(O)PH-group in the substrate slightly affected its affinity for the enzyme but substantially diminished the conversion rate. For phosphonic analogues, containing (HO)2(O)P group, the affinity to the enzyme was decreased considerably while the conversion was completely prevented. Thus, the structural parameters of the acid group are important not only for the affinity for the enzyme, but also for the formation of the catalytically competent conformation of the active site.

[Phosphinic analog of methionine inhibits growth of leucosis cell L1210 and transforms to phosphinic analog of S-adenosylmethionine]. / Fosfinovyi analog metionina tormozit rost leikoznykh kletok L1210 i prevrashchaetsia v fosfinovyi analog S-adenozilmetionina.

A phosphinic analogue of methionine bearing a phosphinic H(OH)(O)P fragment in place of the carboxyl group inhibited the growth of the L1210 cells and was intracellularly transformed to the phosphinic analogue of S-adenosylmethionine.