Molecular mimicry of the receptor-binding domain of the SARS-CoV-2 spike protein: from the interaction of spike-specific antibodies with transferrin and lactoferrin to the antiviral effects of human recombinant lactoferrin.

The pathogenesis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection involves dysregulations of iron metabolism, and although the mechanism of this pathology is not yet fully understood, correction of iron metabolism pathways seems a promising pharmacological target. The previously observed effect of inhibiting SARS-CoV-2 infection by ferristatin II, an inducer of transferrin receptor 1 (TfR1) degradation, prompted the study of competition between Spike protein and TfR1 ligands, especially lactoferrin (Lf) and transferrin (Tf). We hypothesized molecular mimicry of Spike protein as cross-reactivity of Spike-specific antibodies with Tf and Lf. Thus, strong positive correlations (R2 > 0.95) were found between the level of Spike-specific IgG antibodies present in serum samples of COVID-19-recovered and Sputnik V-vaccinated individuals and their Tf-binding activity assayed with peroxidase-labeled anti-Tf. In addition, we observed cross-reactivity of Lf-specific murine monoclonal antibody (mAb) towards the SARS-CoV-2 Spike protein. On the other hand, the interaction of mAbs produced to the receptor-binding domain (RBD) of the Spike protein with recombinant RBD protein was disrupted by Tf, Lf, soluble TfR1, anti-TfR1 aptamer, as well as by peptides RGD and GHAIYPRH. Furthermore, direct interaction of RBD protein with Lf, but not Tf, was observed, with affinity of binding estimated by KD to be 23 nM and 16 nM for apo-Lf and holo-Lf, respectively. Treatment of Vero E6 cells with apo-Lf and holo-Lf (1-4 mg/mL) significantly inhibited SARS-CoV-2 replication of both Wuhan and Delta lineages. Protective effects of Lf on different arms of SARS-CoV-2-induced pathogenesis and possible consequences of cross-reactivity of Spike-specific antibodies are discussed.

Structure-biological activity relationships of myeloperoxidase to effect on platelet activation.

Myeloperoxidase (MPO), an oxidant-producing enzyme of neutrophils, has been shown to prime platelet activity promoting immunothrombosis. Native MPO is a homodimer, consisting of two identical protomers (monomer) connected by a single disulfide bond. But in inflammatory foci, MPO can be found both in the form of a monomer and in the form of a dimer. Beside MPO can also be in complexes with other molecules and be modified by oxidants, which ultimately affect its physicochemical properties and functions. Here we compared the effects of various forms of MPO as well as MPO in complex with ceruloplasmin (CP), a physiological inhibitor of MPO, on the platelet activity. Monomeric MPO (hemi-MPO) was obtained by treating the dimeric MPO by reductive alkylation. MPO was modified with HOCl in a molar ratio of 1:100 (MPO-HOCl). Using surface-enhanced Raman scattering (SERS) spectroscopy we showed that peaks at about 510 and 526 cm-1 corresponded to disulfide bond was recognizable in the SERS-spectra of dimeric MPO, absent in the spectrum of hemi-MPO and less intense in the spectra of MPO-HOCl, which indicates the partial decomposition of dimeric MPO with a disulfide bond cleavage under the HOCl modification. It was shown hemi-MPO to a lesser extent than dimeric MPO bound to platelets and enhanced their agonist-induced aggregation and platelet-neutrophil aggregate formation. MPO modified by HOCl and MPO in complex with CP did not bind to platelets and have no effect on platelet activity. Thus, the modification of MPO by HOCl, its presence in monomeric form as well as in complex with CP reduces MPO effect on platelet function and consequently decreases the risk of thrombosis in inflammatory foci.

Binding of lactoferrin to the surface of low-density lipoproteins modified by myeloperoxidase prevents intracellular cholesterol accumulation by human blood monocytes.

Myeloperoxidase (MPO) is a unique heme-containing peroxidase that can catalyze the formation of hypochlorous acid (HOCl). The strong interaction of MPO with low-density lipoproteins (LDL) promotes proatherogenic modification of LDL by HOCl. The MPO-modified LDL (Mox-LDL) accumulate in macrophages, resulting in the formation of foam cells, which is the pathognomonic symptom of atherosclerosis. A promising approach to prophylaxis and atherosclerosis therapy is searching for remedies that prevent the modification or accumulation of LDL in macrophages. Lactoferrin (LF) has several application points in obesity pathogenesis. We aimed to study LF binding to Mox-LDL and their accumulation in monocytes transformed into macrophages. Using surface plasmon resonance and ELISA techniques, we observed no LF interaction with intact LDL, whereas Mox-LDL strongly interacted with LF. The affinity of Mox-LDL to LF increased with the degree of oxidative modification of LDL. Moreover, an excess of MPO did not prevent interaction of Mox-LDL with LF. LF inhibits accumulation of cholesterol in macrophages exposed to Mox-LDL. The results obtained reinforce the notion of LF potency as a remedy against atherosclerosis.

[Biomarkers of system inflammation in local and diffuse peritonitis]. / Markery sistemnogo vospaleniia pri mestnom i rasprostranennom peritonite.

In cases of any acute surgical abdominal disease the progression of purulent inflammation can lead to local or diffuse peritonitis. The indicators of the degree and specificity of the inflammatory response in blood such as cytokine concentration, neutrophil activity, plasma antioxidant capacity (thiols concentration) could be considered as potential predictors of complications. The luminol-dependent chemiluminescence (CL) response of blood activated by the phorbol ester (PMA), and the concentration of cytokines IL-6, IL-8, IL-10, myeloperoxidase (MPO) and thiols in plasma were measured in patients with uncomplicated condition (group 1, n=8), local peritonitis (group 2, n=9) or diffuse peritonitis (group 3, n=9) at admission to surgery (before surgical operation, b/o), immediately after surgical operation (a/o) and a day after surgery (1 day) as well as in healthy volunteers (norm, n=12). In all time-points the cytokines and MPO concentrations measured by ELISA, in group 3 were higher than in healthy volunteers and in patients in groups 1 and 2. Blood CL demonstrated a more than 5-fold increase above the normal values in all patients, and was also higher in group 2 as compared to group 1 (b/o and a/o). Patients in group 3 had shown both maximum and minimum of CL values, which could be a consequence of neutrophil priming or exhaustion ("immune paralysis"), respectively. The same patients' plasma exhibited low thiol concentration (≤30% vs normal values). In patients with fatal outcomes (group 3, n=2) within a day after surgery, either a decrease of the CL to zero values concurrently with elevated IL-8 and IL-6 concentrations and low thiol levels was observed, or CL exceeded normal values more than 20 times with concurrent complete exhaustion of the plasma thiol pool. No clear dependency between the plasma parameters and neutrophil activity was found. Hence a parameter set for prognosis and/or early diagnosis of infectious complications in acute abdominal pathology should include different biomarkers of the inflammatory response: cytokine profile (IL-6, IL-8, IL-10), MPO and neutrophil activity, antioxidant plasma capacity (e.g., total thiols concentration).

[The dynamics of human total and activated anti-mullerian hormone serum levels in different life periods.]

Here, changes in the serum level of total anti-mullerian hormone (AMH) and its activated form in children of both sexes and women with different reproductive status are investigated. This TGFß superfamily cytokine is known to provide the formation of the male-type reproductive system in mammalian embryogenesis, and regulate folliculogenesis, spermatogenesis and the balance of sex hormones after birth. The biologically active form of the hormone (aAMH) is formed as a result of limited proteolysis of the AMH molecule; it is not reliably known in which tissues and under the action of which enzyme it occurs. The serum level of aAMH seems to be a more informative clinical indicator than the content of total AMH (tAMH), but there are no ELISA systems at the world market that provide direct quantitative detection of aAMH. In this work, quantitative detection of total hormone (tAMH) and its biologically active form (aAMH) in serum was performed using specially developed enzyme immunoassay systems. We showed that in girls, the total serum AMH level, as well as the concentration ratio aAMH / tAMH, practically does not change with age, whereas in boys, there is not only a significant decrease in the total serum AMH level previously described in the literature (Pearson correlation coefficient R = - 0.86, p <0.001), but also in the ratios of the aAMH / tAMH level (R = -0.531, p <0.001). It was also found that in pregnant women, the amount of total AMH and the proportion of aAMH in serum was significantly higher (p <0.01 and p <0.001, respectively) than in the control group women. The obtained results are in good agreement with the available data on the total and activated AMH content in the blood serum of people of different sex and age and indicate a change in the ratio of aAMH / tAMH serum levels in pregnancy. These data may be important both for deepening the understanding of AMH biology and for interpreting the results obtained using AMH detection based diagnostics.

[Neutrophils as a source of factors that increase the length of the inflammatory phase of wound healing in patients with type 2 diabetes mellitus]. / Neitrofily kak istochnik faktorov, uvelichivaiushchikh prodolzhitel'nost' fazy vospaleniia ranevogo protsessa u bol'nykh sakharnym diabetom 2-go tipa.

Oxidative stress and neutrophil activation leading to an increase in myeloperoxidase (MPO), elastase and neutrophil extracellular trap (NET) levels in blood are considered as pathogenic mechanisms responsible for the development of extremity damage in people with type 2 diabetes mellitus (T2DM). The aim of this study was to analyze the relationship between factors, associated with neutrophil activation, and the length of the initial phase of wound healing (the inflammatory phase) in T2DM patients. Patients were divided retrospectively into three groups depending on the damage extent: group 1 (wound on toe) < group 2 (wound on foot) < group 3 (wound on lower leg). Compared to the control group (healthy volunteers), T2DM patients at admission to hospital had significantly (p<0.05) increased levels of blood glucose and glycated hemoglobin (groups 1-3), ESR (groups 1 and 3), blood neutrophil count (groups 2 and 3), plasma MPO concentration (groups 1-3) and blood NET concentration (group 3) and decreased levels of plasma thiols (groups 1-3) and erythrocyte glutathione peroxidase activity (groups 2 and 3). The length of hospital stay after surgical procedures corresponded to the length of the inflammatory phase of the wound healing process and correlated with the number of blood neutrophils in patients before surgery (r=0.72, p<0.05). Leukocytic intoxication index depended on wound area (r=0.59, p<0.05), and it was significantly higher for groups 2 and 3 compared to the control group and group 1. The neutrophil count before surgery in T2DM patients with damage in the lower extremities correlated with the length of the inflammatory phase of wound healing. The correlation found can be attributed to an increase in extracellular MPO and NETs, which, in its turn, results from the activation and degranulation of neutrophils and netosis. Thus, the duration of the inflammatory phase of wound healing depends on specific aspects of systemic inflammation increasing oxidative/halogenative stress and intoxication.

Structural Study of the Complex Formed by Ceruloplasmin and Macrophage Migration Inhibitory Factor.

Macrophage migration inhibitory factor (MIF) is a key proinflammatory cytokine. Inhibitors of tautomerase activity of MIF are perspective antiinflammatory compounds. Ceruloplasmin, the copper-containing ferroxidase of blood plasma, is a noncompetitive inhibitor of tautomerase activity of MIF in the reaction with p-hydroxyphenylpyruvate. Small-angle X-ray scattering established a model of the complex formed by MIF and ceruloplasmin. Crystallographic analysis of MIF with a modified active site supports the model. The stoichiometry of 3 CP/MIF trimer complex was established using gel filtration. Conformity of novel data concerning the interaction regions in the studied proteins with previous biochemical data is discussed.

[Comparative study of the activity of anti-arrhythmia agents in calcium chloride-induced arrhythmia in mice]. / Sravnitel'noe izuchenie aktivnosti antiaritmicheskikh sredstv pri khloridkal'tsievoi aritmii u myshei.

Calcium chloride (280 mg/kg intravenously) provokes arrhythmia, heart fibrillation and death of mice. Pretreatment with isoptin, etmozine, lidocaine, trimecaine, quinidine and novocainamide prevents rhythm disorders and animals' death. Isoptin, etmozine, lidocaine and trimecaine have been found the most effective. Atropine does not produce any protective action. The simplicity and availability of the method described enables one to apply it as a model for search of new antiarrhythmic drugs.

[Pharmacological correction of myocardial ischemia and arrhythmias in reversible coronary blood flow disorders and experimental myocardial infarct in dogs]. / Farmakologicheskaia korrektsiia ishemii miokarda i aritmii pri obratimykh narusheniiakh koronarnogo krovotoka i éksperimental'nom infarkte miokarda u sobak.

The efficacy of the sum of alkaloids of the common fumitory in temporary disorders of the coronary blood flow in the circumflex artery was studied in chronic experiments on 8 dogs. Occlusion of the vessel was caused by means of a remote-controlled loop implanted into the thoracic wall. The arrest of circulation in the artery was attended by the appearance of pathological signs on the ECG in the form of tachycardia, growth of the T wave amplitude, and displacement of the RS-T interval. Preliminary injection of the drug in a dose of 1--2 mg/kg prevented completely or reduced to a great measure the ischemic shifts. In experiments on 6 dogs, 5.2 mg/kg dose of the drug relieved for 20 to 87 min the disorders of rhythm induced by ligation of the interventricular branch of the left coronary artery.