IMMUNE PREDICTORS OF FIBROGENESIS AND THEIR APPLICATION FOR NON-INVASIVE DIAGNOSTICS OF LIVER FIBROSIS IN PATIENTS WITH CHRONIC VIRAL HEPATITIS

The study was devoted to development of non-invasive methods for diagnosis of liver fibrosis in patients with chronic viral hepatitis (CVH). The stepwise logistic multivariate regression analysis of biochemical (PTI, glucose, albumin, AST, LDH) and immunological parameters (the functional activity of neutrophils and monocytes, the relative content of CD8+ T- and CD19+B-cells) was used for the establishment of diagnostic model. Based on the multiple regression analysis we derived a 2 novel Integral Indexes of Liver Fibrosis (IILF-biochem and IILF-immun, respectively). It is shown that IILF-biochem allows, without resorting to liver biopsy to predict the appropriate stage of fibrosis in CHV patients with diagnostic accuracy of 86—87 %, and in combination with IILF — at 97.7 %

SPONTANEOUS AND LPS-INDUCED PRODUCTION OF 26 CYTOKINES SECRETED BY BLOOD CELLS OF PATIENTS WITH LIVER CIRRHOSIS DURING OF CELL THERAPY

The objective of the present study was to assess the level of 26 cytokines secreted by peripheral blood cells of the patients with liver cirrhosis (LC; n = 20) during the cell therapy (CT). All the patients were administered with intravenously injected autologous bone marrow-derived mononuclear cells (MNCs) in a dose of 1.3±0.3 × 109 (Ме 1.0 × 109 ) followed by 14 days later intravenous injection of ex vivo generated mesenchymal stromal cells (MSCs) in a dose of 22.3±5.0 × 106 (Ме 16.0 × 106 ). The patients were examined before the CT, 2-3 days after the administration of MNCs and, then, 2-3 days after the introduction of MSCs. Cytokine-secretory function of peripheral blood cells was evaluated in a 24-hour whole blood cultures both in the absence of any stimulation and in response to lipopolysaccharide (LPS). The control group consisted of 10 healthy donors. The administration of patients’ bone marrow cells (both MNCs and MSC) was safe and well tolerated and caused no any adverse (toxic or allergic) events. Compared with donors, LC patients (especially, with class B+C by Child-Pugh) differed by an initially increased production of several cytokines and chemokines. Actually, there was a statistically significant increase of the spontaneous production of IL-9, MIP-1β, and IP-10, as well as a distinct trend to an increase in TNFα, IL-1ra, IL-4, IL-5, IL-6, IL-13, of MCP-1, MIP-1α, RANTES and Eotaxin. Moreover, the blood cells of LC patients were susceptible to the stimulatory effect of LPS, and the LPS-induced production of 11 out of 26 cytokines (IL-1ra, IL-6, IL-9, IL-15, IL-17, IL-7, IL-8, IP-10, MIP-1α, MIP-1β, Eotaxin) significantly exceeded the normative values. Transplantation of bone marrow MNCs had minimal impact on cytokine production. Meanwhile, the MSCs introduction resulted in a significant decrease in spontaneous and LPS-induced production of, respectively, 20 and 18 analytes including pro-/anti-inflammatory and immunoregulatory cytokines, chemokines and growth factors. The normalization of cytokine-secretory function following transplantation of MSCs revealed in the patients with LC indicates the weakening of an inflammatory activity of circulating blood cells and the decrease in their reactivity to endotoxin. MSC suppressive effect on cytokine production was dose-dependent, and most pronounced in patients with decompensated LC (class B+C by Child-Pugh) of viral etiology

Properties of Calcium Phosphate/Hydrogel Bone Grafting Composite on the Model of Diaphyseal Rat Femur’s Defect: Experimental Study

Background. The problem of bone defects replacement is relevant nowadays, that is why many scientists create new synthetic bone substitutes, but the ideal material has not been found so far. The aims of the study: 1) to determine the suitability of the monocortical defect model in the rat femur diaphysis with additional prophylactic reinforcement with a bone plate for assessing the biological properties of implanted materials using the commercially available ChronOS material as an example; 2) to assess of the osteoconductive properties of composite materials based on poly(ethylene glycol)diacrylate and octacalcium phosphate with architecture Kelvin and gyroid types on the developed model. Methods. A prospective study, level of evidence II. A monocortical defect of the rat femoral diaphysis (length 7 mm) was produced under anaesthesia in aseptic conditions and fixed with a polyetheretherketone plate and six titanium screws. In the control group, the defect was left empty. In other groups, blocks of one of three materials were implanted сhronOS and composites of poly(ethylene glycol)diacrylate and octacalcium phosphate with 3D-printed Kelvin and gyroid architectures. After 3 and 6 weeks, the rats were sacrificed, and histological examination of the defect zone was performed. The amount of newly formed bone tissue was histometricly assessed, followed by statistical processing of the results. Results. All rats have reached the planned endpoint, and there were no infectious complications or loss of fixation. Histological examination of the defect zone revealed minimal bone growth in the Control group, rather slow bone formation in the Gyroid group, and statistically significantly more pronounced bone formation in the pores of the materials in the Kelvin and Chronos groups. Conclusions. Bone defect in this model was not spontaneously filled with bone tissue and allowed us to study the biological properties of bone substitutes (the ability to biodegrade and osteoconductive properties). The osteoconductive properties of a composite material based on poly(ethylene glycol)diacrylate and octacalcium phosphate with a Kelvin architecture are higher than with a gyroid architecture and are comparable to that of the сhronOS

Preclinical studies of antiviral activity of the RPH-137 fusion protein and molnupiravir against COVID-19

Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment.The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection.Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung–body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test.Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells; the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection.Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 infection than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research

Доклинические исследования противовирусной активности гибридного белка RPH-137 и молнупиравира в отношении COVID-19

Finding effective and safe medicines to fight SARS-CoV-2 infection is an urgent task. RPH-137 is an original trap fusion protein against SARS-CoV-2 virus. It comprises the angiotensin-converting enzyme type 2 extracellular domain and the human IgG1 Fc fragment.The aim of the study was to carry out a preclinical evaluation of the efficacy of RPH-137 and molnupiravir against SARS-CoV-2 infection.Materials and methods: the authors analysed RPH-137 expressed in a stable CHO cell line and molnupiravir used as an active pharmaceutical ingredient. Drug-mediated inhibition of virus-induced cytotoxicity was assessed in Vero cell culture. In vivo efficacy assessments were performed in Syrian hamsters. The animals were infected intranasally with SARS-CoV-2 (PIK35 clinical isolate) in the dose of 5 log TCID50. The authors evaluated body weight measurements, lung–body weight ratios, and lung histopathology findings and determined viral RNA levels in oropharyngeal swabs by RT-PCR using the amplification cycle threshold (Ct). The statistical analyses involved one- and two-way ANOVA, Student's t-test, and Mann–Whitney test.Results: RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells; the EC50 values of RPH-137 amounted to 4.69 μg/mL (21.3 nM) and 16.24 μg/mL (73.8 nM) for 50 TCID50 and 200 TCID50, respectively, whereas the EC50 values of molnupiravir were 0.63 μg/mL (1900 nM) for both doses. Intramuscular RPH-137 (30 and 80 mg/kg) had no effect on the infection process in Syrian hamsters. The comparison with the challenge control group showed that intraperitoneal RPH-137 (100 mg/kg) had statistically significant effects on a number of parameters, including a 27% reduction in inflammation and a 30% reduction in the total lesion area of the lungs by Day 7. Intragastric molnupiravir (300 mg/kg twice daily) significantly inhibited SARS-CoV-2 infection.Conclusions: both RPH-137 and molnupiravir inhibited the cytopathic effect of SARS-CoV-2 in Vero cells. In Syrian hamsters, molnupiravir demonstrated a more pronounced inhibition of SARS-CoV-2 infection than RPH-137. However, RPH-137 had statistically significant effects on a range of parameters. This offers additional perspectives for further research.Поиск эффективных и безопасных лекарственных средств для борьбы с коронавирусной инфекцией, вызванной вирусом SARS-CoV-2, является актуальной задачей. RPH-137 – оригинальный гибридный белок-ловушка вируса SARS-CoV-2, состоящий из внеклеточного домена ангиотензинпревращающего фермента 2 типа и Fc-фрагмента IgG1 человека.Цель работы: доклиническая оценка эффективности RPH-137 и молнупиравира в отношении инфекции, вызванной SARS-CoV-2.Материалы и методы: RPH-137 получали в стабильной линии клеток китайского хомячка. В работе использовали субстанцию молнупиравира. Изучение ингибирования вирус-индуцированной цитотоксичности проводили в культуре клеток Vero. В исследовании эффективности in vivo сирийских хомячков заражали интраназально SARS-CoV-2 (вариант ПИК35) в дозе 5 lg ТЦД50. Оценивали массу тела, массовый коэффициент и гистологическую картину легких. В орофарингеальных мазках измеряли содержание вирусной РНК методом ОТ-ПЦР по показателю порогового цикла амплификации Ct. Статистическая обработка: однофакторный и двухфакторный дисперсионный анализ (ANOVA), t-тест Стьюдента, критерий Манна–Уитни.Результаты: RPH-137 и молнупиравир ингибировали цитопатическое действие вируса SARS-CoV-2 в культуре клеток Vero: для RPH-137 EC50=4,69 мкг/мл (21,3 нМ) и 16,24 мкг/мл (73,8 нМ) для доз 50 ТЦД50 и 200 ТЦД50 соответственно, для молнупиравира EC50=0,63 мкг/мл (1900 нМ) для обеих доз вируса. RPH-137 при внутримышечном введении в дозах 30 и 80 мг/кг не оказывал влияния на развитие инфекции у сирийских хомячков. RPH-137 при внутрибрюшинном введении в дозе 100 мг/кг показал статистически значимый эффект по ряду параметров по сравнению с животными контрольной группы (контроль заражения), в том числе вызывая снижение воспалительного процесса и общей площади поражения легких на 7 сут на 27 и 30% соответственно. Молнупиравир при пероральном введении в дозе 300 мг/кг 2 раза в сутки значимо подавлял развитие инфекции, вызванной SARS-CoV-2.Выводы: RPH-137 и  молнупиравир ингибируют цитопатическое действие вируса SARS-CoV-2 в культуре клеток Vero. У сирийских хомячков введение молнупиравира демонстрировало более выраженное подавление инфекции, вызванной SARS-CoV-2, по сравнению с RPH-137. Однако RPH-137 проявлял статистически значимое действие по ряду параметров, что открывает перспективы для его дальнейшего изучения.

Geometric morphometrics reveals shifts in flower shape symmetry and size following gene knockdown of CYCLOIDEA and ANTHOCYANIDIN SYNTHASE

Peer reviewe

CYTOKINE PROFILE IN PATIENTS WITH CHRONIC VIRUS HEPATITIS COMPLICATED WITH FIBROSIS AND CIRRHOSIS

Abstract. In present study, a comparative analysis of spontaneous and LPS-stimulated production of Th1/ Th2 (IFN-γ/IL-4, IL-13), and monocyte-derived pro- and anti-inflammatory cytokines (TNF-α/IL-10) was carried out in blood cell cultures of healthy donors and the patients with chronic viral hepatitis complicated with liver fibrosis and cirrhosis. The patients’ cells exhibited an increased production of both Th1/proinflammatory and Th2/anti-inflammatory cytokines. Meanwhile, hepatitis-induced cirrhotic transformation correlates with enhancement of spontaneous IL-4 and IL-13 production (not observed in fibrosis patients), and progressive increase of spontaneous IFN-γ, TNF-α and IL-10 secretion. In turn, the progression of cirrhosis is connected with increase of pro-inflammatory blood activity and reduction of anti-inflammatory potential. E.g., the patients with grade C cirrhosis have significantly higher index of TNF-α/IL-10 and IFN-γ/IL-10 ratio, as compared to the grade A patients. Moreover, we revealed a positive correlation of TNF-α/IL-10 ratio with cytolysis and severity of cirrhosis (Child-Pugh score), and a reverse correlation between the grade of liver cirrhosis and LPS-induced stimulation index of IL-10. Predominance of pro-inflammatory cytokines, displayed as increased IFN-γ/IL-10 ratio, was also associated with presence of anemia. Hence, the profile of cytokines produced by peripheral blood cells reflects some clinical features in the patients with chronic virus hepatitis complicated with fibrosis and cirrhosis

COMPARATIVE ANALYSIS OF FEMTOSECOND LASER UNITS FEMTO VISUM (RUSSIA) AND FEMTO LDV Z6 (SWITZERLAND). A 3D-DIGITAL ASSESSMENT OF MORPHOMETRIC PARAMETERS OF CORNEAL FLAP IN THE EXPERIMENT

Purpose. To perform a comparative 3D digital morphometric assessment of corneal flap formed using a femtosecond laser Femto Visum and Femto LDV Z6.Material and methods. Experiment was performed on 20 cadaver eyes, on which corneal flaps were formed using the femtosecond laser systems Femto Visum (Optosystems, Russia) (Group 1 – «Femto Visum» – 10 eyes) and Femto LDV Z6 (Ziemer, Switzerland) (Group 2 – «Femto LDV Z6» – 10 eyes). In both groups, the corneal flap was formed on the set parameters: side cut angle – 70°, the flap thickness – 100 µm, diameter of stromal bed – 9.0 mm. By using 3D digital microscope Hirox KH-8700 (Japan) the side cut angle of the flap, the flap thickness and diameter of the stromal bed were measured.Results. With 3D digital estimation we obtained following results in group 1 («Femto Visum»): side cut angle – 74.61±3.09°, the flap thickness – 87.91±6.93 µm, diameter of the stromal bed – 8.88±0.09 mm; in group 2 ( «Femto LDV Z6») – side cut angle – 74.06±3.08°, the flap thickness – 87.92±7.79 µm, diameter of the stromal bed – 8.83±0.06 mm. Those parameters are not statistically different between the two groups (p>0.05, Mann-Whitney U-test).Conclusions. 1. 3D digital microscope Hirox KH-8700 allows precise measurements of morphometric parameters of corneal flap in the coordinates X, Y, Z. 2. Femtosecond laser systems, Femto Visum and Femto LDV Z6, allow us to create the corneal flap such that the side cut angle and the diameter of the corneal stromal bed meet the set parameters, with no statistically significant difference between systems (p>0.05). The flap thickness was thinner than desired, («Femto Visum» and «Femto LDV Z6»), but there was no statistical difference between two groups (p>0.05)