Failure of anti-infectious immune protection is considered a reason for the prolonged course and recurrence of chronic infectious and inflammatory diseases of pelvic organs (PID). Our aim was to evaluate the effect of an original hexapeptide (HP) on negatively altered subpopulations of neutrophil granulocytes (NG) CD11b+CD64-CD32+CD16+ and CD11b+CD64+CD32+CD16+, their phenotype and associated effector functions in immunocompromised women with PID. 35 women (20-40 years old) with PID were studied during the period of clinical exacerbation (study group 1, SG1). Study group 1a (SG1a) consisted of patients who underwent treatment including the HP injections (45 mcg/ mL, 1 ml intramuscularly once a day for 10 days). The comparison group (CG) consisted of 20 conditionally healthy women. The numbers of CD11b+CD64-CD32+CD16+NG and CD11b+CD64+CD32+CD16+NG cell subsets and the density of receptor expression, phagocytic and microbicidal function of NG were determined. In SG1, decreased counts of the major NG subpopulation (CD11b+CD64-CD32+CD16+NG) was revealed (p 0.05), with a trend for increase of minor subset CD11b+CD64+CD32+CD16+NG (p 0.05). In the CD11b+CD64-CD32+CD16+NG subset, we noted a decreased expression of CD16 (1.4-fold), CD11b (2-fold) (p1, 2 0.05). In the minor subset CD11b+CD64+CD32+CD16+NG, the expression densities were decreased in CD16 (1.7-fold), CD11b (2.1-fold, p1, 2 0.05). At the same time, the phagocytic and microbicidal functions of NG were found to be decreased. In the course of immunomodulatory therapy with the HP-based drug, positive changes in immunological parameters were revealed. In SG1a, an increased number of major NC subset was observed, with an increase in the expression density of CD16 by 1.2 times, CD11b by 1.7 times relative to SG1 (p1, 2 0.05). The contents of minor NG subset tended to decrease, along with CD16 expression density reaching the indices of comparison group. CD11b increased 1.3 times relative to SG1 (p 0.05). Higher ratios of actively phagocytizing NG and their killing ability have been registered. Clinically, we observed faster regression of clinical PID exacerbation symptoms and decreased frequency of relapses 6 months after treatment in 88.6% of cases. The positive immunomodulatory effects of the HP-based drug upon altered subsets of CD11b+CD64-CD32+CD16+ and CD11b+CD64+CD32+CD16+ NGs, their phenotype and associated effector functions suggest an opportunity of its usage for the correction of NG dysfunctions in immunocompromised women with PID, thus providing stable clinical and immunological remission and protective effect.

The development of post-ovoid syndrome is the serious consequence of COVID-19. The link of the clinical manifestations of COVID-19 with the cytokine profile remains insufficiently investigated until now. Objective. To study the biomarkers of the incompleteness of the immune hyperinflammatory process in the period of reconvalescence of patients with COVID-19 in order to predict the threat of post-covid syndrome based on the study of the dynamics of changes in serum cytokine levels IL-1b, TNF-α, IL-4, IL-6, IL-8, IL-10, IL-17A, IL-18, VEGF-A and interferons IFN-α, IFN-γ. Patients and methods. IL-1b -4, -6, -8, -10, -17A, -18 cytokines, TNF-α,VEGF-A, IFN-α, IFN-γ have been studied in dynamics,(enzyme immunoassay) in patients with moderate COVID-19 (n = 45, 61 (57; 71) years) in the acute period (study group (SG) 1a), against the background of antiviral therapy (GI1b) and upon discharge from the hospital (SG1b). Comparison group (CG) – 50 conditionally healthy people. Results. It was found that the levels of IL-1β, TNF-α, IL-8 were normalized in GI1b, in 35% of cases IL-6, while the level of cytokines IL-18, IL-17A, VEGF-A remained at the values determined in the acute course of COVID-19 (p > 0.05). Conclusion. Elevated levels of IL-17A, IL-18, VEGF-A in the period of COVID-19 reconvalescence could be prognostic biomarkers indicating the continuing incompleteness of the immune inflammatory process, which determines the need for cytokine monitoring in the post-ovoid period and requires the creation of targeted immunotherapy strategies. Key words: COVID-19, biomarkers, post-COVID syndrome, moderate form, cytokines

Chronic inflammatory diseases of the pelvic organs (pelvic inflammatory disease, PID) in women is among the main understudied problems in gynecology worldwide with adverse medical and socio-economic consequences, thus justifying the need for further study of immunopathogenesis and development of new approaches to treatment. Our objective was to develop new immunotherapeutic approaches to correction of combined disorders of the immune system functioning in immunocompromised women with PID and to evaluate their clinical and immunological efficacy. 55 women aged 20-40 years were examined, i.e., 35 patients with exacerbation of sluggish or recurrent PID, resistant to conventional therapy. Testiung was performed before complex treatment (study group 1 GI- 1) and after the course (study group 2 GI-2). Contents of T and B lymphocytes, natural killer cells (NK) (CYTOMICS FC500, USA), phagocytic and microbicidal functions of neutrophilic granulocytes (NG) were assessed in SG-1 and SG-2 before and 2-3 days after complex treatment with addition of an immunotherapeutic drug based on hexapeptide (HP) at a daily dose of 45 mg/ml intramuscularly for 10 days. In patients from SG-1, a decrease in T cells (CD3+CD19- ) and B cells (CD3-CD19+), a 2-fold increase in the content of NK CD3-CD16+CD56+ was found, along with altered functioning of NG (deficiency of actively phagocytizing NG, a decrease in their digestive function and NADPH-oxidase activity). In SG-2 patients, the treatment was followed by restoration of the T (CD3+CD19-) and B cells (CD3-CD19+), NK cells (CD3-CD16+CD56+), like as an increase in effector functions, i.e., microbial capture by NG and their killing ability due to activation of NADPH oxidases and normalization of microbicidal reserve capacity in the NG cell population. Positive clinical effect included reduction of clinical symptoms in acute period, absence of PID exacerbations over follow-up for 6 months (85.6% of cases). Occasional exacerbations of PID were associated with medical manipulations (5.7%) and unprotected sexual contacts (5.7%). The immunopathogenetically proven approach to correction of combined functional impairment of immune system in the women with PID shows a positive clinical and immunological effect.

Chronic inflammatory diseases of the pelvic organs (CIDPO) in women represent one of the urgent and insufficiently studied problems in gynecology across the world. These disorders are followed by adverse medical and socio-economic consequences, i.e., chronic local inflammatory process, chronic pelvic pain syndrome, ectopic pregnancy, infertility. Due to increasing chronicity and recurrence rates of genital infections and inflammatory diseases, there is a need for further studying the effector and regulatory mechanisms of immune system. Of special relevance are the studies of the receptor transformation in neutrophilic granulocytes (NG), the basic population of antimicrobial defense, with further substantiation of targeted immunomodulatory therapy. Purpose of the present study was to assess transformation of neutrophilic granulocytes from CD11b+CD64-CD32+CD16+ to that CD11b+CD64+CD32+CD16+ phenotype in immunocompromised women with CIDPO exacerbation, as well as to evaluate the possibility of in vitro reprogramming the neutrophile phenotype under the action of recombinant interferon (recIFN2b). Peripheral blood neutrophils were tested in the comparison group of 10 conditionally healthy women 20 to 40 years old, and in 17 women (20-40 years old) with the CIDPO exacerbation (group 1). The in vitro effect of recIFNa2b on the blood neutrophils was evaluated for 17 women with CIDPO (group 2). Flow cytometric technique (FCT, CYTOMICS FC500, Beckman Coulter, USA) was used to determine the number of NGs and cell receptor expression levels of neutrophilic CD11b+CD64-CD32+CD16+NG and CD11b+CD64+CD32+CD16+ subpopulations. In peripheral blood of women with CIDPO exacerbation, an increased expression density of surface membrane molecules was revealed by means of FCT: in the subpopulation CD11b+CD64-CD32+CD16+NG, CD16 proved to be 91.7% higher; in CD11b+CD64+CD32+CD16+NG subpopulation, CD16 was increased by 116%, and CD32 being higher by 81% against the comparison group. In the in vitro system, during the incubation of PB with recIFN2b (group 2), we have revealed an increased number of CD11b+CD64-CD32+CD16+ subpopulation relative to the comparison group and group 1, and significantly increased expression density of CD16 (by 212%); CD11b (by 56%), and CD32 (by 83%) than in comparison group, as well as higher density of CD16 expression by 163%; CD11b (by 223%) compared to group 1. The changes in expression density of membrane molecules was also detected by FCT for the activated subpopulation CD11b+CD64+CD32+CD16+NG, i.e., an increase in CD16 by 232% against control group, and decreased expression density of CD64 by 150% against the background, along with increased density of CD16 expression (by 54%), and CD11b (by 103%), relative to group 1, thus suggesting a reprogramming of negatively transformed NC phenotype. These findings may be considered a positive immunomodulatory effect providing a basis for further research in order to develop new integrated approaches to treatment of CIDPO of various etiologies.

Identification of interrelated innate and sensitive immune responses in COVID-19 is an important step in the development of targeted therapeutic approaches, which is relevant. Objective. To clarify the features of the defect in the function of NK cells, T lymphocytes, the interferon system in patients with moderate and severe COVID-19. Patients and methods. Tests of the peripheral blood of 50 COVID-19 patients aged 61(57–71) and having the moderate and severe disease were performed. The following parameters were measured: the quantity of CD3+CD19–, CD3+CD4+, CD3+CD8+ T lymphocytes, NK – (CD3–CD16+CD56+), and TNK – CD3+CD16+CD56+ with expression density considered membrane receptors (MFI) (FC 500 Beckman Coulter, USA), the levels of IFN-α, IFN-γ, IL-6, TNF-α cytokines (IFA). Results. Combined immunodeficiency associated with quantitative and functional defects in NK, T lymphocytes and their subsets was revealed in moderate and severe COVID-19. An imbalance of cytokines has been established: blockade of the production of IFN-α and IFN-γ against the background of a significant increase in IL-6 and TNF-α, which negatively affects both the number and functionality of the participants in the immune response and is associated with a severe course and poor prognosis of COVID-19. Conclusion. The data obtained demonstrate the need to develop new strategies and tactics for the treatment of COVID-19, including replacement systemic therapy with recombinant IFN-α2b in combination with antioxidants (Viferon®) in adequate therapeutic doses, aimed at restoring the normal functioning of T lymphocytes, NK and the interferon system. Key words: COVID-19, interferons, moderate and severe disease course, NK cells, T lymphocytes

Investigation of molecular mechanisms associated with interferon (IFN) production and receptor function of neutrophil granulocytes (NGs) in COVID-19 is highly relevant because it can be promising in the search for new therapeutic strategies targeting NGs and their reactivity to restore and strengthen the innate immune response against SARS-CoV-2. Objective. To assess the effects of recombinant IFN-α2b on the phenotype of CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119–, and CD16+IFNα/βR1+CD119+ NGs from peripheral blood of patients with COVID-19 in an in vitro experiment. Patients and methods. We analyzed blood samples from 31 patients with a mean age of 61 years (range: 57;71 years) with moderate COVID-19. We assessed the number of CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119–, and CD16+IFNα/βR1+CD119+ NGs, receptor density (FC 500, ‘Beckman Coulter,’ USA), phagocytic activity of NGs before and after incubation with recombinant IFN-α2b. We also measured serum levels of several cytokines, including IFNα, IFNγ, IL-6, IL-8 (ELISA, ‘Vektor-Best’ LLC). The control group comprised 22 adult healthy individuals with a mean age of 58 years (range: 57; 70 years). Results. Patients with moderate COVID-19 demonstrated low serum levels of IFNα and IFNγ along with elevated levels of IL-6 and IL-8. We observed transformation of 3 phenotypes among NG subpopulations: CD16+IFNα/βR1–CD119+, CD16+IFNα/βR1+CD119-, and CD16+IFNα/βR1+CD119+. We observed positive remodulating effects of recombinant IFN-α2b on the number and phenotype of NG subpopulations and their phagocytic activity in our in vitro experiment. Conclusion. Recombinant IFN-α2b demonstrated positive effects in in vitro experiments; therefore, it can be considered in the future as a potential therapeutic tool for moderate COVID-19. Restoration of type I IFN might be an effective treatment option for COVID-19, because it can promote faster virus elimination, restore normal functioning of the IFN system, and have positive regulatory effects on the phenotype of NG subpopulations. Key words: neutrophil granulocytes, subpopulations, COVID-19, phenotype, recombinant interferon α2b

Numerous studies over last decade have shown that functional capacity of neutrophil granulocytes (NG) determines the course and outcome of many diseases. Identification of phenotypic variants of functionally significant NG subpopulations is a new approach allows us to assess the adequacy or deficiency of NG involvement into infectious inflammation processes at molecular level. An opportunity of reorienting a deficient NG subpopulational phenotype in purulent inflammatory diseases due to the rearrangement of the receptor set induced by various immunotropic substances may serve as a key to recovery of normal NG functioning.Our aim was to study the effect of glucosaminylmuramyldipeptide (GMDP) under in vitro conditions upon the phenotypic profile of four functionally significant subpopulations, i.e., CD62L+CD63-NG, CD62L+CD63+NG and CD64-CD32+CD16+CD11b+NG, CD64+CD32+CD16+CD11b+NG, along with assessment of expression density of appropriate membrane molecules and NG microbicidal activity in the children with purulent inflammatory diseases. 90 samples of peripheral blood (PC) were taken from children 2 to 4 years old, including 12 children with minor purulent infection (MPI), and 7 children were studied as conditionally healthy controls. Their peripheral blood was incubated for 60 minutes at 37 °C with GMP (10-6 g/l). Using flow cytometry technique, the relative numbers of some NG subpopulations, i.e., CD64-CD16+CD32+CD11b+NG, CD64+CD16+CD32+CD11b+NG, CD62L+CD63-NG, CD62L+CD63+NG were evaluated, and the phenotype features of each subpopulation were investigated according to the density of appropriate membrane molecule expression (MFI). In parallel, phagocytic and microbicidal activity of NG was tested in these study groups. The obtained data indicate for presence of for distinct NG subpopulations, both in healthy children and in children with MPI. We have revealed phenotypic transformation of the four studied NG subpopulations from MPI patients including disturbed phagocytic and microbicidal functions of the cells. Using of this in vitro system, we have shown that the transformed phenotype of the four functionally significant NG subpopulations of MPI patients was re-arranged under GMDP treatment. At the same time, the number of CD62L+CD63+NG and CD64-CD32+CD16+CD11b+NG subpopulations was increased, along with decreased amounts of CD64+CD32+CD16+CD11b+NG and CD62L+CD63-NG subpopulations, being accompanied by restoration of microbicidal activity of NGs.The obtained data allow us to accomplish current understanding of immunotropic effects of GMDP, and to extend the potential scope of its experimental and clinical application. The new data on GMDP effects revealed by in vitro system, i.e. phenotype rearrangement of functionally significant NG subpopulations CD64-CD16+CD32+CD11b+, CD64+CD16+CD32+CD11b+, CD62L+CD63-, CD62L+CD63+ in atypical purulent inflammatory diseases in children, may be used in the future in order to develop innovative strategies of immunotherapy aiming for correction of NG dysfunction in children with MPI.

Children with clinical signs of immunocompromise have impaired functioning of the immune system. At the same time, the state of subpopulations of multifunctional neutrophil granulocytes (NG), which provide antiviral and antibacterial protection, in such patients has been poorly studied. Objective of the study: to assess the features of the transformation of the phenotype of the three most significant subpopulations of NG, their association with impaired phagocytic and microbicidal activity in immunocompromised children with atypical viral-viral co-infections and bacterial infections, and to clarify their differential diagnostic significance. Materials and methods of research: the phenotypes of NG subpopulations expressing CD64, CD32, CD16, CD11b receptors, phagocytic and microbicidal activity of NG were studied in the peripheral blood of 43 immunocompromised children of both sexes aged of 5–9 years old suffering from recurrent ARVI, atypical chronic mono – or mixed herpes virus infections (HVI) and purulent bacterial infection. 3 study groups were formed: 1st – repeated ARVI/HVI mono, 2nd – repeated ARVI/HVI mixed, 3rd – minor purulent infection (MPI) and a comparison group (20 apparently healthy children). Results: different differentiated transformation of the phenotype of functionally significant subpopulations of NG associated with impaired functional activity of NG as well as with the incidence of viral co-infections (repeated ARVI and recurrent HVI mixed) and the severity of clinical manifestations of MPI has been identified in the studied groups. Conclusion. Еvaluation of the features of transformation of phenotype of NG subpopulations, their effector functions in immunocompromised children with atypical viral-viral co-infections and purulent bacterial infections, will allow in the future to optimize the methods of target immunotherapy aimed at remodulating the negatively transformed phenotype of NG subpopulations, correcting their dysfunctions and, thereby, significantly increase the clinical effectiveness of therapeutic and preventive measures.

The aim of the work was a comprehensive assessment of the system of cytokines and peripheral blood osteocalcin with the establishment of features of their interconnections in children with congenital cleft lip and palate (CCLP) in comparison with the corresponding controls at different age periods. In the peripheral blood of 80 children (0-12 months, 1-3 years, 4-9 years, 10-15 years) with osteocalcin and IL17, IL4, congenital cleft lip and palate and age-appropriate control of healthy individuals (40 people) IL6, IL1β, IFNγ by enzyme immunoassay. An analysis of the data obtained with CCLP revealed significant differences in the dynamics of the relationship between pro-inflammatory (IL1β, IL6, IL17), regulatory (IFNγ), anti-inflammatory (IL4) cytokines and osteocalcin compared with controls. The patterns of changes and effects of IL17, IFNγ, IL4 and osteocalcin in healthy children and in children with congenital cleft lip and palate in postnatal ontogenesis were established. Cytokine dysregulation of immunosteogenesis in CCLP, leading to a significant deficit of osteocalcin in the first year of life due to an imbalance of the cytokine profile: discordant IL17, IFNγ and IL4 were detected. Thus, the data obtained are undoubtedly important in the future for developing new strategies for targeted therapy aimed at normalizing osteocalcin levels at different age periods in children with congenital cleft lip and palate.

Relevance. Neutrophilic granulocytes (NG) are the first cells of the immune system to migrate to the tumor and are actively involved in the implementation of a full-fledged antitumor response through the mechanisms of direct killing of tumor cells, extracellular lysis (NET), and through the activation of antibody-dependent cellular cytotoxicity (ADCC), inhibition of angiogenesis, initiation of other cells with antitumor activity. The aim of the study was to study the effect of cytokines IFN, IFN, G-CSF on the CD62L+CD63- and CD62L+CD63+ subsets and the microbicidal activity of NGs in patients with colorectal cancer (CRC) in vitro. Materials and methods. We studied samples of peripheral blood (PB) of 10 patients of both sexes 38-70 years old with newly diagnosed untreated CRC stage II-III (study group) and 10 healthy volunteers (comparison group). The subsets CD62L+CD63+ NG, CD62L+CD63- NG were assessed by flow cytometry (CYTOMICS FC500, Beckman Coulter, USA), the microbicidal functions of NG were tested by cytochemical methods: activity of NADPH - oxidases, myeloperoxidase (MP), level of cationic protein (CP) in spontaneous tests and under additional stress of S. aureus . The effect of IFN, IFN, G-CSF cytokines on subsets and the microbicidal activity of NG in vitro was studied in both study groups. Microsoft Exel 2016 and StatPlus 2010 were used for statistical processing of the obtained data using nonparametric tests: Me (Q1; Q3), Mann-Whitney U-test and Wilcoxon test . Results . The features of transformation of CD62L+ CD63-NG and CD62L+ CD63+ NG subsets of PB in CRC have been established, that allows to get an idea of the NG ability to roll and readiness to activate the microbicidal arsenal, various defects of spontaneous and induced microbicidal activity of oxygendependent and oxygen-independent mechanisms of NG. The effects of cytokine influence on NG in CRC in vitro have been shown, which indicates the possibility of regulating the receptor and microbicidal functions of NG, and, on the other hand, suggests defects in NG perception of regulatory stimuli, that is confirmed by the progression of tumor growth.

The development of new diagnostic technologies allowed us to expand and deep our understanding of the role of neutrophilic granulocytes (NG) in immune homeostasis and to evaluate the dynamic interrelation between functional potential and phenotypic polarization NG cells in response to inducing signals of intra- and extracellular environment. Until now it is still not completely known how many NG phenotypes are available to realize contents of the granular apparatus, to produce active forms of oxygen, to carry out phagocytosis and cytotoxicity that differ in their properties. Lack of adequate response, hyperactivation or blockade of NG functions leads to the development of infection inflammatory diseases. Term infants with infection inflammatory processes was characterized by increasing of phenotypes CD62L+CD63 +NG, CD64+CD16+CD32+CD11b+NG with different density of the functionally important receptors in congenital pneumonia and neonatal sepsis, which allows assessing the severity of inflammatory process course and role of each of the identified subsets in immunopathogenic of these diseases.

Modern studies have shown a high plasticity and phenotypic diversity of neutrophilic granulocytes (NG) provided by different receptors, which are diagnostic markers for the functional capacity of the cell in the course of their activities. We investigated NG from peripheral blood, obtained from healthy people of both sexes aged from 26 to 66 years. Evaluation of the neutrophil membrane receptor expression was carried out by flow cytometry. The relative amount of neutrophilic granulocytes expressing membrane CD62L, CD63, CD66d receptors and the intensity of their expression were determined according to their fluorescence intensities. The surface NG membrane receptors, i.e., CD62L, CD63, CD66d were studied upon the in vitro experimental influence of the following bacterial peptides: N-formyl-methionyl-leucyl-phenylalanine (FMLP, model 1); glucosaminylmuramyldipeptide (GMDP, model 2), and simultaneous incubation of NG blood with fMLP and GMDP (model 3). The in vitro treatment with fMLP in the in vitro model was used to transform the NG phenotype of conventionally healthy subjects, expressing CD62, CD63, CD66d molecules. The treatment caused a significantly decrease in both CD62L and the CD62L expression in relative amounts of neutrophilic granulocytes with a parallel increase of CD63 expression density. The effect of GMDP on the NG phenotype of conditionally healthy subjects did not change the amount of CD62L+NG and CD63+NG, and did not affect CD62L and CD63 expression density on the surface of NG. However, the amount of CD66d+NG was significantly increased with the unchanged expression of CD66d molecules. GMDP introduced together with the bacterial fMLP peptide was shown to neutralize some features of the NG phenotype transformation caused by fMLP, i.e., the amount of CD62L+ NG was restored by 22 % and the CD62L expression density increased significantly. At the same time, GMDP did not correct the negative effect of fMLP upon the number of CD63+NG and CD66d+NG, and on the CD63 and CD66d expression. Simultaneous addition of fMLP and GMDP did significantly increase the amount of CD66d+NG and expression density of CD63 molecules on the CD63+NG membrane as compared to intact NG of conditionally healthy subjects. The obtained data are important in order to justify some new immunotherapeutic strategies aimed at correction of the negatively transformed NG phenotype, which accompanies some infectious and inflammatory diseases of bacterial etiology with atypical clinical course.

The experimental model of the in vitro transformed phenotype of neutrophilic granulocytes (NG) equipped with receptors CD62L, CD63, CD66d, was created under the influence of N-formyl-methionyl-leucyl-phenylalanine (fMLP). The features of the transformed phenotype of NG were described. The influence of the hexapeptide arginyl-alpha-aspartyl-lysyl-poured-tyrosyl-arginine on untransformed and transformed phenotype of NG was investigated in vitro . It was shown that fMLP reduces the number of NG, expressing the receptor CD62L and reduces the density of expression of this receptor on the surface membrane of NG, moderately increases the density of CD63 and moderately increases the number of CD66dL + NG. Hexapeptide does not influence on the number of untransformed NG, bearing receptors CD62L, CD63, CD66d, and the level of the expression of those molecules. It has been shown the mitigation of the influences of fMLP after the simultaneous incubation NG with fMLP and hexapeptide: the number of CD62L + NG was increased and the density of membrane expression of the molecules of CD62L was increased too. At the same time, hexapeptide did not correct the negative effects of fMLP on the number of CD63 + NG and CD66d + NG, and on level of density of the expression of CD63 and CD66d molecules on the surface membrane of NG. Overall, the results demonstrate the classic immunomodulatory effect of hexapeptide: on the one hand, we saw the absence of any changes of the studying receptors on membrane of the untransformed NG under hexapeptide influences, and on the other hand, it was demonstrated a strong trend of leveling the negative transformational effects of fMLP under hexapeptide influences.