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The study of dysregulatory disorders of the immune system underlying the immunopathogenesis of severe purulent-inflammatory diseases (PIDs) is important for development of new therapeutic tactics of restoring antibacterial defense. Acute peritonitis (AP) is a severe PID of the abdominal cavity, the course of which are dependent on the treatment, cytokine balance and adequate functioning of the immunity. Objective: to evaluate the modulating effects on the immune system and the levels of proinflammatory cytokines of the synthetic thymic hexapeptide, active substance of Imunofan, included in the complex postoperative treatment (CPOT) of immunocompromised children with local AP. Clinical and immunological examination of 20 immunocompromised children aged 5–12 years with local AP was carried out before (study group 1, SG1) and after (study group 1a, SG1a) CPOT including synthetic thymic hexapeptide (Arginyl-alpha-AspartylLysyl-Valyl-Tyrosyl-Arginine, НР), alongside 20 conditionally healthy children (comparison group, SG). The content of T and B lymphocytes, natural killer cells (NK), levels of serum pro-inflammatory cytokines IL-1β, IL-6, TNFα, IL-8, IL-18, phagocytic and microbicidal activity of neutrophils (NG) were assessed. In SG1, before treatment, a decrease in the number of T lymphocytes, T helpers, CTL-lymphocytes, NK and an increase in the level of B lymphocytes was revealed. Defects in the effector functions of NG were determined: impaired bacterial antigen killing and decreased NADPH oxidase activity. It was established that in case of AP in immunocompromised children, the cytokine profile is characterized by overproduction of studied proinflammatory and neutrophil-associated cytokines. After complex treatment including immunomodulatory therapy, there was a restoration of the content of T lymphocytes, T helper cells, TCTL lymphocytes, an increase in the number of NK and decrease in the level of B lymphocytes. In addition, regression of the levels of inflammatory, including neutrophil-associated, cytokines and emergence of effector functions of NG due to restoration of NADPH oxidases activity, was noted. Thus, the restoration of immunological parameters in AP leads to earlier regression of the purulent-inflammatory process in the abdominal cavity and to the absence of postoperative complications. The clinical and immunological effects of the immunomodulatory therapy program with inclusion of the drug based on HP determines the feasibility of its use in the postoperative period in immunocompromised children with local AP.

Post-COVID syndrome (PCS) is characterized by long-term complications and conditions accompanied by neuroimmunoinflammation, and includes chronic fatigue syndrome (CFS) and cognitive disorders (CD), which are often associated with activation of chronic herpesvirus infections (HVI). Timely detection of symptoms and immunodiagnosis of PCS are a priority and are of undoubted interest. Objective: to clarify the levels of serum proand anti-inflammatory cytokines, alpha and gamma interferons in patients with post-COVID syndrome associated with confirmed activation of chronic herpesvirus infections. Patients (n = 60) aged from 18 to 65 years with complaints of manifestations of PCS associated with HVI were studied — the study group (SG). A survey was conducted using a modified scale-questionnaire to assess the severity of PCS symptoms in points from 0 to 4, real-time PCR of HVI (EBV, HSV1/2, VCH6, VCH8, CMV) in saliva and scrapings from the tonsils, determination of the level of IFNα and IFNγ, pro- (TNFα, IL-18, IL-1β, IL-6, IL-17A, IL-8) and anti-inflammatory (IL-4 and IL-10) cytokines in blood serum. Comparison group (CG) — 60 apparently healthy individuals. SG patients with mixed HVI with EBV dominance noted the most pronounced and persistent clinical manifestations of PCS, among which the leading place was occupied by longterm sensations of CFS and CD. A persistent multisystem inflammatory response was identified, confirmed by elevated levels of IL-6 and IL-17A, which caused severe PCS. In post-COVID period, hyperproduction of IL-1β was detected, which was accompanied by clinical manifestations of persistent neuroimmunoinflammation. At the same time, the identified deficiency of IFNα, IFNγ and dysregulatory disorders in the antiviral defense of the immune system in patients with PCS contributed to the activation of HVI. Data on the imbalance of proand anti-inflammatory cytokines in the SG were obtained, which confirms the presence of a persistent multisystem inflammatory reaction with dominance of persistent neuroimmunoinflammation, which causes severe PCS. An imbalance of the cytokine system with IFNα and IFNγ deficiency, associated with the activation of chronic HVI with EBV dominance in the post-COVID period, contributes to the development of neuroimmunoinflammation, which is accompanied by the leading clinical signs of PCS: CFS and CD.

Today, the diagnosis and treatment of severe infectious and inflammatory diseases in newborns, e.g., congenital pneumonia (CP) and neonatal sepsis (NS), present difficult problems. Searching sensitive and specific severity markers of bacterial inflammatory process as well as early and effective treatment are crucial for the outcome and prognosis of these life-threatening diseases. The aim of our study was to assess the effects of intravenous immunoglobulin (IVIG) injections on the negatively transformed subpopulations of neutrophilic granulocytes (NG) СD64-CD16+СD32+СD11b+, СD64+CD16+СD32+СD11b+ and evaluation of their functional activity in newborns with CP and NS. We have observed 38 full-term newborn patients. Group 1 included 19 infants with CP, including 11 children who received conventional therapy and IVIG (group 1.1), and 8 children treated at conventional protocols (group 1.2). Group 2 included 19 children with NS, including 12 children who underwent conventional therapy and IVIG treatment (group 2.1), and 7 children who were subject to conventional therapy (group 2.2). The comparison group consisted of 22 healthy full-term newborns. Testing of NG population included the following parameters: counting the numbers of NG subpopulations which simultaneously expressed CD11b CD64, CD32, CD16, as well as their phenotypic patterns, with regard of the receptor expression density (MFI) using flow cytometric techniques. Moreover, we determined phagocytic and microbicidal activity of the granulocytes. We have revealed negative transformation of СD64-CD16+СD32+СD11b+ and СD64+CD16+СD32+СD11b+ subpopulations of neutrophilic granulocytes in newborns with CP and NS, The diagnostic significance of increased СD64+CD16+СD32+СD11b+NG subpopulation was more pronounced with increasing severity of bacterial infection and inflammatory process, i.e., 18.7-fold in CP, 52.3-fold in NS, along with predominant decrease in expression of appropriate membrane receptors. These phenotypic changes were associated with impaired phagocytic and killing activity of NG. The effect of IVIG on the impaired mechanisms of antibacterial immunity is associated not only with alleviation of IgG deficiency, but also with positive remodeling of negatively transformed subpopulations of СD64-CD16+СD32+СD11b+NG and СD64+CD16+СD32+СD11b+NG, improved effector functions of NG, especially in cases of CP. Thus, following IVIG treatment, a reduced number of СD64-CD16+СD32+СD11b+NG subpopulations was fully recovered in CP, while it increased 1.5 times in NS, and the content of diagnostically significant СD64+CD16+СD32+СD11b+NG subpopulation showed a significantly decrease, both in CP (2-fold) and in NS (2.6-fold). However, this index remained higher than the content of this subpopulation in healthy newborns. At the same time, we have noted the restorative or modulatory effects by changing density of trigger molecules in NG subpopulations. Limitation of the negative NG transformation in their functionally significant subpopulations in newborns with CAP and NS was accompanied by positive clinical effects, i.e., optimization of antibiotic therapy, reduced duration of treatment, and improved mortality rates.

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.

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.

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.

Numerous modern basic research done undeniable fact that neutrophilic granulocytes (NG) are key effector and regulatory circuits both innate and adaptive immunity, and play a crucial role in the pathogenesis of a wide range of diseases. NG have potent receptor repertoire, providing a connection between them, cells of the immune system, as well as communication with endothelial cells, epithelial and other tissues. NG inducing stimuli activate and promote the translocation of cytoplasmic granules and vesicles surface molecules on the cytoplasmic membrane the secretion of a large spectrum of pro-and anti-inflammatory, immunoregulatory cytokines, colony, angiogenic factors and fibrogenic, TNF superfamily members, chemokines, regulatory protein, etc. Chromatin nuclei NG capable of restructuring under the influence of inducing stimuli, which is associated with the expression of multiple cytokine genes. NG receiving complex cytokine influence not only acquire new features, but also in various stages of activation and differentiation processes involved in intracellular intraphagosomalis degranulation and killing of implementing elimination microorganisms and extracellular neutrophil degranulation in the formation neutrophil extracellular traps (NET), while this dying through NETosis. Features NG phenotype and their functional properties, demonstrate the existence of subpopulations of NG with different capabilities: equipment of different receptor, the ability to restructure chromatin expressing cytokine genes and secrete cytokines to implement the contents of the granular system, produce reactive oxygen species, implement cytotoxicity form NET. In our opinion, there subpopulation NG: regulatory; suppressor; proinflammatory — initiating an inflammatory response; inflammation with a positive potential microbicidal (antibacterial, antiviral, antifungal); inflammatory cytotoxic potential of the negative — «aggressive»; anti-inflammation regulating regression; antitumoral — TAN1; pro-tumoral — TAN2; hybrid, combining the characteristics of NG and dendritic cells. The absence of adequate response, or hyperactivation blockade NG functions leads to the development of low-intensity infectious and inflammatory diseases, do not respond to conventional therapy of autoimmune diseases/chronic immune-dependent processes. Remo deling dysfunctions NG — the key to new immunotherapeutic strategies.

Aim. A comprehensive assessment of the various links of immunity in women of reproductive age with genital infectious-inflammatory processes. Materials and methods. The study used peripheral blood of 30 patients with inflammatory diseases of the pelvic organs (IDPO), 30 patients with a viral infection of the urogenital tract (IUT), and peripheral blood of 20 apparently healthy women of reproductive age (Control). The cell immunity was evaluated by the contents of CD3 (+), CD4(+)-, CD8(+)-, CD19(+)-, CD56(+)CD16 (+) lymphocytes, the humoral level – by the concentration of serum immunoglobulins of major classes (IgA, IgM, IgG), the functions of neutrophils – in terms of their phagocytic and oxygen-dependent microbicidal functions. Results. The study of population composition of peripheral blood lymphocytes showed that the absolute and relative levels of lymphocytes in peripheral blood, CD3 (+)-, СD8 (+) lymphocytes, as well as the relative level of CD4 (+) and СD19 (+) lymphocytes, is increased in women with IDPO and IUT relatively to the age reference. Along with this, a strong tendency to decrease in the serum concentration of immunoglobulins A and M and predominant depressive changes in the neutrophilic granulocyte system were revealed in patients suffering from infectious and inflammatory diseases of the genital tract. Conclusion. The revealed immunity disorders in women of reproductive age suffering from IDPO and IUT are pathogenetically significant, and a complex pregravid immunocorrection of defects of phagocytic and microbicidal function of phagocytic cells, as well as certain parameters of cellular (quantitative deficiency of NK- and B-lymphocytes) and humoral (inadequate level of serum IgA, IgM, IgG) immunity are important for maintenance of reproductive potential.

Numerous modern basic research done undeniable fact that neutrophilic granulocytes (NG) are key effector and regulatory circuits both innate and adaptive immunity, and play a crucial role in the pathogenesis of a wide range of diseases. NG have potent receptor repertoire, providing a connection between them, cells of the immune system, as well as communication with endothelial cells, epithelial and other tissues. NG inducing stimuli activate and promote the translocation of cytoplasmic granules and vesicles surface molecules on the cytoplasmic membrane the secretion of a large spectrum of pro-and anti-inf lammatory, immunoregulatory cytokines, colony, angiogenic factors and fibrogenic, TNF superfamily members, chemokines, regulatory protein, etc. Chromatin nuclei NG capable of restructuring under the influence of inducing stimuli, which is associated with the expression of multiple cytokine genes. NG receiving complex cytokine inf luence not only acquire new features, but also in various stages of activation and differentiation processes involved in intracellular intraphagosomalis degranulation and killing of implementing elimination microorganisms and extracellular neutrophil degranulation in the formation neutrophil extracellular traps (NET), while this dying through NETosis. Features NG phenotype and their functional properties, demonstrate the existence of subpopulations of NG with different capabilities: equipment of different receptor, the ability to restructure chromatin expressing cytokine genes and secrete cytokines to implement the contents of the granular system, produce reactive oxygen species, implement cytotoxicity form NET. In our opinion, there subpopulation NG: regulatory; suppressor; proinf lammatory — initiating an inf lammatory response; inf lammation with a positive potential microbicidal (antibacterial, antiviral, antifungal); inf lammatory cytotoxic potential of the negative — “aggressive”; anti-inf lammation regulating regression; antitumoral — TAN1; pro-tumoral — TAN2; hybrid, combining the characteristics of NG and dendritic cells. The absence of adequate response, or hyperactivation blockade NG functions leads to the development of low-intensity infectious and inf lammatory diseases, do not respond to conventional therapy of autoimmune diseases/chronic immune-dependent processes. Remodeling dysfunctions NG — the key to new immunotherapeutic strategies.

The research has shown that the functional state of neutrophilic granulocytes (NG) is determined by the state of NG subpopulation phenotype composition in evaluating the simultaneous expression of CD16, CD32, CD11b membrane markers with considering the density of expressed molecules. The change in NG subpopulations phenotype indicates active or defective inclusion of NG in the immune response in inflammation. Various inducers of endo- and exogenous nature activate NG and promote translocation from cytoplasmic granules and vesicles or expression to the surface cytoplasmic membrane of receptor molecules. In this regard, our interest was in studying the expression peculiarities of CD16, CD32, CD11b NG receptors in patients with atypically occurring bacterial infections (chronic sinusitis) and the possibility of remodeling the NG phenotype under the influence of glucosaminylmuramyl dipeptide (GMDP) and IFNγ in vitro. Subjects of this study were blood samples of patients with chronic sinusitis and conditionally healthy individuals. Flow rate cytometry at CYTOMICS FC500 (Beckman Coulter, USA) was used to evaluate the % NGs expressing CD16, CD32, CD11b, and the fluorescence intensity of these molecules (MFI) before and after incubation with the test substances. Flow cytometry (CYTOMICS FC500, Beckman Coulter, USA) were used to evaluate amount (%) of NGs expressing CD16, CD32, CD11b and mean fluorescence intensity of these molecules (MFI) before and after incubation with the test substances. The studies showed that in patients with chronic sinusitis with persistent recurrent course the presence is characterized by transformed phenotype CD16dimCD32midCD11bbr of CD16+CD32+CD11b+NG subpopulation. We have shown that the revealed changes in the level of expression of trigger membrane receptors do not allow NG to fully engage in inflammatory process and to realize its effector and regulatory functions. Under the influence of GMDP and IFNγ regulatory molecules was demonstrated high mobilization capacity of the studied NGs for functionally significant membrane receptors in realization of various functions.