Search

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.

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.

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.

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.

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.

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.Цель работы - комплексная оценка системы цитокинов и остеокальцина периферической крови с установлением особенностей их взаимосвязей у детей с врожденной расщелиной губы и неба (ВРГН) в сравнении с соответствующими контролями в разные возрастные периоды. В периферической крови ٨٠ детей (٠–12 месяцев, 1–3 года, 4–9 лет, 10–15 лет) с врожденной расщелиной губы и неба и соответствующего по возрасту контроля здоровых лиц (٤٠ человек) проведено определение остеокальцина и IL17, IL4, IL6, IL1β, IFNγ методом иммуноферментного анализа. Анализ полученных данных при ВРГН выявил значительные различия в динамике взаимоотношений провоспалительных (IL1β, IL6, IL17), регуляторного (IFNγ), противовоспалительного (IL4) цитокинов и остеокальцина по сравнении с контролями. Установлены закономерности изменений и влияния IL1٧, IFNγ, IL4 и остеокальцина у условно здоровых детей и у детей с врожденной расщелиной губы и неба в постнатальном онтогенезе. Выявлена цитокиновая дисрегуляция иммуноостеогенеза при ВРГН, ведущая к значительному дефициту остеокальцина на первом году жизни, обусловленная нарушением баланса цитокинового профиля: дискордантностью IL17, IFNγ и IL4. Таким образом, полученные данные, несомненно, важны в дальнейшем для разработки новых стратегий таргетной терапии, направленной на нормализацию уровней остеокальцина в разные возрастные периоды у детей с врожденной расщелиной губы и неба.