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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.

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.