Purinergic signaling has been actively studied over recent years. It happens, due to its involvement in pathogenesis of various diseases and pathologies such as infectious and non-infectious inflammation, tumor growth, graft-versus-host disease, autoimmune disorders, cardiovascular calcification etc. It has been shown that the purinergic system mediates fine tuning of the immune cell populations, like as cytokine and chemokine secretion, elimination of intracellular pathogens and mechanisms of programmed cell death. Novel approaches to therapy and disease prevention are based on studies of appropriate developmental mechanisms, including role of purinergic system in their regulation. This review contains analysis of recent achievements elucidating the role of purinergic regulation in pathogenesis of basic pathological conditions and diseases, as well as trends in development of new therapeutic approaches based on present knowledge.

Interleukin 33 (IL-33) is a member of the IL-1 family, which is widely expressed on all types of cells. IL-33 was identified as a functional ligand for the plasma membrane receptor complex, which is a heterodimer consisting of a membrane bound ST2 receptor (growth stimulating factor). IL-33 is involved in the development of immune response with predominant release of pro-inflammatory T helper type 2 cytokines. IL-33 is widely expressed on various structure-forming cells, such as epithelial, endothelial and smooth muscle cells. Increased expression of IL-33 is observed during necrosis of these cells (after tissue or cell damage), and it is released into extracellular space, and acts as an endogenous danger signal, sending a sort of warnings to neighboring cells and tissues. Recently, many studies have shown that IL-33 can participate in development and progression of fibrosis in various organs. However, it exerts anti-inflammatory effects upon development of other diseases. This review will discuss biological characteristics of IL-33 and a role of the IL-33/ST2 signaling pathway in the development of fibrosis. 

Pre-eclampsia is a multisystemic disease that occurs in the second half of pregnancy, being characterized by the development of hypertension and proteinuria. Pre-eclampsia is still one of the main causes of maternal and neonatal morbidity and mortality. Pre-eclampsia is believed to be a result of complex interactions between maternal and placental factors. However, the exact pathophysiology of this syndrome remains unclear. Intercellular interactions are the basis of fetoplacental development in physiological pregnancy. A special mechanism of intercellular interactions is associated with the release of membranebound extracellular microvesicles by cells. Concentration and molecular composition of extracellular vesicles in biological fluids depend on the producer cell types, as well as the stimuli that trigger their formation. The studies of extracellular vesicles in pre-eclampsia focus on the particles produced by the cells of maternal cardiovascular system (endothelium, smooth muscle cells of blood vessels), and blood (erythrocytes, leukocytes, and platelets), like as by the cells of syncytiotrophoblast. Changed concentrations and molecular composition of these extracellular vesicles can contribute to the pathophysiology of pre-eclampsia, due to increased proinflammatory and procoagulant state occurring during pregnancy. This review focuses, firstly, on characteristics of the extracellular vesicles produced by syncytiotrophoblast, and possible role of their interaction with maternal immune cells, endothelial cells and platelets in the course of developing pre-eclampsia. Understanding the role of extracellular vesicles of syncytiotrophoblast in pathogenesis of pre-eclampsia could suggest an opportunity of providing these results for early and non-invasive diagnostics of placental disorders, as well as for predicting development of this disease.

Ability of mesenchymal multipotent stromal cells (MSCs) to differentiate into several types of mesenchymal tissues allows to consider these cells the main candidates for creating tissue engineering constructions for regenerative medicine. MSCs promote integration of bio-implants into the native bone and stimulate osteogenesis. MSCs are characterized by immunomodulatory properties, due to inflammation control and modification of immune cells. MSCs affect not only the in vivo immune response by preventing immunological rejection of implanted tissue engineering designs, but it can also influence the bone tissue immunity. MSCs play an important role in bone regeneration, by regulating the osteoblastic generation, and suppressing activity of inflammation effectors and osteoclastogenesis. Some pre-clinical and first clinical trials of bone bio-implants colonized with MSC, demonstrate promising outlooks for this strategy in order to obtain tissue engineering constructions for bone regeneration.

A number of studies have shown that distinct common variants of the genes controlling immune/inflammatory response may affect efficiency of chronic lymphocytic leukemia (CLL) treatment. In a recently published paper, we reported polymorphic variants of some immune response genes in CLL patients to be associated with different rates of disease progression. Correlations between the distribution of gene modification profiles in indolent and agressive forms of CLL have been established. The present study describes results of pharmacogenetic studies aimed for identifying associations between the immune response genes polymorphism, and efficacy of FCR treatment regimen in CLL patients. 19 polymorphic loci of 14 immune response genes were studied in 33 patients with CLL who received FCR therapy. The TLR2, TLR3, TLR4, TLR6, TLR9, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-17A, CD14, TNFα, FCGR2A genotypes were determined by polymerase chain reaction with allele-specific primers. CLL patients were divided into several groups depending on the terms of response to FCR treatment, i.e., achieving partial/complete remission after two, four, six courses of treatment, and those who did not respond to the therapy. Statistically significant differences in the distribution of haplotype frequencies were detected for the following genes: IL-1β (C-3953T, p = 0.02-0.009); IL-10 (C-819T, p = 0.04); IL-10 (G-1082A, p = 0.04-0.002-0.006), FCGR2A (His166Arg, p = 0.006); TLR4 (Thr399Ile, p = 0.02); TLR6 (Ser249Pro, p = 0.04); TLR9 (A2848G, p = 0.04-0.007); CD14 (C-159T, p = 0.03). When testing the significance hypothesis by multiple comparisons, the difference for the detected events was confirmed only for IL-10 gene (G-1082A, p < 0.01; χ2 = 20,082). The results show a relationship between the allelic status of the IL-10-1082 gene and the timing of response to FCR therapy, as well as predict a group of patients with primary-resistant CLL before treatment. The role of the relationship between IL-10 gene polymorphism and IL-10 production is discussed in connection with occurrence risk and clinical course of mature B-cell lymphoid malignancies. IL-10 is thought to be a growth factor for normal and transformed human B-lymphocytes, it controls a balance between cellular and humoral immune responses while exerting a pronounced immunosuppressive activity, along with ability to stimulate tumor cell proliferation. A rationale for conducting pharmacogenomic studies in CLL is provided, in order to predict efficiency of a specific drug or their combination in a distinct patient, thus representing chances to detect a factor which may influence success of the therapy since its earlier stage.

Dendritic cells are specialized antigen-presenting cells which are required to involve naive T-lymphocytes into immune response. Dendritic cells are used in numerous research studies, and have also found practical application as a main component of dendritic cell vaccines. In vitro induction of their maturation from monocytes using cytokines is the most widely used method to obtain these cells. In present study, we compared phenotypes of both immature and mature dendritic cells induced from monocytes of adult healthy donors by means of recombinant cytokines provided by different manufacturers. It is shown that recombinant human IL-4 and GM-CSF supplied by sci-store.ru could effectively stimulate in vitro differentiation of human monocytes to immature monocytic dendritic cells. The resulting immature dendritic cells exhibited a typical morphology and phenotype characterized by the absence of CD14 monocyte marker as well as HLA-DR and CD80 costimulatory molecule expression. A fraction of these cells expressed the CD86 costimulatory molecule. The recombinant human IL-6 from sci-store.ru, when used in a mixture of inflammatory mediators (IL-6, IL-1β, TNFα and PGE2), has induced effective terminal differentiation of immature dendritic cells to mature dendritic cells exhibiting typical morphology and phenotype. Virtually all dendritic cells obtained with this reagent were devoid of CD14, and expressed HLA-DR, CD80, CD83 and CD86 at high fluorescence intensity.

Authenticity evaluation of proteins obtained with recombinant DNA technology is an important step in confirming efficacy and safety of the drugs based on them. One of the main ways to assess the authenticity is to compare molecular structure of the test and standard samples using the peptide mapping method with chromatographic separation of the products obtained by enzymatic degradation. Proper selection of a standard reference sample is essential in order to achieve reliable results. A standard sample of Interferon (CRS, Chemical Reference Substances) recommended by the European Agency for the Quality of Medicines for interferon alpha-2b substances containing N-terminal methionine is inappropriate, since the Interferon CRS sample doesn’t contain methionine. We present the results of development of qualification procedure for methionine form of Interferon alfa-2b industrial standard sample (ISS). The range of use for this ISS is authenticity confirmation for the methionine form of interferon alpha-2b substance using peptide mapping method with reverse-phase high-performance liquid chromatography (reverse-phase HPLC). The quality assessment was performed for all the parameters specified by the manufacturer of this candidate substance at the initial stage of qualification procedure, due to changed application area, and changed package size. Further, 30 peptide cards of the ISS candidate substance were obtained after pre-trypsinolysis of the protein followed by validated HPLC method with proven repeatability.

It was shown that the hydrolysis conditions, i.e., the choice of trypsin preparations, may significantly affect the peptide map profile. Therefore, a reference to specific manufacturer and the catalog number of the product should be provided in description of application conditions for the ISS proposed.

A set of eight reference peaks (peaks of comparison) has been justified, as based on evaluation of peptide maps and results of high-resolution mass spectrometry. The peak with maximally stable yield and intensity was selected as the main peak with an established absolute retention time. Two peaks with relative retention times were chosen as essential peaks for evaluation, i.e., the 1st peak containing N-terminal methionine, and the 2nd peak of highest molecular weight with an established amino acid sequence covering 11% of the studied interferon molecule.

We have also qualified ISS parameters expressed as absolute (minimum for one reference peak), and relative (for the remaining reference peaks) retention time periods. Authenticity of the ISS candidate was further confirmed by the peptide mapping method, as compared with interferon CRS reference standard. Their peak patterns proved to be near-similar, except of a peak with eluted peptide containing N-terminal methionine as revealed by high-resolution mass spectrometry

The aim of present study was to investigate relationships between indicators of functional activity of neutrophilic granulocytes, and hemostasis parameters in patients with acute destructive pancreatitis (ADP). The study included thirty-three patients with ADP. 35 healthy persons were examined as a control group. The phagocytosis level in neutrophils was determined by flow cytometry using FITC-labeled staphylococcal protein A. We have calculated the percentage of fluorescent neutrophils as phagocytic index, and average cell fluorescence assumed phagocytic number. The intensity of respiratory burst observed in neutrophil samples was evaluated using chemiluminescence assay. All the persons under study were also tested for blood coagulation and vascular-platelet hemostasis. It was found that the ADP patients with decreased number of phagocytic neutrophils in the blood showed a decrease in respiratory burst intensity in the neutrophils. Moreover, spontaneous and induced synthesis of the primary reactive oxygen species (ROS) in neutrophils of ADP patients proceeded faster than in healthy people, but its intensity was much lower. The maximal level of spontaneous and induced synthesis of secondary ROS in neutrophils of patients was significantly higher than in healthy individuals, but its rapid may be generally characterized by insufficient respiratory burst in these patients. A reduced neutrophil phagocytic activity and kinetic characteristics of primary and secondary ROS synthesis may be attributed to the effects produced by pancreatic enzymes entering blood flow which may alter functional activity of the blood neutrophils. Concerning hemostasis in patients with ADP, some disturbances were found only in the coagulation link which seem to depend on increase in fibrinogen, soluble fibrin monomer complexes and D-dimer in blood plasma, along with reduced antithrombin III levels. Such a change in blood coagulation indexes is typical to inflammatory processes and presumes activation of the coagulation cascade and higher risk of septic complications. In patients with ADP, we have found a significantly increased number of correlations between indicators of functional activity of neutrophils and hemostasis parameters. This analysis revealed a relationship by the patients with ADP reflect some unidirectional changes in functional activity of neutrophils (as phagocytosis and respiratory burst), and blood coagulation parameters (as blood clotting and vascular/ platelet links). The changes in functional activity of neutrophils and the state of hemostasis in the ADP patients, as well as correlations between their alterations are omvolved into the pathogenesis of this disorder, and determine potential mechanisms for evolving complications.

Studies of probable significance of different immunological mediators for the development of chronic allergic inflammation in patients with allergic bronchopulmonary aspergillosis (ABPA) are necessary in order to specify potential targets for therapeutic intervention and timely diagnosis of the disease. The purpose of present study was to determine the features of immune response regulation, and to identify diagnostic markers associated with development of ABPA in patients with bronchial asthma, and to evaluate clinical and immunological efficacy of specific antimycotic therapy.

The study involved 13 patients with ABPA, 14 patients with bronchial asthma with fungal sensitization (BAFS), 17 patients with bronchial asthma (BA) and 12 apparently healthy individuals. Levels of thymic stromal lymphopoietin (TSLP), thymus and activation-regulated chemokine (TARC), IL-8, as well as levels of total IgE and specific IgE to Aspergillus fumigatus (A. fumigatus) were measured in blood serum by enzyme immunoassay; blood eosinophil counts were also made. Monitoring of these immunological markers in the course of antimycotic therapy was carried out.

Significantly higher numbers of eosinophils, increased levels of total IgE and sIgE for A. fumigatus, as well as TARC and IL-8 in serum were revealed in patients with ABPA when compared to the patients with BA. No significant differences in TSLP content were found between the examined groups of patients. A positive correlation between the levels of sIgE to A. fumigatus and contents of TARC and IL-8, numbers of eosinophils, and total IgE levels confirms the important diagnostic value of proinflammatory cytokines in ABPA patients. In the course of itraconazole medication, a positive clinical and immunological dynamics in ABPA patients was revealed. After 12 weeks of therapy, a significant increase in AST, FEV1 and Tiffno respiratory indexes, along with decreased number of eosinophils, total IgE levels, and a trend towards a decrease in TARC and IL-8 levels were documented. This dynamics confirms clinical efficiency of antifungal drugs when treating chronic allergic inflammation in ABPA patients.

Implementation of modern immunological biomarkers, alongside with traditional indicators, will allow to differentially evaluate a probability for ABLA development in patients with bronchial asthma, to present additional evidence for discerning early stages of the disease, and to conclude about the efficiency of the therapy applied.

At present time, the issue of age-dependence of morphological parameters of the thymus blood vessels in members of various terrestrial vertebrates classes has been poorly studied, due to the lack of comparative morphological approaches. Therefore, the main goal of our work was to specify this kind of dependences. We studied samples of thymus glands from vertebrate animals belonging to four classes: Amphibians, Reptilian, Aves, and Mammals, including humans. For the first time, using methods of light microscopy, we performed comparative morphological studies of thymic blood microvessels in animals and humans, looking for age dependence of these differences. It was found the number and area of microcirculatory bed in thymic cortex and medullary substance depends on age, taxonomic class of the animal as well as on the environmental conditions. We have also revealed age-related differences of the intra-organ vasculature in thymus, including large arteries and venous vessels. Some specific age-related changes were found for human thymic vascular bed. On the basis of our studies, we can make conclusions about functional role of age-related changes of thymic blood vessels in immunity. The results of this study are important for basic and applied biology and medicine, being of interest to researches, especially in immunology.

The aim of present study was to evaluate safety and clinical efficacy of inhalatory immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages applied for treatment of patients with organic brain syndrome (OBS).

Materials and methods. The study under the NCT02957123 protocol (www.ClinicalTrails.gov) included thirty patients with OBS of various genesis (10 men and 20 women aged 18 to 81; Me, 62.5 years). Neurological assessment and the levels of 32 cytokines in the blood serum of patients were evaluated before and 2-3 days after completion of inhalation immunotherapy.

Intranasal inhalations of cell-free culture medium of M2 macrophages (2 mL, once a day for 28-30 days) were safe and well tolerated. None of 30 treated patients had severe adverse events and serious treatmentrelated side reactions. One month after starting the inhalations, a positive dynamics in neurological status was noted in all the patients. A marked clinical response was documented in twenty out of thirty patients (67%), which manifested as improvement, according to all scales and questionnaires. The neurological improvement was not reversed over 6 months of follow-up period. In other ten patients (33%), a moderate clinical response was shown as improvement of individual scores. The positive changes were as follows: 1) a 43% decrease in anxiety and depression scores (according to HADS scale, pU = 0.0008); 2) an increase of total motor activity (stability and gait) by 25%, pU = 0.0001); 3) correction of cognitive functions (MoCa test, pU = 0.007); 4) reduced number and intensity of the disease symptoms by 52% (pU = 0.0001). This marked clinical response to immunotherapy is shown to be associated with correction/normalization of serum hepatocyte growth factor (HGF) level.

Conclusion. Inhalation immunotherapy based on intranasal delivery of bioactive factors produced by M2 macrophages can improve neurological and functional recovery in patients with organic brain syndrome. 

The paper presents evidence that the individuals with different blood groups and glucose levels exhibit individual IgE reagin responses to insulin. Thus, the persons with 0 (I) blood group showed the highest values of anti-insulin-specific IgE (124.83±67.9 kE/L) when their glucose level was under 4 mmol/l. Such values are characteristic to the patients with A (II) and B (III) blood groups with normal glucose levels. Among subjects with B (III) blood group showing decreased glucose values (< 4 mmol/L), the lowest insulin induction (0.85±0.05 mkE/ml) was observed, and the IgE production to insulin increased 80-fold, reaching 113.0±56.0 kE/L. At the first ("borderline") stage of carbohydrate metabolism disorders (glucose levels 6.0 to 7.6 mmol/L), the insulin production is increased 3-fold in 0 (I) and A (II) blood groups, whereas insulin levels in persons with B(III) blood group are near-normal (12.3±3.7), and IgE antibodies to insulin show lowest levels (27.2±9.08 kE/L), thus being 5 times lower when compared to the persons with B (III) blood group. However, carriers of all three group antigens and patients with expressed type 2 diabetes respond to increased glucose and insulin levels in blood by decreased production of specific IgE antibodies to insulin.

Сurrent allergen-specific immunotherapy (AIT) is the most pathogenetically justified method of the IgE-dependent treatment of allergic diseases. Subcutaneous (SCIT) and sublingual (SLIT) AIT are currently used in wide clinical practice. Sublingual immunotherapy is characterized by the convenience of use, decreased incidence of systemic and local side effects, and proven clinical efficacy. However, sublingual form of home dust mite allergen for AIT is not yet produced in Russia. Previously, the technology for sublingual form of D. farinae (Der f) mites allergen was developed in our laboratory, and this extract was shown to be non-toxic and free of any significant side effects. We obtained a sublingual form of mixed D. pteronyssinus (Der р)/D. farina mites allergen for sublingually allergen-specific immunotherapy. We studied physico-chemical and immunobiological properties of the Der р and Der f mites phenol-free water-salt mixed extract (WWSME). WWSME was studied after sterilizing filtration. WWSME was analyzed by electrophoresis in polyacrylamide gel in the presence of dodecyl-sodium sulfate and reverse-phase high-performance liquid chromatography. ELISA was performed with polyclonal rabbit serum, obtained by sensitization of animals with recombinant Der f1 and Der f2 allergens. ELISA shows positive reaction of WWSME with IgG-antibodies of rabbits. We studied acute and chronic toxicity of received mixed allergen in laboratory animals. Pathomorphological studies have shown that WWSME has no detectable toxicity and does not cause local irritation in the animals. The WWSME product contains native proteins, retains immunogenic properties, is non-toxic and does not cause significant side effects. This extract can be used as the basis for designing a sublingual form of dust mite mixed allergen, which is especially promising for pediatric applications.