Peer-rewieved medical journal for medical immunology
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Metabolic reprogramming of microglia and astrocytes is a regulatory factor of neuroinflammation in ischemic stroke
Ischemic stroke is one of the most common diseases worldwide, with a high incidence and mortality rate. In the pathological process of ischemia of nervous tissue, neuroinflammation is an important factor that determines the functional prognosis of the outcome of the disease. During the formation of an ischemic focus, microglial cells and astrocytes are activated, which leads to the launch of a cascade of neuroinflammatory reactions that play an important role in the pathophysiology of ischemic stroke. Activated microglial cells and astrocytes are able to form a variety of phenotypes depending on the corresponding parameters of the microenvironment. These phenotypes can have both neurotoxic and neuroprotective effects. On the one hand, when nerve tissue is damaged, glial cells contribute to the removal of cellular debris, maintain ionic homeostasis, regulate the extracellular content of neurotransmitters and ensure the trophism of neurons. On the other hand, microglia and astrocytes can acquire a pro-inflammatory phenotype characterized by the secretion of inflammatory cytokines, which contributes to the progression of neuroinflammation and tissue damage. Thus, astrocytes and microglia undergo both morphological and functional rearrangements, thereby actively participating in neuroinflammation due to the release of pro-inflammatory or anti-inflammatory factors. It is important to note that these rearrangements are associated with metabolic reprogramming, which leads to a change in the activity of metabolic pathways to compensate for the lack of energy and building materials caused by impaired cerebral blood flow. The pro-inflammatory phenotype of microglia is characterized by activation of glycolysis, the pentose phosphate pathway, synthesis of fatty acids and glutamine, whereas the anti-inflammatory phenotype demonstrates increased oxidative phosphorylation and oxidation of fatty acids. Reactive astrocytes are characterized by increased glycolysis, glycogenolysis and reduced glutamate uptake. Recently, there has been increasing evidence that manipulation of glial cell homeostasis can be used to switch from a neurotoxic phenotype to a neuroprotective one. A comprehensive understanding of the basic mechanisms of switching metabolic phenotypes can potentially allow targeted reprogramming of glial cells during the pathological process, which can be used in therapeutic approaches for the treatment of the consequences of ischemic stroke. This review presents current ideas about metabolic reprogramming in astrocytes and microglial cells in the context of pathophysiological processes in cerebral ischemia.
Features of immune response to xenogeneic tissues of cardiac valves and patches: Review of literature
Global studies show that valvular heart disease still takes one of the leading places in the structure of mortality from cardiovascular diseases, being among the major causes of heart failure, including those among the employed population. Xenogeneic tissues are widely used in cardiac surgery, both in biological prosthetic heart valves, and as vascular and intracardiac patches. Modern chemical methods of xenogenic tissue treatment aimed at elimination of its immunogenicity but they do not, however, completely remove xenoantigens from the tissues. The residual carbohydrate antigens are thought to be a trigger of immune response against the animal xenotissues. At the same time, the role of immune response to xenogeneic antigens for induction of inflammation, valve dysfunction, and calcification are under discussion. The aim of this review was to summarize the research data on immune response to xenogeneic tissue implanted into the heart, and to find tools of preventing this immune conflict. Modification of pericardium of large animals by various methods does not entirely remove carbohydrate epitopes from extracellular matrix and cell membranes, which are recognized by pre-existing antibodies of M and G classes. The highly dynamic functioning of xenogeneic biological prostheses increases their antigenicity by reducing the primary cross-linking of extracellular matrix and activating the alternative complement pathway associated with adsorption iC3b complement component on xenogeneic tissue, serving as an opsonin for micro- and macrophages. The inflammatory endotypes of individual patients may be genetically determined by increased synthesis of certain cytokines. In particular, rheumatic heart disease, as a basis for the disorders of mitral heart valves, is characterized by an increase in TNFα, IFNγ and IL-6. Any of these cytokines may be targets for biological therapies aimed at limiting the constitutional inflammatory endotype. The OMICs technologies applied to various studies of biological degradation of xenogeneic heart valve prostheses, their implantation, and wide clinical examination of patients, may help us to find novel variants of immune-inflammatory endotypes leading to dysfunction of bioprostheses, and to identify target molecules for potential inhibition of antixenogeneic immune response.
Viral infection and lipopolysaccharide: Pathological axis “lung–joints” as a potential cause of osteonecrosis
The article concerns the effects of viral infections, e.g., SARS-CoV-2, and bacterial lipopolysaccharide (LPS), a component of the cell wall of Gram-negative microbes, on the development of osteonecrosis via the pathological lung–joint axis. Viral infection causes damage to lung cells and vascular endothelium, and leads to inflammatory and blood clotting disorders, increasing the risk of bone ischemia. LPS, by interacting with TLR4 receptors, enhances the inflammatory response and disrupts the blood supply to bones, thus stimulating resorption and preventing bone formation. In addition, the article highlights the role of lung dysbiosis caused by viral infection, which enhances inflammation and increases permeability of tissue barriers for endotoxins. Appropriate information was searched for the keywords “osteonecrosis and lipopolysaccharide”, “COVID-19 virus and lipopolysaccharide-binding protein”, “viremia and osteonecrosis”, “lung microflora and LPS” in foreign and domestic scientometric databases such as PubMed and eLIBRARY. The presented data suggest that the combined effect of imbalanced LPS-binding systems, impaired pulmonary barrier, viral infection and LPS exposure is an important aggravating pro-inflammatory factor. This combination may propote a predisposal for the full-scale osteonecrosis. This concept seems to be promising for clinical research of potential tools influencing these conditions, either single factors or their combined effects. This study focuses on the variety of effects exerted by viral infections and bacterial LPS upon the bone tissue. Current research shows that influencing these mechanisms may open up new ways for the development of therapeutic strategies. The prospects of using targeted therapies to mitigate the negative effects of these interactions are also being considered. These data emphasize a need for integrated approach in the study and treatment of osteonecrosis, with respect to both infectious and inflammatory components of this disorder.
Antitumor effects of small interfering RNAs
The aim of our work was to summarize the existing results in anticancer therapy with small interfering RNAs (siRNAs), as well as to highlight the role of siRNA as a remodulator of the immune response in oncological diseases. The literature review includes an analysis of research publications from the PubMed, Embase, eLIBRARY, CyberLeninka and Web of Science, CNKI and MEDLINE databases. Sufficient challenges in cancer treatment occur due to the immune tolerance of tumors, their drug resistance, and a number of limitations in usage of conventional treatment schedules. The novel RNA interference (RNAi)-based approaches, may be implemented using small interfering RNA (siRNA) molecules. These methods may offer promising therapeutic strategies that will promote an immunomodulatory effect and targeted suppression of gene expression important for tumor growth. Inhibition of tumor regulatory pathways will affect tumor proliferation and metastasis, while mobilizing antitumor immunity through stimulation of Toll-like receptors, maturation of dendritic cells, and infiltration of cytotoxic T lymphocytes into the tumor. The in vivo studies of this approach in experimental models showed a decrease in the volume of melanoma, breast tumors and hepatocellular carcinoma up to 5 times, suppression of metastasis and increased overall survival. Cancer treatment is among the most significant problems of modern medicine, characterized by high mortality, mechanisms of immune tolerance and frequent emergence of resistance to existing therapeutic approaches. Despite the progress in modern oncoimmunology, usage of checkpoint inhibitors and targeted antitumor agents, the effectiveness of current approaches is limited by immunosuppressive microenvironment of tumors, heterogeneity of malignant cells, adverse effects and toxicity of the drug therapy. RNAi treatment is a promising approach that can simultaneously solve several key problems of oncoimmunology, such as suppression of the driver oncogenes; blocking tumor signaling pathways; as well as activation of innate immunity. The dual effect of RNAi, which consists of a direct effect on tumor cells and immune modulation of the tumor environment, makes RNAi an excellent tool for overcoming tumor immunotolerance and providing a direct cytotoxic antitumor effect.
Platelets and platelet-derived microvesicles: Phenotype, content, impact on endothelial cells
Blood platelets are circulating anuclear structures derived from megakaryocytes. Intercellular adhesion molecules, Toll-like receptors, chemokine and cytokine receptors are represented on their surface. Platelets contain biologically active molecules, including chemokines, cytokines, and growth factors. Due to their cytological features (membranes and secretory granules), the platelets are capable of fast activation and interactions with different cells. Platelets are involved in hemostasis, immune reactions, and angiogenesis, being activated by sub-endothelial components, complement proteins, secretion products from other platelets. The activation is mediated via calcium ions. Upon these events, the platelets change their morphology, release secretory granules and produce microvesicles, a relatively new target of biological research. The aim of this review is a comparative description of platelets and their microvesicles. Platelet-derived microvesicles perform platelets functions and communicate with other cells, including endothelium. Microvesicles represent a promising object of research, and the opportunities of their applications for diagnostics and therapy are being actively studied. Majority of circulating microvesicles are of platelet origin. The platelet-derived microvesicles contain cytokines and other proteins, lipids and nucleic acids (DNA, mRNA, microRNA). Microvesicles bear the surface markers of parental cells; phosphatidylserine is represented on their membrane, which additionally participates in clotting, due to deposition of blood coagulation factors. Under the influence of signals from microenvironment, the composition, phenotype of platelet microvesicles, as well as their functional abilities towards endothelium may vary, depending on the actual stimuli. Their effects upon angiogenesis and regeneration have not been sufficiently elucidated, with controversial effects reported in experimental studies. Increased levels of platelet microvesicles are observed in the disorders accompanied by endothelial dysfunction, thus suggesting their possible participation in these events. The effects of platelets and their microvesicles on endothelium, including activation of various signaling pathways in endothelial cells, still remain the subject of further research.
Effect of avian bursa of Fabricius extract on the compensatory potential of bone marrow, spleen, and thymus cells in cyclophosphamide-induced mice
Malignant neoplasms often arise under immunodeficiency conditions. However, anticancer chemotherapeutic drugs can also cause the development or aggravate immunodeficiency by affecting the bone marrow function. Previous studies have shown that the bioorganocomplex obtained from the chickens’ Fabricius bursa has an antioxidant and immunotropic effect. The aim of this research was to investigate the protective mechanism of the bioorganocomplex “Bursanatal” upon cyclophosphamide-induced immunosuppression in the bone marrow, thymus, spleen of C57Bl/6 mice. Immunodeficient state was induced with single cyclophosphamide injection (200 mg/kg). Histological, morphometric, radiological methods have been used, as well as immunohistochemical staining of the spleen and thymus with CD45 and CD3 antibodies. The studies showed that cyclophosphamide injection affects both peripheral blood and bone marrow. Moreover, neutrophilic, basophilic and erythroid precursor cells are less sensitive to the cyclophosphamide. Subsequently, on the 8 th day their compensatory hyperplasia has been observed. The use of mentioned bioorganocomplex was accompanied by increased counts of leukocytes, erythrocytes and hemoglobin in peripheral blood. Bone marrow cellularity also enriched, associated with active division and maturation of erythroid, myeloid, and lymphoid cells. Bioorganocomplex administration causes a significant decrease of the red pulp area in spleen of mice with cyclophosphamide-induced immunodeficiency. Activation of extramedullary hematopoiesis was found in spleen of treated mice, i.e., the number of colony-forming hematopoietic progenitors was increased. It was also noted that the reactive center areas in the white pulp were reduced, with a significant increase of CD3+ cell contents in red and white pulp under the “Bursanatal” treatment. At the same time, the preparation did not affect thymus pathomorphology.
In vitro effects of paleobacteria exometabolites from permafrost soils on development of immune response
We have tested the effects of metabolites from paleobacteria Bacillus cereus, strain 875 TS, from Pleistocene-Holocene permafrost rocks aiming to assess the mechanisms and features of in vitro immune response in the culture of human peripheral blood mononuclear cells. It was found that paleobacterial exometabolites significantly activate monocyte differentiation into subpopulations of intermediate (CD14+CD16+) and non-classical (CD14loCD16+) monocytes, effector CD4+ and CD8+T lymphocytes, with changes of early (CD69), intermediate (CD25) and late (HLA-DR) activation markers, Treg differentiation (CD3+CD4+CD25hiCD127-). These metabolites also stimulated the synthesis of IFNγ and IL-4 cytokines as compared with control levels. The differential influence of paleobacterial exometabolites upon immunomodulatory activity depended on temperature regimen of their production, i.e., “cold” (obtained from bacteria during their cultivation at 5 °C), “medium-temperature” (22 °C) and “heat” (37 °C) regimens. “Cold” metabolites stimulate predominantly the immune response mechanisms with proinflammatory activity, i.e., differentiation of intermediate CD14+CD16+ monocytes, increased differentiation activity of CD8+T lymphocytes, and synthesis of IFNγ. “Warm” metabolites stimulate mostly the immune response mechanisms with anti-inflammatory activity, namely, differentiation of non-classical CD14loCD16+ monocytes, increased differentiation activity of CD4+T lymphocytes and secretion of IL-4. Another distinctive feature is the ratio of pro- and anti-inflammatory mechanisms, which does not depend on the type of exometabolites. Thus, during the first three days of cell cultivation, the differentiation activity of CD8+T lymphocytes prevails over the differentiation of CD4+T lymphocytes, and the level of IFNγ secretion exceeds the IL-4 amounts. On the third day, there is a significant increase in the Treg level, which is accompanied by a tendency to normalize the balance between IFNγ (Th1) and IL-4 (Th2) by the seventh day. We have observed a clear effect of Treg (CD3+CD4+CD25hiCD127-) on the strength and duration of the immune response. The increase in Treg levels occurs moderately and transiently, which, on the one hand, prevents the excessive development of proinflammatory mechanisms, and on the other hand, does not lead to the development of long-term immune suppression. An increase in the Treg level on days 1-3 is accompanied by a decreased activity of monocyte differentiation into the subsets and the synthesis of proinflammatory IFNγ cytokine. Considering that one of the main functions of induced Treg is the suppression of systemic inflammatory, autoimmune and allergic diseases, increase in their activity under the influence of exometabolites of paleobacteria Bacillus cereus strain 875 TS may serve as a basis for the development of new biopreparations for treatment of a wide range of diseases.
Immunogenic properties of synthetic peptides copying the V3 loop of HIV-1 gp120 envelope protein: effects of various administration routes and poly(I:C) immunoadjvant use
Synthetic peptides provide a promising basis for HIV vaccine development. Following their administration, the immune response is focused only on a specific epitope. Moreover, they are able to activate both humoral and cellular pathways of immune response, being safe and well tolerated. Due to low molecular weight, the synthetic peptides exhibit low immunogenicity, therefore requiring usage of various immunoadjuvants in immunogenic compositions. The V3 loops of gp120 envelope protein are among the main protective epitopes, with a number of monoclonal antibodies with broad neutralizing activity having been obtained to this antigen. We have studied the immunogenicity of peptides copying the V3 loop of the group M HIV-1 virus consensus sequence, and the Russian viral isolate RUA022a2. We have also assessed the possible impact of its administration route (subcutaneously versus intraperitoneally) and usage of an immunoadjuvant. poly(I:C), a synthetic analogue of double-stranded RNA, being a ligand of TLR3 innate immunity receptors, was used as an adjuvant. The studies were conducted on Balb/c mice. It has been shown that the route of administration did not affect an immune response development to the tested peptides. However, earlier production of specific IgG antibodies was observed in the groups treated with immunoadjuvant. At the same time, the antibody titer was slightly higher in the groups where peptides were administered with the adjuvant after the 3rd (last) administration. No differences have been revealed in the isotypes of induced antibodies. IgG1 antibodies were predominantly induced in all groups. Specific IgM antibodies were detected only after 3rd injection of the antigens. The antibody titer did not depend on the administration route, being slightly higher in the groups where peptides were administered with the poly(I:C) adjuvant. The induced antibodies did not exhibit neutralizing activity against the QF495.23.M.EnvA1 isolate. When studying antigen-specific cellular immune activation, the production of IFNγ, the Th1 response marker was detected only in poly(I:С)-treated groups. In addition, a low level of anti-inflammatory cytokine IL-10 was determined in groups where poly(I:С) was included in the immunogenic composition. Moreover, the highest IL-10 level was detected in groups with intraperitoneal administration. Our studies have shown that the use of poly(I:С) adjuvant promotes immune response to the synthetic peptides, thus contributing to earlier induction of specific antibodies as well as switching to the Th1 pathway. The data obtained may be used for development of vaccines against HIV and other viral infections, in order to increase their immunogenicity and ability of inducing a protective immune response.
Effect of iron-molybdenum nanocluster polyoxometalates on the functional activity of macrophages and the state of bone marrow erythroblastic islands
The aim of this study was to determine the mechanisms of action of {Mo72Fe30} on erythropoiesis and hematological parameters in vitro and in vivo. The obtained results showed that neither the nanocluster polyoxometalate {Mo72Fe30} nor its destruction products (DP) – low-molecular-weight iron and molybdenum-containing ions – affected the activity of non-specific esterase in the cytoplasm of central macrophages of bone marrow erythroblastic islands, thus indicating no disruption of endotoxin and xenobiotic detoxification processes carried out by these macrophages. However, administration of the studied substances resulted in a decrease in macrophage phagocytic capacity without altering cellular involvement in phagocytosis. Analysis of erythroblastic island cultures revealed the following patterns: administration of {Mo72Fe30} and its accelerated erythroid cell maturation within the erythroblastic island corona. At 24 hours after their administration, a decreased number of class 3 and involutive erythroblastic islands was observed, due to accelerated maturation and dissociation of erythroblastic islands. This effect was confirmed by an increased number of erythroid cells in the myelogram at 24 hours after these substances administration. A simultaneous increase in the number of reconstructing erythroblastic islands in the groups treated with the polyoxometalate and its DP suggests development of an additional wave of erythropoiesis within the island corona. On day 2 after administration of {Mo72Fe30}, there was an increase in the number of proliferating erythroblastic islands of the class 2 and 3, while the number of class 1 islands remained unchanged. This is attributed to the involvement of new macrophages into erythropoiesis and active erythroid cells proliferation. On day 3, a shift to proliferation of more mature class 2 erythroblastic islands was observed. These data indicate the acceleration of erythrocyte maturation within erythroblastic islands after 3 days of the {Mo72Fe30} administration. A similar trend was observed with the administration of its destruction products. Still, the erythropoiesis-accelerating effect was less pronounced in this series. Analysis of rat bone marrow myelograms revealed an increase in bone marrow cellularity after seven days of administration of the studied substances ({Mo72Fe30} and DP). This increase was attributed, in part, to an increase in the number of erythroid cells and reticulocytes. These data are consistent with peripheral blood parameters in rats. A statistically significant increase in erythrocyte count, hemoglobin levels, and hematocrit values was observed following administration of the studied substances ({Mo72Fe30} and PD). The experimental results suggest a potential stimulatory effect of this compound on erythroid lineage development.
CXCR1, TLR4, and CXCL8: Key Mediators of Pseudomonas aeruginosa Virulence in Wound and Burn Infections
Pseudomonas aeruginosa, an opportunistic pathogen of considerable clinical import, presents a formidable challenge to susceptible individuals, particularly within the confines of nosocomial settings. Those with compromised immune systems, indwelling medical devices such as catheters, and extensive thermal injuries are especially vulnerable to its insidious and often devastating effects. This investigation sought to elucidate the roles of Toll-like receptor 4 (TLR4), Toll-like receptor 5 (TLR5), the chemokine CXCL8, and its cognate receptor CXCR1 in the context of P. aeruginosa infection. Our findings indicate a significant involvement of TLR4 and CXCL8 in the host response to this pathogen. Notably, CXCR1 expression was observed to be downregulated both in cellular models and in whole blood samples obtained from patients afflicted with bacterial infections, specifically those caused by P. aeruginosa. Utilizing antibodies targeting these cell surface molecules, we further explored their influence on bacterial adhesion and the modulation of infection. The application of anti-CXCR1 antibodies resulted in a demonstrable increase in bacterial infection in both T24 and A549 cell lines. Conversely, the administration of anti-TLR4 antibodies exerted an inhibitory effect on P. aeruginosa infection. Anti-CXCL8 antibodies, however, did not elicit a discernible impact on bacterial infection within the initial three hours of exposure. These observations suggest a dichotomous role for these molecules in the host response to P. aeruginosa: CXCR1 appears to function as a negative regulator, while TLR4 appears to act as a positive regulator in the context of bacterial infection.
Clinical and immunological features of PD-1 and Tim-3 expression on monocytes in axial spondyloarthritis
Regulation of inflammation events in inflammatory/autoimmune rheumatic diseases, particularly axial spondyloarthritis, is critically important for alleviating clinical symptoms. Recent studies have shown the key role of PD-1 and Tim-3 inhibitory receptors in regulating the functional phenotype of monocytes in inflammatory/autoimmune diseases. The aim of this study was to investigate the expression of the PD-1 and Tim-3 inhibitory receptors in different subsets of monocytes in patients with axial spondyloarthritis (axSpA), and to evaluate their association with clinical features, as well as with tolerogenic marker Mer tyrosine kinase (MerTK). The expression of PD-1 and Tim-3 in classical, intermediate, and non-classical monocytes was determined by means of multicolor flow cytometry in peripheral blood mononuclear cells in 60 patients with axial spondyloarthritis and 40 healthy donors. The analysis of circulating monocyte subpopulations revealed an increased proportion of classical monocytes in patients, which positively correlated with systemic inflammation markers: erythrocyte sedimentation rate and C-reactive protein. Like as in donor group, the highest expression of PD-1 and Tim-3 in axSpA patients was observed in intermediate and non-classical monocyte subsets. However, the relative proportions of PD-1+ and PD-1+Tim-3+ cells in intermediate and non-classical monocytes were significantly decreased in axSpA patients, whereas Tim-3 expression levels did not differ from the donor values. Notably, the proportion of PD-1+ cells directly correlated with expression of MerTK, a factor mediating the anti-inflammatory activity of monocytes/macrophages in all monocyte subsets. A statistically significant decrease in PD-1 expression and co-expression of PD-1 and Tim-3 was detected in HLA-B27 antigen carriers, patients with advanced and late-stage disease, and those with peripheral axSpA, including hip joint involvement. On the contrary, in HLA-B27-negative patients and those with early/non-radiographic stage disease, PD-1 expression was comparable to donor levels, while Tim-3+ cell contents were increased among classical monocytes. In patients receiving first-line therapy, the decreased PD-1 expression and PD-1+Tim-3+ co-expression directly correlated with disease activity. The obtained data that include a reduced PD-1 expression and PD-1+Tim-3+ co-expression in intermediate and non-classical monocytes in patients with genetic predisposition, advanced disease stages, and peripheral joint involvement, as well as the association between PD-1+ cell number and MerTK expression in all monocyte subsets, suggest a dysregulation of inhibitory checkpoint receptor expression under inflammatory conditions. These findings could presume a diminished anti-inflammatory capacity of monocytes.
Alterations in peripheral blood CD8+T cell subsets in patients with lung sarcoidosis
Sarcoidosis is a systemic inflammatory disorder of unknown etiology characterized by tissue infiltration with macrophages and lymphocytes, including CD8+T cells, and associated non-caseous granuloma formation. The aim of the study was to investigate various peripheral blood CD8+T cells from patients with chronic respiratory sarcoidosis using markers of T cell maturation and ‘polarization’. Peripheral blood samples were collected from 34 patients with newly diagnosed chronic sarcoidosis of respiratory organs with the background of a natural course of disease, and without a history of immunosuppressive therapy. The diagnosis of pulmonary sarcoidosis was performed according to the standard criteria and was confirmed by histological examination for 94.1% of patients. Peripheral venous blood samples from healthy, gender- and age-matched volunteers (n = 40), were used as control specimens. Multicolor flow cytometry revealed that patients with sarcoidosis had decreased levels of CD45RA+CD62L+ ‘naïve’ and CD45RA-CD62L+ central memory CD8+T cells as compared with healthy controls. Moreover, the frequencies of ЕМ1 (CD45RA-CD62L-CD27+CD28+) and pre-effector type 1 (CD45RA+CD62L-CD27+CD28+) cells were also reduced. In order to assess the relevant ‘polarized’ CD8+T cell subsets, we have specified the Tc1 (CCR6-CXCR3+), Tc2 (CCR6-CXCR3-), Tc17 (CCR6+CXCR3-), and double-positive Tc17.1 (CCR6+CXCR3+) cell populations. The relative and absolute numbers of CXCR3-expressing CD8+T cell subsets (Tc1 and Tc17.1 were found to be significantly decreased in patients with sarcoidosis if compared to healthy controls. By contrary, Тс2 CD8+T cell contents were significantly elevated. Furthermore, the relative numbers of Tc1 cells negatively correlated with serum ACE levels (r = -0.456; р = 0.01), whereas Тс2 levels positively correlated with serum ACE levels (r = 0.623; р < 0.001). Thus, our results indicate that CD8+T cells may play a role in pathogenesis of sarcoidosis. More extensive clinical and immunological comparisons are required for further systematization of the obtained data.
The issues of assessing reference ranges for the main subpopulations of blood lymphocytes in children at different ages
The age-related dynamics of main subsets of blood lymphocytes in children has been studied quite well, but the issue of the limits of reference values, especially at an early age, remains actual and important. The paper analyzes results of a direct study dataset of 624 blood samples of healthy children aged 1 week to 17 years and 11 months, residents of Moscow and the central Russian regions who underwent a clinical blood count during routine checkups. The main criterion for the complete inclusion of a child into the study was reliable information about the absence of acute and chronic health disorders. The absolute number of lymphocytes was determined as a complex of indices obtained of automated blood analysis, using 5 Diff technology of flow hemocytometry with fluorescent staining of nucleic acids. The percentage and absolute values of T lymphocytes, T helper cells, T cytotoxic cells, the ratio of helper/cytotoxic lymphocytes, as well as percentages and absolute numbers of B lymphocytes and natural killers were determined in residual blood samples by flow cytometry with 3-4 color staining. 14 age groups were formed with the number of cases from 40 to 64 in each group. The Kolmogorov–Smirnov method confirmed the normal distribution of the obtained numerical indicators, both in the general group and in all distinct age groups. The article provides detailed results of processing the data obtained using nonparametric (median, interquartile range, range from 10 to 90 centiles) and parametric (mean and standard deviation) statistics methods. Based on an estimate of the range of 10-90 centiles, the normal limits for the percentages and absolute values of the main lymphocyte subpopulations were calculated. We have been confirmed the well-known trend for a gradual decrease in the absolute number of lymphocytes and absolute values for all major subpopulations, as well as relatively higher indices of helper/cytotoxic ratio in early age groups. A higher percentage of B lymphocytes was detected in children in the age groups from 2 months to 2 years, which, together with a high total number of lymphocytes, determined fairly high absolute B cell counts in these age groups. The proposed normal ranges were validated on an independent group of 75 children aged 2 to 18 years. The individual indexes of the children in the validation group exceeded the reference values in 4 cases (5.3%, with acceptable level of discrepancies < 10%), which allows us to consider the norms validated. The most debatable findings concern a relatively high absolute level of В lymphocytes in young children which is, however, in good agreement with the pooled data of the world-wide meta-analysis data. The proposed normal ranges are designed in a form that is convenient for practical use.
Correction of immune disorders in acute pyelonefritis at different trimesters of pregnancy
The issues of pathogenetic and restorative therapy in gestational pyelonephritis during pregnancy are relevant being discussed in modern evidence-based medicine. Treatment of these patients is associated with certain controversies, since during pregnancy it is necessary not only to provide for relief of the inflammatory process in maternal urinary system, but also not to affect the foetus by the medication applied. This problem is characterized by an annual increase in morbidity and complications, which often have an extremely negative effect on the course of pregnancy and childbirth at the annual rate of 3-17% of women of childbearing age in Russia. The purpose of our study was to determine the effectiveness of using Viferon in stabilizing immune disorders in gestational pyelonephritis. The study included 80 women (mean age 32.4±3.7 years) divided into groups: control (consisting of healthy non-pregnant female volunteers), two groups with acute pyelonephritis in the 2 nd and 3 rd trimesters of pregnancy, receiving basic therapy, and two groups of pregnant women with a combination of basic treatment and the immunomodulator Viferon. The cytokine spectrum, state of the complement system and functional-metabolic activity of neutrophils were studied in peripheral blood. Blood samples were taken before the start of complex treatment, i.e. immediately upon admission, and after the course of therapy (upon discharge from the hospital), with proven evidence of clinical relief of the disease. More effective relief of immune inflammation of acute pyelonephritis was revealed when using Viferon in the 2 nd and 3 rd trimesters of pregnancy compared to conventional therapy. The proposed treatment method allows for faster normalization of altered immune status parameters and the fastest clinical recovery of patients. This approach in medical practice, along with conventional treatment, is a relevant, safe and effective method for treating acute gestational pyelonephritis, which will reduce the incidence of obstetric and urological complications, reduce the number and costs of hospital stays, improve the quality of life of pregnant women and create a healthy environment for the fetal development.
Bioinformatic analysis of single nucleotide variants in the F12 gene associated with hereditary angioedema
Hereditary angioedema (HAE) is a genetically determined disorder, a primary immunodeficiency involving complement system dysfunction. In most patients, the disease is characterized by a deficiency of C1 inhibitor (type I HAE) or impaired functional activity of the C1 inhibitor (type II HAE). In such cases, the diagnosis is based on laboratory findings. In HAE with normal C1 inhibitor levels and activity, the diagnosis can only be based on family history and/or genetic testing. Among patients with HAE with normal C1 inhibitor, mutations in the F12 gene are most frequently observed, particularly in females. However, mutations with uncertain clinical significance are often identified. Given the limited number of HAE cases, it is not feasible to experimentally determine the clinical relevance of newly discovered polymorphic variants. A potential solution to this problem is the in silico analysis of each novel polymorphism. Hence, the aim of our study was to evaluate the predictive potential of bioinformatic methods in assessing polymorphic variants in the F12 gene. The study was focused on four polymorphic variants: NC_000005.9:g.176831285C>G, NC_000005.9:g.176831258C>G, NC_000005.9:g.176831232G>C, and NC_000005.9:g.176831232G>T, with varying clinical significance statuses. To predict the effect of these polymorphic variants on the F12 protein, we used various Web-based tools employing different algorithms, including SIFT, PolyPhen-2, FATHMM-XF, MutationTaster2021, MutPred2, MUpro, I-Mutant 2, HOPE, and ChimeraX. The in silico approach demonstrated that the NC_000005.9:g.176831232G>C (p.Thr328Arg), and NC_000005.9:g.176831232G>T (p.Thr328Lys) mutations have a pathogenic effect, which is fully consistent with their previously established clinical status. At the same time, the polymorphic variants NC_000005.9:g.176831258C>G (p.Gln319His), and NC_000005.9:g.176831285C>G (p.Arg310Ser) do not appear to be independent causes of the disease. However, their potential role in modifying the clinical phenotype cannot be excluded. Bioinformatic analysis plays a key role in preliminary assessment of clinical significance of newly detected mutations in the F12 gene and provides a more precise identification of pathogenic variants. Integration of bioinformatic tools into diagnostic workflows is essential for determining the cause of disease in patients with hereditary angioedema presenting with normal levels and functional activity of the C1 inhibitor.
Hematopoietic effects of spleen lymphocytes treated with an original anticonvulsant during long-term alcohol consumption
Excessive alcohol consumption has a negative effect on hematopoiesis, which manifests with significant suppression of both blood cell production, and structural changes in hematopoietic precursors, i.e., by suppression of their maturation, up to pancytopenia. One may distinguish between the direct effect of alcohol (toxicity to bone marrow, hematopoietic precursors and mature blood cells), and the indirect action caused by deficiency of trophic factors. Alcohol addicts often develop anemia, due to premature destruction of erythroid cells. Thrombocytopenia, also being an important feature of hematological disorders in alcoholism, results in spontaneous bleeding and petechiae. Chronic alcohol consumption also has a suppressive effect on production and functioning of white blood cells, resulting in poor resistance to bacterial infections. We have previously identified the immunomodulatory properties of an innovative anticonvulsant, meta-chlorobenzhydrylurea (m-CBHU). Its positive effect was determined upon intragastric administration in long-term alcoholized mice, In a recent study, splenic lymphocytes, being in vitro exposed to the mentioned anticonvulsant, have shown a positive psychoneuromodulatory effect during chronic ethanol intoxication. These effects seem to proceed via relatively independent mechanisms. In this study, the effects of intravenous transfusion of m-CBHU-treated spleen lymphocytes on bone marrow hematopoiesis and peripheral blood cells were tested in murine model of chronic alcoholism. In the bone marrow of syngeneic recipients (long-term alcoholized mice), a decreased colony-forming activity of hematopoietic precursors was observed: the population of erythroid precursors was significantly reduced. Decreased counts of granulocyte-macrophage precursors were also detected at a trend level. The only exception was the population of early progenitors, where the number of colonies did not change. In peripheral blood, a decreased number of lymphocytes, platelets, erythrocytes and leukocytes was observed associated with increase in the population of segmented neutrophils, suggesting peripheral inflammation. Lymphocytes pre-cultured with meta-chlorobenzhydryl urea, after intravenous administration to syngeneic long-term alcoholized recipients, had a corrective effect on a number of hematopoietic parameters, which manifested with restoration of the colony-forming activity of bone marrow hematopoietic precursors to the levels comparable to those in intact age-matched mice, along with decrease of segmented neutrophils and restoration of RBC and lymphocyte counts as wells as a tendency for increase in platelet counts in peripheral blood. The data obtained may suggest the efficiency of meta-chlorobenzhydrylurea-modulated lymphocytes in correction of distinct changes in hematopoiesis associated with long-term ethanol intoxication.
The effect of immunization with foreign proteins on the activity of mitochondria in rat thymocytes and splenocytes
Studies of cellular metabolism in development of immunological memory is still relevant since it can help to deepen our knowledge on pathogenesis of immune-inflammatory diseases and provide a basis for improving their prevention and treatment. The aim of the present study was to evaluate the activity of mitochondria in thymocytes and splenocytes after immunization of rats with human albumin. Male Wistar rats were immunized with human albumin for 4 days. The total amount of protein injected was 135 mg. 24 hours after the last injection, we evaluated hematological parameters (general blood test), and mitochondrial activity of thymocytes and splenocytes by measuring rhodamine 6 G fluorescence by means of the Evos M7000 imaging system in RFP mode. We also calculated the mass ratios of the thymus and spleen relative to the body mass of the animals. Statistical analysis included assesment of the median, upper and lower quartiles, and comparisons of hypotheses using the Mann–Whitney U test and correlation analysis according to Pearson’s method. Hypertrophy of the thymus was revealed in animals of the immunized group by 56.9%, p = 0.032, and no changes in the mass of the spleen were observed. A decreased mitochondrial fluorescence was detected in thymocytes by 23.5% (p = 0.037) and splenocytes by 13.7% (p = 0.548) which may be associated with immunosuppressive effect of TGF-β during repeated administration of a foreign protein. Moreover, we have found monocytosis exceeding the control by 59.4%, (p = 0.030), along with thrombocytopenia (by 20.8%, p = 0.045). These changes, were observed in experimental rats thus reflecting reactive changes occurring during immunization. At the early stages after immunization of animals, we have revealed inhibition of energy exchange in thymic and spleen cells. This finding may reflect the effects of inflammatory mediators on mitochondria of lymphocytes and to suggest promising targets for treatment of immuno-inflammatory disorders.
Inhibition of aggressive glioblastoma cell line growth by enhancer rna silencing depends on the knockdown method
Glioblastoma is the most frequent and aggressive brain tumor, with a low survival rate. One of the challenges in treating glioblastoma is the resistance of cells to temozolomide, the main chemical agent used to treat the disease. The STAT3 gene is known to play a significant role in the growth and metastasis of tumor cells. Inhibition of STAT3, or suppression of its expression reduces the resistance of glioblastoma cells to temozolomide. Enhancer RNAs (eRNAs) are non-coding RNAs that are transcribed from enhancer regions. eRNAs can affect the expression of key genes in various biological processes, including carcinogenesis. Previously, we conducted a search for an eRNAs that may potentially affect glioblastoma cell resistance to temozolomide. Knockdown of TMZR1-eRNA (ENSG00000289579) in DBTRG-05MG cell line resulted in decreased STAT3 gene expression and increased cell sensitivity to temozolomide. The effect of enhancer RNA knockdown on STAT3 gene promoter activity has been shown, both alone and together with the enhancer element from which this eRNA is transcribed. The current study was designed to investigate the potency of TMZR1-eRNA in a glioblastoma U-251 cell line. Compared to the previously studied cell line DBTRG-05MG, the U-251 cell line is more aggressive, has a higher proliferation rate and was derived from patient tissue at a later stage of the disease. Knockdown of TMZR1-eRNA by small interfering RNAs resulted in a decrease in STAT3 gene expression. No effect on cell growth was observed both when temozolomide was added and under control conditions of culture medium with DMSO. To study the effects of constitutive (long-term) eRNAs suppression, were have designed U-251 cell cultures expressing short hairpin RNA (shRNA) targeting TMZR1-eRNA and control vector. A significant increase in STAT3 mRNA expression was observed in the obtained cell lines relative to the control. At the same time, viability analysis demonstrated a significant slowdown in the growth rate of the cell line expressing short hairpin RNAs to TMZR1-eRNA compared to the control. A significant effect was observed both when temozolomide was added, as well as in DMSO-containing culture medium.
Evaluation of neutrophil granulocyte respiratory burst in colorectal cancer
Colorectal cancer (CRC) is the third most common cancer in men and the second most common cancer in women. The ambiguity of neutrophils in the implementation of the immune response indicates their transformation into protumor cells in the late stages of the disease. In this regard, the aim of the study was to study the respiratory burst of neutrophilic granulocytes (NG) in the blood of patients with CRC at different stages of the disease and in the dynamics of treatment. The study included 99 patients with CRC in different stages and 112 practically healthy volunteers. The respiratory burst of neutrophils was assessed using chemiluminescent analysis of neutrophil granulocyte activity for 90 minutes on a 36-channel chemiluminescent analyzer “CL3607” (Russia). Statistical data processing was performed using Statistica 10 software packages (StatSoft Inc, USA). Statistical significance of differences was determined using the Mann–Whitney criterion equal to p < 0.05. In patients with CRC, an increase in the intensity of chemiluminescence (CL) and total synthesis of reactive oxygen species (ROS) was recorded both in a state of relative rest and under additional functional load, reflecting the state of the respiratory burst in patients’ neutrophils, both on the 1st day of admission and on the 7th day after surgery. The activation index is increased in patients with CRC at all stages and throughout the examination. Significant deviations in the indices of chemiluminescent activity of neutrophil granulocytes were recorded in patients with CRC: before surgery, an increase in the intensity of spontaneous CL was detected at stages II-IV of the disease, and induced CL at all stages, with the luminescence intensity at the late stage being higher. After surgical treatment, the spontaneous intensity of ROS production at stages I-III normalized, in the induced test, NG patients were still more reactive than the controls. The total ROS production by NG patients with CRC at all stages exceeded the indices of practically healthy people in the spontaneous and induced CL test both upon admission to the hospital and after treatment. The existing reserve capacity for ROS synthesis is also indicated by the increased neutrophil granulocyte activation index in all groups.
Time dynamics of measles antibodies in hospital staff over a five-year period
The incidence of measles has been significantly increased worldwide over recent years. The measles virus is transmitted by aerosol route and is highly contagious. The healthcare staff infected with measles is at particular risk, since they may contribute to the hospital-acquired spread of infection. At the same time, medical personnel are at risk for contact with measles patients and their biological material. The objective of the present study was to assess the contents dynamics of anti-measles antibodies in healthcare workers over a five-year period. We have evaluated the contents of anti-measles IgG antibodies in blood serum, being assayed by ELISA technique in 272 healthcare workers aged 22 to 62 years at the clinics of Samara State Medical University. Statistical processing was performed with StatTech v. 4.1.2. software. The healthcare workers were divided in two age groups: 22 to 44 years, and 45 to 62 years (average age, 43 years old). The baseline level of specific anti-measles IgG was assessed at the 1 st stage of the study in 2018. Individuals who demonstrated negative and questionable results were subjected to additional immunization. At the second stage, specific antibody levels were re-assessed in 2023. In both age groups, the average IgG concentration increased over the five-year period as a result of the 2018 revaccination (0.49 IU in 2023 vs. 0.20 IU in 2018). Any statistically significant differences between age groups could not be detected in 2018. By 2023, the antibody concentrations were higher among those over 45 years of age. In the group without re-immunization, the antibody values showed a trend for decrease over 5 years. The revaccinated group showed an increase in positive results, and a decreased ratio of negative and questionable results. Despite double injection of the anti-measles vaccine in childhood, the specific immunity does not persist in a subgroup of adult persons. The additional immunization of 2018 was, generally, quite effective. Individual characteristics of the host organism, as well as a new coronavirus infection, may affect development of humoral immunity against measles. A regular serology monitoring is necessary to follow the maintenance of anti-measles humoral immunity, especially for the risk-exposed cohorts, including healthcare workers.
Development and validation of an ELISA-based method for determining neutralizing antibodies to pembrolizumab in human serum based on inhibition of the drug binding to its PD-1 target
Monoclonal antibodies (mAbs) are potentially able to trigger undesired humoral immune responses in the patients and develop ADA (anti-drug antibody) to the protein drugs. Neutralizing anti-drug antibodies are among the main factors affecting safety and effectiveness of the therapy. If it is impossible to apply cell-based tests to determine neutralizing antibodies, competitive ligand binding assay may be used as an alternative. Pembrolizumab (Pembro) is a broad-spectrum antitumor drug, being a humanized IgG4 kappa antibody to the programmed cell death receptor-1 (PD-1) that blocks interaction of this receptor with its ligands PD-L1 and PD-L2. Due to some technical issues, cell culture test is not feasible for Pembro, due to high risk of obtaining unreliable results. The aim of our study was to develop and validate a method for detection of neutralizing antibodies to Pembro in human serum based on inhibition of pembrolizumab binding to its PD-1 target. The experimental drug pembrolizumab RPH-075 (R-Pharm) was used in the study. Anti-Pembrolizumab antibodies KRIBIOLISATM Anti-Pembrolizumab (KEYTRUDA®) ELISA, India) were used as a positive control sample for neutralizing antibodies. Determination of antibodies was carried out by ELISA technique using acid dissociation of the immune complex and the Affinity capture elution (ACE) technique. The ELISA method was validated by the following characteristics: selectivity, sensitivity, specificity, “hook” effect, drug tolerance, precision. Due to the use of sample pretreatment approaches (ACE technique) for analysis of neutralizing antibodies, a sensitivity level of 100 ng/mL was achieved in the presence of pembrolizumab at 40 μg/mL. In this paper, a method was substantiated by calculating the cutoff point, sensitivity, and selectivity based on ROC analysis and floating exclusion limit (PSCP) through the average values of optical density NC and LPC in each individual analytical cycle. The developed method for determining neutralizing antibodies to pembrolizumab may be used to assess the undesirable immunogenicity of pembrolizumab at the stage of clinical trials.
Experience in development and application of a technique for studying the ability of neutrophils to form extracellular traps
The phenomenon of neutrophil extracellular trap (NETs) formation, or netosis, plays a key role in pathogenesis of infectious and inflammatory diseases. However, the lack of standardized methods for assessing NETs makes it difficult to correctly interpret the results. The aim of our study was to develop a feasible methodology to investigate the ability of neutrophils to form NETs, which would allow precise identification of netosis-associated phenomena. Neutrophils were isolated from peripheral blood of 15 healthy donors by gradient centrifugation (Ficoll-Verographin, density gradient 1.077-1.105 g/mL). A mixture of bacteria (Lactobacillus reuteri, L. acidophilus, L. rhamnosus, Bifidobacterium longum) was used to stimulate netosis. Propidium iodide (PI), a DNA-intercalating dye, and murine monoclonal antibodies to human CD15/FITC were used to visualize NETs and other fluorescent objects. Luminescence microscopy was performed at excitation wavelengths of 450-480 nm and emission wavelengths of 515 nm. We have registered the following morphological variants: (1) intact neutrophils (bright green, without nuclear staining); (2) activated neutrophils (bright green with a red-orange, PI-stained nucleus); (3) early netosis cells (bright green cells with nuclear material released to one or more sites at cell membrane); (4) NETs: (a) cloud-shaped objects surrounding the neutrophil or its fragments from all sides, being and more than 1.5 times its size, and (b) filamentous forms seen as filament structures exceeding the neutrophil size>2-fold or more. The proportion of each type of observed objects relative to all fluorescent-positive objects was determined. Upon optimization of this method, the key parameters were determined: temperature of cell incubation with the stimulant (37 °C); its concentration (2.5x109 bacteria/ mL), stimulation time (30 min). Testing the reproducibility of this technique showed an acceptable inter-series coefficient of variation (CV): 13.8% for filamentous and 15.2% for cloud-shaped NETs. The proposed method was applied in different groups of patients with certain infectious (pulmonary tuberculosis) and non-infectious pathology (ulcerative colitis, aneurysmal bone cysts). Testing the NET-producing ability of neutrophils in the subjects from these cohorts allows us to conclude that the levels of sensitivity and manifestations for these indexes registered with this technique are quite satisfactory.