New approaches to allergen-specific immunotherapy (ASIT): development of a recombinant vaccine against birch pollen allergy
Birch pollen allergy is widespread in the world and it is the most important problem of public health. Allergy to birch pollen has various clinical manifestations: allergic rhinitis, allergic conjunctivitis, asthma, food allergy, as well as severe life-threatening reactions such as anaphylaxis. The only pathogenetic method of treatment that changes the immune response to a causally significant allergen is allergen-specific immunotherapy (ASIT). The ASIT methods used today have a number of disadvantages: difficulties in standardizing allergens, the risk of adverse events and inconvenient treatment regimens. In recent decades, ASIT molecular strategies have been developed that may allow the development of a vaccine with a minimum of these disadvantages. The review presents novel ASIT molecular strategies and information on the development of an innovative recombinant vaccine for the treatment of birch pollen allergy and cross-food allergy caused by apple allergens.
Unidirectional and multidirectional changes in the expression of membrane-bound TNF receptors on immunocompetent cells in rheumatoid arthritis and bronchial asthma
Introduction. TNF provides control over inflammatory processes in normal conditions and in the development of rheumatoid arthritis (RA) and bronchial asthma (BA). Important regulatory mechanisms are changes in coexpression and expression density of receptors (TNFR1 and TNFR2), which reflect the sensitivity of cells to the action of a mediator and the type of cell response. The question of identifying the expression levels of receptors that are critical for the formation of irreversible changes in the immune system during the development and chronicity of diseases remains open.
Aim of the study was to compare the parameters of expression and coexpression of type 1 and type 2 receptors to TNF on immunocompetent cells in RA and BA patients compared with those of healthy donors and to identify characteristic patterns of parameter changes in chronic inflammatory immune-mediated diseases.
Material and methods. The expression and coexpression of TNFR1 and TNFR2 was determined on mononuclear cells of healthy donors and patients with RA and BA using multicolor flow cytometry. Logistic regression analysis was performed to identify characteristic changes in pathology.
Results. 3 patterns of changes in the expression and co-expression of receptors in diseases have been identified. For 23 indicators, significant changes were demonstrated towards an increase in the levels of expression and co-expression of TNF receptors in both RA and BA, and for 16 indicators – towards a decrease, respectively. 6 parameters had the greatest diagnostic significance, including percent of TNFR1+TNFR2−-cells in CD4+, CD4+/CD45RA+, CD8+ populations; percent of TNFR1+TNFR2+-cells in CD19+, CD8+CD45R0+ populations; and number of TNFR1 on CD4+/CD45R0+-cells.
Conclusion. The study made it possible to establish general trends and characteristic patterns of differences in changes in the expression of TNF receptors in immune-mediated diseases of various etiologies, as well as to establish certain levels of expression associated with a significant increase in the risk of bronchial asthma and rheumatoid arthritis.
The influence of respiratory hypoxic gas mixtures (oxygen-nitrogen and oxygen-nitrogen-argon) in a pressure chamber on the state of the human cellular immunity
Introduction. The development of various fireproof hypoxic argon-containing breathing mixtures and the assessment of their safety for human health is a promising area of modern cosmonautics.
The aim of the work was to study the state of human innate and adaptive immunity during and after prolonged breathing an oxygen-nitrogen-argon gas mixture at elevated pressure.
Material and methods. 6 healthy men from 25 to 42 years of age participated in three experimental series. During the first experimental series, the subjects breathed a hypoxic mixture with the addition of argon at an overpressure of 0.02 MPa. During the second experimental series, the subjects breathed an extremely hypoxic mixture (4.7–5.3 % oxygen) with the addition of argon and an overpressure of 0.2 MPa. During the third experimental series, the subjects breathed a hypoxic mixture without the addition of argon at an overpressure of 0.02 MPa. To assess the state of immunity before, during and after stay in the pressure chamber, the absolute and relative contents of leukocytes, lymphocytes, monocytes, granulocytes, B- and T-lymphocytes, natural killers, as well as their various subpopulations, were determined.
Results. During the first and second experimental series, a day after leaving the pressure chamber, an increase in hematocrit and erythrocyte content in the blood was observed. In the second experimental series, a day after leaving the chamber, an increase in the relative content of monocytes expressing TLR2 and TLR4, an increase in the absolute and relative contents of granulocytes expressing TLR2 and TLR4 and a relative content of granulocytes expressing TLR6 were determined. Also, a day after leaving the chamber in the second experimental series, the relative content of T-lymphocytes with CD3+CD4+ and CD3+CD8+ phenotypes significantly decreased.
Conclusion. The observed changes were short-lived, however, it can be assumed that a longer stay in pressure chamber with an oxygen content of 4.7–5.3 % and an overpressure of 0.2 MPa can cause a pronounced imbalance in the immune system and reduce the protective reserves of the body.
A mouse model of allergic rhinitis mimicking human pathology
Introduction. Allergic rhinitis (AR) is an IgE-mediated inflammation of the upper respiratory tract. More than 400 million people worldwide suffer from this pathology. According to the current concept AR develops through a Th2-dependent mechanism involving cytokines (IL-4, IL-5 and IL-13) and pro-inflammatory cells, primarily eosinophils. Improvement of current therapy, the development of new ways of AR treatment and understanding of pathogenesis of the disease are urgent tasks of biomedicine, which are needed of appropriate animal models.
The aim of the study was to develop a mouse model of allergic rhinitis, which mimics the main features of human pathology.
Material and methods. Sensibilization with the allergen ovalbumin (OVA) was performed by three subcutaneous immunizations at a dose of 20 µg. After the sensitization mice Balb/c were received 25 µl of solution of the same allergen (in concentration 10 mg/ml) intranasally. Nasal hyperreactivity was assessed as frequency of sneezing and nasal rubbing. The levels of serum immunoglobulins and cytokines in the supernatants of lymph node cells were measured by ELISA. The mRNA expression of cytokine genes in the nasal mucosa was determined by quantitative PCR. Histological analysis was used to assess the level of inflammation in the upper respiratory tract.
Results. Subcutaneous immunizations followed by intranasal challenge of mice with an allergen according to the described protocol induced main features of AR, such as increased nasal hyperreactivity (an increase in the number of sneezes by 11 times and scratching of the nose by 8 times, compared to mice of control group), an increased level of allergen specific IgE antibodies. The inflammation of the upper respiratory tract was also observed that expressed in: 5-fold increase in the number cells infiltrating nasal mucosa, 3-fold increase in the area of cell infiltrates, 1.4 times increase in the proportion of mucous secreting goblet cells in the respiratory epithelium. An increased production of Th2-cytokines (IL-4, IL-5 and IL-13) by submandibular lymph node cells and the expression of mRNA of the corresponding genes (Il4, Il5 and Il13) in the nasal mucosa revealed that the mice developed AR phenotype through Th2-dependent mechanisms.
The common AR pharmacotherapy includes antihistamines, antileukotriene and corticosteroids. To test the developed mouse model of AR we conducted an experimental therapy using these drugs. As a result, a significant decrease in main AR manifestations such as nasal hyperreactivity, inflammation of the nasal mucosa and remodeling of the respiratory tract were revealed.
Conclusion. A mouse model of AR mimicking main pathological features was developed: nasal hyperreactivity, increased levels of allergen specific IgE antibodies, infiltration of the nasal mucosa with pro-inflammatory cells. AR phenotype induced in mice developed through Th2-dependent mechanism. Antihistamines, antileukotrienes and corticosteroids were tested on the developed mouse model of AR. These drugs demonstrated effects similar to those in clinical practice, which confirms that the developed mouse model of AR is adequate to clinical observations. The developed model can be used to study the pathogenesis of AR, as well as to test the effectiveness of new drugs.
Reprogramming of myeloid cells of the tumor microenvironment increases the efficiency of CTLA-4 and PD-1 blockade in experimental malignant melanoma immunotherapy
Introduction. Blockade of immune checkpoints (immune checkpoint blockade) for the treatment of patients with malignant neoplasms is successfully and widely implemented in practical oncology. In essence, this direction of immunotherapy is the removal of suppression from T cells using antibodies to the inhibitory receptors CTLA-4 or PD-1 (PD-L1), allowing to reactivate antitumor T-cell immune responses and leading to significant clinical effects. Such immunotherapy saves patients even in the 4th stage of the oncological process. Even though the effectiveness of the checkpoint blockade is not high enough. Complete regression of the disease and complete recovery occur only in a small percentage of patients. The majority of patients with solid tumors who received checkpoint blockade either had no clinical effect or had a partial, relatively short-term effect.
One of the most common reasons preventing effective tumor elimination during checkpoint blockade therapy is that the myeloid cells of the tumor microenvironment create an immunosuppressive microenvironment inside the tumor, thus protecting malignant cells from antitumor immune responses, in particular, from the attack of cytotoxic T and NK cells.
Aim of the work – investigation the possibility of increasing the effectiveness of checkpoint blockade therapy with antibodies to CTLA-4 and PD-1 by simultaneously reprogramming myeloid cells of the tumor microenvironment from their protumor to an antitumor state using a TLR4 agonist.
Material and methods. An experimental model of B16F0 melanoma in C57BL/6J mice was used. Checkpoint blockade was performed simultaneously with two antibodies specific for CTLA-4 and PD-1. Reprogramming of myeloid cells of the tumor microenvironment was induced by intratumoral administration of acidic peptidoglycan (APG, TLR4 agonist). The effectiveness of immunotherapy was assessed by the growth rate of a solid tumor, the tumor mass on day 16 after inoculation of B16F0 cells, the cellular composition of the tumor infiltrate, and also by the intensity of transcription of key genes that determine the polarization and functional activity of myeloid and T cells, purified by sorting from tumor tissue.
Results. It has been shown that combined immunotherapy with both checkpoint blocking antibodies to CTLA-4/PD-1 and a TLR4 agonist significantly suppresses the growth of B16F0 melanoma, reduces the mass of tumors by 3 times by day 16, and induces a powerful infiltration of tumor tissue by CD8+-T cells and NK cells with increased expression of granzyme A and interferon-γ genes, as well as by monocytes and neutrophils polarized into an antitumor state, as evidenced by increased expression of the iNOS gene and the absence of active expression of the arginase-1 gene.
Conclusion. Combination therapy of malignant melanoma in an experimental model with immune checkpoint blockers together with a TLR4 agonist is significantly more effective than monotherapy with the same therapeutic agents used alone.
The intensity of antitumor immune responses to immunization with Multivac vaccines against 4T1 carcinoma in BALB/c mice and B16 melanoma in C57BL/6J mice does not depend on the host genotype and the tissue origin of the tumor
Introduction. Previously, we reported on the new Multivac methodological platform for the development of personalized antitumor vaccines. The strength of immune response depends on the major histocompatibility complex genes and their products presented on the surface of antigen-presenting cells. The nature of antigens and their diversity in the vaccine composition can also strongly influence the intensity of the immune response during vaccination. Antitumor vaccines of the Multivac series, prepared from tumor tissue or malignant tumor cells, contain a large collection of tumor antigens. This suggests that such vaccines will induce intense immune responses and immune memory, regardless of the genotype of the individual and the tissue nature of the tumor.
Aim. In this work we compared the immunogenicity of the Multivac vaccines against two significantly different tumors (mammary gland carcinoma and melanoma) in two inbred mouse strains that differ significantly in their genetic basis and the genes of the major histocompatibility complex, on which the intensity of adaptive immune responses critically depends.
Material and methods. The multi-antigenic vaccines Multivac-1 were prepared from solid 4T1 mammary carcinoma tissue of BALB/c mice (H-2d genotype) or B16F10 melanoma tissue of C57BL/6J mice (H-2b genotype). Multivac-4 vaccines were based on lysates of malignant cells of 4T1-carcinoma or melanoma B16F10 grown under in vitro cell culture conditions. Multi-antigenic lysates of tumor tissue or malignant cells were supplemented with molecular immunoadjuvants from the class of PRR agonists. Systemic immune responses against 4T1 carcinoma and B16F10 melanoma antigens were determined according to the numbers of antitumor T cells in the spleen and the levels of tumor-specific antibodies in the blood serum of mice. Antigen-reactive CD4 and CD8 T memory cells, and T effector cells secreting interferon-γ were analyzed by ELISpot. Antibodies to intracellular antigens of 4T1-carcinoma and B16F10-melanoma were studied by ELISA.
Results. Both BALB/c and C57BL/6J mouse strains responded effectively to immunization with the Multivac-1 vaccine prepared from 4T1 and B16F10 tumor tissues respectively. More than 1000 CD4+ T effector cells, CD4+ T memory cells, CD8+ T memory cells (per 1 million corresponding T cells) were recorded in the spleens of immunized mice. Even more intense immune responses developed in the spleens of BALB/c and C57BL/6J mice immunized with the Multivac-4 vaccine. Vaccination with Multivac-4 caused an intense accumulation of antigen-reactive T cells memory in mice of both genotypes. Immunization of BALB/c against 4T1-carcinoma and C57BL/6J mice against B16F10-melanoma with vaccines of the Multivac series induced the accumulation in blood serum of a large amounts of antitumor antibodies binding antigens of 4T1 carcinoma and B16F10 melanoma cells.
Conclusion. Multivac vaccines based on multi-antigen lysates of tumor tissue or malignant cells of 4T1 carcinoma or B16F10 melanoma demonstrated equally high immunogenicity. The intensity of immune reactions in response to immunization with the Multivac vaccines did not depend on the genotype of the vaccine recipients. BALB/c and C57BL/6J mice exhibited equally high levels of T-cell and antibody responses to the tumor antigens used in the respective vaccines.
The content of platelet-leukocyte coaggregates in the peripheral blood of healthy children of different ages
Introduction. Currently, intercellular interactions are widely studied, which are based on signaling mechanisms mediated by cytokines, adhesion molecules and various components of the vascular wall. The formation of contacts between platelets and leukocytes are important part in the mechanisms that ensure the migration of leukocytes to the area of damage and the development of immune and reparative processes there. It has been established that platelets can interact with neutrophils, monocytes and lymphocytes. In the literature information on the adhesive interaction of platelets with neutrophils, monocytes, lymphocytes and their subpopulations in the blood of healthy children of different age groups is not presented.
The aim of the study – to research the absolute and relative content of platelet coaggregates in the total pool of leukocytes, as well as separately with neutrophils, monocytes, lymphocytes, T- and B-lymphocytes, NK and NKT cells in the peripheral blood of healthy children of different ages.
Material and methods. The object of the study were samples of venous blood of 111 healthy children (boys and girls) aged from 3 months to 14 years old. To detect coaggregates, monoclonal antibodies conjugated with various fluorochromes were used. The parameters under study were determined by flow cytometry.
Results. It was found that the absolute content of platelet-neutrophil coaggregates is minimal in children of the first year of life and maximal in the group of children from 6 to 8 years old. It was found that the absolute content of coaggregates decreased in the total pool of lymphocytes, as well as in all their studied subpopulations, as children grew. With increasing age, the relative content of total platelet-leukocyte coaggregates and platelet-monocyte coaggregates in a particular cell population increased against the background of a constant level of platelet-neutrophil coaggregates. The relative content of platelet-monocyte coaggregates and platelet-neutrophil coaggregates increased in every leucocyte population regarding to PLC with the growth of children, and platelet-lymphocyte coaggregates content, on the contrary, decreased. Physiological crossovers of platelet-neutrophil coaggregates and platelet-lymphocyte coaggregates were found in their relative and absolute content.
Conclusion. The results of the study demonstrate changes in the absolute and relative content of platelet-leukocyte coaggregates in the peripheral blood of healthy children depending on age.
Characteristics of monocyte differentiation and CD163 expression in women with pointed early mission
Introduction. Despite the advances in diagnosis and treatment, reproductive losses before 22 weeks of gestation remain one of the priority problems of modern medicine.
The aim – to assess the character of monocyte differentiation and the level of cells expressing the scavenger receptor CD163 in threatened early miscarriage in dependency of pregnancy outcome.
Material and methods. 115 women at 5–12 weeks of gestation were included into the investigation 80 of them with threatened early miscarriage (main group) and 35 women without signs of threatened miscarriage (comparison group). The relative content of classically activated, intermediate, alternatively activated monocytes expressing the scavenger receptor CD163 in the monocyte gate was assessed using FACScanto II flow cytometer (Becton Dickinson, USA).
Results. In women of the main group, the amount of classically activated and intermediate monocytes did not differ from the parameters of the comparison group and didn’t change depending on the outcome of pregnancy. However, the level of alternatively activated monocytes in women of the main group, whose pregnancy ended with reproductive losses up to 22 weeks of pregnancy, was lower than in the comparison group and in women of the main group, whose pregnancy ended in timely delivery. In women of the main group, the content of classically activated and intermediate monocytes, expressing CD163, did not differ from those in the comparison group and did not depend on the outcome of pregnancy. However, the content of alternatively activated monocytes expressing CD163, in women of the main group, whose pregnancy ended in reproductive losses before 22 weeks of gestation, was higher both in comparison with the parameters of comparison group and those of women of the main group, whose pregnancy ended in timely delivery.
Conclusion. Population of alternatively, activated monocytes, expressing scavenger receptor CD163, might participate in the pathogenesis of threatened early miscarriage, and their peripheral blood levels can be used as the predictor of reproductive losses up to 22 weeks in women with threatened early miscarriage.
Studying of protein-DNA interactions in the cells of immune system using chromatin immunoprecipitation and chromatin immunocleavage
Cells of the immune system respond to infections and other insults by altering expression of hundreds of genes enforcing immune defense. Regulation of gene expression depends on interactions of genomic DNA with proteins, such as histones, transcription factors, RNA polymerases, cofactor proteins. Here, we review two methods of analysing DNA-protein interactions directly in the cells: chromatin immunoprecipitation (ChIP) and chromatin immunocleavage (ChIC). We discuss main technical characteristics and modifications, advantages and disadvantages of either technique. Finally, we present several illustrations of how ChIP and ChIC can be applied in fundamental and clinically oriented immunological studies.
Mast cell involvement in physiological and preeclampsia complicated pregnancy
Mast cells (MCs) are multifunctional tissue-resident cells generated from hematopoietic precursors in the bone marrow, migrate through the circulatory system to the periphery, where they settle on the mucosal surface and in the connective tissue of many organs and complete differentiation and maturation. The phenotypic characteristics of MCs and their functional activity are determined by the local microenvironment. MCs are key inducers and modulators of allergic, anaphylactic and other inflammatory reactions. MCs secrete a wide range of preformed or newly synthesized biologically active products with pro- and anti-inflammatory properties in response to multiple stimuli. Upon activation, MCs secrete histamine, leukotrienes and prostanoids, as well as proteases and a variety of cytokines and chemokines. The wide tissue distribution and ability to respond to many different stimuli suggests the involvement of MCs in many physiological and pathological processes in the body. Data from experimental studies on rodents indicate the involvement of uterine MCs in the processes of blastocyst implantation and placenta formation. An important role of MCs in the processes of remodeling of the uterine spiral arteries, as well as in creating a maternal microenvironment tolerant for the fetus, has also been shown. However to date few studies have been devoted to the contribution of MCs to the pathogenesis of obstetric pathologies including preeclampsia (PE). This review focuses on analyzes the role of MCs in various physiological and pathological conditions, including key pathogenetic mechanisms of preeclampsia.
Frailty: a controversial role of inflammaging
Frailty is one of the main geriatric syndromes, closely associated with human aging. The pathogenesis of frailty has not been sufficiently studied, but the role of inflammaging as a key mechanism in the development of this syndrome is of considerable interest. The review considers the contribution of pro- and anti-inflammatory cytokines associated with the innate immune system, a number of other inflammatory factors to the development of frailty and its main phenotypic manifestations. The controversial role of inflammatory aging in the pathogenesis of frailty is discussed. On the one hand, inflammaging is one of the key features of frailty, but it is not clear whether it is a pathogenetic factor or a consequence of it`s development. On the other hand, in frailty, innate immunity is impaired, expressed in a decrease in the ability to produce pro- and anti-inflammatory cytokines under the influence of TLR ligands, which is a significant predictor of mortality. The need for further search for inflammatory markers of the transition from physiological aging to age-related diseases and syndromes, in particular, to frailty, is emphasized.