Effect of local suppression of Stat3 gene expression in a mouse model of pulmonary neutrophilic inflammation
Introduction. Bronchial asthma (BA) is a chronic inflammatory disease of the airways. For a long time, it was believed that BA developed exclusively according to the Th2-dependent mechanism, however, to date, several phenotypes of the disease have been distinguished, one of which is neutrophilic BA. Neutrophilic BA is characterized by resistance to standard corticosteroid therapy, that need to search for new way of treatment. Neutrophilic inflammation of the lungs is developed under activation of IL-6/STAT3-pathway, that makes the molecular components of this pathway to be the promising targets. One of the technologies by which genes expression can be regulated is RNA interference.
The aim of this study was to evaluate the effects of local suppression of Stat3 gene expression in a mouse model of neutrophilic BA.
Material and methods. During the development of BA features mice were inhaled with a complex of small interfering RNA (siRNA) and a carrier peptide. After the course of inhalations, the severity of BA manifestations was assessed: the level of allergen-specific antibodies, changes in bronchial hyperreactivity, the cellular composition of the bronchoalveolar lavage (BAL) fluid, histopathological alterations in the lungs.
Results. 5 variants of siRNA against Stat3 gene were designed. In vitro experiments established that the variant siSTAT3-1426 significantly (5 times) reduced the expression of the target gene. A complex of siRNA and a carrier peptide LTP down-regulated the expression of Stat3 and Il17a genes in Th17-lymphocytes. In vivo studies in a mouse model of neutrophilic BA revealed, that the inhalations with the complex led to a 2-fold inhibition of Stat3 gene expression in BAL fluid cells, 3-fold decrease in neutrophilic infiltration of the lungs and recovery of the thickness of the airway walls.
Conclusion. The complex of siRNA targeted to Stat3 gene and the carrier peptide was developed. The inhalations with the complex decreased Th17-dependent neutrophilic inflammation of the lungs in neutrophilic BA model in mice, that can be a promising approach to the therapy of neutrophilic corticosteroid-resistant BA.
Synergism of TLR3 and TLR4 agonists during macrophage reprogramming into an antitumor state
Introduction. Solid tumor tissue usually contains many macrophages. These cells actively support the progressive growth of malignant cells, stimulate angiogenesis, promote the growth of the fibrotic scaffold the tumor by activating divisions of fibroblasts and their transformation into myofibroblasts, immune responses inhibition with immunosuppressive substances such as IL-10, TGFβ, IDO, PGE2, and an induction of epithelial-mesenchymal transformation of malignant cells, which allows malignant cells to move freely within tissues (invasive tumor growth) and around the host organism (distant metastases). Macrophages’ plasticity, i.e. their ability for transition from one state to another, is well documented by previous studies. Particularly, tumor-promoting macrophages can be reprogrammed into anti-tumor M1 macrophages. External infection-derived signals acting via pathogen recognition receptors (TLR, NOD, etc.), as well as internal damage-derived signals acting via pro-inflammatory cytokine and other receptors, transfer macrophages into M1 state.
The aim of this study is to analyze the synergism of TLR3- and TLR4-agonists during macrophages reprogramming into their M1 antitumor state.
Material and methods. Macrophages were obtained from the mouse bone marrow using in vitro differentiation in the presence of GM-CSF. Cells were activated with synthetic poly I:C (TLR3 agonist) or acidic peptidoglycan of the plant origin (TLR4 agonist, active component of Immunomax, hereinafter IMM). The activation of TLR3- and TLR4-related downstream signaling pathways was measured according to TBK1/IKKε, ERK1 and ERK2 protein kinases, as well as NF-kB transcription factor phosphorylation intensities. Flow cytometry and Western blotting with specific monoclonal antibodies were used to measure phosphorylation degree of the signaling proteins. The expression intensity of Nos2 and Ifnbl mRNAs was investigated by a quantitative RT-PCR. The cytotoxic activity of macrophages was estimated in their co-culture with 4T1 breast carcinoma cells according to the reduction of the tumor cells number.
Results. A transformation of macrophages into an antitumor state can be induced with a combination of TLR3- and TLR4-agonists, more efficiently than with the same agonists used alone. Both agonists induced significant and rapid activation of TBK1/IKKε, ERK1/2 and NF-kB. Compared to the single agonists, the combined action of TLR3 and TLR4 agonists increased the kinases phosphorylation level by only 15-20 %. Even though the activated genes transcription rate was enhanced synergistically. The combined action of TLR3 and TLR4 agonists increased the level of Nos2 and Ifnbl mRNAs transcription by 2.6-3.5 times as compared to the transcription rate of the same genes in response to each of these agonists used alone. TLR3- and TLR4-agonists also synergistically activated the antitumor properties of macrophages in a cytotoxic test against 4T1 carcinoma cells.
Conclusion. The synergism of TLR3 and TLR4 agonists during reprogramming of macrophages into the M1 state has been described for the 1st time. The synergistic action of agonists leads to a multiple increase in the expression of genes encoding antitumor effector proteins, in particular, iNOS and IFN-p, as well as to a significant increase in the antitumor cytotoxic effect of macrophages. At the level of phosphorylation of TBK1/IKKε and ERK1/2 protein kinases, as well as the NF-kB transcription factor, no synergistic effect of TLR3 and TLR4 agonists was found, which indicates the presence of cooperative interaction between other components of these 2 signaling pathways.
Stimulation of B cells in the IL-21/CD40L system in healthy donors and in patients with common variable immunodeficiency
Introduction. Common variable immunodeficiency (CVID) refers to primary immunodeficiencies. To date, a number of genes have been identified, mutations in which lead to the development of CVID. However, monogenic defects underlie no more than 10 % of CVID cases, and the bulk of CVID is associated with the participation of many genes. Under these conditions, to search for and decipher possible pathogenetic factors, it is effective to use the analysis of functional defects in the activation of B-lymphocytes.
The aim of the study - to search for functional defects in the activation of B-lymphocytes in patients with CVID when stimulated in vitro in the IL-21/CD40L system.
Material and methods. The study involved 14 patients with CVID. The group of comparison consisted of 10 healthy volunteers. Samples of blood mononuclear cells were obtained from peripheral blood and used to activate T cells. Phytohemagglutinin (PHA) or a combination of phorbol myristate acetate (PMA) and ionomycin (Io) were used as activators. CD40L induction on CD4+-cells was assessed using flow cytometry. B cells were isolated from the mononuclear cells by the method of immunomagnetic separation and B cell activation in the IL-21/CD40L system was carried out. Recombinant IL-21 and A549 feeder cells stably transfected with the CD40L gene were used as activating stimuli. The differentiation of B cells into plasmablasts was determined by the proportion of cells with the CD19+CD27+CD38+CD20low phenotype. Subpopulations of naive (IgM+IgD+), unswitched (IgM+CD27+) and switched (IgG+CD27+) B cells were isolated using a flow sorter. The secretion of IgG and IgM was determined using an enzyme immunoassay, and the number of IgG-secreting cells was estimated using the ELISpot method (Enzyme-Linked ImmunoSpot Analysis).
Results. When T cells were activated by polyclonal stimulants, some of the CD4+-cells began to express CD40L. In T cells of healthy volunteers, stimulation with PHA or a combination of PMA and Io resulted in CD40L expression on 10 and 80 % of CD4+-cells, respectively. T cells from patients with CVID showed comparable activation and the proportion of CD4+CD40L+-cells was close to normal. When stimulated in the IL-21/CD40L system, B cells of patients with CVID retained the ability to differentiate into plasmablasts with the CD19+CD27+CD38+CD20low phenotype, but secreted IgG 10 times less than B cells from healthy volunteers.
Сonclusion. In CVID patients the B cell activation pathways associated with IL-21 and CD40L remain intact, and the functional defects observed in CVID are obviously associated with other molecules.
Dermatologic adverse events of cancer immunotherapy with anti-PD-1 and anti-PD-L1 monoclonal antibodies
The article describes major types of dermatologic immune-related adverse events during treatment with PD-1/PD-L1 immune checkpoint inhibitors. We present several clinical cases, describe current topical and systemic therapies of patients with such type of immune-mediated adverse events. In addition, the stepwise approach to the management ofpatients with dermatologic immune-related adverse events based on clinical course and severity grading is presented.
Characterization of cellular immunity in patients with obstructive jaundice caused by cholelithiasis
Introduction. Obstructive jaundice is currently a life-threatening organ dysfunction, accompanied by a dysregulatory immune response to obstruction, hyperbilirubinemia, and an infectious process. The prognosis of the disease depends on the changing cellular responses of innate and adaptive immunity. There is evidence of quantitative changes in T- and B-lymphocytes in obstructive jaundice, however, studies of their use as predictors of the development of an unfavorable prognosis are insufficient.
The aim of the study - estimation of the parameters of the cellular part of adaptive immunity in patients with obstructive jaundice caused by cholelithiasis in dynamics after surgical treatment.
Material and methods. The data of a prospective study of the cells of the immune system of 40 patients with obstructive jaundice due to cholelithiasis and 30 practically healthy volunteers are shown. Blood sampling from patients was carried out in the first 24 hours after admission to the hospital and the establishment of the diagnosis «obstructive jaundice» and on the 7th day after surgery. Evaluation of the level of T-, NK- and B-lymphocytes (CD3+CD19-, CD3CD16/56+, CD3CD19+), subpopulations of T-lymphocytes (CD3+CD4+, CD3+CD8+, CD3+CD4+CD25+) was carried out by the method of flow cytometry using monoclonal antibodies. Determination of the number of Th1- and Th2 cells was assessed indirectly by the presence of cytokines IFN-γ and IL-4 in plasma, which were determined using enzyme-linked immunosorbent assay and a set of monoclonal antibodies produced by ZAO Vector Best (Russia).
Results. On admission, patients with obstructive jaundice showed lymphopenia, a decrease in total T-lymphocytes and their supopulations, a decrease in the number of NK cells, an increase in Treg and IL-4 (p < 0.05). After surgical treatment, the patients retained T-cell immunosuppression, while Treg and cytokines were normalized (p < 0.05).
Conclusion. Significant changes in the adaptive immunity cells were revealed in patients with obstructive jaundice caused by cholelithiasis before surgery, manifested by a decrease in the content of T-lymphocytes and their subpopulations, an imbalance of T-helpers towards Th2 cells, an increase in Treg. In the postoperative period, the suppression of the cellular immunity is preserved due to the additional antigenic load from the surgical intervention itself, while the stabilization of pro-inflammatory and anti-inflammatory cytokines, the normalization of the ratio of Th1/Th2-cells and the number of Treg. Quantification of Treg cells can be a predictor of poor prognosis in these patients.
Photoregulated oligonucleotide detachment as a way to improve the efficiency of polymerase chain reaction with reverse transcription for analysis at the single cell level
Introduction. Due to the improvement of single cell RNA sequencing (scRNA-seq) technologies, a fundamentally new possibility to analyze the expression of groups of genes determining a specific function and allowing to trace the evolution of the cell both under normal conditions and during the development of pathology has appeared. Technologies, by means of which single cells are studied, make it possible to track mutational changes in genes of specific cell populations and find links with the manifestation of specific clinical features. Of greatest interest in this sense are lymphocyte populations and their receptors involved in antigen recognition. However, existing scRNA-seq technologies are not sufficiently effective for studying lymphocyte receptor repertoires. One of the limitations of the method is the insufficient efficiency of cDNA synthesis in the solid phase.
The aim of the study is to develop a technology for the synthesis and photoregulated release of oligonucleotides from the surface of a solid-phase carrier to increase the efficiency of cDNA synthesis from the mRNA of the а-chain T cell receptor gene of individual cells.
Material and methods. We developed a technology of oligonucleotide synthesis on a solid-phase carrier with the possibility of their release into a solution, if required. This process is provided by the incorporation of photosensitive molecules into the oligonucleotide structure at the synthesis stage. The linker is degraded under mild UV irradiation, which does not damage the biopolymer structure, resulting in release of the oligonucleotide sequence into the solution for the reverse transcription priming.
Results. A 39-nucleotide oligonucleotide sequence containing a short spacer, a photoactivated linker, a unique molecular barcode, a site for hybridization of the universal primer, and a fragment specific to the α-chain sequence of the T cell receptor were used. Synthesis was performed using «inverted» amidites in order to correctly orient the oligonucleotide on the carrier, ensuring cDNA synthesis. It was found that incorporation of the photosensitive PC-linker into the oligonucleotide structure, allows releasing all sequences available for UV-induced cleavage from the carrier surface within 5 min. Comparison of the production of the target product with and without photoactivation in the RT reactions showed significant differences in the amount of product obtained. The difference in the amount of cDNA in the two parallel reactions was approximately 7 PCR cycles, which suggests more than 100-fold excess of the initial amount of the specific target in the PC-linker reaction.
Conclusion. As a result of the study, we developed a technology for photoregulated oligonucleotide detachment from a solid carrier. We demonstrated that the PC-linker has no significant effect on the efficiency of polynucleotide chain synthesis and can be used in solid-phase synthesis of long deoxyribonucleotide sequences. Using emulsion PCR, we found that the use of a photodegradable linker significantly increases the efficiency of cDNA synthesis from single cell mRNA.
Inflammation - friend or enemy?
Inflammation and its role in the pathogenesis of numerous diseases is the subject of research and the field for discussions at International and Russian scientific and medical events with the participation of physicians of different specialties. This topic was also discussed at the interdisciplinary panel discussion «Inflammation - friend or enemy?» with the participation of leading experts in the field of immunology, allergology and clinical pharmacology, held within the framework of the 17th International Interdisciplinary Congress on Allergology and Immunology (Moscow, June 23-25, 2021). The interrelation of allergy and the expressed inflammatory process as a result of the action of immune mechanisms underlying allergic diseases in recent years requires increasing attention of the medical community due to the steady increase of the prevalence of allergic diseases. A significant place in the treatment of such diseases still have glucocorticosteroids, the discovery of the structure and biological effects of which was awarded by Nobel Prize in 1950. A wide range of physiological and pharmacological effects of glucocorticoids, in particular, antiinflammatory, nonspecific membrane stabilizing and decongestance, antiallergic and immunosuppressive, antishock, etc., makes these compounds almost universal drugs that are indicated for many pathological conditions at different stages of their development. Special attention during the discussion was paid to the Diprometa® preparation, betamethasone in a convenient pre-filled syringe, which makes its use relevant for a wide range of diseases and conditions.
Practical recommendations for the use of the alpha-glutamyl-tryptophan for respiratory diseases
Respiratory diseases vary widely from acute infections to chronic conditions. Acute diseases of the respiratory tract occupy the overwhelming majority of cases among infectious pathology and each year take on the character of epidemics. In turn, chronic bronchopulmonary diseases, primarily chronic obstructive pulmonary disease (COPD) and bronchial asthma are socially significant and always remain in the center of attention of specialists. Today 65 million of people suffer from COPD in moderate or severe form , bronchial asthma - 235 million of people. It is the most common chronic disease among children . In this case, the layering of respiratory infections on chronic pathology is the main factor leading to an exacerbation of the latter and the progression of the course of the disease.
One of the important causes of morbidity, protracted and complicated course of exacerbations of COPD, biofeedback and pneumonia is the altered immune reactivity of the population of various origins, associated, among other things, with unfavorable environmental factors of the modern world. In this regard, it is advisable to use immunomodulators in terms of correcting changes in immunological parameters against the background of the underlying disease, as well as for the prevention of intercurrent infections stratification and the treatment of exacerbations.
The review presents the results of previous clinical studies on the use of the Timogen® (alpha-glutamyl-tryptophan) for the treatment of bronchopulmonary diseases, both in adult and in children. Practical recommendations are given for the use of alpha-glutamyl-tryptophan in various nosological forms and depending on age.
Substantiation of the strategy of nonspecific immunoprophylaxis of active EBV infection
Introduction. The relevance of the infection caused by the Epstein-Barr virus (EBV) is determined by its ubiquitous spread, the increase in morbidity over the past decades, the lifelong persistence of the pathogen in the host body, a wide range of complications that form somatic pathology. The lack of effective means of therapy and specific prevention makes it necessary to find ways to manage the epidemic process with the help of drugs that have a nonspecific effect on the immune system.
The aim of the work was to substantiate the strategy of non-specific immunoprophylaxis of active EBV-infection.
Material and methods. A systematic review of scientific publications was carried out using the bibliographic databases PubMed, CochraneReviews/CochraneLibrary, eLibrary, Cyberleninka. The development of a strategy for the prevention of EBV infection was carried out on the basis of data on the pathogenesis of the disease and the mechanism of action of drugs.
Results. Potential means of non-specific immunoprophylaxis of active EBV infection can be bacterial lysates and their components, interferon inducers, recombinant interferons, interleukin-2 and -7, immunomodulators of plant origin, vitamins and antioxidants, pre- and probiotics. Taking into account the peculiarities of EBV infection, three directions of prevention are identified (emergency, including post-contact; prevention of complications of active EBV infection; prevention of reactivation of chronic EBV infection), for each of which potential groups of drugs are proposed.
Conclusion. The analysis of domestic and foreign publications made it possible to justify the strategy of non-specific immunoprophylaxis of active EBV infection, based on the impact on different parts of the body’s immune defense in relation to different pathogenetic stages of the disease. The use of the proposed strategy requires further in-depth study.
Immune response to Helicobacter pylori
H. pylori causes pathologies of different severity: asymptomatic infection, gastritis, gastric and duodenal ulcers, adenocarcinoma and MALT-lymphoma. According to available data, the form of the disease depends both on the virulence of the pathogen and on the characteristics of the immune response. It should be noted that in H. pylori infection the role of individual cell groups of the immune system is very ambiguous. Thus, pro-inflammatory T cell subgroups have a certain anti-infectious potential, but at the same time make a significant contribution to the development of inflammatory and destructive processes, while regulatory T cells weaken the clinical manifestations of infection, but contribute to the persistence of the pathogen. The review presents data on the role of various cells involved in the immune response to H. pylori, as well as information on the pathogenesis of lymphomas associated with H. pylori infection.
Problems associated with the manifestation of immunogenicity of biotherapeutic proteins and ways to solve them
The clinical effectiveness of the use of biotechnological medicines (therapeutic/biotherapeutic proteins) is due to the high selectivity of action on certain links in the pathogenesis of the disease due to the specificity of their interaction with a significant target antigen. However, their use is accompanied by the development of serious side effects, in some cases due to the manifestation of unwanted immunogenicity. One of the main clinical manifestations of the formation of an immune response to a medicine is the development of the patient’s non-response to the therapy. The review analyzes the mechanisms of development of primary and secondary nonresponsiveness to the medicine, approaches to elucidating the cause of the manifestations of responsiveness and choosing a strategy for further treatment of patients with this condition. A critical analysis of methodological techniques for assessing the immunogenicity of a medicinal product and detecting anti-drug antibodies (ADA) in blood serum samples from patients is presented.
Adjuvants and immunomodulators in vaccines
Infection diseases remain one of the leading causes of the death toll in the world. Resent achievements in vaccine development made a significant contribution to frequency reduction in incidence of infection diseases. Live and inactivated vaccines contain a weakened pathogen, which provides the highest level of immunological potency of vaccine. However, this negatively affects the safety of the vaccine itself. Subunit vaccine based on fragments of viruses or bacteria. High level of safety is provided by the absence of an infection agent in the vaccine. This fact naturally reduces its ability to induce long-term and post vaccination immunity. Thus, adjuvants are necessary for development of effective vaccines. Originally chemical compounds were used as adjuvants to promote local inflammation and antigen deposition. The effectiveness of this approach has been demonstrated in numerous experiments. Discovery of pattern-recognition receptors (PRRs) shed the light on the role of many well-known adjuvants to activate the innate immune system, what is critically important for triggering the adaptive immunity. Interaction of adjuvant with PRRs results in activation of inflammasome, amplification of the expression of MHC and the cytokine network activation. However there is still no full understanding of adjuvant effects and this fact requires future investigation. The data on the common adjuvants used in contemporary vaccines are analyzed in review. Literature search was conducted through the following science data bases: Web of Science, Pubmed, RSCI.
Tumor xenografts as the model for a preclinical trials of genetically modified cell therapy
Currently, anticancer therapy based on the adoptive transfer of genetically modified cells is being actively developed, which increasingly actualizes tumor xenografts as a model for preclinical testing of these cellular products. The tumor xenografts model is the closest of the existing methods that simulate natural tumor growth since it reproduces a three-dimensional vascularized tumor in the body with maintained homeostasis, metabolism, and possible mechanisms of tolerance to the therapeutic agent. Long-term use of the model has led to its standardization due to the emergence of well-characterized tumor cell lines and inbred mice, as well as in vivo imaging methods that measure the progression of cancer objectively. Modern approaches based on patient-derived tumor xenografts more closely imitate the intratumoral and intertumoral heterogeneity observed in clinical practice, which provides a more adequate assessment of the antitumor potential of the cell products studied. At the moment, the world literature has accumulated a large amount of data on the used tumor cell lines and inbred mice properties and the model methodology, which is necessary to obtain reliable results in the tumor xenograft model. The xenograft tumor biology and the interaction mechanisms of therapeutic genetically modified T cells and the tumor have been studied in sufficient detail, which will also be considered in this work to help in choosing an adequate and informative model for research.