Parameters of innate immunity in common variable immune deficiency and X-linked agammaglobulinemia
Introduction. One of the main means whereby antibodies fulfil their protective function is activation and enhancement of innate defense mechanisms, i.e. complement and phagocytes. Therefore, studying innate immunity is important for understanding pathogenesis and development of novel therapeutic approaches in primary immune deficiencies (PID) with predominantly antibody deficiencies.
Aim – to assess functional parameters of innate immunity in patients with X-linked agammaglobulinemia (XLA) and common variable immune deficiency (CVID), including in patients with different response to replacement therapy with intravenous immunoglobulins.
Material and methods. We studied 38 patients with CVID, 9 patients with XLA and 34 healthy donors. Engulfment and intracellular killing of fluorescein-labelled Staphylococcus aureus, expression of Fcγ receptors (CD16, CD32, CD64) and complement receptors (CD35, CD11b, CD11c) by blood neutrophils and monocytes was assessed using flow cytometry. Production of pro- and anti-inflammatory cytokines IL-1β, IL-6, IL-8, IL-10 and tumor necrosis factor (TNF) was studied using enzyme-linked immunosorbent assay.
Results. In patients with CVID, we observed reduced surface expression of complement receptors by neutrophils, whereas engulfment and killing of St. aureus as well as cytokine production was not different from those in healthy donors. In a subgroup of CVID patients who required additional supportive antibiotics despite adequate replacement therapy, the decrease of complement receptors on neutrophils was more pronounced than in patients who did not require antibiotics. In XLA patients, both complement receptor and CD16 (FcγRIII) expression on neutrophils was reduced, correlating with decreased engulfment of St. aureus in different opsonization conditions. At the same time, intracellular killing of St. aureus in XLA patients was normal, while production of IL-1β, IL-6 and IL-10 by lipopolysaccharide-stimulated blood mononuclear cells was increased as compared to healthy donors.
Conclusions. The discovered abnormalities of innate immunity may contribute to decreased resistance to infections in CVID and XLA.
Biochemical and functional characterization of multimerized proteins containing the CD40L receptor domain
Introduction. In the processes of B-lymphocyte activation and regulation of its activity, an important role is played by the interaction of the CD40 receptor expressed on the surface of the B-lymphocyte and its ligand CD40L located on the surface of the T-helper. The signaling activity of CD40L is most pronounced in the trimerized state, which is provided when the protein is expressed on the surface membrane. To create a feeder-free system for stimulating B-lymphocytes, it is necessary to obtain recombinant multimerized CD40L.
The aim of the study was to produce a soluble oligomerized human CD40L protein suitable for oligoclonal stimulation of B-lymphocytes in vitro.
Material and methods. Two genetic constructs encoding the receptor part of the CD40L molecule fused with two types of sequences were obtained by molecular cloning methods. In one case, it was a human IgG1 Fc fragment coupled to an isoleucine zipper, which was named LIF. In another case, it was the sequence of the collagen like domain of adiponectin, this construct was named LA. Plasmids encoding LIF and LA were transfected into HEK293 cells and the corresponding soluble recombinant proteins were generated. The molecular weight of proteins was determined by immunoprecipitation followed by Western blotting. The functional activity of LIF and LA proteins was determined under conditions of feeder-free stimulation of B-lymphocytes in vitro.
Results. In transiently transfected HEK293 cells, intracellular expression of LIF and LA proteins were observed, as well as the secretion of these proteins into the culture supernatant. After immunoprecipitation and following Western blotting, the LIF preparation migrated on electrophoresis as a band of ~ 55 kDa under reducing and 100 kDa under non-reducing conditions. Under appropriate conditions, the LA preparation migrated as 30 and 85 kDa correspondingly. In the presence of exogenous IL-21, the LA preparation provided B-lymphocyte proliferation comparable to that observed in the feeder system. The B-lymphocyte proliferation index under the action of IL-21 and LIF was three times lower than in the feeder system. Stimulation with LA resulted in a higher proportion of plasmablasts than with LIF. In addition, LA ensured the differentiation of some of the stimulated B-lymphocytes into plasma cells. On the 20th day of stimulation of B lymphocytes in a feeder-free system with LA, secretion of IgM and IgG was observed in supernatants even in cultures containing single B cells.
Conclusion. Recombinant multimerized proteins containing the CD40L receptor domain have good stimulatory activity against peripheral blood B-lymphocytes. CD40L multimerized by fusion with the collagen-like domain of adiponectin exhibits higher physiological activity compared to CD40L multimerized by an IgG Fc-fragment and an isoleucine zipper. The adiponectin-CD40L fusion protein can be used for feeder-free stimulation of single or oligoclonal populations of B-lymphocytes.
Development of experimental mouse xenograft models of human tumors for preclinical in vivo studies of product for cellular immunotherapy
Introduction. In connection with the active development of new directions in cellular immunotherapy, the question of approaches for conducting preclinical studies on safety and efficacy is acute. The most optimal for solving this problem is the use of tumor xenografts to simulate tumor growth in the body while maintaining homeostasis, metabolism and possible mechanisms of tolerance to a therapeutic agent. The effectiveness of this approach to the analysis of the effectiveness of cell preparations led to the need to standardize methodological approaches to the selection of human tumor cell lines and lines of immunodeficient mice.
The purpose of the study was to evaluate the effectiveness of xenograft formation in two strains of immunodeficient mice in order to obtain optimal models for preclinical studies of preparations of modified human cells.
Material and methods. To test the models we used mice of the NRG (NOD.Cg-Rag1tm1Mom Il2rgtm1Wjl/SzJ) and SCID lines and human tumor cell lines SW620 (human colon adenocarcinoma), U-87MG (human epithelial brain tumor), SK-MEL-37 (melanoma), NW-MEL-38 (melanoma) and S6 (melanoma). Experimental animals were injected subcutaneously into the region of the right shoulder blade with 100 µl of tumor cell suspension at doses of 1, 3 and 5 mln/mouse, followed by measurement of the size of the tumor node.
Results. The effectiveness of the formation of xenografts of various human cell lines in mice with immunodeficiency was evaluated. It has been shown that the NRG mice, compared to the SCID mice, has a higher ability to engraft tumor cell lines. And the cell lines themselves differ in the levels of expression of tumor-associated proteins and form a xenograft with different efficiency. The most optimal was the use of NRG mice and the SK-MEL-37 cell line, with a high level of expression of the GD2, NY-ESO-1 and MAGE-A4 antigens. An experiment on the treatment of solid tumors obtained after transplantation of human tumor cell lines into animals using anti-GD2-CAR-T cells showed effectiveness in reducing the size of the tumor node with intratumoral administration of modified human cells.
Conclusion. The results obtained confirm the efficiency of using xenografts in evaluating the effectiveness of the antitumor immune response of modified cell preparations in vivo.
Study of reproductive toxicity of the recombinant allergy vaccine for the treatment of allergy to birch pollen and cross-related allergens
Introduction. The vast majority of allergy vaccines contain natural allergens, because they are produced from extracts of natural raw materials. This limits their use because undesirable effects are often taking place. Moreover, in order to avoid sensitization extract-based vaccines are not used for the prevention of healthy individuals and are not used during pregnancy and lactation. Recently, a recombinant hypoallergenic vaccine ABP-Vax was developed for the treatment and prevention of birch pollen and cross related allergies. Due to its hypoallergenic properties, this vaccine has prophylactic potential and can also potentially be used during pregnancy and lactation.
The aim of the study – experimental study of the effect of the allergy vaccine ABP-Vax on the indices of generative function (fertility) of female rats and on the prenatal and postnatal development of their offspring.
Material and methods. Female rats were repeatedly subcutaneously injected with the ABP-Vax allergy vaccine before mating and during pregnancy. Injections course covered the period before conception and until the lactation. Treated females were mated with intact males and female fertility indices were determined, as well as parameters of the embryotoxic effect of the allergy vaccine during pre- and postnatal period.
Results. Analysis of prenatal development of the offspring did not reveal any signs of allergovaccine embryotoxic effect. During the study of the offspring postnatal development, no effect of the vaccine on physical development was observed. The indices of experimental animals’ reproductive function had normal level.
Conclusion. No negative effects of the allergy vaccine ABP-Vax were established on female rats’ generative function as well as on the pre- and postnatal development of their offspring. The results confirm our assumption about the possibility of using ABP-Vax before conception, during pregnancy and lactation.
Severe polypous rhinosinusitis associated with non-allergic bronchial asthma
Introduction. Chronic rhinosinusitis with nasal polyp (CRSwNP) and bronchial asthma (BA) are heterogeneous inflammatory diseases of the respiratory tract. Despite the fact that BA and CRSwNP are currently considered as separate diseases, they often coexist in one patient and may influence each other. Due to the insufficient efficacy of current therapy of severe BA (especially comorbid with CRSwNP), the urgent task is to search for new approaches of treatment of these diseases, which is impossible without a detailed understanding of the molecular and cellular mechanisms of their pathogenesis.
The aim of the study was to investigate the course characteristics of CRSwNP in patients with severe asthma.
Material and methods. 96 participants were enrolled into 4 groups of 24 in each: group I – patients with hypertrophic rhinitis without BA and atopy (comparison group), group II – patients with CRSwNP, group III – patients with CRSwNP comorbid with allergic BA, group IV – patients with CRSwNP comorbid with non-allergic BA. ELISA was used to determine the concentration of cytokines in supernatants of peripheral blood mononuclear cells (PBMCs) and polyp tissue homogenates. The degree of polyp tissue infiltration with pro-inflammatory cells was assessed by histological analysis. Quantitative PCR was used to evaluate the levels of cytokine gene expression in PBM and polyp tissues. The epithelial cells of the polyps were obtained by flow sorting.
Results. Non-allergic asthma comorbid with CRSwNP is more severe and uncontrolled. CRSwNP is accompanied primarily by local rather than systemic inflammation. Non-allergic BA leads to more aggressive and recurrent course of CRSwNP, and the polyp tissues characterized by a pronounced inflammation with eosinophils and neutrophils. Allergic BA comorbid with CRSwNP is associated with suppression of Th1- and activation of Th2-immune response, as well as with increased expression of the IL25 and TSLP genes in epithelial cells of polyps. At the same time, non-allergic BA comorbid with CRSwNP was characterized by activation of the Th17-immune response and high expression of IL25, TSLP, IL33 and IL37.
Conclusion. BA and CRSwNP are heterogeneous diseases encompassing multiple phenotypes, which arise through distinct molecular mechanisms. Our study revealed the interplay between CRSwNP and BA. Particularly, non-allergic bronchial asthma, primarily, emerges as a factor contributing to a more aggressive course of CRSwNP.
Link between the inflammasome complex and cytokines IL-1β and IL-10 and pathological phenotype of aging in centenarians
Introduction. There are two phenotypes of aging: physiological (or successful) and pathological. The last one is extremely undesirable, accompanied by the development of age-associated pathologies. In recent years, the attention of scientists has been focused on the study of the role of chronic inflammation in the formation of aging.
Aim – study the role of the inflammasome complex NLRP3 (Nod-like receptor), CASP1 (caspase-1) of cytokines IL-1β and IL-10 in the symptoms of the pathological phenotype of aging.
Material and methods. The main group included 56 centenarians. The comparison group consisted of 25 healthy donors. Nucleic acids were isolated from peripheral blood cells. The level of expression of NLRP3, CASP1, IL1B and IL10 genes was determined by real-time polymerase chain reaction. The concentration of IL-1β and IL-10 in sera was estimated by enzyme-linked immunosorbent assay.
Results. The present study revealed overexpression of genes of the inflammasome complex NLRP3 and CASP1 and the pro-inflammatory cytokine IL1B, as well as an increase in the concentration of IL-1β in main group compared to the comparison group, while the concentration of the anti-inflammatory cytokine IL-10 in main group was reduced. In multimorbid centenarians, an increase in the expression of genes NLRP3, CASP, IL1B, IL10 and an increase in the concentration of cytokines IL-1β and IL-10 were revealed compared to centenarians with a low comorbidity index. A high correlation was found between the genes expression levels of the NLRP3, CASP1 and IL1B and the Charlson comorbidity index. A high concentration of cytokines IL-1β and IL-10 was found in centenarians with high comorbidity.
Conclusion. Hyperexpression of genes of the inflammasome complex and cytokines is associated with a high comorbidity index, which may explain the role of these factors in development of age-related diseases and the pathological phenotype of aging. These results can be used to search for early immunological predictors of pathological aging.
Signaling pathways of transcription factors and the role of genes in their regulation in severe acne
Introduction. The pathogenesis of acne, one of the most common skin diseases, is not fully understood and complex, and the inflammatory reaction is one of the predominant in the development of this dermatosis. Recent studies have established that the TLR4/NF-kB/p38 MAPK pathway and the cytokines IL-6, IL-1ß and TNFa are of leading importance in the inflammatory response in acne. The results of the studies show the participation of transcription factors, including NF-kB, in the pathogenesis of a number of diseases, including acne, but these mechanisms have not yet been established.
Aim – is to identify and analyze variants of the nucleotide sequence of genes of the family of transcription factors NF–kB RNF31, CARD14, CARD11, SETBP1, BACH2 in patients with severe acne.
Material and methods. To achieve this goal, a prospective open, non-randomized, single-center comparative study was conducted in 2017–2020. 70 people aged 15 to 46 years (median – 22.1 [10.2; 25.4] year) were under our supervision in clinical conditions at the Skin Diseases and Cosmetology Department of the FDPO FGAOU at the RNIMU named after N.I. Pirogov of the Ministry of Health of the Russian Federation. Molecular genetic diagnostics was performed in all 50 patients of the main and 20 conditionally healthy individuals of the comparison group by the method of high-performance DNA sequencing – next-generation sequencing (NGS) in the laboratory of Molecular Biology of the D. Rogachev NMRC PHOI, MOH of Russia. The results were processed using XLSTAT2019 software. To assess the relationship between nominal and ordinal features, conjugacy tables were built and Pearson’s criterion χ2 was calculated on their basis. To assess risk factors, the odds ratio (odds ratio, OR) was calculated. Statistically significant differences were considered at p < 0.05.
Results. We have identified 5 SNPs in the RNF31 gene, 1 SNPs in the CARD14 gene, 5 SNPs in the CARD11 gene, 2 SNPs in the SETBP1 gene and 2 SNPs in the BACH2 gene (described by us for the first time). According to the results of calculating OR of all the SNPs of the studied genes diagnosed by us in exons with severe acne, 2 SNPs of the BACH2 gene are significantly associated with an increased risk of acne. We identified 10 SNPs in the RNF31 gene in introns and 3 SNPs in the 5'-UTR region, in the CARD14 gene – 4 SNPs in introns and 1 SNPs in the 5'-UTR region, in the CARD11 gene – 6 SNPs in introns, in the SETBP1 and BACH2 genes – 1 SNP in introns. When calculating OR, 2 SNPs of the RNF31 gene and 2 SNPs of the CARD11 gene in introns were significantly associated with an increased risk of acne.
Conclusion. Considering that the transcription factor NF-kB is an important mediator of the C. acnes recognition cascade by TLR 2 and 4, it is possible that there is a violation of the regulation of the expression of key genes associated with inflammation. Alteration of the activation of NF-kB and the TLR-MyD88 signaling cascade may be associated with the pathogenetic mechanism of the prolonged course of severe acne.
Features of cell immunity of healthcare workers in the first wave of the SARS-CoV-2 infection pandemic
Introduction. The role of the immune system in the clinical course of coronavirus infection 2019 (COVID-19) is of great interest. Health care workers are at greater risk of infection than the general population.
Aim of the study – evaluation of post-infection abnormalities in the immune system in recovered employees, identification of immunity vulnerability points, as well as development of appropriate preventive measures.
Material and methods. Cellular immunity indicators were determined by multiparameter flow cytometry in 170 medical workers of the N.N. Blokhin NMRCO of the MOH of Russia during the period of quarantine in March-July 2020. Samples of blood sera was tested by ELISA for the presence of specific IgG antibodies agaist SARS-CoV-2.
Results. The presence of specific anti-SARS-CoV-2 antibodies in employees who had an infection was detected only in 72 % (65/90). A high titer of specific anti-SARS-CoV-2 antibodies was found in 17 % (14/80) of employees who did not have clinical manifestations of a coronavirus infection. There were no statistically significant differences in the parameters of cellular immunity in the groups of recovered and not ill employees with the presence of specific anti-SARS-CoV-2 antibodies or their absence. Correlation analysis revealed a weak direct statistically significant relationship between the titer of specific anti-SARS-CoV-2 antibodies and level CD4+CD25+CD127– cells and an inverse relationship with level CD3+CD4+ cells.
Conclusion. The beginning of the formation of herd immunity in the examined medical workers during the first wave of the pandemic was recorded, regardless of the presence/absence of clinical manifestations of a viral infection prior to the study or specific anti-SARS-CoV-2 antibodies.
Dynamics and functional characteristics of antibodies and memory B cells to SARS-CoV-2 in peripheral blood from COVID-19 patients for up to 16 months
Introduction. The efficiency of the humoral immune response is crucial for pathogen elimination in respiratory viral infections. The humoral response to SARS-CoV-2 infection is achieved by the involvement of B cells in the immune process, leading to the production of specific antibodies. However, there are still insufficient data on the strength of the humoral immune response to SARS-CoV-2 in more than a year after the infection.
Aim – to study the dynamics of post-infection laboratory humoral immunity to SARS-CoV-2 over 16 months after symptoms onset.
Material and methods. 15 healthy volunteers and 87 COVID-19 patients were included in the study. The affected participants were divided into 3 study groups according to the time elapsed from the onset of symptoms to the collection of blood samples for the study (from 14 to 500 days). Specific antibody levels to S1-, S2-, RBD-, N-protein of SARS-CoV-2, virus neutralizing activity and avidity of antibodies, the activity of antibody-dependent phagocytosis and the proportion of S- and RBD-specific memory B cells were determined for all samples.
Results. Antibody levels to S1- and RBD-peptides have been shown to decrease slightly during the first year after infection and then to plateau. Level of N-protein antibodies decreased after six months of symptom onset. The antibody avidity index and antibody-dependent cell phagocytosis gradually increased throughout the observed period, while the neutralizing activity of the sera decreased. A decrease in the neutralizing activity of antibodies for SARS-CoV-2 Delta variant compared to the wild type was also found. The proportion of memory B-cells in peripheral blood gradually increased from the first days of infection, peaked by about 200 days for S-specific cells and by 300 days for RBD-specific cells, and then gradually decreased.
Conclusion. The antibody levels in the peripheral blood and their functionality are markers of the efficacy of the humoral immune response to SARS-CoV-2 infection. We have shown that even after 500 days after symptoms onset more than 2/3 of those who had been infected remained above the protective threshold for neutralizing antibodies to the S1-protein of Wuhan variant of SARS-CoV-2. However, the emergence of viral variants with decreased sensitivity to neutralization makes it necessary to carefully assess the protective capacity of the antibodies.
3D models of malignant tumors – a modern approach to assessing the antitumor properties of macrophages
Tumor tissue forms a complex microenvironment that consists of tumor cells, cells of the immune system, connective tissue cells, and extracellular matrix. The study of such a system requires a special approach that takes into account the contacts between all types of cells and the extracellular matrix. The transition from the usual two-dimensional (2D) cell co-culture on plastic surfaces to three-dimensional (3D) models of malignant tumors makes it possible to more accurately reproduce, under in vitro conditions, the complex interactions between cells as well as between cells and the extracellular matrix which occur only in vivo. Interactions between tumor cells and cells of the immune system have a key influence on the development of the tumor process and on the effectiveness of malignant neoplasm therapy. In this review, we consider main types of 3D models of malignant tumors, paying special attention to those designed to study the interactions of tumor cells and macrophages. Examples of the use of 3D models of malignant tumors for preclinical studies of drugs aimed at modulating macrophage functions are presented.
Cellular technologies in immunotherapy of autoimmune diseases
Currently used methods for treating of autoimmune diseases have some disadvantages that can lead to infectious diseases and cancer, or lack of effectiveness. An important characteristic of autoimmune diseases is the loss of tolerance and induction of an immune response to self-antigens, leading to damage to cells and tissues of the body. Therefore, the development of new methods of therapy aimed at restoring immune tolerance is an urgent and promising task. This review highlights several immunotherapies using mesenchymal stromal cells, tolerogenic dendritic cells and regulatory T cells, as well as new technologies using T-cell receptors and chimeric antigen receptors. Each of these methods has its own advantages and disadvantages.