Type I allergy is mediated by the formation of IgE antibodies to proteins secreted by nonreplicating microorganisms (plant pollen, house dust mites, etc.) that enter the mucous membranes in very low concentrations. The mechanisms and localization of naive B cells’ switching to IgE production have not been fully determined. The aim of this work was to determine the switching site of B cells and the traffic of IgEproducing B cells in mice immunized with a low dose of equimolar mixture of egg proteins Gal d1, Gal d2, and Gal d3. Allergens in saline solution were injected into the withers of mice 9-10 times with an interval of 2-3 days; the total dose was 2.7 µg/mouse. The production of IgE to Gal d proteins in the blood and by B cells isolated from the withers, draining lymph nodes, spleen, and bone marrow of immune mice was analyzed in dynamics after cessation of sensitization. Both in blood and in in vitro cultures, the dominance of IgE changed from the recognition of the LMW Gal d2 during the sensitization of mice to the HMW Gal d3 after sensitization was discontinued. In this model, an IgG memory response appeared only a month after the end of sensitization and recognized only Gal d3. In vitro cultures showed that B cells switched to IgE production locally in the withers with low traffic to the spleen. In the blood serum, IgE titers for all Gal d proteins decreased after the cessation of sensitization and persisted for a long time. A month after the cancellation of the sensitization, a pool of B cells producing IgE in vitro appeared in the spleen. These B-cells died after 20-30 days as no in vitro IgE production was observed later than 85-90 days. The results obtained allowed us to draw several conclusions. B cells switch to IgE synthesis locally at the site of allergen injections. The response was two-phase: LMW Gal d2 was recognized in the early response, while HMW Gal d3 was recognized in the late phase. In this model, the IgG response to HMW Gal d3 was clearly dominant. In conclusion, it has been shown that when the immune system recognizes a mixture of proteins originating from some allergen, the dominance of proteins recognized by both IgE and IgG is observed. Since allergy patients most often do not have IgG antibodies, it can be assumed that in this case an acute phase response, supported by antigen intake, is observed, in which LMW allergens are recognized.

The cellular response to endoplasmic reticulum (ER) stress accompanies plasma cell maturation and is one of triggers and cofactors of the local inflammatory response. Chemical chaperones, low-molecular substances that eliminate pathological ER stress, are proposed as means of treating pathologies associated with ER stress. The aim of this study was to evaluate the effect and mechanisms of influence of chemical chaperones on the humoral response in a low-dose model of allergy. The allergic immune response was induced in BALB/c mice by repeated administration of ovalbumin at a dose of 100 ng for 6 weeks. Some animals were injected with both the antigen and the chemical chaperones, TUDCA (tauroursodeoxycholic acid) or 4-PBA (4-phenylbutyrate). Administration of TUDCA, but not 4-PBA, suppressed production of allergen-specific IgE (a 2.5-fold decrease in titer). None of the chemical chaperones affected the production of specific IgG1. The effect of TUDCA was associated with suppression of the switch to IgE synthesis in regional lymph nodes. This phenomenon was associated with suppressed expression of genes encoding cytokines involved in type 2 immune response, especially Il4 and Il9, which in turn could be caused by suppression of IL-33 release. In addition, TUDCA significantly suppressed expression of the cytokine APRIL, and to a lesser extent, BAFF. Thus, TUDCA inhibition of the allergy-specific IgE production is due to suppression of the release of IL-33 and a decrease in the production of type 2 immune response cytokines, as well as suppression of the expression of the cytokines APRIL and BAFF.

Despite a large number of works focused on the search for the mechanisms of formation of IgE-producing B cells, the question of the relative contribution of germinal centers and extrafollicular foci B cells in this process still remains controversial. Of particular interest is the study of the mechanisms of stimulation of the allergic immune response under the influence of air pollutants. The aim of the work was to study the connection between the adjuvant effect of benzo(a)pyrene (BaP) on the production of specific IgE in a novel low-dose allergy model with changes in the subpopulation composition of B-cells in the tissue of the immunization site and secondary lymphoid organs. Antigen without any stimuli was administrated to one group of BALB/c mice for 9 weeks in a low (0.3 μg) dose. BaP was administrated to another group of mice along with antigens at a dose of 4 ng. B-cell subpopulations were analyzed by flow cytometry. BaP significantly stimulated the production of allergen-specific IgG1 at early (3 weeks) time point, and allergen-specific IgE at late (9 weeks) time point. The aeropollutant increased the content of CD19+CD38–CD95+B220+ germinal center B-cells with the phenotype and their precursors (CD19+CD38+CD95+B220+) with the phenotype in the spleen at early and late time points, but not in the lungs or regional lymph nodes. Under its influence, the content of CD19+CD38–CD95+B220– and CD19+CD38+CD95+B220+ extrafollicular plasmablasts in the spleen at an early time point and in lung tissue at a later time point also increases. In the spleen, BaP increased the content of CD138+CD19–B220+ and CD138+CD19–B220– mature plasma cells, and in regional lymph nodes the content of CD138+CD19+B220– immature plasma cells at a later time point. The adjuvant effect of BaP on the production of specific IgE was largely associated with stimulation of the formation of germinal centers in the spleen and with extrafollicular activation of B cells in lung tissue.

Objective: Despite a large number of works focused on the search for the mechanisms of formation of IgE-producing B-cells, the question of the relative contribution of germinal centers and extrafollicular foci B-cells in this process still remains controversial. Of particular interest is the study of the mechanisms of stimulation of the allergic immune response under the influence of air pollutants. The aim of the work was to study the connection between the adjuvant effect of benzo(a)pyrene (BaP) on the production of specific IgE in a novel low-dose allergy model with changes in the subpopulation composition of B-cells in the tissue of the immunization site and secondary lymphoid organs. Methods: Antigen without any stimuli was administrated to one group of BALB/c mice for 9 weeks in a low (0.3 μg) dose. BaP was administrated to another group of mice along with antigens at a dose of 4 ng. B-cell subpopulations were analyzed by flow cytometry. Results and discussion: BaP significantly stimulated the production of allergen-specific IgG1 at early (3 weeks) time point, and allergen-specific IgE at late (9 weeks) time point. The aeropollutant increased the content of CD19+CD38–CD95+B220+ germinal center B-cells with the phenotype and their precursors (CD19+CD38+CD95+B220+) with the phenotype in the spleen at early and late time points, but not in the lungs or regional lymph nodes. Under its influence, the content of CD19+CD38–CD95+B220– and CD19+CD38+CD95+B220+ extrafollicular plasmablasts in the spleen at an early time point and in lung tissue at a later time point also increases. In the spleen, BaP increased the content of CD138+CD19–B220+ and CD138+CD19–B220– mature plasma cells, and in regional lymph nodes the content of CD138+CD19+B220– immature plasma cells at a later time point. Conclusions: The adjuvant effect of BaP on the production of specific IgE was largely associated with stimulation of the formation of germinal centers in the spleen and with extrafollicular activation of B-cells in lung tissue.

A significant increase in the prevalence of diseases linked with IgE production can be seen in recent years, but the question about the role of TLR receptors in this process remains controversial. According to the hygiene hypothesis, the decrease of the contact of the individual with pathogens that contain PRR receptor ligands in the recent years leads to the development of allergic diseases. The aim of this work was to investigate whether TLR4 and NLRP3 receptor activation contributes to allergen-specific antibody formation. BALB/c mice were immunized according to two different protocols. In the first one, OVA antigen was administered in 0.1 µg dose 2-3 times a week for 6 weeks by subcutaneous route. In the second one, OVA was administered in 0.3 µg dose intranasally in combination with 4 ng of benzo(a)pyrene (BaP) 2 times a week for 8 weeks. In both cases, TLR4 and NLRP3 receptor inhibitors, namely TLR4-IN-C34 in 1 mg/kg dose and CY- 09 in 20 mg/kg dose respectively were also administered to the some of the mice. Specific antibody production was determined by ELISA. Immunization of mice with TLR4-IN-C34 significantly (p 0.01) amplify IgE production (about 2.5 times in comparison with control group), but has no effect on specific IgG1 production in subcutaneous model. Specific IgE titers in the control group immunized without small molecule inhibitor and in the TLR4-IN-C34 group were (3±0.6) × 103 and (8±2) × 103, respectively. In this model, CY-09 administration has no effect on humoral immune response. In the secondary (intranasal) model, BaP significantly increase specific IgE and IgG1 production. CY-09 but not TLR4-IN-C34 administered in combination with BaP significant (p 0.05) and approximately 2 times enhances specific IgE but not IgG1 production. Specific IgE titers in the control group without inhibitor and in the CY-09 group were (2.0±0.4) × 102 and (5.1±0.3) × 102, respectively. So, PRR-activation, in our case activation of TLR4 in the model based on subcutaneous immunization or NLRP3 in the model based on intranasal antigen administration with BaP suppressed the production of allergen-specific IgE, but not IgG1. These data are in consistent with the hygiene theory of allergy development.

IgE-mediated allergic reaction occurs in response to harmless environmental compounds, such as tree and grass pollen, fragments of household microorganisms, etc. To date, the only way to treat IgE-mediated allergy is allergen-specific immunotherapy (ASIT), which consists of a prolonged subcutaneous administration of allergen extracts or recombinant proteins. The long duration of the treatment, the cost and the risk of life-threatening adverse reactions are the main limiting factors for ASIT. The aim of this work was to develop allergen proteins encapsulated in chitosan-based microparticles that can be safely administered at high doses and in a rash protocol. The egg white allergen, Gal d 1 protein, was used as a model antigen. The protein was packed into core–shell type microparticles (MPs), in which the core was formed with succinyl chitosan conjugated to Gal d 1, subsequently coated with a shell formed by quaternized chitosan. The obtained core–shell MPs containing Gal d 1 in the core (Gal-MPs) were non-toxic to macrophage and fibroblast cell lines. At the same time, Gal-MPs were quickly engulfed by bone marrow-derived dendritic cells or RAW264.7 macrophage cells, as was visualized using flow cytometry and confocal microscopy. Encapsulated Gal d 1 was not recognized by Gal d 1-specific IgE in ELISA. Female BALB/c mice were immunized with Gal-MPs subcutaneously three times a week for 2 weeks. Immunization of mice resulted in IgG titers 1250 ± 200 without IgE production. Allergy in control and vaccinated mice was induced by low-dose Gal d 1 injections in the withers of mice. IgE was induced in control-sensitized but not in the vaccinated mice. Thus, preventive vaccination with the encapsulated allergens is safe and rapid; it significantly reduces the risk of IgE production induced by respiratory and oral allergens.

This review is devoted to the analysis of the main mechanisms of the formation of IgE-producing cells in the body and a brief review of the main, most striking candidate agents for use in innovative methods of therapy for IgE-dependent pathologies. Data are presented according to which the role of IgE + plasma cells and various subpopulations of memory B-lymphocytes in the formation and persistence of the state of sensitization to a harmless allergen differs depending on the model system used or the clinical case under study. Therefore, drugs that target signaling pathways involved in the regulation of both plasma cells and memory B-lymphocytes are especially promising in the treatment of allergic diseases. The authors conclude that the components of the cellular response to oxidative stress and related genotoxic stress and ER stress are the most promising as such targets, since (a) all of them directly or indirectly affect the processes that regulate both of these subpopulations; (b) are involved in the process of formation and maintenance of local allergic inflammation. The review presents data pointing to the particular promise of using nanoparticles of noble metals and complexes of rare earth metals of lanthanides in this regard, due to their ability to induce long-term effects in small doses due to changes in the properties of innate immunity cells and long-term accumulation in the body.

In view of increased incidence of IgE-mediated diseases in Russia and worldwide, the efforts of various research groups are focused on studying the mechanisms that trigger the process of switching B-lymphocytes to IgE synthesis upon human exposure to harmless allergens, including the role of various antigen-presenting cells (APCs) in this process. However, the role of distinct APCs upon long-term penetration of low antigen doses via the tissue barriers, is yet poorly understood, as well as specific features of these events upon entrance of the antigen through the subcutaneous adipose tissue which contains tissueassociated lymphoid clusters (TALC).The aim of this work was to determine the relationship between the local accumulation of various APCs in the subcutaneous adipose tissue and development of early IgE production in a clinically relevant experimental model of allergy with long-term administration of low allergen doses. In this experimental model, specific IgE synthesis is induced, with minimal concomitant IgG production, thus mimicking the situation observed in patients with clinically sound allergies. BALB/c mice were immunized for 4 weeks subcutaneously in the withers area or intraperitoneally with low (100 ng) or high (10 μg) doses of the model allergen (ovalbumin). Blood samples were taken weekly from mice for ELISA testing, to determine the production of specific antibodies. Provocation tests were performed with high dose of the allergen, and adipose tissue samples were taken from the site of antigen injection for flow cytometric assays, in order to evaluate the contents of various APC subpopulations. Specific IgE production was induced mainly by subcutaneous injection of the antigen at low doses (100 ng) into the area of withers. When using this experimental regimen, we observed accumulation of classical CD11b + cells in adipose tissue at the withers site, but not in the peritoneal adipose tissue, in absence of CD11b - classical, inflammatory or plasmacytoid, dendritic cells. These findings coincided in time with increased production of specific IgE on days +14 to +21. Accumulation of CD11b + CD11c - macrophages and their CD206 + M2 subpopulations at early terms (days +7 and +21) was also observed only after subcutaneous injection of the antigen into the withers area. The high-dose antigen injection (10 μg) which mediated IgG 1 production to greater extent than production of IgE, led to earlier accumulation of CD11b + classical dendritic cells (on day 7 th ), and to the absence of macrophage accumulation at later stages (day 21 th ). Thus, the early start of specific IgE production upon low-dose injection of the antigen into the subcutaneous adipose tissue may be associated with its presentation by CD11b + classical dendritic cells in the presence of CD11b + CD11c - macrophages.

At the present time, the efforts of many research groups around the world are aimed at finding new factors triggering the allergic sensitization process linked with IgE synthesis to harmless allergens. According to the recent data, production of tissue cytokines is induced in tissue cells by alarmins, thus, in turn, eliciting pro-allergic immune response. Previously we have shown that β-alanine could be a potential alarmin capable to stimulate production of tissue cytokines. The aim of this work was to determine the impact of β-alanine on humoral immune response in low-dose allergy model. BALB/c mice were immunized by recombinant Asp f 2 protein or commercial ovalbumin (OVA) in the withers 3 times a week with or without β-alanine supplementation. To determine the mechanism of β-alanine effect, α-L-alanine, an isomer which is not MrgD receptor ligand, and β-aminoisobutyrate with β-alanine-like affinity to MrgD ligand, were compared. According to our data, β-alanine stimulated specific IgE and IgG1 production in a short-term course (7 immunizations) and enhanced antibody affinity after long-term (14 immunizations) protocol in the case of low-immunogenic protein Asp f 2. In the case of high-immunogenic OVA protein, the impact of β-alanine was significant only upon antibody affinity. Hence, β-alanine accelerates specific IgE production in the case of low-immunogenic protein. The impact of β-alanine on specific IgE production was not linked to specific MrgD receptor activation, because β-aminoisobutyrate, which is the other ligand of this receptor, did not have a similar effect upon humoral immune response. The effect of β-alanine on IgG1 production seems also independent of MrgD receptor, since the common proteinogenic amino acid α-L-alanine also enhanced specific IgG1 production. The effect of β-alanine on humoral immune response could be linked to its non-specific action, e.g., due to its ability to induce oxidative stress through blocking taurine transporter, or due to its ability to stimulate cellular metabolism.

Аллергия I типа опосредована формированием IgE-антител к безвредным веществам. Механизмы возникновения аллергии остаются дискуссионными. Одним из факторов риска может быть снижение защитных функций барьерных тканей. Целью работы было проанализировать гуморальный иммунный ответ на вирус Эпштейна–Барр (ВЭБ) у больных с аллергией на гриб A. alternata и клещей домашней пыли D. farinae (КДП) и у здоровых людей. Известно, что до 90% населения инфицированы ВЭБ. Инфицирование происходит в раннем возрасте параллельно с формированием аллергических реакций. Анализ антител проводили методом иммуно-ПЦР с использованием рекомбинантного белка ВЭБ rEBNA1. Показали, что инфицирование как у больных, так и у доноров происходит в детстве; доля сероположительных по ВЭБ индивидов была сравнимой в группах (75% и 74%). Доля пациентов с высокими титрами IgG1 среди больных с аллергией была ниже (7%) по сравнению с донорами (18%), что соответствует меньшей вирусной нагрузке. У больных с аллергией, но не у здоровых людей, наблюдали снижение титров IgG1 с возрастом (р = 0,037). Кроме того, при аллергии повышены титры IgA1 по сравнению с донорами, однако IgA1-ответы при аллергии на гриб A. alternata и на КДП различались. При аллергии также раньше формировались IgM к ВЭБ. Таким образом, при аллергии быстрее формируется IgM и IgA1 гуморальный ответ, что приводит к снижению с возрастом IgG1-титров.

AIMS:Multicellular tumor spheroids (MCTS) produced by different methods vary in forms, sizes, and properties. The aim of this work was to characterize MCTS formed by six pancreatic cell lines on a non-adherent surface.MATERIALS AND METHODS:Human pancreatic cells were grown in 2D and 3D conditions and compared for the expression of E- and desmosomal cadherins (PCR, confocal microscopy), growth, cell cycling, apoptosis (flow cytometry), and a response to antitumor drugs doxorubicin and gemcitabine (MTT-assay).KEY FINDINGS:Three types of MCTS were identified: BxPC-3, T3M4 formed small number of large and dense spheroids representing type I MCTS; COLO-357 and AsPC-1 generated type II multiple and loose MCTS of different sizes while MiaPaCa-2 and PANC-1 represented type III cultures which grew almost as floating monolayer films. Formation of type I MCTS depended on the simultaneous expression of DSG3 and several DSC proteins; II MCTS expressed solely DSG2-DSC2 but not DSG3, while type III cells either did not express E-cadherin or a pair of DSG and DSC proteins. Cells in type I MCTS but not in types II and III ones quickly became quiescent which correlated with a decrease in the proliferation, increased apoptosis, and a higher resistance to antitumor drugs doxorubicin and gemcitabine.SIGNIFICANCE:Taken collectively, pancreatic cells significantly vary in the expression of desmosomal cadherins, resulting in the formation of MCTS with different characteristics. The sensitivity of MCTS to various drugs depends on the type of cells and the method of spheroid preparation used.

Type I hypersensitivity is mediated by the production of IgE antibodies in response to normally harmless substances. Debate still continues about the mechanisms underlying allergic reactions. Reduced barrier tissue function can be one of the risk factors for allergies. The aim of the present work was to compare the humoral immune response to Epstein-Barr virus in patients allergic to the A. alternata fungus or D. farinae house dust mites and healthy donors. It is known that up to 90% of the world population are infected with EBV. This infection occurs at early age when a child develops allergy. The antibodies were analyzed using immuno-PCR and the recombinant EBV protein rEBNA. We were able to demonstrate that infection occurs at early age in both allergic patients and healthy donors. The proportion of EBP-seropositive individuals was comparable between the groups (75% and 74%). The proportion of patients with high IgG1 titers among patients with allergies was lower (7%) than in healthy donors (18%), suggesting a lower viral load. In patients with allergies (but not in healthy donors) IgG1 titers declined as children grew older (р = 0.037). Besides, IgA1 titers were increased in patients with allergies in comparison with healthy donors, but differed between patients allergic to A. alternata and house dust mites. In allergic individuals, production of IgM against EBV was triggered earlier than in healthy donors. We conclude that IgM production and the IgA1-mediated humoral response occur earlier in patients with allergies, causing a decline in IgG1 titers over time.

A primary response of the innate immune system to a pathogen invasion or tissue damage is the synthesis of tissue cytokines. Interleukins (IL) 25 and 33 or thymic stromal lymphopoietin (TSLP) are produced by epithelial or muscle cells in response to pathogen fragments, i.e., pathogen associated molecular patterns (PAMPs) or endogenous stress factors (danger associated molecular patterns, DAMPs). The goal of this work is to compare the ability of the bacterial peptide N-formyl-met-leu-phe (fMLP) and an endogenous pruritus inducer, amino acid β-alanine, to stimulate the expression of tissue cytokines. It has been shown by quantitative PCR that fMLP stimulates the ex vivo gene expression of TSLP and IL-25 in murine trachea explants, and β-alanine also stimulates IL-33 gene expression. The in vivo level of TSLP and IL-33 has been evaluated by ELISA in lung homogenates 1 and 6 h after intratracheal injection of the stimulants. Both β‑alanine and fMLP induce the release of IL-33 and TSLP from the intracellular depot. It is shown for the first time that β-alanine and fMLP are activators of tissue cytokine synthesis.

Allergy is characterized by IgE production to innocuous antigens. The question whether the switch to IgE synthesis occurs via direct or sequential pathways is still unresolved. The aim of this work was to analyze the distribution of immunoglobulins (Ig) to house dust mite D. farinae and A. alternata fungus in allergic children with primarily established diagnosis and compare it to Epstein-Barr antiviral (EBV) response in the same patients. In allergy patients the only significant difference was found in allergen specific IgE, likely mediated by a direct isotype switch, while antiviral response was dominated by EBV specific IgG and low level of concordant IgA and IgG4 production consistent with a minor sequential Ig switches. Taken collectively, we concluded that sequential isotype switch is likely to be a much rarer event than a direct one.

Intracellular N-acetylglucosaminylmuramyl peptide-binding proteins of murine macrophages and myelomonocytic WEHI-3 cells were characterized. SDS-PAGE and Western blotting revealed proteins with molecular masses of 18, 32 and 34 kDa retaining the ability to specifically bind glucosaminylmuramyl dipeptide. The inhibition analysis demonstrated that only biologically active muramyl peptides but not inactive analogs or fragments of glucosaminylmuramyl dipeptide could inhibit glucosaminylmuramyl dipeptide-binding to these proteins. Purification of these proteins and sequencing of peptides obtained after in-gel trypsin digestion enabled us to identify the above mentioned proteins as histones H1 and H3. These findings suggest that nuclear histones might be target molecules for muramyl peptides.