Interleukin-2 receptor β chain as a possible target for low doses of mafosfamide

The 7-day cytotoxic lymphocytes (CTL) induced in mixed lymphocyte culture express only the chain of the interleukin-2 receptor (IL-2R). In the present study this fact has been confirmed in a murine semi-allogeneic system. The ability of low doses of mafosfamide (Mf) to affect IL-2-induced CTL proliferation has been demonstrated. It was also shown that IL-2 activated resting suppressor cells. The pretreatment of the suppressor cells with either monoclonal antibodies (mAbs) against the p75 chain of IL-2R, or with Mf abolished the suppressive effect of these cells. No restoration of the proliferative response occurred when the anti-IL-2Rα mAb had been used. Flow cytometry analysis of 7-day CTL was carried out with mAbs against the α and β chains of IL-2R. CTL treatment with Mf inhibited anti-IL-2Rβ mAb binding. It may be assumed that the anti-proliferative effects of Mf which have been demonstrated in this paper, were a result of blocking the IL-2R β chain

Heterologous immune responses in health and disease

Immunological memory and tolerance represent major achievements and advantages of adaptive immunity. Organisms bearing adaptive immunity display prominent competitive advantages in the fight against infections. Memory immune cells are preserved for decades and are able to repel a second attack of an infectious agent. However, studies performed in the XXI century have shown that even unrelated pathogens may be quickly and effectively destroyed by memory cells. This type of response is called heterologous so that heterologous immune response is mainly typical to viral infections and other intracellular infections, where T-cells play a lead role in protection. This review will discuss various mechanisms involved in implementing T-cell cross-reactivity, describe molecular prerequisites for heterologous T-cell responses. Experimental evidence of memory T-cell potential to heterologous immune response in mouse models and in human infections are also discussed. Heterologous immune response is an important immune arm in adults and the elderly when the yield of naive cells to the periphery declines due to thymus involution. Along with obvious advantages, heterologous immune response leads to imbalanced memory T-cell repertoire, replacement of immunodominant epitopes with minor ones allowing viruses to evade immune response that results in virus persistence, or, conversely, fulminant infection course. Another threat of heterologous immune response due to switch in dominant repertoire of recognizable epitopes is presented by random self-epitope recognition, which can lead to development of autoimmune pathology. Heterologous immunity can also disrupt drug-induced tolerance in organ and tissue transplants and lead to graft rejection. Heterologous immune response should be taken into consideration while developing and using new vaccines, especially in adults and the elderly

Floquet–Liouville supermatrix approach. II. Intensity‐dependent generalized nonlinear optical susceptibilities

This is the published version, also available here: http://dx.doi.org/10.1063/1.451981We present a practical n o n p e r t u r b a t i v e method for e x a c t treatment of i n t e n s i t y‐d e p e n d e n t generalized nonlinear optical susceptibilities χ(ω) in intense polychromatic fields, valid for arbitrary laser intensities, detunings, and relaxation. By means of the many‐mode Floquet theory, the time‐dependent Liouville equation can be transformed into an equivalent t i m e‐i n d e p e n d e n t infinite‐dimensional Floquet–Liouville supermatrix (FLSM) eigenvalue problem. It is then shown that the nonlinear optical susceptibilities χ(ω) can be completely determined simply from the supereigenvalues and eigenfunctions of the Floquet–Liouvillian  L̂ F . In addition to this exact FLSM approach, we have also presented higher‐order perturbative results, based on the extension of the Salwen’s nearly degenerate perturbation theory, appropriate for somewhat weaker fields and near‐resonant multiphoton processes, but beyond the conventional perturbative or rotating wave approximation (RWA). In the case of two‐level systems, for example, the implementation of Salwen’s method in the time‐independent L̂ F allows the reduction of the infinite‐dimensional FLSM into a 4×4 dimensional effective Hamiltonian, from which essential a n a l y t i c a l formulas for intensity‐dependent χ(ω) can be obtained. These methods are applied to a detailed study of intensity‐dependent spectralline shapes (such as hole burning and extra resonance peaks at the line center, and the effects of saturation, detuning, and radiative and collisional damping, etc.) and subharmonic structures in nonlinear multiple wave mixings χ[(m+1)ω1−mω2] for two‐level systems in intense linearly polarized bichromatic fields

Interleukin-36 family as a novel regulator of inflammation in the barrier tissues

The interleukin-36 (IL-36) family was discerned in the superfamily of interleukin-1 (IL-1) ten years ago. This family includes three isoforms of IL-36α, IL-36β, IL-36γ, which have pro-inflammatory activity and a specific receptor antagonist, IL-36ra, which implements anti-inflammatory function. All of them bind to the same IL-1R6 receptor. The pro-inflammatory isoforms also involve an accessory IL-1RAcP protein into signaling; resulting into conduction of a signal into the cell via the assembling heterodimer receptor. In contrast, IL-36ra inhibits the formation of a heterodimer and blocks the signal transmission. The cytokines of the IL-36 family and appropriate receptors are normally expressed on epithelial cells in barrier tissues such as the respiratory, intestinal tract and skin. Like all cytokines of the IL-1 superfamily, IL-36 is synthesized as inactive form and requires activation, but not due to caspases, but being mediated by neutrophil enzymes, such as cathepsin G, proteinase-3, and elastase, which are constantly present in barrier tissues. In this regard, IL-36 is involved in homeostasis of barrier tissues. Apparently, the IL-36 cytokine system appeared in response to the developing ability of some microorganisms to avoid immune recognition and activation of innate immune response, and, in particular, the IL-1 pro-inflammatory system. An imbalance between the pro- and anti-inflammatory pathways readily causes inflammation in the corresponding tissue. This review discusses participation of cytokines from the IL-36 family in homeostasis of barrier tissues, as well as potential role of the IL-36 family in pathogenesis of bacterial, viral, and fungal skin diseases, atopic dermatitis, autoimmune diseases, such as rheumatoid arthritis, systemic lupus erythematosus, Sjogren's syndrome, ulcerative colitis and Crohn's disease. The role of IL-36 family cytokines in the immunopathogenesis of psoriasis has been well studied. This review is presenting the modern ideas about immune pathogenesis of psoriasis. The special role of cytokines from the IL-36 family was shown both for induction of psoriatic inflammation and evolving a positive feedback loop that supports and enhances the immune component of inflammation, which leads to progression of the disease. Moreover, modern methods of treating psoriasis are discussed, in particular, a possible promising approach to IL-36 blockade, or usage of recombinant IL-36ra for the treatment of psoriatic patients. Experimental studies in this area in mice provide some grounds for optimism

CHANGES IN THE CAPILLARY AND VENOUS BLOOD CYTOKINE PROFILE OF PATIENTS WITH PSORIASIS DEPENDING ON THE TREATMENT

ABSTRACT. Psoriasis is a chronic autoimmune skin disease involving T-cell immunity. The interleukin (IL)-23/IL-17/IL-22 cytokine axis is key in the immunopathogenesis of psoriasis. The role of the IL-36 subfamily regulating inflammation in the skin is shown. Topical preparations are used to treat psoriasis. Objective: to study changes in the cytokine profile of venous and capillary blood taken near the focus of psoriatic inflammation, depending on the treatment with topical preparations. 40 patients with psoriasis, mean age 43.7 years, were examined. Group 1a (20 people) received local treatment with mometasone, Group 1b (20 people) received topical gel containing IL-36 receptor antagonist. 20 healthy people, mean age 46.6 years, consisted the control group 2. Capillary blood was collected from a finger, in patients near the lesion 200 μl in a microvette with EDTA. Venous blood was taken from the cubital vein 3 ml into a vacuum tube with EDTA. The concentration of 15 cytokines in blood plasma was tested by the multiplex method (MagPix, BioRad, USA). The effectiveness of therapy was assessed using the PASI and DLQI indices. At the end of treatment (day 14), the PASI and DLQI indices significantly decreased in both groups. On the 28th day, the PASI index in Group 1a returned to its original level, in group 1b it remained steadily reduced. Before treatment, the levels of all cytokines except IL-10 in the capillary blood of patients with psoriasis were significantly increased compared to Group 2, and the levels of 5 cytokines were increased in the venous blood. After 14 days in Group 1a, the levels of IL-1, IL-4, IL-6, IL-21, IL-22, IL-23, IL-25, IL-33 significantly decreased in capillary blood, and only IL-17F, IL-21, IL-33 and TNF in the venous blood. On the 28th day, the concentrations of almost all cytokines returned to their original level. In Group 1b, on the 14th day, the levels of IFN-γ, IL-1, IL-4, IL-17F, IL-21, IL-22, IL-23, IL-25, IL-33 significantly decreased in capillary blood, and in venous blood - IFN-γ, IL-21, IL-22, IL-23, IL-33. On the 28th day, the concentration continued to decrease, or the level of these cytokines remained reduced, and IL-6 significantly decreased in the vein. Thus, the method for determining the profile of capillary blood cytokines from the area of ​​psoriatic lesions can be used to monitor the effect of treatment in patients with psoriasis

COMPARISON OF DIFFERENT METHODS FOR EVALUATION CELLULAR IMMUNITY TO THE SARS-CoV-2 VIRUS

Most methods for evaluation T-cell immunity are laborious and unsuitable for routine laboratory diagnostics. This encourages researchers to create accessible and reproducible tests. The purpose of the study is to compare three methods for evaluation the level of cellular immune response to antigens of the SARS-CoV-2 virus in patients who have been ill and vaccinated against a new coronavirus infection. Examined: 26 people who had mild or moderate COVID-19 (group 1), 19 people vaccinated twice with Sputnik V, who did not have COVID-19 (group 2), 21 people who had COVID-19 and were twice vaccinated with Sputnik V (group 3) and 14 people who had COVID-19 twice (group 4). Peripheral blood mononuclear cells were isolated by gradient centrifugation. In the first method, mononuclear cells were incubated with the S-protein of the SARS-CoV-2 virus, stained with fluorescently labeled antibodies, the percentage of CD8highCD107a was counted on a BD FACS Canto II flow cytometer. When assessed by the ELISpot method on the “Human IFN-γ ELISpot” kit, IFN-γ production was stimulated by SARS-CoV-2 S-protein, or a mixture of SARS-CoV-2 protein peptides on the “Corona-T-test” kit. There were no significant differences in the level of expression of CD107a on CD8high in groups 1, 2, 3, and 4 and the number of IFN-γ producers per SARS-CoV-2 S-protein on the “Human IFN-γ ELISpot” kit. Production of IFN-γ is significantly lower in group 3 (hybrid immunity) 317.29±19.04 pg/ml than in groups 1 and 2 (post-infection and post-vaccination immunity) 454.95±20.32 and 470.77±26.24 pg /ml. The relative level of IFN-γ-producing cells in group 2 was higher (22.34±3.77) versus 16.83±2.35 in group 1 and 15.46±1.83 in group 3, the relative level of IFN- γ did not differ in these groups. Stimulation with full-length S-protein showed a significant reduction in the number of spots in group 4 (breakthrough immunity) 30.59±2.29 vs. 58.97±4.47 in group 3, and stimulation with a mixture of SARS-CoV-2 peptides in group 4 compared with group 3 revealed a significant increase in the number of IFN-γ-producing cells 86.72±7.20 versus 69.38±5.53 and IFN-γ production 991.25±65.18 pg/ml versus 760.76±50.70 pg/ml and in relative terms, 10.30±2.77 versus 8.61±2.66 and 68.10±9.41 versus 48.35±8.15, respectively. The results of three methods for evaluation the cellular immune response correlate positively with each other, but with different strengths

Gating strategy for plasmablast enumeration after hepatitis B vaccination

B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27++CD38++ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27++CD38++ plasmablast ratio among CD19+ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27+CD71+ population proved to be different from occurrence of classic plasmablasts with CD27++CD38++ phenotype. This finding suggests that the CD27++CD71+population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens.B cell stimulation develops upon vaccination, thus causing occurrence of activated B cells (plasmoblasts) in bloodstream. Similar cells are also observed in some viral infections. The contents of plasmablasts may be a marker of successful vaccination, or a diagnostic feature of ongoing infection. The plasmablasts are normally represented by a small cell subpopulation which is not easy to detect. A study was performed with 15 healthy volunteers who were subjected to a single immunization with a recombinant vaccine against hepatitis B virus. To identify the plasmablasts, we have used labeled antibodies prepared in our laboratory. These reagents were previously validated for counting the plasmablasts. Different gating strategies for plasmablast gating have been compared. Upon staining of lymphocytes from immunized volunteers, we observed a distinct cluster of plasmablasts with CD27++CD38++ phenotype using the following antibody set: CD19-PE, CD3/CD14/CD16-FITC, CD27-PC5.5 and CD38-PC7. Inclusion of a CD20-FITC antibody into the panel caused an increase of CD27++CD38++ plasmablast ratio among CD19+ lymphocytes to > 60%. Upon substitution of CD38 antibody by anti-CD71, a distinct plasmablast cluster was again revealed, which contained ca. 5 per cent В cells. Two strategies for the plasmablast gating using the CD27/ CD38 and CD27/CD71 combinations were compared in dynamics with lymphocyte samples from a single vaccinated volunteer. When applying the CD27/CD38 combination, a sharp and pronounced plasmablast peak was registered on day 7 post-vaccination. With CD27/CD71 combination, the peak was extended between day 7 and day 14 following immunization. Hence, time kinetics of the CD27+CD71+ population proved to be different from occurrence of classic plasmablasts with CD27++CD38++ phenotype. This finding suggests that the CD27++CD71+ population contains both plasmablasts and other types of activated B cells. A minor HBV surface antigen was prepared and labeled with phycoerythrin (HBsAg-PE), thus allowing to quantify the antigen-specific plasmablasts. The results of HBsAg-PE-based detection of antigen-specific cells were in compliance with the data obtained by ELISpot technique. At the present time, we use the original plasmablast gating technique for detection of activated B cells in SARS-CoV-2 infection. At the next step, this technique will be applied to sorting of antigen-specific B cells, thus permitting sequencing of Ig genes and design of novel human antibodies against viral antigens

Comparing humoral immune response in adult measles patients and measles vaccinated subjects

Introduction. The implementation of the WHO Measles Elimination Program has yielded serious results, but in recent years an increase in the incidence rate of this infection has been observed. In particular, according to the WHO, in 2019 vs. 2018 measles morbidity was elevated by 3-fold worldwide. While investigating measles outbreaks among patients, apart from unvaccinated subjects, a substantial group of adults vaccinated in childhood was distinguished. The aim of this work was to examine the characteristics of humoral measles immunity in adult measles patients as well as subjects after measles vaccination. Materials and methods. 50 adult measles patients aged 20 to 55 years were examined. In all patients, the diagnosis was confirmed clinically and by laboratory assays by detecting measles IgM antibodies. The second group consisted of 50 conditionally healthy seronegative age-matched adults, vaccinated with the live measles vaccine (Microgen, Russia). Peripheral blood samples were collected from the cubital vein in total volume of 4 ml on 6±1 day after the onset of rash in patients as well as 6 weeks after vaccination. Specific measles antibodies and their avidity were determined by ELISA using the commercial Avidity: Anti-Measles Viruses ELISA/IgG kit (Euroimmun, Germany). Results. It was shown that people aged 20—35 years more likely suffered from measles than elderly. And it was in this age group that healthy measles seronegative individuals were more abundant. Among vaccinees, 44% responded to vaccination with the primary type of immune response, and 56% responded with the secondary type, while among measles patients, 34% and 66% responded with the primary and secondary type, respectively, as follows from the spectrum of specific IgG subclasses and the antibody avidity assay. The secondary type of immune response indicates that these subjects were apparently vaccinated against measles in childhood, but lost with time long-lived plasma cells producing protective antibodies. While comparing the parameters of specific humoral immunity in groups with acute measles infection (day 6 from the onset of rash) and early convalescents (3 weeks after the onset of rash), it was shown that the level of specific IgG increased threefold in early convalescents (p < 0.01) compared with those at acute phase. The level of specific IgA, on the contrary, decreased from 73.44 (69—75.3) Me/ml to 48.64 (45—56.4) Me/ml, but remained very high. At the same time, the spectrum of specific IgG subclasses shifted from primary immune response (high IgG3 and low IgG1) to secondary response (low IgG3 and high IgG1), which is typical for the response of emerging memory B cells

Diagnostic value of anti-GP2 antibodies determined in serum and coprofiltrates in children with inflammatory bowel disease

Inflammatory bowel diseases (IBD), such  as Crohn’s disease (CD) and  ulcerative colitis (UC), are characterized by chronically recurring inflammation of intestinal wall and are associated with a significant decrease in the  quality  of life. A spectrum of genetic  variants  associated with  Crohn’s disease  is described. Intestinal dysbiosis (DB)  may be the triggering factor of the disease. Glycoprotein 2 (GP2), the main protein of pancreatic zymogen  granules, is secreted  into the intestines with digestive enzymes.  Anti-GP2 antibodies were found in the serum of patients with CD.  The aim of the present  study was to investigate  the levels of anti-GP2 antibodies in serum  and feces of children with IBD  compared with the DB group.  Serums  and coprofiltrates from 110 children (64 boys and 46 girls) at the age of 12.3 (2.6-17.9) years were studied; 36 patients with CD, 30 patients with UC.  A comparison group consisted of 44 patients with DB. IgG and IgA antibodies against GP2 were tested with ELISA. Nonparametric statistics methods are applied, the results are presented as percentages and medians (Me (Q0.25-Q0.75)). The serum levels of anti-GP2 IgA antibodies were 9.97 (3.35-13.45) U/ml for the CD patients, 6.08 (2.71-14.26) U/ml for UC and 2. 94 (2.29-6.41) U/ml for DB. The levels of anti-GP2 IgG antibodies in serum were 6.16 (3.26-18.4) U/ml for CD, 5.26 (2.97-7.52) U/ml for UC, and for DB 5.23 (2.53-8.85) U/ml. The cut-off  threshold concentration for anti-GP2 IgG antibodies was 13.8 U/ml, with sensitivity of 63.2%, specificity 100%, and for IgA 5.63 U/ml, with sensitivity of 60.5% and specificity of 78.8%, thus being lower than the calculated cut-off  for adults (20 U/ml). The levels of anti-GP2 IgG in coprofiltrates in children of comparison group  were 1.99 (1.26-3.04) U/ml; in the  patients with CD, 23.5 (16.15-29.3) U/ml, and  in children with UC, 20.45 (13.63-25.5) units/ml (p < 0.001). The cut-off  value amounted 8.0 U/ml, with 100% sensitivity  and  100% specificity.  Concentrations of anti-GP2 IgA in coprofiltrates of patients with IBD  did not significantly  differ from DB patients. Moreover, the concentration of sIgA in the coprofiltrates of patients with IBD  was significantly  higher than  their level in DB group. The anti-GP2 IgA/sIgA  ratio was significantly lower in patients with CD (0.326 (0.23-0.512)), and UC (0.327 (0.205-0.435)), than in patients with DB (2.332 (1.575-3.523)) (p < 0.001);  the cut-off  level was 0.784, with a sensitivity of 97.7% and specificity  of 98.6%. It is discussed, whether fecal anti-GP2 IgA antibodies should  be considered as protective, supporting intestinal homeostasis, whereas anti-GP2 IgG antibodies are pathogenetically significant  for development of IBD.  Thus, using a non-invasive method for determining anti-GP2 antibodies in stool, when exceeding the cut-off for IgG, and reduction of IgA/sIgA ratio below the cut-off, one may differentiate IBD from DB with a similar symptoms at the onset of disease, with 100% sensitivity and 100% specificity