BackgroundThe development of obesity and related disorders, e.g., type II diabetes (T2D), hypertension, and metabolic disturbances is strongly related to increased levels in proinflammatory cytokines (IL-1, IL-6, and TNF-α). Both IL-6 and TNF-α are secreted by adipocytes and their concentration correlates with the percentage and distribution of fat tissue in the body. Both cytokines are the main factors responsible for the induction of acute phase proteins production (e.g., CRP) and to inflammatory state.ObjectiveTo compare of TNF-α and IL-6 concentrations in serum from obese subjects with those in subjects with normal BMI and to analyze the relation between TNF-α, IL-6, BMI and the inflammatory state as measured by the level of CRP.Material and methodsThe study included 80 obese subject (54 males and 26 females) BMI > 25 kg/m2. A control group consisted of 53 healthy subjects (24 males and 29 females) with BMI < 25 kg/m2. To determine the blood plasma concentration of IL-6 and TNF, commercial ELISA assay kits were used.ResultsThe concentration of IL-6 was lower in the control compared with the obese patients, but a significance difference concerned only female subjects (P = 0.001). TNF-α concentration was significantly higher in all obese subjects (P < 0.001). A higher level of this cytokine was also found in patients with obesity suffering from T2DM. A positive correlation was present between IL-6 and TNF-α concentrations. Only did the IL-6 level correlate with the concentration of CRP in serum.ConclusionsThe study confirmed that increased inflammatory cytokines lead to the persistence of inflammation in obese subjects. However, some other factors, such as gender, may contribute to the development of obesity-related inflammatory states.

Leptin or obesity receptor (Ob-R) is a member of class I cytokine receptor family. Ob-R, expressed in six isoforms, is the product of alternative RNA splicing of db gene. According to its structural differences, the receptor's isoforms are divided into three classes: long, short, and secretory isoforms. A long, fully active isoform of Ob-Rb is expressed mainly in the hypothalamus, where it takes part in energy homeostasis and in the regulation of secretory organs' activity. Ob-Rb is also present on all types of immune cells, involved in innate and adaptive immunity. Short leptin isoforms (Ob-Ra, Ob-Rc, Ob-Rd, and Ob-Re) that contain box 1 motif are able to bind JAK kinases (Janus kinases) as well as to activate some other signal transduction cascades. A soluble isoform (Ob-Re) can regulate serum leptin concentration and serve as a carrier protein delivering the hormone to its membrane receptors and is able to transduce the signal into the cell. JAK/STAT pathway plays the major role in leptin signal transduction through membrane receptors. Among all Ob-R isoforms, only full-length isoform (Ob-Rb) is able to fully transduce activation signal into the cell.

Background: Atopic allergy is among the immune tolerance-related disorders resulting from a failure of the regulatory network. Regulatory T cells (Tregs) play a leading role in the development of homeostasis in the immune system. The aim of this study was to determine the role of Tregs in the pathogenesis of atopic diseases in children by exploring the relationship between Treg frequency, activation markers and the clinical manifestations of the disease. Methods: Twenty allergic and 50 healthy children were enrolled to the study. Peripheral blood mononuclear cells were stained with monoclonal antibodies (anti-CD25-CD4-CD127-FoxP3-CD69-CD71) and evaluated using flow cytometry. Tregs were identified as CD4+CD25^+/highFoxP3+CD127- T cells. Results: The percentage of Tregs in allergic patients (2.3%) was significantly decreased in comparison to healthy controls (4.6%, p = 0.003). The frequency of Tregs in patients with symptoms of atopic dermatitis and/or food allergy (1.7%) was significantly lower than in patients without these symptoms (2.9%, p = 0.04). A significant correlation between the percentage of Tregs and the sIgE serum concentration was observed (p = 0.037). Relative fluorescence intensities of FoxP3 expression in allergic patients were higher than in healthy controls (p = 0.00004). The frequency of CD4+CD25^highCD127-CD71+ cells did not differ between the groups. Conclusions: Tregs display substantial deficiencies in atopic children, especially in children with multiorgan involvement, compared to patients with single organ manifestations. Additionally, there is an association between Tregs and the sIgE serum concentration. Better identification and characterization of Tregs in allergy is needed as they limit responses to foreign antigens, thereby minimizing T cell-mediated immunopathology in allergic diseases.

Studies on neutrophil extracellular traps (NETs) are challenging as neutrophils live shortly and easily become activated. Thus, availability of a cell line model closely resembling the functions of peripheral blood neutrophils would be advantageous. Our purpose was to find a compound that most effectively differentiates human promyelocytic leukemia (HL-60) cells toward granulocyte-like cells able to release NETs. HL-60 cells were differentiated with all-trans retinoic acid (ATRA), dimethyl sulfoxide (DMSO) or dimethylformamide (DMF) and stimulated with phorbol 12-myristate 13-acetate (PMA) or calcium ionophore A23187 (CI). Cell differentiation, phagocytosis and calcium influx were analyzed by flow cytometry. Reactive oxygen species production and NETs release were measured fluorometrically and analyzed microscopically. LC3-II accumulation and histone 3 citrullination were analyzed by western blot. ATRA most effectively differentiated HL-60 cells toward granulocyte-like cells. ATRA-dHL-60 cells released NETs only upon PMA stimulation, DMSO-dHL-60 cells only post CI stimulation, while DMF-dHL-60 cells formed NETs in response to both stimuli. Oxidative burst was induced in ATRA-, DMSO- and DMF-dHL-60 cells post PMA stimulation and only in DMF-dHL-60 cells post CI stimulation. Increased histone 3 citrullination was observed in stimulated DMSO- and DMF-, but not in ATRA-dHL-60 cells. The calcium influx was diminished in ATRA-dHL-60 cells. Significant increase in autophagosomes formation was observed only in PMA-stimulated DMF-dHL-60 cells. Phagocytic index was higher in ATRA-dHL-60 cells than in control, DMSO- and DMF-dHL-60 cells. We conclude that ATRA, DMSO and DMF differentiate HL-60 in different mechanisms. DMF is the best stimulus for HL-60 cell differentiation for NETs studies.

IntroductionThe gut microbiota may be relevant in the development of type 1 diabetes (T1D). We examined the effects of Lactobacillus rhamnosus GG and Bifidobacterium lactis Bb12 on beta-cell function in children with newly diagnosed T1D.Research design and methodsChildren aged 8–17 years with newly (within 60 days) diagnosed T1D were enrolled in a double-blind, randomised controlled trial in which they received L. rhamnosus GG and B. lactis Bb12 at a dose of 109 colony-forming units or placebo, orally, once daily, for 6 months. The follow-up was for 12 months. The primary outcome measure was the area under the curve (AUC) of the C-peptide level during 2-hour responses to a mixed meal.ResultsNinety-six children were randomised (probiotics, n=48; placebo n=48; median age 12.3 years). Eighty-eight (92%) completed the 6-month intervention, and 87 (91%) completed the follow-up at 12 months. There was no significant difference between the study groups for the AUC of the C-peptide level. For the secondary outcomes at 6 months, there were no differences between the study groups. At 12 months, with one exception, there also were no significant differences between the groups. Compared with the placebo group, there was a significantly increased number of subjects with thyroid autoimmunity in the probiotic group. However, at baseline, there was also a higher frequency of thyroid autoimmunity in the probiotic group. There were no cases of severe hypoglycemia or ketoacidosis in any of the groups. No adverse events related to the study products were reported.ConclusionsL. rhamnosus GG and B. lactis Bb12, as administered in this study, had no significant effect in maintaining the residual pancreatic beta-cell function in children with newly diagnosed T1D. It remains unclear which probiotics, if any, alone or in combination, are potentially the most useful for management of T1D.Trial registration numberNCT03032354.

Regulatory T cells (Tregs) play an important role in the suppression of the immune response in lung cancer. Cytotoxic T-lymphocyte antigen 4 (CTLA-4) expressed on T lymphocytes is capable of downregulating cytotoxic T cells and is constitutively expressed on Tregs. Little is known about the population of Tregs with two forms of CTLA-4: surface (s) and intracellular (in) in the lung cancer environment. Th17 cells defined by production of IL-17 have pleiotropic functions in anticancer immune response. Our aim was to detect the elements of immune response regulation in lung cancer in three compartments: by analysis of bronchoalveolar lavage fluid (BALF) from the lung affected by cancer (clBALF), healthy symmetrical lung (hlBALF) and peripheral blood (PB) from the same patient. A total of 54 samples were collected. Tregs, (s)CTLA-4, (in)CTLA-4 were detected by flow cytometry with antibodies against CD4, CD25, Foxp3, CD127, CTLA-4, and concentration of IL-17 was estimated by ELISA. We observed a significantly higher proportion of Tregs in clBALF than in hlBALF or PB (8.5 vs. 5.0 vs. 5.1%, respectively, p < 0.05). The median proportion of (in)CTLA-4+ Tregs was higher in clBALF than in hlBALF or PB (89.0, 81.5, 56.0%, p < 0.05). IL-17 concentration was the highest in clBALF—6.6 pg/ml. We observed a significant correlation between the proportion of Tregs and (in)CTLA-4+ Tregs with IL-17A concentration in clBALF. We confirmed significant differences in the proportion of regulatory elements between cancerous lung and healthy lung and PB and the usefulness of BALF analysis in evaluation of immune response regulation in local lung cancer environment.

No abstract

BackgroundPaediatric respiratory tract infections are among the most common reasons for preschool and school absences and visits to physicians. The disease mainly involves the upper respiratory tract and is associated with fever, cough, sore throat, and running nose. Children with recurrent respiratory infections (RRI), which are defined as more than six serious diseases a year, are a difficult diagnostic challenge. The aim of this study was to assess immunological deviations in laboratory tests performed in children with RRI.Material and methodsIn the retrospective study 25 children suffering from recurrent respiratory tract infection, aged 4.1 ±2.3 years, 13 boys and 12 girls, were involved. For all children chemiluminescence of granulocytes and immunophenotyping of lymphocytes from peripheral blood were examined. An immunophenotype of peripheral blood lymphocytes involved evaluation of T cell, B cells, and NK cells, examined with flow cytometry.ResultsEleven of the studied children had decreased chemiluminescent response to stimulants, normal response was found for nine children, and five children had an increased result of the test. Five of the 25 children had decreased B cells number, and five had decreased number of T cells including decrease of CD4, as well as CD8 positive cells. Children with decreased chemiluminescence had more frequent neutropaenia than children with normal or increased chemiluminescent response, p < 0.05 (exact Fisher test).ConclusionsRecurrent respiratory tract infection could be associated with improper neutrophils response to pathogens, and immunological examination should be performed to find the reason for the increased number of infections in a year.