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IntroductionObesity is thought to be accompanied by chronic, low-grade, inflammation. The adipocytes are present in the subcutaneous and visceral fat tissue and contribute to the bone marrow cell compartment. Therefore, it poses a question whether the factors influencing adipocyte functions also have an impact on the hematopoietic function of the bone marrow. The aim of our study was to evaluate the association between erythrocyte parameters, the proinflammatory Th17 lymphocytes, and IR markers in children with excessive body weight.MethodsA total of 27 overweight/obese and 15 normal-weight children aged 8–18 years were enrolled in the study. The analysis included anthropometric measurements, evaluation of Th17 cell frequency, erythrocyte parameters, and carbohydrate metabolism parameters.ResultsIn overweight/obese children, erythrocyte count (p = 0.00002), hemoglobin (HGB) concentration (p = 0.005), and frequency of Th17 cells (p = 0.048) were higher. Anthropometric parameters correlated with erythrocyte parameters, as well as Th17 cell frequency in all children. The erythrocyte count correlated with the Th17 subset (p = 0.01, r = 0.38). Moreover, in all children, the correlation between erythrocyte and fasting insulin (FI) (p = 0.00005, r = 0.58), HOMA-IR (p = 0.00005, r = 0.58), and QUICKI (p = 0.000042, r = −0.59), as well as between HGB and FI (p = 0.037, r = 0.32), HOMA-IR (p = 0.37, r = 0.32), and QUICKI (p = 0.049, r = −0.31) was found. In the overweight/obese group, erythrocyte count correlated with insulin 2 h after the oral glucose tolerance test (OGTT) (p = 0.04, r = 0.4), while HGB correlated with glucose and insulin 2 h after OGTT (p = 0.018, r = 0.45; p = 0.04, r = 0.44, respectively).ConclusionsOur study confirmed that the erythrocyte parameters are higher in children with obesity and are positively correlated with insulin resistance and proinflammatory Th17 lymphocyte. Thus, it can be concluded that erythrocyte parameters reflect the risk of developing IR in response to chronic inflammation associated with obesity. These are simple, easily accessible, and repeatable tests that, in the assessment of obese patients, may herald the developing metabolic syndrome and serve as a helpful additional tool for assessing the effectiveness of treatment.

Gene therapy perfectly fits in the current needs of medicine for patients with melanoma. One of the major challenges of gene therapy is to increase gene transfer. The role of hyperthermia in the improvement of AAV (adeno-associated virus) transduction efficiency has been indicated. The aim of the present study was to assess the transduction efficacy of melanoma cell lines (A375, G-361, and SK-MEL-1) with the use of the rAAV/DJ mosaic vector under hyperthermia conditions. The analysis of changes in the transduction efficacy and expression of HSPs (heat shock proteins) and receptors for AAV was performed. The transduction was performed at 37 °C and at 43 °C (1 h). Hyperthermia enhanced gene transfer in all the tested cell lines. The most efficient transducing cell line under hyperthermia was A375 (increase by 17%). G361 and SK-MEL-1 cells showed an increase of 7%. The changes in the expression of the AAV receptors and HSPs after hyperthermia were observed. A key role in the improvement of gene transfer may be played by AAVR, HSPB1, HSP6, DNAJC4, HSPD1, HSPA8, HSPA9, HSP90AB1, and AHSA1. This study showed the possibility of the use of hyperthermia as a factor enabling the stimulation of cell transduction with rAAV vectors, thereby providing tools for the improvement in the efficacy of gene therapy based on rAAV.

Abstract The release of neutrophil extracellular traps (NETs) can be either beneficial or detrimental for the host, thus it is necessary to maintain a balance between formation and clearance of NETs. Multiple physiological factors eliciting NET release have been identified, yet the studies on natural signals limiting NET formation have been scarce. Accordingly, our aim was to analyze whether cytokines or immune cells can inhibit NET formation. To that end, human granulocytes were incubated with interleukin (IL)-4, IL-10, transforming growth factor beta-2 or adenosine and then stimulated to release NETs. Additionally, neutrophils were cultured in the presence of natural killer (NK) cells, regulatory T cells (Tregs), pro-inflammatory or anti-inflammatory macrophages (M1 or M2 macrophages), or in the presence of NK/Tregs/M1 macrophages or M2 macrophages-conditioned medium and subsequently stimulated to release NETs. Our studies showed that secretome of M1 and M2 macrophages, but not of NK cells and Tregs, diminishes NET formation. Co-culture experiments did not reveal any effect of immune cells on NET release. No effect of cytokines or adenosine on NET release was found. This study highlights the importance of paracrine signaling at the site of infection and is the first to show that macrophage secretome can regulate NET formation.

IntroductionTwin gestation is related to a higher risk of hypertensive disorders in pregnancy with possible risk stratification depending on chorionicity. It may be related to differences in plasma renin-angiotensin-aldosterone components between monochorionic and dichorionic twin pregnancies. The study aimed to analyze the plasma ANG II and ANG 1-7 concentrations in women with monochorionic and dichorionic twin gestation.MethodsA prospective observational study included 79 women between 32 and 34 weeks of gestation with twin pregnancy (31 with monochorionic gestation and 48 with dichorionic gestation). Angiotensin II and angiotensin 1-7 concentrations were measured in the collected blood samples.ResultsNo significant differences were observed in angiotensin II concentrations between the dichorionic and monochorionic group with significantly higher levels of angiotensin 1-7 being observed in the dichorionic group. Angiotensin 1-7 level was higher than angiotensin II in 20 women (64.5%) in the monochorionic group and in 42 women (87.5%, p=0.01) in the dichorionic group. Higher plasma concentrations of angiotensin II and lower concentrations of angiotensin 1-7 were found in 5 women with gestational hypertension and in 3 with preeclampsia compared to normotensive women.DiscussionIt is the first study investigating angiotensin II and angiotensin 1-7 in twin pregnancies regarding chorionicity. Our results showed that plasma angiotensin 1-7 concentration was related to chorionicity, while plasma angiotensin II level was not. In most women with twin gestation angiotensin 1-7 concentration exceeded the concentration of angiotensin II. A switch in the relation between angiotensin II and angiotensin 1-7 was observed in hypertensive pregnant women.

Pregnancy after organ transplantation is considered high-risk and requires supervision in specialized centers. The impact of immunosuppression on the developing fetus is still the subject of research. It has been shown that it affects lymphocyte populations in the first year of life. For this reason, researchers suggest postponing mandatory infant vaccinations. The aim of the study was to analyze the influence of intrauterine exposure of the fetus to immunosuppression on the immunogenicity of protective vaccinations against selected bacterial pathogens. The ELISA method was used to determine the concentration of post-vaccination IgG antibodies against diphtheria, tetanus, pertussis, tuberculosis, H. influenzae type B, and S. pneumoniae in 18 children of mothers who underwent organ transplantation. The results were compared with the control group (n = 21). A comparison of the incidence of adverse post-vaccination reactions between the analyzed groups was also performed. There were no statistically significant differences in the immunogenicity of the analyzed vaccines between children of mothers who underwent organ transplantation and the age-matched general pediatric population. There were no differences in the incidence of adverse post-vaccination reactions between the analyzed groups. The obtained results do not indicate the need to modify the current protective vaccination schemes against bacterial pathogens in children of mothers who underwent organ transplantation.

BackgroundVitamin D affects adipogenesis, oxidative stress, inflammation, secretion of adipocytokines, lipid metabolism and thermogenesis. Some researchers postulate that those effects could be exerted by the influence of vitamin D on chemerin levels.Aim of the studyWe aimed to investigate if there is a link between serum 25-hydroksyvitamin D [25(OH)D], chemerin and metabolic profile in overweight and obese children before and after vitamin D supplementation.Material and methodsThe prospective study included 65 overweight and obese children aged 9.08-17.5 years and 26 peers as a control. None of the patients in the study group had received vitamin D within the last twelve months before the study.ResultsThe study group had lower baseline 25(OH)D (p

Extracellular vesicles (EVs) have emerged as vital mediators in intracellular communication in the lung microenvironment. Environmental exposure to various triggers (e.g., viruses, allergens) stimulates the EV-mediated cascade of pro-inflammatory responses that play a key role in the asthma pathomechanism. This complex EV-mediated crosstalk in the asthmatic lung microenvironment occurs between different cell types, including airway epithelial cells and immune cells. The cargo composition of EVs mirrors hereby the type and activation status of the parent cell. Therefore, EVs collected in a noninvasive way (e.g., in nasal lavage, serum) could inform on the disease status as a “liquid biopsy”, which is particularly important in the pediatric population. As a heterogeneous disease, asthma with its distinct endotypes and phenotypes requires more investigation to develop novel diagnostics and personalized case management. Filling these knowledge gaps may be facilitated by further EV research. Here, we summarize the contribution of EVs in the lung microenvironment as potential novel players towards precision medicine in the development of asthma. Although rapidly evolving, the EV field is still in its infancy. However, it is expected that a better understanding of the role of EVs in the asthma pathomechanism will open up new horizons for precision medicine diagnostic and therapeutic solutions.

Background Circulating calcification inhibitors: fetuin A (FA) and osteoprotegerin (OPG) together with soluble ligand of receptor activator of nuclear factor kappa-B (sRANKL) have been linked to vascular calcifications and arterial damage. This study aimed to evaluate relationships between FA, OPG, sRANKL, and arterial damage in children with primary hypertension (PH). Methods In this cross-sectional single-center study, calcification inhibitors (FA, OPG, sRANKL) levels were measured in blood samples of 60 children with PH (median age 15.8, IQR: [14.5-16.8] years) and 20 age-matched healthy volunteers. In each participant, peripheral and central blood pressure evaluation (BP) and ambulatory BP monitoring (ABPM) were performed. Arterial damage was measured using common carotid artery intima media thickness (cIMT), pulse wave velocity (PWV), augmentation index (AIx75HR), and local arterial stiffness (ECHO-tracking-ET) analysis. Results Children with PH had significantly higher peripheral and central BP, BP in ABPM, thicker cIMT, higher PWV, and AIx75HR. FA was significantly lower in patients with PH compared to healthy peers without differences in OPG, sRANKL, and OPG/sRANKL and OPG/FA ratios. In children with PH, FA level correlated negatively with cIMT Z-score and ET AIx; sRANKL level correlated negatively with ABPM systolic blood pressure (SBP), SBP load, diastolic BP load, and AIx75HR; OPG/sRANKL ratio correlated positively with SBP load, while OPG/FA ratio correlated positively with ET AIx. In multivariate analysis, FA was a significant determinant of cIMT (mm) and cIMT Z-score. Conclusions This study reveals that in children with primary hypertension, arterial damage is related to lower fetuin A concentrations. Graphical abstract, Author(s): Piotr Skrzypczyk [sup.1] , Anna Stelmaszczyk-Emmel [sup.2] , Michal Szyszka [sup.1] , Anna Ofiara [sup.1] , Malgorzata Panczyk-Tomaszewska [sup.1] Author Affiliations: (1) grid.13339.3b, 0000000113287408, Department of Pediatrics and Nephrology, [...]

The cholera vaccine can protect patients with inflammatory bowel disease (IBD) against both cholera and travelers’ diarrhea. However, both immunosuppressive treatment and IBD can affect its vaccine immunogenicity. The aim of this study was to assess the immunogenicity and safety of the cholera vaccine in children with IBD. Children older than 6 years with diagnosed IBD were enrolled in this multicenter study. All patients were administered two doses of the oral cholera vaccine (Dukoral®). Anti-cholera toxin B subunit IgA and IgG seroconversion rates were evaluated in a group with immunosuppressive (IS) treatment and a group without IS treatment (NIS). Immunogenicity was assessed in 70 children, 79% of whom received IS treatment. Post-vaccination seroconversion was displayed by 33% of children, for IgA, and 70% of children, for IgG. No statistically significant differences were found in the immune responses between the IS and NIS groups: 35% vs. 27% (p = .90), for IgA, and 68% vs. 80.0% (p = .16), for IgG, respectively. One case of IBD exacerbation after vaccination was reported. The oral cholera vaccine is safe. The immunogenicity of the oral cholera vaccine in children with IBD was lower than previously observed in healthy ones. The treatment type does not seem to affect the vaccine immunogenicity.

IntroductionChildhood obesity contributes to the development of cardiovascular diseases. The molecular pathway – receptor activator of nuclear factor-κβ ligand (RANKL), its receptor RANK and osteoprotegerin (OPG) - takes part not only in bone metabolism but is also involved in the atherosclerosis process. RANKL stimulates osteogenic differentiation and calcification of vascular smooth cells. The associations between the OPG-sRANKL system and various cardiovascular risk factors were displayed. We aimed to evaluate the relationships between serum sRANKL (soluble RANKL) levels and the OPG/sRANKL ratio with cardiometabolic risk factors in overweight and obese children.Material and methodsThe study included 70 children with overweight and obesity (mean age 13.0 ± 2.8) and 35 age-matched normal weight, healthy peers as a control group. In all patients, anthropometric measurements and laboratory tests were performed. Additionally, an oral glucose tolerance test (OGTT) was made only in overweight and obese children. Atherogenic and insulin resistance indices were calculated.ResultsOverweight and obese children had lower sRANKL levels compared to the control group (median 276.95 vs 325.90, p=0.011), and consequently a higher OPG/sRANKL ratio (0.02 vs 0.01, p = 0.013). The studied children in the lowest quartile of sRANKL levels had higher body weight, Body Mass Index, waist circumference and increased glucose and insulin levels 60 minutes after OGTT and higher uric acid values compared to children in the highest quartile. In multivariable linear regression analysis sRANKL negatively correlated only with uric acid (β = - 0.508, p = 0.041). No association was found for the OPG/sRANKL ratio.ConclusionExcess fat mass seems to alter the OPG/RANKL ratio mainly by reducing serum sRANKL levels. The correlation between sRANKL and uric acid may suggest a contribution of the OPG-sRANKL system in the cardiometabolic process, but that observation should be confirmed in future studies.

The objective of this work was to compare the quality of FMT preparations made from fresh feces with those made from feces frozen at –30°C without any pre-processing or cryopreservation additives. The research hypothesis was that such preservation protocol (frozen whole stool, then thawed and processed) is equipotent to classical fresh FMT preparation. For that, three complementary methods were applied, including: (i) culturing in aerobic and anaerobic conditions, (ii) measuring viability by flow cytometry, and (iii) next-generation sequencing. Flow cytometry with cell staining showed that the applied freezing protocol causes significant changes in all of the observed bacterial fractions. Alive cell counts dropped four times, from around 70% to 15%, while the other two fractions, dead and unknown cell counts quadrupled and doubled, with the unknown fraction becoming the dominant one, with an average contribution of 57.47% per sample. It will be very interesting to uncover what this unknown fraction is (e.g., bacterial spores), as this may change our conclusions (if these are spores, the viability could be even higher after freezing). Freezing had a huge impact on the structure of cultivable bacterial communities. The biggest drop after freezing in the number of cultivable species was observed for Actinobacteria and Bacilli. In most cases, selected biodiversity indices were slightly lower for frozen samples. PCoA visualization built using weighted UniFrac index showed no donor-wise clusters, but a clear split between fresh and frozen samples. This split can be in part attributed to the changes in the relative abundance of Bacteroidales and Clostridiales orders. Our results clearly show that whole stool freezing without any cryoprotectants has a great impact on the cultivability and biodiversity of the bacterial community, and possibly also on the viability of bacterial cells.

BackgroundObesity has been a growing problem in young patients leading to serious metabolic complications. There are many studies supporting the idea, that obesity should be considered as a chronic inflammation closely associated with immune system alterations. Th17 subpopulation is strongly involved in this process. The aim of our study was to evaluate circulating Th17 cells in overweight and obese children and explore the relationships between Th17 subset and metabolic parameters.MethodsWe evaluated peripheral Th17 cells in fresh peripheral blood samples from 27 overweight and obese and 15 normal-weight children. Th17 cells were identified by flow cytometry using monoclonal antibody and intracellular IL-17A staining. Th17 cells were defined as CD3+CD4+CD196+IL-17Aic+. The analysis involved anthropometric and metabolic parameters measured at baseline and three months after the change of lifestyle and diet. We evaluated the relationship between metabolic parameters and Th17 cells.ResultsIn overweight and obese children we found significantly higher Th17 cells percentage compared to normal weight controls (median 0.097% (0.044 - 0.289) vs 0.041% (0.023 - 0.099), p = 0.048). The percentage of Th17 cells decreased statistically significantly in children who reduced weight after the intervention (0.210% (0.143 - 0.315) vs 0.039% (0.028 - 0.106), p = 0.004). In this group we also noticed statistically significant reduction of TC and LDL-C concentration (p = 0.01, p = 0.04, respectively).ConclusionsObesity in children is associated with increased percentage of peripheral Th17 cells. Weight reduction leads to significant decrease of circulating Th17 cells and improvement of lipid parameters. This significant reduction of proinflammatory Th17 cells is a promising finding suggesting that obesity-induced inflammation in children could be relatively easily reversible.

BackgroundObesity is related to changes in adipokine secretion, activity of adipose tissue macrophages, helper T cells, and regulatory T cells. It has been confirmed that vitamin D has potent anti-inflammatory properties. It contributes to reduction in pro-inflammatory mediators and an increase in anti-inflammatory cytokines. There is also evidence that vitamin D could decrease C-reactive protein (CRP) and affect selected haematological indices.Aim of the StudyWe aimed to evaluate the effect of vitamin D on interleukin (IL)-10, IL-17, CRP, blood leukocyte profile, and platelet (PLT) count in overweight and obese children before and after six months of vitamin D supplementation.Material and MethodsThe study group consisted of 67 overweight and obese children aged 9.08-17.5 years. The control group included 31 normal weight peers age- and sex-matched. None of the studied children had received vitamin D supplementation before the study. Data were analyzed at baseline and after vitamin D supplementation.ResultsThe study group had lower baseline 25(OH)D (p

Background: Pregnancy in women who are organ recipients has long been a controversial issue due to the lack of data on the safety of immunosuppressive drugs for the developing foetus. Scientific data show that the effect of immunosuppressants on the foetus causes an impairment of T and B lymphocyte function and a reduction in their total number. For this reason, some authors recommend delaying the obligatory immunization of infants. The aim of the study is to analyse the impact of chronic immunosuppressive therapy used during pregnancy by women after organ transplantation on the effectiveness of anti-viral vaccinations in the children of these women. Methods: Concentrations of post-vaccination IgG antibodies (measles, HBV, polio) in 18 children of post-transplant mothers (9KTRs; 9LTRs) were determined using the ELISA method. The results were compared with the control group (n = 21). The incidence of vaccination AEs was also analysed. Results: There were no significant differences between the analysed groups in the concentrations of antibodies against HBV, measles and polio (p > 0.05). Conclusions: No difference was observed in the immunogenicity of HBV, polio and measles vaccinations between children of post-transplant mothers and the general population. The immunization of children of post-transplant mothers is safe, and the percentage of adverse post-vaccination events does not differ from the general population. The obtained study results do not indicate the necessity for modifying the vaccination program for HBV, measles, and polio in this group of patients.

Introduction The 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 methods Children 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.Results Ninety-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.Conclusions L. 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 number NCT03032354.

Background: Preeclampsia occurs more often in dichorionic than in monochorionic twin pregnancy. We hypothesize that serum concentrations of biomarkers: placental growth factor (PlGF), serum soluble fms-like tyrosine kinase-1 (sFlt-1), and endoglin (Eng) differ between monochorionic and dichorionic twin pregnancies. Methods: A prospective observational study including 43 monochorionic and 36 dichorionic twin gestation was conducted. Blood samples were collected twice from all participants: between 11 + 0 and 13 + 6 and between 32 + 0 and 34 + 0 weeks of gestation. PlGF, sFlt-1 and Eng were measured using immnunoenzymatic assays. Results: We found a significantly higher concentration of sFlt-1 in dichorionic in comparison to monochorionic pregnancies in both the first and third trimesters. PlGF and sEng levels did not differ between mono- and dichorionic gestation in both study periods. sFlt-1 level was related to twin gestation chorionicity, while PlGF expression was not. PlGF, sFlt-1 and sEng concentrations increased significantly during gestation and were much higher in the third trimester compared to the values measured in the first trimester. Conclusions: Angiogenic biomarkers expression differ between dichorionic and monochorionic twin pregnancy. The sFlt-1 level is related to chorionicity of a twin gestation.

Introduction: Acute kidney injury biomarkers are opening a new era in diagnosing kidney failure. The requirement for a specific and sensitive marker of kidney function is highly desirable in neonates because the diagnostic possibilities in this age group are not sufficient. Recent research show that neutrophil gelatinase-associated lipocalin (NGAL) can have a great potential but there is a wide range of medical conditions, that may influence their expression. The aim of the study was to evaluate the impact of perinatal risk factors on NGAL level in neonates. Material and methods : NGAL was measured in umbilical cord blood and peripheral blood in full term neonates with perinatal risk factors during the first days of life. Results : We found significantly higher umbilical cord blood NGAL levels in neonates with perinatal risk factors (117.69 ng/ml) compared to the control group (64.37 ng/ml). No significant difference in peripheral blood NGAL level was shown between the two groups. Umbilical cord blood NGAL level correlated positively with peripheral blood NGAL level (r = 0.36, p < 0.01). Umbilical cord blood NGAL level was significantly higher in neonates with fetal distress and infection compared to neonates with other perinatal risk factors. Peripheral blood NGAL level was significantly higher in neonates with infection compared to neonates with other perinatal risk factors. Significantly higher umbilical cord blood NGAL levels were seen in neonates born by operative delivery compared to born by natural delivery.

Currently, extracellular vesicles (EVs) have been implicated in the etiopathogenesis of many diseases, including lung disorders, with the possibility of diagnostic and therapeutic applications. The analysis of EV in respiratory tract diseases faces many obstacles, including material collection from airways, standardization of isolation techniques, detection methods, the analysis of their content, etc. This review focuses on the role of extracellular vesicles in the pathogenesis of atopic respiratory diseases, especially asthma, with a special focus on their clinical applicability as a diagnostic tool. We also summarize available laboratory techniques that enable the detection of EVs in various biological materials, with particular emphasis on flow cytometry. The opportunities and limitations of detecting EV in bronchoalveolar lavage fluid (BALF) were also described.

Primary Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infection usually affects preadolescent children or young adults, causing similar clinical presentation. Signs and symptoms are typically mild, and the majority of clinical and laboratory findings resolve spontaneously within one month after onset. In adulthood, the risk of fulminant EBV infection and severe complications is much higher, which may be related to increasing memory CD8+ T-cell population with age. It is still not clear what exactly triggers T-cell clonal proliferation. Animals model studies on heterologous immunity between viruses revealed that pre-existing memory T-cells may contribute to excessive immune response during subsequent infection with a new, unrelated pathogen. A 3.5-year-old girl was admitted to hospital with a suspicion of lymphoproliferative disorder. Peripheral blood smear revealed a massive lymphocytosis (61,600 cells/µl) with 62% share of atypical lymphocytes. The clinical presentation and positive EBV and CMV IgM test strongly suggested infectious mononucleosis syndrome as a result of EBV and CMV coinfection.