SARS-CoV-2 is still a major burden for global health despite effective vaccines. With the reduction of social distancing measures, infection rates are increasing in children, while data on the pediatric immune response to SARS-CoV-2 infection is still lacking. Although the typical disease course in children has been mild, emerging variants may present new challenges in this age group. Peripheral blood mononuclear cells (PBMC) from 51 convalescent children, 24 seronegative siblings from early 2020, and 51 unexposed controls were stimulated with SARS-CoV-2-derived peptide MegaPools from the ancestral and beta variants. Flow cytometric determination of activation-induced markers and secreted cytokines were used to quantify the CD4+ T cell response. The average time after infection was over 80 days. CD4+ T cell responses were detected in 61% of convalescent children and were markedly reduced in preschool children. Cross-reactive T cells for the SARS-CoV-2 beta variant were identified in 45% of cases after infection with an ancestral SARS-CoV-2 variant. The CD4+ T cell response was accompanied most predominantly by IFN-γ and Granzyme B secretion. An antiviral CD4+ T cell response was present in children after ancestral SARS-CoV-2 infection, which was reduced in the youngest age group. We detected significant cross-reactivity of CD4+ T cell responses to the more recently evolved immune-escaping beta variant. Our findings have epidemiologic relevance for children regarding novel viral variants of concern and vaccination efforts.

(1) Background: The introduction of highly effective CFTR-modulating therapies (HEMT) has changed the course of the disease for many people with Cystic Fibrosis (pwCF). Attention previously focused on life-threatening conditions of the respiratory system has broadened, bringing the involvement of the digestive system into the clinical and scientific focus. This emphasized the need for sensitive tools to capture and quantify changes in abdominal symptoms (AS), ideally applying patient-reported outcome measures (PROMs). (2) Methods: The present review focuses on studies addressing AS assessment deriving from the multi-organic abdominal involvement in pwCF. Among 5224 publications retrieved until Nov. 2022, 88 were eligible, and 39 were finally included. (3) Results: The review reveals that for a long time, especially before HEMT availability, AS in pwCF were assessed by single questions on abdominal complaints or non-validated questionnaires. PROMs focusing on quality of life (QOL) including a few GI-related questions were applied. Likewise, PROMs developed and partially validated for other non-CF GI pathologies, such as chronic inflammatory bowel diseases, irritable bowel syndrome, gastroesophageal reflux, constipation, or pancreatitis, were implemented. (4) Conclusions: Only lately, CF-specific GI-PROMs have been developed and validated following FDA guidelines, showing high sensitivity to changes and capturing marked and statistically significant reductions in the burden of AS achieved with HEMT implementation.

Pediatric SARS‐CoV‐2 infection is often mild or asymptomatic and the immune responses of children are understudied compared to adults. Here, we present and evaluate the performance of a two‐panel (16‐ and 17 parameter) flow cytometry‐based approach for immune phenotypic analysis of cryopreserved PBMC samples from children after SARS‐CoV‐2 infection. The panels were optimized based on previous SARS‐CoV‐2 related studies for the pediatric immune system. PBMC samples from seven SARS‐CoV‐2 seropositive children from early 2020 and five age‐matched healthy controls were stained for analysis of T‐cells (panel T), B and innate immune cells (panel B). Performance of the panels was evaluated in two parallel approaches, namely classical manual gating of known subpopulations and unbiased clustering using the R‐based algorithm PhenoGraph. Using manual gating we clearly identified 14 predefined subpopulations of interest for panel T and 19 populations in panel B in low‐volume pediatric samples. PhenoGraph found 18 clusters within the T‐cell panel and 21 clusters within the innate and B‐cell panel that could be unmistakably annotated. Combining the data of the two panels and analysis approaches, we found expected differentially abundant clusters in SARS‐CoV‐2 seropositive children compared to healthy controls, underscoring the value of these two panels for the analysis of immune response to SARS‐CoV‐2. We established a two‐panel flow cytometry approach that can be used with limited amounts of cryopreserved pediatric samples. Our workflow allowed for a rapid, comprehensive, and robust pediatric immune phenotyping with comparable performance in manual gating and unbiased clustering. These panels may be adapted for large multi‐center cohort studies to investigate the pediatric immune response to emerging virus variants in the ongoing and future pandemics.

The situation of limited data concerning the response to COVID-19 mRNA vaccinations in immunocom-promised children hinders evidence-based recommendations. This prospective observational study investigated humoral and T cell responses after primary BNT162b2 vaccination in secondary immunocompromised and healthy children aged 5–11 years. Participants were categorized as: children after kidney transplantation (KTx, n = 9), proteinuric glomerulonephritis (GN, n = 4) and healthy children (controls, n = 8). Expression of activation-induced markers and cytokine secretion were determined to quantify the T cell response from PBMCs stimulated with peptide pools covering the spike glycoprotein of SARS-CoV-2 Wuhan Hu-1 and Omicron BA.5. Antibodies against SARS-CoV-2 spike receptor-binding domain were quantified in serum. Seroconversion was detected in 56% of KTx patients and in 100% of the GN patients and controls. Titer levels were significantly higher in GN patients and controls than in KTx patients. In Ktx patients, the humoral response increased after a third immunization. No differences in the frequency of antigen-specific CD4+ and CD8+ T cells between all groups were observed. T cells showed a predominant anti-viral capacity in their secreted cytokines; however, this capacity was reduced in KTx patients. This study provides missing evidence concerning the humoral and T cell response in immunocompromised children after COVID-19 vaccination.

Introduction: The pathogenesis of biliary atresia (BA) is unclear to date and no therapies targeting immune regulatory pathways exist. Here we characterized potent effector liver tissue resident memory CD8⁺ T cells (Trm) and monocytic cells in children with advanced BA and an age-matched control group to gain insight into BA pathogenesis and immunologic regulation.Methods: Liver explants from 18 children with biliary atresia and 10 with metabolic disease and normal histology were analyzed ex vivo by multicolor flow-cytometry and immunohistochemistry. Cytokines and cytotoxic mediators were quantified by intracellular staining and bead-based arrays in culture supernatant.Results: The frequency of CD103⁺CD69⁺CD8⁺ Trm cells and CD14⁺CD16⁺ monocytes was significantly higher in BA than in the control group. In BA, T cells showed elevated expression of CD103, CD69, CD39 and production of TNF-α and Granzyme-B ex vivo, which could be reproduced in vitro by allowing cell-contact with monocytes.Conclusions: Cytotoxic CD8⁺ Trm cells and intrahepatic monocytes might contribute to tissue destruction in BA. Therapies targeting Trm cells or the TNF-α signaling pathway could be explored to delay progression to cirrhosis in BA.

Background/Objectives: Disrupted intracellular calcium (Ca²⁺_i) regulation and renin–angiotensin system (RAS) activation are pathogenetic factors in diabetic cardiomyopathy, a major complication of type 1 (T1D) and type 2 (T2D) diabetes. This study explored their potential link in diabetic rat hearts. Methods: Experiments were conducted on T1D and T2D Sprague-Dawley rats induced by streptozotocin and fructose-rich diet, respectively. In T1D, rats were treated with Enalapril (Ena) or Losartan (Los) for six weeks, whereas T2D animals received high-dose (HD) or low-dose (LD) Ena for 8 weeks. Heart function was assessed via echocardiography, Ca²⁺_i transients by Indo-1 fluorometry in Langendorff-perfused hearts, and key Ca²⁺_i cycling proteins by Western blot. Data: mean ± SD. Results: Diabetic hearts exhibited reduced contractile performance that was improved by RAS inhibition both in vivo (ejection fraction (%): T1D model: Control: 79 ± 7, T1D: 54 ± 11, T1D + Ena: 65 ± 10, T1D + Los: 69 ± 10, n = 18, 18, 15, 10; T2D model: Control: 73 ± 8, T2D: 52 ± 6, T2D + LDEna: 62 ± 8, T2D + HDEna: 76 ± 8, n = 9, 8, 6, 7) and ex vivo (+dPressure/dt_max (mmHg/s): T1D model: Control: 2532 ± 341, T1D: 2192 ± 208, T1D + Ena: 2523 ± 485, T1D + Los: 2643 ± 455; T2D model: Control: 2514 ± 197, T2D: 1930 ± 291, T2D + LDEna: 2311 ± 289, T2D + HDEna: 2614 ± 268). Analysis of Ca²⁺_i transients showed impaired Ca²⁺_i release and removal dynamics and increased diastolic Ca²⁺_i levels in both models that were restored by Ena and Los treatments. We observed a decrease in sarcoendoplasmic reticulum Ca²⁺-ATPase2a (SERCA2a) expression, accompanied by a compensatory increase in 16Ser-phosphorylated phospholamban (P-PLB) in T2D that was prevented by both LD and HD Ena (expression level (% of Control): SERCA2a: T2D: 36 ± 32, T2D + LDEna: 112 ± 32, T2D + HDEna: 106 ± 30; P-PLB: T2D: 557 ± 156, T2D + LDEna: 129 ± 38, T2D + HDEna: 108 ± 42; n = 4, 4, 4). Conclusions: The study highlights the critical role of RAS activation, most likely occurring at the tissue level, in disrupting Ca²⁺_i homeostasis in diabetic cardiomyopathy. RAS inhibition with Ena or Los mitigates these disturbances independent of blood pressure effects, underlining their importance in managing diabetic heart failure.