Validation of the pharmacokinetic model for anti- TNFα clearance in infants exposed to anti- TNFα during pregnancy.

BACKGROUND AND AIMS: ECCO guideline recommend postponing live attenuated vaccines in infants exposed to anti-Tumor Necrosis Factor alpha (anti-TNFα) in utero until drug clearance. The aim was to validate the predictive performance of the anti-TNFα clearance model. METHODS: Newborns and anti-TNFα concentrations from the prospective PETIT cohort were included. The anti-TNFα clearance model was used to predict all measured concentrations in the PETIT cohort, based on the measured cord blood concentration and the mean population clearance described in the model. Bayesian maximum a posteriori optimization was used to estimate the value of drug monitoring. Predictive capability and drug monitoring were assessed through Mean Absolute Error (MAE), Root mean Squared Prediction Error and Limits of Agreement according to Bland and Altman. RESULTS: Observed drug concentrations after birth were within the 80% prediction interval in 94% of adalimumab samples and 93% of infliximab samples. The anti-TNFα clearance model accurately predicted the concentration at six months after birth with an MAE of 0.03 (SD 0.03) µg/mL for adalimumab and 0.11 (SD 0.18) µg/mL for infliximab based on cord blood concentrations. Addition of an additional sample between 1 and 4 months after birth improved the predictive accuracy for infliximab (MAE 0.05 (SD 0.09)) but not for adalimumab. Guidance for use in clinical practice was formulated. CONCLUSIONS: The validity of the anti-TNFα clearance model is high, and hence can be used to guide clinicians regarding timing of live vaccines in infants exposed to adalimumab or infliximab in utero.

De novo missense variants in RRAGC lead to a fatal mTORopathy of early childhood.

PURPOSE: Mechanistic target of rapamycin (mTOR) complex 1 (mTORC1) regulates cell growth in response to nutritional status. Central to the mTORC1 function is the Rag-GTPase heterodimer. One component of the Rag heterodimer is RagC (Ras-related GTP-binding protein C), which is encoded by the RRAGC gene. METHODS: Genetic testing via trio exome sequencing was applied to identify the underlying disease cause in 3 infants with dilated cardiomyopathy, hepatopathy, and brain abnormalities, including pachygyria, polymicrogyria, and septo-optic dysplasia. Studies in patient-derived skin fibroblasts and in a HEK293 cell model were performed to investigate the cellular consequences. RESULTS: We identified 3 de novo missense variants in RRAGC (NM_022157.4: c.269C>A, p.(Thr90Asn), c.353C>T, p.(Pro118Leu), and c.343T>C, p.(Trp115Arg)), which were previously reported as occurring somatically in follicular lymphoma. Studies of patient-derived fibroblasts carrying the p.(Thr90Asn) variant revealed increased cell size, as well as dysregulation of mTOR-related p70S6K (ribosomal protein S6 kinase 1) and transcription factor EB signaling. Moreover, subcellular localization of mTOR was decoupled from metabolic state. We confirmed the key findings for all RRAGC variants described in this study in a HEK293 cell model. CONCLUSION: The above results are in line with a constitutive overactivation of the mTORC1 pathway. Our study establishes de novo missense variants in RRAGC as cause of an early-onset mTORopathy with unfavorable prognosis.

Group A streptococcal disease in paediatric inpatients: a European perspective.

Group A streptococcal (GAS) disease shows increasing incidence worldwide. We characterised children admitted with GAS infection to European hospitals and studied risk factors for severity and disability. This is a prospective, multicentre, cohort study (embedded in EUCLIDS and the Swiss Pediatric Sepsis Study) including 320 children, aged 1 month to 18 years, admitted with GAS infection to 41 hospitals in 6 European countries from 2012 to 2016. Demographic, clinical, microbiological and outcome data were collected. A total of 195 (61%) patients had sepsis. Two hundred thirty-six (74%) patients had GAS detected from a normally sterile site. The most common infection sites were the lower respiratory tract (LRTI) (22%), skin and soft tissue (SSTI) (23%) and bone and joint (19%). Compared to patients not admitted to PICU, patients admitted to PICU more commonly had LRTI (39 vs 8%), infection without a focus (22 vs 8%) and intracranial infection (9 vs 3%); less commonly had SSTI and bone and joint infections (p < 0.001); and were younger (median 40 (IQR 21-83) vs 56 (IQR 36-85) months, p = 0.01). Six PICU patients (2%) died. Sequelae at discharge from hospital were largely limited to patients admitted to PICU (29 vs 3%, p < 0.001; 12% overall) and included neurodisability, amputation, skin grafts, hearing loss and need for surgery. More patients were recruited in winter and spring (p < 0.001). CONCLUSION: In an era of observed marked reduction in vaccine-preventable infections, GAS infection requiring hospital admission is still associated with significant severe disease in younger children, and short- and long-term morbidity. Further advances are required in the prevention and early recognition of GAS disease. WHAT IS KNOWN: • Despite temporal and geographical variability, there is an increase of incidence of infection with group A streptococci. However, data on the epidemiology of group A streptococcal infections in European children is limited. WHAT IS NEW: • In a large, prospective cohort of children with community-acquired bacterial infection requiring hospitalisation in Europe, GAS was the most frequent pathogen, with 12% disability at discharge, and 2% mortality in patients with GAS infection. • In children with GAS sepsis, IVIG was used in only 4.6% of patients and clindamycin in 29% of patients.

AKT Hyperphosphorylation and T Cell Exhaustion in Down Syndrome.

Down syndrome (DS) is associated with increased susceptibility to infections, auto-immunity, immunodeficiency and haematological malignancies. The exact underlying immunological pathophysiology is still unclear. The immunophenotype and clinical characteristics of DS resemble those of Activated PI3K Delta Syndrome (APDS), in which the PI3K/AKT/mTOR pathway is overactivated. We hypothesized that T cell exhaustion and the hyperactivation of the AKT signalling pathway is also present in immune cells of children with DS. In this observational non-interventional cohort study we collected blood samples of children with DS (n=22) and healthy age-matched controls (n=21) for flowcytometric immunophenotyping, phospho-flow AKT analysis and exhaustion analysis of T cells. The median age was 5 years (range 1-12y). Total T and NK cells were similar for both groups, but absolute values and transitional B cells, naive memory B cells and naive CD4+ and CD8+ T cells were lower in DS. pAKT and AKT were increased for CD3+ and CD4+ T cells and CD20+ B cells in children with DS. Total AKT was also increased in CD8+ T cells. Children with DS showed increased expression of inhibitory markers Programmed cell dealth-1 (PD-1), CD244 and CD160 on CD8+ T cells and increased PD-1 and CD244+ expression on CD4+ T cells, suggesting T cell exhaustion. Children with DS show increased pAKT and AKT and increased T cell exhaustion, which might contribute to their increased susceptibility to infections, auto immunity and haematological malignancies.

Clinical validation of digital biomarkers for paediatric patients with asthma and cystic fibrosis: potential for clinical trials and clinical care.

BACKGROUND: Digital biomarkers are a promising novel method to capture clinical data in a home setting. However, clinical validation prior to implementation is of vital importance. The aim of this study was to clinically validate physical activity, heart rate, sleep and forced expiratory volume in 1 s (FEV1) as digital biomarkers measured by a smartwatch and portable spirometer in children with asthma and cystic fibrosis (CF). METHODS: This was a prospective cohort study including 60 children with asthma and 30 children with CF (aged 6-16 years). Participants wore a smartwatch, performed daily spirometry at home and completed a daily symptom questionnaire for 28 days. Physical activity, heart rate, sleep and FEV1 were considered candidate digital end-points. Data from 128 healthy children were used for comparison. Reported outcomes were compliance, difference between patients and controls, correlation with disease activity, and potential to detect clinical events. Analysis was performed with linear mixed effects models. RESULTS: Median compliance was 88%. On average, patients exhibited lower physical activity and FEV1 compared with healthy children, whereas the heart rate of children with asthma was higher compared with healthy children. Days with a higher symptom score were associated with lower physical activity for children with uncontrolled asthma and CF. Furthermore, FEV1 was lower and (nocturnal) heart rate was higher for both patient groups on days with more symptoms. Candidate biomarkers appeared able to describe a pulmonary exacerbation. CONCLUSIONS: Portable spirometer- and smartwatch-derived digital biomarkers show promise as candidate end-points for use in clinical trials or clinical care in paediatric lung disease.

The Role of the Respiratory Microbiome and Viral Presence in Lower Respiratory Tract Infection Severity in the First Five Years of Life.

Lower respiratory tract infections (LRTIs) in children are common and, although often mild, a major cause of mortality and hospitalization. Recently, the respiratory microbiome has been associated with both susceptibility and severity of LRTI. In this current study, we combined respiratory microbiome, viral, and clinical data to find associations with the severity of LRTI. Nasopharyngeal aspirates of children aged one month to five years included in the STRAP study (Study to Reduce Antibiotic prescription in childhood Pneumonia), who presented at the emergency department (ED) with fever and cough or dyspnea, were sequenced with nanopore 16S-rRNA gene sequencing and subsequently analyzed with hierarchical clustering to identify respiratory microbiome profiles. Samples were also tested using a panel of 15 respiratory viruses and Mycoplasma pneumoniae, which were analyzed in two groups, according to their reported virulence. The primary outcome was hospitalization, as measure of disease severity. Nasopharyngeal samples were isolated from a total of 167 children. After quality filtering, microbiome results were available for 54 children and virology panels for 158 children. Six distinct genus-dominant microbiome profiles were identified, with Haemophilus-, Moraxella-, and Streptococcus-dominant profiles being the most prevalent. However, these profiles were not found to be significantly associated with hospitalization. At least one virus was detected in 139 (88%) children, of whom 32.4% had co-infections with multiple viruses. Viral co-infections were common for adenovirus, bocavirus, and enterovirus, and uncommon for human metapneumovirus (hMPV) and influenza A virus. The detection of enteroviruses was negatively associated with hospitalization. Virulence groups were not significantly associated with hospitalization. Our data underlines high detection rates and co-infection of viruses in children with respiratory symptoms and confirms the predominant presence of Haemophilus-, Streptococcus-, and Moraxella-dominant profiles in a symptomatic pediatric population at the ED. However, we could not assess significant associations between microbiome profiles and disease severity measures.

Low Prevalence of Severe Underlying Pathology in Children With Recurrent Respiratory Tract Infections.

No underlying pathology could be detected in 64% of 208 children presenting with recurrent respiratory tract infections in general pediatric practice. Asthma/preschool wheezing and adenoid hypertrophy were commonly diagnosed. None of the children had a severe primary immunodeficiency or severe pulmonary illness such as cystic fibrosis. Our findings can guide pediatricians in their diagnostic approach of children with respiratory tract infections.

Response to Vaccination in Infants Exposed to Antitumor Necrosis Factor Alpha In Utero.

In this retrospective cohort study, the response to routinely administered Haemophilus influenzae type B vaccine, pneumococcal and pertussis vaccinations in 27 children exposed to antitumor necrosis factor alpha (anti-TNFα) during pregnancy was measured. The overall vaccination response seems comparable for children exposed to anti-TNFα and healthy infants. After primary vaccination series, inadequate response was present in some patients and might be related to exposure to anti-TNFα.

The influence of chest X-ray results on antibiotic prescription for childhood pneumonia in the emergency department.

The aim of this study is to evaluate the influence of chest X-ray (CXR) results on antibiotic prescription in children suspected of lower respiratory tract infections (RTI) in the emergency department (ED). We performed a secondary analysis of a stepped-wedge, cluster randomized trial of children aged 1 month to 5 years with fever and cough/dyspnoea in 8 EDs in the Netherlands (2016-2018), including a 1-week follow-up. We analysed the observational data of the pre-intervention period, using multivariable logistic regression to evaluate the influence of CXR result on antibiotic prescription. We included 597 children (median age 17 months [IQR 9-30, 61% male). CXR was performed in 109/597 (18%) of children (range across hospitals 9 to 50%); 52/109 (48%) showed focal infiltrates. Children who underwent CXR were more likely to receive antibiotics, also when adjusted for clinical signs and symptoms, hospital and CXR result (OR 7.25 [95% CI 2.48-21.2]). Abnormalities on CXR were not significantly associated with antibiotic prescription.Conclusion: Performance of CXR was independently associated with more antibiotic prescription, regardless of its results. The limited influence of CXR results on antibiotic prescription highlights the inferior role of CXR on treatment decisions for suspected lower RTI in the ED. What is Known: • Chest X-ray (CXR) has a high inter-observer variability and cannot distinguish between bacterial or viral pneumonia. • Current guidelines recommend against routine use of CXR in children with uncomplicated respiratory tract infections (RTIs) in the outpatient setting. What is New: • CXR is still frequently performed in non-complex children suspected of lower RTIs in the emergency department • CXR performance was independently associated with more antibiotic prescriptions, regardless of its results, highlighting the inferior role of chest X-rays in treatment decisions.

Technical validity and usability of a novel smartphone-connected spirometry device for pediatric patients with asthma and cystic fibrosis.

BACKGROUND: Diagnosis and follow-up of respiratory diseases traditionally rely on pulmonary function tests (PFTs), which are currently performed in hospitals and require trained personnel. Smartphone-connected spirometers, like the Air Next spirometer, have been developed to aid in the home monitoring of patients with pulmonary disease. The aim of this study was to investigate the technical validity and usability of the Air Next spirometer in pediatric patients. METHODS: Device variability was tested with a calibrated syringe. About 90 subjects, aged 6 to 16, were included in a prospective cohort study. Fifty-eight subjects performed conventional spirometry and subsequent Air Next spirometry. The bias and the limits of agreement between the measurements were calculated. Furthermore, subjects used the device for 28 days at home and completed a subject-satisfaction questionnaire at the end of the study period. RESULTS: Interdevice variability was 2.8% and intradevice variability was 0.9%. The average difference between the Air Next and conventional spirometry was 40 mL for forced expiratory volume in 1 second (FEV1) and 3 mL for forced vital capacity (FVC). The limits of agreement were -270 mL and +352 mL for FEV1 and -403 mL and +397 mL for FVC. About 45% of FEV1 measurements and 41% of FVC measurements at home were acceptable and reproducible according to American Thoracic Society/European Respiratory Society criteria. Parents scored difficulty, usefulness, and reliability of the device 1.9, 3.5, and 3.8 out of 5, respectively. CONCLUSION: The Air Next device shows validity for the measurement of FEV1 and FVC in a pediatric patient population.

Repertoire Sequencing of B Cells Elucidates the Role of UNG and Mismatch Repair Proteins in Somatic Hypermutation in Humans.

The generation of high-affinity antibodies depends on somatic hypermutation (SHM). SHM is initiated by the activation-induced cytidine deaminase (AID), which generates uracil (U) lesions in the B-cell receptor (BCR) encoding genes. Error-prone processing of U lesions creates a typical spectrum of point mutations during SHM. The aim of this study was to determine the molecular mechanism of SHM in humans; currently available knowledge is limited by the number of mutations analyzed per patient. We collected a unique cohort of 10 well-defined patients with bi-allelic mutations in genes involved in base excision repair (BER) (UNG) or mismatch repair (MMR) (MSH2, MSH6, or PMS2) and are the first to present next-generation sequencing (NGS) data of the BCR, allowing us to study SHM extensively in humans. Analysis using ARGalaxy revealed selective skewing of SHM mutation patterns specific for each genetic defect, which are in line with the five-pathway model of SHM that was recently proposed based on mice data. However, trans-species comparison revealed differences in the role of PMS2 and MSH2 in strand targeting between mice and man. In conclusion, our results indicate a role for UNG, MSH2, MSH6, and PMS2 in the generation of SHM in humans comparable to their function in mice. However, we observed differences in strand targeting between humans and mice, emphasizing the importance of studying molecular mechanisms in a human setting. The here developed method combining NGS and ARGalaxy analysis of BCR mutation data forms the basis for efficient SHM analyses of other immune deficiencies.

A 3-Year-Old Girl With a Mediastinal Mass.

CASE PRESENTATION: A 3-year-old girl was referred to a pediatric pulmonologist for dyspnea and recurrent upper respiratory tract infections (RTIs). The patient was born full term to unrelated Dutch parents after an uneventful pregnancy and birth. The year before presentation, she had suffered from pneumonia and > 10 upper RTIs. Apart from the recurrent RTIs, which started in infancy, her medical history was not significant and did not include allergies or eczema. An adenotonsillectomy was performed at the age of 2 years, and she was treated with multiple antibiotic regimens and inhalation therapy with salbutamol and corticosteroids, with no relief of symptoms.

Imaging of Bronchial Pathology in Antibody Deficiency: Data from the European Chest CT Group.

Studies of chest computed tomography (CT) in patients with primary antibody deficiency syndromes (ADS) suggest a broad range of bronchial pathology. However, there are as yet no multicentre studies to assess the variety of bronchial pathology in this patient group. One of the underlying reasons is the lack of a consensus methodology, a prerequisite to jointly document chest CT findings. We aimed to establish an international platform for the evaluation of bronchial pathology as assessed by chest CT and to describe the range of bronchial pathologies in patients with antibody deficiency. Ffteen immunodeficiency centres from 9 countries evaluated chest CT scans of patients with ADS using a predefined list of potential findings including an extent score for bronchiectasis. Data of 282 patients with ADS were collected. Patients with common variable immunodeficiency disorders (CVID) comprised the largest subgroup (232 patients, 82.3%). Eighty percent of CVID patients had radiological evidence of bronchial pathology including bronchiectasis in 61%, bronchial wall thickening in 44% and mucus plugging in 29%. Bronchiectasis was detected in 44% of CVID patients aged less than 20 years. Cough was a better predictor for bronchiectasis than spirometry values. Delay of diagnosis as well as duration of disease correlated positively with presence of bronchiectasis. The use of consensus diagnostic criteria and a pre-defined list of bronchial pathologies allows for comparison of chest CT data in multicentre studies. Our data suggest a high prevalence of bronchial pathology in CVID due to late diagnosis or duration of disease.

Pregnant women with inflammatory bowel disease: the effects of biologicals on pregnancy, outcome of infants, and the developing immune system.

INTRODUCTION: Relapse of inflammatory bowel disease (IBD) during conception and pregnancy has been associated with a negative pregnancy outcome. Therefore, it is advised to maintain drugs in order to prevent relapse. The effect of drugs, which cross the placenta, on children who have been exposed during pregnancy will be discussed in this review. Areas covered: A literature search was performed using the following search terms: inflammatory bowel disease, pregnancy, infant, antitumor necrosis factor alpha, infliximab, adalimumab, golimumab, certolizumab, anti-integrins, vedolizumab, anti-interleukin (IL)-12/23 ustekinumab, placenta, vaccination. Other studies were identified by using references from articles identified through our original literature search. The occurrence of unfavorable pregnancy outcome and congenital malformations does not seem to be increased after exposure to anti-TNFα, but the effects on the developing immune system are largely unknown. For anti-integrins and anti IL-12/23, the numbers of exposed pregnancies are too small to draw any conclusions. Expert commentary: Follow-up of the developing immune system in children exposed to these drugs seems warranted, preferably in a prospective study design.

Immunoglobulin G Fragment Crystallizable Glycosylation After Hematopoietic Stem Cell Transplantation Is Dissimilar to Donor Profiles.

Immunoglobulin G (IgG) fragment crystallizable (Fc) N-glycosylation has a large influence on the affinity of the antibody for binding to Fcγ-receptors (FcγRs) and C1q protein, thereby influencing immune effector functions. IgG Fc glycosylation is known to be partly regulated by genetics and partly by stimuli in the microenvironment of the B cell. Following allogeneic hematopoietic stem cell transplantation (HSCT), and in the presence of (almost) complete donor chimerism, IgG is expected to be produced by, and glycosylated in, B cells of donor origin. We investigated to what extent IgG glycosylation in patients after transplantation is determined by factors of the donor (genetics) or the recipient (environment). Using an IgG subclass-specific liquid chromatography-mass spectrometry method, we analyzed the plasma/serum IgG Fc glycosylation profiles of 34 pediatric patients pre-HSCT and at 6 and 12 months post-HSCT and compared these to the profiles of their donors and age-matched healthy controls. Patients treated for hematological malignancies as well as for non-malignant hematological diseases showed after transplantation a lower Fc galactosylation than their donors. Especially for the patients treated for leukemia, the post-HSCT Fc glycosylation profiles were more similar to the pre-HSCT recipient profiles than to profiles of the donors. Pre-HSCT, the leukemia patient group showed as distinctive feature a decrease in sialylation and in hybrid-type glycans as compared to healthy controls, which both normalized after transplantation. Our data suggest that IgG Fc glycosylation in children after HSCT does not directly mimic the donor profile, but is rather determined by persisting environmental factors of the host.

HLA-DR Expression on Monocyte Subsets in Critically Ill Children.

BACKGROUND: To longitudinally study blood monocyte subset distribution and human leukocyte antigen-DR (HLA-DR) expression on monocyte subsets in children with sepsis, post-surgery and trauma in relation to nosocomial infections and mortality. METHODS: In 37 healthy children and 37 critically ill children (12 sepsis, 11 post-surgery, 10 trauma and 4 admitted for other reasons)-participating in a randomized controlled trial on early versus late initiation of parenteral nutrition-monocyte subset distribution and HLA-DR expression on monocyte subsets were measured by flow cytometry upon admission and on days 2, 3 and 4 of pediatric intensive care unit (PICU) stay. RESULTS: Upon PICU admission, critically ill children had a higher proportion of classical monocytes (CD14++CD16-) than healthy children [PICU 95% (interquartile range [IQR] 88%-98%); controls, 87% (IQR 85%-90%), P < 0.001]. HLA-DR expression was significantly decreased within all monocyte subsets and at all time points, being most manifest on classical monocytes and in patients with sepsis. Percentage of HLA-DR expressing classical monocytes [upon PICU admission 67% (IQR 44%-88%); controls 95% (IQR 92%-98%), P < 0.001], as well as the HLA-DR mean fluorescence intensity [upon PICU admission 3219 (IQR 2650-4211); controls 6545 (IQR 5558-7647), P < 0.001], decreased during PICU stay. Patients who developed nosocomial infections (n = 13) or who died (n = 6) had lower HLA-DR expression on classical monocytes at day 2 (P = 0.002) and day 3 (P = 0.04), respectively. CONCLUSIONS: Monocytic HLA-DR expression decreased during PICU stay and was lower compared with controls on all examined time points, especially on classical monocytes and in children admitted for sepsis. Low HLA-DR expression on classical monocytes was associated with nosocomial infections and death.

Development and Function of Immune Cells in an Adolescent Patient With a Deficiency in the Interleukin-10 Receptor.

OBJECTIVE: Monogenic defects in the interleukin-10 (IL-10) pathway are extremely rare and cause infantile-onset inflammatory bowel disease (IBD)-like pathology. Understanding how immune responses are dysregulated in monogenic IBD-like diseases can provide valuable insight in "classical" IBD pathogenesis. Here, we studied long-term immune cell development and function in an adolescent IL-10 receptor (IL10RA)-deficient patient who presented in infancy with severe colitis and fistulizing perianal disease and is currently treated with immune suppressants. METHODS: Biomaterial was collected from the IL10RA-deficient patient, pediatric patients with IBD, and healthy controls. The frequency and phenotype of immune cells were determined in peripheral blood and intestinal biopsies by flow cytometry and immunohistochemistry. Functional changes in monocyte-derived dendritic cells and T cells were assessed by in vitro activation assays. RESULTS: The IL10RA-deficient immune system developed normally with respect to numbers and phenotype of circulating immune cells. Despite normal co-stimulatory molecule expression, bacterial lipopolysaccharide-stimulated monocyte-derived dendritic cells from the IL10RA-deficient patient released increased amounts of tumor necrosis factor α compared to healthy controls. Upon T-cell receptor ligation, IL10RA-deficient peripheral blood mononuclear cells released increased amounts of T-cell cytokines interferon γ and IL-17 agreeing with high numbers of T-bet and IL-17 cells in intestinal biopsies taken at disease onset. In vitro, the immunosuppressive drug thalidomide used to treat the patient's decreased peripheral blood mononuclear cell-derived tumor necrosis factor production. CONCLUSIONS: With time and during immunosuppressive treatment the IL10RA-deficient immune system develops relatively normally. Upon activation, IL-10 is crucial for controlling excessive inflammatory cytokine release by dendritic cells and preventing interferon γ and IL-17-mediated T-cell responses.

Genetic defects in PI3Kδ affect B-cell differentiation and maturation leading to hypogammaglobulineamia and recurrent infections.

BACKGROUND: Mutations in PIK3CD and PIK3R1 cause activated PI3K-δ syndrome (APDS) by dysregulation of the PI3K-AKT pathway. METHODS: We studied precursor and peripheral B-cell differentiation and apoptosis via flowcytometry. Furthermore, we performed AKT-phosphorylation assays and somatic hypermutations (SHM) and class switch recombination (CSR) analysis. RESULTS: We identified 13 patients of whom 3 had new mutations in PIK3CD or PIK3R1. Patients had low total B-cell numbers with increased frequencies of transitional B cells and plasmablasts, while the precursor B-cell compartment in bone marrow was relatively normal. Basal AKT phosphorylation was increased in lymphocytes from APDS patients and natural effector B cells where most affected. PI3K mutations resulted in altered SHM and CSR and increased apoptosis. CONCLUSIONS: The B-cell compartment in APDS patients is affected by the mutations in PI3K. There is reduced differentiation beyond the transitional stage, increased AKT phosphorylation and increased apoptosis. This B-cell phenotype contributes to the clinical phenotype.

Circulating T Cells of Patients with Nijmegen Breakage Syndrome Show Signs of Senescence.

PURPOSE: The Nijmegen breakage syndrome (NBS) is an inherited genetic disorder characterized by a typical facial appearance, microcephaly, growth retardation, immunodeficiency, and a strong predisposition to malignancies, especially of lymphoid origin. NBS patients have a mutation in the NBN gene which involves the repair of DNA double-strand breaks (DSBs). Here we studied the peripheral T cell compartment of NBS patients with a focus on immunological senescence. METHODS: The absolute numbers and frequencies of the different T cell subsets were determined in NBS patients from young age till adulthood and compared to age-matched healthy individuals (HI). In addition, we determined the expression of senescent T cell markers and the signal joint T cell receptor excision circles (sjTRECs) content. RESULTS: Our results demonstrate that NBS patients have reduced T cell numbers. NBS patients showed lower numbers of αß+ T cells, but normal γδ+ T cell numbers compared to HI. Concerning the αß+ T cells, both CD4+ as well as CD8+ T cells were excessively reduced in numbers compared to aged-matched HI. In addition, NBS patients showed higher frequencies of the more differentiated T cells expressing the senescent cell marker CD57 and did not express co-stimulatory molecule CD28. These effects were already present in the youngest age group. Furthermore, NBS patients showed lower sjTREC content in their T cells possibly indicative of a lower thymic output. CONCLUSIONS: We conclude that circulating T cells from NBS patients show signs of a senescent phenotype which is already present from young age on and which might explain their T cell immune deficiency.