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BACKGROUND: Inborn errors of immunity (IEI) are rare diseases, which makes diagnosis a challenge. A better description of the initial presenting manifestations should improve awareness and avoid diagnostic delay. Although increased infection susceptibility is a well-known initial IEI manifestation, less is known about the frequency of other presenting manifestations.OBJECTIVE: We sought to analyze age-related initial presenting manifestations of IEI including different IEI disease cohorts.METHODS: We analyzed data on 16,486 patients of the European Society for Immunodeficiencies Registry. Patients with autoinflammatory diseases were excluded because of the limited number registered.RESULTS: Overall, 68% of patients initially presented with infections only, 9% with immune dysregulation only, and 9% with a combination of both. Syndromic features were the presenting feature in 12%, 4% had laboratory abnormalities only, 1.5% were diagnosed because of family history only, and 0.8% presented with malignancy. Two-third of patients with IEI presented before the age of 6 years, but a quarter of patients developed initial symptoms only as adults. Immune dysregulation was most frequently recognized as an initial IEI manifestation between age 6 and 25 years, with male predominance until age 10 years, shifting to female predominance after age 40 years. Infections were most prevalent as a first manifestation in patients presenting after age 30 years.CONCLUSIONS: An exclusive focus on infection-centered warning signs would have missed around 25% of patients with IEI who initially present with other manifestations.

Early evidence during the coronavirus disease 2019 (COVID-19) pandemic indicated high levels of interleukin (IL)-6 in patients with severe COVID-19. This led to the off-label use of tocilizumab (TCZ) during the first wave of the pandemic. While the monoclonal antibody blocks IL-6 pathway, its effect on other inflammatory cytokines remains poorly described. To better understand the effect of TCZ on the biological inflammatory profile, we monitored a large panel of inflammatory cytokines in critically ill COVID-19 patients receiving off-label TCZ. Twenty-three patients with polymerase chain reaction-confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection were included in the study, among which 15 patients received TCZ and 8 patients did not. Serum samples were collected for 8 days, before and following TCZ administration or hospital admission for the control group. Serum profile of 12 cytokines (IL-1

Early evidence during the COVID-19 pandemic indicated high levels of IL-6 in patients with severe COVID-19. This led to the off-label use of tocilizumab (TCZ) during the first wave of the pandemic.We aimed to monitor IL-6 and several inflammatory cytokines in critically ill COVID-19 patients receiving off-label TCZ. Fifteen critically ill SARS-CoV-2 PCR confirmed cases were enrolled and serum samples were collected during 8 days, before and following administration of a single dose of TCZ. In parallel, a control group consisting of 8 non-treated COVID-19 patients not receiving TCZ was established. Serum profile of 12 cytokines (IL-1β, -2, -4, -6, -8, -10, -12, -13, -17, -18, TNF-α and INF-γ) and of IL-6R were assessed in these two groups. Although the increased IL-6 concentrations after TCZ infusion were expected, we observed an unexpected increase in IL-1β, -2, -4, -10, -12p70, -18 and IL-6R levels in the treated patients with maximal values reached 2 to 4 days after TCZ. In contrast, no change in cytokine levels was observed in the control group. There was no significant difference in cytokine levels between survivors (TCZ/S) or non-survivors (TCZ/D). This observation suggests that some inflammatory pathways escape IL-6R blockade leading to an increase in several pro-inflammatory cytokines. Our findings could highlight an anti-inflammatory role of IL-6 and may explain why TCZ has failed to improve survival in critically ill COVID-19 patients when given alone.

Background and Aims: Several factors have been reported to affect faecal calprotectin [FC] values, and significant variation in FC concentrations has been observed in inflammatory bowel disease [IBD] patients. We aimed to evaluate FC variability in IBD patients, and to assess the robustness of a single stool punch. Methods: This is a single-centre observational case-control study. Disease activity was assessed using endoscopic and clinical activity scores, as well as C-reactive protein levels. Stool samples were collected twice within a 1 to 6 days interval, and FC was measured on punches and homogenates by fluorometric enzyme immunocapture assay. Results: In all, 260 stool samples were collected from 120 patients. Intrastool variability was low, with an intraclass correlation coefficient for single measures between three punches from a single stool sample of 0.91, and median coefficient of variation [CV] of 17%. CV of two stool samples a few days apart [intra-individual variability] were significantly higher [p, SCOPUS: ar.j, info:eu-repo/semantics/published

SCOPUS: ar.j, info:eu-repo/semantics/published

To better understand vaccine-induced protection and its potential failure in light of recent whooping cough resurgence, we evaluated quantity as well as quality of memory T cell responses in B. pertussis-vaccinated preadolescent children. Using a technique based on flow cytometry to detect proliferation, cytokine production and phenotype of antigen-specific cells, we evaluated residual T cell memory in a cohort of preadolescents who received a whole-cell pertussis (wP; n=11) or an acellular pertussis vaccine (aP; n=13) during infancy, and with a median of 4 years elapsed from the last pertussis booster vaccine, which was aP for all children. We demonstrated that B. pertussis-specific memory T cells are detectable in the majority of preadolescent children several years after vaccination. CD4(+) and CD8(+) T cell proliferation in response to pertussis toxin and/or filamentous hemagglutinin was detected in 79% and 60% of the children respectively, and interferon-γ or tumor necrosis factor-α producing CD4(+) T cells were detected in 65% and 53% of the children respectively. Phenotyping of the responding cells showed that the majority of antigen-specific cells, whether defined by proliferation or cytokine production, were CD45RA(-)CCR7(-) effector memory T cells. Although the time since the last booster vaccine was significantly longer for wP-compared to aP-vaccinated children, their proliferation capacity in response to antigenic stimulation was comparable, and more children had a detectable cytokine response after wP- compared to aP-vaccination. This study supports at the immunological level recent epidemiological studies indicating that infant vaccination with wP induces longer lasting immunity than vaccination with aP-vaccines., Journal Article, SCOPUS: ar.j, info:eu-repo/semantics/published

Detailed phenotypic characterization of B cell subpopulations is of utmost importance for the diagnosis and management of humoral immunodeficiencies, as they are used for classification of common variable immunodeficiencies. Since age-specific reference values remain scarce in the literature, we analysed by flow cytometry the proportions and absolute values of total, memory, switched memory and CD21(-/low) B cells in blood samples from 168 healthy children (1 day to 18 years) with special attention to the different subpopulations of CD21(low) B cells. The percentages of total memory B cells and their subsets significantly increased up to 5-10 years. In contrast, the percentages of immature CD21(-) B cells and of immature transitional CD21(low)CD38(hi) B cells decreased progressively with age, whereas the percentage of CD21(low) CD38(low) B cells remained stable during childhood. Our data stress the importance of age-specific reference values for the correct interpretation of B cell subsets in children as a diagnostic tool in immunodeficiencies.

Antibody deficiencies constitute the largest group of symptomatic primary immunodeficiency diseases. In several patients, mutations in CD19 have been found to underlie disease, demonstrating the critical role for the protein encoded by this gene in antibody responses; CD19 functions in a complex with CD21, CD81, and CD225 to signal with the B cell receptor upon antigen recognition. We report here a patient with severe nephropathy and profound hypogammaglobulinemia. The immunodeficiency was characterized by decreased memory B cell numbers, impaired specific antibody responses, and an absence of CD19 expression on B cells. The patient had normal CD19 alleles but carried a homozygous CD81 mutation resulting in a complete lack of CD81 expression on blood leukocytes. Retroviral transduction and glycosylation experiments on EBV-transformed B cells from the patient revealed that CD19 membrane expression critically depended on CD81. Similar to CD19-deficient patients, CD81-deficient patients had B cells that showed impaired activation upon stimulation via the B cell antigen receptor but no overt T cell subset or function defects. In this study, we present what we believe to be the first antibody deficiency syndrome caused by a mutation in the CD81 gene and consequent disruption of the CD19 complex on B cells. These findings may contribute to unraveling the genetic basis of antibody deficiency syndromes and the nonredundant functions of CD81 in humans., Case Reports, Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

J. Smet, L. Schandene, F. Mascart : Immunobiology clinic and laboratory of vaccinology and mucosal immunity, Hopital Erasme, Universite libre de Bruxelles, Brussels, Belgium, 808, route de Lennik, 1070 Bruxelles, Belgique. M.C. van Zelm, M. van der Burg : Department of immunology, Erasmus MC, University medical center, Rotterdam, Pays-Bas. B. Adams : Department of nephrology, Huderf, Universite libre de Bruxelles, Belgique. fmascart@erasme.ulb.ac.be jsmet@ulb.ac.be Figure 2. Effet du resveratrol dans les cellules de la lignee tumorale humaine SW48. Le resveratrol module le niveau de microARN en ciblant des genes codant des suppresseurs de tumeur et des effecteurs de la signalisation de TGF1 [9]. Voie de signalisation de TGFb, DICER1, PDCD4, PTEN, reparation de l’ADN Resveratrol : anti-oxydant anti-inflammatoire antiproliferatif antimetastatique Prometastatique au stade malin

Community-acquired pneumonia (CAP) is a major cause of morbidity in children. This study estimated the proportion of children with pneumococcal CAP among children hospitalised with CAP in Belgium and describes the causative serotype distribution after implementation of the 7-valent pneumococcal conjugate vaccine. Children 0-14 years hospitalised with X-ray-confirmed CAP were prospectively enrolled in a multicentre observational study. Acute and convalescent blood samples were collected. Pneumococcal aetiology was assessed by conventional methods (blood or pleural fluid cultures with Quellung reaction capsular typing or polymerase chain reaction [PCR] in pleural fluid), and recently developed methods (real-time PCR in blood and World Health Organization-validated serotype-specific serology). A total of 561 children were enrolled. Pneumococcal aetiology was assessed by conventional methods in 539, serology in 171, and real-time PCR in blood in 154. Pneumococcal aetiology was identified in 12.2% (66/539) of the children by conventional methods alone but in 73.9% by the combination of conventional and recently developed methods. The pneumococcal detection rate adjusted for the whole study population was 61.7%. Serotypes 1 (42.3%), 5 (16.0%), and 7F(7A) (12.8%) were predominant. In conclusion, Streptococcus pneumoniae remains the predominant bacteria in children hospitalised for CAP in Belgium after implementation of 7-valent pneumococcal conjugate vaccine, with non-vaccine-serotypes accounting for the majority of cases. The use of recently developed methods improves diagnosis of pneumococcal aetiology., Journal Article, Research Support, Non-U.S. Gov't, SCOPUS: ar.j, info:eu-repo/semantics/published

lmmunoglobulin superfamily (IgSF) domains are conserved structures present in many proteins in eukaryotes and prokaryotes. These domains are well-capable of facilitating sequence variation, which is most clearly illustrated by the variable regions in immunoglobulins (Igs) and T cell receptors (TRs). We studied an antibody-deficient patient suffering from recurrent respiratory infections and with impaired antibody responses to vaccinations. Patient's B cells showed impaired Ca(2+) influx upon stimulation with anti-IgM and lacked detectable CD19 membrane expression. CD19 sequence analysis revealed a homozygous missense mutation resulting in a tryptophan to cystein (W52C) amino acid change. The affected tryptophan is CONSERVED-TRP 41 located on the C-strand of the first extracellular IgSF domain of CD19 and was found to be highly conserved, not only in mammalian CD19 proteins, but in nearly all characterized IgSF domains. Furthermore, the tryptophan is present in all variable domains in Ig and TR and was not mutated in 117 Ig class-switched transcripts of B cells from controls, despite an overall 10% amino acid change frequency. In vitro complementation studies and CD19 western blotting of patient's B cells demonstrated that the mutated protein remained immaturely glycosylated. This first missense mutation resulting in a CD19 deficiency demonstrates the crucial role of a highly conserved tryptophan in proper folding or stability of IgSF domains.

Background Persistent polyclonal B cell lymphocytosis (PPBL) is a rare condition characterized by increased IgM and large excess of B cells with an IgD + CD27 + phenotype. In normal individuals, these cells play a central role in the defense against pneumococcal infection. So far, few studies have characterized humoral immune responses in PPBL patients. We therefore measured IgG directed against S. pneumoniae antigens in a 51 yr-old woman with PPBL before and after vaccination with a pneumococcal 23-valent polysaccharide vaccine. Methods Antibodies against pneumococcal antigens were measured first with an overall immunoassay using microplates coated with the 23-valent pneumococcal vaccine. A serotype-specific test was also performed according to the WHO consensus protocol. Results Despite a large number of IgD + CD27 + cells, our patient had low baseline titers of IgG directed against pneumococcal antigens and did not significantly respond to a 23-valent polysaccharide vaccine against S. pneumoniae . On the contrary, she had good titers of IgG directed against tetanus toxoid. Conclusion IgM + IgD + CD27 + cells which accumulate in this patient with typical PPBL patient failed to perform IgG isotype switch after a polysaccharide vaccine. The potential mechanisms and relationships with the main features of PPBL are discussed. Further studies on a larger number of similar patients are needed.

The tetraspan molecule CD81 is widely expressed on immune cells, such as B-, T-, NK-lymphocytes, monocytes and eosinophils, but also on most stromal and epithelial cells and on hepatocytes. In B-cells it is a member of the CD19 complex (CD19, CD21, CD81, CD225), which is required for signaling together with the B-cell antigen receptor upon antigen recognition. Its functions on other cells are unclear, but murine studies show an antiproliferative role for CD81. On hepatocytes, two different epitopes of CD81 act as a co-receptor for Hepatitis C virus and for Plasmodium infection. We evaluated a 4-year-old girl from consanguineous parents of Moroccan decent. She presented with recurrent infections and an acute nephrotic syndrome: >50% of glomeruli were affected due to focal mesangial hypercellularity. She showed poor weight gain (below 3rd percentile), but normal motor development. Her spleen and liver were enlarged, but function normally. Measurements of retina epithelium and CNS showed no signs of hypercellularity. Although serum IgG levels were strongly decreased (2.4 g/L) and IgM and IgA concentrations low within normal range, she tested positive for anti-platelet antibodies. Flow cytometric immunophenotyping of blood showed normal distribution and absolute numbers of granulocyte, monocyte and lymphocyte subsets; however, no CD19 expression was detected on the patient’s B-cells, whereas CD21 expression levels were normal. The patient carried no mutations in the CD19 and IFITM1 (CD225) genes. Additional immunophenotyping showed that all cells lacked CD81 expression. Sequencing of the CD81 gene showed a homozygous G>A substitution immediately downstream of exon 6 (c.561+1G>A). Spectratyping and quantitative PCR analysis showed clearly reduced total CD81 mRNA expression levels. Nearly all CD81 transcripts contained 13 additional nucleotides downstream of exon 6. This insertion results in a frame-shift and a premature stop (p.Glu188MetfsX13). The hypothetical protein lacks the fourth transmembrane domain. Similar to previously described CD19-deficient patients, our patient had reduced numbers CD5+ B-cells and Ig class switched and non-switched CD27+ memory-B cells. Whereas Vh-Cα and Vh-Cγ transcripts from Ig switched cells contained somatic hypermutations, the response of the patient’s B cells to in vitro stimulation through the B-cell receptor was impaired. The antibody response to rabies, tetanus and pneumococcal vaccinations is currently under investigation, as well as the potential impact of CD81 deficiency on the antigen-specific Th1 and Th2 cytokine production. In conclusion, the here presented CD81 deficiency is a new primary immunodeficiency, which leads to disruption of the CD19 complex and consequent hypogammaglobulinemia comparable to the CD19 deficiency. However, due to the broad tissue distribution, the clinical phenotype is not restricted to the B-cell system. Other organs are affected as well, most likely due to excessive proliferation and hypercellularity, with acute nephritic syndrome as dominant clinical problem. Currently, in vitro studies are being performed to identify whether the CD81 defect directly results in impaired B-cell and T-cell functions and abnormal proliferation of kidney and liver cells.