(Rockford, IL). Fifty or 100 μL of the reconstituted standards or samples of the supernatant medium were isocitrate dehydrogenase inhibitor plated onto wells of plates coated with anti-human primary antibody and then incubated with 50 μL of a biotinylated detection antibody reagent at room temperature for 2 h. At the end of the incubation, the plate was washed three times and 100 μL of streptavidin–horseradish peroxidase solution was added to each well and incubated for 30 min at room temperature. Following another

three washes, 100 μL of tetramethylbenzidine substrate solution was added to each well and the colored product was allowed to develop at room temperature in the dark. After 30 min, 100 μL of stop solution was added and the absorbance of the samples was measured at 450 nm (Golub et al., 2008). As shown in Fig. 1, control wells were incubated with monocytes in serum-free conditioned media (SFCM) and stimulated (or not) by lipopolysaccharide. In the absence of doxycycline and lipopolysaccharide, <50 pg mL−1 of TNF-α was secreted by the monocytes, which was increased to 376.9 pg mL−1 of TNF-α when lipopolysaccharide was added to the culture. When doxycycline was added to the culture of the

lipopolysaccharide-stimulated monocytes in final concentrations of 0.1, 1 and 10 μM, the extracellular TNF-α levels were decreased by 46%, 52% and 71%, respectively. The effect of the same concentrations of doxycycline was much less dramatic on the production of IL-1β (Fig. 2). Monocytes secreted 58 pg mL−1 of IL-1β when lipopolysaccharide was added to the culture. However, when these cells were incubated in the presence of doxycycline at concentrations of 0.1, 1 and 10 μM, the extracellular IL-1β Ibrutinib datasheet levels were

only reduced by 9%, 16% and 16%, respectively. The extracellular levels of MMP-9, a major MMP secreted by monocytes, in the CM from lipopolysaccharide-stimulated monocyte cultures maintained in the presence of 0.1, 1 and 10 μM doxycycline, were initially analyzed by ELISA. Decreased MMP-9 levels were observed; 0.1, 1 and 10 μM doxycycline decreased MMP-9 levels by 18%, 20% and 41%, respectively (Fig. 3). In separate experiments, the monocytes were allowed to mature for 7 days into macrophages and the levels of both MMP-2 (72-kDa gelatinase) and MMP-9 (92-kDa gelatinase) Glycogen branching enzyme were assessed by gelatin zymography (Fig. 4). MMP-9 was consistently found to be more dominant than MMP-2 at days 1, 3 and 7, particularly at the later time periods; the MMP-9 levels progressively increased with the duration of the incubation, while MMP-2 remained constant. Moreover, doxycycline in final concentrations of 0–20 μM inhibited MMP-9 in a dose–response manner, but had no effect on MMP-2. A similar effect of these concentrations of doxycycline was observed when 0.1 μg mL−1 lipopolysaccharide was added to the macrophages in culture. Monocyte-derived macrophages were cultured with lipopolysaccharide in the presence of 0, 10 and 20 μM doxycyline for 2 days.

[7, 37] (Supporting Information Fig. 2B and 3C). Although expression of some alternative activation markers in CD11bloF4/80hi TAMs was affected by Stat1 deficiency, a parallel upregulation of M2 transcripts could be observed in the Stat1-null CD11bhiF4/80lo subset. We hypothesize that this may represent Carfilzomib concentration a compensatory mechanism that should guarantee expression of M2 proteins of potentially vital importance for the tumor such as IL-10 implicated in blunting antitumor

T-cell response [38]. Our findings expand the existing knowledge on the impact of CSF1R signaling on TAM homeostasis [5, 6, 22] by documenting its profound influence on proliferation and/or survival of CD11bloF4/80hi macrophages (Fig. 6). CSF1, postulated to act as a strong M2-polarization factor [5], may also trigger the M2 transcriptional response in these cells (Supporting Information Fig. 2B). In contrast, the transient effects of the CSF1R blockage

on the CD11bhiF4/80lo TAM population (Fig. 6) buy Pembrolizumab may point toward a redundancy between CSF1/CSF1R and other signaling pathways (e.g., IL-4R [17] or GM-CSFR [5, 11, 13]) in the homeostasis of this subset. Here, we report that STAT1 interacts with the promoter of the Csf1 gene and stimulates its expression in tumor cells (Fig. 6). By this means, STAT1 could regulate via CSF1 the expansion of CD11bloF4/80hi TAMs and their M2 phenotype. Despite lower CSF1 levels in Stat1-null tumors, Stat1-deficient TAMs possessed similar proliferation capabilities and did not display enhanced apoptosis when compared with Stat1+/+ infiltrating macrophages (Fig. 5). Furthermore, monocyte recruitment was apparently not controlled by STAT1

(Supporting Information Fig. 5C, 10B and C). We consider the possibility that STAT1-dependent CSF1 fosters the maturation of CD11bhiF4/80lo Org 27569 cells into CD11bloF4/80hi TAMs and by this means accounts for the higher numbers of the later in Stat1-proficient tumors. This hypothesis needs to be further explored. It is also conceivable that the diminished, but still substantial amounts of CSF1 in Stat1-deficient tumors (Fig. 7A and B) can suffice for the development and maintenance of a smaller and less M2-polarized CD11bhiF4/80lo TAM population (Fig. 1 and Supporting Information Fig. 2B) by means of in situ proliferation. However, a more profound interference with the CSF1R signaling through pharmacological inhibition resulted in a depletion of the CD11bloF4/80hi population (Fig. 6). In summary, we provide here a novel insight into ontogeny and homeostasis of TAMs with potential clinical implication, stressing the role of their heterogeneity and their local proliferation and survival fostered by CSF1 production.

IgE antibodies are conspicuous in the response to helminths [15] and other parasites [16, 17], but we are unaware of any study that has examined IgE cDNA transcripts from parasitized individuals. In the light of the accumulating evidence of the unique mutational features of the IgE response in some conditions, we undertook immunogenetic studies of IgE selleck antibodies in a community from the highlands of Papua New Guinea (PNG), where helminth infections are endemic, malaria is increasingly common, but allergic disease is almost unknown [18]. Here, we describe an analysis of sequences derived from 14

rural PNG villagers. To provide suitable data sets for comparison, we also amplified IgG sequences from both PNG and Australian individuals, and because of the possibility that different IgG subclasses could display varying patterns of mutation, selleck chemicals we generated IgG sequences using subclass-specific PCR primers. The

average number of mutations in the IgE sequences of Papua New Guineans was very high and was broadly similar to the number of mutations seen in IgG sequences from the same individuals. Although the extent of IgE mutations was significantly higher than has been reported from studies of allergic individuals, the mean level of IgG mutations reported here is little different to previous reports of IgG sequences from the developed world. The distribution of replacement and silent mutations between framework regions (FRs) and CDRs suggest the involvement of antigen selection in the development of responses of each

IgG subclass, but there was little evidence of selection in the IgE response. Sample processing.  After informed consent, and with the PAK6 approval of both the UNSW Human Research Ethics Committee and the Papua New Guinea Medical Advisory Council, peripheral blood was collected from 14 life-long residents of Masilakaiufa village, Eastern Highlands Province. The donors had no clinical symptoms or history of allergic disease and were aged between 22 and 53 years. Peripheral blood was also collected from 14 residents of Sydney, Australia. Serum was prepared from peripheral blood samples and frozen at −70 °C for later testing. Mononuclear cells were also prepared by density gradient centrifugation and frozen at −70 °C. Serum Ig determination.  Total IgE concentrations were determined for each PNG serum sample by enzyme immunoassay on a UniCAP® 100 system (Pharmacia, Uppsala, Sweden). On initial testing, one IgE sample was out of range (>5000 kU/l). It was re-measured after 1:5 dilution in an Australian serum sample known to have very low IgE (<5 kU/l). Total IgG and IgG subclasses were determined using a BN ProSpec® (Dade Behring, Sydney, Australia) nephelometer. Reference ranges were based on data from healthy adult residents of Sydney, Australia. Sequence amplification.

Additional studies on the role of platelets and IL-1 family members may be important to fully understand their roles in DENV pathogenesis. In summary, strategies that may

limit learn more IL-1 and IL-17 production at local sites of inflammation and viral replication during DENV might represent a step forward in the attenuation of severe manifestations of the disease such as DHF/DSS. In addition, any eventual strategy that allows local release of IL-22 or enhances IL-22 production to counterbalance the up-regulation of IL-17 would also bring a beneficial impact to limit tissue damage and hepatic dysfunction during DHF/DSS. However, further experimental studies are necessary to understand the complex interactions of the virus with the host

cells and the regulation of cytokines, chemokines and other mediators of inflammation including complement, tissue homeostasis and metabolism at large. This is a comprehensive review of DENV biology and research, especially of the different mouse models used to study the pathogenesis of DENV infection. Overall, each mouse model has its advantages and disadvantages and the researcher must carefully select the optimal model to investigate dengue immunopathogenesis and pre-clinical testing of antiviral drugs and vaccines. With a focus on the immune competent mouse model of DENV-2 infection, we described important molecular and cellular mechanisms underlying the exacerbated inflammatory response triggered by uncontrolled viral

replication in mice (Fig. 1). These studies will help to define new potential targets to attenuate disease severity and outcome in patients. Although the P23085 Erlotinib ic50 adapted strain represents progress, further studies are required to define how the altered sequence by this adapted strain influence host–pathogen interactions and to scrutinize the phenotype against the known clinical aspects of DHF/DSS in humans. We acknowledge Dr Mauro Farnesyltransferase M. Teixeira (UFMG, Brazil) and Dr François Trottein (INSERM, Lille, France) for their mentorship and support. Our work was supported by research grants from The Centre National de la Recherche Scientifique (CNRS), Institut National de la Santé et de la Recherche Médicale (INSERM), the French National Research Agency (ANR), Fondation pour la Recherche Médicale (FRM), Fond Européen de Développement Régional (FEDER) and the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq, Brazil). The research on DENV-2 experimental infection was developed and performed under the auspices of the programme INCT em Dengue (Brazil). The authors declare that they have no financial or commercial conflict of interests. “
“Autoimmune diseases are characterized by the body’s ability to mount immune attacks on self. This results from recognition of self-proteins and leads to organ damage due to increased production of pathogenic inflammatory molecules and autoantibodies.

Therefore, the foci stained by anti-SMN antibody have been designated

as Gemini of the Cajal body, or Gems. learn more However, coilin and SMN are colocalized in most of the cell. Therefore, these bodies are indistinguishable in most cell types.[30] It has been reported that Gems are partly colocalized with TDP-43 bodies in cultured cells.[9] In human spinal motor neurons, some Gems are stained with TDP-43, but not all of them.[34] In addition, the depletion of TDP-43 decreases the number of Gems in HeLa cells and mouse spinal motor neurons.[34, 35] A decrease in the number of Gems is also observed in spinal muscular atrophy.[36] Thus, we hypothesized that the loss of nuclear TDP-43 may result in a decrease in the number of Gems in spinal motor neurons with ALS as well. Indeed, our group and others have found that the number of Gems decreased in spinal motor neurons with ALS.[34, 37] However, surprisingly we found that the number of Gems was decreased in spinal motor neurons that still contained nuclear TDP-43.[34] This result raises the possibility that

the decreasing number of Gems precedes the alteration of TDP-43. However, in spinal motor neurons with spinal muscular atrophy, no alteration of TDP-43 has been reported, suggesting that the alteration of TDP-43 precedes the decrease in the number of Gems. Therefore, we propose that disturbance of a function of TDP-43 associated with the formation of Gems precedes the disappearance of TDP-43 from the nucleus (Fig. 1a–c). Accumulating

evidence suggests that 3-mercaptopyruvate sulfurtransferase the disappearance of nuclear TDP-43 precedes the inclusion formation of TDP-43 (Fig. 1d,e).[14] Although Belinostat supplier the mechanism for the disappearance of nuclear TDP-43 is unclear, the dysfunction of TDP-43 might precede their disappearance from the nucleus. Research has shown that TDP-43 regulates its own amounts of product by affecting its own mRNA.[18, 38] Thus, the decreasing amount of nuclear TDP-43 should induce the production of more TDP-43. However, in spinal motor neurons with ALS, nuclear TDP-43 disappears. Therefore, these cells lose TDP-43 function, which is associated with pre-mRNA splicing, including the autoregulation mechanism (Fig. 1a–g). We must consider the possibility that the decreasing number of Gems results from the decreasing number of large motor neurons in ALS, because the number of Gems is positively correlated with the size of the cell.[39, 40] Moreover, large motor neurons are more vulnerable to ALS than small ones.[41] To rule out this possibility, multiple regression analysis should be conducted to investigate whether ALS is an independent factor determining the number of Gems regardless of cell size. If our hypothesis is correct, the next question is whether the decreasing number of Gems results from a direct or indirect function of TDP-43. The number of Gems also declines due to decreasing transcriptional activity.

In comparison with adult cattle, we have previously demonstrated that the immune response of calves involves early IL-12 expression with consequent IFN-γ production, a nitric oxide burst and modulation Selleck Sotrastaurin by IL-10 (6–9). This age-related immunity is dependent upon cellular events within the spleen as splenectomy of calves renders them equally susceptible (5,10). Our studies have utilized a technique to

marsupialize the spleen of calves (11) so that cells could be acquired for ex vivo analysis (microplate assays and flow cytometry) (12–16). Such analyses have proven valuable in determining the function of various splenic cell phenotypes but lack the ability to place these cell populations within their anatomical context which include the marginal zone, red and white pulp (17). Amongst many factors that comprise an effective immune response to haemoparasitic infection, trafficking and interaction of cells within such domains are central (18). Intravital imaging techniques have been used to dynamically study such factors within

superficial lymphoid organs (19,20) and, to a limited extent, also within deeper structures including PF-02341066 ic50 the spleen of mice (21). But current techniques are not well suited to study the spleen of large mammals because of the limits on depth resolution (22). An approach readily applied to the spleen of large mammals is the serial analysis of the distribution of phenotyped cells in tissue sections. Similar to a recent study on the acute immune response of naïve mice to haemoparasitic infection (23), we have applied this technique to the spleen of naïve calves infected with Babesia bovis. The results document acute change in the distribution of several cells thought to be important to the spleen-dependent response of naïve calves to B. bovis

and serve to underscore common themes in the acute response to haemoparasitic infections. In addition, this is the first documented use of magnetic resonance imagery to measure spleen volume in calves. Twelve Holstein–Friesian steer calves were obtained at 8 weeks of age, vaccinated against pathogenic Clostridium species, castrated and dehorned. All animals were cELISA seronegative for Anaplasma marginale (VMRD, Pullman WA, USA) and B. bovis and B. bigemina (24–26). Immune system The care and use of these calves were approved by the Institutional Animal Care and Use Committee at Washington State University (Pullman, WA, USA). At 12 weeks of age, all calves underwent a surgical procedure to marsupialize the spleen (11). When necessary, spleen cell aspirates were obtained under local lidocaine anaesthesia into 60cc syringes containing ACD and prepared for in vitro studies as previously described (14,27). Ten of the twelve calves were inoculated intravenously with 1 × 105 erythrocytes infected with the T2Bo virulent isolate of B. bovis (7).

For a long time, DCs have been shown to contribute to the polarization of the immune response, to elicit an efficacious host defence. However, besides this essential immunostimulatory function of DCs, consolidated findings showed that DCs may act as pivotal players in the peripheral tolerance network by active induction of immunosuppressive T cells and regulation of T-effector cell activity. To understand whether DCs play a role in the tolerance and/or subsequent immunosuppressive mechanisms that occur within the

peritoneal cavity of AE-infected mice, we addressed Dinaciclib manufacturer whether these cells were activated. Previous studies with other helminth models had shown that DCs did not display any new phenotype following stimulation with respective parasite antigens (ES-62, SEA, glycan LNFPIII); thus, DC-dependent Th2 immunity appeared to result from antigen Ferroptosis cancer presentation in the absence of DC maturation (12). Furthermore, it has also been previously shown that immature DCs did not mature upon exposure to unfractionated metacestode proteins of E. multilocularis (13). These findings prompted us to study AE-DC activation and maturation within the peritoneal cavity of AE-infected mice. Therefore, we determined the gene expression levels of selected

cytokines (TGF-β, IL-10 and IL-12) and the expression of surface markers for pe-DCs maturation. As MHC class II (I-a) molecules were weakly expressed, we further investigated the relative gene expression levels of different molecules involved in the newly synthesized MHC class II (I-a) complex and in the formation of MHC class II (I-a)–peptide complexes [class II transactivator factor (CIITA), invariant chain (li), HLA-DM (H-2Ma), class II β-chain (I-aβ) and cathepsin S (Cat-S)] (14). In addition, we verified whether E/S and V/F might

alter MHC class II (I-a) molecules on BMDCs in vitro. The effect of AE-pe-DCs on a Con A-driven Endonuclease proliferation of naïve CD4+ pe-T cells determined whether AE-pe-DCs exhibited more immunosuppressive rather than stimulating properties. If not otherwise stated, all chemical reagents were from Sigma (St Louis, MO, USA) and all media from Gibco BRL (Invitrogen, Carlsbad, CA, USA). Female 6- to 10-week-old C57BL/6 mice were purchased from Charles River GmbH (Germany) and used for secondary infection with E. multilocularis (and as mock-infected control animals). All mice were housed and handled according to the rules of the Swiss regulations for animal experimentation. The parasite used in this study was a cloned E. multilocularis (KF5) isolate maintained by serial passages (vegetative transfer) in C57BL/6 mice (15). Metacestode tissue was obtained from infected mice by aseptic removal from the peritoneal cavity.

The median values of triplets were used to calculate relative expression of each gene according to the ΔΔCt method [50]. Unsedated animals were held in the hands of the researchers and allowed to defecate directly into

a clean tube. Mice were then sacrificed and dissected as described above. The cecum was amputated in the ileocecal junction and at the proximal end of the colon and the distal third Doramapimod cell line cut transversally and collected. The distal two-thirds of the cecum were then cut open longitudinally and luminal contents were gently collected with a disposable spatula. The rest of the luminal contents was washed off the cecum biopsy in three consecutive baths of cold PBS before the biopsy was laid flat with its mucosal side up and the mucosa was scraped off with a disposable rubber cell scrape and collected. All

collected samples were put straight in clean 1.5 mL Eppendorf tubes (Sarstedt, Nümbrect, Germany), snap frozen in liquid nitrogen and kept at −70°C until use. Design of the MITChip, sample processing, and data analysis was performed as described for the HITChip [51]. Briefly, Selleckchem LY2157299 90,000 sequences derived from the mouse intestinal tract were obtained from ARB-Silva database. Design of OTUs was performed with a cutoff of 98% and a total of 1885 OTUs were used for designing probes. Both V1 and V6 regions were exported and divided into three overlapping 24 nucleotide parts. Calculation of the predicted melting temperature (Tm) of the probes was achieved using the SantaLucia algorithm. The sequences of the probes were optimized so that the Tm fitted into a selected 5°C range (60–65°C). Before optimization of the probes, 30% of the probes fitted in the selected region of Tm, while after optimization, this percentage increased to 98%. Redundant probes were screened, and unique probes

(3580) Montelukast Sodium were printed on Agilent slides. The small subunit rRNA gene was amplified from fecal DNA using the primers T7prom-Bact-27-for (5′-TGA ATT GTA ATA C GA CTC ACT ATA GGG GTT TGA TCC TGG CTC AG–3′) and Uni-1492-rev (5′-CGG CTA CCT TGT TAC GAC-3′). Samples were initially denatured at 94°C for 2 min followed by 35 cycles of 94°C (30 s), 52°C (40 s), 72°C (90 s), and a final extension at 72°C for 7 min. The PCR products were purified by using the DNA Clean and Concentrator kit (Zymo Research, Orange, NJ, USA). In vitro transcription was performed at room temperature for 2 h with the Riboprobe System (Promega, La Jolla, USA), 500 ng of the T7–16S rRNA gene amplicon, including a 1:1 mix of rUTP and aminoallyl-rUTP (Ambion Inc.

The TmLIG4-replacement cassette containing nptII was introduced into the wild-type strain TIMM2789 by the ATMT method. Twenty-five G418 resistant-colonies were picked at random and tested for inactivation of the TmLIG4 locus by molecular biological methods. PCR with the primers Tmlig4/GW3F and nptII-RA suggested replacement of TmLIG4 in four clones. Southern blotting analysis confirmed the deletion without any additional

bands (Fig. 1). Two vigorously growing mutants, TmL28 and TmL36, were chosen for subsequent analysis. Microscopic and macroscopic selleck observations of TmL28 and TmL36 strains did not reveal any unique morphology in comparison to the parental strain (data not shown). In addition, they showed the same growth ability on solid medium at various temperatures,

and on media containing chemical mutagens, as the wild-type TIMM2789 (Fig. 3). They displayed normal check details growth activity at 28°C and 37°C and growth inhibition at 42°C (data not shown). When the sensitivities of the TmLIG4Δ mutants and TIMM2789 to several mutagens (EMS, hydroxyurea and phleomycin) were compared, no remarkable differences in growth were observed (Fig. 3). These finding allowed the usage of TmLIG4-disruptant in further experiments. In many fungi, Lig4 plays an essential role in the nonhomologous integration pathway. Deletion of Lig4-encoding genes often leads to an increase in gene replacement frequency. The effects of TmLIG4 inactivation on gene targeting

frequency were estimated at different loci. The wild-type strain TIMM2789 and TmL28 were used as host recipients for these disruption experiments. With homologous fragments nearly 2 kb in length, gene replacement of TmKu80, tnr, TmFKBP12 and TmSSU1 was carried out using a hygromycin B resistance cassette as a dominant selectable marker. First, we attempted to disrupt tnr, which is an areA (31)/nit-2 (32) ortholog, encoding GATA-type transcription factors which activate genes involved in nitrogen catabolite repression. Replacement of tnr causes a decrease in growth activity of T. mentagrophytes on many nitrogen sources (14, 23). In a previous study, we Florfenicol used the wild-type TIMM2789 and TmKu80 disruptant as host cells for tnr inactivation (14). In TIMM2789, the homologous integration frequencies ranged from 3% to 13%, while the HI frequency was about 70% in the TmKu80-lacking strain. In this study, the disruption vector pAg1-tnr/T was introduced into both recipients by ATMT (Fig. 4). A total of 15 hygromycin resistant-colonies were randomly isolated for molecular biological analysis. The HI frequency was 40% in the wild-type and 80% in the TmLIG4Δ mutant (Table 2). Phenotypic analysis of tnrΔ mutants Tmt1 and TmLt8 showed altered growth ability which correlated with the nitrogen sources used (Table 3). Glutamine, glutamate and arginine supported vigorous growth of tnrΔ mutants.

Also, drugs, malignancies and diseases which cause protein and/or lymphocyte loss may cause secondary immunodeficiency; this is more common than unrecognized PID in adults [5]. It is important to eliminate these

Afatinib purchase possibilities before making a definitive diagnosis of PID. Many new PIDs have been identified in the past decades, and more are likely in the near future, so this multi-stage diagnostic protocol will need to be revised from time to time. The key to detect a PID is to consider the possibility. This work was supported in part by the NIHR Biomedical Research Centres funding scheme (K. Gilmour) and BMBF PIDNET (C. Klein), which enabled them to spend time on the multi-stage diagnostic protocol for suspected immunodeficiency. P. Soler Palacín gratefully acknowledges Fabiola Caracseghi for her useful help in reviewing the manuscript. E. de Vries, Department of Paediatrics, Jeroen Bosch Hospital ‘s-Hertogenbosch, the Netherlands; A. Alvarez Cardona, Primary Immunodeficiency Investigation Unit,

Instituto Nacional de Pediatría, Universidad Autónoma de México, Ciudad de Mexico, Mexico; A. H. Abdul Latiff, Division of Clinical Immunology and Paediatrics School of Medicine and Health Sciences, Monash University, Sunway Campus, Malaysia; SCH727965 R. Badolato, Clinica Pediatrica dell’Università di Brescia c/o Spedali Civili, Brescia, Italy; N. Brodszki, Department of Paediatric Immunology, Lund University Hospital, Lund, Sweden; A. J. Cant, Great North Children’s Hospital, Newcastle upon Tyne, UK; J. Carbone, Department of Immunology, Gregorio Marañon Hospital, Madrid, Spain; J. T. Casper, Medical College of Wisconsin, Department of Paediatrics, Immunology/BMT, MACC Fund Research Center, Milwaukee, USA; P. Čižnár,

1st Paediatric Department, Comenius University Medical School, Children’ University Hospital, Bratislava, Slovakia; A. V. Cochino, Racecadotril Department of Paediatrics, University of Medicine and Pharmacy ‘Carol Davila’, Bucharest, Romania; B. Derfalvi, 2nd Department of Paediatrics, Immunology–Rheumatology–Nephrology Unit, Semmelweis University Budapest, Budapest, Hungary; G. J. Driessen, Department of Paediatric Infectious Disease and Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, the Netherlands; R. Elfeky, Department of Pediatrics, Ain Shams University, Cairo, Egypt; D. El-Ghoneimy, Department of Paediatric Allergy & Immunology, Faculty of Medicine, Ain Shams University, Cairo, Egypt; T. Espanol, Immunology Unit, University Hospital Vall d’Hebron, Barcelona, Spain; A. Etzioni, Meyer’s Children Hospital, Faculty of Medicine, Technion, Haifa, Israel; E. Gambineri, Department of Sciences for Woman and Child’s Health, University of Florence, ‘Anna Meyer’ Children’s Hospital, Florence, Italy; K. Gilmour, Camelia Botnar Laboratories, Great Ormond Street for Children NHS Trust, London, UK; L. I. Gonzalez-Granado, Immunodeficiencies Unit, Department of Paediatrics, Hospital 12 octubre, Madrid, Spain; M. N.