Erik-Oliver Glocker

A homozygous CARD9 mutation in a family with susceptibility to fungal infections.

BACKGROUND Chronic mucocutaneous candidiasis may be manifested as a primary immunodeficiency characterized by persistent or recurrent infections of the mucosa or the skin with candida species. Most cases are sporadic, but both autosomal dominant inheritance and autosomal recessive inheritance have been described. METHODS We performed genetic studies in 36 members of a large, consanguineous five-generation family, in which 4 members had recurrent fungal infections and an additional 3 members died during adolescence, 2 after invasive infection of the brain with candida species. All 36 family members were enrolled in the study, and 22 had blood samples taken for DNA analysis. Homozygosity mapping was used to locate the mutated gene. In the 4 affected family members (patients) and the 18 unaffected members we sequenced CARD9, the gene encoding the caspase recruitment domain-containing protein 9, carried out T-cell phenotyping, and performed functional studies, with the use of either leukocytes from the patients or a reconstituted murine model of the genetic defect. RESULTS We found linkage (lod score, 3.6) to a genomic interval on chromosome 9q, including CARD9. All four patients had a homozygous point mutation in CARD9, resulting in a premature termination codon (Q295X). Healthy family members had wild-type expression of the CARD9 protein; the four patients lacked wild-type expression, which was associated with low numbers of Th17 cells (helper T cells producing interleukin-17). Functional studies based on genetic reconstitution of myeloid cells from Card9(-/-) mice showed that the Q295X mutation impairs innate signaling from the antifungal pattern-recognition receptor dectin-1. CONCLUSIONS An autosomal recessive form of susceptibility to chronic mucocutaneous candidiasis is associated with homozygous mutations in CARD9.

Infant colitis—it's in the genes

Department of Immunology, Division of Infection and Immunity, University College London, Royal Free Hospital, London, UK (E-O Glocker MD, M Perro MSc, Prof B Grimbacher MD); Department of Haematology, University Hospital Freiburg, Freiburg, Germany (N Frede); Department of Paediatric Laboratory Medicine (Prof N Sebire MD) and Department of Paediatric Gastroenterology (M Elawad MD, N Shah MD), Great Ormond Street Hospital, University College London, London, UK

IL‐10 and IL‐10 receptor defects in humans

Inflammatory bowel disease (IBD), which includes Crohns disease (CD) and ulcerative colitis (UC), is chronic in nature and is characterized by abdominal pain, diarrhea, bleeding, and malabsorption. It is considered a complex multigenic and multifactorial disorder that results from disturbed interactions between the immune system and commensal bacteria of the gut. Recent work has demonstrated that IBD with an early‐onset within the first months of life can be monogenic: mutations in IL‐10 or its receptor lead to a loss of IL‐10 function and cause severe intractable enterocolitis in infants and small children. Both IL‐10 and IL‐10 receptor deficiency can be successfully treated by hematopoietic stem cell transplantation.

Interleukin-10 and Interleukin-10–Receptor Defects in Inflammatory Bowel Disease

Inflammatory bowel disease (IBD) is a chronic inflammatory disease characterized by abdominal pain, bloody diarrhoea, and malabsorption leading to weight loss. It is considered the result of inadequate control of an excessive reaction of the immune system to the resident flora of the gut. Like other primary immunodeficiencies, IL-10 and IL-10 receptor (IL10R) deficiency present with IBD and demonstrate the sensitivity of the intestine to any changes of the immune system. Both IL-10 and IL10R deficiency cause severe early-onset enterocolitis and can be successfully treated by hematopoietic stem cell transplantation (HSCT).

High secondary resistance to quinolones in German Helicobacter pylori clinical isolates

OBJECTIVES The aim of this study was to update data on levofloxacin/ciprofloxacin and triple resistance (resistance to metronidazole, clarithromycin and levofloxacin/ciprofloxacin) in Helicobacter pylori clinical isolates and to identify the impact of prior eradication therapies on their development. METHODS We tested the antimicrobial susceptibility to amoxicillin, metronidazole, clarithromycin, levofloxacin/ciprofloxacin, tetracycline and rifampicin of 5296 clinical H. pylori strains isolated between 2006 and 2011. Information on prior eradication therapies was gathered and their impact on the development of antimicrobial resistance, in particular to levofloxacin/ciprofloxacin and triple resistance, was analysed. RESULTS From 2006 onwards, both levofloxacin/ciprofloxacin and triple resistance have steadily increased and peaked in 2011 with 29.1% and 18.6%, respectively. Unsuccessful prior eradication attempts proved a major risk factor for resistance development. Patients who had undergone unsuccessful eradication attempts harboured levofloxacin/ciprofloxacin- and triple-resistant isolates significantly more often than untreated individuals (26.7% and 18.1% versus 10.6% and 1.6%). Levofloxacin/ciprofloxacin and triple resistance occurred significantly more often in patients who had received quinolones when compared with patients who had not (44.5% versus 23.1% and 28.7% versus 15.6%). We did not observe any significant differences in resistance rates in the different German federal states. CONCLUSIONS Resistance to levofloxacin/ciprofloxacin and triple resistance have continuously risen and reached worrying numbers. Hence we strongly advise against the use of quinolones in empirical second-line therapies for H. pylori without prior susceptibility testing and/or a carefully taken patient medical history.

Prospective multicentre study on antimicrobial resistance of Helicobacter pylori in Germany

OBJECTIVES Antimicrobial resistance of Helicobacter pylori endangers the successful eradication of the bacteria. The aim of this prospective surveillance study (ResiNet) is to continuously keep antimicrobial resistance of H. pylori in Germany under surveillance and to identify risk factors for its development. METHODS From July 2001 until December 2012, we tested the antimicrobial susceptibility of H. pylori strains isolated from 1651 prospectively enrolled patients. We analysed clinical and epidemiological data and identified risk factors for the development of resistance. RESULTS Average primary resistances were 29.4% for metronidazole, 6.7% for clarithromycin and 3.1% for both antimicrobials. Prior unsuccessful eradication treatments, female sex and country or continent of origin were identified as independent risk factors for development of resistance. CONCLUSIONS H. pylori-positive patients without prior eradication therapy can be treated empirically; antimicrobial susceptibility testing is recommended in previously unsuccessfully treated patients and in patients who have received antimicrobial chemotherapies due to unrelated bacterial infections.

Mucosal antifungal defence: IL-17 signalling takes centre stage

Chronic mucocutaneous candidiasis (CMC) is characterised by an increased susceptibility to infections/colonisation with primarily Candida albicans. This may be either due to a primary immune defect (isolated CMC), or secondary to a variety of underlying diseases and predisposing factors such as HIV infection, diabetes mellitus, neoplasias or the long-term use of broad-spectrum antibiotics or inhaled steroids.1,2 It may, however, also present as a clinical feature of multisystem primary immunodeficiencies including the hyper-immunoglobulin E syndrome (HIES) or autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), which may or may not be complicated by a plethora of other immunological conditions like autoimmunity (for example, autoimmune endocrinopathies) or autoinflammatory diseases (for example, inflammatory bowel disease).3,4 The genetic background of isolated CMC (iCMC) is so far poorly understood. In a recent publication in Science, Puel et al.5 provide evidence that mutations affecting the interleukin (IL)-17 signalling pathway impair immunity of the skin and mucosa and cause an iCMC phenotype. Professor Casanova’s group and collaborators showed in two families with an iCMC phenotype that heterozygous mutations in IL-17F can be dominant-negative and result in reduced IL17 cytokine activity, whereas homozygous mutations in the IL-17 receptor A (IL17RA) may lead to a completely abrogated IL-17A/F signalling. Their results support previous observations that IL-17 and one of its main sources, T helper 17 (Th17) cells, have a major role in the defence against C. albicans. Dendritic cells and macrophages sense components of the fungal cell wall such as 1,3and 1,6-linked b-glucans or a-mannans by C-type lectin receptors including Dectin-1 and -2, and kick off a spleen tyrosine kinase (SYK)/caspase recruitment domain-containing protein (CARD)9-dependent signalling pathway, which leads to the activation of nuclear factor (NF)-kB. NF-kB mediates the induction of pro-IL-1b, which is then processed to the bioactive IL-1b by the caspase-1-containing NACHT-domain-, leucinrich repeat-, and PYD-containing protein (NLRP)3 inflammasome. The Dectin/Syk/ CARD9 signalling is operative in myeloid cells and triggers the production of further cytokines such as tumour necrosis factor a, IL-6 and IL-23, which are critical for the priming and maintenance of Th17 cells.6,7 Th17 cell-deficient mice and mice deficient in IL-17RA showed increased susceptibility to oropharyngeal candidiasis with decreased expression of the antimicrobial peptide b-defensin 3, decreased fungicidal activity of the saliva and an impaired recruitment of neutrophils to the sites of inflammation.8 IL-17 and IL-22, another cytokine typically expressed by Th17 cells, were shown to synergistically upregulate expression of antimicrobial peptides, including b-defensin 2, cathelicidin and calprotectin, thereby improving the resistance of the skin and protecting from infections.9–11 However, in contrast to IL-17, IL-22 seems to be less critical for antimicrobial defence in the oral cavity, but to be more important in the gut and lung. The relevance of Th17 cells in antifungal host defence has been demonstrated in several human diseases: Significantly reduced numbers of Th17 cells were described in patients with CMC, even though the genetic background in those patients remained unknown.12 In addition, patients with CARD9 deficiency revealed increased susceptibility to infections with Candida and dermatophytes and also showed substantially reduced numbers of Th17 cells.13 Moreover, patients with the autosomal-dominant form of the HIES, caused by dominant-negative mutations in signal transducer and activator of transcription 3 (STAT3)—the key transcription factor for Th17 cell priming—have severely reduced to absent numbers of Th17 cells14–16 and hence suffer from recurrent candidiasis due to the dependency of human keratinocytes and bronchial epithelial cells on IL-17 for CXCL8 and human beta-defensin secretion.17 In humans, Th17 cells develop from naive T cells in the presence of IL-1b, IL-6, TGFb and possibly IL-21.18,19 IL-6 activates STAT3 to induce the Th17 lineage defining transcription factor retinoic acid-related RORgt, thereby providing an explanation of why mutations in STAT3 HIES in patients lead to recurrent candidiasis. Another finding reinforced the need for IL-17 and IL-22 in Candida host defence: Patients with CMC and APECED (autoimmune polyendocrinopathy candidiasis ectodermal dystrophy) may produce autoantibodies against IL-17A, IL-17F and IL-22; these neutralizing autoantibodies were not found in healthy controls or patients suffering from different autoimmune disorders.20,21 The hypothesis by Puel et al., that IL-17 is only essential for mucosal immunity against Candida but redundant for other pathogens, remains to be clarified by further research. IL-17 has so far proved to be a very versatile cytokine: Apart from its relevance for antifungal immunity and its Dr E-O Glocker is at the Institute of Medical Microbiology and Hygiene University Hospital, Freiburg, Germany and Professor B Grimbacher is at the Department of Immunology and Molecular Pathology, University College London, Royal Free Hospital, London, UK. E-mail: erik-oliver.glocker@uniklinik-freiburg.de or b.grimbacher@ucl.ac.uk Immunology and Cell Biology (2011) 89, 823–826 & 2011 Australasian Society for Immunology Inc. All rights reserved 0818-9641/11

Outcome of second- and third-line Helicobacter pylori eradication therapies based on antimicrobial susceptibility testing

OBJECTIVES The objective of this study was to assess the outcome of antimicrobial susceptibility-guided therapies in Helicobacter pylori-infected individuals who had undergone unsuccessful prior eradication treatments. METHODS From October 2004 to December 2013, 481 H. pylori-positive patients with prior unsuccessful eradication treatments were administered susceptibility-guided salvage eradication treatments. Six months on, treatment outcome was assessed by urea breath test, stool antigen ELISA, Helicobacter urease test or microbiology and/or histopathology. RESULTS Resistance to metronidazole and clarithromycin was high in patients with prior unsuccessful eradication treatments and was dependent on the number of treatment failures. Susceptibility-guided salvage eradication treatments achieved eradication rates of nearly 70% in these patients. No particular regimen was significantly better than another. CONCLUSIONS Antimicrobial susceptibility testing prevents prescription of inefficient antimicrobials and enables individualized and promising salvage treatments in patients with prior unsuccessful eradication treatments.

Evidence for non-neutralizing autoantibodies against IL-10 signalling components in patients with inflammatory bowel disease

BackgroundInflammatory bowel disease constitutes a heterogeneous group of conditions, whose aetiology is only partly understood. The prevailing hypothesis on its pathogenesis is that IBD is the result of an inadequate immune response to the resident bacterial flora of the intestine. An autoimmune background, however, has been discussed since the 1950s. Lately, it has been shown that failures in interleukin-10 (IL-10) signalling due to IL-10- and IL-10 receptor (IL-10R) mutations result in IBD. Our study aimed at investigating the existence of inhibitory autoantibodies against IL-10 and IL-10R in IBD patients capable of down-modulating IL-10 signalling thereby mimicking IL-10 or IL-10R deficiency.ResultsThirteen IBD patients had IgG autoantibodies against IL-10, IL-10RA and/or IL-10RB, and three patients had IgA autoantibodies against IL-10. However, the absolute OD values of the serum antibodies measured by ELISA were low, there was overall no significant difference between patients and controls, and positive sera had no neutralizing activity.ConclusionNo evidence for an involvement of autoantibodies against IL-10 or IL-10R in the pathogenesis of inflammatory bowel disease could be established.

Targeted Gene Panel Sequencing for Early-onset Inflammatory Bowel Disease and Chronic Diarrhea

Background: In contrast to adult-onset inflammatory bowel disease (IBD), where many genetic loci have been shown to be involved in complex disease etiology, early-onset IBD (eoIBD) and associated syndromes can sometimes present as monogenic conditions. As a result, the clinical phenotype and ideal disease management in these patients often differ from those in adult-onset IBD. However, due to high costs and the complexity of data analysis, high-throughput screening for genetic causes has not yet become a standard part of the diagnostic work-up of eoIBD patients. Methods: We selected 28 genes of interest associated with monogenic IBD and performed targeted panel sequencing in 71 patients diagnosed with eoIBD or early-onset chronic diarrhea to detect causative variants. We compared these results to whole-exome sequencing (WES) data available for 25 of these patients. Results: Target coverage was significantly higher in the targeted gene panel approach compared with WES, whereas the cost of the panel was considerably lower (approximately 25% of WES). Disease-causing variants affecting protein function were identified in 5 patients (7%), located in genes of the IL10 signaling pathway (3), WAS (1), and DKC1 (1). The functional effects of 8 candidate variants in 5 additional patients (7%) are under further investigation. WES did not identify additional causative mutations in 25 patients. Conclusions: Targeted gene panel sequencing is a fast and effective screening method for monogenic causes of eoIBD that should be routinely established in national referral centers.