Pédiatrie 2008. [Pediatrics]

This year we present three papers on recent advances in paediatrics from the fields of neonatology, adolescent medicine and Duchenne muscular dystrophy. 1. Recent studies question the application of pure oxygen for neonatal reanimation and suggest that lower concentrations or even air may be more adequate for the reanimation of most newborns. 2. Bullying is an aggressive, repetitive and intentionally blessing behaviour. It is observed mainly at school and the victims are usually children with a weak personality or children suffering from chronic diseases. The doctor's role is to detect this behaviour and to help protect the victims. 3. The respiratory surveillance of patients with Duchenne muscular dystrophy is the corner-stone of their management. An algorithm allows to time correctly the initiation of non-invasive ventilation and to insure as long as possible a good life quality.

ECIL recommendations for the use of biological markers for the diagnosis of invasive fungal diseases in leukemic patients and hematopoietic SCT recipients.

As culture-based methods for the diagnosis of invasive fungal diseases (IFD) in leukemia and hematopoietic SCT patients have limited performance, non-culture methods are increasingly being used. The third European Conference on Infections in Leukemia (ECIL-3) meeting aimed at establishing evidence-based recommendations for the use of biological tests in adult patients, based on the grading system of the Infectious Diseases Society of America. The following biomarkers were investigated as screening tests: galactomannan (GM) for invasive aspergillosis (IA); β-glucan (BG) for invasive candidiasis (IC) and IA; Cryptococcus Ag for cryptococcosis; mannan (Mn) Ag/anti-mannan (A-Mn) Ab for IC, and PCR for IA. Testing for GM, Cryptococcus Ag and BG are included in the revised EORTC/MSG (European Organization for Research and Treatment of Cancer/Mycoses Study Group) consensus definitions for IFD. Strong evidence supports the use of GM in serum (A II), and Cryptococcus Ag in serum and cerebrospinal fluid (CSF) (A II). Evidence is moderate for BG detection in serum (B II), and the combined Mn/A-Mn testing in serum for hepatosplenic candidiasis (B III) and candidemia (C II). No recommendations were formulated for the use of PCR owing to a lack of standardization and clinical validation. Clinical utility of these markers for the early management of IFD should be further assessed in prospective randomized interventional studies.

Increased Presence of FOXP3+ Regulatory T Cells in Inflamed Muscle of Patients with Active Juvenile Dermatomyositis Compared to Peripheral Blood.

Juvenile dermatomyositis (JDM) is an immune-mediated inflammatory disease affecting the microvasculature of skin and muscle. CD4+CD25+FOXP3+ regulatory T cells (Tregs) are key regulators of immune homeostasis. A role for Tregs in JDM pathogenesis has not yet been established. Here, we explored Treg presence and function in peripheral blood and muscle of JDM patients. We analyzed number, phenotype and function of Tregs in blood from JDM patients by flow cytometry and in vitro suppression assays, in comparison to healthy controls and disease controls (Duchenne's Muscular Dystrophy). Presence of Tregs in muscle was analyzed by immunohistochemistry. Overall, Treg percentages in peripheral blood of JDM patients were similar compared to both control groups. Muscle biopsies of new onset JDM patients showed increased infiltration of numbers of T cells compared to Duchenne's muscular dystrophy. Both in JDM and Duchenne's muscular dystrophy the proportion of FOXP3+ T cells in muscles were increased compared to JDM peripheral blood. Interestingly, JDM is not a self-remitting disease, suggesting that the high proportion of Tregs in inflamed muscle do not suppress inflammation. In line with this, peripheral blood Tregs of active JDM patients were less capable of suppressing effector T cell activation in vitro, compared to Tregs of JDM in clinical remission. These data show a functional impairment of Tregs in a proportion of patients with active disease, and suggest a regulatory role for Tregs in JDM inflammation.

A PiggyBac-mediated approach for muscle gene transfer or cell therapy.

An emerging therapeutic approach for Duchenne muscular dystrophy is the transplantation of autologous myogenic progenitor cells genetically modified to express dystrophin. The use of this approach is challenged by the difficulty in maintaining these cells ex vivo while keeping their myogenic potential, and ensuring sufficient transgene expression following their transplantation and myogenic differentiation in vivo. We investigated the use of the piggyBac transposon system to achieve stable gene expression when transferred to cultured mesoangioblasts and into murine muscles. Without selection, up to 8% of the mesoangioblasts expressed the transgene from 1 to 2 genomic copies of the piggyBac vector. Integration occurred mostly in intergenic genomic DNA and transgene expression was stable in vitro. Intramuscular transplantation of mouse Tibialis anterior muscles with mesoangioblasts containing the transposon led to sustained myofiber GFP expression in vivo. In contrast, the direct electroporation of the transposon-donor plasmids in the mouse Tibialis muscles in vivo did not lead to sustained transgene expression despite molecular evidence of piggyBac transposition in vivo. Together these findings provide a proof-of-principle that piggyBac transposon may be considered for mesoangioblast cell-based therapies of muscular dystrophies.

Assessing dystrophies and other muscle diseases at the nanometer scale by atomic force microscopy.

AIM: Atomic force microscopy nanoindentation of myofibers was used to assess and quantitatively diagnose muscular dystrophies from human patients. MATERIALS & METHODS: Myofibers were probed from fresh or frozen muscle biopsies from human dystrophic patients and healthy volunteers, as well as mice models, and Young's modulus stiffness values were determined. RESULTS: Fibers displaying abnormally low mechanical stability were detected in biopsies from patients affected by 11 distinct muscle diseases, and Young's modulus values were commensurate to the severity of the disease. Abnormal myofiber resistance was also observed from consulting patients whose muscle condition could not be detected or unambiguously diagnosed otherwise. DISCUSSION & CONCLUSION: This study provides a proof-of-concept that atomic force microscopy yields a quantitative read-out of human muscle function from clinical biopsies, and that it may thereby complement current muscular dystrophy diagnosis.

Type I interferon inhibits interleukin-1 production and inflammasome activation.

Type I interferon (IFN) is a common therapy for autoimmune and inflammatory disorders, yet the mechanisms of action are largely unknown. Here we showed that type I IFN inhibited interleukin-1 (IL-1) production through two distinct mechanisms. Type I IFN signaling, via the STAT1 transcription factor, repressed the activity of the NLRP1 and NLRP3 inflammasomes, thereby suppressing caspase-1-dependent IL-1β maturation. In addition, type I IFN induced IL-10 in a STAT1-dependent manner; autocrine IL-10 then signaled via STAT3 to reduce the abundance of pro-IL-1α and pro-IL-1β. In vivo, poly(I:C)-induced type I IFN diminished IL-1β production in response to alum and Candida albicans, thus increasing susceptibility to this fungal pathogen. Importantly, monocytes from multiple sclerosis patients undergoing IFN-β treatment produced substantially less IL-1β than monocytes derived from healthy donors. Our findings may thus explain the effectiveness of type I IFN in the treatment of inflammatory diseases but also the observed "weakening" of the immune system after viral infection.

Preventing invasive candida infections. Where could we do better?

Invasive candidiasis is associated with high mortality rates, ranging from 35% to 60%, in the range reported for septic shock. The epidemiology and pathogenesis of invasive candidiasis differ according to the patient's immune status; the majority of cases in immunocompromised hosts are candidaemia, whereas non-candidaemic systemic candidiasis accounts for the majority of cases in critically ill patients. In contrast to candidaemia, non-candidaemic systemic candidiasis is difficult to prove, especially in critically ill patients. Up to 80% of these patients are colonized, but only 5-30% develop invasive infection. The differentiation of colonization and proven infection is challenging, and evolution from the former to the latter requires seven to 10 days. This continuum from colonization of mucosal surfaces to local invasion and then invasive infection makes it difficult to identify those critically ill patients likely to benefit most from antifungal prophylaxis or early empirical antifungal treatment. Early empirical treatment of non-candidaemic systemic candidiasis currently relies on the positive predictive value of risk assessment strategies, such as the colonization index, candida score, and predictive rules based on combinations of risk factors such as candida colonization, broad-spectrum antibiotics, and abdominal surgery. Although guidelines recently scored these strategies as being supported by limited evidence, they are widely used at bedside and have substantially decreased the incidence of invasive candidiasis.

Dysferlinopathy in Switzerland: clinical phenotypes and potential founder effects.

BACKGROUND: Dysferlin is reduced in patients with limb girdle muscular dystrophy type 2B, Miyoshi myopathy, distal anterior compartment myopathy, and in certain Ethnic clusters. METHODS: We evaluated clinical and genetic patient data from three different Swiss Neuromuscular Centers. RESULTS: Thirteen patients from 6 non-related families were included. Age of onset was 18.8 ± 4.3 years. In all patients, diallelic disease-causing mutations were identified in the DYSF gene. Nine patients from 3 non-related families from Central Switzerland carried the identical homozygous mutation, c.3031 + 2T>C. A possible founder effect was confirmed by haplotype analysis. Three patients from two different families carried the heterozygous mutation, c.1064_1065delAA. Two novel mutations were identified (c.2869C>T (p.Gln957Stop), c.5928G>A (p.Trp1976Stop)). CONCLUSIONS: Our study confirms the phenotypic heterogeneity associated with DYSF mutations. Two mutations (c.3031 + 2T>C, c.1064_1065delAA) appear common in Switzerland. Haplotype analysis performed on one case (c. 3031 + 2T>C) suggested a possible founder effect.

Ectodysplasin/NF-κB Promotes Mammary Cell Fate via Wnt/β-catenin Pathway.

Mammary gland development commences during embryogenesis with the establishment of a species typical number of mammary primordia on each flank of the embryo. It is thought that mammary cell fate can only be induced along the mammary line, a narrow region of the ventro-lateral skin running from the axilla to the groin. Ectodysplasin (Eda) is a tumor necrosis factor family ligand that regulates morphogenesis of several ectodermal appendages. We have previously shown that transgenic overexpression of Eda (K14-Eda mice) induces formation of supernumerary mammary placodes along the mammary line. Here, we investigate in more detail the role of Eda and its downstream mediator transcription factor NF-κB in mammary cell fate specification. We report that K14-Eda mice harbor accessory mammary glands also in the neck region indicating wider epidermal cell plasticity that previously appreciated. We show that even though NF-κB is not required for formation of endogenous mammary placodes, it is indispensable for the ability of Eda to induce supernumerary placodes. A genome-wide profiling of Eda-induced genes in mammary buds identified several Wnt pathway components as potential transcriptional targets of Eda. Using an ex vivo culture system, we show that suppression of canonical Wnt signalling leads to a dose-dependent inhibition of supernumerary placodes in K14-Eda tissue explants.

The Role of Biomarkers for Starting Antifungals in the Intensive Care Unit

Invasive candidiasis is associated with high mortality rates (35% to 60%), similar to the range reported for septic shock. The most common types include candidemia, frequently observed in immunocompromised patients, and noncandidemic systemic candidiasis, which constitutes the majority of cases in critically ill patients. However, they are difficult to prove and a definite diagnosis usually occurs late in the course of the disease, thus contributing to their bad prognosis. Early empirical treatment improves the prognosis and currently relies on the positive predictive value (PPV) of risk-assessment strategies (colonization index, Candida score, predictive rules) based on combinations of risk factors, but it may have also largely contributed to the overuse of antifungal agents in critically ill patients. In this context, non- culture-based diagnostic methods, including specific and nonspecific biomarkers, may significantly improve the diagnosis of invasive candidiasis. Candida DNA and mannan antigen/antimannan antibodies are of limited interest for the diagnosis of invasive candidiasis as they fail to identify noncandidemic systemic candidiasis, despite early positivity in candidemic patients. The utility of 1,3-beta-D-glucan (b-D-glucan), a panfungal cell wall antigen, has been demonstrated for the diagnosis of fungal infections in immunocompromised patients. Preliminary data suggest that it is also detectable early in critically ill patients developing noncandidemic systemic candidiasis. To take advantage of the high negative predictive value of risk-assessment strategies and the early increase in specific fungal biomarkers in high-risk patients, we propose a practical 2-step approach to improve the selection of patients susceptible to benefit from empirical antifungal treatment.

NBAS mutations cause a multisystem disorder involving bone, connective tissue, liver, immune system, and retina.

We report two unrelated patients with a multisystem disease involving liver, eye, immune system, connective tissue, and bone, caused by biallelic mutations in the neuroblastoma amplified sequence (NBAS) gene. Both presented as infants with recurrent episodes triggered by fever with vomiting, dehydration, and elevated transaminases. They had frequent infections, hypogammaglobulinemia, reduced natural killer cells, and the Pelger-Huët anomaly of their granulocytes. Their facial features were similar with a pointed chin and proptosis; loose skin and reduced subcutaneous fat gave them a progeroid appearance. Skeletal features included short stature, slender bones, epiphyseal dysplasia with multiple phalangeal pseudo-epiphyses, and small C1-C2 vertebrae causing cervical instability and myelopathy. Retinal dystrophy and optic atrophy were present in one patient. NBAS is a component of the synthaxin-18 complex and is involved in nonsense-mediated mRNA decay control. Putative loss-of-function mutations in NBAS are already known to cause disease in humans. A specific founder mutation has been associated with short stature, optic nerve atrophy and Pelger-Huët anomaly of granulocytes (SOPH) in the Siberian Yakut population. A more recent report associates NBAS mutations with recurrent acute liver failure in infancy in a group of patients of European descent. Our observations indicate that the phenotypic spectrum of NBAS deficiency is wider than previously known and includes skeletal, hepatic, metabolic, and immunologic aspects. Early recognition of the skeletal phenotype is important for preventive management of cervical instability. © 2015 Wiley Periodicals, Inc.

Tgfbi/Bigh3 silencing activates ERK in mouse retina.

BIGH3 is a secreted protein, part of the extracellular matrix where it interacts with collagen and integrins on the cell surface. BIGH3 can play opposing roles in cancer, acting as either tumor suppressor or promoter, and its mutations lead to different forms of corneal dystrophy. Although many studies have been carried out, little is known about the physiological role of BIGH3. Using the cre-loxP system, we generated a mouse model with disruption of the Bigh3 genomic locus. Bigh3 silencing did not result in any apparent phenotype modifications, the mice remained viable and fertile. We were able to determine the presence of BIGH3 in the retinal pigment epithelium (RPE). In the absence of BIGH3, a transient decrease in the apoptotic process involved in retina maturation was observed, leading to a transient increase in the INL thickness at P15. This phenomenon was accompanied by an increased activity of the pro-survival ERK pathway.