Changes in glial cell activation and extracellular vesicles production precede the onset of disease symptoms in transgenic hSOD1G93A pigs.

SOD1 gene is associated with progressive motor neuron degeneration in the familiar forms of amyotrophic lateral sclerosis. Although studies on mutant human SOD1 transgenic rodent models have provided important insights into disease pathogenesis, they have not led to the discovery of early biomarkers or effective therapies in human disease. The recent generation of a transgenic swine model expressing the human pathological hSOD1G93A gene, which recapitulates the course of human disease, represents an interesting tool for the identification of early disease mechanisms and diagnostic biomarkers. Here, we analyze the activation state of CNS cells in transgenic pigs during the disease course and investigate whether changes in neuronal and glial cell activation state can be reflected by the amount of extracellular vesicles they release in biological fluids. To assess the activation state of neural cells, we performed a biochemical characterization of neurons and glial cells in the spinal cords of hSOD1G93A pigs during the disease course. Quantification of EVs of CNS cell origin was performed in cerebrospinal fluid and plasma of transgenic pigs at different disease stages by Western blot and peptide microarray analyses. We report an early activation of oligodendrocytes in hSOD1G93A transgenic tissue followed by astrocyte and microglia activation, especially in animals with motor symptoms. At late asymptomatic stage, EV production from astrocytes and microglia is increased in the cerebrospinal fluid, but not in the plasma, of transgenic pigs reflecting donor cell activation in the spinal cord. Estimation of EV production by biochemical analyses is corroborated by direct quantification of neuron- and microglia-derived EVs in the cerebrospinal fluid by a Membrane Sensing Peptide enabled on-chip analysis that provides fast results and low sample consumption. Collectively, our data indicate that alteration in astrocytic EV production precedes the onset of disease symptoms in the hSODG93A swine model, mirroring donor cell activation in the spinal cord, and suggest that EV measurements from the cells first activated in the ALS pig model, i.e. OPCs, may further improve early disease detection.

Combined Presence in Heterozygosis of Two Variant Usher Syndrome Genes in Two Siblings Affected by Isolated Profound Age-Related Hearing Loss.

Sensorineural age-related hearing loss affects a large proportion of the elderly population, and has both environmental and genetic causes. Notwithstanding increasing interest in this debilitating condition, the genetic risk factors remain largely unknown. Here, we report the case of two sisters affected by isolated profound sensorineural hearing loss after the age of seventy. Genomic DNA sequencing revealed that the siblings shared two monoallelic variants in two genes linked to Usher Syndrome (USH genes), a recessive disorder of the ear and the retina: a rare pathogenic truncating variant in USH1G and a previously unreported missense variant in ADGRV1. Structure predictions suggest a negative effect on protein stability of the latter variant, allowing its classification as likely pathogenic according to American College of Medical Genetics criteria. Thus, the presence in heterozygosis of two recessive alleles, which each cause syndromic deafness, may underlie digenic inheritance of the age-related non-syndromic hearing loss of the siblings, a hypothesis that is strengthened by the knowledge that the two genes are integrated in the same functional network, which underlies stereocilium development and organization. These results enlarge the spectrum and complexity of the phenotypic consequences of USH gene mutations beyond the simple Mendelian inheritance of classical Usher syndrome.

Rare Missense Variants of the Human ß4 Subunit Alter Nicotinic α3ß4 Receptor Plasma Membrane Localisation.

α3ß4 nicotinic acetylcholine receptors (nARs) are pentameric ligand-gated cation channels that function in peripheral tissue and in the peripheral and central nervous systems, where they are critical mediators of ganglionic synaptic transmission and modulators of reward-related behaviours. In the pentamer, two α3ß4 subunit couples provide ligand-binding sites, and the fifth single (accessory) subunit (α3 or ß4) regulates receptor trafficking from the endoplasmic reticulum to the cell surface. A number of rare missense variants of the human ß4 subunit have recently been linked to nicotine dependence and/or sporadic amyotrophic lateral sclerosis, and altered responses to nicotine have been reported for these variants; however, it is unknown whether the effects of mutations depend on the subunit within the ligand-binding couples and/or on the fifth subunit. Here, by expressing single populations of pentameric receptors with fixed stoichiometry in cultured cells, we investigated the effect of ß4 variants in the fifth position on the assembly and surface exposure of α3ß4 nAChRs. The results demonstrate that the missense mutations in the accessory subunit alone, despite not affecting the assembly of α3ß4 receptors, alter their trafficking and surface localisation. Thus, altered trafficking of an otherwise functional nAChR may underlie the pathogenic effects of these mutations.

Respiratory distress syndrome in preterm infants of less than 32 weeks: What difference does giving 100 or 200 mg/kg of exogenous surfactant make?

BACKGROUND: Surfactant dosing and effective delivery could affect continuous positive airways pressure (CPAP)-failure. Nevertheless, information on exogenous surfactant dosing with current administration methods is limited. OBJECTIVE: To describe the effect of 100 or 200 mg/kg of surfactant as first-line treatment of respiratory distress syndrome in preterm infants of less than 32 weeks gestation. STUDY DESIGN: A retrospective single-center cohort study comparing two epochs, before and after switching from 100 to 200 mg/kg surfactant therapy. RESULTS: Six hundred and fifty-eight of the 1615 infants of less than 32 weeks were treated with surfactant: 282 received 100 mg/kg (S-100) and 376 received 200 mg/kg (S-200). There were no differences between S-100 and S-200 in perinatal data including prenatal corticosteroids, medication use, age at first surfactant administration and respiratory severity before surfactant. The S-200 vs. S-100 had fewer retreatments (17.0% vs. 47.2%, p < 0.001) and a shorter duration of oxygen therapy and mechanical ventilation (315 vs. 339 h, p = 0.018; 37 vs. 118 h, p = 0.000, respectively). There was no difference in postnatal corticosteroid use (S-200 10.0% vs. S-100 11.0%, p = 0.361). Bronchopulmonary dysplasia (BPD) was significantly lower in S-200 vs. S-100 when comparing either the 4 and 6-year periods before and after the dose switch (29.4% vs. 15.7%, p = 0.003, and 18.7% vs. 27.3%, p = 0.024, respectively) CONCLUSIONS: The switch from 100 to 200 mg/kg was associated with a marked reduction in the need for surfactant redosing, respiratory support, and BPD. This information could be important when designing a study in the modern era of less invasive administration as surfactant dosing and its effective delivery may affect the outcome.

Muscle weakness as an additional criterion for grading sarcopenia-related prognosis in patients with cancer.

BACKGROUND: Low muscle strength has been pointed out as a key characteristic of sarcopenia, but the prognostic significance of muscle function next to reduced skeletal muscle mass (SMM) in patients with cancer has been scantily investigated. METHODS: Data on muscle strength by handgrip (HG) dynamometry and total-body SMM estimated by bioelectrical impedance analysis (BIA) of Italian and German patients with cancer observed prospectively until death or censoring were analysed (N = 1076). Patients were stratified in four risk categories based on low HG (<10th percentiles of age and gender-specific normative values) and low total-body SMM according to SMM index cutoffs (<10.75 and <6.75 kg/m2 in men and women, respectively). RESULTS: During a median follow-up of 58 months [25th-75th percentile, 37-60], 566 patients had died. Patients presenting low HG in combination or not with low SMM were characterised by shorter median survival (12.7 vs. 27.2 months, respectively; p < 0.001) compared to those with low SMM/normal HG and normal SMM/normal HG (>60 months for both). After adjusting for sex, age, body mass index and percentage of weight loss, disease's stage, performance status and type of cancer, compared to reference category (normal HG and SMM; N = 210) the hazard ratios were: low SMM/normal HG (N = 342), 0.83 [95% confidence interval, CI, 0.67-1.02] (p = 0.073); normal SMM/low HG (N = 158), 1.19 [95% CI, 1.07-1.32] (p = 0.002); low SMM/low HG (N = 366), 1.39 [95% CI, 1.27-1.53] (p < 0.001). CONCLUSIONS: Muscle weakness was found to be a more powerful predictor of survival than BIA-estimated SMM and should be considered as an additional key feature of sarcopenia in patients with cancer.

The efficacy of immunonutrition in improving tolerance to chemoradiotherapy in patients with head and neck cancer, receiving nutritional counseling: study protocol of a randomized, open-label, parallel group, bicentric pilot study.

BACKGROUND: Nutritional support, including nutritional counseling and oral nutritional supplements (ONSs), has been recommended at the earliest opportunity in head and neck (H&N) cancer patients. The limited available evidence on the efficacy of immunonutrition during chemoradiotherapy (CT-RT) in H&N cancer patients is positive with regard to some secondary endpoints, but is still scanty, particularly with regard to toxicity and treatment tolerance. We hypothesize that early systematic provision of ONSs with a high-protein-high-calorie mixture containing immunonutrients (Impact) compared to standard high-calorie-high-protein nutritional blends, in addition to nutritional counseling, may be beneficial to patients with H&N cancer during CT-RT. Hence, we designed the present study to evaluate the efficacy, in terms of treatment tolerance, toxicity and response, body weight, body composition, protein-calorie intake, quality of life (QoL), fatigue, muscle strength and immunological profile of the early systematic provision of ONSs enriched in immunonutrients compared to isonitrogenous standard blends, in H&N cancer patients undergoing CT-RT. METHODS: This is a pragmatic, bicentric, randomized (1:1), parallel-group, open label, controlled, pilot clinical trial. DISCUSSION: Many efforts are still to be taken to improve the efficacy of nutritional support in oncology. Immunonutrition represents a promising approach also in H&N cancer patients, but the evidence on its efficacy in improving clinical outcomes during CT-RT is still inconclusive. The present pilot study, which guarantees the early provision of nutritional assessment and support to all the enrolled patients in accordance with the recent guidelines and recommendations, could represent one of the first proofs of the clinical effectiveness of early oral immunonutrition in cancer patients undergoing CT-RT and could stimulate further large randomized trials, potentially resulting in the improvement of supportive care quality. TRIAL REGISTRATION: This study is registered on ClinicalTrials.gov Identifier: NCT04611113.

The Link between VAPB Loss of Function and Amyotrophic Lateral Sclerosis.

The VAP proteins are integral adaptor proteins of the endoplasmic reticulum (ER) membrane that recruit a myriad of interacting partners to the ER surface. Through these interactions, the VAPs mediate a large number of processes, notably the generation of membrane contact sites between the ER and essentially all other cellular membranes. In 2004, it was discovered that a mutation (p.P56S) in the VAPB paralogue causes a rare form of dominantly inherited familial amyotrophic lateral sclerosis (ALS8). The mutant protein is aggregation-prone, non-functional and unstable, and its expression from a single allele appears to be insufficient to support toxic gain-of-function effects within motor neurons. Instead, loss-of-function of the single wild-type allele is required for pathological effects, and VAPB haploinsufficiency may be the main driver of the disease. In this article, we review the studies on the effects of VAPB deficit in cellular and animal models. Several basic cell physiological processes are affected by downregulation or complete depletion of VAPB, impinging on phosphoinositide homeostasis, Ca2+ signalling, ion transport, neurite extension, and ER stress. In the future, the distinction between the roles of the two VAP paralogues (A and B), as well as studies on motor neurons generated from induced pluripotent stem cells (iPSC) of ALS8 patients will further elucidate the pathogenic basis of p.P56S familial ALS, as well as of other more common forms of the disease.

Modifications at C(5) of 2-(2-Pyrrolidinyl)-Substituted 1,4-Benzodioxane Elicit Potent α4ß2 Nicotinic Acetylcholine Receptor Partial Agonism with High Selectivity over the α3ß4 Subtype.

A series of diastereomeric 2-(2-pyrrolidinyl)-1,4-benzodioxanes bearing a small, hydrogen-bonding substituent at the 7-, 6-, or 5-position of benzodioxane have been studied for α4ß2 and α3ß4 nicotinic acetylcholine receptor affinity and activity. Analogous to C(5)H replacement with N and to a much greater extent than decoration at C(7), substitution at benzodioxane C(5) confers very high α4ß2/α3ß4 selectivity to the α4ß2 partial agonism. Docking into the two receptor structures recently determined by cryo-electron microscopy and site-directed mutagenesis at the minus ß2 side converge in indicating that the limited accommodation capacity of the ß2 pocket, compared to that of the ß4 pocket, makes substitution at C(5) rather than at more projecting C(7) position determinant for this pursued subtype selectivity.

Selenoprotein N is an endoplasmic reticulum calcium sensor that links luminal calcium levels to a redox activity.

The endoplasmic reticulum (ER) is the reservoir for calcium in cells. Luminal calcium levels are determined by calcium-sensing proteins that trigger calcium dynamics in response to calcium fluctuations. Here we report that Selenoprotein N (SEPN1) is a type II transmembrane protein that senses ER calcium fluctuations by binding this ion through a luminal EF-hand domain. In vitro and in vivo experiments show that via this domain, SEPN1 responds to diminished luminal calcium levels, dynamically changing its oligomeric state and enhancing its redox-dependent interaction with cellular partners, including the ER calcium pump sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA). Importantly, single amino acid substitutions in the EF-hand domain of SEPN1 identified as clinical variations are shown to impair its calcium-binding and calcium-dependent structural changes, suggesting a key role of the EF-hand domain in SEPN1 function. In conclusion, SEPN1 is a ER calcium sensor that responds to luminal calcium depletion, changing its oligomeric state and acting as a reductase to refill ER calcium stores.

Management of Unresectable Metastatic Primitive Myxoid Mesenchymal Tumor of Infancy: A Case Report and Systematic Review of the Literature.

Primitive myxoid mesenchymal tumor of infancy is a rare soft tissue tumor. The present case is one of the most invasive primitive myxoid mesenchymal tumor of infancy reported to date. To our knowledge, it is the first case described with extensive involvement of pelvis and the third described developing metastasis and with an invasion of the spinal canal without evidence of transformation into undifferentiated sarcoma. The patient failed to respond to chemotherapy (CHT). According to the few available data, CHT seems to be more effective in the presence of metastatic disease or increased cellularity. However, CHT, including high-dose ifosfamide, resulted ineffective even after lung metastasis development with pathologic evidence of increased mitotic rate. The management of this case and the data in the literature confirm surgery as the gold standard treatment in this pathology.

The WRB Subunit of the Get3 Receptor is Required for the Correct Integration of its Partner CAML into the ER.

Calcium-modulating cyclophilin ligand (CAML), together with Tryptophan rich basic protein (WRB, Get1 in yeast), constitutes the mammalian receptor for the Transmembrane Recognition Complex subunit of 40 kDa (TRC40, Get3 in yeast), a cytosolic ATPase with a central role in the post-translational targeting pathway of tail-anchored (TA) proteins to the endoplasmic reticulum (ER) membrane. CAML has also been implicated in other cell-specific processes, notably in immune cell survival, and has been found in molar excess over WRB in different cell types. Notwithstanding the stoichiometric imbalance, WRB and CAML depend strictly on each other for expression. Here, we investigated the mechanism by which WRB impacts CAML levels. We demonstrate that CAML, generated in the presence of sufficient WRB levels, is inserted into the ER membrane with three transmembrane segments (TMs) in its C-terminal region. By contrast, without sufficient levels of WRB, CAML fails to adopt this topology, and is instead incompletely integrated to generate two aberrant topoforms; these congregate in ER-associated clusters and are degraded by the proteasome. Our results suggest that WRB, a member of the recently proposed Oxa1 superfamily, acts catalytically to assist the topogenesis of CAML and may have wider functions in membrane biogenesis than previously appreciated.

The Ways of Tails: the GET Pathway and more.

Due to their topology tail-anchored (TA) proteins must target to the membrane independently of the co-translational route defined by the signal sequence recognition particle (SRP), its receptor and the translocon Sec61. More than a decade of work has extensively characterized a highly conserved pathway, the yeast GET or mammalian TRC40 pathway, which is capable of countering the biogenetic challenge posed by the C-terminal TA anchor. In this review we briefly summarize current models of this targeting route and focus on emerging aspects such as the intricate interplay with the proteostatic network of cells and with other targeting pathways. Importantly, we consider the lessons provided by the in vivo analysis of the pathway in different model organisms and by the consideration of its full client spectrum in more recent studies. This analysis of the state of the field highlights directions in which the current models may be experimentally probed and conceptually extended.

VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.

Aerobic Catalytic Features in Photoredox- and Copper-Catalyzed Iodolactonization Reactions.

Mechanistic evaluations and comparison of two important aerobic catalytic oxidation processes, aerobic copper catalysis and photoredox catalysis, are performed. Interesting and distinct catalytic behaviors were observed for a common reaction of iodolactonization of alkenoic acids. Namely, the aerobic copper catalysis requires the formation of a copper carboxylate, whereas the aerobic photoredox catalysis requires the addition of proton sources to proceed to completion. Furthermore, the iodolactone products obtained from these catalytic processes are extensively derivatized to a number of functionalized lactones, including aryl lactones generated from the nickel-catalyzed reductive coupling with aryl halides.

The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

The α3ß4 subtype is the predominant neuronal nicotinic acetylcholine receptor present in the sensory and autonomic ganglia and in a subpopulation of brain neurons. This subtype can form pentameric receptors with either 2 or 3 ß4 subunits that have different pharmacologic and functional properties. To further investigate the role of the fifth subunit, we coexpressed a dimeric construct coding for a single polypeptide containing the ß4 and α3 subunit sequences, with different monomeric subunits. With this strategy, which allowed the formation of single populations of receptors with unique stoichiometry, we demonstrated with immunofluorescence and biochemical and functional assays that only the receptors with 3 ß4 subunits are efficiently expressed at the plasma membrane. Moreover, the LFM export motif of ß4 subunit in the fifth position exerts a unique function in the regulation of the intracellular trafficking of the receptors, their exposure at the cell surface, and consequently, their function, whereas the same export motif present in the ß4 subunits forming the acetylcholine binding site is dispensable.-Crespi, A., Plutino, S., Sciaccaluga, M., Righi, M., Borgese, N., Fucile, S., Gotti, C., Colombo, S. F. The fifth subunit in α3ß4 nicotinic receptor is more than an accessory subunit.

Discrimination between the endoplasmic reticulum and mitochondria by spontaneously inserting tail-anchored proteins.

Tail-anchored (TA) proteins insert into their target organelles by incompletely elucidated posttranslational pathways. Some TA proteins spontaneously insert into protein-free liposomes, yet target a specific organelle in vivo. Two spontaneously inserting cytochrome b5 forms, b5-ER and b5-RR, which differ only in the charge of the C-terminal region, target the endoplasmic reticulum (ER) or the mitochondrial outer membrane (MOM), respectively. To bridge the gap between the cell-free and in cellula results, we analyzed targeting in digitonin-permeabilized adherent HeLa cells. In the absence of cytosol, the MOM was the destination of both b5 forms, whereas in cytosol the C-terminal negative charge of b5-ER determined targeting to the ER. Inhibition of the transmembrane recognition complex (TRC) pathway only partially reduced b5 targeting, while strongly affecting the classical TRC substrate synaptobrevin 2 (Syb2). To identify additional pathways, we tested a number of small inhibitors, and found that Eeyarestatin I (ESI ) reduced insertion of b5-ER and of another spontaneously inserting TA protein, while not affecting Syb2. The effect was independent from the known targets of ESI , Sec61 and p97/VCP. Our results demonstrate that the MOM is the preferred destination of spontaneously inserting TA proteins, regardless of their C-terminal charge, and reveal a novel, substrate-specific ER-targeting pathway.

Proteins and chemical chaperones involved in neuronal nicotinic receptor expression and function: an update.

Neuronal nicotinic ACh receptors (nAChRs) are a family of ACh-gated cation channels, and their homeostasis or proteostasis is essential for the correct physiology of the central and peripheral nervous systems. The proteostasis network regulates the folding, assembly, degradation and trafficking of nAChRs in order to ensure their efficient and functional expression at the cell surface. However, as nAChRs are multi-subunit, multi-span, integral membrane proteins, the folding and assembly is a very inefficient process, and only a small proportion of subunits can form functional pentamers. Moreover, the efficiency of assembly and trafficking varies widely depending on the nAChR subtypes and the cell type in which they are expressed. A detailed understanding of the mechanisms that regulate the functional expression of nAChRs in neurons and non-neuronal cells is therefore important. The purpose of this short review is to describe more recent findings concerning the chaperone proteins and target-specific and target-nonspecific pharmacological chaperones that modulate the expression of nAChR subtypes, and the possible mechanisms that underlie the dynamic changes of cell surface nAChRs. LINKED ARTICLES: This article is part of a themed section on Nicotinic Acetylcholine Receptors. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.11/issuetoc.

Nutritional counseling with or without systematic use of oral nutritional supplements in head and neck cancer patients undergoing radiotherapy.

BACKGROUND: To evaluate the benefit of oral nutritional supplements (ONS) in addition to nutritional counseling in head and neck cancer (HNC) patients undergoing radiotherapy (RT). METHODS: In a single-center, randomized, pragmatic, parallel-group controlled trial (ClinicalTrials.gov: NCT02055833; February 2014-August 2016), 159 newly diagnosed HNC patients suitable for to RT regardless of previous surgery and induction chemotherapy were randomly assigned to nutritional counseling in combination with ONS (N = 78) or without ONS (N = 81) from the start of RT and continuing for up to 3 months after its end. Primary endpoint was the change in body weight at the end of RT. Secondary endpoints included changes in protein-calorie intake, muscle strength, phase angle and quality of life and anti-cancer treatment tolerance. RESULTS: In patients with the primary endpoint assessed (modified intention-to-treat population), counseling plus ONS (N = 67) resulted in smaller loss of body weight than nutritional counseling alone (N = 69; mean difference, 1.6 kg [95%CI, 0.5-2.7]; P = 0.006). Imputation of missing outcomes provided consistent findings. In the ONS-supplemented group, higher protein-calorie intake and improvement in quality of life over time were also observed (P < 0.001 for all). The use of ONS reduced the need for changes in scheduled anti-cancer treatments (i.e. for RT and/or systemic treatment dose reduction or complete suspension, HR=0.40 [95%CI, 0.18-0.91], P = 0.029). CONCLUSION: In HNC patients undergoing RT or RT plus systemic treatment, and receiving nutritional counseling, the use of ONS resulted in better weight maintenance, increased protein-calorie intake, improved quality of life and was associated with better anti-cancer treatment tolerance.

Extended-Spectrum Beta Lactamase-producing Enterobacteriaceae among the pediatric population: who is at risk and why? Results from a single-centre prospective study.

A prospective 18-month case-control study was performed in a tertiary Paediatric Centre in Turin (Italy) to analyse the disease burden and identify risk factors for acquisition of Extended Spectrum Beta Lactamase-producing Enterobacteriaceae (ESBL-pE). Children with ESBL-pE isolation were enrolled as cases, with controls matched according to age, type of pathogen isolated and sample of isolation. Out of 83 children (37 males, mean age 4.7 ± 5.46 years), 45 were identified as infected (54.2%) and 38 as colonised (45.8%) by ESBL-pE. Twenty-nine (64.4%) infectious disease episodes were categorised as community-acquired, 16 (35.6%) as healthcare-associated. Escherichia coli was the most frequently isolated pathogen (52, 62.7%) and the urinary tract the most frequent site involved (26, 57.9%). No deaths occurred, even in bloodstream infection cases. Hospitalisation and exposure to broad-spectrum penicillins and III/IV generation cephalosporins in the 90-day period before bacteria isolation were found to be independent risk factors at multivariate analysis. Immunodepression, prolonged central venous catheter (CVC) and urine catheter stay, and receiving a total parenteral nutrition (TPN) in the previous 30 days were otherwise recognized as potential risk factors at univariated analysis. ESBL-producing Enterobacteriaceae infections are a growing threat even in children. Careful recognition of patients at risk should promote targeted interventions in order to reduce the ESBL burden.