Teresa Silva

Long-term ventilation in children: ten years later.

INTRODUCTION Home mechanical ventilation (HMV) represents a treatment option for patients with chronic respiratory failure and has changed prognosis and survival of many disorders in children. The aim of this study was to characterize a group of children on long-term mechanical ventilation (LTMV) for a period longer than 10 years. METHODS A retrospective analysis was carried out including patients on LTMV for more than 10 years (LTMV-10) in a tertiary pediatric hospital. STATISTICAL ANALYSIS PASW Statistics 18(®). RESULTS Thirty-one children (61% female) belong to the LTMV-10 group. Median age at the beginning of ventilatory support was 3 years (birth to 13 years). Main indications for assisted ventilation were neuromuscular disease (n=12, 39%), metabolic disease (n=7, 23%) and central hypoventilation (n=6, 19%). Volume ventilation was used in 2 children, and positive pressure ventilation in the others, mainly bilevel positive airway pressure (n=25, 81%). Invasive ventilation via tracheostomy was used since the beginning in four cases, and subsequently in two other children. The mean time of ventilatory support was 146 months and the maximum was 219 months. Respiratory morbidity was the most frequent cause of hospitalization and the annual rate of such episodes was 0.17 per child. Global mortality rate was 19%. CONCLUSIONS HMV programs provide necessary and safe assistance for children with severe chronic respiratory failure. As shown in our series, it is possible to be kept on this respiratory support modality for long periods with good compliance and a small number of hospitalizations.

Oxidative stress mediates apoptotic effects of ascorbate and dehydroascorbate in human Myelodysplasia cells in vitro

The Myelodysplastic Syndromes are stem cell heterogeneous disorders characterized by peripheral cytopenias and hypercellular bone marrow, which can evolute to acute leukaemia. Vitamin C can act as an antioxidant, ascorbic acid (AA) donates two electrons and becomes oxidized to dehydroascorbic acid (DHA). Under physiological conditions, vitamin C predominantly exists in its reduced (AA) form but also exists in trace quantities in the oxidized form (DHA). This study evaluates the therapeutic potential of vitamin C in Myelodysplastic Syndromes (MDSs). F36P cells (MDS cell line) were treated with ascorbate and dehydroascorbate alone and in combination with cytarabine. Cell proliferation and viability were assessed by trypan blue assay and cell death was evaluated by optical microscopy and flow cytometry. The role of reactive oxygen species, mitochondrial membrane potential, BAX, BCL-2 and cytochrome C were also assessed. Vitamin C decreases cell proliferation and viability in a concentration, time and administration dependent-manner inducing cell death by apoptosis, which was shown to be associated to an increased in superoxide production, mitochondrial membrane depolarization. These compounds modulate BCL-2, BAX and cytochrome C release. These results suggest that vitamin C induces cell death trough apoptosis in F36P cells and may be a new therapeutic approach in Myelodysplasia.

Selenium compounds induced ROS-dependent apoptosis in myelodysplasia cells.

Several authors have demonstrated the chemoprotective and anti-carcinogenic role of selenium. However, the therapeutic potential of selenium in myelodysplastic syndrome (MDS) as single agent and as co-adjuvant of the current therapies has not been previously studied. Sodium selenite and selenomethionine, alone and in combination with cytarabine, induce a decrease in cell viability in a time-, dose- and administration-dependent manner inducing cell death by apoptosis in F36P cells (MDS cell line). These compounds increased superoxide production and induced mitochondrial membrane depolarization. The increase in BAX/BCL-2 ratio and in the activated caspase 3 expression levels, the decrease in mitochondria membrane potential, as well as the increase in superoxide production, supports the mitochondria contribution on selenium-induced apoptosis. These findings suggest that selenium may offer a new therapeutic approach in myelodysplastic syndrome in monotherapy and/or as co-adjuvant therapy to conventional anti-carcinogenic.

Knocking out of CD38 accelerates development of a lupus-like disease in lpr mice

OBJECTIVES CD38 participates in lymphocyte ontogeny and function and may be involved in autoimmunity. Absence of CD38 accelerates development of non-obese diabetic (NOD) mice diabetes and anti-CD38 antibodies are good markers of human disease. Little is known regarding systemic autoimmunity. Active SLE patients have higher numbers of CD38(+) T and B cells. CD38 is a candidate gene for the murine Lmb2 lupus locus. We aimed to investigate whether CD38 was involved in lupus development. METHODS We developed Cd38(-/-)-Fas(lpr)/Fas(lpr) mice and monitored them for development of a lupus-like disease through measurement of protein excretion in urine, histological assessment of the kidneys, quantification of circulating immunoglobulins and autoantibodies. We have also immunophenotyped 2- and 6-month old Cd38(-/-)-Fas(lpr)/Fas(lpr) mice. RESULTS We found that absence of CD38 accelerated disease development: female Cd38(-/-)-Fas(lpr)/Fas(lpr) mice presented severe proteinuria, GN, deposition of ICs in the renal medulla and increased amounts of circulating immunoglobulin G (IgG), although anti-dsDNA autoantibodies and RF were not significantly increased at disease onset. We have found that Cd38(-/-)-Fas(lpr)/Fas(lpr) male mice, similarly to other murine models of lupus, were able to control disease. Absence of CD38 in lpr mice altered differentiation of T cells and dendritic cells (DC). CONCLUSION Although the role of CD38 in tolerance is still to be elucidated, we provide evidence that it may play an active role in the control of a murine lupus-like disease.

Apoptosis Deregulation Influences Chemoresistance to Azaguanine in Human Leukemic Cell Lines

The involvement of apoptosis in the cytotoxicity mediated by nucleoside analogues, namely azaguanine, and its implication in resistance are not well understood. Using human T-cell acute lymphoblastic leukemia cell lines, sensitive (CEM cells) and resistant to azaguanine (CM3 cells), we observe a decrease in the expression of proapoptotic proteins in CM3 cells, which may be related to the resistance to cell death induced by azaguanine. On the other hand, CM3 cells lack cross resistance with other anticarcinogenic drugs, suggesting that azaguanine may be used alternatively in the presence of chemoresistance. A better knowledge of the apoptotic pathways involved in leukemic cell death resistance may contribute to the development of therapeutic strategies, aimed to prevent chemotherapy resistance.

WITHDRAWN: Long-term ventilation in children: Ten years later

This article has been withdrawn for editorial reasons because the journal will be published only in English. In order to avoid duplicated records, this article can be found at http://dx.doi.org/10.1016/j.rppnen.2014.03.017. The Publisher apologizes for any inconvenience this may cause. The full Elsevier Policy on Article Withdrawal can be found at http://www.elsevier.com/locate/withdrawalpolicy.

Delocalized lipophilic cations as a new therapeutic approach in cancer

Delocalized lipophilic cations (DLCs) penetrate plasma and mitochondrial membranes and accumulate in mitochondria. The higher mitochondrial membrane potentials of neoplastic vs normal cells, in general, account for greater uptake and may be a way to selectively target these cells since. Dequalinium (DQA) is a DLC and so our goal is to evaluate the therapeutic potential of DQA in cancer, namely in B-cell Chronic Lymphocytic Leukaemia (B-CLL), Acute Promyelocytic Leukaemia (APL) and Hepatocellular Carcinoma (HCC). For this we used 3 cell lines, EHEB (B-CLL), HL-60 (APL) and HUH-7 (HCC), to evaluate the effect of different concentrations of DQA either by single dose administration, by daily dose administration and by association with conventional anticarcinogenic agents. Cell viability and death was determined by the resazurin assay, optical microscopy and by flow cytometry. The latter was also used to evaluate the mitochondrial membrane potential, the levels of ROS (H2O2; O2•-) and the antioxidant defense, Reduced Glutathione (GSH), using fluorescent probes. We found that DQA induced a decrease in cell viability inducing cell death by late apoptosis/necrosis in a time, dose and cell type dependent manner, with and IC50 of 2.5, 4.7 and 7.5 μM at 48h of exposure, respectively to HL-60, HUH-7 and EHEB. These effects may be mediated by oxidative stress as we have observed and increase in ROS production and a decrease in GSH levels and in mitochondrial membrane potential. We also observed that if DQA is administered on a daily basis a much lower concentration is required to induce the same effect. On the other hand, the association of DQA with the conventional drug induces a synergistic effect, because lower concentration of both drugs is required to obtain the some effect.

Cystic Fibrosis Newborn Screening in Portugal: PAP Value in Populations with Stringent Rules for Genetic Studies

Newborn screening (NBS) for cystic fibrosis (CF) has been shown to be advantageous for children with CF, and has thus been included in most NBS programs using various algorithms. With this study, we intend to establish the most appropriate algorithm for CF-NBS in the Portuguese population, to determine the incidence, and to contribute to elucidating the genetic epidemiology of CF in Portugal. This was a nationwide three-year pilot study including 255,000 newborns (NB) that were also screened for congenital hypothyroidism (CH) and 24 other metabolic disorders included in the Portuguese screening program. Most samples were collected in local health centers spread all over the country, between the 3rd and 6th days of life. The algorithm tested includes immunoreactive trypsinogen (IRT) determination, pancreatitis associated protein (PAP) as a second tier, and genetic study for cases referred to specialized clinical centers. Thirty-four CF cases were confirmed positive, thus indicating an incidence of 1:7500 NB. The p.F508del mutation was found in 79% of the alleles. According to the results presented here, CF-NBS is recommended to be included in the Portuguese NBS panel with a small adjustment regarding the PAP cut-off, which we expect to contribute to the improvement of the CF-NBS performance. According to our results, this algorithm is a valuable alternative for CF-NBS in populations with stringent rules for genetic studies.

P25 - Hypersensitivity/adverse reactions to antituberculosis drugs – a case report

Case report A ten years old boy was admitted for pleural tuberculosis (negative cultures, positive quantiferon assay, and father under treatment for tuberculosis). On the tenth day of treatment with isoniazid, rifampicin and pyrazinamide he develloped an exuberant urticarial rash, facial oedema, fever, myalgias, oliguria and, later, conjunctival hyperemia. Laboratory results included low platelet count, hypoalbuminemia and hyponatremia. Gradual improvement occured after suspension of treatment. One month later, drugs were gradualy re-introduced (1 drug/week) but several reactions occurred: anaphylaxis 5 hours after rifampicin administration and generalized macular rash after pyrazinamide. Both drugs were suspended. Isoniazid caused an initial light generalized macular exanthema. Streptomycin, ethambutol and ciprofloxacin were introduced and well tolerated. In subsequent evaluation the child was asymptomatic, with normal analytic results and radiologic improvement. After 6 months of successful treatment, he was tested in detail for reactions to the implicated drugs.

Recombinant trail: a synergistic effect in myeloid leukemias.

The tumor necrosis factor (TNF)-related apoptosis inducing ligand (TRAIL/Apo-2L) is a member of the TNF superfamily that trigger and activate 2 death receptors, DR4 and DR5, and 2 decoy receptors, DcR1 and DcR2. Several studies demonstrated that TRAIL in monotherapy can induces cancer cell death cells, but few have been done in leukemias in combination with conventional drugs. The aim of this work is to analyse the potential synergist effect of a recombinant TRAIL (rhTRAIL) in myeloid leukemias. For this, 2 myeloid leukemia cell lines, HL-60 (promyelocitic leukemia) and K-562 (Chronic Myeloid Leukemia) were treated with different concentrations of rhTRAIL as single agent and in combination with ATRA (all-transretinoic acid) and imatinib, respectively. The viability was measure using the trypan blue test and cell death by flow cytometry (FC) and Optical Microscopy. TRAIL and TRAIL-Rs were evaluated by FC. Our results show that rhTRAIL induced a decrease in cell viability inducing cell death, in a time, dose and cell type dependent manner. We observe an IC50 in HL-60 treated for 48h of 250 ng/mL, although in K562 cells, rhTRAIL wasn’t able to induce a significant effect. However, when we previously treated the cells with ATRA or IMATINIB a synergistic effect is observed, mainly in HL60 cells. These results may be correlated with the differential TRAIL receptors expression, namely the presence of the anti-apoptotic TRAIL receptors, in K562 cells. On the other hand, the higher percentage of pro-apoptotic TRAIL receptors may be related with the therapeutic efficacy of rhTRAIL in HL-60 cells. Our study suggests that rhTRAIL can be use as a new therapeutic aproach in APL, as single agent. However, it can potentiate the cytotoxic effect of conventional drugs.