Alterations in the small intestinal wall and motor function after repeated cisplatin in rat.

BACKGROUND: Gastrointestinal adverse effects occurring during cancer chemotherapy are well known and feared; those persisting once treatment has finished are relatively unknown. We characterized the alterations occurring in the rat small intestine, after repeated treatment with cisplatin. METHODS: Male Wistar rats received saline or cisplatin (2 mg kg-1  week-1 , for 5 weeks, ip). Gastric motor function was studied non-invasively throughout treatment (W1-W5) and 1 week after treatment finalization (W6). During W6, upper gastrointestinal motility was also invasively studied and small intestinal samples were collected for histopathological and molecular studies. Structural alterations in the small intestinal wall, mucosa, submucosa, muscle layers, and lymphocytic nodules were histologically studied. Periodic acid-Schiff staining and immunohistochemistry for Ki-67, chromogranin A, and neuronal-specific enolase were used to detect secretory, proliferating, endocrine and neural cells, respectively. The expression of different markers in the tunica muscularis was analyzed by RT/qPCR. KEY RESULTS: Repeated cisplatin induced motility alterations during and after treatment. After treatment (W6), the small intestinal wall showed histopathological alterations in most parameters measured, including a reduction in the thickness of circular and longitudinal muscle layers. Expression of c-KIT (for interstitial cells of Cajal), nNOS (for inhibitory motor neurons), pChAT, and cChAT (for excitatory motor neurons) increased significantly (although both ChATs to a lesser extent). CONCLUSIONS & INFERENCES: Repeated cisplatin induces relatively long-lasting gut dysmotility in rat associated with important histopathological and molecular alterations in the small intestinal wall. In cancer survivors, the possible chemotherapy-induced histopathological, molecular, and functional intestinal sequelae should be evaluated.

Recommendations for the use of in vitro methods to detect specific immunoglobulin E: are they comparable?

Total and specific immunoglobulin (Ig) E can be detected in vitro using several commercially available methods. The largest share of the global market for these methods is held by the ImmunoCAP technique (Thermo Fisher, previously Phadia), Immulite (Siemens), and Hytec-288 (Hycor). Most comparative studies examine Immulite and ImmunoCAP, which differ methodologically but use similar units of measurement relative to the same standard of total IgE (WHO IgE Standard 75/502). Despite their similarity, these kits differ in their quantification of specific IgE, which varies depending on the allergen studied.Thus, specific IgE results obtained with ImmunoCAP and Immulite are not interchangeable. It is important to bear this in mind, especially when determining cutoff points as predictors of a response to oral challenge with specific food allergens. The method used in practice must be the same as the one in the publication guiding clinical decision making. We analyze differences between ImmunoCAP and ISAC microarray, 2 methods from the same manufacturer used to detect IgE to specific proteins (purified or recombinant).The results show that the IgE values obtained with ImmunoCAP are not equivalent to the corresponding values obtained with the ISAC microarray system.

Effect of PI3K- and mTOR-specific inhibitors on spontaneous B-cell follicular lymphomas in PTEN/LKB1-deficient mice.

BACKGROUND: The PI3K-mTOR (phosphoinositide 3-kinase-mammalian target of rapamycin kinase) pathway is activated in the majority of tumours, and there is interest in assessing whether inhibitors of PI3K or mTOR kinase have efficacy in treating cancer. Here, we define the effectiveness of specific mTOR (AZD8055) and PI3K (GDC-0941) inhibitors, currently in clinical trials, in treating spontaneous B-cell follicular lymphoma that develops in PTEN(+/-)LKB1(+/hypo) mice. METHODS: The PTEN(+/-)LKB1(+/hypo) mice were administered AZD8055 or GDC-0941, and the volumes of B-cell follicular lymphoma were measured by MRI. Tumour samples were analysed by immunohistochemistry, immunoblot and flow cytometry. RESULTS: The AZD8055 or GDC-0941 induced ∼40% reduction in tumour volume within 2 weeks, accompanied by ablation of phosphorylation of AKT, S6K and SGK (serum and glucocorticoid protein kinase) protein kinases. The drugs reduced tumour cell proliferation, promoted apoptosis and suppressed centroblast population. The AZD8055 or GDC-0941 treatment beyond 3 weeks caused a moderate additional decrease in tumour volume, reaching ∼50% of the initial volume after 6 weeks of treatment. Tumours grew back at an increased rate and displayed similar high grade and diffuse morphology as the control untreated tumours upon cessation of drug treatment. CONCLUSION: These results define the effects that newly designed and specific mTOR and PI3K inhibitors have on a spontaneous tumour model, which may be more representative than xenograft models frequently employed to assess effectiveness of kinase inhibitors. Our data suggest that mTOR and PI3K inhibitors would benefit treatment of cancers in which the PI3K pathway is inappropriately activated; however, when administered alone, may not cause complete regression of such tumours.

Esplenomegalia familiar como manifestación clínica del síndrome linfoprolifetativo autoinmune / Familial splenomegaly as a first clinical sign of autoimmune lymphoproliferative syndrome

Introducción: El síndrome linfoproliferativo autoinmune es la consecuencia de un defecto genético que compromete la apoptosis de los linfocitos. La linfoproliferación se manifiesta por adenomegalias y/o esplenomegalia crónica. El diagnóstico requiere demostrar el defecto de la apoptosis linfocitaria y el aumento de linfocitos T dobles negativos (TDN) que carecen de CD4 y CD8. Existe riesgo de desarrollar linfomas y enfermedades autoinmunes, sobre todo citopenias. Métodos: Estudiamos a un niño de 14 años con esplenomegalia de varios años de evolución con antecedentes familiares de esplenomegalia y adenomegalias. Se analiza el fenotipo de los linfocitos T y el defecto molecular del gen TNFRSF6 en el niño, su hermana y el padre. Se estudia el defecto de la apoptosis en el niño y su padre. Resultados: En el niño y su padre se confirman el defecto de la apoptosis de los linfocitos, el aumento de linfocitos dobles negativos (el 18 y 5%, respectivamente) y la misma mutación del gen TNFRSF6. La hermana presenta la misma mutación con un 16% de linfocitos doblemente negativos. Comentarios: La esplenomegalia crónica familiar puede ser la única manifestación del síndrome linfoproliferativo autoinmune (AU)

Background: The autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defect in lymphocyte apoptosis. Chronic lymphadenopathy and splenomegaly are the consequence of lymphoproliferation. The diagnosis is based on the assessment of the defective lymphocyte apoptosis and the identification of lymphocyte T subset that are double negative (CD4-CD8-). The susceptibility to lymphoma and autoimmune diseases, mainly blood cytopenias is increased. Methods: We studied a 14 year-old boy with chronic splenomegaly and familial history of splenomegaly and lymphadenopathy. T lymphocyte phenotypes, and molecular defect of TNFRSF6 gene were studied in the child, his sister and his father. Lymphocyte apoptosis was also analysed in the child and his father. Results: The boy and his father showed in vitro apoptosis defects, an increased number of double negative T lymphocytes (18% and 5%, respectively) and the same mutation in the TNFRSF6 gene. His sister had 16% of double negative T lymphocytes and the mutation in the TNFRSF6 gene. Comments: Chronic familial splenomegaly can be the only clinical sign of autoimmune lymphoproliferative syndrome (AU)

[Familial splenomegaly as a first clinical sign of autoimmune lymphoproliferative syndrome]. / Esplenomegalia familiar como manifestación clínica del síndrome linfoprolifetativo autoinmune.

BACKGROUND: The autoimmune lymphoproliferative syndrome (ALPS) is caused by genetic defect in lymphocyte apoptosis. Chronic lymphadenopathy and splenomegaly are the consequence of lymphoproliferation. The diagnosis is based on the assessment of the defective lymphocyte apoptosis and the identification of lymphocyte T subset that are double negative (CD4-CD8-). The susceptibility to lymphoma and autoimmune diseases, mainly blood cytopenias is increased. METHODS: We studied a 14 year-old boy with chronic splenomegaly and familial history of splenomegaly and lymphadenopathy. T lymphocyte phenotypes, and molecular defect of TNFRSF6 gene were studied in the child, his sister and his father. Lymphocyte apoptosis was also analysed in the child and his father. RESULTS: The boy and his father showed in vitro apoptosis defects, an increased number of double negative T lymphocytes (18% and 5%, respectively) and the same mutation in the TNFRSF6 gene. His sister had 16% of double negative T lymphocytes and the mutation in the TNFRSF6 gene. COMMENTS: Chronic familial splenomegaly can be the only clinical sign of autoimmune lymphoproliferative syndrome.

PH domain leucine-rich repeat protein phosphatase 1 contributes to maintain the activation of the PI3K/Akt pro-survival pathway in Huntington's disease striatum.

Dysregulation of gene expression is one of the mechanisms involved in the pathophysiology of Huntington's disease (HD). Here, we examined whether mutant huntingtin regulates the levels of PH domain leucine-rich repeat protein phosphatase 1 (PHLPP1), a phosphatase that specifically dephosphorylates Akt at Ser473. Our results show decreased PHLPP1 protein levels in knock-in models (Hdh(Q111/Q111) mouse striatum and STHdh(Q111/Q111) cells), in the striatum of N-terminal exon-1 mutant huntingtin transgenic mouse models (R6/1; R6/1 : BDNF + or - , R6/2 and Tet/HD94) and in the putamen of HD patients. Quantitative PCR analysis revealed a reduction in PHLPP1 mRNA levels in the striatum of R6/1 compared with wild-type mice. Coincident with reduced PHLPP1 protein levels, we observed increased phosphorylated Akt (Ser473) levels specifically in the striatum. The analysis of the conditional mouse model Tet/HD94 disclosed that after mutant huntingtin shutdown PHLPP1 levels returned to wild-type levels whereas phospho-Akt levels were partially reduced. In conclusion, our results show that mutant huntingtin downregulates PHLPP1 expression. In the striatum, these reduced levels of PHLPP1 can contribute to maintain high levels of activated Akt that may delay cell death and allow the recovery of neuronal viability after mutant huntingtin silencing.

WNT/beta-catenin increases the production of incretins by entero-endocrine cells.

AIMS/HYPOTHESIS: Glucose-dependent insulinotropic peptide (GIP) plays a pivotal role in the regulation of glucose homeostasis. Rates of diet-induced obesity, insulin resistance and type 2 diabetes are decreased when GIP signalling is disturbed in mice, suggesting that GIP plays a role in the onset of type 2 diabetes. WNT signalling is linked to type 2 diabetes and induces synthesis of the other incretin, glucagon-like peptide 1 (GLP-1). GLP-1 analogues improve treatment of type 2 diabetes patients in whom GLP-1 signalling is intact and have captured clinical attention. GIP levels are altered at the onset of type 2 diabetes and later on, while GIP signalling is impaired. Thus, GIP is not a candidate for treatment but might be an important target from a prevention perspective. Hypothesising that hypersecretion of GIP links altered WNT signalling to the onset of type 2 diabetes, we sought to determine whether WNT signalling induces GIP production by entero-endocrine cells. METHODS: RT-PCR and chromatin immunoprecipitation (ChIP) were used to study Gip gene induction. Gip promoter elements mediating WNT/lithium induction were identified (electrophoretic mobility shift assay, co-transfection of deletion mutants, ChIP). RESULTS: Lithium or WNT/beta-catenin signalling enhanced GIP production by entero-endocrine cells through a conserved site in the proximal Gip promoter. Lithium favours lymphoid enhancer factor-1/beta-catenin binding to Gip promoter and diminishes ChIP through T cell factor-4 and histone deacetylase 1. CONCLUSIONS/INTERPRETATION: Lithium and WNT are incretin inducers in general. This work provides a novel link between WNT signalling, obesity and diabetes.

Bax deficiency promotes an up-regulation of Bim(EL) and Bak during striatal and cortical postnatal development, and after excitotoxic injury.

In this study we analyzed whether other members of the Bcl-2 family are regulated in the absence of Bax during the postnatal development of the striatum and cortex and after striatal excitotoxic lesion. Compared with wild-type animals, Bax knockout mice showed region- and time-dependent increases in pro-apoptotic proteins Bak and Bim(EL). Excitotoxicity induced in the adult striatum increased Bim(EL) in both genotypes whereas Bak and Bcl-x(L) were only increased in Bax knockout mice. However, translocation of Bim(EL) protein to the mitochondrial fraction, cytochrome c release and caspase-3 activation were only observed in wild-type striata. Furthermore, analysis of Bim null mutant mice showed that this protein is not essential to excitotoxicity-induced striatal cell death. In conclusion, our results show that in Bax deficient mice Bim(EL) and Bak are specifically regulated during postnatal development, suggesting that these proteins may participate in the compensatory mechanisms triggered in the absence of Bax. In contrast, Bax is required to induce apoptosis after excitotoxicity in the adult striatum.

Mice heterozygous for neurotrophin-3 display enhanced vulnerability to excitotoxicity in the striatum through increased expression of N-methyl-D-aspartate receptors.

The striatum is one of the brain areas most vulnerable to excitotoxicity, a lesion that can be prevented by neurotrophins. In the present study, intrastriatal injection of the N-methyl-d-aspartate receptor (NMDAR) agonist quinolinate (QUIN) was performed in mice heterozygous for neurotrophin-3 (NT3 +/-) or brain-derived neurotrophic factor (BDNF +/-) to analyze the role of endogenous neurotrophins on the regulation of striatal neurons susceptibility to excitotoxic injury. QUIN injection induced a decrease in dopamine- and cyclic AMP-regulated phosphoprotein of 32 kDa (DARPP-32) protein levels that was higher in NT-3 +/- than in BDNF+/- or wild type animals. This enhanced susceptibility was specific for enkephalin- and tachykinin-positive projection neurons, and also for parvalbumin-positive interneurons. However the excitotoxic damage in large interneurons was not modified in NT-3 +/- mice compared with wild type animals. This effect can be related to the regulation of NMDARs by endogenous NT-3. Thus, our results show that there is an age-dependent regulation of NMDAR subunits NR1 and NR2A, but not NR2B, in NT-3 +/- mice. The deficit of endogenous NT-3 induced a decrease in NR1 and NR2A subunits at postnatal day (P) 0 and P3 mice respectively, whereas an upregulation was observed in 12 week old NT-3 +/- mice. This differential effect was also observed after administration of exogenous NT-3. In primary striatal cultures, NT-3 treatment induced an enhancement in NR2A, but not NR2B, protein levels. However, intrastriatal grafting of NT-3 secreting-cells in adult wild type mice produced a down-regulation of NR2A subunit. In conclusion, NT-3 regulates the expression of NMDAR subunits modifying striatal neuronal properties that confers the differential vulnerability to excitotoxicity in projection neurons and interneurons in the striatum.

[Round Table: Severe asthma in pediatrics: treatment of acute crises]. / Mesa Redonda: Asma grave en Pediatría: Tratamiento de las crisis agudas.

Morbidity and mortality derived from asthma continue to be a main public health problem in many countries, in spite of the advances in the knowledge on the disease and its treatment. There are several risk factors for asthma attack which have to be considered in the management of patients in order to prevent exacerbations and mortality. Smooth bronchial muscle constriction and inflammation with oedema of the bronchial wall are the facts that cause airway flow and resistance disturbances, with hyperinflation, leading to a bigger respiratory work. On the other hand, the bronchial obstruction leads to a ventilation-perfusion disequilibrium and hypoxia. At the beginning of the process there is hypocarbia, but when the attack progresses muscle fatigue happens, and retention of CO2, being a sing of alarm (predictive of respiratory failure) a normal and rising PaCO2. The evaluation of an acute asthmatic patient should accomplish a clinical and objective assessment (peak flow rate and saturation of O2), in order to classify the crisis in: mild, moderate or severe. Managing acute asthmatic patient includes: oxygen, bronchodilator ss2 agonists at high and even continuous doses and systemic corticosteroids to prevent the progression and to control inflammation. These procedures should be promptly instituted. Although there is less evidence on their beneficial effects other measures as intravenous aminophylline, nebulized anticholynergics, magnesium sulphate and intravenous ss2 agonists may be used when the conventional therapy is not quickly successful and the patient is in a critical situation, at a real risk of respiratory failure, and in order to avoid mechanical ventilation. If this is finally instituted, controlled hypoventilation with permissive hypercarbia is now recommended, to avoid barotrauma, which used to be a frequent complication when more aggressive attitude was the rule. Interaction between paralytic agents and corticosteroids may produce a miopathy, so the recommendation now is to try not to use paralytic agents, even with profound sedation of needed. Sixty four patients were treated on 77 occasions in the Pediatric Intensive Care Unit of our hospital. They were 0,5 to 13,9 years old, being 50% less than 5 years old. It was the first attack in 9 (14%) patients. The standard management consisted of oxygen, frequently or continuously nebulized salbutamol and intravenous methylprednisolone (1 to 6 mg/kg/day). Furthermore nebulized ipratropium bromide was administered 58 times (75%), as well as intravenous aminophylline 69 (89%), intravenous salbutamol 23 (30%), magnesium sulphate 16 (21%) and ketamine 10 (13%). Antibiotics were given 22 times (29%). Two 15 month old infants received mechanical ventilation in three occasions, and relevant complications happened (pneumothorax and myopathy, and pneumomediastinum and bronchiolitis obliterans respectively). Fifty six patients have been followed for a period of 3 to 110 months (median 48 months), and 16 (29%) have needed high doses (equal to or move than 800 mcg of budesonide or equivalent). There are data on lung function in 36 of them, FEV1 is normal (> 85% of predicted, between 86 and 127) in 26 (78%) and < 85% (65 to 84%) of predicted in 8 (22%) FEV1 rises more than 15% (16 to 23%) in four patients after the inhalation of a ss2 agonist. Inhaled anesthetic agents and heliox have been used in some pediatric cases. After a severe asthma attack the strategy of management should be reviewed, as well as the possible risk factors.

[Latex allergy in children with spina bifida]. / Alergia al látex en niños con espina bífida.

Latex allergy is a frequent happening in children with spina bifida or congenital urologic abnormalities. These patients have multiples exposures to derivatives of latex as part of the management increasing the risk of allergic reactions. We present three children who developed general anaphylactic reaction during urodynamic exploration with a rectal latex catheter. All patients showed positive skin test and positive latex specific IgE test (RAST). A prospective survey of 17 patients with spina bifida at the time of cystometry showed positive RAST levels in 7 (41%). Recommendations for prophylaxis are included, suggesting the avoidance of latex products in children with spina bifida since birth.