Acute and persistent nociceptive paw sensitisation in mice: The involvement of distinct signalling pathways

Aims: Many fundamental pharmacological studies in pain and inflammation have been performed on rats. However, the pharmacological findings were generally not extended to other species in order to increase their predictive therapeutic value. We studied acute and chronic inflammatory nociceptive sensitisation of mouse hind paws by prostaglandin E(2) (PGE(2)) or dopamine (DA), as previously described in rats. We also investigated the participation of the signalling pathways in acute and persistent sensitisation. Main methods: Mechanical sensitisation (hypernociception) induced by intraplantar administrations of PGE(2) or DA was evaluated with an electronic pressure meter. The signalling pathways were pharmacologically investigated with the pre-administration of adenylyl cyclase (AC), cAMP-dependent protein kinase (PKA), protein kinase C epsilon (PKC epsilon), and the extracellular signal-related kinase (ERK) inhibitors. Key findings: Single or 14 days of successive intraplantar injections of PGE(2) or DA-induced acute and persistent hypernociception (lasting for more than 30 days), respectively. The involvement of AC, PKA or PKC epsilon was observed in the acute hypernociception induced by PGE(2), while PKA or PKC epsilon were continuously activated during the period of persistent hypernociception. The acute hypernociception induced by DA involves activation of ERK, PKC epsilon, AC or PKA, while persistent hypernociception implicated ERK activation, but not PKA, PKC epsilon or AC. Significance: In mice, acute and persistent paw sensitisation involves the different activation of kinases, as previously described for rats. This study opens the possibility of comparing pharmacological approaches in both species to further understand acute and chronic inflammatory sensitisation, and possibly associated genetic manipulations. (C) 2009 Elsevier Inc. All rights reserved.

Upregulation of intermediate calcium-activated potassium channels counterbalance the impaired endothelium-dependent vasodilation in stroke-prone spontaneously hypertensive rats

Endothelial dysfunction has been linked to a decrease in nitric oxide (NO) bioavailability and attenuated endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxation. The small (SK(Ca)) and intermediate (IK(Ca)) calcium-activated potassium channels play a key role in endothelium-dependent relaxation. Because the repressor element 1-silencing transcription factor (REST) negatively regulates IK(Ca) expression, we hypothesized that augmented REST and decreased IK(Ca) expression contributes to impaired endothelium-dependent vasodilation associated with hypertension. Acetylcholine (ACh) responses were slightly decreased in small mesenteric arteries from male stroke-prone spontaneously hypertensive rats (SHRSPs) versus arteries from Wistar Kyoto (WKY) rats. Incubation with N-nitro-L-arginine methyl ester (L-NAME; 100 mu mol/L) and indomethacin (100 mu mol/L) greatly impaired ACh responses in vessels from SHRSP. lberiotoxin (0.1 mu mol/L), which is a selective inhibitor of large-conductance K(Ca) (BK(Ca)) channels, did not modify EDHF-mediated vasodilation in SHRSP or WKY. UCL-1684 (0.1 mu mol/L.), which is a selective inhibitor of SKCa channels, almost abolished EDHF-mediated vasodilation in WKY and decreased relaxation in SHRSP. 1-((2-chlorophenyl)diphenylmethyl)-1H-pyrazole (TRAM-34; 10 mu mol/L) and charybdotoxin (0.1 mu mol/L), which are both IKCa inhibitors, produced a small decrease of EDHF relaxation in WKY but completely abrogated EDHF vasodilation in SHRSP. EDHF-mediated relaxant responses were completely abolished in both groups by simultaneous treatment with UCL-1684 and TRAM-34 or charybdotoxin. Relaxation to SK(Ca)/IK(Ca) channels agonist NS-309 was decreased in SHRSP arteries. The expression of SK(Ca) was decreased, whereas IK(Ca) was increased in SHRSP mesenteric arteries. REST expression was reduced in arteries from SHRSP. Vessels incubated with TRAM-34 (10 mu mol/L) for 24h displayed reduced REST expression and demonstrated no differences in IK(Ca). In conclusion, IK(Ca) channel upregulation, via decreased REST, seems to compensate deficient activity of SK(Ca) channels in the vasculature of spontaneously hypertensive rats. (Translational Research 2009; 154:183-193)

Role of the superior colliculus in the expression of acute and kindled audiogenic seizures in Wistar audiogenic rats

P>Purpose: The role of the superior colliculus (SC) in seizure expression is controversial and appears to be dependent upon the epilepsy model. This study shows the effect of disconnection between SC deep layers and adjacent tissues in the expression of acute and kindling seizures. Methods: Subcollicular transections, ablation of SC superficial and deep layers, and ablation of only the cerebral cortex were evaluated in the Wistar audiogenic rat (WAR) strain during acute and kindled audiogenic seizures. The audiogenic seizure kindling protocol started 4 days after surgeries, with two acoustic stimuli per day for 10 days. Acute audiogenic seizures were evaluated by a categorized seizure severity midbrain index (cSI) and kindled seizures by a severity limbic index (LI). Results: All subcollicular transections reaching the deep layers of the SC abolished audiogenic seizures or significantly decreased cSI. In the unlesioned kindled group, a reciprocal relationship between limbic and brainstem pattern of seizures was seen. The increased number of stimuli provoked an audiogenic kindling phenomenon. Ablation of the entire SC (ablation group) or of the cerebral cortex only (ctx-operated group) hampered the acquisition of limbic behaviors. There was no difference in cSI and LI between the ctx-operated and ablation groups, but there was a difference between ctx-operated and the unlesioned kindled group. There was also no difference in cSI between SC deep layer transection and ablation groups. Results of histologic analyses were similar for acute and kindled audiogenic seizure groups. Conclusions: SC deep layers are involved in the expression of acute and kindled audiogenic seizure, and the cerebral cortex is essential for audiogenic kindling development.

Sexual differentiation of cortical spreading depression propagation after acute and kindled audiogenic seizures in the Wistar audiogenic rat (WAR)

Brain excitability diseases like epilepsy constitute one factor that influences brain electrophysiological features. Cortical spreading depression (CSD) is a phenomenon that can be altered by changes in brain excitability. CSD propagation was presently characterized in adult mate and female rats from a normal Wistar strain and from a genetically audiogenic seizure-prone strain, the Wistar audiogenic rat (WAR), both previously submitted (RAS(+)), or not (RAS(-)), to repetitive acoustic stimulation, to provoke audiogenic kindling in the WAR-strain. A gender-specific change in CSD-propagation was found. Compared to seizure-resistant animals, in the RAS- condition, mate and female WARs, respectively, presented CSD-propagation impairment and facilitation, characterized, respectively, by lower and higher propagation velocities (P<0.05). In contraposition, in the RAS(+) condition, mate and female WARs displayed, respectively, higher and tower CSD-propagation rates, as compared to the corresponding controls. In some Wistar and WAR females, we determined estrous cycle status on the day of the CSD-recording as being either estrous or diestrous; no cycle-phase-related differences in CSD-propagation velocities were detected. In contrast to other epilepsy models, such as Status Epilepticus induced by pilocarpine, despite the CSD-velocity reduction, in no case was CSD propagation blocked in WARs. The results suggest a gender-related, estrous cycle-phase-independent modification in the CSD-susceptibility of WAR rats, both in the RAS(+) and RAS(-) situation. (C) 2008 Elsevier B.V. All rights reserved.

CHANGES IN FETAL AND MATERNAL DOPPLER PARAMETERS OBSERVED DURING ACUTE SEVERE HYPERTENSION TREATMENT WITH HYDRALAZINE OR LABETALOL: A RANDOMIZED CONTROLLED TRIAL

We evaluated 16 pregnant women with gestational age between 20 and 32 weeks in acute severe hypertension which were randomly allocated to receive either hydralazine or labetalol. Blood pressure and Doppler ultrasound parameters from maternal uterine and fetal middle cerebral and umbilical arteries were assessed during acute severe hypertension and after treatment. A significant reduction in systolic and diastolic blood pressure was observed in both groups. A significant change in Doppler parameters was observed only in pregnant women who received hydralazine: an increase in uterine arteries resistance index. We concluded that both drugs were highly effective in reducing blood pressure in these women. Despite the observed increase in resistance index of uterine arteries associated with hydralazine, the use of hydralazine and labetalol were not related to any significant changes in fetal Doppler, which is reassuring about the safety of these drugs when treating acute severe hypertension in pregnancy. (E-mail: wpmartins@gmail.com) (C) 2011 World Federation for Ultrasound in Medicine & Biology.

Validation of the National Institutes of Health Stroke Scale, Modified Rankin Scale and Barthel Index in Brazil: The Role of Cultural Adaptation and Structured Interviewing

Background: We aimed to validate three widely used scales in stroke research in a multiethnic Brazilian population. Methods: The National Institutes of Health Stroke Scale (NIHSS), modified Rankin Scale (mRS) and Barthel Index (BI) were translated, culturally adapted and applied by two independent investigators. The mRS was applied with or without a previously validated structured interview. Interobserver agreement (kappa statistics) and intraclass correlation coefficients were calculated. Results: 84 patients underwent mRS (56 with and 28 without a structured interview), 57 BI and 62 NIHSS scoring. Intraclass correlation coefficient was 0.902 for NIHSS and 0.967 for BI. For BI, interobserver agreement was good (kappa = 0.70). For mRS, the structured interview improved interobserver agreement (kappa = 0.34 without a structured interview; 0.75 with a structured interview). Conclusion: The NIHSS, BI and mRS show good validity when translated and culturally adapted. Using a structured interview for the mRS improves interobserver concordance rates. Copyright (C) 2008 S. Karger AG, Basel

Detection of Clonal Immunoglobulin and T-Cell Receptor Gene Rearrangements in Childhood Acute Lymphoblastic Leukemia Using a Low-Cost PCR Strategy

Background Imunoglobulin (Ig) and T cell receptor (TCR) gene rearrangements function as specific markers for minimal residual disease (MRD) which is one of the best predictors of outcome in childhood acute lymphoblastic leukemia (ALL) We recently reported on the prognostic value of MRD during the induction of remission through a simplified PCR method Here we report on gene rearrangement frequencies and offer guidelines for the application of the technique Procedure Two hundred thirty three children had DNA extracted from bone marrow Ig and TCR gene rearrangements were amplified using consensus primers and conventional PCR PCR products were submitted to homo/heteroduplex analysis A computer program was designed to define combinations of targets for clonal detection using a minimum set of primers and reactions Results At least one clonal marker could be detected in 98% of the patients and two markers in approximately 80% The most commonly rear ringed genes in precursor B cell ALL were IgH (75%) TCRD (59%) IgK (55%), and TCRG (54%) The most commonly rearranged genes for TALL were TCRG (100%) and TCRD (24%) The sensitivity of primers was limited to the detection of 1 leukemic cell among 100 normal cells Conclusions We propose that eight PCR reactions per ALL subtype would allow for the detection of two markers in most cases In addition these reactions ire suitable for MRD monitoring especially when aiming the selection of patients with high MRD levels (>= 10(-2)) at the end of induction therapy Such an approach would be very useful in centers with limited financial resources Pediatr Blood Cancer 2010 55 1278-1286 (C) 2010 Wiley Liss Inc

Vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (NOS3) polymorphisms are associated with high relapse risk in childhood acute lymphoblastic leukemia (ALL)

Background: Angiogenesis has been shown as an important process in hematological malignancies. It consists in endothelial proliferation, migration, and tube formation following pro-angiogenic factors releasing, specially the vascular endothelial growth factor (VEGF), which angiogenic effect seems to be dependent on nitric oxide (NO). We examined the association among functional polymorphism in these two angiogenesis related genes: VEGF (-2578C>A, -1154G>A, and -634G>C) and NOS3 (-786T>C, intron 4 b>a, and Glu298Asp) with prognosis of childhood acute lymphoblastic leukemia (ALL). Methods: The genotypes were determined and haplotypes estimated in 105 ALL patients that were divided in 2 groups: high risk (HR) and low risk of relapse (LR) patients. In addition, event-free survival curves according to genotypes were assessed. Results: The group HR compared to the LR showed a higher frequency of the alleles -2578C and -634C and the haplotype CGC for VEGF (0.72 vs. 0.51, p<0.008; 0.47 vs. 0.26, p<0.008; and 42.1 vs. 14.5, p<0.006; respectively) and a lower frequency of the haplotype CbGlu (0.4 vs. 8.8, p<0.006), for NOS3. Conclusion: Polymorphisms of VEGF and NOS3 genes are associated with high risk of relapse, therefore may have a prognostic impact in childhood ALL. (C) 2010 Elsevier B.V. All rights reserved.

Role of the CYP2D6, EPHX1, MPO, and NQO1 Genes in the Susceptibility to Acute Lymphoblastic Leukemia in Brazilian Children

Polymorphic variations of several genes associated with dietary effects and exposure to environmental carcinogens may influence susceptibility to leukemia development. The objective of the present study was to evaluate the effect of the polymorphisms of debrisoquine hydroxylase (CYP2D6), epoxide hydrolase (EPHX1), myeloperoxidase (MPO), and quinone-oxoreductase (NQO1), which have been implicated in xenobiotic metabolism, on the risk of childhood acute lymphoblastic leukemia (ALL). We evaluated the frequency of polymorphisms in the CYP2D6 (*3 and *4), EPHX1 (*2 and *3), MPO (*2), and NQO1 (*2) genes in 206 patients with childhood ALL and in 364 healthy individuals matched for age and gender from a Brazilian population separated by ethnicity (European ancestry and African ancestry), using the PCR-RFLP method. The CYP2D6 polymorphism variants were associated with an increased risk of ALL. The EPHX1, NQO1, and MPO variant genotypes were significantly associated with a reduced risk of childhood ALL. A significantly stronger protective effect is observed when the EPHX1, NQO1, and MPO variant genotypes are combined suggesting that, CYP2D6 polymorphisms may play a role in the susceptibility to pediatric ALL, whereas the EPHX1, NQO1, and MPO polymorphisms might have a protective function against leukemogenesis. Environ. Mal. Mulagen. 51:48-56, 2010. (C) 2009 Wiley-Liss, Inc.

mRNA Expression Profile of Multidrug Resistance Genes in Childhood Acute Lymphoblastic Leukemia. Low Expression Levels Associated With a Higher Risk of Toxic Death

Background. Increased activity of multidrug resistance (MDR) genes has been associated with treatment failure in acute leukemias, although with controversial reports. The objective of the present study was to assess the expression profile of the genes related to MDR: ABCB1, ABCC1, ABCC3, ABCC2, and LRP/MVP in terms of the clinical and biological variable and the survival of children with acute lymphoblastic leukemia (ALL). Procedure. The levels of mRNA expression of the drug resistance genes ABCB1, ABCC1, ABCC3, ABCG2, and LRP/MVP were analyzed by quantitative real-time PCR using the median Values as cut-off points, in consecutive samples from 140 children with ALL at diagnosis. Results. Expression levels of the ABCG2 gene in the patient group as a whole (P=0.05) and of the ABCG2 and ABCC1 genes in patients classified as being at high risk were associated with higher rates of 5-year event-free survival (EFS) (P=0.04 and P=0.01). Expression levels of the ABCG2 gene below the median were associated with a greater chance of death related to treatment toxicity for the patient group as a whole (P=0.009) and expression levels below the median of the ABCG2 and ABCC1 genes were associated with a greater chance of death due to treatment toxicity for the high-risk group (P=0.02 and P=0.03, respectively). Conclusion. The present data suggest a low participation of the drug efflux genes in treatment failure in patients with childhood ALL. However, the low expression of some of these genes may be associated with a higher death risk related to treatment toxicity. Pediatr Blood Cancer 2009;53:996-1004. (C) 2009 Wiley-Liss, Inc.

Polymorphisms of xenobiotic metabolizing enzymes and DNA repair genes and outcome in childhood acute lymphoblastic leukemia

The interindividual variation in the activity of xenobiotic metabolizing enzymes and DNA repair genes could modify an individual`s risk of recurrent malignancy and response to therapy. We investigated whether ALL outcome was related to polymorphisms in genes CYP2D6. MPO, EPHX1, NQO1, TS, XPD and XRCC1 in 95 consecutive ALL children by PCR or PCR-FRLP techniques. Polymorphisms in genes NQO1 and TS were associated with a significantly slow response to induction chemotherapy and NQO1 was also associated with a lower five-year event-free survival. This study suggests that polymorphisms of NQO1 and TS could be important for patient response to induction therapy and for treatment outcome. (C) 2009 Elsevier Ltd. All rights reserved.

Impact of thymidylate synthase promoter and DNA repair gene polymorphisms on susceptibility to childhood acute lymphoblastic leukemia

The aim of this study was to evaluate the frequency of polymorphisms in the TYMS, XRCC1, and ERCC2 DNA repair genes in pediatric patients with acute lymphoblastic leukemia using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism (RFLP) approaches. The study was conducted in 206 patients and 364 controls from a Brazilian population. No significant differences were observed among the analyzed groups regarding XRCC1 codon 399 and codon 194 and ERCC2 codon 751 and codon 312 polymorphisms. The TYMS 3R variant allele was significantly associated with a reduced risk of childhood ALL, represented by the sum of heterozygous and polymorphic homozygous genotypes (odds ratio 0.60; 95% confidence interval 0.37-0.99). The results suggest that polymorphism in TYMS may play a protective role against the development of childhood ALL.

Benefits of the Intermittent Use of 6-Mercaptopurine and Methotrexate in Maintenance Treatment for Low-Risk Acute Lymphoblastic Leukemia in Children: Randomized Trial From the Brazilian Childhood Cooperative Group Protocol ALL-99

Purpose To describe event-free survival (EFS) and toxicities in children with low-risk acute lymphoblastic leukemia (ALL) assigned to receive either continuous 6-mercaptopurine (6-MP) and weekly methotrexate (MIX) or intermittent 6-MP with intermediate-dose MTX, as maintenance treatment. Patients and Methods Between October 1, 2000, and December 31, 2007, 635 patients with low-risk ALL were enrolled onto Brazilian Childhood Cooperative Group for ALL Treatment (GBTLI) ALL-99 protocol. Eligible children (n=544) were randomly allocated to receive either continuous 6-ME/MIX (group 1, n 272) or intermittent 6-MP (100 mg/m(2)/d for 10 days, with 11 days resting) and MIX (200 mg/m(2) every 3 weeks; group 2, n = 272). Results The 5-year overall survival (OS) and EFS were 92.5% +/- 1.5% SE and 83.6% +/- 2.1% SE, respectively. According to maintenance regimen, the OS was 91.4% +/- 2.2% SE (group 1) and 93.6% +/- 2.1% SE (group 2; P=.28) and EFS 80.9% +/- 3.2% SE (group 1) and 86.5% +/- 2.8% SE (group 2; P=.089). Remarkably, the intermittent regimen led to significantly higher EFS among boys (85.7% v 74.9% SE; P=027), while no difference was seen for girls (87.0% v 88.8% SE; P=.78). Toxic episodes were recorded in 226 and 237 children, respectively. Grade 3 to 4 toxic events for groups 1 and 2 were, respectively, 273 and 166 for hepatic dysfunction (P=.002), and 772 and 636 for hematologic episodes (P=.005). Deaths on maintenance were: seven (group 1) and one (group 2). Conclusion The intermittent use of 6-MP and MIX in maintenance is a less toxic regimen, with a trend toward better long-term EFS. Boys treated with the intermittent schedule had significantly better EFS.