Inhibition of the renin-angiotensin system prevents seizures in a rat model of epilepsy

The RAS (renin angiotensin system) is classically involved in BP (blood pressure) regulation and water electrolyte balance, and in the central nervous system it has been mostly associated with homoeostatic processes, such as thirst, hormone secretion and thermoregulation. Epilepsies are chronic neurological disorders characterized by recurrent epileptic seizures that affect 1-3% of the world`s population, and the most commonly used anticonvulsants are described to be effective in approx. 70% of the population with this neurological alteration. Using a rat model of epilepsy, we found that components of the RAS, namely ACE (angiotensin-converting enzyme) and the AT(1) receptor (angiotensin II type I receptor) are up-regulated in the brain (2.6- and 8.2-fold respectively) following repetitive seizures. Subsequently, epileptic animals were treated with clinically used doses of enalapril, an ACE inhibitor, and losartan, an AT(1) receptor blocker, leading to a significant decrease in seizure severities. These results suggest that centrally acting drugs that target the RAS deserve further investigation as possible anticonvulsant agents and may represent an additional strategy in the management of epileptic patients.

Constitutional hypomorphic telomerase mutations in patients with acute myeloid leukemia

Loss-of-function mutations in telomerase complex genes can cause bone marrow failure, dyskeratosis congenita, and acquired aplastic anemia, both diseases that predispose to acute myeloid leukemia. Loss of telomerase function produces short telomeres, potentially resulting in chromosome recombination, end-to-end fusion, and recognition as damaged DNA. We investigated whether mutations in telomerase genes also occur in acute myeloid leukemia. We screened bone marrow samples from 133 consecutive patients with acute myeloid leukemia and 198 controls for variations in TERT and TERC genes. An additional 89 patients from a second cohort, selected based on cytogenetic status, and 528 controls were further examined for mutations. A third cohort of 372 patients and 384 controls were specifically tested for one TERT gene variant. In the first cohort, 11 patients carried missense TERT gene variants that were not present in controls (P<0.0001); in the second cohort, TERT mutations were associated with trisomy 8 and inversion 16. Mutation germ-line origin was demonstrated in 5 patients from whom other tissues were available. Analysis of all 3 cohorts (n = 594) for the most common gene variant (A1062T) indicated a prevalence 3 times higher in patients than in controls (n = 1,110; P = 0.0009). Introduction of TERT mutants into telomerase-deficient cells resulted in loss of enzymatic activity by haploinsufficiency. Inherited mutations in TERT that reduce telomerase activity are risk factors for acute myeloid leukemia. We propose that short and dysfunctional telomeres limit normal stem cell proliferation and predispose for leukemia by selection of stem cells with defective DNA damage responses that are prone to genome instability.

Constitutional Telomerase Mutations Are Genetic Risk Factors for Cirrhosis

Some patients with liver disease progress to cirrhosis, but the risk factors for cirrhosis development are unknown. Dyskeratosis congenita, an inherited bone marrow failure syndrome associated with mucocutaneous anomalies, pulmonary fibrosis, and cirrhosis, is caused by germline mutations of genes in the telomerase complex. We examined whether telomerase mutations also occurred in sporadic cirrhosis. In all, 134 patients with cirrhosis of common etiologies treated at the Liver Research Institute, University of Arizona, between May 2008 and July 2009, and 528 healthy subjects were screened for variation in the TERT and TERC genes by direct sequencing; an additional 1,472 controls were examined for the most common genetic variation observed in patients. Telomere length of leukocytes was measured by quantitative polymerase chain reaction. Functional effects of genetic changes were assessed by transfection of mutation-containing vectors into telomerase-deficient cell lines, and telomerase activity was measured in cell lysates. Nine of the 134 patients with cirrhosis (7%) carried a missense variant in TERT, resulting in a cumulative carrier frequency significantly higher than in controls (P = 0.0009). One patient was homozygous and eight were heterozygous. The allele frequency for the most common missense TERT variant was significantly higher in patients with cirrhosis (2.6%) than in 2,000 controls (0.7%; P = 0.0011). One additional patient carried a TERC mutation. The mean telomere length of leukocytes in patients with cirrhosis, including six mutant cases, was shorter than in age-matched controls (P = 0.0004). Conclusion: Most TERT gene variants reduced telomerase enzymatic activity in vitro. Loss-of-function telomerase gene variants associated with short telomeres are risk factors for sporadic cirrhosis. (HEPATOLOGY 2011;53:1600-1607)

Parathyroid hormone secretion in women in late menopause submitted to EDTA-induced hypocalcemia

Objective: The physiological role of parathormone (PTH) in the maintenance of bone mass in humans has not been fully defined. The main objective of the present study was to evaluate basal and EDTA-stimulated PTH levels in Young women (Group Y = 30.9 years, N = 7) and in women in late menopause (Group M = 64.7 years, N = 7) and their relationship to bone mineral density. Methods: The PTH secretion test was performed by induction of hypocalcemia through intravenous administration of EDTA for 2 h. Blood samples were collected every 10 min and used for ionic calcium and PTH measurements. During the basal period, an additional sample was collected for the determination of osteocalcin, FSH, and estradiol. A sample of early morning second voided urine was collected for analysis of deoxypiridinoline and creatinine Lis well as bone mass density (BMD) was determined by dual X-ray energy absorptiometry (DEXA). Results: The aged patients presented lower femoral BMD (Y = 0.860 g/cm(2) vs. M = 0.690 g/cm(2), P < 0.01), With four of them having a T score lower than - 2.5 S.D. Basal, and during the EDTA infusion, PTH values were similar in both groups. However, among aged volunteers, the rise in PTH levels was higher for subjects with normal bone mass (NM: peak = 236 pg/ml) than for subjects with osteoporosis (OM: peak = 134.4 pg/ml). Conclusions: The present results suggest that PTH can have a modulating effect on the rate of bone loss during late menopause. (c) 2007 Elsevier Ireland Ltd. All rights reserved.

Prostaglandin mediates IL-23/IL-17-induced neutrophil migration in inflammation by inhibiting IL-12 and IFN gamma production

IL-23/IL-17-induced neutrophil recruitment plays a pivotal role in rheumatoid arthritis (RA). However, the mechanism of the neutrophil recruitment is obscure. Here we report that prostaglandin enhances the IL-23/IL-17-induced neutrophil migration in a murine model of RA by inhibiting IL-12 and IFN gamma production. Methylated BSA (mBSA) and IL-23-induced neutrophil migration was inhibited by anti-IL-23 and anti-IL-17 antibodies, COX inhibitors, IL-12, or IFN gamma but was enhanced by prostaglandin E(2) (PGE(2)). IL-23-induced IL-17 production was increased by PGE(2) and suppressed by COX-inhibition or IL-12. Furthermore, COX inhibition failed to reduce IL-23-induced neutrophil migration in IL-12- or IFN gamma-deficient mice. IL-17-induced neutrophil migration was not affected by COX inhibitors, IL-12, or IFN gamma but was inhibited by MK886 (a leukotriene synthesis inhibitor), anti-TNF alpha, anti-CXCL1, and anti-CXCL5 antibodies and by repertaxin (a CXCR1/2 antagonist). These treatments all inhibited mBSA- or IL-23-induced neutrophil migration. IL-17 induced neutrophil chemotaxis through a CXC chemokines-dependent pathway. Our results suggest that prostaglandin plays an important role in IL-23-induced neutrophil migration in arthritis by enhancing IL-17 synthesis and by inhibiting IL-12 and IFN gamma production. We thus provide a mechanism for the pathogenic role of the IL-23/IL-17 axis in RA and also suggest an additional mechanism of action for nonsteroidal anti-inflammatory drugs.

Involvement of the prelimbic prefrontal cortex on cannabidiol-induced attenuation of contextual conditioned fear in rats

Rationale: Systemic administration of cannabidiol (CBD), a non-psychotomimetic component of Cannabis sativa, is able to attenuate cardiovascular and behavioral (freezing) changes induced by re-exposure to a context that had been previously paired with footshocks. The brain sites mediating this effect, however, remain unknown. The medial prefrontal cortex (mPFC) has been related to contextual fear conditioning. Objectives: (1) To verify, using c-Fos immunocytochemistry, if the mPFC is involved in the attenuation of contextual fear induced by systemic administration of CBD; (2) to investigate if direct microinjections of CBD into mPFC regions would also attenuate contextual fear. Results: Confirming previous results systemic administration of CBD (10 mg/kg) decreased contextual fear and associated c-Fos expression in the prefrontal cortex (prelimbic and infralimbic regions). The drug also attenuated c-Fos expression in the bed nucleus of the stria terminalis (BNST). Direct CBD (30 nmol) microinjection into the PL prefrontal cortex reduced freezing induced by re-exposure to the aversively conditioned context. In the infralimbic (IL) prefrontal cortex, however, CBD (30 nmol) produced an opposite result, increasing the expression of contextual fear conditioning. This result was confirmed by an additional experiment where the conditioning session was performed under a less aversive protocol. Conclusion: These results suggest that the PL prefrontal cortex may be involved in the attenuation of contextual fear induced by systemic injection of CBD. They also support the proposition that the IL and PL play opposite roles in fear conditioning. A possible involvement of the BNST in CBD effects needs to be further investigated. (C) 2009 Elsevier B.V. All rights reserved.

Antidepressant-like effects of cannabidiol in mice: possible involvement of 5-HT(1A) receptors

Background and purpose: Cannabidiol (CBD) is a non-psychotomimetic compound from Cannabis sativa that induces anxiolytic- and antipsychotic-like effects in animal models. Effects of CBD may be mediated by the activation of 5-HT(1A) receptors. As 5-HT(1A) receptor activation may induce antidepressant-like effects, the aim of this work was to test the hypothesis that CBD would have antidepressant-like activity in mice as assessed by the forced swimming test. We also investigated if these responses depended on the activation of 5-HT(1A) receptors and on hippocampal expression of brain-derived neurotrophic factor (BDNF). Experimental approach: Male Swiss mice were given (i.p.) CBD (3, 10, 30, 100 mg.kg(-1)), imipramine (30 mg.kg(-1)) or vehicle and were submitted to the forced swimming test or to an open field arena, 30 min later. An additional group received WAY100635 (0.1 mg.kg(-1), i.p.), a 5-HT(1A) receptor antagonist, before CBD (30 mg.kg(-1)) and assessment by the forced swimming test. BDNF protein levels were measured in the hippocampus of another group of mice treated with CBD (30 mg.kg(-1)) and submitted to the forced swimming test. Key results: CBD (30 mg.kg(-1)) treatment reduced immobility time in the forced swimming test, as did the prototype antidepressant imipramine, without changing exploratory behaviour in the open field arena. WAY100635 pretreatment blocked CBD-induced effect in the forced swimming test. CBD (30 mg.kg(-1)) treatment did not change hippocampal BDNF levels. Conclusion and implications: CBD induces antidepressant-like effects comparable to those of imipramine. These effects of CBD were probably mediated by activation of 5-HT(1A) receptors. British Journal of Pharmacology (2010) 159, 122-128; doi:10.1111/j.1476-5381.2009.00521.x; published online 4 December 2009

Increased sympathetic outflow in juvenile rats submitted to chronic intermittent hypoxia correlates with enhanced expiratory activity

Chronic intermittent hypoxia (CIH) in rats produces changes in the central regulation of cardiovascular and respiratory systems by unknown mechanisms. We hypothesized that CIH (6% O(2) for 40 s, every 9 min, 8 h day(-1)) for 10 days alters the central respiratory modulation of sympathetic activity. After CIH, awake rats (n = 14) exhibited higher levels of mean arterial pressure than controls (101 +/- 3 versus 89 +/- 3 mmHg, n = 15, P < 0.01). Recordings of phrenic, thoracic sympathetic, cervical vagus and abdominal nerves were performed in the in situ working heart-brainstem preparations of control and CIH juvenile rats. The data obtained in CIH rats revealed that: (i) abdominal (Abd) nerves exhibited an additional burst discharge in late expiration; (ii) thoracic sympathetic nerve activity (tSNA) was greater during late expiration than in controls (52 +/- 5 versus 40 +/- 3%; n = 11, P < 0.05; values expressed according to the maximal activity observed during inspiration and the noise level recorded at the end of each experiment), which was not dependent on peripheral chemoreceptors; (iii) the additional late expiratory activity in the Abd nerve correlated with the increased tSNA; (iv) the enhanced late expiratory activity in the Abd nerve unique to CIH rats was accompanied by reduced post-inspiratory activity in cervical vagus nerve compared to controls. The data indicate that CIH rats present an altered pattern of central sympathetic-respiratory coupling, with increased tSNA that correlates with enhanced late expiratory discharge in the Abd nerve. Thus, CIH alters the coupling between the central respiratory generator and sympathetic networks that may contribute to the induced hypertension in this experimental model.

Identification of genetic alterations by multiple ligation-dependent probe amplification (MLPA) analysis in oligodendrogliomas

Concurrent deletion at 1p/19q is a common signature of oligodendrogliomas, and it may, be identified in low-grade tumours (grade II) suggesting it represents an early event in the development of these brain neoplasms. Additional non-random changes primarily involve CDKN2A, PTEN and EGFR. Identification of all of these genetic changes has become an additional parameter in the evaluation of the clinical patients` prognosis, including good response to conventional chemotherapy. Multiple ligation-dependent probe amplification (MLPA) analysis is a new methodology that allows an easy identification of the oligodendrogliomas` abnormalities in a single step. No need of the respective constitutional DNA from each patient is another advantage of this method. We used MLPA kits P088 and P105 to determine the molecular characteristics of a series of 40 oligodendrogliomas. Deletions at I p and 19q were identified in 45% and 65% of cases, respectively. Alterations of EGFR, CDKN2A, ERBB2, PTEN and TP53 were also identified in variable frequencies among 7% to 35% of tumours. These findings demonstrate that MLPA is a reliable technique to the detection of molecular genetic changes in oligodendrogliomas.

POSTOPERATIVE ANALGESIA INDUCED BY INTRATHECAL NEOSTIGMINE OR BETHANECHOL IN RATS

P>Cholinergic agonists and acetylcholinesterase inhibitors, such as neostigmine, produce a muscarinic receptor-mediated antinociception in several animal species that depends on activation of spinal cholinergic neurons. However, neostigmine causes antinociception in sheep only in the early, and not late, postoperative period. In the present study, a model of postoperative pain was used to determine the antinociceptive effects of bethanechol (a muscarinic agonist) and neostigmine administered intrathecally 2, 24 or 48 h after a plantar incision in a rat hind paw. Changes in the threshold to punctate mechanical stimuli were evaluated using an automated electronic von Frey apparatus. Mechanical hyperalgesia was obtained following plantar incision, the effect being stronger during the immediate (2 h) than the late post-surgical period. Bethanechol (15-90 mu g/5 mu L) or neostigmine (1-3 mu g/5 mu L) reduced incision-induced mechanical hyperalgesia, the effects of both drugs being more intense during the immediate (2 h) than the late post-surgical period. The ED(50) for bethanechol injected at 2, 24 and 48 h was 5.6, 51.9 and 82.5 mu g/5 mu L, respectively. The corresponding ED(50) for neostigmine was 1.62, 3.02 and 3.8 mu g/5 mu L, respectively. The decline in the antinociceptive potency of neostigmine with postoperative time is interpreted as resulting from a reduction in pain-induced activation of acetylcholine-releasing descending pathways. However, the similar behaviour of bethanechol in the same model points to an additional mechanism involving intrinsic changes in spinal muscarinic receptors.

Mechanisms of Action of Eicosapentaenoic Acid in Bladder Cancer Cells In Vitro: Alterations in Mitochondrial Metabolism, Reactive Oxygen Species Generation and Apoptosis Induction

Purpose: Eicosapentaenoic acid has been tested in bladder cancer as a synergistic cytotoxic agent in the form of meglumine-eicosapentaenoic acid, although its mechanism of action is poorly understood in this cancer. The current study analyzed the mechanisms by which eicosapentaenoic acid alters T24/83 human bladder cancer metabolism in vitro. Materials and Methods: T24/83 human bladder cancer cells were exposed to eicosapentaenoic acid for 6 to 24 hours in vitro and incorporation profiles were determined. Effects on membrane phospholipid incorporation, energy metabolism, mitochondrial activity, cell proliferation and apoptosis were analyzed Reactive oxygen species and lipid peroxide production were also determined. Results: Eicosapentaenoic acid was readily incorporated into membrane phospholipids with a considerable amount present in mitochondrial cardiolipin. Energy metabolism was significantly altered by eicosapentaenoic acid, accompanied by decreased mitochondrial membrane potential, and increased lipid peroxide and reactive oxygen species generation. Subsequently caspase-3 activation and apoptosis were detected in eicosapentaenoic acid exposed cells, leading to decreased cell numbers. Conclusions: These findings confirm that eicosapentaenoic acid is a potent cytotoxic agent in bladder cancer cells and provide important insight into the mechanisms by which eicosapentaenoic acid causes these changes. The changes in membrane composition that can occur with eicosapentaenoic acid likely contribute to the enhanced drug cytotoxicity reported previously in meglumine-eicosapentaenoic acid/epirubicin/mitomycin studies. Dietary manipulation of the cardiolipin fatty acid composition may provide an additional method for stimulating cell death in bladder cancer. In vivo studies using intravesical and dietary manipulation of fatty acid metabolism in bladder cancer merit further attention.

Population structure and mouse-virulence of Toxoplasma gondii in Brazil

Recent studies found that isolates of Toxoplasma gondii from Brazil were biologically and genetically different from those in North America and Europe. However, to date only a small number of isolates have been analysed from different animal hosts in Brazil. In the present study DNA samples of 46 T. gondii isolates from cats in 11 counties in Sao Paulo state, Brazil were genetically characterised using 10 PCR restriction fragment length polymorphism markers including SAG1, SAG2, SAG3, STUB, GRA6, c22-8, c29-2, L358, PKI and Apico. An additional marker, CS3, that locates on chromosome VIIa and has previously been shown to be linked to acute virulence of T. gondii was also used to determine its association to virulence in mice. Genotyping of these 46 isolates revealed a high genetic diversity with 20 genotypes but no clonal Type I, II or III lineage was found. Two of the 46 isolates showed mixed infections. Combining genotyping data in this study with recent reported results from chickens, dogs and cats in Brazil (total 125 isolates) identified 48 genotypes and 26 of these genotypes had single isolates. Four of the 48 genotypes with multiple isolates identified from different hosts and locations are considered the common clonal lineages in Brazil. These lineages are designated as Types BrI, BrII, BrIII and BrIV. These results indicate that the T. gondii population in Brazil is highly diverse with a few successful clonal lineages expanded into wide geographical areas. In contrast to North America and Europe, where the Type II clonal lineage is overwhelmingly predominant, no Type II strain was identified from the 125 Brazil isolates. Analysis of mortality rates in infected mice indicates that Type BrI is highly virulent, Type BrIII is non-virulent, whilst Type BrII and BrIV lineages are intermediately virulent. In addition, allele types at the CS3 locus are strongly linked to mouse-virulence of the parasite. Thus, T. gondii has an epidemic population structure in Brazil and the major lineages have different biological traits. (C) 2007 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved.

Clinical observations of cattle and buffalos with experimentally induced chronic copper poisoning

The susceptibility of cattle and buffalos to chronic copper poisoning (CCP) was compared by using cattle (n = 10) and buffalo (n = 10) steers distributed into two copper supplemented (n = 6) and two control (n = 4) groups. Supplemented animals received 2 mg copper (Cu)/kg body weight daily for one week, with an additional 2 mg weekly until the end of the experiment (day 105). Three liver biopsies (day 0, 45, and 105) were obtained for mineral analyses; clinical examinations and blood samples were obtained every 15 days. Three supplemented cattle and two buffalos with typical manifestations of CCP died. There were no differences in the frequency of mortality between cattle and buffalos; hepatic copper concentration was higher in cattle than buffalos. These findings suggest that buffalos and cattle might be equally susceptible to CCP. However, buffalos accumulate less liver copper than cattle and have a lower threshold of hepatic Cu accumulation, which leads to clinical manifestation of CCP. (c) 2009 Elsevier Ltd. All rights reserved.

Diode Laser Irradiation Effects on the Sealing Ability of Root Canal Sealers

This study aimed to test the hypothesis that dentine alterations induced by 810 nm-diode laser may affect the interaction between root canal sealers and the dentin wall. Seventy-two single root human teeth were selected and root canals were enlarged with K-files. Dentine was treated with 0.5% NaOCl and 17% EDTA-T and irradiated (laser group) by diode laser (810 nm/P = 2.5W/I = 1989 W/cm(2)) or remained non-irradiated (control group). Six samples per group were analyzed by scanning electron microscopy (SEM). The remaining samples of each group were divided into three subgroups (n = 10) and sealed with one of the tested sealers (N-Rickert/AHPlus (TM)/Apexit (R)). Apical leakage was estimated by evaluating penetration of 0.5% methylene-blue dye. SEM analysis revealed that dentine at the apical third in irradiated samples was melted and fusioned whereas non-irradiated samples exhibited opened dentinal tubules. Despite the morphological changes induced by irradiation, laser did not affect the sealing ability of N-Rickert and AHPlus (TM) sealers. However, the length of apical leakage in roots filled with Apexit (R) was lower in irradiated root canals than in non-irradiated samples (p < 0.05). Morphological changes of root canal walls promoted by diode laser irradiation may improve de sealing ability of Apexit (R), a calcium hydroxide-based sealer, suggesting that improved sealing promoted by irradiation may represent an additional factor contributing to the endodontic clinical outcome.