Xanthine oxidase pathway and muscle damage: Insights from McArdle disease

The intent of this review is to summarize current body of knowledge on the potential implication of the xanthine oxidase pathway (XO) on skeletal muscle damage. The possible involvement of the XO pathway in muscle damage is exemplified by the role of XO inhibitors (e.g., allopurinol) in attenuating muscle damage. Reliance on this pathway (as well as on the purine nucleotide cycle) could be exacerbated in conditions of low muscle glycogen availability. Thus, we also summarize current hypotheses on the etiology of both baseline and exertional muscle damage in McArdle disease, a condition caused by inherited deficiency of myophosphorylase. Because myophosphorylase catalyzes the first step of muscle glycogen breakdown, patients are unable to obtain energy from their muscle glycogen stores. Finally, we provide preliminary data from our laboratory on the potential implication of the XO pathway in the muscle damage that is commonly experienced by these patients.

Biological rationale for regular physical exercise as an effective intervention for the prevention and treatment of depressive disorders

Depression is a major medical and social problem. Here we review current body of knowledge on the benefits of exercise as an effective strategy for both the prevention and treatment of this condition. We also analyze the biological pathways involved in such potential benefits, which include changes in neurotrophic factors, oxidative stress and inflammation, telomere length, brain volume and microvessels, neurotransmitters or hormones. We also identify major caveats in this field of research: further studies are needed to identify which are the most appropriate types of exercise interventions (intensity, duration, or frequency) to treat and prevent depression.

Talidomida

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Aconselhamento Farmacêutico em Suplementação Alimentar

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Investigação sobre qualidade de vida e qualidade de vida relacionada com a saúde

Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências Farmacêuticas

Optimizing perioperative analgesia for patients undergoing operative fixation of hip fractures

This PhD thesis describes work carried out on investigation of various interventions with the aim to optimise the anaesthetic management of patients scheduled to undergo operative fixation of hip fractures. We analysed the perioperative effects of continuous femoral nerve block, single preoperative dose of i.v. dexamethasone, the intention to deposit local anaesthetic in different locations around the femoral nerve during ultrasound guided femoral nerve block, continuous spinal anaesthesia and peri-surgical site infiltration with local anaesthetic after surgical fixation of hip fractures. Continuous femoral nerve block provided more effective preoperative analgesia six hours after the insertion of the perineural catheter compared to a standard opiate-based regimen in patients undergoing operative fixation of fractured hip. A single low dose of preoperative dexamethasone in the intervention group decreased pain scores by 75% six hours after the surgery. Both interventions had no major effect on the functional recovery in the first year after the surgical fixation of fractured hip. The results of the ultrasound guided femoral nerve block trial showed no clinical advantage of intending to deposit local anaesthetic circumferentially during performing femoral nerve block. Using the Dixon and Massey’s “up- and-down” method, we demonstrated that intrathecal 0.26 ml of 0.5% bupivacaine provided adequate surgical anaesthesia within 15 minutes in 50% of patients undergoing operative fixation of hip fracture. Finally, we demonstrated that local anaesthetic infiltration had no effect on pain scores 12 hours after the surgical fixation of fractured neck of femur. In addition to this original body of work, a review article was published on femoral nerve block highlighting the use of ultrasound guidance. In conclusion, the results of this thesis offer an insight into interventions aimed at optimising perioperative analgesia in patients scheduled to undergo operative fixation of hip fractures.

Identifying novel molecular mechanisms of ghrelin receptor signalling underlying neural control of food intake: interaction with stress and impulsivity

The gut-hormone, ghrelin, activates the centrally expressed growth hormone secretagogue 1a (GHS-R1a) receptor, or ghrelin receptor. The ghrelin receptor is a G-protein coupled receptor (GPCR) expressed in several brain regions, including the arcuate nucleus (Arc), lateral hypothalamus (LH), ventral tegmental area (VTA), nucleus accumbens (NAcc) and amygdala. Activation of the GHS-R1a mediates a multitude of biological activities, including release of growth hormone and food intake. The ghrelin signalling system also plays a key role in the hedonic aspects of food intake and activates the dopaminergic mesolimbic circuit involved in reward signalling. Recently, ghrelin has been shown to be involved in mediating a stress response and to mediate stress-induced food reward behaviour via its interaction with the HPA-axis at the level of the anterior pituitary. Here, we focus on the role of the GHS-R1a receptor in reward behaviour, including the motivation to eat, its anxiogenic effects, and its role in impulsive behaviour. We investigate the functional selectivity and pharmacology of GHS-R1a receptor ligands as well as crosstalk of the GHS-R1a receptor with the serotonin 2C (5-HT2C) receptor, which represent another major target in the regulation of eating behaviour, stress-sensitivity and impulse control disorders. We demonstrate, to our knowledge for the first time, the direct impact of GHS-R1a signalling on impulsive responding in a 2-choice serial reaction time task (2CSRTT) and show a role for the 5-HT2C receptor in modulating amphetamine-associated impulsive action. Finally, we investigate differential gene expression patterns in the mesocorticolimbic pathway, specifically in the NAcc and PFC, between innate low- and high-impulsive rats. Together, these findings are poised to have important implications in the development of novel treatment strategies to combat eating disorders, including obesity and binge eating disorders as well as impulse control disorders, including, substance abuse and addiction, attention deficit hyperactivity disorder (ADHD) and mood disorders.

Repeated concanavalin A challenge in mice induces an interleukin 10-producing phenotype and liver fibrosis.

Weekly injections of Concanavalin A (Con A) were performed in BALB/c mice to evaluate the pattern of cytokine production and liver injury. High serum levels of tumor necrosis factor alpha (TNF-alpha), interleukin 2 (IL-2), IL-4, and interferon gamma (IFN-gamma) were found in the serum after the first 2 injections of Con A but rapidly decreased from the third injection. Conversely, IL-10 serum levels after repeated Con A challenge increased by 7 times from week 1 to 20. In vivo depletion studies indicated that CD4(+) T cells are essential in IL-10 production. Hepatocyte necrosis was only observed after the first injections of Con A whereas centrilobular inflammatory infiltrates persisted up to 20 weeks. Perisinusoidal liver fibrosis was also increasingly detected in BALB/c mice, whereas no fibrous change was observed in nude mice after 6 weeks of Con A challenge. The number of stellate cells, detected by immunostaining, increased after 20 weeks of Con A injections. Liver cytokine messenger RNA (mRNA) expression after 20 weeks showed expression of transforming growth factor beta1 (TGF-beta1), IL-10, and IL-4 whereas IL-2 was no more expressed. The present study shows that mice repeatedly injected with Con A develop liver fibrosis. The cytokine-release pattern observed after 1 injection of Con A is rapidly shifted towards an immunomodulatory phenotype characterized by the systemic production of large amounts of IL-10.

Critical role of interleukin 5 and eosinophils in concanavalin A-induced hepatitis in mice.

BACKGROUND & AIMS: Eosinophils are observed in several liver diseases, but their contribution in the pathogenesis of these disorders remains poorly investigated. Concanavalin A (Con A)-induced hepatitis is an experimental model of immune-mediated liver injury in which natural killer T (NKT) cells play a critical role through the production of interleukin (IL)-4 and the expression of Fas ligand (FasL). Because activated NKT cells also produce IL-5, a critical cytokine for eosinophil maturation and function, the role of IL-5 was investigated in this model. METHODS: IL-5-deficient mice, eosinophil depletion in wild-type (WT) mice, and NKT cell transfer from WT- or IL-5-deficient mice into NKT cell-deficient mice were used to assess the role of IL-5 and eosinophils. RESULTS: Liver eosinophil infiltrate and IL-5 production were observed after Con A challenge. Liver injury was dramatically reduced in IL-5-deficient or eosinophil-depleted mice. In addition, residual hepatitis observed in Fas-deficient mice was abolished after IL-5 neutralization. Finally, we showed that NKT cells constituted a critical source of IL-5. Indeed, transfer of WT NKT cells to mice lacking NKT cells restored liver injury, whereas transfer of IL-5-deficient NKT cells did not. CONCLUSIONS: These observations highlight the pathologic role of IL-5 and eosinophils in experimental immune-mediated hepatitis.

Dose-effect of interleukin-10 and its immunoregulatory role in Crohn's disease.

BACKGROUND: Interleukin-10 (IL-10) is currently being extensively studied in clinical trials for the treatment of Crohn's disease (CD). Only marginal effects have, however, been reported, and the dose-response curve was bell-shaped contrasting with the reported data from in vitro experiments. AIM: To use another in vitro model to analyze the effect of rhIL-10 and rhIL-4 on the spontaneous mucosal TNF-alpha secretion in patients with CD, and to characterize the phenotype of the cells targeted by rhIL-10. METHODS: Non-inflamed colon biopsies from CD patients were cultured for 16 hours in presence of different concentrations of rhIL-10 or rhIL-4. The numbers of TNF-alpha-secreting cells among isolated lamina propria mononuclear cells (LPMNC) were estimated by Elispot. RESULTS: Both rhIL-10 and rhIL-4 down-regulate TNF-alpha secretion by LPMNC from CD patients, with a more pronounced effect with rhIL-10. These effects were closely linked to the cytokine concentrations used, with a bell-shaped dose-response curve. Residual TNF-alpha secretion, in the presence of optimal rhIL-10 concentration was mainly attributable to CD3+ T cells. In contrast, at higher rhIL-10 concentrations, CD3- cells contributed significantly to the TNF-alpha secretion. CONCLUSIONS: The in vitro model we used, demonstrates that IL-4, but mostly IL-10, efficiently suppresses TNF-alpha secretion in LPMNC from CD patients, with a dose-response curve similar to results obtained in vivo. Resistance at high rhIL-10 concentrations was associated with a change in the phenotype of TNF-alpha-secreting cells.

The neuropeptide pituitary adenylate cyclase activating protein stimulates human monocytes by transactivation of the Trk/NGF pathway.

Transactivation is a process whereby stimulation of G-protein-coupled receptors (GPCR) activates signaling from receptors tyrosine kinase (RTK). In neuronal cells, the neuropeptide pituitary adenylate cyclase-activating polypeptide (PACAP) acting through the GPCR VPAC-1 exerts trophic effects by transactivating the RTK TrkA receptor for the nerve growth factor (NGF). Both PACAP and NGF have pro-inflammatory activities on monocytes. We have tested the possibility that in monocytes, PACAP, as reported in neuronal cells, uses NGF/TrkA signaling pathway. In these cells, PACAP increases TrkA tyrosine phosphorylations through a PI-3kinase dependent but phospholipase C independent pathway. K252a, an inhibitor of TrkA decreases PACAP-induced Akt and ERK phosphorylation and calcium mobilisation resulting in decreases in intracellular H2O2 production and membrane upregulation of CD11b expression, both functions being inhibited after anti-NGF or anti-TrkA antibody treatment. K252a also inhibits PACAP-associated NF-KB activity. Monocytes increase in NGF production is seen after micromolar PACAP exposure while nanomolar treatment which desensitizes cells to high dose of PACAP prevents PACAP-induced TrkA phosphorylation, H2O2 production and CD11b expression. Finally, NGF-dependent ERK activation and H2O2 production is pertussis toxin sensitive. Altogether these data indicate that in PACAP-activated monocytes some pro-inflammatory activities occur through transactivation mechanisms involving VPAC-1, NGF and TrkA-associated tyrosine kinase activity.

The effect of exemestane on serum lipid profile in postmenopausal women with metastatic breast cancer: a companion study to EORTC Trial 10951, 'Randomized phase II study in first line hormonal treatment for metastatic breast cancer with exemestane or tamoxifen in postmenopausal patients'.

BACKGROUND: The impact of aromatase inhibitors (AIs) on non-cancer-related outcomes, which are known to be affected by oestrogens, has become increasingly important in postmenopausal women with hormone-dependent breast cancer. So far, data related to the effect of AIs on lipid profile in postmenopausal women is scarce. This study, as a companion substudy of an EORTC phase II trial (10951), evaluated the impact of exemestane, a steroidal aromatase inactivator, on the lipid profile of postmenopausal metastatic breast cancer (MBC) patients. PATIENTS AND METHODS: The EORTC trial 10951 randomised 122 postmenopausal breast cancer patients to exemestane (E) 25 mg (n = 62) or tamoxifen (T) 20 mg (n = 60) once daily as a first-line treatment in the metastatic setting. Exemestane showed promising results in all the primary efficacy end points of the trial (response rate, clinical benefit rate and response duration), and it was well tolerated with low incidence of serious toxicity. As a secondary end point of this phase II trial, serum triglycerides (TRG), high-density lipoprotein cholesterol (HDL), total cholesterol (TC), lipoprotein a (Lip a), and apolipoproteins (Apo) B and A1 were measured at baseline and while on therapy (at 8, 24 and 48 weeks) to assess the impact of exemestane and tamoxifen on serum lipid profiles. Of the 122 randomised patients, those who had baseline and at least one other lipid assessment are included in the present analysis. The patients who received concomitant drugs that could affect lipid profile are included only if these drugs were administered throughout the study treatment. Increase or decrease in lipid parameters within 20% of baseline were considered as non-significant and thus unchanged. RESULTS: Seventy-two patients (36 in both arms) were included in the statistical analysis. The majority of patients had abnormal TC and normal TRG, HDL, Apo A1, Apo B and Lip a levels at baseline. Neither exemestane nor tamoxifen had adverse effects on TC, HDL, Apo A1, Apo B or Lip a levels at 8, 24 and 48 weeks of treatment. Exemestane and tamoxifen had opposite effects on TRG levels: exemestane lowered while tamoxifen increased TRG levels over time. There were too few patients with normal baseline TC and abnormal TRG, HDL, Apo A1, Apo B and Lip a levels to allow for assessment of E's impact on these subsets. The atherogenic risk determined by Apo A1:Apo B and TC:HDL ratios remained unchanged throughout the treatment period in both the E and T arms. CONCLUSIONS: Overall, exemestane has no detrimental effect on cholesterol levels and the atherogenic indices, which are well-known risk factors for coronary artery disease. In addition, it has a beneficial effect on TRG levels. These data, coupled with E's excellent efficacy and tolerability, support further exploration of its potential in the metastatic, adjuvant and chemopreventive setting.

IL-12 inhibits in vitro immunoglobulin production by human lupus peripheral blood mononuclear cells (PBMC).

Systemic lupus erythematosus (SLE) is a prototypical autoimmune disease characterized by polyclonal B cell activation and by the production of anti-double-stranded (ds) DNA antibodies. Given the inhibitory effects of IL-12 on humoral immune responses, we investigated whether IL-12 displayed such an activity on in vitro immunoglobulin production by SLE PBMC. Spontaneous IgG, IgG1, IgG2, IgG3 and IgM antibody production was dramatically reduced by addition of IL-12. These results were confirmed by Elispot assays detecting IgG- and anti-dsDNA-secreting cells. While IL-6 and TNF titres measured in PBMC supernatants were not modified by addition of IL-12, interferon-gamma (IFN-gamma) titres were up-regulated and IL-10 production down-regulated. Since addition of IFN-gamma did not down-regulate immunoglobulin production and since the inhibitory activity of IL-12 on immunoglobulin synthesis was not suppressed by anti-IFN-gamma antibody, we concluded that the effect of IL-12 on immunoglobulin production was not mediated through IFN-gamma. Our data also argue against the possibility that down-regulation of endogenous IL-10 production was responsible for the effect of IL-12. Thus, inhibition of IL-10 production by IFN-gamma was not accompanied by inhibition of immunoglobulin production, and conversely, restoration of IL-10 production by anti-IFN-gamma antibody did not suppress the inhibitory activity exerted by IL-12 on immunoglobulin production. Taken together, our data indicate that reduction of excessive immunoglobulin and anti-dsDNA antibody production by lupus PBMC can be achieved in vitro by IL-12, independently of IFN-gamma and IL-10 modulation.

Differential inducibility of HLA class I and II antigens by r-IFN gamma in type III bare lymphocyte syndrome.

Case Reports

A binding site for the cyclic adenosine 3',5'-monophosphate-response element-binding protein as a regulatory element in the grp78 promoter.

The 78-kDa glucose-regulated protein (GRP78) is ubiquitously expressed in many cell types. Its promoter contains multiple protein-binding sites and functional elements. In this study we examined a high affinity protein-binding site spanning bp -198 to -180 of the rat grp78 promoter, using nuclear extracts from both B-lymphoid and HeLa cells. This region contains a sequence TGACGTGA which, with the exception of one base, is identical to the cAMP-response element (CRE). Site-directed mutagenesis reveals that this sequence functions as a major basal level regulatory element in hamster fibroblast cells and is also necessary to maintain high promoter activity under stress-induced conditions. By gel mobility shift analysis, we detect two specific protein complexes. The major specific complex I, while immunologically distinct from the 42-kDa CRE-binding protein (CREB), binds most strongly to the grp site, but also exhibits affinity for the CRE consensus sequence. As such, complex I may consist of other members of the CREB/activating transcription factor protein family. The minor specific complex II consists of CREB or a protein antigenically related to it. A nonspecific complex III consists of the Ku autoantigen, an abundant 70- to 80-kDa protein complex in HeLa nuclear extracts. By cotransfection experiments, we demonstrate that in F9 teratocarcinoma cells, the grp78 promoter can be transactivated by the phosphorylated CREB or when the CREB-transfected cells are treated with the calcium ionophore A23187. The differential regulation of the grp78 gene by cAMP in specific cell types and tissues is discussed.