Two decades into the epidemic caused by the human immunodeficiency virus (HIV) and this virus has infected nearly 1% of the world's population. Highly active antiretroviral therapy (HAART) has transformed the epidemic from a death sentence into a chronic infection. However restricted access, complicated regimens, drug resistance, and long-term toxicities are all impediments to life long therapy for all those infected. These factors stress the need for vaccines and immunotherapeutic strategies that may be used in conjunction with antiretroviral drugs for the optimization of patient care. / It is clear that cytotoxic T lymphocytes are potent suppressors of viral replication throughout the clinical course of HIV infection. The research presented in this thesis focuses on the characterization of immune responses at different stages of HIV infection in order to gain a better understanding of the pattern of changes that occur within the host response to virus on and off therapy following infection. / The HIV specific immune response broadens during the first two months of infection, suggesting that while it is induced early during acute infection, the antiviral response evolves to respond to the virus. HIV specific immune responses can be preserved if HAART is initiated while the subject is in early PI, and appears to depend on the preservation of HIV specific CD4+ T cell help. Subjects remaining untreated in PI experience significant perturbations (expansions and contractions) in responses to individual HIV peptides over a 1 year period, which is characterized by an overall contraction in the breadth and persistence in the magnitude of their antiviral effector activity. / New strategies that map the specificity, breadth and magnitude of the HIV specific immune response testing all HIV expressed gene products were evaluated. The novel approach will strengthen conclusions derived from the types of studies conducted for this thesis by detecting all reactivities present in subjects being tested in an unbiased manner. Interferon-gamma (IFN-gamma) secretion in response to epitopes previously unknown to be immunogenic are frequently detected using this screening method in subjects in the chronic phase of infection. Use of this technique demonstrates that HAART-naive HIV+ chronically infected subjects in asymptomatic disease target Gag predominantly and possess variable breadths and magnitudes of responses to HIV. / Taken together, the data presented in this thesis have improved our understanding of the pattern of the evolution of the immune response over time. Data presented here have implications for HIV infected patient management, by providing new information useful for deciding whether the immunological benefit of initiating HAART early in infection outweighs toxicities associated with long term treatment. Finally, new strategies for the detection of HIV specific immune responses will further our ability to characterize the global HIV specific immune responses at all stages of HIV disease.

Oligodendrocytes, the cells that produce and maintain myelin in the central nervous system (CNS), express several muscarinic acetylcholine (ACh) receptor (mAChR) subtypes. Carbachol (CCh), a stable analogue of ACh, stimulates various intracellular signals through mAChRs, including p42-mitogen-activated protein kinase (p42mapk) activation and c-fos gene expression. The present study examined the mAChR subtype(s) and the signaling pathway(s) involved in both responses using purified cultures of oligodendrocyte progenitors from newborn rats. / The M3 mAChR selective antagonist 4-DAMP and its irreversible analogue 4-DAMP-mustard, but not pirenzepine (M1) or methoctramine (M2/M4), prevented p42mapk activation and c-fos mRNA expression. Pretreatment with a phospholipase C (PLC) inhibitor U73122 but not the inactive analogue U73343 blocked both responses, while the phosphoinositide 3-kinase (PI3K) inhibitor wortmannin had no effect, suggesting the involvement of PLC but not PI3K. / In view of the fact that the M3 mAChR mediates p42mapk activation as well as c-fos mRNA expression, the MAPK Kinase (MEK) inhibitor PD 098059 was used to establish a link between both responses. PD 098059 pretreatment caused no significant attenuation in c-fos mRNA levels, demonstrating that this response may not be dependent on p42mapk activation. However, the phospholipase A2 (PLA2) inhibitors quinacrine and AACOCF3 abolished c-fos mRNA expression, implicating arachidonic acid and/or its bioactive eicosanoid metabolites. / In summary, these results demonstrate that in oligodendrocyte progenitors, the M3 mAChR is mediating both p42mapk activation and c-fos gene expression via PLC but not PI3K, whereby c-fos gene expression is not regulated by p42mapk but by PLA2.

Oxygen free radicals have been implicated in several diseases including ischemic stroke, and myocardial infarction. They can trigger chain reactions like peroxidation of membrane phospholipids, leading to osmotic imbalance and cell death. Isoprostanes are stable products of lipid peroxidation that have a constrictor effect on the vasculature and bronchii. As isoprostanes are abundantly generated in tissues under oxidant stress, we have hypothesized that they could be related to endothelial dysfunction observed during ischemia/reperfasion by affecting endothelial cell survival. The effects of 8-iso-PGE2 and 8-iso-PGF2alpha, two abundantly produced isoprostanes, were studied on porcine endothelial cultures and isolated brain microvessels. Cell survival was evaluated by MTT reduction, double staining with DNA-binding fluorochromes and in situ DNA fragmentation labeling, / 8-Iso-PGF2alpha (1--10 nM) induced 20--25% cell death in endothelial cultures after 24 h coincident with similar increase in the number of cells that become permeable to PI. On the contrary, 8-iso-PGE 2 did not affect endothelial cell survival. Approximately 9% of the cells suffered apoptosis. This percentage remained unchanged regardless the treatment. Several observations indicate a role for thromboxane A2 to mediate 8-iso-PGF2alpha-induced death: (1) the levels of thromboxane A2 increased dramatically in endothelial cultures after 8-iso-PGF2alpha-treatment; (2) inhibitors of thromboxane synthase, CGS12970 and U6355A and Ibuprofen, a non-selective inhibitor of cyclooxygenases, reverted the effect of the isoprostane; (3) analogs of thromboxane A2 U46619 and IBOP, reproduce the effect of 8-iso-PGF 2alpha after 24 h. 8-Iso-PGF2alpha also decreased endothelial viability on isolated brain microvessels. These results suggest, that 8-iso-PGF2alpha, might be a direct contributor to ischemia/reperfusion injury.

Calcitonin gene-related peptide (CGRP) is a 37 amino acid peptide that arises from the alternative splicing of the RNA transcript of the calcitonin gene. Both CGRP and its receptors are widely distributed in the peripheral and central nervous systems. Recent data have suggested the existence of at least two major classes of CGRP receptors designated as, CGRP$ sb1$ and CGRP$ sb2$ receptor subtypes. The evidence suggesting the existence of receptor subtypes is based on the differential potencies in several in vitro and in vivo bioassays of the C-terminal fragment, CGRP$ sb{8-37}$ and the linear analogue, (acetamidomethyl-cysteine$ sp{2,7}$) CGRP$ alpha$. The CGRP$ sb1$ is particularly sensitive to the antagonistic properties of CGRP$ sb{8-37}$ whereas the linear analogue acts preferentially as an agonist on the CGRP$ sb2$ receptors. / The primary focus of my thesis was therefore to provide novel evidence for the existence of multiple CGRP receptor sites in peripheral and especially brain tissues. / Taken together, the findings of my thesis support the existence of functionally distinct receptors for CGRP and related peptides in the rat brain. (Abstract shortened by UMI.)

CO2 is an important regulator of cerebral blood flow (CBF). Sustained hypercapnia results in a transient rapid CBF rise in newborns with respiratory disorders; however, contrary to adults, the secondary effects of hypercapnia especially the decrease in blood pH, do not seem to reset (Brubakk et al., 1987). Acute hypercapnia and associated acidosis cause a significant rise in nitric oxide (NO) and prostaglandins (PG) levels, which in turn affect early CBF changes. However, the mechanism(s) and their mediator(s) in prolonged hypercapnic CBF fluctuations remain elusive. Results of this study provide direct evidence that in newborn brain, blood flow exhibits a biphasic response to sustained hypercapnia. PG synthase and NO synthase inhibitors block the secondary increase suggesting an important interaction between these factors. The slow and more sustained profile of the secondary increase suggests an induction of a gene implicated in vasodilation. Indeed, we report an increase in endothelial constitutive NOS (ecNOS) expression, activity and reactivity due to sustained hypercapnia and associated acidosis. We also demonstrate that PGs regulate both ecNOS expression and reactivity during acidosis in cerebral microvasculature. Acidosis does not directly stimulate PG synthase or NO pathways, instead an effect through the endothelial cell membrane seemed more plausible. Blockade of K+ channels, which are important regulators of membrane potential, inhibited the pH-dependent increase in PGs and ecNOS mRNA expression. Moreover, their inhibition blocked the influx of calcium elicited by acidosis in microvascular endothelial cells irrespective of Ca++ channel blockade. We conclude that these findings help in the understanding of the contribution and interaction of distinct factors proposed to regulate CBF in the newborn.

Glucocorticoids and somatostatin both influence a broad spectrum of biological activities and their actions are cooperative in growth control, pancreatic islet function, immune suppression, and stress response, e.g. in vivo studies indicate that glucocorticoids may act through somatostatin to suppress growth, growth hormone secretion and inflammation. Recent studies have suggested that glucocorticoids influence somatostatin production but the precise nature of this effect has remained unclear. In this thesis, I characterized the actions of glucocorticoids on somatostatin gene expression and their molecular mechanisms of action in three consecutive studies. (1) I started with an investigation of the in vivo and in vitro effects of glucocorticoids and found that dexamethasone exerts significant effects on somatostatin peptide and steady state mRNA levels in normal somatostatinoma (1027B$ sb2$) cells. Glucocorticoids stimulate somatostatin production in peripheral tissues (stomach, pancreas, and jejunum) and suppress its biosynthesis in cerebral cortex and hypothalamus. Glucocorticoids induce dose-dependent biphasic effects on steady state somatostatin-mRNA levels in normal rat islet and 1027B$ sb2$ cells, characterized by stimulation at low doses (10$ sp{-10}$ M) and marked inhibition at high doses ($ geq$10$ sp{-7}$ M). This suggests a complex molecular mechanisms of glucocorticoid action on the somatostatin gene involving multi-level regulation. (2) I further discovered that glucocorticoids stimulate somatostatin gene transcription in PC12 (pheochromocytoma) cells transfected with somatostatin promoter-CAT (chloramphenicol acetyl transferase) reporter gene. Dexamethasone induces a dose-dependent 2.2 fold stimulation of somatostatin-CAT expression in PC12 cells and exerts an additive effect on cAMP-induced gene transcription. The dexamethasone effect is abolished in A126-1B2 (protein kinase A-deficient mutant PC12) cells and with CRE (cAMP response element) mutant

Since the formulation of the monoaminergic hypotheses of depression, several investigations have established that the serotonin (5-HT) as well as the noradrenaline (NA) systems are altered by antidepressant treatments. In the last few years, several studies have indicated that interactions between these two systems might also be important in the mechanism of action of antidepressant drugs. We have thus undertaken: (1) to elucidate the nature of some interactions between the 5-HT and NA systems in the rat hippocampus using in vivo electrophysiology and in vitro superfusion techniques; and (2) to determine whether antidepressant treatments alter these interactions. It was found that NA tonically inhibits 5-HT neurotransmission via $ alpha sb2$-adrenergic heteroreceptors. In addition, the presence of 5-HT$ sb3$ receptors facilitating the release of NA was also documented in this brain region. These receptors were shown to be activated by elevated levels of endogenous 5-HT, but did not appear to be activated by basal levels of this neurotransmitter. Long-term administrations of antidepressant drugs commonly known to increase the synaptic concentration of NA, i.e. monoamine oxidase and NA reuptake inhibitors, were found to desensitize the $ alpha sb2$-adrenoceptors that inhibit the release of 5-HT. This effect was also produced by atypical antidepressant drugs that have $ alpha sb2$-adrenergic antagonist properties (e.g. mianserin and idazoxan), but not by other antidepressant treatments, (e.g. selective 5-HT reuptake inhibitors or electroconvulsive shocks). In contrast to $ alpha sb2$-adrenergic heteroreceptors, 5-HT$ sb3$ receptors that enhance the release of NA did not become desensitized following long-term treatments with drugs that elevate the synaptic concentration of their endogenous neurotransmitter. Rather, the responsiveness of these 5-HT$ sb3$ receptors became blunted following an antidepressant treatment (desipramine) that caused a large increase in the r

Introduction. Defects in ion channels and kinase phosphorylation have been reported in streptozotocin (STZ) models of diabetic neuropathy. We hypothesise that changes in phosphorylation may underlie changes in the excitability in the peripheral nerves. / Objective. To examine the effect of pharmacological modulation of electrophysiological parameters in STZ-diabetic mice. / Methods. Electrophysiological measurements were done in sciatic nerves of mice made diabetic with Streptozotocin. / Results. We report that sucrose gap measurements on sciatic nerves of compound action potential conduction velocity, area and refractory period was affected in STZ mice. Treatment with 4-Aminopyridine increased the delayed depolarization area in diabetic more than in control, which could reveal problems with fast potassium channels and/or slow sodium channels. Pharmacological tools showed that regulation of PKC and tyrosine phosphorylation in diabetic mice was disturbed. / Conclusion. These results strongly suggest that aberrant phosphorylation could contribute to the pathological symptoms observed in diabetic neuropathy.

Alzheimer's disease (AD) is a neurological disorder characterized by plaque deposition and an elevated immune response. Epidemiological studies have shown that use of non-steroidal anti-inflammatory drugs (NSAIDs) by the elderly is associated with a decreased relative risk and a delayed onset of AD. Moreover, the apolipoprotein E (apoE) gene has been proven to be a risk factor for AD with apoE &egr;4 AD patients having been found to show lower levels of brain apoE. In the present study, treatment of primary rat mixed glial cell cultures with indomethacin, aspirin, and interleukin-1beta resulted in significant increases in extracellular apoE protein. Furthermore, treatment of primary rat astrocyte cell cultures with aspirin, interleukin-6, and a cyclooxygenase-2 selective aspirin derivative was found to result in increased levels of apoE. Consequently, NSAID-induced increases in apoE protein may enhance apoE-mediated immunosuppression and compensatory synaptic plasticity, potentially resulting in decreased risk and delay of disease onset.

There is an historical debate in which the role and importance of the serotoninergic (5-HT) and noradrenergic (NE) systems as being the key components in the antidepressant response is disputed. Alternatively, this contentious debate has directly led to the hypothesis which posits that a combined pharmacological action on both these systems might result in an enhanced antidepressant efficacy. The development of the dual 5-HT/NE reuptake inhibitor venlafaxine has allowed to put to the test this hypothesis. Hence, clinical insight into this problem was provided by studies that documented a greater efficacy of venlafaxine in major depression when used at high doses. The present research endeavour, undertaken in parallel at the Neurobiological Psychiatry Unit with clinical studies, sought to determine the effects of acute and prolonged treatments with venlafaxine on the rat 5-HT and NE systems such as to suggest a biological substratum for its clinical features. Using in vivo electrophysiological paradigms in the rat dorsal hippocampus, dorsal raphe and locus coeruleus, we have shown that: (1) venlafaxine blocks the reuptake of 5-HT more potently than that of NE; (2) venlafaxine and the SSRI paroxetine are equipotent in blocking the reuptake of 5-HT; (3) the NE reuptake inhibitor desipramine is more potent than venlafaxine in blocking the reuptake of NE. We have documented an important discrepancy between the affinity of venlafaxine for both the 5-HT and NE transporters binding sites and its in vivo reuptake inhibition potencies: the in vivo 5-HT and NE reuptake inhibition potencies of venlafaxine are much greater than could have been predicted from its affinities for the 5-HT and NE transporters. The disclosure of this peculiarity sheds doubt on the exact nature of the mechanism by which venlafaxine blocks both reuptake processes and, ultimately, exerts its antidepressant effect. The complementary use of in vivo electrophysiological and in vitro superfusion paradigm