Tobacco smoking has consistently been the leading cause of preventable diseases and premature deaths. Currently, pharmacological interventions have only shown limited smoking cessation efficacy and sometimes are associated with severe side effects. As an alternative, nicotine vaccines have emerged as a promising strategy to combating nicotine addiction. However, conventional conjugate nicotine vaccines have shown limited ability to induce a sufficiently strong immune response due to their intrinsic shortfalls. In this study, a lipid-poly(lactic-co-glycolic acid) (PLGA) nanoparticle-based next-generation nicotine vaccine has been developed to overcome the drawbacks of conjugate nicotine vaccines. Also, the influence of multiple factors, including nanoparticle size, hapten density, hapten localization, carrier protein, and molecular adjuvants, on its immunogenicity has been investigated. Results indicated that all these studied factors significantly affected the immunological efficacy of the nicotine nanovaccine. First, 100 nm nanovaccine was found to elicit a significantly higher anti-nicotine antibody titer than the 500 nm nanovaccine. Secondly, the high-density nanovaccine exhibited a better immunological efficacy than the low- and medium-density counterparts. Thirdly, the nanovaccine with hapten localized on both carrier protein and nanoparticle surface induced a significantly higher anti-nicotine antibody titer and had a considerably better ability to block nicotine from entering the brain of mice than the nanovaccines with hapten localized only on carrier protein or nanoparticle surface. Fourthly, the nanovaccines carrying cross reactive materials 197 (CRM197) or tetanus toxoid (TT) showed a better immunological efficacy than the nanovaccines using keyhole limpet hemocyanin (KLH) or KLH subunit as carrier proteins. Finally, the co-delivery of monophosphoryl lipid A (MPLA) and Resiquimod (R848) achieved a considerably higher antibody titer and brain nicotine reduction than only using MPLA or R848 alone as adjuvants. Collectively, the findings from this study may lead to a better understanding of the impact of multiple factors on the immunological efficacy of the hybrid nanoparticle-based nicotine nanovaccine. The findings may also provide significant guidance for the development of other drug abuse and nanoparticle-based vaccines. In addition, the optimized lipid-PLGA hybrid nanoparticle-based nicotine nanovaccine obtained by modulating the studied factors can be a promising candidate as the next-generation nicotine vaccine for treating nicotine addiction. / PHD

Thesis (M.Tech.; Homoeopathy)-Dept. of Homoeopathy, Durban Institute of Technology, 2005 xiii, 90 p. : ill. ; 30 cm / A sudden decrease in the use of nicotine containing products, which was used daily for at least several weeks, can cause Nicotine Withdrawal Syndrome (American Psychiatric Association, 1994: 244). The mental symptoms of the withdrawal syndrome includes depressed mood; irritability, frustration, anger, anxiety, difficulty concentrating, restlessness or impatience. The aim of this placebo-controlled double-blind study was to determine the effectiveness of a homoeopathic complex, compared to homoeopathic similimum treatment in the management of tobacco addiction. The complex was based on the selection of those homoeopathic remedies whose symptomology most accurately matched the symptoms associated with smoking cessation.

The critical prompt innate immune response is highly built upon the influx of neutrophils from the blood stream to the site of infection. In the battlefield, neutrophils sense pathogen-associated molecular patterns (PAMPs) through their pattern-recognition receptors (PRRs) to launch a number of responses with the goal to defeat the invading pathogen. Neutrophils’ wide spectrum of responses ranges from reactive oxygen species production (ROS), phagocytosis, cytokine and chemokine secretion, and neutrophil extracellular trap (NET) formation. The NET scaffold is composed of nuclear chromatin which is armed with antimicrobial proteins. DNA traps are able to ensnare and kill microbes in the extracellular space and NET release concurs with cell death of the neutrophil. An increasing body of literature describes that NETs impose deleterious effects on the host itself in addition to their antimicrobial activity. These hazardous effects mainly stem from pro-inflammatory and tissue-destructive activity of NETs. These two diverse outcomes of NETs result in a series of effects on both host and pathogen. Therefore, it seems rational that NET formation is tightly regulated and not happening spontaneously. The opportunistic fungal pathogen Candida albicans captured and killed by NETs. This fungus has the remarkable ability to grow as budding yeast or as filamentous hyphae, and reversibly alternate between these morphotypes. Hyphae are the tissue-destructive, invasive and pro-inflammatory form of C. albicans, whereas yeast is the proliferative, non-invasive form. Hence, it is important to find out how neutrophils discriminate between distinct growth forms of C. albicans and how NET release is regulated in this regard. To assess neutrophils responses towards each growth form of C. albicans, the mere ratio of each fungal morphotypes is an insufficient measure to describe comparable amounts used in infection experiments; we therefore used dry mass of fungal cells to serve as a common denominator for amounts of fungal cells with different morphotypes. As assessment of dry mass is laborious, we developed a quick correlative method, which quantified fungal metabolic activity corresponding to the actual dry mass. We applied this method in consecutive studies investigating the neutrophil responses specific to different morphotypes of C. albicans. Positive and negative regulators of NET formation were investigated for this thesis in a mechanistic fashion. To identify how NET release is negatively regulated during C. albicans infection we focused on anti-inflammatory receptors on neutrophils. We observed that adenosine signals via adenosine receptor reduces the amount of NETs exclusively in response to C. albicans hyphae, the invasive, pro-inflammatory form. We identified adenosine receptor A3 as the responsible receptor suggesting that targeting of adenosine A3 would be a promising approach to control invasive fungal infection, since particularly during immune reconstitution invasive mycoses are frequently accompanied by hyperinflammation which additionally worsens the patient’s state. As unbalanced inflammation is harmful to the host, a situation reflected in autoimmune diseases, such as systemic lupus erythematosus, we aimed to find molecules, which are able to inhibit NET formation. Thus, we introduced the non-toxic agent tempol’’. During ROS-depended stimulation of NET formation via C. albicans and phorbol esters, the stable redox-cycling nitroxide tempol efficiently blocked NET induction. We therefore proposed tempol as a potential treatment during inflammatory disorders where NET formation is out of balance. In quest for positive regulators of NET formation we found the major addictive component of tobacco and electronic cigarettes, nicotine, as compelling direct inducer of NET release. Interestingly, nicotine is associated with exacerbated inflammatory diseases exerting its pro-inflammatory activity via acetylcholine receptor by targeting protein kinase B (known as Akt) activation with no effect on NADPH oxidase complex in a ROS independent fashion. In consideration of neutrophils role in smoking-related diseases we propose targeting Akt could lower the undesirable effect of NET.  In conclusion, this thesis identified new modulators of NET formation in response to fungal infection and more broadly to other NET-inducing stimuli, which might have implications in forthcoming therapies.

published_or_final_version / Dentistry / Doctoral / Doctor of Philosophy

Dissertation submitted in partial compliance with the requirements for the Master's Degree in Technology, Homoeopathy, Technikon Natal, 1996. / The aim acupuncture of this study was to compare the treatment and homoeopathic treatment smoking cessation. The study was a clinical trial, in which an acupuncture treatment group group. Thirty was compared with a homoeopathic treatment volunteers who were anxious to stop smoking participated in the trial and were randomly divided into two groups. One group received acupuncture treatment (n=l5), while the other group received homoeopathic treatment (n=l5) in the form of isotherapy. The study took place over a period of 9 months and each participant completed a 3-month trial period. Prior to the trial period each sUbject completed a set of questionnaires. These ~ncluded the Fagerstrom Tolerance Test which determined their dependence on nicotine; a questionnaire to determine if the subject smoked predominantly for reduction of negative affect, for positive affect or as an habitualaddiction; and a questionnaire to determine the subject's estimation of the health risks of smoking. / M

Pharmacotherapies for tobacco and alcohol cessation are only modestly successful, so it is important to better understand mechanisms underlying their use and abuse. The overarching goal of this research is to assess a6b2 subunit containing nicotinic acetylcholine receptor (a6b2*nAChR; *denotes possible assembly with other subunits) contributions to abuse-related effects of nicotine and alcohol. In the absence of a6b2*nAChR-selective agonists, a6b2*nAChR gain-of-function (a6L9’S) mice provide a tool for selective activation of a6b2*nAChRs. Using the a6L9’S mice together with nicotine doses sub-threshold for stimulation of native nAChRs, these studies tested the hypothesis that activation of a6b2*nAChRs is sufficient to promote neurochemical and behavioral effects relevant to nicotine addiction. Intracranial infusions of an a6b2*nAChR-selective antagonist further tested the neuroanatomical locus of a6b2*nAChR contributions to mesolimbic dopamine (DA) release and nicotine reward behavior. Our in vivo microdialysis and nicotine conditioned place preference (CPP) studies reveal that stimulation of a6b2*nAChRs on ventral tegmental area (VTA) DA neurons, as well as on DA terminals in the nucleus accumbens (NAc) shell support nicotine reward. VTA a6b2*nAChR stimulation is required for elevated basal NAc DA levels in a6L9’S mice, who also show elevated nicotine CPP. These studies also showed elevated anxiety-like behavior in a6L9’S mice, but no change in a6 subunit null mutant (a6KO) mice to suggest that elevated cholinergic tone at a6b2*nAChRs promotes anxiety-like behavior. To better define the molecular make-up of a6b2*nAChRs supporting nicotine reward and anxiety-like behavior, these studies crossed a6L9’S to a4 subunit knockout mice to differentiate (non-a4)a6b2* and a4a6b2*nAChR contributions. (non-a4)a6b2*nAChRs appear to promote nicotine reward behavior, while the a6b2*nAChR subtype that regulates anxiety-like behavior depends on the anxiety assay. Finally, these studies developed a mouse model of oral operant ethanol (EtOH) self-administration and assessed EtOH reinforcement in a6 heterozygous (a6HET) and a6KO mice to characterize the role of a6b2*nAChRs in EtOH reinforcement. EtOH self-administration was similar to wild type mice in a6KO mice, but not a6HET mice, suggesting that expression of a6b2*nAChRs modulates EtOH reinforcement. Together, these preclinical studies implicate a6b2*nAChRs in various abuse-related effects of nicotine and alcohol, identifying this receptor as a potential therapeutic target for treatment of dependence.

Developmental nicotine exposure (DNE) is associated with increased risk of cardiorespiratory, intellectual, and behavioral abnormalities in neonates, and is a risk factor for apnea of prematurity, altered arousal responses and Sudden Infant Death Syndrome. Alterations in nicotinic acetylcholine receptor signaling (nAChRs) after DNE lead to changes in excitatory neurotransmission in neural networks that control breathing, including a heightened excitatory response to AMPA microinjection into the hypoglossal motor nucleus. Here, we report on experiments designed to probe possible postsynaptic and presynaptic mechanisms that may underlie this plasticity. Pregnant dams were exposed to nicotine or saline via an osmotic mini-pump implanted on the 5th day of gestation. We used whole-cell patch clamp electrophysiology to record from hypoglossal motoneurons (XIIMNs) in thick medullary slices from neonatal rat pups (N = 26 control and 24 DNE cells). To enable the translation of our findings to breathing-related consequences of DNE, we only studied XIIMNs that were receiving rhythmic excitatory drive from the respiratory central pattern generator. Tetrodotoxin was used to isolate XIIMNs from presynaptic input, and their postsynaptic responses to bath application of L-glutamic acid (glutamate) and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) were studied under voltage clamp. DNE had no influence on inward current magnitude evoked by either glutamate or AMPA. However, in cells from DNE animals, bath application of AMPA was associated with a right shift in the amplitude distribution (P = 0.0004), but no change in the inter-event interval distribution of miniature excitatory postsynaptic currents (mEPSCs). DNE had no influence on mEPSC amplitude or frequency evoked by glutamate application, or under (unstimulated) baseline conditions. Thus, in the presence of AMPA, DNE is associated with a small but significant increase in quantal size, but no change in the probability of glutamate release.

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Orientador: Ercy Mara Cipulo Ramos / Banca: Dionei Doffinger Ramos / Banca: Mariangela Macchione / Resumo: Introdução: A exposição ao cigarro promove alterações que prejudicam a eficácia do transporte mucociliar. Contudo, a influência da intensidade de exposição, assim como os efeitos da abstinência ao tabagismo sobre essas alterações foram pouco elucidados. Objetivos: Avaliar a influência de diferentes intensidades de exposição ao cigarro sobre o transporte mucociliar e o efeito da cessação do tabagismo sobre o transporte mucociliar nasal em fumantes avaliados durante um período de 180 dias. Casuística e Métodos: Participantes de um programa de cessação ao tabagismo, foram avaliados quanto ao histórico tabagístico, ao nível de dependência à nicotina, à avaliação da função pulmonar (espirometria), a concentração de monóxido de carbono no ar exalado (COex), ao nível de carboxihemoglobina (COHb) e ao transporte mucociliar (tempo de trânsito de sacarina - TTS). Para comparação foi avaliado um grupo... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Introduction: Exposure to cigarette smoke promotes changes that harm the effectiveness of the mucociliary clearance. However, the influence of the intensity of exposure, as well as the effects of abstinence from smoking on these changes is poorly understood. Objectives: To assess the influence of different intensities of exposure to cigarette smoke on mucociliary clearance and the effect of cessation of smoking on nasal mucociliary clearance in smokers evaluated over a period of 180 days. Methods: Participants from a smoking cessation programme, were evaluated about smoking behavior, level of nicotine dependence, lung function (spirometry), the carbon monoxide in exhaled air (exhaled CO), the carboxyhemoglobin (COHb) and mucociliary clearance (saccharin transit time - STT). Was evaluated for comparison... (Complete abstract click electronic access below) / Mestre

Navigation through the environment requires the ability to select relevant information from a multitude of irrelevant stimuli. Under conditions of processing conflict, attention and cognitive control processes bias sensory input based on internal goals. These processes are supported by the interplay of a fronto-parietal attention network that exerts a top-down influence on information processing and a superior temporal network that operates in parallel and that responds in a stimulus-driven manner to behaviorally salient stimuli. It is often reported that nicotine can enhance top-down attention control and reduce distraction. In experiments 1 and 2, the effects of increasing control demands on behavior were assessed using electrophysiological (EEG) and behavioral measures in an auditory number parity decision task with different levels of distraction. Participants made forced choice ‘odd’ or ‘even’ number decisions, while ignoring preceding or simultaneous novel distractors. A group of non-smokers was compared to overnight abstinent smokers (9 hours) and after nicotine intake via 2 mg nicotine tablet or via smoke-inhaled nicotine. The results revealed that preceding distractors impaired task performance due to orienting to and reorienting from the distractor. Simultaneous distractors did not cause orientation of attention (indicated by absence of a P3a Event-Related Potential) and produced smaller increments in response latencies. However, this type of complex novel stimulus initiated processes of memory updating that significantly impaired response sensitivity and accuracy. Nicotine withdrawal enhanced these distraction effects, whereas nicotine intake, particularly via smoking, normalized performance. In experiment 3, dichotic listening performance in a group of non-smokers was compared to abstinent smokers (12 hours) using behavioral, EEG and functional Magnetic Resonance Imaging (fMRI) measures. The perceptual salience of the stimuli was manipulated by systematically varying the Inter-aural Intensity Difference (IID) between them. The analysis pointed to distinct brain networks that differentially activate depending on the level of competition between sensory inputs and these effects were additionally modulated by nicotine withdrawal. Nicotine withdrawal impaired behavioral performance supported by evidence of enhanced use of memory and attention resources, and some evidence of task-independent default mode network activation. Overall, the findings suggest that withdrawal from nicotine, particularly in heavy smokers, is associated with impairments in cognitive control and that subsequent intake of nicotine serves mainly to normalize performance.