No description available.

Activation of nociceptors causes them to secrete neuropeptides, such as substance P (SP) and calcitonin-gene related peptide (CGRP). By reacting with receptors on blood vessels these peptides contribute to inflammation by evoking vasodilation and increasing vascular permeability that allows proteins and fluid to leave the blood vessels (plasma extravasation, PE). These substances can also lead to the sensitization of nociceptors and the resulting positive feedback thereby prolongs inflammation and pain. Thus, blocking the release of neuropeptides may have important therapeutic value in pain conditions where neuropeptides have been implicated. Therefore, the aim of this study was to define the time course of changes in vascular permeability and to test the ability of novel agents to modulate this response. PE and vasodilation was evoked by stimulating the saphenous nerve or by injecting the chemical irritant capsaicin into the rat hindpaw. Changes in blood flow were evaluated with a laser Doppler and digitized image analysis was used to measure changes in reflectance of the skin due to accumulation of extravasated (EB) dye. Analysis of the change in pixel intensity in the digitized images revealed that the magnitude of PE was dependent on the stimulus pulse number. Moreover, the time course of enhanced vascular permeability produced by electrical stimulation was a transient event compared to a much longer response with capsaicin. It was also demonstrated that sumatriptan, (a 5-HT1B/D receptor agonist) and botulinum neurotoxin type-A were effective treatments for capsaicin and saphenous nerve induced vasodilation and PE. Neither drug interfered with activation of the SP or CGRP receptor, which may suggest that both drugs work by inhibiting neuropeptide release. Therefore, this study has described the time course of vascular permeability evoked by two different stimuli and has demonstrated the ability of two novel agents to attenuate these responses.

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation. / Thesis (Master, Anatomy & Cell Biology) -- Queen's University, 2013-07-05 14:37:05.15

Abstract Cordia africana (Boraginaceae) is a tree used in traditional medicine to treat inflammation related conditions and infectious diseases. This study was undertaken with the objectives of establishing the scavenging effect of extracts and fractions of Codia africana on the mediator of inflammation Lipoxygenases (LOX), and some non-biological free radicals such as 2,2-diphenyl-1-picrylhydrazyl (DPPH), the [2, 2-azinobis (3-ethylbenzothiazoline-6-sulfonic acid)] (ABTS) radicals and the Ferric ion reducing antioxidant power (FRAP). Antimicrobial activities, total phenolics/flavonoids and cytotoxicity of extracts of Codia africana were also evaluated. Extracts were obtained by maceration. Anti-inflammatory activity was determined using a LOX-inhibitor screening assay kit according to the manufacturer's instructions. A broth serial micro dilution method was used to determine the minimum inhibitory concentration (MIC) against, Gram-positive and Gram-negative bacteria and Mycobacterium species. The antioxidant activity was determined using free-radical-scavenging assays, and the 3-(4,5- dimethylthiazolyl-2)-2,5- diphenyltetrazolium bromide reduction assay was used for cytotoxicity. Both the extracts of C. africana inhibited LOX enzyme. The most active being the methanol extract of the bark with IC50 value of 55 ± 0.9 μg/ml. Both the extracts of C. africana, had excellent to weak antimicrobial activites (MICs ranging from 32 to 1024 μg/ml) against bacteria. All the extracts had significant (P< 0.05) free-radical scavenging activity (IC50 ranging from 6.79 ± 0.07 to 331.98 ± 0.07 μg/ml). There was a positive correlation between the antioxidant activity and the total flavonoid and total phenolic contents of Cordia africana. The cytotoxicity on Vero cells was low with LC50 of 81.79 ± 13.31 and 99.67 ± 16.10 μg/ml. The results support the use of C. africana leaves in traditional medicine to treat inflammation related conditions and infectious diseases.

Sepsis is the major cause of mortality on intensive care units (ICU) with ~36,000 deaths annually in the UK. Sepsis is a systemic, dysregulated activation of the innate immune system in response to an infection characterised by excessive inflammatory mediator production and oxidative stress. Mitochondrial dysfunction is implicated in sepsis-­‐induced organ dysfunction and death. Zinc is an essential micronutrient with a multitude of biological functions, including anti-­‐inflammatory and antioxidant properties. A relationship has been established between zinc deficiency and severity of sepsis, in which zinc deficiency negatively influences the processes of sepsis leading to organ damage and ultimately death. This study investigated the effect of zinc on sepsis-­‐related mechanisms to evaluate its importance for sepsis pathophysiology. The relationship between zinc levels and sepsis-­‐related molecular mechanisms were investigated in an endothelial cell culture model of sepsis and in blood samples obtained from patients on ICU with and without sepsis. The in vitro study showed no evidence of zinc as an antioxidant or anti-­‐inflammatory agent in endothelial cells exposed to lipopolysaccharide and peptidoglycan, however mitochondrial baseline function was increased in a zinc concentration-­‐dependent manner. Plasma zinc levels were far below normal in all patients and patients with sepsis had lower levels compared to non-­‐infected patients, possibly because they were overall more severely ill. No clear correlations could be established between plasma zinc and markers of inflammation, oxidative stress or disease severity. The lack of anti-inflammatory and antioxidant properties of zinc in the endothelial sepsis model, and the lack of clear correlations between zinc and markers of disease severity, inflammation and oxidative stress in the clinical study, challenges the concept of the importance of zinc in the pathophysiology of sepsis. The prolonged reduction of plasma zinc in all ICU patients prompts to consideration of zinc supplementation to replenish plasma levels and assure sufficient availability to maintain tissue functions.

Micturition requires the precise reciprocal function of the urinary bladder and urethral outlet. Perhaps due to this degree of precision, micturition is prone to dysfunction with injury or disease. One such disease, interstitial cystitis (IC)/painful bladder syndrome (PBS), is characterized by urinary urgency, frequency and pelvic pain. Inflammation has been implicated as a factor in IC/PBS. The overall hypothesis of this project is that urinary bladder inflammation induces expression of inflammatory mediators and changes neurotrophin receptor expression that contribute to functional changes in the urinary bladder. Using a well-characterized rat model of cyclophosphamide (CYP)-induced urinary bladder inflammation, the expression and function of cyclooxygenase-2 (COX-2), a known inflammatory enzyme, and the p75 neurotrophin receptor (p75NTR), involved in neurotrophin signaling, were examined in micturition reflex pathways using neuroanatomical, biochemical, molecular and physiological techniques. Although COX-2 expression is increased in urinary bladder and involved in bladder hyperreflexia after CYP-induced cystitis, the localization and time course of upregulation was not known. We hypothesized increased COX-2 expression in specific tissue compartments (urothelium or smooth muscle) of the urinary bladder with CYPinduced inflammation. Western blotting for COX-2 showed a significant increase in COX-2 expression in both detrusor and urothelium/suburothelium, with the greatest increase in the urothelium/suburothelium. Immunostaining showed increased COX-2 staining in suburothelium with cystitis, co-localized with CD86, a marker for dendritic cells and macrophages. Nerve growth factor (NGF) has been implicated in inflammation, increased voiding frequency and altered sensation in urinary bladder. The specific NGF tyrosine kinase receptor, TrkA, is increased in bladder afferent cells with CYP. NGF also binds p75NTR. The second goals were to examine the expression and functional role of p75NTR in urinary bladder pathways in control and CYP-treated rats. We hypothesized that p75NTR is constitutively expressed in micturition pathways and upregulated with cystitis. With cystitis, p75NTR expression was increased in lumbosacral spinal cord and in bladder afferent cells in dorsal root ganglia. Western blotting for p75NTR showed increased expression in whole urinary bladder with cystitis. Based on bladder function effects of TrkA blockade with cystitis, we hypothesized that p75NTR blockade in the urinary bladder would also decrease bladder hyperreflexia with cystitis. The functional role of p75NTR was studied by intravesical blockade by immunoneutralization with a monoclonal antibody to p75NTR and by PD90780, known to block NGF-p75NTR binding. Both forms of p75NTR blockade significantly decreased bladder capacity in control and CYP-treated rats. Changes in micturition and threshold pressure, and non-voiding contractions were also demonstrated. In conclusion, these dissertation studies demonstrate that CYP-induced bladder inflammation alters expression of inflammatory mediators and neurotrophin receptors in micturition pathways. This altered expression can affect overall urinary bladder function.

Dans cette étude, par une approche de type "génome entier", les profils d'expression génique des macrophages du tissu adipeux viscéral (ATM) et ceux des macrophages issus de la différenciation in vitro de monocytes (MDM) circulants, provenant d’un même individu obèse, ont été comparés à l'état basal. Nos résultats ont permis d’identifier plusieurs voies de signalisation différemment exprimées entre ces deux types cellulaires. En particulier des cytokines inflammatoires et leurs récepteurs, des composants de la matrice extracellulaire de même que des molécules attractantes telles que les chimiokines de type CCL et CXCL, sont fortement exprimés dans les ATM. Fait intéressant, les protéines appartenant à ces voies ont été retrouvées sécrétées dans le milieu conditionné provenant de la culture des ATM, ou en concentrations élevées dans le sérum des patients obèses comparés à celui de patients minces. Nous avons voulu valider les différences observées, en reconstituant l’environnement obésogène des ATM dans le TA grâce à l’utilisation de "co-cultures" indirectes de préadipocytes et de MDM. Dans leur ensemble, nos résultats montrent que les ATM ont un profil génomique et fonctionnel qui ressemble à celui des macrophages associés aux tumeurs (TAM). De plus, nous retrouvons au sein des gènes spécifiquement exprimés par les ATM une surexpression et une activation des facteurs de transcription NF-kB, HIF1a et STAT-3, connus comme impliqués dans le développement des cancers et qui sont à l’origine de la plasticité des TAM. Dans ce contexte, les Liver X Receptors (LXR) a et b, de même que les Peroxisomes Proliferator-Ativated Receptor gamma (PPARg) sont des récepteurs nucléaires exprimés dans les MDM qui apparaissent comme des cibles thérapeutiques de grand intérêt du fait de leurs propriétés anti-inflammatoires. Le second objectif de notre étude a été de savoir, toujours par une approche de type "génome entier", si les ATM et les MDM répondent différemment aux agonistes de LXR. De nouveaux gènes cibles de LXR spécifiques des ATM, ont été identifiés et en particulier nous montrons que les chimiokines ligands CXCL5 et CXCL11 surexprimés dans les ATM (et impliquées dans les cancers), sont négativement régulés par les agonistes de LXR. De plus, notre étude nous a permis d’analyser le rôle des LXR dans la modulation du "cross-talk" entre ATM et adipocytes en évaluant l’influence des facteurs sécrétés par les ATM, préalablement traités ou non par les agonistes des LXR, sur le métabolisme adipocytaire. En effet, nos travaux démontrent que le milieu conditionné des ATM traités par les ligands de LXR restaure partiellement l'expression de gènes impliqués dans la lipogenèse et le métabolisme du glucose e diminue l’expression des gènes impliqués dans la réponse inflammatoire dans les adipocytes.Dans une autre partie de notre étude, la capacité de PPARg et/ou de LXR à réguler l’expression de la visfatine dans les macrophages a été analysée, de même que l’influence de cette régulation sur sa sécrétion et sur la production intracellulaire de NAD+. Au sein du TA, les ATM sont la principale source de visfatine dont l'implication dans le développement de certaines pathologies dont le DT2 est largement reconnue. La visfatine (Nampt) est une adipokine pro-inflammatoire mais aussi une enzyme assurant la synthèse du NAD+, cofacteur ou substrat de nombreuses enzymes cellulaires. Nos résultats permettent de montrer que l’activation de LXR ou de PPARg par leurs ligands entraîne respectivement une diminution ou une augmentation de l'expression génique de la visfatine. L’augmentation de l’expression de l’ARNm de la visfatin suite à l’activation de PPARg s’accompagne d’une augmentation de l’expression protéique et de la sécrétion. / X

In this thesis I show that macrophages (both cell lines and primary cultures) can be transfected by recombinant adenoviruses expressing -galactosidase (A-gal/Av1Bng), that the macrophages become activated by the transfection process and can be easily manipulated to localise to inflamed glomeruli after direct injection into the renal artery of rats with an experimentally induced glomerular inflammation caused by nephrotoxic nephritis. The injection of transfected macrophages reduces the severity of injury in this model of glomerulonephritis as shown by a reduction in the degree of albuminuria. This approach provides a favourable system for gene delivery in inflammatory disease and shows that both the functional properties of the transfected macrophage as well the transgene it is engineered to produce are relevant for in vivo gene transfer. This approach has also been used to determine the effect of macrophages expressing active TGF-1 on the development of glomerular inflammation. TGF-1 expressing macrophages localised efficiently to inflamed glomeruli and produced the cytokine in vivo. These cells produced a reduction in the level of albuminuria compared to unmodified disease but not in comparison to injection of macrophages transfected with adenovirus expressing -galactosidase. In addition there was no alteration in the infiltration by ED1 positive macrophages. Thus TGF-1 expressed in this manner appears unable to significantly modulate glomerular inflammatory disease and the potential reasons for this are discussed. The system I have developed of macrophage transfection and delivery provides a valuable approach to study and modulate inflammatory disease.

University of Macau / Institute of Chinese Medical Sciences

Chronic pain is one of the most significant medical and scientific challenges in western society today. As well as the emotional and physical effects on the individual it proves to be a significant financial burden to society. Studies have shown that chronic pain leads to central sensitisation that is partially regulated by release of proinflammatory molecules within the CNS. Most work has concentrated on the role of the spinal cord and little is known about changes in supraspinal regions. The CFA footpad model was used to investigate the expression of inflammatory mediators in the brain in persistent inflammatory pain. Using RT-PCR, gene expression of inflammatory mediators was measured in various brain regions. Consistent with previous reports at 7 and 14 days post-injection IL1β expression was significantly elevated in the posterior of the brain (p<0.05), TNFα showed differential expression with a significant increase in the posterior ipsilateral brain region at 72 hours (p<0.05). The chemokine CXCL1 was significantly elevated at 6 hours post-injection (p<0.05) suggesting a role for this chemokine in regulation of the acute pain response. Contrary to evidence from the spinal cord, CX3CL1 and its receptor CX3CR1 were down regulated in the brain at 6 and 24 hours post-injection. Differential expression of astrocyte activation was identified by GFAP immunochemistry. Hypoxia has been implicated in neurodegeneration, a process thought to play a role in chronic pain. Here Hypoxia inducible factor (Hif1α) mRNA expression within the brain was not altered in a CFA model of peripheral inflammation. Interestingly, using Hypoxyprobe immunohistochemistry, a higher level of hypoxia was identified in non-injected controls than in CFA treated animals. This is the first evidence of differential chemokine expression in the brain in persistent inflammatory pain and the first study to suggest a potential role for differential oxygenation within the brain in this condition.