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A Mechanistic Investigation of Anesthesia-Induced Spatial Learning Deficits in Aged RatsMawhinney, Lana J 29 April 2011 (has links)
Anesthesia-induced spatial learning impairments in aged rats model postoperative cognitive dysfunction (POCD) in the elderly surgical population. Mechanisms underlying both normal age-related cognitive decline and anesthesia-induced spatial learning deficits in aged rats were investigated. With respect to the involvement of inflammasome activation and age-related cognitive decline, I hypothesized that the aged hippocampus exhibits an elevated activation of inflammasome components contributing to elevated levels of IL-1β in the aged brain. Age-related cognitive decline was identified in a subpopulation of male Fischer 344 rats. Activation of the NLRP1 inflammasome was elevated in the aged brain, contributing to spatial learning deficits in aged rats. With respect to anesthesia-induced spatial learning impairment in aged rats, I hypothesized that an increase in NR2B subunit in the hippocampus and cortex during and following isoflurane anesthesia exposure resulting in spatial learning impairment in aged rats via disruption of downstream signaling molecule, extracellular-signal regulated protein kinase (ERK). Anesthesia exposure resulted in chronic spatial learning impairment in aged rats that were previously unimpaired in spatial learning tasks. Additionally, anesthesia induced elevated levels of N-methyl-D-aspartate (NMDA) receptor NR2B subunit protein expression in aged. It was concluded that various factors contribute to age-related spatial impairment including: NLRP1 inflammasome activation and NMDA receptor NR2B protein expression elevation. It was also concluded that anesthesia exposure exacerbates the elevation in NR2B protein expression in the aged brain, with subsequent disruption of ERK activation leading to chronic spatial learning deficits in aged rats. In the final chapter, a relationship for the interplay between inflammation and NMDA receptor function in the aged brain is discussed. In addition, a novel mechanism for anesthesia-induced cognitive deficits is presented. Therapeutic treatments for cognitive decline and anesthesia-induced cognitive deficits are explored. Finally, future lines of research are proposed.
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The FIIND Domain of Nlrp1b Promotes Oligomerization and Pro-caspase-1 Activation in Response to Lethal Toxin of Bacillus anthracisJoag, Vineet 29 November 2012 (has links)
Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.
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The FIIND Domain of Nlrp1b Promotes Oligomerization and Pro-caspase-1 Activation in Response to Lethal Toxin of Bacillus anthracisJoag, Vineet 29 November 2012 (has links)
Lethal toxin (LeTx) of Bacillus anthracis kills murine macrophages in a caspase-1 and Nod-like-receptor-protein 1b (Nlrp1b)-dependent manner. Nlrp1b detects intoxication, and self-associates to form a macromolecular complex called the inflammasome, which activates the pro-caspase-1 zymogen. I heterologously reconstituted the Nlrp1b inflammasome in human fibroblasts to characterize the role of the FIIND domain of Nlrp1b in pro-caspase-1 activation. Amino-terminal truncation analysis of Nlrp1b revealed that Nlrp1b1100-1233, containing the CARD domain and amino-terminal 42 amino acids within the FIIND domain was the minimal region that self-associated and activated pro-caspase-1. Residues 1100EIKLQIK1106 within the FIIND domain were critical for self-association and pro-caspase-1 activation potential of Nlrp1b1100-1233, but not for binding to pro-caspase-1. Furthermore, residues 1100EIKLQIK1106 were critical for cell death and pro-caspase-1 activation potential of full-length Nlrp1b upon intoxication. These data suggest that after Nlrp1b senses intoxication, the FIIND domain promotes self-association of Nlrp1b, which activates pro-caspase-1 zymogen due to induced pro-caspase-1 proximity.
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Macrophage Activation and Differentiation with Cholesterol CrystalsBurrowes, Hannah Mahony January 2012 (has links)
Cholesterol crystals have been linked to activation of the NLRP3
inflammasome and the formation of foreign body giant cells (FBGCs). It
has been hypothesized that FBGCs have a role in advanced atherosclerotic
plaque formation. This thesis examined the feasibility of producing
stable cultures of FBGCs starting with human monocytes with the
goal to examine pterin production by these cells in comparison to
human monocyte derived macrophages (HMDMs). The study also
investigated the effect of cholesterol crystals on 7,8-dihydroneopterin
(7,8-NP) production and modulation of IL-1β levels in macrophages.
7,8-Dihydroneopterin is a potent antioxidant generated by macrophages
which also down regulates the expression of macrophage scavenger
receptor CD36. The use of alpha-tocopherol and IL-4 as FBGC fusion
mediators was explored. Using these mediators, large numbers of
FBGC were successfully cultured. The rates of fusion achieved in the
cultures were low, and the cells had poor adhesion, which prevented
pterin measurement. FBGC, which are thought to remove crystallized
cholesterol from the plaque, cleared 21% of cholesterol crystal compared
to 50% cleared by HMDM cells. Due to this result, the effect of
cholesterol crystals on pterin production in monocytes and macrophages
was explored. Cholesterol crystals cause inflammation through the
activation of the NLRP3 inflammasome, however, it was unknown
whether they could modulate 7,8-NP production. Cholesterol crystals
caused an intracellular dose-dependent loss of 7,8-NP to its oxidized form,
neopterin, in HMDM cells. Cholesterol crystals induced intracellular
synthesis of 7,8-NP in HMDMs. 7,8-NP was released into the supernatant
and oxidized to neopterin in media. Monocytes treated with cholesterol
crystals released up to 100 nM of neopterin and 120 nM of 7,8-NP in
the media after 48 hours. The combination of IFN- and cholesterol
crystals appeared to inhibit the release of 7,8-NP into the media for the
first 48 hours, after this time 7,8-NP release rapidly increased. The
addition of exogenous 200 μM 7,8-NP showed that in the presence of
monocytes, cholesterol crystals did not cause the oxidation of 7,8-NP to
neopterin, as seen in HMDMs but possibly to 7,8-dihydroxanthopterin
or xanthopterin. The presence of 7,8-NP increased IL-1β expression in
the presence of cholesterol crystals after 24 hours incubation. FBGCs
and the removal of cholesterol crystals may be a key process in the
resolution of atherosclerotic plaques. It appears that cholesterol crystals
are able to modulate inflammatory processes including activation of
the inflammasome and balance of 7,8-dihydroneopterin to the oxidized
neopterin. The infiltrating monocytes may provide antioxidant protection
against the inflammation induced by cholesterol crystals and the activity
of the infammasome.
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NUCLEOSIDE REVERSE TRANSCRIPTASE INHIBITORS ARE ANTI-INFLAMMATORY AND TARGET DRY AGE-RELATED MACULAR DEGENERATIONFowler, Benjamin J 01 January 2014 (has links)
Age-related macular degeneration (AMD) is a principal cause of blindness in the United States and other industrialized nations. An estimated 10 million Americans are afflicted with AMD, which is comparable in scope to the 12 million living with cancer, or the 5 million with Alzheimer’s disease. The prevalence of AMD steadily increases with age, affecting 2% of the population at age 40, and one in four people by age 80. For reasons that are not fully understood, AMD is more common in lightly-pigmented and female populations. Treatment of AMD is largely an unmet need: There are no FDA approved therapies except for a small percentage of individuals with end-stage disease. This dissertation investigates the mechanisms of AMD pathogenesis and offers insight into novel therapeutic strategies for this disease.
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Immunité innée et inflammasome : rôle des signaux de dangers endogènesRiteau, Nicolas 22 September 2011 (has links) (PDF)
La théorie du danger développée par Polly Matzinger stipule que l'attrait principal du système immunitaire ne réside pas dans la distinction entre le soi " à protéger " et le non-soi " à combattre ". Toute situation potentiellement délétère pour l'hôte, avec émission de signaux de dangers endogènes, est à ce titre capable de générer une réponse immunitaire afin de mobiliser les acteurs capables de permettre un retour à une situation basale. L'exposition des poumons de façon répétée à des agents toxiques environnementaux se traduit par une inflammation et une fibrose pulmonaire en condition stérile, c'est-à-dire sans intervention de micro-organisme. L'administration de Bléomycine dans les poumons de souris est un bon modèle pour étudier les molécules endogènes ou signaux de dangers engagés et les voies de signalisations associées. Nous avons identifié l'ATP extracellulaire et l'acide urique, provenant des cellules stressées ou endommagées, comme capables d'induire l'activation d'un complexe protéique cytoplasmique appelé inflammasome Nlrp3. Celui-ci conduit à la maturation de l'interleukine-1β, cytokine pro-inflammatoire. Dans une seconde partie, nous nous sommes intéressés aux mécanismes moléculaires d'activation de l'inflammasome Nlrp3 en réponse à des agents particulaires, responsables notamment de pathologies pulmonaires après inhalation. Nos résultats montrent que de l'ATP endogène est libéré activement par les cellules mises en contact avec des particules. Par un mécanisme autocrine/paracrine l'ATP va ensuite signaler sur plusieurs récepteurs purinergiques membranaires. Cette signalisation purinergique est importante dans la capacité des cellules à produire de l'interleukine-1β mature. Au final, les travaux présentés dans ce manuscrit attestent du rôle critique de molécules endogènes dans la mise en place d'une réponse immunitaire innée basée sur l'activité de l'inflammasome Nlrp3.
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The Role of the Inflammasome During Chlamydia InfectionMcKeithen, Danielle N 29 July 2016 (has links)
Chlamydia trachomatis (C. trachomatis) is the most prevalent sexually transmitted bacteria with devastating reproductive consequences that lead to tubal factor infertility (TFI). Recent studies have implicated apoptosis – associated speck – like protein containing a caspase recruitment domain (ASC) as an adaptor of inflammasomes that stimulate IL – 1β and IL – 18 secretion, pro – inflammatory cytokines with critical functions in host defense against a variety of pathogens. Therefore, for the first time, we are reporting the use of ASC-/- mice in a mouse model of Chlamydia infection that might provide some information on the role of inflammasomes in the pathogenesis of Chlamydia infection. In this study, wild type (WT) and ASC-/- mice were infected with Chlamydia. Infectivity, pathology of the upper genital tract (UGT), and, fertility were evaluated. In addition, expression of ASC – dependent inflammasomes and the activation of immune cells within the genital tract (GT) were studied. Results showed that Chlamydia infectivity in ASC-/- mice was significantly higher (p-/- mice which, when compared to infected WT mice, was exhibited by decrease in average number of pups and percent pregnancy. There was also severe UGT damage in ASC-/- mice compared to WT mice, correlating with the higher number of hydrosalpinx observed on the UGT of Chlamydia infected ASC-/- mice. Furthermore, IL – 1β and IL – 18 production as well as immune cell activation were down regulated in the GT of Chlamydia infected ASC-/- mice. This finding indicates that in absence of ASC, host innate and adaptive immunity is impaired. Results imply that ASC plays a protective role in the mucosal immunity against GT Chlamydia infection.
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Inflammasome activation in ruminant cells infected with Chlamydia abortusDoull, Laura Elizabeth January 2016 (has links)
Chlamydia abortus is the most common known infectious cause of ovine abortion worldwide but is rarely linked with bovine abortion. The reasons for this differential pathogenesis are unknown but may involve differences in innate immune recognition and immune responsiveness. This is supported by the observation that chlamydial abortion in sheep is associated with an inflammatory cytokine/chemokine cascade that is not commonly observed in cattle. Studies with other Chlamydia species have demonstrated that innate inflammatory pathways including inflammasome activation contribute to both pathogen clearance and pathology. Pattern recognition receptors (PRRs) activate these innate immune signalling pathways but are relatively poorly characterized in ruminants. We hypothesize that the ruminant hosts differ in their ability to innately sense C. abortus infection and activate the inflammasome. The main aims of this project were to: analyse PRR expression in innate immune cells; assess cytokine production from innate immune cells in response to C. abortus; investigate the role of PRRs in the induction of innate immune responses to C. abortus; and, conduct RNA-seq analysis on macrophages following infection with C. abortus to identify important immune signalling pathways. Ruminant oro-nasal turbinate cells, monocyte derived dendritic cells (MDDCs) and monocyte derived macrophages (MDMs) express the cell-surface PRRs TLR2 and TLR4 and also the intracellular PRRs NOD 1 and NLRP3. Oro-nasal turbinate cells produce CXCL8 late into the chlamydial developmental cycle independent of IL-1β. In contrast, ruminant MDMs and MDDCs secrete early IL-1β in response to C. abortus infection. In MDMs and MDDCs, live and UV-inactivated C. abortus induced TNF-α and CXCL8 but live infection was required for IL-1β secretion. Therefore, intracellular C. abortus multiplication is necessary to stimulate the IL-1β processing pathway within these cells. In order to determine PRR function, NOD1 and NLRP3 were knocked down in ruminant MDMs using siRNA. In both ovine and bovine MDMs, NOD1 was identified as a factor in C. abortus mediated IL-1β production. NLRP3 knockdown in bovine but not ovine MDMs also reduced IL-1β production, indicating species-specific differences in C. abortus recognition. The RNA-seq analysis of ruminant MDMs identified novel pathways of immune activation by C. abortus and potentially important species-specific differences. An improved understanding of the innate immune pathways activated in susceptible and resistant hosts following C. abortus infection will inform on disease pathogenesis and could contribute to novel chlamydial vaccine design.
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Inflamação, hemólise e ativação celular: importância nos eventos vasculares na anemia falciformeCerqueira, Bruno Antonio Veloso January 2011 (has links)
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Previous issue date: 2011 / Universidade Federal da Bahia. Centro de Pesquisas Gonçalo Moniz. Salvador, Bahia, Brasil / A anemia falciforme (AF) é uma desordem genética caracterizada por hemólise,
estresse oxidativo, infecções recorrentes, estado inflamatório crônico e eventos de oclusão vascular,
com ocorrência de crises de dor, danos teciduais e possibilidade de óbito precoce. Os eventos
vasculares na AF envolvem a participação de hemácias, leucócitos, células endoteliais, plaquetas,
fatores da coagulação e proteínas plasmáticas. Neste estudo, avaliou-se o papel de marcadores
hematológicos, de hemólise, inflamação e de ativação celular na fisiopatologia da ativação endotelial
na AF. Foi realizado um estudo de corte transversal, seguido por caso-controle. Quarenta e cinco (45)
indivíduos com AF em estado estável, com idade média 20,5 anos participaram do estudo. O perfil de
hemoglobinas foi confirmado por HPLC. Os marcadores bioquímicos foram investigados por kits
específicos; a expressão de moléculas na superfície dos leucócitos por citometria de fluxo e a
quantificação de citocinas e moléculas de adesão solúveis por ELISA. Níveis elevados de lactato
desidrogenase (LDH) e bilirrubinas foram associados a eventos de Síndrome Torácica Aguda e úlcera
de perna, respectivamente. A leucocitose foi observada em pacientes com histórico de hepatomegalia
e úlcera de perna. Os pacientes com histórico de terapia transfusional apresentaram concentrações
aumentadas da aspartato aminotransferase (AST) e contagem de reticulócitos. Os níveis diminuídos
de HDL estiveram associados a eventos de sequestro esplênico e esplenomegalia. A expressão do
CD11b em neutrófilos, linfócitos e monócitos foi maior nos pacientes que nos controles; entretanto, a
expressão do CD62L nos neutrófilos foi menor nos pacientes do que nos controles antes e após o
desafio com o lipopolissacarídeo bacteriano (LPS). A expressão do CD11b e CD32 em neutrófilos
teve correlação positiva com plaquetas e com o LDL, respectivamente. A expressão do CD62L em
neutrófilos apresentou correlação negativa com plaquetas. A expressão do CD11b e CD18 em
linfócitos teve correlação positiva com o ácido úrico e com a molécula de adesão vascular solúvel 1
(VCAM-1s), respectivamente. A expressão do CD62L em linfócitos foi negativamente
correlacionada com o número de plaquetas, molécula de adesão intercelular solúvel 1 (ICAM-1s) e
TNF-alfa. A expressão do CD32 em monócitos foi menor nos pacientes que nos controles e
apresentou correlação negativa com a AST e hemoglobina S. A expressão reduzida do CD32 em
monócitos de pacientes foi associada ao aumento da LDH, enquanto que a expressão do CD62L
nessas células apresentou correlação positiva com o HDL e com as concentrações de hemoglobina.
Os níveis de IL-18 e do ácido úrico foram associados a marcadores de hemólise, de disfunção
endotelial e citocinas e podem representar a participação do complexo inflammasome. Os resultados
apresentados sugerem o papel importante de biomarcadores na avaliação do perfil clínico de
pacientes com AF, além de sugerir a provável influência de moléculas expressas na superfície de
leucócitos, da IL-18 e do ácido úrico no processo inflamatório crônico e na gravidade clínica da AF.
Acreditamos que a interação desses biomarcadores envolve processos complexos relacionados à
hemólise, inflamação e ativação celular com importância na avaliação da gravidade dos eventos
vasculares presentes na AF. Em conjunto, todos esses marcadores podem ser considerados como
alvos terapêuticos promissores em intervenções clínicas futuras na doença. / Sickle cell anemia (SCA) is a common, severe monogenetic disorder characterized by
chronic hemolysis, oxidative stress, frequent infections, a chronic inflammatory state and recurrent
occlusions of the microcirculation, resulting in painful crises, organ damage and premature death.
Vaso-occlusive episodes in SCA involve interactions between sickle red blood cells, endothelial
cells, leukocytes, platelets, coagulation factors and plasma proteins. Herein, we evaluated the role of
oxidative stress, inflammation and the profile of cell activation on pathophysiology of vascular
occlusion in SCA. We conducted a cross-sectional study, followed by case control study. Forty-five
SCA patients without general symptoms, median age 20.5 years, were included in this study.
Hemoglobin profile was confirmed by HPLC; biochemical markers by biochemical kits; surface
molecule expressions on leukocytes were determined by flow cytometry and the concentration of
cytokines and soluble adhesion molecules were measured by ELISA kits. The increase serum levels
of lactate dehydrogenase (LDH) and bilirubins were associated with acute thoracic syndrome and leg
ulcer respectively. The clinic aspects like hepatomegaly and leg ulcer were associated with increase
number of leukocyte. The blood transfusion therapy was associated positively with aspartate
aminotransferase (AST) levels and reticulocytes number. The decreased levels of HDL-C were
associated with splenic sequestration and splenectomy. The surface CD11b expression of neutrophils,
lymphocyte and monocytes in patients was higher than in controls; however, CD62L expression on
neutrophils was lower in patients than in controls before and after lipopolysaccharide (LPS)
challenge. CD11b and CD32 expression level on neutrophils were positively correlated with platelet
counts and LDL-C levels respectively. CD62L expression on neutrophils was negatively correlated
with platelet counts. CD11b and CD18 expression levels on lymphocytes were positively correlated
with uric acid and soluble vascular adhesion molecule 1(sVCAM-1) levels respectively. CD62L
expression on lymphocyte was negatively correlated with platelet count, soluble intercellular
adhesion molecule 1 (sICAM-1) and TNF-alpha levels. CD32 expression on monocytes in patients
was lower than in controls, further correlated negatively with AST and hemoglobin S. Decrease
CD32 expression on monocytes was associated with high LDH levels and CD62L expression was
positively correlated with HDL-C and hemoglobin concentration. The IL-18 and uric acid plasma
concentrations were correlated closely with markers of hemolysis, endothelial dysfunction and
cytokine levels, possibly representing the participation of inflammasome complex. We believe that
the interaction of these biomarkers involve complex processes related to hemolysis, inflammation
and cellular activation with importance in assessing severity of the vascular events on the SCA. So,
together, all these biomarkers can be considered as promising therapeutic targets in future clinical
interventions in SCA
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Ion Flux Regulates Inflammasome SignalingJanuary 2015 (has links)
abstract: The NLR family, pyrin domain-containing 3 (NLRP3) inflammasome is essential for the innate immune response to danger signals. Importantly, the NLRP3 inflammasome responds to structurally and functionally dissimilar stimuli. It is currently unknown how the NLRP3 inflammasome responds to such diverse triggers. This dissertation investigates the role of ion flux in regulating the NLRP3 inflammasome. Project 1 explores the relationship between potassium efflux and Syk tyrosine kinase. The results reveal that Syk activity is upstream of mitochondrial oxidative signaling and is crucial for inflammasome assembly, pro-inflammatory cytokine processing, and caspase-1-dependent pyroptotic cell death. Dynamic potassium imaging and molecular analysis revealed that Syk is downstream of, and regulated by, potassium efflux. Project 1 reveals the first identified intermediate regulator of inflammasome activity regulated by potassium efflux. Project 2 focuses on P2X7 purinergic receptor-dependent ion flux in regulating the inflammasome. Dynamic potassium imaging revealed an ATP dose-dependent efflux of potassium driven by P2X7. Surprisingly, ATP induced mitochondrial potassium mobilization, suggesting a mitochondrial detection of purinergic ion flux. ATP-induced potassium and calcium flux was found to regulate mitochondrial oxidative signaling upstream of inflammasome assembly. First-ever multiplexed imaging of potassium and calcium dynamics revealed that potassium efflux is necessary for calcium influx. These results suggest that ATP-induced potassium efflux regulates the inflammasome by calcium influx-dependent mitochondrial oxidative signaling. Project 2 defines a coordinated cation flux dependent on the efflux of potassium and upstream of mitochondrial oxidative signaling in inflammasome regulation. Lastly, this dissertation contributes two methods that will be useful for investigating inflammasome biology: an optimized pipeline for single cell transcriptional analysis, and a mouse macrophage cell line expressing a genetically encoded intracellular ATP sensor. This dissertation contributes to understanding the fundamental role of ion flux in regulation of the NLRP3 inflammasome and identifies potassium flux and Syk as potential targets to modulate inflammation. / Dissertation/Thesis / Doctoral Dissertation Biological Design 2015
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