Published Article / The LPS, endotoxin of Gram-negative organisms in communal growth as compared to pure culture was the focus of this research. The experiment aimed to show pure and communal samples grown in the presence of the extrinsic stresses. The change in toxicity was measured using the Limulus Amoebocyte Lysate (LAL) test. The overall sensitivity of organisms was similar for the same sanitiser and the same detergent. Growth in community was found not to be the arithmetic sum of the individual growth patterns. The detergents had a marked effect on the growth of all samples throughout the growth cycle. This finding reveals that the acceptable refrigeration temperatures still allows for pathogen growth and thus for biofilm formation. The quantification by LAL showed that the enumeration of the food-borne pathogens isolated from households might not be indicative of acclimatisation obtained over short periods of time and the causal stress could turn these organisms into more or less toxic pathogens.

Francisella tularensis is a Gram-negative pathogenic organism that causes the disease tularemia. This disease can be potentially fatal without treatment. Francisella tularensis virulent strains can cause disease in humans with an infectious dose as low as 10 organisms. As a result of this low infectious dose, high mortality, and ease to produce an aerosol inoculum, the Centers for Disease Control and Prevention has classified Francisella tularensis as a Tier I select agent, the highest threat level. Much research has been done to determine the cause for the extreme virulence. However, despite these efforts, little is known about the mechanisms by which Francisella goes undetected inside host cells until it is too late for the host to respond. Researchers in the Jones' laboratory utilized a transposon site hybridization (TraSH) screen with human monocyte derived macrophages (MDMs) as the host cell and an enzyme-linked immunosorbent assay (ELISA) screen of pools of transposon mutants searching for virulence determinants and genes responsible for Francisella capsule or LPS. Through the TraSH screen, our group identified a locus of genes, FTT1236, FTT1237, and FTT1238c as being important for survival within human MDMs. From the mutant library screen using ELISA, I identified the same genes, FTT1236 and FTT1238c. In addition, I also identified wzy, wbtA, FTT0846, and hemH as being involved in LPS and or capsule production. A similar ELISA screen was done by researchers in Apicella laboratory using a different monoclonal antibody that identified insertions in, dnaJ, manB and an intergenic region between FTT0673 and FTT0674c that potentially disrupted LPS and capsule biogenesis. Previously, FTT1236, FTT1237, and FTT1238c mutants were observed by our laboratory to be serum sensitive and activate MDMs by an unknown mechanism. I further characterized these mutant strains by analyzing the changes in the LPS core. I identified core truncations for the FTT1236 and FTT1237 mutants, but not FTT1238c. Combining this new data with previously published work and bioinformatical analysis of the FTT1236, FTT1237 and FTT1238c proteins, I hypothesized that these proteins have functions similar to Waa proteins of other organisms, which are involved in LPS core assembly and O-antigen ligation. With functional complementation and mass spectrometry of LPS preparations, I have designated FTT1236, FTT1237, and FTT1238c as WaaY, WaaZ, and WaaL respectively. In addition to this work characterizing the biochemical functions of these gene products, I examined the effect of mutations in these genes on the virulence of Francisella. In contrast to infection with wild type Schu S4, mice infected either intraperitoneally or intranasally displayed significant inflammatory responses to infection and the strains were significantly attenuated by either route of infection. I also observed that waaY and waaL mutant strains disseminated to the liver and spleen after an intranasal infection despite their lack of O-antigen and capsule. At an i.n. dose of 106 CFU these mutant strains still caused lethal murine infection, but death occurred around day 12 post infection; mice infected with <20 CFU of Schu S4 succumb at day 5 post infection. The cause of the death in mice infected with these mutant strains was pulmonary edema, rather than multiple organ failure induced by Schu S4. Of the additional seven mutant strains identified from the ELISA screens, I characterized their physical phenotypes, virulence defects, and their potential as an attenuated live vaccine. All of these strains were determined to be sensitive to human pooled serum to various degrees. Three of these strains, dnaJ::Tn5, hemH::Tn5, and FTT0673p/prsAp::Tn5 did not have identifiable defects in capsule or LPS biosynthesis, nor were they attenuated in mice. The remaining four strains, FTT0846::Tn5, manB::Tn5, wzy::Tn5, and wbtA::Tn5, were found to have LPS O-antigen and capsule defects, and two of these strains had LPS core defects (FTT0846::Tn5 and manB::Tn5). Each of these four strains was attenuated in mice, when compared to WT. I also tested the ability of mice infected with waaY::TrgTn, waaL::TrgTn, and wbt::Tn5 to be protected from lethal challenges of Schu S4. All three strains provided some level of protection against lethal Schu S4 challenges. In addition, I also tested Francisella LPS and capsule to provide protection against lethal challenges of LVS and Schu S4. I determined that LPS and capsule protected against high doses of LVS, but LPS did not provide any protection when immunized mice were challenged with Schu S4. Interestingly, we observed that mice immunized with capsule were partially protected from lethal Schu S4 challenges. In addition, I observed a novel difference between virulent Francisella strains and LVS, in that virulent strains have O-antigen glycosylated and LVS appears to be lacking this characteristic. Collectively, this work adds to the growing data of the importance of LPS and the role of capsule role in immune evasion as well as the significance of capsule and LPS mutant strains to provide protection against Schu S4.

Abstract Recent studies demonstrated that LPS treated Sprague-Dawley rats induced a reduction (Phase I), followed by an augmentation (Phase II) and a secondary decrease (Phase III) in the power density of vasomotor components (0-0.8 Hz) in systemic arterial pressure signals. The molecular mechanisms underlie the progression toward death in the brain stem is unclear. In order to find out the differentially expressed genes between LPS-treated RVLM and saline-treated RVLM, we used suppression subtractive hybridization,a method commonly used to search differentially expressed genes, and subtractive cDNA library construction. At present, we have found some differentially expressed genes and these genes are up-regulation expression. These genes may be involved in the progression toward death in the rat brain stem.

There are a lot of inflammatory agents, such as substance P, histamine, capsaicin, and lipopolysaccharide¡]LPS¡^. LPS is the component of all gram-negative bacterial cell wall that can stimulate the immune cells to release pro-inflammatory cytokines that can induce systemic acute inflammation and sepsis. The present study sought to investigate the location of leaky microvessels and magnitude of plasma leakage in the rat stomach secretory and non-secretory portions after an intravenous injection of high dose of LPS¡]15 mg/kg¡^. India ink¡]1 ml/kg¡^ and Evans blue¡]30 mg/ml¡^were used as tracer dyes to measure the magnitude of plasma leakage after LPS application. In the whole mounts of the stomach secretory and non-secretory portions with silver staining, the boundaries of endothelial cells and gaps between endothelial cells for plasma leakage in the blood vessels of microcirculation were made visible. Tissue sections were stained with Alcian blue and periodic acid-Schiff reagent to reveal the mucosubstance present in the mucous cells of the stomach secretory tissue. The result of study demonstrated that the magnitude of LPS-induced plasma leakage in the rat stomach secretory and non-secretory portions was larger than that of the vehicle control. Extensive plasma extravasation was found from 5 min to 30 min after LPS injection. Numerous endothelial gaps formed in both postcapillary venules and collecting venules in the rat stomach secretory portion at 5 min and 30 min after LPS, but the number of gaps declined strikingly 60 min after LPS. Endothelial gaps were rare in the stomach of vehicle control. Histological sections of the stomach secretory portion showed that the leaky vessels were present in the serosa and muscularis externa. Administration of LPS also resulted in release of mucus in gastric gland cells. It is concluded that endotoxin-induced increase in plasma leakage correlated with the formation of endothelial gaps, and associated with depletion of mucosubstance from the mucous cells.

Macrophages are mononuclear phagocytic cells that have diverse roles within the body. Tissue specific macrophages, e.g. Kupffer cells, microglia and osteoclasts, have roles in tissue homeostasis, while circulating macrophages play an important role in the innate immune system. Macrophages detect the presence of pathogen associated molecular patterns (PAMPs) via a range of receptors known collectively as pathogen recognition receptors (PRRs). Detection of pathogens causes the macrophages to become ‘activated,’ during which the macrophages undergo extreme morphological and translational changes that enable the pathogen to be neutralised and other immune system components to be recruited. Macrophage activation must be carefully regulated and promptly resolved, as chronic inflammation is damaging to the host. MicroRNAs have emerged as one mechanism by which activation is regulated. MicroRNAs are small, non-coding pieces of RNA that function as a post-transcriptional regulatory mechanism. Their action is exerted through binding with a complementary region in the 3’ untranslated region (3’UTR) of the target mRNA. This binding, facilitated by the ribonuclear protein complex RISC, prevents successful translation of the mRNA into its protein product. MicroRNAs have been shown to function across species, throughout development and during the adult life-span. In the immune system, microRNAs are known to be required for correct formation of germinal centres and normal development of B- and T-cells. MicroRNAs have also been shown to be differentially regulated during macrophage activation with different stimuli. In particular, miR-155, miR-146a and miR-21 are associated with macrophage activation by lipopolysaccharide (LPS). The objective of this work was to further understand the role of microRNAs during macrophage activation with LPS. Two approaches were adopted. Firstly, the regulation of individual microRNAs in LPS-activated bone marrow derived macrophages (BMDMs) was characterised by the use of illumina small RNA sequencing. Secondly, the requirement of the global microRNA population during macrophage biology was investigated through the use of DGCR8 and Dicer knockout systems. In keeping with the large number of changes reported in mRNA translation upon activation, expression of >400 microRNAs were found to be differentially regulated by exposure to LPS. Twelve of these microRNAs were chosen for further study (miR- 142-3p, -146a, -15b, -155, -16, -191, -21, -27b, -30b, -322-5p, -378 and -7a). Individual knock-down of these microRNAs in the RAW264.7 macrophage-like cell line mostly demonstrated subtle, rather than dramatic changes to the activation marker genes studied by RT-QPCR analysis. However, knock-down of miR-146a, -15b, - 155 and -191 were able to significantly alter the expression of the activation marker genes (Tnf-a, Cox2, Cxcl2, Il-6 and Saa3). Interestingly, knock-down of miR-142-3p, miR-146a and miR-155 appeared to show cross-regulation of these microRNAs. The cell index (CI) data suggested that miR-191 and miR-21 influence adhesion in activated macrophages. Studies with the DGCR8 and Dicer knockout systems showed that the global microRNA population was required for successful differentiation of macrophages from embryonic stem cells, and for normal expression of differentiation and activation markers in bone marrow derived macrophages. Overall, these results show that dynamic expression of microRNAs is an integral part of the macrophage response to LPS.

The aim of this thesis was to find optimal anticancer regimen of therapy, based on a naturally occuring polysaccharide laminarin. It was studied on the Mus musculus skin melanoma B16-F10. There were explored an ajuvant effect of LTA. Mixture of laminarin with LPS was applied with various anchorings and was tryed out the possible replacement of LPS. Using the flow cytometry there were determined progress of immune cells during application. This therapy resulted in reduction of tumor volume and the prolongation of overall survival.

Obesity-related metabolic derangements have been linked to toll-like receptor 4 (TLR4), an innate immune system receptor, due to its role in proinflammatory pathways. Lipopolysaccharide (LPS), a gram-negative bacteria cell wall component, is the ligand for TLR4, and has been shown to be elevated in states of metabolic disease. Heightened levels of circulating endotoxin is termed metabolic endotoxemia and has been linked to systemic inflammation which is associated with obesity, type 2 diabetes mellitus (T2DM), and cardiovascular disease (CVD). Immune cells exhibit a protective ability to develop endotoxin tolerance. The objective of this study was to determine if endotoxin tolerance exists in skeletal muscle cells, and if a condition that mimics a state of over nutrition, such as elevated levels of fatty acids, affect this tolerance. To this end, L6 skeletal muscle cells were treated with low (50 pg/mL)- and high (500 ng/mL)-doses of LPS, with and without the presence of free fatty acids (FFAs). Tolerance was assessed by measuring: 1) changes in mRNA expression of interleukin-6 (IL-6) and monocyte chemoattractant-1 (MCP-1) as markers of a pro-inflammatory response; and 2) mRNA levels of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC1-°) and mitochondrial oxidative capacity via an XF24 Flux Analyzer (Seahorse Bioscience) as measures of the metabolic response. Tolerance to LPS was observed in response to low- and high-doses with MCP-1 mRNA transcription but not IL-6. Changes in PGC1-° and mitochondrial OCR exhibited a tolerant effect in response to the high dose of LPS but not the low dose. The addition of free fatty acids to LPS treatments did not prevent the tolerant effects under any conditions. In conclusion, LPS tolerance exists in skeletal muscle cells but appears to differ depending on pro-inflammatory target and LPS concentration. Additionally, fatty acids, in the current model, have no effect on LPS tolerance. / Master of Science

Horses are prone to potentially lethal endotoxemia due to their surrounding fecal containing environment and their predisposition to colic. Their gastrointestinal tract and feces naturally contain gram-negative bacteria. These bacteria express lipopolysaccharide (LPS) on their cell membranes, which is recognized by Toll-like receptor 4 (TLR4). In cases where epithelial barriers are compromised or breached LPS has the potential to enter circulation and cause the inflammatory symptoms seen with endotoxemia. The objective of this study was to determine TLR4 presence and functionality on equine B cells. TLR4 expression on B lymphocytes has been studied in mouse, human and many other mammals, but has not been well characterized in the horse. Humans are highly sensitive to LPS but their B cells express non-functional TLR4. Mice in contrast are highly tolerant of LPS yet their B cells express functional TLR4. Studies in horse have perhaps been limited by the limited array of antibody markers available for use in horse. Anti-human CD21 has previously been shown to mark equine B lymphocytes. We show rat anti-mouse CD45R(B220) mAbs also accurately labels equine B lymphocytes. To investigate TLR4 expression in horses 12 Thoroughbred geldings, ages 5-10, were used for blood collection. By using the density gradient, Lympholyte, lymphocytes were separated from peripheral blood and incubated with or without LPS. B lymphocyte proliferation, TLR4 expression and mRNA changes were examined before or after culture in the presence or absence of LPS. We demonstrate TLR4 is expressed on equine B lymphocytes through the use of a mouse anti-human TLR4 antibody, clone 76B357.1, not previously used in horse. We demonstrated equine B cells fail to proliferate under LPS challenge as opposed to highly proliferative mouse B lymphocytes. However, transcriptional changes were observed in the equine cells within the TLR4 pathway upon treatment with LPS. / Master of Science

Les sepsis sévères et chocs septiques constituent des causes croissantes de morbidité et de mortalité chez les patients hospitalisés. La dysfonction cardio-circulatoire lors d'un choc septique s'avère un élément majeur et initial dans l'induction et l'entretien des défaillances organiques subséquentes. En effet, certaines bactéries et endotoxines déclenchent une séquence d'événements cellulaires qui mènent à une altération des performances cardiaques. Le macrophage migration inhibitory factor (MIF) a récemment été identifié en tant que facteur de dépression cardiaque lors de choc septique. Ainsi, la présente étude révèle que la neutralisation du MIF prévient la dysfonction myocardique induite par l'endotoxine dans un modèle expérimental chez le rat. De façon sous-jacente, le blocage du MIF empêche la hausse d'expression et de production dans le coeur de cytokines inflammatoires paracrines et autocrines (IL-1, IL-6 et TNF-[alpha]) en réponse au LPS. D'un point de vue mécanistique, cette étude démontre que la neutralisation du MIF inhibe l'apoptose des cardiomyocytes provoquée par l'endotoxine. Ainsi, le présent travail met en évidence un mécanisme précis par lequel le MIF influence la dépression cardiaque endotoxinique. Ce mécanisme implique que le blocage du MIF induit une augmentation du ratio protéique cardiaque de Bcl-2/Bax contribuant ainsi à prévenir le relâchement du cytochrome c mitochondrial induit par l'endotoxine. Cette inhibition de la perte de cytochrome c mitochondrial entraîne une réduction de l'activation de la caspase-3. La neutralisation du MIF rétablie la déficience provoquée par le LPS au niveau de la translocation nucléaire de phospho-Akt et par conséquent l'expression du facteur nucléaire de survie cardiaque GATA-4. Cette baisse d'activité caspase-3 et le rétablissement de la translocation/expression des facteurs de survie par le blocage du MIF résultent en une diminution de la fragmentation de l'ADN caractéristique de l'apoptose tardive. Globalement, l'inactivation du MIF lors d'un choc endotoxinique prévient le déséquilibre inflammatoire et apoptotique dans le coeur protégeant ainsi contre la dysfonction myocardique subséquente.

Introduction : Les artères cérébrales de gros calibre du système antérieur, regroupées sous le nom de bifurcation carotidienne intracrânienne, sont les plus affectées par les accidents vasculaires cérébraux artériels néonatals, ces AVC étant localisés dans ce territoire dans 85% des cas. L'hypothèse pathophysiologique classique, mais non prouvée, postule que l'occlusion artérielle est causée par un embole d'origine placentaire. Cette croyance reste controversée par le débalancement de la distribution antérieure versus postérieure des infarctus cérébraux, et l'absence d'infarctus extra-cérébraux associés. Une nouvelle perspective pathophysiologique émerge de l'association épidémiologique entre l'inflammation gestationnelle et l'AVC néonatal. Nous postulons que l'inflammation materno-fœtale, induite par l'exposition gestationnelle aux pathogènes, mène à une vasculite affectant spécifiquement la bifurcation carotidienne puis provoque une thrombose focale. Méthodes : Des rates Lewis gestantes sont injectées avec de la saline ou du lipopolysaccharide (LPS) d'Escherichia coli (200 μg/kg/12h) entre les jours de gestation (G) 21 et 22. Les cerveaux de la progéniture sont prélevés à G21, G22 et au jour postnatal (P)1. Le sang maternel, les placentas et le sang fœtal sont échantillonnés à G21 ou G22. A P1, un stress prothrombotique (photothrombose transcutanée) combiné à une hypoxie (3h30, 8% O2) est appliqué sur les artères cérébrales moyennes pour comparer leur susceptibilité à la thrombose chez les ratons exposés au LPS et ceux non exposés. L'immunohistochimie, l'immunofluorescence et l'ELISA ont détecté les marqueurs inflammatoires maternels, placentaires et fœtaux. Résultats : Les artères intra-crâniennes les plus susceptibles à l'AVC expriment constitutivement plus de marqueurs inflammatoires en comparaison aux artères intra- ou extra-crâniennes non susceptibles à l'AVC périnatal. L'exposition gestationnelle au LPS provoque une inflammation maternelle, placentaire et fœtale associées à une production d'IL-1β, de TNF-α et de MCP-1 ainsi qu'une inflammation artérielle affectant le segment proximal des artères intra-crâniennes fortement susceptible aux AVC néonatals. Les ratons LPS + photothrombose + hypoxie présentent des AVC ischémiques ainsi que des déficits moteurs, qui n'étaient pas détectés lorsque la photothrombose + hypoxie était appliquée sans traitement préalable avec le LPS. Conclusion : les résultats acquits par le biais de notre nouveau modèle animal préclinique supportent notre hypothèse d'une augmentation de la susceptibilité à l'inflammation des artères cérébrales antérieures, et ouvrent une nouvelle avenue physiopathologique vasculitique pour les AVC néonatals.