Nontypeable Haemophilus influenzae (NTHi), Streptococcus pneumoniae and Moraxella catarrhalis are common bacterial agents of otitis media which is a major cause of morbidity in young children. Mucosal immune responses are an integral part of the immune defense against middle ear infection and it is known that certain populations, including Australian Aboriginal children, are highly susceptible to disease. The current study focussed on the development of the mucosal immunity to the three bacterial pathogens in Aboriginal and non-Aboriginal children from birth to two years of age, living in the Kalgoorlie-Boulder region of Western Australia. Salivary and breast milk IgA levels were measured by the enzyme Linked immunosorbent assay. The measured IgA levels, combined with socio-economic, demographic and bacteriological data were analyzed statistically to determine the influential factors on the mucosal IgA response in these children over time. This study found that each antigen-specific IgA examined followed a distinct ontogeny pattern and IgA responses differed significantly according to age, indigenous status and feeding type. Indoors smoke exposure, maternal smoking, and sibling day care attendance had some impact on salivary IgA levels in the children. However, household crowding and the presence of older siblings had the most significant impact on salivary IgA levels for children of different age groups. These two factors were correlated to increased nasophayrngeal colonization by H. influenzae, S. pneumoniae and M. catarrhalis and colonization status was also found to influence salivary IgA levels in the children. No correlation between maternal breast milk IgA levels and child salivary IgA levels was observed. The results suggest that the degree of exposure to environmental factors rather than immunological deficit is responsible for the observed differences in salivary IgA responses between Aboriginal and non-Aboriginal children and modifying these factors could lead to a reduction in the burden of otitis media experienced by the children. Further studies correlating specific salivary IgA levels to diseases such as otitis media will reveal the role of specific salivary IgA responses in the prevention of infection by respiratory pathogens.

An in vitro model of the follicle-associated epithelia that overlie the Peyer's patches of the small intestine was developed and validated to examine the mechanisms of mucosal antigen sampling. This model displays many phenotypic and physiological characteristics of M cells including apical expression of [alpha]5[beta]l integrin and enhanced energy dependent participate transport. CD4+ T-cells were shown to be an important influence on the development of Mlike cells. The model was used to examine the M cell mediated uptake of several putative whole-cell killed bacterial vaccines. Greater numbers of non-typeable Haemophilus influenzae NTHi 289, NTHi 2019, Escherichia coli 075 HMN and Streptococcus pneumoniae were transported by model M cells compared to control Caco-2 enterocyte-like cells. Studies in isolated murine intestine segments confirmed the selective uptake of NTHi 289 and Escherichia coli demonstrating that intestinal mucosal sampling of these antigens is performed by M cells. Pseudomonas aeruginosa was not absorbed as whole cell bacteria but as soluble antigen, as indicated by the presence of bacterial DNA in the cytoplasm of epithelial cells. These results suggest that bacteria such as NTHi and E. coli are sampled by the mucosal immune system in a different manner to that of bacteria such as Pseudomonas. A number of potential cell surface receptors were investigated to identify which molecules are responsible for intestinal uptake whole-cell killed bacteria. Immunofluorescence studies detected the presence of toll-like receptor-2, toll-like receptor-4, PAF-R and [alpha]5[beta]l integrin on in vitro M-like cell cultures. Examinations of murine intestine confirmed the presence of TLR-4 and PAF-R. TLR-4 was found in small quantities and on M cells. In contrast to the M cell model, TLR-2 expression in the murine intestine was sparse. Receptor inhibition experiments provided evidence for the involvement of TLR-4, PAF-R and [alpha]5[beta]l integrin in M cell uptake of killed bacteria both in vitro and in vivo. This thesis has contributed valuable information regarding the mechanisms of uptake of whole-cell killed bacteria by the intestinal mucosal immune system. For the first time, M cell sampling of whole-cell killed bacteria has been demonstrated. Furthermore, the receptors involved in these processes have been identified. This information will be of great use in the development and optimisation of new oral vaccines.

<p>Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. </p><p>In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an <i>in vivo</i> substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.</p>

The correct and timely delivery of immune cells is critical for protection against foreign antigen. In order for cells to access most organs, there are requirements that must be met to facilitate exit from the blood into extravasculature. The initial requirement is selectin-selectin ligand interactions that mediate tethering and rolling to allow shear resistance. For proper selectin-selectin ligand interaction, glycoproteins must be modified by fucosyltransferases –IV and –VII, which adds fucose to an acceptor substrate to form the sialyl-LewisX moiety. Using fucosyltransferase –IV and –VII double knockout (FtDKO) mice, we made several novel observations. Our first observation showed increased numbers of naïve T cells in non-lymphoid organs. To support this observation, we blocked chemokine-mediated entry into lymph nodes (LNs) with pertussis toxin and L-selectin mediated entry with anti-CD62L antibody in WT mice. We also treated WT mice with the S1P1 agonist, FTY720, to retain lymphocytes in LNs. Our results suggested that when access to LN is perturbed, lymphocytes accumulate in non-lymphoid organs. Our second observation showed an enrichment of effector/memory T cells in FtDKO LNs. To determine if effector/memory CD8 T cells were retained in LNs, we transferred naïve and memory CD8 T cells into WT mice then treated the recipient mice with anti-CD62L. We found that LN exit rates of naïve and memory CD8 T cells were similar, but slowed as T cell density decreased. To understand if memory CD8 T cells were using selectin ligand independent mechanisms, we transferred naïve and memory CD8 T cells into WT or FtDKO mice. We found reduced numbers of memory CD8 T cells in LNs, however, their frequency was increased. We explored this result by transferring CFSE labeled memory CD8 T cells. We found that memory CD8 T cells divide more in FtDKO mice compared to WT. These experiments suggested that selectin ligand deficiencies cause increased frequency of effector/memory T cells in LNs due to low density and increased emptiness induced proliferation. Taken together, these findings reveal how selectin ligand deficiencies contribute to T cell accumulation in non-lymphoid organs and elucidate mechanisms of retention in LNs.

The adaptive evolutionary potential of a species or population to cope with omnipresent environmental challenges is based on its genetic variation. Variability at immune genes, such as the major histocompatibility complex (MHC) genes, is assumed to be a very powerful and effective tool to keep pace with diverse and rapidly evolving pathogens. In my thesis, I studied natural levels of variation at the MHC genes, which have a key role in immune defence, and parasite burden in different small mammal species. I assessed the importance of MHC variation for parasite burden in small mammal populations in their natural environment. To understand the processes shaping different patterns of MHC variation I focused on evidence of selection through pathogens upon the host. Further, I addressed the issue of low MHC diversity in populations or species, which could potentially arise as a result from habitat fragmentation and isolation. Despite their key role in the mammalian evolution the marsupial MHC has been rarely investigated. Studies on primarily captive or laboratory bred individuals indicated very little or even no polymorphism at the marsupial MHC class II genes. However, natural levels of marsupial MHC diversity and selection are unknown to date as studies on wild populations are virtually absent. I investigated MHC II variation in two Neotropical marsupial species endemic to the threatened Brazilian Atlantic Forest (Gracilinanus microtarsus, Marmosops incanus) to test whether the predicted low marsupial MHC class II polymorphism proves to be true under natural conditions. For the first time in marsupials I confirmed characteristics of MHC selection that were so far only known from eutherian mammals, birds, and fish: Positive selection on specific codon sites, recombination, and trans-species polymorphism. Beyond that, the two marsupial species revealed considerable differences in their MHC class II diversity. Diversity was rather low in M. incanus but tenfold higher in G. microtarsus, disproving the predicted general low marsupial MHC class II variation. As pathogens are believed to be very powerful drivers of MHC diversity, I studied parasite burden in both host species to understand the reasons for the remarkable differences in MHC diversity. In both marsupial species specific MHC class II variants were associated to either high or low parasite load highlighting the importance of the marsupial MHC class II in pathogen defence. I developed two alternative scenarios with regard to MHC variation, parasite load, and parasite diversity. In the ‘evolutionary equilibrium’ scenario I assumed the species with low MHC diversity, M. incanus, to be under relaxed pathogenic selection and expected low parasite diversity. Alternatively, low MHC diversity could be the result of a recent loss of genetic variation by means of a genetic bottleneck event. Under this ‘unbalanced situation’ scenario, I assumed a high parasite burden in M. incanus due to a lack of resistance alleles. Parasitological results clearly reject the first scenario and point to the second scenario, as M. incanus is distinctly higher parasitised but parasite diversity is relatively equal compared to G. microtarsus. Hence, I suggest that the parasite load in M. incanus is rather the consequence than the cause for its low MHC diversity. MHC variation and its associations to parasite burden have been typically studied within single populations but MHC variation between populations was rarely taken into account. To gain scientific insight on this issue, I chose a common European rodent species. In the yellow necked mouse (Apodemus flavicollis), I investigated the effects of genetic diversity on parasite load not on the individual but on the population level. I included populations, which possess different levels of variation at the MHC as well as at neutrally evolving genetic markers (microsatellites). I was able to show that mouse populations with a high MHC allele diversity are better armed against high parasite burdens highlighting the significance of adaptive genetic diversity in the field of conservation genetics. An individual itself will not directly benefit from its population’s large MHC allele pool in terms of parasite resistance. But confronted with the multitude of pathogens present in the wild a population with a large MHC allele reservoir is more likely to possess individuals with resistance alleles. These results deepen our understanding of the complex causes and processes of evolutionary adaptations between hosts and pathogens. / In einer sich ständig verändernden Umwelt ist es unverzichtbar, sich fortwährend zu verändern und anzupassen. Dabei gründet sich das Anpassungsvermögen oder das evolutionäre Potential einer Art auf ihre genetische Variabilität. In der Krankheitsabwehr ist die Variabilität der Immungene ein besonders wichtiges und effektives Instrument, weil Pathogene sehr vielfältig sind und schnell evolvieren. Im Rahmen meiner Doktorarbeit habe ich mich mit der Variabilität des Immungen-Komplexes MHC (major histocompatibility complex) beschäftigt, der eine Schlüsselrolle in der Immunabwehr bei Vertebraten einnimmt. Anhand verschiedener Arten und Populationen von Kleinsäugern habe ich den Einfluss der MHC Vielfalt auf den Parasitenbefall unter natürlichen Bedingungen untersucht. Dabei interessierte mich insbesondere das Vorkommen geringer MHC Variabilität in Populationen, das möglicherweise eine Folge von Lebensraum-fragmentierung und Isolation ist. Obwohl Beuteltiere eine zentrale Rolle in der Evolution der Säugetiere spielen, ist über ihren MHC bislang nur sehr wenig bekannt. Einige Studien befassten sich mit Labor- oder Zootieren, und deuteten auf geringe oder sogar gar keine Variation im MHC Klasse II bei Beuteltieren hin. Allerdings gab es bislang nahezu keine Studien an frei lebenden Beuteltieren, deshalb war bislang ein natürliches Ausmaß der MHC Variabilität unbekannt. Anhand von zwei endemischen neotropischen Beuteltieren aus dem brasilianischen Küstenregenwald (Gracilinanus microtarsus, Marmosops incanus) habe ich überprüft, ob sich diese geringe MHC Vielfalt unter natürlichen Freilandbedingungen bestätigt. Erstmals konnte ich zeigen, dass der MHC II bei Beuteltieren charakteristische Merkmale positiver Selektion aufweist, die bisher nur von placentalen Säugern, Vögeln und Fischen bekannt waren: Positive Selektion auf spezifischen Aminosäurepositionen, Rekombination und Trans-Species-Polymorphismus. Darüber hinaus unterschieden sich die beiden Beuteltierarten beträchtlich in ihrer MHC II Variabilität. Während M. incanus sich als relativ wenig divers erwies, zeigte G. microtarsus eine zehnmal höhere Vielfalt und widerlegt damit die generelle Gültigkeit der ursprünglich angenommenen geringen MHC II Variabilität bei Beuteltieren. Um diese beachtlichen Diversitätsunterschiede zwischen den beiden Arten zu erklären, habe ich die Parasitenbelastung untersucht. Bei beiden Arten konnte ich nachweisen, dass bestimmte MHC Varianten mit entweder hoher oder niedriger Parasitenbelastung verknüpft waren. Solche Assoziationen spiegeln Pathogen-vermittelte Selektion wider, untermauern die Funktionalität des MHC Klasse II bei Beuteltieren und weisen auf dieselbe Bedeutsamkeit des MHC wie bei placentalen Säuger, Vögeln und Fischen hin. Ich entwickelte zwei alternative evolutionäre Szenarien, unter denen eine geringe MHC Variabilität denkbar ist. Im Szenario des ‘evolutionären Gleichgewichts’ ist geringe MHC Variabilität die Folge eines verminderten Selektionsdruckes durch wenige Parasiten, sodass eine geringe Parasitendiversität zu erwarten ist. Alternativ könnte eine geringe MHC Variabilität aber auch Folge eines kürzlich erlittenen Verlustes an genetischer Variabilität sein, beispielsweise durch ein Flaschenhalsereignis. Unter diesem Szenario des ‘Ungleichgewichts’ wäre bei M. incanus im Falle eines potentiellen Verlustes von Resistenzallelen eine starke Parasitenbelastung zu erwarten. Die parasitologischen Ergebnisse widersprechen dem ersten und deuten eher auf das zweite Szenario. M. incanus war deutlich stärker parasitiert als G. microtarsus, wohingegen die Parasitendiversität bei beiden Arten ungefähr gleich war. Die hohe Parasitenbelastung bei M. incanus ist offenbar weniger der Auslöser als vielmehr eine Folge seiner geringen MHC Vielfalt zu sein. Üblicherweise werden sowohl die Variabilität des MHC als auch seine Verknüpfung mit Parasitenbelastung innerhalb von einzelnen Populationen untersucht, nur selten wird die Variation zwischen Populationen in Betracht gezogen. Um Erkenntnisse auf dieser Ebene zu gewinnen, habe ich den Zusammenhang zwischen genetischer Vielfalt und Parasitenbelastung nicht auf der Ebene des Individuums, sondern auf der Populationsebene anhand der europäischen Gelbhalsmaus (Apodemus flavicollis) erforscht. Dabei wurden Populationen mit unterschiedlicher genetischer Variabilität am MHC und an neutralen genetischen Markern (Mikrosatelliten) betrachtet. Ich konnte nachweisen, dass Populationen, die über ein großes Spektrum verschiedener MHC Allele verfügen, besser gegen starke Parasitenbelastung gewappnet sind als Populationen mit einer geringen Anzahl MHC Allele. In einer MHC-diversen Population ist die Gegenwart von Individuen mit Resistenzallelen deutlich wahrscheinlicher, und damit die Überlebenswahrscheinlichkeit der Population. Diese Ergebnisse erweitern und vertiefen unsere Erkenntnisse zu die komplexen evolutionären Vorgängen und Mechanismen zwischen Wirt und Parasit in ihrem fortwährenden Wettstreit.

This study aimed to investigate the effects of commercially available natural (sugar cane molasses, white sugar and brown sugar) and artificial (Canderel™, Equal™, Natreen™, Sweetex™, Splenda™ and Swheet™) sweeteners on various physiological systems. The artificial sweeteners tested in this study may be categorised into their respective groups based on their primary ingredient. The brands Canderel™ and Equal™ contain aspartame, Natreen™ and Sweetex™ consist of saccharin and Splenda™ and Swheet™ are composed of sucralose. The inclusion of artificial or natural sweeteners in the human diet has been continually debated and their implication in the development of certain diseases has raised concern regarding their safe use. Therefore, it is necessary that these food products be subjected to a battery of tests to determine adverse effects on human health.

Immune cells like NK cells, T cells, neutrophils and mast cells store high amounts of <u>gr</u>anule <u>s</u>erine <u>p</u>rote<u>ases</u>, graspases. Graspases are encoded from the mast cell chymase locus. The human locus holds four genes: α-chymase, cathepsin G, and granzymes H and B. In contrast, the mouse locus contains at least 14 genes. Many of these belong to subfamilies not found in human, e.g. the Mcpt8-family. These differences hamper functional comparisons of graspases and of immune cells in the two species. Studies of the mast cell chymase locus are therefore important to better understand the mammalian immune system. In this thesis, the evolution of the mast cell chymase locus was analysed by mapping the locus in all available mammalian genome sequences. It was revealed that one single ancestral gene founded this locus probably over 215 million years ago. This ancestor was duplicated more than 185 million years ago. One copy evolved into the α-chymases, whereas the second copy founded the families of granzymes B and H, cathepsin G, Mcpt8 and duodenases. Different subfamilies were later remarkably expanded in particular mammalian lineages, e.g. the Mcpt8- and Mcpt2-subfamilies in the rat. Four novel members of these families were identified in rat mucosal mast cells. Rat and mouse mast cells express numerous different graspases, whereas human and dog mast cells express only one graspase, chymase. To better understand mast cell functions in these species, one member of the mouse Mcpt8-family, mMCP-8, and human and dog chymase were studied. The preferred substrate sequence was analysed by substrate phage display. mMCP-8 remains yet enigmatic, although it is probably proteolytically active. Dog and human chymase, interestingly, have common preferences in certain substrate positions, but differ in others. These two chymases may have coevolved with an in vivo substrate that is conserved only in the positions with a common preference. We also obtained evidence that substrate positions on either side of the scissile bond influence each other. This kind of interactions can only be detected with a method investigating both sides simultaneously, such as substrate phage display.

Influenza causes annual global epidemics and severe morbidity and mortality. The influenza virus evolves to escape from immune system antibodies that bind to it. The immune system produces influenza virus specific antibodies by VDJ recombination and somatic hypermutation. In this dissertation, we analyze the mechanism of influenza virus evolution and immune system dynamics using theoretical modeling and computational simulation. The first half of this thesis discusses influenza virus evolution. The epidemiological data inspires a novel sequence-based antigenic distance measure for subtypes H1N1 and H3N2 virus, which are superior to the conventional measure using hemagglutination inhibition assay. Historical influenza sequences show that the selective pressure increases charge in immunodominant epitopes of the H3 hemagglutinin influenza protein. Statistical mechanics and high-performance computing technology predict fixation tendencies of the H3N2 influenza virus by free energy calculation. We introduce the notion of entropy from physics and informatics to identify the epitope regions of H1-subtype influenza A with application to vaccine efficacy. We also use entropy to quantify selection and diversity in viruses with application to the hemagglutinin of H3N2 influenza. Using the bacterial E. coli as a model, we show the evidence for recombination contributing to the evolution of extended spectrum β-lactamases (ES-BLs) in clinical isolates. A guinea pig experiment supports the discussion on influenza virus evolution. The second half of the thesis discusses immune system dynamics. We design a two-scale model to describe correlation in B cell VDJ usage of zebrafish. We also introduce a dynamical system to model original antigenic sin in influenza. This dissertation aims to help researchers understand the interaction between influenza virus and the immune system with a quantitative approach.

Fishes in the superorder Ostariophysi, including fathead minnows (Pimephales promelas), possess specialized epidermal club cells that contain an alarm substance. Damage to these cells, as would occur during a predator attack, causes the release of the alarm substance and can indicate the presence of actively foraging predators to nearby conspecifics. For nearly 70 years, research involving epidermal club cells has focused on the alarm substance and the role it plays in predator/prey interactions. However, recent studies have indicated that there may be a connection between epidermal club cells and the fish immune system. Fish increase investment in epidermal club cells upon exposure to skin penetrating pathogens and parasites. In this study I tested for differences in epidermal club cell investment by fathead minnows exposed to the immunosuppressive effects of the glucocorticoid hormone cortisol. In experiment 1, fathead minnows were exposed to either a single intraperitoneal injection of corn oil or no injection at all. The purpose of this experiment was to determine whether corn oil, the vehicle for cortisol injections in later experiments, had an effect on epidermal club cell density. The treatments had no effect on epidermal club cell size, cell area, or epidermal thickness. In experiment 2, skin extract was prepared from the skin of corn oil injected and non injected fathead minnows as in experiment 1 to determine whether corn oil had an effect on the epidermal club cell alarm substance concentration. The treatments showed no significant differences in observed anti-predator behaviour, including change in shelter use, dashing and freezing. In experiment 3, fathead minnows were exposed to either a single intraperitoneal injection of cortisol or corn oil. The purpose of this experiment was to determine whether cortisol, a known immunosuppressant, had an effect on epidermal club cell investment. Fathead minnows exposed to a single cortisol injection had significantly reduced respiratory burst activity of kidney phagocytes indicating that there was suppression of the innate immune system. Furthermore, cortisol treated fathead minnows showed significantly lower numbers of epidermal club cells. The treatments had no effect on individual epidermal club cell area, epidermal thickness and serum cortisol levels after 12 days. The results from this experiment suggest that pharmacological cortisol injections in fathead minnows have a suppressive effect on the fish innate immune system. Furthermore, the findings that cortisol induced immunosuppression also influences epidermal club cell investment provides support for the hypothesis that epidermal club cells may function as part of the fish immune system.

Respiratory diseases are a major cause of human morbidity and mortality and are a leading cause of economic loss to livestock producers. The respiratory tract is constantly in contact with dust, bacteria, fungi, viruses and other pathogenic agents that are found in the air. Normally, the body has the ability to clear these foreign particles. However, physiological and environmental stresses can impair airway defense mechanisms resulting in establishment of pulmonary infections. The microbes and their products engage various receptors in the lung to activate epithelium, endothelium, macrophages, neutrophils and other cells. The activation of inflammatory cascade in the lung results in recruitment of neutrophils, damage to air-blood barrier and development of edema. Although there have been significant advances in our understanding of mechanisms of lung inflammation, there have been a lack of any significant advances in the development of new therapeutics to manage lung disease, which may suggest that our understanding of the inflammatory mechanisms is still incomplete. Pentraxin 3 (PTX3) is an innate immune protein which has been implicated in a diverse range of inflammatory processes, such as recruitment of cells and production of cytokines. PTX3 is an acute phase protein, with low or undetectable levels in the circulation of healthy humans and animals, and rapid, dramatic increase in inflammatory diseases. The expression and function of this protein has not been characterized in the lungs of domestic animal species. Because of potential implications of PTX3 in lung inflammation, I studied the expression of PTX3 in normal and inflamed lungs of calves, pigs, horses, foals and humans. Lungs from all of these species showed expression of PTX3 in airway epithelium, alveolar septa, vascular endothelium and inflammatory cells. Western blot performed on homogenates from normal and inflamed lungs from calves and pigs show an increased expression of PTX3 in inflamed lungs (P<0.05). Because protein function is influenced by its location in the cell, I clarified the subcellular expression of PTX3 with immuno-electron microscopy on normal and inflamed calf and horse lungs. PTX3 was localized on pulmonary intravascular macrophages, monocytes, neutrophils and, unexpectedly, platelets. PTX3 was also present in the nuclei of neutrophils, monocytes and pulmonary intravascular macrophages. Neutrophils are critical regulators of acute lung inflammation. Having observed PTX3 in neutrophils, I investigated the effect of E. coli lipopolysaccharide-induced activation on PTX3 in neutrophils in vitro. Neutrophils challenged with E. coli LPS were examined at 30, 60, 90 and 120 minutes after the treatment. Normal peripheral blood neutrophils showed PTX3 expression. Neutrophils activated with LPS appeared ruffled and showed loss of PTX3 expression at 30 minutes followed by recovery of the expression. Western blots performed on normal and activated neutrophil homogenates did not show any differences (P=0.05). Collectively, the data show PTX3 in normal and inflamed lungs across multiple species. PTX3 was also detected in normal and activated neutrophils. While the function of intriguing localization of PTX3 in the nuclei as well as in platelets is not known, the similarity of expression across the species suggest a role for PTX3 in lung inflammation.