Cannabinoids suppress Th1 immunity in a variety of models including infection with the intracellular pathogen Legionella pneumophila (Lp). To examine the cellular mechanism of this effect, mouse bone marrow-derived dendritic cells (DCs) were studied following infection and drug treatment. DCs produced high levels of IL-12p40 following Lp infection. THC suppressed this cytokine response in a concentration-dependent manner and the endocannabinoids 2-arachidonoyolglycerol and virodhamine less potently suppressed cytokine production. DCs expressed mRNA for cannabinoid receptor 1 (CB1), CB2, and transient receptor potential vanilloid type 1 (TRPV1); furthermore, inhibition of Gi signaling by adding pertussis toxin completely attenuated the suppression induced by low concentrations of THC but not at high concentrations. In addition, the THC suppression was partially attenuated in DC cultures from CB1 and CB2 knockout mice and in cultures from normal mice co-treated with THC and cannabinoid receptor antagonists. Cytokine suppression was not attenuated by pretreatment with the TRPV1 antagonist capsazepine, suggesting that Gi signaling and cannabinoid receptors, but not TRPV1, are involved in THC-induced suppression of DC potential to polarize the development of naïve T cells to be Th1 cells. Besides IL-12, THC suppressed other DC polarizing characteristics such as the expression of MHC class II and co-stimulatory molecules CD86 and CD40, as well as the Notch ligand Delta 4. However, THC treatment did not affect other DC functions such as intracellular killing of Lp and Lp-induced apoptosis. Testing the capacity of THC to suppress DC polarizing function with T cells showed that DCs infected in vitro with Lp were able to immunize mice when injected prior to a lethal Lp infection; however, the immunization potential along with Th1 cytokine production was attenuated by THC treatment of the cells at the time of in vitro infection. In addition, THC-treated and Lp-infected DCs poorly stimulated primed splenic CD4 T cells in culture to produce IFN-gamma (IFN-y); however, this stimulating deficiency was reversed by adding recombinant IL-12p40 protein to the cultures. In conclusion, the data suggest that THC inhibits Th1 polarization by targeting essential DC functions such as IL-12p40 secretion and the maturation and expression of co-stimulatory and polarizing molecules.

Early Growth Response Genes (EGR) is a family of four transcription factors containing a unique zinc finger domain. EGR-2 and EGR-3 are important for hindbrain development and myelination. These transcription factors are also necessary for lymphocyte function however, the mechanisms are still unclear. Previous findings have shown that EGR-2cKO mice develop lupus-like autoimmune disease with high levels of pro-inflammatory cytokines despite showing normal T and B cell proliferation after mitogenic stimulation. Therefore we established the CD2-EGR-2-/-EGR-3-/- mouse model to explore the phenotype, susceptibility to autoimmune disease and relevant lymphocyte function. We discovered that CD2-EGR-2-/-EGR-3-/- mice developed severe systemic autoimmune disease and expressed high levels of inflammatory cytokines. More importantly we discovered a novel finding that CD2-EGR-2-/-EGR-3-/- T and B cells had impaired cell proliferation after mitogenic stimulation. Further investigations revealed that the molecular mechanism defected in the T cell receptor signalling pathway is due to a dysfunction in Activator Protein-1 (AP-1). AP-1 is a heterodimeric protein composed of AP-1 family members including Jun, Atf and Fos. Our data shows that EGR-2 and EGR-3 directly bind with the Atf family member Batf, which prevents Batf’s inhibitory function on AP-1 activation. This research demonstrates that EGR-2 and EGR-3 intrinsically regulate chronic inflammation and also positively regulate antigen receptor activation. In conclusion EGR-2 and EGR-3 are essential for providing optimal immune responses, whilst limiting inflammatory immunopathology. We propose that this new model could be used for studying autoimmune disease.

I locate my dissertation at the critical intersection of philosophy, medical discourse and literature, and anchor it around five intertwining concepts: modernity, subjectivity, masculinity, immunity and Frenchness. I contend that immunity, as a concept at which life and law converge, offers an alternative and largely overlooked episteme shaping contemporary French literary consciousness as a primary regulator/negotiator between health and sickness, belonging and not belonging, volition and involition, and, finally, self and other. I treat immunity metaphorically and scientifically, and then trace the episteme through the works of three French authors—Émile Zola, Albert Camus and Hervé Guibert—all of whom adopt the medical novel as a way of addressing the relationship of the individual to society and to the self. Anne-Marie Moulin frames the immunological revolution as an ever-evolving "semantic event." In this vein, I devote my first chapter to examining how immunity instituted itself as a common trope of "becoming" embraced—and left naturalized—by post-structural thinkers grappling with their corporal limits. This rhetorical turn culminates in Jean-Luc Nancy's characterization of the immune system as the body’s “physiological signature," inhibiting the potential of man to transcend his biology. In my second chapter, I move from the metaphor of immunity to a brief exposition of the history of the science, ending my survey with Elie Metchnikoff (and his legacy), the "father" of cellular immunology who envisioned the internal body as a dynamic, every-changing structure. I focus the next three chapters of my study on literary examples where the male protagonist’s immunity has been compromised. For my first two examples—Le Docteur Pascal by Emile Zola and La Peste by Albert Camus—I analyze the portrait of the supposedly immune doctor, considering what the “costs and benefits" of this immunity are and how this "exceptional status" is destabilized. Then, in my last chapter, I switch perspectives from the doctors to the patient, examining the texts of Hervé Guibert who, I argue, models his writing strategy on the retrovirus’s tactics, challenging literary conventions so as better to exteriorize his experience and “contaminate” (in the etymological sense as "touch together") his readers. / Romance Languages and Literatures

Vaccines are one of the most effective advances in medical science and continue to be developed for applications against infectious diseases, cancers, and autoimmunity. A common strategy for vaccine construction is the use of viral vectors derived from various virus families, with Adenoviruses (Ad) and Poxviruses (Pox) being extensively used. Studies utilizing viral vectors have shown a broad variety of vaccine-elicited immune response phenotypes. However, innate immune stimulation elicited by viral vectors and its possible role in shaping these vaccine-elicited adaptive immune responses remains unclear. Here we show that Ad and Pox vectors display profound intra- and inter-group differences in innate immune cytokine and chemokine elicitation. The CD46-utilizing vectors Ad35, Ad26, and Ad48 induced greater anti-viral and proinflammatory cytokines and chemokines relative to Ad5 in vaccinated rhesus monkeys and stimulated human PBMC. Ad fiber protein, as well as other capsid components, influenced resultant Ad vector innate stimulatory phenotypes. Analysis of human sera from Ad26-vaccinated volunteers showed similar anti-viral and proinflammatory cytokine and chemokine elicitation. Mechanistic analysis of Ad innate immune stimulation showed greater amounts Ad35 and Ad26, and small amounts of Ad5, traffic to the late endosome following infection. Innate immune stimulation by all three was reduced by inhibition of endosomal acidification, Cathepsin B, and Caspase-1, suggesting a common set of innate immune sensors triggered by Ads between 0-6 hours post-infection, in agreement with trafficking data showing Ad vector colocalization in the late endosome at similar time points. These data suggest a model mechanism explaining differences in observed Ad vector innate immune stimulation phenotypes. Similar to results obtained with Ad vectors, analysis of innate cytokine and chemokine responses elicited by Pox vectors ALVAC, MVA, and NYVAC showed that all three were distinct, with the canarypox-based vector ALVAC eliciting a unique potent proinflammatory response. Together these results reveal surprising and pronounced differences in innate immune stimulatory properties of viral vectors. Furthermore, these results could lead to possible strategies for targeted construction of vaccines for desired innate immune phenotypes, and have profound implications on vaccine design against infectious diseases and cancers, as well as gene therapy.

A synthesis is described for a number of 16,16-dimethyl analogs of the prostacyclin and related 6-keto prostaglandin types. Included are l6, l6-dimethyl-PGI2 sodium salt (XLIV), 6α-l6, l6-dimethyl-PGI1(XLV), 6β-l6, l6-dimethyl-PGI1 (XLVII), 6-keto-l6, l6-dimethyl-PGF1-α (XLIX), and 6-keto-l6, l6-dimethyl PGE1 (LV). Done, but not included, is the activity for these analogs in the blood platelet aggregation inhibition assay. This activity was consistently less than for the corresponding 16, 16-dihydro compounds.

published_or_final_version / Microbiology / Master / Master of Philosophy

Whole-genome studies of rates of protein evolution show that genes underlying reproduction and immunity tend to evolve faster than other genes, consistent with the frequent action of positive selection. The evolution of immunity has been well-studied at the interspecific level, but much remains unknown about the population-level dynamics of immunity. This project described genetic variation at immunity and non-immunity loci as well as variation among levels of infection for diverse pathogens in a natural population of mice from Tucson. Analysis of autosomal and X-linked loci in the native range of Mus domesticus, the species from which Tucson mice are primarily descended, revealed low levels of variation consistent with a recent population expansion, resulting in a slight excess of rare alleles across the genome. Genetic variation among a set of classical inbred strains represented a small fraction of wild variation. An overlapping set of genes sequenced in mice from Tucson revealed that there is significant introgression from Mus castaneus. After controlling for gene flow, Tucson mice showed evidence of a mild bottleneck that produced a slight excess of intermediate frequency alleles, but did not result in a dramatic loss of genetic variability. Most of the 15 pathogens and parasites studied in Tucson were found at low to intermediate frequency, and most mice had one to three infections, suggesting that there are many opportunities for host-pathogen coevolution, and a possible role for coinfection. A study of Fv-4, which confers resistance to murine leukemia viruses, confirmed that the resistance allele originated in M. castaneus and is now found at intermediate frequency in Tucson after introduction through gene flow. Finally, a study of the recently duplicated Ceacam1 and Ceacam2 genes, previously shown to be involved in resistance to mouse hepatitis virus (MHV), revealed that a gene conversion event moved a suite of mutations from Ceacam2 to Ceacam1. An elevated rate of protein evolution showed that Ceacam2 had experienced positive selection after duplication. Interestingly, there was no association between MHV antibody presence and Ceacam1 genotype in Tucson. This project showed that gene flow and gene conversion mediated resistance to infections in wild mice.

Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In Drosophila, one major defense mechanism is the production of a battery of antimicrobial peptides (AMPs). The expression of AMPs is primarily regulated at the level of transcription and constitutes both constitutive expression in a tissue-specific manner and inducible systemic expression in response to infection. The aim of my thesis has been to investigate the regulation of AMP gene expression at different levels. I have studied a novel cis-regulatory element, Region 1 (R1) found in the proximal promoter of all Cecropin genes in Drosophila melanogaster, as well as in other species of Drosophila. We found that the R1 element was important for the expression of CecropinA1 (CecA1) both in vitro and in vivo. A signaling-dependent R1-binding activity (RBA) was identified in nuclear extracts from Drosophila cells and flies. The molecular nature of the RBA, has despite considerable effort, not yet been identified. I also have studied the role of the JNK pathway in transcriptional regulation of AMP genes. The role of the JNK pathway in the regulation of AMP genes has long been elusive, however, in this study we showed that the pathway is directly involved in the expression of AMP genes. Analysis of cells mutant for JNK pathway components showed severely reduced AMP gene expression. Fur-thermore, over-expression of a JNK pathway-inhibitor also inhibited AMP gene expression. Lastly, I have studied transcription factors that have not previously been implicated in transcriptional regulation of AMP genes. In a yeast screen, three members of the POU family of transcription factors were identified as regulators of CecA1. Two of them, Drifter (Dfr) and POU do-main protein 1 (Pdm1) were further characterized. We showed that Dfr was able to promote AMP gene expression in the absence of infection, suggest-ing it to play a role in constitutive expression of AMP genes. Indeed, down-regulation of Dfr expression using RNAi severely reduced the constitutive expression of AMP genes in the male ejaculatory duct. We also identified an enhancer element important for Dfr-mediated expression of CecA1. Pdm1, on the other hand, was shown to be important for the systemic expression of AMP genes. In Pdm1 mutant flies, several AMP genes are systemically expressed even in the absence of infection, suggesting that Pdm1 works as a repressor of those genes. However, at least on AMP gene, AttacinA (AttA) requires Pdm1 for its expression, suggesting that Pdm1 works as an activator for this gene. Upon infection, Pdm1 was rapidly degraded, but, regenerated shortly after infection. We propose that the degradation of Pdm1 is important for the activation of the Pdm1-repressed genes and that regeneration sup-ports the expression of AttA.

The fruit fly Drosophila melanogaster has been a powerful model system in which to study the immune response. When microorganisms breach the mechanical barrier of the insect, phagocytosing cells and a battery of induced antimicrobial molecules rapidly attack them. These antimicrobial peptides can reach micromolar concentrations within a few hours. This immediate response is reminiscent of the mammalian innate immune response and utilizes transcription factors of the NF-κB family. We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the Drosophila immune response to microbes. Relish mutant flies have a severely impaired immune response to Gram-negative (G-) bacteria and some Gram-positive (G+) bacteria and fungi and succumb to an otherwise harmless infection. The main reason for the high susceptibility to infection is that these mutant flies fail to induce the antimicrobial peptide genes. The cellular responses appear to be normal. Relish is retained in the cytoplasm in an inactive state. We designed a set of expression plasmids to investigate the requirements for activation of Relish in a hemocyte cell line after stimulation with bacterial lipopolysaccharide. Signal-induced phosphorylation of Relish followed by endoproteolytic processing at the caspase-like target motif in the linker region released the inhibitory ankyrin-repeat (ANK) domain from the DNA binding Rel homology domain (RHD). Separation from the ANK domain allowed the RHD to move into the nucleus and initiate transcription of target genes like those that encode the inducible antimicrobial peptides, likely by binding to κB-like sites in the promoter region. By studying the immune response of the Relish mutant flies in combination with mutants for another NF-κB-like protein, Dorsal-related immunity factor (Dif), we found that the Drosophila immune system can distinguish between various microbes and generate a differential response by activating the Toll/Dif and Imd/Relish pathways. The recognition of foreign microorganisms is believed to occur through pattern recognition receptors (PRRs) that have affinity for selective pathogen-associated molecular patterns (PAMPs). We found that the Drosophila PRRs can recognize G- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G+ bacteria such as Micrococcus luteus and Bacillus megaterium and fungal PAMPs from Beauveria bassiana and Geotrichum candidum. This thesis also investigates the expression of the antimicrobial peptide genes, Diptericin B and Attacin C, and the putative intracellular antimicrobial peptide gene Attacin D, and explores a potential evolutionary link between them.

Vaccination against Marek's disease (MD) is greatly enhanced by host genetic resistance. Genes of the growth hormone (GH) axis have been reported to affect the ontogeny and effector functions of cells of the immune system. Two strains of White Leghorn chickens bred for contrasting homozygous markers in the GH and GH receptor (GHR) genes were challenged with MDV. Contrasts for the significant interaction between marker genotype and tissue indicated that the GH/GHR marker genotype caused a shift in the distribution of the viral load in lymphoid tissues in the two strains. The analysis suggests that genetic variations in genes of the GH axis may differentially affect the host response to MDV replication in lymphoid tissues. Regarding the early time course of infection, at day 6 the viral load was highest in the thymus, while at day 10, it was highest in the spleen, indicating that the virus may have accumulated in the spleen or was continuing to replicate in this tissue. (Abstract shortened by UMI.)