The drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim was investigated using an in vitro culture system, an in vivo mouse model, and a clinical trial in HIV-infected patients. We hypothesized that combination exposure causes immune cell populations in the bone marrow to undergo apoptotic cell death, and that the toxicity would affect the host response to an infectious stimulus. Mice were dosed with zidovudine, sulfamethoxazole-trimethoprim, the combination of both drugs, or vehicle only control via oral gavage. Focusing on B-lineage cells in the bone marrow, we determined that cells of the rapidly cycling, early pre-B cell subset are targeted, as well as pro-B cells earlier in development. This toxicity was found to be cell cycle dependent, with an increase in percentage of cells in the S/G2/M phases of the cycle. In vitro experiments using the drugs in a bone marrow culture system demonstrated that the effect of cytotoxicity with combination exposure is synergistic and concentration-dependent. The mechanism of apoptosis that is induced appears to be caspase-independent. To measure host response in mice, animals treated with zidovudine plus sulfamethoxazole-trimethoprim were infected with Pneumocystis murina pneumonia, and the group that received the combination of agents had a blunted antigen-specific IgG response, possibly due to a decreased number of B cells and activated B cells in the draining lymph nodes of the lungs. A clinical trial was conducted in HIV-infected patients, dividing subjects into groups receiving zidovudine, sulfamethoxazole-trimethoprim, the combination of both, or neither agent. Upon vaccination with the influenza vaccine, the combination treatment group had a blunted humoral response, with reduced antigen-specific serum IgG titers as compared to the control group. We conclude that the drug-drug interaction involving zidovudine and sulfamethoxazole-trimethoprim is clinically-significant, and clinicians must consider this toxicity when treating patients with these agents concurrently.

Pro-inflammatory cytokines have been implicated in the pathophysiology and maintenance of depression. This study investigated the effects of a short mindfulness intervention on pro-inflammatory correlates of depression (IL-6 and TNF-α) and selfreported psychological health. Sixty-four college females were assigned to a four-week mindfulness training group or a contact-control group. Cytokines and psychological health were assessed at baseline, post-treatment, and 3-month follow-up (mindfulness group only). IL-6 and TNF-α significantly decreased from baseline to post-treatment in the mindfulness group only; these changes were sustained at 3-month follow-up. No between-group differences in psychological health emerged. Although reductions in proinflammatory cytokines in the mindfulness condition were not attributable to psychological changes, they may serve to protect against the development of future depressive episodes.

Secreted surface proteins are an innate immune defense component employed by animals to inhibit invading microbes. Surface proteins have not been documented in plants, even though the aerial leaf surface, or phylloplane, is a major site of pathogen ingress. We have discovered novel proteins, termed phylloplanins, which accumulate on leaf surfaces of Nicotiana tabacum, and we have isolated the gene Phylloplanin that is unique in gene databases. Natural and E. coli-expressed phylloplanins inhibit spore germination and limit leaf infection by the oomycete pathogen Peronospora tabacina. We investigated the site of phylloplanin biosynthesis using biochemical techniques. These techniques included radiolabeling of detached trichome glands, radiolabeling of epidermal peels, analysis of leaf water washes of various Nicotiana plants, and examination of guttation fluid, leaf vein contents, and extracellular fluid. From these experiments, we tentatively conclude that phylloplanins are produced by hydathodes, or an unknown surface secreting system, but not by glandular secreting trichomes. Future experiments with the phylloplanin promoter, whose elucidation is described herein, and its fusion to a reporter gene (GUS or GFP), will undoubtedly provide further insight into the location of phylloplanin biosynthesis and deposition. We suggest that the hydrophobic nature of phylloplanins aids in their dispersal over the leaf surface. Phylloplanins constitute a first-point-of-contact, rapid response, innate immune deterrent to pathogen establishment on N. tabacum leaf surfaces, and are the first studied representatives of a novel protein class in the plant kingdom. Further study of leaf surface proteins is justified to understand further their roles in plant defense, and to investigate their potential in agricultural biotechnology. Additionally, we describe miscellaneous observations we have made during the course of this research. Low molecular mass proteins (as yet uncharacterized) are washed from leaf surfaces of sunflower, soybean, and other plants. Pathogenesis-related (PR-)-5a, a known antifungal protein, was found to be present on the leaf surfaces of healthy plants, although its function there remains unknown. A phylloplanin homologue from Arabidopsis appears to be antibacterial. Further study of this protein is warranted. We note that proteins can also be recovered from N. tabacum root surfaces, or the rhizoplane, but we have not further characterized these proteins. In summary, novel surface-accumulated proteins, termed phylloplanins, and the gene encoding these have been discovered in N. tabacum. An antifungal function for phylloplanins is reported, and evidence was found for a unique mechanism of surface deposition.

The gastrointestinal tract houses one of the most dense and diverse communities of bacteria on the planet. The mutualistic relationship between the host and commensal microbe permits the microbe an ideal environment to grow and provides the host with increased caloric intake, maturation of the adaptive immune system, and resistance against invading pathogens. To maintain a system in which both parties benefit, the epithelium has evolved numerous strategies to ensure epithelial cells respond to microbes appropriately and that potentially hazardous commensals remain distanced from the soma proper. Breakdown of these propitiating mechanisms elicits unchecked inflammation and can lead to pathology and reduction of host fitness. We show that oral and intestinal epithelial cells respond to the circulating hormone adiponectin in the presence of bacterial constituents, and that adiponectin has the potential to downregulate NF-κB signaling. We also show many commensal bacteria have no effect on TNF or IL-8 proinflammatory gene expression in intestinal cells. Commensals within the family Enterobacteriaceae can increase TNF and IL-8 expression, but also expression of the NF-κB regulator A20 and MAPK phosphatase MKP-1. Importantly, Enterobacteriaceae also increased expression of the IgA transporter pIgR. In the mouse model, we show pIgR expression along the intestinal epithelium is necessary for SIgA accumulation in the outer mucus layer where commensal bacteria reside. Loss of the mucus layer, but not pIgR is sufficient to allow direct bacterial-epithelial cell contact and induce spontaneous inflammation along the colon. Secretory IgA is supplied maternally through breast milk early in life to compensate for the neonate’s inability to produce sufficient endogenous amounts. By utilizing a breeding scheme in which mouse dams were unable to provide their offspring with SIgA, we show the necessity of maternally-supplied SIgA to control bacterial invasion to mesenteric lymph nodes before weaning. In addition, 8-10 week old adult offspring not receiving SIgA as neonates showed both a unique intestinal microbiota and different patterns of intestinal epithelial cell gene expression with and without chemically-induced acute colitis. In summary, we reveal new mechanisms the mammalian host utilizes in order to maintain peace between the commensal microbe and host immune system.

<p>Insects rely solely on innate immune reactions for protection against infect-ing microbes in their environment. In <i>Drosophila</i>, 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 <i>Drosophila.</i> 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 <i>Drosophila</i> 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.</p>

<p>The fruit fly <i>Drosophila melanogaster</i> 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. </p><p>We have generated loss-of-function mutants of the NF-κB-like transcription factor Relish in order to investigate Relish's role in the <i>Drosophila</i> 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. </p><p>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. </p><p>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 <i>Drosophila</i> 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 <i>Drosophila</i> PRRs can recognize G^- bacteria as a group. Interestingly, the PRRs are specific enough to distinguish between peptidoglycans from G⁺ bacteria such as <i>Micrococcus luteus</i> and <i>Bacillus megaterium </i>and fungal PAMPs from <i>Beauveria bassiana</i> and <i>Geotrichum candidum</i>. </p><p>This thesis also investigates the expression of the antimicrobial peptide genes, <i>Diptericin B</i> and <i>Attacin C</i>, and the putative intracellular antimicrobial peptide gene <i>Attacin D</i>, and explores a potential evolutionary link between them.</p>

I investigated the humoral immune response to streptococcal cell walls (SCW) in arthritis susceptible Lewis and resistant Fisher rats. All rats were given a single intraperitoneal injection of either SCW or saline (controls). Rats were sacrificed, three rats per time point, over an eleven week period and serum was collected for ELISA. SCW injected Lewis rats produced anti-SCW antibody, whereas control rats did not. Anti-SCW antibody was significantly elevated over controls between days 14-28 (post injection). Both saline and SCW injected Fisher rats produced anti-SCW antibody, but with different kinetics. Anti-SCW antibody increased by day 7 and remained elevated over controls till day 21, after which there was no difference. ELISA were designed to determine the SCW epitope(s) recognized by anti-SCW antibody. Formamide extracts of SCW, peptidoglycan and polysaccharide, were investigated along with the terminal epitope of polysaccharide, N-acetyl-D-glucosamine, and the peptidoglycan precursor peptide. The data revealed that anti-SCW antibody was directed against a combined SCW epitope, given the lack of significant binding to any of the SCW epitopes tested. Isotype analysis of anti-SCW antibody revealed that the Lewis response was composed primarily of IgG2a whereas the Fisher response was composed primarily of IgM. Binding of rat IgG isotypes to whole streptococcus, SCW, peptidoglycan, and polysaccharide was investigated, given the possibility of background binding by the streptococcal Fc-receptor. Streptococcal binding of rat IgG was specific for IgG2c and the polysaccharide portion of SCW was necessary for binding. Passive immunization of naive Lewis rats with antibody from rats with active arthritis was ineffective at transferring the disease. However, subcutaneous injection of affinity purified anti-SCW antibody or IgG into Lewis rats, followed twenty-four hours later by a single intraperitoneal injection of SCW, suppressed the acute phase and inhibited the chronic disease. IgM rheumatoid factor (RF) was present in the serum of both saline and SCW injected Lewis and Fisher rats. However, SCW injection only induced a significant increase in IgM RF (between days 3-7) in Lewis rats. Passive immunization of Fisher rats with affinity purified IgM RF (from Lewis serum), three days post SCW injection, was ineffective at inducing arthritis.

No description available.

Human peripheral blood monocytes provide a model for the in vivo exposure to, and immune functional damage caused by chronic UVB exposure at the skin surface. Retinoids and carotenoids are known immune function enhancers; they can also prevent cellular toxic product formation caused by UVB exposure. Application of these compounds in vitro may prevent functional damage to monocytes. Monocytes were exposed in vitro to UVB, then assayed for cytotoxic, phagocytic, and antigen presenting abilities. Phagocytic activity was protected from UVB damage by exposure to these compounds; cytotoxic activity was not altered by UVB exposure, but increased by retinoid or carotenoid exposure. Antigen presentation was not affected by either the UVB or these compounds. Protection of phagocytic function was not due to release of activating monokines or prostaglandins. Instead, the cell membrane antioxidant properties of these retinoids or carotenoids were the factors that protected the monocyte from phagocytic damage caused by UVB exposure.

Neutrophils migrate rapidly towards a site of inflammation and mediate bacterial killing through highly regulated pathways that involve the phagocytosis of bacteria and the generation of reactive oxygen species by the NADPH oxidase complex. The Rac small GTPases have prominent roles in the regulation of neutrophil signaling pathways but the research strategies used to analyze their functions in live cells have been limited, since neutrophils are terminally differentiated and difficult to manipulate genetically. In this thesis, I describe a novel high efficiency protocol for transiently transfecting neutrophils that allowed me to investigate the roles of Rac1 and Rac2 in neutrophils in a completely new way, in real time. Using this technique, I show that a bacterial protein known to inhibit chemotaxis in vitro, selectively inhibits Rac1 activation downstream of fMLP stimulation and inhibits neutrophils polarization. Further dissecting the roles of Rac isoforms, I used various approaches to show that Rac1 and Rac2 differentially regulate free-barbed end (FBE) formation downstream of the fMLP receptor. Rac1 is responsible for ~30% of FBE whereas Rac2 is the regulator of FBE formation (~70%) through the activation of cofilin and Arp2/3. Finally, these observations led to the analysis of the mechanisms underlying the Rac1 and Rac2 functions. I show that membrane charge determines Rac1 and Rac2 differential localization during phagocytosis and chemotaxis iii based on their different aminoacid residues in the polybasic domain. This mechanism depends on lipid metabolism and the accumulation of negatively charged lipids at cellular membranes. During chemotaxis, neutrophils have a polarized accumulation of negatively charged lipids at the leading edge membrane that selectively recruit Rac1. In contrast, the lipid metabolism that occurs at the phagosome membrane decreases its negativity and selectively recruits Rac2. All together, this thesis describes the study of primary neutrophil functions from a new angle and adds some valuable information to the comprehension of effective neutrophil activation based on the analysis of Rac isoforms.