Orientador: Maria Marluce dos Santos Vilela / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-11T01:20:12Z (GMT). No. of bitstreams: 1 Zorzeto_TatianeQueiroz_M.pdf: 1695884 bytes, checksum: 267f0534bc256440762ad9d78bc6402d (MD5) Previous issue date: 2008 / Resumo: A associação temporal de eventos adversos de variada gravidade à imunização com pertussis celular total (DTP) tem estimulado o desenvolvimento de vacinas antipertussis menos reatogênicas. Este ensaio clínico fase I visou à avaliação da imunogenicidade da vacina pertussis celular modificada pela extração do lipopolissacarídeo (DTPm) em comparação com a vacina convencional (DTP). Um total de 234 lactentes foi imunizado aos dois, quatro e seis meses de idade com DTPm ou DTP. Os títulos de anticorpos para os componentes pertussis, tétano, difteria e hepatite B foram determinados um mês após a terceira dose de vacina. A proliferação de células T CD3+ foi avaliada por citometria de fluxo após seis dias de cultivo de células mononucleares de sangue periférico estimuladas com células inativadas de B. pertussis ou com fitohemaglutinina (PHA). Células CD4+, CD8+ e TCR ?d+ foram identificadas no gate de blastos. Os níveis de IFN-?, TNF-a, IL-4 e IL-10 no sobrenadante de cultura foram quantificados por ensaio imunoenzimático (ELISA). A vacina modificada DTPm mostrou-se inferior à DTP quanto ao título de anticorpos antipertussis, mas não houve diferença de resposta aos outros componentes vacinais avaliados. A porcentagem líquida de blastos sob estímulo da B. pertussis foi menor no grupo que recebeu três doses de DTPm (mediana de 3,9% para DTPm e 6,2% para DTP, p=0,029), mas as freqüências de células CD4+, CD8+ e ?d+ em proliferação e as concentrações de citocinas não diferiram entre os grupos. A vacina DTPm não apresentou, portanto, imunogenicidade similar à da vacina DTP convencional nos ensaios laboratoriais / Abstract: Concerns about systemic reactions after immunization with whole-cell pertussis (wP) have stimulated efforts to produce less reactogenic vaccines. This phase I comparative trial aimed the efficacy evaluation of a cellular pertussis vaccine with low lipopolysaccharide (LPS) content (wPlow) in comparison to conventional wP vaccine. A total of 234 infants was vaccinated at 2, 4, and 6 months with conventional wP or wPlow. Serum antibody titers to pertussis, diphtheria, tetanus and hepatitis B were measured 1 month after the third dose of vaccine. Proliferation of CD3+ T cells was evaluated by flow cytometry after 6 days of peripheral blood mononuclear cells culture, with heat-killed B. pertussis or phytohemagglutinin (PHA) stimulation. CD4+, CD8+ and TCR ?d+ cells were identified in the gate of blast lymphocytes. IFN-?, TNF-a, IL-4 and IL-10 levels in supernatants were determined by ELISA. wPlow was inferior to wP in terms of anti-pertussis titers, but there was no diference in other serum antibody evaluations. Net percent blasts in cultures with B. pertussis was lower in the group vaccinated with wPlow (medians of 3.9% and for wPlow and 6.2% for wP; p=0.029), but the frequency of proliferating CD4+, CD8+ and ?d+ cells and the cytokine concentrations in supernatants were similar between vaccination groups. Therefore, wPlow wasn't as imunogenic as conventional wP in experimental evaluations / Mestrado / Saude da Criança e do Adolescente / Mestre em Saude da Criança e do Adolescente

Le monoxyde d'azote (NO) est une molécule produite par l'ensemble des cellules des voies aériennes. Sa synthèse à partir de la L-arginine fait appel à un groupe d'enzymes : les NO synthases (NOS). Il existe trois isoformes de NOS exprimées par des cellules ayant des fonctions diverses conférant ainsi au NO produit une spécificité d'action dépendante de la cellule et de la « situation » ayant entraîné sa synthèse. Les travaux effectués dans le cadre de cette thèse explorent des rôles différents (bronchoréactivité, immunitaires) du NO produit par les voies aériennes ainsi que la mesure du NO dans le contexte de l'anesthésie – réanimation. L'objectif du premier travail était de « revisiter » la fonction régulatrice du tonus bronchique dans le cadre d'une pathologie respiratoire, l'objectif du second travail était méthodologique (étude des sources anatomiques du NO expiré chez le patient sous ventilation mécanique), et l'objectif du troisième travail était d'utiliser la mesure validée dans le second travail pour évaluer les fonctions immunes du NO en réanimation.Premier travail. La compétition entre les NO synthases et les arginases pour leur substrat commun, la L-arginine, pourrait être impliquée dans régulation de la réactivité et du remodelage bronchique chez le patient atteint de BPCO. Le but du premier travail était d'évaluer la relation entre expression de cette balance enzymatique, et les effets pharmacologiques de l'inhibition des NOS et des arginases sur la réactivité bronchique ex vivo à l'acétylcholine de patients sans et avec une BPCO peu sévère. Pour cela, nous avons étudié les bronches de 22 patients. Des études immunohistochimiques nous ont permis de mettre en évidence une expression bronchique NOS-2 plus importante chez les patients BPCO comparée aux patients non BPCO. De plus, les études pharmacologiques réalisées en cuve à organe ont permis de mettre en évidence une tension bronchique de base plus importante chez les BPCO, corrélée à l'expression de la NOS-2 et au degré d'obstruction bronchique (VEMS). L'utilisation d'inhibiteur des NOS diminuait cette tension de base. Nous avons démontré ainsi qu'une augmentation de l'expression NOS-2 chez le BPCO était impliquée dans l'augmentation du tonus bronchique de base et dans l'obstruction bronchique.Second travail. Les variations de NO expiré après chirurgie cardiaque avec circulation extracorporelle (CEC) demeurent controversées. Le but de ce deuxième travail était de déterminer quelle source de NO expiré (bronchique ou alvéolaire) était modifiée après CEC, et d'étudier les effets de la ventilation mécanique pendant la CEC sur ces variations. Nous avons étudié ainsi 32 patients ventilés ou non durant une chirurgie cardiaque avec CEC. Nous avons observé une diminution significative du NO expiré d'origine bronchique après la CEC. Cette diminution n'était pas observée lors du maintien durant la CEC d'une ventilation avec pression expiratoire positive (PEEP). Ce travaille permettait de conclure que la diminution du NO bronchique après la CEC pouvait être liée à une occlusion des petites voies aériennes. Cette atteinte de petites voies aériennes était prévenue par la PEEP.Troisième travail. Enfin, dans ce troisième travail, nous avons émis l'hypothèse que la mesure du NO produit par les voies aériennes (NO expiré et nasal) pouvait constituer un marqueur prédictif de survenue d'infection nosocomiale (fonctions immunitaires du NO). Dans une étude observationnelle chez 45 patients de réanimation ventilés (15 patients ont développé une infection nosocomiale), le NO nasal était le seul marqueur significativement plus bas chez les patients développant une infection nosocomiale (le NO expiré, les dosages d'IL6 et d'IL10 ainsi que le score SOFA n'étaient pas différents entre les deux groupes). Un NO nasal inférieur à 148 ppb 72 heures après l'admission du patient, permettait de prédire la survenue d'une infection nosocomiale avec une sensibilité et une spécificité de 80% et de 70% respectivement et un odds ratio de 2.7. Le développement de ce bio marqueur simple à mesurer permettrait de mettre en place des stratégies préventives (immunonutrition avec de la L-arginine). / In the respiratory tract, NO is produced by a wide variety of cell types and is generated via oxidation of l-arginine that is catalyzed by the enzyme NO synthase (NOS). NOS exists in three distinct isoforms: neuronal NOS (NOS-1), inducible NOS (NOS-2), and endothelial NOS (NOS-3). NO derived from the constitutive isoforms of NOS (NOS-1 and NOS-3) and other NO-adduct molecules (nitrosothiols) have been shown to be modulators of bronchomotor tone. On the other hand, NO derived from NOS-2 seems to be a proinflammatory mediator with immunomodulatory effects. This thesis explores the physiological and pathophysiological role of endogenous nitric oxide in the airways, and the clinical aspects of monitoring nitric oxide in exhaled air of patients with respiratory disease.First Study: competition between nitric oxide synthases (NOSs) and arginases for their common substrate l-arginine could be involved in the regulation of cholinergic airway reactivity and subsequent airway remodeling. The aims of this study were to evaluate the relationships between the expression of this enzymatic balance and the effects of NOS and arginase inhibition on bronchoconstrictive response to acetylcholine of patients without and with early chronic obstructive pulmonary disease (COPD). Twenty-two human bronchi were investigated for immunohistochemistry and modulation of acetylcholine-induced airway constriction. Significantly increased expression of NOS2 in immunoblots of bronchial tissue and staining in smooth muscle cells was evidenced in patients with COPD compared with control subjects. Forced expiratory volume in 1 s (FEV1) and NOS2 expression were negatively correlated. Pharmacological experiments demonstrated that resting tension was elevated in COPD compared with control subjects and was positively correlated with the expression of NOS2. The sole effect of the specific arginase inhibitor Nomega-hydroxy-nor-L-arginine was to decrease sensitivity in COPD patients, whereas NG-nitro-L-arginine methyl ester unexpectedly decreased resting tension because of a non-cGMP-dependent effect. In conclusion, an upregulation of NOS2 expression in COPD patients is involved in airway tone regulation and functional airflow limitation, whereas increased arginase activity is involved in airway sensitivity.Second Study: the change in exhaled NO after cardio-pulmonary bypass remains controversial. The aims were to determine whether exhaled NO sources (alveolar or bronchial) are modified after bypass, and whether mechanical ventilation (MV) settings during bypass modify exhaled NO changes. Thirty-two patients were divided into three groups: without MV during bypass and positive end-expiratory pressure (PEEP) (n=12), dead space MV without PEEP (n=10) and dead space MV with PEEP (n=10). Alveolar NO concentration and bronchial NO flux were calculated before and 1h after surgery using a two-compartment model of NO exchange developed in spontaneous breathing patients. Whereas a significant decrease in bronchial NO was found after bypass in the two groups without PEEP during bypass, this decrease was not observed in patients with dead space ventilation with PEEP. Alveolar NO was not significantly modified whatever the ventilation settings. In conclusion, the impairment of bronchial NO seemed related to airway closure since dead space mechanical ventilation with PEEP prevented its decrease.Third Study: the development of biomarkers able to predict the occurrence of nosocomial infection could help manage preventive strategies, especially in medical patients whose degree of acquired immunosuppression may be variable. We hypothesized that the NO fraction present in the airways (upper and lower) of critically ill patients under mechanical ventilation could constitute such a biomarker. We conducted an observational study in a medical intensive care unit. Forty-five patients (26 men; 72 [25th-75th percentiles] years [56-82]; Simplified Acute Physiology Score II, 63 [50-81], 14 infected) under mechanical ventilation (>3 days) underwent on day 1 and day 3 of their stay: nasal and exhaled (partitioned in bronchial and alveolar sources) bedside NO measurements, determination of urine NO end products and plasma cytokine (IL-6, IL-10) concentrations, and Sequential Organ Failure Assessment score calculation. Nosocomial infection incidence was recorded during the 15 subsequent days. Fifteen patients (33%) acquired a nosocomial infection. Nasal NO was the only marker significantly different between patients with and without subsequent infection (day 1, 52 ppb [20-142] vs. 134 [84-203], P = 0.038; day 3, 98 ppb [22-140] vs. 225 [89-288], P = 0.006, respectively). Nasal NO fraction 148 ppb or less at day 3 had an 80% sensitivity, a 70% specificity, and an odds ratio of 2.7 (95% confidence interval, 1.9-3.8) to predict acquisition of nosocomial infection. Nasal NO seems to be a relatively sensitive and specific biomarker of subsequent nosocomial infection acquisition.

Macrophages, phagocytic cells of the immune system involved in host defence, homeostasis and development, are controlled and influenced by a variety of growth factors. In mammals, the colony stimulating factor 1 (CSF1) is a secreted cytokine that controls macrophages survival, proliferation and differentiation. It acts through the CSF1 receptor (CSF1R), a transmembrane receptor tyrosine kinase, expressed mainly in mononuclear phagocytes. Mammalian CSF1R is found exclusively at the surface of the mononuclear phagocytes and their progenitors. CSF1R-/- knockout mice display more severe phenotypes than the CSF1-deficient mice, thus suggesting the existence of another CSF1R ligand. Indeed, recent studies have shown that interleukin 34 (IL34) also binds to and activates CSF1R and regulates monocyte viability in vitro. While the exact role of this protein is yet to be fully elucidated, studies in mammals thus far implied its involvement in embryogenesis and development. CSF1R system is highly conserved within vertebrates and has been identified in variety of mammals. Chicken has been used extensively as a model for vertebrate development and to identify fundamental biological processes. Previous studies by colleagues in the lab demonstrated that the CSF1R system is conserved in the chicken, where it controls the generation of monocytes and tissue macrophages. This thesis provides a thorough evolutionary and structural analysis to fully demonstrate the similarities and differences between avian and mammalian CSF1R systems. The primary objective of this thesis was the comparative functional and structural analyses of the three proteins in birds and mammals, using evolutionary and experimental approaches. Here the presence of CSF1, CSF1R and IL34 genes and protein products is identified in a number of evolutionary diverse birds, indicating that the system is well maintained within the group. Avian genes were cloned and sequenced or otherwise extracted from different databases, and the mammalian sequences were gathered from available online sources. Whilst the gene regulation and the differential expression of the mammalian CSF1R, CSF1 and IL34 are reasonably well understood, they have not been extensively studied in birds. Preliminary comparison between these two groups provided in this thesis suggests a number of similar patterns are involved in regulation of avian CSF1R system. The mammalian CSF1/CSF1R and IL34/CSF1R ligand:receptor peptide interface has been previously resolved and was used to model similar structures in the chicken. The models were then utilised to determine which amino acids are involved in receptor binding in birds. The apparent lack of cross-species reactivity between the chicken CSF1 and zebra finch CSF1R provided a basis for an experimental validation of the in silico binding site predictions. Altogether the structural modelling, evolutionary analysis and experimental confirmation provided sufficient proof for the location of avian CSF1/CSF1R interface. Finally, an extensive bioinformatics analysis has been performed on both the coding DNA and the protein structures of the CSF1R system. The results uniformly showed that IL34 remains under purifying selection in both groups. CSF1 is diverse amongst most mammalian species, while avian CSF1 is only positively selected along particular lineages. This implies the rapid evolution of mammalian CSF1, probably in response to the selection pressure from pathogens. Contrasting situation is found in the CSF1R. Whilst mammalian CSF1R remains positively selected only along particular branches, avian CSF1R presents a number of pervasively positively selected sites, found mostly in the extracellular domains of the receptor. That suggests that in birds it is the receptor, not CSF1, which remains under strong selective pressure. These indicates that birds employ a unique way of competing in the hostpathogen arms race, suggesting the existence of yet unknown pathogen-encoded protein interacting with the avian receptor.

The pathogenicity of mycobacteria is directly related to their ability to survIve within macrophages, thereby circumventing host defense responses. This ability to resist degradation in macrophage phagosomes/lysosomes derives in large part from the complex structure of the cell wall of Mycobacterium tuberculosis. Surface exposure of lipid and glycolipid components of the mycobacterial cell wall is considered to be a major factor in the virulence of the pathogen by orchestrating the dialogue with host cells. Their interactions and modulating properties on host macrophage functions may contribute to our understanding of the pathogenesis of tuberculosis. In this study the modulating properties on macrophage functions by the major mycobacterial cell wall lipids, mycolic acids, were investigated. The investigation focused not only on the physical changes induced in macrophages as a result of the interaction with mycolic acids but also on the modulation of macrophage functions involved in innate and adaptive immunity. It was concluded that MA was involved both in mechanisms of pathogenesis of M tuberculosis, as in induction of protective immunity. By opening up some of the secrets of pathogenesis and immunity of tuberculosis, it provided new avenues for research to pursue a timeous and efficient solution to the disease. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2005. / Biochemistry / unrestricted

Inflammatory airway disease (IAD) is a common performance limiting pulmonary disorder in young racehorses in training. Although the precise aetiopathogenesis is poorly understood, proposed mechanisms include opportunistic bacterial infections and/or suboptimal air-hygiene. Since alveolar macrophages (AMs) are the first line defence in the lungs of mammalian species, they may constitute an appropriate therapeutic target cell in the treatment and the prevention of opportunistic airway infections. This thesis aimed to investigate the basic biology of the equine AM. A series of experiments were conducted to investigate the function and phenotype of this cell and comparisons made with equine macrophages derived from other anatomical sites and macrophage datasets derived from other species. The lung environment is unique, and may direct a unique phenotype and function compared with macrophages derived from other sites. Macrophages were isolated from the lungs, peritoneal cavity and other regions of healthy horses. Excellent cell recovery was demonstrated and associated with good viability, RNA yield and a demonstrable response to several stimuli, both when fresh and following cryopreservation. AMs produced tumor necrosis factor alpha (TNFα) when stimulated with lipopolysaccharide (LPS), polyinosinic-polycytidylic acid (Poly IC) and heat-killed Salmonella typhimurium and were actively phagocytic. By comparison, peritoneal macrophages (PMs) did not respond to these inducers and lacked phagocytic activity. In contrast to AMs, which showed high expression of the specific macrophage markers cluster of differentiation (CD) 14, CD163 and toll-like receptor 4 (TLR4), PMs lacked CD14. Moreover, gene expression analysis revealed an alternative macrophage activation for AMs, whereas PM showed a hybrid macrophage activation potentially attributed to the phenomenon of endotoxin tolerance. The response of equine AMs to LPS was analysed using microarrays. There was significant change in the expression of 240 genes. Those that were upregulated included well known inflammatory genes such as TNFα, IL1A and CXCL6. The pattern of response more closely resembled human and pig macrophages than mouse, including the LPS-induced expression of STAT4, IDO, IL7R genes and the failure to produce nitrite in response to LPS. These data suggest that the horse may represent a suitable animal model for human macrophage-associated lung inflammation, and conversely that data from humans may translate to horses. A final aim of this study was to investigate the effect of exercise on equine AM function. Therefore, AMs were isolated from bronchoalveolar lavage samples obtained from Standardbred racehorses at rest and during the training period and microarray analysis performed. Despite important limitations of the study, a few mechanisms at the molecular level were detected which may be involved in the development of either training-associated symptoms of, or susceptibility to IAD. Overall, this thesis aims to improve our understanding of equine macrophage biology and to provide useful information regarding the role of AMs in exercise-associated inflammation. Moreover, the findings presented here may help to inform future preventative pharmacological and/or managemental interventions for IAD.

Maternal tolerance during pregnancy increases the risk of infection with certain intracellular pathogens such as Salmonella enterica serovar Typhimurium (S.Tm). Systemic S.Tm infection during pregnancy in normally resistant 129X1/SvJ mice, with a functional natural resistance-associated macrophage protein-1 (Nramp1), leads to severe placental infection followed by fetal and maternal death. We hypothesized infection-induced inflammatory trophoblast cell death contributes to adverse pregnancy outcomes. We therefore investigated the kinetics of systemic and oral S.Tm infection in wild-type and gene deficient mice with defects in specific inflammatory pathways. Systemic infection with S.Tm resulted in preferential placental replication compared to other tissues in Nramp1+/+ mice. At 24 hours, <25% of individual placentas per mouse were infected, progressively increasing to >75% by 72 hours which correlated with a steady increase in resorption rates. Moreover, placental infection was associated with increased neutrophils, macrophages and natural killer cells whereas neutrophil numbers in the spleen remained unchanged, suggesting dichotomous modulation of inflammation in the systemic compartment compared to the feto-maternal interface. Oral infection resulted in systemic dissemination of the bacteria, substantial placental colonization and fetal loss five days post-infection in C57BL/6J mice. Systemic infection in pregnant cell death deficient Rip3-/-Nramp1+/+ mice (with defective necroptosis) resulted in decreased fetal demise relative to Nramp1+/+ and Caspase-1,11-/-Nramp1+/+ mice (with defective pyroptosis) suggesting a role for necroptotic inflammation. This study provides insight into the kinetics and mechanism of inflammation and cell death during placental S.Tm infection. Such studies may assist in the rational management of foodborne pathogens contracted during pregnancy.

Mammals are constantly challenged by numerous pathogens that pose a threat to their health. Upon infection, retroviruses quickly integrate their genome into that of their host thereby permanently modifying it. Protein members of the APOBEC3 (A3) family exhibit cytidine deaminase activity that specifically acts on single-stranded DNA to deaminate deoxycytidine bases into deoxyuridines. This process is potentially mutagenic because uracil directs the insertion of adenine on the opposite DNA strand. High levels of mutations induced by A3 proteins in the retroviral genome ultimately inactivate progeny viruses. However, under conditions where low levels of A3 proteins are present, sub-lethal mutagenesis can occur and is generally believed to be beneficial for the virus. Powerful and affordable techniques designed to detect rare deamination events generated by these deaminases along the full length of retroviral genomes are therefore essential. Through the course of my studies, I developed such a new tool that I called HyperHRM which was instrumental to my project’s success. In addition to the antiretroviral affects of their catalytic activity, some members of the A3 family have the ability to hinder reverse transcription independently of their enzymatic properties. Yet, the details underlying the deamination-independent restriction by the proteins remain unclear. Through my work, I have advanced our current understanding of this elusive process by defining the essential role for RNA-binding in the inhibition of the early steps of infection by APOBEC3G (A3G). I also demonstrate that the ability to bind RNA is important for the selection of DNA dinucleotides targeted for deamination by A3 enzymes. Based on the premise that the DNA context for deamination may alter viral fitness in various ways, I then investigated the gene inactivation potency of different A3 based on their preferred DNA substrate. My experiments showed that mutations introduced in a 5'CC context by A3G are much more lethal for the virus because of the high frequency of termination codons that are generated. I therefore clearly established that deamination target specificity has a strong influence on the overall restriction potency of A3 proteins and demonstrated that such specificity was linked to the ability of A3 proteins to bind RNA. Finally, in addition to retroviruses, mobile elements such as retrotransposons can also lead to genomic instability if not properly controlled. The A3 protein family has been shown to play a crucial role in the restriction of these elements through a mechanism that is not believed to require the enzymatic activity of the proteins, although the details of the restriction mechanism are not yet understood. Here, I provide molecular insights on the potential mechanism of retrotransposon restriction by showing that the RNA-binding properties of the enzymes are not involved in the restriction of L1 retrotransposition. A complete elucidation of the modes of restriction employed by the A3 could lead to the development of a new generation of antiretroviral drugs. Overall, my research has led to the design of a new research tool to detect and quantify A3-induced mutations in retroviruses, but more importantly, it has enabled a better understanding of how the RNA-binding abilities of A3 proteins play an essential role in the overall restriction potency of retroviruses and retrotransposons.

The innate immune system is the first line of host defense against external exposures. During these initial encounters, antigen presenting cells - specifically monocytes and macrophages - modulate further inflammatory responses. Macrophages exist along a spectrum of phenotypic programs; on the inflammatory M1 end they enhance immune activity while on the anti-inflammatory M2 end they suppress further immune activation. Furthermore, within M2 macrophages there exist many subpopulations, namely M2a and M2d, each with specific roles during infection or exposure. We sought to compare the epigenetic profiles of these subpopulations of macrophages to determine key regulatory gene networks and factors that could be exploited for therapeutic benefit.While traditionally viewed as primitive and nonspecific, a growing body of clinical and experimental evidence argues the innate immune system develops memory as a result of previous exposures, allowing the innate system to respond with enhanced and broad immunological protection upon exposure to a secondary stimulus. This biological process of innate immunity has been termed trained immunity. Trained immunity shares many phenotypic and epigenetic characteristics with adaptive immune memory; however, one of the starkest distinctions is the propensity of trained immunity to develop against heterologous stimuli. Innate memory is not antigen specific, frequently protecting the host against unrelated organisms.

DNA immunization, or the use of antigen-expressing DNAs to raise immune responses, represents a novel approach to the study and manipulation of immune responses. In this dissertation, we examine the role of antigen expression at the target site, the role of antigen presentation by bone marrow-derived cells, and the effect of secretion of antigen on DNA-raised responses in mice. Immunizations were conducted using either gene gun delivery of DNA to the epidermis or intramuscular (i.m.) saline injections. To examine the role of antigen expression at the target site, we excised target sites at different time points following immunization. We immunized with plasmid DNA expressing three different forms of antigens: influenza hemagglutinin H1, human growth hormone and influenza nucleoprotein NP (membrane-bound, secreted and intracellular, respectively). We hypothesized that antigen expression at the target site would be essential in initiating immune responses. We demonstrate here that the target site plays different roles in gene gun and i.m. immunizations. We found that the skin target site played an essential role in eliciting maximal antibody and cytotoxic T lymphocyte (CTL) responses by gene gun immunization, although low-level responses can be raised independent of the target site. In contrast, the muscle target site was not essential for eliciting maximal immune responses following i.m. immunization. We suggest that gene gun immunization results in transfection of keratinocytes and bone marrow-derived Langerhans cells at the target site, and these cells together initiate maximal responses. In i.m. immunizations, on the other hand, nonmuscle cells at distal sites, perhaps bone marrow-derived cells in lymphoid tissues, become transfected and are sufficient for initiation of maximal responses. We also examined the role of antigen presentation by bone marrow-derived cells in initiation of CTL responses to influenza NP following gene gun and i.m. immunization. We hypothesized that antigen presentation by bone marrow-derived cells would be involved in initiation of CTL responses. To test this hypothesis, irradiated F1 mice of MHC class I H-2bxd haplotype were reconstituted with bone marrow from either H-2b or H-2d donors, creating two sets of bone marrow chimeric mice (H-2b → H-2bxd and H-2d → H-2bxd, respectively). We immunized the two sets of bone marrow chimeric mice and determined the MHC haplotype restriction of the induced CTL responses using H-2b- or H-2d-restricted peptides of NP. We found that the CTL responses initiated following gene gun and i.m. immunization were restricted to the haplotype of the bone marrow donor. In H-2b→ H-2bxd chimeric mice, CTL responses were restricted to H-2b, while in H-2d→ H-2bxd chimeric mice, CTL responses were restricted to H-2d. Thus, antigen presentation by bone marrow-derived cells, and not by skin or muscle cells, initiates CTL responses following both gene gun and i.m. immunization. Finally, we examined the effect of secretion of a DNA-expressed antigen on antibody responses. We hypothesized that a secreted antigen would raise greater antibody responses than a membrane-bound antigen, due to easier access of a soluble antigen to lymphoid tissues and to uptake by professional antigen-presenting cells and by antigen-specific B cells. We immunized mice with plasmid DNA expressing either a secreted or the normal membrane-bound form of influenza hemagglutinin H1. We found that secretion of H1 (sH1) did not result in enhanced antibody responses, with sH1 appearing to be less effective than H1. We suggest that the effectiveness of DNA immunization with membrane-bound H1 in raising maximal antibody responses may be due to MHC class II presentation of H1 via an endogenous pathway, resulting from direct transfection of bone marrow-derived APCs. We also found that secretion of H1 influenced the predominant IgG subclass of antibody responses raised by i.m. immunization. Secreted H1 raised predominantly IgG1 responses and H1 raised predominantly IgG2a responses. The IgG1 response to sH1 following i.m. immunization was IL-4 dependent, suggesting that the response to sH1 had a T-helper type 2 phenotype. We propose a model for the mechanism of initiation of immune responses by DNA immunization based on our results and taking them within the context of results from other investigators in the field. We propose that DNA immunization may initiate immune responses primarily by the direct transfection of bone marrow-derived cells that then express and present the DNA vaccine-encoded antigen. However, antigen expression by nonhemopoietic cells, particularly in skin, may play a role in raising maximal responses.

Antimicrobial peptides (AMPs) are innate host defense peptides that protect against pathogenic microbes by neutralizing toxins or via a direct killing mechanism. AMPs are classified based on their physical properties such as charge, structure, and binding motifs. Here we investigated the antimicrobial and immune-modulating effects of the Regenerating Islet-Derived Protein 3 (REG3) family and LL-37 REG3 peptides are C-type lectins and have been demonstrated to have antimicrobial activity against Gram-positive bacteria by binding to sugars on the peptidoglycan membrane of these bacteria. A similar strategy is also employed by the lectin Surfactant Protein-D which has been shown to bind and neutralize Influenza A Virus (IAV). REG3 peptides were shown to be expressed in the lungs of mice infected with IAV. We observed reduction of IAV infected cells when IAV was pre-incubated with an Escherichia coliexpressed recombinant version of human REG3A peptide. This peptide also modified interaction of IAV with primary human neutrophils. However, these effects were lost when using a mammalian cell expressed recombinant REG3A. A second member of the REG3 family, REG3G, showed minimal inhibition of IAV infection. While the mechanism remains unclear, LL-37 has demonstrated killing activity against a spectrum of microbes including IAV. Previous work from our group identified the core domain of LL-37 responsible for IAV neutralization. In addition, our group showed that LL-37 modulates interaction of IAV with neutrophils. Here we tested three modified versions of LL-37 that retain the overall size and charge of LL-37, but with modifications in the core domain reducing hydrophobicity. We observed that these mutants retain IAV killing activity across multiple strains. In addition, these mutants retain the modulation of IAV induced neutrophil responses. We also found that the compounds sodium butyrate and Entinostat, which can upregulate endogenous expression of LL-37, have variable effects in IAV infection. We believe these findings will aid in the development of LL-37 derivatives to expand the repertoire of antimicrobials.