Despite the availability of live attenuated measles virus vaccines, a large number of measles-associated deaths occur among infants in developing countries during the "window of susceptibility" (age 4-9 months). During this period declining maternal antibody titers are no longer protective against wild-type measles virus (MV) and impede successful immunization with the live attenuated vaccines. Therefore, the development of a safe vaccine that would induce protective immunity in the presence of maternally derived MV-specific antibodies in young infants and would close the "window of susceptibility" is desirable. Since adenoviruses have been shown as suitable vaccine candidates capable of eliciting potent protection against mucosal infectious diseases, the ability of an adenovirus-vectored anti-measles vaccine to elicit robust immune responses against MV was assessed in this study. Mice immunized intramuscularly or intranasally with a combination of human adenovirus serotype 5 (Ad5) recombinants expressing MV hemagglutinin (H) and fusion (F) glycoproteins developed MV-specific neutralizing antibody titers similar for both routes of immunization. However, intramuscular immunization of mice with Ad5 recombinants resulted in induction of a predominant T helper type (Th1) immune response, whereas intranasal immunization induced a balanced Th1/Th2 immune response. Furthermore, intranasal immunization resulted in increased titers of MV-specific immunoglobulin A (IgA) in lungs in comparison to intramuscularly immunized animals. The ability of the Ad5 recombinants to induce protective immune responses in cotton rats by different routes of administration was also evaluated. Cotton rats that received a single dose of the Ad5 recombinants intramuscularly or intranasally experienced a rise in MV-specific neutralizing antibody titers and reduction of the viral RNA load in the lung tissue after intranasal MV challenge. In addition, the largest reduction in viral replication was observed in the group of cotton rats inoculated with the Ad5 recombinants intranasally. Based on these observations, the Ad5-based vaccine appears to be a suitable candidate against measles. Furthermore, a capability of purified globular head domain of MV H protein produced in a human cell line to induce MV-specific immune responses in mice was tested. Subcutaneous immunization of mice with the recombinant protein alone resulted in both humoral and cell-mediated immunity, characterized by the production of MV-specific serum IgG and neutralizing antibodies, as well as interferon-gamma and interleukin 5 (IL-5) production by in vitro restimulated splenocytes. The former responses were further enhanced by formulation of the protein with aluminium hydroxide. However, very low numbers of INF-gamma secreting cells and low levels of IgG2a in the serum suggested a Th2 immune response. Novel adjuvants (Th1-directing), as well as MV F protein should be considered for the inclusion into the vaccine formulations to induce more balanced Th1/Th2 immune responses against measles.

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.

The immunomodulatory effects of Trichinella spiralis or Trichinella pseudospiralis soluble antigen extracts were examined in an effort to characterize the differences in immune responses seen during these Trichinella infections. The newborn larvae extracts of either parasite exhibited similar potency for stimulating macrophage PGE production; however, the muscle larvae extracts of T. pseudospiralis stimulated greater levels of PGE than did the muscle larvae extracts of T. spiralis. These data clearly indicate that Trichinella antigens possess immunomodulatory capabilities.

Vaccination against intracellular pathogens requires generation of pool of memory T cells, which can respond upon infection and mediate immune responses by either killing of infected host cells or induce killing mechanisms in infected cells. T cell-inducing vaccines aim to deliver the antigen to antigen presenting cells (APCs) by presenting on MHC molecules thus bridging innate and adaptive immunity. The intracellular pathogen Edwardsiella ictaluri causes enteric septicemia of catfish (ESC), which is a devastating disease in catfish industry. E. ictaluri can survive in professional phagocytes and use them as an infection source. Two new live attenuated vaccine (LAV) strains, EiDELTAevpB and ESC-NDKL, were developed by our group. However, the role of LAVs in phagocytosis, bacterial killing, and antigen presentation is unexplored. Therefore, further research is necessary to determine immune responses in channel catfish against LAVs. The long-term goal of this project is to identify immunological APC-dependent mechanisms that underscore E. ictaluri pathogenesis to enable development of effective control strategies for ESC. The overall goal of this project is to assess the role of three professional APCs, dendritic cells (DCs), macrophages and B cells in the LAV-induced innate and adaptive immune responses in catfish. The central hypothesis is that efficacious LAV strains will enhance phagocytosis and microbial killing, and promote the generation of T cells that regulate and control protective B cell-mediated immunity. The rationale for this research is that more detailed knowledge about phenotype and function of catfish APCs will not only help gain insight into the evolution of vertebrate adaptive immune system but will provide valuable information for development and optimization of immunotherapies and vaccination protocols for aquaculture use. In this study, we first identified DC-like cells in immune-related organs of catfish and assessed their expression patterns in lymphoid organs of catfish in E. ictaluri infection. Although WT strain induces the functional inability of DC-like cells in migration and maturation, LAVs strains promote the migration and maturation of DC-like cells for antigen presentation. Two LAVs enhanced the phagocytosis and killing activity in catfish macrophages and B cells. Also, LAVs induce high expression of T cell-related genes without causing inflammation.

Summary of the work Abdominal aortic aneurysm (AAA) is a chronic degenerative disease that usually affects men over 65 years with an estimated prevalence of 5%. Aneurysm rupture represents a catastrophic event which carries a mortality rate of almost 90%. Current therapeutic options for AAAs measuring 5.5 cm in diameter or larger are based on prophylactic surgery, including conventional open reconstruction and endovascular stent-graft insertion. For patients with small asymptomatic AAAs (4.0 up to 5.5 cm in diameter), evidence from two recent large randomized controlled trials indicates no long-term survival benefit from immediate elective surgical repair as compared to imaging surveillance until aneurysm expands to 5.5 cm. This highlights the need for development of novel medical management strategies, including selective pharmacologic approaches, directed at preventing aneurysm expansion. In this regard, it is expected that a detailed knowledge of the pathobiology of human AAA lesion and a better understanding of pathophysiological mechanisms underlying initiation and progression of aneurysmal degeneration, particularly the specific involvement of T lymphocytes, will have special relevance to this challenging issue. Inflammatory and helper T-cell responses in abdominal aortic aneurysm : controversial issues Innate and inflammatory responses to endovascular versus open AAA repair. The occurrence of early acute systemic inflammatory responses after conventional open AAA repair is widely recognized and is thought to lead to the development of organ dysfunction and multiple organ failure, responsible for a large proportion of morbidity and mortality associated with aortic surgery. New therapeutic strategies designed to avoid ischemia-reperfusion injury related to aortic cross-clamping and to minimize the degree of tissue damage have thus been developed recently. Specifically, the advent of endovascular techniques has radically extended management options for patients with AAA. Although the method is believed to offer a clear short-term benefit over open repair, notably as regards restricted perioperative haemodynamic parameter fluctuations, reduced blood loss, briefer duration of surgery, shorter hospital stay, and lower 30-day mortality and complication rates, conflicting data are available regarding the exact nature and extent of the inflammatory events arising after such endoluminal procedures ; while several authors have indeed reported that endovascular AAA repair can determine a less intense and extensive inflammatory response, others have unexpectedly observed that the method may elicit a strong inflammatory response, the so-called « postimplantation syndrome ». Adaptive cellular immune responses in human aneurysmal aortic lesion. The inflammatory nature of AAA disease has long been suggested by the presence of a great number of CD4+ T lymphocytes in the outer media and adventitia of human AAA lesion. Interestingly, such infiltrating T-cell populations may have significant implications in the process of aneurysm dilation, since cytokines produced by T cells, notably IFN-gamma, have previously been shown to modulate production of matrix-degrading enzymes by resident macrophages and to induce apoptosis of medial SMCs. Through these key pathological mechanisms, T cells could potentially contribute to orchestrate aortic wall connective tissue disordered remodeling and degradation, and promote extensive disruption of elastic media, ultimately leading to aneurysmal degeneration. Nevertheless, despite their relative abundance in human AAA wall tissues, there is limited and controversial information as regards the functional profile of lesional lymphocytes, the exact nature of aortic wall adaptive cellular responses, and the etiologic role of T cells and their cytokines in initiation and progression of the aneurysmal process. Indeed, both Th1-type and Th2-type responses have been identified in human studies and experimental animal models of AAA. Aims of the work The main objectives of our work were to explore the innate and adaptive cellular immune responses in human AAA. In the first part of our work, we aimed to examine prospectively innate and inflammatory responses arising in a non-randomised cohort of patients undergoing endovascular versus open AAA repair. In the second part of our work, we focused our efforts on characterizing the nature of adaptive cellular immune responses and the phenotypic and functional repertoire of T cells in human AAA wall tissues obtained from a consecutive series of patients undergoing open AAA repair. Specifically, we sought to determine whether type 1 or type 2 responses occur predominantly in advanced AAA lesion. Main experimental findings Limited inflammatory response after endovascular AAA repair. Serial peripheral venous blood samples were collected preoperatively, immediately after declamping or insertion of endograft, and after 1, 3, 6, 12, 24, 48, and 72 hours. We first examined the acute phase reaction and liberation of complement cascade products using turbidimetric method and nephelometry. We found that endovascular repair produced lower postoperative CRP, leucocytosis, neutrophilia, and C3d/C3 ratio as compared to open surgery. We next analyzed surface expression of activation markers on peripheral CD3+ T cells using flow cytometry. We observed a strong upregulation of CD38 after open but not endovascular repair. Analysis of CD69 and CD25 molecules revealed no perioperative fluctuations in any group. We then investigated release of various circulating soluble cell adhesion molecules, proinflammatory cytokines, and chemokines using enzyme-linked immunosorbent assays. We demonstrated that both procedures are characterized by similar increases in ICAM-1 and IL-6 levels. Finally, tendency towards high levels of TNF-alpha and IL-8 was detected in endovascular repair, but data failed to reach statistical significance. Predominance of type 1 CD4+ T cells in human aneurysmal aortic lesion. We have developed a tissue enzymatic digestion and cell extraction procedure to isolate intact mononuclear cells from aortic wall segments. This original cell isolation protocol enabled us to examine ex vivo the presence, phenotype, and cytokine secretion profile of infiltrating T lymphocytes freshly isolated from human AAA tissues for comparison with their circulating counterparts using flow cytometry. We found that both populations of infiltrating CD4+ and CD8+ T cells display a unique activated memory phenotype, as assessed by an increased expression of CD69 and HLA-DR activation antigens, downregulation of CD62L molecule, and predominant expression of the CD45RO isoform characteristics of memory cells. In addition, we identified the presence in human aneurysmal aortic wall lesion of CD4+ T cells producing high levels of IFN-gamma but not IL-4, reflecting their type 1 nature. In an additional series of experiments, cytokine gene expression was determined in whole aneurysmal and non-diseased aortic samples using LightCycler-based quantitative real-time reverse transcription-polymerase chain reaction. The molecular basis of type 1 or type 2 dominant responses was further specified by analyzing mRNA levels of transcription factors specifically involved in Th1 or Th2 differentiation such as T-bet and GATA-3. We demonstrated that aneurysmal aortic specimens exhibit high transcript levels of IFN-gamma but not IL-4, and consistently overexpressed the IFN-g-promoting cytokine IL-12 and the type 1-restricted transcription factor T-bet, further establishing the prominent type 1 nature of aortic wall responses. Moreover, such selective tissue expression of IL-12 and T-bet in the vessel microenvironment points to a potential role for these signals in directing aortic wall responses towards a type 1 phenotype. Conclusions Our findings indicate that endovascular AAA repair is associated with a lesser degree of acute phase reaction, peripheral T-cell activation, and release of complement proteins as compared to conventional open surgery, suggesting that the innate and inflammatory responses to AAA repair are significantly attenuated by the endovascular approach as compared to the traditional open reconstruction. These results support the view that the endoluminal procedure represents an attractive alternative to open surgery for the treatment of large aneurysms. On the other hand, we have demonstrated that Th1 cell infiltrates predominate in human end-stage AAA lesion. These observations are relevant for helping clarify the pathobiology of human AAA tissues and defining prospects for the prevention of aneurysm expansion. Indeed, identification of such infiltrating populations of IFN-gamma-producing CD4+ T cells not only provide new insights into the pathogenesis of the disorder, but could also serve as a basis for the development of novel medical management strategies directed at preventing aneurysm formation and progression, including therapeutic approaches based on the modulation of aortic wall responses and designed to selectively target T-cell activation and cytokine production. In this respect, the present work provides experimental evidence in support of the emerging concept that, although multifactorial, aneurysm disease may be regarded as a Th1-driven immunopathological condition, and suggests that strategies targeting IFN-gamma could be a particularly exciting and fruitful avenue for further investigation. Ongoing clinical and basic research in these areas can be expected to yield design of promising pharmacologic approaches to control AAA expansion. From a clinical perspective, such efforts have the potential to dramatically influence both the outcome and management of this common and life threatening condition.

La transfusion sanguine est un traitement essentiel à la survie de millions de patients. Son principal risque immunologique est l’allo-immunisation post-transfusionnelle. Elle se traduit par la production d’allo-anticorps contre des antigènes de globules rouges (GR) conduisant à des hémolyses post-transfusionnelles. Les mécanismes à l’origine de la tolérance des GR ou de son inhibition lors de l’allo-immunisation sont mal connus. Ainsi, mes travaux de thèse, portant sur la compréhension de ces effets, se sont articulés en trois parties avec 1/ l’étude des conditions optimales aux réponses allo-immunes, 2/ l’étude des effets d’une stratégie thérapeutique utilisant un anticorps monoclonal et 3/ l’étude des effets immunomodulateurs, incluant la tolérance, médiée par des composants présents dans les concentrés de globules rouges (CGR).Afin d’étudier l’allo-immunisation, nous avons utilisé le modèle murin. Nous montrons qu’une variation du délai entre la transfusion et la stimulation du TLR3 impacte la réponse immune dans la rate. Une activation importante des lymphocytes T CD4+ (LT CD4+) allo-réactifs accompagnée d’une production accrue d’allo-anticorps ont été montrées à 7 jours de délai. Afin de limiter l’allo-immunisation, l’utilisation d’un anticorps anti-CD20 déplétant les lymphocytes B (LB) montre une altération des LB mais surtout des LT CD4+ impliqués dans le processus d’induction de l’allo-immunisation. Enfin, la modification du phénotype des cellules dendritiques CD11c+ de la rate des souris transfusées, observée hors contexte inflammatoire, suggère une maturation incomplète à l’origine d’une tolérance antigénique. Pour finir, l’analyse de différents composants présents dans les CGR confirme l’existence de microparticules (MPs) lymphocytaires. Ces MPs présentent des molécules inhibitrices et pourraient donc être impliquées dans la tolérance des antigènes transfusés.En conclusion, mes travaux montrent la coopération des DCs avec les LT CD4+ permettant celle des LT CD4+ avec les LB pour induire une réponse immune. Comme toute réponse humorale, nous confirmons que l’allo-immunisation fait intervenir des DCs, des LT CD4+ et des LB. Ces résultats ouvrent de nouvelles voies de recherche pour mieux caractériser l’allo-immunisation en particulier chez les patients drépanocytaires qui sont les plus touchés. / Red blood cell (RBC) transfusion is a life-saving treatment for millions of patients. However, its main immunological risk is RBC alloimmunization resulting in antibody production against RBC antigen. Alloimmunization can lead to severe complications threatening the life of the patient. The mechanisms explaining RBC alloimmunization are poorly understood. Therefore, my doctoral work aiming at understanding transfusion effects, was subdivided into three parts with 1/ the study of optimal conditions for alloimmune responses, 2/ the impact of a therapeutic strategy using a monoclonal antibody to inhibit alloimmunization and 3/ the study of immunomodulatory effects of transfusion, including tolerance, through components present in the RBC concentrates.We also used HOD murine model for the study of alloimmunization to show an impact of the delay between the TLR3 agonist injection and the transfusion on immune responses against RBCs. At 7 days of delay, we have demonstrated an important alloreactive CD4+ T-cell activation and a wider alloantibody production. Furthermore, B-cell depletion, using a monoclonal anti-CD20 antibody, revealed potential changes on LB implicated in alloimmunization induction and mostly on alloreactive CD4+ T cells. We finally observed a modification of splenic CD11c+ DC phenotype from transfused mice out of a TLR context. This suggest an incomplete maturation that could explain antigen-specific tolerance. The investigation of several components in RBC concentrates confirmed the presence of microparticules (MPs) issued from T lymphocytes. These MPs carry inhibitory markers and could thus inhibit DC activation to induce antigen-specific tolerance.Therefore, my doctoral work highlights the implication and the cooperation of DCs with CD4+ T cells to allow cellular cooperation between CD4+ T cells and B cells for immune response induction. As in any humoral response, we confirmed that RBC alloimmunization involves DCs, CD4+ T cells and B cells. In addition, these results are opening up new areas of investigation for a better characterization of alloimmunization in particular in the most affected patients, the SCD patients.

No description available.

Background: Allergic diseases are characterised by dysregulated immune responses. The first manifestation of the atopic phenotype is often food allergy, with symptoms like eczema. Food allergy in children is generally outgrown before 3 years of age, but a temporary food elimination diet is often advocated. The prevalence of allergic diseases has increased in affluent countries during the last decades, possibly as a consequence of a changed lifestyle leading to decreased microbial load. Aim: To investigate humoral, mucosal and cell-mediated immunity in association to allergy and allergy development in young children and relate this to environmental factors. Subjects: Two cohorts of children were investigated; 1) Children from countries with high (Sweden) and low (Estonia) prevalence of allergy that were followed prospectively from birth to 5 years of age. 2) Infants with eczema and suspected food allergy that were followed prospectively to 4 ½ years of age. Methods: Endotoxin levels were analysed in house dust samples. Antibodies were measured in serum and saliva samples with ELISA. Food allergen induced cytokine responses were analysed in mononuclear cells. Results: The microbial load, delineated as endotoxin levels, was higher in house dust from Estonia than Sweden and was, in Swedish children, inversely associated with sensitisation and clinical symptoms of allergy. The decreased microbial load in Sweden may have an impact on mucosal immune responses as different IgA antibody patterns were observed in Sweden and Estonian children with much lower secretory (S)IgA antibody levels and high proportion of non-SIgA, i.e. IgA antibodies lacking the secretory component, in the Swedish children. Moreover, low levels of SIgA were associated with clinical symptoms in sensitised children. High IgG4 antibody levels to food allergens during infancy were associated with faster tolerance development in food allergic children. Cytokine responses by mononuclear cells after allergen stimulation was upregulated with age in children with prolonged food allergy, but not in children who develop tolerance before 4 ½ years of age, possibly because of the prolonged elimination diet in the former group. Summary: Reduced microbial exposure in affluent countries may affect the mucosal immune responses during infancy, possibly resulting in an increased risk of developing allergic disease. High levels of IgG4 antibodies during infancy are associated with faster achievement of tolerance in food allergic children. Allergen elimination during infancy may result in a dysfunctional cytokine response.

Two different feeding trials were conducted to evaluate the effects of dietary supplementation with the dairy/yeast prebiotic GroBiotic-A (GBA) to Nile tilapia diets. A nutritionally complete basal diet was supplemented with GBA at either 1 or 2% of dry weight, and all three diets were fed to triplicate groups of juvenile fish in two consecutive trials. Trial 1 continued for 8 weeks, while Trial 2 was conducted for 5 weeks to more specifically assess immunological responses, intestinal characteristics and disease resistance of tilapia. At the conclusion of Trial 1, there were no differences in weight gain (WG) or feed efficiency (FE) among fish fed the three diets. However, fish fed the diet with GBA at 2% had significantly increased survival and noticeably elevated levels of plasma lysozyme compared to fish fed the basal diet or the diet with GBA at 1%. Similarly, at the conclusion of Trial 2, WG and FE were unaffected by GBA supplementation; however, fish fed the diet with GBA at 2% also exhibited elevated plasma lysozyme as well as significantly (P < 0.05) increased levels of extracellular superoxide anion production (EX-SOAP) by macrophages. Dendrogram analysis of denaturing gradient gel electrophoresis (DGGE) images detected a significantly different microbial community within the intestine of fish fed the diet with GBA at 2% compared to fish fed the basal diet and diet with GBA at 1%. None of the experimental diets resulted in significant improvements to survival after exposure to Streptococcus iniae due to within treatment variability. However, fish fed the diet with GBA at 2% did tend to experience reduced mortality (12.5%) as compared to fish fed the basal diet (35%). Thus, supplementation of GBA at 2% of diet did alter the gut microbiota of tilapia and enhanced immunological responses and disease resistance to S. iniae.

Hypoxia can result in brain injury. Hypoxic brain injury can also result in excess stress hormones and activated immune responses. In this study, we examined multiple spontaneous motor behaviors, concentrations of stress hormones, and gene expression of immune responses in rats after perinatal hypoxia. Hypoxic animals exhibited impaired spontaneous motor behaviors in several tests. Perinatal hypoxia also caused increased levels of stress hormones and altered expression of genes associated with adaptive and innate immunity at different time points after hypoxia exposure. Findings demonstrate stress hormones and immune responses are available to play an important role in perinatal brain injury and can impact delayed behavioral development.