Allergen-reactive CD4+ T cells are implicated in the pathogenesis of allergic disease. Peptide immunotherapy (PIT) involves therapeutic administration of short immunodominant peptides from within the protein allergen to which CD4+ T cell responses are directed. This approach can induce tolerance of allergen-reactive CD4+ T cells, while negating the risk of severe allergic reactions associated with whole allergen specific immunotherapy. PIT therefore holds promise as a diseasemodifying treatment for allergic patients. However, further information regarding the mechanisms of action of PIT are required to aid translation to the allergy clinic. Chicken ovalbumin (OVA) is a commonly used model allergen in mouse models of allergic airways inflammation (AAI). Trackable, T cell receptor transgenic T cells recognizing the immunodominant 323-339 peptide of OVA (pOVA) allow mechanistic investigation of PIT in response to pOVA. This thesis investigated the hypothesis that strong, systemic T cell responses induced by intravenous administration of soluble pOVA will induce i) tolerance to pOVA and ii) linked suppression to any additional OVA T cell epitopes, hence improving OVA-induced AAI. Contrary to the hypothesis, intravenous pOVA PIT did not improve disease in a C57BL/6 model of OVA-induced AAI. Models of OVA-induced allergic sensitisation and AAI were therefore developed incorporating trackable CD4+ pOVA-reactive T cells (OT-II cells). pOVA PIT induced tolerance of these cells in an allergic sensitisation setting, but had limited impact on the overall OVA response. Yet, in a model of AAI driven solely by Th2 polarised CD4+ OT-II cells, pOVA PIT did improve disease. It was concluded that, in non-transgenic C57BL/6 mice, CD4+ T cells responding to additional epitope(s) within OVA were important in driving disease and that these T cells were not subject to linked suppression following pOVA PIT. Using a panel of overlapping peptides constituting the sequence of OVA, a novel CD4+ epitope within OVA was characterised. The effects of PIT using pOVA in combination with a peptide containing this additional epitope on OVA-induced AAI were then assessed. Findings from this project therefore hold importance for future mechanistic work surrounding PIT in allergic disease.

published_or_final_version / abstract / Surgery / Master / Master of Philosophy

Cancer is a worldwide health problem and despite advances in traditional treatments i.e. surgery, chemotherapy and radiotherapy, the cure rate remains disappointing for some cancers. Different novel therapeutic strategies are being developed. In this thesis two nontraditional cancer therapy approaches are studied: gene therapy using viral vectors and antitumour vaccination with dendritic cell - tumour cell (DC/TC) hybrids. We have developed a novel ELISPOT titration method for viral vectors that is based on the actual expression of the transgene in target cells. This method was developed with recombinant parvovirus MVM-IL2, but it should be adaptable for other vectors carrying expression cassettes for secreted transgene products for which antibodies are available. The ELISPOT titration method allows for faster and better quantification of transducing units present in vector stocks as opposed to titration by in situ hybridisation (annexe I). The MVMIL2 vector has shown an anti-tumour effect against melanoma in an immunocompetent mouse model (annexe IV). Previous work concerns photodynamic inactivation of adenoviral vectors for biosafety and an in vivo study in which a synergistic effect of antiangiogenesis gene therapy combined with radiotherapy could be shown (annexes V and VI). DC/TC hybrids have been proposed as cancer vaccines for their simultaneous expression of antigen presentation machinery and tumour associated antigens. Hybrids are classically generated by polyethylene glycol (PEG) or electrofusion. These methods however require special skills and equipment and cause rather high cell lethality. Fusion via the expression of viral fusogenic membrane glycoproteins (FMG), such as the vesicular stomatitis virus-G (VSV-G) (annexe III) or the Gibbon ape Leukemia Virus (GaLV) FMG, have recently been described. We have mainly focussed on the latter. Transduction of cells with GaLV-FMG proved to be a limiting step for an efficient generation of hybrids. On the other hand, constitutive expression of GaLV-FMG leads to lethal syncytia formation in human cells. Therefore we developed a novel fusion strategy for the generation of DC/TC cell hybrids that involves the use of a non-human fusogenic cell line that constitutively expresses the GaLV-FMG. With this method we were able to generate reproducible yields of DC/TC triparental hybrids. The formation of tri-parental hybrids via the fusogenic cell line is an interesting alternative to existing DC/TC fusion methods because of its simplicity and its flexibility in the choice of fusion partners, i.e. autologous or allogeneic DCs and tumour cells. Moreover, the tri-parent hybrid system offers the possibility to further enhance the immune response by the addition of transgenes that code for immuno-modulating factors to the fusogenic cell line (annexe II).

Cancer is one of the most pervasive health problems in the world today. Despite major advances in its treatment in recent decades, conventional therapies have seen limited success. Aggressive, drug-resistant cancer cells can reemerge after treatment, resulting in relapse. Immunotherapy, a strategy that utilizes a patient's own immune system to specifically destroy cancer cells, is a potential solution to this problem. Immunotherapy, however, is limited by multiple mechanisms of cancer-induced immunosuppression. One of the most important of these mechanisms is the induction of Treg, which are capable of suppressing multiple arms of the anti-cancer immune response. In the current study, we evaluated strategies to hinder the deleterious function of Treg on cancer immunotherapy. First, we determined that imatinib mesylate could inhibit Treg function in vivo and in vitro and increase the efficacy of dendritic cell-based immunization against an imatinib-resistant lymphoma. Then, searching for further methods to inhibit Treg, we found that Th-1 cells were capable of inhibiting Treg function and synergizing with a tumor lysate vaccine to treat leukemia. This process was dependent on IFN-γ secretion by the Th-1 cells. While investigating the influence of Th-1 on Treg and the resulting immunomodulatory effects of these cells in vivo, we discovered that they were capable of promoting the non-conventional direct tumor killing function of DC. We determined that Th-1 induce the cytotoxic function of bone marrow-derived DC generated with GM-CSF and IL-4 by a mechanism dependent on IFN-γ. Finally, because our results indicate that the antigen presenting function of KDC may depend upon their cytotoxic ability, and since DC generated with IL-15 have been reported to be more efficient APC than those generated with IL-4, we evaluated their ability to also function as direct tumor cell killers. We found that while IL-15 DC can indeed kill tumor cells, only LPS and not IFN-γ was capable of inducing this capability. These findings contribute to both arms of anti-cancer immunity by impairing immunosuppression with imatinib and Th-1, and promoting anti-tumor immunity with KDC. This double-pronged approach may contribute to further strategic advances in the field of cancer immunotherapy.

African animal trypanosomosis or nagana is a disease in livestock caused by various species of protozoan parasites belonging to the genus Trypanosoma particularly T. congolense, T. vivax and T. b. brucei. Nagana is the most important constraint to livestock and mixed crop-livestock farming in tropical Africa. Trypanosomes undergo part of their developmental life in their insect vector, the tsetse fly and part in their mammalian host. Measures for eradicating the continent of the tsetse fly vector include insecticidal spraying, targeting and trapping. Vaccine development has been hampered by the generation of an inexhaustible collection of variant surface glycoproteins that trypanosomes possess and allow for evasion of the host immune system. Anti-disease vaccines aimed at reducing the symptoms of the disease rather than killing the parasite itself have been demonstrated as an alternative approach. Trypanotolerant cattle are able to protect themselves from the disease-associated symptoms. They are able to mount a better antibody response to the CATL-like cysteine peptidase, TcoCATL, compared to trypanosusceptible breeds. Bovine trypanosomosis, however, continues to be controlled primarily by trypanocidal compounds such as isometamidium chloride, homidium and diaminazene that have been developed more than 50 years ago and consequently drug resistance is widespread. Trypanosomal cysteine peptidases have also been proven to be effective targets for chemotherapeutics. TcrCATL, inhibited by the vinyl sulfone pseudopeptide inhibitor K11777, was effective in curing or alleviating T. cruzi infection in preclinical proof-of-concept studies and has now entered formal preclinical drug development investigation. Understanding enzymatic as well as structural characteristics of pathogenic peptidases is the first step towards successful control of the disease. To date no such characterisation of the major cysteine peptidases from T. vivax has been conducted. Although the major cysteine peptidase from T. vivax, TviCATL, has not been proven as a pathogenic factor yet, its high sequence identity with the pathogenic counterparts such as TcrCATL and TcoCATL hold much speculation for TviCATLs role in pathogenocity. In the present study, native TviCATL was isolated from T. vivax Y486, purified and characterised. TviCATL showed to have a general sensitivity to E-64 and cystatin and has a substrate specificity defined by the S2 pocket. TviCATL exhibited no activity towards the CATB-like substrate, Z-Arg-Arg-AMC but was able to hydrolyse Z-Phe-Arg-AMC, the CATL-like substrate. Leu was preferred in the P2 position and basic and non-bulky hydrophobic residues were accepted in the P1 and P3 positions respectively. Similar findings were reported for TcoCATL. The substrate specificity of TviCATL and TcoCATL does argue for a more restricted specificity compared to TcrCATL. This was based on the Glu333 in TcrCATL substituted with Leu333 in TviCATL and TcoCATL. In the case of TcrCATL, the Glu333 allows for the accommodation of Arg in the P2 position. Like other trypanosomal cysteine peptidases, TviCATL was inhibited by both chloromethyl ketones, Z-Gly-Leu-Phe-CMK and H-D-Val-Phe-Lys-CMK. Determining further structural and functional characteristics as well as whether TviCATL, like the T. congolense homolog, TcoCATL, acts as a pathogenic factor, would be important information to the designing of specific chemotherapeutic agents. To date, TcrCATL and TbrCATL (from T. b. rhodesiense) are the only trypanosomal CATL-like cysteine peptidases been crystallised and their tructures solved. This advantage has allowed for the directed design of synthetic peptidase inhibitors. The crystal structure of TcoCATL will be of major significance to the design of specific chemotherapeutic agents. Furtherrmore, understanding the dimeric conformation of TcoCATL is important for vaccine design as immune responses are likely to recognise the dimer specific epitopes. In the current study, the catalytic domain of TcoCATL and TviCATL, were recombinantly expressed in Pichia pastoris and purified to homogeneity. The T. congolense cysteine peptidase pyroglutamyl peptidase (PGP), also proven to be pathogenic in T. b. brucei, was recombinantly expressed in E. coli BL21 (DE3) cells and also purified to homogeneity. Purified cysteine peptidases along with previously purified TcoCATL dimerisation mutants, TcoCATL (H43W) and TcoCATL (K39F; E44P), possessing mutated residues involved in TcoCATL dimerisation, as well as the mutant proenzyme TcoCATL (C25A), were screened for crystallisation conditions using the Rigaku robotic crystallisation suite. One-dimensional needle-like crystals were found for TcoCATL (K39F; E44P). Optimisation of the TcoCATL (K39F; E44P) crystals were analysed for X-ray diffraction. The poor diffraction pattern prompted further optimisations for better crystal quality, which is presently underway. The crystal structure of TcoCATL, with some of the residues involved in dimerisation mutated, will be pivotal in understanding the dimerisation model. Furthermore, the information about the structure will be valuable for vaccine design and chemotherapeutics development. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2011.

Senile plaques (Aβ) and neurofibrillary tangles (tau) are pathological hallmarks of Alzheimer’s disease (AD). If and how the formation of these deposits are mechanistically linked remains mainly unknown. In recent years, the focus has shifted from insoluble protein deposits to soluble aggregates of Aβ and tau. Protofibrils are large soluble Aβ oligomers which were linked to AD by the discovery of the Arctic AβPP mutation. Treatment of young tg-ArcSwe mice with an Aβ protofibril-selective antibody, mAb158, cleared protofibrils, prevented amyloid plaque deposition and protected cultured cells from protofibril-mediated toxicity. This suggests that Aβ protofibrils are necessary for the formation of Aβ deposits. Functional assessment of tg-ArcSwe mice in IntelliCage demonstrated hippocampal-dependent behavioral deficits such as memory/learning impairments, hyperactivity and perseverance behavior. Learning impairments did not correlate to Aβ-measures but to calbindin, which might be a good marker for Aβ-mediated neuronal dysfunction. Splicing of exon 10 in the tau gene differs between human and mouse brain. Exon 10 is part of the microtubule-binding domains which helps to maintain microtubule stability and axonal transport, functions vital to neuronal viability. Axonal transport dysfunction has been proposed as a common pathway of Aβ and tau pathogenesis in AD. Generation of a novel tau mouse model with absence of exon 10 led to age-dependent sensorimotor impairments which may relate to dysfunctions in cerebellum. No tau pathology was evident suggesting that a trigger of tau fibrillization e.g. a human Aβ or tau aggregate is needed. Generation of AβPPxE10 bitransgenic mice with no exon 10 showed lower Aβ plaque burden. Possibly changes in microtubule function lead to altered intracellular AβPP transport and Aβ production. Initiation of tau pathology in AβPPxE10 mice might require a certain type of Aβ-aggregates which is not produced or exist at too low concentration in transgenic mouse brain. In summary, the Aβ protofibril-selective antibody was found to be a promising treatment for AD. The IntelliCage system was proven to be useful for functional evaluation of AβPP mice. Exon 10 in tau was shown to affect sensorimotor functions and Aβ pathology in bitransgenic mice by mechanisms that deserve further investigation.

by Wong Chun-kwok. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1993. / Includes bibliographical references (leaves 233-246). / ABSTRACT --- p.I / ACKNOWLEDGEMENTS --- p.V / ABBREVIATIONS --- p.VI / PUBLICATIONS --- p.IX / CHAPTER / Chapter 1. --- GENERAL INTRODUCTION --- p.1 / Chapter 1.1 --- EFFECTOR CELLS MEDIATING ANTI一TUMOR IMMUNITY --- p.3 / Chapter 1.1.1 --- CYTOTOXIC T LYMPHOCYTES --- p.4 / Chapter 1.1.2 --- MACROPHAGES --- p.4 / Chapter 1.1.3 --- NATURAL KILLER CELLS --- p.5 / Chapter 1.1.4 --- LYMPHOKINE ACTIVATED KILLER CELLS --- p.7 / Chapter 1.1.5 --- TUMOR-INFILTRATING LYMPHOCYTES --- p.8 / Chapter 1.2 --- BIOLOGICAL RESPONSE MODIFIERS : THE NEW IMMUNOTHERAPY --- p.9 / Chapter 1. 3 --- CYTOKINES AS BIOLOGICAL RESPONSE MODIFIERS IN CANCER THERAPY --- p.12 / Chapter 1.3.1 --- INTERFERONS --- p.12 / Chapter 1.3.2 --- TUMOR NECROSIS FACTOR-ALPHA --- p.13 / Chapter 1.3.3 --- INTERLEUKIN-1 --- p.15 / Chapter 1.3.4 --- INTERLEUKIN-2 --- p.16 / Chapter 1.3.5 --- GRANULOCYTES /MACROPHAGES COLONY-STIMULATING FACTORS --- p.16 / Chapter 1.3.6 --- EPIDERMAL GROWTH FACTOR --- p.17 / Chapter 1.3.7 --- TRANSFORMING GROWTH FACTOR-BETA --- p.17 / Chapter 1.4 --- BIOACTIVE POLYSACCHARIDES FROM CHINESE MEDICINAL HERBS ACT AS BIOLOGICAL RESPONSE MODIFIERS --- p.18 / Chapter 2. --- AIM AND SCOPE OF INVESTIGATION --- p.27 / Chapter 3. --- MATERIALS AND METHODS --- p.30 / Chapter 3.1 --- MATERIALS --- p.30 / Chapter 3.2 --- METHODS --- p.39 / Chapter (I) --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF PSEUDOSTELLARIA HETEROPHYLLA" / Chapter 3.2.1 --- Hot water extraction and stepwise alcohol precipitation --- p.39 / Chapter 3.2.2 --- "Determination of carbohydrate, protein, uronic acid contents" --- p.41 / Chapter 3.2.3 --- Gel filtration --- p.41 / Chapter 3.2.4 --- Anion-exchange chromatography --- p.41 / Chapter 3.2.5 --- Paper chromatography --- p.42 / Chapter 3.2.6 --- Gas liquid chromatography --- p.43 / Chapter 3.2.7 --- Determination of molecular weight by high performance liquid chromatography --- p.44 / Chapter 3.2.8 --- SDS-polyacrylamide gel electrophoresis --- p.44 / Chapter 3.2.9 --- Determination of the bio´ؤtoxicity of samples --- p.46 / Chapter 3.2.10 --- Treatment of samples with sodium periodate or acetic acid --- p.46 / Chapter (II) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON LYMPHOCYTES / Chapter 3.2.11 --- Isolation and preparation of cells --- p.48 / Chapter 3.2.12 --- In vitro lymphocyte transformation assay --- p.50 / Chapter 3.2.13 --- Mixed lymphocyte culture --- p.50 / Chapter 3.2.14 --- Depleting mouse T cells by anti-Thy-1.2 antibody plus complement treatment --- p.51 / Chapter 3.2.15 --- Depleting mouse B cells by anti-mouse B cell antibody plus complement treatment --- p.51 / Chapter 3.2.16 --- Haemolytic plaque assay --- p.52 / Chapter 3.2.17 --- Delayed-type hypersensitivity --- p.53 / Chapter 3.2.18 --- Immunofluorescent assay for interleukin-2 receptor expression --- p.54 / Chapter 3.2.19 --- Assay of murine interleukin-2 --- p.55 / Chapter (III) --- ASSAYS OF IMMUNOMODULATORY ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA ON MACROPHAGES / Chapter 3.2.20 --- Assay of murine interleukin-1 --- p.55 / Chapter 3.2.21 --- In vivo migration of macrophages --- p.56 / Chapter 3.2.22. --- Assay of phagocytic activity of peritoneal macrophages --- p.56 / Chapter 3.2.23 --- Northern blotting of mRNA of β-actin gene extracted from peritoneal exudate cells --- p.57 / Chapter (IV) --- ASSAYS OF ANTI-TUMOR ACTIVITIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.24 --- Assay of anti-tumor activity in vitro --- p.62 / Chapter 3.2.25 --- Assay of anti-tumor activity in vivo --- p.63 / Chapter 3.2.26 --- Priming effect of different fractions for the induction of TNF-α in mice --- p.63 / Chapter 3 .2.27 --- In vitro stimulation of TNF-α release from resting peritoneal macrophages --- p.64 / Chapter 3.2.28 --- Effects of P. heterophylla polysaccharides on TNF-α and IFN-gamma production as well as EAT growth in vivo --- p.64 / Chapter 3.2.29 --- Macrophage-mediated cytostatic activity --- p.65 / Chapter 3 2.30 --- Assay of lymphokine-activated killer cell activity --- p.66 / Chapter 3 2.31 --- Assay of natural killer cell activity --- p.67 / Chapter 3.2.32 --- Assay of tumor-infiltrating lymphocytes --- p.68 / Chapter (V) --- ASSAYS FOR THE EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON THE PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / Chapter 3.2.33 --- Assay of proliferation of murine bone marrow cells --- p.69 / Chapter 3.2.34 --- Assay of differentiation of murine bone marrow cells --- p.70 / Chapter 3.2.35 --- Assay of differentiation of Ml cells --- p.71 / Chapter 3.2.36 --- Induction of GM-CSF from bone marrow cells and Ml cells --- p.71 / Chapter (VI) --- ASSAYS OF THE IMMUNORESTORATIVE PROPERTIES OF PSEUDOSTELLARIA HETEROPHYLLA / Chapter 3.2.37 --- Immunorestoration in tumor-bearing mice --- p.72 / Chapter 3.2.38 --- Immunorestoration in aged mice --- p.72 / Chapter 3.2.39 --- Immunorestoration in cyclophosphamide- treated mice --- p.73 / Chapter 3.2.40 --- Statistical analysis --- p.73 / Chapter 4. --- "EXTRACTION, FRACTIONATION AND CHARACTERIZATION OF MITOGENIC FRACTIONS FROM PSEUDOSTELLARIA HETEROPHYLLA" / INTRODUCTION --- p.74 / RESULTS --- p.76 / Chapter 4.1 --- Extraction and fractionation of Pseudostellaria heterophylla --- p.76 / Chapter 4.2 --- Gel filtration and anion-exchange chromatography --- p.76 / Chapter 4.3 --- Characterization of bioactive fractions from Pseudostellaria heterophylla --- p.79 / Chapter 4.4 --- Mitogenic activity of fraction PH-I on murine lymphocytes in vitro --- p.96 / Chapter 4.5 --- Mitogenic effect of PH-I on murine lymphocytes in vivo --- p.102 / Chapter 4.6 --- Effect of PH-I on polyclonal B cell activation --- p.102 / Chapter 4.7 --- Adjuvant effect of PH-I on antibody response to SRBC in vivo --- p.106 / Chapter 4.8 --- Evidences to support the mitogenic activity of PH-I is due to its polysaccharide rather than due to the contamination by LPS --- p.106 / Chapter 4.9 --- The effects of PH-I on IL-2 production and IL-2 receptor expression on murine lymphocytes in vitro --- p.110 / Chapter 4.10 --- The mitogenic activity of the purified fractions on murine lymphocytes in vitro --- p.110 / Chapter 4.11 --- Adjuvant effect of PH-I Ab on antibody response to SRBC in vivo --- p.116 / Chapter 4.12 --- Mitogenic effect of PH-I C on murine lymphocytes in vivo --- p.116 / Chapter 4.13 --- Evidences to support the mitogenic activity of PH-I Ab is due to its polysaccharide rather than due to the contamination by LPS --- p.122 / DISCUSSION --- p.122 / Chapter 5. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF ALCOHOL- INSOLUBLE FRACTION (PH-I) FROM THE HOT WATER EXTRACT OF PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.133 / RESULTS --- p.135 / Chapter 5.1 --- Effect of PH-I on cytokine production --- p.135 / Chapter 5.2 --- In vivo activation of macrophages by PH-I --- p.135 / Chapter 5.3 --- Effect of PH-I on the activation of β-actin gene transcription in peritoneal macrophages --- p.142 / Chapter 5.4 --- Effect of PH-I on the in vitro growth of various tumor cell lines --- p.142 / Chapter 5.5 --- Immunorestoration of PH-I on the mitogenic response in EAT-bearing mice --- p.147 / DISCUSSION --- p.147 / Chapter 6. --- IMMUNOMODULATING AND ANTI-TUMOR ACTIVITIES OF PURIFIED FRACTIONS SEPARATED FROM PSEUDOSTELLARIA HETEROPHYLLA / INTRODUCTION --- p.154 / RESULTS --- p.157 / Chapter 6.1 --- In vitro anti-tumor activities of P. heterophylla --- p.157 / Chapter 6.2 --- In vivo anti-tumor activities of P. heterophylla --- p.165 / Chapter 6.3 --- Effect of P. heterophylla fractions on induction of delayed-type hypersensitivity --- p.165 / Chapter 6.4 --- Effect of PH-I fraction on the cytotoxic alloreactive T lymphocytes in vitro --- p.165 / Chapter 6.5 --- Effect of P. heterophylla on the production of TNF-α and IFN-gamma --- p.170 / Chapter 6.6 --- Effect of P. heterophylla on the activation of macrophages --- p.176 / Chapter 6.7 --- "Effect of P. heterophylla on the activation of NK, LAK and TIL" --- p.181 / Chapter 6.8 --- The effect of combined treatment of EAT-bearing mice with P. heterophylla amd Mur-TNF-α on the growth of EAT cells in vivo --- p.181 / Chapter 6 9 --- Immunorestorative activities of P. heterophylla in aged mice and cyclophosphamide-treated mice --- p.187 / DISCUSSION --- p.187 / Chapter 7. --- EFFECTS OF PSEUDOSTELLARIA HETEROPHYLLA ON PROLIFERATION AND DIFFERENTIATION OF MURINE BONE MARROW CELLS AND MYELOID LEUKAEMIC Ml CELLS / INTRODUCTION --- p.200 / RESULTS --- p.202 / Chapter 7.1 --- Effect of P. het erophyl1a on the proliferation and differentiation of murine bone marrow cells --- p.202 / Chapter 7 .2 --- Effects of P. heterophyl la on the proliferation and differentiation of murine myeloid leukaemia Ml cells --- p.205 / Chapter 7 .3 --- Effects of P. heterophylla on GM-CSF production by bone marow cells and myeloid leukaemia Ml cells --- p.214 / DISCUSSION --- p.218 / Chapter 8. --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.223 / BIBLIOGRAPHY --- p.233

Trois bactéries du genre Yersinia sont pathogènes pour l’homme : Yersinia pestis (bacille de la peste), et les bactéries entéropathogènes Yersinia pseudotuberculosis et Yersinia enterocolitica. Yersinia pestis est responsable de plus de 20 000 cas humains de peste déclarés à l’Organisation Mondiale de la Santé (OMS) ces dix dernières années dans différents foyers en Afrique, Asie et Amériques. Considérée aujourd’hui à tort comme une maladie du passé, elle est au contraire classée parmi les maladies réémergentes Même si elle ne se présente plus sous la forme d’épidémies massives, elle pose encore au monde actuel d’importants défis de par son extrême gravité, sa rapidité de dissémination, une apparition de résistances aux antibiotiques et une éventuelle utilisation terroriste du bacille. Dans ce contexte, l’immunothérapie contre Y. pestis pourrait être une bonne alternative pour traiter la peste bubonique et pulmonaire. Un des objectifs de cette thèse était de produire des anticorps monoclonaux murins contre trois protéines de l’injectisome (YscF, YscC et LcrV), un facteur de virulence clé des Yersinia. Les anticorps obtenus ont été caractérisés et pour certains leurs épitopes identifiés. Par la suite, en collaboration avec Elisabeth Carniel à l’Institut Pasteur, leur pouvoir neutralisant a été évalué in vivo dans un modèle murin de peste bubonique. Ces mêmes anticorps monoclonaux, produits contre les protéines de l’injectisome sont en cours d’évaluation pour la mise au point d’un test de diagnostic rapide de Y. pestis dans différents fluides et échantillons biologiques. Yersinia pseudotuberculosis et Yersinia enterocolitica sont présentes dans le monde entier et sont transmises par contamination à partir de viande de porc mal cuite, de lait ou produits laitiers et de végétaux, ou par contact avec des animaux porteurs sauvages ou domestiques. Une transmission interhumaine par voie fécale-orale est également possible. Ces bactéries sont responsables très fréquemment d’infections entériques. Cependant leur recherche dans les coprocultures n’est pas réalisée de façon systématique en laboratoires d’analyses médicales du fait de leur croissance lente et difficile sur les milieux usuels, ce qui rend leur isolement à partir de fèces difficile. De plus, les procédures de routine sont coûteuses et longues. Cela entraine probablement une sous-estimation de l’incidence des infections à Yersinia entéropathogènes, la prescription de traitements non adaptés et la réalisation d’appendicectomies non nécessaires, d’où la nécessité de développer des tests de diagnostic rapides, spécifiques, sensibles et faciles à utiliser. Un des objectifs de cette thèse était de produire un panel d‘anticorps monoclonaux murins contre les principaux biotypes et sérotypes pathogènes de Y. pseudotuberculosis et Y. enterocolitica pour le développement de tests de diagnostic immunologiques (ELISA et tests bandelettes) répondant aux caractéristiques recherchées et utilisables directement avec des échantillons biologiques humains. / Three bacteria of the genus Yersinia are pathogenic for the human: Yersinia pestis (the plague bacillus) and the enteropathogenic bacteria: Yersinia pseudotuberculosis and Yersinia enterocolitica. Yersinia pestis is responsible for more than 20,000 human cases of plague declared to the World Health Organization (WHO) during the ten last years in different areas from Africa, Asia and America. Mistakenly considered today as a disease from the past, on the contrary, the plague is re-emerging. Even if it doesn’t occur as a massive epidemic, it still lays down a challenge to the world for its important severity, its quick spreading, the appearance of antimicrobial resistance and a potential use for terrorism. Under the circumstances, the immunotherapy against Y. pestis could be a good option to treat bubonic and pneumonic plague. One the aims of this thesis was to produce murine monoclonal antibodies against the three proteins of the injectisom (YscF, YscC, LcrV), a key virulence factor of Yersinia. The obtained antibodies were characterized and for certain, the epitopes were identified. Then, in collaboration with Elisabeth Carniel from Institut Pasteur, their therapeutic effect was evaluated in vivo with a bubonic plague model in mice. The antibodies generated against the proteins from the injectisom are now evaluated in a diagnosis test for a fast detection of Y. pestis in different biological samples. Yersinia enterocolitica and Yersinia pseudotuberculosis, the two enteropathogenic Yersinia species for humans, have a worldwide distribution and are among the most frequent agents of human diarrhea in temperate and cold countries. However, research of enteropathogenic Yersinia is not consistently performed in medical laboratories because of their specific growth characteristics, which makes their isolation from the stool samples difficult. Moreover, current procedures for isolation are expensive and time consuming, which leads to underestimation of the incidence of yersiniosis and prescriptions of inappropriate antibiotic treatments. One the aims of this thesis was to produce different murine monoclonal antibodies against the main pathogenic biotypes and serotypes of Y. pseudotuberculosis and Y. enterocolitica for the development of fast, sensitive, specific and easy-to-use immunoassays (ELISA and dipsticks), useful for both human and veterinary diagnosis.

Cette thèse a pour objectif d’identifier et caractériser des biomarqueurs liés aux traitements d’immunothérapie allergénique (ITA). En particulier, le travail s’est porté sur les biomarqueurs prédictifs de l’efficacité du traitement : leur estimation avant la prise médicamenteuse permettrait d’évaluer les bénéfices cliniques à l’issue de l’ITA. À partir d’échantillons de sérum collectés avant ITA et provenant de patients inclus dans une étude clinique contrôlée, randomisée et dirigée contre les pollens de graminées, une méthode d’analyse protéomique différentielle (la 2D-DIGE) a permis d’identifier une protéine comme candidat biomarqueur prédictif de l’efficacité de l’ITA. Dans un premier temps, les variants moléculaires de la protéine identifiée ont été caractérisés. L’analyse en spectrométrie de masse de la protéine a permis d’identifier les modifications post-Traductionnelles associées aux isoformes plus abondants dans le sérum des patients pour lesquels une réponse positive à l’ITA est observée. Une approche protéomique de quantification relative a été mise en œuvre par LC-MS/MS sans marquage, et a permis d’identifier des peptides de la protéine d’intérêt, portant des modifications post-Traductionnelles spécifiques, associés à un bénéfice clinique accru en fin d’ITA. Dans un deuxième temps, l’implication de la fétuine-A dans la physiopathologie de l’allergie a été étudiée. Des modèles cellulaires humains ont mis en évidence le caractère essentiel des acides sialiques portés par la protéine pour l’activation de la voie TLR4, dans les mécanismes de l’immunité innée initiant la réaction allergique. In vivo, les modèles murins d’asthme allergique développés ont en revanche montré la fonction anti-Inflammatoire de la protéine. La fétuine-A a donc un rôle ambivalent, mais est indubitablement liée à la régulation de l’inflammation allergique. La validation des candidats biomarqueurs peptidiques de la protéine dans d’autres cohortes cliniques représente une future étape clé, qui permettrait d’envisager l’usage de ces marqueurs en clinique, pour améliorer la sélection des patients les plus susceptibles de répondre à l’ITA. / This thesis aimed at identifying and characterizing predictive biomarkers associated with allergen-Specific immunotherapy (AIT) efficacy. In particular, we were focused on biomarkers predictive of AIT efficacy: quantifying such markers before treatment would allow estimating the final clinical benefit. For this purpose, serum samples were collected before AIT from patients included in a double-Blind, placebo controlled clinical study against grass pollen allergy. Their analysis by means of differential proteomics (2D-DIGE) pointed out a protein, named fetuin-A, as a candidate biomarker predictive of AIT efficacy. First, the protein fetuin-A isoforms were extensively characterized by mass spectrometry, and specific post-Translational modifications (PTMs) were associated to the isoforms more abundant in sera from patients positively responding to AIT. A second proteomic approach allowed identifying fetuin-A peptides, differentially expressed among groups of patients. Some peptides from the candidate protein, bearing specific post-Translational modifications (PTMs), are associated with an increased clinical benefit at the end of the treatment. In a second part, we studied the involvement of the fetuin-A during the course of allergic inflammation. Human cellular assays highlighted that sialic acids on the protein PTMs are essential to activate the TLR4 pathway, and inducing innate immune mechanisms linked to allergy. In vivo, murine models of allergic asthma showed an opposite anti-Inflammatory function of the protein. Thus, the protein fetuin-A is still ambivalent, but is undoubtedly linked to the regulation of allergic inflammation. Validation of peptides candidate biomarkers in larger clinical cohorts is of utmost interest, since using such biomarkers in clinics would improve patients’ selection and therefore clinical benefit from the treatment.

Les cellules Natural Killer (NK) sont de grands lymphocytes granuleux capables de rapidement éliminer des cellules tumorales et des cellules infectées par des virus sans immunisation au préalable. Au cours de ma thèse, j’ai analysé plusieurs paramètres impliqués dans la reconnaissance et la lyse des cellules de mélanome par les NK. J’ai montré à partir d’analyses ex vivo que les NK sanguines de patients atteints de mélanome métastatique (stade III-IV) présentent un faible potentiel lytique. Cependant, de telles NK provenant de patients mélanomes de tout stade clinique activées in vitro par de l’IL-2 lysent efficacement des lignées de mélanome métastatique. L’analyse du phénotype de NK circulantes de patients stade IV a montré une diminution de l’expression du récepteur activateur NKp46/NCR1 comparé aux NK de donneurs sains. J’ai également montré une corrélation positive entre l’expression du NKp46 à la surface des NK et la durée du stade IV. Pour caractériser les NK infiltrant le mélanome, J’ai analysé ex vivo les NK infiltrant des ganglions métastatiques (GG) provenant de 25 patients en stade III. Les GG de patients mélanomes contiennent une population unique de NK CD56brightCD16+ représentant 50% des NK dans ces GG qui expriment fortement les récepteurs NK NCR, NKG2D, KIRs et produisent une plus forte proportion de perforin comparée aux NK CD56brightCD16- ganglionnaires. Les NK immunsélectionnées à partir de GG et activées avec de l’IL-2 ou de l’IL-15 lysent rapidement et efficacement des lignées cellulaires de mélanome. Elles sont caractérisées par des capacités lytiques supérieures aux NK sanguines. De plus, afin d’évaluer l’impact des NK au cours du mélanome, j’ai analysé in situ les NK infiltrant des ganglions sentinelles positive et négatif ainsi que des tumeurs cutanées primaires. Les NK sont faibles dans les GS ; cependant nous avons montré que le nombre de NK infiltrant ces ganglions sentinelles est associé à une plus forte rechute à cinq ans des patients. Les cellules NK infiltrant les tumeurs cutanées sont présentes préférentiellement dans la zone peritumorale et sont très rares dans la tumeur.Chez les patients atteints de mélanome, les NK sanguines et infiltrant les tumeurs ont des caractéristiques phénotypiques et fonctionnelles différentes. Une meilleure compréhension de telles différences doit être prise en compte, ainsi la biologie des NK et de leur modulation au cours du cancer est nécessaire pour développer une stratégie thérapeutique à base de cellules NK efficace. / Cytotoxic immune effectors can control the development and growth of certain solid tumours. Among these cytotoxic effectors, NK cells are capable of rapidly eliminating tumour cells and virus-infected cells without prior immunization.The objectives of my thesis were to evaluate the potential role of NK cells in the immune response against melanoma. First, I have characterized the functional status of blood NK cells from melanoma patients at different stages of the disease. I showed that ex vivo NK cells from most advanced stage III-IV patients display low lytic potential. However, IL-2-activated NK cells from patients efficiently lyse melanoma cells and that independently to the clinical stage. Moreover, the expression of the activating receptor NKp46/NCR1 by blood NK cells was decreased in stage IV patients compared to healthy donors, and a positive correlation between NKp46 expression by NK cells and the duration of stage IV was found. I have also characterized ex vivo NK cells infiltrating metastatic lymph nodes (M-LN) from stage III melanoma patients. I have identified in M-LN a unique subpopulation of mature CD56brightCD16+ NK cells that expressed higher NCR, NKG2D, KIRs, and perforin levels than CD56brightCD16- NK cells counterpart. NK cells from M-LN activated with IL-2 or IL-15, rapidly lysed metastatic melanoma cell lines with higher efficiency than autologous blood NK cells. Finally, to determine if NK cells display a prognostic value, I analysed by immunohistochemistry NK cells and other immune cells infiltrating positive and negative sentinel lymph nodes (SLN). SLN are characterized by high densities of macrophages and endothelial cells, even higher in SLN+. Few NK cells and Granzyme B+ cells infiltrate SLNs while CD8+ T cells are numerous. Moreover, numbers of NK cells in SLN correlated with higher rate of 5 year-relapse of patients. Compared to SLN, primary cutaneous melanomas contain high numbers of NK cells that are preferentially localized in the periphery of the tumour and are not related to the Breslow. My findings showed that in melanoma patients, circulating and tumour infiltrating NK cells display unique phenotypic and functional characteristics, indicating that tumour may alter their function. However, they respond to cytokine activation and acquire antitumor lytic potential. In the new landscape of melanoma treatment, NK cells are worthy to be considered for combined treatment with BRAF inhibitors.