Leung Yiu Kwong, Michael. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 155-161). / Acknowledgements --- p.i / Abbreviations --- p.ii / Aim and scope of this dissertation --- p.v / Abstract --- p.vi / Table of contents --- p.viii / Introduction --- p.1 / Chapter 1.1 --- Introduction --- p.2 / Chapter 1.2 --- Tumor Biology --- p.3 / Chapter 1.3 --- The Defence Mechanisms --- p.4 / Chapter 1.3.1 --- Non-specific defence mechanisms --- p.5 / Chapter 1.3.2 --- Specific defence mechanisms --- p.6 / Chapter 1.4 --- Effector Mechanisms in Anti-tumor Immunity --- p.7 / Chapter 1.4.1 --- B-cell --- p.8 / Chapter 1.4.2 --- "Natural killer (NK) cells (Non-T, Non-B)" --- p.8 / Chapter 1.4.3 --- Macrophages --- p.9 / Chapter 1.4.4 --- Cytolytic T-lymphocytes (CTLs) --- p.10 / Chapter 1.5 --- Cancer Treatment --- p.10 / Chapter 1.5.1 --- Surgery --- p.10 / Chapter 1.5.2 --- Radiotherapy --- p.12 / Chapter 1.5.3 --- Drug therapy --- p.12 / Chapter 1.5.4 --- Gene therapy --- p.13 / Chapter 1.5.5 --- lmmunotherapy --- p.13 / Chapter 1.6 --- Non-cytotoxic Antitumor Polysaccharides of Fungi --- p.14 / Chapter 1.6.1 --- Yeast polysaccharides --- p.14 / Chapter 1.6.2 --- Lichen polysaccharides --- p.15 / Chapter 1.6.3 --- Fungal polysaccharides --- p.18 / Chapter 1.7 --- Fungi and their Polysaccharides --- p.20 / Chapter 1.7.1 --- Reserve carbohydrates --- p.20 / Chapter 1.7.2 --- Structural polysaccharides --- p.21 / Chapter 1.8 --- The Architecture of the Fungal Cell Wall --- p.22 / Materials and Methods --- p.26 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- Animals --- p.27 / Chapter 2.1.2 --- Mushrooms --- p.27 / Chapter 2.1.3 --- "Buffers, culture media and chemicals" --- p.27 / Chapter 2.1.4 --- Cell lines --- p.34 / Chapter 2.2 --- Methods --- p.35 / Chapter 2.2.1 --- Screening of β-(l→3)-D-glucan --- p.35 / Chapter 2.2.2 --- Extraction and Fractionation of Flammulina velutipes --- p.35 / Chapter 2.2.3 --- Characterisation of Flammulina velutipes --- p.38 / Chapter 2.2.3.1 --- The determination of carbohydrate content of F.V fractions --- p.38 / Chapter 2.2.3.2 --- The determination of protein content of F.V. fractions --- p.39 / Chapter 2.2.3.3 --- The determination of uronic acid content of F.V fractions --- p.39 / Chapter 2.2.3.4 --- The determination of component sugar units of F.V fractions --- p.39 / Chapter 2.2.3.5 --- Periodate uptake of F.V. fractions --- p.40 / Chapter 2.2.3.6 --- Limulus amebocyte lysate (LAL) coagulation assay --- p.40 / Chapter 2.2.3.7 --- The digestion of F.V. fractions with laminarinase --- p.41 / Chapter 2.2.3.8 --- The Secondary and tertiary structure determination of FH and SFA1 --- p.42 / Chapter 2.2.3.9 --- Molecular weight estimation of FH and SFA1 --- p.43 / Chapter 2.2.3.10 --- Vascular dilation and hemorrhage (VDH) activity of F.V. fractions / Chapter 2.2.4 --- Isolation and preparation of cells --- p.43 / Chapter 2.2.4.1 --- Bone marrow cell --- p.43 / Chapter 2.2.4.2 --- Peritoneal exudate cell (PEC) --- p.44 / Chapter 2.2.4.3 --- Splenocytes --- p.44 / Chapter 2.2.4.4 --- Depleting mouse T-cells by anti-mouse T-cell antigen antibody plus complement treatment --- p.45 / Chapter 2.2.4.5 --- Depleting mouse B-cells by Cedarlane column kit --- p.45 / Chapter 2.2.5 --- Assays for the cytotoxicity of Flammulina velutipes --- p.45 / Chapter 2.2.5.1 --- Brine shrimp assay --- p.45 / Chapter 2.2.5.2 --- In vitro cytotoxicity of FH and SFA1 on bone marrow cells of female BALB/c mice --- p.46 / Chapter 2.2.5.3 --- In vivo cytotoxicity of FH and SFA1 on female BALB/c mice --- p.47 / Chapter 2.2.6 --- "Assays for the immunomodulatory activities of Flamm""lina velutipes" --- p.47 / Chapter 2.2.6.1 --- In vitro mitogenic activities of FH and SFA1 on murine lymphocytes --- p.47 / Chapter 2.2.6.2 --- In vitro mitogenic activities of FH and SFA1 with PMB on murine lymphocytes --- p.48 / Chapter 2.2.6.3 --- In vitro mitogenic activities of FH and on T-cell depleted murine lymphocytes --- p.48 / Chapter 2.2.6.4 --- In vitro mitogenic activities of FH on B-cell depleted murine lymphocytes --- p.49 / Chapter 2.2.6.5 --- In vitro co-mitogenic activitiy of FH and SFA1 on murine lymphocytes --- p.49 / Chapter 2.2.6.6 --- In vitro mitogenic activities of FH and SFA1 on murine bone marrow cells --- p.50 / Chapter 2.2.6.7 --- In vivo mitogenic activities of FH and SFA1 on murine lymphocytes --- p.50 / Chapter 2.2.6.8 --- Effect of FH and SFA1 on the enhancement of first antibody production of SRBC immunised mice --- p.51 / Chapter 2.2.6.9 --- Effect of FM and SFA1 on the in vitro phagocytic activity of murine macrophage --- p.51 / Chapter 2.2.6.10 --- Effect of FM and SFA1 on the in vivo phagocytic activity of murine macrophage --- p.51 / Chapter 2.2.6.11 --- In vivo migration of macrophage in FH- and SFAl-treated mice --- p.53 / Chapter 2.2.6.12 --- Effect of FH and SFA1 on the enhancement of murine PEC cytostatic activity --- p.53 / Chapter 2.2.6.13 --- Effect of FH and SFA1 on the Fc receptor expression of peritoneal exudate cells --- p.54 / Chapter 2.2.6.14 --- Effect of FH and SFA1 on murine serum cytokine level --- p.55 / Chapter 2.2.6.15 --- Effect of FH and SFA1 on murine serum TNF level --- p.55 / Chapter 2.2.6.16 --- Effect of FH and SFA1 on the augmentation of SRBC lysing ability of murine serum --- p.56 / Chapter 2.2.7 --- Assays for the anti-tumor activities of Flammulina velutipes --- p.57 / Chapter 2.2.7.1 --- In vitro anti-tumor activity of FH and SFA1 --- p.57 / Chapter 2.2.7.2 --- Effect of FH and SFA1 on the growth of murine transplantable tumor invivo --- p.58 / Chapter 2.2.8 --- Statistical analysis --- p.59 / "Screening, Purification, Fractionation and Characterisation of β-(l→3)-D-glucan(s) from Flammulina velutipes" --- p.60 / Introduction --- p.61 / Results --- p.62 / Chapter 3.1 --- Screening of β-(l→3)-D-Glucan --- p.62 / Chapter 3.2 --- Extraction and Fractionation of Flammulina velutipes --- p.62 / Chapter 3.3 --- The Determination of Carbohydrate Content of F.V. Fractions --- p.65 / Chapter 3.4 --- The Determination of Protein Content of F.V. Fractions --- p.65 / Chapter 3.5 --- The Determination of Uronic Acid Content of F.V. Fractions --- p.69 / Chapter 3.6 --- The Determination of Component Sugar Units of F.V. Fractions --- p.69 / Chapter 3.7 --- Periodate Uptake of F.V. Fractions --- p.69 / Chapter 3.8 --- Limulus Amebocyte Lysate (LAL) Coagulation Assay --- p.73 / Chapter 3.9 --- The Digestion of F.V. Fractions with Laminarinase --- p.73 / Chapter 3.10 --- The Secondary and tertiary Structure Determination of FH and SFA1 --- p.80 / Chapter 3.11 --- Molecular Weight Estimation of FH and SFA1 --- p.82 / Chapter 3.12 --- "Vascular Dilation and Hemorrhage (VDH) Activity of FH, SFA1 and lFA1" --- p.82 / Discussion --- p.90 / The Toxicity of Flammulina velutipes --- p.96 / Introduction --- p.97 / Results --- p.97 / Chapter 4.1 --- Lack of Cytotoxicity of Flammulina velutipes to Brine Shrimp --- p.97 / Chapter 4.2 --- Lack of Cytotoxicity of Flammulina velutipes to Murine Bone Marrow Cells --- p.99 / Chapter 4.3 --- Lack of Cytotoxicity of Flammulina velutipes to Mouse --- p.99 / Discussion --- p.102 / The Immunomodulatory Activities of Flammulina velutipes --- p.103 / Introduction --- p.104 / Results --- p.105 / Chapter 5.1 --- Effect of Flammulina velutipes on Murine Lymphocytes --- p.105 / Chapter 5.2 --- Effect of Flammulina velutipes on Murine Macrophage --- p.115 / Chapter 5.3 --- Effect of Flammulina velutipes on Murine Serum Cytokine and Complement Level --- p.125 / Discussion --- p.133 / The Anti-tumor Activities of Flammulina velutipes --- p.136 / Introduction --- p.137 / Results --- p.137 / Chapter 6.1 --- In Vitro Anti-Tumor Activity of FH and SFA1 --- p.137 / Chapter 6.2 --- Effect of FH and SFAI on the Growth of Murine TransplantableTumors --- p.138 / Discussion --- p.145 / General Discussion --- p.146 / General Discussion and Future Perspectives --- p.147 / References --- p.154

by Tang Yan Chi. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1999. / Includes bibliographical references (leaves 156-168). / Abstracts in English and Chinese. / ACKNOWLEDGEMENTS --- p.I / ABBREVIATIONS --- p.II / ABSTRACT --- p.V / ABSTRACT (CHINESE) --- p.VII / TABLE OF CONTENT --- p.1 / GENERAL INTRODUCTION --- p.4 / Chapter 1.1. --- Immunotherapy in Cancer --- p.4 / Chapter 1.1.2. --- Cytokines used in Immunotherapy in Cancer --- p.5 / Chapter 1.1.2.1. --- Interferons (IFNs) --- p.6 / Chapter 1.1.2.2. --- interleukins (ILs) --- p.11 / Chapter 1.1.2.3. --- Tumor Necrosis Factor-Alpha (TNF-α) --- p.14 / Chapter 1.2. --- Immunomodulatory and Anti-tumor Activities of Plant Polysaccharides --- p.17 / Chapter 1.3. --- Previous Studies on the Capsular Polysaccharides (CPS) from Klebsiella pneumoniae --- p.21 / Chapter 1.4. --- Activation of TNF-α Production from Macrophages by Bacterial Polysaccharides --- p.26 / Chapter 1.5. --- Chemotherapy of Cancer --- p.29 / Chapter 1.5.1. --- Types of Chemotherapeutic Drugs --- p.29 / Chapter 1.5.1.1. --- Alkylating Agents --- p.29 / Chapter 1.5.1.2. --- Plant Alkaloids --- p.31 / Chapter 1.5.1.3. --- Anti-metabolites --- p.32 / Chapter 1. 5.1.4. --- Antitumor Antibiotics --- p.32 / Chapter 1.5.2. --- Chemotherapeutic Drugs in this Project --- p.34 / Chapter 1.5.2.1. --- actinomycin D (ACT D) --- p.34 / Chapter 1.5.2.2. --- Cyclophosphamide (CY) --- p.35 / Chapter 1.5.2.3. --- Doxorubicin (Dox) --- p.37 / Chapter CHAPTER TWO --- AIM AND SCOPE OF THIS DISSERTATION --- p.40 / Chapter CHAPTER THREE --- MATERIALS AND METHODS --- p.42 / Chapter 3.1 --- MATERIALS --- p.42 / Chapter 3.1.1. --- Animals --- p.42 / Chapter 3.1.2. --- Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.42 / Chapter 3.1.3. --- Cell lines --- p.43 / Chapter 3.1.4. --- "Buffers, culture media and chemicals" --- p.43 / "POLYCLONAL RABBIT ANTI-ACTIVE ERK1/2, ANTI-ACTIVE JNK 1/2 AND ANTI-ACTIVE P38WERE FROM PROMEGA. POLYCLONAL RABBIT ANTI-TOTAL JNK1, ANTI-TOTAL P38WERE FROM SANTA CRUZ. MONOCLONAL ANTI-MOUSE TOTAL ERK1 AND POLYCLONAL ANTI-RABBIT TOTAL ERK2 WERE PURCHASED FROM ZYMED" --- p.50 / Chapter 3.2. --- METHODS --- p.51 / Chapter 3.2.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides (CPS) from Klebsiella pneumoniae" --- p.51 / Chapter 3.2.1.1. --- Extraction and Purification of K1 and K24 Klebsiella Capsular Polysaccharides (CPS) --- p.51 / Chapter 3.2.1.2. --- Gel filtration of K1 and K24 Klebsiella Capsular Polysaccharides --- p.52 / Chapter 3.2.1.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.52 / Chapter 3.2.3. --- Assay for Macrophage Activating Activities of Klebsiella Capsular Polysaccharides --- p.55 / Chapter 3.2.3.1. --- Tumour Necrosis Factor-α (TNF-α) Production by Peritoneal Macrophages --- p.55 / Chapter 3.2.3.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.56 / Chapter 3.2.4. --- Assay for Anti-Tumor Activities of Klebsiella Capsular Polysaccharides --- p.63 / Chapter 3.2.4.1. --- Assay of in vivo Suppression of EAT growth by K1 and K24 Capsular Polysaccharides --- p.63 / Chapter 3.2.4.2. --- Assay of the Effect of CPS on the Survival of EAT-Bearing Mice --- p.64 / Chapter 3.2.5. --- "Assay for Anti-Tumor Activities of Combined Treatment of Klebsiella Capsular Polysaccharides and Chemotherapeutic Drugs (Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox)" --- p.65 / Chapter 3.2.5.1. --- "Assay of the Cytotoxic Effect of Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on wehi-164 cell line" --- p.65 / Chapter 3.2.5.2. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on WEHI-164 cell line in vitro" --- p.66 / Chapter 3.2.5.3. --- Assay for Anti-Tumor Effect of Chemotherapeutic Drugs (Act D，CY and Dox) on EAT-bearing Mice in vivo --- p.67 / Chapter 3.2.5.4. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.68 / Chapter 3.2.5.5. --- "Assay of the Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the survival of eat-bearing mice in vivo" --- p.72 / Chapter CHAPTER FOUR --- "EXTRACTION, PURIFICATION & CHARACTERIZATION OF KLEBSIELLA K1 & K24 CAPSULAR POLYSACCHARIDES" --- p.76 / Chapter 4.1. --- Extraction and Purification of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24 --- p.76 / Chapter 4.2. --- Gel Filtration Chromatography of K1 and K24 Capsular Polysaccharides --- p.77 / Chapter 4.3. --- Characterization of K1 and K24 Capsular Polysaccharides --- p.81 / Chapter CHAPTER FIVE --- EFFECT OF THE K1 & K24 CAPSULAR POLYSACCHARIDES ON THE MACROPHAGE ACTIVITIES --- p.83 / Chapter 5.1. --- Effect ofK1 and K24 Capsular Polysaccharides on the in vitro induction of TNF- α Production of Murine Peritoneal Macrophages --- p.83 / Chapter 5.2. --- Effect of Capsular Polysaccharides on the activities of MAPK family in murine macrophages --- p.87 / Chapter CHAPTER SIX --- ANTI-TUMOR ACTIVITIES ON MURINE TUMOR CELL LINES OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES --- p.94 / Chapter 6.1. --- Effect of K1 and K24 Capsular Polysaccharides on the In vivo Anti-tumor Activities on EAT-bearing Mice --- p.94 / Chapter 6.2. --- In vivo Anti-tumor Effect on the Survival of EAT-Bearing Mice by Treatment of K1 and K24 CPS --- p.97 / Chapter CHAPTER SEVEN --- "ANTI-TUMOR ACTIVITIES OF COMBINED TREATMENT OF KLEBSIELLA K1 &K24 CAPSULAR POLYSACCHARIDES AND CHEMOTHERAPEUTIC DRUGS: ACTINOMYCIN D, CYCLOPHOSPHAMIDE AND DOXORUBICIN" --- p.100 / Chapter 7.1. --- "Cytotoxic Effect Chemotherapeutic Drugs： Actinomycin D (Act D), Cyclophosphamide (CY) and Doxorubicin (Dox), on WEHI-164 cell line" --- p.100 / Chapter 7.2. --- "Cytotoxic Effect of the Combined Treatment of the CPS and the Chemotherapeutic Drugs (Act D, CY and Dox), on WEHI-164 cell line" --- p.104 / Chapter 7.3. --- "Anti-Tumor Effect of Chemotherapeutic Drugs (Act D, CY and Dox) on EAT-bearing Mice in vivo" --- p.111 / Chapter 7.4. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the EAT Growth in vivo" --- p.115 / Chapter 7.5. --- "Combined Treatment of the CPS and Chemotherapeutic Drugs (Act D, CY and Dox) on the Survival of EAT-bearing Mice in vivo" --- p.122 / Chapter CHAPTER EIGHT --- GENERAL DISCUSSION --- p.135 / Chapter 8.1. --- "Extraction, Purification and Characterization of K1 and K24 Capsular Polysaccharides from Klebsiella pneumoniae Serotype 1 and Serotype 24" --- p.135 / Chapter 8.2. --- Effect of K1 and K24 Capsular Polysaccharides on the Macrophage Activation --- p.140 / Chapter 8.3. --- Anti-tumor Activities on Murine Tumor Cell LInes of Klebsiella K1 and K24 Capsular Polysaccharides --- p.145 / Chapter 8.4. --- "Anti-tumor Activities of Combined Treatment of K1 and K24 Capsular Polysaccharides and Chemotherapeutic Drugs： Actinomycin D, Cyclophosphamide and Doxorubicin" --- p.148 / Chapter CHAPTER NINE --- CONCLUSIONS AND FUTURE PERSPECTIVES --- p.152 / BIBLIOGRAPHY --- p.156

Clostridium difficile is a major problem as the aetiological agent of antibiotic associated diarrhoea. The mechanism by which the bacterium colonises the gut is poorly understood, but undoubtedly involves a myriad of components present on the bacterial surface. The aims of this study were to further define roles for selected surface proteins using a knockout approach, to evaluate the feasibility of surface protein based immunotherapeutics and to obtain structural information using X-ray crystallography. Mutants of cell wall-binding domain (PFam04122) containing proteins CD1036, CD2735, CD2784, Cwp66, CD2791, Cwp84, CD2795 and the flagella cap (FliD) were created. Mutants were characterised with regard to growth, sporulation, toxin production, adhesion in vitro, and, for the Cwp84 mutant, using the in vivo hamster model. The surface-located cysteine protease, Cwp84, was found to play a key role in maturation of the C. difficile S-layer, yet the Cwp84 mutant still caused disease with a similar pathology to the wildtype. Culture supernatant levels of toxin A were increased in CD2735, Cwp66, CD2791, CD2795 and particularly in Cwp84 and FliD 24 hr cultures, while CD2735, Cwp66, CD2791, CD2795 mutants also showed reduced adherence to Caco-2 cells compared to the wild-type. Passively administered immunotherapy, generated to low pH surface protein extracts of the C. difficile R20291 strain, did not protect hamsters from challenge with the cognate strain. Structural studies were undertaken on the surface proteins CD2791, Cwp66 and CD2767. Crystallisation conditions were identified for a recombinant N-terminal domain of CD2767 and an X-ray data set collected to 2 Å, although the structure was not solved by molecular replacement. Together these results further our knowledge of C. difficile surface proteins, although further work is required to identify which surface proteins play key roles in vivo during infection.

L’allergie ou l’hypersensibilité de type I est une réponse inappropriée du système immunitaire à une substance étrangère à l’organisme, nommée « allergène ». L’immunothérapie allergénique (ITA) est actuellement le seul traitement sur le marché qui permet de traiter l’étiologie de la maladie allergique par opposition aux traitements symptomatiques qui diminuent temporairement les manifestations allergiques. Son action consiste à réduire la sensibilité de l’organisme vis-à-vis de l’allergène en modulant progressivement la réponse immunitaire dirigée contre ce dernier. L’objectif de cette thèse était de définir des biomarqueurs d’efficacité clinique utilisables dans le cadre des traitements de l’ITA. La stratégie de recherche est basée sur une hypothèse qui consiste à suggérer que les cellules dendritiques (DCs) sont impliquées dans le succès de l’immunothérapie. En particulier, nous supposons que le traitement induit une baisse des DCs de type 2 (DC2), qui induisent des lymphocytes T auxiliaires de type 2 (TH2), et une augmentation des DCs régulatrices (DCreg), qui induisent des lymphocytes T régulateurs. La première partie de cette thèse a consisté à mettre au point des conditions de culture induisant des DC2. Pour cela, un criblage de molécules biologiques et pharmacologiques a été entrepris sur les DCs dérivées des monocytes afin d’induire in vitro des DC2 et a conduit à la mise au point d’un mélange de plusieurs molécules, dont certaines sont impliquées dans les mécanismes de l’allergie. Le phénotype des DC2 obtenu a été étudié ainsi que la polarisation des lymphocytes T induite après co-cultures en comparaison avec des DCs de type 1 (DC1) et des DCreg.La deuxième partie de cette thèse a consisté à analyser, à l’échelle moléculaire, les différents types de DCs induites (DC1, DC2 et DCreg). Pour cela, deux techniques ont été utilisées, une analyse transcriptomique par puces à ADN et une analyse protéomique par spectrométrie de masse sans marquage, pour comparer le transcriptome et le protéome des DCs induites. Le différentiel d’expression des marqueurs les plus pertinents a été validé au niveau transcriptionnel et protéique.Dans la troisième partie de cette thèse, le suivi des marqueurs dans des cellules du sang de patients allergiques traités ou non par ITA lors d’une étude clinique randomisée, contrôlée, en double aveugle, a permis de définir six nouveaux candidats biomarqueurs d’efficacité de l’immunothérapie, dont trois spécifiques des DC2 et trois autres spécifiques des DCreg. Ces marqueurs pourront être suivis lors des traitements d’ITA pour distinguer les patients répondeurs des non-répondeurs. / Allergy or type I hypersensitivity is an inappropriate response of the immune system to a foreign substance in the body, called "allergen". Allergen immunotherapy (AIT) is currently the only treatment on the market that can handle the etiology of allergic disease versus symptomatic treatments that temporarily reduce allergic manifestations. Its action is to reduce the sensitivity of the body against allergens.The aim of this thesis was to define biomarkers of clinical efficacy of AIT. The research strategy is based on the following hypothesis: dendritic cells (DCs) are involved in the success of immunotherapy. In particular, we assume that the treatment induces a decrease in DCs type 2 (DC2), which induce type 2 helper T cells, and an increase of regulatory DCs (DCreg), which induce regulatory T cells.First, we defined optimal culture conditions inducing the polarization of in vitro immature monocyte-derived DCs (MoDCs) toward a DC2 pattern. After screening several biological and pharmaceutical agents, we selected a cocktail of six molecules with some of them are pro-allergenic molecules. The phenotype of those DC2 cells and the CD4+ T cell polarization induced after coculture were characterized extensively in comparison with type 1 DC (DC1) and DCreg.In a second part, we compared the transcriptomes and the proteomes of MoDCs polarized into DC1, DC2 and DCreg by using cDNA microarrays together with label-free mass spectrometry. The differential expression of the most relevant markers was confirmed at the transcriptional and protein level. In the third part, markers were also followed in the peripheral blood from allergic patients enrolled in a randomized, double-blind, placebo-controlled AIT study. The expression of three DC2 markers was down-regulated and of three DCreg markers was up-regulated in patients who responded to the treatment and correlated with clinical efficacy. These markers could be used as follow-up read-outs of AIT efficacy in order of to discriminate responders from nonresponders.

INTRODUCTION: Melanoma is the most deadly skin cancer with mortality dependent on the extent and location of metastases. Lymphatic metastasis occurs early in melanoma, and tumor-associated lymphatic vessel area correlates with melanoma progression. Recently, the discovery of checkpoint inhibitors has drastically changed the treatment strategy and survival rates in melanoma. Neuropilin-2 (NRP2) is a potential common target in melanoma cells, tumor-associated lymphatic endothelial cells (LECs) and tumor-infiltrating lymphocytes (TILs). NRP2 is a cell surface receptor with competing stimulatory ligands (VEGF-A/-C) and inhibitory ligands (SEMA3F/G). AIM: The goal of this study was to investigate the role of NRP2 in both melanoma cells and the melanoma microenvironment (LECs, TILs) and to examine the effect of semaphorin 3F (SEMA3F) on the tumor cells as well as an immune modulator. RESULTS: Mouse and human melanocytes expressed NRP2 but not other vascular endothelial growth factor (VEGF) receptor tyrosine kinases in vitro and in vivo. NRP2 protein expression, as analyzed by immunohistochemistry, was upregulated in human metastatic melanoma sections. Treatment of melanoma cells in vitro with SEMA3F inhibited migration and phosphorylation of protein kinase B (AKT) but did not inhibit cell viability. SEMA3F also increased programmed cell death-ligand 1 (PD-L1) expression in melanoma. Syngeneic B16F10 melanoma did not grow in global NRP2 knockout (KO) mice but did grow in wild-type mice. In addition, mice inoculated with B16F10 were treated with SEMA3F or phosphate-buffered saline (PBS) by mini-osmotic pumps. Resulting tumors were analyzed histologically for microvessel density and presence of TILs (number and subtype). CONCLUSIONS: Expression of NRP2 protein positively correlated with melanoma progression in human patient samples. NRP2 functions differently in melanoma tumor cells than in host stromal cells (endothelial cells [ECs], LECs). In melanoma, NRP2 is not a VEGF receptor but responds to the ligand, SEMA3F. Alternately, NRP2 appears to be an important VEGF-A/-C co-receptor in tumor-associated angiogenesis and lymphangiogenesis, as demonstrated in the NRP2 transgenic mice studies. SEMA3F inhibited tumor cell migration but increased PD-L1 expression. Systemic treatment with purified SEMA3F protein in melanoma preclinical trials inhibited melanoma growth and microvessel density. Taken together, these results suggest that exploiting the NRP2/SEMA3F signaling axis may be a novel treatment strategy to be used in combination with existing immunotherapy in melanoma. / 2020-07-03T00:00:00Z

Emerging evidence suggests the immune system has a role in preventing cancer, and in advanced cancer evidence of immune dysfunction is widespread. This project focused on cytotoxic T lymphocyte antigen 4 (CTLA4), a key negative regulator of T cell activation found on dedicated regulatory T cells (Treg) and activated T lymphocytes, and asked whether modulation of immune control with anti-CTLA4 blockade led to significant anti-tumour activity. Clinical and laboratory investigation of anti-CTLA4 blockade using tremelimumab in a phase II trial of second-line therapy in advanced oesophageal and gastric adenocarcinomas was combined with an attempt to establish a suitable pre-clinical model based on therapeutic vaccination against the tumour associated antigen (TAA) 5T4.Eighteen patients received tremelimumab. Most drug-related toxicity was mild but there was a single death due to bowel perforation. Four patients had stable disease with clinical benefit; one achieved a partial response after eight cycles (25.4 months) and remains well on study after four years. Markers of regulatory phenotype, forkhead box protein 3 (FoxP3) and CTLA4, doubled transiently in CD4+CD25high lymphocytes in the first month after tremelimumab before returning to baseline. In contrast, CTLA4 increased in CD4+CD25low/negative lymphocytes throughout the cycle of treatment. Post-treatment expanded Treg expressed FoxP3 without interleukin-2 and their defining suppressive function was not abolished despite prolonged anti-CTLA4 blockade. De novo proliferative responses to TAA 5T4 (8 of 18 patients) and carcinoembryonic antigen (CEA; 5 of 15) were detected. Patients with a post-treatment CEA proliferative response had median survival of 17.1 months compared to 4.6 months for non-responders (p=0.002). Baseline interleukin-2 release after T lymphocyte activation was higher in patients with clinical benefit and toxicity. Heterologous mouse 5T4 (m5T4) vaccination showed some evidence of weak therapeutic benefit, but all tumour models investigated had rapidly lethal kinetics. Specific m5T4 immune responses could be detected by serum antibody ELISA and IFN-gamma ELISPOT assays in naive animals but were lower frequency than published responses to h5T4, and were further attenuated in tumour-bearing animals. The addition of anti-CTLA4 blockade did not result in significant augmentation of m5T4 specific immunity after vaccination in non tumour-bearing animals and combination treatment was ineffective as therapy in this autologous model. Results are discussed in the context of emerging immunotherapeutics in melanoma and prostate cancer. In the absence of supportive data from the model system it would not be appropriate to pursue combination heterologous 5T4 vaccine with anti-CTLA4 blockade, but in view of the unusual durability of the best response to tremelimumab, and in vitro evidence of enhanced proliferative responses to relevant TAA, further investigation of drug activity may be warranted in metastatic gastric and oesophageal second-line treatment.

Objetivo: O objetivo desse estudo foi avaliar alterações na resposta clínica e imunológica ao camarão após a imunoterapia com Dermatophagoides pteronyssinus. Métodos: Selecionou-se 35 indivíduos alérgicos a Dermatophagoides pteronyssinus (Der p), os quais foram submetidos a testes cutâneos de leitura imediata para ácaros, baratas, camarão, tropomiosina recombinante, além de cão, gato e fungos. A detecção de IgE espcífica in vitro foi feita para o ácaro, camarão, barata americana e para suas tropomiosinas. Em todos, avaliou-se reatividade clínica ao camarão através de provocação oral. Dez pacientes foram alocados para o grupo controle, e 25 foram submetidos à imunoterapia alérgeno específica para o ácaro. Os testes cutâneos e a dosagem de IgE sérica específica foram repetidas após a indução da imunoterapia, e após 1 ano do início. A reatividade clínica ao camarão foi reavaliada no final do estudo pela provocação oral. Resultados: No grupo dos pacientes que foram submetidos à imunoterapia, observamos diminuição na reatividade nos testes cutâneos e dosagem de IgE específica para Der p, camarão e tropomiosina recombinante. Dos 10 pacientes com testes cutâneos positivos para camarão, 4 foram negativos na dosagem após um ano de imunoterapia (p= 0,04). Quanto à dosagem sérica de IgE para camarão, dos 9 positivos no início, 6 ficaram negativos (p= 0,014). Nenhum paciente submetido a imunoterapia desenvolveu nova sensibilização para camarão. Não houve alteração na reatividade clínica ao camarão após imunoterapia. Conclusão: A imunoterapia para Dermatophagoides pteronyssinus foi acompanhada de diminuição da reatividade imunológica para camarão e clinicamente não houve alteração da sensibilidade a camarão / Objective: The objective of this study was to determine changes in clinical and immunological response to shrimp after immunotherapy with Dermatophagoides pteronyssinus. Methods: We studied 35 allergic subjects to Dermatophagoides pteronyssinus (Der p), submitted to skin tests to mites, cockroach, shrimp, recombinant tropomyosin, and dog, cat and fungi. The detection of serum specific IgE was performed to mite, shrimp, and tropomyosin from American cockroach. In all patients, the clinical reactivity to shrimp was assessed through oral challenge. Ten patients were allocated to the control group, and 25 were submitted to immunotherapy for mite. Skin tests and determination of serum specific IgE were repeated after the induction of immunotherapy (3-4 months) and 1 year after of beginning of the treatment. The clinical reactivity to shrimp was assessed again at the end of the study by oral challenge. Results: In the group of patients who were undergoing immunotherapy, we observed decreased reactivity in the skin tests and specific IgE levels to Der p, shrimp and recombinant tropomyosin. Among the 10 patients with positive skin tests to shrimp, 4 were negative when assessed after one year of immunotherapy (p = 0.04). About serum specific IgE to shrimp, from the 9 positive reactors in the beginning of treatment, 6 became negative (p= 0.014). There was no change in clinical reactivity to shrimp after immunotherapy. Conclusion: The immunotherapy for Dermatophagoides pteronyssinus was accompanied by decreased immune reactivity to shrimp and clinically there was no change in sensitivity to shrimp

Human γδ T cells expressing Vγ2Vδ2 TCRs monitor foreign- and self-prenyl pyrophosphate metabolites in isoprenoid biosynthesis to mediate immunity to microbes and tumors. Vγ2Vδ2 cells have been used for adoptive cancer immunotherapy with some partial and complete remissions. Most trials have used continuous zoledronate exposure to expand Vγ2Vδ2 cells. Zoledronate inhibits farnesyl pyrophosphate synthase causing isopentenyl pyrophosphate to accumulate that then stimulates Vγ2Vδ2 cells. Because zoledronate exposure is toxic, we hypothesized that a short period of exposure would reduce T cell toxicity but still be sufficient for monocytes uptake. Supporting this hypothesis, pulse zoledronate exposure with IL-2 resulted in more uniform expansion of Vγ2Vδ2 cells with higher purity and cell numbers as compared with continuous exposure. These Vγ2Vδ2 cells also had higher levels of CD107a and perforin and slightly increased tumor cytotoxicity. Importantly, adoptive immunotherapy with Vγ2Vδ2 cells derived by pulse stimulation controlled human PC-3 prostate cancer cells in immunodeficient NSG mice significantly better than those derived by continuous stimulation. Pulse zoledronate stimulation of Vγ2Vδ2 cells with IL-15 also resulted in higher purity and cell numbers. Like with CD8 αβ T cells, IL-15 preserved early memory Vγ2Vδ2 T cell subsets better than IL-2. However, despite this fact, adoptive immunotherapy with Vγ2Vδ2 cells derived with IL-15 showed similar inhibition of PC-3 tumor growth as those derived with IL-2. Thus, pulse zoledronate stimulation maximizes the purity, quantity, and quality of expanded Vγ2Vδ2 cells. This simple modification to existing protocols would likely enhance the effectiveness of adoptively transferred Vγ2Vδ2 T cells.

CD40 is a TNF receptor superfamily (TNFRSF) member central to the development of many aspects of the adaptive immune response. CD40 signaling promotes adaptive immunity in part by inducing the expression of cytokines, chemokines, and various adhesion and co-stimulatory molecules. The family of cytoplasmic adapter proteins, the TNFR-associated factors (TRAFs), serve as major mediators of TNFRSF pathways. CD40 regulates itself in part via the signaling induced degradation of TRAF2 and TRAF3. However, the effect of CD40-induced TRAF degradation on other TRAF dependent pathways is unknown. Here I provide evidence that CD40-mediated degradation of TRAFs 2 and 3 also influences the responsiveness of immune cells to CD40-independent, TRAF2- and 3-dependent pathways. LMP1 is a functional mimic of CD40, but signals to B lymphocytes in an amplified and sustained manner. LMP1 contributes to the development of B cell lymphoma in immunosuppressed patients, and may exacerbate flares of certain autoimmune diseases. The cytoplasmic (CY) domain of LMP1 binds TRAF2 with lower avidity than the CY domain of CD40, and TRAF2 is needed for CD40-mediated degradation of TRAFs 2 and 3. LMP1 doesn't induce TRAF degradation, and employs TRAF3 as a positive mediator of cell signaling, whereas CD40 signals are inhibited by TRAF3. Here, I tested the hypothesis that relative affinity for TRAF2, and/or distinct sequence differences in the TRAF2/3 binding sites of CD40 vs. LMP1, controls the disparate ways in which CD40 and LMP1 use TRAFs 2 and 3. The results revealed that TRAF binding affinity and TRAF binding site sequence dictate a distinct subset of CD40 vs. LMP1 signaling properties. The E3 ubiquitin ligases, cIAP1 and cIAP2, have been reported to play a crucial role in CD40 signaling. Because LMP1 is a mimic of CD40 signals, I hypothesized that LMP1 requires the cIAPs for signaling. To elucidate the role of the cIAPs in CD40 and LMP1 signaling, I specifically depleted the cIAPs and found that the cIAPs are differentially utilized in CD40 and LMP1 signaling. I also sought to further the understanding of the molecular underpinnings of how CD40, but not LMP1 signaling induces TRAF2 and TRAF3 degradation upon signaling. To do this, I investigated the ability of various CD40 and LMP1 mutants to induce TRAF degradation in distinct TRAF or cIAP deficient models. I found that neither a high TRAF2 binding potential nor the presence of the cIAP molecules are required for this process. Thus, this work reveals important insights into the molecular mechanisms of and role of CD40-mediated TRAF degradation in the immune system.

One major therapeutic target for preventing and treating Alzheimer's Disease (AD) is removal of excess β-amyloid (Aβ) from the brain. Both active and passive immunotherapies targeting Aβ have proven effective in reducing brain Aβ levels and improving cognitive function in mouse transgenic models of AD. However, these approaches can induce adverse neuropathologic effects and immunologic over-activation. Indeed, clinical trials of active Aβ immunotherapy in AD patients were halted due to development of meningoencephalitis, apparently resulting from wide-spread neuroinflammation. Here we show that a more restricted and specific immune re-activation through a single adoptive transfer of Aβ-specific T cells can provide long-term benefits to APPsw+PS1 transgenic mice that last at least 1 1/2 months. Aβ-sensitive splenocytes and lymphocytes were generated in normal mice, re-stimulated with Aβ in vitro, and then adoptively transferred into cognitively-impaired APPsw+PS1 mice. Compared to control transgenic mice through 1 1/2 month post-infusion, those mice that received Aβ-sensitive T cells exhibited a reversal of pre-infusion working memory impairment and demonstrated superior basic mnemonic processing. Step-wise forward Discriminant Function Analysis of behavioral results clearly demonstrated that T cell infused mice performed comparably to wild-type non-transgenics, further emphasizing the extent of cognitive benefit this therapeutic technique afforded. Importantly, a global inflammatory response did not accompany these benefits. Though no overall reductions in Aβ deposition were noted for T cell recipient mice, a subset of T cell infused mice that benefited most in cognitive function had reduced hippocampal burdens, suggesting that hippocampal Aβ burdes did play a role in determining performance capabilities of these mice. Since chronically high levels of beta-amy loid such as those found in APPsw+PS1 mice cause immune hypo-responsive/tolerance to Aβ, our results indicate that adoptive transfer of Aβ-sensitive T-cells can supercede such immune tolerance to Aβ, and may provide a safe, long-lasting therapy for AD.