Experiments were done to determine the effect of recombinant interleukin 2 (rIL-2) on mononuclear cells (MC) of newborns and adults. MC were tested for (1) ability to lyse Daudi cells in a 51Cr release assay, (2) cell surface markers using monoclonal antibodies and flow cytometer analysis, and (3) cell types as determined by differential cell counts. Without rIL-2 adults show greater cytotoxicity than newborns in vitro. Incubation with rIL-2 dramatically increased the cytotoxicity expressed with cord blood and adult MC showing equivalent responses. Differences in cell surface markers between newborns and adults prior to rIL-2 exposure were in agreement with those previously published. This study did not demonstrate changes in phenotypes after exposure to rIL-2. Slight changes in differential cell counts occurred after increased incubation periods and rIL-2 exposure.

Submitted in complete fulfillment of the requirements for the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2012. / Immunomodulation using plants is of primary interest in scientific communities because it provides an alternative to conventional chemotherapy for a wide range of diseases. It is based on the ability of the plants to effectively modulate immune functions, thus being able to promote positive health and maintain the body’s resistance to infection. This research is aimed to evaluate the immunomodulatory potential of fourteen traditional leafy vegetables from Kwa-Zulu Natal, South Africa on human peripheral blood mononuclear cells (PBMC). In this study the methanolic and aqueous extracts were screened for lymphocyte proliferation using the MTT assay. The cytokine response was evaluated by measuring the secretion of interleukin 10 (IL-10) and interferon-gamma (IFN-γ) using the ELISA assay. The subpopulation of T cells viz., CD4+, CD8+, NK and B cells were measured by flow cytometry. Most of the methanolic extracts stimulated PBMC’s whilst a few suppressed lymphocyte proliferation. Most of the aqueous extracts were inactive. The methanolic extracts of Amaranthus hybridus and Centella asiatica stimulated PBMC’s and showed an increase in IFN-γ secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-1 immune response and stimulated cell mediated immunity. The methanolic extracts of Asystasia gangetica, Bidens pilosa, Emex australis, Justicia flava Momordica balsamina, Oxygonum sinuatum, Senna occidentalis and Sonchus oleraceous and the aqueous extracts of Amaranthus spinosus and Asystasia gangetica, Ceratotheca triloba, Oxygonum sinuatum, Physalis viscosa and Sonchus oleaceous stimulated PBMC’s and showed an increase in IL-10 secretion and the CD8+ cytotoxic T cells and B cells. Thus, they induced the Tc-2 immune response and stimulated humoral immunity. Also, the methanolic extracts of Amaranthus spinosus and Ceratotheca triloba and the aqueous extracts of Bidens pilosa and Justicia flava increased both IL-10 and IFN-γ secretion and the CD8+ vii cytotoxic T cells indicating the stimulation of both the Tc1 and Tc2 cytokine profiles. The elevated secretion of IFN-γ and IL-10 caused by the extracts can be attributed to the CD8+ cytotoxic T cells and B cells. The findings of this study show that leafy vegetables hold promise as immunomodulatory candidates. They may enhance cell-mediated immune functions by a pro-inflammatory response whilst some can promote humoral immune functions by means of an anti-inflammatory response. Further investigation should be considered on the effect of the extracts on other immune parameters. / National Research Foundation

published_or_final_version / abstract / Microbiology / Master / Master of Philosophy

Interleukin-l0 (IL-10) helps to limit the duration of potentially harmful inflammatory responses but has also been implicated in the persistence of a number of chronic viral infections. This thesis aimed to investigate the phenotype and function of mv -specific IL-l0-producing cells in chronic HIV-I infection, and the effect of IL-10 blockade on responses to candidate HIV -I vaccines. A cytokine capture assay was used to determine the HIV -specific cellular sources of IL- 10 in PBMC from 55 chronically infected individuals. A rare subset of CD8+ T cells was found to be the major HIV -I Gag-specific IL-10-producing population; these cells were restricted to ART-naive individuals and did not express the regulatory T cell markers CD25 or FoxP3 but could co-express IFN-y. A proportion of the population (median 48% and 9% respectively) expressed the P7 chain of the gut-homing integrin a4p7 and the chemokine receptor CXCR3, which mediates lymphocyte migration to sites of inflammation. Experimental depletion of Gag-specific IL-10+ CD8+ T cells did not affect T cell activation, or the production of cytokines such as IL-2 or IFN-y during short-term culture. However, depletion was associated with a significant increase in CD38 expression on CDI4+ monocytes, a trend towards increased HLA-DR expression on the same cells, and a significant increase in the concentration of the pro-inflammatory cytokine IL-6 in culture supernatants. There was also a significant increase in the number of HIV-infected (p24 antigen+) CD4+ T cells in cultures depleted of Gag- specific IL-10+ CD8+ T cells after 3 days, indicating that this population may contribute to control of viral replication. In order to determine the effect of IL-10 blockade on vaccine immunogenicity, IL-10R blocking antibody was administered to BALB/c mice prior to immunisation with two mV-I candidate vaccines, HIVA and HIVconsv. IL-10R blockade resulted in a trend towards increased IFN-y production by CD8+ T cells in response to the dominant H (Env) and P (Pol) epitopes of HIV A, and a significant increase in IFN-y ELISPOT responses to the subdominant Gl (Gag) epitope of HIV consv in vitro. Collectively, these data suggest that IL-10 producing cell populations may play critical but different roles in chronic infection and vaccination. Further research into how the timing of IL-10 responses affects disease outcome may allow IL-IO blockade to be explored as a therapeutic strategy in humans

Choi, Man Wing. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 126-139). / Abstracts in English and Chinese. / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Antigen presenting cells (APC) in Immune systems --- p.1 / Chapter 1.1.1 --- Dendritic cells --- p.2 / Chapter 1.1.1.1 --- Differentiation of dendritic cells in mice --- p.2 / Chapter 1.1.1.2 --- Maturation of dendritic cells --- p.3 / Chapter 1.1.1.3 --- Stimulation and polarization of T cells stimulated by dendritic cells --- p.6 / Chapter 1.1.2 --- Monocyte and macrophage --- p.7 / Chapter 1.1.2.1 --- Differentiation of monocyte and macrophage in humans --- p.7 / Chapter 1.1.2.2 --- Changes involved in differentiation of monocytes into macrophages --- p.9 / Chapter 1.2 --- "Isolation, structure and activity of mushroom polysaccharides" --- p.13 / Chapter 1.2.1 --- Sources of mushroom polysaccharides --- p.13 / Chapter 1.2.2 --- Extraction methods --- p.14 / Chapter 1.2.3 --- Structure-Activity Relationship (SAR) of mushroom polysaccharides --- p.15 / Chapter 1.2.4 --- Previous studies on immunomodulatory effects of mushroom sclerotial polysaccharides --- p.18 / Chapter 1.3 --- Recognition of β-glucan by specific receptors --- p.20 / Chapter 1.3.1 --- Complement Receptor 3 (CR3) --- p.22 / Chapter 1.3.1.1 --- Introduction of CR3 --- p.22 / Chapter 1.3.1.2 --- Expressions of CR3 to recognize fungi --- p.22 / Chapter 1.3.2 --- Dectin-1 --- p.24 / Chapter 1.3.2.1 --- Introduction of Dectin-1 --- p.24 / Chapter 1.3.2.2 --- Structure of Full-length Dectin-1 --- p.26 / Chapter 1.3.2.2.1 --- Isoforms of Dectin-1 in Mice --- p.28 / Chapter 1.3.2.2.2 --- Isoforms of Dectin-1 in Humans --- p.28 / Chapter 1.3.2.3 --- Immune responses triggered by of Dectin-1 --- p.29 / Chapter 1.3.3 --- Toll-like 2 receptor (TLR2) --- p.31 / Chapter 1.3.3.1 --- Introduction of TLR2 --- p.31 / Chapter 1.3.3.2 --- Structure of TLR2 --- p.33 / Chapter 1.3.3.3 --- Immune responses triggered by TLR2 --- p.34 / Chapter 1.4 --- Research Objectives --- p.35 / Chapter Chapter 2 --- Materials and Methods --- p.38 / Chapter 2.1 --- Materials --- p.38 / Chapter 2.1.1 --- Mushroom sclerotia --- p.38 / Chapter 2.1.1.1 --- Polysaccharide extraction from mushroom sclerotia --- p.38 / Chapter 2.1.2 --- Antibodies and reagents --- p.41 / Chapter 2.1.3 --- Human acute leukocyte monocytic cell line and culture medium --- p.42 / Chapter 2.1.4 --- Preparation of murine bone marrow-derived immature dendritic primary cells (immature BMDCs) --- p.43 / Chapter 2.2 --- Methods --- p.45 / Chapter 2.2.1 --- Chemical Analysis --- p.45 / Chapter 2.2.1.1 --- Measurement of monosaccharide profile --- p.45 / Chapter 2.2.1.1.1 --- Acid depolymerisation --- p.45 / Chapter 2.2.1.1.2 --- Neutral sugar derivatization --- p.45 / Chapter 2.2.1.1.3 --- Gas chromatography (GC) --- p.46 / Chapter 2.2.1.2 --- Determination of total sugar by phenol-sulfuric acid method --- p.47 / Chapter 2.2.1.3 --- Determination of protein content by Lowry-Folin Method --- p.48 / Chapter 2.2.1.4 --- Size exclusion chromatography by high pressure liquid chromatography (HPLC) --- p.49 / Chapter 2.2.1.5 --- Endotoxin detection --- p.50 / Chapter 2.2.2 --- Measurement of Bioactivities --- p.51 / Chapter 2.2.2.1 --- Trypan blue exclusion assay --- p.51 / Chapter 2.2.2.2 --- MTT cell proliferation assay --- p.51 / Chapter 2.2.2.3 --- BrdU cell proliferation assay --- p.53 / Chapter 2.2.2.4 --- Expression of cell surface markers --- p.54 / Chapter 2.2.2.5 --- Phagocytosis / Endocytosis of FITC-labeled dextrans --- p.55 / Chapter 2.2.2.6 --- Nitric oxide production assay --- p.55 / Chapter 2.2.2.7 --- Reactive oxygen species production --- p.57 / Chapter 2.2.2.8 --- Determination of cytokine profile using cytokine antibody array --- p.58 / Chapter 2.2.2.9 --- Cell cycle analysis --- p.59 / Chapter 2.2.2.10 --- Expression of surface receptors --- p.60 / Chapter 2.2.2.11 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results and Discussion --- p.61 / Chapter 3.1 --- Chemical characteristics of sclerotial polysaccharides --- p.61 / Chapter 3.1.1. --- The yield of sclerotial polysaccharides --- p.61 / Chapter 3.1.2 --- Total carbohydrate content of sclerotial polysaccharides --- p.65 / Chapter 3.1.3 --- Protein content of sclerotial polysaccharides --- p.66 / Chapter 3.1.4 --- Monosaccharide profiles of sclerotial polysaccharides from PR by gas chromatography (GC) --- p.66 / Chapter 3.1.5 --- Molecular weight of sclerotial polysaccharides from PR by size exclusion chromatography (SEC) --- p.69 / Chapter 3.1.6 --- Endotoxin test --- p.73 / Chapter 3.2 --- Immune responses for human monocytic cell line THP-1 --- p.74 / Chapter 3.2.1 --- MTT cell viability assay --- p.74 / Chapter 3.2.2 --- BrdU cell proliferation assay --- p.75 / Chapter 3.2.3 --- Change in cell morphology of THP-1 --- p.79 / Chapter 3.2.4 --- Phenotypic maturation of THP-1 --- p.81 / Chapter 3.2.5 --- Up-regulated phagocytic ability of THP-1 --- p.84 / Chapter 3.2.6 --- Increased nitrite production in THP-1 --- p.86 / Chapter 3.2.7 --- Production of reactive oxygen species --- p.88 / Chapter 3.2.8 --- Human cytokines profile array --- p.90 / Chapter 3.2.9 --- Cell cycle analysis --- p.93 / Chapter 3.2.10 --- Surface receptors expression --- p.95 / Chapter 3.2.11 --- Summary --- p.98 / Chapter 3.3 --- Immune responses for murine immature BMDCs --- p.102 / Chapter 3.3.1 --- Inhibition effects on murine immature BMDCs --- p.102 / Chapter 3.3.2 --- Change in cell morphology of murine immature BMDCs --- p.103 / Chapter 3.3.3 --- Phenotypic maturation of murine immature BMDCs --- p.105 / Chapter 3.3.4 --- Down-regulation of endocytosis in murine immature BMDCs --- p.106 / Chapter 3.3.5 --- Increased nitrite production --- p.109 / Chapter 3.3.6 --- Decreased expression of CD 11c in PRW-treated immature BMDCs --- p.109 / Chapter 3.3.7 --- Cytokine profile detection --- p.112 / Chapter 3.3.8 --- Surface receptors expression --- p.116 / Chapter 3.3.9 --- Summary --- p.119 / Chapter Chapter 4 --- Conclusion and future works --- p.123 / Appendix --- p.125 / References --- p.126

Guo, Cuixia. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 75-85). / Abstracts in English and Chinese. / Thesis committee --- p.ii / Abstract --- p.iii / 摘要 --- p.iv / Acknowledgment --- p.v / List of Tables --- p.vi / List of Figures --- p.vii / List of Abbreviations --- p.viii / Chapter 1. --- Introduction --- p.1 / Chapter 1.1 --- Introduction to immune system --- p.1 / Chapter 1.2 --- Immune effecter cells --- p.1 / Chapter 1.2.1 --- Macrophage --- p.1 / Chapter 1.2.2 --- Dendritic Cells (DCs) --- p.5 / Chapter 1.3 --- Immunomodulatory and antitumor activities of mushrooms --- p.8 / Chapter 1.3.1 --- Introduction to mushroom --- p.11 / Chapter 1.3.2 --- Mushroom polysaccharides --- p.11 / Chapter 1.3.3 --- Mushroom β-glucan --- p.14 / Chapter 1.4 --- The receptors for polysaccharides associated with immune effecter cells --- p.16 / Chapter 1.4.1 --- CR3 --- p.16 / Chapter 1.4.2 --- Dectin-1 --- p.18 / Chapter 1.4.3 --- TLR2 --- p.19 / Chapter 1.5 --- Nuclear factor-kappa B (NF-kB) activation --- p.19 / Chapter 1.6 --- Previous studies on mushroom sclerotium --- p.20 / Chapter 1.6.1 --- Pleurotus tuber-regium (PT) --- p.20 / Chapter 1.6.2 --- Polyporus rhinocerus (PR) --- p.21 / Chapter 1.7 --- Objectives --- p.21 / Chapter 2. --- Materials and Methods --- p.23 / Chapter 2.1 --- Materials --- p.23 / Chapter 2.1.1 --- Mushroom sclerotia --- p.23 / Chapter 2.1.2 --- Animal --- p.23 / Chapter 2.1.3 --- Cell lines --- p.24 / Chapter 2.2 --- Methods --- p.24 / Chapter 2.2.1 --- Hot water extraction --- p.24 / Chapter 2.2.2 --- Measurement of monosaccharide profile --- p.25 / Chapter 2.2.2.1 --- Acid depolymerization --- p.25 / Chapter 2.2.2.2 --- Neutral sugar derivatization --- p.25 / Chapter 2.2.2.3 --- Gas chromatography (GC) --- p.26 / Chapter 2.2.3 --- Determination of molecular weight by size exclusion chromatography (SEC) --- p.27 / Chapter 2.2.4 --- Determination of total sugar by phenol-sulfuric acid method --- p.28 / Chapter 2.2.5 --- Determination of protein content by Lowry-Folin method --- p.28 / Chapter 2.2.6 --- Detection of endotoxin --- p.29 / Chapter 2.2.7 --- Immunomodulatory activities induced in RAW264.7 cell line and murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.1 --- Isolation of murine peritoneal macrophages (PMs) --- p.30 / Chapter 2.2.7.2 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.30 / Chapter 2.2.7.3 --- Phagocytic uptake --- p.31 / Chapter 2.2.7.4 --- Reactive Oxygen Species (ROS) generation --- p.32 / Chapter 2.2.7.5 --- Nitric Oxide (NO) production --- p.32 / Chapter 2.2.7.6 --- Inducible Nitric Oxide Synthase (iNOS) expression --- p.32 / Chapter 2.2.7.6.1 --- Cell lysates preparation --- p.33 / Chapter 2.2.7.6.2 --- Determination of protein concentrations --- p.33 / Chapter 2.2.7.6.3 --- Western blot --- p.34 / Chapter 2.2.7.7 --- Tumor Necrosis Factor-alpha (TNF-α) production --- p.36 / Chapter 2.2.8 --- DC cell marker determination --- p.37 / Chapter 2.2.9 --- Nuclear factor kappa B (NF-kB) activation --- p.37 / Chapter 2.2.10 --- Determination of the expression of existing cell surface β-glucan receptors --- p.37 / Chapter 2.2.11 --- Statistical methods --- p.38 / Chapter 3. --- Results --- p.39 / Chapter 3.1 --- Yield and chemical composition of mushroom sclerotial polysaccharides --- p.39 / Chapter 3.2 --- Endotoxin examination --- p.41 / Chapter 3.3 --- Monosaccharide profiles of PTW and PRW by GC --- p.41 / Chapter 3.4 --- Molecular weight profile by size exclusion chromatography (SEC) --- p.43 / Chapter 3.5 --- Immunomodulatory activities induced in RAW264.7 cells and murine peritoneal macrophages (PMs) --- p.46 / Chapter 3.5.1 --- Detection of cell surface antigens on RAW 264.7 cells and PMs --- p.46 / Chapter 3.5.2 --- Phagocytic uptake --- p.49 / Chapter 3.5.3 --- ROS generation --- p.53 / Chapter 3.5.4 --- NO production --- p.56 / Chapter 3.5.5 --- iNOS expression --- p.59 / Chapter 3.5.6 --- TNF-α production --- p.60 / Chapter 3.5.7 --- Morphological changes of cells --- p.62 / Chapter 3.5.8 --- DC cell marker determination --- p.64 / Chapter 3.6 --- Receptors expression on RAW 264.7 cells and PMs --- p.66 / Chapter 3.7 --- NF-kB activation --- p.68 / Chapter 3.8 --- Discussion --- p.70 / Chapter 4. --- Conclusions and Future Works --- p.73 / Chapter 5. --- References --- p.75

Psychological conditions, including stress, compromise immune defenses. Although this concept is not novel, the molecular mechanism behind it remains unclear. Neuropeptide Y (NPY), regulates anxiety and is a part of the stress response. The NPY system also modulates immune functions such as cytokine release, cell migration, and innate immune cell activity. Postganglionic sympathetic nerves innervating lymphoid organs release NPY, which together with other peptides activate five receptors (Y1, Y2, Y4, Y5, and y6). Additionally, immune cells themselves release NPY following activation. Previous studies have shown that Y1 mediates NPY-immune effects and data presented here shows expression of Y1 on a wide range of immune cells. Results presented in this thesis, using Y1-deficient mice (Y1-/-), have uncovered a novel role for Y1 on immune cells. NPY acts endogenously to inhibit T cell activation whereas Y1-/- T cells are hyper-responsive to activation and trigger severe colitis after transfer into lymphopenic mice. Thus, signalling through the Y1 receptor on T cells inhibits T cell activation and controls the magnitude of T cell responses. Paradoxically, in Y1-/- mice, T cell differentiation to Th1 T cells appears to be defective as these mice were resistant to T helper type 1 (Th1) cell???mediated inflammatory responses and showed reduced levels of the Th1 cell???promoting cytokine interleukin 12 and reduced interferon ?? production. This defect was due to functionally impaired antigen presenting cells (APCs). Y1-deficient APCs are defective in their ability to produce Th1-promoting cytokines and present antigens to T cells and consequently, Y1-/- mice had reduced numbers of effector T cells. Key reciprocal bone marrow chimera experiments indicated that this effect is intrinsic to immune cells and not driven by other Y1-expressing cell types. These results demonstrate a fundamental bimodal role for the Y1 receptor in the immune system, serving as a strong negative regulator on T cells as well as a key activator of APC function. The findings presented in this thesis uncover a sophisticated molecular mechanism regulating immune cell functions and thus adds to a growing number of signalling pathways shared by the immune and nervous system.

Three experiments were conducted to study the influence of dietary fatty acids on the production performance and immune response of chickens. In experiment I, forty day-old broiler chicks were fed diets containing 5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), flax oil (Diet III) or fish oil (Diet IV). No significant differences (P>0.05) were observed between the live weight of birds. The liver tissue total fat content was lower (P<0.05) in treatment I and II. The fatty acid composition of breast and thigh muscle, liver, heart, pericardial fat, plasma, splenocytes and gut associated lymphoid tissue differed (P<0.05) between treatments. The thiobarbituric acid reactive substances (TBARS) of breast and thigh muscle, liver and heart tissue were lower (P<0.05) in Diet I fed birds. Serum antibody activity was decreased (P<0.05) in Diet II fed birds. In experiment II, 120 day-old broiler chicks were fed diets containing 3.5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), linseed oil (Diet III) or fish oil (Diet IV). Body weight gain was higher (P<0.05) in Diets III and IV compared to Diets I and II fed birds. Feed intake was increased (P<0.05) in Diet IV fed birds. Birds fed Diets III and IV had higher (P<0.05) n-3 fatty acids in all tissues studied. A preferential incorporation of CLA was observed in spleen mononuclear cells. TBARS were higher (P<0.05) in the breast and thigh muscle of Diet IV fed birds. Serum anti-BSA antibody content was higher (P<0.05) in birds fed Diets III and IV. Delayed type hypersensitivity (DTH) response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. In experiment III, 120 layer birds were fed diets containing 3% of CLA+animal fat (Diet I), sunflower oil (Diet II), canola+flax oil (Diet III) or fish oil (Diet IV). Egg production, feed consumption and feed efficiency did not differ (P>0.05) among treatments. Birds fed Diets III and IV had higher content of n-3 fatty acids in eggs. Eggs from hens fed Diet I incorporated higher (P<0.05) CLA and saturated fatty acids with a concomitant reduction in (P<0.05) monounsaturated fatty acid content. A preferential incorporation of CLA was observed in eggs over other tissues. TBARS were higher (P<0.05) in breast and thigh muscle of Diet IV fed birds. Egg TBARS content did not differ (P>0.05) among treatments. Serum and yolk anti-BSA antibody contents were higher (P<0.05) in birds fed Diets III and IV. DTH response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. Feeding n-3 fatty acids increased antibody mediated immune response while n-6 fatty acids and CLA increased cell mediated immune response. / Graduation date: 2003

Not available

The purpose of this study was to examine the influence of physical activity on cytokine production in healthy older males. Twelve males (six very active, six less active) ages 65 to 74 were recruited for this study. Blood was obtained at rest and serum concentrations for MIP-I a, IL-1 ra, IL-6 and IL-10 were measured. No difference was found in MIP-la and IL-Ira concentrations between the two groups. The serum concentration of IL-6 was significantly lower (p = 0.016) in the very active group compared to the less active group. The very active group had a significantly higher (p = 0.016) concentration of IL-10 compared to the less active group. The beneficial influence of physical activity on cytokine production is clinically important because of the role of IL-6 and IL-10 in disease development in older adults. Through influencing cytokine concentrations, our results provide further insights into the role of physical activity in attenuating the effects of aging. / School of Physical Education