Dissertations / Theses on the topic 'Immune function'

Within the UK severe sepsis is responsible for 29% of intensive care admissions and carries a mortality of 44.7%. Decades of research have failed to deliver a single clinically successful immune modulating therapeutic agent. These failings may be explained by fundamental methodological challenges of sepsis laboratory investigations, namely diagnostic uncertainty, an indeterminate onset and the identification of an immunologically similar control population. The underlying hypothesis for this thesis is that the translational investigation of surgical patients may overcome many of these methodological challenges, since surgical trauma generates a homogenous inflammatory insult at a planned time to a carefully phenotyped human population. The biological basis for modelling sepsis with traumatic injury is discussed. Firstly, I reviewed the current literature to demonstrate the methodological advantages of studying surgical patients as a surrogate for sepsis. Next, I performed an observational study of neutrophil immune function following major elective surgery which identified a reduced neutrophil respiratory burst and changes in cell surface immune receptor expression. This impairment of activated neutrophil immune function was associated with resting neutrophil mitochondrial dysfunction, namely a raised mitochondrial membrane potential and increased production of reactive oxygen species. Using two different models of mitochondrial dysfunction I demonstrated that neutrophil respiratory burst may be regulated by altered mitochondrial functionality. Finally, I provide evidence that the cytoplasmic target for this mitochondrial signal is the enzyme pyruvate kinase M2, which through oxidative inhibition reduces the production of the respiratory burst substrate NADPH by limiting flow of glucose through the hexose monophosphate shunt. In summary, major elective surgery provides a translational model of human sepsis. Using this model, I demonstrate impairment of the neutrophil respiratory burst, and provide evidence that this is mediated through neutrophil mitochondrial dysfunction which promotes oxidative inhibition of the glycolytic regulatory enzyme pyruvate kinase.

The research in this thesis is concerned with the effect of chronic stress, caregiving and bereavement, and ageing on immune and endocrine parameters. First, there was no difference in serum anti-CMV antibody titre between younger caregivers and matched controls, but CMV seropositive caregivers with more negative health behaviours had higher CMV antibody titre. Second, there was no difference in neutrophil function between caregivers and controls in both younger and older group, while only younger caregivers showed a higher serum cortisol:cortisone ratio than controls. Further, those caregivers that reported higher anxiety and burden symptoms had lower neutrophil phagocytosis. Third, caregivers had more senescent KLRG1\(^+\) T cells than controls, but comparable number of “exhausted” PD-1\(^+\) T cells and thymic output. Finally, young bereaved adults showed similar neutrophil function and serum cortisol and DHEAS levels as non-bereaved controls, whereas older bereaved adults had impaired neutrophil function and a higher cortisol:DHEAS ratio. These findings suggest that chronic stress can have differential effects on immune and endocrine parameters, but in some cases, presence of immunosenescence is required for immune decrements to be observed. Further, they emphasise the importance of focusing on the individual's response to chronic stress rather than their chronic stress status, per se.

The millions of bioactive compounds found within animal venoms are a veritable war chest of novel immunotherapeutics. Discoveries have resulted in six venom derived drugs currently approved by the FDA for diseases such as blood disorders and diabetes. Venom-derived compounds are known to be highly potent and highly selective for ion channels as well as other key biological pathways. To date only an infinitesimally small fraction of venom-derived compounds have been investigated for medical applications. The aim of this project was the systematic scan of a vast venom biorepository (over 300 different venoms; including 217 different spider venoms, 92 snake venoms, 33 cone snail venoms and 2 jellyfish venoms, and 21 venom peptides) to characterize novel venom-derived peptides with immunomodulatory function. Using resting and stimulated human leucocytes, we performed high throughput, multiplex scanning of venom activity using Cytometric Bead Array readout (CBA). During my doctorate, this experimental platform was optimized and used for venom and the venom fraction screening. The CBA scanning allowed the detection and quantification of nine different human cytokines/chemokines. In Chapter 1, I provide an overview of what is currently known around immune modulation using venoms. Here, I detail previously described immunomodulation from snake, scorpion, bee and sea anemone venoms/toxins and provide an outline of what is known in the field. In Chapter 2, I detailed the breadth of materials and methods used in this doctorate including immunology and proteomics methodologies. In Chapter 3, I present the mass CBA scanning across spider, snake, jellyfish, and cone snail on primary human leucocytes. In this Chapter, I present cytokine/chemokine mastergraphs of IL-1β, IL-6, IL-8, IL-10, TNF-α, IFN-γ and MIP-1β release from primary human leucocytes after the addition of venom. In this Chapter, I also present mastergraphs of cytokine release from PMA/Ionomycin-stimulated primary human leucocytes after the addition of venom. These experiments identified T cell “accelerants” that enhanced cytokine production such as the spider P57 Badumna Insignis, P332 Pamphobeteus wuschi and the snake Boiga Dendrophila. In Chapter 3, I also present mastergraphs of cytokine/chemokine release from LPS-stimulated primary human leucocytes after the addition of venom. These experiments identified myeloid cell “accelerants” with enhanced cytokine production such as Pseudonaja affinis and Psudonaja Nuchalis. In this Chapter, I used principal component analysis (PCA) to identify venoms with similar cytokine/chemokine release patterns. I found that in general the PCA clustered venoms that produced pro-inflammatory cytokines IL-1β, IL-6, IL-8, MIP-1β and the anti-inflammatory cytokine IL-10 in one group and venoms that produced T cell related cytokines such as IL-2, IL-4, TNF-α and IFN-γ in a different group. I next performed Pearson’s correlation between cytokine/chemokine release and known LD50 data from venom. This was to investigate whether there was a correlation between release of a particular cytokine and lethality of the venom. I observed a strong positive correlation between IL-10 and intraperitoneal administered LD50 in the presence of Phorbol 12-Myristate 13-Acetate (PMA) and Ionomycin (r=0.9325) (P ≤ 0.05). In Chapter 4, I identified the top five venoms that enhanced the immune response mediated by cytokines (P204, P175, P332, P57 and P168) by testing venoms across multiple genetically unrelated donors. I attempted to map immunologically active peptides in the most bioactive venoms using Reversed Phase-High Performance Liquid Chromatography (RP-HPLC), CBA, Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS), transcriptomics and Edman degradation. The sequences of three original peptides derived from spider venom were identified. In Chapter 5, I investigated the ability of venoms and venom-derived peptides to augment the human immune response in vitro. Here, I used monocyte and dendritic cell stimulation, mixed lymphocyte reactions and dextran uptake to quantify antigen presenting cell activation profiles, T cell stimulation and antigen processing. I found that the venom peptides 16 and 19 increased the CD83 expression marker and the peptide P332 increased dendritic cell antigen uptake. In Chapter 6, I provide an overall summary of my findings and discuss how they fit in the wider literature. In this Chapter I also discuss possible future directions of my findings to progress research to the preclinical phase. In summary, I anticipate this research will help catalyse development of new classes of venom-derived peptide based immune stimulatory compounds to help augment vaccine effectiveness and enhance the effects of current and future immunotherapeutic strategies for cancer, infectious disease and autoimmunity.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environment and Sc
Science, Environment, Engineering and Technology
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CD4 is a co-receptor for binding of T cells to antigen-presenting cells (APC) and the primary receptor for human immunodeficiency virus-I. The disulfide bond in the second extracellular domain (D2) of CD4 is reduced on the cell surface, which leads to formation of disulfide-linked homodimers. A large conformational change must take place in D2 to allow for formation of the disulfide-linked dimer. Domain swapping of D2 is the most likely candidate for the conformational change leading to formation of two disulfide-bonds between Cysl30 in one monomer and Cysl59 in the other one (Cys133 and Cysl62 in the mouse CD4). Thus, we hypothesized that the domain swapping of D2 in CD4 regulates its co-receptor function of antigenspecific T cell activation. We found that mild reduction of the extracellular part of human CD4 resulted in formation of disulfide-linked dimers. We then tested the functional significance of dimer formation for co-receptor function using the engineered Jurkat T cell system by expressing wild-type or disulfide-bond mutant mouse CD4. Eliminating the D2 disulfide bond markedly impaired CD4's coreceptor function as assessed by antigen-specific IL-2 production. Exogenous wild type thioredoxin, but not redox-inactive thioredoxin, could inhibit the CD4-mediated IL-2 production, suggesting that the redox state of D2 disulfide bond is controlled by this oxidoreductase. Furthermore, structural modeling of the complex ofthe T cell receptor and domain-swapped CD4 dimer bound to class II major histocompatibility complex and antigen supports the domain-swapped dimer as the immune co-receptor. The known involvement of D4 residues Lys318 and Gln344 in dimer formation isalso accommodated by this model. These findings imply that disulfide-linked dimeric CD4 is the preferred functional co-receptor for binding to APC. Strategies to promote dimerisation of CD4 should, therefore, enhance the immune response, while inhibiting dimer formation is predicted to be immunosuppressive.

Host defense against infection and disease relies on the reciprocal communication between the immune and neuroendocrine systems where sex hormones exert negative and positive feedback actions on immune functions. Indeed, sex hormones have been implicated in gender dimorphic immune response and in the potentiation of immune-related disorders. The female hormone estrogen plays a role as an immunomodulator and may exert immunosuppressive and immunostimulatory effects. Though many studies focus on estrogen’s role in immunity within the female reproductive tract and autoimmunity, the modulatory effects of estrogen on vaccine responses are largely unexplored. The insufficient efficacy of some vaccines in certain target populations, as for example the elderly population, is well recognized. Hormones fluctuate throughout an individual’s life, and females in particular undergo several necessary reproductive (pregnancy and menopause) and lifestyle (oral contraceptive use) changes which involve sex hormones. Vaccine efficacy might be influenced by endogenous estrogen levels or by exogenous estrogen administration. Therefore, in the pursuit of improved vaccine efficacy, it is necessary to consider such hormonal factors and their contribution to immune status. We have studied estrogen’s role in modulation of vaccine responses using a mouse ovariectomy model where exogenous estrogen delivery can be controlled. Our studies included two different types of vaccines, a bacterial toxoid formulation and a bacterial secreted protein formulation. Results from these studies indicate that estrogen enhances vaccine-specific antibody production by likely supporting a general TH2 pathway and also modulates expression of genes encoding molecules critical in innate immune signaling and required for development of proper adaptive immune responses and antigen clearance through antibody-mediated mechanisms. The level at which estrogen modulates antibody responses appears to be dependent on the route of vaccine administration. The enhancement of specific humoral responses may involve mechanisms involving TLR2 and antibody Fc receptor expression on macrophages, cells that link innate and adaptive immune responses. Advances in our understanding of the relationship between sex hormones and the immune system may provide new insights into the mechanisms by which hormones act and thus may be exploited to guide the design of future vaccine strategies.

In this thesis, the pleiotropic effects of statins on the disruption of prenylation and membrane rafts were investigated in immune effector cells. T cells and NK cells were extracted from various groups of patients treated with statins and proliferation and cytotoxicity respectively, measured ex vivo. In a study of patients with cardiovascular disease, a 23% reduction in T cell proliferation and 43% reduction in NK cytotoxicity were observed. In a normal volunteer study where healthy subjects received simvastatin (40mg per day) for 4 weeks, a comparable reduction in T cell proliferation was not apparent, however a 30% reduction in NK cytotoxicity was observed. In vitro statin treatment further reduced proliferation and cytotoxicity whereas addition of farnesyl and geranylgeranyl transferase inhibitors had little or no effect. In conclusion, this thesis has demonstrated that statins have a multitude of applications and effects. The pleiotropism of statins due to reduced prenylation was observed in vitro by western blot in multiple signalling pathways and confirmed with the use of FT and GGT inhibitors in these pathways. However, the lack of functional effect of FT and GGTIs indicated that prenylation has a lesser impact on the functions of immune effector cells than cholesterol depletion of rafts. The comparable results obtained with MßCB indicated that the reduction of cholesterol in the membrane by statins was disrupting rafts and therefore disrupting signalling pathways to a greater extent than prenylation inhibition. It is however likely to be a combination of both processes that contributes to the pleiotrophic effect of statins.

This project investigated the effect of β2-adrenergic receptor (β2-AR) stimulation on the function of cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells in vitro. Catecholamines have been previously shown to increase efflux of these cells into the blood, but the effects on cell function are unclear. In this thesis three aspects of function have been addressed. The results of the studies presented in this thesis showed that: (1) β2-adrenergic stimulation by salmeterol reduced the percentage of IFN-γ producing CD4+ and CD8+ T cells activated by Staphylococcus Aureus enterotoxin type B (SEB) superantigen, cytomegalovirus lysate (CMV) or CMV pp65 (pp65) recombinant protein. (2) salmeterol, at high concentrations, increased rolling behaviour and decreased stationary behaviour of peripheral blood lymphocytes (PBLs) on human microvascular cell line (HMEC-1) and on vascular cell adhesion molecule-1 (VCAM-1), in both flow and static assays. (3) adrenergic stimulation impaired the activation and cytotoxic function of CD8+ T and NK cells, as indicated by lower expression of CD107a (a marker of CD8+ T and NK cell activation and function) following incubation of peripheral blood mononuclear cells (PBMCs) with human erythromyeloblastoid leukemia (K562) cell line or MHC class I chain-related gene A (MICA*009) transfected Chinese hamster ovary cells (T-CHO) was analysed. The results presented in this thesis showed that adrenergic stimulation influences a number of cellular functions, such as those related to migration, cytokine production and cytotoxic function. Together the above studies may contribute to our understanding about how stress affects the ability of the cytotoxic cells.

Thesis (PhD)--Stellenbosch University, 2015.
ENGLISH ABSTRACT: Introduction In the era of antiretroviral therapy (ART), people living with Human Immunodeficiency Virus (HIV) now have prolonged life spans. An emerging trend of non- acquired immune deficiency syndrome (AIDS) related complications now prevails in the aging HIV infected population. Increased levels of inflammation and chronic immune activation are associated with HIV infection. In the era of ART people living with HIV are at an increased risk of cardiovascular disease (CVD). Platelets play a pivotal role in both inflammation and immune activation and upon activation platelets degranulate and secrete various inflammatory, coagulatory and adhesion molecules. Activated platelets express surface P-selectin (CD62P) and are a key component of the coagulation pathway and serve as a link between inflammation and thrombosis. Activated platelets have been implicated in inflammatory and cardiovascular disease and have been identified as immune cells that play a crucial role in pathogen recognition and modulation of immune cells during infections. Several antiviral and antibacterial properties of platelet alpha granule contents have been established. Platelet aggregometry remains the most widely used technique to evaluate platelet function even though this technique is limited by many pre-analytical variables. Platelet flow cytometry on the other hand offers a rapid measurement of platelet function in their physiological environment with minimal artefactual activation. Few studies have however reported on standardized methods to evaluate platelet function in the context of HIV. Platelet function remains unclear and data on HIV infected treatment naïve individuals remains scarce. The aim of this project was to examine the relationship between platelet function and immune activation in patients with HIV Materials and methods This study consisted of five sub-studies, firstly platelet indices and levels of platelet activation were determined in a cohort of 330 participants (185 HIV infected ARV naïve and 145 uninfected healthy controls) using; flow cytometry and haemotology analyzers. The relationship between these indices and markers of platelet activation, disease progression and immune activation were assessed. Furthermore, levels of platelet activation and aggregation were evaluated in a cohort of 82 participants (41 HIV infected (ARV naïve) individuals and 41 uninfected healthy controls), using a novel whole blood flow cytometry based functional assay. These baseline levels were then correlated with markers of immune activation and disease progression in HIV. In a subsequent study, platelet function in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls was evaluated using flow cytometry. Platelet response was measured post stimulation with adenosine diphosphate (ADP) at concentrations known to induce reversible (0.04mM) and irreversible (0.2mM) platelet aggregation. In order to assess platelet function in HIV, platelet response was evaluated in a cohort consisting of 58 HIV infected (ARV naïve) and 38 uninfected controls. Platelets were activated using varying concentrations of ADP, arachidonic acid (AA) and collagen and platelet function was measured using flow cytometry. Levels of circulating platelet leukocyte aggregates (PLAs) were also measured using flow cytometry in a cohort consisting of 35 HIV-infected (ARV naïve) individuals and 32 uninfected healthy controls. Associations between PLAs, immune activation and disease progression in HIV infected individuals were determined. The final study evaluated platelet aggregates, platelet derived microparticles (PMPs) and microparticles (MPs) in a cohort consisting of 46 HIV infected (ARV-naïve) and 40 uninfected healthy controls. Associations between MPs, PMPs, platelet aggregates and markers of immune activation and disease progression were evaluated. Results HIV infected individuals showed decreased mean platelet volume levels (HIV mean 7.91 ± 0.85 vs. 8.52 ± 1.12, p<0.0001) that directly correlated with CD4 counts (r=-0.2898, p=0.0075) and viral load (r=0.2680, p=0.0177). Platelet distribution width (PDW) levels directly correlated (r=0.3455, p=0.0362) with active coagulation and inversely correlated (r=-0.3666, p=0.0463) with platelet aggregation. HIV infected individuals showed increased levels of platelet activation (%CD62P median 11.33[5.96-29.36] vs. control group 2.48[1.56-6.04], p=0.0001). In HIV, platelet function is retained and platelets showed increased response to submaximal concentrations of endogenous agonists. HIV infected individuals showed increased levels of circulating platelet monocyte aggregates (25.26[16.16-32.28] vs. control group 14.12[8.36- 18.83], p=0.0001) that directly correlated with markers of immune activation; %CD38/8 (r=0.54624, p=0.0155), viral load (r=0.633, p<0.009). Furthermore we report on increased levels of circulating MPs (median %MPs 1.7[0.95-2.83] vs. Control group 1.12[0.63-1.57], p=0.0160); PMPs (median %PMPs 26.64[11.33-36.62] vs. Control group 20.02[18.08-26.08], p=0.0133); activated PMPs (median CD62P MFI 3.81[3.46-4.54] vs. Control group 3.41[3.16-3.6], p=0.0037) and platelet aggregates (Median %CD62P 14.10[5.49-39.94] vs. Control group 0.17[0.10-10.99], p= 0.0097) in HIV infected asymptomatic individuals. Conclusion This study supports the potential use of the MPV and PDW as readily available markers of platelet activation and immune activation in HIV. We also showed elevated levels of activated platelets in HIV infected individuals that were hyper responsive to endogenous agonists in a concentration dependent manner. Platelet flow cytometry is a rapid and valuable technique in the evaluation of platelet function in HIV. The measurement of platelet function using flow cytometry allows the evaluation of platelet signalling pathways that may be modified in HIV infected individuals. Lastly we describe an optimized whole blood flow cytometry based assay for the evaluation of circulating microparticles (MPs), platelet derived microparticles (PMPs) and levels of activated platelets and aggregates which mimics the in vivo physiological environment of MPs. To the best of our knowledge, this study is the first to report on a novel approach in evaluating platelet function in HIV using a series of optimised whole blood flow cytometry based platelet assays. In addition, minimal work has been performed previously on platelet function in the context of HIV-infection; and particularly in a cohort of asymptomatic, untreated patients as defined for this study.
AFRIKAANSE OPSOMMING: Inleiding In die era van antiretrovirale terapie (ART), het mense wat met die menslike immuniteitsgebreksvirus (MIV) leef, het nou 'n verlengde lewensduur. 'N opkomende neiging van nie-verworwe immuniteitsgebreksindroom (vigs) heers nou in die verouderende MIV-besmette bevolking. Verhoogde vlakke van inflammasie en chroniese immuun aktivering word geassosieer met MIV-infeksie en in die era van ART loop mense wat met MIV leef, 'n verhoogde risiko van kardiovaskulêre siekte (KVS). Plaatjies speel 'n belangrike rol in beide inflammasie en immuun aktivering en met aktivering degranulate en skei plaatjies verskeie inflammatoriese, coagulatory en adhesie molecule af. Geaktiveerde plaatjies druk oppervlak P-selectin (CD62P) is 'n belangrike komponent van die stollings weg en dien as 'n skakel tussen inflammasie en trombose. Geaktiveerde plaatjies is in beide inflammasie en kardiovaskulêre siekte betrokke en is geïdentifiseer as immuun selle wat 'n deurslaggewende rol speel in die patogeen erkenning en modulasie van immuun selle tydens infeksies. Verskeie antivirale en antibakteriese eienskappe van plaatjie alpha korrel inhoud is vasgestel. Plaatjie aggregometry bly die mees gebruikte tegniek om plaatjie funksie te evalueer, alhoewel hierdie tegniek is beperk deur baie pre-analitiese veranderlikes. Plaatjie vloeisitometrie aan die ander kant bied 'n vinnige meting van plaatjie funksie in hul fisiologiese omgewing met 'n minimale artefactual aktivering. Min studies het egter berig op gestandaardiseerde metodes om plaatjie funksie in die konteks van MIV te evalueer. Plaatjie funksie is steeds onduidelik en data oor MIV besmet behandeling naïef individue bly skaars. Die doel van hierdie projek was om die verhouding tussen die plaatjie funksie en immuun aktivering in pasiënte met MIV te ondersoek. Materiaal en metodes Hierdie studie het bestaan uit vyf sub-studies. In die eerste plekis plaatjie indekse en vlakke van plaatjie aktivering bepaal in 'n groep van 330 deelnemers (185 MIV-besmette ARV naïef en 145 onbesmette gesonde kontrole) met behulp van vloeisitometrie en hematologie ontleders. Die verhouding tussen hierdie indekse en merkers van plaatjie aktivering, die siekte se progressive en immuun aktivering is beoordeel. Verder is die vlakke van plaatjie aktivering en samevoeging in 'n groep van 82 deelnemers (41 MIV-besmette (ARV naïef) individue en 41 onbesmette gesonde kontrole) geëvalueer, met behulp van 'n nuwe vol bloed vloeisitometrie gebaseerde funksionele toets. Hierdie basislyn vlakke is dan gekorreleer met merkers van immuun aktivering en die progreessie van die siekte in MIV. In 'n daaropvolgende studie, is plaatjie funksie in 'n groep wat bestaan uit 58 MIV besmet te (ARV naïef) en 38 onbesmette beheer geëvalueer met behulp van vloeisitometrie. Plaatjie reaksie is na stimulasie gemeet met adenosine diphophate (ADP) by konsentrasies bekend omkeer (0.04mM) te oorreed en onomkeerbaar (0.2mm) plaatjie aggregasie. Ten einde plaatjie funksie in MIV te evalueer, is plaatjie reaksie in 'n groep wat bestaan uit 58 MIV-besmette (ARV naïef) en 38 onbesmette kontrole geëvalueer. Die plaatjies is geaktiveer deur gebruik te maak van wisselende konsentrasies van ADP, is aragidoonsuur (AA) en kollageen en plaatjie funksie gemeet met behulp van vloeisitometrie. Vlakke van sirkulerende plaatjie leukosiet gemiddeldes is ook gemeet met behulp van vloeisitometrie in 'n groep wat bestaan uit 35 MIV-positiewe (ARV naïef) individue en 32 onbesmette gesonde kontrole. Assosiasies tussen leukosiet gemiddeldes, immuun aktivering en die progressive van ie siekte in MIV-besmette individue is ook bepaal. Die finale studie het plaatjie-gemiddeldes, plaatjie afgelei mikrodeeltjies en mikrodeeltjies geëvalueer in 'n groep wat bestaan uit 46 MIV besmet (ARV-naïewe) en 40 onbesmette gesonde kontrole. Assosiasies tussen mikrodeeltjies, plaatjie afgelei, plaatjie gemiddeldes en merkers van immuun aktivering en die siekte se progressie is geëvalueer. Resultate MIV-besmette individue het gedaalde gemiddelde plaatjie volume vlakke getoon (HIV gemiddelde 7,91 ± 0,85 8,52 ± 1,12 teen, p <0,0001) wat direk gekorreleer het met CD4-tellings (r = -0,2898, p = 0,0075) en virale (r = 0,2680, p = 0,0177). Plaatjie verspreiding breedte vlakke het direk gekorreleer met (r = 0,3455, p = 0,0362) met 'n aktiewe koagulasie en omgekeerd gekorreleer (r = -0,3666, p = 0,0463) met plaatjie aggregasie. MIV-besmette individue het verhoogde vlakke van plaatjie aktivering getoon (% CD62P mediaan 11,33 [5,96-29,36] teen kontrole groep 2,48 [1,56-6,04], p = 0,0001). In MIV, was plaatjie funksie behou en plaatjies het 'n verhoogde reaksie op submaksimale konsentrasies van endogene agoniste getoon. MIVbesmette individue het verhoogde vlakke van sirkuleer plaatjie monosiet-gemiddeldes gedemonstreer (25.26 [16,16-32,28] teen kontrole groep 14,12 [8,36-18,83], p = 0,0001) wat direk gekorreleer het met merkers van immuun aktivering; % CD38 / 8 (r = 0,54624, p = 0,0155), virale lading (r = 0,633, p <0,009). Verder rapporteer ons op verhoogde vlakke van sirkulerende mikrodeeltjies (mediaan% LP 1.7 [0,95-2,83] teen kontrole groep 1,12 [0,63-1,57], p = 0,0160); PMPs (mediaan% PMPs 26,64 [11,33-36,62] teen kontrole groep 20,02 [18,08-26,08], p = 0,0133); geaktiveer PMPs (mediaan CD62P MFI 3,81 [3,46-4,54] teen kontrole groep 3,41 [3,16- 3,6], p = 0,0037) en plaatjie gemiddeldes (Mediaan% CD62P 14,10 [5,49-39,94] teen 0.17 [0,10- 10,99], p= 0.0097) in MIV besmet asimptomatiese individue. Gevolgtrekking Hierdie studie ondersteun die potensiële gebruik van die MPV en PDW as waardevolle geredelik waardevolle merkers van plaatjie aktivering en immuun aktivering in MIV. Ons het ook getoon verhoogde vlakke van geaktiveer de plaatjies in MIV-besmette individue getoon wat hyper reageer op endogene agoniste was in 'n konsentrasie-afhanklike wyse. Plaatjie vloeisitometrie is 'n vinnige en waardevolle tegniek in die evaluering van plaatjie funksie in MIV. Die meting van plaatjie funksie gebruik vloei cytometry maak die evaluering van plaatjie sein paaie wat in MIVgeïnfekteerde individue verander moontlik. Laastens het ons beskryf 'n hele bloed vloeisitometrie gebaseer de toets vir die evaluering van sirkulerende mikrodeeltjies, plaatjie afgelei mikrodeeltjies en vlakke van geaktiveer plaatjies en gemiddeldes wat lyk soos die in vivo fisiologiese omgewing van MP's. Na die beste van ons kennis, is hierdie studie die eerste om te rapporteer oor 'n nuwe benadering in die evaluering van plaatjie funksie in MIV met behulp van 'n reeks van new hele bloed vloeisitometrie gebaseer de plaatjie toetse. Daarbenewens is minimale werk voorheen uitgevoer op die plaatjie funksie in die konteks van MIV-infeksie; en veral in 'n groep van asimptomatiese, onbehandelde pasiënte soos vir hierdie studie. Hierdie projek het bewyse bygevoeg tot die teorie dat plaatjies, in MIV, kan 'n skakel wees tussen die aktiewe inflammatoriese reaksie en die toename in die aantal trombotische en kardiovaskulêre siekte waargeneem in pasiënte wat met hierdie siekte saamleef.

Prion protein (PrP) is the only factor known to be essential in the pathogenesis of the transmissible spongiform encephalopathies (TSEs) or prion diseases. The cellular isoform (PrPc), a GPI-anchored sialoglycoprotein of unknown function, has an identical primary structure to the disease-associated conformer (PrPSc). Thus, animals are tolerant to PrPSc and TSEs do not trigger a classical immune response. Vaccine development for human TSEs requires elucidation of the immunodominant human T cell epitopes within PrP. Further, successful immunotherapy requires that the function of PrPc in lymphocytes is understood, as therapeutic targeting of prion protein risks interfering with immune function. Peripheral blood leukocytes from healthy donors were cultured with PrP sequence peptides to elicit proliferative and cytokine responses. Responses were seen to peptides clustered around the position 129 polymorphism and the C-terminus, in accordance with a predictive algorithm. The substitution of methionine by valine at position 129 altered both epitope immunogenicity and cytokine profile. Studies in murine T cell activation models demonstrated transcriptional and late surface protein upregulation of PrPc. Memory T cells expressed higher PrPc levels than naive cells and there was also a strong correlation at both protein and transcriptional levels between expression of PrPc and the regulatory T cell marker, Foxp3. Embryonic deletion of Prnp did not lead to deficits in T cell conjugation, proliferation or cytokine production, although memory cell numbers were slightly reduced. In PrP*7" mice regulatory T cells developed normally but may have enhanced suppressor function. However, neither PrP ablation nor anti-PrP monoclonal antibodies altered the phenotype of T cell mediated autoimmune disease. These findings demonstrate that tolerance to PrP is not complete in humans and raise the prospect of generating protective immunity through vaccination. However, PrPc is a potentially important memory, regulatory and T cell activation antigen, therapeutic disruption of which may precipitate immunopathology.

Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal storage disease caused by a mutation in the NPC1 or NPC2 genes. The functions of the proteins these genes encode are not fully understood, but are thought to be involved in cholesterol egress from the acidic compartment. The pathogenic cascade in proposed to begin with sphingosine accumulation followed by reduction of acidic store calcium levels. This results in impairing intracellular trafficking and storage of multiple lipid substrates in the late endosome/lysosomal compartment including cholesterol and multiple sphingolipids. Although clinically characterized by progressive neurodegeneration, there is also evidence of altered innate immune responses, such as neuroinflammation, that promotes disease progression. In this thesis, we initiated an investigation into whether phagocytosis, an important innate immune activity and the process by which particles are ingested by professional phagocytes, is defective in NPC. Our results indicate a global defect in phagocytosis that is not particle or size specific. We further found evidence of altered phagocytosis in vitro and in vivo that is likely contributing to NPC disease pathology and disease progression and may contribute to iron deficiency and Crohn's disease susceptibility in NPC patients. We next analysed lipid content on the plasma membrane. We found evidence to support the hypothesis that intracellular accumulation of lipids (e.g.glycosphingolipids) in NPC directly contributes to abnormal actin dynamics at the plasma membrane, leading to defective formation of phagocytic cups. When we reintroduced certain glycosphingolipids exogenously to the cells we were able to partially rescue the phagocytic defect suggesting that GM1 ganglioside may be a regulator of actin: plasma membrane contact sites. Lastly, we present evidence showing that some current NPC therapeutics partially correct the phagocytic defect. Taken together, this thesis provides insights into a novel aspect of NPC pathogenesis (innate immune dysfunction) and provides new knowledge of underlying immune defects in NPC, that in the future may act as novel targets for treatments.

Inhibited immune responses have been observed following occupational, inadvertent, or therapeutic exposure to chemically diverse xenobiotics. In the present studies, preliminary data were generated showing limited but significant systemic immunotoxicity following low-level topical exposure to the pyrethroid insecticide, permethrin (formerly not considered an immunotoxicant). Permethrin was applied to the shaved dorsal interscapular region of C57Bl/6N mice at doses of 0.5, 1.5, or 5.0 μl/day. The highest of these doses was approximately equal to 215 μg/kg/day, which is about seven times the estimated daily human exposure in individuals wearing permethrin treated clothing for insect protection. Mice were thus exposed to permethrin daily for 10 or 30 consecutive days, or every other day for 7 or 14 exposures. Body weight was not affected by the treatment. However thymic weight was decreased and splenic weight increased 2 days after termination of the topical exposure. Histopathology of immune organs showed no significant changes. Splenic macrophages showed significantly depressed chemiluminescent responses up to 10 days following termination of exposure, but macrophage phagocytic activity was not affected. Cell surface markers of thymocytes, splenocytes and bone marrow cells were not affected. Antibody production as shown by plaque forming cell (PFC) assay decreased significantly at 10 days after dosing termination. Taken together, these data indicate that low-level topical permethrin exposure may produce systemic immunotoxicity.
Master of Science

Crohn's Disease (CD) is a debilitating condition characterised by chronic intermittent intestinal inflammation. More than 90 genetic polymorphisms are associated with CD susceptibility, including several in genes of the innate immune system. Here I present a series of experiments designed to enhance our knowledge of the roles of CD-associated polymorphisms in pathogenesis. Many therapeutic regimens are employed in CD treatment, but patients' responses to treatment and disease progression vary widely. There is great interest in studying whether analysis of patients' genotype at CD-associated polymorphisms can be used to predict their disease course, and guide clinical decision-making. To answer these questions, it is essential to be able routinely and cost- effectively to genotype patients at the full range of known CD-associated polymorphisms. The first project presented here describes the design and initial successful testing of a CD-specific genotyping microarray for use in genotype-phenotype studies. The polymorphism most strongly associated with CD susceptibility is in the pattern recognition receptor NOD2; the remaining experiments presented here study the function of NOD2 in primary human monocyte-derived Dendritic Cells (DCs). First, a microarray study is presented which characterises global transcriptional responses to NOD2 stimulation in DCs. NOD2 stimulation is shown to enhance transcriptional changes induced by Toll-Like Receptor 2 stimulation, and NOD2-mediated transcriptional regulation is shown to be lost in DCs expressing CD-associated NOD2 variants. Second, experiments are presented which describe development of a new protocol for proteomic analysis of post-translational protein modifications, and which identify a number of novel candidate targets of NOD2 signalling in DCs. Finally, a project is presented which demonstrates for the first time that NOD2 stimulation induces autophagy in DCs, in an NF-kB and RIPK2-dependent pathway. CD-associated polymorphisms in NOD2 and ATG 16Ll abolish NOD2-mediated autophagy induction, resulting in impaired bacterial handling and antigen presentation.

Plasmacytoid dendritic cells (pDCs) regulate innate and adaptive immunity. Neurotrophins and their receptors control the function of neuronal tissue. In addition, they have been demonstrated to be part of the immune response but little is known about the effector immune cells involved. We report, for the first time, the expression and immune-regulatory function of the low affinity neurotrophin receptor p75 neurotrophin receptor (p75NTR) by the antigen-presenting pDCs, mediated by toll-like receptor (TLR) 9 activation and differential phosphorylation of interferon regulatory factor 3 and 7. The modulation of p75NTR on pDCs significantly influences disease progression of asthma in an ovalbumin-induced mouse model mediated by the TLR9 signaling pathway. p75NTR activation of pDCs from patients with asthma increased allergen-specific T cell proliferation and cytokine secretion in nerve growth factor concentration-dependent manner. Further, p75NTR activation of pDCs delayed the onset of autoimmune diabetes in RIP-CD80GP mice and aggravated graft-versus-host disease in a xenotransplantation model. Thus, p75NTR signaling on pDCs constitutes a new and critical mechanism connecting neurotrophin signaling and immune response regulation with great therapeutic potential for a variety of immune disorders.

Thesis (Ph. D.)--West Virginia University, 2007.
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Omega-3 fatty acids (ω-3 FA, including eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]), are essential polyunsaturated fatty acids which are correlated with lower incidence of chronic diseases. DHA and EPA can be enzymatically converted to resolvins, protectins and maresins, which play important roles in resolution of inflammation. Additionally, ω-3 FA can also directly activate surface receptors, namely the long-chain free fatty acid receptors GPR40 and GPR120, two GPCRs with poorly investigated biology. Using real-time PCR analysis, GPR40 transcript in human neutrophils was detected; the protein expression was also confirmed by flow cytometry and image stream analysis. Expression of GPR40 protein was up-regulated after stimulation with platelet-activating factor (PAF, 10nM) or leukotriene B4 (LTB4, 10nM) for 10 minutes. I utilised the selective agonist GW9508 to investigate the biology of GPR40. Tested on human neutrophils, GW9508 elevated intracellular calcium when applied within the 0.1-10μM range. The up-regulation of GPR40 expression by pro-inflammatory stimuli suggested to us potential regulatory roles for this receptor during inflammation. I then showed that 1 and 10μM GW9508 increased neutrophil chemotaxis in response to the cytokine IL-8 (30ng/ml). In addition, GPR40 activation by GW9508 enhanced phagocytosis of E. coli by human neutrophils by approximately 50% when tested at 0.1 and 1μM. Moreover, GW9508-neutrophil stimulation augmented microvesicle release and delayed apoptosis after stimulation. Finally, I demonstrated that GPR40 is expressed in inflammatory cells isolated from murine arthritic joints, such as neutrophils, macrophages and inflammatory monocytes. KBN-serum induced arthritic mice developed a more severe disease when treated prophylactically with GW9508 (10mg/kg, i.p. treated from day 0, daily), characterized by a higher clinical score and increased oedema when compared to vehicle control mice. Therapeutic intervention with GW9508 at the peak of the disease (day 5) delayed the resolution of arthritis. In summary, the data suggest that activation of GPR40 by GW9508 enhances neutrophil activation, up regulating the pro-inflammatory properties of this cell type, and therefore, exacerbating experimental inflammatory arthritis.

Prolonged exercise has been associated with depressed immune function, and hence an increased risk of infection. However, several nutritional supplements may reduce or overcome this problem. Thus, the aims of this thesis were to investigate the effects of some nutritional supplements on athletes immune function. In study 1 (Chapter 3), effects of several vaccine stimulant dose on whole blood culture cytokine production was carried out to determine effective vaccine stimulant dose; which was found to be between a dilution of 4000 (dose 4) and 1000 (dose 6) of the original vaccine. This finding was used for the other studies (Chapter 4 and 5). In addition, the relationship between data obtained from Evidence Investigator analyser and enzyme linked-immuno-sorbent assay (ELISA) for IL-10 was analysed and the results show a positive strong correlation between them. In study 2 (Chapter 4), in vitro effects of various immunomodulatory nutritional compounds on antigen-stimulated whole blood culture cytokine production was investigated and it was found that caffeine and quercetin showed tendency towards decrease cytokine production as the doses were increased. On the other hand, an upward trend was evident with kaloba, where high dose of kaloba seemed to increase the cytokine production. Since kaloba appeared to act as an immunostimulant in vitro, its effects on the immune response to prolonged exercise were examined in study 3 (Chapter 5). However, 7 days kaloba supplementation (20 mg of the root extract) did not alter athletes immune response although prolonged moderate intensity exercise significantly decreased S-IgA secretion rate and concentration post-exercise with the values returning to baseline by 1 h post-exercise. A 14-strain probiotic supplement effects on salivary antimicrobial proteins at rest and in response to an acute bout of prolonged exercise was investigated in study 4 (Chapter 6). Unfortunately, 30 days supplementation of the 14-strain probiotic appeared not enough to induce any significant effects on salivary antimicrobial proteins. Lastly, in study 5 (Chapter 7), the effects of a Lactobacillus probiotic on healthy people, who tend to have a higher than normal incidence of infection due to exercise stress-induced immune impairment was studied. In summary, this 16-week intervention study on 267 athletes found that regular ingestion of the probiotic reduced the extent to which training was negatively affected in endurance athletes when infection was present, and increased both S-IgA concentration and secretion rate over time. But it did not appear to reduce URTI incidence or the duration and severity of URTI episodes. Two major confounding factors, namely the unexpectedly low incidence of URTI during the winter period and the lower baseline S-IgA in the probiotic group may have prevented potential beneficial effects of probiotic supplementation from being identified.

Many college students exercise individually or participate in collegiate and intramural sports in addition to fulfilling their stressful academic requirements. The combination of accumulated stress and vigorous exercise could result in an impaired immune system, prompting the onset of disease and absences in class and sports practice. Twenty-six male and female participants aged 18 to 23 were recruited for this study. Over the course of an academic semester, participants completed weekly electronic surveys documenting stress levels, aerobic exercise, and symptoms related to upper respiratory tract infections. Participants were evaluated at four different time points (Baseline, Post-Midterm Exam, Baseline Reassessment, and Post-Final Exam) for body fat percentage, cardiovascular fitness, heart rate, blood pressure, and a 10mL blood draw. Blood samples were used to measure blood glucose, cortisol, IL-6, and CD11b levels. Analysis of cortisol and IL-6 concentrations required ELISA kits for protein quantification in plasma samples. CD11b levels in peripheral blood mononuclear cell samples were measured by Western Blot analysis. There was a significant increase in blood pressure during the final exam compared to rest for systolic (p=0.005) and diastolic (p=0.004) blood pressures. There was a significant decrease in anxiety during the final exam compared to anxiety during the mid-term exam (p=0.022). The acute stress of an exam was strong enough to illicit physiologic blood pressure change, but the chronic stress throughout the semester was not intense enough did not illicit physiologic or immune responses. The volume of aerobic exercise in the vigorous workout group was not great enough to influence immune responses nor disease incidence.

Major Histocompatibility Complex (MHC) receptors serve a critical role in self/non-self recognition through the presentation of peptide antigen to circulating T lymphocytes and are also believed to play a role in mate selection. Through the development of antibodies to MHC homologues in trout, this report demonstrates the presence of MHC expression in germ cells, as well as a soluble form in seminal fluid. What role these immune molecules may perform in reproduction and mate selection is discussed. In addition, as ectotherms, fish are often subjected to low temperatures. Previous data indicates that the expression of these genes is abolished by low temperatures. Employing these same antibodies, this report further demonstrates that trout maintain the expression of MH I and its critical light chain component, beta-2-microglobulin when subjected to 2oC for 10 days. Expression of the MH II receptor sub-units however, was sensitive to both confinement stress and low-temperature in vivo, as well as to factors secreted from a known fungal pathogen in cultured macrophage. As the cause of "winter kill", Saprolegniales cultures induced homotypic aggregation and pro-inflammatory gene expression in the macrophage cell line, RTS11 as well as down-regulation of MH II. Though no evidence of fungal toxins was evident, fungal spore size appeared to exceed macrophage phagocytic capabilities. Taken together, such a loss of MH II expression at low temperature may allow for establishment of fungal and bacterial diseases and that upon the return to warmer temperatures, saprolegniales have the ability to maintain MH II down-regulation and evade immune recognition. Concurrent to the study of MH expression, this report includes the first cloning and characterization of calreticulin (CRT) in fish. Like its mammalian homologue and primary chaperone to MHC receptors and other immune proteins, trout CRT appears to be a single copy gene with ubiquitous tissue distribution, displaying anomalous migration as a doublet with relative molecular mass of 60kD. Despite its promoter containing endoplasmic reticulum stress elements (ERSE), trout CRT expression did not increase upon treatment with several calcium homeostasis antagonists. Treatment of peripheral blood leukocytes with phytohemaglutinin did reveal a qualitative increase in cell surface expression, as seen in mammals; however, cellular protein levels did not change, suggesting that, in trout, CRT function may be regulated through cellular sub-localization, rather than through changes in gene expression, as it is in mammals.