Aquaculture is known as a major food-producing industry and Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) are the major cultured species in Scotland. However, disease outbreaks in aquaculture have been reported and are commonly associated with intensive fish farming, which results in a tremendous cost in the industry. Hence, understanding what immune-related genes and cells are present, their responses, mechanisms and functions in these farmed animals is a first requirement for potent vaccine design, selection of disease-resistant breeds and disease outbreak prevention. The innate immune system is the first line of defence against microbes which use germline-encoded pattern recognition receptors (PRRs) to recognise specific, conserved and constitutive products of invading pathogens, called pathogen-associated molecular patterns (PAMPs) that are important for survival of the microorganism and are thus hard for the microorganism to change. This thesis focuses on the identification and characterisation of a family of PRR called toll-like receptors (TLRs) and a TLR accessory protein UNC93B1 using different approaches. In Chapters 2, 3 and 5, eleven TLR genes and UNC93B1 were identified from Atlantic salmon whole-genome shotgun (WGS) contigs. These genes were cloned and sequenced and their putative domain structure, gene synteny and homology to other genes were determined by bioinformatics analysis. In addition, the constitutive expression profile of these genes was examined in different tissues from healthy salmon using real-time PCR. The potential modulation of these genes was examined in different in vitro and in vivo models which provide information to help understand the role(s) of these genes during inflammation or in the immune responses against pathogens. Several of these TLRs are so-called non-mammalian TLRs (TLR19, TLR20a and TLR20d) and are therefore particularly interesting to study. The sub-cellular localization was also investigated in TLR-GFP expression plasmid transfected Salmon Head Kidney-1 (SHK-1) cells. Lastly, attempts were made to develop a Human Embryonic Kidney (HEK) 293T cell line based platform to study TLR signalling and ligand specificity (Chapter 4).

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Thesis (MSc)--Stellenbosch University, 2003. / ENGLISH ABSTRACT: The immune system matures progressively from infancy to adulthood, thus children may differ from adults in their immune function. The immature immune system demonstrates a higher naive to memory T cell ratio, defective macrophage function and antigen presentation which, cumulatively, results in diminished production of cytokines such as IFN-y. This cytokine has been shown to play a pivotal role in protection against Mycobacterium tuberculosis (M. tuberculosis) disease. Other cytokines, such as IL-12 and TNF-a, are also involved in the defence against M. tuberculosis. Epidemiological evidence suggests an agerelated incidence of tuberculosis (TB) irrespective of prevalence in a given region. Reports in the literature also demonstrate depressed immune responses in TB patients, at diagnosis, (before TB therapy) with subsequent improvement after TB therapy. The aims of this study were to optimise a whole blood assay in order to characterise immune responses, as measured by proliferation and cytokine production, in TB patients (after TB therapy) and healthy controls of different ages. Immune responses of TB patients would also be compared, before, and after TB therapy. A total of 68 subjects were included in this study. These comprised 27 TB patients and 41 healthy Mantoux positive controls. All subjects were stratified into two age groups: <12 years and >12 years. Diluted whole blood was cultured and stimulated with the mitogen, phytohaemagglutinin (PHA) and the specific mycobacterial antigen, purified protein derivative (PPD) to measure proliferation and IFN-y, IL-2, TNF-a and IL-10 production in the supernatant of cultures. Age was a significant variable for the following PHA-stimulated cytokines: IFN-y, TNF-a and IL-10. Proliferation and IL-2 production after PHA stimulation did not demonstrate any relationship with age. None of the PPD-stimulated proliferative or cytokine responses demonstrated any correlation with age. Concentrations of PHA- and PPD-induced IFN-y for all subjects (patients and controls) were increased “after therapy”, compared to “before therapy”. This phenomenon could possibly be due to maturation in the capacity of the immune system to produce this cytokine. Patients >12yrs demonstrated improvement in all proliferative and cytokine responses (except for PPD-induced IL-2 and TNF-a) “after therapy”, compared to “before therapy”. This is probably a valid finding and is thus in accordance with the literature. The whole blood assay is a simple, non-laborious assay that, according to the literature, produces results that seem to correlate well with that of conventionally used PBMCs. Age appears to be an important variable in the quantitative assessment of cellular immune responses (when the mitogen, PHA is used as a stimulant) and immune responses of older TB patients appear to improve after TB therapy, compared to before TB therapy. / AFRIKAANSE OPSOMMING: Die immuunsisteem matureer stelselmatig van kind na volwassene. Dus sal kinders se immuniteit verskil van volwassenes s’n. Die immature immuunsisteem het ‘n hoer nai'witeit vir geheue T-sel verhouding, defektiewe makrofaag funksie en antigeen presentering wat gesamentlik lei tot verminderde produksie van sitokiene soos byvoorbeeld IFN-y. Daar is bewys dat hierdie sitokien ‘n deurslaggewende rol speel in die beskerming teen Mycobacterium tuberculosis (M. tuberculosis). Ander sitokiene, soos IL-12 en TNF-a speel ook ‘n rol in die beskerming teen M. tuberculosis. Epidemiologiese data dui aan dat daar ‘n ouderdomverwante insidensie van tuberkulose (TB) is sonder dat dit beinvloed word deur die voorkoms van TB in ‘n sekere area. Verslae in die literatuur wys ook op onderdrukte immuniteitrespons in TB-pasiente by diagnose (voor TB-behandeling) met uiteindelike verbetering na TB-behandeling. Die doel van hierdie studie was om ’n volbloed metode te optimaliseer in ’n poging om die immuunrespons te karakteriseer soos gemeet met behulp van proliferasie en sitokien produksie by TB-pasiente (na TB-behandeling) en gesonde kontrole persone van verskillende ouderdomme. Die immuunrespons van TB-pasiente word ook vergelyk voor en na TBbehandeling. ‘n Totaal van 68 gevalle is vir die studie gebruik. Dit sluit in 27 TB-pasiente en 41 gesonde Mantoux positiewe kontroles. A1 die gevalle is in twee ouderdomsgroepe verdeel: <12 jaar en >12 jaar. Kulture is gemaak van verdunde volbloed en gestimuleer met phytohaemaglutinin (PHA) en gesuiwerde proteien derivaat (purified protein derivative-PPD) om proliferasie en IFN-y, IL- 2, TNF-a en IL-10- produksie in die supernatant van die kulture te meet. Ouderdom was ‘n beduidende veranderlike vir die volgende PHA-gestimuleerde sitokiene: IFN-y, TNF- a en IL-10. Daar was geen korrelasie tussen proliferasie en IL-2-produksie na PHA-stimulasie aan die een kant en ouderdom aan die ander kant nie. Geen van die PPDgestimuleerde proliferasie response of sitokien response het enige korrelasie met ouderdom getoon nie. Konsentrasies van PHA- en PPD-geinduseerde IFN-y vir alle gevalle (pasiente en kontrole) was verhoog “na behandeling”, vergeleke met “voor behandeling”. Hierdie fenomeen kan moontlik toegeskryf word aan maturasie in die vermoe van die immuunsisteem om sitokiene te vervaardig. Pasiente >12 jaar het bewyse getoon van verbetering in alle proliferasie en sitokien response (behalwe vir PPD-gei'nduseerde IL-2 en TNF-a) “na behandeling”, vergeleke met “voor behandeling”. Dit is waarskynlik ‘n geldige bevinding en is dus in ooreenstemming met verslae in die literatuur. Die volbloed metode is ‘n eenvoudige metode wat nie baie arbeidsintensief is nie, wat volgens die literatuur, resultate lewer wat goed korreleer met die konvensionele gebruik van perifere bloed mononukliere selle (PBMC’s). Dit wil voorkom asof ouderdom ‘n belangrike veranderlike is in die kwantitatiewe beoordeling van sellulere immuunrespons (wanneer PHA gebruik word as ‘n stimulant), en of die immuunrespons van ouer TB-pasiente verbeter na TB-behandeling in vergeleke met die respons voor TB-behandeling.

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