Evaluation of multiple cytokine levels to improve our understanding of protective immune responses against Tuberculosis and to develop novel diagnostic methods

Phalane, Khutso Gemina (2013-03)

Thesis (MScMedSc)--Stellenbosch University, 2013.

Thesis

ENGLISH ABSTRACT: Important steps towards the global control of Tuberculosis include the improvement of diagnosis, the development of effective vaccines and the identification of correlates of protection/protective immunity to Mycobacterium tuberculosis. This study has of three objectives: 1. To validate the findings of a previous study that showed increased levels of IL-1β and decreased levels of IL-17 in children who are exposed to tuberculosis but remain uninfected compared to those who are exposed/infected and unexposed/uninfected. 2. To define the protective immunological phenotype in children with negative IGRA’s and TST following exposure to Mycobacterium tuberculosis. 3. To evaluate a number of cytokines in both serum and saliva samples of identified tuberculosis cases and controls for their diagnostic potential and to evaluate saliva as a possible new diagnostic sample type. The study designs were as follows: Objectives1, and 2: Children with documented tuberculosis exposure and with Mycobacterium tuberculosis infection as assessed through interferon gamma release assays, children with exposure but no infection and a control group with no exposure nor infection were investigated. These participants were selected according to their exposure and infection phenotypes from a larger TB household contact study that was conducted in communities in Cape Town. Whole blood was stimulated in QuantiFeron tubes overnight and ten cytokines were measured in antigen stimulated and unstimulated supernatants by Luminex multiplex Immunoassay. Differential production of cytokines in the three groups was evaluated. Objective 3. Saliva and serum samples were collected from thirty eight adults with suspected tuberculosis who were recruited from a community health centre in Cape Town, after which the levels of thirty three host markers were evaluated in the samples using the Luminex platform. The main findings of the studies included: 1. Increased levels of IL-1β and decreased levels of IL-17 in children who are tuberculosis exposed but remain uninfected compared to those who are exposed/infected and unexposed/uninfected could not be confirmed. 2. Immune responses other than IFN-γ are different in children with different exposure and infection phenotypes. Higher IL-23 and IL-33 levels in children with tuberculosis exposure without subsequent Mycobacterium tuberculosis infection compared to children with no exposure were shown. 3. In both the tuberculosis cases and controls, the levels of most markers were above the minimum detectable limit in both serum and saliva, but marker levels were not consistently higher in one sample type. The levels of fractalkine , IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF and IL-5 in saliva, and those of IL-6, IL-2, SAP and SAA in serum, were significantly higher in tuberculosis patients, in comparison to the levels obtained in those without active tuberculosis (p<0.05). The area under the ROC curve was ≥ 0.70 for most of these markers, thereby confirming their diagnostic potential for TB disease. The work presented in this thesis has identified markers that may grant an improved understanding on the mechanisms that are associated with protection against Mycobacterium tuberculosis in children. The preliminary results presented show that the identification of host markers in saliva is possible and the utility of saliva for the development of rapid immune-based tests for active tuberculosis is promising.

AFRIKAANSE OPSOMMING: Noemenswaardige vooruitgang in die globale beheer van Tuberkulose is onderworpe aan verbeterde diagnose, die ontwikkeling van doeltreffende vaksienes en die identifikasie van aanwysers van immuniteit teen Mycobacterium tuberculosis. Die doel van hierdie studie is: 1. Om die bevindinge van ‘n vorige studie te bevestig, waar verhoogde vlakke van IL-1β en verlaagde vlakke van IL-17 waargeneem is in kinders wat aan tuberkulose blootgestel is, maar nie geïnfekteer is nie. Hierdie bevindinge was in vergelyking met geïnfekteerde en nie-blootgestelde kinders. 2. Om ‘n beskermende immunologiese fenotipe te definieer in kinders met negatiewe IGRA’s en TST, na blootstelling aan Mycobacterium tuberculosis. 3. Om sekere sitokines, in beide serum en speeksel monsters van tuberkulose gevalle en kontroles, te evalueer as potensiële diagnosemiddels, asook die moontlikheid dat speeksel kan dien as ‘n nuwe diagnostiese monstertipe. Die studieraamwerk was as volg: Doel 1 &2:Die volgende groepe was onder meer ondersoek – Kinders blootgestel aan tuberkulose en wat gevolglik geïnfekteer is, soos vasgestel deur interferon gamma vrystellingstoetse; kinders wat wel blootgestel is maar nie geïnfekteer is nie en ‘n kontrolegroep wat geen blootstelling aan Mycobacterium tuberculosis gehad het nie. Hierdie individue is geselekteer volgens hul blootstellingsprofiel en infeksiefenotipes, uit ‘n groter blootstellingstudie op Kaapse huishoudings. Heelbloed is oornag gestimuleer en tien sitokiene is gemeet in antigeen-gestimuleerde en ongestimuleerde supernatante, deur middel van Luminex multipleks Immunotoetse. Differensiële produksie van sitokienes in hierdie groepe is gevolglik geëvalueer Doel 3: Speeksel en serummonsters van 38 volwassenes met vermeende tuberkulose, is versamel en die vlakke van drie en dertig gasheermerkers is gemeet deur middel van die Luminex platvorm. Die hoof bevindinge van hierdie studie sluit in: 1.Vehoogde vlakke van IL-1β en verlaagde vlakke van IL-17 kon nie bevestig word in die verskeie kindergroepe (Sien doel 1) nie. 2. Die immuunrespons, uitsluitend die IFN- γ respons, is veskillend in kinders met uiteenlopende blootstelling en infeksiefenotipes. Hoër vlakke van IL-23 en IL-33 is gevind in kinders wat blootgestel is aan tuberkulose, maar nie geïnfekteer is nie, in teenstelling met nie-blootgestelde kinders.. 3. In beide die pasiënte en kontroles was die meeste sitokienvlakke hoër as die minimum meetbare limiet in beide speeksel en serummonsters, hoewel merkervlakke nie konstant hoër was in enige van die twee monstertipes nie. Die vlakke van fractalkine, IL-17, IL-6, IL-9, MIP-1β, CRP, VEGF en IL-5 in speeksel en IL-6, IL-2, SAP en SAA in serum, was merkbaar hoër in tuberkulosepasiënte, in vergelyking met vasgestelde vlakke in individue sonder aktiewe tuberkulose. (p<0.05). Die oppervlak onder die ROC kurwe was ≥ 0.70 vir die meerderheid van die merkers. Dit is ‘n sterk aanduiding dat hierdie merkers potensiaal het as diagnostiese merkers vir tuberkulose. Hierdie navorsing het merkers geïdentifiseer wat die begrip van die megansime waarmee beskerming teen Mycobacterium tuberculosis gebied word in kinders, verbreed. Hierdie voorlopige resultate dui aan dat die identifikasie van gasheermerkers in speeksel moontlik is en dat speeksel moontlik kan dien as ‘n proefkonyn vir die ontwikkeling van immuungebaseerde sneltoetse vir die diagnose van aktiewe tuberkulose.

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