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.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:sun/oai:scholar.sun.ac.za:10019.1/53457 |
| Date | 04 1900 |
| Creators | Bowers, Desiree Ann |
| Contributors | Van Helden, P., Stellenbosch University. Faculty of Medicine & Health Sciences. Dept. of Medicine. |
| Publisher | Stellenbosch : Stellenbosch University |
| Source Sets | South African National ETD Portal |
| Language | en_ZA |
| Detected Language | English |
| Type | Thesis |
| Format | 149 p : ill. |
| Rights | Stellenbosch University |
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