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The evolution of the Mycobacterium tuberculosis proteome in response to the development of drug resistanceFortuin, Suereta 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: This study is the first of its kind to highlight the importance of using the latest state of
the art technology available in the field of proteomics as a complementary tool to
characterize the proteome of members of the Mycobacterium tuberculosis Beijing
lineage which have been linked to outbreaks and drug resistance of Tuberculosis
(TB).
Our label-free comparative analysis of two closely related M. tuberculosis strains with
different transmission patterns and levels of virulence highlighted numerous factors
that may alter metabolic pathways leading to hyper-virulence whereby the strain was
able to rapidly replicate in the host and cause extensive disease. This comparative
analysis clearly demonstrated that both instrumentation and analysis software impacts
on the number of proteins identified and thereby the interpretation of the proteomic
data. These proteomes also served as substrates for the discovery of phosphorylation
sites, a field of research that reflects a significant knowledge gap in the field of M.
tuberculosis. By using differential separation techniques in combination with the
state of the art mass spectrometry we described the phosphorylation sites on 286
proteins. This was the first study to document phosphorylation of tyrosine residues in
M. tuberculosis. By this means, our data set further extend and complement previous
knowledge regarding phosphorylated peptides and phosphorylation sites in M.
tuberculosis. Using advanced mass spectrometry methods we further investigated the impact of the
in vivo evolution of rifampicin resistance on the proteome of a rifampicin-resistant
strain containing a S531L rpoB mutation. We identified the presence of overabundant
proteins which could provide novel insight into potential compensatory mechanisms that the bacillus uses to reduce susceptibility to anti-TB drugs. Our
findings suggest that proteins involved in a stress response may relate to an altered
physiology enabling the pathogen to tolerate and persist when exposed to anti-TB
drugs. Together this suggests that structural changes in the RNA polymerase
precipitated a cascade of events leading to alterations of metabolic pathways. In
addition, we present the first comprehensive analysis of the effect of rifampicin on the
proteome of a rifampicin resistant M. tuberculosis isolate suggesting that rifampicin
continues to influence the biology of M. tuberculosis despite the presence of an rpoB
mutation. Our analysis showed alterations in the cell envelope composition and
allowing the bacterium to survive in a metabolically dormant/persistent growth state.
The results presented in this study illustrate the full potential of using a proteomic
approach as a complementary molecular technique to select promising candidate
molecules and genes for further characterization using the tools of molecular biology. / AFRIKAANSE OPSOMMING: Die huidige studie is ‘n eerste van sy soort, deur die nuutste gevorderde tegnologie in
die proteomika veld te gebruik. Die proteoom van lede van die Mycobacterium
tuberculosis Beijing stam, wat die oorsaak is van tuberkulose (TB) uitbrake en ook
weerstandige TB, is gekarakteriseer.
Ons merkervrye vergelykende analise van twee naby verwante M. tuberculosis
stamme met verskillende vlakke van oordraagbaarheid en virulensie, beklemtoon
verskeie faktore wat metaboliese paaie mag verander, wat kan ly tot hiper-virulensie,
wat die TB-stam in staat stel om vinniger te repliseer in die gasheer en ‘n uitgebreide
siektetoestand kan veroorsaak. Die analise het duidelik gewys dat die toerusting wat
gebruik word, sowel as die sagteware ‘n invloed kan hê op die hoeveelheid proteïne
wat geïdentifiseer kan word en daardeur intrepretasie van proteomika data kan
beïnvloed. Hierdie proteome dien as substrate vir die ondekking van fosforilasie
setels, ‘n veld van navorsing wat dui op ‘n gaping in ons kennis van M. tuberculosis.
Deur gebruik te maak van differensiële skeidingstegnieke en moderne spektrometrie
beskryf ons fosforileringsetels in 286 proteine. Hierdie is die eerste studie wat
fosforilasie van tirosien residue in M. tuberculosis beskryf. Hierdeur komplimenteer
en brei ons data die huidige kennis oor gefosforileerde peptiede en fosforilasie setels
in M. tuberculosis uit. Deur gebruik te maak van gevorderde massa spektrometriese tegnieke het ons verder
ook die impak van in vivo evolusie van rifampicin weerstandigheid op die proteoom
van ‘n rifampicin weerstandige TB-stam met die algemene S531L rpoB mutasie
ondersoek. Ons het proteïne geïdentifiseer wat in groot hoeveelhede voorkom en kan nuwe insigte gee tot potensiele kompenserende meganismes wat deur die bacillus
gebruik word om vatbaarheid vir anti-TB middels te verminder. Ons bevindings dui
daarop dat proteïene betrokke in ‘n stresreaksie mag lei tot ‘n verandering in fisologie
wat die patogeen in staat stel om anti-TB middels te verdra en te volhard in die
teenwoordigheid van sulke middels. Saam impliseer dit dat ‘n ketting van gebeure
wat lei tot veranderinge in metaboliese paaie, word vooraf gegaan deur strukturele
veranderinge in die RNS polimerase. Tesame hiermee bied ons ook die eerste
omvattende analise aan van die effek wat rifampicin op die proteoom van ‘n
rifampicin weerstandige M. tuberculosis isolaat het, en wat aan die hand doen dat
rifampicin voordurend die biologie van M. tuberculosis beïnvloed, ten spyte van die
teenwoordigheid van ‘n rpoB mutasie. Ons analise dui op veranderinge in die
samestelling van die selomhulsel wat die bakterie toelaat om te oorleef in ‘n
metabolies dormante staat.
Die resultate wat in hierdie studie aangebied word illustreer die volle potensiaal van
‘n proteomiese benadering as komplementêre molekulêre tegniek om belowende
kandidaat molekules en gene te kies vir verdere karakterisering, deur gebruik te maak
van molekulêre tegnieke. / The National Research Foundation (RSA), / Norwegian Research Council (Norway) / National Institute of Health –Forgarty (USA) / Southern Africa Consortium for Research Excellence-Welcome Trust (SACORE) (United Kingdom) / Kwazulu-Natal Research Institute for Tuberculosis and HIV (K-RITH) (USA)
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Population structure, host cell interactions and pathogenesis of Staphylococcus aureus strains isolated at Tygerberg hospital, South AfricaOosthuysen, Wilhelm Frederick 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Numerous studies conducted internationally have identified and described several endemic methicillin-susceptible Staphylococcus aureus (MSSA) clones. However, only some of these clones are associated with methicillin resistance (CC5, CC8, CC22, CC30 and CC45). To date, studies reporting on the population structure of S. aureus isolated in South Africa represent limited demographic areas, focus on methicillin-resistant S. aureus (MRSA) only and have displayed little emphasis on virulence. This study was undertaken to elucidate the population structure of S. aureus isolated from specific clinical sources at Tygerberg hospital, and to investigate specific host-pathogen interactions of representative isolates.
Consecutive non-repetitive clinical S. aureus isolates were collected over one year (September 2009/2010) with patient demographics and limited clinical information. Strains were typed by PFGE and molecular markers (spa, multi-locus sequence typing (MLST), agr, Staphylococcal Chromosome Cassette mec and Panton-Valentine leukocidin (PVL)). Representative isolates were selected and investigated for the presence of virulence genes, adherence (to immobilised fibronectin [Fn], fibrinogen [Fg], collagens IV [CnIV] and VI [CnVI]), cellular invasion and cell death induction. Statistical association were determined between all in vitro results and methicillin-resistance, clonality, patient HIV status and bacterial PVL status.
Fifteen percent of the isolates (n = 367) were MRSA. Forty four present of isolates were PVL+. agr I-IV and SCCmec I-V were identified. The MSSA population was diverse: ST22 (dominant), ST1865 and ST121 were PVL+. ST45, ST1863 and ST15 were PVL-. PVL- MRSA were diverse: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II and ST22-MRSA-IV. The genes fnbA/B (fibronectin-binding protein A/B), clfA/B (clumping factor A/B), eap (extracellular adherence protein), nuc (nuclease), coa (coagulase) and hld (delta toxin) were detected in all representative isolates.
The CC8 and CC6 isolates adhered strongly to all ligands (100-700% of control, ligand dependent), while isolates of CC45, CC22 and CC88 adhered strongly only to Fg and Fn. The CC30, CC15, and CC12 isolates adhered extremely strongly to CnIV (>300%) and CC8, CC15, and C6 to CnVI (>200%). Isolates from CC30, CC8, CC15, CC6, CC12, CC97, CC88 and CC45 were highly invasive (>100%). ST121 was non-invasive (>50%). Isolates of CC5, CC30 and CC121 were non-cytotoxic (<50%), while isolates of CC22, CC8, CC15, CC45 and CC88 were very cytotoxic (>70%). No significant difference was observed in adherence or cell death induction of MRSA vs. MSSA clones or between isolates from HIV+ vs. HIV- persons. PVL- isolates displayed higher cellular invasiveness than PVL+ isolates.
The presence of ST612-MRSA-IV, ST22-MRSA-V and ST8-MRSA-V points to local SCCmec acquisition, as we found MSSA isolates with the same spa types. Numerous MSSA clones were prevalent, but do not appear to have a major common genetic background with MRSA. PVL was highly prevalent among MSSA, indicating acquisition of PVL genes independently of SCCmec. The abilities to adhere to specific immobilised ligands in vitro were diverse and grouped with the genetic background, while the vast majority of isolates were invasive and induced significant cell death.
We can conclude that the population of S. aureus at Tygerberg hospital is composed of a vast number of MSSA and MRSA clones, which display varying patters of adherence to selected ligands and of which, the majority clones are invasive and cytotoxic. / AFRIKAANSE OPSOMMING: Talle internasionale studies het verskeie endemiese metisillien vatbare Staphylococcus aureus (MSSA) klone geïdentifiseer en beskryf. Slegs 'n paar van hierdie klone word geassosieer met metisillien weerstandigheid (Klonale kompleks (KK) 5, KK8, KK22, KK30 en KK45). Studies oor die bevolking struktuur van S. aureus geïsoleer in Suid-Afrika is tot dusver beperk tot demografiese gebiede, fokus slegs op metisillien-weerstande S. aureus (MRSA) en het min klem op virulensie geplaas. Hierdie studie is onderneem om die bevolking struktuur van S. aureus, geïsoleer vanaf spesifieke kliniese bronne, in die pasiëntpopulasie van Tygerberg-hospitaal te ondersoek en om ondersoek in te stel na spesifieke gasheer-patogeen interaksies van verteenwoordigende isolate.
Opeenvolgende, nie-herhalende en suiwer kliniese S. aureus isolate is versamel oor ´n periode van een jaar (September 2009/2010), tesame met pasiënt demografiese- en beperkte kliniese inligting. Stamme is deur PFGE en molekulêre merkers (spa, MLST, agr, SCCmec en PVL) beskryf. Verteenwoordigende isolate is gekies en ondersoek vir die teenwoordigheid van virulensie gene, aanhegting ( aan geïmmobiliseerde fibronektien [Fn], fibrinogeen [Fg], kollageen IV [CnIV] en kollageen VI [CnVI]), sellulêre indringing en die induksie van seldood. Statistiese assosiasies is bepaal tussen alle in vitro resultate en methicillin-weerstandigheid, klonaliteit, pasiënt MIV status en bakteriese PVL status.
Fyftien persent van die isolate (n = 367) was MRSA. Vier-en-veertig van die isolate was PVL+. agrI-IV en SCCmec I-V is geïdentifiseer. Die MSSA bevolking was divers: ST22 (dominant), ST1865 en ST121 PVL +. ST45, ST1863 en ST15 was PVL+. PVL- MRSA was divers: ST612-MRSA-IV (dominant), ST5-MRSA-I, ST239-MRSA-III, ST36-MRSA-II en ST22-MRSA-IV. Die gene fnbA/B (fibronektien A/B), clfA/B (klontings faktor A/B), eap (ekstrasellulêre aanhegtings protein), nuc (nukease), coa (koagulase) en hld (delta toksien) was aangetref in alle verteenwoordigende isolate.
Isolate van KK8 en KK6 het sterk aan alle ligande (100-700% van kontrole, ligand-afhanklike) aangeheg, terwyl isolate van KK45, KK22 en KK88 slegs sterk aand fibronektien en fibrinogeen aangeheg het. Isolate van KK30, KK15, en KK12 het baie sterk aan CnIV (> 300%) aangeheg en KK8, KK15, en KK6 and CnVI (> 200%). Isolate van KK30, KK8, KK15, KK6, KK12, KK97, KK88 en KK45 was hoogs indringend (> 100%). ST121 was nie-indringende (> 50%). Isolate van KK5, KK30 en KK121 was nie-sitotoksiese (<50%), terwyl isolate van KK22, KK8, KK15, KK45 en KK88 baie sitotoksies was (> 70%).
Geen betekenisvolle verskil is waargeneem in die aanhegting of seldood induksie van MRSA teenoor MSSA klone of tussen isolate van MIV+ teenoor MIV- persone nie. PVL- isolate het hoër sellulêre indringing as PVL+ isolate vertoon.
Die teenwoordigheid van ST612-MRSA-IV, ST22-MRSA-V en ST8-MRSA-V verwys na die plaaslike verwerwing van SCCmec, aangesien ons MSSA isolate beskryf het met dieselfde spa-tipes. Talle MSSA klone was algemeen, maar het nie 'n beduidende genetiese agtergrond met MRSA vertoon nie. PVL was baie algemeen onder MSSA isolate en die PVL gene is dalk onafhanklik van SCCmec verkry.
Die vermoë om aan spesifieke geïmmobiliseer ligande in vitro aan te heg was divers en groepeer met die genetiese agtergrond, terwyl die meerderheid van die isolate indringend was en kon betekenisvolle sel dood veroorsaak.
Ons kan aflei dat die bevolking van S. aureus by die Tygerberg hospitaal saamgestel is uit 'n groot aantal van MSSA en MRSA klone, wat verskillende patrone van aanhegting aan geselekteerde ligande vertoon en waarvan die meeste klone indringende en sitotoksies is. / DFG/NRF International Research and Training Group (IRTG) 1522 “HIV and associated infectious diseases in Southern Africa” / National Research Foundation / Medical Research Council, Medi-Clinic / Harry Crossley Fund (Stellenbosch University) / Stellenbosch University
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Ischaemic preconditioning : an investigation of the patterns of kinase activation and protein expression profiles during reperfusion in the rat heartHattingh, Susanna Maria (Suzel) 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: Introduction: Coronary heart disease (CHD) is the leading cause of death
worldwide with 3.8 million men and 3.4 million women dying globally each year.
Although existing myocardial reperfusion strategies such as thrombolysis and
percutaneous coronary intervention (PCI), if applied in a timely manner, limit
myocardial infarct size, the mortality and morbidity remains significantly high.
Ischaemic preconditioning (IPC) may offer the potential to attenuate myocardial
ischaemia/reperfusion injury through cardioprotective signaling pathways which is
recruited at the time of myocardial reperfusion, thereby improving clinical
outcomes in patients with coronary artery disease.
Ischaemic preconditioning is a phenomenon whereby short intermittent episodes
of coronary occlusion followed by reperfusion protect the myocardium against a
subsequent period of sustained ischaemia. This protection is reflected in the
limitation of infarct size and improved functional recovery of the ischaemic heart
during reperfusion. Despite intensive research efforts, the promise of an effective
cardioprotective strategy using the endogenous protective mechanisms of the
heart which underlies IPC, has not yet been materialized. Although progress has
been made in terms of signaling mechanisms in the preconditioned heart, the
identification of the myocardial reperfusion phase as the critical “window” for
cardioprotection, requires the elucidation of the signal transduction pathways
during the reperfusion phase after IPC.
In view of the above, the aims of the present study were to investigate:
i. the involvement of the RISK pathway and p38 MAP kinase pathway in IPC
during early and late reperfusion
ii. the involvement of heat shock protein-27 (HSP-27), heat shock protein-70
(HSP-70), GSK-3β, CAMKII, AMPK and the transcription factor CREB in
the context of IPC during early reperfusion
iii. the involvement of autophagy and apoptosis during early and late
reperfusion after IPC iv. the correlation of the protein kinases with the hemodynamic parameters of
the heart
v. the mechanism of IPC by means of two-dimensional (2D) proteomics
Methods: The isolated perfused working rat heart model was used with
functional recovery as end-point. Hearts were preconditioned (IPC) for 3x5 min
global ischaemia, alternated with 5 min reperfusion. Hearts were subjected to 25
min sustained global ischaemia, followed by 5, 10, 15 or 30 min reperfusion when
hearts were snap-frozen for western blotting analysis. Alternatively, hearts were
reperfused for 30 min to record hemodynamic parameters and measure
functional recovery. Non-preconditioned (Non-IPC) hearts were stabilized for 30
min and subjected to 25 min sustained global ischaemia followed by 5, 10, 15 or
30 min reperfusion when hearts were snap-frozen. Alternatively Non-IPC hearts
were reperfused for 30 min to serve as control for the 30 min reperfused IPC
group. Activation of the protein kinases was determined by western blotting
analysis.
For the proteomic study mitochondrial and cytosolic proteins were isolated from
heart tissue and separated in the first dimension by isoelectric focusing, followed
by separation in the second dimension by two dimensional gel electrophoresis.
The PD Quest software programme was used to identify significantly expressed
protein spots. Protein spots of interest were excised and subjected to in-gel
digestion and the resulting peptides were analysed by mass spectrometry.
Proteins were identified by Mascot and the Swiss Prot database.
Results: Western blotting analysis demonstrated that the RISK pathway and p38
MAPK are activated very early in reperfusion, but the activation is not sustained
during the reperfusion period. Autophagy is also upregulated during this early
reperfusion phase; it is attenuated in the middle reperfusion phase and increase
for a second peak of upregulation in the late reperfusion phase. In addition, we
identified CAMKII as a novel marker of functional recovery in IPC after
reperfusion. The proteomic analysis identified twenty differentially expressed mitochondrial
and thirty six differentially expressed cytosolic proteins between Non-IPC and IPC hearts. Functions ascribed to the majority of these individual proteins were
directly related to cardiac metabolism.
Conclusion: Activation of the majority of the protein kinases investigated in the
present study is associated with the hemodynamic parameters of the heart
instead of functional recovery. Results indicated that the variable signaling
patterns could be attributed to differences in heart rate and the effect thereof
(ejection fraction, minimum and maximum rate of contraction), as a result of
sympathetic stimulation due to psychological stress in the animals before
slaughtering. Proteomics results demonstrated that IPC hearts which failed after
ischaemia /reperfusion are metabolically compromised and “worse off” compared
to non-IPC hearts. / AFRIKAANSE OPSOMMING: Inleiding: Koronêre hartsiekte is die vernaamste oorsaak van sterftes wêreldwyd
met 3.8 miljoen mans en 3.4 miljoen vrouens wat jaarliks sterf. Alhoewel
bestaande miokardiale herperfusie strategieë soos trombolise en perkutane
koronêre intervensie (PKI), wanneer betyds toegepas, miokardiale infarktgrootte
beperk, bly mortaliteit en morbiditeit steeds hoog. Isgemiese prekondisionering
(IPK) beskik oor die potensiaal om miokariale isgemie/herperfusie skade te
verminder deur beskermende seinoordragpaaie tydens miokardiale herperfusie te
aktiveer en sodoende die pasiënte wat aan koronêre arterie siekte ly, se
prognose te verbeter.
Isgemiese prekondisionering verwys na die verskynsel waartydens kort episodes
van isgemie opgevolg deur herperfusie, die miokardium teen ‘n daaropvolgende
langdurige isgemiese insident beskerm. Hierdie beskerming word gereflekteer in
die beperking van infarktgrootte en verbeterde funksionele herstel van die
isgemiese hart tydens herperfusie. Ten spyte van intensiewe navorsingspogings
is die presiese meganisme van endogene beskerming tydens IPK nog nie ten
volle ontrafel nie. Die identifisering van die miokardiale herperfusie fase se
kritiese “vensterperiode” van beskerming, noodsaak ‘n volledige analise van die
seinoordragpaaie wat geaktiveer word tydens die herperfusie fase na IPK.
In die lig van bogenoemde, was die doel van die huidige studie om die volgende
te ondersoek:
i. die betrokkenheid van die RISK seinoordragpad en p38 MAP kinase
tydens vroeë en laat herperfusie na IPK
ii. die betrokkenheid van “heat shock protein-27” (HSP-27), “heat shock
protein- 70” (HSP-70), GSK -3β, CAMKII, AMPK en die transkripsie faktor,
CREB, in die konteks van IPK tydens vroeë herperfusie
iii. die betrokkenheid van outofagie en apoptose tydens vroeë en laat
herperfusie na IPK
iv. die korrelasie van die proteïenkinases met die hemodinamiese parameters
van die hart v. die meganisme van IPK deur middel van twee dimensionele proteomika
Metodes: Die geïsoleerde werkende rothart model, met funksionele herstel as
eindpunt, is gebruik. Harte is geprekondisioneer (IPK) met 3x5 min globale
isgemie, afgewissel met 5 min herperfusie. Daarna is harte blootgestel aan 25
min volgehoue globale isgemie, gevolg deur 5, 10, 15 of 30 min herperfusie,
waartydens harte gevriesklamp is. Alternatiewelik, is harte blootgestel aan 30 min
herperfusie ten einde funksionele herstel te meet en hemodinamiese parameters
te registreer. Nie-geprekondisioneerde (Non-IPK) harte is gestabiliseer vir 30
min, waarna dit onderwerp is aan 25 min volgehoue globale isgemie, gevolg deur
5, 10, 15 of 30 min herperfusie, waartydens harte gevriesklamp is vir westelike
klad analise. Alternatiewelik, is Non-IPK harte onderwerp aan 30 min herperfusie
om te dien as kontrole vir die 30 min IPK groep. Aktivering van die
proteïenkinases is bepaal deur westelike klad analise.
Vir die proteomiese studie, is onderskeidelik mitokondriale en sitosoliese
proteïene geïsoleer en geskei in die eerste dimensie met behulp van isoelektriese
fokusering, gevolg deur skeiding in die tweede dimensie met behulp
van twee dimensionele gel elektroforese. Die PDQuest sagteware program is
gebruik om proteïenkolle te identifiseer wat statisties beduidende verskille toon.
Proteïenkolle van belang is uitgesny en onderwerp aan in-gel tripsinering en die
peptiede wat sodoende verkry is, is deur middel van massa spektrometrie
geanaliseer. Proteïene is geïdentifiseer deur Mascot en die Swiss Prot databasis. Resultate: Westelike klad analise het aangetoon dat die RISK pad en p38 MAPK
geaktiveer is tydens vroeë herperfusie, maar die aktivering word nie volgehou
tydens die hele herperfusie periode nie. Outofagie word gestimuleer tydens die
vroeë herperfusie fase; dit word onderdruk in die middel herperfusie fase en
bereik ‘n tweede piek van stimulering in die laat herperfusie fase. Die
proteomiese analise het onderskeidelik twintig differensieel gereguleerde
mitokondriale proteïene en ses en dertig differensieel gereguleerde sitosoliese
proteïene geïdentifiseer tussen Non-IPK en IPK. Die grootste persentasie van
hierdie proteïene is direk betrokke by miokardiale energie metabolisme. CAMKII
is geidentifiseer as ‘n unieke merker van funksionele herstel in IPK tydens
reperfusie. Gevolgtrekking: Aktivering van die meeste van die proteïenkinases wat
ondersoek is in die huidige studie, is geassosieer met die hemodinamiese
parameters van die hart, in plaas van funksionele herstel. Die resultate het
aangetoon dat die varierende patrone van kinase aktivering toegeskryf kan word
word aan verskille in harttempo en die effek daarvan (ejeksie fraksie, minimum
en maksimum tempo van kontraksie), as gevolg van simpatiese stimulasie
toegeskryf aan sielkundige stres in die diere voor slagting. Proteomiese analise
het getoon dat IPK harte wat faal na isgemie/reperfusie metabolies
gekompromiseer is en “slegter daaraan toe” is, in vergelyking met Non-IPK harte.
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The interaction between human spermatozoa and its homologous zona pellucida : scientific advances and clinical significanceOehninger, Sergio C. 12 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2002. / ENGLISH ABSTRACT: Infertility is a very common problem worldwide. Recent data have shown that disorders of the
male represent the most common single defined cause of infertility. This proposal examines
the clinical significance and fundamental physiological aspects of human gamete interaction.
These studies are focused on the assessment of the cellular-molecular mechanisms involved in
human sperm binding to its homologous zona pellucida resulting in the physiologic induction
of the acrosome reaction. We have developed and validated in vitro bioassays that assess
specific steps of the fertilization process that are critical for early embryo development. The
results of our translational research have already had a significant impact on the overall
evaluation of male infertility and on the clinical management of the infertile man in the
assisted reproduction arena. Furthermore, the unveiling of the basic mechanisms involved in
human gamete interaction will ultimately allow for both (i) the development of new male
reproductive diagnostic capabilities and (ii) the design of improved and safer therapies aiding
conception in childless couples suffering from male infertility. / AFRIKAANSE OPSOMMING: Menslike onvrugbaarheid is 'n algemene wêreldwye probleem en onlangse data toon aan dat
die manlike factor die grootste enkel bydraende factor tot hierdie toestand is. Die werk loods
'n intensiewe ondersoek na die kliniese betekenis en basiese fisiologiese aspekte wat 'n rol
tydens spermsel en eisel interaksie speel. Hoofstuk 3 fokus op die sellulêre en molekulêre
meganismes wat betrokke is tydens spermsel en eisel binding wat gevolglik lei tot
akrosoomreaksie van die spermsel. Die werk verteenwoordig die resultate van 10 jaar se
navorsing tussen die kandidaat en die promoter. Dit gee oorsprong aan 'n reeks bio-toetse wat
die bevrugtingsproses koriografiese ontleed en verskaf dus 'n stap-vir-stap uiteenseting van
menslike bevrugting en gevolglike embrio ontwikkeling. Die resultate in Hoostuk 4 bring
vernuwing in die begrippe van die manlike faktor en die rol in die kinderlose huwelik. Die
resulate soos in Hoofstuk 3 en 4 uiteengesit, vorm nie net die basis vir die moontlike
ontwikkeling van nuwe diagnostiese benaderings tot die hantering van die man nies maar
speel oojk 'n rol die daarstelling van verbeterde terapeutiese hantering van die kinderlose
egpaar. Hoofstuk 5 gee kortliks riglyne en aanbevelings tot opsigte van die gebruik van die
spermsel-zona pellucida bindingstoets en akrosomreaksie. Die kandidaat bevel aan dat die
genoemde twee bio-toetse deel van die laboratorium ondersoeke van die man gebruik moet
word.
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Hypothalamic-pituitary-adrenal axis suppression in asthmatic children on corticosteroidsZollner, Ekkehard Werner Arthur 12 1900 (has links)
Thesis (PhD)-- Stellenbosch University, 2013. / ENGLISH ABSTRACT: Although the effect of inhaled corticosteroids (ICS) on the hypothalamic- pituitary-adrenal
axis (HPA) has been regarded as a “benign physiological response”, a survey published in
2002 suggested that adrenal crisis is more common in asthmatic children on ICS than
previously thought. Relying on clinical features to detect chronic adrenal insufficiency
secondary to corticosteroids may not be wise, as these are non-specific and can therefore
easily be missed. Accurate biochemical assessment of the whole axis to detect subclinical
HPA suppression (HPAS) is thus desirable. A review of the literature indicates that basal
adrenal function tests, including plasma cortisol profiles, do not identify which children can
appropriately respond to stress. There is no evidence to suggest that the degree of the
physiological adjustment of the HPA to ICS and/or nasal steroids (by reducing basal cortisol
production), predicts HPAS. Cortisol profiles should therefore only be used to demonstrate
differences in systemic activity of various ICS and delivery devices. Only two tests,
considered as gold standard adrenal function tests [the insulin tolerance test (ITT) and the
metyrapone test] can assess the integrity of the whole axis. / AFRIKAANSE OPSOMMING: Die outeurs van ´n opname wat in 2002 gepubliseer is stel voor dat ´n bynierkrisis meer
algemeen by asmatiese kinders, wat inhalasie kortikosteroïede ontvang, voorkom as wat
voorheen gedink is. Dit is strydig met die gevestigde opvatting dat die effek van IKS op die
hipotalamiese-hipofise-bynier-as (HHB) ’n “goedaardige fisiologiese reaksie” is. Die kliniese
kenmerke van kroniese bynierontoereikendheid sekondêr tot die gebruik van kortikosteroïede
(KS) is nie-spesifiek en gevolglik onbetroubaar. ´n Akkurate biochemiese toets van
subkliniese HBB onderdrukking (HHBO) sou gevolglik waardevol wees. ´n Literatuur oorsig
toon dat toetse van basale bynierfunksie, insluitend plasma kortisol (K) profiele, nie kinders
uitken wat toepaslik op stres sal reageer nie. Daar is geen bewyse dat die graad van
fisiologiese aanpassing van die HHB, soos aangedui deur laer K-vlakke, na die gebruik van
IKS en/of nasale steroïede (NS), HHBO voorspel nie. Serum K profiele is dus slegs van
waarde om die sistemiese aktiwiteit van verskillende IKS en toedieningsstelsels te ondersoek.
Slegs twee toetse, naamlik die insulien toleransie toets (ITT) en die metyrapone -(MTP)-toets
(wat beide as die goue standaard van bynier funksie beskou word), kan die integriteit van die
hele as meet. / Stellenbosch University / Medical Research Council / SA Thoracic Society / Harry Crossley Foundation / Red Cross Children’s Hospital.
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Toll-like receptor genes and their pathway : role in susceptibility to pulmonary tuberculosis in a South African populationLucas, Lance Andrew 03 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: The communicable disease tuberculosis (TB) is responsible for millions of deaths each year, on a
global scale. At present the contribution of host genetics in TB is generally accepted and, together
with environmental aspects (e.g. nutrition and crowding) and the causative bacterium,
Mycobacterium tuberculosis (M. tuberculosis); it will possibly have a hand in the outcome of
disease. Clearly, TB is a multifaceted disease and the repercussions for studying genetic
susceptibility are that many genes will potentially be implicated.
To date a variety of genes such as NRAMP1 and HLA have been implicated in influencing the host
response to TB, albeit with varying effects in different populations. Some of the more recently
implicated genes are the pattern recognition receptors, the Toll-like receptors (TLRs). Genetic
variation in theses genes has been associated with a myriad of different diseases, including those of
an infectious nature, such as TB. In the case of TB, TLR2 is the most prominent candidate with
TLR8 and 9 more recently implicated. One of the more well known genes implicated with TB is the
vitamin D receptor (VDR), as the antimicrobial gene cathelicidin (CAMP), one of the most
important agents of mycobacterial killing, has a VDR response element in its promoter. TLR2, VDR
and CAMP are all connected in a complex pathway essential for the host defence against M.
tuberculosis.
Nine single nucleotide polymorphisms (SNPs) in three TLR genes (TLR2,8 & 9) were investigated
via a case-control approach to determine their potential role in human genetic susceptiblity to TB in
the Coloured population of South Africa. The effect of the VDR polymorphism Cdx2 on the
expression of cathelicidin mRNA and protein expression was also investigated.
Three genes were found to contribute significantly to genetic host susceptibility in the Coloured
population of South Africa. An allelic association (p = 0.031) was observed for the TLR8 (located
on the X-chromosome) SNP rs3761624, with the A-allele being more prominent in females. Four
haplotypes of TLR8 were found to be significantly linked to TB susceptibility with the three SNP
haplotype rs3761624-rs3764879-rs3764880, specifically the allelic combination of G/C/A [p =
0.004, OR = 2.67(95% CI: 1.90-3.74)], showing a marked association (p = 0.001). The TLR9 introexon2
boundary SNP rs352139 was significantly associated with TB susceptiblity on a genotypic (P
= 0.02) and allelic scale [p = 0.05, OR=0.70; (95% CI: 0.55–0.90)], with the T allele more frequent in controls. The TLR9 two SNP haplotype consisting of rs5743836 and rs352139 was linked (p =
0.037) to TB susceptibility, specifically the combination of the alleles A/T [p = 0.013, OR=0.71;
(95% CI: 0.55–0.92)]. No gene-gene interaction between TLR2, TLR8 and TLR9 was observed. No
significant conclusions could be drawn from the analysis of the mRNA and protein expression of
CAMP in samples harbouring the different genotypes of the VDR polymorphism Cdx2.
Genetic variations in the TLR8 and 9 genes were identified as potential factors that influence
genetic host susceptibility to tuberculosis in the Coloured population of South Africa. / AFRIKAANSE OPSOMMING: Die oordragbare siekte tuberkulose (TB) is elke jaar verantwoordelik vir miljoene sterftes
wêreldwyd. Die invloed van die gasheer genoom op TB vatbaarheid word huidiglik aanvaar,
tesame met die invloed van omgewingsfaktore (dieet, oorbevolking ens.) en die bakterium
Mycobacterium tuberculosis (M. tuberculosis). Dit is duidelik dat TB ‘n veelvlakkigesiekte is wat
beïnvloed sal word deur ‘n menigte verskillende gene.
‘n Verskeidenheid gene is al betrek by TB genetiese vatbaarheid, onder andere NRAMP1 en HLA,
hoewel hul effekte in uiteenlopende bevolkings verskil. Sommige van die onlangse gene wat betrek
is in TB genetiese vatbaarheid is die Toll-like reseptore (TLRs). Genetiese variasie in hierdie gene
is geassosieer met ‘n wye verskeidenheid van siektes, insluitend aansteeklik van aard, onder andere
TB. In die geval van TB speel TLR2 ‘n prominente rol, terwyl TLR8 en TLR9 meer onlangs
geïmpliseer is. Een van die meer bestudeerde TB vatbaarheidsgene is die vitamiene D reseptor
geen (VDR). VDR is direk betrokke in die uitdrukking van die anti-mikrobiale geen cathelicidin
(CAMP),’n integrale komponent in die vernietiging van mikobakterieë. Die CAMP geen het ‘n
VDR respons-element in sy promotor. Die TLR2, VDR en CAMP gene word verbind deur ‘n
komplekse netwerk wat integraal is tot die liggaam se vermoë om TB af te weer.
Nege enkel nukleotied polimorfismes (ENPs) in drie gene (TLR2,8 & 9) is vir hierdie studie
ondersoek, deur gebruik te maak van ‘n pasiënt-kontrole assosiasiestudies, om te bepaal watter rol
hul speel in genetiese vatbaarheid vir TB in die Kleurling bevoling van Suid-Afrika, al dan nie. Die
invloed van die VDR polimorfisme Cdx2 op die uitdrukking van die mRNS (boodskapper
ribonukleïensuur) en proteïen van die geen CAMP is ook ondersoek.
Ons het gevind dat drie gene beduidend bygedra het tot genetiese vatbaarheid vir TB in die
Kleurling populasie. ‘n Alleel verwante assosiasie (p = 0.031) was gevind vir die TLR8 SNP
rs3761624, waar die A-alleel meer algemeen was in vroue. Vier haplotipes vir TLR8 het
beduidende assosiasies met TB vatbaarheid getoon. Die drie SNP haplotipe rs3761624-rs3764879-
rs3764880, spesifiek die alleel kombinasie C/G/A [p = 0.004, OR = 2.67(95% CI: 1.90-3.74)] het
sterk assosiasie (p = 0.001) met TB getoon. Die TLR9 intron-ekson2 grens SNP, rs352139 het
beduidende assosiasie met TB getoon op ‘n genotipiese (p = 0.02) sowel as alleliese skaal [p =
0.05, OR=0.70; (95% CI: 0.55–0.90)], met die T alleel meer algemeen in kontroles. Die twee SNP haplotipe bestaande uit rs5743836 en rs352139 het TB vatbaarheid beïnvloed (p = 0.037), spesifiek
die alleliese kombinasie van A/T [p = 0.013, OR=0.71; (95% CI: 0.55–0.92)]. Geen
noemenswaardige interaksies tussen TLR2, 8 en 9 is gevind nie. So ook is geen beduidende
resultate gevind vir die effek van die VDR SNP Cdx2 op die uitdrukking van CAMP mRNS en
proteïen nie.
Genetiese variasie in die TLR8 en 9 gene is geïdentifiseer as moontlike faktore wat gasheer
genetiese vatbaarheid vir TB in die Kleurlingbevolking van Suid-Afrika beïnvloed. / WW Roome Trust
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Genetic studies on susceptibility to pulmonary tuberculosis mediated by MARCO, SP-D and CD14 : molecules affecting uptake of mycobacterium tuberculosis into macrophagesWagman, Chandre K. 03 1900 (has links)
Thesis (MScMedSc)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: South Africa is ranked amongst the top tuberculosis (TB) burden countries in the world and
the Western Cape has a particularly high incidence of the disease. Previous studies have
showed that several genes may play crucial roles in susceptibility to TB. In this study, we
investigated the role of three genes previously associated with susceptibility to TB and
progression to disease. These genes were Surfactant protein D (SFTPD), Macrophage
receptor with collagenous structure (MARCO) and CD14. The proteins from these genes bind
M. tuberculosis and are involved in the uptake of the bacteria into macrophages.
The study investigated the role of ten polymorphisms from SFTPD and MARCO within a
South African Coloured (SAC) population, where tuberculosis is highly prevalent. A casecontrol
study design was used and polymorphisms were genotyped with Taqman®
genotyping assays and amplification refractory mutation system polymerase chain reaction
(ARMS-PCR). The results were analysed for association with disease, linkage disequilibrium,
haplotypes and gene-gene interactions. Allele and genotype frequencies were also determined
which allowed for comparisons to other populations.
Five SNPs were associated with TB: two in SFTPD (rs1923537; rs2255326) and three in
MARCO (rs1318645; rs3943679; rs2119112). The associated SNPs were located in regions
other than exons and the effects of polymorphisms in these regions are not well understood
but studies in other genes have shown them to play a functional role. Gene-gene interaction
analysis showed that polymorphisms interacted with each other within and between genes,
illustrating the importance of epistasis and the complexity of the genetic influences on TB.
In addition to the case-control association studies, the role of the rs2569190 promoter SNP in
CD14 was assessed. Gene-expression analysis was conducted with qPCR and a reporter gene
assay and results from both of these approaches showed that individuals with the TT
genotype had a twofold greater expression level than individuals with the CC genotype.
Previously, the TT genotype has been associated with stronger promoter activity and
expression of soluble CD14 in serum. Since the TT genotype was present at a higher
frequency in the control group, we speculate that greater expression of CD14 may contribute
to a more TB resistant phenotype. The work presented in this study illustrates the importance of the host genetic component of
TB. Genetic studies will eventually revolutionize the current treatment regime as the
identification of vulnerable individuals and populations will aid in the development of
personalised medicines. / AFRIKAANSE OPSOMMING: Suid-Afrika is een van die top tuberkulose (TB) lande in die wêreld en die Wes-Kaap het
veral ‘n hoë insidensie van die siekte. Vorige studies het gewys dat verskeie gene bydra to die
vatbaarheid vir tuberkulose. In hierdie studie het ons drie gene, wat voorheen vir vatbaarheid
vir tuberkulose en progressie na die siekte ondersoek is, bestudeer. Hierdie gene is Surfactant
protein D (SFTPD), Macrophage receptor with collagenous structure (MARCO) en CD14.
Die proteïene van hierdie gene bind M. tuberculosis en is betrokke in die opname van die
bakterieë in die makrofages.
Hierdie studie het tien polimorfismes van SFTPD en MARCO in die Suid-Afrikaanse
Kleurlingbevolking (SAK), wat ‘n hoë TB insidensie het, getoets. Pasiënt-kontrole assosiasie
studies is gedoen en polimorfismes is gegenotipeer met Taqman® genotiperingsisteem en die
amplifikasie refraktoriese mutasie sisteem polimerase ketting reaksie (ARMS-PCR). Die
resultate is geanaliseer vir assosiasies met TB, koppelings disekwilibrium, haplotipes en
geen-geen interaksies. Alleel en genotype frekwensies is ook bepaal en vergelyk met die van
ander bevolkings.
Vyf enkel nukleotied polimorfismes (ENPs) is met TB geassosieer: twee in SFTPD
(rs1923537; rs2255326) en drie in MARCO (rs1318645; rs3943679; rs2119112). Die
geassosieerde ENPs was nie in eksons nie. Die effek van polimorfismes in areas anders as
eksons word nie goed verstaan nie, maar studies het bewys dat hulle wel ‘n funksionele rol
kan hê. Geen-geen interaksie analise het gewys dat polimorfismes interaksies met mekaar
binne sowel as tussen gene gehad het, wat die belangrikheid van epistase en die kompleksiteit
van genetiese invloede op TB illustreer.
Tesame met die pasiënt-kontrole assosiasie studies is die rol van die rs2569190 promoter
ENP in CD14 ook ondersoek. Geenuitdrukkingsanalise is gedoen met qPKR en
rapporteerder geen toetse. Die resultate van beide hierdie benaderings het gewys dat
individue met die TT genotipe twee keer soveel uitdrukkingsvlakke gehad het as individue
met die CC genotipe. Die TT genotipe is voorheen geassosieer met sterk promoter aktiwiteit
en die uitdrukking van oplosbare CD14 in serum. Aangesien die TT genotipe meer in die kontrolegroep gevind is, spekuleer ons dat die hoër uitdrukking van CD14 kan bydra tot ‘n
meer TB weerstandbiedende fenotipe.
Hierdie werk illustreer die belangrikheid van die gasheer genetiese komponent in TB.
Genetiese studies sal in die toekoms die huidige behandeling regime revolusioneer, aangesien
die identifikasie van individue en bevolkings met ‘n hoë risiko om TB te ontwikkel sal bydra
tot die ontwikkeling van persoonlike medisynes. / The National Research Foundation (NRF); the South African Medical Research Council
(MRC); Stellenbosch University and the Harry Crossley Foundation.
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The impact of the steroid hormones medroxyprogesterone acetate, cortisol and progesterone on protective immunity to tuberculosisKleynhans, Leanie 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2012. / Bibliography / ENGLISH ABSTRACT: Most individuals latently infected with Mycobacterium tuberculosis (Mtb) contain the infection by a
balance of effector and regulatory immune responses. However, this balance can be influenced by
steroid hormones such as glucocorticoids (GCs), which are known to increase the risk of reactivation of
TB. The contraceptive medroxyprogesterone acetate (MPA), which also possesses selective
glucocorticoid activity, is widely used in developing countries with approximately 60% of women on
contraceptives using MPA in our study cohort. Therefore, our aim was to investigate the effect of this
hormone on protective immune responses to BCG in HIV negative household contacts of active TB
patients. When PBMCs of TB household contacts were stimulated with BCG in the presence of 10 μM
MPA; this hormone displayed both glucocorticoid as well as progestogenic properties. Similarly to
cortisol, MPA suppressed antigen specific expression of a range of cytokines including IL-1α, IL-1ra, IL-
17, TNFα, IL-5 and IFNγ. Dose response curves showed that MPA can also alter expression of some
cytokines at lower contraceptive doses (in the nano molar range). To assess whether this effect of MPA
in vitro also occurs in women using this hormone as contraceptive the PBMCs of MPA users and controls
were stimulated with BCG and the levels of up to 29 different cytokines measured by luminex analysis.
PBMCs of MPA users produced significantly lower levels of cytokines involved in immune responses
against Mtb such as IL-12p40, IL-1α, IL-10, IL-13 and G-CSF, which corresponds with lower numbers of
circulating monocytes observed in these women. These findings warrant further investigation and clinical
trials should investigate the risk of progression from latent to active TB disease in women using this
contraceptive. These trials, however, require a large number of participants and are prohibitively
expensive; therefore it was decided to setup an Mtb/MPA mouse model to determine the effect of MPA on
the disease outcome. BALB/c and C57BL/6 mice were injected with a weekly dose of one mg MPA or
PBS and infected with 30 colony forming units of Mtb H37Rv one week after commencing the hormonal
treatment. Both strains were included to establish which strain best represents the human model. Three
and eight weeks post infection the MPA treated C57BL/6 mice had a significantly higher bacterial load in
their lungs compared to untreated mice, whereas no difference was found in the bacterial loads of the
BALB/c mice. MPA treated C57BL/6 mice had significantly lower serum levels of IL-10 and G-CSF and
MPA treated BALB/c mice lower serum levels of IFNγ, when compared to untreated mice. Furthermore,
cells isolated from the MLNs of MPA treated C57BL/6 mice, produced significantly less TNFα, significantly
more IP-10 and less IL-10 in response to PPD, while MLN cells of MPA treated BALB/c mice produced
significantly less IFNγ, IL-2, IL-17, GM-CSF and MCP-1. Data of the C57BL/6 mouse strain correlated
with our human data and can it therefore be said that the C57BL/6 mouse strain, together with the serum
concentration of MPA used in these experiments, is a good model to determine the effect of MPA in the
context of a low dose Mtb infection. To conclude MPA use could therefore alter susceptibility to TB, TB
disease severity as well as change the efficacy of new BCG-based vaccines, especially prime-boost
vaccine strategies which may be administered to adult of adolescent women in the future. / AFRIKAANSE OPSOMMING: Die meeste mense wat latent met Mycobacterium tuberculosis (Mtb) geïnfekteer is, hou die infeksie onder
beheer deur ʼn balans te handhaaf tussen effektor en regulatoriese immuunresponse. Hierdie balans kan
egter beïnvloed word deur steroïedhormone soos glukokortikoïede (GCs), wat bewys is om die risiko van
die heraktivering van TB te verhoog. Die voorbehoedmiddel medroksiprogesteroon-asetaat (MPA), wat
ook selektiewe glukokortikoïed-aktiwiteit toon, word wyd gebruik in ontwikkelende lande en omtrent 60%
van die vrouens in ons studie-bevolking wat voorbehoedmiddels gebruik, gebruik MPA. Om dié rede wou
ons die effek van hierdie hormoon op die beskermende immuun-response teenoor M.bovis Bacilli
Calmette-Guérin (BCG) in HIV negatiewe huishoudelike kontakte (HHKe) van pasiënte met aktiewe TB
ondersoek. Ons het gevind dat wanneer perifere bloed mononukleêre selle (PBMSe) met BCG
gestimuleer word in die teenwoordigheid van 10 μM MPA, hierdie hormoon beide glukokortikoïede en
progesterogeniese eienskappe toon. Soos kortisol het MPA die antigeenspesifieke-uitdrukking van ʼn
reeks sitokiene, insluitend IL-1α, IL-1ra, IL-17, TNFα, IL-5 en IFNγ, onderdruk. Respons kurwes wat
verskillende konsentrasies van hormoon insluit, het getoon dat MPA ook by laer (nano-molare) dosisse
die uitdrukking van sommige sitokiene kon verander. Om te bepaal of hierdie in vitro effek van MPA ook
in vrouens wat MPA as voorbehoedmiddel gebruik voorkom, het ons PBMSe van MPA-gebruikers and
kontroles met BCG gestimuleer en die vlakke van tot 29 verskillende sitokiene met behulp van Luminexanalise
gemeet. PBMSe van MPA-gebruikers produseer beduidende laer vlakke van IL-12p40, IL-1α, IL-
10, IL-13 en G-CSF, wat elk in imuunafweerreaksies teen Mtb betrokke is. Die afname in dié sitokiene
het gepaard gegaan met laer hoeveelhede sirkulerende monosiete. Ons resultate regverdig verdere
ondersoeke en kliniese proewe behoort die risiko van progressie vanaf latente tot aktiewe TB in vrouens
wat hierdie voorbehoedmiddel gebruik te bepaal. Sulke proewe vereis egter groot getalle deelnemers en
is skrikwekkend duur, om die rede het ons besluit om ʼn Mtb/MPA muis-model op te stel om sodoende die
algehele effek van MPA op die uitkoms van die siekte te bepaal. BALB/c en C57BL/6 muise is met ʼn
weeklikse dosis van een mg MPA of sout oplossing ingespuit en een week na die aanvang van die
hormoon behandeling met 30 kolonie-vormende eenhede Mtb H37Rv geïnfekteer. Beide muis tipes was
ingesluit om sodoende te bepaal watter tipe die mens data die beste verteenwoordig. Drie en agt weke
na die infeksie het die MPA-behandelde C57BL/6 muise ‘n beduidende hoër bakteriële lading in hul longe
gehad as die onbehandelde muise, maar was daar geen verskil in die bakteriële ladings in die longe van
die BALB/c muise nie. MPA-behandelde C57BL/6 muise het beduidende laer serumvlakke van IL-10 en
G-CSF gehad, terwyl MPA-behandelde BALB/c muise laer serumvlakke van IFNγ gehad het. Verder het
ons gevind dat die geisoleerde limfosiete van MPA-behandelde C57BL/6 muise beduidend minder TNFα,
beduidend meer IP-10 en minder IL-10 geproduseer het na stimulasie met PPD, terwyl die limfosiete van
MPA-behandelde BALB/c muise beduidend minder IFNγ, IL-2, IL-17, GM-CSF en MCP-1 geproduseer
het. Data van die C57BL/6 muise stem ooreen met die van ons mens studie en ons kan dus vermeld dat
die C57BL/6 muise, tesame met die spesifieke serumkonsentrasie van MPA wat gebruik is, ʼn goeie
model is om die effek van MPA in die konteks van ʼn lae-dosis Mtb-infeksie te bestudeer. MPA gebruik
kan dus die vatbaarheid vir TB, asook die erns van die siekte verander en kan ook die effektiwiteit van
nuwe BCG-gebaseerde entstowwe, veral prima-hupstoot enstowwe, wat moontlik in die nabye toekoms
vir volwasse en adolessente vroue toegedien kan word, verander.
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Lipoatrophy in HIV-infected children on antiretroviral therapyInnes, Steven Eugene Vere 03 1900 (has links)
Thesis (PhD)--Stellenbosch University, 2013. / Bibliography / ENGLISH ABSTRACT: Introduction:
Lipoatrophy is a common adverse effect of stavudine and this effect is strongly dose-dependent. Stavudine remains the most commonly used paediatric antiretroviral drug in sub-Saharan Africa, yet when the current study began in 2009, the prevalence and severity of lipoatrophy in children on antiretroviral therapy in sub-Saharan Africa had never been studied. The development of lipoatrophy may have serious and far-reaching consequences for patients and their families. The off-label stavudine dosing method, prescribed to children whose caregivers do not have access to a refrigerator, in which the contents of an adult capsule is mixed into tap water, has potential for over-dosing or under-dosing. In addition, children on stavudine continue to be exposed to a disproportionately high dose out of line with the reduced adult dose.
Aims:
1. a) To investigate the prevalence and risk factors for lipoatrophy in HIV-infected children in Southern Africa
b) To identify a simple anthropometric screening tool to detect early lipoatrophy in children
2. To validate the off-label stavudine dosing method prescribed to children whose caregivers do not have access to a refrigerator, with a view to reducing the recommended dose and thereby the side-effects.
Methods:
1. a) We recruited pre-pubertal children on antiretroviral therapy from a family HIV clinic in our facility. Lipoatrophy was identified by two experienced paediatric HIV clinicians using a standardized grading scale. A dietician performed dietary assessment and anthropometric
measurements. Previous antiretroviral exposures were recorded. A subset of recruits received Dual-Energy X-ray Absorbtiometry scanning.
b) Anthropometric measurements in children with and without lipoatrophy were compared using multivariate linear regression adjusting for age and gender. The most discerning anthropometric variables underwent Receiver Operating Characteristic curve analysis to identify the most appropriate diagnostic cut-off.
2. a) Accuracy of the standard off-label stavudine dosing method was investigated using high-performance liquid chromatography to recover active drug from solutions made up using the prescribed method. This was compared to the stated drug content of the capsules.
b) Bioavailability was investigated by performing a randomized crossover pharmacokinetic study wherein healthy HIV-seronegative adult volunteers received one of two generic stavudine capsule formulations, either intact or mixed in water using the prescribed method. Plasma stavudine concentrations were assayed by liquid chromatography tandem mass spectrometry.
Results:
1. a) Prevalence of lipoatrophy was 36%, and incidence was 12% per person-year. Adjusted odds ratio for developing lipoatrophy was 1.9 (CI: 1.3–2.9) for each additional year of accumulated exposure to standard-dose stavudine.
b) Baseline biceps skin-fold thickness correlated well with maximum lipoatrophy grading score at any site, giving a partial correlation coefficient of 0.33 (p=0.0006), and a receiver operating characteristic area-under-curve value of 0.75 (CI: 0.64 – 0.84). Biceps skin-fold thickness <5mm at baseline had a sensitivity of 89% (CI: 67–100%) and a negative predictive value of 97% (CI: 91–100%) for predicting which children would go on to develop lipoatrophy by 15 month follow-up. Specificity was 60% (CI: 46–75%) and positive predictive value was 32% (CI: 14–50%).
2. a) Recovery of active drug from solution was 97.1%, 97.4% and 93.8% for the proprietary and two generic formulations respectively.
b) Pharmacokinetic parameters of the off-label dosing method were well within the target range of intact capsule dosing for both generics.
Conclusions:
1. a) The prevalence and incidence of lipoatrophy in pre-pubertal children on antiretroviral therapy in South Africa is high. Cumulative exposure to standard-dose stavudine was the greatest risk factor for lipoatrophy.
b) Biceps skin-fold thickness provided reasonable sensitivity and specificity to detect and predict lipoatrophy in pre-pubertal children on antiretroviral therapy.
2. The off-label dosing method for stavudine prescribed to children whose caregivers do not have access to a refrigerator is reasonably accurate and is bioequivalent to intact capsule administration. / AFRIKAANSE OPSOMMING: Inleiding:
Lipoatrofie is 'n algemene nadelige uitwerking van stavudien en hierdie effek is sterk dosis-afhanklike. Stavudien bly die mees algemeen gebruikte paediatriese antiretrovirale medikasie in sub-Sahara Afrika, maar toe ons studie begin het, was lipoatrofie in kinders op antiretrovirale terapie in sub-Sahara Afrika nog nooit voorheen bestudeer nie. Die ontwikkeling van lipoatrofie kan ernstige en verreikende gevolge vir die pasiënt en hul familie hê. Die af-etiket stavudien dosering metode voorgeskryf aan kinders wie se versorgers nie toegang tot 'n yskas het nie het 'n aansienlike potensiäal vir oor-dosering of onder-dosering. Daarbenewens, is kinders op stavudien blootgestel aan 'n disproporsionele hoë dosis uit-pas met die verminderde volwasse dosis.
Doelwitte:
1. a) Om ondersoek in te stel na die voorkoms en risiko faktore vir lipoatrofie in MIV-geïnfekteerde kinders in Suid Afrika
b) Om 'n eenvoudige antropometriese instrument te identifiseer om vroeë lipoatrofie op te spoor in kinders op antiretrovirale medikasie
2. Om die af-etiket stavudien dosering metode wat voorgeskryf is aan kinders wie se versorgers nie toegang tot 'n yskas het nie te valideer, met 'n oog op die vermindering van die aanbevole dosis
Metodes:
1. a) Ons het 'n groep van onder-puberteitsjarige kinders op antiretrovirale terapie gewerf uit 'n familie MIV kliniek in ons fasiliteit. Lipoatrofie is geïdentifiseer deur twee ervare MIV pediaters deur gebruik van 'n gestandaardiseerde gradering skaal. 'n Diëetkundige het diëet assessering en
antropometriese metings uitgevoer. Vorige antiretrovirale blootstellings is aangeteken. In 'n subset was Dual-energie X-straal Absorbtiometry (DXA) skandering uitgevoer.
b) Antropometriese metings in kinders met en sonder lipoatrofie is vergelyk met behulp van meerveranderlike lineêre regressie aangepas vir ouderdom en geslag. Die mees kieskeurige antropometriese veranderlikes het Receiver Operating Curve analise ondergaan om die mees geskikte diagnostiese afgesnypunt te identifiseer.
2. a) Akkuraatheid is ondersoek deur gebruik te maak van hoë werkverrigting vloeistofchromatografie om aktiewe medikasie vanuit oplossings te herstel, wat gemeng is soos aangedui deur die voorgeskrewe af-etiket dosering metode.
b) Biobeskikbaarheid is ondersoek deur die uitvoering van 'n ewekansige oorgesteekde farmakokinetiese studie waarin gesonde MIV- negatiewe volwasse vrywilligers een van twee generiese stavudien kapsule formulerings ontvang het, óf heel of in water gemeng soos aangedui deur die voorgeskrewe af-etiket dosering metode. Plasma stavudien konsentrasies is gemeet deur vloeistofchromatografie tandem massaspektrometrie.
Uitslae:
1. a) Voorkoms van lipoatrofie was 36%, en insidensie was 12% per persoon-jaar. Aangepaste Odds ratio vir die ontwikkeling van lipoatrofie was 1,9 (CI: 1,3-2,9) vir elke addisionele jaar van opgehoopte blootstelling aan standaard dosis stavudien.
b) Biceps vel-vou dikte <5mm het 'n sensitiwiteit van 89% (CI: 83-96%) en 'n negatiewe voorspellende waarde van 90% (CI: 84-96%) vir die opsporing en voorspelling van lipoatrofie.
2. a) Herwinning van aktiewe medikasie uit oplossings was 97,1%, 97,4% en 93,8% vir die oorspronklike en twee generiese formulerings onderskeidelik.
b) Farmakokinetiese parameters van die af-etiket dosering metode was wel binne die teikenband van ongeskonde kapsule dosering vir beide generiese formulerings.
Gevolgtrekkings:
1. a) Die voorkoms van lipoatrofie in onder-puberteitsjarige kinders op antiretrovirale terapie in Suid-Afrika is hoog. Die bedrag stavudien waaraan kinders blootgestel is moet hersien word. Die standaard stavudien dosis vir kinders moet herge-evalueer word.
b) Biceps vel-vou dikte het redelike goeie sensitiwiteit en spesifisiteit om lipoatrofie op te spoor en te voorspel.
2. Die af-etiket dosering metode vir stavudien voorgeskryf aan kinders wie se versorgers nie toegang tot 'n yskas het nie is redelik akkuraat en is bio-ekwivalent aan ongeskonde kapsule administrasie.
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Possible mechanisms for levosimendaninduced cardioprotectionGenis, Amanda 12 1900 (has links)
Thesis (MScMedSc (Biomedical Sciences. Medical Physiology))--Stellenbosch University, 2008. / Background and purpose. To limit ischaemic injury, rapid restoration of coronary
blood flow is required, which will in turn reduce infarct size. However, reperfusion
itself causes myocyte death – a phenomenon termed lethal reperfusion-induced
injury, which limits protection of the ischaemic myocardium. Thus the reperfusion
of irreversibly damaged myocytes may accelerate the process of cell necrosis.
Additive protection of the ischaemic myocardium in the form of adjunct therapy
remains a topic of intensive research. Levosimendan, a calcium sensitizing agent
with positive inotropic effects has in several studies been found to alleviate the
damaging effects of reperfusion injury. Levosimendan has been shown to be a
KATP channel opener. These channels have been implicated to play an important
role in ischaemic preconditioning (IPC). With this knowledge, the aim of this study
was to determine whether levosimendan and IPC have certain cardioprotective
mechanisms in common and whether protection with pharmacological
preconditioning could be elicited with levosimendan. In this study, we investigated
whether: 1) the isolated guinea pig heart could be protected by ischaemic
preconditioning (IPC) and postconditioning (IPostC), 2) the heart could be
pharmacologically pre- and postconditioned, using levosimendan (LPC & LPostC),
3) a combination of IPC & LPC had an additive protective effect on the heart, 4)
the KATP (both mitochondrial and sarcolemmal) channels are involved in this
protection and 5) the pro-survival kinases of the RISK (reperfusion injury salvage
kinase) pathway are involved.
Experimental approach. Isolated perfused guinea pig hearts were subjected to
three different IPC protocols (1x5, 2x5 and 3x5 minutes of ischaemia) or
levosimendan (0.1μM) preconditioning, before coronary artery occlusion (CAO –
40min@36.5ºC), followed by 30 minutes of reperfusion. Hearts were also
subjected to a combination of IPC & LPC, to establish whether they had additive
protective effects. In addition, hearts were pre-treated with levosimendan directly
before induction of sustained ischaemia (without washout of the drug –
levosimendan pre-treatment (LPT)) for 10min. With the postconditioning protocol,
iii
the hearts were subjected to 3x30second cycles of ischaemia/reperfusion or
levosimendan/vehicle. In a separate series of experiments, hearts were treated
with KATP channel blockers (for both sarcolemmal & mitochondrial), before LPC,
LPT and LPostC. The endpoints that were measured were: cardiac reperfusion
function, myocardial infarct size and RISK pathway expression and
phosphorylation (PKB/Akt and extracellular signal-regulated kinase – ERK42/44).
Results. IPC, IPostC, LPC & LPostC decreased myocardial infarct size
significantly compared with their controls (21.9±2.2%, 21.4±2.2%, 20.6±3.1% and
20.6±1.8% respectively vs. 46.4±1.8% for controls, p<0.05). The combination of
IPC & LPC had no additive protective effect. Pre-treating the hearts with
levosimendan (without washout), before index ischaemia, proved to be the most
effective method of cardioprotection (infarct size: 5.8±0.9% vs. 46.4±1.8% for
controls, p<0.001). With LPT a significant increase (p < 0.05 vs. control) in
phosphorylation of ER42/44 was also observed. An increase in the activity of one
of the RISK pathway kinases, ERK42/44 seems to be one of the reasons for LPT’s
efficacy. Treating the hearts with KATP channel blockers before subjecting them to
LPC, LPT & LPostC abolished the protective effects induced by levosimendan,
suggesting a role for the sarcolemmal and mitochondrial KATP channels in
levosimendan-induced cardioprotection.
Conclusions and implications. 1) Isolated guinea pig hearts could be pre- and
postconditioned within the setting of ischaemia, 2) Hearts could be
pharmacologically pre- and postconditioned with levosimendan, 3) levosimendan
pre-treatment is the most effective way to reduce infarct size, possibly acting by
increasing the phosphorylation of ERK42/44, 4) Myocardial protection was not
increased by combining IPC & LPC (suggesting similar mechanisms of protection),
5) LPC, LPT and LPostC were abolished by both sarcolemmal and mitochondrial
KATP channel blockers.
.LPC and especially LPT, could be useful before elective cardiac surgery while
LPostC may be considered after acute coronary artery events.
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