Application of Becton Dickinson FACSTM Combinatorial Antibody Profile (FACSTM CAP) technology to the identification of efficiency of tuberculosis therapy

Smith, Bronwyn Kerry (2015-03)

Thesis (MSc)--Stellenbosch University, 2015

Thesis

ENGLISH ABSTRACT : Currently the treatment of individuals with active Mycobacterium tuberculosis (Mtb) infection involves a standard six-month multi-drug regimen, impacting negatively on treatment adherence, which in turn fuels multi- and extensive drug resistant TB. However, some patients may not require the full six-month regimen due to less extensive disease or rapid early treatment response. The identification of these patients has been problematic but would allow significant cost savings and may impact positively on treatment adherence if treatment duration could be shortened and if this subgroup constituted a significant portion of patients. The aim of this project was to identify peripheral blood lymphocyte surface markers through a proprietary technology, FACSTM CAP by Becton Dickinson Technologies, to investigate the change in expression during the course of treatment with potential treatment monitoring utility. Peripheral blood mononuclear cells (PBMCs) were isolated from TB patients (n=33), healthy community controls (n=11) and other lung disease controls (OLD, n=9) at diagnosis of disease, week 4 (after commencement of treatment) and week 24 (end of treatment, EOT). Antibodies to 252 surface markers were used to stain PBMCs, the cells were fixed in 2% paraformaldehyde and data acquired on a FACS Calibur flow cytometer. Post-acquisition compensation and analysis was performed using FlowJo software. The analysis was performed by gating on the lymphocytes and overlaying sample plots on isotype controls. Statistics analysis included repeated measures ANOVA, paired t-test and independent t-test. Comparisons were made between the expression levels of patient time points (diagnosis, week 4 and week 24) and participant groups (TB, healthy community controls and OLD controls). Sample wells that provided an uncertain demarcation of the positive and negative expression population were flagged and excluded from analysis. After the application of the Bonferroni correction, results revealed five overall treatment response markers (CD120b, CD126, CD62L, CD48 and CD29) that were significantly different (p-value <0.0002) when comparing expression levels at TB diagnosis and EOT (week 24) samples. A comparison of expression between TB at diagnosis and healthy community controls showed a significant difference for four markers (CD48, CD18, CD126 and fMLPr). Due to the application of the stringent Bonferroni correction, only these few markers were found to be statistically significant therefore all markers with a p-value <0.01 prior to Bonferroni correction, were included for analysis with Ingenuity Pathway Analysis (IPA) and Qlucore Omics Explorer software. IPA identified 23 biological pathways that were associated with two or more markers with significant changes during treatment. The top nine pathways are discussed and included the inflammatory response, cell migration, differentiation and maturation and crosstalk between cells of the innate and adaptive immune responses. In conclusion, this project resulted in the identification of three promising biologically significant surface markers that require further validation as candidates for biomarkers of TB treatment response. Future studies will investigate the most promising markers, including those that showed a trend for differences after the Bonferroni correction, in a candidate biomarker project with a new cohort of TB patients undergoing treatment.

AFRIKAANSE OPSOMMING : Die roetine behandeling van individue met aktiewe Mikobakterium tuberkulosa (Mtb) infeksie, behels ses maande van multi-middel terapie, ʼn tydperk wat gevolglik negatief impakteer op behandelingsgetrouheid en dus bydra tot multi- en ekstensiewe middelweerstandige TB. Dit mag egter wees dat sommige pasiënte, as gevolg van minder verspreide infeksie of ʼn versnelde reaksie op vroeë behandeling, nie die volle ses maande-lange behandeling benodig nie. Alhoewel die identifikasie van sulke pasiënte problematies is, kan dit beduidende kostebesparings meebring en moontlik ook ʼn positiewe impak op behandelingsgetrouheid hê indien behandelingsduur verkort kan word en indien dié subgroep ʼn beduidende deel van die pasiënte uitmaak. Die doel van die huidige projek was om perifere bloed-limfosiet oppervlaksmerkers te identifiseer met behulp van ʼn patente tegnologie, FACSTM CAP van Becton Dickinson, om sodoende die verandering in merker uitdrukking tydens die verloop van behandeling te ondersoek vir moontlike gebruik as behandelings monitering toepassing. Perifere bloed mononukleêre selle (PBMSe) is geïsoleer van TB pasiënte (n=33), gesonde kontroles (n=11) en kontroles met ander longsiektes (OLD, n=9) tydens diagnose van siekte, week 4 (na begin van behandeling) en week 24 (einde van behandeling, EOT). Teenliggame is gebruik om 252 seloppervlaksmerkers van die PBMSe te merk, die selle is met 2% paraformaldehied gefikseer en die data op ʼn FACS Calibur vloeisitometer verkry. FlowJo sagteware is gebruik vir na-verkryging-kompensasie en analise wat gedoen is deur die limfosiete te selekteer, gevolg deur oorlegging van isotipe-kontroles. Statistiese analises het herhaalde metings-ANOVA, die gepaarde en onafhanklike t-toetse ingesluit. Vergelykings is getref tussen die uitdrukkingsvlakke van verskillende pasiënt-metings (diagnose, 4 weke en 24 weke) en deelnemende groepe (TB, gesonde kontroles en OLD kontroles). Proefdata wat nie tussen die positiewe en negatiewe uitdrukkingspopulasie kon onderskei nie, is van die analise uitgesluit. Na toepassing van die Bonferroni-korreksie het die resultate getoon dat vyf algehele behandelingsrespons-merkers (CD120b, CD126, CD62L, CD48 en CD29) beduidend verskil (p-waarde <0.0002) wanneer die uitdrukkingsvlakke tussen die TB diagnose en EOT (24 weke) tydstip vergelyk is. Vergelyking van merker uitdrukking tussen TB (by diagnose) en gesonde kontroles het ʼn beduidende verskil vir 4 merkers (CD48, CD18, CD126 enfMLPr) aangetoon. Aangesien slegs hierdie merkers statisties beduidend was na toepassing van die streng Bonferroni-korreksie is alle merkers met ʼn p-waarde <0.01 voor Bonferroni-korreksie ingesluit vir analise met Ingenuity Pathway Analysis (IPA) en Qlucore Omics Explorer sagteware. IPA het 23 biologiese paaie geïdentifiseer wat geassosieer is met twee of meer merkers met beduidende veranderinge tydens behandeling. Die belangrikste nege paaie word bespreek en sluit in die inflammatoriese respons, sel-migrasie, -differensiasie, -maturasie en kruiskommunikasie tussen selle van die ingebore en sellulêre immuun sisteme. Om op te som, hierdie projek het drie belowende biologies beduidende oppervlaksmerkers geïdentifiseer wat verdere ondersoek as kandidaatbiomerkers van TB behandelingsrespons, regverdig. Toekomstige studies sal die mees belowende merkers, insluitende daardie wat ʼn tendens in verskille na Bonferroni-korreksie getoon het, navors in ʼn kandidaat-biomerkerprojek met ʼn nuwe populasie TB pasiënte gedurende TB behandeling.

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