Theses and Dissertations (Immunology)

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Investigation of peripheral blood immune cell subsets and cytokine levels in females living with breast cancer before and after first-phase chemotherapy
(University of Pretoria, 2025-01) Worsley, Catherine M.; Meyer, Pieter Willem Adriaan; u19012251@tuks.co.za; Chicken, Nicole Ambrin
Introduction: Breast cancer is the most frequently diagnosed cancer in females in sub-Saharan Africa and up to 80% of cases are stage 3 or 4 tumours. Triple-negative breast cancer (TNBC) accounts for up to 20% of all breast cancer cases and is aggressive, highly metastatic, tends to be diagnosed in younger women, and has a 5-year survival rate of 60%. This breast cancer subtype has limited treatment options due to its hormone insensitivity and heightened proliferative potential. The tumour microenvironment is a key driver of tumourigenesis and consists of different cancer cell clones, normal stromal cells, cytokines, chemokines, growth factors, and immune cells. Manipulation of the tumour microenvironment by cancer cells to promote tumour progression and metastasis has been observed in both breast and other cancer types and affects treatment response and patient prognosis. Immune cells and cytokines within the tumour microenvironment and the circulation may play a pro- or anti-tumourigenic role in TNBC and therefore act as potential surrogate biomarkers. The aim of this study was to compare immune cell subsets and cytokine levels in peripheral blood between healthy donors and TNBC patients, and in patients before and after first-phase (at least 3 cycles) of chemotherapy. Methods: Twenty-three patients recently diagnosed with TNBC and ten healthy donors consented to participate in this study. Patients were recruited from a private clinic in Johannesburg, Gauteng with their consent. Blood was collected prior to treatment and after first-phase chemotherapy. Full blood counts, patient demographics, and treatment response were recorded. The percentage of circulating CD4+ and CD8+ T-cells, B-cells, natural killer (NK) cells, monocytes, and regulatory T-cells (Tregs) as well as the expression of various cell markers was evaluated by flow cytometry and compared between participant groups. The concentrations of vascular endothelial growth factor (VEGF), granulocyte-macrophage colony stimulating factor (GM-CSF), growth and differentiation factor 15 (GDF-15), Interleukin-6, -8, and -10 (IL-6, -8, -10) and interferon gamma-induced protein (IP-10) were evaluated in participant plasma by enzyme-linked immunosorbent assays (ELISAs) and the results were compared between participant groups. These were also compared to treatment outcomes. Results: This study showed an increase in the percentage of CD14+ monocytes and CD56high NK cells, and a decrease in CD19+ B-cells after first-phase chemotherapy, while no significant differences in cell percentages were seen between healthy donors and treatment-naïve patient groups. The expression of CD206 was increased in B-cells and monocytes, while HLA-DR expression was increased on NK cells after first-phase chemotherapy. While little difference in the median Treg percentage was observed between patient groups, changes in the number of Tregs were observed after first-phase treatment when looking at individual patients, suggesting that observations may be patient-specific. An increase in expression of CD39, CD45RA, Helios, and CD127 was observed in Tregs after first-phase treatment. The concentrations of VEGF and GM-CSF were similar between patient and healthy donor groups, while IL-6, -8, and -10, and IP-10 and GDF-15 were increased in the post first-phase treatment plasma. Conclusion: This study provides valuable insights into the dynamic changes in immune cell subsets and cytokine levels in TNBC patients undergoing chemotherapy. The observed alterations in T-cells, B-cells, NK cells, monocytes, and Tregs, along with their associated markers, suggest a complex interplay between the immune system and cancer progression. The increased levels of specific cytokines post-treatment highlight potential biomarkers for monitoring treatment response and disease progression. These findings contribute to our understanding of the immune landscape in TNBC and may inform future strategies for immunotherapy and personalized treatment approaches. Further research is warranted to elucidate the functional implications of these changes and their potential as prognostic indicators or therapeutic targets in TNBC management.
The role of micro-RNA in the diagnosis of HIV-associated immune reconstitution inflammatory syndrome
(University of Pretoria, 2024-12-06) Meyer, Pieter Willem Adriaan; Rossouw, Theresa M.; dvruler@gmail.com; Van Ruler, Divan
Immune Reconstitution Inflammatory Syndrome (IRIS) arises from the restoration of antigen-specific immune responses following antiretroviral therapy (ART) and presents in two forms: paradoxical and unmasking IRIS. Currently, no definitive diagnostic method exists for IRIS, and diagnosis relies on clinical symptoms in the context of immunological and virological responses to ART. This study hypothesized that specific microRNAs (miRNAs) involved in viral control and inflammation could be informative in identifying IRIS. We investigated the expression of 10 candidate miRNAs (miR-155, miR-132a, miR-10a, miR-146a, miR-146b, miR-181a, miR-27a, and miR-375, miR-16-5p, miR-191-5p) in 28 HIV-positive patients before ART, at approximately six months post-ART, and upon development of inflammatory symptoms. Significant differential expression of miR-146a and miR-181a was observed in IRIS patients compared to those without IRIS. Using pre-ART CD4 count and viral load data, in addition to miR-146a and miR-181a expression data, 7 patients who presented with ambiguous cases could be classified into IRIS and non-IRIS groups. Of these 7 patients, 3 were classified as having developed IRIS. These findings suggest that miR-146a and miR-181a may serve as potential biomarkers for diagnosing or predicting IRIS, though further research is required to confirm their clinical utility.
Establishing an in-house real-time polymerase chain reaction assay to quantify T-cell receptor excision circles and kappa-deleting recombination excision circles for use in screening newborn babies for inborn errors of immunity
(University of Pretoria, 2024-12) Kwofie, Luyanda Laura Illicia; Worsley, Catherine M.; Meyer, Pieter Willem Adriaan; sharsingo@gmail.com; Singo, Shudufhadzo Sharon
Inborn errors of immunity (IEI) are genetic disorders that hinder immune responses and are underdiagnosed, particularly in low-to-middle income countries. These disorders occur in one in every 500 live births. Early detection is crucial for preventing health complications. The need for early detection of IEI has led to the development of newborn screening (NBS) programmes; however, many countries lack routine NBS due to financial and technological challenges. The present study aimed to develop an in-house screening technique for T-cell receptor excision circles (TREC) and kappa-deleting recombination excision circles (KREC) quantification using real-time polymerase chain reaction (PCR) to screen for IEI in newborn babies. We developed an in-house real-time PCR technique to detect and quantify TREC and KREC in human immunodeficiency virus (HIV)-exposed uninfected (HEU) and HIV-unexposed uninfected (HUU) neonates, using dried blood spot (DBS) samples collected and preserved as part of the Siyakhula study cohort. Deoxyribonucleic acid (DNA) was extracted from DBS samples, and TREC and KREC genes were simultaneously detected using a newly developed real-time PCR technique. Results revealed that HUU infants had statistically significant higher TREC concentrations (P-value = 0.006) than HEU infants, however, no significant difference in KREC concentrations were noted between the two cohorts. Statistical significance was determined by a P-value <0.05. These results indicate reduced TREC levels in immunologically vulnerable babies (HEU). The TREC and KREC assays showed significant potential as biomarkers for IEI screening. Importantly, the simultaneous detection and quantification of TREC/KREC is a cost-effective method for IEI screening.
Identification of allelic variants implicated in neonatal encephalopathy with suspected hypoxic ischaemic encephalopathy and consequential cerebral palsy in individuals of African origin
(University of Pretoria, 2025-01) Pepper, Michael Sean; Joubert, Fourie; Mellet, Juanita; u15280854@tuks.co.za; Ryder, Megan A. (Ashley); Holborn, Megan A. (Ashley)
Neonatal encephalopathy with suspected hypoxic ischaemic encephalopathy (NESHIE) is a form of brain injury occurring in neonates due to a shortage of blood flow and consequential oxygen delivery to the brain around the time of birth. Cases of NESHIE often result in severe outcomes including permanent neurological disability or death. NESHIE is a significant health issue in Africa with incidences of up to 35.2 cases per 1000 live births reported. Despite the high incidence of NESHIE in Africa, research on the genetics of NESHIE has primarily focused on individuals of European, Asian and Latin American descent. To date, no African-specific genetic studies on NESHIE have been published. To lay a foundation for future African-specific genetic case-control studies on NESHIE, this study aimed to determine the allele frequencies and predicted effects of variants within NESHIE genes of interest in the general African population. As a preliminary step, genetic findings on NESHIE from other global populations were catalogued and used to prioritise genes for further study based on their strength of association with NESHIE and involvement in diseases with similar phenotypes. Following the selection of genes of interest, variants within these genes were identified using African-specific sequencing data from the 1000 Genomes Project and the Human Genome Diversity Project. The frequencies and effects of these variants were then analysed to (i) assess the comparability of genetic findings between African populations and European, Asian and Latin American populations, and (ii) identify variants in the genes of interest present in African populations that may warrant further research on their potential involvement in the genetics of NESHIE due to their known or predicted association with disease. The findings from this study, along with future African-specific research on the genetics of NESHIE, will help determine whether genetic factors contribute to the high incidence of NESHIE in African populations. If such genetic factors are identified, these factors could aid healthcare workers in predicting which neonates are at risk of severe outcomes and facilitate the development of personalised treatment plans.
The haematological profile of a treatment-naïve HIV-positive cohort : a pilot study
(University of Pretoria, 2024) Durandt, Chrisna; Mellet, Juanita; Moodley, Vanessa; tsungaimash@icloud.com; Mashingaidze, Tsungai Vesta
Human immunodeficiency virus (HIV) infection has been observed to significantly impact both the immune system and haematopoiesis. Haematopoiesis is the regulated process of producing the cellular components of blood with the haematopoietic stem and progenitor cells (HSPCs) as the cells of origin. Haematopoiesis occurs mainly in the bone marrow (BM). Haematopoietic stem and progenitor cells are necessary for the maintenance of steady-state haematopoiesis and a fully functional immune system. In HIV-negative individuals, it has been noted that a small proportion of HSPCs consistently migrate from the BM into the peripheral circulation. Under stress conditions such as infection with HIV, steady-state haematopoiesis is disrupted, and downstream HSPC differentiation activity becomes dysregulated. Literature reports that dysregulated HSPC differentiation leads to increased levels of the immunosuppressive myeloid-derived suppressor cells (MDSCs) in HIV infection, with no evidence of MDSCs in healthy individuals. To better understand this disruption, we investigated whether a link exists between the immature circulating HSPCs, heterogeneous MDSCs, and the haematological profile of treatment-naïve HIV-positive and HIV-negative (control) groups, respectively. This was performed using peripheral blood donated by consenting treatment-naïve HIV-positive and HIV-negative (control) participants. The frequency and phenotype of the circulating HSPCs in a treatment-naïve HIV-positive cohort was determined and compared to an HIV-negative (control) cohort using flow cytometry. This study found a significant decrease in circulating HSPCs in the treatment-naïve HIV-positive group in comparison to the HIV-negative (control) group with no difference in the phenotype between the two groups. Furthermore, HIV-mediated disruption of haematopoiesis often results in haematological abnormalities, such as cytopenias. Cytopenias arising from HIV infection are associated with increased morbidity and mortality. A full blood count with a white cell differential was performed to investigate the extent of the disruption. We observed anaemia and thrombocytopenia within the HIV-positive group. Neither leukopenia nor neutropenia were observed in this study. Additionally, significant reductions were observed in the white and red cell counts, haemoglobin, and absolute neutrophil counts among the HIV-positive group, in comparison to the HIV-negative (control) group. The exact mechanism of HIV-mediated cytopenias is not clear, but scientific evidence strongly suggests that HIV disrupts the BM milieu which impacts on the function of HSPCs. Therefore, the functionality of circulating HSPCs were also investigated with the colony forming unit (CFU) assay. The HSPCs differentiated to produce CFU-granulocyte, erythrocytes, monocyte, megakaryocyte (GEMM), CFU-granulocyte, monocyte (GM), and burst forming units-erythrocyte (BFU-E). No phenotypic differences were observed between HIV-positive and HIV-negative (control) cohorts. However, when investigating the impact of HIV on the immune system using flow cytometry, various immune cell populations, including the cluster of differentiation (CD)4+ T cells were significantly decreased in the HIV-positive group when compared to the HIV-negative (control) group. Whereas the CD8+ T cells were significantly increased in the HIV-positive group compared to the HIV-negative (control) group. Furthermore, this study did not observe increased levels of MDSCs in the HIV-positive group as hypothesised. In summary, the study observed decreased frequencies of circulating HSPCs, which is indicative of abnormal haematopoiesis and subsequent BM disruption. The BM disruption trickles down into haematological indices and supports the onset of cytopenias.
Characterisation of umbilical cord blood derived haematopoietic stem and progenitor cells from HIV exposed infants that are HIV negative at birth
(University of Pretoria, 2024-06) Pepper, Michael Sean; Mellet, Juanita; candice_hendricks@outlook.com; Hendricks, Candice Laverne
Umbilical cord blood (UCB) has been used for approximately 30 years as a source of haematopoietic stem and progenitor cells (HSPCs) for haematopoietic stem cell transplantation (HSCT). Advantages include less stringent allele matching requirements and lower immunogenicity compared to other stem cell (SC) sources. The greatest disadvantage is the lower HSPC number leading to slower haematopoietic recovery. This has led to its use primarily in paediatric patients who are able to benefit from the administration of one unit as opposed to two or more units as would be required in adults to achieve similar outcomes. Multiple in vitro HSPC expansion techniques have been used to overcome this barrier and have been shown to decrease time to engraftment. In South Africa (SA) however, many challenges exist in accessing HSCT, which include but are not limited to infrastructure, human resource barriers, SC donors and a high prevalence of human immunodeficiency virus (HIV). There is also no public UCB bank and thus UCB HSCT is rare. The HIV prevalence presents a major challenge as approximately 30-40% of expectant mothers are infected with HIV nationally. With a mother to child transmission (MTCT) rate of 1-2%, the vast majority of infants born to HIV positive mothers, and thus their UCB, are HIV negative. The impact of maternal HIV associated chronic immune activation and antiretroviral (ARV) exposure on the immunophenotype, expansion ability and function of UCB HSPCs from HIV exposed but uninfected infants (HEU infants), and similarities to or differences from HSPCs from HIV unexposed uninfected (HUU) infants, merits investigation. In this study, UCB HSPCs from HIV exposed infants born from virologically suppressed mothers, and who test HIV PCR negative at birth (HEU infants), were characterised by immunophenotype, differentiation capacity, expansion ability, and gene expression, and compared to an HIV negative control group. Immunophenotyping was performed by flow cytometry on freshly isolated CD34+ HSPCs to determine different CD34+ subsets. Expansion was performed using cytokines in the presence or absence of StemRegenin-1 (SR1), to determine the expansion ability of these cells. SR1 is a small-molecule aryl hydrocarbon receptor antagonist that causes an exponential increase in CD34+ HSPCs cultured in vitro. The optimal dose for expansion, as well as confirmation of an increase in the HSPC population, has been determined in our laboratory. Colony forming unit (CFU) assays were used as an indicator of differentiation capacity of freshly isolated CD34+ HSPCs, and the gene expression profile of unexpanded CD34+ cells was determined by microarray analysis. Additionally, the effect of ARVs, individually and in combinations as seen in maternal ARV fixed-drug combination (FDC) tablets, on UCB HSPCs from HUU infants, was determined by assessing impact on immunophenotype, expansion and differentiation capacity. This combined approach has been valuable in portraying differences between CD34+ cells from HEU infants compared to HUU infants, which may have implications for (a) the possible utility of HSPCs from these infants for HSCT, a currently unexplored area meriting research, and (b) the haematological profile of HEU infants.
Investigating the direct infection of ex vivo bone marrow-derived and peripheral blood-derived haematopoietic stem/progenitor cells by HIV-1
(University of Pretoria, 2023) Durandt, Chrisna; Potgieter, Johan J.C.; priyalmistry03@gmail.com; Mistry, Priyal
Human immunodeficiency virus (HIV-1) infection remains a significant global public health concern, particularly in sub-Saharan Africa where the majority of HIV infections are concentrated. HIV-1 is known to target the host immune system. However, HIV-infected patients also often present with haematological abnormalities such as cytopenias. Haematopoietic stem/progenitor cells (HSPCs) give rise to all blood cell types and are crucial for maintaining continuous production of blood cells throughout life via the process of haematopoiesis. HSPCs have been investigated in the context of HIV-1 infection with HIV-1 being suggested to negatively affect the functioning of HSPCs through various mechanisms. This leads to impaired haematopoiesis resulting in the manifestation of HIV-associated cytopenias. While not the primary targets of HIV-1, the direct infection of HSPCs is proposed as one of the direct mechanisms by which HIV-1 interacts with HSPCs thereby disrupting optimal haematopoiesis. Uncertainty surrounds whether HSPCs are susceptible to direct infection by HIV-1 as no consensus has been reached regarding this topic. Moreover, the phenotype of HSPCs that may be prone to HIV-1 infection has not been elucidated. The direct infection of bone marrow (BM)-derived HSPCs was investigated in this project by determining if HSPCs from HIV-infected patients harbour HIV-1 proteins. The phenotypic profile of HSPCs that harboured HIV-1 proteins was also established. Firstly, the HIV-associated receptors, namely cluster of differentiation 4 (CD4), C-X-C motif chemokine receptor 4 (CXCR4) and C-C motif chemokine receptor 5 (CCR5) of BM-derived HSPCs from HIV-positive and HIV-negative controls was investigated as an indicator of their susceptibility to HIV-1 infection. CD4 was expressed by HSPCs that were maturing into haematopoietic progenitors with these cells also co-expressing at least one of the co-receptors (CXCR4 or CCR5) required for viral entry. Secondly, the intracellular HIV-1 p24 expression of BM-derived HSPCs was determined. This was done by performing the HIV-flow assay on HSPCs from the BM aspirates of HIV-positive patients. Intracellular p24 was detected in a subset of haematopoietic progenitors. Findings thus indicated that haematopoietic progenitors, of both the lymphoid and myeloid lineage, are prone to direct HIV-1 infection. In addition, the absolute count and sub-population distribution of HSPCs in the BM aspirates of HIV-positive and HIV-negative patients was also investigated. BM aspirates of HIV-positive patients were observed to have elevated HSPCs counts with a myeloid-bias HSPC sub-population distribution. In conclusion, the present study provides evidence of a plausible mechanism involved in the development of HIV-associated cytopenias. It also sheds light on the HIV-induced changes to HSPCs sub-population distribution in the BM compartment of HIV-infected individuals.
Sodium, potassium adenosine triphosphatase as a potential target of the anti-tuberculosis agents, clofazimine and bedaquiline
(University of Pretoria, 2023) Cholo, Moloko C.; Steel, Helen Carolyn; u11283026@tuks.co.za; Mmakola, Khomotso Madimetsa Shelboy
BACKGROUND: Tuberculosis (TB) is a disease caused by the acid-fast bacterium, Mycobacterium tuberculosis (M. tuberculosis). It is currently the leading cause of morbidity and mortality worldwide due to a single infectious agent. One of the main contributing factors to the burden of the disease is an alarming increase in the number of drug-resistant tuberculosis (DR-TB) cases. Recently, an efficient chemotherapeutic regimen, containing two second-line drugs, namely clofazimine (CFZ) and bedaquiline (BDQ), has been introduced. The use of these antibiotics has been associated with prolongation of the cardiac QT interval (this is measurement of the heart rate shown on the electrocardiogram), which leads to fatal cardiac arrhythmias. However, the mechanism by which these agents cause cardiac arrhythmia remains unknown. In this context, CFZ has been reported to inhibit the activity of the sodium, potassium-adenosine triphosphatase (Na+,K+-ATPase) enzyme in T lymphocytes, while the effects of BDQ on Na+,K+-ATPase have, to date, been unexplored. Importantly, however, both CFZ and BDQ target K+ channels in human mammalian cells including those of the mononuclear leukocytes (MNLs) and cardiac muscle cells (cardiomyocytes: CMs). Despite this, the effect of these two antibiotics on Na+,K+-ATPase of cardiomyocytes has not been described. AIM AND OBJECTIVES: The aim of the current study was to evaluate the effects of CFZ and BDQ on the activity of Na+,K+-ATPase of MNLs and rat cardiomyocytes (RCMs), by determining the activity of Na+,K+-ATPase via inorganic phosphate (Pi) concentration measurements, quantification of intracellular ATP levels and cellular viability. METHODS: The MNLs were isolated from healthy adult volunteers while the RCMs were obtained commercially. The MNLs and RCMs were treated with varying concentrations of CFZ and BDQ individually and in combinations (0.15 - 5 mg/L). Thereafter, Pi concentrations (using a Na+,K+-ATPase activity assay), ATP levels (using a colorimetric ATP assay) and number of viable cells (measured flow cytometrically) were determined. RESULTS: All antibiotic assays demonstrated inhibition in Na+,K+-ATPase activity in a dose-response related manner. Clofazimine was found to have the greatest inhibitory effect on the Na+,K+-ATPase activity in MNLs followed by BDQ while the effect was attenuated when the two antibiotics were used in combination. However, in RCMs, the greatest inhibitory effect of the antibiotics was demonstrated by combinations of the two antibiotics, followed by CFZ and BDQ alone, which however, demonstrated a comparable effect. In both cell lines, the inhibitory effect on Na+,K+-ATPase activity by the antibiotics was associated with an increase in ATP concentrations. Additionally, these effects were followed by a decline in cellular viability in the case of MNLs while a slight increase in cellular viability was observed in the RCMs for all antibiotic treatments. However, there was a decrease in cellular viability of the RCMs when the antibiotics were used in combination at their highest concentration of 5 mg/L. CONCLUSION: The results of the current study illustrate the implication of the Na+,K+-ATPase in the beneficial effects of CFZ and BDQ on the immunomodulatory roles of the MNLs while, importantly, demonstrating the potentially detrimental effects of these antibiotics, especially BDQ and, more notably, when the two antibiotics are used in combination, on the viability of the cardiomyocytes via targeting the Na+,K+-ATPase pump. This may contribute to the risk on individuals administered these drugs of developing cardiac arrhythmia.
The role of Solute Carrier Family 7 Member 8 (SLC7A8) in adipogenesis in vitro and in a murine model of obesity
(University of Pretoria, 2023) Ambele, Melvin; Pepper, Michael Sean; u10228188@tuks.co.za; Pitere, Reabetswe
Obesity is a pandemic affecting both adults and children with an increasing annual prevalence. Adipogenesis, a process in which adipocyte precursors differentiate into mature adipocytes, is considered an important process in identifying molecular determinants that could be targeted to modulate lipid accumulation and adipocyte hypertrophy, thereby combating obesity. Over the past two decades various studies have been conducted to investigate genes that are central to the adipogenesis process. The one limitation has been that most of the studies used animal models and cell lines for this purpose which may possibly be different to how the process is regulated in humans. To overcome this challenge, Ambele et al., 2016 performed an in vitro transcriptome analysis of human adipose-derived stromal cells (ASCs) undergoing adipogenic differentiation. Various genes at various phases of differentiation were identified but one gene of interest was the SLC7A8 which was transiently expressed and was highly upregulated in the early phases of adipogenesis. The SLC7A8 gene encodes LAT2 which is a neutral amino acid transporter. It belongs to a superfamily of proteins that have been implicated in obesity and/or adipogenesis. Since SLC7A8 had not been previously described in the context of adipogenesis and obesity, -it was necessary to elucidate its function in this context using a murine model of diet-induced obesity (DIO). Wildtype and knockout Slc7a8 mice fed on high-fat and control diets were monitored over a 14-week period and various analyses were performed at different time points. Further, human fASCs were differentiated into mature adipocytes in the presence/absence of 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), an inhibitor of LAT2/SLC7A8. The results obtained from the study showed that in conditions of DIO, Slc7a8 knockout mice had significantly reduced weight gain, improved glucose tolerance, reduced inflammation due to macrophage infiltration and decreased adipocyte hypertrophy in different adipose depots when compared with the wildtype. Lipid accumulation in other peripheral non-lipid storage tissues and organs such as the liver was reduced in the knockout model. Further, the possible mechanism of hypotrophy prevention in Slc7a8 knockout mice was investigated by measuring the expression of genes involved in lipid transport and metabolism and the effect on different plasma metabolites. The observations demonstrated that attenuation of adipocyte hypertrophy in knockout mice differed across the adipose tissue depots, i.e., hypotrophy in the perigonadal (pWAT) and brown adipose tissues (BAT) is due to increased lipolysis, in addition to browning (in BAT), with reduced lipid uptake in the mesenteric adipose tissue (mWAT). Adipocyte hypotrophy in Slc7a8 knockout mice resulted to a significantly lower and higher leptin and adiponectin levels, respectively, as well as reduced plasma levels of the proinflammatory cytokines IL-α, IL-6, IL-7, MIP-1α, and elevated levels of the anti-inflammatory cytokines IL-5, IL-13, and G-CSF in comparison to the wildtype. The inhibition of SLC7A8 function in human ASCs undergoing adipogenic differentiation led to reduced adipogenic capacity with a reduction in lipid droplet formation in mature adipocytes. This was accompanied by downregulation of important adipogenic genes such as PPARϒ, FABP4 and CD36 in response to SLC7A8 function inhibition. Moreover, the timing of inhibition of SLC7A8 function appeared to be critical as inhibition on the day of induction (day 0) suppressed white adipogenesis while inhibition on day 3 post adipogenic induction both suppressed white adipogenesis and promoted white adipose tissue browning activity through increase expression of PRDM16. Overall, this study demonstrates that SLC7A8 is important in obesity development and that its function is important in the production and maturation of adipocytes. Furthermore, the results suggest that SLC7A8 may serve as a potential therapeutic target for anti-obesity drug development with great promise for improving metabolic health.
The effect of cigarette smoke extract on the activity of clarithromycin with anti-pseudomonal agents on growth and biofilm formation of Pseudomonas aeruginosa
(University of Pretoria, 2023) Cholo, Moloko C.; Richards, G.A.; u19360305@tuks.co.za; Sekalo, Lebogang
The present study was undertaken with the initial objective of investigating the effects of six primary anti-pseudomonal antibiotics, namely amikacin, cefepime, ciprofloxacin, meropenem, piperacillin and tazobactam, on the planktonic growth of, and formation of biofilm by three different strains of the resilient respiratory pathogen, Pseudomonas aeruginosa [two drug-sensitive strains: the wild-type reference strain, PAO1(WT), and a clinical isolate (DS), as well as a multidrug resistant (MDR) clinically isolated variant of the pathogen]. These agents were investigated individually and in combination with the macrolide antibiotic, clarithromycin. Although all three test strains of P. aeruginosa are resistant to clarithromycin, this agent was included as an adjunct to the conventional anti-pseudomonal agents because of its inhibitory effects on various virulence factors of the pathogen. Following acquisition of the minimal inhibitory concentrations (MICs) of the individual anti-pseudomonal agents with respect to planktonic growth of, and biofilm formation by all three strains of P. aeruginosa, these experiments were repeated using the test anti-pseudomonal antibiotics in combination with clarithromycin. These results showed that amikacin, cefepime, ciprofloxacin and meropenem individually were potent inhibitors of the growth and formation of biofilm of the two susceptible strains of the P. aeruginosa, while, as expected, the MDR strain was highly resistant. When used in combination with clarithromycin, however, synergistic interactions with amikacin, cefepime and ciprofloxacin were observed, while additive activity was observed with the MDR strain. This phase of the study was followed by investigating the effects of exposure of all three strains of P. aeruginosa to cigarette smoke condensate (CSC) on the anti-pseudomonal activities of amikacin, cefepime and ciprofloxacin individually and in combination with clarithromycin. Although brief exposure of all three strains of the pathogen to CSC had modest, albeit variable, augmentative effects on bacterial planktonic growth and biofilm formation, the antimicrobial activities of amikacin, cefepime and ciprofloxacin, both individually and in combination with clarithromycin, were unaffected by exposure to CSC.
Influence of mesenchymal stromal cells and 2-methoxyestradiol in a murine model of spontaneous mammary carcinoma
(University of Pretoria, 2023) Ambele, Melvin; Pepper, Michael Sean; Durandt, Chrisna; kimberlypeta23@gmail.com; Peta, Kimberly Thando
Breast cancer (BC) is the most prevalent cancer in females and the leading cause of cancer deaths. Treatment options include mastectomy, chemotherapy, and radiotherapy. While these treatments can improve 5-year survival rates and reduce recurrence risk, they also affect healthy cells and not are effective for metastatic BC. To address these limitations, alternative therapies targeting only cancerous cells such as mesenchymal stromal/stem cell (MSC) therapy and a novel chemotherapeutic agent, 2-methoxyestradiol (2-ME), have been explored. MSCs have the ability to “home” to the tumour microenvironment (TME) and either promote or suppress tumour progression. Previous studies resulted in inconsistent results because of varied experimental designs including xenograft models that yielded conflicting results due to cross-species variations, limiting their interpretation. To overcome this, an isogenic mouse model of spontaneous BC was utilized to investigate the effect of MSCs on BC development. MSCs isolated from FVB/N mouse adipose tissue (mASC) were administered to heterozygous FVB/N-Tg(MMTV-PyVT)634Mul/J female mice that develop palpable mammary tumours. While no significant change in mammary tumour mass and volume was observed with mASC treatment, necrosis in lung lesions increased. Also, there was reduced number of CD163+ M2 macrophages and increased CD3+ T cells in the lungs but not mammary tumours in treatment group. Vegfr1, cd105 and mtdh were downregulated in the lungs suggesting an anti-tumour effect, potentially due to the presence of trapped mASCs. Overall, 13 of the measured cytokines were higher in the mASC treated group. These findings indicate that mASCs have an anti-tumour effect on pulmonary metastatic BC. The effect of 2-ME, a compound known for its anti-proliferative and anti-angiogenic properties, on the different stages of BC tumour development, is still unknown and was therefore investigated. The effects of 2-ME treatment on early- and late-stage BC were compared. While 2-ME treatment of early-stage BC led to reduced tumour necrosis with increased mass and volume of mammary tumours, a greater number of necrotic lesions and CD163 macrophages were observed in pulmonary metastatic tumours. Conversely, 2-ME treatment of late-stage BC inhibited tumour growth, increased CD3+ T cells and induced tumour necrosis. However, survival rates were not improved. Cytokine measurements of v early-stage BC indicated that 2-ME may have a pro-rumour effect. These findings suggest that 2-ME treatment has an antitumour effect on late-stage BC but does not enhance survival while no significant benefits were observed with 2-ME treatment of early-stage BC. Interestingly, 2-ME treatment before the appearance of palpable tumours resulted in a significant increase in tumour mass. This pro-tumour activity was accompanied by lower numbers of CD3+ T cells in the TME and elevated levels of the pro-inflammatory cytokine interleukin (IL)-1β. However, 2-ME treatment also led to fewer CD163+ macrophages in the TME, increased tumour necrosis, increased IL-10, and reduced IL-6 and IL-27 levels. This suggests that 2-ME may promote tumour development at the onset and early stages of BC development. In summary, BC is a complex disease with various stages, including tumour initiation, growth, progression and metastasis, and treatment effectiveness varies according to cancer stage. While mASCs show promise in treating pulmonary metastatic BC, 2-ME demonstrates an antitumour effect in late-stage BC but lacks efficacy in early-stage BC. Understanding the diverse responses to these treatments is crucial for developing targeted therapies that can effectively combat BC at different stages of progression.
In vitro osteogenic differentiation of mesenchymal stem cells
(University of Pretoria, 2022) Pepper, Michael Sean; Durandt, Chrisna; jmmollentze33@gmail.com; Mollentze, Jamie
The use of adipose-derived stromal/stem cells (ASCs) continues to increase in the field of regenerative medicine and other clinical applications. Adipose tissue can be collected in a less invasive procedure, when compared to bone marrow aspirations, from patients undergoing cosmetic liposuction or abdominoplasty procedures either as an aspirate or as intact tissue. Adipose tissue is seen as medical waste that would otherwise been discarded. ASCs are seen as the one of the most promising stem cell populations for tissue regeneration as they can be harvested with relative ease, can yield large quantities, grow under standard cell culture conditions, differentiate into multiple lineages, and secrete various cytokines. One of the clinical applications of ASCs is their role in enhancement of bone regeneration. To achieve osteogenic differentiation of ASCs, the ASCs are exposed to a differentiation cocktail containing β-glycerophosphate, ascorbate-2-phosphate and dexamethasone. Currently there is no consensus regarding the most optimal osteogenic differentiation medium for in vitro osteogenic differentiation of mesenchymal stromal/stem cells (MSCs), and the concentrations of the stimulating factors vary amongst published osteogenic induction media. It is for this reason we tested 3 previously published osteogenic differentiation media with varying concentrations of β-glycerophosphate, ascorbate-2-phosphate and dexamethasone. The success of the different differentiation cocktails was assessed using two osteogenic assays namely Alizarin Red S (ARS) and alkaline phosphatase (ALP) which were used to quantify the amount of calcified product and ALP enzyme activity respectively. Of the three differentiation media, one differentiation medium that produced the best osteogenic differentiation was chosen for further downstream testing. Foetal bovine serum (FBS) has been the gold standard for medium supplementation when expanding ASCs ex vivo despite the many disadvantages associated with its use, such as batch-to-batch variability, the presence of xenogenic proteins, possibility of zoonotic disease transmission, and ethical concerns regarding animal welfare to name a few. Furthermore, for ASCs to be used in a clinical setting, they need to comply with Good Manufacturing Practice (GMP) guidelines, which strongly advise against the use of FBS in clinical-grade cell therapy products. For this reason, researchers and clinicians continue to seek optimal and GMP compliant alternatives to FBS. Human alternatives to FBS will not only overcome FBS-associated disadvantages, but also more accurately mimic the environmental niche thus making the cell therapy product more physiologically compatible and consequently more reliable when applied clinically. The current study explored the use of two human alternatives to FBS namely platelet-rich plasma (PRP) and pooled human platelet lysate (pHPL) for use in osteogenic differentiation of ASCs in vitro. ASCs were expanded and differentiated in medium supplemented with either FBS, PRP or pHPL. Again, the two osteogenic assays, ARS and ALP were used to determine the success of osteogenic differentiation. To examine the kinetics of osteogenic gene expression, RNA was isolated at 4 time points during the differentiation period (days 0, 7, 14 and 21) and quantitative reverse transcription polymerase chain reaction (RT-qPCR) was performed. We found that both human alternatives were superior to FBS supplemented medium in terms of the amount of calcified bone product produced and the rate at which osteogenesis occurred. RT-qPCR data revealed that cells grown in FBS take longer to switch from a proliferative to an osteogenic differentiation state. Results revealed that PRP and pHPL are effective substitutes to FBS when differentiating ASCs into osteoblasts, and that this can effectively be assessed in the therapeutic setting. The aim of this study is to optimize and standardize protocols to differentiate MSCs into osteoblasts.
Development of a high-throughput screening assay to evaluate compound libraries for the modulation of adipogenesis
(University of Pretoria, 2022) Pepper, Michael Sean; Durandt, Chrisna; Ambele, Melvin; rachel26364@gmail.com; Giles, Rachel
The aim of this project was to develop assays that will allow for high-throughput screening to monitor adipogenesis. Such an assay may be useful for the identification of potential drug targets during the process of adipogenesis, which may result in discovering novel solutions to combat obesity and its related comorbidities. The main question for this study is whether adipogenesis can be assessed in smaller well sizes (such as the 96-well plate) with confidence using the specified methods.
Resistance and immune activation profiles in HIV-1 subtype C-infected subjects failing antiretroviral therapy
(University of Pretoria, 2014) Cassol, Sharon; De Oliveira, Tulio; Glen.Malherbe@gmail.com; Malherbe, Glen Pierre
No abstract
Isolation and characterization of the cytotoxicity, intracellular bioactivity and mechanism of antimycobacterial action of Euclea natalensis-derived naphthoquinones
(University of Pretoria, 2010) Lall, Namrita; Anderson, Ross; veneesha@ul.ac.za; Thaver, Veneesha
The major cause of HIV-related mortality in Sub-Saharan countries is pulmonary tuberculosis (TB), which is an escalating threat due to the emergence of multidrug resistant (MDR) and extremely multidrug resistant (XDR) TB. There is clearly an urgent requirement for the identification of novel, affordable anti-TB (as well as anti-HIV) drugs. This study was undertaken with the objective of isolating and characterizing the antimycobacterial potential of 3 naphthoquinones, i.e. neodiospyrin, diospyrin and 7- methyljuglone present in the roots of Euclea natalensis. The laboratory research included: i) isolation of diospyrin and neodiospyrin, from the roots of E. natalensis; ii) assessment of the cytotoxicity of these agents and synthetic 7-methyljuglone for eukaryotic cells (Vero and THP-1 cells); iii) determination of the intracellular bioactivities of the naphthoquinones against the H37Rv strain of Mycobacterium tuberculosis (MTB); and iv) mechanistic studies designed to investigate the effects of the test agents on cation (K+/ Ca2+) fluxes and energy metabolism (ATP levels) in MTB and M. smegmatis. With respect to the first objective, the naphthoquinones (diospyrin and neodiospyrin) were isolated from crude methanol extracts of crushed roots using chromatography and spectroscopic analysis. The yields of the compounds were 0.16 %, 0.32 %, and 0.12 % for neodiospyrin, diospyrin (isolated from plant) and synthetic 7-methyljuglone (synthesised in laboratory), respectively. The effects of the compounds (0.3-50μg/ml) on the viability of Vero and THP-1 cells were measured using the XTT assay (sodium 3’-[1-(phenyl amino-carbonyl)-3, 4 tetrazolium]-bis-[4-methoxy-6-nitro] benzene sulfonic acid hydrate) based on cellular metabolic activity. All 3 test compounds were found to possess cytotoxic activity at 1.5- 12.5g/ml) for both cell lines. Intracellular bioactivity of the test agents was measured using MTB-infected THP-1 cells as a surrogate for infected human macrophages. Following exposure of the MTBinfected cells to the test naphthoquinones, at a concentration range of 6.25-25g/ml, for 5 days, the cells were lysed and the viability of MTB in the lysates was then measured using the BACTEC radiometric system. All 3 test agents were found to be bioactive intracellularly, with complete inhibition of growth detected at 12.5, 25, and 6.25g/ml in the case of neodiospyrin, diospyrin, and synthetic 7-methyljuglone respectively. The effects of the 3 naphthoquinones on mycobacterial cation fluxes were measured according to the magnitude of uptake of 86Rb+ (a surrogate for K+) and 45Ca2+, while ATP was measured using a chemiluminescence procedure. None of the test agents was found to affect Ca2+ uptake by the bacteria. However, all 3 test agents were found to be potent inhibitors of uptake of K+ by MTB and M. smegmatis, with inhibition detected at submicrogram concentrations of these agents. All 3 test agents, especially synthetic 7- methyljuglone, were found to interfere with energy metabolism in MTB, manifested as decreases in mycobacterial ATP levels. Synthetic 7-methyljuglone which has the lowest MIC value for MTB (0.5μg/ml), and which was the most potent inhibitor of energy metabolism in MTB, shows promise as a potential anti-TB agent.These agents also are of potential value in drug modelling, possibly in the design of novel anti-TB agents which selectively target mycobacterial K+ transporters.
The roles of the potassium-uptake systems, Trk and Kdp, in the extracellular and intracellular growth of Mycobacterium tuberculosis
(University of Pretoria, 2020) Cholo, Moloko C.; aymangassim@gmail.com; Osman, Ayman Gassim Elamin
In his thesis, The roles of the potassium-uptake systems, Trk and Kdp, in the extracellular and intracellular growth of Mycobacterium tuberculosis, Mr Osman investigated the roles of the major potassium-uptake transporters utilized by the dangerous bacterial pathogen, Mycobacterium tuberculosis, during bacterial growth. This necessitated generating an Mtb triple-gene-knockout strain in which these potassium transporter-encoding genes were selectively inactivated using a homologous recombination procedure, whereafter the functional roles of these transporters were probed by phenotypic characterisation of the mutant strain extracellularly in planktonic and biofilm cultures and intracellularly in macrophages. He demonstrated that the mutagenesis tools were effective in generating an Mtb triple-gene-knockout mutant strain. Deletion of these transporters affected bacterial growth in the three environments, which manifested as an increase in planktonic growth, attenuation of biofilm formation and a decrease in macrophage intracellular survival. The findings demonstrated the essentiality of these bacterial transporters during various bacterial growth stages, underscoring their potential as novel drug and vaccine targets.
Optimising the efficacy of clofazimine against biofilm-encased Mycobacterium tuberculosis
(University of Pretoria, 2020) Cholo, Moloko C.; Steel, Helen Carolyn; mashelesa2@gmail.com; Mashele, Sizeka Aubrey
BACKGROUND : The chemotherapy of tuberculosis (TB) patients is administered for a six to nine-month period consisting of an intensive phase during the first two months with four primary anti-TB drugs, rifampicin (RMP), isoniazid (INH), ethambutol (EMB) and pyrazinamide (PZA), followed by a continuation phase during the remaining four to seven months with RMP and INH. During the intensive phase the active-replicating organisms (AR) are effectively and rapidly eliminated (99% killing), while the slow-replicating (SR) / non-replicating (NR) populations are targeted during the continuation phase. These latter bacterial populations respond poorly to treatment and are often associated with disease reactivation and relapse in treated patients, highlighting the necessity of identifying effective antimicrobial agents against these bacteria. Clofazimine (CFZ) has demonstrated high antimycobacterial activities against the AR, SR and NR microbial populations in vitro. However, its effects in combination with the primary drugs against these bacteria have not been demonstrated. AIMS AND OBJECTIVES : To investigate the antimycobacterial activity of CFZ in combination with primary anti-TB drugs against the AR and SR organisms isolated in planktonic and biofilm-forming cultures respectively, by evaluating their inhibitory and bactericidal activities. METHODS : The inhibitory activities of CFZ and three primary anti-TB drugs viz. RMP, INH and EMB were evaluated individually and in combination using minimum inhibitory concentration (MIC) and fractional inhibitory concentration index (FICI) determinations by the microtitre Alamar blue assay (MABA) and biofilm formation/ and crystal violet quantification for planktonic and biofilm cultures respectively. The bactericidal activities of these various combinations of the test agents were evaluated using minimum bactericidal concentration (MBC) and fractional bactericidal concentration index (FBCI) determinations by colony-counting procedures. RESULTS : In planktonic cultures, CFZ demonstrated a high inhibitory (MIC: 0.15 μg/mL), but low bactericidal activity (MBC: 5 μg/mL). In combination with primary anti-TB drugs, CFZ demonstrated synergistic inhibitory activities in combination with RMP and INH individually, as well as when the two antibiotics were used together. With respect to bactericidal activity, CFZ exhibited synergistic activity only in a two-drug combination with RMP. Synergistic activities were also demonstrated in a two-drug combination of RIF and INH and in a three-drug combination of these two antibiotics with EMB. However, in biofilm-forming cultures, CFZ demonstrated high inhibitory and bactericidal activities, achieving equal MIC and MBC values of 0.15 μg/mL. All CFZ-containing anti-TB drug combinations exhibited synergistic effects, with high activities being shown in combinations containing RIF and INH. CONCLUSION : CFZ exhibited synergistic effects in combination with primary anti-TB drugs against both planktonic and biofilm-forming cultures, showing potential benefit in promoting treatment outcome when used in TB chemotherapy.
Hematopoietic stem and progenitor cell heterogeneity and susceptibility to HIV-1
(University of Pretoria, 2019) Pepper, Michael Sean; juanitamellet@yahoo.co.uk; Mellet, Juanita
Umbilical cord blood (UCB) is a rich source of hematopoietic stem and progenitor cells (HSPCs). There are however limitations to using UCB as a regular source for hematopoietic stem cell transplantation (HSCT). The number of CD34+ HSPCs is limited, while a minimum number of CD34+ HSPCs is required for HSCT, which cannot always be achieved. New developments in HSCT are currently underway to expand current applications and improve safety and efficacy. This necessitates efficient ex vivo expansion of these cells to therapeutic numbers. HSCT is being investigated in therapies for non-hematopoietic disorders with the goal of replacing diseased cells or tissue with healthy cells. HSPC-based gene therapy strategies are becoming attractive applications of corrective ex vivo gene transfer given the reconstitutive potential of HSCT. The success of these strategies for the treatment of monogenic disorders resulted in the application of HSPC gene therapy being considered for other diseases such as the human immunodeficiency virus (HIV). The optimal isolation method was determined for increased HSPC purity and viability by testing two different methods, magnetic activated cell sorting (MACS) and fluorescent activated cell sorting (FACS). FACS was considered optimal for our purposes and was used to isolate CD34+ HSPCs for subsequent experiments. Different commercially available serum-free media were tested and compared to standard medium supplemented with foetal bovine serum (FBS). All commercial serum-free media outcompeted the standard medium based on viability and proliferation. Building on the previous work, StemSpan ACF was used to test combinations of cytokines for their expansion potential. The combination containing FLT3L, SCF, TPO, IL-3 and G-CSF resulted in the greatest expansion of HSPCs. The effect of StemRegenin-1 (SR1) on the expansion of HSPCs was explored by adding SR1 to the above-mentioned cytokine combinations. This resulted in minor effects on HSPC expansion based on viability and immunophenotype. Similarly, it resulted in only two significantly downregulated genes, cytochrome P450, family 1, subfamily B, polypeptide 1 (CYP1B1) and erythrocyte membrane protein band 4.1-like 3 (EPB41L3), in both CD34+ and CD34– cells compared to non-treated controls. The use of CD34+ HSPCs exclusively expanded with SR1 would be beneficial in cases where the HSPC cell dose of the initial harvested cell therapy product is suboptimal and therefore not a feasible option for HSCT on its own. Single-cell RNA sequencing was performed on CD34+ HSPCs and four populations were identified, which is in line with previous publications. HSPC gene therapy is a promising approach to treat HIV. However, this type of approach would require the presence of significant numbers of long-term repopulating HSPCs to enable successful long-term engraftment of gene-modified cells. One aspect that could result in this approach not succeeding is the presence of proviral DNA in HSPCs. It would therefore be important to identify a population of HSPCs that is resistant to HIV infection. It was therefore investigated whether HSPCs from leukapheresis products are susceptible to infection with HIV and whether a subset of HSPCs exists that is resistant to infection to use in HIV gene therapies. Unfortunately, this could not be achieved due to loss of viability of HSPCs from leukapheresis products.
Characterisation of adipose-derived stromal cell heterogeneity
(University of Pretoria, 2019) Pepper, Michael Sean; elizewol@yahoo.com; Wolmarans, Elize
Human adipose-derived stromal cells (hASCs) have gained increasing attention in the past decade as a potential cell therapeutic product. hASCs are classified as multipotent, fibroblast-like, plastic-adherent cells that can easily be expanded in vitro and has the ability to differentiate into multiple cell lineages. A distinct advantage of hASCs is that large numbers of cells can be extracted with minor donor site morbidity. This has sparked the worldwide growth of a new research field and industry. There is however, still much to learn about these cells before they can be used with confidence in a clinical setting. An inherent characteristic of hASCs that is not well understood is their heterogeneity. Freshly isolated hASCs consists of various sub-populations of cells. Whether this heterogeneity is beneficial or detrimental with respect to the potential therapeutic effect of hASCs is unknown. Thus, a great deal of effort is currently being made to understand the heterogeneous nature of hASCs. In the attempt to characterise the heterogeneity of hASCs in this study, two different approaches were used. The SP assay was used in an attempt to identify a sub-population of cells with greater dye efflux capability than the rest of the hASC population. We also examined levels of ABC transporter gene and protein expression (known to cause the SP phenotype). Results revealed a sub-population of cells with efflux ability present in hASCs. Results also provided novel insight into the expression of ABC transporter genes and the expression pattern of ABC transporter proteins in hASCs. The second approach used was the first study, to the author’s knowledge, that attempted to characterize the heterogeneity of hASCs at P2 using whole transcriptome analysis at the single-cell level. Results revealed the presence of a minimum of two and a maximum of five sub-populations in hASCs at P2. In addition, one of the sub-populations identified is believed to be contractile cells such as pericytes and/or vascular smooth muscle cells (vSMCs). Another aspect that needs to be taken into consideration in order to use hASCs in a clinical setting, is the proliferation and maintenance of the isolated cells ex vivo. Many research groups are moving away from foetal bovine serum (FBS) as the standard serum supplement in order to make the isolation and expansion of cells compliant to good manufacturing practice. Suggested replacements for FBS are human blood alternatives which can create a culture environment that more accurately resembles the human environment. The human alternatives suggested include human serum (HS), platelet-rich plasma (PRP), platelet-poor plasma (PPP), freshly frozen plasma (FFP) and platelet lysate (PL). This was, to the author’s knowledge, the first head to head comparison done between FBS and all the listed human alternatives simultaneously. Results revealed that all the different human alternatives were able to sustain hASCs in vitro with no effect on the viability or immunophenotypic profiles of the cells. Results also revealed PL and PRP to cause a significant increase in the proliferation rate of hASC.
Effects of cigarette smoke condensate on clarithromycin-mediated inhibition of biofilm formation and related alterations in resistance gene expression by Streptococcus pneumonia
(University of Pretoria, 2019) Steel, Helen C. ; Cockeran, Riana; Dix-Peek, Thérèse; matapa.kg@gmail.com; Matapa, Kgashane Given
Streptococcus pneumoniae (the pneumococcus) is a Gram-positive bacterium that frequently colonises the nasopharynx of healthy humans. In susceptible hosts, especially children under 5 years with underdeveloped immune systems and the elderly, this asymptomatic colonisation can lead to development of severe disease such as pneumonia and meningitis. In many cases, disease severity is linked to the inability of the infection to be successfully treated, possibly due to formation of bacterial biofilms. In this context, cigarette smoking, which is a well-recognised risk factor for development of severe pneumococcal disease, also promotes biofilm formation by various types of bacterial pathogens. Notwithstanding poor penetration of biofilms by antibiotics, bacteria within biofilms are exposed to low levels of antibiotics, which promote gene modifications that mediate antibiotic resistance. However, little is known about the effects of exposure of the pneumococcus to cigarette smoke on the induction of pre-existing antibiotic resistance genes, specifically those that mediate resistance to macrolide antibiotics. In addition to measuring bacterial growth and biofilm formation, the research described in this dissertation was designed primarily to investigate the effects of exposure of different strains of the pneumococcus to cigarette smoke condensate (CSC) on the expression of genes which mediate resistance to macrolide antibiotics, specifically the erm(B) and mef(A) genes. The bacterial strains used were 172 (macrolide-susceptible), 521 [macrolide-resistant, mef(A) efflux pump-expressing] and 2507 [macrolide-resistant, erm(B) ribosomal methylase-expressing], all belonging to serotype 23F. In addition, the effects of exposure of all three strains of the pneumococcus to CSC on the expression of the SP2003 gene were also investigated. This gene encodes an ABC-type transporter, expression of which has been linked to antibiotic resistance. All three strains of the pneumococcus were exposed to CSC (80 and 160 μg/mL) and sub-minimal concentrations of clarithromycin individually or in combination, followed by measurement of growth, biofilm formation and gene expression. Bacterial growth was measured using spectrophotometric and colony counting procedures, biofilm formation by a crystal violet-based spectrophotometric method, and gene expression [(mef(A), erm(B) and SP2003)] using real-time qPCR. Exposure of all three strains of the pneumococcus to either CSC, at both concentrations used, or to clarithromycin alone, resulted in a transient inhibition of growth which persisted for several hours and was followed by a rebound. Exposure to combinations of the antibiotic and CSC resulted in prolongation of the lag phase, particularly in the case of strain 172. Augmentation of biofilm formation was observed following exposure of all three strains of the pneumococcus to CSC, while exposure of strain 172 to clarithromycin inhibited biofilm formation, which was partly attenuated by CSC. In the case of gene expression, exposure to clarithromycin alone caused significant upregulation of expression of the macrolide-resistance genes, mef(A) and erm(B), by strains 521 and 2507 respectively, as expected. Exposure of strain 2507 to the combination of clarithromycin and CSC resulted in significant augmentation of expression of the erm(B) gene relative to the expression level noted with clarithromycin alone. This augmentative effect of CSC on gene expression was not, however, evident in the case of the mef(A) gene. In addition, and somewhat surprisingly, exposure of strain 2507 to CSC only at 160 μg/mL resulted in a significant increase in erm(B) gene expression. In the case of the SP2003 gene, exposure of all three strains of the pneumococcus to CSC resulted in significant upregulation of this gene, probably as a stress response linked to elimination of smoke-derived toxicants, while exposure to clarithromycin alone resulted in modest upregulation, compatible with a role for SP2003 in mediating macrolide resistance. In conclusion, the pathogen-targeted effects of CSC described in this dissertation provide additional insights into the mechanisms by which cigarette smoking impacts negatively on the outcome of pneumococcal infections by undermining the therapeutic efficacy of macrolide antibiotics.

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