Theses and Dissertations (Immunology)

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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 C.; u11283026@tuks.co.za; Mmakola, Khomotso Madimetsa Shelboy
ABSTRACT 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 C.; 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. Aim 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.
Transcriptional analysis of the host response to HIV-1 infection in CD4+ T lymphocytes monocytes and macrophages
(University of Pretoria, 2019) Pepper, Michael Sean; Durandt, Chrisna; kitcat1121@hotmail.com; Wickham, Catherine Heather
The human immunodeficiency virus (HIV) is the causative agent of acquired immune deficiency syndrome (AIDS), a condition characterized by depletion of CD4+ T cells and other immune system dysfunctions. Due to its widespread geographic distribution and ease of 4 transmission, HIV has become a serious global healthcare concern. HIV-1 subtype C (HIV-1-C) 5 is of particular interest since it is the most rapidly expanding and is especially prevalent in 6 southern Africa. In addition, HIV-1-C is poorly studied in comparison to other subtypes. Despite HIV being extensively researched, much is still unknown about the host cell response 8 to infection at a molecular level and how this might be exploited for therapeutic intervention. Therefore, the purpose of this study was to use microarray-based transcriptomic analysis to gain an unbiased perspective of the host cell response in CD4+ T lymphocytes and macrophages when exposed to primary HIV-1-C viruses of different tropisms (R5-tropic, X4- tropic, dual-tropic). In order to achieve this overall aim, we first needed to develop and optimize protocols for the culture of primary HIV-1-C strains. A p24 ELISA assay was used to confirm successful viral replication, followed by functional titration using the GHOST reporter cell line. We found that the efficiency of replication of primary viral isolates was highly stochastic. While many of the strains cultured suffered severe losses of infectivity, we managed to produce infective stocks of dual-tropic and X4-tropic isolates. We next aimed to optimize cell culture protocols for CD4+ T lymphocytes and macrophages that would ensure maximal susceptibility to HIV. Since activation of CD4+ T cells is reported to enhance viral replication, we assessed the efficacy of various activation protocols. We found that antibody- mediated activation was highly successful. Flow cytometric analysis revealed increased cell proliferation and CD25 expression. In addition, CD4+ T-cell activation dramatically increased CXCR4 surface expression while CCR5 expression was diminished. Infection experiments with our dual-tropic isolate confirmed that these cells were susceptible to infection. In order to optimize macrophage cell culture conditions, we compared the effects of differentiation under the influence of different growth factors (M-CSF or GM-CSF). Successful differentiation was determined by phenotypic analysis using flow cytometry coupled with a functional phagocytosis assay. Additional analysis of co-receptor expression revealed extremely low levels of CXCR4 and relatively high CCR5 expression in both macrophage populations. The differentiated macrophages were completely refractory to infection with our dual-tropic isolate, making them unsuitable for further experiments. The final component of the study was the gene expression analysis itself, which was performed using activated CD4+ T lymphocytes exposed to a dual-tropic isolate (CM9). Differential gene expression analysis revealed altered expression patterns in a relatively small but functionally diverse group of genes involved in apoptosis, deubiquitination, transcriptional regulation and immune system functions. Apoptosis and deubiquitination were identified as statistically overrepresented functional pathways in our subsequent GO-based analysis. Comparison of our findings with previous studies performed using HIV-1-B seems to indicate that while many of the genes observed in our study have not specifically been detected previously, they do tend to belong to similar pathways.
The role of Pref-1 in in vitro adipogenic differenctiation of mesenchymal stromal/stem cells
(University of Pretoria, 2019) Durandt, Chrisna; Pepper, Michael Sean; carinacrdasilva@gmail.com; Da Silva, Carina Corte-Real
Obesity is characterized by an excessive amount of body fat which can negatively affect health as it is a risk factor for various non-communicable diseases such as cardiovascular disease and diabetes, amongst others. A better understanding of the process of fat cell formation, a process known as adipogenesis, would aid in finding sustainable solutions to this growing global health challenge. The ability of human derived mesenchymal stromal/stem cells (hMSCs) to differentiate into adipocytes holds great promise to serve as an in vitro model to study human adipogenesis. It is known from the literature that hMSCs from different sources within the body may differ in their differentiation potential. It is well reported that human Wharton’s jelly derived MSCs (hWJSCs) have reduced adipogenic differentiation capacity when compared to hMSCs isolated from adipose tissue. hMSCs isolated from adipose tissue are referred to as adipose-derived mesenchymal stromal/stem cells (hASCs). The poor adipogenic differentiation potential of hWJSCs has also been observed in our group. We also observed elevated pre-adipocyte factor 1 (Pref-1) Mrna levels in hWJSCs. The observed elevated Pref-1 mRNA levels in hWJSCs led to the hypothesis that this may be responsible for hWJSCs poor adipogenic differentiation capacity. Pref-1 is a transmembrane protein capable of being cleaved to give rise to a 50 kDa soluble protein which is thought to be an important adipogenic inhibitor by preventing cells from maturing into adipocytes thereby keeping them in a pre-adipocyte state. It was therefore important to confirm whether the elevated Pref-1 gene expression observed translated into higher levels of Pref-1 protein in hWJSCs when compared to hASCs. The aim of this study was to further investigate a previous observation in our group of elevated Pref-1 gene expression levels in hWJSCs undergoing adipogenesis. This will be done by examining the association between Pref-1 protein expression levels in hWJSCs and hASCs and their respective in vitro adipogenic differentiation potential. The cells were induced to undergo adipogenesis for a period of 21 days and monitored on days 0, 1, 3, 7, 14 and 21. During this time period, the adipogenic differentiation potential of the cells was assessed by means of flow cytometry (quantitative) and microscopy (qualitative). The level of Pref-1 protein expression was investigated using various techniques which included flow cytometry, ELISA and western blot assays. Gene expression was determined by reverse transcription quantitative polymerase chain reaction (RT-qPCR). As previously determined, hWJSCs displayed poor adipogenic differentiation despite the upregulation of peroxisome proliferator activated receptor gamma (PPARγ). Flow cytometry data revealed hASCs to have greater levels of Pref-1 protein expression on the cell surface. We were unable to detect any Pref-1 protein in the cell culture supernatant collected at each time point using ELISA, but western blot data suggested that there may be more soluble Pref-1 protein in the cell culture supernatant obtained from hWJSCs. Gene expression data also revealed higher levels of Pref-1 mRNA in hWJSCs. However, the amount of Pref-1 protein detected was very low and no statistical significance could be determined between Pref-1 expression in hASCs versus hWJSCs in any of the assays. The in vivo data obtained by immunohistochemistry shows that both adipose tissue and umbilical cord express Pref-1 around blood vessels. The umbilical cord does however have additional Pref-1 staining that is not associated with blood vessels. The stromal cells in the Wharton’s jelly displayed positive Pref-1 staining. How this finding correlates with the in vitro results obtained is unclear. Comparing the adipogenic differentiation potential of hWJSCs to hASCs did however allow for an interesting observation. HASCs were able to upregulate CD36 (a fatty acid translocase) during the adipogenic differentiation period, while this was not observed for hWJSCs. The low levels of Pref-1 protein expression combined with the upregulation of PPARγ in hWJSCs thus raises the question as to whether other regulatory elements, currently unknown, downstream of PPARγ, suppress the upregulation of endstage adipogenic genes and consequently prevent the induced hWJSCs from acquiring the phenotypic characteristics associated with mature adipocytes.
Investigating the homing and healing properties of adipose-derived mesenchymal stromal cells using a model of wound repair
(University of Pretoria, 2019) Pepper, Michael Sean; Pittet-Cuénod, Brigitte; Modarressi, Ali; Martinou, Jean-Claude; karlienkallmeyer@gmail.com; Kallmeyer, Karlien
Adult mesenchymal stromal cells show promise therapeutically due to their multipotent differentiation capacity, immunomodulatory properties, paracrine signalling and ability to migrate to sites of injury. The potential use of adipose-derived stromal cells (ASCs) as a cellular therapy for treating delayed healing, non-healing and chronic wounds, their optimal route of administration, bio-distribution and fate needs to be investigated. Therefore, this study aimed first to establish the location and survival of systemically and locally administered ASCs during wound repair under physiological conditions (model 1) and second, the effect of locally administered ASCs during wound repair under pathological conditions of hyperglycaemia and ischemia (model 2). A dual tracking approach was used to follow firefly luciferase (Fluc) and green fluorescent protein (GFP) expressing ASCs by bioluminescence imaging (BLI) and histological analysis. The immuno-phenotype and differentiation capacity of rat ASCs transduced to express Fluc and GFP were assessed before the cells were used in the wound repair models. In model 1, full-thickness bilateral wounds were created on the dorsal aspect of the hind paws in healthy rats and two treatment modes were assessed: a single dose of 2 x 106 ASCs or NaCl administered systemically into the tail vein or 2 x 105 ASCs injected locally around each wound. Animals were followed by digital photography, BLI and sacrificed for histological analysis. In model 2, ischemia was induced unilaterally by resection of the femoral artery in hyperglycaemic rats before full-thickness bilateral wounds were created and 2 x 105 ASCs or NaCl administered locally around each non-ischemic and ischemic wound. Animals were followed by digital photography and sacrificed for histology and immunohistochemistry (IHC). Haematoxylin/eosin (H/E) staining as well as Masson’s trichrome staining and IHC for alpha-smooth muscle actin (αSMA), ionised calcium binding adaptor molecule 1 (Iba1) and GFP were performed on sample sections. Wound closure time and the contraction/epithelialisation ratio were assessed in both models. Transduced ASCs maintained their immuno-phenotype and differentiation capacity. Under physiological conditions, systemically administered ASCs were filtered out in the lungs, whereas locally administered ASCs survived at the injection site and migrated into the wound bed. Wound closure was accelerated by 5 and 7 days with systemic and local ASC treatment Page | iv respectively compared to control animals, and this was the result of increased epithelialisation. Under pathological conditions, locally administered ASCs significantly enhanced wound closure in non-ischemic and ischemic wounds by 9 days compared to control wounds. Semi-quantitative analysis revealed that ASC treatment led to enhanced cellularity in the wound. No changes in collagen deposition, vascularisation (as determined by αSMA staining) or macrophage infiltration were observed between ASC treated and control groups. However, αSMA staining was detected earlier and remained higher at wound closure in the former without enhancing wound closure by contraction. Despite the limited systemic homing capacity of ASCs, wound healing was improved. Locally injected ASCs migrated from the wound edge into the wound bed where they promoted wound repair. Under pathological conditions, ASCs enhanced wound closure. A significant increase in wound cellularity was observed, possibly through a mechanism of paracrine signalling that recruited more immune regulating and tissue repair cells into the granulation tissue. Administration of ASCs for delayed healing wounds show promise as a cellular treatment for enhancing wound repair. Key words: Adipose-derived mesenchymal stromal cells (ASCs), in vivo imaging, bioluminescence imaging (BLI), green fluorescent protein (GFP), firefly luciferase (Fluc), re-epithelialisation, contraction, wound healing, wound repair, homing
Functional characterisation of gonadotropin-releasing hormone-estrogen conjugates
(University of Pretoria, 2019-11-12) Millar, Robert Peter; Newton, Claire; Andreson, Ross; u27107052@tuks.co.za; Leijenaar, Stacey-Lee
Prostate cancer (PC) is the second most commonly occurring cancer in men, and the fourth most common commonly occurring cancer overall. Almost all PC begins in an androgen-dependent state with androgen deprivation therapy (ADT) an effective treatment at this stage. PC can overtime develop into an androgen independent state at which point it can no longer be treated with ADT. The focus of this research is on androgen dependent PC and ADT. The hypothalamic–pituitary–gonadal (HPG) axis, controlled by gonadotropin releasing hormone (GnRH) is responsible for regulating reproduction, puberty and the production of spermatozoa and androgens in men. GnRH analogues (which downregulate the axis) are the foremost ADT agents. GnRH analogues may also have direct anti-proliferative effects in some cancers. However, ADT has negative side effects including loss of bone mass, hot flushes and loss of libido, due to a concomitant decrease in estrogen which is synthesised from androgen. We hypothesise that a molecule which retains GnRH receptor (GnRHR) activation while replacing estrogen activity which activates the estrogen receptors (ERs) may be beneficial. Conjugates of GnRH analogue (GnRHag) with 17β-Estradiol (E2C) or genistein (GenC) were studied examining GnRH and estrogen activity in vitro to evaluate their potential as novel PC therapeutics. Synthesis of the conjugates was commissioned from a commercial company. GnRHR activation was tested in HEK 293T cells by determining the generation of inositol phosphate in cells expressing GnRHR. ER activation was determined in MCF-7 cells using an E-screen assay in a cell line expressing ERs. Anti-proliferative effects were assessed in PC cell lines by crystal violet assay. Potential bone-protective capability was measured by ability to inhibit RANKL-induced osteoclast differentiation of Raw 264.7 macrophages. E2C and GenC elicited potent stimulation of GnRHR. The conjugates also had estrogenic activity similar to the unconjugated estrogen and phytoestrogen in the E-screen assay. Their estrogenic activities were confirmed by their ability to inhibit osteoclast differentiation to the same degree as unconjugated 17β-Estradiol and genistein. No direct antiproliferative effects, by the conjugates or GnRH, on PC cells were observed, indicating that GnRHR may not be expressed in these cell lines. The demonstration that E2C and GenC displayed GnRHR and ER activities similar to their unconjugated counterparts suggests they may be efficacious as ADT agents with reduced side effects of estrogen deprivation. Key Words: GnRH analogues, Prostate Cancer, Androgen Deprivation Therapy, Estrogen Deficiency, RANKL, Hot Flushes, Libido, Bone Loss

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