Browsing by Author "Davids, Lester M"

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    Human scalp hair: geometry, biochemistry, growth parameters and mechanical characteristics
    (2016) Mkentane, Kwezikazi; Khumalo, Nonhlanhla P; Davids, Lester M
    Scalp hair is increasingly being used as a testing substrate for toxins and monitoring treatment adherence. The biochemistry of human hair is assumed to be similar; however, a recent study reported higher concentration of lipids in African hair. The effect of hair curvature, if any, on drug incorporation (e.g. lipid soluble drugs) is unknown. Racial description of hair morphology is unscientific. A geometric classification of hair into 8 groups (I-straight to VIII-tightly curly) was recently proposed, however its reliability has not been confirmed. The aim of this thesis was to investigate the reliability of the geometric classification (and to assess whether it could be improved) and investigate relationships between morphology and other hair characteristics. Virgin hair was collected from 128 volunteers using a standardized protocol. Geometric measurements of hair using published templates were conducted for classification. Reliability was assessed using Kappa statistics. Characteristics assessed included mechanical properties (miniature tensile tester), growth rate and hair density (TrichoScan® trichogram), biochemistry (Vanillin assay for lipids and Fourier Transform Infrared adsorption) and imaging (Electron and Fluorescent Light Microscopy). Inter-observer agreement was poor for 8-groups (k=0.418) but improved for 6-groups (k=0.671). The intra-observer agreement also improved [ranges: k=0.444 to 0.648 (8-groups) and k=0.599 to 0.836 (6-groups)]. The yield strength of all hair groups was higher than reported for racially grouped samples. Curly hair groups had lower growth rates and tensile strengths. The TrichoScan based growth rate was for fastest for the straightest (0.72±0.3 cm/month) and slowest for the curliest (0.39 ± 0.2 cm/month) hair. No correlation with biochemistry was detected for either the 8 or 6-group classification, although a trend toward higher absorption of lipid (C-H) bands was noted for curly hair. A supervised statistical approach applied to 4 hair groups using the FTIR data improved classification success to 79% (range: 69% - 88%), which needs confirmation but would be more objective than using race for hair testing in Medicine and Forensic Science. This thesis supports a geometric classification with fewer groups (6, based kappa statistics and 4, based on biochemistry); it is also the first to report correlations between hair geometry, biochemistry and physical properties.
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    An in vitro investigation into the pigmentary phenotype of melanocytes and keratinocyte co-cultures to improve wound healing
    (2013) Chang, Ju-Wei; Davids, Lester M
    On healing, partial-thickness burn wounds usually result in depigmentation of the skin. This is due to the loss of melanocytes. The lack of pigmentation in the healed wound is particularly prominent in dark-skinned individuals and could result in serious psychosocial consequences such as low self-esteem, stigmatisation and discrimination among sufferers. Methods aimed at investigating rapid and efficient repigmentation in wounded skin are therefore pertinent. The aims of this study were two-fold: i) To promote melanin synthesis in human skin cells using different ratios of human melanocytes (Mc) to keratinocytes (Kc) in an in vitro co-culture system, in order to ensure pigmentation of the skin and, ii) To understand cellular mechanisms that contribute basic scientific knowledge towards clinically improved wound healing.
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    Investigating the by-stander effect of Hypericin induced photodynamic therapy on human skin cells
    (2014) Popovic, Ana; Davids, Lester M
    Skin cancer is the most common cancer worldwide, and its incidence rate in South Africa is increasing. Photodynamic therapy (PDT) has been shown to be an effective treatment modality, through topical administration, for treatment of non-melanoma skin cancers. Our group investigates hypericin-induced PDT (HYP-PDT) for the treatment of both non-melanoma and melanoma skin cancers. However, a prerequisite for effective cancer treatments is efficient and selective targeting of the tumoral cells with minimal collateral damage to the surrounding normal cells, as it is well know that cancer therapies have bystander effects on normal cells in the body, often causing undesirable side effects. PDT can induce a bystander effect, defined as indirect damaged induced into adjacent cells either via intercellular gap junctions or via diffusible ROS released in the microenvironment. It is therefore important to know the effects of HYP-PDT on the normal cell population surrounding the non-melanoma skin cancer or melanoma tumor. The aim of this project was to investigate the cellular and molecular effects of HYP-PDT on normal primary human keratinocytes (Kc), melanocytes (Mc) and fibroblasts (Fb) in an in vitro tissue culture model thus representing both the epidermal and dermal cellular compartments of human skin.
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    An investigation into the synergistic action of chemotherapy and photodynamic therapy in resistant skin cancers
    (2015) Nsole Biteghe, Fleury Augustin; Davids, Lester M
    Melanoma is a form of skin cancer, arising from epidermal cells of the melanocyte lineage, which undergo a series of transformations and genetic alterations that may give rise to both pigmented and unpigmented melanoma. Melanoma represents 4% of all skin cancers but due to its aggressive nature, it accounts for 80% of death among skin cancer patients. South Africa has a melanoma incidence rate that is second worldwide to only Australia. In melanoma; non-metastatic primary tumours are treated by surgical resection. However, metastatic melanoma is highly resistant to conventional radio and chemotherapy, thus reducing the median life of patient's diagnosis with the metastatic form to about 7-9months. Given the implications of the pigment in failure of chemotherapy, two human metastatic pigmented and unpigmented melanoma cell lines were used to investigate the mechanisms underlying chemo-resistance. During the course of this study, the first aim was to determine the concentration of the chemotherapeutic drug dacarbazine (DTIC) causing fifty percent decrease in melanoma cell viability (LD50), then to investigate the possible synergism of hypericin activated-photodynamic therapy in reducing (HYP-PDT) melanoma cell viability, when combined with chemotherapy. In addition we wanted to assess the morphology and the clonogenic capacity of the melanoma cells, after the different treatments and further investigate the ATP-binding cassette (ABC) transporters (ABCB5/1 & ABCG2) expression profile, before and after chemotherapeutic (DTIC) and combination therapy (DTIC+HYP-PDT) treatments.
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    Mitochondrial targeting of wild-type and mutant human protoporphyrinogen oxidase (PPOX)
    (2003) Davids, Lester M; Meissner, Peter; Corrigall, Anne
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    Skin cells as a tool in genetic diagnosis of Duchenne muscular dystrophy
    (2016) Tyers, Lynn; Esterhuizen, Alina; Davids, Lester M
    Duchenne muscular dystrophy (DMD) is the most common and severe of the dystrophies, with an incidence of 1 in 3500 live male births, worldwide. Becker Muscular dystrophy (BMD) has a lower incidence of approximately 1 in 17500 births, a milder progression and longer life expectancy. Many advancements have been made in the development of gene-based therapies for the treatment of D/BMD, however, these treatments require genetic confirmation of the disease which continues to present a significant diagnostic challenge. The current standard for RNA-based analysis requires obtaining an invasive, often distressing, muscle biopsy. This dissertation investigated the utility of human autologous epidermal melanocyte and dermal fibroblast cell cultures for use as a tool for genetic confirmation of D/BMD from a much less invasive shave skin biopsy. Methodologies included immunohistochemical, immunocytochemical, Western blot, qPCR analysis and cDNA sequencing. The results suggest that melanocytes and fibroblasts express the full length muscle isoform of dystrophin, although at differing levels, and that melanocytes could potentially be used as an alternative for the genetic confirmation of D/BMD.
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    St John's Wort (Hypericum perforatum L.) photomedicine: hypericin-photodynamic therapy induces metastatic melanoma cell death
    (Public Library of Science, 2014) Kleemann, Britta; Loos, Benjamin; Scriba, Thomas J; Lang, Dirk; Davids, Lester M
    Hypericin, an extract from St John's Wort ( Hypericum perforatum L. ), is a promising photosensitizer in the context of clinical photodynamic therapy due to its excellent photosensitizing properties and tumoritropic characteristics. Hypericin-PDT induced cytotoxicity elicits tumor cell death by various mechanisms including apoptosis, necrosis and autophagy-related cell death. However, limited reports on the efficacy of this photomedicine for the treatment of melanoma have been published. Melanoma is a highly aggressive tumor due to its metastasizing potential and resistance to conventional cancer therapies. The aim of this study was to investigate the response mechanisms of melanoma cells to hypericin-PDT in an in vitro tissue culture model. Hypericin was taken up by all melanoma cells and partially co-localized to the endoplasmic reticulum, mitochondria, lysosomes and melanosomes, but not the nucleus. Light activation of hypericin induced a rapid, extensive modification of the tubular mitochondrial network into a beaded appearance, loss of structural details of the endoplasmic reticulum and concomitant loss of hypericin co-localization. Surprisingly the opposite was found for lysosomal-related organelles, suggesting that the melanoma cells may be using these intracellular organelles for hypericin-PDT resistance. In line with this speculation we found an increase in cellular granularity, suggesting an increase in pigmentation levels in response to hypericin-PDT. Pigmentation in melanoma is related to a melanocyte-specific organelle, the melanosome, which has recently been implicated in drug trapping, chemotherapy and hypericin-PDT resistance. However, hypericin-PDT was effective in killing both unpigmented (A375 and 501mel) and pigmented (UCT Mel-1) melanoma cells by specific mechanisms involving the externalization of phosphatidylserines, cell shrinkage and loss of cell membrane integrity. In addition, this treatment resulted in extrinsic (A375) and intrinsic (UCT Mel-1) caspase-dependent apoptotic modes of cell death, as well as a caspase-independent apoptotic mode that did not involve apoptosis-inducing factor (501 mel). Further research is needed to shed more light on these mechanisms.
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    The use of in vitro 2d co-culture models to determine the optimal keratinocyte: melanocyte ratio to be used in the development of pigmented 3d skin model
    (2015) Lebeko, Maribanyana Robert; Davids, Lester M
    Burn injuries are among the most devastating of all injuries and a major global public health crisis, with fire related burns accounting for approximately 265 000 deaths annually. The African continent, most especially Sub-Saharan Africa, has the second highest mortality rates (15% of global mortality rates). In South Africa, 3.2 % of the total population sustains burn injuries, with 50 % of these cases as children under the age of20 years. Studies have also shown that most of these incidences are prevalent within the age groups of 0-5 years, and account for the 3rd most common cause of mortality in children under the age of 15 years. In depth knowledge and understanding of cellular facets of wound healing has allowed for a greater stance in the interventions aimed at circumventing problems associated with development of effective wound defects treatment regimen. Burn treatment options are largely dependent on the degree and extensiveness of burns. A wide body of literature exists with regards to traditional as well as current treatment options. These include, for instance the use of various forms of skin auto-grafts. Despite such great success with all kinds of innovative ideas surrounding the use of autologous skin grafting, lack of available donor sites for skin grafts still remains a problem, more so in cases where patients suffer burns spanning more than 70% TBSA. This therefore has inspired the design and use of bioengineered skin substitutes as well as cultured/non-cultured autologous epidermal cells. Unfortunately, to date, no tissue engineering technique has fully been able to recapitulate the anatomy and physiology of the skin, or has attained the biological stability as well as achieving the aesthetic outcome. Several hurdles are yet to be overcome to achieve this. Amongst many, inclusion of melanocytes, other skin appendages as well as potential progenitor cells is some of the attributes of an ideal 3D skin equivalent. Therefore pigmented 3D skin constructs are of great interest as they address not only the issues of complete wound healing, but also the aesthetic outcomes. In light of this, correct keratinocyte to melanocyte ratios are also of great importance in designing such pigmented 3D constructs. Therefore the major aim of this study was to isolate skin melanocytes and keratinocytes, and co-culture them at different ratios in order to attain optimal pigment production and/or consequent improved wound healing outcome. To determine the best keratinocyte to melanocyte ratio to use in developing pigmented3D skin constructs, the following co-culture ratios were used: 5:1, 10:1 and 20:1.Proliferation assays were employed to further elucidate the growth dynamics of both human skin melanocytes and keratinocytes in either mono- or co-culture system. Secondly, FACS was used to develop a reliable technique to be used to separate the two cell types from a co-culture system in order to perform downstream analyses. Thirdly, to establish the roles of the co-cultured cells in wound healing (with regards to proliferation and migration), scratch wound healing assays were employed. Lastly, FACS was used to infer the effect of such ratios on pigment production. Our results demonstrated that keratinocytes, compared to melanocytes mono-cultures have higher proliferation capacity. On the contrary melanocyte's proliferation is up-regulated by the presence of keratinocytes in a co-culture, whereas higher numbers of melanocytes in co-culture with keratinocytes resulted in less proliferative keratinocyte phenotype. The FACS separation technique worked excellently in identifying keratinocyte population from melanocytes, with an almost 100% accuracy. This is shown by melanocytes being sorted as 93% of MART-1 + cells in a mono-culture, followed by an approximately 5:1 separation of keratinocytes from melanocytes (77% Kc and 17% Mc). In vitro scratch assays demonstrated that none of the co-culture ratios was significantly superior with regards to wound healing capacities and pigment production, in the absence of fibroblast-conditioned medium. In conclusion, the 5:1 co-culture ratio seemed to yield a non-significant, yet best outcome with regards to wound healing capacity (only in the presence of fibroblast-derived factors), thus conferring it as a potential optimal ratio of keratinocytes to melanocytes, to be used in development of our pigmented 3D constructs.