UWCScholar :: Browsing by Author "Fisher, David"

Browsing by Author "Fisher, David"

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Carica papaya seed extract slows human sperm
(Elsevier, 2019) Ghaffarilaleh, Vahid; Fisher, David; Henkel, Ralf
Ethnopharmacological relevance: Traditional healers use Carica papaya seeds as a remedy for diseases and as a contraceptive for men and abortion in women. Material and methods: Semen samples from 35 healthy men were allowed to liquefy and subsequently incubated for 60 min in Human Tubular Fluid medium containing 1% bovine serum albumin with aqueous C. papaya seed extract at concentrations of zero, 0.025, 0.25, 2.5, 25, 250 and 2500 μg/ml. Afterwards, sperm were washed and used for assessment of capacitation and acrosome reaction, DNA fragmentation, vitality, motility, reactive oxygen species (ROS) and mitochondrial membrane potential (MMP). Results: The extract showed no effects on straight-line velocity, linearity, straightness, beat-cross frequency and the percentage of capacitated, acrosome-reacted sperm. In contrast, vitality, total motility, progressive motility, curvilinear velocity, average-path velocity and the percentages of hyper-activated, ROS-positive and MMP-intact sperm decreased significantly (P < 0.05), while the percentage of DNA-fragmented sperm increased (P < 0.05). Conclusions: Our data show that aqueous C. papaya seed extract significantly and negatively affects sperm motility parameters crucial for fertility; and thus, poses as a likely candidate for male contraception.
Dexamethasone's impact on LPS-induced inflammation of blood-brain barrier endothelial cells
(University of the Western Cape, 2023) Jeftha, Tershlin; Fisher, David
The blood-brain barrier (BBB) serves as a protective and regulatory barrier between the blood and the brain. Microvascular endothelial cells provide homeostatic regulation of the central nervous system (CNS). BBB integrity is compromised by inflammation, which contributes to a wide range of neurological disorders. It has been shown that glucocorticoids (GCs) have anti-inflammatory functions on a variety of cells. A particular example is Dexamethasone (Dex), which is used for the treatment of inflammatory conditions and has recently been found to be effective in attenuating COVID-19. Aim: The purpose of this study was to determine whether low or high concentrations of Dex can attenuate the inflammatory response induced by LPS on the in vitro BBB model.
Differential effects of normoxic versus hypoxic derived breast cancer paracrine factors on brain endothelial cells
(MPDI, 2021) Rado, Mariam; Flepisi, Brian; Fisher, David
The blood-brain barrier (BBB) is a central nervous system protective barrier formed primarily of endothelial cells that regulate the entry of substances and cells from entering the brain. However, the BBB integrity is disrupted in disease, including cancer, allowing toxic substances, molecules, and circulating cells to enter the brain. This study aimed to determine the mitochondrial changes in brain endothelial cells co-cultured with cancer cells. Method: Brain endothelial cells (bEnd.3) were co-cultivated with various concentrations of breast cancer (MCF7) conditioned media (CM) generated under normoxic (21% O2 ) and hypoxic conditions (5% O2 ). The mitochondrial activities (including; dehydrogenases activity, mitochondrial membrane potential (∆Ψm), and ATP generation) were measured using Polarstar Omega B.M.G-Plate reader. Trans-endothelial electrical resistance (TEER) was evaluated using the EVOM system, followed by quantifying gene expression of the endothelial tight junction (ETJs) using qPCR. Results: bEnd.3 cells had reduced cell viability after 72 h and 96 h exposure to MCF7CM under hypoxic and normoxic conditions.
The effect of methamphetamine on the blood-testis barrier
(University of the Western Cape, 2018) Zabida, Omer Saleh; Fisher, David
Introduction The blood-testis barrier (BTB) is formed by tight junctions between adjacent Sertoli cells. The barrier formed by these tight junction helps to create a specialized environment for spermatogenesis and provide an immunological barrier to protect developing germ cells. Methamphetamine (Meth) is known as neurotoxin however, its effects on the male reproductive system, especially on Sertoli cells and, the BTB are not well established. Therefore, this study aimed to determine the effects of Meth on the TM4 mouse testis Sertoli cell line and on the integrity of the BTB permeability. Materials and Methods This study investigated the effect of selected concentrations of Meth (0.1 μM, 1 μM, 10 μM, 20 μM and 100 μM) on TM4 mouse testis Sertoli cell line for 24 until 96 hours, using two treatments: an “acute” study (24 hrs exposure) and a “chronic” study, where treatment occurred on a daily basis over 96 hrs. The following parameters were investigated: viability, cell proliferation, mitochondrial activity, monolayer permeability.
The effects of Ethanol and Aspalathus linearis on immortalized mouse brain endothelial cells (bEnd5)
(University of the Western Cape, 2015) Thomas, Kelly Angelique; Fisher, David; Gamieldien, Kareemah
The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of t h e Rf extract was able to partially alleviate excess ROS-induced effects.
The effects of ethanol and aspalathus linearis on immortalized mouse brain endothelial cells (bEnd5)
(University of the Western Cape, 2015) Thomas, Kelly Angelique; Fisher, David; Gamieldien, Kareemah
The blood brain barrier (BBB) is a signaling interface between the blood and the central nervous system (CNS), which prohibits the entry of harmful blood-borne substances into the brain micro-environment, thus maintaining brain homeostasis. The crucial role of the BBB is protecting the CNS, which may adversely be affected by alcohol. The central component of the BBB, endothelial cells (ECs), regulates BBB transport by regulating the permeability both transcellularly and through their paracellular junctions, by structures called tight junctions (TJs) that are composed of proteins. The aim of this study was to investigate the in vitro effects of ethanol (EtOH) and fermented rooibos (Rf) on a monolayer of bEnd5 mouse brain ECs, by determining the effects of EtOH and Rf on bEnd5 (i) cell viability (ii) cell proliferation (iii) rate of cell division (iv) cell toxicity (v) claudin-5 transcription (vi) permeability across a monolayer of bEnd5 ECs and (vii) morphology, for a selected experimental timeline of 24, 48, 72, and 96hrs. We then investigated if the simultaneous exposure of Rf and EtOH could reverse or alleviate the EtOHinduced effects on the bEnd5 ECs. EtOH metabolism induces oxidative stress and results in a range of adverse physiological effects. Aspathalus linearis (rooibos) contains many phenolic compounds, of which the main antioxidant activity is attributed to aspalathin. Our underlining hypothesis is that the antioxidants in an aqueous rooibos extract may therefore protect against the potential oxidant damaging effects of alcohol on the BBB. Cells were exposed for 24hrs to selected concentrations of EtOH (25mM and 100mM), a concentration of Rf containing equivalent of 1.9nM aspalathin, and the combinations of EtOH and Rf. Cell viability and cell toxicity was determined, while cell proliferation and rate of cell division was estimated using the trypan blue exclusion assay. Real time quantitative PCR was implemented to quantify claudin-5 transcription, normalized against housekeeping genes, GAPDH and HPRT. Transepithelial electrical resistance (TEER) was measured using the Ohm Millicell-electrical resistance system, while bEnd5 monolayer morphology was analysed using the Zeiss scanning electron microscope. Both concentrations of EtOH led to an overall decrease in cell viability, and a decreased number of live cells across 72hrs. Consistent with this, EtOH resulted in increased cell toxicity across the 96hr experimental timeframe and a diminished rate of cell division. The transcription of claudin-5 in bEnd5 ECs exposed to 25mM and 100mM EtOH varied dramatically across the 96hr timeframe. While 25mM EtOH resulted in an overall decrease in TEER, cells exposed to 100mM EtOH only decreased TEER between 48 and 96hrs. Morphologically, both concentrations of EtOH led to compromised paracellular spaces as endorsed by high definition SEM analysis. The administration of Rf on its own resulted in an initial decrease in viability, followed by recovery between 72 and 96hrs. Exposure to Rf diminished live cell numbers at 72 and 96hrs, accompanied by a compromised rate of cell division and an overall increase in cell toxicity. In addition, Rf down-regulated claudin-5 transcription across the course of the experiment, particularly between 24 and 48hrs. In alignment with this, Rf also led to an increase in BBB permeability from 24 to 96hrs. However, SEM studies were not able to discriminate any differences between control and Rf treated cells. Our study showed that the BBB could be protected against the adverse effects of EtOH, and this at the plasma concentration induced by 500ml’s of Rooibos tea. The simultaneous exposure of Rf and EtOH was able to negate the effects of EtOH on cell viability, cell proliferation, and cell toxicity but exacerbated the effects of EtOH on claudin-5 transcription and paracellular permeability. Morphologically, co-exposure with Rf only reversed the effects of 25mM EtOH while exacerbating the effects of 100mM EtOH at 96hrs. In conclusion, EtOH was shown to be detrimental to the integrity of bEnd5 ECs, and the addition of a minuscule quantity of the Rf extract was able to partially alleviate excess ROS-induced effects.
Exosomes form tunneling nanotubes (TUNTs) in the blood-brain barrier: A nano-anatomical perspective of barrier genesis
(Frontiers Media, 2022) Mentor, Shireen; Fisher, David
The blood-brain barrier (BBB) is a robust interface between the blood and the central nervous system. Barrier type endothelium is able to limit paracellular (PC) movement, relegating molecular flux to the transendothelial pathways of brain endothelial cells (BECs). It is, therefore, apparent that any leakage via the PC shunts would effectively nullify the regulation of molecular flux across the transcellular pathways. The application of higher-resolution scanning electron microscopy (HR-SEM) illuminates the heterogenous, morphological profile that exists on the surface of BEC membranes and the relationship between these ultrastructures during the molecular construction of the PC space between adjacent BECs. In this study developing BEC monolayers were grown on mixed, cellulose esters insert membranes in a bicameral system.
Face-to-face versus online-based lectures: A covid-19 induced study on assessments
(Frontiers Media, 2022) Fisher, David; Solomons, Deon; Makhathini, Khayelihle Brian
There is much debate regarding the impact of COVID-induced lockdown on the standard of assessments, mainly since students were assessed at home via an online assessment platform. Regular orthodox lectures and assessments were carried out during the first term, while the strictly enforced South African COVID lockdown warranted that 2nd term lectures and assessments were based online. This created the fortunate control conditions to statistically compare orthodox face-to-face with online-home-based assessments.
High-resolution insights into the in vitro developing blood-brain barrier: Novel morphological features of endothelial nanotube function
(Frontiers Media, 2021) Mentor, Shireen; Fisher, David
High-resolution electron microscopy (HREM) imaging of the in vitro blood-brain barrier (BBB), is a promising modality for investigating the dynamic morphological interplay underpinning BBB development. The successful establishment of BBB integrity is grounded in the brain endothelial cells (BEC’s) ability to occlude its paracellular spaces of brain capillaries through the expression of the intercellular tight junction (TJ) proteins. The impermeability of these paracellular spaces are crucial in the regulation of transcellular transport systems to achieve homeostasis of the central nervous system. To-date research describing morphologically, the dynamics by which TJ interaction is orchestrated to successfully construct a specialized barrier remains undescribed. In this study, the application of HREM illuminates the novel, dynamic and highly restrictive BEC paracellular pathway which is founded based on lateral membrane alignment which is the functional imperative for the mechanical juxtapositioning of TJ zones that underpin molecular bonding and sealing of the paracellular space.
The in vitro effects of pure and street methamphetamine on the proliferation and cell cycle of mouse brain endothelial (bend5) cells
(2012) Mafunda, Patrick Siyambulela; Fisher, David; Gamieldien, K.
The blood-brain barrier (BBB) is an interface between the brain parenchyma and the circulating system. This barrier plays a vital role in protecting the CNS by restricting free paracellular diffusion of molecules from the systemic circulation. Methamphetamine (MA) is a highly addictive psychostimulant and has demonstrated neurotoxic properties as well as the ability to compromise the BBB. MA exposure is strongly linked with increased oxidative stress which can result in a decrease in the integrity of the BBB.The aim of this study was to investigate in vitro effects of pure and street MA "tik" on DNA proliferation and cell cycles in mouse brain endothelial (bEnd5) cells. Trypan blue was used to determine effects of MA (0.0001M-1mM) on cell viability and % cell growth. The Cell Titer Glo® luminescent assay and nonradioactive analogue, 5-bromo-2'-deoxyuridine (BrdU) was used to detect ATP and DNA levels, respectively. Cell cycles (propidium iodide incorporation) were analysed using flow cytometry. Statistical analysis was performed using Wilcoxin Rank Sum Test in which P<0.05 was denoted as significant. Results of this study showed that: 1. Viability of bEnd5 cells exposed to all selected concentrations of MA were unaffected when compared to controls (P>0.05). 2. % Cell growth was suppressed by MA exposure at 96hrs in comparison to that of controls (P≤0.03). 3. Cells exposed to MA had significant higher ATP concentrations than control cells at 96hrs (P ≤.0.03) 4. DNA synthesis was markedly suppressed in cells exposed to pure MA and street MA sample 4 (P≤0.03), while was similar and higher in cells exposed to street MA sample 1 (P=0.39), and street MA sample 2 and 3 (P≤0.04), respectively at 96hrs. 5. bEnd5 cell were arrested between 72 and 96hrs at the G1-S phase. In conclusion, this study demonstrated pure and illicit samples of MA obtained from forensic police did not affect the viability of bEnd5 cells, however resulted in the significant suppression of their cell numbers. This growth inhibition may be due to MA-induced cell cycle arrest at the G1-S phase. The study also showed that compounds found in the samples of street MA produced results significantly different to that of pure MA.
Investigating novel aspects of the blood-brain barrier using high resolution electron microscopy
(University of the Western Cape, 2022) Mentor, Shireen; Fisher, David
The blood-brain barrier (BBB) is a restrictive interface located between the blood circulation and the central nervous system (CNS), regulating the homeostatic environment of the neuronal milieu, by controlling the permeability of the cerebrovasculature. Currently, we cannot fully comprehend the regulatory features and the complexity of BBB morphology to allow for intervention clinically. The thesis consists of four publications. The methodology paper proposes a novel experimental design to visualize the morphological architecture of immortalized mouse brain endothelial cell lines (bEnd3/bEnd5). The brain endothelial cells (BECs) were grown on cellulose matrices and fixed in 2.5 % glutaraldehyde in preparation for visualization of the paracellular (PC) spaces between adjacent BECs, employing high-resolution electron microscopy (HREM), with vested interest in the morphological profile of the developing BEC.
The Ism between endothelial cilia and endothelial nanotubules is an evolving concept in the genesis of the BBB
(MDPI, 2022) Mentor, Shireen; Fisher, David
The blood–brain barrier (BBB) is fundamental in maintaining central nervous system (CNS) homeostasis by regulating the chemical environment of the underlying brain parenchyma. Brain endothelial cells (BECs) constitute the anatomical and functional basis of the BBB. Communication between adjacent BECs is critical for establishing BBB integrity, and knowledge of its nanoscopic landscape will contribute to our understanding of how juxtaposed zones of tight-junction protein interactions between BECs are aligned. The review discusses and critiques types of nanostructures contributing to the process of BBB genesis. We further critically evaluate earlier findings in light of novel high-resolution electron microscopy descriptions of nanoscopic tubules.
The paracrine effect of hypoxic and normoxic cancer secretion on the proliferation of brain endothelial cells (bend.3)
(MDPI, 2022) Rado, Mariam; Fisher, David
: Background: This study aimed to investigate the disruption of cell cycle phases of bEnd.3 cells exposed to cancer paracrine secretion. Cancer cells have been reported to use the secretion of paracrine factors to compromise the endothelial barrier to prepare for their passage into the parenchyma. As cancer cells are known to act differently under conditions of hypoxia, we investigated how conditional media (CM) derived from breast and glioblastoma cells incubated under conditions of normoxia and hypoxia would affect proliferation of brain endothelial cells (bEnd.3). Methods: Brain endothelial cells (bEnd.3) were cultivated with normoxic and hypoxic CM generated from breast cancer MCF7 cells and glioblastoma U-87 cells. Cell proliferation was evaluated using the trypan blue exclusion assay and phases of the cell cycle were evaluated using flow cytometry. Results: bEnd.3 proliferations was suppressed more aggressively with hypoxic CM after 72 and 96 h; cell cycle analysis showed that paracrine treatment tended to prevent BECs from entering the G2 phase, thus suppressing cell division.
The relationship between seminal leukocytes, oxidative status in the ejaculate, and apoptotic markers in human spermatozoa
(Taylor & Francis, 2013) Mupfiga, Cleyson; Fisher, David; Kruger, Thinus; Henkel, Ralf
The aim of this study was to investigate the relationship between seminal leukocytes, reactive oxygen species (ROS) production in the ejaculate, and markers of apoptosis in human spermatozoa. Semen samples were collected from 60 patients attending fertility clinics at the Reproductive Biology Unit at Tygerberg Academic Hospital and Vincent Pallotti Hospital, Cape Town, South Africa. The concentration of seminal leukocytes was determined and was correlated with ROS production in the ejaculate, the percentage of superoxide (·O2 )- and hydrogen peroxide (H2O2)-positive spermatozoa, glutathione activation in the ejaculate, and with markers of apoptosis in spermatozoa, namely cysteine-dependent aspartate-directed proteases (caspase)-3/7 activation, mitochondrial membrane potential (ΔΨm), and the percentage of terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL)-positive sperm. Significant correlations with the concentration of seminal leukocytes were found for ROS production in the ejaculate, the percentage of ·O2 -positive spermatozoa, and caspase-3/7 activation in the ejaculate. Leukocytospermic samples showed significantly higher ROS production, percentage of ·O2 -positive sperm, GSH activation, and caspase-3/7 activation compared to non-leukocytospermic samples. The percentage of ·O2 -positive sperm was significantly correlated with sperm ΔΨm and caspase-3/7 activation in the ejaculate. Sperm ΔΨm and TUNEL-positive sperm did not correlate with seminal leukocyte concentration. Data demonstrate that high seminal leukocyte concentrations that leads to increased seminal ROS production, and is also associated with caspase activation in the male germ cell and increased mitochondrial ROS production. The latter could possibly be a result of disturbed ΔΨm. The activation of caspase-3/7 could then follow the increased intrinsic superoxide levels due to depleted intrinsic glutathione (GSH). These cellular events might not directly and immediately lead to DNA fragmentation as an endpoint of apoptosis because of topological hindrances.
Therapeutic potential of natural products in schistosomiasis-associated liver fibrosis
(Frontiers Media SA, 2024) Liu, Cuiling; Fisher, David; Pronyuk, Khrystyna
Schistosomiasis is a parasitic disease that endangers human health and social development. The granulomatous reaction of Schistosoma eggs in the liver is the main cause of hepatosplenomegaly and fibrotic lesions. Anti liver fibrosis therapy is crucial for patients with chronic schistosomiasis. Although Praziquantel is the only clinical drug used, it is limited in insecticide treatment and has a long-term large-scale use, which is forcing the search for cost-effective alternatives. Previous research has demonstrated that plant metabolites and extracts have effective therapeutic effects on liver fibrosis associated with schistosomiasis. This paper summarizes the mechanisms of action of metabolites and some plant extracts in alleviating schistosomiasis-associated liver fibrosis. The analysis was conducted using databases such as PubMed, google scholar, and China national knowledge infrastructure (CNKI) databases. Some plant metabolites and extracts ameliorate liver fibrosis by targeting multiple signaling pathways, including reducing inflammatory infiltration, oxidative stress, inhibiting alternate macrophage activation, suppressing hepatic stellate cell activation, and reducing worm egg load. Natural products improve liver fibrosis associated with schistosomiasis, but further research is needed to elucidate the effectiveness of natural products in treating liver fibrosis caused by schistosomiasis, as there is no reported data from clinical trials in the literature.

Browsing by Author "Fisher, David"

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