Browsing by Author "Katz, Arieh A"

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    Exercise intervention alters HDL subclass distribution and function in obese women
    (BioMed Central, 2018-10-10) Woudberg, Nicholas J; Mendham, Amy E; Katz, Arieh A; Goedecke, Julia H; Lecour, Sandrine
    Background Obesity is associated with a change in high-density lipoprotein (HDL) function and subclass. Exercise training reduces cardiovascular risk in obese patients. We aimed to explore the effect of an exercise training stimulus on HDL functionality and subclass in obese women. Methods Thirty-two obese black South African women were randomly assigned to exercise (combined aerobic and resistance exercise) or control (no exercise) conditions for 12-weeks. Pre- and post-testing included venous blood sampling for analysis of lipid profile and HDL functionality, by measuring cellular cholesterol efflux capacity, reduction in endothelial vascular cell adhesion molecule (VCAM) expression (anti-inflammatory function), paraoxonase (PON) (antioxidative function) and platelet activating factor acetylhydrolase (PAF-AH) activities (anti-thrombotic function). PON-1 and PAF-AH expression were determined in serum and in isolated HDL using Western blotting. Levels of large, intermediate and small HDL subclasses were measured using the Lipoprint® system. Results Exercise training resulted in a decrease in body mass index (− 1.0 ± 0.5% vs + 1.2 ± 0.6%, p = 0.010), PON activity (− 8.7 ± 2.4% vs + 1.1 ± 3.0%, p = 0.021), PAF-AH serum expression (− 22.1 ± 8.0% vs + 16.9 ± 9.8, p = 0.002), and the distribution of small HDL subclasses (− 10.1 ± 5.4% vs + 15.7 ± 6.6%, p = 0.004) compared to controls. Exercise did not alter HDL cellular cholesterol efflux capacity and anti-inflammatory function. Conclusions These results demonstrate the potential for exercise training to modify HDL subclass distribution and HDL function in obese women. Trial registration Clinical trials number: PACTR201711002789113 .
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    Exogenous Vimentin Supplementation Transiently Affects Early Steps during HPV16 Pseudovirus Infection
    (2021-12-10) Carse, Sinead; Lang, Dirk; Katz, Arieh A; Schäfer, Georgia
    Understanding and modulating the early steps in oncogenic Human Papillomavirus (HPV) infection has great cancer-preventative potential, as this virus is the etiological agent of virtually all cervical cancer cases and is associated with many other anogenital and oropharyngeal cancers. Previous work from our laboratory has identified cell-surface-expressed vimentin as a novel HPV16 pseudovirus (HPV16-PsVs)-binding molecule modulating its infectious potential. To further explore its mode of inhibiting HPV16-PsVs internalisation, we supplemented it with exogenous recombinant human vimentin and show that only the globular form of the molecule (as opposed to the filamentous form) inhibited HPV16-PsVs internalisation in vitro. Further, this inhibitory effect was only transient and not sustained over prolonged incubation times, as demonstrated in vitro and in vivo, possibly due to full-entry molecule engagement by the virions once saturation levels have been reached. The vimentin-mediated delay of HPV16-PsVs internalisation could be narrowed down to affecting multiple steps during the virus’ interaction with the host cell and was found to affect both heparan sulphate proteoglycan (HSPG) binding as well as the subsequent entry receptor complex engagement. Interestingly, decreased pseudovirus internalisation (but not infection) in the presence of vimentin was also demonstrated for oncogenic HPV types 18, 31 and 45. Together, these data demonstrate the potential of vimentin as a modulator of HPV infection which can be used as a tool to study early mechanisms in infectious internalisation. However, further refinement is needed with regard to vimentin’s stabilisation and formulation before its development as an alternative prophylactic means.
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    Filamin a binds to CCR2B and regulates its internalization
    (Public Library of Science, 2010) Minsaas, Laura; Planagumà, Jesús; Madziva, Michael; Krakstad, Beate F; Masià-Balagué, Míriam; Katz, Arieh A; Aragay, Anna M
    The chemokine (C-C motif) receptor 2B (CCR2B) is one of the two isoforms of the receptor for monocyte chemoattractant protein-1 (CCL2), the major chemoattractant for monocytes, involved in an array of chronic inflammatory diseases. Employing the yeast two-hybrid system, we identified the actin-binding protein filamin A (FLNa) as a protein that associates with the carboxyl-terminal tail of CCR2B. Co-immunoprecipitation experiments and in vitro pull down assays demonstrated that FLNa binds constitutively to CCR2B. The colocalization of endogenous CCR2B and filamin A was detected at the surface and in internalized vesicles of THP-1 cells. In addition, CCR2B and FLNa were colocalized in lamellipodia structures of CCR2B-expressing A7 cells. Expression of the receptor in filamin-deficient M2 cells together with siRNA experiments knocking down FLNa in HEK293 cells, demonstrated that lack of FLNa delays the internalization of the receptor. Furthermore, depletion of FLNa in THP-1 monocytes by RNA interference reduced the migration of cells in response to MCP-1. Therefore, FLNa emerges as an important protein for controlling the internalization and spatial localization of the CCR2B receptor in different dynamic membrane structures.
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    Investigating excitatory GABAergic signalling & benzodiazepine resistance in an in vitro model of status epilepticus
    (2018) Burman, Richard J; Raimondo, Joseph V; Katz, Arieh A
    Status epilepticus (SE) describes a state of persistent seizures which are unrelenting. First- line treatment for status epilepticus uses a group of drugs, the benzodiazepines, that promote the action of the major inhibitory neurotransmitter within the brain, gamma (γ)-aminobutyric acid (GABA). In a subset of patients however, benzodiazepines prove to be ineffective in terminating SE. Previous data from in vitro models has demonstrated that during single seizures, instead of being inhibitory, activation of the GABAA receptor can have an excitatory effect on neurons. To date, it is unknown whether this shift in GABAergic function contributes to SE, nor how it may modulate the anticonvulsant properties of benzodiazepines. In this thesis I explore the role of excitatory GABAergic signaling in an in vitro model of SE and how this may affect the anticonvulsant efficacy of the benzodiazepine, diazepam. Firstly, I confirm that benzodiazepine-resistant SE is prevalent in a South African paediatric population. Secondly, consistent with its established mechanism of action, I show that diazepam enhances GABAAR synaptic currents. Thirdly, using the in vitro 0 Mg²⁺ model of status epilepticus I show that whilst early application of diazepam has anticonvulsant properties, this is lost when the drug is applied during prolonged epileptiform activity. Fourthly, to investigate this phenomenon I use optogenetic activation of GABAergic interneurons to show that interneurons can drive epileptiform discharges during SE-like activity in vitro. Finally, I confirm that during seizure-like events there is a transient shift in GABAergic signaling that is caused by activity driven changes in the transmembrane Cl⁻ gradient. This thesis provides insight into how excitatory GABAergic signaling during prolonged seizures may contribute towards benzodiazepine resistance in SE. I believe that these results are relevant for understanding of the pathophysiology of SE and may help inform optimal treatment protocols for this condition.
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    Kaposi's sarcoma: Genetic subtypes and clinical correlation in a South African population
    (2017) Isaacs, Thuraya; Todd, Gail; Katz, Arieh A
    Human herpes virus 8 (HHV8) is the aetiological agent of all forms of Kaposi's sarcoma (KS). Seven major subtypes (A, B, C, D, E, F, Z) based on genetic variability of open reading frame (ORF)-K1, have been identified. Numerous studies point to differing tumorigenic and pathogenic properties of the HHV8 subtypes. The study objective was to determine the prevalence of the HHV8 subtypes in a cohort of clinical and histologically confirmed KS in Cape Town, South Africa, and analyse associations between the different subtypes, clinico- epidemiological forms and clinical presentation of KS. The clinical data was prospectively collected and recorded on a body diagram and with photographs. Demographic data was retrospectively collected from clinical records. Tissue biopsies were taken for ORF-K1 subtyping. Out of a cohort of 103, eighty six patients were subtyped; 81 AIDS (aquired immune deficiency syndrome)-KS and 5 African endemic. Subtype A5 (42/86) and B2 (16/86) predominated. B1, B3, A1 and A4 subtypes were identified in 10/86, 9/86, 4/86 and 1/86 patients respectively. A5, B1, B2 and B3 were found in African blacks and individuals of mixed ancestry, while subtypes A1 and A4 are found only in whites and individuals of mixed ancestry. Subtype A5 was associated with >10 KS lesions at presentation in the AIDS-cohort (32/38, p=0,050), but not in the African endemic patients (2/4, p=0,600). Subtypes A1 and A4 were less likely to be associated with poor risk tumour extension (p=0,031) and A1 was associated with lower likelihood of lower limb involvement (p=0,004).
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    Kisspeptin regulation of genes involved in cell invasion and angiogenesis in first trimester human trophoblast cells
    (Public Library of Science, 2014) Francis, Víctor A; Abera, Aron B; Matjila, Mushi; Millar, Robert P; Katz, Arieh A
    The precise regulation of extravillous trophoblast invasion of the uterine wall is a key process in successful pregnancies. Kisspeptin (KP) has been shown to inhibit cancer cell metastasis and placental trophoblast cell migration. In this study primary cultures of first trimester human trophoblast cells have been utilized in order to study the regulation of invasion and angiogenesis-related genes by KP. Trophoblast cells were isolated from first trimester placenta and their identity was confirmed by immunostaining for cytokeratin-7. Real-time quantitative RT-PCR demonstrated that primary trophoblast cells express higher levels of GPR54 (KP receptor) and KP mRNA than the trophoblast cell line HTR8Svneo. Furthermore, trophoblast cells also expressed higher GPR54 and KP protein levels. Treating primary trophoblast cells with KP induced ERK1/2 phosphorylation, while co-treating the cells with a KP antagonist almost completely blocked the activation of ERK1/2 and demonstrated that KP through its cognate GPR54 receptor can activate ERK1/2 in trophoblast cells. KP reduced the migratory capability of trophoblast cells in a scratch-migration assay. Real-time quantitative RT-PCR demonstrated that KP treatment reduced the expression of matrix metalloproteinase 1, 2, 3, 7, 9, 10, 14 and VEGF-A, and increased the expression of tissue inhibitors of metalloproteinases 1 and 3. These results suggest that KP can inhibit first trimester trophoblast cells invasion via inhibition of cell migration and down regulation of the metalloproteinase system and VEGF-A.
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    Seminal plasma enhances cervical adenocarcinoma cell proliferation and tumour growth in vivo
    (Public Library of Science, 2012) Sutherland, Jason Robert; Sales, Kurt J; Jabbour, Henry N; Katz, Arieh A
    Cervical cancer is one of the leading causes of cancer-related death in women in sub-Saharan Africa. Extensive evidence has shown that cervical cancer and its precursor lesions are caused by Human papillomavirus (HPV) infection. Although the vast majority of HPV infections are naturally resolved, failure to eradicate infected cells has been shown to promote viral persistence and tumorigenesis. Furthermore, following neoplastic transformation, exposure of cervical epithelial cells to inflammatory mediators either directly or via the systemic circulation may enhance progression of the disease. It is well recognised that seminal plasma contains an abundance of inflammatory mediators, which are identified as regulators of tumour growth. Here we investigated the role of seminal plasma in regulating neoplastic cervical epithelial cell growth and tumorigenesis. Using HeLa cervical adenocarcinoma cells, we found that seminal plasma (SP) induced the expression of the inflammatory enzymes, prostaglandin endoperoxide synthase (PTGS1 and PTGS2), cytokines interleukin (IL) -6, and -11 and vascular endothelial growth factor-A(VEGF-A). To investigate the role of SP on tumour cell growth in vivo, we xenografted HeLa cells subcutaneously into the dorsal flank of nude mice. Intra-peritoneal administration of SP rapidly and significantly enhanced the tumour growth rate and size of HeLa cell xenografts in nude mice. As observed in vitro, we found that SP induced expression of inflammatory PTGS enzymes, cytokines and VEGF-A in vivo. Furthermore we found that SP enhances blood vessel size in HeLa cell xenografts. Finally we show that SP-induced cytokine production, VEGF-A expression and cell proliferation are mediated via the induction of the inflammatory PTGS pathway.
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    Seminal plasma induces the expression of IL-1α in normal and neoplastic cervical cells via EP2/EGFR/PI3K/AKT pathway
    (2014-08-08) Adefuye, Anthonio O; Sales, Kurt J; Katz, Arieh A
    Abstract Background Cervical cancer is a chronic inflammatory disease of multifactorial etiology usually presenting in sexually active women. Exposure of neoplastic cervical epithelial cells to seminal plasma (SP) has been shown to promote the growth of cancer cells in vitro and tumors in vivo by inducing the expression of inflammatory mediators including pro-inflammatory cytokines. IL-1α is a pleotropic pro-inflammatory cytokine induced in several human cancers and has been associated with virulent tumor phenotype and poorer prognosis. Here we investigated the expression of IL-1α in cervical cancer, the role of SP in the regulation of IL-1α in neoplastic cervical epithelial cells and the molecular mechanism underlying this regulation. Methods and results Real-time quantitative RT-PCR confirmed the elevated expression of IL-1α mRNA in cervical squamous cell carcinoma and adenocarcinoma tissue explants, compared with normal cervix. Using immunohistochemistry, IL-1α was localized to the neoplastically transformed squamous, columnar and glandular epithelium in all cases of squamous cell carcinoma and adenocarcinomas explants studied. We found that SP induced the expression of IL-α in both normal and neoplastic cervical tissue explants. Employing HeLa (adenocarcinoma) cell line as a model system we identified PGE2 and EGF as possible ligands responsible for SP-mediated induction of IL-1α in these neoplastic cells. In addition, we showed that SP activates EP2/EGFR/PI3kinase-Akt signaling to induce IL-1α mRNA and protein expression. Furthermore, we demonstrate that in normal cervical tissue explants the induction of IL-1α by SP is via the activation of EP2/EGFR/PI3 kinase-Akt signaling. Conclusion SP-mediated induction of IL-1α in normal and neoplastic cervical epithelial cells suggests that SP may promote cervical inflammation as well as progression of cervical cancer in sexually active women.
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    The garlic compound ajoene covalently binds vimentin, disrupts the vimentin network and exerts anti-metastatic activity in cancer cells
    (2019-03-20) Kaschula, Catherine H; Tuveri, Rosanna; Ngarande, Ellen; Dzobo, Kevin; Barnett, Christopher; Kusza, Daniel A; Graham, Lisa M; Katz, Arieh A; Rafudeen, Mohamed S; Parker, M Iqba; Hunter, Roger; Schäfer, Georgia
    Background Garlic has been used for centuries for its flavour and health promoting properties that include protection against cancer. The vinyl disulfide-sulfoxide ajoene is one of the phytochemicals found in crushed cloves, hypothesised to act by S-thiolating reactive cysteines in target proteins. Methods Using our fluorescently labelled ajoene analogue called dansyl-ajoene, ajoene’s protein targets in MDA-MB-231 breast cancer cells were tagged and separated by 2D electrophoresis. A predominant band was identified by MALDI-TOF MS/MS to be vimentin. Target validation experiments were performed using pure recombinant vimentin protein. Computational modelling of vimentin bound to ajoene was performed using Schrödinger and pKa calculations by Epik software. Cytotoxicity of ajoene in MDA-MB-231 and HeLa cells was measured by the MTT assay. The vimentin filament network was visualised in ajoene-treated and non-treated cells by immunofluorescence and vimentin protein expression was determined by immunoblot. The invasion and migration activity was measured by wound healing and transwell assays using wildtype cells and cells in which the vimentin protein had been transiently knocked down by siRNA or overexpressed. Results The dominant protein tagged by dansyl-ajoene was identified to be the 57 kDa protein vimentin. The vimentin target was validated to reveal that ajoene and dansyl-ajoene covalently bind to recombinant vimentin via a disulfide linkage at Cys-328. Computational modelling showed Cys-328 to be exposed at the termini of the vimentin tetramer. Treatment of MDA-MB-231 or HeLa cells with a non-cytotoxic concentration of ajoene caused the vimentin filament network to condense; and to increase vimentin protein expression. Ajoene inhibited the invasion and migration of both cancer cell lines which was found to be dependent on the presence of vimentin. Vimentin overexpression caused cells to become more migratory, an effect that was completely rescued by ajoene. Conclusions The garlic-derived phytochemical ajoene targets and covalently modifies vimentin in cancer cells by S-thiolating Cys-328. This interaction results in the disruption of the vimentin filament network and contributes to the anti-metastatic activity of ajoene in cancer cells.