Browsing by Author "Katz, Arieh"

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    Characterisation of human surfactant protein A and recombinant human vimentin in their modulation of HPV16 pseudovirus infection
    (2019) Carse, Sinead; Schafer, Georgia; Katz, Arieh
    Infection by oncogenic human papillomavirus (HPV) is the primary cause of cervical cancer, where low-and middle-income countries (LMIC) have the highest incidence. Prophylactic HPV vaccines exist but LMIC have limited access. Therefore, alternative preventative measures against HPV infection and cervical cancer progression are needed. Two human proteins have been identified in our laboratory that modulate HPV16 pseudovirus (HPV16-PsVs) infection in vitro, namely surfactant protein A (SP-A) and recombinant human vimentin (rhVim). Previous work suggested SP-A mediated immune recognition of HPV since SP-A-coated HPV16- PsVs enhanced viral uptake by RAW264.7 murine macrophages. These initial observations were confirmed using a murine C57BL/6 cervicovaginal challenge model: pre-incubation of HPV16- PsVs with purified human SP-A significantly reduced the level of HPV16-PsV infection in vivo. Moreover, when isolated cells from female reproductive tracts of naïve C57BL/6 mice were incubated with HPV16-PsVs and stained for selected innate immune cell populations by flow cytometry, significant increases in viral uptake by eosinophils, neutrophils, monocytes and macrophages were observed over time using SP-A-pre-coated virions compared to control particles. Compared to SP-A mediated modulation of HPV infection through activation of innate immune responses, rhVim was suggested to directly interfere with HPV entry into host cells. Indeed, supplementation with non-filamentous rhVim resulted in decreased viral uptake by NIKS cells which was confirmed in vivo using the murine C57BL/6 cervicovaginal HPV16-PsVs challenge model. Co-localisation analysis employing confocal imaging, revealed that rhVim-coated HPV16- PsVs co-localised, to a lesser degree, with surface-expressed heparan sulphate proteoglycans (HSPGs) than control particles. Removal of surface HSPGs on NIKS cells decreased HPV16-PsVs cell surface binding and internalisation, while pre-incubation of HPV16-PsVs with rhVim decreased viral particle binding and internalisation to a greater extent. This indicates that rhVim may modulate HPV16 infection by interfering with its attachment to HSPGs as well as viral engagement with the yet unknown entry receptor(s). In summary, both SP-A and vimentin modulate HPV16-PsVs infection by different mechanisms. These in vivo studies strongly confirm previous in vitro observations, rendering both proteins potentially suitable for further development into possible candidates for use in topical microbicides, which may provide protection against new HPV infections.
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    Characterisation of Kaposi's sarcoma-associated herpesvirus (KSHV)-driven pathology and disease outcome in HIV infected South African patients
    (2020) Blumenthal, Melissa; Schafer, Georgia; Katz, Arieh
    Kaposi's sarcoma-associated herpesvirus (KSHV), a gamma-herpesvirus with a particularly high seroprevalence in Sub-Saharan Africa (SSA), is the etiological agent of the endothelial tumour Kaposi's sarcoma (KS), the most common acquired immunodeficiency syndrome (AIDS)-related malignancy worldwide and particularly in SSA. It also causes primary effusion lymphoma (PEL), multicentric Castleman disease (MCD) and KSHV inflammatory cytokine syndrome (KICS). AIDS-related deaths have declined, due to global scale-up of antiretroviral therapy (ART). However, the vast majority of these occurred in SSA, where tuberculosis (TB) is the leading cause of mortality among human immunodeficiency virus (HIV)-infected individuals, accounting for a third of all AIDS-related deaths. The exceptionally high burden of suspected TB in SSA causes misdiagnosis or delayed diagnosis of diseases mimicking TB, such as several pathologies associated with KSHV. KSHV infection is essential but insufficient for the development of KS and other KSHV-associated pathologies; precipitating factors, such as HIV-related immune suppression and potentially genetic predisposition, are required. The erythropoietin-producing hepatocellular carcinoma (Eph) receptor A2 protein (EPHA2) tyrosine kinase receptor is a promising candidate for studies on genetic variants as it potentially acts on two levels: susceptibility to KSHV infection (being one of the key receptors utilised by KSHV for cell entry and intracellular trafficking) and susceptibility to KS development (being implicated in oncogenesis). Despite the high seroprevalence in SSA, the contribution of dysregulated KSHV lytic replication or host KSHV receptor variations to disease outcome in HIV-infected patients is unknown. We hypothesised that KSHV lytic reactivation plays yet unrecognised roles for morbidity and mortality in high HIV settings and to this end, we conducted a cohort study of 682 HIV-positive critically ill patients admitted to Khayelitsha Day Hospital, South Africa, investigated for TB, and followed for 12-weeks to ascertain vital status. We demonstrated that elevated blood KSHV viral load (VL) was a strong predictor of death in hospitalised HIV-infected patients without microbiologically proven TB. Further, we identified and validated variants in the EPHA2 protein tyrosine kinase and sterile alpha motif domains that were significantly associated with susceptibility to infection, KS development and/or KSHV VL in 300 South African HIV-infected patients, by aggregate by-gene analysis. In order to elucidate the functional significance of the identified EPHA2 missense mutations, we knocked out endogenous EPHA2 by CRISPR/Cas9 in the human endothelial cell line, HuARLT2, and reintroduced the wild type and mutant EPHA2 open reading frames by lentiviral transduction. These engineered cells were assessed for baseline EPHA2 phosphorylation levels and susceptibility to KSHV infection utilising recombinant KSHV in binding, internalisation and infection assays. We found that the EPHA2 mutant c.2254T>C (p.Leu700Pro) in the tyrosine kinase domain, associated with KS in our patient cohort, was deficient in tyrosine phosphorylation and less permissive to rKSHV infection when introduced as a single mutation or as a double mutant together with c.2257A>C (p.Asp701Ala) which was found to be in linkage disequilibrium with it. Another tyrosine kinase domain variant, c.2688G>S (p.Ala845Pro), found to be overrepresented among KS patients, had enhanced baseline tyrosine phosphorylation levels. These findings validated the patient-derived data on the molecular level by assigning functional consequences to some mutants which might have implications for the development of future biomarkers predicting KS susceptibility in high-risk populations. In summary, this novel research contributes to the understanding of KSHV-associated pathology and disease outcome. It identified KSHV VL as a potential biomarker to predict KSHV-associated diseases and mortality and assessed the contribution of KSHV entry receptor EPHA2 variations to KSHV-associated pathologies, with potential clinical implications, by facilitating the development of novel diagnostic and surveillance tools.
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    Characterization of signalling cross-talk between the EP2 and FP receptors in endometrial epithelial cells
    (2009) Abera, Aron Berhanie; Katz, Arieh; Jabbour, Henry
    Uterine fibroids are benign tumors that arise from the smooth-muscle uterine cells (myometrium) and are the most common uterine disorder occurring in as many as 30% of women over 35 years of age. Despite their frequent occurrence, the etiology of uterine fibroids is not well elucidated. Several studies have shown that numerous tumors can be regulated by cyclooxygenase (COX) enzyme products but their role in uterine fibroids is not well established. The initial aim of the study was to determine the expression level of COX enzymes and prostaglandin receptors in fibroids and autologous myometrium samples from women with fibroids. Real-Time reverse-transcriptase polymerase chain reaction (RT-PCR) revealed that the expression of COX enzymes, EP1, EP2 and EP4 prostanoid receptors and prolactin were not significantly altered while the EP3 subtype receptor was significantly down-regulated in fibroids compared to adjacent myometrium samples. The EP3 receptor has a protective role in tumor development suggesting the role for down-regulation of the receptor in uterine fibroids pathology. In addition, the expression of COX enzymes, prostaglandin receptors and prostaglandin-mediated genes were assessed in endometrium samples from women with and without uterine fibroids in different stages of the menstrual cycle. COX-2 and interleukin-8 (IL-8) mRNA expressions were significantly higher in both proliferative stage and early-mid secretory, EP2 receptor and IL-11 were elevated in the proliferative stage, vascular endothelial growth factor (VEGF) was highly expressed in the early-mid secretory phase while FP receptor was up-regulated in all stages of the menstrual cycle in endometrium samples from women with fibroids. These data suggest that up-regulation of COX-2 and prostaglandin receptors (EP2 and FP) in endometrium can induce expression of angiogenic and mitogenic factors such as VEGF, IL-8 and IL-11 which might act in a paracrine manner on neighboring myometrial/fibroid tissue to promote angiogenesis and facilitate tumor growth. XVII Furthermore, since EP2 and FP receptors were up-regulated in the proliferative phase of endometrium from uterine fibroid patients and the receptors are co-expressed in endometrial adenocarcinoma (Ishikawa) cells, this study investigated a possible cross-talk that influences intracellular signalling by using Ishikawa cells stably expressing the EP2 and FP receptors (FPEP2 cells) as a model cell line. Real-Time RT-PCR, Western blot analysis and immunofluorescence microscopy confirmed stable expression of the EP2 and FP receptors in FPEP2 cells localized to the perinuclear and plasma membrane. Using FPEP2 cells, the integrated effect of Butaprost (EP2 receptor ligand) and PGF (FP receptor ligand) co-administration on inositol phosphate (IP3) and adenosine 3-,5-cyclic monophosphate (cAMP) release was assessed to study a possible heterologous-interaction or cross-talk between the EP2 and FP receptors. The study showed that in FPEP2 cells, PGF alone does not alter cAMP production, but in combination with Butaprost augments EP2 receptor-mediated cAMP release. PGF-mediated potentiation of cAMP release was abolished by antagonism of the FP receptor, inhibition of phospholipase C (PLC) and IP3-receptor whereas inhibition of protein kinase C (PKC) had no effect suggesting the cross-talk is mediated by FP receptor activation of IP3 release. Moreover, inhibition of calcium effectors using calmodulin antagonist (W7) or Ca2+/calmodulin-dependent kinase II (CaMK-II) inhibitor (KN-93) abolished PGF potentiation of Butaprost-mediated cAMP release. Using short interfering RNA (siRNA) molecules targeted against the adenylyl cyclase 3 (AC3) isoform, the study showed the isoform to be responsible for the cross-talk between the FP and EP2 receptors. In order to determine the integrative effects of the EP2 and FP receptors co-activation on gene expression, a whole genome array profiling in FPEP2 cells in response to Butaprost and/or PGF was performed. The gene array revealed 228 genes that are regulated by co-activation of the EP2 and FP receptors that are involved in cell morphology, proliferation and differentiation. XVIII In addition, co-activation of EP2 and FP receptors with their respective ligands enhanced or repressed a set of EP2 receptor-regulated genes. One of the genes identified, SAT1 (Spermidine/ N1-acetyltransferase), was regulated by the EP2 and FP receptors cross-talk via the calcium sensitive AC3 isoform. SAT1, with known role in regulation of tumorigenesis was also up-regulated in the proliferative stage of endometrium samples from women with uterine fibroids suggesting the EP2 and FP receptor cross-talk characterized in vitro can also happen in vivo. In conclusion, this study reports that COX-2, EP2 and FP receptors, VEGF, IL-8, IL-11 and SAT1 are up-regulated in endometrium from women with uterine fibroids. These genes play a major role in development of fibroids by facilitating angiogenesis and cell growth and by inhibiting apoptosis via autocrine/paracrine mechanisms. In addition, this study demonstrates that co-activation of the EP2 and FP receptors results in enhanced release of cAMP via the FP receptor-G +-q-Ca2+-calmodulin pathway by activating the calcium-sensitive AC3 isoform and modulates a molecular switch which alters the trans-activation of a subset single-receptor induced genes that have important functions in the pathogenesis of reproductive pathologies.
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    The differential expression of Kiss1, MMP9 and angiogenic regulators across the feto-maternal interface of healthy human pregnancies: implications for trophoblast invasion and vessel development
    (Public Library of Science, 2013) Matjila, Mushi; Millar, Robert; van der Spuy, Zephne; Katz, Arieh
    Genes involved in invasion of trophoblast cells and angiogenesis are crucial in determining pregnancy outcome. We therefore studied expression profiles of these genes in both fetal and maternal tissues to enhance our understanding of feto-maternal dialogue. We investigated the expression of genes involved in trophoblast invasion, namely Kiss1, Kiss1 Receptor (Kiss1R) and MMP9 as well as the expression of angiogenic ligands Vascular Endothelial Growth Factor-A ( VEGF-A) and Prokineticin-1 ( PROK1 ) and their respective receptors (VEGFR1, VEGFR2 and PROK1R ) across the feto-maternal interface of healthy human pregnancies. The placenta, placental bed and decidua parietalis were sampled at elective caesarean delivery. Real-time RT-PCR was used to investigate transcription, while immunohistochemistry and western blot analyses were utilized to study protein expression. We found that the expression of Kiss1 (p<0.001), Kiss1R (p<0.05) and MMP9 (p<0.01) were higher in the placenta compared to the placental bed and decidua parietalis. In contrast, the expression of VEGF-A was highest in the placental bed ( p<0.001 ). While VEGFR1 expression was highest in the placenta (p<0.01), the expression of VEGFR2 was highest in the placental bed (p<0.001). Lastly, both PROK1 (p<0.001) and its receptor PROK1R (p<0.001) had highest expression in the placenta. Genes associated with trophoblast invasion were highly expressed in the placenta which could suggest that the influence on invasion capacity may largely be exercised at the fetal level. Furthermore, our findings on angiogenic gene expression profiles suggest that angiogenesis may be regulated by two distinct pathways with the PROK1/PROK1R system specifically mediating angiogenesis in the fetus and VEGFA/VEGFR2 ligand-receptor pair predominantly mediating maternal angiogenesis.
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    The effect of seminal fluid on TBx2 and TBX3 expression and activity in cervical cancer cells
    (2017) Cooper, George William; Katz, Arieh; Prince, Sharon
    Cervical cancer is one of the most common female cancers in Africa, both in terms of incidence and mortality, and is disproportionately prevalent in developing nations due to a lack of adequate access to healthcare. While new vaccine technologies are rapidly reducing the incidence of Human Papilloma Virus (HPV) infection, the primary causative agent of cervical cancer, new cases continue to accumulate in the developing world. Beyond the role of HPV in the early stages of cancer development, the molecular aetiology of this disease is poorly understood. Frequent exposure to seminal fluid (SF), the liquid component of semen, has been proposed as a potential driver of oncogenesis in cervical cancers and has been shown to exacerbate some aspects of cervical cancers. While some of the cellular signaling pathways responsible for these phenomena have been identified, much remains to be elucidated. We hypothesized that TBX2 and TBX3, two highly homologous transcription factors frequently implicated in other cancers, may be responsible for mediating some of the effects of SF on cervical cancer cells. We established that TBX3 protein is significantly overexpressed in both primary cervical adenocarcinomas and squamous cell carcinomas compared to normal tissue. SF was shown to increase expression of both TBX2 and TBX3 mRNA in HeLa and CaSki, but not C-33 A, cervical cancer cell lines. Furthermore, SF upregulated TBX3 protein expression in both of these cell lines. In contrast, TBX2 protein was undetectable in these cell lines. In addition, our results showed that SF treatment of HeLa cells increases the expression of the known TBX3 target gene, p21CIP1/WAF1 (p21), while having no effect on PTEN expression. Transient knockdown of TBX3 resulted in decreased p21 expression in SF-treated cells suggesting that SF upregulation of p21 is dependent on TBX3. This is the first study to investigate TBX3 protein expression in primary cervical tissues and SF regulation of TBX3. However, further research is required in order to elucidate the role of SF-induced TBX3 in cervical cancer development. The identification of the role of TBX3 in cervical cancer development could aid in the development of more effective treatments for cervical cancers and could potentially impact sexual health policy recommendations for women with cervical cancer.
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    Expression and functional role of cyclooxygenase enzymes in cervical carcinoma
    (2001) Sales, Kurt Jason; Katz, Arieh; Jabbour, Henry
    Cervical cancer is considered an important clinical problem in sub-Saharan Africa. Recent studies have suggested that epithelial tumors may be regulated by cyclooxygenase enzyme products. The purpose of this thesis was to determine the expression, localisation and possible functional role of cyclooxygenase enzymes in cervical carcinomas. The initial aim of the study was to determine whether cyclooxygenase-1 and cyclooxygenase-2 expession and prostglandin E₂ synthesis are up-regulated in cervical cancers. Real-time quantitative reverse-transcriptase polymerase chain reaction and Western blot analysis confirmed cyclooxygenase-1 and cyclooxygenase-2 ribonucleic acid and protein expression in all cases of squamous cell carcinoma and adenocarcinoma investigated. In contrast, minimal expression of cyclooxygenase-1 or cyclooxygenase-2 was detected in histologically normal cervix. Immunohistochemical analyses localised the site of cyclooxygenase-1 and cyclooxygenase-2 expression and prostaglandin E₂ synthesis to neoplastic epithelial cells of all squamous cell carcinomas and adenocarcinomas studied.
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    Functional consequences of South African mutations of the HIV-1 co-receptor, CCR5
    (2007) Folefoc, Asongna Theresia Forkem; Flanagan, Colleen; Katz, Arieh
    Four mutations of the CCR5 receptor have been identified in the South African population, but the effects of these mutations on CCR5 function and HIV infection are unknown. We have used in vitro methods to assess the ffect of the mutations, Asp2Val, Leu107Phe, Arg225Gln and Arg225stop, on CCR5 interactions with chemokine ligands and HIV.
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    The impact of EPHA2 polymorphism on KSHV infectivity and KS prevalence among HIV/AIDS patients in South Africa
    (2017) Blumenthal, Melissa Jayne Walcott; Schäfer, Georgia; Katz, Arieh
    Kaposi's Sarcoma (KS) is the most common Acquired Immune Deficiency Syndrome (AIDS)-related malignancy globally and is of particular significance in sub-Saharan Africa where, due to the Human Immunodeficiency Virus (HIV) epidemic, KS is the cause of significant morbidity and mortality. The oncogenic Kaposi's Sarcoma-associated herpes virus (KSHV) is the etiological agent of KS. Although KSHV seroprevalence in sub-Saharan Africa is high, not all AIDS patients develop KS, suggesting that host genetic factors contribute to susceptibility. The infection mechanism of KSHV in endothelial cells has recently been elucidated and highlights Eph Receptor A2 (EPHA2) as a specific host receptor for virus entry. Furthermore, EPHA2 has been implicated in oncogenesis and is upregulated in a number of cancers including KS. We therefore hypothesised that mutations in the KSHV host receptor's coding region could result in an altered EPHA2 that could affect susceptibility to KSHV infection and/or KS development among HIV/AIDS patients. To test our hypothesis, we studied three groups of HIV positive South African patients, namely patients with KS and patients without KS who were KSHV positive or KSHV negative. KS status was determined clinically and KSHV seroconversion was assessed using a combination of ELISAs to KSHV lytic antigen K8.1 and latency-associated nuclear antigen in patient plasma samples. All patients with KS were found to be KSHV seropositive as expected, while 45.45% of HIV positive patients without KS were found to be KSHV seropositive. From patient blood cells, we extracted genomic DNA and subsequently PCR amplified and sequenced the coding region of EPHA2, before comparing these sequences to the NCBI reference by multiple alignment. A number of variants were identified throughout the EPHA2 coding region and assessed statistically for association with KSHV susceptibility and/or KS prevalence. A novel heterozygous transition (c.2727C>T), which is predicted to result in the substitution of Cysteine for Arginine at amino acid position 858 in the functionally important tyrosine kinase domain, was identified as statistically associated with KSHV susceptibility as well as KS prevalence. Three additional missense variants (c.2254T>C, c.2257A>C and c.2688G>C) occurring in the tyrosine kinase domain and one occurring in the sterile-α-motif (c.2990G>T), a putative protein interaction domain, were found to be statistically associated with KS prevalence. This is the first study to investigate polymorphism in EPHA2 in HIV/AIDS patients in relation to susceptibility to KSHV infection and/or KS prevalence. The identification of variants in the KSHV entry receptor, EPHA2, opens new doors for the development of biomarkers involved in prognosis and treatment of KSHV-associated pathologies.
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    Interactions of GPR54 and GPR147 receptors with RF-amide ligands
    (2014) Hendrikse, Megan; Katz, Arieh; Millar, Robert
    G protein-coupled receptors play a key role in cellular signaling by transducing extracellular signals via G proteins to elicit intracellular responses. Studies have provided evidence supporting the role of the GPCR GPR54 and its cognate peptide ligand, kisspeptin (an RFamide peptide), in the regulation of reproduction. Kisspeptin and GPR54 play a critical role in the control of the hypothalamic-pituitary-gonadal axis by regulating gonadotropin-releasing hormone secretion. Despite the physiological importance of GPR54/kisspeptin signalling, the GRP54 residues important for receptor activation and signalling have not been extensively investigated. Another hypothalamic peptide, gonadotropin inhibiting hormone (also known as RFamide-related peptide), which interacts with the GPCR GPR147, has been found to inhibit GnRH-induced gonadotropin release and is therefore also of importance in control of the HPG axis. As many of the RFamide and RFamide-related receptors and ligands can be promiscuous, there is the potential for crosstalk between the GPR54/kisspeptin and GRP147/RFRP systems (or other RFamides) which may be of importance in the regulation of reproduction. GPR54 chimeras and point mutants were constructed in order to investigate the residues important for kisspeptin binding and receptor activation. The data obtained indicate that the acidic residues within the extracellular loops of GPR54 contribute to cell surface receptor expression and play a role in receptor signalling. In order to investigate the interactions of kisspeptin/RFRP peptides at GPR147 and GPR54, binding and activation of these receptors was studied with a range of ligands and their analogs. In addition to RFRP and its analogs, kisspeptin and several kisspeptin analogs were found to act as agonists at GRP147. In contrast, of all the ligands tested, only kisspeptin was able to bind to GPR54 with high affinity and elicit a response, thus indicating that GPR54 has high specificity for kisspeptin in contrast to the more promiscuous GPR147. These data demonstrate the therapeutic potential of kisspeptin analogs, for the inhibition of gonadotropin secretion and treatment of sex steroid hormone disease. In addition, these data have identified ligand and receptor residues important for binding and activation of GRP54/GRP147 which may aid development of new analogs targeting these receptors and highlighted the importance of testing these analogs for receptor specificity.
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    Investigating the immune modulatory properties of kisspeptin: implications for pregnancy
    (University of Cape Town, 2020) Botha, Stefan Marc; Katz, Arieh; Matjila Mushi
    Pregnancy is dependent on the development of maternal immune tolerance to the genetically foreign fetus. During pregnancy the mother's immune reactivity and energy metabolism undergoes significant changes and the levels of certain hormones in peripheral blood are significantly increased. Hormones are important regulators of the functional activity of the immune system and immune cells within. Hormones secreted by the placenta, protect the fetus from the maternal immune response of the mother, emphasizing their immunomodulatory effects. Therefore, hormonal regulation is essential for the functional activity of immune cells. There is evidence that the hormone, kisspeptin, plays a role in the development of immune tolerance during pregnancy based on its role in the regulation of the adaptive T regulatory (aTreg)/T-helper 17 (Th17) cells, induction of the enzyme indoleamine 2,3-dioxygenase (IDO) and regulation of monocyte function during pregnancy. In addition, kisspeptin has been implicated in the regulation of specific cytokines during pregnancy. It is crucial to maintain an appropriate cytokine balance at the maternal– fetal interface as well as in circulation. Several pregnancy-related disorders have been associated with a variation in Th1/Th2/Th17 cytokines and aTreg cell subsets. Kisspeptin has been implicated in regulating cytokines IL-10 and IL-17A as well as aTreg and Th17 cells which are significant role players in immune tolerance during pregnancy. However, its effect on other pro- and anti-inflammatory cytokines remain unknown. Therefore, more research is required to better understand the role of kisspeptin in the development of immune tolerance during pregnancy. The hypothesis of this study is that kisspeptin alters the expression of anti-and pro-inflammatory cytokines and may thus influence the establishment of immune tolerance in pregnancy. To test this hypothesis, we used a previously established in vitro peripheral blood mononuclear cell (PBMC) Mycobacterium tuberculosis (Mtb) infection assay model as well as a newly established in vitro infection model using lipopolysaccharide (LPS)-stimulated whole blood. Protein expression analysis of selected pro- and anti-inflammatory cytokines was performed on PBMC infected with Mtb and on whole blood cells stimulated with LPS in the absence and presence of kisspeptin-10 for different times. The cytokines levels were measured by luminex multiplex assay and sandwich ELISA, respectively. Results from the PBMC infection assay showed a varied but not statistically significant effect of kisspeptin-10 on selected pro- and anti-inflammatory cytokine expression at 2 hours post-infection. However, there was a suggestion of an inhibitory effect of kisspeptin-10 on selected pro- and anti-inflammatory cytokine expression, macrophage inflammatory protein (MIP)-1α, MIP-1β, tumour necrosis factor (TNF)-α, granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin (IL)-10, after 24 hours which was not observed at 6 days post-infection. Results from the whole blood stimulation assay suggested an inhibitory effect of kisspeptin-10 on selected LPS-induced pro-inflammatory cytokines (IL-1β, TNF-α, IL-6) whilst generally not having an effect on selected anti-inflammatory cytokines (IL-10). Overall this study suggests, based on the lack of statistically significant data, a potential immunomodulatory effect of kisspeptin-10 based on the observed inhibition of pro-inflammatory cytokines. Investigating and developing an understanding of key regulators and mechanisms of maternal immune tolerance may help researchers understand the pathophysiological mechanisms underlying certain pregnancy-related disorders. This was a pilot study aimed at characterising the effect of kisspeptin stimulation on cytokines and chemokines responses. Manipulation of regulatory hormones such as kisspeptin could represent a potentially novel approach in the treatment of various pregnancy-related disorders including preeclampsia and unexplained recurrent miscarriage.
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    The molecular pathways mediating the role of cyclooxygenase enzymes and prostaglandins in cervical neoplasias
    (2005) Muller, Melissa; Jabbour, Henry; Katz, Arieh
    Cervical Carcinoma is one of the leading causes of cancer-related death in women. The prevalence of this disease is particularly high in South Africa, occurring on average, in 60 out of every 100 000 women. Previous studies have demonstrated over-expression of cyclooxygenase-2 enzyme and enhanced synthesis of prostanoids, such as prostaglandin E2, in cervical carcinomas. Prostaglandin E2 mediates its effects by interacting with one of four receptors termed EPI-4. Expression and signalling of EP receptors, including EP4, are elevated in cervical carcinomas. The initial aim of this study was to localise the site of expression of EP4 receptors in cervical squamous cell- and adenocarcinomas. Immunohistochemical analysis performed on paraffm wax-embedded cervical tissue sections localised the site of EP4 receptor expression to the neoplastic epithelial cells of all squamous cell carcinomas and adenocarcinomas studied. Minimal EP4 receptor immunoreactivity was detected in normal cervix. The site of localisation of the EP4 receptor within the epithelial compartment suggested that prostaglandin E2 may act in an autocrine/paracrine manner to modulate epithelial cell function and promote tumourigenesis. In addition to endogenous prostaglandin E2, EP receptors in cervical carcinomas can be activated by seminal plasma prostaglandins. Prostaglandin concentration in seminal plasma is 10,000 times higher than that found at a site of inflammation, and prostaglandin E2 is the predominant type of prostaglandin detected in semen. In order to investigate the potential activation of the EP4 receptor by prostaglandin E2 or seminal plasma prostaglandins, we developed an EP4-overexpressing adenocarcinoma cell model system using HeLa (cervical carcinoma) cells.Using this model system the signal transduction pathways activated by prostaglandin E2-or seminal plasma-EP4 receptor interaction in HeLa wild type and EP4 receptor overexpressing(EP4S) He La cells were investigated. Treatment of EP4S cells with seminal plasma or prostaglandin E2 resulted in a rapid accumulation of cAMP (p<;0.001) and phosphorylation of ERK1I2 (p<;O.OOI) in EP4S compared with wild-type cells. This elevated phosphorylation of ERK1I2 is inhibited by co-treatment of cells with chemical inhibitors of MEK (PD98059), epidermal growth factor receptor tyrosine kinase (AGI478) or EP4-selective receptor antagonist (ONO-AE2-227). We next investigated the target genes activated by seminal plasma or prostaglandin E2-EP4 ligand-receptor interaction. Treatment of EP4S cells with seminal plasma or prostaglandin E2 also resulted in elevated expression of the twnourigenic gene, cyclooxygenase-2 (p<;O.OOI), and two genes associated with angiogenesis, vascular endothelial growth factor (p<;O.OOI) and basic fibroblast growth factor (p<;O.05). Expression of these genes was inhibited by co-treatment of cells with seminal plasma or prostaglandin E2 and the MEK inhibitor, the epidermal growth factor receptor tyrosine kinase inhibitor or the EP4-selective receptor antagonist.
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    The role of Gai in the Gonadotropin-releasing hormone (GnRH) receptor inhibition of cell proliferation
    (2011) Phillips, Pumza Samantha; Katz, Arieh
    The activation of Gonadotropin-releasing hormone receptor (GnRHR) by the GnRH ligand has been shown to mediate antiproliferative effects in extra-pituitary cells and in reproductive cancer cell lines. The GnRHR couples to Gαq in pituitary gonadotropes. However, the GnRHR expressed in reproductive cancer cell lines is thought to couple to Gαi. Recent evidence also suggests that the antiproliferative effects may be mediated via Gαq in these cells. Therefore our study involved determining the role of Gαi in the antiproliferative effects mediated by the GnRHR. The results suggest that the Gαi pathway could play a role in mediating the antiproliferative effects of GnRH.
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    The role of kisspeptin and its cognate receptor GPR54 in normal and abnormal placentation
    (2015) Matjila, Mushi Johannes; van der Spuy, Zephne Margaret; Katz, Arieh; Millar, Robert
    Poor invasion of trophoblast cells in early pregnancy has been associated with preeclampsia and intrauterine growth restriction as well as other adverse pregnancy outcomes such as miscarriage, preterm birth and intrauterine death. Hypertensive disorders of pregnancy, including pre-eclampsia are one of the leading causes of maternal mortality in South Africa (Third report on Confidential Enquiries into Maternal Deaths in South Africa (2002-2004)) and the rest of the world. The currently accepted mechanism underlying the development of preeclampsia implicates poor trophoblast invasion and inadequate transformation of the maternal spiral arteries. Despite extensive research in this area, the control of trophoblast invasion and early placental development remains poorly understood. A whole host of factors such as oxygen tension, activation of matrix metalloproteinases (MMPs), angiogenic factors (VEGF-A) and immunological factors such as TNF alpha, interleukins and TGFβ have been shown to be involved in the control of trophoblast invasion. Our knowledge of the molecular details of pregnancy is unfortunately limited to in-vitro experiments and animal studies. Recently kisspeptins and their cognate receptor GPR-54 originally involved in tumour metastasis suppression and regulation of puberty, have been implicated in the inhibition of trophoblast invasion. Expression levels of kisspeptin and its receptor in trophoblast cells are highest in the first trimester, when control of trophoblast invasion is critical, and lower towards term.
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    The role of seminal plasma in cervical carcinoma
    (2010) Sutherland, Jason Robert; Katz, Arieh; Jabbour, Henry
    Cervical cancer is a worldwide public health problem with in excess of 370,000 cases being reported each year. It is the leading cause of death from cancer among women in the developing world where 80% of cases occur. Human papilloma virus (HPV) has been identified as the main causative factor linked with the development and progression of cancer of the cervix, although other factors are known to exist and include genital warts, consenting to sex at an early age, smoking, long term use of contraceptive pills and multiple sexual partners.
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    The role of the chicken gonadotropin-releasing hormone receptor C-terminal tail in expression and coupling
    (2000) Lopes, John; Katz, Arieh
    The role of the carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was determined by testing the activity of a series of chicken gonadotropinreleasing hormone receptors with progressive deletions in their carboxyl terminus. The 55 amino acid carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor was progressively truncated, resulting in cS320STOP, cR330STOP, cS337STOP, cS346STOP, cT35ISTOP, cD356STOP, cS366STOP and cC375STOP truncated mutants, which were all tested in parallel with the wild type chicken gonadotropin-releasing hormone receptor. Truncation of the entire carboxy terminal tail from the chicken gonadotropin-releasing hormone receptor, cS320STOP abolished gonadotropin-releasing hormone binding and gonadotropin-releasing hormone-induced inositol phosphate production. The loss of gonadotropin-releasing hormone binding by the cS320STOP-truncated mutant suggests that this receptor is possibly not expressed on the cell membrane, which might be due to improper receptor folding by cS320STOP. The carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might therefore be required for proper folding of newly formed chicken gonadotropin-releasing hormone receptors and expression of these receptors on the cell membrane. The cR330STOP mutant had a maximal gonadotropin-releasing hormone binding of ~12%, which is the lowest receptor expression detected. The amino acid region between P³¹⁹ and L³²⁹ might therefore play a role in receptor expression. Progressive increase in the carboxy terminal tail from L³²⁹ resulted in progressive increase in the receptor expression. Maximal gonadotropin-releasing hormone binding levels reached wild type levels at truncation of the cGnRHR at S³⁶⁶. These results indicate that the first 45 amino region, ie. between P³¹⁹ and S³⁶⁶ of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail contains elements that promote receptor expression. Gonadotropin-releasing hormone-induced inositol phosphate production was enhanced for all the truncated receptors except cR330STOP and cS337STOP, though all the truncated receptors had coupling efficiency values larger than the wild type chicken gonadotropin-releasing hormone receptor. This enhanced inositol phosphate production might be due to an increased coupling efficiency between the truncated chicken gonadotropin-releasing hormone receptors and the aq111-type G-protein. However, none of the truncated chicken gonadotropin-releasing hormone receptors have left-shifted EC50 values, indicating that coupling efficiency did not increase. Alternatively, a loss or retardation in receptor desensitization and/ or internalization for the truncated chicken gonadotropin-releasing hormone receptor mutants might be responsible for the enhanced gonadotropin-releasing hormone-induced inositol phosphate production by the truncated chicken gonadotropin-releasing hormone receptors. The chicken gonadotropin-releasing hormone receptor has a highly conserved cysteine residue in position 328 that might be palmitoylated. Replacing this cysteine in the chicken gonadotropin-releasing hormone receptor with an alanine [cC328A] increased receptor expression 2 fold, reduced maximal inositol phosphate production to ~69% and severely impaired coupling efficiency to 30% relative to the wild type levels. This finding indicates that C³²⁸ might be palmitoylated and is required for receptor coupling. In conclusion, the ammo terminal region of the chicken gonadotropin-releasing hormone receptor carboxy terminal tail increases receptor expression, either by affecting the transport of newly synthesized chicken gonadotropin-releasing hormone receptors to the plasma membrane and/or the proper folding of this receptor. The intracellular carboxy terminal tail of the chicken gonadotropin-releasing hormone receptor might play a negative role in G-protein coupling. However, the enhanced inositol phosphate production from the truncated chicken gonadotropin-releasing hormone receptors could be due to reduced internalization and/ or desensitization of the carboxy terminal truncated receptors. Point-mutation of C³²⁸ to A resulted in decreased coupling suggesting that C³²⁸ may be a palmitoylation site and might play a role in coupling or desensitization.
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    Testing Revonsuo's Threat simulation theory of dreaming
    (2005) Malcolm-Smith, Susan; Jabbour, Henry; Katz, Arieh
    Revonsuo's Threat Simulation Theory of dreaming asserts that dreaming was selected during human evolution because it has the adaptive function of providing a threat-free context in which threat perception and avoidance can be rehearsed. This study aimed to test the prediction that the threat simulation mechanism will activate differently depending on waking exposure to ecologically valid threat cues. It also compared the impact of waking threat events on dream content with that of waking positive events, as TST asserts that only threat impacts on dream content. Data was collected from three contexts: a high threat context (the Western Cape in South Africa; n=208); a medium threat context (a black southern university in the US; n=34); and a low threat context (North Wales; n=116). Questionnaires included a Most Recent Dream report, details of exposure to walking threatening and positive events, and dreams of such events.
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    The role of seminal fluid in cervical squamous carcinoma progression: Impact on cell proliferation, EMT, motility and gene expression
    (2023) Mkwanazi, Nonkululeko; Katz, Arieh; Leaner Virna
    Cervical cancer is the leading cause of cancer related deaths and the second most common cancer amongst South African women. The key cause for cervical cancer development is sexual transmission and persistent infection with high-risk Human Papillomavirus (HPV). However, it takes several years from infection to cervical cancer development, suggesting that other factors contribute to the disease. Exposure of neoplastic epithelial cells to Seminal Fluid (SF) has been shown to promote cell proliferation in culture and growth of explants in mice injected with HeLa cervical adenocarcinoma cells. Since the majority of cervical cancer cases are squamous cell carcinoma, in this study, we examined the effect of SF on cancer cell proliferation, EMT, motility and gene expression using two squamous cell carcinoma cell line model systems, SiHa and Me180. This study shows that SF significantly enhanced cell proliferation in both cell lines. Using confocal microscopy and phalloidin staining, it was further shown that SF caused morphological changes and induced stress fibre formation. SF upregulated the expression of EMT transcription factors Snail, Twist and ZEB1. EMT induction was confirmed by the increase of N-cadherin and a decrease in E-cadherin protein expression. Additionally, results showed that the induction of EMT transcription factors Snail, Twist and ZEB1 by SF occurs via EP4 receptor, ERK1/2 and COX signaling pathways. To investigate the effect of SF on migration and invasion, transwell migration assays were used. SF significantly enhanced directional cell migration and invasion of SiHa and Me180 cells. Cell invasion was associated with an increase in MMP-2 and MMP-9. SF also induced proinflammatory and angiogenic gene expression in cervical squamous carcinoma cells. SF mediated induction of inflammatory and angiogenic genes was shown to be associated with AP-1 and NFkB transcription factors. A small molecule inhibitor of nuclear import, INI-43 inhibited the nuclear localization and activity of SF activated NF-kB as well as the expression of SF induced inflammatory and angiogenic genes. Employing ectocervical tissue biopsies, SF caused the upregulation of EMT transcription factors, MMPs, inflammatory and angiogenic genes. Taken together, these results suggest that SF may play a role in promoting EMT and enhances the migratory and invasive potential of cervical squamous cell carcinoma. These findings together implicate SF as a possible factor that may promote cervical cancer progression.
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    The role of surfaceant protein A in immunity to HPV16 pseudovirus infection
    (2018) Ujma, Sylvia; Schäfer, Georgia; Katz, Arieh; Horsnell, William
    Infection by oncogenic human papillomavirus (HPV) is known to be the causative agent for the development of various anogenital cancers, including cervical cancer. Worldwide, the majority of cervical cancer cases occur in less developed regions, and while prophylactic vaccines exist to combat HPV infection, they are largely unattainable in these areas. Therefore, alternative preventative measures against HPV infection are needed to help eradicate cervical cancer over time. Since HPV employs multiple mechanisms to evade the host immune response, a proposed method for preventing infection may be by enhancing HPV recognition by the immune system. Surfactant proteins A and D (SP-A and SP-D) are innate immune proteins with a variety of functions including recognition and opsonisation of pathogens. They are primarily found in the lung, but have also been shown to be expressed at other sites of the body, including the female reproductive tract. It was hypothesised that SP-A and/or SP-D may enhance immune recognition of HPV, thereby preventing infection. To assess this hypothesis, co-immunoprecipitation and flow cytometry experiments were performed to determine whether SP-A and/or SP-D bind to HPV16 pseudovirions (HPV16- PsVs). SP-A was shown to bind to HPV16-PsVs as well as enhance viral uptake by RAW264.7 murine macrophages, while SP-D bound HPV16-PsVs weakly and had no effect on viral uptake. To confirm these observations and to assess whether SP-A had an effect on HPV16- PsVs infection in vivo, a well-established, but not yet available murine HPV16-PsVs cervicovaginal challenge model system was set up at UCT. It was determined that neither naïve nor C57BL/6 mice challenged with HPV16-PsVs expressed SP-A in the female genital tract. However, under the experimental conditions established herein, pre-incubation of HPV16-PsVs with purified SP-A at a 1:10 weight per weight ratio resulted in a reduction in infection. This study is the first to describe a biochemical and functional association of HPV16 virions with the innate immune molecule SP-A. In the long term, these observations may contribute to the development of topical microbicides incorporating recombinant fragments of SP-A to reduce the burden of new HPV infections.
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    β-arrestin interacting domains on the type II gonadotropin-releasing hormone (GnRH) receptor
    (2006) Nkwayana, Nonhlanhla; Katz, Arieh; Katz, Arieh
    Over-expression of β-arrestin 1 in COS-l cells revealed that the mammalian type GnRH receptor can internalise in a β-arrestin dependent manner whereas the internalisation of the mammalian type I GnRH receptor is β-arrestin independent. investigate which domains on the mammalian type II GnRH receptor are required for β~arrestin dependent internalisation, chimeric receptors were created.