Jan G Nel | University of Pretoria (original) (raw)

Papers by Jan G Nel

This article has been accepted for publication and undergone full peer review but has not been th... more This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as

Frontiers in Pharmacology, Nov 20, 2018

Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the... more Although the inclusion of the cationic amphiphilic, anti-mycobacterial agent, clofazimine, in the chemotherapeutic regimens of patients with multidrug-resistant tuberculosis (TB) has contributed to improved outcomes, concerns remain about the cardiotoxic potential of this agent. Accordingly, the current study was undertaken with the primary objective of investigating the effects of clofazimine, on the reactivity of human platelets in vitro, a seemingly unexplored, mechanism of cardiotoxicity. Platelet-rich plasma (PRP) prepared from the blood of healthy, adult humans was treated with clofazimine (0.625-10 mg/L), or the primary anti-TB agents, isoniazid and rifampicin (at final concentrations of 5 and 10 mg/L), followed by addition of either adenosine 5-diphosphate (ADP) or thrombin and measurement of platelet activation according to the magnitude of expression of CD62P (P-selectin), as well as the CD62P-mediated formation of heterotypic neutrophil:platelet (NP) aggregates, using flow cytometry. Clofazimine, but neither isoniazid nor rifampicin, caused dose-related potentiation of both ADP-and thrombin-activated expression of CD62P by platelets, achieving statistical significance at threshold concentrations of 0.625 and 2.5 mg/L, respectively, as well as significant formation of N:P aggregates. These stimulatory effects of clofazimine on platelet activation were partly attenuated by pre-treatment of PRP with the membrane-stabilizing agent, α-tocopherol, possibly consistent with a membrane-disruptive mechanism. In conclusion, clofazimine, at concentrations within the therapeutic range, augments platelet activation in vitro, probably by a mechanism linked to membrane destabilization. If operative in vivo, these pro-thrombotic activities of clofazimine may predispose for development of microvascular occlusion, exacerbating an already existing high risk for development of TB-associated cardiovascular disease.

South African Journal of Science, Jan 29, 2016

The human innate immune system is indispensable for protection against potentially invasive micro... more The human innate immune system is indispensable for protection against potentially invasive microbial and viral pathogens, either neutralising them or containing their spread until effective mobilisation of the slower, adaptive (specific), immune response. Until fairly recently, it was believed that the human innate immune system possessed minimal discriminatory activity in the setting of a rather limited range of microbicidal or virucidal mechanisms. However, recent discoveries have revealed that the innate immune system possesses an array of novel pathogen recognition mechanisms, as well as a resourceful and effective alternative mechanism of phagocyte (predominantly neutrophil)-mediated, anti-infective activity known as NETosis. The process of NETosis involves an unusual type of programmed, purposeful cell death, resulting in the extracellular release of a web of chromatin heavily impregnated with antimicrobial proteins. These structures, known as neutrophil extracellular traps (NETs), immobilise and contribute to the eradication of microbial pathogens, ensuring that the anti-infective potential of neutrophils is sustained beyond the lifespan of these cells. The current review is focused on the mechanisms of NETosis and the role of this process in host defence. Other topics reviewed include the potential threats to human health posed by poorly controlled, excessive formation of NETs, specifically in relation to development of autoimmune and cardiovascular diseases, as well as exacerbation of acute and chronic inflammatory disorders of the airways.

Thorax, Nov 15, 2016

from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera sa... more from BR patients (n = 115), categorised by bronchiectasis severity index (BSI) scores and sera samples from HV controls (n = 26) Results Endobronchial biopsies from BR airways had a significantly (p < 0.05) higher number of blood vessels per mm of basement membrane than HV samples (18 and 9 blood vessels/ mm basement membrane respectively). Stimulation of HV neutrophils with a variety of molecules (PMA, fMLP, LPS, TNF-a etc.) resulted in a significant increase in VEGF secretion compared to unstimulated (p < 0.05). Although elevated VEGF was found in some patient samples there was no significant correlation between sera/sputa VEGF and individual patient BSI scores. Conclusion The increased presence of vascular tissue seen in BR could indicate a pro-angiogenic airway environment in BR. The in vitro data collected also show that a variety of stimulants can initiate secretion of VEGF by neutrophils. However, our data does not suggest that VEGF levels in sera or sputa can be used to predict disease severity.

Frontiers in Immunology, Feb 26, 2021

Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concern... more Although bedaquiline has advanced the treatment of multidrug-resistant tuberculosis (TB), concerns remain about the cardiotoxic potential of this agent, albeit by unexplored mechanisms. Accordingly, we have investigated augmentation of the reactivity of human platelets in vitro as a potential mechanism of bedaquiline-mediated cardiotoxicity. Platelet-rich plasma (PRP) or isolated cells prepared from the blood of healthy, adult humans were treated with bedaquiline (0.625-10 µg/ml), followed by activation with adenosine 5'-diphosphate (ADP), thrombin or the thromboxane A 2 receptor agonist (U46619). Expression of platelet CD62P (P-selectin), platelet aggregation, Ca 2+ fluxes and phosphorylation of Akt1 were measured using flow cytometry, spectrophotometry, fluorescence spectrometry, and by ELISA procedures, respectively. Exposure to bedaquiline caused dose-related inhibition of ADP-activated, but not thrombin-or U46619-activated, expression of CD62P by platelets, achieving statistical significance at a threshold concentration of 5 µg/ml and was paralleled by inhibition of aggregation and Ca 2+ mobilization. These ADP-selective inhibitory effects of bedaquiline on platelet activation were mimicked by wortmannin, an inhibitor of phosphatidylinositol 3-kinase (PI3-K), implicating PI3-K as being a common target of both agents, a contention that was confirmed by the observed inhibitory effects of bedaquiline on the phosphorylation of Akt1 following activation of platelets with ADP. These apparent inhibitory effects of bedaquiline on the activity of PI3-K may result from the

International Journal of Molecular Sciences, Apr 11, 2018

Pneumolysin (PLY), a member of the family of Gram-positive bacterial, cholesterol-dependent, β-ba... more Pneumolysin (PLY), a member of the family of Gram-positive bacterial, cholesterol-dependent, β-barrel pore-forming cytolysins, is the major protein virulence factor of the dangerous respiratory pathogen, Streptococcus pneumoniae (pneumococcus). PLY plays a major role in the pathogenesis of community-acquired pneumonia (CAP), promoting colonization and invasion of the upper and lower respiratory tracts respectively, as well as extra-pulmonary dissemination of the pneumococcus. Notwithstanding its role in causing acute lung injury in severe CAP, PLY has also been implicated in the development of potentially fatal acute and delayed-onset cardiovascular events, which are now recognized as being fairly common complications of this condition. This review is focused firstly on updating mechanisms involved in the immunopathogenesis of PLY-mediated myocardial damage, specifically the direct cardiotoxic and immunosuppressive activities, as well as the indirect pro-inflammatory/pro-thrombotic activities of the toxin. Secondly, on PLY-targeted therapeutic strategies including, among others, macrolide antibiotics, natural product antagonists, cholesterol-containing liposomes, and fully humanized monoclonal antibodies, as well as on vaccine-based preventive strategies. These sections are preceded by overviews of CAP in general, the role of the pneumococcus as the causative pathogen, the occurrence and types of CAP-associated cardiac complication, and the structure and biological activities of PLY.

International Journal of Laboratory Hematology, Apr 17, 2018

To determine if the current set of evaluation criteria used for dilute Russel Viper Venom Time (d... more To determine if the current set of evaluation criteria used for dilute Russel Viper Venom Time (dRVVT) investigations in the routine laboratory meet expectation and identify possible shortcomings. Methods All dRVVT assays requested from January 2015 to December 2015, were appraised in this crosssectional study. The raw data panels were compared to the new reference interval, established in 2016, to determine the sequence of assays that should have been performed. The interpretive comments were audited, and false-negative reports identified. Interpretive comments according to three interpretation guidelines were compared. The reagent cost per assay was determined and reagent cost wastage, due to redundant tests, was calculated. Results Only ~9% of dRVVT results authorized during 2015, had an interpretive comment included in the report. ~15% of these results were false negative interpretations. There is a significant statistical difference in interpretive comments between the three interpretation methods. Redundant mixing tests resulted in R 7 477.91 (~11%) reagent cost wastage in 2015. Conclusions We managed to demonstrate very evident deficiencies in our own practice and managed to establish a standardised workflow that will potentially render our service more efficient and cost effective, aiding clinicians in making improved treatment decisions and diagnoses. Furthermore, it is essential that standard operating procedures be kept up-to-date and executed by all staff in the laboratory.

Journal of Immunotoxicology, Nov 17, 2022

South African Medical Journal, Sep 10, 2019

The 2017 World Health Organization report [1] indicates that ~37 million people are infected with... more The 2017 World Health Organization report [1] indicates that ~37 million people are infected with human immunodeficiency virus (HIV) in sub-Saharan Africa. Southern and eastern sub-Saharan Africa are most affected by the pandemic, contributing 53% of global HIV-infected cases. With ~7 million persons infected (12.6% of the total population), South Africa (SA) carries the highest HIV-associated disease burden in this region. [1] As its name indicates, HIV targets the immune system, resulting in progressive immune dysfunction. HIV infection leads not only to a weakened immune system, but also impacts negatively on the haematopoietic system of infected individuals. This is not surprising, as a close link exists between the haematopoietic and immune systems. This open-access article is distributed under Creative Commons licence CC-BY-NC 4.0.

Medical technology SA, 2017

Flow cytometry is a globally accepted diagnostic tool used for the rapid identification of cells ... more Flow cytometry is a globally accepted diagnostic tool used for the rapid identification of cells based on their surface and intracellular antigens, especially for the diagnosis of haematological malignancies. The aim of this study was to evaluate the requests received for flow cytometric immunophenotyping and to create a profile of diagnoses. In 2014 data regarding indications and diagnoses were captured from request forms received and final diagnosis reports issued by the Tshwane Academic Division (TAD) of the National Health Laboratory Service (NHLS). A total of 1234 requests were received over the one year period, of which 80.4% were performed and 16.8% were rejected. The most common indications were leukaemia, lymphoma and cytopenia. Nineteen percent of requests received contained no correct indication or clinical history. In total, 103 and 153 diagnoses were established based on peripheral blood and bone marrow aspirate specimens respectively. Samples were mostly rejected due t...

Thorax, 2015

with FK506 inhibits maturation in this context. This suggests an inhibitory effect of FK506 on in... more with FK506 inhibits maturation in this context. This suggests an inhibitory effect of FK506 on innate antigen presentation to Tcells and may impair the adaptive immune response to invasive aspergillosis in lung transplants recipients.

European Respiratory Journal, 2015

Introduction: NET formation, driven by the process of NETosis, represents a novel strategy utilis... more Introduction: NET formation, driven by the process of NETosis, represents a novel strategy utilised by cells of the innate immune system, especially neutrophils, to trap and dispose of extracellular pathogens. Some bacterial pathogens, such as the pneumococcus appear to be adept not only at evading NETs, but also subverting them to promote persistence and dissemination. Aims and objectives: The aim of the current study was to investigate the potential of the pneumococcal toxin, Ply, to activate NETosis. Methods: Isolated human blood neutrophils were exposed to recombinant Ply (5 and 10 ng.ml -1 ) for 30 and 60 min at 37°C after which NET formation was measured using the following combination of procedures: i) spectrofluorimetry using the fluorophore, sytox orange (5 μM), as well as NanoDrop® technology to detect extracellular DNA; ii) fluorescence microscopy using DAPI (nuclear stain) and anti-citrullinated histone monoclonal antibodies to visualise nets; and iii) flow cytometry using vibrant dye cycle ruby to detect loss of intracellular DNA. Results: Exposure of neutrophils to Ply resulted in significant increases in the numbers of NET-forming cells (4.3±0.9%, 14.3±0.2%, 16.5±1.5%, for the control and systems treated with 5 and 10 ng.ml -1 Ply respectively, P 0.0001) which was paralleled by significant ( P 0.05) time-related increases in the release of extracellular DNA and citrullinated histones in the setting of loss of intracellular DNA and retention of cell viability. Conclusions: Ply induces vital NETosis in human neutrophils, which may contribute to either host defence or disease severity depending on the intensity of the inflammatory response during pneumococcal infection.

Journal of Immunotoxicology

Frontiers in Immunology, 2021

Clinical and experimental immunology, Jan 8, 2016

The primary objective of the current study was to investigate the potential of the pneumococcal t... more The primary objective of the current study was to investigate the potential of the pneumococcal toxin, pneumolysin (Ply), to activate neutrophil extracellular trap (NET) formation in vitro. Isolated human blood neutrophils were exposed to recombinant Ply (2.5-20 ng.ml(-1) ) for 30-90 min at 37°C and NET formation measured using the following procedures to detect extracellular DNA: i) flow cytometry using Vybrant Dye Cycle Ruby; ii) spectrofluorimetry using the fluorophore, Sytox® Orange (5 μM); iii) and NanoDrop® technology. These procedures were complemented by fluorescence microscopy using DAPI (nuclear stain) in combination with anti-citrullinated histone monoclonal antibodies to visualise nets. Exposure of neutrophils to Ply resulted in relatively rapid (detected within 30-60 min), statistically significant (p<0.05) dose- and time-related increases in the release of cellular DNA impregnated with both citrullinated histone and myeloperoxidase. Microscopy revealed that NETosis ...

Journal of Infection, 2017

Highlights  Pneumolysin (Ply) activates production of PAF and thromboxane A 2 (TxA 2) by neutrop... more Highlights  Pneumolysin (Ply) activates production of PAF and thromboxane A 2 (TxA 2) by neutrophils.  Ply also promotes formation of pro-thrombotic neutrophil:platelet (NP) aggregates.  Ply-mediated aggregate formation is independent of PAF and TxA 2.  P-selectin (CD62P) and protease-activated receptor 1 are involved in Ply-induced NP aggregation.  Ply-mediated NP aggregate formation may contribute to pulmonary and myocardial injury.

Pharmaceuticals

This study probed the differential utilization of P2Y1 and P2Y12 receptors in mobilizing CD62P (P... more This study probed the differential utilization of P2Y1 and P2Y12 receptors in mobilizing CD62P (P-selectin) from intracellular granules following activation of human platelets with adenosine 5′-diphosphate (ADP, 100 µmol·L−1) Platelet-rich plasma (PRP) was prepared from the blood of adult humans. CD62P was measured by flow cytometry following activation of PRP with ADP in the absence and presence of the selective antagonists of P2Y1 and P2Y12 receptors, MRS2500 and PSB0739 (both 0.155–10 µmol·L−1), respectively. Effects of the test agents on ADP-activated, CD62P-dependent formation of neutrophil:platelet (NP) aggregates were also measured by flow cytometry, while phosphatidylinositol 3-kinase (PI3K) activity was measured according to Akt1 phosphorylation in platelet lysates. Treatment with MRS2500 or PSB0739 at 10 µmol·L−1 almost completely attenuated (94.6% and 86% inhibition, respectively) ADP-activated expression of CD62P and also inhibited NP aggregate formation. To probe the me...

Lung, 2016

This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating huma... more This study has explored the role of the pneumococcal toxin, pneumolysin (Ply), in activating human platelets. Following exposure to Ply [10-80 nanograms (ng)/ml], platelet activation and cytosolic Ca 2+ concentrations were measured flow cytometrically according to the level of expression of CD62P (P-selectin) and spectrofluorimetrically respectively. Exposure to Ply resulted in marked upregulation of expression of platelet CD62P, achieving statistical significance at concentrations of 40 ng/ml and higher (p<0.05), in the setting of increased influx of Ca 2+. These potentially pro-thrombotic actions of Ply were attenuated by depletion of Ca 2+ from the extracellular medium, or by exposure of the cells to a pneumolysoid devoid of pore-forming activity. These findings are consistent with a mechanism of Plymediated platelet activation involving sub-lytic pore formation, Ca 2+ influx, and mobilization of CD62P-expressing α-granules, which, if operative in vivo, may contribute to the pathogenesis of associated acute lung and myocardial injury during invasive pneumococcal disease.

International Journal of Laboratory Hematology