Faculty of Health Sciences
School of Medicine
Department of Immunology
Selected Highlights from Research Findings
Professor Sharon Cassol joined the department of immunology in January 2007 to spear-head the establishment of a new research program focused on HIV-1 mucosal pathogenesis. This programme is designed to fill a critical niche in basic science and answer important unresolved questions that are fundamental to the development of prevention and treatment strategies that are safe, affordable and maximally effective against HIV-1 subtype C viruses in southern Africa. The program combines transitional research with infrastructure and capacity building, and is designed to: 1) bring together a distinguished and highly skilled international research team; 2) tie together and expand existing research at UP; 3) link basic science themes to clinical applications and 4) stimulate a multi-disciplinary, international approach to transitional basic science research. Major progress has been made in all areas. A highly productive and dedicated research team has been assembled. This team includes clinician scientists specialized in the care and treatment of HIV-/AIDS (T Rosseau), gastroenterologists [S van der Merwe, O Mwantembe, R Bond], a molecular virologist [S Cassol], immunologists [R Anderson, G Poli (Italy), M Alfano (Italy), E Cassol (Italy/South Africa), B Patterson (USA) and R Shattock (UK)], pathologists [T Slavik, L Vago (Italy)], a bioinformaticist [T de Oliveira] and a statistician [P Rheeder]. Two PhD students received training in advanced bioinformatics, several exchange students from Holland were training in clinical gastroenterology, a collaborative agreement was established with the San Raffaele Research Institute in Milan, and a flow-based Centre of Excellence for the quantification of intracellular virus in the gastrointestinal tract is under development with B. Patterson at Stanford. A total of fifteen HIV-1-infected, treatment-naïve patients have been enrolled, biopsied at multiple sites using a novel double balloon endoscopic procedure and treated with antiretroviral therapy (ART) and (or) antibiotics. With the exception of a single patient who was non-compliant, all patients have shown a favorable clinical response to antibiotics and/or ART, and a marked increase in their weight and CD4+ T-cell counts, including four patients with previously undiagnosed parasitic infections [cryptosporidium (n = 3), schistosoma (n= 1)] Important new findings relate to the severe, but differential, loss of CD4+ T-cells in different regions of the gastrointestinal tract, the role of macrophages in polarizing the immunological and activation status of the gut, and the effect of ART on the early restoration of CD4+ and CD8+ effector T-cells. It is expected that, over the next few years, these studies will lead to a wealth of new clinically-relevant information that will have major implications not only for treatment of HIV-1, but also for vaccine and microbicide development.
Contact person: Prof SA Cassol.
This study was designed to investigate the effects of platinum, as hydrogen hexachloroplatinate on the production of reactive oxygen species (ROS) by human neutrophils in vitro. ROS were measured by lucigenin-enhanced chemiluminescence (LECL). Addition of Pt to neutrophils was accompanied by a lag phase of about one minute, followed by a linear dose-related increase in LECL, which peaked at around four minutes and achieved statistical significance at concentrations of 0.025 AM Pt and higher. Interestingly, Pt-mediated enhancement of LECL was not associated with meaningful alterations in neutrophil oxygen consumption, assembly of NADPH oxidase, or cytosolic Ca2+ and was completely attenuated by superoxide dismutase and inhibitors of NADPH oxidase, but not by catalase or scavengers of hydroxyl radical, and was undetectable with cells from individuals with chronic granulomatous disease. Exposure of a1-proteinase inhibitor to Pt-treated neutrophils resulted in inactivation of elastase-inhibitory capacity, underscoring the potential toxicity of neutrophil/Pt interactions. The pro-oxidative actions of Pt were mimicked by palladium (Pd), but not by cisplatin or rhodium. These observations demonstrate that Pt and Pd potentiate the reactivity, as opposed to the generation of neutrophil-derived oxidants, an activity that may contribute to airway inflammation in occupationally and possibly environmentally exposed individuals.
Contact person: Prof AJ Theron.
This study was undertaken to investigate the effects of vanadium in the +2, +3, +4, and +5 valence states on superoxide generation, myeloperoxidase (MPO) activity, and hydroxyl radical formation by activated human neutrophils in vitro, using lucigenin-enhanced chemiluminescence (LECL), autoiodination, and electron spin resonance with 5,5-dimethyl-l-pyrroline N-oxide as the spin trap, respectively. At concentrations of up to 25 AM, vanadium, in the four different valence states used, did not affect the LECL responses of neutrophils activated with either the chemoattractant, N-formyl-l-methionyl-l-leucyl-l-phenylalanine (1 AM), or the phorbol ester, phorbol 12-myristate 12-acetate (25 ng/ml). However, exposure to vanadium in the +2, +3, and +4, but not the +5, valence states was accompanied by significant augmentation of hydroxyl radical formation by activated neutrophils and attenuation of MPO-mediated iodination. With respect to hydroxyl radical formation, similar effects were observed using cell-free systems containing either hydrogen peroxide (100 AM) or xanthine/xanthine oxidase together with vanadium (+2, +3, +4), while the activity of purified MPO was inhibited by the metal in these valence states. These results demonstrate that vanadium in the +2, +3, and +4 valence states interacts prooxidatively with human neutrophils, competing effectively with MPO for hydrogen peroxide to promote formation of the highly toxic hydroxyl radical.
Contact person: Prof AJ Theron.
The human neutrophil is the most abundant type of leucocyte in blood and is a key component in host defence against pathogenic bacteria and other types of microorganism. Transient increases in cytosolic calcium (Ca2+) precede, and are a pre-requisite for activation of the protective activities of these cells. Neutrophils utilise both intracellular stored Ca2+, as well as extracellular Ca2+ for this purpose. Importantly, the Ca2+-dependent activation of neutrophils should be brief and protective, with rapid restoration of Ca2+ homeostasis and a return to basal metabolic activity of the cells. However, if the level of activation of the cells is excessive and/or sustained, this leads to hyperacute chronic inflammation with resultant inflammation-mediated tissue damage and disease. Many disease states are associated with excessive/chronic activation of neutrophils, including some types of bronchial asthma, chronic obstructive pulmonary disease, cystic fibrosis, and rheumatoid arthritis. As a novel strategy to control neutrophil-mediated inflammation and tissue damage, calcium handling by activated neutrophils has been identified by researchers in the Department of Immunology as being a multi-step process which presents several different targets, including those involved in Ca2+ mobilization, Ca2+ clearance, and store-refilling, which are amenable to pharmacological manipulation. During 2007, we have identified several novel targets for neutrophil-directed anti-inflammatory chemotherapy, all of which are involved in Ca2+ release and handling during exposure of the cells to pro-inflammatory stimuli. In some cases, target enhancement (of activity) improves the efficiency of clearance of Ca2+ from the cytosol of activated neutrophils (endomembrane Ca2+-ATPase, NADPH oxidase, myeloperoxidase, protein kinase C), while in others, target inhibition improves the efficiency of Ca2+ clearance (Na+/Ca2+ exchanger, 5’-lipoxygenase). This is a particularly important research programme, given the broad involvement of neutrophils in the immunopathogenesis of acute and chronic diseases of both infective and non-infective origin, as well as the unresponsiveness of these cells to almost all currently available, conventional anti-inflammatory agents (including corticosteroids). Some of the most important findings of our research conducted over the past year are as follows:
Leukotriene B4, generated endogenously by neutrophils during exposure to a primary, receptor-mediated stimulus, causes a secondary wave of Ca2+ mobilization/influx which not only sustains elevated cytosolic Ca2+ levels, but also prolongs the Ca2+-dependent pro-inflammatory activities of the cells. Clearly, LTB4 is a key target for anti-inflammatory chemotherapy, either by inhibition of synthesis or by receptor antagonism.
The cysteinyl leukotrienes, LTC4 and LTD4, sensitize (prime) human neutrophils to hyperreact on exposure to a primary stimulus (e.g. chemoattractant). Importantly, neutrophils do not synthesize cysteinyl leukotrienes, but do possess membrane receptors for these bioactive lipids. Antagonists of cysteinyl leukotriene receptors, in addition to their conventional smooth muscle-targeted therapeutic activities, may also possess anti-inflammatory properties.
We have recently demonstrated that in addition to type 4 phosphodiesterase, neutrophils also possess high level activity of type 3 phosphodiesterase (PDE). This has important implications for anti-inflammatory chemotherapy. In the case of neutrophils, PDE-directed anti-inflammatory strategies are likely to require inhibition of both types 3 and 4 PDE to achieve maximal therapeutic efficiency, and not solely type 4 PDE as is currently believed to be the case.
We have also found that the cysteinyl leukotriene receptor antagonist, montelukast, which is used primarily in the treatment of mild-to-moderate bronchial asthma, possesses additional previously-unrecognized, anti-inflammatory activities. We have found that montelukast possesses potent non-specific PDE inhibitory activity which affects both cAMP and cGMP PDEs. This observation has important implications, not only for the therapeutic applications and mechanisms of action of montelukast, but for future design of neutrophil-directed anti-inflammatory strategies in general.
Contact person: Prof R Anderson.
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