Elmarie Myburgh - Academia.edu (original) (raw)

Papers by Elmarie Myburgh

The copyright of this thesis vests in the author. No quotation from it or information derived fro... more The copyright of this thesis vests in the author. No quotation from it or information derived from it is to be published without full acknowledgement of the source. The thesis is to be used for private study or noncommercial research purposes only. Ie Exchange ttl!' fund me throughout stud i:unily ti-iends, Thank With all you in my Ii nothing seems ill

The FASEB Journal, Jan 3, 2018

Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasi... more Leishmania major is the causative agent of the neglected tropical disease, cutaneous leishmaniasis. In the mouse, protective immunity to Leishmania is associated with inflammatory responses. Here, we assess the dynamics of the inflammatory responses at the lesion site during experimental long-term, low-dose intradermal infection of the ear, employing noninvasive imaging and genetically modified L. major. Significant infiltrates of neutrophils and monocytes occurred at 1-4 d and 2-4 wk, whereas dermal macrophage and dendritic cell (DC) numbers were only slightly elevated in the first days. Quantitative whole-body bioluminescence imaging of myeloperoxidase activity and the quantification of parasite loads indicated that the Leishmania virulence factor, inhibitor of serine peptidase 2 (ISP2), is required to modulate phagocyte activation and is important for parasite survival at the infection site. ISP2 played a role in the control of monocyte, monocyte-derived macrophage, and monocyte-derived DC (moDC) influx, and was required to reduce iNOS expression in monocytes, monocyte-derived cells, and dermal DCs; the expression of CD80 in moDCs; and levels of IFN-g in situ. Our findings indicate that the increased survival of L. major in the dermis during acute infection is associated with the down-regulation of inflammatory monocytes and monocyte-derived cells via ISP2.-Goundry, A

Experimental Parasitology, Nov 1, 2010

An understanding of host-parasite interplay is essential for the development of therapeutics and ... more An understanding of host-parasite interplay is essential for the development of therapeutics and vaccines. Immunoparasitologists have learned a great deal from 'conventional' in vitro and in vivo approaches, but recent developments in imaging technologies have provided us (immunologists and parasitologists) with the ability to ask new and exciting questions about the dynamic nature of the parasite-immune system interface. These studies are providing us with new insights into the mechanisms involved in the initiation of a Leishmania infection and the consequent induction and regulation of the immune response. Here, we review some of the recent developments and discuss how these observations can be further developed to understand the immunology of cutaneous Leishmania infection in vivo.

Infection and Immunity, Dec 1, 2008

The role of CD4 ؉ T-cell interleukin-4 (IL-4) receptor alpha (IL-4R␣) expression in T helper 2 (T... more The role of CD4 ؉ T-cell interleukin-4 (IL-4) receptor alpha (IL-4R␣) expression in T helper 2 (TH2) immune responses has not been defined. To examine this role, we infected CD4 ؉ T-cell IL-4R␣ knockout (KO) mice with the parasitic nematode Nippostrongylus brasiliensis, which induces strong host TH2 responses. Although N. brasiliensis expulsion was not affected in CD4 ؉ T-cell IL-4R␣ KO mice, the associated lung pathology was reduced. Infected CD4 ؉ T-cell IL-4R␣ KO mice showed abrogation of airway mucus production. Furthermore, CD4 ؉ T-cell IL-4R␣ KO mouse lungs contained reduced numbers of lymphocytes and eosinophils. Restimulation of pulmonary region-associated T-cell populations showed that TH2 cytokine responses were disrupted. Secretion of IL-4, but not secretion of IL-13 or IL-5, from mediastinal lymph node CD4 ؉ T cells was reduced in infected CD4 ؉ T-cell IL-4R␣ KO mice. Restimulation of tissue-derived CD4 ؉ T cells resulted in equivalent levels of IL-4 and IL-13 on day 7 postinfection (p.i.) in control and CD4 ؉ T-cell IL-4R␣ KO mice. By day 10 p.i. the TH2 cytokine levels had significantly declined in CD4 ؉ T-cell IL-4R␣ KO mice. Restimulation with N. brasiliensis antigen of total lung cell populations and populations with CD4 ؉ T cells depleted showed that CD4 ؉ T cells were a key TH2 cytokine source. These data demonstrated that CD4 ؉ T-cell IL-4 responsiveness facilitates eosinophil and lymphocyte recruitment, lymphocyte localization, and TH2 cytokine production in the allergic pathology associated with N. brasiliensis infections.

Progress in Neurobiology, Sep 1, 2017

Rapid progress is being made in understanding the roles of the cerebral meninges in the maintenan... more Rapid progress is being made in understanding the roles of the cerebral meninges in the maintenance of normal brain function, in immune surveillance, and as a site of disease. Most basic research on the meninges and the neural brain is now done on mice, major attractions being the availability of reporter mice with fluorescent cells, and of a huge range of antibodies useful for immunocytochemistry and the characterization of isolated cells. In addition, two-photon microscopy through the unperforated calvaria allows intravital imaging of the undisturbed meninges with sub-micron resolution. The anatomy of the dorsal meninges of the mouse (and, indeed, of all mammals) differs considerably from that shown in many published diagrams: over cortical convexities, the outer layer, the dura, is usually thicker than the inner layer, the leptomeninx, and both layers are richly vascularized and innervated, and communicate with the lymphatic system. A membrane barrier separates them and, in disease, inflammation can be localized to one layer or the other, so experimentalists must be able to identify the compartment they are studying. Here, we present current knowledge of the functional anatomy of the meninges, particularly as it appears in intravital imaging, and review their role as a gateway between the brain, blood, and lymphatics, drawing on information that is scattered among works on different pathologies. ________________________________________________________________________________ 4.2.3.2. Sympathetic innervation of the dura. 4.2.3.3. Parasympathetic innervation of the dura. 4.2.4. Lymph vessels in the cortical dura. 4.3. The leptomeninx 4.3.1. Innervation of the leptomeninx 5. Vascular dynamics in the meninges 5.1. Motor control of pial vessels. 5.2. Vascular dynamics in the dura. 5.2.1. Vasomotor control. 5.2.2. Extravasation of plasma proteins from dural vessels. 5.2.3. A note on headaches. 6. Extravascular transport of molecules within the cranium. 6.1. The 'macrocirculation' of CSF. 6.1.1. Secretion of CSF by the choroid plexuses. 6.1.2. Flow of CSF from ventricles to the surface of the cortex. 6.1.3. Efflux of CSF from the cranium. 6.2. Long-distance transport of solutes in the dorsal cortex. 6.2.1. Is there net flow of CSF along paravascular spaces? 6.2.2. A critique of the 'glymphatic' circuit. 6.3. Clearance of exogenous and endogenous molecules from the cortex. 6.4. Pathways for CSF in the meninges. 7. Where are we? Identifying locations in the meninges. 7.1. Meningeal landmarks for intravital imaging. 7.2. Mechanical separation of the dura, arachnoid and pia in rats and mice. 8. Immune cells in the meninges in health and disease. 8.1. Mast cells 8.2. Neutrophils 8.3. Microglia. 8.4. Perivascular immune cells. 8.5. Meningeal macorphages. 8.6. Dendritic cells. 8.7. T cells 8.8. Antigen presentation in the meninges. 8.9. Communication between the dura and CSF. 9. Conclusion.

Methods, Aug 1, 2017

A wide range of viral and microbial infections are known to cause meningitis, and there is eviden... more A wide range of viral and microbial infections are known to cause meningitis, and there is evidence that the meninges are the gateway to pathogenic invasion of the brain parenchyma. Hence observation of these regions has wide application to understanding host-pathogen interactions. Interactions between pathogens and cells of the immune response can be modified by changes in their environment, such as suppression of the flow of blood and lymph, and, particularly in the case of the meninges, with their unsupported membranes, invasive dissection can alter the tissue architecture. For these reasons, intravital imaging through the unperforated skull is the method of choice. We give a protocol for a simple method of two-photon microscopy through the thinned cortical skull of the anesthetized mouse to enable real-time imaging with sub-micron resolution through the meninges and into the superficial brain parenchyma. In reporter mice in which selected cell types express fluorescent proteins, imaging after infection with fluorescent pathogens (lymphocytic choriomeningitis virus, Trypanosoma brucei or Plasmodium berghei) has shown strong recruitment to the cortical meninges of immune cells, including neutrophils, T cells, and putative dendritic cells and macrophages. Without special labeling, the boundaries between the dura mater, the leptomeninx, and the parenchyma are not directly visualized in intravital two-photon microscopy, but other landmarks and characteristics, which we illustrate, allow the researcher to identify the compartment being imaged. While most infectious meningitides are localized mainly in the dura mater, others involve recruitment of immune cells to the leptomeninx.

Journal of Immunology, Dec 15, 2001

IL-12p35 ؊/؊ p40 ؊/؊ mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin ... more IL-12p35 ؊/؊ p40 ؊/؊ mice are highly susceptible to Mycobacterium bovis bacillus Calmette-Guérin (BCG) or Mycobacterium tuberculosis infection. In this study IL-12p35 ؊/؊ mice, which are able to produce endogenous IL-12p40, cleared M. bovis BCG and showed reduced susceptibility to pulmonary M. tuberculosis infection, which was in striking contrast to the outcome of mycobacterial infection in IL-12p35 ؊/؊ p40 ؊/؊ mice. Resistance in wild-type and IL-12p35 ؊/؊ mice was accompanied by protective granuloma formation and Ag-specific delayed-type hypersensitivity responses, which were impaired in susceptible IL-12p35 ؊/؊ p40 ؊/؊ mice. Furthermore, IL-12p35 ؊/؊ mice, but not IL-12p35 ؊/؊ p40 ؊/؊ mice, mounted Ag-specific Th1 and cytotoxic T cell responses. In vivo therapy with rIL-12p40 homodimer restored the impaired delayed-type hypersensitivity responses in M. bovis BCG-infected IL-12p35 ؊/؊ p40 ؊/؊ mice and reverted them to a more resistant phenotype. Together, these results show evidence for a protective and agonistic role of endogenous and exogenous IL-12p40 in mycobacterial infection, which is independent of IL-12p70.

PLOS Pathogens, 2007

The third author's name was incorrectly listed as Claire J. Hoving. The correct name is J. Claire... more The third author's name was incorrectly listed as Claire J. Hoving. The correct name is J. Claire Hoving.

bioRxiv (Cold Spring Harbor Laboratory), Oct 21, 2022

Leishmania parasites undergo differentiation between various proliferating and non-1 dividing for... more Leishmania parasites undergo differentiation between various proliferating and non-1 dividing forms to adapt to changing host environments. The mechanisms that link 1 environmental cues with the parasite's developmental changes remain elusive. Here, 1 we report that Leishmania TORC1 is a key environmental sensor for parasite 1 differentiation in the sand fly-stage promastigotes and for replication of mammalian-1 stage amastigotes. We show that Leishmania RPTOR1, interacts with TOR1 and 2 LST8. We investigate TORC1 function by conditional deletion of RPTOR1, where 2 under nutrient rich conditions RPTOR1 depletion results in decreased protein 2 synthesis and growth, G1 cell cycle arrest and premature differentiation from 2 proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. 2 These parasites cannot develop into proliferative amastigotes in the mammalian 2 host, or respond to nutrients to differentiate to proliferative retroleptomonads, which 2 are required for their blood-meal induced amplification in sand flies and enhanced 2 mammalian infectivity. RPTOR1-dependent TORC1 functionality represents a critical 2 mechanism for driving parasite growth and proliferation.

Methods in molecular biology, 2020

Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei ... more Traditional animal models for human African trypanosomiasis rely on detecting Trypanosoma brucei brucei parasitemia in the blood. Testing the efficacy of new compounds in these models is cumbersome because it may take several months after treatment before surviving parasites become detectable in the blood. To expedite compound screening, we have used a Trypanosoma brucei brucei GVR35 strain expressing red-shifted firefly luciferase to monitor parasite distribution in infected mice through noninvasive wholebody bioluminescence imaging. This protocol describes the infection and in vivo bioluminescence imaging of mice to assess compound efficacy against T. brucei during the two characteristic stages of disease, the hemolymphatic phase (stage 1) and the encephalitic or central nervous system phase (stage 2).

Science

Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infection... more Millions who live in Latin America and sub-Saharan Africa are at risk of trypanosomatid infections, which cause Chagas disease and human African trypanosomiasis (HAT). Improved HAT treatments are available, but Chagas disease therapies rely on two nitroheterocycles, which suffer from lengthy drug regimens and safety concerns that cause frequent treatment discontinuation. We performed phenotypic screening against trypanosomes and identified a class of cyanotriazoles (CTs) with potent trypanocidal activity both in vitro and in mouse models of Chagas disease and HAT. Cryo–electron microscopy approaches confirmed that CT compounds acted through selective, irreversible inhibition of trypanosomal topoisomerase II by stabilizing double-stranded DNA:enzyme cleavage complexes. These findings suggest a potential approach toward successful therapeutics for the treatment of Chagas disease.

SUMMARYLeishmaniaparasites undergo differentiation between various proliferating and non-dividing... more SUMMARYLeishmaniaparasites undergo differentiation between various proliferating and non-dividing forms to adapt to changing host environments. The mechanisms that link environmental cues with the parasite’s developmental changes remain elusive. Here, we report thatLeishmaniaTORC1 is a key environmental sensor for parasite differentiation in the sand fly-stage promastigotes and for replication of mammalian-stage amastigotes. We show thatLeishmaniaRPTOR1, interacts with TOR1 and LST8. We investigate TORC1 function by conditional deletion ofRPTOR1, where under nutrient rich conditions RPTOR1 depletion results in decreased protein synthesis and growth, G1 cell cycle arrest and premature differentiation from proliferative promastigotes to non-dividing mammalian-infective metacyclic forms. These parasites cannot develop into proliferative amastigotes in the mammalian host, or respond to nutrients to differentiate to proliferative retroleptomonads, which are required for their blood-meal ...

Molecular Immunology, Mar 1, 2008

Human IL-4Ralpha binds to mouse gammac resulting in a chimeric receptor specific for human IL-4 b... more Human IL-4Ralpha binds to mouse gammac resulting in a chimeric receptor specific for human IL-4 but not mouse IL-4, providing in principle an inducible hIL-4 system. We investigated the in vitro and in vivo characteristics of human IL-4Ralpha transgenic mice on a mouse IL-4Ralpha-deficient background (hIL-4Ralpha Tg/mIL-4Ralpha(-/-)). The integrity of lymphocyte-specific hIL-4Ralpha expression in hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice was demonstrated by FACS analysis. This was confirmed in functional studies as lymphocytes responded to recombinant hIL-4 but not mIL-4 or mIL-13 in proliferation and T helper differentiation assays, demonstrating species-specificity and inducibility of the chimeric receptor in vitro. We then infected transgenic mice with Nippostrongylus brasiliensis, known to induce a strong Type 2 response in wild-type mice. As expected hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice were unable to expel N. brasiliensis worms which confirms unresponsiveness in non-lymphocytes. However they developed a Th2 cytokine and IgE response in the absence of induction with hIL-4. These results suggested that lymphocyte-specific IL-4Ralpha responsiveness was still present in vivo. Neutralization of endogenous mIL-4 resulted in inhibition of N. brasiliensis-induced Th2 cytokine and total IgE production in hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice suggesting that mIL-4 was involved. Intercrossing hIL-4Ralpha Tg/mIL-4Ralpha(-/-) mice with mIL-4(-/-)/mIL-13(-/-) mice completely abrogated Type 2 responses in N. brasiliensis infections. Together, these data demonstrate that mIL-4 triggered the hIL-4Ralpha/mgammac chimeric receptor in vivo.

SUMMARYMany parasites of significant public health importance assume skin residency without causi... more SUMMARYMany parasites of significant public health importance assume skin residency without causing overt pathlogy. How immune and stromal cells respond to such “cryptic” infections and how exposure to UVB alters such responses in poorly understood. We combined scRNA-seq, spatial transcriptomics and inferential network analysis to address these questions in a model of cryptic skin infection byLeishmania donovani. In infected C57BL/6 mice, p-selectin and CXCL12 interactions dominate intercellular communication between leucocytes, fibroblast and endothelial cells, but effector T cell function remains muted. Following UVB exposure, increased numbers of IFNγ+CD4+Th1 cells and NK cells enter the skin, communicating with stromal cells via CCL5-CCR5 and LFA-1-ICAM1/2. However, spatial mapping indicated that Th1 cells and macrophages occupied distinct niches after UVB exposure, likely limiting effector function. Our data provide the first holistic view of the immune landscape during cryptic...

Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sick-ness, act... more Peripheral infection by Trypanosoma brucei, the protozoan responsible for sleeping sick-ness, activates lymphocytes, and, at later stages, causes meningoencephalitis. We have videoed the cortical meninges and superficial parenchyma of C56BL/6 reporter mice in-fected with T.b.brucei. By use of a two-photon microscope to image through the thinned skull, the integrity of the tissues was maintained. We observed a 47-fold increase in CD2+ T cells in the meninges by 12 days post infection (dpi). CD11c+ dendritic cells also increased, and extravascular trypanosomes, made visible either by expression of a fluorescent protein, or by intravenous injection of furamidine, appeared. The likelihood that invasion will spread from the meninges to the parenchyma will depend strongly on whether the trypanosomes are below the arachnoid membrane, or above it, in the dura. Making use of optical signals from the skull bone, blood vessels and dural cells, we conclude that up to 40 dpi, the extra-vascular ...

Kenji Izuhara, Frank Brombacher and Masato KuboKatsuhiko Hayashi, Kazuhiko Arima, Elmarie Myburgh... more Kenji Izuhara, Frank Brombacher and Masato KuboKatsuhiko Hayashi, Kazuhiko Arima, Elmarie Myburgh, Noriyasu Seki, Mayumi Miyazaki, Wataru Suzuki,http://www.jimmunol.org/content/172/10/6158J Immunol€2004; 172:6158-6166; ;Referenceshttp://www.jimmunol.org/content/172/10/6158.full#ref-list-1This article cites 42 articles, 14 of which you can access for free at: Subscriptionshttp://jimmunol.org/subscriptionsInformation about subscribing to The Journal of Immunology is online at: Permissionshttp://www.aai.org/ji/copyright.htmlSubmit copyright permission requests at: Email Alertshttp://jimmunol.org/cgi/alerts/etocReceive free email-alerts when new articles cite this article. Sign up at:

Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed ... more Conditional gene deletion using dimerizable Cre recombinase (DiCre) is so far the best developed system for the phenotypic analysis of essential genes in Leishmania species. Here, we describe a protocol for the generation of a conditional gene deletion mutant and the subsequent inducible deletion of a target gene. Leishmania parasites are genetically modified to express two inactive Cre subunits (DiCre) and a single LoxP-flanked version of a target gene in a context where both endogenous copies of the gene have been deleted. Treatment with rapamycin dimerizes the DiCre subunits, resulting in activation of the enzyme, recombination between the LoxP sites, and excision of the LoxP-flanked target gene. Subsequent phenotyping allows for the analysis of essential gene function.

Lymphocyte-specific reconstitution of ILARu was recently established by intercrossing lymphocyte-... more Lymphocyte-specific reconstitution of ILARu was recently established by intercrossing lymphocyte-specific human IL-4Ra transgenic mice with mIL-4Ra-deficient mice. I~uman ILARu may bind to mouse yc resulting in a chimeric receptor specific for human I LA but not mouse I L-4. This provides us with an inducible I L-4 system. The aim of this study was to investigate the in vitro and in vivo characteristics of ollr novel hlLARu Tg/mIL-4RumOllse model. The integrity of the lymphocyte-specific hIL-4R(x expression in h1L-4Ra -rglmILARcxmice was demonstrated by FACS analysis. Lymphocytes responded to hILA but not mILA or mlL-13 in proliferation and T helper differentiation assays, demonstrating the species-specificity and inducibility of the chimeric receptor in vitro. Non-lymphocytes like macrophages were unresponsive to mlL-4 and mlL-13 as assayed by IfN-y/LPS-induced NO production and arginase activity. The in vivo characteristics of h1L-4Ru Tg/mIL-4Ramice were studied using 2 murine dis...