Browsing by Department "Division of Immunology"
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- ItemOpen AccessAllergenicity of latex rubber products used in South African dental schools(2009) Mabe, Dikeledi O; Singh, Tanusha S; Bello, Braimoh; Jeebhay, Mohammed F; Lopata, Andreas L; Wadee, AhmedBackground: Allergens from latex products in healthcare settings have been known to trigger latex induced allergic reactions in healthcare workers (HCWs). There is a need to quantify individual latex allergens in products in order to assess the allergenicity of latex products used in health care settings, so as to minimize the risk of sensitisation to these proteins. Methods: Fourteen latex examination gloves representing six brands (powdered and non-powdered) and five dental rubber dams from five dental academic institutions were analysed for latex allergens and total protein. Total protein content was determined using the BIORAD DC protein assay kit and natural rubber allergen levels using a capture ELISA assay specific for hev b 1, hev b 3, hev b 5 and hev b 6.02. Results: Hev b 6.02 was found in higher concentrations than other NRL allergens in the products analysed. Hev b 5 content ranged from 0 to 9.2µg/g and hev b 6.02 from 0.09 to 61.5µg/g of sample. Hev b 1 levels were below the detection limit (DL) for 79% of the samples (15/19). Dental dams showed higher allergen levels (median: 80.91µg/g) in comparison to latex gloves (median: 11.34µg/g). Powdered rubber samples also showed higher allergen levels (median: 40.54µg/g) compared to non-powdered samples (median: 5.31µg/g). A statistically significant correlation was observed between total protein and total allergen (r=0.74, p<0.001) concentrations. Conclusion Natural rubber latex (NRL) allergen concentrations differ significantly by product and brand. This study has demonstrated that NRL allergens in latex containing products used in South African dental institutions are present at sufficiently high levels to pose an allergic health risk.
- ItemOpen AccessAlterations in preconception, antenatal, and postnatal maternal gut microbiota influence offspring intestinal microbiota and immunity(2017) Nyangahu, Donald D; Jaspan, Heather B; Horsnell, WilliamMaternal microbiota during pregnancy, as well as maternal disease state, may impact offspring gut bacterial colonisation. Here, we explore the impact of maternal antibiotics during gestation and/or nursing on offspring gut microbiota. Further, we investigate the effect of preconception helminth infections on maternal and infant gut microbiota. For maternal antibiotic experiments, dams were fed vancomycin, polymyxin B, or both, in drinking water during gestation, nursing or gestation plus nursing, and their offspring microbiota analysed at 14 days of life, alongside immunity in the spleens. Offspring born to vancomycin treated mothers had significantly higher relative abundance of Proteobacteria and Tenericutes while maternal oral polymyxin B led to significantly lower abundance of Proteobacteria and Deferribacteres in infants. Maternal oral vancomycin led to significant reduction in proportions of infant central memory CD4+ T cells (CD4+CD44hiCD62Lhi) regardless of antibiotic timing. Effector memory CD4+ T cells were significantly lower in pups born to dams treated with polymyxin B while nursing while proportions of central memory CD4 T cells were significantly increased in gestation only or gestation plus nursing pups. In addition, oral vancomycin in dams during nursing resulted in significantly reduced proportions of both total and follicular B cells in offspring born to antibiotic treated dams. Pups born to Vancomycin treated mothers had a significant delay in growth when infected with Respiratory Syncytial Virus (RSV). On the other hand, pups born to mothers treated with Polymyxin B during gestation or gestation plus nursing were susceptible to Nippostrongylus brasiliensis (Nb) infection. In the second study, we infected female BALB/c mice with 500Nb L3 three weeks prior to mating and examined the effect of preconception helminth infection on offspring microbiota and immunity. Preconception Nb infections led to alterations of maternal gut microbiota during pregnancy. In addition, we observed dramatic differences in offspring microbiota in pups born to previously helminth infected dams. Coriobacteriaceae were predominant in pups born to previously Nb infected dams when compared to uninfected dams. Overall, manipulation of maternal microbiota during gestation or lactation profoundly impacts offspring growth, intestinal microbiota and immunity to RSV and helminths.
- ItemOpen AccessCancer cell behaviour following parasite exposure(2018) Jacobs, Brittany-Amber; Smith, Katherine; Prince, SharonInfectious diseases, including helminthiases, are estimated to cause 16.1% of global cancer cases. While certain helminths are conclusive causes of cancer, others have been shown to reduce the disease. It is currently unknown why differing helminth infections promote or prevent cancer development and progression, or which cellular mechanisms are altered following exposure. Using several in vitro and in vivo techniques, this study aimed to determine the effect that certain helminths have on the progression of cervical and colorectal cancer. The results revealed that antigen from the hookworm Nippostrongylus brasiliensis significantly reduced cervical cancer cell migration and the expression of two markers of metastasis: vimentin and N-cadherin. Importantly, N. brasiliensis antigen significantly lowered the expression of cell-surface vimentin, while decreasing Human Papillomavirus type16 pseudovirion internalization. In vivo infection with N. brasiliensis significantly decreased vimentin expression within the female genital tract, confirming the relevance of these in vitro findings. Furthermore, exposure to antigen from the gastrointestinal nematode Heligmosomoides polygyrus decreased the in vitro proliferation of human and mouse colorectal cancer cells and simultaneously increased the expression of cell cycle regulator proteins, p53 and p21. Surprisingly, while antigen from H. polygyrus inhibited human colorectal cancer cell migration, it had the opposite effect on mouse colorectal cancer cells, suggesting that its impact on colorectal cancer migration may be, at the very least, species dependent. Using a syngeneic tumour model, the excretory-secretory product from H. polygyrus was shown to significantly increase tumour growth and the expansion of regulatory T cells and neutrophils in the tumour. Similarly, in a model of colitis-associated colorectal cancer this antigen significantly worsened pathology in a TGF-β dependent manner. Undoubtedly, the knowledge gained from this study will contribute to the limited understanding about helminths and the effect that these parasites have on cancer progression.
- ItemOpen AccessCellular immune ontogeny and birth transcriptome in HIV-exposed uninfected infants(2021) Kiravu, Agano; Gray, Clive; Jaspan, HeatherBackground. In some regions of Sub-Saharan Africa, up to 30% of newborns are born to mothers infected with human immunodeficiency virus (HIV). Maternal antiretroviral treatment (ART) has reduced vertical transmission to lower than 1%. Despite the success of prevention of mother-tochild transmission (PMTCT) programmes, a large number of children born to these mothers are exposed to HIV and antiretrovirals (ARVs) in utero yet remain uninfected. These individuals, known as children who are HIV-exposed and uninfected (cHEU), succumb to higher rates of disease morbidity compared to children who are HIV-unexposed (cHU) which suggests altered immunity in the cHEU. Differences in the numbers and function of cells of the innate and adaptive immune system have been documented in cHEU—though not consistently. While vaccine-induced antibody responses are robust in cHEU, data on potential cell mediated perturbations to vaccine antigens remains conflicting. This is in part due to inherent inter-cohort variation and differences in ART therapy strategies, feeding practices between cohorts and the assays used measure cellmediated responses. We leveraged two independent cohorts from Nigeria and South Africa of mother-infant pairs receiving antenatal and postnatal care all under Option B+ PMTCT. All HEU infants received pre-exposure prophylaxis for 6 weeks and the majority were exclusively breastfed until 6 months of age. We applied the same assays in both cohorts to test the hypotheses that HEU have altered T cell immunity compared to HU controls and distinct transcriptomic signatures at birth. These were tested in three distinct aims: 1) To identify transcriptional signatures at baseline that delineate cHEU from cHU 2) To compare the expression of surface marker broadly defining activated or regulatory phenotypes and the expression of intracellular markers of T cell function between cHEU and cHU over the first 9 months of life. 3) To characterise how differences in the immunising strains of Bacille Calmette-Gu'erin (BCG), the first vaccine received in these infants, impacts T cell immunity to both mycobacterial and non-mycobacterial antigens in cHEU and cHU. Methods. Two birth cohorts from Jos, Nigeria and Cape Town (CT), South Africa were recruited into this study as part of a larger parent study that aims to identify biological determinants of protection from mother-to-child transmission of HIV (Innate, Adaptive and Mucosal Immune Responses in Infants/INFANT study: HREC 285/2012). Infant blood was collected at several time points from birth to 36 weeks of life for immunological assays. Whole blood, collected at birth was preserved in PAXgene fluid for downstream messenger ribonucleic acid (mRNA) transcript analyses. Other whole blood samples were fixed and cryopreserved either directly ex vivo or after re-stimulation within 1 hour of phlebotomy with BCG, Tetanus Toxoid (TT), Bordetella pertussis (BP) antigens and Phytohemagglutinin (PHA). Multi-parameter flow cytometry was used to measure batched whole blood samples for (i) markers of T cell regulation and activation directly ex vivo, markers of T cell gut homing and proliferation—a proxy for HIV susceptibility, and (ii) vaccine-induced Th1 cytokine expression (IFN-, TNF-a, IL-2) and memory maturation. Cytokine responses were profiled for polyfunctionality by SPICE analysis and complemented by the COMPASS algorithm. Transcriptional profiling of whole blood at birth was done by RNA sequencing and differentially expressed genes were reported for absolute fold change of normalized counts were < 1.5 with FDR set at 0.05 using the DESeq2 package in R. Gene-set enrichment analysis (GSEA) was used to identify enriched or repressed gene pathways for absolute normalised effect sizes < 1.5 with FDR set at 0.05. Longitudinal analyses used a mixed effects ANOVA with time and HIV exposure as explanatory variables. Cross-sectional analyses comparing HIV exposure groups used Wilcoxon Ranked Sum Test, with p< 0.05 considered significant after multiple correction adjustment by Holm's step-down method. Results. Aim 1: A small set of DEGs were found between HEUs and HU groups at birth, 3 of which were upregulated and 12 that were downregulated. Among the upregulated genes, two are homologues of the arrestins: ARRDC4 (2.3 fold, adjusted p-adj< 0.001) and TXNIP (1.4 fold, padj< 0.001). Gene-set enrichment analysis however, showed no significant enrichment or suppression of gene pathways in HEUs. Aim 2: HIV/ARV exposure did not have an interaction effect with age (all time points) in explaining the frequencies of T cell markers ex vivo in a mixed-effects model. In cross-sectional unadjusted analyses however, trends towards increased median frequencies of markers of activation in the HEU group compared to HU controls were observed for specific ages: at birth (%CD8+HLA-DR+: 0.12 vs. 0.01, p=0.05), at week 7 (%CD8+CD25+: 0.13 vs. 0.04, p=0.01 and %CD8+HLA-DR+: 0.84 vs. 0.07, p=0.01) and at week 36 (%CD8+CD25+: 0.52 vs. 0.03, p< 0.001 and %CD8+HLA-DR+: 0.81 vs. 0.17, p=0.003). When adjusting for multiple comparisons, only CD25 expression remained significant on CD8+ T cells at week 36 (p-adj =0.04). The magnitudes of cytokine responses by T cells to vaccine antigens did not differ between HEU and HU infants however, transient differences in the polyfunctional profile of cells was observed at week 1 for mycobacterial-specific Th1 profiles in CT infants (p=0.002) by SPICE analysis. There were later differences at week 7 for BP-specific Th1 profiles in Jos infants (p=0.01) and at week 36 for BP-specific Th1 profiles in CT infants (p=0.03). The more robust COMPASS algorithm only detected a trend towards increased polyfunctional scores to BP responses in CT infants at week 36 (p=0.03). Aim 3: BCG immunising strain impacted the magnitudes and quality of responses to mycobacterial and non-mycobacterial vaccine antigens irrespective of HIV exposure status. Most significantly, at week 7, BCG-Denmark induced higher mycobacterial-specific frequencies of CD4 Th1 cytokines compared to Bulgarian (p< 0.001) and Russian strains (and (p< 0.001). BCGDenmark induced greater triple cytokine profiles to mycobacterial antigen compared to Bulgarian (p< 0.001) and Russian (p< 0.001) strains in SPICE analyses and the resulted were confirmed by COMPASS algorithm polyfunctional scores. Furthermore, BCG-Denmark significantly enhanced antigenicity to TT and BP vaccines. Conclusion. Transient differences exist in the frequencies of CD25 expressing CD8 T cells between HEU and HU groups, however other readouts of immunity suggest that in the context of effective PMTCT and exclusive breastfeeding practices, HEU infants are indistinguishable from their HIV unexposed peers.
- ItemOpen AccessCharacterisation of fibrinogen and fibrin proteolysis by the neutrophil membrane(1999) Kirsch, Richard; Shephard, EnidRecent studies have identified a novel 600 kDa neutrophil membrane associated protease which degrades fibrinogen, fibrin and C-reactive protein (CRP) during incubation of these ligands with phorbol 12-myristate 13-acetate (PMA, 5-10 ng/ml) stimulated neutrophils. This proteolysis is predominantly an extracellular event which occurs through a ligand dependent release of this protease from the neutrophil. Degradation products arising from this proteolysis not only become neutrophil associated but influence a number of important processes occurring in inflammation and coagulation. The aim of the present 'study was to purify and further characterize this protease and investigate the location of the neutrophil associated fibrinogen and fibrin degradation products. Whilst enzyme purification procedures were unsuccessful, several observations made during these attempts suggested that the neutrophil membrane associated proteolytic activity displayed similar characteristics to proteases of the azurophil granule. The proteolytic activity of the membrane was concluded from inhibitor profiles, zymography, and the apparent molecular mass values and hydrophobicity of the fibrinogen degradation products that it generated, to be the composite action of the azurophil granule proteases, human neutrophil elastase, cathepsin G and possibly proteinase 3. Electron microscopy analysis of PMA stimulated neutrophils incorporated within fibrin clots revealed morphological changes suggestive of neutrophil degranulation, and the proteolytic activity released by these cells was shown to be identical to that of azurophil granule proteases with respect to the apparent molecular mass values of the fibrin products that it generated. Immunoelectron microscopy revealed minimal internalization of fibrin like material during this process suggesting that neutrophil mediated fibrinolysis under these conditions is predominantly an extracellular event. Immunoelectron microscopy was used to localise fibrinogen degradation products previously reported to be associated with the neutrophil following incubation with fibrinogen. This revealed neutrophil associated fibrinogen products to be intracellular. Internalisation appears to be the result of pinocytosis which is stimulated in the presence of PMA. Although internalisation may be enhanced by an initial interaction of fibrinogen with the neutrophil membrane, a large proportion of uptake occurs via the fluid phase. Both intact and degraded forms of fibrinogen can associate with the neutrophil. Internalised material is rapidly degraded intracellularly into low molecular weight products which are partially released into the surrounding medium. This intracellular degradation, however, contributes minimally to the overall degradation of fibrinogen by neutrophils; the major pathway is extracellular. The demonstration in this· study, that the previously identified fibrinogen- fibrin- and CRP-degrading activity of the neutrophil membrane is due to azurophil granule proteases co-incides with numerous recent reports suggesting that membrane bound forms of these proteases, due to their ability to evade naturally occurring protease inhibitors, are the biologically relevant forms of these proteases. The membrane expression of azurophil granule proteases has recently been shown to be under the control of a variety of inflammatory mediators. Thus, neutrophil mediated degradation of fibrinogen, fibrin and CRP in vivo may be tightly controlled by the regulated expression of azurophil granule proteases on the neutrophil membrane.
- ItemOpen AccessCharacterisation of innate fungal recognition in the lung(Public Library of Science, 2012) Faro-Trindade, Inês; Willment, Janet A; Kerrigan, Ann M; Redelinghuys, Pierre; Hadebe, Sabelo; Reid, Delyth M; Srinivasan, Naren; Wainwright, Helen; Lang, Dirk M; Steele, ChadThe innate recognition of fungi by leukocytes is mediated by pattern recognition receptors (PRR), such as Dectin-1, and is thought to occur at the cell surface triggering intracellular signalling cascades which lead to the induction of protective host responses. In the lung, this recognition is aided by surfactant which also serves to maintain the balance between inflammation and pulmonary function, although the underlying mechanisms are unknown. Here we have explored pulmonary innate recognition of a variety of fungal particles, including zymosan, Candida albicans and Aspergillus fumigatus , and demonstrate that opsonisation with surfactant components can limit inflammation by reducing host-cell fungal interactions. However, we found that this opsonisation does not contribute directly to innate fungal recognition and that this process is mediated through non-opsonic PRRs, including Dectin-1. Moreover, we found that pulmonary inflammatory responses to resting Aspergillus conidia were initiated by these PRRs in acidified phagolysosomes, following the uptake of fungal particles by leukocytes. Our data therefore provides crucial new insights into the mechanisms by which surfactant can maintain pulmonary function in the face of microbial challenge, and defines the phagolysosome as a novel intracellular compartment involved in the innate sensing of extracellular pathogens in the lung.
- ItemOpen AccessCharacterisation of mucosal tissue in the foreskin after voluntary medical male circumcision(2016) Harryparsad, Rushil; Gray, Clive M; Olivier, Abraham JacobusBackground: Medical Male Circumcision (MMC) reduces the risk of HIV-1 acquisition by up to 60% as shown in a number of randomized controlled trials in Uganda, Kenya and South Africa. MMC has also been shown to reduce the prevalence of other sexually transmitted infections (STIs) like Herpes Simplex Virus (HSV) -2 and Human Papillomavirus (HPV) by 25% and 35% respectively. Asymptomatic STIs may elevate the risk of HIV-1 acquisition by recruiting HIV-1 target cells to the foreskin. The higher permeability of the inner foreskin may play a role in HIV-1 acquisition as well as the number of target cells present in the foreskin. The more inflamed inner foreskin may be increasing the risk of a productive HIV-1 infection. The aims of this dissertation was to a) examine the levels of keratinisation in the inner and outer foreskins after MMC; b) investigate the number of Langerhans, Ki67+ and CD4+ T cells in the inner and outer foreskin and c) identify the impact of asymptomatic STIs on the numbers and proliferative capacity of foreskin-resident Langerhans and CD4+ T cells.
- ItemOpen AccessCharacterization of a plasminogen activator from human melanoma cells cultured in vitro(1982) Heussen, Christa; Heussen, Christa; Dowdle, Eugene BIn this thesis I describe the work that I have done on the isolation and characterization of a plasminogen activator, Mel-PA, that is released by human melanoma cells cultured in vitro. This enzyme was compared to the urinary plasminogen activator, urokinase. The human melanoma cell line, RPMI-7272, (also referred to as the "Bowes" melanoma cell line) released large amounts of Mel-PA into the surrounding medium when cultured under serum-free conditions. A subline of these cells (Bowes II) was developed that was capable of continuous growth in the absence of serum. These cells released only one type of plasminogen activator with a molecular weight of approximately 70 000 daltons. A technique was developed in which plasminogen activators were separated electrophoretically and detected in polyacrylamide gel slabs containing the co-polymerized substrates, plasminogen and gelatin. The technique was compared with the zymographic procedure developed by Granelli-Piperno and Reich (62) using fibrin-plasminogen-agarose underlays. Mel-PA was concentrated and partially purified by affinity chromatography on benzamidine-sepharose. This preparation was used to prepare rabbit antisera to the enzyme. These antibodies inhibited the activity of plasminogen activators released by all melanoma cells but had no effect on urokinase. Antibodies to urokinase had no effect on Mel-PA. A survey of human plasminogen activators and their distribution by immunochemical and electrophoretic techniques showed that tissue extracts and body fluids, with the exception of normal urine, contained mixtures of Mel-PA- and urokinase-type enzymes. Urine of patients with some types of renal disease also contained a Mel-PA type enzyme. A study of the distribution of plasminogen activators in tissues and body fluids obtained from a number of animals showed that all mammals examined had two immunochemically distinct plasminogen activators that corresponded, in their distribution, to the urokinase-like and Mel-PA-like enzymes of man. Antibodies to human Mel-PA cross-reacted with the corresponding enzyme in all mammals tested, whereas antibodies to human urokinase were species specific. The seeds of the South African legume, Erythrina latissima, contain a 20 000 dalton protein that functioned as an inhibitor of Mel-PA, plasmin, and trypsin, but had no effect on urokinase. During its reaction with the enzymes the inhibitor was cleaved by Mel-PA and trypsin but not by urokinase. The susceptible bond was straddled by an intrachain disulphide bridge. The inhibitor bound reversibly to Mel-PA and could therefore be used to develop an affinity reagent for a one-step purification procedure for Mel-PA in melanoma cell harvest fluids. Purified preparations of Mel-PA c0uld be shown to comprise both active enzyme (two chain form) and pro-enzyme (one chain form). The one chain form could be converted to the two-chain form by treatment with plasmin. It could also be shown that fibrinogen and fibrin contained a contaminating protease that was capable of converting pro-Mel-PA to Mel-PA. A comparative study of the kinetic behaviour of Mel-PA and urokinase showed numerous differences between the catalytic activities of these two enzymes. Mel-PA was capable of binding to fibrinogen insolubilized on a plastic surface whereas urokinase did not. The presence of fibrinogen enhanced the plasminogen activating activity of Mel-PA but had no effect on urokinase activity.
- ItemOpen AccessCharacterization of CD8 T cell responses in Mycobacterium Tuberculosis infection(2011) Moshi, Noell Dominika; Day, Cheryl; Hanekom, Willem AThe aim of this project was to compare the breadth and magnitude of CFP10 and ESAT6-specific CD8 T cell responses in individuals with latent Mycobacterium tuberculosis (MTB) infection (LTBI) and active TB disease, and further define MTB-specific CD8 T cell phenotypes associated with latent infection and active disease. Ex vivo IFN? Elispots and proliferation assays were used to identify immunodominant ESAT6 and CFP10 15mer peptides targeted by CD8 T cells in LTBI and TB donors. A multiparameter flow cytometry panel was designed and optimized to assess turnover, susceptibility to apoptosis and terminal differentiation/senescence in CD8 T cells from TB and LTBI donors. Bcl-2, Ki67,CD95, CD57, CD127 and IFNγ were thus measured in each group.
- ItemRestrictedChinks in the armor of the HIV-1 Envelope glycan shield: implications for immune escape from anti-glycan broadly neutralizing antibodies(Elsevier, 2017-01-15) Moyo, Thandeka; Ferreira, Roux-cil; Davids, Reyaaz; Sonday, Zarinah; Moore, Penny L; Travers, Simon A; Wood, Natasha T; Dorfman, Jeffrey RobertGlycans on HIV-1 Envelope serve multiple functions including blocking epitopes from antibodies. We show that removal of glycan 301, a major target of anti-V3/glycan antibodies, has substantially different effects in two viruses. While glycan 301 on Du156.12 blocks epitopes commonly recognized by sera from chronically HIV-1-infected individuals, it does not do so on CAP45.G3, suggesting that removing the 301 glycan has a smaller effect on the integrity of the glycan shield in CAP45.G3. Changes in sensitivity to broadly neutralizing monoclonal antibodies suggest that the interaction between glycan 301 and the CD4 binding site differ substantially between these 2 viruses. Molecular modeling suggests that removal of glycan 301 likely exposes a greater surface area of the V3 and C4 regions in Du156.12. Our data indicate that the contribution of the 301 glycan to resistance to common neutralizing antibodies varies between viruses, allowing for easier selection for its loss in some viruses.
- ItemOpen AccessComplement component C6 deficiency and susceptibility to Neisseria Meningitidis infection(2004) Orren, Ann; Potter, Paul CMeningococcal disease remains one of the most serious bacterial infections in both Western and developing countries. Despite recent advances in treatment the mortality rate remains at about 12%.1 There is a group of South Africans who are particularly vulnerable to this disease. They are individuals with genetically determined deficiencies of individual terminal complement proteins, in particular of the sixth component of complement (C6).2 The human complement system forms part of the humeral immune system and consists of a series of proteins.3 The interaction of antigen (such as components of bacterial outer membranes) and antibody leads to activation of the first component C1q, and consequent activation of part or all of the complement cascade. This has a number of biological effects including the formation on the membrane of the membrane attack complex (MAC) from components C5b, C6, C7, C8 and C9. The MAC is able to mediate lysis of some mammalian cells such as red blood cells, as well as lysis of bacteria and certain viruses. This action of complement has been recognised for many years as playing a major role in defence against infection.4 In addition, MAC action is often sublytic on nucleated host cells, and this interaction can sometimes stimulate cellular biosynthesis and act in a pro-inflammatory manner.5 Many other complement proteins, and products of complement activation, also interact with the cellular immune system and the inflammatory system. If activation is caused by pathological processes such as ischaemia, complement can be an important contributor to host tissue necrosis.6 The effects of complement activation are very complex and can be detrimental as well as beneficial.
- ItemOpen AccessDelayed goblet cell hyperplasia, acetylcholine receptor expression, and worm expulsion in SMC-specific IL-4Ralpha-deficient mice(Public Library of Science, 2007) Horsnell, William G C; Cutler, Antony J; Hoving, J Claire; Mearns, Helen; Myburgh, Elmarie; Arendse, Berenice; Finkelman, Fred D; Owens, Gary K; Erle, Dave; Brombacher, FrankInterleukin 4 receptor α (IL-4Rα) is essential for effective clearance of gastrointestinal nematode infections. Smooth muscle cells are considered to play a role in the type 2 immune response-driven expulsion of gastrointestinal nematodes. Previous studies have shown in vitro that signal transducer and activator of transcription 6 signaling in response to parasitic nematode infection significantly increases smooth muscle cell contractility. Inhibition of the IL-4Rα pathway inhibits this response. How this response manifests itself in vivo is unknown. In this study, smooth muscle cell IL-4Rα-deficient mice (SM-MHC Cre IL-4Rα −/lox ) were generated and characterized to uncover any role for IL-4/IL-13 in this non-immune cell type in response to Nippostrongylus brasiliensis infection. IL-4Rα was absent from α-actin-positive smooth muscle cells, while other cell types showed normal IL-4Rα expression, thus demonstrating efficient cell-type-specific deletion of the IL-4Rα gene. N. brasiliensis -infected SM-MHC Cre IL-4Rα −/lox mice showed delayed ability to resolve infection with significantly prolonged fecal egg recovery and delayed worm expulsion. The delayed expulsion was related to a delayed intestinal goblet cell hyperplasia, reduced T helper 2 cytokine production in the mesenteric lymph node, and reduced M3 muscarinic receptor expression during infection. Together, these results demonstrate that in vivo IL-4Rα-responsive smooth muscle cells are beneficial for N. brasiliensis expulsion by coordinating T helper 2 cytokine responses, goblet hyperplasia, and acetylcholine responsiveness, which drive smooth muscle cell contractions.
- ItemOpen AccessDeletion of IL-4Ralpha on CD4 T cells renders BALB/c mice resistant to Leishmania major infection(Public Library of Science, 2007) Radwanska, Magdalena; Cutler, Antony J; Hoving, J Claire; Magez, Stefan; Holscher, Christoph; Bohms, Andreas; Arendse, Berenice; Kirsch, Richard; Hunig, Thomas; Alexander, JamesAuthor Summary Leishmaniasis is a disease induced by a protozoan parasite and transmitted by the sandfly. Several forms of infection are identified, and the different diseases have wide-ranging symptoms from localized cutaneous sores to visceral disease affecting many internal organs. Animal models of human cutaneous leishmaniasis have been established in which disease is induced by infecting mice subcutaneously with Leishmania major. Different strains of inbred mice have been found to be susceptible or resistant to L. major infection. "Healer" C57BL/6 mice control infection with transient lesion development. The protective response to infection in this strain is dominated by type 1 cytokines inducing parasite killing by nitric oxide. Conversely, "nonhealer" BALB/c mice are unable to control infection and develop nonhealing lesions associated with a dominant type 2 immune response driven by cytokines IL-4 and IL-13. However, mice deficient in IL-4/IL-13 signaling are not protected against development of cutaneous leishmaniasis. Here we describe a BALB/c mouse where the ability to polarize to a dominant type 2 response is removed by cell-specific deletion of the receptor for IL-4/IL-13 on CD4 + T cells. These mice are resistant to L. major infection similar to C57BL/6 mice, which highlights the role of T helper 2 cells in driving susceptibility and the protective role of IL-4/IL-13 signaling in non-CD4 + T cells in BALB/c mice.
- ItemOpen AccessThe detection of occult metastatic disease in patients with cutaneous melanoma(1999) Hanekom Gideon S; Kidson, SusanThe ability to identify melanoma patients with progressive disease is central to efficient management. The challenge therefore, is to develop prognostic markers and techniques which will allow the identification of those patients whom, at the time of primary tumor diagnosis, already have micrometastases (occult or clinically undetectable metastases). The use of the reverse transcription-PCR (RT-PCR) technique for the detection of circulating melanoma cells (CMCs) is potentially a powerful tool for identifying those patients at risk for developing metastases. The first aim of this study was to develop a more sensitive, reproducible, cost effective and clinically applicable assay and to eliminate the problem of false positives. A combined RT-PCR assay for tyrosinase mRNA, a marker specific for melanoma cells, was developed and tested. It was shown that the assay can reproducibly detect a single, viable melanoma cell in 10-15 ml of peripheral blood. Furthermore, a simple but effective procedure was developed to prevent carryover contamination. It was found that the chance of obtaining normal melanocyte contamination with the needle prick during blood sampling was only 2% and that illegitimate transcription does not contribute to sporadic false positives. The second aim of this study was to determine whether the early detection of CMCs is of any clinical value to monitor melanoma progression. Peripheral blood samples from 143 patients with primary melanoma (PM) were analysed by RT-PCR for the presence of tyrosinase mRNA. Seven percent (10/143) of the patients with PM had detectable CMCs. The percentage of PCR-positive patients was higher for stage II patients (9.0%) compared to stage I (5.3%) but the difference was not significant. A significantly higher percentage (P < 0.05) PCR-positive patients were found to have tumors greater than 1.5 mm thick and with ulceration present. Although this finding supports the notion that tumor thickness and ulceration are the two most significant prognostic factors, it was not possible at this stage, to link this directly to a poor prognosis since the majority of the PCR-positive patients have not yet (within four years) developed metastatic disease. However, the data does indicate that cells from tumors greater than 1.5 mm thick and with ulceration have a greater propensity to enter the circulation but that these cells do not necessarily have the ability to establish metastases. The results suggest that the detection rate of 9% for patients with stage II disease is much lower than would be expected, since 23.9% (16/67) of the stage II patients subsequently developed metastases. Of these 16 patients, only one was PCR-positive, one week before the metastases became clinically evident. Thus, the current technique fails to predict the likelihood of developing metastatic disease (P = 0.3485). The other nine PCR-positive patients had not yet developed metastases after a median follow-up period of four years. It is concluded that the current technique for the detection of CMCs is of limited clinical value to predict the likelihood of metastasis in patients with PM. It is suggested that other anatomic compartments, such as sentinel lymph nodes, should be explored for the identification of patients at risk for developing metastases. The third aim of this study was to determine whether high or low plasma levels and/or activity of plasminogen activator inhibitor type 1 (PAI1) correlate with the presence of metastatic disease in patients with melanoma. PAI1 is considered to be the main regulator of fibrinolytic activity in blood and has been identified as a key enzyme in the metastasis and vascularization of solid tumors. A unique enzyme-linked immunosorbant assay was developed to measure both the total amount of PAI1 in plasma as well as the active fraction of the inhibitor. This novel assay was then used to analyse and compare the plasmatic PAI1 levels and activity of a group of patients with advanced melanoma (AM) with a group of patients with primary disease and a control population. There was no statistical difference in the total plasmatic PAI1 levels between the controls and patients with PM and AM (P = 0.6199). In contrast, there was a significant difference in the active fraction of PAI1 between the controls and patients with PM or AM (P = 0.0076). A value of less than 44% active PAI1 was shown to be clinically meaningful by linear discriminant analysis. This means that a melanoma patient with a plasmatic PAI1 activity value less than 44% will have a 50% chance of harbouring metastases. Of the patients with PM, 19% had PAI1 activity values less than 44%, which strongly supports further investigations to determine whether plasmatic PAI1 activity levels might be predictive of metastatic disease. The false positive rate was 2.6%. It is speculated that this reduction in the active fraction of PAI1 for patients with AM might be attributed to tumor-derived tissue plasminogen activator and/or other melanoma-derived proteases or factors. The last section of this study describes several monoclonal antibodies (Mabs) that were developed against PAI1 in order to obtain useful reagents to study the regulatory functions of PAI1 in the metastasis and vascularization of solid tumors. The baculovirus expression system was used to express human PAI1 in insect cells and the crude infected cell population was used as the immunogen in mice. This approach was followed since the Escherichia coli-derived recombinant molecule elicited a poor immune response. A unique panel of anti-PAI1 Mabs was developed that were characterized with regard to their use for immunoblotting, immunofluorescence and immunocytochemistry. One of these antibodies blocked the binding of PAI1 to vitronectin and inhibited the activity of the inhibitor. Finally, two of these Mabs turned out to be extremely valuable and were used to develop a novel microtiter plate assay for measuring the active fraction of PAI1 in biological fluids by making use of Mabs against different epitopes of PAI1.
- ItemOpen AccessDetermination of the role of cytokines using gene deficient mice in African trypanosomiasis infection(2008) Barkhuizen, Mark; Brombacher, Frank; Magez, SefanAfrican trypanosomiasis encompasses diseases caused by pathogenic trypanosomes, infecting both humans and animals alike. To determine the immunological role of IL=12 family members in Trypanosoma brucei brucei, Trypanosoma evansi and Trypanosoma congolense infections, IL-12p35¯/¯, IL-12p40¯/¯ and IL-12p35¯/¯/p40¯/¯ mice were used. While the two latter mouse strains lack all IL-12 homologues, IL-12p35¯/¯ mice still produce IL-12p80 homodimers and IL-23. In infection with T.b. brucei and T.evansi; IL-12p35¯/¯, IL-12p40¯/¯ or IL-12p35¯/¯/p40¯/¯ mice were susceptible to both these pathogens, demonstrated by increased mortality compared to wild type C57BL/6 mice. The different IL-12 deficient mouse strains showed similar mortality kinetics, suggesting that IL-12p70 but not the IL-12p80 homodimer or IL-23 plays a crucial role in survival. Similarly, parasitemia control was reduced in the absence of IL-12p70. While plasma levels of IgM and IgG2c were similar between IL-12 deficient mice and wild type mice, IF-γ production. As IFN-γR¯/¯ mice were also highly susceptible to both T.b. brucei and T. evansi, IL-12p70-dependent IFN-γ production seems to be important mechanism involved in resistance against both these pathogens.
- ItemOpen AccessDifferential adaptation of Candida albicans in vivo modulates immune recognition by dectin-1(Public Library of Science, 2013) Marakalala, Mohlopheni J; Vautier, Simon; Potrykus, Joanna; Walker, Louise A; Shepardson, Kelly M; Hopke, Alex; Mora-Montes, Hector M; Kerrigan, Ann; Netea, Mihai G; Murray, Graeme IAuthor Summary Dectin-1 is a pattern recognition receptor recognising the fungal cell-wall component, β-glucan, and plays an essential role in controlling C. albicans infections in both mouse and man. Candida albicans is part of the normal human microflora, yet is capable of causing superficial mucosal infections as well as life-threatening invasive diseases, particularly in patients whose immune function is compromised. Here we found that the contribution of Dectin-1 is limited to specific strains of C. albicans ; effects which are due to the differential adaptation of these pathogens during infection. Importantly, C. albicans strains showed variations in both the composition and nature of their cell walls, and it was these differences which influenced the role of Dectin-1. Crucially, we found that we could alter the fungal cell wall, and subsequent interactions with the host, using antifungal drugs. These findings have substantial implications for our understanding of the factors contributing to human susceptibility to infections with C. albicans , but also treatment strategies.
- ItemOpen AccessEffect of standard tuberculosis treatment on plasma cytokine levels in patients with active pulmonary tuberculosis(Public Library of Science, 2012) Riou, Catherine; Peixoto, Blas Perez; Roberts, Lindi; Ronacher, Katharina; Walzl, Gerhard; Manca, Claudia; Rustomjee, Roxana; Mthiyane, Thuli; Fallows, Dorothy; Gray, Clive MBACKGROUND: Sputum Mycobacterium tuberculosis (Mtb) culture is commonly used to assess response to antibiotic treatment in individuals with pulmonary tuberculosis (TB). Such techniques are constrained by the slow growth rate of Mtb, and more sensitive methods to monitor Mtb clearance are needed. The goal of this study was to evaluate changes in plasma cytokines in patients undergoing treatment for TB as a means of identifying candidate host markers associated with microbiologic response to therapy. METHODS: Twenty-four plasma cytokines/chemokines were measured in 42 individuals diagnosed with active pulmonary TB, 52% were HIV co-infected. Individuals, undergoing a 26-week standard TB treatment, were followed longitudinally over 18 months and measurements were associated with HIV status and rates of sputum culture conversion. RESULTS: Plasma concentrations of interferon-inducible protein-10 (IP-10) and vascular endothelial growth factor (VEGF) were significantly reduced upon TB treatment, regardless of HIV status. By the end of treatment, IP-10 concentrations were significantly lower in HIV negative individuals when compared to HIV-positive individuals (p = 0.02). Moreover, in HIV negative patients, plasma VEGF concentrations, measured as early as 2-weeks post TB treatment initiation, positively correlated with the time of sputum conversion (p = 0.0017). No significant changes were observed in other studied immune mediators. CONCLUSIONS: These data suggest that VEGF plasma concentration, measured during early TB treatment, could represent a surrogate marker to monitor sputum culture conversion in HIV uninfected individuals.
- ItemOpen AccessEffects of delayed BCG vaccination on cellular immune responses in HIV-exposed infants(2014) Tchakoute, Christophe Toukam; Jaspan, Heather B; Gray, CliveIncludes abstract. Includes bibliographical references.
- ItemOpen AccessThe effects of fibroblast growth factor-2 (FGF-2) on haematopoietic cells and the identification of those cells expressing FGF receptors(2002) Burger, Patricia E; Wilson, E LBibliography: leaves 125-150.
- ItemOpen AccessEvaluating the neutralizing antibody response to HIV-1 membrane proximal external regional; Implications for vaccine design(2014) Jacob, Rajesh Abraham; Dorfman, JeffreyInducing broadly neutralizing antibodies targeting the HIV-1 envelope is thought to be crucial for developing an effective vaccine. The Membrane Proximal External Region (MPER) within the HIV- 1 gp41 envelope is a promising vaccine target. The MPER is highly conserved, functionally constrained, facilitates virus fusion and is targeted by broadly neutralizing monoclonal antibodies. The objectives of this research were 1) To evaluate the neutralization breadth of antibodies induced by epitopes within the MPER compared to the PG9/16-site in chronically HIV-1-infected individuals, 2) to identify neutralization resistant HIV-1 isolates (using plasma samples infected with the same subtype) and to characterize their sensitivity to anti-MPER antibodies and 3) to determine the accessibility of the MPER to HIV-1 induced polyclonal anti-MPER antibodies in a highly neutralization resistant virus (253-11; CRF02_AG subtype).