OpenUCT :: Browsing by Subject "Immunology"

Browsing by Subject "Immunology"

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Open Access
Characterisation of fibrinogen and fibrin proteolysis by the neutrophil membrane
(1999) Kirsch, Richard; Shephard, Enid
Recent 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.
Open Access
Characterization of a plasminogen activator from human melanoma cells cultured in vitro
(1982) Heussen, Christa; Heussen, Christa; Dowdle, Eugene B
In 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.
Open Access
Determination of the role of cytokines using gene deficient mice in African trypanosomiasis infection
(2008) Barkhuizen, Mark; Brombacher, Frank; Magez, Sefan
African 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.
Open Access
The 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 L
Bibliography: leaves 125-150.
Open Access
The functional characterisation of the C-type lectin : Clecsf8
(2011) Graham, Lisa Maria; Brown, Gordon
Clecsf8 is a poorly characterised member of the "Dectin-2 cluster" and was originally thought to be expressed exclusively by macrophages. In this study it was demonstrated that Clecsf8 is primarily expressed by peripheral blood neutrophils and monocytes.
Open Access
Immune and allergic responses to Anisakis pegreffii, with focus on the roles of IL-4, IL-13 and the IL-4 receptor alpha
(2007) Nieuwenhuizen, Natalie; Lopata, Andreas; Brombacher, Frank
The fish-parasitizing nematode Anisakis pegreffii induces gastrointestinal disease and allergy when ingested by humans, and can cause occupational allergy in seafood processing workers. The present study examines immune and allergic responses to A. pegreffii in wildtype and gene deficient mice, with special focus on interleukin(IL)-4, IL-13, and the IL-4 receptor alpha (IL-4Rα). Experimental murine models of Anisakis infection, Anisakis-induced anaphylaxis and Anisakis-induced dermatitis were established in order to gain insight into the immune responses generated against Anisakis and unravel mechanisms of allergic disease.
Open Access
Immune parameters as predictors of response to maintenance therapy in low grade non-Hodgkin's lymphoma patients
(1998) Watkins, Marcia Linda Vivienne; Ress, Stanley; Lukey, Pauline
The non-Hodgkin's lymphomas (NHL) are a heterogeneous group of malignancies characterised by the uncontrolled proliferation of lymphoid cells. The Working Formulation divides these neoplasms into low, intermediate and high-grade categories according to their natural history. Low grade NHL (LG-NHL) is clinically indolent whereas high grade NHL is more aggressive. Most LG-NHL patients respond well to chemotherapy, but are rarely cured, making death from LG-NHL virtually inevitable. In this study on LG-NHL patients, all patients received combination chemotherapy for 6 weeks, which consisted of cyclophosphamide, oncovin (vincristine) and prednisone (COP). After this treatment patients received either oral cyclophosphamide or alpha-interferon (α-IFN) as maintenance treatment for a period of two years. The ability to predict patients' treatment response at diagnosis would be extremely helpful for both the patient and the clinician. This would enable appropriate therapy to be instituted at diagnosis, whether it be cyclophosphamide or α-IFN, which might increase the subsequent survival of these patients, as this would prevent patients from receiving treatment to which they would not respond. Furthermore, patients who were unlikely to respond to either of these two treatments could be targeted for alternative treatment or form part of a clinical research trial. LG-NHL patients were assessed at diagnosis for a number of immune parameters. These included: - natural killer activity (NKA), α-IFN enhanced NKA, mitogen and antigen proliferation, lymphokine activated kill against both KS62 and Daudi targets, phenotypic analysis of circulating lymphocytes, assessment of IL-2 levels after mitogen stimulation of lymphocytes, as well as the determination of plasma IL-2 levels. All these above-mentioned parameters were serially monitored over time in an attempt to predict early relapse. A statistically significant reduction in percentage of circulating CD3+ and CD8+ cells and an increase in percentage NK cells was found in patients at diagnosis as compared to normal controls. A reduction in percentage of circulating NK cells over time appeared to be a good prognostic indicator and an increase in percentage NK cells a poor prognostic indicator in this group of patients, although this was not statistically significant. When the in vitro α-IFN enhanced NKA was indicated as a percentage of NKA, a negative correlation appeared to exist between this in vitro response to α-IFN and the in vivo response, although this was not statistically significant (i.e. those patients showing the least α-IFN enhanced NKA were those that responded clinically and those with the highest α-IFN augmented NKA either relapsed or transformed to a higher grade of NHL). By incorporating the percentages of circulating lymphocytes present in the peripheral blood, into the multivariate discriminant analysis, it was possible to derive formulae to enable the prediction of response to either α-IFN or cyclophosphamide. This was a particularly exciting and apparently novel finding. The work presented here has therefore established both a possible negative indicator of α-IFN response (a high in vitro α-IFN response) and a positive indicator (the formula generated in the multivariate discriminant analysis using the flow cytometric phenotypic analysis). By making using of both of these factors, it would increase the chances of patients being selected for appropriate forms of treatment and minimise the chances of patients suffering a relapse. The Kaplan-Meier curve indicated that cyclophosphamide treatment was more beneficial than α-IFN therapy in this group of patients, although this did not attain statistical significance. Verification of all of these above-mentioned findings would need further confirmation in a larger study.
Open Access
Immunological analysis of pericardial tuberculosis
(2011) Matthews, Kerryn; Wilkinson, Katalin; Wilkinson, Robert J; Mayosi, Bongani
Pericardial tuberculosis (TB) offers a relevant human model to study TB at the site of disease and to determine the effect of HIV-1 infection. 96 Patients with pericardial TB were recruited into this study, 68 of whom were HIV-1 infected. Where clinically indicated, pericardiocentesis was performed to drain pericardial fluid and blood was drawn. The data derived from the study provide novel insight into the immune response in the pericardium to TB infection. Furthermore, HIV-1 infection caused a dysregulation of the immune response.
Open Access
Immunological evaluation of HIV-negative invasive fungal disease at Groote Schuur Hospital, Cape Town, South Africa
(2019) Onyango, Vonwicks Czelestakov; Peter, Jonathan; Dlamini, Sipho
Background The majority of invasive fungal disease in South African hospitals is HIV-related or associated with another secondary immunodeficiency e.g. haematopoietic stem cell transplant. After excluding secondary immunodeficiency, a detailed immune work-up can lead to a diagnosis of primary immunodeficiency. Objective To detail an appropriate step-wise immunological work-up for a series of patients with invasive fungal diseases and possible underlying primary immune deficiency. Methods Detailed review of all culture- or histologically confirmed cases of invasive fungal disease (IFD) at Groote Schuur Hospital between 2007-2017. Step-wise immunological work-up of IFD patients with no secondary immunodeficiency. Clinical characteristics and step-wise immunological profiles were evaluated. Results Sixty-seven adults with IFD were identified; 72% (48/67) were HIV-related. 8/19 HIVnegative cases were either deceased (4) or lost-to-follow-up (4). Work-up of the remaining 11 cases found five with non-HIV secondary immunodeficiencies (Lupus, liver transplant, endstage renal failure and haematological malignancy). A primary immunodeficiency was suspected in six cases, but 1 case of cutaneous sporotrichosis was excluded; with five cases (4 with disseminated Cryptococcus neoformans and 1 with cerebral aspergillosis) undergoing detailed immune work-up. A case of idiopathic CD4 lymphopenia was diagnosed; but all other cases had no evidence of neutrophil or a cell-mediated immune defect; including investigations of naïve and memory T-cell subsets and cytokine responses to PHA and candida. All cases were noted to have low baseline vaccine responses and Vitamin D deficiency. Conclusion Invasive fungal disease is predominantly associated with HIV and secondary immunodeficiency in South Africa. Known primary immunodeficiencies can be identified with basic immune work-up; but no obvious functional immune defect is evident in the majority of these cases.
Open Access
Investigating the role of IL-4/IL-13 and their receptors in ulcerative colitis
(2010) Hoving, J Claire; Brombacher, Frank
Ulcerative colitis (UC) is a heterogeneous inflammatory bowel disease (IBD) associated with chronic inflammation of the gastrointestinal tract. Characterized by genetic and immunological abnormalities, UC has overly aggressive T-cell responses to commensal bacteria eventually leading to disease pathology. UC is distinguished from Crohn's disease, another form of IBD, in that it is driven by a T helper type 2 (Th2) immune response. Oxazolone-induced colitis is a mouse model resembling UC presenting with inflammation limited to the distal colon and mixed neutrophil/lymphocyte infiltration in the superficial layer of the mucosa. The Th2 cytokines interleukin (IL)-4 and IL-13 are associated with the onset of oxazolone colitis and both signal through a common IL-4 receptor-alpha chain (IL-4R +-). Neutralizing these cytokines prevents or ameliorates disease significantly, while neutralizing IL-12 exacerbates disease symptoms. As many aspects of the mechanisms involving Th2 cytokines in colitis remain undefined, the aim of this study was to investigate the role of IL-4 and IL-13 and the receptors through which they signal in oxazolone-induced colitis. Previous studies have highlighted a role for IL-4 and IL-13 in mediating oxazolone colitis. We show that while IL-13-deficient BALB/c mice were protected from disease onset, IL-4R +- deficient BALB/c mice developed exacerbated disease symptoms.
Open Access
Investigating transmembrane TNF and transmembrane p55TNFR mediated signaling in host immune function during Mycobacterium tuberculosis infection
(2010) Dambuza, Ivy M; Jacobs, Muazzam
The importance of TNF-TNFR signaling in immunity against M. tuberculosis has been established. The aims of this study were to characterize the functions of membrane-bound TNF (Tm-TNF) and soluble TNF (solTNF) and to investigate the role of membrane-bound p55TNFR signaling as well as the in vivo significance of TNFR shedding in host immune responses during infection with M. tuberculosis H37Rv. To address this, mice expressing only the membrane-bound TNF or membrane-bound p55TNFR were exposed to a low dose of M. tuberculosis H37Rv by aerosol inhalation infection. The results presented in this dissertation illustrate that Tm-TNF mice were able to control acute M. tuberculosis infection but succumbed to chronic exposure to M. tuberculosis with pneumonia. We demonstrate that Tm-TNF mice displayed heightened pulmonary macrophage activation reflected by enhanced cell surface expression of MHC-II, CD80 and CD86 as well as enlargement of granulomas. Furthermore, our results show that solTNF has a regulatory function that modulates the magnitude of Th1 immune responses during acute and chronic stages of the infection. The evaluation of the functions of Tm-TNF and solTNF in host immune function in the presence of an established mycobacteria-specific immune response was carried out using a 'drug-based' M. tuberculosis reactivation model. Here, mice that were challenged with a low dose of M. tuberculosis were exposed to INH-RIF treatment for six weeks in drinking water, after which therapy was withdrawn and immune responses during reactivation were analyzed. Our results demonstrate that complete absence of TNF resulted in host susceptibility to recrudescence tuberculosis in the presence of a mycobacteria-specific immune response. TNF deficient mice were unable to suppress bacilli growth and formed diffused granulomas and succumbed early to reappearing tuberculosis compared to WT mice. By contrast, we show that Tm-TNF was sufficient for containment of reappearing mycobacterial growth and sustaining immune pressure in a manner comparable to WT control mice. xii Lastly, the analysis of host immune responses in mice expressing a non-sheddable p55TNFR revealed that persistent p55TNFR cell surface expression does not afford better protection to low dose M. tuberculosis infection. However, we observed a transient elevation in the frequency of pulmonary CD11b+/MHC-II+ cells in mice expressing a non-sheddable p55TNFR relative to WT mice as well as reduced cell surface expression of CD44 on CD4+ T cells. We also found that pulmonary IL-12p70 and TNF concentrations were elevated whereas IFNÎ³ levels were reduced in mice expressing a non-sheddable p55TNFR relative to WT mice. Furthermore, data presented here describe the in vivo functional significance of p75TNFR shedding. We demonstrate using a double mutant mouse strain that in the absence of p75TNFR, mice expressing a non-sheddable p55TNFR display enhanced ability to control M. tuberculosis infection.
Open Access
Investigation of immune responses in different mouse models of allergic asthma
(2008) Kirstein, Frank; Lopata, Andreas; Brombacher, Frank; Horsnell, William
Allergies are a common chronic disease and considerably decrease the quality of life for affected individuals. Understanding the immune responses during allergic diseases is essential for both diagnosis and the development of effective therapies. The route of sensitisation to allergens is one factor that influences the immune response and the outcome of allergic diseases and both human and animal studies have highlighted IL-4Ra as an important component in the induction of allergy. The aim of this study was to investigate the contributions of the route of sensitisation to allergens with focus on the significance of cell specific expression of IL-4Ra in the onset of allergy. The route of sensitization to Anisakis pegreffii influences the outcome of experimental allergic asthma: Worldwide, increasing numbers of allergies to the fish parasite Anisakis pegreffii are reported. Anisakis can cause allergies after accidental infection of humans and in the occupational environment. Currently it is not clear if different exposure routes to Anisakis affect the development of allergic asthma and if they have an influence on the immune response. To address these questions, the present study investigated immune responses and disease development after Anisakis live infection and after nasal sensitisation in a mouse model of allergic airway disease. We showed that the route of sensitisation influences the outcome of Anisakis pegreffii induced allergic asthma and demonstrated important contributions of IL-4Ra to the underlying immune response. Alternatively activated macrophages are not necessary for the development of experimental allergic lung inflammation: Development of alternatively activated macrophages (AAM) is induced by signals of IL-4Ra. Alternatively activated macrophages (AAM) are a feature of allergic asthma in clinical and experimental investigations but their role in the development of allergy is not defined. To address this, a model of acute allergic asthma was used to compare mice deficient in AAM (LysMcrelL-4Ra-110x mice) with control mice. We found that the presence of AAM at early stages of allergic airway inflammation these cells was not required for the onset of the disease. Smooth muscle IL-4Ra is not required for experimental allergic asthma: In vitro studies have suggested that IL-4Ra signalling on airway smooth muscle cells (ASMC) is critical for airway irrflammation and airway hyperresponsiveness. Using mice deficient for IL-4Ra in ASMC, the in vivo effects of impaired IL-4Ra signalling in ASMC on the outcome of asthmatic disease were investigated. The impairment of IL-4Ro: on SMC had no effect on major aetiological markers of allergic asthma. These findings suggest that IL-4Ra responsiveness in airway SMC during the acute phase of allergic asthma is not critical for the outcome of the disease. Conclusions: The present study showed the importance of the route of sensitisation and IL4Ra in the development of allergy to Anisakis pegreffii. The use of in vivo models of experimental allergic asthma revealed that the route of sensitisation can influence the underlying immune response of the disease. Furthermore, by using mice with cell specific deficiencies in IL-4Ra it was demonstrated that expression of this receptor on smooth muscle cells and macrophages is not essential for the development of acute experimental allergic airway disease, as it has been previously suggested.
Open Access
Investigation the immunological response elicited to the gastrointestinal nematode pinworm (Syphacia obvelata)
(2006) Michels, Chesney Elroy; Brombacher, Frank
It is important to emphasize with the advance of biotechnology and increased global exchange of animals and animal products, the risks of introducing adventitious infections. Previous studies of specitic-pathogen-free mouse colonies have identified the presence of infectious agents in 10-35% of research institutions investigated. Prevalence was higher among non-SPF mice with pinworm reported in 70% of institutions housing rodents under these conditions. Pinworm, a gastrointestinal (GI) nematode is commonly found in laboratory animals. The direct transmission of the parasite by contaminated food, water and bedding result in their continual re-exposure to the host, making the control of pinworm in animal holdings quite difficult. Syphacia obvelata, mouse pinworm, has been shown to interfere with research goals in several experimental models. In this study, we show the consequence of a pinworm outbreak in a transgenic barrier facility and define the immune response elicited in BALB/c mice. Infection with S. obvelata induced a transient Th2-type immune response with elevated cytokine production and parasite-specific IgG1. In contrast, HALB/c mice, deficient for IL-13, lL-4/13 or IL-4Ra showed chronic disease with more than 100-fold higher parasite burden, increased IFN-y production, parasite-specific IgG2b and a default Th2 response. Notably, infected lL-4-/- BALB/c mice showed only slight elevated parasite burden compared to controls, suggesting that IL-13 plays the dominant role in the control of S. obvelata. Furthermore, no significant eosinophilia, mastocytosis or goblet cell hyperplasia was induced. In a well-established ovalbumin (Ova) anaphylaxis model, we show that mice infected with S. obvelata induce a more severe anaphylactic reaction, with consistently greater temperature decline than their non-infected counterparts. Analysis of spleen cells further revealed a marked reduction of Ova-specific Th2 cytokines, highlighting the importance of pinworm free experimental mice. Finally, we generated anti-S. obvelata antibody to optimize the detection ELISA and identified target epitopes for future analysis. In conclusion, we identify the T helper immune response induced to S. obvelata and demonstrate the importance of IL-13 for the expulsion of the GI nematode. We show that S. obvelata induces a non-protective immune response to a common food allergen and confirm that the pinworm-specific ELISA is an effective diagnostic tool for detecting pinworm infected mice.
Open Access
Investigations of cellular immune mechanisms to malaria during pregnancy in a malaria holoendemic region of Western Kenya
(2003) Othoro, Caroline; Ryffel, Bernhard
Women during pregnancy in holoendemic regions of malaria are at an increased risk for peripheral malaria infections with potential for developing placental malaria. The immunological basis of protection and pathogenesis are incompletely understood. This thesis investigates both processes. Research on maternal placental immune responses necessitates the collection of reliable placental intervillous blood; an appropriate method for placental blood collection was therefore ﬁrst determined. Five documented methods of collection (perfusion, incision, biopsy, tissue grinding and prick) were compared for foetal blood contamination and mononuclear cell profiles using ﬂow cytometry. Placental blood collection by prick was established as the most appropriate method and was subsequently used for further immunological investigations.
Open Access
Involvement of endothelial cells and macrophages in mycobacterial infections
(2002) Nkhahle, Senate; Ryffel, Bernhard; Mpagi, Joseph
Tuberculosis remains to be a leading infectious cause of death worldwide. This is in spite of the BCG vaccine against tuberculosis that has been in use for over 80 years as well as several chemotherapies. Mycobacterium tuberculosis, the causative agent of tuberculosis, is a facultative intracellular pathogen targeting host cells, predominantly macrophages, to establish an infection. Endothelial cells form a barrier that has to be crossed by the bacilli in the establishment, and subsequent dissemination of mycobacterial infection. The present study was undertaken to investigate the phagocytosis of mycobacteria by endothelial cells and macrophages and the subsequent activation of these host cells after infection with the bacilli. Endothelial cells, obtained from the human umbilical vein (HUVEC) were infected with BCG-GFP under various conditions and uptake determined by means of ﬂow cytometry. Endothelial cells phagocytised mycobacteria in a dose- and a time-dependant manner. Exposure of mycobacteria to serum opsonins enhanced the uptake of the bacilli by endothelial cells. However, heat killing of mycobacteria inhibited its uptake by endothelial cells. Data from the fluorescent microscope showed the association of BCG-GFP signal with endothelial cells detected on the FACS caliber. Analysis by confocal microscopy conﬁrmed internalisation of endothelial cells by mycobacteria. Endothelial cells were further investigated for an acquired phenotype following infection with mycobacteria. CD31, a marker for endothelial cells, was neither down regulated nor up regulated. However, ICAM-1 expression, one of the adhesion molecules was down regulated upon infection of endothelial cells with mycobacteria. No TNF-α and IL-6 were detected in culture supernatants of infected endothelial cells. Macrophages obtained from murine bone marrow, phagocytosed mycobacteria in both a dose- and a time-dependant manner. Unlike endothelial cells, heat killing of mycobacteria did not obliterate their uptake by macrophages. However, macrophages preferentially phagocytosed viable mycobacteria in a 3h infection period, but not in an 18h period. Macrophages from the Mac-l mouse strain, lacking a phagocytic receptor 3 (CR3), were included in this study. The uptake of pathogenic mycobacteria, H37Rv by macrophages from Mac-1, was reduced in cell cultures infected for 4 hours but not those infected at 1 and 2 hours. Similarly, reduced uptake of avirulent mycobacteria strains H3 7Ra and BCG in the absence of CR3 was pronounced in cell cultures infected for longer periods. The activation state macrophages acquire after infection with mycobacteria was investigated with respect to the expression of MHC glycoproteins, and secretion of IL-10 and IL-12. The activation state of macrophages with respect to these parameters studied is critical in the interaction with T-lymphocytes, for subsequent containment of mycobacteria infection. Production of IL-12, a critical Th-1 cytokine, was proportional to MOI, and enhanced by viability of pathogenic mycobacteria. Furthermore, prior exposure of mycobacteria to serum opsonins inhibited the secretion of IL-12, while exposure of the bacilli to bronchoalveolar factors greatly enhanced it. IL-10 production by infected macrophages was on the other hand inhibited by prior exposure of mycobacteria to both serum and bronchoalveolar ﬂuid factors. Macrophages consitutively expressed MHC I. After infection with pathogenic mycobacteria, cells positive for MHC I, were hardly detected in macrophage cultures infected with mycobacteria that had been exposed to fresh serum and bronchoalveolar fluid opsonins. MHC II on the other hand, was not consitutively expressed on macrophages. Following infection with pathogenic mycobacteria, the highest percentage of cells positive for the antibody against MHC II were observed in macrophage cultures infected both without any opsonin and in the presence of bronchoalveolar ﬂuid factors. - In conclusion, the present study demonstrates that endothelial cells bind and internalise mycobacteria. That they get activated as evidenced in the down-regulation of ICAM-1 following infection with mycobacteria. Thus endothelial cells may not just be a passive, physical barrier but host cells that may have an active role in mycobacterial infection. Macrophages in comparison to endothelial cells were more effective in phagocytosis of mycobacteria in a time- and dose-dependant manner, differentiating themselves as professional phagocytes in the internalisation of heat-killed bacteria where the endothelial cells lacked the ability for the uptake of mycobacteria.
Open Access
Is air pollution a risk factor for rheumatoid arthritis?
(2015) Essouma, Mickael; Noubiap, Jean Jacques N
Rheumatoid arthritis is a chronic inflammatory debilitating disease triggered by a complex interaction involving genetic and environmental factors. Active smoking and occupational exposures such as silica increase its risk, suggesting that initial inflammation and generation of rheumatoid arthritis-related autoantibodies in the lungs may precede the clinical disease. This hypothesis paved the way to epidemiological studies investigating air pollution as a potential determinant of rheumatoid arthritis. Studies designed for epidemiology of rheumatoid arthritis found a link between traffic, a surrogate of air pollution, and this disease. Furthermore, a small case–control study recently found an association between wood smoke exposure and anticyclic citrullinated protein/peptide antibody in sera of patients presenting wood-smoke-related chronic obstructive pulmonary disease. However, reports addressing impact of specific pollutants on rheumatoid arthritis incidence and severity across populations are somewhat conflicting. In addition to the link reported between other systemic autoimmune rheumatic diseases and particulate matters/gaseous pollutants, experimental observation of exacerbated rheumatoid arthritis incidence and severity in mice models of collagen-induced arthritis after diesel exhaust particles exposure as well as hypovitaminosis D-related autoimmunity can help understand the role of air pollution in rheumatoid arthritis. All these considerations highlight the necessity to extend high quality epidemiological researches investigating different sources of atmospheric pollution across populations and particularly in low-and-middle countries, in order to further explore the biological plausibility of air pollution’s effect in the pathogenesis of rheumatoid arthritis. This should be attempted to better inform policies aiming to reduce the burden of rheumatoid arthritis.
Open Access
LncRNA discovery in the Listeria monocytogenes infection model
(2015) Magagula, Loretta Q; Mhlanga, Musa; Brombacher, Frank
A growing body of evidence indicates that long noncoding RNAs (lncRNAs), the most abundant noncoding RNA (ncRNA) species of the pervasively transcribed mammalain genome, have functional roles in the gene regulation of an array of cellular processes. These observations have since discredited the long standing central dogma formulated by Franscis Crick in 1958, which states that genetic output is entirely conducted by protein. Recent studies collectively indicate that lncRNAs play important functional roles in the transcriptional regulation of a wide array of cellular processes. In the last year alone, a handful of studies have identified lncRNAs linc-Cox2, Lethe, PACER and THRIL as central players in host cell innate immune response against microbial infection. These discoveries and the vast numbers of uncharacterized lncRNAs identified by high-throughput nextgeneration transcriptome sequencing technologies, set a precedence for further investigation and characterization of lncRNAs in infection biology. Importantly, lncRNAs may serve as important diagnostic markers of infection as well as therapeutic targets. These aspect of lncRNAs field although extensively being explored in cancer research, have been neglected in infection biology, particularly in microbial infection. In this study, next-generation technologies were used to identify subtle vairations in transcriptional activity, with particular emphasis to lncRNA differential expression, and uncover their physiological relevance during Listeria monotocytogenes infection. To this end, an RNA-Seq dataset of Listeria-infected HeLa cells was subjected to several variations of data analysis lncRNA discovery pipelines. Potential lncRNA functioning was hypothesized using the Rinn & Chang "guilt by association" approach in which lncRNA functioning was hypothesized based on the known functions of tightly co-expressed protein coding mRNAs. "Guilty" lncRNAs were then knocked down in the HeLa cells using transcription activator-like nucleases (TALENs) to validate their candidacy as infection-regulating lncRNAs. Preliminary investigations conducted in this study have revealed potential Listeria infectionregulating lncRNA candidates. Furthermore, we explored the use of the physiologically relevant cellular model of iPSC-MDMs to validate identified lncRNA candidates. This work provides a framework for lncRNA discovery from RNA-Seq data by iterative and intergrative analysis.
Open Access
Lymphocyte-specific reconstitution of IL-4Ra-deficient mice : characterization and infectious disease studies
(2006) Myburgh, Elmarie; Brombacher, Frank
Lymphocyte-specific reconstitution of IL-4Ra was recently established by intercrossing lymphocyte-specific human IL-R4a transgenic mice with mIL-4Ra-deficient mice. Human IL-4Ra may bind to mouse yc resulting in a chimeric receptor specific for human IL-4 but not mouse IL-4. This provides us with and inducible IL-4 system. The aim of this study was to investigate in vitro and in vivo characteristics of our novel hlL-4Ra Tg/mIL-4Ra+/- mouse model.
Open Access
Male genital tract versus blood HIV-1 compartmentalization and selection: the first step of the transmission bottleneck?
(2019) Kariuki, Samuel Mundia; Dorfman, Jeffrey Robert; Williamson, Carolyn
Introduction Sexual transmission of HIV-1 accounts for more than 80% of all the transmissions globally. After transmission, approximately 80% of the newly disseminated infections can be traced to a single variant, which comes from the minor HIV-1 population within the transmitting donor. This has led to the widely accepted idea of an HIV-1 transmission bottleneck. The nature of this bottleneck is not fully understood. Many studies working on understanding the nature of the transmitted virus have reported discordant traits of transmitted/founder viruses compared to viral isolates from chronically infected individuals. Such studies therefore lacked analysis of the intermediate step between these two populations: HIV-1 from the genital tract of the donors: where viruses are on their way to transmission to a new recipient. Importantly, numerous prior studies have shown that there is compartmentalization of HIV-1 populations between the general circulation and the genital tract, raising the possibility that the genital tract is an important selective environment. Collectively, prior studies of genital tract compartmentalization in males detected compartmentalization in about half of the donors studied, although by using techniques with limited depth of sampling than that employed in our study. The virus that establishes disseminated infection in a new recipient is selected. However, the extent to which this selection occurs before, during or after crossing the mucosal surfaces of the recipient is less clear – the period during which transmission selection could extend far back to the donor, and the donor’s genital tract. In other words, it is not clear as to what extent the transmission bottleneck occurs during compartmentalization of viral populations in the genital tract tissues. The design of an effective vaccine and other intervention strategies will rely upon the understanding the nature of the transmitted virus as this is the virus that must be targeted. This thesis Compartmentalization of minor variants cannot be tracked by techniques previously used to describe compartmentalization between the genital tract and the blood circulation. We therefore used deep sequencing-based techniques to further understand compartmentalization of viral populations between blood and the male genital tract. In addition, we tested the sensitivity of variants to a range of entry and other inhibitors in order to explore possible changes in function that may arise when viral variants grow in the shifted selective milieu of the genital tract. We further hypothesized that this change of selective milieu as HIV-1 moves from blood into the genital tract may lead to viral variants in semen that are sensitive to autologous neutralization because such sensitive variants may be able to grow in the genital tract, which is presumably partially or completely shielded from antibodies. Because the viral populations in semen comes from a site that may be relatively protected from antibodies, they may be permitted to evolve differently in the relative absence of antibody pressure. It is possible that evolution of the virus within the genital tract is a significant part of the change the virus undergoes on its way to establishment of a new disseminated infection in the new recipient. We considered this possibility because even some small molecules like those of some antiretroviral drugs do not penetrate the genital tract effectively under some circumstances, raising the possibility that antibodies might not always penetrate in all areas of the genital tract. This thesis had three objectives: 1. To evaluate HIV-1 compartmentalization in blood and the male genital tract using next generation sequencing to understand the nature of viral populations in these anatomical sites in greater detail. 2. To identify the differences in sensitivity of blood and semen variants to entry inhibitors to obtain information about differences in function between HIV-1 populations in blood vs the male genital tract. 3. To compare neutralization sensitivities of viral variants compartmentalized in blood and semen by testing their sensitivity to neutralization by autologous antibodies. As a control, we measured sensitivity to a pool of clade-matched heterologous sera to determine if any observed difference was due to global changes in neutralization sensitivity. Methods Study participants Forty-four HIV-1 seropositive men were recruited and then requested to donate blood and semen samples at ANOVA Health’s Ivan Toms clinics at Woodstock and Green Point or through their mobile clinic in Khayelitsha, all in Cape Town, South Africa. Viral loads from blood and semen and CD4+ T cell counts from blood were measured. HIV-1 was enriched from semen using a Nycodenz gradient, and then concentrated using ultracentrifugation. Chapter 2: HIV-1 Compartmentalization in blood and semen Next generation sequencing on Illumina paired-end Miseq platform was performed. To our knowledge there is no published study that has used this technique to study male genital tract HIV-1 variants in chronically infected male donors, although there is one that does so for acutely infected donors. We argue here that this is a superior method of sampling populations in blood and the male genital tract. In particular, it allowed us to more accurately track minor populations within each compartment. Additionally, the use of PrimerID approach allowed us to more clearly identify clonal amplification events in the HIV-1 populations. Sequencing was performed on either the V3 or C3-V5 region of the HIV-1 envelope gene from paired blood and semen samples from 11 donors. To evaluate compartmentalization, populations from blood and semen were compared using three standard techniques, Slatkin Maddison Test (SMT), Wrights measure of population subdivision (FST) and nearest neighbour statistic (Snn). Clonal amplification and results of modelling a lower depth of sampling are also presented. Chapter 3: Sensitivity of blood and semen variants to entry inhibitors and changes in function From three subjects who exhibited the highest extent of compartmentalization, full-length envelope clones derived from semen and blood RNA were made using limiting dilution PCR (single genome amplification), which provided the advantage of minimizing PCR-based artificial recombination. A high fidelity Taq polymerase was also used to minimize base-substitution errors. An average of 10 clones were isolated per compartment. Pseudoviruses were then constructed from the full-length envelope clones from blood and semen. The sensitivities of these pseudoviruses were tested against HIV-1 entry inhibitors; Maraviroc, PSCRANTES, enfurvirtide (T-20) and JM2987. Maraviroc and PSC-RANTES are CCR5 inhibitors while JM2987 is a CXCR4 inhibitor. Enfurvirtide (T-20) is a fusion inhibitor blocking the virus from entering cells. The full-length clones used to make the pseudoviruses were also sequenced and genomic variations in variable loop characteristics (length and number of potential glycosylation sites) between blood and semen compared. Chapter 4: Sensitivity of blood and semen variants to autologous and heterologous antibodies To study the differences in sensitivity of blood and semen variants to antibodies, pseudoviruses cloned from semen RNA and blood RNA (above) were tested for their sensitivities to donor antibodies collected at the same time the samples were collected or to a pool of HIV-1 subtype C sera. Results Objective 1: Viral compartmentalization via next generation sequencing HIV-1 populations were compartmentalized in all the 11 donors studied but to varying extents. Donor SVB043 had the most compartmentalized viral populations between blood and the male genital compartment using all the three measures of compartmentalization. Further analysis of the phylogenetic trees revealed that some clusters contained either purely blood or semen sequences, even in trees generated from analysis of donors with weakest compartmentalization. This might explain the viral compartmentalization signal in these weakly compartmentalized donors. To mimic reduced sampling depth, subsampling of the Illumina Miseq data with a small number of sequences was done. This analysis revealed that viral compartmentalization between blood and male genital tract would likely (>50% estimated likelihood) have been detected in only 5/11 (45%) of the donors, a proportion which is very similar to the aggregate proportion from previous studies that had used single genome amplification (SGA) analysis. This means that the difference in detecting HIV-1 compartmentalization in this thesis vs previous studies can be explained by the depth of sequencing achieved here and that there is no evidence that the dynamics of the viral populations studied in this thesis were different from those previously studied. In addition, the most recent common ancestor of semen variants was mostly located in blood, indicating the male genital tract was seeded by incoming variants from blood. Clonal amplification was also observed in all the 11 study participants and it was a characteristic of variants from blood and the male genital tract and its frequency did not obviously correlate with the severity of compartmentalization. In sum, blood and male genital tract HIV-1 compartmentalization and clonal amplification is present in most or all HIV-1 infected males but was not detected in all individuals in previous studies when using techniques with lower depth of sampling. Objective 2: Sensitivities of blood and semen variants to entry inhibitors and variable loop characteristics Viral variants from the most compartmentalized donors had variations in sensitivities to entry inhibitors; although the direction of the difference was inconsistent. Donor SVB043 who had the most severely compartmentalized viral populations between blood and semen, had semen viruses that were 1.67 (95%CI 1.08 – 2.56) times more resistant to maraviroc (p=0.024) while SVB008 which was the second most compartmentalized donor, had semen isolates that were 4.8 (95%CI 2.76 – 8.28) times more sensitive to inhibition by maraviroc (p < 0.0001). The meaning of this discrepancy is not entirely clear. It could mean that trait(s) that are selected for in genital tract variants over blood circulation variants are linked to the CCR5 binding region, and that the linked CCR5 genotype was carried along with the selected trait(s). There were no differences in sensitivity to maraviroc between blood and semen clones for donor SVB049 (p=0.847); although this donor on further investigation was found to have functional levels of efavirenz in his blood (3µg/ml, which were within the therapeutic range of 1-4µg/ml) indicating that he was likely on antiretroviral therapy (ART). This was not known to the clinic staff at the clinic at which he was known to receive care and was recruited to this study. The direction of sensitivities to PSC_RANTES (another CCR5 inhibitor) was concordant to that observed for maraviroc for donors SVB008 and SVB049 but not for donor SVB043 where semen variants were 1.67 (95%CI 1.08 – 2.56) times less sensitive than blood variants to maraviroc, with no detected difference in sensitivity to PSC_RANTES (p = 0.783). This discrepancy for donor SVB043 probably reflects the difference in mode of action between Maraviroc and PSC_RANTES. The change in envelope conformation over movement from blood into the genital tract presumably affected the maraviroc binding site and not PSC_RANTES. All the clones from blood and semen for the three most viral compartmentalized donors were resistant to CXCR4 inhibitor suggesting that they were all R5 tropic viruses. There were no differences in sensitivities of blood and semen viruses to fusion inhibitor T-20. The findings here suggest a changed viral envelope conformation/structure for the viruses in the male genital tract. The discordance suggests that the selected trait over movement of virus from blood into genital tract is linked or close to CCR5 binding site but itself does not involve binding to CCR5 coreceptor. Differences in length and number of glycosylation sites were found between variants from blood and those from the genital tract but the direction of the difference was also inconsistent. Donor SVB043 who had the most compartmentalized blood and seminal variants had semen variants that had longer and more glycosylated envelopes. Donor SVB008 who had the second most compartmentalized blood and semen variants had no difference in variable loop length, but semen variants were less glycosylated. This therefore shows that selection for some of the previous reported traits of acute viral isolates may have started in the genital tract in a subset of the donors. Objective 3: Sensitivities of blood and semen variants to autologous and heterologous antibodies Viral populations compartmentalized in blood and the male genital compartment displayed a range of sensitivities to autologous and heterologous neutralization. Donor SVB043 who had the most compartmentalized viral populations between blood and the genital tract; had semen clones that were 1.75 (95%CI 1.11-2.78) times more sensitive to autologous neutralization compared to blood clones (p = 0.018). In contrast, donors SVB008 and SVB049 who exhibited substantial compartmentalization, but to a lesser extent than that found in donor SVB043, showed no differences in sensitivities of blood and semen variants to autologous serum. Neutralization sensitivity to a pool of heterologous subtypematched sera revealed no differences in sensitivities between clones from blood and semen for donors SVB043 and SVB008. Interpretation of results from donor SVB049 are clouded by the donor’s ART use. Overall, these results suggest that, in some individuals, a shift in selective milieu of the genital tract virus occurs. This is presumably due to partial or complete shielding of the genital tract tissue from circulating antibodies, and this shielding shape the populations of HIV-1 variants available for transmission from some but not all individuals. Overall conclusions Our data add to the existing knowledge of existence of distinct viral populations between blood and the male genital tract of chronically HIV-1 infected donors. Importantly, and for the first time, we present evidence that HIV-1 compartmentalization between blood and the male genital tract is present in most or all donors, and that some clones are severely compartmentalized even in donors who exhibit very mild compartmentalization. It appears that viral compartmentalization and clonal amplification in these anatomical sites may be present in most individuals but remained undetected in some individuals in previous studies due to the lower depth of sampling applied. We observed a discordance in entry inhibitor sensitivities and variable loop characteristics between blood vs semen variants among different donors. This may suggest a changed envelope conformation over importation of virus from blood into the genital tract. This change seems to be near or linked to the co-receptor binding site but does not appear to directly involve the co-receptor binding tested in this thesis. This interpretation also may explain the discordance in viral characteristics for the virus establishing infection reported in other studies. This thesis also shows that some of these traits of the transmitted/founder virus relating to neutralization sensitivity, sensitivity to entry inhibitors and variable loop characteristics may originate in and/or be enhanced by transition through the genital tract on the way to becoming a founder virus. These results are important in understanding how the populations in the genital compartment are selected, giving rise to the population of HIV-1 that is available for transmission to a new individual. An understanding of the dynamics of HIV-1 populations prior to and during transmission is important for vaccine design and other intervention strategies.
Open Access
Mycobacterium tuberculosis and trypanosoma brucei as models for the TLR-dependent activation of the innate immune system
(2005) Drennan, Michael B; Ryffel, Bernhard; Magez, Stefan
Includes bibliographical references.