Browsing by Subject "pathology"

Now showing 1 - 8 of 8
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    Open Access
    Early-life immunity and susceptibility to Mycobacteria
    (2018) Logan, Erin; Horsnell, William; Hatherill, Mark; Cunningham, Adam F
    The naïve and not-yet developed infant immune system exhibits heightened susceptibility to external factors (e.g pathogens), and is shaped by these and others, such as maternal immunity. However, we do not yet fully understand their impact on development of infant immunity. A better understanding of these effects would benefit children world-wide, but especially those in low-middle income countries (LMIC), where increased exposure to pathogens due to poorer living conditions highlights the necessity of robust early-life immunity. Mycobacterium tuberculosis (Mtb) and helminths are pathogens co-endemic in many LMIC and cause significant morbidity and mortality in children. Infant immune responses to these pathogens, whether during standalone infection, co-infection or resulting from maternal infection are not fully understood. To contribute to this knowledge gap, we investigated early-life immune responses, how they relate to childhood Mtb/helminth infection and how they are affected by maternal infectious history and immunity. Analysis of clinical humoral responses revealed total IgG that increased significantly between baseline and tuberculosis (TB) investigation in infants who did not acquire Mtb infection; these infants also exhibited raised levels of measles-specific IgG and BCG-specific IgG2. No active helminth infections were detected, but the presence of Ascaris lumbricoides- and Trichuris trichiura-specific class-switched antibodies indicated prior exposure. No association was found between helminth-specific humoral responses and risk of Mtb infection, nor with maternal helminth-specific humoral responses. Conversely, data from murine experiments revealed a protective effect of maternal helminth infection (Nippostrongylus brasiliensis) on BCG infection in offspring, with reduced lung bacterial burden and increased numbers of activated CD4+ T cells and B cells. Maternal Nb infection may have a synergistic effect on BCG vaccination, as BCGvaccinated/infected pups from Nb-infected mothers had reduced lung bacterial burdens, increased CD4+ T cell and B cell responses and increased IFNγ-producing CD4+ T cells. Findings from this study suggest that childhood vaccines could provide heterologous protection against unrelated pathogens such as Mtb. The murine data suggest a protective effect of maternal helminth infection against BCG infection in offspring, but no similar finding was observed with the clinical data. The clear protective effect of maternal Nb infection during offspring BCG infection warrants a more in-depth clinical study addressing the functional effects of maternal helminth infection on Mtb infection outcome in infants.
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    Open Access
    Impact of the parasitic helminth Schistosoma mansoni on host anti-viral vaccine responses: proof of concept from the anti-polio vaccine
    (2022) Musaigwa, Fungai; Nono, Komguep Justin; Brombacher, Frank
    Schistosomiasis is a devastating and neglected tropical disease caused by Schistosoma spp. parasites. The disease remains greatly neglected by global health control programmes thus classified as a top neglected tropical disease of humankind. One of the most common species of these parasites, Schistosoma mansoni, causes hepatosplenic schistosomiasis and distributes widely in sub-Saharan Africa. Schistosoma mansoni is physiologically debilitating and potentially deadly with a known potential as a master regulator of the host immune responses. However, the mechanistic bases and the scope of this immunoregulatory potential of S. mansoni still remain poorly understood. This study explored the influence of S. mansoni-driven schistosomiasis on vaccine-induced memory immunity in schoolchildren from a rural endemic area of Cameroon and in laboratory mice. As a proof of concept, a well-established anti-viral vaccination programme with wide global coverage, i.e., anti-polio vaccination, was evaluated for its sustainability in the face of schistosomiasis in human and mouse hosts. Our findings using the poliovirus specific enzyme-linked immunosorbent assay revealed a schistosomiasis-associated impairment of polio specific serologic antibody memory responses in previously vaccinated schoolchildren and mice, as judged by significantly reduced serum anti-polio IgG antibody levels. To explore our findings further, cellular evaluations were conducted using flow cytometry in mouse modes of vaccination and schistosomiasis. The reduction of anti-polio elicited antibody responses was paralleled by the general depletion, through reduced survival and increased cell death, of bone marrow plasma cells and plasma blasts in schistosomiasis-diseased mice. Notably, schistosomiasis reduced memory T cell responses as well in vaccinated hosts and considerably impaired the expression of survival markers on antibody-producing long term plasma cells in the bone marrow. When attempting to control the disease by administration of the sole commercially available drug for schistosomiasis control, praziquantel, the parasite-driven depletion of the plasma cell and memory T cell compartment was restored followed by a partial recovery of antibody-producing abilities in vaccinated hosts. Taken together, our findings unprecedentedly demonstrate how schistosomiasis might negatively influence the efficacy and potentially the effectiveness of anti-viral vaccine memory immunity. Additionally, we present findings on how strategic therapeutic interventions against schistosomiasis might positively influence vaccine-elicited anti-viral immunity in schistosomiasis-diseased hosts. Our results have robust implications for future more informed deployment of effective vaccination campaigns, beyond the sole consideration of coverage and encourage frequent mass drug administration of praziquantel in schistosomiasis-endemic areas to ensure the sustainability of vaccine elicited responses thus protection of the hosts against vaccine preventable diseases.
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    Open Access
    Investigating the opinions on telephonic advanced maternal age genetic counselling
    (2024) Bayley, Samantha; Laing, Nakita; Malope, Malebo; Wessels, Tina-Marié
    Background: The research on telephonic genetic counselling (GC) services has increased since 2020. However, there is still limited research on the experience and opinions of this service from the patients' perspective. This is particularly true in low to middle-income settings such as South Africa. The advanced maternal age (AMA) telephonic GC service at Groote Schuur Hospital (GSH), has been implemented since COVID-19 regulations were enforced in 2020. This study aims to investigate the opinions and experiences of the patients on this telephonic service. Methods: This qualitative study used individual semi-structured interviews, both in-person and telephonic, and followed a phenomenological approach. The data were analysed using thematic data analysis. Results: The participants (n=9) had varying opinions about the telephonic GC service offered through GSH. The information gathering process varied for the participants; especially the differing Midwife Obstetric Units (MOUs) referrals and the information given. Some participants found online resources helpful, but not all participants felt the same. Overall, the participants felt the information given by the GC service was informative and useful. An important outcome of the research was a general trust between the GC trainee and participants but a distrust between other health care professionals and the participants. Numerous factors influenced decision-making concerning invasive testing including participants' fears and seeking control or having a sense of control based on if you would cope with having a child with DS. Conclusion: The distrust in the healthcare system can have a significant impact on patients' understanding and decision-making in a GC session. Overall, there are benefits and barriers to be aware of, but most participants found the GC session informative and allowed them to make informed decisions. This research included a limited sample size, which prevents the generalizability of these findings.
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    Investigating the T cell-specific role of Interleukin-4 receptor alpha (IL-4Rα) in tuberculosis (TB)
    (2024) Rousseau, Robert Pierre; Parihar, Suraj P; Riou, Catherine; Brombacher, Frank
    Protective immunity to tuberculosis (TB) is dependent on the ability of the host to mount a robust T cell response. The main effector T cells which contribute to protection in TB include T helper (Th) Th1 and Th17 T cells. The Th2 response, associated with IL-4Rα mediated signalling, remains largely overlooked in TB. The role of IL-4Rα in TB appears to be different according to cell type. Deletion of IL-4Rα on macrophages has no effect on disease, but deletion of the receptor on B cells, leads to better control. This thesis aimed to explore the T cell-specific role of IL-4Rα in TB disease by making use of T cell specific knockout model, iLCKCreIL-4Rα-/lox. We found that absence of IL-4Rα on T cells results in a delay in the recruitment of T cells to the lung. This was demonstrated by decreased CD4 and CD8 T cell numbers during acute infection compared to littermate controls. Consequently, the bacilli are able to better establish infection and proliferate in the lung, shown by increases in lung mycobacterial burden at both acute and chronic stages of infection. However, no differences are observed in the spleen, indicating deletion of IL-4Rα does not have a role in the dissemination of TB. In the absence of IL-4Rα, T cells express higher amounts of T-bet or RORγt transcription factors, indicating stronger Th1 and Th17 responses, respectively. The stronger pro-inflammatory responses do not clear the pathogen, and instead contribute to immunopathology. During chronic infection, we observed higher amounts of IL-17 as well as a corresponding increase in neutrophils, which in turn lead to a decrease in alveolar free space. The promotion of increased Th1 CD4 T cells resulted in greater amounts of terminally differentiated (CD44+KLRG1+ ) which have a poor proliferative capacity despite secreting large amounts of IFN-γ. KLRG1 expression is also associated with a reduced ability to migrate into the lung parenchyma, the site of disease. We also found that these (CD44+KLRG1+ ) expressed reduced amounts of CXCR3, CD69, and CD103, which are all markers associated with poorer migration into the lung parenchyma. Importantly, these factors result in decreased survival in T cell-specific IL-4Rα mice. In conclusion this study demonstrates that absence of IL-4Rα on T cells promotes a predominant Th1/Th17 response, that results in delayed recruitment into the lung tissue, which ultimately proves detrimental for the host.
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    Retrospective analysis of the utilisation of DNA analyses in the identification of human remains at Salt River Mortuary (Cape Town, South Africa)
    (2024) Kambowo, Sophy Celine; Heathfield, Laura Jane; Reid, Kate
    Post-mortem human identification is crucial for medico-legal investigations and for social justice. Unfortunately, many people remain unidentified, particularly in developing countries. The use of forensic DNA profiling is a reliable method for human identification and was legislated in South Africa in January 2015. However, the use and success of DNA as an identification tool at Salt River Mortuary (SRM) in South Africa are unknown. Medico-legal case files were reviewed from all 3696 cases admitted in 2015, to evaluate the use of DNA in identifying human remains at SRM immediately after the implementation of the ‘DNA Act'. While 213 (5.76 %) cases were admitted in 2015 without an alleged identity, 221 individuals had no confirmed identity following post-mortem and identification attempts. DNA samples (for identification and/or investigative purposes) were taken in a total of 490 cases, yet concerningly, these samples represented many people with a confirmed identity, and not all individuals without an (alleged) identity. Of the 221 unidentified human remains, only 62 (28 %) were afforded DNA analysis, suggesting an underutilisation of DNA analysis requests. This observation highlights a potential missed opportunity to leverage DNA technology more comprehensively in cases where conventional identification methods prove insufficient. Further, DNA results were only obtained for 64 cases (13.06 %) with results from 426 cases still outstanding seven years later. Where reports were available, identification via familial matching was successful in 95.31 % of cases (n = 61/64), leading to a success rate of 12.45 % (n = 61/490) for DNA as an identification tool. This poor success rate in 2015 could be attributed to several factors including: inadequate sampling post-mortem possibly due to unclear guidelines about which state authority was responsible for sampling, the low chances of a match on the National Forensic DNA Database due to its infancy (and thus containing few reference DNA profiles), next-of-kin not providing reference samples due to lack of awareness or fear of authorities, or inadequate infrastructure or access to resources. This study highlights the need for intentional and consistent sampling of unidentified human remains and calls for larger efforts to encourage next-of-kin to provide reference samples to increase the chances of identification.
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    UCT Digital Pathology collection
    (2014-09-22) University of Cape Town. Department of Clinical Laboratory Sciences
    An online catalogue with thousands of pathology specimens used as a teaching collection. Detailed cases and exhibitions are available. This website gives electronic access to several thousand pathology specimens in our pathology teaching collection. It is intended for use by undergraduate and postgraduate students in the health sciences. There are currently three main catalogues for (1) the anatomical pathology collection (2) the forensic pathology collection and (3) the obstetrics and gynaecology collection. (A paediatric pathology section is in the pipeline). This is an historical collection (begun in the 1920’s) so the cataloguing is rather old fashioned. The specimens are catalogued by organ or system e.g. “kidneys” and then by broad pathological category e.g. “neoplasms”. Each specimen has a brief description and commentary along with good quality photographs. The emphasis is on macroscopic pathology; we are aiming to include more radiographic imaging and also microscopy going forward. The site also includes a more detailed section with “student cases” that are useful as teaching cases, "specialist cases" presenting unusual cases, and a few online exhibitions around special topics. The website is a work in progress so much of our material is still in the process of being reviewed and uploaded. For all that use the website, please be respectful of all the specimens and their images. Although anonymous now, they originate from real patients whose diseases were often distressing, painful and fatal.
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    Open Access
    Using immunofluorescence techniques to Identify T cells in the foreskin tissue after medical male circumcision
    (2022) Sebaa, Shorok; Gray, Clive; Chigorimbo-Tsikiwa, Nyaradzo
    Background: Medical Male Circumcision (MMC) plays an important role in reducing the risk of acquiring sexually transmitted infections (STIs) such as Human papilloma virus (HPV), Herpes simplex type 2 (HSV-2) and HIV-1. The foreskin tissue (FS) is a site abundant in Langerhans cells (LCs), macrophages and T helper cells that express CD4 and CCR5 that are target markers for HIV1 binding and viral infection. The foreskin tissue may also contribute chemokines and cytokines including those that promote inflammation such as IL-17, IL-1β, IL-8, MCP-1 and MIG. The inner foreskin has been shown to contain higher levels of CD4+CCR5+ cells and thus more susceptible to HIV infection compared to the outer foreskin. It was demonstrated that the majority of chemokines measured were highly expressed in the inner foreskin compared to the outer foreskin including CCL27 which was approximately 7-fold higher in the inner foreskin compared to the outer foreskin, in congruent with the higher density of CD4+CCR5+ observed in the epithelium of the inner foreskin. In this study, we hypothesized that CCL27 upregulation in the inner foreskin triggers the recruitment of CD4+ T cells to the epithelium of the foreskin tissue. This could subsequently lead to increased susceptibility to infections in the inner foreskin tissue. The aims of this dissertation were: 1) to measure the impact of CCL27 on the recruitment of CD4+ T cells to the epithelium of the foreskin tissue using immunofluorescence imaging. 2) to compare manual counting and semi-automated method for counting dually positive cells. 3) to use multiparameter flow cytometry to characterize the cells recruited under the influence of CCL27. Methodology: Inner foreskin tissue (n=11) and outer foreskin tissue (n=4) explants were treated with either TNFα or CCL27 and evaluated using immunofluorescence imaging to quantify the levels of CD3 and CD4 expressing cells. Dually positive CD3+CD4+ cells were counted manually using softworx software on the Deltavision microscope and with semi-automated counting using PIPSQUEAK on ImageJ. TNFα and CCL27 treated inner and outer FS cells were immunophenotyped using polychromatic flow cytometry to measure and compare the densities of Th17 and Th22 cells under the influence of the chemokines. Results: Exogenous exposure of inner foreskin tissue explants to TNFα showed a significant increase in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin (p=0.035) from 78.90 cells/mm2 (IQR: 33.02-127.50) in the unstimulated inner foreskin explants to 134.80 cells/mm2 (IQR: 109.30-206.60). Similarly, the addition of exogenous CCL27 resulted in the median density of CD3+CD4+ T cells in the epithelium of the inner foreskin to increase from the unstimulated inner foreskin (value above) to 164.80 cells/mm2 (IQR: 140.30-184.90, p=0.008). No significant difference was observed in the median density of CD3+CD4+ T cells in the outer foreskin tissue explants after exposure to TNFα and CCL27 (36.50 cells/mm2 , IQR: 18.29-96.65 in the unstimulated tissues compared to 65.12 cells/mm2 , IQR: 7.30-202.80 in the TNFα stimulated tissues; p>0.999 and 24 cells/mm2 , IQR: 11.35-149.40 for the CCL27 stimulated tissues; p=0.686). The median density of CD3+CD4+ T cells in the epithelium of unstimulated inner foreskin tissue showed a trend of an increase from the unstimulated outer foreskin tissue but was not statistically different (127.50 cells/mm2 , IQR: 89.22-219.50 in the inner foreskin compared to 36.52 cells/mm2 , IQR:18.29-96.65 in the outer foreskin explants; p=0.057). When comparing the cell counting methods: manual counting vs semi-automated counting, we observed that the manual counting method estimated higher numbers of dually positive cells compared to the semiautomated method in samples measuring 200 cells/mm2 . Despite these differences, there was strong correlation (R=0.782, p0.999) in CCL27 treated explants. The median frequency of Th22 cells in the inner foreskin in the unstimulated tissue explants was 8.80% (IQR: 1.68-12.60%) vs 5.30% (IQR: 0.96-7.67%, p=0.250) in TNFα treated explants and 4.90% (IQR:0.75-7.39%, P=0.125) in CCL27 treated explants. Meanwhile, the median frequency of Th17 cells in the outer foreskin in the unstimulated tissue explants was 21.60% (IQR: 15.40-37.33%) vs 28.20% (IQR: 14.60-39.40%, P=0.750) in TNFα treated explants and 22.90% (IQR:22.90-29.50%, p>0.999) in CCL27 treated explants. The median frequency of Th22 cells in the outer foreskin in the unstimulated tissues was 4.67% (IQR: 2.30-12.90%) vs 5.37% (IQR: 5.34- 7.58%, P=0.750) in TNFα treated tissues and 4.45% (IQR:3.64-5.98%, p>0.999) in CCL27 treated tissues. Furthermore, FS cells isolated using Dispase had significantly lower median frequencies of cells expressing CCR6 (18.35%, IQR:1.33-28.30%) compared to whole tissue controls (41.90%, IQR: 22.46-67%, p=0.031). This impacted the characterization of CD4+ T cell subsets in FS cells and limited our ability to adequately phenotype and measure the impact of TNFα and CCL27 on FS-derived cells using flow cytometry. Conclusion: This study demonstrates that exogenous exposure of FS to TNFα and CCL27 increased the density of CD3+CD4+ T cells in the epithelium of the inner but not the outer foreskin tissue. It was noteworthy that the density of CD3+CD4+ in the epithelium of the inner foreskin was higher than the outer in the unstimulated tissues, suggesting that the proinflammatory environment in the inner FS potentially leads to higher density of T cells in the inner FS even without exogenous stimulation. These results suggest a possible mechanism for recruiting HIV target cells in the inner foreskin tissue associated with higher levels of CCL27 that recruits HIV-1 target T cells during inflammatory responses. A limitation to this conclusion is the small sample size in the outer foreskin. The study also shows potential bias depending on the method used to quantify dually positive cells, whereby semi-automated counting underestimated the densities of CD3+CD4+ T cells compared to manual counting and therefore careful consideration is required when selecting the quantification method. Furthermore, there were no significant difference in the frequencies of Th17 and Th22 cells after exposure to TNFα and CCL27 using flow cytometry. The effects of Dispase on cell surface marker expression and the low cell yield across the experiments impacted the characterization of Th17 and Th22 using flow cytometry and thus limiting capacity to determine how CCL27 influences these T cell subsets.
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    Open Access
    Visualising the Mycobacterial Mutasome
    (2018) Reiche, Michael Anton; Warner, Digby
    An SOS-inducible DNA repair system has been linked to transient hyper-mutation and the development of drug resistance in Mycobacterium tuberculosis. Previous work has established that this “mycobacterial mutasome” comprises the specialist DNA polymerase, DnaE2, and accessory factors of unknown function, ImuA′ and ImuB. However, the molecular interactions and sub-cellular recruitment dynamics enabling mutasome function remain poorly understood. Here, a panel of fluorescent strains of M. smegmatis was developed to investigate expression and subcellular localization of ImuA′ and ImuB in live mycobacteria exposed to genotoxic agents. Using fluorescence microscopy, it was observed that, during prolonged genotoxic stress, single M. smegmatis cells exhibited an elongated cell phenotype and apparent aneuploidy – potentially providing an environment for recombination between differentially mutated chromosomes. Furthermore, ImuB was seen to associate with the dnaNencoded β clamp in discrete foci during mutagenic DNA repair. In contrast, ImuA′ did not exhibit similar localization and instead appeared to diffuse throughout the bacillus. A mutant ImuB protein deficient in the β clamp-binding motif failed to colocalize with the β clamp, reinforcing the inferred essentiality of the ImuB-β clamp protein-protein interaction for mutasome recruitment and induced mutagenesis. Additionally, exposure of M. smegmatis to griselimycin, a novel β clamp-targeting natural product antibiotic, prevented ImuB-β clamp co-localization during SOS induced mutagenesis, an observation confirmed by superresolution, threedimensional interferometric photo-activated light microscopy. These results establish the capacity of griselimycin to inhibit DNA replication as well as prevent DNA damage-induced mutagenesis by disrupting mutasome assembly and activity. Notably, this differentiates griselimycin from other inhibitors of DNA metabolic function which carry the often-unavoidable liability of accelerating drug-resistance by inducing mutagenic DNA repair. In turn, it suggests the potential application of griselimycin as an anti-evolution agent in novel therapeutic regimens designed to protect existing tuberculosis drugs.