Browsing by Author "Naidoo, Kewreshini Kasturi."

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Assessment of the diversity of bacteria and methanogenic Archaea in Zebra faeces.
(2013) Naidoo, Kewreshini Kasturi.; Schmidt, Stefan.
The need to develop a renewable, environmentally friendly source of energy has become a primary focus in modern science, with bio gas showing considerable potential. Interest in the methanogenic Archaea has therefore grown in recent years and extensive studies have been carried out to investigate the population diversity in various habitats. Presently, there are only a few studies that have evaluated the microbial communities inhabiting the gastrointestinal tract of wildlife native to southern Africa. This study aimed to investigate the microbial diversity, in particular the bacterial and methanogen communities involved in fermentative digestion in the gastrointestinal tract of zebra. Assessment of the microbial diversity in zebra faeces included both culture-based techniques and nucleic acid targeting analysis via 16S rRNA gene sequencing. Quantitative analysis using selected solid media revealed high counts for aerobic and anaerobic Bacteria (7.51x108 and 2.45x109/gram of faecal sample respectively). The majority of aerobic colonies that were detected exhibited Bacillus-like morphology. Nucleic acid based analysis of the diversity of both Bacteria and methanogenic Archaea in zebra faecal material was performed. Both manual and kit based extractions were used for DNA isolation in order to compare the efficiency of the two methods. Results show that a vigorous mechanical treatment was best for the release of DNA from the faecal matter. Amplification of target gene regions was carried out using established primer pairs (ARCH69F/ARCH915R and EUB338F/EUB907R) for methanogen and bacterial DNA respectively. Amplified 16S rRNA gene regions were cloned into a high copy number vector and random clones were selected for evaluation. Clones containing the target gene were further analysed by ARDRA and were assigned to a specific phylotype. Two bacterial (105 clones in total) and three methanogen (178 clones in total) clone libraries were constructed, of which 24 phylotypes were established for Bacteria and 25 for methanogenic Archaea. A representative of each phylotype was analysed by sequencing and further phylogenetic analysis was conducted. Six bacterial phylotypes, which represented 56% of all bacterial clones, exhibited 99% sequence similarity to Bacillus species. Six methanogen phylotypes, which exhibited 99% sequence similarity to the hydrogenotrophic species Methanobrevibacter gottschalkii strain PG, were established to be predominant in zebra faeces. These phylotypes represented 71% of all archaeal clones selected for analysis in this study.
Impact of Duffy Antigen Receptor for Chemokines (DARC)-null linked Neutropenia on Neutrophil and Natural Killer cell Function in HIV-1 Infection.
(2018) Naidoo, Kewreshini Kasturi.; Thobakgale-Tshabalala, Christina Fanesa.
Sub-Saharan Africa carries a disproportionate burden of the HIV-1 epidemic. The Duffy Antigen Receptor for Chemokines (DARC)-null polymorphism is a predictor of ethnic neutropenia which commonly occurs in persons of African ethnicity and is thought to account for up to 11% of HIV-1 infections on the African continent. Neutrophils are recognised for their killing mechanisms and have been noted for their regulatory mechanisms in recent years. For example, a role for neutrophils in natural killer (NK) cell priming in the periphery has been suggested, and neutrophil deficiency has been implicated in contributing to NK cell immaturity and dysfunction. While the DARC-null genotype is well associated with lower absolute neutrophil counts (ANCs), studies that assess the effects of the polymorphism on neutrophil functionality are lacking and the impact of DARC-null linked neutropenia on HIV disease progression is debatable. Furthermore, the influence of DARC-null neutropenia on the NK cell compartment is unknown. In this cross sectional pilot study, we assessed the impact of the DARC-null trait on neutrophil effector functions and also characterised NK cell profiles in Zulu/Xhosa African individuals from a high incidence HIV setting in Durban, South Africa. We hypothesised that in the context of the DARC-null genotype and lower ANCs in our cohort participants, neutrophils would have impaired functionality and would be unable to efficiently prime NK cells; thus affecting NK cell maturation and function, and altering NK cell homeostatic activities such as survival and proliferation. We further hypothesised that the impaired cellular responses in DARC-null individuals would be more prominent in HIV-1 infected individuals compared to HIV negative individuals. Neutrophil killing mechanisms were measured in HIV-1 chronically infected (n=22) individuals and HIV negative (uninfected) controls (n=20). For assessment of key neutrophil effector functions, isolated neutrophils were evaluated for Fc receptor-mediated phagocytosis following uptake of IgG opsonised beads using flow cytometry; reactive oxygen species (ROS) emission was measured by chemi-luminesce after activation of neutrophils with phorbol 12-myristate 13-acetate (PMA). Activated neutrophils were also visualised by fluorescent microscopy for neutrophil extracellular trap (NET) quantification. Assessment of the NK cell compartment in chronically HIV-1 infected (n=18) and uninfected (n=20) individuals using multi-parametric flow cytometry determined NK cell subsets, maturation profiles, cytokine production and degranulation. Annexin V and propidium iodide assays were used to determine NK cell survival, whilst CFSE staining was used to examine cytokine-activated NK cell proliferation. Study subjects were genotyped for the DARC trait using TaqMan allelic discrimination assays and ANCs were measured by full blood count. Our findings confirmed a high prevalence of the DARC-null allele in the African population and the polymorphism was significantly associated with lower ANCs. Neutrophil functional analysis detected rapid and higher phagocytic activity in the absence of DARC at 10 minutes (p=0.05 and p=0.009) and 60 minutes (p=0.05 and p=0.07) in HIV negative and HIV-1 infected subjects respectively. ROS and NET production in neutrophils were mostly unaffected by DARC negativity irrespective of HIV status. The only exception to this was a reduction in NET production in neutrophils from DARC-null HIV infected subjects (p=0.04) following prolonged in vitro stimulation. In the NK cell compartment, individuals showed similar NK cell counts irrespective of HIV status. In HIV negative individuals, a marked reduction of total NK cell counts was noted in the absence of DARC (p=0.006) and this correlated with lower ANCs (p=0.002) and a weak trend towards higher CD56 bright subset proportions was noted in DARC-null individuals (p=0.08). HIV negative DARC-null subjects also displayed a less mature NK cell phenotype with higher proportions of hypo-responsive KIR-NKG2A- NK cells (p=0.06) and lower frequencies of terminally differentiated CD57 (p=0.02) expressing NK cells. However, this immature phenotype did not translate to differences in expression of NK cell activation markers CD69 or HLA-DR and exhaustion marker PD-1 by DARC state. Furthermore, no differences in relation to NK cell degranulation and cytokine production were detected in the absence of DARC in HIV negative subjects. In contrast to HIV negative individuals, HIV-1 infected subjects displayed NK cell subset redistribution marked by higher CD56 negative NK cells, marginally higher frequencies of less mature NK cells, accompanied by higher expression of activation and exhaustion markers and lower cytolytic potential. However, these observed phenotypic and functional differences were lost upon DARC stratification in HIV-1 infected persons. Lastly, examination of NK cell survival capacity demonstrated only marginal differences during HIV infection in the absence of DARC (p=0.09); no changes were detected in NK cell proliferation by DARC state in HIV negative or infected individuals. Together our data suggests that the DARC-null polymorphism and lower ANCs does not adversely affect neutrophil activity irrespective of HIV status. We also show that while HIV negative individuals with the DARC-null genotype displayed reduced NK cell counts with a less mature phenotype, the condition did not compromise NK cell functionality or homeostatic activities. Furthermore, no significant differences were exhibited in the context of DARC during HIV infection, suggesting that any advantage that the DARC-positive trait may offer pre-infection is lost in chronic infection. DARC-null associated neutropenia is considered a mild condition and thus our findings support reports that the effect of ethnic neutropenia is not as pronounced as exhibited in severe neutropenia. Overall the data presented here provides mechanistic evidence behind the asymptomatic clinical characteristics associated with benign ethnic neutropenia.markers.
Interleukin 1-Beta (IL-1β) production by innate cells following TLR stimulation correlates with TB recurrence in art-treated HIV-infected patients.
(Wolters Kluwer Health., 2017) Thobakgale, Christina Fanesa.; Naidoo, Kewreshini Kasturi.; McKinnon, Lyle R.; Werner, Lise.; Samsunder, Natasha.; Abdool Karim, Salim Safurdeen.; Ndung'u, Peter Thumbi.; Altfeld, Marcus.; Naidoo, Kogieleum.
Abstract available.