The inhibition of M-MLV and HIV-1 reverse transcriptase by polyphenols extracted from the resurrection plant Myrothamnus flabellifolia (Welw.)

Includes abstract.Includes bibliographical references (leaves 80-90).Polyphenols have been shown to exhibit anti-viral activity in vitro, making them a promising starting point for the development of HIV treatment drugs. The main objective of this thesis was to assess the inhibitory effect of polyphenols extracted from Myrothamnus flabellifolia (Welw.) on M-MLV and HIV-I reverse transcriptases. The first part of the thesis was an attempt to isolate 3,4,5 tri-O-galloylquinic acid, the major polyphenol found in Namibian Myrothamnus flabellifolia plants. This polyphenol was successfully purified by column chromatography (Sephadex LH-20) and its purity was confirmed by HPLC and MALDI-TOF mass spectrometry. The second part of this thesis involved the development of a polymerase enzyme activity assay based on ethidium bromide fluorescence. A calibration curve for quantification of DNA was therefore prepared from the ethidium bromide fluorescence of Calf Thymus DNA. Results demonstrated that Calf Thymus DNA was a good standard for estimating the amount of cDNA synthesised during reverse transcription, thus enabling the monitoring of both M-ML V and HIV -1 reverse transcriptase activity. The reverse transcriptase activity assay was optimised using a poly (rA) template, an oligo (dTb primer and dTTP as a substrate. It was observed that the rate of catalysis for M-ML V and HIV -1 RTs decreased with increase in the concentration of dTTP, which suggested substrate inhibition. A decrease in M-MLV RT activity with increased substrate concentration was found to be due to depletion of Mg2+ ions by dTTP. True substrate inhibition was however observed for HIV-I RT, and analysis of the observed kinetics suggested the formation of an ineffective enzyme substrate complex with two substrate molecules binding to HIV -1 reverse transcriptase. A Hill coefficient of one was obtained at low dTTP concentration and less than one at high dTTP concentration, suggesting zero and negative cooperativity respectively. The final part of this thesis tested the inhibitory effect of pure and crude polyphenol fractions on the activity of M-MLV and HIV-1 RTs. Results showed that all polyphenol fractions inhibited M-ML V and HIV -I reverse transcriptase activity, with the highest inhibitory activity demonstrated by the fraction that contained pure 3,4,5 tri-O-galloylquinic acid. The 50 % inhibitory concentrations of 3,4,5 tri-O-galloylquinic acid was 0.5 μM for M-MLV RT and 34 μM for HIV-I RT. Lineweaver-Burk plots showed that 3,4,5 tri-O-galloylquinic acid inhibited both enzymes non-competitively. Pure non-competitive inhibition was observed for M-MLV RT and mixed non-competitive inhibition for HIV-I RT. Results showed that the binding of 3,4,5 tri-O-galloylquinic acid to M-MLV RT was irreversible, suggesting strong binding under the conditions tested. 3,4,5 Tri-O-galloylquinic acid, however, bound to HIV-I RT reversibly. A comparison of catalytic efficiencies showed that M-MLV RT was more efficient than HIV -1 RT under saturating substrate concentrations with Kcat (min-¹) values of II ± 3 and 1.31 ± 0.02 respectively. M-MLV RT and HIV-¹ RT were, however, equally efficient under limiting substrate concentrations with Kcat/Km (min-¹M-¹) values of 1.1 ± 0.3 x 10⁴ and 1.2 ± 0.2 x 10⁴ respectively

Human alveolar macrophages predominately express combined classical M1 and M2 surface markers in steady state.

Alveolar macrophages (AM) are critical to the homeostasis of the inflammatory environment in the lung. Differential expression of surface markers classifies macrophages to either classically (M1) or alternatively activated (M2). We investigated the phenotype of human alveolar macrophages (AM) in adults living in two different geographical locations: UK and Malawi. We show that the majority of AM express high levels of M1 and M2 markers simultaneously, with the M1/M2 phenotype being stable in individuals from different geographical locations. The combined M1/M2 features confer to AM a hybrid phenotype, which does not fit the classic macrophage classification. This hybrid phenotype may confer to alveolar macrophages an ability to quickly switch between M1 or M2 associated functions allowing for appropriate responses to stimuli and tissue environment

Piliation of Invasive Streptococcus pneumoniae Isolates in the Era before Pneumococcal Conjugate Vaccine Introduction in Malawi

The pneumococcal pilus has been shown to be an important determinant of adhesion and virulence in mouse models of colonization, pneumonia, and bacteremia. A pilus is capable of inducing protective immunity, supporting its inclusion in next-generation pneumococcal protein vaccine formulations. Whether this vaccine target is common among pneumococci in sub-Saharan Africa is uncertain. To define the prevalence and genetic diversity of type I and II pili among invasive pneumococci in Malawi prior to the introduction of the 13-valent pneumococcal conjugate vaccine (PCV13) into routine childhood immunization, we examined 188 Streptococcus pneumoniae isolates collected between 2002 and 2008 (17% serotype 1). In this region of high disease burden, we found a low frequency of invasive piliated pneumococci (14%) and pilus gene sequence diversity similar to that seen previously in multiple global pneumococcal lineages. All common serotypes with pilus were covered by PCV13 and so we predict that pilus prevalence will be reduced in the Malawian pneumococcal population after PCV13 introduction

B cell, CD8 + T cell and gamma delta T cell infiltration alters alveolar immune cell homeostasis in HIV-infected Malawian adults

HIV infection is associated with increased risk to lower respiratory tract infections (LRTI). However, the impact of HIV infection on immune cell populations in the lung is not well defined. We sought to comprehensively characterise the impact of HIV infection on immune cell populations in the lung. : Twenty HIV-uninfected controls and 17 HIV-1 infected ART-naïve adults were recruited from Queen Elizabeth Central Hospital, Malawi. Immunophenotyping of lymphocyte and myeloid cell populations was done on bronchoalveolar lavage fluid and peripheral blood cells. : We found that the numbers of CD8 T cells, B cells and gamma delta T cells were higher in BAL fluid of HIV-infected adults compared to HIV-uninfected controls (all p<0.05). In contrast, there was no difference in the numbers of alveolar CD4 T cells in HIV-infected adults compared to HIV-uninfected controls (p=0.7065). Intermediate monocytes were the predominant monocyte subset in BAL fluid (HIV-, 63%; HIV+ 81%), while the numbers of classical monocytes was lower in HIV-infected individuals compared to HIV-uninfected adults (p=0.0006). The proportions of alveolar macrophages and myeloid dendritic cells was lower in HIV-infected adults compared to HIV-uninfected controls (all p<0.05). : Chronic HIV infection is associated with broad alteration of immune cell populations in the lung, but does not lead to massive depletion of alveolar CD4 T cells. Disruption of alveolar immune cell homeostasis likely explains in part the susceptibility for LRTIs in HIV-infected adults

Performance of molecular methods for the detection of Salmonella in human stool specimens [version 2; peer review: 2 approved]

Background: The relationship between asymptomatic Salmonella exposure within the gastrointestinal tract and Salmonella bacteraemia is poorly understood, in part due to the low sensitivity of stool culture and the lack of validated molecular diagnostic tests for the detection of Salmonella in the stool. The study aimed to determine a reliable molecular diagnostic test for Salmonella in stool specimens. Methods: We optimised an in-house monoplex real-time polymerase chain reaction (PCR) for the detection of Salmonella ttr and InvA genes in stool by including a selenite broth pre-culture step for Salmonella before DNA extraction and validated their specificity against other local common pathogens. Then we assessed their performance against a well-validated multiplex PCR targeting the same ttr and InvA genes and against stool culture using clinical stool specimens collected from a cohort of 50 asymptomatic healthy Malawian children that were sampled at 1-month intervals over 12 months. We employed a latent Markov model to estimate the specificities and sensitivities of PCR methods. Results: Ttr and InvA primers were both able to detect all the different Salmonella serovars tested and had superior limits of detection when DNA was extracted after selenite pre-culture. Ttr sensitivity and specificity for monoplex-PCR were (99.53%, 95.46%) and for multiplex-PCR (90.30%, 99.30%) respectively. InvA specificity and specificity for using monoplex-PCR was (95.06%, 90.31%) and multiplex-PCRs (89.41%, 98.00%) respectively. Sensitivity and specificity for standard stool culture were 62.88% and 99.99%, respectively. Culture showed the highest PPV (99.73%), and monoplex-ttr had the highest NPV (99.67%). Conclusion: Test methods demonstrated high concordance, although stool culture and monoplexed ttr primers had superior specificity and sensitivity, respectively. The use of selenite pre-enrichment step increased Salmonella detection rate. Taken together, molecular detection methods used here could be used to reveal the true extent of both asymptomatic and symptomatic Salmonella exposure events

Recombination in Streptococcus pneumoniae Lineages Increase with Carriage Duration and Size of the Polysaccharide Capsule.

Streptococcus pneumoniae causes a high burden of invasive pneumococcal disease (IPD) globally, especially in children from resource-poor settings. Like many bacteria, the pneumococcus can import DNA from other strains or even species by transformation and homologous recombination, which has allowed the pneumococcus to evade clinical interventions such as antibiotics and pneumococcal conjugate vaccines (PCVs). Pneumococci are enclosed in a complex polysaccharide capsule that determines the serotype; the capsule varies in size and is associated with properties including carriage prevalence and virulence. We determined and quantified the association between capsule and recombination events using genomic data from a diverse collection of serotypes sampled in Malawi. We determined both the amount of variation introduced by recombination relative to mutation (the relative rate) and how many individual recombination events occur per isolate (the frequency). Using univariate analyses, we found an association between both recombination measures and multiple factors associated with the capsule, including duration and prevalence of carriage. Because many capsular factors are correlated, we used multivariate analysis to correct for collinearity. Capsule size and carriage duration remained positively associated with recombination, although with a reduced P value, and this effect may be mediated through some unassayed additional property associated with larger capsules. This work describes an important impact of serotype on recombination that has been previously overlooked. While the details of how this effect is achieved remain to be determined, it may have important consequences for the serotype-specific response to vaccines and other interventions. IMPORTANCE: The capsule determines >90 different pneumococcal serotypes, which vary in capsule size, virulence, duration, and prevalence of carriage. Current serotype-specific vaccines elicit anticapsule antibodies. Pneumococcus can take up exogenous DNA by transformation and insert it into its chromosome by homologous recombination. This mechanism has disseminated drug resistance and generated vaccine escape variants. It is hence crucial to pneumococcal evolutionary response to interventions, but there has been no systematic study quantifying whether serotypes vary in recombination and whether this is associated with serotype-specific properties such as capsule size or carriage duration. Larger capsules could physically inhibit DNA uptake, or given the longer carriage duration for larger capsules, this may promote recombination. We find that recombination varies among capsules and is associated with capsule size, carriage duration, and carriage prevalence and negatively associated with invasiveness. The consequence of this work is that serotypes with different capsules may respond differently to selective pressures like vaccines

Circulating microparticles are increased amongst people presenting with HIV and advanced immune suppression in Malawi and correlate closely with arterial stiffness: a nested case control study

Background: We aimed to investigate whether circulating microparticle (CMPs) subsets were raised amongst people presenting with human immunodeficiency virus (HIV) and advanced immune suppression in Malawi, and whether they associated with arterial stiffness. Methods: Antiretroviral therapy (ART)-naïve adults with a new HIV diagnosis and CD4 <100 cells/µL had microparticle characterisation and carotid femoral Pulse Wave Velocity (cfPWV) at 2 weeks post ART initiation. HIV uninfected controls were matched on age, systolic blood pressure (BP) and diastolic BP in a 1:1 ratio.  Circulating microparticles were identified from platelet poor plasma and stained for endothelial, leucocyte, monocyte and platelet markers. Results: The median (IQ) total CMP count for 71 participants was 1 log higher in HIV compared to those without (p<0.0001) and was associated with arterial stiffness (spearman rho 0.47, p<0.001). In adjusted analysis, every log increase in circulating particles showed a 20% increase in cfPWV (95% confidence interval [CI] 4 – 40%, p=0.02). In terms of subsets, endothelial and platelet derived microparticles were most strongly associated with HIV. Endothelial derived E-selectin+ CMPs were 1.3log-fold higher and platelet derived CD42a+ CMPs were 1.4log-fold higher (both p<0.0001). Endothelial and platelet derived CMPs also correlated most closely with arterial stiffness (spearman rho: E-selectin+ 0.57 and CD42a 0.56, both p<0.0001). Conclusions: Circulating microparticles associate strongly with arterial stiffness among people living with HIV in Malawi. Endothelial damage and platelet microparticles are the predominant cell origin types and future translational studies could consider prioritising these pathways

Circulating microparticles are increased amongst people presenting with HIV and advanced immune suppression in Malawi and correlate closely with arterial stiffness: a nested case control study [version 2; peer review: 2 approved, 1 approved with reservations]

BACKGROUND: We aim to investigate whether circulating microparticle (CMPs) subsets were raised amongst people presenting with a new diagnosis of HIV and advanced immune suppression in Malawi, and whether they associated with arterial stiffness. Methods: Microparticle characterisation and carotid femoral Pulse Wave Velocity (cfPWV) were carried out in a cohort of adults with a new HIV diagnosis and CD4 <100 cells/µL at 2 weeks post ART initiation. HIV uninfected controls were matched on age, systolic BP and diastolic BP in a 1:1 ratio. Circulating microparticles were identified from platelet poor plasma and stained for endothelial, leucocyte, monocyte and platelet markers. METHODS: Microparticle characterisation and carotid femoral Pulse Wave Velocity (cfPWV) were carried out in a cohort of adults with a new HIV diagnosis and CD4 <100 cells/µL at 2 weeks post ART initiation. HIV uninfected controls were matched on age, systolic BP and diastolic BP in a 1:1 ratio. Circulating microparticles were identified from platelet poor plasma and stained for endothelial, leucocyte, monocyte and platelet markers. RESULTS: The median (IQ) total CMP count for 71 participants was 1 log higher in HIV compared to those without (p<0.0001) and was associated with arterial stiffness (spearman rho 0.47, p<0.001). In adjusted analysis, every log increase in circulating particles showed a 20% increase in cfPWV (95% CI 4 – 40%, p=0.02). In terms of subsets, endothelial and platelet derived microparticles were most strongly associated with HIV. Endothelial derived E-selectin+ CMPs were 1.3log-fold higher and platelet derived CD42a+ CMPs were 1.4log-fold higher (both p<0.0001). Endothelial and platelet derived CMPs also correlated most closely with arterial stiffness [spearman rho: E-selectin+ 0.57 and CD42a 0.56, both p<0.0001). CONCLUSIONS: Circulating microparticles associate strongly with arterial stiffness among PLWH in Malawi. Endothelial and platelet microparticles are the predominant cell origin types, indicating that platelet driven endothelial dysfunction pathways warrant further investigation in HIV associated arterial stiffness