Prospective monitoring reveals dynamic levels of T cell immunity to Mycobacterium tuberculosis in HIV infected individuals

Monitoring of latent Mycobacterium tuberculosis infection may prevent disease. We tested an ESAT-6 and CFP-10-specific IFN-γ Elispot assay (RD1-Elispot) on 163 HIV-infected individuals living in a TB-endemic setting. An RD1-Elispot was performed every 3 months for a period of 3-21 months. 62% of RD1-Elispot negative individuals were positive by cultured Elispot. Fluctuations in T cell response were observed with rates of change ranging from -150 to +153 spot-forming cells (SFC)/200,000 PBMC in a 3-month period. To validate these responses we used an RD1-specific real time quantitative PCR assay for monokine-induced by IFN-γ (MIG) and IFN-γ inducible protein-10 (IP10) (MIG: r?=?0.6527, p?=?0.0114; IP-10: r?=?0.6967, p?=?0.0056; IP-10+MIG: r?=?0.7055, p?=?0.0048). During follow-up 30 individuals were placed on ARVs and 4 progressed to active TB. Fluctuations in SFC did not correlate with CD4 count, viral load, treatment initiation, or progression to active TB. The RD1-Elispot appears to have limited value in this setting.     

Cost-Effectiveness of Tenofovir Instead of Zidovudine for Use in First-Line Antiretroviral Therapy in Settings without Virological Monitoring

Background

The most recent World Health Organization (WHO) antiretroviral treatment guidelines recommend the inclusion of zidovudine (ZDV) or tenofovir (TDF) in first-line therapy. We conducted a cost-effectiveness analysis with emphasis on emerging patterns of drug resistance upon treatment failure and their impact on second-line therapy.

Methods

We used a stochastic simulation of a generalized HIV-1 epidemic in sub-Saharan Africa to compare two strategies for first-line combination antiretroviral treatment including lamivudine, nevirapine and either ZDV or TDF. Model input parameters were derived from literature and, for the simulation of resistance pathways, estimated from drug resistance data obtained after first-line treatment failure in settings without virological monitoring. Treatment failure and cost effectiveness were determined based on WHO definitions. Two scenarios with optimistic (no emergence; base) and pessimistic (extensive emergence) assumptions regarding occurrence of multidrug resistance patterns were tested.

Results

In the base scenario, cumulative proportions of treatment failure according to WHO criteria were higher among first-line ZDV users (median after six years 36% [95% simulation interval 32%; 39%]) compared with first-line TDF users (31% [29%; 33%]). Consequently, a higher proportion initiated second-line therapy (including lamivudine, boosted protease inhibitors and either ZDV or TDF) in the first-line ZDV user group 34% [31%; 37%] relative to first-line TDF users (30% [27%; 32%]). At the time of second-line initiation, a higher proportion (16%) of first-line ZDV users harboured TDF-resistant HIV compared with ZDV-resistant viruses among first-line TDF users (0% and 6% in base and pessimistic scenarios, respectively). In the base scenario, the incremental cost effectiveness ratio with respect to quality adjusted life years (QALY) was US$83 when TDF instead of ZDV was used in first-line therapy (pessimistic scenario: US$ 315), which was below the WHO threshold for high cost effectiveness (US$ 2154).

Conclusions

Using TDF instead of ZDV in first-line treatment in resource-limited settings is very cost-effective and likely to better preserve future treatment options in absence of virological monitoring.     

Spatial pattern analysis and competition between Acacia karroo trees in humid savannas

It is unclear whether inter-tree competition plays a role in determining the woody plant cover of humid savannas. Spatial point-pattern analysis can give insights to the underlying processes affecting the individuals in a population. We assessed the spatial patterns of Acacia karroo trees from savannas in KwaZulu-Natal, using conventional nearest neighbour analysis and second-order spatial statistics such as Ripley??s K- and L-functions, and the univariate and bivariate O-ring statistics. We predicted that juvenile trees would be spatially aggregated, because of facilitation between shrubs when zones of overlap are relatively small, while adult trees would be regularly spaced because of the effects of density-dependent mortality (i.e. consistent with the honeycomb rippling model). We found that juvenile trees were more aggregated than expected by chance, and the overall spatial distribution of all trees was also found to be aggregated, with no evidence of regularity among large individuals. Nearest neighbour analysis, however, revealed significant positive correlations between the sum of the distances to the four nearest neighbours and the sum of the canopy diameters of the target tree and its four nearest neighbours, indicating the presence of competition. In sum, these findings suggest that competitive interactions between A. karroo trees at these sites are relatively weak, and result in decreased performance (smaller canopy diameters) rather than mortality, thus preventing a regular pattern of tree distribution. We advocate the use of both methods of detecting competitive interactions in the field, especially if the effects of competition are too subtle to result in differential mortality.     

Selectivity of stop codon recognition in translation termination is modulated by multiple conformations of GTS loop in eRF1

Translation termination in eukaryotes is catalyzed by two release factors eRF1 and eRF3 in a cooperative manner. The precise mechanism of stop codon discrimination by eRF1 remains obscure, hindering drug development targeting aberrations at translation termination. By solving the solution structures of the wild-type N-domain of human eRF1 exhibited omnipotent specificity, i.e. recognition of all three stop codons, and its unipotent mutant with UGA-only specificity, we found the conserved GTS loop adopting alternate conformations. We propose that structural variability in the GTS loop may underline the switching between omnipotency and unipotency of eRF1, implying the direct access of the GTS loop to the stop codon. To explore such feasibility, we positioned N-domain in a pre-termination ribosomal complex using the binding interface between N-domain and model RNA oligonucleotides mimicking Helix 44 of 18S rRNA. NMR analysis revealed that those duplex RNA containing 2-nt internal loops interact specifically with helix α1 of N-domain, and displace C-domain from a non-covalent complex of N-domain and C-domain, suggesting domain rearrangement in eRF1 that accompanies N-domain accommodation into the ribosomal A site.     

Screening of selected ethnomedicinal plants from South Africa for larvicidal activity against the mosquito Anopheles arabiensis

ABSTRACT: BACKGROUND: This study was initiated to establish whether any South African ethnomedicinal plants (indigenous or exotic), that have been reported to be used traditionally to repel or kill mosquitoes, exhibit effective mosquito larvicidal properties. METHODS: Extracts of a selection of plant taxa sourced in South Africa were tested for larvicidal properties in an applicable assay. Thirty 3rd instar Anopheles arabiensis larvae were exposed to various extract types (dichloromethane, dichloromethane/methanol) (1:1), methanol and purified water) of each species investigated. Mortality was evaluated relative to the positive control Temephos (Mostop; Agrivo), an effective emulsifiable concentrate larvicide. RESULTS: Preliminary screening of crude extracts revealed substantial variation in toxicity with 24 of the 381 samples displaying 100% larval mortality within the seven day exposure period. Four of the high activity plants were selected and subjected to bioassay guided fractionation. The results of the testing of the fractions generated identified one fraction of the plant Toddalia asiatica as being very potent against the An. arabiensis larvae. CONCLUSION: The present study has successfully identified a plant with superior larvicidal activity at both the crude and semi pure fractions generated through bio-assay guided fractionation. These results have initiated further research into isolating the active compound and developing a malaria vector control tool.     

Expression of a versatile DC-targeting fusion protein using an Adenovirus expression system

The importance of viral and tumour vaccines in eliciting elicit strong CD8+ T-cell responses has been widely acknowledged. Strategies exploring ways to enhance CD8+ T-cell responses have been developed, including targeting of vaccine antigens to dendritic cell (DC) receptors to access to the cross presentation pathway. Many DC endocytic receptors could potentially lead to augmented CD8+ T-cell responses if antigens were targeted directly to them, however only a few receptors have been explored because current targeting reagents are limited in the number of receptors that they are able to target. Consequently, this study describes the production and purification of a streptavidin-fusion protein that provides a versatile and efficient means to target antigen to more than one DC receptor. A model antigen gene, CMV pp65, and a streptavidin core gene, were spliced together using an overlap-extension PCR technique. The resulting fusion gene was cloned into a vector allowing expression in an Adenovirus-based expression system. Expression was verified and optimised before Ni-NTA affinity chromatography purification. Evaluation of pp65-streptavidin immunogenicity revealed that it elicits similar levels of CD8+ T-cell proliferative responses as pp65 and is able to effectively target specific DC receptors when used in addition to biotinylated receptor-specific antibodies. Additionally, enhancement of CD8+ T-cell responses was shown after directing pp65-strep to selected DC receptors in preliminary in vitro experiments. Collectively, this highlights the ease of production of a streptavidin-fusion protein, and demonstrates its use as a promising strategy to evaluate numerous DC receptors as potential targets in vaccine strategies.     

Aerobic degradation of 2,4-dichlorophenoxyacetic acid and other chlorophenols by <Emphasis Type="Italic">Pseudomonas</Emphasis> strains indigenous to contaminated soil in South Africa: Growth kinetics and degradation pathway

Three indigenous pseudomonads, Pseudomonas putida DLL-E4, Pseudomonas reactans and Pseudomonas fluorescens, were isolated from chlorophenol-contaminated soil samples collected from a sawmill located in Durban (South Africa). The obtained isolates were tested for their ability to degrade chlorophenolic compounds: 2,4-dichlorophenoxyacetic acid (2,4-D), 2,4-dichlorophenol (2,4-DCP) and 2,4,6-trichlorophenol (2,4,6-TCP) in batch cultures. The isolates were found to effectively degrade up to 99.5, 98.4 and 94.0% with a degradation rate in the range of 0.67–0.99 (2,4-D), 0.57–0.93 (2,4-DCP) and 0.30–0.39 (2,4,6-TCP) mgL^–1 day^–1 for 2,4-D; 2,4-DCP and 2,4,6-TCP, respectively. The degradation kinetics model revealed that these organisms could tolerate up to 600 mg/L of 2,4-DCP. Catechol 2,3-dioxygenase activity detected in the crude cell lysates of P. putida DLL-E4 and P. reactans was 21.9- and 37.6-fold higher than catechol 1,2-dioxygenase activity assayed, suggesting a meta-pathway for chlorophenol degradation by these organisms. This is also supported by the generally high expression of C23O gene (involved in meta-pathway) relative to tfdC gene (involved in ortho-pathway) expression. Results of this study will be helpful in the exploitation of these organisms and/or their enzymes in bioremediation strategies for chlorophenol-polluted environment.     

Antimycobacterial,docking and molecular dynamic studies of pentacyclic triterpenes from Buddleja saligna leaves

Buddleja saligna (family Buddlejaceae) is a medicinal plant endemic to South Africa. Two isomeric pentacyclic triterpenes, oleanolic acid and ursolic acid, were isolated from the leaves of B. saligna using silica gel column chromatography. Compounds oleanolic acid and ursolic acid were subjected to derivatization with acetic anhydride in the presence of pyridine to obtain oleanolic acid-3-acetate and ursolic acid-3-acetate, respectively. The structures of these compounds were fully characterized by detailed nuclear magnetic resonance (NMR) investigations, which included ¹H and ¹³C NMR. Molecular docking studies predicted the free binding energy of the four triterpenes inside the steroid binding pocket of Mycobacterium tuberculosis fadA5 thiolase compared to a reported inhibitor. Thus, their ability to inhibit the growth of M. tuberculosis was predicted and was confirmed to possess significant antimycobacterial activity when tested against Mycobacterium smegmatis, M. tuberculosis H₃₇Rv (ATCC 25177), clinical isolates of multi-drug-resistant M. tuberculosis (MDR-TB) and extensively drug-resistant M. tuberculosis (XDR-TB) using the Micro Alamar Blue Assay. Ursolic acid was isolated from this plant for the first time.     

Insulin-stimulated serine/threonine phosphorylation of the insulin receptor: paucity of threonine 1348 phosphorylation in vitro indicates the involvement of more than one serine/threonine kinase in vivo.

Immunoaffinity-purified insulin receptors were used to analyse and compare the serine/threonine sites phosphorylated on the insulin receptor in vitro (isolated receptor) with the insulin-stimulated phosphorylation in vivo (intact cells in culture). In vivo, insulin-stimulation resulted in the appearance of three phosphoserine-containing phosphopeptides and a distinct phosphothreonine peptide (threonine 1348). In vitro, similar phosphoserine peptides were observed but the phosphothreonine peptide was absent. These results indicate that multiple serine sites are phosphorylated in vivo and in vitro and that an additional protein kinase mediates insulin-stimulated insulin receptor threonine phosphorylation in vivo.