Trends in First-Line Antiretroviral Therapy in Asia: Results from the TREAT Asia HIV Observational Database

Background

Antiretroviral therapy (ART) has evolved rapidly since its beginnings. This analysis describes trends in first-line ART use in Asia and their impact on treatment outcomes.

Methods

Patients in the TREAT Asia HIV Observational Database receiving first-line ART for ≥6 months were included. Predictors of treatment failure and treatment modification were assessed.

Results

Data from 4662 eligible patients was analysed. Patients started ART in 2003–2006 (n = 1419), 2007–2010 (n = 2690) and 2011–2013 (n = 553). During the observation period, tenofovir, zidovudine and abacavir use largely replaced stavudine. Stavudine was prescribed to 5.8% of ART starters in 2012/13. Efavirenz use increased at the expense of nevirapine, although both continue to be used extensively (47.5% and 34.5% of patients in 2012/13, respectively). Protease inhibitor use dropped after 2004. The rate of treatment failure or modification declined over time (22.1 [95%CI 20.7–23.5] events per 100 patient/years in 2003–2006, 15.8 [14.9–16.8] in 2007–2010, and 11.6 [9.4–14.2] in 2011–2013). Adjustment for ART regimen had little impact on the temporal decline in treatment failure rates but substantially attenuated the temporal decline in rates of modification due to adverse event. In the final multivariate model, treatment modification due to adverse event was significantly predicted by earlier period of ART initiation (hazard ratio 0.52 [95%CI 0.33–0.81], p = 0.004 for 2011–2013 versus 2003–2006), older age (1.56 [1.19–2.04], p = 0.001 for ≥50 years versus <30years), female sex (1.29 [1.11–1.50], p = 0.001 versus male), positive hepatitis C status (1.33 [1.06–1.66], p = 0.013 versus negative), and ART regimen (11.36 [6.28–20.54], p<0.001 for stavudine-based regimens versus tenofovir-based).

Conclusions

The observed trends in first-line ART use in Asia reflect changes in drug availability, global treatment recommendations and prescriber preferences over the past decade. These changes have contributed to a declining rate of treatment modification due to adverse event, but not to reductions in treatment failure.     

The effects of Eucalyptus terpenes on hepatic cytochrome P450 CYP4A, peroxisomal Acyl CoA oxidase (AOX) and peroxisome proliferator activated receptor alpha (PPARalpha) in the common brush tail possum (Trichosurus vulpecula)

Eucalyptus leaves contain a high proportion of essential oils comprising of a complex mixture of monoterpenes such as 1,8-cineole, alpha-pinene, d-limonene and p-cymene. In this study, hepatic levels of microsomal lauric acid hydroxylase and peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activities were examined in livers of possums given an artificial diet consisting of the above monoterpenes for 10 days. These values were compared with those of possums fed a control diet containing only fruits and cereals. Peroxisomal cyanide-intensive palmitoyl coenzyme A oxidative activity was significantly higher in livers of treated possums relative to that of control possums (2.96+/-0.93 vs. 0.98+/-0.88 nmol/mg protein per min, P<0.01) (mean+/-S.D., n=4). A small increase in microsomal lauric acid hydroxylase activity was observed in the treated possum in comparison with the control group (4.40+/-0.85 vs. 3.60+/-0.48 nmol/mg protein per min) (mean+/-S.D., n=4). A higher NAD/ NADP ratio was observed in treated possums as compared with control possums (4.73+/-0.65 vs. 3.51+/-0.64 nmol/mg protein per min, P<0.05) (mean+/-S.D., n=4). No other statistically significant differences in pyridine nucleotide contents were found between control and treated possums. Northern blot analysis of mRNA from rat, human, terpene treated and control possum livers, using the corresponding koala cDNA probes, detected a more intense acyl CoA oxidase (AOX) mRNA band in livers of terpene fed possums. Negligible differences in the intensity of CYP4A and PPARalpha mRNA bands were observed between the two groups. These data suggest that Eucalyptus terpenes elevate hepatic AOX expression in possums.     

Conservation of Structure and Protein-Protein Interactions Mediated by the Secreted Mycobacterial Proteins EsxA,EsxB, and EspA

Mycobacterium tuberculosis EsxA and EsxB proteins are founding members of the WXG100 (WXG) protein family, characterized by their small size (∼100 amino acids) and conserved WXG amino acid motif. M. tuberculosis contains 11 tandem pairs of WXG genes; each gene pair is thought to be coexpressed to form a heterodimer. The precise role of these proteins in the biology of M. tuberculosis is unknown, but several of the heterodimers are secreted, which is important for virulence. However, WXG proteins are not simply virulence factors, since nonpathogenic mycobacteria also express and secrete these proteins. Here we show that three WXG heterodimers have structures and properties similar to those of the M. tuberculosis EsxBA (MtbEsxBA) heterodimer, regardless of their host species and apparent biological function. Biophysical studies indicate that the WXG proteins from M. tuberculosis (EsxG and EsxH), Mycobacterium smegmatis (EsxA and EsxB), and Corynebacterium diphtheriae (EsxA and EsxB) are heterodimers and fold into a predominately α-helical structure. An in vivo protein-protein interaction assay was modified to identify proteins that interact specifically with the native WXG100 heterodimer. MtbEsxA and MtbEsxB were fused into a single polypeptide, MtbEsxBA, to create a biomimetic bait for the native heterodimer. The MtbEsxBA bait showed specific association with several esx-1-encoded proteins and EspA, a virulence protein secreted by ESX-1. The MtbEsxBA fusion peptide was also utilized to identify residues in both EsxA and EsxB that are important for establishing protein interactions with Rv3871 and EspA. Together, the results are consistent with a model in which WXG proteins perform similar biological roles in virulent and nonvirulent species.The WXG100 (WXG; pfam06013) proteins are a class of effector molecules found in gram-positive bacteria (26). WXG proteins are characterized by their small size (∼ 100 amino acids [aa]) and the presence of a WXG motif, or its structural equivalent, near the midpoint of their primary sequence (26). Bioinformatic analyses have shown that one WXG gene is frequently positioned near, or directly adjacent to, a second, related, WXG gene (14). The gene pairs characterized thus far encode proteins that associate to form 1:1 complexes (20, 31). The WXG proteins were once thought to be restricted to the mycobacteria, but homologues have now been detected in species of Bacillus, Listeria, Streptomyces, and Corynebacterium, among others, and the Pfam server lists >89 distinct WXG-encoding species and strains (10).The identification of WXG proteins encoded by the pathogens Mycobacterium tuberculosis (15, 17, 19, 36), Mycobacterium marinum (13), and Staphylococcus aureus (5) has created significant interest in the proteins'' biological activity. Nevertheless, these proteins are not a priori virulence factors (39), since organisms expressing WXG proteins are not necessarily capable of causing disease. In addition to pathogenesis, the WXG proteins are associated with processes as disparate as zinc homeostasis (24) and conjugal gene transfer (9, 11). A model for the mechanism(s) of action of these proteins that includes an explanation for their apparent functional versatility is at present lacking. One reason for this ambiguity may be the near-absence of studies comparing virulence-associated and non-virulence-associated WXG proteins, which is a goal of this study.The M. tuberculosis secreted virulence factors EsxA (also called ESAT-6, or Rv3875) and EsxB (CFP-10; Rv3874) are the founding members of the WXG family, and M. tuberculosis derivatives defective in EsxA and EsxB are attenuated (17, 19, 36). The results of biochemical and structural studies indicate that EsxA and EsxB form a tightly associated heterodimer, EsxAB (25, 30, 31). The M. tuberculosis genome contains 23 WXG genes, named esxA to esxW, and the majority of these are expressed as tandem pairs (26). Of the pairs, five, including esxA and esxB, are contained within larger, highly conserved genetic loci, called esx-1 to esx-5 (Fig. ​(Fig.1).1). These loci have been the focus of much research, since mutants of esx-1 are attenuated, and esx-3 and esx-5 are necessary for in vitro growth of M. tuberculosis and M. marinum (1, 2, 32-34). The esx loci are proposed to encode secretory apparatuses dedicated to the secretion of their cognate WXG proteins (1).Open in a separate windowFIG. 1.Genetic map of the esx-1 loci of M. tuberculosis and M. smegmatis. The M. tuberculosis esx-1 genes discussed in the text are indicated by white arrows, as are their M. smegmatis homologues. The M. tuberculosis map also shows the Rv3884 and Rv3885 genes, which are part of the adjacent esx-2 locus. pRD1-2F9 is the cosmid that was used to create an esx-1-specific prey library. pRD1-2F9 includes the Rv3860 to Rv3885 genes, thus encompassing the entire esx-1 locus and part of esx-2. The four genes below the M. smegmatis map include defective insertion sequences (ISs) inserted into MSMEG_0075.Although the majority of genes required for the secretion of the EsxAB heterodimer are encoded from within esx-1, additional non-esx-1 genes are necessary for secretion. In particular, one M. tuberculosis locus, esp, encodes three proteins essential for EsxAB secretion (12, 23). The first gene of the operon encodes a protein, EspA, that is cosecreted with EsxAB via the ESX-1 apparatus (12). Although no direct physical evidence has been presented, the inference from the interdependent cosecretion of the three proteins is that they likely form a complex, which is secreted by the ESX-1 apparatus. In this paper we provide the first genetic evidence that these three proteins interact.The lack of a genetic assay for the study of ESX-1 activity in M. tuberculosis has hindered the identification of all of the protein components of the apparatus and all of the substrates that it secretes. However, the fast-growing, nonpathogenic organism Mycobacterium smegmatis has a conserved esx-1 locus that is essential for DNA transfer, and we have exploited this requirement for genetic studies (9). These analyses have shown that the M. smegmatis ESX-1 apparatus is functionally related to that of M. tuberculosis (11) and that M. smegmatis encodes non-esx-1 genes necessary for the secretion of the EsxAB heterodimer, including orthologues of EspA (9).Here we have examined whether the secondary and quaternary structures of M. tuberculosis EsxA and EsxB are prototypical for other, functionally distinct and evolutionarily distant members of the WXG family (Fig. ​(Fig.2A).2A). Comparisons were made to homologues encoded by M. smegmatis (esxA and esxB), Corynebacterium diphtheriae (esxA and esxB), and an additional non-virulence-related pair from M. tuberculosis (esxG and esxH, encoded from the esx-3 locus). Structural characterization of these proteins establishes that their secondary and quaternary structures are conserved, with each pair folding into a predominately α-helical structure and associating to form a heterodimer. We next devised and tested the utility of a novel strategy to identify proteins that interact specifically with these WXG heterodimers. This involved fusing EsxB and EsxA to create a biomimetic heterodimer for use in mycobacterial two-hybrid experiments. We reasoned that the use of this unique bait would allow the detection of proteins that interact with both components of the native heterodimer and that these proteins would normally go undetected in the conventional, single-protein two-hybrid screens. Indeed, using this approach, we identified novel protein partners of M. tuberculosis EsxBA (MtbEsxBA). We show for the first time that EspA proteins from M. tuberculosis and M. smegmatis interact with the EsxBA heterodimer (from both species) but not with EsxA or EsxB alone. We also provide evidence for promiscuity between the different M. tuberculosis ESX apparatuses by showing that EsxBA, encoded by esx-1, can interact with Esx proteins encoded by esx-2. Taken together, our studies suggest that the WXG proteins possess similar structures and properties, regardless of the host species and the apparent biological function.Open in a separate windowFIG. 2.Sequence alignment of WXG proteins characterized in this study and the strategy used to facilitate their expression. (A) Amino acid sequence alignment of four pairs of WXG proteins. Conserved sequences are in boldface, and the signature WXG motif is indicated with asterisks. Three residues in Rv3874 (EsxB) and a single residue in Rv3875 (EsxA) are underlined; they are the sites of amino acid substitutions discussed in the text that abrogate Rv3871 interactions. (B) (Bottom) Scheme for coexpression of tandemly arranged WXG genes. (Top) The ribbon cartoon (30) shows how the two monomers are freed from the expressed fusion protein by thrombin cleavage (scissors) at the peptide tether (balls and sticks).     

Ligand recognition by A‐class Eph receptors: crystal structures of the EphA2 ligand‐binding domain and the EphA2/ephrin‐A1 complex

Ephrin (Eph) receptor tyrosine kinases fall into two subclasses (A and B) according to preferences for their ephrin ligands. All published structural studies of Eph receptor/ephrin complexes involve B‐class receptors. Here, we present the crystal structures of an A‐class complex between EphA2 and ephrin‐A1 and of unbound EphA2. Although these structures are similar overall to their B‐class counterparts, they reveal important differences that define subclass specificity. The structures suggest that the A‐class Eph receptor/ephrin interactions involve smaller rearrangements in the interacting partners, better described by a ‘lock‐and‐key’‐type binding mechanism, in contrast to the ‘induced fit’ mechanism defining the B‐class molecules. This model is supported by structure‐based mutagenesis and by differential requirements for ligand oligomerization by the two subclasses in cell‐based Eph receptor activation assays. Finally, the structure of the unligated receptor reveals a homodimer assembly that might represent EphA2‐specific homotypic cell adhesion interactions.     

Suppressive Subtractive Hybridization of and Differences in Gene Expression Content of Calcifying and Noncalcifying Cultures of Emiliania huxleyi Strain 1516

The marine coccolithophorid Emiliania huxleyi is a cosmopolitan alga intensely studied in relation to global carbon cycling, biogeochemistry, marine ecology, and biomineralization processes. The biomineralization capabilities of coccolithophorids have attracted the attention of scientists interested in exploiting this ability for the development of materials science and biomedical and biotechnological applications. Although it has been well documented that biomineralization in E. huxleyi is promoted by growth under phosphate-limited conditions, the genes and proteins that govern the processes of calcification and coccolithogenesis remain unknown. Suppressive subtractive hybridization (SSH) libraries were constructed from cultures grown in phosphate-limited and phosphate-replete media as tester and driver populations for reciprocal SSH procedures. Positive clones from each of the two libraries were randomly selected, and dot blotting was performed for the analysis of expression patterns. A total of 513 clones from the phosphate-replete library and 423 clones from the phosphate-limited library were sequenced, assembled, and compared to sequences in GenBank using BLASTX. Of the 103 differentially expressed gene fragments from the phosphate-replete library, 34% showed significant homology to other known proteins, while only 23% of the 65 differentially expressed gene fragments from the phosphate-limited library showed homology to other proteins. To further assess mRNA expression, real-time RT-PCR analysis was employed and expression profiles were generated over a 14-day time course for three clones from the phosphate-replete library and five clones from the phosphate-limited library. The fragments isolated provide the basis for future cloning of full-length genes and functional analysis.     

The ‘Densitometric Image Analysis Software’ and Its Application to Determine Stepwise Equilibrium Constants from Electrophoretic Mobility Shift Assays

Current software applications for densitometric analysis, such as ImageJ, QuantityOne (BioRad) and the Intelligent or Advanced Quantifier (Bio Image) do not allow to take the non-linearity of autoradiographic films into account during calibration. As a consequence, quantification of autoradiographs is often regarded as problematic, and phosphorimaging is the preferred alternative. However, the non-linear behaviour of autoradiographs can be described mathematically, so it can be accounted for. Therefore, the ‘Densitometric Image Analysis Software’ has been developed, which allows to quantify electrophoretic bands in autoradiographs, as well as in gels and phosphorimages, while providing optimized band selection support to the user. Moreover, the program can determine protein-DNA binding constants from Electrophoretic Mobility Shift Assays (EMSAs). For this purpose, the software calculates a chosen stepwise equilibrium constant for each migration lane within the EMSA, and estimates the errors due to non-uniformity of the background noise, smear caused by complex dissociation or denaturation of double-stranded DNA, and technical errors such as pipetting inaccuracies. Thereby, the program helps the user to optimize experimental parameters and to choose the best lanes for estimating an average equilibrium constant. This process can reduce the inaccuracy of equilibrium constants from the usual factor of 2 to about 20%, which is particularly useful when determining position weight matrices and cooperative binding constants to predict genomic binding sites. The MATLAB source code, platform-dependent software and installation instructions are available via the website http://micr.vub.ac.be.     

Proteomic profiling of aphid Macrosiphum euphorbiae responses to host-plant-mediated stress induced by defoliation and water deficit

Abiotic and biotic host-plant stress, such as desiccation and herbivory, may strongly affect sap-sucking insects such as aphids via changes in plant chemicals of insect nutritional or plant defensive value. Here, we examined (i) water deprivation and (ii) defoliation by the beetle Leptinotarsa decemlineata as stresses indirectly affecting the aphid Macrosiphum euphorbiae via its host plant Solanum tuberosum. For plant-induced stress, aphids were reared on healthy vs. continuously stressed potato for 14 days (no watering; defoliation maintained at approximately 40%). Aphid performance under stress was correlated with metabolic responses monitored by profiling of the aphid proteome. M. euphorbiae was strongly affected by water stress, as adult survival, total aphid number and biomass were reduced by 67%, 64%, and 79%, respectively. Aphids performed normally on defoliated potato, indicating that they were unaffected or able to compensate any stress induced by plant defoliation. Stressed aphid proteomes revealed 419-453 protein spots, including 27 that were modulated specifically or jointly under each kind of host-plant stress. Reduced aphid fitness on water-stressed plants mostly correlated with modulation of proteins involved in energy metabolism, apparently to conserve energy in order to prioritize survival. Despite normal performance, several aphid proteins that are known to be implicated in cell communication were modulated on defoliated plants, possibly suggesting modified aphid behaviour. The GroEL protein (or symbionin) of the endosymbiont Buchnera aphidicola was predominant under all conditions in M. euphorbiae. Its expression level was not significantly affected by aphid host-plant stresses, which is consistent with the high priority of symbiosis in stressed aphids.     

CD8 T cell help for innate antitumor immunity

Innate immunity is considered to initiate adaptive antitumor responses. We demonstrate that monoclonal CD8 T lymphocytes reactive to tumor Ag P1A on P815 mastocytoma cells provide essential "help" to NK cells for rejection of P1A-deficient tumors. RAG-deficient mice have normal NK cells but do not reject either tumor. Reconstitution of these mice with P1A-specific T cells conferred resistance to both P1A-expressing and -deficient tumor cells provided they were present at the same site. Elimination of Ag-negative tumor variants required both activated T and NK cells. Gene expression profiling of NK cells infiltrating P1A-positive tumors in mice with specific CD8 T cells demonstrated an activated effector phenotype. However, CD8 T cell help to NK cells appeared ineffective for P1A-negative variants separated from the P1A-positive tumor. Local tumor Ag-specific T cell-NK cell collaboration results in the elimination of tumor cells whether they express or not the T cell tumor Ag epitope, thus containing the emergence of tumor escape variants before metastasis.     

Dopaminergic neuron loss and up-regulation of chaperone protein mRNA induced by targeted over-expression of alpha-synuclein in mouse substantia nigra

Several transgenic mouse lines with altered alpha-synuclein expression have been developed that show a variety of Parkinson's disease-like symptoms without specific loss of dopaminergic neurons. Targeted over-expression of human alpha-synuclein using viral-vector mediated gene delivery into the substantia nigra of rats and non-human primates leads to dopaminergic cell loss and the formation of alpha-synuclein aggregates reminiscent of Lewy bodies. In the context of these recent findings, we used adeno-associated virus (AAV) to over-express wild type human alpha-synuclein in the substantia nigra of mice. We hypothesized that this over-expression would recapitulate pathological hallmarks of Parkinson's disease, creating a mouse model to further characterize the disease pathogenesis. Recombinant AAV expressing alpha-synuclein was stereotaxically injected into the substantia nigra of mice, leading to a 25% reduction of dopaminergic neurons after 24 weeks of transduction. Furthermore, examination of mRNA levels of stress-related proteins using laser capture microdissection and quantitative PCR revealed a positive correlation of Hsp27 expression with the extent of viral transduction at 4 weeks and a positive correlation of Hsp40, Hsp70 and caspase 9 with the extent of viral transduction at 24 weeks. Taken together, our findings suggest that targeted over-expression of alpha-synuclein can induce pathology at the gross anatomical and molecular level in the substantia nigra, providing a mouse model in which upstream changes in Parkinson's disease pathogenesis can be further elucidated.