Subsidized optimal ART for HIV-positive temporary residents of Australia improves virological outcomes: results from the Australian HIV Observational Database Temporary Residents Access Study.

INTRODUCTION: HIV-positive (HIV+) temporary residents living in Australia legally are unable to access government subsidized antiretroviral treatment (ART) which is provided via Medicare to Australian citizens and permanent residents. Currently, there is no information systematically being collected on non-Medicare eligible HIV+ patients in Australia. The objectives of this study are to describe the population recruited to the Australian HIV Observational Database (AHOD) Temporary Residents Access Study (ATRAS) and to determine the short- and long-term outcomes of receiving (subsidized) optimal ART and the impact on onwards HIV transmission. METHODS: ATRAS was established in 2011. Eligible patients were recruited via the AHOD network. Key HIV-related characteristics were recorded at baseline and prospectively. Additional visa-related information was also recorded at baseline, and updated annually. Descriptive statistics were used to describe the ATRAS cohort in terms of visa status by key demographic characteristics, including sex, region of birth, and HIV disease status. CD4 cell count (mean and SD) and the proportion with undetectable (<50 copies/ml) HIV viral load are reported at baseline, 6 and 12 months of follow-up. We also estimate the proportion reduction of onward HIV transmission based on the reduction in proportion of people with detectable HIV viral load. RESULTS: A total of 180 patients were recruited to ATRAS by June 2012, and by July 2013 39 patients no longer required ART via ATRAS, 35 of whom became eligible for Medicare-funded medication. At enrolment, 63% of ATRAS patients were receiving ART from alternative sources, 47% had an undetectable HIV viral load (<50 copies/ml) and the median CD4 cell count was 343 cells/µl (IQR: 222-479). At 12 months of follow-up, 85% had an undetectable viral load. We estimated a 75% reduction in the risk of onward HIV transmission with the improved rate of undetectable viral load. CONCLUSIONS: The immunological and virological improvements highlight the importance of supplying optimal ART to this vulnerable population. The increase in proportion with undetectable HIV viral load shows the potentially significant impact on HIV transmission in addition to the personal health benefit for each individual.

Glycosylation of sputum mucins is altered in cystic fibrosis patients.

Cystic fibrosis (CF) is characterized by chronic lung infection and inflammation, with periods of acute exacerbation causing severe and irreversible lung tissue damage. We used protein and glycosylation analysis of high-molecular mass proteins in saline-induced sputum from CF adults with and without an acute exacerbation, CF children with stable disease and preserved lung function, and healthy non-CF adult and child controls to identify potential biomarkers of lung condition. While the main high-molecular mass proteins in the sputum from all subjects were the mucins MUC5B and MUC5AC, these appeared degraded in CF adults with an exacerbation. The glycosylation of these mucins also showed reduced sulfation, increased sialylation, and reduced fucosylation in CF adults compared with controls. Despite improvements in pulmonary function after hospitalization, these differences remained. Two CF children showed glycoprotein profiles similar to those of CF adults with exacerbations and also presented with pulmonary flares shortly after sampling, while the remaining CF children had profiles indistinguishable from those of healthy non-CF controls. Sputum mucin glycosylation and degradation are therefore not inherently different in CF, and may also be useful predictive biomarkers of lung condition.

Marked differences in the structures and protein associations of lymphocyte and monocyte CD4: resolution of a novel CD4 isoform.

The structures, molecular interactions and functions of CD4 in a subset of T lymphocytes have been well characterized. The CD4 receptors of other cell types have, however, been poorly documented. We have previously shown that lymphocytes and monocytes/macrophages differ in their expression of CD4 monomers and dimers. In the present study, we have shown further significant differences. Variability in the blocking of CD4 mAb binding by sulfated polyanions indicated differences in exofacial CD4 structures. In contrast to the well-documented 55 kDa monomers in lymphocytic cells, monocytic cells were found to coexpress two monomer isoforms: the 55 kDa form and a novel 59 kDa species. Experimental uncoupling of CD4 disulfides indicated that the oxidized 55 kDa monomer could be converted to the 59 kDa form. This was achieved by chemical reduction of purified native or recombinant CD4, or in cell transfection experiments by mutation of cysteine to alanine in domain 1 (D1) (Cys16 or Cys84) and in domain 4 (D4) (Cys303 or Cys345). All of these modifications promote CD4 distension on SDS-PAGE analysis and indicate that, when CD4 inter-beta-sheet disulfides in the D1 and D4 Ig folds are disrupted, there is an unravelling of the oxidized form to an extended 59 kDa unfolded state. We hypothesize that this may be a transition-state, structural-intermediate in the formation of disulfide-linked homodimers. Also identified were CD4-tyrosine kinase dissimilarities in which lymphocyte CD4 associated with Lck, but monocyte CD4 associated with HcK. These findings show that there is complex heterogeneity in structures and interactions in the CD4 of T lymphocytes and monocytes.

Proteomic analysis of sputum from adults and children with cystic fibrosis and from control subjects.

RATIONALE: Recurrent pulmonary exacerbations are associated with progressive lung disease in cystic fibrosis (CF). Current definitions of an exacerbation, although not precisely defined, include new/worsening symptoms, declining lung function, and/or changing radiologic appearance. Early diagnosis of exacerbations by rapid noninvasive means should expedite therapeutic intervention, thereby minimizing lung damage. OBJECTIVES: To identify biomarkers of lung exacerbation for point-of-care monitoring of CF lung disease progression. METHODS: Saline-induced sputum was collected from adults with CF with an exacerbation and requiring hospitalization (FEV(1) < 60%), a subset of these adults at hospital discharge, children with stable CF and preserved lung function (FEV(1) > 70%), and control subjects (FEV(1) > 80%). Sputum was arrayed by two-dimensional electrophoresis and differentially expressed proteins were identified by proteomic analysis. MEASUREMENTS AND MAIN RESULTS: Sputum profiles from adults with CF with an exacerbation were characterized by extensive proteolytic degradation and influx of inflammation-related proteins, with some adults with CF approaching a "healthy" protein profile after hospitalization. Two children with CF showed profiles and biomarker expression resembling those of adults with an exacerbation. Levels of differentially expressed myeloperoxidase, cleaved alpha(1)-antitrypsin, IgG degradation, interleukin-8, and total protein concentration, together with their correlation to FEV(1), were statistically significant. Statistical correlation analyses indicated that changes in myeloperoxidase expression and IgG degradation were the strongest predictors of FEV(1). CONCLUSIONS: We identified extensive protein degradation and differentially expressed proteins as biomarkers of inflammation relating to pulmonary exacerbations. Prediction of exacerbation onset and more precise evaluation of the extent of resolution with treatment could be achieved by including biomarkers in standard assessment.

Optimal replication and the importance of experimental design for gel-based quantitative proteomics.

Quantitative proteomic studies, based on two-dimensional gel electrophoresis, are commonly used to find proteins that are differentially expressed between samples or groups of samples. These proteins are of interest as potential diagnostic or prognostic biomarkers, or as proteins associated with a trait. The complexity of proteomic data poses many challenges, so while experiments may reveal proteins that are differentially expressed, these are often not significant when subjected to rigorous statistical analysis. However, this can be addressed through appropriate experimental design. A good experimental design considers the impact of different sources of variation, both analytical and biological, on the statistical importance of the results. The design should address the number of samples that must be analyzed and the number of replicate gels per sample, in the context of a particular minimum difference that one is seeking to achieve. In this study, we explore the ways to improve the quality of protein expression data from 2-DE gels, and describe an approach for defining the number of samples required and the number of gels per sample. It has been developed for the simplest of situations, two groups of samples with variation at two levels: between samples and between gels. This approach will also be useful as a guide for more complex designs involving more than two groups of samples. We describe some Internet-accessible tools that can assist in the design of proteomic studies.

An immunoproteomic approach for identification of clinical biomarkers for monitoring disease: application to cystic fibrosis.

Circulating antibodies can be used to probe protein arrays of body fluids, prepared by two-dimensional gel electrophoresis, for antigenic biomarker detection. However, detected proteins, particularly low abundance antigens, often remain unidentifiable due to proteome complexity and limiting sample amounts. Using a novel enrichment approach exploiting patient antibodies for isolation of antigenic biomarkers, we demonstrate how immunoproteomic strategies can accelerate biomarker discovery. Application of this approach as a means of identifying biomarkers was demonstrated for cystic fibrosis (CF) lung disease by isolation and identification of inflammatory-associated autoantigens, including myeloperoxidase and calgranulin B from sputum of subjects with CF. The approach was also exploited for isolation of proteins expressed by the Pseudomonas aeruginosa strain PA01. Capture of PA01 antigens using circulating antibodies from CF subjects implicated in vivo expression of Pseudomonas proteins. All CF subjects screened, but not controls, were immunoreactive against immunocaptured Pseudomonas proteins, representing stress (GroES and ferric iron-binding protein HitA), immunosuppressive (thioredoxin), and alginate synthetase pathway (nucleoside-diphosphate kinase) proteins, implicating their clinical relevance as biomarkers of infection.

Marked structural and functional heterogeneity in CXCR4: separation of HIV-1 and SDF-1alpha responses.

CXCR4, the chemotactic cell receptor for SDF-1alpha, is essential for immune trafficking and HIV infection. CXCR4 is remarkably heterogeneous and the purpose of this study was to better identify the isoforms expressed by cells and compare their structure and function. We found that cells express either a predominant isoform or multiple isoforms. These were best resolved on SDS-PAGE using sucrose-gradient-fractionated, triton-insoluble, membrane extracts. We hypothesized that glycosyl modification may underpin some of this heterogeneity and that cell isoform(s) differences may underscore CXCR4's multiple cell functions. A comparison of wild-type (WT) and dual N-linked glycosylation site, N11A/N176A, mutant CXCR4 expressed in 3T3 and HEK-293 cells served to implicate variabilities in glycosylation and oligomerization in almost half of the isoforms. Immunoprecipitation of CXCR4 revealed monomer and dimer non-glycosylated forms of 34 kDa and 68 kDa from the N11A/N176A mutant, compared with glycosylated 40 kDa and 47 kDa and 73 kDa and 80 kDa forms from WT. The functional specificity of isoform action was also implicated because, despite CEMT4 cells expressing high levels of CXCR4 and 11 different isoforms, a single 83 kDa form was found to bind gp120 for HIV-1 IIIB infection. Furthermore, comparative studies found that in contrast to SDF-1alpha-responsive Nalm-6 cells that expressed similar levels of a single isoform, CEMT4 cells did not show a Ca(++) flux or a chemotactic response to SDF-1alpha. Thus, CXCR4 can differ both structurally and functionally between cells, with HIV-1 infection and chemotaxis apparently mediated by different isoforms. This separation of structure and function has implications for understanding HIV-1 entry and SDF-1alpha responses and may indicate therapeutic possibilities.

Mucin glycosylation changes in cystic fibrosis lung disease are not manifest in submucosal gland secretions.

SMG (submucosal gland) secretions are a major component of the airway surface liquid, are associated with innate immunity in the lung, and have been reported to be altered in lung disease. Changes in lung mucosal glycosylation have been reported in CF (cystic fibrosis), which may be responsible for differential bacterial binding to glycosylated components in the lung mucosa and hence increased pre-disposition to pulmonary infection. Glycoproteomic analysis was performed on SMG secretions collected from explanted bronchial tissue of subjects with severe lung disease, with and without CF, and controls without lung disease. Mucins MUC5B and MUC5AC were shown to be the dominant high-molecular-mass glycoprotein components, with a minor non-mucin glycoprotein component, gp-340, also present. Oligosaccharides containing blood-group determinants corresponding to subjects' blood type were abundant on MUC5B/MUC5AC, as were Lewis-type epitopes and their sialylated analogues, which are ligands for pathogens and leucocytes. No significant differences were found in the glycosylation of MUC5B/MUC5AC or gp-340 between CF and non-CF subjects with severe lung disease, implying that CF does not influence SMG secretion mucin glycosylation in end-stage lung disease. There were also no significant differences found in the glycosylation of these components in severe lung disease compared with non-diseased lungs. This suggests that previously reported changes in the glycosylation of respiratory glycoconjugates in CF, and other pulmonary conditions, are not due to the glycosylation of components in SMG secretions, but may involve other secretions, responses or extracellular factors.

High throughput peptide mass fingerprinting and protein macroarray analysis using chemical printing strategies.

We describe a chemical printer that uses piezoelectric pulsing for rapid, accurate, and non-contact microdispensing of fluid for proteomic analysis of immobilized protein macroarrays. We demonstrate protein digestion and peptide mass fingerprinting analysis of human plasma and platelet proteins direct from a membrane surface subsequent to defined microdispensing of trypsin and matrix solutions, hence bypassing multiple liquid-handling steps. Detection of low abundance, alkaline proteins from whole human platelet extracts has been highlighted. Membrane immobilization of protein permits archiving of samples pre-/post-analysis and provides a means for subanalysis using multiple chemistries. This study highlights the ability to increase sequence coverage for protein identification using multiple enzymes and to characterize N-glycosylation modifications using a combination of PNGase F and trypsin. We also demonstrate microdispensing of multiple serum samples in a quantitative microenzyme-linked immunosorbent assay format to rapidly screen protein macroarrays for pathogen-derived antigens. We anticipate the chemical printer will be a major component of proteomic platforms for high throughput protein identification and characterization with widespread applications in biomedical and diagnostic discovery.