The Immune Response to Mycobacterium tuberculosis in HIV-1-Coinfected Persons.

Globally, about 36.7 million people were living with HIV infection at the end of 2015. The most frequent infection co-occurring with HIV-1 is Mycobacterium tuberculosis-374,000 deaths per annum are attributable to HIV-tuberculosis, 75% of those occurring in Africa. HIV-1 infection increases the risk of tuberculosis by a factor of up to 26 and alters its clinical presentation, complicates diagnosis and treatment, and worsens outcome. Although HIV-1-induced depletion of CD4+ T cells underlies all these effects, more widespread immune deficits also contribute to susceptibility and pathogenesis. These defects present a challenge to understand and ameliorate, but also an opportunity to learn and optimize mechanisms that normally protect people against tuberculosis. The most effective means to prevent and ameliorate tuberculosis in HIV-1-infected people is antiretroviral therapy, but this may be complicated by pathological immune deterioration that in turn requires more effective host-directed anti-inflammatory therapies to be derived.

PathIntegrate: Multivariate modelling approaches for pathway-based multi-omics data integration.

As terabytes of multi-omics data are being generated, there is an ever-increasing need for methods facilitating the integration and interpretation of such data. Current multi-omics integration methods typically output lists, clusters, or subnetworks of molecules related to an outcome. Even with expert domain knowledge, discerning the biological processes involved is a time-consuming activity. Here we propose PathIntegrate, a method for integrating multi-omics datasets based on pathways, designed to exploit knowledge of biological systems and thus provide interpretable models for such studies. PathIntegrate employs single-sample pathway analysis to transform multi-omics datasets from the molecular to the pathway-level, and applies a predictive single-view or multi-view model to integrate the data. Model outputs include multi-omics pathways ranked by their contribution to the outcome prediction, the contribution of each omics layer, and the importance of each molecule in a pathway. Using semi-synthetic data we demonstrate the benefit of grouping molecules into pathways to detect signals in low signal-to-noise scenarios, as well as the ability of PathIntegrate to precisely identify important pathways at low effect sizes. Finally, using COPD and COVID-19 data we showcase how PathIntegrate enables convenient integration and interpretation of complex high-dimensional multi-omics datasets. PathIntegrate is available as an open-source Python package.

A Phase 2A Trial of the Safety and Tolerability of Increased Dose Rifampicin and Adjunctive Linezolid, With or Without Aspirin, for Human Immunodeficiency Virus-Associated Tuberculous Meningitis: The LASER-TBM Trial.

BACKGROUND: Drug regimens that include intensified antibiotics alongside effective anti-inflammatory therapies may improve outcomes in tuberculous meningitis (TBM). Safety data on their use in combination and in the context of human immunodeficiency virus (HIV) are needed to inform clinical trial design. METHODS: We conducted a phase 2, open-label, parallel-design, randomized, controlled trial to assess the safety of high-dose rifampicin, linezolid, and high-dose aspirin in HIV-associated TBM. Participants were randomized (1.4:1:1) to 3 treatment arms (1, standard of care [SOC]; 2, SOC + additional rifampicin [up to 35 mg/kg/d] + linezolid 1200 mg/d reducing after 28 days to 600 mg/d; 3, as per arm 2 + aspirin 1000 mg/d) for 56 days, when the primary outcome of adverse events of special interest (AESI) or death was assessed. RESULTS: A total of 52 participants with HIV-associated TBM were randomized; 59% had mild disease (British Medical Research Council (MRC) grade 1) vs 39% (grade 2) vs 2% (grade 3). AESI or death occurred in 10 of 16 (63%; arm 3) vs 4 of 14 (29%; arm 2) vs 6 of 20 (30%; arm 1; P = .083). The cumulative proportion of AESI or death (Kaplan-Meier) demonstrated worse outcomes in arm 3 vs arm 1 (P = .04); however, only 1 event in arm 3 was attributable to aspirin and was mild. There was no difference in efficacy (modified Rankin scale) between arms. CONCLUSIONS: High-dose rifampicin and adjunctive linezolid can safely be added to the standard of care in HIV-associated TBM. Larger studies are required to determine whether potential toxicity associated with these interventions, particularly high-dose aspirin, is outweighed by mortality or morbidity benefit. CLINICAL TRIALS REGISTRATION: NCT03927313.

Host transcriptomic signatures of tuberculosis can predict immune reconstitution inflammatory syndrome in HIV patients.

Immune reconstitution inflammatory syndrome (IRIS) can be a complication of antiretroviral therapy (ART) in patients with advanced HIV, but its pathogenesis is uncertain. In tuberculosis (TB) endemic countries, IRIS is often associated with mycobacterial infections or Bacille-Calmette-Guerin (BCG) vaccination in children. With no predictive or confirmatory tests at present, IRIS remains a diagnosis of exclusion. We tested whether RISK6 and Sweeney3, validated immune-based blood transcriptomic signatures for TB, could predict or diagnose IRIS in HIV+ children and adults. Transcripts were measured by RT-qPCR in BCG-vaccinated children and by microarray in HIV+ adults with TB including TB meningitis (TBM). Signature scores before ART initiation and up to IRIS diagnosis were compared between participants who did or did not develop IRIS. In children, RISK6 and Sweeney3 discriminated IRIS cases from non-IRIS controls before ART, and at diagnosis. In adults with TB, RISK6 discriminated IRIS cases from controls after half-week on ART and at TB-IRIS onset. In adults with TBM, only Sweeney3 discriminated IRIS cases from controls before ART, while both signatures distinguished cases from controls at TB-IRIS onset. Parsimonious whole blood transcriptomic signatures for TB showed potential to predict and diagnose IRIS in HIV+ children and adults.

Single sample pathway analysis in metabolomics: performance evaluation and application.

BACKGROUND: Single sample pathway analysis (ssPA) transforms molecular level omics data to the pathway level, enabling the discovery of patient-specific pathway signatures. Compared to conventional pathway analysis, ssPA overcomes the limitations by enabling multi-group comparisons, alongside facilitating numerous downstream analyses such as pathway-based machine learning. While in transcriptomics ssPA is a widely used technique, there is little literature evaluating its suitability for metabolomics. Here we provide a benchmark of established ssPA methods (ssGSEA, GSVA, SVD (PLAGE), and z-score) alongside the evaluation of two novel methods we propose: ssClustPA and kPCA, using semi-synthetic metabolomics data. We then demonstrate how ssPA can facilitate pathway-based interpretation of metabolomics data by performing a case-study on inflammatory bowel disease mass spectrometry data, using clustering to determine subtype-specific pathway signatures. RESULTS: While GSEA-based and z-score methods outperformed the others in terms of recall, clustering/dimensionality reduction-based methods provided higher precision at moderate-to-high effect sizes. A case study applying ssPA to inflammatory bowel disease data demonstrates how these methods yield a much richer depth of interpretation than conventional approaches, for example by clustering pathway scores to visualise a pathway-based patient subtype-specific correlation network. We also developed the sspa python package (freely available at https://pypi.org/project/sspa/ ), providing implementations of all the methods benchmarked in this study. CONCLUSION: This work underscores the value ssPA methods can add to metabolomic studies and provides a useful reference for those wishing to apply ssPA methods to metabolomics data.

M. tuberculosis infection of human iPSC-derived macrophages reveals complex membrane dynamics during xenophagy evasion.

Xenophagy is an important cellular defence mechanism against cytosol-invading pathogens, such as Mycobacterium tuberculosis (Mtb). Activation of xenophagy in macrophages targets Mtb to autophagosomes; however, how Mtb is targeted to autophagosomes in human macrophages at a high spatial and temporal resolution is unknown. Here, we use human induced pluripotent stem cell-derived macrophages (iPSDMs) to study the human macrophage response to Mtb infection and the role of the ESX-1 type VII secretion system. Using RNA-seq, we identify ESX-1-dependent transcriptional responses in iPSDMs after infection with Mtb. This analysis revealed differential inflammatory responses and dysregulated pathways such as eukaryotic initiation factor 2 (eIF2) signalling and protein ubiquitylation. Moreover, live-cell imaging revealed that Mtb infection in human macrophages induces dynamic ESX-1-dependent, LC3B-positive tubulovesicular autophagosomes (LC3-TVS). Through a correlative live-cell and focused ion beam scanning electron microscopy (FIB SEM) approach, we show that upon phagosomal rupture, Mtb induces the formation of LC3-TVS, from which the bacterium is able to escape to reside in the cytosol. Thus, iPSDMs represent a valuable model for studying spatiotemporal dynamics of human macrophage-Mtb interactions, and Mtb is able to evade capture by autophagic compartments.

Sugar-Assisted Cryopreservation of Stem Cell-Laden Gellan Gum-Collagen Interpenetrating Network Hydrogels.

Tissue engineering involves the transplantation of stem cell-laden hydrogels as synthetic constructs to replace damaged tissues. However, their time-consuming fabrication procedures are hurdles to widespread application in clinics. Fortunately, similar to cell banking, synthetic tissues could be cryopreserved for subsequent central distribution. Here, we report the use of trehalose and gellan gum as biomacromolecules to form a cryopreservable yet directly implantable hydrogel system for adipose-derived stem cell (ADSC) delivery. Through a modified cell encapsulation method and a preincubation step, adequate cryoprotection was afforded at 0.75 M trehalose to the encapsulated ADSCs. At this concentration, trehalose demonstrated lower propensity to induce apoptosis than 10% DMSO, the current gold standard cryoprotectant. Moreover, when cultured along with trehalose after thawing, the encapsulated ADSCs retained their stem cell-like phenotype and osteogenic differentiation capacity. Taken together, this study demonstrates the feasibility of an "off-the-shelf" biomacromolecule-based synthetic tissue to be applied in widespread tissue engineering applications.

Pathway analysis in metabolomics: Recommendations for the use of over-representation analysis.

Over-representation analysis (ORA) is one of the commonest pathway analysis approaches used for the functional interpretation of metabolomics datasets. Despite the widespread use of ORA in metabolomics, the community lacks guidelines detailing its best-practice use. Many factors have a pronounced impact on the results, but to date their effects have received little systematic attention. Using five publicly available datasets, we demonstrated that changes in parameters such as the background set, differential metabolite selection methods, and pathway database used can result in profoundly different ORA results. The use of a non-assay-specific background set, for example, resulted in large numbers of false-positive pathways. Pathway database choice, evaluated using three of the most popular metabolic pathway databases (KEGG, Reactome, and BioCyc), led to vastly different results in both the number and function of significantly enriched pathways. Factors that are specific to metabolomics data, such as the reliability of compound identification and the chemical bias of different analytical platforms also impacted ORA results. Simulated metabolite misidentification rates as low as 4% resulted in both gain of false-positive pathways and loss of truly significant pathways across all datasets. Our results have several practical implications for ORA users, as well as those using alternative pathway analysis methods. We offer a set of recommendations for the use of ORA in metabolomics, alongside a set of minimal reporting guidelines, as a first step towards the standardisation of pathway analysis in metabolomics.

Rationalisation of Antifungal Properties of α-Helical Pore-Forming Peptide, Mastoparan B.

The high mortality associated with invasive fungal infections, narrow spectrum of available antifungals, and increasing evolution of antifungal resistance necessitate the development of alternative therapies. Host defense peptides are regarded as the first line of defense against microbial invasion in both vertebrates and invertebrates. In this work, we investigated the effectiveness of four naturally occurring pore-forming antimicrobial peptides (melittin, magainin 2, cecropin A, and mastoparan B) against a panel of clinically relevant pathogens, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida glabrata. We present data on the antifungal activities of the four pore-forming peptides, assessed with descriptive statistics, and their cytocompatibility with cultured human cells. Among the four peptides, mastoparan B (MB) displayed potent antifungal activity, whereas cecropin A was the least potent. We show that MB susceptibility of phylogenetically distant non-candida albicans can vary and be described by different intrinsic physicochemical parameters of pore-forming α-helical peptides. These findings have potential therapeutic implications for the design and development of safe antifungal peptide-based drugs.

LigaSure™ haemorrhoidectomy and the risk of postoperative bleeding.

AIM: LigaSure™ is an electro-surgical device that has increasingly been utilized in haemorrhoid surgery. However, recent literature has highlighted a possible increased risk of delayed postoperative bleeding following LigaSure haemorrhoidectomy (LH). We aim to evaluate the rates of postoperative bleeding following LigaSure compared to Ferguson (closed) haemorrhoidectomy (FH). METHODS: A retrospective cohort study was undertaken at our single academic safety-net county hospital from August 2016 through July 2019 evaluating patients who received FH or LH. Patient demographics, surgical data, postoperative emergency department visit for pain or bleeding within 30 days and resulting transfusion requirement, and rates of readmission and interventions within 30 days were collected. RESULTS: Sixty-one FH and 66 LH patients were identified. The groups had no difference in demographics. The LH group and FH group had similar rates of postoperative emergency department visits (29% vs. 23%, P = 0.454), as well as visits for bleeding (20% vs. 11%, P = 0.204). The average operating time was also significantly shorter with LH (14.5 min vs. 24.9 min, P ≤ 0.001). On multivariate analysis, male sex (OR 7.28, 95% CI 1.88-28.25) and haemorrhoid grade ≤2 (OR 4.64, 95% CI 1.31-16.49) were significantly associated with postoperative bleeding on multivariate analysis. Use of LH was not independently associated with postoperative bleeding risk (OR 1.89, 95% CI 0.70-5.11). CONCLUSIONS: LH and FH have similar risks for postoperative bleeding and other complications. Male sex and haemorrhoid Grades 1 or 2 may be associated with increased postoperative bleeding risk. Excisional haemorrhoidectomy should be undertaken with caution for male patients with lower internal haemorrhoid grades.

Complement pathway gene activation and rising circulating immune complexes characterize early disease in HIV-associated tuberculosis.

The transition between latent and active tuberculosis (TB) occurs before symptom onset. Better understanding of the early events in subclinical disease will facilitate the development of diagnostics and interventions that improve TB control. This is particularly relevant in the context of HIV-1 coinfection where progression of TB is more likely. In a recent study using [18F]-fluoro-2-deoxy-d-glucose positron emission/computed tomography (FDG-PET/CT) on 35 asymptomatic, HIV-1-infected adults, we identified 10 participants with radiographic evidence of subclinical disease, significantly more likely to progress than the 25 participants without. To gain insight into the biological events in early disease, we performed blood-based whole genome transcriptomic analysis on these participants and 15 active patients with TB. We found transcripts representing the classical complement pathway and Fcγ receptor 1 overabundant from subclinical stages of disease. Levels of circulating immune (antibody/antigen) complexes also increased in subclinical disease and were highly correlated with C1q transcript abundance. To validate our findings, we analyzed transcriptomic data from a publicly available dataset where samples were available in the 2 y before TB disease presentation. Transcripts representing the classical complement pathway and Fcγ receptor 1 were also differentially expressed in the 12 mo before disease presentation. Our results indicate that levels of antibody/antigen complexes increase early in disease, associated with increased gene expression of C1q and Fcγ receptors that bind them. Understanding the role this plays in disease progression may facilitate development of interventions that prevent this, leading to a more favorable outcome and may also be important to diagnostic development.

Comparative cardiovascular risk in users versus non-users of xanthine oxidase inhibitors and febuxostat versus allopurinol users.

OBJECTIVES: The aim of this study is to determine major adverse cardiovascular events (MACE) and all-cause mortality comparing between xanthine oxidase inhibitors (XOIs) and non-XOI users, and between allopurinol and febuxostat. METHODS: This is a retrospective cohort study of gout patients prescribed anti-hyperuricemic medications between 2013 and 2017 using a territory-wide administrative database. XOI users were matched 1:1 to XOI non-users using propensity scores. Febuxostat users were matched 1:3 to allopurinol users. Subgroup analyses were conducted based on colchicine use. RESULTS: Of the 13 997 eligible participants, 3607 (25.8%) were XOI users and 10 390 (74.2%) were XOI non-users. After propensity score matching, compared with non-users (n = 3607), XOI users (n = 3607) showed similar incidence of MACE (hazard ratio [HR]: 0.997, 95% CI, 0.879, 1.131; P>0.05) and all-cause mortality (HR = 0.972, 95% CI 0.886, 1.065, P=0.539). Febuxostat (n = 276) users showed a similar risk of MACE compared with allopurinol users (n = 828; HR: 0.672, 95% CI, 0.416, 1.085; P=0.104) with a tendency towards a lower risk of heart failure-related hospitalizations (HR = 0.529, 95% CI 0.272, 1.029; P=0.061). Concurrent colchicine use reduced the risk for all-cause mortality amongst XOI users (HR = 0.671, 95% 0.586, 0.768; P<0.001). CONCLUSION: In gout patients, XOI users showed similar risk of MACE and all-cause mortality compared with non-users. Compared with allopurinol users, febuxostat users showed similar MACE and all-cause mortality risks but lower heart failure-related hospitalizations.

Multi-parametric system for risk stratification in mitral regurgitation: A multi-task Gaussian prediction approach.

BACKGROUND: We hypothesized that a multi-parametric approach incorporating medical comorbidity information, electrocardiographic P-wave indices, echocardiographic assessment, neutrophil-to-lymphocyte ratio (NLR) and prognostic nutritional index (PNI) calculated from laboratory data can improve risk stratification in mitral regurgitation (MR). METHODS: Patients diagnosed with mitral regurgitation between 1 March 2005 and 30 October 2018 from a single centre were retrospectively analysed. Outcomes analysed were incident atrial fibrillation (AF), transient ischemic attack (TIA)/stroke and mortality. RESULTS: This study cohort included 706 patients, of whom 171 had normal inter-atrial conduction, 257 had inter-atrial block (IAB) and 266 had AF at baseline. Logistic regression analysis showed that age, hypertension and mean P-wave duration (PWD) were significant predictors of new-onset AF. Low left ventricular ejection fraction (LVEF), abnormal P-wave terminal force in V1 (PTFV1) predicted TIA/stroke. Age, smoking, hypertension, diabetes mellitus, hypercholesterolaemia, ischemic heart disease, secondary mitral regurgitation, urea, creatinine, NLR, PNI, left atrial diameter (LAD), left ventricular end-diastolic dimension, LVEF, pulmonary arterial systolic pressure, IAB, baseline AF and heart failure predicted all-cause mortality. A multi-task Gaussian process learning model demonstrated significant improvement in risk stratification compared to logistic regression and a decision tree method. CONCLUSIONS: A multi-parametric approach incorporating multi-modality clinical data improves risk stratification in mitral regurgitation. Multi-task machine learning can significantly improve overall risk stratification performance.

What types of real-world evidence studies do U.S. commercial health plans cite in their specialty drug coverage decisions?

PURPOSE: To examine the RWE U.S. commercial health plans cite in their specialty drug coverage decisions. METHODS: We used the Tufts Medical Center Specialty Drug Evidence and Coverage Database to identify specialty drug coverage decisions (n = 7267) issued by 17 large commercial health plans. We categorized the clinical evidence plans cited in these coverage decisions (n = 5227) as randomized controlled trials (RCTs), RWE studies, and other clinical studies (studies other than RCT or RWE study). We categorized RWE studies with respect to study type, for example, case series, studies based on medical records, and so on. We compared the frequency that plans cited different categories of RWE, cited RWE for different diseases, and cited RWE for drugs on the market for different time periods. RESULTS: RWE comprised 16% of cited clinical studies. Health plans cited RWE with different frequencies (5%-31% of the cited clinical evidence). Overall, plans cited RWE categorized as medical records most often (26% of cited RWE studies). Plans varied in the frequency they cited different RWE categories. Plans most frequently cited RWE for gastroenterological diseases (35% of clinical study citations) and least frequently for respiratory diseases (11% of clinical study citations). Plans cited RWE more for drugs that have long been on the market. CONCLUSIONS: Health plans varied with respect to the number and types of RWE studies they cited in their specialty drug coverage decisions. Plans cited RWE more often for some diseases than others, and cited more RWE for older drugs.

Electrographic indices in migraine patients: A systematic review and meta-analysis.

BACKGROUND AND AIM: Migraine patients can exhibit autonomic dysregulation, in turn leading to cardiac conduction and repolarization abnormalities. This systematic review and meta-analysis evaluated the electrocardiographic changes in migraineurs. METHOD: PubMed and Embase databases were searched for human studies using the search terms 'migraine' and 'electrocardiogram' until 15th December 2018, identifying 108 and 131 studies. RESULTS: Thirteen studies involving 667 migraineurs and 208 normal subjects included (mean age=30.7, total male percentage=19.8%) were included. A longer mean QTc interval (standard mean difference=7.89, 95% confidence interval=[3.29, 12.49], p=0.0008) and higher frequency of QTc prolongation (risk ratio [RR]=6.23, [2.86-13.58], p<0.00001), but no difference in PR-interval (SMD=4.33, [-3.90-12.56], p=0.30) were observed during migraine attacks compared to pain-free periods. P-wave dispersion was higher in migraine patients compared to controls (mean difference=3.62, [1.03-6.21], p=0.006). RR-interval were statistically indistinguishable between migraine patients and controls (SMD=0.08, [-0.65-0.81], p=0.83), or between migraineurs with and without aura (SMD=-0.03, [-0.44-0.38], p=0.89). Deep breathing ratio was significantly lower in migraineurs compared to controls (SMD=-0.27, 95% CI=[-0.46, -0.08], p=0.006) but similar between migraineurs with and without aura (SMD=-0.04, [-0.27-0.19], p=0.74). No significant difference in Valsalva ratio is found between migraineurs and controls (SMD=0.10, [-0.32-0.53], p=0.63) or between migraineurs with and without aura (SMD=-0.17, [-0.40-0.06], p=0.14). Root mean square of successive differences (RMSSD) (SMD=-0.07, [-1.10-0.95], p=0.89) and standard deviation of NN intervals (SDNN) (SMD=-0.10, [-0.61-0.41], p=0.71) did not significantly differ between migraine patients and controls. CONCLUSION: Electrocardiographic alterations are observed in migraine patients compared to controls, especially during migraine attacks.

Rapid Antibiotic Combination Testing for Carbapenem-Resistant Gram-Negative Bacteria within Six Hours Using ATP Bioluminescence.

To guide the timely selection of antibiotic combinations against carbapenem-resistant Gram-negative bacteria (CR-GNB), an in vitro test with a short turnaround time is essential. We developed an in vitro ATP bioluminescence assay to determine effective antibiotic combinations against CR-GNB within 6 h. We tested 42 clinical CR-GNB strains (14 Acinetobacter baumannii, 14 Pseudomonas aeruginosa, and 14 Klebsiella pneumoniae strains) against 74 single antibiotics and two-antibiotic combinations. Bacteria (approximately 5 log10 CFU/ml) were incubated with an antibiotic(s) at 35°C; ATP bioluminescence was measured at 6 h and 24 h; and the measurements were compared to viable counts at 24 h. Receiver operating characteristic (ROC) curves were used to determine the optimal luminescence thresholds (TRLU) for distinguishing between inhibitory and noninhibitory combinations. The areas under the 6-h and 24-h ROC curves were compared using the DeLong method. Prospective validation of the established thresholds was conducted using 18 additional CR-GNB. The predictive accuracy of TRLU for the 6-h ATP bioluminescence assay was 77.5% when all species were analyzed collectively. Predictive accuracies ranged from 73.7% to 82.7% when each species was analyzed individually. Upon comparison of the areas under the 6-h and 24-h ROC curves, the 6-h assay performed significantly better than the 24-h assay (P < 0.01). Predictive accuracy remained high upon prospective validation of the 6-h ATP assay (predictive accuracy, 79.8%; 95% confidence interval [CI], 77.6 to 81.9%), confirming the external validity of the assay. Our findings indicate that our 6-h ATP bioluminescence assay can provide guidance for prospective selection of antibiotic combinations against CR-GNB in a timely manner and may be useful in the management of CR-GNB infections.

Increased, Durable B-Cell and ADCC Responses Associated with T-Helper Cell Responses to HIV-1 Envelope in Macaques Vaccinated with gp140 Occluded at the CD4 Receptor Binding Site.

Strategies are needed to improve the immunogenicity of HIV-1 envelope (Env) antigens (Ag) for more long-lived, efficacious HIV-1 vaccine-induced B-cell responses. HIV-1 Env gp140 (native or uncleaved molecules) or gp120 monomeric proteins elicit relatively poor B-cell responses which are short-lived. We hypothesized that Env engagement of the CD4 receptor on T-helper cells results in anergic effects on T-cell recruitment and consequently a lack of strong, robust, and durable B-memory responses. To test this hypothesis, we occluded the CD4 binding site (CD4bs) of gp140 by stable cross-linking with a 3-kDa CD4 miniprotein mimetic, serving to block ligation of gp140 on CD4+ T cells while preserving CD4-inducible (CDi) neutralizing epitopes targeted by antibody-dependent cellular cytotoxicity (ADCC) effector responses. Importantly, immunization of rhesus macaques consistently gave superior B-cell (P < 0.001) response kinetics and superior ADCC (P < 0.014) in a group receiving the CD4bs-occluded vaccine compared to those of animals immunized with gp140. Of the cytokines examined, Ag-specific interleukin-4 (IL-4) T-helper enzyme-linked immunosorbent spot (ELISpot) assays of the CD4bs-occluded group increased earlier (P = 0.025) during the inductive phase. Importantly, CD4bs-occluded gp140 antigen induced superior B-cell and ADCC responses, and the elevated B-cell responses proved to be remarkably durable, lasting more than 60 weeks postimmunization.IMPORTANCE Attempts to develop HIV vaccines capable of inducing potent and durable B-cell responses have been unsuccessful until now. Antigen-specific B-cell development and affinity maturation occurs in germinal centers in lymphoid follicles through a critical interaction between B cells and T follicular helper cells. The HIV envelope binds the CD4 receptor on T cells as soluble shed antigen or as antigen-antibody complexes, causing impairment in the activation of these specialized CD4-positive T cells. We proposed that CD4-binding impairment is partly responsible for the relatively poor B-cell responses to HIV envelope-based vaccines. To test this hypothesis, we blocked the CD4 binding site of the envelope antigen and compared it to currently used unblocked envelope protein. We found superior and durable B-cell responses in macaques vaccinated with an occluded CD4 binding site on the HIV envelope antigen, demonstrating a potentially important new direction in future design of new HIV vaccines.

Inflammasome Activation Underlying Central Nervous System Deterioration in HIV-Associated Tuberculosis.

Tuberculous meningitis (TBM) is a frequent cause of meningitis in individuals with human immunodeficiency virus (HIV) infection, resulting in death in approximately 40% of affected patients. A severe complication of antiretroviral therapy (ART) in these patients is neurological tuberculosis-immune reconstitution inflammatory syndrome (IRIS), but its underlying cause remains poorly understood. To investigate the pathogenesis of TBM-IRIS, we performed longitudinal whole-blood microarray analysis of HIV-infected patients with TBM and reflected the findings at the protein level. Patients in whom TBM-IRIS eventually developed had significantly more abundant neutrophil-associated transcripts, from before development of TBM-IRIS through IRIS symptom onset. After ART initiation, a significantly higher abundance of transcripts associated with canonical and noncanonical inflammasomes was detected in patients with TBM-IRIS than in non-IRIS controls. Whole-blood transcriptome findings complement protein measurement from the site of disease, which together suggest a dominant role for the innate immune system in the pathogenesis of TBM-IRIS.

Understanding the cholesterol metabolism-perturbing effects of docosahexaenoic acid by gas chromatography-mass spectrometry targeted metabonomic profiling.

PURPOSE: Over the past few decades, docosahexaenoic acid (DHA) has gained special attention for management of cholesterol-associated metabolic disorders and neurodegenerative diseases such as Alzheimer's disease (AD) owing to its neuroprotective, anti-inflammatory and hypolipidemic properties. Several epidemiological studies have reported the effect of DHA in reducing the risk of developing AD by lowering cholesterol. Hypercholesterolemia is a pro-amyloidogenic factor influencing the enzymatic processing of amyloid-ß precursor protein (AßPP) to toxic ß-amyloid. However, the mechanism by which DHA modulates the cholesterol pathway has not been established. Thus, the objective of this study was to investigate the mechanism of regulation of cholesterol metabolism by DHA in an AßPP695 overexpressing AD cell model. METHODS: A gas chromatography/mass spectrometry method was developed and validated for the targeted profiling of 11 cholesterol metabolites in DHA-treated Chinese hamster ovary wild-type (CHO-wt) and AßPP695 overexpressing (CHO-AßPP695) cells. The differential metabolite profiles between DHA- and vehicle-treated groups were further analyzed using fold change values of the ratio of concentration of metabolites in CHO-AßPP695 to CHO-wt cells. Effect of DHA on key rate-limiting enzymatic activities within the cholesterol pathway was established using biochemical assays. RESULTS: Our results showed that DHA reduced the levels of key cholesterol anabolites and catabolites in CHO-AßPP695 cells as compared to CHO-wt cells. Further enzymatic studies revealed that the cholesterol-lowering effect of DHA was mediated by regulating HMG-CoA reductase and squalene epoxidase enzyme activities. CONCLUSION: We demonstrate for the first time the dual effects of DHA in inhibiting HMG-CoA reductase and squalene epoxidase and modulating the sterol biosynthesis axis of the cholesterol pathway in AßPP695 overexpressing AD. Our novel findings underscore the potential of DHA as a multi-target hypocholesterolemic agent for the prophylaxis of AD and other cholesterol-associated diseases.