The Influence Of Host Genetics And Different <i>Mycobacterium tuberculosis</i> Strains On Macrophage Functions And Clinical Outcome Of Tuberculosis Disease

Only 5-10 % of Mycobacterium tuberculosis (Mtb) infected individuals develop active pulmonary TB (PTB), and around Mtb isolates are associated with Mtb lineages and distinct host immune responses. I developed assays using ligand coated beads and Mtb reporter strain to measure the macrophage antimicrobial activities. Our assays were able to detect the variation in macrophage activities among different individuals. I investigated the macrophage antimicrobial functions and its association with different TB phenotypes. I measured the macrophage activities in 43 latent TB (LTB) and active TB (ATB) cases combining 54 PTB and 60 TBM patients. Our results showed that macrophages treated with ligands displayed higher antimicrobial activities in LTB than ATB, but no difference between PTB and TBM. Whereas, in Mtb-infected macrophages from both LTB and ATB, proteolysis was reduced due to the modulation of Mtb and there was no difference in their ability to control Mtb. Our data also indicated that the antimicrobial activities of macrophages were ligand-specific or pathway-dependent. The influence of host genetics on TB susceptibility was examined by the association of variants on phagocytic genes using a case-control study with 450 TBM, 450 PTB and 450 controls. Heterozygotes of Macrophage receptor with collagenous structure (MARCO) single nucleotide polymorphisms (SNPs) were associated with increased TB susceptibility, abnormal chest X-ray, infection by Beijing strains and also with impaired macrophage phagocytosis of ligand coated beads. The virulence of Mtb strains was examined by observing cell lysis of macrophages infected with 159 Mtb strains. Virulence phenotype was grouped as low, moderate and high. High virulence was associated with Beijing lineage, reduced TNF-α and IL-6 and increased IL-1β concentration. Overall, this thesis provides new insights into the influence of host and bacterial factors on TB susceptibility. It also provides a foundation for further studies on factors influencing TB susceptibility

Safety and efficacy of fluoxetine on functional outcome after acute stroke (AFFINITY): a randomised, double-blind, placebo-controlled trial

Background Trials of fluoxetine for recovery after stroke report conflicting results. The Assessment oF FluoxetINe In sTroke recoverY (AFFINITY) trial aimed to show if daily oral fluoxetine for 6 months after stroke improves functional outcome in an ethnically diverse population. Methods AFFINITY was a randomised, parallel-group, double-blind, placebo-controlled trial done in 43 hospital stroke units in Australia (n=29), New Zealand (four), and Vietnam (ten). Eligible patients were adults (aged ≥18 years) with a clinical diagnosis of acute stroke in the previous 2–15 days, brain imaging consistent with ischaemic or haemorrhagic stroke, and a persisting neurological deficit that produced a modified Rankin Scale (mRS) score of 1 or more. Patients were randomly assigned 1:1 via a web-based system using a minimisation algorithm to once daily, oral fluoxetine 20 mg capsules or matching placebo for 6 months. Patients, carers, investigators, and outcome assessors were masked to the treatment allocation. The primary outcome was functional status, measured by the mRS, at 6 months. The primary analysis was an ordinal logistic regression of the mRS at 6 months, adjusted for minimisation variables. Primary and safety analyses were done according to the patient's treatment allocation. The trial is registered with the Australian New Zealand Clinical Trials Registry, ACTRN12611000774921. Findings Between Jan 11, 2013, and June 30, 2019, 1280 patients were recruited in Australia (n=532), New Zealand (n=42), and Vietnam (n=706), of whom 642 were randomly assigned to fluoxetine and 638 were randomly assigned to placebo. Mean duration of trial treatment was 167 days (SD 48·1). At 6 months, mRS data were available in 624 (97%) patients in the fluoxetine group and 632 (99%) in the placebo group. The distribution of mRS categories was similar in the fluoxetine and placebo groups (adjusted common odds ratio 0·94, 95% CI 0·76–1·15; p=0·53). Compared with patients in the placebo group, patients in the fluoxetine group had more falls (20 [3%] vs seven [1%]; p=0·018), bone fractures (19 [3%] vs six [1%]; p=0·014), and epileptic seizures (ten [2%] vs two [<1%]; p=0·038) at 6 months. Interpretation Oral fluoxetine 20 mg daily for 6 months after acute stroke did not improve functional outcome and increased the risk of falls, bone fractures, and epileptic seizures. These results do not support the use of fluoxetine to improve functional outcome after stroke

Effects of Size and Surface Properties of Nanodiamonds on the Immunogenicity of Plant-Based H5 Protein of A/H5N1 Virus in Mice

Nanodiamond (ND) has recently emerged as a potential nanomaterial for nanovaccine development. Here, a plant-based haemagglutinin protein (H5.c2) of A/H5N1 virus was conjugated with detonation NDs (DND) of 3.7 nm in diameter (ND4), and high-pressure and high-temperature (HPHT) oxidative NDs of ~40–70 nm (ND40) and ~100–250 nm (ND100) in diameter. Our results revealed that the surface charge, but not the size of NDs, is crucial to the protein conjugation, as well as the in vitro and in vivo behaviors of H5.c2:ND conjugates. Positively charged ND4 does not effectively form stable conjugates with H5.c2, and has no impact on the immunogenicity of the protein both in vitro and in vivo. In contrast, the negatively oxidized NDs (ND40 and ND100) are excellent protein antigen carriers. When compared to free H5.c2, H5.c2:ND40, and H5.c2:ND100 conjugates are highly immunogenic with hemagglutination titers that are both 16 times higher than that of the free H5.c2 protein. Notably, H5.c2:ND40 and H5.c2:ND100 conjugates induce over 3-folds stronger production of both H5.c2-specific-IgG and neutralizing antibodies against A/H5N1 than free H5.c2 in mice. These findings support the innovative strategy of using negatively oxidized ND particles as novel antigen carriers for vaccine development, while also highlighting the importance of particle characterization before use

LTA4H genotype is associated with susceptibility to bacterial meningitis but is not a critical determinant of outcome.

Adjunctive dexamethasone saves lives in the treatment of tuberculous meningitis but this response is influenced by the patient's LTA4H genotype. Despite less certain benefit, adjunctive dexamethasone is also frequently used in the treatment of pyogenic bacterial meningitis, but the influence of LTA4H genotype on outcomes has not been previously investigated. We genotyped the LTA4H promoter region SNP (rs17525495) in 390 bacterial meningitis patients and 751 population controls. rs17525495 was associated with susceptibility to bacteriologically confirmed bacterial meningitis (P = 0.01, OR 1.27 95% confidence interval [CI] 1.05-1.54) but did not influence clinical presentation, disease severity or survival following dexamethasone treatment

<i>LTA4H</i> genotypes, dexamethasone and survival in bacterial meningitis patients.

<p>Kaplan-Meier estimates of survival among BM patients stratified by their rs17525495 genotype in, (A) all BM patients (bacterially confirmed and probable), (B) all BM patients who received placebo, (C) all BM patients who received dexamethasone (D) definite BM patients (bacterially confirmed), (E) definite BM patients who received placebo and (F) definite BM patients who received dexamethasone. Solid line denotes TT, dash line denotes CT, and dot-dash line denotes CC genotype group. Comparisons of survival between genotypes were based on Cox regression models adjusted for the randomized treatment group</p

rs17525495 is associated with susceptibility to bacterial meningitis.

<p>^<i>a</i>frequency</p><p>^<i>b</i>cytosine</p><p>^<i>c</i>thymine</p><p>^<i>d</i>additive model</p><p>^<i>e</i>odds ratio (95% confidence interval)</p><p>^<i>f</i>general model; a genotypic model where one genotype group (e.g. CC group) is baseline</p><p>^<i>g</i>estimates of odds for general model, comparing baseline (CC) to CT</p><p>^<i>h</i>estimates of odds for general model comparing baseline (CC) to TT</p><p>^<i>i</i>recessive model</p><p>^<i>j</i>Hardy Weinberg Equilibrium</p><p>^<i>k</i>definite. NB: data for dominant and heterozygote advantage model were not significant and are not shown here.</p><p>rs17525495 is associated with susceptibility to bacterial meningitis.</p

FLASH-TB: an Application of Next-Generation CRISPR to Detect Drug Resistant Tuberculosis from Direct Sputum

Offering patients with tuberculosis (TB) an optimal and timely treatment regimen depends on the rapid detection of Mycobacterium tuberculosis (Mtb) drug resistance from clinical samples. Finding Low Abundance Sequences by Hybridization (FLASH) is a technique that harnesses the efficiency, specificity, and flexibility of the Cas9 enzyme to enrich targeted sequences. Here, we used FLASH to amplify 52 candidate genes probably associated with resistance to first- and second-line drugs in the Mtb reference strain (H37Rv), then detect drug resistance mutations in cultured Mtb isolates, and in sputum samples. 92% of H37Rv reads mapped to Mtb targets, with 97.8% of target regions covered at a depth ≥ 10X. Among cultured isolates, FLASH-TB detected the same 17 drug resistance mutations as whole genome sequencing (WGS) did, but with much greater depth. Among the 16 sputum samples, FLASH-TB increased recovery of Mtb DNA compared with WGS (from 1.4% [IQR 0.5-7.5] to 33% [IQR 4.6-66.3]) and average depth reads of targets (from 6.3 [IQR 3.8-10.5] to 1991 [IQR 254.4-3623.7]). FLASH-TB identified Mtb complex in all 16 samples based on IS1081 and IS6110 copies. Drug resistance predictions for 15/16 (93.7%) clinical samples were highly concordant with phenotypic DST for isoniazid, rifampicin, amikacin, and kanamycin [15/15 (100%)], ethambutol [12/15 (80%)] and moxifloxacin [14/15 (93.3%)]. These results highlighted the potential of FLASH-TB for detecting Mtb drug resistance from sputum samples

Human genetic variation in VAC14 regulates Salmonella invasion and typhoid fever through modulation of cholesterol.

Risk, severity, and outcome of infection depend on the interplay of pathogen virulence and host susceptibility. Systematic identification of genetic susceptibility to infection is being undertaken through genome-wide association studies, but how to expeditiously move from genetic differences to functional mechanisms is unclear. Here, we use genetic association of molecular, cellular, and human disease traits and experimental validation to demonstrate that genetic variation affects expression of VAC14, a phosphoinositide-regulating protein, to influence susceptibility to Salmonella enterica serovar Typhi (S Typhi) infection. Decreased VAC14 expression increased plasma membrane cholesterol, facilitating Salmonella docking and invasion. This increased susceptibility at the cellular level manifests as increased susceptibility to typhoid fever in a Vietnamese population. Furthermore, treating zebrafish with a cholesterol-lowering agent, ezetimibe, reduced susceptibility to S Typhi. Thus, coupling multiple genetic association studies with mechanistic dissection revealed how VAC14 regulates Salmonella invasion and typhoid fever susceptibility and may open doors to new prophylactic/therapeutic approaches

Tryptophan metabolism determines outcome in tuberculous meningitis: a targeted metabolomic analysis

Background: Cellular metabolism is critical for the host immune function against pathogens, and metabolomic analysis may help understand the characteristic immunopathology of tuberculosis. We performed targeted metabolomic analyses in a large cohort of patients with tuberculous meningitis (TBM), the most severe manifestation of tuberculosis, focusing on tryptophan metabolism. Methods: We studied 1069 Indonesian and Vietnamese adults with TBM (26.6% HIV-positive), 54 non-infectious controls, 50 with bacterial meningitis, and 60 with cryptococcal meningitis. Tryptophan and downstream metabolites were measured in cerebrospinal fluid (CSF) and plasma using targeted liquid chromatography–mass spectrometry. Individual metabolite levels were associated with survival, clinical parameters, CSF bacterial load and 92 CSF inflammatory proteins. Results: CSF tryptophan was associated with 60-day mortality from TBM (hazard ratio [HR] = 1.16, 95% confidence interval [CI] = 1.10–1.24, for each doubling in CSF tryptophan) both in HIV-negative and -positive patients. CSF tryptophan concentrations did not correlate with CSF bacterial load nor CSF inflammation but were negatively correlated with CSF interferon-gamma concentrations. Unlike tryptophan, CSF concentrations of an intercorrelating cluster of downstream kynurenine metabolites did not predict mortality. These CSF kynurenine metabolites did however correlate with CSF inflammation and markers of blood–CSF leakage, and plasma kynurenine predicted death (HR 1.54, 95% CI = 1.22–1.93). These findings were mostly specific for TBM, although high CSF tryptophan was also associated with mortality from cryptococcal meningitis. Conclusions: TBM patients with a high baseline CSF tryptophan or high systemic (plasma) kynurenine are at increased risk of death. These findings may reveal new targets for host-directed therapy. Funding: This study was supported by National Institutes of Health (R01AI145781) and the Wellcome Trust (110179/Z/15/Z and 206724/Z/17/Z)