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1.
Nicotinamide phosphoribosyltransferase as a biomarker for the diagnosis of infectious pleural effusions.
Huang, J, Guo, L, Kang, HW, Lv, D, Lin, W, Li, CF, Huang, XQ, Ding, QL
Scientific reports. 2021;(1):21121
Abstract
Nicotinamide phosphoribosyltransferase (NAMPT) has been reported to be involved in infectious diseases, but it is unknown whether it plays a role in infectious pleural effusions (IPEs). We observed the levels of NAMPT in pleural effusions of different etiologies and investigated the clinical value of NAMPT in the differential diagnosis of infectious pleural effusions. A total of 111 patients with pleural effusion were enrolled in the study, including 25 parapneumonic effusions (PPEs) (17 uncomplicated PPEs, 3 complicated PPEs, and 5 empyemas), 30 tuberculous pleural effusions (TPEs), 36 malignant pleural effusions (MPEs), and 20 transudative effusions. Pleural fluid NAMPT levels were highest in the patients with empyemas [575.4 (457.7, 649.3) ng/ml], followed by those with complicated PPEs [113.5 (103.5, 155.29) ng/ml], uncomplicated PPEs [24.9 (20.2, 46.7) ng/ml] and TPEs [88 (19.4, 182.6) ng/ml], and lower in patients with MPEs [11.5 (6.5, 18.4) ng/ml] and transudative effusions [4.3 (2.6, 5.1) ng/ml]. Pleural fluid NAMPT levels were significantly higher in PPEs (P < 0.001) or TPEs (P < 0.001) than in MPEs. Moreover, Pleural fluid NAMPT levels were positively correlated with the neutrophil percentage and lactate dehydrogenase (LDH) levels and inversely correlated with glucose levels in both PPEs and TPEs, indicating that NAMPT was implicated in the neutrophil-associated inflammatory response in infectious pleural effusion. Further, multivariate logistic regression analysis showed pleural fluid NAMPT was a significant predictor distinguishing PPEs from MPEs [odds ratio (OR) 1.180, 95% confidence interval (CI) 1.052-1.324, P = 0.005]. Receiver-operating characteristic (ROC) analysis demonstrated that NAMPT was a promising diagnostic factor for the diagnosis of infectious effusions, with the areas under the curve for pleural fluid NAMPT distinguishing PPEs from MPEs, TPEs from MPEs, and IPEs (PPEs and TPEs) from NIPEs were 0.92, 0.85, and 0.88, respectively. In conclusion, pleural fluid NAMPT could be used as a biomarker for the diagnosis of infectious pleural effusions.
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The Synergistic Effects of the Glutathione Precursor, NAC and First-Line Antibiotics in the Granulomatous Response Against Mycobacterium tuberculosis.
Teskey, G, Cao, R, Islamoglu, H, Medina, A, Prasad, C, Prasad, R, Sathananthan, A, Fraix, M, Subbian, S, Zhong, L, et al
Frontiers in immunology. 2018;:2069
Abstract
Mycobacterium tuberculosis (M. tb), the causative bacterial agent responsible for tuberculosis (TB) continues to afflict millions of people worldwide. Although the human immune system plays a critical role in containing M. tb infection, elimination proves immensely more challenging. Consequently, there has been a worldwide effort to eradicate, and limit the spread of M. tb through the conventional use of first-line antibiotics. Unfortunately, with the emergence of drug resistant and multi-drug resistant strains of M. tb the archetypical antibiotics no longer provide the same ascendancy as they once did. Furthermore, when administered, these first-line antibiotics commonly present severe complications and side effects. The biological antioxidant glutathione (GSH) however, has been demonstrated to have a profound mycobactericidal effect with no reported adverse consequences. Therefore, we examined if N-Acetyl Cysteine (NAC), the molecular precursor to GSH, when supplemented in combination with suboptimal levels of standalone first-line antibiotics would be sufficient to completely clear M. tb infection within in vitro derived granulomas from healthy subjects and individuals with type 2 diabetes (T2DM). Our results revealed that by virtue of immune modulation, the addition of NAC to subprime levels of isoniazid (INH) and rifampicin (RIF) was indeed capable of inducing complete clearance of M. tb among healthy individuals.
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Detection and Quantification of Mycobacterium tuberculosis in the Sputum of Culture-Negative HIV-infected Pulmonary Tuberculosis Suspects: A Proof-of-Concept Study.
Madico, G, Mpeirwe, M, White, L, Vinhas, S, Orr, B, Orikiriza, P, Miller, NS, Gaeddert, M, Mwanga-Amumpaire, J, Palaci, M, et al
PloS one. 2016;(7):e0158371
Abstract
RATIONALE Rapid diagnosis of pulmonary tuberculosis (TB) is critical for timely initiation of treatment and interruption of transmission. Yet, despite recent advances, many patients remain undiagnosed. Culture, usually considered the most sensitive diagnostic method, is sub-optimal for paucibacillary disease. METHODS We evaluated the Totally Optimized PCR (TOP) TB assay, a new molecular test that we hypothesize is more sensitive than culture. After pre-clinical studies, we estimated TOP's per-patient sensitivity and specificity in a convenience sample of 261 HIV-infected pulmonary TB suspects enrolled into a TB diagnostic study in Mbarara, Uganda against MGIT culture, Xpert MTB/RIF and a composite reference standard. We validated results with a confirmatory PCR used for sequencing M. tuberculosis. MEASUREMENTS AND RESULTS Using culture as reference, TOP had 100% sensitivity but 35% specificity. Against a composite reference standard, the sensitivity of culture (27%) and Xpert MTB/RIF (27%) was lower than TOP (99%), with similar specificity (100%, 98% and 87%, respectively). In unadjusted analyses, culture-negative/TOP-positive patients were more likely to be older (P<0·001), female (P<0·001), have salivary sputum (P = 0·05), sputum smear-negative (P<0.001) and less advanced disease on chest radiograph (P = 0.05). M. tuberculosis genotypes identified in sputum by DNA sequencing exhibit differential growth in culture. CONCLUSIONS These findings suggest that the TOP TB assay is accurately detecting M. tuberculosis DNA in the sputum of culture-negative tuberculosis suspects. Our results require prospective validation with clinical outcomes. If the operating characteristics of the TOP assay are confirmed in future studies, it will be justified as a "TB rule out" test.
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Adenovirus type 35-vectored tuberculosis vaccine has an acceptable safety and tolerability profile in healthy, BCG-vaccinated, QuantiFERON(®)-TB Gold (+) Kenyan adults without evidence of tuberculosis.
Walsh, DS, Owira, V, Polhemus, M, Otieno, L, Andagalu, B, Ogutu, B, Waitumbi, J, Hawkridge, A, Shepherd, B, Pau, MG, et al
Vaccine. 2016;(21):2430-2436
Abstract
In a Phase 1 trial, we evaluated the safety of AERAS-402, an adenovirus 35-vectored TB vaccine candidate expressing 3 Mycobacterium tuberculosis (Mtb) immunodominant antigens, in subjects with and without latent Mtb infection. HIV-negative, BCG-vaccinated Kenyan adults without evidence of tuberculosis, 10 QuantiFERON(®)-TB Gold In-Tube test (QFT-G)(-) and 10 QFT-G(+), were randomized 4:1 to receive AERAS-402 or placebo as two doses, on Days 0 and 56, with follow up to Day 182. There were no deaths, serious adverse events or withdrawals. For 1 AERAS-402 QFT-G(-) and 1 AERAS-402 QFT-G(+) subject, there were 3 self-limiting severe AEs of injection site pain: 1 after the first vaccination and 1 after each vaccination, respectively. Two additional severe AEs considered vaccine-related were reported after the first vaccination in AERAS-402 QFT-G(+) subjects: elevated blood creatine phosphokinase and neutropenia, the latter slowly improving but remaining abnormal until study end. AERAS-402 was not detected in urine or throat cultures for any subject. In intracellular cytokine staining studies, curtailed by technical issues, we saw modest CD4+ and CD8+ T cell responses to Mtb Ag85A/b peptide pools among both QFT-G(-) and (+) subjects, with trends in the CD4+ T cells suggestive of boosting after the second vaccine dose, slightly more so in QFT-G(+) subjects. CD4+ and CD8+ responses to Mtb antigen TB10.4 were minimal. Increases in Adenovirus 35 neutralizing antibodies from screening to end of study, seen in 50% of AERAS-402 recipients, were mostly minimal. This small study confirms acceptable safety and tolerability profiles for AERAS-402, in line with other Phase 1 studies of AERAS-402, now to include QFT-G(+) subjects.
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Serum indoleamine 2,3-dioxygenase activity is associated with reduced immunogenicity following vaccination with MVA85A.
Tanner, R, Kakalacheva, K, Miller, E, Pathan, AA, Chalk, R, Sander, CR, Scriba, T, Tameris, M, Hawkridge, T, Mahomed, H, et al
BMC infectious diseases. 2014;:660
Abstract
BACKGROUND There is an urgent need for improved vaccines to protect against tuberculosis. The currently available vaccine Bacille Calmette-Guerin (BCG) has varying immunogenicity and efficacy across different populations for reasons not clearly understood. MVA85A is a modified vaccinia virus expressing antigen 85A from Mycobacterium tuberculosis which has been in clinical development since 2002 as a candidate vaccine to boost BCG-induced protection. A recent efficacy trial in South African infants failed to demonstrate enhancement of protection over BCG alone. The immunogenicity was lower than that seen in UK trials. The enzyme Indoleamine 2,3-dioxygenase (IDO) catalyses the first and rate-limiting step in the breakdown of the essential amino acid tryptophan. T cells are dependent on tryptophan and IDO activity suppresses T-cell proliferation and function. METHODS Using samples collected during phase I trials with MVA85A across the UK and South Africa we have investigated the relationship between vaccine immunogenicity and IDO using IFN-γ ELISPOT, qPCR and liquid chromatography mass spectrometry. RESULTS We demonstrate an IFN-γ dependent increase in IDO mRNA expression in peripheral blood mononuclear cells (PBMC) following MVA85A vaccination in UK subjects. IDO mRNA correlates positively with the IFN-γ ELISPOT response indicating that vaccine specific induction of IDO in PBMC is unlikely to limit the development of vaccine specific immunity. IDO activity in the serum of volunteers from the UK and South Africa was also assessed. There was no change in serum IDO activity following MVA85A vaccination. However, we observed higher baseline IDO activity in South African volunteers when compared to UK volunteers. In both UK and South African serum samples, baseline IDO activity negatively correlated with vaccine-specific IFN-γ responses, suggesting that IDO activity may impair the generation of a CD4+ T cell memory response. CONCLUSIONS Baseline IDO activity was higher in South African volunteers when compared to UK volunteers, which may represent a potential mechanism for the observed variation in vaccine immunogenicity in South African and UK populations and may have important implications for future vaccination strategies. TRIAL REGISTRATION Trials are registered at ClinicalTrials.gov; UK cohort NCT00427830, UK LTBI cohort NCT00456183, South African cohort NCT00460590, South African LTBI cohort NCT00480558.
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All-trans retinoic acid-triggered antimicrobial activity against Mycobacterium tuberculosis is dependent on NPC2.
Wheelwright, M, Kim, EW, Inkeles, MS, De Leon, A, Pellegrini, M, Krutzik, SR, Liu, PT
Journal of immunology (Baltimore, Md. : 1950). 2014;(5):2280-2290
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Abstract
A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the mechanism is unclear. In this study, we demonstrate that vitamin A-triggered antimicrobial activity against M. tuberculosis requires expression of NPC2. Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3 (1,25D3), the biologically active forms of vitamin A and vitamin D, respectively, indicates that ATRA and 1,25D3 induce mechanistically distinct antimicrobial activities. Stimulation of primary human monocytes with ATRA did not result in expression of the antimicrobial peptide cathelicidin, which is required for 1,25D3 antimicrobial activity. In contrast, ATRA triggered a reduction in the total cellular cholesterol concentration, whereas 1,25D3 did not. Blocking ATRA-induced cellular cholesterol reduction inhibits antimicrobial activity as well. Bioinformatic analysis of ATRA- and 1,25D3-induced gene profiles suggests that NPC2 is a key gene in ATRA-induced cholesterol regulation. Knockdown experiments demonstrate that ATRA-mediated decrease in total cellular cholesterol content and increase in lysosomal acidification are both dependent upon expression of NPC2. Expression of NPC2 was lower in caseous tuberculosis granulomas and M. tuberculosis-infected monocytes compared with normal lung and uninfected cells, respectively. Loss of NPC2 expression ablated ATRA-induced antimicrobial activity. Taken together, these results suggest that the vitamin A-mediated antimicrobial mechanism against M. tuberculosis requires NPC2-dependent expression and function, indicating a key role for cellular cholesterol regulation in the innate immune response.
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CD40 ligand and interferon-γ induce an antimicrobial response against Mycobacterium tuberculosis in human monocytes.
Klug-Micu, GM, Stenger, S, Sommer, A, Liu, PT, Krutzik, SR, Modlin, RL, Fabri, M
Immunology. 2013;(1):121-8
Abstract
The ability of T cells to activate antimicrobial pathways in infected macrophages is essential to host defence against many intracellular pathogens. Here, we compared the ability of two T-cell-mediated mechanisms to trigger antimicrobial responses against Mycobacterium tuberculosis in humans, CD40 activation and the release of interferon-γ (IFN-γ). Given that IFN-γ activates a vitamin D-dependent antimicrobial response, we focused on induction of the key components of this pathway. We show that activation of human monocytes via CD40 ligand (CD40L) and IFN-γ, alone, and in combination, induces the CYP27b1-hydroxylase, responsible for the conversion of 25-hydroxyvitamin D (25D) to the bioactive 1,25-dihydroxyvitamin D (1,25D), and the vitamin D receptor (VDR). The activation of the vitamin D pathway by CD40L and IFN-γ results in up-regulated expression of the antimicrobial peptides, cathelicidin and DEFB4, as well as induction of autophagy. Finally, activation of monocytes via CD40L and IFN-γ results in an antimicrobial activity against intracellular M. tuberculosis. Our data suggest that at least two parallel T-cell-mediated mechanisms, CD40L and IFN-γ, activate the vitamin D-dependent antimicrobial pathway and trigger antimicrobial activity against intracellular M. tuberculosis, thereby contributing to human host defence against intracellular infection.
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Oral intake of phenylbutyrate with or without vitamin D3 upregulates the cathelicidin LL-37 in human macrophages: a dose finding study for treatment of tuberculosis.
Mily, A, Rekha, RS, Kamal, SM, Akhtar, E, Sarker, P, Rahim, Z, Gudmundsson, GH, Agerberth, B, Raqib, R
BMC pulmonary medicine. 2013;:23
Abstract
BACKGROUND We earlier showed that 4-phenylbutyrate (PB) can induce cathelicidin LL-37 expression synergistically with 1,25-dihydroxyvitamin D3 in a lung epithelial cell line. We aimed to evaluate a therapeutic dose of PB alone or in combination with vitamin D3 for induction of LL-37 expression in immune cells and enhancement of antimycobacterial activity in monocyte-derived macrophages (MDM). METHODS Healthy volunteers were enrolled in an 8-days open trial with three doses of PB [250 mg (Group-I), 500 mg (Group-II) or 1000 mg (Group-III)] twice daily (b.d.) together with vitamin D3 {5000 IU once daily (o.d.)}, PB (500 mg b.d.) (Group-IV) or vitamin D3 (5000 IU o.d.) (Group-V), given orally for 4 days. Blood was collected on day-0, day-4 and day-8; plasma was separated, peripheral blood mononuclear cells (PBMC), non-adherent lymphocytes (NAL) and MDM were cultured. LL-37 transcript in cells and peptide concentrations in supernatant were determined by qPCR and ELISA, respectively. In plasma, 25-hydorxyvitamin D3 levels were determined by ELISA. MDM-mediated killing of Mycobacterium tuberculosis (Mtb) (H37Rv) was performed by conventional culture method. RESULTS MDM from Group-II had increased concentration of LL-37 peptide and transcript at day-4, while Group-I showed increased transcript at day-4 and day-8 compared to day-0 (p < 0.05). Both Group-I and -II exhibited higher levels of transcript on day-4 compared to Group-III and Group-V (p < 0.035). Increased induction of peptide was observed in lymphocytes from Group-II on day-4 compared to Group-I and Group-IV (p < 0.05), while Group-IV showed increased levels on day-8 compared to Group-I and Group-III (p < 0.04). Intracellular killing of Mtb on day-4 was significantly increased compared to day-0 in Group-I, -II and -V (p < 0.05). CONCLUSION The results demonstrate that 500 mg b.d. PB with 5000 IU o.d. vitamin D3 is the optimal dose for the induction of LL-37 in macrophages and lymphocytes and intracellular killing of Mtb by macrophages. Hence, this dose has potential application in the treatment of TB and is now being used in a clinical trial of adults with active pulmonary TB (NCT01580007).
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A broad profile of co-dominant epitopes shapes the peripheral Mycobacterium tuberculosis specific CD8+ T-cell immune response in South African patients with active tuberculosis.
Axelsson-Robertson, R, Loxton, AG, Walzl, G, Ehlers, MM, Kock, MM, Zumla, A, Maeurer, M
PloS one. 2013;(3):e58309
Abstract
We studied major histocompatibility complex (MHC) class I peptide-presentation and nature of the antigen-specific CD8+ T-cell response from South African tuberculosis (TB) patients with active TB. 361 MHC class I binding epitopes were identified from three immunogenic TB proteins (ESAT-6 [Rv3875], Ag85B [Rv1886c], and TB10.4 [Rv0288], including amino acid variations for Rv0288, i.e., A10T, G13D, S27N, and A71S for MHC allotypes common in a South African population (e.g., human leukocyte antigen [HLA]-A*30, B*58, and C*07). Inter-allelic differences were identified regarding the broadness of the peptide-binding capacity. Mapping of frequencies of Mycobacterium tuberculosis (M. tb) antigen-specific CD8+ T-cells using 48 different multimers, including the newly constructed recombinant MHC class I alleles HLA-B*58:01 and C*0701, revealed a low frequency of CD8+ T-cell responses directed against a broad panel of co-dominant M. tb epitopes in the peripheral circulation of most patients. The antigen-specific responses were dominated by CD8+ T-cells with a precursor-like phenotype (CD45RA+CCR7+). The data show that the CD8+ T-cell response from patients with pulmonary TB (prior to treatment) is directed against subdominant epitopes derived from secreted and non-secreted M. tb antigens and that variant, natural occurring M. tb Rv0288 ligands, have a profound impact on T-cell recognition.
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Arginine as an adjuvant to chemotherapy improves clinical outcome in active tuberculosis.
Schön, T, Elias, D, Moges, F, Melese, E, Tessema, T, Stendahl, O, Britton, S, Sundqvist, T
The European respiratory journal. 2003;(3):483-8
Abstract
Nitric oxide (NO) is involved in the host defence against tuberculosis (TB). Patients with TB exhibit increased catabolism and reduced energy intake. Thus the hypothesis for this study was that restoring a relative deficiency in the amino acid arginine, the substrate for mycobactericidal NO production, would improve the clinical outcome of TB by increasing NO production. In a randomised double-blind study, patients with smear-positive TB (n = 120) were given arginine or placebo for 4 weeks in addition to conventional chemotherapy. Primary outcomes were sputum conversion, weight gain, and clinical symptoms after week 8. Secondary outcomes were sedimentation rate and levels of NO metabolites, arginine, citrulline, and tumour necrosis factor-a. Compared with the human immunodeficiency virus (HIV)-/TB+ placebo group, the HIV-/TB+ patients in the arginine group showed significant improvement, defined as increased weight gain, higher sputum conversion rate and faster reduction of symptoms, such as cough. The arginine level increased after week 2 in the HIV-/TB+ arginine group (100.2 microM (range 90.5-109.9) versus 142.1 microM (range 114.1-170.1)) compared with the HIV-/TB+ placebo group (105.5 microM (range 93.7-117.3) versus 95.7 microM (range 82.4-108.9)). HIV seroprevalence was 52.5%. No clinical improvement or increase in serum arginine was detected in arginine supplemented HIV+/TB+ patients compared with placebo. Arginine is beneficial as an adjuvant treatment in human immunodeficiency virus-negative patients with active tuberculosis, most likely mediated by increased production of nitric oxide.