Protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages.

Bacillus Calmette-Guérin (BCG), the only licensed tuberculosis vaccine, provides non-specific protection against non-tuberculosis diseases that is mediated by trained immunity, a functional reprogramming mediated by innate immune memory. Here, we present a protocol for analyzing BCG-induced trained immunity in murine bone marrow-derived macrophages (BMDMs). We describe steps for preparing BCG single bacterial suspensions, isolating BMDM cells, and the training process. This protocol can assist researchers to conveniently utilize BMDM cells to study trained immunity. For complete details on the use and execution of this protocol, please refer to Xu et al.1.

Antifungal activity of Cinnamaldehyde derivatives against fluconazole-resistant Candida albicans.

BACKGROUND: Candida albicans is an opportunistic pathogen commonly found in human mucous membranes. In light of the escalating challenge posed by antibiotic resistance of C. albicans strains worldwide, it is an urgently necessary to explore alternative therapeutic options. OBJECTIVE: This study aims to assess the efficacy of two Cinnamaldehyde derivatives, 2-Cl Cinnamaldehyde (2-Cl CA) and 4-Cl Cinnamaldehyde (4-Cl CA), against C. albicans through both in vitro experiments and in vivo murine models and to evaluate their potential as new drug candidates for treating C. albicans. METHODS AND RESULTS: The minimum inhibitory concentrations (MICs) of Cinnamaldehyde 2-Cl and 4-Cl benzene ring derivatives against C. albicans were 25 µg/mL. Time-killing experiments revealed that both Cinnamaldehyde derivatives exhibited fungicidal activity against C. albicans at concentrations of 5 MIC and 10 MIC. In the checkerboard experiment, 4-Cl CA did not show any antagonistic effect when combined with first-line antifungal drugs. Instead, it exhibited additive effects in combination with nystatin. Both 2-Cl and 4-Cl CA demonstrated inhibitory activity against C. albicans biofilm formation, especially at 8 MIC and 16 MIC concentrations. In C. albicans biofilm eradication experiments, although high drug concentrations of 2-Cl and 4-Cl CA were unable to eradicate the biofilm completely, they were still effective in killing C. albicans cells within the biofilm. Moreover, sub-inhibitory concentrations of 4-Cl CA (ranging from 5 to 20 µg/mL) significantly inhibited cell aggregation and hyphal formation. Furthermore, 4-Cl CA effectively inhibited intracellular C. albicans infection in macrophages. Lastly, the effectiveness of 4-Cl CA was evaluated in a mouse model of hematogenous disseminated candidiasis caused by C. albicans, which revealed that 4-Cl CA significantly reduced fungal burden and improved mouse survival compared to the untreated controls. CONCLUSION: The 4-Cl CA exhibited inhibitory effects against C. albicans through both in vivo and in vitro models, demonstrating its therapeutic potential as a promising new drug candidate for treating drug-resistant candidiasis albicans.

Regulatory role of Mycobacterium tuberculosis MtrA on dormancy/resuscitation revealed by a novel target gene-mining strategy.

Introduction: The unique dormancy of Mycobacterium tuberculosis plays a significant role in the major clinical treatment challenge of tuberculosis, such as its long treatment cycle, antibiotic resistance, immune escape, and high latent infection rate. Methods: To determine the function of MtrA, the only essential response regulator, one strategy was developed to establish its regulatory network according to high-quality genome-wide binding sites. Results and discussion: The complex modulation mechanisms were implied by the strong bias distribution of MtrA binding sites in the noncoding regions, and 32.7% of the binding sites were located inside the target genes. The functions of 288 potential MtrA target genes predicted according to 294 confirmed binding sites were highly diverse, and DNA replication and damage repair, lipid metabolism, cell wall component biosynthesis, cell wall assembly, and cell division were the predominant pathways. Among the 53 pathways shared between dormancy/resuscitation and persistence, which accounted for 81.5% and 93.0% of the total number of pathways, respectively, MtrA regulatory genes were identified not only in 73.6% of their mutual pathways, but also in 75.4% of the pathways related to dormancy/resuscitation and persistence respectively. These results suggested the pivotal roles of MtrA in regulating dormancy/resuscitation and the apparent relationship between dormancy/resuscitation and persistence. Furthermore, the finding that 32.6% of the MtrA regulons were essential in vivo and/or in vitro for M. tuberculosis provided new insight into its indispensability. The findings mentioned above indicated that MtrA is a novel promising therapeutic target for tuberculosis treatment since the crucial function of MtrA may be a point of weakness for M. tuberculosis.

A multistage protein subunit vaccine as BCG-booster confers protection against Mycobacterium tuberculosis infection in murine models.

The eradication of tuberculosis remains a global challenge. Despite being the only licensed vaccine, Bacillus Calmette-Guérin (BCG) confers limited protective efficacy in adults and individuals with latent tuberculosis infections (LTBI). There is an urgent need to develop novel vaccines that can enhance the protective effect of BCG. Protein subunit vaccines have garnered significant research interest due to their safety and plasticity. Based on previous studies, we selected three antigens associated with LTBI (Rv2028c, Rv2029c, Rv3126c) and fused them with an immunodominant antigen Ag85A, resulting in the construction of a multistage protein subunit vaccine named A986. We evaluated the protective effect of recombinant protein A986 adjuvanted with MPL/QS21 as a booster vaccine for BCG against Mycobacterium tuberculosis (Mtb) infection in mice. The A986 + MPL/QS21 induced the secretion of antigen-specific Th1 (IL-2+, IFN-γ+ and TNF-α+) and Th17 (IL-17A+) cytokines in CD4+ and CD8+ T cells within the lung and spleen of mice, while also increased the frequency of central memory and effector memory T cells. Additionally, it also induced the enhanced production of IgG antibodies. Compared to BCG alone, A986 + MPL/QS21 boosting significantly augmented the proliferation of antigen-specific multifunctional T cells and effectively reduced bacterial load in infected mice. Taken together, A986 + MPL/QS21 formulation induced robust antigen-specific immune responses and provided enhanced protection against Mtb infection as a booster of BCG vaccine.

Multi-omics analysis reveals that linoleic acid metabolism is associated with variations of trained immunity induced by distinct BCG strains.

Trained immunity is one of the mechanisms by which BCG vaccination confers persistent nonspecific protection against diverse diseases. Genomic differences between the different BCG vaccine strains that are in global use could result in variable protection against tuberculosis and therapeutic effects on bladder cancer. In this study, we found that four representative BCG strains (BCG-Russia, BCG-Sweden, BCG-China, and BCG-Pasteur) covering all four genetic clusters differed in their ability to induce trained immunity and nonspecific protection. The trained immunity induced by BCG was associated with the Akt-mTOR-HIF1α axis, glycolysis, and NOD-like receptor signaling pathway. Multi-omics analysis (epigenomics, transcriptomics, and metabolomics) showed that linoleic acid metabolism was correlated with the trained immunity-inducing capacity of different BCG strains. Linoleic acid participated in the induction of trained immunity and could act as adjuvants to enhance BCG-induced trained immunity, revealing a trained immunity-inducing signaling pathway that could be used in the adjuvant development.

Establishment of an in vitro model of monocyte-like THP-1 cells for trained immunity induced by bacillus Calmette-Guérin.

BACKGROUND: Mycobacteria bloodstream infections are common in immunocompromised people and usually have disastrous consequences. As the primary phagocytes in the bloodstream, monocytes and neutrophils play critical roles in the fight against bloodstream mycobacteria infections. In contrast to macrophages, the responses of monocytes infected with the mycobacteria have been less investigated. RESULTS: In this study, we first established a protocol for infection of non-adherent monocyte-like THP-1 cells (i.e. without the differentiation induced by phorbol 12-myristate 13-acetate (PMA) by bacillus Calmette-Guérin (BCG). Via the protocol, we were then capable of exploring the global transcriptomic profiles of non-adherent THP-1 cells infected with BCG, and found that NF-κB, MAPK and PI3K-Akt signaling pathways were enhanced, as well as some inflammatory chemokine/cytokine genes (e.g. CCL4, CXCL10, TNF and IL-1ß) were up-regulated. Surprisingly, the Akt-HIF-mTOR signaling pathway was also activated, which induces trained immunity. In this in vitro infection model, increased cytokine responses to lipopolysaccharides (LPS) restimulation, higher cell viability, and decreased Candida albicans loads were observed. CONCLUSIONS: We have first characterized the transcriptomic profiles of BCG-infected non-adherent THP-1 cells, and first developed a trained immunity in vitro model of the cells.

Natural products in anti-tuberculosis host-directed therapy.

Given that the disease progression of tuberculosis (TB) is primarily related to the host's immune status, it has been gradually realized that chemotherapy that targets the bacteria may never, on its own, wholly eradicate Mycobacterium tuberculosis, the causative agent of TB. The concept of host-directed therapy (HDT) with immune adjuvants has emerged. HDT could potentially interfere with infection and colonization by the pathogens, enhance the protective immune responses of hosts, suppress the overwhelming inflammatory responses, and help to attain a state of homeostasis that favors treatment efficacy. However, the HDT drugs currently being assessed in combination with anti-TB chemotherapy still face the dilemmas arising from side effects and high costs. Natural products are well suited to compensate for these shortcomings by having gentle modulatory effects on the host immune responses with less immunopathological damage at a lower cost. In this review, we first summarize the profiles of anti-TB immunology and the characteristics of HDT. Then, we focus on the rationale and challenges of developing and implementing natural products-based HDT. A succinct report of the medications currently being evaluated in clinical trials and preclinical studies is provided. This review aims to promote target-based screening and accelerate novel TB drug discovery.

A multistage Sendai virus vaccine incorporating latency-associated antigens induces protection against acute and latent tuberculosis.

One-quarter of the world's population is infected with Mycobacterium tuberculosis (Mtb). After initial exposure, more immune-competent persons develop asymptomatic latent tuberculosis infection (LTBI) but not active diseases, creates an extensive reservoir at risk of developing active tuberculosis. Previously, we constructed a novel recombinant Sendai virus (SeV)-vectored vaccine encoding two dominant antigens of Mtb, which elicited immune protection against acute Mtb infection. In this study, nine Mtb latency-associated antigens were screened as potential supplementary vaccine candidate antigens, and three antigens (Rv2029c, Rv2028c, and Rv3126c) were selected based on their immune-therapeutic effect in mice, and their elevated immune responses in LTBI human populations. Then, a recombinant SeV-vectored vaccine, termed SeV986A, that expresses three latency-associated antigens and Ag85A was constructed. In murine models, the doses, titers, and inoculation sites of SeV986A were optimized, and its immunogenicity in BCG-primed and BCG-naive mice were determined. Enhanced immune protection against the Mtb challenge was shown in both acute-infection and latent-infection murine models. The expression levels of several T-cell exhaustion markers were significantly lower in the SeV986A-vaccinated group, suggesting that the expression of latency-associated antigens inhibited the T-cell exhaustion process in LTBI infection. Hence, the multistage quarter-antigenic SeV986A vaccine holds considerable promise as a novel post-exposure prophylaxis vaccine against tuberculosis.

A topochemical reaction induced the formation of Bi2S3 micro-straws from a Bi-MOF for an ultra-long Zn storage life.

Aqueous zinc ion batteries (ZIBs) are considered as promising energy storage devices in the post-lithium-ion era, due to their high energy density, low cost, high safety, and environmental benignity, however their commercialization is hindered by the sluggish diffusion kinetics of cathode materials due to the large hydrate Zn2+ radius. In this work, we propose a unique structure inheritance strategy for preparing Bi2S3 micro-straws in which a metal-organic framework (MOF) denoted as Bi-PYDC (PYDC2- = 3,5-pyridinedicarboxylate) with a string of [Bi2O2]2+ chains is judiciously selected as the structure-directing template to induce the formation of micro-straws based on a topochemical reaction. The distinctive hollow structure significantly enhances the ionic storage kinetics. Impressively, the obtained battery exhibits an ultra-long cycle life of more than 10 000 cycles at a current density of 1 A g-1 while maintaining a capacity of more than 153.4 mA h g-1. In addition, the Zn2+ insertion/extraction mechanism of Bi2S3 micro-straws is also investigated by multiple analytical methods, revealing the involvement of Zn2+ rather than H+ in the electrochemical storage process. This work may lead a new direction for constructing high performance cathodes of Zn-ion batteries through a MOF-based structure-directing template.

Substrate DNA Promoting Binding of Mycobacterium tuberculosis MtrA by Facilitating Dimerization and Interpretation of Affinity by Minor Groove Width.

In order to deepen the understanding of the role and regulation mechanisms of prokaryotic global transcription regulators in complex processes, including virulence, the associations between the affinity and binding sequences of Mycobacterium tuberculosis MtrA have been explored extensively. Analysis of MtrA 294 diversified 26 bp binding sequences revealed that the sequence similarity of fragments was not simply associated with affinity. The unique variation patterns of GC content and periodical and sequential fluctuation of affinity contribution curves were observed along the sequence in this study. Furthermore, docking analysis demonstrated that the structure of the dimer MtrA-DNA (high affinity) was generally consistent with other OmpR family members, while Arg 219 and Gly 220 of the wing domain interacted with the minor groove. The results of the binding box replacement experiment proved that box 2 was essential for binding, which implied the differential roles of the two boxes in the binding process. Furthermore, the results of the substitution of the nucleotide at the 20th and/or 21st positions indicated that the affinity was negatively associated with the value of minor groove width precisely at the 21st position. The dimerization of the unphosphorylated MtrA facilitated by a low-affinity DNA fragment was observed for the first time. However, the proportion of the dimer was associated with the affinity of substrate DNA, which further suggested that the affinity was actually one characteristic of the stability of dimers. Based on the finding of 17 inter-molecule hydrogen bonds identified in the interface of the MtrA dimer, including 8 symmetric complementary ones in the conserved α4-ß5-α5 face, we propose that hydrogen bonds should be considered just as important as salt bridges and the hydrophobic patch in the dimerization. Our comprehensive study on a large number of binding fragments with quantitative affinity values provided new insight into the molecular mechanism of dimerization, binding specificity and affinity determination of MtrA and clues for solving the puzzle of how global transcription factors regulate a large quantity of target genes.

Advances in protein subunit vaccines against tuberculosis.

Tuberculosis (TB), also known as the "White Plague", is caused by Mycobacterium tuberculosis (Mtb). Before the COVID-19 epidemic, TB had the highest mortality rate of any single infectious disease. Vaccination is considered one of the most effective strategies for controlling TB. Despite the limitations of the Bacille Calmette-Guérin (BCG) vaccine in terms of protection against TB among adults, it is currently the only licensed TB vaccine. Recently, with the evolution of bioinformatics and structural biology techniques to screen and optimize protective antigens of Mtb, the tremendous potential of protein subunit vaccines is being exploited. Multistage subunit vaccines obtained by fusing immunodominant antigens from different stages of TB infection are being used both to prevent and to treat TB. Additionally, the development of novel adjuvants is compensating for weaknesses of immunogenicity, which is conducive to the flourishing of subunit vaccines. With advances in the development of animal models, preclinical vaccine protection assessments are becoming increasingly accurate. This review summarizes progress in the research of protein subunit TB vaccines during the past decades to facilitate the further optimization of protein subunit vaccines that may eradicate TB.

Proteinase K-pretreated ConA-based ELISA assay: a novel urine LAM detection strategy for TB diagnosis.

Objectives: Lipoarabinomannan (LAM), an abundant cell wall glycolipid of mycobacteria including Mycobacterium tuberculosis (Mtb), is a promising TB diagnostic marker. The current commercially available urine LAM assays are not sufficiently sensitive, and more novel detection strategies are urgently needed to fill the current diagnostic gap. Methods: A proteinase K-pretreated Concanavalin A (ConA)-based ELISA assay was developed. Diagnostic performance was assessed by several bacterial strains and clinical urine samples. Results: The limit of detection (LoD) of the assay against ManLAM was 6 ng/ml. The assay reacted strongly to Mtb H37Rv and M. bovis BCG, intermediately to M. smegmatis mc2155, and weakly to four non-mycobacteria pathogens. This method could distinguish TB patients from healthy controls (HCs) and close contacts (CCs) in 71 urine samples treated with proteinase K, which increases urine LAM antibody reactiveness. In TB+HIV+ and TB+HIV- patients, the sensitivity was 43.8 and 37.5%, respectively, while the specificity was 100.0%. The areas under ROC curves (AUCs) were 0.74 and 0.82, respectively. Conclusion: This study implies that ConA can be paired with antibodies to detect LAM. Proteinase K treatment could effectively enhance the sensitivity by restoring the reactiveness of antibodies to LAM.

Electron-Rich Triazine-Conjugated Microporous Polymers for the Removal of Dyes from Wastewater.

Conjugated microporous polymers (CMP) as porous functional materials have received considerable attention due to their unique structures and fascinating properties for the adsorption and degradation of dyes. Herein, a triazine-conjugated microporous polymer material with rich N-donors at the skeleton itself was successfully synthesized via the Sonogashira-Hagihara coupling by a one-pot reaction. These two polymers had Brunauer-Emmett-Teller (BET) surface areas of 322 and 435 m2g-1 for triazine-conjugated microporous polymers (T-CMP) and T-CMP-Me, respectively. Due to the porous effects and the rich N-donor at the framework, it displayed a higher removal efficiency and adsorption performance compared to cationic-type dyes and selectivity properties for (methylene blue) MB+ from a mixture solution of cationic-type dyes. Furthermore, the T-CMP-Me could quickly and drastically separate MB+ and (methyl orange) MO- from the mixed solution within a short time. Their intriguing absorption behaviors are supported by 13C NMR, UV-vis absorption spectroscopy, scanning electron microscopy, and X-ray powder diffraction studies. This work will not only improve the development of porous material varieties, but also demonstrate the adsorption or selectivity of porous materials for dyes from wastewater.

Diagnosis of extrapulmonary tuberculosis by ultrasound-guided biopsy: A retrospective comparison study.

Objective: To compare the diagnostic performance of laboratory assays on the ultrasound-guided core needle biopsy samples for diagnosis of extra-pulmonary tuberculosis (EPTB) in HIV-positive and HIV-negative patients. Methods: A total of 217 patients suspected to have EPTB underwent lesion biopsy from 2017 to 2020. Results of laboratory tests on the biopsy and non-biopsy samples were collected with clinical data for retrospective analysis of test utility. The calculated diagnostic accuracy of the tests was stratified according to the specimen types and HIV status. Results: The cohort contained 118 patients with a final positive diagnosis of extrapulmonary tuberculosis (EPTB group, 54.4%) and 99 finally diagnosed as without TB (non-EPTB group, 45.6%). The risk factor for EPTB was HIV co-infection (OR 2.22, 95% CI 1.17-4.28, p = 0.014). In biopsy samples, GeneXpert (Xpert) showed higher sensitivity (96.6% [91.6-98.7], p < 0.0001) than culture (56.1% [47.0-64.9]). Regardless of HIV status, Xpert had the highest sensitivity (>95%) and specificity (nearly 100%) of any methods. In non-biopsy samples, only T-SPOT.TB (T-SPOT) showed higher sensitivity than culture (90.9% [62.3-99.5] vs 35.3% [17.3-58.7], p = 0.0037). Furthermore, the sensitivities of Xpert were lower in non-biopsy samples (60.0% [23.1-92.9], p = 0.022) than in biopsy samples (100% [86.7-100]). Even in smear-negative biopsy samples, Xpert still had higher sensitivity than culture and retained high specificity (100% [95.7-100]). Conclusion: Superior performance of Xpert in diagnosing EPTB was observed regardless of HIV status and specimen types. Nevertheless, the biopsy samples still substantially facilitated the accurate diagnosis of extrapulmonary tuberculosis.

Trisodium citrate as a modulation additive to increase the cycling capability of a Bi2S3 cathode in a zinc-ion battery.

3D Bi2S3 materials were prepared by the trisodium citrate (Na3Cit)-assisted solvothermal method and applied to aqueous zinc ion batteries (AZIBs) to explore the effect of the electrode material morphology on the electrochemical performance. As the concentration of Na3Cit increases, the 3D assembly morphology evolves from coral-like to sphere-like to snowflake-like structures. The electrochemical test results show that the electrode materials of various morphologies possess excellent cycle life, but the specific capacity varies greatly depending on the morphology. Impressively, the Bi2S3-1.2 electrode has the best electrochemical performance, with a capacity of 203.5 mA h g-1 after 4000 charge/discharge cycles at 0.5 A g-1. Furthermore, the Bi2S3-1.2 electrode delivers an ultralong lifetime of over 10 000 cycles with a capacity of 150.2 mA h g-1 at 1 A g-1. This work demonstrates a feasible route to prepare ultra-long cycle life AZIBs.

An Updated Review on MSMD Research Globally and A Literature Review on the Molecular Findings, Clinical Manifestations, and Treatment Approaches in China.

Mendelian susceptibility to mycobacterial disease (MSMD) arises from a group of rare inherited errors of immunity that result in selective susceptibility of otherwise healthy people to clinical disease caused by low virulence strains of mycobacteria, such as Mycobacterium bovis Bacille Calmette-Guérin (BCG) and environmental mycobacteria. Patients have normal resistance to other pathogens and no overt abnormalities in routine immunological and hematological evaluations for primary immunodeficiencies. At least 19 genes and 34 clinical phenotypes have been identified in MSMD. However, there have been no systematic reports on the clinical characteristics and genetic backgrounds of MSMD in China. In this review, on the one hand, we summarize an update findings on molecular defects and immunological mechanisms in the field of MSMD research globally. On the other hand, we undertook a systematic review of PubMed (MEDLINE), the Cochrane Central Register of Controlled Trials (CENTRAL), Web of Science, EMBASE, CNKI, and Wanfang to identify articles published before Jan 23, 2022, to summarize the clinical characteristics, diagnosis, treatment, and prognosis of MSMD in China. All the English and Chinese publications were searched without any restriction on article types.

Association of in vitro fertilization with maternal and perinatal outcomes among pregnant women with active tuberculosis: A retrospective hospital-based cohort study.

Background: Study on effect of fertilization methods on maternal and perinatal outcomes with respect to TB during pregnancy was scarce. This study aimed to analyze maternal and perinatal outcomes in active TB cases after in vitro fertilization (IVF) treatment vs. normal pregnancy. Methods: Clinical data of 80 pregnant women with active TB hospitalized at Shanghai Public Health Clinical Center between June 1st, 2014 and November 30th, 2020 were extracted and retrospectively analyzed. History of receiving IVF was recorded at admission and its association with maternal and perinatal outcomes were assessed using multivariable logistic regression models with adjustment for potential confounders. Results: Of the 80 pregnant women with active TB, 28 (35.0%) received IVF treatment and 52 (65.0%) did not receive IVF treatment. After adjusting for potential confounders, receiving IVF was associated with worse maternal and perinatal outcomes, including maternal criticality (21.4 vs. 2.0%, adjusted OR = 28.3, P = 0.015), miliary TB (89.3 vs. 13.5%, adjusted OR = 75.4, P < 0.001), TB meningitis (32.1 vs. 7.7%, adjusted OR = 6.2, P = 0.010), and perinatal mortality (64.3 vs. 28.8%, adjusted OR = 9.8, P = 0.001). Conclusion: The additional risk of TB to women receiving IVF treatment is a public health challenge specific to countries with a high tuberculosis burden. Increased awareness of latent tuberculosis infection in women receiving IVF treatment is needed.