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.

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.

A Rare Case of Craniocervical Penetrating Injury by a Steel Bar.

RATIONALE: Non-missile penetrating injuries caused by foreign bodies, such as knives or sharp wood, are infrequent. We report a 49-year-old male suffering from severe craniocervical penetrating injury by a steel bar was successfully treated by surgery. CHIEF COMPLAINT: The male patient was a 49-year-old builder. Although working on the construction site, an approximately 60 cm steel bar penetrated the patient's brain vertically through the left top of the head presenting with unconsciousness and intermittent irritability. DIAGNOSIS: Computed tomography of the head showed the entrance and exit of the skull damaged by the steel bar. Three-dimensional reconstruction showed that the steel bar entered the skull from the posterior left coronal suture and penetrated the ipsilateral occipital bone, about 5 cm into the neck soft tissue. INTERVENTION: We successfully performed the operation and removed the steel bar. OUTCOMES: The patient was followed up for 5 years; muscle strength returned to normal. LESSONS: Penetrating injuries caused by steel bars are rare, which always cause severe intracranial injury combined with peripheral tissue injury, by sharing our experience in the treatment of this rare case, we hope to provide a reference for similar injuries in the future.

Compound Cocktail Inhibits Influenza Viral Pneumonia via Phospholipase Cγ1 Phosphorylation-Related Necroptosis and Partial Autophagy in Natural Killer Cells.

Influenza viral infections are prone to global outbreaks and cause pneumonia in affected populations. High morbidity and mortality caused by pneumonia occur during an influenza pandemic. Antivirals or control of inflammation is the primary means of influenza treatment. A compound cocktail composed of arctiin, daidzein, glycyrrhizic acid, and liquiritin inhibited mouse pneumonia resulting from a PR8 viral infection and caused a weight gain after oral administration. Natural killer cell activating receptors, both Ly49D and Ly49H in the lungs, were increased in the treatment in mice. In H3N2 virus-infected natural killer-92MI cells, the cocktail treatment had different effects on phosphorylation sites of phospholipase Cγ1 (PLCγ1) and killed infected cells through necroptosis or late apoptosis, in which RIP3 was increased and both caspase-3 and phosphorylated-JNK in the cells were downregulated. Acid phosphatase activity in viral-infected natural killer-92MI cells was induced by the compound cocktail treatment, which could be related to the p62 decrease in natural killer-92MI cells. In addition, an autophagic flux induction was observed in alveolar basal epithelial cells (A549). Protein p65, but not phosphorylated-p65, was significantly decreased by the treatment. Our results indicate that the compound cocktail strengthened the phosphorylation of PLCγ1-related necroptosis and partial autophagy in natural killer cells, which could yield an inhibitory effect on viral pneumonia in influenza.

Oxidation of dCTP contributes to antibiotic lethality in stationary-phase mycobacteria.

Growing evidence shows that generation of reactive oxygen species (ROS) derived from antibiotic-induced metabolic perturbation contribute to antibiotic lethality. However, our knowledge of the mechanisms by which antibiotic-induced oxidative stress actually kills cells remains elusive. Here, we show that oxidation of dCTP underlies ROS-mediated antibiotic lethality via induction of DNA double-strand breaks (DSBs). Deletion of mazG-encoded 5-OH-dCTP-specific pyrophosphohydrolase potentiates antibiotic killing of stationary-phase mycobacteria, but did not affect antibiotic efficacy in exponentially growing cultures. Critically, the effect of mazG deletion on potentiating antibiotic killing is associated with antibiotic-induced ROS and accumulation of 5-OH-dCTP. Independent lines of evidence presented here indicate that the increased level of DSBs observed in the ΔmazG mutant is a dead-end event accounting for enhanced antibiotic killing. Moreover, we provided genetic evidence that 5-OH-dCTP is incorporated into genomic DNA via error-prone DNA polymerase DnaE2 and repair of 5-OH-dC lesions via the endonuclease Nth leads to the generation of lethal DSBs. This work provides a mechanistic view of ROS-mediated antibiotic lethality in stationary phase and may have broad implications not only with respect to antibiotic lethality but also to the mechanism of stress-induced mutagenesis in bacteria.

The Role of KLRG1 in Human CD4+ T-Cell Immunity Against Tuberculosis.

Background: KLRG1 is a marker of terminally differentiated CD8+ T cells in viral infection, but its role in human Mycobacterium tuberculosis infection remains elusive. Methods: A set of cohorts of patients with tuberculosis was designed, and the expression profiles and functions of KLRG1+CD4+ T cells were determined with and without antibody blocking. Results: KLRG1 expression on CD4+ T cells was significantly increased in patients with active tuberculosis, compared with healthy controls and patients without tuberculosis. Upon M. tuberculosis-specific stimulation, the ability to secrete interferon γ, interleukin 2, and tumor necrosis factor α was significantly greater in KLRG1-expressing CD4+ T cells than in their KLRG-negative counterparts and was accompanied by a decreased proportion of regulatory T cells and increased Akt signaling. However, KLRG1-expressing CD4+ T cells had a shorter life-span, which was associated with a higher apoptosis rate but a similar proliferative response. Blockade of KLRG1 signaling significantly enhanced interferon γ and interleukin 2 secretion without affecting either cell apoptosis or multiplication. Addition of a specific Akt inhibitor prevented this increased cytokine response, implicating the Akt signaling pathway. Conclusions: Our study delineated the profile of KLRG1+CD4+ T cells in patients with tuberculosis and suggests that M. tuberculosis infection drives CD4+ T cells to acquire increased effector function in a terminally differentiated state, which is restrained by KLRG1 via KLRG1/Akt signaling pathway.

Sendai Virus Mucosal Vaccination Establishes Lung-Resident Memory CD8 T Cell Immunity and Boosts BCG-Primed Protection against TB in Mice.

Accumulating evidence has shown the protective role of CD8+ T cells in vaccine-induced immunity against Mycobacterium tuberculosis (Mtb) despite controversy over their role in natural immunity. However, the current vaccine BCG is unable to induce sufficient CD8+ T cell responses, especially in the lung. Sendai virus, a respiratory RNA virus, is here engineered firstly as a novel recombinant anti-TB vaccine (SeV85AB) that encodes Mtb immuno-dominant antigens, Ag85A and Ag85B. A single mucosal vaccination elicited potent antigen-specific T cell responses and a degree of protection against Mtb challenge similar to the effect of BCG in mice. Depletion of CD8+ T cells abrogated the protective immunity afforded by SeV85AB vaccination. Interestingly, only SeV85AB vaccination induced high levels of lung-resident memory CD8+ T (TRM) cells, and this led to a rapid and strong recall of antigen-specific CD8+ T cell responses against Mtb challenge infection. Furthermore, when used in a BCG prime-SeV85AB boost strategy, SeV85AB vaccine significantly enhanced protection above that seen after BCG vaccination alone. Our findings suggest that CD8+ TRM cells that arise in lungs responding to this mucosal vaccination might help to protect against TB, and SeV85AB holds notable promise to improve BCG's protective efficacy in a prime-boost immunization regimen.

Variable Virulence and Efficacy of BCG Vaccine Strains in Mice and Correlation With Genome Polymorphisms.

Bacille Calmette-Guérin (BCG), an attenuated strain of Mycobacterium bovis, is the only vaccine available for tuberculosis (TB) control. However, BCG is not an ideal vaccine and has two major limitations: BCG exhibits highly variable effectiveness against the development of TB both in pediatric and adult populations and can cause disseminated BCG disease in immunocompromised individuals. BCG comprises a number of substrains that are genetically distinct. Whether and how these genetic differences affect BCG efficacy remains largely unknown. In this study, we performed comparative analyses of the virulence and efficacy of 13 BCG strains, representing different genetic lineages, in SCID and BALB/c mice. Our results show that BCG strains of the DU2 group IV (BCG-Phipps, BCG-Frappier, BCG-Pasteur, and BCG-Tice) exhibit the highest levels of virulence, and BCG strains of the DU2 group II (BCG-Sweden, BCG-Birkhaug) are among the least virulent group. These distinct levels of virulence may be explained by strain-specific duplications and deletions of genomic DNA. There appears to be a general trend that more virulent BCG strains are also more effective in protection against Mycobacterium tuberculosis challenge. Our findings have important implications for current BCG vaccine programs and for future TB vaccine development.

Electrochemical construction of three-dimensional porous Mn3O4 nanosheet arrays as an anode for the lithium ion battery.

Three-dimensional (3D) porous Mn3O4 nanosheet arrays were constructed via an electrodeposition followed by high temperature annealing using 3D porous Cu, prepared by a facile electroless plating method, as the substrate. The 3D pores and voids between the nanosheet arrays were able to provide rapid ion transfer channels, as well as accommodating the volumetric changes of Mn3O4 during the electrochemical cycling. Electrons can directly exchange between the substrate and the nanosheet units, avoiding curving and the long transfer distance in conventional electrodes constructed using casting technology. Furthermore, the nanosheets were transformed into the architecture with smaller sub-nanosheets on the pristine nanosheets after 1 cycle, facilitating ion transferring, and were thoroughly transformed into smaller sub-nanosheets after 1000 cycles but without obvious exfoliation, assuring good electrical contact between the active particles and substrate. Based on the above unique characteristics, the 3D porous Mn3O4 nanosheet arrays could be directly used as a binder-free and conductive-agent-free electrode to deliver ultrahigh electrochemical performance that is much better than achieved in previous reports. The first reversible capacity was 1166.3 mA h g(-1) and remained 667.9 mA h g(-1) after 1000 cycles at 1.0 A g(-1). Also, the reversible capacities at high current densities of 10.0 A g(-1) and 20.0 A g(-1) remained high at 416.1 and 216.7 mA h g(-1), respectively.

Mycobacterial MazG safeguards genetic stability via housecleaning of 5-OH-dCTP.

Generation of reactive oxygen species and reactive nitrogen species in phagocytes is an important innate immune response mechanism to eliminate microbial pathogens. It is known that deoxynucleotides (dNTPs), the precursor nucleotides to DNA synthesis, are one group of the significant targets for these oxidants and incorporation of oxidized dNTPs into genomic DNA may cause mutations and even cell death. Here we show that the mycobacterial dNTP pyrophosphohydrolase MazG safeguards the bacilli genome by degrading 5-OH-dCTP, thereby, preventing it from incorporation into DNA. Deletion of the (d)NTP pyrophosphohydrolase-encoding mazG in mycobacteria leads to a mutator phenotype both under oxidative stress and in the stationary phase of growth, resulting in increased CG to TA mutations. Biochemical analyses demonstrate that mycobacterial MazG can efficiently hydrolyze 5-OH-dCTP, an oxidized nucleotide that induces CG to TA mutation upon incorporation by polymerase. Moreover, chemical genetic analyses show that direct incorporation of 5-OH-dCTP into mazG-null mutant strain of Mycobacterium smegmatis (Msm) leads to a dose-dependent mutagenesis phenotype, indicating that 5-OH-dCTP is a natural substrate of mycobacterial MazG. Furthermore, deletion of mazG in Mycobacterium tuberculosis (Mtb) leads to reduced survival in activated macrophages and in the spleen of infected mice. This study not only characterizes the mycobacterial MazG as a novel pyrimidine-specific housecleaning enzyme that prevents CG to TA mutation by degrading 5-OH-dCTP but also reveals a genome-safeguarding mechanism for survival of Mtb in vivo.

Enhanced protective efficacy against Mycobacterium tuberculosis afforded by BCG prime-DNA boost regimen in an early challenge mouse model is associated with increased splenic interleukin-2-producing CD4 T-cell frequency post-vaccination.

The development of improved vaccines and vaccination strategies against Mycobacterium tuberculosis has been hindered by a limited understanding of the immune correlates of anti-tuberculosis protective immunity. Simple measurement of interferon-γ frequency or production per se does not provide adequate prediction of immune protection. In this study, we examined the relationship between T-cell immune responses and protective efficacy conferred by the heterologous vaccination strategy, bacillus Calmette-Guérin (BCG) prime-Ag85A DNA boost (B/D), in an early challenge mouse model of pulmonary tuberculosis. The results demonstrated that mice vaccinated with the B/D regimen had a significantly reduced bacillary load compared with BCG-vaccinated mice, and the reduction in colony-forming units was associated with decreased pathology and lower levels of inflammatory cytokines in the infected lungs. Further analysis of immunogenicity showed that the superior protection afforded by the B/D regimen was associated with significantly increased frequency of splenic interleukin-2 (IL-2) -producing CD4 T cells and increased IL-2 production when measured as integrated mean fluorescence intensity post-vaccination as well. These data suggest that measurement of elevated frequency of IL-2-producing CD4 T cells or IL-2 production in the spleens of vaccinated mice can predict vaccine efficacy, at least in the B/D strategy, and add to the accumulating body of evidence suggesting that BCG prime-boost strategies may be a useful approach to the control of M. tuberculosis infection.

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.

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.

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.

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.

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.