PANoptosis is a recently identified, inflammatory programmed cell death pathway that amalgamates features of pyroptosis, apoptosis, and necroptosis, executed via multiprotein complexes called PANoptosomes. Increasing evidence highlights PANoptosis has emerged as a potentially pivotal factor in the pathogenesis of several diseases, including viral and bacterial infections and malignancies. Given its extensive pathophysiological relevance, this review systematically explores the conceptual underpinnings, molecular mechanisms, and structural composition of PANoptosome. Special attention is devoted to elucidating the pathophysiological interactions between PANoptosis and tuberculosis (TB), with the aim of developing integrated diagnostic-therapeutic strategies and targeted pharmaceutical innovations for TB. Tuberculosis, a respiratory infectious disease caused by Mycobacterium tuberculosis(M.tb), continues to pose a significant global health challenge.

Non-tuberculous mycobacterial pulmonary disease (NTM-PD) poses significant diagnostic challenges due to its clinical similarity to pulmonary tuberculosis (PTB). This study aimed to compare the clinical characteristics, immune status and lower respiratory tract microbiome profiles of NTM-PD and PTB patients. A total of 113 NTM-PD patients and 105 PTB patients were enrolled. The clinical features, laboratory parameters, and comorbidity profiles were analyzed. Bronchoalveolar lavage fluid (BALF) samples were subjected to bacterial culture and targeted next-generation sequencing (tNGS) for microbiome characterization. Microbiome distributions were then compared across disease groups, host characteristics, immune cell subsets, and NTM species. Compared with PTB patients, NTM-PD patients were older and exhibited lower rates of smoking, alcohol use, interferon-gamma release assay (IGRA) positivity, red blood cell (RBC) count, hemoglobin (Hb) and albumin levels, but a higher erythrocyte sedimentation rate (ESR). Hemoptysis, bronchiectasis, and chronic obstructive pulmonary disease (COPD) were more common among NTM-PD patients, while lymphadenopathy and diabetes were more frequent among PTB patients. BALF microbiome analysis revealed distinct profiles: Pseudomonas aeruginosa and Aspergillus spp. were more frequently detected in NTM-PD patients, while Neisseria spp. and Streptococcus viridans predominated in PTB patients. Reduced CD4⁺ T cell counts were associated with a higher detection rate of Pseudomonas aeruginosa and Candida spp. in NTM-PD patients. The most prevalent NTM species were the M. avium complex (MAC) (62.37

BACKGROUND:The global tuberculosis (TB) epidemic remains severe. We aimed to develop a therapeutic DNA vaccine as an adjunct to TB treatment to improve efficacy. METHODS:The W545 DNA vaccine was constructed using the M. tuberculosis (MTB) antigens Ag85A and Rv1419, integrated with epitopes from the Ag85B, Rv3407, and Rv2628. Bioinformatics tools were used to predict and analyze the physicochemical properties, structure modelling and molecular docking, epitopes (HTL, CTL, and B-cell), safety, population coverage, and simulated immunization of the W545 vaccine protein. Animal studies were then performed to evaluate the vaccine's immunogenicity by measuring Th1-type immune responses (IFN-γ, IL-2) and IgG antibody levels, as well as its therapeutic efficacy in reducing lung inflammation and pathological damage in a murine TB model. RESULTS:The vaccine protein is a 70 kDa hydrophilic protein with a half-life of 30 h, an instability index of 43.33, and strong affinity to Toll-like receptor (TLR) 2 and TLR4. It contains 397 helper T cell (HTL) epitopes, 248 cytotoxic T cell (CTL) epitopes, and 27 B cell epitopes, with broad population coverage (global: 99.7 %, Chinese: 97.6 %). The W545 vaccine significantly induced a Th1-type immune response, producing high levels of IFN-γ (5.38 pg/ml ± 0.89 pg/ml) and IgG antibodies (OD450: 0.13 ± 0.06). It also reduced the lung weight index, tissue lesions, and severity in the murine TB model. CONCLUSION:The W545 DNA vaccine effectively induces a Th1-type immune response, alleviates pathological damage, and demonstrates potential as an immunotherapeutic agent. Bioinformatics analysis provides valuable guidance for vaccine design and optimization.

Tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a major infectious disease worldwide. Despite the availability of anti-TB drugs, the emergence of drug resistance, the need for prolonged treatment duration and the occurrence of side effects highlight the urgent need for new therapeutic strategies. The c-Jun N-terminal kinase (JNK) signaling pathway, which is an important member of the mitogen-activated protein kinase (MAPK) family, plays a crucial role in regulating cellular stress responses, inflammation, apoptosis, autophagy, and ferroptosis. Excessive JNK activation can induce uncontrolled inflammation, tissue damage, and chronic immune activation. In contrast, insufficient activation may impair the host’s defense, facilitating Mtb immune evasion and persistence. Such alterations disrupt the delicate immune equilibrium essential for effective pathogen clearance and host protection. This review summarizes the molecular mechanisms through which Mtb manipulates the JNK signaling pathway to disrupt host immunity, emphasizing its roles in metabolic reprogramming, apoptosis, autophagy, and ferroptosis. In addition, this review discusses potential therapeutic strategies targeting the JNK pathway, including the development of selective JNK inhibitors, with a focus on their prospects in TB treatment. Progress has been made in elucidating the role of JNK signaling pathway in TB, but further research is required to clarify its specific mechanisms and evaluate the safety and efficacy of JNK-targeted interventions. Continued exploration of this pathway may provide new targets and strategies for TB therapy.

BACKGROUND:Mycobacterium tuberculosis (MTB) Ag85A has become a component of multiple new tuberculosis vaccines. It is necessary to evaluate the immunogenicity, biological distribution, and safety of ag85a plasmid DNA (pDNA) to lay the foundation for the design of new vaccines. METHOD:Chronic toxicity test: cynomolgus monkeys were injected intramuscularly with different doses of ag85a pDNA, and the vaccine absorption kinetics, tissue distribution, and toxicity were observed. Their immune function was evaluated. Acute toxicity test: Mice were injected intramuscularly 0.5 ml saline, and injected intramuscularly and intravenously 0.5 mg/0.5 ml ag85a pDNAs, respectively. The toxicity and death of the mice were observed continuously for 14 days. Allergic test: Guinea pigs were intraperitoneally injected with different doses of ag85a pDNA. After stimulation, the allergic reaction and its severity were observed. RESULTS:Chronic and acute toxicity tests demonstrated that ag85a pDNA injections caused no clinical symptoms or tissue damage. Repeated intramuscular injections in cynomolgus monkeys enhanced specific Th1 immune responses, with pDNA rapidly entering the bloodstream and its concentration positively correlating with dosage. After 8 weeks, ag85a gene was detected only in muscles, myocardium, iliac lymph nodes, and blood. Guinea pig allergy tests showed no weight changes or allergic reactions, even after multiple sensitizations. CONCLUSIONS:The ag85a pDNA showed good safety in cynomolgus monkeys, mice, and guinea pigs, and induced high levels of antibodies and T-cell responses, making it a candidate antigen for the construction of a new tuberculosis vaccine.

Rifampin (RIF) resistance in Mycobacterium tuberculosis (M.tb) is primarily caused by mutations in the rpoB gene. Rapid and accurate detection of RIF resistance is critical for effective tuberculosis (TB) control. Nucleotide matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is an emerging technology used to detect RIF resistance-associated rpoB mutations in 210 M.tb clinical isolates, including 107 RIF-sensitive and 103 RIF-resistant strains, as determined by phenotypic drug susceptibility testing (DST). DNA sequencing was used as the reference method to validate nucleotide MALDI-TOF MS results. Nucleotide MALDI-TOF MS demonstrated a sensitivity of 93.2%, specificity of 98.1%, and an overall accuracy of 95.7% compared to phenotypic DST. The Kappa value between nucleotide MALDI-TOF MS and phenotypic DST was 0.91, indicating excellent agreement. DNA sequencing confirmed that nucleotide MALDI-TOF MS successfully identified RIF resistance-associated mutations, particularly in codons 450, 445, and 435 of the rpoB gene. Among the 61 isolates analyzed by DNA sequencing, nucleotide MALDI-TOF MS and sequencing results were consistent for 52 of 56 RIF-resistant strains and all five RIF-sensitive strains, with an overall concordance of 93.4%. Importantly, nucleotide MALDI-TOF MS accurately detected heteroresistance in eight isolates (14.3%), confirmed by sequencing. These results support that nucleotide MALDI-TOF MS is a rapid, accurate, and reliable method for detecting rpoB mutations associated with RIF resistance in M.tb. Its high concordance with DNA sequencing, excellent diagnostic performance, and ability to identify heteroresistance highlight its potential as a valuable tool for early TB diagnostics and improve the precision of chemotherapy regimen development.IMPORTANCEThe emergence of multidrug-resistant tuberculosis (MDR-TB) and rifampin-resistant tuberculosis (RR-TB) poses a significant challenge to global tuberculosis (TB) control efforts. Rifampin (RIF) resistance is a critical marker for MDR-TB, which requires more complex, prolonged, and costly treatment regimens. Early and accurate detection of RIF resistance is crucial for effective TB control. This study evaluates the performance of nucleotide MALDI-TOF MS, an innovative technology, for detecting RIF resistance-associated mutations in the rpoB gene. The method demonstrates high sensitivity (93.2%) and specificity (98.1%), with the added advantage of identifying heteroresistance, capabilities that are lacking in conventional methods. These capabilities are crucial for early diagnosis, guiding personalized treatment regimens, and curbing the transmission of drug-resistant TB. The findings demonstrate that nucleotide MALDI-TOF MS provides a rapid, high-throughput, and cost-effective alternative for detecting rpoB gene mutations associated with RIF resistance.

mRNA vaccines for infectious diseases prevent diseases by stimulating the body to produce specific immune responses through mRNA molecules encoding pathogenic proteins. Compared to traditional vaccines (e.g., inactivated, live attenuated, subunit, recombinant protein and viral vectors), mRNA vaccines offer several advantages including high safety, potent efficacy, scalable large-scale production, and cost-effectiveness. mRNA vaccines have demonstrated significant potential in combating infectious diseases since their inception. In particular, during the 2019 Coronavirus Disease (COVID-19) pandemic, the mRNA vaccines delivered with lipid nanoparticles (LNPs) have been developed by BioNTech and Moderna, their exceptional protective efficacy and extensive clinical application further proved the rapid responsiveness of mRNA vaccines in addressing emerging infectious diseases. This success has brought mRNA vaccines back into the spotlight of the scientific community. This article reviews the molecular biological basis, delivery systems, and immune mechanisms of mRNA vaccines, as well as the progress of research and clinical trials related to mRNA vaccines targeting the COVID-19 virus, influenza virus, rabies virus, Zika virus, human immunodeficiency virus, and Mycobacterium tuberculosis (M.tb), while also discussing the current challenges faced in the application of mRNA vaccines. These discussions provide a theoretical foundation and practical guidance for the future development of mRNA vaccines targeting bacterial infectious diseases such as tuberculosis (TB).

Background: Tuberculosis (TB) is a respiratory infectious disease, and the current TB vaccine has low local lung protection. We aim to optimize immune pathways to improve the immunogenicity of vaccines. Methods: In the immunogenicity study, 50 BALB/c mice were randomly divided into the following: (1) phosphate buffered saline (PBS)+intramuscular injection combined with electroporation (EP) group (100 μL), (2) pVAX1+EP group (50 μg/100 μL), (3) ag85ab+EP group (50 μg/100 μL), (4) pVAX1+pulmonary delivery (PD) group (50 μg/50 μL), and (5) ag85ab+PD group (50 μg/50 μL). Immunization was given once every 2 weeks for a total of three times. The number of IFN-γ-secreting lung and spleen lymphocytes was determined by enzyme-linked immunospot assay (ELISPOT). The levels of Th1, Th2, and Th17 cytokines in the culture supernatants of lung and spleen lymphocytes were detected with the Luminex method. The proportion of FoxP3 regulatory T cells in splenocytes was determined by flow cytometry. The levels of IgG-, IgG1-, and IgG2a-specific antibodies in plasma and IgA antibody in bronchoalveolar lavage fluid (BALF) were determined by enzyme-linked immunosorbent assay (ELISA). Results: The PD and EP routes of Mycobacterium tuberculosis (M. tb) ag85ab DNA vaccine can effectively induce the responses of IFN-γ-secreting lung and spleen lymphocytes, and induce dominant Th1 and Th17 cell immune responses. The PD route can induce earlier, greater numbers and stronger responses of pulmonary effector T cells, with higher levels of the specific antibody IgA detected in BALF. High levels of the specific antibodies IgG, IgG1, and IgG2α were detected in the plasma of mice immunized by the EP route. Conclusions: The PD route of DNA vaccines can more effectively stimulate the body to produce strong cellular and mucosal immunity than the EP route, especially local cellular immunity in the lungs, which can provide early protection for the lungs. It can significantly improve the immunogenicity of the ag85ab DNA vaccine, suggesting a feasible and effective approach to DNA immunization.

BackgroundThe pathogenic mechanism underlying Mycobacterium tuberculosis (MTB) remains elusive, posing challenges to its diagnosis and treatment. Cuproptosis is a newly identified mechanism of cell death. This study explores the role of cuproptosis-related genes (CRGs) in pulmonary tuberculosis (PTB) to uncover potential diagnostic biomarkers and therapeutic targets.MethodsDifferentially expressed gene (DEG) analysis and weighted gene co-expression network analysis (WGCNA) were carried out using the GSE83456 dataset. PTB-associated DEGs were intersected with CRGs to identify PTB-related CRGs. Subsequent analyses included functional enrichment, gene interaction, and protein-protein interaction (PPI) network construction. Hub CRGs were screened out via least absolute shrinkage and selection operator (LASSO) regression and random forest (RF) algorithms. Diagnostic models were subsequently constructed and validated. The associations of immune cell infiltration and pathway with the identified hub genes were evaluated through single-sample gene set enrichment analysis (ssGSEA) and CIBERSORT. Hub gene expressions were validated in the GSE42834 and GSE89403 datasets, as well as by RT-qPCR and Western blot (WB) in PTB and extrapulmonary tuberculosis (EPTB) patients. The GSE89403 dataset and gene expression profiling were leveraged to analyze the differential expression of hub genes and their dynamic changes during treatment.ResultsSeven PTB-related CRGs were significantly upregulated, were significantly upregulated, among which ASPHD2, GK, and GCH1 were identified as hub genes. These genes exhibited high expression levels in patients with PTB and EPTB, with marked reductions observed following treatment. Notable alterations in immune cell infiltration and immune function in PTB patients were closely related to these hub genes, suggesting activation of innate immune responses and suppression of adaptive immune function.ConclusionThe cuproptosis hub genes ASPHD2, GK, and GCH1 influence the pathogenesis of PTB, and possibly serve as novel diagnostic biomarkers and therapeutic targets.

ObjectiveTuberculous meningitis (TBM) is the most severe form of tuberculosis (TB). The purpose of this study was to explore the relationship between the number of natural killer (NK) cells and adaptive immune status, and disease severity in TBM patients.MethodsWe conducted a retrospective study on 244 TB patients and 146 healthy control subjects in the 8th Medical Center of the PLA General Hospital from March 2018 and August 2023.ResultsThe absolute count of NK cells in the peripheral blood of TBM patients was significantly lower than that in normal controls (NC), latent tuberculosis infection (LTBI), and non-severe TB (NSTB) patients (p < 0.05). The proportion of TBM patients (48.7%) with a lower absolute count of NK cells than the normal reference value was significantly higher than that in NC (5.2%) and LTBI groups (4.0%) (p < 0.05), and slightly higher than that in NSTB group (36.0%) (p > 0.05). The absolute counts of lymphocyte subsets in TBM combined with other active TB group, etiology (+) group, IGRA (−) group, and antibody (+) group were lower than that in simple TBM group, etiology (−) group, IGRA (+) group, and antibody (−) group, respectively. The CD3+ T, NK, and B cells in BMRC-stage III TBM patients were significantly lower than those in stage I and stage II patients (p < 0.05). The counts of CD3+ T, CD4+ T, and B cells in the etiology (+) group were significantly lower than those in the etiology (−) group (p < 0.05).ConclusionThe absolute counts of lymphocyte subsets in the peripheral blood of TBM patients were significantly decreased, especially in NK cells. The reduction of these immune cells was closely related to the disease severity and had a certain correlation with cellular and humoral immune responses. This study helps to better understand the immune mechanism of TBM and provides reliable indicators for evaluating the immune status of TBM patients in clinical practice.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 M. tuberculosis (MTB) H37Rv-infected BALB/c mice were treated with PBS, 10μg, 50μg, 100μg, and 200μg ag85a/b DNA vaccine delivered by IM and EP three times at two-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from 3 mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive PCR (RT-qPCR) and the GEO database. After MTB infection, most of the up-regulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine, for example, Iapp, Scg2, Chga, Amy2a5, etc, and most of the down-regulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of alveolar epithelial cell, for example, Sftpc, Sftpd, Pdpn, etc. Most of the abnormally up-regulated or down-regulated DE genes in the TB model group were recovered in the 100μg and 200μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway down-regulated and Rap1 signal pathway up-regulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action target and mechanism of IM and EP are highly consistent. Tuberculosis infection caused rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.

ABSTRACTBackgroundVaccination is one of the effective measures to prevent latent tuberculosis infection (LTBI) from developing into active tuberculosis (TB). Applying bioinformatics methods to pre‐evaluate the biological characteristics and immunogenicity of vaccines can improve the efficiency of vaccine development.ObjectivesTo evaluate the immunogenicity of TB vaccine W541 and to explore the application of bioinformatics technology in TB vaccine research.MethodsThis study concatenated the immunodominant sequences of Ag85A, Ag85B, Rv3407, and Rv1733c to construct the W541 DNA vaccine. Then, bioinformatics methods were used to analyze the physicochemical properties, antigenicity, allergenicity, toxicity, and population coverage of the vaccine, to identify its epitopes, and to perform molecular docking with MHC alleles and Toll‐like receptor 4 (TLR4) of the host. Finally, the immunogenicity of the vaccine was evaluated in animal experiments.ResultsThe W541 vaccine protein is a soluble cytoplasmic protein with a half‐life of 1.1 h in vivo and an instability index of 45.37. It has good antigenicity and wide population coverage without allergenicity and toxicity. It contains 138 HTL epitopes, 73 CTL epitopes, 8 linear and 14 discontinuous B cell epitopes, and has a strong affinity for TLR4. Immune simulations have shown that it can effectively stimulate innate and adaptive immune responses. Animal experiments confirmed that the W541 DNA vaccine could effectively activate Th1‐ and Th17‐type immune responses, producing high levels of IFN‐γ and IL‐17A, but could not significantly increase antibody levels.ConclusionThe W541 DNA vaccine can induce strong cellular immune responses. However, further optimization of the vaccine design is needed to make the expressed protein more stable in vivo. Bioinformatics analysis could reveal the physicochemical and immunological information of vaccines, which is critical for guiding vaccine design and development.

Gastroesophageal reflux related chronic cough (GERC), is a common type of chronic cough. Drug treatment is the first choice. But some patients are tired of taking medicine everyday and some patients can not benefit from drugs. For these patients, fundoplication may be the most effective method. However, the importance of fundoplication in treating GERC is undervalued, and there is very few meta-analysis looking into the effect and safety of fundoplication in treating GERC. To solve this question, we performed this meta-analysis. The PRISMA strategy was used for this study. Our study was registered with PROSPERO (ID: CRD42021251072). We searched PubMed, Medline, Web of Science, and the Cochrane databases from 1990 to December of 2022. The meta-analysis was performed with Review Manager 5.4 and Stata 14. After selection and exclusion, 15 articles out of 672 were included. The meta-analysis showed that the cure rate of laparoscopic fundoplication in treating GERC was 58% (95%CI: 52%–65%), with I2 = 45%; and the effective rate was 86% (95%CI: 80%–93%), with I2 = 0%. Laparoscopic fundoplication is effective for the most of GERC patients; however, when the goal is to cure GERC completely, a relatively conservative attitude should be taken. In terms of safety, laparoscopic fundoplication is quite reliable offered by skilled surgeons.

Traditional vaccines have played an important role in the prevention and treatment of infectious diseases, but they still have problems such as low immunogenicity, poor stability, and difficulty in inducing lasting immune responses. In recent years, the nucleic acid vaccine has emerged as a relatively cheap and safe new vaccine. Compared with traditional vaccines, nucleic acid vaccine has some unique advantages, such as easy production and storage, scalability, and consistency between batches. However, the direct administration of naked nucleic acid vaccine is not ideal, and safer and more effective vaccine delivery systems are needed. With the rapid development of nanocarrier technology, the combination of gene therapy and nanodelivery systems has broadened the therapeutic application of molecular biology and the medical application of biological nanomaterials. Nanoparticles can be used as potential drug-delivery vehicles for the treatment of hereditary and infectious diseases. In addition, due to the advantages of lung immunity, such as rapid onset of action, good efficacy, and reduced adverse reactions, pulmonary delivery of nucleic acid vaccine has become a hot spot in the field of research. In recent years, lipid nanocarriers have become safe, efficient, and ideal materials for vaccine delivery due to their unique physical and chemical properties, which can effectively reduce the toxic side effects of drugs and achieve the effect of slow release and controlled release, and there have been a large number of studies using lipid nanocarriers to efficiently deliver target components into the body. Based on the delivery of tuberculosis (TB) nucleic acid vaccine by lipid carrier, this article systematically reviews the advantages and mechanism of liposomes as a nucleic acid vaccine delivery carrier, so as to lay a solid foundation for the faster and more effective development of new anti-TB vaccine delivery systems in the future.

Our previous research developed a novel tuberculosis (TB) DNA vaccine ag85a/b that showed a significant therapeutic effect on the mouse tuberculosis model by intramuscular injection (IM) and electroporation (EP). However, the action mechanisms between these two vaccine immunization methods remain unclear. In a previous study, 96 Mycobacterium tuberculosis (MTB) H37 Rv-infected BALB/c mice were treated with phosphate-buffered saline, 10, 50, 100, and 200 μg ag85a/b DNA vaccine delivered by IM and EP three times at 2-week intervals, respectively. In this study, peripheral blood mononuclear cells (PBMCs) from three mice in each group were isolated to extract total RNA. The gene expression profiles were analyzed using gene microarray technology to obtain differentially expressed (DE) genes. Finally, DE genes were validated by real-time reverse transcription-quantitive polymerase chain reaction and the GEO database. After MTB infection, most of the upregulated DE genes were related to the digestion and absorption of nutrients or neuroendocrine (such as Iapp, Scg2, Chga, Amy2a5), and most of the downregulated DE genes were related to cellular structural and functional proteins, especially the structure and function proteins of the alveolar epithelial cell (such as Sftpc, Sftpd, Pdpn). Most of the abnormally upregulated or downregulated DE genes in the TB model group were recovered in the 100 and 200 μg ag85a/b DNA IM groups and four DNA EP groups. The pancreatic secretion pathway downregulated and the Rap1 signal pathway upregulated had particularly significant changes during the immunotherapy of the ag85a/b DNA vaccine on the mouse TB model. The action targets and mechanisms of IM and EP are highly consistent. Tuberculosis infection causes rapid catabolism and slow anabolism in mice. For the first time, we found that the effective dose of the ag85a/b DNA vaccine immunized whether by IM or EP could significantly up-regulate immune-related pathways and recover the metabolic disorder and the injury caused by MTB.

Bacille Calmette-Guérin (BCG) vaccination can confer nonspecific protection against heterologous pathogens. However, the underlying mechanisms remain mysterious. We show that mice vaccinated intravenously with BCG exhibited reduced weight loss and/or improved viral clearance when challenged with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2 B.1.351) or PR8 influenza. Protection was first evident between 14 and 21 d post-vaccination and lasted ∼3 months. Notably, BCG induced a biphasic innate response and robust antigen-specific type 1 helper T cell (TH1 cell) responses in the lungs. MyD88 signaling was essential for innate and TH1 cell responses, and protection against SARS-CoV-2. Depletion of CD4+ T cells or interferon (IFN)-γ activity before infection obliterated innate activation and protection. Single-cell and spatial transcriptomics revealed CD4-dependent expression of IFN-stimulated genes in lung myeloid and epithelial cells. Notably, BCG also induced protection against weight loss after mouse-adapted SARS-CoV-2 BA.5, SARS-CoV and SHC014 coronavirus infections. Thus, BCG elicits integrated organ immunity, where CD4+ T cells feed back on tissue myeloid and epithelial cells to imprint prolonged and broad innate antiviral resistance.

Background: Verapamil enhances the sensitivity of Mycobacterium tuberculosis to anti-tuberculosis (TB) drugs, promotes the macrophage anti-TB ability, and reduces drug resistance, but its mechanism is unclear. Herein, we have investigated the effect of verapamil on cytokine expression in mouse peritoneal macrophages. Methods: Macrophages from mice infected with M. tuberculosis or S. aureus were cultured with verapamil, the cytokines were detected by enzyme-linked immunosorbent assay, and the RNA was measured with quantitative real-time polymerase chain reaction and agarose gel electrophoresis. The intracellular calcium signaling was measured by confocal microscopy. Results: Significantly higher levels of NF-κB, IL-12, TNF-α, and IL-1β were observed after TB infection. The levels of NF-κB and IL-12 increased when verapamil concentration was less than 50 μg/ml, but decreased when verapamil concentration was greater than 50μg/ml. With the increase in verapamil concentration, TNF-α and IL-1β expressed by macrophages decreased. The L-type calcium channel transcription significantly increased in M. tuberculosis rather than S. aureus-infected macrophages. Furthermore, during bacillus Calmette-Guerin (BCG) infection, verapamil stimulated a sharp peak in calcium concentration in macrophages, while calcium concentration increased mildly and decreased smoothly over time in the absence of verapamil. Conclusions: Verapamil enhanced macrophage immunity via the NF-κB pathway, and its effects on cytokine expression may be achieved by its regulation of intracellular calcium signaling.

目的:对抗结核中药牛贝消核提取物进行血清药物化学初步研究,通过分析入血成分,探讨牛贝消核发挥药效的物质基础.方法:2018年4月至2019年6月在中国人民解放军总医院第八医学中心全军结核病研究所制备桔梗、白及、鱼腥草和牛蒡子标准品溶液及牛贝消核供试品;55只小鼠随机分为11组,每组5只:1～5组为牛贝消核提取物组,每只小鼠每天给予1.25 mg/g牛贝消核提取物灌胃;6～10组为牛蒡苷组,每只小鼠每天给予0.3mg/g牛蒡苷灌胃;11组为空白对照组,给予蒸馏水灌胃;连续灌胃7 d.于末次给药后0.5、1、2、4、6 h小鼠眼眶后静脉丛取血,制备血清供试品溶液.采用高效液相色谱法(high-performance liquid chromatography,HPLC)分析各中药标准品、牛贝消核提取物供试品及各组小鼠给药后血清供试品溶液,分析色谱图中各物质保留时间;然后参照对照品初步鉴定牛贝消核提取物的入血成分.结果:通过牛贝消核提取物和各药材标准品重叠色谱图中可以看出,牛贝消核提取物保留时间为7.1 min处的色谱峰所对应的物质来源于桔梗,保留时间为13.5 min、15.5 min、21.2 min处的色谱峰所对应的物质来源于白及,保留时间为16.3 min的色谱峰所对应的物质来源于牛蒡子,并进一步鉴定为牛蒡苷化合物.牛贝消核提取物和牛蒡苷给药后血清样本与空白血清比较,均在17.5～22.5 min发现有吸收峰.结论:HPLC分析可作为牛贝消核中桔梗、白及、鱼腥草和牛蒡子提取的质量控制方法,检出的牛贝消核提取物血中移行成分可能是牛蒡苷代谢产物,其余药效成分入血微量未检出.

BACKGROUND:Tuberculosis (TB) is a global infectious disease, but there is no ideal vaccine against TB except the Bacille Calmette-Guérin (BCG) vaccine.METHODS:Herein, 25 candidate peptides were predicted from four antigens of Mycobacterium tuberculosis based on their high-affinity binding capacity for the human leukocyte antigen (HLA) DRB1∗0101. Three T-helper 1 (Th1) immunodominant peptides (Ag85B12-26, CFP2112-26, and PPE18149-163) were identified by ELISPOT assays in the humanized C57BL/6 mice. They resulted in a novel Th1 peptide-based vaccine ACP named by the first letter of the three peptides. In addition, the protective efficacy was evaluated in humanized or wild-type C57BL/6 mice and the humoral and cellular immune responses were confirmed in vitro.RESULTS:Compared with the PBS group, the ACP vaccinated mice showed slight decreases in colony-forming units (CFUs) and pathological lesions. However, when using it as a booster, the ACP vaccine did not significantly enhance the protective efficacy of BCG in humanized or wild-type mice. Interestingly, we found that ACP vaccination significantly increased the number of interferon-γ positive (IFN-γ+) T lymphocytes and the levels of IFN-γ cytokines as well as antibodies. Furthermore, the IL-2 level was significantly higher in humanized mice prime-boosted with BCG and ACP.CONCLUSIONS:Our results suggested that ACP vaccination could stimulate higher levels of cytokines and antibodies but failed to improve the protective efficacy of BCG in mice, indicating that the secretion level of IFN-γ may not be positively correlated with the protection efficiency of the vaccine. These findings provided important information on the feasibility of a peptide vaccine as a booster for enhancing the protective efficacy of BCG.

目的 建立半定量法检测十二烷基硫酸钠(sodium dodecyl sulfate,SDS)残留量并进行方法学验证,用于结核DNA疫苗的质量控制.方法 样本中残留的SDS与吖啶橙形成复合物,用Spectra Max 340PC微孔板检测器测定该复合物的吸光度.通过定量法测定SDS的残留量时,样品的吸光度不在标准曲线线性范围内,采用对比供试品与对照溶液的吸光度的半定量法,判断供试品中SDS的残留量.结果 该方法回收率范围为102％～105％,不同人员、不同时间测定结果一致,该法受辅料、测试温度、萃取时间的影响较小,测定的3批供试品SDS残留量均小于0.02％.结论 建立的方法准确度、中间精密度、专属性和耐用性较好,可用于结核DNA疫苗中SDS残留量的快速检测.