[Development of a flow cytometry method for detection of bovine multi-cytokines].

This study aims to develop a method to detect bovine multi-cytokines based on flow cytometry. Previously we have prepared and screened monoclonal antibodies against bovine cytokines IFN-γ, IL-2, TNF-α, IP-10 and MCP-1. These bovine cytokine monoclonal antibodies were fluorescently labeled, and the combination of antibody and cell surface molecules were used to develop the method for detecting bovine multi-cytokines. Subsequently, the developed method was used to determine the cytokine expression profile of Mycobacterium bovis BCG infected bovine peripheral blood mononuclear cells in vitro, and evaluate the cytokine expression level of peripheral blood CD4+ T cells of tuberculosis-positive cattle. The bovine multi-cytokine flow cytometry detection method can effectively determine the cytokine expression of BCG-infected bovine peripheral blood T lymphocytes. Among them, the expression levels of IFN-γ, IL-2, and TNF-α continue to increase after 40 hours of infection, while the expression levels of IP-10 and MCP-1 decreased. The combined detection of IFN-γ, IL-2, and TNF-α on CD4+ T lymphocytes in peripheral blood of cattle can effectively distinguish tuberculosis-positive and tuberculosis-negative samples. This method may facilitate evaluating the level of cellular immune response after bovine pathogen infection and vaccine injection.

Mycobacterium bovis BCG increase the selected determinants of monocyte/macrophage activity, which were diminished in response to gastric pathogen Helicobacter pylori.

High antibiotic resistance of gastric pathogen Helicobacter pylori (Hp) and the ability to escape the host immune response prompt searching for therapeutic immunomodulators. Bacillus Calmette-Guerin (BCG) vaccine with Mycobacterium bovis (Mb) is a candidate for modulation the activity of immunocompetent cells, and onco-BCG formulation was successfully used in immunotherapy of bladder cancer. We determined the influence of onco-BCG on the phagocytic capacity of human THP-1 monocyte/macrophage cells, using the model of Escherichia coli bioparticles and Hp fluorescently labeled. Deposition of cell integrins CD11b, CD11d, CD18, membrane/soluble lipopolysaccharide (LPS) receptors, CD14 and sCD14, respectively, and the production of macrophage chemotactic protein (MCP)-1 were determined. Furthermore, a global DNA methylation, was also assessed. Human THP-1 monocytes/macrophages (TIB 202) primed or primed and restimulated with onco-BCG or Hp, were used for assessment of phagocytosis towards E. coli or Hp, surface (immunostaining) or soluble activity determinants, and global DNA methylation (ELISA). THP-1 monocytes/macrophages primed/restimulated with BCG showed increased phagocytosis capacity towards E. coli fluorescent particles, elevated expression of CD11b, CD11d, CD18, CD14, sCD14, increased MCP-1 secretion and DNA methylation. Preliminary results indicate that BCG mycobacteria may also induce the phagocytosis of H. pylori by THP-1 monocytes. Priming or priming and restimulation of monocytes/macrophages with BCG resulted in an increased activity of these cells, which was negatively modulated by Hp.

Mycobacterial phosphodiesterase Rv0805 is a virulence determinant and its cyclic nucleotide hydrolytic activity is required for propionate detoxification.

Cyclic AMP (cAMP) signaling is essential to Mycobacterium tuberculosis (Mtb) pathogenesis. However, the roles of phosphodiesterases (PDEs) Rv0805, and the recently identified Rv1339, in cAMP homeostasis and Mtb biology are unclear. We found that Rv0805 modulates Mtb growth within mice, macrophages and on host-associated carbon sources. Mycobacterium bovis BCG grown on a combination of propionate and glycerol as carbon sources showed high levels of cAMP and had a strict requirement for Rv0805 cNMP hydrolytic activity. Supplementation with vitamin B12 or spontaneous genetic mutations in the pta-ackA operon restored the growth of BCGΔRv0805 and eliminated propionate-associated cAMP increases. Surprisingly, reduction of total cAMP levels by ectopic expression of Rv1339 restored only 20% of growth, while Rv0805 complementation fully restored growth despite a smaller effect on total cAMP levels. Deletion of an Rv0805 localization domain also reduced BCG growth in the presence of propionate and glycerol. We propose that localized Rv0805 cAMP hydrolysis modulates activity of a specialized pathway associated with propionate metabolism, while Rv1339 has a broader role in cAMP homeostasis. Future studies will address the biological roles of Rv0805 and Rv1339, including their impacts on metabolism, cAMP signaling and Mtb pathogenesis.

Targeted Next-Generation Sequencing of Flat Urothelial Lesions Reveals Putative Pathobiological Pathways, Potential Biomarkers, and Rational Therapeutic Targets.

Flat urothelial lesions are controversial diagnostic and prognostic urologic entities whose importance relies mainly on their ability to progress to muscle-invasive tumors via urothelial carcinoma in situ (CIS). However, the carcinogenetic progression of preneoplastic flat urothelial lesions is not well established. Moreover, predictive biomarkers and therapeutic targets of the highly recurrent and aggressive urothelial CIS lesion are lacking. Using a targeted next-generation sequencing (NGS) panel of 17 genes directly involved in bladder cancer pathogenesis, we investigated alterations of genes and pathways with clinical and carcinogenic implications on 119 samples of flat urothelium, including normal urothelium (n = 7), reactive atypia (n = 10), atypia of unknown significance ( n = 34), dysplasia ( n = 23), and CIS (n = 45). The majority of the flat lesions were tumor-associated but grossly/microscopically or temporally separated from the main tumor. Mutations were compared across flat lesions and concerning the concomitant urothelial tumor. Associations between genomic mutations and recurrence after intravesical bacillus Calmette-Guerin treatment were estimated with Cox regression analysis. TERT promoter mutations were highly prevalent in intraurothelial lesions but not in the normal or reactive urothelium, suggesting that it is a critical driver mutation in urothelial tumorigenesis. We found that synchronous atypia of unknown significance-dysplasia-CIS lesions without concomitant papillary urothelial carcinomas had a similar genomic profile that differed from atypia of unknown significance-dysplasia lesions associated with papillary urothelial carcinomas, which harbored significantly more FGFR3, ARID1A, and PIK3CA mutations. KRAS G12C and ERBB2 S310F/Y mutations were exclusively detected in CIS and were associated with recurrence after bacillus Calmette-Guerin treatment (P = .0006 and P = .01, respectively). This targeted NGS study revealed critical mutations involved in the carcinogenetic progression of flat lesions with putative pathobiological pathways. Importantly, KRAS G12C and ERBB2 S310F/Y mutations were identified as potential prognostic and therapeutic biomarkers for urothelial carcinoma.

Mycobacterium tuberculosis induces delayed lipid droplet accumulation in dendritic cells depending on bacterial viability and virulence.

Tuberculosis remains a global health threat with high morbidity. Dendritic cells (DCs) participate in the acute and chronic inflammatory responses to Mycobacterium tuberculosis (Mtb) by directing the adaptive immune response and are present in lung granulomas. In macrophages, the interaction of lipid droplets (LDs) with mycobacteria-containing phagosomes is central to host-pathogen interactions. However, the data available for DCs are still a matter of debate. Here, we reported that bone marrow-derived DCs (BMDCs) were susceptible to Mtb infection and replication at similar rate to macrophages. Unlike macrophages, the analysis of gene expression showed that Mtb infection induced a delayed increase in lipid droplet-related genes and proinflammatory response. Hence, LD accumulation has been observed by high-content imaging in late periods. Infection of BMDCs with killed H37Rv demonstrated that LD accumulation depends on Mtb viability. Moreover, infection with the attenuated strains H37Ra and Mycobacterium bovis-BCG induced only an early transient increase in LDs, whereas virulent Mtb also induced delayed LD accumulation. In addition, infection with the BCG strain with the reintroduced virulence RD1 locus induced higher LD accumulation and bacterial replication when compared to parental BCG. Collectively, our data suggest that delayed LD accumulation in DCs is dependent on mycobacterial viability and virulence.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype.

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14 C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial-Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy.

A Hydrazine-Hydrazone Adamantine Compound Shows Antimycobacterial Activity and Is a Probable Inhibitor of MmpL3.

Tuberculosis remains an important cause of morbidity and mortality throughout the world. Notably, an important number of multi drug resistant cases is an increasing concern. This problem points to an urgent need for novel compounds with antimycobacterial properties and to improve existing therapies. Whole-cell-based screening for compounds with activity against Mycobacterium tuberculosis complex strains in the presence of linezolid was performed in this study. A set of 15 bioactive compounds with antimycobacterial activity in vitro were identified with a minimal inhibitory concentration of less than 2 µg/mL. Among them, compound 1 is a small molecule with a chemical structure consisting of an adamantane moiety and a hydrazide-hydrazone moiety. Whole genome sequencing of spontaneous mutants resistant to the compounds suggested compound 1 to be a new inhibitor of MmpL3. This compound binds to the same pocket as other already published MmpL3 inhibitors, without disturbing the proton motive force of M. bovis BCG and M. smegmatis. Compound 1 showed a strong activity against a panel ofclinical strains of M. tuberculosis in vitro. This compound showed no toxicity against mammalian cells and protected Galleria mellonella larvae against M. bovis BCG infection. These results suggest that compound 1 is a promising anti-TB agent with the potential to improve TB treatment in combination with standard TB therapies.

Structure-based in silico and in vitro Analysis Reveals Asiatic Acid as Novel Potential Inhibitor of Mycobacterium tuberculosis Maltosyl Transferase.

AIMS: The present study aimed to search for novel potent inhibitor(s) against the recently discovered maltosyltransferase (GlgE) target of M.tb. BACKGROUND: GlgE belongs to an α-amylase family and catalyzes the elongation of cytosolic branched α-glucan. Inactivation of M.tb. GlgE results in DNA damage and rapid death of M.tb. due to the accumulation of a toxic altosyl donor, maltose-1-phosphate (M1P), suggesting that GlgE is an intriguing target for inhibitor design. METHODS: 1000 natural compounds were compiled from public databases and literature through virtual screening, of which 25 compounds were found to satisfy all drug-likeness properties and ADME/ toxicity criteria, followed by molecular docking with GlgE. Compound(s) showing the lowest binding energy was further subjected to molecular dynamics simulation (MDS) and in vitro analysis. RESULTS: Molecular docking analysis allowed the selection of 5 compounds withsignificant binding affinity to GlgE targets. Amongst these compounds, asiatic acid exhibited the lowest binding energy (-12.61 kcal/mol). The results of 20-ns MDS showed that asiatic acid formed a stable complex with GlgE. Additionally, asiatic acid exhibited in vitro anti-mycobacterial activity against M.tb. H37Ra, M. bovis BCG, and M. smegmatis strains. CONCLUSION: The study reveals asiatic acid as a promising anti-mycobacterial agent that might emerge as a novel natural anti-TB lead molecule in the future.

Effects of dietary supplementation of different levels of vitamin B12 on the liver metabolism of laying hens.

BACKGROUND: Vitamin B12 plays an important role in lipid, protein, carbohydrate and nucleic acid metabolism. We investigated the effect of supplementing layers' diets with different vitamin B12 levels on liver metabolism using a liquid chromatography-mass spectrometry-based metabolomic approach to observe and analyse wide-target metabolomics in the liver. RESULTS: We assigned hens to three groups, namely blank control group without vitamin B12 diet (BCG), normal control group with 25 µg kg-1 vitamin B12 (NCG) and vitamin B12 supplement group I with 100 µg kg-1 vitamin (VBSG I). The VBSG I group layers had higher (P < 0.05) vitamin B12 concentration than those from other groups. The egg yolk vitamin B12 concentration increased (P < 0.01) with the increasing vitamin B12 dietary supplemental level. Between the NCG versus BCG, VBSG I versus BCG, and VBSG I versus NCG groups, 11, 20 and 11 metabolites were significantly changed, respectively. The KEGG pathway of vitamin B6 metabolism was significantly impacted in the NCG layers than those from BCG; seven and five pathways were significantly impacted in the VBSG I layers compared with those from BCG and NCG, including pyrimidine metabolism, vitamin B6 metabolism, glycerophospholipid metabolism, etc. CONCLUSION: We concluded that 25 µg kg-1 vitamin B12 supplementation in corn-soybean meal-based layer diet increased the egg yolk vitamin B12 concentration and impacted the vitamin B6 metabolic pathway, and 100 µg kg-1 of it increased the egg yolk and liver vitamin B12 concentrations and impacted vitamin B6 , lipid, nucleic acid and amino acid metabolic pathways. © 2022 Society of Chemical Industry.

Characterization of O-mannosylated proteins profiling in bacillus Calmette-Guérin via gel-based and gel-free approaches.

Posttranslational modifications (PTMs) could influence many aspects of protein behavior and function in organisms. Protein glycosylation is one of the major PTMs observed in bacteria, which is crucial for functional regulations of many prokaryotic and eukaryotic organisms. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been recognized as an indispensable tool in the global fight against tuberculosis (TB) worldwide over several decades. Nevertheless, analysis of glycoprotein profiles of BCG has not been clearly investigated. In this study, we performed O-mannosylated protein analysis in BCG bacteria using gel-based and gel-free approaches. In total, 1,670 hexosylated peptides derived from 754 mannosylated proteins were identified. Furthermore, 20 novel protein products supported by 78 unique peptides not annotated in the BCG database were detected. Additionally, the translational start sites of 384 proteins were confirmed, and 78 proteins were validated through the extension of translational start sites based on N-terminus-derived peptides. The bioinformatic analysis of the O-mannosylated proteins was performed and the expression profiles of four randomly selected proteins were validated through Western blotting. A number of proteins involved in metabolic pathways, including the tricarboxylic acid cycle, glycolysis, oxidative phosphorylation, and two-component system, are discussed. Taken together, these results offer the first O-mannosylated protein analysis of a member of mycobacteria reported to date by using complementary gel-based and gel-free approaches. Some of the proteins identified in this study have important roles involved in metabolic pathways, which could provide insight into the immune molecular mechanisms of this recognized vaccine strain.

Phenolic glycolipid-1 of Mycobacterium leprae is involved in human Schwann cell line ST8814 neurotoxic phenotype

Leprosy is a chronic infectious disease caused by Mycobacterium leprae infection in Schwann cells. Axonopathy is considered a hallmark of leprosy neuropathy and is associated with the irreversible motor and sensory loss seen in infected patients. Although M. leprae is recognized to provoke Schwann cell dedifferentiation, the mechanisms involved in the contribution of this phenomenon to neural damage remain unclear. In the present work, we used live M. leprae to infect the immortalized human Schwann cell line ST8814. The neurotoxicity of infected Schwann cell-conditioned medium (SCCM) was then evaluated in a human neuroblastoma cell lineage and mouse neurons. ST8814 Schwann cells exposed to M. leprae affected neuronal viability by deviating glial 14C-labeled lactate, important fuel of neuronal central metabolism, to de novo lipid synthesis. The phenolic glycolipid-1 (PGL-1) is a specific M. leprae cell wall antigen proposed to mediate bacterial­Schwann cell interaction. Therefore, we assessed the role of the PGL-1 on Schwann cell phenotype by using transgenic M. bovis (BCG)-expressing the M. leprae PGL-1. We observed that BCG-PGL-1 was able to induce a phenotype similar to M. leprae, unlike the wild-type BCG strain. We next demonstrated that this Schwann cell neurotoxic phenotype, induced by M. leprae PGL-1, occurs through the protein kinase B (Akt) pathway. Interestingly, the pharmacological inhibition of Akt by triciribine significantly reduced free fatty acid content in the SCCM from M. leprae- and BCG-PGL-1-infected Schwann cells and, hence, preventing neuronal death. Overall, these findings provide novel evidence that both M. leprae and PGL-1, induce a toxic Schwann cell phenotype, by modifying the host lipid metabolism, resulting in profound implications for neuronal loss. We consider this metabolic rewiring a new molecular mechanism to be the basis of leprosy neuropathy. (AU)

Intravescical instillation of Calmette-Guérin bacillus and COVID-19 risk.

It has been theorized that Calmette-Guérin bacillus may prevent or reduce the severity of COVID-19 through a nonspecific stimulation of the immune system. A preliminary assessment of SARS-CoV-2 infection rates and outcomes among 2803 individuals affected with high risk non-muscle-invasive bladder cancer and treated with intra-bladder instillation of BCG, showed no evidence of a protective effect. However, the interpretation of these data need some caution, due to the low prevalence of infection (<1%) observed within this population, along with the fact that intra-bladder administration cannot mirror the usual intradermal administration of BCG, in particular in patients partially immunocompromised. Confirmation by larger prospective studies is required.

Proteomic characterization of a second-generation version of the BCGΔBCG1419c vaccine candidate by means of electrospray-ionization quadrupole time-of-flight mass spectrometry.

Tuberculosis (TB) is the most important infectious disease worldwide, based on the number of new cases and deaths reported by the World Health Organization. Several vaccine candidates against TB have been characterized at preclinical and clinical levels. The BCGΔBCG1419c vaccine candidate, which lacks the BCG1419c gene that encodes for a c-di-GMP phosphodiesterase, provides improved efficacy against chronic TB, reactivation from latent-like infection and against chronic TB in the presence of type 2 diabetes in murine models. We previously reported that compared with wild type BCG, BCGΔBCG1419c changed levels of several proteins. Here, using a label-free proteomic approach, we confirmed that a novel, second-generation version of BCGΔBCG1419c maintains changes in antigenic proteins already reported, and here we further found differences in secreted proteins, as well as that this new BCGΔBCG1419c version modifies its production of proteins involved in redox and nitrogen/protein metabolism compared with wild type BCG. This work contributes to the proteomic characterization of a novel vaccine candidate that is more effective against TB than parental BCG in diverse murine models.

BCG Vaccination Induces Long-Term Functional Reprogramming of Human Neutrophils.

The tuberculosis vaccine bacillus Calmette-Guérin (BCG) protects against some heterologous infections, probably via induction of non-specific innate immune memory in monocytes and natural killer (NK) cells, a process known as trained immunity. Recent studies have revealed that the induction of trained immunity is associated with a bias toward granulopoiesis in bone marrow hematopoietic progenitor cells, but it is unknown whether BCG vaccination also leads to functional reprogramming of mature neutrophils. Here, we show that BCG vaccination of healthy humans induces long-lasting changes in neutrophil phenotype, characterized by increased expression of activation markers and antimicrobial function. The enhanced function of human neutrophils persists for at least 3 months after vaccination and is associated with genome-wide epigenetic modifications in trimethylation at histone 3 lysine 4. Functional reprogramming of neutrophils by the induction of trained immunity might offer novel therapeutic strategies in clinical conditions that could benefit from modulation of neutrophil effector function.

Transcriptional portrait of M. bovis BCG during biofilm production shows genes differentially expressed during intercellular aggregation and substrate attachment.

Mycobacterium tuberculosis and M. smegmatis form drug-tolerant biofilms through dedicated genetic programs. In support of a stepwise process regulating biofilm production in mycobacteria, it was shown elsewhere that lsr2 participates in intercellular aggregation, while groEL1 was required for biofilm maturation in M. smegmatis. Here, by means of RNA-Seq, we monitored the early steps of biofilm production in M. bovis BCG, to distinguish intercellular aggregation from attachment to a surface. Genes encoding for the transcriptional regulators dosR and BCG0114 (Rv0081) were significantly regulated and responded differently to intercellular aggregation and surface attachment. Moreover, a M. tuberculosis H37Rv deletion mutant in the Rv3134c-dosS-dosR regulon, formed less biofilm than wild type M. tuberculosis, a phenotype reverted upon reintroduction of this operon into the mutant. Combining RT-qPCR with microbiological assays (colony and surface pellicle morphologies, biofilm quantification, Ziehl-Neelsen staining, growth curve and replication of planktonic cells), we found that BCG0642c affected biofilm production and replication of planktonic BCG, whereas ethR affected only phenotypes linked to planktonic cells despite its downregulation at the intercellular aggregation step. Our results provide evidence for a stage-dependent expression of genes that contribute to biofilm production in slow-growing mycobacteria.

Clofazimine enhances the efficacy of BCG revaccination via stem cell-like memory T cells.

Tuberculosis (TB) is one of the deadliest diseases, claiming ~2 million deaths annually worldwide. The majority of people in TB endemic regions are vaccinated with Bacillus Calmette Guerin (BCG), which is the only usable vaccine available. BCG is efficacious against meningeal and disseminated TB in children, but protective responses are relatively short-lived and fail to protect against adult pulmonary TB. The longevity of vaccine efficacy critically depends on the magnitude of long-lasting central memory T (TCM) cells, a major source of which is stem cell-like memory T (TSM) cells. These TSM cells exhibit enhanced self-renewal capacity as well as to rapidly respond to antigen and generate protective poly-functional T cells producing IFN-γ, TNF-α, IL-2 and IL-17. It is now evident that T helper Th 1 and Th17 cells are essential for host protection against TB. Recent reports have indicated that Th17 cells preserve the molecular signature for TSM cells, which eventually differentiate into IFN-γ-producing effector cells. BCG is ineffective in inducing Th17 cell responses, which might explain its inadequate vaccine efficacy. Here, we show that revaccination with BCG along with clofazimine treatment promotes TSM differentiation, which continuously restores TCM and T effector memory (TEM) cells and drastically increases vaccine efficacy in BCG-primed animals. Analyses of these TSM cells revealed that they are predominantly precursors to host protective Th1 and Th17 cells. Taken together, these findings revealed that clofazimine treatment at the time of BCG revaccination provides superior host protection against TB by increasing long-lasting TSM cells.

Synergistic effects of combined vaccination with BCG and influenza vaccines on spatial cognition and hippocampal plasticity in rats.

Previous study has demonstrated the neurobeneficial role of BCG and influenza vaccines. Based on this, our study concentrated on the synergistic effects on development of central nervous system by combined vaccination with BCG and influenza vaccines in rats. Our results displayed that pups combinedly vaccinated with BCG and influenza vaccines showed a significant enhance in spatial cognition, induction of LTP, hippocampal neurogenesis and morphology of dendritic spines compared with pups vaccinated with BCG solely. Furthermore, combined vaccination with BCG and influenza vaccines showed higher expression of BDNF, IGF-1, IL-4, IFN-γ and lower IL-1ß, TNF-α and IL-6 than BCG. Taken together, combined vaccination with BCG and influenza vaccines presented synergistic effects on spatial cognition and hippocampal plasticity in rats.

A Single ssDNA Aptamer Binding to Mannose-Capped Lipoarabinomannan of Bacillus Calmette-Guérin Enhances Immunoprotective Effect against Tuberculosis.

Because Mycobacterium bovis, termed bacillus Calmette-Guérin (BCG), the only available used tuberculosis (TB) vaccine, retains immunomodulatory properties that limit its protective immunogenicity, there are continuous efforts to identify the immunosuppression mechanism as well as new strategies for improving the immunogenicity of BCG. Here, an ssDNA aptamer "antibody" BM2 specifically bound to the mannose-capped lipoarabinomannan (ManLAM) of BCG was selected. BM2 significantly blocked ManLAM-mannose receptor (MR) binding, triggered ManLAM-CD44 signaling, and enhanced M1 macrophage and Th1 activation via cellular surface CD44 in vitro and in vivo. BM2 enhanced immunoprotective effects of BCG against virulent Mycobacterium tuberculosis H37Rv infection in mice and monkeys models. Thus, we report a new mechanism of the interaction between ManLAM and CD44 on macrophages and CD4(+) T cells and reveal that ManLAM-binding membrane molecule CD44 is a novel target for the enhancement of BCG immunogenicity, and BM2 has strong potential as an immune enhancer for BCG.

Impact of Mycobacterium tuberculosis RD1-locus on human primary dendritic cell immune functions.

Modern strategies to develop vaccines against Mycobacterium tuberculosis (Mtb) aim to improve the current Bacillus Calmette-Guerin (BCG) vaccine or to attenuate the virulence of Mtb vaccine candidates. In the present study, the impact of wild type or mutated region of difference 1 (RD1) variants on the immunogenicity of Mtb and BCG recombinants was investigated in human primary dendritic cells (DC). A comparative analysis of transcriptome, signalling pathway activation, maturation, apoptosis, cytokine production and capacity to promote Th1 responses demonstrated that DC sense quantitative and qualitative differences in the expression of RD1-encoded factors--ESAT6 and CFP10--within BCG or Mtb backgrounds. Expansion of IFN-γ producing T cells was promoted by BCG::RD1-challenged DC, as compared to their BCG-infected counterparts. Although Mtb recombinants acted as a strong Th-1 promoting stimulus, even with RD1 deletion, the attenuated Mtb strain carrying a C-terminus truncated ESAT-6 elicited a robust Th1 promoting phenotype in DC. Collectively, these studies indicate a necessary but not sufficient role for the RD1 locus in promoting DC immune-regulatory functions. Additional mycobacterial factors are likely required to endow DC with a high Th1 polarizing capacity, a desirable attribute for a successful control of Mtb infection.

[miR-144 regulates BCG- and rapamycin-induced autophagy by targeting Atg4a in RAW264.7 cells].

OBJECTIVE: To study the effect of Bacilli Calmette-Guerin (BCG) on miRNAs (miR-21, miR-181a, miR-155 and miR-144) in RAW264.7 cells, and with miR-144 as an example, to verify the relationship of miR-144 and Atg4a, an autophagy-related gene, and study the mechanism underlying the regulatory effect of miR-144 on autophagy in the process of Mycobacterium tuberculosis infection. METHODS: RAW264.7 cells were treated respectively with starvation (12 hours), 50 ng/mL rapamycin (2 hours) and 10 nmol/L 3-methyl adenine (3-MA, 12 hours), and then stimulated with BCG for 12, 24 or 48 hours. The macrophages were collected for total RNA extraction. The expression levels of miR-21, miR-181a, miR-155 and miR-144 were detected by real-time quantitative PCR (qRT-PCR). Thereafter, we constructed the recombinant plasmids pMIR-Report-Atg4a and pMIR-Report-Atg4a mut. The targeting effect of miR-144 on Atg4a gene was verified by the dual-luciferase reporter assay system, Western blotting and qRT-PCR. RESULTS: After BCG stimulated RAW264.7 cells, the expression levels of miR-21, miR-155 and miR-144 were up-regulated, and miR-181a expression was down-regulated. The expressions of miR-21, miR-144, miR-155 and miR-181a increased 64 times, 52 times, 14 times and 1 times in the rapamycin group, respectively; the expressions of miR-21, miR-144, miR-155 and miR-181a decreased 1.22 times, 1.05 times, 1.54 times and 12.5 times in the 3-MA group, respectively. The dual-luciferase reporter assay system and Western blotting demonstrated that miR-144 could suppress Atg4a expression by targeting the specific 3'-untranslated region (3'UTR) sequence of Atg4a gene. CONCLUSION: miR-144 can directly inhibit the autophagy-related gene Atg4a expression and participate in the regulation of autophagy process in Mycobacterium tuberculosis infection.