Use of DosR and Rpf antigens from Mycobacterium tuberculosis to screen for latent and relapse tuberculosis infection in a tuberculosis endemic community of Huainan City.

The dormancy survival regulator (DosR) antigens upgraded during latency and resuscitation-promoting factors (Rpfs) expressed over the reactivation from dormant Mycobacterium tuberculosis (M. tuberculosis) could be used to diagnose tuberculosis (TB) at different stages. We performed a retrospective cohort study based on four groups, including healthy controls (HCs), active tuberculosis infections (ATBs), latent tuberculosis infections (LTBIs), and relapse tuberculosis infections (RTBs) enrolled between November 2020 and June 2021. Compared to the fusion protein E6-C10, combined with early secreted antigenic target 6 kDa (ESAT-6) and culture filtrate of 10 kDa (CFP-10), the DosR- or Rpf-encoded antigens could not elicit significant IFN-γ concentration for the diagnosis of ATB. Of note, the DosR antigens produce significantly more antigen-specific IFN-γ in LTBIs than Rpfs, and the levels of antigen-specific IFN-γ elicited in RTBs stimulated by Rpfs were higher than the DosR antigens. Among the DosR antigens, Rv2003c was the most immunogenic in diagnosing LTBIs, followed by Rv2007c and Rv2005c. As far as Rpfs are concerned, Rv0867c was the best antigen to identify RTBs, followed by Rv2389c and Rv1009. Both Rv2450c and Rv1884c showed relatively limited IFN-γ concentration in RTBs. Besides, the selected DosR antigens and Rpfs showed ideal specificity and inadequate sensitivity, which could have been enhanced by the fusion antigens prepared by the DosR antigens or Rpfs, respectively. The results of this study can provide more accurate detection methods for LTBIs and RTBs and could be used for screening the dormant M. tuberculosis throughout reactivation.

IRAK-M alters the polarity of macrophages to facilitate the survival of Mycobacterium tuberculosis.

BACKGROUND: Intracellular bacterium, Mycobacterium tuberculosis (M. tb), infects specifically macrophages as host cells. IRAK-M, a member of IRAK family, is a negative regulator in TLR signaling and specifically expresses in monocytes and macrophages. The role of IRAK-M in intracellular growth of M. tb and macrophage polarization was explored, for deeply understanding the pathogenesis of M. tb, the significance of IRAK-M to innate immunity and pathogen-host interaction. METHODS: IRAK-M expression was detected in M. tb infected macrophages and in human lung tissue of pulmonary tuberculosis with immunofluorescence staining, Western blot and immunohistochemistry. IRAK-M knock-down and over-expressing cell strains were constructed and intracellular survival of M. tb was investigated by acid-fast staining and colony forming units. Molecular markers of M1-type (pSTAT1 and iNOS) and M2-type (pSTAT6 and Arg-1) macrophages were detected using Western blot in IRAK-M knockdown U937 cells infected with M. tb H37Rv. U937 cells were stimulated with immunostimulant CpG7909 into M1 status and then infected with M. tb H37Rv. Expression of IRAK-M, IRAK-4 and iNOS was detected with immunofluorescence staining and Western blot, to evaluate the effect of IRAK-M to CpG directed M1-type polarization of macrophages during M. tb infection. Molecules related with macrophage's bactericidal ability such as Hif-1 and phosphorylated ERK1/2 were detected with immunohistochemistry and Western blot. RESULTS: IRAK-M increased in M. tb infected macrophage cells and also in human lung tissue of pulmonary tuberculosis. IRAK-M over-expression resulted in higher bacterial load, while IRAK-M interference resulted in lower bacterial load in M. tb infected cells. During M. tb infection, IRAK-M knockdown induced M1-type, while inhibited M2-type polarization of macrophage. M1-type polarization of U937 cells induced by CpG7909 was inhibited by M. tb infection, which was reversed by IRAK-M knockdown in U937 cells. IRAK-M affected Hif-1 and MAPK signaling cascade during M. tb infection. CONCLUSIONS: Conclusively, IRAK-M might alter the polarity of macrophages, to facilitate intracellular survival of M. tb and affect Th1-type immunity of the host, which is helpful to understanding the pathogenesis of M. tb.

A live attenuated BCG vaccine overexpressing multistage antigens Ag85B and HspX provides superior protection against Mycobacterium tuberculosis infection.

Tuberculosis (TB) remains one of the most menacing infectious diseases, although attenuated Mycobacterium bovis Bacillus Calmette-Guerin (BCG) vaccine has been widely used to protect children against primary TB. There are increasing evidences that rapid growing and dormant Mycobacterium tuberculosis (M. tuberculosis) coexist in vivo after infection. However, BCG vaccine only elicits cell-mediated immune responses to secretory antigens expressed by rapid growing pathogen. BCG vaccine is thus unable to thwart the reactivation of latent tuberculosis infection (LTBI), and its protection wanes over age after neonatal immunization. In order to extend its ability for a durable protection, a novel recombinant BCG (rBCG) strain, named rBCG::XB, was constructed by overexpressing immunodominant multistage antigens of Ag85B and HspX, which are expressed by both rapid replicating and dormant M. tuberculosis. Long-term protective effect and immunogenicity of rBCG::XB were compared with the parental BCG in vaccinated C57BL/6 mice. Our results demonstrated that rBCG::XB provided the stronger and long-lasting protection against M. tuberculosis H37Rv intranasal infection than BCG. The rBCG::XB not only elicited the more durable multistage antigen-specific CD4(+)Th1-biased immune responses and specific polyfunctional CD4(+)T cells but also augmented the CD8(+) CTL effects against Ag85B in vivo. In particular, higher levels of CD4(+) TEM and CD8(+) TCM cells, dominated by IL2(+) CD4(+) and CD8(+) TCM cells, were obtained in the spleen of rBCG::XB vaccinated mice. Therefore, our findings indicate that rBCG::XB is a promising candidate to improve the efficacy of BCG.

Long-Term Immunogenicity and In Vitro Prophylactic Protective Efficacy of M. tuberculosis Fusion Protein DR2 Combined with Liposomal Adjuvant DIMQ as a Boosting Vaccine for BCG.

The resuscitation of dormant Mycobacterium tuberculosis is an important cause of adult tuberculosis (TB) transmission. According to the interaction mechanism between M. tuberculosis and the host, the latency antigen Rv0572c and region of difference 9 (RD9) antigen Rv3621c were selected in this study to prepare the fusion protein DR2. Stimulating clinically diagnosed active tuberculosis infections (i.e., TB patients), latent tuberculosis infections, and healthy controls confirmed that T lymphocytes could recognize DR2 protein in the peripheral blood of TB-infected individuals more than subcomponent protein. The DR2 protein was then emulsified in the liposome adjuvant dimethyl dioctadecyl ammonium bromide, and imiquimod (DIMQ) was administered to C57BL/6 mice immunized with Bacillus Calmette-Guérin (BCG) vaccine to evaluate their immunogenicity. Studies have shown that DR2/DIMQ, a booster vaccine for BCG primary immunization, can elicit robust CD4+ Th1 cell immune response and predominant IFN-γ+ CD4+ effector memory T cells (TEM) subsets. Furthermore, the serum antibody level and the expression of related cytokines increased significantly with the extension of immunization time, with IL2+, CD4+, or CD8+ central memory T cells (TCM) subsets predominant in the long term. This immunization strategy showed matched prophylactic protective efficacy by performing in vitro challenge experiment. This result provides robust evidence that the novel subunit vaccine prepared by fusion protein DR2 combined with liposomal adjuvant DIMQ is a promising TB vaccine candidate for further preclinical trials as a booster vaccine for BCG.

Enhanced immunogenicity of the tuberculosis subunit Rv0572c vaccine delivered in DMT liposome adjuvant as a BCG-booster.

COVID-19 has affected the progress made in the prevention and treatment of tuberculosis (TB); hence, the mortality of tuberculosis has risen. Different strategies-based novel TB vaccine candidates have been developed. This study identifies strategies to overcome the limitations of Bacille Calmette-Guérin (BCG) in preventing latent infection and reactivation of TB. The latency antigen Rv0572c was selected based on the mechanism of interaction between Mycobacterium tuberculosis and its host. The rRv0572c protein was used to stimulate whole blood samples derived from patients with clinically diagnosed active TB (ATBs) or latent TB infections (LTBIs) and healthy control (HCs) donors, confirming that this protein can be recognized by T cells in patients with TB, especially LTBIs. C57BL/6 mice were used to investigate the immunogenicity of the rRv0572c protein emulsified in the liposome adjuvant dimethyldioctadecylammonium [DDA], monophosphoryl lipid A [MPLA], trehalose-6, 6'-dibehenate [TDB] (DMT). The results demonstrated that rRv0572c/DMT could boost BCG-primed mice to induce antigen-specific CD4+ T cell production and generate functional T cells dominated by antigen-specific CD8+ T cells. The rRv0572c/DMT vaccine could also trigger limited Th2 humoral immune responses. These findings suggest that rRv0572c/DMT is a potential subunit vaccine candidate that can be used as a booster vaccine for BCG.

Identification of novel biomarkers and candidate small-molecule drugs in cutaneous melanoma by comprehensive gene microarrays analysis.

Background: Melanoma is a pernicious skin cancer with high aggressiveness. This study aimed to identify potential novel biomarkers associated with the prognosis and pathogenesis of cutaneous melanoma and to explore new targeted drugs for melanoma. Methods: Two Gene Expression Omnibus (GEO) microarray datasets, GSE3189 and GSE7553 were combined to analyze the differentially expressed genes (DEGs). To better understand the DEGs in the melanoma pathogenesis, we performed gene enrichment analyses and established a protein-protein interaction network (PPI). The survival analyses for key genes were conducted based on the GEPIA platform. Finally, we mined the CMap database to explore potential small-molecule drugs to target the obtained DEGs. Results: In short, we identified 500 DEGs between cutaneous melanoma samples and normal samples. The PPI network was established with 349 nodes and 1251 edges. Signaling pathway analysis showed that these genes play a vital role in ECM-receptor interactions, the PPAR signaling pathway and pathways in cancer. Eight DEGs with a relatively high degree of connectivity (CDC45, CENPF, DTL, FANCI, GINS2, HJURP, TPX2 and TRIP13) were selected as hub-genes that remarkably correlated to a poor survival rate. Based on 500 DEGs, 20 small-molecule drugs that potentially target genes with abnormal expression in cutaneous melanoma were obtained from the CMap database. Among these compounds, we found that menadione has the greatest therapeutic value for melanoma. Conclusions: In conclusion, we identified the 8 candidate biomarkers and potential key signaling pathways in cutaneous melanoma through comprehensive microarray analyses. The identified candidate drugs have provided several directive significances for the synthesis medicine for melanoma.

Enhanced tuberculosis clearance through the combination treatment with recombinant adenovirus-mediated granulysin delivery.

Rationale: Tuberculosis (TB) remains the leading cause of death among infectious diseases worldwide. Poor compliance of TB patients to the lengthy treatment increases the risk of relapse and leads to the emergence of multidrug-resistant and extensively drug-resistant TB (MDR-TB and XDR-TB). More effective therapies for TB are urgently needed. We hypothesized that granulysin-mediated clearance of M. tuberculosis parasited inside and outside of alveolar macrophages in presumptive infected hosts might enhance the chemotherapeutic efficacy on TB. Methods: Recombinant adenovirus type 5 (rAd5) based therapeutic vaccines rAdhGLi and rAdhGLs (rAds) were respectively developed to express intracellular and extracellular granulysin. The ex vivo bactericidal effects of rAdhGLi and rAdhGLs were evaluated on U937 and RAW264.7 cells. The efficacy of immunotherapy with both rAdhGLi and rAdhGLs on TB SCID mice, or immunotherapy combined with chemotherapy on drug-susceptible TB or MDR-TB mouse models were further evaluated. Results: rAdhGLs, as well as rAdhGLi, showed a direct bactericidal effect on extracellular or intracellular M. tuberculosis H37Rv and MDR-TB clinical strains, respectively. Immunotherapy with a dose of 109 PFU of rAdhGLi and 109 PFU of rAdhGLs demonstrated a more significant bactericidal effect on M. tuberculosis H37Rv infected SCID mice and prolonged their survival periods than rAdhGLi or rAdhGLs alone. More importantly, chemotherapy combined with rAds immunotherapy shortened the chemotherapeutic duration to 4 months on M. tuberculosis H37Rv infected mice and prevented the relapse. Combination of rAds with chemotherapy on MDR-TB mice also more significantly decreased organ bacterial load than their single use. Conclusions: Delivery of granulysin by recombinant adenovirus to the infected lung could enhance the clearance of TB in vivo and might be a promising adjunct therapeutic vaccine for TB and MDR-TB.

miR-199a-5p suppresses epithelial- mesenchymal-transition in anaplastic thyroid carcinoma cells via targeting Snail signals.

MicroRNAs (miRNAs) have been validated to play prominent roles in the occurrence and development of anaplastic thyroid carcinoma (ATC). miR-199a-5p was previously reported to act as a tumor suppressor or oncomiRNA in various types of cancer. However, its accurate expression, function, and mechanism in ATC remain unclear. Here, we find that miR-199a-5p is significantly downregulated in ATC tissues compared with adjacent non-cancerous tissues. Overexpression of miR-199a-5p significantly inhibits migration and invasion of ATC cells in vitro, and lung metastasis in vivo. Importantly, miR-199a-5p suppresses epithelial-mesenchymal transition (EMT) both in vitro and in vivo by targeting Snail. Taken together, this study reveals that miR-199a-5p is critical to the EMT progression in ATC cells. Targeting the pathway described here may be a novel approach for inhibiting metastasis of ATC.

Hepatoprotective effect of essential oils of Nepeta cataria L. on acetaminophen-induced liver dysfunction.

Nepeta cataria L. has long been used in folk food and medicine for several functions. Essential oils (EOs) were extracted from Nepeta cataria L. by supercritical fluid extraction. The results of animal experiments showed that EOs from Nepeta cataria L. significantly attenuated acetaminophen-induced liver damage. Further study confirmed that EOs were able to increase mRNA expression of uridine diphosphate glucuronosyltransferases (UGTs) and sulfotransferases (SULTs), as well as inhibit CYP2E1 activities, and thereby suppressed toxic intermediate formation. Nrf-2 activation might be involved in EOs-induced up-regulation of Phase II enzymes. Collectively, our data provide evidence that EOs protect the liver against acetaminophen-induced liver injury mainly by accelerating acetaminophen harmless metabolism, implying that EOs can be considered as a potential natural resource to develop hepatoprotective agent.

ECSM2, an endothelial specific VE-cadherin binding protein, has a tyrosine phosphorylation site essential to cell migration.

Endothelial cell-specific molecule 2 (ECSM2) is a transmembrane protein located in cell-cell junction of endothelial cells (EC). ECSM2 was determined to play an important role in vascular development, EC migration, apoptosis and proliferation, however, no functional domains were determined in intracellular and extracellular region of ECSM2. In current work, functional domains of ECSM2, the relationship of ECSM2 with other endothelial specific protein such as VE-cadherin and the role of ECSM2 in neovascular diseases were determined. It was shown that the 54th amino acid residue of ECSM2 extracellular domain was a tyrosine phosphorylation site, whose mutation led to the loss of EGF-induced tyrosine phosphorylation and inhibition of cell migration. In primary human umbilical vein endothelial cells, ECSM2 bound with VE-cadherin and VEGF stimulation enhanced their binding. In hepatocellular carcinoma, ECSM2 expression was increased, as compared with para-cancerous tissue. This data firstly revealed the functional sites of ECSM2, the crosstalk between ECSM2 and other endothelial cell specific molecules, the expression of ECSM2 in tissues of angiogenesis diseases, thus providing understanding about ECSM2 in depth.

Heterologous Boost Following Mycobacterium bovis BCG Reduces the Late Persistent, Rather Than the Early Stage of Intranasal Tuberculosis Challenge Infection.

Adults are the leading population affected by tuberculosis (TB) epidemic and death. Developing an effective vaccine against adult TB is urgently needed. Mycobacterium bovis Bacillus Calmette-Guerin (BCG) prime-heterologous boost strategy has been explored extensively to protect adults against primary TB infection, but the majority of experimental regimens have not improved the protection primed by the BCG vaccine. The reason attributed to the failure remains unknown. In this study, CTT3H-based vaccines, namely DMT adjuvanted CTT3H subunit or DNA vaccine (pCTT3H-DMT), and recombinant adenovirus rAdCTT3H were constructed. Protective efficacy and immunogenicity of BCG prime-CTT3H based boosters were compared in C57BL/c mice models of primary or late persistent TB infection. Similar protective efficacy against early intranasal infection was provided by different CTT3H-based vaccines alone in vaccinated mice, and their protection was inferior to that of the BCG vaccine. In addition, CTT3H-based heterologous boosters did not enhance the protection conferred by the BCG vaccine against primary infection. However, all of these three boosters provided stronger protection against late persistent TB infection than BCG alone, regardless of vaccine types. Although BCG prime-boosters elicited Th1-biased responses to the antigen CTT3H, the number of CTT3H-sepcific IFN-γ-expressing TEM (CD62LloCD44hi) and IL-2-expressing TCM (CD62LhiCD44hi) cells in the spleen was not improved before exposure to Mycobacterium tuberculosis infection. In contrast, IFN-γ+ TEM and IL-2+ TCM cells in spleens, especially in lungs were significantly increased in BCG prime-boosters after exposure vaccination. Our results indicate that BCG prime-boost strategy might be a promising measure for the prevention against late persistent TB infection by induction of IFN-γ+ TEM and IL-2+ TCM cells in the lung, which can be used as alternative biomarkers for guiding the clinical practice and future development of TB vaccine for adults.

A Multistage Subunit Vaccine Effectively Protects Mice Against Primary Progressive Tuberculosis, Latency and Reactivation.

Adult tuberculosis (TB) is the main cause of TB epidemic and death. The infection results mainly by endogenous reactivation of latent TB infection and secondarily transmitted by exogenous infection. There is no vaccine for adult TB. To this end, we first chose antigens from a potential antigenic reservoir. The antigens strongly recognized T cells from latent and active TB infections that responded to antigens expressed by Mycobacterium tuberculosis cultured under different metabolic states. Fusions of single-stage polyprotein CTT3H, two-stage polyprotein A1D4, and multistage CMFO were constructed. C57BL/6 mice vaccinated with DMT adjuvant ed CMFO (CMFO-DMT) were protected more significantly than by CTT3H-DMT, and efficacy was similar to that of the only licensed vaccine, Bacillus Calmette-Guérin (BCG) and A1D4-DMT in the M. tuberculosis primary infection model. In the setting of BCG priming and latent TB infection, M. tuberculosis in the lung and spleen was eliminated more effectively in mice boosted with CMFO-DMT rather than with BCG, A1D4-DMT, or CTT3H-DMT. In particular, sterile immunity was only conferred by CMFO-DMT, which was associated with expedited homing of interferon-gamma+CD4+ TEM and interleukin-2+ TCM cells from the spleen to the infected lung. CMFO-DMT represents a promising candidate to prevent the occurrence of adult TB through both prophylactic and therapeutic methods, and warrants assessment in preclinical and clinical trials.

Mycobacterium tuberculosis multistage antigens confer comprehensive protection against pre- and post-exposure infections by driving Th1-type T cell immunity.

There is an urgent need for a vaccine against tuberculosis (TB) that is more effective than the current sole licensed option. However, target antigens of Mycobacterium tuberculosis with the vaccine potential remain elusive. Five immunodominant antigens with characteristic expressions at the stages of primary infection (Ag85A), the regulation of nutrition and metabolism when transferring from rapid growth to latency (PhoY2 and Rv3407), latency (Rv2626c), and reactivation (RpfB) were selected to construct the fusion polyprotein WH121, which has better immunogenicity and protection than each multistage antigen. DMT adjuvanted WH121 vaccinated C57BL/6 mice could confer persistent and significant protection against the respiratory challenge with 80 CFU of virulent M. tuberculosis H37Rv at 9 and 18 weeks after immunization, as the BCG vaccine did. Moreover, WH121/DMT could boost the BCG primed mice against post-exposure infection, and more significantly inhibit the growth of M. tuberculosis in the spleen than BCG repeat vaccination. The protection elicited by WH121/DMT is attributed to the WH121-specific Th1-type biased immune responses, characterized by increased antigen-specific IgG2a/IgG1 ratio and high levels of IFN-γ secreted by the splenocytes of vaccinated mice. In particular, high levels of IFN-γ+ TEM cells in the spleen are an effective biomarker for the vaccine-induced early protection, and the persistent protection mainly depends on the increasing IL-2+IFN-γ+CD4+ and CD8+ T cells, especially IL-2+ TCM cells. These findings demonstrate that multistage-specific antigens might be promising targets for the next generation TB vaccine, and a combination of these antigens such as WH121/DMT is required for further preclinical evaluation.