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Background/purpose: The long-term outcomes of implants placed in grafted sinuses using recombinant human bone morphogenetic protein-2 (rhBMP-2) are unclear. This study aimed to compare 3- and 5-year implant survival rates and marginal bone loss (MBL) during functional loading. Materials and methods: In this retrospective study, we analyzed 63 implants inserted after maxillary sinus floor augmentation (MSFA) in 45 patients between January 2016 and April 2019. The outcome variables were: 1) 3- and 5-year cumulative survival rates of the implants and 2) MBL after functional loading. Other assessed variables included patient demographic information, preoperative residual bone height (RBH), surgical site, implant length and diameter, graft material, healing period before loading, prosthetic type, and opposing dentition. Results: The cumulative 3- and 5-year survival rates of the implants were 100% in the rhBMP-2 group and 95.5% and 86.4% in the non-rhBMP-2 group, respectively. The average 3- and 5-year MBL were 1.14 ± 0.67 mm, 1.30 ± 0.74 mm in the rhBMP-2 group and 1.68 ± 0.90 mm, 2.27 ± 1.29 mm in the non-rhBMP-2 group, respectively. Significant differences were observed between 3 and 5 years between the two groups. Conclusion: Addition of the rhBMP-2 to the graft materials positively affects implant placement in the grafted maxillary sinus in terms of implant survival and MBL when preoperative RBH is unfavorable.

Abstract Background Mycobacterium tuberculosis is the causative agent of tuberculosis (TB), and its pathogenicity is associated with its ability to evade the host defense system. The secretory form of the chorismate mutase of M. tuberculosis (TBCM, encoded by Rv1885c) is assumed to play a key role in the pathogenesis of TB; however, the mechanism remains unknown. Methods A tbcm deletion mutant (B∆tbcm) was generated by targeted gene knockout in BCG to investigate the pathogenic role of TBCM in mice or macrophages. We compared the pathogenesis of B∆tbcm and wild-type BCG in vivo by measuring the bacterial clearance rate and the degree of apoptosis. Promotion of the intrinsic apoptotic pathway was evaluated in infected bone marrow-derived macrophages (BMDMs) by measuring apoptotic cell death, loss of mitochondrial membrane potential and translocation of pore-forming proteins. Immunocytochemistry, western blotting and real-time PCR were also performed to assess the related protein expression levels after infection. Furthermore, these findings were validated by complementation of tbcm in BCG. Results Deletion of the tbcm gene in BCG leads to reduced pathogenesis in a mouse model, compared to wild type BCG, by promoting apoptotic cell death and bacterial clearance. Based on these findings, we found that intrinsic apoptosis and mitochondrial impairment were promoted in B∆tbcm-infected BMDMs. B∆tbcm down-regulates the expression of Bcl-2, which leads to mitochondrial outer membrane permeabilization (MOMP), culminating in cytochrome c release from mitochondria. Consistent with this, transcriptome profiling also indicated that B∆tbcm infection is more closely related to altered mitochondrial-related gene expression than wild-type BCG infection, suggesting an inhibitory role of TBCM in mitochondrial dysfunction. Moreover, genetic complementation of B∆tbcm (C∆tbcm) restored its capacity to inhibit mitochondria-mediated apoptotic cell death. Conclusions Our findings demonstrate the contribution of TBCM to bacterial survival, inhibiting intrinsic apoptotic cell death of macrophages as a virulence factor of M. tuberculosis complex (MTBC) strains, which could be a potential target for the development of TB therapy.

Despite the great diversity of malonate semialdehyde decarboxylases (MSADs), one of five subgroups of the tautomerase superfamily (TSF) found throughout the biosphere, their distribution among strains within the genus Mycobacterium remains unknown. In this study, we sought to investigate the phylogenetic distribution of MSAD genes of mycobacterial species via genome analysis of 192 different reference Mycobacterium species or subspecies retrieved from NCBI databases. We found that in a total of 87 of 192 strains (45.3%), MSAD-1 and MSAD-2 were distributed in an exclusive manner among Mycobacterium species except for 12 strains, including Mycobacterium chelonae members, with both in their genome. Of note, Mycobacterium strains better adapted to the host and of high virulence potential, such as the Mycobacterium tuberculosis complex, Mycobacterium leprae, Mycobacterium marinum, Mycobacterium ulcerans, and Mycobacterium avium subsp. paratuberculosis, had no orthologs of MSAD in their genome, suggesting MSAD loss during species differentiation in pathogenic slow-growing Mycobacterium. To investigate the MSAD distribution among strains of M. avium subspecies, the genome sequences of a total of 255 reference strains from the four subspecies of M. avium (43 of subspecies avium, 162 of subspecies hominissuis, 49 of subspecies paratuberculosis, and 1 of subspecies silvaticum) were further analyzed. We found that only 121 of 255 strains (47.4%) had MSADs in their genome, with none of the 49 M. avium subsp. paratuberculosis strains having MSAD genes. Even in 13 of 121 M. avium strains with the MSAD-1 gene in their genome, deletion mutations in the 98th codon causing premature termination of MSAD were found, further highlighting the occurrence of MSAD pseudogenization during species or subspecies differentiation of M. avium. In conclusion, our data indicated that there are two distinct types of MSADs, MSAD-1 and MSAD-2, among strains in the Mycobacterium genus, but more than half of the strains, including pathogenic mycobacteria, M. tuberculosis and M. leprae, have no orthologs in their genome, suggesting MSAD loss during host adaptation of pathogenic mycobacteria. In the future, the role of two distinct MSADs, MSAD-1 and MSAD-2, in mycobacterial pathogenesis or evolution should be investigated.

IntroductionSkin vaccination using dissolving microneedle patch (MNP) technology for transdermal delivery is a promising vaccine delivery strategy to overcome the limitations of the existing vaccine administration strategies using syringes. To improve the traditional microneedle mold fabrication technique, we introduced droplet extension (DEN) to reduce drug loss. Tuberculosis remains a major public health problem worldwide, and BCG revaccination had failed to increase the protective efficacy against tuberculosis. We developed an MNP with live Mycobacterium paragordonae (Mpg) (Mpg-MNP) as a candidate of tuberculosis booster vaccine in a heterologous prime-boost strategy to increase the BCG vaccine efficacy.Materials and methodsThe MNPs were fabricated by the DEN method on a polyvinyl alcohol mask film and hydrocolloid-adhesive sheet with microneedles composed of a mixture of mycobacteria and hyaluronic acid. We assessed the transdermal delivery efficiency by comparing the activation of the dermal immune system with that of subcutaneous injection. A BCG prime Mpg-MNP boost regimen was administered to a mouse model to evaluate the protective efficacy against M. tuberculosis.ResultsWe demonstrated the successful transdermal delivery achieved by Mpg-MNP compared with that observed with BCG-MNP or subcutaneous vaccination via an increased abundance of MHCII-expressing Langerin+ cells within the dermis that could migrate into draining lymph nodes to induce T-cell activation. In a BCG prime-boost regimen, Mpg-MNP was more protective than BCG-only immunization or BCG-MNP boost, resulting in a lower bacterial burden in the lungs of mice infected with virulent M. tuberculosis. Mpg-MNP-boosted mice showed higher serum levels of IgG than BCG-MNP-boosted mice. Furthermore, Ag85B-specific T-cells were activated after BCG priming and Mpg-MNP boost, indicating increased production of Th1-related cytokines in response to M. tuberculosis challenge, which is correlated with enhanced protective efficacy.DiscussionThe MNP fabricated by the DEN method maintained the viability of Mpg and achieved effective release in the dermis. Our data demonstrate a potential application of Mpg-MNP as a booster vaccine to enhance the efficacy of BCG vaccination against M. tuberculosis. This study produced the first MNP loaded with nontuberculous mycobacteria (NTM) to be used as a heterologous booster vaccine with verified protective efficacy against M. tuberculosis.

For clinical application by dendritic cell (DC)-based cancer immunotherapy, a proper adjuvant system to elicit a strong anticancer immune response is needed. Here, we investigated the potential of chorismate mutase (TBCM, Rv1885c), a putative Mycobacterium tuberculosis (TB) virulence factor, as an immunoadjuvant in DC-based tumor immunotherapy. First, we found that TBCM functionally activated DCs by upregulating costimulatory molecules, increasing the secretion of proinflammatory cytokines, enhancing migration and inducing the Th1-type immune response in a dose-dependent manner via TLR4-mediated signaling. In addition, subcutaneous injection of TBCM-activated DCs loaded with cell lysates led to reduced tumor mass, enhanced mouse survival and lowered tumor incidence in lung carcinoma (LLC) cell-bearing mice. This is mainly mediated by functional cytotoxic T lymphocyte-mediated oncolytic activity and inhibition of cancer proliferation- and metastasis-related genes. Moreover, TBCM-induced DCs can also generate memory CD4 T cells and exert long-term tumor prevention effects. In conclusion, our findings suggest that TBCM (Rv1885c), a novel TLR4 agonist, could be used as an immunoadjuvant for DC-based cancer immunotherapy.

At present, concerns that the recent global emergence of SARS-CoV-2 variants could compromise the current vaccines have been raised, highlighting the urgent demand for new vaccines capable of eliciting T cell-mediated immune responses, as well as B cell-mediated neutralizing antibody production. In this study, we developed a novel recombinant Mycobacterium paragordonae expressing the SARS-CoV-2 receptor-binding domain (RBD) (rMpg-RBD-7) that is capable of eliciting RBD-specific immune responses in vaccinated mice. The potential use of rMpg-RBD-7 as a vaccine for SARS-CoV-2 infections was evaluated in in vivo using mouse models of two different modules, one for single-dose vaccination and the other for two-dose vaccination. In a single-dose vaccination model, we found that rMpg-RBD-7 versus a heat-killed strain could exert an enhanced cell-mediated immune (CMI) response, as well as a humoral immune response capable of neutralizing the RBD and ACE2 interaction. In a two-dose vaccination model, rMpg-RBD-7 in a two-dose vaccination could also exert a stronger CMI and humoral immune response to neutralize SARS-CoV-2 infections in pseudoviral or live virus infection systems, compared to single dose vaccinations of rMpg-RBD or two-dose RBD protein immunization. In conclusion, our data showed that rMpg-RBD-7 can lead to an enhanced CMI response and humoral immune responses in mice vaccinated with both single- or two-dose vaccination, highlighting its feasibility as a novel vaccine candidate for SARS-CoV-2. To the best of our knowledge, this study is the first in which mycobacteria is used as a delivery system for a SARS-CoV-2 vaccine.

Background Macrophage migration inhibitory factor (MIF) is a pleotropic inflammatory cytokine that is overexpressed in a number of cancer types including most types of human cancer. Inhibition of MIF signaling can restore anticancer immune responses in tumor microenvironments. In this study, we aimed to develop a therapeutic vaccine capable of inhibiting tumor development by inducing anti-MIF immune responses.Methods We introduced a recombinant Mycobacterium smegmatis (rSmeg-hMIF-hIL-7) vaccine that could deliver a fusion protein of human macrophage migration inhibitory factor (MIF) and interleukin 7, which could act as a target antigen and as an adjuvant of cancer vaccine, respectively. We checked the anticancer potential of the vaccine in a tumor-bearing mouse model.Results We found that rSmeg-hMIF-hIL-7 showed enhanced oncolytic activity compared with PBS, BCG or Smeg in MC38-bearing mice, and there was an increase in the humoral and cell-mediated immune responses against MIF. rSmeg-hMIF-hIL-7 can also induce a neutralizing effect regarding MIF tautomerase activity in the serum of vaccinated mice. We also found downregulation of MIF, CD74, and CD44, which are related to the MIF signaling pathway and PI3K/Akt and MMP2/9 signaling, which are regulated by MIF in the tumor tissue of rSmeg-hMIF-hIL-7-vaccinated mice, suggesting a significant role of the anti-MIF immune response to rSmeg-hMIF-hIL-7 in its anticancer effect. In addition, rSmeg-hMIF-hIL-7 treatment led to enhanced activation of CD4+ and CD8+ T cells in the tumor regions of vaccinated mice, also contributing to the anticancer effect. This trend was also found in LLC-bearing and PanO2-bearing mouse models. In addition, rSmeg-hMIF-hIL-7 treatment exerted an enhanced anticancer effect with one of the immune checkpoint inhibitors, the anti-PD-L1 antibody, in a tumor-bearing mouse model.Conclusions In conclusion, our data showed that rSmeg-hMIF-hIL-7 exerts a strong antitumor immune response in mice, possibly by inhibiting the MIF-dependent promotion of tumorigenesis by the anti-MIF immune response and via enhanced cytotoxic T cell recruitment into tumor microenvironments. We also found that it also exerted an enhanced anticancer effect with immune checkpoint inhibitors. These results suggest that rSmeg-hMIF-hIL-7 is a potential adjuvant for cancer immunotherapy. This is the first report to prove anticancer potential of immunotherapeutic vaccine targeting immune response against MIF.

A coronavirus SARS-CoV-2, which has caused the pandemic viral pneumonia disease COVID-19, significantly threatens global public health, highlighting the need to develop effective and safe vaccines against its infection. In this study, we developed a novel DNA vaccine candidate against SARS-CoV-2 by expressing a chimeric protein of its receptor-binding domain (RBD) fused to a 33-bp sequence (11 aa) from the hepatitis B virus (HBV) preS1 region with a W4P mutation (W4P-RBD) at the N-terminal region and evaluated its immunogenicity. In vitro transfection experiments in multiple cell lines demonstrated that W4P-RBD vs. wild-type RBD protein (W-RBD) led to enhanced production of IL-6 and TNFα at the transcription and translation levels, suggesting the adjuvant potential of N-terminal HBV preS1 sequences for DNA vaccines against SARS-CoV-2. W4P-RBD also led to enhanced production of IgG and IgA, which can neutralize and block SARS-CoV-2 infection in both blood sera and bronchoalveolar lavage (BAL) fluid from the lung in vaccinated mice. Additionally, W4P-RBD led to an enhanced T-cell-mediated cellular immune response under S1 protein stimulation. In summary, W4P-RBD led to robust humoral and cell-mediated immune responses against SARS-CoV-2 in vaccinated mice, highlighting its feasibility as a novel DNA vaccine to protect against SARS-CoV-2 infection.