Yujiro Hirose

Trophic Effects and Regenerative Potential of Mobilized Mesenchymal Stem Cells from Bone Marrow and Adipose Tissue as Alternative Cell Sources for Pulp/Dentin Regeneration:

Dental pulp stem cell (DPSC) subsets mobilized by granulocyte-colony-stimulating factor (G-CSF) are safe and efficacious for complete pulp regeneration. The supply of autologous pulp tissue, however, is very limited in the aged. Therefore, alternative sources of mesenchymal stem/progenitor cells (MSCs) are needed for the cell therapy. In this study, DPSCs, bone marrow (BM), and adipose tissue (AD)-derived stem cells of the same individual dog were isolated using G-CSF-induced mobilization (MDPSCs, MBMSCs, and MADSCs). The positive rates of CXCR4 and G-CSFR in MDPSCs were similar to MADSCs and were significantly higher than those in MBMSCs. Trophic effects of MDPSCs on angiogenesis, neurite extension, migration, and anti-apoptosis were higher than those of MBMSCs and MADSCs. Pulp-like loose connective tissues were regenerated in all three MSC transplantations. Significantly higher volume of regenerated pulp and higher density of vascularization and innervation were observed in response to MDPSCs compared to MBMSC and MADSC transplantation. Collagenous matrix containing dentin sialophosphoprotein (DSPP)-positive odontoblast-like cells was the highest in MBMSCs and significantly higher in MADSCs compared to MDPSCs. MBMSCs and MADSCs, therefore, have potential for pulp regeneration, although the volume of regenerated pulp tissue, angiogenesis, and reinnervation, were less. Thus, in conclusion, an alternative cell source for dental pulp/dentin regeneration are stem cells from BM and AD tissue.

Therapeutic Potential of Dental Pulp Stem Cell Secretome for Alzheimer's Disease Treatment: An In Vitro Study.

The secretome obtained from stem cell cultures contains an array of neurotrophic factors and cytokines that might have the potential to treat neurodegenerative conditions. Alzheimers disease (AD) is one of the most common human late onset and sporadic neurodegenerative disorders. Here, we investigated the therapeutic potential of secretome derived from dental pulp stem cells (DPSCs) to reduce cytotoxicity and apoptosis caused by amyloid beta (Aβ) peptide. We determined whether DPSCs can secrete the Aβ-degrading enzyme, neprilysin (NEP), and evaluated the effects of NEP expression in vitro by quantitating Aβ-degrading activity. The results showed that DPSC secretome contains higher concentrations of VEGF, Fractalkine, RANTES, MCP-1, and GM-CSF compared to those of bone marrow and adipose stem cells. Moreover, treatment with DPSC secretome significantly decreased the cytotoxicity of Aβ peptide by increasing cell viability compared to nontreated cells. In addition, DPSC secretome stimulated the endogenous survival factor Bcl-2 and decreased the apoptotic regulator Bax. Furthermore, neprilysin enzyme was detected in DPSC secretome and succeeded in degrading Aβ 1–42 in vitro in 12 hours. In conclusion, our study demonstrates that DPSCs may serve as a promising source for secretome-based treatment of Alzheimers disease.

Injection of Dental Pulp Stem Cells Promotes Healing of Damaged Bladder Tissue in a Rat Model of Chemically Induced Cystitis

Dental pulp stem cells (DPSCs) are reported as sources of mesenchymal stem cells (MSCs). MSCs are used as cell therapy options for various diseases. The present study examined the healing effects of DPSC injection on damaged bladder tissue in a chemically induced cystitis rat model. Cystitis was induced by hydrochloride injection into the bladder of female F344/NSlc rats. On the following day, DPSCs suspended in phosphate-buffered saline (PBS) were injected into the bladder and maintained for 1 h (DPSC injection group), while PBS alone was injected as the standard for comparison (PBS injection group). After 2 days following injection, considerable submucosal edema, vascular structure destruction, hemorrhage, and inflammatory cell invasion were observed both in the DPSC and PBS injection groups, with no difference in their degree of submucosal edema and hemorrhage. Six days after injection, vascular structure regeneration was observed in both groups; however, unlike the DPSC injection group, the PBS injection group showed traces of submucosal edema and hemorrhage. These results correlated with tissue concentrations of myeloperoxidase (MPO) and the inflammatory cytokines IL-1β, IL-6, and TNF-α. Furthermore, the intercontraction interval was prolonged, and the frequency of nociceptive behaviors was reduced in the DPSC injection group compared with the PBS injection group. DPSCs were found on the bladder epithelium until day 3 after injection. In the DPSC-conditioned media (CM), the trophic factors FGF-2, VEGF, and the C-C and C-X-C families of chemokines were detected. The results of DPSC injection into the cystitis rat model suggested that the injected cells promote the healing of the damaged bladder tissue by exerting various trophic effects while localizing on the bladder epithelium and that MSC injection is a potential novel therapy for interstitial cystitis/painful bladder syndrome.

Zinc metalloproteinase ZmpC suppresses experimental pneumococcal meningitis by inhibiting bacterial invasion of central nervous systems

ABSTRACT Streptococcus pneumoniae is a leading cause of bacterial meningitis. Here, we investigated whether pneumococcal paralogous zinc metalloproteases contribute to meningitis onset. Findings of codon-based phylogenetic analyses indicated 3 major clusters in the Zmp family; ZmpA, ZmpC, and ZmpB, with ZmpD as a subgroup. In vitro invasion assays of human brain microvascular endothelial cells (hBMECs) showed that deletion of the zmpC gene in S. pneumoniae strain TIGR4 significantly increased bacterial invasion into hBMECs, whereas deletion of either zmpA or zmpB had no effect. In a mouse meningitis model, the zmpC deletion mutant exhibited increased invasion of the brain and was associated with increased matrix metalloproteinase-9 in plasma and mortality as compared with the wild type. We concluded that ZmpC suppresses pneumococcal virulence by inhibiting bacterial invasion of the central nervous system. Furthermore, ZmpC illustrates the evolutional theory stating that gene duplication leads to acquisition of novel function to suppress excessive mortality.

Evolutionary inactivation of a sialidase in group B Streptococcus

Group B Streptococcus (GBS) is a leading cause of bacterial sepsis and meningitis in newborns. GBS possesses a protein with homology to the pneumococcal virulence factor, NanA, which has neuraminidase (sialidase) activity and promotes blood-brain barrier penetration. However, phylogenetic sequence and enzymatic analyses indicate the GBS NanA ortholog has lost sialidase function – and for this distinction we designate the gene and encoded protein nonA/NonA. Here we analyze NonA function in GBS pathogenesis, and through heterologous expression of active pneumococcal NanA in GBS, potential costs of maintaining sialidase function. GBS wild-type and ΔnonA strains lack sialidase activity, but forced expression of pneumococcal NanA in GBS induced degradation of the terminal sialic acid on its exopolysaccharide capsule. Deletion of nonA did not change GBS-whole blood survival or brain microvascular cell invasion. However, forced expression of pneumococcal NanA in GBS removed terminal sialic acid residues from the bacterial capsule, restricting bacterial proliferation in human blood and in vivo upon mouse infection. GBS expressing pneumococcal NanA had increased invasion of human brain microvascular endothelial cells. Thus, we hypothesize that nonA lost enzyme activity allowing the preservation of an effective survival factor, the sialylated exopolysaccharide capsule.

Composition of salivary microbiota in elderly subjects

Frailty is gaining attention worldwide with the aging of society. Despite the potential lethality and multiple signs and symptoms in affected individuals, preclinical detection of early manifestations leading to frailty syndrome have not been established. We speculated that the composition of the oral microbiota is associated with general frailty, as well as a relationship between gut microbiota and general health condition. In the present study, we investigated the salivary microbiota composition in samples from healthy and frail elderly individuals using 16S rRNA sequencing analysis for characterization. We found a significant difference in diversity between elderly individuals living in a nursing home (EN) and healthy control (HC) subjects, as well as in the microbiota composition at the phyla level. A supervised orthogonal partial least squared discriminant analysis (OPLS-DA) revealed a significant difference in clear classification trend between the EN and HC groups, with all observations falling within the Hotellings T2 (0.95) ellipse, with model fitness parameters of R2(cum) = 0.937 and Q2(cum) = 0.888, respectively. In addition, the score plots by unsupervised principal component analysis (PCA) showed a clear classification trend in both groups. Our findings suggest that general frailty is associated with oral microbiota composition and formation.

Identification of evolutionarily conserved virulence factor by selective pressure analysis of Streptococcus pneumoniae

Streptococcus pneumoniae is a pathogen that poses one of the greatest threats to human health, causing pneumonia, sepsis, and meningitis. Here, we investigated the evolutionary selective pressures on 16 pneumococcal choline-binding cell-surface proteins. Phylogenetic and molecular analyses revealed that the cbpJ and lytA genes had the highest codon rates under negative selection among those examined. Although LytA is an important virulence factor inducing pneumococcal autolysis, the role of CbpJ in pneumococcal pathogenesis is unknown. We therefore examined whether CbpJ acts as a virulence factor in vitro and in vivo. In neutrophil bactericidal assays, the cbpJ mutant showed reduced survival as compared to wild-type (WT) S. pneumoniae. Mice intranasally infected with a cbpJ mutant strain showed a lower bacterial burden in bronchoalveolar lavage fluid and higher survival rate than those infected with WT cells, whereas no differences were observed between strains by intravenous infection. Thus, molecular evolutionary analysis is a powerful tool that reveals the importance of virulence factors in real-world infection and transmission, since calculations are performed based on bacterial genome diversity following transmission of infection in an uncontrolled population. In addition, evolutionarily conserved virulence factors are candidate therapeutic targets or vaccine antigens.

Competence-induced protein Ccs4 facilitates pneumococcal invasion into brain tissue and virulence in meningitis

ABSTRACT Streptococcus pneumoniae is a major pathogen that causes pneumonia, sepsis, and meningitis. The candidate combox site 4 (ccs4) gene has been reported to be a pneumococcal competence-induced gene. Such genes are involved in development of S. pneumoniae competence and virulence, though the functions of ccs4 remain unknown. In the present study, the role of Ccs4 in the pathogenesis of pneumococcal meningitis was examined. We initially constructed a ccs4 deletion mutant and complement strains, then examined their association with and invasion into human brain microvascular endothelial cells. Wild-type and Ccs4-complemented strains exhibited significantly higher rates of association and invasion as compared to the ccs4 mutant strain. Deletion of ccs4 did not change bacterial growth activity or expression of NanA and CbpA, known brain endothelial pneumococcal adhesins. Next, mice were infected either intravenously or intranasally with pneumococcal strains. In the intranasal infection model, survival rates were comparable between wild-type strain-infected and ccs4 mutant strain-infected mice, while the ccs4 mutant strain exhibited a lower level of virulence in the intravenous infection model. In addition, at 24 hours after intravenous infection, the bacterial burden in blood was comparable between the wild-type and ccs4 mutant strain-infected mice, whereas the wild-type strain-infected mice showed a significantly higher bacterial burden in the brain. These results suggest that Ccs4 contributes to pneumococcal invasion of host brain tissues and functions as a virulence factor.

Comparison of trophic factors secreted from human adipose-derived stromal vascular fraction with those from adipose-derived stromal/stem cells in the same individuals

Cell therapy for several different diseases has been experimentally investigated for regeneration of damaged tissues. Adipose tissue in the human body is abundant, and can be easily and safely harvested in large quantities, thus it has attracted attention as a source for cell therapy. Stromal vascular fraction (SVF) is currently a major source for cell therapy after isolation from adipose tissues without the need for culture using a Celution System (CytoriTherapeutics) [1,2]. In our previous studies, we found that periurethral injection of autologous SVFs may represent a safe and feasible treatment modality for male stress urinary incontinence [3,4]. However, characterization of the SVFs used in our experiments has yet to be reported. Mesenchymal stromal cells (MSCs) are well known as a potential source for cell therapy and their therapeutic effect reportedly occurs through secretion of trophic factors [5,6]. SVFs are composed of heterogeneous cell populations, including adipose-derived stromal/stem cells (ASCs), granulocytes, monocytes, lymphocytes, endothelial cells and pericytes [7]. Unfortunately, there is scant information available from comprehensive cytokine or chemokine quantitative analyses of conditioned medium samples isolated from SVFs. In this letter, we show trophic factors released by SVFs and compared them with those released by ASCs in the same individuals. SVFs were previously isolated from tissues obtained from three male subjects (77, 69 and 75 years old) with a Celution System and used in our clinical research [3]. For the present study, SVFs were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS; AusGenex) for isolation of ASCs and SVFs were also cultured in Roswell Park Memorial Institute (RPMI) 1640 medium (Sigma-Aldrich) supplemented with 10% FBS as culture of SVFs in 24-well plates. ASCs were detached after reaching 60– 70% confluence by incubation with 0.05% trypsinethylenediaminetetraacetic acid (EDTA; Life Technologies) and subcultured at a 1:4 dilution under the same conditions. Furthermore, after reaching 60–70% confluence following the second passage (Figure 1A), the culture medium was switched to DMEM without serum and medium samples were collected 24 h later. For SVFs, on day 2 of the primary culture, floating cells and culture media were collected and centrifuged, then the cell pellets were suspended in RPMI 1640 without serum and re-plated in 24well plates containing adherent cells (Figure 1B), then medium samples were collected after 24 h. Collected medium samples from both ASCs and SVFs were concentrated approximately 20-fold by use of an Amicon Ultra-15 centrifugal filter unit with an Ultracel-3 membrane (Millipore). The concentrated samples were placed in 96-well plates for analyses of