Time-course study of genetic changes in periodontal ligament regeneration after tooth replantation in a mouse model.

This research focused on analyzing gene expression changes in the periodontal ligament (PDL) after tooth re-plantation to identify key genes and pathways involved in healing and regeneration. Utilizing a mouse model, mRNA was extracted from the PDL at various intervals post-replantation for RNA sequencing analysis, spanning from 3 to 56 days. The results revealed significant shifts in gene expression, particularly notable on day 28, supported by hierarchical clustering and principal component analysis. Gene ontology (GO) enrichment analysis highlighted an upregulation in olfactory receptor and G protein-coupled receptor signaling pathways at this time point. These findings were validated through reverse transcription-quantitative PCR (RT-qPCR), with immunochemical staining localizing olfactory receptor gene expression to the PDL and surrounding tissues. Moreover, a scratch assay indicated that olfactory receptor genes might facilitate wound healing in human PDL fibroblasts. These results underscore the importance of the 28-day post-transplant phase as a potential "tipping point" in PDL healing and regeneration. In conclusion, this research sheds light on the potential role of olfactory receptor genes in PDL regeneration, providing a foundation for developing new therapeutic approaches in tooth replantation and transplantation, with broader implications for regenerative medicine in oral health.

Autologous concentrated growth factor mediated accelerated bone healing in root-end microsurgery: A multicenter randomized clinical trial.

Introduction: Concentrated growth factor (CGF) is a new-generation autologous platelet concentrate that promotes tissue regeneration and has anti-inflammatory properties. This randomized multicenter trial aimed to evaluate the effects of CGF on bone healing in combination with root-end microsurgery. Methods: Healthy adult patients indicated for root-end microsurgery were randomly assigned to either the CGF or control (no CGF implantation) groups. CGF was implanted into the bone cavity after root-end filling with mineral trioxide aggregate. Clinical and periapical radiographic evaluations were conducted at 1, 3, 6, and 12 months postoperatively, with follow-up cone-beam computed tomography (CBCT) at 6 months. The lesion volume reduction rate was calculated based on data from the preoperative and follow-up CBCT images. Results: A total of 24 patients were enrolled. The treatment success rate was 91.7% and 83.3% on 12-month periapical radiography and 6-month CBCT, respectively, without a significant difference between the two groups. The lesion volume reduction rate in the CGF group (75.6%) was significantly higher than that in the control (61.0%) group. Conclusions: Autologous CGF in conjunction with root-end microsurgery accelerated lesion reduction as observed on CBCT. Administering autologous blood products to stimulate healing in addition to removing the source of infection appears to be a promising treatment option for root-end microsurgery.

Porphyromonas gingivalis Tyrosine Phosphatase Php1 Promotes Community Development and Pathogenicity.

Protein-tyrosine phosphorylation in bacteria plays a significant role in multiple cellular functions, including those related to community development and virulence. Metal-dependent protein tyrosine phosphatases that belong to the polymerase and histindinol phosphatase (PHP) family are widespread in Gram-positive bacteria. Here, we show that Porphyromonas gingivalis, a Gram-negative periodontal pathogen, expresses a PHP protein, Php1, with divalent metal ion-dependent tyrosine phosphatase activity. Php1 tyrosine phosphatase activity was attenuated by mutation of conserved histidine residues that are important for the coordination of metal ions and by mutation of a conserved arginine residue, a key residue for catalysis in other bacterial PHPs. The php1 gene is located immediately downstream of the gene encoding the bacterial tyrosine (BY) kinase Ptk1, which was a substrate for Php1 in vitro Php1 rapidly caused the conversion of Ptk1 to a state of low tyrosine phosphorylation in the absence of discernible intermediate phosphoforms. Active Php1 was required for P. gingivalis exopolysaccharide production and for community development with the antecedent oral biofilm constituent Streptococcus gordonii under nutrient-depleted conditions. In contrast, the absence of Php1 had no effect on the ability of P. gingivalis to form monospecies biofilms. In vitro, Php1 enzymatic activity was resistant to the effects of the streptococcal secreted metabolites pABA and H2O2, which inhibited Ltp1, an enzyme in the low-molecular-weight (LMW) phosphotyrosine phosphatase family. Ptk1 reciprocally phosphorylated Php1 on tyrosine residues 159 and 161, which independently impacted phosphatase activity. Loss of Php1 rendered P. gingivalis nonvirulent in an animal model of periodontal disease. Collectively, these results demonstrate that P. gingivalis possesses active PHP and LMW tyrosine phosphatases, a unique configuration in Gram-negatives which may allow P. gingivalis to maintain phosphorylation/dephosphorylation homeostasis in multispecies communities. Moreover, Php1 contributes to the pathogenic potential of the organism.IMPORTANCE Periodontal diseases are among the most common infections of humans and are also associated with systemic inflammatory conditions. Colonization and pathogenicity of P. gingivalis are regulated by signal transduction pathways based on protein tyrosine phosphorylation and dephosphorylation. Here, we identify and characterize a novel component of the tyrosine (de)phosphorylation axis: a polymerase and histindinol phosphatase (PHP) family enzyme. This tyrosine phosphatase, designated Php1, was required for P. gingivalis community development with other oral bacteria, and in the absence of Php1 activity P. gingivalis was unable to cause disease in a mouse model of periodontitis. This work provides significant insights into the protein tyrosine (de)phosphorylation network in P. gingivalis, its adaptation to heterotypic communities, and its contribution to colonization and virulence.

Streptococcus gordonii programs epithelial cells to resist ZEB2 induction by Porphyromonas gingivalis.

The polymicrobial microbiome of the oral cavity is a direct precursor of periodontal diseases, and changes in microhabitat or shifts in microbial composition may also be linked to oral squamous cell carcinoma. Dysbiotic oral epithelial responses provoked by individual organisms, and which underlie these diseases, are widely studied. However, organisms may influence community partner species through manipulation of epithelial cell responses, an aspect of the host microbiome interaction that is poorly understood. We report here that Porphyromonas gingivalis, a keystone periodontal pathogen, can up-regulate expression of ZEB2, a transcription factor which controls epithelial-mesenchymal transition and inflammatory responses. ZEB2 regulation by P. gingivalis was mediated through pathways involving ß-catenin and FOXO1. Among the community partners of P. gingivalis, Streptococcus gordonii was capable of antagonizing ZEB2 expression. Mechanistically, S. gordonii suppressed FOXO1 by activating the TAK1-NLK negative regulatory pathway, even in the presence of P. gingivalis Collectively, these results establish S. gordonii as homeostatic commensal, capable of mitigating the activity of a more pathogenic organism through modulation of host signaling.

p62 Plays a Specific Role in Interferon-γ-Induced Presentation of a Toxoplasma Vacuolar Antigen.

Also known as Sqstm1, p62 is a selective autophagy adaptor with a ubiquitin-binding domain. However, the role of p62 in the host defense against Toxoplasma gondii infection is unclear. Here, we show that interferon γ (IFN-γ) stimulates ubiquitin and p62 recruitment to T. gondii parasitophorous vacuoles (PVs). Some essential autophagy-related proteins, but not all, are required for this recruitment. Regardless of normal IFN-γ-induced T. gondii clearance activity and ubiquitination, p62 deficiency in antigen-presenting cells (APCs) and mice diminishes the robust IFN-γ-primed activation of CD8(+) T cells that recognize the T. gondii-derived antigen secreted into PVs. Because the expression of Atg3 and Irgm1/m3 in APCs is essential for PV disruption, ubiquitin and p62 recruitment, and vacuolar-antigen-specific CD8(+) T cell activation, IFN-γ-mediated ubiquitination and the subsequent recruitment of p62 to T. gondii are specifically required for the acquired immune response after PV disruption by IFN-γ-inducible GTPases.

RabGDIα is a negative regulator of interferon-γ-inducible GTPase-dependent cell-autonomous immunity to Toxoplasma gondii.

IFN-γ orchestrates cell-autonomous host defense against various intracellular vacuolar pathogens. IFN-γ-inducible GTPases, such as p47 immunity-related GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), are recruited to pathogen-containing vacuoles, which is important for disruption of the vacuoles, culminating in the cell-autonomous clearance. Although the positive regulation for the proper recruitment of IRGs and GBPs to the vacuoles has been elucidated, the suppressive mechanism is unclear. Here, we show that Rab GDP dissociation inhibitor α (RabGDIα), originally identified as a Rab small GTPase inhibitor, is a negative regulator of IFN-γ-inducible GTPases in cell-autonomous immunity to the intracellular pathogen Toxoplasma gondii. Overexpression of RabGDIα, but not of RabGDIß, impaired IFN-γ-dependent reduction of T. gondii numbers. Conversely, RabGDIα deletion in macrophages and fibroblasts enhanced the IFN-γ-induced clearance of T. gondii. Furthermore, upon a high dose of infection by T. gondii, RabGDIα-deficient mice exhibited a decreased parasite burden in the brain and increased resistance in the chronic phase than did control mice. Among members of IRGs and GBPs important for the parasite clearance, Irga6 and Gbp2 alone were more frequently recruited to T. gondii-forming parasitophorous vacuoles in RabGDIα-deficient cells. Notably, Gbp2 positively controlled Irga6 recruitment that was inhibited by direct and specific interactions of RabGDIα with Gbp2 through the lipid-binding pocket. Taken together, our results suggest that RabGDIα inhibits host defense against T. gondii by negatively regulating the Gbp2-Irga6 axis of IFN-γ-dependent cell-autonomous immunity.

mTOR Complex Signaling through the SEMA4A-Plexin B2 Axis Is Required for Optimal Activation and Differentiation of CD8+ T Cells.

Mammalian target of rapamycin (mTOR) plays crucial roles in activation and differentiation of diverse types of immune cells. Although several lines of evidence have demonstrated the importance of mTOR-mediated signals in CD4(+) T cell responses, the involvement of mTOR in CD8(+) T cell responses is not fully understood. In this study, we show that a class IV semaphorin, SEMA4A, regulates CD8(+) T cell activation and differentiation through activation of mTOR complex (mTORC) 1. SEMA4A(-/-) CD8(+) T cells exhibited impairments in production of IFN-γ and TNF-α and induction of the effector molecules granzyme B, perforin, and FAS-L. Upon infection with OVA-expressing Listeria monocytogenes, pathogen-specific effector CD8(+) T cell responses were significantly impaired in SEMA4A(-/-) mice. Furthermore, SEMA4A(-/-) CD8(+) T cells exhibited reduced mTORC1 activity and elevated mTORC2 activity, suggesting that SEMA4A is required for optimal activation of mTORC1 in CD8(+) T cells. IFN-γ production and mTORC1 activity in SEMA4A(-/-) CD8(+) T cells were restored by administration of recombinant Sema4A protein. In addition, we show that plexin B2 is a functional receptor of SEMA4A in CD8(+) T cells. Collectively, these results not only demonstrate the role of SEMA4A in CD8(+) T cells, but also reveal a novel link between a semaphorin and mTOR signaling.

Selective and strain-specific NFAT4 activation by the Toxoplasma gondii polymorphic dense granule protein GRA6.

Toxoplasma gondii infection results in co-option and subversion of host cellular signaling pathways. This process involves discharge of T. gondii effector molecules from parasite secretory organelles such as rhoptries and dense granules. We report that the T. gondii polymorphic dense granule protein GRA6 regulates activation of the host transcription factor nuclear factor of activated T cells 4 (NFAT4). GRA6 overexpression robustly and selectively activated NFAT4 via calcium modulating ligand (CAMLG). Infection with wild-type (WT) but not GRA6-deficient parasites induced NFAT4 activation. Moreover, GRA6-deficient parasites failed to exhibit full virulence in local infection, and the treatment of WT mice with an NFAT inhibitor mitigated virulence of WT parasites. Notably, NFAT4-deficient mice displayed prolonged survival, decreased recruitment of CD11b(+) Ly6G(+) cells to the site of infection, and impaired expression of chemokines such as Cxcl2 and Ccl2. In addition, infection with type I parasites culminated in significantly higher NFAT4 activation than type II parasites due to a polymorphism in the C terminus of GRA6. Collectively, our data suggest that GRA6-dependent NFAT4 activation is required for T. gondii manipulation of host immune responses to maximize the parasite virulence in a strain-dependent manner.

Role of mouse and human autophagy proteins in IFN-γ-induced cell-autonomous responses against Toxoplasma gondii.

IFN-γ mediates cellular innate immunity against an intracellular parasite, Toxoplasma gondii, by inducing immunity-related GTPases such as p47 IFN-γ-regulated GTPases (IRGs) and p65 guanylate-binding proteins (GBPs), which also participate in antibacterial responses via autophagy. An essential autophagy protein, Atg5, was previously shown to play a critical role in anti-T. gondii cell-autonomous immunity. However, the involvement of other autophagy proteins remains unknown. In this study, we show that essential autophagy proteins differentially participate in anti-T. gondii cellular immunity by recruiting IFN-γ-inducible GTPases. IFN-γ-induced suppression of T. gondii proliferation and recruitment of an IRG Irgb6 and GBPs are profoundly impaired in Atg7- or Atg16L1-deficient cells. In contrast, cells lacking other essential autophagy proteins, Atg9a and Atg14, are capable of mediating the anti-T. gondii response and recruiting Irgb6 and GBPs to the parasites. Although IFN-γ also stimulates anti-T. gondii cellular immunity in humans, whether this response requires GBPs and human autophagy proteins remains to be seen. To analyze the role of human ATG16L1 and GBPs in IFN-γ-mediated anti-T. gondii responses, human cells lacking ATG16L1 or GBPs were generated by the Cas9/CRISPR genome-editing technique. Although both ATG16L1 and GBPs are dispensable for IFN-γ-induced inhibition of T. gondii proliferation in the human cells, human ATG16L1 is also required for the recruitment of GBPs. Taken together, human ATG16L1 and mouse autophagy components Atg7 and Atg16L1, but not Atg9a and Atg14, participate in the IFN-γ-induced recruitment of the immunity-related GTPases to the intracellular pathogen.

CREBH determines the severity of sulpyrine-induced fatal shock.

Although the pyrazolone derivative sulpyrine is widely used as an antipyretic analgesic drug, side effects, including fatal shock, have been reported. However, the molecular mechanism underlying such a severe side effect is largely unclear. Here, we report that the transcription factor CREBH that is highly expressed in the liver plays an important role in fatal shock induced by sulpyrine in mice. CREBH-deficient mice were resistant to experimental fatal sulpyrine shock. We found that sulpyrine-induced expression of cytochrome P450 2B (CYP2B) family genes, which are involved in sulpyrine metabolism, in the liver was severely impaired in CREBH-deficient mice. Moreover, introduction of CYP2B in CREBH-deficient liver restored susceptibility to sulpyrine. Furthermore, ectopic expression of CREBH up-regulated CYP2B10 promoter activity, and in vivo knockdown of CREBH in wild-type mice conferred a significant resistance to fatal sulpyrine shock. These data demonstrate that CREBH is a positive regulator of CYP2B in response to sulpyrine administration, which possibly results in fatal shock.

A cluster of interferon-γ-inducible p65 GTPases plays a critical role in host defense against Toxoplasma gondii.

Interferon-γ (IFN-γ) is essential for host defense against intracellular pathogens. Stimulation of innate immune cells by IFN-γ upregulates ∼2,000 effector genes such as immunity-related GTPases including p65 guanylate-binding protein (Gbp) family genes. We show that a cluster of Gbp genes was required for host cellular immunity against the intracellular parasite Toxoplasma gondii. We generated mice deficient for all six Gbp genes located on chromosome 3 (Gbp(chr3)) by targeted chromosome engineering. Mice lacking Gbp(chr3) were highly susceptible to T. gondii infection, resulting in increased parasite burden in immune organs. Furthermore, Gbp(chr3)-deleted macrophages were defective in IFN-γ-mediated suppression of T. gondii intracellular growth and recruitment of IFN-γ-inducible p47 GTPase Irgb6 to the parasitophorous vacuole. In addition, some members of Gbp(chr3) restored the protective response against T. gondii in Gbp(chr3)-deleted cells. Our results suggest that Gbp(chr3) play a pivotal role in anti-T. gondii host defense by controlling IFN-γ-mediated Irgb6-dependent cellular innate immunity.

Hypoglycaemic neuropathy: microvascular changes due to recurrent hypoglycaemic episodes in rat sciatic nerve.

Intensive diabetes treatment causes a considerable increase in the number of severe hypoglycaemic episodes which could aggravate the progression of diabetic neuropathy. However, the effect of repeated hypoglycaemic episodes on nerve morphology has never been previously investigated. The aims of the present study were: (i) to establish a rat model of recurrent episodes of severe hypoglycaemia, and (ii) to assess morphological changes after repeated hypoglycaemic episodes in rat sciatic nerves. We induced hypoglycaemic episodes, blood glucose level <3.0 mmol/l for 3 h, by injecting regular insulin intravenously on 4 consecutive days. We found endothelial swelling of endoneurial microvessels at the thigh level of sciatic and tibial nerves 24 h after four daily episodes of hypoglycaemia. Endothelial swelling was confirmed by vascular morphometry which showed significantly increased endothelial and pericyte areas. No obvious abnormalities were seen on nerve fibres. In conclusion, recurrent hypoglycaemic episodes cause early vascular anomalies in endoneurial microvessels in rat sciatic nerves without any observable changes in nerve fibres.

Drug use disorders in Japanese eating disorder patients.

A previous questionnaire study suggested that drug use disorder (DUD: abuse/dependence on drugs, other than alcohol) in Japanese eating disorder (ED) patients was less prevalent than in Western countries, although eating and drug use disorders have spread simultaneously in Western countries. However, the precise prevalence and comorbidity features remain unknown. Subjects consisted of 62 patients with anorexia nervosa restricting type; 48 patients with anorexia nervosa binge eating/purging type; and 75 patients with bulimia nervosa purging type. The Japanese version of the Structured Clinical Interview for DSM-III-R; the Structured Clinical Interview for DSM-III-R Personality Disorders; and the supplement module of the Schedule for Affective Disorders and Schizophrenia-Lifetime version were used for the interview. Sixteen (8.6%, 95% CI = 4.6-12.7%) patients had lifetime diagnoses of DUD. Drugs were solvent fumes or benzodiazepines, and only one patient had been dependent on methamphetamine. More than half of the patients with lifetime DUD diagnoses were multi-impulsivitists. On multivariate analysis, DUD was significantly linked with childhood parental loss, history of conduct disorder and borderline personality disorder. Thus, the prevalence of DUD in Japanese ED patients was indeed lower than that in Western countries. However, similar comorbidity was found in ED patients with DUD compared with that of those in Western countries. The current study suggests that ED and DUD have different origins, although they share the feature of impulsivity. Further study in the general population is needed to clarify these issues.