Scaffold-free bone-like 3D structure established through osteogenic differentiation from human gingiva-derived stem cells.

Introduction & objectives: Stem cell therapy for regenerative medicine has been sincerely investigated, but not still popular although some clinical trials show hopeful results. This therapy is suggested to be a representative candidate such as bone defect due to the accident, iatrogenic resection oncological tumor, congenital disease, and severe periodontitis in oral region. Recently, the Bio-3D printer "Regenova®" has been introduced as an innovative three-dimensional culture system, equipped scaffold-free bio-assembling techniques without any biomaterials. Therefore, we expected a mount of bone defect could be repaired by the structure established from this Bio-3D printer using osteogenic potential stem cells. Material & methods: The gingival tissue (1x1 mm) was removed from the distal part of the lower wisdom tooth of the patients who agreed our study. Human Gingival Mesenchymal Stem Cells (hGMSCs) were isolated from this tissue and cultured, since we confirmed the characteristics such as facile isolation and accelerated proliferation, further, strong potential of osteogenic-differentiation. Spheroids were formed using hGMSC in 96-well plates designed for low cell adhesion. The size of the spheroids was measured, and fluorescent immunostaining was employed to verify the expression of stem cell and apoptosis marker, and extracellular matrix. Following four weeks of bone differentiation, µCT imaging was performed. Calcification was confirmed by alizarin red and von Kossa staining. Fluorescent immunostaining was utilized to assess the expression of markers indicative of advanced bone differentiation. Results: We have established and confirmed the spheroids (∼600 µm in diameter) constructed from human GMSCs (hGMSCs) still maintain stem cell potentials and osteogenic differentiation abilities from the results that CD73 and not CD34 were expressed as stem cell positive and negative marker, respectively. These spheroids were pilled up like cylindal shape to the "Kenzan" platform of Bio-3D printer and cultured for 7days. The cylindal structure originated from compound spheroids were tried to differentiate into bone four weeks with osteogenic induction medium. The calcification of bio-3D printed bone-like structures was confirmed by alizarin red and Von Kossa staining. In addition, µCT analysis revealed that the HU (Hounsfield Unit) of the calcified structures was almost identical to that of trabecular bone. Immunofluorescent staining detected osteocalcin expression, a late-stage bone differentiation marker. Conclusion: For the first time, we have achieved the construction of a scaffold-free, bone-like luminal structure through the assembly of spheroids comprised of this hGMSCs. This success is sure to be close to the induction of clinical application against regenerative medicine especially for bone defect disease.

Luteolin Is a Potential Immunomodulating Natural Compound against Pulpal Inflammation.

Aim: The present study evaluated the therapeutic effects of luteolin in alleviating pulpitis of dental pulp- (DP-) derived microvesicles (MVs) via the inhibition of protein kinase R- (PKR-) mediated inflammation. Methodology. Proteomic analysis of immortalized human dental pulp (DP-1) cell-derived MVs was performed to identify PKR-associated molecules. The effect of luteolin on PKR phosphorylation in DP-1 cells and the expression of tumor necrosis factor-α (TNF-α) in THP-1 macrophage-like cells were validated. The effect of luteolin on cell proliferation was compared with that of chemical PKR inhibitors (C16 and 2-AP) and the unique commercially available sedative guaiacol-parachlorophenol. In the dog experimental pulpitis model, the pulps were treated with (1) saline, (2) guaiacol-parachlorophenol, and (3) luteolin. Sixteen teeth from four dogs were extracted, and the pulp tissues were analyzed using hematoxylin and eosin staining. Immunohistochemical staining was performed to analyze the expression of phosphorylated PKR (pPKR), myeloperoxidase (MPO), and CD68. Experimental endodontic-periodontal complex lesions were established in mouse molar through a silk ligature and simultaneous MV injection. MVs were prepared from DP-1 cells with or without pretreatment with 2-AP or luteolin. A three-dimensional microcomputed tomography analysis was performed on day 7 (n = 6). Periodontal bone resorption volumes were calculated for each group (nonligated-ligated), and the ratio of bone volume to tissue volume was measured. Results: Proteomic analysis identified an endogenous PKR activator, and a protein activator of interferon-induced PKR, also known as PACT, was included in MVs. Luteolin inhibited the expressions of pPKR in DP-1 cells and TNF-α in THP-1 cells with the lowest suppression of cell proliferation. In the dog model of experimental pulpitis, luteolin treatment suppressed the expression of pPKR-, MPO-, and CD68-positive cells in pulp tissues, whereas guaiacol-parachlorophenol treatment caused coagulative necrosis and disruption. In a mouse model of endodontic-periodontal complex lesions, luteolin treatment significantly decreased MV-induced alveolar bone resorption. Conclusion: Luteolin is an effective and safe compound that inhibits PKR activation in DP-derived MVs, enabling pulp preservation.

Factors associated with the need for long-term total parenteral nutrition in survivors of acute superior mesenteric artery occlusion.

BACKGROUND: Acute superior mesenteric artery (SMA) occlusion is an uncommon condition associated with high mortality. If extensive bowel resection is performed for patients with acute SMA occlusion and the patient survives, long-term total parenteral nutrition (TPN) may be needed due to short bowel syndrome. This study examined factors associated with the need for long-term TPN after the treatment of acute SMA occlusion. METHODS: We retrospectively analyzed 78 patients with acute SMA occlusion. Patients were abstracted from a Japanese database from institutions with at least 10 patients with acute SMA occlusive disease from January 2015 through December 2020 RESULTS: Among the initial cohort there were 41/78 survivors. Of these, 14/41 (34%) required permanent TPN who were compared with those who did not require long-term TPN (27/41, 66%). Compared to patients in the non-TPN group, those in the TPN group had significantly shorter remaining small intestine (90.7 cm vs. 218 cm, P<0.01), more patients with time from onset to intervention >6 hours (P=0.02), pneumatosis intestinalis on enhanced computed tomography scan (P=0.04), ascites (Odds Ratio 11.6, P<0.01), and a positive smaller superior mesenteric vein sign (P= 0.03). These were considered significant risk factors for needing long-term TPN. Age, gender, underlying disease, presence of peritoneal sign, presence of shock requiring vasopressors, site of obstruction (proximal vs. distal), and initial treatment (surgery vs. interventional radiology vs. thrombolytic therapy) were not significantly different between the two groups. Long-term TPN was significantly associated with longer hospital stay (52 vs. 35 days, P=0.04). Multivariate analysis identified the presence of ascites as an independent risk factor for needing long-term TPN. CONCLUSION: The need for permanent TPN after treatment of acute SMA occlusion is significantly associated with longer hospital stay, longer time to intervention, and characteristic imaging findings (pneumatosis intestinalis, ascites, Smaller SMV sign). Ascites is an independent risk factor. LEVEL OF EVIDENCE: III.

A Rare Case of Extensive Basal Cell Carcinoma With Invasion of the Humerus: Excision and Immediate Reconstruction With Fibula Allograft, Plating, and Latissimus Dorsi Flap.

Basal cell carcinoma (BCC) is the most common skin cancer, and most of the reports have involved the head and neck, but it is rare for it to be highly invasive, with an invasion of long bone being extremely rare. A 73 year old woman presented with a giant BCC on her right arm. Magnetic resonance imaging suggested the involvement of the right humerus. Biopsy confirmed the nodular type of BCC. The patient underwent BCC excision including hemicortical humerus excision with fibula allograft and latissimus dorsi flap with a split-thickness skin graft. Excluding a transient radial nerve palsy, the patient's postoperative course was otherwise uncomplicated. Although BCC invasion into the long bone is extremely rare, the gold standard treatment is, as a rule, en bloc surgical resection with a wide variety of reconstructive techniques. This treatment is only possible through the collaboration of general surgery, orthopedics, and plastic surgery.

Horseshoe Abscess as a Rare Complication After Transanal Hemorrhoid Dearterialization With Mucopexy: A Case Report.

Transanal hemorrhoidal dearterialization (THD) is a minimally invasive procedure that has gained popularity as a treatment for symptomatic hemorrhoids. It involves ligating the arterial blood supply to the hemorrhoidal plexus. Compared to conventional ligation or resection, THD is associated with less postoperative bleeding and pain, allowing for same-day surgery discharge. Horseshoe abscess is a rare but known complication of anorectal surgery, characterized by an abscess that extends around the anal canal, often involving the ischiorectal fossa and adjacent structures. Although horseshoe abscesses have been reported after various anorectal surgeries, including hemorrhoidectomy, their occurrence following THD has not been well-documented in the literature. A 72-year-old male underwent THD for rectal prolapse with internal hemorrhoids and presented to the hospital on postoperative day 6 with severe rectal pain. A computed tomography (CT) scan revealed a large complex horseshoe perirectal abscess with fluid and air and significant rectal wall thickening. A rectal examination under anesthesia confirmed the presence of purulent drainage from the anus, and surgical drainage of the abscess was performed. The patient received antibiotics and analgesics and experienced a favorable recovery. The exact pathophysiology of a horseshoe abscess following THD remains unclear, and the incidence and risk factors associated with this complication are not well-established. Moreover, there has yet to be a consensus on the optimal management of horseshoe abscesses after THD, whether through surgical or medical approaches. This case emphasizes the importance of considering horseshoe abscess as a potential complication of THD and highlights the need for further research to understand better its incidence, risk factors, and optimal management strategies.

Clinical features and outcomes of spine surgery in patients with ankylosing spondylitis.

OBJECTIVES: The study aimed to comprehend the clinical features and outcomes of surgical treatments for spinal disorders in patients with ankylosing spondylitis. METHODS: This retrospective study enrolled patients with ankylosing spondylitis who underwent spine surgery between 2000 and 2019 in our facility. RESULTS: Thirteen patients with ankylosing spondylitis underwent spine surgeries. The mean age was 56.2 years, and the mean disease duration was 25.1 years at the time of surgery. Nine patients had vertebral fracture, two had kyphotic deformity, and two had myelopathy due to the spinal ligament ossification. Fracture cases included five patients with secondary pseudarthrosis/delayed palsy due to conservative treatment failure. Spinal fixation was performed in all patients. Pedicle subtraction osteotomy for kyphosis and laminectomy for myelopathy were also conducted. All patients improved after surgeries. One patient with kyphotic deformity underwent additional surgery of bilateral hip prosthesis, which resulted in better spine alignment. Four cases of perioperative complications were observed. CONCLUSION: Myelopathy was newly found as the aetiology requiring surgery in patients with ankylosing spondylitis. This summarized case series could help physicians to identify patients with surgically treatable spinal disorders among patients with ankylosing spondylitis.

miR-1260b inhibits periodontal bone loss by targeting ATF6ß mediated regulation of ER stress.

The expression profiles of exosomal microRNAs (miRNAs) are regulated by the microenvironment, and appropriate priming with mesenchymal stem cells (MSCs) is one of the strategies to enhance the paracrine potency of MSCs. Our previous work demonstrated that exosomes from tumor necrosis factor (TNF)-α-primed human gingiva-derived MSCs (GMSCs) could be a therapeutic tool against periodontitis, and that TNFα-inducible exosomal miR-1260b is essential for the inhibition of alveolar bone loss. However, the precise molecular mechanism underlying miR-1260b-mediated inhibition of osteoclastogenesis is not yet fully understood. Here, we found that the activating transcription factor (ATF)-6ß, a novel miR-1260b-targeting gene, is critical for the regulation of osteoclastogenesis under endoplasmic reticulum (ER) stress. An experimental periodontal mouse model demonstrated that induction of ER stress was accompanied by enhanced ATF6ß expression, and local administration of miR-1260b and ATF6ß siRNA using polyethylenimine nanoparticles (PEI-NPs) significantly suppressed the periodontal bone resorption. In periodontal ligament (PDL) cells, the ER stress inducer, tunicamycin, enhanced the expression of the receptor activator of NF-κB ligand (RANKL), while miR-1260b-mediated downregulation of ATF6ß caused RANKL inhibition. Furthermore, the secretome from miR-1260b/ATF6ß-axis-activated PDL cells inhibited osteoclastogenesis in human CD14+ peripheral blood-derived monocytes. These results indicate that the miR-1260b/ATF6ß axis mediates the regulation of ER stress, which may be used as a novel therapeutic strategy to treat periodontal disease.

Extracellular vesicles derived from GMSCs stimulated with TNF-α and IFN-α promote M2 macrophage polarization via enhanced CD73 and CD5L expression.

Immunoregulatory properties of mesenchymal stem cell (MSC)-derived extracellular vesicles (EVs) are promising. Gingival tissue-derived MSCs (GMSCs) have unique immunoregulatory capacity and secrete large amounts of EVs. Recent findings suggest that priming MSCs with inflammatory stimuli is an effective strategy for cell-free therapy. However, the precise mechanism by which the contents of EVs are customized has not been fully elucidated. Here, we show that EVs derived from GMSCs primed with a combination of two pro-inflammatory cytokines, tumor necrosis factor-α (TNF-α) and interferon-α (IFN-α), synergistically promote anti-inflammatory M2 macrophage polarization by increasing the expression of cluster of differentiation 73 (CD73) and CD5 molecule-like (CD5L). Expression of CD73 by TNF-α/IFN-α stimulation was transcriptionally upregulated by the activation of mammalian target of rapamycin signaling and nuclear translocation of hypoxia-inducible factor 1α in GMSCs. TNF-α/IFN-α treatment also significantly increased the expression of CD5L mRNA via the transcription factor DNA-binding protein inhibitor ID3 and liver X receptor. Interestingly, exosomal CD5L is a prerequisite for the synergistic effect of EVs-mediated M2 macrophage polarization. These results indicate that combined pre-licensing with TNF-α and IFN-α in GMSCs is ideal for enhancing the anti-inflammatory function of EVs, which contributes to the establishment of a therapeutic tool.

Knowledge and Attitude towards Cervical Cancer and Human Papillomavirus among Pharmacists in Japan.

OBJECTIVE: The objective of this study was to assess the knowledge and attitude towards cervical cancer and human papillomavirus (HPV) among pharmacists in Japan. METHODS: Questionnaires were disseminated to 788 pharmacists employed by the Tsuruha Holdings Inc. A total of 617 pharmacists responded, generating a response rate of 78.3%. RESULT: Of the 362 females and 255 males, vaccination rates were 14.4% and 0.8%, respectively. In terms of cervical cytology, 35.1% of females received it once every two years, and 26.2% received it irregularly. As for HPV testing, 12.2% of females received it once every two years, and 16.6% received it irregularly. The rate of "school curriculum" as an information source was significantly higher among younger pharmacists; while "internet", "media", "training seminar for pharmacist", "advertisement in medical institution", "internal manual", and "others" were significantly higher among older pharmacists. The proportion of pharmacists with knowledge on general questions, except for those about HPV testing, was significantly higher among females than males. The vaccination rates of younger pharmacists were significantly higher than those of older pharmacists. The screening rates of cervical cytology were significantly higher among older than younger pharmacists, and also among those with at least 10 years of experience than those with less. There were no differences in the screening rates of HPV testing according to age or pharmacist experience. CONCLUSION: The proportion of pharmacists with knowledge about cervical cancer and HPV significantly varied depending on sex, age, and experience as a pharmacist. This study suggested that spreading the knowledge about cervical cancer and HPV might be effective for increasing the rates of cervical cancer screening.

Combined application of geranylgeranylacetone and amelogenin promotes angiogenesis and wound healing in human periodontal ligament cells.

Amelogenin directly binds to glucose-regulated protein 78 (Grp78). Cell migration activity is expected to increase when human periodontal ligament cells (hPDLCs) overexpressing Grp78 are treated with amelogenin. Geranylgeranylacetone (GGA) is a drug that induces the expression of heat shock protein and is routinely used to treat gastric ulcers. Here, we investigated the changes in the properties and behavior of hPDLCs in response to treatment with GGA and the synergistic effects of amelogenin stimulation in hPDLCs pretreated with GGA for the establishment of a novel periodontal tissue regenerative therapy. We observed that GGA treatment increased Grp78 protein expression in hPDLCs and enhanced cell migration. Microarray analysis demonstrated that increased Grp78 expression triggered the production of angiopoietin-like 4 and amphiregulin, which are involved in the enhancement of angiogenesis and subsequent wound healing via the activation of hypoxia-inducible factor 1α and peroxisome proliferator-activated receptors as well as the phosphorylation of cAMP response element-binding protein and protein kinase A. Moreover, the addition of recombinant murine amelogenin (rM180) further accelerated hPDLC migration and tube formation of human umbilical vein endothelial cells due to the upregulation of interleukin-8 (IL-8), monocyte chemotactic protein 1, and IL-6, which are also known as angiogenesis-inducing factors. These findings suggest that the application of GGA to gingival tissue and alveolar bone damaged by periodontal disease would facilitate the wound healing process by inducing periodontal ligament cells to migrate to the root surface and release cytokines involved in tissue repair. Additionally, supplementation with amelogenin synergistically enhanced the migratory capacity of these cells while actively promoting angiogenesis. Therefore, the combined application of GGA and amelogenin may establish a suitable environment for periodontal wound healing and further drive the development of novel therapeutics for periodontal tissue regeneration.

Exosomes from TNF-α-treated human gingiva-derived MSCs enhance M2 macrophage polarization and inhibit periodontal bone loss.

Mesenchymal stem cell (MSC)-derived exosome plays a central role in the cell-free therapeutics involving MSCs and the contents can be customized under disease-associated microenvironments. However, optimal MSC-preconditioning to enhance its therapeutic potential is largely unknown. Here, we show that preconditioning of gingival tissue-derived MSCs (GMSCs) with tumor necrosis factor-alpha (TNF-α) is ideal for the treatment of periodontitis. TNF-α stimulation not only increased the amount of exosome secreted from GMSCs, but also enhanced the exosomal expression of CD73, thereby inducing anti-inflammatory M2 macrophage polarization. The effect of GMSC-derived exosomes on inflammatory bone loss were examined by ligature-induced periodontitis model in mice. Local injection of GMSC-derived exosomes significantly reduced periodontal bone resorption and the number of tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts, and these effects were further enhanced by preconditioning of GMSCs with TNF-α. Thus, GMSC-derived exosomes also exhibited anti-osteoclastogenic activity. Receptor activator of NF-κB ligand (RANKL) expression was regulated by Wnt5a in periodontal ligament cells (PDLCs), and exosomal miR-1260b was found to target Wnt5a-mediated RANKL pathway and inhibit its osteoclastogenic activity. These results indicate that significant ability of the TNF-α-preconditioned GMSC-derived exosomes to regulate inflammation and osteoclastogenesis paves the way for establishment of a therapeutic approach for periodontitis.

Knowledge of Cervical Cancer and Human Papillomavirus among Japanese Women.

BACKGROUND: The combination of human papillomavirus (HPV) vaccination and cervical cancer tests are globally recommended. Although knowledge regarding cervical cancer and HPV and experience of HPV vaccination are reportedly closely associated, the associations between knowledge and frequency of cervical cancer testing are unclear. METHODS: We conducted a questionnaire survey regarding the knowledge of cervical cancer and HPV and experience of HPV vaccination and frequency of cervical cancer testing including cervical cytology and HPV testing. RESULTS: Among 99 women who visited Tsuruha Festa, most of the 77 non-medical workers who received information on cervical cancer and HPV through the Internet were not more likely to have knowledge about cervical cancer and HPV than were 12 medical workers who had gotten information in vocational school or university curriculum. The rates of HPV vaccination, cervical cytology, and HPV testing were 4.0%, 14.1%, and 4.0%, respectively, among participants and did not differ significantly according to participant job and age. Knowledge about cervical cancer and HPV was associated with experience of HPV vaccination and frequency of cervical cytology and was not associated with frequency of HPV testing. CONCLUSIONS: We observed insufficient knowledge about cervical cancer and HPV among non-medical workers as well as low HPV vaccination, cervical cytology, and HPV testing rates, and knowledge about cervical cancer and HPV to which frequency of cervical cancer testing were partially related. Therefore, the government should take measures to enhance public awareness about cervical cancer and HPV through social media such as the Internet.
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Improving contrast and spatial resolution in crystal analyzer-based x-ray dark-field imaging: Theoretical considerations and experimental demonstration.

PURPOSE: This paper describes and experimentally validates a methodology for improving contrast and spatial resolution of the x-ray dark-field imaging (XDFI) by cutting the monochromator-collimator asymmetrically and thinning the Laue angle analyzer. METHODS: We measure the spatial resolution of our XDFI setup using a test object consisting of wolfram tungsten meshes and compare it with the theoretical prediction. Using x-ray dark-field computed tomography of breast cancer specimens (lobular carcinoma in situ), we demonstrate that the resolution of XDFI is sufficient for histopathologic analysis. RESULTS: Our experimental results show that the overall spatial resolution of XDFI can be improved by approximately a factor of 2 when these modifications are implemented. The reconstructed images of breast cancer specimens provide sufficient details for radiologic histopathology. CONCLUSIONS: By cutting the monochromator-collimator and thinning the Laue angle analyzer, XDFI can achieve the resolution sufficient for radiologic histopathology.

Amelogenin Downregulates Interferon Gamma-Induced Major Histocompatibility Complex Class II Expression Through Suppression of Euchromatin Formation in the Class II Transactivator Promoter IV Region in Macrophages.

Enamel matrix derivatives (EMDs)-based periodontal tissue regenerative therapy is known to promote healing with minimal inflammatory response after periodontal surgery, i. e., it promotes wound healing with reduced pain and swelling. It has also been reported that macrophages stimulated with amelogenin, a major component of EMD, produce various anti-inflammatory cytokines and growth factors. We previously found that stimulation of monocytes with murine recombinant M180 (rM180) amelogenin suppresses major histocompatibility complex class II (MHC II) gene expression using microarray analysis. However, the detailed molecular mechanisms for this process remain unclear. In the present study, we demonstrated that rM180 amelogenin selectively downmodulates the interferon gamma (IFNγ)-induced cell surface expression of MHC II molecules in macrophages and this mechanism mediated by rM180 appeared to be widely conserved across species. Furthermore, rM180 accumulated in the nucleus of macrophages at 15 min after stimulation and inhibited the protein expression of class II transactivator (CIITA) which controls the transcription of MHC II by IFNγ. In addition, reduced MHC II expression on macrophages pretreated with rM180 impaired the expression of T cell activation markers CD25 and CD69, T cell proliferation ability, and IL-2 production by allogenic CD4+ T lymphocytes in mixed lymphocyte reaction assay. The chromatin immunoprecipitation assay showed that IFNγ stimulation increased the acetylation of histone H3 lysine 27, which is important for conversion to euchromatin, as well as the trimethylation of histone H3 lysine 4 levels in the CIITA promoter IV (p-IV) region, but both were suppressed in the group stimulated with IFNγ after rM180 treatment. In conclusion, the present study shows that amelogenin suppresses MHC II expression by altering chromatin structure and inhibiting CIITA p-IV transcription activity, and attenuates subsequent T cell activation. Clinically observed acceleration of wound healing after periodontal surgery by amelogenin may be partially mediated by the mechanism elucidated in this study. In addition, the use of recombinant amelogenin is safe because it is biologically derived protein. Therefore, amelogenin may also be used in future as an immunosuppressant with minimal side effects for organ transplantation or MHC II-linked autoimmune diseases such as type I diabetes, multiple sclerosis, and rheumatoid arthritis, among others.

Varenicline aggravates atherosclerotic plaque formation in nicotine-pretreated ApoE knockout mice due to enhanced oxLDL uptake by macrophages through downregulation of ABCA1 and ABCG1 expression.

Varenicline is a widely used and effective drug for smoking cessation. We previously reported that varenicline aggravates atherosclerosis in apolipoprotein E knockout (ApoE KO) mice. However, it remains unknown whether varenicline affects cardiovascular events in patients with nicotine addiction. Here, we examined the effect of varenicline on atherosclerotic plaque formation in nicotine-pretreated ApoE KO mice and oxidized low-density lipoprotein (oxLDL) uptake in nicotine-treated peritoneal macrophages. Varenicline caused significant progression of plaque formation in the whole aorta and aortic root and further accelerated the increased formation of a macrophage-rich plaque area in the aortic root in nicotine-pretreated ApoE KO mice. Varenicline (10 µM) enhanced oxLDL uptake in peritoneal macrophages. Furthermore, this treatment significantly further lowered the decreased protein levels of ATP-binding cassette (ABC) transporter without affecting the expression of scavenger receptors LOX-1 and CD36 in RAW264.7 cells treated with 100 nM nicotine. Varenicline enhanced nicotine-induced oxLDL uptake in macrophages through decreased expression of cholesterol efflux transporters ABCA1 and ABCG1 and thereby progressed atherosclerotic plaque formation. Taken together, we tentatively conclude that nicotine exposure before and/or during varenicline treatment can aggravate varenicline-increased atherosclerotic plaque formation and progression. Therefore, this enhanced risk requires special consideration when prescribing varenicline to smoker patients.

Dental pulp cell-derived powerful inducer of TNF-α comprises PKR containing stress granule rich microvesicles.

It is well known that dental pulp tissue can evoke some of the most severe acute inflammation observed in the human body. We found that dental pulp cells secrete a factor that induces tumor necrosis factor-α production from macrophages, and designated this factor, dental pulp cell-derived powerful inducer of TNF-α (DPIT). DPIT was induced in dental pulp cells and transported to recipient cells via microvesicles. Treatment of dental pulp cells with a PKR inhibitor markedly suppressed DPIT activity, and weak interferon signals were constitutively activated inside the cells. In recipient macrophages, stimulation with DPIT-containing supernatants from pulp cells resulted in activation of both nuclear factor-κB and MAP kinases like JNK and p38. Proteomics analyses revealed that many stress granule-related proteins were present in supernatants from dental pulp cells as well as microvesicle marker proteins like GAPDH, ß-actin, HSPA8, HSPB1, HSPE1, and HSPD1. Furthermore, giant molecule AHNAK and PKR were detected in microvesicles derived from dental pulp cells, and gene silencing of AHNAK in dental pulp cells led to reduced DPIT activity. Thus, it appeared that the core protein of DPIT was PKR, and that PKR was maintained in an active state in stress granule aggregates with AHNAK and transported via microvesicles. The activity of DPIT for TNF-α induction was far superior to that of gram-negative bacterial endotoxin. Therefore, we, report for the first time, that active PKR is transported via microvesicles as stress granule aggregates and induces powerful inflammatory signals in macrophages.

Structural instability of IκB kinase ß promotes autophagic degradation through enhancement of Keap1 binding.

IKKß, an essential kinase of NF-κB signaling, is composed of an N-terminal kinase domain (KD) and a C-terminal scaffolding domain, containing a ubiquitin-like domain (ULD). The Hsp90 chaperon has special responsibility for folding of protein kinases including IKKß. Here, we found that Hsp90 inhibition induced IKKß degradation, which is partially mediated by Keap1. Geldanamycin (GA), a Hsp90 inhibitor, enhances association of IKKß with Keap1 through the binding site in KD, and translocates IKKß to detergent-insoluble fractions leading to its autophagic degradation. An electrophile tBHQ suppressed Keap1-mediated proteasomal Nrf2 degradation but not autophagic IKKß degradation. Substitution mutation of Leu353 to Ala in the ULD destabilizes IKKß, enhances its association with Keap1, translocates it to detergent-insoluble fractions, and causes its autophagic degradation. These results suggest that Keap1 is involved in the degradation of structural destabilized IKKß and negative regulation of NF-κB under proteotoxic stress.

Distinct B subunits of PP2A regulate the NF-κB signalling pathway through dephosphorylation of IKKß, IκBα and RelA.

PP2A is composed of a scaffolding subunit (A), a catalytic subunit (C) and a regulatory subunit (B) that is classified into four families including B, B', B'' and B'''/striatin. Here, we found that a distinct PP2A complex regulates NF-κB signalling by dephosphorylation of IKKß, IκBα and RelA/p65. The PP2A core enzyme AC dimer and the holoenzyme AB'''C trimer dephosphorylate IKKß, IκBα and RelA, whereas the ABC trimer dephosphorylates IκBα but not IKKß and RelA in cells. In contrast, AB'C and AB''C trimers have little effect on dephosphorylation of these signalling proteins. These results suggest that different forms of PP2A regulate NF-κB pathway signalling through multiple steps each in a different manner, thereby finely tuning NF-κB- and IKKß-mediated cellular responses.

Amelogenin induces M2 macrophage polarisation via PGE2/cAMP signalling pathway.

OBJECTIVES: Amelogenin, the major component of the enamel matrix derivative (EMD), has been suggested as a bioactive candidate for periodontal regeneration. Apart from producing a regenerative effect on periodontal tissues, amelogenin has also been reported to have an anti-inflammatory effect. However, the precise molecular mechanisms underlying these effects remain unclear. In the present study, we examined the immunomodulatory effects of amelogenin on macrophages. DESIGN: Human phorbol 12-myristate 13-acetate (PMA)-differentiated U937 macrophages and CD14+ peripheral blood-derived monocytes (PBMC)-derived macrophages were stimulated with recombinant amelogenin (rM180). After performing a detailed microarray analysis, the effects of rM180 on macrophage phenotype and signal transduction pathways were evaluated by real-time polymerase chain reaction, enzyme-linked immunosorbent assay, confocal microscopy and flow cytometry. RESULTS: The microarray analysis demonstrated that rM180 increased the expression of anti-inflammatory genes in lipopolysaccharide (LPS)-challenged macrophages after 24h, while it temporarily up-regulated inflammatory responses at 4h. rM180 significantly enhanced the expression of M2 macrophage markers (CD163 and CD206). rM180-induced M2 macrophage polarisation was associated with morphological changes as well as vascular endothelial growth factor (VEGF) production. rM180 enhanced prostaglandin E2 (PGE2) expression, and the activation of the cAMP/cAMP-responsive element binding (CREB) signaling pathway was involved in amelogenin-induced M2 macrophage polarisation. Blocking of PGE2 signaling by indomethacin specifically abrogated rM180 with or without LPS-induced M2 shift in PBMC-derived macrophages. CONCLUSION: Amelogenin could reprogram macrophages into the anti-inflammatory M2 phenotype. It could therefore contribute to the early resolution of inflammation in periodontal lesions and provide a suitable environment for remodeling-periodontal tissues.