Artificial intelligence-based analysis of the spatial distribution of abnormal computed tomography patterns in SARS-CoV-2 pneumonia: association with disease severity.

BACKGROUND: The substantial heterogeneity of clinical presentations in patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pneumonia still requires robust chest computed tomography analysis to identify high-risk patients. While extension of ground-glass opacity and consolidation from peripheral to central lung fields on chest computed tomography (CT) might be associated with severely ill conditions, quantification of the central-peripheral distribution of ground glass opacity and consolidation in assessments of SARS-CoV-2 pneumonia remains unestablished. This study aimed to examine whether the central-peripheral distributions of ground glass opacity and consolidation were associated with severe outcomes in patients with SARS-CoV-2 pneumonia independent of the whole-lung extents of these abnormal shadows. METHODS: This multicenter retrospective cohort included hospitalized patients with SARS-CoV-2 pneumonia between January 2020 and August 2021. An artificial intelligence-based image analysis technology was used to segment abnormal shadows, including ground glass opacity and consolidation. The area ratio of ground glass opacity and consolidation to the whole lung (GGO%, CON%) and the ratio of ground glass opacity and consolidation areas in the central lungs to those in the peripheral lungs (GGO(C/P)) and (CON(C/P)) were automatically calculated. Severe outcome was defined as in-hospital death or requirement for endotracheal intubation. RESULTS: Of 512 enrolled patients, the severe outcome was observed in 77 patients. GGO% and CON% were higher in patients with severe outcomes than in those without. Multivariable logistic models showed that GGO(C/P), but not CON(C/P), was associated with the severe outcome independent of age, sex, comorbidities, GGO%, and CON%. CONCLUSION: In addition to GGO% and CON% in the whole lung, the higher the ratio of ground glass opacity in the central regions to that in the peripheral regions was, the more severe the outcomes in patients with SARS-CoV-2 pneumonia were. The proposed method might be useful to reproducibly quantify the extension of ground glass opacity from peripheral to central lungs and to estimate prognosis.

Combination Therapy with EGFR Tyrosine Kinase Inhibitors and TEAD Inhibitor Increases Tumor Suppression Effects in EGFR Mutation-positive Lung Cancer.

EGFR-tyrosine kinase inhibitors (TKI) are the first-line therapies for EGFR mutation-positive lung cancer. EGFR-TKIs have favorable therapeutic effects. However, a large proportion of patients with EGFR mutation-positive lung cancer subsequently relapse. Some cancer cells survive the initial treatment with EGFR-TKIs, and this initial survival may be associated with subsequent recurrence. Therefore, we aimed to overcome the initial survival against EGFR-TKIs. We hypothesized that yes-associated protein 1 (YAP1) is involved in the initial survival against EGFR-TKIs, and we confirmed the combined effect of EGFR-TKIs and a YAP1-TEAD pathway inhibitor. The KTOR27 (EGFR kinase domain duplication) lung cancer cell lines established from a patient with EGFR mutation-positive lung cancer and commercially available PC-9 and HCC827 (EGFR exon 19 deletions) lung cancer cell lines were used. These cells were used to evaluate the in vitro and in vivo effects of VT104, a TEAD inhibitor. In addition, YAP1 involvement was investigated in pathologic specimens. YAP1 was activated by short-term EGFR-TKI treatment in EGFR mutation-positive lung cancer cells. In addition, inhibiting YAP1 function using siRNA increased the sensitivity to EGFR-TKIs. Combination therapy with VT104 and EGFR-TKIs showed better tumor-suppressive effects than EGFR-TKIs alone, in vitro and in vivo. Moreover, the combined effect of VT104 and EGFR-TKIs was observed regardless of the localization status of YAP1 before EGFR-TKI exposure. These results suggest that combination therapy with the TEAD inhibitor and EGFR-TKIs may improve the prognosis of patients with EGFR mutation-positive lung cancer.

Hydraulic calcium silicate-based root canal sealers mitigate proinflammatory cytokine synthesis and promote osteogenesis in vitro.

Background/purpose: The mineralized tissue-inductive ability and anti-inflammatory properties of hydraulic calcium silicate-based (HCSB) sealers have not been fully elucidated. This study aimed to evaluate the effects of the HCSB sealers Bio-C sealer (BioC), Well-Root ST (WST), and EndoSequence BC sealer (BC), on osteoblastic differentiation/mineralization and proinflammatory cytokine synthesis by macrophages. Materials and methods: Diluted extracts of set sealers or calcium chloride solutions of approximately equivalent Ca2+ concentrations were applied to a mouse osteoblastic cell line (Kusa-A1 cells) and lipopolysaccharide-stimulated mouse macrophage cell line (RAW264.7 cells). Expressions of osteoblastic markers in Kusa-A1 cells and proinflammatory cytokines in RAW264.7 cells were evaluated by reverse transcription-quantitative polymerase chain reaction and enzyme-linked immunosorbent assays. Mineralized nodules were detected by Alizarin red S staining. Cell proliferation was assessed by WST-8 assay and cell attachment on set sealers was examined by scanning electron microscopy. Results: The three sealer extracts significantly upregulated osteocalcin and osteopontin mRNA, and promoted significant mineralized nodule formation in Kusa-A1 cells. The three sealer extracts significantly downregulated the mRNA expressions of interleukin (IL)-1α, IL-1ß, IL-6, and tumor necrosis factor (TNF)-α and protein levels of IL-6 and TNF-α in RAW264.7 cells. Calcium chloride solutions induced osteoblastic differentiation/mineralization. AH Plus Jet (a control sealer) extract did not. The three HCSB sealers did not interfere with the growth and attachment of Kusa-A1 cells. Conclusion: BioC, WST, and BC were biocompatible, upregulated osteoblastic differentiation/mineralization, and downregulated proinflammatory cytokine expression. Ca2+ released from HCSB sealers might be involved, at least in part, in the induction of osteoblastic differentiation/mineralization.

CD47 polymorphism for predicting nivolumab benefit in patients with advanced non­small­cell lung cancer.

Immune checkpoint inhibitors (ICIs), such as nivolumab, play an essential role in non-small-cell lung cancer (NSCLC) treatment. Programmed death ligand-1 has been used as a predictive biomarker for the efficacy of ICI treatment in patients with NSCLC; however, its predictive value is considered insufficient. Therefore, there is an urgent need for better predictive biomarkers. The present study focused on the CD47 molecule, which is associated with macrophages and tumor immunity. The study aimed to investigate the association between CD47 single nucleotide polymorphism (SNP) and the therapeutic effect of nivolumab in patients with NSCLC. The CD47 SNP genotypes and clinical outcomes were retrospectively analyzed in 164 patients with NSCLC treated with nivolumab at Kyoto University Hospital (Kyoto, Japan). Patients with the G/G genotype of the CD47 SNP rs3804639 had significantly longer progression-free survival than those with the G/T or T/T genotypes [2.6 months vs. 2.1 months, hazard ratio (HR), 0.70; P=0.026]. Moreover, the G/G genotype of the CD47 SNP rs3804639 was associated with a significantly longer median overall survival than the G/T or T/T genotypes of the CD47 SNP rs3804639 (24.8 months vs. 12.0 months, HR, 0.64; P=0.021). In conclusion, CD47 polymorphism may be a novel predictive biomarker of nivolumab efficacy in patients with advanced NSCLC.

Remote Electronic Effect of Chiral N-Heterocyclic Carbene Catalyst on an Asymmetric Benzoin Reaction.

A remote electronic effect of chiral aminoindanol-derived N-heterocyclic carbene catalyst on an asymmetric benzoin reaction was investigated. The catalyst bearing remote electron-withdrawing substituents increased enantioselectivity of the reaction at the cost of the reaction rate. DFT calculations rationalized the increased enantioselectivity.

Coupling of Cortical Hyperintense Signals and Increased Glucose Metabolism in a Case of Anti-GABAA Receptor Antibody-associated Encephalitis.

We herein report a case of anti-gamma aminobutyric acid type A receptor antibody-associated encephalitis (anti-GABAA-RE) with progressive aphasia and generalized tonic-clonic seizures. Cerebral magnetic resonance imaging (MRI) showed cortical brain lesions coupled with hypermetabolism on fluorodeoxyglucose-positron emission tomography. After two courses of methylprednisolone pulse therapy, improvements in neurological symptoms without sequelae and the total disappearance of MRI lesions were observed. Upon encountering patients with refractory status epilepticus, multifocal cerebral MRI lesions, and suspected autoimmune encephalitis, especially in cases with thymoma, it would be prudent to suspect anti-GABAA-RE and consider the evaluation of anti-GABAA receptor antibody and methylprednisolone pulse therapy.

Yes-associated protein 1 mediates initial cell survival during lorlatinib treatment through AKT signaling in ROS1-rearranged lung cancer.

Tyrosine kinase inhibitors (TKIs) that target the ROS proto-oncogene 1, receptor tyrosine kinase (ROS1) gene have shown dramatic therapeutic effects in patients with ROS1-rearranged non-small-cell lung cancer (NSCLC). Nevertheless, advanced ROS1-rearranged NSCLC is rarely cured as a portion of the tumor cells can survive the initial stages of ROS1-TKI treatment, even after maximum tumor shrinkage. Therefore, understanding the mechanisms underlying initial cell survival during ROS1-TKI treatment is necessary to prevent cell survival and achieve a cure for ROS1-rearranged NSCLC. In this study, we clarified the initial survival mechanisms during treatment with lorlatinib, a ROS1 TKI. First, we established a patient-derived ezrin gene-ROS1-rearranged NSCLC cell line (KTOR71). Then, following proteomic analysis, we focused on yes-associated protein 1 (YAP1), which is a major mediator of the Hippo pathway, as a candidate factor involved in cell survival during early lorlatinib treatment. Yes-associated protein 1 was activated by short-term lorlatinib treatment both in vitro and in vivo. Genetic inhibition of YAP1 using siRNA, or pharmacological inhibition of YAP1 function by the YAP1-inhibitor verteporfin, enhanced the sensitivity of KTOR71 cells to lorlatinib. In addition, the prosurvival effect of YAP1 was exerted through the reactivation of AKT. Finally, combined therapy with verteporfin and lorlatinib was found to achieve significantly sustained tumor remission compared with lorlatinib monotherapy in vivo. These results suggest that YAP1 could mediate initial cell resistance to lorlatinib in KTOR71 cells. Thus, combined therapy targeting both YAP1 and ROS1 could potentially improve the outcome of ROS1-rearranged NSCLC.

Sarcomatoid malignant pleural mesothelioma treated with nivolumab: A case series.

Immune checkpoint therapy (ICT) with nivolumab has been widely used to treat malignant pleural mesothelioma (MPM) since clinical trials confirmed its efficacy. However, only a few clinical trials have been conducted for the treatment of sarcomatoid MPM, which is a rare histological type of MPM. Additionally, clinical reports of sarcomatoid MPM are scarce. Therefore, the benefits and risks of nivolumab treatment for sarcomatoid MPM remain unclear. The present report describes the treatment of 3 cases of sarcomatoid MPM (all 3 were men) with nivolumab monotherapy. In all three cases, nivolumab was effective despite variations in the duration of treatment, although side effects were observed in 2 patients. Programmed death ligand 1 (PD-L1) expression was positive in all 3 cases. In particular, the patient with the highest PD-L1 expression had the most rapid response of the 3 patients, and the effect lasted as long as those of the other 2, despite receiving the smallest number of doses of nivolumab. It has been reported that sarcomatoid MPM tends to respond poorly to chemotherapy and express higher levels of PD-L1 than epithelial MPM; thus, ICT may be necessary in these cases. This case series suggests that ICT with nivolumab is a promising treatment option for sarcomatoid MPM.

Biocompatibility and pro-mineralization effect of tristrontium aluminate cement for endodontic use.

Background/purpose: Tristrontium aluminate (S3A) is a hydraulic cement with setting behavior similar to that of mineral trioxide aggregate (MTA). This study examined the biological effects of S3A on mouse dental papilla cells (MDPs) in vitro and on rat exposed pulps in vivo. Materials and methods: Extracts of S3A and MTA were prepared by immersing each cement in ultrapure water. MDPs were cultured with S3A or MTA extracts, and cell proliferation was evaluated with a tetrazolium-salt assay. Attachment of MDPs on the set cements was examined with scanning electron microscopy (SEM). mRNA expression of bone morphogenic protein (Bmp2), osteocalcin (Oc) and osteopontin (Opn) in MDPs exposed to S3A or MTA extracts was determined with reverse transcription-quantitative polymerase chain reaction. Mineralized nodule formation was evaluated with Alizarin Red S staining. Simulated body fluid (SBF)-dipped S3A was examined with SEM and energy dispersive X-ray analysis (EDX). Exposed molar pulps of male Wistar rats capped with S3A or MTA were histologically examined. Results: S3A extract did not inhibit proliferation of MDPs. Set S3A and MTA exhibited attachment of MDPs on their surface. S3A extract showed significantly higher mineralized nodule formation and mRNA expression of Bmp2, Oc, and Opn than did MTA extract. SBF-dipped S3A exhibited formation of surface precipitates, which were composed of Ca, P, Sr, and Al. Direct pulp capping with S3A and with MTA induced mineralized tissue repair of the exposed pulp. Conclusion: S3A possesses biocompatibility and pro-mineralization effects comparable to those of MTA.

Kinetics of LYVE-1-positive M2-like macrophages in developing and repairing dental pulp in vivo and their pro-angiogenic activity in vitro.

Tissue-resident macrophages expressing lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) are found in multiple tissues and organs. We aimed to evaluate the dynamics and biological functions of LYVE-1+ macrophages in dental pulp during post-injury tissue remodeling. Immunofluorescence staining of mouse embryos revealed that LYVE-1+ macrophages colonized dental pulp before birth. In mature rat molar dental pulp, LYVE-1+ macrophages were the main subset of macrophages expressing CD163, an M2 marker, and were distributed throughout the tissue. In response to dental pulp injury induced by cavity preparation, LYVE-1+ macrophages quickly disappeared from the affected area of the pulp and gradually repopulated during the wound healing process. RAW264.7 mouse macrophages cultured with a mixture of macrophage colony-stimulating factor, interleukin-4, and dexamethasone increased LYVE-1 expression, whereas lipopolysaccharide-stimulation decreased LYVE-1 expression. Enforced expression of Lyve1 in RAW264.7 cells resulted in increased mRNA expression of matrix metalloproteinase 2 (Mmp2), Mmp9, and vascular endothelial growth factor A (Vegfa). Lyve1-expressing macrophages promoted the migration and tube formation of human umbilical vein endothelial cells. In conclusion, LYVE-1+ tissue-resident M2-like macrophages in dental pulp showed dynamism in response to pulp injury, and possibly play an important role in angiogenesis during wound healing and tissue remodeling.

Hypoxia-inducible factor 1α induces osteo/odontoblast differentiation of human dental pulp stem cells via Wnt/ß-catenin transcriptional cofactor BCL9.

Accelerated dental pulp mineralization is a common complication in avulsed/luxated teeth, although the mechanisms underlying this remain unclear. We hypothesized that hypoxia due to vascular severance may induce osteo/odontoblast differentiation of dental pulp stem cells (DPSCs). This study examined the role of B-cell CLL/lymphoma 9 (BCL9), which is downstream of hypoxia-inducible factor 1α (HIF1α) and a Wnt/ß-catenin transcriptional cofactor, in the osteo/odontoblastic differentiation of human DPSCs (hDPSCs) under hypoxic conditions. hDPSCs were isolated from extracted healthy wisdom teeth. Hypoxic conditions and HIF1α overexpression induced significant upregulation of mRNAs for osteo/odontoblast markers (RUNX2, ALP, OC), BCL9, and Wnt/ß-catenin signaling target genes (AXIN2, TCF1) in hDPSCs. Overexpression and suppression of BCL9 in hDPSCs up- and downregulated, respectively, the mRNAs for AXIN2, TCF1, and the osteo/odontoblast markers. Hypoxic-cultured mouse pulp tissue explants showed the promotion of HIF1α, BCL9, and ß-catenin expression and BCL9-ß-catenin co-localization. In addition, BCL9 formed a complex with ß-catenin in hDPSCs in vitro. This study demonstrated that hypoxia/HIF1α-induced osteo/odontoblast differentiation of hDPSCs was partially dependent on Wnt/ß-catenin signaling, where BCL9 acted as a key mediator between HIF1α and Wnt/ß-catenin signaling. These findings may reveal part of the mechanisms of dental pulp mineralization after traumatic dental injury.

Integrative analysis of gut microbiome and host transcriptomes reveals associations between treatment outcomes and immunotherapy-induced colitis.

Immune checkpoint inhibitors (ICIs) are widely used to treat various malignancies. Although the gut microbiome is known to influence the efficacy of ICIs on epithelial tumors, the functional interactions between gut taxa and colonic mucosa remain poorly understood. Here we performed transcriptomic profiling and 16S rRNA sequencing to investigate the relationships between mucosal gene expression and microbial composition with ICI responses and gastrointestinal immune-related adverse events (GI irAEs). In responders, genes related to DNA repair and cell cycle signatures were enriched in responders whereas signatures related to innate immune response, NFAT and IFN-γ signaling pathways were enriched in nonresponders. Gut microbial composition revealed an association between moderate GI irAE and favorable response to ICI therapy. Favorable therapeutic responses to ICI and GI irAE treatments were associated with taxa classified as Enterobacteriaceae and were related to ribonucleoprotein complex biogenesis, cytokine-mediated signaling pathway, tRNA metabolic process, and ribonucleoprotein complex assembly in the colon. These findings open new perspectives for improving the efficacy and safety of cancer immunotherapy.

Evaluation of the anti-inflammatory effects of surface-reaction-type pre-reacted glass-ionomer filler containing root canal sealer in lipopolysaccharide-stimulated RAW264.7 macrophages.

A prototype surface-reaction-type pre-reacted glass-ionomer (S-PRG) filler containing root canal sealer (S-PRG sealer) exhibits bioactive potential by releasing multiple ions. This study explored the suppressive effects and modes of action of S-PRG sealer extracts on proinflammatory cytokine expression in lipopolysaccharide (LPS)-stimulated RAW264.7 cells. Expression of proinflammatory cytokines was evaluated by RT-qPCR and ELISA. Expression of phosphorylated nuclear factor-kappa B (p-NF-kB) p65 was evaluated by western blotting. S-PRG sealer extracts significantly downregulated mRNA expression levels of interleukin (IL)-1α, IL-6, and TNF-α in LPS-stimulated RAW264.7 cells; the extracts also reduced the levels of IL-6 protein and p-NF-kB. In order to verify that Zn2+ was responsible for downregulation of proinflammatory cytokine expression, N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine (TPEN) was used as a heavy metal chelator with strong affinity for Zn2+. These effects were mitigated by TPEN. The application of ZnCl2 reproduced the actions of S-PRG sealer extracts. These data suggest that S-PRG sealer has anti-inflammatory potential involving heavy metal ions such as Zn2+.

Evaluation of the cytocompatibility of methacrylate resin-based root canal sealers with osteoblast-like cells.

This study compared the cytocompatibilities of three methacrylate resin-based root canal sealers [MetaSEAL Soft (MSS), Hybrid Root SEAL (HRS), and Superbond Sealer (SBS)] in either freshly mixed or set conditions using the Kusa A1 osteoblastic cell line. The three sealers and an epoxy resin-based sealer (AH Plus; AHP) were extracted in culture medium; cell growth and osteogenic properties were analyzed. Cell adhesion on set sealers was analyzed with scanning electron microscopy. The respective extents of cell growth were as follows in freshly mixed and set sealer extracts: SBS>MSS>AHP>HRS and SBS=AHP>MSS>HRS. Light irradiation of MSS and HRS increased the cell growth of set sealer extracts. Set SBS, MSS, and AHP did not alter expression of osteogenic genes or formation of mineralized nodules. Attached cells were observed only on SBS. In conclusion, the four sealers exhibited varying degrees of compatibility to osteoblasts; SBS and HRS were the most and least compatible, respectively.

Multiple mutations in RNA polymerase ß-subunit gene (rpoB) in Streptomyces incarnatus NRRL8089 enhance production of antiviral antibiotic sinefungin: modeling rif cluster region by density functional theory.

Streptomyces incarnatus NRRL8089 produces the antiviral, antifungal, antiprotozoal nucleoside antibiotic sinefungin. To enhance sinefungin production, multiple mutations were introduced to the rpoB gene encoding RNA polymerase (RNAP) ß-subunit at the target residues, D447, S453, H457, and R460. Sparse regression analysis using elastic-net lasso-ridge penalties on previously reported H457X mutations identified a numeric parameter set, which suggested that H457R/Y/F may cause production enhancement. H457R/R460C mutation successfully enhanced the sinefungin production by 3-fold, while other groups of mutations, such as D447G/R460C or D447G/H457Y, made moderate or even negative effects. To identify why the rif cluster residues have diverse effects on sinefungin production, an RNAP/DNA/mRNA complex model was constructed by homology modeling and molecular dynamics simulation. The 4 residues were located near the mRNA strand. Density functional theory-based calculation suggested that D447, H457, and R460 are in direct contact with ribonucleotide, and partially positive charges are induced by negatively charged chain of mRNA.

A Novel Bioactive Endodontic Sealer Containing Surface-Reaction-Type Prereacted Glass-Ionomer Filler Induces Osteoblast Differentiation.

Surface­reaction­type prereacted glass-ionomer (S­PRG) fillers exhibit bioactive properties by the release of multiple ions. This study examined whether a novel endodontic sealer containing S­PRG fillers (PRG+) has the capacity to induce osteoblast differentiation. Kusa­A1 osteoblastic cells were cultured with extracts of PRG+, PRG- (an experimental sealer containing S­PRG­free silica fillers), AH Plus (an epoxy-resin­based sealer), and Canals N (a zinc-oxide noneugenol sealer). Cell viability and mineralized nodule formation were determined using WST­8 assay and Alizarin red staining, respectively. Osteoblastic-marker expression was analyzed with RT­qPCR and immunofluorescence. Phosphorylation of extracellular signal­regulated kinase (ERK) and p38 mitogen­activated protein kinase (MAPK) was determined with Western blotting. Extracts of freshly mixed PRG+, PRG-, and AH Plus significantly decreased cell growth, but extracts of the set samples were not significantly cytotoxic. Set PRG+ significantly upregulated mRNAs for alkaline phosphatase and bone sialoprotein (IBSP) compared to set PRG-, and upregulation was blocked by NPS2143, a calcium­sensing receptor antagonist. Set PRG+ significantly accelerated IBSP expression, mineralized nodule formation, and enhanced the phosphorylation of ERK and p38 compared with set PRG-. In conclusion, PRG+ induced the differentiation and mineralization of Kusa­A1 cells via the calcium-sensing receptor-induced activation of ERK and p38 MAPK.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Author Correction: Strontium ranelate promotes odonto-/osteogenic differentiation/mineralization of dental papillae cells in vitro and mineralized tissue formation of the dental pulp in vivo.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

HIF1α inhibits LPS-mediated induction of IL-6 synthesis via SOCS3-dependent CEBPß suppression in human dental pulp cells.

Hypoxia-inducible factor 1 alpha (HIF1α) is a transcriptional factor that plays a key role in the regulation of various molecules expressed in hypoxic conditions. Ischemic/hypoxic conditions are regarded as a distinct characteristic of dental pulp inflammation due to the encasement of pulp tissue within the rigid tooth structure. This study was performed to examine the role of HIF1α in the regulation of interleukin (IL)-6, a proinflammatory cytokine expressed in inflamed dental pulp, in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). LPS stimulation promoted the expression of IL-6 in hDPCs, while HIF1α suppressed the expression of IL-6. Moreover, HIF1α induced suppressor of cytokine signaling 3 (SOCS3) expression in LPS-stimulated hDPCs, and SOCS3 activity led to downregulate expression of CCAAT enhancer-binding protein beta (CEBPß), an inducer of IL-6. LPS stimulation promoted HIF1α expression in hDPCs and mouse pulp tissue explants cultured under hypoxic conditions. These findings suggest that HIF1α negatively regulates IL-6 synthesis in LPS-stimulated hDPCs via upregulation of SOCS3 and subsequent downregulation of CEBPß.

Anti-inflammatory roles of microRNA 21 in lipopolysaccharide-stimulated human dental pulp cells.

microRNAs are small noncoding RNA molecules that regulate RNA silencing and posttranscriptional gene expression, and many microRNAs are involved in inflammatory processes. In particular, microRNA 21 (miR-21) is upregulated in inflammatory environment and reported to induce anti-inflammatory responses. However, the involvement of miR-21 in pulpal inflammation and the precise mechanisms of anti-inflammatory reactions induced by miR-21 remain unclear. We hypothesized that miR-21-5p expression is induced in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs) and that miR-21-5p downregulates the proinflammatory cytokine expression in LPS-stimulated hDPCs. We found that miR-21-5p was upregulated in LPS-stimulated hDPCs concomitant with elevated proinflammatory cytokine expression and nuclear factor-kappa B (NF-κB) phosphorylation. miR-21-5p and cytokine expression were downregulated by BAY11-7085 and caffeic acid phenylethyl ester (CAPE), specific and potent NF-κB inhibitors. Enforced expression of miR-21-5p downregulated the Toll-like receptor (TLR)/NF-κB signaling via reducing the expression of TNF receptor-associated factor 6 (TRAF6) and programmed cell death 4 (PDCD4), which further induced the decrease of proinflammatory cytokine expression. hDPCs forcibly overexpressing miR-21-5p downregulated the LPS-induced expression of TNF receptor-associated factor 6 (TRAF6; a component of the Toll-like receptor [TLR]/NF-κB signaling pathway), programmed cell death 4 (PDCD4, a positive regulator of the TLR/NF-κB signaling pathway), and proinflammatory cytokines. In contrast, miR-21-5p inhibitor-transfected hDPCs upregulated the expression of TRAF6, PDCD4, and inflammatory cytokines following LPS stimulation. These findings suggest that miR-21-5p expression was induced by the NF-κB signaling pathway, which was in turn negatively regulated by miR-21-5p via downregulation of TRAF6 and PDCD4 expression in LPS-stimulated hDPCs.