Establishment and Characterization of Immortalized Human Dermal Papilla Cells Expressing Human Papillomavirus 16 E6/E7.

Primary human dermal papilla cells (HDPCs) are often preferred in studies on hair growth and regeneration. However, primary HDPCs are limited by their reduced proliferative capacity, decreased hair induction potential, and extended doubling times at higher passages. To overcome these limitations, pTARGET vectors containing human papillomavirus16 (HPV16) E6/E7 oncogenes were transfected into HDPCs and selected using G-148 to generate immortalized cells here. HPV16 E6/E7 oncogenes were efficiently transfected into primary HDPCs. Immortalized HDPC showed higher proliferative activity than primary HDPC, confirming an increased proliferation rate. Expression of p53 and pRb proteins was downregulated by E6 and E7, respectively. E6/E7 expressing HDPC cells revealed that cyclin-dependent kinase (CDK) inhibitor p21 expression was decreased, while cell cycle-related genes and proteins (CDK2 and cyclin E) and E2F family genes were upregulated. Immortalized HDPCs maintained their responsiveness to Wnt/ß-catenin pathway and hair follicle formation capability, as indicated by their aggregative properties and stemness. E6/E7 immortalized HDPCs may facilitate in vitro hair growth and regeneration studies.

Phorbal-12-mysristate-13-acetate-induced inflammation is restored by protectin DX through PPARγ in human promonocytic U937 cells.

AIMS: Protectin DX (PDX), a specialized pro-resolving mediator, is an important pharmaceutical compound with potential antioxidant and inflammation-resolving effects. However, the fundamental mechanism by which PDX's ameliorate chronic inflammatory diseases has not yet been elucidated. This study aims to evaluate the anti-inflammatory properties and PPARγ-mediated mechanisms of PDX in phorbal-12-mysristate-13-acetate (PMA)-stimulated human promonocytic U937 cells. MAIN METHODS: We confirmed the effects of PDX on expressions of pro-inflammatory cytokines, mediators, and CD14 using conventional PCR, RT-qPCR, ELISA, and flow cytometry. Using western blotting, immunofluorescence, and reactive oxygen species (ROS) determination, we observed that PDX regulated PMA-induced signaling cascades. Molecular docking analysis and a cellular thermal shift assay were conducted to verify the interaction between PDX and the proliferator-activated receptor-γ (PPARγ) ligand binding domain. Western blotting was then employed to explore the alterations in PPARγ expression levels and validate PDX as a PPARγ full agonist. KEY FINDINGS: PDX attenuated protein and mRNA expression levels of interleukin-6, tumor necrosis factor-α, and cyclooxygenase-2 in PMA-treated U937 cells. PDX acts as a PPARγ agonist, exerting a modulating effect on the ROS/JNK/c-Fos signaling pathways. Furthermore, PDX reduced human monocyte differentiation antigen CD14 expression levels. SIGNIFICANCE: PPARγ exhibits pro-resolving effects to regulate the excessive inflammation. These results suggest that PDX demonstrates the resolution of inflammation, indicating the potential for therapeutic targeting of chronic inflammatory diseases.

Human IL-32θA94V mutant attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 via binding to cell surface receptor integrin αVß3 and αVß6 in TNF-α-stimulated HUVECs.

Interleukin-32 (IL-32), first reported in 2005, and its isoforms have been the subject of numerous studies investigating their functions in virus infection, cancer, and inflammation. IL-32θ, one of the IL-32 isoforms, has been shown to modulate cancer development and inflammatory responses. A recent study identified an IL-32θ mutant with a cytosine to thymine replacement at position 281 in breast cancer tissues. It means that alanine was also replaced to valine at position 94 in amino acid sequence (A94V). In this study, we investigated the cell surface receptors of IL-32θA94V and evaluated their effect on human umbilical vein endothelial cells (HUVECs). Recombinant human IL-32θA94V was expressed, isolated, and purified using Ni-NTA and IL-32 mAb (KU32-52)-coupled agarose columns. We observed that IL-32θA94V could bind to the integrins αVß3 and αVß6, suggesting that integrins act as cell surface receptors for IL-32θA94V. IL-32θA94V significantly attenuated monocyte-endothelial adhesion by inhibiting the expression of Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in tumor necrosis factor (TNF)-α-stimulated HUVECs. IL-32θA94V also reduced the TNF-α-induced phosphorylation of protein kinase B (AKT) and c-jun N-terminal kinases (JNK) by inhibiting phosphorylation of focal adhesion kinase (FAK). Additionally, IL-32θA94V regulated the nuclear translocation of nuclear factor kappa B (NF-κB) and activator protein 1 (AP-1), which are involved in ICAM-1 and VCAM-1 expression. Monocyte-endothelial adhesion mediated by ICAM-1 and VCAM-1 is an important early step in atherosclerosis, which is a major cause of cardiovascular disease. Our findings suggest that IL-32θA94V binds to the cell surface receptors, integrins αVß3 and αVß6, and attenuates monocyte-endothelial adhesion by suppressing the expression of ICAM-1 and VCAM-1 in TNF-α-stimulated HUVECs. These results demonstrate that IL-32θA94V can act as an anti-inflammatory cytokine in a chronic inflammatory disease such as atherosclerosis.

Unsaturated oxidated fatty acid 12(S)-HETE attenuates TNF-α expression in TNF-α/IFN-γ-stimulated human keratinocytes.

Chronic skin inflammatory diseases are associated with abnormal immune responses characterized by skin barrier dysfunction. Keratinocytes participate in immune homeostasis regulated by immune cells. Immune homeostasis dysfunction contributes to the pathogenesis of skin diseases mediated by pro-inflammatory cytokines and chemokines, such as tumor necrosis factor (TNF)-α, which are produced by activated keratinocytes. 12(S)-Hydroxy eicosatetraenoic acid [12(S)-HETE], an arachidonic acid metabolite, has anti-inflammatory properties. However, the role of 12(S)-HETE in chronic skin inflammatory diseases has not been elucidated yet. In this study, we investigated the effect of 12(S)-HETE on TNF-α/interferon (IFN)-γ-induced pro-inflammatory cytokine and chemokine expression. Our data showed that 12(S)-HETE modulates TNF-α mRNA and protein expression in TNF-α-/IFN-γ-treated human keratinocytes. Molecular docking analyses demonstrated that 12(S)-HETE bound to extracellular signal-regulated kinase (ERK)1/2, thus preventing ERK activation and downregulating phosphorylated ERK expression. We also demonstrated that 12(S)-HETE treatment inhibited IκB and ERK phosphorylation and nuclear factor (NF)-κB, p65/p50, and CCAAT/enhancerbindingproteinß (C/EBPß) translocation. Overall, our results showed that 12(S)-HETE attenuated TNF-α expression and secretion by inhibiting the mitogen-activated protein kinase ERK/NF-κB and C/EBPß signaling pathways. Overall, these results suggest that 12(S)-HETE effectively resolved TNF-α-induced inflammation.

Cinnamomum verum extract inhibits NOX2/ROS and PKCδ/JNK/AP-1/NF-κB pathway-mediated inflammatory response in PMA-stimulated THP-1 monocytes.

BACKGROUND: Cinnamomum verum J. Presl (Cinnamon) is widely used in the food and pharmaceutical industries. C. verum exhibits various biological activities. However, it is unclear whether C. verum can inhibit NOX, a major source of ROS generation, and exert anti-inflammatory and antioxidant effects in PMA-stimulated THP-1 cells. PURPOSE: This study investigates the anti-inflammatory and antioxidant effects of C. verum in PMA-stimulated THP-1 cells. METHODS: The MeOH extract of C. verum was analyzed using UPLC-QTOF/MS. Anti-inflammatory and antioxidant effects of C. verum extract were examined by DCF-DA staining, immunofluorescence staining, RT-PCR, and immunoblotting in PMA-stimulated THP-1 cells. RESULTS: C. verum and its components, cinnamic acid and coumarin, significantly attenuated the expression of IL-1ß, IL-8, CCL5, and COX-2 in PMA-stimulated THP-1. C. verum decreased ROS levels via NOX2 downregulation, as well as ameliorated plasma membrane translocation of PKCδ and decreased JNK phosphorylation. Besides, C. verum suppressed the nuclear translocation of AP-1 and NF-κB, which modulates diverse pro-inflammatory genes. CONCLUSION: C. verum effectively inhibits inflammation and oxidative stress during monocyte-macrophage differentiation and downregulates inflammatory mediators via NOX2/ROS and PKCδ/JNK/AP-1/NF-κB signaling.

Comparison of the recovery and respiratory effects of aminophylline and doxapram following total intravenous anesthesia with propofol and remifentanil.

STUDY OBJECTIVE: To compare the effects of aminophylline and doxapram on recovery, respiration, and bispectral index (BIS) values in patients after total intravenous anesthesia (TIVA) with propofol and remifentanil. DESIGN: Prospective, randomized, blinded clinical trial. SETTING: Operating room of a university hospital. PATIENTS: 90 adult, ASA physical status 1 and 2 patients scheduled for elective laparoscopic vaginal hysterectomy. INTERVENTIONS: TIVA was performed with the induction target of remifentanil 3 ng/mL and propofol 6 µg/mL, followed by the maintenance target of remifentanil 1-3 ng/mL and propofol 3-5 µg/mL at the effect site, and with BIS scores in 40-50 range. Patients were randomized to three groups to receive intravenous (IV) aminophylline 3 mg/kg (n = 30), IV doxapram 1 mg/kg (n = 30), or normal IV saline (control; n = 30). MEASUREMENTS AND MAIN RESULTS: After administration of the study drugs, return to spontaneous ventilation differed significantly among the three groups. The times to eye opening and hand squeezing on verbal command were similar. The time to extubation was shortened in both the doxapram and aminophylline groups (P < 0.05). Tidal volumes were increased in the doxapram group at 5-14 minutes and the aminophylline group at 5-12 minutes (P < 0.05). Respiratory rates were increased at 2 to 8 minutes and then showed a decrease at the 12 to 14-minute mark in both the doxapram and aminophylline groups (P < 0.05). No difference was noted between the two groups. BIS values were increased in both the doxapram and aminophylline groups at 4-10 minutes (P < 0.05). Heart rates were increased in the doxapram group for the first 8 minutes and at 1-2 minutes in the aminophylline group (P < 0.05). CONCLUSION: Aminophylline 3 mg/kg or doxapram 1 mg/kg shortened the time to spontaneous ventilation and improved early recovery from TIVA without appreciable side effects. The more rapid emergence correlates with higher BIS values when compared with the saline control group. The arousal and respiratory effects of aminophylline were comparable to those of doxapram.