In vitro C3 mRNA Expression in Pemphigus Vulgaris: Complement Activation is Increased by IL-1α and TNF-α

1999 ◽  
Vol 3 (3) ◽  
pp. 140-144 ◽  
Author(s):  
Claudio Feliciani ◽  
Paola Toto ◽  
Paolo Amerio ◽  
Pierluigi Amerio
Keyword(s):  
Mrna Expression ◽  
Complement Activation ◽  
In Vitro Study ◽  
Peak Level ◽  
Pemphigus Vulgaris ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Tnf Α

Background: Pemphigus vulgaris (PV) is a potentially life-threatening disease, characterized immunohistologically by IgG deposits and complement activation on the surface of keratinocytes. Complement activation has been implicated in the pathogenesis with C3 deposits in about 90% of patients. Objective: In order to further elucidate the role of complement in PV and to define which cytokines play a role in C3 mRNA expression, we performed an in vitro study in human keratinocytes. Methods: Normal human epidermal keratinocytes (NHuK) were incubated with PV serum and C3 mRNA was measured. We previously had shown that IL-1α and TNF-α are expressed in PV in vivo and in vitro. Since cytokines are able to modulate complement activation, mRNA expression was evaluated in a similar experiment after pretreatment using antibodies against IL-1α and TNF-α. Results: Incubation of NHuK with PV sera caused their detachment from the plates after 20–30 minutes with a complete acantholysis within 12 hours. An early C3 mRNA expression was seen after 30 minutes with a peak level after 1 hour. Blocking studies, using antibodies against human IL-1α and TNF-α in NHuK together with PV-IgG, showed reduction of in vitro induced acantholysis and inhibition of C3 mRNA expression. Conclusions: This study supports the hypothesis that complement C3 is important in PV acantholysis and that complement activation is increased by IL-1α and TNF-α.

scholarly journals Impact of isoflavone genistein on psoriasis in in vivo and in vitro investigations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Katarzyna Bocheńska ◽  
Marta Moskot ◽  
Elwira Smolińska-Fijołek ◽  
Joanna Jakóbkiewicz-Banecka ◽  
Aneta Szczerkowska-Dobosz ◽  
...  
Keyword(s):  
Mrna Level ◽  
Response Characteristic ◽  
Epidermal Keratinocytes ◽  
Serious Adverse Events ◽  
Human Epidermal Keratinocytes ◽  
Good Tolerability ◽  
Tnf Α ◽  
Pi3k Activation

AbstractGenistein is applied worldwide as an alternative medicament for psoriasis (Ps) because of its anti-inflammatory activity and perceived beneficial impact on the skin. Hereby, we report our in vivo and in vitro investigations to supplement scientific research in this area. The reduction of clinical and biochemical scores in mild to moderate Ps patients taking genistein, its safety, good tolerability with no serious adverse events or discontinuations of treatment, no dose-limiting toxicities, negligible changes in pharmacodynamic parameters and remarkable serum interleukin level alterations were documented in this study. A certain regression of the Ps phenotype was visible, based on photo-documented Ps lesion evaluation. Through in vitro experiments, we found that genistein reduced IL-17A and TNF-α induced MAPK, NF-κB, and PI3K activation in normal human epidermal keratinocytes. Moreover, at the mRNA level of genes associated with the early inflammatory response characteristic for Ps (CAMP, CCL20, DEFB4A, PIK3CA, S100A7, and S100A9) and key cellular signalling (MTORC1 and TFEB), we showed that this isoflavone attenuated the increased response of IL-17A- and TNF-α-related pathways. This allows us to conclude that genistein is a good candidate for Ps treatment, being attractive for co-pharmacotherapy with other drugs.

scholarly journals 5-Aminolevulinic Acid-Based Photodynamic Therapy Pretreatment Mitigates Ultraviolet A-Induced Oxidative Photodamage

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hui Hua ◽  
Jiawei Cheng ◽  
Wenbo Bu ◽  
Juan Liu ◽  
Weiwei Ma ◽  
...  
Keyword(s):  
Aminolevulinic Acid ◽  
Epidermal Keratinocytes ◽  
Control Group ◽  
Hacat Cells ◽  
Ultraviolet A ◽  
Hairless Mice ◽  
Human Epidermal Keratinocytes ◽  
Experimental Group

Aim. To determine whether 5-aminolevulinic acid-based photodynamic therapy (ALA-PDT) is effective in combating ultraviolet A- (UVA-) induced oxidative photodamage of hairless mice skin in vivo and human epidermal keratinocytes in vitro. Methods. In in vitro experiments, the human keratinocyte cell line (HaCaT cells) was divided into two groups: the experimental group was treated with ALA-PDT and the control group was left untreated. Then, the experimental group and the control group of cells were exposed to 10 J/m2 of UVA radiation. ROS, O2− species, and MMP were determined by fluorescence microscopy; p53, OGG1, and XPC were determined by Western blot analysis; apoptosis was determined by flow cytometry; and 8-oxo-dG was determined by immunofluorescence. Moreover, HaCaT cells were also treated with ALA-PDT. Then, SOD1 and SOD2 were examined by Western blot analysis. In in vivo experiments, the dorsal skin of hairless mice was treated with ALA-PDT or saline-PDT, and then, they were exposed to 20 J/m2 UVA light. The compound 8-oxo-dG was detected by immunofluorescence. Conclusion. In human epidermal keratinocytes and hairless mice skin, UVA-induced oxidative damage can be prevented effectively with ALA-PDT pretreatment.

scholarly journals Bakuchiol Suppresses Inflammatory Responses Via the Downregulation of the p38 MAPK/ERK Signaling Pathway

2019 ◽  
Vol 20 (14) ◽  
pp. 3574 ◽  
Author(s):  
Hye-Sun Lim ◽  
Yu Jin Kim ◽  
Bu-Yeo Kim ◽  
Soo-Jin Jeong
Keyword(s):  
Mrna Expression ◽  
P38 Mapk ◽  
Inflammatory Responses ◽  
Mitogen Activated Protein Kinase ◽  
Enzyme Linked Immunosorbent Assay ◽  
Mice Model ◽  
Tnf Α ◽  
Cox 2

The purpose of the present study was to evaluate the effects of bakuchiol on the inflammatory response and to identify the molecular mechanism of the inflammatory effects in a lipopolysaccharide (LPS)-stimulated BV-2 mouse microglial cell line and mice model. The production of prostaglandin E2 (PGE2), tumor necrosis factor-α (TNF-α), and interleukin-6 (IL-6) was measured by enzyme-linked immunosorbent assay. The mRNA expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), TNF-α, and IL-6 was measured using reverse transcription–polymerase chain reaction analysis. Mitogen-activated protein kinase (MAPK) phosphorylation was determined by western blot analysis. In vitro experiments, bakuchiol significantly suppressed the production of PGE2 and IL-6 in LPS-stimulated BV-2 cells, without causing cytotoxicity. In parallel, bakuchiol significantly inhibited the LPS-stimulated expression of iNOS, COX-2, and IL-6 in BV-2 cells. However, bakuchiol had no effect on the LPS-stimulated production and mRNA expression of TNF-α or on LPS-stimulated c-Jun NH2-terminal kinase phosphorylation. In contrast, p38 MAPK and extracellular signal-regulated kinase (ERK) phosphorylation were inhibited by bakuchiol. In vivo experiments, Bakuchiol reduced microglial activation in the hippocampus and cortex tissue of LPS-injected mice. Bakuchiol significantly suppressed LPS-injected production of TNF-α and IL-6 in serum. These results indicate that the anti-neuroinflammatory effects of bakuchiol in activated microglia are mainly regulated by the inhibition of the p38 MAPK and ERK pathways. We suggest that bakuchiol may be beneficial for various neuroinflammatory diseases.

scholarly journals Gps2, a Protein Partner for Human Papillomavirus E6 Proteins

2001 ◽  
Vol 75 (1) ◽  
pp. 151-160 ◽  
Author(s):  
Yan Yan Degenhardt ◽  
Saul J. Silverstein
Keyword(s):  
Transcriptional Activation ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Papilloma Virus ◽  
Hpv E6 ◽  
Yeast Two Hybrid System ◽  
Protein Partners ◽  
Normal Human

ABSTRACT We have used the yeast two-hybrid system to screen a cDNA library prepared from normal human epidermal keratinocytes and identified protein partners for human papilloma virus (HPV) E6 proteins. A clone that encoded Gps2 interacted with E6 proteins from HPVs of high and low oncogenic risk. The specificity of these reactions was verified and the regions of E6 that were required for interaction were mapped. Steady-state and pulse-chase analyses of cells cotransfected with DNAs expressing E6 from either HPV6 or HPV18 and Gps2 demonstrated that the E6 proteins induced the degradation of Gps2 in vivo but not in vitro. Gps2 exhibited transcriptional activation activity, and high-risk E6 suppressed this activity.

The effects in vitro of TNF-α and its antagonist ‘etanercept’ on ejaculated human sperm

2017 ◽  
Vol 29 (6) ◽  
pp. 1169 ◽  
Author(s):  
Nicola A. Pascarelli ◽  
Antonella Fioravanti ◽  
Elena Moretti ◽  
Giacomo M. Guidelli ◽  
Lucia Mazzi ◽  
...  
Keyword(s):  
Mitochondrial Function ◽  
In Vitro Study ◽  
Human Sperm ◽  
Dna Integrity ◽  
Sperm Function ◽  
Sperm Viability ◽  
Tnf Α ◽  
The Impact

Tumour necrosis factor (TNF)-α is primarily involved in the regulation of cell proliferation and apoptosis; in addition it possesses pro-inflammatory properties. Anti-TNF-α strategies involve either administration of anti-TNF-α antibody or soluble TNF receptor to mop up circulating TNF-α. Etanercept, a recombinant human TNF-α receptor, was found to be effective in the treatment of rheumatoid arthritis. The impact of TNF-α inhibitors on human fertility is of notable interest. This in vitro study investigated the effect of different concentrations of TNF-α and etanercept used alone or in combination on sperm viability, motility, mitochondrial function, percentage of apoptosis and chromatin integrity in swim-up selected human spermatozoa. A negative effect of TNF-α (300 and 500 ng mL–1) and etanercept (from 800 µg mL–1 to 2000 µg mL–1) individually on sperm viability, motility, mitochondrial function, percentage of apoptotic spermatozoa and sperm DNA integrity was demonstrated. However, at concentrations of 100 and 200 µg mL–1, etanercept can block, in a significant way, the toxic effects of TNF-α (500 ng mL–1) on studied sperm characteristics. Our results confirm that TNF-α has a detrimental effect on sperm function and suggest, for the first time, that etanercept may counteract the in vitro toxic action of TNF-α. This data appears to be quite promising, although further studies, both in vivo and in vitro, are needed to understand the exact mechanism of action of TNF-α and TNF-α antagonists on sperm function.

Curcumin-loaded niosomes downregulate mRNA expression of pro-inflammatory markers involved in asthma: an in vitro study

Nanomedicine ◽  
2020 ◽  
Vol 15 (30) ◽  
pp. 2955-2970
Author(s):  
Jin-Ying Wong ◽  
Zhao Yin Ng ◽  
Meenu Mehta ◽  
Shakti D Shukla ◽  
Jithendra Panneerselvam ◽  
...  
Keyword(s):  
Mrna Expression ◽  
Inflammatory Markers ◽  
In Vitro Release ◽  
Physicochemical Characterization ◽  
In Vitro Study ◽  
Physicochemical Characteristics ◽  
Vitro Study ◽  
Tnf Α ◽  
Vitro Release

Aim: In this study, curcumin was encapsulated in niosomes (Nio-Curc) to increase its effectiveness for the treatment of asthma. Materials & methods: The formulation underwent various physicochemical characterization experiments, an in vitro release study, molecular simulations and was evaluated for in vitro anti-inflammatory activity. Results: Results showed that Nio-Curc had a mean particle size of 284.93 ± 14.27 nm, zeta potential of -46.93 and encapsulation efficacy of 99.62%, which demonstrates optimized physicochemical characteristics. Curcumin release in vitro could be sustained for up to 24 h. Additionally, Nio-Curc effectively reduced mRNA transcript expression of pro-inflammatory markers; IL-6, IL-8, IL-1β and TNF-α in immortalized human airway basal cell line (BCi-NS1.1). Conclusion: In this study, we have demonstrated that Nio-Curc mitigated the mRNA expression of pro-inflammatory markers in an in vitro study, which could be applied to treatment of asthma with further studies.

scholarly journals Rapamycin Attenuates Aldosterone-Induced Tubulointerstitial Inflammation and Fibrosis

2015 ◽  
Vol 35 (1) ◽  
pp. 116-125 ◽  
Author(s):  
Bin Wang ◽  
Wei Ding ◽  
Minmin Zhang ◽  
Hongmei Li ◽  
Yong Gu
Keyword(s):  
Mrna Expression ◽  
Epithelial Mesenchymal Transition ◽  
Kidney Diseases ◽  
Immunofluorescent Staining ◽  
Sprague Dawley ◽  
Mesenchymal Transition ◽  
Tnf Α ◽  
Tubulointerstitial Inflammation

Background/Aim: Aldosterone (Aldo), a mediator of kidney fibrosis, is implicated in the pathogenesis of chronic kidney diseases (CKD). The aim of this study was to evaluate the regulatory role of rapamycin (Rap) in Aldo-induced tubulointerstitial inflammation and fibrosis. Methods: Uninephrectomized, Sprague-Dawley rats were given 1% NaCl (salt) to drink and were randomized to receive treatment for 28 days as follows: vehicle infusion (control), 0.75 μg/h Aldo subcutaneous infusion, or Aldo infusion plus 1 mg/kg/day of Rap by intraperitoneal injection. The effect of Rap on Aldo-induced fibrosis and renal inflammation was investigated using Masson's technique, immunohistochemistry, and western blotting. The effects of Rap on the Aldo-induced epithelial-mesenchymal transition (EMT) process and on TNF-α mRNA expression and secretion in cultured HK-2 cells were investigated by immunofluorescent staining, western blot, qRT-PCR and ELISA. Results: An in vivo study indicated that signaling by the mammalian target of Rap (mTOR) was activated in rats in the Aldo group compared to controls, as indicated by up-regulated expression of p-mTOR and p-S6K. In addition, the inflammatory response increased, as evidenced by increases in inflammatory markers (MCP-1, ICAM-1, F4/80), and the accumulation of extracellular matrix (ECM), as indicated by increased collagen I and fibronectin expression and pro-fibrogenic gene (PAI-1 and TGF-β1) expression. These changes were attenuated by Rap treatment. An in vitro study showed that Rap significantly suppressed the Aldo-induced EMT process and TNF-α mRNA expression and secretion in cultured HK-2 cells. Conclusions: Rap can ameliorate tubulointerstitial inflammation and fibrosis by blocking mTOR signaling. Tubular cells may be a major cell type involved in this physiologic process.

scholarly journals Vitamin E (α-Tocopherol) Exhibits Antitumour Activity on Oral Squamous Carcinoma Cells ORL-48

2016 ◽  
Vol 16 (3) ◽  
pp. 414-425 ◽  
Author(s):  
Rahayu Zulkapli ◽  
Fathilah Abdul Razak ◽  
Rosnah Binti Zain
Keyword(s):  
Vitamin E ◽  
Antitumour Activity ◽  
In Vitro Study ◽  
Ultrastructural Changes ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
High Accumulation ◽  
Squamous Carcinoma Cells ◽  
Oral Squamous Carcinoma

Cancers involving the oral cavity, head, and neck regions are often treated with cisplatin. In cancer therapy, the main target is to eliminate unwanted cancerous cells. However, reports on the nonselective nature of this drug have raised few concerns. Incorrect nutritional habits and lifestyle practices have been directly linked to cancer incidence. Nutrients with antioxidant activity inhibit cancer cells development, destroying them through oxidative stress and apoptosis. α-tocopherol, the potent antioxidant form of vitamin E is a known scavenger of free radicals. In vitro study exhibited effective antitumor activity of α-tocopherol on ORL-48 at 2.5 ± 0.42 µg/mL. Cisplatin exhibited stronger activity at 1.0 ± 0.15 µg/mL, but unlike α-tocopherol it exhibited cytotoxicity on normal human epidermal keratinocytes at very low concentration (<0.1 µg/mL). Despite the lower potency of α-tocopherol, signs of apoptosis such as the shrinkage of cells and appearance of apoptotic bodies were observed much earlier than cisplatin in time lapse microscopy. No apoptotic vesicles were formed with cisplatin, instead an increased population of cells in the holoclone form which may suggest different induction mechanisms between both agents. High accumulation of cells in the G0/G1 phase were observed through TUNEL and annexin V-biotin assays, while the exhibition of ultrastructural changes of the cellular structures verified the apoptotic mode of cell death by both agents. Both cisplatin and α-tocopherol displayed cell cycle arrest at the Sub G0 phase. α-tocopherol thus, showed potential as an antitumour agent for the treatment of oral cancer and merits further research.

scholarly journals Effects of Helichrysum bracteatum flower extracts on UVB irradiation-induced inflammatory biomarker expression

2019 ◽  
Vol 3 (1) ◽  
Author(s):  
Yun Jeong Kim ◽  
Ji Hyun Seok ◽  
Waiting Cheung ◽  
Sung-Nae Lee ◽  
Hyun Hee Jang ◽  
...  
Keyword(s):  
Inflammatory Responses ◽  
Statistical Significance ◽  
Ultraviolet B ◽  
Epidermal Keratinocytes ◽  
Dpph Assay ◽  
Human Epidermal Keratinocytes ◽  
Tnf Α ◽  
Cox 2 ◽  
Helichrysum Bracteatum

Abstract Background The present study aimed to investigate the anti-inflammatory activity of Helichrysum bracteatum (H. bracteatum) flower extracts in vitro. Methods H. bracteatum flowers were extracted with water, ethanol and 1,3-butylene glycol, and the anti-oxidative activities of the extracts were measured using a 2,2-diphenyl-1-picrylhydrazyl (DPPH) assay. The inhibition of the expression of inflammation-related genes, including tumour necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and cyclooxygenase-2 (COX-2), was evaluated in vitro using reverse transcription-PCR in ultraviolet B (UVB)-irradiated human epidermal keratinocytes (HEKa cells). To investigate the inhibitory effects of H. bracteatum flower extracts on UVB-induced inflammatory responses in HEKa cells, the production of nitric oxide (NO) and TNF-α was measured using enzyme-linked immunosorbent assays. Results were expressed as the mean ± standard deviation; statistical significance was calculated using the Student’s t-test. Results The DPPH assay results showed that H. bracteatum flower extracts have good anti-oxidative effects and inhibited the expression of inflammation-related genes IL-6, COX-2 and TNF-α. Moreover, the production of NO and TNF-α was inhibited by H. bracteatum flower extracts. Conclusions These findings indicate that H. bracteatum flower extracts have efficacy against UVB-induced inflammation-related gene expression.

Histamine enhances the production of human β-defensin-2 in human keratinocytes

2007 ◽  
Vol 293 (6) ◽  
pp. C1916-C1923 ◽  
Author(s):  
Naoko Kanda ◽  
Shinichi Watanabe
Keyword(s):  
Mrna Expression ◽  
Wound Repair ◽  
Human Keratinocytes ◽  
Serine Phosphorylation ◽  
Epidermal Keratinocytes ◽  
Mrna Levels ◽  
Tnf Α ◽  
Ifn Γ ◽  
Microbial Defense

The anti-microbial peptide human β-defensin-2 (hBD-2), produced by epidermal keratinocytes, plays pivotal roles in anti-microbial defense, inflammatory dermatoses, and wound repair. hBD-2 induces histamine release from mast cells. We examined the in vitro effects of histamine on hBD-2 production in normal human keratinocytes. Histamine enhanced TNF-α- or IFN-γ-induced hBD-2 secretion and mRNA expression. Histamine alone enhanced transcriptional activities of NF-κB and activator protein-1 (AP-1) and potentiated TNF-α-induced NF-κB and AP-1 activities or IFN-γ-induced NF-κB and STAT1 activities. Antisense oligonucleotides against NF-κB components p50 and p65, AP-1 components c-Jun and c-Fos, or H1 antagonist pyrilamine suppressed hBD-2 production induced by histamine plus TNF-α or IFN-γ. Antisense oligonucleotide against STAT1 only suppressed hBD-2 production induced by histamine plus IFN-γ. Histamine induced serine phosphorylation of inhibitory NF-κBα (IκBα) alone or together with TNF-α or IFN-γ. Histamine induced c-Fos mRNA expression alone or together with TNF-α, whereas it did not further increase c-Jun mRNA levels enhanced by TNF-α. Histamine induced serine phosphorylation of STAT1 alone or together with IFN-γ, whereas it did not further enhance IFN-γ-induced tyrosine phosphorylation of STAT1. The histamine-induced serine phosphorylation of STAT1 was suppressed by MAPKK (MEK) inhibitor PD98059. These results suggest that histamine stimulates H1 receptor and potentiates TNF-α- or IFN-γ-induced hBD-2 production dependent on NF-κB, AP-1, or STAT1 in human keratinocytes. Histamine may potentiate anti-microbial defense, skin inflammation, and wound repair via the induction of hBD-2.

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