interleukin-8 and thiazolyl-blue

Endometriosis affects about 10-15% women for reproductive age, but it is not currently curable and the underlying etiology for this disease is still not clear. In the present study, functions and mechanisms of miR-182 and RELA in endometriosis were investigated. BAY 11-7082 was used to block NF-κB pathway. qRT-PCR, ELISA and western blot assays were employed to evaluate the expressions of miR-182 and RELA, inflammatory factors and epithelial-mesenchymal transition (EMT)-related markers, and activation of NF-κB pathway. MTT, wound healing or Transwell assays were used to evaluate the cell proliferation, migration and invasion capacities. Bioinformatic and dual-luciferase reporter assays were carried out to analyze the interaction between miR-182 and RELA. MiR-182 expression was decreased, while RELA was increased as developed from normal to eutopic and ectopic status, which was accompanied by upregulated inflammatory factors and EMT-related proteins. RELA was directly targeted by miR-182 in human endometrial stromal cells. Overexpression of RELA increased inflammation-associated and EMT-related markers expression, while miR-182 upregulation decreased the expression of these genes in a dose-dependent manner, which finally attenuated the proliferation, migration and invasion capacities of endometrial stromal cells through deactivation of NF-κB signaling pathway. Moreover, co-overexpression of RELA reversed the above effects induced by miR-182. In a word, miR-182 directly targeted RELA and inhibited proliferation, migration, invasion, EMT and inflammation of endometrial stromal cells through deactivation of NF-κB signaling pathway in endometriosis. These results provide new insights into the interaction between miR-182 and NF-κB pathway and their potential as therapeutic targets for treatment of endometriosis.

Topics: Cell Movement; Cell Proliferation; Computational Biology; Endometriosis; Endometrium; Epithelial Cells; Epithelial-Mesenchymal Transition; Female; Humans; Inflammation; Interleukin-6; Interleukin-8; MicroRNAs; Neoplasm Invasiveness; NF-kappa B p50 Subunit; Signal Transduction; Stromal Cells; Tetrazolium Salts; Thiazoles; Transcription Factor RelA; Transfection; Treatment Outcome; Wound Healing

Toxoplasmosis is a widely distributed parasitic protozoan disease, caused by Toxoplasma gondii (T. gondii). High prevalence of toxoplasmosis and limitations of conventional treatments lead to a search for new therapeutic drugs. Lycosin-I is a linear peptide, derived from the venom of the spider Lycosa singoriensis. The aim of the present study was to determine the anti-parasitic effect of lycosin-Ι against T. gondii. In vitro, the anti-T. gondii activities of lycosin-Ι were evaluated by MTT assay, trypan blue exclusion assay, cell counting assay and plaque assay. Cytokines of IL-6 and IL-8 were measured by quantitative PCR. In addition, the structures of tachyzoites treated with lycosin-Ι were also observed by scanning and transmission electron microscopy. In vivo, mice were challenged with parasites treated by lycosin-I. The results revealed that lycosin-Ι had shown a significant ability to inhibit T. gondii invasion and proliferation. Cytokines of IL-6 and IL-8 were reduced by lycosin-Ι at transcription level in human foreskin fibroblast (HFF) cells infected with T. gondii tachyzoites, but they were increased compared to non-infected cells. For tachyzoites, lycosin-Ι induced their cell membrane alterations with formation of invaginations, some of them appeared to be vacuolated in their cytoplasm. Moreover, lycosin-Ι had prolonged the survival time of mice by controlling T. gondii proliferation. In conclusion, our present study provides the first evidence for anti-T. gondii by using the spider peptide lycosin-Ι. These findings suggest that lycosin-Ι is a potential alternative agent for the treatment of toxoplasmosis.

Topics: Animals; Antimicrobial Cationic Peptides; Cell Count; Cell Membrane; Cells, Cultured; Coccidiostats; Female; Fibroblasts; Humans; Interleukin-6; Interleukin-8; Male; Mice; Microscopy, Electron, Scanning; Microscopy, Electron, Transmission; Real-Time Polymerase Chain Reaction; Spider Venoms; Tetrazolium Salts; Thiazoles; Toxoplasma; Trypan Blue

Serine protease inhibitor elafin (E) and its precursor, trappin-2 (Tr), have been associated with mucosal resistance to HIV-1 infection. We recently showed that Tr/E are among principal anti-HIV-1 molecules in cervicovaginal lavage (CVL) fluid, that E is ∼130 times more potent than Tr against HIV-1, and that Tr/E inhibited HIV-1 attachment and transcytosis across human genital epithelial cells (ECs). Since herpes simplex virus 2 (HSV-2) is a major sexually transmitted infection and risk factor for HIV-1 infection and transmission, we assessed Tr/E contribution to defense against HSV-2. Our in vitro studies demonstrated that pretreatment of endometrial (HEC-1A) and endocervical (End1/E6E7) ECs with human Tr-expressing adenovirus (Ad/Tr) or recombinant Tr/E proteins before or after HSV-2 infection resulted in significantly reduced virus titers compared to those of controls. Interestingly, E was ∼7 times more potent against HSV-2 infection than Tr. Conversely, knockdown of endogenous Tr/E by small interfering RNA (siRNA) significantly increased HSV-2 replication in genital ECs. Recombinant Tr and E reduced viral attachment to genital ECs by acting indirectly on cells. Further, lower viral replication was associated with reduced secretion of proinflammatory interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-α) and decreased NF-κB nuclear translocation. Additionally, protected Ad/Tr-treated ECs demonstrated enhanced interferon regulatory factor 3 (IRF3) nuclear translocation and increased antiviral IFN-β in response to HSV-2. Lastly, in vivo studies of intravaginal HSV-2 infection in Tr-transgenic mice (Etg) showed that despite similar virus replication in the genital tract, Etg mice had reduced viral load and TNF-α in the central nervous system compared to controls. Collectively, this is the first experimental evidence highlighting anti-HSV-2 activity of Tr/E in female genital mucosa.

Topics: Adenoviridae; Analysis of Variance; Animals; Blotting, Western; Cell Line, Tumor; Chlorocebus aethiops; DNA Primers; Elafin; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Female; Fluorescent Antibody Technique; Gene Knockdown Techniques; Genetic Vectors; Herpes Genitalis; Herpesvirus 2, Human; Humans; Interferon Regulatory Factor-3; Interleukin-8; Mice; Mice, Transgenic; NF-kappa B; Real-Time Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha; Vero Cells; Viral Load; Virus Attachment; Virus Replication

Although Helicobacter pylori (Hp) plays an important role in the pathogenesis of chronic gastritis and gastric ulcer, little is known about the probable mechanisms of these types of gastrointestinal damage. To determine the precise mechanisms involved in ulcer formation, immune responses in patients with gastric ulcer (GUP) caused by Hp infection (Hp(+)) were compared with those of other gastritis patients (GP). The sensitivity and proliferation of peripheral blood mononuclear cells (PBMNCs) obtained from patients were evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay against exposure with complex Hp crude antigen (HPCA) and mitogen (phytohemagglutinin, PHA). Production of inflammatory cytokines, including interleukin (IL)-1β and IL-8, in serum and supernatants of PBMNCs were then measured by ELISA. It was found that, after stimulation with PHA, both IL-8 and IL-1β concentrations in sera and supernatants as well as proliferation and sensitivity were statistically greater in GUP Hp(+) than GP Hp(-) . Furthermore, HPCA inhibited the proliferation of PBMNCs dose-dependently; however, it stimulated IL-8 and IL-1β production in supernatants of mononuclear cells. Therefore, the up-regulated concentrations of IL-8 and IL-1β may have been caused by increase in the size of mononuclear cell subpopulations or in their cytokine secretory activity, indicating the greatest cell responsiveness in GUP Hp(+) patients. These results suggest that tissue damage and ulcers occur in patients who produce more IL-8 and IL-1β than patients who do not develop ulcers; the former consequently have more activated immune cells at the site of infection. Therefore, both host responses and Hp virulence factors may be involved in the development of gastric ulcers.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cell Proliferation; Cells, Cultured; Enzyme-Linked Immunosorbent Assay; Female; Helicobacter Infections; Helicobacter pylori; Humans; Interleukin-1beta; Interleukin-8; Leukocytes, Mononuclear; Male; Middle Aged; Staining and Labeling; Stomach Ulcer; Tetrazolium Salts; Thiazoles; Young Adult

Camphorquinone (CQ) is a photoinitiator that triggers polymerization of light-curing materials such as dental adhesives and composites. CQ does not become a part of the polymer network, suggesting that CQ can be leached out into surrounding environment including dental pulp and exert adversary effects on tissues. In order to understand the mechanisms of CQ-induced side effects, we investigated the effect of CQ on cell viability, cytokine secretion, and odontogenic differentiation of dental pulp stem cells in vitro.. Cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay after CQ exposure. Western blotting was performed for p16(INK4A), p21(WAF1), and p53. Secretory cytokines were evaluated using the membrane-enzyme-linked immunosorbent assay as well as conventional and quantitative reverse-transcription polymerase chain reaction. The effects of CQ on odontogenic differentiation were evaluated using alkaline phosphatase and alizarin red S staining methods.. CQ treatment suppressed the proliferation of DPSCs and induced the expression of p16(INK4A), p21(WAF1), and p53. Levels of proinflammatory cytokines (eg, interleukin 6, interleukin 8, and matrix metalloproteinase-3 [MMP3]) were increased by CQ treatment. CQ also inhibited odontogenic differentiation and mineralization capacities of DPSC and MC3T3-E1 cells.. Our study showed that CQ may trigger pulpal inflammation by inducing proinflammatory cytokine production from the pulpal cells and may impair odontogenic differentiation of dental pulp cells, resulting in pulpal irritation and inflammation.

Topics: 3T3 Cells; Alkaline Phosphatase; Animals; Anthraquinones; Blotting, Western; Camphor; Cell Differentiation; Cell Survival; Coloring Agents; Cyclin-Dependent Kinase Inhibitor p16; Cyclin-Dependent Kinase Inhibitor p21; Cytokines; Dental Materials; Dental Pulp; Humans; Inflammation Mediators; Interleukin-6; Interleukin-8; Materials Testing; Matrix Metalloproteinase 3; Methacrylates; Mice; Odontogenesis; Photoinitiators, Dental; Tetrazolium Salts; Thiazoles; Tooth Calcification; Tumor Suppressor Protein p53

Although inflammation may be a physiological defense process, imbalanced neuroinflammation has been associated with the pathophysiology of brain disorders, including major depression and schizophrenia. Activated glia releases a variety of pro-inflammatory cytokines that contribute to neuronal dysfunction. Elevated levels of S100B, a glia derived protein, have been observed in the serum and CSF of schizophrenic patients suggesting a glial role in the disease. We evaluated whether S100B secretion (in C6 glioma cells and hippocampal slices in Wistar rats) could be directly modulated by the main inflammatory cytokines (IL-1β, TNF-α, IL-6 and IL-8) altered in schizophrenia, as well as the possible involvement of mitogen-activated protein kinase (MAPK) pathways in these responses. We also investigated the effects of typical and atypical antipsychotic drugs on glial cytokine-induced S100B release. Our results suggest that S100B secretion is increased by pro-inflammatory cytokines via MAPK and that oxidative stress may be a component of this modulation. These results reinforce the idea that the S100B protein is involved in the inflammatory response observed in many brain diseases, including schizophrenia. Moreover the antipsychotics, haloperidol and risperidone, were able to inhibit the secretion of S100B following IL-6 stimulation in C6 glioma cells.

Topics: Animals; Antipsychotic Agents; Cell Line, Tumor; Cytokines; Enzyme-Linked Immunosorbent Assay; Glial Fibrillary Acidic Protein; Glioma; Glutathione; Haloperidol; Hippocampus; Immunohistochemistry; Interleukin-1beta; Interleukin-6; Interleukin-8; L-Lactate Dehydrogenase; Nerve Growth Factors; Neuroglia; Nitric Oxide; Oxidative Stress; Rats; Rats, Wistar; Reactive Oxygen Species; Risperidone; S100 Calcium Binding Protein beta Subunit; S100 Proteins; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

To evaluate morphological features, cell growth and interleukin-6 (IL-6) and interleukin-8 (IL-8) secretion in expanded ex vivo human dental pulp mesenchymal stem cells (DP-MSCs) after exposure to 2-hydroxyethyl methacrylate (HEMA)..   Dental pulp mesenchymal stem cells were derived from the dental pulps of 10 young donors. After in vitro isolation, DP-MSCs were treated with 3 and 5 mmol L(-1) HEMA, and after 24, 48 and 72 h of incubation, their morphological features, cell growth, IL-6 and IL-8 secretion were analysed. Differences in the cell growth and in the interleukin secretion were analysed for statistical significance with two-way anova tests and the Holm-Sidak method for multiple comparisons..   Dental pulp mesenchymal stem cells revealed a decrease in cell growth with both treatments (P < 0.05), more evident at 5 mmol L(-1) . Microscopic analysis displayed extensive cytotoxic effects in treated cells, which lost their fibroblastoid features and became retracted, even roundish, with a large number of granules. An up-regulation of IL-6 and IL-8 in treated cells cytokines was evident (P < 0.05)..   2-Hydroxyethyl methacrylate exhibited cytotoxicity, inhibited cell growth and induced morphological changes in cultured DP-MSCs. Moreover, in treated samples, an up-regulation of soluble mediators of inflammation such as IL-6 and IL-8 cytokines was found. The direct application of HEMA potentially induces an inflammation process that could be the starting point for toxic response and cell damage in DP-MSCs.

Topics: Adolescent; Cell Culture Techniques; Cell Proliferation; Cell Shape; Cell Survival; Coloring Agents; Cytoplasmic Granules; Dental Materials; Dental Pulp; Dose-Response Relationship, Drug; Gene Expression Regulation; Humans; Interleukin-6; Interleukin-8; Mesenchymal Stem Cells; Methacrylates; Tetrazolium Salts; Thiazoles; Time Factors; Trypan Blue

In numerous clinical and experimental studies, preservatives present in eye drops have had detrimental effects on ocular epithelial cells. The aim of this study was to compare the cytotoxic and inflammatory effects of the preservative polyquaternium-1 (PQ-1) containing Travatan (travoprost 0.004%) and Systane Ultra eye drops with benzalkonium chloride (BAK) alone or BAK-preserved Xalatan (0.005% latanoprost) eye drops in HCE-2 human corneal epithelial cell culture.. HCE-2 cells were exposed to the commercial eye drops Travatan, Systane Ultra, Xalatan, and the preservative BAK. Cell viability was determined using colorimetric MTT (3-(4,5-dimethyldiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay and by release of lactate dehydrogenase (LDH). Induction of apoptosis was measured with a using a colorimetric caspase-3 assay kit. DNA binding of the nuclear factor kappa B (NF-κB) transcription factor, and productions of the proinflammatory cytokines, interleukins IL-6 and IL-8, were determined using an enzyme-linked immunosorbent assay (ELISA) method.. Cell viability, as measured by the MTT assay, declined by up to 50% after exposure to Travatan or Systane Ultra solutions which contain 0.001% PQ-1. BAK at 0.02% rather than at 0.001% concentration evoked total cell death signs on HCE-2 cells. In addition, cell membrane permeability, as measured by LDH release, was elevated by sixfold with Travatan and by a maximum threefold with Systane Ultra. Interestingly, Travatan and Systane Ultra activated NF-κB and elevated the secretion of inflammation markers IL-6 by 3 to eightfold and IL-8 by 1.5 to 3.5 fold, respectively, as analyzed with ELISA.. Eye drops containing PQ-1 evoke cytotoxicity and enhance the NF-κB driven inflammation reaction in cultured HCE-2 cells. Our results indicate that these harmful effects of ocular solutions preserved with PQ-1 should be further evaluated in vitro and in vivo.

Topics: Apoptosis; Benzalkonium Compounds; Caspase 3; Cell Line; Cell Membrane Permeability; Cell Survival; Cloprostenol; Cornea; Epithelial Cells; Humans; Interleukin-6; Interleukin-8; L-Lactate Dehydrogenase; Latanoprost; NF-kappa B; Ophthalmic Solutions; Polymers; Preservatives, Pharmaceutical; Prostaglandins F, Synthetic; Tetrazolium Salts; Thiazoles; Travoprost

Psoriasis, characterized by circumscribed, red, thickened plaques with an overlying silver-white scale, is a common T-cell-mediated chronic inflammatory skin disease. Although hydrogen sulfide (H(2)S) has been shown to be a signaling molecule with both pro- or anti-inflammatory effects, its relationship with psoriasis has not been elucidated. In the present study, 15 patients with chronic progressive psoriasis and 15 healthy volunteers were investigated. Serum H(2)S levels in psoriasis patients were significantly lower than those of healthy controls (16.69 ± 5.47 μM vs. 34.5 ± 6.39 μM). In contrast, serum levels of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8) were significantly higher in psoriasis patients than healthy controls (22.88 ± 6.24 pg/ml vs. 12.07 ± 3.68 pg/ml; 61.47 ± 8.21 pg/ml vs. 31.54 ± 13.73 pg/ml; and 39.43 ± 8.56 pg/ml vs. 20.55 ± 6.45 pg/ml, respectively). The serum H(2)S levels negatively correlated with clinical disease severity. Furthermore, treatment of HaCaT human keratinocytes with TNF-α increased the levels of nitric oxide (NO), IL-6 and IL-8 (32.21 ± 5.71 μM vs. 3.22 ± 0.98 μM; 203.96 ± 13.16 pg/ml vs. 13.57 ± 3.75 pg/ml; and 301.24 ± 30.17 pg/ml vs. 29.06 ± 10.91 pg/ml, respectively) in the culture media. Exogenous H(2)S inhibited the TNF-α-mediated upregulation of NO, IL-6 and IL-8 in a dose-dependent manner. In addition, H(2)S inhibited TNF-α-mediated activation of p38, extracellular-signal-regulated kinase and nuclear factor kappa B. In conclusion, H(2)S may play a protective role in the pathogenesis of psoriasis. H(2)S-releasing agents may be promising therapeutics for psoriasis.

Topics: Anti-Inflammatory Agents; Blotting, Western; Cell Line; DNA Primers; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Humans; Hydrogen Sulfide; Interleukin-6; Interleukin-8; Keratinocytes; Nitric Oxide; Nitrites; Psoriasis; Real-Time Polymerase Chain Reaction; Reverse Transcriptase Polymerase Chain Reaction; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

There is increasing interest in the development of new in vitro tissue models. In this study, a tissue model of periodontal ligament (PDL) was established by 3-D-culturing human PDL cells in a thin sheet of porous poly (lactic-co-glycolic acid) scaffold. Growth of the model was evidenced by MTT assay and various microscopies. After being subjected to static compression of 5 ~ 35 g/cm(2) for 6 hrs, the RANKL mRNA expression was significantly up-regulated by force ≥ 25 g/cm(2) in the model. After being subjected to static compression of 25 g/cm(2) for 6 ~ 72 hrs, the mRNA expression of PTHrP, IL-11, IL-8, and FGF-2, potential osteoclastogenesis inducers, was significantly up-regulated in the model, which was further verified by the compression of human PDL in vivo. However, when human gingival fibroblasts were substituted for PDL cells in the model, almost no osteoclastogenesis inducers were up-regulated by compression. This tissue model can serve as an effective tool for the study of PDL mechanoresponse.. periodontal ligament, PDL; periodontal ligament cells, PDLCs; poly (lactic-co-glycolic acid), PLGA; orthodontic tooth movement, OTM; extracellular matrix, ECM.

Topics: Adolescent; Biocompatible Materials; Biomechanical Phenomena; Cell Culture Techniques; Cell Survival; Coloring Agents; Fibroblast Growth Factor 2; Fibroblasts; Gingiva; Humans; Interleukin-11; Interleukin-8; Lactic Acid; Microscopy, Fluorescence; Osteoclasts; Parathyroid Hormone-Related Protein; Periodontal Ligament; Polyglycolic Acid; Polylactic Acid-Polyglycolic Acid Copolymer; RANK Ligand; Stress, Mechanical; Tetrazolium Salts; Thiazoles; Time Factors; Tissue Scaffolds; Tooth Movement Techniques; Up-Regulation

The pathogenesis of disease resulting from exposure to diesel exhaust particles (DEP) is often studied using cultured lung cells. Frequently, researchers expose cells to DEP by spiking a suspension of particles in liquid onto the apical surface. This is not representative of in vivo exposure, where aerosols are deposited onto cell surfaces at the air-liquid interface (ALI). Inertial impaction provides an opportunity to deliver high doses of particles with aerodynamic diameters>∼1 μm to the surface of cells in seconds in a reproducible and predictable manner. A custom device was constructed to deposit DEP aerosols onto the surface of Calu-3 and A549 cells grown at the ALI. The pro-inflammatory and toxic cellular response to exposure to the deposited DEP aerosols was measured and compared to the response of cells exposed to DEP as suspensions. Calu-3 cells showed evidence of an oxidative stress response for both exposure types, while there was strong evidence to suggest that the method of aerosol delivery was harmful to the A549 cells.

Topics: Aerosols; Air Pollutants; Atmosphere Exposure Chambers; Cell Line, Tumor; Cell Survival; Equipment Design; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-8; L-Lactate Dehydrogenase; Lung; Oxidative Stress; Reactive Oxygen Species; Tetrazolium Salts; Thiazoles; Toxicity Tests; Vehicle Emissions

Methyl thiazolyl tetrazolium (MTT) and interleukin-8 (IL-8) assays are common colorimetric methods to measure mitochondrial activity and drug induced pro-inflammatory factors. However, many reports have described how MTT absorbance and cytokine adsorption could limit their applicability in evaluating the cytotoxicity of nanomaterials. In this study, we used an acid-containing isopropanol complex as a substitute for dimethyl sulfoxide (DMSO) solvent to dissolve MTT formazan, which was expected to diminish the absorbance of nano-ZnO at 570 nm where maximum absorbance for the MTT formazan was detected. In addition, we used a serum-containing medium to prevent the possible effects of IL-8 protein adsorption in the nano-ZnO and nano-TiO2. The results showed that the modified method by using acid-containing isopropanol step in MTT assay, nano-ZnO exposed to human lung epithelial cells had the lowest cell viability (from 12.5 to 50 microg mL(-1)) and EC50 value (8.4 microg mL(-1)) comparing with the conventional MTT protocol or adding phosphate buffered saline (PBS) to wash cells. The reason for this was the acid-containing isopropanol completely dissolved nano-ZnO with no additional absorbance when compared to the background solvent at 570 nm. On the other hand, the IL-8 protein had a marked influence on the adsorption of nano-TiO2 in the serum-free medium. While only at 100 microg mL(-1) of nano-ZnO, an influence on the adsorption of IL-8 was observed. This could be attributed to the different charges on the surface of nanomaterials. This problem could be overcome through the addition of fetal bovine serum (FBS) to the medium.

Topics: 2-Propanol; Adsorption; Biological Assay; Cell Line, Tumor; Cell Survival; Dimethyl Sulfoxide; Humans; Interleukin-8; Metal Nanoparticles; Spectrophotometry; Tetrazolium Salts; Thiazoles; Titanium; Toxicity Tests; Zinc Oxide

To test a new multiple endpoint analysis (MEA) including occludin gene expression for screening the ocular irritation potential of tear substitutes on human corneal epithelium (HCE), an in vitro model proposed to limit the use of animal testing in pre-clinical studies.. Four chemically-preserved and two non chemically-preserved tear substitutes were tested after acute (24h, 24h+24h post incubation) and repeated applications (for 72h) and compared to the positive control, benzalkonium chloride (BAK) at 0.1% and 0.01%, by assessing complementary parameters. Cellular viability was evaluated using MTT, histomorphologic analysis was performed on H&E stained vertical sections, IL-8 release was measured by ELISA, and occludin gene expression was quantified using qRT-PCR.. Cellular viability was moderately reduced by Perborate and Polyquad-preserved tear substitutes and dramatically reduced by BAK and by Thiomersal and Oxyd preserved tear substitutes. Thiomersal also increased IL-8 release. Occludin expression profiles were modified by the four chemically-preserved tear substitutes and by the mechanically-preserved Comod, but not by the mechanically-preserved Abak. The behavior of BAK and tear substitutes led us to propose a prediction model for the classification of different levels of irritants, mainly based on the occludin transcriptional study.. The versatility and sensitivity of the HCE model allowed the modeling of cumulative effects that may approach conditions obtained after long term application of tear substitutes. Thus, the modified MEA proposed in this study represents a valuable tool for in vitro eye irritation assessment with the power to detect mild irritants and subclinical eye irritant potential.

Topics: Cell Survival; Coloring Agents; Drug Evaluation, Preclinical; Enzyme-Linked Immunosorbent Assay; Eye Diseases; Fluorescent Antibody Technique; Gene Expression; Genetic Markers; Humans; In Vitro Techniques; Interleukin-8; Irritants; Membrane Proteins; Occludin; Ophthalmic Solutions; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles

Biopolymer chitosan (beta-1,4-d-glucosamine) comprises the copolymer mixture of N-acetylglucosamine and glucosamine. The natural biocompatibility and biodegradability of chitosan have recently highlighted its potential use for applications in wound management. Chemical and physical modifications of chitosan influence its biocompatibility and biodegradability, but it is unknown as to what degree. Hence, the biocompatibility of the chitosan porous skin regenerating templates (PSRT 82, 87 and 108) was determined using an in vitro toxicology model at the cellular and molecular level on primary normal human epidermal keratinocytes (pNHEK). Cytocompatibility was accessed by using a 3-[4,5-dimethyl-2-thiazolyl]-2,5-diphenyl tetrazolium bromide (MTT) assay from 24 to 72h. To assess the genotoxicity of the PSRTs, DNA damage to the pNHEK was evaluated by using the Comet assay following direct contact with the various PSRTs. Furthermore, the skin pro-inflammatory cytokines TNF-alpha and IL-8 were examined to evaluate the tendency of the PSRTs to provoke inflammatory responses. All PSRTs were found to be cytocompatible, but only PSRT 108 was capable of stimulating cell proliferation. While all of the PSRTs showed some DNA damage, PSRT 108 showed the least DNA damage followed by PSRT 87 and 82. PSRT 87 and 82 induced a higher secretion of TNF-alpha and IL-8 in the pNHEK cultures than did PSRT 108. Hence, based on our experiments, PSRT 108 is the most biocompatible wound dressing of the three tested.

Topics: Biocompatible Materials; Cell Survival; Cells, Cultured; Chitosan; Coloring Agents; Comet Assay; Humans; Interleukin-8; Keratinocytes; Materials Testing; Mutagenicity Tests; Mutagens; Porosity; Regeneration; Skin, Artificial; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

Changes in epithelial cell activity and the production of pro-inflammatory cytokines were examined utilizing an organotypic culture system as an in vitro model to study the effects of radiation on oral keratinocytes to simulate what is thought to occur in radiation-induced oral mucositis. Monolayer cultures of oral keratinocyte were irradiated by varying the dose. Cell injury was assessed using a colony forming efficiency (CFE) assay. Third passage oral keratinocytes were seeded onto AlloDerm to form a 3D construct of an ex vivo produced oral mucosa equivalent (EVPOME) which was irradiated with 0, 1, 3 and 8Gy. Formalin-fixed sections of the EVPOME were used for histology and immunohistochemistry to examine proliferative capacity. Epithelial cell viability of EVPOME was measured by MTT assay. Spent culture medium was used to determine post-radiation pro-inflammatory cytokine production. Basal cells became more swollen and pyknotic as radiation increased, implying loss of cell viability also determined by MTT assay. The number of Ki-67 immunopositive cells and CFE showed negative correlation with radiation, indicating loss of cell proliferative capacity. The production of pro-inflammatory cytokines, IL-1alpha and IL-8, tended to increase in a radiation dose dependent manner. The EVPOME lacking submucosal cellular components was a useful model.

Topics: Biocompatible Materials; Cell Adhesion; Cell Count; Cell Culture Techniques; Cell Proliferation; Cell Shape; Cell Survival; Collagen; Coloring Agents; Dose-Response Relationship, Radiation; Female; Humans; Inflammation Mediators; Interleukin-1alpha; Interleukin-8; Keratinocytes; Ki-67 Antigen; Male; Mouth Mucosa; Radiation Dosage; Stomatitis; Tetrazolium Salts; Thiazoles; Tissue Scaffolds

The Food and Drug Administration requires an accurate determination of the dose and potency of tissue-engineered or combination products as is required for drugs. This needs to be done as a rapid, quantitative, and noninvasive measurement of biologic function/activity in a way so as not to perturb the tissue-engineered product being developed. The aim of this study was to correlate constitutive release of cytokine(s) from unstimulated cells, at different stages of development, within a 3-dimensional (3D) organotypic ex vivo produced oral mucosa equivalent (EVPOME) to be used for intraoral grafting, with oral keratinocyte cell viability of the EVPOME.. Tissue culture medium was assayed with an enzyme-linked immunosorbent assay from monolayer culture of oral keratinocytes and a 3D EVPOME to determine the constitutive release of interleukin (IL) 1alpha, IL-6, IL-8, and vascular endothelial growth factor (VEGF). VEGF messenger ribonucleic acid expression by oral keratinocytes within the 3D EVPOME was detected by in situ hybridization at days 4, 7, and 11. The number of viable oral keratinocytes within the EVPOME was extrapolated from VEGF release by use of a modified MTT assay.. Both VEGF release level and the number of viable cells in the monolayer cultures and 3D EVPOME as measured by MTT assay significantly increased in a time-dependent manner (P < .001, r = 0.743).. These results suggest that the increasing detectable levels of VEGF associated with the increasing number of viable cells in the EVPOME may provide a useful noninvasive/nondestructive means of assessing both cellular viability (dose) and biologic function/activity (potency) of a combination cell-based device such as the EVPOME.

Topics: Cell Survival; Coloring Agents; Culture Media, Conditioned; Enzyme-Linked Immunosorbent Assay; Humans; In Situ Hybridization; Interleukin-1alpha; Interleukin-6; Interleukin-8; Interleukins; Keratinocytes; Mouth Mucosa; Organ Culture Techniques; RNA, Messenger; Tetrazolium Salts; Thiazoles; Time Factors; Tissue Engineering; Tissue Scaffolds; Vascular Endothelial Growth Factor A

In the present study, we investigated the anti-inflammatory effects of a Kampo medicine Shosaikoto (TJ-9) using in vitro periodontal disease model, in which human gingival fibroblasts (HGFs) treated with lipopolysaccharide (LPS) from Porphyromonas gingivalis (PgLPS) produce IL-6, IL-8 and prostaglandin E2 (PGE2). Treatment with PgLPS (10 ng/ml), TJ-9 (up to 1 mg/ml) and their combinations for 24 h did not affect the viability of HGFs. Moreover, TJ-9 did not alter LPS-induced IL-6 and IL-8 productions. However, TJ-9 significantly suppressed LPS-induced PGE2 production in a dose-dependent manner but TJ-9 alone did not affect basal PGE2 level. Western blotting demonstrated that TJ-9 decreased cyclooxygenase-2 (COX-2) expression in a dose-dependent manner but not phospholipase A2. Moreover, TJ-9 selectively and dose-dependently inhibited COX-2 activity. These results suggest that TJ-9 decreased PGE2 production by inhibition of both COX-2 expression and activity and that TJ-9 may be useful to improve gingival inflammation in periodontal disease.

Topics: Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Survival; Cyclooxygenase 1; Cyclooxygenase 2; Dinoprostone; Dose-Response Relationship, Drug; Drugs, Chinese Herbal; Enzyme-Linked Immunosorbent Assay; Fibroblasts; Gingiva; Gingivitis; Humans; Indicators and Reagents; Interleukin-6; Interleukin-8; Lipopolysaccharides; Macrophages; Medicine, Kampo; Monocytes; Porphyromonas gingivalis; Tetrazolium Salts; Thiazoles

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells.

Topics: Angiogenesis Inducing Agents; Animals; Cell Line, Tumor; Cell Proliferation; Corneal Surgery, Laser; Disease Models, Animal; Enzyme-Linked Immunosorbent Assay; Gene Expression Regulation, Neoplastic; Glioblastoma; Humans; Interleukin-8; Mice; N-Formylmethionine Leucyl-Phenylalanine; Receptors, Formyl Peptide; Tetrazolium Salts; Thiazoles; Time Factors; Transplantation, Heterologous; Vascular Endothelial Growth Factor A

Increased indoor air concentrations of volatile organic compounds (VOC) have been shown to contribute to the risk of respiratory and allergic diseases. The aim of this study was to investigate the inflammatory potential of single VOC and mixtures using an in vitro model. TNF-alpha stimulated human lung epithelial cells (A549) were exposed to VOC (1 ng/m3-100g/m3) via gas phase. After 20 h of exposure cytotoxicity and the release of the pro-inflammatory molecules monocyte chemoattractant protein-1 (MCP-1), Interleukin-6 (IL-6) and IL-8 was analysed. Exposure of A549 cells to chlorobenzene, styrene or m-xylene increased the MCP-1 production within the indoor relevant concentration range (1-25,000 microg/m3), higher concentrations increased the secretion of IL-8. Mixtures of aromatic compounds caused comparable effects to the single compounds on MCP-1 and IL-8 with a shift to lower concentration ranges. Neither the aliphatic compounds n-nonane, n-decane, n-undecane, n-dodecane, n-tridecane, and methylcyclopentane nor the mixture of these VOC showed any effects on MCP-1 and IL-8 production. Cytotoxic effects were not observed. These results show that aromatic, but no aliphatic compounds stimulate the release of pro-inflammatory mediators from lung epithelial cells. When aromatic compounds were mixed the sensitivity of lung cells to these compounds was increased.

Topics: Cell Line; Cell Line, Tumor; Cell Survival; Chemokine CCL2; Drug Interactions; Enzyme-Linked Immunosorbent Assay; Epithelial Cells; Humans; Hydrocarbons; Hydrocarbons, Aromatic; Inflammation; Interleukin-6; Interleukin-8; Lung; Organic Chemicals; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha; Volatilization

This study was carried out to assess the effects of chromium and nickel upon isolated keratinocytes as an in vitro model of human skin. Keratinocytes were isolated from healthy volunteer skin samples of unknown metal sensitivity (n=10) and were compared with cells from patient biopsies of known metal sensitivity (n=7). Cells were dosed with a concentration range of nickel and chromium (0-10,000 microM) and cellular mitochondrial activity, viability, metal uptake and cytokine release were measured. Responses of primary versus passaged keratinocytes were also compared. Toxicity data from primary and passaged keratinocytes was statistically analysed by the non-linear Hill Plot model. Results showed that hexavalent chromium was significantly more cytotoxic, associated more with keratinocytes and induced a dose dependant release of IL-1alpha compared to nickel. Significant differences were observed between primary and passaged keratinocytes with regard to the toxicity of chromium and nickel and variation of response. No differences were observed in the cytotoxicity or cytokine release induced by chromium or nickel for the known sensitised biopsy patient samples (n=4) compared to patch test negative controls (n=3). The results from this study suggest human keratinocytes in vitro respond very differently to chromium and nickel.

Topics: Algorithms; Benzimidazoles; Cell Separation; Cell Survival; Cells, Cultured; Chromium; Cytokines; Data Interpretation, Statistical; Dermatitis, Contact; Enzyme-Linked Immunosorbent Assay; Fluorescent Dyes; Humans; Interleukin-1alpha; Interleukin-8; Keratinocytes; Mitochondria; Nickel; Propidium; Skin Tests; Tetrazolium Salts; Thiazoles

Inflammation probably plays a significant role in perinatal brain injury. To study the contribution of locally produced cytokines, the effect on cell death of addition of IL-8 and MCP-1 or antibodies to these, and the impact of acidosis, human postmitotic NT2-N neurons were exposed to 3 h of hypoxia and glucose deprivation and reoxygenated for 21 h. After 3 h of hypoxia with neutral medium, IL-8 was significantly increased compared to controls (150 (100-250)% vs. 100 (85-115)%, p=0.023). After 21 h of neutral reoxygenation, both IL-8 (380 (110-710)% vs. 150 (85-260)%, p=0.041) and monocyte chemoattractant protein-1 (MCP-1) (650 (440-2000)% vs. 310 (230-340)%, p=0.007) were significantly increased compared to controls. After 3 h of hypoxia, both IL-8 (p=0.002) and MCP-1 (p=0.008) were significantly lower in cells with acidotic compared with cells with neutral medium. Acidosis during reoxygenation, however, significantly increased IL-8 release, whereas MCP-1 release was diminished. Similar effects of acidosis were seen in normoxic controls. The cells also secreted RANTES and IP-10, but not 8 other cytokines tested. We found no effect on cell death, measured by MTT assay, of addition of IL-8, MCP-1 or antibodies to these. We conclude that human NT2-N neurons release IL-8 and MCP-1 during 21 h of reoxygenation after 3 h of hypoxia. Acidosis led to a differential effect on IL-8 and MCP-1, with increased IL-8 and decreased MCP-1, both during reoxygenation and in normoxic controls. IL-8 and MCP-1 had no effect on cell death.

Topics: Acidosis; Antibodies; Cell Hypoxia; Cell Line; Chemokine CCL2; Chemokine CCL5; Dose-Response Relationship, Drug; Fluorescent Antibody Technique; Gene Expression Regulation; Glucose; Humans; Hypoxia; Interleukin-8; Neurofilament Proteins; Neurons; Oxygen; Statistics, Nonparametric; Tetrazolium Salts; Thiazoles; Time Factors

The Korean genuine medicine "Seonghyangjeongkisan" (SHJKS) has long been used for various cerebrovascular diseases. However, very little scientific investigation has been carried out. Cytokines involved in the regulation of inflammatory reactions and immune responses may play a role in the pathogenesis of cerebral infarction (CI). The aim of the present study is to elucidate how SHJKS modulates the inflammatory reaction in lipopolysaccaride (LPS) plus phytohaemagglutinin (PHA)-stimulated peripheral mononuclear cells (PBMCs) from CI patients. The amount of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-6, and IL-8 in PBMC culture supernatant was significantly increased in the LPS plus PHA treated cells compared to unstimulated cells. SHJKS inhibited the TNF-alpha, IL-1beta, IL-6, and IL-8 production in dose dependent manner. Maximal inhibition rate of the TNF-alpha, IL-1beta, IL-6, and IL-8 by SHJGS (1.0 mg/ml) was 68.01 +/- 0.28% (P < 0.01), 52.11 +/- 0.56 % (P < 0.01), 53.42 +/- 0.46 % (P < 0.01), and 46.70 +/- 0.37% (P < 0.05), respectively. In addition, we show that SHJKS suppressed nuclear factor (NF)-kappaB activation induced by LPS plus PHA, leading to suppression of IkappaB-alpha phosphorylation and degradation. These results suggest that SHJKS might have regulatory effects on LPS plus PHA-induced cytokine production and NF-kappaB activation, which might explain its beneficial effect in the treatment of CI.

Topics: Aged; Anti-Inflammatory Agents; Biotransformation; Blotting, Western; Cell Nucleus; Cells, Cultured; Cerebral Infarction; Chromatography, High Pressure Liquid; Cytokines; Cytoplasm; Female; Humans; Indicators and Reagents; Interleukin-1beta; Interleukin-6; Interleukin-8; Lipopolysaccharides; Male; Middle Aged; Monocytes; NF-kappa B; Phytohemagglutinins; Plant Extracts; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

CXCL8 (previously known as Interleukin-8), a member of the alpha-chemokine family of chemotactic cytokines, stimulates intestinal neutrophil activation and chemotaxis. As intestinal epithelial cells have been recently shown to produce CXCL8, the aim of this study was to identify functional activities of CXCL8 on intestinal epithelial cells.. The expression of CXCL8 receptors CXCR1 and CXCR2 was assessed by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells. The effects of CXCL8 on intestinal epithelial proliferation were assessed with colorimetric MTT assays and the effects on epithelial restitution with an in vitro migration model using Caco-2 and HT-29 cells.. While the expression of both CXCR1 mRNA and protein could be demonstrated by RT-PCR and FACS analysis in human Caco-2 and HT-29 cells, no expression of CXCR2 was observed in these cell lines. Colorimetric MTT assays revealed that CXCL8 does not modulate cell proliferation in HT-29 and Caco-2 cells. In contrast, CXCL8 significantly enhanced intestinal epithelial migration in an in vitro migration model of HT-29 and Caco-2 cells. Enhancement of intestinal epithelial cell migration by CXCL8 was partially CXCR1-dependent and TGFbeta-independent.. CXCL8 exerts functional effects on intestinal epithelial cells that may be relevant for intestinal inflammation and mucosal healing.

Topics: Caco-2 Cells; Cell Line, Tumor; Cell Movement; Cell Proliferation; Cell Separation; Chemotaxis; Colon; Coloring Agents; Cytokines; Epithelial Cells; Flow Cytometry; Humans; Inflammation; Interleukin-8; Intestinal Mucosa; Mucous Membrane; Receptors, Interleukin-8A; Receptors, Interleukin-8B; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Tetrazolium Salts; Thiazoles; Time Factors; Transforming Growth Factor beta

We performed this study to determine the relationship between activation of nuclear factor (NF)-kappaB and inhibition of keratinocyte growth by anthralin, which not only might be useful for a better understanding of the role of NF-kappaB in the pathogenesis of psoriasis, but also indicate whether the inflammatory reaction induced by anthralin is inseparable from its antipsoriatic activity. The involvement of NF-kappaB was assessed using the antipsoriatic drugs leflunomide and triptolide (T0) as effectors, since they can inhibit NF-kappaB activation induced by anthralin. The results showed that the inhibition of keratinocyte growth by anthralin was not related to the activation of NF-kappaB. Using sodium salicylate, a known NF-kappaB inhibitor, further confirmed this conclusion. Thus it might be possible to inhibit the inflammatory response induced by anthralin via repression of NF-kappaB activation. We found that leflunomide or T0 could significantly inhibit the mRNA overexpression of interleukin-8 and intercellular adhesion molecule-1 in keratinocytes induced by anthralin. Taken together, our data indicate that the growth inhibition of anthralin is related to the NF-kappaB-independent signaling pathway, and that leflunomide or T0 could control proinflammatory cytokine expression induced by anthralin via inhibiting the activation of NF-kappaB.

Topics: Anthralin; Anti-Inflammatory Agents, Non-Steroidal; Blotting, Western; Cell Proliferation; Cells, Cultured; Diterpenes; Enzyme Inhibitors; Epoxy Compounds; Humans; I-kappa B Proteins; Immunosuppressive Agents; Intercellular Adhesion Molecule-1; Interleukin-8; Isoxazoles; Keratinocytes; Leflunomide; NF-kappa B; Phenanthrenes; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Signal Transduction; Tetrazolium Salts; Thiazoles

Plant medications have been applied to treat pains from various types of arthritis in Korea. Rheumatoid arthritis (RA) is well known to be a chronic autoimmune/inflammatory disease that leads to progressive joint damage and cartilage destruction. Accumulation and activation of mast cells have been demonstrated in rheumatoid synovial tissue. Because infiltrated mast cells and their mediators may contribute to the initiation and progression of the inflammatory process and matrix degradation of RA, we tested the inhibitory effects of "Cool-Cool" (CC, Cool-X-A), an Oriental medication, on the production and migration of major inflammatory cytokines in mast cells. CC was treated in vitro before activation of human mast cell line (HMC-1) with phorbol 12-myristate 13-acetate, and the cytotoxicity of CC was assessed by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazoliumbromide assay. CC had no cytotoxic effects on HMC-1 cell viability. The inhibitory effects on cytokine production were monitored by enzyme-linked immunosorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR). CC inhibited not only the secretion but also the expression of TNF-alpha and IL-8 in HMC-1 cells. CC also suppressed migration of mast cells induced by stem cell factor. These findings may help in understanding the mechanism of action of this herbal medication, leading to the control of mast cells in inflammatory conditions like RA.

Topics: Cell Movement; Cell Survival; Cells, Cultured; Chemotaxis; Cytokines; Depression, Chemical; Drugs, Chinese Herbal; Humans; Interleukin-8; Mast Cells; Reverse Transcriptase Polymerase Chain Reaction; Stem Cell Factor; Tetradecanoylphorbol Acetate; Tetrazolium Salts; Thiazoles; Tumor Necrosis Factor-alpha

In the present study, reconstructed human epidermis (RHE) was used as an in vitro model to discriminate 1-chloro-2,4-dinitrobenzene (DNCB), nickel sulfate (NiSO(4)), oxazolone (OXA), 2,4-dinitrofluorobenzene (DNFB) and 2,4,6-trinitrobenzenesulfonic acid (TNBS) as skin sensitizers from benzalkonium chloride (BC), benzoic acid (BA) and sodium lauryl sulfate (SLS) as skin irritants. Our criteria were (a) the differential IL-1alpha and IL-8 synthesis and release (b) cytotoxicity assessment by MTT assay. When the RHE are topically treated with the sensitizers, very low levels of extra- and intracellular IL-1alpha are observed although they induce significant cytotoxicity. In contrast, they exhibit a sharp maximum of IL-8 release. In the presence of the tested irritants, we observe the inverse cytokine release profile, although they induce dose-dependent cytotoxicity profiles similar to those observed with the sensitizers. Finally, IL-1alpha mRNA upregulation is observed after topical application of both sensitizers and irritants, but only the latter significantly increase extracellular IL-1alpha. In conclusion, our results suggest that the associated determination of IL-8, with IL-1alpha, and MTT conversion are at least necessary to discriminate and classify, in a single assay, irritant and sensitizing agents and represent a potential in vitro alternative to two classical in vivo assays.

Topics: Administration, Topical; Animal Testing Alternatives; Biological Assay; Biomarkers; Coloring Agents; Culture Techniques; Epidermal Cells; Epidermis; Humans; Interleukin-1; Interleukin-8; Irritants; Predictive Value of Tests; Skin Irritancy Tests; Tetrazolium Salts; Thiazoles

There is still no evidence whether human peritoneal mesothelial cells (HPMC) from patients with end-stage renal failure are altered in cell viability or show a different pattern of the release of proinflammatory cytokines. Also the serum of patients with uremia may contain substances stimulating the cytokine release of HPMC.. The IL-1beta-induced IL-6/IL-8 release of HPMC from healthy donors and from patients with end-stage renal disease (ESRD) were measured before the start of chronic peritoneal dialysis (PD) and during PD therapy. Additionally the influence of uremic and non-uremic serum on IL-6 and IL-8 release of normal HPMC was studied. Cell viability was assessed by MTT assay and by the measurement of intracellular ATP (chemoluminescence assay). HPMC were obtained from the following patient groups: (1) non-uremic control patients (n = 7); (2) patients with ESRD undergoing PD catheter implantation for the first time (n = 7), and (3) patients on PD undergoing catheter exchange for noninfectious reasons (n = 6). Pooled human serum from PD patients and normal controls were used for stimulation experiments. HPMC from different donors were grown to confluence (second passage) and then stimulated with IL-1beta (1,000 pg/ml in M199) for 24 h. IL-6 and IL-8 concentrations were measured in the supernatant by ELISA. Additionally uremic and non-uremic sera were incubated with HPMC from normal donors for 24 h with a subsequent 24-hour IL-1beta stimulation. Mesothelial cell protein mass was determined by the Bradford reagent.. Non-uremic patients and ESRD patients did not differ with regard to the global cell viability of HPMC according to MTT assay activity or the intracellular ATP concentration. However, HPMC from uremic patients produced more IL-8 on IL-1beta stimulation than the non-uremic controls (group 2, 53.5 +/- 15.7 pg/microg; group 3, 70.5 +/- 27.3 pg/microg vs. group 1, 24.0 +/- 11.8 pg/microg). HPMC from patients on chronic PD additionally released significantly more IL-6 (30.5 +/- 13.8 pg/microg) on IL-1beta stimulation than uremic patients before the onset of PD (6.2 +/- 2.6 pg/microg; p < 0.01). Incubation of normal HPMC with the serum from uremic donors produced an enhanced stimulated IL-8 release compared to the exposition with normal control serum (50.6 +/- 6.1 vs. 20.8 +/- 2.9 pg/microg; p < 0.01).. HPMC from uremic patients more readily release IL-8 on stimulation with IL-1beta. On chronic PD treatment IL-6 release was further enhanced. Not further classified serum components in uremia also enhance IL-6 and IL-8 release of HPMC.

Topics: Adenosine Triphosphate; Adult; Cell Survival; Cells, Cultured; Cytokines; Enzyme-Linked Immunosorbent Assay; Epithelium; Female; Humans; Immunohistochemistry; Interleukin-1; Interleukin-6; Interleukin-8; Kidney Failure, Chronic; Luminescent Measurements; Male; Middle Aged; Peritoneal Cavity; Protein Biosynthesis; Tetrazolium Salts; Thiazoles; Uremia

The cytokines IL-6, initially recognized as a regulator of immune and inflammatory response and IL-8, a potential regulator of angiogenesis, also regulate the growth of many tumor cells. Human cancer cells selected for multidrug resistance to common chemotherapeutic agents demonstrate increased expression of IL-6 and IL-8. To determine whether IL-6 or IL-8 overexpression contributes directly to the drug resistant phenotype, IL-6 or IL-8 cDNA were introduced into the paclitaxel sensitive human osteosarcoma cell line U-2OS using the pIRESneo bicistronic expression vector. Interleukin-6 and IL-8 transfectants were selected for either high IL-6 or IL-8 secretion and evaluated in drug resistance assays. Two IL-6 and two IL-8 secreting clones express IL-6 or IL-8 levels of 10 ng/ml and 1 ng/ml in culture, while parental U-2OS and pIRESneo vector transfected control cells express IL-6 and IL-8 levels of 0.005 ng/ml and 0.1 ng/ml, respectively. MTT cytotoxicity with IL-6 transfected cells demonstrates a five-fold increase in resistance to paclitaxel and a four-fold increase in resistance to doxorubicin as compared to U-2OS. There are no changes in mitoxantrone or topotecan resistance in the IL-6 transfectants as compared to parental U-2OS. Northern analysis of IL-6 transfectants demonstrates that the resistant phenotype is not related to increased levels of MDR-1, MRP-1, or LRP. Western analysis also confirms that P-glycoprotein levels are not altered in IL-6 transfectants. Further supporting an MDR-1 independent mechanism of drug resistance, verapamil cannot reverse paclitaxel resistance in transfected cells, findings further supported by rhodamine 123 exclusion data. Treatment of IL-6 transfected cells with paclitaxel, compared with drug-sensitive parental U-2OS, shows U-2OS(IL-6) are significantly more resistant to apoptosis induced by paclitaxel and exhibit decreased proteolytic activation of caspase-3. In contrast U-2OS(IL-8) transfectants demonstrate no appreciable increase in paclitaxel resistance when compared with parental cells. In summary, while both IL-6 and IL-8 are overexpressed in paclitaxel resistant cell lines, only IL-6 has the potential to contribute directly to paclitaxel and doxorubicin resistance in U-2OS. This resistance is through a non-MDR-1 pathway.

Topics: Antineoplastic Agents; Antineoplastic Agents, Phytogenic; ATP Binding Cassette Transporter, Subfamily B, Member 1; Blotting, Northern; Blotting, Western; Caspase 3; Caspases; Cell Division; DNA, Complementary; Dose-Response Relationship, Drug; Drug Resistance, Neoplasm; Enzyme Activation; Enzyme-Linked Immunosorbent Assay; Genetic Vectors; Humans; Interleukin-6; Interleukin-8; Mitoxantrone; Osteosarcoma; Paclitaxel; Phenotype; RNA; Tetrazolium Salts; Thiazoles; Time Factors; Topotecan; Transfection; Tumor Cells, Cultured

Interleukin 1alpha (IL-1alpha) is an important regulatory cytokine, the release of which after an injury can induce activation of transcription factors nuclear factor (NF)kappaB and activator protein (AP-1), which promote expression of genes involved in cell survival, proliferation, and angiogenesis. IL-1alpha is expressed autonomously by head and neck squamous cell carcinomas (HNSCCs) and a variety of other cancers, raising the possibility that IL-1alpha may serve as an autocrine factor that stimulates the activation of prosurvival transcription factors and target genes in cancer. In this study, we examined the role of IL-1alpha in the activation of NFkappaB and AP-1, the expression of proangiogenic cytokine IL-8, and in the survival and proliferation of HNSCC cell lines. HNSCCs were found to secrete and respond to functional IL-1alpha, in that culture supernatant from a high IL-1alpha-secreting line, UM-SCC-11B, could induce secretion of cytokine IL-8 by a low IL-1alpha-secreting line, UM-SCC-9; and the induction of IL-8 secretion could be blocked by the anti-IL-1alpha-neutralizing antibody or the IL-1 receptor antagonist (IL-1RA). Furthermore, IL-1alpha could induce the expression of IL-8 through an autocrine mechanism, in that transfection of UM-SCC-9 cells with a plasmid encoding IL-1alpha resulted in the increased coexpression of IL-1alpha and IL-8; whereas transfection with a plasmid encoding IL-1RA lacking the secretory leader sequence led to the decreased coexpression of IL-1alpha and IL-8. IL-1alpha was found to induce coexpression of IL-8 through the activation of NFkappaB and AP-1, in that mutation of the NFkappaB site within the IL-8 promoter abolished autocrine- and recombinant IL-1alpha-induced IL-8 reporter gene activity, whereas mutation in AP-1 partially decreased IL-8 reporter gene activity in UM-SCC-9 cells. Intracellular expression of IL-1RA decreased NFkappaB reporter gene activity, indicating that endogenously expressed IL-1alpha contributes to constitutive NFkappaB activation in this HNSCC line. Expression of IL-1alpha affected survival of UM-SCC-9, inasmuch as transfection of cells with plasmid encoding IL-1alpha or IL-1RA led to the increased or decreased survival of cells cotransfected with a beta-galactosidase reporter gene, respectively. IL-1alpha was also found to promote the increased growth of UM-SCC-9 cells in vitro. We demonstrate that exogenous and endogenous IL-1alpha contributes to the transcriptional activation of NFk

Topics: Carcinoma, Squamous Cell; Cell Division; Cell Survival; Coloring Agents; Enzyme-Linked Immunosorbent Assay; Genes, Reporter; Genetic Vectors; Head and Neck Neoplasms; Humans; Interleukin-1; Interleukin-8; Mutation; NF-kappa B; Plasmids; Recombinant Proteins; Tetrazolium Salts; Thiazoles; Time Factors; Transcription Factor AP-1; Transcriptional Activation; Transfection; Tumor Cells, Cultured

Thermal injury by short pulses (1-30 s) of relatively high temperature (50-68 degrees C) was investigated in normal human epidermal keratinocytes (NHEK). NHEK were cultured on plastic cover-slips and dipped in medium held at various temperatures. Survival assessed by methylthiazol tetrazolium reduction assay at 6 days postheating demonstrated an inverse time-temperature relationship that indicated that most cells could survive after a 1-s, 60 degrees C exposure or a 30-s, 55 degrees C exposure. Arrhenius plots of the data indicated major transition points for cell injury at 50 and 60 degrees C. Heat shock protein 70 (HSP70) and interleukin-8 (IL-8) were both induced by elevation of temperature between 50 and 60 degrees C for as short a time as 1 s. HSP70 synthesis stimulated by short, high pulses of heat appeared to induce thermotolerance. These results demonstrate that brief exposure to relatively high temperature can induce HSP70 and IL-8 synthesis in keratinocytes.

Topics: Cell Survival; Cells, Cultured; Coloring Agents; Epidermal Cells; Epidermis; Female; Hot Temperature; HSP70 Heat-Shock Proteins; Humans; Interleukin-8; Keratinocytes; Kinetics; Mammaplasty; Tetrazolium Salts; Thiazoles; Time Factors