Effects In Epithelial Cells Research Articles

L-Alanine promotes anti-infectious properties of Bacillus subtilis S-2 spores via the germination receptor gerAA.

Bacillus species, which have two cell-type forms (vegetative cells and spores), demonstrate a variety of probiotic functions in animal feed additives and human nutrition. We previously found that the probiotic effect of Bacillus subtilis S-2 spores with high germination response to L-alanine was specifically enhanced by the L-alanine pretreatment. The germination response of Bacillus is highly associated with the germination receptors of spores. However, how L-alanine-induced germination of spores exerts anti-infectious effect in epithelial cells remains unclear. In this study, we constructed the mutant strain of B. subtilis S-2 with germination receptor gerAA knockout to further explore the role of spore germination in resisting pathogen infection to cells. The differential probiotic effects of B. subtilis S-2 and S-2ΔgerAA spores pretreated with L-alanine were evaluated in intestinal porcine epithelial cells (IPEC-J2) or Caco2 cells infected with enterotoxigenic Escherichia coli (ETEC) or following IL-1β stimulation. The results showed that the germination response of the S-2ΔgerAA spores to L-alanine was significantly reduced. Compared with the S-2ΔgerAA spores, the L-alanine-induced germination of B. subtilis S-2 spores significantly increased the activity of anti-adhesion of ETEC to IPEC-J2 cells and reduced the expression of inflammatory factors and cell receptors. L-alanine induction also significantly promoted the expression of autophagy-related proteins in the B. subtilis S-2 spores. These findings demonstrate that the gerAA germination receptor is essential for the probiotic function of Bacillus spores and that L-alanine treatment promotes the anti-infectious properties of the germinated spores in porcine intestinal epithelial IPEC-J2 cells. The result suggests the importance of germination receptor gerAA in helping spore germination and enhancing anti-infectious activity. The findings in the study benefit to screening of potential Bacillus probiotics and increasing probiotic efficacy induced by L-alanine as an adjuvant.

717 TCF-4 negatively regulates IL-17C In human keratinocytes and in a mouse model of psoriasis

IL-17C is a keratinocyte-derived cytokine that promotes and amplifies innate defense in various inflammatory skin diseases including psoriasis. However, little is known about the mechanisms regulating IL-17C expression or its downstream proinflammatory effects in epithelial cells that promote psoriasis inflammation. Here we identify a critical role for the transcription factor TCF4 in the negative regulation of IL17C expression. RNAseq analyses of psoriasis patient lesional skin (n=99) revealed a negative correlation (r=-0.43, p=6.4E-06) between TCF4 and IL17C, and immunohistochemistry of lesional psoriasis skin confirmed that increases in IL-17C correspond to decreases in nuclear TCF4.

103 Differences in Estradiol Levels Following Burn Injury

IntroductionThe American Burn Association estimates over one million people with burn injuries in the US need medical care, with around 4,500 cases ending in mortality each year. Mortality is due to bacterial infection as a consequence of severe cytokine dysregulation and impaired wound healing. Previous research has shown females have worse outcomes than males following burn injury, but the reasoning is unknown. Estradiol is involved in the regulation of local and systemic interleukin-6 (IL-6) levels, which has been demonstrated by others to positively correlates with poor outcomes following burn. We hypothesize that concentrations of Estrogen can create a pro-inflammatory effect in epithelial cells, which is controlled in a negative feedback loop.MethodsWe utilized plasma from 1) human burn patients and 2) our murine model of burn injury, in which C57BL/6 mice are exposed to a 20% total body surface area burn injury and 3) an in vitro cell model using human Oral Epithelial Cells (OECs) and human Airway Epithelial Cells (AECs).ResultsWe measured human estradiol levels 1-3 days after burn injury and murine estradiol levels 3 and 7 days after injury by ELISA. In humans there were differences in levels at these timepoints (p< 0.05). In mice we observed a difference in the change in murine estradiol levels between males and females 7 days after injury (p < 0.01). We simulated a wound by removing the cell insert and treating cells with concentrations of estradiol (0.1 nM, 1.0 nM & 250 nM). Images were taken at 0, 6 & 24 H and analyzed using Fiji to observe wound closure. Supernatant was removed from cells at 24 H and analyzed for IL-6 levels (a key pro-inflammatory cytokine linked to poor wound healing after burn) via ELISA. mRNA was isolated from cells and analyzed for IL-6, TNFα & VEGF (important regulators of wound healing) via PCR. We observed an increase in percent wound closure in the AECs, and no difference in OECs. We observed an increase in IL-6 production following stimulation with Estradiol and LPS at different rates for each cell type and corresponding reduction in TNFα and VEGF expression. We also described different Estradiol Receptors in the two cell types.ConclusionsTaken together, these data suggest burn injury upregulates estradiol in both humans and mice following burn injury whereby this is more robust in females at later stages of burn injury and thus contributing to female-associated poor clinical outcomes.

TGFβ Signaling in the Pancreatic Tumor Microenvironment.

Simple SummaryThere is currently no effective treatment for patients with advanced pancreatic ductal adenocarcinoma (PDAC). Transforming Growth Factor β (TGFβ) signaling has been implicated in several hallmark features of PDAC pathobiology, and TGFβ inhibitors are beginning to show promise in the treatment of PDAC. Here, we discuss the known roles of TGFβ signaling in the pancreatic tumor microenvironment, as well as clinical trials evaluating TGFβ pathway inhibitors in PDAC patients.Pancreatic ductal adenocarcinoma (PDAC) is associated with poor clinical outcomes, largely attributed to incomplete responses to standard therapeutic approaches. Recently, selective inhibitors of the Transforming Growth Factor β (TGFβ) signaling pathway have shown early promise in the treatment of PDAC, particularly as a means of augmenting responses to chemo- and immunotherapies. However, TGFβ is a potent and pleiotropic cytokine with several seemingly paradoxical roles within the pancreatic tumor microenvironment (TME). Although TGFβ signaling can have potent tumor-suppressive effects in epithelial cells, TGFβ signaling also accelerates pancreatic tumorigenesis by enhancing epithelial-to-mesenchymal transition (EMT), fibrosis, and the evasion of the cytotoxic immune surveillance program. Here, we discuss the known roles of TGFβ signaling in pancreatic carcinogenesis, the biologic consequences of the genetic inactivation of select components of the TGFβ pathway, as well as past and present attempts to advance TGFβ inhibitors in the treatment of PDAC patients.

Antiviral Drug Screen of Kinase Inhibitors Identifies Cellular Signaling Pathways Critical for SARS-CoV-2 Replication

The emergence of a highly contagious novel coronavirus, SARS-CoV-2 that causes COVID-19, has precipitated the current global health crisis. However, our knowledge of the mechanisms of COVID-19 pathogenesis is very limited which has hampered attempts to develop targeted antiviral strategies. We developed a medium-throughput primary drug screening system and identified a small molecule library of 34 of 430 protein kinase inhibitors that were capable of inhibiting viral cytopathic effect in epithelial cells. These drug inhibitors are all in various stages of clinical trials. We detected key proteins involving in cellular signaling pathways mTOR-PI3K-AKT, ABL-BCR/MAPK, and DNA-Damage Response pathways that are critical for SARS-CoV-2 infection. A drug-protein interaction based secondary screen confirmed compounds such as Berzosertib (VE-822) and Nilotinib with anti SARS-CoV-2 activity, in which Berzosertib demonstrated potent antiviral activity in a human epithelial cell line and human induced pluripotent stem cell-derived cardiomyocytes. Our study highlights key promising kinase inhibitors to constrain SARS-CoV-2 replication as a host-directed therapy in the treatment of COVID-19 and provides an important mechanism of host-pathogen interactions.Funding: This research was funded by UCLA DGSOM and Broad Stem Cell Research Center institutional award (OCRC #20-1) to V.A. and California Institute for Regenerative Medicine Discovery Award (DISC2COVID19-11764) to B.G and (TRAN1COVID19-11975) to V.A., an institutional training grant (T32 HL116273) to A.S., and an American Heart Association Rapid Response Grant to A.S and C.N.S. Conflict of Interest: The authors declare no competing financial interests.Ethical Approval: This study was performed in strict accordance with the recommendations of UCLA.

Regulation of epithelial-mesenchymal transition and organoid morphogenesis by a novel TGF\u03b2-TCF7L2 isoform-specific signaling pathway

Alternative splicing contributes to diversification of gene function, yet consequences of splicing on functions of specific gene products is poorly understood. The major transcription factor TCF7L2 undergoes alternative splicing but the biological significance of TCF7L2 isoforms has remained largely to be elucidated. Here, we find that the TCF7L2 E-isoforms maintain, whereas the M and S isoforms disrupt morphogenesis of 3D-epithelial cell-derived organoids via regulation of epithelial-mesenchymal transition (EMT). Remarkably, TCF7L2E2 antagonizes, whereas TCF7L2M2/S2 promotes EMT-like effects in epithelial cells induced by transforming growth factor beta (TGFβ) signaling. In addition, we find TGFβ signaling reduces the proportion of TCF7L2E to TCF7L2M/S protein in cells undergoing EMT. We also find that TCF7L2 operates via TGFβ-Smad3 signaling to regulate EMT. Collectively, our findings unveil novel isoform-specific functions for the major transcription factor TCF7L2 and provide novel links between TCF7L2 and TGFβ signaling in the control of EMT-like responses and epithelial tissue morphogenesis.

Geranium and Lemon Essential Oils and Their Active Compounds Downregulate Angiotensin-Converting Enzyme 2 (ACE2), a SARS-CoV-2 Spike Receptor-Binding Domain, in Epithelial Cells.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), also known as coronavirus disease-2019 (COVID-19), is a pandemic disease that has been declared as modern history’s gravest health emergency worldwide. Until now, no precise treatment modality has been developed. The angiotensin-converting enzyme 2 (ACE2) receptor, a host cell receptor, has been found to play a crucial role in virus cell entry; therefore, ACE2 blockers can be a potential target for anti-viral intervention. In this study, we evaluated the ACE2 inhibitory effects of 10 essential oils. Among them, geranium and lemon oils displayed significant ACE2 inhibitory effects in epithelial cells. In addition, immunoblotting and qPCR analysis also confirmed that geranium and lemon oils possess potent ACE2 inhibitory effects. Furthermore, the gas chromatography-mass spectrometry (GC–MS) analysis displayed 22 compounds in geranium oil and 9 compounds in lemon oil. Citronellol, geraniol, and neryl acetate were the major compounds of geranium oil and limonene that represented major compound of lemon oil. Next, we found that treatment with citronellol and limonene significantly downregulated ACE2 expression in epithelial cells. The results suggest that geranium and lemon essential oils and their derivative compounds are valuable natural anti-viral agents that may contribute to the prevention of the invasion of SARS-CoV-2/COVID-19 into the human body.

Helicobacter pylori VacA, a distinct toxin exerts diverse functionalities in numerous cells: An overview.

Helicobacter pylori, gastric cancer-causing bacteria, survive in their gastric environment of more than 50% of the world population. The presence of H.pylori in the gastric vicinity promotes the development of various diseases including peptic ulcer and gastric carcinoma. H.pylori produce and secret Vacuolating cytotoxin A (VacA), a major toxin facilitating the bacteria against the host defense system. The toxin causes multiple effects in epithelial cells and immune cells, especially T cells, B cells, and Macrophages. This review describes the diverse functionalities of protein toxin VacA. The specific objective of this review is to address the overall structure, mechanism, and functions of VacA in various cell types. The recent advancements are summarized and discussed and thus conclusion is drawn based on the overall reported evidences. The searched articles on H.pylori VacA were evaluated and limited up to 66 articles for this review. The articles were divided into four major categories including articles on vacA gene, VacA toxin, distinct effects of VacA toxin, and their effects on various cells. Based on these studies, the review article was prepared. This review describes an overview of how VacA is secreted by H.pylori and contributes to colonization and virulence in multiple ways by affecting epithelial cells, T cells, Dendritic cells, B cells, and Macrophages. The reported evidence suggests that the comprehensive outlook need to be developed for understanding distinctive functionalities of VacA.

ATM-activated autotaxin (ATX) propagates inflammation and DNA damage in lung epithelial cells: a new mode of action for silica-induced DNA damage?

Silica exposure is a common risk factor for lung cancer. It has been claimed that key elements in cancer development are activation of inflammatory cells that indirectly induce DNA damage and proliferative stimuli in respiratory epithelial cells. We studied DNA damage induced by silica particles in respiratory epithelial cells and focused the role of the signaling enzyme autotaxin (ATX). A549 and 16 bronchial epithelial cells (16HBE) lung epithelial cells were exposed to silica particles. Reactive oxygen species (ROS), NOD-like receptor family pyrin domain containing-3 (NLRP3) inflammasome activation, ATX, ataxia telangiectasia mutated (ATM), and DNA damage (γH2AX, pCHK1, pCHK2, comet assay) were end points. Low doses of silica induced NLRP3 activation, DNA damage accumulation, and ATM phosphorylation. A novel finding was that ATM induced ATX generation and secretion. Not only silica but also rotenone, camptothecin and H2O2 activated ATX via ATM, suggesting that ATX is part of a generalized ATM response to double-strand breaks (DSBs). Surprisingly, ATX inhibition mitigated DNA damage accumulation at later time points (6-16 h), and ATX transfection caused NLRP3 activation and DNA damage. Furthermore, the product of ATX enzymatic activity, lysophosphatidic acid, recapitulated the effects of ATX transfection. These data indicate an ATM-ATX-dependent loop that propagates inflammation and DSB accumulation, making low doses of silica effective inducers of DSBs in epithelial cells. We conclude that an ATM-ATX axis interconnects DSBs with silica-induced inflammation and propagates these effects in epithelial cells. Further studies of this adverse outcome pathway may give an accurate assessment of the lowest doses of silica that causes cancer.

Effects of gasoline and ethanol-gasoline exhaust exposure on human bronchial epithelial and natural killer cells in vitro

Air pollution exposure, including passenger car emissions, may cause substantial respiratory health effects and cancer death. In western countries, the majority of passenger cars are driven by gasoline fuel. Recently, new motor technologies and ethanol fuels have been introduced to the market, but potential health effects have not been thoroughly investigated.We developed and verified a coculture model composed of bronchial epithelial cells (ECs) and natural killer cells (NKs) mimicking the human airways to compare toxic effects between pure gasoline (E0) and ethanol-gasoline-blend (E85, 85% ethanol, 15% gasoline) exhaust emitted from a flexfuel gasoline car. We drove a steady state cycle, exposed ECs for 6h and added NKs. We assessed exhaust effects in ECs alone and in cocultures by RT-PCR, flow cytometry, and oxidative stress assay.We found no toxic effects after exposure to E0 or E85 compared to air controls. Comparison between E0 and E85 exposure showed a weak association for less oxidative DNA damage after E85 exposure compared to E0. Our results indicate that short-term exposure to gasoline exhaust may have no major toxic effects in ECs and NKs and that ethanol as part of fuel for gasoline cars may be favorable.

Detecção do virus ‘HoBi’-like (BVDV-3) em bovino no semiárido do Estado da Paraíba

RESUMO: Objetivou-se descrever os aspectos clínicos e anatomopatológicos, e a identificação viral de um caso de infecção pelo vírus ‘Hobi’-like (BVDV-3) em bovino do semiárido paraibano, Nordeste do Brasil. Um bovino, fêmea, três meses de idade, foi levado ao Hospital Veterinário da UFCG apresentando salivação, dificuldade de apreensão do teto, falta de apetite, fezes escuras e em pouca quantidade. Diante da piora do quadro clínico optou-se por sua eutanásia in extremis, seguida da realização da necropsia e coleta de material para histopatologia. Histologicamente, nas mucosas do trato digestivo, havia edema, degeneração balonosa, necrose e infiltrado inflamatório, que foi observado na face dorsal da língua e no seu epitélio mais profundo. A imunohistoquímica de amostras de extremidade de pavilhão auricular demonstrou marcação antigênica positiva e pela RT-PCR foi possível detectar RNA viral do BVDV no soro sanguíneo, cujo efeito citopático em células epiteliais de rim bovino da linhagem “Madin Darby bovine kidney” (MDBK) não foi observado. O sequenciamento do gene 5’NCR demonstrou que o vírus isolado estaria mais relacionado ao ‘Hobi’-like (BVDV-3). Após a confirmação do diagnóstico foram coletadas amostras de soro dos 23 animais do rebanho para sorologia por ELISA indireto, sendo constatada 69,6% (16/23) de soropositividade. A identificação deste novo caso de infecção por ‘Hobi’-like na Paraíba reafirma a necessidade de um monitoramento regular para BVDV na região para detecção precoce da infecção dos rebanhos e adoção de medidas eficazes de prevenção e controle.

TGFβ Signaling in the Pancreatic Tumor Microenvironment Promotes Fibrosis and Immune Evasion to Facilitate Tumorigenesis.

In early pancreatic carcinogenesis, TGFβ acts as a tumor suppressor due to its growth-inhibitory effects in epithelial cells. However, in advanced disease, TGFβ appears to promote tumor progression. Therefore, to better understand the contributions of TGFβ signaling to pancreatic carcinogenesis, we generated mouse models of pancreatic cancer with either epithelial or systemic TGFBR deficiency. We found that epithelial suppression of TGFβ signals facilitated pancreatic tumorigenesis, whereas global loss of TGFβ signaling protected against tumor development via inhibition of tumor-associated fibrosis, stromal TGFβ1 production, and the resultant restoration of antitumor immune function. Similarly, TGFBR-deficient T cells resisted TGFβ-induced inactivation ex vivo, and adoptive transfer of TGFBR-deficient CD8(+) T cells led to enhanced infiltration and granzyme B-mediated destruction of developing tumors. These findings paralleled our observations in human patients, where TGFβ expression correlated with increased fibrosis and associated negatively with expression of granzyme B. Collectively, our findings suggest that, despite opposing the proliferation of some epithelial cells, TGFβ may promote pancreatic cancer development by affecting stromal and hematopoietic cell function. Therefore, the use of TGFBR inhibition to target components of the tumor microenvironment warrants consideration as a potential therapy for pancreatic cancer, particularly in patients who have already lost tumor-suppressive TGFβ signals in the epithelium. Cancer Res; 76(9); 2525-39. ©2016 AACR.

IFN-γ-induced JAK/STAT, but not NF-κB, signaling pathway is insensitive to glucocorticoid in airway epithelial cells.

Although the majority of patients with asthma are well controlled by inhaled glucocorticoids (GCs), patients with severe asthma are poorly responsive to GCs. This latter group is responsible for a disproportionate share of health care costs associated with asthma. Recent studies in immune cells have incriminated interferon-γ (IFN-γ) as a possible trigger of GC insensitivity in severe asthma; however, little is known about the role of IFN-γ in modulating GC effects in other clinically relevant nonimmune cells, such as airway epithelial cells. We hypothesized that IFN-γ-induced JAK/STAT-associated signaling pathways in airway epithelial cells are insensitive to GCs and that strategies aimed at inhibiting JAK/STAT pathways can restore steroid responsiveness. Using Western blot analysis we found that all steps of the IFN-γ-induced JAK/STAT signaling pathway were indeed GC insensitive. Transfection of cells with reporter plasmid showed IFN-γ-induced STAT1-dependent gene transcription to be also GC insensitive. Interestingly, real-time PCR analysis showed that IFN-γ-inducible genes (IIGs) were differentially affected by GC, with CXCL10 being GC sensitive and CXCL11 and IFIT2 being GC insensitive. Further investigation showed that the differential sensitivity of IIGs to GC was due to their variable dependency to JAK/STAT vs. NF-κB signaling pathways with GC-sensitive IIGs being more NF-κB dependent and GC-insensitive IIGs being more JAK/STAT dependent. Importantly, transfection of cells with siRNA-STAT1 was able to restore steroid responsiveness of GC-insensitive IIGs. Taken together, our results show the insensitivity of IFN-γ-induced JAK/STAT signaling pathways to GC effects in epithelial cells and also suggest that targeting STAT1 could restore GC responsiveness in patients with severe asthma.

The Effect of Omnaris® on the Inflammatory Reaction and Mucin Gene Expression from Nasal Polyp Epithelial Cells

Received December 13, 2013 Revised May 12, 2014 Accepted June 9, 2014 Address for correspondence Seung-Heon Shin, MD Department of OtolaryngologyHead and Neck Surgery, School of Medicine, Catholic University of Daegu, 33 Duryugongwon-ro 17-gil, Nam-gu, Daegu 705-718, Korea Tel +82-53-650-4530 Fax +82-53-650-4533 E-mail hsseung@cu.ac.kr Background and ObjectivesZZGlucocorticoids are a potent anti-inflammatory agent. The authors conducted this study to investigate the effect of Omnaris on suppression of inflammation induction and mucin gene expression in nasal polyp epithelial cells. Subjects and MethodZZPrimary nasal polyp epithelial cells were stimulated by 5 ug/mL of streptococcal enterotoxin B (SEB) and lipopolysaccharide (LPS). To determine the effects of Omnaris, cells were pretreated with 200, 100, 10, 1 ng/mL of Omnaris. The anti-inflammatory effect of epithelial cells were confirmed by measuring interleukin (IL)-6, IL-8, and granulocyte-macrophage colony stimulating factor (GM-CSF), and mucin gene expressions were determined by real time PCR for MUC4, MUC5AC, MUC5B, MUC8. ResultsZZSEB and LPS enhanced the production of IL-6, IL-8, and GM-CSF from nasal polyp epithelial cells. The increased cytokine levels were significantly suppressed by Omnaris at 100 and 10 ng/mL. The expressions of MUC4, MUC5AC, MUC5B, MUC8 mRNA, and MUC4 mRNA were increased by SEB and LPS, respectively. The increased expression of these mucin genes were significantly suppressed by 100, 10, and 1 ng/mL of Omnaris. ConclusionZZOmnaris significantly suppressed the production of chemical mediators and mucin gene expression, which indicated that Omnaris is effective in improving and treating inflammatory diseases in the nasal cavity. Korean J Otorhinolaryngol-Head Neck Surg 2014;57(9):603-9

Effect of the Potent Antiviral 1‐Cinnamoyl‐3,11‐Dihydroxymeliacarpin on Cytokine Production by Murine Macrophages Stimulated with HSV‐2

The limonoid 1-cinnamoyl-3,11-dihydroxymeliacarpin (CDM) isolated from leaf extracts of Melia azedarach L, has potent antiherpetic effect in epithelial cells. Since Meliacine, the partially purified extract source of CDM, has therapeutic effect on murine genital herpes, the potential use of CDM as microbicide against herpetic infections was studied here. To determine the cytotoxic effect of CDM, the MTT assay and acridine orange staining of living cells were performed. The antiherpetic action of CDM was measured by plaque reduction assay, and the immunomodulatory effect was determined by measuring the cytokine production using a bioassay and ELISA method. The results presented here showed that CDM inhibited Herpes Simplex Virus type 2 (HSV-2) multiplication in Vero cells but did not affect its replication in macrophages which were not permissive to HSV infection. In macrophages, levels of TNF-α, IFN-γ, NO, IL-6 and IL-10 were increased by CDM used alone or in combination with HSV-2. Besides, CDM not only synergized TNF-α production combined with IFN-γ, but also prolonged its expression in time. Results indicate that CDM inhibits HSV-2 multiplication in epithelial cells and also increases cytokine production in macrophages, both important actions to the clearance of infecting virus in the mouse vagina.

Sex Steroids Regulate Epithelial–Stromal Cell Cross Talk and Trophoblast Attachment Invasion in a Three-Dimensional Human Endometrial Culture System

Human embryo implantation involves a complex network of molecular signaling that is modulated by endocrine and paracrine pathways. Here, we performed studies using a unique and recently developed three-dimensional (3D) implantation model, characterized by an endometrium-like 3D culture system and Jar cell-derived spheroids mimicking the embryo/trophoblast. The aims were to investigate the effects of 17β estradiol (E2) and medroxyprogesterone acetate (MPA) on (1) the interaction between epithelial and stromal cells, and (2) the attachment and invasion of trophoblast cells. We observed that epithelial and stromal cells in the 3D culture were ERα⁺, ERβ⁺, and PR⁺. Decidualization was confirmed by enhanced prolactin gene expression on day 7 of E2 plus MPA treatment. An effect of epithelial cells on the decidualization of stromal cells was indicated by significantly higher levels of prolactin mRNA expression in the 3D culture compared to stromal cells grown within the fibrin-agarose gel matrix. On the other hand, the relative gene expressions of E-cadherin and IL-1β in epithelial cells of the 3D culture under decidualization conditions significantly differed from those in epithelial cells grown over the fibrin-agarose gel matrix without stromal cells, pointing to regulation of epithelial cells by the stroma. The attachment rate of Jar spheroids to the 3D was significantly increased by E2 plus MPA treatment. Analyses of Z-stack confocal and stained optic microscopic images demonstrated that Jar spheroids breached the epithelial cell monolayer, invaded, and were embedded into the 3D matrix in response to decidualization signals. In summary, the newly bioengineered system provides a unique model for studying interactions between the different endometrial cell compartments, via soluble-paracrine signals as well as cell-to-cell interactions, and is a useful tool to study early embryonic implantation events.

Differential activation of the inflammasome in THP-1 cells exposed to chrysotile asbestos and Libby “six-mix” amphiboles and subsequent activation of BEAS-2B cells

Inflammatory responses of THP-1 cells (macrophage cell line) exposed to chrysotile asbestos (Chry) and Libby six-mix (LIB) and the subsequent impact on bronchial epithelial cells were determined. Direct treatment of THP-1 cells with Chry caused cell death, activation of caspase-1 and release of IL-1β, while the addition of caspase-1 inhibitor, Z-YVAD-FMK, reduced IL-1β, suggesting that Chry activated the caspase-1 mediated Nod-like receptor protein 3 (NLRP3) inflammasome; by comparison, LIB had less effects on all of these parameters. Expression of antioxidant enzymes, protein oxidation and nitration, and lipid peroxides in THP-1 cells treated with the two particles suggest that LIB generated more reactive oxygen species (ROS) than the same dose of Chry. Differences in fiber length and surface area suggest a possible role for particulate size in the differential activation of the inflammasome. BEAS-2B cells, representing the bronchial epithelium, treated with supernatants of medium from Chry- or LIB-treated THP-1 cells (conditioned medium) activated the MAPK cascade, increased phosphorylation of ERK and Cot (MAP3K8), increased AP-1 binding activity and induced IL-6 release. To verify that IL-1β from THP-1 cells was responsible for activation of BEAS-2B, conditioned medium with added IL-1Ra, an IL-1β antagonist, was applied to BEAS-2B. Results show that IL-1Ra attenuated effects of conditioned medium, supporting a role of IL-1β, as a secondary mediator, in the transduction of inflammatory signaling from the macrophage to epithelial cells. The effects of LIB-conditioned medium appeared to be less dependent on IL-1β. In conclusion, Chry and LIB induce differential inflammatory responses in THP-1 cells that subsequently lead to differential effects in epithelial cells.

Tiam1-regulated osteopontin in senescent fibroblasts contributes to the migration and invasion of associated epithelial cells

The tumor microenvironment undergoes changes concurrent with neoplastic progression. Cancer incidence increases with aging and is associated with tissue accumulation of senescent cells. Senescent fibroblasts are thought to contribute to tumor development in aging tissues. We have shown that fibroblasts deficient in the Rac exchange factor Tiam1 promote invasion and metastasis of associated epithelial tumor cells. Here, we use a three-dimensional culture model of cellular invasiveness to outline several steps underlying this effect. We find that stress-induced senescence induces decreased fibroblast Tiam1 protein levels and increased osteopontin levels, and that senescent fibroblast lysates induce Tiam1 protein degradation in a calcium- and calpain-dependent fashion. Changes in fibroblast Tiam1 protein levels induce converse changes in osteopontin mRNA and protein. Senescent fibroblasts induce increased invasion and migration in co-cultured mammary epithelial cells. These effects in epithelial cells are ameliorated by either increasing fibroblast Tiam1 or decreasing fibroblast osteopontin. Finally, in seeded cell migration assays we find that either senescent or Tiam1-deficient fibroblasts induce increased epithelial cell migration that is dependent on fibroblast secretion of osteopontin. These findings indicate that one mechanism by which senescent fibroblasts promote neoplastic progression in associated tumors is through degradation of fibroblast Tiam1 protein and the consequent increase in secretion of osteopontin by fibroblasts.

Neutralizing effect of green tea epigallocatechin‐3‐gallate on nicotine‐induced toxicity and chemokine (C‐C motif) ligand 5 secretion in human oral epithelial cells and fibroblasts

Tobacco use has been identified as the most important environmental risk factor for periodontitis. The aim of this study was to investigate the effect of green tea epigallocatechin-3-gallate on the nicotine-induced toxic and inflammatory responses in oral epithelial cells and gingival fibroblasts. The effect of nicotine, alone and in combination with the lipopolysaccharide of Aggregatibacter actinomycetemcomitans, on the viability of oral epithelial cells and fibroblasts was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide colorimetric assay. The ability of epigallocatechin-3-gallate to neutralize the nicotine-induced cytotoxicity was then investigated. The nicotine-induced cytokine secretion in epithelial cells and the inhibitory effect of epigallocatechin-3-gallate were determined by enzyme-linked immunosorbent assay. Our results indicated that nicotine caused a dose-dependent loss of viability in both epithelial cells and fibroblasts. A mixture of nicotine and A. actinomycetemcomitans lipopolysaccharide demonstrated additive instead of synergistic effects on loss of cell viability. Pretreatment of cells with epigallocatechin-3-gallate efficiently neutralized the nicotine-induced toxic effects in epithelial cells and fibroblasts. It also dose dependently inhibited the nicotine-induced secretion of chemokine (C-C motif) ligand 5 by epithelial cells. The present study suggests that epigallocatechin-3-gallate, the major polyphenol in green tea, may represent a novel preventive/therapeutic agent for smoking-related periodontitis.

Carbofuran administration induces genotoxic effects in epithelial cells across crypt–villus axis in rat intestine

Oral intake of carbofuran (2,3-dihydro-2,2-dimethyl-7-benzofuranyl N-methyl carbamate), as contaminant of water and foods comprises a major source of carbofuran exposure to the digestive system in mammals. Thus in the present study, the genotoxic effects of carbofuran on enterocytes across crypt–villus axis was investigated in rats using micronucleus test and comet assay. Carbofuran was administered to rats, daily in the dose of 4.0 mg/kg body weight for 7 days or 2.8 mg/kg body weight for 30 days. Micronucleus test revealed an increase ( p < 0.001) in the frequency of micro-nucleated enterocytes in villus tip (VT), mid villus (MV) and crypt base (CB) regions in intestine of rats exposed to carbofuran for 7 or 30 days compared to the respective controls. Comet assay revealed an increase ( p < 0.001) in tail moment, tail length, % DNA and mean intensities of enterocytes all across the villus height in pesticide treated rats. Maximum DNA damage was observed in CB cells upon carbofuran treatment. The observed DNA damage was ∼6–9 times in carbofuran fed rats for 30 days in contrast to those given pesticide for 7 days (∼4–5 times). These findings suggest that carbofuran may have genotoxic effects in the rat intestine.