Cell Inflammatory Mediators Research Articles

Organotypic model of the gingiva for studying bacterial and viral pathogens implicated in periodontitis

ABSTRACT Background Three-dimensional (3D) tissue models bridge the gap between conventional two-dimensional cell cultures and animal models. The aim of this study was to develop an organotypic 3D gingival (OTG) model to provide a tool to investigate bacterial and viral pathogens in periodontitis. Methods The OTG model composed of gingival fibroblasts (GFs) and telomerase-immortalized gingival keratinocytes (TIGKs) was constructed and applied to study infections by Porphyromonas gingivalis and herpes simplex virus 1 (HSV-1). Immunohistochemical staining, confocal microscopy, qPCR, titration techniques, and colony-forming unit counts were applied to interrogate epithelial markers expression, monitor P. gingivalis and HSV-1 presence, and evaluate the immune response along with the efficiency of antimicrobial drugs. Results The OTG model resembled the morphology of the human gingiva. During infection, both pathogens penetrated deep into the tissue and persisted for a few days with P. gingivalis also forming a biofilm on the cell surface. The infection triggered the expression of inflammatory mediators in cells and both pathogens were efficiently eliminated by specific antimicrobials. Conclusions Presented OTG model constitutes a simple and convenient tool to study the interaction between bacterial and viral pathogens within the gingival tissue, including penetration, persistence and biofilm formation. It is also suitable to examine the efficiency of antimicrobial drugs.

Bronchoalveolar lavage fluid cytology and airway hyper-reactivity in clinically normal horses.

To characterise the relationship between bronchoalveolar lavage fluid (BALF) cytology and pulmonary function testing with histamine bronchoprovocation (HBP) methods in a population of clinically normal horses. Cross-sectional study METHODS: Clinically normal adult horses (n = 33) underwent pulmonary function testing and HBP with a commercial flowmetric plethysmography system. BAL was performed 1-5 days later. Statistical analysis was used to assess associations between BALF cell concentration, relative inflammatory cell percentages and categorisation, and airway hyper-reactivity (AHR). AHR (PC35 ≤ 8 mg/mL) was demonstrated in 17 (52%) of the horses. Using current definitions, BALF cytology was consistent with inflammatory airway disease in 14 (42%) of the horses and 7 of those demonstrated either mastocytic and/or eosinophilic responses. There was no correlation between total inflammatory cell counts or relative percentage and AHR. No statistical association was found between BALF inflammatory cell categories and AHR. A direct association between cytological evidence of airway inflammation and AHR was not identified in this population of clinically normal horses. Determining the presence and measuring inflammatory cell mediators in BALF may more accurately reflect AHR. In addition, normal values for cell proportions in BALF may vary between different populations of horses and more appropriate regional reference ranges should be established.

Expression of T helper cell-associated inflammatory mediator mRNAs in cells of bronchoalveolar lavage fluid samples and oxygen concentration in arterial blood samples from healthy horses exposed to hyperbaric oxygen.

OBJECTIVE To evaluate the mRNA expression of T helper (Th)1, Th2, and Th17 cell-associated inflammatory mediators in cells of bronchoalveolar lavage fluid samples collected from healthy horses exposed to hyperbaric oxygen (HBO) and to monitor blood oxygen concentration during and following HBO therapy. ANIMALS 8 healthy horses. PROCEDURES In a randomized controlled crossover design study, each horse was exposed (beginning day 1) to 100% oxygen at a maximum of 3 atmospheres absolute (304 kPa) daily for 10 days or ambient air at atmospheric pressure in the HBO chamber for an equivalent amount of time (control). Bronchoalveolar lavage fluid samples were collected on days 0 and 10. After validation of candidate reference genes, relative mRNA expressions of various innate inflammatory, Th1 cell-derived, Th2 cell-derived (including eotaxin-2), Th17 cell-derived, and regulatory cytokines were measured by quantitative PCR assays. For 3 horses, arterial blood samples were collected for blood gas analysis during a separate HBO session. RESULTS The optimal combination of reference genes was glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine ribosyltransferase, and ribosomal protein L32. Compared with day 0 findings, expression of eotaxin-2 mRNA was significantly lower (0.12-fold reduction) and the percentage of neutrophils in bronchoalveolar lavage fluid samples was significantly lower on day 10 when horses received HBO therapy. Values of Pao2 rapidly increased (> 800 mm Hg) but immediately decreased to pretreatment values when HBO sessions ended. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that HBO therapy does not increase mRNA expression of inflammatory cytokines, but reduces eotaxin-2 mRNA transcription. The Pao2 increase was transient with no cumulative effects of HBO.

Kinetics of chemotaxis, cytokine, and chemokine release of NR8383 macrophages after exposure to inflammatory and inert granular insoluble particles

Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators.NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR.Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription.In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.

Characterization of 2‐Chlorofatty Acid Signaling Pathways

Monocytes and macrophages are key inflammatory cell mediators in the response to infection and tissue injury. Monocytes and macrophages produce a respiratory burst, resulting in the release of reactive oxygen species. Upon activation of these leukocytes, myeloperoxidase (MPO) is released. MPO catalyzes the conversion of hydrogen peroxide into hypochlorous acid (HOCl). HOCl then is able to target the vinyl ether bond of plasmalogens, an abundant class of phospholipids. This results in the production of a novel class of chlorinated lipids, including 2‐chlorofatty aldehyde (2‐ClFALD) and its oxidation product, 2‐chlorofatty acid (2‐ClFA). 2‐Chlorofatty acids have been shown to accumulate in activated human neutrophils and monocytes, as well as the plasma of rats subjected to lipopolysaccharide treatment or the cecal ligation and puncture sepsis model. The present study is designed to identify signaling mechanisms mediated by 2‐ClFA. Initial studies used dynamic mass redistribution (DMR) assays, which is a label‐free functional cell‐based technology that measures whole cell response to ligands. DMR analyses demonstrated that the 2‐ClFA, 2‐chloropalmitic acid, selectively stimulates THP‐1 cells and not HEK293 cells. Furthermore, the dose‐response for 2‐chloropalmitic acid was striking compared to the lack of response to equimolar concentrations of the non‐chlorinated fatty acid, palmitic acid. CCAAT/enhancer‐binding protein homologous protein (CHOP) mRNA increases selectively in response to 2‐chloropalmitic acid compared to palmitic acid in both RAW 264.7 and THP‐1 cells. CHOP mRNA expression in RAW 264.7 cells is inhibited by Bisindolylmaleimide, SB203580, and U0126 indicating 2‐ClFA elicited CHOP expression is PKC, p38 and MEK1/2 dependent. Thus, THP‐1 and RAW 264.7 cells are ideal cells to use for expression analysis and DMR to further elucidate the mechanisms and receptor by which 2‐ClFA elicits cell responses.Support or Funding InformationNational Institutes of Health Grant GM115553

Neural effects of inflammation, cardiovascular disease, and HIV: Parallel, perpendicular, or progressive?

The pervasive reach of the inflammatory system is evidenced by its involvement in numerous disease states. Cardiovascular disease, marked by high levels of circulating inflammatory mediators, affects an estimated 83.6 million Americans. Similarly, human immunodeficiency virus (HIV) produces a paradoxical state of generalized immune activity despite widespread immunosuppression, and affects 35 million people worldwide. Patients living with HIV (PLWH) suffer from inflammatory conditions, including cardiovascular disease (CVD), at a rate exceeding the general population. In this combined disease state, immune mechanisms that are common to both CVD and HIV may interact to generate a progressive condition that contributes to the exacerbated pathogenesis of the other to the net effect of damage to the brain. In this review, we will outline inflammatory cell mediators that promote cardiovascular risk factors and disease initiation and detail how HIV-related proteins may accelerate this process. Finally, we examine the extent to which these comorbid conditions act as parallel, perpendicular, or progressive sequela of events to generate a neurodegenerative environment, and consider potential strategies that can be implemented to reduce the burden of CVD and inflammation in PLWH.

Role of inflammatory mediators in cells of patients with recurrent miscarriage

Role of inflammatory mediators in cells of patients with recurrent miscarriage

Association of phosphodiesterase 4 polymorphism (rs702553) with blood pressure in the African American Study of Kidney Disease and Hypertension Genomics Study

Hypertension is a risk factor for diseases such as stroke, heart and kidney disease. Phosphodiesterase (PDE) enzymes play a significant role in regulating inflammation and there is some association between increased inflammatory cell mediators and development of hypertension. Previous research has shown the single nucleotide polymorphism (rs702553) on the PDE4D gene to be associated with stroke and carotid atherosclerosis. This research analyzed the association of rs702553 with baseline mean arterial blood pressure (MAP) in a subset of the African American Study of Kidney Disease and Hypertension Cohort. Data analysis identified baseline diuretic use as an interaction term in the association between this polymorphism and MAP. Compared with participants with AA/AT genotypes, those with a TT genotype at rs702553 had significantly lower baseline MAP among study participants on a diuretic (P=0.02). To our knowledge, the influence of rs702553 on final PDE4D gene expression has not yet been studied. Additional clinical and in-vitro studies are needed to better understand the biological mechanisms from gene expression to enzyme translation that affect blood pressure.

Tumour necrosis factor α secretion induces protease activation and acinar cell necrosis in acute experimental pancreatitis in mice

BackgroundAcute pancreatitis has long been considered a disorder of pancreatic self-digestion, in which intracellular activation of digestive proteases induces tissue injury. Chemokines, released from damaged pancreatic cells then attract inflammatory...

Supramolecular polymer micelles self-assembled from α-cyclodextrin and PLLA–PCL based copolymers

Accumulation of macrophages and neutrophil granulocytes in the lung are key events in the inflammatory response to inhaled particles. The present study aims at the time course of chemotaxis in vitro in response to the challenge of various biopersistent particles and its functional relation to the transcription of inflammatory mediators.NR8383 rat alveolar macrophages were challenged with particles of coarse quartz, barium sulfate, and nanosized silica for one, four, and 16 h and with coarse and nanosized titanium dioxide particles (rutile and anatase) for 16 h only. The cell supernatants were used to investigate the chemotaxis of unexposed NR8383 macrophages. The transcription of inflammatory mediators in cells exposed to quartz, silica, and barium sulfate was analyzed by quantitative real-time PCR.Challenge with quartz, silica, and rutile particles induced significant chemotaxis of unexposed NR8383 macrophages. Chemotaxis caused by quartz and silica was accompanied by an elevated transcription of CCL3, CCL4, CXCL1, CXCL3, and TNFα. Quartz exposure showed an earlier onset of both effects compared to the nanosized silica. The strength of this response roughly paralleled the cytotoxic effects. Barium sulfate and anatase did not induce chemotaxis and barium sulfate as well caused no elevated transcription.In conclusion, NR8383 macrophages respond to the challenge with inflammatory particles with the release of chemotactic compounds that act on unexposed macrophages. The kinetics of the response differs between the various particles.

Phosphoinositide 3-Kinase γ Regulates Airway Smooth Muscle Contraction by Modulating Calcium Oscillations

Phosphoinositide 3-kinase gamma (PI3Kgamma) has been implicated in the pathogenesis of asthma, but its mechanism has been considered indirect, through release of inflammatory cell mediators. Because airway smooth muscle (ASM) contractile hyper-responsiveness plays a critical role in asthma, the aim of the present study was to determine whether PI3Kgamma can directly regulate contractility of ASM. Immunohistochemistry staining indicated expression of PI3Kgamma protein in ASM cells of mouse trachea and lung, which was confirmed by Western blot analysis in isolated mouse tracheal ASM cells. PI3Kgamma inhibitor II inhibited acetylcholine (ACh)-stimulated airway contraction of cultured precision-cut mouse lung slices in a dose-dependent manner with 75% inhibition at 10 muM. In contrast, inhibitors of PI3Kalpha, PI3Kbeta, or PI3Kdelta, at concentrations 40-fold higher than their reported IC(50) values for their primary targets, had no effect. It is noteworthy that airways in lung slices pretreated with PI3Kgamma inhibitor II still exhibited an ACh-induced initial contraction, but the sustained contraction was significantly reduced. Furthermore, the PI3Kgamma-selective inhibitor had a small inhibitory effect on the ACh-stimulated initial Ca(2+) transient in ASM cells of mouse lung slices or isolated mouse ASM cells but significantly attenuated the sustained Ca(2+) oscillations that are critical for sustained airway contraction. This report is the first to show that PI3Kgamma directly controls contractility of airways through regulation of Ca(2+) oscillations in ASM cells. Thus, in addition to effects on airway inflammation, PI3Kgamma inhibitors may also exert direct effects on the airway contraction that contribute to pathologic airway hyper-responsiveness.

신화학물질 HS-1580 유도체(HS-1580 HS-1581 HS-1582)의 항염증 효과

HS-1580 series (HS-1580, HS-1581, HS-1582) can produce anti-imflammatory effects were synthesized from the marine algae extraction in 2,3,6-tribromo-4,5-dihydroxy benzyl methyl ether (TDB). Raw 264.7 cells were pre-treated with 1 μg/μl lipopolysaccharide (LPS) and later treated with HS-1580 series. These cells of inflammatory mediators were tested as well. Nitric oxide (NO) is related to autoimmune disease and is produced by inducible NOS (iNOS). When treated with HS-1580 series, the product of NO will reduce in a dose-dependent manner. HS-1580 series significantly inhibit the iNOS protein expression. Cyclooxygenase (COX) involves with the various physiologic events and catalyzes in prostaglandin. HS-1580 series also inhibit the COX-2 protein expression as well as pro-inflammatory cytokines production such as tumor necrosis factor-α (TNF-α) and interluekin-1β (IL-1β). These upcoming results suggest that HS-1580 series have anti-inflammatory effects in Raw 264.7 cells by inhibiting such as iNOS, COX-2, TNF-α and IL-1β as inflammatory mediators.

Effects of Clarithromycin on Inflammatory Cell Mediator Release and Survival

Background: Clarithromycin exhibits anti-inflammatory as well as antimicrobial activity, leading to decreased symptoms of asthma and chronic sinusitis. The mode of anti-inflammatory effects of clarithromycin on inflammatory cells is not well understood. We hypothesized that clarithromycin inhibits inflammatory cell mediator release and survival. Methods: We investigated the effects of this drug on survival and mediator release from mast cells, eosinophils and neutrophils. Results: Human eosinophil and neutrophil respiratory burst was inhibited by up to 54% after 1–2 h pretreatment with 100 µg/ml clarithromycin. Similar doses of erythromycin did not affect respiratory burst responses in these cells. Clarithromycin at doses of up to 100 µg/ml had no effect on granule-derived mediators released from mast cells and neutrophils. However, we found that clarithromycin (100 µg/ml) induced cell death in mast cells and eosinophils after 16–48 h incubation. Conclusion: Clarithromycin inhibited inflammatory cell mediator release and survival, which may enhance its ability to reduce the symptoms of chronic sinusitis and asthma.

Multiple Pulmonary Leiomyomatous Hamartomas Associated With a Bronchogenic Cyst in a Man

Multiple pulmonary leiomyomatous hamartomas (MPLHs) are extremely rare lesions. We present a case of MPLHs associated with a bronchogenic cyst in a symptomless, 46-year-old man. Previously, MLPHs have been reported to occur in men; therefore, not all cases of MPLHs represent metastases from a uterine smooth muscle tumor. Because these lesions represent a tumorlike overgrowth of normal tissue, we think that leiomyomatous hamartoma is an appropriate term to designate this entity. The possible influence of inflammatory cell mediators in the pathogenesis of MPLHs remains speculative.

A CD36-initiated Signaling Cascade Mediates Inflammatory Effects of β-Amyloid

beta-Amyloid accumulation is associated with pathologic changes in the brain in Alzheimer's disease and has recently been identified in plaques of another chronic inflammatory disorder, atherosclerosis. The class B scavenger receptor, CD36, mediates binding of fibrillar beta-amyloid to cells of the monocyte/macrophage lineage, including brain macrophages (microglia). In this study, we demonstrate that in microglia and other tissue macrophages, beta-amyloid initiates a CD36-dependent signaling cascade involving the Src kinase family members, Lyn and Fyn, and the mitogen-activated protein kinase, p44/42. Interruption of this signaling cascade, through targeted disruption of Src kinases downstream of CD36, inhibits macrophage inflammatory responses to beta-amyloid, including reactive oxygen and chemokine production, and results in decreased recruitment of microglia to sites of amyloid deposition in vivo. The finding that engagement of CD36 by beta-amyloid initiates a Src kinase-dependent production of inflammatory mediators in cells of the macrophage lineage reveals a novel receptor-mediated pro-inflammatory signaling pathway of potential therapeutic importance.

Adenovirus infection induces microglial activation: involvement of mitogen‐activated protein kinase pathways

Non‐replicating adenovirus (Ad) vectors represent effective tools for long‐term gene expression in the CNS, but they also elicit inflammation. The cellular and molecular mechanisms of such a response are not well‐understood. We show here that infection with Ad causes an activation of microglial cells, the key cells involved in inflammatory and immune‐regulatory functions in the brain. Exposure of cultured rat brain microglia to Ad resulted in an induced production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and the pro‐inflammatory cytokine, TNFα. The roles of signal transduction pathways believed to be involved in microglial activation, in particular, mitogen‐activated protein kinases (MAPKs) and nuclear factor κB (NFκB) were explored by determining their activation in response to Ad infection and by testing the effects of specific pharmacological inhibitors. It was found that Ad strongly activates extracellular signal‐regulated kinase (ERK) and to a lesser extent, p38 MAPK but not NFκB. The kinase inhibitor i.e. PD98059, specific for the ERK pathway, potently inhibits and, in combination with the p38 MAPK inhibitor, SB203580, almost completely abolishes Ad‐induced expression of iNOS and TNFα. The results suggest that Ad uses cellular signal transduction machinery, in particular, the ERK pathway to elicit microglial activation and that increased production by these cells of inflammatory mediators may primarily contribute to CNS inflammatory responses commonly seen in models of gene therapy using Ad vectors.Acknowledgements: Supported by NINDS grant # NS41035‐01.

Adenovirus infection induces microglial activation: involvement of mitogen-activated protein kinase pathways

Non-replicating adenovirus vectors (AdV) represent effective tools for long-term gene expression in the central nervous system (CNS), but they also elicit inflammation. The cellular and molecular mechanisms of such a response are not understood. In the present study, we show that infection with AdV causes activation of microglial cells, the key cells involved in inflammatory and immune-regulatory functions in the brain. Exposure of cultured rat brain microglia to AdV resulted in an induced production of nitric oxide (NO) and the expression of inducible nitric oxide synthase (iNOS) and the pro-inflammatory cytokine, TNFα. The roles of signal transduction pathways believed to be involved in microglial activation in particular, mitogen-activated protein kinases (MAPKs) and nuclear factor κB (NFκB) were explored by determining their activation in response to AdV infection and by testing the effects of specific pharmacological inhibitors. It was found that AdV strongly activates extracellular signal-regulated kinase (ERK) and to a lesser extent, p38 MAPK but not NFκB. Addition of the kinase inhibitor, i.e. PD98059 (specific for the ERK pathway), inhibits and, in combination with the p38 MAPK inhibitor, SB203580, drastically suppresses AdV-induced expression of iNOS and TNFα. The results suggest that AdV uses cellular signal transduction machinery, in particular the MAPK pathways, to elicit microglial activation and that increased production by these cells of inflammatory mediators may primarily contribute to CNS inflammatory responses commonly seen in models of gene therapy using AdV vectors.

Association between decreased pulmonary endothelial cell thrombomodulin and local fibrin deposition in pneumonia.

Thrombomodulin (TM) plays an important role in anticoagulation by forming a complex with thrombin, which subsequently activates protein C. TM is inactivated and downregulated by inflammatory cell mediators. This study examined whether bronchopneumonia is associated with changes in TM immunoreactivity, and whether a decrease in TM is accompanied by evidence of hypercoagulability, i.e. local deposition of fibrin. Double antibody staining for TM and fibrin was performed on lung tissue sections from patients who had died of pneumonia and from patients who had died rapidly, secondary to trauma. Inflammatory changes were assessed histologically and immunohistochemically using antibodies against interleukin-1alpha, tumor necrosis factor-alpha, and myeloperoxidase. Areas with bronchopneumonia exhibited markedly decreased endothelial TM staining of alveolar walls and small vessels. These changes were associated with prominent fibrin immunoreactivity. Some areas exhibited mild to moderate inflammation with little fibrin deposition and variable amounts of TM in adjacent vessels. This study is the first to relate changes of TM immunoreactivity levels to fibrin deposition in a human disease process. These data may have implications for pulmonary pathophysiology in patients with bronchopneumonia.

Proinflammatory potential of the airway epithelium in bronchial asthma.

Histopathological studies of asthmatic airways removed postmortem or by bronchial biopsy show marked inflammatory changes, notably epithelial cell disruption and damage, and the presence of large numbers of eosinophils. The epithelial damage is seen in mild, asymptomatic asthmatics as well as in patients who have died in status asthmaticus. Damage to the epithelium may also correlate with bronchial hyperreactivity. The epithelium has been suggested to be a target for inflammatory cell mediators and cytokines. Recently, the airway epithelium has itself been shown to produce and release several proinflammatory mediators and cytokines, and to express adhesion molecules for inflammatory cells. The epithelium, thus, may actively participate in the inflammatory changes in asthma, where it may be a source as well as a target. Drug therapy aimed at preventing inflammatory changes in the epithelium, such as cytokine and adhesion molecule expression, may be an important step forward in halting disease progression in asthma.

Effect of inflammatory cell mediators on M 2 muscarinic receptors in the lungs

Acetylcholine released from vagal nerve endings constricts airways by stimulating M 3 muscarinic receptors on the airway smooth muscle. At the same time, released acetylcholine feeds back onto inhibitory M 2 muscarinic autoreceptors on the nerve endings, limiting further release of acetylcholine. Loss of function of these M 2 receptors increases vagally-mediated bronchoconstriction after viral airway infections, exposure to ozone, or antigen inhalation. Viral infections may decrease M 2 receptor function by inducing inflammation or via direct damage to the receptors as a result of cleavage of sialic acid residues by viral neuraminidase. Inflammation appears to be critical in the loss of M 2 receptor function after ozone exposure. Antigen-induced loss of M 2 receptor function can be reversed acutely by administering the poly-anionic substances heparin or poly-l-glutamate, possibly by binding and neutralizing positively charged eosinophil proteins. Such positively charged eosinophil proteins, particularly major basic protein, may be acting as endogenous inhibitors at the M 2 receptors, as can be demonstrated in in vitro ligand binding studies.