Journal articles: 'Cytomix'

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Nelin, Leif D., Hilary A. White, Yi Jin, Jennifer K. Trittmann, Bernadette Chen, and Yusen Liu. "The Src family tyrosine kinases src and yes have differential effects on inflammation-induced apoptosis in human pulmonary microvascular endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 9 (May 1, 2016): L880—L888. http://dx.doi.org/10.1152/ajplung.00306.2015.

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Endothelial cells are essential for normal lung function: they sense and respond to circulating factors and hemodynamic alterations. In inflammatory lung diseases such as acute respiratory distress syndrome, endothelial cell apoptosis is an inciting event in pathogenesis and a prominent pathological feature. Endothelial cell apoptosis is mediated by circulating inflammatory factors, which bind to receptors on the cell surface, activating signal transduction pathways, leading to caspase-3-mediated apoptosis. We hypothesized that yes and src have differential effects on caspase-3 activation in human pulmonary microvascular endothelial cells (hPMVEC) due to differential downstream signaling effects. To test this hypothesis, hPMVEC were treated with siRNA against src (siRNAsrc), siRNA against yes (siRNAyes), or their respective scramble controls. After recovery, the hPMVEC were treated with cytomix (LPS, IL-1β, TNF-α, and IFN-γ). Treatment with cytomix induced activation of the extracellular signal-regulated kinase (ERK) pathway and caspase-3-mediated apoptosis. Treatment with siRNAsrc blunted cytomix-induced ERK activation and enhanced cleaved caspase-3 levels, while treatment with siRNAyes enhanced cytomix-induced ERK activation and attenuated levels of cleaved caspase-3. Inhibition of the ERK pathway using U0126 enhanced cytomix-induced caspase-3 activity. Treatment of hPMVEC with cytomix induced Akt activation, which was inhibited by siRNAsrc. Inhibition of the phosphatidylinositol 3-kinase/Akt pathway using LY294002 prevented cytomix-induced ERK activation and augmented cytomix-induced caspase-3 cleavage. Together, our data demonstrate that, in hPMVEC, yes activation blunts the ERK cascade in response to cytomix, resulting in greater apoptosis, while cytomix-induced src activation induces the phosphatidylinositol 3-kinase pathway, which leads to activation of Akt and ERK and attenuation of apoptosis.

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Rodriguez, Luis A., Arezoo Mohammadipoor, Lucero Alvarado, Robin M. Kamucheka, Amber M. Asher, Leopoldo C. Cancio, and Ben Antebi. "Preconditioning in an Inflammatory Milieu Augments the Immunotherapeutic Function of Mesenchymal Stromal Cells." Cells 8, no. 5 (May 15, 2019): 462. http://dx.doi.org/10.3390/cells8050462.

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Multipotent mesenchymal stromal cells (MSCs) have emerged as potent therapeutic agents for multiple indications. However, recent evidence indicates that MSC function is compromised in the physiological post-injury milieu. In this study, bone marrow (BM)- and adipose-derived (AD)-MSCs were preconditioned in hypoxia with or without inflammatory mediators to potentiate their immunotherapeutic function in preparation for in vivo delivery. Human MSCs were cultured for 48 h in either normoxia (21% O2) or hypoxia (2% O2) with or without the addition of Cytomix, thus creating 4 groups: (1) normoxia (21%); (2) Cytomix-normoxia (+21%); (3) hypoxia (2%); and (4) Cytomix-hypoxia (+2%). The 4 MSC groups were subjected to comprehensive evaluation of their characteristics and function. Preconditioning did not alter common MSC surface markers; nonetheless, Cytomix treatment triggered an increase in tissue factor (TF) expression. Moreover, the BM-MSCs and AD-MSCs from the +2% group were not able to differentiate to chondrocytes and osteoblasts, respectively. Following Cytomix preconditioning, the metabolism of MSCs was significantly increased while viability was decreased in AD-MSCs, but not in BM-MSCs. MSCs from both tissues showed a significant upregulation of key anti-inflammatory genes, increased secretion of IL-1 receptor antagonist (RA), and enhanced suppression of T-cell proliferation following the Cytomix treatment. Similarly, following a lipopolysaccharide challenge, the Cytomix-treated MSCs suppressed TNF-α secretion, while promoting the production of IL-10 and IL-1RA. These preconditioning approaches facilitate the production of MSCs with robust anti-inflammatory properties. AD-MSCs preconditioned with Cytomix under normoxia appear to be the most promising therapeutic candidates; however, safety concerns, such as thrombogenic disposition of cells due to TF expression, should be carefully considered prior to clinical translation.

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Scharte, Marion, Xiaonan Han, Daniel J. Bertges, Mitchell P. Fink, and Russell L. Delude. "Cytokines induce HIF-1 DNA binding and the expression of HIF-1-dependent genes in cultured rat enterocytes." American Journal of Physiology-Gastrointestinal and Liver Physiology 284, no. 3 (March 1, 2003): G373—G384. http://dx.doi.org/10.1152/ajpgi.00076.2002.

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Cellular adaptation to hypoxia depends, in part, on the transcription factor hypoxia-inducible factor-1 (HIF-1). Normoxic cells exposed to an inflammatory milieu often manifest phenotypic changes, such as increased glycolysis, that are reminiscent of those observed in hypoxic cells. Accordingly, we investigated the effects of cytomix, a mixture containing IFN-γ, TNF, and IL-1β on the expression of HIF-1-dependent proteins under normoxic and hypoxic conditions. Incubation of intestine-derived epithelial cells (IEC-6) under 1% O2increased HIF-1 DNA binding and expression of aldolase A, enolase-1, and VEGF mRNA. Incubation of normoxic cells with cytomix for 48 h also markedly increased HIF-1 DNA binding and expression of mRNAs for these proteins. Incubation of hypoxic cells with cytomix did not inhibit HIF-1 DNA binding or upregulation of HIF-1-dependent genes in response to hypoxia. Neither cytomix nor hypoxia increased steady-state levels of HIF-1α mRNA. Incubation of IEC-6 cells with cytomix induced nitric oxide (NO·) biosynthesis, which was blocked if the cultures containedl- NG-(1-iminoethyl)lysine hydrochloride (l-NIL). Treatment with l-NIL, however, failed to significantly alter aldolase A, enolase-1, and VEGF mRNA levels in normoxic cytomix-treated cells. Proinflammatory cytokines activate the HIF-1 pathway and increase expression of glycolytic genes in nontransformed rat intestinal epithelial cells, largely through an NO·-independent mechanism.

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Zhu, Y. K., X. D. Liu, C. M. Sköld, T. Umino, H. J. Wang, J. R. Spurzem, T. Kohyama, R. F. Ertl, and S. I. Rennard. "Synergistic neutrophil elastase-cytokine interaction degrades collagen in three-dimensional culture." American Journal of Physiology-Lung Cellular and Molecular Physiology 281, no. 4 (October 1, 2001): L868—L878. http://dx.doi.org/10.1152/ajplung.2001.281.4.l868.

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Proteolytic degradation of extracellular matrix is thought to play an important role in many lung disorders. In the current study, human lung fibroblasts were cast into type I collagen gels and floated in medium containing elastase, cytomix (combination of tumor necrosis factor-α, interleukin-1β, and interferon-γ), or both. After 5 days, gel collagen content was determined by measuring hydroxyproline. Elastase alone did not result in collagen degradation, but in the presence of fibroblasts, elastase reduced hydroxyproline content to 75.2% ( P < 0.01), whereas cytomix alone resulted in reduction of hydroxyproline content to 93% ( P < 0.05). The combination of elastase and cytomix reduced hydroxyproline content to 5.2% ( P < 0.01). α1-Proteinase inhibitor blocked this synergy. Gelatin zymography and Western blot revealed that matrix metalloproteinase (MMP)-1, -3, and -9 were induced by cytomix and activated in the presence of elastase. Tissue inhibitor of metalloproteinase (TIMP)-1 and -2 were also induced by cytomix but were cleaved by elastase. We conclude that a synergistic interaction between cytomix and elastase, mediated through cytokine induction of MMP production and elastase-induced activation of latent MMPs and degradation of TIMPs, can result in a dramatic augmentation of collagen degradation. These findings support the notion that interaction among inflammatory mediators secreted by mononuclear cells and neutrophils can induce tissue cells to degrade extracellular matrix. Such a mechanism may contribute to the protease-anti-protease imbalance in emphysema.

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Thompson, David C., Stephanie E. Porter, Alison K. Bauer, Kumuda C. Das, Brandon Ou, Lori Dwyer-Nield, Carl W. White, and Alvin M. Malkinson. "Cytokine-induced nitric oxide formation in normal but not in neoplastic murine lung epithelial cell lines." American Journal of Physiology-Lung Cellular and Molecular Physiology 274, no. 6 (June 1, 1998): L922—L932. http://dx.doi.org/10.1152/ajplung.1998.274.6.l922.

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Cytomix, a mixture of interferon-γ, tumor necrosis factor-α, and interleukin-1β, induces nitric oxide (NO) production in lung epithelial cell lines. It is not known whether neoplastic transformation alters a cell’s ability to form NO in response to cytokines. The present study investigated NO formation in two murine lines of immortalized “normal” (nontumorigenic) lung epithelial cells of alveolar type II origin, E10 and C10, and their sibling spontaneous transformants, E9 and A5. Nontumorigenic cells elaborated much more NO after cytomix exposure than did their tumorigenic counterparts. NO production was prevented by inhibiting protein synthesis and NO synthase and attenuated by dexamethasone. Northern and Western blot analyses of inducible NO synthase (iNOS) demonstrated cytomix-induced induction of iNOS only in nontumorigenic cells. The deficiency in NO production in tumorigenic cells was not associated with reduced iNOS mRNA stability or with differences in cytomix-induced nuclear factor-κB activation. Although cytomix caused a greater production of NO in E10 cells than in E9 cells, the same treatment induced equivalent proliferation in both cell lines. These results indicate a specific deficiency in cytokine-induced NO synthesis in transformed murine lung epithelial cells relative to their normal progenitor cells and provide a model for investigating iNOS regulation.

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Liu, Shiguang, Donna B. Stolz, Penny L. Sappington, Carlos A. Macias, Meaghan E. Killeen, Jyrki J. Tenhunen, Russell L. Delude, and Mitchell P. Fink. "HMGB1 is secreted by immunostimulated enterocytes and contributes to cytomix-induced hyperpermeability of Caco-2 monolayers." American Journal of Physiology-Cell Physiology 290, no. 4 (April 2006): C990—C999. http://dx.doi.org/10.1152/ajpcell.00308.2005.

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High-mobility group box 1 (HMGB1), a cytokine-like proinflammatory protein, is secreted by activated macrophages and released by necrotic cells. We hypothesized that immunostimulated enterocytes might be another source for this mediator. Accordingly, Caco-2 cells or primary mouse intestinal epithelial cells (IECs) were incubated with “cytomix” (a mixture of TNF, IL-1β, and IFN-γ) for various periods. HMGB1 in cell culture supernatants was detected by Western blot analysis and visualized in Caco-2 cells with the use of fluorescence confocal and immunotransmission electron microscopy. Caco-2 cells growing on filters in diffusion chambers were stimulated with cytomix for 48 h in the absence or presence of anti-HMGB1 antibody, and permeability to fluorescein isothiocyanate-dextran (average molecular mass, 4 kDa; FD4) was assessed. Cytomix-stimulated Caco-2 cells secreted HMGB1 into the apical but not the basolateral compartments of diffusion chambers. Although undetectable at 6 and 12 h after the start of incubation with cytomix, HMGB1 was present in supernatants after 24 h of incubation. HMGB1 secretion by Caco-2 monolayers also was induced when the cells were exposed to FSL-1, a Toll-like receptor (Tlr)-2 agonist, or flagellin, a Tlr5 agonist, but not lipopolysaccharide, a Tlr4 agonist. Cytomix also induced HMGB1 secretion by primary IECs. Cytoplasmic HMGB1 is localized within vesicles in Caco-2 cells and is secreted, at least in part, associated with exosomes. Incubating Caco-2 cells with cytomix increased FD4 permeation, but this effect was significantly decreased in the presence of anti-HMGB1 antibody. Collectively, these data support the view that HMGB1 is secreted by immunostimulated enterocytes. This process may exacerbate inflammation-induced epithelial hyperpermeability via an autocrine feedback loop.

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Subasinghe, Wasanthi, Ismail Syed, and Anjaneyulu Kowluru. "Phagocyte-like NADPH oxidase promotes cytokine-induced mitochondrial dysfunction in pancreatic β-cells: evidence for regulation by Rac1." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 300, no. 1 (January 2011): R12—R20. http://dx.doi.org/10.1152/ajpregu.00421.2010.

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Reactive oxygen species (ROS) are important mediators of cellular signal transduction cascades such as proliferation, migration, and apoptosis. Chronic exposure of isolated β-cells to proinflammatory cytokines elevates intracellular oxidative stress leading to the demise of pancreatic β-cells culminating in the onset of diabetes. Although the mitochondrial electron transport chain is felt to be the primary source of ROS, several lines of recent evidence suggest that phagocyte-like NADPH oxidase plays a central role in cytokine-mediated ROS generation and apoptosis of β-cells. However, the precise mechanisms underlying the regulation of NADPH oxidase remain unknown. To address this, insulin-secreting INS 832/13 cells were treated with cytomix (IL-1β, IFN-γ, and TNF-α; 10 ng/ml each) for different time intervals (0–24 h). A significant, time-dependent increase in NADPH oxidase activation/intracellular ROS production, p47phox subunit, but not p67phox subunit, expression of the phagocyte-like NADPH oxidase were demonstrable under these conditions. Furthermore, siRNA-p47phox transfection or exposure of INS 832/13 cells to apocynin, a selective inhibitor of NADPH oxidase, markedly attenuated cytomix-induced ROS generation in these cells. Cytomix-mediated mitochondrial dysfunction in INS 832/13 cells was evident by a significant loss of mitochondrial membrane potential (MMP) and upregulated caspase 3 activity. Cytomix treatment also caused a transient (within 15 min) activation of Rac1, a component of the NADPH oxidase holoenzyme. Furthermore, GGTI-2147 and NSC23766, known Rac1 inhibitors, not only attenuated the cytomix-induced Rac1 activation but also significantly prevented loss of MMP (NSC23766 > GGTI-2147). However, NSC23766 had no effect on cytomix-induced NO generation or caspase 3 activation, suggesting additional regulatory mechanisms might underlie these signaling steps. Together, these findings suggested that Rac1-mediated regulation of phagocyte-like NADPH oxidase contributes to cytokine-mediated mitochondrial dysfunction in the β-cell.

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Burke-Gaffney, A., and P. G. Hellewell. "Endogenous nitric oxide limits cytokine-induced damage of murine lung epithelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 272, no. 4 (April 1, 1997): L707—L713. http://dx.doi.org/10.1152/ajplung.1997.272.4.l707.

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This study investigated whether endogenous nitric oxide (NO) limits cytokine-induced damage to the murine lung epithelial cell line LA-4. NO production was assessed as nitrite using the Griess reaction, and cell damage was assessed using ethidium homodimer-1. Cytotoxicity was first detected after a 24-h incubation with a combination of tumor necrosis factor-alpha, interleukin-1beta, and interferon-gamma (cytomix). Nitrite production increased to 78.0 +/- 0.5 nmol/10(6) cells at 24 h. Coincubation of LA-4 with cytomix and NO synthase inhibitors, aminoguanidine (3-1,000 microM) and N(G)-monomethyl-L-arginine (10-1,000 microM), but not N(G)-monomethyl-D-arginine, or a soluble guanylate cyclase inhibitor, 1H-[1,2,4] oxadiazole [4,3-a] quinoxalin-1-one, reduced cytomix-induced nitrite production and increased cytotoxicity up to twofold (24 h). Removal of L-arginine from the medium increased damage; reintroduction of 1,000 microM L-arginine, but not D-arginine, reversed this. In aminoguanidine-treated cells, replacement of NO with an NO donor, S-nitrosoglutathione (30 microM), reversed, in part, the cell damage observed in aminoguanidine/cytomix-treated cells. These results suggest that endogenous NO limits cytokine-induced lung epithelial damage.

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Panas, Donna, Fadi H. Khadour, Csaba Szabó, and Richard Schulz. "Proinflammatory cytokines depress cardiac efficiency by a nitric oxide-dependent mechanism." American Journal of Physiology-Heart and Circulatory Physiology 275, no. 3 (September 1, 1998): H1016—H1023. http://dx.doi.org/10.1152/ajpheart.1998.275.3.h1016.

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Proinflammatory cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-γ; Cytomix) depress myocardial contractile work partially by stimulating expression of inducible nitric oxide (NO) synthase (iNOS). Because NO and peroxynitrite inhibit myocardial O2 consumption (MV˙o 2), we examined whether this mechanism contributes to reduced cardiac work. In control isolated working rat hearts, cardiac work was stable for 60 min, followed by a decline from 60 to 120 min, without change in MV˙o 2. Cardiac efficiency (work/MV˙o 2) was therefore reduced from 60 to 120 min. Cytomix shortened the onset (within 20–40 min) and enhanced the depression in cardiac work and efficiency and inhibited MV˙o 2 after 80 min. Mercaptoethylguanidine (MEG), an iNOS inhibitor and peroxynitrite scavenger, or the glucocorticoid dexamethasone (Dex) abolished the effects of Cytomix. iNOS expression was increased 10-fold by Cytomix and abolished by Dex but not MEG. That cytokine-induced depression in cardiac work precedes the reduction in MV˙o 2 suggests, at least in the early response, that NO and/or peroxynitrite may not impair heart function by inhibiting mitochondrial respiration but reduce the heart’s ability to utilize ATP for contractile work.

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Supinski, Gerald S., Alexander P. Alimov, Lin Wang, Xiao-Hong Song, and Leigh A. Callahan. "Calcium-dependent phospholipase A2 modulates infection-induced diaphragm dysfunction." American Journal of Physiology-Lung Cellular and Molecular Physiology 310, no. 10 (May 15, 2016): L975—L984. http://dx.doi.org/10.1152/ajplung.00312.2015.

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Calpain activation contributes to the development of infection-induced diaphragm weakness, but the mechanisms by which infections activate calpain are poorly understood. We postulated that skeletal muscle calcium-dependent phospholipase A2 (cPLA2) is activated by cytokines and has downstream effects that induce calpain activation and muscle weakness. We determined whether cPLA2 activation mediates cytokine-induced calpain activation in isolated skeletal muscle (C2C12) cells and infection-induced diaphragm weakness in mice. C2C12 cells were treated with the following: 1) vehicle; 2) cytomix (TNF-α 20 ng/ml, IL-1β 50 U/ml, IFN-γ 100 U/ml, LPS 10 μg/ml); 3) cytomix + AACOCF3, a cPLA2 inhibitor (10 μM); or 4) AACOCF3 alone. At 24 h, we assessed cell cPLA2 activity, mitochondrial superoxide generation, calpain activity, and calpastatin activity. We also determined if SS31 (10 μg/ml), a mitochondrial superoxide scavenger, reduced cytomix-mediated calpain activation. Finally, we determined if CDIBA (10 μM), a cPLA2 inhibitor, reduced diaphragm dysfunction due to cecal ligation puncture in mice. Cytomix increased C2C12 cell cPLA2 activity ( P < 0.001) and superoxide generation; AACOCF3 and SS31 blocked increases in superoxide generation ( P < 0.001). Cytomix also activated calpain ( P < 0.001) and inactivated calpastatin ( P < 0.01); both AACOCF3 and SS31 prevented these changes. Cecal ligation puncture reduced diaphragm force in mice, and CDIBA prevented this reduction ( P < 0.001). cPLA2 modulates cytokine-induced calpain activation in cells and infection-induced diaphragm weakness in animals. We speculate that therapies that inhibit cPLA2 may prevent diaphragm weakness in infected, critically ill patients.

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Tan, Xiahui, Najwa Khalil, Candice Tesarik, Karunasri Vanapalli, Viki Yaputra, Hatem Alkhouri, Brian G. G. Oliver, Carol L. Armour, and J. Margaret Hughes. "Th1 cytokine-induced syndecan-4 shedding by airway smooth muscle cells is dependent on mitogen-activated protein kinases." American Journal of Physiology-Lung Cellular and Molecular Physiology 302, no. 7 (April 1, 2012): L700—L710. http://dx.doi.org/10.1152/ajplung.00167.2011.

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In asthma, airway smooth muscle (ASM) chemokine secretion can induce mast cell recruitment into the airways. The functions of the mast cell chemoattractant CXCL10, and other chemokines, are regulated by binding to heparan sulphates such as syndecan-4. This study is the first demonstration that airway smooth muscle cells (ASMC) from people with and without asthma express and shed syndecan-4 under basal conditions. Syndecan-4 shedding was enhanced by stimulation for 24 h with the Th1 cytokines interleukin-1β (IL-1β) or tumor necrosis factor-α (TNF-α), but not interferon-γ (IFNγ), nor the Th2 cytokines IL-4 and IL-13. ASMC stimulation with IL-1β, TNF-α, and IFNγ (cytomix) induced the highest level of syndecan-4 shedding. Nonasthmatic and asthmatic ASM cell-associated syndecan-4 protein expression was also increased by TNF-α or cytomix at 4–8 h, with the highest levels detected in cytomix-stimulated asthmatic cells. Cell-associated syndecan-4 levels were decreased by 24 h, whereas shedding remained elevated at 24 h, consistent with newly synthesized syndecan-4 being shed. Inhibition of ASMC matrix metalloproteinase-2 did not prevent syndecan-4 shedding, whereas inhibition of ERK MAPK activation reduced shedding from cytomix-stimulated ASMC. Although ERK inhibition had no effect on syndecan-4 mRNA levels stimulated by cytomix, it did cause an increase in cell-associated syndecan-4 levels, consistent with the shedding being inhibited. In conclusion, ASMC produce and shed syndecan-4 and although this is increased by the Th1 cytokines, the MAPK ERK only regulates shedding. ASMC syndecan-4 production during Th1 inflammatory conditions may regulate chemokine activity and mast cell recruitment to the ASM in asthma.

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Goolaerts, Arnaud, Nadia Pellan-Randrianarison, Jérôme Larghero, Valérie Vanneaux, Yurdagül Uzunhan, Thomas Gille, Nicolas Dard, Carole Planès, Michael A. Matthay, and Christine Clerici. "Conditioned media from mesenchymal stromal cells restore sodium transport and preserve epithelial permeability in an in vitro model of acute alveolar injury." American Journal of Physiology-Lung Cellular and Molecular Physiology 306, no. 11 (June 1, 2014): L975—L985. http://dx.doi.org/10.1152/ajplung.00242.2013.

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Mesenchymal stromal cells (MSCs) or their media (MSC-M) were reported to reverse acute lung injury (ALI)-induced decrease of alveolar fluid clearance. To determine the mechanisms by which MSC-M exert their beneficial effects, an in vitro model of alveolar epithelial injury was created by exposing primary rat alveolar epithelial cells (AECs) to hypoxia (3% O2) plus cytomix, a combination of IL-1β, TNF-α, and IFN-γ. MSC-M were collected from human MSCs exposed for 12 h to either normoxia (MSC-M) or to hypoxia plus cytomix (HCYT-MSC-M). This latter condition was used to model the effect of alveolar inflammation and hypoxia on paracrine secretion of MSCs in the injured lung. Comparison of paracrine soluble factors in MSC media showed that the IL-1 receptor antagonist and prostaglandin E2 were markedly increased while keratinocyte growth factor (KGF) was twofold lower in HCYT-MSC-M compared with MSC-M. In AECs, hypoxia plus cytomix increased protein permeability, reduced amiloride-sensitive short-circuit current (AS- Isc), and also decreased the number of α-epithelial sodium channel (α-ENaC) subunits in the apical membrane. To test the effects of MSC media, MSC-M and HCYT-MSC-M were added for an additional 12 h to AECs exposed to hypoxia plus cytomix. MSC-M and HCYT-MSC-M completely restored epithelial permeability to normal. MSC-M, but not HCYT-MSC-M, significantly prevented the hypoxia plus cytomix-induced decrease of ENaC activity and restored apical α-ENaC channels. Interestingly, KGF-deprived MSC-M were unable to restore amiloride-sensitive sodium transport, indicating a possible role for KGF in the beneficial effect of MSC-M. These results indicate that MSC-M may be a preferable therapeutic option for ALI.

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Johnson, Bruce A., Bruce R. Pitt, and Paul Davies. "Pulmonary arterial smooth muscle cells modulate cytokine- and LPS-induced cytotoxicity in endothelial cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 278, no. 3 (March 1, 2000): L460—L468. http://dx.doi.org/10.1152/ajplung.2000.278.3.l460.

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Cytokines and lipopolysaccharide (LPS) are known to be injurious to vascular endothelial cells (ECs), but the influence of adjacent vascular smooth muscle cells (SMCs) on this injury is unknown. Exposure of cultured rat (RPMECs) or human (HPMECs) pulmonary microvascular ECs on tissue culture plastic to a mixture of cytokines (interleukin-1β, tumor necrosis factor-α, and interferon-γ) and LPS (cytomix) resulted in a significant increase in51Cr release to 35–40%. When unstimulated RPMECs were cocultured with cytomix-pretreated rat pulmonary microvascular SMCs (RPMSMCs) there was an increase in 51Cr release to 8.4%, which was nitric oxide dependent. However, when RPMECs or HPMECs were stimulated in direct contact with their respective SMCs, rather than a further increase in cytomix-induced injury (e.g., >35–40%), 51Cr release decreased to <10%. This cytoprotection was fully reproduced with fixed RPMSMCs, and partially reproduced by plating HPMECs on gelatin. These data show that the direct toxicity of a cytokine and endotoxin mixture on cultured ECs can be reduced by contact with vascular smooth muscle.

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Alrashdan, Yazan A., Hatem Alkhouri, Emily Chen, Daniel J. Lalor, Maree Poniris, Sheridan Henness, Christopher E. Brightling, et al. "Asthmatic airway smooth muscle CXCL10 production: mitogen-activated protein kinase JNK involvement." American Journal of Physiology-Lung Cellular and Molecular Physiology 302, no. 10 (May 15, 2012): L1118—L1127. http://dx.doi.org/10.1152/ajplung.00232.2011.

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CXCL10 (IP10) is involved in mast cell migration to airway smooth muscle (ASM) bundles in asthma. We aimed to investigate the role of cytokine-induced MAPK activation in CXCL10 production by ASM cells from people with and without asthma. Confluent growth-arrested ASM cells were treated with inhibitors of the MAPKs ERK, p38, and JNK and transcription factor NF-κB, or vehicle, and stimulated with IL-1β, TNF-α, or IFN-γ, alone or combined (cytomix). CXCL10 mRNA and protein, JNK, NF-κB p65 phosphorylation, and Iκ-Bα protein degradation were assessed using real-time PCR, ELISA, and immunoblotting, respectively. Cytomix, IL-1β, and TNF-α induced CXCL10 mRNA expression more rapidly in asthmatic than nonasthmatic ASM cells. IL-1β and/or TNF-α combined with IFN-γ synergistically increased asthmatic ASM cell CXCL10 release. Inhibitor effects were similar in asthmatic and nonasthmatic cells, but cytomix-induced release was least affected, with only JNK and NF-κB inhibitors halving it. Notably, JNK phosphorylation was markedly less in asthmatic compared with nonasthmatic cells. However, in both, the JNK inhibitor SP600125 reduced JNK phosphorylation and CXCL10 mRNA levels but did not affect CXCL10 mRNA stability or Iκ-Bα degradation. Together, the JNK and NF-κB inhibitors completely inhibited their CXCL10 release. We concluded that, in asthmatic compared with nonasthmatic ASM cells, JNK activation was reduced and CXCL10 gene expression was more rapid following cytomix stimulation. However, in both, JNK activation did not regulate early events leading to NF-κB activation. Thus JNK and NF-κB provide independent therapeutic targets for limiting CXCL10 production and mast cell migration to the ASM in asthma.

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Comer, Brian S., Blanca Camoretti-Mercado, Paul C. Kogut, Andrew J. Halayko, Julian Solway, and William T. Gerthoffer. "MicroRNA-146a and microRNA-146b expression and anti-inflammatory function in human airway smooth muscle." American Journal of Physiology-Lung Cellular and Molecular Physiology 307, no. 9 (November 1, 2014): L727—L734. http://dx.doi.org/10.1152/ajplung.00174.2014.

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MicroRNA (miR)-146a and miR-146b are negative regulators of inflammatory gene expression in lung fibroblasts, epithelial cells, monocytes, and endothelial cells. The abundance of cyclooxygenase-2 (COX-2) and IL-1β is negatively regulated by the miR-146 family, suggesting miR-146a and/or miR-146b might modulate inflammatory mediator expression in airway smooth muscle thereby contributing to pathogenesis of asthma. To test this idea we compared miR-146a and miR-146b expression in human airway smooth muscle cells (hASMCs) from nonasthmatic and asthmatic subjects treated with cytomix (IL-1β, TNF-α, and IFNγ) and examined the miRNAs' effects on COX-2 and IL-1β expression. We found that cytomix treatment elevated miR-146a and miR-146b abundance. Induction with cytomix was greater than induction with individual cytokines, and asthmatic cells exhibited higher levels of miR-146a expression following cytomix treatment than nonasthmatic cells. Transfection of miR-146a or miR-146b mimics reduced COX-2 and IL-1β expression. A miR-146a inhibitor increased COX-2 and IL-1β expression, but a miR-146b inhibitor was ineffective. Repression of COX-2 and IL-1β expression by miR-146a correlated with reduced abundance of the RNA-binding protein human antigen R. These results demonstrate that miR-146a and miR-146b expression is inducible in hASMCs by proinflammatory cytokines and that miR-146a expression is greater in asthmatic cells. Both miR-146a and miR-146b can negatively regulate COX-2 and IL-1β expression at pharmacological levels, but loss-of-function studies showed that only miR-146a is an endogenous negative regulator in hASMCs. The results suggest miR-146 mimics may be an attractive candidate for further preclinical studies as an anti-inflammatory treatment of asthma.

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Su, W. Y., B. J. Day, B. H. Kang, J. D. Crapo, Y. C. Huang, and L. Y. Chang. "Lung epithelial cell-released nitric oxide protects against PMN-mediated cell injury." American Journal of Physiology-Lung Cellular and Molecular Physiology 271, no. 4 (October 1, 1996): L581—L586. http://dx.doi.org/10.1152/ajplung.1996.271.4.l581.

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A calcium-independent type II nitric oxide (NO) synthase has been localized in lung epithelial cells; however, the function of NO. released by epithelial cells is unclear. We hypothesized that epithelial-derived NO may affect the interactions between polymorphonuclear neutrophils (PMN) and the alveolar epithelium and studied PMN adhesion and cytotoxicity to lung epithelial cells. A dose- and time-dependent production of NO. by L2 cells can be induced by a mixture of inflammatory mediators (cytomix) containing lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. Increased NO. production by L2 cells was associated with decreased 51Cr release by the epithelial cells after they were incubated with activated PMN. Addition of NG-monomethyl-L-arginine or oxyhemoglobin reversed the protective effects of cytomix, suggesting that increased NO. production by L2 cells was responsible for the decreased 51Cr release. However, PMN adhesion and intercellular adhesion molecule-1, a major adhesion molecule involved in PMN adhesion to epithelium, were increased by cytomix. We conclude that NO. released by lung epithelial cells was involved in protecting epithelial cells from PMN-mediated cytotoxicity. NO.-mediated protection of lung epithelial cells occurred in spite of PMN adhesion being increased, suggesting that reduced adhesion is not required for NO.-mediated inhibition of PMN cytotoxicity.

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Khan, Abrar U., Russell L. Delude, Yong Y. Han, Penny L. Sappington, Xianonan Han, Joseph A. Carcillo, and Mitchell P. Fink. "Liposomal NAD+ prevents diminished O2consumption by immunostimulated Caco-2 cells." American Journal of Physiology-Lung Cellular and Molecular Physiology 282, no. 5 (May 1, 2002): L1082—L1091. http://dx.doi.org/10.1152/ajplung.00358.2001.

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Accumulating data support the view that sepsis is associated with an acquired intrinsic derangement in the ability of cells to consume O2, a phenomenon that has been termed “cytopathic hypoxia.” We sought to use an in vitro “reductionist” model system using cultured cells stimulated with proinflammatory cytokines to test the hypothesis that cytopathic hypoxia is mediated, at least in part, by depletion of intracellular levels of NAD+/NADH secondary to activation of the nuclear enzyme poly(ADP-ribose) polymerase (PARP). We measured O2 consumption by Caco-2 enterocytes growing on microcarrier beads after cells were incubated for 24 h under control conditions or with cytomix, a mixture of tumor necrosis factor-α, interleukin-1β, and interferon-γ. Immunostimulated cells consumed O2 at about one-half the rate of control cells, but this effect was largely prevented if any one of the following pharmacological agents was present during the period of incubation with cytomix: 4,5-dihydroxy-1,3-benzene disulfonic acid, a superoxide radical anion scavenger; 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide, a nitric oxide scavenger; 5,10,15,20- tetrakis-[4-sulfonatophenyl]-porphyrinato-iron[III], a peroxynitrite (ONOO−) decomposition catalyst; urate, an ONOO− scavenger; 3-aminobenzamide, a PARP inhibitor; or N-(6-oxo-5,6-dihydrophenanthridin-2-yl)- N,N-dimethylacetamide HCl, a chemically dissimilar and more potent PARP inhibitor. The decrease in O2 uptake induced by cytomix was associated with decreased cellular levels of NAD+/NADH. The decrease in cellular NAD+/NADH content and the decrease in O2 uptake induced by cytomix were completely abrogated if liposome-encapsulated NAD+ was added to the cultures during immunostimulation. Empty liposomes also increased O2 uptake by immunostimulated Caco-2 cells, but much less effectively than liposomes containing NAD+. These data are consistent with the view that enterocytes exposed to proinflammatory cytokines consume less O2 due to NAD+/NADH depletion secondary to activation of PARP by ONOO− or other oxidants.

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Miki, Keita, Abhai Kumar, Runkuan Yang, Meaghan E. Killeen, and Russell L. Delude. "Extracellular activation of arginase-1 decreases enterocyte inducible nitric oxide synthase activity during systemic inflammation." American Journal of Physiology-Gastrointestinal and Liver Physiology 297, no. 4 (October 2009): G840—G848. http://dx.doi.org/10.1152/ajpgi.90716.2008.

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Liver dysfunction secondary to severe inflammation is associated with the release of enzymes normally sequestered within hepatocytes. The purpose of these studies was to test the hypothesis that these enzymes are released, at least in part, to modulate potentially deleterious inflammatory processes in distant tissues like the gut. Human Caco-2BBe enterocyte-like cells were exposed to cytomix (IFN-γ, TNF-α, and IL-1β) in the absence or presence of human liver cytosol (LC). Nitric oxide (NO•) and inducible nitric oxide synthase (iNOS) protein production were measured by the Griess assay and Western analysis, respectively. Cytomix induced the expression of iNOS and release of NO•. LC protein (400 μg/ml) added to the basal compartment but not apical compartment completely blocked the release of NO• but only slightly decreased the magnitude of iNOS protein induction. Ultrafiltration and ultracentrifugation studies demonstrated that microsome-associated arginase-1 activity was the iNOS-suppressing activity in LC. Liver arginase required activation by a <10-kDa factor that was present in supernatants of cytomix-stimulated cells. The selective iNOS inhibitor l- N6-(1-iminoethyl)-lysine·2HCl prevented production of this factor. The biotin switch assay detected increased S-nitrosylation of arginase-1 after incubation with supernatants from immunostimulated Caco-2 cells. Serum from endotoxemic mice contained significantly greater arginase activity compared with serum from control mice. Furthermore, the ratio of mucosal monomeric to dimeric iNOS increased in endotoxemic mice compared with controls. Thus reciprocal activation of arginase-1 and modulation of mucosal iNOS activity may be protective because it would be expected to decrease NO•-dependent intestinal barrier dysfunction on that basis.

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Supinski, Gerald S., Alexander P. Alimov, Lin Wang, Xiao-Hong Song, and Leigh A. Callahan. "Neutral sphingomyelinase 2 is required for cytokine-induced skeletal muscle calpain activation." American Journal of Physiology-Lung Cellular and Molecular Physiology 309, no. 6 (September 15, 2015): L614—L624. http://dx.doi.org/10.1152/ajplung.00141.2015.

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Calpain contributes to infection-induced diaphragm dysfunction but the upstream mechanism(s) responsible for calpain activation are poorly understood. It is known, however, that cytokines activate neutral sphingomyelinase (nSMase) and nSMase has downstream effects with the potential to increase calpain activity. We tested the hypothesis that infection-induced skeletal muscle calpain activation is a consequence of nSMase activation. We administered cytomix (20 ng/ml TNF-α, 50 U/ml IL-1β, 100 U/ml IFN-γ, 10 μg/ml LPS) to C2C12 muscle cells to simulate the effects of infection in vitro and studied mice undergoing cecal ligation puncture (CLP) as an in vivo model of infection. In cell studies, we assessed sphingomyelinase activity, subcellular calcium levels, and calpain activity and determined the effects of inhibiting sphingomyelinase using chemical (GW4869) and genetic (siRNA to nSMase2 and nSMase3) techniques. We assessed diaphragm force and calpain activity and utilized GW4869 to inhibit sphingomyelinase in mice. Cytomix increased cytosolic and mitochondrial calcium levels in C2C12 cells ( P < 0.001); addition of GW4869 blocked these increases ( P < 0.001). Cytomix also activated calpain, increasing calpain activity ( P < 0.02), and the calpain-mediated cleavage of procaspase 12 ( P < 0.001). Procaspase 12 cleavage was attenuated by either GW4869 ( P < 0.001), BAPTA-AM ( P < 0.001), or siRNA to nSMase2 ( P < 0.001) but was unaffected by siRNA to nSMase3. GW4869 prevented CLP-induced diaphragm calpain activation and diaphragm weakness in mice. These data suggest that nSMase2 activation is required for the development of infection-induced diaphragm calpain activation and muscle weakness. As a consequence, therapies that inhibit nSMase2 in patients may prevent infection-induced skeletal muscle dysfunction.

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Tan, X., Y. A. Alrashdan, H. Alkhouri, B. G. G. Oliver, C. L. Armour, and J. M. Hughes. "Airway smooth muscle CXCR3 ligand production: regulation by JAK-STAT1 and intracellular Ca2+." American Journal of Physiology-Lung Cellular and Molecular Physiology 304, no. 11 (June 1, 2013): L790—L802. http://dx.doi.org/10.1152/ajplung.00356.2012.

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In asthma, airway smooth muscle (ASM) chemokine (C-X-C motif) receptor 3 (CXCR3) ligand production may attract mast cells or T lymphocytes to the ASM, where they can modulate ASM functions. In ASM cells (ASMCs) from people with or without asthma, we aimed to investigate JAK-STAT1, JNK, and Ca2+ involvement in chemokine (C-X-C motif) ligand (CXCL)10 and CXCL11 production stimulated by interferon-γ, IL-1β, and TNF-α combined (cytomix). Confluent, growth-arrested ASMC were treated with inhibitors for pan-JAK (pyridone-6), JAK2 (AG-490), JNK (SP-600125), or the sarco(endo)plasmic reticulum Ca2+ATPase (SERCA) pump (thapsigargin), Ca2+ chelator (BAPTA-AM), or vehicle before and during cytomix stimulation for up to 24 h. Signaling protein activation as well as CXCL10/CXCL11 mRNA and protein production were examined using immunoblot analysis, real-time PCR, and ELISA, respectively. Cytomix-induced STAT1 activation was lower and CXCR3 ligand mRNA production was more sensitive to pyridone-6 and AG-490 in asthmatic than nonasthmatic ASMCs, but CXCL10/CXCL11 release was inhibited by the same proportion. Neither agent caused additional inhibition of release when used in combination with the JNK inhibitor SP-600125. Conversely, p65 NF-κB activation was higher in asthmatic than nonasthmatic ASMCs. BAPTA-AM abolished early CXCL10/CXCL11 mRNA production, whereas thapsigargin reduced it in asthmatic cells and inhibited CXCL10/CXCL11 release by both ASMC types. Despite these inhibitory effects, neither Ca2+ agent affected early activation of STAT1, JNK, or p65 NF-κB. In conclusion, intracellular Ca2+ regulated CXCL10/CXCL11 production but not early activation of the signaling molecules involved. In asthma, reduced ASM STAT1-JNK activation, increased NF-κB activation, and altered Ca2+ handling may contribute to rapid CXCR3 ligand production and enhanced inflammatory cell recruitment.

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Costanzo, M., V. Cesi, F. Palone, M. Pierdomenico, E. Colantoni, B. Leter, R. Vitali, A. Negroni, S. Cucchiara, and L. Stronati. "Krill oil, vitamin D and Lactobacillus reuteri cooperate to reduce gut inflammation." Beneficial Microbes 9, no. 3 (April 25, 2018): 389–99. http://dx.doi.org/10.3920/bm2017.0078.

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Current research into original therapies to treat intestinal inflammation is focusing on no-drug therapies. KLD is a mixture of krill oil (KO), probiotic Lactobacillus reuteri (LR), and vitamin D (VitD3). The aim of this study was to assess in vitro and in vivo the potential cooperative effects of KLD in reducing gut inflammation. Colorectal adenocarcinoma cell lines, CACO2 and HT29, and C57BL/6 mice were used for in vitro and in vivo analyses, respectively. Cells were exposed to cytomix (interferon gamma + tumour necrosis factor alpha (TNF-α)) to induce inflammation or co-exposed to cytomix and KO, LR and VitD3 alone or to cytomix and KLD. Animals were treated for 7 days with dextran sodium sulphate (DSS) to induce colitis or with DSS and KLD. In vitro assays: F-actin expression was analysed by immunofluorescence; scratch test and trans-epithelial electric resistance test were performed to measure wound healing; adhesion/invasion assays of adhesive and invasive Escherichia coli (AIEC) bacteria were made; mRNA expression of TNF-α, interleukin (IL)-8 and vitamin D receptor (VDR) was detected by quantitative PCR. In vivo assays: body weight, clinical score, histological score and large intestine weight and length were estimated; mRNA expression of TNF-α, IL-1β, IL-6, IL-10 by quantitative PCR; VDR expression was detected by quantitative PCR and immunohistochemistry. In vitro: KLD restores epithelial cell-cell adhesion and mucosal healing during inflammation, while decreases the adhesiveness and invasiveness of AIEC bacteria and TNF-α and IL-8 mRNA expression and increases VDR expression. In vivo: KLD significantly improves body weight, clinical score, histological score and large intestine length of mice with DSS-induced colitis and reduces TNF-α, IL-1β and IL-6 mRNA levels, while increases IL-10 mRNA and VDR levels. KLD has significant effects on the intestinal mucosa, strongly decreasing inflammation, increasing epithelial restitution and reducing pathogenicity of harmful commensal bacteria.

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Cheadle, Gerald A., Todd W. Costantini, Nicole Lopez, Vishal Bansal, Brian P. Eliceiri, and Raul Coimbra. "Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown." PLoS ONE 8, no. 7 (July 1, 2013): e69042. http://dx.doi.org/10.1371/journal.pone.0069042.

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Cheadle, G. A., T. W. Costantini, A. Hageny, J. G. Putnam, N. Lopez, B. Eliceiri, V. Bansal, and R. Coimbra. "Enteric Glia Cells Attenuate Cytomix-Induced Intestinal Epithelial Barrier Breakdown." Journal of Surgical Research 172, no. 2 (February 2012): 202–3. http://dx.doi.org/10.1016/j.jss.2011.11.272.

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24

Wang, Lefeng, Sanjay Mehta, Michael Brock, and Sean E. Gill. "Inhibition of Murine Pulmonary Microvascular Endothelial Cell Apoptosis Promotes Recovery of Barrier Function under Septic Conditions." Mediators of Inflammation 2017 (2017): 1–15. http://dx.doi.org/10.1155/2017/3415380.

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Sepsis is characterized by injury of the pulmonary microvasculature and the pulmonary microvascular endothelial cells (PMVEC), leading to barrier dysfunction and acute respiratory distress syndrome (ARDS). Our recent work identified a strong correlation between PMVEC apoptosis and microvascular leak in septic mice in vivo, but the specific role of apoptosis in septic PMVEC barrier dysfunction remains unclear. Thus, we hypothesize thatPMVEC apoptosis is likely required for PMVEC barrier dysfunction under septic conditions in vitro. Septic stimulation (mixture of tumour necrosis factorα, interleukin 1β, and interferonγ[cytomix]) of isolatedmurinePMVEC resulted in a significant loss of barrier function as early as 4 h after stimulation, which persisted until 24 h. PMVEC apoptosis, as reflected by caspase activation, DNA fragmentation, and loss of membrane polarity, was first apparent at 8 h after cytomix. Pretreatment of PMVEC with the pan-caspase inhibitor Q-VD significantly decreased septic PMVEC apoptosis and was associated with reestablishment of PMVEC barrier function at 16 and 24 h after stimulation but had no effect on septic PMVEC barrier dysfunction over the first 8 h. Collectively, our data suggest that early septic murine PMVEC barrier dysfunction driven by proinflammatory cytokines is not mediated through apoptosis, but PMVEC apoptosis contributes to late septic PMVEC barrier dysfunction.

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Wu, Huaxing, Guonian Wang, Shuai Li, Mingyue Zhang, Hulun Li, and Kun Wang. "TNF-α- Mediated-p38-Dependent Signaling Pathway Contributes to Myocyte Apoptosis in Rats Subjected to Surgical Trauma." Cellular Physiology and Biochemistry 35, no. 4 (2015): 1454–66. http://dx.doi.org/10.1159/000373965.

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Background: The accumulation of cytokines in the plasma after trauma can induce myocyte apoptosis. We aimed to identify which cytokine(s) present in the plasma responsible for myocyte apoptosis, and delineated the signal transduction mechanism in rats subjected to surgical trauma. Methods: Rats were randomized into two groups: control and trauma groups, which was divided into five subgroups: posttraumatic 0, 3, 6, 12, and 24 h subgroups. Cardiomyocytes isolated from traumatized rats were incubated with one of the factors for 12 h (normal plasma; Cytomix; TNF-α; IL-1β; IFN-γ; trauma plasma; anti-TNF-α antibody; SB203580). Myocyte apoptosis, cytokine levels, and MAPKs activation, as the primary experimental outcomes, were measured by TUNEL, flow cytometry, ELISA and Western blot, respectively. Results: Myocyte apoptosis was induced by surgical trauma during the early stage after trauma. Accompanying this change, plasma TNF-α, IL-1β, and IFN-γ levels were elevated in traumatized rats. Incubation of traumatized cardiomyocytes with cytomix or TNF-α alone induced myocyte apoptosis, and increased the activation of p38 and ERK1/2. Myocyte apoptosis and p38 activation were elevated in traumatized cardiomyocytes with trauma plasma, and these increases were partly abolished by anti-TNF-α antibody or SB203580. Conclusion: Our study demonstrated that there exists the TNF-α-mediated-p38-dependent signaling pathway that contributed to posttraumatic myocyte apoptosis of rats undergoing surgical trauma.

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Gutierrez, H. H., B. R. Pitt, M. Schwarz, S. C. Watkins, C. Lowenstein, I. Caniggia, P. Chumley, and B. A. Freeman. "Pulmonary alveolar epithelial inducible NO synthase gene expression: regulation by inflammatory mediators." American Journal of Physiology-Lung Cellular and Molecular Physiology 268, no. 3 (March 1, 1995): L501—L508. http://dx.doi.org/10.1152/ajplung.1995.268.3.l501.

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Nitric oxide (.NO) is a short-lived mediator that can be induced by different cytokines and lipopolysaccharide (LPS) in a variety of cell types and produces many physiological and metabolic changes in target cells. In the current study, we show that a combination of cytokines, LPS, and zymosan-activated serum (ZAS; called for convenience cytomix Z) induces production of high concentrations of the NO oxidation products nitrite (NO2-) and nitrate (NO3-) by cultured rat fetal lung epithelial type II cells in a time-dependent fashion. Interferon-gamma and tumor necrosis factor-alpha alone did not significantly affect .NO synthesis, whereas ZAS, LPS, and interleukin-1 beta caused only a modest increase in formation of .NO oxidation products. Production of NO2- and NO3- was inhibited by NG-monomethyl-L-arginine and cyclohexmide. After exposure of these cells to a combination of the above cytokines, Escherichia coli LPS, and ZAS (cytomix Z), enhanced inducible nitric oxide synthase (iNOS) expression was indicated by an elevation in steady-state mRNA specific for iNOS (via Northern blot analysis) and increased immunofluorescence for iNOS after cell permeabilization, incubation with anti-iNOS antibody, and treatment with Cy3.18-conjugated rabbit-specific antibody. The extent of inflammatory mediator-induced.NO production by alveolar epithelium, which exceeds that of other lung cell types, reveals new insight into mechanisms of pulmonary host defense and pathways of free radical-mediated lung injury.

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Frieri, M., and A. Capetandes. "Human Keratinocytes Secrete TNFa Stimulated With Cytomix and D. Pteronyssinus Extract." Journal of Allergy and Clinical Immunology 119, no. 1 (January 2007): S263. http://dx.doi.org/10.1016/j.jaci.2006.12.397.

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Baer, Koch, Hickmann, Schubert, Cinatl, Hauser, and Geiger. "Isolation, Characterization, Differentiation and Immunomodulatory Capacity of Mesenchymal Stromal/Stem Cells from Human Perirenal Adipose Tissue." Cells 8, no. 11 (October 29, 2019): 1346. http://dx.doi.org/10.3390/cells8111346.

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Mesenchymal stromal/stem cells (MSCs) are immature multipotent cells, which represent a rare population in the perivascular niche within nearly all tissues. The most abundant source to isolate MSCs is adipose tissue. Currently, perirenal adipose tissue is rarely described as the source of MSCs. MSCs were isolated from perirenal adipose tissue (prASCs) from patients undergoing tumor nephrectomies, cultured and characterized by flow cytometry and their differentiation potential into adipocytes, chondrocytes, osteoblasts and epithelial cells. Furthermore, prASCs were stimulated with lipopolysaccharide (LPS), lipoteichoic acid (LTA) or a mixture of cytokines (cytomix). In addition, prASC susceptibility to human cytomegalovirus (HCMV) was investigated. The expression of inflammatory readouts was estimated by qPCR and immunoassay. HCMV infection was analyzed by qPCR and immunostaining. Characterization of cultured prASCs shows the cells meet the criteria of MSCs and prASCs can undergo trilineage differentiation. Cultured prASCs can be induced to differentiate into epithelial cells, shown by cytokeratin 18 expression. Stimulation of prASCs with LPS or cytomix suggests the cells are capable of initiating an inflammation-like response upon stimulation with LPS or cytokines, whereas, LTA did not induce a significant effect on the readouts (ICAM-1, IL-6, TNFα, MCP-1 mRNA and IL-6 protein). HCMV broadly infects prASCs, showing a viral load dependent cytopathological effect (CPE). Our current study summarizes the isolation and culture of prASCs, clearly characterizes the cells, and demonstrates their immunomodulatory potential and high permissiveness for HCMV.

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Farley, K. S., L. Wang, and S. Mehta. "Septic pulmonary microvascular endothelial cell injury: role of alveolar macrophage NADPH oxidase." American Journal of Physiology-Lung Cellular and Molecular Physiology 296, no. 3 (March 2009): L480—L488. http://dx.doi.org/10.1152/ajplung.90201.2008.

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A significant role for alveolar macrophages (AM) in the pathophysiology of sepsis-induced acute lung injury (ALI) has been shown; however, the mechanisms behind AM-related lung injury remain relatively uncertain. We examined the role of AM nicotinamide adenine dinucleotide phosphate (NADPH) oxidase in pulmonary endothelial cell septic injury. NADPH oxidase is one of the major sources of cellular reactive oxygen species and has been implicated in endothelial injury in ALI. Pulmonary microvascular endothelial cells (PMVEC) monolayers were grown on Transwell inserts and incubated with wild-type and NADPH oxidase-deficient AM in the presence or absence of cytomix (equimolar TNF-α, IL-1β, and IFN-γ). Injury to the monolayers was assessed by trans-PMVEC Evans blue (EB)-labeled albumin flux. We found AM under cytomix stimulation caused significant EB-albumin flux across the PMVEC monolayers, and this effect was attenuated by the genetic deletion of AM NADPH oxidase. The pharmacological inhibition of AM NADPH oxidase with apocynin and PR-39 also significantly reduced AM-dependent PMVEC injury. In the AM-PMVEC cocultures, we also assessed PMVEC injury through measurement of protein oxidation and lipid peroxidation. AM were shown to cause a significant increase in these markers of PMVEC injury, which was also attenuated by the inhibition of NADPH oxidase or through the use of NADPH oxidase-deficient AM. PMVEC NADPH oxidase was shown not to significantly contribute to PMVEC injury in our studies. From our findings we have concluded that AM NADPH oxidase is crucial for the septic increase in pulmonary vascular permeability.

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Cetin, Selma, Faisal Qureshi, Orkan Ergun, Jun Li, Jeffrey Upperman, Henri Ford, and David Hackam. "CYTOMIX IMPAIRS INTESTINAL EPITHELIAL RESTITUTION BY DECREASING PHOSPHORYLATION OF FOCAL ADHESION KINASE." Shock 21 (June 2004): 16. http://dx.doi.org/10.1097/00024382-200406002-00046.

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Supinski, Gerald S., Xinying Ji, Wenyi Wang, and Leigh A. Callahan. "The extrinsic caspase pathway modulates endotoxin-induced diaphragm contractile dysfunction." Journal of Applied Physiology 102, no. 4 (April 2007): 1649–57. http://dx.doi.org/10.1152/japplphysiol.00377.2006.

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The mechanisms by which infections induce diaphragm dysfunction remain poorly understood. The purpose of this study was to determine which caspase pathways (i.e., the extrinsic, death receptor-linked caspase-8 pathway, and/or the intrinsic, mitochondrial-related caspase-9 pathway) are responsible for endotoxin-induced diaphragm contractile dysfunction. We determined 1) whether endotoxin administration (12 mg/kg IP) to mice induces caspase-8 or -9 activation in the diaphragm; 2) whether administration of a caspase-8 inhibitor ( N-acetyl-Ile-Glu-Thr-Asp-CHO, 3 mg/kg iv) or a caspase-9 inhibitor ( N-acetyl-Leu-Glu-His-Asp-CHO, 3 mg/kg iv) blocks endotoxin-induced diaphragmatic weakness and caspase-3 activation; 3) whether TNF receptor 1-deficient mice have reduced caspase activation and diaphragm dysfunction following endotoxin; and 4) whether cytokines (TNF-α or cytomix, a mixture of TNF-α, interleukin-1β, interferon-γ, and endotoxin) evoke caspase activation in C2C12 myotubes. Endotoxin markedly reduced diaphragm force generation ( P < 0.001) and induced increases in caspase-3 and caspase-8 activity ( P < 0.03), but failed to increase caspase-9. Inhibitors of caspase-8, but not of caspase-9, prevented endotoxin-induced reductions in diaphragm force and caspase-3 activation ( P < 0.01). Mice deficient in TNF receptor 1 also had reduced caspase-8 activation ( P < 0.001) and less contractile dysfunction ( P < 0.01) after endotoxin. Furthermore, incubation of C2C12 cells with either TNF-α or cytomix elicited significant caspase-8 activation. The caspase-8 pathway is strongly activated in the diaphragm following endotoxin and is responsible for caspase-3 activation and diaphragm weakness.

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Chromova, E. A., E. A. Akhmatova, S. A. Skhodova, I. A. Semochkin, V. G. Khomenkov, N. K. Akhmatova, and M. P. Kostinov. "EFFECT OF INFLUENZA VACCINES ON SUBPOPULATIONS OF BLOOD DENDRITIC CELLS." Journal of microbiology, epidemiology and immunobiology 93, no. 5 (October 28, 2016): 23–28. http://dx.doi.org/10.36233/0372-9311-2016-5-23-28.

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Aim. Study the effect of Vaxigrip split, Influvac subunit and Grippol plus immune-adjuvanted vaccines on the content of myeloid (mDC) and plasmacytoid (pDC) dendritic cells (DC) in blood of vaccinated healthy women. Materials and methods. Blood of 30 healthy women aged 18-50 years was studied at days 7 and 30 after the vaccination. pDC (CD14+CD16-/CD85k(ILT3)-PE/ CD123-PC5) and mDC (CD14+CD16-/CD85k(ILT3)-PE/CD33-PC5) immune phenotyping was carried out using mAbs (Beckman Coulter, France) and flow cytometer Cytomix FC-500 (Beckman Coulter, USA). Results. Use of unadjuvanted vaccines Vaxigrip and Influvac resulted in an increase of the numbers of mDC and pDC (p

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Sappington, Penny L., Xiaonan Han, Matthew E. Fink, Runkuan Yang, Russell L. Delude, and Mitchell P. Fink. "ETHYL PYRUVATE (EP) AMELIORATES CYTOMIX-INDUCED HYPERPERMEABILITY IN CACO-2 HUMAN ENTEROCYTIC MONOLAYERS." Critical Care Medicine 30, Supplement (December 2002): A12. http://dx.doi.org/10.1097/00003246-200212001-00045.

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Bastarache, Julie A., Ling Wang, Zhengming Wang, Kurt H. Albertine, Michael A. Matthay, and Lorraine B. Ware. "Intra-alveolar tissue factor pathway inhibitor is not sufficient to block tissue factor procoagulant activity." American Journal of Physiology-Lung Cellular and Molecular Physiology 294, no. 5 (May 2008): L874—L881. http://dx.doi.org/10.1152/ajplung.00372.2007.

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The alveolar compartment in acute lung injury contains high levels of tissue factor (TF) procoagulant activity favoring fibrin deposition. We previously reported that the alveolar epithelium can release TF procoagulant activity in response to a proinflammatory stimulus. To test the hypothesis that the alveolar epithelium further modulates intra-alveolar fibrin deposition through secretion of an endogenous inhibitor to TF, tissue factor pathway inhibitor (TFPI), we measured TFPI levels in edema fluid (EF) from patients with acute respiratory distress syndrome. To determine whether the alveolar epithelium can release TFPI, both full-length TFPI and truncated TFPI were measured (ELISA) in pulmonary edema fluid from patients with acute respiratory distress syndrome (ARDS) and a control group of patients with hydrostatic pulmonary edema (HYDRO). TFPI protein was also measured in conditioned media (CM) and cell lysates (CL) from human alveolar epithelial cells (A549) after exposure to cytomix (TNF-α, IL-1β, IFN-γ). TFPI protein levels were higher in pulmonary edema fluid from patients with ARDS vs. HYDRO. TFPI protein was increased in CM and did not change in CL after cytomix treatment; TFPI mRNA levels (RT-PCR) did not change. Despite the high levels of TFPI, both the EF and CM retained significant TF procoagulant activity as measured by plasma recalcification time. The majority of intra-alveolar TFPI was in a truncated, inactive form, whereas the majority of TFPI released from cells was full length, suggesting different mechanisms of inactivation. In summary, the alveolar epithelium releases TFPI in response to an inflammatory stimulus but does not increase TFPI gene transcription or protein production. Levels of intra-alveolar TFPI in ARDS are not sufficient to block intra-alveolar TF procoagulant activity due to truncation and inactivation of intra-alveolar TFPI.

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Bruda, N. L., B. J. Hurlbert, and G. E. Hill. "Aprotinin Reduces Nitric Oxide Production in Vitro and in Vivo in a Dose-Dependent Manner." Clinical Science 94, no. 5 (May 1, 1998): 505–9. http://dx.doi.org/10.1042/cs0940505.

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1. Cardiopulmonary bypass is associated with an increase in nitric oxide concentrations, and plasma levels of tumour necrosis factor and interleukin-1. Aprotinin, a serine protease inhibitor, commonly used during cardiopulmonary bypass to reduce blood loss, has been demonstrated to exhibit significant anti-inflammatory effects during and after cardiopulmonary bypass. 2. Airway nitric oxide was measured during cardiopulmonary bypass in 10 controls (Group 1), 10 subjects receiving half-dose aprotinin (Group 2) and 10 patients receiving full-dose aprotinin (Group 3). In vitro, a murine bronchial epithelial cell line (LA-4) was cultured with cytomix (a combination of tumour necrosis factor, interleukin-1, and (γ-interferon) with and without aprotinin in increasing concentrations. Nitrite concentrations, the stable and measureable end-product of nitric oxide oxidative metabolism, were measured in the culture supernatant by chemiluminescence. 3. Airway nitric oxide concentrations were increased after 50 min cardiopulmonary bypass compared with that measured at 5 min in controls (53 ± 5 versus 29 ± 3 ppb, P < 0.05) but not in the aprotinin-treated groups (25 ± 4 versus 14 ± 5, Group 2; 21 ± 6 versus 15 ± 3 ppb, Group 3). 4. In a dose-dependent manner, nitrite levels (means ± S.E.M.) were significantly reduced by aprotinin at 500 and 1000 units/ml when compared with cells cultured in the presence of cytomix alone (P < 0.05). 5. These data demonstrate that aprotinin, in a dose-responsive manner, reduces nitric oxide production in vivo and reduces cytokine-induced nitrite production by murine bronchial epithelial cells in vitro. Since increased airway nitric oxide is found in inflammatory lung diseases, like asthma, and anti-inflammatory therapy reduces the concentration of airway nitric oxide, these data support the concept that aprotinin is anti-inflammatory during cardiopulmonary bypass.

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de Courcey, F., A. V. Zholos, H. Atherton-Watson, M. T. S. Williams, P. Canning, H. L. Danahay, J. S. Elborn, and M. Ennis. "Development of primary human nasal epithelial cell cultures for the study of cystic fibrosis pathophysiology." American Journal of Physiology-Cell Physiology 303, no. 11 (December 1, 2012): C1173—C1179. http://dx.doi.org/10.1152/ajpcell.00384.2011.

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Cultured primary epithelial cells are used to examine inflammation in cystic fibrosis (CF). We describe a new human model system using cultured nasal brushings. Nasal brushings were obtained from 16 F508del homozygous patients and 11 healthy controls. Cells were resuspended in airway epithelial growth medium and seeded onto collagen-coated flasks and membranes for use in patch-clamp, ion transport, and mediator release assays. Viable cultures were obtained with a 75% success rate from subjects with CF and 100% from control subjects. Amiloride-sensitive epithelial Na channel current of similar size was present in both cell types while forskolin-activated CF transmembrane conductance regulator current was lacking in CF cells. In Ussing chambers, cells from CF patients responded to UTP but not to forskolin. Spontaneous and cytomix-stimulated IL-8 release was similar (stimulated 29,448 ± 9,025 pg/ml; control 16,336 ± 3,308 pg/ml CF; means ± SE). Thus nasal epithelial cells from patients with CF can be grown from nasal brushings and used in electrophysiological and mediator release studies in CF research.

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Byrnes, Declan, Claire H. Masterson, Jack Brady, Senthilkumar Alagesan, Hector E. Gonzalez, Sean D. McCarthy, Juan Fandiño, Daniel P. O’Toole, and John G. Laffey. "Differential Effects of Cytokine Versus Hypoxic Preconditioning of Human Mesenchymal Stromal Cells in Pulmonary Sepsis Induced by Antimicrobial-Resistant Klebsiella pneumoniae." Pharmaceuticals 16, no. 2 (January 19, 2023): 149. http://dx.doi.org/10.3390/ph16020149.

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Background: Pulmonary sepsis is a leading cause of hospital mortality, and sepses arising from antimicrobial-resistant (AMR) bacterial strains are particularly difficult to treat. Here we investigated the potential of mesenchymal stromal cells (MSCs) to combat established Klebsiella pneumoniae pneumosepsis and further evaluated MSC preconditioning and pre-activation methods. Methods: The potential for naïve and preconditioned MSCs to enhance wound healing, reduce inflammation, preserve metabolic activity, and enhance bacterial killing was assessed in vitro. Rats were subjected to intratracheal K. pneumoniae followed by the intravenous administration of MSCs. Physiological indices, blood, bronchoalveolar lavage (BAL), and tissues were obtained 72 h later. Results: In vitro assays confirmed that preconditioning enhances MSC function, accelerating pulmonary epithelial wound closure, reducing inflammation, attenuating cell death, and increasing bacterial killing. Cytomix-pre-activated MSCs are superior to naïve and hypoxia-exposed MSCs in attenuating Klebsiella pneumosepsis, improving lung compliance and oxygenation, reducing bacteria, and attenuating histologic injuries in lungs. BAL inflammatory cytokines were reduced, correlating with decreases in polymorphonuclear (PMN) cells. MSCs increased PMN apoptosis and the CD4:CD8 ratio in BAL. Systemically, granulocytes, classical monocytes, and the CD4:CD8 ratio were reduced, and nonclassical monocytes were increased. Conclusions: Preconditioning with cytokines, but not hypoxia, enhances the therapeutic potential of MSCs in clinically relevant models of K. pneumoniae-induced pneumosepsis.

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Valet, G., J. F. Leary, and A. Tárnok. "Cytomics-New technologies: Towards a human cytome project." Cytometry Part A 59A, no. 2 (May 17, 2004): 167–71. http://dx.doi.org/10.1002/cyto.a.20047.

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Han, Xiaonan, Mitchell P. Fink, Takashi Uchiyama, Runkuan Yang, and Russell L. Delude. "Increased iNOS activity is essential for pulmonary epithelial tight junction dysfunction in endotoxemic mice." American Journal of Physiology-Lung Cellular and Molecular Physiology 286, no. 2 (February 2004): L259—L267. http://dx.doi.org/10.1152/ajplung.00187.2003.

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A murine endotoxemia model and cultured Calu-3 monolayers were used to test the hypothesis that excessive nitric oxide (NO) production secondary to induction of inducible NO synthase (iNOS) is a key factor leading to altered tight junction (TJ) protein expression and function in the pulmonary epithelium. C57Bl/6J mice were injected with either Escherichia coli 0111:B4 lipopolysaccharide (LPS; 2 mg/kg) or vehicle. Twelve hours later, leakage of FITC-dextran ( Mr 4 kDa; FD4) from blood into bronchoalveolar lavage fluid was significantly increased in endotoxemic but not control mice. This decrease in bronchoalveolar barrier function was associated with upregulation of iNOS protein expression and NF-κB activation in lung tissue. Expression of the TJ proteins, zonula occludens (ZO)-1, ZO-2, ZO-3, and occludin, as assessed by immunoblotting and/or immunofluorescence, decreased in lung after the injection of mice with LPS. Treatment of endotoxemic mice with an isoform-selective iNOS inhibitor [l- N6-(1-iminoethyl)lysine; l-NIL] ameliorated LPS-induced changes in TJ protein expression and preserved bronchoalveolar epithelial barrier function. Incubating Calu-3 bronchiolar epithelial monolayers with cytomix (a mixture of 1,000 U/ml IFN-γ, 10 ng/ml TNF-α, and 1 ng/ml IL-1β) increased permeability to FD4, but adding l-NIL prevented this effect. These results suggest that decreased expression and mistargeting of TJ proteins in lung after systemic inflammation may be NO dependent.

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Sangiorgi, Claudia, Davide Vallese, Isabella Gnemmi, Fabio Bucchieri, Bruno Balbi, Paola Brun, Angelo Leone, et al. "HSP60 activity on human bronchial epithelial cells." International Journal of Immunopathology and Pharmacology 30, no. 4 (October 4, 2017): 333–40. http://dx.doi.org/10.1177/0394632017734479.

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HSP60 has been implicated in chronic inflammatory disease pathogenesis, including chronic obstructive pulmonary disease (COPD), but the mechanisms by which this chaperonin would act are poorly understood. A number of studies suggest a role for extracellular HSP60, since it can be secreted from cells and bind Toll-like receptors; however, the effects of this stimulation have never been extensively studied. We investigated the effects (pro- or anti-inflammatory) of HSP60 in human bronchial epithelial cells (16-HBE) alone and in comparison with oxidative, inflammatory, or bacterial challenges. 16-HBE cells were cultured for 1–4 h in the absence or presence of HSP60, H2O2, lipopolysaccharide (LPS), or cytomix. The cell response was evaluated by measuring the expression of IL-8 and IL-10, respectively, pro- and anti-inflammatory cytokines involved in COPD pathogenesis, as well as of pertinent TLR-4 pathway mediators. Stimulation with HSP60 up-regulated IL-8 at mRNA and protein levels and down-regulated IL-10 mRNA and protein. Likewise, CREB1 mRNA was up-regulated. H2O2 and LPS up-regulated IL-8. Experiments with an inhibitor for p38 showed that this mitogen-activated protein kinase could be involved in the HSP60-mediated pro-inflammatory effects. HSP60 showed pro-inflammatory properties in bronchial epithelial cells mediated by activation of TLR-4-related molecules. The results should prompt further studies on more complex ex-vivo or in-vivo models with the aim to elucidate further the role of those molecules in the pathogenesis of COPD.

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Arpino, Valerie, Sanjay Mehta, Lefeng Wang, Ryan Bird, Marta Rohan, Cynthia Pape, and Sean E. Gill. "Tissue inhibitor of metalloproteinases 3-dependent microvascular endothelial cell barrier function is disrupted under septic conditions." American Journal of Physiology-Heart and Circulatory Physiology 310, no. 11 (June 1, 2016): H1455—H1467. http://dx.doi.org/10.1152/ajpheart.00796.2015.

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Sepsis is associated with dysfunction of microvascular endothelial cells (MVEC) leading to tissue edema and multiple organ dysfunction. Metalloproteinases can regulate MVEC function through processing of cell surface proteins, and tissue inhibitor of metalloproteinases 3 (TIMP3) regulates metalloproteinase activity in the lung following injury. We hypothesize that TIMP3 promotes normal pulmonary MVEC barrier function through inhibition of metalloproteinase activity. Naive Timp3−/−mice had significantly higher basal pulmonary microvascular Evans blue (EB) dye-labeled albumin leak vs. wild-type (WT) mice. Additionally, cecal-ligation/perforation (CLP)-induced sepsis significantly increased pulmonary microvascular EB-labeled albumin leak in WT but not Timp3−/−mice. Similarly, PBS-treated isolated MVEC monolayers from Timp3−/−mice displayed permeability barrier dysfunction vs. WT MVEC, evidenced by lower transendothelial electrical resistance and greater trans-MVEC flux of fluorescein-dextran and EB-albumin. Cytomix (equimolar interferon γ, tumor necrosis factor α, and interleukin 1β) treatment of WT MVEC induced significant barrier dysfunction (by all three methods), and was associated with a time-dependent decrease in TIMP3 mRNA and protein levels. Additionally, basal Timp3−/−MVEC barrier dysfunction was associated with disrupted MVEC surface VE-cadherin localization, and both barrier dysfunction and VE-cadherin localization were rescued by treatment with GM6001, a synthetic metalloproteinase inhibitor. TIMP3 promotes normal MVEC barrier function, at least partially, through inhibition of metalloproteinase-dependent disruption of adherens junctions, and septic downregulation of TIMP3 may contribute to septic MVEC barrier dysfunction.

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42

Supinski, Gerald S., Xinying Ji, and Leigh Ann Callahan. "The JNK MAP kinase pathway contributes to the development of endotoxin-induced diaphragm caspase activation." American Journal of Physiology-Regulatory, Integrative and Comparative Physiology 297, no. 3 (September 2009): R825—R834. http://dx.doi.org/10.1152/ajpregu.90849.2008.

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We previously demonstrated that endotoxin-induced sepsis results in caspase 8-mediated diaphragmatic dysfunction. The upstream signaling pathways modulating diaphragm caspase 8 activation in response to endotoxin administration are, however, unknown. The purpose of the present study was to test the hypothesis that the JNK (Jun N-terminal Kinase) pathway is activated in the diaphragm during sepsis and contributes to sepsis-induced diaphragm caspase 8 activation. Endotoxin was administered to intact animals to model the effects of sepsis. We first assessed the time course of JNK activation after endotoxin (12 mg/kg ip) administration to mice. We then determined whether JNK inhibitor administration ( 30 μm/kg ip SP600125) could prevent caspase 8 activation and diaphragm weakness in endotoxin-treated mice. Experiments were then repeated comparing the effects of endotoxin on control and transgenic JNK knockout mice. We finally determined whether cytomix (LPS, TNFα, IL1β, and IFN-γ) exposure activated caspase 8 in C2C12 muscle cells and whether caspase 8 activation was attenuated by either chemical inhibition of JNK (30 μM SP600125) or transfection with a dominant negative JNK construct. We found that endotoxin activated diaphragm JNK ( P < 0.001) and increased active caspase 8 ( P < 0.01). Inhibition of JNK with SP600125 or by use of JNK-deficient animals prevented diaphragm caspase 8 activation ( P < 0.01) and prevented diaphragm weakness ( P < 0.05). JNK inhibition also prevented caspase 8 activation in cytokine-treated muscle cells ( P < 0.001). These data implicate JNK activation as a major factor mediating inflammation-induced skeletal muscle caspase 8 activation and weakness.

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Li, Shuzhuang, Xiangying Jiao, Ling Tao, Huirong Liu, Yue Cao, Bernard L. Lopez, Theodore A. Christopher, and Xin L. Ma. "Tumor necrosis factor-α in mechanic trauma plasma mediates cardiomyocyte apoptosis." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 3 (September 2007): H1847—H1852. http://dx.doi.org/10.1152/ajpheart.00578.2007.

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Mechanical traumatic injury causes cardiomyocyte apoptosis and cardiac dysfunction. However, the signaling mechanisms leading to posttraumatic cardiomyocyte apoptosis remains unclear. The present study attempted to identify the molecular mechanisms responsible for cardiomyocyte apoptosis induced by trauma. Normal cardiomyocytes (NC) or traumatic cardiomyocytes (TC; isolated immediately after trauma) were cultured with normal plasma (NP) or traumatic plasma (TP; isolated 1.5 h after trauma) for 12 h, and apoptosis was determined by caspase-3 activation. Exposure of TC to NP failed to induce significant cardiomyocyte apoptosis. In contrast, exposure of NC to TP resulted in a greater than twofold increase in caspase-3 activation ( P < 0.01). Incubation of cardiomyocytes with cytomix (a mixture of TNF-α, IL-1β, and IFN-γ) or TNF-α alone, but not with IL-1β or IFN-γ alone, caused significant caspase-3 activation ( P < 0.01). TP-induced caspase-3 activation was virtually abolished by an anti-TNF-α antibody, and TP isolated from TNF-α−/− mice failed to induce caspase-3 activation. Moreover, incubation of cardiomyocytes with TP upregulated inducible nitric oxide (NO) synthase (iNOS)/NADPH oxidase expression, increased NO/superoxide production, and increased cardiomyocyte protein nitration (measured by nitrotyrosine content). These oxidative/nitrative stresses and the resultant cardiomyocyte caspase-3 activation can be blocked by neutralization of TNF-α (anti-TNF-α antibody), inhibition of iNOS (1400W), or NADPH oxidase (apocynin) and scavenging of peroxynitrite (FP15) ( P < 0.01). Taken together, our study demonstrated that there exists a TNF-α-initiated, cardiomyocyte iNOS/NADPH oxidase-dependent, peroxynitrite-mediated signaling pathway that contributes to posttraumatic myocardial apoptosis. Therapeutic interventions that block this signaling cascade may attenuate posttraumatic cardiac injury and reduce the incidence of secondary organ dysfunction after trauma.

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44

Valet, G�nter. "Human cytome project, cytomics, and systems biology: The incentive for new horizons in cytometry." Cytometry Part A 64A, no. 1 (2005): 1–2. http://dx.doi.org/10.1002/cyto.a.20120.

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Kakoullis, Stylianos, Russell L. Delude, and Mitchell P. Fink. "IMPAIRED O2 CONSUMPTION (VO2) IN HEP3B HEPATOCYTE-LIKE CELLS INCUBATED WITH CYTOMIX (TNF + IL-1β + IFN-γ): PHARMACOLOGICAL RESCUE BY AGENTS THAT SCAVENGE PEROXYNITRITE (ONOO-) OR INHIBIT POLY(ADP-RIBOSE) POLYMERASE (PARP)." Critical Care Medicine 30, Supplement (December 2002): A52. http://dx.doi.org/10.1097/00003246-200212001-00181.

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46

Gomase, Virendra, and Somnath Tagore. "Cytomics." Current Drug Metabolism 9, no. 3 (March 1, 2008): 263–66. http://dx.doi.org/10.2174/138920008783884731.

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47

Bound, S. A., K. M. Jones, and M. J. Oakford. "Integrating Cytolin into a chemical thinning program for red ‘Delicious’ apple." Australian Journal of Experimental Agriculture 37, no. 1 (1997): 113. http://dx.doi.org/10.1071/ea96065.

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Summary. The thinning efficiency of Cytolin and its interaction with the blossom thinner naphthalene acetic acid (NAA) and the post-bloom thinner CyLex (a formulation of 6-benzyladenine) were examined in a trial on Hi-Early red ‘Delicious’ apples in northern Tasmania. Cytolin was applied as either a single or split application at 4 concentrations (25, 40, 55, 70 mg/L). The split application was followed by a low rate of NAA at full bloom or by CyLex as a post-bloom thinner. While the higher concentrations of Cytolin achieved some thinning it was not sufficient. Satisfactory thinning levels, with a corresponding increase in fruit size, were achieved by the addition of either NAA or CyLex to the program. There was no consistent difference in thinning effect or fruit size between the single and split applications of Cytolin. High concentrations of Cytolin followed by NAA resulted in a high proportion of pygmy fruit but there were no pygmy fruit at the lower Cytolin rates. In general, split applications of Cytolin improved fruit typiness better than single applications. Cytolin alone had no effect on seed numbers, whereas addition of either NAA or CyLex to the program reduced seed numbers. Fruit firmness was improved by the CyLex treatments. Return bloom decreased with increasing concentration of Cytolin. Thinning and fruit quality can be improved with the recommended label rate of Cytolin (25 mg/L) applied as a split application (12.5 mg/L applied at king petal stage and 12.5 mg/L 3 days later) followed by 3 mg/L NAA at full bloom or by CyLex as a post-bloom thinner. The improvement in fruit firmness and increase in soluble solids by CyLex compared with NAA may make this combination preferable for long-term storage fruit although further assessment is needed.

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48

Kumar, G., and S. Naseem. "EMS induced intercellular chromatin transmigration in Papaver somniferum L." Czech Journal of Genetics and Plant Breeding 49, No. 2 (May 16, 2013): 86–89. http://dx.doi.org/10.17221/85/2012-cjgpb.

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The phenomenon of chromatin migration was observed during microsporogenesis in an ethyl methane sulphonate (EMS) treated population of poppy, which is an important medicinal plant. Cytomixis occurred through a cytoplasmic channel or by direct fusion of pollen mother cells (PMCs); the former was more recurring than the latter. The process was associated with irregular meiosis. PMCs with differing chromosome numbers from the normal diploid number (2n = 22) through cytomixis may lead to the production of aneuploid and polyploid gametes. An increase in the concentration of EMS had a positive effect on the percentage of PMCs showing cytomixis. In addition to cytomixis, other chromosomal abnormalities were also found. Cytomixis along with the related chromosomal abnormalities largely affected the post-meiotic products resulting in some pollen sterility.

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Valet, G. "Cytomics, the human cytome project and systems biology: top-down resolution of the molecular biocomplexity of organisms by single cell analysis." Cell Proliferation 38, no. 4 (August 2005): 171–74. http://dx.doi.org/10.1111/j.1365-2184.2005.00342.x.

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50

Alijanpoor, Behnaz, and Masoumeh Safaeishakib. "CYTOMIXIS AND MEIOTIC ABNORMAL BEHAVIOR RELATED IN SOME SELECTED SPECIES OF THE GENUS Salvia L. (Lamiaceae) FROM IRAN." Acta Scientifica Malaysia 6, no. 2 (2022): 34–37. http://dx.doi.org/10.26480/asm.02.2022.34.37.

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In this research, the cytogenetic characteristics, phases of meiosis, abnormality during meiosis, variety of pollen grains, and cytomixis phenomenon have been studied. Populations of 10 species belonging to Salvia L. have been collected from different geographical regions of Iran. Cytomixis was recorded during meiosis I in three species of Salvia (S. nemorosa, S. staminea, S. verticellata) of the Lamiaceae family. Cytomixis and other associated meiotic abnormalities such as chromatin stickiness, clumping of metaphase -1, laggards, bridges, aberrations anaphase I, II, and micronuclei in the species reported into pollen sterility and some variable sizes. It can be said all of these are dependent on each other to create some abnormalities. Cytomixis and chromosome migration occurre in diploid and polyploid meiocytes.

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Journal articles on the topic 'Cytomix'

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