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Endotracheal Intubation in Mice <em>via</em> Direct Laryngoscopy Using an Otoscope

Mice, both wildtype and transgenic, are the principal mammalian model in biomedical research currently. Intubation and mechanical ventilation are ...

Va San Diego Healthcare System

University of California, San Diego

The yin and the yang of 5-lipoxygenase pathway activation.

What about the role of the US Food and Drug Administration?

We examined expression of the 5-lipoxygenase activating protein (FLAP), which is critical for inflammatory cell leukotriene synthesis. A 3.4-kb segment of the FLAP gene 5'-untranslated region accounted for a 22-fold increase in promoter activity when transfected into the monocyte-like cell line, THP-1, and demonstrated no activity in non-inflammatory cells. Virtually all of the promoter activity was mediated by the first 134 bp upstream of the transcription start site, a region that contains CCAAT/enhancer-binding proteins (C/EBP) consensus binding sites, at -36 to -28 bp (distal) and -25 to -12 bp (proximal). DNase I footprint analyses demonstrated THP-1 nuclear extract proteins bind to the proximal site. Electrophoretic mobility shift assay analyses revealed that C/EBP alpha, delta, and epsilon bind to the proximal site and C/EBP alpha and epsilon bind to the distal site, constitutively. Transfection studies indicated that mutation of both the proximal and distal sites decreased constitutive FLAP promoter activity. Overexpression of C/EBP alpha, beta, and delta transactivated promoter activity and increased native FLAP mRNA accumulation. Mutation of both C/EBP sites essentially abolished promoter induction by C/EBP overexpression. Tumor necrosis factor (TNF) alpha induced FLAP mRNA expression, FLAP promoter activity, and C/EBP alpha, delta, and epsilon binding to the proximal and distal promoter consensus sites. Chromatin immunoprecipitation assays demonstrated that C/EBP alpha, delta, and epsilon bound to this region of the 5'-untranslated region, whereas C/EBP beta does not bind even under conditions of overexpression and stimulation. We conclude that the FLAP gene is transactivated by members of the C/EBP family of transcription factors in inflammatory cells and that these factors play an important role in FLAP gene induction by TNFalpha.

5-lipoxygenase-activating protein gene expression. Key role of CCAAT/enhancer-binding proteins (C/EBP) in constitutive and tumor necrosis factor (TNF) alpha-induced expression in THP-1 cells.

We studied the effects of LPS on cysteinyl leukotriene (LT) synthesis and LTC(4) synthase expression in mononuclear phagocytes. Conditioning of the monocyte-like cell line, THP-1, with LPS for 7 days resulted in significantly decreased ionophore-stimulated LTC(4) release. The putative LPS receptor, Toll-like receptor 4, was expressed in THP-1 cells. LPS down-regulated LTC(4) synthase mRNA in THP-1 cells in a dose- and time-dependent manner, with down-regulation observed as early as 4 h. Conditioning of actinomycin D-treated cells with LPS resulted in no change in the rate of LTC(4) synthase mRNA decay. LPS treatment of THP-1 cells, transiently transfected with a LTC(4) synthase promoter (1.35 kb)-reporter construct, decreased promoter activity. Neutralization of TNF-alpha and inhibition of mitogen-activated protein kinase kinase/extracellular signal-regulated kinase did not inhibit the effect of LPS. Treatment of cells with a Toll-like receptor 4-blocking Ab and an inhibitor of NF-kappaB activation resulted in inhibition of the LPS effect, while activation of NF-kappaB and p50/p65 overexpression down-regulated the LTC(4) synthase gene. LPS down-regulates cysteinyl LT release and LTC(4) synthase gene expression in mononuclear phagocytes by an NF-kappaB-mediated mechanism.

Lipopolysaccharide down-regulates the leukotriene C4 synthase gene in the monocyte-like cell line, THP-1.

Lipopolysaccharide down-regulates leukotriene C4 synthase gene expression in a cell-specific manner in the monocyte-like cell line, THP-1.

Regulation of leukotriene C4 synthase gene expression by SP1 and SP3 in mononuclear phagocytes.

Significant glomerular vasoconstriction and production of reactive oxygen species has been known to occur with exposure to anti-glomerular basement membrane antibody (AGBM-Ab) in the rat model. Previously published studies have demonstrated that such effects can be reduced by therapy with phentolamine, an alpha-adrenergic antagonist. It was hypothesized that antioxidant pretreatment with water-soluble probucol would improve glomerular hemodynamics 60 to 90 min after the administration of AGBM-Ab. These relationships were examined with both in vivo renal micropuncture and in vitro studies in rats. Single-nephron GFR (SNGFR) decreased markedly in untreated rats after AGBM-Ab as a result of afferent and efferent arteriolar vasoconstriction with consequent reductions in nephron plasma flow (SNPF) and decreases in the glomerular ultrafiltration coefficient (LpA). Basal SNGFR was increased, and SNGFR was significantly higher after AGBM-Ab in probucol-treated versus untreated rats. This finding was due solely to higher values for SNPF and prevention of afferent arteriolar constriction. A reduction in LpA after AGBM-Ab was not prevented by probucol treatment. In vitro analyses of glomeruli revealed reduced myeloperoxidase activity in antioxidant-treated rats. Lipoxygenase activity and leukotriene products, however, were not changed by antioxidant therapy, yet vasoconstriction was prevented. H(2)O(2) generation before and after formyl-methionyl-leucyl-phenylalanine stimulation was significantly reduced before and after AGBM-Ab in glomeruli harvested from rats that were treated with the antioxidant. Antioxidant therapy in this model of AGBM-Ab injury did not prevent reductions in LpA, an index of glomerular membrane damage, but did prevent afferent arteriolar vasoconstriction. Reactive oxygen species generation was reduced by probucol. The specific mechanisms whereby antioxidant therapy ameliorates glomerular hemodynamic effects will be defined in additional studies and is likely to involve either enhanced vasodilator or diminished vasoconstrictor activity.

Mechanisms of glomerular immune injury: effects of antioxidant treatment.

While effective for the prevention and treatment of allergic rhinitis (AR) symptoms, currently available medications do not reverse allergen specific hypersensitivities. Therefore, pharmacotherapeutics are not curative and their daily use is often required for years. These investigations were conducted to determine whether immunostimulatory sequence oligodeoxynucleotide (ISS-ODN) delivery protects previously sensitized mice from AR hypersensitivity responses and modulates their allergen specific immune profiles. Mice were first sensitized with ovalbumin (OVA) and alum, twenty-four hr before beginning a series of seven daily intranasal (i.n.) allergen challenges, subsets of mice received a single i.n. or intradermal (i.d.) dose of ISS-ODN or control oligodeoxynucleotide (C-ODN), a single intraperitoneal (i.p.) injection of dexamethasone (DXM), or no intervention. Mice receiving i.d. or i.n. ISS-ODN were found to have attenuated immediate and late phase effector cell responses to i.n. OVA challenge. Specifically, ISS-ODN treated mice had less histamine and cysteinyl leukotriene release and eosinophilic inflammation in their nasal passages than mice treated with C-ODN. In addition, splenocytes from ISS-ODN but not C-ODN treated mice displayed attenuated OVA-specific interleukin (IL)-4, IL-5, and IL-13 but increased interferon-gamma responses. Finally, ISS-ODN was generally a more effective treatment than DXM, both in blunting AR hypersensitivity responses and in shifting T helper 2 Th2-biased immune parameters towards Th1 dominance. As ISS-ODN delivery rapidly attenuated effector cell responses in this AR model in an allergen independent manner, the present results suggest that therapy with ISS-ODN alone may be an effective alternative to corticosteroid medications for the clinical management of AR.

Allergen-independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis.

We examined induced expression of the 5-lipoxygenase-activating protein (FLAP), which is critical for leukotriene synthesis in mononuclear phagocytes. Prolonged exposure to the bacterial component, lipopolysaccharide (LPS), increased FLAP gene transcription, mRNA expression, and protein expression in the human monocyte-like THP-1 cell line. Activation and inhibition of the NF-kappaB pathway modulated LPS induction of FLAP gene expression. An NF-kappaB-mediated mechanism of action was supported by overexpression of dominant-negative IkappaBalpha and p50/p65 proteins. EMSA/supershift and DNase I footprint analyses revealed that p50 binds to an NF-kappaB site located in the proximal FLAP promoter, while chromatin immunoprecipitation assays demonstrated that LPS induced binding of p50 but not of p65. Moreover, EMSA/supershift analyses demonstrated that LPS induced time-dependent binding of THP-1 nuclear extracts (containing p50) to this promoter region. Mutation of the NF-kappaB site decreased basal promoter activity and abolished the p50- and p65-associated induction. EMSA/supershift analyses also demonstrated that LPS induced binding of THP-1 nuclear extracts [containing CCAAT/enhancer binding protein (C/EBP)-alpha, -delta, and -epsilon] to a C/EBP site located adjacent to the NF-kappaB site in the FLAP promoter. We conclude that LPS enhances FLAP gene expression via both NF-kappaB- and C/EBP-mediated transcriptional mechanisms in mononuclear phagocytes.

Lipopolysaccharide induces 5-lipoxygenase-activating protein gene expression in THP-1 cells via a NF-kappaB and C/EBP-mediated mechanism.

Sodium nitroprusside is used to treat hypertensive emergencies and acute heart failure. It acts by releasing nitric oxide (NO), a highly potent vasodilator, but unfortunately, for each NO molecule released, five cyanide ions are released. Thus, nitroprusside therapy is limited by cyanide toxicity. Therefore, a cyanide scavenger could be beneficial when administering nitroprusside. Hydroxocobalamin, which has a relatively high binding affinity for cyanide, has been shown to reduce cyanide levels in nitroprusside-treated patients. Cobinamide, the penultimate precursor in hydroxocobalamin biosynthesis, has a much greater affinity for cyanide than cobalamin, and binds two cyanide ions. We now show that cobinamide is highly effective in neutralizing cyanide ions released by nitroprusside in cultured mammalian cells, Drosophila melanogaster, and mice. Cobinamide also binds NO, but at molar concentrations 2.5-5 times that of nitroprusside, it did not decrease NO concentrations or the physiological effectiveness of nitroprusside. We conclude that cobinamide could be a valuable adjunct to nitroprusside therapy.

The cobalamin precursor cobinamide detoxifies nitroprusside-generated cyanide.

A limited number of nitric oxide (NO)-generating drugs are available for clinical use for acute and chronic conditions. Most of these agents are organic nitrates, which do not directly release NO; tolerance to the drugs develops, in part, as a consequence of their conversion to NO. We synthesized nitrosyl-cobinamide (NO-Cbi) from cobinamide, a structural analog of cobalamin (vitamin B12). NO-Cbi is a direct NO-releasing agent that we found was stable in water, but under physiologic conditions, it released NO with a half-life of 30 mins to 1 h. We show in five different biological systems that NO-Cbi is an effective NO-releasing drug. First, in cultured rat vascular smooth muscle cells, NO-Cbi induced phosphorylation of vasodilator-stimulated phosphoprotein, a downstream target of cGMP and cGMP-dependent protein kinase. Second, in isolated Drosophila melanogaster Malpighian tubules, NO-Cbi-stimulated fluid secretion was similar to that stimulated by Deta-NONOate and a cGMP analog. Third, in isolated mouse hearts, NO-Cbi increased coronary flow much more potently than nitroglycerin. Fourth, in contracted mouse aortic rings, NO-Cbi induced relaxation, albeit to a lesser extent than sodium nitroprusside. Fifth, in intact mice, a single NO-Cbi injection rapidly reduced blood pressure, and blood pressure returned to normal after 45 mins; repeated NO-Cbi injections induced the expected fall in blood pressure. These studies indicate that NO-Cbi is a useful NO donor that can be used experimentally in the laboratory; moreover, it could be developed into a vasodilating drug for treating hypertension and potentially other diseases such as angina and congestive heart failure.

Nitrosyl-cobinamide, a new and direct nitric oxide releasing drug effective in vivo.

Some Pseudomonas aeruginosa strains are cyanogenic, and cyanide may contribute to the bacterium's virulence. Using human isolates of P. aeruginosa, we have shown that Drosophila melanogaster suspended above cyanogenic strains become motionless and develop bradycardia and that flies injected with cyanogenic bacterial strains die more rapidly than those injected with noncyanogenic strains. Flies exposed to cyanogenic strains had high cyanide and low adenosine triphosphate (ATP) concentrations in body extracts, and treatment with a cyanide antidote equalized survival of flies injected with cyanogenic and noncyanogenic strains. P. aeruginosa PAO1 strain with a mutation in the hydrogen cyanide synthase gene cluster was much less toxic to flies than the parental cyanogenic strain or 2 knock-in strains. Transgenic flies overexpressing rhodanese, which detoxifies cyanide by converting it to thiocyanate, were resistant to cyanide and the increased virulence of cyanogenic strains. We conclude that D. melanogaster is a good model for studying cyanide produced by P. aeruginosa.

Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster.

Asthma remains a major health problem worldwide that has increased in developed countries. Much of the focus in asthma research in the past has been on adaptive, antigen-dependent immune responses. Recent work suggests that the innate, non-antigen-dependent immune system plays a critical role in asthma pathogenesis. Here we will highlight innate receptors and cells in the context of allergic responses. Reviewing animal models and human studies, we focus on interactions of innate and adaptive immunity.

Innate immunity and asthma.

Cyanide is a rapidly acting cellular poison, primarily targeting cytochrome c oxidase, and is a common occupational and residential toxin, mostly via smoke inhalation. Cyanide is also a potential weapon of mass destruction, with recent credible threats of attacks focusing the need for better treatments, as current cyanide antidotes are limited and impractical for rapid deployment in mass casualty settings.

Cobinamide is superior to other treatments in a mouse model of cyanide poisoning.

Cyanide is a component of smoke in residential and industrial fires, and accidental exposure to cyanide occurs in a variety of industries. Moreover, cyanide has the potential to be used by terrorists, particularly in a closed space such as an airport or train station. Current therapies for cyanide poisoning must be given by intravenous administration, limiting their use in treating mass casualties.

Article	Year
The yin and the yang of 5-lipoxygenase pathway activation. Molecular pharmacology\| PubMed ID: 12130669	2002
What about the role of the US Food and Drug Administration? Chest\| PubMed ID: 12426302	2002
5-lipoxygenase-activating protein gene expression. Key role of CCAAT/enhancer-binding proteins (C/EBP) in constitutive and tumor necrosis factor (TNF) alpha-induced expression in THP-1 cells... The Journal of biological chemistry\| PubMed ID: 12571239	2003
Lipopolysaccharide down-regulates the leukotriene C4 synthase gene in the monocyte-like cell line, THP-1. Journal of immunology (Baltimore, Md. : 1950)\| PubMed ID: 12574384	2003
Lipopolysaccharide down-regulates leukotriene C4 synthase gene expression in a cell-specific manner in the monocyte-like cell line, THP-1. Chest\| PubMed ID: 12629014	2003
Regulation of leukotriene C4 synthase gene expression by SP1 and SP3 in mononuclear phagocytes. Advances in experimental medicine and biology\| PubMed ID: 12664565	2002
Mechanisms of glomerular immune injury: effects of antioxidant treatment. Journal of the American Society of Nephrology : JASN\| PubMed ID: 12819234	2003
Allergen-independent immunostimulatory sequence oligodeoxynucleotide therapy attenuates experimental allergic rhinitis. Immunology\| PubMed ID: 15312141	2004
Lipopolysaccharide induces 5-lipoxygenase-activating protein gene expression in THP-1 cells via a NF-kappaB and C/EBP-mediated mechanism. American journal of physiology. Cell physiology\| PubMed ID: 15625306	2005
The cobalamin precursor cobinamide detoxifies nitroprusside-generated cyanide. Experimental biology and medicine (Maywood, N.J.)\| PubMed ID: 17526771	2007
Nitrosyl-cobinamide, a new and direct nitric oxide releasing drug effective in vivo. Experimental biology and medicine (Maywood, N.J.)\| PubMed ID: 18040067	2007
Cyanide produced by human isolates of Pseudomonas aeruginosa contributes to lethality in Drosophila melanogaster. The Journal of infectious diseases\| PubMed ID: 18199034	2008
Innate immunity and asthma. Proceedings of the American Thoracic Society\| PubMed ID: 19387027	2009
Cobinamide is superior to other treatments in a mouse model of cyanide poisoning. Clinical toxicology (Philadelphia, Pa.)\| PubMed ID: 20704457	2010
The combination of cobinamide and sulfanegen is highly effective in mouse models of cyanide poisoning. Clinical toxicology (Philadelphia, Pa.)\| PubMed ID: 21740135	2011

Timothy D. Bigby

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