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GCs are highly effective in treating a wide range of inflammatory diseases but are limited in their ability to control neutrophilic lung inflammation in conditions such as COPD. Neutrophil apoptosis, a central feature of inflammation resolution, is delayed in response to microenvironmental cues, such as hypoxia and inflammatory cytokines, present at inflamed sites. GCs delay neutrophil apoptosis in vitro, and this may therefore limit the ability of GCs to control neutrophilic inflammation. This study assesses the effect GCs have on hypoxia- and inflammatory cytokine-induced neutrophil survival. Human neutrophils were treated with GCs in the presence or absence of GM-CSF or inflammatory macrophage-CM at a range of oxygen concentrations (21-1% oxygen). Neutrophil apoptosis and survival were assessed by flow cytometry and morphological analysis and neutrophil function, by stimulus-induced shape change and respiratory burst. Dexamethasone promoted neutrophil survival at 21%, 10%, and 5% oxygen but not at 1% oxygen. Interestingly, GM-CSF and inflammatory CM increased neutrophil survival significantly, even at 1% oxygen, with cells remaining functionally active at 96 h. Dexamethasone was able to reduce the prosurvival effect of GM-CSF and inflammatory CM in a hypoxic environment. In conclusion, we found that GCs do not augment neutrophil survival in the presence of severe hypoxia or proinflammatory mediators. This suggests that GCs would not promote neutrophil survival at sites of inflammation under these conditions.

Inflammation is a beneficial host reaction to tissue damage and has the essential primary purpose of restoring tissue homeostasis. Inflammation plays a major role in containing and resolving infection and may also occur under sterile conditions. The cardinal signs of inflammation dolor, calor, tumor and rubor are intrinsically associated with events including vasodilatation, edema and leukocyte trafficking into the site of inflammation. If uncontrolled or unresolved, inflammation itself can lead to further tissue damage and give rise to chronic inflammatory diseases and autoimmunity with eventual loss of organ function. It is now evident that the resolution of inflammation is an active continuous process that occurs during an acute inflammatory episode. Successful resolution requires activation of endogenous programs with switch from production of pro-inflammatory towards pro-resolving molecules, such as specific lipid mediators and annexin A1, and the non-phlogistic elimination of granulocytes by apoptosis with subsequent removal by surrounding macrophages. These processes ensure rapid restoration of tissue homeostasis. Here, we review recent advances in the understanding of resolution of inflammation, highlighting the pharmacological strategies that may interfere with the molecular pathways which control leukocyte survival and clearance. Such strategies have proved beneficial in several pre-clinical models of inflammatory diseases, suggesting that pharmacological modulation of the resolution process may be useful for the treatment of chronic inflammatory diseases in humans., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The technical limitations of isolating neutrophils without contaminating leukocytes, while concurrently minimizing neutrophil activation, is a barrier to determining specific neutrophil functions. We aimed to assess the use of FACS for generating highly pure quiescent neutrophil populations in an antibody-free environment. Peripheral blood human granulocytes and murine bone marrow-derived neutrophils were isolated by discontinuous Percoll gradient and flow-sorted using FSC/SSC profiles and differences in autofluorescence. Postsort purity was assessed by morphological analysis and flow cytometry. Neutrophil activation was measured in unstimulated-unsorted and sorted cells and in response to fMLF, LTB4, and PAF by measuring shape change, CD62L, and CD11b expression; intracellular calcium flux; and chemotaxis. Cytokine production by human neutrophils was also determined. Postsort human neutrophil purity was 99.95% (sem=0.03; n=11; morphological analysis), and 99.68% were CD16(+ve) (sem=0.06; n=11), with similar results achieved for murine neutrophils. Flow sorting did not alter neutrophil activation or chemotaxis, relative to presorted cells, and no differences in response to agonists were observed. Stimulated neutrophils produced IL-1β, although to a lesser degree than CXCL8/IL-8. The exploitation of the difference in autofluorescence between neutrophils and eosinophils by FACS is a quick and effective method for generating highly purified populations for subsequent in vitro study.

Neutrophils play a pivotal role in the pathophysiology of multiple human inflammatory diseases. Novel pharmacological strategies which drive neutrophils to undergo programmed cell death (apoptosis) have been shown to facilitate the resolution of inflammation. Both the cyclin-dependent kinase inhibitor (CDKi) R-roscovitine and nitric oxide (NO) have been shown to enhance apoptosis of neutrophils and possess pro-resolution of inflammation properties. In order to search for new multi-target pro-resolution derivatives, here we describe the design, synthesis and investigation of the biological potential of a small series of hybrid compounds obtained by conjugating R-roscovitine with two different NO-donor moieties (compounds 2, 9a, 9c). The synthesized compounds were tested as potential pro-resolution agents, with their ability to promote human neutrophil apoptosis evaluated. Both compound 9a and 9c showed an increased pro-apoptotic activity when compared with either R-roscovitine or structurally related compounds devoid of the ability to release NO (des-NO analogues). Inhibition of either NO-synthase or soluble guanylate cyclase did not affect the induction of apoptosis by the R-roscovitine derivatives, similar to that reported for other classes of NO-donors. In contrast the NO scavenger PTIO prevented the enhanced apoptosis seen with compound 9a over R-roscovitine. These data show that novel compounds such as CDKi-NO-donor hybrids may have additive pro-resolution of inflammation effects., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Background: Activation of NADPH oxidase is required for neutrophil extracellular trap (NET) formation. Protein kinase C (PKC) is an upstream mediator of NADPH oxidase activation and thus likely to have a role in NET formation., Methods: Pharmacological inhibitors were used to block PKC activity in neutrophils harvested from healthy donor blood., Results: Pan PKC inhibition with Ro-31-8220 (p<0.001), conventional PKC inhibition with Go 6976 (p<0.001) and specific PKCβ inhibition with LY333531 (p<0.01) blocked NET formation in response to PMA. Inhibition of novel and atypical PKC had no effect. LY333531 blocked NET induction by the diacylglycerol analogue OAG (conventional PKC activator) (p<0.001)., Conclusions: Conventional PKCs have a prominent role in NET formation. Furthermore PKCβ is the major isoform implicated in NET formation.

Neutrophil apoptosis and subsequent nonphlogistic clearance by surrounding phagocytes are key to the successful resolution of neutrophilic inflammation, with dysregulated apoptosis reported in multiple human inflammatory diseases. Enhancing neutrophil apoptosis has proresolution and anti-inflammatory effects in preclinical models of inflammation. Here we investigate the ability of the flavones apigenin, luteolin, and wogonin to induce neutrophil apoptosis in vitro and resolve neutrophilic inflammation in vivo. Human neutrophil apoptosis was assessed morphologically and by flow cytometry following incubation with apigenin, luteolin, and wogonin. All three flavones induced time- and concentration-dependent neutrophil apoptosis (apigenin, EC=12.2 μM; luteolin, EC=14.6 μM; and wogonin, EC=28.9 μM). Induction of apoptosis was caspase dependent, as it was blocked by the broad-spectrum caspase inhibitor Q-VD-OPh and was associated with both caspase-3 and caspase-9 activation. Flavone-induced apoptosis was preceded by down-regulation of the prosurvival protein Mcl-1, with proteasomal inhibition preventing flavone-induced Mcl-1 down-regulation and apoptosis. The flavones abrogated the survival effects of mediators that prolong neutrophil life span, including lipoteichoic acid, peptidoglycan, dexamethasone, and granulocyte-macrophage colony stimulating factor, by driving apoptosis. Furthermore, wogonin enhanced resolution of established neutrophilic inflammation in a zebrafish model of sterile tissue injury. Wogonin-induced resolution was dependent on apoptosis in vivo as it was blocked by caspase inhibition. Our data show that the flavones induce neutrophil apoptosis and have potential as neutrophil apoptosis-inducing anti-inflammatory, proresolution agents.

Terminally differentiated neutrophils are short-lived but the key effector cells of the innate immune response, and have a prominent role in the pathogenesis and propagation of many inflammatory diseases. Delayed apoptosis, which is responsible for their extended longevity, is critically dependent on a balance of intracellular survival versus pro-apoptotic proteins. Here, we elucidate the mechanism by which the cyclin-dependent kinase (CDK) inhibitor drugs such as R-roscovitine and DRB (5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole) mediate neutrophil apoptosis. We demonstrate (by a combination of microarray, confocal microscopy, apoptosis assays and western blotting) that the phosphorylation of RNA polymerase II by CDKs 7 and 9 is inhibited by R-roscovitine and that specific effects on neutrophil transcriptional capacity are responsible for neutrophil apoptosis. Finally, we show that specific CDK7 and 9 inhibition with DRB drives resolution of neutrophil-dominant inflammation. Thus, we highlight a novel mechanism that controls both primary human neutrophil transcription and apoptosis that could be targeted by selective CDK inhibitor drugs to resolve established inflammation.

Dysregulation of inflammation is central to the pathogenesis of innumerable human diseases. Understanding and tracking the critical events in inflammation are crucial for disease monitoring and pharmacological drug discovery and development. Recent progress in molecular imaging has provided novel insights into spatial associations, molecular events and temporal sequelae in the inflammatory process. While remaining a burgeoning field in pre-clinical research, increasing application in man affords researchers the opportunity to study disease pathogenesis in humans in situ thereby revolutionizing conventional understanding of pathophysiology and potential therapeutic targets. This review provides a description of commonly used molecular imaging modalities, including optical, radionuclide and magnetic resonance imaging, and details key advances and translational opportunities in imaging inflammation from initiation to resolution., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: Eosinophils not only defend the body against parasitic infection but are also involved in pathological inflammatory allergic diseases such as asthma, allergic rhinitis and contact dermatitis. Clearance of apoptotic eosinophils by macrophages is a key process responsible for driving the resolution of eosinophilic inflammation and can be defective in allergic diseases. However, enhanced resolution of eosinophilic inflammation by deliberate induction of eosinophil apoptosis using pharmacological agents has not been previously demonstrated. Here we investigated the effect of a novel cyclin-dependent kinase inhibitor drug, AT7519, on human and mouse eosinophil apoptosis and examined whether it could enhance the resolution of a murine model of eosinophil-dominant inflammation in vivo., Methodology/principal Findings: Eosinophils from blood of healthy donors were treated with AT7519 and apoptosis assessed morphologically and by flow-cytometric detection of annexin-V/propidium iodide staining. AT7519 induced eosinophil apoptosis in a concentration dependent manner. Therapeutic administration of AT7519 in eosinophil-dominant allergic inflammation was investigated using an established ovalbumin-sensitised mouse model of allergic pleurisy. Following ovalbumin challenge AT7519 was administered systemically at the peak of pleural inflammation and inflammatory cell infiltrate, apoptosis and evidence of macrophage phagocytosis of apoptotic eosinophils assessed at appropriate time points. Administration of AT7519 dramatically enhanced the resolution of allergic pleurisy via direct induction of eosinophil apoptosis without detriment to macrophage clearance of these cells. This enhanced resolution of inflammation was shown to be caspase-dependent as the effects of AT7519 were reduced by treatment with a broad spectrum caspase inhibitor (z-vad-fmk)., Conclusions: Our data show that AT7519 induces human eosinophil apoptosis and enhances the resolution of a murine model of allergic pleurisy by inducing caspase-dependent eosinophil apoptosis and enhancing macrophage ingestion of apoptotic eosinophils. These findings demonstrate the utility of cyclin-dependent kinase inhibitors such as AT7519 as potential therapeutic agents for the treatment of eosinophil dominant allergic disorders.

Small-cell lung cancer (SCLC) is a particularly aggressive cancer, which metastasises early. Despite initial sensitivity to radio- and chemo-therapy, it invariably relapses, so that the 2-year survival remains less than 5%. Neuropeptides particularly arginine vasopressin (AVP) and gastrin-releasing peptide (GRP) act as autocrine and paracrine growth factors and the expression of these and their receptors are a hallmark of the disease. Substance-P analogues including [D-Arg1,D-Phe5,D-Trp7,9,Leu11]-substance-P (SP-D) and [Arg6,D-Trp7,9,NmePhe8]-substance-P (6-11) (SP-G) inhibit the growth of SCLC cells by modulating neuropeptide signalling. We show that GRP and V1A receptors expression leads to the development of a transformed phenotype. Addition of neuropeptide provides some protection from etoposide-induced cytotoxicity. Receptor expression also leads to an increased sensitivity to substance-P analogue-induced growth inhibition. We show that SP-D and SP-G act as biased agonists at GRP and V1A receptors causing blockade of Gq-mediated Ca2+ release while directing signalling to activate ERK via a pertussis toxin-sensitive pathway. This is the first description of biased agonism at V1A receptors. This unique pharmacology governs the antiproliferative properties of these agents and highlights their potential therapeutic potential for the treatment of SCLC and particularly in tumours, which have developed resistance to chemotherapy.

A critical evaluation of the available information demonstrates that reactions to natural color additives are rare. Studies of turmeric and carotenoid pigments administered in mixtures with other food colorings failed to definitely identify reactions to either color additive. For carotenoids, the one case report of an adverse reaction was not conclusive. An anaphylactic reaction to saffron does suggest an IgE-mediated reaction, but the high use of saffron as compared with this single report of an adverse reaction suggests that sensitivity to saffron is extremely rare. Numerous reports of reactions to grapes or grape products have been reported in the literature, but no reports of sensitivities to grape skin extract or grape color extract were found. In rare cases, annatto dye may provoke a severe, adverse reaction in individuals with an uncommon hypersensitivity, and may aggravate the symptoms of patients suffering from recurrent urticaria. In its long history of use, there has been only one reported case of anaphylaxis resulting from the ingestion of annatto. Studies designed to investigate the role of annatto in recurrent urticaria sufferers were limited due to the absence of double-blind challenge and placebo controls. A number of cases of adverse reactions to carmine following ingestion have been reported in the literature. These adverse reactions suggest an IgE-mediated hypersensitivity. In many of the reported cases, the cause of sensitization to carmine was topical exposure from the use of carmine-containing cosmetics or occupational exposure to carmine and not from ingestion of carmine-containing foods and beverages. Following sensitization, affected individuals would be sensitive to carmine and the amounts present in foods and beverages could elicit allergic reactions. It is not known whether all individuals with carmine sensitivity induced through topical use are sensitive to the ingestion of carmine in foods. However, reactions to carmine solely because of ingestion are likely to be exceedingly rare due to the low use levels of carmine in foods and beverages. Despite their widespread use in food products, few reports of allergic reactions following ingestion have been reported for the majority of natural color additives. It is concluded that the ingestion of natural color additives presents a very low risk of provoking adverse reactions.

This publication is the fourth in a series of safety evaluations performed by the Expert Panel of the Flavour and Extract Manufacturers' Association (FEMA). In 1993, the Panel initiated a comprehensive program to re-evaluate the safety of more than 1700 GRAS flavouring substances under conditions of intended use. In this review, scientific data relevant to the safety evaluation of trans-anethole (i.e. 4-methoxypropenylbenzene) as a flavouring substance is critically evaluated by the FEMA Expert Panel. The evaluation uses a mechanism-based approach in which production of the hepatotoxic metabolite anethole epoxide (AE) is used to interpret the pathological changes observed in different species and sexes of laboratory rodents in chronic and subchronic dietary studies. Female Sprague Dawley rats metabolize more trans-anethole to AE than mice or humans and, therefore, are the most conservative model for evaluating the potential for AE-induced hepatotoxicity in humans exposed to trans-anethole from use as a flavouring substance. At low levels of exposure, trans-anethole is efficiently detoxicated in rodents and humans primarily by O-demethylation and omega-oxidation, respectively, while epoxidation is only a minor pathway. At high dose levels in rats, particularly females, a metabolic shift occurs resulting in increased epoxidation and formation of AE. Lower activity of the "fast" acting detoxication enzyme epoxide hydrolase in the female is associated with more pronounced hepatotoxicity compared to that in the male. The continuous intake of high dose levels of trans-anethole (i.e. cumulative exposure) has been shown in dietary studies to induce a continuum of cytotoxicity, cell necrosis and cell proliferation. In chronic dietary studies in rats, hepatotoxicity was observed when the estimated daily hepatic production of AE exceeded 30 mg AE/kg body weight. In female rats, chronic hepatotoxicity and a low incidence of liver tumours were reported at a dietary intake of 550 mg trans-anethole/kg body weight/day. Under these conditions, daily hepatic production of AE exceeded 120 mg/kg body weight. Additionally, neither trans-anethole nor AE show any evidence of genotoxicity. Therefore, the weight of evidence supports the conclusion that hepatocarcinogenic effects in the female rat occur via a non-genotoxic mechanism and are secondary to hepatotoxicity caused by continuous exposure to high hepatocellular concentrations of AE. trans-Anethole was reaffirmed as GRAS (GRASr) based on (1) its low level of flavour intake (54 microg/kg body weight/day); (2) its metabolic detoxication pathway in humans at levels of exposure from use as a flavouring substance; (3) the lack of mutagenic or genotoxic potential; (4) the NOAEL of 120 mg trans-anethole/kg body weight/day in the female rat reported in a 2 + -year study which produces a level of AE (i.e. 22 mg AE/kg body weight/day) at least 10,000 times the level (0.002 mg AE/kg body weight day) produced from the intake of trans-anethole from use as a flavouring substance; and (5) the conclusion that a slight increase in the incidence of hepatocellular tumours in the high dose group (550 mg trans-anethole/kg body weight/day) of female rats was the only significant neoplastic finding in a 2+ -year dietary study. This finding is concluded to be secondary to hepatotoxicity induced by high hepatocellular concentrations of AE generated under conditions of the study. Because trans-anethole undergoes efficient metabolic detoxication in humans at low levels of exposure, the neoplastic effects in rats associated with dose-dependent hepatotoxicity are not indicative of any significant risk to human health from the use of trans-anethole as a flavouring substance.