Development and validation of an assay method for the determination of trifluoroacetic acid in a cyclosporin-like drug.

An improved method for the assay of trifluoroacetic acid (TFA) in a cyclosporin-like drug substance is presented, based on ion chromatography with suppressed conductivity detection. Column fouling by the drug molecule is avoided by use of a sample preparation method in which the drug substance is precipitated at alkaline pH whilst the TFA remains in solution. The new method requires a smaller sample mass than a previous method based on headspace-GC-FID whilst achieving an improvement in sensitivity. During validation, the method's performance was found to be consistent with usual acceptance criteria, and the method was found to be robust in routine use.

Expanding the substantial interactome of NEMO using protein microarrays.

Signal transduction by the NF-kappaB pathway is a key regulator of a host of cellular responses to extracellular and intracellular messages. The NEMO adaptor protein lies at the top of this pathway and serves as a molecular conduit, connecting signals transmitted from upstream sensors to the downstream NF-kappaB transcription factor and subsequent gene activation. The position of NEMO within this pathway makes it an attractive target from which to search for new proteins that link NF-kappaB signaling to additional pathways and upstream effectors. In this work, we have used protein microarrays to identify novel NEMO interactors. A total of 112 protein interactors were identified, with the most statistically significant hit being the canonical NEMO interactor IKKbeta, with IKKalpha also being identified. Of the novel interactors, more than 30% were kinases, while at least 25% were involved in signal transduction. Binding of NEMO to several interactors, including CALB1, CDK2, SAG, SENP2 and SYT1, was confirmed using GST pulldown assays and coimmunoprecipitation, validating the initial screening approach. Overexpression of CALB1, CDK2 and SAG was found to stimulate transcriptional activation by NF-kappaB, while SYT1 overexpression repressed TNFalpha-dependent NF-kappaB transcriptional activation in human embryonic kidney cells. Corresponding with this finding, RNA silencing of CDK2, SAG and SENP2 reduced NF-kappaB transcriptional activation, supporting a positive role for these proteins in the NF-kappaB pathway. The identification of a host of new NEMO interactors opens up new research opportunities to improve understanding of this essential cell signaling pathway.

Lipoxin A4: anti-inflammatory and anti-angiogenic impact on endothelial cells.

Lipoxins (LX) are a class of eicosanoid that possesses a wide spectrum of antiinflammatory and proresolution bioactions. Here we have investigated the impact of the endogenously produced eicosanoid LXA(4) on endothelial cell inflammatory, proliferative, and antigenic responses. Using HUVECs we demonstrate that LXA(4) inhibits vascular endothelial growth factor (VEGF)-stimulated inflammatory responses including IL-6, TNF-alpha, IFN-gamma and IL-8 secretion, as well as endothelial ICAM-1 expression. Interestingly, LXA(4) up-regulated IL-10 production from HUVECs. Consistent with these antiinflammatory and proresolution responses to LXA(4), we demonstrate that LXA(4) inhibited leukotriene D(4) and VEGF-stimulated proliferation and angiogenesis as determined by tube formation of HUVECs. We have explored the underlying molecular mechanisms and demonstrate that LXA(4) pretreatment is associated with the decrease of VEGF-stimulated VEGF receptor 2 (KDR/FLK-1) phosphorylation and downstream signaling events including activation of phospholipase C-gamma, ERK1/2, and Akt.

Identification of polyubiquitin binding proteins involved in NF-kappaB signaling using protein arrays.

Attachment of ubiquitin to proteins represents a central mechanism for the regulation of protein metabolism and function. In the NF-kappaB pathway, binding of NEMO to polyubiquitinated substrates initiates the pathway in response to cellular stimuli. Other polyubiquitin binding proteins can antagonize this pathway by competing with NEMO for polyubiquitin. We have used protein arrays to identify polyubiquitin binding proteins that regulate NF-kappaB activity. Using polyubiquitin as bait, protein arrays were screened and polyubiquitin binders identified. Novel polyubiquitin binders AWP1, CALCOCO2, N4BP1, RIO3, TEX27, TTC3, UBFD1 and ZNF313 were identified using this approach, while known NF-kappaB regulators including NEMO, A20, ABIN-1, ABIN-2, optineurin and p62 were also identified. Overexpressed AWP1 and RIO3 repressed NF-kappaB activity in a manner similar to optineurin, while siRNAs directed against AWP1 and RIO3 also reduced NF-kappaB activity. TNFalpha-dependent degradation of IkappaBalpha was also suppressed by overexpression of AWP1 and RIO3, possibly due to the polyubiquitin binding activity of these proteins. Protein array screening using polyubiquitin enabled rapid identification of many known and novel polyubiquitin binding proteins and the identification of novel NF-kappaB regulators.

Aromatic lipoxin A4 and lipoxin B4 analogues display potent biological activities.

Lipoxins are a group of biologically active eicosanoids typically formed by transcellular lipoxygenase activity. Lipoxin A4 (LXA4) and Lipoxin B4 (LXB4) biosynthesis has been detected in a variety of inflammatory conditions. The native lipoxins LXA4 and LXB4 demonstrate potent antiinflammatory and proresolution bioactions. However, their therapeutic potential is compromised by rapid metabolic inactivation by PG dehydrogenase-mediated oxidation and reduction. Here we report on the stereoselective synthesis of aromatic LXA4 and LXB4 analogues by employing Sharpless epoxidation, Pd-mediated Heck coupling, and diastereoselective reduction as the key transformations. Subsequent biological testing has shown that these analogues display potent biological activities. Phagocytic clearance of apoptotic leukocytes plays a critical role in the resolution of inflammation. Both LXA4 analogues (1R)-3a and (1S)-3a were found to stimulate a significant increase in phagocytosis of apoptotic polymorphonuclear leukocytes (PMN) by macrophages, with comparable efficacy to the effect of native LXA4, albeit greater potency, while the LXB4 analogue also stimulated phagocytosis with a maximum effect observed at 10-11 M. LX-stimulated phagocytosis was associated with rearrangement of the actin cytoskeleton consistent with that reported for native lipoxins. Using zymosan-induced peritonitis as a murine model of acute inflammation (1R)-3a significantly reduced PMN accumulation.

Annexin-1 and peptide derivatives are released by apoptotic cells and stimulate phagocytosis of apoptotic neutrophils by macrophages.

The resolution of inflammation is a dynamically regulated process that may be subverted in many pathological conditions. Macrophage (Mphi) phagocytic clearance of apoptotic leukocytes plays an important role in the resolution of inflammation as this process prevents the exposure of tissues at the inflammatory site to the noxious contents of lytic cells. It is increasingly appreciated that endogenously produced mediators, such as lipoxins, act as potent regulators (nanomolar range) of the phagocytic clearance of apoptotic cells. In this study, we have investigated the intriguing possibility that apoptotic cells release signals that promote their clearance by phagocytes. We report that conditioned medium from apoptotic human polymorphonuclear neutrophils (PMN), Jurkat T lymphocytes, and human mesangial cells promote phagocytosis of apoptotic PMN by Mphi and THP-1 cells differentiated to a Mphi-like phenotype. This prophagocytic activity appears to be dose dependent, sensitive to the caspase inhibitor zVAD-fmk, and is associated with actin rearrangement and release of TGF-beta1, but not IL-8. The prophagocytic effect can be blocked by the formyl peptide receptor antagonist Boc2, suggesting that the prophagocytic factor(s) may interact with the lipoxin A(4) receptor, FPRL-1. Using nanoelectrospray liquid chromatography mass spectrometry and immunodepletion and immunoneutralization studies, we have ascertained that annexin-1 and peptide derivatives are putative prophagocytic factors released by apoptotic cells that promote phagocytosis of apoptotic PMN by M[phi] and differentiated THP-1 cells. These data highlight the role of annexin-1 and peptide derivatives in promoting the resolution of inflammation and expand on the therapeutic anti-inflammatory potential of annexin-1.

Lipoxins and annexin-1: resolution of inflammation and regulation of phagocytosis of apoptotic cells.

Phagocytosis of apoptotic cells plays a pivotal role in developmental processes and in the resolution of inflammation. Failed or delayed clearance of apoptotic cells can result in chronic inflammation. Furthermore, clearance of apoptotic cells leads to release of anti-inflammatory cytokines. Recent evidence has shown that endogenous mediators can regulate such processes. In this article, we will review the recognition and signaling mechanisms involved in the phagocytosis of apoptotic cells as well as the role of endogenous compounds that play a relevant role in the modulation of inflammation. The first of these endogenous local mediators to be described are lipoxins (LXs). LXs and aspirin-triggered LXs (ATLs) are considered to act as "braking signals" in inflammation, limiting the entrance of leukocytes to the site of inflammation through inhibition of neutrophil and eosinophil trafficking. LXs are actively involved in resolution of inflammation, stimulating nonphlogistic phagocytosis of apoptotic cells by macrophages. LXA4 and ATLs elicit cellular responses by interacting with a G protein -coupled receptor (ALXR) that is expressed in various cell types. ALXR, originally identified as a low-affinity N-formyl-methionyl-leucyl-phenylalanine receptor-like 1, can bind pleiotropic ligands, i.e., both lipid and peptides, including the glucocorticoid-inducible protein, annexin-1. Interestingly, a role for annexin-1 in phagocytosis has recently emerged. Understanding the role and mechanism of the powerful anti-inflammatory and proresolution actions of endogenous compounds can be a useful tool in the development of potential therapeutics in resolving inflammatory diseases.