LITAF mediation of increased TNF-α secretion from inflamed colonic lamina propria macrophages.

Dysregulation of TNF-α in lamina propria macrophages (LPM) is a feature of inflammatory bowel diseases (IBD). LPS-Induced-TNF-Alpha-Factor (LITAF) is a transcription factor that mediates TNF-α expression. To determine whether LITAF participates in the mediation of TNF-α expression in acutely inflamed colonic tissues, we first established the TNBS-induced colonic inflammation model in C57BL/6 mice. LPM were harvested from non-inflamed and inflamed colonic tissue and inflammatory parameters TNF-α and LITAF mRNA and protein levels were measured ex-vivo. LPM from TNBS-treated mice secreted significantly more TNF-α at basal state and in response to LPS than LPM from untreated mice (p<0.05). LITAF mRNA and protein levels were elevated in LPM from TNBS compared with untreated animals and LPS further increased LITAF protein levels in LPM from inflamed tissue (P<0.05). To further confirm the role of LITAF in acutely inflamed colonic tissues, TNBS-induced colonic inflammation was produced in LITAF macrophage specific knockout mice (LITAF mac -/- mice) and compared to wild type (WT) C57BL/6. Twenty four hours following TNBS administration, colonic tissue from LITAF mac -/- mice had less MPO activity and reduced colonic TNF-α mRNA then WT C57BL/6 mice (p<0.05). LPM harvested from LITAF mac -/- secreted significantly less TNF-α in response to LPS than wild type (WT) C57BL/6 (p<0.05). This study provides evidence that LITAF contributes to the regulation of TNF-α in LPM harvested following acute inflammation or LPS treatment paving the way for future work focusing on LITAF inhibitors in the treatment of TNF-α-mediated inflammatory conditions

Structure-Activity Relationships for 1-Alkyl-3-(1-naphthoyl)indoles at the Cannabinoid CB1 and CB2 Receptors: Steric and Electronic Effects of Naphthoyl Substituents. New Highly Selective CB2 Receptor Agonists

In an effort to improve indole-based CB2 cannabinoid receptor ligands and also to develop SAR for both the CB1 and CB2 receptors, 47 indole derivatives were prepared and their CB1 and CB2 receptor affinities were determined. The indole derivatives include 1-propyl- and 1-pentyl-3-(1-naphthoyl)indoles both with and without a 2-methyl substituent. Naphthoyl substituents include 4- and 7-alkyl groups as well as 2-, 4-, 6-, 7-methoxy and 4-ethoxy groups. The effects of these substituents on receptor affinities are discussed and structure–activity relationships are presented. In the course of this work three new highly selective CB2 receptor agonists were identified, 1-propyl-3-(4-methyl-1-naphthoylindole (JWH-120), 1-propyl-2-methyl-3-(6-methoxy-1-naphthoylindole (JWH-151), and 1-pentyl-3-(2-methoxy-1-naphthoylindole (JWH-267). GTPγS assays indicated that JWH-151 is a full agonist at CB2, while JWH-120 and JWH-267 are partial agonists. Molecular modeling and receptor docking studies were carried out on a set of 3-(4-propyl-1-naphthoyl)indoles, a set of 3-(6-methoxy-1-naphthoyl)indoles and the pair of N-pentyl-3-(2-methoxy-1-naphthoyl)indoles. Docking studies indicated that the CB1 receptor affinities of these compounds were consistent with their aromatic stacking interactions in the aromatic microdomain of the CB1 receptor

CCDC 248000: Experimental Crystal Structure Determination

Related Article: J.W.Huffman, G.Zengin, Ming-Jung Wu, Jianzhong Lu, G.Hynd, K.Bushell, A.L.S.Thompson, S.Bushell, C.Tartal, D.P.Hurst, P.H.Reggio, D.E.Selley, M.P.Cassidy, J.L.Wiley, B.R.Martin|2005|Bioorg.Med.Chem.|13|89|doi:10.1016/j.bmc.2004.09.050,An entry from the Cambridge Structural Database, the world’s repository for small molecule crystal structures. The entry contains experimental data from a crystal diffraction study. The deposited dataset for this entry is freely available from the CCDC and typically includes 3D coordinates, cell parameters, space group, experimental conditions and quality measures

LPM harvested from LITAF macrophage specific knockout mice (LITAF mac -/- mice) produce less TNF-α than LPM harvested from C57BL/6 mice.

<p><b><i>A</i></b>) Western blot of LITAF and Actin confirming the knockout of LITAF protein in peritoneal macrophages harvested from LITAF mac -/- and C57BL/6 mice. <b><i>B</i></b>) TNF-α secretion from inflamed LPM isolated from colonic tissues of LITAF mac -/- (N = 6) or wildtype C57BL/6 (N = 12) following LPS stimulation (24 hours). Data are expressed as % Inflamed C57BL/6 group ± SEM. * Statistical difference (p<0.05), # statistical difference (p<0.001).</p

LITAF protein levels are increased in LPM harvested from inflamed colonic tissue and further increased in inflamed LPM following LPS (1 ug/mL) stimulation (N = 5).

<p><b><i>A</i></b>) Western blot of LITAF levels and GAPDH levels in freshly harvested LPM from Non-Inflamed and Inflamed colonic tissue. The graph shows the optical density (OD) of LITAF levels normalized to GAPDH levels. * Statistical difference from Non-Inflamed group (p<0.05). B) Western blot of LITAF levels and β-Actin levels in LPM harvested from inflamed colonic tissue stimulated with LPS ex vivo compared to LPM harvested from inflamed colonic tissue, but not treated with LPS (Non-treated). The graph show OD of LITAF levels normalized to β-Actin levels. * Statistical difference (p<0.05).</p

LITAF macrophage specific knockout mice (LITAF mac -/-) show reduced changes in bodyweight and MPO following TNBS administration.

<p><b><i>A</i></b>) Following TNBS (3.0 mg) administration to LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 6), bodyweights were measured 24 hours later. Data are expressed as % initial (day 0) bodyweight. * Statistical difference (p<0.05), # statistical difference (p<0.001). B) Following TNBS (3.0 mg) administration to LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 6), colonic tissue was collected and assessed for MPO activity. Data are expressed as % Non-inflamed MPO activity. ^# Statistical difference (p<0.001).</p

TNF-α message levels from inflamed colonic tissue harvested from LITAF macrophage specific mice (LITAF mac -/- mice) is reduced compared to C57BL/6 mice.

<p>TNF-α message levels were measured in colonic tissue following TNBS administration (0.3 mg) for 24 hrs in LITAF mac -/- mice (N = 6) and C57BL/6 mice (N = 12). Data are expressed as % Non-Inflamed control ± SEM. * Statistical difference (p<0.05).</p

TNBS-induced inflammation in C57BL/6 Mice.

<p>Inflammation was induced by TNBS (1.5 mg, in 50% ethanol solution) and animals were monitored for: A) Colonic tissue was collected and assessed for MPO activity (N = 4, except Day 1, N = 3). Data are expressed as % Day 0 Control ± SEM. ^# Statistical difference (p<0.001). <b><i>B</i></b>) Representative histological images of colonic tissue from C57BL/6 mice 24 hours following TNBS administration showing colonic inflammation (Mag 100×).</p