Behavioral consequences of the downstream products of ethanol metabolism involved in alcohol use disorder.

Research concerning Alcohol Use Disorder (AUD) has previously focused primarily on either the behavioral or chemical consequences experienced following ethanol intake, but these areas of research have rarely been considered in tandem. Compared with other drugs of abuse, ethanol has been shown to have a unique metabolic pathway once it enters the body, which leads to the formation of downstream metabolites which can go on to form biologically active products. These metabolites can mediate a variety of behavioral responses that are commonly observed with AUD, such as ethanol intake, reinforcement, and vulnerability to relapse. The following review considers the preclinical and chemical research implicating these downstream products in AUD and proposes a chemobehavioral model of AUD.

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.

Carcinoembryonic antigen-stimulated THP-1 macrophages activate endothelial cells and increase cell-cell adhesion of colorectal cancer cells.

The liver is the most common site for metastasis by colorectal cancer, and numerous studies have shown a relationship between serum carcinoembryonic antigen (CEA) levels and metastasis to this site. CEA activates hepatic macrophages or Kupffer cells via binding to the CEA receptor (CEA-R), which results in the production of cytokines and the up-regulation of endothelial adhesion molecules, both of which are implicated in hepatic metastasis. Since tissue macrophages implicated in the metastatic process can often be difficult to isolate, the aim of this study was to develop an in vitro model system to study the complex mechanisms of CEA-induced macrophage activation and metastasis. Undifferentiated, human monocytic THP-1 (U-THP) cells were differentiated (D-THP) to macrophages by exposure to 200 ng/ml phorbol myristate acetate (PMA) for 18 h. Immunohistochemistry showed two CEA-R isoforms present in both U- and D-THP cells. The receptors were localized primarily to the nucleus in U-THP cells, while a significant cell-surface presence was observed following PMA-differentiation. Incubation of D-THP-1 cells with CEA resulted in a significant increase in tumor necrosis factor-alpha (TNF-alpha) release over 24 h compared to untreated D-THP-1 or U-THP controls confirming the functionality of these cell surface receptors. U-THP cells were unresponsive to CEA. Attachment of HT-29 cells to human umbilical vein endothelial cells significantly increased at 1 h after incubation with both recombinant TNF-alpha and conditioned media from CEA stimulated D-THP cells by six and eightfold, respectively. This study establishes an in vitro system utilizing a human macrophage cell line expressing functional CEA-Rs to study activation and signaling mechanisms of CEA that facilitate tumor cell attachment to activated endothelial cells. Utilization of this in vitro system may lead to a more complete understanding of the expression and function of CEA-R and facilitate the design of anti-CEA-R therapeutic modalities that may significantly diminish the metastatic potential of CEA overexpressing colorectal tumors.

A fluorescence-based assay for fatty acid amide hydrolase compatible with high-throughput screening.

A novel fluorescent assay to continuously monitor fatty acid amide hydrolase (FAAH) activity that is simple, sensitive, and amenable to high-throughput screening (HTS) of compound libraries is described in this article. Stable Chinese hamster ovary (CHO) cell lines expressing either human FAAH or an inactive mutant, FAAH-S241A, were established. Arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a novel fluorogenic substrate for FAAH, was designed and synthesized. FAAH catalyzes the hydrolysis of AAMCA to generate arachidonic acid and a highly fluorescent 7-amino, 4-methyl coumarin (AMC). The assay was done at 25 degrees C by incubating whole cell or microsomal preparations from FAAH-expressing cells with AAMCA. Release of AMC was monitored continuously using a fluorometer. Microsomal FAAH catalyzed the hydrolysis of AAMCA with an apparent K(m) of 0.48muM and V(max) of 58pmolmin(-1)mgprotein(-1). The assay is specific for FAAH given that microsomes prepared from cells expressing FAAH-S241A or vector alone had no significant activity against AAMCA. Furthermore, the activity was inhibited by URB-597, an FAAH-specific inhibitor, in a concentration-dependent manner with an IC(50) of 33.5nM. The assay was optimized for HTS and had a Z' value ranging from 0.7 to 0.9. The assay is also compatible with ex vivo analysis of FAAH activity.