Use of Early Passage Fetal Intestinal Epithelial Cells in Semi–High-Throughput Screening Assays: An Approach to Identify New Innate Immune System Adjuvants

Abstract

Innate immune system stimulants (innate adjuvants) offer complementary approaches to vaccines and antimicrobial compounds to increase host resistance to infection. The authors established fetal bovine intestinal epithelial cell (BIEC) cultures to screen natural product and synthetic compound libraries for novel mucosal adjuvants. They showed that BIECs from fetal intestine maintained an in vivo phenotype as reflected in cytokeratin expression, expression of antigens restricted to intestinal enterocytes, and induced interleukin-8 (IL-8) production. BIECs could be infected by and support replication of bovine rotavirus. A semi-high-throughput enzyme-linked immunosorbent assay-based assay that measured IL-8 production by BIECs was established and used to screen commercially available natural compounds for novel adjuvant activity. Five novel hits were identified, demonstrating the utility of the assay for selecting and screening new epithelial cell adjuvants. Although the identified compounds had not previously been shown to induce IL-8 production in epithelial cells, other known functions for 3 of the 5 were consistent with this activity. Statistical analysis of the throughput data demonstrated that the assay is adaptable to a high-throughput format for screening both synthetic and natural product derived compound libraries. (Journal of Biomolecular Screening 2006:664-671)

The Effects of Psychostimulant Drugs on Blood Brain Barrier Function and Neuroinflammation

“…Cytokines [e.g., tumor necrosis factor alpha (TNFα), interleukin (IL)-1β and IL-6] and the CCL2 chemokine are increased in serum, neuronal tissue, and cerebrospinal fluid in several CNS disorders including traumatic brain injury (Morganti-Kossman et al, 1997), HIV-associated encephalitis (Cartier et al, 2005), and Huntington disease (Stolp and Dziegielewska, 2009). These neuroinflammatory factors likely contribute to BBB breakdown occurring in these disorders through activity at their receptors on BECs and other BBB cell types (Buckner et al, 2006). Peripheral immune cells can also trigger increased production of inflammatory factors (Verma and Szmitko, 2006; Fletcher et al, 2009), neurotransmitters (e.g., glutamate), neurotrophic factors (e.g., vascular endothelial growth factor), and proteases (e.g., matrix metallopeptidase (MMP-9; Petty and Lo, 2002) from BECs and astrocytes.…”

Primary Leukocyte Screens for Innate Immune Agonists

“…The most commonly used high-throughput screening (HTS) technique for identification of TLR agonists is measurement of NFκB activity in a cell line (typically HEK293) overexpressing a specific TLR gene. 10,11 However, this technique is relatively insensitive; is prone to false positives from non-TLR-specific NFκB activation; is limited to the identification of immunostimulants that bind to a single, specific TLR; and has been shown not to fully recapitulate the in vivo responsiveness of TLRs. 12 Other assay systems for the measurement of TLR activation have been reported, 11,13 although these assays have been employed primarily for cell biology or mechanism of action studies and are not well suited for screening or structure-activity relationship applications.…”

A mast cell degranulation screening assay for the identification of novel mast cell activating agents

“…Others have used a semi-high-throughput screening assay 37 to identify possible adjuvant compounds based on induction of interleukin 8 (IL-8) by bovine epithelial cells. Screening a natural compound library containing 280 compounds revealed 3 compounds that consistently induced IL-8 production by bovine intestinal epithelial cells.…”

“…Screening a natural compound library containing 280 compounds revealed 3 compounds that consistently induced IL-8 production by bovine intestinal epithelial cells. Our mast cell degranulation screening assay also utilizes live cells but has an important difference when compared to the epithelial cell screening assay used by others 37 . The epithelial screening assay depends upon the incubation of the cells with the test compound for 24 hours followed by testing of the culture supernatant for IL-8 produced in response to the test compound.…”