Identification of gene expression indicators for thyroid axis disruption in a Xenopus laevis metamorphosis screening assay. Part 1. Effects on the brain.

Thyroid hormones (TH), thyroxine (T(4)) and 3,5,3'-triiodothyronine (T(3)), play crucial roles in regulation of growth, development and metabolism in vertebrates and their actions are targets for endocrine disruptive agents. Perturbations in TH action can contribute to the development of disease states and the US Environmental Protection Agency is developing a high throughput screen using TH-dependent amphibian metamorphosis as an assay platform. Currently this methodology relies on external morphological endpoints and changes in central thyroid axis parameters. However, exposure-related changes in gene expression in TH-sensitive tissue types that occur over shorter time frames have the potential to augment this screen. This study aims to characterize and identify molecular markers in the tadpole brain. Using a combination of cDNA array analysis and real time quantitative polymerase chain reaction (QPCR), we examine the brain of tadpoles following 96 h of continuous exposure to T(3), T(4), methimazole, propylthiouracil, or perchlorate. This tissue was more sensitive to T(4) rather than T(3), even when differences in biological activity were taken into account. This implies that a simple conversion of T(4) to T(3) cannot fully account for T(4) effects on the brain and suggests distinctive mechanisms of action for the two THs. While the brain shows gene expression alterations for methimazole and propylthiouracil, the environmental contaminant, perchlorate, had the greatest effect on the levels of mRNAs encoding proteins important in neural development and function. Our data identify gene expression profiles that can serve as exposure indicators of these chemicals.

Modulation of the TLR-mediated inflammatory response by the endogenous human host defense peptide LL-37.

The sole human cathelicidin peptide, LL-37, has been demonstrated to protect animals against endotoxemia/sepsis. Low, physiological concentrations of LL-37 (< or =1 microg/ml) were able to modulate inflammatory responses by inhibiting the release of the proinflammatory cytokine TNF-alpha in LPS-stimulated human monocytic cells. Microarray studies established a temporal transcriptional profile and identified differentially expressed genes in LPS-stimulated monocytes in the presence or absence of LL-37. LL-37 significantly inhibited the expression of specific proinflammatory genes up-regulated by NF-kappaB in the presence of LPS, including NFkappaB1 (p105/p50) and TNF-alpha-induced protein 2 (TNFAIP2). In contrast, LL-37 did not significantly inhibit LPS-induced genes that antagonize inflammation, such as TNF-alpha-induced protein 3 (TNFAIP3) and the NF-kappaB inhibitor, NFkappaBIA, or certain chemokine genes that are classically considered proinflammatory. Nuclear translocation, in LPS-treated cells, of the NF-kappaB subunits p50 and p65 was reduced > or =50% in the presence of LL-37, demonstrating that the peptide altered gene expression in part by acting directly on the TLR-to-NF-kappaB pathway. LL-37 almost completely prevented the release of TNF-alpha and other cytokines by human PBMC following stimulation with LPS and other TLR2/4 and TLR9 agonists, but not with cytokines TNF-alpha or IL-1beta. Biochemical and inhibitor studies were consistent with a model whereby LL-37 modulated the inflammatory response to LPS/endotoxin and other agonists of TLR by a complex mechanism involving multiple points of intervention. We propose that the natural human host defense peptide LL-37 plays roles in the delicate balancing of inflammatory responses in homeostasis as well as in combating sepsis induced by certain TLR agonists.