Gene expression profiles in auricle skin as a possible additional endpoint for determination of sensitizers: A multi-endpoint evaluation of the local lymph node assay.

The murine local lymph node assay (LLNA) is widely used to test chemicals to induce skin sensitization. Exposure of mouse auricle skin to a sensitizer results in proliferation of local lymph node T cells, which has been measured by in vivo incorporation of H3-methyl thymidine or 5-bromo-2'-deoxyuridine (BrdU). The stimulation index (SI), the ratio of the mean proliferation in each treated group to that in the concurrent vehicle control group, is frequently used as a regulatory-authorized endpoint for LLNA. However, some non-sensitizing irritants, such as sodium dodecyl sulfate (SDS) or methyl salicylate (MS), have been reported as false-positives by this endpoint. In search of a potential endpoint to enhance the specificity of existing endpoints, we evaluated 3 contact sensitizers; (hexyl cinnamic aldehyde [HCA], oxazolone [OXA], and 2,4-dinitrochlorobenzene [DNCB]), 1 respiratory sensitizer (toluene 2,4-diisocyanate [TDI]), and 2 non-sensitizing irritants (MS and SDS) by several endpoints in LLNA. Each test substance was applied to both ears of female CBA/Ca mice daily for 3 consecutive days. The ears and auricle lymph node cells were analyzed on day 5 for endpoints including the SI value, lymph node cell count, cytokine release from lymph node cells, and histopathological changes and gene expression profiles in auricle skin. The SI values indicated that all the test substances induced significant proliferation of lymph node cells. The lymph node cell counts showed no significant changes by the non-sensitizers assessed. The inflammatory findings of histopathology were similar among the auricle skins treated by sensitizers and irritants. Gene expression profiles of cytokines IFN-γ, IL-4, and IL-17 in auricle skin were similar to the cytokine release profiles in draining lymph node cells. In addition, the gene expression of the chemokine CXCL1 and/or CXCL2 showed that it has the potential to discriminate sensitizers and non-sensitizing irritants. Our results suggest that multi-endpoint analysis in the LLNA leads to a better determination of the sensitizing potential of test substances. We also show that the gene expression of CXCL1 and/or CXCL2, which is involved in elicitation of contact hypersensitivity (CHS), can be a possible additional endpoint for discrimination of sensitizing compounds in LLNA.

Immunosuppressive potential of bardoxolone methyl using a modified murine local lymph node assay (LLNA).

2-Cyano-3, 12-dioxooleana-1, 9-dien-28-oic acid methyl ester (CDDO-Me; bardoxolone methyl) is one of the synthetic oleanane triterpenoids (SOs). It is known that it is the strongest Nrf2/ARE signaling inducer of SOs and slightly inhibits immune response. Little was known about the immunomodulatory action of CDDO-Me in vivo. We assessed its immunosuppressive potential by using the modified mouse lymph node assay (LLNA) including immunosuppression-related gene expression analysis. In the modified LLNA, CDDO-Me showed a significant decrease in lymph node weight and changes in expressions of the immunosuppression-related genes, Zfp459 and Fmo2. It has been already reported that a decrease in lymph node weight was induced by several types of immunosuppressive chemicals such as calcineurin inhibitors, antimetabolites, steroids, and alkylators. In addition, changes in Zfp459 and Fmo2 expression was reported in response after only treatment of antimetabolites. From these results, CDDO-Me is considered to have an immunosuppressive action and similar mechanism to antimetabolites.

Synthesis of novel IP agonists via N-aminoethyl cyclic amines prepared by decarboxylative ring-opening reactions.

An efficient synthesis of a highly potent and selective IP (PGI(2) receptor) agonist that is not structurally analogous to PGI(2) is described. This synthesis is accomplished through the following key steps: Nucleophilic ring-opening of 3-(4-chlorophenyl)-oxazolidin-2-one prepared by a one-pot procedure with 4-piperidinol and selective O-alkylation of 1-(2-(4-chlorophenylamino)ethyl)piperidin-4-ol. The obtained compound is a potent and selective IP agonist displaying a long duration of action.

Novel gene markers of immunosuppressive chemicals in mouse lymph node assay.

The murine local lymph node assay (LLNA) is an immunologically based test of the sensitizing potential of immunotoxicants, but also evaluates immunosuppressive potential with good sensitivity and specificity. We conducted the LLNA with calcineurin inhibitors (tacrolimus and cyclosporin A), antimetabolites (methotrexate and azathioprine), steroids (dexamethasone and prednisolone), and an alkylator (cyclophosphamide). We performed a comprehensive analysis of results of gene expression in lymph nodes obtained in the LLNA using a highly sensitive DNA chip, 3D-Gene™, and the quantitative reverse transcription-polymerase chain reaction (qPCR). Zfp459 expression increased in all animals treated with immunosuppressive chemicals. Ltf, Cbll1 and Lias expression changed specifically in response to the calcineurin inhibitors, Fmo2 and 9630033F20Rik in response to the antimetabolites, Krt8, Gjb1, Hmha1 and Sfrs7 in response to the steroids, and Gbp1 and Mup5 in response to the alkylator. Therefore, these genes were considered novel markers for immunosuppression and their expression could be used to evaluate the mechanism of action of immunosuppressive chemicals.

Molecular and electrophysiological characteristics of K+ conductance sensitive to acidic pH in aortic smooth muscle cells of WKY and SHR.

Changes in K(+) conductances and their contribution to membrane depolarization in the setting of an acidic pH environment have been studied in myocytes from aortic smooth muscle cells of spontaneously hypertensive rats (SHR) compared with those from Wistar-Kyoto (WKY) rats. The resting membrane potential (RMP) of aortic smooth muscle at extracellular pH (pH(o)) of 7.4 was significantly more depolarized in SHR than in WKY rats. Acidification to pH(o) 6.5 made this difference in RMP between SHR and WKY rats more significant by further depolarizing the SHR myocytes. Large-conductance Ca(2+)-activated K(+) (BK) currents, which were markedly suppressed by acidification, were larger in aortic myocytes of SHR than in those of WKY rats. In contrast, acid-sensitive, non-BK currents were smaller in SHR. Western blot analyses showed that expression of BK-alpha- and -beta(1) subunits in SHR aortas was upregulated and comparable with those in WKY rats, respectively. Additional electrophysiological and molecular studies showed that pH- and halothane-sensitive two-pore domain weakly inward rectifying K(+) channel (TWIK)-like acid-sensitive K(+) (TASK) channel subtypes were functionally expressed in aortas, and TASK1 expression was significantly higher in WKY than in SHR. Although the background current through TASK channels at normal pH(o) (7.4) was small and may not contribute significantly to the regulation of RMP, TASK channel activation by halothane or alkalization (pH(o) 8.0) induced significant hyperpolarization in WKY but not in SHR. In conclusion, the larger depolarization and subsequent abnormal contractions after acidification in aortic myocytes in the setting of SHR hypertension are mainly attributable to the larger contribution of BK current to the total membrane conductance than in WKY aortas.