Development of tolerance to endotoxin prevents sustained hyper inflammation during systemic infections. Here we report for the first time that chronic morphine treatment tempers endotoxin tolerance resulting in persistent inflammation, septicemia and septic shock. Morphine was found to down-regulate endotoxin/LPS induced miR-146a and 155 in macrophages. However, only miR-146a over expression, but not miR-155 abrogates morphine mediated hyper-inflammation. Conversely, antagonizing miR-146a (but not miR-155) heightened the severity of morphine-mediated hyper-inflammation. These results suggest that miR-146a acts as a molecular switch controlling hyper-inflammation in clinical and/or recreational use of morphine.

To investigate expression of monocyte chemoattractant protein-1 (MCP-1) in the ovine corpus luteum, a partial cDNA was produced by reverse transcription-polymerase chain reaction. This cDNA was 89% identical to that reported for bovine MCP-1 mRNA. In experiment 1, steady-state concentrations of mRNA encoding MCP-1 were measured in pools of luteal tissue collected on Days 3, 6, 9, 12, and 15 of the estrous cycle (estrus = O; n = 4/day). There were no differences in mRNA concentrations for MCP-1 among any of the days studied (p = 0.43). In experiment 2, midluteal-phase corpora lutea were collected from ewes at 0 (untreated), 2, 4, 8, and 16 h after administration of a luteolytic dose of prostaglandin F2alpha (PGF2alpha; n = 4/time point). Concentrations of MCP-1 mRNA were undetectable in untreated controls, were detectable at 2 h post-treatment, had increased 4 and 8 h after administration of PGF2alpha when compared to those at 2 h (p < 0.05), and were decreased 16 h after administration of PGF2alpha when compared to those at 4 h (p < 0.05). In situ hybridization for MCP-1 mRNA combined with immunocytochemical labeling of tissue inhibitor of metalloproteinase-1 (TIMP-1) in large luteal cells was used to determine whether the steroidogenic cells that have PGF2alpha receptors express MCP-1 mRNA in response to PGF2alpha. Messenger RNA encoding MCP-1 and TIMP-1 were not colocalized, indicating that MCP-1 was not expressed by large steroidogenic luteal cells during luteolysis.

To determine whether prostaglandin (PG) F(2alpha) had a dose-dependent effect upon secretion of progesterone, oligonucleosome formation, or loss of luteal weight, ewes on Day 9 or 10 of the estrous cycle were administered 0, 3, 10, or 30 mg PGF(2alpha) per 60 kg BW (i.v.), and luteal tissue was collected 9 and 24 h after injection. All doses of PGF(2alpha) decreased (P < 0. 05) concentrations of progesterone in sera by 9 h; however, in ewes treated with 3 mg PGF(2alpha), concentrations of progesterone were similar to control values at 24 h and higher (P < 0.05) than those in the 10- or 30-mg groups. Concentrations of progesterone in sera over all dose levels were highly correlated to luteal concentrations of mRNA encoding steroidogenic acute regulatory protein (P < 0.001), cytochrome P450 side-chain cleavage (P < 0.02), and 3beta-hydroxysteroid dehydrogenase (P < 0.01). Corpora lutea collected at 24 h from ewes treated with the 10- and 30-mg doses of PGF(2alpha) weighed less (P < 0.05) than those from controls. Oligonucleosomes were not present in luteal tissues from control ewes. Surprisingly, all doses of PGF(2alpha)-induced oligonucleosomes in a majority of animals at 9 h and in a majority of ewes treated with 10 and 30 mg of PGF(2alpha) at 24 h. In conclusion, 3 mg of PGF(2alpha) per 60 kg BW transiently decreased serum concentrations of progesterone and induced oligonucleosome formation, but did not result in reduced luteal weight. The 10- and 30-mg doses of PGF(2alpha) decreased secretion of progesterone and induced oligonucleosome formation and luteolysis.

Aberrant expression of the B7-H1/PD-L1 immunomodulatory molecule is evident in many forms of cancer. The evidence for increased protein levels of B7-H1/PD-L1 comes largely from immunohistochemistry analysis of surgical and biopsy specimens. While several studies attempt to correlate B7-H1/PD-L1 expression with poor prognosis, there are studies that conclude no correlation based on technical difficulties associated with detecting B7-H1/PD-L1 protein expression on the cell surface. We were able to quantifiably measure soluble B7-H1/PD-L1 in conditioned medium from tumor cell cultures using two new immunoassays, a Quantikine® ELISA and a Simple Plex™ Assay. We show that breast, prostate, glioma, and non-Hodgkin’s lymphoma cells express various levels of B7-H1/PD-L1 which correlate to their PTEN or proliferation status. This is in agreement with previous findings showing that increases in B7-H1/PD-L1 expression levels are frequently coincident with loss of the PTEN tumor suppressor or increased activation of the phosphoinositide 3-kinase (PI 3-K) pathway. The androgen-responsive LNCaP prostate cancer cell line does not exhibit measurable B7-H1/PD-L1 in any instance, while the androgen-independent prostate cancer cell line PC-3 expresses measurable B7-H1/PD-L1 that is further inducible with PMA treatment. In all cell lines included in our study, incubation with PMA increases the level of B7-H1/PD-L1 released into the culture media, while pretreatment with the PI 3-K inhibitor, LY294002, abrogates this increase. Importantly, the Simple Plex platform is able to quantify very low levels of B7-H1/PD-L1 that are below detection by ELISA. Our data suggest that combining the Quantikine ELISA and Simple Plex platforms is a powerful strategy for reliably and quantitatively assessing soluble B7-H1/PD-L1 levels. Citation Format: Justin Haworth, Rainer Grant, Samuel Thayer, Melissa Floren, Nathan Steere, Isabel O'Brien, Paul Younge, Michael Anderson, Greta Wegner, Kathryn Brumbaugh. B7-H1/PD-L1 soluble expression in multiple cancer subtypes: High sensitivity measurement by immunoassay [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3978. doi:10.1158/1538-7445.AM2017-3978