Eric W. Rowe

Effects of nanosized titanium dioxide on innate immune system of fathead minnow (Pimephales promelas Rafinesque, 1820).

Effects of nanosized (<100 nm) titanium dioxide (TiO(2)) particles on fish neutrophils and immune gene expression was investigated using the fathead minnow (Pimpehales promelas). Expanded use of TiO(2) in the cosmetic industry has increased the potential exposure risk to aquatic ecosystems and human health. Effects of nano-TiO(2) on neutrophil function of the fathead minnow was investigated using oxidative burst, neutrophil extracellular traps (NETs) release and degranulation of primary granules. The innate immune gene expression was determined with quantitative PCR (qPCR). Application of 0.1 μg mL(-1) of nano-TiO(2) in vitro stimulated oxidative burst and NET release. Intraperitoneal injection of 10 μg g(-1) of nano-TiO(2) caused a significant decrease in oxidative burst, NETs release and degranulation (21%; 11%; and 30%, decrease, respectively). Fish exposed to nano-TiO(2) for 48 h in vivo had significantly increased expression of interleukin 11, macrophage stimulating factor 1, and neutrophil cytosolic factor 2 (4; 2.5; and 2 fold increase, respectively). Nano-TiO(2) has potential to interfere with the evolutionary conserved innate immune system responses, as evidenced with observed changes in gene expression and neutrophil function. This finding encourages the use of fish models in the studies of nanoparticle immunotoxicity. The lowest significant response concentration studied in vitro is four times greater than the estimated environmental concentration for TiO(2) (0.025 μg mL(-1)) causing concern about potential impact of nano-TiO(2) on aquatic animals and ecosystems.

Hydroxylated fullerenes inhibit neutrophil function in fathead minnow (Pimephales promelas Rafinesque, 1820).

Hydroxylated fullerenes act as potent inhibitors of cytochrome P450-dependent monooxygenases, and are reported to be very strong antioxidants quenching reactive oxygen species (ROS) production. Effects of nanosized hydroxylated fullerenes on fish neutrophil function and immune gene transcription was investigated using fathead minnow (Pimephales promelas). Neutrophil function assays were used to determine the effects of fullerene exposure in vitro and in vivo on oxidative burst, degranulation and extracellular trap (NETs) release, and the innate immune gene transcription was determined with quantitative PCR (qPCR). Application of fullerenes (0.2-200 microgmL(-1)in vitro) caused concentration dependent inhibition of oxidative burst and suppressed the release of NETs and degranulation of primary granules (up to 70, 40, and 50% reduction in activity compared to non-treated control, respectively). Transcription of interleukin 11 and myeloperoxidase genes was significantly increased and transcription of elastase 2 gene was significantly decreased in fish exposed to hydroxylated fullerenes for 48h in vivo (12 and 3 fold increase, and 5 fold decrease, respectively). Observed changes in gene transcription and neutrophil function indicate potential for hydroxylated fullerenes to interfere with the evolutionary conserved innate immune system responses and encourages the use of fish models in studies of nanoparticle immunotoxicity.

Development of Functional Neurons from Postnatal Stem Cells In Vitro

In order for stem cells to fulfill their clinical promise, we must understand their developmental transitions and it must be possible to control the differentiation of stem cells into specific cell fates. To understand the mechanism of the sequential restriction and multipotency of stem cells, we have established culture conditions that allow the differentiation of multipotential neural stem cells from postnatal stem cells. We used immunocytochemistry, fluorescence microscopy, and calcium imaging to demonstrate that progeny of adult rat neural stem cells develop into functional neurons that release excitatory neurotransmitters. We also found that the nontoxic heavy chain fragment of tetanus toxin, a toxin that targets neurons with high specificity, retained the specificity toward neural stem cell–derived neurons. These studies show that neural stem cells derived from adult tissues retain the potential to differentiate into functional neurons with morphological and functional properties of mature central nervous system neurons.

Effects of Leptin on Intracellular Calcium Concentrations in Isolated Porcine Somatotropes

Leptin, the product of the obese gene, is a protein that is secreted primarily from adipocytes. Leptin can influence the function of the pituitary gland through its action on the hypothalamus, but it can also directly act at the level of the pituitary gland. The ability of leptin to induce an increase in intracellular Ca2+ concentration ([Ca2+]i) in somatotropes was examined in dispersed porcine pituitary cells using a calcium imaging system. Somatotropes were functionally identified by the application of human growth hormone releasing hormone. Leptin increased [Ca2+]i in porcine somatotropes in a dose-dependent manner. The application of 100 nM leptin for 3 min did not have a significant effect on [Ca2+]i, while a 3-min application of 1 µM leptin increased [Ca2+]i in about 50% of the somatotropes (p < 0.01). The application of a second leptin challenge (1 µM) evoked a response in only 18% of the observed somatotropes. The stimulatory effect of leptin was abolished in low calcium saline and blocked by nifedipine, an L-calcium channel blocker, suggesting an involvement of calcium channels. Pretreatment of the cultures with AG 490, a specific Janus kinase inhibitor, and with SB 203580, a mitogen-activated protein kinase (MAP kinase) inhibitor, abolished the increase in [Ca2+]i evoked by leptin. In the presence of Nω-nitro-L-arginine methyl ester (L-NAME), a nitric oxide synthase (NOS) inhibitor, the magnitude of the increase in [Ca2+]i evoked by 1 µM leptin was not significantly changed. However, in the presence of L-NAME only 24% of the somatotropes responded to leptin, while in parallel control cultures 70% of the somatotropes responded to leptin. These results imply an involvement of Janus kinase/signal transducer and activator or transcription, MAP kinase and NOS-signaling pathways in the stimulatory effect of leptin on porcine somatotropes.

Echinacea-induced cytosolic Ca2+ elevation in HEK293

BackgroundWith a traditional medical use for treatment of various ailments, herbal preparations of Echinacea are now popularly used to improve immune responses. One likely mode of action is that alkamides from Echinacea bind to cannabinoid type 2 (CB2) receptors and induce a transient increase in intracellular Ca2+. Here, we show that unidentified compounds from Echinacea purpurea induce cytosolic Ca2+ elevation in non-immune-related cells, which lack CB2 receptors and that the Ca2+ elevation is not influenced by alkamides.MethodsA non-immune human cell line, HEK293, was chosen to evaluate E. purpurea root extracts and constituents as potential regulators of intracellular Ca2+ levels. Changes in cytosolic Ca2+ levels were monitored and visualized by intracellular calcium imaging. U73122, a phospholipase C inhibitor, and 2-aminoethoxydiphenyl borate (2-APB), an antagonist of inositol-1,4,5-trisphosphate (IP3) receptor, were tested to determine the mechanism of this Ca2+ signaling pathway. E. purpurea root ethanol extracts were fractionated by preparative HPLC, screened for bioactivity on HEK293 cells and by GC-MS for potential constituent(s) responsible for this bioactivity.ResultsA rapid transient increase in cytosolic Ca2+ levels occurs when E. purpurea extracts are applied to HEK293 cells. These stimulatory effects are phospholipase C and IP3 receptor dependent. Echinacea-evoked responses could not be blocked by SR 144528, a specific CB2 receptor antagonist, indicating that CB2 is not involved. Ca2+ elevation is sustained after the Echinacea-induced Ca2+ release from intracellular Ca2+ stores; this longer-term effect is abolished by 2-APB, indicating a possible store operated calcium entry involvement. Of 28 HPLC fractions from E. purpurea root extracts, six induce cytosolic Ca2+ increase. Interestingly, GC-MS analysis of these fractions, as well as treatment of HEK293 cells with known individual and combined chemicals, indicates the components thought to be responsible for the major immunomodulatory bioactivity of Echinacea do not explain the observed Ca2+ response. Rather, lipophilic constituents of unknown structures are associated with this bioactivity.ConclusionsOur data indicate that as yet unidentified constituents from Echinacea stimulate an IP3 receptor and phospholipase C mediation of cytosolic Ca2+ levels in non-immune mammalian cells. This pathway is distinct from that induced in immune associated cells via the CB2 receptor.