Previous studies have shown that the mechanical wounding of 3-week-old cultured rat astrocytes results in cell proliferation and hypertrophy resembling astrocyte responses to a brain injury in vivo. We now report the effects of basic fibroblast growth factor (bFGF) and an anti-bFGF antibody on astrocyte morphology, proliferation, and migration following in vitro wounding of confluent secondary cultures. Addition of bFGF (20 ng/ml) to wounded cultures induced morphological changes characteristic of differentiation in wounded and nonwounded areas of the culture. Combined treatment with bFGF and an anti-bFGF antibody (100 micrograms/ml) prevented this effect. Astrocyte proliferation along the edges of a scratch wound was at maximum 24 hr after wounding in cells growing in Eagle's minimum essential medium (EMEM) containing 10% serum. Low serum concentration and treatment with dibutyryl cyclic adenosine monophosphate (dbc-AMP) reduced injury-associated astrocyte proliferation. Addition of bFGF to cultures in EMEM with serum increased astrocyte proliferation at 18 and 24 hr after wounding. This effect was reduced considerably by treatment of cultures with bFGF in combination with an anti-bFGF antibody. The combined treatment and the antibody alone reduced cell division to a level lower than in control cultures. Twenty-four hr following wounding, astrocytes along the edges of the wound exhibited extension of thick, flat processes into the wound area. At 3 and 5 days after wounding, a bodily migration of astrocytes into the wounded area was observed. Addition of bFGF significantly increased astrocyte migration 1 day after wounding, with maximum effect on day 3 and no subsequent increase on day 5. A combination of bFGF and anti-bFGF antibody as well as the antibody alone reduced astrocyte migration to a level lower than in controls. Immunohistochemical localization and isoform pattern of bFGF in astrocytes did not change with dbc-AMP treatment or wounding. We conclude that mechanically wounded confluent astrocytes respond to bFGF added to the culture medium by enhancing cell division, differentiation, and migration. In addition, the results of the antibody treatment also suggest a role for endogenous bFGF in astrocyte proliferation and migration elicited by wounding in vitro. These results support the notion that in vivo, both bFGF released by injury and endogenous bFGF synthesized by astrocytes, contribute to the cellular responses that lead to astrogliosis.

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BackgroundThe purpose of the present study was to investigate activation of inflammatory markers in human macrophages derived from the U937 cell line after exposure to particulate matter (PM) collected on dairy farms in California and to identify the most potent components of the PM.MethodsPM from different dairies were collected and tested to induce an inflammatory response determined by the expression of various pro-inflammatory genes, such as Interleukin (IL)-8, in U937 derived macrophages. Gel shift and luciferase reporter assays were performed to examine the activation of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and Toll-like-receptor 4 (TLR4).ResultsMacrophage exposure to PM derived from dairy farms significantly activated expression of pro-inflammatory genes, including IL-8, cyclooxygenase 2 and Tumor necrosis factor-alpha, which are hallmarks of inflammation. Acute phase proteins, such as serum amyloid A and IL-6, were also significantly upregulated in macrophages treated with PM from dairies. Coarse PM fractions demonstrated more pro-inflammatory activity on an equal-dose basis than fine PM. Urban PM collected from the same region as the dairy farms was associated with a lower concentration of endotoxin and produced significantly less IL-8 expression compared to PM collected on the dairy farms.ConclusionThe present study provides evidence that the endotoxin components of the particles collected on dairies play a major role in mediating an inflammatory response through activation of TLR4 and NF-κB signaling.

Over the past 30 yr, individual California dairy operations have grown in size; however, little is known about the distribution and determinants of particulate matter (PM) concentrations on these dairies. Elevated exposure to PM is associated with respiratory and cardiovascular health effects, particularly in occupational settings. The purpose of this study was to quantify the concentrations of PM and all inhalable PM (0-100 µm) on California dairies. Samplers were placed at various locations (e.g., milking parlor, grain storage area, drylot corral, and freestall barns) on 13 different dairies to collect PM and all inhalable PM during the 2008 summer months. The PM and all inhalable PM concentrations varied between different areas on a dairy and from dairy to dairy. Geometric mean concentrations for PM and inhalable PM were 24 µg m (range, 2-116 µg m) and 332 μg m (range, 74-1690 µg m). A key variable for explaining variation in PM concentrations with a mixed effects model was regional background ambient concentrations of PM No significant differences were observed in mean concentrations between upwind and downwind fence line concentrations (adjusted geometric mean ratio [AGMR] = 0.7; 95% CI, 0.4-1.3), although significant differences were found between upwind and central location mean values (AGMR = 0.5; 95% CI, 0.3-0.8; < 0.01). These results indicate dairy PM sources and, thus, elevated occupational exposure. Covariates, such as the age of the dairy and number of cows in the freestall barn and drylot corral, were important variables in explaining PM concentration variability. Levels of PM were lower compared with dairies in other U.S. states and other countries.