Inflammatory responses in epithelia: endotoxin-induced IL-6 secretion and iNOS/NO production are differentially regulated in mouse mammary epithelial cells.

BACKGROUND: IL-6 is a pro-inflammatory cytokine that signals via binding to a soluble or membrane bound receptor, while nitric oxide (NO), an oxidative stress molecule, diffuses through the cell membrane without a receptor. Both mediators signal through different mechanisms, yet they are dependent on NFκB. We proposed that both mediators are co-induced and co-regulated in inflamed mammary epithelial cells. METHODS: SCp2 mammary epithelial cells were treated with bacterial endotoxin (ET) for different time periods and analyzed for induction of IL-6 secretion and NO production by ELISA and Griess reaction, respectively. The expression of IL-6 and induced NO synthase (iNOS) was assayed by real time PCR and/or western immunoblots, and the activation of NFκB was assayed by immunobinding assay. To investigate the role of mammary cell microenvironment (cell-substratum or interaction of mammary epithelial cell types; critical to mammary development, function, and disease) in modulation of the inflammatory response, SCp2 cells were cultured with or without extracellular matrix (EHS) or in coculture with their myoepithelial counterpart (SCg6), and assayed for ET-induced IL-6 and NO. RESULTS: Endotoxin induced NFκB activation at 1 h after ET application. IL-6 secretion and NO production were induced, but with unexpected delay in expression of mRNA for iNOS compared to IL-6. NFκB/p65 activation was transient but NFκB/p50 activation persisted longer. Selective inhibition of NFκB activation by Wedelolactone reduced ET-induced expression of IL-6 mRNA and protein but not iNOS mRNA or NO production, suggesting differences in IL-6 and iNOS regulation via NFκB. SCp2 cells in coculture with SCg6 but not in presence of EHS dramatically induced IL-6 secretion even in the absence of ET. ET-induced NO production was blunted in SCp2/SCg6 cocultures compared to that in SCp2 alone. CONCLUSIONS: The differential regulation of IL-6 and iNOS together with the differential activation of different NFκB dimers suggest that IL-6 and iNOS are regulated by different NFκB dimers, and differentially regulated by the microenvironment of epithelial cells. The understanding of innate immune responses and inflammation in epithelia and linkage thereof is crucial for understanding the link between chronic inflammation and cancer in epithelial tissues such as the mammary gland.

Putting microbes to work in sequence: recent advances in temperature-phased anaerobic digestion processes.

Methane biogas production through anaerobic digestion (or biomethanation) is one of the few technologies that both produce bioenergy and protect the environment. When the focus of anaerobic digestion (AD) is shifted from primarily wastewater treatment to bioenergy production, efficiency and process stability become critical to the economic viability of AD technologies. Temperature-phased anaerobic digestion (TPAD) is a promising process that can significantly enhance both digestion efficiency and process robustness. A TPAD system separates the conventional AD process into two phases, so both phases can be optimized according to their individual functional needs. In the first, thermophilic phase, the often rate-limiting hydrolysis step of polymeric feedstock is accelerated by elevated temperatures, while in the second, mesophilic phase, the fastidious syntrophic acetogens and methanogens are provided with permissive conditions where inhibitions to key guilds (e.g., syntrophic acetogens and methanogens) are attenuated. Although large-scale TPAD systems have not been applied widely, researchers have demonstrated the potential superiority of TPAD systems over single-stage digesters and other AD processes with enhanced VS (volatile solids) and pathogen removal; increased methane yield, process stability, OLR (organic loading rate); shorter HRT (hydraulic retention time); decreased foaming and short-chain fatty acids in effluent.

Evaluations of different hypervariable regions of archaeal 16S rRNA genes in profiling of methanogens by Archaea-specific PCR and denaturing gradient gel electrophoresis.

Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

In vivo effects of bifidobacteria and lactoferrin on gut endotoxin concentration and mucosal immunity in Balb/c mice.

The aim of the present study was to examine the effects of oral supplementation of newborn Balb/c mice with bifidobacteria (B. infantis, B. bifidum) and iron-free apo-lactoferrin (bovine, human) on gut endotoxin concentration and mucosal immunity. Endotoxin concentration was measured in ileocecal filtrates at 7, 14, 21, and 28 days postdelivery by a quantitative limulus amebocyte lysate test. While endotoxin levels in bifidobacteria-fed mice showed a steady rise over time, they were consistently lower than that observed in control animals. Results of lactoferrin supplementation varied depending on the specific time point, but overall by day 28, all treatment groups showed lower intestinal endotoxin concentrations compared to saline fed animals. Neither bifidobacteria nor lactoferrin stimulated an increase in B or T cells, or in cytokine production (IL-6, TNF-alpha, INF-gamma), in Peyer's patches as measured by flow cytometry. Bifidobacteria and lactoferrin were well tolerated as dietary supplements and showed promising potential to reduce gut endotoxin levels.

In vitro growth responses of bifidobacteria and enteropathogens to bovine and human lactoferrin.

A series of in vitro experiments was performed to test the ability of bovine and human lactoferrin to influence the growth of the gram-positive probiotic bacteria, Bifidobacterium bifidum, Bifidobacterium infantis, and Lactobacillus acidophilus, as well as the gram-negative enteric bacteria, E. coli O157:H7 and Salmonella typhimurium. None of the lactoferrin preparations stimulated the growth of the tested strains. However, iron-free apo-lactoferrin (bovine and human) and 66% iron-saturated bovine lactoferrin dramatically slowed the growth of E. coli O157:H7 in single culture experiments, while 98% iron-saturated preparations had no effect. In coculture experiments of B. infantis and E. coli, the iron-limited preparations of lactoferrin also slowed the growth of the latter without inhibiting the bifidobacteria. These results suggest that lactoferrin in iron-limited forms may have the potential to be combined with probiotic bacteria in biotherapeutic products, which could help balance human gut microflora and limit the overgrowth of certain enteric microorganisms.