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Clostridium difficile is the main cause of hospital-acquired diarrhea and colitis known as C. difficile-associated disease (CDAD).With increased severity and failure of treatment in CDAD, new approaches for prevention and treatment, such as the use of probiotics, are needed. Since the pathogenesis of CDAD involves an inflammatory response with a massive influx of neutrophils recruited by interleukin (IL)-8, this study aimed to investigate the probiotic effects of Lactobacillus spp. on the suppression of IL-8 production in response to C. difficile infection.
We screened Lactobacillus conditioned media from 34 infant fecal isolates for the ability to suppress C. difficile-induced IL-8 production from HT-29 cells. Factors produced by two vancomycin-resistant lactobacilli, L. rhamnosus L34 (LR-L34) and L.casei L39 (LC-L39), suppressed the secretion and transcription of IL-8 without inhibiting C. difficile viability or toxin production. Conditioned media from LR-L34 suppressed the activation of phospho-NF-κB with no effect on phospho-c-Jun. However, LC-L39 conditioned media suppressed the activation of both phospho-NF-κB and phospho-c-Jun. Conditioned media from LR-L34 and LC-L39 also decreased the production of C. difficile-induced GM-CSF in HT-29 cells. Immunomodulatory factors present in the conditioned media of both LR-L34 and LC-L39 are heat-stable up to 100°C and > 100 kDa in size.
Our results suggest that L. rhamnosus L34 and L. casei L39 each produce factors capable of modulating inflammation stimulated by C. difficile. These vancomycin-resistant Lactobacillus strains are potential probiotics for treating or preventing CDAD.
Keywords:Lactobacillus; Clostridium difficile; Probiotic; IL-8; Anti-inflammatory
Clostridium difficile is a gram-positive, spore-forming anaerobe that causes antibiotic-associated diarrhea, colitis, and pseudomembranous colitis in humans [1,2]. C. difficile-associated disease (CDAD) is acquired in association with the disruption and alteration of the gut microbiota [3]. The frequency and severity of primary CDAD are increasing; as well as recurrent cases and infections refractory to standard antibiotic therapy [4]. C. difficile toxins, toxin A (TcdA, 308 kDa) and toxin B (TcdB, 270 kDa), are the main virulence factors contributing to intestinal tissue damage and severe inflammation [5]. Both toxins disrupt the actin cytoskeleton and tight junctions of intestinal epithelial cells [6,7], and cause apoptotic and necrotic cell death [8,9]. C. difficile toxins, TcdA and TcdB, induce the release of chemokines, like IL-8, from intestinal epithelial cells [10-12]. In addition to IL-8, TcdA induces human epithelial cells to secrete other CXC chemokines, including growth-related oncogene (GRO)-α and neutrophil activating protein-78 (ENA-78), along with the CC chemokine, monocyte chemoattractant protein (MCP)-1 [12]. The disruption of tight junctions is thought to enable TcdA and TcdB to enter the laminar propria and submucosa to induce immune cells to secrete chemokines, proinflammatory cytokines, and mediators which promote proinflammatory and cytotoxic effects [4,13]. Proinflammatory cytokines, especially IL-1β, also act on epithelial cells to increase IL-8 secretion and upregulate intercellular adhesion molecule-1 (ICAM-1) expression, leading to increased ICAM-1 and neutrophil CD11/CD18 receptor-dependent neutrophil adhesion [14].
A prominent feature of CDAD results from the release of IL-8 from intestinal epithelial cells that causes a massive influx of neutrophils into the colonic mucosa [4,5,15]. Neutrophil-derived inflammatory mediators exert toxic effects on epithelial cells, causing congestion and edema of the mucosa and epithelial cell damage [14,16]. IL-8 is a major CXC chemokine that mediates neutrophil recruitment, activation, and adhesion. IL-8 potency relies on high binding affinity for neutrophil surface receptors, CXCR1 and CXCR2; which activate chemotaxis [17,18] and robust effector functions [19,20], and trigger the upregulation of adhesion molecules CD11/CD18 that facilitate transendothelial migration and subsequent tissue infiltration [14,21].
CDAD is most often associated with the disruption of a healthy intestinal microbiome after the administration of antibiotics [3]. Potential biotherapies for the treatment and prevention of CDAD are probiotics. In particular, candidate probiotics are capable of enhancing microbiome stability, interfering with pathogen activity, modulating the immune system, and are intrinsically resistant to broad-spectrum antibiotics [22,23]. Meta-analyses of randomized controlled trials [24,25] support the efficacy of probiotic Saccharomyces boulardii and Lactobacillus rhamnosus GG in the prevention of CDAD. Placebo-controlled clinical trials have shown that lyophilized S. boulardii decreased diarrhea associated with β-lactam antibiotics [26], and when used in combination with metronidazole or vancomycin, S. boulardii significantly reduced recurrent episodes in patients with CDAD [27,28]. The probiotic effects of S. boulardii resulted from a secreted protease that interferes with receptor binding of C. difficile toxins A and B to brush borders of human colonic epithelial cells [29] and the downstream modulation of host mitogen-activated protein kinase (MAPK) signaling pathway [30]. In vitro studies with probiotic bacteria show that Lactobacillus delbrueckii ssp. bulgaricus B-30892 inhibits the cytotoxic effects and adhesion of C. difficile to Caco-2 cells [31], while specific strains of Bifidobacterium spp. and Lactobacillus spp. have antagonistic effects on the production of C. difficile toxins A and B [32].
In the present study, we hypothesized that specific strains of Lactobacillus species isolated from healthy hosts produce factors that suppress toxigenic C. difficile-induced IL-8 production. We investigated the probiotic effect of 34 Lactobacillus infant-fecal isolates on the suppression of IL-8 production from colonic epithelial cells stimulated by C. difficile. Conditioned media of two vancomycin-resistant isolates, L. rhamnosus L34 and L. casei L39, suppressed IL-8 production at the level of transcription without inhibiting C. difficile growth or toxin production. Large (>100 kDa) heat-stable, soluble factors are suggested to be responsible for the observed IL-8 suppression.
Human-derived Lactobacillus spp. produce factors that inhibit IL-8 and GM-CSF production by C. difficile-stimulated colonic epithelial cells
Lactobacillus conditioned media (LCM) from thirty-four human-derived Lactobacillus isolates (Additional file 1) were screened for the ability to suppress pro-inflammatory cytokine production from C. difficile-stimulated colonic epithelial cells. LCM from 3 of 34 Lactobacillus isolates, L. rhamnosus L34 (LR-L34), L. rhamnosus L35 (LR-L35), and L. casei L39 (LC-L39), significantly suppressed IL-8 production by approximately 50% or greater when compared to media control (Figure 1A). These three IL-8-suppressing isolates did not stimulate IL-8 production when co-cultured with HT-29 cells in the absence of C. difficile (data not shown). Only a single isolate of L. casei was included in this study (Additional file 1), and the third L. rhamnosus isolate in this collection, strain L31, did not suppress IL-8 in the same assay (Figure 1A). In a previous study [33], we sequenced and compared the genomes of L. rhamnosus L31, L34, and L35. Due to the fact that LR-L34 and LR-L35 were isolated from the same host, had similar colony morphology, were nearly identical at the nucleotide level and similarly genetically distinct from strain LR-L31, we determined that they were most likely independent isolates of the same strain. Therefore, the remaining experiments in this study were conducted excluding LR-L35.
Additional file 1. Infant feces-derived Lactobacillus spp. used in this study.
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Figure 1. Infant feces-derived Lactobacillus spp. produce factors that suppress pro-inflammatory cytokine production by C. difficile-stimulated HT-29 intestinal epithelial cells. LCM from specific strains of human-derived lactobacilli were found to significantly inhibit IL-8 production by HT-29 cells stimulated with C. difficile. Cells were stimulated with C. difficile in the presence of LCM for 24 h. (A) IL-8 production was monitored by ELISA, and (B) GM-CSF production was monitored by a Luminex premixed cytokine assay by Millipore. The results were from three independent experiments in triplicate for figure (A) and one experiment in triplicate for figure (B) and are expressed as the mean ± SEM, **p-value < 0.01 as compared to medium control.
LCM from LR-L34 and LC-L39 were screened for effects on fourteen additional cytokines in a multi-plex luminex assay. In addition to IL-8, C. difficile stimulated GM-CSF production, but affected no other cytokines tested (Additional file 2). GM-CSF is an important pro-inflammatory cytokine that allows neutrophils to persist at sites of inflammation [34,35] and enhances the chemotactic response of neutrophils to IL-8 [36]. In addition to IL-8, LCM from both anti-inflammatory lactobacilli strains significantly inhibited the production of GM-CSF (Figure 1B). LCM from a different strain of a matched species of L. rhamnosus, strain L31, not only had no effect on IL-8 production, but also did not inhibit GM-CSF production in the same assay (Additional file 2).
Additional file 2. Production of cytokines and chemokines by C. difficile-stimulated HT-29 cells.
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Further characterization of isolates LR-L34 and LC-L39 showed that they are resistant to both vancomycin and metronidazole (both with MIC > 256 μg/mL), two drugs commonly used to treat C. difficile infection in humans [37,38]. The anti-inflammatory effect of soluble factors produced by LR-L34 and LC-L39 is not unique to HT-29 cells as LCM from both strains also suppresses IL-8 production greater than 50% from human colonocytes (Caco-2) stimulated by C. difficile (Additional file 3). Trypan blue dye exclusion indicated that HT-29 and Caco-2 cell viability (>90%) was not compromised by the presence of any LCM tested (data not shown). Furthermore, neither viability of (Additional file 4) nor toxin production by (Additional file 5) C. difficile was negatively affected by the presence of LCM. All further in vitro experimentation was carried out using the C. difficile-HT-29 cell co-culture model.
Additional file 3. Infant feces-derived Lactobacillus spp. produce factors that suppress pro-inflammatory cytokine production by C. difficile-stimulated Caco-2 intestinal epithelial cells. LCM from human-derived lactobacilli were found to significantly inhibit IL-8 production from Caco-2 cells stimulated with C. difficile. Cells were stimulated with C. difficile in the presence of LCM for 24 h and IL-8 production was monitored by ELISA. The results were from three independent experiments in triplicate and are expressed as the mean ± SEM, **p-value <0.01 as compared to medium control.
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Additional file 4. C. difficile viability was not affected by Lactobacillus conditioned media. IL-8 suppression did not result from LCM effects on the antagonism of C. difficile growth. C. difficile B2-CU-0001-54 was assayed for viability after co-incubation with LCM and HT-29 cells. Counts of isolated C. difficile B2-CU-0001-54 colonies from co-culture assays with and without LCM treatment from three independent experiments in triplicate were compared and results are reported as mean ± SD. No significant difference in viability of C. difficile was seen by LCM treatment as compared to medium control.
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Additional file 5. Lactobacillus conditioned media had no effect on C. difficile toxins in co-culture assay. IL-8 suppression did not result from attenuated toxin production and endocytosis to HT-29 cells. Extracellular and intracellular toxin concentrations were determined from LCM-treated, C. difficile-stimulated HT-29 cells. Cell culture supernatants or lysates of C. difficile and HT-29 cells were collected, spotted onto PVDF membranes and assayed in succession with mouse anti-TcdA or anti-TcdB monoclonal antibodies. The presence of toxin was determined colorimetrically and toxin concentrations were calculated by ChemiDoc™ XRS (Bio-Rad, Philadelphia, USA). The results were from three independent experiments in triplicate and are expressed as the mean ± SEM. No significant difference in intracellular toxin concentrations was seen by LCM treatment as compared to medium control. (A) Whole cell lysates, (B) culture supernatant of HT-29 cells, (C) the ratio between spot intensity of C. difficile co-culture with LCM and C. difficile with MRS.
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L. rhamnosus L34 and L. casei L39 affect IL-8 gene transcription through decreased activity of NF-κB and c-Jun
The effects of soluble factors produced by LR-L34 and LC-L39 on IL-8 transcription were determined by IL-8 gene-specific quantitative RT-PCR. C. difficile-stimulated HT-29 cells were treated with LCM for 4 h prior to total RNA isolation. When compared to media control, treatment with LCM from LR-L34 or LC-L39 resulted in an approximate 0.5-fold down-regulation (p-value < 0.001) of IL-8 mRNA concentration relative to gapdh (Figure 2).
Figure 2. Lactobacillus soluble factors suppress IL-8 transcription in HT-29 cells. IL-8 gene expression was determined in C. difficile-stimulated HT-29 cells after 4 h incubation with medium control or LCM from either LR-L34 or LC-L39. Quantitative real-time PCR was conducted with primers specific to IL-8 and GAPDH transcripts. Gene expression data were normalized to housekeeping gene, GAPDH. Fold change ratios of IL-8 (LCM strain/medium control) from one experiment in triplicate were calculated, and results represent the mean ± SEM, ***p-value < 0.001 as compared to medium control.
Transcriptional regulation of IL-8 is mediated via NF-κB and AP-1 by the upstream phosphorylation of subunits p65 and c-Jun, which result in the respective activation of NF-κB and AP-1 and subsequent downstream transcription of IL-8 [39-42]. To determine whether or not LR-L34 or LC-L39 produce factors that affect activation of NF-κB or c-Jun, C. difficile-stimulated HT-29 cells were treated with LCM from either LR-L34 or LC-L39 for 15 and 30 min, then were assayed for effects on phosphorylated NF-κB (p-NF-κB) and c-Jun (p-c-Jun) concentrations by western blot. LCM from LR-L34 decreased p-NF-κB by 47.93% at 30 min only (p-value <0.001, Figure 3A), and did not affect p-c-Jun at either time point (Figure 3B). A 15 min treatment with LC-L39 LCM resulted in decreased concentrations of p-NF-κB (18.62%, p-value <0.01, Figure 3A) and p-c-Jun (38.19%, p-value <0.001, Figure 3B) while a 30 min treatment resulted in a 43.19% decrease in p-NF-κB (p-value <0.001, Figure 3A).
Figure 3. Human-derived Lactobacillus spp. suppress activation of C. difficile-induced transcription factors. Concentrations of activated NF-κB (A) and c-Jun (B) were determined by western blot on whole cell lysates of HT-29 cells stimulated with C. difficile with or without medium control or LCM treatment. Concentrations were measured at 15 and 30 min using antibodies corresponding to p-NF-κB p65, NF-κB p65, β-actin, p-c-Jun, and c-Jun. Relative protein concentrations were determined by densitometry, and activated transcription factors were normalized to their non-activated counterpart (p-NF-κB p65 (Ser 536) to NF-κB p65; p-c-Jun to c-Jun). The results were from three independent experiments in duplicate and are expressed as the mean ± SEM, *p-value <0.05 and **p-value <0.01.
The anti-inflammatory factor(s) produced by L. rhamnosus L34 and L. casei L39 is heat-stable and greater than 100 kDa in size
Heat tolerance and size prediction of anti-inflammatory factors in LCM from LR-L34 and LC-L39 were characterized as follows. LCM from LR-L34 and LC-L39 were heated to 100°C for 15 min, 30 min, and 1 h and then tested for the ability to suppress C. difficile-induced IL-8 production in HT-29 cells. The ability to suppress IL-8 production in this assay was retained at all time points (Figure 4A). Size fractionation of LR-L34 and LC-L39 LCM was performed using 30 kDa and 100 kDa Amicon® Centrifugal Filters and filtrates containing factors <30 kDa and <100 kDa did not suppress IL-8 activity, while fractions containing factors >100 kDa retained the anti-IL-8 activity in each case (Figure 4B).
Figure 4. IL-8 suppression by LCM after heat treatment and size fractionation. Soluble factors in LCM from LR-L34 and LC-L39 were assayed for heat stability and size prediction. LCM from L. rhamnosus L34 and L. casei L39 was either heated to boiling for various time points (A) or size fractionated by filtration (B) and the effects on IL-8 production by C. difficile-stimulated HT-29 cells was evaluated by ELISA. The results were from three independent experiments in triplicate and are expressed as the mean ± SEM, **p-value <0.01 and ***p-value <0.001.
Standard therapy for treating CDAD with metronidazole and vancomycin is effective, but the association of these drugs with high relapse rates represents a major health problem [43-45]. Recent reports estimate the rate of recurrence after an initial episode of CDAD to be 13-38% [46-49]. Standard therapy for recurrent CDAD has been modified to include probiotics like L.rhamnosus GG, L. plantarum 299v, or S. boulardii which reduce the rate of recurrence around 20-30% [28,50,51]. Health promoting effects of lactobacilli include the stabilization of indigenous microbial populations, protection against intestinal infection, modulation of the immune system, and effects on gene expression in the human mucosa [52-54]. In addition, a recent systematic review and meta-analysis indicated that probiotics given concurrently with antibiotics reduced the risk of antibiotic-associated diarrhea and C. difficile infection [55]. A key characteristic of the pathophysiology of CDAD is an inflammatory response with a marked neutrophil accumulation resulting from secreted IL-8 [14-16,21]. Lactobacilli are known to inhibit IL-8 production by intestinal epithelial cells stimulated with bacterium-derived LPS [56] but have not yet been associated with the modulation of C. difficile-induced IL-8 production.
We identified two Lactobacillus isolates from a library of infant feces-derived Lactobacillus spp. that can significantly suppress IL-8 production by C. difficile-stimulated HT-29 cells. IL-8 suppression by LR-L34 and LC-L39 did not result from negative effects on C. difficile viability as colony counts from the co-culture supernatants of either C. difficile alone, or in combination with MRS, or LCM from LR-L34 or LC-L39, were not significantly different (Additional file 4). Trejo et al. [32] demonstrated that spent culture supernatants of B. bifidum 5310 and L. plantarum 83114 diminished the production of toxin A and toxin B by C. difficile ATCC 9689 and clinical isolate 117. IL-8 suppression by LR-L34 and LC-L39 seems not to result from attenuated toxin production and endocytosis by HT-29 cells. No significant difference of intracellular toxin concentrations in C. difficile and HT-29 cells was seen by LCM treatment as compared to medium control (Additional file 5). However, we cannot exclude the possibility that LCM of LR-L34 and LC-L39 interfere with toxin self-cleavage in the endosome prior to entering the cytosol which induces several downstream inflammatory consequences [57]. It is likely that C. difficile-secreted toxins were endocytosed by HT-29 cells as a very small amount of toxin was present in tissue culture medium at 24 h of the co-culture assay.
LR-L34 and LC-L39 suppressed IL-8 gene transcription at 4 h after co-culture with HT-29 cells which is in agreement with a report of Imaoka et al. [58] for B. bifidum strain Yakult. The IL-8 gene promoter contains binding sites for transcription factors such as NF-κB and AP-1 [59], which control the transcription of IL-8 in C. difficile-stimulated HT-29 colonic epithelial cells [40,60]. LCM from LR-L34 decreased p-NF-κB, and did not affect p-c-Jun while LC-L39 LCM decreased both of p-NF-κB and p-c-Jun, with greater effects on pNF-κB. Modulation of pro-inflammatory signaling pathways by probiotic bacteria and yeasts have been demonstrated previously. Ma et al. [61] reported that L. reuteri inhibited TNF-induced IL-8 production in both T84 and HT-29 intestinal epithelial cells by inhibiting nuclear translocation of NF-κB. S. boulardii modifies host cell pro-inflammatory signaling pathways during bacterial infection by blocking the activation of NF-κB and MAPK [62-64]. Our data showed that C. difficile stimulated GM-CSF production in addition to IL-8 and conditioned media generated from LR-L34 and LC-L39 can also suppress GM-CSF production. The ability to suppress GM-CSF of these lactobacilli potentially enhances their anti-inflammatory effects on C. difficile infection.
Probiotic factors can vary from organism to organism in regards to activity, size, and stability. Sougioultzis et al.[65] reported that S. boulardii probiotic yeast produce a heat stable, <1 kDa soluble, anti-inflammatory compound that blocks NF-κB activation and NF-κB-mediated IL-8 gene expression in both HT-29 colonic epithelial cells and THP-1 monocytes. L. rhamnosus GG secretes p40 (40 kDa) and p75 (75 kDa) proteins that activate EGFR and downstream PI3K/Akt and PKC signaling that modulate intestinal epithelial cell survival and growth [66]. Castagliuolo et al.[29] reported that a 54-kDa protease of S. boulardii can inhibit the effect of C. difficile toxins A and B in HT-29 cells. L. reuteri ATCC PTA 6475 produces the small molecule, histamine, which inhibits TNF production in THP-1 monocytes via PKA and ERK signaling [67]. Active substances in LCM of LR-L34 and LC-L39 were found to be heat-stable and >100 kDa. Although LR-L34 and LC-L39 also inhibit TNF production by THP-1 monocytes, they do not produce histamine (data not shown). Therefore the TNF-suppressive mechanism of action of these lactobacilli must be different from L. reuteri, and further investigation is needed to characterize the soluble factors produced by these specific Lactobacillus isolates. The discovery of vancomycin-resistant LR-L34 and LC-L39 with IL-8-suppressing ability in this study offers us potential probiotic strains for combating CDAD. These strains may harbor other probiotic properties, including the ability to replenish normal microbiota, a key factor for treating CDAD as shown by the recent successes with intestinal microbiome transplant therapies [68,69].
We have demonstrated the probiotic effect of two vancomycin-resistant strains, L. rhamnosus L34 and L. casei L39, on the suppression of IL-8 production from C. difficile-stimulated colonic epithelial cells. These strains suppressed IL-8 production by inhibiting activation of transcription factors for IL-8 gene expression without inhibiting C. difficile growth or toxin production. Our data also suggest that heat-stable, >100 kDa factors are responsible for IL-8 suppression. These vancomycin-resistant Lactobacillus strains are potential probiotics for treating or preventing CDAD.
Bacterial strains and culture conditions
Thirty-four Lactobacillus spp. isolated from infant feces were analyzed in this study (Additional file 1). All lactobacilli were routinely cultured in an anaerobic chamber (Concept Plus, Ruskinn Technology, UK) (10% CO2, 10% H2, and 80% N2) for 24 h at 37°C in de Man, Rogosa, Sharpe (MRS) medium (Oxoid, England).
A C. difficile isolate, designated strain B2-CU-0001-54 was obtained from feces of an infected patient positive for C. difficile toxins A and B by VIDAS® Clostridium difficile A & B (Biomérieux, France) at the Department of Microbiology, Faculty of Medicine, Chulalongkorn University. This strain is positive for TcdA and TcdB as determined by PCR for toxin A and B genes [70] and the reactivity with mouse anti-TcdA and anti-TcdB monoclonal antibodies (Meridian Life Science, Inc.). C. difficile B2-CU-0001-54 was routinely cultured anaerobically on Brucella agar (Oxoid, England) at 37°C for 48 h. Cells were harvested, re-suspended in McCoy's medium, and adjusted to a McFarland 6 standard (1.8×109 cells/mL) prior to co-culture with HT-29 cells. This study was approved by the Ethics Committee of Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand (COA no.617/2011, IRB no.246/54). Written informed parental consent for fecal samples was obtained from participants.
Minimum inhibitory concentration assay
The minimum inhibitory concentration of vancomycin on LR-L34 and LC-L39 was determined by the broth microdilution procedure as previously described [71]. Briefly, 100 μl of lactobacilli (final concentration 1 × 106 CFU/mL or 1 × 105 CFU/well) were inoculated into the wells of a 96 well plate containing 100 μl of vancomycin in serial 2-fold dilutions from 256 to 0.125 μg/mL, then were cultured anaerobically for 48 h. Optical density was measured at 600 nm using a microplate reader Multiskan® EX (Thermo Scientific, USA). The results were compared with growth control (lactobacilli alone) and the endpoint of MIC is the concentration where no growth or a reduction in growth by 90%, is observed.
Preparation of Lactobacillus conditioned media
LCM were prepared as previously described [72]. Briefly, 24 h cultures were adjusted to an OD600 0.1 and incubated anaerobically for 48 h. Supernatants were collected, filtered with 0.22 μm Millex-GV Filter Units (Millipore, USA), and 500 μL aliquots were concentrated by Eppendorf Vacufuge® vacuum concentrator (Eppendorf North America, USA) at 60°C for 2.5 h. Pellets were resuspended in an equal volume of McCoy's 5a modified medium (Gibco-Invitrogen, Carlsbad, CA, USA) or Eagle's Minimal Essential Medium (Gibco-Invitrogen, USA) and stored at −20°C until further analysis.
Cell lines and culture conditions
Human colonic epithelial cells, HT-29 or Caco-2, were obtained from the American Type Culture Collection (ATCC HTB-38 and HTB-37, respectively; Manassas, VA, USA). HT-29 cells were maintained in McCoy's 5a modified medium supplemented with 10% (v/v) heat-inactivated fetal bovine serum (Gibco-Invitrogen, USA) at 37°C under 5% CO2 for 48 h. Caco-2 cells were maintained in Eagle's Minimal Essential Medium supplemented with 20% (v/v) heat-inactivated fetal bovine serum at 37°C under 5% CO2 for 72 h. Adherent cells were detached with 0.25% (v/v) Trypsin (Gibco-Invitrogen, USA) in 1 mM EDTA (Gibco-Invitrogen, USA) and resuspended in their respective medium. Resuspensions of each cell type were used in subsequent C. difficile co-culture assays.
LCM treatment and C. difficile co-culture with colonic epithelial cells
Colonic epithelial cells were treated with LCM and stimulated to produce IL-8 by co-culture with C. difficile. HT-29 (2.0 × 104 cells) or Caco-2 cells (5.0 × 104 cells) were pre-incubated for 24 h in a 96-well format as described above. LCM (5% v/v) was added with or without the subsequent addition of viable C. difficile B2-CU-0001-54 (6.0 × 106 CFU/well with HT-29 cells or 4.5 × 107 CFU/well with Caco-2 cells) and co-incubated for an additional 24 h. Cell culture supernatants were collected by centrifugation (125 × g, 4°C for 7 min) and stored at -20°C until further use.
Effects of LCM on C. difficile viability
To ensure IL-8 suppression did not result from the antagonism of C. difficile growth by soluble factors in LCM, C. difficile B2-CU-0001-54 was assayed for viability after co-incubation with LCM and HT-29 cells. Briefly, co-culture supernatants were serially diluted and cultured anaerobically on Brucella agar (Oxoid, England) at 37°C for 48 h. Counts of isolated C. difficile B2-CU-0001-54 colonies from co-culture assays with and without LCM treatment were compared.
Effects of LCM on C. difficile toxins in the co-culture assay
To determine whether Lactobacillus spp. produce factors that suppress C. difficile toxin secretion, intracellular and secreted toxin concentrations from co-culture assays were determined as previously described [32] with the following modifications. Secreted toxin was measured from LCM-treated, C.difficile-stimulated HT-29 cell supernatants, which were concentrated 10-fold by speed vacuum drying and spotted onto polyvinylidene fluoride (PVDF) membranes (Bio-Rad, Philadelphia, USA). Blocked membranes were incubated in succession with mouse anti-TcdA or anti-TcdB monoclonal antibodies (Meridian Life Science, Inc.), biotinylated goat anti-mouse IgG, and extravidin-alkaline phosphatase (Sigma-Aldrich, St. Louis, MO, USA). The presence of toxin was determined colorimetrically with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate (NBT/BCIP) solution (Sigma-Aldrich, St. Louis, MO, USA). Intracellular toxin concentrations were determined from LCM-treated, C.difficile-stimulated HT-29 cell pellets. Pellets from 20 ml of cell culture were washed, resuspended in 2 ml cell culture medium, and lysed by sonication (Vibra-Cell™, Sonics & Materials, Inc., USA). Cell lysates were collected, 100 μl spotted onto PVDF membranes, and analyzed for the presence of toxin as described above. Toxin concentrations were calculated by ChemiDoc™ XRS (Bio-Rad, Philadelphia, USA).
Effects of LCM on cytokine production by ELISA and Luminex assays
Supernatants from co-culture assays were tested for the effects of soluble factors produced by Lactobacillus on IL-8 production and other related cytokines. IL-8 concentrations in LCM-treated, C. difficile-stimulated HT29 or Caco-2 cell co-culture supernatants were measured using a Human CXCL8/IL-8 ELISA kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. To determine whether Lactobacillus spp. can modulate cytokines other than IL-8 in this assay, cell supernatants from C. difficile-stimulated HT-29 cells in the presence or absence of LCM were screened by a Human Cytokine/Chemokine-Premixed 14-plex kit (Millipore, Billerica, MA) in a Luminex 100 system (Luminex Corporation, Austin, TX) for quantification of the analytes GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 (p70), IL-13, MCP-1, and TNF-α. Analytes at concentrations exceeding the minimum detectable dose were evaluated and raw data were obtained with MasterPlex CT version 22.214.171.124 and analyzed with MasterPlex QT version 126.96.36.199 (Hitachi MiraiBio, San Francisco, CA).
Analysis of IL-8 gene transcription by qPCR
The effects of LCM on the transcription of IL-8 were determined by qPCR as previously described [58] with the following modifications. HT-29 cells (8.0×105 cells) were pre-incubated in a 24-well format as outlined above. Cells were LCM-treated (5% v/v) with or without the subsequent addition of viable C. difficile B2-CU-0001-54 (2.4×108 CFU/well) at 37°C, 5% CO2 for 4 h. Total RNA was extracted from HT-29 cells using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. Synthesis of cDNA was completed using the SuperScript® VILO™ cDNA Synthesis kit (Invitrogen), and qPCR was performed in a LightCycler® 2.0 (Roche, Germany) for 45 cycles of: 15 s at 94°C, 25 s at 60°C, and 25 s at 72°C. The amplified product was detected using Light Cycler® Fast Start DNA MasterPLUS SYBR Green I (Roche, Germany) at 530 nm. The following primers were used to amplify cDNA fragments: IL-8 forward primer (5′-ACACTGCGCCAACACAGAAATTA-3′), IL-8 reverse primer (5′-TTTGCTTGAAGTTTCACTGGCATC-3′); GAPDH forward primer (5′-GCACCGTCAAGGCTGAGAAC-3′), GAPDH reverse primer (5′-ATGGTGGTGAAGACGCCAGT-3′). IL-8 gene expression, relative to GAPDH, was calculated according to the 2-ΔΔCp method [73].
Examination of cell signaling pathways by quantitative western blot
Changes in the NF-κB signaling pathway coordinating with LCM treatment were analyzed by western blot as previously described [30] with minor modifications. LCM-treated, C. difficile-stimulated HT-29 cells were lysed in a radioimmunoprecipitation assay (RIPA) buffer (50 mM TrisHCl, pH7.4, 150 mM NaCl, 10% NP-40, 0.5% Na-DOC, 0.1% SDS). Protein concentrations from cell lysates were measured using the Pierce® BCA protein assay kit (Pierce Biotechnology, Illinois, USA). Cell extracts were fractionated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred onto PVDF membranes (Bio-Rad, Philadelphia, USA), and blocked (10% non-fat milk in TBST (50 mM Tris, pH 7.5, 0.15 M NaCl, 0.05% Tween 20). Blocked membranes were incubated with mouse antibodies against NF-κB (p65), phospho-NF-κB (p65), c-Jun, phospho-c-Jun and β-actin (Santa Cruz Biotechnology, California, USA), washed with TBST and treated with horseradish peroxidase-labeled goat anti-mouse secondary antibodies for 1 h. Peroxidase signals were detected and imaged by ChemiDoc™ XRS (Bio-Rad, Philadelphia, USA).
Characterization of IL-8 suppressive factors in LCM
Lactobacillus anti-inflammatory factors responsible for suppressing IL-8 production from C. difficile-stimulated colonic epithelial cells were assessed for thermal stability and size estimation. Heat stability was assessed by heating LCM to 100°C for 15 min, 30 min, and 1 h. The sizes of active factors present in LCM were estimated by 30 kDa and 100 kDaAmicon® Ultra-4 Centrifugal Filters (Merck, Massachusetts, USA). After each treatment, LCM was tested for the ability to suppress IL-8 production from C. difficile-treated HT-29 cells using techniques outlined above.
Statistical analyses
All experiments were performed at least in triplicate and the results were reported as mean ± standard deviation or standard error of mean (SEM). The data were analyzed in Microsoft Excel using the Student's t-test with one-tailed distribution and considered statistically significant at a p-value ≤ 0.05, unless otherwise stated.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
JKS, JV, and ST conceived and designed the study. PB designed and performed the experiments. SV carried out Luminex assay. JKS, JV, and ST supervised and provide funding for the research. PB, JKS, and ST wrote the manuscript. All authors read and approved the final manuscript.
We would like to graciously acknowledge Tor Savidge for his advice, guidance, and generosity, and extend our gratitude to both the Texas Children's Microbiome Center and the Functional Genomics & Microbiome Core for providing equipment and resources for a fruitful collaboration.
This work was supported by the Thailand Research Fund through the Royal Golden Jubilee PhD Program (PHD/0295/2550 to PB); the Thai Government Research Budget for Fiscal year 2009 and 2010; the Rachadapisek Sompoj research fund, Faculty of Medicine, Chulalongkorn University (Grant No. RA51/1and RA55/20 to ST). Funding was also provided in part by the Texas Children's Microbiome Center, Department of Pathology at Texas Children's Hospital; and the National Institutes of Health, National Cancer Institute (Grant No. U01 CA170930 to JV); by the National Institute of Diabetes and Digestive and Kidney Diseases (Grant Nos. UH3 DK083990 to JV, P30 DK56338, and R01 DK065075 to JV); and by the National Center for Complementary and Alternative Medicine (Grant No. R01 AT004326 to JV).
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text:
GMC Sonoma Problem Report
GMC Sonoma Power Door Locks Stop Working
(14 reports)
The power door locks may stop working in one or more doors due to a failed actuator. Replacement of the failed actuator should correct this concern.
Just bought truck I ordered new remote door opener, When programming after I cycle the ignition switch twice and release the door unlock switch. I hear 2 clicks but the door locks do not move. And I can not program remote. I put a new battery in remote. Nothing, tried 8 times. email@example.com - nickey.jones
haven't repaired yet. - coachestruck
it wontr lock all the way - Visitor
Related Items:
I can't seem to find a short, the fuse is fine, both headlights are fairly new, the switch seems to have power and works, the dash lights work fine, dome light works good, tail lights/signals/brake...
On vehicles with intermittent windshield wipers, the wiper motor circuit board may fail and c...
It started by working now and then mainly inthe mornings, now the mileage dislay quite working...
My speedometer does not work and I have replaced the sensonr. Any helpful hints?
This is a 1999 GMC Sonoma 2.2 4cylinder engine,both of these quit working at the same time.

subset: commoncrawl


text:
Youngstown State University's faculty union walked out of contract negotiations this week, and the negotiating committee believes the administration is trying to drive the process to fact-finding, according to a memo distributed Wednesday to membership by the union's chief negotiator.
"Our new president is inheriting a mess," Gabriel Palmer-Fernandez wrote in the memo which was obtained by The Vindicator.
The new president, Jim Tressel, officially starts his job July 1.
Ron Cole, a YSU spokesman, declined comment, citing a news blackout regarding negotiations.
Palmer-Fernandez, a professor and director of the Dr. James Dale Ethics Center at YSU, also declined comment, citing the blackout.
The memo says that in the first negotiation meeting, the university's chief negotiator declared the sides were at an impasse.
"I assumed then that his use of the term 'impasse' was just one of those verbal mistakes one sometimes makes, one of those unintended words that come out of one's mouth and cannot be placed back in," Palmer-Fernandez wrote in the memo.
Palmer-Fernandez, writes that in hindsight, however, it should have signaled the intention to drive the process to fact-finding and then to bargain down from the report once it's issued.
On Tuesday, the union walked out of contract negotiations.
Despite meeting 10 times for a total of more than 40 hours, the two sides reached agreement on only four noncontroversial issues.
When the union proposed items meant to "recognize excellence, boost morale and extend respect to faculty at no financial cost," the administration rejected the proposals without providing reasons, the memo says.
On May 13, the union, the YSU-Ohio Education Association, presented a comprehensive proposal, walked the administrative side through it, and told them they expected a comprehensive counterproposal.
"On May 14, we again stressed the importance of a comprehensive counterproposal — e.g., that we cannot negotiate in any informed manner if all they give us is a dribble of an article here and other there," Palmer-Fernandez wrote.
On Tuesday, the administration gave proposals on three articles.
"The faculty team caucused, and on our return to the table we informed them that they were unprepared to negotiate, absent [a] comprehensive package," he wrote.
Another session is set for today.
"The end game for administration is to drive us to fact-finding," Palmer-Fernandez wrote. "We know this. The administration's chief negotiator has made it clear that, by his account, fact-finding is a normal part of negotiations. We believe otherwise."
Annette Burden, union president, says she's still hopeful.
"I can't speak for the negotiating team," she said. "I'm always hopeful."
But Burden said Palmer-Fernandez's memo is a fair and accurate assessment of what's been happening.
The union has twice requested mediation, but the administration declined.
"The administrative team delays bargaining process at all costs, and the cost in August might be very high," Palmer-Fernandez wrote. "Unless administration takes a different attitude in negotiations we might find ourselves in a situation that could have been avoided."
Three years ago, negotiations grew heated with the union initially calling and then canceling a strike.
Obviously this memo reports the union side of the story and I am sure there is spin to this, but it is consistent with what we heard elsewhere about YSU leadership being off track and not knowing what it is doing.
If YSU doesn't start on time, then every day that the students have to wait to go to school. Their tuition should be reduced for each and every day they can't attend.
I see another strike on the horizon. The faculty is the backbone of the institution. Without faculty you have no university. They have sat back and watched top administrators take gross raises while they have had their wages froze or marginally increased. Remember that support staff is also in negotiations. A repeat of three years ago is coming. Except I believe this time the faculty will go out and this time it will be ugly. The BOT needs to get to the business of getting YSU back on track or there will be no YSU in the near future. YSU being the only state university in Ohio that does not have full time faculty with a masters could be part of this problem. Allot of money to be saved there. Full time tenured profs with doctorate degrees don't need to be teaching basket weaving 101.
Must been some nice comments for the Vindy to remove them.
billdog, you do realize that something like 30 percent of courses at YSU are taught by masters only staff, right? Many are full-time, just not tenure track, which allows the university to pay them less. That said, accreditation standards do require a healthy number of PHDs. There are some schools where the majority of faculty are masters holding full-time employees-- community colleges.
Good question--the number changes daily. Also, its a structural deficit not an operational deficit which both of the un-chosen presidential candidates pointed out.
@KnightCap: I think the last number in the Vindy was $7 million. Yes, after all those things. Administrative and athletic expenses were increased after telling the rest of the university that cuts were needed. Priorities have gotten all messed up.
Seriouslee, the only masters level full time faulty at YSU are faculty that their discipline does not offer a Ph.d. The rest are considered limited service faculty. They are not full time. Are not offered health care and are not guaranteed a return semester to semester. If the person doing the hiring decides her/his friend or relative, needs a job, so long. I know many that have done and do these jobs. Just because some have been there for eons, doesn't make them full time. As a matter of fact there was an article not to long ago in the Vindy about those employees getting their hours cut do to The Affordable Health Care Act. If they exceed a certain number of hours YSU would have to offer them health care. So, many may be on campus, been there decades, but few are not full time university employees. They do pay into the pension system and retire out of OPERS, but during their time on campus they were never considered full time by the university.


text:
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Catherine Howard
Painting - Acrylic On Canvas
This regal lion was named after my brother, Teddy Howard. 1956 - 2000. He was the most wonderful brother and man, kind, sensitive, generous and loving. These traits live on in his four wonderful daughters. Oh... and he loved lions and anything to do with Narnia by C.S. Lewis.
October 19th, 2012
Viewed 265 Times - Last Visitor from Beverly Hills, CA on 06/30/2015 at 12:31 PM
Sales Sheet
More from Catherine Howard
Cieszyn, Sl - Poland
Beautiful acrylic Catherine! L/F
Thank you for your lovely comment, Dariusz!
Firenze - Italy
Bello effetto complimenti.
Thank you, Mario for your lovely comment.
Suffield, CT - United States
Catherine, this is a beautiful painting with a beautiful description. Voted/f
Thank you, Anne...both for the kind comment and for the appreciation of the description.
Cambridge, ON - Canada
Wonderful work, Catherine! I'm sure your brother would have loved this regal lion - what a marvelous tribute! v/f
Thank you, Hanne.. I think he would like it also.
Iola, WI - United States
Catherine, I totally love the "big cats" and this acrylic beauty is awesome! just love it! fave and vote
Randy.. This is a special one for me and I truly appreciate your comments!
Rehoboth, MA - United States
Just beautiful!
Thank you, Joan!
Anaheim, CA - United States
Magnificent. v.
Thanks for the lovely comment and v, Angela!
Denver, CO - United States
You put remarkable feeling into this one.
Your comments are really appreciated, Beverley!
Van Vleck, TX - United States
Beautiful Lion. Wonderful painting in honor of your brother. f/v
Thank you Jimmie... your comments mean a lot to me,
Lyon - France
Beautiful painting of this regal lion, Catherine...And such a lovely homage to your brother...I'm deeply touched. f&v
Mona... I truly appreciated your kind and understanding comment. Thank you.
Depoe Bay, OR - United States
Beautiful painting, Catherine! Wonderful tribute to your brother!
Awww... thanks Glenna, he was very special.
Fort Worth, TX - United States
nice work! v
Thanks Joan!
USA, US - United States
Nice painting of a magnificent animal! v
Thank you so much Kami for the visit and support!
Merritt Island, Fl - United States
Ooops...forgot to say that I favorited and voted.
Thank you so much, AnnaJo.. for your wonderful comments and f/v... and I love your paintings!
Merritt Island, Fl - United States
Such a beautiful nobel tribute to your brother! This is a lovely painting. The textures are marvelous. The eyes are commanding and yet gentle. Thanks for your kind words about my paintings.
Boise, ID - United States
voted .f
Thank you so much, Sandi.
Lawrenceburg, TN - United States
Great painting, Catherine!! v
Thank you so much, Jk... for the lovely comment and the v.
Shawnee, Ks - United States
beautiful work. v
Thank you Liane.. I really appreciate your visit!
Coral Springs, FL - United States
I love Theodore. Great job. v
Thank you Gerry!
Fort Worth, TX - United States
love it, love the texture too. the eyes are wonderful, and that's what always makes the portrait! v
Thank you Joan.. I always look forward to your great comments.
Franklin Square, NY - United States
Catherine, Fantastic wildlife painting with amazing details of the King of the Jungle. Love the way you have expressed the eyes, and given him that large mane around his head. Excellent details in the face, with great colors. Fantastic painting so well done with capturing the right colors to express this beautiful subject. Excellent painting, voted, google, tweet and FB promoted and chosen as my Pay It Forward in the Self Taught Artist group
John... I am overwhelmed and honoured and grateful for your more than generous comments... you have totally made my day! Thank you for all the promotions also!
Pike Road, AL - United States
Fantastic work Catherine, v/f!
Thank you so much, Leslie!
Jackson, MI - United States
Beautiful work Catherine!! You are very talented!! f/v
Lee, I consider you to be one of my favourite artists here, so your comment is high praise indeed! thank you so much
New Westminster, BC - Canada
Majestic! f/v
Awww... thanks for all your support, Teresa!
Bolton, ON - Canada
Thank you, Glenna!
Depoe Bay, OR - United States
Beautiful Painting, Catherine!!! V/G+
Plymouth, MA - United States
A beautiful painting and a heartfelt tribute to your brother, Teddy. Voted/Favored.
Thank you so much, Janice! I really appreciate your visit and kind comments.
Fort Worth, TX - United States
already voted, G+ today
Thank you Joan, for the return visit and g 1 today!
Stetson, ME - United States
Stunning! v
Thanks Cindi!
Heiskell, Te - United States
a great tribute to your brother, such a wonderful painting, Thanks for sharing your talent Cathy.
Thank you Terry, it is an honour to receive such a compliment.
Prophetstown, IL - United States
Magnificent work!!
Diane, thank you for your lovely comment and visit!
Riverside, CA - United States
Beautiful painting! v
Thank you Sophia!
Fort Worth, TX - United States
very nice v
I really appreciate your comments and v, Joan!
Odessa, On - Canada
Definitely one of your best works Catherine! This painting makes a very touching tribute to a brother who was obviously very close to you. Great job!
Richard... your opinion means a great deal to me... thank you for your kind and positive comments.
Santa Fe, NM - United States
Hello Dear Catherine, Happy Winter Solstice 2012! I am in love with this image of yours and the beauty, regality, presence and wisdom that emanates. With many blessings to you and your beautiful brother-in-spirit from the Sangre de Cristo Mountains, Susanne
What an absolutely wonderful comment, Susanne! Thank you so much for the visit!
Bolton, ON - Canada
Thank you Talya... it makes me happy to know that your wonderful son's name is Teddy!
Wasilla, AK - United States
My eldest son's name is Teddy. and he is a gorgeous courageous boy just as you have captured in this lion! V
Chicago, IL - United States
What a gorgeous painting and a loving tribute to your brother :)). v/f
Awww.... thanks Anne, I truly appreciate that you read the description and for the v/f.
Bolton, ON - Canada
Lump in my throat Cheri.... thank you, always love your comments and thanks also for the vote.
New Orleans, LA - United States
Catherine, since I am a "Leo", I can appreciate Theodore's perfectly regal pose and demeanor. It's a wonderful tribute to your brother. voted
St. Louis, MO - United States
What a beautiful painting Catherine. Love your expression. v/f
Ah Genevieve, thank you for the wonderful comments and vote... this painting has a special meaning for me so I really appreciate you liking it!
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Beverly Hills, CA (United States)
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1. Trim the ends of the parsnips, peel them.
2. Using a mandoline or sharp knife, cut the parsnips into wafer-thin slices. (The final slices should be wafer thin and translucent, if using a mandoline)
3. In a large deep pan, place all the oil for frying and heat it.
4. Test it by dipping in a parsnip slice. When it starts sizzling, the oil is ready.
5. Fry the parsnip slices, a large handful at a time, stirring them with a slotted spoon. (Be careful not to add too much at a time)
6. The oil will vigorously bubble until all the water has been fried out of the parsnip slices. When the bubbling stops completely and the parsnip slices are just floating around in the oil, using a spoon remove them from the oil and set them on an absorbent kitchen paper to drain the excess oil.
7. Sprinkle salt over the chips, if desired.
8. Serve right away.


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3/26/2014 (1 year ago)
Catholic Online (www.catholic.org)
We have witnessed an ominous trend in the United States. Classical, faithful Christians who uphold the unbroken teaching of the Church and the scriptures concerning the dignity of every human life and the truth concerning marriage as solely between one man and one woman and formative of family, are being squeezed out of commerce.
Catholic Online (www.catholic.org)
3/26/2014 (1 year ago)
Published in U.S.
Keywords: Hobby Lobby, Conestaga Wood, Free exercise, Religious Freedom, HHS Mandate, Common Good Foundation, Common Good Alliance, Catholic Online, Deacon Keith Fournier
WASHINGTON,DC (Catholic Online) - On Tuesday, March 25, 2014, at the United States Supreme Court, one of the most important cases concerning the Fundamental Right to Religious Liberty will be argued. The case is styled Hobby Lobby and Conestoga Wood Specialties Corp. v. Kathleen Sebelius.
The excellent Lawyers from the Becket Fund for Religious Liberty represent Hobby Lobby. Hobby Lobby is a for profit business dedicated to Christian principles in the operation of its business.
We have witnessed an ominous trend in the United States.
Classical, faithful Christians who uphold the unbroken teaching of the Church and the scriptures concerning the dignity of every human life and the truth concerning marriage as solely between one man and one woman and formative of family, are being squeezed out of commerce.
This is not the first time in our two thousand year history as a Christian people that we have faced the reality of having to avoid some aspects of commerce because of the corruption of the cultures in which we live.
It is not our first time facing the hostility of worldly Caesars demanding that we bend the knee to their claims. It is simply that few thought it would happen in the United States of America with its robust protection of religious freedom embedded in its founding documents.
We need to expose and oppose this abuse of power by defending all of the Rights protected by the US Constitution, including the free exercise of religion, the right to free speech, free association and the right to participate in commerce.
However, we also need to be aware that morally coherent Christians, who live a unity of life and believe that their faith informs every aspect of their life, are at the beginning of a period of growing persecution. We must pray, band together and act.
The hostility being faced by Hobby Lobby is an affront to all of us. It calls all faithful Christians, Catholic, Protestant or Orthodox, to solidarity. They filed a lawsuit back in September of 2012 which can be read in its entirety here.
You can find an excellent update on the many cases challenging the HHS Mandate which seeks to compel Christian employers to (directly or indirectly) provide Chemical abortifacients, sterilization and contraceptives here.
This attempt by the Obama Administration to compel compliance with this unjust edit violates the Constitution and the fundamental right to Religious freedom. It is a blatant violation of the First Amendment to the Bill of Rights to the US Constitution.
Many of my readers know that I spent much of a thirty three year legal career as a Constitutional Lawyer. I continue to join with others who are engaged in this critical work on important cases.
I do so through Common Good Foundation and its legal defense fund. Common Good Foundation and Common Good Alliance joined in an Amicus Curiae or Friend of the Court Brief in this important case.
So did Catholic Online.
Here are the descriptions of the three organizations from that Amicus brief:
Common Good Foundation is a 501(c)(3) organization. Common Good Alliance is a 501(c)(4) organization. Founded by Keith A. Fournier, a Catholic apologist and constitutional lawyer, both organizations are informed by classical Christian social teaching and committed to building a culture of life, family, and freedom, while affirming classical Christian teaching, including that concerning religious freedom.
Common Good Foundation and Common Good Alliance each have a Legal Defense Fund that provides legal advocacy and support in cases concerning the organizations' missions. To perform their missions, Common Good Foundation and Common Good Alliance rely upon a robust interpretation of the religious liberty protected by the Free Exercise Clause and RFRA. Common Good Foundation and Common Good Alliance are concerned that a decision rejecting the plaintiffs' claims in this case would unduly restrict the protection RFRA provides for religious liberty.
Catholic Online is a business that serves Catholics and all people of good will by providing content over its integrated media network. Catholic Online operates its business in fidelity with the Catholic Church's teachings and is thus committed to building a culture of life, family, freedom, and solidarity. Catholic Online affirms classical Christian teaching, including that concerning religious freedom.
Catholic Online's business mission depends upon a proper interpretation of the religious liberty protected by the Free Exercise Clause and RFRA. Catholic Online is concerned that a decision rejecting the plaintiffs' claims in this case would unduly restrict the protection RFRA provides for religious liberty.
Here is the question which was briefed:
The individual plaintiffs in these actions, the Hahns and the Greens, seek to operate their closely-held, family-run businesses consistently with their Christian faith. These plaintiffs, based on that faith, believe that abortion is morally wrong and contrary to their faith and also that to facilitate abortion by providing their employees with health insurance that covers abortifacient drugs and devices is morally wrong and contrary to their faith.
The HHS mandate at issue in these cases subjects the Hahns' and the Greens' businesses to millions of dollars in penalties if the Hahns and the Greens do not direct their businesses to provide employees with health insurance that covers abortifacients. In effect, the mandate commands the Hahns and the Greens to do what their faith tells them is a sin or subject their businesses to substantial financial penalties.
The question before this Court is the following: Does the HHS mandate impose a "substantial burden" on the Hahns' and the Greens' exercise of religion as that term is used in the Religious Freedom Restoration Act, 42 U.S.C. § 2000bb-1(a) and in First Amendment jurisprudence.
We ask every reader of Catholic Online to pray that the United States Supreme Court upholds the fundamental Right to Religious Freedom. We will keep you up to date on this vital case.
St. Barbara
St. Gerard
St. Jude
St. George
St. Joseph
St. Christopher
St. Anthony
St. Florian
St. Isidore the Farmer
St. Benedict
St. Matthew
St. Joseph of Cupertino
St. Clare
St. John the Baptist
St. Francis of Assisi
St. Brigid of Ireland
By Deacon Keith Fournier
The Declaration of Independence was the birth certificate of the United States of America, a Nation often called the American Experiment because of the very fact that it was founded upon principles. The words are still memorized by schoolchildren and can bring a ... continue reading
By Deacon Keith A Fournier
What conclusion, dear brethren, do you come to? Surely it was not by chance that this chosen disciple, was missing in the first place? Or that on his return he heard, that hearing he doubted, that doubting he touched, and that touching he believed? It was by ... continue reading
By Matt Hadro, CNA/EWTN News
As bishops warn of current threats to religious liberty in the United States, Catholics can take heart that such challenges have faced them before, and they have persevered, one Church historian says. Washington D.C. (CNA/EWTN News) - "It's okay to realize that ... continue reading
By Catholic Online (NEWS CONSORTIUM)
It was bound to happen. After the Supreme Court ruled same-sex unions legal in all 50 states, other proponents of non-traditional arrangements are clamoring for equality under the law. Nathan Collier, along with his wives Victoria and Christine have applied at ... continue reading
By Talia Ramos (NEWS CONSORTIUM)
To kick off the summer break and celebrate Father's day, a family went aboard a cruise that ended with a traumatizing accident. As the cruise started to sail back to the harbor, a group of dolphins started to swim alongside the boat. However, as the passengers started ... continue reading
By Catholic Online (NEWS CONSORTIUM)
Episcopalians voted overwhelmingly this week to allow religious weddings for same-sex couples. The Episcopal Church has long been on the forefront of equal rights for homosexuals, ever since electing it first openly gay bishop 10 years ago. LOS ANGELES, ... continue reading
By Catholic Online (NEWS CONSORTIUM)
It appears that an important bit of counter-terrorism may have landed into the wrong hands. The Federal Bureau of Investigation, or FBI is installing command centers around the nation to prepare for possible terror attacks for the coming Fourth of July holiday ... continue reading
By Linky C. (NEWS CONSORTIUM)
A total of 10,000 American troops remain in Afghanistan. These troops believe they have been forgotten by the Obama administration, as the US Government shifts its attention to ISIS. MUNTINLUPA, PHILIPPINES (Catholic Online) - The US Troops, 10,000 men and women left ... continue reading
By Talia Ramos (NEWS CONSORTIUM)
Researchers recently discovered helium leaking from a giant fault in the Earth's crust near Los Angeles, as well as discovering an increase in chance for California to have an 8.0 magnitude earthquake. MUNTINLUPA, PHILIPPINES (Catholic Online) - They did not expect the ... continue reading
By Linky C. (NEWS CONSORTIUM)
Three days before the United States' Independence Day, the FBI has already began setting up command centers in all of the 56 field offices across the country prior to the fourth. The U.S. Government is preparing for possible terrorist attacks and terrorist threats that ... continue reading


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Madlenka (Paperback)
List Price: $7.99
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A trip around a city block is like a trip around the world
Peeking out through a die-cut window on the jacket, Madlenka invites the reader to enter her world. And what a world it is On the surface, it looks like an ordinary city block, but as we meet Madlenka's neighbors -- the French baker, the Indian news vendor, the Italian ice-cream man, the Latin American grocer, a retired opera singer from Germany, an African American school friend, and the Asian shopkeeper -- and look through die-cut windows to the images and memories they have carried from old country to new, we can see that Madlenka's block is as richly varied as its inhabitants. And why is Madlenka going around the block, jumping for joy? Her tooth is loose, and she wants everyone to know
"Madlenka" is a 2000 New York Times Book Review Notable Children's Book of the Year.
About the Author
Peter Sis is an internationally acclaimed illustrator, author, and filmmaker. He was born in Brno, Czechoslovakia, and attended the Academy of Applied Arts in Prague and the Royal College of Art in London. Peter is a seven-time winner of The New York Times Book Review Best Illustrated Book of the Year, a two-time Boston Globe-Horn Book Award Honoree, and has won the Society of Illustrators Gold Medal twice. Peter's books, "Starry Messenger: Galileo Galilei", T"ibet through the Red Box", and "The Wall: Growing Up Behind the Iron Curtain" were all named Caldecott Honor books by the American Library Association. "The Wall" was also awarded the Robert F. Sibert Medal.
In addition, Peter Sis is the first children's book illustrator to win the prestigious MacArthur Fellowship. He was chosen to deliver the 2012 May Hill Arbuthnot Honor Lecture for the Association for Library Service to Children. Peter won the 2012 Hans Christian Andersen Award. This award is considered the most prestigious in international children's literature, given biennially by the International Board on Books for Young People.
Peter Sis lives in the New York City area with his wife and children.
Praise For…
"For Madlenka, who has just discovered that she has a loose tooth, the block is a world unto itself, a familiar, self-contained place and also a realm of infinite newness and variety, always reassuringly the same and yet constantly changing its shape, color and texture. Her adventure is perfectly ordinary and completely magical, like the wonderful, scary experience of losing a tooth. . . . The book's design is ingenious, a kind of two-dimensional hypertext of maps and windows that allows you to flip from the cartography of real places to a geography of pure imagination. . . . A book to be read slowly and repeatedly."—The New York Times Book Review "As he did with Tibet Through the Red Box, Sís takes readers to exotic lands, yet continues to bring them back to the comfort of what they know. . . . When Madlenka returns home and tells her parents that she 'went all around the world,' readers will feel that they, too, have been armchair travelers, delivered safely home in Sís's capable hands."—Publishers Weekly
"Visually stunning . . . The real magic comes in the cleverly cut-away windows in each storefront through which children glimpse complex, global dreamscapes. Madlenka journeys through these mystical places, too, and it is these surreal, wordless stories-within-the-story that will excite a wide range of children, launching them in their own imagined departures."—Booklist, Boxed Review
"[Sís] incorporates simple and telling details—real and imaginary—into his rich and sophisticated art. Illustrations are intricate, providing plenty to see on every turn of the page. Several ingenious cut-outs provide literal peeks into this international city."—The Christian Science Monitor
"This brief story captures all of Sís's most distinguished qualities of dreamlike mystery while remaining accessible to younger children. The book is meticulously designed . . . The exquisite double-page spreads invite close inspection . . . Undeniably clever, well-intentioned, and beautiful."—Kirkus Reviews
"In Sís's hands, this is a journey filled with mystical creatures and magical symbols, as the child is greeted by an international panoply of merchants and neighbors. . . . The stark white background around the child contrasts effectively with the dark ink-and-watercolor scenes once the threshold has been crossed. Groups will be captivated by the concept and the drama provided by the die-cuts and the fantastic settings. Individuals will pore over the many details, delighting in the emergence of forms and meaning provided by close inspection. An odyssey made all the more wondrous by pairing a big moment in a small child's life with the happenings in the cosmos."—School Library Journal


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Originally written on WHYGAVS? | Last updated 11/14/11
Neal Huntington joined with Brian Cashman and Theo Epstein (I know!) this weekend at Buster Olney's "Going to Bat for Vermont Farmers" benefit to help the region recover from Hurricane Irene, and Jed from Sox Detox was there to chronicle the event. He wrote that Huntington, who's inclusion probably stems from the fact that he's a New Hampshire native, held his own pretty well with the big-market guys and in a brief chat with him afterwards discussed everything from trading Joel Hanrahan to Rod Barajas to Pedro Alvarez to Starling Marte to pitcher development. It's definitely worth your time to check out both links. Ryan Doumit reportedly turned down a 1-year/$3 million offer from the Dodgers over the weekend. That's quite a bit less than the Pirates would've owed him on his team option and even less than the Pirates are paying Rod Barajas, so it's possible that quite a few clubs have downgraded Doumit due to his poor defense behind the plate and inability to stay healthy. Jerry Cras...
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HDVShort for High Definition Video, HDV is a video format, commonly used in camcorders, that allows high-definition footage to be recorded onto standard DV tapes. HDV uses Long GOP MPEG2 compression. The HDV format includes both 720p and 1080i specifications. The HDV standard was established by Canon Inc., Sharp Corp., Sony Corp., and the Victor Company of Japan, Ltd.
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The Not So Big Life: Making Room for What Really MattersPaperback
List price $23.35
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- Publisher: Broadway Books (A Division of Bantam Doubleday Del
- Format: Paperback | 304 pages
- Dimensions: 130mm x 201mm x 18mm | 259g
- Publication date: 13 February 2008
- Publication City/Country: New York
- Edition statement: Reprint
- Illustrations note: black & white illustrations
- Sales rank: 206,573
Product description
Have you ever found yourself asking, Is this all there is to life? Or wondering if this bigger life you have created is actually a better life? And do you wonder how it all got so out of control? In her groundbreaking bestseller The Not So Big House, architect Sarah Susanka showed us a new way to inhabit our houses by creating homes that were better-not bigger, Now, in The Not So Big Life, Susanka takes her revolutionary philosophy to another dimension by showing us a new way to inhabit our lives, Most of us have lives that are as cluttered with unwanted obligations as our attics are cluttered with things. The bigger-is-better idea that triggered the explosion of McMansions has spilled over to give us McLives. For many of us, our ability to find the time to do what we want to do has come to a grinding halt. Now we barely have time to take a breath before making the next call on our cell phone, while at the same time messaging someone else on our Blackberry. Our schedules are chaotic and overcommitted, leaving us so stressed that we are numb, yet we wonder why we cannot fall asleep at night. In The Not So Big Life, Susanka shows us that it is possible to take our finger off the fast-forward button, and to our surprise we find how effortless and rewarding this change can be. We do not have to lead a monastic life or give up the things we love. In fact, the real joy of leading a not so big life is discovering that the life we love has been there the entire time. Through simple exercises and inspiring stories, Susanka shows us that all we need to do is make small shifts in our day-subtle movements that open our minds as if we were finally opening the windows tolet in fresh air. The Not So Big Life reveals that form and function serve not only architectural aims but life goals as well. Just as we can tear down interior walls to reveal space, we can tear down our fears and assumptions to open up new possibilities. The result is that we quickly discover we have all the space and time we need for the things in our lives that really matter. But perhaps the greatest reward is the discovery that small changes can yield enormous results. In her elegant, clear style, Susanka convinces us that less truly is more-much more. From the Hardcover edition.
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Sarah Susanka is a bestselling author, architect, and cultural visionary. Her "build better, not bigger" approach to residential architecture has been embraced across the country, and her "Not So Big" philosophy has sparked an international dialogue, evolving beyond our houses and into how we inhabit our lives. In addition to sharing her insights with Oprah Winfrey and Charlie Rose, Susanka has been named a "Fast 50" innovator by "Fast Company," a "top newsmaker" by "Newsweek," and an "innovator in American culture" by "U.S. News & World Report." She is a member of the College of Fellows of the American Institute of Architects and a Senior Fellow of the Design Futures Council. The author of seven books, Susanka resides in North Carolina. Visit her at www.notsobig.com. "From the Hardcover edition."


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Due to a Mississippi High School Activities Association rule change, soccer players throughout Starkville are forced with a heavy decision when it comes to where they will play.
There is a good bit experience and depth on the Mississippi State defensive line, but the key word this year is consistency.
In his second stint with the Bulldogs as the defensive line coach, David Turner inherits a vast amount of talent and big bodies.
Although there's some experience with the defensive line, last Saturday in practice his squad took a step back.
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