Unstimulated Control Research Articles

Modulation of protease-activated receptor expression by Porphyromonas gingivalis in human gingival epithelial cells.

BackgroundProtease-activated receptors (PARs) are G-protein-coupled receptors with an active role in mediating inflammation, pain and other functions. The oral pathogen Porphyromonas gingivalis (P. gingivalis) secretes proteases that activate PARs. The aim of this study was to elucidate the role of PARs in the pathogenesis of chronic periodontitis by expression analysis of PARs in human gingival epithelial cells (GECs) before and after P. gingivalis supernatants treatment.MethodsGECs were isolated from healthy human gingival tissue samples. The expression of PARs in GECs was determined by reverse transcription-polymerase chain reaction (RT-PCR) and flow cytometry. The effect of P. gingivalis proteases was investigated by quantitative real-time reverse transcription polymerase chain reaction (QRT-PCR) and flow cytometry.ResultsPAR-1, PAR-2, and PAR-3 were expressed in GECs. PAR-4 was not found by both RT-PCR and flow cytometry. Analysis of gene expression using QRT-PCR showed an up-regulation of PAR-2 mRNA in comparison to the untreated control cells (P < 0.05). In contrast, the mRNA expressions of PAR-1 and PAR-3 were significantly down-regulated (P > 0.05) in response to P. gingivalis supernatant compared to that in unstimulated control cells. This effect was abrogated by the protease inhibitor TLCK (P < 0.05). The results of flow cytometry indicated PARs protein levels consistent with mRNA levels in the results of QRT-PCR.ConclusionsOur study shows that PAR-1, PAR-2 and PAR-3 are expressed in GECs. P. gingivalis proteases play a role in the regulation of innate immune responses in GECs. GECs use PARs to recognize P. gingivalis and mediate cell responses involved in innate immunity.

Effects of Functional Electrical Stimulation on Denervated Laryngeal Muscle in a Large Animal Model.

Bilateral vocal fold paralysis (BVCP) is a life-threatening condition that follows injury to the Recurrent Laryngeal nerve (RLn) and denervation of the intrinsic laryngeal musculature. Functional electrical stimulation (FES) enables restoration and control of a wide variety of motor functions impaired by lower motor neuron lesions. Here we evaluate the effects of FES on the sole arytenoid abductor, the posterior cricoarytenoid (PCA) muscle in a large animal model of RLn injury. Ten horses were instrumented with two quadripolar intramuscular electrodes in the left PCA muscle. Following a 12-week denervation period, the PCA was stimulated using a once-daily training session for 8 weeks in seven animals. Three animals were used as unstimulated controls. Denervation produced a significant increase in rheobase (P < 0.001). Electrical stimulation produced a 30% increase in fiber diameter in comparison with the unstimulated control group (33.9 ± 2.6 µm FES+, 23.6 ± 4.2 µm FES-, P = 0.04). A trend toward a decrease in the proportion of type 1 (slow) fibers and an increase in type 2a (fast) fibers was also observed. Despite these changes, improvement in PCA function at rest was not observed. These data suggest that electrical stimulation using a relatively conservative set of stimulation parameters can reverse the muscle fiber atrophy produced by complete denervation while avoiding a shift to a slow (type 1) fiber type.

Impact of cyclic mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff fibroblasts

Mechanical stimulation plays an important role in the development and remodelling of tendons. The aim of the study was to evaluate the effects of mechanical stimulation on the expression of extracellular matrix proteins in human primary rotator cuff (RC) fibroblasts. RC fibroblasts were isolated from patients with degenerative RC tears and characterized using flow cytometry and immunohistochemistry. Cells were stimulated using the Flexcell FX5K™ Tension System. The stimulation regime was a uniaxial sinusoidal waveform with 10% elongation and a frequency of 0.5Hz, whereby each cycle consists of 10-s strain and 30-s relaxation. Data were normalized to mechanically unstimulated control groups for every experimental condition. RT-qPCR was performed to determine relative mRNA levels, and collagen production was measured by a colorimetric assay. The positive expression of CD91 and CD10, and negativity for CD45 and CD4 confirmed the fibroblast phenotype of RC primary cells. RT-qPCR revealed that 10% continuous cyclic strain for 7 and 14days induced a significant increase in the mRNA expression both on the matrix metalloproteinases MMP1, MMP3, MMP13, and MMP14 and on the extracellular matrix proteins decorin, tenascin-C, and scleraxis. Furthermore, mechanically stimulated groups produced significantly higher amounts of total collagen. These results may contribute to a better understanding of strain-induced tendon remodelling and will form the basis for the correct choice of applied force in rehabilitation after orthopaedic surgery. These findings underline the fact that early passive motion of the joint in order to induce remodelling of the tendon should be included within a rehabilitation protocol for rotator cuff repair.

Abstract 2313: Characterizing intratumoral cellular heterogeneity in human glioblastoma xenografts by 30+ parameter single-cell mass cytometry

Abstract Introduction: Glioblastoma tumors contain dramatic intratumoral heterogeneity at the level of cellular phenotypes. High-parameter single-cell platforms such as mass cytometry offer an opportunity to simultaneously measure tumor subpopulations with unexpected combinations of differentiation markers, as well as stromal cells, vasculature, and infiltrating immune cells. Methods: A subcutaneous patient-derived xenograft from an adult with glioblastoma was enzymatically digested into a single-cell suspension. Half of the cells were treated with epidermal growth factor (EGF) at 20 ng/mL for 15 minutes. Both samples were then stained with a 32-marker antibody panel and measured by single-cell mass cytometry. The antibody panel included surface-expressed markers of stemness and differentiation, as well as intracellular markers to monitor cell cycle, apoptosis, and growth factor signaling. Unsupervised spectral clustering was used to segregate &amp;gt;200,000 tumor cells into 13 tumor cell subsets. Inducible signaling phenotypes were quantified in these subsets by comparing binding of phospho-specific antibodies in the stimulated sample, relative to the unstimulated control. Results: Many previously reported markers of cancer stem cells were detectable, but they did not agree on a single population of cells. Although the CD133+ fraction was rare (&amp;lt;5% of cells), it contained three distinct subpopulations of phenotypically distinct cells. The most informative markers for discriminating the tumor subsets were markers of stemness (i.e. CD133, CD44, Sox2) or integrin expression (i.e. CD49e, CD49f, CD51). The primitive cells (i.e. Sox2+) exhibited higher basal kinase activity across multiple pathways, including markers of cell division (i.e. Histone H3 pSer28). Short-term stimulation with EGF induced distinct responses in each of the different CD133+ subsets. Conclusions: Single-cell phenotyping by mass cytometry produced rich developmental and functional profiles of glioblastoma cell subsets. Subpopulations of primitive (CD133+) cells can exist in a single glioblastoma tumor and exhibit distinct signaling responses to external stimulation. Altogether, these results validate the use of this platform for investigation of the intratumoral cellular heterogeneity of solid neural tumors with this platform. Work is underway to identify correlations between intratumoral cellular phenotypes and upstream driver mutations in a larger cohort. Citation Format: Erin F. Simonds, Garry P. Nolan, William A. Weiss. Characterizing intratumoral cellular heterogeneity in human glioblastoma xenografts by 30+ parameter single-cell mass cytometry. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2313. doi:10.1158/1538-7445.AM2015-2313

Abstract 4278: Soy isoflavones and their metabolites modulate IL-12-induced NK cell IFN-γ production

Abstract Soybeans and foods derived from them are a rich source of phytochemcials that have gained attention due to their ability to affect obesity, metabolism, cancer, and inflammation. Soy isoflavones (genistein and daidzein) have also been shown to be differentially metabolized between individuals. This is presumably due to differences in gut microbiota, and can lead to production of terminal isoflavone metabolites such as O-desmethylangolensin (ODMA) and equol. In a prior phase-I clinical study conducted by our group, patients with prostate cancer received a novel soy-enriched bread product. Analysis of blood and urine following soy intervention revealed that men clustered into distinct groups based on their ability to metabolize daidizein into either ODMA or equol. Reduced pro-inflammatory cytokines and percentages of Tregs and MDSC were also observed in the blood of patients after 8 weeks of soy bread intervention as compared to baseline. Given these differences, we hypothesized that individual isoflavones and metabolites would have unique immunomodulatory effects upon human natural killer (NK) cell function. Although a limited number of prior studies have evaluated how soy isoflavones or their metabolites modulate inflammation and different immune subsets (monocytes, macrophages, DCs), their role in NK cell biology remains poorly defined. Understanding the role of soy isoflavones and their metabolites in regulating NK cell activity is critical due to NK cells’ importance in the anti-tumor response and immunosurveillance. We found that the soy isoflavones and their metabolites did not affect the viability of healthy donor PBMC (n = 3) at concentrations as high as 25μM. However, pre-treatment of healthy donor PBMC (n = 3) with genistein (mean 7.1 ng/ml ±1.3) or equol (mean 6.7 ng/ml ±1.8) for 4 hours and then stimulated with IL-12/18 for 72 hours decreased IFN-γ production compared to unstimulated controls (mean 26.7 ng/ml ±16.1). In contrast, daidzein and ODMA had no effect. We next utilized intracellular flow cytometry to determine the cellular subset(s) that were modulated by soy isoflavones or their metabolites. These data revealed that only genistein and equol decreased the percentage of IL-12/18 induced IFN-γ producing NK cells (CD56+CD3-) by 30% and 53%, respectively. There was no observed difference in other IFN-γ producing immune subsets (T and NKT cells) when cultured with the soy isoflavones or metabolites. Furthermore, we found soy isoflavones and metabolites altered in vitro cytotoxicity of human IL-12 stimulated NK cells. Ongoing studies in our laboratory are elucidating the precise signaling pathways downstream of IL-12 in human NK cells that are modulated in response to soy isoflavones and their bioactive metabolites. This study provides critical insight regarding the impact of dietary soy on NK cell mediated responses potentially informing the use of soy during chemopreventive or immune based therapies. Citation Format: Thomas A. Mace, Samantha King, Matthew Farren, Elizabeth McMichael, Steven Scoville, William E. Carson, Gregory Young, Jennifer Thomas-Ahner, Kenneth M. Riedl, Steven Schwartz, Steven K. Clinton, Gregory Lesinski. Soy isoflavones and their metabolites modulate IL-12-induced NK cell IFN-γ production. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4278. doi:10.1158/1538-7445.AM2015-4278

Valvular interstitial cells suppress calcification of valvular endothelial cells.

Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. We previously proposed that valvular endothelial cells (VECs) replenish injured adult valve leaflets via endothelial-to-mesenchymal transformation (EndMT); however, whether EndMT contributes to valvular calcification is unknown. We hypothesized that aortic VECs undergo osteogenic differentiation via an EndMT process that can be inhibited by valvular interstitial cells (VICs). VEC clones underwent TGF-β1-mediated EndMT, shown by significantly increased mRNA expression of the EndMT markers α-SMA (5.3 ± 1.2), MMP-2 (13.5 ± 0.6) and Slug (12 ± 2.1) (p < 0.05), (compared to unstimulated controls). To study the effects of VIC on VEC EndMT, clonal populations of VICs were derived from the same valve leaflets, placed in co-culture with VECs, and grown in control/TGF-β1 supplemented media. In the presence of VICs, EndMT was inhibited, shown by decreased mRNA expression of α-SMA (0.1 ± 0.5), MMP-2 (0.1 ± 0.1), and Slug (0.2 ± 0.2) (p < 0.05). When cultured in osteogenic media, VECs demonstrated osteogenic changes confirmed by increase in mRNA expression of osteocalcin (8.6 ± 1.3), osteopontin (3.7 ± 0.3), and Runx2 (5.5 ± 1.5). The VIC presence inhibited VEC osteogenesis, demonstrated by decreased expression of osteocalcin (0.4 ± 0.1) and osteopontin (0.2 ± 0.1) (p < 0.05). Time course analysis suggested that EndMT precedes osteogenesis, shown by an initial increase of α-SMA and MMP-2 (day 7), followed by an increase of osteopontin and osteocalcin (day 14). The data indicate that EndMT may precede VEC osteogenesis. This study shows that VICs inhibit VEC EndMT and osteogenesis, indicating the importance of VEC-VIC interactions in valve homeostasis.

Inflammatory and Immune Activation in Intestinal Myofibroblasts Is Developmentally Regulated.

We previously demonstrated that intestinal myofibroblasts from immature tissue produce excessive IL-8 in response to Escherichia coli lipopolysaccharide (LPS) compared to cells from mature tissue. However, it is unknown whether other cytokines and TLR agonists contribute to this developmentally regulated response. The aim of this study was to further characterize differences in inflammatory signaling in human primary intestinal fibroblasts from fetal (FIF) and infant (IIF) tissue and examine their potential to activate the adaptive immune response in vitro. Cytokine profiles of LPS-stimulated FIF and IIF were assessed by cytokine profile array. IL-8, IL-6, and IL-10 production in response to TLR2, TLR2/6, TLR4, and TLR5 agonists was determined by quantitative ELISA. The potential of activated myofibroblasts to activate adaptive immunity was determined by measuring surface class II MHC expression using flow cytometry. LPS-stimulated FIF produced a distinct proinflammatory cytokine profile consisting of MCP-1, GRO-alpha, IL-6, and IL-8 expression. FIF produced significant IL-8 and IL-6 in response to TLR4 agonist. IIF produced significant levels of IL-8 and IL-6 in the presence of TLR5 and TLR2 agonists. IFN-γ-treated FIF expressed greater HLA-DR levels compared to unstimulated controls and IFN-γ- and LPS-treated IIF. Activated FIF produce a more diverse inflammatory cytokine profile and greater levels of IL-8 and IL-6 in response to TLR4 stimulation compared to IIF. FIF express class II MHC proteins associated with activation of the adaptive immune response. These data suggest that FIF may contribute to bacterial-associated gut inflammation in the immature intestine.

OC-036 Stimulated expression of ileal fibroblast growth factor 19 by bile acids is impaired in patients with primary and secondary bile acid diarrhoea

<h3>Introduction</h3> Bile acid diarrhoea (BAD) results from excess luminal bile acids (BA) in the colon. Most BAs are reabsorbed at the terminal ileum. Increased colonic BA occur secondary to ileal disease or resection (common in Crohn’s disease [CD]) leading to BA malabsorption and secondary BAD (SBAD). Primary BAD (PBAD) occurs in the absence of overt ileal disease or malabsorption. The peptide FGF19 is produced from enterocytes in response to BA absorption and maintains homeostasis of the BA pool size by inhibiting hepatic BA synthesis. Impaired ileal FGF19 production may underlie the excess faecal BA observed in PBAD and may be contributory in CD as a result of increased hepatic BA synthesis. We aimed to investigate whether BA-induced expression of FGF19 in the ileum is reduced in patients with PBAD or CD. <h3>Method</h3> Patients attending for colonoscopy (n = 58) were prospectively recruited and gave informed consent to give additional ileal biopsies. 14 patients had CD (4 with previous right hemi colectomy and 10 without), 16 patients had unexplained diarrhoea and had SeHCAT tests, and 28 controls. Groups of 2–3 biopsies (explants) were incubated separately for 6 h with either BA-free culture media (unstimulated controls), chenodeoxycholate (CDCA) or glyco-CDCA (GCDCA), both at 50 µM. After RNA extraction and cDNA synthesis, FGF19 expression was quantified relative to GAPDH by RT-PCR. <h3>Results</h3> In patients with unexplained diarrhoea SeHCAT 7d retention values were &lt;15% in 7 patients, indicating primary BAD, and &gt;15% in 9 who were normal diarrhoea controls. A positive correlation was found between SeHCAT retention and the magnitude of the fold change in FGF19 expression stimulated by either CDCA (r = 0.50, n = 16, p = 0.02) or by GCDCA (r = 0.76, n = 7, p = 0.02). The median CDCA stimulated fold change in FGF19 expression was significantly lower in CD compared to controls (87, n = 12 vs 251, n = 28, p = 0.03) and a similar non-significant trend was observed for median GCDCA stimulated expression (97, n = 10 vs 156, n = 13, p = 0.18). <h3>Conclusion</h3> Ileal explants from patients with lower SeHCAT 7d retention had a lower response to both CDCA and GCDCA. Patients with CD had lower responses to CDCA. As uptake of GCDCA is dependent on carrier-mediated apical BA uptake but CDCA is not, this implies abnormalities in the intracellular ileal FGF19 response to BA. A reduced capacity to induce ileal FGF19 expression in response to absorbed BA is proposed to increase faecal BA due to increased hepatic BA synthesis in primary BAD. In secondary BAD an impaired ileal BA FGF19 response may lead to an increase in BA synthesis and faecal BA that is disproportionate to the degree of ileal BA malabsorption. <h3>Disclosure of interest</h3> None Declared.

Optogenetic control of nerve growth.

Due to the limited regenerative ability of neural tissue, a diverse set of biochemical and biophysical cues for increasing nerve growth has been investigated, including neurotrophic factors, topography, and electrical stimulation. In this report, we explore optogenetic control of neurite growth as a cell-specific alternative to electrical stimulation. By investigating a broad range of optical stimulation parameters on dorsal root ganglia (DRGs) expressing channelrhodopsin 2 (ChR2), we identified conditions that enhance neurite outgrowth by three-fold as compared to unstimulated or wild-type (WT) controls. Furthermore, optogenetic stimulation of ChR2 expressing DRGs induces directional outgrowth in WT DRGs co-cultured within a 10 mm vicinity of the optically sensitive ganglia. This observed enhancement and polarization of neurite growth was accompanied by an increased expression of neural growth and brain derived neurotrophic factors (NGF, BDNF). This work highlights the potential for implementing optogenetics to drive nerve growth in specific cell populations.

The Effect of Progestins on Tumor Necrosis Factor α-Induced Matrix Metalloproteinase-9 Activity and Gene Expression in Human Primary Amnion and Chorion Cells In Vitro.

Current treatment modalities for preventing preterm premature rupture of membranes are limited, but progestins may play a role. Tumor necrosis factor α (TNFα) enhances matrix metalloproteinase-9 (MMP-9) gene expression and activity in fetal membranes, contributing to membrane weakening and rupture. We previously demonstrated that progestins attenuate TNFα-induced MMP-9 activity in a cytotrophoblast cell line. However, whether they have a similar effect in primary amnion and chorion cells of fetal membranes is unknown. In this study, we evaluated the effect of progestins on basal and TNFα-induced MMP-9 activity and gene expression in primary chorion and amnion cells harvested from the fetal membranes of term nonlaboring patients. Primary amnion and chorion cells were isolated from fetal membranes obtained from term uncomplicated nonlaboring patients following elective cesarean delivery (n = 11). Confluent primary amnion and chorion cell cultures were both pretreated with vehicle (control), progesterone (P4), 17α-hydroxyprogesterone caproate (17P), or medroxyprogesterone acetate (MPA) at 10 M concentration for 6 hours followed by stimulation with TNFα at 10 ng/mL for an additional 24 hours. Cell cultures pretreated with the vehicle only served as the unstimulated control and the vehicle stimulated with TNFα served as the stimulated control. Both controls were assigned a value of 100 units. Cell culture medium was harvested for MMP-9 enzymatic activity quantification using gelatin zymography. Total RNA was extracted for quantifying MMP-9 gene expression using real-time quantitative PCR. Basal MMP-9 activity and gene expression data were normalized to the unstimulated control. TNFα-stimulated MMP-9 activity and gene expression were normalized to the stimulated control. The primary outcome was the effect of progestins on TNFα-induced MMP-9 enzymatic activity in term human primary amnion and chorion cells in vitro. Secondary outcomes included the effect of progestin therapy on TNFα-induced MMP-9 gene expression and on basal MMP-9 activity and gene expression in primary amnion and chorion cells in vitro. Primary cells were harvested from 11 patients. Compared with the unstimulated control, TNFα increased MMP-9 activity (P = 0.005 versus control in primary amnion cells and P < 0.001 versus control in primary chorion cells) and MMP-9 gene expression (P = 0.030 versus control in primary amnion cells, P < 0.001 versus control in primary chorion cells). Compared with the unstimulated controls, MPA, but not P4 or 17P, reduced basal MMP-9 activity [mean difference (95% CI) -49.6 (-81.9, -17.3) units, P = 0.001] and gene expression [mean difference (95% CI) -53.4 (-105.9, -0.9) units, P = 0.045] in primary amnion cells. Compared with the stimulated control, MPA also reduced TNFα-induced MMP-9 activity [mean difference (95% CI) -69.0 (-91.8, -46.3) units, P < 0.001] and gene expression [mean difference (95% CI) -86.0 (-120.7, -51.3) units, P < 0.001] in primary amnion cells. Progestin pretreatment had no significant effect on basal or TNFα-induced MMP-9 activity and gene expression in primary chorion cells. The inhibitory effect of MPA on both basal and TNFα-induced MMP-9 activity and gene expression in primary amnion cells demonstrate a possible mechanism by which progestins may prevent fetal membrane weakening leading to preterm premature rupture of membranes.

Oncostatin M promotes epithelial barrier dysfunction and is elevated in eosinophilic mucosal disease (HYP7P.255)

Abstract Epithelial barrier dysfunction is thought to play a role in many mucosal diseases including asthma, chronic rhinosinusitis (CRS), and eosinophilic esophagitis (EoE). Oncostatin M (OSM), was elevated in both mRNA (28.3 fold, p&amp;lt;.01, n=12) and protein (4.4 fold, p&amp;lt;.05, n=12-19) in the nasal polyps of CRS patients compared to control tissue. OSM protein was also elevated in bronchoalveolar lavage (BAL) of allergic asthma (AA) patients following segmental allergen challenge (SAC) vs. saline challenge (14 fold, p&amp;lt;.0001). Additionally, OSM mRNA was elevated in esophageal biopsies of EoE patients vs. controls, (3 fold, p&amp;lt;.01). OSM stimulation of differentiated airway epithelium induced barrier dysfunction, as measured by decreased transepithelial electrical resistance (63% reduction, p&amp;lt;.0001, n=11) and increased permeability to 10kD dextran (2.45 fold, p&amp;lt;.05, n=5). Staining for the tight junction protein, occludin showed that OSM stimulation of differentiated airway epithelium impaired the organization of the tight junctions compared to unstimulated controls (n=5). Levels of OSM protein in tissue lysates from CRS and controls correlated with levels of α2-macroglobulin, a marker of epithelial leak, in matched nasal secretions (r=.5055, p&amp;lt;.01). Additionally, OSM in the BAL following SAC in AA patients correlated with serum albumin, another marker of epithelial leak (r=.8062, p&amp;lt;.001). These results suggest that OSM may play a role in epithelial barrier dysfunction in mucosal disease.

616. Disc Gene Therapy: Development of a Novel Inducible System To Regulate Expression of the Therapeutic Transgene TIMP1

Introduction: Gene therapy using the anti-catabolic factor Tissue Inhibitor of Metalloproteinase-1 (hTIMP1) has shown promising efficacy in treating disc degeneration. However, a major limitation of the current gene therapy technique is the use of a constitutive promoter to drive transgene expression that results in uncontrolled and excessive expression of therapeutic gene product which could result in undesirable side effects. To overcome this important limitation, we successfully developed a new gene therapy recombinant adeno-associated viral (rAAV)vector, rAAV-NFkB-hTIMP1, in which the therapeutic recombinant human TIMP1 gene is placed under the control of a promoter containing the NF-kB element. The rAAV-NFkB-hTIMP1 construct is designed to produce the therapeutic gene product TIMP1 only when the cells experience stress which activates the NF-kB pathway. Hence we expect using this strategy that hTIMP1 will be selectively expressed in cells under inflammatory conditions typically found in active degenerating discs and not under normal conditions, in which the therapeutic gene was not needed. In this study we tested our hypothesis that rabbit annulus fibrosus cells transfected with aserotype 2 of rAAV vector carrying a hTIMP1 gene will not express high level of hTIMP1 unless stimulated with the IL-1b.Methods: Cultured rabbit annulus fibrosus (rAF) cells were divided into six groups; “Control” (cell only); “rAAV-CMV-hTIMP1(cells transfected with AAV-CMV-hTIMP1 plasmid DNA)” and “rAAV-NFkB-hTIMP1(cells transfected with AAV-NFkB-hTIMP1 plasmid DNA)” treated with and without IL-1b (n=4 for each group). Production of hTIMP1 was determined by RT-PCR. To determine if MMP activity was inhibited by the produced TIMP1, MMP activity assay was measured by following cleavage of a fluorogenic substrate.Results: RT-PCR analysis demonstrated that the level of hTIMP1 transcription from cells and culture media transfected with rAAV-NFkB-hTIMP1 construct was greater than in the IL-1b stimulated condition compared to unstimulated control. The result shows that the level of MMP activity was decreased compared to baseline levels or cells exposed to IL-1.Discussion: The RT-PCR result showed cells transfected with AAV-NFkB-hTIMP1 plasmid DNA had significant increase in the hTIMP-1 mRNA expression after stimulation with IL-1b. These results demonstrate the novelty of this strategy in which the NF-kB element containing promoter ensures that the transgene hTIMP1 is expressed highly only under conditions of inflammation typically found in injured and degenerating discs which would act to block MMP-mediated matrix proteolytic destruction. Low levels of hTIMP1 were also observed under unstimulated conditions and the bioenergetic consequences of the cell are unclear.

Electrical Pulse Stimulation Induced Changes On Lipid, Mitochondrial, Glut4 And Ampk Content In Human Myotubes

It has been previously shown that human myotubes retain certain in vivo characteristics of the donors. Furthermore, we have shown that Electrical pulse stimulation (EPS), an in vitro exercise mimetic, increases mitochondrial and lipid content in cultured human myotubes after 24 hr. of stimulation. PURPOSE: We aimed to examine the EPS induced adaptations to lipid, mitochondrial, Glucose transporter 4 (GLUT4) and total 5’ Adenosine Monophosphate Activated Protein Kinase (AMPK) content using human myotubes from lean donors. Additionally, we investigated the impact of cell harvest time (immediate vs. 24 hr. after the EPS cessation) on the above-mentioned parameters. METHODS: EPS was applied to myotubes for 60 min (bipolar pulses of 100 Hz, 30V, every 5 seconds), 24 hr. or 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Mitochondrial and lipid content were measured by immunohistochemistry using primary antibody for complex IV and bodipy green dye, respectively. A microplate fluorometer was used to quantify fluorescence signals. GLUT4 and AMPK were measured using western immunoblotting. RESULTS: Lipid in myotubes was significantly higher (p<0.05) after 24 hr early harvest EPS compared to unstimulated control. There was no significant changes in lipid content with late harvest EPS. Mitochondrial content was significantly higher (p<0.05) after acute, 24 hr. as well as 48 hr. early harvest EPS, but was significantly lower (p<0.05) after acute late harvest EPS, compared to unstimulated controls. GLUT4 protein content was higher after acute and 24 hr. early harvest and 48 hr. late harvest EPS. Total AMPK was higher after 24 hr. early harvest and 48 hr. late harvest. AMPK was lower after 24 hr. late harvest. CONCLUSION: These findings suggest 24 hours of EPS treatment lead to increase in lipid, mitochondria, GLUT4 and AMPK protein content in human myotubes cultured from lean donors. Additionally, cell-harvesting time a reflection of post-exercise recovery is a key factor for these adaptations.

Multi-parametric analysis of phagocyte antimicrobial responses using imaging flow cytometry

We feature a multi-parametric approach based on an imaging flow cytometry platform for examining phagocyte antimicrobial responses against the gram-negative bacterium Aeromonas veronii. This pathogen is known to induce strong inflammatory responses across a broad range of animal species, including humans. We examined the contribution of A. veronii to the induction of early phagocyte inflammatory processes in RAW 264.7 murine macrophages in vitro. We found that A. veronii, both in live or heat-killed forms, induced similar levels of macrophage activation based on NF-κB translocation. Although these macrophages maintained high levels of viability following heat-killed or live challenges with A. veronii, we identified inhibition of macrophage proliferation as early as 1h post in vitro challenge. The characterization of phagocytic responses showed a time-dependent increase in phagocytosis upon A. veronii challenge, which was paired with a robust induction of intracellular respiratory burst responses. Interestingly, despite the overall increase in the production of reactive oxygen species (ROS) among RAW 264.7 macrophages, we found a significant reduction in the production of ROS among the macrophage subset that had bound A. veronii. Phagocytic uptake of the pathogen further decreased ROS production levels, even beyond those of unstimulated controls. Overall, this multi-parametric imaging flow cytometry-based approach allowed for segregation of unique phagocyte sub-populations and examination of their downstream antimicrobial responses, and should contribute to improved understanding of phagocyte responses against Aeromonas and other pathogens.

Sa1779 Soluble Plantain (Banana) Fibre Inhibits Epithelial Cell Damage in Response to Clostridium difficile and Its Toxins

G A A b st ra ct s fecal BA loss, which occurs in the absence of any absorptive defect within the terminal ileum, is due to increased BA synthesis, resulting from impaired FGF19 ileal production or hepatic actions. We aimed to investigate whether BA-induced expression of FGF19 in the ileum is reduced in patients with low SeHCAT retention.Methods: Patients being investigated for unexplained diarrhea (n=16) were prospectively recruited and gave informed consent at colonoscopy to give additional ileal biopsies. All had SeHCAT tests. Groups of 2-3 biopsies (explants) were incubated separately for 6h with either BA-free culture media (unstimulated controls), chenodeoxycholate (CDCA) or glyco-CDCA (GCDCA), both at 50uM. After RNA extraction and cDNA synthesis, FGF19 expression was quantified relative to GAPDH by RT-PCR. Results: SeHCAT 7d retention values were 15% in 9 who were normal diarrhea controls. Previous studies had shown no significant difference in baseline unstimulated FGF19 expression between these groups and this was confirmed with the unstimulated control samples (p=0.76). Median stimulation overall was 184-fold with CDCA and 373-fold with GCDCA. A positive correlation was found between SeHCAT retention and the magnitude of the fold change in FGF19 expression stimulated by either CDCA (r=0.50, n=16, p=0.02) or by GCDCA (r=0.76, n=7, p=0.02). These relationships were still present when the fold changes were normalized to the baseline FGF19 expression found in the unstimulated explants. Conclusion: Ileal explants from patients with lower SeHCAT 7d retention have a lower response to both CDCA and GCDCA. As uptake of GCDCA is dependent on carrier-mediated apical BA uptake but CDCA is not, this implies there is no defect in apical BA uptake, but abnormalities elsewhere in the FGF19 response. A reduced capacity to induce ileal FGF19 expression in response to absorbed BA is proposed to lead to the excess BA production and fecal BA losses found in primary BAD.

CD4+CD25+ Invariant Natural Killer T Cells Are Enriched in Cord Blood: Implication on Immune-Regulation

unstimulated control. The surface expression of CCR5 was measured separately by flow cytometry. Results: Twenty-four reduced-intensity alloSCT recipients received MVC 300 mg b.i.d. orally from day -3 to day +90 on an ongoing phase II study. Recipient bloodwas tested on days -6 (beforeMVC), 0 and 14, and donor bloodwas tested on day 0. We observed significant variability in surface CCR5 expression on T-cells in donors (range 0e9%) and recipients (range 0e49%). Recipient surface CCR5 expression increased from d-6 to d0 on both CD4 (mean 3.6 to 7.3%, p1⁄40.005) and CD8 (mean 13.5 to 30.6%, p1⁄40.005) T-cells, consistent with blockade of receptor internalization. CD8 T-cell pCCR5 levels increased in response to CCL4 stimulation on d -6 (p1⁄40.01) and decreased in response to MVC (p1⁄40.002), compared to unstimulated blood (Fig. 2). A decrease in pCCR5 levels in response to MVC was observed evenwithout CCL4 stimulation, reflecting loss of background CCR5 activity (p1⁄40.0002). In contrast, d0 pCCR5 levels failed to increase in response to CCL4 (p1⁄40.32), nor did they decrease in response to additional MVC (p1⁄40.13), mirroring effective in vivo blockade of CCR5. The magnitude of increase in CD8 T-cell pCCR5 levels in response to CCL4 stimulation on d-6 correlated with CCR5 surface expression (r1⁄40.55, p1⁄40.02). The same was true of CD4 T-cells (r1⁄40.76, p1⁄40.0004). On d0, however, the ability to stimulate CCR5 inversely correlated with CCR5 surface expression for both CD8 (r1⁄4-0.46, p1⁄40.04) and CD4 (r1⁄4-0.62, p1⁄40.004), suggesting that greater CCR5 surface upregulation may serve as an indicator for complete receptor blockade. Conclusions: Intracellular phosphorylation of CCR5 in response to stimulation is efficiently blocked by MVC in patients undergoing alloSCT, as measured by a novel phosphoflow assay. Upregulation of surface CCR5 may indicate effective CCR5 blockade and should be explored as a biomarker for drug response.

Measuring naturally acquired ex vivo IFN-γ responses to Plasmodium falciparum cell-traversal protein for ookinetes and sporozoites (CelTOS) in Ghanaian adults.

BackgroundA malaria vaccine that targets the sporozoite/liver stage parasites could potentially prevent blood stage infection and the associated clinical symptoms. Identification of sporozoite/liver stage antigens is, therefore, crucial for the development of effective vaccines. Cell-traversal protein for ookinetes and sporozoites (CelTOS) is a highly conserved antigen involved in sporozoite motility and hepatocyte invasion and has been shown to induce significant IFN-γ production in PBMCs from radiation-attenuated sporozoite-immunized malaria-naïve individuals. The aim of this study was to ascertain whether such CelTOS-specific recall responses are also induced in individuals with natural exposure to Plasmodium falciparum.MethodsEx vivo IFN-γ responses to 15mer overlapping peptide pools covering the entire sequence of CelTOS and five other candidate antigens, CSP, AMA1, MSP1, TRAP and LSA1, were characterized using PBMCs from 35 malaria exposed adults. Responses to four CelTOS peptide pools (CelTp1, CelTp2, CelTp3 and CelTp4), a pool containing peptides from the entire CelTOS antigen (CelTTp), and pools comprised of overlapping peptides from each of the other five malaria antigens were assessed by ex vivo ELISpot assay. A positive IFN-γ response for stimulants was defined by two criteria; a stimulation index of two or greater relative to the unstimulated control, and a difference of 10 or greater in spot forming cells between stimulant and the unstimulated control.ResultsOf the 35 volunteers tested, five had positive IFN-γ recall responses against the four different CelTOS pools while four volunteers made responses against the CelTTp pool; six volunteers were, therefore, positive with CelTOS. By contrast, six volunteers responded to AMA1, seven to LSA1, 15 to MSP1 and two volunteers responded against CSP and TRAP.ConclusionsThese results suggest natural malaria transmission induces CelTOS-specific ex vivo IFN-γ in Ghanaian adults and that the frequency of these responses was similar to those of other previously characterized malaria antigens. These findings support the further evaluation of CelTOS as a pre-erythrocytic candidate antigen for inclusion in a potential multi-antigen vaccine.Electronic supplementary materialThe online version of this article (doi:10.1186/s12936-014-0539-5) contains supplementary material, which is available to authorized users.

Whole platelets induces macrophage programming toward a repair profile characterized by high IL-10 production

Whole platelets induces macrophage programming toward a repair profile characterized by high IL-10 production

Alcohol intoxication following muscle contraction in mice decreases muscle protein synthesis but not mTOR signal transduction.

Alcohol (ethanol [EtOH]) intoxication antagonizes stimulation of muscle protein synthesis and mammalian target of rapamycin (mTOR) signaling. However, whether the anabolic response can be reversed when alcohol is consumed after the stimulus is unknown. A single bout of electrically stimulated muscle contractions (10 sets of 6 contractions) was induced in fasted male C57BL/6 mice 2hours prior to alcohol intoxication. EtOH was injected intraperitoneally (3g/kg), and the gastrocnemius/plantaris muscle complex was collected 2hours later from the stimulated and contralateral unstimulated control leg. Muscle contraction increased protein synthesis 28% in control mice, while EtOH abolished this stimulation-induced increase. Further, EtOH suppressed the rate of synthesis ~75% compared to control muscle irrespective of stimulation. This decrease was associated with impaired protein elongation as EtOH increased the phosphorylation of eEF2 Thr(56) . In contrast, stimulation-induced increases in mTOR protein complex-1 (mTORC1) (S6K1 Thr(421) /Ser(424) , S6K1 Thr(389) , rpS6 Ser(240/244) , and 4E-BP1 Thr(37/46) ) and mitogen-activated protein kinase (MAPK) (JNK Thr(183) /Tyr(185) , p38 Thr(180) /Tyr(182) , and rpS6S(235/236) ) signaling were not reversed by acute EtOH. These data suggest that EtOH-induced decreases in protein synthesis in fasted mice may be independent of mTORC1 and MAPK signaling following muscle contraction and instead due to the antagonistic actions of EtOH on mRNA translation elongation. Therefore, EtOH suppresses the contraction-induced increase in protein synthesis, and over time has the potential to prevent skeletal muscle hypertrophy induced by repeated muscle contraction.

Abstract 13389: Early Matrix Metalloproteinase-9 Inhibition Stimulates Neutrophil Infiltration and Delays Neutrophil Apoptosis Post-Myocardial Infarction in Mice

Matrix metalloproteinase-9 (MMP-9) increases in the first week post-MI, and targeted deletion of the MMP-9 gene attenuates cardiac remodeling post-myocardial infarction (MI), but whether MMP-9 inhibition early post-MI is effective therapeutic strategy has not been evaluated. Adult male C57BL/6J mice (3-6 months old, n=80) were subjected to coronary artery ligation. Saline or MMP-9 inhibitor (MMP-9i; 0.03 μg/day) was delivered at 3 h post-MI, and the mice were sacrificed at days 1 or 7 post-MI. Infarct area was similar in the saline (57±1%) and MMP-9i (55±1%) groups (p=0.22), indicating equal initial injury. The 7 day post-MI survival rates showed no significant difference between saline and MMP-9i groups (p=0.54). MMP-9 inhibition resulted in a 31±1% reduction in MMP-9 activity at day 1 post-MI (p&lt;0.05 vs. saline). Surprisingly, MMP-9 inhibition worsened LV function at day 7 post-MI, as shown by ejection fractions of 13±1% for saline vs. 8±1% for MMP-9i (p&lt;0.05). Of 84 inflammatory genes examined in the day 7 infarct, 15 pro-inflammatory genes were increased and one decreased in the MMP-9i group (all p&lt;0.05 vs. saline). Because MMP-9i showed increased inflammation at day 7, we measured neutrophil numbers. The saline group showed increased neutrophils at day 1, with numbers declining by day 7 due to induction of apoptosis. In MMP-9i, neutrophils were 2-fold higher at day 1 and persisted at day 7 for an 18-fold difference from saline, indicating prolonged neutrophil presence. Isolated blood neutrophils stimulated with active MMP-9 showed increased levels of pro-apoptotic markers Bcl and Bax compared to unstimulated control (both p&lt;0.05). Interestingly, neutrophils stimulated with active MMP-9 and treated with MMP-9i showed increased levels of anti-apoptotic markers Mcl and Xiap (both p&lt;0.05) compared to MMP-9 treated cells. This suggests that prolonged neutrophil presence with the MMP-9 inhibition was due to a dramatic reduction in neutrophil apoptosis. In conclusion, MMP-9 inhibition starting immediately post-MI decreased neutrophil apoptosis leading to increased inflammation and resulting in exacerbated LV dysfunction. Cat: 515 - Apoptosis and Necrosis