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The systemic inflammatory response to infection is the leading cause of mortality in North American intensive-care units. Although much is known about inflammatory mediators, the relationships between microregional inflammation, microvascular heterogeneity, hypoxia, hypoxia-inducible gene expression, and myocardial dysfunction are unknown. Male Sprague-Dawley rats were injected intraperitoneally with LPS to test the hypothesis that sepsis-induced local inflammation and increased microvascular heterogeneity are spatially and temporally associated with hypoxia, hypoxia-inducible gene expression, and decreased left-ventricular contractility. Using a combination of three-dimensional microvascular imaging, tissue Po2, and pressure-volume conductance measurements, we found that 5 h after LPS, minimum oxygen-diffusion distances increased (P < 0.05), whereas tissue oxygenation and contractility both decreased (P < 0.05) in the left ventricle. Real-time RT-PCR analysis revealed that the hypoxia-inducible genes hypoxia-inducible factor (HIF)-1α, VEGF, and glucose transporter (GLUT) 1 were all upregulated (P < 0.05) in the left ventricle. Tissue regions expressing ICAM-1, obtained by using laser-capture microdissection, had increased HIF-1α and GLUT1 (P < 0.05) gene expression. VEGF gene expression was more diffuse. In LPS rats, GLUT1 gene expression correlated (P < 0.05) with left-ventricular contractility. In 5-h hypoxic cardiomyocytes, we found strong transient HIF-1α, weak VEGF, and greater prolonged GLUT1 gene expression. By comparison, the HIF-1α-GLUT1 gene-induction pattern was reversed in the left ventricle of LPS rats. Together, these results show that LPS induces hypoxia in the left ventricle associated with increased microvascular heterogeneity and decreased contractility. HIF-1α and GLUT1 gene induction are related to a heterogeneous ICAM-1 expression and may be cardioprotective during the onset of septic injury.
- sepsis
- microcirculation
- inflammation
- heart
sepsis, the systemic inflammatory response to infection, is the leading cause of death in North American intensive-care units. As many patients die annually from sepsis-related cardiovascular collapse or multiple organ failure as do from acute myocardial infarction (1). Inadequate oxygen supply is probably the most important pathophysiological mechanism leading to myocardial dysfunction (25) and may play an important role in sepsis pathophysiology and decreased heart function, because progressive hypoxia reduces left ventricle contractility (42). Although direct evidence of tissue hypoxia within the septic heart has been lacking (4, 22, 23), evidence of diminished blood flow autoregulation (5) and loss of myocardial capillary density (6) suggest that a maldistribution of capillary blood flow (17) exists within the septic heart and that microvascular oxygen transport (8) has been compromised. As a consequence of sepsis-induced microvascular dysfunction, oxygen supply may be inadequate at the microregional level to support normal heart metabolism and function.
Although initial cytoprotective mechanisms to hypoxia involve protein modifications, a prolonged adaptive response to hypoxia requires change in gene expression (25). The master regulator of oxygen homeostasis is the nuclear transcription factor hypoxia-inducible factor-1 (HIF-1) (37, 43). HIF-1 is composed of a constitutively expressed β-subunit (HIF-1β) and an oxygen-dependent α-subunit (HIF-1α) (25, 36). The HIF-1 system regulates over 70 genes containing hypoxia-response elements in their promoter regions, including glucose transporter (GLUT) 1 and VEGF (46). HIF-1 activity is regulated by expression of the HIF-1α subunit, which is controlled by the balance between its synthesis, which is modulated by inflammatory mediators (10, 18), and its degradation, which is modulated by oxygen-dependent ubiquitination and proteasomal degradation under normoxic conditions (36, 39, 47).
During the onset of sepsis, the complex relationships between microregional inflammation, microvascular heterogeneity, hypoxia, hypoxia-inducible gene expression, and organ function are not well understood in general nor in the heart specifically. We hypothesize that sepsis induces local inflammation and increased microvascular heterogeneity, which is spatially and temporally associated with hypoxia and hypoxia-inducible gene expression, which in turn modulates heart function. To address this hypothesis, we studied the left ventricle of rats injected with LPS compared with controls. First, we imaged the myocardial microvasculature in three dimensions by using laser-scanning confocal microscopy. On the basis of these images, we determined whether LPS altered microvascular oxygen transport by increasing oxygen-diffusion distances in the left ventricle. Second, we directly measured changes in myocardial tissue oxygenation by using a fiber optic Po2 probe. Third, we determined whether this degree of myocardial hypoxia could stabilize the HIF-1α subunit and trigger hypoxia-inducible gene expression of HIF-1α, VEGF, and GLUT1 in isolated cardiomyocytes and the left ventricle. Fourth, by assessing microregional hypoxia-inducible gene expression in tissue expressing ICAM-1, we were able to characterize gene expression as either homogeneous or heterogeneous relative to a local marker of tissue inflammation. Finally, we examined the relationship between hypoxia-inducible gene expression and decreased heart contractility, a clinically important sepsis event.
Animal model of endotoxemia.
Nonfasting male Sprague-Dawley rats (250–300 g) were anesthetized with 3% isoflurane, injected intraperitoneally with LPS (10 mg/kg, Escherichia coli serotype 0111:B4; Sigma, St. Louis, MO), and allowed to recover. Five hours after LPS administration, animals were reanesthetized and placed on a heating pad to maintain internal core temperature between 36 and 37°C. This normotensive model was used to investigate the acute effects of the systemic inflammatory response to LPS on myocardial function, microvascular and ICAM-1 heterogeneity, tissue oxygenation, and hypoxia-inducible gene expression. White blood cell and platelet counts were assessed as markers of systemic inflammation, and ICAM-1 expression was used as a marker of local tissue inflammation. Global oxygen transport parameters, hematocrit, and hemoglobin were determined from venous blood (CELL-DYN 3700; Abbott Laboratories, Abbott Park, IL), and arterial oxygen saturation was measured by using pedal-pulse oximetry (Nonin Medical, Plymouth, MN). All investigations conformed with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH publication no. 85–23, revised 1996), and all experimental protocols were approved by the University of British Columbia Animal Care Committee.
Heart function and left ventricular contractility.
Five hours after LPS administration, a pressure-volume micromanometer conductance catheter (Millar Mikro-Tip 2.0; Millar Instruments, Houston, TX) was advanced into the left ventricle via the right carotid artery of the anesthetized rat. Measurements of left-ventricular pressure and volume were acquired at steady state and during compression of the inferior vena cava. Data were recorded by using the ARIA P-V conductance system (Millar Instruments). Maximum elastance (Emax), a measure of heart contractility relatively independent of cardiac preload and afterload (38), and measures of ventricular pressures and volumes, heart rate, and cardiac output were determined by using PVAN v. 2.9 data-analysis software (Millar Instruments). The P-V catheter was calibrated against known blood volumes according to manufacturer instructions. Following catheter measurement, the heart was excised and 30 mg of left ventricle tissue was harvested in RNA lysis buffer (Qiagen, Mississauga, ON, Canada) and stored at −80°C for target gene-expression analysis (HIF-1α, VEGF, GLUT1) and correlation with heart function (Emax).
Myocardial microvascular geometry.
Change in myocardial microvascular geometry, a determining factor of microvascular heterogeneity and tissue hypoxia (20, 21), was determined by measuring the minimum oxygen-diffusion distance between tissue and the nearest capillary segment. In a separate experiment, heparin was administered (4000 U/kg ip) to the anesthetized animal 5 h after LPS injection, and the heart was removed and mounted on a Langendorff perfusion system. Circulating blood cells and fibrinogen were washed out by infusion of Krebs solution for 3–5 min. Once the draining solution ran clear, the heart was fixed, and microvascular endothelial membranes were labeled under constant 75-cmH2O pressure by infusion of 2% paraformaldehyde containing 0.8 mg/40 ml of the fluorescent probe DiOC18 (Molecular Probes, Eugene, OR).
A sample of mid-left-ventricle tissue was excised by using a vibratome (Leica VT1000S) and was optically sectioned at 1-μm intervals (60 × 512 × 512 pixels) by using laser-scanning confocal microscopy (×40 dry/0.6 numerical aperture objective; Bio-Rad Radiance Plus). The resulting image stack was deconvolved by using a theoretical point-spread function (XCOSM v. 2.5; Washington University, St. Louis, MO; http://www.ibc.wustl.edu/xcosm). With the use of three-dimensional reconstructions of microvascular networks, minimum oxygen-diffusion distances were calculated on the basis of shortest tissue element voxel-to-capillary distance, as previously described (12). Because distributions were skewed, we calculated the median, 75th percentile, and fraction of myocardium with minimum oxygen-diffusion distances >7 μm, because myocardium farthest from the nearest capillary would be at a relatively higher risk of hypoxia.
Myocardial tissue oxygenation.
To determine whether LPS induced myocardial hypoxia by 5 h, a third group of animals was used. In this experiment, the chest of mechanically ventilated animals (Harvard small animal ventilator; Harvard Apparatus, Holliston, MA) was opened by left-sided thracotomy, and a 230-μm-diameter-tip fluorescent-tissue Po2 probe (OxyLite; Oxford Optronics, Oxford, UK) was inserted into the left-ventricular myocardium to a depth of 1–2 mm, which limited tissue damage. A 20-gauge needle was used to guide the probe into the heart muscle and was withdrawn before recording. During the procedure, heart rate and oxygen saturation were monitored. Six 20-s measurements were obtained as quickly as possible and were averaged over the time interval (13). The probe was precalibrated by the manufacturer and was only used if readings were ±10% of true Po2 at 0 and 21% oxygen.
In vitro kinetics of cardiomyocyte expression of hypoxia-inducible genes.
Having found that LPS reduced myocardial tissue Po2 to 5 mmHg, we simulated this effect in cardiomyocytes to elaborate the kinetics of HIF-1α protein stabilization and gene expression of the target genes HIF-1α, VEGF, and GLUT1. Cardiomyocytes (65,000/ml) were isolated from healthy animals, as previously described (15), and were placed in a hypoxic chamber (37°C, 5% CO2 with a Po2 = 5 mmHg, balance N2). Cells were washed, and the MEM medium was exchanged with medium equilibrated to chamber conditions. Because LPS increases the TNF-α level in plasma (6) and triggers the expression of TNF-α in cardiomyocytes (31), and because TNF-α is also associated with decreased cardiomyocyte contractility (15), we assessed whether TNF-α (50 ng/ml) could also upregulate hypoxia-inducible gene expression under normoxic conditions. Additionally, because we found that LPS induced myocardial hypoxia, cardiomyocytes were treated with TNF-α under hypoxic conditions (a situation that may exist in the septic myocardium) to assess whether a synergistic effect existed between hypoxia and TNF-α stimulation with respect to hypoxia-inducible gene expression. Cells were harvested at 0, 1, 2, 3.5, and 5 h in RLT lysis buffer (Qiagen) and were stored at −80°C for later quantitative real-time RT-PCR measurement of mRNA expression.
In vivo myocardial expression of hypoxia-inducible genes during controlled hypoxia.
A separate group of animals was used as a positive in vivo myocardial ischemia control. In this case, mechanically ventilated animals (Harvard small animal ventilator) were subjected to left anterior descending (LAD) coronary artery ligation. After 5 h, hearts were excised and 30 mg of left ventricle was harvested in RLT lysis buffer and stored at −80°C for later quantitative real-time RT-PCR measurement of mRNA expression.
Microregional relationship between ICAM-1 and hypoxia-inducible gene expression.
Because LPS stimulates expression of myocardial ICAM-1, a marker of local tissue inflammation, we sought to evaluate the spatial relationship between local inflammation and hypoxia-inducible gene expression. In an additional experiment, 5 h after LPS administration, hearts were removed, covered with optimal cutting temperature compound embedding medium (RA Lamb), and flash frozen in isopentane. The heart was then cut into alternating 8-μm-thick left ventricle sections for immunohistochemical identification of ICAM-1 regions and corresponding tissue sampling using laser capture microdissection.
To identify ICAM-1 regions for laser-capture microdissection, even-numbered sections were fixed in acetone (10 min), blocked with Universal Block (15 min; DAKO, Fort Collins, CO), and incubated with mouse anti-rat ICAM-1 antibody [2 h at room temperature at 1/400 in Tris-buffered saline (TBS-1%) BSA; PharMingen, San Diego, CA]. Washed sections (TBS, pH 7.6) were incubated with fluorescently labeled secondary rabbit anti-mouse antibody (30 min at 1/20 in TBS-1% BSA; DAKO) and were examined by fluorescent microscopy. To sample myocardial tissue from overexpressed ICAM-1 areas, corresponding odd-numbered tissue sections were treated with 75% ethanol (30 s), transferred to diethyl pyrocarbonate water (5 s), stained with 100 μl HistoGene staining kit (Arcturus Biosciences, Mountain View, CA), and placed in diethyl pyrocarbonate water (5 s). Water was removed by successive treatments in 75, 95, and 100% ethanol (30, 30, and 300 s, respectively), and the ethanol was removed by xylene (5 min). Regions of ICAM-1 myocardial tissue were sampled by using an Arcturus PixCell II laser-capture microdissection system (Arcturus Bioscience) operating at 50-mW laser power with a laser spot of 7.5 μm and 450-μs duration. Tissue samples were harvested in RLT lysis buffer and were stored at −80°C for later quantitative real-time RT-PCR measurement of mRNA expression.
Quantitative real-time RT-PCR.
Two micrograms of RNA, isolated by using a mini-column (Qiagen), was assayed by using QuantiTect SYBR Green one-step RT-PCR (Qiagen). Quantitative real-time RT-PCR was performed by using an ABI 7900 (Applied Biosystems, Foster City, CA) with the sense and antisense primers listed in Table 1. All target genes were evaluated simultaneously under the same reaction conditions: 50°C for 30 min, 9°C for 15 min, and then 40 cycles of 94°C for 15 s, 60°C for 30 s, and 72°C for 30 s. Samples were analyzed by quantification software (SDS 2.1; Applied Biosystems). Products were confirmed by electrophoresis on a 3% agarose gel stained with ethidium bromide. For each treatment group, HIF-1α, VEGF, and GLUT1 gene expressions were first normalized to 18s ribosomal mRNA. To assess differences between groups, the normalized gene-expression data were then expressed relative to the corresponding normalized control gene-expression data (i.e., gene induction is the ratio of treatment to control gene expression) and were compared on the basis of differences in gene induction.
HIF-1α protein expression.
HIF-1α protein from isolated cardiomyocytes was quantified by using Western blot. Briefly, cardiomyocytes were harvested in lysis buffer (in mM: 20 Tris·HCl, pH 7.5, 150 NaCl, 1 EDTA, 1 EGTA, 2.5 pyrophosphate, and 1 Na3VO4) containing a protease inhibitor cocktail (Roche Diagnostics, Mannheim, Germany). Twenty micrograms of protein was loaded on a 7.5% SDS-PAGE gel, electrophoresed (80 V for 30 min followed by 110 V for 90 min), transferred to nitrocellulose membrane (20 V for 16 h), blocked with 5% nonfat milk in TBS-Tween 20 for 1 h at room temperature, and incubated with anti HIF-1α primary antibody (Novus Biologicals, Littleton, CO) in TBS-Tween 20 (5% BSA) at 4°C overnight. The membrane was incubated with horseradish peroxidase-conjugated anti-mouse IgG for 1 h at room temperature and was imaged (ImageJ; Amersham Biosciences, Little Chalfont, UK) to quantify the signal.
Statistical analysis.
All values are reported as means (SD), unless otherwise stated. P values <0.05 were considered statistically significant. Student's t-tests were used to assess differences between LPS and control groups. One-way ANOVA was used to test for differences in minimum oxygen-diffusion distance distributions and mRNA expression of HIF-1α, VEGF, and GLUT1 genes under different conditions. The Holm-Sidak test was used to test all multiple pair-wise comparisons between groups. Repeated-measures ANOVA was used to assess differences in protein and gene expression over time in isolated cardiomyocyte experiments. Linear regression was used to assess the correlation between Emax and gene expression. All statistical tests were performed by using SigmaStat 3.0 (Systat Software, Richmond, CA).
Effect of LPS on heart function.
Five hours after administration of LPS, rat heart rate increased 21.4%(SD 2.7; P < 0.05) without significant changes in either left-ventricular preload (end-diastolic pressure or volume) or afterload (end-systolic pressure). The most striking effect of LPS on heart function was a 47% (SD 20; P < 0.05) decrease in left-ventricular contractility, as measured by Emax (Fig. 1, Table 2). There were no differences, however, in cardiac output, arterial oxygen saturation, hematocrit, or hemoglobin concentration between LPS and control rats. Both leukocyte and platelet counts decreased in LPS rats relative to control (P < 0.05), consistent with the onset of a systemic inflammatory response (Table 2). The survival rate was 75% in LPS-treated rats (n = 32) compared with 100% in controls (n = 20) and 100% in the LAD group (n = 4).
Myocardial microvascular geometry and tissue oxygenation.
Whereas global oxygen-delivery parameters remained unchanged, changes in microvascular geometry indicated that microvascular heterogeneity had increased in the left ventricle relative to control. On the basis of confocal imaging of the myocardial microcirculation, we found the minimum oxygen-diffusion distance increased from 3.4 (SD 0.5) in control to 4.3 μm (SD 0.6) in LPS (P < 0.05, as shown in Fig. 2C, inset); moreover, minimum oxygen-diffusion distances >7 μm doubled from 10.7 (SD 4.4) to 22.4% (SD 6.6; P < 0.05; Fig. 2). Having found changes in microvascular heterogeneity that would affect the distribution of red blood cell and oxygen flow within the heart, and consequently tissue oxygenation, it was important to determine whether the heart became hypoxic during the onset of endotoxemia. Using a Po2 probe inserted into the left ventricle, we found that myocardial tissue Po2 decreased 47% (SD 17) in LPS-treated animals compared with control [from 9.6 (SD 4.1) to 4.8 mmHg (SD 2.2); P < 0.05; Fig. 2E], indicating that the left ventricle was hypoxic.
Gene-expression kinetics under hypoxic and inflammatory stimulation.
Because LPS induced left-ventricular hypoxia at 5 h, we evaluated the effect of a similar degree of hypoxia (Po2 = 5 mmHg) on hypoxia-inducible gene expression in isolated rat cardiomyocytes by determining the kinetics of HIF-1α protein stabilization and HIF-1α, VEGF, and GLUT1 gene expression (Fig. 3). HIF-1α protein and HIF-1α mRNA expression both increased transiently (P < 0.05) within the first 1–2 h, returning to baseline by 5 h. In contrast, VEGF mRNA increased slowly by twofold (P < 0.05), whereas GLUT1 mRNA increased rapidly by sixfold (P < 0.05) over the 5-h period. Because LPS increases the expression of TNF-α in cardiomyocytes (31), and because plasma levels of TNF-α increase following LPS administration (6), cardiomyocytes were also exposed to TNF-α under normoxic conditions (Fig. 3, B and E). Under normoxic conditions, TNF-α caused a similar, but delayed, transient HIF-1α protein and HIF-1α mRNA response relative to the hypoxic response (Fig. 3, A and D). And although there was no difference in the kinetics of VEGF gene expression between hypoxic and normoxic TNF-α conditions, because a similar twofold increase was found, GLUT1 gene expression was attenuated under normoxic TNF-α conditions relative to hypoxia (threefold compared with sixfold; P < 0.05). When cardiomyocytes were subjected to combined hypoxia and TNF-α stimulation (Fig. 3, C and F), the kinetics of HIF-1α protein stabilization and gene expression of HIF-1α, VEGF, and GLUT1 all resembled that of the response to hypoxia alone. Under hypoxic conditions, HIF-1α protein was stabilized to a greater degree than the HIF-1α gene was induced (Fig. 3A). In terms of gene expression, the hypoxia-inducible response was heterogeneous, because HIF-1α gene expression was the strongest early indicator of hypoxia, whereas GLUT1 gene expression was the strongest indicator of prolonged hypoxia.
Comparison of hypoxic gene expression under hypoxic and inflammatory conditions.
Having associated increased microvascular heterogeneity with myocardial tissue hypoxia 5 h after LPS administration, and having assessed gene expression in isolated cardiomyocytes under hypoxic conditions, we asked whether hypoxia-induced gene expression was similarly spatially heterogeneous and related to areas of tissue inflammation in the rat heart 5 h after LPS administration. Using immunohistochemistry to identify regions of ICAM-1 protein expression, we found ICAM-1 to be heterogeneously overexpressed by fivefold (P < 0.05) in endotoxemic rat hearts (Fig. 4). In myocardial tissue regions with ICAM-1 overexpression, both HIF-1α and GLUT1 gene induction were higher (P < 0.05) than in non-ICAM-1 tissue background, whereas there was no difference in VEGF gene induction (Fig. 5). Comparing all treatment groups at the 5-h time point (Fig. 5), HIF-1α gene induction was greatest in ICAM-1 regions of endotoxemic hearts, VEGF gene induction was elevated to the same degree in all treatment groups, and GLUT1 gene induction was highly variable between groups. Compared with ICAM-1 regions of endotoxemic hearts, GLUT1 gene expression was increased relative to non-ICAM-1 background (P < 0.05), comparable with that in LAD control hearts but less than that observed in hypoxic cardiomyocytes (P < 0.05; Fig. 5).
Vector analysis of hypoxia gene expression.
We used normalized gene expressions of HIF-1α, VEGF, and GLUT1 (relative to control) expressed as vectors to define a hypoxia-inducible gene expression "signature." We then calculated the fractional similarity between our experimental conditions as the inner product of these vectors. (This is the cosine of the angle between the expression vectors. If the two vectors line up exactly, the cosine is 1, whereas if one vector does not overlap at all with the other, the cosine is 0.) We found that gene expression of hypoxic cardiomyocytes was 96% similar to gene expression in heart tissue following LAD occlusion [Po2 = 1.5 mmHg (SD 0.7)]. Thus this pattern defines a hypoxia-inducible gene-expression signature. The hypoxia-inducible gene signature found in the left ventricle of LPS hearts was 78% similar to the LAD condition, 42% similar to hypoxic cardiomyocytes, 83% similar to TNF-α-stimulated cardiomyocytes, and 75% similar to ICAM-1-expressing regions.
Correlation between heart contractility and gene expression.
Of the three hypoxia-inducible target genes examined in this study (HIF-1α, VEGF, and GLUT1), only GLUT1 was found to have a significant correlation with left-ventricular contractility, as measured by Emax, 5 h after LPS administration (Fig. 6). Although both VEGF and HIF-1α expression showed trends toward a correlation with Emax (P = 0.08 and P = 0.15, respectively), no significant relationships were detected.
In this study, the key findings were that LPS increased oxygen-diffusion distances and decreased tissue oxygenation in the left ventricle while simultaneously increasing hypoxia-inducible HIF-1α, VEGF, and GLUT1 gene expression and decreasing left ventricle contractility. Gene induction was not homogeneous, but rather both HIF-1α and GLUT1 were overexpressed in heterogeneous regions of ICAM-1 expression, whereas VEGF was not. Five hours after administration of LPS, we found that GLUT1 gene expression correlated with left-ventricular contractility, suggesting that increased glucose metabolism may play a role in heart function during endotoxemia. The results also suggest that increased hypoxia-inducible gene expression, induced by both hypoxic and nonhypoxic signaling pathways, may be cardioprotective.
Myocardial microvascular heterogeneity and tissue oxygenation.
Under normal hemodynamic conditions, cardiac output is such that global and local oxygen supply exceeds local oxygen demand. At the microregional level, the microcirculation regulates and distributes red blood cells and oxygen throughout the tissue to maintain tissue oxygen homeostasis (8). However, despite normal or enhanced cardiac output during sepsis, distribution and regulation of local tissue oxygen delivery are compromised by decreased functional capillary density (6, 7, 11, 17, 21, 26) and diminished conducted microvascular vasoconstriction (29). In our study, although LPS had no effect on global oxygen transport parameters, local myocardial microvascular oxygen transport was disturbed. We found evidence of increased oxygen-diffusion distances within the left ventricle, consistent with findings that LPS decreases functional capillary density in the heart (6).
Loss of capillary density is mediated in part by leukocyte adhesion (6, 19) and reduced erythrocyte deformability (7). These events occur early in the onset of sepsis and cause physical plugging of individual capillaries in both skeletal and heart muscle tissue (6, 7, 17). No data yet exists that fully characterizes the nature of the microvascular injury in the in vivo septic heart. In septic skeletal muscle, however, in vivo studies have revealed a complex microvascular derangement that increases microvascular heterogeneity by altering microvascular geometry, flow distribution, and oxygen diffusion. This is manifested by loss of functional capillary density and increased oxygen-diffusion distances (consistent with our findings in the endotoxemic rat heart) (7, 11, 17, 26), as well as increased fast flow or shunting of red blood cell flow through some capillaries and reversal of red blood cell flow in others (8, 17) and, perhaps most importantly, an inability of the microcirculation to regulate capillary red blood cell flow (17) to match local oxygen supply with local oxygen demand.
Consistent with experimental findings, theoretical models and biosimulations (20, 21, 41) predict that increased microvascular heterogeneity will decrease tissue oxygen extraction in the heart (14, 16, 22) and will increase tissue hypoxia in skeletal muscle (2, 3, 21). In our study, we found the left ventricle of control rats to have a baseline tissue Po2 of 9.6 mmHg (SD 1.0), consistent with myocardial Po2 measurements made in mice by using electron paramagnetic resonance (EPR) spectroscopy (49) and in piglets by using phosphorescence (33). Five hours after LPS administration, tissue oxygenation decreased in the left ventricle, consistent with increased NADH levels in endotoxemic rat hearts (50). We found no differences in cardiac output, arterial oxygen saturation, or hemoglobin concentration, indicating that global oxygen delivery was unaffected by LPS. Because blood flow remains constant or increases in endotoxemic rat hearts (5, 27, 40), the myocardial tissue hypoxia observed in our study was not likely due to hypoperfusion; rather, decreased capillary density, increased microvascular heterogeneity, and mismatch of microregional oxygen supply to increased oxygen demand (32) appear to be important factors.
Hypoxia-inducible gene expression in the endotoxemic heart.
Having established that LPS induced myocardial hypoxia in the rat left ventricle 5 h after administration, we investigated whether this degree of hypoxia was sufficient to upregulate the hypoxia-inducible genes HIF-1α, VEGF, and GLUT1. In isolated rat cardiomyocytes, under hypoxic conditions similar to those observed in the endotoxemic rat heart, we found that both HIF-1α protein and HIF-1α mRNA were rapidly and transiently increased. This was consistent with the HIF-1α response in hypoxic macrophages (10), human monocytes (18), human HeLa cells (24), and rat liver (35), but contrary to findings in Hep 3B or HeLaS3 cells subjected to 0.5% oxygen for 4 h (45). In our study, a similar but delayed HIF-1α response was observed when normoxic cardiomyocytes were treated with TNF-α. This indicates that HIF-1α is transcriptionally regulated in rat cardiomyocytes via both hypoxic and nonhypoxic inflammatory signaling pathways. This is significant because LPS induces both increased myocardial TNF-α expression (31) and increased plasma TNF-α levels (6), as well as myocardial tissue hypoxia. In contrast to the synergistic effect between LPS and hypoxia on HIF-1α induction in human monocytes (18), we found no synergistic effect between TNF-α and hypoxia on HIF-1α gene expression in isolated cardiomyocytes. Five hours after LPS treatment, HIF-1α gene expression was increased in myocardial regions with ICAM-1 staining, indicating that the tissue inflammatory response was temporally and spatially associated with hypoxia-inducible gene expression during the onset of sepsis.
The "classical response" to hypoxia involves upregulation of the GLUT1 glucose transporter and glycolytic enzymes through a HIF-1-dependent mechanism (9), as well as translocation of GLUT4 and increased glucose uptake. Increased GLUT1 gene expression is also part of a coordinated response to preserve neonatal cardiomyocyte viability under conditions of metabolic inhibition (47) and may reflect a state of myocardial hibernation (28). In our study, we found the GLUT1 gene to be strongly induced in hypoxic rat cardiomyocytes. And although GLUT1 gene expression was less responsive to TNF-α, this nevertheless indicated that GLUT1 was transcriptionally regulated in cardiomyocytes via both hypoxic and nonhypoxic inflammatory signaling pathways. Similar to HIF-1α, GLUT1 was overexpressed in ICAM-1 regions of the endotoxemic left ventricle, again indicating that hypoxia-inducible gene expression was related to tissue inflammation. GLUT1 expression also correlated with left-ventricular contractility, suggesting that increased GLUT1 transport and enhanced glucose metabolism were beneficial to heart function during the onset of endotoxemia. However, because Emax is a multifactorial process, additional factors including upregulation of HIF-1α (although this study was underpowered to detect a correlation) and glycolytic enzymes (35) may also be involved in modulating heart function.
In addition to increasing glycolytic capacity, hypoxia also stimulates the growth of new blood vessels to enhance local oxygen delivery by upregulating VEGF (48). In our study, we found VEGF gene expression in hypoxic cardiomyocytes to be slowly upregulated and weaker than either HIF-1α or GLUT1 and to be similarly induced by both hypoxia and TNF-α. Consistent with HIF-1α and GLUT1, VEGF was transcriptionally regulated in cardiomyocytes via both hypoxic and nonhypoxic inflammatory signaling pathways. Contrary to HIF-1α and GLUT1, VEGF was not overexpressed in ICAM-1 regions in the left ventricle of LPS-treated rats, although it was overexpressed overall in the septic heart, indicative of a more diffuse VEGF response throughout the left ventricle. Because both VEGF and GLUT1 genes contain hypoxia-response elements in their respective promoter regions, we expected to find similar degrees of gene induction. The differences in degree, spatial, and temporal gene induction suggest that additional transcription factors besides HIF-1 (44, 46) were governing VEGF and GLUT1 gene expression during the onset of endotoxemia. Of note is that both heart function and sublingual microvascular perfusion density improve in patients who recover from sepsis (34). Whether the microcirculation clears or VEGF production mediates remodeling of myocardial capillary density, which in turn improves heart function, is unknown.
On the basis of vector analysis of target gene expression at 5 h, we found that the HIF-1α, VEGF, and GLUT1 hypoxia-inducible gene signature in the endotoxemic (LPS-treated) left ventricle most closely resembled both the positive in vivo hypoxic LAD control and the isolated cardiomyocytes treated with TNF-α. This was a somewhat unexpected result, given that LPS induced hypoxia in the left ventricle. Nevertheless, it suggests that inflammatory mediators, including TNF-α, may be a factor in the initiation of hypoxia-inducible gene expression during the onset of sepsis, consistent with our findings in isolated cardiomyocytes. Increased HIF-1α and GLUT1 gene induction in ICAM-1 regions also suggests that local inflammation, possibly mediated by leukocyte plugging, is temporally and spatially associated with hypoxia-inducible gene expression and microregional hypoxia.
Study limitations.
It is possible that the ex vivo microvascular labeling procedure underestimated the degree of stopped flow in the heart. Although we found a significant increase in the minimum oxygen-diffusion distance in LPS hearts, consistent with in vivo loss of functional capillary density in skeletal muscle (17, 26), the in vivo minimum oxygen-diffusion distance may have been greater. Due to the relatively large Po2 probe diameter, tissue-oxygenation measurements include oxygen sources from local microvessels as well as tissue, and thus local tissue Po2 may be lower than reported.
The pattern of high HIF-1α and low GLUT1 gene induction in the left ventricle 5 h after LPS administration is the inverse situation of the low HIF-1α and high GLUT1 gene induction found in isolated hypoxic cardiomyocytes after 5 h. A possible explanation for this discrepancy is that hypoxia was delayed by several hours following the administration of LPS. In the context of a progressive septic microvascular injury, it is conceivable that early inflammatory mediators initiate hypoxia-inducible gene expression in the heart, whereas later tissue hypoxia, resulting from increased microvascular heterogeneity, either sustains or expands the response. The subsequent adaptive cardiomyocyte response to local inflammatory and hypoxic microregional conditions may facilitate preservation and eventual recovery of organ function, provided the host is not overwhelmed by the septic injury.
This study was supported by an operating grant from the Canadian Institutes of Health Research. R. M. Bateman is a Heart and Stroke Foundation, Michael Smith Foundation for Health Research, and Canadian Institutes of Health Research/Heart and Stroke Foundation of Canada IMPACT Strategic Training Postdoctoral Fellow. K. R. Walley is a Michael Smith Foundation for Health Research Distinguished Scholar.
We acknowledge the help of Amrit Samra with tissue sectioning, Shelley Dai with real time RT-PCR, Yingjin Wang with animal experiments, and Dr. Elaine Humphrey for discussions regarding confocal microscopy.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
- Copyright © 2007 by the American Physiological Society


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TORONTO - Apparently theres a third certainty in life, besides death and taxes.
And that is ... if you dare criticize Phil Kessel or Joffrey Lupul, on Twitter or in print, Leafs Nation goes bananas.
Which is fine. Fans are allowed to wear blinders and blinkers and whatever. But its a living, breathing reality that whether its Tyler Bozak or Tim Connolly at centre, Kessel and Lupul end up running around in their own end far too often, pretty well every game, sometimes with disastrous results, as was the case on Tuesday in a 3-2 loss to the Ottawa Senators in the Battle of Ontario.
Two Ottawa goals were scored with Kessel and Lupul on the ice. (Originally the game summary had them on for all three Ottawa goals, but that was changed afterwards). Yes, the dynamic duo broke out on Tuesday offensively, picking up points for the first time in four games, as Lupul scored on a great set-up by Kessel. But they have to work on their defensive game in their own end. If they dont, this Leafs team will miss the playoffs once again.
Kessel and Lupul are not there for defence, but they have to be much more responsible in their own end. Thats why, even as they sit near the top in NHL scoring, their plus/minus rating isnt great Kessel has 49 points in 45 games but is only
plus-1, while Lupul, who has 48 points, is a plus-3. Against the Sens, they both finished the game a minus-1.
Rookie defenceman Jake Gardiner made his return to action after being benched the past four games, and looked impressive, finishing the game a plus-2, with three blocked shots. Not that Gardiner was playing poorly before the DNDs started piling up. He was looking tired. When hes not fatigued, he brings an offensive touch the Leafs blue line which has been missing since dare I say this Tomas Kaberle in his prime ... James Reimer had a chance to show Ron Wilson that he is still in the mix as the clubs No.1 goaltender, but he dropped the ball against the Senators even though it appears as though he was interfered by Jason Spezza on Ottawas second goal (Wilson described it as a leg lock). Expect Wilson to start riding Jonas Gustavsson on a regular basis, until the Monster goes in a slump. The bottom line is, neither goaltender has been outstanding for an extended period of time this season, and thats what the Leafs need to make a run at a playoff spot. Reimer hasnt won in his past four starts ... The Battle of Ontario is often more hype than substance, but Tuesdays game was a hard-fought, exciting tilt, probably because there are playoff implications ... The Leafs penalty kill is now 12-for-12 over seven games. Remember when Wilson was getting vilified for that stat? ... The Leafs were out-hit 39-24 and Luke Schenn lead the Leafs with eight. Second on the Leafs was Bozak with four. Not enough hitting by Leafs defence ... Dion Phaneuf was unavailable to the media after the game because he was getting treatment, according to a Leafs media guy. But Wilson later said: He wasnt hurt, we wouldnt have played him if he was hurt. He was probably pissed off.
The Leafs have had trouble scoring, but decided to bench forward Nazem Kadri in place of tough guy Jay Rosehill. Not sure if they was a great move. Kadri doesnt have any points in his past five games, but you cant blame him. The young forward has made a number of great plays to set up his linemates, who failed to convert. Still think hed be a great fit on the first power-play unit with Kessel and Lupul ... Senators goaltender Craig Anderson allowed a goal first shot, but was outstanding in the final two periods, as the Leafs out-shot the Senators 39-21.
Blue Jays third baseman Brett Lawrie wears a Mats Sundins No.13 Leafs Jersey for his Twitter pic, while teammate J.P. Arencibias Twitter pic is a shot of him with the Leafs Tyler Bozak and Colby Armstrong, two partners of his social media group, TeamUnit ... Again, Leafs keyboard guy Jimmy Holmstrom was up to his old tricks before the Senators game, playing the song Signs by the Five Man Electrical Band a group from Ottawa.


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NICOLAS Ladislao Fedor Flores, aka Miku, has a few notable notches on his metaphorical goalpost.
The 27-year-old Venezuelan is a man who has scored a double against Real Madrid at the Bernabeu last season, and beaten an Argentina side containing Lionel Messi in a World Cup qualifying tie in Puerto La Cruz. But there was only one stadium in the world which could hold a candle to the atmosphere he experienced at Celtic Park during Wednesday night's Champions League opener against Benfica. The Maracana Stadium in Rio de Janeiro ("it is much bigger than Celtic Park, but the people are the same," Miku says). In two years' time, he hopes to be gracing that revamped grand arena of the world game as part of the 2014 World Cup finals.
But it wasn't just glamour matches which brought Miku to Scotland on loan. It is legitimate to question quite how so illustrious a player alighted in the SPL, but after his recent low-key existence at mid-table La Liga side Getafe, the South American enjoyed the reaction to the defeat at St Johnstone as much as the goalless draw against Benfica.
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"I have played for big teams," he adds, "Valencia was a big team who fought for championships. But when I came here I wanted to come to a club which must fight in all games for the win. At Getafe, you won, lost or drew and nothing happened. Here the first game we lost and it was terrible, the media, the trainer, the people, everyone was very angry. My agent said I could come here or go to another place, but Celtic has a big history and big players in the past and the present and they play always to win. I want that."
Miku speaks excellent English but some aspects of life in Scotland have been lost in translation – Emilio Izaguirre helps out the best he can – though he has already reached an understanding with Neil Lennon, the manager who joins a distinguished list of the player's former coaches which includes Michael Laudrup, Michel, Ronald Koeman, and Unai Emery – the very man who will be plotting his downfall with Spartak Moscow in the next Champions League matchday. "[Lennon] is a manager with a different mentality than most British managers," says Miku. "When I looked at games when I was young in Venezuela or in Spain you see all the teams here play with the long pass and fighting for the second phase and then after that they start to play. But when I arrived here and he told me the long pass is one way, but it is not the only way. We want to play with the ball on the grass and make it more enjoyable for the public. It is better for me."
In contrast to the high-octane environment of the Champions League or even the routine nature of the SPL, comes Tuesday night's Scottish Communities League Cup tie against Raith Rovers. Lennon, who regards his new frontman as a "class act on and off the field", would love to offer some game time to his other new boys Lassad Nouinoui and Efe Ambrose, not to mention emerging talents like Tony Watt, Dylan McGeouch, Paul Slane and even Rabiu Ibrahim. But this is the only domestic trophy he has yet to get his hands on as a manager and he won't be leaving anything to chance, having had the first division side watched last week. "I'll want to put a strong team out," explains Lennon. "I don't want any banana skins."


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Carly G
The total wild Justden is willing to amateurepornos about enormous breasts. Mackenzie has small pillows and likes total lewd sex movies with total graceless fucker The total graceless Carri has a tight coosie and wants to try something new Valiery is eager to amateurepornos with unblushinglover and a very niceslut Sex film about the obscene Rizzorio that is breaking taboos Gorgeia has giant boobs and would be happy to very brazenly amateure pornos with horny fellow. Hardcore-movies about the brazen Reanna that wants to try something new.
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Carly G.
Cameo Collection Volume 2
- Carly G.
Threesomes, foursomes, and moresomes! This non-stop fuck-fest of horny British girls includes DP's, anal, facials and hot girl-on-girl action. See two girls drain the cum from six guys as they satisfy their every desire. Cum in for the girls, and stick around for the hot fucking action! - Carly G.
Cameo Collection Volume 3
- Carly G.
Hard pounding sex the way you want it! Deep sucking oral, full face creaming, lusty DP, two-some, three-some, four-some and other nasties are shown in crystal clear detail! These horny babes are ready to please and the guys end up as pleased as you'll be after watching this fuck crazy movie! It's how the Europeans do their sex! - Carly G.
Cameo Collection Volume 4
- Carly G.
Two pool cleaners fuck a housewife, a secretary gets a pounding from two superiors, a model gets fucked from both ends by a couple of art students, five factory workers bang a timid, female coworker in a tool room and finally three customers fuck a sales assistant before she gets it up the ass! All this and more - and you'll see why the Europeans love their jobs! - Carly G.
Cameo Collection Volume 5
- Carly G.
A horny dreamer is group fucked by four masked men. A couple moving into a house enjoy humping and tonguing. A peeper watches a couple fuck, and then he screws the wife in the ass! At a photo shoot, two glamour models have strap-on lesbo sex. A stud spies on a couple fucking, and then he takes a turn. A housewife does it with three building inspectors. Cum take a peek at this latest collection! - Carly G.
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The Ballantine's Urban Freestyle has been one of the most surprising Freestyle invitational events of the pre-seasson. Some of the best and well known national and intenational riders, like Freddy Kalbermatten, Stefan Gimpl, Flo Mausser, Jonas Emery, o Iker Fernandez came to Barcelona searching for some good runs at the Big of Montjuich and the famous Barcelona party culture!!
The large amount of riders and it's high quality forced the organization to establish classifications runs on friday. In this way, more than 40 riders where invited to the Urban Freestyle, of which only few like Peyo Mongelos, Faust Punsola, Dani Fernandez, Mauricio Penarroya, Isaac Verges, Igor Dominguez, Hugo Morales y Andy Barrosso passed through the final during Saturday and were selected to messure themselves with the wildcards: Jonas Emery, Stefan Gimpl, Iker Fernandez, Freddy Kalbermatten, Heinz Lohle, David Lopez, Morgan Lefacheur, Tato Chiala y Pajaro Lasagabaster.
Most of the international "pros clan", came to Barcelona to enjoy the party ambience and discover the famous skating spots!! Riders like Jonas Emery, Freddy Kalbermatten, Heinz Lohle o Stefan Gimpl showed their best during day, and night, arriving exhausted to the finals!! But this was not an excuse for Freedy performing a perfect switch BS 720 or Jonas executing Rippeyflip 1260 with lost of air. Flo Mauser also gave his best with a perfect Cab 900, Gimpl executed two 900 fr with perfect landings and Iker showed his best style with his switch bk5!
Spanish outstanding riders where David Lopez giving a hard time with lots of air bs rodeo, also Daniel Sastre, with a two perfect landed cab 5 cork or Isaac Verges with fr 5, demonstrating their upgrading abilities.
1. Jonas Emery (Rossignol, Billabong-Sui)
2. Flo Mausser (F2, Billabong-Aut)
3. Stefan Gimpl (F2, O'Neill-Aut)
4. Iker FErnandez (Burton, Vlcom-Spa)
5. Freddy Kalbermatten (Burton-Sui)
6. Bjorn Hartweger (Salomon, Volcom-Aut)
7. David Lopez (Burton-Spa)
8. Tato Chiala (Burton-Ita)
9. Julien Harricot (Burton-Fra)
10. Daniel Sastre (Atomic, Protest-Spa)


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See the little goblin, see his little feet, see his little nosey-wosey, isn't the goblin sweet?
I like to play the auction house a bit. I'm nowhere near as good as some of the goblins out there, and I don't try to be, but I enjoy watching my gold supply go up. On my home server I have close to 40,000 gold server wide, epic flying for my four 80s that hang around there, my main character has over 100 vanity pets, both the Traveler's Tundra Mammoth and the motorbike. Basically, I make enough to have the frills and still have some leftovers, which is nice. So naturally, on this new server I'm trying to piece together a bit of gold. Of course, I'm nowhere near as good at it as Tobold (I think he was upto about 400g at level 17 last time we asked him, from his Northrend fishing venture), but so far I've made around 160 gold and I'm level 30, which isn't too bad.
My method is quite simple really. I don't use any auction house mods at the moment because, honestly, I deleted my WTF, Cache, and Interface folders a while back and couldn't be bothered to reconfigure. So my auctions take me a little longer than they should do for now but I guess I'll fix that eventually. I sell things I've picked up while playing, I buy raw epic gems, cut them, and resell them, and occasionally I use my Leatherworking to make an item and sell it, though recently that has been less successful. I undercut by 1 copper only, and my bid will be, generally, no lower than a gold less than the buyout price, in some situations I will set the bid price to the lowest I'm willing to sell the item for, though I only really do this with high selling items such as rare pets.
Sometimes, if I can be bothered, I'll finish off quests in Northrend on various alts, do a few dailies, perhaps grind a little, which makes me a bit of gold and gives me something to do but mostly I don't bother. It's not necessary these days. The buying and reselling market makes me enough gold to not have to worry about it.
Starting again on Argent Dawn has given me a fresh perspective. No high level professions to help with gold, no heirlooms, just a character screen, a couple of gold from the guild bank if you feel you need it, and a pinch of initiative. I love thinking of ways to make money without the easy options!
I've tried buying items from the vendor and reselling to the auction house, some work well, some don't. On my first day I sold a 12 slot herb bag for 50 gold, which paid for my 4x 16 slot bags, I haven't sold one since. Prairie Dog Whistles sell quite nicely. However, the other faction pets, mind you I haven't tried Dragonhawks yet, and various recipes don't. It's all trial and error. Certain things will eventually sell, for profession levelling or whatnot. Things like cloth and low level gems. I've sold a few Moss Agates and stacks of Linen Cloth, and the odd green I've picked up too.
I've been pickpocketting whatever I can for the spare copper, sometimes you find some interesting things, a few gems and a Pickled Murlocs Head for example, when I was in BFD. I've also been keeping my lockpicking levelled up, which has proved useful for locked chests, lockboxes, and again, the spare bit of copper.
It's looking to me like the Wool Cloth market is going to be the best money maker, I've considered levelling a death knight to 60, just so I have something to farm with, but we'll see. Perhaps doing the first few quests in Hellfire Peninsula would be a good idea.
A large part of my attempted gold making is having a character to send all of my unwanted items to, namely, my bank alt. My bank alt on Vashj was created with a random name in order to do my auctions in private, none of this, "I bought a gem from you today!" Which just ends with me feeling guilty. I keep its identity a secret! No guilt, and I can get stuff done without offending people. My bank alt on Terenas is a level 15 rogue I made in TBC and abandoned. I used her because she was close to a city and already had a few bags.
In my opinion, I think the best spots for bank alts are Orgrimmar, and Ironforge, because not only are the bank/auction house/mailbox fairly close together, it's mostly running in a straight line.
Another great thing about bank alts is dressing them, I've seen a lot of people running about in Tuxedos and Pimp Sticks. Though I prefer casual dress. Each to their own. All in all, take care of your bank alts, because they're people too.


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GAINESVILLE - Gainesville-based Internet marketing and web development company, Full Media, recently hired five new team members for it's Gainesville office.
Founded in 2008, Full Media also has offices in Greenville, South Carolina, and Chattanooga, Tennessee.
Santiago Gomez, Alan Vitek and Matthew Martin are graphic designers.
Lauren Ables and Nicole Penno have joined the company's Internet marketing team as Internet marketing analysts.


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After weekends away and travelling I decided to do something closer to home. It's Easter weekend and my friend Lucy is coming up from Leeds for a wee visit. I knew exactly what I wanted to do.
Doing a bungee jump has been on the list for a long time. I have been adventurous over the years because I have skydived, done a canyon swing, white water rafted, swam with wild dolphins, but something had always stopped me doing the head first, tied at your ankles jump. It had to be rectified and when better to do that than the year of my 30th.
The Highland fling bungee jump at Kilicrankie (near Pitlochry) fitted the bill. It gets rave reviews from trip advisor and was only a two hour drive away. I of course asked if Lucy would accompany me to hold my hand and perhaps distract me from the butterflies on the way up. She obliged along with my other friend Claire. So on we went on our road trip on what was a gorgeous Saturday morning, a perfect day for a jump.
We arrived at the Kilicrankie visitor centre and bungee sign in an hour early. Which was good actually, got checked in and sat in the sun while Lucy then decided she would jump with me, she just had a fear of missing out. Not that I was complaining, I welcomed her presence.
We get taken to the bridge, the site of the jump and get harnessed up, butterflies grumbling, we then climb up two ladders onto the bridge itself. It's all grilled so you can see directly below you, yup, nerves have well and truly surfaced now. Then we follow our guides to the centre where you can see the lower level platform where the actual jump takes place. Wringing my hands now. Nervous energy.
We watch two guys from our six strong group jump and then it was my turn to climb down the ladder. Deep breaths. Although I have to say my nerves are holding. I'm chatting away to Jason as he sorts the ankle straps, just enjoying the view. I guess as I was so determined to do this that was still the main feeling, determination and excitement of getting it done. Fulfilling this personal goal.
So the time comes and the main guy steps forward, he introduces himself, Matt, or was it Mike?! If I'm honest my brain isn't processing much other than trying to not think about what I'm about to do! You penguin shuffle forward, baby steps out onto ledge, he then tells you to walk to edge with your toes just over. Arms stretched out, trying not to look at the drop below, the gorgeous water and tree tops. He counts to three and that's your cue to jump.
So I did.
Well after a deep breath of course. I can't believe it! Straight off, no thinking just doing. I can't even begin to describe it. It was incredible. Sheer adrenaline rush. Then you're just swinging! Upside down and disorientated. I still can't quite believe it even as I write this. It's a bizarre feeling. It then feels like an eternity for the second rope to reach you, which you've then to clip on to your waist harness whilst your hands are shaking uncontrollably. I think for me that was the worst part, the hanging around with no control. Then slowly you get pulled back up. You're standing in front of the crew, they've unhooked you, bungee off, ankle straps off, up the ladders and you're watching the rest of the group. Surreal does not even cover it. It was amazing, almost outer body experience. Did that actually just happen...Did I fling myself off a bridge and now sitting back comfortably, well almost comfortably with the other crazies?
Watching Lucy go after me was a bit scary, although I was still buzzing from my jump, I knew how scared she was so I'm really proud of her. When she's back beside the jumpees she's shaking from head to toe, I guess she had a different experience from me and as she would tell you herself, she hated it, and would not do it again. However I wouldn't say no to another jump but I'm also not planning one. All I know is I felt great afterwards. I definitely walked a bit taller all weekend.
The fabulous weekend continued with a celebratory night out. Just want to say thanks to Claire for photography skills and joining the road trip and also to Lucy for coming up to visit, I had a blast!
As for Highland fling bungee, thank you for another tick off my list. I do have one gripe, which I'm not alone if you read reviews. You pay for the jump, then add on photos or videos etc, I went for photos. However you don't get a hard copy on day and you need to go through rigmarole of a download to get them stored on your computer. I thought for the money you're spending it should be more substantial. Anyway, I got my photos and I loved my bungee. The team were fantastic and kept the nerves for myself and Lucy. So overall a great day out.
Kilicrankie bungee jump, number 8 flinging fantastic!


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I'm having the same issue. When you do your next documentation additions, can you provide examples of how the switches and options should be used? Thanks! I've tried to put the auto accept of the host key in many places using a script file with no succes so I decided to go to my command line. Here's my command
open sftp:user:email@example.com /hostkey=ssh-rsa 1024 46:62:9d:86:45:d0:b9:b2:b4:0f:61:a2:af:40:8a:ea
When I execute this I receive a "too many parameters for command 'open'" error.


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This news release is available in Spanish.
Researchers at the Laboratorio de Accesibilidad Audiovisual (Laboratory for Audiovisual Accessibility) in Universidad Carlos III of Madrid's Science Park have created a system that analyzes the contents of subtitles on DTTV. According to the data that is automatically gathered, 30 percent of channels fail to comply with current legislation regarding subtitling and accessibility.
The objective of this innovation, called SAVAT, is to automate the control of subtitled content on Digital Terrestrial Television (DTTV). To do so, the system records the signals of all channels and analyzes the subtitles, so it can then check the number of hours subtitling was emitted, its speed, the coherence of the signal and the accuracy of spelling, among other parameters. Its creators, researchers at the Centro Español del Subtitulado y la Audiodescripción (CESyA - the Spanish Center for Subtitling and Audio Description, a center that comes under the Real Patronato sobre Discapacidad - the Royal Trust for the Handicapped - managed by UC3M), have been working at the UC3M Laboratory for Audiovisual Accessibility of the Technology Center for the Handicapped and Dependents (Laboratorio de Accesibilidad Audiovisual del Centro de Tecnologías para la Discapacidad y Dependencia) to develop their own algorithm for detecting and calculating with greater than 95% precision the time that subtitles are available on each working television channel.
One of the keys to the system is that it carries out monitoring autonomously. "Before, it was very complicated to verify the presence or absence of subtitling on television, because with 30 channels with national range, inspection of more than 5000 hours of programming per week had to be done using human intervention," the researchers explain. "That kind of work can only realistically be done by automating it, and before SAVAT there was no tool in the world that was capable of doing that," indicates Mercedes de Castro, CESyA's technical coordinator.
Failure to comply with legislation
All of the information that this tool provides is stored in a database that can then be used to generate daily reports, and can even activate alarms when the obligations established by the Ley General de la Comunicación Audiovisual (LGCA - General Law on Audiovisual Communication) are not being met. The minimum levels of accessibility set by this regulation establish that public channels must emit 90% of their programming with subtitles (with 75% being the level for private channels). Regarding audio description and sign language, public television stations must emit 10 hours weekly using one of these services (2 hours weekly on private stations). During 2013 these levels have been slightly lower, as the LGCA provides for a transition period that will end on December 31st. After that, the stations will be obliged to reach the established levels, and incompliance will have to be detected within time frames of ten days. "SAVAT's protocol regarding the detection of incompliance of the LGCA includes recording the sequences of DVB/TDT signals that are necessary for proof during any future legal suit against the offending channels," explain the researchers. According to the data that they have collected, approximately 30 percent of stations currently fail to comply with the obligations established by the LGCA with regard to subtitling; the figure is greater with regard to audio description and sign language.
SAVAT can be sold as a service or product. In Spain, since 2012 it has been analyzing the broadcasts of the eight multiplex television stations with nationwide coverage. The results that have been obtained via the SAVAT system have enabled CESyA to become the model body for monitoring audiovisual accessibility in Spain. Both the incompliance alarms and the storage of proof constitute valuable tools for regulating bodies (the Ministry for Industry and the Commission on the Telecommunications Market), the representatives of associations related to the handicapped (CERMI, CNSE, FIAPAS, etc.) and stations with local, autonomic and national broadcasts themselves. The next step, according to its creators, will be the international marketing of the system, since it can be put to use in any of the 86 countries that have adopted DVB digital television standards (all of the countries in Europe, part of Africa, South America, Asia and Oceania).
The Laboratory for Audiovisual Accessibility in UC3M's Science Park is developing technology to guarantee individual's access to the information society. Among its services are web access (structure and contents), access to cinema, DTTV and to the theater (prototype of subtitles, tools) and accessible doors.
Further information:
Laboratorio de Accesibilidad Audiovisual Parque Científico UC3M (Laboratory for Audiovisual Accessibility in UC3M's Science Park)
Centro Español del Subtitulado y la Audiodescripción (CESyA - Spanish Center for Subtitling and Audio Description):
Centro de Tecnologías para la Discapacidad y Dependencia UC3M (Technology Center for the Handicapped and Dependents:


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Comment #1 Wednesday, June 30, 2010 11:18 AM
Nice and......calm. Love your shots and skins. How's Crown Point doing? I was through there going to Cedar Lake last week. Other than Valpo it's my favorite NWI place. My Boss lives there.
Comment #2 Wednesday, June 30, 2010 11:46 AM
Thanks Chasbo!
Crown Point's been hot & rainy, but the weather's been ...calm... (ahem) the last few days.
Comment #3 Wednesday, June 30, 2010 2:32 PM
I know what you mean about the rainy. So tired of the tree branches on the ground and the sirens going off! Using today to wash windows etc. Big Holiday weekend coming up!
Comment #5 Thursday, July 1, 2010 8:48 AM
crown point? another hoosier? today.....wincustomize. tomorrow.....the world!!
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Police examine the dragster after the crash. Photographer: Owen Humphreys/PA
Top Gear is renowned for its daredevil antics, but is showing Richard Hammond's near-fatal crash a macho stunt too far?
Top Gear presenter Jeremy Clarkson has said that the BBC will broadcast footage of the 288mph dragster crash that left co-presenter Richard Hammond fighting for his life. "Half the world wants to see the crash, so I'm sure we will show it," said the outspoken autophile.
Hammond, who has yet to comment on the BBC's proposal, suffered serious head injuries as a result of the crash but made a full recovery. Shortly after the accident, he told the Daily Mirror newspaper that he was "gutted" that his injuries weren't more impressive. "I've been through hell and I've got nothing to show for it except a chipped tooth," he said.
Would it be right for the BBC to broadcast the crash in full? Or has the "Lad's Mag" culture of the show gone too far and is it in bad taste to show an accident that nearly cost a presenter his life and required emergency medical treatment? Good clean fun or ghoulish rubbernecking ... give us your views.


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Reaginic antibody
Revision as of 21:16, 3 October 2005 by WikiConvertor (Talk)
reaginic antibody --> homocytotropic antibody
(Science: immunology) A type of antibody which is able to attach itself to the surfaces of cells without needing specific combining sites. A homocytotropic antibody will only bind to cells which are from the same species as itself.
Please contribute to this project, if you have more information about this term feel free to edit this page.


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(NECN) - Medway, Mass. police are asking for your help finding a man they say exposed himself to a high school student.
The suspect is described as a white male in his 40's.
Authorities say a girl was walking home Monday when a man in a silver car drove by and asked for directions.
When the girl turned to answer, she saw the man was naked from the waist down and exposing himself.
She ran home, and the man drove away.
Published at 6:14 PM EST on Feb 28, 2014


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South Face of Pine Mountain...Rare glimpse of Nelson Big Horn Sheep
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View of the South face of Pine mountain from the saddle between Pine Mountain and Dawson Peak. Spotted a few of the elusive Nelson Big Horn Sheep on the face highlighted by the red boxes. The box on the left includes a ewe and lamb. It is believed the population is down to less than 100 in Angeles Forest from nearly 800 in the 80's.
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I have quite a few others if interested. I am looking for a 100% complete set with instructions and no sun damage or play damage. I do not care about the box. I also have some pretty good minifigure and other sets. Let me know if you have one and what you may be interested in. I also have Batman Ultimate Collector Set.
I also have batman UCS set mint in a mint box. It is perfect.


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There is this old fuck sitting here talking really loudly. Like I can hear every word he utters.
So far he has hit on "wetbacks" an how to solve the "immigration problem" and how we should give "them guns and let them kill each other."
Then he starts on President Obama being "half black" and then says, "Hell I ain't racist. I think everyone should own one."
Now he's talking about women getting jobs "on their backs."
I am trying so hard not to get arrested by telling the fucker off.
Why haven't you moved away from him or just left where you are?
You do realize you can't fix stupid, right?
I'd certainly put passing a huge test above dealing with nuts like this.
AJ's MOM
"Truth has no special time of its own. Its hour is now - always." - Albert Schweitzer
Me: BW - Him: 200+ # tumor removed 7/09
One AMAZING DS - 34
Then he starts on President Obama being "half black" and then says, "Hell I ain't racist. I think everyone should own one."
OMG...unfortunately, he sounds like some members of my family.
If someone keeps stabbing you in the back, then quit handing them the knife.
ok, I'm done
Back to your studying ok??
He does that for shock factor. He's looking for a fight. That is ignorance at it's best. Move your seat, if possible. What an asshole.
I've encountered the vile pigs of which you speak... it sometimes blows my mind how they feel the climate is such that they can spew ignorant bile publicly. I ponder the psychology of those who feel the need to denigrate whole groups of people...like do they feel so powerless and small that they're desperate to have others be "less than" themselves? I don't know, but I can empathize with how obnoxious it is to have to hear it.
These, in a nutshell.
As Neil DeGrasse Tyson (a favorite in our household) points out, children aren't born with racism, intolerance, bigotry, cultural insensitivity, racial arrogance/pride, religion, or superstition. To a baby, a black cat is just a cat, and a black person is just a person. It's what we, as parents, teach our children that form their impressions of the world.
For example, I noted that before we pulled our DS out of his daycare, he was being harassed by three or four other children, all of whom happened to be of African descent. One of his best friends in the class happened to be as white as he was.
Now, the reject SouthernGal was having to suffer through would draw an immediate correlation here -- where there isn't one, considering that my son was the racial minority at this daycare facility, which means it's incredibly likely that any child who would interact with him, for good or for bad, would be of African descent. His other best friend from this school happens to be of non-Caucasian descent, too, so there goes the other leg of Ol' Grampaw Racist's little working hypothesis.
I've thought of this in two different ways.
1. When the majority is the clear majority, they don't have to fight or voice their own rights, because they drown out all other dissenting noise. When the majority is no longer the clear majority, they now have to fight to be heard over the rest of the people trying to express their opinions (who have been trying to express opinions for a lot longer, natch), which means they start becoming the squeaky wheels themselves (I personally love the hypocrisy when a white male pundit takes to the airwaves to combat something that an NAACP pundit, for example, or some other non-representative of race, culture, or religion expresses either a personal opinion or an opinion on behalf of the organization they represent, and the white guy extends the other pundit's opinion to encompass the entirety of the sector of the population they themselves are afraid of, which is then interpreted as the opinion of all staunchy white males, ad nauseam). They feel a perceived threat, so they react with hate-speech, loudly, to counteract any reasonable argument.
2. The other part of it is exactly as sad12008 says. There's a reason that people who take to the pulpit to bash homosexuals tend to focus on male-male homosexuality in particular. They're afraid, either because they're closet cases themselves or because they personally find it icky, and they happen to have supporting doctrine to fluff their speech. For every redneck hillbilly white-trash racist homophobe and inner city gangbanger thug, there are at least a dozen more-well-informed people of all genders and colors in the spectrum who can remove themselves from their own personal habits and fears and make rational, reasoned decisions about what to do with themselves. And, furthermore, I happen to know that these people aren't the ones who hang around Piggly Wiggly, just waiting for a camera crew or reporter to come by so that they can inject their own agenda into the regional dynamic. Most of the people who aren't prejudiced are simply too busy actually living their lives to be quoted, televised, recorded, or otherwise put on the record as to how much shit they don't give about racism, sexism, or religious intolerance because the acts of working, parenting, and being a part of their community take up too much of their time. We hear from the ones who speak loudest, not the ones who speak with the most conviction.
[This message edited by KBeguile at 10:41 AM, April 24th (Wednesday)]
He does that for shock factor. He's looking for a fight.
I wouldn't be inclined to believe he gave it near as much thought as you have.
When people spew ridiculous garbage that has removed any doubt as to their intellectual capabilites , I always think of those penguins in Madagascar. "Smile and wave, boys. Smile and wave." It's all you can do.
t/j - Being from the South, I hate it when these "vile pigs" (and that's an insult to pigs) surface from the sewer and give us all a bad name. There's vile pigs in the North, but the media just luuuuurves it when there's a drawl involved. . .
This is when my Dixie Carter wants to surface and land my well-polished pumps in his scrotum (as such exists).
Honey Badger don't care. - Randall
So yeah, I'd have to speak my piece and then move away from him.
Sometimes it's appreciated, sometimes it's not. But I know I've done the right thing.
I actually did say something.
I thought about letting it go and just leaving. But honestly, by the time he got around to talking about a former employee of the restaurant and what a "bitch" she was because she was a lesbian, I just couldn't take it any more.
I was seated on the other side of the restaurant from him and I could hear his every word because he was that LOUD.
So on my way out, after I threw away my half-eaten breakfast, I stopped by his table. He was talking to the on-duty manager, by the way.
I said, "I couldn't help but hear every word you've uttered since I walked in because you were talking so loudly. In less than 20 minutes you managed to denigrate blacks, women, and gays. You completely ruined my breakfast, and my entire morning. You are a vile, disgusting, and vulgar excuse for a human being."
As I was walking out his response was to say, "What a dyke bitch! Did you see that hair?"
Yes, that's right you jackhole. Because I have very short hair I must be a "dyke." I thought about telling him that I am not, in fact, a dyke. I thought about explaining that I am bisexual (not homosexual) and that if I were to place a label on myself I would be a "blue jean femme" - that is something between a dyke (butch) and lipstick (feminine) lesbian. But I figured such subtleties would be completely lost on his tiny brain.
I swear he owes an apology to all of the trees who tirelessly make the oxygen he uses to be so disgusting.
[This message edited by SouthernGal at 2:22 PM, April 24th (Wednesday)]
I'm sure there were others around who wanted to say something, but were to shy, or scared.
It may not change this idiot one bit.
Standing ovation! Vile pig.
eta: I have very short hair, and therefore must also be a dyke bitch!! And we won't even go into the different nationalities in my background (that are probably ALSO in his!).
[This message edited by PippaPeach6 at 2:25 PM, April 24th (Wednesday)]
It won't change him. He was probably 70+, and probably fancies himself a real "good ol' boy" who thinks that all Southern men should be just like him.
I just keep hoping that he and his ilk are a dying breed and that someday the labels we stick on one another will all say the same thing - "Human."
Thank-you for your chutzpah!
And I hope you did well on the exam!
Nice try however.
Divorced 2009, Closing on house Nov 2011 -
No longer waiting for the other "she" to drop.


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