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Traumatic injury to the central nervous system results in the disruption of the blood brain/spinal barrier, followed by the invasion of cells and other components of the immune system that can aggravate injury and affect subsequent repair and regeneration. Although studies of chronic neuroinflammation in the injured spinal cord of animals are clinically relevant to most patients living with traumatic injury to the brain or spinal cord, very little is known about chronic neuroinflammation, though several studies have tested the role of neuroinflammation in the acute period after injury. The present study characterizes a novel cell preparation method that assesses, quickly and effectively, the changes in the principal immune cell types by flow cytometry in the injured spinal cord, daily for the first 10 days and periodically up to 180 days after spinal cord injury. These data quantitatively demonstrate a novel time-dependent multiphasic response of cellular inflammation in the spinal cord after spinal cord injury and are verified by quantitative stereology of immunolabelled spinal cord sections at selected time points. The early phase of cellular inflammation is comprised principally of neutrophils (peaking 1 day post-injury), macrophages/microglia (peaking 7 days post-injury) and T cells (peaking 9 days post-injury). The late phase of cellular inflammation was detected after 14 days post-injury, peaked after 60 days post-injury and remained detectable throughout 180 days post-injury for all three cell types. Furthermore, the late phase of cellular inflammation (14–180 days post-injury) did not coincide with either further improvements, or new decrements, in open-field locomotor function after spinal cord injury. However, blockade of chemoattractant C5a-mediated inflammation after 14 days post-injury reduced locomotor recovery and myelination in the injured spinal cord, suggesting that the late inflammatory response serves a reparative function. Together, these data provide new insight into cellular inflammation of spinal cord injury and identify a surprising and extended multiphasic response of cellular inflammation. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury could be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.
Traumatic injury to the central nervous system results in disruption of the blood brain/spinal barrier, facilitating the entrance of immune cells and proteins. The cellular immune response has garnered increasing attention because of accumulating evidence suggesting a contributing role for immune cells in spinal cord injury pathology and repair. The infiltration of neutrophils (polymorphonuclear leucocytes, PMNs) or macrophages/microglia exacerbates poor recovery from traumatic brain and spinal cord injury (Giulian and Robertson, 1990; Popovich et al., 1999; Weaver et al., 2000; Hains et al., 2001; Noble et al., 2002; Saville et al., 2004; Stirling et al., 2004). Likewise, central nervous system-reactive T cells have been suggested to exacerbate axonal injury, demyelination and functional loss (Popovich et al., 1996b; Howe et al., 2007). Conversely, macrophages/microglia and T cells can also secrete factors that promote neuroprotection and/or neuroregeneration after spinal cord injury (Guth et al., 1994; Rapalino et al., 1998; Moalem et al., 1999; Hauben et al., 2001; Li et al., 2001; Crutcher et al., 2006; Yin et al., 2006). These contrasting immune functions may support a bimodal role for immune cells in the injured spinal cord.
Although a few studies have shed light onto the possible roles of immune cells in the injured central nervous system, the underlying mechanisms are complex and remain unclear. Growth factors and protease inhibitors released by macrophages or T cells are thought to promote neuro regeneration (Cantini and Carraro, 1995; La Fleur et al., 1996; Jameson et al., 2002; Yin et al., 2006). Conversely, conditioned-media collected from PMNs or macrophages has been shown to promote neurotoxicity in vitro (Flavin et al., 1997; Brana et al., 1999; Nguyen et al., 2007). Although specific macrophage-derived neurotoxic factors have not been identified, we have shown that matrix metalloproteinases, reactive oxygen species and tumour necrosis factor-α released by PMNs all promote neurotoxicity in vitro (Nguyen et al., 2007). Additionally, physical PMN-to-neuron contact has been shown to enhance neurotoxicity (Dinkel et al., 2004). As similar mechanisms are likely to be involved in immune cell function after spinal cord injury, understanding the kinetics of cellular inflammation after spinal cord injury is essential for the development of effective therapeutic interventions.
Previous studies on kinetics of the cellular immune response to spinal cord injury have generally focused on a subset of time points, ranging from 1 to 42 days post-injury (dpi), which represent a relatively narrow range of assessment within the lifespan of animals with spinal cord injury. These studies have shown that PMNs infiltrate the spinal cord within hours after injury and remain detectable at the injury epicentre at 42 dpi (Taoka et al., 1997; Royo et al., 1999; de Castro et al., 2004). In contrast, macrophages/microglia and T cells infiltrate the spinal cord from 3 to 14 dpi (Saville et al., 2004; Jones et al., 2005; Wu et al., 2005; Conta and Stelzner, 2008), remaining detectable for at least 42 dpi (Guizar-Sahagun et al., 1994; Kigerl et al., 2006). In parallel with data from rodent studies, Fleming et al. (2006) described the presence of PMNs, macrophages/microglia and T cells in the post-mortem human spinal cord up to 12 months after injury (Fleming et al., 2006), although the study design prohibited quantitative comparisons. Critically, longer time points and finer quantitative assessments of neuroinflammation are needed to understand the kinetics of neuroinflammation in the injured spinal cord fully, as it relates to functions of specific cell types.
Most previous studies have characterized cellular inflammation in the injured central nervous system using histological techniques that cannot always provide true quantitative analysis. Flow cytometry is an alternative method to quantify immune cells in the injured spinal cord tissue quickly and effectively. Historically, flow cytometry has been used to quantify immune cells collected from blood or dissociated from the spleen or thymus, and only a few studies have attempted to quantify immune cells in the injured spinal cord by flow cytometry using fresh dissociated cord tissue or cells separated by the Percoll gradient system (Gonzalez et al., 2003; Tjoa et al., 2003; Stirling and Yong, 2008). In this study, we describe a novel and alternative cell preparation methodology using the OptiPrep gradient system (Fisher Scientific) to separate cells effectively from lipid/myelin debris and assess cellular inflammation by flow cytometry. Additionally, we demonstrate the reliability and sensitivity of the OptiPrep gradient-purified cell preparation by (i) testing the sensitivity of this gradient purified cell preparation in comparison with preparation of samples by enzymatic dissociation alone; (ii) assessing changes in PMN and macrophage/microglial infiltration in response to injury severity and (iii) comparing flow cytometric data of cellular infiltration with quantitative stereology of immunolabelled spinal cord sections at selected time points. Critically, we also report the first characterization of acute and chronic cellular inflammation after spinal cord injury to include a complete time course for PMNs, macrophages/microglia and T cells over a period ranging from 2 h to 180 dpi, identifying a novel second phase of cellular inflammation. The second phase did not coincide with changes in motor recovery after spinal cord injury. However, reduction of delayed C5a-mediated inflammation reduced locomotor recovery and myelination in the injured spinal cord, suggesting that chronic inflammation is reparative. Findings from this study demonstrate, for the first time, a temporally distinct second phase of cellular inflammation in the injured central nervous system and reveal an important multiphasic component of neuroinflammation that may be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions for spinal cord injury.
Additional experimental details and selected methods are given in the online supplementary material.
Female Sprague Dawley rats received T9 spinal contusions with the Infinite Horizons Impactor (Precision Instuments, Lexington, KY) (Nguyen et al., 2008). For graded contusion injury experiments, contusion injuries of 150, 200 or 250 kilodyne (kd) were administered (n = 5/group). A 200 kd force was used for all other experiments including the flow cytometric timecourse (n = 3–5/time point, Table 1), stereologic quantification timecourse (1 dpi: n = 6; 7 dpi: n = 7; 28 dpi: n = 6; 90 dpi: n = 7), 1 and 7 day C5a receptor antagonist (Ra) efficacy (n = 5/group/time point), C5aRa depletion (n = 11/group) and 91 day openfield locomotor assessment (n = 12). Animals used for histology were perfused with phosphate-buffered saline-buffered 4% paraformaldehyde, and tissue from spinal cord segments containing the injury epicentre were dissected from spinal roots (T6–T12) for subsequent immunolabelling. Animals used for flow cytometric analyses (three to five animals per group or time point) were sacrificed by CO2 asphyxiation, tissue from spinal segments T8-T10 was rapidly dissected and placed in Hank's buffered saline solution (HBSS) on ice. All work was conducted with the approval of the Institutional Animal Care and Use Committee at the University of California, Irvine.
Functional recovery was assessed using the Basso, Beattie and Bresnahan locomotor rating (BBB) scale (Basso et al., 1995). Two experienced raters, blinded to experimental treatment, observed open-field locomotion in 4 min testing periods at 2 and 7 dpi, and then weekly until sacrifice.
For flow cytometry, spinal cord segments T8–T10 were mechanically dissociated with fine scissors in HBSS. Cells were retrieved by centrifugation and enzymatically dissociated with 2.5 mg trypsin and 5 mg collagenase in 5 ml Dulbecco's modified Eagle's medium for 20 min at 37°C before trituration with a Pasteur pipette. Ten ml Dulbecco's modified Eagle's medium plus 10% foetal bovine serum was added before filtering with a 40 µm cell strainer. The pellet was resuspended in 6 ml of HBSS and overlayed on an OptiPrep gradient (Krickhahn et al., 2001; Bagamery et al., 2005). The OptiPrep gradient was constructed first by diluting OptiPrep (Fisher Scientific) 1:1 with 3-(N-morpholino)propanesulfonic acid (MOPS) buffer (0.15 M NaCl, 10 mM MOPS, pH 7.4). Four dilutions were made with 350, 250, 200 or 150 µl OptiPrep/MOPS and added to HBSS for a final volume of 1 ml. The four solutions were carefully placed in layers in a 15 ml conical tube with the least dilute at the bottom and the most dilute on the top (Fig. 1A). Dissociated spinal cells in HBSS (6 ml) were layered on top of the OptiPrep gradient before centrifugation (15 min, 1900 rpm, 20°C), separating the cell solution into distinct layers with debris on top, followed by three layers of neurons, and inflammatory cells, glia and red blood cells in the pellet. The debris layer was carefully aspirated. The remaining cells were washed and resuspended in HBSS for immunolabelling.
For immunohistochemistry, fixed T6–T12 spinal cord segments were cryoprotected in 20% sucrose in phosphate-buffered saline-buffered 4% paraformaldehyde overnight at 4°C, frozen in isopentane at −56°C and then stored at −80°C. Coronal sections (30 µm) of spinal cord tissue were cut on a sliding microtone. Serial sections were collected in phosphate-buffered saline with sodium azide (0.02%) and stored at 4°C until use.
Cells collected from spinal cord preparations were pelleted and resuspended in 0.85% ammonium chloride for 5 min to lyse red blood cells, blocked for 30 min in normal rabbit or goat serum, and incubated for 1 h with primary antibodies or isotype immunoglobulin (Ig)Gs diluted in HBSS for 1 h [rabbit anti-PMN fluorescein isothiocyanate (FITC), Accurate Chemical and Scientific; mouse anti-rat granulocytes (RP1) RPE, BD Pharmingen; mouse anti-rat ED1, Serotec; mouse anti-rat CD11b, Serotec; mouse anti-rat CD3, Serotec; rabbit IgG1 FITC, MP Biomedicals; mouse IgG1 RPE, Serotec; mouse IgG2a, BD Pharmingen]. Non-conjugated antibodies required a 1 h incubation with conjugated secondary antibody (Alexa 488 or 555 goat anti-mouse IgG1 or IgG2a, Invitrogen) solution diluted in HBSS. Cells were washed twice after each step and then resuspended in HBSS after the final step. Samples were analysed on a fluorescence-activated cell sorting Calibur (Becton-Dickinson) flow cytometer using Cell Quest software. Five thousand events were read for all samples. Data analysis was completed with Summit (DakoCytomation). Flow cytometric gates were set using control IgG isotype labelled cells or spinal cord cells from uninjured control animals to set baseline values for normalization across time points. Control IgG isotype-labelled cells from the injured spinal cords show very little difference in flow cytometric reading from the spinal cord cells of uninjured control animals (Nguyen et al., 2008). All antibodies used for flow cytometry were diluted 1:100 prior to use. The mean values of positively labelled cells were expressed as per cent (SEM) of the entire sample.
In some conditions, cells were adhered to slides by centrifugation (Cytospin, Shandon, USA) and immunolabelled. Briefly, cells (106/ml) were fixed with acetone for 10 min, quenched in 0.3% hydrogen peroxide for 15 min, blocked (3% bovine serum albumin, 0.05% Tween-20), incubated in primary antibody (mouse anti-CD43, 1:500, Serotec; or rabbit anti-β-tubulin III, 1:1000, Covance), followed by secondary antibody (goat anti-rabbit IgG conjugated with Alexa Fluor 488, 1:1000, Invitrogen; or biotinylated donkey anti-mouse IgG, 1:500, Jackson Immunoresearch) followed by ABC and 3,3'-diaminobenzidine exposure as described below. Antibody control slides were processed identically, except that incubation with primary antibody was omitted.
Immunohistochemistry of spinal cord sections was performed as previously described (Anderson et al., 1996). Primary antibodies were targeted against CD43 (1:1000, Serotec), ED1 (1:1200, Serotec), CD3 (1:1200, Serotec) and myelin basic protein (1:250, Chemicon). Species-specific affinity-purified biotinylated secondary antibodies were applied (1:500, Jackson Immunoresearch), followed by incubation in ABC reagent (Vector Laboratories) and development with diaminobenzidine (Vector Laboratories) before methyl green nuclear counterstaining.
Sprague Dawley rats were given 200 kd T9 contusion injuries and sacrificed 1 (n = 6), 7 (n = 7), 28 (n = 6) and 90 (n = 7) dpi. The injured spinal cord (T6–T12) was dissected and 30 μm serial spinal cord sections (1:24 sampling) were used for immunohistochemistry as described above for PMNs, macrophages/microglia or T cells. An Olympus BX51 microscope with motorized stage and StereoInvestigator (Version 6.3 Software, Microbrightfield, Williston, VT, USA) were used to estimate stereologically the number of PMNs and T cells with the optical fractionator probe (Hooshmand et al., 2009). Macrophage/microglia volume was determined with the Cavalieri estimator probe.
Animals received 200 kd T9 contusions, as described above, and were given either a C5a receptor (CD88) antagonist {C5aRa, PMX205, (HC)-[OP(D-Cha)WR], 10 mg/kg p.o., Promics Ltd} (n = 5) or water (n = 5) as a vehicle control twice daily for 1 or 7 days until sacrifice, for flow cytometric determination of PMN or macrophage/microglia infiltration to the injured spinal cord, respectively (Woodruff et al., 2006).
A separate cohort of Sprague Dawley rats received 200 kd T9 contusions as described above. The effect of sub-acute to chronic inflammation was tested by administering the C5aRa (n = 11) or vehicle control (n = 11) twice daily, as above, for 14–28 dpi until sacrifice. Rats were assessed for locomotor recovery with the BBB scale. Following sacrifice, injured spinal cords were harvested for immunohistochemistry.
Comparisons of PMN or macrophage/microglial accumulation in the injured spinal cord in response to graded injuries were performed using one-way ANOVAs with post hoc Tukey's multiple comparison tests with Prism 4.0 (Graphpad, Inc.). Correlations were calculated using the actual force applied by the Infinite Horizons Impactor with the linear regression function of Prism 4.0 (Graphpad, Inc.). A post hoc power analysis (ANOVA, single factor design) was used to determine statistical power in the graded injury experiments (n = 3–5) to validate flow cytometric sample sizes (Supplementary Table S1). For the timecourse experiments, changes over time were assessed with one-way ANOVA following by post hoc Bonferroni's multiple comparison tests (Table 2). Statistical significance was defined as P < 0.05. All data are presented as mean ± standard error mean (SEM).
The use of flow cytometry to quantify immune cells in the central nervous system is complicated by lipid/myelin content and debris. In addition to interfering with antibody staining and specificity, myelin debris can have similar size and granulation properties to immune cells, decreasing measurement sensitivity and accuracy (Lipton et al., 2005).
We have developed a novel cell preparation procedure that effectively removes myelin debris and improves the sensitivity of flow cytometry to detect changes in cellular inflammation in the injured spinal cord. The ability of OptiPrep gradient isolation to remove myelin debris and improve PMN detection by flow cytometry was tested 1 dpi. Myelin debris was removed from each sample by centrifugation through an Optiprep density gradient (Fig. 1A). Following centrifugation, sample contents separated into distinct layers, in which myelin and most debris were separated from neurons, glia, red blood cells and immune cells. Alternatively, identically dissected spinal cords were enzymatically dissociated without removal of myelin debris. There was a shift in the position of events for samples isolated with OptiPrep gradient purification compared to enzymatic dissociation alone (Fig. 1B). However, immunolabelled slide preparations of samples from both methods contained equivalent numbers of PMNs and neurons (Fig. 1E), suggesting the observed shift in the position of events following OptiPrep purification was due to a reduction in myelin debris. Together, these observations prompted the prediction that debris removal by OptiPrep purification may lead to improved flow cytometric detection of PMNs.
Accordingly, PMNs were elevated in the injured spinal cord 1 dpi for both cell preparations; however, the percentage of PMNs detected in samples with intact myelin debris was only a fraction (0.5%) of the percentage of PMNs detected (5.1%) in OptiPrep purified samples (Fig. 1C and D). These data suggest that myelin debris in tissue preparations can obscure flow cytometric readings and demonstrate that removal of myelin debris enhances the sensitivity of immune cell detection in the injured spinal cord.
Flow cytometric analysis does not permit morphological criteria to distinguish cell types further; therefore, characterization of antibody specificity is important. Anti-CD3 is a pan-T cell marker for both flow cytometry and immunocytochemistry (Tanaka et al., 1989; Kappelmayer et al., 2000). Anti-PMN and anti-ED1 have been used extensively for immunocytochemistry, but have not been widely used for flow cytometry. Accordingly, the specificity of these antibodies was tested by comparing labelling of peritoneal PMNs, alveolar macrophages and cells in the injured spinal cord with antibodies used in this and previous studies. PMNs isolated from peritoneal cavities were positive for both anti-PMN FITC and an alternative PMN antibody, anti-granulocyte (RP1) RPE (Supplementary Fig. S1A and B), but were negative for anti-ED1 Alexa-555 (Supplementary Fig. S1C and D). Similarly, alveolar macrophages (ATCC) were positive for both anti-ED1 Alexa-488 and an alternative macrophage antibody, anti-CD11b Alexa-555 (Supplementary Fig. S1E and F), but were not detected by anti-PMN FITC (Supplementary Fig. S1G and H). These data demonstrate the specificity of the antibodies for PMNs and macrophages in vitro. Next we tested antibody specificity in the injured spinal cord, predicting minimal cross-reactivity between anti-PMN or anti-ED1 antibodies at 1 and 7 dpi. As predicted, 6.12% of cells in the injured spinal cord were positive for anti-PMN FITC but not for anti-ED1 Alexa-555 (0.20%) 1 dpi (Supplementary Fig. S2A and B). In contrast, 6.46% of cells in the injured spinal cord were positive for anti-ED1 Alexa-555 at 7 dpi, and only a small percentage of cells were positive for anti-PMN FITC (Supplementary Fig. S2C and D). Importantly, very few cells were double-positive for anti-PMN FITC and anti-ED1 Alexa 555 at 1 dpi (0.98%) and 7 dpi (0.92%) as compared to respective IgG controls (0.86% and 1.08%), suggesting that these antibodies are specific and accurate in detecting PMNs and macrophages/microglia after spinal cord injury.
Injury severity is correlated with locomotor recovery (Scheff et al., 2003; Nishi et al., 2007) as well as PMN infiltration (Tjoa et al., 2003). Therefore, we tested the ability of our flow cytometric technique to detect changes in cellular inflammation in the injured spinal cord in response to graded injury severities.
Changes in cellular inflammation in the injured spinal cord were assessed in response to graded injuries after 1 dpi (PMNs, Fig. 2A) or 7 dpi (macrophages/microglia, Fig. 2B), based on the peak infiltration of these cells (Popovich et al., 1997; Carlson et al., 1998). Although five animals were assigned to each injury group, between three and five cell samples were recovered successfully for analysis. Statistical power was calculated using post hoc power analysis (ANOVA, single factor design) (Cohen, 1988). The effect size was large and statistical power adequate for both PMN (f = 1.360, power = 0.950) and macrophage/microglia (f = 0.954, power = 0.753) graded injury data sets (Supplementary Table S1), confirming the sample sizes were sufficient.
One-way ANOVA comparison of PMN infiltration into the spinal cord following graded contusions revealed a significant effect [ANOVA: F(2,10) = 10.69, P = 0.0042]. Following mild spinal cord injury, 3.3% of total cells in the injured spinal cord were PMNs 1 dpi, significantly less than after severe spinal cord injury (6.4% PMNs) (Tukey's multiple comparison tests: P < 0.01), but not moderate spinal cord injury (4.8% PMNs) (Fig. 2A). Linear regression analysis of naïve control, mild spinal cord injury, moderate spinal cord injury and severe spinal cord injury groups revealed a significant, positive correlation between injury force and PMN detection (Fig. 2C, r2 = 0.71, P = 0.0001). Restriction of the regression analysis to injury groups, excluding naïve controls, also yielded a significant, positive correlation (r2 = 0.67, P = 0.0007).
Similarly, one-way ANOVA comparison of macrophage/microglia accumulation after graded contusions revealed a significant effect [ANOVA: F(2,10) = 5.752, P = 0.0218] across mild, moderate and severe spinal cord injury animal groups. After mild spinal cord injury, 6.1% of cells in the injured spinal cord were ED1+ macrophages/microglia at 7 dpi, significantly less than after severe spinal cord injury (10.4% ED1+ cells) (Tukey's multiple comparison tests, P < 0.05), but not moderate spinal cord injury (8.8% ED1+ cells). As with PMNs, macrophage/microglia accumulation in the injured spinal cord was significantly correlated with injury force (Fig. 2D, r2 = 0.65, P = 0.0005). Restriction of the regression analysis to injury groups, excluding naïve controls, also yielded a significant correlation between injury force and macrophage/microglia detection (r2 = 0.40, P = 0.02).
As the first immune cells to infiltrate the injured spinal cord, PMNs are thought to affect injury acutely within hours or days following spinal cord injury. The early time course of PMN infiltration generated by flow cytometry confirmed immunohistochemical data from previous studies (Popovich et al., 1997; Carlson et al., 1998; Kigerl et al., 2006), showing that PMN infiltration peaked 1 dpi (Fig. 3A and B). However, although PMN number decreased after 1 dpi, PMNs persisted in the cord for up to 6 months after spinal cord injury (Fig. 3B). These data suggest continuing PMN infiltration because PMNs are absent in the uninjured spinal cord, do not replicate in tissue and exhibit a tissue half-life of ~48 h (Cartwright et al., 1964). Persistence of PMNs was confirmed by immunohistochemistry 1 dpi (Fig. 3C), 14 dpi (Fig. 3D) and 90 dpi (Fig. 3E). Quantitative stereologic analysis showed elevated PMN numbers at 1 dpi (536 177 ± 135 556), 28 dpi (59 906 ± 7958) and 90 dpi (64 782 ± 12 642), confirming the chronic persistence of PMNs in the injured spinal cord (Fig. 3B).
Unlike PMNs, macrophages/microglia were not detected in the injured spinal cord until 3 dpi, and peaked at 7 dpi (Fig. 4A, B and D). Interestingly, macrophages/microglia were detected at very low levels 14 dpi. The population increased after 14 dpi, peaked for the second time 60 dpi and remained elevated throughout 180 dpi (Fig. 4A and B). While not detected by flow cytometry 14 dpi, immunohistochemistry detected some macrophages/microglia localized at the injury epicentre (Fig. 4E). Macrophages/microglia were detected again by flow cytometry 21 dpi, as macrophage/microglia counts began to increase for the second time (Fig. 4B). Moreover, a striking 15.9% of all cells counted by the cytometer were macrophages/microglia at 90 dpi, as represented in parallel by immunohistochemistry in Fig. 4F. Accordingly, the biphasic response of macrophages/microglia was confirmed by quantitative stereology, which showed elevated ED1+ macrophage/microglia at 7 dpi (6.270 mm3 ± 0.783), a significant decrease at 28 dpi (1.650 mm3 ± 0.342) and a significant rise in cell number at 90 dpi (20.034 mm3 ± 1.649) (Fig. 4B). These novel findings implicate macrophages/microglia as a significant part of a prolonged, multiphasic response after spinal cord injury.
Similar to macrophages/microglia, T cell infiltration was predicted to peak 7 dpi (Popovich et al., 1997); however, flow cytometry did not detect any change in T cell number in the first 7 dpi, though an elevated number of T cells was detected at 9 dpi (1.6%) (Fig. 5A and B). In contrast, 40% of cells were positive for CD3 in spleen tissue (Fig. 5B). While T cell number was decreasing by 10 dpi, a persistent T cell response was observed throughout 180 dpi, at which time 4.4% of cells were labelled for CD3 (Fig. 5B). The persistence of T cells in the injured spinal cord was verified by quantitative stereology, showing elevated T cell numbers in the spinal cord at 7 dpi (4008 ± 913), 28 dpi (1252 ± 109) and 90 dpi (2243 ± 744) (Fig. 5C and E). Few T cells can be found in the parenchyma of the uninjured spinal cord, as there are no resident T cell populations, though a very low number of cells can be found in the subarachnoid space (Popovich et al., 1997; Sroga et al., 2003). Therefore, the detection of T cells in the injured spinal cord by both methodologies suggests an ongoing T cell response, perhaps indicative of increased immune surveillance.
Together, these data demonstrate a time-dependent multiphasic response of cellular inflammation after spinal cord injury (Fig. 6A); the initial phases of cellular inflammation were composed of the early peak of PMNs 1 dpi, followed by a peak of ED1+ macrophages/microglia 7 dpi and T cells 9 dpi, while the later phases were composed of all three cellular populations rising after 14 dpi and persisting throughout 180 dpi, with a notable second peak of macrophages/microglia at 60 dpi.
Acute (0–7 dpi) blockade of PMN (Taoka et al., 1997; Tonai et al., 2001) or monocyte/macrophage (Popovich et al., 1999; Gris et al., 2004) infiltration results in improvement of locomotor function. We have reported novel evidence for an extended second phase of inflammation, raising the direct question of whether the later phase of cellular inflammation is associated with a decline in locomotor function. Accordingly, we investigated the relationship between functional locomotor recovery and the kinetics of cellular inflammation after spinal cord injury using the BBB scale from 1 to 91 dpi using a separate cohort of rats that received a 200 kd contusion spinal cord injury at the T9 vertebral level.
Initially after trauma, hindlimb locomotor function was minimal (BBB score at 1 dpi = 0.5 ± 0.14) (Fig. 6B). As previously reported (Basso et al., 1995), BBB performance improved steadily from 2 to 7 dpi, beginning to plateau at 14 dpi. Coincidently, PMN, macrophage/microglia and T cell numbers peaked at 1, 7 and 9 dpi (Fig. 6A), respectively, before motor recovery plateaued (Fig. 6B). In contrast, the second phase of inflammation, ranging from 14 to 180 dpi, did not coincide with changes in BBB performance, suggesting that the later phase may not have an independent effect on open-field locomotor function after spinal cord injury.
Previously, we have shown that the complement cascade is activated following spinal cord injury (Anderson et al., 2004). Complement activation results in the cleavage of C5, releasing C5a, a potent chemoattractant for PMNs, macrophages/microglia, T cells and other immune cells. It is predicted that blocking receptor–ligand interactions with C5aRa will reduce immune cell recruitment to the site of injury (Woodruff et al., 2006). To test this hypothesis, rats with spinal cord injury were given C5aRa or water twice daily for 1 or 7 dpi and sacrificed to assess PMN or macrophage/microglial infiltration to the spinal cord. While no difference in PMN presence was detected at 1 dpi (data not shown), C5aRa-treated animals (3.5% ± 0.2) had significantly fewer macrophages/microglia at 7 dpi than vehicle-treated animals (5.7% ± 0.6) (Student's t-test, P = 0.05) (Supplementary Fig. S3).
Having confirmed the efficacy of C5aRa in reducing macrophage/microglia presence in the injured spinal cord, our next aim was to deplete immune cells after spinal cord injury to test their role in mediating damage and/or repair following spinal cord injury. As many studies have assessed the role of acute inflammation, generally yielding improved locomotor function and/or pathology (Popovich et al., 1999; Tonai et al., 2001; Gris et al., 2004; Trivedi et al., 2006), we focused our experiment on the sub-acute to chronic phase of inflammation, as its role has yet to be defined. This period is dominated by macrophages/microglia (Fig. 6A), whose presence we were able to reduce in the injured cord with the C5aRa. Injured rats were treated from 14 to 28 days with C5aRa or vehicle. The control animals exhibited greater locomotor recovery than C5aRa-treated animals (Fig. 7A). Repeated measures ANOVA revealed main effects of drug treatment [F(1,20) = 5.66, P = 0.027], time [F(5,20) = 438.80, P < 0.0001] and drug treatment by time interaction [F(5,20) = 2.60, P = 0.030]. Bonferroni post hoc t-tests showed control rats significantly outperformed C5aRa-treated rats at 21 (vehicle: 15.8 ± 0.9, C5aRa: 13.3 ± 1.0, P < 0.01) and 28 dpi (vehicle: 13.5 ± 0.8, C5aRa: 11.4 ± 0.3, P < 0.05). Chi-square analysis revealed that significantly more control rats achieved frequent coordination or better (BBB score > 12) at 28 dpi than C5aRa-treated rats (P = 0.03, χ2 = 4.701). Animals were sacrificed 28 dpi and spinal cord myelin basic protein quantified (Fig. 7C–H), revealing that C5aRa-treated rats had less myelination caudal to the injury epicentre than controls (Fig. 7B, two-tailed t-tests, 1.44 µm from epicentre: P = 0.016, 2.16 µm from epicentre: P = 0.004). Together, these data suggest that the late inflammatory response after 14 dpi improves functional recovery and myelination post-spinal cord injury.
The data presented here provide new insight into the cellular immune response to spinal cord injury, including a detailed picture of the daily dynamics of cellular inflammation. We used a novel OptiPrep gradient cell preparation method combined with flow cytometry to assess the principal immune cell types present in the injured spinal cord. Removal of myelin debris by the OptiPrep gradient method enhanced detection of PMNs compared to enzymatic dissociation alone. Additionally, the OptiPrep method allowed detection of a graded increase in both PMNs at 1 dpi and macrophages/microglia at 7 dpi, showing a quantitative linear relationship between the cellular inflammatory response and spinal cord injury severity. The persistent multiphasic inflammatory response of PMNs, macrophages/microglia and T cells observed here by flow cytometry was supported by quantitative stereologic data at a subset of time points. Each time point in the flow cytometric timecourse was normalized to uninjured animals, providing confirmation within this study that the uninjured spinal cord has minimal immune cells, as previously reported (Popovich et al., 1997; Nguyen et al., 2008). Some temporal differences that were statistically significant in the flow cytometric data set failed to yield significant differences in the stereologic data set (Table 2). Nonetheless, the stereologic data demonstrate the chronic presence of PMNs, macrophages/microglia and T cells. Together, the data revealed a multiphasic response of cellular inflammation after spinal cord injury.
Consistent with previous reports, the first cellular inflammation phase was comprised principally of PMNs (peaking 1 dpi), macrophages/microglia (peaking 7 dpi) and T cells (peaking 9 dpi). A novel and prolonged second phase of cellular inflammation was first observed after 14 dpi and persisted throughout 180 dpi, with a dramatic peak in the macrophage/microglial response at 60 dpi that doubled the magnitude of the earlier peak. Cellular inflammation was lowest for all three cell populations 14 dpi, suggesting a clear division between the phases of inflammation.
Later peaks of cellular inflammation after 14 dpi did not coincide with changes in locomotor function, suggesting ongoing inflammation is insufficient to affect locomotor recovery at this scale of analysis. In this regard, it is possible that more subtle changes in function occur during this timeframe but require more sensitive methodology. Interestingly, the rat contusion spinal cord injury syrinx shrinks from 14 to 28 dpi (Scheff et al., 2003), suggesting a relationship between the second phase of the cellular inflammatory response and tissue reorganization. Accordingly, the hypothesis that this second phase of macrophage/microglial response plays a role in preventing further loss of function must be considered. In this case, one would predict that reducing cellular inflammation during this period would result in a decline in locomotor function. Accordingly, blocking sub-acute to chronic inflammation via C5aRa resulted in reduced functional recovery and myelination in the injured spinal cord. The data presented here are unique in that this is the first study to test the role of delayed inflammation, providing novel insight into the multiphasic immune response following spinal cord injury.
Data from the current and previous studies suggest that cellular inflammation has complex, time-dependent functions. Our data show that PMNs, despite a reduction in number after peaking at 1 dpi, persisted for many months, suggesting that they may have a long-term role in promoting damage and/or repair. PMNs are thought to aggravate acute spinal cord injury within several dpi and have been previously demonstrated to promote neuronal cell death in culture through PMN-to-neuron contact or release of toxic factors (Dinkel et al., 2004; Nguyen et al., 2007). However, potentially PMNs have alternative roles in the long term as they secrete growth factors and cytokines that can promote scar formation/cytogenesis after spinal cord injury (Cicco et al., 1990; Jablonska et al., 2005; Nguyen et al., 2007). Moreover, PMNs express complement proteins, such as C1q, which may play a complex role in the injured microenvironment (Hogasen et al., 1995; Nguyen et al., 2008). The extended presence of PMNs suggests a long-term role after spinal cord injury, which may be very different from the conventional role suggested for acute PMNs.
In parallel with these observations, the role of macrophages/microglia, detected in two distinct phases of inflammation in the present study, may also be complex and time-dependent. The first phase of macrophages/microglia, which peaked 7 dpi, exacerbated spinal cord injury (Popovich et al., 1999). However, the role of the second phase, which peaked 60 dpi, is not known. The data presented here suggest that early chronic inflammation from 14 to 28 dpi modulates myelination in the injured spinal cord. The successful replacement of myelin damaged by spinal cord injury is a biphasic process of debris removal followed by remyelination that may hinge on interactions between the central nervous system and the immune system. Myelin degradation after spinal cord injury results in debris around the injury site, which may inhibit oligodendrocyte progenitor cell maturation (Robinson and Miller, 1999; Kotter et al., 2006). Additionally, macrophages have also been shown to modify the microenvironment, causing an upregulation of factors that promote oligodendrocyte progenitor cell proliferation (fibroblast growth factor-2 and interleukin-1β) and maturation (transforming growth factor-β1 and insulin-like growth factor-1), although factors that may inhibit successful remyelination are also upregulated (transforming growth factor-α and Scya4) (Diemel et al., 1998; Kotter et al., 2005; Setzu et al., 2006). The net effect of these signalling molecules probably determines how oligodendrocyte progenitor cells respond to demyelinated axons, and while there is clear evidence that inflammation aids in remyelination (Foote and Blakemore, 2005; Setzu et al., 2006), the exact mechanism remains unknown.
Interestingly, infiltrated macrophages have been shown to switch from pro-inflammatory to anti-inflammatory over time (Arnold et al., 2007; Villalta et al., 2009). While the role of macrophages in spinal cord injury has been debated (Rapalino et al., 1998; Popovich et al., 2003), the data presented here suggest a functional switch over time, though we did not specifically examine macrophage phenotype. Nonetheless, a thorough understanding of the dual role of these cells in both pro- and anti-inflammatory functions is critical for a complete interpretation of the data.
Unlike PMNs and macrophages/microglia, T cells were scarcely represented in the injured spinal cord. However, the number of cells does not necessarily reflect their importance in affecting the pathophysiology of spinal cord injury. While present in lower numbers, T cells could play an important role in injury and repair by modulating the function and recruitment of both innate and adaptive immune cells after spinal cord injury (Hendrix and Nitsch, 2007; Ankeny et al., 2009).
The issue of which factor is driving the multiphasic kinetics of cellular inflammation after spinal cord injury has not been resolved, although at least three possibilities have been identified. Normally, the spinal cord is a site of relative immunoprivilege; traumatic injury causes an opening of the blood–brain barrier by 3 dpi, which is mostly intact at 14 dpi, and open again at 28 dpi (Popovich et al., 1996a). These periods coincide with our observations of increased immune cell presence acutely after injury, low levels at 14 dpi and increasing again beginning 21 dpi. An increased permeability of the blood–brain barrier would allow immune cells to easily enter the spinal cord from the circulation, although specific cues are also likely to be involved. One such cue could be the complement anaphylatoxin, C5a, a strong recruiter of PMNs, macrophages/microglia and T cells (Klos et al., 2009). Local complement activation in neurons and glia, resulting in C5a production, has been demonstrated as early as 1 dpi and persists to at least 42 dpi (Anderson et al., 2004). Furthermore, PMNs have been shown to express a subset of complement proteins following spinal cord injury (Nguyen et al., 2008), while macrophages expressed the entire complement cascade in vitro (Johnson and Hetland, 1988). It is unlikely that complement proteins are the only factor to recruit immune cells to the injured spinal cord. It has been suggested that a homeostatic balance of pro- and anti-inflammatory cytokines modulates the recruitment of inflammatory cells after central nervous system injury (Lenzlinger et al., 2001; Stoll et al., 2002). Pro-inflammatory cytokines are expressed mostly within days after mouse spinal cord injury, though tumour necrosis factor-α has a second phase of expression beginning 14 dpi and lasting to at least 28 dpi (Pineau and Lacroix, 2007). The expression pattern of anti-inflammatory cytokines after spinal cord injury has not yet been thoroughly investigated, nor has any direct link between cytokine production and leucocyte recruitment.
The temporal and quantitative analysis of cellular inflammation presented here has provided new insight into the dynamics of the spinal cord injury microenvironment, and identified for the first time an extended multiphasic response of cellular inflammation. The development of a novel OptiPrep gradient cell preparation method combined with flow cytometry to assess the principal immune cell types present in the injured spinal cord allowed detection of a graded increase in both PMNs and macrophages/microglia, showing a quantitative linear relationship between the cellular inflammatory response and spinal cord injury severity. Flow cytometric quantification as conducted here also allowed accurate comparison of data across multiple time points, which was verified by quantitative stereology, identifying a time-dependent multiphasic response of cellular inflammation after spinal cord injury. Furthermore, reduction of delayed C5a-mediated macrophage/microglial infiltration after spinal cord injury resulted in decreased functional recovery, suggesting a reparative role for chronic inflammation in this model. Understanding the role of this multiphasic response in the pathophysiology of spinal cord injury is likely to be critical for the design and implementation of rational therapeutic treatment strategies, including both cell-based and pharmacological interventions.
National Institute of Neurological Disorders and Stroke (RO1 NS43428-01 to A.J.A.); Paralysis Project of America (PPA-32574 to H.X.N.); California Institute for Regenerative Medicine (CIRM) Stem Cell Training Award (T1-00008 to H.X.N. and D.L.S.); training program in molecular and cellular neuroscience at UC Irvine (T32 NS007444 to K.D.B.); University of California Alliance for Graduate Education and the Professoriate fellowship (NSF HRD0450366 to D.L.S.).
Supplementary material is available at Brain online.
The authors thank Rebecca Nishi, MS, and the technicians of the Christopher and Dana Reeve Foundation Animal Core Facility at UC Irvine for their help in animal surgery. They also thank Amir Shbeeb, Jon Steller, Viktoria Nikolova and Thaddeus J. O'Barr for general technical assistance.
PubMed Central Canada is a service of the Canadian Institutes of Health Research (CIHR) working in partnership with the National Research Council's national science library in cooperation with the National Center for Biotechnology Information at the U.S. National Library of Medicine(NCBI/NLM). It includes content provided to the PubMed Central International archive by participating publishers.


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Home >> Silver Tone Metallic Shank Round Flat Tip Shaped T Head Diamond Mounted Point Grinder Buffing Bit Tool 3mm x 8mm 4 Pcs
|Product Name
|Diamond Mounted Point
|Head Design
|Round Shaped T Head
|Metal, Diamond
|Head Diameter
|8mm/ 0.3"
|Shank Diameter
|3mm/ 0.12"
|Total Length
|45mm/ 1.8"
|Silver Tone
|Package Content
|4 Pcs x Diamond Mounted Point
- Round T shaped nose, diamond coated head.
- Can be installed on the electronic grinder, hanging mill and hand electric drill (not included here).
- Used for sculpturing, dressing, precision grinding and internal grinding carbide in ceramic, glass, precious stone, alloy and many other abrasive materials.
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subset: commoncrawl


text:
It's All about the Services: Developing Custom Applications for Microsoft SharePoint Server 2010 Using Microsoft ASP.NET, WCF, and REST
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Many developers use services to build and integrate applications and line-of-business systems with SharePoint. With SharePoint 2010, developers now have a wider array of options that include ASP.NET, Windows Communication Foundation (WCF), and RESTful services. Come get a demo-rich walkthrough of each option and discuss where each of the services might be appropriate and what you stand to gain by using them.
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Here are some suggested organisations that offer expert advice on adoption.
Accidentally, shockingly pregnant after adopting 2 dd's.(28 Posts)
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Curly Love - Do You and Your Significant Other Look Alike?
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Join Date: Jun 2011
Posts: 23
Wednesday, November 30, 2011 at 06:36PM
: Curly Love - Do You and Your Significant Other Look Alike?
Hey there, Curlies! Tracey here!
So, inspired by a recent visit to the
- seriously, if you don't know what that is and you even kind of like fashion, click that link! - which featured
a pair of couples in Tokyo
who were soooo PERFECT for each other, I thought - whoa wait! According to like, the whole world, couples are often attracted to each other based on similarities - and throughout the relationship end up looking more and more like one another - so for us curlies, do we date men who look like us? Right down to the natural hair on our heads?
I can't say that I don't! See the picture below is me and my (EX)boyfriend from a few years ago - my last boyfriend to say the least - and um, despite all of our not-alike-at-all issues, we did have the same hair! (I'm just now realizing...)
So ladies, you tell me - and please send me pictures too! (email@example.com) - do you date men who look like you?
Me and the Ex (Sorry for the weird backside photo - but you can see our hair here best!) Ah, La France -
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Crab Chowder
Recipe by weekend cooker
a very flavorful chowder for a cool night. This I got last year while visiting a relative in Florida.
Top Review by bluemoon downunder
A very tasty crab chowder though I admit I did make a few adjustments to meet our taste preferences. I used all the listed vegetables and added a diced onion, four cloves of minced garlic and two sliced leeks. I kept the proportion of liquid the same - as I like my soups thicker rather than thinner - used all the stock specified, but used only 1 cup of low-fat milk, and in place of the other two cups of milk, I used 1 cup of plain yogurt and 1 cup white wine. (I like to squeeze wine into soup recipes if I can find the space for it!) I also used only one tablespoon of melted butter and if I make this again - which is highly likely - I think I'll cut the butter back even more. Not that I don't love butter; just that I try not using it where I know we won't really miss it. Guided by the Zaar Kitchen Dictionary, I added some slivered almonds just before serving. Yummy! Thanks for sharing this recipe, weekend cooker. You do need to adjust the cooking time at the beginning of the recipe: it currently reads as 13 minutes but in the directions this is clearly stated as being a slow-cooker recipe! Made for 1-2-3 Hit Wonders.
See Discussion:2 Reviews
Ingredients Nutrition
- 2 small zucchini, thinly sliced
- 1 red sweet bell pepper, julienned
- 2 celery ribs, diagonally sliced
- 1 medium potato, cubed
- 2 tablespoons butter, melted
- 1 (10 ounce) can chicken broth
- 1 teaspoon seasoning salt
- 2 tablespoons cornstarch
- 3 cups milk
- 2 (6 ounce) cans crabmeat, divided, picked
- 1 (3 ounce) package cream cheese, cut up
Directions See How It's Made
- Place zucchini, bell pepper, celery, potato, butter, broth, and seasoned salt in slow cooker.
- Stir cornstarch into milk, stir and pour into slow cooker.
- Cover and cook on low for 3-4 hours.
- Turn heat to high, add crabmeat and cream cheese and stir until cream cheese melts.


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Nab-paclitaxel in Metastatic Breast Cancer Patients Failing Solvent Based Taxane (Tiffany)
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- How to Read a Study Record
|Metastatic Breast Cancer
|Drug: Nab-Paclitaxel
|Phase 2
|Study Type:
|Study Design:
|Intervention Model: Single Group Assignment
Masking: Open Label
Primary Purpose: Treatment
|Official Title:
|A Multicenter Non-randomized Phase II Study to Evaluate Nab-paclitaxel in Metastatic Brest Cancer Patients Failing a Solvent Based Taxane as (Neo-)Adjuvant Treatment (Tiffany)
- Overall response rate (ORR) [ Time Frame: 3 years ]
- Toxicity of the therapy [ Time Frame: 3 years ]Number and percent of patients with Adverse Events (any Grade and Grade 3/4).
- Clinical benefit rate (CBR) in patients with measurable disease [ Time Frame: 3 years ]
- Duration of response [ Time Frame: 3 years ]
- Progression-free survival (PFS) [ Time Frame: 3 years ]
- Overall survival (OS) [ Time Frame: 3 years ]
- SPARC expression rate of the tissue of the primary and/or metastatic tumor [ Time Frame: 3 years ]
- Compliance of the therapy [ Time Frame: 3 years ]Number and percent of the patients with treatment modifications (dose delay / dose reduction / premature dicontinuation)
|Estimated Enrollment:
|Study Start Date:
|July 2011
|Study Completion Date:
|October 2012
|Primary Completion Date:
|October 2012 (Final data collection date for primary outcome measure)
Drug: Nab-Paclitaxel
Nab-Paclitaxel 125 mg/m2 i.v. over 30 min. weekly in 5 out of 6 weeks
Given until progression, unacceptable toxicity, patient's request or withdrawal from Study
Primary Objective:
To determine overall response rate (ORR) and to exclude that it is 20% or lower.
Secondary Objectives:
- To determine compliance and toxicity of the therapy.
- To determine clinical benefit rate (CBR) in patients with measurable disease.
- To determine duration of response.
- To determine progression-free survival (PFS).
- To determine overall survival.
- To assess biomarkers, e.g. SPARC expression in the tissue of the primary or metastatic tumor.
|Ages Eligible for Study:
|18 Years and older (Adult, Senior)
|Sexes Eligible for Study:
|Accepts Healthy Volunteers:
Inclusion Criteria:
- Written informed consent for all study procedures according to local regulatory requirements prior to beginning specific protocol procedures.
- Complete baseline documentation must be submitted via the web-based data collection system MedCODES to the GBG Forschungs GmbH.
- Diagnosis of locally advanced or metastatic hormone-sensitive or insensitive, HER2-negative or -positive breast cancer.
- Relapse within ≤ 12 months after completing (last day of last cycle) (neo-) adjuvant chemotherapy.
- Documented relapse of either a measurable or a non-measurable lesion according to the modified RECIST criteria.
- Previous neoadjuvant or adjuvant treatment with a solvent based taxane (paclitaxel or docetaxel) irrespective of dose and duration.
- Prior endocrine treatment for metastatic / advanced disease is allowed.
- Complete radiological and clinical tumor assessment within 4 weeks prior to registration performed as clinically indicated.
- Age ≥ 18 years.
- ECOG Performance Status ≤ 2 (irrespective of restrictions due to breast cancer).
- Laboratory requirements:Absolute neutrophil count (ANC) >= 1.5 x 109/L., Platelets >= 100 x 109/L., Hemoglobin >= 9 g/dL (>= 5.6 mmol/L)., Prothrombin time (PT) or international normalized ratio (INR) <= 1.2x ULN (upper normal limit), Partial thromboplastin time (PTT) <= 1.2x ULN, Total bilirubin < 1.5x ULN, ASAT (SGOT) and ALAT (SGPT) <= 2.5x ULN (concomitant elevations in serum bilirubin and ASAT/ALAT above 1.0x ULN are not permitted), Creatinine clearance >= 50 mL/min), Urine Protein to Creatinine Ratio (UPC) < 1 (if UPC >= 1, then 24-hour urine protein must be < 1 g).
- Normal cardiac function confirmed by ECG.
A female either of: 1) Non-childbearing potential, i.e. physiologically incapable of becoming pregnant because of history of hysterectomy, bilateral oophorectomy (ovariectomy), bilateral tubal ligation or postmenopausal status.
2) Childbearing potential with a negative pregnancy test (urine or serum)within 2 weeks prior to registration, preferably as close to the first dose as possible, and agrees to use adequate contraception.
Acceptable contraceptive methods, when used consistently and in accordance with both the product label and the instructions of the physician, are as follows:
An intrauterine device with a documented failure rate of less than 1% per year, Vasectomised partner who is sterile prior to the female subject's entry and is the sole sexual partner for that female, Complete abstinence from sexual intercourse for 14 days before exposure to the investigational product, through the dosing period, and for at least 21 days after the last dose of investigational product, Double-barrier contraception (condom with spermicidal jelly, foam suppository, or film; diaphragm with spermicide; or male condom and diaphragm with spermicide).
- Female subjects who are lactating should discontinue nursing prior to the first dose of study drug and should refrain from nursing throughout the treatment period and for 14 days following the last dose of study drug.
Exclusion Criteria:
- Known or suspected hypersensitivity reaction to the investigational compounds or incorporated substances.
- (Neo-)adjuvant therapy not containing a solvent based taxane.
- (Neo-)adjuvant therapy with nab-paclitaxel.
- Concurrent hormonal therapy for cancer.
- Life expectancy less than 3 months.
- Pre-existing peripheral neuropathy of > grade 2 (per CTCAE).
- Pre-existing grade 3 or 4 diarrhea.
- Presence of uncontrolled infection.
- Any serious and/or unstable pre-existing medical, psychiatric, or other condition that could interfere with the subject's safety, provision of informed consent, or compliance to study procedures.
- Concurrent specific systemic anti-tumor treatment or treatment with experimental compounds during study treatment.
Please refer to this study by its ClinicalTrials.gov identifier: NCT01416558
|GBG Forschungs GmbH
|Neu-Isenburg, Germany, 63263
|Responsible Party:
|German Breast Group
|ClinicalTrials.gov Identifier:
|NCT01416558 History of Changes
|Other Study ID Numbers:
2011-000075-13 ( EudraCT Number )
|Study First Received:
|August 11, 2011
|Last Updated:
|May 21, 2013
Keywords provided by German Breast Group:
German Breast Group
GBG Forschungs GmbH
Breast Cancer
Metastatic Breast Cancer
Taxane Failure
Additional relevant MeSH terms:
Breast Neoplasms
Neoplasms by Site
Breast Diseases
Skin Diseases
Albumin-Bound Paclitaxel
Antineoplastic Agents, Phytogenic
Antineoplastic Agents
Tubulin Modulators
Antimitotic Agents
Mitosis Modulators
Molecular Mechanisms of Pharmacological Action
ClinicalTrials.gov processed this record on June 23, 2017


text:
WATCH: Cutest Blue Ivy Moments Captured Online
During Beyonce's performance Jay Z and daughter Blue Ivy sat in the audience to watch Beyonce on stage for the Video Music Awards.
Blue Ivy made hearts melt when she danced to "Flawless" or when she presented her mom with the Vanguard award shouting "mommy" into the microphone.
Relive these brief but moving moments below:
Photo Credit: Getty Images


text:
Google has dropped the price of additional Google Drive storage by a considerable margin.
Users on the 15GB plan continue to pay nothing, of course, but everything above that is a fair bit cheaper now.
If you want 100GB of cloud storage space, you'll now only have to pay $1.99 per month. Previously, it was $4.99.
Those who require 1TB of storage will make the most impressive savings though. That's now $9.99 per month, which is down from $49.99. You can pay for more, but that should be enough for the vast majority of people.
Google explains a terabyte as "enough storage for you to take a selfie twice a day for the next 200 years and still have room left over."
Google Drive isn't just an online holding place for your files, of course. Your storage allowance is also used by your Gmail account to hold emails and attachments. Google Photos, too, uses Drive to hold your auto-uploaded mobile snaps in the cloud.
All of a sudden, that free allowance is starting to sound a little meagre, and these new low expansion costs a whole lot more appealing.
If you've already upgraded to one of these packages in the past, don't worry - your monthly charge will drop automatically. Google has claimed that these price cuts have been made possible "thanks to a number of recent infrastructure improvements."
Read More: Best mobile phones 2014


text:
Track My Order
Frequently Asked Questions
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Can you recommend a header to allow servos to be plugged into the module?
No public wish lists :(
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text:
2012-13 Rajon Rondo SP OC Auto Limited Private SIgnings On Card Short Print
- Sold for: Start Free Trial or Sign In to see what it's worth.
- Item Category: Sports
- Source: eBay
- Sold Date: Jan 19, 2013
- Channel: Auction House
You are bidding on aRajon Rondo ON CARD AUTO from 2012-13 Panini Limited. This card is in great condition and great for any Rondo or Celtics fan! Great card to have for one of the top Point Guards in the NBA! If you have any questions please feel free to PM me. Shipping only to US and Canada. Payment due within 3 days of auction ending.


text:
scarface or godfather for me then conan
scarface or godfather for me then conan
The Godfather is a masterpiece... Scarface a classic and any Arnie film is worth a watch, good shout!
arnie is underated in my eyes he has made some classics
All about Commando! so many mistakes in that film but so entertaining
Stand by me and lost boys are films I can watch over and over again
ttt for conan
MMA is not a charity
the business
"The more you sweat in peace, the less you bleed in war"
Gladiator or Infernal Affairs (first one)
lost boys is sick , think casino is underated great film


text:
About FrankCullotta.com, the website
I'm Mark Anthony. Host of The The Vegas Tourist.com Podcast. Welcome to the unofficial fan site for Frank Cullotta
It's hard for some to understand how a good ol' boy from the Midwest could build a fan site for a man like Frank Cullotta. It wasn't exaclty on my list of things to do when we got the call asking if we were interested in his story.
We first met Frank Cullotta through his co-author, Dennis Griffin. My co-host and I were privileged (yes, privileged) to read an advanced copy of Frank's biography Cullotta: The Life of a Chicago Criminal, Las Vegas Mobster and Government Witness (True Crime). After reading the book, we were able to arrange to meet and interview the man along with his FBI counterpart, Dennis Arnoldy.
After the interview first posted, we were deluged with requests, comments and letters (good and bad). People wanted more information on Frank, his life, his crimes and about that era of Las Vegas history. Even now, it is one of our most downloaded shows!
It was at that point that I knew a website needed to be built. Not to glorify his past, the crimes that he committed or the lives that he took. He lived his life, confessed to his sins, he served his time and he paid his penance for that. Nor did I build this website to glorify or glamorize his former profession.
If you read his story, you will understand the fascination people have with him, what he did and how he lived through it all. His life plays out before your eyes in living color. Frank is not one to sugar coat anything. You see the decisions he made, the reasons he did what he did as well as the consequences we all pay for our decisions.
You also see a lot of life lessons in his story as well. The power and importance of friendships, the value of information. Living by a code of honor, ethics and knowing that your word is your bond.
So consider this a website built by a friend, for the fans with an educational edge built in.
FYI: Please understand that this is a fan site. Not maintained nor controlled by Frank Cullotta. Do not use this as a way to get connected with Frank Cullotta. Any requests to connect with Frank may or may not be forwarded to him or his representatives…


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Flavored Oyster Crackers RecipeRead Reviews Be the first to add a review
5 3 4
These jazzed-up oyster crackers created by our Test Kitchen have such great flavor, we bet you'll have trouble not eating them all at once! With Parmesan cheese and seasoning from a soup mix, they're a sure-fire hit with any soup.
Featured In: 43 Nut-Free School Snacks
- 2 packages (10 ounces each) oyster crackers
- 1/2 cup canola oil
- 1/4 cup grated Parmesan cheese
- 1 envelope savory herb with garlic soup mix
- Place the crackers in a large bowl. Combine the oil, cheese and soup mix; pour over crackers and toss gently. Transfer to two ungreased 15-in. x 10-in. x 1-in. baking pans.
- Bake at 350° for 5-7 minutes, stirring once. Cool. Store in an airtight container. Yield: 12 cups.
Originally published as Flavored Oyster Crackers in Quick Cooking January/February 2006, p42
Reviews for Flavored Oyster Crackers
Please Log In or Join to add a rating and review.
Click stars to rate
Any changes to your rating or review will appear where you originally posted your review
Reviewed Oct. 27, 2013
"Very good taste, everyone liked this snack."
Reviewed Oct. 23, 2012
"This is the best one I have found using oyster crackers. Much better than the one using Ranch dressing."
Reviewed Mar. 18, 2010
"My family loves this! It is easy to make and does not cost a lot. It satisfies the munchies and is delicious!"


text:
Your Account
Have fun and get great shots with your new Canon T5i/700D
Nothing matches the thrill of a new camera! And with this book in hand, you'll find getting up to speed with your T5i/700D is easy. Can't wait to start shooting? Take the Quick Tour first, and then explore all the controls and settings, the touchscreen, the HDR option, and low-light modes. Learn how to edit and share your photos. Take this guide along as you experiment with different shooting situations, and make the most of your new Rebel.
Inside – your free gray and color checker card to help you achieve accurate white balance and color
© 2016, O'Reilly Media, Inc.
(707) 827-7019(800) 889-8969
All trademarks and registered trademarks appearing on oreilly.com are the property of their respective owners.


text:
Your Dog, Your Life
The Scoop
Dog News, Politics
Bark-Out-Loud Humor
Doggie Style
Gear and Glam
Wildebeest creates the perfect present for new dog parents.
Dog Breeds
Foods & Treats
Health & Care
Behavior & Training
Dogs & Cleaning
Dog of the Day
Photo Comments
Hey guys my mommy is now designing frenchie (and other dogs) stuff. It is great, you have to see it!!
Alright frenchie friends..... I have to share my love for the meetups. If you do not know what a meetup is you are not living life!!
I am part of a local meetup here in WA. We have been meeting at Great Dog by Northgate while it is cold out. You should join if you aren't already a member so we can play, there are a lot of others that go!
If you live in another state, there are frenchie meetups all over and most likely one near you. Just visit meetup.com and look for one. If there is not one, start one up, the monthly site fee is worth all the fun it will become!!!!!!
We searched and searched for a frenchie, and right when we started having some setbacks with one of the breeders, we got an email from Whitney's breeder about her puppies. She sent photos and Whitney was the one we picked out right away. I flew to San Francisco one morning and met Whitney and her breeder mommy at the airport. I really had to try not to cry when I spotted them. Whitney was perfect and her breeder Martha is great and such a wonderful woman. Then Whit and I waited in the airport for hours waiting for our yet again 'late flight' she didn't make a peep our entire time in the airport until the end. We waited to be the last ones on the plane, the long line was formed around where we were sitting, all of a sudden. bark, bark, bark, bark it was the cutest yip. It startled me cause she was so quiet the whole time, then the whole line started laughing. Whitney did excellent on the flight, the couple next to us said they never would have known I had an animal traveling with me if they hadn't seen her when we first boarded (she had her head peeking out of the top of the bag). After our plane landed Whitney had her first ride in her new car seat her grandma had bought for her (zebra print-safety seat). When we got home Tony said well are you going to put her down? hahaha
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Tinh nng mi trong phin ban ZingMe 1.5:- ZingMp3: Tich hp tim kim, nghe cac bai nhac trc tuyn trn Zing Mp3, PlayList cua ban, ...- ZingNews: Cho phep oc tin tc cp nht ti tng phut trn Zing News.- Nng cp tinh nng chup anh.New features in Zing Me version 1.5:- Search, listen to hottest songs from the Zing Mp3, and your Play list.- Read daily latest news from Zing News.- Camera bugs fixed and performance improvements.


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Elizabeth Banks: Making movies is a gamble
Elizabeth Banks says producing a hit movie is always a gamble.
The actress' company, Brownstone Productions, produced last year's breakout hit Pitch Perfect.
Despite the success of the musical comedy, Elizabeth is sceptical that the film industry is suddenly more accepting of funny movies starring women.
"Predicting what content is going to fly is like looking into a crystal ball," Elizabeth told Marie Claire's work issue. "I try not to say, 'Yeah, Bridesmaids opened the door to make more movies about women.' I mean, did it? I don't know, where are they?"
The 39-year-old runs Brownstone with her husband Max Handelman, with whom she has two children. She explained how she juggles producing, acting and raising her kids, who are both under the age of three.
"It's all about managing people," she explained. "You know, women in Hollywood, we don't get action franchises and superheroes. The rom-coms that made Julia Roberts, Sandra Bullock, and Reese Witherspoon stars are out of favour right now - nobody is making them. In this business, if you are an ambitious person, you don't wait for people to hand you opportunities. You have to make a way for yourself."
The go-getting star says maintaining control has been part of her long-term career plan. She has passed up big-money role offers in the past because they didn't offer her the security she was after.
"I was offered a two-year contract on a soap opera right after I graduated," she revealed. "It would have paid off all of my student loans, and it was in New York where I wanted to live. I didn't even have an agent at the time, but I signed with a guy who said to me, 'You got a soap opera on your first day, let's see what else you can get.'"
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Online Evaluation
North Carolina Department of Agriculture and Consumer Services (NCDA & CS) has a nationally leading Geographic Information System (GIS) that has been 15 years in development. The system, called the Multi-Hazard Threat Database (MHTD), has been used for eradication of swine pseudorabies as well as Avian Influenza (AI). GIS has emerged as a proven decision support tool that has gained a critical role in managing emergencies. In the 2002 AI outbreak, North Carolina eradicated the disease with only three commercial flocks affected and GIS was a critical component of that successful response. GIS enhances epidemiological investigation by improving disease tracking through integration of laboratory data and premises identification, routing, quarantine management, communication and analysis. Providing end-user access to the power of GIS has been one of the priorities of NCDA & CS for several years. To this end, North Carolina has employed several tools to provide viewing and editing functionality to the MHTD using private sector products produced by ESRI and Google.
The use of GIS in North Carolina has not been limited to livestock applications. Since 2001, the MHTD has become an asset as an all-hazards planning, preparedness and response tool for other sectors of agriculture as well as a resource for other state agencies. Utilizing GIS helps determine scale of emergency or event, estimate rate of spread or progression of a disease, assist in distribution of resources (personnel and equipment), identify at-risk populations and rapidly communicate complex information. These advantages improve situational awareness of any incident and assist in rapid recovery.
Martin A. Zaluski, DVMDirector, Emergency Programs DivisionNorth Carolina Department of Agriculture and Consumer ServicesRaleigh, North Carolina
Health professionals including administrators, veterinarians, environmentalists and emergency response personnel who are interested in new technologies to utilize in surveillance and response activities.
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Voice Recognition - Phone Mode
I have a 2014 GC Limited and the voice recognition success rate for initiating phone calls is VERY poor. Anyone else experiencing this? Is there a known fix?
Thanks in advance...
Re: Voice Recognition - Phone Mode
just picked up my summit yesterday, but i did notice that it struggled initiating phone calls
Re: Voice Recognition - Phone Mode
As far as what?
I have a 2012, but sometimes uConnect will not recognize entries in my address book and I have no idea why.
Re: Voice Recognition - Phone Mode
i only made a few calls, but when it did struggle, it's like it didn't even hear me. reminds me of my kids. the 2011 ram i traded would mix up my choices but it never had a problem hearing me
I would agree there are issue with the voice recognition. But the phone features works fine as far as volume adjustment and sound quality, and I've never had a problem with the system not recognizing my phone.
Re: Voice Recognition - Phone Mode
Clearly there is an issue. Question is where is the fix?
Re: Voice Recognition - Phone Mode
I agree. The voice recognition is abysmal. In order to call my wife, Mary, I have to say marry as the uConnect voice says it. It's even worse on the last name. It "speaks" letters that it added to the name which are not in the name. Asking it to call anyone by name is an adventure. It gives me various options, none of which are remotely close to what I have asked for. Me: Call Jeff. It: Do you mean Franklin? :mad: I sure wish it ran off Google voice.
Re: Voice Recognition - Phone Mode
I have no issues with voice dail in a 2013 with the 730n. In fact, I'd go so far as to say that it works great and I use it often.
Re: Voice Recognition - Phone Mode
I would like to use it but given the systems poor performance I'm not
Often it is how thing are pronounced. As I have pointed out in other threads it is often the difference between saying Mobil like the gas station vs mobile like the phone. If you refer to the commands list posted for UConnect it also works better.
Re: Voice Recognition - Phone Mode
I have a 2012 with the same issue. It has difficulty recognizing names and simple yes and no commands without shouting at it. Then it may still take 2 or 3 tries. Anyone have a good fix?
Re: Voice Recognition - Phone Mode
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Make ordinary photos look fantastic with 60+ animated filter effects. Whether you're on your own, with a friend, with family, or with however many people there are, Wow!Filter will preserve and add magic to your wonderful moments. Not all moments are fun, but Wow!Filter always is! Pie face game filter is waiting for you! Now we even have prom queen live filter to match your prom dress and fire dragon live filter to clean your bad mood, more fun than SnapChat
【Venus Filter: Discover the Goddess Within You】
Take a selfie, let Venus Filter detect your face, work its magic and get the look of your dreams, including Elf, Flora, Bow, Boheme and even Audrey Hepburn.Reveal your true beauty and attract others without even trying
+ Added " The other vision " filters, let's see this beautiful world from the perspective of animals!
+ Added 10 English fonts. Editing photos is so interesting!
+ Added " photo replacement " feature in grid function. Editing photos is so easy!
+ Improved eye enlarging and face sliming effect in the retouch feature, let you become a beauty easily!
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Download Instructions: PREMIUM features Unlocked