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It is well known that enteric bacterial antigens drive the development of chronic colitis in a variety of different mouse models of the inflammatory bowel diseases (IBD). The objective of this study was to evaluate the role of gut-associated lymphoid tissue (GALT; Peyer's patches, isolated lymphoid follicles), mesenteric lymph nodes (MLNs) and spleen in the pathogenesis of chronic colitis in mice.
Surgical as well as genetic approaches were used to generate lymphopenic mice devoid of one or more of these lymphoid tissues. For the first series of studies, we subjected recombinase activating gene-1-deficient mice (RAG−/−) to sham surgery (Sham), mesenteric lymphadenectomy (MLNx), splenectomy (Splx) or both (MLNx/Splx). In a second series of studies we intercrossed lymphotoxinβ-deficient (LTβ−/−) mice with RAG−/− animals to generate LTβ−/− × RAG−/− offspring that were anticipated to contain functional MLNs but be devoid of GALT and most peripheral lymph nodes. Flow purified naïve (CD4+CD45RBhigh) T-cells were adoptively transferred into the different groups of RAG−/− recipients to induce chronic colitis.
We found that at 3–5 wks following T-cell transfer, all four of the surgically-manipulated RAG−/− groups (Sham, MLNx, Splx and MLNx/Splx) developed chronic colitis that was similar in onset and severity. Flow cytometric analysis revealed no differences among the different groups with respect to surface expression of different gut-homing markers nor were there any differences noted in IFN-γ and IL-17 generation by mononuclear cells isolated among these surgically-manipulated mice. Although we anticipated that LTβ−/− × RAG−/− mice would contain functional MLNs but be devoid of GALT and peripheral lymph nodes (PLNs), we found that LTβ−/− × RAG−/− mice were in fact devoid of MLNs as well as GALT and PLNs. Adoptive transfer of CD45RBhigh T-cells into LTβ−/− × RAG−/− mice or their littermate controls (LTβ+/+ × RAG−/−) induced rapid and severe colitis in both groups.
Taken together, our data demonstrate that: a) neither the GALT, MLNs nor PLNs are required for induction of chronic gut inflammation in this model of IBD and b) T-and/or B-cells may be required for the development of MLNs in LTβ−/− mice.
The intestinal tract is colonized by more than 100 trillion (1014) microorganisms, with the vast majority residing within the colonic lumen.1,2 The gut-associated lymphoid tissue (GALT; Peyer's patches [PPs], isolated lymphoid follicles) as well as the gut-draining mesenteric lymph nodes (MLNs) play important roles in mounting mucosal immune responses to invading pathogenic and/or commensal microorganisms while minimizing the tissue inflammation and systemic immune activation that may arise from these protective responses.3–11 Studies from several different laboratories suggest that commensal bacterial antigens continuously gain access to the intestinal interstitium where they are endocytosed by intestinal dendritic cells (DCs) and then transported from the lamina propria and PPs to the draining MLNs.6–8,12 Using carefully regulated, low-level cellular immune responses to these nonpathogenic bacteria, the MLNs provide for both anatomical and functional compartmentalization of the adaptive immune responses, thereby generating a "firewall" to the systemic dissemination of these microorganisms.6,7 The mechanisms by which the cellular elements of the GALT and MLNs mediate these distinct and seemingly opposing functions are only now beginning to be revealed. Although there is good evidence demonstrating that MLNs play a critical role mounting and regulating mucosal immune responses to enteric bacteria, the evidence demonstrating a role for PPs as a major inductive site for adaptive immune responses is somewhat vague and ill-defined.6–8,13 Recent work by Worbs et al13 demonstrate that the MLNs (but not PPs) are required for the induction of systemic tolerance to orally administered luminal antigens. Those investigators convincingly showed that ovalbumin-loaded DCs are transported from the gut interstitium and PPs to the MLNs by the afferent lymphatics, where tolerance to these luminal antigens is induced. There is an accumulating literature suggesting that a break in tolerance to enteric antigens may be responsible for the induction of the chronic intestinal inflammation observed in patients who suffer from the inflammatory bowel diseases (IBDs; Crohn's disease; ulcerative colitis).14–17 Indeed, the role of commensal bacteria in the pathogenesis of experimental IBD has been well characterized in number of different animal models of chronic gut inflammation.14,16,18 Although the etiology of these chronic inflammatory disorders has not been definitively elucidated, there is accumulating evidence to suggest that chronic gut inflammation arises from a complex interaction among genetic, immune, and environmental factors.16,18,19 A growing body of experimental and clinical data suggest that chronic gut inflammation arises from a dysregulated immune response to enteric bacterial antigens.
An immunologically important yet undefined aspect of disease pathogenesis in animal models of IBD is the anatomic location(s) where naïve T cells encounter enteric antigens to yield colitogenic effector cells. It has been assumed that naïve T cells migrate to the GALT/MLNs, where they are primed and polarized to yield T helper 1 (Th1) and/or Th17 effector cells. These colitogenic effector cells then exit the GALT and/or MLNs by way of the efferent lymphatic vessels, enter into the systemic circulation, and home to the gut where they initiate intestinal inflammation.15–18,20 In reality, there have been few studies that have assessed the role of the GALT and/or MLNs in the induction of chronic gut inflammation. Spahn et al21 demonstrated that dextran sulfate sodium (DSS)-induced colitis is more severe in mice devoid of MLNs, suggesting an overall suppressive function of these gut-draining lymph nodes, whereas Dohi et al22 showed an exacerbated inflammatory infiltrate and Th1 response in PP- and colonic patch-deficient mice exposed to intrarectal trinitrobenzene sulfonic acid (TNBS) dissolved in ethanol. In contrast to these studies, work by Makita et al23 suggests that neither the GALT, MLNs, spleen, nor peripheral lymph nodes were required for the induction (or suppression) of chronic colitis in mice. These investigators intercrossed lymphotoxin-α deficient (LTα−/−) mice with recombinase activating gene-2 deficient (RAG-2−/−) animals to generate LTα−/− × RAG−/− double-deficient progeny that are devoid of all organized lymphoid tissue.23 They found that adoptive transfer of naive or antigen-experienced (activated/memory) CD4+ T cells obtained from the lamina propria of mice with active colitis to LTα−/− × RAG-2−/− recipients induced colonic inflammation; however, the onset and severity of disease was delayed when compared to the disease induced by T-cell transfer to littermate controls (i.e., LTα+/+ × RAG-2−/− recipients). Although compelling, unanswered questions remain. For example, it is unclear how a global defect in LTα during development of lymphopenic RAG−/− mice affects lymphotoxin receptor-β expression and subsequent lymphotoxin signaling events associated with T-cell trafficking, priming, and polarization following adoptive transfer of naïve T-cells. Therefore, the objectives of this study were to evaluate, using both surgical as well as genetic approaches, the role of gut-associated and secondary lymphoid tissue in the conversion of naïve T-cells to colitogenic Th1/Th17 effector cells and their induction of chronic colitis. We present data demonstrating that neither the spleen, GALT, MLNs nor peripheral lymph nodes (PLNs) are required for the induction of chronic gut inflammation.
Healthy wildtype (WT; C57BL/6) and recombinase activating gene-1 deficient (RAG−/−) mice were purchased from the Jackson Laboratories (Bar Harbor, ME), whereas lymphotoxin-β-deficient (LTβ−/−; C57Bl/6) mice were obtained from Dr. Nancy Ruddle (Yale University).24 LTβ−/− mice were intercrossed with RAG−/− animals to generate LTβ−/− × RAG−/− double-deficient offspring that were anticipated to possess functional MLNs but be devoid of GALT (isolated lymphoid follicles, PPs) and most secondary lymphoid tissue.4,9,10,21,24,25 Animals were maintained on 12/12-hour light/dark cycles in standard animal cages with filter tops under specific pathogen-free conditions in our animal care facility at Louisiana State University (LSU) Health Sciences Centers and given standard laboratory rodent chow and water ad libitum. All experimental procedures involving the use of animals were reviewed and approved by the Institutional Animal Care and Use Committee of LSU Health Sciences Center and performed according to the criteria outlined by the National Institutes of Health.
For some experiments, the MLNs and/or spleen were surgically removed from RAG−/− mice by performing a mesenteric lymphadenectomy (MLNx) and/or splenectomy (Splx) using previously published protocols.13,23 Briefly, 6–8-week-old mice were anesthetized using isoflurane during surgery. A ventral incision was made on the abdomen and the intestinal loops were gently reflected to the right to expose the mesentery. A dissecting microscope was used to visualize the lymphatic vessels emerging from the rudimentary MLNs. The MLNs were excised by blunt dissection and incisions were closed with sutures. For splenectomy, a lateral incision was performed on the left abdomen. The spleen was removed after blood vessel ligation and incisions were closed with sutures. Sham-operated mice (Sham) were treated identically; however, neither the MLNs nor the spleen were removed. Following surgery mice were allowed to recover for 2 weeks. Confirmation that the MLNs and/or spleen had been removed was determined by stereomicroscopy at 8 weeks following T-cell transfer or when the animals lost >15% of their original body weight and were euthanized.
The following antibodies were purchased either from BD Pharmingen (San Diego, CA) or eBioscience (San Diego, CA) for cell isolation and flow cytometric analysis: CD45RB-FITC, CD4-APC, CD103-FITC, CD29-PE, lymphocyte Peyer's patch adhesion molecule-1 (LPAM-1)-biotin + streptavidin-phycoerythrin (SA-PE)-Texas Red, CD25-FITC, CD49d-PE, CD4-PE-Cy5, IFN-γ-FITC, IL-17A-PE, and CD4-PerCP.
Chronic colitis was induced via adoptive transfer of naïve CD4+CD45RBhigh T cells derived from WT donors using our previously published method.26,27 Briefly, spleens were removed from donor WT mice and teased into single cell suspensions in phosphate-buffered saline (PBS) with 4% fetal bovine serum (FBS; FACS buffer) using the frosted sides of glass microslides. CD4+ T cells were enriched by negative selection using Dynal Mouse CD4 Negative Isolation Kit (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. Enriched CD4+ T cells were labeled with anti-CD4 and anti-CD45RB antibodies and sorted into CD4+CD45RBhigh and CD4+CD45RBlow fractions by two-color sorting on a FACS Aria (Becton-Dickinson, San Jose, CA). The CD45RBhigh and CD45RBlow populations were defined as the 40% of cells exhibiting the brightest CD45RB staining and the 15% of cells with the dimmest CD45RB expression, respectively, and were found to be >98% pure on post-sort analysis. RAG−/− mice were intraperitoneally injected with 0.5 × 106 cells of either CD4+CD45RBhigh or CD4+CD45RBlow (as a healthy control group) T cells suspended in 500 µL PBS. Clinical evidence of disease (e.g., losing body weight and loose stool/diarrhea) was monitored and recorded weekly from the time of the injection.
At 8 weeks following T-cell reconstitution or when animals lost >15% of their original body weight, RAG−/− mice were euthanized and the colons were removed, cleaned of fecal material, and scored for macroscopic evidence of inflammation using our previously published scoring criteria.26,27 Briefly, normal colonic morphology was assessed a score of 0; mild bowel wall thickening in the absence of visible hyperemia was assigned a score of 1; moderate bowel wall thickening and hyperemia was given a score of 2; severe bowel wall thickening with rigidity and marked hyperemia was assigned a score of 3; and severe bowel wall thickening with rigidity, hyperemia, and colonic adhesions was given a score of 4. Colonic length and weight were also measured to calculate weight-to-length ratios, which provide a quantitative index of inflammation.27 In addition to macroscopic inflammation, representative sections of distal colon were placed in 10% PBS-formalin, fixed overnight at 4°C, embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E). Blinded histopathological evaluation of the colons was performed by a pathologist unaware of the treatment groups using our published scoring criteria.27
Lymphocytes were obtained from the spleen and/or MLNs (when present) as well as the colonic lamina propria and analyzed by flow cytometry as previously described.26–28 Briefly, spleens were removed from RAG−/− mice reconstituted with either CD4+CD45RBhigh T cells or CD4+CD45RBlow T cells and teased into a single cell suspension using the frosted ends of two glass slides in 4% FACS buffer on ice. Colonic lamina propria mononuclear cells (LPMCs) were prepared by the digestion of the finely minced intestinal pieces remaining after intestinal intraepithelial lymphocyte (IEL) isolation in RPMI-1640, 4% FBS, and collagenase type VIII (200 U/mL, Sigma, St. Louis, MO) for 40 minutes at 250 rpm in a 37°C shaker. Cells from the spleen and cLP were further enriched by centrifugation over a 44%/70% Percoll gradient. The cell pellet was washed and resuspended in FACS buffer and viable cells were counted using a solution of 0.4% Trypan blue in 1× PBS. For surface antigen staining, 1 × 106 cells were placed in individual wells of a 96-well plate, incubated first with Fc receptor (FcR) block (CD16/CD32), and then stained with the appropriate antibody cocktails. After staining, cells were fixed for 20 minutes on ice in freshly prepared 2% ultrapure formaldehyde (Polysciences, Warrington, PA) and surface expression levels of CD103, CD29, LPAM-1, CD25, and CD49d on CD4+ T cells were analyzed the next day on the FACS-Calibur or LSRII (BD Biosciences). Absolute numbers of CD4+ T cells in the spleen and colonic lamina propia of reconstituted animals were calculated by multiplying the total number of viable cells isolated from each tissue by the percentage of total cells positive for CD4 as determined by flow cytometric analysis. Flow cytometry data were analyzed using FlowJo software (Tree Star, Ashland, OR).
Colonic lamina propria mononuclear cells were prepared as described above and 1.5 × 105 cells were cultured for 16 hours in 200 µL of complete RPMI-1640 medium at 37°C and 5% CO2 in anti-CD3ε monoclonal antibody (mAb) (BD Pharmingen)-coated 96-well flat-bottom plates containing soluble anti-CD28 mAb (eBioscience, 1 µg/mL final concentration). BD GolgiStop (BD Pharmingen) was added for the last 6 hours. Cells were harvested, stained with anti-CD4, permeabilized using the BD cytofix/cytoperm kit (BD Pharmingen), stained with IFN-γ and IL-17A mAb, and analyzed for intracellular cytokine production by flow cytometry as previously described.27 To determine extracellular cytokine production, colonic LPMCs (1.0 × 105 cells) were cultured for 16 hours in 200 µL of complete RPMI-1640 medium at 37°C and 5% CO2 in an anti-CD3ε mAb-coated 96-well flat-bottom plate with 1 µg/mL soluble anti-CD28 mAb. The supernatants were collected and stored at −80°C until analyzed. IFN-γ and IL-17A levels in the supernatants were determined by sandwich enzyme-linked immunosorbent assay (ELISA) using the commercial kits Mouse IFN-γ or IL-17A ELISA (eBioscience) as described by the manufacturer.
Data are presented as means ± standard error of the mean (SEM). Statistical analyses were performed using one-way analysis of variance (ANOVA) followed by Dunnett's post-hoc test. A probability value (P value) of P < 0.05 was considered significant.
As a first step toward defining the roles of MLNs and/or spleen in enteric antigen-induced conversion of naïve T-cells to yield colitogenic effector cells, RAG−/− mice were subjected to sham, MLNx, and/or Splx surgeries prior to adoptive transfer of naïve CD4+CD45RBhigh T cells. For comparison, a group of sham-operated RAG−/− mice were reconstituted with CD4+CD45RBlow T cells. Beginning at 3–5 weeks following T-cell transfer, mice in the different groups reconstituted with CD4+CD45RBhigh T-cells began to lose body weight and developed visible signs of disease including hunched-over appearance, loose stools, and piloerection (Fig. 1). In contrast, RAG−/− mice that received CD4+CD45RBlow T cells appeared healthy and never lost weight throughout the 8-week observation period (Fig. 1). Macroscopic evidence of colonic inflammation (hyperemia, bowel wall thickening, and adhesions) at 8 weeks following T-cell transfer (or when mice lost >15% of their original body weight) was significantly increased in all four of the CD4+CD45RBhigh→RAG−/− groups compared to the healthy CD4+CD45RBlow→RAG−/− group (Fig. 2A). No statistically significant differences were noted among the different CD4+CD45RBhigh→RAG−/− groups. In addition to macroscopic colon scores, we also quantified the colon weight-to-length (W/L) ratio for each animal, as our previous studies have demonstrated this to be a quantitative index of colonic inflammation that correlates well with blinded histopathological analysis.27 We observed significant increases in the W/L ratios in the four CD4+CD45RBhigh→RAG−/− groups when compared to the CD4+CD45RBlow→RAG−/− controls; however, no statistical differences were identified among the four CD4+CD45RBhigh→RAG−/− groups (Fig. 2B). Blinded histopathological evaluation of the colons revealed that virtually all of the animals in the four CD4+CD45RBhigh→RAG−/− groups developed moderate-to-severe disease characterized by extensive transmural infiltration of polymorphonuclear leukocytes (PMNs), mononuclear leukocytes (monocytes, lymphocytes), bowel wall thickening, goblet cell loss, abnormal crypt architecture, and crypt abscesses when compared to their healthy CD4+CD45RBlow→RAG−/− controls (Fig. 3). Histopathology scores were found to be significantly increased in the Sham, Splx, and MLNx + Splx groups when compared to the CD4+CD45RBlow→RAG−/−group. Although the histopathology score for the MLNx group was 5-fold greater than that of the CD4+CD45RBlow→RAG−/−, this value did not achieve statistical significance. Nevertheless, no significant differences were observed among these four groups, suggesting that neither the onset nor the severity of chronic colitis is dependent on the presence of the MLNs and/or the spleen.
Having established that neither the MLNs nor the spleen are required for induction of chronic colitis, we wished to determine how the absence of MLNs and/or the spleen affected T-cell accumulation and induction of gut-homing marker expression on T cells obtained from the colonic lamina propria of the different mice. We found that the numbers of CD4+ T cells within the colonic lamina propria of all four CD4+CD45RBhigh→RAG−/− groups were significantly greater when compared to their CD4+ CD45RBlow→RAG−/− controls (Fig. 4). Although we noted a trend for fewer T cells in the Splnx group, this difference was not statistically different from the other three groups that received CD4+CD45RBhigh T cells. We next examined how the absence of MLNs and/or spleen affected the surface expression of CD49d (α4 integrin), CD29 (β1 integrin), LPAM-1 (α4β7 integrin), CD103 (αE integrin), and CD25 on CD4+ T cells obtained from colonic lamina propria of reconstituted RAG−/− mice. We observed that CD49d expression decreased from ≈43% in the CD4+CD45RBlow→RAG−/− to ≈17, 18, 27, and 26% in the sham, MLNx, Splx, and MLNx+Splx groups, respectively (Fig. 5). This same trend was also observed for CD49d expression when expressed as median fluorescence intensity (MFI; Fig. 6). A similar trend was observed for CD103 expression in that the MFI was decreased by 30–40% in the four CD4+CD45RBhigh→RAG−/− groups when compared to the CD4+CD45RBlow→RAG−/− controls (Fig. 6). A large decrease (>80%) in CD25 surface expression was observed in all four of the CD4+CD45RBhigh→RAG−/− groups when compared to the healthy CD4+CD45Rlow→RAG−/− controls (Figs. 5, ,6).6). Conversely, although the expression of the gut-homing integrin α4β7 (LPAM-1) was relatively low compared to freshly isolated WT T cells (data not shown), it was increased significantly in the four CD4+CD45RBhigh→RAG−/− groups when compared to the noncolitic CD4+CD45RBlow→RAG−/− group (Figs. 5, ,6).6). Finally, we observed little or no alteration in CD29 expression among the five different groups (Figs. 5, ,66).
Because MLNs and/or spleen is/are thought to be important for enteric antigen-dependent priming and polarization of naïve T cells to yield disease-producing effector cells, we investigated how the absence of MLNs and/or the spleen affected this process. To do this we stimulated LPMCs obtained from the colonic lamina propria of reconstituted RAG−/− mice with plate-bound CD3ε and soluble CD28 mAbs ex vivo, and quantified the intracellular production of IFN-γ and IL-17A in CD4+ T cells using flow cytometry. We observed that the percent of CD4+ T cells expressing IFN-γ increased from 16% in the CD4+CD45RBlow→RAG−/− animals to ≈35, 42, 40, and 31% in the sham, MLNx, Splx, and MLNx+Splx groups, respectively (Fig. 7). In addition, we found that the percent of CD4+ T-cells expressing IL-17A ranged from 15% in T cells isolated from CD4+CD45RBlow→RAG−/− mice to ≈8, 15, 7, and 8% in the sham, MLNx, Splx, and MLNx+Splx groups, respectively (Fig. 7). Finally, we observed that the percent of T cells producing both IFN-γ and IL-17A increased from 2% in the CD4+CD45RBlow→RAG−/− animals to ≈5, 10, 4, and 5% in the sham, MLNx, Splx, and MLNx+Splx mice, respectively (Fig. 7). In addition to intracellular cytokine production, we also quantified extracellular release of the two cytokines following ex vivo activation of LPMCs obtained from the colons of the different mice. We found that the production of IFN-γ increased from very low levels in LPMCs obtained from CD4+CD45RBlow→RAG−/− mice (57 pg/105 cells) to levels ranging from 4220 to >5500 pg/105 cells for the sham, MLNx, Splx, and MLNx+Splx CD4+CD45RBhigh→RAG−/− animals (Fig. 8). Although the percentage of CD4+ expressing IL-17A was not increased in colitic mice, the secreted levels of this cytokine were significantly increased from 133 pg/105 cells for cells obtained from CD4+CD45RBlow→RAG−/− to >1100 pg/105 cells for cells taken from all four CD4+ CD45RBhigh→RAG−/− groups (Fig. 8). No significant differences were observed in the levels of the cytokines among these four groups (Fig. 8).
Data obtained from the studies presented above suggested that surgical removal of both the MLNs and spleen had little effect on enteric antigen-dependent priming and polarization of naïve T-cells to yield colitogenic effector (Th1/Th17) cells. Indeed, adoptive transfer of naïve T-cells into MLNx and/or Splx RAG−/− recipients induced chronic colitis with an onset and severity very similar to sham-operated controls. It could be argued that other lymphoid tissues such as the GALT and/or PLNs could substitute for the MLNs and spleen in promoting the conversion of naïve T cells to disease-producing effector cells. Therefore, we next determined whether adoptive transfer of naïve T cells into LTβ−/− × RAG−/− recipients developed disease, as these mice would be predicted to possess functional MLNs but lack PPs, isolated lymphoid follicles (ILFs) and most PLNs.4,9,10,21,24,25 Surprisingly, and in contrast to what has been described for LTβ−/− mice, LTβ−/− × RAG−/− lacked GALT (ILFs, PPs), PLNs, and MLNs (Fig. 9A). Furthermore, we found that adoptive transfer of naïve T-cells into LTβ−/− × RAG−/− mice induced moderate to severe colitis that began to develop within 2–3 weeks following T-cell transfer (Fig. 9B). This accelerated disease was also observed in their littermate LTβ+/+ × RAG−/− controls as well. The reason for this accelerated disease does not appear to be classical graft versus host disease since we transferred syngeneic WT T-cells into LTβ−/− × RAG−/− and LTβ+/+ × RAG−/− recipients.
The literature contains numerous studies demonstrating that the GALT and/or MLNs are the major inductive sites where naïve T-cells encounter enteric bacterial antigens such that the appropriate immune response may be initiated. Thus, it has been assumed that the GALT and/or MLNs play critical roles in enteric antigen-induced chronic gut inflammation.20,29 Previous studies have suggested that MLNs may function to limit or suppress the induction of the erosive, self-limiting colitis induced by toxic chemicals,21 whereas another study suggested that neither the GALT, MLNs, nor the peripheral lymph nodes are required for the induction of chronic colitis in RAG−/− mice reconstituted with naive or antigen-experienced (activated) CD4+ T cells.23 In the latter study, investigators used a model very similar to our own.23 Although a provacative study, Makita et al23 did not assess how a systemic defect in LTα expression during the development of the lymphopenic RAG−/− mice might affect LT receptor-β expression and LT signaling events associated with T-cell trafficking, priming, and polarization following adoptive transfer of T cells, especially if the MLN environment is important for regulating immune responses to commensal bacterial antigens. Therefore, we surgically removed the MLNs and/or spleen in healthy RAG−/− to evaluate the roles of these gut-draining and peripheral lymphoid tissues in the pathogenesis of T-cell-induced colitis. Although our initial focus was the MLNs, we also wished to determine how the absence of the spleen might affect the onset and severity of chronic gut inflammation in this mouse model of IBD. It is quite possible that commensal enteric antigens may gain access to the systemic circulation where splenic DCs would endocytose, process and present these gut-derived antigens to naïve T cells, thereby promoting their conversion to colitogenic effector cells within the spleen.8 Indeed, Eksteen et al30 have recently demonstrated that splenic DCs can enhance expression of gut-homing surface proteins CCR9 and α4β7 on T-cells. Data obtained in the current study clearly demonstrate that neither the MLNs nor the spleen are required for induction of chronic gut inflammation. In addition, we show that the absence of MLNs and/or spleen did not affect expression of the different T-cell-associated integrins nor did their absence alter cytokine production of T-cells obtained from the colonic lamina propria of colitic mice. Of note however, was the significant decrease in CD25 expression on T-cells obtained from the four CD45RBhigh→RAG−/− groups compared to their healthy CD45RBlow→RAG−/− counter-parts (Figs. 5, ,6).6). The reasons for the decreased expression of the IL-2 receptor are not clear at the present time but may be related to the fact that the large majority of transferred CD45RBlow T-cells express CD25 whereas CD45RBhigh T-cells do not. Another interesting observation noted in the current study was that both intracellular as well as secreted levels of IFN-γ from LPMCs obtained from the colons of all four CD45RBhigh→RAG−/− groups were ≈3-fold greater than for IL-17A (Figs. 7, ,8),8), suggesting a skewing toward a more Th1-like inflammation in this model. Taken together, these data suggest that neither the MLNs nor spleen are required for the conversion of naïve T cells to their disease-producing phenotype. These data are somewhat surprising given the large body of work demonstrating that enteric antigen-loaded DCs migrate from the intestinal lamina propria and PPs to the MLNs by way of the afferent lymphatics in order to induce immune responses to commensal bacteria.5–8,12,13,31,32 However, it could be argued that other gut-associated or secondary lymphoid tissues may substitute as inductive sites for naïve T-cell priming and polarization. Therefore, we generated RAG−/− mice that we assumed would possess functional MLNs but lack PPs, ILFs, and most PLNs.4,9,10,21,24,25 Surprisingly, and in contrast to what has been described for LTβ−/− mice, LTβ−/− × RAG−/− lacked GALT (ILFs, PPs), PLNs, and MLNs (Fig. 9A). The mechanisms responsible for this novel finding are not clear; however, it may be that T- and/or B-cells are required for MLN development in the absence of LTβ. Indeed, one report demonstrated that deletion of the T-cell-associated LTβ receptor ligand (LIGHT) in LTβ−/− mice resulted in a dramatic decrease in the size and numbers of MLNs.33 Nevertheless, we found that adoptive transfer of naïve T cells into LTβ−/− × RAG−/− induced rapid and severe colitis that was virtually identical to the colitis induced in their littermate RAG−/− controls (i.e., LTβ+/+ × RAG−/−). Unlike Makita et al, we did not observe a delay in onset in the LTβ−/− × RAG−/− mice compared to their RAG−/− littermates, but rather, observed an acceleration of disease in both groups of mice. The reasons for the accelerated disease in both groups of mice are not clear at the present time but do not appear to be some form of graft versus host disease (GvHD) because: 1) recipients were reconstituted with syngeneic C57Bl/6 T-cells, and 2) the mice never exhibited the classic signs of GvHD such as loss of body hair, facial swelling, and dermatitis. It may be that intercrossing these two mutant mice produced offspring with enhanced expression of one or more sensitivity factors or generated progeny with reduced expression of certain protective genes. Studies to identify the mechanisms responsible for this accelerated disease are currently under way. Taken together, these data demonstrate that neither the GALT, MLNs nor PLNs are required for the induction of chronic colitis induced by adoptive transfer of naïve T-cells into lymphopenic recipients. The anatomic locations where naïve T-cells are converted to colitogenic effector cells by enteric antigens remain to be defined.
Supported by PO1-DK43785 (Project 1, Animal Models Core and Histopathology Core).
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Definition of aspergilliform
: like a brush; specifically : resembling the sporophore of Aspergillus an aspergilliform stigma
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Origin and Etymology of aspergilliform
International Scientific Vocabulary aspergill- (from New Latin aspergillus) + -iform
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Posted on October 14th, 2011 by
Preoperative chemotherapy or radiation – take your pick or both – improves overall survival of patients with large, soft-tissue sarcomas of the extremities, retrospective studies from two cancer centers suggest. Looking at a cohort of 112 patients, investigators at the Mayo Clinic in Scottsdale, Ariz., saw no significant differences by treatment type in local control rates or overall survival.
Oncology Report, Neil Osterweil, 10/14/11
Tags: Cancer, Mayo Clinic Arizona
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Chocoholics rushed to Tesco supermarkets across the country earlier this week after a pricing error occurred on its range of Terry's Chocolate Orange.
Bargain-hunters hurried to the store as news of the glitch spread across the internet, and a dedicated Facebook page entitled 'Terry's Chocolate Orange glitch – I was there!' was soon set up.
Reports of the exact pricing of the Kraft Food-owned chocolates at the time of the error vary from 25 to 30 pence, as one customer claimed to have bought 55 for £15 – making a saving of £133.
Some quick-thinking customers have chosen to sell on the goodies in order to make a profit, with one happy shopper claiming on Facebook to have sold "nearly half" of the 246 she had purchased.
Earlier this year, the grocer caused a stampede to its Scottish stores as consumers learnt that a pricing error led boxes of beer to be sold at almost half price.
Last month, the leading supermarket launched a value campaign that slashed prices on thousands of its products, as the company seeks to offer value for money during the fragile economic climate.
This week's pricing glitch was soon resolved by Tesco, which has not released details of how much money was lost as a result nor how many products were unwittingly sold at the massively reduced price.
A spokesman commented that the grocer sells thousands of products, adding: "This was a system glitch and we stopped it as soon as we were made aware of it."
The original deal was featured on the Hot UK Deals website, which said on Friday that the thread had been viewed 145,459 times and had received more than 1,100 customer comments.


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Many children with cardiac tumors display no symptoms, but for those that do, the symptoms typically imitate many other cardiovascular conditions, which can make them more challenging to diagnose. Depending on the tumor location, size and growth rate, a child may experience:
Causes of tumors vary; however, for the most part, they are believed to be the result of abnormal growth of heart tissue cells. A small percentage of tumors are genetically inherited, which can be detected with genetic testing. But, generally, tumors develop without any family history.
We are grateful to have been ranked #1 on U.S. News & World Report's list of the best children's hospitals in the nation for the third year in a row, an honor we could not have achieved without the patients and families who inspire us to do our very best for them. Thanks to you, Boston Children's is a place where we can write the greatest children's stories ever told."


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After having waved the flag for Windows 8 I was unimpressed when it came to doing a refresh.
The options are clear, you can either refresh and save your files or go for a clean refresh which takes all the unwanted files out of your hard drive and makes it clean.
I started this process with advice from many sites who advise you to do a re install after you buy a new laptop to take away all the unwanted rubbish associated with a new Laptop or PC.
The mere click of a button can now re install your windows platform, gone are the days of putting in a windows CD to re-install gone are the days of sectioning off your hard drive to re install windows its a darn sight easier these days, however they fail to tell you just how long the refresh takes.
I started the process at 3 o'clock in the afternoon and by 10 pm at night it was still only around 30 percent, the refresh was taking at least an hour every 6 percent I was looking at the early hours of the morning before it would be complete.
I went to various sites AFTER I started the process which was a BIG mistake, as per usual I jumped in with two feet without researching the process first . If I had known it was gonna take up to 48 hours to complete I would not have bothered.
I stayed up later than normal hoping it would complete quicker than the advised times indicated by other users, alas it took nearly 9 hours to complete, I swore after that I would never do this again unless I was forced to.
So a warning to other users of Windows 8 who fancy doing a clean install, be aware that this can take up to 48 hours to complete, which is ridiculous in this day and age.
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Open Plan Khyber Pass Office!
181, Khyber Pass Road, Grafton, Auckland City
Price On Application
- Proposed Usage
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If 181 Khyber Pass Roaddoesn't suit your requirements, please don't hesitate to contact the team on the details listed below. We are actively speaking with landlords of all property types and sizes and would love to help you find your perfect space.
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Special Press Release from Silver Dollar City:
Special Last Blast Of Summer Opportunities At Silver Dollar City Attractions:
Moonlight Madness, July 27 – August 9: Throughout Moonlight Madness, Silver Dollar City will be open all day and into the night. July 27 – August 9, the park will be open from 9:30 a.m. until 10 p.m. for summer night thrills on the thirty themed rides and attractions from high-speed coasters such as the explosive-PowderKeg, to the mega-splashing American Plunge.
Enjoy KidsFest all day and stay all night with you day pass or season passORCome after 5 p.m. for only $20 for night ride thrills.


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Free Application for Federal Student Aid (Part 2)
Hi again. I've got a little bit more Information you'll need to complete and file your FAFSA; remember, the best place to do this is online at fafsa.gov -- Remember that first F stands for FREE, if someone is asking you to pay, you're not at the right place.
In the last video we went over filing the FAFSA each year, getting a PIN for your electronic signature, and why it's important to get the FAFSA filed EARLY.
In this video we'll cover dependent vs independent filing, types of financial aid available, and different sources for aid.
Dependent Vs. Independent Filing:
A lot of students ask about how to file as an independent student for financial aid purposes so that parent's information does not have to be included on the FAFSA.
If you answer yes to any of these you are considered an independent student for financial aid purposes:
- Will you be 24 on January 1st of the year you complete the FAFSA?
- Are you married?
- Are you a master, or doctoral student?
- Are you a veteran of the U.S. armed forces?
- Are you currently on active duty in the U.S. armed forces?
- Do you have children or dependents who receive more than half of their financial support from you?
If none of these questions apply to you, then you should apply as a dependent student and include parent information. The FAFSA form will help guide you through the process.
So next I want to talk about the Types of financial aid:
There are 3 types of Financial Aid: The Three types are Loans, Grants, and Departmental Scholarships
Loans - these are funds that eventually have to be paid back. The FAFSA determines the type of loans you may be eligible to receive.
Grants - these are a gift of money, and do not have to be paid back. The FAFSA form determines what grants you could be eligible for depending on your Expected Family Contribution (remember that?)
The final type is Departmental Scholarships - these are a gift of money and can be awarded based on grades, talent, or excellence in a certain field, like Athletics, Dance, Drama, or Music. These funds do not have to be paid back. Check with the individual academic departments for scholarship information.
Sources of Aid:
No matter where you receive financial aid from, the main sources come from federal, state, and other institutional funds. The FAFSA is THE primary vehicle for most sources of financial aid.
The UNCG Financial Aid office always awards the most desirable aid first- grants and scholarships- to students who qualify, because we encourage students to borrow as little as possible, -to only borrow what they need. A lot of students receive scholarships from local organizations and civic groups, from churches, and from parents' employers. If a student gets financial aid from an outside organization, they need to notify the financial aid office about the funds so we can add it to their financial aid. Hopefully this will reduce the loan amounts. The more you can get in outside aid, the less you'll have to borrow. That's DEFINITELY what we're shooting for.
Special circumstances:
If there is a significant or dramatic change in your family's financial situation since the most recent tax year, you should contact the Financial Aid Office of the school you are attending or planning to attend to discuss your options. In the event of unemployment or some other unfortunate circumstance that has resulted in a dramatic change in the financial situation from the most recent tax year, contact your Financial Aid Office as soon as possible.
Important Message for students:
It's important for you to be actively involved in the financial aid process, to understand what's going on. Sometimes students don't even know they have loans in their names because the parent took over the financial aid process in the first year. As a mature young adult, you must be aware of the rising cost of a quality education and how you plan to pay for it. All communication from the school, the federal government, and the state, goes to you, the student. You need to pay attention to this communication because your parents don't receive it. If you have been sent a student aid report that's been rejected because a signature is missing and you don't do anything about it, well... you'll end up without financial aid. This is your education and your future! Do NOT be afraid to ask questions.


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- published: 02 Jan 2017
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A film, also called a movie, motion picture or photoplay, is a series of still images which, when shown on a screen, creates the illusion of moving images due to the phi phenomenon. This optical illusion causes the audience to perceive continuous motion between separate objects viewed rapidly in succession. A film is created by photographing actual scenes with a motion picture camera; by photographing drawings or miniature models using traditional animation techniques; by means of CGI and computer animation; or by a combination of some or all of these techniques and other visual effects. The word "cinema", short for cinematography, is often used to refer to the industry of films and filmmaking or to the art of filmmaking itself. The contemporary definition of cinema is the art of simulating experiences to communicate ideas, stories, perceptions, feelings, beauty or atmosphere by the means of recorded or programmed moving images along with other sensory stimulations.
The process of filmmaking is both an art and an industry.
Priyanka Chopra (pronounced [prɪˈjaːŋkaː ˈtʃoːpɽaː]; born 18 July 1982) is an Indian film actress, singer, producer and philanthropist, and the winner of the Miss World pageant of 2000. Through her film career, she has become one of Bollywood's highest-paid actresses and one of the most popular and high-profile celebrities in India. Chopra has been cited by various media outlets as the world's sexiest Asian woman, as well as one of the most fashionable Indian celebrities. She has received numerous awards and nominations, including a National Film Award, and five Filmfare Awards, and was awarded the Padma Shri, the fourth highest civilian award, by the Government of India in 2016.
Although Chopra initially aspired to study engineering or psychiatry, she accepted offers to join the Indian film industry, which came as a result of her pageant wins, making her acting debut in the Tamil film Thamizhan in 2002. The following year, she starred in The Hero, her first Hindi film release, and followed it with the box-office hits Andaaz and Mujhse Shaadi Karogi. She later earned wide critical praise for her breakout role in the 2004 thriller Aitraaz. By 2006, Chopra had established herself as a leading actress of Hindi cinema with leading roles in the highly successful films Krrish and Don. The 2008 drama Fashion marked a turning point in her career, earning her critical acclaim as well as the National Film Award for Best Actress and the Filmfare Award for Best Actress. She subsequently gained recognition for her versatility in portraying a range of unconventional characters in films such as Kaminey (2009), What's Your Raashee? (2009), 7 Khoon Maaf (2011), Barfi! (2012), Mary Kom (2014), Dil Dhadakne Do (2015), and Bajirao Mastani (2015), all of which garnered her wide critical acclaim and several accolades. In 2015, she began starring as Alex Parrish on the ABC drama Quantico, becoming the first South Asian woman to headline an American network series.
Full Length LP is the debut album by the Huntington Beach, California punk rock band Guttermouth, released in 1991 by Dr. Strange Records. It introduced the band's style of fast, abrasive punk rock with tongue-in-cheek humor and sarcastic lyrics. The album was originally released as an LP but was repackaged the following year as a CD including tracks from the band's first 2 EPs Puke and Balls, as well as the previously unreleased tracks "Malted Vomit" and "Ghost." It was re-released again in 1996 by Nitro Records under the title The Album Formerly Known as Full Length LP.
The album proved to be a success for the band, expanding their fan base and giving them opportunities to play shows all over southern California alongside other popular punk rock bands. An animated music video was made for the song "1, 2, 3…Slam!" and played on local punk rock and skateboarding video programs. Many of the songs from Full Length would remain staples in the band's live set throughout their career.
Ram Charan is an Indian film actor, dancer, producer, businessman and entrepreneur, who works in Telugu cinema.He won two state Nandi Awards, two Filmfare Awards South, two CineMAA Awards, and one Santosham Best Actor Award. He is popularly known as Mega Power Star of Telugu Cinema. Ram Charan ranked #69 in 2013 India's Forbes list with earnings of ₹12.67 crore (US$1.9 million).
Ram Charan was born to Telugu actor Chiranjeevi and Surekha. He has two sisters. He is the grandson of Allu Rama Lingaiah and the nephew of Nagendra Babu, Pawan Kalyan, and Allu Aravind.He is the cousin of Allu Arjun, Varun Tej and Sai Dharam Tej. Charan got married to Upasana Kamineni, the vice-chairman of Apollo Charity and chief editor of B Positive magazine, in Hyderabad on 1 December 2011. Upasana is the granddaughter of Prathap C. Reddy, the Executive Chairman of the Apollo Hospitals. Subsequently, they got married on 14 June 2012 at the Temple Trees Farm House.
Ram Charan is an equestrian, and entrepreneur. He owns the polo team Ram Charan Hyderabad Polo Riding Club. He serves on the board of directors of MAA TV.
Science fiction is a genre of speculative fiction dealing with imaginative concepts such as futuristic settings, futuristic science and technology, space travel, time travel, faster than light travel, parallel universes and extraterrestrial life. Science fiction often explores the potential consequences of scientific and other innovations, and has been called a "literature of ideas." It usually eschews the supernatural, and unlike the related genre of fantasy, historically science fiction stories were intended to have at least a faint grounding in science-based fact or theory at the time the story was created, but this connection has become tenuous or non-existent in much of science fiction.
Science fiction is difficult to define, as it includes a wide range of subgenres and themes. Author and editor Damon Knight summed up the difficulty, saying "science fiction is what we point to when we say it", a definition echoed by author Mark C. Glassy, who argues that the definition of science fiction is like the definition of pornography: you do not know what it is, but you know it when you see it.
Watch this full movie on my channel and please do not forget to subscribe. This movie is Zanjeer of ram charan, Priyanka Chopra, Sanjay datt, etc. Watch more movies :- https://www.youtube.com/watch?v=gGRHMQ1SLBM Keyword :- Zanjeer, Zanjeer full movie, Zanjeer full movie in hindi, Zanjeer full movie in hindi dubbed, Best action movies, 2017 latest movies, Best movies of 2017, Hindi dubbed movies, Hollywood movies in hindi dubbed, Action movies in hindi, Hindi dubbed, Hindi dubbed had movies, Ram charan movies in hindi, Priyanka Chopra movies, Priyanka Chopra movies in dubbed , Sanjay datt movies in hindi, Ram charan, Priyanka Chopra, Sanjay datt, movies in hindi, Latest movies of ram charan, Latest movies of Priyanka Chopra, Latest movies of ram charan in hindi, Best 2016 movies, 201...
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If you're new, Subscribe! → http://bit.ly/Subscribe-to-Looper From action throwbacks to kid-friendly fare, hard-hitting dramas to game-changing horror, this year has already delivered a bumper crop of excellent flicks for film lovers. We're still just a few pages into the calendar, but let's take a quick look back at the best movies of 2017 so far... The LEGO Batman Movie | 0:17 John Wick Chapter 2 | 1:07 Hidden Figures | 1:51 Patriots Day | 2:25 Split | 3:04 The Founder | 3:44 Get Out | 4:23 Logan | 5:05 Kong: Skull Island | 5:43 Read more here → http://www.looper.com/43864/best-movies-2017-far/ Film and TV Theories https://www.youtube.com/playlist?list=PLOzaghBOlEsf1EjjXEqYZ3cr1Va9eP68j 12 Film Theories That Change Everything https://www.youtube.com/watch?v=zpwRA9KQnWU&list=PLOzaghB...
If you want to Watch more FREE Movies Online?.. VISIT http://mobstarink.com/WatchFreeMoviesOnline NOW! Please SUBSCRIBE to Our Channel NOW If You LOVE MOVIES? MobStar Movies proudly present the official full movie to the drama flick "Gotti" - Starring Armand Assante, Anthony Quinn, William Forsythe. Director Robert Harmon. http://www.youtube.com/watch?v=GPD0ezmvdNE
Watch my tribute to the art of movie posters : https://www.youtube.com/watch?v=Oyh5e9VqB78 -- This is a supercut of selected scenes from Netflix TV serie "Stranger" Things associated with scenes from 1970-1980's movies. Spot all the references of the producers of the serie : - The Goonies - Alien - E.T the Extra-Terrestrial - Firestarter - Poltergeist - Close encounters of the third kind - A nightmare on Elm Street - Explorers - The Shining -Stand by me - Carrie - Commando Have you found more references ? Tell me in comments. http://ulyssethevenon.com
What does silence say? A video essay by Filmscalpel. More at http://fandor.com/keyframe
Our friends at FOX gave us the opportunity to develop the image of an action movies and entertainment channel. This was a great chance to show how much we love adrenaline! The branding consisted in various navigation pieces and brand animations and IDs in which action was magnified and taken to the limit. Hit the play button and hold on tight, 'cause to the rhythm of the Beasty Boys this channel will blow your mind! Directed by: Plenty Art Director: Pablo Alfieri Animation Director: Mariano Farias --- 3D Design: Andres Reisinger, Andres Rossi, Sebastian Curi & Mariano Farias 3D Animation: Hernan Estevez, Sebastian Curi, Leandro Giorni & Mariano Farias 3D Rendering & Lighting: Andres Reisinger, Sebastian Curi y Daniel Bel 3D Modeling: Daniel Bel, Leandro Giorni & Andres Reisinger ID Storyb...
Read the full story here: http://gizmodo.com/how-criterion-collection-brings-movies-back-from-the-de-1501343511 video: Michael Hession and Nicholas Stango for Gizmodo / 2014 music: Twinkie by Podington Bear http://gizmodo.com/how-criterion-collection-brings-movies-back-from-the-de-1501343511
Agency Sky Creative Director Steven Hore Production Company MPC Creative Director Lee Parker VFX Producer Sophie Hogg VFX MPC Senior Designer and Creative Direction Will MacNeil VFX 3D Lead Greg McKneally VFX 2D Lead and Star Wars Advisor Marcus Dryden Motion Design Team Matthew Campbell, William Lanham, Abel Reverter, Mantas Grigaitis & Sam Keehan VFX Team Gayatri Patel, Baptiste Lebouc, Petter Steen, Vittal Kuntla, Anbarasu E, Gerard Dunleavy, Sumesh Chammancheri, Luca Maccarelli, Bibin Balan P, Emma Taylor, Michelle Tolo, Akshay Sunil Wadwalkar, Manjunath Ramakrishnaiah & Martin Joas Grade MPC Colourist George K
"The Fruits of Dystopia" is a short film about having fun in a less than perfect world. Cyrus Sutton explores an escape from modern trappings through excerpts from classic dystopian novels "1984," A Brave New World," and Fahrenheit 451." About www.Korduroy.TV: Korduroy.TV is the intersection between creativity and the outdoors. Offering a platform for independent surf culture, we've been named "best surf blog in the world" by Surfer Magazine. Korduroy is pumping out fresh content daily in collaboration with filmmakers and artists from around the world relevant to the DIY outdoor experience. FEATURING Steve Caballero Peter Hewitt Nick Rosza Dane Reynolds Ryan Burch Harrison Roach Pat Towersy George Trimm Devon Howard Foster Huntington Kameron Brown Geoff Lawton Luke Davis ADDITIONAL FO...
"Turn the volume up & display HD fullscreen mode for a deluxe experience." I hope you enjoy watching this selection as much as I did editing it. : ) My intention was to create a short video, a taster of the scenes that captivated me from an early age (80s onwards). I did use 160 VFX Movies (LIST: http://bit.ly/1gDTvM5). Short version: http://www.youtube.com/watch?v=aYDM1y-2TU0 Song: Thomas Bergersen "Rada" (Extended) Vocals by Vladislava Vasileva ------- Copyright Info: Be aware this channel is only for promotion purpose. I don't take any credits for music, cinematics or any kind of picture. All music belongs to the original creators. Fair use is codified at Section 107 of the Copyright Act. Under the fair use doctrine, it is not an infringement to use the copyrighted works of anot...
UPDATE 05/28/2012: exchanged source video for 720p version, no big changes to the content, only small corrections to framing (4:3 --16:9), audio cleanup and 2 or 3 small changes to the edit. due to the 500MB upload limit for free members the bitrate is still rather low at 3200, so don't expect a massive quality boost.... i also changed the title and added "rather" and "(believable)" to clear up some misunderstandings as to why i didn't add this or that ;) --- it's a little rough around the edges and could have used a day or two to refine the content, but i guess it's informative enough to share. FOR EDUCATIONAL PURPOSES ONLY, NO COPYRIGHT INFRINGEMENT INTENDED. ALL COPYRIGHTED MATERIAL IS OWNED BY ITS RESPECTIVE COPYRIGHT HOLDERS. THIS VIDEO IS COMMENTARY IN NATURE UNDER THE "FAIR USE" ...
Scenes you've seen … or have you? Using Dissolve stock footage – and the magic of editing – we've created this impressionistic homage to some classic movies. Can you name all 14 films? See and license the clips used at dissolve.com/scenes Try your luck with Scenes You've Seen 2: Award Winners Edition: https://vimeo.com/119382931 Test your indie cred with Scenes You've Seen 3: Indie Films Edition: https://vimeo.com/140098318 Music: "Retro Smash Hit" by Pat Andrews, licensed from Audiosocket http://bit.ly/retrosmash If you would like to publish or broadcast this video, please contact us at firstname.lastname@example.com or on Twitter at @dissolve.
Shot by my grandfather on his Bell & Howell Filmo using 16mm Kodachrome film stock. This footage was taken a year after the California theme park opened. It was quite a surprise going through this and seeing my grandmother meeting Walt Disney himself! I've been doing my best to find out what they were shooting at the front of the park. So far I've been unsuccessful. Transferred to HD (1080PsF 23.98) myself off a Spirit and Color Corrected using a da Vinci 2k Plus. Slowed to 18fps to match the original film speed.
My woman so lonely
My woman so fine
My flower, don't worry
You will shine
A marriage, the secret
The shadows behind
Pianos are playing
But as long as your hero's in the movies
And the turtle is safe in the Caribbean sea
While the power of nations set the rules
Still I've got no one lying next to me
Red carpets, no windows
She longs to escape
Her secret against
But as long as your hero's in the movies
And the turtle is safe in the Caribbean sea
While the power of nations set the rules
Still there is no one lying next to me
And the turtle is safe in the Caribbean sea
While the power of nations make the rules
Still there is none lying next to me
I have got none lying next to me


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Little research has been done to study the function of emotion in English vocabulary learning by Chinese learners when experts, teachers and students pay increasing attention to vocabulary teaching skills and learning strategies. This book is to explore through experiments whether emotion aroused by pictures of emotional associations will affect the English vocabulary learning of Chinese learners. In the first section of the experiment, recognition task was required after pictures of squares together with the target word lists for memorization were presented to subjects. In the second section participants were divided into two groups before pictures of positive and negative associations were presented respectively to them with the target word lists for memorization. Our study suggests that emotion aroused by pictures of emotional associations do affect word memorization, that vocabulary memorization would be promoted by appropriate anxiety, and that a relaxed or pleasant mood would impede, or at least, would not facilitate vocabulary learning.


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Come to Learn. Come to Network. Or Both!
Insights, Information and ConnectionsNo matter where you are on your career path as an HR professional, HRMAC offers programs - throughout multiple locations around Chicagoland - to accelerate your learning and enable you to expand your network.
HRMAC offers both free and paid events (discounted for members) that provide thought leadership and resources to help you and your Human Resources organization have greater impact as leaders and as business partners.
Educational events
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3/15/2017 » 4/26/2017
HRCI HR Fundamentals Online Prep Spring Course - (aPHR)
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New Data Mining Blogs
January 20, 2010 by Sandro Saitta
Filed under: Uncategorized
Filed under: Uncategorized
The new year hasn't changed the aims of Data Mining Research. One of them is to present new data mining blogs to you. In this post, let me introduce two new data mining related blogs:
- Blog by bruno: This blog covers a very large number of topics including web data analysis and data visualization. Bruno also has an interesting list of data exploration tools.
- Datamining-blog: The focus of this blog is on data mining for CRM marketing. An interesting point about this blog is that it is written both in English and German.
I wish all the best to these blogs and I welcome them on the data mining list of blogs.
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Oh look at that, what are we doing now? ANOTHER commentary on a 3DS game... Yeah Yeah we know, its a dying system with jumped up mobile games (with shittier Graphics) we PROMISE we'll do something on a proper system soon! But for now: shut up and shove this in your eyeholes, dicklips.
Well well, Richie look at that, what the fuck are you trying to pull? Not only are you talking about yet another 3DS game but you are talking about one of those "download only" ones from months ago.
Yeah well... Its fucking cheap on the eShop, deal with it, if you have a problem that... then seriously dude, take a long hard look at your life and the things that are important to you, reflect for a while on your priorities, because frankly its our fucking blog, and we do what the fuck we want! We have been resident in this little corner of the internet for seven years, we write pure gold, maybe it doesn't make sense to you now. But some day it will, don't think we don't have this planned, no sirree, we have it all worked out and you "dear reader" are nothing but a pawn along for a free ride.
Anyways... AOTFM: ATT as I like to call it. Is a glorified visual novel, it has a great atmosphere and ambiance created but the sights and sounds of the "game". It genuinely makes me pine for a time when I lived as a kid in 1970s Japan... Which I did not! That is how impressively that atmosphere encapsulates you! The "game" or "light entertainment experience" requires no skill, talent or generates a challenge. It simply is. I like it, it was a relaxing fun experience, my only qualms with it are, that it was a bit short, I could not care about the tacked on card game thing.
Definitely worth £2.69
Love and apparently the Vita is full of shit like this, yet I refuse to buy one!?!
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Maharashtra Balushashi
Crisp on the outside, soft on the inside, wearing a light golden colour the Balushahi is a fulfilling delight for the craving sweet tooth. It is similar to a glazed doughnut in terms of ingredients, but differs in texture and taste. In South India, a similar pastry is known as Badushah.
The balls made out of flour (maida), clarified butter (ghee) and yogurt are deep fried in ghee and dunked in the goodness of some rich sugar syrup. The Balushahi is a famous north Indian sweet dish that can be stored at room temperature and served anytime.
They are said to exhibit the traits of Mughal cuisine, possibly reflecting the trade and territorial relationships of the Mughal empire with the south.The first indication is the name itself. Dishes which have "shahi" which comes from the Persian "Shah" meaning royal, invariably have their origins in Mughlai cuisine which is a style of Indian cooking that came with the Mughal invaders or tradespeople from Persia. The "Shahi" cuisine is also incredibly rich usually, and the Balushahi is nothing if not rish considering the ghee in it! Also, the deep-fried pastry dipped in sugar syrup is reminiscent of sweets from the Middle East.
Nutrition facts per 1 piece:
Calories 132
Total Fat 7.0g
Saturated Fat 1.1g
Trans Fat 0.0g
Cholesterol 0mg
Sodium 5mg
Potassium 50mg
Total Carbohydrates 16.4g
Dietary Fiber 1.0g
Sugars 9.9g
Protein 1.6g
Calcium 2%
Iron 2%
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Ecotour Guide
"Take only photographs, leave only footprints". So goes the motto of ecotour guides everywhere. From Himalayan mountain scapes to local arboretums, the idea that we should visit nature in a gentle and unobtrusive manner is quickly spreading through tourist culture.
Ecotour guides enable people to enjoy and learn about the natural, historical and cultural characteristics of unique environments while preserving the nature and beauty of those sites. They are responsible for leading, guiding and teaching tourists as well as helping to stimulate the economic development opportunities in local communities.
Ecotour guides function in the same way that regular tour guides or adventure tour guides do. Except, instead of showing people historically significant locations or guiding people on exciting bungee jumps, ecotour guides help tourists appreciate animals and nature in a new way. Often, this means using bikes and hiking boots instead of cars, rowboats and canoes instead of motorboats, and touring parks out of season, to lessen the stress visitors have on natural environments. They often travel in smaller groups.
These differences can allow for a more rewarding tour. With only 10 tourists on a dolphin-watching trip, in a small, quiet boat, dolphins and other sea animals might be more willing to surface and visit with the tourists, while a larger boat with 60 tourists might just scare them off.
Ecotour guides are often environmental activists, who want to share their love of undisturbed nature with others. They have a tenderness for nature, and a respect for the animals and plants that live within it. They are concerned with issues like urban encroachment, development, agribusiness, and pollution. Yet, they still want to travel and visit natural landscapes. They do so by touring responsibly, promoting meaningful contact between tourists and the natural world. They lead long hikes and camping trips, and short walks to waterfalls. During their tours, they educate tourists on responsible behaviors, human-imposed dangers, and encourage them to use their new knowledge and appreciation of nature to educate others about the importance of ecotourism.
Different levels of government usually support ecotourism initiatives. As well as providing a new source of economic growth for areas, ecotours preserve natural resources, promote conservation, and support sustainable development. Ecotours raise money for developing parks, environmental services, and for natural resource management. They even sometimes clean up messes made by other people (some ecotour guides lead trips up Mount Everest with the intention of cleaning up empty oxygen canisters left on the mountain by climbers).
Ecotours are only some of the many tours offered by tour groups and travel agencies. But tourists who travel with ecotour guides learn wonderful things and witness natural occurrences which, without the efforts of these environmentalists, could shortly disappear.
Interests and Skills
Typical Tasks
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Long Term Career Potential
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Bureau of Labor Statistics, U.S. Department of Labor, Occupational Outlook Handbook, 2014-15 Edition, http://www.bls.gov/ooh/
Bureau of Labor Statistics, U.S. Department of Labor, Occupational Employment Statistics, 2002, http://www.bls.gov/oes/2002/oes_nat.htm
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