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Altered Dynamics of Candida albicans Phagocytosis by Macrophages and PMNs When Both Phagocyte Subsets Are Present
Fiona M. Rudkin, Judith M. Bain, Catriona Walls, Leanne E. Lewis, Neil A. R. Gow, Lars P. Erwig
Antonio Cassone, Editor
Fiona M. Rudkin
Judith M. Bain
Catriona Walls
Leanne E. Lewis
Neil A. R. Gow
Lars P. Erwig
Antonio Cassone
An important first line of defense against Candida albicans infections is the killing of fungal cells by professional phagocytes of the innate immune system, such as polymorphonuclear cells (PMNs) and macrophages. In this study, we employed live-cell video microscopy coupled with dynamic image analysis tools to provide insights into the complexity of C. albicans phagocytosis when macrophages and PMNs were incubated with C. albicans alone and when both phagocyte subsets were present. When C. albicans cells were incubated with only one phagocyte subtype, PMNs had a lower overall phagocytic capacity than macrophages, despite engulfing fungal cells at a higher rate once fungal cells were bound to the phagocyte surface. PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested. In contrast, when both phagocyte subsets were studied in coculture, the two cell types phagocytosed and cleared C. albicans at equal rates and were equally susceptible to killing by the fungus. The increase in macrophage susceptibility to C. albicans-mediated killing was a consequence of macrophages taking up a higher proportion of hyphal cells under these conditions. In the presence of both PMNs and macrophages, C. albicans yeast cells were predominantly cleared by PMNs, which migrated at a greater speed toward fungal cells and engulfed bound cells more rapidly. These observations demonstrate that the phagocytosis of fungal pathogens depends on, and is modified by, the specific phagocyte subsets present at the site of infection.
IMPORTANCE Extensive work investigating fungal cell phagocytosis by macrophages and PMNs of the innate immune system has been carried out. These studies have been informative but have examined this phenomenon only when one phagocyte subset is present. The current study employed live-cell video microscopy to break down C. albicans phagocytosis into its component parts and examine the effect of a single phagocyte subset, versus a mixed phagocyte population, on these individual stages. Through this approach, we identified that the rate of fungal cell engulfment and rate of phagocyte killing altered significantly when both macrophages and PMNs were incubated in coculture with C. albicans compared to the rate of either phagocyte subset incubated alone with the fungus. This research highlights the significance of studying pathogen-host cell interactions with a combination of phagocytes in order to gain a greater understanding of the interactions that occur between cells of the host immune system in response to fungal invasion.
Candida species collectively represent the fourth leading cause of nosocomial bloodstream infections in the United States (1, 2). Within the Candida genus, Candida albicans is the major human fungal pathogen, accounting for approximately 50% of candidemia cases (3, 4). This fungus ordinarily colonizes mucosal surfaces of the skin and gastrointestinal and vulvovaginal tracts of healthy individuals. Around 75 million women suffer at least four episodes of Candida vaginitis each year, and AIDS-associated esophagitis is extremely common in untreated HIV-positive individuals (3). This opportunistic pathogen can also cause life-threatening systemic infections, with associated mortality rates of 30 to 60% in patients who have implanted medical devices, have been administered immunosuppressant drugs, or have been subjected to severe trauma (5).
An important first line of defense against Candida infections is provided by the innate immune system through the recruitment of professional phagocytes, such as polymorphonuclear cells (PMNs) and macrophages, to the site of infection (5–8). Studies to date have recognized the importance of macrophages and neutrophils in host defense against fungal pathogens and suggest a dominant role for the PMNs (9–11). This is reflected in the observation that neutropenic patients and patients with peroxidase deficiency are highly susceptible to invasive candidiasis (5, 12, 13). In particular, patients with chronic granulomatous disease, displaying impaired neutrophil function, are especially prone to systemic fungal infections (12–15). Additionally, in a mouse model where splenic macrophages were eliminated, mice demonstrated increased susceptibility to experimental disseminated candidiasis (16). Despite studies highlighting the clinical importance of phagocytes in anti-Candida infection, the process of fungal cell phagocytosis remains poorly understood at the mechanistic level.
The first step in the phagocytosis process involves macrophages and PMNs recognizing pathogen-associated molecular patterns (PAMPs) present in the fungal cell wall through pattern recognition receptors (PRRs) localized on the phagocytic cell membrane, in endosomes, and in the cytoplasm (17–19). Engagement of these receptors enables the phagocyte to directly engulf and destroy fungal cells within the phagolysosome by a number of oxidative and nonoxidative mechanisms, including the production of toxic reactive oxygen species (ROS) and reactive nitrogen species (RNS), expression of various antimicrobial peptides, and the activities of hydrolytic enzymes (5, 6, 17, 20–23). Furthermore, fungi and bacteria induce the formation of neutrophil extracellular traps (NETs) by activated PMNs, which can entrap and kill fungi and bacteria extracellularly (24). PAMP-PRR interactions also facilitate indirect killing of Candida by triggering the induction of proinflammatory cytokines and chemotactic factors which serve to activate other arms of the host immune system and aid in the clearance of Candida from the body (6, 17, 18).
Extensive work has been carried out identifying the PRRs and downstream signaling pathways that are involved in phagocyte recognition of fungal cells (6, 9, 23). These studies have identified the Toll-like receptors (TLRs) and C-type lectin receptors (CLRs) as the major PRRs involved in C. albicans PAMP recognition (6, 17, 25). Other studies have revealed how the overall phagocytic process in macrophages and PMNs is affected by fungal cell wall composition, morphogenesis, and species (5, 8, 26, 27). For example, Keppler-Ross et al. demonstrated that J774.1 macrophages preferentially phagocytosed Candida glabrata and Saccharomyces cerevisiae over C. albicans and that these macrophages displayed a strong preference for C. albicans yeast cells rather than hyphal cells (26). In addition, C. albicans mutants deficient in cell wall N- and O-linked glycans negatively affected the capacity of PMNs to phagocytose C. albicans (5). Such studies have been informative but have investigated phagocytosis under conditions where only one immune cell type and one fungal cell type were present.
By breaking down the phagocytic process into distinct stages (migration, recognition, engulfment, phagosome maturation, and killing), we have been able to determine the effect of specific factors, such as cell morphology and cell wall composition, on individual stages of the phagocytosis process (8, 28). In doing so, novel insights into the mechanisms that govern effective phagocytic clearance have been revealed. For example, dynamic imaging was used to demonstrate nonlytic expulsion of hyphal C. albicans cells from macrophages (29) and C. albicans-mediated inhibition of cell division in macrophages undergoing mitosis (30).
In this study, we assessed stage-specific C. albicans phagocytosis by human monocyte-derived macrophages and human PMNs in isolation with C. albicans and when both phagocyte subsets were present. Through this dual approach, we revealed marked differences in the behaviors of cells and in the outcomes of the interaction between PMNs and macrophages with C. albicans during phagocytosis. We show a lower overall capacity of PMNs to phagocytose fungal cells, an increased susceptibility of PMNs to C. albicans-mediated killing when phagocytes were incubated alone with C. albicans, and a marked increase in C. albicans-mediated macrophage killing when both types of phagocytes were cocultured with C. albicans.
Comparison of macrophage- and PMN-mediated C. albicans phagocytoses.Phagocytosis of C. albicans by macrophages and PMNs is critical for elimination of fungal infections from the body; however, the relative roles that they play in this process and the interactions that occur between these two cell types remain to be elucidated. We first quantified the dynamics of C. albicans phagocytosis by PMNs or macrophages in cell cultures using pure populations of only one phagocyte type. To do this, human monocyte-derived macrophages and human PMNs were challenged with live C. albicans CAI4-CIp10. Live-cell video microscopy using our standard phagocytosis assay (28) recorded the total number of C. albicans cells taken up by individual macrophages and PMNs over a 6-h period. Video S1 in the supplemental material shows the interactions between C. albicans and macrophages, and Videos S2 to S4 show the interactions between C. albicans and PMNs. An uptake event was defined as the complete engulfment of one C. albicans cell by either a macrophage or a PMN following cell-cell contact. Figure 1A to C and D to F show snapshots of the overall clearance of C. albicans cells throughout the 6 h of the assay by macrophages and PMNs, respectively. At a multiplicity of infection (MOI) of 1, there was no significant difference between the macrophages and PMNs in terms of the average numbers of C. albicans cells taken up (Table 1) or the percentages of cells taking up at least one fungal cell (65.0 ± 3.6 and 62.7 ± 10.4, means ± standard deviations [SD] [n = 300], for macrophages and PMNs, respectively) (Fig. 1G). However, when the MOI was increased to 3, the average C. albicans cell uptake by macrophages was significantly greater than for PMNs (Table 1) (P < 0.05). Furthermore, the percentage of macrophages taking up at least one fungal cell was significantly greater than for PMNs (85.0 ± 3.6 and 68.7 ± 4.0, respectively) (Fig. 1H). Thus, at low concentrations of C. albicans cells, both phagocytes were equally as effective in taking up fungal cells; however, when C. albicans yeast cells were in excess, macrophages took up a greater number of C. albicans cells than PMNs did.
Video S1
Macrophages incubated alone with C. albicans for 6 h (macrophages are stained red, and C. albicans cells are stained green). Download Video S1, WMV file, 3.4 MB.
Copyright © 2013 Rudkin et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 3.0 Unported license.
Neutrophils incubated alone with C. albicans for 6 h (neutrophils are stained red, and C. albicans cells are stained green). Download Video S2, MPG file, 9 MB.
Neutrophils incubated alone with C. albicans over 3 h taken with a spinning-disk microscope (neutrophils are stained red, and C. albicans cells are stained green). Download Video S3, WMV file, 4.2 MB.
Three-dimensional video demonstrating C. albicans engulfment by neutrophils using a spinning-disk microscope (neutrophils are stained red, and C. albicans cells are stained green). Download Video S4, WMV file, 18.5 MB.
Uptake of live C. albicans cells by macrophages and PMNs. (A to F) Snapshots from live-cell video microscopy experiments tracking the phagocytosis of live wild-type C. albicans cells by human monocyte-derived macrophages (A to C) and human PMNs (D to F) over 6 h. Phagocytes (red) with live wild-type C. albicans cells (green) at 0 min, 180 min, and the end of the 6 h. Bars, 30 µm. (G and H) Numbers of C. albicans cells ingested by macrophages and PMNs at the 1:1 (G) and 3:1 (H) C. albicans cell/phagocyte ratios. Bars represent percentages of macrophages or PMNs that ingested a defined number of C. albicans cells.
Uptake of live C. albicans cells by macrophages and PMNsa
Human PMNs engulf C. albicans more rapidly than macrophages do.Uptake of target yeast cells is influenced by the migration of phagocytes toward the site at which fungal cells are located and the time taken from the establishment of cell-cell contact to complete uptake of C. albicans. As in our previous studies, we define the latter as the rate of engulfment (28). We examined whether there were differences in the rates of C. albicans engulfment between macrophages and PMNs by live-cell video microscopy and image analysis (Fig. 2A to C and D to F for macrophages and PMNs, respectively). First, at MOIs of both 1 and 3, PMNs engulfed C. albicans cells at a significantly higher rate than macrophages (Table 2; Fig. 2G and H). Videos S1 to S3 in the supplemental material highlight this observed difference in rates of engulfment between macrophages and PMNs. Furthermore, at both MOIs, approximately 12 to 15% of C. albicans cells bound to a macrophage took longer than 30 min to be internalized, compared with 5% of cells bound to PMNs (Fig. 2G and H). Interestingly, when macrophage activities at the different MOIs were compared, it was observed that macrophages engulfed C. albicans cells at a markedly higher rate at a MOI of 3 than at an MOI of 1 (Table 2). This accelerated uptake of yeast cells did not occur for PMNs at higher MOIs (Table 2).
Macrophage and PMN engulfment of live C. albicans cells. Shown are snapshots taken from live-cell video microscopy capturing the various stages of C. albicans engulfment by macrophages (A to C) and PMNs (D to F). (A and D) Phagocyte (red, ☆) and C. albicans cell (green) prior to cell-cell contact; (B and E) cells once cell-cell contact had been established; (C and F) C. albicans within the phagocyte following ingestion; (G and H) times taken for macrophages and PMNs to ingest live wild-type C. albicans cells at the 1:1 (G) and 3:1 (H) C. albicans cell/phagocyte ratios. The rate of engulfment was defined as the time taken from initial cell-cell contact to complete ingestion of C. albicans cells by the phagocyte. Bars represent the percentages of uptake events (n = 3).
Rate of engulfment of live C. albicans cells by macrophages and PMNsa
In summary, PMNs engulfed C. albicans at a significantly higher rate than macrophages did at both a low and a high MOI, and the rate of macrophage engulfment was affected by the concentration of fungal cells.
PMNs are more susceptible than macrophages to killing by C. albicans.It has been shown previously that C. albicans can form hyphae inside phagocytes, often resulting in phagocyte rupture and death (8, 31, 32). We examined this phenomenon in more detail to determine whether there were differences between the different phagocytic cell types in this respect. It was observed that at both low and high MOIs, a significantly greater number of PMNs than macrophages were killed by C. albicans during the course of the phagocytosis assay (Fig. 3A and B). As expected, at the higher MOI, significantly more macrophages were killed than at the lower MOI (Fig. 3C). In contrast, rates of PMN killing were similar at both MOIs (Fig. 3D). The percentages of macrophages and PMNs killed increased with the number of fungal cells being taken up; however, it was observed that at both MOIs, when macrophages and PMNs had taken up the same number of C. albicans cells, a significantly greater proportion of PMNs were killed (Fig. 4A and B). This may reflect the smaller average size of PMNs (8.3 µm ± 1.1 µm) than macrophages (21.2 µm ± 3.5 µm). Furthermore, the data revealed that >90% of PMNs that took up 3 or more fungal cells were killed, whereas a high proportion (~40%) of macrophages survived even after ingesting up to 7 fungal cells. Thus, in these assays, PMNs were more susceptible to C. albicans-mediated killing than macrophages, irrespective of the number of C. albicans cells ingested.
Killing of macrophages and PMNs by live C. albicans cells over a 6-h period. (A and B) Percentages of macrophages and PMNs killed by C. albicans at the 1:1 (A) and 3:1 (B) C. albicans cell/phagocyte ratios; (C and D) comparisons of C. albicans-mediated phagocyte killings at MOIs for macrophages (C) and PMNs (D). Values are means ± SD (n = 3). *, P < 0.05; **, P < 0.01.
C. albicans-mediated phagocyte killing in relation to the number of C. albicans cells taken up. (A and B) Percentages of phagocytes killed after taking up a defined number of C. albicans cells at the 1:1 (A) and 3:1 (B) C. albicans cell/phagocyte ratios. Bars represent mean percentages of phagocytes killed ± SD (n = 3). *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Phagocytosis and killing dynamics in mixed-phagocyte cocultures.For coculture assays, an MOI of 3 C. albicans cells for all phagocytes (macrophages plus PMNs, with macrophages and PMNs at a 1:1 ratio) was used. C. albicans uptake and engulfment by macrophages and PMNs was quantified, and C. albicans-mediated phagocyte killing was measured (see Video S5 in the supplemental material).
Macrophages and neutrophils incubated together with C. albicans in the coculture assay for 6 h (macrophages are stained blue, neutrophils are stained red, and C. albicans cells are stained green). Download Video S5, WMV file, 2.6 MB.
When phagocytes were cultivated with an excess of C. albicans cells, macrophages took up more fungal cells than PMNs did. We examined whether the same was true when macrophages and PMNs were present in coculture. Interestingly, no significant difference between macrophages and PMNs was observed in C. albicans uptake rates (Table 1 and Fig. 5A to C). Additionally, approximately 22% of PMNs did not take up any C. albicans cells, compared with 33% of macrophages (Fig. 5D). Thus, when both cell types were present, macrophages and PMNs contributed approximately equally to clearance of C. albicans.
Uptake of live C. albicans cells in combined macrophage-PMN cultures. (A to C) Images taken from live-cell video microscopy experiments tracking the phagocytosis of live C. albicans cells by macrophages and PMNs in the combined assay over 6 h. Shown are macrophages (blue) and PMNs (red) with live C. albicans cells (green) at 0 min, 180 min, and the end of the 6 h. Bar, 30 µm. (D) Numbers of C. albicans cells ingested by macrophages and PMNs in the combined assay. Experiments were carried out an MOI of 3. Bars represent percentages of macrophages or PMNs that ingested a defined number of C. albicans cells (n = 3).
Human PMNs engulf C. albicans more rapidly than macrophages do in mixed-phagocyte cocultures.When both phagocyte subsets were present, PMNs engulfed C. albicans at a significantly higher rate than macrophages (Table 2 and see Video S5 in the supplemental material). Furthermore, 38% of C. albicans cells took longer than 30 min to be engulfed by macrophages, whereas only 1% of fungal cells took longer than 30 min to be ingested by PMNs (Fig. 6). The average rate of C. albicans engulfment by PMNs was similar to that observed when the PMNs were studied in isolation (Table 2 and Fig. 2H and 6). In contrast, macrophage engulfment of C. albicans was significantly delayed in the coculture assay, with macrophages taking an average of 14.4 ± 0.6 min to engulf fungal cells, compared to 8.1 ± 0.4 min in the absence of PMNs (Table 2 and Fig. 2H and 6). Thus, the rate of C. albicans engulfment by macrophages decreased when PMNs were present, whereas for the PMNs, the rate of engulfment was unaffected by the presence of macrophages and was significantly higher both in the presence and in the absence of macrophages.
Macrophage and PMN engulfment of live C. albicans cells in combined cultures. Shown are the times taken for macrophages and PMNs to ingest live C. albicans cells in the coculture assay. Rate of engulfment was defined as the time taken from initial cell-cell contact to complete ingestion of C. albicans by the phagocyte. Experiments were carried out at a MOI of 3. Macrophages and PMNS were present at a 1:1 ratio. Bars represent the percentages of uptake events (n = 3).
C. albicans-mediated macrophage killing increases in mixed-phagocyte cocultures.In the coculture assay, C. albicans-mediated macrophage killing was significantly increased from the level observed when macrophages were cultivated in isolation with C. albicans (Fig. 7A). Indeed, there was no difference in rates of C. albicans-mediated killing of macrophages and PMNs when both cell types were present (Fig. 7B). This contrasts with the observation that PMNs were significantly more susceptible to killing by fungal cells when PMNs and macrophages were cultured in isolation (Fig. 3B). Additionally, the percentage of macrophages killed after taking up 2 or more fungal cells in the coculture assay was >80%, compared to approximately 25% when macrophages were incubated alone with C. albicans (Fig. 4B and 7C).
Killing of macrophages and PMNs by live C. albicans cells in the combined assay over 6 h. (A) Comparison of C. albicans-mediated killing of macrophages in the combined assay and that in isolation; (B) percentages of macrophages and PMNs killed by C. albicans in the combined culture assay; (C) percentages of phagocytes killed after taking up a defined number of C. albicans cells in the combined assay. Experiments were carried out at a 3:1 ratio of C. albicans cell to total phagocytes. Values/bars represent means ± SD (n = 3). *, P < 0.05; **, P < 0.01.
PMNs migrate more rapidly toward C. albicans cells.To investigate this increase in C. albicans-mediated macrophage killing, we analyzed the migration of macrophages and PMNs toward fungal cells. C. albicans cells formed germ tubes rapidly under the conditions of the experiment; therefore, the time taken for a phagocyte to locate and migrate toward a fungal cell influenced whether it was taken up early in the yeast form or later on as a germ tube. It is reasonable to speculate that this may ultimately affect the susceptibility of the phagocyte to C. albicans-mediated killing. Tracking diagrams (Fig. 8A and B) illustrate the distances traveled and the directionality and velocity of PMNs and macrophages in the coculture assay in the presence of wild-type C. albicans. In these diagrams, symbols indicate the locations of macrophages or PMNs at 1-min intervals, and arrows represent directionality. Quantitative analysis of the average track revealed a 20-fold-higher average track velocity of PMNs than of macrophages toward C. albicans cells (0.007 ± 0.004 µm/s [n = 3] for macrophages versus 0.13 ± 0.07 µm/s [n = 3] for PMNs) (see Video S5 in the supplemental material). Thus, in the combined phagocyte assays, PMNs migrated at a greater velocity than macrophages toward C. albicans.
Phagocyte migration toward C. albicans. Shown are tracking diagrams illustrating the distances traveled, directionality, and velocity of macrophages (A, blue) and PMNs (B, red) in pursuit of live C. albicans cells in the combined assay. Tracks represent the movement of individual phagocytes relative to their starting position, symbols indicate the locations of phagocytes at 1-min intervals, and arrows represent directionality.
This faster migration of PMNs toward fungal cells resulted in earlier clearance of yeast cells by PMNs, leaving the remaining Candida cells time to germinate before being engulfed by macrophages as hyphae. Indeed, in the coculture assays, macrophages took up a significantly greater proportion of hyphal cells (relative to yeast cells) in the coculture assay than they did when in isolation (72.4% ± 9.6% and 59.3% ± 2.7% [means ± SD, n = 300] of hyphal cells were taken up by macrophages in coculture and isolation assays, respectively [data not shown]), and this coincided with increased C. albicans-mediated macrophage killing. These data are consistent with previous reports which indicate that uptake of hyphal cells enhances phagocyte killing (8). In addition, this increase in hyphal uptake was accompanied by a lower rate of engulfment than when macrophages were incubated alone with C. albicans (Table 2). Furthermore, in some uptake events, we observed continued extension of C. albicans hyphae during the engulfment process (see Video S6 in the supplemental material). These are all factors which are likely to have contributed to enhanced macrophage killing. Thus, in the presence of PMNs, macrophages were more susceptible to killing by the fungus.
C. albicans hyphal extension during uptake by a macrophage (the macrophage is stained blue, and the C. albicans cell is stained green). Download Video S6, WMV file, 3.2 MB.
C. albicans is a major life-threatening human fungal pathogen that causes infections associated with high morbidity and high mortality (2). Professional phagocytes, such as macrophages and PMNs, provide an important line of defense against invasive candidiasis by directly engulfing and destroying fungal cells. We conducted a detailed comparison of stage-specific phagocytosis of C. albicans by PMNs and that by macrophages in isolation and when both phagocyte subsets were present and competing for fungal cells. We observed profound differences in (i) migration, (ii) rate of engulfment, (iii) overall uptake, and (iv) phagocyte survival when C. albicans yeast cells interacted with mixtures of macrophages and PMNs rather than individual phagocyte subsets. Nonprofessional phagocytes, such as dendritic and endothelial cells, also contributed to the overall clearance of C. albicans in vivo and will be the subject of future studies.
PMNs are considered the primary mediators of phagocytic clearance (9–11, 31, 33). Indeed, neutropenia is a major predisposing factor in clinical studies for developing invasive candidiasis (21, 22). Furthermore, recent in vitro studies suggest that the onset of hyphal formation and subsequent escape of fungal cells is inhibited inside human neutrophils (33). This may suggest that PMNs not only mediate the majority of C. albicans uptake but also are less susceptible than macrophages to killing by C. albicans following phagocytosis. Surprisingly, we observed that when only one immune cell type was present, macrophages, not PMNs, had the greater capacity for C. albicans uptake despite engulfing fungal cells at a lower rate after establishing cell-cell contact. However, when both cell types were present, macrophages and PMNs contributed approximately equally to clearance of C. albicans. Our data illustrate that uptake rates are affected not only by the fungal/phagocyte ratio but also by the proportion of the phagocyte subsets encountered. This suggests that the overall phagocytic capacity in single phagocyte assays is not determined simply by the number PMNs and macrophages but is rather a consequence of the percentage of phagocytes contributing to uptake. In vivo, the contribution of phagocyte subsets to overall uptake would also be influenced further by factors such as chemokine and cytokine concentrations and spatial considerations such as location of the fungal infiltrate, the numbers of phagocytes, and accessibility to the infection sites. Interestingly, observations from a larval zebrafish model of disseminated candidiasis where phagocytosis of Candida cells was imaged in vivo showed that macrophage-like cells took up a significantly greater number of C. albicans yeast cells than neutrophils did (17).
A number of studies have previously established that C. albicans can form filaments inside macrophages and that hyphal formation is essential for C. albicans escape and phagocyte killing (8, 30–32). In this study, we examined this phenomenon further and observed that PMNs were significantly more susceptible than macrophages to C. albicans-mediated killing when incubated in isolation with C. albicans. Indeed, in vitro, nearly 50% of macrophages that took up 5 or more fungal cells survived, whereas 100% of PMNs that took up 3 or more fungal cells were killed following continued growth of hyphae inside the host cell. However, when both macrophages and PMNs were studied in coculture, macrophage survival following C. albicans phagocytosis was significantly impaired, and the rate of C. albicans-mediated killing of macrophages increased to the same level as that of PMNs incubated alone with C. albicans.
We showed here that PMNs not only migrated more rapidly than macrophages toward C. albicans cells but also engulfed C. albicans cells at a significantly higher rate once cell-cell contact was established. The differential activation and expression of PRRs displayed on macrophages and PMNs may contribute to the observed differences in rates of engulfment seen in these myeloid cells in the present study. This is in keeping with the results of a number of studies that have established that immune cell migration and fungal cell recognition is influenced by the relative levels of expression and specificities of PRRs displayed on the immune cell surface (34,35). The observations also mirror findings from our previous work showing that the glycosylation status of the C. albicans cell wall and therefore repertoire of PAMPs affects the migration, engulfment, and killing of neutrophils (5) and macrophages (28).
Taken together, the data from the current study suggest that when both macrophages and PMNs are presented to C. albicans in a coculture, PMNs mediate the majority of early C. albicans yeast cell uptake events due to their ability to migrate more rapidly toward fungal cells and then engulf bound fungal cells at a higher rate than macrophages do. As a consequence, macrophages may encounter a higher proportion of hyphal cells, which they engulf at a lower rate. Both uptake of a higher proportion of hyphal C. albicans cells and increased hyphal length at the time of uptake is reportedly associated with host cell death (8, 28, 31, 32), which may explain the increase in macrophage death seen here in the coculture assay compared to that when macrophages were incubated alone with C. albicans.
This study highlights the critical role that macrophages and PMNs play in C. albicans phagocytosis and demonstrates that major kinetic parameters related to the rate and efficiency of phagocytosis and killing are multifactorial, depending on the presence of multiple cell types and a temporal program of events that ultimately defines the outcome of the predation of fungal cells by different phagocyte cell types. The implications of this study are that extrapolations of complex interactions between pathogen cells and the myeloid cells involved in innate immunity have to be made in the knowledge that cellular interactions are significantly affected by the proximity of other cell types and dynamic changes in the predator/prey cell type ratio.
C. albicans strains and growth conditions.C. albicans serotype A strain CAI4 + CIp10 was used for all experiments and was the progenitor of the mutant strains used in our previous studies (28). This strain was obtained from glycerol stocks stored at −80°C and plated on synthetic complete medium lacking uracil (SC−Ura). SC−Ura plates consisted of 6.9 g yeast nitrogen base without amino acids (Formedium, Norfolk, United Kingdom), 1 ml 1 M NaOH (BDH Chemicals, VWR International, Leicestershire, United Kingdom), 10 ml 1% (wt/vol) adenine hemisulfate salt (Sigma, Dorset, United Kingdom), 50 ml 40% d-glucose (Fisher Scientific, Leicestershire, United Kingdom), 50 ml 4% SC−Ura dropout (Formedium, Norfolk, United Kingdom), and 2% (wt/vol) technical agar (Oxoid, Cambridge, United Kingdom) made up to 1,000 ml in distilled H2O.
Preparation of human monocyte-derived macrophages.Human monocyte-derived macrophages were isolated from the blood of healthy volunteers. Peripheral venous blood was collected in EDTA-coated tubes, pooled, and diluted 1:3 in Hanks' balanced salt solution (HBSS). Lymphoprep (Axis-Shield, Norway) kits were used to separate peripheral blood mononuclear cells (PBMCs) from whole blood. The PBMC layer was washed and resuspended in DMEM (Lonza, Slough, United Kingdom) supplemented with 200 U/ml penicillin-streptomycin antibiotics (Invitrogen, Paisley, United Kingdom) and 2 mM l-glutamine (Invitrogen, Paisley, United Kingdom). Serum was separated from blood using standard methods and heat inactivated at 56°C for 20 min before use. PBMCs were seeded at 10 × 106 in 2 ml supplemented DMEM containing 10% autologous human serum onto glass-based imaging dishes (PAA Imaging gas chromatography [GC] dishes; The Cell Culture Company, GE Healthcare, France) and incubated at 37°C with 5% CO2 for 2 h to allow for monocyte adherence to the dish. After 2 h, the supernatant containing the floating lymphocytes was removed and replaced with 2 ml warm supplemented DMEM containing 10% autologous human serum. Dishes were incubated at 37°C with 5% CO2, and medium was changed on days 3 and 6. Cells were used in imaging experiments on day 7. Immediately prior to phagocytosis experiments, supplemented DMEM was replaced with prewarmed supplemented serum-free CO2-independent medium (Gibco, Invitrogen, Paisley, United Kingdom) containing 1 µM LysoTracker red DND-99 (Invitrogen, Paisley, United Kingdom) when macrophages were the only immune cell type present or 1 µM LysoTracker blue DND-22 (Invitrogen, Paisley, United Kingdom) when both macrophages and PMNs were present to distinguish between the two immune cell types. LysoTracker red and blue DND are fluorescent dyes that stain acidic compartments in live cells, enabling tracking of these cells during phagocytosis and phagolysosome maturation.
Preparation of human PMNs.Human PMNs were isolated from the blood of healthy volunteers and used in phagocytosis assays within 3 h of isolation. Peripheral venous blood was collected in EDTA-coated tubes, pooled, and diluted 1:2 in sterile PBS. Histopaque-1119 (Sigma, Dorset, United Kingdom) was added to a conical tube and carefully overlaid with the same volume of Histopaque-1077 (Sigma, Dorset, United Kingdom) to create a double-density gradient. An equal volume of diluted blood was carefully layered on top of the upper Histopaque gradient, and tubes were centrifuged at 700 × g for 40 min at room temperature to allow for separation of PBMCs and PMNs from erythrocytes. Serum and PBMC layers were aspirated and discarded, and the PMN layer was washed once in PBS. For hypotonic lysis of erythrocytes carried over into the PMN fraction, 3 ml of 0.2% NaCl solution was added to cells and mixed gently. After 30 to 45 s, 3 ml of 1.6% NaCl solution was added, mixed gently, and then diluted to 50 ml in PBS before centrifugation at 350 × g for 10 min at room temperature. Cells were then washed in PBS and resuspended in prewarmed supplemented serum-free CO2-independent medium containing 1 µM LysoTracker red DND‑99 (Invitrogen, Paisley, United Kingdom) and used immediately in phagocytosis assays.
Preparation of FITC-stained C. albicans.C. albicans colonies were grown in SC−Ura medium and incubated overnight at 30°C with 200-rpm shaking. Live C. albicans cells were stained for 10 min at room temperature in the dark with 1 mg/ml fluorescein isothiocyanate (FITC) (Sigma, Dorset, United Kingdom) in 0.05 M carbonate-bicarbonate buffer (pH 9.6) (BDH Chemicals, VWR International, Leicestershire, United Kingdom). Following incubation, cells were washed three times in 1× PBS to remove any residual FITC and finally resuspended in 1× PBS.
Live-cell video microscopy phagocytosis assays.Phagocytosis assays were performed using our standard protocol, with modifications (8, 28). Samples of live FITC-stained wild-type C. albicans (CA14-CIp10) cells were added to 1 × 106 LysoTracker red DND-99-stained human monocyte-derived macrophages or 1 × 106 LysoTracker red DND-99-stained human PMNs in glass-based imaging dishes at a multiplicity of infection (MOI) of either 1 or 3. For phagocytosis assays where both macrophages and PMNs were present at a 1:1 ratio, live, FITC-stained wild-type C. albicans (CA14-CIp10) cells were added to glass-based imaging dishes containing 5 × 105 macrophages stained with 1 µM LysoTracker blue DND-22, and 5 × 105 PMNs stained with 1 µM LysoTracker red DND-99 at an MOI of 3 (ratio of C. albicans cells to total phagocytes, 3:1). In the coculture experiments, macrophages and PMNs were not always isolated from the same donors. Video microscopy was performed using a DeltaVision Core microscope (Applied Precision, WA, USA) in a 37°C chamber, and images were captured at 1-min intervals over a 6-h period using an EMCCD camera. At least three independent experiments were performed for both macrophages and PMNs incubated alone with C. albicans, and at least 3 videos were analyzed from each experiment using softWoRx Explorer image analysis software (Applied Precision). The same setup was used for the coculture assays where both macrophages and PMNs were present. One hundred phagocytes were selected at random from each experiment and analyzed individually at 1-min intervals over a 6-h period. Measurements taken included C. albicans uptake, defined as the number of C. albicans cells taken up by an individual phagocyte over the 6-h period, the time point at which cell-cell contact was established, and the time point at which a C. albicans cell was fully engulfed; these measurements were used to determine the rate of C. albicans engulfment, defined as the time taken from establishment of cell-cell contact to complete ingestion of a C. albicans cell (a fungal cell was considered to have been fully ingested when its FITC fluorescent signal was lost, indicating that the fungal cell was now inside the phagocyte and not merely bound to the phagocyte cell surface). The viability of the phagocytes over the 6-h period was defined as the percentage of macrophages or PMNs from the total macrophage or PMN population, respectively, that had been killed by specific time points. In the coculture assays, macrophages and PMNs were also analyzed separately in this manner, so the percentage of macrophages killed by certain time points in the coculture assays represents the percentage of macrophages out of the total macrophage population sampled that had been killed. Velocity 6.2 imaging analysis software was used to track phagocyte migration at 1-min intervals throughout the 6-h phagocytosis assay. The software enabled high-throughput analysis of phagocyte migration, providing detailed information on the distances traveled, directionality, and velocity of hundreds of individual phagocytes. Data were subsequently displayed in tracking diagrams and used to calculate the mean track velocity and track lengths of phagocytes cultured with C. albicans (28).
Means values and standard deviations were calculated. Unpaired, two-tailed t tests and two-way analysis of variance (ANOVA) followed by Bonferroni multiple-comparison tests were used to determine statistical significance.
F.M.R. was supported by a SULSA BioSkape studentship. L.P.E. and N.A.R.G. acknowledge funding by the Wellcome Trust (grants 080088, 086827, 075470, and 099215) and by an FP7-2007-2013 grant agreement (HEALTH-F2-2010-260338-ALLFUN).
Accepted 30 September 2013
Published 29 October 2013
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mBio Oct 2013, 4 (6) e00810-13; DOI: 10.1128/mBio.00810-13
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COSA TI PIACE DI PIU?
-means "What do you like the most" Di piu-The most (Di-used as a definite article).
-means "It's a thing/something that.." Una cosa (a thing/something).
-means "There was less" C'era (ci era) means "There was".
-"I did my school here" Ho fatto (used when you made/finished something).
-Capito means "Understood" (also used interrogatively) e.g. "Understood?../Got it?..".
-an Idiom meaning "Few choices/shortage of cash or something".
-means "Frequently/always end up"(turn out to be in a particular place or situation).
E BELLO CHE ENTRI E..
-means "It's nice that you walk/join in..".
-means "The art of getting by".
-means "There is no excuse to keep" same as "No Excuses" in English.
-means "We'll manage to get by".
-Italian way of saying "I'm leaving now/ I'm outta (out of) here".
-means "If one makes it".
-(Expression of acceptance) same as "Then so be it" in English.


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About Tilburg University
Organization Tilburg University
Tilburg University operates in the area of the humanities and social sciences. More than 900 scientists work at one of our five Schools, focusing on economics, business and entrepreneurship, social and behavioral sciences, law and public administration, the humanities and digital sciences, and theology.
More than 600 employees in the seven Divisions of University Services support the Schools.
The Executive Board is the university's managing committee. It is in charge of the university's administrative affairs and is responsible for efficient management. The Executive Board is accountable to the Board of Governors.
Dr. K.M. (Koen) Becking
President of the Executive Board
'The social context is becoming increasingly important for the university, also for our funding. As a result, we must increasingly often ask ourselves how we can make a difference for that social context. Without selling our souls to the business sector, of course, because independence is crucial in science.'
Read the vision and profile of Koen Becking
Rector Magnificus
Paulina Snijders
'I feel at home in the academic world and feel inspired by the enthusiasm of researchers and teachers and by the interaction with students who want to get the most out of their program. Business operations is a business that is close to my heart. I want to contribute to further developing the support of the primary process.'
Read the vision and profile of Paulina Snijders
Read the speeches by members of the Executive Board
Tilburg University is a so-called 'special university'. Unlike the state universities, Tilburg University comes under a private-law foundation. The Board of Governors functions as a supervisory council and monitors the objectives of the foundation, including its open Catholic identity.
The Board of Governors
Mr. H. Reumkens, LLM (chairman)
Mr. W.I.I. van Beek
Professor W.M.P. van der Aalst
Professor E. van Sliedregt
Mr. J.H.P.M. van Lange
More information can be obtained from the official secretary to the Board of Governors: Mr. Dr. Martijn Nolen LLM, tel. 013 - 466 2248 or the secretariat of the Board of Governors Ms. Anja de Bie, tel. 013 - 466 2327.
Schools and institutes
Five Schools form part of Tilburg University.
Tilburg School of Economics & Management
Tilburg Law School
Tilburg School of Social and Behavioral Sciences
Tilburg School of Humanities and Digital Sciences
Tilburg School of Theology
Tilburg University research is conducted within the Schools' graduate schools or within one of its many research institutes. Each institute specializes in one or more areas of expertise; there are School-based and multidisciplinary institutes. In the latter type, specialists from various disciplines work together. Always with a view to innovative science.
Visit the institutes in the overview
Support and staff
University Services consists of seven Divisions and provides support for research, education, and valorization.
Honorary Doctorates
Exceptional scientific accomplishments can be rewarded with a honorary degree. This title can also be bestowed upon a person who rendered exceptional community services. The Honorary Doctorates are awarded at anniversaries and at special occasions during a ceremony on the Dies Natalis at Tilburg University.
See the Honorary Doctorates
Participative decision-making
Students and employees contribute to thinking, discussing, and deciding on Tilburg University's policies.
Read more about participation
Find someone at Tilburg University
Open day and other study orientation activities
Working at Tilburg University
Get your PhD
Contact Tilburg University
Do you have a question about studying in Tilburg?
Are you a student at Tilburg University?
Do you have a question for our press officers?
Do you have a question about collaborating with Tilburg University?
Are you looking for a specific expert or expertise?
Do you have another type of question?

redpajama_set_name: RedPajamaCommonCrawl


Although it is often said that breakfast is the most important meal of the day, we cannot consider other meals as less important. In today's busy world, we often forget to eat regularly, and we cannot find the time for healthy dinner and lunch, so we grab the first thing at hand. Tasty and healthy main meals can be however quick and easy to prepare, and you do not even have to be an experienced cook. Just have a look at my fit recipes for dinners; and moreover, you will surely appreciate easy lunch recipes as well.
When we say healthy lunch, many people automatically think of a boring salad. Vegetables are inseparable part of diet recipes, but a good meal should be slightly more varied. Satisfying legume salads, vegetable meals, pasta, meat meals or recipes with quinoa or tofu are also a great choice. Additionally, you should not forget to prepare a nutritionally valuable dinner; otherwise, a hunger can get you in the middle of the night and your healthy-lifestyle dream will disappear. Healthy dinner should be richer on proteins rather than carbohydrates. Do not hesitate to try out my fish or chicken healthy dinner recipes.
I am sure you will love my healthy lunch and dinner recipes.


Razer's 3-screen laptop is a sight to behold—but will it be worth it?
It boasts 51 inches of display and around 25 million pixels.
You've probably seen gamers playing on three monitors simultaneously, but what about on a laptop with three displays?
Razer's Project Valerie, announced at CES, is a gaming PC with three 17-inch 4K displays. Yes, a single personal computer with three full-sized screens.
How does Razer pull off the magic? By doing what you'd probably have guessed: creating a really thick and heavy laptop with two foldable displays. The two complementary displays retract into slots behind the main display, and slide out when deployed, automatically positioning and aligning themselves with the main screen. This is all achieved with a pair of mechanical hinges.
All three 17.3-inch displays feature 4K resolution, and come with the same gaming-optimized technology as the company's flagship Blade Pro. In fact, the radical PC is similar to the Pro in a number of ways, also equipped with Nvidia's beastly GTX 1080 graphics, and Razer's in-house mechanical keyboard with Chroma lighting.
The laptop weighs 10 to 12 pounds and is 1.5 inches thick, figures we haven't seen in years.
This is surprisingly not the first laptop with more than one display. The folks at PCWorld dug into their archives and rediscovered the 2011 Lenovo ThinkPad W700ds, a gaming PC with a 17-inch screen and an auxiliary 10.6-inch display.
Razer has not provided battery life estimates for the Valerie, though it is hard to even imagine it running without being plugged in. The company has also not detailed pricing, but given the single-display Blade Pro starts at well over $3,000… Well, just convince yourself you're pulling out money for a car.


I've used the 'Caviar' technique before, but using the beads from a local craft store. This is the first Accessorize beauty product I'd used, and was pretty excited. The beads came in a little container with a pointed applicator which was perfect for applying the beads, it's pretty much identical to the MUA packaging. The nail varnish 'Lilac Sorbet' wasn't bad quality. It only took 2 coats to achieve a good colour, but I felt I was waiting a while for it to dry - I'm not the most patient of people! You can get these sets for £5 from Superdrug - And I do believe they're on a special offer at £3, which is a bargain for a nail varnish AND some nail art beads.
To start using this set, I first took away the front of the plastic packaging as I thought it was ideal for catching the left over beads. I simply painted my nail with the two coats it recommended, put my nail over the tray and then shook the beads all over the nail until it was fully covered. It took about 10 minutes to fully dry.
It was so easy putting the remains back in the pot too, the tray was a perfect size to tip all the beads back in. After I'd done that I then found a clear nail varnish and just went slightly over the tips of the beaded nail, just to secure the beds in case the end of the nail got caught. This is such and easy thing to do, and makes your nails look amazing in minutes. In the past, 'Caviar Nails' only lasted me up to 2-3 days before the beads started to fall off, so it's not the most sustainable nail look. Overall this look took my about 15 minutes, including drying time, so if you have time to spare, this is such a fun look to do. I loved the finished look. It was very girly and somehow reminded me of sweets! I love this innocent look, and it really catches the eye. I can't wait to wear these out and show my claws off! There are many bead nail kits out there, and I really enjoyed using this. At the moment they are £3, so it's hard not to say no. Let me know in the comments what you think of this trend, and if you've ever tried it. Thank you so much for reading!
Ooh I'm incredibly tempted to try this myself. I've been trying to get back into nail art but I'm always rushed for time, but this seems very time efficient and effective!
I love the combination of the pink polish with the beads! It looks like a cupcake! :D I like the caviar trend but I'm not sure that I could wear it because I am such a texture person that I would end up just rubbing my fingers on my nail all day! I'm doing that with my glitter polish from my last nail of the day post! Great post!
I've got the MUA ones and I love them, I think they look really effective for an easy nail art!
Ah you're the exception then! :P lucky! I also have an MUA one, can't wait to try it out!
Using the plastic tray to catch the beads is simply genius! I tried this look but it didn't even last 1 day due to my job. It's a very pretty look for a night out though.
I love accessorize stuff I think I am going to have to try this!


text:
East Belfast
East Belfast 'stabbing' leaves man in hospital
A male has been arrested
Nathan Hanna
PSNI (Image: Stock)
A man has been hospitalised after an alleged stabbing incident in East Belfast in the early hours of this morning.
Just before 1.30am on Sunday police received a report that a 33-year-old male had been stabbed in Global Crescent.
He was taken to hospital for treatment to his shoulder.
A PSNI spokesman said: "Police have arrested a man [aged 30] on suspicion of grievous bodily harm with intent and possession of offensive weapon with intent to commit an indictable offence. He remains in police custody at this time.
Abbey Park Belfast incident sees masked gang attack man with hammers
"Detective Sergeant Gardiner is appealing for anyone who was in the vicinity of Global Crescent early this morning and who witnessed the alleged incident to contact detectives in Musgrave on 101 quoting reference 129 28/10/18.
"Alternatively, information can also be provided to the independent charity Crimestoppers on 0800 555 111, which is 100% anonymous and gives people the power to speak up and stop crime."
UPDATE: The 30-year-old man has been released on bail pending further enquiries.


text:
Oregon Prioritizes Student Health and Wellness in School Report Cards
What is chronic absenteeism and why is it important?
Report cards aren't just for students — they can also be an important tool for sharing eye-opening information about your school overall. Under No Child Left Behind, in order to receive key funding for schools, states must prepare and publish annual report cards for each school that detail performance, promote transparency and accountability and empower both parents and educators to hold schools to higher standards.
While the vast majority of these report cards focus on academic indicators, such as students' performance on standardized tests, a handful of states and school districts (including Illinois and Colorado) have publicly recognized the connection between health and learning by including health and wellness indicators in their school report cards. Including health and wellness measures on school report cards can help school principals, teachers, staff and parents understand the important connection between health and learning and support healthier school environments.
Today, we have the opportunity to highlight a recent successful effort that took place in Oregon to include a health and wellness indicator in the state's school report card and achievement compacts (a district-level component of Oregon's educational accountability model).
In 2012, the Healthy Kids Learn Better Coalition lobbied hard to convince the Oregon Education Investment Board (OEIB) to include a health and wellness indicator in the district-level reports, called "achievement compacts." Staff at the OEIB and in the Governor's office told the coalition that they only had one chance to include a health and wellness indicator in the achievement compacts and that the indicator must reflect information schools were already collecting and be something that educators would understand.
The group chose chronic absenteeism. Not only did the OEIB include chronic absenteeism in the achievement compacts, but the state Department of Education decided to include it in its model state school report card, as well. Now schools and districts across the state are reporting the percentage of students that are chronically absent.
So what is chronic absenteeism and why is it important?
A student is chronically absent when they miss 10 percent or more of the school year (equivalent to 18 days out of a 180-day school year) regardless of whether absences are excused or unexcused. Chronic absence in the first years of school is associated with
lower academic performance in subsequent grades. For example, according to the National Center for Children in Poverty, chronic absence in kindergarten is associated with lower academic performance in first grade, especially for Latino children. In addition, low-income children who were chronic absentees in kindergarten had the lowest performance in reading and math in fifth grade.
In Oregon, chronic absenteeism is a very real and big problem. According to the Oregonian (the major daily newspaper in Portland, Oregon), last school year, about 100,000 Oregon students missed at least 3½ weeks of school. This means that nearly one in five Oregon students missed at least 10 percent of the school year and were considered chronically absent.
The inclusion of chronic absenteeism on the achievement compacts and new Oregon school report card is an important step towards changing that. While Oregon's new report cards do not include why students are absent, we do know that asthma is the leading cause of absenteeism in Oregon and dental pain is the second biggest cause. Members of the Healthy Kids Learn Better Coalition hope that closely tracking absenteeism rates will motivate school districts to look closer at students' underlying health needs.
"Attendance reflects a lot of health-related barriers to learning," said Kasandra Griffin, Food and School Health Policy Manager at Upstream Public Health and now Chair of the Healthy Kids Learn Better Coalition. "There are obviously other reasons why kids miss school… but poor physical, emotional and/or social health are probably the leading causes of absenteeism."
The inclusion of chronic absenteeism on the achievement compacts and report cards is already generating attention. "Including chronic absenteeism in these accountability models is increasing awareness of chronic absenteeism as an issue that educators should watch," said Griffin. "Many educators were unaware their school had a problem with chronic absenteeism and are now beginning to ask what they can to do address it."
Next up, Griffin and her team hope to develop a better understanding of why students in Oregon are chronically absent and how to address the problem. The goal would be to eventually develop and share toolkits and resources with principals and school staff for improving attendance and supporting the needs of chronically absent students. Upstream Public Health is currently seeking funding for that effort.
We commend the Healthy Kids Learn Better Coalition and Upstream Public Health for their work to elevate the connection between health and learning in Oregon.
Guest Blog: Partnering with USDA to Bring More Good Food to Schools
At the Farm to Cafeteria Conference, Power in Community


Packed with seasoned ground beef, refried beans and all the classic toppings, these crowd-ready tacos make a meal everyone will love.
Heat oven to 400°F. Spray bottom of 13x9-inch (3-quart) baking dish with cooking spray. Place taco shells standing up and together in dish; set aside.
In 10-inch nonstick skillet, cook beef and onion over medium-high heat 5 to 7 minutes, stirring frequently, until beef is thoroughly cooked; drain. Stir in taco seasoning mix and water; heat to boiling. Reduce heat; simmer uncovered 2 to 3 minutes, stirring frequently, until mixture has thickened. Remove from heat.
To assemble, spread about 2 tablespoons refried beans in bottom of each shell. Top each with heaping 1/4 cup beef mixture and 2 tablespoons shredded cheese. Bake 5 to 7 minutes or until cheese is melted. Top with remaining ingredients, if desired.
Make it bold! Try other varieties of Old El Paso™ Stand 'N Stuff taco shells, like bold ranch, bold spicy Cheddar or bold nacho cheese.
Make these tacos a complete meal by adding a side of your favorite rice, such as cilantro-lime rice. Simply cook rice as desired; stir in fresh cilantro leaves and freshly squeezed lime juice with a dash of salt. Delicious!


When you have a craving and nothing else will due, it just has to be Ben & Jerry's. Offering more unique, irresistible flavors than you could ever hope for, Ben & Jerry's at Hard Rock Hotel & Casino Biloxi is the only place to get your sweet tooth its satisfaction. Choose from any of your delicious dairy concoctions or discover something new, from frozen yogurt to ice cream and an assortment of sorbets, there's something for everybody at Ben & Jerry's.


Discussion in 'CycleChat Cafe' started by yenrod, 24 Oct 2007.
I wished someone to be in prison once. And he ended up their.
How did you fit him up?
OMG - really didn't need/want to hear that.
I think it's just an opportunity you choose to take as opposed to one you don't.
My considered scientific opinion is that it's a load of bollix to think that things are somehow preordained. Just a way of passing the buck when things go wrong maybe!
Show me a believer in fate and I will show you an insane person.
My considered scientific opinion is that it's a load of bollix to think that things are somehow preordained.
Fate is the common-law wife of Karma and they both live on a hill with the Daily Llama.
So you don't believe in the p&*c%^7e fairy then.
Oh yes I do, every one believes in the puncture fairy surely?


An adjusted 2019 race course GPS track will be updated in late February 2019.
We race Towards Fishtail – the Matterhorn-like one – along this forested ridge top.
The start and finish of both races is in the village of Dhampus.
One thing you will notice in Nepal is that the hills and mountains are big. The rivers that flow away from the mountains cut deep. This means if you want to get close to the mountains in Nepal, you have to do some climbing.
From the start you get some traversing descent on trails and tracks. This is a great warm up and the first 13 km will pass easily. You meet a section of jeep track which lasts for about 4 km which facilitates the small amount of climbing you have to do.
Most of this section is lightly shaded by trees and passes through several farming villages.
We (you) are lucky with this course in that you get 90% of the climb done in one go. How lucky! But this is a monster of a climb, almost 2000m of unrelenting climb in a tangle of forest. At about 26.6 km you come out of the forest – it ends abruptly – and you're above the tree line. That means if it is clear, you've got some amazing views. As mentioned, you will notice the altitude so can take this section as a hike.
After taking some time (and pictures) at the checkpoint, you have 2000m to descend over 22 km. Some of this is slightly technical in the forest and some is going to be nicely runnable. It's going to be hard on your legs, same some energy for a stylish finish.


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National football champions
Prime Minister Phuc welcomes national football squad
December 22, 2018 by vietnamplus Leave a Comment
Prime Minister Nguyen Xuan Phuc welcomed Vietnamese national football squad, the champion of the AFF Suzuki Cup 2018, in Hanoi on December 21, praising their excellent performance at the regional tournament. The Prime Minister spoke highly of Coach Park Hangseo, members of the coaching group, especially the football players for their achievement. On this occasion, the Prime Minister presented a friendship medal to Coach Park Hang Seo and the Labour medal, first class to the national football squad. Striker Nguyen Quang Hai was presented the Labour medal, second class for his contribution to the squad's achievements. 15 other members received the labour medal, third class.-VNA … [Read more...] about Prime Minister Phuc welcomes national football squad
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Hà Nội become national U21 champions
November 18, 2018 by vietnamnews Leave a Comment
U21 Hà Nội won the National U21 Champs. — Photo baovinhlong.com.vnViet Nam News HÀ NỘI — Hà Nội were crowned National U21 Champions on Saturday at the Tự Do Stadium in Huế. The team from the capital defeated rivals Becamex Bình Dương 1-0 to claim the title after attacking from the whistle. The strategy was rewarded with a goal from Nguyễn Thành Chung of Hà Nội in the 10th minute, which proved to be the decider. Chung was named man of the match, and said he was ecstatic to hear he had also been voted player of the tournament. "I will try my best at the upcoming National Sports Games to win a plave on the national U22 side. I feel like I only gave 85 per cent during this tournament, so I need to do more," he said. Top scorer went to striker Phan Thanh Hậu of HAGL with three goals. Hà Nội has number of young footballers playing for the national U19, … [Read more...] about Hà Nội become national U21 champions
Filed Under: sports u21 champions league, national boxing champion, national football champions, national title champions, champions league u21, u21 youth champions league, bosnia u21 national team, u21 6 nations, national champions, college national champions, National Champion, NCAA National Champion, SCCA National Champions, football national champions, ncaaf national champions, national ncaa football champions, ucf national champions shirt, why ucf is not national champion, why ucf is not national champions, u of l 2013 national champions, when was alabama national champions, champion course at pga national resort & spa, national hot dog eating champion, ucf national champions, umass 1998 national champions, Vietnam, Vietnam News, news, National U21 Championships, Politics, Business, Economy, Society, Life, Sports, Environment, Your Say, English Through the News, Magazine, vietnam war, current news, Vietnamese to english, tin viet nam, latest news today, english newspapers, the vietnam war, news latest, today news headlines, nation news paper, today breaking news, vietnamese culture, vietnam history, bao vietnam, vietnam economy, today headlines, national news headlines, vietnam war summary, vietnam culture, vietnam government, news headline, daily nation today, newspaper headlines today, news website, báo online, headlines news, news site, war in vietnam, tin vietnam, vietnam people, vietnam today, vietnamese news, tin viet nam net, viet to english, news headlines for today, news paper online, national news in english, current news headlines, vietnam war history, english news papers, top news headlines, today hot news, english news headlines, vietnam conflict, up to the minute news, english daily, viet news, news highlights, today international news, govt news, the vietnam war summary, vietnam exports, việt nam, bao vn net, news.vn, baovietnam, thongtanxavietnam, vietnam plus, vietnam news agency
Vietnam's national football team trains for AFF Cup in Laos
November 5, 2018 by vietnamplus Leave a Comment
Vietnam's national football team had a gentle training session in Vientiane on November 5 (Photo: VNA) Vientiane (VNA) – Vietnam's national football team had a gentle training session in Vientiane on November 5 afternoon to prepare for the upcoming match against Laos within the framework of the ASEAN Football Federation (AFF) Suzuki Cup 2018. Regarding the match against the host Laos in Group A on November 8, defender Tran Dinh Trong said: "Of course, the Vietnamese team's target is to win the upcoming match, but this is an away match so it will not be easy for us. The whole team will make every effort to accomplish the target set by the coaches." Earlier, Vietnam's head coach Park Hang-seo announced the list of 25 players preliminary selected to compete at the AFF Suzuki Cup 2018. The national premier league's defending champions Hanoi contributes seven players including defenders Tran Dinh Trong and Doan Van Hau, … [Read more...] about Vietnam's national football team trains for AFF Cup in Laos
Filed Under: Culture Sports netherland women's national football team, ex england women's national football team roster, algeria women's national football team, cameroon women's national football team, laos national football team, greece national football team, cambodia national football team, germany national football team, england national football team, philippines national football team, germany under 21 national football team, iceland national football team, israel national football team, palestine national football team, ireland national football team, lithuanian national football team, managing virtual teams training, national cyber forensics and training alliance, college top ten football teams, nationally recognized personal training certification, dominican national soccer team, best junior college football teams, nationally accredited personal training certification, australia competes at their second football world cup, #META_Keywords, Culture - Sports, training session, Laos, head coach Park Hang-seo, vietnamplus, Vietnam News Agency,
National football team receives more support
July 23, 2018 by vietnamplus Leave a Comment
At the ceremony to announce to renew Yanmar's sponsorship held on July 23 (Photo: VNA) Hanoi (VNA) - Japan-based Yanmar company will continue to be the main sponsor for the national men's football team and U23 team, heard a ceremony to renew the sponsorship on July 23. At the event, Secretary General of the Vietnam Football Federation (VFF) Le Hoai Anh lauded the company's efforts since March 2015 to improve practice environment, contributing to the development of the Vietnamese national football team. According to Director General of the company Takehito Yamaoka, Yanmar will also sponsor the ASEAN Football Federation (AFF) Suzuki Cup 2018. The final round of the tournament will be held from November 8 to December 15. Vietnam are in Group A with Malaysia, Myanmar, Laos and Cambodia. Yanmar hopes that with its sponsorship, the Vietnam national men's football team will harvest new successes in the future, especially towards the goal of becoming the … [Read more...] about National football team receives more support
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24 teams to compete in 2018 national football cup
April 21, 2018 by sggpnews Leave a Comment
At a ceremony held in Hanoi on April 20, VPF also revealed that Nguyen Quang Hai, a member of the Vietnamese team competing at the Asian Football Confederation (AFC) U23 Championship in China in January, is the official representative of the football cup. The tournament will be named the Vietnam National Football Cup – White Lion 2018 and is scheduled to run until October 14. The teams will compete in knockout format in the qualifying round and round of 16, and in two-legged tie starting from the quarter-final. The cooperation of the VPF and MCH is hoped to foster Vietnam's sport sector and football development in particular, VPF Chairman and General Director Tran Anh Tu said. The champion will walk home with a prize worth VND1 billion ($43,900), while the runner-up receives half of that amount. In addition, the two teams will be Vietnam's representatives at the Asian Football Confederation Cup.Vietnamnews Tags: 24 teams 2018 national football cup Asian Football … [Read more...] about 24 teams to compete in 2018 national football cup
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An atrial septal defect is a hole in the wall between the two upper chambers (right and left atriums) of the heart. A transcatheter procedure is a minimally-invasive way to fix the hole. During this procedure, a device is implanted to seal the hole. As your child gets better, the device will trigger the heart tissue to grow. It will slowly grow over the hole.
If a child is born with a hole between the upper chambers of the heart, the blood can flow backward into the right side of the heart and into the lungs. This triggers the heart to work harder. Over time, this can lead to damage to blood vessels in the lungs and congestive heart failure . This is done to fix the hole.
Most children will have good results.
Talk to the doctor about ways to manage things that may raise your child's risk of problems such as chronic disease such as diabetes or obesity. Low birth weight or a recent infection may raise the risk of problems.
You will be told whether your child needs to stop taking certain medicine. Ask when your child should stop eating or drinking before the surgery.
General anesthesia will be used. It will block any pain and keep your child asleep through the surgery.
Your child will get IV fluid and medicines through a vein in the arm. A tube will be inserted either in the arm or groin. Next, electrodes will be placed on the chest. They will send information to the EKG machine, allowing the heart to be monitored.
A catheter will be placed in the blood vessel and advanced so the end is in the heart. Dye will be injected to allow the doctor to view x-ray images of the heart. An echocardiogram may also be used. Before the hole can be covered, the doctor will need to find out the size of the hole. A tube with a balloon attached to it will be sent to the upper chambers of the heart. The balloon will inflate and measure the hole.
When the size of the defect is known, another tube will be sent to the heart. This tube will have a device attached. There are many types of devices. Some are able to open so that the hole is covered on both sides. Others open like an umbrella to cover the defect. After the device is placed, the tube will be removed. Lastly, a bandage will be placed over the site.
Your child will have pain. Your child will be given pain medicine.
The usual length of stay is 2 to 4 days. In some cases, your child may be able to go home as soon as the next day. The doctor may choose to keep your child longer if there are problems.
Do tests, such as an EKG, chest x-ray, and blood tests.
Have your child lie still and flat for several hours. This is to prevent bleeding.
Place a pressure bandage to reduce bleeding.
Encourage your child to drink plenty of fluids to flush the dye from his body.
Give pain medicine to ease discomfort.
Atrial septal defect. Cove Point Foundation website. Available at: http://www.pted.org/?id=atrialseptal4. Updated January 24, 2017. Accessed June 26, 2018.
Atrial septal defect. Kids Health—Nemours Foundation website. Available at: http://kidshealth.org/parent/medical/heart/asd.html. Accessed September 2016. Accessed June 26, 2018.
Open-heart surgery. Cincinnati Children's Hospital website. Available at: http://www.cincinnatichildrens.org/health/heart-encyclopedia/treat/surg/open.htm. Updated June 2015. Accessed June 26, 2018.


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