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Anatomic Localization of Immature and Mature Dendritic Cells in an Ectopic Lymphoid Organ: Correlation with Selective Chemokine Expression in Rheumatoid Synovium
Guillaume Page, Serge Lebecque and Pierre Miossec
J Immunol May 15, 2002, 168 (10) 5333-5341; DOI: https://doi.org/10.4049/jimmunol.168.10.5333
Guillaume Page
*Department of Immunology and Rheumatology and Institut National de la Sante et de la Recherche Médicale Unité 403, Hôpital Edouard Herriot, Lyon, France; and
Serge Lebecque
†Schering-Plough Laboratory for Immunological Research, Dardilly, France
Pierre Miossec
It remains to be clarified whether dendritic cells (DC) reach the rheumatoid arthritis (RA) synovium, considered an ectopic lymphoid organ, as mature cells or undergo local maturation. We characterized by immunohistochemistry the DC subsets and used tonsils as a control. Immature and mature DC were defined by CD1a and DC-lysosome-associated membrane protein/CD83 expression, respectively. Immature DC were mainly detected in the lining layer in RA synovium. Mature DC were exclusively detected in the lymphocytic infiltrates. The DC-lysosome-associated membrane protein/CD1a ratio was 1.1 in RA synovium and 5.3 in tonsils, suggesting the relative accumulation of immature DC in RA synovium. We then focused on the expression of CCL20/CCR6 and CCL19/CCR7, CCL21/CCR7 chemokine/receptor complex, which control immature and mature DC migration respectively. A close association was observed between CCL20-producing cells and CD1a+ cells, suggesting the contribution of CCL20 to CCR6+ cell homing. Conversely, CCL21 and CCL19 expression was only detected in perivascular infiltrates. The association among CCL19/21-producing cells, CCR7 expression, and mature DC accumulation is in line with the roles of these chemokines in mature CCR7+ DC homing to lymphocytic infiltrates. The role of DC in disease initiation and perpetuation makes chemokines involved in DC migration a potential therapeutic target.
Dendritic cells (DC)2 are the most potent APCs and play a central role in the processing and presentation of Ags to T cells during the immune response (1). After Ag exposure, immature DC migrate to draining lymph nodes where they differentiate into mature DC to present Ags to T cells (1).
DC are found in the rheumatoid arthritis (RA) synovium and are likely to play an important role in its pathogenesis. This conclusion is supported by the histological observations demonstrating MHC class II+ APC clustered with T cells around blood vessels, sometimes in germinal center (GC) structures, and the association of HLA-DR alleles with disease severity (2, 3, 4, 5, 6). However, the function of DC according to phenotype and subset in RA synovium remains to be clarified, in particular regarding their mode of migration and accumulation (7, 8, 9, 10). Recently, differentiated DC expressing nuclear RelB have been detected in RA synovium, predominantly located in perivascular mononuclear cell aggregates (11).
RA synovium has several, but not all, of the characteristics of lymphoid organs with regard to cellular composition and organization (12), but it is unknown whether DC reach the synovium as mature cells or undergo local maturation. In particular, an accumulation of immature DC could be secondary to inflammation. Such studies are difficult to perform because of the low frequency of DC in such tissue. In addition, synovium dissociation with enzymes may affect phenotypes and functions with a difficult recovery of such rare cells. Accordingly, we have selected immunohistochemical techniques to investigate the different DC subsets found in RA synovium. We used several newly defined markers of the immature and mature DC subsets. CD1a, first described as a marker of Langerhans cells (LC) in skin epithelium, was later described as a marker of immature DC (13). Conversely, DC-lysosome-associated membrane protein (LAMP) and CD83 have been associated with more mature DC subsets. In vitro studies of DC differentiation have shown that CD83 expression appears before that of DC-LAMP, suggesting that CD83+ DC are less mature than DC-LAMP+ DC (14, 15). We selected tonsils, an active lymphoid organ, as a control. We analyzed DC frequency and localization with the aim of studying the respective immature/mature DC compartmentalization pattern in RA synovium. Such compartmentalization has been observed in breast carcinoma tissue where immature DC were detected within the tumor and mature DC in peritumoral areas (16).
To study the mode of migration of DC, we also looked at the expression of several chemokines and their associated receptors. Macrophage-inflammatory protein-3α, a CC chemokine, recently renamed CCL20 (17), and its receptor CCR6 (18) are critical for the recruitment of immature DC (19). CCL20 plays a major role in epithelial colonization by LC in response to inflammation (20). Macrophage-inflammatory protein-3β/CCL19 and 6-chemokine/CCL21 contribute through their associated receptor CCR7 to the accumulation of Ag-loaded mature DC in T cell-rich areas of lymphoid organs (19). These chemokines attract in vitro-derived DC following CCR7 induction by CD40 ligand, TNF-α, or LPS stimulation (19, 21). In situ detection of CCL19 showed its expression within the T cell areas of secondary lymphoid organs (22), with CCL21 having a wider distribution.
In this work we report the coexistence of both immature and mature DC subsets in RA synovium. The close association between immature CD1a+ DC and CCL20-producing cells suggests the contribution of CCL20 to the homing of immature CCR6+ cells into RA synovium. Conversely, the colocalization of CCL21/CCL19, CCR7, and mature DC in perivascular infiltrates suggests a role for these chemokines in the homing of mature CCR7+ DC in lymphocytic infiltrates.
Collection of samples
Synovial samples were obtained from 12 patients with RA, defined according to the revised criteria of the American College of Rheumatology (formerly the American Rheumatism Association) (23). To assess inflammatory synovium, samples were obtained from patients undergoing knee or wrist synovectomy, and not at a late stage, such as for joint replacement. Tonsils were obtained from children undergoing tonsillectomy.
The study was first initiated with frozen sections. However, immunostaining using frozen specimens, despite good Ag preservation, was not sufficient to clearly define the morphology of DC and to allow their quantification. Accordingly, an effort was made to develop immunostaining in paraffin-embedded sections, which allows a better preservation of histological patterns. Samples were fixed in 4% phosphate-buffered paraformaldehyde and then embedded in paraffin. Four-micrometer sections were cut and mounted on glass slides (adhesive slides, SuperFrost; CML, Nemours, France). To detect Ag expression in paraffin-embedded sections, Ag retrieval procedures were performed, including incubation in either citrate buffer (10 mM, pH 6) or EDTA buffer (1 mM, pH 8), followed by microwave oven incubation (three times for 3 min each time).
Following staining with hematoxylin, samples were classified according to the following criteria (24): diffuse infiltration of T cells and B cells and absence of lymphoid organization (n = 4), T cell-B cell aggregates without GC (n = 5), and presence of GC (n = 3).
Detection of DC subsets
For single staining, an immunoperoxidase technique using 3-amino 9-ethylcarbazole (AEC; red color; DAKO, Glostrup, Denmark) or 3,3′-diaminobenzidine tetrahydrochloride (DAKO) as chromogen was performed. Briefly, endogenous peroxidase activity was blocked with 3% hydrogen peroxide. Sections were incubated with several mouse mAbs recognizing the following molecules (see also Table I⇓): CD1a (IgG2b; BD Biosciences, Pont de Claix, France), CD11c (IgG2b; CamFolio, San Jose, CA), Langerin (IgG1; Schering-Plough, Dardilly, France), CD40 (IgG1; Schering-Plough), HLA-DR (IgG2b; BioSource, Camarillo, CA), CD83 (IgG2a; Immunotech, Marseilles, France), and DC-LAMP (IgG1; Immunotech). (Refs. 13, 14, 15, 25, 26, 27, 28 , see Table I⇓ for details). After overnight incubation at 4°C and washing, the sections were incubated with biotinylated anti-mouse IgG, followed by streptavidin-peroxidase (DAKO). The peroxidase was developed by AEC, and Mayer hematoxylin (DAKO) was used as the counterstain. In negative control sections the primary Abs were omitted, or irrelevant Ab was applied at the same concentration as the primary Ab.
Markers and Abs used for DC subset detection
In double-step immunohistochemical staining, after an initial blocking with rat serum, avidin-biotin (DAKO), and BSA, primary Ab to DC-LAMP was followed by rat biotinylated anti-mouse IgG1 and streptavidin-peroxidase. Peroxidase was developed by 3,3′-diaminobenzidine tetrahydrochloride. Mouse mAb to CD1a was followed by rat anti-mouse IgG2a/2b (BD Biosciences) and mouse alkaline phosphatase anti-alkaline phosphatase (DAKO). Alkaline phosphatase was revealed using Fast Blue as chromogen (blue color; Vector Laboratories, Burlingame, CA). In negative control sections, one of the two primary Abs was omitted.
Detection of chemokines and their associated receptors
For detection of chemokines, goat polyclonal Abs to CCL19, CCL20, or CCL21 (R&D Systems, Abingdon, U.K.) were used, followed by biotinylated rabbit anti-goat IgG (DAKO), and was developed using streptavidin-peroxidase. CCR6 and CCR7 were detected using anti-CCR6 (R&D Systems) and anti-CCR7 (BD Biosciences) mouse mAbs.
In double-step immunohistochemical staining, after an initial blocking with rabbit serum, avidin-biotin, and BSA, primary Ab to CCL20 was added, followed by rabbit biotinylated anti-goat IgG and streptavidin-peroxidase. Peroxidase was developed by AEC. Mouse mAb to CD1a (IgG2b) was followed by rat anti-mouse IgG2a/2b (BD Biosciences) and mouse alkaline phosphatase anti-alkaline phosphatase (DAKO). Alkaline phosphatase was revealed using Fast Blue as chromogen (blue color; Vector Laboratories).
Quantification of positive cells
Because of the high heterogeneity of RA synovium and the low frequency of DC subsets, quantification was performed with a method first used for the estimation of new blood vessel formation in breast tumor (29). This method allows quantification of cells in hot spots, defined in this study as synovium areas containing the highest density of positive cells. Accordingly, two hot spots per rheumatoid synovium section were selected. In each spot positive cells were counted in 10 consecutive high-power fields (×500). One field corresponded to 0.3 mm2, and thus one spot to 3 mm2. The number of positive cells per two hot spots was averaged, and results are expressed as the number of positive cells per square millimeter.
Results were expressed as the mean ± SD. Levels of marker expression were compared using the nonparametric Mann-Whitney U test between RA synovium and tonsils and between subsets of RA synovium.
Expression of HLA-DR, CD11c, and CD40 in RA synovium
We investigated DC subsets in sections obtained from 12 RA patients and used tonsils, an active secondary lymphoid organ as a control. We used several mAbs against specific and nonspecific DC markers (Table I⇑). HLA-DR, CD11c, and CD40, known to be expressed by DC, were detected in all RA synovium samples. HLA-DR, CD11c, and CD40 were highly expressed in every sample (44 ± 11, 32 ± 11, and 35 ± 12 positive cells/mm2, using the hot spot analysis, respectively; Table II⇓). Similar expression was observed in tonsils (not significantly different vs RA synovium). These markers were detected in many cell types other than DC, such as T cells, B cells, and synoviocytes, and localized in the lining and sublining layers as well as in infiltrates.
Quantification of DC subsets in RA synovium and tonsilsa
Immature DC are present within RA synovium
We then focused on more DC-specific markers. Immature DC were defined by CD1a expression. Immature CD1a+ DC were detected in 10 of 12 RA synovium samples. The number of CD1a+ cells per square millimeter in RA synovium ranged from 1 to 26 cells (mean ± SD, 7 ± 7; Table II⇑). In tonsils, that number was ∼2-fold higher (15 ± 2 positive cells/mm2), but without reaching significance (not significantly different vs RA synovium).
The LC subset, also expressing CD1a marker in skin epithelium (13), has been characterized by the specific expression of langerin/DCGM4. This represents a marker of epithelial tissue. As expected, no LC were found in RA synovium. Conversely, LC in tonsils (14 ± 2 positive cells/mm2; p < 0.001 vs RA synovium) were exclusively present in the epithelium. Such finding excludes the migration of epithelial-derived DC to the synovium.
In RA synovium immature CD1a+ DC were preferentially localized in the lining (inset of Fig. 1⇓A) or sublining layer as well as at the periphery of perivascular infiltrates (Fig. 1⇓A). CD1a+ cells were detected in the lining layer of all 10 CD1a+ samples, whereas immature DC in perivascular infiltrates were present in only 4 of 12 samples (patients 4, 8, 10, and 12). In tonsils, CD1a+ DC were detected in the T cell zone and the epithelium (Fig. 1⇓B).
Independent subsets of immature CD1a+ DC and mature DC-LAMP+ and CD83+ DC are detected in RA synovium and tonsils. Immunostaining of paraffin-embedded RA synovium and tonsils sections using mouse anti-CD1a mAb shows CD1a+ cells (red) in the perivascular infiltrates (A) and the lining layer (inset of A) in RA synovium and in the epithelium and the T cell zone of tonsils (B). Immature CD1a+ DC presented a dendritic-like morphology characterized by a few processes from the cell body (A). CD83+ cells (red) in RA synovium are detected at the periphery of the perivascular or lymphocytic infiltrates in RA synovium (C) and in GC and T cell zone in tonsils (D). Mature CD83+ DC have a dendritic-like appearance (inset of C). DC-LAMP+ cells (red) are found inside the lymphocytic infiltrates in RA synovium (E) and with a strong expression in the T cell areas of tonsils (F). DC-LAMP+ DC have a dendritic-like morphology, with very short or long veils (inset of E). Double staining with anti-DC-LAMP (brown) and anti-CD1a (blue) Abs shows a lack of coexpression of DC-LAMP and CD1a in RA synovium (G) and tonsils (H). Magnification: A, C, D, E, G, H, and inset of A, ×400; B and F, ×250; inset of C and F, ×1000.
Most of the immature CD1a+ DC showed a different dendritic morphology compared with the stellate shape of LC in skin epithelium, with large veils expanding in every direction from the cell body with long (10 μm) and thin processes. In RA synovium, immature CD1a+ DC had only a few long processes (Fig. 1⇑A).
Mature DC are preferentially localized in the perivascular or lymphocytic infiltrates
Mature DC were analyzed for the expression of CD83 and DC-LAMP. In vitro studies of DC differentiation have shown that CD83 expression appears before that of DC-LAMP, suggesting that CD83+ DC are less mature than DC-LAMP+ DC (15). CD83+ cells were detected in 9 of 12 samples, and DC-LAMP+ cells were found in 11 of 12 samples. The mean numbers of CD83+ cells and DC-LAMP+ cells per square millimeter in RA synovium were 5 ± 6 and 8 ± 5, respectively (Table II⇑). In tonsils the expression of CD83 and DC-LAMP was 7-fold higher (35 ± 7 positive cells/mm2; p < 0.001 vs RA synovium) and 10-fold higher (79 ± 3 positive cells/mm2; p < 0.001 vs RA synovium) compared with RA synovium, and the DC-LAMP/CD83 ratio was 79/35 = 2.3, indicating a relative accumulation of the most mature subset. In RA synovium, that ratio was 8/5 = 1.6 (not significant vs tonsils).
Mature CD83+ cells were mainly detected at the periphery of the perivascular or lymphocytic infiltrates (Fig. 1⇑C). In contrast, DC-LAMP+ DC were not observed at the periphery, but inside the lymphocytic infiltrates (Fig. 1⇑E), the site of interactions between mature DC and lymphocytes. In tonsils, DC-LAMP+ DC were exclusively detected in the T cell zone (Fig. 1⇑F), whereas CD83+ cells were found in both T cell and GC zones (Fig. 1⇑D).
In synovium, most of the mature DC also had a dendritic-like morphology (inset of Fig. 1⇑, C and E). Some of the DC-LAMP+ or CD83+ cells had a less common aspect, with a plasmacytoid-like appearance and a remote nucleus in a large cytoplasm (Fig. 1⇑E, inset).
To further clarify the dichotomy between immature and mature DC subsets in RA synovium, double staining using anti-DC-LAMP and anti-CD1a Abs was performed. The lack of coexpression of DC-LAMP and CD1a confirms the presence of independent immature and mature DC subsets (Fig. 1⇑, G and H). The DC-LAMP/CD1a ratio was 8/7 = 1.1 in RA synovium vs 79/15 = 5.3 in tonsils (p < 0.001 vs RA synovium), suggesting the relative accumulation of immature DC in RA synovium.
CCL20 is expressed in RA synovium
To study the mode of migration of DC in response to chemokines, we first analyzed the expression pattern of CCL20 and its receptor CCR6, which is known to control the migration of immature DC. In RA synovium CCL20 was mainly expressed in the lining layer (Fig. 2⇓A), but also in the perivascular infiltrates (Fig. 2⇓C). CCL20 expression showed a large degree of heterogeneity between the different RA synovium samples. In 4 of 12 samples no CCL20 expression was detected. In the eight CCL20+ RA synovium, the densities of CCL20+ cells per square millimeter in the lining layer and/or perivascular infiltrates were 9 ± 9 and 9 ± 11, respectively, with a large degree of heterogeneity (Table III⇓). In samples 11 and 12, CCL20 was only expressed in the lining layer. In tonsils, CCL20-producing cells were more common (38 ± 5 positive cells/mm2; p < 0.001 vs RA synovium) and were exclusively detected in the epithelial crypts (Fig. 2⇓E).
CCL20 and its associated receptor CCR6 are detected near CD1a+ cells in the lining layer and the perivascular infiltrates in RA synovium and tonsils. Immunostaining with anti-CCL20 (A and C) and anti-CCR6 (B and D) Abs of serial paraffin-embedded RA synovium sections shows the association between CCL20-producing cells and CCR6+ cells in the lining layer, as well as in the perivascular infiltrates. In tonsils CCL20 expression is found only in epithelial crypts (E), and that of its associated receptor, CCR6, is found in the T cell zone adjacent to the CCL20-producing cells (F). Double staining with anti-CCL20 (red) and anti-CD1a (blue) Abs shows the association of CCL20-producing cells and immature CD1a+ cells in the lining layer (G) and in perivascular infiltrates (H). Such an association is also observed in tonsils where immature CD1a+ cells are adjacent to CCL20-expressing epithelial cells (I). Magnification for all: ×400.
Quantification of chemokines and their associated receptors in RA synovium and tonsilsa
In RA synovium CCL20-producing cells detected in the lining layer extend the demonstration of secretion of CCL20 by synoviocytes (30). In tonsils CCL20-producing cells in epithelial crypts were identified as epithelial cells characterized by their elongated shape.
CCR6, the receptor for CCL20, is highly expressed in RA synovium
CCR6 expression was detected in all RA synovium samples. CCR6 was highly expressed (39 ± 20 positive cells/mm2) in lymphocytic infiltrates and at the periphery of blood vessels (Fig. 2⇑D). In some cases CCR6 was also observed in the lining layer where CCL20 was highly expressed (Fig. 2⇑B). In tonsils, CCR6 was highly detected (66 ± 6 positive cells/mm2; not significant vs RA synovium) in the T cell zone, and accumulation of CCR6+ cells was directly adjacent to CCL20-expressing epithelial cells (Fig. 2⇑F). In both RA synovium and tonsils, CCR6-expressing cells had various morphotypes, including cells with a dendritic morphology.
Close association between CCL20-producing cells and immature CD1a+ DC
As observed in Table II⇑, the lack of CCL20 expression was associated with the absence (samples 3 and 9) or low number (patients 6 and 7) of CD1a+ cells. This suggested a link between the accumulation of immature CD1a+ DC and the expression of CCL20 in RA synovium. This observation was extended by double staining with anti-CCL20 and anti-CD1a Abs. In both RA synovium and tonsils we observed a close association between immature CD1a+ DC and CCL20-producing cells. In tonsils, CCL20+ cells in epithelial crypts localized near, but not in close contact with, CD1a+ cells (Fig. 2⇑I). In RA synovium this association was mainly detected in the lining layer (Fig. 2⇑G) and to a lesser degree in perivascular infiltrates (Fig. 2⇑H).
CCL19 and CCL21 are mainly localized in perivascular infiltrates
We then looked at CCL19 and CCL21 expression, the CCR7 ligands known to control the migration of mature DC. In RA synovium the frequencies of CCL21+ and CCL19+ cells were 13 ± 14 and 7 ± 10/mm2, respectively (Table III⇑). Staining of CCL21+ cells was more intense than that of CCL19+ cells. As observed for CCL20, the expression of CCL21 and CCL19 revealed a large degree of heterogeneity between samples. No expression of CCL21 and CCL19 was detected in samples characterized by a diffuse infiltrate without organization of the lymphocytic aggregates (patients 3, 6, 7, and 9). All samples with such features (patients 1, 2, 5, 8, and 11) showed the expression of CCL21, whereas CCL19 was only detected in two of those samples (patients 1 and 2). Both chemokines were detected in samples showing lymphocytic infiltrates with GC (patients 4, 10, and 12). More CCL21+ and CCL19+ cells were found in samples with GC (33 ± 8 and 22 ± 3 positive cells/mm2, respectively) than in samples without GC (11 ± 3 and 3 ± 5 positive cells/mm2, respectively; p < 0.001 and p < 0.001 vs samples with GC).
In RA synovium CCL21 and CCL19 were mainly localized in perivascular or lymphocytic infiltrates (Fig. 3⇓, A and B). We also observed the expression of CCL21 and CCL19 in endothelium cells (Fig. 3⇓, C and D). In tonsils, CCL21 (82 ± 5 positive cells/mm2; p < 0.001 vs RA synovium) and CCL19 (43 ± 5 positive cells/mm2; p < 0.001 vs RA synovium) were highly present and detected in T cell areas, with a wider distribution for CCL21 (Fig. 3⇓, G and H). They were expressed around GC.
CCL19 and CCL21 are detected in perivascular infiltrates and vascular endothelium in RA synovium and tonsils. CCR7, their associated receptor, is expressed in the lymphocytic infiltrates. Immunostaining of paraffin-embedded sections was performed with anti-CCL21 (A and C, RA synovium; G, tonsils) and anti-CCL19 (B and D, RA synovium; H, tonsils) Abs. CCL21+ (A) and CCL19+ cells (B) are found in the perivascular infiltrates, with a stronger expression for CCL21. Some vascular endothelial cells in RA synovium are also CCL21+ (C) or CCL19+ (D), again with a higher intensity for CCL21. In tonsils CCL21 (G) is highly expressed only in the T cell zone, where CC19-producing cells are less common (H). Staining using anti-CCR7 Ab (brown) shows CCR7+ cells in the lymphocytic infiltrates in RA synovium (E). Some CCR7+ cells have a dendritic morphology (inset of E). In tonsils, most CCR7+ cells are detected in the GC and some in the T cell zone (F), with a dendritic morphology (inset of F). Immunostaining of serial RA synovium sections with anti-CCR7 (I), anti-DC-LAMP (J), and anti-CCL21 (K) Abs shows that CCL21, a chemoattractant for mature DC, and its associated receptor, CCR7, colocalize in the lymphocytic infiltrates with mature DC. Magnification: A–C, inset of E and F, and I–K ×400; G and H, ×100; E and F, ×250; D, ×600.
These observations suggested a link between the expression of CCL21 or CCL19 and the organization of lymphoid infiltrates in GC.
CCR7, the receptor for CCL21 and CCL19, is expressed in lymphocytic infiltrates
CCR7 expression was detected in 8 of 12 RA synovium samples at a frequency of 10 ± 11/mm2 and at 31 ± 4/mm2 in tonsils (p < 0.005 vs RA synovium). As observed for CCL19 and CCL21, CCR7 was only detected in samples characterized by lymphocytic aggregates with or without GC. In RA synovium, CCR7+ cells were mainly detected in lymphocytic infiltrates and rarely near a vascular endothelium without any infiltrate (Fig. 3⇑E). Some of them had a dendritic morphology (Fig. 3⇑E, inset). In tonsils most of the CCR7+ cells localized in GC and the T cell zone (Fig. 3⇑F), some also with a dendritic morphology (Fig. 3⇑F, inset).
Staining of serial sections with anti-CCR7, anti-CCL21, and anti-DC-LAMP indicated the close association between CCL21+ cells (Fig. 3⇑K), CCR7+ cells (Fig. 3⇑I), and mature DC (Fig. 3⇑J). This observation is in line with an interaction between the presence of mature CCR7+ DC and that of CCL21- and CCL19-producing cells.
We demonstrate in this work the presence in RA synovium of independent immature and mature DC subsets using the CD1a- and DC-LAMP/CD83-specific markers, respectively. We focused on these three markers as reflecting the different steps of in vitro DC differentiation, with CD1a and DC-LAMP linked to early and late stages of differentiation, respectively, CD83 expression appearing before that of DC-LAMP.
Compared with tonsils, accumulation of DC in RA synovium was lower, with a relative accumulation of immature DC. In tonsils, the mature/immature ratios (DC-LAMP/CD1a) were 5.3 and 1.1 in RA synovium. Such a difference may lead to functional changes between a lymphoid organ in a normal position (in this study, the tonsils) or an ectopic position (in this study, the synovium). Immature DC are highly efficient in Ag uptake. In the context of RA, further clarification is difficult because the causative Ag remains unknown. Immature DC could take up Ag directly in synovium, as suggested by the presence of a long process from CD1a+ cells, but immature DC could have already loaded Ag before their entry into the synovium. Conversely, mature DC are highly efficient APC and were localized in lymphocytic infiltrates of the synovium, where they can interact with T and B cells. Such interaction could occur at various stages of the RA process (31).
Immature CD1a+ DC were localized in the lining or sublining layers, as well as in the perivascular infiltrates. Mature DC were mainly detected in perivascular infiltrates. In RA synovium, detection of both immature and mature DC subsets in perivascular infiltrates argues for the lack of compartmentalization, such as is observed for breast carcinoma tissue, where immature DC were found within the tumor and mature DC in peritumoral areas (16). However, in RA synovium different localization patterns were observed for DC-LAMP+ and CD83+ DC in these infiltrates. CD83+ cells, considered less mature than DC-LAMP+ cells, were exclusively detected at the periphery, whereas the fully mature DC-LAMP+ cells were also observed inside the lymphocytic infiltrates. Differential localization of CD83+ and DC-LAMP+ cells could reflect the effect of maturation on the response of DC to the chemokines that control their homing into infiltrates.
Such differences in the mature/immature ratio suggest changes in the chemokine contribution. Indeed, chemokines are nonspecific soluble factors that control the migration of DC and many other cells into the synovium. We analyzed the expression of CCL20, the ligand of CCR6, and CCL19 and CCL21, the ligands of CCR7. These chemokines have been classified as chemoattractant for in vitro-derived immature and mature DC, respectively (19, 32). It was critical to demonstrate whether such findings are in line with the in vivo situation. The close association between CCL20-producing cells and CD1a+ immature DC observed in this study was indeed an in situ confirmation of these previous results obtained in vitro. CCL20 was highly expressed in the lining layer, extending our recent results where in vitro incubation of synoviocytes with IL-1, IL-17, and TNF-α was associated with an up-regulation of CCL20 production (30). Synoviocytes are predominant in the lining layer and represent the leading CCL20-producing cells, similar to keratinocytes in psoriasis skin and epithelial cells in tonsil crypts, which are the sites of CCL20 production (19, 20, 33). The expression of CCR6 near the lining layer suggests that it represents one site of entry of immature CCR6+ DC into the RA synovium. Indeed, such DC were observed inside the lining layer. However, high endothelial venules should also be considered a site of immature DC entry, as shown by the high expression of CCR6 and the close association between CCL20-producing cells and CD1a+ cells in the perivascular infiltrates.
Compared with tonsils, where mature DC represent the major DC subset, the RA synovium was characterized by the relative accumulation of immature DC. This suggests a relative defect in DC maturation in RA synovium. When considering the various cytokines used for in vitro generation of hemopoietic CD34+-derived DC (GM-CSF and TNF-α) and monocyte-derived DC (GM-CSF and IL-4) (34, 35), the lack of IL-4 in RA synovium contrasts with the high levels of GM-CSF and TNF-α (36). The defect in DC differentiation compared with tonsils could be linked to the lack of IL-4 leading to defective Ag processing. In addition, T cell-derived cytokines are characterized by high IL-17 and low IL-4 production, resulting in a proinflammatory cytokine pattern (37). IL-10, another anti-inflammatory cytokine known to inhibit the capacity of synovial macrophages to function as APC (38), had no immunosuppressive effect on rheumatoid synovial fluid DC compared with peripheral blood DC (39). These results indicate the contribution of synovium cytokine microenvironment that results in complex changes in DC recruitment and function.
The potent local defect of maturation of mature DC in RA synovium led to evaluation of the expression of CCR7 and its ligands, CCL19 and CCL21, known chemoattractants for in vitro-derived mature DC. The detection of both chemokines in vascular endothelium and perivascular infiltrates suggests that migration of mature CCR7+ DC occurs from blood to RA synovium through vascular endothelium. The expression of CCL19 and CCL21 in RA samples with GC and their absence in samples with diffuse infiltrates suggest that these chemokines are implicated in GC formation and migration of mature DC in these lymphocytic aggregates. A similar conclusion was reached with the B cell-attracting chemokine-1 (40). However, stronger expression of CCL21 compared with CCL19 in RA synovium may indicate a greater contribution of CCL21 to the migration of mature DC. In addition, CCL19 expression (5 of 12 samples) was less common than that of CCL20 production (9 of 12). When considering the quantification of their associated receptors, the CCR6/CCR7 ratio was 39/10 = 3.9 in RA synovium, suggesting a higher contribution of CCL20/CCR6 to that of CCR7 and its ligands, CCL19 and CCL21, in the homing of DC. Such differences could result in a reduced migration of mature DC from blood. The combined results observed here are depicted in Fig. 4⇓, which represents a model of DC subsets and migration patterns in RA synovium.
A model of DC subsets and migration patterns in RA synovium. In response to local inflammation and the production of proinflammatory cytokines (1) following an unknown event, immature DC are attracted to the synovium in response to the local production of CCL20 in the perivascular infiltrates and the lining layer (2). CCR6+ immature DC migrate from blood through vascular endothelium and possibly also from synovial fluid through the lining layer. It is also possible that CCR6+ cells migrate to synovial fluid toward CCL20 expressed within the lining layer (3). The presence of mature DC in synovium results from the combined effects of cell interactions and cytokine microenvironment (4). A defect in production of differentiation factors, such as IL-4, may favor a relative accumulation of immature DC. However, the detection of CCL19 and CCL21 in perivascular infiltrates and vascular endothelium argues for a direct migration of CCR7+ mature DC from blood into the RA synovium, where they can interact with lymphocytes, leading to potent local lymphocyte activation.
Chemokines acting as chemoattractants of DC are key factors in stimulating immunity against tumor Ags. Adenovirus-mediated gene transfer of CCL20 to murine tumors induced the local accumulation of DC, leading to tumor-specific cellular immunity and suppression of tumor (41). Vaccination of mice with the CCL19-transduced C3L5 resulted in the rejection of CCL19-transduced tumor, showing an antitumor activity for CCL19 (42). The role of DC in disease initiation and perpetuation makes DC subsets a potential therapeutic target in RA. This also suggests the control of chemokines involved in DC migration. The link between CCL20 production and inflammation makes this chemokine a potential therapeutic target.
↵1 Address correspondence and reprint requests to Dr. Pierre Miossec, Clinical Immunology Unit, Departments of Immunology and Rheumatology, Hôpital Edouard Herriot, 69437 Lyon Cedex 03, France. E-mail address: miossec{at}univ-lyon1.fr
↵2 Abbreviations used in this paper: DC, dendritic cell; AEC, 3-amino 9-ethylcarbazole; GC, germinal center; LAMP, lysosome-associated membrane protein; LC, Langerhans cell; RA, rheumatoid arthritis.
Received October 11, 2001.
Accepted February 25, 2002.
Copyright © 2002 by The American Association of Immunologists
Banchereau, J., R. M. Steinman. 1998. Dendritic cells and the control of immunity. Nature 392: 245
Itoh, J., K. Kinjoh, A. Ohyama, M. Nose, M. Kyogoku. 1992. Application of two-color immunofluorescence staining to demonstration of T cells and HLA-DR-bearing cells in rheumatoid synovitis. J. Histochem. Cytochem. 40: 1675
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De Saint-Vis, B., J. Vincent, S. Vandenabeele, B. Vanbervliet, J. J. Pin, S. Ait-Yahia, S. Patel, M. G. Mattei, J. Banchereau, S. Zurawski, et al 1999. A novel lysosome-associated membrane glycoprotein, DC-LAMP, induced upon DC maturation, is transiently expressed in MHC class II compartment. Immunity 9: 325
Bell, D., P. Chomarat, D. Broyles, G. Netto, G. M. Harb, S. Lebecque, J. Valladeau, J. Davoust, K. A. Palucka, J. Banchereau. 1999. In breast carcinoma tissue, immature dendritic cells reside within the tumor, whereas mature dendritic cells are located in peritumoral areas. J. Exp. Med. 190: 1417
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Liao, F., R. L. Rabin, C. S. Smith, G. Sharma, T. B. Nutman, J. M. Farber. 1999. CC-chemokine receptor 6 is expressed on diverse memory subsets of T cells and determines responsiveness to macrophage inflammatory protein 3α. J. Immunol. 162: 186
Dieu, M. C., B. Vanbervliet, A. Vicari, J. M. Bridon, E. Oldham, S. Aït-Yahia, F. Brière, A. Zlotnik, S. Lebecque, C. Caux. 1998. Selective recruitment of immature and mature dendritic cells by distinct chemokines expressed in different anatomic sites. J. Exp. Med. 188: 373
Dieu-Nosjean, M. C., C. Massacrier, B. Homey, B. Vanbervliet, J. J. Pin, A. Vicari, S. Lebecque, C. Dezutter-Dambuyant, D. Schmitt, A. Zlotnik, et al 2000. Macrophage inflammatory protein 3α is expressed at inflamed epithelial surfaces and is the most potent chemokine known in attracting Langerhans cell precursors. J. Exp. Med. 192: 705
Yanagihara, S., E. Komura, E. Nagafune, H. Watarai, Y. Yamaguchi. 1998. EBI1/CCR7 is a new member of dendritic cell chemokine receptor that is up-regulated upon maturation. J. Immunol. 161: 3096
Ngo, V. N., H. L. Tang, J. G. Cyster. 1998. Epstein-Barr virus-induced molecule 1 ligand chemokine is expressed by dendritic cells in lymphoid tissues and strongly attracts naive T cells and activated B cells. J. Exp. Med. 188: 181
Arnett, F. C., S. M. Edworthy, D. A. Bloch, D. J. McShane, J. F. Fries, N. S. Cooper, L. A. Healey, S. R. Kaplan, M. H. Liang, H. S. Luthra, et al 1988. The American Rheumatism Association 1987 revised criteria for the classification of rheumatoid arthritis. Arthritis Rheum. 31: 315
Klimiuk, P. A., J. J. Goronzy, J. Björnsson, R. D. Beckenbaugh, C. M. Weyand. 1997. Tissue cytokine patterns distinguish variants of rheumatoid synovitis. Am. J. Pathol. 151: 1311
Lenz, A., M. Heine, G. Schuler, N. Romani. 1993. Human and murine dermis contain dendritic cells: isolation by means of a novel method and phenotypical and functional characterization. J. Clin. Invest. 92: 2587
Inaba, K., M. Inaba, M. Witmer-Pack, K. Hatchcock, R. Hodes, R. M. Steinman. 1995. Expression of B7 costimulator molecules on mouse dendritic cells. Adv. Exp. Med. Biol. 378: 65
Fearnley, D. B., A. D. McLellan, S. I. Mannering, B. D. Hock, D. N. Hart. 1997. Isolation of human blood dendritic cells using the CMRF-44 monoclonal Ab: implications for studies on antigen-presenting cell function and immunotherapy. Blood 89: 3708
Valladeau, J., V. Duvert-Frances, J. J. Pin, C. Dezutter-Dambuyant, C. Vincent, C. Massacrier, J. Vincent, K. Yoneda, J. Banchereau, C. Caux, et al 1999. The monoclonal Ab DCGM4 recognizes langerin, a protein specific of Langerhans cells, and is rapidly internalized from the cell surface. Eur. J. Immunol. 29: 2695
Bertin, N., P. Clezardin, R. Kubiak, L. Frappart. 1997. Thrombospondin-1 and -2 messenger RNA in normal, benign, and neoplastic breast tissues: correlation with prognostic factors, tumor angiogenesis and fibroblastic desmoplasia. Cancer Res. 57: 396
Chabaud, M., G. Page, P. Miossec. 2001. Enhancing effect of IL-1, IL17, and TNF-α on MIP-3α production in rheumatoid arthritis: regulation by soluble receptors and Th2 cytokines. J. Immunol. 167: 6015
Thomas, R., P. E. Lipsky. 1996. Presentation of self peptides by dendritic cells: possible implications for the pathogenesis of rheumatoid arthritis. Arthritis Rheum. 39: 183
Kellermann, S. A., S. Hudak, E. R. Oldham, Y. J. Liu, L. M. McEvoy. 1999. The CC chemokine receptor-7 ligands CCL21 and macrophage inflammatory protein-3β are potent chemoattractants for in vitro- and in vivo-derived dendritic cells. J. Immunol. 162: 3859
Homey, B., M. C. Dieu-Nosjean, A. Wiesenborn, C. Massacrier, J. J. Pin, E. Oldham, D. Catron, M. E. Buchanan, A. Muller, R. deWaal Malefyt, et al 2000. Up-regulation of macrophage inflammatory protein-3α/CCL20 and CC chemokine receptor 6 in psoriasis. J. Immunol. 164: 6621
Caux, C., B. Vanbervliet, C. Massacrier, C. Dezutter-Dambuyant, B. de Saint-Vis, C. Jacquet, K. Yoneda, S. Imamura, D. Schmitt, J. Banchereau. 1996. CD34+ hematopoietic progenitors from human cord blood differentiate along two independent dendritic cell pathways in response to GM-CSF+TNFα. J. Exp. Med. 184: 695
Caux, C., C. Massacrier, B. Dubois, J. Valladeau, C. Dezutter-Dambuyant, I. Durand, D. Schmitt, S. Saeland. 1999. Respective involvement of TGF-β and IL-4 in the development of Langerhans cells and non-Langerhans dendritic cells from CD34+ progenitors. J. Leukocyte Biol. 66: 781
Miossec, P., M. Naviliat, A. Dupuy d'Angeac, J. Sany, J. Banchereau. 1990. Low levels of interleukin 4 and high levels of transforming growth factor β in rheumatoid synovitis. Arthritis Rheum. 33: 1180
Chabaud, M., J. M. Durand, N. Buchs, F. Fossiez, G. Page, L. Frappart, P. Miossec. 1999. Human interleukin-17: a T cell-derived proinflammatory cytokine produced by the rheumatoid synovium. Arthritis Rheum. 42: 963
Mottonen, M., P. Isomaki, R. Saario, P. Toivanen, J. Punnonen, O. Lassila. 1998. Interleukin-10 inhibits the capacity of synovial macrophages to function as antigen-presenting cells. Br. J. Rheumatol. 37: 1207
MacDonald, K. P., A. R. Pettit, C. Quinn, G. J. Thomas, R. Thomas. 1999. Resistance of rheumatoid synovial dendritic cells to the immunosuppressive effect of IL-10. J. Immunol. 163: 5599
Shi, K., K. Hayashida, M. Kaneko, J. Hashimoto, T. Tomita, P. E. Lipsky, H. Yoshikawa, T. Ochi. 2001. Lymphoid chemokine B cell-attracting chemokine-1 (CXCL13) is expressed in germinal center of ectopic lymphoid follicles within the synovium of chronic arthritis patients. J. Immunol. 166: 650
Fushimi, T., A. Kojima, M. A. S. Moore, R. G. Crystal. 2000. Macrophage inflammatory protein 3α transgene attracts dendritic cells to established murine tumors and suppresses tumor growth. J. Clin. Invest. 105: 1383
Braun, S. E., K. Chen, R. G. Foster, C. H. Kim, R. Hromas, M. H. Kaplan, H. E. Broxmeyer, K. Cornetta. 2000. The CC chemokine CKβ/MIP-3β/ELC/Exodus 3 mediates tumor rejection of murine breast cancer cells through NK cells. J. Immunol. 164: 4025
Vol. 168, Issue 10
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The Journal of Immunology May 15, 2002, 168 (10) 5333-5341; DOI: 10.4049/jimmunol.168.10.5333
Differential Susceptibility to Staphylococcal Superantigen (SsAg)-Induced Apoptosis of CD4+ T Cells from Atopic Dermatitis Patients and Healthy Subjects: The Inhibitory Effect of IL-4 on SsAg-Induced Apoptosis
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Q: Meromorphic function on the Riemann surfaces Let $V$ be a Riemann surface, $x\in V$, and $B:=B(x,r)$ some small ball (in a local chart). It is well known that there is a meromorphic function $f$ on $V$ with the only pole at $x$. What I'd like to ask of is if there is a meromorphic f on V that has a pole at x and additionally such that $|f|<1$ outside $B$?

A: For open surfaces, there are counterexamples. The first one was constructed by P. Myrberg:
Ueber die analytische Fortsetzung von beschrankten Funktionen, Ann. Acad. Sci. Fenn., Ser. A. I N:o 58 (1949)
Since this paper is difficult to obtain (and written in German), I refer to another paper
Heins, Maurice,
Riemann surfaces of infinite genus.
Ann. of Math. (2) 55 (1952), 296–317,
Which proves an even stronger result: there is an open Riemann surface, such that if you remove a disk from it, then on the remaining surface every non-constant meromorphic function takes all complex values, except at most two of them.

redpajama_set_name: RedPajamaStackExchange


LWVMA has joined the Transportation for Massachusetts (T4MA) coalition. T4MA is a nonpartisan, diverse coalition of Bay State organizations working together to create safe, convenient, and affordable transportation choices for everyone in Massachusetts. Its 30 members have expertise in transportation, affordable housing, social justice, public health, the environment, planning and smart growth. Member organizations include the Conservation Law Foundation, MASSPIRG, Metropolitan Planning Area Council, and the MBTA Advisory Board. See the coalition website www.t4ma.org.
T4MA is committed to reducing greenhouse gas emissions by providing low-carbon transportation choices throughout the commonwealth. It also works to ensure that the transportation system provides a path to economic opportunity for those without cars, like youth, seniors, and low-income residents. It supports maintaining and growing an affordable and robust public transportation system, completing streets with bikeways and sidewalks, and developing walkable neighborhoods around transit.
T4MA's major issues reflect LWVMA's transportation position adopted in May 2013, particularly: Support a transportation system to move people and goods which includes a variety of transportation modes, with emphasis on increased public transportation services and other viable alternatives to reduce vehicle miles traveled; is efficient, convenient, and cost effective; is safe and secure; serves all segments of the population and diverse geographic needs; minimizes harmful effects on the environment; is integrated with land use; and is supported by extensive public education.
Our transportation system is a vital part of our economy, providing economic opportunity and ensuring everyone can get where they need to go.
Massachusetts needs a transportation network that offers safe, affordable, and convenient choices to our residents and businesses, including public transportation, sidewalks, and bikeways.
People without cars, including youth, seniors and low-income residents, should have the opportunity to get to the schools and jobs that will help them pursue their dreams.
Transportation investments should enhance mobility, expand economic opportunity, reduce pollution and greenhouse gases, and support healthy, sustainable communities.
Public stewards of transportation funds should be held accountable for wisely, fairly and transparently using public dollars.


text:
fast feed
Google Launches Its Own Wi-Fi Router
[Photo: via OnHub]
By Sarah Lawson 1 minute Read
Google just announced in a blog post that it's entering the Wi-Fi router business with its own version of what used to be the most universally hated part of connecting to the Internet.
OnHub, Google's sleek new wireless router, will go for $200 and is so attractive, it's meant to sit right in plain view–where it'll also deliver the best possible connection.
Google is co-producing the router with TP-LINK, a Chinese manufacturer of computer networking products. Later this year, Google plans to also work with Asus as a manufacturing partner.
OnHubPhoto: via Google
OnHub setup and management comes through the Google On app for Android and iOS, which allows users to monitor bandwidth by device, run network checks, and text passwords to friends. OnHub searches the airwaves and automatically selects the best channel for the fastest connection, according to the blog post. It also automatically downloads software and security updates without interrupting Wi-Fi service and allows users to prioritize a device so that important activities get the fastest speed.
The router is a sound infrastructural move for Google, which is constantly adding to its Internet-based product suite. A stronger, more streamlined Internet connection means more bandwidth for using Google's products.
According to Google's post, the router will be able to support Bluetooth Smart Ready, Weave, and 802.15.4 connections.
OnHub is available for presale now and will be available in the U.S. and Canada in the coming weeks, according to Google.
Somewhere, your old router is sputtering in revolt.

redpajama_set_name: RedPajamaCommonCrawl


TORONTO – Gleyber Torres barely hit in the spring after coming back from season-ending Tommy John surgery on his left elbow, and started the 2018 campaign in the minors.
But after rediscovering his swing and having an unlikely power surge following his promotion to the big leagues, the 21-year-old rookie wunderkind is headed to the All-Star Game — even though he likely won't play due to a right hip strain.
Torres, the midseason favorite for AL Rookie of the Year, is one of four Yankees headed to the midsummer classic on July 17 in D.C., joined by Aaron Judge, Luis Severino and Aroldis Chapman on the American League squad.
Torres is only the fourth Yankee to be named to the AL All-Star team at 21 years old or younger, joining Joe DiMaggio (age 21 in 1936), Mickey Mantle (ages 20-21 from 1952-53) and Willie Randolph (age 21 in 1976).
Giancarlo Stanton is part of the Final Vote, and can make it if the fans who have regularly booed him in the Bronx stuff the online ballot boxes this week.
Aaron Boone broke the news to Torres in a fun way on Sunday.
The Bombers were 10-9 when Torres was called up. Now, they're 58-29, boosted by their second baseman who has done most of his damage in the No. 9 hole, blasting 15 homers – already four more than he had in his best season in the minors — and posting a .905 OPS.
Torres wasn't the only Yankee who overcame adversity to get to play on the big stage in our nation's capital.
Judge hit .179 in 2016, the same year Severino went 0-8 with an 8.50 ERA as a starter. But both players have proven that their breakout 2017 seasons and All-Star selections were no fluke. Judge has hit 25 homers to go along with a .970 OPS, while Severino is 14-2 with a 2.12 ERA, and a deserving candidate to start for the AL.
Chapman lost his closer job briefly last season after signing a five-year, $86 million deal to return to the Bronx. But this season, he's 24-for-25 in save opportunities while pitching to a 1.42 ERA. This is his fifth trip to the All-Star Game and first as a Yankee.
Despite 18 straight scoreless appearances, Dellin Betances struggled mightily at the beginning of the season, and will not appear in his fifth consecutive All-Star Game. Aaron Hicks and Miguel Andujar also made compelling cases, though they fell short as well.


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Lincoln 1st XV travel to Stamford today in the semi-final of the County Cup.
After last week's nail-biting victory the Lincoln Outfit are in a confident mood to take on their higher league opponents.
Mix and Match at 3pm today on Siren 107.3 FM will keep you up to date with the latest score.


A hopeful candidate for Raymondville City Commission who had outstanding traffic court fines has paid fines mentioned in last week's Chronicle but now must clear up 20 more outstanding fines or warrants in Willacy County and other counties if she plans to have her named placed on the ballot.
Erica Reyes Rubalcaba, 34, has outstanding warrants or unpaid fines as far away as El Paso totaling between $4,000 and $5,000, Municipal Court Judge Felicita Gutierrez said on Monday.
She did pay the amounts listed in last week's Chronicle but did so by mail. If she had paid them in person, she would have learned there are additional fees because she was paying them late after pleading guilty to traffic offenses and promising to pay the fines earlier, then failing to appear in court or paying the amounts owed, the judge said.
"She (also) has five citations in Willacy County Justice of the Peace Court No. 5 and six more in Precinct No. 2," the judge said. "She has three in the City of Harlingen; one each in Cameron County Precinct 3 and Precinct 4; she has three in Combes; and one in Cameron County Precinct 2," Gutierrez said.
Reyes Rubalcaba also has warrants in Pecos and El Paso County dating back to 2009 and 2003, Gutierrez said.
The warrants in Willacy County are all misdemeanors, but it is obvious that Reyes Rubalcaba does not have a valid driver's license, the municipal judge said. In a posting on social media, she was driving while recording her video message, the judge said.
If she is pulled over by a law enforcement officer, she is subject to arrest, the judge said. Driving without a valid license would be another offense.
City of Raymondville employees must have valid driver's licenses to hold their jobs, Gutierrez said.
Infractions listed against Reyes Rubalcaba (her listed name varies in some of the cases) include infractions such as no insurance, speeding, bail jumping and failure to appear in court, as well as unpaid fines in cases in which she had pleaded guilty, the judge said.
City Manager Eleazar "Yogi" Garcia said Reyes Rubalcaba did make a payment this week on her property taxes.
Reyes Rubalcaba was not immediately available for comment on Tuesday.


Last night just a few miles from my home a ten year old little girl was shot and killed as she sat in her living room, it was a drive-by. A few nights before this one a six year old child after buying bubble gum walked out of a convience store with her Daddy was shot and killed, a few days prior the school shootings in Washington. I don't know how many more but I am sure many that we don't hear about on our local news, I only know it wrenches my heart each time.
I don't cope well with hearing or reading of these deaths, each time a father dies in an auto accident or a mother passes from breast cancer my heart hurts and I begin to cry. It is a loss of a loved one to someone somewhere. There are all types of memes and even books or counselors to help with grief and perhaps I am too sensitive to death of others because I have experienced so much of it, I only know my heart aches and I want so badly to reach out with a hug and a helping hand yet I don't as I can barely hold onto my sanity at such times.
I find it quite difficult to have the right words to say to those that have lost a child or loved one be it brother, sister etc. What others struggle to say at such times, sounds so trite to my ears although I know all are meant from the deep recesses of ones heart and they want to help the ones left; I just cannot speak words of comfort as my tears flow from my cheeks with my pain of loss. It has been four years this February since I lost my daughter, you would think by now I would be able to support some semblance of decorum but I cannot, there must be something wrong with me that I am no longer able to act "normally" for others going through their loss.


text:
Hospital in Queens plans a $6.8 million renovation
Robin D. Schatz
Propertyshark.com
Elmhurst Hospital Center
Elmhurst Hospital Center, a 545-bed facility in the Health + Hospitals system, is proposing a $6.8 million renovation to construct a second-floor addition to its emergency department.
It will serve as the new home of its Adult Partial Hospitalization Program, or PHP, an outpatient intensive and structured program to stabilize adults with mental health and substance abuse disorders, who at the time of admission are experiencing acute psychiatric symptoms of their mental illness.
The project, which is being funded through a Capital Restructuring Financing Program grant, replaces a previous application and increases the costs because of information-technology infrastructure construction expenses, according to its certificate-of-need application with the state Department of Health.
Relocating the PHP next to the emergency department will improve care coordination, Elmhurst said. The emergency department is also adjacent to the hospital's Comprehensive Psychiatric Emergency program, allowing for "a warm hand-off" of patients who can be better served in the PHP, according to the application.
The space where the PHP is presently located, in Elmhurst's H building, will be used for a planned expansion of its outpatient mental health and substance-use-disorder clinics to better integrate primary care and behavioral health care.
De Blasio says fentanyl-laced heroin warrants war on drugs
Trumpcare may benefit this entrepreneur
City's busiest emergency room will distribute naloxone to treat heroin overdoses
Bloodletting: City's public hospital system fires 396 employees
New York-area hospitals resuscitate profits


A cool option for weekends in the city, Givenchy's black leather sandals have been made in Italy and punctuated with the label's signature chunky chains. They have a slight heel and cushioned insole for added comfort and look best with cropped pants or jeans.
Shown here with: Givenchy T-shirt, Marques' Almeida Track pants, Givenchy Clutch.


text:
Q: Dynamic height for a child div when the position is relative It seems a duplicated question but unfortunately I didn't find answers for this simple scenario when the Position is Relative.
I have a parent div with two child divs:

where both parent and child divs have a position:relative;
A Jsfiddle example is HERE
So my question is almost like THIS question but the position is not absolute here.
How do I make the height of content_rdivider div the same as the height of the parent div?

A: try this
http://jsfiddle.net/9c4MK/15/
CSS
#content {
    position: relative;
    background-color: #FFF;
    width: 975px;
    padding: 0px 0px 0px 0px;
    background-repeat: no-repeat;
    background-position: bottom left;
    border: solid 1px #d7d7d7;
    border-bottom: none;
    margin-left: auto;
    margin-right: auto;
    direction: ltr;
    overflow: hidden;
    height: auto;
    display: table;
}
#content_right {
    width: 180px;
    text-align: right;
    display: table-cell;
}
#content_rdivider {
    width: 10px;
    text-align: center;
    background-color: red;
    position: relative;
    height: 100%;
    display: table-cell;
}


text:
Q: jQuery add variable to Parameters of function Beginners question about adding variables from a split result to a function.
I have split[1] which contains the number for the accordion to be active.
this is the function:
jQuery("#termine").accordion({ autoHeight: false,
                               active: 1,
                               header: '> div.termin-item > div.termin-header'
                             });

Here I would like to replace the active: "1" with the variable split[1]
any hints on how to add that variable to that list of parameters?

A: Perhaps this:
jQuery("#termine").accordion({
    autoHeight: false,
    active: split[1],
    header: '> div.termin-item > div.termin-header'
});

A: Use this...
jQuery("#termine").accordion({
    autoHeight: false,
    active: parseInt(split[1], 10),
    header: '> div.termin-item > div.termin-header'
});

It converts the string value split[1] into an int and passes it as the active parameter.


"The plan is to die young but as late as possible"
Throughout the 20th century, global lifespan doubled, a trend that continued into the 21st century. If you were lucky enough to be born in 2007 or later you have a 50% chance of seeing your 100th birthday. If you are around 50 years old just now you can reasonably expect to reach 83. I happen to be in between these two age groups but I'll tell you one thing for sure, I have no intention to kick the bucket any time before hitting a full century and then some. Although there are no guarantees, here's a few helpful hints and tips on how I plan to do it.
I heard of this magic little pill. It improves strength, endurance, muscle quality and function. It reduces risk of heart disease, cancer, diabetes, anxiety and stress. It even helps with concentration, self-esteem and weight management. The pill comes in different forms, sometimes it's easy to take and sometimes it's hard to swallow. Whatever form it comes in, it always makes me feel satisfied, relaxed and happy. That's why I make it a priority to take this magic little pill 6 or 7 days a week.
Exercise being good for us is nothing new. In fact there is documented evidence dating back to 1949 when Jerry Morris studied London's double decker bus staff. He noticed that the drivers, who were static most of the day, had a much higher incidence of heart disease than the conductors who were walking up and down stairs numerous times a day. He extended his study to the postal service and noticed the same pattern. Employees sitting behind counters had a much higher incidence of heart disease than those delivering post on foot. His study was published in The British Medical Journal in 1958.
Your body is constantly adapting. Like an organic garden hosting billions of bacteria and a river of red blood cells generating millions of new cells per second. Your body strives to reach a balance known as homeostasis. As we progress through life we are always either growing or decaying. For the most part, you have the choice whether to "catabolise"- breakdown molecules or "anabolise" - manufacture new cells. So how do we maximise growth and minimalise decay?
Firstly, take your medicine as prescribed above. Simply moving more and sitting less can have a profound influence on reducing your risk of early death. In 2009, a study of over 17,000 people showed that no matter how active you are, the more you sit down, the more you increase your risk of chronic disease. So even if you are a regular gym bunny, but you sit in an office all day, make sure you take plenty of active breaks. Every 55 minutes go for a 5 minute walk.
Recovery is where it all happens. Exercise of all forms produces a stress response from the body. Applying appropriate levels of stress over time with enough recovery, will encourage the body to adapt and you become stronger and fitter. Without recovery, you simply drain your battery.
Doctors these days report that fatigue is one of the most common complaints and is often characterised by lack of motivation, lethargy and sleepiness. Sound familiar? Wearable technology is fast becoming the next global fitness trend. They can measure a whole host of markers including daily activity, heart rate and sleep. Wearable technology by itself won't drive behaviour change but I find it can be useful to draw attention to key areas of life such as sleep, and seeing improvements can be rewarding and often helps to promote behaviour change. I use a device called Firstbeat which measures sleep quantity and quality as well as exercise and stress, by measuring heart rate variability (beat to beat variations in heart rate) as well as basic heart rate and movement. (More on heart rate variability another time.) There's a whole host of evidence linking poor sleep to a higher incidence of chronic disease and premature death.
Research has shown that Blue light from television and mobile devices suppresses melatonin production by as much as 22% which could negatively affect sleep. We also know that alcohol inhibits sleep quality so phones off 30 minutes before bedtime and cut down on the booze. After all bedrooms are for more important things than watching television (ahem).
So as we progress through life and through the 21st century, you have the decision to make. Do you want to merely improve your lifespan or do you want to improve your "health- span?" I want to improve my enjoyment span. So whatever that entails is the most important thing...Being Happy.


Haynesley Benn (right) and Mackie Holder during just concluded campaign.
Former government minister Haynesley Benn is fooling himself if he thinks he will ever be elected to represent the place of his birth in the House of Assembly.
And this is regardless of whether or not his cousin and longstanding St. Peter MP, Owen Arthur, is his opponent.
That's the firm belief of well-known St. Peter personality and Arthur's friend and advisor of many years, Mackie Holder, speaking in the wake of recent statements from Benn, who failed to defeat Arthur for a second consecutive time in the February 21 general election.
"Arthur will not run again, but Haynesley will not win a seat in St. Peter. Haynesley is my friend and I really don't want to attack him, but Haynesley is saying that the DLP caused him to lose, the DLP did not cause him to lose, he would lose anyhow," Holder told Barbados TODAY.
Speaking on Sunday during a meeting of his Democratic Labour Party branch at the All Saints Skills Training Centre at Mile-And-A-Quarter, Benn criticised his party's treatment of the northern constituency, and said he intended to be the candidate whenever the next election was held there.
"The party must therefore be informed, as soon as we have our business meeting, … that Haynesley Benn intends to be the candidate next time, whether there's a by-election or a general election. And don't try to entertain the thought of doing what you did in St. James North or what you did in St. Peter in 1991," he warned.
Just yesterday after he was sworn in as St. Peter MP, Arthur told the media he intended to serve his full five-year parliamentary term.
"I will never step down from St. Peter, I was elected to serve and it would be a slap in the face of the people of St. Peter for me to say I am giving up. I asked to be elected and I don't feel that you should do those things, (if) you are asked to be elected you serve," he said at Government House.
Today, Holder, who has been involved in Arthur's election campaigns since the 1980s, and was campaign manager for new St. George South MP Dwight Sutherland, said while Benn was right about the DLP's long neglect of St. Peter, he was wrong if he thought he would ever be a victorious election candidate there.
"The performance of the DLP in St. Peter is the epitome of the neglect of one government against a segment of people. The history is there, Errol Barrow said that Boscobelle is off the map and Black Bess is behind God's back. In other words they don't care about you and this has always been the lot of St. Peter under the DLP, they just do nothing," Holder said.
"He is claiming a road building project which was part of the Pleasant Hall to Speightstown rehabilitation project, they just did not get around to completing the one from Ashton Hall, which was really the cutting down of Station Hill, but to claim it is just ingenious.


The pink beige striped summer cream espadrille makes you look cool. Because of its color and design, your always on the trend. The sturdy striped cotton canvas, hand stitched to the sole, comes from the city of Valencia.


Hi! My name is Mrs. Lisa Jensen (formally known as Ms. Craft) and I will be your child's teacher this year. This is my fourth year teaching at Rosedell Elementary and I love being part of such a wonderful school! I have lived in the Santa Clarita Valley my whole life and even graduated from Bouquet Canyon Elementary many years ago.
My background in education includes graduating with my Bachelor of Arts degree in English from the University of Southern California (USC). After my undergraduate education, I received my multiple subject teaching credentials from California State University at Northridge and National University. I completed my Master's of Arts degree in Teaching with an emphasis in Educational Technology from National University.
I have been a teacher in some aspect or another for the past 13 years. Before becoming a 4th grade teacher here at Rosedell Elementary, I was a 4th grade intervention teacher, a 3rd grade teacher, a substitute teacher and a home hospital teacher.
At home, I am a mother to two sweet girls who are 9 and 11 years old. They keep me busy with their school and extracurricular activities. I also love animals and personally I have three basset hound dogs, one guinea pigs and a fish.
If you have any questions or concerns please email me at ljensen@saugususd.org. I am looking forward to a great school year!