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CXCL10+ T cells and NK cells assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes during Mycobacteria-enhanced colitis
Udai P Singh1,
Rajesh Singh2,
Shailesh Singh2,
Russell K Karls3,
Frederick D Quinn3,
Dennis D Taub4 &
James W Lillard Jr1,2
The role of Mycobacteria in the etiology of Crohn's disease (CD) has been a contentious subject for many years. Recently, our laboratory showed that spontaneous colitis in IL-10-/- mice is driven in part by antigens (Ags) conserved in Mycobacteria. The present study dissects some of the common cellular and molecular mechanism that drive Mycobacteria-mediated and spontaneous colitis in IL-10-/- mice.
We show that serum from inflammatory bowel disease (IBD) patients contain significantly higher levels of Mycobacterium avium paratuberculosis-specific IgG1 and IgG2 antibodies (Abs), serum amyloid A (SAA) as well as CXCR3 ligands than serum from healthy donors. To study the cellular mechanisms of Mycobacteria-associated colitis, pathogen-free IL-10-/- mice were given heat-killed or live M. avium paratuberculosis. The numbers of mucosal T cells, neutrophils, NK/NKT cells that expressed TNFα, IFN-γ, and/or CXCL10 were significantly higher in mice that received live Mycobacteria than other groups. The numbers of mucosal CXCR3+, CXCL9+, CXCL11+ and/or IFN-γ+ dendritic cells (DCs) were also significantly higher in M. avium paratuberculosis-challenged mice, than compared to control mice.
The present study shows that CD and UC patients mount significant Mycobacteria-specific IgG1 > IgG2 and CXCR3 ligand responses. Several cellular mechanisms that drive spontaneous colitis also mediate Mycobacteria-enhanced colitis in IL-10-/- mice. Similar to IL-10-/- mice under conventional housing, we show that Mycobacteria-challenge IL-10-/- mice housed under otherwise pathogen-free conditions develop colitis that is driven by CXCR3- and CXCR3 ligand-expressing leukocytes, which underscores another important hallmark and molecular mechanism of colitis. Together, the data show that Mycobacteria-dependent host responses, namely CXCL10+ T cells and NK cells, assist in the recruitment and activation of CXCR3+ and CXCL11+ leukocytes to enhance colitis of susceptible hosts.
Although increasing evidence suggests that intestinal flora is involved in the pathogenesis of IBD, to date no specific bacterial pathogen has been identified as the cause of this disease. The role of Mycobacteria in the etiology of IBD has been a contentious subject for many years. The possible association between M. avium paratuberculosis and CD in humans was first suggested nearly 100 years ago because Johne's disease (JD) and CD have similar histological and pathological features with affected tissues containing granulomas [1]. Interest in a causal association between JD and CD was stimulated in the 1980s after M. avium paratuberculosis was isolated from material taken from patients with CD [1, 2]; subsequent studies confirmed these results [3, 4]. This premise is further supported by successful CD treatment with anti-mycobacterials containing macrolide antibiotics [5] and from the sero-reactivity of the CD patient serum samples against M. avium paratuberculosis [6]. Recently, we reported that humoral and cellular responses during spontaneous colitis in IL-10-/- mice are driven in part by conserved Ags in Mycobacterium species [7]. Together, these studies form a wide body of evidence that supports the potential role for Mycobacteria in CD. However, an incomplete understanding of the cellular mechanisms responsible for the pathology of colitis makes this conclusion uncertain.
There is a consensus that the mucosa of CD patients is dominated by Th1 cell producing inflammatory cytokines [8, 9]. CXCR3 interactions with CXCL9, CXCL10 and CXCL11 are important for the selective homing of Th1 effectors cells [10], which mediate mucosal immunity, inflammation, and colitis [11, 12]. IL-10-/- mice develop spontaneous colitis that has similarities to human CD at ~3 months of age under conventional housing conditions which can be abrogated by anti-CXCL10 Ab treatment. The present study explores the CXCR3 axis and cellular mechanisms that drive Mycobacteria-associated colitis in IL-10-/- mice. We also report that serum from both CD and UC patients contain significantly higher Mycobacteria-specific IgG1 and IgG2 Ab responses as well as CXCL9, CXCL10, CXCL11 and SAA levels than compared to normal healthy donors. These innate and adaptive immune responses coincide with Mycobacteria-specific humoral and cellular responses and modulation of mesenteric lymph node (MLN), Peyer's patch (PP) and lamina propria (LP) Th1-associated cytokines and CXCR3 ligands expressed by DCs, T cells, neutrophils and NK/NKT cell subsets following Mycobacteria challenge of otherwise specific pathogen-free mice.
Immunogens
M. avium paratuberculosis strain Ben (CIP 103966), a clinical isolate from a CD patient, was obtained from the American Type Culture Collection (ATCC# 43544). Bacteria were cultured in Middlebrook 7H9 broth supplemented with 10% ADC (BD/Difco) and 2 μg/ml mycobactin J (Allied Monitor) to an OD580 of 0.5 and frozen. The viable titer of these stocks was determined by thawing replicates, serial dilution in culture medium and plating on Middlebrook 7H10 agar supplemented with 2 μg/ml of mycobactin J. For detection of Mycobacteria-specific responses, M. avium paratuberculosis bacilli were fixed with 2% paraformaldehyde.
Sera collection
Sera from 62 CD and 88 UC female patients and 32 normal healthy female donors were collected by Clinomics Biosciences, Inc. The ages of the patients were 20 to 41 years. Diagnoses were based on clinical, radiological, endoscopic, and histological criteria. These patients did not receive any steroid treatment before blood was drawn. All subjects gave written informed consent and the study was approved by the Clinomics Biosciences Research Ethics Committee, (Pittsfield, MA, USA). Subsequently, the University of Louisville Institutional Review Board (IRB) approved the use of these diagnostic specimens in accordance with the Department of Health and Human Service Policy for the Protection of Human Research Subjects 45 CFR 46.101(b) 2 and use of archived de-identified materials.
Mycobacterium-specific Ab detection by ELISA
Mycobacteria-specific IgG Ab responses in the serum of human patients were measured by ELISA, according to manufacturer's protocol (Invitrogen-Zymed). Briefly, 96-well Falcon ELISA plates (Fisher Scientific) were coated with 100 μl of 1 μg/ml of heat-killed and paraformaldehyde-fixed M. avium paratuberculosis in coating buffer (sodium carbonate-bicarbonate buffer) or serially diluted human IgG1, IgG2, IgG3, and IgG4 isotype controls overnight at 4°C and blocked with 200 μl of 10% FBS (Atlanta Biologicals) in PBS (FBS-PBS) for 2 hours at room temperature. Individual samples were added and serially diluted in FBS-PBS. After 4 hours incubation at room temperature, the plates were washed (3 times), and the concentrations of IgG subclass Abs were determined by the addition 100 μl of biotin-conjugated mouse anti-human γ1 (Clone 8c/6-39 at 12.5 ng/ml), γ2 (Clone HP-6014 at 125 ng/ml), γ3 (Clone HP-6050 at 12.5 ng/ml), and γ4 (Clone HP-6025 at 50 ng/ml) heavy-chain-specific monoclonal Abs. After the incubation and washing steps, 100 μl of 0.5 μg/ml horse radish peroxidase (HRP)-conjugated anti-biotin Ab (Vector Labs) in FBS-PBS was added to IgG subclass Ab detection wells, and the plates were incubated for 2 hours at room temperature. Following incubation, the plates were washed 6 times and developed by adding 100 μl of ABTS solution and read at 415 nm. The Mycobacterium-specific ELISA assays were capable of detecting 8 pg/ml for human IgG subclass samples.
Chemokine analysis by ELISA
Serum concentrations of human CXCL9, CXCL10 and CXCL11 were determined by ELISA (R&D Systems), according to the manufacturer's instructions. In brief, 96-well ELISA plates were coated with capture Abs, and the plates were incubated overnight at room temperature. After blocking, 100 μl of sample or standards were added to each well, and the plates were incubated for 2 hours at room temperature. Then, 100 μl of detection Abs solution was added to each well, and the plates were further incubated for 2 hours at room temperature. After washing the plates, 100 μl of streptavidin-HRP solution was added, and the plates were incubated for 20 min in the dark. Next, 100 μl of substrate solution was added to the plates, which were incubated for an additional 20 min at room temperature in the dark. Finally, 50 μl of the stop solution was added, and the plates were read at an optical density of 450 nm after 30 min using λ corrections of 540 and 570 nm. The ELISA assays were capable of detecting >10 pg/ml of each chemokine.
SAA ELISA
SAA levels were determined by ELISA kit (Biosource International, Camarillo, CA). In brief, 50 μl of SAA-specific mAb solution was used to coat micro-titer strips to capture SAA. Serum samples and standards were added to wells and incubated for 2 hr at RT. After washing in the assay buffer, the HRP-conjugated anti-SAA mAb solution was added, and the plates were incubated for 1 hr at 37°C. After washing, 100 μl tetra-methyl-benzidine (TMB) substrate solution was added, and the reaction was stopped after incubation for 15 minute at RT. After the addition of stop solution, the plates were read at an optical density of 450 nm.
Animals and M. avium paratuberculosis challenge
Female 4 to 5 weeks old IL-10-/- mice, on B6 background, were purchased from Jackson Laboratories (Bar Harbor, ME) and used to establish a colony at the Morehouse School of Medicine animal facility. The animals were housed and maintained in isolator cages under specific-pathogen-free housing conditions. The guidelines proposed by the Committee for the Care of Laboratory Animal Resources Commission of Life Sciences-National Research Council were followed to minimize animal pain and distress. To determine the cellular responses enhanced by Mycobacteria, groups of naïve IL-10-/- mice received 200 μl of 104 CFU live or heat-killed (pasteurized; 65°C for 2 hours) M. avium paratuberculosis in cream milk or cream milk alone (control) by oral gavage. Mice were sacrificed by CO2 inhalation 14 weeks post inoculation. Each experimental group consisted of 5 to 10 mice.
Cell isolation
Spleens and MLN from individual mice were mechanically dissociated, and red blood cells were lysed with ACK lysis buffer (Sigma), 14 weeks after challenge. Single cell suspensions of the spleens and MLN were passed through a sterile wire screen (Sigma). Cell suspensions were washed twice in RPMI 1640 and stored on ice in complete medium containing 10% FBS until use. Cells from the LP and PP were isolated as described previously [13]. After removal, PP were kept on ice-cold RPMI containing 2% FBS. Intestines were cut into 1 cm stripes and stirred in PBS containing 1 mM EDTA at 37°C for 30 min. In brief, the intestinal LP was isolated by digesting intestinal tissue in collagenase type IV (Sigma) in RPMI 1640 (collagenase solution), for 45 min with stirring at 37°C. After each 45-minute interval, the released cells were centrifuged and stored in complete medium, and remaining mucosal pieces were replaced with fresh collagenase solution. LP lymphocytes were further purified using a discontinuous percoll (Pharmacia, Uppsala, Sweden) gradient, collecting between the 40/75% percoll interface. Leukocytes were maintained in a complete medium, which consisted of RPMI 1640 supplemented with 10 ml/L of nonessential amino acids (Mediatech, Washington, DC), 1 mM sodium pyruvate (Sigma), 10 mM HEPES (Mediatech), 100 U/ml penicillin, 100 μg/ml streptomycin, 40 μg/ml gentamycin (Elkins-Sinn, Inc., Cherry Hill, NJ), 50 μM mercaptoethanol (Sigma), and 10% FBS (Atlanta Biologicals).
Flow cytometry analysis
Leukocytes from the spleen, MLN, PP, and LP were isolated as described above and stained to measure ex vivo expression of chemokines and cytokines, 14 weeks after challenge. Cells were first treated with Fc block (BD PharMingen, San Diego CA) for 15 min at 4°C, washed with staining buffer, and then stained with CY-, FITC- or PE-conjugated anti-CD3 (145-2C11), -CD4 (H129.19), -CD8 (LY-2 53-6.7), -CD11b (M1/70), -CD11c (HL3), and -NK1.1 (PK136) (BD-PharMingen, San Diego CA) for 30 min with shaking. The cells were washed 2 times with staining buffer and resuspended with BD Cytofix/Cytoperm (BD-PharMingen) solution for 10 min each. The cells were again washed 2 times in BD perm/wash solution at 4°C for 10 min each. The fixed, permeabilized cells were then resuspended in a pre-determined APC-conjugated anti-CXCL10, CXCL9, CXCL11, TNF-α, IFN-γ, or CXCR3 Ab for 30 min at 4°C in the dark. Lymphocytes were then washed with FACS staining buffer (PBS with 1% BSA) and fixed in 2% paraformaldehyde and analyzed by flow cytometry (Becton Dickinson, San Diego, CA).
Intestinal tissues were preserved using Streck™ fixative (Streck Laboratories, LaVista, NE) and embedded in paraffin. Fixed tissues were sectioned at 6 μm and stained with hematoxylin and eosin for microscopic examination. Intestinal lesions were multi-focal and of variable severity. The grades given to intestinal sections that took into account the number of lesions as well as their severity. A score (0 to 4) was given, based on the criteria described previously [11]. The summation of these scores provided a total colonic disease score per mouse. The disease score could range from 0 to a maximum of 12 (with Grade 4 lesions in ascending, transverse, and descending colon segments).
The data expressed as the mean ± SEM and compared using a two-tailed paired student's t-test or an unpaired Mann Whitney U-test. The results were analyzed using the Statview II statistical program (Abacus Concepts, Inc., Berkeley, CA) and Microsoft Excel (Microsoft, Seattle, WA) for Macintosh computers. Single-factor and two-factor variance ANOVA analyses were used to evaluate groups and subgroups, respectively. Hence, results were considered statistically significant if p values were < 0.01.
Changes in IgG subclass Ab profile in IBD patients
Previously, we have shown that serum Mycobacteria-specific IgG2a Ab responses were higher in IL-10-/- mice with spontaneous colitis, than compared to control mice [7]. This previously described imbalance of the Th1 > Th2 cytokine levels as well as humoral response (IgG2a > > IgG1) suggested that a Th1-biased host response is associated with the progression of colitis. To further test this hypothesis in human subjects, we measured levels of Mycobacteria-specific IgG subclass Abs in the sera of IBD patients and healthy donors. While total IgG1, IgG2, IgG3, and IgG4 subclass Abs were significantly higher in the sera of IBD patients compared to healthy donors (data not shown), the profile of the IgG humoral response in IBD patients also revealed increases in Mycobacteria-specific IgG1 and IgG2 Abs (Figure 1). These responses in CD patients were significantly higher than in UC patients or normal healthy donors. CXCR3 ligands were also increased in these samples than compared to healthy donors. These results suggest that IBD patients have higher CXCL9, CXCL10, and CXCL11 levels and Mycobacteria-specific IgG1 and IgG2 Ab responses. These findings correlate well with our previous findings showing higher levels of Mycobacteria-specific IgG2a and CXCR3 ligands during spontaneous colitis in IL-10-/- mice under conventional housing [7].
Serum CXCR3 ligands and mycobacterial -specific Ab responses in IBD patients. Sera from 62 CD and 88 UC female patients and 32 normal healthy female donors, not undergoing any treatment, were isolated and evaluated for the presence of CXCR3 ligands (i.e., CXCL9, CXCL10, and CXCL11) and mycobacterial-specific IgG1, IgG2, IgG3 and IgG4 Abs. These levels were determined by ELISAs capable of detecting 10 > pg/ml of these ligands. The data presented are concentrations ± SEM. Asterisk(s) indicate statistically significant differences, i.e., p < 0.01, between healthy donors and IBD patients.
Characteristics of colitis progression
We also measured the SAA levels of IBD patients and healthy donors; CD patients displayed significantly higher SAA levels compared to other groups, which correlated with the higher Mycobacteria-specific IgG2a levels described above. Colitis progression in IL-10-/- mice closely corresponds with increased SAA levels (≥ 250 μg/ml) and with reduction in body weight, compared with the initial body weight [9, 11, 12]. The results of the present study show that mice challenged with live Mycobacteria in otherwise specific pathogen-free conditions experienced a significant rise in SAA levels when compared to similar mice challenged with heat-killed Mycobacteria or control mice (Figure 2). Despite the significant increase in SAA and more pronounced reduction in body mass, both live and heat-killed Mycobateria challenged IL-10-/- mice developed histologically similar colitis than compared to controls, as illustrated by significantly higher colitis disease scores (Table 1). The intestinal tissues of mice challenged with Mycobacteria showed higher increases in leukocyte infiltrates, which consisted of lymphocytes and occasionally polymorphonuclear cells as well as a higher frequency of lymphoid follicles in live versus heat-killed Mycobacteria-challenged groups (Figure 3). In short, colitis was more aggressive in mice that received live Mycobacteria, as noted by multi-focal lesions and aggregates of leukocyte infiltrates in the large intestines, than compared to control mice.
Change in SAA levels in IBD patients and in IL-10-/- mice after mycobacterial challenge. Sera from 62 CD and 88 UC female patients and 32 normal healthy female donors, not undergoing any treatment, were isolated and evaluated for the presence of SAA. IL-10-/- mice on B6 background, received 200 μl of cream milk alone (○; control) or cream milk containing 104 CFU of live (●) or heat-killed (▲) M. avium paratuberculosis. SAA levels during Mycobacteria-enhanced colitis as well as IBD patients and healthy donors were measured by ELISA. Experimental groups consisted of 5 mice, and experiments were repeated 3 times. The data presented are the mean ± SEM concentration of SAA. Asterisks indicate statistically significant differences, i.e., p < 0.01, between control and Mycobacteria-treated groups or healthy donors and IBD patients.
Intestinal histological characteristics of IL-10-/- mice challenged with Mycobacteria. IL-10-/- mice on B6 background, received 200 μl of cream milk alone (○; control) or cream milk containing 104 CFU of live (●) or heat-killed (▲) M. avium paratuberculosis. After sacrifice, intestines were fixed, sectioned at 6 μm, and stained with hematoxylin and eosin. Sections were examined by light microscopy. Experimental groups consisted of 5 mice and experiments were repeated 3 times.
Table 1 Histological evaluation of Mycobacteria-enhanced colitis in IL-10-/- mice
CXCR3, CXCL11, IFN-γ, and/or TNF-α expression by CD4+ and CD8+ T cells
To address the leukocyte subsets responsible for colitis progression, we assessed the ex vivo expression of chemokines and cytokines by MLN-, PP-, and LP-derived cell subsets in IL-10-/- mice challenged with live or heat-killed M. avium paratuberculosis bacilli. The numbers of CD4+ and CD8+ T cells from MLN, PP, and LP lymphocytes that expressed CXCR3, IFN-γ, and TNF-α from mice challenged with live bacilli were significantly higher than similar mice challenged with heat-killed bacilli or the control group. Dramatic increases in the number of PP CXCL11+ T cells, PP and LP CXCR3+ T cells and PP TNF-α+ T cells as well as MLN TNF-α+ CD8+ T cell subsets were observed in mice challenged with live bacilli than compared to other groups (Figure 4). PP, MLN, and LP IFN-γ+ CD4+ T cells as well as MLN and LP IFN-γ+ CD8+ T cells were significantly elevated after livemycobacterial challenge. These results imply that intestinal inflammation is driven in part by effector site-derived (PP and LP) CXCR3+ TNF-α+ CD4+ and CD8+ T cells along with similar cells from PP that produce CXCR3 ligands as well as MLN (inductive site) IFN-γ+ T cells to presumably support Th1-biased colitis.
Changes in the T cell populations expressing CXCR3, IFN-γ, TNF-α, and CXCR3 ligands in IL-10-/- mice after mycobacterial challenge. MLN, PP, and LP lymphocytes were isolated from IL-10-/- mice challenged with cream milk alone (control) or 104 CFU of heat-killed or live M. avium paratuberculosis in cream milk. Changes in the frequency of LP CXCR3+ CD4+ T cells from heat-killed or live Mycobacteria-challenged and control mice (Panel A). Changes in the number (± SEM) of total IFN-γ-, TNF-α-, CXCR3-, CXCL9-, CXCL10- or CXCL11-expressing CD4+ and CD8+ T cell lymphocytes from control mice as well as heat-killed or live Mycobacteria-challenged IL-10-/- mice are shown in Panel B. Asterisks indicate statistically significant differences, i.e., p < 0.01, between control and Mycobacteria-treated groups.
Alteration of DC cytokine and CXCR3 ligands expression following Mycobacteria challenge
To further address the adaptive versus innate leukocyte subsets responsible for Mycobacteria-enhanced colitis in IL-10-/- mice, we next assessed myeloid and plasmacytoid DC phenotypes from MLN, PP, and LP after disease onset. PP and MLN DCs expressed elevated levels of CXCL9 and CXCL11, but not CXCL10, during Mycobacteria-enhanced colitis as indicated by their relative mean fluorescent intensities (Figure 5A). Interestingly, both DC subsets heterogeneously expressed CXCL9. The number of PP CXCR3+ DCs was significantly higher in live Mycobacteria-challenged mice than in other groups (Figure 5B). However, the number of MLN CXCR3+ plasmacytoid DCs was significantly higher in mycobacterial-challenged mice than in other groups of mice. The numbers of MLN and LP DCs subsets expressing CXCL9 and CXCL11 after the onset of colitis were significantly increased during live Mycobacteria-enhanced colitis than in the other groups with the exception of MLN CXCL10+ myeloid DCs that were elevated following live bacterial challenge. While the DCs subsets studied did not appear to significantly express TNF-α, there were significantly more numbers of MLN and PP CD8α+ DCs and only PP-derived CD8α- DCs that expressed IFN-γ from mice with live Mycobacteria-enhanced colitis, than compared to other groups. LP plasmacytoid DCs from mice receiving live bacteria were also increasingly IL-12p40+, than compared to other groups.
Changes in CD8α + and CD8α - CD11b+CD11c+ cells expressing CXCR3, IFN-γ, TNF-α, IL-12p40, and CXCR3 ligands in IL-10-/- mice after mycobacterial challenge. MLN, PP, and LP lymphocytes were isolated from IL-10-/- mice challenged with cream milk alone (control) or cream milk containing 104 CFU of live or heat-killed M. avium paratuberculosis. Changes in the mean fluorescent intensities of CXCL9, CXCL10, and CXCL11 expressed by MLN CD8α+ and CD8α- CD11b+CD11c+ cells from control mice (shaded histogram) and heat-killed (dotted outlined histogram)- or live Mycobacteria (solid outlined histogram)-challenged mice are shown in Panel A. Changes in the number (± SEM) of CXCR3-, IFN-γ-, TNF-α-, IL-12p40-, CXCL9-, CXCL10- or CXCL11-expressing CD8α+ and CD8α- CD11b+CD11c+ cells from control mice as well as live or heat-killed Mycobacteria-challenged IL-10-/- mice are shown in Panel B. Asterisks indicate statistically significant differences, i.e., p < 0.01, between control and Mycobacteria-treated groups.
CXCR3, IFN-γ, TNF-α, IL-12, CXCL9, CXCL10 and CXCL11 expression by NK and NKT cells during Mycobacteria-enhanced colitis
NK cells act as regulators of CD4+ T cell-driven colitis [14]. While neither NK nor NKT cell subsets largely expressed CXCR3, there were substantially more CXCL10+ NK cells in the MLN, PP, and LP during Mycobacteria-enhanced colitis, than compared to other groups (Figure 6). IL-12p40 expression and the number of NK cell subsets were elevated in mice challenged with live Mycobacteria than compared to mice challenged with heat-killed Mycobacteria or control mice. Whereas, greater numbers of IFN-γ+ NK and NKT cells were confined to the MLN and LP in live Mycobacteria-challenged group than compared to other groups. In contrast, we noticed a modest decrease in the number of PP CXCR3+, IFN-γ+, CXCL9+ and CXCL11+ NK cells during live Mycobacteria-enhanced colitis. These results suggest that together NK and NKT cells express CXCL10, IL-12p40, TNF-α and IFN-γ to recruit and activate CXCR3+ leukocytes to inductive and effector sites during Mycobacteria-enhanced colitis.
Changes in NK and NKT cells of IL-10-/- mice challenged with Mycobacteria. MLN, PP, and LP lymphocytes were isolated from IL-10-/- mice challenged with cream milk alone (control) or cream milk containing 104 CFU of live or heat-killed M. avium paratuberculosis. Changes in the mean fluorescent intensities of IL-12p40, IFN-γ, and TNF-α expressed by LP CD3+ and CD3- NK1.1+ cells from control mice (shaded histogram) or heat-killed (dotted outlined histogram)- and live MAP (solid outlined histogram)-challenged mice are shown in Panel A. Changes in the number (± SEM) of CXCR3-, IFN-γ-, TNF-α-, IL-12p40-, CXCL9-, CXCL10- or CXCL11-expressing by CD3+ and CD3- NK1.1+ cells from control mice and live or heat-killed Mycobacteria-challenged IL-10-/- mice are shown in Panel B. Asterisks indicate statistically significant differences, i.e., p < 0.01, between control and Mycobacteria-treated groups.
Th1-associated cytokines, CXCR3 and CXCR3 ligands expressed by neutrophils
Neutrophils are among the first cells to arrive at sites of inflammation and infection. These cells also have the capacity to release CXCL10 [15] and are present during Mycobacteria-enhanced colitis. LP neutrophils significantly expressed TNF-α, in a heterogenous fashion, following live mycobacterial challenge (Figure 7A). TNF-α+ neutrophils were largely retained in the LP during disease when compared to other groups. MLN and LP, but not PP or spleen, contained higher numbers of TNF-α+ and IFN-γ+ neutrophils during Mycobacteria-enhanced colitis (Figure 7B). In contrast, higher numbers of IL-12p40+ and CXCL10+ LY6G+ cells were found in PP and spleen, but not in MLN of LP from live Mycobacteria-challenged mice relative to control mice and heat-killed Mycobacteria-challenged mice. However, greater numbers of CXCL9+ and CXCL11+ neutrophils were found in MLNs of live bacteria-challenged mice, than compared to other groups. These data suggest that neutrophils are compartmentalized and tightly regulated to differentially express CXCR3 ligands, CXCR3 and Th1-promoting cytokines during Mycobacteria-driven colitis.
Changes in cell populations of neutrophils during Mycobacteria -enhanced colitis. Spleen, MLN, PP, and LP lymphocytes were isolated from IL-10-/- mice challenged with cream milk alone (control) or cream milk containing 104 CFU of live or heat-killed M. avium paratuberculosis. Changes in the mean fluorescent intensities of IL-12p40, IFN-γ, and TNF-α expressed by LP CD3- LY6G+ cells from control mice (shaded histogram) or heat-killed (dotted outlined histogram)- and live Mycobacteria (solid outlined histogram)-challenged mice are shown in Panel A. Changes in the number (± SEM) of CXCR3-, IFN-γ-, TNF-α-, IL-12p40-, CXCL9-, CXCL10- or CXCL11-expressed by CD3- LY6G+ cells from control mice and live or heat-killed Mycobacteria-challenged IL-10-/- mice are shown in Panel B. Asterisks indicate statistically significant differences, i.e., p < 0.01, between control and Mycobacteria-treated groups.
Although there is no recognized or agreed on infectious agent for IBD, interest in M. avium paratuberculosis as a possible causative began due to similar signs and symptoms of JD and CD. For example, chronic granulomatous and intramural inflammation is common in both CD and JD; however, the precise pathophysiologic mechanisms sustaining inflammation in these diseases are imprecisely known. In confirmation with others [16, 17], we show an increase in Mycobacteria-specific IgG1 and IgG2 Abs in sera from patients with active CD or UC. While the cellular mechanism(s) and response(s) mediating Mycobacteria-enhanced IBD have not been entirely demonstrated, recently we showed that colitis in IL-10-/- mice was driven in part by 85B Ag-specific CD4+ T cell responses [7]. As M. avium paratuberculosis is a mycobactin auxotroph and is not known to replicate outside a host, spontaneous colitis of IL-10-/- mice upon transfer to conventional housing is possibly the result of exposure to Mycobacterium species, which are commonly found in soil and water. We illustrate several cellular mechanisms that may drive Mycobacteria-enhanced colitis in IL-10-/- mice. Mycobacteria-challenged IL-10-/- mice housed under otherwise pathogen-free conditions develop colitis that is driven by CXCR3- and corresponding ligand(s)-expressing leukocytes, which underscores another important hallmark or molecule mechanism of colitis.
The expression of CXCR3 and its ligands (CXCL10 >> CXCL11 or CXCL9) by mucosal T cells significantly increased in mice challenged with live Mycobacteria than compared with similar mice challenged with heat-killed Mycobacteria or controls. Our results also suggest plasmacytoid > > myeloid DCs express CXCL9 and CXCL11, but less CXCL10 as well as IFN-γ to recruit, differentiate, and expand Th1 cells to mediate colitis. NK cell and neutrophil subsets were compartmentalized in mucosal inductive and/or effector site to further foster adaptive Th1 responses through IFN-γ and IL-12p40 production as well as TNF-α expression during colitis. Relatively high levels of serum TNF-α and IFN-γ as well as Ag-specific Th1 responses were also noted in this model [7]. These findings suggest an association between Mycobacteria and CD patients as well as IL-10-/- murine colitis.
It is well established that IL-12 drives Th1 differentiation and subsequent IFN-γ production [18]. We demonstrate that local and/or systemic expression of the TNF-α, IFN-γ, and CXCR3 ligands are increased during live Mycobacteria-enhanced colitis. TNF-α is a proinflammatory cytokine produced by leukocytes at local sites of inflammation [19]. This cytokine is also important for the activation of granulocytes and fibroblasts during CD [20]. LP lymphocytes produce significant amounts of TNF-α in IBD tissues and secretary fluids [21–23]. Perhaps the production of CXCR3 ligands along with IFN-γ and TNF-α during colitis progression creates a situation in which Th1 cells are recruited, activated, further differentiated, and expanded to initiate or maintain disease. While TNF-α produced by CD4+ T cells is neither sufficient nor required for the induction of murine colitis, its production by Ag-presenting cells is essential for the histopathological and clinical signs of colitis [24]. We show that NK cell subsets and neutrophils, compared to T cells, expressed the greatest levels of TNF-α during Mycobacteria-mediated colitis, which is also a hallmark of spontaneous murine colitis.
The presence of CXCL10 is considered an important signal for selective homing and/or activation of effector cells, which preferentially accumulate at inflammatory sites [25]. Previously, we showed CXCL10 enhanced CD28 and CD40 expression by Ag-activated CD4+ T cells [12] and DC activation [26]. Plasmacytoid DCs have an inherent capacity to produce large amounts of IFN-γ and DC activation is a critical step and is reported to be higher in CD patients [27]. Intestinal LP DCs prime T cells and home to MLN for further activation and clonal expansion of T cells [28, 29]. DCs can be found in the intestinal mucosal [30] and have also been shown to produce and respond to CXCR3 ligands [31]. In the present study, we show that CXCL9 and CXCL11 are predominantly produced by DCs in the MLN as well as in PP and to a lesser degree in the intestinal LP. The selective expression of CXCL9 and CXCL11 by plasmacytoid and myeloid DCs suggests a differential role for these cells in the attraction of specific lymphocyte subsets [10] as well as in the preferential recruitment and activation of Th1 cells [11, 32]. In a recent study, mature DC-derived CXCR3 ligands were shown to be essential in Th1 cell retention in draining lymph nodes and to optimize Th1-mediated immune responses [33].
NK cells have been shown to be involved in the differentiation of naïve CD4+ T cells into Th1 cells [34]. The current study suggests NK and NKT cells collaborate to produce CXCL10, IL-12p40, TNF-α, and IFN-γ to recruit and stimulate CXCR3+ leukocytes in either inductive or effector sites during colitis. In IBD, neutrophil activation, migration, and degranulation are important effector mechanisms of intestinal damage [35]. It has been reported that human neutrophils have the capacity to release CXCL10 [15]. In the present study, changes in the number of neutrophils that expressed CXCR3, IFN-γ, CXCL9, and CXCL11 in the MLN, IL-12p40 and CXCL10 in PP as well as IFN-γ and TNF-α in LP of mice during Mycobacteria-enhanced colitis suggest that neutrophil subsets are differentially compartmentalized and regulated to propagate colitis.
Activation of toll-like receptors (TLRs) leads to the induction of antimicrobial pathways central to innate defense as well as upregulation of Ag presentation molecules and secretion of cytokines that influence the nature of the subsequent adaptive immune response. To this end, TLR signal transduction requires MyD88 [36, 37]; MyD88-/- mice are highly susceptible to infection by M. tuberculosis and colitis [38]. TLR-activated DCs generally favor Th1 responses due in large part to the production of IL-12 [39, 40]. Mycobacteria cell constituents have long been recognized as powerful immunologic adjuvants. It is plausible that Mycobacteria stimulate TLRs to promote colitis in IL-10-/- mice, which presents a scenario where mycobacterial recognition via TLR and MyD88 activate DCs to control Th1 polarization and expansion.
Live orally administered M. avium paratuberculosis do not readily colonize or extensively replicate in the murine host. The ability of this bacterial species to colonize and replicate in man is also questionable. In fact, B6 mice are only modestly susceptible to orally administered M. avium paratuberculosis [41]. Granulomatous lesions containing acid-fast bacteria develop in mesenteric lymph nodes 11 months after oral challenge using 109 CFU. In this study, 104 CFU of live M. avium paratuberculosis initiated the development of colitis, but there was no evidence of infection by histology (data not shown) or bacterial culture [7]. Presumably, IL-10 deficiency or a host that elicits Th1-biased immune responses to microbial challenge supports the development of colitis with either undetectable or no growth of M. avium paratuberculosis.
We speculate that innate (i.e., TLR agonists) and adaptive (e.g., 85B protein) immunity antigens are better presented by live M. avium paratuberculosis or recognized by the Th1-biased host to modulate relevant cellular and molecular mechanisms required by colitis development. Indeed, persistent presentation of flora present in conventional housing is required for colitis in IL-10-/- B6 mice. Hence, heat-killed mycobacteria may not have allowed for this sustained presentation and expression in the host. Additional studies using antibiotics along with or without live or heat-killed bacteria (e.g., Citrobacter rodenticum, Mycobacterium avium hominissuis, etc.) will be necessary to reveal the precise requirements for Mycobacteria-induced colitis.
We can conclude that IBD patients have higher CXCL9, CXCL10, and CXCL11 levels and Mycobacteria-specific IgG1 and IgG2 Ab responses. These findings correlate with our previous findings that show higher levels of Mycobacteria-specific IgG2a and CXCR3 ligands occur during spontaneous colitis in IL-10-/- mice [7]. In short, colitis was more aggressive in IL-10-/- mice that received live Mycobacteria, than compared to similar mice that received heat-killed bacilli or vehicle, housed under specific pathogen-free housing. It can be surmised that IL-12p40+ TNF-α+ IFN-γ+ NK and NKT cells express CXCL10 to recruit and activate CXCR3+ leukocytes to inductive and effector sites during Mycobacteria-enhanced colitis (Figure 8). As with these innate immunity cells, neutrophils are compartmentalized in the MLN and tightly regulated to differentially express CXCL9 and CXCL11, CXCR3 and Th1-associated cytokines during Mycobacteria-mediated colitis. These results further show that murine colitis is driven in part by effector site-derived CXCR3+ TNF-α+ CD4+ and CD8+ T cells as well as inductive site IFN-γ+ T cells to support Th1-biased colitis. This response is enhanced or maintained by high numbers of MLN and PP CD8α+ IFN-γ+ DCs, PP-derived CD8α- IFN-γ+ DCs, and IL-12p40+ LP plasmacytoid DCs. While additional studies will be required to ascertain the precise role of Mycobacteria in the induction and progression of colitis, we report that live mycobacterial challenge accelerates chronic colitis in IL-10-/- mice.
Possible cellular and molecular mechanisms of Mycobacteria -mediated colitis. The differential production of CXCR3 ligands and their recognition by CXCR3+ cells are involved in Mycobacteria-enhanced colitis. Regulatory T cells (Tr1) are critical to maintain tolerance or homeostasis in the presence of commensal flora. In a host (e.g., IL-10-/- or NOD2/CARD15 or TLR polymorphisms) that is deficient in the regulation of tolerance, inactive dendritic cells (iDC) mature and aid in the differentiation of precursor T helper cells (pT) to Th1 cells. These Th1 cells express CXCR3, TNF-α, and IFN-γ, while mature dendritic cells (mDC) and other activated antigen-presenting cells express CXCR3, CXCL9, CXCL11, and IL-12p40 to support Th1 development as well as the recruit of CXCR3- and CXCL10-expressing cells (e.g., CD8+ T cells, polymorphonuclear cells (PMN), natural killer (NK) and NK T cells (NKT)) for the propagation and recurrence of IBD, which correlates with increases in SAA, CXCR3 ligands, and anti-Mycobacteria IgG1 and IgG2 Abs.
BrdU:
5-Bromo-2'-deoxy-uridine
colony forming unit
CXCL9:
MIG, monokine-induced by IFN-γ
CXCL10:
IP-10, IFN-γ-inducible protein 10
I-TAC, IFN-γ-inducible T cell-α chemoattractant
DCs:
JD:
Johne's disease
lamina propria
MLN:
mesenteric lymph nodes
pDCs:
plasmacytoid dendritic cells
Peyer's patches
SAA:
TLR:
toll-like receptor
UC:
ulcerative colitis.
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Bonecchi R, Bianchi G, Bordignon PP, D'ambrosio D, Lang R, Borsatti A, Sozzani S, Allavena P, Gray PA, Mantovani A, Sinigaglia F: Differential expression of chemokine receptors and chemotactic responsiveness of type 1 T helper cells (Th1s) and Th2s. J Exp Med. 1998, 187: 129-34. 10.1084/jem.187.1.129.
Singh UP, Singh S, Taub DD, Lillard JW: Inhibition of IFN-gamma-inducible protein-10 abrogates colitis in IL-10(-/-) mice. J Immunol. 2003, 171: 1401-6.
Singh UP, Singh S, Weaver CT, Iqbal N, McGhee JR, Lillard JW: IFN-γ-inducible chemokines enhance adaptive immunity and oolitis. J Interferon Cytokine Res. 2003, 23: 591-600. 10.1089/107999003322485099.
Lillard JW, Boyaka PN, Taub DD, McGhee JR: RANTES potentiates antigen-specific mucosal immune responses. J Immunol. 2001, 166: 162-9.
Fort MM, Leach MW, Rennick DM: A role for NK cells as regulators of CD4+ T cells in a transfer model of colitis. J Immunol. 1998, 161: 3256-61.
Cassatella MA, Gasperini S, Calzetti F, Bertagnin A, Luster AD, McDonald PP: Regulated production of the interferon-gamma-inducible protein-10 (IP-10) chemokine by human neutrophils. Eur J Immunol. 1997, 27: 111-5. 10.1002/eji.1830270117.
Olsen I, Wiker HG, Johnson E, Langeggen H, Reitan LJ: Elevated antibody responses in patients with Crohn's disease against a 14-kDa secreted protein purified from Mycobacterium avium subsp. paratuberculosis. Scan J Immunol. 2001, 53: 198-203. 10.1046/j.1365-3083.2001.00857.x.
El-Zaatari FA, Naser SA, Hulten K, Burch P, Graham DY: Characterization of Mycobacterium paratuberculosis p36 antigen and its seroreactivities in Crohn's disease. Curr Microbiol. 1999, 39: 115-9. 10.1007/s002849900430.
Trinchieri G: Interleukin-12: a cytokine produced by antigen-presenting cells with immunoregulatory functions in the generation of T-helper cells type 1 and cytotoxic lymphocytes. Blood. 1994, 84: 4008-27.
van Deventer SJ: Chemokine production by intestinal epithelial cells: a therapeutic target in inflammatory bowel disease?. Aliment Pharmacol Ther. 1997, 11 (Suppl 3): 116-20. discussion 120-1.
Mouser JF, Hyams JS: Infliximab: a novel chimeric monoclonal antibody for the treatment of Crohn's disease. Clin Ther. 1999, 21: 932-42. 10.1016/S0149-2918(99)80015-0. discussion 931.
Breese EJ, Michie CA, Nicholls SW, Murch SH, Williams CB, Domizio P, Walker-Smith JA, MacDonald TT: Tumor necrosis factor alpha-producing cells in the intestinal mucosa of children with inflammatory bowel disease. Gastroenterology. 1994, 106 (6): 1455-66.
Reimund JM, Wittersheim C, Dumont S, Muller CD, Kenney JS, Baumann R, Poindron P, Duclos B: Increased production of tumour necrosis factor-alpha interleukin-1 beta, and interleukin-6 by morphologically normal intestinal biopsies from patients with Crohn's disease. Gut. 1996, 39: 684-9. 10.1136/gut.39.5.684.
Baumgart DC, McVay LD, Carding SR: Mechanisms of immune cell-mediated tissue injury in inflammatory bowel disease. Int J Mol Med. 1998, 1 (2): 315-32.
Corazza N, Eichenberger S, Eugster HP, Mueller C: Nonlymphocyte-derived tumor necrosis factor is required for induction of colitis in recombination activating gene (RAG)2(-/-) mice upon transfer of CD4(+)CD45RB(hi) T cells. J Exp Med. 1999, 190: 1479-91. 10.1084/jem.190.10.1479.
Sallusto F, Lanzavecchia A, Mackay CR: Chemokines and chemokine receptors in T-cell priming and Th1/Th2-mediated responses. Immunol Today. 1998, 19: 568-74. 10.1016/S0167-5699(98)01346-2.
Singh UP, Singh S, Singh R, Cong Y, Taub DD, Lillard JW: CXCL10-producing mucosal CD4+ T cells, NK and NKT cells are associated with chronic colitis in IL-10-/- mice, which can be abrogated by anti-CXCL10 antibody inhibition. J Interferon Cytokine Res. 2008, 28: 31-43. 10.1089/jir.2007.0059.
te Velde AA, van Kooyk Y, Braat H, Hommes DW, Dellemijn TA, Slors JF, van Deventer SJ, Vyth-Dreese FA: Increased expression of DC-SIGN+IL-12+IL-18+ and CD83+IL-12-IL-18- dendritic cell populations in the colonic mucosa of patients with Crohn's disease. Eur J Immunol. 2003, 33: 143-51. 10.1002/immu.200390017.
Iwasaki A, Kelsall BL: Localization of distinct Peyer's patch dendritic cell subsets and their recruitment by chemokines macrophage inflammatory protein (MIP)-3alpha, MIP-3beta, and secondary lymphoid organ chemokine. J Exp Med. 2000, 191: 1381-94. 10.1084/jem.191.8.1381.
Sozzani S, Sallusto F, Luini W, Zhou D, Piemonti L, Allavena P, Van Damme J, Valitutti S, Lanzavecchia A, Mantovani A: Migration of dendritic cells in response to formyl peptides, C5a, and a distinct set of chemokines. J Immunol. 1995, 155: 3292-95.
MacPherson GG, Jenkins CD, Stein MJ, Edwards C: Endotoxin-mediated dendritic cell release from the intestine. Characterization of released dendritic cells and TNF dependence. J Immunol. 1995, 154: 1317-22.
Dieu-Nosjean MC, Vicari A, Lebecque S, Caux C: Regulation of dendritic cell trafficking: a process that involves the participation of selective chemokines. J Leukoc Biol. 1999, 66: 252-62.
Taub DD, Longo DL, Murphy WJ: Human interferon-inducible protein-10 induces mononuclear cell infiltration in mice and promotes the migration of human T lymphocytes into the peripheral tissues and human peripheral blood lymphocytes-SCID mice. Blood. 1996, 87: 1423-31.
Sasaki S, Yoneyama H, Suzuki K, Suriki H, Aiba T, Watanabe S, Kawauchi Y, Kawachi H, Shimizu F, Matsushima K, Asakura H, Narumi S: Blockade of CXCL10 protects mice from acute colitis and enhances crypt cell survival. Eur J Immunol. 2002, 32: 3197-205. 10.1002/1521-4141(200211)32:11<3197::AID-IMMU3197>3.0.CO;2-1.
Romagnani S: Induction of TH1 and TH2 responses: a key role for the 'natural' immune response?. Immunol Today. 1992, 13: 379-81. 10.1016/0167-5699(92)90083-J.
Verspaget HW, Pena AS, Weterman IT, Lamers CB: Diminished neutrophil function in Crohn's disease and ulcerative colitis identified by decreased oxidative metabolism and low superoxide dismutase content. Gut. 1988, 29: 223-8. 10.1136/gut.29.2.223.
Medzhitov R, Preston-Hurlburt P, Kopp E, Stadlen A, Chen C, Ghosh S, Janeway CA: MyD88 is an adaptor protein in the hToll/IL-1 receptor family signaling pathways. Mol Cell. 1998, 2: 253-8. 10.1016/S1097-2765(00)80136-7.
Adachi O, Kawai T, Takeda K, Matsumoto M, Tsutsui H, Sakagami M, Nakanishi K, Akira S: Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function. Immunity. 1998, 9: 143-50. 10.1016/S1074-7613(00)80596-8.
Shi S, Nathan C, Schnappinger D, Drenkow J, Fuortes M, Block E, Ding A, Gingeras TR, Schoolnik G, Akira S, Takeda K, Ehrt S: MyD88 primes macrophages for full-scale activation by interferon-gamma yet mediates few responses to Mycobacterium tuberculosis. J Exp Med. 2003, 198: 987-97. 10.1084/jem.20030603.
Trinchieri G: Interleukin-12 and the regulation of innate resistance and adaptive immunity. Nat Rev Immunol. 2003, 3: 133-46. 10.1038/nri1001.
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Veazey RS, Taylor HW, Horohov DW, Krahenbuhl JL, Oliver JL, Snider TG: Histopathology of C57BL/6 mice inoculated orally with Mycobacterium paratuberculosis. J Comp Pathol. 1995, 113: 75-80. 10.1016/S0021-9975(05)80071-4.
The content of this manuscript benefited from many fruitful conversations with members of the University of Georgia at Athens, National Institute on Aging and the University of Louisville. This study was supported in part by funds provided by the Smith & Lucille Gibson Endowment in Medicine, the University of Louisville, American Digestive Health Foundation/American Gastroenterological Association Research Scholar Award and National Institute of Health grants DK58967, RR03034, MD00525, and GM08248. This research was also supported in part by the Intramural Research Program of the National Institute on Aging, National Institutes of Health.
Department of Microbiology, Biochemistry, & Immunology, Morehouse School of Medicine, Atlanta, GA, USA
Udai P Singh
& James W Lillard Jr
Department of Microbiology & Immunology, University of Louisville, Louisville, KY, USA
Rajesh Singh
, Shailesh Singh
Department of Infectious Diseases, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Russell K Karls
& Frederick D Quinn
Laboratory of Immunology, National Institute of Aging, Gerontology Research Center, Baltimore, MD, USA
Dennis D Taub
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Correspondence to James W Lillard Jr.
UPS carried-out all animal studies, quantified serum CXCR3 ligands, performed flow cytometry acquisition and analyzed data with the assistance of SS and RS. RKK and FDQ cultured Mycobacterium avium paratuberculosis, determined the corresponding CFU for challenge. DDT coordinated and performed the CXCR3 ligand and SAA serum ELISA of IBD patients. JWL conceived the study, participated in its design with all authors, coordinated and helped to draft the manuscript with the assistance of all authors. All authors read and approved the final manuscript.
Inflammatory Bowel Disease Patient
Mesenteric Lymph Node
CXCR3 Ligand
Lamina Propria Lymphocyte


text:
import unittest
import os

from baanprint import bwprint

class TestBwprint(unittest.TestCase):
    def test_plugin_count(self):
        plugin_dir = './baanprint/plugins/'
        yapsy_files = len([f for f in os.listdir(plugin_dir)
                          if f.endswith('.yapsy-plugin')])
        plugins = bwprint.get_plugins()
        self.assertIsInstance(plugins, list)
        self.assertEqual(len(plugins), yapsy_files)

#class TestDocTypeDetection(unittest.TestCase):
#    def test_type_detection(self):
#        path = './tests/purchase_orders.bpf'
#        self.assertEqual('PurchaseOrder', bwprint.testtype(path))

class TestConvert(unittest.TestCase):
    def test_convert(self):
        path = './tests/purchase_orders.bpf'
        bwprint.convert(path, './tests/purchase_orders.pdf')
        self.assertTrue(os.path.exists('./tests/purchase_orders.pdf'))

    def test_add_template(self):
        pdf = './tests/purchase_orders.pdf'
        output = './tests/purchase_orders_w_logo.pdf'
        template = './tests/template.pdf'
        bwprint.add_template(pdf, template, output)
        self.assertTrue(os.path.exists('./tests/purchase_orders_w_logo.pdf'))

redpajama_set_name: RedPajamaGithub


Well, folks, there you have it. Marouane Fellaini has left Manchester United Football Club, with the Red Devils taking to Twitter to confirm the news earlier today.
The midfielder now heads to China, to play for Shandong Luneng Taishan FC, where he'll be a huge hit amongst the fans due to the sheer global brand that Fellaini is.
It's tough to summarise his time at Old Trafford, because the fans never really took to him due to his style of play. However, he deserves huge credit for the fact that he has always given 100% for the badge.
After signing a new deal just last summer it looked like Fellaini would be sticking around for a while yet. However, after the change-in-manager, Solskjaer's plan seemingly was always to get him out of the exit door during the current transfer window.
I now genuinely hope that Fellaini goes on to enjoy success and happiness out in China. I respect his play, I respect him as a person and as the old saying goes, once a red, always a red. His contributions will always be fondly remembered, particularly all those semi-final goals!
Good luck and thanks for everything, Marouane!
Manchester United Football Club have reportedly cooled their interest in Antoine Griezmann. That's according to the BBC, who say the 26-year-old from Atletico Madrid is no longer a priority transfer target.


October | 2018 | Movies On UP!
To cap off our month-long Halloween celebration, as promised earlier this week here's a longer discussion of our favorite horror movie from 2017, Andy Muschietti's It.
One last list to go this October and it's our five faves from last year. It's a good list full of a bunch of great movies, especially our number one. To find out what it is, listen up!
To continue the theme of classic horror movies receiving a new coat of paint, Luca Guadagnino's Suspiria hits select theaters this weekend. Since Dave has never sat down to experience Dario Argento's 1977 masterpiece if not now, when?
Dave and I are back with another October list to fill The Void in your lives. This time, it our five favorites from 2016. No need to Hush, we're screaming them from the mountains!
After 40 years, David Gordon Green and Danny McBride are finally bringing us a proper Halloween sequel and after 6 or so years, Dave and I finally bring you a proper Halloween episode.
Horror month rolls on with our five faves from 2015. As it turns out, both Dave and I had a surprise number one. Find out what it is right along with us.


text:
Film Review: Spider-Man: Homecoming (Jon Watts, 2017) ★★★½
(Courtesy of Sony Pictures)
Following his cameo with the Avengers in Civil War, a young Peter Parker (Tom Holland) returns home under the supervision of Tony Stark (Robert Downey Jr). Struggling to adapt to a normal routine, Peter is eager to prove himself as a superhero and goes in search of criminals. However, when a vengeful arms dealer (Michael Keaton) threatens to reveal Peter's true identity, his powers are truly put to the test.
As the third official revival of the Spider-Man franchise, it's safe to say that this superhero needs no introduction. Now back in the hands of it's rightful owners, the Marvel Cinematic Universe picks up exactly where we left it. Having stolen Captain America's shield in some airport shenanigans, Peter has now returned to life with his Aunt May (Marisa Tomei). But having been fitted with a state-of-the-art superhero suit from Tony Stark, Peter is eager to get out and fight crime; tackling bank robbers and petty thieves on the streets of New York. At the same time, Peter picks up on a gang of arms dealers selling something beyond your usual weaponry, Peter stumbles upon arms dealer Adrian Toomes (Keaton).
Forget everything you've seen before. Unlike the Spider-Man introduced by Sam Raimi back in 2002, this Peter Parker is a refreshing take on the geeky highschooler. Peter is obviously Spider-Man in secret, he is also your average kid who just wants to fit in and is constantly battling his own social awkwardness. It is a combination of this and Holland's performance that makes the character so endearing and (equally) amusing. From shooting at the airport fight from Captain America: Civil War on his phone to building a Death Star with his best friend, there is something far more relatable in Holland's representation then we have seen previously. Further to this, Peter is barely in control of his powers and despite his super-duper suit, seems to fall flat on his face on more than one occasion.
As with the majority of superhero narratives, audiences are naturally expected to suspend disbelieve in several instances. True to it's genre conventions, Spider-Man: Homecoming does follow a refreshing take on the comic-book template, there are some very minor plot holes which perhaps are best taken with a pinch of salt (or just ignored entirely. For example, Peter disappears constantly throughout the film - even on school trips when he's meant to be supervised by a teacher. Yet no one thinks to ask where he's been. As such, the genre suggests it is far more of a comedy over anything else.
Much like Ant-Man (Peyton Reed, 2015), the film focuses far more on humour - but this is possibly it's biggest success. Besides the laughs there is also a great deal of heart with some thrill and action thrown in for good measure. Whilst it is quite clearly constructed of your standard narrative building blocks, this does in no way take anything away from the enjoy-ability of the film. However, if you're looking for the darkness and grit of Captain America: The Winter Soldier (Joe and Anthony Russo, 2014), you'll most likely be disappointed...or wonderfully surprised.
Director, Jon Watts, is somewhat of a rookie when it comes to blockbuster movies. Best known for the Kevin Bacon crime/thriller, Cop Car (2015), he makes a seemingly natural step in to the Marvel Cinematic Universe with comedic references from Ferris Bueller and beloved comic book heroes. Just as he pitted children against an adult villain in Cop Car, Watts showcases the same dynamic here when Peter is forced to fight against Adrian Toomes, aka the Vulture.
Sporting his fluffy-collared bomber jacket, we are immediately introduced to Spider-Man's new nemesis. Opening the film to Adrian's backstory of a failing business, it seems that Marvel has attempted to give this particular villain a real purpose; besides 'just wanting to watch the world burn' or seek revenge. Instead, the Vulture is all about the money. Having suffered financial loss directly at the hands of Stark's company, he and his crew now scavenge for alien technology, left behind from The Avengers (Joss Whedon, 2012), and turning them in to deadly weapons for sale on the black market. Sadly, whilst Keaton himself is always compelling, his character felt a little rushed in part. In the CGI heavy finale in which The Vulture and Spider-Man must battle it out, it felt somewhat drawn out and anti-climatic. Keaton's Vulture does, however, provide the movie with some very cool scenes (no spoilers) including his bird-like armour and amusing one-liners. Overall, a worthy opponent for Peter Parker.
All in all, it may not be perfect but it's arguably the best Spider-Man we've seen so far. Including in this success is a brief performance from Zendaya (as Peter's quick witted classmate, Michelle) and a short lived but ever present cameo from Iron-Man (Robert Downey Jr.) himself. It's safe to say that the film gets progressively more enjoyable as it goes on. The story may be nothing new and neither are the characters but you can find comfort in this funny, refreshing take on a legendary superhero narrative. Spider-Man: Homecoming is in cinemas from July 5th and gets a Sophie star rating of 4 out of 5 stars.
Right, that's it for another film review. Are you seeing Spider-Man: Homecoming this week? What are you most looking forward to? As always, I love reading your comments so please do leave me lots of lovely ones down below.
Until next time fellow film fans,
Posted by Sophie Elizabeth at Monday, July 03, 2017
Labels: female film bloggers uk, london film bloggers, marvel bloggers, Marvel film, Marvel movies, spiderman homecoming review, spiderman movie review, spiderman tom holland
Gil Friday, 7 July 2017 at 23:28:00 BST
I'm glad of the positive review as I will be seeing it this weekend :) I'll probably watch Baby Driver again, too since a lot of the other summer films have gotten negative reviews. I hope you're doing all right, Sophie!
Sophie Athawes Saturday, 8 July 2017 at 09:01:00 BST
Ah great! You'll have to let me know what you think. I was very tempted to give it 4 stars because it is very good but there's just a few very minor flaws that probably don't matter but I couldn't unsee haha. I think everyone will really enjoy it! That's great, I really loved Baby Driver even more the second time around. Hope you have a good weekend :)
Gil Sunday, 9 July 2017 at 20:23:00 BST
Now that I've seen it, I understand what you said about the comedy throughout the film. I thought it worked very well as a John Hughes-inspired-teen comedy with a superhero twist :) Oh, Baby Driver was definitely worth another watch! I'm looking forward to Dunkirk later on in July. It'll be good to see Christopher Nolan's direction again :)
Sophie Athawes Monday, 10 July 2017 at 21:18:00 BST
Most definitely - I totally agree. There's a lot of Ferris Bueller type references which I love. With Baby Driver, I'm so pleased I could see it twice - I spotted so much more detail and just LOVED it.
I should be seeing Dunkirk this Thursday - I'm thinking it could be my film of the year so I have very high expectations. I hope it lives up to it :) Will be reviewing it shortly!
Lyn Duncan Saturday, 8 July 2017 at 09:11:00 BST
I just seen this last night! Totally agree it's the best Spider-Man so far, I got a bit bored of the others and I went to this with no expectations as he's my least favourite super hero but it was surprisingly enjoyable and really fun! Loved his friend Ned and was also crushing on Aunt May!!
I seen Baby Driver last week and loved that as well! Don Draper gone rogue with weapons! You summed up John Hamm perfectly! I love him!! It was like nothing I'd seen before, loved all the music in it and the car chases were super fun!
Great reviews as always! Xxx
Ahhh! I'm so glad you liked it. I did wonder if you'd see it :) Haha omg I know. I love that her being hot is like an on going joke in both Civil War and Homecoming.
Ah don't get me started on Jon Hamm in that movie. He is FAR TOO good looking. Thank you so so much for taking the time to read them babe, I really appreciate it xx
Ashley Christie Saturday, 15 July 2017 at 10:33:00 BST
I finally saw Spider-Man and I loved it! I completely agree with the comedy you mentioned here and I can see why it reminds you of Ant-Man now. I think it is my favorite of the Spider-Man films although I really liked Tobey Maguire XD
Ashley Christie
Sophie Athawes Saturday, 15 July 2017 at 19:00:00 BST
Thanks for reading Hun! Glad you liked it! Ah see I wasn't much of a fan of Tobey Maguire but they're very different :) xx
Robyn Vernon Saturday, 15 July 2017 at 18:56:00 BST
I'm glad you liked this film! I really liked the Spider Man with Andrew Garfield and Emma Stone so I was apprehensive as to whether I would like this one but I will definitely be adding this to my list of movies to watch!
Robyn // http://www.midnightandlace.co.uk
Thanks for reading Hun. Yeah I liked the Andrew Garfield ones too so I think you'll like this too :) Tom Holland is really good xx
Summer Hanif Saturday, 15 July 2017 at 20:12:00 BST
I haven't watched this yet but from trailers etc I think I'll like it. Iron Man is probably my favourite avenger too so that's a bonus x
www.owlsinthesummer.blogspot.co.uk
Sophie Athawes Sunday, 16 July 2017 at 16:18:00 BST
Yeah Iron-Man is good in it although he's not in it for long. I'd definitely recommend it :) xx
Kimberly Saturday, 15 July 2017 at 22:32:00 BST
I am absolutely desperate to see this film! I'm so ready for a fresh take on the whole storyline and with a hint of comedy value to it. Fantastic review x
Yaass!! Go see it :) It's very funny and I much prefer it to the other Spider-Man films so far x

redpajama_set_name: RedPajamaCommonCrawl


text:
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DeVincent, Sam (15)
Warshaw, Isadore (12)
Ellington, Duke (8)
National Museum of American History (U.S.). Division of Musical History (6)
Smithsonian Folklife Festival (6)
Du Bois, W. E. B. (William Edward Burghardt) (5)
Goodman, Benny (Benjamin David) (5)
Hartley, Marsden (5)
Armstrong, Louis (4)
Chase, William Merritt (4)
Henri, Robert (4)
National Museum of American History (U.S.). Division of Community Life (4)
Scurlock Studio (Washington, D.C.) (4)
Scurlock, Addison N. (4)
Scurlock, George H. (Hardison) (4)
Scurlock, Robert S. (Saunders) (4)
Smithsonian Institution. Center for Folklife and Cultural Heritage (4)
Washington, Booker T. (4)
Armory Show (1913: New York, N.Y.) (3)
Avery, Milton (3)
Cole, Nat King (3)
Davies, Arthur B. (Arthur Bowen) (3)
Ellsworth, Scott, Dr. (3)
Fitzgerald, Ella (3)
Howard University (3)
Lipchitz, Jacques (3)
Marin, John (3)
Mexicans (3)
Cherokee Indians (2)
Kootenai Indians (2)
10th Naval District Steel Band (1)
Aboriginal Australians (1)
Acoma Indians (1)
Afro-Caribbean cults (1)
Ainu (1)
Almerico, Tony, 1905- (1)
Animal sounds (1)
Appalachians (People) (1)
Arab Americans (1)
Arctic peoples (1)
Ast, Anita (1)
Athapascan Indians (1)
Audio Engineering Society (1)
Aymara Indians (1)
Bach, Johann Sebastian, 1685-1750 (1)
Bannock Indians (1)
Caribbean Area (4)
Shaw (Washington, D.C.) (4)
Boston (Mass.) (2)
Brooklyn (New York, N.Y.) (2)
Disneyland (California) (2)
Harlem (New York, N.Y.) (2)
Hollywood (Los Angeles, Calif.) (2)
Ivoryton (Conn.) (2)
Eliot Elisofon Photographic Archives, National Museum of African Art (5)
Freer Gallery of Art and Arthur M. Sackler Gallery Archives (2)
Smithsonian American Art Museum, Research and Scholars Center (2)
Query: Sheet music -- advertising
313 records — Page 30 of 32
Leo Castelli Gallery
215.9 Linear feet
AAA.leocast
The Leo Castelli Gallery records measure 215.9 linear feet and 0.001 GB and date from circa 1880-2000, with the bulk of the materials dating from the gallery's founding in 1957 through Leo Castelli's death in 1999. The major influence of dealer Leo Castelli and his gallery on the development of mid-to-late twentieth century modern art in America is well-documented through business and scattered personal correspondence, administrative files, exhibition files, extensive artists' files and printed materials, posters, awards and recognitions, photographs, and sound and video recordings. Also included are records for the subsidiary firms of Castelli Graphics and Castelli/Sonnabend Tapes and Films.
Central Files
The Freer Gallery of Art Central Files are comprised mostly of the documentation of routine office procedures. Included in the daily routine were: filling orders for facsimiles of photographs, responding to reference requests seeking information about art and artifacts falling under the domain of the Freer's expertise, the Freer granting ...
Scurlock Studio Records, Subseries 4.5: Black and white negatives in cold storage arranged by job number
Custom Craft
Scurlock Studio (Washington, D.C.)
Scurlock, Addison N., 1883-1964
Scurlock, George H. (Hardison), 1919-2005
NMAH.AC.0618.S04.05
Archives Center, National Museum of American History
The materials document the orders placed by the clients of the Scurlock Studio. The photographs primarily depict individual portrait sittings but there are also portraits of children, groups, and other subjects.
Ruth Landes papers
Boas, Franz, 1858-1942
Wallis, Ruth Sawtell, 1895-1978
Wagley, Charles, 1913-1991
26.5 Linear feet ((63 document boxes and 1 oversized box))
Most of Ruth Landes's papers relate directly or indirectly to Landes's American Indian research, her work in Brazil, and her study of bilingualism. There is also a considerable amount of material that relates to her experiences (sometimes fictionalized) at Fisk University. There is only small amount of material related to her other interests. Her collection also has material of and relating to the Brazilian folklorist and journalist Edison Carneiro. There is also noteworthy material concerning Herbert Baldus, Ruth Benedict, Elmer C. Imes, Charles S. Johnson, and Robert E. Park. There is a large amount of printed and processed materials in the collection, mainly in the form of newspaper clippings and a collection of scholarly papers.
William Louis Abbott Papers
1.81 cu. ft. (3 document boxes) (1 half document box) (3 microfilm reels)
The papers of William Louis Abbott consist of letters to Smithsonian curators and specimen lists, 1892-1917; letters to his mother and sister, 1887-1923; photographs taken by Abbott in the field, 1890-1923; and some miscellaneous material. In describing the individual pieces of this collection, particular attention was paid to material relati...
Smithsonian Institution. Office of the Secretary
167.20 cu. ft. (5 record storage boxes) (324 document boxes) (10 microfilm reels)
Record Unit 50
These records document part of the secretarial administration of Alexander Wetmore and the whole of Leonard Carmichael's tenure. During this period the Smithsonian Institution's capacity to carry out research in its traditional scientific disciplines was substantially strengthened. At the same time the Institution was able to invest much ...
Guide to the Beatrice Medicine papers
Medicine, Beatrice
1914, 1932-1949, 1952-2003 (bulk dates, 1945-2003).
28 Linear feet (65 document boxes, 1 box of oversize materials, 1 box of ephemera, 1 shoebox of index cards, 1 map drawer)
The Beatrice Medicine papers, 1913-2003 (bulk 1945-2003), document the professional life of Dr. Beatrice "Bea" Medicine (1923-2005), a member of the Standing Rock Sioux tribe, anthropologist, scholar, educator, and Native rights activist. The collection also contains material collected by or given to Medicine to further her research and activism interests. Medicine, whose Lakota name was Hinsha Waste Agli Win, or "Returns Victorious with a Red Horse Woman," focused her research on a variety of topics affecting the Native American community: 1) mental health, 2) women's issues, 3) bilingual education, 4) alcohol and drug use, 5) ethno-methodologies and research needs of Native Americans, and 6) Children and identity issues. The collection represents Medicine's work as an educator for universities and colleges in the United States and in Canada, for which she taught Native American Studies courses. Additionally, because of the large amount of research material and Medicine's correspondence with elected U.S. officials and Native American leaders, and records from Medicine's involvement in Native American organizations, the collection serves to represent issues affecting Native Americans during the second half of the 20th century, and reflects what Native American leaders and organizations did to navigate and mitigate those issues. Collection materials include correspondence; committee, conference, and teaching material; ephemera; manuscripts and poetry; maps; notes; periodicals; photographs; training material; and transcripts.
The Garden Club of America collection
Garden Club of America
circa 1920-present
37,000 Slides (35mm slides)
33 Linear feet ((garden files))
3,000 Lantern slides
AAG.GCA
This collection contains over 37,000 35mm slides, 3,000 glass lantern slides and garden files that may include descriptive information, photocopied articles (from journals, newspapers, or books), planting lists, correspondence, brochures, landscape plans and drawings. Garden files were compiled by Garden Club of America (GCA) members for most of the gardens included in the collection. Some gardens have been photographed over the course of several decades; others only have images from a single point in time. In addition to images of American gardens, there are glass lantern slides of the New York Flower Show (1941-1951) and trips that GCA members took to other countries, including Mexico (1937), Italy, Spain, Japan (1935), France (1936), England (1929), and Scotland. A number of the slides are copies of historic images from outside repositories including horticultural and historical societies or from horticultural books and publications. The GCA made a concerted effort in the mid-1980s to acquire these images in order to increase its documentation of American garden history. Because of copyright considerations, use of these particular images may be restricted.
Myron Bement Smith Collection
Smith, Myron Bement, 1897-1970
192 Linear feet
FSA.A.04
Freer Gallery of Art and Arthur M. Sackler Gallery Archives
The Myron Bement Smith collection consists of two parts, the papers of Myron Bement Smith and his wife Katharine and the Islamic Archives. It contains substantial material about his field research in Italy in the 1920s and his years working on Islamic architecture in Iran in the 1930s. Letters describe the milieu in which he operated in Rochester NY and New York City in the 1920s and early 1930s; the Smiths' life in Iran from 1933 to 1937; and the extensive network of academic and social contacts that Myron and Katharine developed and maintained over his lifetime. The Islamic Archives was a project to which Smith devoted most of his professional life. It includes both original materials, such as his photographs and notes, and items acquired by him from other scholars or experts on Islamic art and architecture. Smith intended the Archives to serve as a resource for scholars interested in the architecture and art of the entire Islamic world although he also included some materials about non-Islamic architecture.
Black and Decker Collection
1 Cubic foot (5 boxes)
NMAH.AC.1441
Approximately 150 photographic negatives, 54 photographic prints, and two rolls of film negatives documenting the history of the Black and Decker Corporation, from a few years after its founding in Baltimore, Maryland, in 1910, through the 1990s.


text:
Top tech firms pitch their perks at London jobs exhibition
By Kate Dubinski
Helping women connect with jobs in the city?s tech sector was the focus of a meeting in Barrie Tuesday night. FILE PHOTO
It's a matchmaking event, job fair and foosball tournament — all in one.
In a world where a competitive salary and benefits are the baseline, tech companies have to shine to sell themselves to potential talent.
Enter Techvibes Tech Fest, where executives from London's best tech companies will pitch their perks to employees.
"We know that talent recruitment is a concern for many tech companies in London," said Kapil Lakhotia, president and chief executive of London Economic Development Corp., which is bringing the event to London.
"It's really a unique recruitment event. It's happened in Toronto and Vancouver, but we're really excited that London is the next place where it's going on."
London developers and innovators will get to watch companies like Big Viking Games, GoodLife Fitness, StarTech.com and Inner Geek pitch themselves to potential recruits.
"We try to have the best culture in London," said Colton Hathaway, founder of Inner Geek, which will pitch its web and mobile jobs.
"We offer a competitive salary and benefits, obviously, and catered lunches, plus unlimited paid vacation, flexible work hours and access to Inner Geek events," Hathaway said.
"The tech industry says there's a gap in talent, but it really is very competitive for those top students and with Techvibes you get national exposure," he added.
"Our philosophy has always been that if we take care of our people, our people will take care of our products."
Inner Geek has grown from a one-man company last May to one with 16 employees today. It's hiring.
Previous Tech Fest events, in Toronto and Vancouver, have featured ice cream sundaes, popcorn machines, giant Lego, ping-pong and foosball.
It's a way for London's top employers to connect with top talent, Lakhotia said.
Several hundred candidates will get exposure to these companies and hopefully some successful matches will be made."
kate.dubinski@sunmedia.ca
Fast fest facts
What: Techvibes Tech Fest
When: Oct. 28, 6 p.m. to 9:30 p.m.
Where: London Music Hall
Why: Brings together top tech companies to pitch their open job positions to top tech talent.


Once in a while, a new menswear collection comes along that's feel-good, carefully crafted, super comfortable and very saleable. And so we were pleased to discover Olefant, a sportswear collection that might best be described as Edgy Preppy or Classic Cool (or as one retail account put it, "Vineyard Vines meets Ted Baker"). But however you describe it, it's a great new brand targeting better independents and select department stores that won't be found online or in their own company stores. Their dynamic team is headed up by design director Jonathan Whaley, head stylist Iva Anthony, and Scott Ludmar in sales; Whaley and Ludmar have worked together for many years at various upscale brands and have also produced better private labels).
Olefant is privately owned and totally vertical, with factories in China and India so the prices afford retailers nice markups (most pants – in chino, dobby or twill – are $31 cost for a suggested $125 retail; shirts average $28 for $100). But more impactful than price is the product itself: well designed using beautiful fabrics (much of it performance) and fabric combinations, great detailing (much of it hidden so it's not too contrived) and terrific colors.
And if you like the fall '19 collection, wait 'til you see spring 2020 (in the works at this writing)!
Scott Ludmar (left) and Jonathan Whaley in the Olefant showroom.


Behind the restored 18th century facade, 22 rooms - 19 doubles, 2 individuals, and one suite - are tucked away, distributed among 6 floors.
Luminous and inviting, the rooms have a private bathroom, extra large bed, direct line telephone, satellite television, radio, Internet connection, lock box for hire, and adjustable central air/heat.
Additionally, among its facilities the hotel has an entry hall with elevator, 24 hour reception area offering lock box service, currency exchange, lounge and conference room, bar, and air conditioned restaurant.
Likewise, it also offers laundry service, medical attention services, Internet connection, as well as parking spaces and garage.


Not only it is easy to carry and store, but with a lifting capacity of 440lb /(200kg) it's in a class of its own. The 24-volt battery pack can be recharged in less than 2 hours. It comes with a unique feature called the "Quick Releaser." Combined with our reacher, it becomes a user-friendly option that swiftly disengages the strap for easy, safe and quick suspension and removal of the lift.


Description "33 [age] ; Tanner ; 75 [value of personal estate owned] ; South Carolina [place of birth]."
Source Includes data from the 1860 Census Population Schedules, Clarksville Post Office, Red River County, Texas, Series: M653, Roll: 1303, p. 123 (stamped), entry 8.
Artisan or Artist Luna, Franklin.


- All heroes from the regular modes are now available in the PBT Hero Pool.
* Exception: Circe (due to the potential of undesirable bugs being produced with her ultimate).
- Ally illusions now have a green tint on the minimap, and their unit circles are 50% smaller on the minimap.
- Holding the "Alt" button down now displays the neutral creep respawn zones.
- No longer takes a target to activate. Now automatically throws a goblin towards the closest visible enemy within 600 range, prioritising heroes.
* Cannot be activated if no valid targets are within range.
- Max number of charges (goblins) reduced from 7 to 1/2/3/4.
- Goblins are no longer upgradeable.
* No more green/red goblins, they all remain blue.
- Goblins deal 70/90/110/130 Magic Damage and apply a 10/15/20/25% Movement Speed Slow for 2 seconds when thrown.
- Projectile speed decreased from 1000 to 700.
- Charge refresh time decreased from 8 to 5 seconds.
- Upon use, rushes to the target location at 800 speed and explodes the area, dealing 360/520/680 Magic Damage to enemies within range and applying 4 stacks of Conscription to each enemy if you have it leveled.
- Magic Damage per second changed from 40 to 30/50/70/90.
- Mana Cost per second reduced from 15 to 5.
- Cooldown increased from 30 seconds to 60/55/50/45 seconds.
- Mana Cost increased from 60 to 80.
- Magic Damage per second reduced from [4/8/12/16 + 30% of Attack Damage] to [4/8/12/16 + 25% of Attack Damage].
- Base Strength reduced from 25 to 22.
- Base Armor reduced from 5.5 to 3.5.
- Staff of the Master effect only: Fixed the ability to go on cooldown the moment the hammer is thrown (i.e. not when Hammerstorm teleports to the target).
+ The Four Symbols were thought to be mythological creatures who dwelled among the constellations, but as the future of Newerth teeters on the brink of disaster, these honorable beings reveal themselves to be not only real, but also incredibly powerful.
- The frozen climate of the North may be endless and unwelcoming to some, but the lovely Black Tortoise of the North adores it. The time spent indoors allows her to gather much wisdom and spend countless hours studying the gorgeous, powerful crystals she finds buried beneath the snow with the help of her snake companion. The two of them make a formidable pair in battle, with the Tortoise providing a very somber, studied approach to any who threaten her people or seek to steal her gems, while the snake prefers to be much more of a...well...snake.
- The Vermilion Bird of the South embodies the full beauty of peak summer, when the scorching heat and dry conditions offer ample opportunities for a good cleansing fire to sweep through and rid the land of undesirable inhabitants. Now that she has set foot upon Newerth, this includes the corrupted Hellbourne, who cannot help but admire the elegance of the Vermilion Bird and her graceful flames, even as they are engulfed by them.
- The Plume Riders of Fuathmoor bring a dazzling display of color and flair to an otherwise dreary landscape of fog, bogs and hummocks. The village children and offspring of the Poison-Plumed Harriers are paired at a young age to foster trust and an uncanny, unspoken communication that allows them to fly in perfect, graceful harmony. Originally used only for ceremonies to honor warriors heading off to battle and welcome back the lucky survivors, they were forced to evolve into warriors themselves when the Hellbourne rose and no survivors returned from the slaughter. Now, the riders and harriers use both spear and poison feathers to drive the corrupted out of their land, and they sing of the day when they can be part of the ceremony celebrating the end of this terrible war.
- The Exit icon on the login page is now more distinguishable from the background.
- Heroes that become unselectable (e.g. Fayde's Shadow Walk ability in PBT) will no longer be de-selected if you're the owner of that hero and have multiple units selected in a group that includes that specific hero.
- Fixed the Geojuice avatar to have correct sight range while burrowed.
- Hammer Throw's projectile is now properly blocked by Moraxus' Arcane Shield ability.
- Taunt is now considered a StatusDisable to allow its residual Silenced/Perplexed debilitating effects to be properly removed by Berzerker's Carnage.
- Preda Tori avatar's attack animation is now properly synchronized with the actual attack impact time.
- Prison Break Staff of the Master effect now applies the correct level of Shackled to the target.
- Ensnare now has proper cast mechanics (as opposed to having the non-interrupting & inherit-movement cast mechanics).
- Typhoon's auxiliary (non-damaging) effects are now properly set to Superior Magic.
- Now properly allows out of range casts if targeting a position.


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You arranged for the DEA to come and arrest you, which was nice, because it was right around Christmas time and you didn't want them filling you with bullets on the Lord's big day.
"It's his birthday," I reminded you. We were sitting on the couch and sometimes you got Christmas and Easter confused there.
Your lap was covered in Christmas cards. You picked one out and handed it to me respectfully with two hands. "Have a cool drink of water," you said. The card had a picture of ice cubes on it. I was worried you were holding a bullet in your mouth and was trying to get closer to check when the men broke down my door.
Click here to see constraints of this project, and strange videos.
Amelia Gray is the author of AM/PM, published by Featherproof Books. Her book Museum of the Weird is coming out in October 2010 through FC2. For other publications, see here.


Wemo achieves what many smart plug manufacturers are going for, and they've done it near flawlessly. You're augmenting your smart home, making it more interconnected to your daily life, to your devices that are in constant use, and your expectations are that there will be no complications. Wemo upholds those expectations.
Their mini smart plug stays compact without dangling out of the socket. They kept the connectivity hardware and case as lightweight as possible, so even the wobbliest of outlets can easily be stabilized with Wemo's mini smart plug.
Wemo connects to your Google Assistant, it can communicate with Alexa and Siri, and just about any smart hub on the market. If you're going to truly master your home and make it work for you, you need to be able to control your appliances and small electronics with a simple voice command.
It's compact, it allows scheduling through the free app, and that app allows you to connect an endless amount of smart plugs to your single device. That's going to come in handy with another feature, one that works fantastically for home defense: randomizing the lights.
Don't want to let potential criminals know when you go to sleep? Wemo will automatically shuffle your lights, turning some on, some off, and keeping it unpredictable so that burglars never know when you've quieted down for the night. It works when you're sleeping, so you can rest easy. This feature is a major bonus if you're heading out of town on a trip, and want to maintain the appearance that someone is always home.
The beauty of Wemo is that is isn't piggybacking on a smart hub: if you don't have one, you can still manage every single function from your phone, including commanding multiple plugs at the same time. Compact, inexpensive, and a stellar way to add a layer of security and automation to your home without going through an installation process.


Hi Jeff! I watched the end of Arrieta's no-hitter last night, and with two outs in the ninth and his team losing 16-0, a Reds player (Schebler) went from first to second base. The announcer said "That may be the greatest example of fielders' indifference in the history of the game," and it occurred to me that he might be right. Can you think of any way in which the stakes of that "steal" could be lower?
Also, if you could be any lava mountain, would you be a good lava mountain or an evil lava mountain? I'd probably be an outer space one because that seems pretty cool!
Lorenzo Cain- is he settling back into a 8HR / 30SB / .290 BA?
Will HOF voters start to change their stance on PED users once the hypocrisy of MLB becomes more obvious? It seems like MLB had a pretty good idea as to what was happening at the time, allowed it to happen, but now has taken a revisionist historical stance and doesn't really hold themselves at all accountable.
too early to tell if mookie's selling out for power? he murdered that ball yesterday.
How close is Jake Arrieta from being the best pitcher in baseball?
Of course the lowest FIP and xFIP on the Cubs pitching staff after last night belongs to Jake Ar.....wait.....Kyle Hendricks? How does one explain that?
CarGo's exit velocity looks massive...does this suggest he can repeat his crazy second half numbers from last year?
Things are tough here in MN today. What, if anything, should we be excited about?
What do the Giants do at this point to right the ship?
I don't think anything is too broken. Defend a little better? They'll be okay.
Are you buying the "Harvey is fixable" line or is something else afoot?
Andrew McCutchen is a slow starter. His April is generally not good relative to the rest of his months. How I concerned should I be about his increased O-Swing% and lack of contact both outside the zone and in the zone?
Jeff, explain the Cardinals. I'll hang up and listen.
I didn't even realize Aledmys Diaz is batting .385. All right!
The Cardinals do almost everything right. Except for that one thing, they're baseball's model organization.
How much does strength of schedule matter when evaluating performances right now? For example, Nationals look GREAT. Their schedule....OOF.
Like, Bryce Harper is great, but Aaron Nola is the only good pitcher he's homered off so far. His opponents have sucked. Of *course* his numbers are insane.
How can my Sox bat the DH eighth and not do something about this lifeless lineup?!?!? They were refunded $13M and did nothing. Jerry (whom I love) should be called out, imo.


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text:
La  d'infanterie territoriale est le nom d'une unité de l'armée française.

Chefs de la  d'infanterie territoriale
  - : général Joppé
 -
  - : général Dhers

La Première Guerre mondiale

Composition au cours de la guerre
Mobilisée dans la .

 Régiment d'infanterie territoriale de  à
 Régiment d'infanterie territoriale de  à
 Régiment d'infanterie territoriale d' à
 Régiment d'infanterie territoriale d' à
 Régiment d'infanterie territoriale d' à
 Régiment d'infanterie territoriale d' à
 Régiment d'infanterie territoriale d' à
 Régiment d'infanterie territoriale d'avril à
 Régiment d'infanterie territoriale d'avril à

1914
  –
Transport par V.F., de Saint-Servan, à Valognes; garde du littoral dans cette région.
À partir du , transport par mer et par V.F. dans la région du Havre; travaux et défense.
À partir du , transport par mer à Dunkerque.

 7 –
Mouvement vers Ypres.
À partir du , occupation et défense de la région de Zillebeke, Pilkem; travaux.
, combat vers Bikschote.

  –
Relève d'une division britannique vers Langemark et Steenstrate.
Engagée dans la Bataille d'Ypres: combats vers Weidendreft, Mangelaare et Kortekeer Cabaret.

  –
Retrait du front; repos vers Killem, puis travaux vers Elverdinge et Dikkebus.

  –
Mouvement vers le front et occupation d'un secteur sur le canal de l'Yser, vers le pont de Knocke, le pont de Drie Grachten et Kortekeer Cabaret,réduit à droite, le , jusque vers la maison du Passeur.

1915
 24 –
Retrait du front (relève par l'Armée belge), et repos vers Proven

  –
Mouvement vers le front et occupation d'un secteur vers Poelkapelle et Steenstrate.

  –
Retrait du front et repos vers Socx.

  –
Mouvement vers le front et occupation d'un secteur vers Langemarck et Steenstrate.
, attaque allemande par gaz; violentes contre-attaques françaises; puis occupation, par des éléments, d'un nouveau secteur sur l'Yser au nord de Boezinge.

  –
Retrait du front et repos vers Rexpoëde (éléments en secteur vers Steenstrate et Boesinghe).

  –
Mouvement vers le front et occupation d'un secteur vers Steenstrate et Boesinghe, en liaison avec les armées belges et britanniques.

1916
  –
Retrait du front; stationnement vers Rexpoëde: des éléments de la  D.T. participent à l'organisation de la défense de Dunkerque et à l'occupation du secteur du   CA.

  –
Occupation d'un secteur vers Steenstraate et Boesinghe.

 5 –
Retrait du front, et à partir du , transport par V.F. dans la région d'Estrées-Saint-Denis; repos.

  –
Mouvement vers le front et occupation d'un secteur vers Belval et l'Oise, réduit à gauche, le , jusque vers l'Ecouvillon.

  –
Retrait du front; repos vers Grandfresnoy et Sacy-le-Grand.
À partir du , travaux dans les régions de Villers-Cotterêts, de Senlis, de Ressons-sur-Matz et de Creil.

1917
  –
Instruction au camp de Pontarmé.

 12 –
Mouvement vers Compiègne, puis occupation d'un secteur entre l'Oise et l'Ecouvillon.

 17 –
Poursuite de l'ennemi (Repli allemand): prise de Noyon.

   avril
Travaux dans la région de Noyon.

  –
Regroupement vers Roye et Porquericourt; transformation de la  D.T. en   D.I. active.

Rattachements
Affectation organique: Isolée, d' à

Sources

Articles connexes
Ordre de bataille de l'armée française le  août 1914

Notes

Division d'infanterie territoriale de l'Armée de terre française
Unité d'infanterie française dissoute
Unité ou formation militaire créée en 1914
Unité ou formation militaire dissoute en 1917

redpajama_set_name: RedPajamaWikipedia


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