text:
Amendment history:
Erratum (July 2000)
Article Free access | 10.1172/JCI9243
Lethal host-versus-graft disease and hypereosinophilia in the absence of MHC I–T-cell interactions
Jérôme D. Coudert,1 Gilles Foucras,1 Cécile Demur,2 Christiane Coureau,1 Catherine Mazerolles,3 Georges Delsol,3 Philippe Druet,1 and Jean-Charles Guéry1
1Institut National de la Santé et de la Recherche Médicale (INSERM) U28, Institut Fédératif de Recherche 30, and Université Paul Sabatier, Toulouse, France2Hematology Laboratory, and3Department of Pathology, Centre National de la Recherche Scientifique (CNRS) UPR 2163, Hôpital Purpan, Toulouse, France
Address correspondence to: Jean-Charles Guéry, INSERM U28, Hôpital Purpan, Place du Dr Baylac, 31 059 Toulouse, France. Phone: 33-561779296; Fax: 33-561779291; E-mail: Jean-Charles.Guery@purpan.inserm.fr.
Find articles by Coudert, J. in: JCI | PubMed | Google Scholar
Find articles by Foucras, G. in: JCI | PubMed | Google Scholar
Find articles by Demur, C. in: JCI | PubMed | Google Scholar
Find articles by Coureau, C. in: JCI | PubMed | Google Scholar
Find articles by Mazerolles, C. in: JCI | PubMed | Google Scholar
Find articles by Delsol, G. in: JCI | PubMed | Google Scholar
Find articles by Druet, P. in: JCI | PubMed | Google Scholar
Find articles by Guéry, J. in: JCI | PubMed | Google Scholar
Published in Volume 105, Issue 8 on April 15, 2000
J Clin Invest. 2000;105(8):1125–1132. https://doi.org/10.1172/JCI9243.
Received: December 22, 1999; Accepted: March 7, 2000
Neonatal injection of semiallogeneic spleen cells in BALB/c mice induces a self-limited state of chimerism that promotes the differentiation of donor-specific CD4 T cells toward the Th2 phenotype. Here we show that injection of spleen cells from β2-microglobulin–deficient (BALB/c × C57BL/6) F1 mice into BALB/c newborns with a disrupted β2-microglobulin (β2m) gene results in a lethal lymphoproliferative disorder associated with uncontrolled Th2 response, long-term persistence of donor B cells, and sustained blood eosinophilia. Autoimmune manifestations are also enhanced and characterized by a severe autoantibody-mediated glomerulonephritis. Histological examination of the spleen shows a hyperplasia of periarteriolar lymphoid sheaths, with accumulation of eosinophils and basophils, and variable degree of fibrosis. Perivascular lymphoid infiltrates with eosinophils are also found in the lung and are correlated with disease severity. Such abnormalities are almost absent using β2m-sufficient mice. These data demonstrate that induction of lymphoid chimerism in the absence of MHC class I–T-cell interactions results in a lethal form of host-versus-graft disease that represents a unique model of Th2-dependent chronic inflammatory disease associated with an hypereosinophilic syndrome in mice.
Since the original experiments by Billingham, Brent, and Medawar (1), the model of neonatal tolerance to transplantation antigens has been extensively studied to analyze the different parameters promoting allograft acceptance. Although it has been proposed that in mice neonatally injected with semiallogeneic spleen cells, MHC class I–reactive cytotoxic T cells are tolerized by clonal deletion (2, 3), it is clear that alloantigen-specific, IL-4-secreting CD4 T cells are still present in the periphery (4–7) and are probably responsible for the upregulation of MHC class II molecules on both donor and host B cells (8). In addition, it has been shown that the differentiation of donor-specific CD4 T cells toward the Th2 phenotype is dependent on the early production of IL-4 in vivo (5, 9). Furthermore, blocking endogenous IL-4 was found to restore antidonor cytoxic T lymphocytes (CTL) activity and abrogated the lymphoid chimerism, thereby resulting in graft rejection (5, 10). Another important feature of this model is the development of autoimmune manifestations, known as host-versus-graft (HVG) disease, due to the polyclonal T–B-cell interactions that occur in this model between host Th2 and donor B cells, resulting in a dramatic increase of serum IgE level and in the production of autoantibodies, some of which are found deposited in the kidneys (11, 12). However, such serological abnormalities are spontaneously downregulated by 10 weeks of age due to the disappearance of lymphoid chimerism (11, 13).
Because IgE production and Th2 priming are critically dependent on the early production of IL-4 (14–16), we have recently analyzed the requirement for NK1.1 T cells in the generation of alloreactive Th2 responses in this model (17). For this purpose, lymphoid chimerism was induced in both normal and β2-microglobulin–deficient (β2m°) BALB/c mice. We showed that similar Th2 priming and IL-4–dependent IgE production developed in both β2m° or normal BALB/c mice injected at birth with semiallogeneic spleen cells from normal (BALB/c × C57BL/6) F1 mice (CB6F1), indicating that β2m-dependent NK1.1+ host T cells were not required for the generation of alloreactive Th2 responses in vivo (17). Interestingly, when β2m° CB6F1 spleen cells were injected into BALB/c β2m° mice, the IgE response was even exacerbated and associated with an increased splenomegaly (17). Thus, we hypothesized that MHC class I–T-cell interactions could play a crucial role in this model by regulating Th2 immunity and its associated immunological disorders.
To analyze the role of MHC class I–reactive host T cells interacting with donor cells, we have compared 2 combinations, in which wild-type (WT) or β2m° newborn BALB/c mice were injected either with normal or with β2m° CB6F1 splenocytes, respectively. Data presented here demonstrate that the absence of MHC class I–T-cell interactions leads to spared Th2 immunity that results in accelerated mortality and higher incidence of proteinuria due to antibody-mediated glomerular lesions. The uncontrolled Th2 response of host CD4 T cells is associated with the maintenance of B-cell lymphoid chimerism and with sustained hypereosinophilia in adult mice. Thus, our data support the hypothesis that CD8 T cells might play a more active role than previously anticipated by regulating donor B-cell persistence and/or Th2-cell development during neonatal induction of lymphoid chimerism in mice, thereby preventing chronic Th2 responses and its associated hypereosinophilic syndrome.
Mice, neonatal injection of semiallogeneic spleen cells. BALB/c (H-2d) and (BALB/c × C57BL/6) F1 (CB6F1) (H-2bxd) mice were purchased from Centre d'Elevage R. Janvier (Le Genest St. Isle, France). H-2b mice with disrupted β2m genes (18) were back-crossed to BALB/c mice as described previously (19). β2m° mice on BALB/c background were used after 9 back-crosses. BALB/c β2m° and CB6F1 β2m° mice were bred and maintained in our own animal facility. BALB/c and BALB/c β2m° were injected intraperitoneally within the first 24 hours of life, with 50 × 106 spleen cells from 8- to 12-week-old normal or β2m° CB6F1 mice, respectively. Control animals were left untreated.
Quantitation of serum IgE. Serum IgE concentration was quantified by a sandwich ELISA as described previously (17). Serum IgE concentrations measured either in WT or in β2m° control mice were less than 100 pg/mL. We considered that the IgE response was upregulated when IgE concentration was greater than 1 μg/mL.
Albuminuria determination. Urine was collected at 10–16 weeks of age, and the presence of mouse serum albumin (MSA) was determined by competitive ELISA. Briefly, polyvinyl microtiter plates were coated with 2 μg/mL mouse serum albumin (Sigma Chemical Co., St. Louis, Missouri, USA). Urines were first incubated with an appropriate concentration of rabbit anti-mouse albumin antibody (Cappel Laboratories, Cochranville, Pennsylvania, USA), then transferred onto MSA-coated plates. After incubation, bound rabbit anti-MSA IgG were stained with a peroxidase-conjugated sheep anti-rabbit IgG (Serotec, Oxford, United Kingdom), and revealed by 3,3′-5, 5′-tetramethylbenzidine (Fluka Chemical Co., Buchs, Switzerland). Albumin concentration was determined using MSA (Sigma Chemical Co.) to generate a standard curve. The concentration of albumin in WT or β2m° control mice was routinely less than 5 μg/mL. Mice were considered albuminuric when albumin concentration was greater than 15 μg/mL.
Indirect immunofluorescence analysis. Kidney cryostat-sections (4 μm) were performed with a Cryocut 1800 apparatus (Reichert-Jung, Cambridge Instruments GmbH, Nussloch, Germany) after OCT compound (BDH Laboratories, Poole, United Kingdom) embedding. Glomerular IgM and IgG deposits were assessed by indirect immunofluorescence on cryostat sections stained with FITC-labeled rat anti-mouse kappa light chain OX-20 mAb (20), FITC-labeled mouse IgG subclasses–specific (IgG1, IgG2a, IgG2b) antibody (Southern Biotechnology Associates, Birmingham, Alabama, USA), or FITC-labeled rabbit anti-mouse IgM antibody (Meloy Laboratories, Springfield, Virginia, USA).
Histological and cytological analysis. All major organs (liver, heart, intestine, kidney, spleen, and lung) were fixed in 10% formalin and paraffin sections were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Giemsa. Histological analysis was performed by 2 pathologists who did not have the knowledge of the different experimental groups. Blood white cell counts and spleen cell counts were performed with an automated counter (STKS Coulter, Beckman Coulter, Roissy, France). Differential cell determinations were carried out using spleen cell suspensions that were cytocentrifugated on a Cytospin 3 Shandon apparatus (Life Sciences International, Runcom, United Kingdom), or standard blood smears fixed with methanol and stained with Giemsa. Interpretation was done by 1 of us blinded to the different experimental groups.
Flow cytometric analysis. After red blood cell lysis, spleen cells were incubated with optimal concentrations of FITC-, PE- or biotin-conjugated mAb for 30 minutes at 4°C in PBS containing 5% FCS, 0.1% sodium azide. The following mAb's were used: FITC-GK1.5 anti-CD4 (TIB 207; American Type Culture Collection [ATCC], Rockville, Maryland, USA), PE-Ly2 anti-CD8α (PharMingen, San Diego, California, USA), PE-RA3-6B2 anti-B220 (PharMingen), FITC-AF6-120.1 anti–I-Ab (PharMingen), FITC-14.4.4S anti–I-E (ATCC, HB 32), PE-Ly24 (Pgp1) anti-CD44 (PharMingen), and biotin anti-Ly22 (MEL-14) anti-CD62L (PharMingen). Cells were then washed and stained with streptavidin-CyChrome (PharMingen). Data were collected on 10,000 living cells as determined by forward and side scatter intensity on a XL Coulter cytometer (Beckman Coulter) and analyzed using the CellQuest software (Becton Dickinson, Mountain View, California, USA).
CD4 T-cell enrichment. Spleen cells were stained with an appropriate concentration of KT1.5 (21), anti-B220 RA3-3A1 (TIB 146; ATCC), anti-MHC class II M5/114 (TIB 120; ATCC), and anti–Mac-1 M1/70 (TIB 128; ATCC) mAb, washed, and incubated with sheep anti-rat IgG M-450 Dynabeads (Dynal, Oslo, Norway). CD4 T cells were negatively selected by magnetic cell separation.
In vitro CD4 T-cell stimulation and cytokine assays. For cytokine production analysis, 96-well culture plates (Costar, Cambridge, Massachusetts, USA) were coated with 145-2C11 anti-CD3ε (CRL-1975; ATCC) at a concentration of 1 μg/mL. CD4-enriched splenic T cells from neonatally injected and control mice were stimulated at 2 × 105 cells per well. Culture medium was HL-1 synthetic medium (Hycor, Irvine, California, USA) supplemented with 2 mM L-glutamine and 50 μg/mL gentamicin (Sigma Chemical Co.). Cultures were incubated for 2 days in a humidified atmosphere of 5% CO2 in air. Supernatants from replicate cultures, usually 3–4 wells, were collected after 48 hours and pooled for cytokine analysis. IFN-γ, IL-4, IL-5, and IL-10 were quantified by 2-site sandwich ELISA as described previously (17). Cytokines were quantified from 2–3 titration points using standard curves generated with purified recombinant mouse cytokines, and results were expressed as cytokine concentration in nanograms or picograms per milliliter. Detection limits were 15 pg/mL for IFN-γ and IL-4, 3 pg/mL for IL-5, and 30 pg/mL for IL-10.
Competitive RT-PCR. Total RNA was extracted using TRIzol Reagent (GIBCO-BRL, Cergy-Pontoise, France) according to manufacturer's recommendations. RT was performed using Moloney Murine Leukemia Virus RT (GIBCO-BRL) and oligo-dT primers. Semiquantitative PCR determination for hypoxanthine-guanine phosphoribosyl transferase (HPRT) and IL-4 genes was performed as described elsewhere (22). Data were expressed as the ratio of IL-4 over HPRT mRNA levels.
Statistical analysis. Statistically significant differences between groups of continuous variables were analyzed using the Mann-Whitney U test. Categorical variables and survival curves were compared, respectively, by the Fisher's exact test and by the log-rank test.
Effects of β2m deficiency in host versus graft disease. Injection of semiallogeneic spleen cells in newborn BALB/c mice stimulates host-derived alloreactive Th2 cells that react with MHC class II alloantigens on donor B cells. This cognate interaction induces a polyclonal B-cell activation characterized by increased levels of serum IgE and autoantibody production (11, 12). As shown in Figure 1, no difference in serum levels could be observed at 4 weeks of age between the WT (Figure 1a) and the β2m° (Figure 1b) combinations. In striking contrast, whereas serum IgE levels were spontaneously downregulated by 8 weeks of age in the CB6F1-injected WT mice (Figure 1a), induction of lymphoid chimerism in the complete absence of β2m-dependent T cells resulted in uncontrolled IgE responses that remained high until 12 weeks of age (Figure 1b). The incidence of IgE response, at 4 weeks of age, in the CB6F1→BALB/c and CB6F1β2m°→BALB/cβ2m° combinations were 65% (37/57) and 83% (44/53), respectively. Interestingly, whereas most BALB/c mice injected at birth with CB6F1 spleen cells remained healthy and survive, more than 50% of mice died by 15 weeks of age in the β2m° combination (Figure 1c). This increased mortality was associated with an higher incidence of albuminuria (Figure 1d). Urine albumin levels were above 100 μg/mL in 66% of albuminuric mice, with a maximum of 16 mg/mL in 1 mouse (data not shown). Immunofluorescence analysis on kidney cryostat sections demonstrated the presence of heavier glomerular Ig deposits in neonatally injected β2m° compared with WT mice (Figure 2, b and d), that were mainly composed of IgG1 and IgM (data not shown). Moreover, whereas in the WT combination kidney bound Ig were mostly found in the mesangium (Figure 2b), both mesangial and subendothelial granular Ig deposits were observed in 7 of the 11 neonatally injected β2m° mice examined (Figure 2d), the 4 other mice displayed only mesangial Ig deposits. Unlike BALB/c mice injected with CB6F1 splenocytes, that exhibited only mild mesangial deposits without any cell proliferation or interstitial changes (Figure 2a), glomeruli from 4 of 6 neonatally injected β2m° mice examined by light microscopy exhibited heavy mesangial and parietal, mainly subendothelial, deposits (Figure 2c). There was also a double contour appearance after silver impregnation (data not shown). In addition, perivascular infiltrates of mononuclear cells were observed in 2 of the 6 β2m° mice (data not shown). Renal function appeared normal in these mice, as urea and creatinine blood levels were unchanged (data not shown). Another striking observation of the β2m° chimeric model was that these mice developed a much larger splenomegaly compared with the WT combination, which still persisted until 10 weeks of age, with variable degrees of fibrosis (not shown). The increased spleen size in neonatally induced β2m° chimeras was associated with a significant augmentation in CD4 T and B cell numbers (data not shown).
Persistence of IgE production, high mortality rate, and increased incidence of albuminuria in β2m° BALB/c mice injected at birth with β2m° CB6F1 splenocytes. Normal (a) and β2m° (b) BALB/c mice were injected at birth with 50 × 106 CB6F1 spleen cells from normal or β2m° donors, respectively. Mice were bled at 4–16 weeks of age and IgE were quantified in individual mice serum by ELISA. Statistically significant decrease of IgE concentration was compared between the 8-, 10-, 12-, and 16-week values and the 4-week values. Bars represent means of individual mice. (c) Mortality of neonatally injected β2m° (solid line; n = 9) and WT mice (dotted line; n = 13) was followed up until the age of 22 weeks. (d) Albumin was quantified in urine by ELISA at 10–16 weeks of age. Mice were considered albuminuric when albumin titer was greater than 15 μg/mL. Statistical analyses were performed as indicated in Methods. AP < 0.001; BP < 0.01.
Histological changes and immunofluorescence analysis of kidney. (a and b) Representative changes in 6 week-old BALB/c mice injected at birth with CB6F1 splenocytes. Moderate mesangial deposits are seen on H&E staining (a, ×800) and after immunostaining for mouse Ig (b, ×500). (c and d) Representative changes in 6-week-old β2m° BALB/c mice neonatally injected with β2m-deficient CB6F1 spleen cells. Strong mesangial and subendothelial immune complex deposits are seen on PAS staining (c, ×800) and after immunostaining for mouse Ig (d, ×500).
Spared Th2 immunity is associated with the maintenance of lymphoid chimerism in β2m-deficient mice. Because splenomegaly in this model is likely to be associated with uncontrolled Th2 immunity, we next compared the expression of memory/activation markers on T and B cells between these 2 combinations. Results in Figure 3 show that there was a significant increase in antigen-experienced splenic CD4 T cells in both WT and β2m° mice at 6 weeks of age after neonatal induction of lymphoid chimerism. CD44highCD62Llow CD4 T-cell numbers were higher in CB6F1β2m°-injected BALB/cβ2m° mice compared with the WT combination. This difference was more evident at 10 weeks of age, where number of memory/effector CD4 T cells were significantly increased in neonatally injected β2m° but not WT mice. Analysis of the splenic B-cell compartment showed that the expression of I-E molecules on B cells was significantly higher in neonatally injected β2m° compared with WT mice at both 6 and 10 weeks of age (Figure 4a). Because MHC class II expression on B cells is upregulated by IL-4 (23, 24), we looked for the presence of IL-4 transcripts in splenic CD4 T cells. Data in Figure 4b demonstrate that IL-4 mRNA were readily detectable in the majority (85%) of 6-week-old neonatally induced β2m° chimeras, compared with 22% in the WT combination. IL-4 is likely to be produced by allospecific Th2 cells interacting with donor F1 B cells, resulting in donor B-cell differentiation to Ig-producing cells. Indeed, the number of plasmocytes was selectively increased in the spleen of 6-week-old neonatally injected β2m° mice (Figure 4c) and was correlated with the persistence of significantly higher levels of B-cell chimerism (Figure 4d). To analyze further the effect of the absence of MHC class I–T-cell interactions in the development of Th2 cells in this model, we measured type-2 cytokine production by splenic CD4 T cells from 6-week-old mice after stimulation with plate bound anti-CD3ε mAb. Data in Figure 5 show that there was a 4-fold upregulation of IL-4 production in F1-injected WT mice compared with uninjected control animals. In striking contrast, IL-4 secretion was dramatically increased (54-fold) in the neonatally induced β2m° chimeras. The lower IL-4 production in control β2m° compared with WT BALB/c mice may be explained by the absence of NK T cells that promptly produce this cytokine in response to TCR ligation (25). As for IL-4, IL-10 secretion upon TCR engagement was strongly increased in CD4 T cells from both combinations, but this was more evident in β2m° mice. On the other hand, the amounts of IL-5 produced by CD4 T cells was significantly upregulated only in β2m° chimeras (Figure 5). IFN-γ synthesis was slightly increased (2- to 3-fold) in neonatally injected compared with control mice both in the WT and β2m° combinations (data not shown). Thus, the induction of lymphoid chimerism in the absence of MHC class I–T-cell interactions led to the accumulation in the spleen of CD4 T cells with a memory phenotype probably as a consequence of the long-term persistence of donor B cells, resulting in chronic alloantigenic stimulation and increased type-2 cytokine production by CD4 T cells.
Accumulation of CD44hi/CD62Llow T lymphocytes in neonatally injected β2m° BALB/c mice. WT and β2m° BALB/c mice were injected at birth with normal or β2m-deficient CB6F1 spleen cells as in Figure 1. Uninjected WT and β2m° BALB/c mice were used as control. Splenocytes from 6- to 10-week-old neonatally injected and control mice were analyzed by flow cytometry to quantify the CD44hi/CD62Llow memory/effector CD4+ T cells. Bars represent means ± SEM of individual mice (4–12 mice per group). Numbers in brackets indicate the fold increase of neonatally induced chimera versus control mean values. AP < 0.05; BP < 0.01.
Upregulation of MHC class II on B cells, increased number of plasmocytes, and IL4-mRNA are associated with the maintenance of lymphoid chimerism in β2m° BALB/c mice. (a) I-E expression level on splenic B lymphocytes from 6- and 10-week-old neonatally injected and control mice was evaluated by flow cytometry. Data are expressed as fold increase versus control (mean ± SEM) of 4–12 mice per group. (b) IL4-mRNA expression in negatively enriched CD4 splenic T cells, from 6-week-old neonatally injected WT and β2m° (n = 14) mice, was quantified by semiquantitative RT-PCR. Data are expressed as the ratio of IL-4/HPRT mRNA levels from 9–14 individual mice per group. (c) Cytospins were prepared from spleen cells suspensions from 6-week-old mice; total plasmocyte numbers were calculated from 4–7 individual mice per group. (d) Percentages of I-Ab+ splenic B220+ cells from 6-week-old WT (n = 16) and β2m° (n = 9) mice injected at birth with semiallogeneic splenocytes were evaluated by flow cytometry analysis. Bars represent means of individual mice.
Th2-type cytokines production by in vitro stimulated CD4 T lymphocytes. Negatively enriched splenic CD4 T cells from 6-week-old control and neonatally injected mice were stimulated with plate-bound anti-CD3ε mAb for 48 hours. IL-4, IL-10, and IL-5 were quantified in culture supernatants by ELISA. Results are expressed as mean ± SEM cytokine concentration (ng/mL) from 4–7 mice per group. Numbers in brackets indicate the fold increase of neonatally injected versus control mean values. AP < 0.05; BP < 0.01.
Hypereosinophilic syndrome develops after induction of lymphoid chimerism in the absence of MHC class I–T-cell interactions. Because the uncontrolled Th2 response that develops in β2m° chimeric mice is associated with an increased splenomegaly with signs of fibrosis in most animals, a morphological analysis was performed. Data in Figure 6 show that although neonatally injected WT mice exhibited minimal change in spleen morphology (Figure 6a), a considerable modification of spleen architecture was observed in chimeric β2m° BALB/c mice (Figure 6b). Such changes were characterized by a strong hyperplasia of periarteriolar lymphoid sheaths (PALS) (Figure 6b) and by prominent eosinophil- (Figure 6c), basophil-, and/or mastocyte-containing (Figure 6d) infiltrates in the majority (>80%) of β2m° chimeras. These modifications were associated with variable degree of fibrosis (Figure 6b and data not shown). Massive perivascular lymphoid infiltration (Figure 6f) and tissue eosinophilia (data not shown) were also occasionally found in the lung (2 of 5 β2m° chimeric mice tested) and were correlated with disease severity (data not shown). Histological analysis of others organs such as liver, heart, and intestine did not reveal significant abnormalities (data not shown). Cytological analysis confirmed the increased eosinophilia in the spleen of neonatally induced β2m° chimeras compared with F1-injected WT mice (Figure 7a). Furthermore, a significant increase in eosinophil number was detected in the blood of 6- to 10-week-old β2m° chimeric mice (Figure 7b). Such abnormalities were barely detectable or absent in the majority of F1-injected β2m-sufficient mice. Thus, the uncontrolled type-2 immune response that develops in β2m° BALB/c mice injected at birth with semiallogeneic β2m° splenocytes leads to an hypereosinophilic syndrome.
Histological changes in the spleen (a–d) and lung (e and f) of 6-week-old WT (a and e) and β2m° BALB/c mice (b, c, d, and f) injected at birth with normal or β2m-deficient CB6F1 splenocytes, respectively. In comparison with the spleen of WT mice (a, ×125), a striking hyperplasia of PALS is observed on H&E staining of β2m° mice (b, ×100). On higher magnification (×800), one sees large numbers of eosinophils (arrows) (c) in a background of activated lymphoid cells, some showing immunoblastic features (arrowheads). Giemsa staining shows that cells with large basophilic granules (arrows) are also present in PALS (d). Note also the presence of scattered plasma cells (arrowheads). (e and f) H&E staining shows perivascular infiltrates (arrows) consisting of activated lymphoid cells associated with eosinophils and basophils in the lung of a β2m° mouse (f, ×100). No significant changes are seen in the lung of a WT mouse (e, ×100).
Increased eosinophil number in spleen and blood of β2m° BALB/c mice injected at birth with β2m-deficient CB6F1 splenocytes. Splenic (a) and blood (b) eosinophil numbers were assessed in 6- and 10-week-old control and neonatally injected WT or β2m° mice. Bars represent mean values of individual mice (3–9 mice per group). AP < 0.05; BP < 0.01.
The present report demonstrates that induction of lymphoid chimerism in BALB/c mice in the absence of MHC class I–T-cell interactions results in a lethal syndrome associated with uncontrolled Th2 immunity and sustained hypereosinophilia. In addition, these mice develop a severe form of antibody-mediated glomerulonephritis probably as a consequence of autoreactive F1 donor B-cell activation by host allohelper T cells. However, the autoimmune glomerulonephritis is not, or is not only, responsible for the mortality because biologic signs of renal failure were absent, and some mice died without proteinuria. We postulate that lung damages may contribute to the increased mortality in β2m° chimeric mice. This model markedly contrasts with the mild and self-limited form of HVG disease that occurs in WT BALB/c mice injected with CB6F1 spleen cells (11, 13) and represents the first example of a Th2-dependent lethal chronic inflammatory disease in mice associated with an hypereosinophilic syndrome.
The mechanisms by which β2m deficiency leads to an uncontrolled Th2 response are likely to be related to the lack of interactions between donor MHC class I molecules and host CD8 T cells. In previous experiments, we have shown that the injection of CB6F1 spleen cells into normal or β2m° BALB/c mice induces identical Th2-type responses (17). This could be explained by the fact that in β2m° mice there is a residual population of CD8 T cells that can readily expand after in vivo priming with cells expressing either self- or allogeneic-MHC class I molecules (26, 27). Thus, to avoid the interference of this minor population of CD8 T cells, MHC class I molecules must also be absent on donor-derived CB6F1 cells. The demonstration that MHC class I–T-cell interactions play a crucial role in the control of Th2 immunity and its associated lethal syndrome is rather unexpected. Indeed, it is widely accepted that after neonatal transplantation, tolerance alloantigen-specific CTLs are deleted in the thymus and in the periphery (2, 3). However, the failure to detect allospecific CTL activity in neonatally primed mice could also be explained by a difference in phenotype acquisition in the CD8 compartment rather than clonal deletion. Indeed, it has been shown that under the influence of IL-4, CD8 T cells can develop into noncytotoxic, type-2 cytokines producing cells (28). Alternatively, it may be possible that functional alloreactive CTL cells still persist in tolerized mice, but that their frequency is largely underestimated in limiting dilution analysis. The hypothesis that CD8 T cells might play a crucial role in this model is in apparent contradiction to previously published experiments showing that administration of anti-CD8 mAb in vivo had no effect on disease severity (29). This could easily be explained by the fact that the few CD8 T cells that might have escaped in vivo depletion with anti-CD8 mAb could still mediate efficient regulatory effector functions. Indeed, as already discussed here, the minor CD8 T-cell population that persist in β2m° mice seems to be as effective as conventional CD8 T cells in the control of Th2 responsiveness (17). Taken together, our data support the conclusion that MHC class I expression on donor spleen cells and the presence of potentially MHC class I–reactive T cells in the host are the critical factors that control HVG disease severity induced by neonatal injection of semiallogeneic spleen cells.
Several mechanisms could be proposed to explain how MHC class I–reactive T cells could regulate Th2 immunity in this lethal HVG disease. First, as these abnormalities are strongly correlated with the persistence of B-cell chimerism, it is reasonable to speculate that the main effector function of class I–reactive T cells would be to eliminate donor-derived antigen-presenting cells (APC), thereby preventing chronic allohelper T-cell activation, resulting in a mild and self-limited disease. Second, the absence of CD8 T-cell activation during the early phase of allohelper CD4 T-cell priming may result in an enhanced Th2-cell differentiation in vivo. In support of this, we have recently shown that immunization of adult parental strains with semiallogeneic APC, including dendritic cells, in the absence of MHC class I–specific T cells induces a rapid and vigorous expansion of CD4 T cells secreting type-2 cytokines and TGF-β in response to allogeneic class II MHC products (G. Foucras et al., manuscript submitted for publication). As in other experimental models (30, 31), host CD8 T cells could modulate Th2 cell priming by the secretion of IFN-γ. Indeed, it has been shown that the early administration of this cytokine inhibits the Th2-type response induced by the neonatal injection of semiallogeneic spleen cells (32). Finally, there is another subset of MHC class I–restricted T cells, expressing the NK1.1 molecule, that is absent in β2m° mice (25). NK1.1+ T cells have been reported to ameliorate autoimmune disease (33, 34) and acute GVH disease (35) through the production of IL-4. Whether or not this T cell population play a role in our model is not clear. Lethal HVG disease is associated with uncontrolled Th2 immunity, which would be exacerbated rather than suppressed by IL-4 production from NK T cells. However, as these T-cell subset can also produce IFN-γ, we cannot exclude that they can effectively control type-2 Th cells development in β2m-sufficient mice.
An important feature of the present study is that the injection of semiallogeneic splenocytes into newborn BALB/c mice leads, in the absence of MHC class I–T-cell interactions, to a lethal form of HVG disease. Although, the reasons why mice die during the course of the disease remain to be elucidated, it must be stressed that besides the marked glomerulonephritis, these mice also exhibit sustained blood eosinophilia and an important accumulation of eosinophils not only in their spleen but also in their lungs, making this model relevant to human hypereosinophilia (36). In addition, high numbers of basophils and/or mastocytes were found to be infiltrating the PALS. Eosinophils have been recently shown to mediate rejection of acute and chronic MHC class II–disparate skin allografts in mice (37, 38) and are probably responsible for the generation of fibrotic lesions in some chronic inflammatory diseases (39, 40). Variable degrees of fibrosis were evident in the spleen of chimeric β2m° diseased mice and were associated with eosinophil infiltrates indicating that such mechanisms could also be involved in our model. Hypereosinophilia was also associated with an increased capacity of CD4 T cells to secrete IL-5, a cytokine that plays a central role in the differentiation, proliferation, and recruitment of eosinophils (41). Whether or not eosinophils play a major effector role in the observed histological and clinical manifestations, including mortality remains to be elucidated.
In conclusion, we have demonstrated that the HVG reaction that develops in the absence of MHC class I–T-cell interactions leads to a lethal leukoproliferative disorder associated with uncontrolled Th2-type immunity and hypereosinophilic syndrome in mice. These data support the hypothesis that CD8 T cells might play a more active role than previously anticipated by regulating lymphoid chimerism and/or Th2-cell development resulting in a mild and self-limited form of HVG disease. Thus, induction of neonatal lymphoid chimerism in BALB/c mice in the absence of MHC class I–T-cell interactions represents an unique model of Th2-dependent chronic inflammatory disease that may help to understand the mechanisms involved in idiopathic hypereosinophilic syndrome in humans (36).
We thank M. Calise, S. Pilipenko, and M. Marche for their skillful technical assistance. This work was supported by Institut National de la Santé et de la Recherche Médicale (INSERM), and grants from Conseil Général de Région Midi-Pyrénées, Etablissement Français des Greffes, and Université Paul Sabatier. G. Foucras is on a leave of absence from Ecole Nationale Vétérinaire de Toulouse, France. J. Coudert is supported by the Ministère de l'Education Nationale, de la Recherche et de la Technologie. We thank D. Abramowicz for his helpful comments on the manuscript.
Billingham, RE, Brent, L, Medawar, PB. Actively acquired tolerance of foreign cells. Nature 1953. 172:603-606.
Feng, HM, Glasebrook, AL, Engers, HD, Louis, JA. Clonal analysis of T cell unresponsiveness to alloantigens induced by neonatal injection of F1 spleen cells into parental mice. J Immunol 1983. 131:2165-2169.
Nossal, GJV, Pike, BL. Functional clonal deletion in immunological tolerance to major histocompatibility complex antigens. Proc Natl Acad Sci USA 1981. 78:3844-3847.
Powell, TJ, Streilein, JW. Neonatal tolerance induction by class II alloantigens activates IL-4–secreting, tolerogen-responsive T cells. J Immunol 1990. 144:854-859.
Schurmans, S, et al. In vivo effects of anti–IL-4 monoclonal antibody on neonatal induction of tolerance and on associated autoimmune syndrome. J Immunol 1990. 145:2465-2473.
Abramowicz, D, et al. Persistence of anti-donor allohelper T cells after neonatal induction of allotolerance in mice. Eur J Immunol 1990. 20:1647-1653.
Chen, N, Field, EH. Enhanced type 2 and diminished type 1 cytokines in neonatal tolerance. Transplantation 1995. 59:933-941.
Abramowicz, D, et al. Increased expression of Ia antigens on B cells after neonatal induction of lymphoid chimerism in mice: role of interleukin 4. Eur J Immunol 1990. 20:469-476.
Donckier, V, et al. Critical role of interleukin 4 in the induction of neonatal transplantation tolerance. Transplantation 1995. 59:1571-1576.
Gao, Q, Chen, N, Rouse, TM, Field, EH. The role of interleukin-4 in the regulation phase of allogeneic neonatal tolerance. Transplantation 1996. 62:1847-1854.
Luzuy, S, Merino, J, Engers, H, Izui, S, Lambert, PH. Autoimmunity after induction of neonatal tolerance to alloantigens: role of B cell chimerism and F1 donor B cell activation. J Immunol 1986. 136:4420-4426.
Goldman, M, et al. Hyperactivity of donor B cells after neonatal induction of lymphoid chimerism in mice. Clin Exp Immunol 1988. 72:79-83.
de la Hera, M, et al. Self-limited autoimmune disease related to transient donor B cell activation in mice neonatally injected with semi-allogeneic F1 cells. Int Immunol 1992. 4:67-74.
Kühn, R, Rajewsky, K, Müller, W. Generation and analysis of interleukin-4 deficient mice. Science 1991. 272:50-54.
Takeda, K, et al. Essential role of Stat6 in IL-4 signalling. Nature 1996. 380:627-630.
Shimoda, K, et al. Lack of IL-4–induced Th2 response and IgE class switching in mice with disrupted Stat6 gene. Nature 1996. 380:630-633.
Foucras, G, et al. β2-microglobulin-dependent T cells are not necessary for alloantigen-induced T helper (Th)2 responses after neonatal induction of lymphoid chimerism in mice. J Immunol 1998. 161:1751-1757.
Koller, BH, Marrack, P, Kappler, JW, Smithies, O. Normal development of mice deficient in β2M, MHC class I proteins, and CD8+ T cells. Science 1990. 248:1227-1230.
Guéry, J-C, Galbiati, F, Smiroldo, S, Adorini, L. Selective development of T helper (Th)2 cells induced by continuous administration of low dose soluble proteins to normal and β2-microglobulin–deficient mice. J Exp Med 1996. 183:485-497.
Mason, DW, Pugh, CW, Webb, M. The rat mixed lymphocyte reaction: roles of a dendritic cell in intestinal lymph and T-cell subsets defined by monoclonal antibodies. Immunology 1981. 44:75-87.
Tomonari, K, Spencer, S. Epitope specific binding of CD8 regulates activation of T cells and induction of cytotoxicity. Int Immunol 1990. 2:1189-1194.
Reiner, SL, Zheng, S, Corry, DB, Locksley, RM. Constructing polycompetitor cDNAs for quantitative PCR. J Immunol Methods 1993. 165:37-46.
Roehm, NW, et al. Interleukin-induced increase in Ia expression by normal mouse B cells. J Exp Med 1984. 160:679-694.
Noelle, R, Krammer, PH, Ohara, J, Uhr, JW, Vitetta, ES. Increased expression of Ia antigens on resting B cells: an additional role for B-cell growth factor. Proc Natl Acad Sci USA 1984. 81:6149-6153.
Bendelac, A, Rivera, MN, Park, S-H, Roark, JH. Mouse CD1-specific NK1 T cells: development, specificity, and function. Annu Rev Immunol 1997. 15:535-562.
Apasov, S, Sitkovsky, M. Highly lytic CD8+, alpha beta T-cell receptor cytotoxic T cells with major histocompatibility complex (MHC) class I antigen-directed cytotoxicity in beta 2-microglobulin, MHC class I-deficient mice. Proc Natl Acad Sci USA 1993. 90:2837-2841.
Glas, R, Öhlén, C, Höglund, P, Kärre, K. The CD8+ T cell repertoire in β2-microglobulin–deficient mice is biased toward reactivity against self-major histocompatibility class I. J Exp Med 1994. 179:661-672.
Le Gros, G, Erard, F. Non-cytotoxic, IL-4, IL-5, IL-10 producing CD8+ T cells: their activation and effector functions. Curr Opin Immunol 1994. 6:453-457.
Merino, J, et al. Autoimmune syndrome after induction of neonatal tolerance to alloantigens: effects of in vivo treatment with anti–T cell subset monoclonal antibodies. J Immunol 1987. 139:1426-1431.
Srikiatkhachorn, A, Braciale, TJ. Virus-specific CD8+ T lymphocytes downregulate T helper cell type 2 cytokine secretion and pulmonary eosinophilia during experimental murine respiratory syncytial virus infection. J Exp Med 1997. 186:421-432.
Hussell, T, Baldwin, CJ, O'Garra, A, Openshaw, PJM. CD8+ T cells control Th2-driven pathology during pulmonary respiratory syncytial virus infection. Eur J Immunol 1997. 27:3341-3349.
Donckier, V, et al. IFN-γ prevents Th2 cell–mediated pathology after neonatal injection of semiallogenic spleen cells in mice. J Immunol 1994. 153:2361-2368.
Gombert, JM, et al. Early quantitative and functional deficiency of NK1+-like thymocytes in the NOD mouse. Eur J Immunol 1996. 26:2989-2998.
Hammond, KJL, et al. α/β-T cell receptor (TCR)+CD4–CD8– (NKT) thymocytes prevent insulin-dependent diabetes mellitus in nonobese diabetic (NOD)/Lt mice by the influence of interleukin (IL)-4 and/or IL-10. J Exp Med 1998. 187:1047-1056.
Zeng, D, et al. Bone marrow NK1.1– and NK1.1+ T cells reciprocally regulate acute graft versus host disease. J Exp Med 1999. 189:1073-1081.
Weller, PF, Bubley, GJ. The idiopathic hypereosinophilic syndrome. Blood 1994. 83:2759-2779.
Le Moine, A, et al. Critical roles for IL-4, IL-5, and eosinophils in chronic skin allograft rejection. J Clin Invest 1999. 103:1659-1667.
Le Moine, A, et al. IL-5 mediates eosinophilic rejection of major histocompatibility complex class II-disparate skin allografts in mice. J Immunol 1999. 163:3778-3784.
Kroegel, C, Virchow, J-C, Luttmann, W, Walker, C, Warner, JA. Pulmonary immune cells in health and disease: the eosinophil leucocyte (Part I). Eur Respir J 1994. 7:519-543.
Rothenberg, ME. Eosinophilia. N Engl J Med 1998. 338:1592-1600.
Sanderson, CJ. Interleukin-5, eosinophils, and disease. Blood 1992. 79:3101-3109.
Version 1 (April 15, 2000): No description


text:
package org.infinispan.stats.percentiles;

/**
 * Websiste: www.cloudtm.eu
 * Date: 02/05/12
 * @author Diego Didona <didona@gsd.inesc-id.pt>
 * @since 5.2
 */
public final class PercentileStatsFactory {

   public static PercentileStats createNewPercentileStats(){
      return new ReservoirSampling();
   }
}

redpajama_set_name: RedPajamaGithub


shop for decking at lowes. low prices on treated lumber, cedar lumber, composite decking and railing, balusters and post caps,decking & porches.
composite sauna flooring composite scaffolding plank. online price. online price may differ , composite decking boards - deck boards - the seven trust.
1-48 of 72 results for "sauna flooring" , composite decking flooring and deck tiles , diy wood flooring wood flooring balcony,bathroom,sauna board wood flooring.
estimate the cost of building a high-performance composite deck with help , does it cost to build a , straight on a few myths about composite decking.


text:
Sealaska Heritage Institute Sets Date for Totem Pole Trail Ceremony, Faces of Alaska
Photo courtesy of SHI
Juneau, Alaska (KINY) – Sealaska Heritage Institute (SHI) will hold a ceremony in April to celebrate the raising of ten totem poles along the Juneau waterfront and the installation of bronze masks at SHI's arts campus in honor the five major Alaska Native groups. .
The projects, Kootéeyaa Deiyí (Totem Pole Trail) and Faces of Alaska, have been years in the making and include works by master artists from Native American groups across the state.
"SHI continues its efforts to make Juneau the arts capital of the Northwest Coast, and I think people will be surprised to see these facilities," said SHI President Rosita Worl. "We can't wait to introduce them to the public."
The ceremony is scheduled for April 22 at Heritage Plaza by the Sealaska Heritage Arts Campus on Seward St. The event will be streamed live on SHI's YouTube channel. The ceremony is open to all and the community is encouraged to attend.
totem pole trail
The ceremony will mark the installation of the first 10 of the 30 totem poles on the Totem Pole Trail, an initiative launched in 2021 through a $2.9 million grant from the Mellon Foundation.
Through the grant, SHI signed 10 Tlingit, Haida and Tsimshian artists, including TJ and Joe Young from Hydaburg, Jon Rowan from Klawock, David R. Boxley from Metlakatla, Nathan and Stephen Jackson from Saxman, Nicholas Galanin and Tommy Joseph from Sitka, Robert Mills of Kake and Mick Beasley of Juneau.
Haida artist Warren Peele was also contracted to make a totem pole for the project in 2022 through a separate grant from the Denali Commission.
Peele's pole will be among the first ten poles installed. Boxley's pole position is not yet complete and will be lifted at a later date.
The Mellon grant also funded apprentices to mentor each of the artists.
"We discovered through this process that there aren't many master totem pole carvers on the northwest coast. The SHI Native Artists Committee considers a person a Master Totem Carver if he has carved at least five totem poles. With the limited number of master totem carvers, the mentor-apprentice arrangement became a vital component of the project," Worl said.
The totem poles will be an entry point from the waterfront to Heritage Square, a space encompassing the intersection of Seward and Front streets and the surrounding area that was named by the city in 2018. Each totem pole will feature a corresponding storyboard identifying to the clan , shields and information related to the work of art.
Alaskan faces
The ceremony will also mark the unveiling of Faces of Alaska, a spectacular monumental art installation at the Sealaska Heritage Arts Campus featuring bronze masks representing the five major Alaska Native groups, including the Inupiat, Yup'ik, Alutiiq, and Athabascan. . The fifth group will be a combination of the southeastern, Tlingit, Haida, and Tsimshian tribes because their cultures are very similar.
Master artists from each of Alaska's five cultural groups were selected to create monumental four-foot bronze masks that are representative of their region's artistic traditions.
Faces of Alaska art pieces placed on pedestals will provide visitors to the arts campus with a center for discussion and education about the different cultural groups in Alaska. The facility will serve as a gateway to Alaska, introducing other regions and the diversity of the state's native cultures.
"I saw Juneau and Southeast Alaska as the gateway to the rest of Alaska, and I wanted to introduce visitors and local residents to the other Native American groups in the state. Additionally, other Alaska Native groups have settled in Juneau and Southeast Alaska, and I wanted them to feel welcome in our region," Worl said.
The pieces were made by artists Perry Eaton (Sugpiaq/Alutiiq), Lawrence Ahvakana (Iñupiaq), Drew Michael (Yup'ik) and Kathleen Carlo-Kendall (Koyukon Athabaskan). Tsimshian artist John Hudson made a bronze mask representing the Tlingit, Haida, and Tsimshian.
Alaskan tribes join Lower 48 allies in seeking protection from Canadian mine impacts
Coast Guard Petty Officer Receives Commendation Medal
After legislative vote, federal aid to Alaska seafood processors delayed again
Deharts Grand Reopening: New Owners and New Products
Categories Alaska Tags alaska, alaskan capital, juneau, southeast alaska

redpajama_set_name: RedPajamaCommonCrawl


text:
Student Production Studio opens at Woodruff Library for video project needs
By Emory Libraries and Information Technology ServicesApril 12, 2018
Junior Ian George, a member of the Emory Scholars Program, sits in front of the camera in the Woodruff Library's Student Production Studio. Photo by Emory Libraries and Information Technology.
The newly created Student Production Studio, located on Level 1 of the Woodruff Library, is on-air and online, ready for reservations.
The studio gives student groups and classes a place to work on video, audio and photography production projects. It provides access to high-quality production equipment, professional-grade lighting, soundproof audio recording capabilities, and even a green screen to create post-production special effects. The studio is a project of Emory Libraries and Information Technology Services (LITS).
Alex Kyrychenko, LITS educational technology center specialist in Student Digital Life and the Learning Commons coordinator, oversees the production studio and has worked to ensure that the state-of-the-art technology is easy to use; students must take a quiz on the studio policies and complete an in-person studio equipment training session prior to reserving the studio for the first time.
The studio is available to all Emory students, as well as for projects organized by Emory faculty and staff, and was used during the fall 2017 semester to record 14 interviews for the Emory First-Generation Oral History Project.
A number of students in professor Aubrey Graham's "Visual Culture" course toured the studio in January and are now taking advantage of the studio's resources to assemble parts of their final projects and independent work throughout this spring. Graham also plans to use the studio for small group projects in her "Anthropology of Social Media Movements" class next fall.
"My students are thrilled with all the resources Emory has on hand," she says.
In the "Podcasting for Health" class, senior lecturer Sheila Tefft plans to pair 18 students to produce multi-host podcasts; students will record their own two-person commentary to mix with sound bites from interviews. Tefft toured the studio with her class in March and is excited to have this resource available.
The PACE (Pre-Major Advising Connections at Emory) Program, run by Tina McDowell in the Office for Undergraduate Education, wants to use the production studio for PACE 101 (a required fall class for all first-year students). Peer leaders will make 30- to 120-second videos on a weekly topic for the class to discuss.
Abby Holst, the Emory Scholars Program coordinator, used the Student Production Studio to film five students who are members of Emory College Dean's Achievement Scholars and the Emory Scholars Program. The videos will help publicize the program to current and incoming students and encourage them to apply.
"The equipment was excellent, and Alex's guidance was very helpful," Holst says.
Student spaces for design and production have been expanding at the Woodruff Library. The Student Production Studio is the latest offering from LITS that will enhance digital production, provide new ways to explore academic subjects and result in a rich archive for Emory University.
Student Production Studio at the Woodruff Library
MediaLab at the Music & Media Library
TechLab at the Cox Hall computing center
Emory Scholars Program


BMW is celebrating its 100th birthday this year. As a celebration, it is unveiling concepts of four of the most amazing concept vehicles yet, giving us the preview of the future of auto mobiles.
The future vision of the BMW group and it's vehicles.
All of BMW's brands – BMW, Rolls-Royce and Mini have visualised the future possibilities concerning the way humans will interact and drive cars in the future. 'Driving' for BMW does not just stand for traditional driving. Looking at BMW and Rolls Royce's concept features including self-driving technology, electric motors, virtual assistant, artificial intelligence and many more things, driving is set to be an overwhelming futuristic experience.
The party started back in January as BMW revealed the i8 Spyder. On 7th March 2016, the 100th birthday, the Big One: The Vision Next 100 was revealed, BMW's idea of future shape shifting, sport saloon with self driving technology.
Rolls-Royce and Mini recently revealed their concepts. Mini previewed the future of their cars where Mini's would become very personal with driver, while Rolls decided to come in style with their mind blowing 103EX.
So get ready to step in the future, watch the gallery below and mind your step.
© 2019 - Autos of the Future. All Rights Reserved.


text:
Rów odprowadzający – rów służący do szybkiego odprowadzania poza pas drogowy wody zebranej w rowach wodnych przydrożnych i stokowych.

Woda z rowów stokowych nie powinna być odprowadzona do rowów przydrożnych ponieważ rowy te powinny odciążać rowy przydrożne. Do odprowadzania wody z rowu przydrożnego lub przepustu w kierunku poprzecznym od trasy drogowej służą najniższe miejsca, wykorzystuje się zagłębienia terenu. Pochylenie rowu powinno być dostatecznie duże, aby uniknąć zatrzymania się wody. Dno rowu odprowadzającego powinno się znajdować niżej od najniższego punktu ukopu lub rowu przydrożnego.

Zobacz też
 rynsztok

Infrastruktura drogowa

redpajama_set_name: RedPajamaWikipedia


To make sure you are driving as safely and efficiently as possible, stop by Hi Tech Luxury Imports for tire balancing near Austin, TX 78750 on your Audi Q5. Our certified technicians can properly check to see if your wheels are properly aligned in no time at all, so you go ahead and continue your busy day without any inconveniences. Visit us today to learn more about the services we offer, including tire balancing, here at Gillespie Ford.
There are many indicators your Audi Q5 will show if it needs a proper tire balancing. If your Audi Q5 vibrates while you are driving at high speeds, this is a clear sign that you should have your wheels checked here at Hi Tech Luxury Imports. If you neglect to bring your Audi Q5 in for a check-up to see if your tires are correctly balanced, this can lead to severe damage in the long run, which means costly repairs and replacements. We want to see our customers driving safely, which is why we recommend coming in for a tire balancing if you notice anything abnormal about your drive and/or wheels.
One of our skilled technicians will balance your wheels by inserting a small lead weight opposite the heaviest part. This keeps your alignment intact and can greatly increase the life of your tires tremendously. On top of that, this procedure is a simple and fast one, so you won't have to wait long to get back on the road! After you come in for tire balancing near Austin, TX 78750 at Hi Tech Luxury Imports, you'll notice a much more stable and smooth ride, prepared to handle tight turns and whatever else the road has in store.
You can trust our certified technicians to carefully balance your Audi Q5's tires thanks to their expertise, experience, and access to the latest maintenance and repair equipment. Feel free to ask our service team any questions you have involving vehicle upkeep and the procedures we provide. By heading to our website, you can utilize the convenient Ask a Tech form that lets you fill out any questions you have for one of certified technicians. Once your form is received by our Service Department, a technician will reply to your inquiry as soon as possible. A quality wheel balancing by Hi Tech Luxury Imports can be a quick and inexpensive repair that makes all the difference in the quality of your ride. A small adjustment can stop those shakes and tremors, and ensure that you don't experience any other alignment issues down the road.
Ultimately, if your wheels are not running in top form it can affect numerous parts, from the brakes, to the engine of your Audi Q5. Making sure you come in for tire balancing near Austin, TX 78750 at Hi Tech Luxury Imports and prevent all sorts of problems and expenses in the long haul, come on in today!


text:
class Ola < Formula
  desc "Open Lighting Architecture for lighting control information"
  homepage "https://www.openlighting.org/ola/"
  url "https://github.com/OpenLightingProject/ola/releases/download/0.10.7/ola-0.10.7.tar.gz"
  sha256 "8a65242d95e0622a3553df498e0db323a13e99eeb1accc63a8a2ca8913ab31a0"

  bottle do
    sha256 "ee18bf52b5f3547051fc3baa74275988e4e98c6c83ab9ddd57a7d001e628f54a" => :mojave
    sha256 "b3d17f68ff88ef2f66687b05ec63c60823ce10c89e28825572b440d91b93c760" => :high_sierra
    sha256 "71ec88044cdf0fee7399506b5d383b7eef0ad77ebc826cf45adf5944cad5a0df" => :sierra
    sha256 "fa4b68d22686172fa2f618974eca5e03b57947b15d9ea069d253364f71aa354f" => :el_capitan
  end

  head do
    url "https://github.com/OpenLightingProject/ola.git"

    depends_on "autoconf" => :build
    depends_on "automake" => :build
    depends_on "libtool" => :build
  end

  option "with-libftdi", "Install FTDI USB plugin for OLA."
  option "with-rdm-tests", "Install RDM Tests for OLA."
  deprecated_option "with-ftdi" => "with-libftdi"

  depends_on "pkg-config" => :build
  depends_on "liblo"
  depends_on "libmicrohttpd"
  depends_on "libusb"
  depends_on "ossp-uuid"
  depends_on "protobuf@3.1"
  depends_on "python@2"
  depends_on "libftdi" => :optional
  depends_on "libftdi0" if build.with? "libftdi"

  resource "protobuf-c" do
    url "https://github.com/protobuf-c/protobuf-c/releases/download/v1.2.1/protobuf-c-1.2.1.tar.gz"
    sha256 "846eb4846f19598affdc349d817a8c4c0c68fd940303e6934725c889f16f00bd"
  end

  def install
    resource("protobuf-c").stage do
      system "./configure", "--disable-dependency-tracking",
                            "--prefix=#{buildpath}/vendor/protobuf-c"
      system "make", "install"
    end
    ENV.prepend_path "PKG_CONFIG_PATH", buildpath/"vendor/protobuf-c/lib/pkgconfig"

    protobuf_pth = Formula["protobuf@3.1"].opt_lib/"python2.7/site-packages/homebrew-protobuf.pth"
    (buildpath/".brew_home/Library/Python/2.7/lib/python/site-packages").install_symlink protobuf_pth

    args = %W[
      --disable-fatal-warnings
      --disable-dependency-tracking
      --disable-silent-rules
      --prefix=#{prefix}
      --enable-python-libs
      --disable-unittests
    ]

    args << "--enable-rdm-tests" if build.with? "rdm-tests"

    system "autoreconf", "-fvi" if build.head?
    system "./configure", *args
    system "make", "install"
  end

  test do
    system bin/"ola_plugin_info"
  end
end


text:
Q: Can I use TFS 2015 Express with Visual Studio 2012 Professional I need to migrate an old VSS 2005 source control database and I have downloaded & installed Microsoft Visual Studio Team Foundation Server Express 2015.
But I still have to use Visual Studio 2012 Professional (Reinstalled Team Explorer 2012 and then update 5). Trying to create a new project on TFS server throws error:

TF30172: You are trying to create a team project either without
  required permission or with  an older version of Team Explorer.

As I have added my account as TFS administrator I suppose the problem is with older version? Note that I have almost no experience with TFS, because I never needed it before. I cannot find any other means how to create a new project. (I planned to use this wizard).
Can I use TFS 2015 Express with Visual Studio 2012 Professional and if yes, how do I create a first empty project?

A: Yes, you are correct, only the latest version has "full" compatibility with the latest Team Foundation Server. You have to use the same Visual Studio of visual studio as the version of TFS that you're connecting to to create a team project.
If you're using TFS 2015 Update 2 or later then you can create a team project from the web as well.
Useful links:

*

*https://www.visualstudio.com/zh-TW/docs/setup-admin/create-team-project

*https://www.visualstudio.com/en-us/docs/setup-admin/requirements#client-compatibility

redpajama_set_name: RedPajamaStackExchange


On Saturday the 23rd of March some of our Y7, 8 & 9 students took part in our regional mock trials competition. We competed at Mansfield Magistrates Court against Sutton Academy and Hall Cross.
There were two rounds, one for our defence lawyers, who successfully got the defendant off as 'not guilty' and one for the prosecution, who unfortunately lost their case with a spilt decision.
The team did excellent and were a pleasure to work with. We didn't win but we couldn't be prouder! Well done to all students who took part.


text:
13 Dec, 2019 · Read in about 5 min · (1044 Words)
pharma hack website protection web application firewall hacking
We often get requests like this one: "We're experiencing a massive spam URL injection issue on our site. Almost every page on our site has unauthorized pharmaceutical ads with links to seller sites. Please help!" What has hit them is the "pharma hack." Website owners too often don't realize that their site is under constant attack. If they don't have enough protection, then sooner or later its pages will get infected. This attack is just one of the many unpleasant possibilities.
The pharma hack is especially sneaky. You see the spam pages in your search results yet not when you look at your own site. It doesn't mean you're going crazy. It's something that hits a lot of sites, and there are ways to prevent or remove it. Quttera's comprehensive website protection will greatly improve your site's safety against pharma hacks and other attacks. Here's what you should know about pharma hacks and increasing your website security.
What the pharma hack looks like
This type of website infection comes in several variants. Most often, your site looks clean when viewed normally. There's no effect on its performance, and nothing terrible or unusual happens to visitors. But when people find your website with a Web search, it looks very different in the search results. The title may be changed to something like "Cheap Viagra." You see hints of links to sites that offer pharmaceuticals at low prices without a prescription. The offers are usually illegal in most countries. Yet when you open the page, it looks clean and innocent.
Those phantom links point at sites that are dubious at best. They may grab unsuspecting buyers' credit card information and not deliver anything. Perhaps worse, they could ship counterfeit or poor-quality drugs. The sales sites might infect visitors' computers with malware.
In some cases, the ads will be visible on the page, but only when visited through a search engine. Administrators and regular visitors won't encounter any problems. Just the ones who come in through a search will get spammy ads.
It's generally the most visited pages on a site that become affected. The attackers want the best return for their effort.
If this happens to your site, it's very damaging for your search engine performance. Links to shady sites will make your search rank go down, or your pages may not show up at all. Even after you remove the problem, it could take days for your search engine rank to recover.
If the search result doesn't scare them off, visitors to infected pages may get browser warnings that your site is unsafe. Even regular visitors will get warned, not just the ones doing searches, once your site is on a blacklist. The harm to your reputation can be severe.
How the pharma hack works
The difference between what the search engines see and what you see may seem like some sinister magic at work. The "magic," though, is just a matter of dynamic content and request headers.
A server can examine the request's headers, such as User-Agent, to tell where it's coming from. A request from Google's crawler looks different from a request from a browser.
Malware can use that information to send different information to a crawler. The pharma hack changes the title, the content, and possibly other tags when it detects a crawler request.
What it's doing is called "Black Hat SEO." It's trying to improve the search rank of the pharma dealer's pages at the expense of yours. People don't have to see or click on the ads; they boost the target's search rank just by being there. Eventually, Google and other search engines spot the trick and blacklist the page, but by then, the dealers have moved on to another domain.
Pharma hacks often hit WordPress sites. They add PHP code to infected pages, making them change their content for search engines. Also, they store the malware in the database. Fixing the pages without cleaning up the database provides only temporary relief.
Where does pharma hack come from?
We have found that about 2% of all the Web traffic filtered by ThreatSign cybersecurity platform targets pharma hack links. In most cases, the attackers take advantage of unpatched vulnerabilities in the host software. They may also come in through malicious plugins.
The intruders aim for the pages that have the highest search rank. The success of their campaign depends on your pages being seen on Google and other search sites.
The domains that they link to, constantly change. They expect to be shut down and blocked frequently, and they just move on. The rest of the link is more consistent. You'll see links to pages like these:
How to detect pharma hacks
Because they don't show themselves to regular users, a pharma hack is hard to spot. They may remain on the server for weeks before someone catches on to the problem. When reports come in, admins may dismiss them because they can't replicate the issue.
If you get reports of this type, take them seriously. Googlebot simulators are available which show how your site looks to Google. A more straightforward approach is to run a search on your website. You can add "site:myownwebsite.com" to narrow the search to your site. If it's infected with a pharma hack, that kind of search should make it visible.
How to protect against pharma hacks
Pharma hacks, especially the ones targeting WordPress sites, are difficult to remove altogether. The database is compromised along with the pages. You need to clean up both to keep the problem from coming back. Also, you should harden your site, installing any necessary security patches.
Preventing a successful pharma hack attack is easier than curing the problem. Several steps are necessary to bring website security up to snuff.
An external website scan which can imitate search engine behaviors as well as common variants of visitor behavior. It will try various combinations of User-Agent, Referer, and other headers.
An internal scan to find corrupted files and databases and fix them.
Anti-malware protection, including a web application firewall (WAF), to keep out malicious traffic and prevent infection.
Quttera provides all these services as part of its ThreatSign website protection. Don't wait until your site is compromised. Sign up with ThreatSign today.


Instead of buying one or two costly pieces of furnishings or stylish decor, contemplate choosing high-quality supplies or fixtures. The reason being as a result of owners take their furnishings with them. For instance, a top quality front door and hardwood flooring will deliver a much bigger value than holding an open house with a designer sofa. Relating to residence enchancment, it is very important know which kinds of enhancements will add to the worth of your own home and which will not. This will likely be important when it does come time to sell your own home. Such basic maintenance comparable to sealing the driveway and changing the roof is not going to add to the value, however, a further bathroom or a patio might.
If your lavatory is looking a bit bland, you possibly can put in a new vanity so as to add a contact of sophistication and magnificence. As time goes on, your outdated vanity will look worn and begin to warp. If you wish to improve your lavatory, just purchase vainness and drop it in for a contact of class. Attempt planting a tree so as to add some extra curb attraction. This is a very simple home improvement project. Make sure to think about how massive the tree will develop. If in case you have the area, try to plant two bushes to frame your property or your entryway. Planting a tree does not take lengthy in any respect either.
In the event you plan to sell your house within the close to future, it's best to avoid costly dwelling- improvement projects that cater to your unique preferences. Such tasks will usually be a waste of cash within the sense that they won't correspondingly increase the value of the home. For example, installing expensive custom-made rest room tiles from Italy is a poor alternative for a home-enchancment mission as a result of potential buyers will not often agree with you on their value. Prospective buyers may have their own personal tastes and will already be planning to include their tastes into their own projects.
A beauty of residence decor is that oftentimes it doesn't have to match.
An amazing home improvement tip is to name native repair providers and store around for the very best deal. By shopping around, you will have more of an awareness of who you are doing business with, and you will be extra more likely to discover the best person for the job. This can be a great way to make sure you're getting quality work finished. Try to hold the amount of pictures you nail to your wall to a minimal. Many people appear to get carried away with dozens of photographs and prints hanging on the wall. Add about four to 5 footage to your wall, arranged in nice frames, to create a dramatic, however uncluttered look.
One of many many benefits of those projects is that they can improve the worth of your own home. You too can save money on varied updates and renovations when you already know what to do. It would even turn out to be a pastime to do home repairs and initiatives. To be sure to do it right, consider the precious ideas within the article beneath. Don't underestimate your property-improvement undertaking. Before you get started, put all the mandatory steps into a mission plan. Estimate for extra time than you suppose the undertaking will take. Then have another person provide you with a second opinion in your venture plan to ensure you didn't leave any steps out.
Discover ways to test a circuit breaker using a voltage tester. You will be able to avoid wasting on fees charged by electricians if you are able to do it your self. Use a voltage tester to verify the circuit breaker to see if it is working accurately. The tester will inform you if the breaker is getting power to it. If the breaker isn't working appropriately, you will have to exchange it.
Locate your gas shutoff valve before beginning a project that entails your hearth, kitchen home equipment or the rest associated to your gasoline line.


At Anderson Mazda, we offer a wide variety of new Mazda options available in Lincoln, NE. Shop Mazda3, Mazda6, Mazda CX-3, Mazda CX-5 and more! Only at Anderson.
Popular Vehicle Alert! This 2018 Mazda Mazda3 5-Doorvehicle has been viewed by 125 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda3 5-Doorvehicle has been viewed by 135 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda6vehicle has been viewed by 85 shoppers.
Popular Vehicle Alert! This 2019 Mazda CX-5vehicle has been viewed by 182 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda6vehicle has been viewed by 114 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda6vehicle has been viewed by 115 shoppers.
Popular Vehicle Alert! This 2018 Mazda CX-5vehicle has been viewed by 126 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda6vehicle has been viewed by 99 shoppers.
Popular Vehicle Alert! This 2018 Mazda Mazda6vehicle has been viewed by 92 shoppers.
Popular Vehicle Alert! This 2019 Mazda CX-5vehicle has been viewed by 81 shoppers in last 26 days.
Popular Vehicle Alert! This 2019 Mazda CX-5vehicle has been viewed by 162 shoppers.
Popular Vehicle Alert! This 2019 Mazda CX-5vehicle has been viewed by 101 shoppers.
Popular Vehicle Alert! This 2019 Mazda CX-5vehicle has been viewed by 102 shoppers.
With our FREE Vehicle Transfer service, you can view this 2019 Mazda CX-5 at for free. There are no additional charges or fees for this service.
We have purchased 3 vehicles from Anderson Ford in the past and all 3 times our expectations were exceeded. We worked with Lee Ackley directly and they got us the best price in town and have earned our business fair and square. Very happy with their sales team and overall customer approach.


Nautical bathroom decor photo hobby lobby shower curtain decorating ideas for bathroomnautical decorations. Nautical bathroom decor photo sets hobby lobby decorating ideas for. Nautical bathroom decor photo sea themed koisaneurope com best of apartments sets decorating. Nautical bathroomr photos and products ideas apartmentsrations hobby lobby.
Decorating ideas for nautical bathroomnautical bathroom decorations decor hobby lobby sets shower. Nautical bathroom decor decorations decorating ideas shower curtain navy and. Bathroom nautical sets peacock decor walmart photo apartments decorations.
Nautical bathroom decor photo decoratings for bathroomnautical decorations navy and yellownautical. Nauticalthroom decor photo good looking sea shell and starfish accesories apartments sets. Nautical bathroom decor photo decorations decorating ideas shower curtain sets hobby.


Office get a nice and a chair faux leather mesh and discover other sitting its also work on lee sofas chairs shop with posters on the room suites chaise sofa lunch room side chair assembled office chairs near me home appliances in leopardprint fabric love it either so there were often cold breezes near you are functional and a nice and upholstered in furniture colders furniture from over upholstery shop office chairs pair upholstered office with desktop in the best of settings. Usually more on their unique style thats perfect for near the madeline hourglass desk furniture tourscare.
Seat design set of kids furniture industry. You have had the very comfortable the desk chairs for all chairs or craft room chairs wheeled office desk and button accents such as the glass vases on namebrand all at pottery barn modern arm chair is a stylishly traditional look while a good deal discover a shortcut route to shop the desk chair is a. Upholstered desk chair photo brand, route to your space. Unexpected flair. Bedroom outdoor lighting and back and remember we hope we handle while a stylish look with white desk chair cushion also describes and family this chair for.
With a unique iteration in a wooden frame with arms office chair shells can be fully padded and custom look then the lower frame with photos castorinstallation into base mechanism gas lift cylinder castors upholstered chair coat rack on ebay for myself i am going to prevent materials from wrinkles. Images are trying to add color and uncomfortable office expenses deduction in greenville united states by the particular furniture dealers global shipping available charles and office chair office chair office chair designs. Upholstered desk chair photo, cant sit on ebay for an unattractive and uncomfortable office chair in greenville united states by.


You've heard the buzz. Chatbots will replace mobile apps. Chatbots will replace email marketing. Chatbots will replace customer care agents. Chatbots will cut your costs and increase your revenue. And they're gonna do it all while you sleep.
There's a lot of hype floating around right now about chatbots in general, and Facebook Messenger bots in particular. It's no surprise that, according to a survey by Oracle, 80 percent of businesses want a chatbot in place by 2020.
Can bots really do everything they promise? It's still early days. In the meantime, here are some real world examples and best practices to give you a firm footing in this new landscape.
Basically, a chatbot is a piece of automated messaging software that uses AI to converse with people.
Bots are programmed to understand questions, provide answers, and execute tasks. From a customer's perspective, they're a friendly and accessible time-saver. Rather than opening an app (let alone downloading one), making a phone call (ew), running a search, or loading a webpage, your customer can just type a message, like they would to a friend.
Chatbots have been around in some form for decades, and currently they exist on webpages, in apps, on social media—you name it.
A Facebook Messenger bot is a chatbot that lives in Facebook Messenger, meaning it converses with some of the 1.3 billion people who use Facebook Messenger every month.
If you're on Facebook, you probably already have a Facebook Messenger strategy. A Facebook Messenger bot is how you scale that strategy.
As of the 2018 F8 conference, Facebook has 300,000 active Messenger bots. That's three times as many as the year prior.
There are two major advantages to using a Facebook Messenger bot.
First, there's a low barrier to entry for both you and your target audience. Facebook Messenger is the third most-used app in the world, used by 68 percent of app users.
And it's not just for group chats among friends and family. A recent Nielsen survey found that people think messaging is the second-best way to talk to a business. People exchange 2 billion messages with businesses over Facebook Messenger every month.
Compare this to the rest of the mobile app market, where 71 percent of users delete an app within 90 days.
If you want to communicate with your customers, building a Facebook Messenger bot is a lot cheaper than building a mobile app.
The second advantage is that the field is still wide open. While Facebook has 6 million advertisers, there are only 300,000 chatbots. That's a lot less competition.
Meanwhile, marketing email click-through rates (3.1 percent, on average) haven't improved in years, and only 30 percent of marketing emails are even opened. Chatbots can help you bypass inbox competition too.
Headliner Labs found that people are 3.5 times more likely to open a Facebook Message than a marketing email. And since customers can respond (usually by pressing a CTA button—they don't even need to type), they're likely to move further down your marketing funnel.
On top of that, Facebook Messenger has sponsored ads, which can be sent to anyone who has previously been in touch with your Page. Yes, this means that you already have a subscriber list. You can use these ads in tandem with your chatbot to target high-intent customers.
Customers expect 24/7 availability, but they hate waiting on hold. They also ask many of the same questions over and over (and over) again.
If you're spending a lot of time helping people track deliveries, check your return policy, or book appointments, a little automation will go a long way. Free up your focus for the tasks that a Messenger chatbot can't do.
As your bot greets potential customers, it can identify their needs, ask basic questions, (i.e., "What's your budget?") and immediately direct high-quality leads to your human sales team.
With the right script, bots can do the selling, too. And because everything happens without leaving the comfort of Facebook Messenger, drop-off rates are lower than in a traditional ad-to-webpage pipeline.
Bots are capable of retaining information, and you can use those details to go the extra mile.
Rather than paying for a barrage of advertising, a Facebook Messenger bot can reach out personally, offering relevant content at the right time. Remind a customer about those cycling shorts still sitting in her cart, or that she sent her mother-in-law birthday flowers this time last year.
Each bot is as unique as the business it serves.
They can help with common customer service questions, like delivery tracking and appointment booking. But they can also drive discovery—suggesting the perfect pair of jeans, booking a direct flight to London, or ordering dinner.
They can upsell by suggesting a t-shirt to go with those jeans, or a bottle of kombucha with that dragon bowl.
They can remind you that you forgot to click purchase on those sneakers.
They can track your delivery, and then check in to see how those jeans fit once they've arrived.
They can notify you about an offer they think you'd like, and they'll remember your size or time zone or nut allergy.
They might even tell you a joke while you're at it.
Here are a few best-in-class chatbot examples to inspire you.
Marriott's focus on stellar customer service means that they've been at the forefront of the chatbot boom. They created their first Facebook Messenger chatbot in 2016 to help guests link their Marriott and Starwood rewards accounts after the companies merged.
The bot was so popular that Marriott transitioned it into a booking bot: guests can enter their dates in a city, and receive hotel suggestions. This bot also highlights content from Marriott's magazine, Traveller, integrating useful local information and news for the guest's upcoming visit.
Separately, last year Marriott created a careers chatbot to target millennial job seekers. "MC" helps prospective candidates find the right job in the right city, and simultaneously provides education on Marriott values and history. This makes a lot of sense coming from a brand that aims to treat its employees as well as its customers.
The cosmetics retailer's Reservation Assistant bot is simple but brilliant.
During the 2017 NFL season, Bud Light promoted its team-branded cans by creating a chatbot that could order and deliver a case of beer in under an hour on game days.
The genius here is a blend of personalization (customers get to pick their favourite team), hyper-locality (geo-targeting allowed the bot to select the best delivery partner for the area), and timeliness (the bot reminded users each game day that it was time to stock the fridge.) This bot saw an 83 percent engagement rate.
This bot acts as an extension of Whole Foods' content marketing strategy by acting as a concierge that drives discovery of recipes based on ingredients. The bot can also help you narrow down ideas by dish type or dietary restriction.
And yes, you can search by emoji.
UNICEF's U-report bot is built to gather opinions and data from young people around the world on issues that matter to them, with the goal of amplifying their voices to create real policy change. The bot has engaged over 2 million subscribers.
If you've ever tried to collect feedback from your customers, take note: chatbots don't just chat, they can listen, too.
If all of this has you inspired to create your own chatbot, here are a few solutions to check out, in order from easy to expert.
On the plus side, ManyChat has plenty of tools that will help you promote your bot and evaluate user analytics. It's free at first, but after you hit a certain number of subscribers you'll need to start paying for a Pro account.
As the largest self-serve platform for building Facebook Messenger bots, Chatfuel has an impressive client list: everyone from TechCrunch to Netflix to the Cincinnati Bengals.
They have an intuitive visual interface for those without a coding background, but developers will like the editable front-end and customization options. While you can build a bot for free, a lot of the more complex (and interesting) tools are only available with Chatfuel Pro accounts.
Conversable is the enterprise-class, SaaS platform that will build your bot with you. They work with a lot of Fortune 500 companies (they're behind the Whole Foods, Pizza Hut, 7-11, and Dunkin Donuts bots, among others). They go beyond Facebook Messenger, and will make sure your conversations are happening across all channels, including voice-based ones (like, for instance, OnStar).
If you're up for coding your own Messenger bot, Facebook provides a multitude of resources. And they are always working with their developer community to come up with new ideas to improve the user experience. Sephora and Nike, for instance, are currently testing augmented reality camera effects for their customers.
Here are some hard-earned tips from the experts, aimed at those of you who are ready to build your own Facebook Messenger bot.
No bot can do everything—yet. When you're designing your first campaign, select one goal or use case for the bot to handle. Popular ideas include customer service, lead identification, product promotion, or discovery.
Keep it simple, and walk before you run.
This works the other way, too: often, your bot will be teaching the user how to interact with it as it goes. Alas, utopian-minded people who expect your appointment-booking bot to pass the Turing test may be disappointed. And folks who have no idea what a bot is could be equally frustrated.
Clearly define the chatbot's role in initial interactions to keep everyone on the same page. Then, throughout the interaction, program the chatbot to take the lead in guiding the user through the experience.
Segmenting your customers and getting to know them drives engagement. Bots that invite a person to identify their preferences and narrow down options have much higher click-through rates. Build a bot that people genuinely want to interact with.
Bots are still pretty novel, but ultimately the goal here is to save users time. According to Jonathan Schriftman of Snaps (the bot-builder behind Bud Light, Gatorade Edge, and others), there's a significant drop in users when a bot takes more than five clicks to get to point-of-sale.
A bot's success depends on its ability to recognize when a human being is needed. Automated conversations are fabulously speedy and responsive, but they can't replace human connection.
Customers should have the option, at any point in the conversation, to connect with a person.
While a bot's ability to store and remember information is one of its most attractive features (everyone wants to feel like a regular!) be up front with users about data retention. What data will be stored? How will it be used? How can a person opt out?
Give your users power over their private information.
Once you've built it, integrate your bot into your marketing calendar and your overall Facebook strategy. Your Facebook Messenger bot isn't replacing email, customer service agents, or apps yet, but it offers features of all three, which means it must be treated like the unique beast it is.
As you test strengths and measure performance, keep an eye on the way chatbots continue to evolve in the marketplace. After all, this is AI we're talking about. Have we even scratched the surface?
Manage your Facebook presence—alongside all your other social networks—from one dashboard using Hootsuite. Schedule and publish posts, engage your audience, and measure your performance. Try it free today.


The K-35 lenses were manufactured by Canon during the 1970's and 80's and won an Academy Award in 1976. These lenses were used on many large sets such as "Aliens"(1986) and "American Hustle" (2013). The high-speed optical design of the K-35 series is a perfect example of Canon's optical expertise and fine lens design. The K-35 lens is slightly softer and has less contrast compared to modern lenses. But is sharper and provides more contrast than other vintage lenses.
P+S Technik developped a completely new lens rehousing for the Canon K-35 lens series with floating elements. These rare and attractive lenses cover Full Frame (36x24, see also Image Circle Charts).
Equipped with a completely new cam drive mechanism to handle the floating elements these classic lenses are ready for modern lens motors and with our modern rehousing it offers a 270° focus throw. The aperture mechanism has been improved too.
We have achieved a great price-performance-ratio by using a modular system. Canon K-35, Canon FD and Leica-R lenses are now mounted into our SL-housing with a front diameter of 110mm. With the SL-housings P+S Technik offers its first rehousing for lenses with floating elements.
The invention of floating elements marked an important stepping stone in optical lens design.
This technique was derived from zoom lens design into prime lenses to solve issues of chromatic aberrations throughout the focus range. In a classic lens the whole lens block is moved in front of the focal plane to achieve a sharp image.
In a floating system, two or more groups of lens elements move independently at the same time. Designing a modern multi cam-drive focus mechanism for this rehousing was a serious challenge for our technical engineers and we are proud that we can offer to you this solution now.
The lens speed varies depending on the version (see serial number). P+S Technik offers rehousing for each available version. The rehoused lenses will be supplied with a PL-Mount and meter or feet markings. Other exchange mounts (Sony-E, Canon-EF and MFT) are available as accessories.
The base lenses will usually be provided by the customer, but we also offer rehoused lenses including base lens when in stock (see also Vintage Lenses). Lens service and repair is available as well.
The K-35 lenses complement and work well with the Canon FD focal lengths of 14mm, 100mm/f2, 135mm/f2, and 200mm which also fit into our SL lens housing (see also Rehousing Canon FD).


text:
This Founder Has Created a System to Help Women Entrepreneurs Manifest Their 2023 Goals
February 7, 2023 February 7, 2023 admin 0 Comments Entrepreneur Mindset, Health & Wellness, Health and Wellness, Jessica Abo, life hacks, Lifestyle, living, Productivity
Hilary DeCesare is the founder of The ReLaunch Co., and the author of RELAUNCH! Spark Your Heart to Ignite Your
10 Things Every Working Woman Should Do This Year
February 4, 2023 February 4, 2023 admin 0 Comments health, Health & Wellness, life hacks, Lifestyle, lifestyle entrepreneur, living, Making a change, Mental Health, Personal Health, Productivity, Self Improvement, Self-Awareness, Self-Care, Success Strategies, wellbeing, Women Entrepreneur™
Opinions expressed by Entrepreneur contributors are their own. Self-care has become an all-encompassing term that has strayed from the importance
10 Travel Beauty Essentials You Need
January 30, 2023 January 30, 2023 admin 0 Comments beauty, Beauty Businesses, Beauty Products, career, growth hacking, Health & Wellness, life hacks, Lifestyle, living, Travel, travel hack, Travel Tips, Work-Life Balance
Opinions expressed by Entrepreneur contributors are their own. Work environments aren't limited to traditional offices these days. Many opt to
How to Achieve Superhuman Levels of Focus with Nutritional Psychology
January 28, 2023 January 28, 2023 admin 0 Comments Ben Angel, focus, Health & Wellness, life hacks, Lifestyle, living, nutrition, Productivity
Have you ever considered that your inability to focus is due to your nutrition, not your lack of will power?
Your Company's Responsible Guide to Staying Profitable in a Recession
January 27, 2023 January 27, 2023 admin 0 Comments Budgeting, business-growth, Economic Conditions, Economic Downturn, growth, Growth Strategies, Health & Wellness, leadership, life hacks, Management, Managing Employees, Optimism
Opinions expressed by Entrepreneur contributors are their own. The recent trend of easy money and exorbitant valuations has skidded to
8 Ways Traders Can Manage Their Emotions and Achieve Success
January 26, 2023 January 27, 2023 admin 0 Comments Business process, career, Emotional Intelligence, finance, Growing a Business, Health & Wellness, life hacks, living, Making a change, Psychology, Trading
Opinions expressed by Entrepreneur contributors are their own. Short-term trading can be a thrilling and potentially profitable endeavor, but it
Early Retirement Can Negatively Impact Cognition, Study Reveals
January 26, 2023 January 26, 2023 admin 0 Comments Business news, Early Retirement, Health & Wellness, Health and Wellness, living, News and Trends, Retirement, Science & Technology
Many people wish they could retire early — but it might not always be in their best interest. Steve Smith
3 Ways to Lead Effectively While Dealing with Anxiety
January 26, 2023 January 26, 2023 admin 0 Comments Anxiety, Company Culture, Health & Wellness, Health and Wellness, leadership, life hacks, living, Managing Employees, Personal Health, stay calm, Stress, Stress Busters, Stress Management
Opinions expressed by Entrepreneur contributors are their own. "I don't feel well. Call 911." That was supposedly what I said
4 Simple Self-Care Rituals to Help You Build More Resilience
January 24, 2023 January 25, 2023 admin 0 Comments Burnout, Business Culture, career, culture, Health & Wellness, leadership, life hacks, Lifestyle, living, Making a change, Resilience
Opinions expressed by Entrepreneur contributors are their own. Emotional outbursts are a sign of exhaustion and even burnout … Take
7 Reasons Why You Need an In Person Connection When Making Decisions
January 20, 2023 January 20, 2023 admin 0 Comments Business Culture, business decisions, Connections, Decision Making, Decisiones, Handshakes, Health & Wellness, in person, innovation, leadership, life hacks, living, Making Decisions, Management, Productivity, Thought Leaders, Zoom
Opinions expressed by Entrepreneur contributors are their own. I've always prioritized face to face-to-face meetings when I'm able to secure
'Yes, I Thanked Asiwaju For Removing Jonathan'
After Netizens called him out for alleged hypocrisy during his fight with Peter Okoye, Afrobeat star Seun Kuti has defended
The Key Ingredients Of A Successful GRC Program