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Immunology and Microbiology | January 2012
αβ TCR+ T Cells, but Not B Cells, Promote Autoimmune Keratitis in B10 Mice Lacking γδ T Cells
Rebecca L. O'Brien; Jennifer L. Chain; M. Kemal Aydintug; Dawn Bohrer-Kunter; Yafei Huang; Ian R. Hardy; John C. Cambier; Kevin Lahmers; Tanja Nuhsbaum; Richard Davidson; Deming Sun; Willi K. Born
Rebecca L. O'Brien
From the Integrated Department of Immunology, National Jewish Health, and University of Colorado School of Medicine, Denver, Colorado;
Jennifer L. Chain
M. Kemal Aydintug
Dawn Bohrer-Kunter
Biological Resource Center, National Jewish Health, Denver, Colorado;
Yafei Huang
Ian R. Hardy
John C. Cambier
Kevin Lahmers
the Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington;
Tanja Nuhsbaum
the Veterinary Referral Center of Colorado/Ophthalmology, Englewood, Colorado;
Department of Ophthalmology, University of Colorado School of Medicine, Denver, Colorado and
Deming Sun
the Doheny Eye Institute, Department of Ophthalmology, University of Southern California, Los Angeles, California.
Willi K. Born
Corresponding author: Rebecca L. O'Brien, National Jewish Health, Denver, CO, 80206; obrienr@njhealth.org.
Investigative Ophthalmology & Visual Science January 2012, Vol.53, 301-308. doi:10.1167/iovs.11-8855
Rebecca L. O'Brien, Jennifer L. Chain, M. Kemal Aydintug, Dawn Bohrer-Kunter, Yafei Huang, Ian R. Hardy, John C. Cambier, Kevin Lahmers, Tanja Nuhsbaum, Richard Davidson, Deming Sun, Willi K. Born; αβ TCR+ T Cells, but Not B Cells, Promote Autoimmune Keratitis in B10 Mice Lacking γδ T Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(1):301-308. doi: 10.1167/iovs.11-8855.
Purpose.: To investigate additional factors in the spontaneous development of keratitis previously reported in B10.TCRδ−/− female mice.
Methods.: The study tested whether susceptible B10.TCRδ−/− mice have dry eyes compared with resistant B6.TCRδ−/− females and also rederived the B10.TCRδ−/− strain to test for the role of an infectious agent. Also assessed was whether adoptive transfer of αβ T cells from autoimmune mice induced keratitis in resistant mice. In addition, a potential role was examined for B cells or autoantibodies by B-cell inactivation, and the role of female hormones was tested by ovariectomy. Finally, the study investigated whether adoptive transfer of Vγ1+ γδ T cells confers protection.
Results.: Tear production in B10.TCRδ−/− females was actually higher than in B6.TCRδ−/− controls. Rederived B10.TCRδ−/− mice still developed keratitis. Keratitis was induced in resistant mice after adoptive transfer of αβ T cells from keratitic donors. Inactivation of B cells from susceptible mice had no effect on the development of keratitis. Ovariectomy did not significantly reduce disease in B10.TCRδ−/− females. Adoptive transfer of Vγ1+ cells from wild-type donors reduced keratitis in B10.TCRδ−/− females.
Conclusions.: Neither low tear levels nor ovarian hormones contribute to spontaneous keratitis in B10.TCRδ−/− female mice, nor does it appear to depend on an infectious agent carried vertically in this strain. However, αβ T cells from keratitic hosts are sufficient to induce disease in the resistant B10.TCRβ−/−δ−/− strain. Autoaggressive αβ T cells in the absence of Vγ1+ T cells in B10.TCRδ−/− mice may be insufficiently checked to prevent disease.
The role of γδ T cells as immunoregulatory cells has been documented in many different settings, but in the eye, these cells appear to be of particular importance. Mice that lack or have been depleted of γδ T cells do not develop anterior chamber-associated immune deviation (ACAID) 1 –3 and also reject allogeneic corneal grafts much more readily than do γδ T-cell-sufficient mice, 2 pointing to the role of these cells in maintaining tolerance to antigens normally present in the eye. Mice with herpes stromal keratitis, an infectious disease that eventually progresses to autoimmune keratitis after the virus (HSV) has been cleared (reviewed in Ref. 4), have been shown to be particularly susceptible to progression to HSV infection of the brain if they lack γδ T cells, 5 consistent with the idea that γδ T cells normally downregulate immune responses that are evoked in the cornea and thus prevent inflammatory damage that leads to this complication. Increases in γδ T cells during autoimmune disorders of the human eye have also been noted, including Behçet's disease 6 and ocular cicatricial pemphigoid, 7,8 as well as in chronic corneal graft rejection, 8 which suggests that γδ T cells play a similar regulatory role in the human eye. We recently reported that, in female mice of the C57BL/10 background, which lack γδ T cells because of genetic disruption of the TCR-δ constant region (B10.TCRδ−/− mice), keratitis develops spontaneously, such that by 18 weeks of age, 70% to 80% of adult females show evidence of disease. 9 The development of keratitis is dependent on the B10 background, because mice with the same genetic defect but having instead the closely related C57BL/6 background do not develop keratitis. The disease is also much more prevalent in females than in males. Our previous study additionally indicated that male hormones do not protect against keratitis, because orchiectomized males show no increase in disease incidence, but that αβ T cells appear to play a role in the development of keratitis, because mice depleted of αβ T cells with a monoclonal antibody or treated with the immunosuppressive drug cyclosporine, developed keratitis at a reduced level.
In this article, we investigate additional factors that could play a role in this spontaneous eye disease, including dry eye, ovarian hormones, an insidious infectious component, and autoimmune αβ T cells and B cells. Of these, our results indicate that only autoaggressive αβ T cells play a role in inducing keratitis. Moreover, Vγ1+ γδ T cells provide some resistance against development of the disease.
C57Bl/10J (B10) mice, C57BL/6J (B6) mice, and B6.TCRδ−/− mice 10,11 were either newly obtained from The Jackson Laboratory (Bar Harbor, ME) or maintained in our colony from Jackson Laboratory stock. The B10.TCRδ−/− and B10.TCRβ−/−δ−/− strains were backcrossed in our facility, as previously described. 9 The work described in this article was reviewed and approved by the National Jewish Institutional Animal Care and Use Committee and adhered to the guidelines in the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.
Keratitis Scoring
The keratitis was scored once every 2 weeks by gross observation of the mice, and a severity score was assigned for each side, as previously described. 9 The maximum possible score for an individual mouse was 10.
Tear Measurements
To approximate the Schirmer test used clinically, we used phenol red–impregnated cotton thread (Zone-Quick thread tear test; Oasis, Glendora, CA). Mice to be tested were anesthetized briefly with isoflurane vapor, and the thread was applied to the outer canthus of each eye for 60 seconds. The area wetted was then recorded (in millimeters) for each, according to the scale present on the thread. The mice were tested at 6 to 10 weeks of age, a time when B10.TCRδ−/− mice are not likely to have developed keratitis. Any mice with obvious keratitis at the time of testing were excluded from the analysis.
Rederivation of the B10.TCRδ−/− Strain
The rederivation was performed in the barrier of the National Jewish Health animal facility, which is designed to prevent transfer of any potential infectious agents that could be accidentally introduced from the regular (SPF) animal facility. Personnel must shower before entering the barrier from another National Jewish animal room, and all equipment and supplies are sterilized before transfer into the barrier. Personnel working in the barrier wear a uniform, shoe covers, hair cover, face mask, and gloves. The mice are housed in ventilated units, and the cages are opened and manipulated in laminar flow hoods. All mice housed or admitted to the barrier are rederived into the facility, except for certified mice from The Jackson Laboratory.
To rederive the strain, female B10.TCRδ−/− mice of 3 to 4 weeks of age were superovulated by giving each an IP injection of pregnant mare serum gonadotropin (0.1 mL of 50 IU/mL in PBS) and, 46 to 48 hours later, an IP injection of human chorionic gonadotropin (0.1 mL of 50 IU/mL in PBS). Four to 7 hours later, each female was added to a cage containing either an experienced male B10.TCRδ−/− breeder mouse or a male 7 weeks of age or older that had been exposed to females for approximately 24 hours, followed by a 2- to 3-day rest. After 15 to 18 hours, the females were checked for the presence of a vaginal plug. Positive females were euthanized 2 days later. The embryos were removed from the oviducts, placed in cyropreservation medium in straws, and slow frozen in a cryomed, controlled-rate freezer (Biocool; SP Scientific, Warminster, PA). The embryos remained frozen for approximately 10 months, and the strain was then rederived in the barrier facility into SPF foster mothers newly obtained from The Jackson Laboratory. The embryos were thawed and injected, as described previously, 12 into the oviduct of an outbred ICR female made pseudopregnant by copulation with a vasectomized male, confirmed 3 days previously by the presence of a vaginal plug.
Rederived pups were maintained in the barrier facility. After reaching 8 weeks of age, two rederived females were bred with C57BL/10 wild-type males newly obtained from The Jackson Laboratory to produce F1 offspring that were +/− at the TCR-δ locus and then interbred to produce F2 pups. All breeding and maintenance were performed in the barrier facility for the duration of the experiment.
To identify F2 offspring that were TCRδ−/−, the F2 mice were bled from the tail vein within 1 month of weaning, collecting up to 6 drops of blood into 2.5 mL of balanced salt solution containing 0.0038 USP units of heparin (Sigma-Aldrich, St. Louis, MO). After RBC lysis in 9 mL of Gey's solution, the suspension was underlaid with 0.5 mL FBS and spun at 1200 rpm for 8 minutes. Blood T cells in the pellet were then enriched by nylon wool passage, 13 using a 0.25-g nylon wool column in a 3-mL syringe. The presence of γδ T cells was confirmed by two-color flow cytometry with an anti-mouse CD3 monoclonal antibody (KT3 14 ) and an anti-mouse TCR-δ monoclonal antibody (GL3 15 ). Female F2 mice having no detectable γδ T cells were deemed TCRδ−/−. All rederived B10.TCRδ−/− females were scored for keratitis after reaching 18 weeks of age.
Adoptive T-Cell Transfers
For keratitic αβ T-cell adoptive transfers, splenic T cells from keratitic B10.TCRδ−/− females having an overall score of at least 4 were used as donors. Spleens were homogenized in cell culture medium containing antibiotics 16 plus 5% FBS, and the RBCs were lysed with Gey's solution. The cells were then passed over nylon wool to enrich for T cells 13 and resuspended in sterile balanced saline plus 5% heat-inactivated FBS (Atlanta Biologicals, Lawrenceville, GA). Then, 1 to 2 × 106 were injected into the tail vein of 6- to 12-week-old B10.TCRβ−/−δ−/− female hosts. Keratitis develops spontaneously in B10.TCRβ−/−δ−/− mice at a low rate (∼20% of females by 18 weeks of age 9 ). Any that had discernible keratitis at the time of adoptive transfer were excluded from the experiment.
For Vγ1+ T cell transfers, spleen cells from normal B10 donors were similarly prepared as described above. Spleen cell suspensions were treated with 40 μg/mL 2.4G2 monoclonal antibody 17 plus 20 μg/mL mouse gamma globulin (Jackson ImmunoResearch) for approximately 60 minutes at 4°C to block Fc-receptors, washed once, and then incubated with a biotinylated monoclonal antibody specific for Vγ1 (2.11 18 ) for 15 minutes at 4°C. The Vγ1+ cells were next purified using streptavidin magnetic beads with MS minicolumns (MACS; Miltenyi Biotec, Auburn, CA), in accordance with the manufacturer's instructions. In some experiments, the eluted cells were passed a second time over another MS column, to improve purity. An aliquot was then stained with an FITC-labeled anti-γδ TCR monoclonal antibody (GL3 19 ) plus streptavidin-PE and analyzed by flow cytometry to assess the purity of the Vγ1+ cells. Among the live lymphocytes in various preparations, Vγ1+ cells ranged from 41% to 79%. In all cases, less than 1% represented Vγ1-negative γδ TCR+ cells. Purified Vγ1+ cells were then adoptively transferred into 5- to 9-week-old B10.TCRδ−/− female hosts with no evident keratitis at the time of injection by tail vein inoculation, injecting 1.7 to 3.25 × 105 Vγ1+ cells per mouse.
For CD4+ αβ T-cell transfers, spleen cells from normal B10 mice were purified over nylon wool columns as described above, and the eluted cells were incubated in a cocktail of biotinylated monoclonal antibodies recognizing mouse CD8α, 20 CD19 (eBioscience, San Diego, CA), NK1.1, 21 and TCR-γδ, 19 to label CD8+ αβ T cells, B cells, NK cells, and γδ T cells, respectively. The labeled cells were then incubated with streptavidin beads (MACS; Miltenyi Biotec) and passed over LD columns (MACS; Miltenyi Biotec) to remove all labeled cells together. An aliquot of unbound negatively selected cells was then taken for flow cytometric analysis and stained with an FITC-labeled anti-αβ TCR monoclonal antibody plus anti-CD4-PE (eBioscience). Of the live lymphocytes, 96% were αβ TCR+ and CD4+. Purified CD4+ cells were then adoptively transferred into 7- to 12-week-old B10.TCRβ−/−δ−/− female hosts with no evident keratitis at the time of injection, by tail vein inoculations, injecting 0.3 to 1.0 × 106 purified CD4+ cells per mouse.
B-Cell Inactivation
B10.TCRδ−/− females without evident keratitis at 7 weeks of age were treated with a monoclonal antibody that inactivates B cells by serial injection of the hamster anti-mouse CD79β (Igβ) monoclonal antibody HM79. This antibody, diluted in balanced saline, was injected at a dose of 200 μg per mouse into the tail vein at 7, 8, 9, 11, 13, 15, and 17 weeks of age. This antibody has been shown to efficiently inactivate and, with repeated doses, to deplete B cells in vivo. 22,23 Two weeks after the final antibody injection, the keratitis was scored. The sham-treated controls were instead injected on the same schedule with normal Syrian hamster gamma globulin (Jackson ImmunoResearch, West Grove, PA) diluted to the same concentration. In one experiment, the mice were euthanized after the treatment and their spleens removed for flow cytometric analysis. B cells from these animals were stained using a monoclonal antibody against CD19 (biotinylated; eBioscience) plus streptavidin-PE (InVitrogen, Carlsbad, CA). T cells were also stained as a positive control with an FITC-labeled anti-CD3ε monoclonal antibody (KT3 14 ).
Ovariectomy
Six- to 8-week-old B10.TCRδ−/− females were weighed and anesthetized with isoflurane. The hair in the midback region was shaved, and the skin was disinfected with 70% ethanol. Ointment (DuoLube; Bausch & Lomb, Rochester, NY) was applied to the eyes with a cotton swab (Q-tip; Unilever, New York, NY), to ensure that they did not dry out excessively during surgery, and a steady flow of isoflurane in air and oxygen was supplied via a vaporizer by placing the mouse's nose in a nose cone. A horizontal dorsal incision approximately 1 cm long was made on the side through the skin about halfway between the ribs and the base of the tail and another through the underlying muscle layer into the peritoneal cavity. The ovary and surrounding fatty tissue were withdrawn, the ovary exposed, the oviduct tied off with a resorbable suture (4-0 self-absorbing, braided Vicryl), and the ovary excised. Two to three sutures were used to close the internal incision, and 10-mm wound clips were applied to close the skin. The second ovary was then exposed on the other side and removed similarly. For sham-ovariectomized controls, the ovaries were briefly exposed, then reinserted into the opening and the mouse closed up. The mice were treated with buprenorphine at 1 mg/kg by subcutaneous injection just before surgery and every 12 to 16 hours for the first 48 hours after surgery. Wound clips were removed 10 to 14 days after surgery. The keratitis in the mice was scored until 18 to 19 weeks of age. Any mice that had discernible keratitis before the time of surgery were excluded from the analysis.
Differences between tear levels in the different groups were analyzed with a one-tailed Student's t-test. Differences in the incidence of keratitis between two groups were analyzed with a two-tailed Fisher's exact test. Because keratitis scores were assessed by an assigned rank, the differences in average keratitis score were analyzed with a nonparametric one-tailed Wilcoxon rank sum or Mann-Whitney U test (all analyses: Prism; GraphPad, San Diego, CA).
The High Incidence of Keratitis in B10.TCRδ−/− versus B6.TCRδ−/− Female Mice Is Not the Result of Reduced Tear Production
In humans, keratitis is often associated with keratoconjunctivitis sicca, or dry eye. 24 We therefore hypothesized that a failure to produce an adequate amount of tears could contribute to the susceptibility of B10.TCRδ−/− females to keratitis, compared with B6.TCRδ−/− females. We used a method described previously to approximate the Schirmer test used clinically in humans 25 for comparing tear production in the two strains. However, we found that in fact the B10-background mice produced higher tear levels. This was also true of wild-type B10 versus B6 mice, and the difference was significant for both comparisons (Fig. 1). Thus, low tear production does not predispose the B10.TRCδ−/− strain to the development of keratitis.
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Keratitis-susceptible B10.TCRδ−/− mice had higher tear levels than did keratitis-resistant B6.TCRδ−/− mice. Tear levels were measured in both eyes of individual mice, and the average for each group is shown. Errors bars, SEM. The total number examined in each group is indicated in parentheses.
Rederived B10.TCRδ−/− Mice Still Show a High Incidence of Spontaneous Keratitis
To test whether an infectious agent transmitted to the offspring at birth could cause keratitis in B10.TCRδ−/− mice, we rederived this strain by removing embryos at the eight-cell stage from pregnant mothers and implanting them into uteri of pseudopregnant mothers of an unrelated strain. The rederivation was performed in a barrier facility in the National Jewish Health animal facility, which is designed to prevent potential transfer of infectious agents from the regular SPF facility, and the rederived strain was maintained in the barrier. Two females were obtained from the rederivation, both of which developed keratitis after 18 weeks of age, suggesting that rederived B10.TCRδ−/− females are similarly apt to develop keratitis. To obtain more for this analysis, these two females were cross-bred with C57BL/10 males obtained from The Jackson Laboratory, and the offspring were then intercrossed to produce F2 progeny. The F2 mice having two defective TCRδ loci were then identified by staining peripheral blood T cells with an anti-TCR-γδ monoclonal antibody. To prevent any potential reinfection, the B10.TCRδ−/− F2 progeny were maintained in the barrier facility until they reached 18 weeks of age, and then the keratitis was scored. As can be seen (Fig. 2), the rederived B10.TCRδ−/− females developed keratitis at a rate that was not significantly different from that of the original B10.TCRδ−/− females housed in the regular facility during the same period. Thus, we conclude that the disease probably does not depend on the transmission of an unknown infectious agent. This conclusion adds support to our previous hypothesis that the keratitis stems instead from an autoimmune attack that develops against the normally immune-privileged cornea.
Rederivation of the B10.TCRδ−/− strain did not significantly reduce the incidence of keratitis. Individual keratitis scores by 18 weeks are shown for the rederived B10.TCRδ−/− females versus females of the original B10.TCRδ−/− strain tested over approximately the same time period. The number of individual mice screened in each group is shown in parentheses. The small difference between means was not significant (NS).
Adoptive Transfer of αβ T Cells from Keratitic Donors Increases the Frequency of Keratitis in B10.TCRβ−/−δ−/− Hosts
In many autoimmune diseases, there is evidence that T cells, particularly CD4+ αβ T cells, induce the development of disease (reviewed in Ref. 26), and our previous results also suggest a role for αβ T cells in the keratitis that develops in B10.TCRδ−/− mice. 9 To test this notion more directly, we examined whether keratitis could be induced in resistant B10.TCRβ−/−δ−/− mice by adoptive transfer of αβ T cells from keratitic B10.TCRδ−/− donors. As shown in Figure 3A, we found that these αβ T cells were indeed sufficient to induce keratitis. Unexpectedly, we also found that the mice that received these αβ T cells underwent a dramatic weight loss (not shown). A similar weight loss was also noted when enriched normal CD4+ αβ T cells from B10 donors were adoptively transferred into B10.TCRβ−/−δ−/− hosts, although these mice did not develop keratitis (Fig. 3B). Thus, the weight loss in both cases may be mediated by CD4+ αβ T cells, but does not appear to be caused by an autoimmune attack by keratitis-inducing αβ T cells.
(A) Adoptively transferred αβ T cells from keratitic B10.TCRδ−/− donors induce keratitis in B10.TCRβ−/−δ−/− hosts. Average final keratitis scores of mice at 18 to 19 weeks of age are shown for 8 B10.TCRβ−/−δ−/− hosts into which αβ T cells from keratitic B10.TCRδ−/− donors were transferred, and for 6 B10.TCRβ−/−δ−/− controls that were sham treated at the same time with the cell diluent only. Scores obtained for individual mice are indicated by each symbol with the mean and SD bars superimposed as horizontal lines. P > 0.05 was regarded as nonsignificant (NS). (B) Adoptively transferred CD4+ αβ T cells from normal B10 donors do not induce keratitis in B10.TCRβ−/− δ−/− hosts. Average keratitis scores are indicated as in (A), for seven B10.TCRβ−/−δ−/− hosts into which αβ T cells from normal B10 donors were transferred and for seven B10.TCRβ−/−δ−/− controls that were sham-treated at the same time with the cell diluent only.
Inactivation of B Cells from B10.TCRδ−/− Females Has No Effect on the Development of Keratitis
Autoantibodies play a pathologic role in several autoimmune diseases, and the elimination of B cells during the course of the disease can therefore ameliorate the symptoms. 27 Our finding that adoptively transferred αβ T cells from keratitic hosts can induce disease (Fig. 3) does not eliminate a possible role for B cells in B10.TCRδ−/− keratitis, because the transferred T cells could potentially act as helper cells for B cells producing anticorneal autoantibodies. Alternatively, the T-cell preparation could contain contaminating B cells at sufficient levels to induce keratitis, if the B cells are potentially pathogenic. To test whether B cells could play a role in the spontaneous keratitis of B10.TCRδ−/− mice, we treated B10.TCRδ−/− females periodically with an antibody against a pan-B-cell antigen to keep the peripheral B cells depleted or in an inactive state, beginning at a young age before keratitis developed. As shown in Figures 4A and 4B, there was no effect of this treatment on keratitis, since mice treated with normal hamster IgG developed disease at a similar rate and severity as those treated with the B-cell-depleting antibody. We confirmed the efficacy of the antibody treatment by staining spleen cells from B-cell-inactivated versus sham-treated mice with an anti-CD19 monoclonal antibody, 2 weeks after the final injection, which showed that the overall B-cell counts were still reduced by an average of more than 25-fold (Fig. 4C). We therefore conclude that B cells do not play a role in the development of spontaneous keratitis in B10.TCRδ−/− mice.
B-cell inactivation does not affect the incidence or severity of keratitis in B10.TCRδ−/− females. (A) The average keratitis scores for mice treated with anti-CD79β antibody or sham-treated with hamster IgG as a control are shown. Each column indicates the average at a given age. Error bars, SD. The total number of mice tested in each group is shown in parentheses. (B) The incidence of keratitis of the mice in (A) at 18 to 19 weeks, shown as the percentage of mice in the group that developed keratitis compared with the total number in the group. The difference was not significant (NS). (C) The average number of CD19+ cells obtained from spleens of mice shown in (A) was calculated from the percentage determined by flow cytometry, as determined for individual mice. Error bars, SE.
The Incidence of Keratitis Is Not Significantly Decreased after Ovariectomy
In both humans and mice, many autoimmune diseases are more prevalent in females than in males. 28 Our previous results indicated that a lack of male hormones cannot explain the higher prevalence of keratitis in B10.TCRδ−/− females compared to males. 9 However, female hormones could instead promote the development of autoaggressive lymphocytes, or in some other way act to increase susceptibility to keratitis. Therefore, we tested whether ovariectomized B10.TCRδ−/− females are less likely to develop keratitis than sham-ovariectomized controls. As shown in Figure 5, we found only a slight, nonsignificant difference between ovariectomized mice and sham-ovariectomized controls. Thus, the presence of ovarian hormones cannot explain why keratitis is six to seven times more prevalent in female than in male B10.TCRδ−/− mice.
Ovariectomy does not significantly affect the incidence of keratitis in B10.TCRδ−/− females. (A) The average keratitis scores for ovariectomized B10.TCRδ−/− mice and for sham-ovariectomized B10.TCRδ−/− mice are shown. Each column indicates the average score at a given age. Error bars, SE. The total number of mice tested in each group is shown in parentheses. The small difference was not significant at any age. (B) The disease incidence for each group shown in (A) at various ages, calculated as the percentage of mice with keratitis divided by the total number in the group. Again, the small difference was not significant at any age.
Adoptive Transfer of Vγ1+ Cells from Wild-Type Donors Reduced the Incidence of Keratitis in B10.TCRδ−/− Females
We had shown that adoptive transfer of γδ T cells from wild-type B10 donors could reduce the incidence of keratitis in B10.TCRδ−/− females. However, different γδ T cell subsets have distinct functional properties (reviewed in Ref. 29). In other disease models, we and others have shown that Vγ1+ γδ T cells have an anti-inflammatory effect. 30 –33 We therefore tested whether purified Vγ1+ γδ T cells from B10 donors are sufficient to reduce the incidence of keratitis when adoptively transferred to B10.TCRδ−/− female hosts. As shown in Figure 6, reconstitution of these mice with Vγ1+ cells indeed reduced the average disease incidence by approximately twofold. This transfer did not result in any weight loss in the hosts, unlike the transfer of αβ T cells into B10.TCRβ−/−δ−/− hosts (Fig. 3).
Vγ1+ γδ T cells from the spleens of normal B10 donors can suppress the incidence of keratitis in B10.TCRδ−/− hosts. Average final keratitis scores at 18 to 19 weeks of age are shown for 12 B10.TCRδ−/− mice, into which Vγ1+ cells from normal B10 donors were transferred, and for 12 sham-treated controls that received only cell diluent. Scores obtained for individual mice are indicated by each symbol, with the mean and SE bars superimposed as horizontal lines.
The B10.TCRδ−/− mice develop what appears to be autoimmune keratitis spontaneously, rather than after infection or immunization, and as such they represent an intriguing model for investigating immune dysregulation in the eye. In this study, we attempted to determine additional factors that contribute to the susceptibility of these mice to keratitis.
Our observation that B10.TCRδ−/− and B10 mice on average have higher tear levels than do B6.TCRδ−/− and B6 mice (Fig. 1) was the opposite of what we had expected to find, since dry eye is often associated with keratitis. Conceivably, the opposite could be true, that excess tear levels are in some way pathologic, if the mice tend to build up a pool of stagnant tears due to an obstruction of the nasolacrimal duct, which allows bacteria to grow abnormally, as sometimes occurs congenitally in young children. 34 Another possibility is that the tears produced by B10 mice, though more copious, may lack something in their chemical composition that sets the stage for keratitis. Finally, the increased tear level in B10-background mice could be symptomatic of a preexisting corneal irritation due to unknown causes, which often goes on to develop into overt keratitis when γδ T cells are absent. We plan to further investigate these possibilities, because the difference between the two strains was quite striking. That B10.TCRδ−/− mice with keratitis go on to develop dry eye seems unlikely, because we found no evidence of lacrimal gland inflammation or degeneration in histologic sections. 9
Keratitis can result from infection of the eye with either a viral or bacterial agent, and in particular herpes simplex virus 35,36 and Pseudomonas aeruginosa 37 have been studied in this regard. Both types 1 and 2 herpes simplex virus are known to be transmissible during birth in rare instances in humans. 38 Therefore, it seemed possible that a congenital eye infection may in part be responsible for the high frequency with which keratitis develops in B10.TCRδ−/− females. Our finding that rederived B10.TCRδ−/− females kept in a barrier facility and shielded from direct or indirect contact with the original strain still developed keratitis at approximately the same frequency as did the original strain (Fig. 2) indicates that congenital infection is unlikely to play a role in bringing about the disease.
Indirect evidence that spontaneous keratitis in the B10.TCRδ−/− strain is promoted by αβ T cells, based on the results of four different experiments, was presented in our prior study. 9 First, depleting B10.TCRδ−/− females of αβ T cells by injection of a TCRβ-specific monoclonal antibody reduced the incidence of disease. Second, when we crossed B10.TCRδ−/− mice with B10.TCRβ−/− mice to generate B10.TCRβ−/−δ−/− mice, which are able to produce neither αβ nor γδ T cells, this double-knockout strain had a substantially reduced tendency to develop keratitis. Third, treatment of B10.TCRδ−/− females with cyclosporine, which inhibits the activation of T cells, also reduced the incidence of disease. Fourth, we were able to detect both CD4+ and CD8+ αβ T cells infiltrating the corneas of B10.TCRδ−/− mice with keratitis. Our finding that the adoptive transfer of enriched αβ T cells derived from keratitic donors promotes the development of keratitis in B10.TCRβ−/−δ−/− hosts (Fig. 3) provides direct evidence that αβ T cells mediate the disease. Therefore, the prevalent mechanism by which keratitis develops in B10.TCRβ−/−δ−/− mice is most likely T-dependent autoimmunity. However, since not only wild-type B10 mice but also B10.TCRβ−/−δ−/− mice show a low incidence of spontaneous keratitis, there must be another mechanism that can sometimes lead to this disease. The αβ T cells do not appear to promote keratitis by helping autoreactive B cells to differentiate and secrete anticorneal autoantibodies, because B-cell inactivation by monoclonal antibody treatment did not affect the rate or severity of disease in B10.TCRδ−/− mice (Fig. 4). An unexpected observation from the adoptive transfer experiments with αβ T cells was that the host mice suffered a profound weight loss. This was evident even when the mice were given αβ T cells from normal B10 mice, rather than from keratitic B10.TCRδ−/− donors (Fig. 3B). However, the B10 strain has been noted to carry genes that promote susceptibility to autoimmunity, in contrast to B6 strain, 39 so it is possible that the weight loss reflects other autoimmunities which develop after the transfer of αβ T cells.
The greatly increased susceptibility of B10.TCRδ−/− females compared with males is reminiscent of many other autoimmune diseases, in which females are also more often affected. Our previous results suggested that this difference in susceptibility is not due to a protective effect stemming from male hormones, as was found previously in experimental autoimmune encephalomyelitis, 40 because orchiectomizing B10.TCRδ−/− males did not result in an increase in keratitis. The preponderance of autoimmune disease in females has alternatively been attributed to the ability of estrogens to stimulate immune cells, influencing processes such antigen presentation, lymphocyte activation, and cytokine gene expression. 28 However, ovariectomy of B10.TCRδ−/− mice, which greatly diminishes female hormone levels, had no significant effect on the rate or incidence of keratitis (Fig. 5). Another possible explanation is that the susceptibility of females versus males stems from immune-relevant genes that are encoded on the X chromosome, which tend to be overexpressed in females and can lead to the expansion of autoimmune lymphocytes. 41
γδ T cells normally appear to prevent keratitis from developing in B10 mice, and our results from this study suggest that the suppressive γδ T cells may be a Vγ1+ subset (Fig. 6). Certain Vγ1+ cells have been shown to be potent producers of IL-4, 42 –45 and increased levels of IL-4 could mediate the effect reported here after the adoptive transfer of Vγ1+ cells. Whether Vγ1+ cells are the γδ T cells that normally mediate protection against the development of keratitis remains to be seen, because the artificially introduced cells could prevent the development of keratitis in another way. Indeed, a subset of γδ T cells has been described that normally resides in the limbus of the murine eye and plays a role in corneal wound healing. 46 –48 Virtually all the T cells in the limbus express the γδ TCR. This restricted distribution implies that they have a very specific role to play, and it seems likely that the limbal γδ T cells protect the cornea against autoimmune attack. Whether the limbal γδ T cells are in fact Vγ1+ is currently unknown, however.
Supported by National Eye Institute Grant R21EY019328-01A2 (RLO).
Disclosure: R.L. O'Brien, None; J.L. Chain, None; M.K. Aydintug, None; D. Bohrer-Kunter, None; Y. Huang, None; I.R. Hardy, None; J.C. Cambier, None; K. Lahmers, None; T. Nuhsbaum, None; R. Davidson, None; D. Sun, None; W.K. Born, None
The authors thank James Gross and Jennifer Matsuda for rederivation of the B10.TCRδ−/− strain, Sarah Heyborne for help in breeding the mice, and Desiree Garcia for assistance in screening the rederived mice.
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Li Z Burns AR Rumbaut RE Smith CW . γδ T cells are necessary for platelet and neutrophil accumulation in limbal vessels and efficient epithelial repair after corneal abrasion. Am J Pathol. 2007;171:838–845. [CrossRef] [PubMed]
Copyright © Association for Research in Vision and Ophthalmology
Alpha-internexin as a Corneal Autoantigen in Spontaneous Autoimmune Keratitis Mouse Model
Induction of Experimental Autoimmune Keratitis by Adoptive Transfer of Human Corneal Antigen–Specific T-Cell Line
Experimental Autoimmune Keratitis Induced in Rats by Anti–Cornea T-cell Lines
Reactivation of Uveitogenic T Cells by Retinal Astrocytes Derived from Experimental Autoimmune Uveitis-Prone B10RIII Mice
Osteopontin Is Proinflammatory in Experimental Autoimmune Uveitis
From Other Journals
GCAP1, Rab6, and HSP27: Novel Autoantibody Targets in Cancer-Associated Retinopathy and Autoimmune Retinopathy
Effective Arrestin–Specific Immunotherapy of Experimental Autoimmune Uveitis with RTL: A Prospect for Treatment of Human Uveitis
Dynamic coding of border-ownership in visual cortex
Eye Anatomy and Disorders


Most staff client check-boxes are "sticky" — if you select or deselect them, that status persists. For example, Auto-Print, which will print the relevant receipts automatically in certain functions, is sticky. If you select it on one login, it will persist for future logins until you uncheck the box.
Fast Item Add is another "sticky" check box that makes it possible to add volume and item records from the MARC editor.


A concept project for the 25th anniversary of Casabella uses microbial electrolysis to generate power from the bacteria it picks up.
Limbo's adjustable wheels can be expanded, contracted, or lowered to give it fast ground coverage, help it clean close to walls, and climb up stairs. The robotic floor cleaner also powers itself through the bacteria it consumes.


Live in the City – the summer gigs 2016!
The 4th edition of Live in the City (Miasto na żywo) – concerts taking place in the public space as part of the Gdańsk Outdoor Gallery – is about to begin. This year, Dolne Miasto will witness such acts as Band A, Asia i Koty, Hoszpital and Popsysze, while DJ Wika and DJ Czarna are going to play in Nowy Port. The first gig is on 23 July!
Live in the City began in 2013. The idea was to organise gigs in unusual locations: in yards, rather than within gallery walls and music clubs. The project counts with the participation of artists whose concerts may interest both local inhabitants and guests from other part of the city or tourists visiting Gdańsk. The objective is to create meeting-points for the local inhabitants, revive Dolne Miasto and Nowy Port, overcome stereotypes surrounding these districts and convince accidental passers-by to become interested in art and contemporary music, and to take part in artistic events more often.
23 July 2016, 8 p.m.
Band A was formed by four musicians from the Tricity area, all of whom are remarkable artistic personalities in their own right. Their meeting during a Sopot jam session – almost accidental and burdened with no expectations – resulted in the idea to play together. Their first concert during the "Nasiono Sunrise" festival was recorded – the organizers liked it so much that Karol Schwarz of Nasiono Recorde released it as the group's first album. They are really well received live: during the first concert promoting the album, the entire stock was sold. Their music is a volcano of energy and emotion combined with the unconventional style of each of the instrumentalists. It is a result of improvisation and joint inspirations. The group are: Krzysztof Stachura (guitar), Michał Gos (percussion), Maciek Szkudlarek (bass), Tomasz Gadecki (saxophone). A combination of avant-garde, alternative, jazz and rock makes them unique both on the Polish and international scene.
TOMASZ GADECKI – saxophone player (b. 1971 in Gdynia). He spent the last decade in various improvised projects. Co-founder of such groups as Olbrzym i kurdupel, Sambar, Band A free, 250 kg (ćwierć tony), TRC and Helaton. He collaborates with a number of musicians in Poland and abroad. He composes music for theatre productions and is a session musician in many projects, such as Pure Phase Ensemble, Dreadless Lions, Galago, Artur Maćkowiak, Contemporary Noise Sextet and 7FAZ. Together with Anna Haracz, he plays in Acoustic Body – a duo combining music and dance. Improvisation is a very important element of his art, which is why he often works with performers, painters and poets.
Joanna Bielawska became known to the Polish audience in 2012, when she released her debut album "Miserable Miaow." In it, she presented her fascination with songs, acoustic melodies and electronic influences, which were combined in a number of melancholy, yet distinct pieces. The artist, performing under the moniker Asia i Koty, composes all tracks herself, plays electric guitar and uses loopers. This is minimalist songwriting, enriched with complex harmonies and mantra-like repetitions, whose most important element is the voice: poignant, clear and mature.
Bielawska's music is both delicate and dark, song-based and open to musical experimentation. Suffice it to say that the artist took part in 4 releases by Folder and after her debut album collaborated on such records as "Nasiono Swap Singers", "Hi, Mom!" by Karol Schwarz All Start and two live albums by Pure Phase Ensemble recorded during the first and third edition of the SpaceFest festival. She also started two further electronica-inspired projects: Morgaine Fay duo, based on ambient/noise synthesisers and electric guitar, and techno-inspired solo project Kthx NFZ.
The artist's latest album "Sing" was released on 2 April by Nasiono – a Tricity label she's been faithful to since the beginning of her career. As the title suggests, it's an album about singing and it's the voice which plays the main part here. "Sing" also documents the artist's struggle with depression, low self-esteem and lack of self-confidence. Asia i Koty combines various inspirations here, the most tangible being Angel Olsen, Cat Power or Julianna Barwick. The music is based on electric guitars, but Bielawska also reaches for acoustic guitar, piano, loops her instruments and enhances them with subtle beats.
06 August 2016, 8 p.p.m.
Hoszpital was formed in March 2012 in Poznań. Until this year, the trio released an EP in 2014 and their debut album in 2015. A year after their first album, Hoszpital are showing their new face. No longer that of a shy lover, conquering girls' hearts with melancholy and poems by Andrzej Bursa, but that of an agitated dancer at a sad disco. "Horor" consists of ten loud, energetic pieces, which take on the form of an accelerating racing car ("Taśma") or brittle, Lady Pank-esque ballads ("Niebywałość"). The record provides an original mix of rock and 80s new wave influences with contemporary trends (swagger-like riff in "Problemy", drums occasionally bringing to mind the drum'n'bass culture). The crazy and dirty side of the songs was brought out by producer Paweł Cieślak from Hasselhoff Studio in Łódź. The entire album is pieces together by the strong, vibrating voice of Michał Bielawski, while the emotional depth of his dithers on life and love is enhanced by Joanna Bielawska's backing vocals (the artist's wife).
Hoszpital are: Michał Bielawski (vocals, guitar), Adrian Borucki (drums) and Paweł Swiernalis (bass).
14 August 2016, 8 p.m.
DJ Wika is the stage pseudonym of 77-year-old Wirginia Szmyt – the oldest DJ in Poland. DJ Wika is a social activist and co-organiser of parades for seniors. She used to work as a pedagogue in centres for troubled youth, and began her music career when she retired. Since 2011, she has taken part in Intergenerational Dances – a cycle aimed at familiarising senior citizens with contemporary music and introducing young people to music their grandparents listened to. DJ Wika overturns stereotypes surrounding old age. The concert is going to form part of the annual Cud nad Martwą Wisłą event organised by Łaźnia CCA in Nowy Port, where the Łaźnia 2 CCA building is located.
27 August 2016, 8 p.m.
Popsysze to rock and rollowe trio, które powstało w 2008 roku w Trójmieście. Grają w nim Jarosław Marciszewski (gitara, wokal), Jakub Świątek (perkusja) i Sławek Draczyński (bas). Zespół swój debiutancki album "Popstory" wydał w 2012 roku, premiera drugiej płyty o nazwie "Popsute" odbyła się 16.01.2015 roku. Jesienią 2016 planowane jest wydanie trzeciego albumu.
Popsysze is a rock'n'roll trio formed in 2008 in Tricity. The band is composed of Jarosław Marciszewski (guitar, vocals), Jakub Świątek (drums) and Sławek Draczyński (bass). Their debut album "Popstory" was released in 2012, and the second album "Popsute" premiered in January 2015. They plan to release their third album in autumn 2016.
Popsysze like simple rock'n'roll and melodies, but they also like to 'spoil' their songs by improvising and leaving the song formula in favour of more loose compositions. Their second album was inspired by the beginnings of hard rock and stoner rock, but the band remains faithful to simple rock'n'roll forms, occasionally edging towards improv and psychedelia. Most texts on the second album are in Polish.
The project is organised within The Outdoor Gallery of the City of Gdansk.


text:
Schools under strain as Covid causes at least 58,000 pupil absences last week
Covid in Scotland: At least 58,000 pupil absences last week
By John-Paul Holden
There were tens of thousands of Covid-linked pupil absences last week, figures show.
Covid caused at least 58,000 pupil absences last week amid rising fears over keeping schools open in the face of the surge in the Omicron variant.
The first national snapshot of post-Christmas attendance, which covers January 5 and 6, came as calls grew for an earlier decision on arrangements for this year's exams.
While the vast majority of schools appear to be open as planned, unions are increasingly concerned about disruption and the hit to educational quality.
There are reports that staff shortages have resulted in large groups of pupils being herded into areas such as canteens or halls, with some establishments sending S4-6 students home.
One headteacher said he knew of schools where absences had forced senior management to ask entire year groups to learn at home for the remainder of January.
The Herald has also been told that teachers, headteachers and specialist support staff are being pulled away from their normal duties to look after children or provide general supply cover.
Union leaders warned previously that keeping schools open at all costs when absence rates are high could damage education and create safety risks. They have also argued that short periods of remote learning can provide a more coherent learning experience.
Seamus Searson, general secretary at the Scottish Secondary Teachers' Association (SSTA), said: "At the moment we're in a bit of a crisis. We haven't got enough bodies to put in front of classes."
Seamus Searson said schools were experiencing a range of pressures.
The latest figures show 39,799 pupils were not in school for Covid-related reasons on January 6. This is one of the highest totals since September 8 last year, even though education establishments in a number of local authority areas had still to reopen following the Christmas break. There were 18,789 absences on January 5.
Statistics also reveal there was a huge leap in coronavirus-linked pupil absences between December 16 and 17 - from 43,163 to 83,414.
The Scottish Government did not explain why it thought the number had increased so dramatically. However, rapidly rising infection rates fuelled by the Omicron variant are likely to have contributed.
Anecdotal reports suggest that parents keeping children off to cut the risk of self-isolation over Christmas may also have been a factor.
Local authority data, including figures for teacher absences, will be available later this week. However, with fears growing that the return to school will create fresh chains of transmission, statistics obtained from individual councils suggest pressures are already significant.
In South Lanarkshire, schools were dealing with 236 Covid-linked teacher absences on January 5 – well over double the number recorded for December 14.
And, although most schools reopened as planned after the Christmas holiday, union bosses stressed that challenges on the ground were huge.
Mr Searson said the SSTA had become aware of reports that staffing pressures are leading to large groups of pupils being supervised together in facilities such as school halls.
He also warned some schools were sending senior phase students home and that teachers had been losing preparation and marking time so they could provide emergency cover.
"It's been going on for a good while now, probably months, because the priority is to keep the schools open and keep the children in school," Mr Searson added. "An emergency is one day or two days at the most. Emergency can't be for weeks and months.
"And our headteacher and depute members are losing all their management time – all the time they should be running the school, they're using it to cover classes individually or grouping them together in a hall or a canteen."
ASL staff in Edinburgh are to be used as supply cover in mainstream schools.
Our sister title the Herald has also learned that additional support for learning (ASL) staff in Edinburgh are set to be used as supply cover in mainstream council schools in an effort to alleviate strains caused by Covid-linked teacher shortages. Rotas for the measure are in place until the February holidays.
It comes after we told how those working in the capital's ASL service were being redeployed to plug gaps in special schools. Council bosses said individuals would only be asked to cover if absolutely necessary and to avoid closures or asking pupils to learn at home.
Mr Searson said: "That's been happening for a while – that's exactly the same sort of measure that takes place already in secondary schools. If there's a shortage of teachers and the class needs to be covered, then the ASL teachers are taken off their duties to do that. It's very unfair on the youngsters that need that support."
Concerns over disruption to education come amid increasing pressure for an early decision on holding exams and potentially providing additional help for preparation. Such assistance could include advance sight of some topics that will feature in question papers.
Critics say waiting until late March to make an announcement would be unacceptable.
Daniel Wyatt, rector of Kelvinside Academy in Glasgow, said: "I – as I'm sure many working in education in Scotland will be – am dismayed a decision on whether or not to go ahead with exams could be left until March. It's far too late.
"Put simply, it is not acceptable and shows complete disregard for the mental health of pupils and staff following two years of disruption, distraction, and disappointment, all against a backdrop of coping with the impact of the pandemic."
He added: "I appreciate it can never be 100%, particularly in this climate, but the educational community deserves more certainty in what the future holds, and there simply must be more positive messaging for pupils and indeed all involved in education in Scotland.
"Exams must go ahead unless there is a significant health concern far greater than we are currently experiencing with the Omicron variant."
There is growing pressure for early decisions on this year's exams.
In a social media post, Rod Grant, headmaster at Clifton Hall School near Edinburgh, said: "We have learned that a decision regarding exams won't potentially be made until the end of March.
"The Government needs to stop dithering on such issues because ultimately it is our children who suffer from being in a constant state of flux, as do our teachers. I appreciate the contextual difficulties within which we are operating but we need to put those difficulties to one side and make a decision.
"If the Government is worried that exams may not go ahead then just cancel them now. That would be fair to our children. Perhaps, however, our Government is waiting to see what England does – that seems to be the way political decisions in Scotland are currently being handled."
He added: "As a Head, I am really, really tired of the constantly shifting sands on which we are meant to operate... We already know of schools that have sent home entire year groups for the remainder of January because of the absence of teachers."
Echoing calls from School Leaders Scotland president Jon Reid, Mr Searson also said he favoured a speedy move towards adopting the contingency plan that would give teachers and pupils notice of some topics that will be assessed in exams.
A Scottish Government spokesperson said: "Our priority is to keep schools safely open and to minimise further disruption to education. We know the next few weeks will be challenging for pupils, staff and parents and we are monitoring the situation very closely.
"Safety guidance for schools was strengthened in light of Omicron and updated to reflect changes to self-isolation requirements. We are working with local authorities to keep a close eye on staffing levels in schools and we will keep the need for any further measures under close review.
"As set out in August last year, exams will go ahead if it is safe to do so. They will only be cancelled if public health advice says it isn't safe for them to take place – for example, if there are restrictions on gatherings. If this were to happen, awards would be made on teachers' judgements based on normal in-year assessment.
"Due to the unpredictability of the pandemic, we cannot know now what the public health advice will be for exams in the spring, but learners, parents and teachers will be told of any changes to plans as soon as possible."
Education Secretary Shirley-Anne Somerville is under pressure to make an earlier decision on the 2022 exams diet.
A Scottish Qualifications Authority spokesperson said: "The clear intention is for exams to take place in 2022. They will only be cancelled by the Scottish Government if public health advice restricts physical gatherings at the time of the exams.
"The Covid-19 Education Recovery Group is closely monitoring levels of learner and teacher absences, and school closures, and SQA will be guided by these in deciding whether to provide learners with additional support for exam revision.
"It is important to remember that the significant modifications already made to courses this year are designed to address the ongoing disruption to learning and teaching."

redpajama_set_name: RedPajamaCommonCrawl


Banning single use plastic bags is a step in the right direction, but it is a long way from fixing the issues of pollution in our oceans.
Of course, the green groups are rejoicing the ban as a major win against pollution, but if they actually got out on the water they'd realised they couldn't be further from the truth.
You don't have to go far. If you want to see where the real issue is just go down to your local creek.
I did just that the other day, taking the dogs for a walk I headed to my nearest creek in suburban Sydney.
Clambering in through the mangrove forest I was quickly surrounded by plastic pollution.
There weren't many single use plastic shopping bags, but there were endless drink bottles and food packaging, as well polystyrene, and endless disposable coffee cups.
This creek flows straight into the Parramatta River and ultimately into the ocean.
It's a sad fact that all our drains from roads, carparks and pathways spill straight into our waterways - so much for a first-world country. I guess out of sight, out of mind!
Banning plastic bags is a start, but let's face it, it's a Band-Aid fix to appease the inner-city greens and their guilt for living in the most changed environment on earth.
The real issue is swept under the carpet, or more appropriately, down the drain, right underneath them.
Urban run-off is an ugly truth and a massive issue in all urban areas, but especially Sydney with its population explosion.
Everything that ends up on the ground pretty much finds its way into our waterways after a bit of rain, and ultimately into our oceans, from rubbish to oils and chemicals.
There is a link here with our ill thought-out marine parks.
Anglers are locked out, many fishos are the only ones who clean up the rubbish in isolated areas. At the same time the proponents of lock outs ignore those dreaded stormwater drains pouring rubbish straight into the marine parks.
It really is frustrating because I want healthy waterways and yet I seem to be constantly fighting for common sense.
It's time we started focusing on the big issues and doing something that counts.
Looking after habitat by reducing litter is the first step to a healthy waterway and everyone needs to contribute. I carry my own drink bottle and coffee mug.
It's a small thing, but just imagine if everyone did it - now that would be a massive reduction in rubbish in coffee cups alone.
1 Always leave your fishing spots better than you found them.
2 Complain to your local, state and federal members to get our waterways cleaned up.
3 Take your own coffee mug and reusable water bottle.


text:
from __future__ import absolute_import

import mock
from datetime import timedelta
from django.conf import settings
from django.core import mail
from django.core.urlresolvers import reverse
from django.db import connection
from django.utils import timezone
from sentry.constants import MINUTE_NORMALIZATION
from sentry.db.models.fields.node import NodeData
from sentry.models import (
    Project, ProjectKey, Group, Event, Team,
    GroupTagValue, GroupCountByMinute, TagValue, PendingTeamMember,
    LostPasswordHash, Alert, User, create_default_project)
from sentry.testutils import TestCase, fixture
from sentry.utils.compat import pickle
from sentry.utils.strings import compress

class ProjectTest(TestCase):
    fixtures = ['tests/fixtures/views.json']

    def setUp(self):
        self.project = Project.objects.get(id=1)

    def test_migrate(self):
        project2 = Project.objects.create(name='Test')
        self.project.merge_to(project2)

        self.assertFalse(Project.objects.filter(pk=1).exists())
        self.assertFalse(Group.objects.filter(project__isnull=True).exists())
        self.assertFalse(Event.objects.filter(project__isnull=True).exists())
        self.assertFalse(GroupTagValue.objects.filter(project__isnull=True).exists())
        self.assertFalse(GroupCountByMinute.objects.filter(project__isnull=True).exists())
        self.assertFalse(TagValue.objects.filter(project__isnull=True).exists())

        self.assertEquals(project2.group_set.count(), 4)
        self.assertEquals(project2.event_set.count(), 10)
        assert not GroupTagValue.objects.filter(project=project2).exists()
        assert not project2.groupcountbyminute_set.exists()
        assert not TagValue.objects.filter(project=project2).exists()

class ProjectKeyTest(TestCase):
    def test_get_dsn(self):
        key = ProjectKey(project_id=1, public_key='public', secret_key='secret')
        with self.Settings(SENTRY_URL_PREFIX='http://example.com'):
            self.assertEquals(key.get_dsn(), 'http://public:secret@example.com/1')

    def test_get_dsn_with_ssl(self):
        key = ProjectKey(project_id=1, public_key='public', secret_key='secret')
        with self.Settings(SENTRY_URL_PREFIX='https://example.com'):
            self.assertEquals(key.get_dsn(), 'https://public:secret@example.com/1')

    def test_get_dsn_with_port(self):
        key = ProjectKey(project_id=1, public_key='public', secret_key='secret')
        with self.Settings(SENTRY_URL_PREFIX='http://example.com:81'):
            self.assertEquals(key.get_dsn(), 'http://public:secret@example.com:81/1')

    def test_get_dsn_with_public_endpoint_setting(self):
        key = ProjectKey(project_id=1, public_key='public', secret_key='secret')
        with self.Settings(SENTRY_PUBLIC_ENDPOINT='http://public_endpoint.com'):
            self.assertEquals(key.get_dsn(public=True), 'http://public@public_endpoint.com/1')

    def test_get_dsn_with_endpoint_setting(self):
        key = ProjectKey(project_id=1, public_key='public', secret_key='secret')
        with self.Settings(SENTRY_ENDPOINT='http://endpoint.com'):
            self.assertEquals(key.get_dsn(), 'http://public:secret@endpoint.com/1')

    def test_key_is_created_for_project_with_existing_team(self):
        user = User.objects.create(username='admin')
        team = Team.objects.create(name='Test', slug='test', owner=user)
        project = Project.objects.create(name='Test', slug='test', owner=user, team=team)
        assert project.key_set.filter(user__isnull=True).exists() is True

    def test_key_is_created_for_project_with_new_team(self):
        user = User.objects.create(username='admin')
        project = Project.objects.create(name='Test', slug='test', owner=user)
        assert project.key_set.filter(user__isnull=True).exists() is True

class PendingTeamMemberTest(TestCase):
    def test_token_generation(self):
        member = PendingTeamMember(id=1, team_id=1, email='foo@example.com')
        with self.Settings(SECRET_KEY='a'):
            self.assertEquals(member.token, 'f3f2aa3e57f4b936dfd4f42c38db003e')

    def test_token_generation_unicode_key(self):
        member = PendingTeamMember(id=1, team_id=1, email='foo@example.com')
        with self.Settings(SECRET_KEY="\xfc]C\x8a\xd2\x93\x04\x00\x81\xeak\x94\x02H\x1d\xcc&P'q\x12\xa2\xc0\xf2v\x7f\xbb*lX"):
            self.assertEquals(member.token, 'df41d9dfd4ba25d745321e654e15b5d0')

    def test_send_invite_email(self):
        team = Team(name='test', slug='test', id=1)
        member = PendingTeamMember(id=1, team=team, email='foo@example.com')
        with self.Settings(SENTRY_URL_PREFIX='http://example.com'):
            member.send_invite_email()

            self.assertEquals(len(mail.outbox), 1)

            msg = mail.outbox[0]

            self.assertEquals(msg.to, ['foo@example.com'])

class LostPasswordTest(TestCase):
    @fixture
    def password_hash(self):
        return LostPasswordHash.objects.create(
            user=self.user,
        )

    def test_send_recover_mail(self):
        with self.Settings(SENTRY_URL_PREFIX='http://testserver'):
            self.password_hash.send_recover_mail()
            assert len(mail.outbox) == 1
            msg = mail.outbox[0]
            assert msg.to == [self.user.email]
            assert msg.subject == '[Sentry] Password Recovery'
            url = 'http://testserver' + reverse('sentry-account-recover-confirm',
                args=[self.password_hash.user_id, self.password_hash.hash])
            assert url in msg.body

class AlertTest(TestCase):
    @fixture
    def params(self):
        return {
            'project_id': self.project.id,
            'message': 'This is a test message',
        }

    @mock.patch('sentry.models.alert.has_trending', mock.Mock(return_value=True))
    @mock.patch('sentry.models.Group.objects.get_accelerated')
    def test_does_add_trending_events(self, get_accelerated):
        get_accelerated.return_value = [self.group]
        alert = Alert.maybe_alert(**self.params)
        assert alert is not None
        get_accelerated.assert_called_once_with([self.project.id], minutes=MINUTE_NORMALIZATION)
        assert list(alert.related_groups.all()) == [self.group]

class GroupIsOverResolveAgeTest(TestCase):
    def test_simple(self):
        group = self.group
        group.last_seen = timezone.now() - timedelta(hours=2)
        group.project.update_option('sentry:resolve_age', 1)  # 1 hour
        assert group.is_over_resolve_age() is True
        group.last_seen = timezone.now()
        assert group.is_over_resolve_age() is False

class CreateDefaultProjectTest(TestCase):
    def test_simple(self):
        user, _ = User.objects.get_or_create(is_superuser=True, defaults={
            'username': 'test'
        })
        Team.objects.filter(project__id=settings.SENTRY_PROJECT).delete()
        Project.objects.filter(id=settings.SENTRY_PROJECT).delete()

        create_default_project(created_models=[Project])

        project = Project.objects.filter(id=settings.SENTRY_PROJECT)
        assert project.exists() is True
        project = project.get()
        assert project.owner == user
        assert project.public is False
        assert project.name == 'Sentry (Internal)'
        assert project.slug == 'sentry'
        team = project.team
        assert team.owner == user
        assert team.slug == 'sentry'

class EventNodeStoreTest(TestCase):
    def test_does_transition_data_to_node(self):
        group = self.group
        data = {'key': 'value'}

        query_bits = [
            "INSERT INTO sentry_message (group_id, project_id, data, logger, level, message, checksum, datetime)",
            "VALUES(%s, %s, %s, '', 0, %s, %s, %s)",
        ]
        params = [group.id, group.project_id, compress(pickle.dumps(data)), 'test', 'a' * 32, timezone.now()]

        # This is pulled from SQLInsertCompiler
        if connection.features.can_return_id_from_insert:
            r_fmt, r_params = connection.ops.return_insert_id()
            if r_fmt:
                query_bits.append(r_fmt % Event._meta.pk.column)
                params += r_params

        cursor = connection.cursor()
        cursor.execute(' '.join(query_bits), params)

        if connection.features.can_return_id_from_insert:
            event_id = connection.ops.fetch_returned_insert_id(cursor)
        else:
            event_id = connection.ops.last_insert_id(
                cursor, Event._meta.db_table, Event._meta.pk.column)

        event = Event.objects.get(id=event_id)
        assert type(event.data) == NodeData
        assert event.data == data
        assert event.data.id is None

        event.save()

        assert event.data == data
        assert event.data.id is not None

        node_id = event.data.id
        event = Event.objects.get(id=event_id)

        Event.objects.bind_nodes([event], 'data')

        assert event.data == data
        assert event.data.id == node_id

redpajama_set_name: RedPajamaGithub


text:
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UPT – Oporto Global University, is a private higher education institution, ISO 9001 certified, recognized by the Portuguese Government and the Portuguese Agency for Assessment and Accreditation of Higher Education - A3ES. UPT's Research unit, IJP, is certified by the Portuguese funding agency for research, FCT.
Created in June of 1986, UPT – Oporto Global University located in Oporto, known as the 'Cidade Invicta', Oporto gave Portugal its name. Located in the north of Portugal, on the right bank of the River Douro, Oporto is the country's second largest city situated within an urban area of over 1.3 million inhabitants.
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UPT is organized in four Departments: Law, Economics, Management and Informatics, Heritage and Tourism and Psychology and Education that teach 1st, 2nd and 3rd cycle programmes and post graduations, structured according to the Bologna Process. UPT aims to be a model in higher education, taking the advantages of the different department's synergies. More than acquiring theoretical knowledge, the students have the chance to develop practical work and research projects, developing different kinds of skills that prepare them for jobs in future career and for living in a competitive global society.
Polytechnic Institute Of Bragança
The Polytechnic Institute of Bragança (IPB) is a Portuguese University of Applied Sciences with 7000 students and 500 teachers and researchers. It has an internationalization programme with widely recognized success, including the annual mobility of more than 900 incoming and outgoing students and 300 teaching staff members. Nowadays the IPB is a multicultural institution where 20% of its students are international students from all over the World. IPB is also recognized by its performance in applied research and regional engagement.
The Polytechnic Institute of Bragança (IPB) is a Portuguese Higher Education Public Institution with 7000 undergraduate and master students, embracing a wide area of knowledge and technology, including agriculture sciences, arts and sports, education and teachers' training, informatics and engineering, administration and management, health, communication and tourism.
The IPB belongs to the European Network for Universities of Applied Sciences (UASnet), which main objectives include the transferability of professional skills and the integration of applied research in their professional and technological education mission. IPB has also an internationalization programme with widely recognized success, including the annual mobility of more than 900 incoming and outgoing students and 300 teaching staff members, as the result of the collaboration with several higher education institutions from all over the World. Nowadays the IPB is a multicultural institution, where 20% of its students are international students.
In 2011, the European Commission has awarded the ECTS Label to the IPB, acknowledging the quality of the information provided in terms of the degree programmes offered and the management of student mobility within the European Higher Education Area. In 2013, IPB has been awarded also the DS Label. The purpose of the Diploma Supplement (DS) is to provide sufficient independent data to improve the international transparency and fair academic and professional recognition of qualifications.
Besides the academic programmes taught in the Portuguese language, the IPB also offers bachelor and master programmes fully lectured in English, as well as Portuguese language and culture courses for foreign students.
Polytechnic Institute Of Coimbra
Polytechnic Institute of Coimbra is a Public Higher Education Institution located in the West centre of Portugal, in the city known for centuries as the "city of students".
One of the ten largest and major institutions of public higher education in Portugal, with six units
Escola Superior Agrária (Coimbra Agriculture School) (ESAC)
Escola Superior de Educação (Coimbra Education School) (ESEC)
Escola Superior de Tecnologia da Saúde (Coimbra Health School) (ESTeSC)
Escola Superior de Tecnologia e Gestão de Oliveira do Hospital (Oliveira do Hospital Management and Technology School) (ESTGOH
Instituto Superior de Contabilidade e Administração (Coimbra Business School) (ISCAC)
Instituto Superior de Engenharia (Coimbra Institute of Engineering) (ISEC)
Covering a huge diversity of training areas, from agriculture and environment, education, communication, tourism, arts, management, accounting and marketing, to health and engineering.
The Polytechnic of Coimbra offers Higher Professional Degree Programmes and Vocational Education, Bachelor's Degrees, Post-graduations and Master's Degrees, representing a vital part of the city and playing a critical role in local development and national progress.
Quality education – where the practical component is strongly supported by solid theory training, with a constant concern to adapt training to market demands –, a close connection with companies, incentive to entrepreneurship and internationalisation are central pillars of its education, assuring successful careers to its graduates, as well as high employability rates.
Social Service is another core organisational unit responding to the students' needs by means of general support, scholarships, board and accommodation, medical services and sports practice.
The Polytechnic of Coimbra represents:
- Modernity
- Knowledge
- Quality
- Employability
- Citizenship
- Solidarity
- Multiculturalism
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Polytechnic Institute Of Leiria
The Polytechnic Institute of Leiria (IPL) is a public institution of Higher Education that offers courses of training on an Undergraduate Degree, Masters, Post-Graduate, Technology Specialization and Preparation Courses for Access to Higher Education level. It began its activity in 1980 and is present in the Leiria and West region through its five schools, divided in five campus, located in the cities of Leiria (School of Education and Social Sciences, School of Technology and Management and School of Health Sciences), Caldas da Rainha (School of Fine Art and Design) and Peniche (School of Tourism and Maritime Technology). IPL offers Undergraduate e master's degrees courses in the areas of: Art and Design, Law studies, Education and Communication, Engineering and Technology, Tourism and Health.
Polytechnic Institute Of Santarem
The Polytechnic Institute of Santarém is a polytechnic public higher education institution, at the service of society, committed to the high level qualification of the citizens, destined to the production and diffusion of the knowledge, creation, transmission and diffusion of the knowledge of professional nature, Science, technology, the arts, guided research and experimental development, emphasizing centrality in the student and the surrounding community, within an international frame of reference.
It is recognized as a pole of development and a reference in training, culture and research developed in the region, created in the 70's, currently comprises five High Schools, four in the city of Santarém and one in the city of Rio Maior.
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Tag: Radio Pakistan
Radio Pakistan DG not allowed to attend office by protesting employees
[ 0 ] September 28, 2018
ISLAMABAD: Despite an announcement by the government that the plan to lease out the headquarters of Radio Pakistan has been shelved, angry employees on Thursday forced the director general out of his office to lodge their protest, forcing the information minister to issue a statement saying that action would be taken against those involved in […]
Govt delays leasing out Radio Pakistan premises by one-month
ISLAMABAD: The Pakistan Tehreek-e-Insaf (PTI) led government has given one month's time to the management of Radio Pakistan to table a plan to bring out the state-run broadcaster from the financial crisis it is currently facing. The federal government had given directives t lease out Pakistan Broadcasting Corporation headquarters building in the federal capital. However, the one […]
Radio Pakistan employees protest against govt decision to lease out PBC HQs'
Employees of Radio Pakistan on Monday held a peaceful demonstration in Islamabad and other Units of the organization across the country in protest against the decision of the government to lease out Pakistan Broadcasting Corporation Headquarters building. Employees of all the wings of the national broadcaster participated in the demonstrations and demanded the government to […]
Govt to lease out Radio Pakistan HQ
ISLAMABAD:Federal Minister for Information, Broadcasting and National Heritage Fawad Chaudhry on Wednesday said the building of Radio Pakistan will be given on lease to build Pak Media University and in this regard it has asked for proposal from the department concerned. While responding over a letter shared in the official Whatsapp group of Ministry of […]
Rashid inaugurates Radio Pakistan's new transmitter in Karachi
Minister for information and Broadcasting Pervaiz Rashid inaugurated 10 KW medium wave transmitter at Radio Pakistan Karachi today. Director General Radio Pakistan Saba Mohsin and other officials concerned were also present on the occasion. Our Karachi correspondent Altaf Pirzado reports that the transmitter has been installed by Radio Pakistan from its own resources at a […]
Death anniversary of Riaz Ahmed Khan observed
The fifth death anniversary of the legend English newscaster of Radio Pakistan, Riaz Ahmed Khan was observed on Saturday. Riaz Ahmed Khan joined Central News Organisation of Radio Pakistan in December 1970 and superannuated in April 1997. It was his passion for news reading that he kept on reading news bulletins of Radio Pakistan even […]
Radio Pakistan turned into vibrant medium: DG
Director General, Radio Pakistan, Syed Muhammad Imran Gardezi says Radio Pakistan has been turned into a vibrant medium to promote peace, interfaith harmony and culture, to educate people in peace time and natural disasters. In a message on the occasion of World Radio Day, he said, with access to 98 percent population of the country, […]
World Radio Day being observed today
Radio Pakistan is airing special programmes to mark World Radio Day being observed today [Saturday] to highlight the role of this medium. The objective of the World Radio Day observed on February 13 is to raise greater awareness among the public about the importance of radio, encourage decision makers to establish and provide access to […]
Research, reference section inaugurated at Radio Pakistan
ISLAMABAD – A new research and reference section has been set up in News and Current Affairs Channel of Radio Pakistan to help make current affairs programs credible, relevant, and comprehensive. Director General Radio Pakistan, Syed Muhammad Imran Gardezi inaugurated the section here Thursday. Speaking on the occasion, he said it has been the hallmark […]


What comes after Pokémon X and Y? Well, if Y follows X, then it would make sense for the next game in the series to be titled Pokémon Z. Mainline Pokémon games typically release in pairs, and are commonly followed by a "third" game: Pokémon Red and Blue had Yellow, Gold and Silver had Crystal, Ruby and Sapphire had Emerald, Diamond and Pearl had Platinum and finally, Black and White had… well, not a third game, but rather a direct sequel of sorts in Black 2 and White 2. Pokémon X and Y, on the other hand, has had neither a "third game", nor a sequel of any sort. Why could that be?
The short answer is, it's almost certainly not happening. With no official confirmations or even leaks, there is no reason to believe that Pokémon Z is or was ever under development. That ship has probably sailed, and Game Freak is already hard at work developing the next pair of Pokémon games: Sun and Moon. Pokémon Z presents a whole new region to explore, complete with new Pokémon to train and capture. As such, it is very unlikely that the company will take one step back and return to Kalos. This rings especially true now that the Nintendo 3DS is likely on its way out. If you were looking for Pokémon Z news, then well, that's the long and short of it.
If it had indeed been in development, the Pokémon Z release date would likely have been somewhere around last year. A cursory glance at the release dates of previous games in the series shows that the 'third' game or sequel has always released 2 years after the original pair's Japanese release. With Pokémon X and Y having released in 2013, the Pokémon Z release date should have been in 2015. Regardless, the game has failed to materialise, and it's time to move on to Pokémon Sun and Moon, or Pokémon GO (or both!).


text:
December 22, 2016 January 10, 2017 Sauce
We caught The 1975 at Bournemouth BIC, and here's our conclusion…
There was no better way to reach the end of 2016 than catching The 1975 at Bournemouth BIC last night. Although it wasn't the first time we saw the 'Love Me' indie rockers perform, we were blown away by their performance and ability to get the crowd buzzing before even setting foot on the stage. Although many people presume The 1975 to be a relatively new band, they have been around for nearly 15 years before rising to stardom the world over.
As we looked around the sweaty crowd, the BIC was jam packed with fans of all ages. And after a quick set change from their support act, The Japanese House (who, FYI, you must also check out), the lights dimmed and the excitement became uncontrollable.
'Love Me'? We LOVE them.
The 1975 kicked things off with 'Love Me' and 'UGH!', wowing the crowd with mirrors and an epic light show. Unlike many bands, The 1975 don't necessarily need the fancy tricks and gimmicks behind them, but in this case they added an air of magic to their performance.
Matt Healy played to the crowd, singing into the camera and making full use of the space available around him. The screens on either side of the stage aided those a little further back to get a full view, although made the show feel more like a festival than an intimate gig.
With a set list as strong as theirs, The 1975 were on full power from start to finish, with a minor break for Matt Healy to discuss what's going on in the world around us – including the election of Trump. Despite some of the fans around us falling into an age group of 10-13 and remaining perplexed at this discussion, their level of interaction remained totally committed to his every word.
Related: Look Out For... Eagulls.
Not forgetting hit singles 'Girls' and 'Chocolate', The 1975 eventually brought the show to a close on 'The Sound' before exiting the stage.
Until next time, guys.
Review // Gary Numan Portsmouth Pyramids 24/9/16
Review // Palms And Pelicans The Joiners 21/5/16
Review // Wolf Alice Southampton Guildhall 23/09/1...
We Love // Green Or Blue.
Entertainment 1975, BIC, Bournemouth, Chocolate, Concert, Gig, Gig Review, Live Review, Matty, Music, Music Review, Show, The 1975
← Review // GFW Clothing Boxers.
Panic! The Life Of A Millennial. →
http://sauce-magazine.co.uk/review-1975-bournemouth-bic/">


We can offer Capital Connection deliveries from Derby, Nottingham and Leicester for any size of parcel. From an envelope to a Pallet into London.
Most deliveries can be achieved within a 2.5 hour time frame, with Derby Sameday Couriers offering a dedicated vehicle for your specific job.
Envelopes can be delivery extremely quickly, which is ideal for contracts and transactions where paperwork is needed quick.


Another morning shot in Bryce Canyon.
I can FEEL the warmth in this photo. BEAUTIFUL!!!
Rich, sumptuous colour I even love the lens flare, it really adds to the 'heat' of the scene.
Deffinately the photo I needed to see on this Fridged Wis. Day!!!!
I can almost hear my brother sneak up behind me and then yell "Don't FALL!" as I peer over the edge. Those hexagons on the lense flare are cool. Very nice.
Thoughts of: Ed Little, Jr.
Yes, the light is great...what I first noticed.
Love this shot Ryan. There's something almost Biblical about it and I can hear the ground rustling under my footsteps. Wonderful place ~ and you chose a neat way to capture it.
With this picture you have captured the magnificance of our dawn experience at Bryce Canyon, Ryan. This brings back great memories of that morning!


text:
•September 11, 2017
•Vallarta Daily News
•Arts & Culture, Local Entertainment
Celebrate Mexican Independence Day with dinner and a show at Incanto
Come celebrate Mexican Independence Day with dinner and a show at Incanto! Master Chef Jorge will prepare a special Mexican Independence Day menu on Friday, Sept.15, featuring traditional Chiles en nogada from a secret family recipe dating back to the 1800's. Using all fresh ingredients made from scratch, his not-too-sweet traditional dish of a meat/fresh fruit stuffed poblano chile covered in a creamy, nutty white sauce and topped with pomegranate seeds is a perfect way to celebrate! The special also includes a welcome cocktail known as 'Bandarita' made of lemon, tequila and Sangrita shots in the colors of the Mexican flag, rich Poblano Chile Soup (non-spicy) and Dulce de Papaya for dessert. The cost is $295 MXN per person and includes entertainment by Spanish guitarist/singer Gabriel Reyes. And for $400 MXN, make it dinner and a show with hit drag comedy/music revue Pearl's Gurlz, as seen recently on the hit TV series Bachelor in Paradise. It stars local drag icons Angelica, Diva, Maury & Ghia and is hosted by the hilarious Aunt Pearl in the cabaret theater. Audiences are loving it with raucous laughter and non-stop fun. Pearl's Gurlz plays on Fridays at 8:30 P.M.
Tongo is a recently discovered local musician who is taking talent to the next level at Incanto! A former fireman and lifeguard, he plays Latin rhythms/vocals, featuring Cuban and Chilean influences on handmade instruments. Originally from Guadalajara, he is a self-taught musician, having learned by watching and playing with other musicians over the past 20 years. During that time, he developed not only his musical and vocal abilities, but also his skills as a master craftsman, creating his own instruments including flutes and drums. Tongo plays on Wednesdays at 7:30 P.M. in the piano bar.
One night only, Thursday, Sept. 21, Spanish guitarist/singer Gabriel Reyes partners with popular trio Piel Canela, featuring Eduardo Leon and Fernando González, in 'Gabriel Reyes La Flor De La Piel Canela' in the cabaret theater. A night of Spanish music, they will play many of your favorite songs from a vast repertoire of classics and popular modern hits. Their combined talents are sure to create an outstanding evening of artistic collaboration, music and entertainment. Tickets are now available at Incanto's website.
Paco Ojeda presents PechaKucha Night Volume 2 at Incanto on Sept. 23 at 8:20 P.M. PechaKucha Nights began in the early 2000's in Tokyo, when two British architects began organizing meetings with their colleagues to present and share their most recent projects using 20 images and speaking about them for 20 seconds each. They called it pechacucha, a Japanese word that describes the sound of whispering. Also referred to as "20×20", these bi-lingual events are new to Puerto Vallarta. Proposals are now being accepted for presentations in English and Spanish until Friday, September 15. For more information and updates, please visit and like Incanto Vallarta's Facebook page. Also, please visit their official website, www.pechakucha.org. The cost is $40 MXN pesos (donation) per person. Tickets are now available at Incanto's website.
On Tuesday Sept. 12, guest showmen/entertainers Pedro Islas and Nick Olsina (of the Three Tenors, now touring the region) will sing many of your favorites in the piano bar starting at 7:30pm. Open Mic is on Thursdays at 7pm hosted by Tracy Parks with Chris Kenny on guitar. Bingo with Pearl fundraisers are held on Saturdays at 5pm, and Loteria with Pearl and Bev begins every third Saturday of the month starting Sept. 16 at 5pm. Singer/songwriter Chris Kenny plays solo on Saturdays at 8pm, followed by Spanish guitarist/singer Gabriel Reyes at 10pm. He also plays on Fridays starting at 7pm. Piel Canela, featuring guitarists Eduardo Leon and Fernando Huerta González with percussionist Samuel Renteria, plays on Sundays at 7:30. Also on Sundays at 8pm in the piano bar, is Salsa Dancing and instruction by Francisco Ledesma. Enjoy cold A/C and 2×1 premium tequila cocktails/shots all day/night in September (offer excludes cabaret theater). No cover charge in the piano bar.
Tickets are available at www.IncantoVallarta.com. Hours 9:00am -11:30pm Tues.- Sun. The cafe is open for casual dining daily, rain or shine, with piano music by Salvatore starting at 10am. Happy Hour 2-6pm. For event inquiries or group bookings contact General Manager, Gilberto Figueroa at [email protected]. Incanto is located at Insurgentes 109 (at the Rio Cuale). Box Office opens at 9am. Call 322 223 9756 for reservations. Closed Mondays.
Related: Celebrate Mexican Independence in Puerto Vallarta, Piel Canela adds Sunday nights at Incanto


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