Green listed-a CRISPR screen tool.

Motivation: Genome editing using versions of the bacterial CRISPR/Cas9 system can be used to probe the function of selected genes in any organism. Green Listed is a web-based tool that rapidly designs custom CRISPR screens targeting sets of genes defined by the user. It could thus be used to design screens targeting for example all genes differentially expressed during a specific stimuli or all genes related to a specific pathway or function, as well as to generate targeted secondary screens following a large-scale screen. Availability and Implementation: The software, including a demo function as well as explanatory texts and videos, is available through greenlisted.cmm.ki.se . Contact: fredrik.wermeling@ki.se.

Loss of Functionally Redundant p38 Isoforms in T Cells Enhances Regulatory T Cell Induction.

The evolutionarily conserved protein kinase p38 mediates innate resistance to environmental stress and microbial infection. Four p38 isoforms exist in mammals and may have been co-opted for new roles in adaptive immunity. Murine T cells deficient in p38α, the ubiquitously expressed p38 isoform, showed no readily apparent cell-autonomous defects while expressing elevated amounts of another isoform, p38ß. Mice with T cells simultaneously lacking p38α and p38ß displayed lymphoid atrophy and elevated Foxp3+ regulatory T cell frequencies. Double deficiency of p38α and p38ß in naïve CD4+ T cells resulted in an attenuation of MAPK-activated protein kinase (MK)-dependent mTOR signaling after T cell receptor engagement, and enhanced their differentiation into regulatory T cells under appropriate inducing conditions. Pharmacological inhibition of the p38-MK-mTOR signaling module produced similar effects, revealing potential for therapeutic applications.

Immunoglobulin E induces VEGF production in mast cells and potentiates their pro-tumorigenic actions through a Fyn kinase-dependent mechanism.

BACKGROUND: High concentrations of plasmatic IgE have been related to distinct systemic inflammatory conditions that frequently predispose individuals to hypersensitivity reactions. Although effects of IgE have been suggested to relay on the low-intensity activation of distinct effector elements of the immune system, such as mast cells (MC), experimental evidence on the role of IgE-induced production of inflammatory mediators on specific pathologies is scarce. MC are an important component in tumor microenvironment where they seem to secrete a number of immunomodulatory and angiogenic mediators, such as the Vascular Endothelial Growth Factor (VEGF) by not well-described mechanisms. In this work, we investigated the effect of monomeric IgE (in the absence of antigen) on the production of VEGF in MC, analyzed if monomeric IgE could exacerbate the pro-tumorigenic properties of that cell type and characterized some of the molecular mechanisms behind the effects of IgE on VEGF production and tumor growth. METHODS: For in vitro studies, murine bone marrow-derived mast cells (BMMCs) were used. Pharmacological inhibitors and phosphorylation of key elements controlling VEGF secretion and protein translation were used to characterize the mechanism of VEGF production triggered by IgE.In vivo, the effect of a single i.v. administration of monomeric IgE on B16 melanoma tumor weight, intratumoral blood vessel formation and tumor-associated MC was assessed in four groups of mice: MC-proficient (WT), MC-deficient (Wsh), Wsh reconstituted with MC derived from WT mice (Wsh Rec WT) and Wsh reconstituted with MC derived from Fyn -/- mice (Wsh Rec Fyn -/-). RESULTS: Monomeric IgE induced VEGF secretion through a Fyn kinase-dependent mechanism and modulated de novo protein synthesis modifying the activity of the translational regulator 4E-BP1 in BMMCs. In vivo, monomeric IgE increased melanoma tumor growth, peritumoral MC and blood vessel numbers in WT but not in Wsh mice. The positive effects of IgE on melanoma tumor growth were reproduced after reconstitution of Wsh mice with WT but not with Fyn -/- BMMCs. CONCLUSION: Our data suggest that monomeric IgE, in the absence of antigen, induces VEGF production in MC and in vivo contributes to melanoma tumor growth through a Fyn kinase-dependent mechanism.

Pyritinol reduces nociception and oxidative stress in diabetic rats.

The purpose of this study was to assess the antinociceptive and antiallodynic effect of pyritinol as well as its possible mechanism of action in diabetic rats. Streptozotocin (50 mg/kg) injection caused hyperglycemia within 1 week. Formalin-evoked flinching was increased in diabetic rats as compared to non-diabetic rats. Oral acute administration of pyritinol (50-200 mg/kg) dose-dependently reduced flinching behavior in diabetic rats. Moreover, prolonged administration of pyritinol (12.5-50 mg/kg, every 2 days for 2 weeks) reduced formalin-induced nociception. 1H-[1,2,4]-oxadiazolo [4,3-a] quinoxalin-1-one (ODQ, a guanylyl cyclase inhibitor, 2 mg/kg, i.p.), but not naltrexone (a non-selective opioid receptor antagonist, 1 mg/kg, s.c.) or indomethacin (a non-selective cycloxygenase inhibitor, 5 mg/kg, i.p.), blocked the pyritinol-induced antinociception in diabetic rats. Given alone ODQ, naltrexone or indomethacin did not modify formalin-induced nociception in diabetic rats. Oral acute (200 mg/kg) or prolonged (25 mg/kg, every 2 days for 2 weeks) administration of pyritinol significantly reduced streptozotocin-induced changes in free carbonyls, dityrosine, malondialdehyde and advanced oxidative protein products. Four to 8 weeks after diabetes induction, tactile allodynia was observed in the streptozotocin-injected rats. On this condition, oral administration of pyritinol (50-200 mg/kg) reduced tactile allodynia in diabetic rats. Results indicate that pyritinol is able to reduce formalin-induced nociception and tactile allodynia in streptozotocin-injected rats. In addition, data suggest that activation of guanylyl cyclase and the scavenger properties of pyritinol, but not improvement in glucose levels, play an important role in these effects.