Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire, and Cappuccino

The actin nucleation factors Spire and Cappuccino regulate the onset of ooplasmic streaming in Drosophila1-5. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here we demonstrate that Cappuccino and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC, and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, while SpireC is a potent crosslinker. We show that SpireD binds to Cappuccino and inhibits Factin/ microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments

Targeted GAS6 Delivery to the CNS Protects Axons from Damage during Experimental Autoimmune Encephalomyelitis

Growth arrest-specific protein 6 (GAS6) is a soluble agonist of the TYRO3, AXL, MERTK (TAM) family of receptor tyrosine kinases identified to have anti-inflammatory, neuroprotective, and promyelinating properties. During experimental autoimmune encephalomyelitis (EAE), wild-type (WT) mice demonstrate a significant induction of Gas6, Axl, and Mertk but not Pros1 or Tyro3 mRNA. We tested the hypothesis that intracerebroventricular delivery of GAS6 directly into the CNS of WT mice during myelin oligodendrocyte glycoprotein (MOG)-induced EAE would improve the clinical course of disease relative to artificial CSF (ACSF)-treated mice. GAS6 did not delay disease onset, but significantly reduced the clinical scores during peak and chronic EAE. Mice receiving GAS6 for 28 d had preserved SMI31(+) neurofilament immunoreactivity, significantly fewer SMI32(+) axonal swellings and spheroids and less demyelination relative to ACSF-treated mice. Alternate-day subcutaneous IFNß injection did not enhance GAS6 treatment effectiveness. Gas6(-/-) mice sensitized with MOG35-55 peptide exhibit higher clinical scores during late peak to early chronic disease, with significantly increased SMI32(+) axonal swellings and Iba1(+) microglia/macrophages, enhanced expression of several proinflammatory mRNA molecules, and decreased expression of early oligodendrocyte maturation markers relative to WT mouse spinal cords with scores for 8 consecutive days. During acute EAE, flow cytometry showed significantly more macrophages but not T-cell infiltrates in Gas6(-/-) spinal cords than WT spinal cords. Our data are consistent with GAS6 being protective during EAE by dampening the inflammatory response, thereby preserving axonal integrity and myelination.This work was supported by the National Multiple Sclerosis Society (Grant RG 4046-A6) and the National Institutes of Health (Grant R21 NS079144-01, Neuropathology Training Grant T32 NS007098, and Cellular and Molecular Biology and Genetics Training Grant T32 GM007491).Peer Reviewe

Efficacy of an in-home test kit in reducing dust mite allergen levels: results of a randomized controlled pilot study

<p><i>Background</i>: Dust mite allergens can induce allergic sensitization and exacerbate asthma symptoms. Although dust mite reduction and control strategies exist, few asthmatics employ them. <i>Objectives</i>: We examined whether an in-home test kit, which quantifies dust mite allergen levels, resulted in behavioral changes in implementation and maintenance of mite reduction strategies and helped reduce allergen levels in homes of dust mite-sensitive children. <i>Methods</i>: We enrolled 60 households of children aged 5–15 with parent-reported dust mite allergy into a randomized controlled trial. Intervention homes (<i>N</i> = 30) received educational material about reducing dust mites and test kits at 1, 2, 5 and 8 months. Control homes (<i>N</i> = 30) received only educational material. At baseline, 6 and 12 months, study staff visited all homes, collected dust samples from three locations and obtained information about parents’ mite reduction behaviors by questionnaire. Allergen concentrations (<i>Der f 2/Der p2</i>) in dust were assessed by immunoassays. After adjusting for visit and location, allergen concentrations in intervention and control homes were compared using mixed effects model analysis. <i>Results</i>: In the intervention homes, allergen concentrations in the child’s bedroom and living room floors were significantly reduced over time compared to control homes. Although not all location-specific differences in allergen concentrations were statistically significant, combining data across locations, there was a differential reduction in allergen concentrations in the intervention group versus the control group (<i>p</i> = 0.02). <i>Conclusion</i>: The use of in-home test kits along with education may beneficially influence behaviors and attitudes toward dust mite reduction strategies and help reduce residential dust mite allergen levels.</p

Serial stimulation of invariant natural killer T cells with covalently stabilized bispecific T cell engagers generates anti-tumor immunity while avoiding anergy

CD1d-restricted invariant natural killer T cells (iNKT cells) mediate strong anti-tumor immunity when stimulated by glycolipid agonists. However, attempts to develop effective iNKT cell agonists for clinical applications have been thwarted by potential problems with dose-limiting toxicity and by activation-induced iNKT cell anergy, which limits the efficacy of repeated administration. To overcome these issues, we developed a unique bispecific T cell engager (BiTE) based on covalent conjugates of soluble CD1d with photoreactive analogs of the glycolipid α-galactosylceramide. Here we characterize the in vivo activities of iNKT cell-specific BiTEs and assess their efficacy for cancer immunotherapy in mouse models using transplantable colorectal cancer or melanoma tumor lines engineered to express human Her2 as a tumor-associated antigen. Systemic administration of conjugated BiTEs stimulated multiple iNKT cell effector functions including cytokine release, secondary activation of NK cells, and induction of dendritic cell maturation and also initiated epitope spreading for tumor-specific CD8+ cytolytic T cell responses. The anti-tumor effects of iNKT cell activation with conjugated BiTEs were further enhanced by simultaneous checkpoint blockade with antibodies to CTLA-4, providing a potential approach for combination immunotherapy. Multiple injections of covalently stabilized iNKT cell-specific BiTEs activated iNKT cells without causing iNKT anergy and exhaustion, thus enabling repeated administration for effective and nontoxic cancer immunotherapy regimens