Human cerebrospinal fluid monoclonal LGI1 autoantibodies increase neuronal excitability

OBJECTIVE: Leucine-rich glioma-inactivated 1 (LGI1) encephalitis is the second most common antibody-mediatedencephalopathy, but insight into the intrathecal B-cell autoimmune response, including clonal relationships, isotype dis-tribution, frequency, and pathogenic effects of single LGI1 antibodies, has remained limited. METHODS: We cloned, expressed, and tested antibodies from 90 antibody-secreting cells (ASCs) and B cells from thecerebrospinalfluid (CSF) of several patients with LGI1 encephalitis. RESULTS: Eighty-four percent of the ASCs and 21% of the memory B cells encoded LGI1-reactive antibodies, whereasreactivities to other brain epitopes were rare. All LGI1 antibodies were of IgG1, IgG2, or IgG4 isotype and had under-gone affinity maturation. Seven of the overall 26 LGI1 antibodies efficiently blocked the interaction of LGI1 with itsreceptor ADAM22 in vitro, and their mean LGI1 signal on mouse brain sections was weak compared to the remaining,non–ADAM22-competing antibodies. Nevertheless, both types of LGI1 antibodies increased the intrinsic cellular excit-ability and glutamatergic synaptic transmission of hippocampal CA3 neurons in slice cultures. Interpretation: Our data show that the patients’intrathecal B-cell autoimmune response is dominated by LGI1 anti-bodies and that LGI1 antibodies alone are sufficient to promote neuronal excitability, a basis of seizure generation.Fundamental differences in target specificity and antibody hypermutations compared to the CSF autoantibody reper-toire in N-methyl-D-aspartate receptor encephalitis underline the clinical concept that autoimmune encephalitides arevery distinct entities

Silencing of IQGAP1 by shRNA inhibits the invasion of ovarian carcinoma HO-8910PM cells in vitro

<p>Abstract</p> <p>Background</p> <p>IQGAP1 is a scaffolding protein and overexpressed in many human tumors, including ovarian cancer. However, the contribution of IQGAP1 to invasive properties of ovarian cancer cells remains unknown. Here, we investigated the effect of IQGAP1-specific short hairpin RNA (shRNA) expressing plasmids on metastatic potential of ovarian cancer HO-8910PM cells.</p> <p>Methods</p> <p>We used RT-PCR and Western blot analysis to characterize expression of IQGAP1 in three human ovarian cancer-derived cell lines SK-OV-3, HO-8910 and HO-8910PM. We then determined whether expression of endogenous IQGAP1 correlated with invasive and migratory ability by using an in vitro Matrigel assay and cell migration assay. We further knocked down IQGAP1 using shRNA expressing plasmids controlled by U1 promoter in HO-8910PM cells and examined the proliferation activity, invasive and migration potential of IQGAP1 shRNA transfectants using MTT assay, in vitro Matrigel-coated invasion assay and migration assay.</p> <p>Results</p> <p>IQGAP1 expression level seemed to be closely associated with the enhanced invasion and migration in ovarian cancer cell lines. Levels of both IQGAP1 mRNA and protein were significantly reduced in HO-8910PM cells transfected with plasmid-based IQGAP1-specific shRNAs. RNAi-mediated knockdown of IQGAP1 expression in HO-8910PM cells resulted in a significant decrease in cell invasion and migration.</p> <p>Conclusion</p> <p>Our findings support the hypothesis that IQGAP1 promotes tumor progression and identify IQGAP1 as a potential therapeutic strategy for ovarian cancer and some other tumors with over-expression of the IQGAP1 gene.</p

Extracts of Feijoa Inhibit Toll-Like Receptor 2 Signaling and Activate Autophagy Implicating a Role in Dietary Control of IBD

Inflammatory bowel disease (IBD) is a heterogeneous chronic inflammatory disease affecting the gut with limited treatment success for its sufferers. This suggests the need for better understanding of the different subtypes of the disease as well as nutritional interventions to compliment current treatments. In this study we assess the ability of a hydrophilic feijoa fraction (F3) to modulate autophagy a process known to regulate inflammation, via TLR2 using IBD cell lines

The role of prostaglandin E2 (PGE 2) in toll-like receptor 4 (TLR4)-mediated colitis-associated neoplasia

<p>Abstract</p> <p>Background</p> <p>We have previously found that TLR4-deficient (TLR4-/-) mice demonstrate decreased expression of mucosal PGE ₂and are protected against colitis-associated neoplasia. However, it is still unclear whether PGE ₂is the central factor downstream of TLR4 signaling that promotes intestinal tumorigenesis. To further elucidate critical downstream pathways involving TLR4-mediated intestinal tumorigenesis, we examined the effects of exogenously administered PGE ₂in TLR4-/- mice to see if PGE ₂bypasses the protection from colitis-associated tumorigenesis.</p> <p>Method</p> <p>Mouse colitis-associated neoplasia was induced by azoxymethane (AOM) injection followed by two cycles of dextran sodium sulfate (DSS) treatment. Two different doses of PGE ₂(high dose group, 200 μg, n = 8; and low dose group, 100 μg, n = 6) were administered daily during recovery period of colitis by gavage feeding. Another group was given PGE ₂during DSS treatment (200 μg, n = 5). Inflammation and dysplasia were assessed histologically. Mucosal Cox-2 and amphiregulin (AR) expression, prostanoid synthesis, and EGFR activation were analyzed.</p> <p>Results</p> <p>In control mice treated with PBS, the average number of tumors was greater in WT mice (n = 13) than in TLR4-/- mice (n = 7). High dose but not low dose PGE ₂treatment caused an increase in epithelial proliferation. 28.6% of PBS-treated TLR4-/- mice developed dysplasia (tumors/animal: 0.4 ± 0.2). By contrast, 75.0% (tumors/animal: 1.5 ± 1.2, P < 0.05) of the high dose group and 33.3% (tumors/animal: 0.3 ± 0.5) of the low dose group developed dysplasia in TLR4-/- mice. Tumor size was also increased by high dose PGE ₂treatment. Endogenous prostanoid synthesis was differentially affected by PGE ₂treatment during acute and recovery phases of colitis. Exogenous administration of PGE ₂increased colitis-associated tumorigenesis but this only occurred during the recovery phase. Lastly, PGE ₂treatment increased mucosal expression of AR and Cox-2, thus inducing EGFR activation and forming a positive feedback mechanism to amplify mucosal Cox-2.</p> <p>Conclusions</p> <p>These results highlight the importance of PGE ₂as a central downstream molecule involving TLR4-mediated intestinal tumorigenesis.</p

The NLRP3 inflammasome functions as a negative regulator of tumorigenesis during colitis-associated cancer

Colitis-associated cancer (CAC) is a major complication of inflammatory bowel diseases. We show that components of the inflammasome are protective during acute and recurring colitis and CAC in the dextran sulfate sodium (DSS) and azoxymethane + DSS models. Mice lacking the inflammasome adaptor protein PYCARD (ASC) and caspase-1 demonstrate increased disease outcome, morbidity, histopathology, and polyp formation. The increased tumor burden is correlated with attenuated levels of IL-1β and IL-18 at the tumor site. To decipher the nucleotide-binding domain, leucine-rich-repeat-containing (NLR) component that is involved in colitis and CAC, we assessed Nlrp3 and Nlrc4 deficient mice. Nlrp3−/− mice showed an increase in acute and recurring colitis and CAC, although the disease outcome was less severe in Nlrp3−/− mice than in Pycard−/− or Casp1−/− animals. No significant differences were observed in disease progression or outcome in Nlrc4−/− mice compared with similarly treated wild-type animals. Bone marrow reconstitution experiments show that Nlrp3 gene expression and function in hematopoietic cells, rather than intestinal epithelial cells or stromal cells, is responsible for protection against increased tumorigenesis. These data suggest that the inflammasome functions as an attenuator of colitis and CAC

Palmitoylation Regulates Epidermal Homeostasis and Hair Follicle Differentiation

Palmitoylation is a key post-translational modification mediated by a family of DHHC-containing palmitoyl acyl-transferases (PATs). Unlike other lipid modifications, palmitoylation is reversible and thus often regulates dynamic protein interactions. We find that the mouse hair loss mutant, depilated, (dep) is due to a single amino acid deletion in the PAT, Zdhhc21, resulting in protein mislocalization and loss of palmitoylation activity. We examined expression of Zdhhc21 protein in skin and find it restricted to specific hair lineages. Loss of Zdhhc21 function results in delayed hair shaft differentiation, at the site of expression of the gene, but also leads to hyperplasia of the interfollicular epidermis (IFE) and sebaceous glands, distant from the expression site. The specific delay in follicle differentiation is associated with attenuated anagen propagation and is reflected by decreased levels of Lef1, nuclear β-catenin, and Foxn1 in hair shaft progenitors. In the thickened basal compartment of mutant IFE, phospho-ERK and cell proliferation are increased, suggesting increased signaling through EGFR or integrin-related receptors, with a parallel reduction in expression of the key differentiation factor Gata3. We show that the Src-family kinase, Fyn, involved in keratinocyte differentiation, is a direct palmitoylation target of Zdhhc21 and is mislocalized in mutant follicles. This study is the first to demonstrate a key role for palmitoylation in regulating developmental signals in mammalian tissue homeostasis

LGI2 Truncation Causes a Remitting Focal Epilepsy in Dogs

Peer reviewe

Rapid Suppression of Activated Rac1 by Cadherins and Nectins during De Novo Cell-Cell Adhesion

Cell-cell adhesion in simple epithelia involves the engagement of E-cadherin and nectins, and the reorganization of the actin cytoskeleton and membrane dynamics by Rho GTPases, particularly Rac1. However, it remains unclear whether E-cadherin and nectins up-regulate, maintain or suppress Rac1 activity during cell-cell adhesion. Roles for Rho GTPases are complicated by cell spreading and integrin-based adhesions to the extracellular matrix that occur concurrently with cell-cell adhesion, and which also require Rho GTPases. Here, we designed a simple approach to examine Rac1 activity upon cell-cell adhesion by MDCK epithelial cells, without cell spreading or integrin-based adhesion. Upon initiation of cell-cell contact in 3-D cell aggregates, we observed an initial peak of Rac1 activity that rapidly decreased by ∼66% within 5 minutes, and further decreased to a low baseline level after 30 minutes. Inhibition of E-cadherin engagement with DECMA-1 Fab fragments or competitive binding of soluble E-cadherin, or nectin2alpha extracellular domain completely inhibited Rac1 activity. These results indicate that cadherins and nectins cooperate to induce and then rapidly suppress Rac1 activity during initial cell-cell adhesion, which may be important in inhibiting the migratory cell phenotype and allowing the establishment of initially weak cell-cell adhesions

Airway smooth muscle as a target of asthma therapy: history and new directions

Ultimately, asthma is a disease characterized by constriction of airway smooth muscle (ASM). The earliest approach to the treatment of asthma comprised the use of xanthines and anti-cholinergics with the later introduction of anti-histamines and anti-leukotrienes. Agents directed at ion channels on the smooth muscle membrane (Ca(2+ )channel blockers, K(+ )channel openers) have been tried and found to be ineffective. Functional antagonists, which modulate intracellular signalling pathways within the smooth muscle (β-agonists and phosphodiesterase inhibitors), have been used for decades with success, but are not universally effective and patients continue to suffer with exacerbations of asthma using these drugs. During the past several decades, research energies have been directed into developing therapies to treat airway inflammation, but there have been no substantial advances in asthma therapies targeting the ASM. In this manuscript, excitation-contraction coupling in ASM is addressed, highlighting the current treatment of asthma while proposing several new directions that may prove helpful in the management of this disease