Viability of SH-SY5Y cells is associated with purinergic P2 receptor expression alterations

To investigate the role of metabotrophic purinergic P2Y receptors in neuroblastoma cell survival, expression of P2 receptors by normal mouse (C57BL/6) brain and human neuroblastoma SH-SY5Y cells was investigated by Western blot and real time PCR studies. Viability of SH-SY5Y cells treated with purinergic receptor antagonists suramin and pyridoxal-phosphate-6-azophenyl-2′,4′-disulfonate (PPADS) was evaluated by MTT assay and flow cytometry. In the brain samples of C57BL/6 mice, expressions of P2Y4 and P2X7 were significantly reduced, whereas that of P2Y1 was significantly elevated in an age-dependent manner. SH-SY5Y cell viability was significantly reduced and necrotic cell rates were mildly increased by 400 μM suramin and 100 μM PPADS treatment. Antagonist treatment downregulated P2Y1, P2Y2 and P2Y4 and upregulated P2Y6, P2Y12 and P2X7 mRNA levels in SH-SY5Y cells on the 24th hour. These alterations were abolished for all P2 receptors except P2Y1 in the 48th hour. P2Y receptors are expressed by both normal mouse brain and human neuroblastoma cells. Purinergic receptor antagonism interferes with neuroblastoma viability through elevation of necrotic cell death and modulation of P2 receptor expression. P2Y receptors might thus be useful targets for future anti-tumor treatment trials

Genetic Association of a Gain-of-Function IFNGR1 Polymorphism and the Intergenic Region LNCAROD/DKK1 With Behcet's Disease

Objective. Behçet’s disease is a complex systemic inflammatory vasculitis of incompletely understood etiology. This study was undertaken to investigate genetic associations with Behçet’s disease in a diverse multiethnic population.Methods. A total of 9,444 patients and controls from 7 different populations were included in this study. Genotyping was performed using an Infinium ImmunoArray- 24 v.1.0 or v.2.0 BeadChip. Analysis of expression data from stimulated monocytes, and epigenetic and chromatin interaction analyses were performed.Results. We identified 2 novel genetic susceptibility loci for Behçet’s disease, including a risk locus in IFNGR1(rs4896243) (odds ratio [OR] 1.25; P = 2.42 × 10−9) and within the intergenic region LNCAROD/DKK1 (rs1660760) (OR 0.78; P = 2.75 × 10−8). The risk variants in IFNGR1 significantly increased IFNGR1 messenger RNA expression in lipopolysaccharide- stimulated monocytes. In addition, our results replicated the association (P 30 genetic susceptibility loci with a suggestive level of association (P < 5 × 10−5), which will require replication. Finally, functional annotation of genetic susceptibility loci in Behçet’s disease revealed their possible regulatory roles and suggested potential causal genes and molecular mechanisms that could be further investigated.Conclusion. We performed the largest genetic association study in Behçet’s disease to date. Our findings reveal novel putative functional variants associated with the disease and replicate and extend the genetic associations in other loci across multiple ancestries

Autoimmune mechanisms in focal epilepsy of unknown cause

The manifestation of immunological findings in diverse disorders presenting with seizures points to autoimmunity and inflammation in the etiology of epilepsy. Typical examples of autoimmunity-associated epilepsy are autoimmune encephalitis, Rasmussen encephalitis, and glutamic acid decarboxylase (GAD) antibody-positive temporal lobe epilepsy (TLE). The last entity is a typical example of antibody-positive focal epilepsy of unknown cause (FEUC). One of the most prominent findings emphasizing the coexistence of epilepsy and autoimmunity is the detection of anti-neuronal antibodies in patients manifesting with seizures. Emergence of antibody-producing plasma cells in the early course of GAD-antibody-positive TLE and induction of seizures in rodents upon intracerebral administration of N-methyl-D-aspartate receptor antibodies indicate that anti-neuronal antibodies may play a causal (rather than bystander) role in the induction of seizures. By contrast, innate immunity of the central nervous system (CNS) and infiltrating cytotoxic T-cells appear to participate in Rasmussen encephalitis and autoimmune encephalitis with antibodies to intracellular antigens. In addition, repetitive seizures may activate glial cells through the release of damage-associate molecular pattern mediators and activation of toll-like receptors, which in turn leads to disrupted blood–brain barrier and increased cerebral infiltration of peripheral blood immune cells. In conclusion, complex interactions of humoral and cellular immunity in the CNS appear to cause or at least contribute to seizure induction in FEUC. The nature of these interactions has recently started to be understood. Investigation of these mechanisms is substantial for the discovery of new treatment strategies and biomarkers in epilepsy

Long Remission in Muscle-Specific Kinase Antibody-Positive Juvenile Myasthenia

The clinical course of anti-muscle specific kinase-positive myasthenia in children has been little reported. Described here is the case of an 8 year-old boy who presented with ptosis and generalized weakness, which resolved within I month without any immunomodulatory treatment. This spontaneous remission lasted 6 years and was then followed by a relapse with bulbar symptoms. The patient was put on plasmapheresis; after initial benefit, symptoms worsened and serum antibody levels persisted. This case constitutes an example of the variability of the clinical presentation, course, and therapeutic response in anti-muscle specific kinase-positive myasthenia in childhood. (C) 2009 by Elsevier Inc. All rights reserved

IL-12 and IL-10 polymorphisms and their effects on cytokine production

Interleukin (IL)-12 is an inducer of differentiation of T helper (Th) cells towards Th1, whereas IL-10 is mainly an anti-inflammatory cytokine inhibiting Th1 functions. Single nucleotide polymorphisms (SNPs) in the regulatory sequences of genes are presumed to be associated with the differential production of cytokines. One IL12B 3′untranslated region (UTR) and five of IL10 gene promoter region SNPs were screened in 152 individuals by genotyping. IL-10 and IL-12 secretion of lipopolysaccharide (LPS), purified protein derivative (PPD) and Staphylococcus aureus Cowan strain I (SAC) stimulated peripheral blood mononuclear cells (PBMCs) was determined. The frequencies of the less frequent IL12B +16974 C, IL10 -2763 A -3575 A, -1082 G, -819 T and -592 A alleles were 27.4, 32.2, 25.9, 14.8, 9.3 and 8.6%, respectively. Individuals CC homozygous at IL12B 3′UTR had significantly higher IL-12 secretion levels from LPS and PPD stimulated PBMCs than AC heterozygotes (P = 0.03 and P = 0.02) or AA homozygotes (P = 0.02 and P = 0.05, respectively). IL10 -2763 and -3575 SNPs did not show any effect on in vitro secretion levels, whereas the association of proximal promoter -1082 SNP with IL-10 production is confirmed. IL10 proximal and distal promoter SNP distribution with estimated haplotype variations has implicated considerable similarities with the Caucasian populations in Turkey. © 2005 Elsevier Ltd. All rights reserved

Immunization with Recombinantly Expressed LRP4 Induces Experimental Autoimmune Myasthenia Gravis in C57BL/6 Mice

Background: Myasthenia gravis (MG) is an autoimmune disease of the neuromuscular junction (NMJ), characterized with muscle weakness. While MG develops due to acetylcholine receptor (AChR) antibodies in most patients, antibodies to muscle-specific receptor tyrosine kinase (MuSK) or low-density lipoprotein receptor-related protein 4 (LRP4) may also be identified. Experimental autoimmune myasthenia gravis (EAMG) has been previously induced by both LRP4 immunization and passive transfer of LRP4 antibodies.Objective: Our aim was to confirm previous results and to test the pathogenic effects of LRP4 immunization in a commonly used mouse strain C57BL/6 (B6) using a recombinantly expressed human LRP4 protein.Methods: B6 mice were immunized with human LRP4 in CFA, Torpedo Californica AChR in CFA or only CFA. Clinical and pathogenic aspects of EAMG were compared among groups.Results: LRP4- and AChR-immunized mice showed comparable EAMG clinical severity. LRP4-immunized mice displayed serum antibodies to LRP4 and NMJ IgG and complement factor C3 deposits. IgG2 was the dominant anti-LRP4 isotype. Cultured lymph node cells of LRP4- and AChR-immunized mice gave identical pro-inflammatory cytokine (IL-6, IFN- and IL-17) responses to LRP4 and AChR stimulation, respectively.Conclusion: Our results confirm the EAMG-inducing action of LRP4 immunization and identify B6 as a LRP4-EAMG-susceptible mouse strain. Demonstration of complement fixing anti-LRP4 antibodies in sera and complement/IgG deposits at the NMJ of LRP4-immunized mice indicates complement activation as a putative pathogenic mechanism. We have thus developed a practical LRP4-induced EAMG model using a non-conformational protein and a widely available mouse strain for future investigation of LRP4-related MG