CARD15/NOD2, CD14 and Toll-like 4 Receptor Gene Polymorphisms in Saudi Patients with Crohn's Disease

Crohn's disease (CD) is a multifactorial disease with a genetic component and an observed association with genes related to the innate immune response. Polymorphisms in the CARD15/NOD2 gene, in addition to functional variants of the toll-like receptor-4 (TLR4) and CD14 genes, have been associated with the development of Crohn's disease. There is no information about the frequency of these polymorphisms in the Saudi population. We examined the frequency of the three major CARD15/NOD2 risk alleles (Leu1007fsinsC, Arg702Trp, and Gly908Arg) and the TLR4 (Thr399Il) polymorphism as well as a functional polymorphism in the promoter of the CD14-159C/T in 46 Saudi CD patients and 50 matched controls. Genotyping was performed by allele-specific PCR or by restriction fragment length polymorphism (PCR-RFLP) analysis. The mutant genotype frequencies of the Leu1007fsinsC, Arg702Trp and Gly908Arg in the patient group were 6.5, 21.7 and 6.5%, respectively, compared with frequencies of 0, 4 and 2%, respectively, in the control group. There were 15 patients who carried the mutant alleles for all three CARD15/NOD2 variants, Leu1007fsinsC, Arg702Trp and Gly908Arg, while none of the control candidates carried the three alleles. This genetic study provides evidence that the three major CARD15/NOD2 variant alleles and the CD14 -159C/T polymorphism are associated with Crohn's disease (CD) susceptibility in the Saudi population; however, there is no evidence that the TLR4 (Thr399Il) or CARD15/NOD2 polymorphisms can be considered risk factors for Crohn's disease.     

Phosphorylation of Histone H2AX in the Mouse Brain from Development to Senescence

Phosphorylation of the histone H2AX (γH2AX form) is an early response to DNA damage and a marker of aging and disease in several cells and tissues outside the nervous system. Little is known about in vivo phosphorylation of H2AX in neurons, although it was suggested that γH2AX is an early marker of neuronal endangerment thus opening the possibility to target it as a neuroprotective strategy. After experimental labeling of DNA-synthesizing cells with 5-bromo-2-deoxyuridine (BrdU), we studied the brain occurrence of γH2AX in developing, postnatal, adult and senescent (2 years) mice by light and electron microscopic immunocytochemistry and Western blotting. Focal and/or diffuse γH2AX immunostaining appears in interkinetic nuclei, mitotic chromosomes, and apoptotic nuclei. Immunoreactivity is mainly associated with neurogenetic areas, i.e., the subventricular zone (SVZ) of telencephalon, the cerebellar cortex, and, albeit to a much lesser extent, the subgranular zone of the hippocampal dentate gyrus. In addition, γH2AX is highly expressed in the adult and senescent cerebral cortex, particularly the piriform cortex. Double labeling experiments demonstrate that γH2AX in neurogenetic brain areas is temporally and functionally related to proliferation and apoptosis of neuronal precursors, i.e., the type C transit amplifying cells (SVZ) and the granule cell precursors (cerebellum). Conversely, γH2AX-immunoreactive cortical neurons incorporating the S phase-label BrdU do not express the proliferation marker phosphorylated histone H3, indicating that these postmitotic cells undergo a significant DNA damage response. Our study paves the way for a better comprehension of the role of H2AX phosphorylation in the normal brain, and offers additional data to design novel strategies for the protection of neuronal precursors and mature neurons in central nervous system (CNS) degenerative diseases.     

Metal-Sulfate Induced Generation of ROS in Human Brain Cells: Detection Using an Isomeric Mixture of 5- and 6-Carboxy-2',7'-Dichlorofluorescein Diacetate (Carboxy-DCFDA) as a Cell Permeant Tracer

Evolution of reactive oxygen species (ROS), generated during the patho-physiological stress of nervous tissue, has been implicated in the etiology of several progressive human neurological disorders including Alzheimer's disease (AD) and amylotrophic lateral sclerosis (ALS). In this brief communication we used mixed isomers of 5-(and-6)-carboxy-2',7'-dichlorofluorescein diacetate (carboxy-DCFDA; C(25)H(14)C(l2)O(9); MW 529.3), a novel fluorescent indicator, to assess ROS generation within human neuronal-glial (HNG) cells in primary co-culture. We introduced pathological stress using the sulfates of 12 environmentally-, industrially- and agriculturally-relevant divalent and trivalent metals including Al, Cd, Cu, Fe, Hg, Ga, Mg, Mn, Ni, Pb, Sn and Zn. In this experimental test system, of all the metal sulfates analyzed, aluminum sulfate showed by far the greatest ability to induce intracellular ROS. These studies indicate the utility of using isomeric mixtures of carboxy-H(2)DCFDA diacetates as novel and highly sensitive, long-lasting, cell-permeant, fluorescein-based tracers for quantifying ROS generation in intact, metabolizing human brain cells, and in analyzing the potential epigenetic contribution of different metal sulfates to ROS-generation and ROS-mediated neurological dysfunction.