The P2X 7 receptor is a ligand-gated cation-selective channel that mediates ATP-induced apoptosis of cells of the immune system. We and others have shown that P2X 7 is nonfunctional both in lymphocytes and monocytes from some subjects. To study a possible genetic basis we sequenced DNA coding for the carboxyl-terminal tail of P2X 7 . In 9 of 45 normal subjects a heterozygous nucleotide substitution (1513A3 C) was found, whereas 1 subject carried the homozygous substitution that codes for glutamic acid to alanine at amino acid position 496. Surface expression of P2X 7 on lymphocytes was not affected by this E496A polymorphism, demonstrated both by confocal microscopy and immunofluorescent staining. Monocytes and lymphocytes from the E496A homozygote subject expressed nonfunctional receptor, whereas heterozygotes showed P2X 7 function that was half that of germline P2X 7 . Results of transfection experiments showed that the mutant P2X 7 receptor was nonfunctional when expressed at low receptor density but regained function at a high receptor density. This density dependence of mutant P2X 7 function was also seen on differentiation of fresh monocytes to macrophages with interferon-␥, which up-regulated mutant P2X 7 and partially restored its function. P2X 7 -mediated apoptosis of lymphocytes was impaired in homozygous mutant P2X 7 compared with germline (8.6 versus 35.2%). The data suggest that the glutamic acid at position 496 is required for optimal assembly of the P2X 7 receptor.Purinergic P2X 7 receptors are ligand-gated cation channels, present on cells of the immune and hemopoietic system, that have been shown to mediate the ATP-induced apoptotic death of monocytes (1), macrophages (2), and lymphocytes (3, 4). The P2X 7 receptor family has two transmembrane domains with intracellular amino and carboxyl termini and an oligomeric structure in the plasma membrane based on trimeric or larger complexes of identical subunits (5). Moreover, the P2X7 receptor does not appear to form heteropolymers with other P2X subtypes (6). The genes for both the rat and human P2X 7 receptors have now been cloned and show extensive homology (30 -40%) with the other members of the P2X receptor family, although P2X 7 differs in having a long carboxyl terminus of 240 amino acids from the inner membrane face (7). The genomic structure of P2X 7 consists of 13 exons, with exon 12 and exon 13 coding for the C-terminal tail of this molecule. There is strong evidence that this long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor, because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye YoPro-1 (8). Studies of P2X 7 of macrophages or lymphocytes as well as of human embryonic kidney cells (HEK-293) expressing the cDNA for P2X 7 have shown features that are most unusual for a channel. These include the slow further dilatation following channel opening (9) and the activation of various proteases including membrane metalloproteases (10) and intracellular caspases (2, 11). The fully ...

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The P2X(7) receptor is an ATP-gated cation channel expressed in immune cells and plays a role in proinflammatory cytokine release from monocytes and macrophages. This study investigated the coinheritance of 12 functionally relevant single nucleotide polymorphisms (SNPs) in the human P2X(7) gene (P2RX7), and the functional effect of each singly and in combination was assessed by measurements of ATP-induced currents and ethidium(+) uptake. Genotyping of 3430 Caucasian subjects identified 4 common haplotypes in addition to the common (wild-type) P2X(7)-1. Two haplotypes (denoted P2X(7)-2 and P2X(7)-4) contained various combinations of gain-of-function SNPs. P2X(7)-4 was identified uniquely by the Gln-460 to Arg polymorphism (rs2230912). When expressed in HEK-293 cells, recombinant P2X(7)-2, and P2X(7)-4 haplotypes displayed a 3-fold and 5-fold increase, respectively, in receptor function compared to the wild-type P2X(7)-1. Both P2X(7) haplotypes contained the Ala-348>Thr polymorphism (rs1718119), and this mutation was critical for the gain-of-function effect. Peripheral blood monocytes and erythrocytes from subjects homozygous for gain-of-function P2X(7) haplotypes exhibited increased ATP-induced ethidium(+) uptake and (86)Rb(+) efflux, respectively, and this correlated with increased IL-1beta secretion from LPS-primed monocytes. Inheritance of these P2X(7) haplotypes predisposing to increased proinflammatory cytokine secretion may be important in genetic association studies of inflammatory, infectious, and psychiatric disorders.

This paper presents an inertial microfluidic device with a simple serpentine micro-channel to continuously separate particles with high performance. Separation of micro/nano-particles has a variety of potential applications in biomedicine and industry. Among the existing separation technologies, a label-free technique without the use of antibody affinity, filter or centrifugation is highly desired to ensure minimal damage and alteration to the cells. Inertial microfluidics utilising hydrodynamic forces to separate particles is one of the most suitable label-free technologies with a high throughput. Our separation concept relies on size-based differential equilibrium positions of the particles perpendicular to the flow. Highly efficient separation is demonstrated with particles of different sizes. The results indicate that the proposed device has an integrative advantage to the existing microfluidic separation techniques, taking accounts of purity, efficiency, parallelizability, footprint, throughput and resolution. Our device is expected to be a good alternative to conventional separation methods for sample preparation and clinical diagnosis.

The P2X 7 receptor is a ligand-gated channel that is highly expressed on mononuclear cells and that mediates ATP-induced apoptosis of these cells. Wide variations in the function of the P2X 7 receptor have been observed, in part because of a loss-of-function polymorphism that changes Glu-496 to Ala without affecting the surface expression of the receptor on lymphocytes. In this study a second polymorphism (Ile-568 to Asn) has been found in heterozygous dosage in three of 85 normal subjects and in three of 45 patients with chronic lymphocytic leukemia. P2X 7 function was measured by ATPinduced fluxes of Rb ؉ , Ba 2؉ , and ethidium ؉ into various lymphocyte subsets and was decreased to values of ϳ25% of normal. The expression of the P2X 7 receptor on lymphocytes was approximately half that of normal values as measured by the binding of fluorescein-conjugated monoclonal antibody. Transfection experiments showed that P2X 7 carrying the Ile-568 to Asn mutation was non-functional because of the failure of cell surface expression. The differentiation of monocytes to macrophages with interferon-␥ up-regulated P2X 7 function in cells heterozygous for the Ile-568 to Asn mutation to a value around 50% of normal. These data identify a second loss-of-function polymorphism within the P2X 7 receptor and show that Ile-568 is critical to the trafficking domain, which we have shown to lie between residues 551 and 581.The purinergic P2X 7 receptor is a ligand-gated channel, selective for cationic permeants, which has a wide distribution including cells of the immune and hemopoietic system (1, 2). Activation of this receptor by brief exposure to extracellular ATP opens a channel that allows Ca 2ϩ and Na ϩ influx and K ϩ efflux and that initiates a cascade of intracellular downstream events. These include the stimulation of phospholipase D (3, 4), the activation of membrane metalloproteases (5-7), and the stimulation of intracellular caspases, which eventually lead to the apoptotic death of the target cell (8, 9). P2X 7 activation also leads to extensive membrane blebbing (10), which is a typical morphological feature of the apoptotic process. P2X 7 receptors have two transmembrane domains with intracellular amino and carboxyl termini, and the P2X 7 receptor differs from other members of the P2X receptor family in having a long carboxyl terminus of 240 amino acids from the inner membrane face (11). This long carboxyl terminus is necessary for the permeability properties of the P2X 7 receptor because truncation of this tail abolishes ATP-induced uptake of the fluorescent dye Yo-Pro-1 (12). P2X 7 has an oligomeric structure in the membrane based on trimeric or larger complexes of identical subunits (13,14), and there is evidence that P2X 7 interacts with a number of structural and adhesion proteins in a complex at the cell surface (15). Phosphorylation of a tyrosine at amino acid 343 of the P2X 7 primary structure has been proposed as being important for maintaining the full activity of the P2X 7 channel (15). A number of regulatory d...

The 1513C allele increases susceptibility to extrapulmonary TB, and this defect is associated with the reduction in the capacity of macrophages to kill M. tuberculosis.

The P2X(7) receptor is a ligand-gated cation channel that is highly expressed on mononuclear leukocytes and that mediates ATP-induced apoptosis and killing of intracellular pathogens. There is a wide variation in P2X(7) receptor function between subjects, explained in part by four loss-of-function polymorphisms (R307Q, E496A, I568N, and a 5'-intronic splice site polymorphism), as well as rare mutations. In this study, we report the allele frequencies of 11 non-synonymous P2X(7) polymorphisms and describe a fifth loss-of-function polymorphism in the gene (1096C --> G), which changes Thr(357) to Ser (T357S) with an allele frequency of 0.08 in the Caucasian population. P2X(7) function was measured by ATP-induced ethidium(+) influx into peripheral blood lymphocytes and monocytes and, when compared with wild-type subjects, was reduced to 10-65% in heterozygotes, 1-18% in homozygotes, and 0-10% in compound heterozygotes carrying T357S and a second loss-of-function polymorphism. Overexpression of the T357S mutant P2X(7) in either HEK-293 cells or Xenopus oocytes gave P2X(7) function of approximately 50% that of wild-type constructs. Differentiation of monocytes to macrophages, which also up-regulates P2X(7), restored P2X(7) function to near normal in cells heterozygous for T357S and to a value 50-65% of wild-type in cells homozygous for T357S or compound heterozygous for T357S/E496A. However, macrophages from subjects that are compound heterozygous for either T357S/R307Q or T357S/stop codon had near-to-absent P2X(7) function. These functional deficits induced by T357S were paralleled by impaired ATP-induced apoptosis and mycobacteria killing in macrophages from these subjects. Lymphocytes, monocytes, and macrophages from subjects homozygous for T357S or compound heterozygous for T357S and a second loss-of-function allele have reduced or absent P2X(7) receptor function.

The human P2X7 receptor is a two-transmembrane ionotropic receptor which has a ubiquitous distribution and is most highly expressed on immune cells. In macrophages and similar myeloid cells primed by lipopolysaccharide (LPS), activation of P2X7 by extracellular ATP opens a cation channel/pore allowing massive K+ efflux associated with processing and secretion of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. A variety of other downstream effects follows P2X7 activation over several minutes including shedding of certain surface molecules, membrane blebbing, microvesicle/exosome release and apoptosis of the cell. High concentrations of ATP (>100 µM) are required to activate P2X7 but it remains unclear where these levels exist, other than in inflammatory foci or confined spaces such as in bone. A variety of potent selective antagonists of P2X7 activation have recently become available, allowing clinical trials to be undertaken in inflammatory and immune-mediated disorders. Proteomic studies have shown that P2X7 exists as a large multiprotein complex which includes non-muscle myosin heavy chain and other elements of the cytoskeleton. In the absence of its ATP ligand and serum, P2X7 has an alternate function in the recognition and phagocytosis of non-opsonized foreign particles, including bacteria and apoptotic cells. The P2RX7 gene has many polymorphic variants and isoforms which increase or decrease function of the receptor. Genetic association studies have linked loss-of-function polymorphisms with reactivation of latent tuberculosis as well as symptomatic infection with certain other obligate intracellular pathogens. The many roles involving P2X7 suggest that this receptor is essential to fundamental aspects of the innate immune response.