No abstract

The nucleotide receptor P2X7 is expressed by most leukocytes and initiates signaling events that amplify numerous LPS responses. We tested the hypothesis that loss-of-function polymorphisms in the human P2X7 gene predispose to the production of an anti-inflammatory mediator balance. Accordingly, we developed a novel P2X7 pore assay in whole blood that magnifies the activity from wild-type alleles and preserves the gene dosage effect for the 1513 C polymorphism (AA, 69 ± 4; AC, 42 ± 4; and CC, 6 ± 1-fold stimulation). Thirty of 200 healthy individuals were identified as having low P2X7 pore activity. Seven low pore subjects were 1513 CC, 3 and 11 participants had the other known variants 946 GA and 1729 TA respectively; the remaining 9 volunteers likely have novel polymorphisms. Because platelets are a large source of extracellular ATP during inflammation, whole blood was treated ex vivo with Salmonella typhimurium LPS in the absence of exogenous nucleotides. LPS-stimulated whole blood from individuals in the low pore activity group generated reduced plasma levels of TNF-α (p = 0.036) and higher amounts of IL-10 (p < 0.001) relative to the high pore controls. This reduction in the TNF-α to IL-10 ratio persisted to at least 24 h and is further decreased by cotreatment with 2-methylthio-ATP. The ability of P2X7 polymorphisms to regulate the LPS-induced TNF-α to IL-10 ratio suggests that 15% of healthy adults may exhibit anti-inflammatory mediator responses during major infectious perturbations of the immune system, which can be predicted by P2X7 pore activity.

No objective antitumor activity was detected with single agent vorinostat in this setting; however, it yields TTP in relapsed NSCLC similar to that of other targeted agents. Further studies in NSCLC should focus on combining vorinostat with other antitumor agents.

Background: Innate immune response amplification is achieved by leukocyte expression of the purinergic nucleotide receptor P2X 7 , an extracellular nucleotidegated pore. Previously, low P2X 7 pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharidestimulated tumor necrosis factor-␣ to interleukin-10, of relevance to a variety of infectious and inflammatory disorders. We hypothesized that evaluation of participants with discordance between the P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype. Methods: Comparison of whole-blood pore results with restriction fragment length polymorphism data for known loss-of-function genotypes from 200 healthy participants optimized the diagnostic threshold for low pore activity by ROC curve analysis. We identified novel alleles and inferred haplotypes by sequencing outlier genomic templates and by linkage analysis. Results: With a refined threshold of low activity, a normal pore result had only a 2% probability of association with known loss-of-function variants. By contrast, the positive predictive value of low pore activity was 59% for identifying known alleles. DNA samples from this discordant group contained 28 P2X7 sequence variations. Linkage analysis demonstrated that A1513C, T1729A, and G946A are inherited independently from one another, although these loss-of-function variants are

The purpose of this study was to determine the role of the CD11b-dependent respiratory burst in neutrophil oxidant generation and activation, interleukin-8 (IL-8) production, and myofiber damage after muscle stretch injury by using the monoclonal antibody M1/70 to block this pathway. Twelve male New Zealand White rabbits were randomly assigned to a treatment group: M1/70 (n = 6), IgG isotype control (n = 3), or saline control (n = 3). After intravenous injection of the assigned agent under gas anesthesia, a standardized single-stretch injury was created in the right tibialis anterior, whereas the left tibialis anterior underwent a sham surgery. Blood-borne neutrophil oxidant generation and CD11b receptor density and plasma IL-8 levels were measured pre- and 24 h postinjury. Damage was assessed histologically at the hematoma site by counting torn myofibers. M1/70 group demonstrated decreased blood-borne neutrophil oxidant generation (P < 0.05) and CD11b receptor density (P < 0.05), an increase in plasma IL-8 concentration (P < 0.01), and less torn myofibers (P < 0.01) compared with IgG isotype or saline control groups. These data indicate that 1). CD11b-dependent respiratory burst is a major source of oxidants produced by the neutrophil, and that treatment with M1/70 2). attenuates neutrophil activation status, 3). increases plasma IL-8 concentration, and 4). minimizes myofiber damage 24 h postmuscle stretch injury.

Rationale: Upper respiratory tract infection is a guideline accepted risk domain for the loss of asthma control. The ionotrophic nucleotide receptor P2X 7 regulates compartmentalized acute inflammation and the immune response to airway pathogens. Objectives: We hypothesized that variability in P2X 7 function contributes to neutrophilic airway inflammation during a cold and thereby is linked to acute asthma. Methods: Research volunteers with asthma were enrolled at the onset of a naturally occurring cold and monitored through convalescence, assessing symptoms, lung function, and airway inflammation. P2X 7 pore activity in whole blood samples was measured using a genomically validated flow cytometric assay. Measurements and Main Results: Thirty-five participants with mild to moderate allergic asthma were enrolled and 31 completed all visits. P2X 7 pore function correlated with the change in nasal lavage neutrophil counts during the cold (R s 5 0.514, P 5 0.004) and was inversely related to the change in asthma symptoms (R s 5 -0.486, P 5 0.009). The change in peak expiratory flow recordings, precold use of inhaled corticosteroids, and P2X 7 pore function were multivariate predictors of asthma symptoms (P 5 0.001, , 0.001 and 5 0.003 respectively). Attenuated P2X 7 activity was associated with the risk of losing asthma control (crude odds ratio, 11.0; 95% confidence interval, 1.1-106.4) even after adjustment for inhaled corticosteroids and rhinovirus (odds ratio, 15.0). Conclusions: A whole blood P2X 7 pore assay robustly identifies participants with loss-of-function genotypes. Using this assay as an epidemiologic tool, attenuated P2X 7 pore activity may be a novel biomarker of virus-induced loss of asthma control.

Flow cytometry was evaluated as a method of assessing in vitro the effects of leukocytes on blood-stage Plasmodium falciparum. Hydroethidine is converted by metabolizing cells to ethidium, a nucleic acid fluorochrome. After incubation with hydroethidine, viable and dead leukocytes and parasitized and uninfected erythrocytes could all be identified on the basis of fluorescence intensity and size. Leukocytes can therefore be eliminated from further analysis; this allows assessment, at any parasite developmental stage, of the level of parasitemia within erythrocytes in the presence of any of several types of leukocytes. Whether leukocytes actually kill intraerythrocytic parasites can therefore be determined and the level of cytotoxicity can be assessed. The ability of leukocytes to prevent merozoites from invading new erythrocytes, i.e., inhibition of parasite invasion, can also be assessed by this method. When erythrocytes containing schizont-stage parasites were cocultured with different leukocyte populations and the level of parasitemia was determined after merozoite release and invasion, only cultures containing ␥␦ T cells inhibited parasite invasion. The different blood-stage forms of the parasite vary in nucleic acid content, which allows each of the developmental stages to be distinguished by flow cytometry; this permits assessment of changes in parasite development in the presence of leukocytes. Monocyte-derived macrophages (MDMs) appeared to have an effect on parasite development. In this instance, when erythrocytes containing ring-form parasites were cocultured with MDMs and harvested 24 h later, the parasites in cultures containing MDMs were at the late schizont stage, whereas parasites in control cultures were early trophozoites; this finding suggests that MDMs accelerate parasite development. Together, these results indicate that flow cytometry is potentially useful for measuring the following effects mediated by leukocytes: (i) level of cytotoxicity, (ii) changes in parasite development, and (iii) inhibition of parasite invasion.

Okadaic acid (OA), a toxin from the black sponge Halicondria okadai, is a specific inhibitor of serine/threonine protein phosphatases 1 (PP1) and 2A (PP2A). OA is a tumor promoter but also induces apoptosis in some tumor cell lines. In this study, we determined whether ras mutation and/or p53 status are characteristics associated with the cell's sensitivity to the induction of apoptosis by OA. Several cell lines that differed in ras and p53 mutations were treated with OA (10-100 nM). At 24 to 48 h after treatment, the percentage of cells undergoing apoptosis was quantitated. The cell lines with mutations in either H-ras (human bladder carcinoma cell line T24 and mouse keratinocyte cell line 308), or K-ras (human colon carcinoma cell lines DLD-1 and HCT116; human prostate cancer cell lines LNCaP and PC-3; human lung cancer cell lines Calu-6 and SKLU-1; and human pancreatic cancer cell line MIAPaCa2) were more sensitive to OA-induced apoptosis (3- to 10-fold) than the cell lines that lacked the ras mutation (mouse epidermal cell lines C50 and JB6; murine fibroblast cell line NIH3T3; human colon cancer cell line HT29; human kidney epithelial cell line Hs715.K; and human pancreatic cancer cell line Bx-PC3). Similarly, using isogenic cell lines we found that overexpression of mutated H-ras in NIH3T3 and in SV40 immortalized human uroepithelial cells (SVHUC) enhanced their sensitivity to undergo apoptosis in response to OA treatment. The T24, DLD-1, SKLU-1, Calu-6, and MIAPaCa2 cell lines express mutated p53. The SVHUC as well as their ras-transfected counterparts have inactive p53 due to complex formation between large "T" antigen and p53. Taken together, these results imply that OA-induced apoptosis may involve a p53-independent pathway. The transfectants (NIH3T3-ras and SVHUC-ras), which express mutated H-ras, have up-regulated PP2A activity. OA treatment inhibited in vivo the levels of PP1 and PP2A activity, and induced apoptosis in SVHUC-ras and other cell lines. We conclude that OA-induced cell death pathway in ras-activated cell lines may involve a cross talk between PP1 and PP2A and ras signaling pathways. In light of the present results, the current theory that OA promotes mouse skin tumor formation by selective expansion of initiated cells that harbor ras mutations needs reevaluation.