The function of GPI-anchored proteins in T cell development, activation and regulation of homeostasis

Many glycosylphosphatidyl-inositol-anchored proteins (GPI-AP) are expressed on T lymphocytes. Ligand or mAb-mediated aggregation of all GPI-AP tested to date results in the initiation of signal transduction pathways via the activation of src family protein tyrosine kinases. Src family kinases co-localise with GPI-AP in specialised sub-domains of the plasma membrane, referred to as detergent insoluble membrane microdomains (DIGS), which are thought to function as signalling platforms. GPI-AP may play a role in the regulation of T cell clonal expansion and effector functions at multiple levels, including the initiation of T cell activation through the antigen receptor complex, the regulation of ongoing responses supported by persisting antigen, as well as proliferative responses to the major T cell growth factor, IL-2. Evidence supporting the role of GPI-AP in the regulation of T cell development, activation and homeostasis is discussed, as well as insights provided by studies in humans and mice lacking GPI-AP.     

Potassium-Induced Stimulation of Glutamate Uptake in Mouse Cerebral Astrocytes: The Role of Intracellular pH

Abstract: The Na⁺-glutamate cotransporters are believed to countertransport OH^? and K⁺. Previous evidence that the velocity of glutamate uptake can exceed the acid extrusion capacity of astrocytes raised the question of whether intracellular pH can become rate limiting for glutamate uptake. Cytoplasmic buffering capacity and acid extrusion in astrocytes are partially HCO₃^? dependent. Also, it was reported recently that raising extracellular [K⁺] alkalinizes astrocyte cytoplasm by an HCO₃^?-dependent mechanism. Here, we have compared glutamate uptake in HCO₃^?-buffered and HCO₃^?-depleted solutions at varying [K⁺]. We observed a pronounced stimulation of glutamate uptake by extracellular K⁺ (3–24 mM) that was substantially HCO₃^? dependent and affected preferentially the uptake of high concentrations (>25 µM) of glutamate. Stimulation of uptake by low extracellular [K⁺] (1.5–3 mM) was less dependent on HCO₃^?. Potassium-induced stimulation of uptake was weaker in rat astrocyte cultures than in mouse. The effects of Ba²⁺ and amiloride on glutamate uptake, as well as the HCO₃^?-dependent stimulatory effects of K⁺ and the species difference, all related consistently to effects on intracellular pH. The effects on uptake, however, were much larger than predicted by the associated changes in electrochemical gradient of OH^?. A “bimodal” scheme for glutamate transport can account qualitatively for the observed correlation between intracellular pH and velocity of glutamate uptake.     

Susceptibility of apple clearwing moth larvae,Synanthedon myopaeformis (Lepidoptera: Sesiidae) to Beauveria bassiana and Metarhizium brunneum

Apple clearwing moth larvae, Synanthedon myopaeformis (Lepidoptera: Sesiidae) were found to be susceptible to infection by two entomopathogenic fungi: an indigenous fungus isolated from S. myopaeformis cadavers and identified as Metarhizium brunneum (Petch); and Beauveria bassiana isolate GHA. In laboratory bioassays, larvae exhibited dose related mortality after exposure to both the M. brunneum and Beauveria bassiana with 7 day LC₅₀'s of 2.9×10⁵ and 3.4×10⁵ spores/mL, respectively. Larval mortalities caused by the two isolates at 1×10⁶ spores/mL were not significantly different and 73% of the M. brunneum-treated, and 76% of the B. bassiana-treated larvae were dead 7 days post treatment, with LT₅₀'s of 5.5 and 5.1 days, respectively.     

Investigation into the role of phosphatidylserine in modifying the susceptibility of human lymphocytes to secretory phospholipase A(2) using cells deficient in the expression of scramblase

Normal human lymphocytes resisted the hydrolytic action of secretory phospholipase A(2) but became susceptible to the enzyme following treatment with a calcium ionophore, ionomycin. To test the hypothesis that this susceptibility requires exposure of the anionic lipid phosphatidylserine on the external face of the cell membrane, experiments were repeated with a human Burkitt's lymphoma cell line (Raji cells). In contrast to normal lymphocytes or S49 mouse lymphoma cells, most of the Raji cells (83%) did not translocate phosphatidylserine to the cell surface upon treatment with ionomycin. Those few that did display exposed phosphatidylserine were hydrolyzed immediately upon addition of phospholipase A(2). Interestingly, the remaining cells were also completely susceptible to the enzyme but were hydrolyzed at a slower rate and after a latency of about 100s. In contradistinction to the defect in phosphatidylserine translocation, Raji cells did display other physical membrane changes upon ionomycin treatment that may be relevant to hydrolysis by phospholipase A(2). These changes were detected by merocyanine 540 and trimethylammonium diphenylhexatriene fluorescence and were common among normal lymphocytes, S49 cells, and Raji cells. The levels of these latter effects corresponded well with the relative rates of hydrolysis among the three cell lines. These results suggested that while phosphatidylserine enhances the rate of cell membrane hydrolysis by secretory phospholipase A(2), it is not an absolute requirement. Other physical properties such as membrane order contribute to the level of membrane susceptibility to the enzyme independent of phosphatidylserine.     

Associations of Fibroblast Growth Factor-23 with Markers of Inflammation,Insulin Resistance and Obesity in Adults

Introduction

Elevated fibroblast growth factor-23 (FGF23) is an established marker of cardiovascular disease. The underlying reason(s) for the rise accompanying cardiovascular health decline are unclear. Prior studies have shown that FGF23 concentrations are associated with markers of inflammation and insulin resistance but they have been limited by a focus on persons with chronic kidney disease (CKD) and lack of race and sex diversity. The objective of this study was to examine the associations of FGF23 and markers of inflammation, insulin resistance, and anthropometrics in a large cohort of community-dwelling adults.

Methods

Associations of FGF23 with markers of inflammation [interleukin-6 (IL-6), IL-10, high sensitivity-CRP (hsCRP)], insulin utilization [resistin, adiponectin, homeostatic model assessment of insulin resistance (HOMA-IR)] and anthropometrics [BMI and waist circumference (WC)] were examined cross-sectionally in a 1,040 participants randomly selected from the Reason for Geographic and Racial Differences in Stroke (REGARDS) Study, a national study of black and white adults ≥45 years. Effect modification by race and CKD status was tested, and stratified models were analyzed accordingly.

Results

Median FGF23 concentration was 69.6 RU/ml (IQR: 53.2, 102.7). Higher quartiles of FGF23 were associated with higher mean concentrations of IL-6, IL-10, hsCRP and resistin (P _trend<0.001 for all). There were no significant differences in HOMA-IR, adiponectin concentrations, BMI, or WC across FGF23 quartiles in the crude analyses. CKD significantly modified the relationships between FGF23 and inflammatory markers, HOMA-IR, BMI and WC (P ≤ 0.01 for all). In linear regression models adjusted for sociodemographic and clinical variables, FGF23 was positively associated with IL-6, hsCRP, IL-10, HOMA-IR, BMI and WC in individuals without CKD, but not among individuals with CKD. Additionally, FGF23 was positively associated with resistin irrespective of CKD status.

Conclusions

Elevated FGF23 concentrations may be considered a biomarker for decline in metabolic function among individuals with normal kidney function.     

A case of mistaken identity,Opuntia abjecta,long-lost in synonymy under the Caribbean species,O. triacantha,and a reassessment of the enigmatic O. cubensis

Opuntia abjecta and O. militaris have been placed in synonymy under the Caribbean species O. triacantha for the past 30 years. Recent molecular phylogenetic evidence has shown, however, that O. abjecta and O. triacantha are actually in two very different clades suggesting that the Floridian endemic O. abjecta should be recognized as a distinct species. Here, we summarize major morphological differences between O. abjecta and O. triacantha. We also include new sequence data from the rare Cuban taxon, O. militaris, in a molecular phylogenetic analysis to determine its relationship to O. triacantha and O. abjecta. We discuss the putative hybrid taxa O. cubensis and O. ochrocentra, which currently are treated as synonyms. We also show through analysis of morphological and molecular characters that these two taxa were derived from two independent origins from divergent maternal progenitors, confirming that O. ochrocentra should not be treated as synonymous with O. cubensis. A key is provided for identifying these taxonomically confusing taxa and their close relatives. This study emphasizes the distinctions among O. abjecta, O. militaris, and O. triacantha and illustrates that extreme caution must be employed when using herbarium specimens for identifying species of Opuntia. It also indicates that broad phytogeographic assumptions regarding species’ relationships in Opuntia may sometimes be misleading. Hybridization and polyploidy are common in Opuntia and have played a role in the formation of new species in this group as well. A neotype is here designated for O. triacantha.     

Diagnostic accuracy of the TIMI risk score in patients with chest pain in the emergency department: a meta-analysis

Background

The Thrombolysis in Myocardial Infarction (TIMI) risk score uses clinical data to predict the short-term risk of acute myocardial infarction, coronary revascularization or death from any cause. It was originally developed for use in patients with unstable angina or non–ST-elevation myocardial infarction. We sought to expand the clinical application of the TIMI risk score by assessing its prognostic accuracy in patients in the emergency department with potential acute coronary syndromes.

Methods

We searched five electronic databases, hand-searched reference lists of included studies and contacted content experts to identify articles for review. We included prospective cohort studies that validated the TIMI risk score in emergency department patients. We performed a meta-regression to determine whether a linear relation exists between TIMI risk score and the cumulative incidence of cardiac events.

Results

We included 10 prospective cohort studies (with a total of 17 265 patients) in our systematic review. Data were available for meta-analysis in 8 of the 10 studies. Of patients with a score of zero, 1.8% had a cardiac event within 30 days (sensitivity 97.2%, 95% CI 96.4–97.8; specificity 25.0%, 95% CI 24.3–25.7; positive likelihood ratio 1.30, 95% CI 1.28–1.31; negative likelihood ratio 0.11, 95% CI 0.09–0.15). Meta-regression analysis revealed a strong linear relation between TIMI risk score (p < 0.001) and the cumulative incidence of cardiac events.

Interpretation

Although the TIMI risk score is an effective risk stratification tool for patients in the emergency department with potential acute coronary syndromes, it should not be used as the sole means of determining patient disposition.Chest pain is a common presenting complaint in the emergency department that requires efficient risk stratification, timely initiation of treatment in high-risk patients and safe determination of patient disposition. Several studies have been published that stratify the risk of patients in the emergency department with chest pain.^1–5 However, only the Thrombolysis in Myocardial Infarction (TIMI) risk score, which was initially developed for use in patients with unstable angina or non–ST-segment elevation myocardial infarction or both,⁶ has been broadly validated in several independent emergency department populations with chest pain and thus constitutes the highest level of evidence available.The TIMI risk score assigns each of seven predictors a value of one point, allowing stratification of patients into one of eight prognostic categories (Box 1).⁶ The clinical end points are acute myocardial infarction, coronary revascularization and death from any cause.

Box 1.?Predictor variables included in the TIMI risk score^*

• Age of more than 65 years
• Three or more risk factors for atherosclerosis
• Known coronary artery disease
• Two or more episodes of anginal chest pain in the preceding 24 hours
• Acetylsalicylic acid use in the seven days before hospitalization
• ST-segment deviation of 0.05 mV or more
• Elevated cardiac markers

A robust estimate of the performance of the TIMI risk score obtained from a systematic review may prove useful to both clinicians and researchers. Clinicians would have a reliable quantitative estimate of a patient’s short-term risk of a cardiac event. This could be used as an adjunct to clinical acumen and as a tool to communicate risk to patients in a shared decision-making model of care.⁷ Researchers would also have an estimate of the prognostic accuracy of the TIMI risk score derived from different practice settings and patient populations that represent a wide variety of ethnic backgrounds. This estimate may serve as a useful baseline for comparison as emerging clinical prediction rules and imaging modalities continue to refine our approach to diagnosis and risk stratification in patients in the emergency department with potential acute coronary syndromes.We conducted a comprehensive systematic review and meta-analysis to assess the methodological quality and prognostic performance of studies that had prospectively validated the TIMI risk score in patients in the emergency department.     

Allergic airways disease develops after an increase in allergen capture and processing in the airway mucosa

Airway mucosal dendritic cells (AMDC) and other airway APCs continuously sample inhaled Ags and regulate the nature of any resulting T cell-mediated immune response. Although immunity develops to harmful pathogens, tolerance arises to nonpathogenic Ags in healthy individuals. This homeostasis is thought to be disrupted in allergic respiratory disorders such as allergic asthma, such that a potentially damaging Th2-biased, CD4(+) T cell-mediated inflammatory response develops against intrinsically nonpathogenic allergens. Using a mouse model of experimental allergic airways disease (EAAD), we have investigated the functional changes occurring in AMDC and other airway APC populations during disease onset. Onset of EAAD was characterized by early and transient activation of airway CD4(+) T cells coinciding with up-regulation of CD40 expression exclusively on CD11b(-) AMDC. Concurrent enhanced allergen uptake and processing occurred within all airway APC populations, including B cells, macrophages, and both CD11b(+) and CD11b(-) AMDC subsets. Immune serum transfer into naive animals recapitulated the enhanced allergen uptake observed in airway APC populations and mediated activation of naive allergen-specific, airway CD4(+) T cells following inhaled allergen challenge. These data suggest that the onset of EAAD is initiated by enhanced allergen capture and processing by a number of airway APC populations and that allergen-specific Igs play a role in the conversion of normally quiescent AMDC subsets into those capable of inducing airway CD4(+) T cell activation.     

Molecular phylogeny, biogeography, and systematics of Dicerandra (Lamiaceae), a genus endemic to the southeastern United States

Dicerandra, an endemic mint of the southeastern United States, comprises nine species, all of which are threatened or endangered and restricted to sandhill vegetation and a mosaic of scrub habitats. Molecular phylogenetic analyses of Dicerandra based on data from the nuclear and plastid genomes for all 13 taxa of the genus, identified two strongly supported clades, corresponding to the four annual and to the five perennial species of Dicerandra. However, the nuclear and plastid trees were incongruent in their placement of two perennial taxa, D. cornutissima and D. immaculata var. savannarum, perhaps due to ancient hybridization or to lineage sorting. Based on these analyses, the widespread D. linearifolia is not monophyletic, with populations of D. linearifolia var. linearifolia falling into either western or eastern clades. The western clade, comprising populations of D. linearifolia var. linearifolia and var. robustior, occurs in an area drained by rivers flowing toward the Gulf of Mexico, whereas the eastern clade, comprising populations of D. linearifolia var. linearifolia, D. densiflora, D. odoratissima, and D. radfordiana (i.e., all the annual species), occupies a region drained by rivers flowing to the Atlantic Ocean. Although this pattern of genetic differentiation between populations from these two river drainages has been documented in several animal species, it has not previously been reported for plants. A revised subgeneric classification is presented to reflect the annual and perennial clades.     

Modification of platelet proteins by malondialdehyde: prevention by dicarbonyl scavengers

The thromboxane synthase converts prostaglandin H₂ to thromboxane A₂ and malondialdehyde (MDA) in approximately equimolar amounts. A reactive dicarbonyl, MDA forms covalent adducts of amino groups, including the ε-amine of lysine, but the importance of this reaction in platelets was unknown. Utilizing a novel LC/MS/MS method for analysis of one of the MDA adducts, the dilysyl-MDA cross-link, we demonstrated that dilysyl-MDA cross-links in human platelets are formed following platelet activation via the cyclooxygenase (COX)-1/thromboxane synthase pathway. Salicylamine and analogs of salicylamine were shown to react with MDA preferentially, thereby preventing formation of lysine adducts. Dilysyl-MDA cross-links were measured in two diseases known to be associated with increased platelet activation. Levels of platelet dilysyl-MDA cross-links were increased by 2-fold in metabolic syndrome relative to healthy subjects, and by 1.9-fold in sickle cell disease (SCD). In patients with SCD, the reduction of platelet dilysyl-MDA cross-links following administration of nonsteroidal anti-inflammatory drug provided evidence that MDA modifications of platelet proteins in this disease are derived from the COX pathway. In summary, MDA adducts of platelet proteins that cross-link lysines are formed on platelet activation and are increased in diseases associated with platelet activation. These protein modifications can be prevented by salicylamine-related scavengers.