Determination of Cellular Lipids Bound to Human CD1d Molecules

CD1 molecules are glycoproteins that present lipid antigens at the cell surface for immunological recognition by specialized populations of T lymphocytes. Prior experimental data suggest a wide variety of lipid species can bind to CD1 molecules, but little is known about the characteristics of cellular ligands that are selected for presentation. Here we have molecularly characterized lipids bound to the human CD1d isoform. Ligands were eluted from secreted CD1d molecules and separated by normal phase HPLC, then characterized by mass spectroscopy. A total of 177 lipid species were molecularly identified, comprising glycerophospholipids and sphingolipids. The glycerophospholipids included common diacylglycerol species, reduced forms known as plasmalogens, lyso-phospholipids (monoacyl species), and cardiolipins (tetraacyl species). The sphingolipids included sphingomyelins and glycosylated forms, such as the ganglioside GM3. These results demonstrate that human CD1d molecules bind a surprising diversity of lipid structures within the secretory pathway, including compounds that have been reported to play roles in cancer, autoimmune diseases, lipid signaling, and cell death.     

Platelet thrombus formation in patients with end-stage renal disease before and after hemodialysis as measured by the total thrombus-formation analysis system

Background

Patients with end-stage renal disease (ESRD) receiving hemodialysis (HD) often experience bleeding. However, mechanisms behind this bleeding tendency are incompletely understood but may involve platelet dysfunction. We, therefore, studied platelet-dependent thrombus formation in flowing whole blood inside a microchip coated with collagen, and its association with circulating von Willebrand factor (VWF).

Methods

Blood samples were obtained in 22 patients before and after HD. The area under the 10 min flow pressure curve in a microchip (AUC10) reflecting total platelet thrombogenicity was measured, using the Total Thrombus-formation Analysis System (T-TAS01). AUC10?<?260 indicates platelet dysfunction. VWF activity and antigen in plasma were also assayed.

Results

VWF levels were moderately elevated and increased further after HD (P?<?0.01 or lower). In contrast, AUC10 before and after HD was?<?260 in 17/22 patients and?<?130 in 15/22 patients, with no statistically significant difference in pre- vs post-HD measurements, indicating reduced platelet thrombogenicity, but with some variability as 5/22 patients showed normal platelet responsiveness. AUC10 and VWF activity or antigen levels in plasma were not correlated, either before or after HD.

Conclusions

Most ESRD patients display moderate-to-severe platelet dysfunction as assessed by shear-induced platelet-dependent thrombus formation with T-TAS01. HD does not influence platelet function despite HD-induced elevations in VWF. T-TAS01 should be further evaluated as a tool in the assessment of bleeding risk in patients on HD.

Graphical abstract

     

Cross‐reactivity between tick and wasp venom can contribute to frequent wasp sensitization in patients with the α‐Gal syndrome

Backgroundα‐Gal syndrome (AGS) is a food allergy with severe delayed allergic reactions, mediated by IgE‐reactivity to galactose‐α1,3‐galactose (α‐Gal). AGS is strongly associated with tick bites. An increased incidence of venom sensitization has been found in AGS patients. Here, we evaluated the frequency of wasp sensitization in Swedish AGS patients and the possible cross‐reactivity between wasp venom and tick proteins.MethodsSera from 136 Swedish AGS patients and 29 wasp‐positive non‐AGS control sera were analyzed for IgE‐reactivity against wasp venom (Vespula spp.), the European tick Ixodes ricinus (Streptavidin ImmunoCAP), α‐Gal and total IgE by ImmunoCAP. The presence of α‐Gal on wasp venom proteins (Vespula vulgaris) was investigated by western blot (WB), and possible cross‐reactivity between wasp venom and tick proteins by enzyme‐linked immunosorbent assay and WB. Involvement of cross‐reactive carbohydrate domains (CCDs) was also assessed.ResultsWasp sensitization was present in 54% of AGS patients, although the IgE levels were low. Wasp sensitized patients had higher IgE levels to α‐Gal and total IgE levels compared to non‐wasp sensitized AGS patients. α‐Gal was not detected in wasp venom, but cross‐reactivity between wasp and tick proteins was demonstrated which was not dependent on CCDs. The same cross‐reactivity was also observed in the control sera. Furthermore, 17 putative cross‐reactive peptides were identified using an in silico approach.ConclusionsFor the first time, cross‐reactivity between wasp venom and tick proteins has been described. This may be a reason why the majority of Swedish AGS patients, who have all been tick bitten, are also sensitized against wasp.     

Cytotoxic guaianolide from Anthemis segetalis (Asteraceae)

Two epimeric guaianolides were isolated from the aerial parts of Anthemis segetalis. These compounds have not been detected in any Anthemis species so far. One of them was tested for cytotoxicity against human cervical cancer (HeLa) and murine melanoma (B16) cell lines showing appreciable activity. Copyright © 2009 John Wiley & Sons, Ltd.     

N-Telopeptide of Type II Collagen Interacts with Annexin V on Human Chondrocytes

Type II collagen binds to chondrocytes through integrins and annexin V. While the potential integrin binding sites have been identified, it is unclear which domains bind to annexin V. Proteolytic fragments of collagen are known to modulate cell signaling pathways resulting in degradation of articular cartilage; it is unknown whether annexin V binds to the fragments. The focus of our study was to determine the binding of type II collagen and its fragments to chondrocytes using flow cytometry and fluorescence microscopy. The N-telopeptide binds to annexin V, whereas the C-telopeptide and triple helical peptides do not. These data suggest that the binding of the N-telopeptide of type II collagen is through annexin V, whereas binding of the C-telopeptide and the triple helical peptide to the surface of chondrocytes are potentially facilitated through other collagen receptors, such as integrins or cell-associated matrix proteins.     

Prognostic Value of Coronary Flow Reserve in Asymptomatic Moderate or Severe Aortic Stenosis with Preserved Ejection Fraction and Nonobstructed Coronary Arteries

Aim: Patients with moderate and severe aortic stenosis (AS) and without obstructive epicardial coronary disease have been shown to have an impairment of coronary flow reserve (CFR). We investigated the prognostic significance of CFR in predicting death during mid‐to‐long‐term follow‐up in asymptomatic patients with moderate/severe AS, preserved ejection fraction (EF), and with nonobstructed coronary arteries. Method and Result: A total of 127 patients with moderate or severe AS (effective orifice area of 1.5 cm² or less), mean age 66 ± 11 were enrolled in this prospective study. The median follow‐up was 32 ± 7 months. All patients had standard Doppler echo study, coronary angiography, and adenosine‐stress transthoracic Doppler echo for CFR measurement. Univariate analysis showed that diabetes mellitus, CFR, aortic valve area (AVA), maximal velocity (V_max), mean pressure gradient (P_mean), energy loss index (ELI), aortic valve resistance (AVR), NT‐proBNP, E/E′, valvulo‐arterial impedance (Z_va), and stroke work loss (SWL) were associated (P < 0.05) with death. Multivariable logistic regression analysis revealed that only Z_va and CFR were independent predictors of death, with the CFR being the single strongest predictor (Table 2). Using receiver operating characteristics (ROC) analysis, the CFR value of 1.85 had the highest accuracy in predicting the death during mid‐to‐long‐term follow‐up (area under the curve; AUC 0.890, P = 0.009, sensitivity 96.3%, specificity 75%; 95% CI 0.287–0.946; Fig. 1). The Z_va value of 5.52 Hg/mL per m had a sensitivity 70.0% and specificity 72.0% (AUC 0.766, 95% CI 0.587–0.946; P = 0.005). Conclusion: This study demonstrates that CFR has a prognostic value in patients with asymptomatic moderate or severe AS with preserved EF and nonobstructed coronary arteries.