No treatment for low-risk thrombocythaemia:results from a prospective study

Essential thrombocythaemia (ET) is a chronic myeloproliferative disorder characterized by the occurrence of thromboembolic episodes, particularly in patients aged > 60 years or with a previous history of thrombosis, and/or by haemorrhages in patients with an exceedingly high platelet count. In these subgroups of patients the use of cytoreductive therapy is beneficial in terms of risk/benefit ratio. Only limited anecdotal data are available on the thrombotic or haemorrhagic risk and survival in young asymptomatic ET patients with a platelet count < 1500 × 10⁹/l. Therefore the optimal management of these patients is unknown.
To assess the incidence of thrombosis and haemorrhages in this group of patients we carried out a prospective observational study in a cohort of 65 patients with ET, aged < 60 years, with no history of thrombosis or haemorrhage and platelet count < 1500 × 10⁹/l, and in 65 age- and sex-matched controls. Patients were not treated with cytoreductive therapy until the occurrence of thrombosis or haemorrhage. Arterial or venous thrombotic events were objectively documented both in cases and in controls.
The median follow-up was 4.1 years, with an incidence of thrombosis in patients and controls of 1.91 and 1.50 cases/100 patient-years, respectively. The age- and sex-adjusted risk rate ratio was 1.43 (95% CI 0.37–5.4). Only three minor haemorrhagic episodes occurred in patients, with an incidence of 1.12 cases/100 patient-years.
Pregnancy and surgery were not associated with thrombosis in these patients.
We conclude that the thrombotic risk in young ET patients, with no thrombotic history and a platelet count < 1500 × 10⁹/l, is not increased compared to the normal population and that a conservative therapeutic approach should therefore be considered in these patients.     

The actin cytoskeleton modulates the activation of iNKT cells by segregating CD1d nanoclusters on antigen-presenting cells

Invariant natural killer T (iNKT) cells recognize endogenous and exogenous lipid antigens presented in the context of CD1d molecules. The ability of iNKT cells to recognize endogenous antigens represents a distinct immune recognition strategy, which underscores the constitutive memory phenotype of iNKT cells and their activation during inflammatory conditions. However, the mechanisms regulating such “tonic” activation of iNKT cells remain unclear. Here, we show that the spatiotemporal distribution of CD1d molecules on the surface of antigen-presenting cells (APCs) modulates activation of iNKT cells. By using superresolution microscopy, we show that CD1d molecules form nanoclusters at the cell surface of APCs, and their size and density are constrained by the actin cytoskeleton. Dual-color single-particle tracking revealed that diffusing CD1d nanoclusters are actively arrested by the actin cytoskeleton, preventing their further coalescence. Formation of larger nanoclusters occurs in the absence of interactions between CD1d cytosolic tail and the actin cytoskeleton and correlates with enhanced iNKT cell activation. Importantly and consistently with iNKT cell activation during inflammatory conditions, exposure of APCs to the Toll-like receptor 7/8 agonist R848 increases nanocluster density and iNKT cell activation. Overall, these results define a previously unidentified mechanism that modulates iNKT cell autoreactivity based on the tight control by the APC cytoskeleton of the sizes and densities of endogenous antigen-loaded CD1d nanoclusters.It is well-established that different populations of T lymphocytes can recognize not only peptides in the context of major histocompatibility complex (MHC) class I (MHCI) and MHCII molecules but also, foreign and self-lipids in association with CD1 proteins (1), antigen-presenting molecules that share structural similarities with MHCI molecules. Of five CD1 isoforms, CD1d restricts the activity of a family of cells known as invariant natural killer T (iNKT) cells because of their semiinvariant T-cell receptor (TCR) use (1). To date, the exogenous glycolipid α-GalactosylCeramide (α-GalCer) represents the best characterized CD1d-restricted agonist for iNKT cells (2). Unlike conventional peptide-specific T cells, iNKT cells react against CD1d⁺ antigen-presenting cells (APCs) in the absence of exogenous antigens, a feature defined as autoreactivity (3). iNKT cell autoreactivity underpins the constitutive memory phenotype of iNKT cells and their ability to be activated during a variety of immune responses from infections to cancer and autoimmunity (1). Some of the endogenous antigens known to elicit iNKT cell autoreactivity belong to glycosphingolipid families, with a mix of α- and β-anomeric configurations (4–7). How iNKT cell autoreactivity is fine-tuned to prevent autoimmunity is subject of much investigation. Previous results have shown that exposure of APCs to Toll-like receptor (TLR) agonists enhances iNKT cell autoreactivity (8, 9), consistent with the proposed mechanism by which ligand availability is regulated by lysosomal glycosidases (4, 6).The recent application of advanced optical techniques (10–13) in combination with substrate patterning and functionalization (14, 15) is providing detailed information on how the lateral organization of a variety of molecules located on both sides of the immunological synapse contributes to controlling T-cell activation. Specifically, single-molecule dynamic approaches and superresolution optical nanoscopy experiments have provided indisputable proof that many receptors on the cell membrane organize in small nanoclusters before ligand activation (16). Membrane nanodomains enriched in cholesterol and sphingolipids (17), protein–protein interactions (18), and interactions between transmembrane proteins and the cytoskeleton (19, 20) have been all implicated in regulating receptor dynamics and nanoclustering. An emerging concept attributes the actin cytoskeleton the ability of imposing barriers or fences on the cell membrane, restricting the lateral mobility of transmembrane proteins (19–21). This transient restriction would, in turn, increase the local concentration of transmembrane proteins, leading to protein nanoclusters. For instance, it has been shown that the actin cytoskeleton promotes the dimerization rate of EGF receptors and facilitates ligand binding and signaling activation (18, 22). Confinement of CD36 has also been observed as a result of its diffusion along linear channels dependent on the integrity of the cortical cytoskeleton (23). This constrained diffusion promotes CD36 clustering, influencing CD36-mediated signaling and internalization. A similar mechanism has been proposed for the maintenance of MHCI clusters on the cell membrane by the actin cytoskeleton, with loss of MHCI clustering resulting in a decreased CD8 T-cell activation (24, 25).Recent confocal microscopy studies have revealed that the association between agonist-loaded CD1d molecules and lipid rafts might contribute to the regulation of iNKT cell activation (26). This elegant study for the first time, to our knowledge, linked the spatial organization of CD1d molecules on the cell membrane of APCs with the activation profile of iNKT cells. However, it remains unclear whether the results of these experiments obtained using mouse cells can be extended to human cells and whether additional insights can be obtained by using higher-resolution microscopy. Indeed, it is not yet known whether surface-expressed CD1d molecules exist as monomers or nanoclusters and whether the actin cytoskeleton might regulate CD1d lateral organization and iNKT cell activation. Interestingly, it has been recently reported that the actin cytoskeleton impairs antigen presentation by CD1d and that disruption of F actin or inhibition of the ρ-associated protein kinase enhances CD1d-mediated antigen presentation (27). These results suggest that the actin cytoskeleton might regulate, in a not yet known manner, antigen presentation by CD1d molecules.Here, we combined dual-color single-molecule dynamic approaches with superresolution optical nanoscopy to characterize for the first time, to our knowledge, the spatiotemporal behavior of CD1d on living human myeloid cells. We find that α-GalCer–loaded human CD1d (hCD1d) molecules are organized in nanoclusters on the cell membrane of APCs. We report that the actin cytoskeleton prevents enhanced hCD1d nanoclustering by hindering physical encountering between hCD1d diffusing nanoclusters, thus reducing basal iNKT cell activation. Furthermore, we observed an increase in nanocluster density on activation of APCs with inflammatory stimuli, such as TLR stimulation, mirroring the increased iNKT cell stimulation. Notably, even during inflammation, the actin cytoskeleton retains an important role to limit hCD1d cluster size and iNKT cell activation. Overall, our results suggest that regulation of CD1d nanoclustering through the actin cytoskeleton represents a previously unidentified mechanism to fine-tune peripheral iNKT cell autoreactivity.     

Clinical usefulness of desmopressin for prevention of surgical bleeding in patients with symptomatic heterozygous factor XI deficiency

Heterozygous factor XI (FXI) deficiency is sometimes associated with a significant bleeding tendency. Fresh frozen plasma or FXI concentrates are the mainstay of treatment in patients with a clear bleeding history, especially prior to surgery. However, these treatments are not completely free of risk. Furthermore, thrombosis has been reported in patients with FXI deficiency infused with FXI concentrate. No data are available on the possible efficacy of desmopressin in these patients.   Two patients with a clear bleeding history associated with FXI deficiency and no additional haemostatic defects agreed to be treated with desmopressin before carpal tunnel surgery and dental extraction. The reduced basal FXI activity and antigen levels slightly increased after infusion, reaching borderline values. No bleeding was observed after surgical procedures.   Desmopressin treatment seems a reasonable and useful choice in symptomatic, heterozygous FXI-deficient patients, thus reducing the cost of treatment, the risk of transmission of blood-borne viruses, and of thrombosis.     

Natural Killer Cell Alloreactivity in Haploidentical Hematopoietic Stem Cell Transplantation

Natural killer (NK) cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented because NK cells coexpress inhibitory receptors (killer cell immunoglobulin-like receptors [KIR]) that recognize groups of (self) major histocompatibility complex class I alleles. Because KIRs are clonally distributed, the NK cell population in any individual are constituted of a repertoire with a variety of class I specificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express the class I alleles that block them. After haploidentical hematopoietic transplantation, NK cell-mediated donor-versus-recipient alloresponses reduce the risk of relapse in acute myeloid leukemia patients while improving engraftment and protecting against graft-versus-host disease. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes, and, in some cases, functional assessment of donor NK clones identify haploidentical donors who are able to mount donor-versus-recipient NK alloreactions.     

Natural killer cells as a therapeutic tool in mismatched transplantation

Natural-killer-cell-mediated, donor-vs-recipient alloresponses occur following transplantation of human-leukocyte-antigen (HLA)-haplotype-mismatched haematopoietic stem cells. Natural killer (NK) cell alloreactivity reduces the risk of relapse in acute myeloid leukaemia patients, while improving engraftment and protecting against graft-vs-host disease. NK cells are primed to kill by several activating receptors. NK cell killing of autologous cells is prevented as NK cells co-express inhibitory receptors (killer cell Ig-like receptors, KIR) that recognize groups of (self) major histocompatibility complex class I alleles. As KIRs are distributed clonally, the NK cell population in any individual constitutes a repertoire with different allospecificities. NK cells in the repertoire mediate alloreactions when the allogeneic targets do not express class I alleles that block them. High-resolution molecular HLA typing of recipient and donor, positive identification of donor KIR genes and, in some cases, functional assessment of donor NK clones will identify haplo-identical donors who are able to mount donor-vs-recipient NK alloreactions.     

Inhibition of platelet function with synthetic peptides designed to be high-affinity antagonists of fibrinogen binding to platelets.

We have constructed synthetic peptides modeled on the sequences of (i) Arg-Gly-Asp, present in fibrinogen, fibronectin, and von Willebrand factor, and of (ii) the fibrinogen gamma chain (gamma 400-411) His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly-Asp-Val. The concentration of each peptide that inhibits 50% of 125I-labeled fibrinogen binding to thrombin-stimulated platelets (IC50) was then determined. The IC50 for (gamma 400-411) was 48-180 microM at a fibrinogen concentration of 60 micrograms/ml. A substitution of arginine for alanine at position 9 decreased the IC50 to 14.5 microM. Arginine substitutions for all other residues on the amino-terminal side of the peptide Arg9-Gly-Asp-Val resulted in an IC50 of 0.4-0.8 microM, and the IC50 of the peptide Arg13-Gly-Asp-Val was 0.2-0.3 microM. This contrasts with an IC50 of 200 microM for Arg5-Gly-Asp-Val-Arg4 and an IC50 greater than 1 mM for the peptide Arg12. The inhibitory effect resulted primarily in a decreased affinity of fibrinogen binding to platelets, although the number of available binding sites had also decreased. Binding was completely inhibited. At concentrations between 10 and 18 microM, Arg9-Gly-Asp-Val blocked all ADP-induced aggregation in citrated platelet-rich plasma. The peptide Tyr-His-His-Lys-Arg-Lys-Arg-Lys-Gln-Arg-Gly-Asp-Val was labeled with 125I to quantitate its binding to thrombin-stimulated platelets; at saturation, 59,990 molecules were bound per cell (Kd = 3.8 X 10(-7) M). These modified synthetic peptides bind to platelets with the same affinity as does intact fibrinogen and inhibit platelet function. The increased affinity of these modified peptides is greater than 20-fold that of peptides comprised of only native sequences and is a prerequisite for the potential antithrombotic use of these agents.     

Tensile destruction test as an estimation of partial proteolysis in fibrin clots

We report a technique devised to evaluate the effects of partial proteolysis on the mechanical characteristics of acellular non-cross-linked fibrin clots. The destruction technique applies coaxial tension on mechanically preconditioned cylindrical molded clots and measures the number of mechanical failures vs the total number of samples at a given load (2, 3, and 4 grams force). We used different plasmin concentrations (0, 0.01, 0.02, 0.04, and 0.08 U/mL) in the bathing medium to cause partial proteolysis. We monitored the fibrinolysis process by measuring the amount of protein released in the bathing medium. Our results showed no difference in the creep function in all the groups studied. We compare our technique with compaction, a commonly used mechanical technique that compresses the sample by centrifugation, and found that our technique is capable of detecting minor changes of fibrinolysis (the results of the least square fit for the destruction test at 2 grams force, as a function of plasmin concentration, has a coefficient of determination of R(2) = 0.55), while compaction did not show a statistically significant difference in the same conditions, suggesting that each individual fibrin fiber bears load only under tension. Our findings suggest that when the fibers are cleaved their capacity to withstand stress is seriously challenged; thus, in principle, tensile destruction test can detect a minimal degree of proteolysis.     

Influence of a commercial tattoo ink on protein production in human fibroblasts

Tattooing is an ancient art and is still widely practiced all over the world. Since the biocompatibility of tattoo dyes has not been well researched, we studied the toxicity of a commercial tattoo ink, commonly used in tattoo lab and esthetic centers, on human fibroblasts. To test cell viability, MTT assays were carried out and scanning electron microscopy to visualize changes in the cell surface after the dye exposure was performed. A possible influence of the pigment on the expression of procollagen α1 type I protein was visualized by western blotting analysis. The results showed a reduction in cell viability, and electron microscopy demonstrated an unmodified cell surface completely covered by pigment particles. Western blotting analysis demonstrated a clear interference of the pigment on the expression of procollagen α1 type I protein. These data demonstrated that the commercial tattoo dye has a time-dependent effect on protein expression. A possible connection of the influence of the tattoo ink with clinical effects is discussed.     

Contribution of distinct adhesive interactions to platelet aggregation in flowing blood.

Aggregation of blood platelets contributes to the arrest of bleeding at sites of vascular injury, but it can occlude atherosclerotic arteries and precipitate diseases such as myocardial infarction. The bonds that link platelets under flow conditions were identified using confocal videomicroscopy in real time. Glycoprotein (GP) Ibalpha and von Willebrand factor (vWF) acted in synergy with alphaIIbbeta3 and fibrinogen to sustain platelet accrual at the apex of thrombi where three-dimensional growth resulted in increasing shear rates. The specific function of distinct adhesion pathways in response to changing hemodynamic conditions helps to explain hemostatic and thrombotic processes.