Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release

Ploidy could be the key to understanding megakaryocyte (MK) biologyand platelet production. Human CD34⁺ cells purified fromumbilical cord blood (CB) and peripheral blood (PB) were investigatedon their capability to give rise, in a serum-free medium containingthrombopoietin, to MKs and platelets. CB-MKs showed reducedpolyploidization and platelet number compared with PB-MKs, but asimilar membrane phenotype. Most CB-MKs showed a 2N content of DNA(~80%) and only 2.6% had 8N, whereas 40% of the PB cells had 8N ormore. Platelets were substantially released in PB culture from day 12;at day 14 the CB-derived MKs were able to release platelets although ata reduced level (~35%), correlating with their reduced size. Adirect correlation was demonstrated by sorting polyploid cells fromPB-MKs and evaluating the platelets released in the supernatant.Furthermore, the study analyzed the expression and distribution ofcyclin D3 and cyclin B1. Cyclin D3 protein was increased in PB incomparison to CB-MKs; in PB culture most cells rapidly became positive,whereas in CB-derived cells cyclin D3 expression was evident only fromday 9 and in a reduced percentage. Cyclin B1 was essentially localizedat the nuclear level in the CB and was expressed during the wholeculture. In PB-MKs, at day 9, a reduction was observed, correlatingwith an advanced ploidy state. The data indicate the inability of the CB-MKs to progress in the endomitotic process and a direct correlation between DNA content and platelet production.     

Secondary prevention of venous thromboembolism: A role for low-molecular-weight heparin.

BACKGROUND: After a short initial course of heparin therapy, patients with venous thrombo-embolism (VTE) require continuing anticoagulant therapy for several months after hospital discharge. At present, two small-scale studies have compared the efficacy and safety of low-molecular-weight heparin (LMWH) with warfarin in the secondary prevention of VTE. PATIENTS AND METHODS: We studied 654 consecutive patients, 202 with pulmonary embolism (PE) and 452 patients with deep vein thrombosis (DVT) of the lower limbs. 220/654 patients (34%) were considered to have some contraindications to coumarin, and were discharged on LMWH (dalteparin, Fragmin((R)), 10, 000 IU s.c. once daily). The remaining 434/654 patients were asked to choose between either coumarin or LMWH: 190 patients preferred LMWH and 244 coumarin. Patients were followed up for a 3-month (DVT patients) or 6-month (PE patients) period. RESULTS: 14/654 patients (2%) developed recurrent VTE while on anticoagulant therapy. One in every three recurrent episodes was PE (which was fatal in 2/5 patients), and half of the recurrent DVT were located in the contralateral leg. We failed to find any differences between patients receiving LMWH and those on coumarin therapy, but recurrences were more common in patients with cancer (hazard ratio: 17.15; 95% CI: 4.0-73.5; p < 0.001). 21 patients (3.3%) bled (major bleeding 5 patients; minor bleeding 16). Bleeding was more common in patients on coumarin therapy (hazard ratio: 3.14; 95% CI: 1.20-8.22; p = 0.02). CONCLUSIONS: Long-term LMWH therapy proved to be both effective and safe in the long-term treatment of VTE. Thus, we suggest long-term LMWH therapy should be considered for patients with contraindications to coumarin, or those with difficulties in coming to laboratory control.     

Central human B cell tolerance manifests with a distinctive cell phenotype and is enforced via CXCR4 signaling in hu-mice

Central B cell tolerance, the process restricting the development of many newly generated autoreactive B cells, has been intensely investigated in mouse cells while studies in humans have been hampered by the inability to phenotypically distinguish autoreactive and nonautoreactive immature B cell clones and the difficulty in accessing fresh human bone marrow samples. Using a human immune system mouse model in which all human Igκ⁺ B cells undergo central tolerance, we discovered that human autoreactive immature B cells exhibit a distinctive phenotype that includes lower activation of ERK and differential expression of CD69, CD81, CXCR4, and other glycoproteins. Human B cells exhibiting these characteristics were observed in fresh human bone marrow tissue biopsy specimens, although differences in marker expression were smaller than in the humanized mouse model. Furthermore, the expression of these markers was slightly altered in autoreactive B cells of humanized mice engrafted with some human immune systems genetically predisposed to autoimmunity. Finally, by treating mice and human immune system mice with a pharmacologic antagonist, we show that signaling by CXCR4 is necessary to prevent both human and mouse autoreactive B cell clones from egressing the bone marrow, indicating that CXCR4 functionally contributes to central B cell tolerance.

The bone marrow tissue is the major site of postnatal B cell development in both mice and humans (1, 2). B lymphopoiesis begins when hematopoietic stem cells (HSCs) differentiate into common lymphoid progenitors that commit to the B cell lineage by developing into precursors of B cell progenitors (3). These precursors undergo a step-by-step differentiation process that is accompanied by the rearrangement of V, D, and J gene segments at the immunoglobulin (Ig) heavy chain locus first and then, after a heavy (H) chain is expressed, of V and J segments at the Ig light chain loci, Igκ and Igλ (4). The de novo synthesized H and L Ig chains pair with the signaling molecules CD79A and CD79B to form a B cell receptor (BCR) that is expressed on the cell surface as IgM and that functions as antigen receptor (5). These newly generated IgM⁺ B cells, which are identified as immature B cells, undergo further differentiation into transitional B cells that enter the circulation and complete their maturation in the spleen (6).A feature of B cell development is that the assembly of the Ig H and L chain genes occurs through random reassortment of numerous V(D)J gene segments, which facilitates the generation of a vast number of antibody specificities, one per each developing B cell (4). While this feature is crucial to the development and maintenance of a B cell population and antibody repertoire capable of recognizing any pathogen, the disadvantage is that the majority of these V(D)J gene sequences encode antibodies that are self-reactive (7, 8). It is well established that the entry of newly generated autoreactive B cell clones into the peripheral tissue is restricted by the physiological process of tolerance, a process that evolved to reduce the chance of autoantibody responses and autoimmunity. Indeed, antibody repertoire studies in mice and humans as well as studies with Ig transgenic and knock-in mice have amply demonstrated that the transition from the bone marrow immature B cell stage to the peripheral B cell stage is accompanied by a significant decrease (about twofold) in the frequency of autoreactive clones (8, 9), and this is because clones with BCRs that exhibit high avidity for self-antigens are prevented from entering the peripheral B cell population (10–12).The process of central B cell tolerance has been mainly characterized in mouse models where it operates via the maintenance of RAG1/2-mediated VJ recombination at the light chain loci (i.e., receptor editing) and by inducing cell death (i.e., clonal deletion) in clones in which receptor editing fails to provide a nonautoreactive specificity within a few days (13–16). A major bottleneck in studying central B cell tolerance in humans is the difficulty of acquiring fresh bone marrow samples and the inability to identify and distinguish autoreactive from nonautoreactive B cell clones. Single-cell cloning of Ig genes from newly formed bone marrow B cells that emigrated into the blood, together with the expression and testing of the antibodies they encode, have provided estimates of the efficiency of central B cell tolerance in humans (7, 17). These studies have elegantly shown that central B cell tolerance is significantly less efficient in many autoimmune patients, and particularly in those with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA), type-1 diabetes (T1D), and Sjögren’s syndrome (18–22). These studies have further shown that the genetic variant R620W of the PTPN22 protein tyrosine phosphatase, a variant linked to a higher risk for the development of autoimmunity, is also associated with higher frequencies of autoreactive/polyreactive clones among the new emigrant transitional B cells, revealing a defective central tolerance checkpoint in individuals carrying this risk allele (23).To elucidate mechanisms of development and tolerance of human B cells, our group has investigated human immune system humanized mice (HIS hu-mice) in which human B lymphocytes develop subsequent to the engraftment of human umbilical cord blood HSCs (24, 25). With this intent, we have previously created a HIS hu-mouse model in which all mouse cells express a synthetic membrane-bound self-antigen (Hcκ) that reacts at high avidity with developing human Igκ⁺ B cells (26). In this model, all human κ⁺ B cells are autoreactive and undergo central tolerance in the bone marrow via a mix of receptor editing and clonal deletion (26).In the present study we aimed to exploit this HIS hu-mouse model to discover markers that distinguish human autoreactive immature B cells from nonautoreactive cells, as well as to identify pathways that contribute mechanistically to the enforcement of central B cell tolerance. Our data show that human autoreactive immature B cells contrast from nonautoreactive cells by up-regulating CD69 and CXCR4 while downmodulating the expression of IgM, CD19, CD81, and BAFFR as well as maintaining lower ERK activation. Cells with a similar phenotype, although more subtle, were also observed within the developing immature B cell population of human bone marrow specimens. Moreover, small differences in the expression of these markers and in the amount of sera autoantibodies were found in HIS hu-mice generated with HSCs from some donors genetically predisposed to autoimmunity. Finally, our data demonstrate that retention of human autoreactive B cells in the bone marrow does not rely on CD69 expression while is critically dependent on CXCR4 signaling, indicating that the CXCR4–CXCL12 pathway enforces central B cell tolerance.     

Radial versus femoral approach for percutaneous coronary diagnostic and interventional procedures; Systematic overview and meta-analysis of randomized trials

OBJECTIVES: We sought to compare, through a meta-analytic process, the transradial and transfemoral approaches for coronary procedures in terms of clinical and procedural outcomes. BACKGROUND: The radial approach has been increasingly used as an alternative to femoral access. Several trials have compared these two approaches, with inconclusive results. METHODS: The MEDLINE, CENTRAL, and conference proceedings from major cardiologic associations were searched. Random-effect odds ratios (ORs) for failure of the procedure (crossover to different entry site or impossibility to perform the planned procedure), entry site complications (major hematoma, vascular surgery, or arteriovenous fistula), and major adverse cardiovascular events (MACE), defined as death, myocardial infarction, emergency revascularization, or stroke, were computed. RESULTS: Twelve randomized trials (n = 3,224) were included in the analysis. The risk of MACE was similar for the radial versus femoral approach (OR 0.92, 95% confidence interval [CI] 0.57 to 1.48; p = 0.7). Instead, radial access was associated with a significantly lower rate of entry site complications (OR 0.20, 95% CI 0.09 to 0.42; p < 0.0001), even if at the price of a higher rate of procedural failure (OR 3.30, 95% CI 1.63 to 6.71; p < 0.001). CONCLUSIONS: The radial approach for coronary procedures appears as a safe alternative to femoral access. Moreover, radial access virtually eliminates local vascular complications, thanks to a time-sparing hemostasis technique. However, gaining radial access requires higher technical skills, thus yielding an overall lower success rate. Nonetheless, a clear ongoing trend toward equalization of the two procedures, in terms of procedural success, is evident through the years, probably due to technologic progress of materials and increased operator experience.     

Human Infection with Highly Pathogenic A(H7N7) Avian Influenza Virus,Italy, 2013

During an influenza A(H7N7) virus outbreak among poultry in Italy during August–September 2013, infection with a highly pathogenic A(H7N7) avian influenza virus was diagnosed for 3 poultry workers with conjunctivitis. Genetic analyses revealed that the viruses from the humans were closely related to those from chickens on affected farms.     

Differential effects of high-carbohydrate and high-fat diets on hepatic lipogenesis in rats

Purpose

Hepatic fatty acid synthesis is influenced by several nutritional and hormonal factors. In this study, we have investigated the effects of distinct experimental diets enriched in carbohydrate or in fat on hepatic lipogenesis.

Methods

Male Wistar rats were divided into four groups and fed distinct experimental diets enriched in carbohydrates (70 % w/w) or in fat (20 and 35 % w/w). Activity and expression of the mitochondrial citrate carrier and of the cytosolic enzymes acetyl-CoA carboxylase and fatty acid synthetase were analyzed through the study with assessments at 0, 1, 2, 4, and 6 weeks. Liver lipids and plasma levels of lipids, glucose, and insulin were assayed in parallel.

Results

Whereas the high-carbohydrate diet moderately stimulated hepatic lipogenesis, a strong inhibition of this anabolic pathway was found in animals fed high-fat diets. This inhibition was time-dependent and concentration-dependent. Moreover, whereas the high-carbohydrate diet induced an increase in plasma triglycerides, the high-fat diets determined an accumulation of triglycerides in liver. An increase in the plasmatic levels of glucose and insulin was observed in all cases.

Conclusions

The excess of sucrose in the diet is converted into fat that is distributed by bloodstream in the organism in the form of circulating triglycerides. On the other hand, a high amount of dietary fat caused a strong inhibition of lipogenesis and a concomitant increase in the level of hepatic lipids, thereby highlighting, in these conditions, the role of liver as a reservoir of exogenous fat.