Sera from patients with heparin-induced thrombocytopenia generate platelet-derived microparticles with procoagulant activity: an explanation for the thrombotic complications of heparin-induced thrombocytopenia

Heparin-induced thrombocytopenia is characterized by moderate thrombocytopenia and thrombotic complications, whereas quinine/quinidine-induced thrombocytopenia usually presents with severe thrombocytopenia and bleeding. Using flow cytometry and assays of procoagulant activity, we investigated whether sera from patients with these immune drug reactions could stimulate normal platelets to generate platelet-derived microparticles with procoagulant activity. Sera or purified IgG from patients with heparin-induced thrombocytopenia stimulated the formation of platelet-derived microparticles in a heparin-dependent fashion. Further studies showed that heparin-induced thrombocytopenia sera also produced a marked increase in procoagulant activity. In contrast, sera from patients with quinine- or quinidine-induced thrombocytopenia did not generate platelet-derived microparticles nor generate increased procoagulant activity. However, quinine/quinidine-induced thrombocytopenia sera produced a significant increase in the binding of IgG to platelets in a drug-dependent fashion, whereas sera from patients with heparin-induced thrombocytopenia demonstrated no drug-dependent binding of IgG to platelets. We also observed increased levels of circulating microparticles in patients with acute heparin-induced thrombocytopenia compared with control patients. Our observations indicate that the generation of procoagulant platelet-derived microparticles in vivo is a plausible explanation for the thrombotic complications observed in some patients with heparin-induced thrombocytopenia.     

A humanised tissue‐engineered bone model allows species‐specific breast cancer‐related bone metastasis in vivo

Bone metastases frequently occur in the advanced stages of breast cancer. At this stage, the disease is deemed incurable. To date, the mechanisms of breast cancer‐related metastasis to bone are poorly understood. This may be attributed to the lack of appropriate animal models to investigate the complex cancer cell–bone interactions. In this study, two established tissue‐engineered bone constructs (TEBCs) were applied to a breast cancer‐related metastasis model. A cylindrical medical‐grade polycaprolactone‐tricalcium phosphate scaffold produced by fused deposition modelling (scaffold 1) was compared with a tubular calcium phosphate‐coated polycaprolactone scaffold fabricated by solution electrospinning (scaffold 2) for their potential to generate ectopic humanised bone in NOD/SCID mice. While scaffold 1 was found not suitable to generate a sufficient amount of ectopic bone tissue due to poor ectopic integration, scaffold 2 showed excellent integration into the host tissue, leading to bone formation. To mimic breast cancer cell colonisation to the bone, MDA‐MB‐231, SUM1315, and MDA‐MB‐231BO breast cancer cells were cultured in polyethylene glycol‐based hydrogels and implanted adjacent to the TEBCs. Histological analysis indicated that the breast cancer cells induced an osteoclastic reaction in the TEBCs, demonstrating analogies to breast cancer‐related bone metastasis seen in patients.     

Peripheral blood accessory cells modulate committed colony-forming units but not 5-week cobblestone-area-forming cell outgrowth from CD34+ cells

While cellular modulation in vitro of committed hematopoietic stem cell (HSC) growth has been known for some time, less is known about the effect of accessory cells (AC) on the growth of more immature HSC. We have examined the effect of peripheral blood (PB) AC on hematopoiesis by coculturing enriched PB CD34+ cells (>96% pure) with different quantities of CD34 cells (<1% contamination) harvested from 10 breast cancer patients. As expected colony growth was predominantly present in the CD34+ fractions, in which colony forming units granulocyte-macrophage (CFU-GM) varied between 89-3289/10(5) (median 1422/10(5) seeded cells) and week 5 cobblestone area forming cells (CAFC) between 64-1330/10(5) (median 427/10(5) seeded cells). Few CFU-GM (0-27/10(5) seeded cells) and no week 5 CAFC (0-1/10(5) seeded cells) were present in the CD34 fractions. The addition of PB CD34 cells to cultures of CD34+ cells resulted in a considerable variation in the cloning efficiency at the CFU-GM level, and the extent of modulation within the single patient was inconsistent between the different CD34+/CD34 cell mixtures. Overall the stimulatory effect was more pronounced than inhibition and on average the CFU-GM formation per CD34+ cell seeded increased 3 fold (stimulatory effect ranged between 3-17 fold and decreases between 2-10 fold). In contrast, the cloning efficiency at the week 5 CAFC level of differentiation remained unaffected by the addition of different amounts of CD34 cells (the stimulatory effect was maximally 3-fold and inhibition 3-fold). We conclude that while the CFU assay is modulated by the presence of AC, the CAFC assay is more robust and can be employed as a reliable and reproducible tool for HSC measurement.     

A continuous decline in menarcheal age in Denmark

We report a renewed decline in mean menarcheal age in a large Danish sample after a period with a halt in the trend towards earlier age at menarche in many North European countries. In our study based on retrospective data from six different samples constituting 42784 women, we find a continuously declining mean menarcheal age in Denmark among women born in the years 1964-1973. In a sample of textile workers born in the years 1939-1968 (n = 12605) we find a 1 year higher mean menarcheal age. This indicates that menarcheal age is still delayed in certain groups in Denmark. This leaves the possibility that the menarcheal age could fall even further in the future.     

New population-based exome data are questioning the pathogenicity of previously cardiomyopathy-associated genetic variants

Cardiomyopathies are a heterogeneous group of diseases with various etiologies. We focused on three genetically determined cardiomyopathies: hypertrophic (HCM), dilated (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC). Eighty-four genes have so far been associated with these cardiomyopathies, but the disease-causing effect of reported variants is often dubious. In order to identify possible false-positive variants, we investigated the prevalence of previously reported cardiomyopathy-associated variants in recently published exome data. We searched for reported missense and nonsense variants in the NHLBI-Go Exome Sequencing Project (ESP) containing exome data from 6500 individuals. In ESP, we identified 94 variants out of 687 (14%) variants previously associated with HCM, 58 out of 337 (17%) variants associated with DCM, and 38 variants out of 209 (18%) associated with ARVC. These findings correspond to a genotype prevalence of 1:4 for HCM, 1:6 for DCM, and 1:5 for ARVC. PolyPhen-2 predictions were conducted on all previously published cardiomyopathy-associated missense variants. We found significant overrepresentation of variants predicted as being benign among those present in ESP compared with the ones not present. In order to validate our findings, seven variants associated with cardiomyopathy were genotyped in a control population and this revealed frequencies comparable with the ones found in ESP. In conclusion, we identified genotype prevalences up to more than one thousand times higher than expected from the phenotype prevalences in the general population (HCM 1:500, DCM 1:2500, and ARVC 1:5000) and our data suggest that a high number of these variants are not monogenic causes of cardiomyopathy.     

To test or not: A registry-based observational study of an online decision support for prostate-specific antigen tests

BackgroundWatchful waiting is an essential part in the handling of patients with prostate cancer (PC). More effective disease management may be achieved by this strategy. Correct use of prostate-specific antigen (PSA) test is crucial and the general practitioner (GP) may therefore benefit from access to a clinical decision support system (CDSS) that focuses on this challenge. There are many barriers to the use of CDSSs. The aim of the present paper is to study if such barriers may be overcome by granting GPs easier access to a web-based CDSS via a hyperlink in the GPs’ electronic medical record system (EMR).MethodsIn the present population-based observational registry study with an intervention and control group, we created a web-based CDSS that was made accessible to GPs via hyperlink inserted into the EMR medical chart contents. The intervention was introduced 1 January 2011. Our outcome measure was the number of age-standardised PSA test rates per 1000 men per practice during three equally sized periods of 6 months within the study period from 1 January 2010 to 30 June 2011.ResultsWe found that none of the differences between intervention and control groups were statistically significant.ConclusionsProviding GPs with access to a CDSS to aid their decision to use the PSA, had no measurable effect on the GPs’ PSA testing behaviour.     

Cripto‐1 Expression in Glioblastoma Multiforme

Human glioblastoma multiforme (GBM) is an aggressive cancer with a very poor prognosis. Cripto‐1 (CR‐1) has a key regulatory role in embryogenesis, while in adult tissue re‐expression of CR‐1 has been correlated to malignant progression in solid cancers of non‐neuronal origin. As CR‐1 expression has yet to be described in cerebral cancer and CR‐1 is regulated by signaling pathways dysregulated in GBM, we aimed to investigate CR‐1 in the context of expression in GBM. The study was performed using enzyme‐linked immunosorbent assay (ELISA), Western blotting, polymerase chain reaction (PCR) and immunohistochemistry to analyze the blood and tissue from 28 GBM and 4 low‐grade glioma patients. Within the patient cohort, we found high CR‐1 protein levels in blood plasma to significantly correlate with a shorter overall survival. We identified CR‐1 in different areas of GBM tissue, including perivascular tumor cells, and in endothelial cells. Collectively, our data suggest that CR‐1 could be a prognostic biomarker for GBM with the potential of being a therapeutic target.