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Pseudothrombocytopenia remains a challenge in the haematological laboratory. The pre-analytical problem that platelets tend to easily aggregate in vitro, giving rise to lower platelet counts, has been known since ethylenediamine-tetra acetic acid EDTA and automated platelet counting procedures were introduced in the haematological laboratory. Different approaches to avoid the time and temperature dependent in vitro aggregation of platelets in the presence of EDTA were tested, but none of them proved optimal for routine purposes. Patients with unexpectedly low platelet counts or flagged for suspected aggregates, were selected and smears were examined for platelet aggregates. In these cases patients were asked to consent to the drawing of an additional sample of blood anti-coagulated with a magnesium additive. Magnesium was used in the beginning of the last century as anticoagulant for microscopic platelet counts. Using this approach, we documented 44 patients with pseudothrombocytopenia. In all cases, platelet counts were markedly higher in samples anti-coagulated with the magnesium containing anticoagulant when compared to EDTA-anticoagulated blood samples. We conclude that in patients with known or suspected pseudothrombocytopenia the magnesium-anticoagulant blood samples may be recommended for platelet counting.

Background It has recently been proposed that the M-type phospholipase A2 receptor (PLA2R1) acts as a tumour suppressor in certain malignancies including mammary cancer. Considering that DNA methylation is an important regulator of gene transcription during carcinogenesis, in the current study we analyzed the PLA2R1 expression, PLA2R1 promoter methylation, and selected micro RNA (miRNA) levels in normal human mammary epithelial cells (HMEC) and cancer cell lines. Methods Levels of PLA2R1 and DNA methyltransferases (DNMT) specific mRNA were determined using real-time RT-PCR. Methylation specific-high resolution melting (MS-HRM) analysis was utilized to quantify the methylation degree of selected CpG sites localized in the promoter region of the PLA2R1 gene. Expression of miRNA was tested using miScript Primer Assay system. Results Nearly complete methylation of the analyzed PLA2R1 promoter region along with PLA2R1 gene silencing was identified in MDA-MB-453 mammary cancer cells. In MCF-7 and BT-474 mammary cancer cell lines, a higher DNA methylation degree and reduced PLA2R1 expression were found in comparison with those in normal HMEC. Synergistic effects of demethylating agent (5-aza-2′-deoxycytidine) and histone deacetylase inhibitor (trichostatin A) on PLA2R1 transcription in MDA-MB-453 cells confirmed the importance of DNA methylation and histone modification in the regulation of the PLA2R1 gene expression in mammary cells. Furthermore, significant positive correlation between the expression of DNMT1 and PLA2R1 gene methylation and negative correlation between the cellular levels of hsa-mir-141, −181b, and -181d-1 and the expression of PLA2R1 were identified in the analyzed cells. Analysis of combined z-score of miR-23b, −154 and -302d demonstrated a strong and significant positive correlation with PLA2R1 expression. Conclusions Our data indicate that (i) PLA2R1 expression in breast cancer cells is controlled by DNA methylation and histone modifications, (ii) hypermethylation of the PLA2R1 promoter region is associated with up-regulation of DNMT1, and (iii) hsa-miR-23b, −154, and −302d, as well as hsa-miR-141, −181b, and −181d-1 are potential candidates for post-transcriptional regulation of PLA2R1 expression in mammary cancer cells.

Epigenetic changes provide a frequent mechanism for transcriptional silencing of genes in cancer cells. We previously established that epigenetic mechanisms are important for control of group IIA phospholipase A(2) (PLA2G2A) gene transcription in human DU-145 prostate cells. In this study, we analyzed the involvement of such mechanisms in the regulation of five sPLA(2) isozymes and the M-type receptor of sPLA(2) (sPLA(2)-R) in human leukemic Jurkat cells. These cells constitutively expressed sPLA(2)-IB, sPLA(2)-III, sPLA(2)-X, and sPLA(2)-R but not sPLA(2)-IIA and sPLA(2)-V. Transcription of sPLA(2)-IIA and sPLA(2)-V was, however, detected after exposure of cells to the DNA demethylating agent, 5-aza-2'-deoxycytidine (5-aza-dC). Expression of sPLA(2)-IIA was further enhanced by additional exposure to interferon-gamma and blocked by inhibitors of specificity protein 1, nuclear factor kappaB, and Janus kinase/signal transducer and activator of transcription-dependent pathways. Sequence analysis and methylation-specific polymerase chain reaction of bisulfite-modified genomic DNA revealed two 5'-CpG sites (-111 and -82) in the sPLA(2)-IIA proximal promoter that were demethylated after 5-aza-dC treatment. These sites may be involved in the DNA binding of specificity protein 1 and other transcription factors. Similar findings after treatment of human U937 leukemia cells with 5-aza-dC indicate that this mechanism of PLA2G2A gene silencing is not restricted to Jurkat and DU-145 cells. These data establish that regulation of sPLA(2)-IIA and sPLA(2)-V in Jurkat and other cells involves epigenetic silencing by DNA hypermethylation.

Research generally supported a positive effect of competition intensity on financial commitment into news production within U.S. markets. The authors test this assumption across European newspaper markets by applying fuzzy set qualitative comparative analysis. Although oligopolistic too, European newspaper markets reveal a somewhat different pattern. The market condition “high number of competitors” in combination with “high competition intensity” is a sufficient condition for financial commitment across the different markets in this study.

Commercialization of the media refers to the increasing importance of market considerations in the daily work of media professionals. Media organizations need to find ways to maintain the quality promise of their brands as an economically successful business. The increasing market orientation of media companies challenges their dedication to good journalism. The media brand identity approach is a good starting point from which to analyze this decision making in a commercialized and complex environment. By introducing the MBAC (“media, brands, actors, and communication”) model, a new approach is offered toward better theoretical understanding of brand identity-driven decision making. Responsibilities and tensions between the journalistic and the business side of news production are presented, and the complex environment in which decisions are made, implemented, and defended are elaborated on.

It was with great interest that we read the recent recommendations regarding the treatment of acquired hemophilia A published in Haematologica1 and followed the subsequent discussion.2,3 The described treatment algorithm for this disease, which occurs at an incidence of 1.5/million/year,4 stresses the importance of early eradication of the acquired fVIII-inhibitor. Using prednisolone alone or in combination with cyclophosphamide, this approach will be successful in 30–50% of affected individuals.5 As Huth-Kuhne pointed out, the CD20-antibody rituximab may be an attractive alternative in non-responders or patients with contraindications to steroids.6 However, concerns have been raised because of rituximab side-effects including associations with progressive multifocal leukencephalopathy (PML).7 Furthermore, as Mannucci et al.2 emphasized, the use of this drug is hampered by its substantial costs. Restricting the administered dose to the lowest effective level, seems to be a reasonable strategy to minimize potential risks and costs of rituximab treatment. We report a 66-year old male AHA-patient successfully treated with single low-dose rituximab. The patient presented with multiple hematomas and severe anemia after recovering from a respiratory infection. Prolonged activated partial-thromboplastin-time (aPTT) in combination with undetectable fVIII-activity due to an fVIII-inhibitor was observed. There was no clinical history of any prior hemorrhages nor a family history of bleeding. Clinical and imaging examinations could not reveal any pathological findings suggesting an idiopathic cause of AHA. Prednisolone (1 mg/kg) was started immediately and 2 units of packed red blood cells had to be administered. Afterwards, bleeding ceased, correlating with an increase in fVIII-activity to 28%. Steroids were completely tapered over the next weeks and the patient was lost to follow-up. However, during the following year the patient experienced recurrent episodes of minor bleeding. Seventeen months after the first onset of AHA, he again presented at our institution for follow-up. He reported no spontaneous bleeding episodes; however, fVIII-activity was 15% translating into an fVIII-inhibitor of 133 BU (Bethesda Units). Two months later, severe limb-bleeding requiring surgical treatment occurred after a minor accident. FVIII-inhibitor could still be detected at high levels (79 BU). Because of impaired wound-healing, steroids were deemed contraindicated. Therefore, given our experience with patients with auto-immune diseases other than AHA,8 inhibitor-eradication treatment was started with a single flat dose of 100 mg rituximab. Treatment was well tolerated and three weeks later complete B-cell depletion could be demonstrated (Figure 1). There was no reoccurrence of significant bleeding and even an accident was tolerated without complications. At last presentation, nearly ten months after rituximab treatment, the patient had only a low residual fVIII-inhibitor activity, in spite of a normal B-cell count. FVIII-levels stabilized at a level of 15 to 20% and aPTT was never again found to be prolonged after rituximab treatment. Figure 1. Clinical course overview. Time since diagnosis in months (x-axis). fVIII-inhibitor titer in Bethesda Units (BU) (left y-axis). B-cell proportion (%) (right y-axis). R: time of rituximab application. The clinical course of our patient demonstrates that complete B-cell depletion can be achieved with low-dose rituximab in AHA. We are aware that given the persistently low fVIII-level, our patient does not meet the strict criteria for inhibitor eradication proposed by Huth-Kuhne. On the other hand, we have to acknowledge that he experienced no further bleeding and had inhibitor-titers near the detection limit. We, therefore, believe that future clinical trials should incorporate this low-dose regimen rather than the classical lymphoma-dose (e.g. 375 mg/m2) in the treatment of acquired hemophilia A.

Changes in the TV industry and usage patterns create the necessity to reevaluate the production and insertion of commercials into the program. We analyze how industry professionals value the effectiveness of traditional TV commercials and examine if increasingly hybrid advertising formats integrating the commercial message into the editorial content are chosen. Contrary to the common assumption that the classical TV commercial is about to disappear, our structured interviews reveal that it is still considered the most important advertising format and cannot be substituted. The future is seen in integrated intra- and inter-medial campaigns using both traditional and hybrid advertising formats.

The present study shows that the IFN-gamma-mediated upregulation of secretory phospholipase A2 of group IIA (sPLA2-IIA) in HASMC and HepG2 cells is synergistically increased after simultaneous inhibition of glycogen synthase kinase-3beta (GSK-3beta) by indirubin-3'-monoxime, 5-iodo or AR-A014418. The effect of GSK-3beta inhibition was dose- and time-dependent and can be further augmented by its concomitant incubation with Clostridium difficile toxin B, an inhibitor of small Rho proteins, or H-1152, an inhibitor of Rho-associated kinase. Using AG-490 and caffeic acid phenethyl ester (CAPE), it is further demonstrated that the effect of GSK-3beta inhibition on sPLA2-IIA expression depends on Janus kinase-2 and NF-kappaB-signaling.

Background Increasing evidences show that beyond its role in coagulation, endothelial protein C receptor (EPCR) interferes with carcinogenesis. Pro-carcinogenic effects of EPCR were linked with a raised generation of activated protein C (aPC) and anti-apoptotic signalling. This study was carried out to analyze the expression, cell surface exposition, and shedding of EPCR in normal and malignant prostate cell lines. Results EPCR expression is up-regulated both at the mRNA and protein levels in invasive prostate DU-145 and PC-3 cells in comparison to normal prostate epithelial cells (PrEC) and less-invasive LNCaP cells. Release of soluble EPCR (sEPCR) is induced by 12-myristate 13-acetate, ionomycin, H2O2, and disruptor of lipid rafts in PrEC, DU-145, and PC-3 cells. Furthermore, interleukin-1β (IL-1β) and tumor necrosis factor-α (TNF-α), but not interleukin-6 or interferon-γ increase sEPCR release. In LNCaP cells, neither pharmacological agents nor IL-1β or TNF-α result in a significant increase of sEPCR release. The effects of IL-1β and TNF-α on EPCR shedding in DU-145 cells are mediated by MEK/ERK 1/2, JNK, and p38 MAPK signalling cascades. In PC-3 cells, however, the MEK/ERK 1/2 pathway is down-regulated and incubation with cytokines did not elevate the phosphorylated ERK-1/2 fraction as in the case of DU-145 cells. Treatment with 4-aminophenylmercuric acetate (APMA), an activator of metalloproteases, causes a disproportionately large increase of sEPCR release in DU-145 and PC-3 cells, compared to PrEC and LNCaP cells. Finally, an increased release of sEPCR mediated by APMA treatment is shown to be connected with reduced generation of activated protein C indicating the functionality of EPCR in these cells. Conclusions The study demonstrates a number of substantial differences in expression and shedding of EPCR in prostate cancer cell lines in comparison with normal cells that may be relevant for understanding the role of this receptor in carcinogenesis.