Shiga toxin of enterohaemorrhagic Escherichia coli directly injures developing human erythrocytes.

Haemolytic anaemia is one of the characteristics of life-threatening extraintestinal complications in humans during infection with enterohaemorrhagic Escherichia coli (EHEC). Shiga toxins (Stxs) of EHEC preferentially damage microvascular endothelial cells of the kidney and the brain, whereby occluded small blood vessels may elicit anaemia through mechanical erythrocyte disruption. Here we show for the first time that Stx2a, the major virulence factor of EHEC, is also capable of direct targeting developing human erythrocytes. We employed an ex vivo erythropoiesis model using mobilized CD34(+) haematopoietic stem/progenitor cells from human blood and monitored expression of Stx receptors and Stx2a-mediated cellular injury of developing erythrocytes. CD34(+) haematopoietic stem/progenitor cells were negative for Stx2a receptors and resistant towards the toxin. Expression of Stx2a-binding glycosphingolipids and toxin sensitivity was apparent immediately after initiation of erythropoietic differentiation, peaked for basophilic and polychromatic erythroblast stages and declined during maturation into orthochromatic erythroblasts and reticulocytes, which became highly refractory to Stx2a. The observed Stx-mediated toxicity towards erythroblasts during the course of erythropoiesis might contribute, although speculative at this stage of research, to the anaemia caused by Stx-producing pathogens.

Utilisation of blood components in cardiac surgery: a single-centre retrospective analysis with regard to diagnosis-related procedures.

BACKGROUND: More blood components are required in cardiac surgery than in most other medical disciplines. The overall blood demand may increase as a function of the total number of cardiothoracic and vascular surgical interventions and their level of complexity, and also when considering the demographic ageing. Awareness has grown with respect to adverse events, such as transfusion-related immunomodulation by allogeneic blood supply, which can contribute to morbidity and mortality. Therefore, programmes of patient blood management (PBM) have been implemented to avoid unnecessary blood transfusions and to standardise the indication of blood transfusions more strictly with aim to improve patients' overall outcomes. METHODS: A comprehensive retrospective analysis of the utilisation of blood components in the Department of Cardiac Surgery at the University Hospital of Münster (UKM) was performed over a 4-year period. Based on a medical reporting system of all medical disciplines, which was established as part of a PBM initiative, all transfused patients in cardiac surgery and their blood components were identified in a diagnosis- and medical procedure-related system, which allows the precise allocation of blood consumption to interventional procedures in cardiac surgery, such as coronary or valve surgery. RESULTS: This retrospective single centre study included all in-patients in cardiac surgery at the UKM from 2009 to 2012, corresponding to a total of 1,405-1,644 cases per year. A blood supply was provided for 55.6-61.9% of the cardiac surgery patients, whereas approximately 9% of all in-patients at the UKM required blood transfusions. Most of the blood units were applied during cardiac valve surgery and during coronary surgery. Further surgical activities with considerable use of blood components included thoracic surgery, aortic surgery, heart transplantations and the use of artificial hearts. Under the measures of PBM in 2012 a noticeable decrease in the number of transfused cases was observed compared to the period from 2009 to 2011 before implementation of the PBM initiative (red blood cells p < 0.002; fresh frozen plasma p < 0.0006; platelets p < 0.00006). CONCLUSION: Until now, cardiac surgery comes along with a significant blood supply. By using a case-related data evaluation programme, the consumption of each blood component can be linked to clinical performance groups and, if necessary, to individual patients. Based on the results obtained from this retrospective analysis, prospective studies are underway to begin conducting target / actual performance comparisons to better understand the individual decision-making by the attending physicians with respect to transfusions.

Utilisation of Blood Components in Trauma Surgery: A Single-Centre, Retrospective Analysis before and after the Implementation of an Educative PBM Initiative.

BACKGROUND: The aim of our single-centre retrospective study presented here is to further analyse the utilisation of allogeneic blood components within a 5-year observation period (2009-2013) in trauma surgery (15,457 patients) under the measures of an educational patient blood management (PBM) initiative. METHODS: After the implementation of the PBM initiative in January 2012, the Institute of Transfusion Medicine und Transplantation Immunology educates surgeons and nurses at the Department of Trauma Surgery to avoid unnecessary blood transfusions. A standardised reporting system was used to document the utilisation of blood components carefully for the most frequent diagnoses and surgical interventions in trauma surgery. These measures served as basis for the implementation of an interdisciplinary systematic exchange of information to foster decision-making processes in favour of patient blood management. RESULTS: Since January 2012, the proportion of patients who received a transfusion as well as the number of transfused red blood cell (RBC) (7.3%/6.4%; p = 0.02), fresh frozen plasma (FFP) (1.7%/1.3%; p < 0.05) and platelet (PLT) (1.0%/0.5%; p < 0.001) units were reduced as a result of our PBM initiative. However, among the transfused patients, the number of administered RBC, FFP and PLT units did not decrease significantly. Overall, patients who did not receive transfusions were younger than transfused patients (p = 0.001). The subgroup with the highest probability of blood transfusion administered included patients with intensive care and long-term ventilation (before/after implementation of PBM: RBC 81.5%/75.9%; FFP 33.3%/20.4%; PLT 24.1%/13.0%). Only a total of 60 patients of 531 patients suffering multiple traumas were massively transfused (before/after implementation of PBM: RBC 55.6%/49.8%; FFP 28.4%/20.4%; PLT 17.6%/8.9%). CONCLUSION: According to our educational PBM initiative, at least the proportion of trauma patients who received allogeneic blood transfusions could be reduced significantly. However, in case of blood transfusions, the total consumption of RBC, FFP and PLT units remained stable in both time periods. This phenomenon might indicate that the actual need of blood transfusions rather depends on the severity of trauma-related blood loss, the coagulopathy rates or the complexity of the surgical intervention which mainly determines the intra-operative blood loss. Taken together, educational training sessions and systematic reporting systems are suitable measures to avoid unnecessary allogeneic blood transfusions and to continuously improve their restrictive application.

Biomonitoring using dried blood spots: detection of ochratoxin A and its degradation product 2'R-ochratoxin A in blood from coffee drinkers.

SCOPE: In this study, human exposure to the mycotoxin ochratoxin A (OTA) and its thermal degradation product 2'R-ochratoxin A (2'R-OTA, previously named as 14R-Ochratoxin A [22]) through coffee consumption was assessed. LC-MS/MS and the dried blood spot (DBS) technique were used for the analysis of blood samples from coffee and noncoffee drinkers (n = 50), and food frequency questionnaires were used to document coffee consumption. METHODS AND RESULTS: For the detection of OTA and 2'R-OTA in blood, a new sensitive and efficient sample preparation method based on DBS was established and validated. Using this technique 2'R-OTA was for the first time detected in biological samples. Comparison between coffee drinkers and noncoffee drinkers showed for the first time that 2'R-OTA was only present in blood from the first group while OTA could be found in both groups in a mean concentration of 0.21 µg/L. 2'R-OTA mean concentration was 0.11 µg/L with a maximum concentration of 0.414 µg/L. Thus, in average 2'R-OTA was approx. half the concentration of OTA but in some cases even exceeded OTA levels. No correlation between the amounts of coffee consumption and OTA or 2'R-OTA levels was observed. CONCLUSION: The results of this study revealed for the first time a high exposure of coffee consumers to 2'R-OTA, a compound formed from OTA during coffee roasting. Since little information is available regarding toxicity and possible carcinogenicity of this compound, further OTA monitoring in blood including 2'R-OTA is advisable.

Influence of Red Blood Cell Storage Time on Clinical Course and Cytokine Profile in Septic Shock Patients.

BACKGROUND: Previous investigations have suggested beneficial effects of fresh versus stored red blood cell transfusion in critically ill patients. The present study investigates the effects of red blood cell storage time on the clinical course and hemodynamic and laboratory parameters in patients with septic shock. PATIENTS AND METHODS: 18 patients with septic shock received 2 erythrocyte units stored for ? 7 days (n = 8) or > 7 days (n = 10). The sequential organ failure assessment (SOFA) score was calculated for 7 days. Hemodynamic parameters (cardiac index, extravascular lung water) were determined using transpulmonary thermodilution. Laboratory parameters (lactate, base excess, C-reactive protein, procalcitonin, IL-1Β, IL-6, TNF-α, sVCAM-1, sICAM-1) were monitored before and 1, 3, 6, 12, 24, and 48 h after transfusion. The Mann-Whitney-U test and Neumann test were used for group comparison and trend assessment, respectively. RESULTS: We failed to observe significant differences with respect to SOFA scores between patients receiving fresh or stored erythrocytes. However, a significant trend towards an improvement in the SOFA score was found in the group receiving fresh erythrocytes (p < 0.01). No significant differences in hemodynamic or laboratory parameters were found between both groups. CONCLUSION: While the present findings do not provide clear-cut evidence supporting beneficial effects of fresh red blood cells in septic shock, they warrant larger randomized studies to confirm or refute such effects.

Retrospective Analysis of the Blood Component Utilization in a University Hospital of Maximum Medical Care.

BACKGROUND: Demographic data illustrate clearly that people in highly developed countries get older, and the elderly need more blood transfusions than younger patients. Additionally, special extensive therapies result in an increased consumption of blood components. Beyond that the aging of the population reduces the total number of preferably young and healthy blood donors. Therefore, Patient Blood Management will become more and more important in order to secure an increasing blood supply under fair-minded conditions. METHODS: At the University Hospital of Münster (UKM) a comprehensive retrospective analysis of the utilization of all conventional blood components was performed including all medical and surgical disciplines. In parallel, a new medical reporting system was installed to provide a monthly analysis of the transfusional treatments in the whole infirmary, in every department, and in special blood-consuming cases of interest, as well. RESULTS: The study refers to all UKM in-patient cases from 2009 to 2011. It clearly demonstrates that older patients (>60 years, 35.2-35.7% of all cases, but 49.4-52.6% of all cases with red blood cell (RBC) transfusions, 36.4-41. 6% of all cases with platelet (PTL, apheresis only) transfusions, 45.2-48.0% of all cases with fresh frozen plasma (FFP) transfusions) need more blood products than younger patients. Male patients (54.4-63.9% of all cases with transfusions) are more susceptible to blood transfusions than female patients (36.1-45.6% of all cases with transfusions). Most blood components are used in cardiac, visceral, and orthopedic surgery (49.3-55.9% of all RBC units, 45.8-61.0% of all FFP units). When regarding medical disciplines, most transfusions are administered to hematologic and oncologic patients (12.9-17.7% of all RBC units, 9.2-12.0% of all FFP units). The consumption of PTL in this special patient cohort (40.6-50.9% of all PTL units) is more pronounced than in all other surgical or in non-surgical disciplines. CONCLUSION: The results obtained from our retrospective analysis may help to further optimize the responsible and medical indication-related utilization of blood transfusions as well as the recruitment of blood donors and their timing. It may be also a helpful tool in order to avoid needless transfusions and transfusionassociated adverse events.

Combined immunochemotherapy in selected patients with metastatic renal cell carcinoma: HLA class II genotype can help to predict response to therapy.

A number of new agents have been approved for systemic therapy of metastatic renal cell carcinoma (mRCC) recently. Thereby, prognostic factors may aid in predicting the effectiveness of various treatment modalities in individual cases. Aim of this study was to determine the value of human leukocyte antigen (HLA) class II characteristics in predicting response of mRCC to combined immunochemotherapy (ICT). A retrospective study of 29 patients with mRCC treated with ICT was performed: 17 patients (group A) with long-term remission and 12 (group B) with progressive disease after ICT. DNA was used for high resolution typing of HLA-DRB1, -DRB3, -DRB4, -DRB5, -DQA1, and -DQB1. Statistical evaluation started with Classification and Regression Trees analysis. The assignment of single alleles to the groups was then aggregated to create a classification on a patients' basis. Finally, the accuracy of this test algorithm was evaluated. HLA-DRB1 (DRB1*0301*0401*0402*0407*1101*1501=progression) was the strongest discriminator between the 2 groups. The test algorithm defined all patients with at least one of these DRB1 alleles to be progressive after ICT. Thus, 12 of 12 patients of group B could have been identified as progressive (sensitivity=100%). However, only 10 of 17 patients of group A would have been identified as responding (specificity=58%). Thus, the test had a positive and negative predictive value of 63% and 100%, respectively. Approximately 5% to 10% of all patients with mRCC are able to benefit from ICT with long-term remission. HLA class II characteristics may aid in identifying this small subgroup of patients with mRCC.

Cellular prion proteins in human platelets show a phenotype different to those in brain tissues.

Prion diseases are characterized by high accumulation of infectious prion proteins (PrP(Sc)) in brains. PrP(Sc) are propagated by the conversion of host-encoded cellular prion proteins (PrP(C)) which are essential for developing the disease but are heterogeneously expressed in brains. The disease can be transmitted to humans and animals through blood and blood products, however, little attention has been given to molecular characterization of PrP(C) in blood cells. In this presented study, we characterized phenotypically PrP(C) of platelets (plt) and characterized the proteins regarding their glycobanding profiles by quantitative immunoblotting using a panel of monoclonal antibodies. The glycosylation patterns of plt and brain PrP(C) were compared using the ratios of di-, mono-, and non-glycosylated prions. The detergent solubility of plt and brain PrP(C) was also analyzed. The distinct banding patterns and detergent solubility of plt PrP(C) differed clearly from the glycosylation profiles and solubility characteristics of brain PrP(C). Plt PrP(C) exhibited single or only few prion protein types, whereas brain PrP(C) showed more extensive banding patterns and lower detergent solubility. Plt PrP(C) are post-translational modified differently from PrP(C) in brain. These findings suggest other or less physiological functions of plt PrP(C) than in brain.

The adult stem cell marker Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via Notch-1 and p21WAF1/CIP1.

The RNA-binding protein Musashi-1 has been proposed to maintain stem cell function during development and regenerative processes as a modulator of the Notch-1 signaling pathway. Musashi-1 expression is upregulated in endometrial carcinoma, however, its pathogenetic role in this tumor entity is unknown. Here we investigate the functional impact and mode of action of Musashi-1 on endometrial carcinoma cell behaviour in vitro. Aldehyde dehydrogenase-1 activity and side population (SP) measurement by Hoechst dye exclusion revealed that the Ishikawa endometrial carcinoma cell line contains a pool of putative cancer stem cells. Musashi-1 expression is 20.8-fold upregulated in SP+ compared to SP- and equally distributed between ALDH+ and ALDH- cell pools. siRNA-mediated knockdown of Musashi-1 mRNA expression lead to an altered expression of the signaling receptor Notch-1 and its downstream targets, the transcription factor Hes-1 and the cell cycle regulators p21(WAF1/CIP1) and cyclin B1, as determined by Western blotting and quantitative real-time PCR. Flow cytometric and ELISA analyses revealed that Musashi-1-mediated modulation of these factors exerted an antiproliferative effect on the cell cycle, and increased apoptosis in endometrial carcinoma cells. We conclude that Ishikawa cells contain a subpopulation of cells with stem cell-like properties. Musashi-1 modulates endometrial carcinoma cell cycle progression and apoptosis via the stemness-related factors Notch-1, Hes-1 and p21(WAF1/CIP1) , thus emerging as a novel future target for endometrial carcinoma therapy.

An improved PCR method for detection of HIV-1 proviral DNA of a wide range of subtypes and recombinant forms circulating globally.

Proviral DNAs are being measured increasingly as a marker of the efficacy of highly active anti-retroviral therapy (HAART) and is accepted for the early diagnosis of perinatal HIV-1 infections. This requires a standardized test which enables the detection of a wide range of subtypes worldwide including O, N and circulating recombinant forms (CRFs). Based on a previous publication, a PCR - Test for HIV-1 provirus detection in peripheral blood mononuclear cells (PBMCs) was developed. Blood samples from 80 individuals infected with HIV-1 and 20 persons negative for HIV-1&2 from Africa and Germany were tested for the presence of HIV-1 provirus DNA. The primer system used enables the detection of proviral DNA despite the high concentrations of human DNA. The limit of detection was determined to be 5 copies per 10(5) cells. All 20 samples from persons negative for HIV were negative for HIV-1 proviral DNA while provirus DNA was amplified from 76 of the 80 (95%) samples from persons infected with HIV. The amplified products were detected by gel-electrophoresis, flow cytometry and real-time PCR. All three detection systems provided the same results.

Differential roles for membrane-bound and soluble syndecan-1 (CD138) in breast cancer progression.

The heparan sulfate proteoglycan syndecan-1 (Sdc1) modulates cell proliferation, adhesion, migration and angiogenesis. Proteinase-mediated shedding converts Sdc1 from a membrane-bound coreceptor into a soluble effector capable of binding the same ligands. In breast carcinomas, Sdc1 overexpression correlates with poor prognosis and an aggressive phenotype. To distinguish between the roles of membrane-bound and shed forms of Sdc1 in breast cancer progression, human MCF-7 breast cancer cells were stably transfected with plasmids overexpressing wild-type (WT), constitutively shed and uncleavable forms of Sdc1. Overexpression of WT Sdc1 increased cell proliferation, whereas overexpression of constitutively shed Sdc1 decreased proliferation. Fibroblast growth factor-2-mediated mitogen-activated protein kinase signaling was reduced following small-interfering RNA (siRNA)-mediated knockdown of Sdc1 expression. Constitutively, membrane-bound Sdc1 inhibited invasiveness, whereas soluble Sdc1 promoted invasion of MCF-7 cells into matrigel matrices. The latter effect was reversed by the matrix metalloproteinase inhibitors N-isobutyl-N-(4-methoxyphenylsufonyl) glycyl hydroxamic acid and tissue inhibitor of metalloproteinase (TIMP)-1. Affymetrix microarray analysis identified TIMP-1, Furin and urokinase-type plasminogen activator receptor as genes differentially regulated in soluble Sdc1-overexpressing cells. Endogenous TIMP-1 expression was reduced in cells overexpressing soluble Sdc1 and increased in those overexpressing the constitutively membrane-bound Sdc1. Moreover, E-cadherin protein expression was downregulated in cells overexpressing soluble Sdc1. Our results suggest that the soluble and membrane-bound forms of Sdc1 play different roles at different stages of breast cancer progression. Proteolytic conversion of Sdc1 from a membrane-bound into a soluble molecule marks a switch from a proliferative to an invasive phenotype, with implications for breast cancer diagnostics and potential glycosaminoglycan-based therapies.

A new affordable flow cytometry based method to measure HIV-1 viral load.

Most commercially available assays for diagnosis of HIV infection have shown shortcomings in the detection and quantification of rare genotypes of the virus. Most of the assays do not detect subtype O (outlier) and/or N (nonmajor, nonoutlier) or new circulating recombinant forms (CRFs), which are becoming more important in sub-Saharan Africa. Furthermore, the commonly available tests require costly measuring devices and expensive test kits, which are not easily affordable for developing countries. This study was designed to explore solutions to the problem of viral load assays in developing countries. Two forward primers, digoxygenin (DIG) and dinitrophenol (DNP) labeled, and one biotin (BIO) labeled reverse primer were used to amplify both, the HIV-1-5'LTR (long terminal repeat) region and an internal standard sequence. The two polymerase chain reaction (PCR)-products were captured by anti-DIG and anti-DNP antibody coated microparticles. Flow cytometric analyses were carried out after labeling with streptavidin-R-phycoerythrine. The primer system used recognized all HIV-1 subtypes. A coamplified internal standard warranted the functionality of the PCR and allows reproducible viral load measurements. Two drawbacks of current viral load measurements are overcome by the flow cytometry based test described hereof. First, all known worldwide relevant HIV-1 subtypes including subtypes O, N, and new CRFs are quantifiable with high sensitivity (50 to >1 x 10(6) copies per PCR). Second, the cost per test can be reduced to less than 12 US$ instead of the current 50-100 US$. Additionally, the test described in this report offers the possibility to perform complete monitoring program (CD4 T-cell count, CD4% and viral load) for the first time, with the same device for HIV-infected persons.

The Impact of the German Tissue Act on the Manufacturing of Autologous and Allogeneic Stem Cell Preparations.

SUMMARY: Cellular therapeutic agents considerably contribute to the optimal treatment of patients with hematological malignancies such as leukemia or nonhematological disorders. Over the last 50 years especially the transplantation of autologous and allogeneic stem cells from different sources after high-dose or myeloablative chemotherapy became a well-established standard therapy that cures or alleviates the symptoms in more than 50,000 patients/year worldwide. In the near future, the current progress in fundamental research on stem cells and immunobiology will allow for the clinical implementation of novel advanced cellular therapies, including gene therapeutic options. The European and German legislation have realized the need of international regulations for improved standardization and harmonization of stem cell transplants, associated cell-therapeutic agents as well as various tissue-engineered preparations in the emerging field of regenerative medicine. The Tissue Directive 2004/23/EC, issued and ratified by the European Parliament in March 2004, and its national transition into the German Tissue Act which came into force in July 2007 define the quality and safety standards for the donation, procurement, testing, processing, preservation, storage, and distribution of human tissues and cells. These standards are of high relevance to ensure the efficient prevention of the transmission of viral and nonviral infectious pathogens and to achieve the same safeguards as in the population's blood supply. This review discusses the pros and cons of the new legislation and argues for keeping the administrative and regulative demands in reasonable limits and for offering innovative approaches of cellular therapies to the European citizens.

IgG2 containing IgM-IgG immune complexes predominate in normal human plasma, but not in plasma of patients with warm autoimmune haemolytic anaemia.

The different physicochemical and sterical properties of IgG subclasses may favour a selective enrichment of defined IgG subclasses in IgM-IgG immune complexes (IC) of human plasma under physiological conditions. Such enrichment of IgG subclasses in IgM-IgG IC of plasma may differ from the normal IgG subclass distribution in plasma itself, and contribute to the physiological functions of IgM-IgG IC. Systematic studies on the IgG subclass distribution in IgM-IgG IC in humans are lacking. Using specific analytical techniques to characterise IgM-IgG IC in human plasma (i.e. fast protein liquid chromatography, enzyme-linked immunosorbent assay, affinity biosensor technology), and taking warm autoimmune haemolytic anaemia (WAIHA) of humans as a disease model, we here demonstrate that: (i) IgG2 is the predominant IgG subclass in IgM-IgG IC under physiological conditions, (ii) the predominance of IgG2 within IgM-IgG IC may get lost in polyclonal IgG-mediated autoimmune disease and (iii) the IgG subclass distribution in IgM-IgG IC influences the interaction between IC and blood cells involved in antigen presentation. The data presented here therefore extend the physiological function of IgG2, which is the protective immune response towards carbohydrate antigens in bacterial infections, and suggest IgG2-dependent regulation of immune responses to self-immunoglobulin in humans. The disturbed IgG subclass distribution in IgM-IgG IC of patients with WAIHA might influence activity of self-reactive B cells involved in the pathophysiology of the disease.

Influence of isotypes of disease-associated autoantibodies on the expression of natural autoantibody repertoires in humans.

Current understanding of immune network interactions mediated by immunoglobulins focuses on the role of idiotypes expressed on antibody variable regions. Idiotype interactions account significantly for the functional integrity of natural self-reactive antibody repertoires, whereas immunoglobulin isotypes are not considered to direct natural autoimmunity. Autoimmune thrombocytopenic purpura (AITP), a bleeding disorder caused by clonally restricted platelet-specific autoantibodies of the IgM or IgG isotype, is an excellent model to investigate the impact of isotype differences of immunoglobulins on the selection of natural self-reactive antibody repertoires in humans. Using specific analytical techniques to characterize the natural self-reactive antibody repertoire (i.e. quantitative immunoblotting, affinity biosensor technology), we here demonstrate that isotype differences of disease-associated autoantibodies are associated with altered natural self-reactive antibody repertoires in humans. Our data suggest that regulation of natural autoreactivity by antibody isotype might occur under certain conditions. The control of natural self-reactive antibody repertoires by immunoglobulin isotypes at a supraclonal level may provide a structural basis for non-organ-specific broad alterations of natural self-reactive antibody repertoires in organ-specific autoimmune diseases.