Michael B. Fischer

Binding of disease-associated prion protein to plasminogen

Transmissible spongiform encephalopathies are associated with accumulation of PrPSc, a conformer of a cellular protein called PrP C. PrPSc is thought to replicate by imparting its conformation onto PrPC (ref. 1), yet conformational discrimination between PrPC and PrPSc has remained elusive. Because deposition of PrPSc alone is not enough to cause neuropathology, PrPSc probably damages the brain by interacting with other cellular constituents. Here we find activities in human and mouse blood which bind PrPSc and prion infectivity, but not PrPC. We identify plasminogen, a pro-protease implicated in neuronal excitotoxicity, as a PrPSc-binding protein. Binding is abolished if the conformation of PrPSc is disrupted by 6M urea or guanidine. The isolated lysine binding site 1 of plasminogen (kringles I–III) retains this binding activity, and binding can be competed for with lysine. Therefore, plasminogen represents the first endogenous factor discriminating between normal and pathological prion protein. This unexpected property may be exploited for diagnostic purposes.

Assessment of a six gene panel for the molecular detection of circulating tumor cells in the blood of female cancer patients

BackgroundThe presence of circulating tumor cells (CTC) in the peripheral blood of cancer patients has been described for various solid tumors and their clinical relevance has been shown. CTC detection based on the analysis of epithelial antigens might be hampered by the genetic heterogeneity of the primary tumor and loss of epithelial antigens. Therefore, we aimed to identify new gene markers for the PCR-based detection of CTC in female cancer patients.MethodsGene expression of 38 cancer cell lines (breast, ovarian, cervical and endometrial) and of 10 peripheral blood mononuclear cell (PBMC) samples from healthy female donors was measured using microarray technology (Applied Biosystems). Differentially expressed genes were identified using the maxT test and the 50% one-sided trimmed maxT-test. Confirmatory RT-qPCR was performed for 380 gene targets using the AB TaqMan® Low Density Arrays. Then, 93 gene targets were analyzed using the same RT-qPCR platform in tumor tissues of 126 patients with primary breast, ovarian or endometrial cancer. Finally, blood samples from 26 healthy women and from 125 patients (primary breast, ovarian, cervical, or endometrial cancer, and advanced breast cancer) were analyzed following OncoQuick enrichment and RNA pre-amplification. Likewise, hMAM and EpCAM gene expression was analyzed in the blood of breast and ovarian cancer patients. For each gene, a cut-off threshold value was set at three standard deviations from the mean expression level of the healthy controls to identify potential markers for CTC detection.ResultsSix genes were over-expressed in blood samples from 81% of patients with advanced and 29% of patients with primary breast cancer. EpCAM gene expression was detected in 19% and 5% of patients, respectively, whereas hMAM gene expression was observed in the advanced group (39%) only. Multimarker analysis using the new six gene panel positively identified 44% of the cervical, 64% of the endometrial and 19% of the ovarian cancer patients.ConclusionsThe panel of six genes was found superior to EpCAM and hMAM for the detection of circulating tumor cells in the blood of breast cancer, and they may serve as potential markers for CTC derived from endometrial, cervical, and ovarian cancers.

Circulating microparticles carry oxidation-specific epitopes and are recognized by natural IgM antibodies

Oxidation-specific epitopes (OSEs) present on apoptotic cells and oxidized low density lipoprotein (OxLDL) represent danger-associated molecular patterns that are recognized by different arcs of innate immunity, including natural IgM antibodies. Here, we investigated whether circulating microparticles (MPs), which are small membrane vesicles released by apoptotic or activated cells, are physiological carriers of OSEs. OSEs on circulating MPs isolated from healthy donors and patients with ST-segment elevation myocardial infarction (STE-MI) were characterized by flow cytometry using a panel of OSE-specific monoclonal antibodies. We found that a subset of MPs carry OSEs on their surface, predominantly malondialdehyde (MDA) epitopes. Consistent with this, a majority of IgM antibodies bound on the surface of circulating MPs were found to have specificity for MDA-modified LDL. Moreover, we show that MPs can stimulate THP-1 (human acute monocytic leukemia cell line) and human primary monocytes to produce interleukin 8, which can be inhibited by a monoclonal IgM with specificity for MDA epitopes. Finally, we show that MDA+ MPs are elevated at the culprit lesion site of patients with STE-MI. Our results identify a subset of OSE+ MPs that are bound by OxLDL-specific IgM. These findings demonstrate a novel mechanism by which anti-OxLDL IgM antibodies could mediate protective functions in CVD.

Enrichment of circulating tumor cells from a large blood volume using leukapheresis and elutriation: Proof of concept†

The aim of this study was to determine the applicability of a sequential process using leukapheresis, elutriation, and fluorescence‐activated cell sorting (FACS) to enrich and isolate circulating tumor cells from a large blood volume to allow further molecular analysis.

Inhibition of Receptor-Dependent and Receptor-Independent Generation of the Respiratory Burst in Human Neutrophils and Monocytes by Human Serum IgA

ABSTRACT: An important feature of the role of IgA in protection against infection and disease at the level of the mucosal surfaces might be the elimination of pathogens without induction of a strong inflammatory reaction. In the present study we addressed the question whether IgA has a regulatory effect on the generation of reactive oxygen intermediates in human neutrophils and monocytes (i.e. the respiratory burst). Cells were stimulated with heat-inactivated Haemophilus influenzae type b or phorbol myristate acetate, stimuli known to use different recognition structures or signal transduction pathways. Concentrations of IgA as low as 10 mg/L significantly inhibited the receptor-dependent Haemophilus influenzae-induced respiratory burst in granulocytes, as assessed by measuring luminol-enhanced chemiluminescence. Furthermore, IgA had a dose-dependent inhibitory effect on the receptor-independent induction of the respiratory burst, as examined by flow cytometry in monocytes and granulocytes activated with phorbol myristate acetate. Our results therefore indicate that inhibition of receptor-ligand interaction is not a sufficient explanation for the IgA-mediated modulation of the respiratory burst in human phagocytic cells. In addition, IgA might directly regulate the activation of the respiratory burst at the level or downstream of protein kinase C activation. By modulating the release of mediators of inflammation such as reactive oxygen intermediates, the inflammatory response could be down-regulated at the level of the mucosal surfaces, thereby preventing the development of sequelae of an exaggerated inflammatory response potentially leading to local or systemic pathology.

Platelet size, fibrinogen and lipoprotein(a) in coronary heart disease

BackgroundAn increase in mean platelet volume has been reported to be associated with arterial thrombosis and myocardial infarction. A larger mean platelet volume has been regarded as an independent risk factor for recurrent myocardial infarction. We therefore investigated whether it is also increased in patients with coronary heart disease examined a few days before cardiac surgery. MethodsFour hundred and twenty-six patients with coronary heart disease who were waiting for cardiac surgery and 125 healthy individuals were included in the study. Mean platelet volume and other platelet parameters were obtained from a routine blood count procedure using a flow cytometric haematology analyser. ResultsMean platelet volume did not differ significantly between patients and controls; however, as expected from the literature, patients had significantly elevated levels of fibrinogen, cholesterol, triglyceride, apolipoprotein B and apolipoprotein(a). Furthermore, we observed no significant difference in mean platelet volume between patients without myocardial infarction and those who had survived at least one myocardial infarction. ConclusionOur findings suggest that, using a routine laboratory procedure, mean platelet volume cannot be used as a predictive marker for coronary heart disease or myocardial infarction.

Observing cellulose biosynthesis and membrane translocation in crystallo

Many biopolymers, including polysaccharides, must be translocated across at least one membrane to reach their site of biological function. Cellulose is a linear glucose polymer synthesized and secreted by a membrane-integrated cellulose synthase. Here, in crystallo enzymology with the catalytically active bacterial cellulose synthase BcsA–BcsB complex reveals structural snapshots of a complete cellulose biosynthesis cycle, from substrate binding to polymer translocation. Substrate- and product-bound structures of BcsA provide the basis for substrate recognition and demonstrate the stepwise elongation of cellulose. Furthermore, the structural snapshots show that BcsA translocates cellulose via a ratcheting mechanism involving a ‘finger helix’ that contacts the polymer’s terminal glucose. Cooperating with BcsA’s gating loop, the finger helix moves ‘up’ and ‘down’ in response to substrate binding and polymer elongation, respectively, thereby pushing the elongated polymer into BcsA’s transmembrane channel. This mechanism is validated experimentally by tethering BcsA’s finger helix, which inhibits polymer translocation but not elongation.

Vascular morphogenesis by adult bone marrow progenitor cells in three-dimensional fibrin matrices

The neovascularization of tissues is accomplished by two distinct processes: de novo formation of blood vessels through the assembly of progenitor cells during early prenatal development (vasculogenesis), and expansion of a pre-existing vascular network by endothelial cell sprouting (angiogenesis), the main mechanism of blood vessel growth in postnatal life. Evidence exists that adult bone marrow (BM)-derived progenitor cells can contribute to the formation of new vessels by their incorporation into sites of active angiogenesis. Aim of this study was to investigate the in vitro self-organizing capacity of human BM mononuclear cells (BMMNC) to induce vascular morphogenesis in a three-dimensional (3D) matrix environment in the absence of pre-existing vessels. Whole BMMNC as well as the adherent and non-adherent fractions of BMMNC were embedded in fibrin gels and cultured for 3-4 weeks without additional growth factors. The expression of hematopoietic-, endothelial-, smooth muscle lineage, and stem cell markers was analyzed by immunohistochemistry and confocal laser-scanning microscopy. The culture of unselected BMMNC in 3D fibrin matrices led to the formation of cell clusters expressing the endothelial progenitor cell (EPC) markers CD133, CD34, vascular endothelial growth factor receptor (VEGFR)-2, and c-kit, with stellar shaped spreading of peripheral elongated cells forming tube-like structures with increasing complexity over time. Cluster formation was dependent on the presence of both adherent and non-adherent BMMNC without the requirement of external growth factors. Developed vascular structures expressed the endothelial markers CD34, VEGFR-2, CD31, von Willebrand Factor (vWF), and podocalyxin, showed basement-membrane-lined lumina containing CD45+ cells and were surrounded by alpha-smooth muscle actin (SMA) expressing mural cells. Our data demonstrate that adult human BM progenitor cells can induce a dynamic self organization process to create vascular structures within avascular 3D fibrin matrices suggesting a possible alternative mechanism of adult vascular development without involvement of pre-existing vascular structures.

Prevalence of factor XII (Hageman factor) deficiency among 426 patients with coronary heart disease awaiting cardiac surgery.

BackgroundSeveral case reports of myocardial infarction in patients with factor XII deficiency have been published. In the present study we investigated the prevalence of this condition. MethodsFactor XII activity (one-stage clotting assay), fibrinogen (derived method), and lipoprotein (a) (enzyme-linked immunosorbent assay) were measured in the plasma of 426 consecutive patients with coronary heart disease awaiting cardiac surgery. ResultsAmong the 426 patients, 44 (10.3 %) were found to be moderately deficient in factor XII (factor XII activity 17–50%, antigen 15–57%). The prevalence of factor XII deficiency was significantly higher (P < 0.0001) among patients with coronary heart disease than among 300 healthy blood donors (2.3%). Among coronary heart disease patients with this deficiency, elevated levels of fibrinogen, lipoprotein (a), and blood pressure were no more prevalent than in those without the deficiency; nor were cigarette smoking or a positive family history of thromboembolism more prevalent. ConclusionsCoronary heart disease patients showed a 10% prevalence of factor XII deficiency. However, the pattern of atherosclerotic risk factors did not differ between patients with or without the deficiency.

Activated platelets increase fibrinolysis of mesenchymal progenitor cells.

Bone regeneration is initiated by the formation of a blood clot. Activated platelets within this fibrin‐rich matrix release signaling molecules that can attract mesenchymal progenitor cells. To gain insight into the cellular mechanism by which activated platelets can support the immigration of mesenchymal progenitors, we have tested the hypothesis that platelet‐released signaling molecules increase the capacity of bone marrow stromal cells (BMSC) to activate plasminogen. We report herein that platelet‐released supernatants (PRS) elevate total urokinase‐type plasminogen activator (uPA) and total plasminogen activator inhibitor‐1 (PAI‐1) levels in BMSC, as assessed by immunoassay. Quantitative polymerase chain reaction showed an upregulation of uPA, uPA receptor, and PAI‐1. Zymography and kinetic analysis based on casein hydrolysis revealed enhanced activity of cell‐associated uPA upon exposure of BMSC to PRS. Inhibiting c‐Jun N‐terminal kinase (JNK) and phosphatidylinositol 3‐kinase (PI3K) signaling reduced uPA production and decreased plasminogen activation. Corresponding Western blot analysis showed increased phosphorylation of JNK and AKT in BMSC treated with PRS. These results suggest that activated platelets can enhance the plasminogen activation capacity of mesenchymal progenitors through the stimulation of uPA production, requiring JNK and PI3K/AKT signaling. By this mechanism platelets may contribute to the organization of the blood clot during bone regeneration.