Radionuclide accumulation in near-shore sediments along the Bulgarian Black Sea coast

The accumulation of radionuclides in Black Sea marine ecosystems was investigated by low level gamma spectrometry. Artificial as well as natural radionuclides were determined in bottom sediments samples from 35 reference locations along the Bulgarian Black Sea coast, evenly distributed from the Rumanian to the Turkish border including the main Black Sea resorts and rivers. The measurement of radionuclides in sea bed sediments was carried out during six consecutive seasons using a HPGe detector. The data obtained show that the nuclide concentrations depend strongly on the sediment nature. Results for sandy sediments are within close range, while those for slime and silt vary to a much greater extent. The radionuclide content in the sandy sediments of the main Black Sea resorts is at the lowest limit of the determined values. Small seasonal changes of radionuclide concentration in sandy sediments were observed while greater variations in slime and silt occur. From the data obtained ¹³⁴Cs/¹³⁷Cs and ¹³⁷Cs_meas/¹³⁷Cs_Chern ratios are calculated to determine the Chernobyl part of the measured ¹³⁷Cs. The activities determined in the sediments for natural radionuclides correspond to those cited in the literature for natural levels, showing no additional anthropogenic contamination. A data base for the nuclide concentration values was created which will enable the modeling of radionuclide transfers by estimation of their concentration variations, accumulation and influence on the marine ecosystems.     

Epitope mapping of a PrP(Sc)-specific monoclonal antibody: identification of a novel C-terminally truncated prion fragment

Monoclonal antibodies (mAbs) against prion proteins (PrPs) are indispensable in research and diagnosis of prion diseases, however the majority of these bind both the cellular (PrP^C) and the disease-associated (PrP^Sc) isoforms. According to the widely accepted protein-only hypothesis the two isoforms share the same sequence, but differ in their conformation. In the present study we set to determine the critical binding residues of our PrP^Sc-specific mAbs with the view of discerning which residues play a key role in the conformational transition between PrP^C and PrP^Sc. Focussing on the V5B2 mAb that provided differential labelling of prion-affected tissue from individuals positive for transmissible spongiform encephalopathies, we performed alanine scanning and phage-display epitope mapping to elucidate the antigenic determinants of this mAb and gain insight into its specificity on a molecular level. We observed that instead of discriminating between the two prion protein isoforms based on conformational differences, V5B2 binds a previously uncharacterized C-terminally truncated form of PrP^Sc that ends with the residue Y226, which we named PrP226*. The addition of a single C-terminal amino-acid residue completely abolished V5B2 binding, while Western blots using recombinant full-length PrPs and PrPs terminating at Y226 confirmed that the V5B2 mAb discriminates between the two based on their difference in length.     

Angiogenesis: a prognostic determinant in pancreatic cancer?

Angiogenesis has been associated with disease progression in many solid tumours, however the statement that tumours need angiogenesis to grow, invade and metastasise seems no longer applicable to all tumours or to all tumour subtypes. Prognostic studies in pancreatic cancer are conflicting. In fact, pancreatic cancer has been suggested an example of a tumour in which angiogenesis is less essential for tumour progression. The aim of the present study was therefore to measure angiogenesis in two anatomically closely related however prognostically different types of pancreatic cancer, pancreatic head and periampullary cancer, and investigate its relation with outcome. Vessels were stained by CD31 on original paraffin embedded tissue from 206 patients with microscopic radical resection (R0) of pancreatic head (n=98) or periampullary cancer (n=108). Angiogenesis was quantified by microvessel density (MVD) and measured by computerised image analysis of three randomly selected fields and investigated for associations with recurrence free survival (RFS), cancer specific survival (CSS), overall survival (OS) and conventional prognostic factors. MVD was heterogeneous both between and within tumours. A higher MVD was observed in periampullary cancers compared with pancreatic head cancers (p<.01). Furthermore, MVD was associated with lymph node involvement in pancreatic head (p=.014), but not in periampullary cancer (p=.55). Interestingly, MVD was not associated with RFS, CSS or with OS. In conclusion, angiogenesis is higher in periampullary cancer and although associated with nodal involvement in pancreatic head cancer, pancreatic cancer prognosis seems indeed angiogenesis independent.     

Intact Doxil is taken up intracellularly and released doxorubicin sequesters in the lysosome: Evaluated by in vitro/in vivo live cell imaging

Doxil, also known as Caelyx, is an established liposomal formulation of doxorubicin used for the treatment of ovarian cancer, sarcoma and multiple myeloma. While showing reduced doxorubicin related toxicity, Doxil does not greatly improve clinical outcome. To become biologically active, doxorubicin needs to be released from its carrier. Uptake and breakdown of the liposomal carrier and subsequent doxorubicin release is not fully understood and in this study we explored the hypothesis that Doxil is taken up by tumor cells and slowly degraded intracellularly. We investigated the kinetics of liposomal doxorubicin (Doxil) in vitro as well as in vivo by measuring cytotoxic effect, intracellular bioavailability and fate of the carrier and its content. To prevent fixation artifacts we applied live cell imaging in vitro and intravital microscopy in vivo. Within 8 h after administration of free doxorubicin, 26% of the drug translocated to the nucleus and when reaching a specific concentration killed the cell. Unlike free doxorubicin, only 0.4% of the doxorubicin added as liposomal formulation entered the nucleus. Looking at the kinetics, we observed a build-up of nuclear doxorubicin within minutes of adding free doxorubicin. This was in contrast to Doxil showing slow translocation of doxorubicin to the nucleus and apparent accumulation in the cytoplasm. Observations made with time-lapse live cell imaging as well as in vivo intravital microscopy revealed the liposomal carrier colocalizing with doxorubicin in the cytoplasm. We also demonstrated the sequestering of liposomal doxorubicin in the lysosomal compartment resulting in limited delivery to the nucleus. This entrapment makes the bioavailable concentration of Doxil-delivered doxorubicin significantly lower and therefore ineffective as compared to free doxorubicin in killing tumor cells.     

Cell surface expression of an endoplasmic reticulum resident heat shock protein gp96 triggers MyD88-dependent systemic autoimmune diseases

Heat shock proteins have been implicated as endogenous activators for dendritic cells (DCs). Without tissue distress or death, these intracellular molecules are inaccessible to surface receptor(s) on DCs, possibly to avoid uncontrolled DC activation and breakdown of immunologic tolerance. We herein addressed this hypothesis in transgenic mice by enforcing cell surface expression of gp96, a ubiquitous heat shock protein of the endoplasmic reticulum. Although a pan-specific promoter is used for transgene expression, neither the expression level nor the tissue distribution of the endogenous gp96 was altered by this maneuver. However, cell surface gp96 induced significant DC activations and spontaneous lupus-like autoimmune diseases, even though the development/functions of lymphocytic compartments were unaltered. Using a bone marrow chimera approach, we further demonstrated that both DC activation and autoimmunity elicited by cell surface gp96 are dependent on the downstream adaptor protein MyD88 for signaling by Toll/IL-1 receptor family. Our study not only established the proinflammatory property of cell surface gp96 in vivo, but also suggested a chronic stimulation of DCs by gp96 as a pathway to initiate spontaneous autoimmune diseases.     

Lack of recombinant factor VIII B‐domain induces phospholipid vesicle aggregation: implications for the immunogenicity of factor VIII

Factor VIII (FVIII) is a multidomain blood plasma glycoprotein. Activated FVIII acts as a cofactor to the serine protease factor IXa within the membrane‐bound tenase complex assembled on the activated platelet surface. Defect or deficiency in FVIII causes haemophilia A, a severe hereditary bleeding disorder. Intravenous administration of plasma‐derived FVIII or recombinant FVIII concentrates restores normal coagulation in haemophilia A patients and is used as an effective therapy. In this work, we studied the biophysical properties of clinically potent recombinant FVIII forms: human FVIII full‐length (FVIII‐FL), human FVIII B‐domain deleted (FVIII‐BDD) and porcine FVIII‐BDD bound to negatively charged phospholipid vesicles at near‐physiological conditions. We used cryo‐electron microscopy (Cryo‐EM) as a direct method to evaluate the homogeneity and micro‐organization of the protein‐vesicle suspensions, which are important for FVIII therapeutic properties. Applying concurrent Cryo‐EM, circular dichroism and dynamic light scattering studies to the three recombinant FVIII forms when bound to phospholipid vesicles revealed novel properties for their functional, membrane‐bound state. The three FVIII constructs have similar activity, secondary structure distribution and bind specifically to negatively charged phospholipid membranes. Human and porcine FVIII‐BDD induce strong aggregation of the vesicles, but the human FVIII‐FL form does not. The proposed methodology is effective in characterizing and identifying differences in therapeutic recombinant FVIII membrane‐bound forms near physiological conditions, because protein‐containing aggregates are considered to be a factor in increasing the immunogenicity of protein therapeutics. This will provide better characterization and development of safer and more effective FVIII products with implications for haemophilia A treatment.