Wouter W. van Solinge

Exosome mimetics: a novel class of drug delivery systems

The identification of extracellular phospholipid vesicles as conveyors of cellular information has created excitement in the field of drug delivery. Biological therapeutics, including short interfering RNA and recombinant proteins, are prone to degradation, have limited ability to cross biological membranes, and may elicit immune responses. Therefore, delivery systems for such drugs are under intensive investigation. Exploiting extracellular vesicles as carriers for biological therapeutics is a promising strategy to overcome these issues and to achieve efficient delivery to the cytosol of target cells. Exosomes are a well studied class of extracellular vesicles known to carry proteins and nucleic acids, making them especially suitable for such strategies. However, the considerable complexity and the related high chance of off-target effects of these carriers are major barriers for translation to the clinic. Given that it is well possible that not all components of exosomes are required for their proper functioning, an alternative strategy would be to mimic these vesicles synthetically. By assembly of liposomes harboring only crucial components of natural exosomes, functional exosome mimetics may be created. The low complexity and use of well characterized components strongly increase the pharmaceutical acceptability of such systems. However, exosomal components that would be required for the assembly of functional exosome mimetics remain to be identified. This review provides insights into the composition and functional properties of exosomes, and focuses on components which could be used to enhance the drug delivery properties of exosome mimetics.

Microvesicles and exosomes: Opportunities for cell-derived membrane vesicles in drug delivery

Cell-derived membrane vesicles (CMVs) are endogenous carriers transporting proteins and nucleic acids between cells. They appear to play an important role in many disease processes, most notably inflammation and cancer, where their efficient functional delivery of biological cargo seems to contribute to the disease progress. CMVs encompass a variety of submicron vesicular structures that include exosomes and shedding vesicles. The lipids, proteins, mRNA and microRNA (miRNA) delivered by these vesicles change the phenotype of the receiving cells. CMVs have created excitement in the drug delivery field, because they appear to have multiple advantages over current artificial drug delivery systems. Two approaches to exploit CMVs for delivery of exogenous therapeutic cargoes in vivo are currently considered. One approach is based on engineering of natural CMVs in order to target certain cell types using CMVs loaded with therapeutic compounds. In the second approach, essential characteristics of CMVs are being used to design nano-scaled drug delivery systems. Although a number of limiting factors in the clinical translation of the exciting research findings so far exist, both approaches are promising for the development of a potentially novel generation of drug carriers based on CMVs.

Expression but incomplete maturation of progastrin in colorectal carcinomas.

BACKGROUND To evaluate the hypothesis that gastrin is a local growth factor in colonic carcinomas, the expression of gastrin messenger RNA (mRNA) and peptides were examined in five human colon carcinoma cell lines, 12 solid colon carcinomas, and normal colonic tissue. METHODS Northern analysis, reverse-transcription PCR, and a library of sequence-specific radioimmunoassays were the principal methods. RESULTS Cell lines, tumors, and normal tissue all expressed a gastrin mRNA of 0.7 kilobases, and all cell lines contained incompletely processed progastrin (range, 17-54 fmol/10(6) cells). Two cell lines secreted progastrin into the media (LoVo, 25 +/- 3 pmol/L; HCT116; 12 +/- 2 pmol/L). Normal colonic tissue and all the solid tumors also contained progastrin, the concentration being higher in tumors (range, 0.4-2 pmol/g) than in normal tissue (range, 0.1-0.2 pmol/g). Only one tumor contained carboxyamidated gastrins. CONCLUSIONS Normal and neoplastic colonic mucosa both express the gastrin gene, but the posttranslational phase of expression is attenuated. The incomplete processing and low level of expression suggest that autocrine gastrin secretion has only minor significance for normal adult and most neoplastic colonic tissue.

Linking Laboratory and medication data: new opportunities for pharmacoepidemiological research

Abstract Transfer of automated laboratory data collected during routine clinical care from the laboratory information system into a database format that enables linkage to other administrative (e.g., patient characteristics) or clinical (e.g., medication, diagnoses, procedures) data provides a valuable tool for clinical epidemiological research. It allows the investigation of biochemical characteristics of diseases, therapeutic effects and diagnostic and/or prognostic markers for disease with easy access and at relatively low cost. To this end, the Utrecht Patient Oriented Database (UPOD), an infrastructure of relational databases comprising data on patient characteristics, laboratory test results, medication orders, hospital discharge diagnoses and medical procedures for all patients treated at the University Medical Centre Utrecht since January 2004, was established. Current research within UPOD is focused on the innovative linkage of laboratory and medication data, which, for example, makes it possible to assess the quality of pharmacotherapy in clinical practice, to investigate interference between laboratory tests and drugs, to study the risk of adverse drug reactions, and to develop diagnostic and prognostic markers or algorithms for adverse drug reactions. Although recently established, we believe that UPOD broadens the opportunities for clinical pharmacoepidemiological research and can contribute to patient care from a laboratory perspective. Clin Chem Lab Med 2007;45:13–9.

Highly efficient depletion strategy for the two most abundant erythrocyte soluble proteins improves proteome coverage dramatically.

In-depth human erythrocyte proteome studies are severely hampered by the presence of hemoglobin and carbonic anhydrase-1, which account for more than 98% of the total erythrocyte soluble protein content. We developed a specific depletion approach that resulted in a drastic increase in the number of identified proteins. This depletion technique is valuable for proteome studies of human erythrocyte disorders with unknown etiology and of tissue samples that contain blood.

Quantitative erythrocyte membrane proteome analysis with Blue-Native/SDS PAGE

The erythrocyte membrane plays a pivotal role in erythrocyte functioning. Many membrane protein aberrations are known that result in hemolytic anemia, however, the origin of numerous disorders is not known to date. To extend the current set of diagnostic tools, we used a novel proteome-wide approach to quantitatively analyze membrane proteins of healthy donor and patient erythrocytes. Blue-native PAGE has proven to be a powerful tool for separation of membrane proteins and their complexes, but has hitherto not been applied to erythrocyte membranes to find biomarkers. Using this technique, we detected almost 150 protein spots, from which more than 500 proteins could be identified by LC-MS/MS. Further, we successfully assessed the potential of using CyDye labeling to quantify the membrane proteins. Our final goal was to determine if this approach is suited to detect protein level changes in disordered erythrocyte membranes, and we could successfully confirm that erythrocyte spectrin levels were dramatically decreased for a hemolytic anemia patient. This approach provides a new tool to detect potential biomarkers and can contribute to an improved understanding of the causes of erythrocyte membrane defects in patients suffering from hemolytic anemia.

When blood transfusion medicine becomes complicated due to interference by monoclonal antibody therapy

Monoclonal antibodies (MoAbs) are increasingly integrated in the standard of care. The notion that therapeutic MoAbs can interfere with clinical laboratory tests is an emerging concern that requires immediate recognition and the development of appropriate solutions. Here, we describe that treatment of multiple myeloma patients with daratumumab, a novel anti‐CD38 MoAb, resulted in false‐positive indirect antiglobulin tests (IATs) for all patients for 2 to 6 months after infusion. This precluded the correct identification of irregular blood group antibodies for patients requiring blood transfusion.

The selective NLRP3-inflammasome inhibitor MCC950 reduces infarct size and preserves cardiac function in a pig model of myocardial infarction.

Aims Myocardial infarction (MI) triggers an intense inflammatory response that is associated with infarct expansion and is detrimental for cardiac function. Interleukin (IL)-1β and IL-18 are key players in this response and are controlled by the NLRP3-inflammasome. In the current study, we therefore hypothesized that selective inhibition of the NLRP3-inflammasome reduces infarct size and preserves cardiac function in a porcine MI model. Methods and results Thirty female landrace pigs were subjected to 75 min transluminal balloon occlusion and treated with the NLRP3-inflammasome inhibitor MCC950 (6 or 3 mg/kg) or placebo for 7 days in a randomized, blinded fashion. After 7 days, 3D-echocardiography was performed to assess cardiac function and Evans blue/TTC double staining was executed to assess the area at risk (AAR) and infarct size (IS). The IS/AAR was lower in the 6 mg/kg group (64.6 ± 8.8%, P = 0.004) and 3 mg/kg group (69.7 ± 7.2%, P = 0.038) compared with the control group (77.5 ± 6.3%). MCC950 treatment markedly preserved left ventricular ejection fraction in treated animals (6 mg/kg 47 ± 8%, P = 0.001; 3 mg/kg 45 ± 7%, P = 0.031; control 37 ± 6%). Myocardial neutrophil influx was attenuated in treated compared with non-treated animals (6 mg/kg 132 ± 72 neutrophils/mm2, P = 0.035; 3 mg/kg 207 ± 210 neutrophils/mm2, P = 0.5; control 266 ± 158 neutrophils/mm2). Myocardial IL-1β levels were dose-dependently reduced in treated animals. Conclusions NLRP3-inflammasome inhibition reduces infarct size and preserves cardiac function in a randomized, blinded translational large animal MI model. Hence, NLRP3-inflammasome inhibition may have therapeutic potential in acute MI patients.

Display of GPI-anchored anti-EGFR nanobodies on extracellular vesicles promotes tumour cell targeting

Background Extracellular vesicles (EVs) are attractive candidate drug delivery systems due to their ability to functionally transport biological cargo to recipient cells. However, the apparent lack of target cell specificity of exogenously administered EVs limits their therapeutic applicability. In this study, we propose a novel method to equip EVs with targeting properties, in order to improve their interaction with tumour cells. Methods EV producing cells were transfected with vectors encoding for anti-epidermal growth factor receptor (EGFR) nanobodies, which served as targeting ligands for tumour cells, fused to glycosylphosphatidylinositol (GPI) anchor signal peptides derived from decay-accelerating factor (DAF). EVs were isolated using ultrafiltration/size-exclusion liquid chromatography and characterized using western blotting, Nanoparticle Tracking Analysis, and electron microscopy. EV–tumour cell interactions were analyzed under static conditions using flow cytometry and under flow conditions using a live-cell fluorescence microscopy-coupled perfusion system. Results V analysis showed that GPI-linked nanobodies were successfully displayed on EV surfaces and were highly enriched in EVs compared with parent cells. Display of GPI-linked nanobodies on EVs did not alter general EV characteristics (i.e. morphology, size distribution and protein marker expression), but greatly improved EV binding to tumour cells dependent on EGFR density under static conditions. Moreover, nanobody-displaying EVs showed a significantly improved cell association to EGFR-expressing tumour cells under flow conditions. Conclusions We show that nanobodies can be anchored on the surface of EVs via GPI, which alters their cell targeting behaviour. Furthermore, this study highlights GPI-anchoring as a new tool in the EV toolbox, which may be applied for EV display of a variety of proteins, such as antibodies, reporter proteins and signaling molecules.

Management of Gene Promoter Mutations in Molecular Diagnostics

BACKGROUND Although promoter mutations are known to cause functionally important consequences for gene expression, promoter analysis is not a regular part of DNA diagnostics. CONTENT This review covers different important aspects of promoter mutation analysis and includes a proposed model procedure for studying promoter mutations. Characterization of a promoter sequence variation includes a comprehensive study of the literature and databases of human mutations and transcription factors. Phylogenetic footprinting is also used to evaluate the putative importance of the promoter region of interest. This in silico analysis is, in general, followed by in vitro functional assays, of which transient and stable transfection assays are considered the gold-standard methods. Electrophoretic mobility shift and supershift assays are used to identify trans-acting proteins that putatively interact with the promoter region of interest. Finally, chromatin immunoprecipitation assays are essential to confirm in vivo binding of these proteins to the promoter. SUMMARY Although promoter mutation analysis is complex, often laborious, and difficult to perform, it is an essential part of the diagnosis of disease-causing promoter mutations and improves our understanding of the role of transcriptional regulation in human disease. We recommend that routine laboratories and research groups specialized in gene promoter research cooperate to expand general knowledge and diagnosis of gene-promoter defects.