Bodo Lachmann

Validation of in vitro cell culture models of the blood–brain barrier: Tightness characterization of two promising cell lines

In the course of the validation of blood-brain barrier in vitro models the aim of this work was to characterize two promising continuous cell lines with regard to their tightness properties. PBMEC/C1-2 and ECV304 cells were cultured in several media with different compositions on Transwell inserts. Inducibility and functionality of tightness were investigated by transendothelial electrical resistance (TEER) and by transport studies with transcellular marker diazepam, glycine antagonist Bu101 and paracellular marker APTS-dextran. Inducibility, expression and localization of tight junctional proteins were assessed by western blotting and immunofluorescence microscopy. Presence of factors derived from glioma cell line C6 resulted in increased TEER in both cell lines. Comparison to APTS-dextran data across Caco-2 layers emphasized that correlations between permeability of the paracellular marker and TEER are dependent on each investigated cell line and the corresponding growth medium. Presence and inducibility of tight junctional proteins ZO-1 and Occludin were proven for ECV304 layers. Cell line ECV304 seemed to be suitable for TEER dependent transport studies, whereas PBMEC/C1-2 showed higher potential for P-gP studies.

Two-dimensional electrophoresis of recombinant human erythropoietin: A future method for the European Pharmacopoeia?

Quality assurance of recombinant protein drugs concerning identity and purity represents a difficult task, in particular, when post‐translational modifications lead to a heterogeneous mixture of biomolecules. We chose Neorecormon® (rh‐EPO, Roche) for our studies to demonstrate the efficiency of two‐dimensional electrophoresis (2‐DE) to analyse post‐translationally modified recombinant drugs. More than 40 protein spots in the range from isoelectric point (pI) 3.5–4.5 and 32–45 kDa could be separated. Enzymatic deglycosylation revealed that the heterogeneity of the protein pattern is mainly caused by variations in glycosylation. In comparison to the separately performed isoelectric focusing and sodium dodecyl sulfate‐polyacrylamide gel electrophoresis, as requested by the European Pharmacopoeia, we see a great synergy to use 2‐DE for the analysis of rh‐EPO. A by far higher resolution can be achieved, allowing an improved differentiation of the various rh‐EPO glycoforms. Sequential deglycosylation of sialic acids, N‐glycosides and the O‐glycoside lead to significant shifts both in apparent relative molecular mass and pI. Comparing the 2‐DE patterns of rh‐EPO before and after deglycosylation allows on the one hand valuable information to be gained on the glycosylation of the recombinant protein and shows on the other hand how significantly the 2‐DE protein pattern can be influenced by the glycosylation. As the equipment for the performance of 2‐DE has improved significantly over the last decade, we see 2‐DE as a reliable method, which should be approved for the routine quality assurance of recombinant drugs and also recommended for the European Pharmacopoeia.

Induction of Apoptosis by Vitamin D Metabolites and Analogs in a Glioma Cell Line

Gliomas are the most common malignant tumors in brain. Recent studies demonstrate the capacity of 1alpha,25(OH)2D3 to specifically induce cell death (apoptosis) in model glioma cell lines and in primary cultures from tumor tissue, but not in primary astrocytes. In spite of this promising activity, a broad therapeutic application of vitamin D metabolites and analogs is still restricted because of their poor bioavailability and their hypercalcemic actions. Compared to 1alpha,25(OH)2D3, its natural 3alpha-epimer exhibits far higher metabolic stability and a reduced calcemic effect. Focusing on a possible therapeutic advantage of the 3alpha-conformation, we have examined the apoptotic potential of a representative set of vitamin D analogs, each of them in the 3alpha- and 3beta-conformation, and of natural vitamin D metabolites in the rat C6 glioma cell line. Exposure of these cells to the synthetic analogs resulted in all cases in a pronounced reduction of cell density (tested by incorporation of neutral red) and induction of apoptosis, monitored by staining nuclei with Hoechst 33258 dye and by following DNA fragmentation by capillary electrophoresis. The 3alpha-epimers showed equivalent or even higher activity on C6 cells than their respective 3beta forms. For their potent effects on growth and apoptosis of tumor cells and their high metabolic stability combined with a low calcemic potential, we speculate that these 3a-epimers could provide advantages for a prospective treatment of glioma.

A Novel Tool to Characterize Paracellular Transport: The APTS-Dextran Ladder

PurposeThe aim of this work was to develop an easy, manageable, and precise analytic tool to describe the tightness of cell layers by a molecular weight ladder.MethodsDextrans were labeled by reductive amination with fluorescent 8-aminopyrene-1,3,6-trisulfonate (APTS). This mixture, including the internal standard diazepam, was used for transport studies in Transwell models using Caco-2, ECV304, and PBMEC/C1–2 cell lines. Samples were analyzed by fluorimetry, capillary electrophoresis, and reverse-phase high-performance liquid chromatography.ResultsFollowing this approach, a logarithm correlation of R2 = 0.8958 between transepithelial electrical resistance (TEER) and APTS–dextran permeability was shown. In addition, a TEER-dependent permeability pattern could be observed including each single fraction from free APTS, APTS–glucose up to APTS–dextran consisting of 35 glucose units. The TEER-independent permeability coefficients of diazepam and confocal laser scanning microscopy images confirmed the paracellular transport of APTS–dextran.ConclusionsAll in all, the developed APTS–dextran ladder is a useful tool to characterize cell layer tightness and especially to describe paracellular transport ways and the extent of leakiness of cell layers (for blood–brain barrier or intestinal studies) over time—applying a wide array from smaller to larger molecules at the same time to refine TEER, sucrose, or Evans blue measurements.

Transport Rankings of Non-Steroidal Antiinflammatory Drugs across Blood-Brain Barrier In Vitro Models

The aim of this work was to conduct a comprehensive study about the transport properties of NSAIDs across the blood-brain barrier (BBB) in vitro. Transport studies with celecoxib, diclofenac, ibuprofen, meloxicam, piroxicam and tenoxicam were accomplished across Transwell models based on cell line PBMEC/C1-2, ECV304 or primary rat brain endothelial cells. Single as well as group substance studies were carried out. In group studies substance group compositions, transport medium and serum content were varied, transport inhibitors verapamil and probenecid were added. Resulted permeability coefficients were compared and normalized to internal standards diazepam and carboxyfluorescein. Transport rankings of NSAIDs across each model were obtained. Single substance studies showed similar rankings as corresponding group studies across PBMEC/C1-2 or ECV304 cell layers. Serum content, glioma conditioned medium and inhibitors probenecid and verapamil influenced resulted permeability significantly. Basic differences of transport properties of the investigated NSAIDs were similar comparing all three in vitro BBB models. Different substance combinations in the group studies and addition of probenecid and verapamil suggested that transporter proteins are involved in the transport of every tested NSAID. Results especially underlined the importance of same experimental conditions (transport medium, serum content, species origin, cell line) for proper data comparison.

Influence of image-analysis software on quantitation of two-dimensional gel electrophoresis data

Image analysis of two‐dimensional gels is a crucial step in a proteomic workflow and has a direct impact on obtained qualitative and quantitative data. Since the analysis is a complex process and creates large data amounts, the use of a respective software is inevitable. There are only a few papers published addressing the issue of analysis‐based variance; therefore, our aim was to highlight the discrepancy of received results when different commercially available image‐tools are used for gel analysis especially in terms of comparability of the obtained outcome when the same digital image set is used. A set of six gels (three replicates per group) of real‐life samples was created and examined with two different versions of PD‐Quest (Bio‐Rad) (version 6.1 and its update version 8.0) and with an external image‐tool Delta 2D (Decodon) (version 3.6). Replicate groups were analyzed and compared with each other with regard to volume ratios of a group of significantly changed spots. The study points out significant variations among results depending on the software package used, underlining the importance of a careful investigation of post‐experimental processes to receive comparable and reliable results.

A proteomic study reveals unspecific apoptosis induction and reduction of glycolytic enzymes by the phosphorothioate antisense oligonucleotide oblimersen in human melanoma cells

The question of specificity and the elucidation of the exact molecular mechanism of action of post-transcriptional gene silencing agents are two major challenges for their establishment as therapeutics. A proteomic off-target effect study (2-DE with MS) in combination with DIGE comparing the phosphorothioate antisense oligonucleotide oblimersen (Genasense, G3139) to a Bcl-2-targeting siRNA-sequence on human melanoma cells showed that additional off-target effects contribute to the apoptotic effect of oblimersen. When both oligonucleotides were transfected with lipofectamine 2000, only oblimersen increased apoptosis as determined by annexin staining and caspase activity measurement. In contrast to the highly specific siRNA, the expression level of a number of proteins was found to be altered after oblimersen treatment. Several proteins linked to apoptosis and stress response, among those galectin-1, cofilin-1, GRP78, HSP60, nucleophosmin, and peroxiredoxins, were identified and found to be down-regulated after oblimersen treatment. A down-regulation of enolase-1 and three other glycolytic enzymes indicates a reversion of the cancer-related Warburg effect. The observed effects may be caused by a phosphorothioate mediated blockage of the mitochondrial voltage dependent anion channel (VDAC).

Comparison of two-dimensional gel electrophoresis patterns and MALDI-TOF MS analysis of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab

The principal objective of this study was the evaluation of two-dimensional gel electrophoresis (2-DE) in combination with MALDI-TOF MS, after tryptic digest with regard to suitability for qualitative characterization and identification of therapeutic recombinant monoclonal antibodies trastuzumab and rituximab. Moreover, the impact of post-translational modifications of these glycoproteins on the electrophoresis behavior has been evaluated. 1-D SDS-PAGE, in reducing and non-reducing conditions, and 2-DE were used for the assessment of M(r) and the monitorization of deglycosylation efficiency. In addition, 2-DE was used for the determination of pIs. 2-DE gels revealed characteristic glycoprotein migration behavior, highly complex spot pattern, typical for recombinant monoclonal antibodies. N-linked oligosaccharides were released with PNGase F; enzymatic desialination was studied with sialidase and carboxypeptidase B was used for the study of lysine truncation. Peptide spots resolved in 2-DE gels were in gel tryptically digested, resulting peptides were subjected to MALDI-TOF MS analysis and peptide mass fingerprinting (PMF) has been used for the identity confirmation of both monoclonal antibodies.

Comparison of aragonitic molluscan shell proteins.

Acidic macromolecules, as a nucleation factor for mollusc shell formation, are a major focus of research. It remains unclear, however, whether acidic macromolecules are present only in calcified shell organic matrices, and which acidic macromolecules are crucial for the nucleation process by binding to chitin as structural components. To clarify these questions, we applied 2D gel electrophoresis and amino acid analysis to soluble shell organic matrices from nacre shell, non-nacre aragonitic shell and non-calcified squid shells. The 2D gel electrophoresis results showed that the acidity of soluble proteins differs even between nacre shells, and some nacre (Haliotis gigantea) showed a basic protein migration pattern. Non-calcified shells also contained some moderately acidic proteins. The results did not support the correlation between the acidity of soluble shell proteins and shell structure.

Quality Control and Stability Studies with the Monoclonal Antibody, Trastuzumab: Application of 1D- vs. 2D-Gel Electrophoresis

Recombinant monoclonal antibodies (rmAbs) are medicinal products obtained by rDNA technology. Consequently, like other biopharmaceuticals, they require the extensive and rigorous characterization of the quality attributes, such as identity, structural integrity, purity and stability. The aim of this work was to study the suitability of gel electrophoresis for the assessment of charge heterogeneity, post-translational modifications and the stability of the therapeutic, recombinant monoclonal antibody, trastuzumab. One-dimensional, SDS-PAGE, under reducing and non-reducing conditions, and two-dimensional gel electrophoresis were used for the determination of molecular mass (Mr), the isoelectric point (pI), charge-related isoform patterns and the stability of trastuzumab, subjected to stressed degradation and long-term conditions. For the assessment of the influence of glycosylation in the charge heterogeneity pattern of trastuzumab, an enzymatic deglycosylation study has been performed using N-glycosidase F and sialidase, whereas carboxypeptidase B was used for the lysine truncation study. Experimental data documented that 1D and 2D gel electrophoresis represent fast and easy methods to evaluate the quality of biological medicinal products. Important stability parameters, such as the protein aggregation, can be assessed, as well.