Quantification of interactions between drug leads and serum proteins by use of "binding efficiency"

To develop efficient and reliable methods for prediction of serum protein binding of drug leads, the kinetic characteristics for the interactions between selected compounds and human serum albumin and α₁-acid glycoprotein have been explored using a surface plasmon resonance biosensor. Conventional methods for quantification of interactions (i.e., using rate constants or affinities determined on the basis of a reasonable mechanistic model) were applicable for only a few of the compounds. The affinity of a primary interaction and the contribution of lower affinity secondary interactions could be estimated for some compounds, but the affinity of many compounds could not be quantified by either of these methods. To have a quantification method that could be used for all compounds, independent of affinity and complexity of interaction mechanisms, the concept of “binding efficiency,” analogous to “catalytic efficiency” used for enzymes, was developed. It allowed the quantification of the binding of compounds interacting with weak affinity and for which saturation is not reached within a concentration range where the compound is soluble or when the influence of interactions with secondary sites makes interpretations difficult. In addition, compounds with large fractional binding can be identified by this strategy and simply quantified relative to reference compounds. This approach will enable ranking and identification of structure–activity relationships of compounds with respect to their serum protein binding profile.     

Lack of collagen XVIII long isoforms affects kidney podocytes, whereas the short form is needed in the proximal tubular basement membrane

Collagen XVIII is characterized by three variant N termini, an interrupted collagenous domain, and a C-terminal antiangiogenic domain known as endostatin. We studied here the roles of this collagen type and its variant isoforms in the mouse kidney. Collagen XVIII appeared to be in a polarized orientation in the tubular basement membranes (BMs), the endostatin domain embedded in the BM, and the N terminus residing at the BM-fibrillar matrix interface. In the case of the glomerular BM (GBM), collagen XVIII was expressed in different isoforms depending on the side of the GBM. The orientation appeared polarized here, too, both the endothelial promoter 1-derived short variant of collagen XVIII and the epithelial promoter 2-derived longer variants having their C-terminal endostatin domains embedded in the BM and the N termini at the respective BM-cell interfaces. In addition to loosening of the proximal tubular BM structure, the Col18a1(-/-) mice showed effacement of the glomerular podocyte foot processes, and microindentation studies showed changes in the mechanical properties of the glomeruli, the Col18a1(-/-) glomeruli being ～30% softer than the wild-type. Analysis of promoter-specific knockouts (Col18a1(P1/P1) and Col18a1(P2/P2)) indicated that tubular BM loosening is due to a lack of the shortest isoform, whereas the glomerular podocyte effacement was due to a lack of the longer isoforms. We suggest that lack of collagen XVIII may also have disparate effects on kidney function in man, but considering the mild physiological findings in the mutant mice, such effects may manifest themselves only late in life or require other compounding molecular changes.     

Immunologic response to the survivin-derived multi-epitope vaccine EMD640744 in patients with advanced solid tumors

Purpose

Survivin is a member of the inhibitor-of-apoptosis family. Essential for tumor cell survival and overexpressed in most cancers, survivin is a promising target for anti-cancer immunotherapy. Immunogenicity has been demonstrated in multiple cancers. Nonetheless, few clinical trials have demonstrated survivin-vaccine-induced immune responses.

Experimental design

This phase I trial was conducted to test whether vaccine EMD640744, a cocktail of five HLA class I-binding survivin peptides in Montanide^® ISA 51 VG, promotes anti-survivin T-cell responses in patients with solid cancers. The primary objective was to compare immunologic efficacy of EMD640744 at doses of 30, 100, and 300 μg. Secondary objectives included safety, tolerability, and clinical efficacy.

Results

In total, 49 patients who received ≥2 EMD640744 injections with available baseline- and ≥1 post-vaccination samples [immunologic-diagnostic (ID)-intention-to-treat] were analyzed by ELISpot- and peptide/MHC-multimer staining, revealing vaccine-activated peptide-specific T-cell responses in 31 patients (63 %). This cohort included the per study protocol relevant ID population for the primary objective, i.e., T-cell responses by ELISpot in 17 weeks following first vaccination, as well as subjects who discontinued the study before week 17 but showed responses to the treatment. No dose-dependent effects were observed. In the majority of patients (61 %), anti-survivin responses were detected only after vaccination, providing evidence for de novo induction. Best overall tumor response was stable disease (28 %). EMD640744 was well tolerated; local injection-site reactions constituted the most frequent adverse event.

Conclusions

Vaccination with EMD640744 elicited T-cell responses against survivin peptides in the majority of patients, demonstrating the immunologic efficacy of EMD640744.     

Functional and Structural Properties of a Novel Protein and Virulence Factor (Protein sHIP) in Streptococcus pyogenes

Streptococcus pyogenes is a significant bacterial pathogen in the human population. The importance of virulence factors for the survival and colonization of S. pyogenes is well established, and many of these factors are exposed to the extracellular environment, enabling bacterial interactions with the host. In the present study, we quantitatively analyzed and compared S. pyogenes proteins in the growth medium of a strain that is virulent to mice with a non-virulent strain. Particularly, one of these proteins was present at significantly higher levels in stationary growth medium from the virulent strain. We determined the three-dimensional structure of the protein that showed a unique tetrameric organization composed of four helix-loop-helix motifs. Affinity pull-down mass spectrometry analysis in human plasma demonstrated that the protein interacts with histidine-rich glycoprotein (HRG), and the name sHIP (streptococcal histidine-rich glycoprotein-interacting protein) is therefore proposed. HRG has antibacterial activity, and when challenged by HRG, sHIP was found to rescue S. pyogenes bacteria. This and the finding that patients with invasive S. pyogenes infection respond with antibody production against sHIP suggest a role for the protein in S. pyogenes pathogenesis.     

Analysis of Alpha-Synuclein in Malignant Melanoma – Development of a SRM Quantification Assay

Globally, malignant melanoma shows a steady increase in the incidence among cancer diseases. Malignant melanoma represents a cancer type where currently no biomarker or diagnostics is available to identify disease stage, progression of disease or personalized medicine treatment. The aim of this study was to assess the tissue expression of alpha-synuclein, a protein implicated in several disease processes, in metastatic tissues from malignant melanoma patients. A targeted Selected Reaction Monitoring (SRM) assay was developed and utilized together with stable isotope labeling for the relative quantification of two target peptides of alpha-synuclein. Analysis of alpha-synuclein protein was then performed in ten metastatic tissue samples from the Lund Melanoma Biobank. The calibration curve using peak area ratio (heavy/light) versus concentration ratios showed linear regression over three orders of magnitude, for both of the selected target peptide sequences. In support of the measurements of specific protein expression levels, we also observed significant correlation between the protein and mRNA levels of alpha-synuclein in these tissues. Investigating levels of tissue alpha-synuclein may add novel aspect to biomarker development in melanoma, help to understand disease mechanisms and ultimately contribute to discriminate melanoma patients with different prognosis.     

Investigation of the potential for microbial contamination of deep granitic aquifers during drilling using 16S rRNA gene sequencing and culturing methods

Total number of bacteria, viable counts of aerobic and anaerobic heterotrophic bacteria and 16S rRNA gene diversity were investigated during drilling of three boreholes in the walls of the Äspö hard rock laboratory tunnel, at depths ranging from 380 to 446 m below sea level. Water samples were taken from the drill water source, the drilling equipment and from the drilled boreholes. The drill water was kept under nitrogen atmosphere and all equipment was steam cleaned before the start of a new drilling. Total and viable counts of bacteria in the drilled boreholes were several orders of magnitude lower than in the samples from the drilling equipment, except for sulphate reducing bacteria. A total of 158 16S rRNA genes that were cloned from the drill water source, the drilling equipment and the drilled boreholes were partially sequenced. The drilled boreholes generally had a 16S rRNA diversity that differed from what was found in samples from the drilling equipment. Several of the sequences obtained could be identified on genus level as one of the genera Acinetobacter, Methylophilus, Pseudomonas and Shewanella. In conclusion, the tubing used for drill water supply constituted a source of bacterial contamination to the rest of the drilling equipment and the boreholes. The results show, using molecular and culturing methods, that although large numbers of contaminating bacteria were introduced to the boreholes during drilling, they did not establish in the borehole groundwater at detectable levels.     

Determination of dimethylated arginines in human plasma by high-performance liquid chromatography

Using high-performance liquid chromatography (HPLC) with multigradient elution, (asymmetric-DMA, ADMA) and (symmetric-DMA, SDMA) can be separated from human plasma samples. The dimethylarginine compounds in plasma, after extraction with a cation-exchange column, are converted to fluorescent derivatives with o-phthaldialdehyde (OPA) in an alkaline medium and the derivatives are separated simultaneously within 50 min on a reversed-phase column (Ultracarb 3 ODS(20)). The recoveries of ADMA and SDMA are over 80% and the method permits quantitative determination of dimethylated arginines at concentrations as low as 0.1 μmol/l in human plasma.     

A robust and reproducible animal serum-free culture method for clinical-grade bone marrow-derived mesenchymal stromal cells

Efficient xenofree expansion methods to replace fetal bovine serum (FBS)-based culture methods are strongly encouraged by the regulators and are needed to facilitate the adoption of mesenchymal stromal cell (MSC)-based therapies. In the current study we established a clinically-compliant and reproducible animal serum-free culture protocol for bone marrow-(BM-) MSCs based on an optimized platelet-derived supplement. Our study compared two different platelet-derived supplements, platelet lysate PL1 versus PL2, produced by two different methods and lysed with different amounts of freeze–thaw cycles. Our study also explored the effect of a low oxygen concentration on BM-MSCs. FBS-supplemented BM-MSC culture served as control. Growth kinetics, differentiation and immunomodulatory potential, morphology, karyotype and immunophenotype was analysed. Growth kinetics in long-term culture was also studied. Based on the initial results, we chose to further process develop the PL1-supplemented culture protocol at 20 % oxygen. The results from 11 individual BM-MSC batches expanded in the chosen condition were consistent, yielding 6.60 × 10⁹ ± 4.74 × 10⁹ cells from only 20 ml of bone marrow. The cells suppressed T-cell proliferation, displayed normal karyotype and typical MSC differentiation potential and phenotype. The BM-MSCs were, however, consistently HLA-DR positive when cultured in platelet lysate (7.5–66.1 %). We additionally show that culture media antibiotics and sterile filtration of the platelet lysate can be successfully omitted. We present a robust and reproducible clinically-compliant culture method for BM-MSCs based on platelet lysate, which enables high quantities of HLA-DR positive MSCs at a low passage number (p2) and suitable for clinical use.