Renopathological Microstructure Visualization from Formalin Fixed Kidney Tissue by Matrix-Assisted Laser/Desorption Ionization-Time-of-Flight Mass Spectrometry Imaging.

Understanding early stage renal malfunctions with regard to the glomerular filtration processes is essential for nephropathological prescreening strategies and intervention at an early stage. Mass spectrometry imaging (MSI) in combination with histopathology can provide an universal analytical approach. Proteomic and lipidomic aspects of glomerular biocompositions were applied for micro-structural differentiation in healthy rat kidney samples. Usability of commonly used tissue embedding media and the compatibility of histological staining and fixation methods were of interest. It was demonstrated that ultra-thin tissue samples (500 nm, 1 and 10 µm) can be used for lipid and peptide-based differentiation at the glomerular resolution level in formalin-fixed tissue samples in combination with preceding histological staining for correlating optical and molecular mass images.

Modulation of plasma complement by the initial dose of epirubicin/docetaxel therapy in breast cancer and its predictive value.

BACKGROUND: Despite the widespread use of neoadjuvant chemotherapy in breast cancer patients, prediction of individual response to treatment remains an unsolved clinical problem. Particularly, administration of an inefficient chemotherapeutic regimen should be avoided. Therefore, a better understanding of the molecular mechanisms underlying response to neoadjuvant chemotherapy is of particular clinical interest. Aim of the present study was to test whether neoadjuvant chemotherapy with epirubicin/docetaxel induces early changes in the plasma proteome of breast cancer patients and whether such changes correlate with response to therapy. METHODS: Plasma samples of 25 breast cancer patients obtained before and 24 h after initiation of epirubicin/docetaxel-based neoadjuvant chemotherapy were analysed using two-dimensional differential gel electrophoresis (2D-DIGE). Protein spots found to be differentially expressed were identified using mass spectrometry and then correlated with the pathological response after six cycles of therapy. Markers identified in a discovery set of patients (n=12) were confirmed in an independent validation set (n=13). RESULTS: 2D-DIGE revealed 33 protein spots to be differentially expressed in response to chemotherapy, including the complement factors C1, C3 and C4, inter-α-trypsin inhibitor, α-1-antichymotrypsin and α-2-Heremans-Schmid glycoprotein (AHSG). With respect to cytokines, only interleukin (IL)-6, IL-10 and soluble intracellular adgesion molecule 3 (sICAM3) were minimally modulated. Moreover, two protein spots within the complement component C3 significantly correlated with response to therapy. CONCLUSION: We have identified acute phase proteins and the complement system as part of the early host response to epirubicin/docetaxel chemotherapy. As complement C3 cleavage correlates with the efficacy of docetaxel/epirubicin-based chemotherapy, it has the potential as an easily accessible predictive biomarker.

Near-infrared spectroscopic study on guest-host interactions among G0-G7 amine-terminated poly(amidoamine) dendrimers and porous silica materials for simultaneously determining the molecular weight and particle diameter by multivariate calibration techniques.

The guest-host interactions of poly(amidoamine) (PAMAM) dendrimers and porous silica surfaces were investigated by near-infrared (NIR) diffuse reflection spectroscopy. G0-G7 of amine-terminated PAMAM (PAMAM-NH2) dendrimers were analyzed comprising early, mid, and late generations. For early stages, the adsorption process of the partly protonated dendrimers to the negatively charged silica surface strongly depends on the size/shape characteristics of the guest (PAMAM-NH2 dendrimers) and host (porous silica) materials. G0-G4 (15-45 A) show smaller particle sizes than the pore diameter of the silica (60 A) and thus have access to the interior surface of the host material. For mid and later stages (G5-G7; 54-81 A) only low amounts of the dendrimers adsorb to the silica surface due to the inaccessibility to the interior surface. The loading capacity of the silica material with adsorbed PAMAM-NH(2) was evaluated by means of capillary zone electrophoresis (CZE), whereas deviations from the theoretical to the effective particle size and molecular weight (MW) was determined by gas-phase electrophoretic mobility molecular analysis (GEMMA) and matrix-assisted laser desorption/ionization linear time-of-flight mass spectrometry (MALDI-lin TOF-MS). Deviations from the theoretical to the actual values showed a maximum of 13.8% and 28.0% for the particle size and MW, respectively. The NIR absorption spectra show a distinct band at 4932 cm(-1) (nu(sym) (NH) + amide II) due to the adsorbed dendrimers. It was found that the absorbance tends to increase with decreasing generation number. On this basis multivariate calibration was performed with the theoretical data and the data obtained by GEMMA and MALDI-lin TOF-MS. All in all, the calculated partial least-squares regression (PLSR) model containing the GEMMA/MALDI-lin TOF-MS reference values showed better results than the models exclusively calculated from the theoretical values. This indicates that the theoretical values do not imply the structural imperfections arising during the synthesis that may be present in the PAMAM-NH2 dendrimers.

Lectin bioconjugates trigger urothelial cytoinvasion--a glycotargeted approach for improved intravesical drug delivery.

A unique structural and functional configuration renders the human urothelium, one of the hardest to overcome biological barriers, and accounts for critical shortcomings in the adjuvant localized therapy of bladder cancer and other severe medical conditions. Strategies to improve intravesical drug absorption are urgently sought, but so far have hardly adopted biorecognitive delivery vectors that are more specifically tailored to the natural characteristics of the target site. The efficient cytoinvasion of uropathogenic bacteria, mediated via a mannose-directed FimH lectin adhesin, and malignancy-dependent differences in bladder cell glycosylation point to considerable unrealized potential of lectins as targeting vectors on the molecular/functional and recognitive level. Here, we outline the ability of wheat germ agglutinin (WGA) to induce endocytosis of conjugated payload in human urothelial SV-HUC-1 cells after stable adhesion to internalizing receptors. A panel of model bioconjugates was prepared by covalently coupling one to six WGA units to fluorescein-labeled bovine serum albumin (fBSA). Cytoadhesive capacity was found to directly correlate to the degree of modification up to a critical threshold of on average three targeting ligands per conjugate. The highly specific, glycan-triggered interaction proved essential for endosomal sorting and was followed by rapid (<60min) and extensive (>40%) internalization. fBSA/WGA bioconjugates were processed analogously to the free lectin, irrespective of the significantly higher molecular weight (100-300kD). Durable entrapment of conjugates in acidic, perinuclear compartments without kiss-and-run recycling to the plasma membrane was found in both single cells and monolayers. Our results assign promising potential to glycotargeted delivery concepts in the intravesical setting and offer new perspectives for the application of complex biologicals in the urinary tract.