Identification of novel neutralizing single-chain antibodies against vascular endothelial growth factor receptor 2

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF 165 in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents. © 2011 International Union of Biochemistry and Molecular Biology, Inc

Antimicrobial Peptides and Skin: A Paradigm of Translational Medicine

Antimicrobial peptides (AMPs) are small, cationic, amphiphilic peptides with broad-spectrum microbicidal activity against both bacteria and fungi. In mammals, AMPs form the first line of host defense against infections and generally play an important role as effector agents of the innate immune system. The AMP era was born more than 6 decades ago when the first cationic cyclic peptide antibiotics, namely polymyxins and tyrothricin, found their way into clinical use. Due to the good clinical experience in the treatment of, for example, infections of mucus membranes as well as the subsequent understanding of mode of action, AMPs are now considered for treatment of inflammatory skin diseases and for improving healing of infected wounds. Based on the preclinical findings, including pathobiochemistry and molecular medicine, targeted therapy strategies are developed and first results indicate that AMPs influence processes of diseased skin. Importantly, in contrast to other antibiotics, AMPs do not seem to propagate the development of antibiotic-resistant micro-organisms. Therefore, AMPs should be tested in clinical trials for their efficacy and tolerability in inflammatory skin diseases and chronic wounds. Apart from possible fields of application, these peptides appear suited as an example of the paradigm of translational medicine for skin diseases which is today seen as a `two-way road' - from bench to bedside and backwards from bedside to bench. Copyright (c) 2012 S. Karger AG, Base

Identification of Markers that Distinguish Monocyte-Derived Fibrocytes from Monocytes, Macrophages, and Fibroblasts

The processes that drive fibrotic diseases are complex and include an influx of peripheral blood monocytes that can differentiate into fibroblast-like cells called fibrocytes. Monocytes can also differentiate into other cell types, such as tissue macrophages. The ability to discriminate between monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions could be beneficial in identifying therapies that target either stromal fibroblasts or fibrocytes. and in sections from human lung. We found that markers such as CD34, CD68, and collagen do not effectively discriminate between the four cell types. In addition, IL-4, IL-12, IL-13, IFN-γ, and SAP differentially regulate the expression of CD32, CD163, CD172a, and CD206 on both macrophages and fibrocytes. Finally, CD49c (α3 integrin) expression identifies a subset of fibrocytes, and this subset increases with time in culture.These results suggest that discrimination of monocytes, macrophages, fibrocytes, and fibroblasts in fibrotic lesions is possible, and this may allow for an assessment of fibrocytes in fibrotic diseases

Cystic schwannoma of the maxillary sinus

Objective: Although 25-45% of all schwannomas are reported to occur in the head and neck region, nasal cavity and paranasal sinus involvement is rare, with 32 such cases described till 1999. Of these cases, only three were cystic. Herein we present a cystic schwannoma of the maxillary sinus of a 66-year-old, otherwise healthy male patient. Methods: The tumor tissue was routinely processed, embedded in paraffin, and stained with H&E. Immunostaining was performed for S-100 protein, epithelial membrane antigen (EMA) and cytokeratin. Results: A mass with sudden enlargement, inhomogenous enhancement with cystic areas, S-100 positive membrane-like structures lining the cystic cavity of an otherwise classical schwannoma were the main features encountered in our case. Conclusion: We recommend that cystic schwannoma should be kept in mind in the differential diagnosis of cystic masses of the maxilla. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity.

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders

Novel short peptides isolated from phage display library inhibit vascular endothelial growth factor activity.

Signal transduction through the vascular endothelial growth factor (VEGF)-VEGF receptor (VEGFR) pathway has a pivotal importance in angiogenesis, and has therefore become a prime target in antitumor therapy. In search for peptides antagonizing VEGF binding to its receptors, we screened a random heptamer library displayed on phage for peptides that bind the whole VEGF165 molecule and inhibit VEGF dependent human umbilical vein endothelial cell (HUVEC) proliferation. Two selected peptides with sequences WHLPFKC and WHKPFRF were synthesized. Biacore and matrix-assisted laser desorption/ionization timeof- flight mass spectrometry analysis indicated that these peptides bind the VEGF homodimer in a concentration- dependent manner, with micromolar affinity, and with a 2:1 peptide:VEGF stoichiometry. They inhibited HUVEC proliferation in vitro by 77 and 55%, respectively. Taken together, our results indicate that these peptides could be potent inhibitors of angiogenesis. Furthermore, we show that the peptide- VEGF binding properties can be quantified, a prerequisite for the further optimization of binders