We recently demonstrated that a human recombinant scFv, L19, reacting with the ED-B domain of fibronectin, a marker of angiogenesis, selectively targets tumoral vasculature in vivo. Using the variable regions of L19, we constructed and expressed a human "small immunoprotein" (SIP) and a complete human IgG1 and performed biodistribution studies in tumor-bearing mice to compare the blood clearance rate, in vivo stability and performance in tumor targeting of the 3 L19 formats [dimeric scFv (scFv) 2 , SIP and IgG1]. The accumulation of the different antibody formats in the tumors studied was a consequence of the clearance rate and in vivo stability of the molecules. Using the SIP, the %ID/g in tumors was 2-5 times higher than that of the (scFv) 2 , reaching a maximum 4 -6 hr after injection. By contrast, the accumulation of IgG1 in tumors constantly rose during the experiments. However, due to its slow clearance, the tumor-blood ratio of the %ID/g after 144 hr was only about 3 compared to a ratio of 10 for the (scFv) 2 and 70 for the SIP after the same period of time. The different in vivo behavior of these 3 completely human L19 formats could be exploited for different diagnostic and/or therapeutic purposes, depending on clinical needs and disease. Furthermore, the fact that ED-B is 100% homologous in human and mouse, which ensures that L19 reacts equally well with the human and the murine antigen, should expedite the transfer of these reagents to clinical trials. © 2002 Wiley-Liss, Inc. Key words: antibody formats; tumor vasculature; tumor targeting; clinical applications; cancer diagnosis and therapyDespite their enormous potential as therapeutic agents, monoclonal antibodies (mAbs) of nonhuman origin have not performed as well as expected in clinical trials as a result of their immunogenicity, 1,2 poor pharmacokinetic properties 3,4 and inefficiency in recruiting effector functions. 5,6 The recent prospect of isolating human antibody fragments from phage display libraries 7-10 transcends these problems, revitalizing studies and rekindling hopes of using these reagents to treat major diseases. Indeed, these molecules should serve as ideal building blocks for novel diagnostic and therapeutic tools. 11,12 Furthermore, these antibodies can be "matured" to reach affinities in the picomolar range, 13 desirable, if not necessary, for their clinical use. 14,15 Clinical applications of human antibody fragments for the selective delivery of diagnostic or therapeutic agents nonetheless require highly specific targets. In the case of tumors, the most popular targets are cell-surface antigens, which are usually neither abundant nor stable. On the other hand, during tumor progression the microenvironment surrounding tumor cells undergoes extensive modification that generates a "tumoral environment" that could ultimately represent a suitable target for antibody-based tumor therapy. 16 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Mol...

We sought to enhance the selective toxicity of tumor necrosis factor ␣ (TNF␣) to permit its systemic use in cancer therapy. Because ligand-targeted therapeutics have proven successful in improving the selective toxicity of drugs, we prepared a fusion protein (L19mTNF␣) composed of mouse TNF␣ and a high-affinity antibody fragment (L19 scFv) to the extradomain B (ED-B) domain of fibronectin, a marker of angiogenesis. L19mTNF␣ was expressed in mammalian cells, purified, and charac- IntroductionDuring tumor progression the microenvironment surrounding tumor cells undergoes extensive modifications that generate a "tumoral environment" which could ultimately represent a suitable target for antibody-based tumor therapy. 1 In fact, the concept that the altered tumor microenvironment is itself a carcinogen that can be targeted is increasingly gaining consensus. Molecules that are able to effectively deliver therapeutic agents to the tumor microenvironment thus represent promising and important new tools for cancer therapy. [1][2][3] Fibronectin is an extracellular matrix (ECM) component that is widely expressed in a variety of healthy tissues and body fluids. Different fibronectin (FN) isoforms can be generated by the alternative splicing of the FN pre-mRNA, a process modulated by cytokines and extracellular pH. [4][5][6][7] The complete type III repeat extradomain B (ED-B) may be entirely included or omitted in the FN molecule. 8 ED-B is highly conserved in different species, having 100% homology in all mammalians thus far studied (human, rat, mouse) and 96% homology with a similar domain in chicken. The FN isoform containing ED-B (B-FN) is undetectable immunohistochemically in healthy adult tissues, with the exception of tissues undergoing physiologic remodeling (eg, endometrium and ovary) and during wound healing. 5,9 By contrast, its expression in tumors and fetal tissues is high. 5 Furthermore, it was demonstrated that B-FN is a marker of angiogenesis 10,11 and that endothelial cells invading tumor tissues migrate along ECM fibers containing B-FN. 12 We reported on the possibility to selectively target tumoral vasculature, both in experimental tumor models and in patients with cancer, using a human recombinant antibody, L19 scFv, specific for B-FN. [12][13][14][15][16][17][18][19] This observation paved the way for the antibody's use in both in vivo diagnostic (immunoscintigraphy) and therapeutic approaches entailing the selective delivery of radionuclides or toxic agents to tumoral vasculature. In addition, Birchler et al 20 showed that L19, chemically coupled to a photosensitizer, selectively accumulates in the newly formed blood vessels of the angiogenic rabbit cornea model and, after irradiation with near infrared light, mediates the complete and selective occlusion of ocular neovasculature. More recently, Nilsson et al 21 reported that the immunoconjugate of L19 with the extracellular domain of tissue factor mediates selective infarction in different types of murine tumor models. Furthermore, the cytokines int...

Different fibronectin (FN) isoforms are generated by the alternative splicing of the primary FN transcript.We previously demonstrated that the isoform containing the extra domain B sequence of fibronectin (B-FN), a complete type-III-homology repeat, is a marker of angiogenesis that accumulates around neovasculature only during angiogenic processes. We produced a single-chain human recombinant antibody (scFv), L19, which reacts specifically with B-FN and selectively targets tumor vasculature in vivo. We used this scFv and an antibody against a pan-endothelial marker (Factor VIII) in a double-staining procedure on specimens of low-and high-grade astrocytomas to determine the percentage of B-FN-positive vessels, (denominating the resulting value angiogenic index [AI]). Compared to vascular density and proliferative activity (evaluated using antibodies to Factor VIII and Ki67, respectively), AI correlated better with tumor grade (1.6 ؎ 2.6% and 92.0 ؎ 8.7% of B-FNpositive vessels in low-and high-grade astrocytomas, respectively) and was a more precise diagnostic tool than either of the two conventional methods. In fact, discriminating analysis using these three parameters showed that only AI accurately classified 100% of the cases studied, compared to 64% and 89% correctly diagnosed by vascular density and of proliferating cells, respectively. Tumors cannot grow without a pronounced remodeling of the extracellular matrix (ECM) of the surrounding normal tissue in which the tumor grows. Remodeling of the ECM takes place through two main processes: proteolytic degradation of the normal tissue's pre-existing ECM components, and neo-synthesis of new ECM components by both neoplastic and stromal cells. The interplay between these two processes results in the generation of a neoplastic ECM whose components may differ, qualitatively and quantitatively, from their nonneoplastic counterparts. Moreover, this provisional ECM also provides a permissive environment for tumor progression of which angiogenesis is a pivotal step.

Hungarian sour cherries (SC) are excellent source of anthocyanin (concentrations (100–300 mg in 100 g fresh fruit) and melatonin (0.15 mg in 100 g fresh fruit), but other flavonoid derivatives also can be isolated by aqueous alcoholic extraction. We have developed a new process for extracting non-extractable procyanidines bound to the membrane, proteins, and fibers. These compounds were seperated with UHPLC-MS methods, and the structure of individual components were identified on the basis of their mass fragmentation spectra. The antioxidant capacity of soluble and non-soluble antioxidants were measured with ferric reducing antioxidant power (FRAP), 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity (DPPH), trolox equivalent antioxidant capacity (TEAC) assays, and compared to the new measurement methods of water-soluble antioxidant capacity (ACW), lipid-soluble antioxidant capacity (ACL). Furthermore, total phenolic content (TPC) and total procyanidin content (PAC) were determinated. As a result of our investigation, we found that the solvent combination, where in the first step is water–ethanol (1:1), then 100% ethanol were suitable for the extraction of the extractable antioxidants. However, the chemiluminescence method that is based on the elimination of the superoxide radical is more accurate than other colorimetric methods which measure antioxidant capacity.

Diabetes mellitus (DM)-related morbidity and mortality are steadily rising worldwide, affecting about half a billion people worldwide. A significant proportion of diabetic cases are in the elderly, which is concerning given the increasing aging population. Proper nutrition is an important component in the effective management of diabetes in the elderly. A plethora of active substances of plant origin exhibit potency to target the pathogenesis of diabetes mellitus. The nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. In this study, the effect of Hungarian sour cherry, which is rich in anthocyanins, on hyperglycemia-induced endothelial dysfunction was tested using human umbilical cord vein endothelial cells (HUVECs). HUVECs were maintained under both normoglycemic (5 mM) and hyperglycemic (30 mM) conditions with or without two concentrations (1.50 ng/µL) of anthocyanin-rich sour cherry extract. Hyperglycemia-induced oxidative stress and inflammatory response and damaged vasorelaxation processes were investigated by evaluating the level of reactive oxygen species (ROS) and gene expression of four proinflammatory cytokines, namely, tumor necrosis factor-alpha (TNF- α), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1α (IL-1α), as well as the gene expression of nitric oxide synthase (NOS) endothelin-1 (ET-1) and endothelin-converting enzyme-1 (ECE-1). It was found that hyperglycemia-induced oxidative stress was significantly suppressed by anthocyanin-rich sour cherry extract in a concentration-dependent manner. The gene expression of the tested proinflammatory cytokines increased under hyperglycemic conditions but was significantly reduced by both 1 and 50 ng/µL anthocyanin-rich sour cherry extract. Further, although increased ET-1 and ECE-1 expression due to hyperglycemia was reduced by anthocyanin-rich sour cherry extract, NOS expression was increased by the extract. Collectively, these data suggest that anthocyanin-rich sour cherry extract could alleviate hyperglycemia-induced endothelial dysfunction due to its antioxidant, anti-inflammatory, and vasorelaxant effects.

In the present study, we monitor the adsorption−desorption kinetics and adsorbed layer structure of the bacterial protein flagellin in the presence of Hofmeister salts by a surface sensitive label-free optical biosensor (optical waveguide lightmode spectroscopy, OWLS). The recorded OWLS data were analyzed by a computer code using a set of coupled differential equations modeling the adsorption−desorption process. By supposing reversibly and irreversibly adsorbed protein states with different adsorption footprints, the kinetic data could be perfectly fitted. We revealed that the proteins adsorbing in the presence of kosmotropic salts had smaller footprints, leading to a more oriented and densely packed layer. Kosmotropic salts increased both the adsorption rate constant and the transition rate constants from the reversibly to the irreversibly adsorbed state. In contrast, chaotropic salts increased the desorption rate constant and led to decreased adsorbed mass and a more loosely packed film. Neither circular dichroism spectroscopy in bulk solutions or Fourier transform infrared spectroscopy of surface-adsorbed flagellins could reveal significant structural changes due to the presence of the Hofmeister salts, and supported our conclusions about the adsorption mechanism. On the basis of the measured kinetic and structural data (footprints of adsorbed proteins), we developed a model to calculate the protein−water-substrate interfacial tension in the presence of Hofmeister salts, and compared the experimentally obtained values with related literature data. The calculated values are consistent with previously published data of surface tension changes, andto the best of our knowledgerepresent the first experimental results for this quantity.

The anthocyanin content of Hungarian sour cherry is remarkable based on our preliminary investigations. Nutraceutical and pharmaceutical effects of anthocyanins have been extensively studied. The objective of this work was to investigate the the effect of purified sour cherry extract using human umbilical cord vein endothelial cells (HUVECs) as the inflammatory model. HUVECs were isolated by enzymatic digestion and characterized by flow cytometry. The optimal concentration range of sour cherry extract was selected based on MTT, apoptosis, and necrosis assays. Cells were divided into three groups, incubating with M199 medium as control, or with lipopolysaccharide (LPS) or with LPS plus anthocyanin extract (ACE). The effect of sour cherry extract on oxidative stress, pro-inflammatory factors, and arachidonic pathway was investigated. An amount of 50 μg/mL ACE (ACE50) was able to increase the level of glutathione and decrease the ROS, thereby improving the unbalanced redox status in inflammation. ACE50 lowered pro-inflammatory cytokine levels including Interleukin-6 (IL-6), regulated on activation, normal T cell expressed and secreted (RANTES), granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor alpha (TNF-α). ACE50 affected the arachidonic acid pathway by reducing the LPS-induced enzyme expression (cyclooxygenase-1, cyclooxygenase-2, and prostacyclin synthase). The extract under investigation seems to have a pleiotropic effect including anti-oxidative, anti-inflammatory, hemostatic, and vasoactive effects. Our results indicate that purified sour cherry extract could reduce the LPS-induced inflammatory response, thereby improving endothelial dysfunction.

Increased permeability of the epithelial and endothelial cell layers results in the onset of pathogenic mechanisms. In both cell types, cell–cell connections play a regulatory role in altering membrane permeability. The aim of this study was to investigate the modulating effect of anthocyanin-rich extract (AC) on TJ proteins in inflammatory Caco-2 and HUVEC monolayers. Distribution of Occludin and zonula occludens-1 (ZO-1) were investigated by immunohistochemical staining and the protein levels were measured by flow cytometry. The mRNA expression was determined by quantitative real-time PCR. The transepithelial electrical resistance (TEER) values were measured during a permeability assay on HUVEC cell culture. As a result of inflammatory induction by TNF-α, redistribution of proteins was observed in Caco-2 cell culture, which was reduced by AC treatment. In HUVEC cell culture, the decrease in protein and mRNA expression was more dominant during inflammatory induction, which was compensated for by the AC treatment. Overall, AC positively affected the expression of the examined cell-binding structures forming the membrane on both cell types.