Immunocompatibility and non-thrombogenicity of gelatin-based hydrogels.

Immunocompatibility and non-thrombogenicity are important requirements for biomedical applications such as vascular grafts. Here, gelatin-based hydrogels formed by reaction of porcine gelatin with increasing amounts of lysine diisocyanate ethyl ester were investigated in vitro in this regard. In addition, potential adverse effects of the hydrogels were determined using the "Hen's egg test on chorioallantoic membrane" (HET-CAM) test and a mouse model.The study revealed that the hydrogels were immunocompatible, since complement activation was absent and a substantial induction of reactive oxygen species generating monocytes and neutrophils could not be observed in whole human blood. The density as well as the activation state of adherent thrombocytes was comparable to medical grade polydimethylsiloxane, which was used as reference material. The HET-CAM test confirmed the compatibility of the hydrogels with vessel functionality since no bleedings, thrombotic events, or vessel destructions were observed. Only for the samples synthesized with the highest LDI amount the number of growing blood vessels in the CAM was comparable to controls and significantly higher than for the softer materials. Implantation into mice showed the absence of adverse or toxic effects in spleen, liver, or kidney, and only a mild lymphocytic activation in the form of a follicular hyperplasia in draining lymph nodes (slightly increased after the implantation of the material prepared with the lowest LDI content). These results imply that candidate materials prepared with mid to high amounts of LDI are suitable for the coating of the blood contacting surface of cardiovascular implants.

Model to evaluate the impact of hospital-based interventions targeting false-positive blood cultures on economic and clinical outcomes.

BACKGROUND: Blood culture contamination (BCC) increases length of stay (LOS) and leads to unnecessary antimicrobial therapy and/or hospital-acquired conditions (HACs). AIM: To quantify the magnitude of additional LOS, costs to hospitals and society, and harm to patients attributable to BCC. METHODS: A retrospective matched survival analysis was performed involving hospitalized patients with septicaemia-compatible symptoms. BCC costs, HACs and potential savings were calculated based on the primary LOS data, a modified Delphi process and published sources. The cost analysis compared standard care with interventions for reducing BCC, and estimated annual economic and clinical consequences for a typical hospital and for the USA as a whole. FINDINGS: Patients with BCC experienced a mean increase in LOS of 2.35 days (P=0.0076). Avoiding BCC would decrease costs by $6463 [$4818 from inpatient care (53% of which was from reduced LOS) and 26% from reduced antibiotic use]. Annually, in a typical 250- to 400-bed hospital, employing phlebotomists would save $1.3 million and prevent 24 HACs (including two cases of Clostridium difficile infection); based on clinical efficacy evidence, use of the studied initial specimen diversion device (ISDD) would save $1.9 million and prevent 34 HACs (including three cases of C. difficile infection). In the USA, the respective strategies would prevent 69,300 and 102,900 HACs (including 6000 and 8900 cases of C. difficile infection) and save $5 and $7.5 billion. CONCLUSION: Costs and clinical burdens associated with false-positive cultures are substantial and can be reduced by available interventions, including phlebotomists and use of ISDD.

Synthesis and radiopharmacological evaluation of 64Cu-labeled bombesin analogs featuring a bis(2-pyridylmethyl)-1,4,7-triazacyclononane chelator.

The bifunctional chelating agent 2-[4,7-bis(2-pyridylmethyl)-1,4,7-triazacyclononan-1-yl]acetic acid, DMPTACN-COOH, has been found to bind strongly to copper(II), resulting in a radiocopper(II)-ligand complex that exhibits high in vivo stability. The pendant carboxylic acid group enables this derivative to be conjugated to the N-terminal amino acid residues of peptides. Exploiting this, two stabilized bombesin (BBN) derivatives, ßAla-ßAla-[Cha(13),Nle(14)]BBN(7-14) and ßhomo-Glu-ßAla-ßAla-[Cha(13),Nle(14)]BBN(7-14) have been coupled to DMPTACN-COOH and radiolabeled with the positron emitter copper-64 ((64)Cu-1 and (64)Cu-3). The in vitro binding characteristics of the [(64)Cu]Cu-labeled bombesin conjugates in gastrin-releasing peptide receptor (GRPR) over-expressing prostate cancer (PC-3) cells have been evaluated. Biodistribution studies performed in Wistar rats indicate a specific uptake in the GRPR-rich pancreas and rapid renal elimination for both (64)Cu-1 and (64)Cu-3. Small animal PET imaging studies performed in NMRI nu/nu mice bearing the human prostate tumor PC-3 demonstrated a very high degree of tumor accumulation for (64)Cu-1 and (64)Cu-3. Incorporation of a single additional glutamic acid residue within the spacer between bombesin and the radiolabeled complex ((64)Cu-3) leads to a higher tumor-to-muscle uptake ratio (amounting to >30 at 100 min post injection) compared to (64)Cu-1.

Anti-tumor effects of peptide analogs targeting neuropeptide hormone receptors on mouse pheochromocytoma cells.

Pheochromocytoma is a rare but potentially lethal chromaffin cell tumor with currently no effective treatment. Peptide hormone receptors are frequently overexpressed on endocrine tumor cells and can be specifically targeted by various anti-tumor peptide analogs. The present study carried out on mouse pheochromocytoma cells (MPCs) and a more aggressive mouse tumor tissue-derived (MTT) cell line revealed that these cells are characterized by pronounced expression of the somatostatin receptor 2 (sst2), growth hormone-releasing hormone (GHRH) receptor and the luteinizing hormone-releasing hormone (LHRH) receptor. We further demonstrated significant anti-tumor effects mediated by cytotoxic somatostatin analogs, AN-162 and AN-238, by LHRH antagonist, Cetrorelix, by the cytotoxic LHRH analog, AN-152, and by recently developed GHRH antagonist, MIA-602, on MPC and for AN-152 and MIA-602 on MTT cells. Studies of novel anti-tumor compounds on these mouse cell lines serve as an important basis for mouse models of metastatic pheochromocytoma, which we are currently establishing.

Factors influencing the formation of small dense low-density lipoprotein particles in dependence on the presence of the metabolic syndrome and on the degree of glucose intolerance.

BACKGROUND: Small dense low-density lipoprotein (LDL) particles are known to be especially atherogenic. Several mechanisms are involved in this atherogenicity. AIMS: We wanted to look for the presence of small dense LDL particles depending on gender, metabolic syndrome (MS) and different degrees of glucose intolerance. Moreover, we looked for anthropometric factors and factors of lipid and carbohydrate metabolism that are associated with changes in the LDL size. RESULTS: We studied 752 persons (330 males, 422 females; age 40 +/- 17 years). LDL particle size was estimated with polyacrylamide gel electrophoresis. Males had smaller LDL particles than females. Probands with the MS had smaller LDL particles than those without this syndrome. With rising plasma triglyceride (TG) levels more small dense LDL particles were seen. The highest proportion of these small dense LDL particles was observed in the subgroup of type 2 diabetic patients. In the whole material, the mean LDL diameter was correlated negatively with plasma TG and very low-density lipoprotein components (TG, cholesterol and proteins) and positively with high-density lipoprotein cholesterol. In a linear stepwise regression analysis different significant factors influencing the LDL size were found in the whole population, in normoglycaemic probands, in persons with impaired glucose tolerance, in type 2 diabetic patients and in type 2 diabetic patients injecting insulin. CONCLUSIONS: Our data point to different mechanisms of the formation of small dense LDL particles in dependence on the degree of glucose intolerance. Moreover, the target values for plasma TG should be set lower.

Glycoxidised LDL isolated from subjects with impaired glucose tolerance increases CD36 and peroxisome proliferator-activator receptor gamma gene expression in macrophages.

AIMS/HYPOTHESIS: Glycoxidised LDL has been implicated in the pathogenesis of atherosclerosis, a major complication of diabetes. Since atherogenesis may occur at an early stage of diabetes, we investigated whether circulating LDL isolated from subjects with IGT (n = 20) showed an increased glycoxidation status and explored the proatherogenic effects of LDL samples on macrophages. SUBJECTS AND METHODS: We investigated LDL modifications using GC-MS. Murine macrophages were incubated with LDL samples for 1 h, and then mRNA expression rates of the scavenger receptors CD36 and scavenger receptor class B type 1 (SCARB1, formerly known as SR-BI) and transcription factor peroxisome proliferator-activator receptor gamma (PPARgamma) were quantified by real-time RT-PCR. RESULTS: The GC-MS experiments revealed that oxidative modifications of proline, arginine, lysine and tyrosine residues in apolipoprotein B100 were three- to fivefold higher in LDL samples from IGT subjects compared with those from NGT subjects (n = 20). Moreover, LDL glycoxidation estimated by both Nepsilon-(carboxymethyl)lysine (CML) and Nepsilon-(carboxyethyl)lysine (CEL) residues was increased more than ninefold in LDL from IGT subjects compared with samples from NGT subjects. Compared with NGT LDL, IGT LDL elicited a significantly higher CD36 (p < 0.05) and PPARG (p < 0.05) gene expression, whereas SCARB1 mRNA expression was not affected. CONCLUSIONS/INTERPRETATION: These data suggest that IGT is associated with increased glycoxidation of circulating LDL, which might contribute to the conversion of macrophages into a proatherogenic phenotype.

Neurotensin receptors in adeno- and squamous cell carcinoma.

BACKGROUND: Peptide receptors seem to be good markers for receptor targeting because of their overexpression in human cancer. Understanding the role of receptors and their cognate ligands, they are currently used for both diagnosis and therapy. Candidates playing a key role in tumor biology are the neurotensin receptors (NTR). The expression of NTR in HT-29 cells (human colon adenocarcinoma cell line), FaDu cells (human squamous cell carcinoma cell line) and in corresponding tumor xenografts on nude mice, was investigated. MATERIALS AND METHODS: Quantitative RT-PCR of the three receptor subtypes was carried out to study mRNA expression. Receptor protein expression was analyzed by immunohistochemistry with specific antibodies for the three known neurotensin receptors NTR1, NTR2 and NTR3. RESULTS: Analysis of receptor mRNA revealed a strong expression of NTR3 and a weak expression of NTR1 and NTR2 in cultured cells and xenografts. Examining the protein levels, a strong signal for NTR1 was detected in tumor cells and xenografts and only a weak signal was detected for NTR3. CONCLUSION: Since the receptor protein is targeted in vivo, the enhanced protein expression of NTR1 in xenografts could be a useful tool for molecular targeting with radioligands and for further characterization of the carcinogenic process.

Glucose-induced enhancement of hemin-catalyzed LDL oxidation in vitro and in vivo.

Growing evidence indicates that oxidative modification of low-density lipoprotein (LDL) is increased in diabetes mellitus; however, the mechanism(s) of this phenomenon is still unclear. gamma-Glutamyl semialdehyde (gammaGSA) is a product of hemin (Fe(3+)-protoporphyrin IX)-catalyzed oxidation of apolipoprotein B-100 (apoB- 100) proline and arginine residues. On reduction, gammaGSA forms 5-hydroxy-2-aminovaleric acid (HAVA). This report describes the application of sensitive HAVA assay, to characterize gammaGSA formation in LDL under normo- and hyperglycemic conditions, both in vitro and in vivo. In vitro studies revealed that apoB-100 proline and arginine residues are not oxidized to HAVA by HOCl or the myeloperoxidase/hydrogen peroxide (H(2)O(2)) oxidation system. Cu(2+), Cu(2+)/H(2)O(2), and Fe(2+) induced only minor HAVA formation. In contrast, the hemin oxidation system appeared reactive toward LDL apoB-100 proline and arginine residues. The resulting significant HAVA formation was specifically inhibited by a redox-inert ferric iron chelator. Glucose further enhanced hemin-induced increase in relative electrophoretic mobility of LDL and apoB-100 HAVAformation. In vivo we observed elevated concentrations of HAVA in LDL apoB-100 in patients with impaired glucose tolerance and with manifest diabetes mellitus. In conclusion, glucose promotes iron-mediated oxidation of apoB- 100 proline and arginine residues via a superoxide-dependent mechanism, thus rendering the LDL particles more atherogenic. The findings (a) identify a potential mechanism of enhanced atherogenesis in subjects with diabetes mellitus and (b) support the value of HAVA as a specific marker of LDL apoB-100 oxidation. Antioxid. Redox Signal. 7, 1507-1512.

Small animal positron emission tomography in food sciences.

Positron emission tomography (PET) is a 3-dimensional imaging technique that has undergone tremendous developments during the last decade. Non-invasive tracing of molecular pathways in vivo is the key capability of PET. It has become an important tool in the diagnosis of human diseases as well as in biomedical and pharmaceutical research. In contrast to other imaging modalities, radiotracer concentrations can be determined quantitatively. By application of appropriate tracer kinetic models, the rate constants of numerous different biological processes can be determined. Rapid progress in PET radiochemistry has significantly increased the number of biologically important molecules labelled with PET nuclides to target a broader range of physiologic, metabolic, and molecular pathways. Progress in PET physics and technology strongly contributed to better scanners and image processing. In this context, dedicated high resolution scanners for dynamic PET studies in small laboratory animals are now available. These developments represent the driving force for the expansion of PET methodology into new areas of life sciences including food sciences. Small animal PET has a high potential to depict physiologic processes like absorption, distribution, metabolism, elimination and interactions of biologically significant substances, including nutrients, 'nutriceuticals', functional food ingredients, and foodborne toxicants. Based on present data, potential applications of small animal PET in food sciences are discussed.

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies.

The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer's disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 ((18)F) by conjugation with N-succinimidyl-4-[(18)F]fluorobenzoate ([(18)F]SFB). Radiolabelling of both nLDL and oxLDL using [(18)F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [(18)F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [(18)F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [(18)F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.

Synthesis and biodistribution of an 18F-labelled resveratrol derivative for small animal positron emission tomography.

Resveratrol (3,4',5-trihydroxy-trans-stilbene) is a naturally occurring phytoalexin and polyphenol existing in grapes and various other plants, and one of the best known 'nutriceuticals'. It shows a multiplicity of beneficial biological effects, particularly, by attenuating atherogenic, inflammatory, and carcinogenic processes. However, despite convincing evidence from experimental and clinical studies, data concerning the role of resveratrol and other members of the large polyphenols family for human health is still a matter of debate. One reason for this is the lack of suitable sensitive and specific methods, which would allow direct assessment of biodistribution, biokinetics, and the metabolic fate of these compounds in vivo. The unique features of positron emission tomography (PET) as a non-invasive in vivo imaging methodology in combination with suitable PET radiotracers have great promise to assess quantitative information on physiological effects of polyphenols in vivo. Herein we describe the radiosynthesis of an (18)F-labelled resveratrol derivative, 3,5-dihydroxy-4'-[(18)F]fluoro-trans-stilbene ([(18)F]-1), using the Horner-Wadsworth-Emmons reaction as a novel radiolabelling technique in PET radiochemistry for subsequent functional imaging of polyphenol metabolism in vivo. In a typical "three-step/one-pot" reaction, (18)F-labelled resveratrol derivative [(18)F]-1 could be synthesized within 120-130 min including HPLC separation at a specific radioactivity of about 90 GBq/mumol. The radiochemical yield was about 9% (decay-corrected) related to [(18)F]fluoride and the radiochemical purity exceeded 97%. First radiopharmacological evaluation included measurement of biodistribution ex vivo and positron emission tomography (PET) studies in vivo after intravenous application of [(18)F]-1 in male Wistar rats using a dedicated small animal PET camera with very high spatial resolution. Concordantly with data on bioavailability and metabolism of native resveratrol from the literature, these investigations revealed an extensive uptake and metabolism in the liver and kidney, respectively, of [(18)F]-1. This study represents the first investigation of polyphenols in vivo by means of PET.

Biodistribution and catabolism of 18F-labelled isopeptide N(epsilon)-(gamma-glutamyl)-L-lysine.

Isopeptide bonds between the epsilon-amino group of lysine and the gamma-carboxamide group of glutamine are formed during strong heating of pure proteins or, more important, by enzymatic reaction mediated by transglutaminases. Despite the wide use of a microbial transglutaminase in food biotechnology, up to now little is known about the metabolic fate of the isopeptide N(epsilon)-(gamma-glutamyl)-L-lysine. In the present study, N-succinimidyl-4-[(18)F]fluorobenzoate was used to modify N(epsilon)-(gamma-glutamyl)-L-lysine at each of its two alpha-amino groups, resulting in the 4-[(18)F]fluorobenzoylated derivatives, for which biodistribution, catabolism, and elimination were investigated in male Wistar rats. A significant different biochemical behavior of the two labelled isopeptides was observed in terms of in vitro stability, in vivo metabolism as well as biodistribution. The results suggest that the metabolic fate of isopeptides is likely to be dependent on how they are reabsorbed - free or peptide bound.

[Oxidatively modified lipoproteins and their antibodies in patients with antiphospholipid syndromeand systemic lupus erythematosus]. / Oxidativ modifizierte Lipoproteine und deren Antikörper bei Patienten mit Antiphospholipidsyndrom und Systemischem Lupus erythematodes.

The antiphospholipid syndrome (APS) with its typical clinical manifestations of recurrent thrombosis and fetal loss is biochemically defined by the presence of circulating antiphospholipid antibodies (aPL). The disease pattern has raised special interest as a possible link between autoimmunity and atherosclerosis. aPL, oxidized low density lipoproteins (oxLDL), and antibodies to oxLDL (Anti-oxLDL) are suggested to play an important role in atherogenesis. In the present study we compared the serum levels of oxLDL and Anti-oxLDL in APS patients (20 subjects with primary APS; 14 subjects with secondary APS) and nonAPS subjects (24 phenotypically healthy controls samples and 12 patients with systemic lupus erythematosus [SLE]) and investigated associations of the above mentioned parameters with the intima-media thickness (IMT), a clinical surrogate parameter of atherosclerosis.SLE patients with and without APS showed significantly increased levels of Anti-oxLDL as compared to the controls group (p = 0.038 and p = 0.007, respectively). In contrast, oxLDL levels were not significantly different between the controls group and patients. The Anti-oxLDL levels correlated significantly with anticardiolipin (p = 0.002) and beta(2)-glycoprotein I antibodies (p < 0.048), both from IgG isotype. Only SLE patients without APS revealed a significantly elevated production of reactive oxygen species indicating an increased proatherogenic oxidative stress in the circulation (p < 0.002). In the patient groups, the circulating levels of oxLDL and Anti-oxLDL showed no association with atherosclerosis as estimated by IMT. In conclusion, our experimental data do not support the concept of oxidative stress-induced accelerated atherosclerosis in APS patients.

The effect of garlic on arteriosclerotic nanoplaque formation and size.

OBJECTIVE: In an in vitro biosensor model (PCT/EP 97/05212), the interplay between different lipoproteins in arteriosclerotic nanoplaque formation, as well as aqueous garlic extract (0.2-5.0 g/l from LI 111 powder) as a possible candidate drug against arterio/atherosclerosis were tested within the frame of a high throughput screening. METHODS: The processes described below were studied by ellipsometric techniques quantifying the adsorbed amount (nanoplaque formation) and layer thickness (nanoplaque size). A thorough description of the experimental setup has been given previously. RESULTS: Proteoheparan sulfate (HS-PG) adsorption to hydrophobic silica was monoexponential and after approximately 30 min constant. The addition of 2.52 mmol/l Ca2+ led to a further increase in HS-PG adsorption because Ca2+ was bound to the polyanionic glycosaminoglycan (GAG) chains thus screening their negative fixed charges and turning the whole molecule more hydrophobic. Incubation with 0.2 g/l aqueous garlic extract (GE) for 30 min did not change the adsorption of HS-PG. However, the following addition of Ca2+ ions reduced the increase in adsorption by 50.8% within 40 min. The adsorption of a second Ca2+ step to 10.08 mmol/l was reduced by even 82.1% within the next 40 min. Having detected this inhibition of receptor calcification, it could be expected that the build-up of the ternary nanoplaque complex is also affected by garlic. The LDL plasma fraction (100 mg/dl) from a healthy probationer showed beginning arteriosclerotic nanoplaque formation already at a normal blood Ca2+ concentration, with a strong increase at higher Ca2+ concentrations. GE, preferably in a concentration of 1 g/l, applied acutely in the experiment, markedly slowed down this process of ternary aggregational nanoplaque complexation at all Ca2+ concentrations used. In a normal blood Ca2+ concentration of 2.52 mmol/l, the garlic induced reduction of nanoplaque formation and molecular size amounted to 14.8% and 3.9%, respectively, as compared to the controls. Furthermore, after ternary complex build-up, GE similar to HDL, was able to reduce nanoplaque formation and size. The incubation time for HDL and garlic was only 30 min each in these experiments. Nevertheless, after this short time the deposition of the ternary complex decreased by 6.2% resp. 16.5%, i.e. the complex aggregates were basically resolvable. CONCLUSIONS: These experiments clearly proved that garlic extract strongly inhibits Ca2+ binding to HS-PG. In consequence, the formation of the ternary HS-PG/LDL/Ca2+ complex, initially responsible for the 'nanoplaque' composition and ultimately for the arteriosclerotic plaque generation, is decisively blunted.

Analysis of 6-hydroxy-2-aminocaproic acid (HACA) as a specific marker of protein oxidation: the use of N(O,S)-ethoxycarbonyl trifluoroethyl ester derivatives and gas chromatography/mass spectrometry.

An alteration of low density lipoprotein (LDL) apolipoprotein (apo) B-100 structure by direct oxidative modification is an important mechanism involved in atherogenesis. There is difficulty in quantifying this type of modification because a lack of specific assays. The use of N(O,S)-ethoxycarbonyl trifluoroethyl amino acid esters for a rapid and sensitive determination of 6-hydroxy-2-aminocaproic acid (HACA), a highly specific marker of metal catalyzed protein oxidation, by using standard gas chromatography/electron impact mass spectrometry, is discussed. The derivatives are formed by the unlabored reaction of amino acids with ethylchloroformate plus trifluoroethanol plus pyridine. Femtomole levels of HACA can be reproducible measured in different LDL preparations subjected to oxidative damage in the presence of iron or copper. HACA determination compares well with the measurement of carbonyl groups that are generally accepted as a nonspecific index of protein oxidation. Thus, the method could prove to be a sensitive assay for studying specific apoB-100 modification.

Measurement of 5-hydroxy-2-aminovaleric acid as a specific marker of metal catalysed oxidation of proline and arginine residues of low density lipoprotein apolipoprotein B-100 in human atherosclerotic lesions.

gamma-Glutamyl-semialdehyde (gammaGSA) is a major product of the metal catalysed oxidation of apolipoprotein B-100 (apoB-100) proline and arginine residues. On reduction, gammaGSA forms 5-hydroxy-2-aminovaleric acid (HAVA). This report describes the application of HAVA measurement to characterise the formation of gammaGSA in low density lipoprotein (LDL) recovered from human atherosclerotic lesions. HAVA concentrations were greatly increased in LDL from early (mean, 10.25; SD, 3.49 mol/mol apoB-100; p < 0.01), intermediate (mean, 11.18; SD, 2.37 mol/mol apoB-100; p < 0.01), and advanced (mean, 9.91; SD, 2.15 mol/mol apoB-100; p < 0.01) lesions, when compared with LDL from normal aortic tissue (mean, 0.05; SD, 0.01 mol/mol apoB-100). These findings support the hypothesis that pathways involving metal catalysed oxidation of LDL apoB-100 are of pathological importance in atherogenesis.