In Vivo Microbial Targeting of 99mTc-Labeled Human ß-Defensin-3 in a Rat Model of Infection.

OBJECTIVE: Differentiation of infection from aseptic inflammation represents a major clinical issue. None of the commercially available compounds (labeled granulocytes, antigranulocyte antibodies, Ga-citrate, labeled immunoglobulin G, F-FDG) is capable of this differentiation, producing a nonnegligible false-positive rate. Recently, our group reported on a reliable labeling procedure of the antimicrobial peptide human ß-defensin 3 (HBD-3) with Tc. The aim of this study was to evaluate in vivo Tc-HBD-3 uptake in a rat model of infection. METHODS: Recombinant HBD-3 was radiolabeled with Tc. Radiolabeling yield and specific activity of the compound were calculated. Chromatographic behavior and biological activity of Tc-HBD-3 were also assessed. An experimental model involving Staphylococcus aureus-induced infection and carrageenan-induced aseptic inflammation was performed in 5 Wistar rats. Serial planar scintigraphic acquisitions were performed from 15 to 180 minutes after Tc-HBD-3 intravenous administration. Radiotracer uptake was evaluated qualitatively and semiquantitatively as a target-to-nontarget ratio. RESULTS: Radiolabeling yield of Tc-HBD-3 was 70% with a specific activity of 6 to 8 MBq/µg. A significant and progressive Tc-HBD-3 uptake was observed in the site of S. aureus-induced infection, with a maximum average target-to-nontarget ratio of 5.7-fold higher in the infection site compared with an inflammation site observed at 140 minutes. CONCLUSIONS: In vivo imaging with Tc-HBD-3 in a rat model of S. aureus-induced infection demonstrated favorable uptake in the infection site compared with sterile inflammation and background. These promising results, together with previous ex vivo uptake and toxicity assessment, suggest the potential of Tc-HBD-3 as a novel agent for specific infection imaging.

Toxicity assessment of (99m)technetium-labeled human beta-defensin-3 in CD1 mice.

OBJECTIVE: Human beta-defensin-3 (HBD-3) is an antimicrobial peptide which is up-regulated during inflammation. Based on the previously demonstrated capacity of technetium-99m ((99m)Tc) labelled HBD-3 of distinguishing infection from inflammation in rats, we have decided to collect information on the potential toxicity of the tracer in view of its possible use for imaging in humans. MATERIALS AND METHODS: Recombinant HBD-3 underwent labeling with (99m)Tc. The CD1 mice were selected as standard rodent species. Ten mice, 5 male and 5 female, were subjected to physical examination and housed in a dedicated room in 5 per cage. After 9 days pre-test period, all mice were weighted for dose adjustment and received intravenously 6mcg/mouse of (99m)Tc-HBD-3. Mortality was recorded daily, while body weight was registered once a week. Clinical observation of animals was performed daily for sickness symptoms due to the drug treatment. At day 19 a second dose of 6mcg/mouse (99m)Tc-HBD-3, was administered. Twenty-four hours after the second dose (day 20) the animals were euthanized. A piece of liver, kidneys, heart and lungs was collected for histopathological analysis. RESULTS: Our results showed that the labelled-HBD-3 dose did not induce significant toxicity in mice. Of course these parameters were not sufficient to authorize use in humans. This non-toxic dose of HBD-3 when translated from animals to humans resulted in an equivalent dose of approximately 25 times higher than that needed for imaging. CONCLUSION: Our non toxicity data of using (99m)Tc-beta-defensin-3 in mice offer a further indication in favour of the clinical use of this radiopharmaceutical in all cases where discrimination between infection and inflammation is needed.

Effect of external magnetic field on IV 99mTc-labeled aminosilane-coated iron oxide nanoparticles: demonstration in a rat model: special report.

Among the most interesting applications of ferromagnetic nanoparticles (NPs) in medicine is the potential for localizing pharmacologically or radioactively tagged agents directly to selected tissues selected by an adjustable external magnetic field. This concept is demonstrated by the application external magnetic field on IV Tc-labeled aminosilane-coated iron oxide NPs in a rat model. In a model comparing a rat with a 0.3-T magnet over a hind paw versus a rat without a magnet, a static acquisition at 45 minutes showed that 27% of the administered radioactivity was in the area subtended by the magnet, whereas the liver displays a percentage of binding of 14% in the presence of the magnet and of 16% in the absence of an external magnetic field. These preliminary results suggest that the application of an external magnetic field may be a viable route for the development of methods for the confinement of magnetic NPs labeled with radioactive isotopes targeted for predetermined sites of the body.

Analysis of pregnancy-associated plasma protein A production in human adult cardiac progenitor cells.

IGF-binding proteins (IGFBPs) and their proteases regulate IGFs bioavailability in multiple tissues. Pregnancy-associated plasma protein A (PAPP-A) is a protease acting by cleaving IGFBP2, 4, and 5, regulating local bioavailability of IGFs. We have previously shown that IGFs and IGFBPs are produced by human adult cardiac progenitor cells (haCPCs) and that IGF-1 exerts paracrine therapeutic effects in cardiac cell therapy with CPCs. Using immunofluorescence and enzyme immunoassays, we firstly report that PAPP-A is produced and secreted in surprisingly high amounts by haCPCs. In particular, the homodimeric, enzymatically active, PAPP-A is secreted in relevant concentrations in haCPC-conditioned media, while the enzymatically inactive PAPPA/proMBP complex is not detectable in the same media. Furthermore, we show that both homodimeric PAPP-A and proMBP can be detected as cell associated, suggesting that the previously described complex formation at the cell surface does not occur easily, thus positively affecting IGF signalling. Therefore, our results strongly support the importance of PAPP-A for the IGFs/IGFBPs/PAPP-A axis in CPCs biology.

Human amniotic glycodelin actively regulates changes in ß-catenin immunoreactivity in cultured human umbilical vein endothelial cells (HUVEC).

OBJECTIVE: Assessment of the capacity of Glycodelin A (GdA) to modulate the aggregation of cultured human umbilical vein endothelial cells. METHODS: Highly purified Glycodelin A (GdA) from late first trimester amniotic fluid has been added to cultured cells and its biological activity has been observed with immunofluorescent staining of ß-catenin molecules. RESULTS: GdA induces translocation of ß-catenin molecules promoting cell-to-cell adhesion and formation of adherents junctions through cytoskeletal reorganization. CONCLUSION: These data provide further mechanistic insight into the specificity of cell-to-cell adhesion, thus corroborating the role of GdA in promoting angiogenesis.

First trimester PAPP-A levels associated with early prediction of pregnancy induced hypertension.

OBJECTIVE: Previous studies have suggested an association between low levels of first trimester serum Pregnancy Associated Plasma Protein-A (PAPP-A) and the occurrence of hypertension in pregnancy (PIH). The purpose of this study was to determine the predictive value of maternal PAPP-A levels in the identification of women at risk of PIH. METHODS: Maternal serum PAPP-A was measured between 11-13 + 6 wks' gestation, as part of the first trimester screening of aneuploidies. Our study included only singleton pregnancies (973 cases) over a three years period. PAPP-A levels were expressed as gestational age-specific multiples of the median (MoM). Hypertension in pregnancy was documented by standard criteria. RESULTS: One hundred and eleven pregnant women developed hypertension (8.9%). In these patients, PAPP-A levels ranged from 0.53 to 1.08 MoM. After performing a backward stepwise regression equation and a ROC curve procedure, a PAPP-A MoM value <0.8 was able to significantly predict PIH (p < 0.001, area under the ROC curve 83%, sensitivity 68%, specificity 86%, 95 degrees CI 0.57-0.94). CONCLUSION: Low levels of serum PAPP-A (?0.8 MoM) may be a potential resource in order to early screen pregnant women at increased risk of hypertension in pregnancy.

Microbial targeting of 99mTc-labeled recombinant human beta-defensin-3 in an animal model of infection: a feasibility pilot study.

UNLABELLED: Human beta-defensin-3 (HBD-3) is an antimicrobial peptide with bactericidal effects on many gram-positive and gram-negative bacteria and some yeast species and, if radiolabeled, might be used to distinguish bacterial infection from sterile inflammation. The goals of the present study were to develop methods for radiolabeling HBD-3 with (99m)Tc and to perform preliminary investigations on (99m)Tc-labeled HBD-3 as a means to evaluate induced infection in an animal model. To this purpose, Staphylococcus aureus-induced infection was used to evaluate the capability of (99m)Tc-HBD-3 to distinguish infection from aseptic inflammation in rats. METHODS: Twenty to 40 microg of recombinant HBD-3 were labeled with (99m)Tc(+) hexa-coordinated with 3 molecules of CO and H(2)O and separated by a column from free (99m)Tc. (99m)Tc-HBD-3 was added to cultures of a bacterial suspension of S. aureus and Escherichia coli to evaluate in vitro antibacterial activity. A bacterial suspension of S. aureus and a carrageenan solution were used to induce infection and sterile inflammation, respectively, in opposite thighs of 9 adult rats. Three separate experiments were performed on groups of 3 rats each. The animals received different doses of (99m)Tc-HBD-3 injected through a cannula into the jugular vein. After sacrifice of the animals, tissue samples were obtained from sites of infection, inflammation, and control muscle (left foreleg) at 1, 3, and 5 h after (99m)Tc-HBD-3 administration. Tissue samples were weighed and then counted in a well-counter. Simultaneously, 1 mL of a standard solution of (99m)Tc-HBD-3 corresponding to each administered dose was counted. RESULTS: (99m)Tc-HBD-3 retained antibacterial activity. Radioactivity in tissue samples from the infected sites was significantly higher than that in samples of either induced inflammation or normal control muscle (ratio, approximately 3:1) at 3 and 5 h after injection, whereas similar radioactivity counts were observed for tissue samples from aseptic inflammation sites and normal control muscle. CONCLUSION: In this investigation, (99m)Tc-HBD-3 retained antibacterial activity and successfully distinguished infection from aseptic inflammation in adult rats.

Structural and kinetic effects of mobile phone microwaves on acetylcholinesterase activity.

The present study provides evidence that "in vitro" simple exposure of an aqueous solution of electric eel acetylcholinesterase (EeAChE; EC 3.1.1.7.) to cellular phone emission alters its enzymatic activity. This paper demonstrates, by combining different experimental techniques, that radio frequency (RF) radiations irreversibly affect the structural and biochemical characteristics of an important CNS enzyme. These results were obtained by using a commercial cellular phone to reproduce the reality of the human exposition. This experimental procedure provided surprising effects collected practically without experimental errors because they were obtained comparing native and irradiated sample of the same enzyme solution. Although these results cannot be used to conclude whether exposure to RF during the use of cellular phone can lead to any hazardous health effect, they may be a significant first step towards further verification of these effects on other "ex vivo" or "in vivo" biological systems.

One site on the apoB-100 specifically binds 17-beta-estradiol and regulates the overall structure of LDL.

The major protein component (apoB-100) of low-density lipoprotein (LDL) is known as a multipotential molecule the several functional regions of which can all be affected by key structural modifications driven by specific domains. Based on our previous report on structural and conformational modifications of apoB-100 in the presence of 17-beta-estradiol (E2), we characterized the interaction between E2 and the apoB-100 and further explored the induced alterations in terms of the structural arrangement of the whole LDL particle. We report evidence for the existence on apoB-100 of a single specific and saturable binding site for E2, the occupancy of which modifies the overall structure of the protein, inducing an increase in the alpha-helix fraction. As a consequence, the structure of the LDL particle is deeply perturbed, with a change in the arrangement of both the outer shell and lipid core and an overall volume shrinkage. The evidence of a regulation of apoB-100 structure by a physiological ligand opens new perspectives in the study of the biological addressing of the LDL particle and suggests a novel rationale in the search for mechanisms underlying the beneficial role of E2 in decreasing the risk of early lesions in atherosclerosis.