Production of NA by endothelial NO-synthase: an in vitro versus in vivo study.

Endothelial-derived relaxing factor (EDRF) is secreted by different endothelia in vivo. It is synthesised by endothelial NO-synthase (eNOS). Despite numerous works, its identity is not fully understood. Here the production of NA, a nitroso-arginine, which was shown to be synthesised by brain NO-synthase (bNOS), was studied in eNOS preparations. NA was quantified by reductive differential pulse voltammetry (RDPV) during its irreversible electrochemical transformation to N-hydroxy-arginine (NHA). Using microelectrodes, NA and nitrite were simultaneously measured in pure recombinant eNOS giving similar enzyme activity. NA was detected at the surface of human endothelial cells (HUVEC) and disappeared when D-arginine was introduced in the culture medium. NA production by endothelium tissue was studied in rat corpus cavernosum using voltammetric microelectrodes. NA concentration at the endothelium surface was linked to vasodilatation measured by laser Doppler induced by acetylcholine injection. LNMA ic injection induced NA disappearance. These preliminary new experiments suggested that NA could be the endogenous nitroso-compound presented early as EDRF.

Production of hydrogen peroxide in rat corpus cavernosum: an on-line study with microvoltammetric electrodes.

Continuous measurements were realized in rat corpus cavernosum using microvoltammetric captor. After inhibition of endothelial NO-synthase by L-NMA vasodilatation was induced by intracavernous injection of acetylcholine. Blood flow of rat penis was monitored by laser Doppler. A new peak appeared in reduction. It was identified as hydrogen peroxide by its peak potential, by intracavernous injection of hydrogen peroxide or catalase. Intracavernous injection of various pharmacological agents permitted to demonstrate that its origin was due to endothelial NAD(P)H oxidases. DPI inhibited its synthesis; NADH and NADPH enhanced it. Intracavernous injection of diethyldithiocarbamic acid gave the disappearance of hydrogen peroxide peak and appearance of the superoxide peak. This preliminary study showed that when the L-arginine pathway was inhibited, the NAD(P)H oxidase pathway functioned in rat corpus cavernosum for vasodilatation.

Capillary electrophoresis of the electrochemical oxidation products of Ng-hydroxy-l-arginine at physiological pH.

Ng-hydroxy-l-arginine has been described as an intermediate in the nitric oxide pathway. Its electrochemical oxidation was studied by capillary electrophoresis using either electrospray ionisation mass spectrometry or diode array detection. Three stable end products and unstable intermediates were found using these techniques. These results permitted to propose an electrochemical oxidation scheme of this molecule.

Evaluation of (99m)Tc-ciprofloxacin scintigraphy in a rabbit model of Staphylococcus aureus prosthetic joint infection.

UNLABELLED: Ciprofloxacin, a quinolone antibiotic drug, binds to DNA topoisomerase IV and DNA gyrase of various bacteria. Thus ciprofloxacin labeled with (99m)Tc could potentially act as a specific marker allowing discrimination between infection and sterile inflammation. We evaluated these properties on a rabbit model of prosthetic joint infection previously validated. We compared the images obtained in 2 groups of animals: rabbits with infected (G1; n = 6) and uninfected (G2; n = 7) prosthesis. METHODS: Partial right-knee arthroplasty was performed on 13 New Zealand White female rabbits, with a tibial silicone-elastomer implant fitting into the intramedullary canal of the tibia. After the surgical wound was closed, 10(7) cfu of a clinical strain of methicillin-susceptible Staphylococcus aureus were injected into the joint in G1 rabbits. G2 rabbits were injected with sterile saline. No antibiotic therapy was given to the animals. (99m)Tc-ciprofloxacin planar imaging was performed on days 5, 12, and 19 after surgery, and after 3 mo in 1 uninfected rabbit. Images were obtained 1, 4, and 24 h after injection (147 +/- 13 MBq). RESULTS: In G1, increased right knee (99m)Tc-ciprofloxacin uptake was observed in 3 of 5 rabbits on day 5, and in all cases on days 12 and 19. Killing of the animals revealed purulent arthritis, osteitis, and tibial myelitis. In G2, significant right-knee uptake was found on days 12 and 19 in 5 of 6 rabbits, and after 3 mo in 1; all sets of images were negative in 1 animal. Bacteriologic studies after the animals were killed were negative in G2. Mean right/left knee uptake ratios on day 19 (4-h images) were 1.8 +/- 0.4 in G1 versus 1.4 +/- 0.3 in G2 (not significant). Late images did not discriminate between infected and uninfected arthroplasty. CONCLUSION: Results of (99m)Tc-ciprofloxacin imaging in rabbits with infected/uninfected knee prosthesis suggest good sensitivity but lack of specificity for the detection of S. aureus infection.

Inability of 99mTc-ciprofloxacin scintigraphy to discriminate between septic and sterile osteoarticular diseases.

UNLABELLED: Ciprofloxacin labeled with (99m)Tc specifically binds to various bacteria. Thus, it potentially constitutes a specific marker allowing discrimination between septic arthritis/osteomyelitis and aseptic osteoarticular diseases. The aim of this prospective study was to evaluate such properties in patients with skeletal diseases. METHODS: We prospectively investigated 2 groups of patients: patients with suspected osteoarticular infections (G1, n = 16) and a control group of patients with a presentation of osteoarticular diseases and no sign suggestive of infection (G2, n = 11). All had clinical, biologic, and radiologic evaluations and had 1-, 4-, and 24-h images from (99m)Tc-ciprofloxacin scintigraphy (370 MBq) before planned biopsy or surgery. For 23 patients, the scintigraphic results were compared with histologic and bacteriologic analyses of biopsy tissue samples; for 4 patients, the scintigraphic results were compared with the findings from 23 +/- 5 mo of follow-up. RESULTS: In G1, (99m)Tc-ciprofloxacin findings were true-positive in all 11 infected sites, true-negative in 2 cases, and false-positive in 3. In G2, (99m)Tc-ciprofloxacin was true-negative in 4 cases and false-positive in 7. Neither the location of (99m)Tc-ciprofloxacin activity nor its intensity or kinetics between 1, 4, and 24 h allowed discrimination between infection and aseptic diseases (sterile loosened joint replacement, pseudoarthrosis, or arthrosis). Sensitivity, specificity, and accuracy were 100%, 37.5%, and 63%. CONCLUSION: (99m)Tc-Ciprofloxacin scintigraphy showed good sensitivity and a high negative predictive value for the detection of bone and joint infection, but it did not discriminate between infected and aseptic osteoarticular diseases in symptomatic patients referred for surgery.

Imaging apoptosis with (99m)Tc-annexin-V in experimental subacute myocarditis.

UNLABELLED: 99mTc-Annexin-V (ANX), which allows in vivo detection of apoptotic cells, is potentially a promising noninvasive tool to diagnose myocarditis. To test this assumption, we compared the myocardial uptake of ANX (imaging and quantitative autoradiography) in experimental subacute myocarditis (Wistar Bonn/Kobori rats [WBN/Kob]) and in normal Wistar rats. WBN/Kob is an inbred strain of Wistar rat in which myocardial injury mimicking subacute catecholamine-induced myocarditis spontaneously develops (course duration, 18 mo). The apoptotic myocardial rates were determined by immunohistochemical studies. METHODS: Fourteen WBN/Kob rats (8-10 mo old) and 12 control rats were injected with ANX (7.4 MBq/100 g). Ten-minute anterior planar thoracic images (matrix, 128 x 128) were obtained using a pinhole collimator, 1 and 4 h after injection. Heart-to-lung activity ratios were calculated on the scintigrams. Four hours after ANX injection, quantitative autoradiography of myocardial slices was performed, as well as histologic studies with hematoxylin-eosin and with a staining assay specific for apoptotic cells. RESULTS: Heart-to-lung activity ratios were higher in WBN/Kob rats than in control rats on 4-h images (2.07 +/- 0.07 vs. 1.66 +/- 0.06, P = 0.0007). Autoradiographic studies showed moderate diffuse, homogeneous myocardial ANX uptake significantly higher in WBN/Kob rats than in control rats: 54 +/- 4 versus 37 +/- 3 counts/mm(2) (P < 0.007). The apoptotic rate, evaluated with an apoptotic cell-staining assay, was 0.51% +/- 0.14% of cells in WBN/Kob rats versus 0.0042% +/- 0.0008% in control rats (P < 0.008). CONCLUSION: Compared with control rats, rats with subacute myocarditis mimicking catecholamine-induced myocarditis showed increased ANX myocardial uptake. This suggests a potential role for ANX imaging in the diagnosis of myocarditis.

A brain nitric oxide synthase study in the rat: production of a nitroso-compound NA and absence of nitric oxide synthesis.

The products of brain NO-synthase (NOS) were studied by different analytical techniques with the same incubation conditions. Voltammetric techniques used a micro cell containing NOS and its substrate (10 mM arginine). Using porphyrin microelectrodes with differential pulse amperometry nitric oxide (NO) was not detected when nafion membrane was present (less than 0.3 muM). Nitrite was detected with the same microelectrode without membrane (0.42 mM). Differential pulse voltammetry (DPV) with micro carbon electrode detected a nitroso-compound (NA) in reduction (1 mM) and not NO. In oxidation the observed DPV peak was due to nitrite (0.43 mM). Citrulline was detected by high performance liquid chromatography (0.51 mM). Using Diels Alder reaction in NOS preparation a NA-cycloadduct was observed by capillary electrophoresis (0.2 mM) and mass spectrometry (0.22 mM). Diels Alder reaction is the reaction of the identification of the nitroso group. NA-cycloadduct degradation by retro Diels Alder reaction gave equimolar concentrations of citrulline and nitrite without NO production. These observations lead us to affirm that NOS synthesizes NA.

Radiolabeled fucoidan as a p-selectin targeting agent for in vivo imaging of platelet-rich thrombus and endothelial activation.

UNLABELLED: P-selectin expression is involved in the pathophysiology of biologically active arterial thrombus and endothelial activation after a transient ischemic event. Fucoidan is a polysaccharidic ligand of P-selectin, with a nanomolar affinity. In the present study, we propose a new approach of P-selectin molecular imaging based on radiolabeled fucoidan. METHODS: Two kinds of experimental models were selected to evaluate the ability of radiolabeled fucoidan to detect P-selectin expression: platelet-rich arterial thrombi (vegetations of infective endocarditis and arterial mural thrombus) and myocardial ischemia-reperfusion. These 2 settings were chosen because they were clinically relevant, and both were associated with an important overexpression of platelet and endothelial P-selectin, respectively. RESULTS: (99m)Tc-fucoidan SPECT was able to detect the presence of platelet-rich arterial thrombi in all animals, with a median target-to-background ratio of 5.2 in vegetations of endocarditis and 3.6 in mural aneurysmal thrombus, and to detect a persistent endothelial activation at 2 h after reperfusion. In this latter model, the magnitude of the signal was correlated with the extent of myocardium that underwent transient ischemia. The sensitivity of selectivity of the uptake and retention of (99m)Tc-fucoidan in both settings was excellent. CONCLUSION: This study supports (99m)Tc-fucoidan as a relevant imaging agent for in vivo detection of biologic activities associated with P-selectin overexpression, such as arterial thrombus and ischemic memory. Given the reported wide availability at a low cost, and its low toxicity, fucoidan seems to overcome some of the limitations of previous P-selectin-targeted imaging agents.

Non-invasive molecular imaging of fibrosis using a collagen-targeted peptidomimetic of the platelet collagen receptor glycoprotein VI.

BACKGROUND: Fibrosis, which is characterized by the pathological accumulation of collagen, is recognized as an important feature of many chronic diseases, and as such, constitutes an enormous health burden. We need non-invasive specific methods for the early diagnosis and follow-up of fibrosis in various disorders. Collagen targeting molecules are therefore of interest for potential in vivo imaging of fibrosis. In this study, we developed a collagen-specific probe using a new approach that takes advantage of the inherent specificity of Glycoprotein VI (GPVI), the main platelet receptor for collagens I and III. METHODOLOGY/PRINCIPAL FINDINGS: An anti-GPVI antibody that neutralizes collagen-binding was used to screen a bacterial random peptide library. A cyclic motif was identified, and the corresponding peptide (designated collagelin) was synthesized. Solid-phase binding assays and histochemical analysis showed that collagelin specifically bound to collagen (Kd 10(-7) M) in vitro, and labelled collagen fibers ex vivo on sections of rat aorta and rat tail. Collagelin is therefore a new specific probe for collagen. The suitability of collagelin as an in vivo probe was tested in a rat model of healed myocardial infarctions (MI). Injecting Tc-99m-labelled collagelin and scintigraphic imaging showed that uptake of the probe occurred in the cardiac area of rats with MI, but not in controls. Post mortem autoradiography and histological analysis of heart sections showed that the labeled areas coincided with fibrosis. Scintigraphic molecular imaging with collagelin provides high resolution, and good contrast between the fibrotic scars and healthy tissues. The capacity of collagelin to image fibrosis in vivo was confirmed in a mouse model of lung fibrosis. CONCLUSION/SIGNIFICANCE: Collagelin is a new collagen-targeting agent which may be useful for non-invasive detection of fibrosis in a broad spectrum of diseases.

Evaluation of 99mTc-UBI 29-41 scintigraphy for specific detection of experimental Staphylococcus aureus prosthetic joint infections.

PURPOSE: (99m)Tc-UBI 29-41 (UBI), an antimicrobial peptide, specifically targets bacteria. We tested the ability of UBI to discriminate between infected and uninfected prosthetic joints using a rabbit model previously validated. METHODS: Left knee arthroplasty was performed on 20 New Zealand rabbits, then 10(7) cfu of S. aureus (n = 12) or sterile saline (n = 8) was injected into the joint. On days 9 and 20 after surgery, planar UBI scintigraphy was performed in six infected and four uninfected rabbits, 1 h and 4 h p.i. (150 MBq), on a gamma camera. Operated-to-normal knee activity ratio (ONKR) was calculated on each scintigram. Then, after sacrifice, tissue samples of both knees were counted in a gamma counter. RESULTS: One rabbit injected with sterile saline had cutaneous infection at sacrifice and was excluded from analysis. ONKR was higher in infected than in uninfected animals 4 h p.i. 20 days after surgery: 1.75 +/- 0.48 vs 1.13 +/- 0.11, p = 0.04. From 1 h to 4 h p.i., ONKR increased in 9/12 infected and 0/7 uninfected animals. According to UBI uptake intensity and kinetics, scintigraphy was truly positive in all infected cases on day 9 and in four of six infected cases on day 20. It was truly negative in two of three sterile inflamed prosthetic knees on day 9, and in all cases on day 20. Biodistribution studies revealed increased UBI uptake in periprosthetic tissues in all animals 9 days after surgery, and only in infected animals on day 20. CONCLUSION: In this experimental study, (99m)Tc-UBI 29-41 scintigraphy permitted the early detection of acute prosthetic joint infection, and exclusion of infection in chronic sterile prosthetic joint inflammation.

Acquired gentamicin resistance by permeability impairment in Enterococcus faecalis.

Enterococci are intrinsically resistant to low levels of aminoglycosides. We previously selected in vitro and in vivo Enterococcus faecalis with intermediate-level resistance to gentamicin that did not abolish synergism with a cell-wall-active agent (E. Aslangul et al., Antimicrob. Agents Chemother. 49:4144-4148, 2005). The aim of this study was to investigate the mechanism of resistance to gentamicin in the 1688-G3 third-step mutant (MIC, 512 microg/ml) of E. faecalis JH2-2. No mutations were found in the genes for L6 ribosomal protein and the four copies of 16S rRNA. Production of a known aminoglycoside-modifying enzyme was unlikely due to the distinct resistance phenotype and absence of the corresponding genes. Efflux was also unlikely since ethidium bromide MICs were similar for JH2-2 and 1688-G3 and since the pump inhibitors reserpine and verapamil had no effect on gentamicin resistance in both strains. To study gentamicin accumulation, we developed a nonisotopic method based on a fluorescent polarization immunoassay. Impaired gentamicin accumulation was observed in 1688-G3 compared to JH2-2 and was only partially reversible by the N,N'-dicyclohexylcarbodiimide (DCCD) uncoupler agent. The lower sensitivity of 1688-G3 to DCCD suggested alteration of the FoF1-ATPase. However, no mutations were detected in the structural genes (atp) for the Fo channel and no difference in transcript levels of atpB and atpE was found between 1688-G3 and JH2-2. Our data are compatible with acquisition of intermediate-level gentamicin resistance by uptake impairment in E. faecalis.

Paradoxic activation of the renin-angiotensin system in twin-twin transfusion syndrome: an explanation for cardiovascular disturbances in the recipient.

Despite advances in treatment, twin-to-twin transfusion syndrome (TTTS) still carries a high risk for perinatal mortality and morbidity. Simple blood transfer from the donor to the recipient twin cannot explain all of the features of this disease, in particular the recipient's hypertensive cardiomyopathy. We report a case in which TTTS resulted in preterm delivery with early neonatal death of both twins, allowing assessment of the renin angiotensin system (RAS) status of each fetus, both by cord blood renin and aldosterone assay and by renal immunohistochemistry. The donor had severe oliguria/oligohydramnios, whereas the recipient, in addition to severe polyuria/polyhydramnios, had cardiomyopathy, atrioventricular regurgitation, and ascites. Although immunohistochemistry demonstrated that renal secretion of renin was up-regulated in the donor and down-regulated in the recipient, cord blood levels of renin and aldosterone were similar, with high renin levels in both twins. This observation supports the hypothesis that despite renal RAS down-regulation, the recipient is exposed to RAS effectors elaborated in the donor and transferred via placental shunts. This may contribute to cardiomyopathy and hypertension in the recipient, which cannot be accounted for by hypervolemia alone. We thus hypothesized that in TTTS, the recipient's hypertensive cardiomyopathy could be due to a mechanism similar to the classical model of hypertension referred to as "2 kidneys-1 clip." Thus the hypovolemic donor twin, comparable to the clipped kidney, produces vasoactive hormones that compromise the recipient, comparable to the normal kidney, causing hypertension and cardiomyopathy.