Effects of long-term active immunization with the second extracellular loop of human ß1- or ß3-adrenoceptors in thoracic aorta and mesenteric arteries in Lewis rats.

OBJECTIVE: To evaluate whether active immunization producing ß1- or ß3-antibodies (ß1-ABs and ß3-ABs) detected in sera of patients with dilated cardiomyopathies has deleterious effects on vascular reactivity in Lewis rat thoracic aorta (TA) and small mesenteric arteries (SMA). DESIGN AND METHOD: Lewis rats were immunized for 6months with peptidic sequences corresponding to the second extracellular loop of ß1- and ß3-adrenoceptors (ARs). During the immunization, systolic blood pressure (SBP) was monitored using the tail cuff method. The vascular reactivity of immunized rats was assessed by ex vivo studies on SMA and TA using various ß-AR agonists, phenylephrine and KCl. RESULTS: The immunizations producing functional ß1-ABs and ß3-ABs did not affect the SBP. However, in TA from ß1-AR-immunized rats, the relaxations mediated by dobutamine and salbutamol were significantly impaired in comparison with adjuvant rats whereas nebivolol-induced relaxation was not modified. Moreover, phenylephrine and KCl-mediated contractions were enhanced in these rats. In contrast, immunization with ß3-AR peptide led to the increase of relaxations induced by dobutamine in TA but did not change those induced by salbutamol and nebivolol. Surprisingly, in SMA from both rats immunized with ß1- or ß3-peptides, relaxations induced by the various ß-agonists were not changed whereas phenylephrine and KCl-mediated contractions were impaired. CONCLUSIONS: Our study shows that ß1- and ß3-ABs can affect vascular reactivity. ß1-ABs would have a pathogenic action whereas ß3-ABs would have a beneficial effect on aorta reactivity.

Cardiac effects of long-term active immunization with the second extracellular loop of human ß1- and/or ß3-adrenoceptors in Lewis rats.

ß1- and ß3-adrenoceptor (AR) auto-antibodies were detected in patients with dilated cardiomyopathy. Many studies have shown that ß1-AR auto-antibodies with partial agonist-like effect play an important role in the pathogenesis of this disease. Moreover, a recent study carried out in our laboratory has shown that ß3-AR antibodies (ß3-ABs), produced in rats, were able to reduce cardiomyocyte contractility via ß3-AR activation. The aims of this study were (1) to investigate, in isolated cardiomyocytes from rabbit, the role of Gi proteins in the ß3-ABs-induced cardiac negative inotropy, (2) to determine whether ß3-ABs may exhibit ß3-AR antagonistic property which is characteristic of partial agonists, and (3) to determine whether long-term active immunization producing both ß1-ABs and/or ß3-ABs leads to the development of cardiac dysfunction in Lewis rats. Lewis rats were immunized for 6 months with peptidic sequences corresponding to the second extracellular loop of human ß3-AR and/or ß1-AR. Agonistic effect of ß3-ABs was evaluated on electrically field-stimulated isolated cardiomyocytes from adult rabbit by measuring the cell shortening. Echocardiography and ex vivo isolated perfused heart studies were conducted on immunized rats. Finally, ß-AR expression was quantified by immunofluorescence and RT-qPCR. SR58611A (10 nM), a preferential ß3-AR agonist, and purified ß3-ABs (25 µg/ml) induced a decrease in cell shortening (-39.71±4.9% (n=10) and -17.06±3.9% (n=10) respectively). This effect was significantly inhibited when the cardiomyocytes were preincubated with pertussis toxin (0.3 µg/ml), a Gi protein inhibitor (p<0.05). In addition, SR58611A-mediated negative inotropic effect was decreased when cardiomyocytes were preincubated with ß3-ABs (p<0.0001). Echocardiography revealed a decrease in the fractional shortening and ejection fraction in rats immunized against ß1-AR and both ß1- and ß3-AR. However, the study on isolated heart showed a decrease of the isoproterenol-induced lusitropic and inotropic effects in the 3 groups of immunized rats. These systolic and diastolic dysfunctions are correlated with a decrease in the expression of ß1-ARs and an increase of ß3-ARs in rats immunized against the ß1-AR and an increase of both ß3-AR and ß1-AR in rats immunized against the ß3-AR. For the first time, these results showed that ß3-ABs had a ß3-AR partial agonist-like activity which might play a role in the pathogenesis of cardiac dysfunction.

[Detection of B19 parvovirus in plasma pools before solvent-detergent treatment of plasma: AFSSAPS and EFS Aquitaine-Limousin's experience]. / Détection du génome viral du parvovirus B19 dans les pools de plasmas servant à la préparation du plasma frais congelé traité pour viro-atténuation par solvant-détergent : expérience de l'Afssaps et de l'EFS Aquitaine-Limousin.

Since 1998, the Aquitaine-Limousin branch of the French Blood Institute has set up a parvovirus B19 (PV B19) systematic screening on each unit of plasma to be treated by solvent-detergent procedure for virus inactivation. Parvovirus B19 nucleic acid systematic testing in plasma pools became mandatory since 2005 (European monograph "Human plasma" - pooled and treated for virus inactivation). The French competent state authority (AFSSAPS) has decided to introduce this test as a part of the external quality control of labile blood products. This process is related to the harmonization of quality control practice realised on blood products in Europe even if the human plasma pooled and treated for virus inactivation by solvent-detergent is considered in France as a blood labile component. Implementation of this test required a validation step and a close cooperation between AFSSAPS and Aquitaine-Limousin blood transfusion centre. Validation consisted in perfecting a semi-quantitative, real-time nucleic acid testing method with automated extraction. This collaborative study leads us to control 1642 plasma pools. All the results were under the threshold of 10,0 IU/microL. AFSSAPS's results were in agreement with those of Aquitaine-Limousin's blood transfusion center who carry out the parvovirus B19 screening both on fresh frozen plasma units composing the pool and on plasma pools.

Validation by image analysis of a turbidimetric method to study calcium oxalate crystallization.

Numerous studies of calcium oxalate crystal formation have been carried out in the past two decades. In the present study, experiments were carried out to validate a turbidimetric method allowing to assess the calcium oxalate crystallization process. This method is quick and reproducible and can be used to quantify the inhibition of calcium oxalate crystal growth by various compounds. An experimental method of validation has been developed, which consisted in filtering solutions pure or containing modifiers at given crystallization times, photographing the filters used on scanning electron microscopy and analyzing the images using mathematical methodology. The results obtained through image analysis, namely crystal density (mean particle number per unit volume) and mean area, were correlated with the turbidimetric parameters. This finding was consistent with the qualitative examination of the photographs. Moreover, the morphological differences in crystals observed on the photographs were confirmed by the calculated length/width ratio. One can therefore assume that inhibition of calcium oxalate crystal growth is at least, partly explained by surface adsorption phenomena, which may add to complex formation.

A new approach to studying inhibitors of calcium oxalate crystal growth.

The nucleation and crystal growth of calcium oxalate (CaOx) were studied at pH 5.5 using turbidimetric measurements at 620 nm of suspensions produced by mixing calcium chloride and sodium oxalate (initial conditions: Ca, 3 x 10(-3) M; Ox, 0.5 x 10(-3) M). CaOx crystallization kinetics were defined first by the induction time ti and then by the slope of turbidity as a function of time during the interval corresponding to a correlation coefficient r2 > 0.99. The technique described requires only a small amount of material, is quick, convenient, and can be used to study inhibitors of CaOx crystallization by comparing ti and the rate of crystal growth in the presence and absence of inhibitors. The effects on CaOx crystal growth of several low molecular weight compounds, i.e. di- and tricarboxylic acids, were examined. The majority of these compounds were inhibitors of crystal growth, the greatest effect being seen with citric acid (50% inhibition in the presence of 1.5 x 10(-3) M citric acid), isocitric acid (50% inhibition in the presence of 0.75 x 10(-3) M isocitric acid) and pyrophosphate (30% inhibition in presence of 0.15 x 10(-3) M pyrophosphate). The inhibitors' behaviour regarding the medium was studied without any assumptions about their possible mechanisms of action. Measurements of ionized calcium before and after the reaction, as well as the observation of crystals by scanning electron microscopy, allowed us to formulate the hypothesis that the effect of citric acid and tartaric acid can be attributed mainly to ion pairing, in contrast to that of pyrophosphate and the other carboxylic acids.