Javier Ruiz

Surfactant-induced release of liposomal contents. A survey of methods and results.

A systematic approach to the phenomenon of surfactant-dependent release of liposomal contents has been attempted. A variety of methods have been comparatively studied. The influence of the size of the entrapped molecule, nature of the surfactant, composition of bilayers and sonication of liposomes have been considered separately. In order to compare different results, a parameter has been defined, R50, as the phospholipid/surfactant mole ratio producing 50% release of the entrapped solute. This parameter appears to be, to a large extent, independent of time and liposome concentration. Surfactant-induced release of liposomal contents does not occur as a result of breakdown of phospholipid bilayers, but is rather a different phenomenon, occurring at detergent concentrations substantially lower (2-5 times) than solubilization. The required amount of surfactant appears to increase with the size of the entrapped solute. R50 depends clearly on the nature of the soluble amphiphile, but there is no obvious relationship with its critical micellar concentration. Liberation of vesicle content also depends on bilayer composition: phospholipids have various effects on the stability of the membrane, while the hydrophobic peptide, gramicidin A, appears to have little influence. Cholesterol is interesting, since at equimolar proportions with phosphatidylcholine, it decreases the stability of bilayer towards Triton X-100, while increasing it in the presence of cholate. Sonication also exerts an influence on the surfactant-dependent release of vesicle contents; it appears to decrease the bilayer stability, so that lower detergent concentrations are required to liberate the entrapped solutes. Finally, it should be noted that, although the decrease in self-quenching of 6-carboxyfluorescein is a convenient method for the study of solute liberation, glucose release, as detected by enzymatic methods, may be more reliable for accurate measurements.

Non-adrenoceptor [3H]idazoxan binding sites (I2-imidazoline sites) are increased in postmortem brain from patients with Alzheimer's disease ☆

The I2-imidazoline site (a non-adrenergic mitochondrial site for which a glial location has been proposed and that is associated with the B-form of the enzyme monoamine oxidase) was evaluated in postmortem cortical membranes from 9 subjects with Alzheimers disease (AD) and 9 matched-controls by using [3H]idazoxan (0.6-30 nM) in the presence of 10(-6) M (-)-adrenaline to prevent binding to alpha 2-adrenoceptors. In AD the density (Bmax) of I2-imidazoline sites was significantly higher (+63%) than in controls whereas no differences were apparent in affinity values (Kd). The results support the hypothesis that the I2 imidazoline site has a major location on glial (astrocyte) cells.

Possible involvement of a fibroblast growth factor 9 (FGF9)–FGF receptor-3-mediated pathway in adult pig retinal ganglion cell survival in vitro

The expression and potential roles of fibroblast growth factors (FGF) and their cognate FGF receptors (FGFR) in adult mammalian retinal ganglion cells (RGC) are poorly known. We show that FGFR-3 and FGFR-4 are especially pronounced on RGC and amacrine cell bodies in adult pig inner retinae both in vivo and in vitro. Western blotting revealed distinct profiles for each receptor. Expression of each FGFR and effects of the preferred ligand for FGFR-3, FGF9, upon RGC survival and neurite outgrowth were examined in primary retinal cell cultures: whereas there was no stimulation of neuritogenesis, RGC survival was promoted in a dose-dependent manner (ED(50) approximately 500 pg/ml, mean maximal increase of 60%) and could be completely blocked by addition of FGF9 neutralising antibody. Experiments with three additional FGF (FGF1, FGF2, and FGF4) showed no stimulation of RGC survival above control levels. Taken together, these data suggest that the ligand-receptor couple FGF9-FGFR-3 may function to promote survival of adult mammalian RGC, and their application might be beneficial in retinal degenerative diseases such as glaucoma.

Increased 3H. raclopride binding sites in postmortem brains from schizophrenic violent suicide victims

The specific binding of the D2-dopamine receptor antagonist radioligand [3H] raclopride was quantitated in the postmortem caudate and frontal cortex from schizophrenic suicide victims and control subjects. In schizophrenic suicides the density of binding sites (Bmax) was higher (40%,P<0.05) in the caudate, whereas it did not change in the cortex as compared to those in controls. The apparent dissociation constants (Kd) were also found increased both in caudate (24%) and cortex (75%) from schizophrenics, but these apparent decreases in receptor affinity did not reach statistical significance. The mean Bmax value in drug-free schizophrenic suicides (n=3) did not differ from the Bmax value in neuroleptic drug-treated schizophrenics (n=7) but it was found increased in respect to control subjects (n=9). No differences in [3H] raclopride binding were observed between non-schizophrenic suicide victims (n=4) and matched controls (n=4), suggesting that the modifications of D2-dopamine receptors in schizophrenia are not related to suicide.

Characterization of [ 3 H]idazoxan binding sites on human platelets

It has been shown previously that cyclosporin A enhances platelet aggregation responses, particularly to adenosine diphosphate (ADP). In this investigation platelet responses to ADP in the presence of cyclosporin A and pimecrolimus (SDZ ASM 981), a new cell selective inhibitor of inflammatory cytokines, were determined. Measurements were performed in whole blood using a sensitive platelet counting method and in platelet-rich plasma (PRP) using a Biola laser aggregometer. The latter monitors both aggregate formation and aggregate size. In vitro studies were performed using recombinant hirudin as anticoagulant in order that physiological concentrations of divalent cation concentrations were maintained. Studies using both methods confirmed an enhanced aggregation response to ADP in the presence of cyclosporin A. In contrast, aggregation responses were not enhanced in the presence of pimecrolimus, either in PRP or in whole blood where a slight reduction of ADP-induced aggregation was seen at concentrations of pimecrolimus >10(-6) M. The effects of cyclosporin A and pimecrolimus on ADP-induced calcium mobilisation in platelets were determined using a flow cytometric method. A significant increase in intracellular calcium mobilisation was seen in the presence of cyclosporin A but not in the presence of pimecrolimus. Enhanced platelet aggregation responses to ADP observed in the presence of cyclosporin A may be a consequence of enhanced ADP-induced calcium mobilisation.Human platelets possess at least two non-adrenoceptor binding sites pharmacologically distinct from the f 2 -adrenoceptors. The effects of various imidazol(in)es on platelet aggregation have suggested that these compounds may interact with these non-adrenoceptor binding sites on platelets. [ 3 H]Idazoxan is an antagonist of the f 2 -adrenoceptors frequently used to characterize imidazoline I 2 receptors. We evaluated the binding of [ 3 H]idazoxan to human platelet membranes. In saturation experiments [ 3 H]idazoxan (1.25-32 nM) recognized a single, saturable binding site with high affinity. However, competition assays revealed the presence of f 2A -adrenoceptors and a non-adrenoceptor minor population (25-39%) recognized with high affinity by the imidazoline drug with low affinity for f 2 -adrenoceptors 2-BFI. After the addition of (-)adrenaline (5 w M) to mask f 2 -adrenoceptors, competition curves against [ 3 H]idazoxan binding were biphasic. The imidazoline I 1 receptor-selective drugs, efaroxan and rilmenidine, recognized the minor component with high affinity, whereas the imidazoline I 2 receptor-selective drugs, guanabenz and 2-BFI, bound with high affinity to the major component. Further masking experiments in the presence of efaroxan (2 w M) or guanabenz (1 w M) confirmed that [ 3 H]idazoxan labels two non-adrenoceptor binding sites pharmacologically compatible with imidazoline I 1 and I 2 receptors as well as f 2A -adrenoceptors in human platelets.Despite the increased safety of blood components, achieved through improved donor selection and testing, transfusion recipients remain at risk of transfusion-associated diseases. Transfusion of cellular blood components has been implicated in transmission of viral, bacterial and protozoan diseases. Investigators have studied a myriad of processes for pathogen depletion and/or inactivation. No successful treatments, apart the leukodepletion, have already been identified for red cells and platelets. And more, several evidences indicate that platelets play a key role in host defence against infection. High levels of pathogens were added to single-donor platelet concentrates (PC) containing 3 to 5 10(11) platelets in 300 ml. The infectivity of each pathogen was measured with established biologic assays. The following levels of pathogen inactivation were achieved : >10(2.63) plaque-forming units (PFU) per ml of adenovirus 5 (ADV5), >10(5.6) PFU per ml of Poliovirus 1 (P1) and >10(4.1) PFU per ml of vaccinia virus (VaV). In conclusion, the PC show a potential virucidal effect. This inactivation process has been found with bacteria and still remains unknown for viruses.A platelet substitute, Synthocytes, is being developed for the prevention and treatment of thrombocytopenia. Synthocytes are composed of fibrinogen adsorbed on heat stabilised albumin microcapsules of defined size. The purpose of this study was to perform experiments in vitro to investigate the capacity of Synthocytes to interact with platelets, one of the means through which Synthocytes may contribute to haemostatic plug formation in vivo. Synthocytes were found to interact with platelets as shown by platelet aggregation assays and measurements of [(14)C]5HT release from platelets in whole blood and platelet-rich plasma. Platelet-Synthocytes co-aggregate formation was demonstrated directly using flow cytometry and the presence of activated platelets in these co-aggregates was demonstrated using an antibody to P-selectin. Synthocytes enhanced platelet responsiveness to conventional aggregating agents such as ADP. Indeed, antagonists of the action of ADP on platelets inhibited the direct effects of Synthocytes on platelets in whole blood, as did a GPIIb/IIIa antagonist. Enhancement of annexin V binding was also observed, indicative of increased pro-coagulant activity. Experiments performed with control microcapsules (lacking fibrinogen) confirmed the importance of fibrinogen in the interactions that occurred. The results suggest that fibrinogen on the surface of Synthocytes can interact with GPIIb/IIIa on platelets to induce platelet activation, secretory activity and aggregation, and that ADP contributes to this process. This initial interaction renders platelets more susceptible to the stimulatory effects of other platelet-activating agents. It is considered likely that in the clinical setting of thrombocytopenia any interaction between Synthocytes and residual platelets that are present may contribute to primary haemostasis.