A model eye for fluorescent characterization of retinal cultures and tissues.

Many human neural or neurodegenerative diseases strongly affect the ocular and retinal environment showing peculiar alterations which can be employed as specific disease biomarkers. The noninvasive optical accessibility of the retina makes the ocular investigation a potentially competitive strategy for screening, thus the development of retinal biomarkers is rapidly growing. Nevertheless, a tool to study and image biomarkers or biological samples in a human-like eye environment is still missing. Here we report on a modular and versatile eye model designed to host biological samples, such as retinal cultures differentiated from human induced pluripotent stem cells and ex-vivo retinal tissue, but also suited to host any kind of retinal biomarkers. We characterized the imaging performance of this eye model on standard biomarkers such as Alexa Fluor 532 and Alexa Fluor 594.

Optical computation of a spin glass dynamics with tunable complexity.

Spin glasses (SGs) are paradigmatic models for physical, computer science, biological, and social systems. The problem of studying the dynamics for SG models is nondetermistic polynomial-time (NP) hard; that is, no algorithm solves it in polynomial time. Here we implement the optical simulation of an SG, exploiting the N segments of a wavefront-shaping device to play the role of the spin variables, combining the interference downstream of a scattering material to implement the random couplings between the spins (the [Formula: see text] matrix) and measuring the light intensity on a number P of targets to retrieve the energy of the system. By implementing a plain Metropolis algorithm, we are able to simulate the spin model dynamics, while the degree of complexity of the potential energy landscape and the region of phase diagram explored are user defined, acting on the ratio [Formula: see text] We study experimentally, numerically, and analytically this Hopfield-like system displaying a paramagnetic, ferromagnetic, and SG phase, and we demonstrate that the transition temperature [Formula: see text] to the glassy phase from the paramagnetic phase grows with α. We demonstrate the computational advantage of the optical SG where interaction terms are realized simultaneously when the independent light rays interfere on the detector's surface. This inherently parallel measurement of the energy provides a speedup with respect to purely in silico simulations scaling with N.

Production of antigen-specific human IgGs by in vitro immunization.

BACKGROUND: We previously developed in vitro immunization based on a fusion protein containing the transcriptional transactivator (Tat) of human immunodeficiency virus and a double domain, called ZZ, derived from protein A of Staphylococcus aureus. In this approach, naïve human peripheral blood mononuclear cells (PBMCs) trigger a specific IgM antibody (Ab) response in the presence of ZZTat. In the present study, we attempted to raise a specific IgG Ab response. RESULTS: We found that PBMCs incubated with ZZTat and a mixture containing anti-CD40, IL4 and IL21 secrete anti-Tat IgG Abs in their supernatants, indicating that the cytokine cocktail provides an isotypic switch. Then, we deciphered the Tat determinant involved in the phenomenon and found that it is located in the region 22-57 and that, within this region, the cysteine-rich domain and the basic residues play a crucial role. Finally, we prepared a fusion protein containing a fragment derived from the NY-ESO-1 cancer/testis antigen (Ag) and showed that PBMCs incubated with ZZfNY-ESO-1Tat trigger a specific anti-fNY-ESO-1 IgG Ab response, which demonstrates the possibility of transferring immunizing ability to an Ag unrelated to Tat. CONCLUSION: Our ZZTat-based in vitro immunization approach that offers the possibility to raise an IgG Ab response against NY-ESO-1 might represent a valuable first stage for the generation of fully human IgG specific Abs.

Comparison of methods for the fabrication and the characterization of polymer self-assemblies: what are the important parameters?

The ability to self-assemble was evaluated for a large variety of amphiphilic block copolymers, including poly(ethyleneoxide-b-ε-caprolactone), poly(ethyleneoxide-b-d,l-lactide), poly(ethyleneoxide-b-styrene), poly(ethyleneoxide-b-butadiene) and poly(ethyleneoxide-b-methylmethacrylate). Different methods of formation are discussed, such as cosolvent addition, film hydration or electroformation. The influence of experimental parameters and macromolecular structures on the size and morphology of the final self-assembled structures is investigated and critically compared with the literature. The same process is carried out regarding the characterization of these structures. This analysis demonstrates the great care that should be taken when dealing with such polymeric assemblies. If the morphology of such assemblies can be predicted to some extent by macromolecular parameters like the hydrophilic/hydrophobic balance, those parameters cannot be considered as universal. In addition, external experimental parameters (methods of preparation, use of co-solvent, …) appeared as critical key parameters to obtain a good control over the final structure of such objects, which are very often not at thermodynamic equilibrium but kinetically frozen. A principal component analysis is also proposed, in order to examine the important parameters for forming the self-assemblies. Here again, the hydrophilic/hydrophobic fraction is identified as an important parameter.

Production of human antibodies by in vitro immunization using a fusion protein containing the transcriptional transactivator of HIV-1.

Antigen-specific activation of human B cells represents a key step for the production of monoclonal antibodies. Several approaches have been developed over the last thirty years in order to improve the process of lymphocyte activation in vitro. In the present study, we investigated whether the transcriptional transactivator (Tat) of human immunodeficiency virus, which possesses numerous biological activities, is able to trigger antibody secretion when incubated with human peripheral blood mononuclear cells. No such effect was observed when using Tat as a free protein. However, we found a significant IgM antibody production when Tat was previously fused to a double domain, called ZZ, derived from protein A of Staphylococcus aureus. The effect was also observed when the fusion protein, called ZZTat101, was incubated with purified B cells, indicating that the phenomenon does not require T-cell help. Antibody secretion was observed in the absence of cytokines that are usually used during in vitro immunization experiments, indicating that ZZTat101 provides the signals required for the initiation of the immune response. Antibody secretion was observed using a ZZTat mutant, containing only the Tat residues 22 to 57, called ZZTat22-57, indicating that this region is sufficient to initiate the immune response. In contrast, the effect was not found with a ZZTat22-57 mutant devoid of the seven Tat cysteines located between residues 22 and 37, demonstrating that these residues play a crucial role in the phenomenon. Our results pave the way to the development of a new in vitro immunization method based on antigens associated with ZZTat.

Preauricular incision outlining during a face-lift: a step-by-step description.

UNLABELLED: The data on the proper way to establish the preauricular incision (PAI) line and manage skin tension during lifting are limited. Undoubtedly, proper tissue handling represents a true challenge during a lifting procedure and is a crucial step in avoiding disfiguration. The authors describe their approach to PAI demarcation and local skin tension management performed in 165 cases since 2007. LEVEL OF EVIDENCE V: This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors at www.springer.com/00266.

Condensation in disordered lasers: theory, 3D+1 simulations, and experiments.

The complex processes underlying the generation of a coherent emission from the multiple scattering of photons and wave localization in the presence of structural disorder are still mostly unexplored. Here we show that a single nonlinear Schrödinger equation, playing the role of the Schwalow-Townes law for standard lasers, quantitatively reproduces experimental results and three-dimensional time-domain parallel simulations of a colloidal laser system.

Effects of the beta3-adrenoceptor (Adrb3) agonist SR58611A (amibegron) on serotonergic and noradrenergic transmission in the rodent: relevance to its antidepressant/anxiolytic-like profile.

SR58611A is a selective beta(3)-adrenoceptor (Adrb3) agonist which has demonstrated antidepressant and anxiolytic properties in rodents. The present study confirmed the detection of Adrb3 mRNA transcript in rodent brain sub-regions and evaluated the effect of SR58611A on serotonergic and noradrenergic transmission in rats and mice in an attempt to elucidate the mechanism(s) underlying these properties. SR58611A (3 and 10 mg/kg, p.o.) increased the synthesis of 5-HT and tryptophan (Trp) levels in several rodent brain areas (cortex, hippocampus, hypothalamus, striatum). Moreover, SR58611A (10 mg/kg, p.o.) increased the release of 5-HT assessed by in vivo microdialysis in rat prefrontal cortex. Systemic (3 mg/kg, i.v.) or chronic administration of SR58611A (10 mg/kg, p.o.), in contrast to fluoxetine (15 mg/kg, p.o.), did not modify the activity of serotonergic neurons in the rat dorsal raphe nucleus. The increase in 5-HT synthesis induced by SR58611A was not observed in Adrb3s knockout mice, suggesting a selective involvement of Adrb3s in this effect. SR58611A (3 and 10 mg/kg, p.o.) did not modify norepinephrine synthesis and metabolism but increased its release in rat brain. Repeated administration of SR58611A (10 mg/kg, p.o.) did not modify basal norepinephrine release in rat prefrontal cortex whereas it prevented its tail-pinch stress-induced enhancement similarly to reboxetine (15 mg/kg, p.o.). Finally SR58611A increased the firing rate of noradrenergic neurons in the rat locus coeruleus following systemic (3 mg/kg, i.v.) or local (0.01 and 1 microM) but not chronic (10 mg/kg, p.o.) administration. These results suggest that the anxiolytic- and antidepressant-like activities of SR58611A involve an increase of brain serotonergic and noradrenergic neurotransmissions, triggered by activation of Adrb3s.

Effects of the vasopressin (V1b) receptor antagonist, SSR149415, and the corticotropin-releasing factor 1 receptor antagonist, SSR125543, on FG 7142-induced increase in acetylcholine and norepinephrine release in the rat.

Arginine vasopressin and corticotropin-releasing factor are two neuroactive peptides that regulate hypothalamic-pituitary-axis and associated stress response. While the potential antidepressant and anxiolytic profiles of corticotropin-releasing factor 1 antagonists have been well studied, the concept of blockade of vasopressin system as another approach for the treatment of emotional processes has only been made available recently by the synthesis of the first non-peptide antagonist at the V1b receptor, SSR149415. In the present study SSR149415 has been compared with the corticotropin-releasing factor 1 antagonist SSR125543 and with anxiolytic and antidepressant drugs on the response of hippocampal cholinergic and cortical noradrenergic systems to the anxiogenic benzodiazepine receptor inverse agonist FG 7142. Acute (0.3-10 mg/kg, i.p.) and long-term administration (10 mg/kg, i.p., 21 days) of SSR149415 and SSR125543 reduced the FG 7142-induced increase in extracellular concentrations of acetylcholine in the hippocampus of anesthetized rats measured by microdialysis. By contrast acute and long-term administration of SSR149415 failed to reduce the FG 7142-induced increase in the release of norepinephrine in the cortex of freely moving rats. The present results demonstrate that the two compounds have similar profiles in a model of activation by an anxiogenic drug of the hippocampal cholinergic system and they suggest that SSR149415 and SSR125543 may have anti-stress anxiolytic and antidepressant effects via a mechanism of action different from classical benzodiazepine ligands and noradrenergic antidepressants.

2-Chloro-N-[(S)-phenyl [(2S)-piperidin-2-yl] methyl]-3-trifluoromethyl benzamide, monohydrochloride, an inhibitor of the glycine transporter type 1, increases evoked-dopamine release in the rat nucleus accumbens in vivo via an enhanced glutamatergic neurotransmission.

2-Chloro-N-S-phenyl 2S-piperidin-2-yl methyl]-3-trifluoromethyl benzamide, monohydrochloride (SSR504734) is a potent and selective inhibitor of the glycine transporter type 1, which increases central N-methyl-D aspartate glutamatergic tone. Since glutamate has been shown to play a role in the regulation of the dopaminergic system in dopamine-related disorders, such as schizophrenia, we investigated the possibility that SSR504734 may modify the basolateral amygdala-elicited stimulation of dopamine release in the nucleus accumbens via an augmentation of glutamate receptor-mediated neurotransmission. First, our data confirmed that SSR504734 is an inhibitor of GlytT1. In the nucleus accumbens of anesthetized rat, SSR504734 (10 mg/kg, i.p.) induced an increase of extracellular levels of glycine as measured by microdialysis coupled with capillary electrophoresis with laser-induced fluorescence detection. Second, the data demonstrated that SSR504734 (10 mg/kg, i.p.) enhanced the facilitatory influence of glutamatergic afferents on dopamine neurotransmission in the nucleus accumbens. Using an electrochemical technique, we measured dopamine release in the nucleus accumbens evoked by an electrical stimulation of the basolateral amygdala. SSR504734 facilitated dopamine release evoked by a 20 or a 40 Hz frequency basolateral amygdala stimulation. This facilitatory effect was dependent on glutamatergic tone, as intra-nucleus accumbens application of 6-7-dinitroquinoxaline-2,3-dione (10(-3) M) or DL-2-amino-5-phosphonopentanoic acid (10(-3) M), alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid and N-methyl-D aspartate receptors antagonists, respectively, inhibited dopamine release evoked by basolateral amygdala stimulation. Furthermore DL-2-amino-5-phosphonopentanoic acid co-administrated with SSR504734 hampered the dopamine-evoked release facilitation. These data underline the in vivo implication of the glycine uptake mechanism in the control of subcortical glutamate/dopamine interactions.

Cotransport-induced instability of membrane voltage in tip-growing cells.

A salient feature of stationary patterns in tip-growing cells is the key role played by the symports and antiports, membrane proteins that translocate two ionic species at the same time. It is shown that these cotransporters destabilize generically the membrane voltage if the two translocated ions diffuse differently and carry a charge of opposite (same) sign for symports (antiports). The orders of magnitude obtained for the time and length scale are in agreement with experiments. A weakly nonlinear analysis characterizes the bifurcation.

Contribution of the Nernst potential to stiffness constants: the asymmetrical case.

Inside biological membranes, one of the fundamental functions of active proteins such as pumps is to generate some electrochemical gradient across the membrane and then, to establish a new stationary state. The membrane electric potential generated by activity modifies the stiffness constants of the membrane. A spontaneous curvature appears if the inner and outer Debye lengths are different. The corresponding characteristic radius falls in the range from 0.08 microm to 50 microm. The bending elastic modulus is always increased. This effect is only noticeable in the limit of large Debye length from 0.5 microm to 0.09 microm. For a Nernst potential of 100 mV and a Debye length of 0.2 microm, the bending modulus can reach 40k(B)T.

Pattern formation of stationary transcellular ionic currents in Fucus.

Stationary and nonstationary spatiotemporal pattern formations emerging from the cellular electric activity are a common feature of biological cells and tissues. The nonstationary ones are well explained in the framework of the cable model. Inversely, the formation of the widespread self-organized stationary patterns of transcellular ionic currents remains elusive, despite their importance in cell polarization, apical growth, and morphogenesis. For example, the nature of the breaking symmetry in the Fucus zygote, a model organism for the experimental investigation of embryonic pattern formation, is still an open question. Using an electrodiffusive model, we report here an unexpected property of the cellular electric activity: a phase-space domain that gives rise to stationary patterns of transcellular ionic currents at finite wavelength. The cable model cannot predict this instability. In agreement with experiments, the characteristic time is an ionic diffusive one (<2 min). The critical radius is of the same order of magnitude as the cell radius (30 microm). The generic salient features are a global positive differential conductance, a negative differential conductance for one ion, and a difference between the diffusive coefficients. Although different, this mechanism is reminiscent of Turing instability.

Endogenous neurotensin down-regulates dopamine efflux in the nucleus accumbens as revealed by SR-142948A, a selective neurotensin receptor antagonist.

SR-142948A belongs to the second generation of potent, selective, non-peptide antagonists of neurotensin receptors. It was used to investigate the role of endogenous neurotensin in the regulation of dopamine efflux in the nucleus accumbens and striatum of anaesthetized and pargyline-treated rats. All the data were obtained using in vivo electrochemistry. Electrically evoked (20 Hz, 10 s) dopamine efflux was monitored by differential pulse amperometry, whereas variations in basal (tonic) dopamine efflux were monitored by differential normal pulse voltammetry. Like the first-generation compound SR-48692, SR-142948A did not affect the tonic and evoked dopamine efflux, but dose-dependently enhanced haloperidol (50 microg/kg, i.p.) induced facilitation of the electrically evoked dopamine release in the nucleus accumbens. In contrast to SR-48692, SR-142948A dose-dependently potentiated haloperidol (50 microg/kg, i.p.) induced increase in the basal dopamine level in the nucleus accumbens. This potentiating effect did not appear in the striatum. When dopaminergic and/or neurotensinergic transmissions were modified by a higher dose of haloperidol (0.5 mg/kg, i.p.), apomorphine, amphetamine or nomifensine, SR-142948A pre-treatment affected only the effect of apomorphine on the basal dopamine level in the nucleus accumbens. These results strengthen the hypothesis that endogenous neurotensin could exert a negative control on mesolimbic dopamine efflux.

Comparative effects of neurotensin, neurotensin(8-13) and [D-Tyr(11)]neurotensin applied into the ventral tegmental area on extracellular dopamine in the rat prefrontal cortex and nucleus accumbens.

Ejections of 10(-5)-10(-3)M neurotensin into the ventral tegmental area increased dopamine efflux measured by electrochemical approaches in the prefrontal cortex of anaesthetized rats. In the same conditions, the effects evoked on dopamine efflux by 10(-5)M neurotensin(8-13) and [D-Tyr(11)]neurotensin were different from each other and depended on the explored area: the prefrontal cortex and the caudal and rostral nucleus accumbens. In the prefrontal cortex, neurotensin(8-13) was as potent as neurotensin, whereas [D-Tyr(11)]neurotensin was ineffective. In the caudal nucleus accumbens, when considering the initial intensity of the effect, neurotensin(8-13) and neurotensin appeared more potent than [D-Tyr(11)]neurotensin. In contrast, in the rostral nucleus accumbens, neurotensin(8-13) was less potent than [D-Tyr(11)]neurotensin and neurotensin. These results support the differential involvement of two pharmacologically distinct neurotensin receptor entities on ventral tegmental area neurons in the modulation of mesolimbic and mesocortical dopaminergic activity.

Recombinant Staphylococcus strains as live vectors for the induction of neutralizing anti-diphtheria toxin antisera.

We have investigated whether the nonpathogenic gram-positive bacteria Staphylococcus xylosus and S. carnosus can display a whole domain of a toxic protein on their surface and if such vectors are suitable for immunization of BALB/c mice. The nucleotide sequence encoding the receptor-binding domain (DTR; amino acids 382 to 535) of diphtheria toxin (DT) was inserted into plasmids pSE'mp18ABPXM and pSPPmABPXM, which were designed to display heterologous proteins on S. xylosus and S. carnosus cell surfaces, respectively. Western blot analysis of the resulting bacterial lysates indicates that DTR is produced by each expression system. However, analysis of rabbit anti-DTR antisera binding to the transformed live bacteria shows that DTR is not displayed on the surface of S. xylosus cells whereas it is efficiently exposed on S. carnosus. A significant anti-DT antibody response was raised in BALB/c mice immunized intraperitoneally with S. carnosus displaying DTR, and the antisera abolished DT cytotoxicity on Vero cells. Thus, only S. carnosus can display a whole domain of a toxic protein and represents a potential vector for humoral vaccination.

Presentation of antigen in immune complexes is boosted by soluble bacterial immunoglobulin binding proteins.

Using a snake toxin as a proteic antigen (Ag), two murine toxin-specific monoclonal antibodies (mAbs), splenocytes, and two murine Ag-specific T cell hybridomas, we showed that soluble protein A (SpA) from Staphylococcus aureus and protein G from Streptococcus subspecies, two Ig binding proteins (IBPs), not only abolish the capacity of the mAbs to decrease Ag presentation but also increase Ag presentation 20-100-fold. Five lines of evidence suggest that this phenomenon results from binding of an IBP-Ab-Ag complex to B cells possessing IBP receptors. First, we showed that SpA is likely to boost presentation of a free mAb, suggesting that the IBP-boosted presentation of an Ag in an immune complex results from the binding of IBP to the mAb. Second, FACS analyses showed that an Ag-Ab complex is preferentially targeted by SpA to a subpopulation of splenocytes mainly composed of B cells. Third, SpA-dependent boosted presentation of an Ag-Ab complex is further enhanced when splenocytes are enriched in cells containing SpA receptors. Fourth, the boosting effect largely diminishes when splenocytes are depleted of cells containing SpA receptors. Fifth, the boosting effect occurs only when IBP simultaneously contains a Fab and an Fc binding site. Altogether, our data suggest that soluble IBPs can bridge immune complexes to APCs containing IBP receptors, raising the possibility that during an infection process by bacteria secreting these IBPs, Ag-specific T cells may activate IBP receptor-containing B cells by a mechanism of intermolecular help, thus leading to a nonspecific immune response.

Increasing immunogenicity of antigens fused to Ig-binding proteins by cell surface targeting.

Fusion of antigenic proteins to Ig-binding proteins such as protein A from Staphylococcus aureus and its derived ZZ fragment is known to increase immunogenicity of the fused Ag in vivo. To shed light on the origin of this effect, we used snake toxins as Ags and observed that 1) fusion of toxins to ZZ enhanced their presentation to a toxin-specific T cell hybridoma (T1B2), using A20 B lymphoma cells, splenocytes, or peritoneal exudate cells as APCs; 2) this enhancement further increased when the number of fused Ig-binding domains varied from two with ZZ to five with protein A; and 3) the phenomenon vanished when the fusion protein was preincubated with an excess of free ZZ or when P388D1 monocytes cells were used as APCs. Therefore, ZZ-fused toxins are likely to be targeted to surface Igs of APCs by their ZZ moiety. Furthermore, ZZ-alpha and toxin alpha stimulated similar profiles of toxin-specific T cells in BALB/c mice, suggesting a comparable processing and presentation in vivo for both toxin forms. To improve the targeting efficiency, ZZ-alpha was noncovalently complexed to various Igs directed to different cell surface components of APCs. The resulting complexes were up to 10(3)-fold more potent than the free toxin at stimulating T1B2. Also, they elicited both a T cell and an Ab response in BALB/c mice, without the need of any adjuvant. This simple approach may find practical applications by increasing the immunogenicity of recombinant proteins without the use of adjuvant.

Towards a recombinant vaccine against diphtheria toxin.

Two recombinant fragments of diphtheria toxin (DT) were fused to an engineered tandem repeat of the immunoglobulin (Ig) binding domain of protein A, called ZZ. These fragments are (i) the receptor binding domain (DTR), which comprises amino acids 382 to 535 of DT, and (ii) a linear peptide (DT(168-220)) which comprises residues 168 to 220 of the loop between fragment A and fragment B of DT. The fusion proteins were produced in Escherichia coli and purified by affinity chromatography. In vitro experiments showed that the DTR domain is responsible for the capacity of ZZ-DTR to bind to Vero cells and is capable of inhibiting the cytotoxicity of DT for these cells. These findings suggest that DTR binds to the cell surface receptors of DT and hence adopts a conformation that is similar to that of the receptor binding domain of DT. We compared the capacities of ZZ-DTR, ZZ-DT(168-220), and a chemically detoxified form of DT currently used for vaccination to elicit antibodies in rabbits. The toxoid was more immunogenic than ZZ-DT(168-220), which in turn was more immunogenic than ZZ-DTR. However, ZZ-DT(168-220) antiserum was poorly efficient at neutralizing DT cytotoxicity on Vero cells, whereas ZZ-DTR antiserum was only 15-fold less potent than anti-DT antisera.

Structural basis of antibody cross-reactivity: solution conformation of an immunogenic peptide fragment containing both T and B epitopes.

A synthetic octadecapeptide with the amino acid sequence of residues 23-40 of toxin alpha from Naja nigricollis, cyclized with a disulfide bridge between residues 23 and 40, induces antibodies that cross-react with toxin alpha. We report a structural analysis of this peptide in aqueous solution using NMR spectroscopy and molecular modeling. Structures compatible with the 151 obtained NMR distance restraints were generated using a random simulated annealing protocol followed by restrained high-temperature dynamics and energy minimization. The generated structures are compared with that of the corresponding sequence in the native toxin. The two stretches 23-28 and 37-40 adopt a canonical beta-strand structure in the toxin but are disordered in the peptide. The region 28-36 is ordered in both the peptide and the toxin. Residues 28-30 and 34-36 adopt beta-strand structures in the toxin but loop structures in the peptide. Residues 30-33 form a reverse turn in both the peptide and the toxin. Residues Val-27, Trp-28, Ile-35, and Ile-36 form a hydrophobic cluster. The similar, reverse-turn fold of residues 30-33 in the peptide and the toxin may be associated with the immunogenic cross-reactivity.