Towards a social and context-aware multi-sensor fall detection and risk assessment platform.

For elderly people fall incidents are life-changing events that lead to degradation or even loss of autonomy. Current fall detection systems are not integrated and often associated with undetected falls and/or false alarms. In this paper, a social- and context-aware multi-sensor platform is presented, which integrates information gathered by a plethora of fall detection systems and sensors at the home of the elderly, by using a cloud-based solution, making use of an ontology. Within the ontology, both static and dynamic information is captured to model the situation of a specific patient and his/her (in)formal caregivers. This integrated contextual information allows to automatically and continuously assess the fall risk of the elderly, to more accurately detect falls and identify false alarms and to automatically notify the appropriate caregiver, e.g., based on location or their current task. The main advantage of the proposed platform is that multiple fall detection systems and sensors can be integrated, as they can be easily plugged in, this can be done based on the specific needs of the patient. The combination of several systems and sensors leads to a more reliable system, with better accuracy. The proof of concept was tested with the use of the visualizer, which enables a better way to analyze the data flow within the back-end and with the use of the portable testbed, which is equipped with several different sensors.

Targeting neonatal ischemic brain injury with a pentapeptide-based irreversible caspase inhibitor.

Brain protection of the newborn remains a challenging priority and represents a totally unmet medical need. Pharmacological inhibition of caspases appears as a promising strategy for neuroprotection. In a translational perspective, we have developed a pentapeptide-based group II caspase inhibitor, TRP601/ORPHA133563, which reaches the brain, and inhibits caspases activation, mitochondrial release of cytochrome c, and apoptosis in vivo. Single administration of TRP601 protects newborn rodent brain against excitotoxicity, hypoxia-ischemia, and perinatal arterial stroke with a 6-h therapeutic time window, and has no adverse effects on physiological parameters. Safety pharmacology investigations, and toxicology studies in rodent and canine neonates, suggest that TRP601 is a lead compound for further drug development to treat ischemic brain damage in human newborns.

Targeted Vpr-derived peptides reach mitochondria to induce apoptosis of alphaVbeta3-expressing endothelial cells.

The HIV-1 encoded apoptogenic protein Vpr induces mitochondrial membrane permeabilization (MMP) via interactions with the voltage-dependent anion channel (VDAC) and the adenine nucleotide translocator (ANT). We have designed a peptide, TEAM-VP, composed of two functional domains, one a tumor blood vessel RGD-like 'homing' motif and the other an MMP-inducing sequence derived from Vpr. When added to isolated mitochondria, TEAM-VP interacts with ANT and VDAC, reduces oxygen consumption and overcomes Bcl-2 protection to cause inner and outer MMP. TEAM-VP specifically recognizes cell-surface expressed alpha(V)beta(3) integrins, internalizes, temporarily localizes to lysosomes and progressively co-distributes with the mitochondrial compartment with no sign of lysosomal membrane permeabilization. Finally TEAM-VP reaches mitochondria of angiogenic endothelial cells to induce mitochondrial fission, dissipation of the mitochondrial transmembrane potential (DeltaPsi(m)), cytochrome c release and apoptosis hallmarks. Hence, this chimeric peptide constitutes the first example of a virus-derived mitochondriotoxic compound as a candidate to kill selectively tumor neo-endothelia.

Structural basis of the cross-reaction between an antibody to the Trypanosoma cruzi ribosomal P2beta protein and the human beta1 adrenergic receptor.

Antibodies from patients with Chagas heart disease and monoclonal antibodies (or mAb) to the carboxy-terminal end (B cell epitope R13) of the ribosomal P2beta protein of Trypanosoma cruzi (TcP2beta) cross-react with the beta1 adrenergic receptor (beta1-AR). Two single-chain Fv fragments (scFv) C5 and B7 derived from the variable regions of the anti-R13 mAb 17.2 were expressed. scFv C5 was a dimer and bound to TcP2beta with an affinity of K(d) = 8 nM, whereas scFv B7 was monomeric and had less affinity than scFv C5 for TcP2beta, K(d) = 46 nM. The affinity constant of scFv C5 to the second extracellular loop of the human beta1-AR was of 10 microM. Moreover, scFv C5 induced an increase in cAMP levels of CHO-K cells transfected with the human beta1-AR; scFv B7 had no effect but blocked isoproterenol stimulation. The agonist-like activity of scFv C5 and the antagonist activity of scFv B7 were both confirmed in vivo on heart beating frequency after their passive transfer to mice. Molecular modeling of the variable region of mAb 17.2 indicated which amino acids were likely to be involved in recognizing both peptide EDDDMGFGLF, derived from the R13 epitope of TcP2beta, and peptide ESDEARRCYN from the second extracellular loop of the human beta1-AR. It is plausible that the recently described cross-reaction of mAb 17.2 with rhodopsin can also be explained by this model. The physiological effects of this type of anti-T. cruzi antibodies may increase the liability of patients with Chagas disease.

The N-terminus of HIV-1 Tat protein is essential for Tat-TAR RNA interaction.

The human HIV transactivator protein Tat is essential for efficient viral transcription that occurs by a complex mechanism involving interaction of Tat with the TAR RNA element. This interaction appears to require the mediation of a cellular protein, cyclin T1. However, the possibility that Tat and TAR associate in a binary Tat-TAR complex has been little investigated. Using a chemically synthesized active Tat protein, the kinetic and equilibrium parameters of its interaction with TAR were determined by surface plasmon resonance technology. Independently of partner and method of immobilization onto the sensor chip, the association (k(a) = 5-9 x 10(5) M(-1) s(-1)) and dissociation rate constants (k(d) = 1.7-4.3 x 10(-3) s(-1)) yielded similar equilibrium dissociation constants (K(d) = 2-8 nM). A truncated peptide encompassing residues 30-86 of Tat did not bind to TAR at all. We conclude that Tat can form a high-affinity complex with TAR in the absence of cyclin T1 and that the N-terminal domain of Tat is essential for this interaction, suggesting a conformational link between this domain and the basic domain of Tat. These results are important in our quest for developing therapeutic compounds that impair viral replication.

Structural and functional complexity of the humoral response against the Trypanosoma cruzi ribosomal P2 beta protein in patients with chronic Chagas' heart disease.

High levels of antibodies against the C-terminus of the Trypanosoma cruzi TcP2 beta ribosomal protein, defined by the peptide EEEDDDMGFGLFD, named R13, have been measured in sera from patients with chronic Chagas' Heart Disease (cChHD). These antibodies also recognize an epitope on the second extracellular loop of the beta 1-adrenergic receptor, inducing a functional response on cardiomyocytes. The aim of this study was to gain novel insights into the structural basis of this cross-reactivity as well as to evaluate the origin of anti-M2- cholinergic receptor antibodies, which are also commonly found in cChHD patients. To address these questions we immunopurified anti-R13 antibodies and studied the structural requirements of epitope recognition. Results showed that the immunopurified antibodies recognized a conformation of R13 in which the third Glu residue was essential for binding, explaining their low affinity for the mammalian homologue (peptide H13: EESDDDMGFGLFD). Alanine mutation scanning showed individual variations in epitope recognition in each of the studied patients. The importance of a negatively charged residue at position 3 for the recognition of anti-R13 antibodies was further confirmed by competition experiments using a Ser3-phosphorylated H13 analogue, which had 10 times more affinity for the anti-R13 antibody than the native H13 peptide. Moreover, anti-R13 antibodies stimulated either the beta 1-adrenergic or the M2-cholinergic receptor, in strict agreement with the functional properties of the IgG fractions from which they derived, demonstrating that the same parasite antigen may generate antibody specificities with different functional properties. This may be a clue to explain the high variability of electrophysiological disturbances found in cChHD.

Immunogenicity of polymerizable synthetic peptides derived from a vaccine candidate against schistosomiasis: the asparaginyl endopeptidase (Sm32).

The asparaginyl endopeptidase (Sm32) is expressed in the gastrodermal cells of the schistosome gut and in the head glands of the cercariae. Possibly, Sm32 hydrolyzes pro-proteins involved in the degradation of host hemoglobin [Parasitol. Today 12 (1996) 125]. Preliminary evidences using an Sj32/Sm32 murine vaccine have shown a profound effect on oviposition and worm burden [Chin. J. Schist. Control. 7 (1995) 72; Bull. Human Med. Univ. 24 (1999) 225; Vaccine 20 (2002) 439]. The importance of Sm32 as a novel vaccine candidate is based on the possibility of preventing the maturation of other cathepsins and/or preventing schistosome skin invasion. We studied the immunogenicity of polymerizable peptides derived from Sm32 to select potential protective epitopes. Sm32 prediction of T and B epitopes and homology studies with human legumain were performed. Among the variety of factors that influence the antibody response, we specifically examined the effect of: (i) genetic background of mouse strain, inbred (C57BL/6) versus outbred (Swiss) mice; and (ii) vaccination with a single peptide versus pool of peptides. Swiss mice raised antibodies to three different regions of the Sm32, as tested by the Multiple Antigen Blot Assay (MABA): 182-215 (peptides IMT-70 and 72), 244-273 (IMT-64) and 336-355 (IMT-66). None of these regions were immunogenic for C57BL/6. On the contrary, other peptides, IMT-4 (21-40), IMT-12 (101-120) and IMT-26 (292-313) were highly immunogenic for this inbred strain. Only Swiss mice immunized with a single peptide (IMT-64 and 72) or with three different pools of IMT-peptides (Pool A-II: 14, 16, 18, 70, 72, 89; pool A-III: 22, 64, 24, 26, 28 and pool A-V: 64, 66, 28, 70, 72) recognized the original protein in a crude extract of the worm antigen by Western blot. Peptides IMT-64, 14 and 26 were responsible for this recognition. In general, the vaccination with pool of peptides was more immunogenic for both mouse strains. Predicted B cell epitopes, with hydrophilicity scores over +10 (IMT-12, 64, 26) were always immunogenic after either single or combined peptide vaccination. Sm32 sequences 41-80 (IMT-6 and 8), 141-160 (IMT-16) and 182-215 (IMT-70 and 72) were nearly identical to the corresponding human legumain regions and should be excluded from the human vaccine. We can conclude that the regions of Sm32 that were recognized by antibodies of mice immunized with polymerizable peptides depended on the mice strain and on the hydrophilicity score of the peptides.

A novel retro-inverso gonadotropin-releasing hormone (GnRH) immunogen elicits antibodies that neutralize the activity of native GnRH.

GnRH vaccines have been successfully used for the inhibition of gonadotropin secretion and gonadal function. As an alternative to native GnRH, retro-inverso (RI) GnRH might be an improved immunogen. The RI peptides are composed of D-amino acids assembled in the reverse order (C to N terminus) in relation to the parent L peptide. These peptides are immunogenic and can produce high titers of antibodies that bind the parent peptide with high affinity and specificity. We show that RI-GnRH peptides conjugated to ovalbumin as well as unconjugated RI-GnRH elicit high titers of anti-GnRH antibodies in rabbits and mice. Antibodies were affinity purified and shown by ELISA to be selective for mammalian GnRH compared with GnRH II and [Gln(8)]GnRH. The binding kinetics of antibody-peptide interactions was determined using biosensor technology (BIACORE). The purified anti-GnRH antibodies inhibited GnRH-stimulated signal transduction in COS-1 cells expressing the human GnRH receptor. Immunization of mice with unconjugated and conjugated RI-GnRH peptide, in the absence of complete Freund's adjuvant, produced antisera that cross-reacted with mammalian GnRH. As RI peptides are resistant to cleavage by proteolytic enzymes, they are potentially orally active. The ability of RI-GnRH peptides to produce antibodies to GnRH without conjugation and without Freund's complete adjuvant constitutes a novel vaccine with improved properties of potential application in animal management and sex hormone-dependent cancers.

Agonist-like activity of antibodies directed against the second extracellular loop of the human cardiac serotonin 5-HT4(e) receptor in transfected COS-7 cells.

We have previously reported that antipeptide antibodies directed against the second extracellular loop of the cardiac h5-HT4 receptor could block the activation of the L-type Ca channel in human atrial cardiomyocytes. In this paper we investigate the immunological and physiological activity of these antibodies, in a cell system expressing a larger amount of receptors than the atrial cells. The recombinant receptor was expressed at the surface of COS-7 cells under an active form (serotonin, EC50 = 1.81 x 10(-7) M), at a high level (375 +/- 25 fmol receptor/mg total protein) and was able to bind a specific ligand (GR113808) with a high affinity (Kd = 0.28 +/- 0.05 nM). In this system, the same anti-peptide antibodies used for the cardiac cells induced an "agonist-like" effect on the recombinant h5-HT4 receptor. These results are in line with those shown for others G-protein coupled receptors, as adrenoreceptors. In addition, this work showed that the effect of the antibodies is not only dependent on the epitopic region recognised but also on the molecular density and/or the cellular environment of the target receptors. Finally, our results support the hypothesis that the h5-HT4 receptor could be a new target for autoantibodies in patients with atrial arrhythmia.

Immunomodulatory properties of cystatins.

Cystatins are natural tight-binding reversible inhibitors of cysteine proteases. Because these cysteine proteases exist in all living organisms and because they are involved in various biological and pathological processes, the control of these protease functions by cystatins is of cardinal importance. Cystatins are found in mammals but cystatin-like molecules are also present in mammals and parasites. In the immune system, cystatins modulate cathepsin activities and antigen presentation. They also induce tumor necrosis factor alpha and interleukin 10 synthesis, and they stimulate nitric oxide production by interferon gamma-activated murine macrophages. In turn, nitric oxide has inhibitory activity on cysteine proteases, especially those from parasitic protozoa. Cystatins isolated from parasitic nematodes also have immunomodulatory activities that are distinguishable from those induced by lipopolysacharide-like molecules from endosymbiotic bacteria. On the whole, cystatins and cystatin-like molecules belong to a new category of immunomodulatory molecules. Doubtless increasing data will improve our knowledge of this property, leading to practical applications in immunotherapy.

Immunogenicity of synthetic peptides from the Sm31 antigen (cathepsin B) of the Schistosoma mansoni adult worms.

The chemical synthesis of peptides may simplify the production of molecules for diagnosis of Schistosoma mansoni. Seventeen polymeric, 20-amino acids long, peptides comprising the entire Sm31 molecule of the adult worm, were synthesized under the t-boc strategy and their immunogenicity was evaluated. Of these, 10 peptides were immunogenic in rabbits. The peptides containing the sequence Gly74-Ser93 (peptide IMT-172) and the sequence Val154-Ala173 (peptide IMT-180) were responsible for the recognition of the Sm31 molecule by Western blot. This was confirmed by the specific inhibition of recognition of each molecule with the homologous peptide. Additionally, antibodies against these peptides strongly fixed to the adult worm gut. The present results, together with the strong immunogenicity shown for the adult worm 31 kDa antigen, establish the basis for the development of an immunodiagnostic method using synthetic peptides.

cGMP-mediated inhibition of cardiac L-type Ca(2+) current by a monoclonal antibody against the M(2) ACh receptor.

The effects of a monoclonal antibody (B8E5) directed against the second extracellular loop of the muscarinic M(2) receptor were studied on the L-type Ca(2+) currents (I(Ca,L)) of guinea pig ventricular myocytes using the whole cell patch-clamp technique. Similar to carbachol, B8E5 reduced the isoproterenol (ISO)-stimulated I(Ca,L) but did not significantly affect basal I(Ca,L). Atropine blocked the inhibitory effect of B8E5. The electrophysiological parameters of ISO-stimulated I(Ca,L) were not modified in presence of B8E5. Inhibition of I(Ca,L) by B8E5 was still observed when intracellular cAMP was either enhanced by forskolin or maintained constant by using a hydrolysis-resistant cAMP analog (8-bromoadenosine 3',5'-cyclic monophosphate) or by applying the phosphodiesterase inhibitor IBMX. The effect of B8E5 was mimicked by 8-bromoguanosine 3',5'-cyclic monophosphate, a potent stimulator of cGMP-dependent protein kinase, and prevented by a selective inhibitor of nitric oxide-sensitive guanylyl cyclase [1H-(1,2,4)oxadiazolo[4,3-a]quinoxaline-1-one]. These results indicate that the antibody B8E5 inhibits the beta-adrenergic-stimulated I(Ca,L) through activation of the M(2) muscarinic receptor and further suggest that the antibody acts not via the classical pathway of decreasing intracellular cAMP, but rather by increasing cGMP.

A monoclonal antibody against the immunodominant epitope of the ribosomal P2beta protein of Trypanosoma cruzi interacts with the human beta 1-adrenergic receptor.

Monoclonal antibodies were raised against a recombinant ribosomal P2beta protein of Trypanosoma cruzi. One of these reacted with the C terminus of this protein (peptide R13, EEEDDDMGFGLFD) and epitope mapping confirmed that this epitope was the same as the one defined by the serum of immunized mice, and similar to the previously described chronic Chagas' heart disease (cChHD) anti-P epitope. Western blotting showed that the monoclonal antibody recognized the parasite ribosomal P proteins, as well as the human ribosomal P proteins. Electron microscopy showed that it stained different structures in parasite and human cells. Interestingly, surface plasmon resonance measurements indicated that the affinity for the parasite ribosomal P protein epitope (R13) was five times higher than for its human counterpart (peptide H13, EESDDDMGFGLFD). Since the human epitope contained an acidic region (EESDD) similar to the AESDE peptide recognized by cChHD patients in the second extra-cellular loop of the human beta1-adrenergic receptor, the biological activity of the antibody was assessed on neonatal rat cardiomyocytes in culture. The monoclonal antibody had an agonist-like effect. These results, together with the fact that the monoclonal reacted in Western blots with the different isoforms of the heart beta1-adrenergic receptor, confirm the possible pathogenic role of antibodies against the parasite ribosomal P protein based on their cross-reaction with the human beta1-adrenergic receptor.

Differential recognition of epitopes present on monomeric and oligomeric forms of gp160 glycoprotein of human immunodeficiency virus type 1 by human monoclonal antibodies.

The mechanism of infectivity neutralization of human immunodeficiency virus type 1 (HIV-1) by Ig is poorly understood. Three human monoclonal antibodies (mAbs 1b12, 2G12 and 2F5) that are able to neutralize primary isolates of HIV-1 in vitro have been shown to act synergistically. In the present study this synergy was analyzed by measuring the epitope accessibility and binding kinetics for these three mAbs with respect to monomeric and oligomeric env protein gp160 IIIB using surface plasmon resonance. The results indicate that oligomerization of gp160 affects the accessibility of some of the epitopes recognized by the mAbs and provide some insight into the mechanism of synergy between different anti-(HIV-1) mAbs.

Heuristic statistical analysis of fluorescence fluctuation data with bright spikes: application to ligand binding to the human serotonin receptor expressed in Escherichia coli cells.

A statistical method for the analysis of fluorescence fluctuation amplitudes including bright spikes is presented. This situation arises e. g. when fluorescent ligands interact with receptors carrying multiple binding sites. The technique gives information on the amount of bound ligand in solution, making it a complementary technique to fluorescence correlation spectroscopy analysis, which cannot be applied in this situation. Two simple statistical tests are proposed that can discriminate between fluorescence intensities originating from free ligands or complexes. The performance of the two tests is evaluated and compared on mixtures of a fluorophore and fluorophore-coated beads that mimic the behaviour of multi-liganded complexes. An application to ligand binding to the serotonin receptor, expressed on Escherichia coli cells, is also provided. Specific binding of a fluorophore to this receptor, as well as competition with several ligands, is assessed.

Induction of neonatal lupus in pups of mice immunized with synthetic peptides derived from amino acid sequences of the serotoninergic 5-HT4 receptor.

We have previously suggested that the recognition of a cross-reactive epitope on the 5-HT4 receptor and the 52-kDa SSA/Ro protein by serotonin-antagonizing autoantibodies could explain the electrophysiological symptoms of congenital heart block in neonatal lupus. To confirm this hypothesis, we immunized female mice with four synthetic peptides corresponding to the recognized epitopes. All mice developed anti-peptide antibodies, which cross-reacted with the Ro52 and 5-HT4 receptor peptides and recognized both cognate proteins. Peptide-immune mice were mated. The pups from mice immunized with the Ro52 peptides had no symptoms of neonatal lupus apart from bradycardia. However, pups from mice immunized with the 5-HT4 receptor peptides and bradycardia, atrioventricular block of type I or II, longer QT intervals, skin rashes and neuromotor problems. The 5-HT4 receptor was detectable in the different fetal tissues affected (heart, skin and brain) by immunohistochemistry. Hearts from diseased pups were less developed and showed disorganized myocardial hyperplasia, compared to the normal littermates. These results demonstrate that the serotoninergic 5-HT4 receptor is the antigenic target of physiopathological autoantibodies in neonatal lupus.

Inhibitory activity of antibodies against the human atrial 5-HT(4)receptor.

Antibodies directed against the second extracellular loop of G protein-coupled receptors have been shown to exert "agonist-like" activities. In order to test the hypothesis that this is a general phenomenon, antibodies were raised in rabbits against a synthetic peptide corresponding to the second extracellular loop of the newly sequenced human cardiac 5-HT(4)receptor. The antibodies were affinity-purified and shown to recognize the 5-HT(4)receptor in immunoblots of Chinese hamster ovary (CHO) cells expressing the receptor. The antibodies had no intrinsic effect but could depress the activation of L -type calcium channel induced by serotonin in human atrial cells. This antagonist-like effect was exerted both by intact IgG and by Fab fragments. These results are physiologically important since it has been shown that the 5-HT(4)receptor could be a target for autoantibodies in mothers at risk of giving birth to children with neonatal atrio-ventricular block.

Control of mitochondrial membrane permeabilization by adenine nucleotide translocator interacting with HIV-1 viral protein rR and Bcl-2.

Viral protein R (Vpr), an apoptogenic accessory protein encoded by HIV-1, induces mitochondrial membrane permeabilization (MMP) via a specific interaction with the permeability transition pore complex, which comprises the voltage-dependent anion channel (VDAC) in the outer membrane (OM) and the adenine nucleotide translocator (ANT) in the inner membrane. Here, we demonstrate that a synthetic Vpr-derived peptide (Vpr52-96) specifically binds to the intermembrane face of the ANT with an affinity in the nanomolar range. Taking advantage of this specific interaction, we determined the role of ANT in the control of MMP. In planar lipid bilayers, Vpr52-96 and purified ANT cooperatively form large conductance channels. This cooperative channel formation relies on a direct protein-protein interaction since it is abolished by the addition of a peptide corresponding to the Vpr binding site of ANT. When added to isolated mitochondria, Vpr52-96 uncouples the respiratory chain and induces a rapid inner MMP to protons and NADH. This inner MMP precedes outer MMP to cytochrome c. Vpr52-96-induced matrix swelling and inner MMP both are prevented by preincubation of purified mitochondria with recombinant Bcl-2 protein. In contrast to König's polyanion (PA10), a specific inhibitor of the VDAC, Bcl-2 fails to prevent Vpr52-96 from crossing the mitochondrial OM. Rather, Bcl-2 reduces the ANT-Vpr interaction, as determined by affinity purification and plasmon resonance studies. Concomitantly, Bcl-2 suppresses channel formation by the ANT-Vpr complex in synthetic membranes. In conclusion, both Vpr and Bcl-2 modulate MMP through a direct interaction with ANT.