Biomedical subjects

J Grassi

Publications and source records attributed to J Grassi.

At least 37 records · Page 2Linked to original sources

PubMed2000

Synaptic prion protein immuno-reactivity in the rodent cerebellum.

The cellular prion protein PrP(c) is a neurolemmal glycoprotein essential for the development of the transmissible spongiform encephalopathies. In these neurodegenerative diseases, host PrP(c) is converted to infectious protease-resistant isoforms PrP(res) or prions. Prions provoque predictable and distinctive patterns of PrP(res) accumulation and neurodegeneration depending on the prion strain and on regional cell-specific properties modulating PrP(c) affinity for infectious PrP(res) in the host brain. Synaptolysis and synaptic accumulation of PrP(res) during PrP-related diseases suggests that the synapses could be primary sites able to propagate PrP(res) and neurodegeneration in the central nervous system. In the rodent cerebellum, the present light and electron microscopic immuno-cytochemical analysis shows that distinct types of synapses display differential expression of PrP(c), suggesting that synapse-specific parameters could influence neuroinvasion and neurodegeneration following cerebral infection by prions. Although the physiological functions of PrP(c) remain unknown, the concentration of PrP(c) almost exclusively at the Purkinje cell synapses in the cerebellum suggests its critical involvement in the synaptic relationships between cerebellar neurons in agreement with their known vulnerability to PrP deficiencies.

Animals↗

PubMed2000

Toward antibody-catalyzed hydrolysis of organophosphorus poisons.

We report here our preliminary results on the use of catalytic antibodies as an approach to neutralizing organophosphorus chemical weapons. A first-generation hapten, methyl-alpha-hydroxyphosphinate Ha, was designed to mimic the approach of an incoming water molecule for the hydrolysis of exceedingly toxic methylphosphonothioate VX (1a). A moderate protective activity was first observed on polyclonal antibodies raised against Ha. The results were further confirmed by using a mAb PAR 15 raised against phenyl-alpha-hydroxyphosphinate Hb, which catalyzes the hydrolysis of PhX (1b), a less toxic phenylphosphonothioate analog of VX with a rate constant of 0.36 M(-1) x min(-1) at pH 7.4 and 25 degrees C, which corresponds to a catalytic proficiency of 14,400 M(-1) toward the rate constant for the uncatalyzed hydrolysis of 1b. This is a demonstration on the organophosphorus poisons themselves that mAbs can catalytically hydrolyze nerve agents, and a significant step toward the production of therapeutically active abzymes to treat poisoning by warfare agents.

Animals↗

PubMed2000

Evaluation of a high IgE-responder mouse model of allergy to bovine beta-lactoglobulin (BLG): development of sandwich immunoassays for total and allergen-specific IgE, IgG1 and IgG2a in BLG-sensitized mice.

An animal model of food allergy represents an important tool for studying the mechanisms of induction and repression of an allergic reaction, as well as for the development of an immunotherapy to prevent or minimize such an adverse reaction. IgE and IgG1 (Th2 response) vs. IgG2a (Th1 response) are good markers for the induction of an allergic response in mice. Nevertheless, while the total serum concentrations of these isotypes are easy to measure using classical sandwich immunoassays, this is not the case for allergen-specific isotypes. To develop an animal model of allergy to bovine beta-lactoglobulin (BLG), we set up quantitative assays for total and for allergen-specific IgE, IgG1 and IgG2a. Microtiter plates coated either with anti-isotype antibodies (Abs) or with allergen were used for Ab capture, while anti-isotype Fab' fragments coupled to acetylcholinesterase were used for visualization. These assays of anti-BLG specific Abs are original in two ways. First, assay calibration is performed using anti-BLG specific mAbs, thus allowing good quantification of the different isotypes and subclasses of serum antibodies. Second, the detection of all anti-BLG specific Abs, i.e., those recognizing both the native and denatured forms of the protein, is achieved through indirect coating of BLG using biotin-streptavidin binding. The present assays are quantitative, specific to the isotype (cross-reactivity <0.5%), very sensitive (detection limit in the 10 pg/ml range), and reproducible (coefficient of variation less than 10%). Applied to the humoral response in mice sensitized with BLG adsorbed on alum, these assays proved to be a very useful tool for monitoring high IgE-responder mice following BLG immunization, and for an immunotherapy directed at polarizing the immune response.

Adsorption↗

PubMed2000

Real-time monitoring of the hybridization reaction: application to the quantification of oligonucleotides in biological samples.

We describe here a competitive hybridization assay using TRACE technology which can be used for real-time monitoring of oligonucleotide hybridization. This assay quantifies all kinds of oligonucleotides in biological fluids without extraction. The assay makes use of two different probes and involves a fluorescent transfer process. As fluorescence measurements are not destructive, they can be sequentially repeated, thereby allowing comparison of the hybridization kinetics and binding strength of chemically modified backbone oligonucleotides (>0.5 nM) in biological media. The assay was validated for pharmacokinetic analysis of phosphodiester and phosphorothioate oligonucleotides in plasma and in different organs (liver, kidneys, lungs, spleen) at low concentrations (0.4 mg/kg, corresponding to clinical doses). Respective sensitivities for phosphodiester and phosphorothioate were 0.2 and 0.8 pmol/ml in plasma and 2 and 8 pmol/g in tissues, which allow to recover intact phosphorothioate sequences in some organs even after 24 h.

Animals↗

PubMed2000

Specific determination of the proteinase K-resistant form of the prion protein using two-site immunometric assays. Application to the post-mortem diagnosis of BSE.

The aim of this work was to establish an immunological test suitable for specifically detecting PrPres in tissues from animals or humans developing TSEs. We chose to use as detection method a conventional two-site immunometric assay (sandwich immunoassay) because over the last 20 years this technique has clearly been shown to be more sensitive and specific than other tests. We have established numerous two-site immunometric assays based on the use of monoclonal antibodies and suitable for measurement of PrPsen in various mammalian species (human, bovine, ovine, mouse and hamster). A detection limit below 100 pg/ml was estimated from standard curves established using ovine recombinant PrP. PrPres was selectively detected by processing samples (currently brain homogenates) to enable specific purification and concentration of PrPres, which was finally solubilized by a strong denaturing treatment. This sample-processing procedure can be achieved within 30 minutes. The capacity of this test to detect bovine PrPres was estimated in the framework of an evaluation study organized by the Directorate-General XXIV of the European Commission during May 1999. On this occasion, a blind test on 1400 brain stem samples taken from either healthy (1000) or BSE-infected (300) cows demonstrated 100% sensitivity and specificity. In addition, dilution experiments showed that the test can significantly detect PrPres in homogenates diluted 1/300 and was at least as sensitive as a conventional bioassay performed on mice.

Animals↗

PubMed2000

A monoclonal antibody directed against the neurokinin-1 receptor contains a peptide sequence with similar hydropathy and functional properties to substance P, the natural ligand for the receptor.

Monoclonal antibody (mAb) PS12, obtained using the complementary peptide methodology, mimics the neuropeptide substance P (SP) in recognizing the SP-binding domain of the neurokinin-1 receptor (NK1R) and eliciting production of polyclonal antibodies cross-reacting with SP with a high affinity (Déry et al., 1997. J. Neuroimmunol. 76, 1-9). The aim of the present study was to investigate which structural features of mAb PS12 might account for this molecular mimicry. Cloning and sequencing of variable regions of both light (VL) and heavy (VH) chains of this 'SP-like' antibody did not indicate any primary sequence homology between SP and any antibody region. Instead, they revealed a striking similarity between the hydropathic profile of SP and that of an 11-amino-acid region in the light chain encompassing the second complementarity determining region (CDR2). When applied to CHO cells expressing the human NK1R, a synthetic extended 17-amino-acid peptide (denoted CDR2L) corresponding to this VL region inhibited the high-affinity binding of radiolabeled SP and antagonized the SP-induced inositol phosphate production. Moreover, a re-examination of the sequences of several antibodies that previously served in the design of CDR-derived bioactive peptides indicated that these antibodies also carried the hydropathic image of the respective ligands that they mimic. In agreement with previous observations on artificial synthetic peptides, our data thus suggest that the molecular mimicry between natural proteins (i.e. antibody and hormone, for example) could be understood on a structural level directly related, at least in part, to hydropathic homology. These results could then guide the search for bioactive paratope-derived peptides of potential pharmacological interest. We also observed inverse hydropathy between multiple CDRs of mAb PS12 (including CDR3H and CDR3L) and the peptide epitope, confirming the importance of hydropathic complementarity in antigen-antibody interactions.

Amino Acid Sequence↗

PubMed2000

Successful transmission of three mouse-adapted scrapie strains to murine neuroblastoma cell lines overexpressing wild-type mouse prion protein.

Propagation of the agents responsible for transmissible spongiform encephalopathies (TSEs) in cultured cells has been achieved for only a few cell lines. To establish efficient and versatile models for transmission, we developed neuroblastoma cell lines overexpressing type A mouse prion protein, MoPrP(C)-A, and then tested the susceptibility of the cells to several different mouse-adapted scrapie strains. The transfected cell clones expressed up to sixfold-higher levels of PrP(C) than the untransfected cells. Even after 30 passages, we were able to detect an abnormal proteinase K-resistant form of prion protein, PrP(Sc), in the agent-inoculated PrP-overexpressing cells, while no PrP(Sc) was detectable in the untransfected cells after 3 passages. Production of PrP(Sc) in these cells was also higher and more stable than that seen in scrapie-infected neuroblastoma cells (ScN2a). The transfected cells were susceptible to PrP(Sc)-A strains Chandler, 139A, and 22L but not to PrP(Sc)-B strains 87V and 22A. We further demonstrate the successful transmission of PrP(Sc) from infected cells to other uninfected cells. Our results corroborate the hypothesis that the successful transmission of agents ex vivo depends on both expression levels of host PrP(C) and the sequence of PrP(Sc). This new ex vivo transmission model will facilitate research into the mechanism of host-agent interactions, such as the species barrier and strain diversity, and provides a basis for the development of highly susceptible cell lines that could be used in diagnostic and therapeutic approaches to the TSEs.

Animals↗

PubMed1999

New insight into abnormal prion protein using monoclonal antibodies.

Studies of abnormal prion protein (PrPres) are hindered by the lack of specific monoclonal antibodies (mAbs), and the relationships between PrPres, infectivity, and strain specificity in prion diseases are still subject to debate. We have studied PrPres with new mAbs produced against PrP in mice using various immunization strategies. PrPres was analyzed by Western blot with different prion strains in various hosts. Differences in the electrophoretic pattern of human PrPres revealed by these antibodies provide new insight into PrPres cleavage by proteases and interpretation of strain typing. This study confirms that the N-terminal extremity of PrPres is differentially sensitive to proteases. Conversely, the C-terminal extremity, which resists proteolysis, seems to be abnormally detectable by antibodies in ultrastructural studies. This work confirms the highly complex role of PrPres in prion diseases and provides new tools which will be made available to facilitate progress in qualitative and quantitative studies of PrP.

Amino Acid Sequence↗

PubMed1999

A novel cellular prion protein isoform present in rapid anterograde axonal transport.

We studied the axonal transport of PrP(C) in hamster retinal and sciatic nerve axons. Our results show that a novel 38kDa form is the predominant form in rapid anterograde axonal transport while the 36kDa and 33kDa PrP(C) forms, abundant in nerve and brain, appear to be either stationary or slowly transported. We did not detect any significant retrograde transport of PrP(C). These results show that 38kDa PrP(C) is the form exported from the cell body to the axonal compartment where it may represent the precursor to the more abundant PrP(C) forms after its modification in nerve fibres or terminals.

Animals↗

PubMed1999

Conformational flexibility of the acetylcholinesterase tetramer suggested by x-ray crystallography.

Acetylcholinesterase, a polymorphic enzyme, appears to form amphiphilic and nonamphiphilic tetramers from a single splice variant; this suggests discrete tetrameric arrangements where the amphipathic carboxyl-terminal sequences can be either buried or exposed. Two distinct, but related crystal structures of the soluble, trypsin-released tetramer of acetylcholinesterase from Electrophorus electricus were solved at 4.5 and 4.2 A resolution by molecular replacement. Resolution at these levels is sufficient to provide substantial information on the relative orientations of the subunits within the tetramer. The two structures, which show canonical homodimers of subunits assembled through four-helix bundles, reveal discrete geometries in the assembly of the dimers to form: (a) a loose, pseudo-square planar tetramer with antiparallel alignment of the two four-helix bundles and a large space in the center where the carboxyl-terminal sequences may be buried or (b) a compact, square nonplanar tetramer that may expose all four sequences on a single side. Comparison of these two structures points to significant conformational flexibility of the tetramer about the four-helix bundle axis and along the dimer-dimer interface. Hence, in solution, several conformational states of a flexible tetrameric arrangement of acetylcholinesterase catalytic subunits may exist to accommodate discrete carboxyl-terminal sequences of variable dimensions and amphipathicity.

Acetylcholinesterase↗

PubMed1999

The binding sites of inhibitory monoclonal antibodies on acetylcholinesterase. Identification of a novel regulatory site at the putative "back door".

We investigated the target sites of three inhibitory monoclonal antibodies on Electrophorus acetylcholinesterase (AChE). Previous studies showed that Elec-403 and Elec-410 are directed to overlapping but distinct epitopes in the peripheral site, at the entrance of the catalytic gorge, whereas Elec-408 binds to a different region. Using Electrophorus/rat AChE chimeras, we identified surface residues that differed between sensitive and insensitive AChEs: the replacement of a single Electrophorus residue by its rat homolog was able to abolish binding and inhibition, for each antibody. Reciprocally, binding and inhibition by Elec-403 and by Elec-410 could be conferred to rat AChE by the reverse mutation. Elec-410 appears to bind to one side of the active gorge, whereas Elec-403 covers its opening, explaining why the AChE-Elec-410 complex reacts faster than the AChE-Elec-403 or AChE-fasciculin complexes with two active site inhibitors, m-(N,N, N-trimethyltammonio)trifluoro-acetophenone and echothiophate. Elec-408 binds to the region of the putative "back door," distant from the peripheral site, and does not interfere with the access of inhibitors to the active site. The binding of an antibody to this novel regulatory site may inhibit the enzyme by blocking the back door or by inducing a conformational distortion within the active site.

Acetylcholinesterase↗

PubMed1999

A solid-phase immobilized epitope immunoassay (SPIE-IA) permitting very sensitive and specific measurement of angiotensin II in plasma.

We have developed a new enzyme immunometric assay for angiotensin II (AII) based on SPIE-IA technology (solid-phase immobilized epitope-immunoassay). A monoclonal antibody with optimal properties (mAb3 131) was selected from a series of 19 anti-AII mAbs. The mAb had to be purified from ascitic fluid in a specific manner in order to remove endogenous AII from the antibody-binding sites. We established a sensitive (minimum detectable concentration 0.5 pg/ml) and precise (CV below 15% in the 2-100 pg/ml range) SPIE-IA. Using different AII-related peptides, we observed that this new assay has a specificity profile that compares favourably with the corresponding competitive immunoassay. We have used the assay to measure AII in 42 plasma samples, and demonstrated a good correlation with values obtained using a commercial radioimmunoassay. Assay specificity was supported by HPLC fractionation experiments, confirming the absence of interference induced by endogenous AII-related products.

Acetylcholinesterase↗

PubMed1999

Identification in the NK1 tachykinin receptor of a domain involved in recognition of neurokinin A and septide but not of substance P.

The three mammalian tachykinins, substance P (SP), neurokinin A (NKA) and neurokinin B (NKB), exert their physiological effects through specific receptors, NK1, NK2 and NK3, respectively. However, homologous binding studies have recently demonstrated that, contrary to the generally accepted belief, NKA could bind NK1 receptor with high affinity (Hastrup and Schwartz, 1996). Using COS-7 cells expressing the human NK1 receptor, we show that two simultaneous point mutations (E193L and V195R) in a restricted five amino acid sequence (the (193-197) region), selected because of its hydropathic complementarity with the common C-terminal extremity of tachykinins, abolish both the high-affinity binding and highly potent biological activity of NKA, without affecting those of SP. In addition, the same mutations also suppressed the high functional activity of septide, a synthetic SP atypical agonist ([pGlu6-Pro9] SP 6-11). These results suggest that the (193-197) region, located at the end of the second extracellular loop of the receptor, could be part of a common high-affinity binding domain for both NKA and septide, distinct from the SP binding site.

Amino Acid Sequence↗

PubMed1999

Cross-linking of 17 beta-estradiol to monoclonal antibodies by direct UV irradiation: application to an enzyme immunometric assay.

Ultraviolet irradiation was used to cross-link 17 beta-estradiol directly to monoclonal anti-17 beta-estradiol antibodies coated on 96-well microtiter plates. Cross-linking efficiency was directly correlated with both irradiation energy and wave-length. The best results were obtained at 254 (10 J/cm2, 45-min irradiation) and 312 nm (40 J/cm2, 160-min irradiation). The irradiation fully denatured both individual molecules (i.e., 17 beta-estradiol and monoclonal anti-17 beta-estradiol antibody), but 17 beta-estradiol was at least partly protected when immunologically bound to the paratope of the antibody. Four different monoclonal anti-17 beta-estradiol antibodies yielded positive results, demonstrating that this photo-cross-linking has considerable potential. We used this original approach to develop a new enzyme immunometric assay of 17 beta-estradiol based on our previously described immunometric procedure, solid-phase immobilized epitope immunoassay, which uses chemical agents to cross-link haptens via amino groups to specific antibodies. The assay was specific (no cross-reactivity with other natural steroids), precise, and sensitive (detection limit of 38 pg/mL in human serum). It correlated well with two competitive commercial immunoassays when tested on 40 human sera.

Antibodies, Monoclonal↗

PubMed1999

A sensitive sandwich enzyme immunoassay for calcitonin gene-related peptide (CGRP): characterization and application.

Thirty mouse monoclonal antibodies (mAbs) directed against rat calcitonin gene-related peptide-alpha (CGRP-alpha) have been obtained. These mAbs are classified in 2 groups, one recognizing the peptide N-terminus and the other binding the C-terminus. A two-site immunometric assay was developed using mAb CGRP-83 as capture antibody, whereas mAb CGRP-72 acts as tracer, covalently labeled with enzyme acetylcholinesterase. This assay appeared sensitive (limit of detection: 2 pg/ml) and precise, allowing quantitative measurement of all human and murine CGRP isoforms. The assay was used to determine specific concentrations of CGRP in different rat, mice and guinea pig samples. The validity of the test was demonstrated by HPLC fractionation experiments.

Amino Acid Sequence↗

PubMed1999

Pharmacokinetic analysis of 6-monoamino-beta-cyclodextrin after intravenous or oral administration to rats using a specific enzyme immunoassay.

We have developed a highly sensitive enzyme immunoassay for 6-monoamino-beta-CD (mono(6-amino-6-deoxy)cyclomaltoheptaose) and its parent compound (beta-CD) with a detection limit in the 100 pg/mL range. The polyclonal antibodies obtained are highly specific for the beta-cyclodextrin core and do not recognize other cyclic cyclodextrins (i.e., alpha- and gamma-CD) or linear analogues. This enzyme immunoassay can be used to quantify 6-monoamino-beta-CD in rat urine and plasma. Using this immunoassay, we have evaluated the main pharmacokinetic parameters of 6-monoamino-beta-CD after iv administration to the rat of a 25 mg/kg dose. Since this method is strictly specific to the native beta-CD form, we have demonstrated that the molecule rapidly disappeared from plasma but is probably distributed in the tissues. The urinary route appears as the predominant way of elimination since almost all the administered drug is recovered in urine. Finally, analysis of the same molecule after oral administration to the rat (25 mg/kg) demonstrates low plasma levels and that about 1% of the administered dose is excreted in urine. These experiments demonstrate the high stability of the beta-CD core irrespective of the method of administration. This immunological method could provide relevant information on the fate of beta-CD and some derivatives for drug delivery using different modes of administration (oral, parenteral, transmucosal, or dermal).

Administration, Oral↗

PubMed1999

A new specific enzyme immunoassay allowing an efficient pharmacokinetic evaluation of gamma-cyclodextrin after intravenous administration to rats.

PURPOSE: Because of its ability to form complexes with drugs, gamma-cyclodextrin is of great potential value in pharmaceutical formulations. The biological fate of y-cyclodextrin must therefore be considered in safety evaluation, using sensitive and specific methods applicable to biological fluids. METHODS: Antibodies were raised against gamma-cyclodextrin, allowing the development of a new enzyme immunoassay. The analytical characteristics of this assay were evaluated. Rats were given a single intravenous 25 mg/kg dose of gamma-cyclodextrin. Plasma and urine samples were collected and assayed. RESULTS: This new enzyme immunoassay was sensitive (limit of detection close to 94 pg/mL) and suitable for quantification of gamma-cyclodextrin in urine and plasma after methanol extraction. The use of different linear and cyclic compounds demonstrated the high specificity of the assay. After i.v. administration, the concentration of gamma-cyclodextrin rapidly decreased in the plasma while the molecule was probably distributed into the tissues. Although urinary elimination predominates, only 50% of the injected gamma-cyclodextrin was recovered in urine, suggesting enzymatic degradation and/or tissular storage. CONCLUSIONS: This assay may provide important information on the fate of gamma-cyclodextrin inclusion complexes dedicated to drug-delivery using various modes of administration (oral, parenteral, transmucosal or dermal).

Acetylcholinesterase↗

PubMed1999

Ultrastructural evidence for dendritic release of acetylcholinesterase in the rat substantia nigra.

Morphological evidence for dendritic secretion of acetylcholinesterase (AChE) in rat substantia nigra--a physiologically known phenomenon--was searched by means of a modified cytochemical method devised for fine localization of AChE activity at the electron microscopic level. DAB precipitate was observed in cluster of small vesicles in contact with the plasma membrane and in the extracellular space in the vicinity of the vesicles. Single coated or uncoated large vesicles filled with stained material were found in the cytoplasm of the dendrites at distance from or in contact with the plasma membrane. Immunoperoxidase staining with specific anti-serum against rat AChE gave similar localization of AChE. These results suggest that AChE is released from the dendrites of the nigral neurons by a process of vesicular exocytosis and captured by endocytosis. The relation of this process to a putative release from the smooth endoplasmic reticulum remains to be elucidated.

Acetylcholinesterase↗