Biomedical subjects

P Moullier

Publications and source records attributed to P Moullier.

44 records · Page 3Linked to original sources

PubMed1988

Angiotensin II (AII)-related idiotypic network. I. Common occurrence of AII internal image on rabbit anti-idiotypic antibodies.

Internal images of foreign Ag have been demonstrated in a variety of systems as anticipated by the idiotypic network theory formulated by Jerne. However, they seem to be of rare occurrence. In order to estimate the actual frequency of antibodies bearing internal images (Ab2-beta) of angiotensin II (AII), a phylogenetically conserved peptide made up of eight amino acids, nine rabbits were immunized with affinity or protein A purified anti-AII antibodies (Ab1) from allotype-matched rabbits. Four of nine antiidiotypic antibodies (Ab2) exhibited internal image-like reactivity. They recognized all the polyclonal Ab1 tested, whatever the species (rabbit, mouse, guinea pig). In addition, they were strongly reactive with three mAb specific for a carboxy terminus epitope on AII (mAb 110, 199, and 211) and with a fourth monoclonal Ab1 (133) identifying a more central epitope. Advantage was taken of this reactivity with mAb1 to purify Ab2-beta by affinity chromatography of Ab2 on Sepharose 4B covalently linked to the three monoclonal Ab1 specific for the carboxy terminus epitope. The eluate displayed typical internal image properties: 1) it reacted with all the polyclonal Ab1 tested, 2) this reaction was completely abolished by AII, and 3) rabbits and mice immunized with the eluate all produced Ab1. The AII related idiotypic network is thus characterized by high frequency and immunogenicity of AII internal images. In addition, reactivity of the latter with monoclonal Ab1 indicates variable expression on Ab2-beta of the epitopes defined by the mAb on the nominal Ag.

Angiotensin II↗

PubMed1987

Lymphocyte expression of a 90 kD brush border antigen.

This study analyses the expression by rat lymphocytes of six renal brush border (BB) antigens defined by a set of monoclonal antibodies and demonstrates that a 90 kD protein (gp 90), synthesized by BB and glomeruli is found on the surface of T and B cells. The other antigens, including the 330 kD protein involved in Heymann's nephritis--a model of epimembranous glomerulonephritis--are not detectable on the surface of lymphocytes. Immunochemical studies indicate that gp 90 and the related protein immunoprecipitated from thymocyte membranes co-migrate in SDS-PAGE. Quantitative binding analysis shows that the number of antigenic sites is in the same order of magnitude on thymocytes, spleen and bone-marrow lymphocytes as well as on two lines of pre-B and pre-T cells, but considerably lower on a highly differentiated helper T cell line.

Animals↗

PubMed1986

Comparative binding of wheat germ agglutinin and its succinylated form on lymphocytes.

We have compared the binding parameters of native wheat germ agglutinin (WGA) and its succinylated form (SWGA) to rat lymphocytes. Scatchard plots were obtained with the fluoresceinated lectins in a concentration range of 10 nM to 0.1 mM. Association and dissociation rate parameters were also measured. The following differences were observed: at low concentration of WGA, binding is positively cooperative with a Hill coefficient of 1.75, whereas binding of SWGA is not. The numbers of high-affinity sites are respectively (2.5 +/- 0.8) X 10(6) and (6.4 +/- 1.3) X 10(5) for WGA and SWGA. Association constants were found to be (4.7 +/- 1.7) X 10(6) l mol-1 for WGA and (1.42 +/- 0.36) X 10(7) l mol-1 for SWGA, which is 35 times higher than for native WGA. Neuraminidase treatment decreases the Hill coefficient as well as the number of sites involved in the cooperative binding of native WGA. Equilibrium data were obtained at three temperatures to determine the thermodynamic parameters (delta H degree and delta S degree). These results are indicative of an oligomerization process dynamically formed at the membrane level before tight binding of the lectin to its receptors could occur.

Animals↗

PubMed1986

Effect of in vivo gamma-irradiation on the binding of wheat germ agglutinin on lymphocyte plasma membranes.

Using quantitative fluorimetry with fluoresceinated wheat germ agglutinin, we have been able to investigate in vivo gamma radiation-induced damage at the outer membrane level of rat splenic lymphocytes, namely damage to the glucosidic moieties of membrane glycoproteins and glycolipids. This paper demonstrates that below an irradiation level of 1 gray (Gy), removal of sialic acid is the major feature leading to new exposed specific binding sites for wheat germ agglutinin, since this lectin is specific for sialic acid and N-acetyl-D-glucosamine. Our studies also suggest that above 1 Gy of irradiation more internal damage occurs, since we observed a striking decrease in wheat germ agglutinin binding sites.

Animals↗

PubMed1986

Characterization of monoclonal antibodies specific for human Tamm-Horsfall protein.

Fifteen monoclonal antibodies have been produced to human Tamm-Horsfall protein (THP), identifying at least seven distinct epitopes. The antibodies have been used to isolate from serum an immunoreactive protein which comigrates with urinary THP. In addition, the antibodies may prove useful to set up an immunoenzymoassay for urinary THP as well as for immunoaffinity purification.

Antibodies, Monoclonal↗

PubMed1986

Epitope diversity of angiotensin II analysed with monoclonal antibodies.

The antigenic heterogeneity of angiotensin II (AII) was studied with monoclonal antibodies. Twelve antibodies were produced and characterized. Association constants for AII varied from 1.2 X 10(8) to 1.1 X 10(10) M-1. The fine specificity of the Mab was studied by immunoenzymoassay using solid-phase AII. Using AII analogues in binding inhibition experiments, three groups of specificity could be characterized: (1) five antibodies reacted only with peptides in which phenylalanine is the carboxy terminal aminoacid; for two of these antibodies, tyrosine4 is closely associated with the binding site, since iodine labelling suppresses reactivity; (2) two antibodies also required phenylalanine in position 8, but, in addition, reacted with AI, a decapeptide in which phenylalanine is not terminal; (3) five antibodies reacted with analogues in which phenylalanine had been substituted for another amino acid. In addition, studies in which binding of a biotinylated Mab to solid-phase AII was analysed in the presence of various unlabelled Mab suggest further antigenic heterogeneity of AII.

Angiotensin I↗

PubMedDate not supplied

Baculovirus vector requires electrostatic interactions including heparan sulfate for efficient gene transfer in mammalian cells.

BACKGROUND: Recently, several reports have described the ability of recombinant baculoviruses to transduce a variety of mammalian cells. Yet, mechanisms involved in baculovirus entry in those cells remain largely unexplored, particularly at the primary binding step of the virions to the cell membrane. METHODS: This report focused on the primary virus-cell interactions that lead to in vitro transduction of human 293 cells using a polyhedrin-deleted baculovirus harboring a CMV-driven beta-galactosidase gene (BacLacZ). RESULTS: Infection rate monitored for 8 h and transduction rate with a multiplicity of infection of up to 800 were, both, non-saturable. Temperatures from 37 degrees C to 4 degrees C dramatically impaired BacLacZ but not adenovirus cell attachment. Competitive infections performed with an excess of a non LacZ-expressing baculovirus hardly competed at a 1/1 ratio. Consistent with an adsorptive binding process onto the cell surface, interactions through electrostatic charges between both viral and cell membranes appeared to be critical for BacLacZ transduction. The addition of polybrene to the cells prior to or during the infection prevented both virus binding and LacZ gene transfer, suggesting the involvement of negatively charged epitopes exposed at the cell surface. The simultaneous presence of the highly charged heparin abrogated BacLacZ binding to the cell surface and subsequent gene transfer. Lastly, direct in vitro binding of BacLacZ to heparin but not BSA columns could be demonstrated after elution of infectious BacLacZ virus in high salt molarity. CONCLUSION: Electrostatic charges play a critical role during the first step in mammalian cell transduction mediated by a recombinant baculovirus.

Animals↗

PubMedDate not supplied

Efficient recombinant adeno-associated virus production by a stable rep-cap HeLa cell line correlates with adenovirus-induced amplification of the integrated rep-cap genome.

BACKGROUND: A possible procedure for the production of clinical grade recombinant adeno-associated virus type 2 (rAAV) would include the use of packaging cell lines, harboring the rep-cap genes and the vector, combined with a replication defective adenoviral plasmid to provide the helper activities. Several studies have already shown that rAAV can be efficiently assembled by infecting the stable packaging cell line with adenovirus. However, the direct comparison with an adenoviral plasmid has never been reported. METHODS: To investigate this point, a clone of HeLa and 293 cells harboring one to two rep-cap copies per cell genome (HeRC32 and 293RC21, respectively) were generated. Recombinant AAV was produced by transiently transfecting the AAVCMVLacZ vector and supplying the adenoviral helper activities by either wild-type adenovirus or an adenoviral plasmid (pAdc). As a control, rAAV was similarly produced from naive Hela and 293 cells additionally transfected with a rep-cap plasmid. RESULTS: Despite satisfactory rAAV yields from Hela and 293 cells, we show that those from HeRC32 and 293RC21 cells dramatically decrease when adenovirus is replaced by the adenoviral plasmid (pAdc). The analysis performed to identify the factors hampering efficient rAAV assembly by HeRC32 cells in the presence of pAdc shows that: (1) while upon adenovirus infection the integrated rep-cap genome undergoes a dramatic amplification leading to a 100-fold increase in the rep-cap copy number, no amplification is detected upon transfection of pAdc; (2) in pAdc-transfected HeRC32 cells, the intracellular localization of the adenovirus E4orf6 and E1B-55kDa proteins is abnormal as compared to adenovirus-infected cells. CONCLUSIONS: This study documents that stable rep-cap cells lines are severely hampered for rAAV assembly when a replicative adenovirus is substituted with an adenoviral plasmid. Furthermore, our results also suggest that the lack of amplification of the rep-cap genes, eventually combined with the altered distribution of the adenoviral proteins, E4orf6 and E1B-55kDa, is related to the low rAAV yields observed under these conditions.

Adenovirus E1B Proteins↗