Léna Edelman

Secretory component: a new role in secretory IgA-mediated immune exclusion in vivo.

Secretory immunoglobulin (Ig) A (SIgA) is essential in protecting mucosal surfaces. It is composed of at least two monomeric IgA molecules, covalently linked through the J chain, and secretory component (SC). We show here that a dimeric/polymeric IgA (IgA(d/p)) is more efficient when bound to SC in protecting mice against bacterial infection of the respiratory tract. We demonstrate that SC ensures, through its carbohydrate residues, the appropriate tissue localization of SIgA by anchoring the antibody to mucus lining the epithelial surface. This in turn impacts the localization and the subsequent clearance of bacteria. Thus, SC is directly involved in the SIgA function in vivo. Therefore, binding of IgA(d/p) to SC during the course of SIgA-mediated mucosal response constitutes a crucial step in achieving efficient protection of the epithelial barrier by immune exclusion.

A novel patatin-like gene stimulated by drought stress encodes a galactolipid acyl hydrolase.

A cDNA (Vupat1) encoding a predicted 43 kDa protein was isolated from drought‐stressed cowpea (Vigna unguiculata) leaves. It has homology with patatin, a potato tuber storage protein with lipolytic acyl hydrolase activity. The recombinant protein VUPAT1 expressed in the baculovirus system displays preferentially galactolipid acyl hydrolase activity. Phospholipids are very slowly hydrolyzed and apparently triacylglycerols are not deacylated. Vupat1 promoter contains putative drought‐inducible sequences. Northern blots showed that gene expression is stimulated by drought stress and is more pronounced in a drought‐sensitive cultivar than in a drought‐tolerant one. An involvement in drought‐induced galactolipid degradation is proposed for VUPAT1.

PATTERNS OF EPSTEIN-BARR VIRUS LATENT AND REPLICATIVE GENE EXPRESSION IN EPSTEIN-BARR VIRUS B CELL LYMPHOPROLIFERATIVE DISORDERS AFTER ORGAN TRANSPLANTATION

B cell lymphoproliferative disorders arising in organ transplant recipients (B cell posttransplant lymphoproliferative disorders [PTLD]) are generally associated with EBV. In previous reports, B cell PTLD were shown to express the full pattern of EBV latent genes, as in vitro-established lymphoblastoid cell lines. Although viral linear DNA was detected in 40% of lymphoproliferative disorders from immunocompromised hosts, immunophenotypic studies failed to detect late EBV replicative antigens. The aim of this study was to investigate the relationship of EBV latent gene expression in B cell PTLD to morphology, clonality, and immunophenotype, and to examine the replicative state of EBV in malignant cells. For this purpose, 9 cases of EBV-related B cell PTLD were analyzed. Immunoglobulin gene rearrangements were detected by Southern blot analysis. The presence of EBV was assessed by Southern blot and by in situ hybridization. B cell differentiation antigens, adhesion and activation molecules, and EBV latent and replicative gene expression were studied using immunohistochemistry techniques. We demonstrated that EBV-related B cell PTLD exhibited varying patterns of latent viral gene expression. Higher levels of adhesion molecules were detected in latent membrane protein 1 (LMP1) or LMP1 plus EBV nuclear antigen 2 (EBNA2)-positive tumors than in LMP1 and EBNA2-negative tumors. In contrast, there was no relationship between CD21 and CD23 expression and latent EBV phenotype. Activation of the EBV replicative cycle was highlighted by BamHI Z left frame 1 expression in 5 of 9 cases. Less frequent expression of late viral proteins suggested that the initiation of the EBV lytic cycle might not always lead to virions production.

Obtaining a functional recombinant anti-rhesus (D) antibody using the baculovirus-insect cell expression system.

The cloning and production of a human anti‐rhesus (Rh) D monoclonal antibody (mAb) using the baculovirus–insect cell expression system is described. This monoclonal recombinant antibody R.D7C2 derived from a human parental IgMλ immunoglobulin was obtained after immortalization of lymphocytes by Epstein–Barr virus (EBV). The human heavy (VH) and light (VL) variable regions were cloned from the parental cell line and genetically fused to the human constant IgG1 heavy (H) and light (L) chain genes (γl and λ, respectively). A recombinant baculovirus was constructed that directs the co‐expression of genes encoding both genetically fused heavy and light chains under the control of two late and strong baculovirus promoters. After infecting the Spodoptera frugiperda (Sf9) insect cell line with this baculovector, a complete IgGl mAb was secreted in the culture medium indicating that each immunoglobulin chain was correctly processed and assembled with a functional glycosylation into a tetrameric form. In vitro analysis showed that the functional properties of R.D7C2 using agglutination tests were efficient for the specific recognition of Rh‐D‐positive red blood cells (RBC). In addition, R.D7C2 showed effector functions of the γl heavy chain resulting in the lysis of Rh+ papain RBC by an antibody‐directed cellular cytotoxicity mechanism. These results demonstrate that R.D7C2 can be produced in the baculovirus–insect cell expression system as a source for potential therapeutic application in the treatment of the haemolytic disease of the newborn.

Human monoclonal antibodies specific for the rabies virus glycoprotein and N protein

Human monoclonal antibodies to rabies virus were established by Epstein-Barr virus infection of peripheral blood lymphocytes collected from a rabies-vaccinated donor, and fusion with a heteromyeloma line. Two human monoclonal antibodies, HUM1 and HUM2, both IgG2, reacted with the envelope glycoprotein of the rabies virus. The antibody HUM1 neutralized rabies virus (lyssavirus serotype 1) and Mokola virus (lyssavirus serotype 3), but did not neutralize European bat lyssavirus, suggesting that some common antigenicity exists between the glycoproteins of serotypes 1 and 3. In addition, this antibody neutralized a series of viruses resistant to neutralization by antibodies recognizing, in a murine system, antigenic sites I, II and III; however, it failed to neutralize viruses altered at site VI, indicating that human monoclonal antibody HUM1 is directed against antigenic site VI. The other human anti-glycoprotein antibody, HUM2, neutralized the European bat lyssavirus in addition to serotypes 1 and 3, but none of the resistant variant viruses altered at the sites mentioned above. A third human monoclonal antibody, HUM3 (IgM), was reactive with the internal nucleoprotein of the rabies virus. This antibody contained a murine light chain corresponding to the cytoplasmic murine chain not secreted in the heteromyeloma line. The potential use of monoclonal antibodies in post-exposure treatment of rabies is discussed.

High level of Bcl-2 counteracts apoptosis mediated by a live rabies virus vaccine strain and induces long-term infection

We report here that rabies virus strains, currently used to immunize wildlife against rabies, induce not only caspase-dependent apoptosis in the human lymphoblastoid Jurkat T cell line (Jurkat-vect), but also a caspase-independent pathway involving the apoptosis-inducing factor (AIF). In contrast, a strain of neurotropic RV that does not induce apoptosis did not activate caspases or induce AIF translocation. Bcl-2 overproduction in Jurkat T cells (Jurkat-Bcl-2) abolished both pathways. ERA infection and production were similar in Jurkat-vect and Jurkat-Bcl-2 cells, indicating Bcl-2 has no direct antiviral effects. Bcl-2 production is naturally upregulated by day 3 in ERA-infected Jurkat-vect cultures. The increase in Bcl-2 levels seems to be controlled by the virus infection itself and results in the establishment of long-term, persistently infected cultures that continue to produce virus. Thus, in infections with live RV vaccine strains, infected cells may be productive reservoirs of virus in the long term. This may account for the high efficacy of live rabies vaccines.

Dynamic analysis of apoptosis in primary cortical neurons by fixed- and real-time cytofluorometry.

We describe here a cytofluorometric technology for the characterization of decision, execution, and degradation steps of neuronal apoptosis. Multiparametric flow cytometry was developped and combined to detailled fluorescence microscopy observations to establish the chronology and hierarchy of death-related events: neuron morphological changes, mitochondrial transmembrane potential (Δ Ψm) collapse, caspase-3 and -9 activation, phosphatidyl-serine exposure, nuclear dismantling and final plasma membrane permeabilization. Moreover, we developped a reliable real-time flow cytometric monitoring of Δ Ψm and plasma membrane integrity in response to neurotoxic insults including MPTP treatment. Taking advantage of recently developped specific fluorescent probes and a third generation pan-caspase inhibitor, this integrated approach will be pertinent to study the cell biology of neuronal apoptosis and to characterize new neuro-toxic/protective molecules.

Characterization of a novel monoclonal antibody (V58A4) raised against a recombinant NH2-terminal heparin-binding fragment of human endothelial cell thrombospondin.

We report herein the characterization of a mouse monoclonal antibody (Mab) raised against the recombinant NH2‐terminal heparin‐binding domain (rHBD) of human endothelial cell thrombospondin (TSP). The antibody, a IgG1 (κ), hereafter referred to as V58A4, reacted with two rHBD, TSPN18 and TSPN28 (i.e. 18 kDa and 28 kDa, respectively) with an affinity constant of 1.33 × 10−8 M. However, V58A4 failed to recognize native or deglycosylated forms of TSP purified from platelets or endothelial cells, as well as a 25–30 kDa HBD fragment produced by limited proteolysis of native TSP. In contrast, Mab V58A4 was shown to react with larger HBD fragments (50–60 kDa) that were present in platelet or endothelial cell extracts and could be retained on a heparin—Sepharose column at low salt concentrations. These fragments also reacted with MA‐II, a mouse Mab (IgG1), which recognizes both rHBD and HBD as well as intact TSP. Thus, V58A4 Mab appears to selectively recognize naturally occurring HBD fragments of TSP and may thus prove to be useful for detecting TSP proteolysis in situ under various physiopathological conditions.

Mapping of 5-HT-moduline binding sites in guinea-pig brain by film and digital autoradiography

5-HT-moduline is a cerebral tetrapeptide [Leu-Ser-Ala-Leu] that was recently isolated from bovine brain tissue and shown to interact specifically with 5-HT1B receptors, particularly in rodents. The pharmacological properties of 5-HT1B receptors in rodents are different from those in other species. In order to better understand the role of this peptide in non-rodent species, we determined the distribution of 5-HT-moduline binding sites in guinea-pig brain using both the film autoradiography and digital autoradiography with a newly developed high resolution beta-imaging techniques. We found that 5-HT-moduline binding sites were expressed in various brain regions. Quantitative analysis showed that densities of binding sites were similar to those observed previously in rat brain. Regions with the highest labelling included cortex, septum, hippocampus and some regions of basal ganglia. Our results extend previous data and show that 5-HT-moduline interacts with the two forms of 5-HT1B receptors that are distinct pharmacologically. By this interaction, 5-HT-moduline may play an important role in regulating the functional activity of 5-HT1B receptors, thereby contributes to the pathophysiology of serotonergic transmission.

Molecular characterization of a monoclonal murine anti-blood group A antibody

A mouse monoclonal anti-human blood group A antigen (AC12, mu, kappa) has been generated and sequenced in order to analyze the immunoglobulin genes used to generate antibodies with anti-human blood group A specificity. Mice were immunized with human type A RBC. Anti-A producing hybridomas were detected by agglutination against human type A RBC. Total cellular RNA was extracted from hybridomas cells. PCR amplification and sequencing of anti-A heavy and light chain cDNAs were performed. The VH and VK sequences of antibody AC12 were shown to be very homologous to that used by other antibodies recognizing carbohydrates as well as glycoproteins, peptides or haptens constituting self antigens as well as nonself antigens. The VH sequence of antibody AC12 presented important homology with a previously reported monoclonal anti-blood group B antibody. The antibody AC12 also presented homology with the VH and VK sequences of a previously reported human anti-blood group A antibody which contributes additional evidence in favor of a restricted usage of V segments by antibodies directed against red blood antigens.