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B Charreau

Publications and source records attributed to B Charreau.

At least 19 recordsLinked to original sources
PubMed2001

Antigraft antibody-mediated expression of metalloproteinases on endothelial cells. Differential expression of TIMP-1 and ADAM-10 depends on antibody specificity and isotype.

Endothelial cell (EC) interaction with antigraft antibodies (Abs) mediates EC injury and activation involved in vascular graft rejection. The aim of this study was to identify EC genes regulated in response to antigraft Ab binding that contribute to the endothelium alterations implicated in graft rejection or survival. By means of RNA differential display, 13 cDNA fragments corresponding to genes differentially expressed in ECs incubated with antigraft Abs were identified. Among these cDNAs were found the tissue inhibitor of metalloproteinase-1 (TIMP-1) and a desintegrin and metalloproteinase (ADAM-10). We demonstrated that TIMP-1 and ADAM-10 mRNA and protein expression was rapidly upregulated in ECs in response to antigraft Ab binding. Our data showed that TIMP-1 was upregulated in response to human IgG but not IgM and anti-galactosyl (Gal) alpha1-3Gal human xenogeneic Abs. In contrast, upregulation of ADAM-10 in ECs was shown to be mostly mediated by anti-Galalpha1-3Gal IgM Abs. Specific effects of human IgG and IgM xenogeneic Abs on endothelial transcripts indicate that different isotypes and specificities of Abs may mediate different EC changes. Our results suggest that interaction of ECs with antigraft Abs, according to their specificity, selectively induces synthesis and release of metalloproteinases and inhibitors, controlling proteolytic processes and immunological events that respectively contribute to graft rejection or survival.

Amyloid Precursor Protein Secretases↗
PubMed2001

Different mechanisms mediate the rejection of porcine neurons and endothelial cells transplanted into the rat brain.

In order to investigate the early cellular responses mediating xenograft rejection in the brain, porcine aortic endothelial cells (PAEC) or porcine fetal mesencephalic neurons (PNEU) were transplanted into the striatum of LEW.1A rats. PAEC were detected with a specific anti-beta1 integrin antibody, and PNEU with an anti-porcine neurofilament antibody, or an antibody recognizing the NeuN antigen. PAEC grafts were massively infiltrated within 24 h by OX42-positive cells, which may correspond to polymorphonuclear (PMN) cells or macrophages. At that moment, the graft contained numerous cells expressing the inducible isoform of NO-synthase (iNOS). Infiltration by ED1-positive macrophages was effective after three days. The beta1-integrin labeling decreased from that time-point to day 7 post-implantation, and vanished after 11 days. Although some OX8-positive cells were present around the graft as soon as 3 days after transplantation, cells expressing the T-cell receptor (TCR)-beta chain infiltrated the graft after 7 days and their number remained low. A strong, diffuse OX8-and ED1-positive immunoreactive material remained in the scar up to the third week. In striking contrast, PNEU grafts remained poorly infiltrated by OX42- or ED1-positive cells during the first two weeks. A massive infiltration by macrophages and TCRbeta-positive lymphocytes occurred after 3 weeks. Natural killer (NK) cells were more scarce. The inflammation territory enlarged, and blood vessels were overloaded with macrophages or lymphocytes. Nevertheless, the graft contained NeuN-positive nuclei and neurites harbouring the porcine neurofilament protein. Hence, rejection was not completed at this time-point. These results suggest that the rapid rejection of PAEC is mainly driven by macrophages and possibly PMN cells, unlike PNEU, whose rejection is delayed and also involves lymphocytes. Differences in immunogenicity of grafted cells and/or patterns of production of pro-inflammatory cytokines may account for these contrasted rejection kinetics.

Animals↗
PubMed2000

Cyclosporine inhibits class II major histocompatibility antigen presentation by xenogeneic endothelial cells to human T lymphocytes by altering expression of the class II transcriptional activator gene.

BACKGROUND: Cyclosporine (CsA) is currently given to recipients of vascularized xenografts as part of the immunosuppressive regimen required to prevent the hyperacute rejection phase. The effects of CsA on non-lymphoid immune cells, such as endothelial cells (ECs), have not been well characterized and sometimes seem contradictory, because both protective and adverse effects have been reported. In the present study, we investigated in vitro whether CsA could alter the antigenicity of activated porcine aortic endothelial cells (PAECs) by reducing class I and class II MHC antigen expression. METHODS: The effect of CsA on MHC antigen expression during tumor necrosis factor (TNF)-alpha- or lymphocyte-mediated PAEC activation was evaluated in vitro by flow cytometry and correlated to the ability of porcine ECs to promote human T lymphocyte proliferation. The effect of CsA on class II MHC antigen mRNA expression was also analyzed and related to class II transcriptional activator (CIITA) mRNA expression. RESULTS: Flow cytometry analysis showed that TNF-alpha-mediated induction of class II MHC antigen expression on PAECs was completely inhibited by CsA, whereas expression of class I MHC was reduced by 50%. The inhibition was dose dependent (at drug concentrations ranging from 2.5 microg/ml to 20.0 microg/ml) and was consistently observed at all time points (24-72 hr) during the activation period. Decreased MHC antigen expression dramatically reduced the ability of PAECs to further promote human T-cell proliferation. Similar levels of inhibition were achieved using an anti-porcine class II MHC blocking monoclonal antibody. Pretreatment of PAECs with CsA for 4 hr before coculture with human peripheral blood leukocytes efficiently blocked the induction on PAECs of E-selectin and class II MHC antigens and inhibited overexpression of class I antigens. Semiquantitative reverse transcriptase-polymerase chain reaction experiments showed that CsA markedly reduced the steady-state level of porcine class II (SLA-DRA and SLA-DQA) mRNA at 16 hr, compared with PAECs stimulated with TNF-alpha alone. The reduced level of class II MHC mRNA was associated with a lack of CIITA expression at this time point, suggesting that CsA could alter transcription or promote the rapid decay of CIITA mRNA. CONCLUSION: Our study indicates that CsA could play a role in preventing porcine MHC antigens being directly presented to human T lymphocytes by xenogeneic ECs.

Animals↗
PubMed2000

Association of glucocorticoids and cyclosporin A or rapamycin prevents E-selectin and IL-8 expression during LPS- and TNFalpha-mediated endothelial cell activation.

BACKGROUND: Endothelial cell (EC) activation plays an important role in inflammation, hemostasis, and organ rejection of allogeneic and xenogeneic transplantation. These processes leads to rapid and transient up-regulation of proinflammatory molecules, such as the adhesion molecule E-selectin and the chemotactic cytokine IL-8. The purpose of this study was to investigate the specific effects of several major and potentially synergistic immunosuppressive drugs-cyclosporin A (CsA), rapamycin (Rap), and glucocorticoids (GC)-on lipopolysaccharide (LPS)- or tumor necrosis factor (TNF)alpha-induced EC activation METHODS: The ability of immunosuppressive drugs, used alone or in combination, to prevent in vitro TNFalpha- and LPS-induced expression of E-selectin and interleukin 8 on porcine ECs, as well as their effect on leukocyte-EC interaction, were investigated. In addition, we studied the in vivo effect of these drugs after i.v. administration of recombinant TNFalpha to rats. RESULTS: At high concentrations, which correspond to the acceptable experimental levels in primate xenograft recipients, CsA, Rap, and GC individually inhibited E-selectin protein induction in a dose-dependent manner in cultured porcine ECs treated with LPS with an additive effect when the drugs were associated. The pattern of drug-mediated inhibition was related to the stimulus used to activate ECs (i.e., LPS vs. TNFalpha). Reduced expression of E-selectin on ECs activated in the presence of the tested immunosuppressive drugs correlated with a weaker adhesion of human U937 cells to ECs. Messenger RNA analysis demonstrated that the presence of CsA, Rap, and GC during EC activation inhibited E-selectin and interleukin 8 at the gene expression level. LPS-mediated induction of IbetaBalpha expression was not observed in ECs treated with CsA, whereas GC reduced its transcripts by approximately 50%. It is interesting that in vivo studies confirmed that CsA and GC inhibited EC activation at therapeutic doses (1 mg/kg and 10 mg/kg for GC and CsA, respectively) and showed that the combination of CsA and GC efficiently prevents TNFalpha-mediated induction of E-selectin on cardiac ECs. CONCLUSION: Our data show that, besides their specific immunosuppressive effects on T cells, CsA, Rap, and GC can efficiently contribute to the attenuation of EC activation in vivo and the resulting inhibition is enhanced by the association of CsA with GC.

Animals↗
PubMed1999

Characterization of two monoclonal antibodies against porcine VCAM-1.

Immunization of mice with TNF alpha-activated porcine endothelial cells led to the characterization of two monoclonal antibodies (MAbs), 5F3 and 8A7, specific for porcine VCAM-1. Upon flow cytometry, both antibodies increasingly labeled endothelial cells according to their degree of activation. They bound a band of MW 80 kDa on Western blots of endothelial cells, which is the apparent molecular weight of porcine VCAM-1. It was determined by surface plasmon resonance that the antibodies are directed to different antigenic sites. It was also found that 5F3 competes for binding the antigen with a MAb previously characterized as binding domain 1 of porcine VCAM-1. Subsequently, 5F3, but not 8A7, was found to inhibit the adhesion of human B lymphocyte Ramos cells to porcine endothelial cells in vitro. These antibodies, which do not cross-react with human VCAM-1, might be useful for diagnostic or therapeutic purposes in xenotransplantation.

Animals↗
PubMed1999

Protection against hyperacute xenograft rejection of transgenic rat hearts expressing human decay accelerating factor (DAF) transplanted into primates.

BACKGROUND: Production of transgenic pigs for multiple transgenes is part of a potential strategy to prevent immunological events involved in xenograft rejection. Use of a genetically engineerable rodent as a donor in primates could allow testing in vivo of the effects of different transgenes on controlling xenograft rejection. As a first step in the development of a donor containing multiple transgenes, transgenic rats for human decay-accelerating factor (DAF) were used as heart donors to test their resistance against complement (C)-mediated rejection by non-human primates. MATERIALS AND METHODS: Transgenic rats were generated by using a construct containing the human DAF cDNA under the transcriptional control of the endothelial cell (EC)-specific human ICAM-2 promoter. DAF expression was evaluated by immunohistology and by FACS analysis of purified ECs. Resistance of transgenic hearts against C-mediated damage was evaluated by ex vivo perfusion with human serum and by transplantation into cynomolgus monkeys. RESULTS: Immunohistological analysis of DAF expression in several organs from two transgenic lines showed uniform expression on the endothelium of all blood vessels. ECs purified from transgenic hearts showed 50% DAF expression compared to human ECs and >70% reduction of C-dependent cell lysis compared to control rat ECs. Hemizygous transgenic hearts perfused with human serum showed normal function for >60 min vs. 11. 2 +/- 1.7 min in controls. Hemi- or homozygous transgenic hearts transplanted into cynomolgus monkeys showed longer survival (15.2 +/- 7 min and >4.5 hr, respectively) than controls (5.5 +/- 1.4 min). In contrast to hyperacutely rejected control hearts, rejected homozygous DAF hearts showed signs of acute vascular rejection (AVR) characterized by edema, hemorrhage, and an intense PMN infiltration. CONCLUSIONS: We demonstrate that endothelial-specific DAF expression increased heart transplant survival in a rat-to-primate model of xenotransplantation. This will aid in the analysis of AVR and of new genes that may inhibit this form of rejection, thus helping to define strategies for the production of transgenic pigs.

Animals↗
PubMed1998

Intracellular expression in pig cells of anti-alpha1,3galactosyltransferase single-chain FV antibodies reduces Gal alpha1,3Gal expression and inhibits cytotoxicity mediated by anti-Gal xenoantibodies.

BACKGROUND: The carbohydrate structure Gal alpha1,3Gal expressed on pig cells is the major antigen recognized by xenoreactive natural antibodies in the higher primates. In xenotransplantation, natural antibodies binding to that structure initiate hyperacute rejection, and the anti-Gal alpha1,3Gal antibodies that are elicited probably take part in later phases of vascularized graft rejection. This epitope also appears to be involved in innate cellular responses. Inactivation of alpha1,3 galactosyltransferase in transgenic pigs would certainly lead to the success of xenotransplantation, but gene knockout in pigs is not feasible yet. METHODS: As a novel strategy to inhibit alpha1,3 galactosylation, we generated recombinant single-chain Fv (ScFv) antibodies directed against pig alpha1,3-galactosyltransferase and evaluated the effect of their intracellular expression on enzyme activity and Gal alpha1,3Gal expression. RESULTS: After in vitro transfection in pig cells, the scFv antibody anti-pig alpha1,3-galactosyltransferase reduced the amount or function of enzyme by up to 70% as evidenced by immunofluorescence and measurement of cell-associated activity. Consequently, Gal alpha1,3Gal on cell membranes was reduced to the same extent. This led to a profound (more than 90%) reduction in the cytotoxicity involving anti-Gal antibodies and complement. CONCLUSION: Although not sufficient to knock out the overall human anti-pig natural xenoreactivity, intracellular expression of the scFv antibody anti-alpha1,3-galactosyltransferase in pig cells significantly decreases the amount of Gal alpha1,3Gal and could be important to protect cells from elicited antibodies as well as from innate effectors.

Animals↗
PubMed1998

Anti-adenovirus immune responses in rats are enhanced by interleukin 4 but not interleukin 10 produced by recombinant adenovirus.

Recombinant adenoviruses can be used for in vivo gene transfer with great efficiency. However, the duration of transgene expression and the possibility of readministering the virus are severely limited by the host anti-adenovirus immune response, which is controlled mainly by cytokine networks. Adenoviruses encoding IL-4 (AdIL-4) or IL-10 (AdIL-10) were administered to rats through the portal vein and the anti-adenovirus immune response was studied. As compared with administering adenoviruses without transgene (Addl324) or with the lacZ gene (AdlacZ), AdIL-4, but not AdIL-10, resulted in a significant increase in leukocytes in the liver, with a predominance of macrophages that peaked on days 7 and 14 after gene transfer and gradually returned to normal by day 28. AdIL-4 induced a significant increase in both neutralizing and ELISA-detected anti-adenovirus antibodies, whereas AdIL-10 caused an increase in ELISA-detected antibodies alone. Anti-adenovirus antibodies were predominantly of Th1-dependent immunoglobulin subclasses in rats receiving Addl324, AdlacZ, or AdIL-10, whereas animals receiving AdIL-4 showed a predominance of Th2-dependent immunoglobulin subclasses. Type 1 (IFN-gamma) and type 2 (IL-5) cytokines were increased only in livers from rats receiving AdIL-4. Rats receiving AdIL-4 showed increased anti-adenovirus cytotoxic T lymphocyte activity and CD8+ cell depletion prevented leukocyte infiltration in the liver. These results show that IL-4 increases local and systemic immune responses against recombinant adenoviruses.

Adenoviridae↗
PubMed1998

Transcriptional and posttranscriptional regulation of alpha 1,3-galactosyltransferase in activated endothelial cells results in decreased expression of Gal alpha 1,3Gal.

Gal alpha 1,3Gal carbohydrate residues are present in the glycoproteins and glycolipids of lower mammals, and appear to be involved in the binding specificity of several membrane receptors. We report here that endothelial cells stimulated with lipopolysaccharide or inflammatory cytokines modulate their expression of UPD-Gal: beta-D-Gal alpha 1,3-galactosyltransferase (alpha 1,3GT), the Golgi enzyme that attaches a galactose in alpha 1,3 configuration to an N-acetyllactosamine acceptor. Upon activation, the steady state level of mRNA is transiently increased, the modifications being paralleled by a transcriptional regulation of the gene. Cell-associated enzyme activity, on the other hand, falls rapidly after activation, before being up- and downregulated with kinetics that parallel those of the mRNA, and after 3 days reaches a level representing 40-60% of the activity in cells before activation. Overall Gal alpha 1,3Gal expression at the cell surface follows enzyme activity, except that it is insensitive to the rapid and transient reduction of activity occurring shortly after activation. This reduced alpha 1,3GT activity in stimulated EC is correlated with lower stability of the protein, and with a switch in the expression of the isoform pattern, isoform 1 being predominant in resting cells whereas after activation it is isoform 2 that predominates. The two isoforms, however, appear to have similar intrinsic stability, so that the reduced stability of the enzyme in activated EC probably results from an induced proteolytic degradation pathway.

Amino Sugars↗
PubMed1998

Characterization of a murine monoclonal antibody specific for swine beta1 integrin.

Murine monoclonal antibodies were raised against porcine platelets in order to provide tools for investigating interactions of human blood cells and natural antibodies with porcine tissues. Hybridomas were screened by cellular ELISA on porcine platelets and endothelial cells. Positive clones were tested by flow cytometry for reactivity with isolated endothelial cells. One clone, NaM160-1A3, produced an antibody that stained porcine but not human endothelial cells and lymphocytes. The antibody bound to a 116 kDa glycoprotein on Western blot of both platelets and endothelial cells. The antigen was purified from a platelet lysate by affinity chromatography, first on a ConA column and then on a column presenting the immobilized NaM160-1A3 antibody. Two glycoproteins were obtained: one (116 kDa) was recognized by the antibody and one (150 kDa) was not. The 116 kDa protein had an internal decapeptide identical with human beta 1 integrin, and the 150 kDa protein had an internal amino acid sequence belonging to porcine alpha 2 integrin. Therefore, the NaM160-1A3 antibody was directed against porcine beta 1 integrin and allowed the purification of the complex alpha 2 beta 1, also termed Very Late Antigen 2 (VLA-2). It did not recognize human beta 1 integrin.

Amino Acid Sequence↗
PubMed1997

Adenovirus-mediated gene transfer in rat liver of interleukin 4 but not interleukin 10 produces severe acute hepatitis.

Several immune responses are either limited to or concentrated in a given organ. Cytokines produced during ongoing immune responses have organ-localized effects that can be only partially mimicked upon their systemic delivery. Recombinant adenoviruses are efficient vectors to induce transient organ-localized cytokine expression. This allows in vivo analysis of the effects of cytokines produced spatially and temporally in a manner comparable to that observed during immune responses. The authors generated recombinant adenovirus for rat IL-4 (AdIL-4) and IL-10 (AdIL-10) to analyse the in vivo effects of these two important immunoregulatory molecules after gene transfer in the liver. It was first established that AdIL-4 and AdIL-10 were able to direct the production of biologically active cytokines by different rat cell types in vitro. Intraportal injection of doses of up to 10(10) pfu of AdIL-10 or control non-coding recombinant adenovirus were well tolerated, and hepatic histology showed only mild alterations. Conversely, animals receiving more than 2.5 x 10(9) pfu of AdIL-4 showed dose-dependent mortality, with clinical signs of hepatic dysfunction. Liver histology in animals receiving 2.5 x 10(9) pfu of AdIL-4 showed severe acute hepatitis with maximal lesions between day 7 and 14 and almost complete normalization by day 28 after gene transfer. The leukocyte infiltrate was composed primarily of mononuclear cells, but eosinophils and mast cells were significantly increased as compared to control animals. Hepatic function was also altered in animals that received AdIL-4, with kinetics similar to that of histological lesions. Our study describes a model for investigating cytokine function in vivo through liver-localized transgene expression mediated by adenoviral vectors and demonstrates that liver production of IL-4 but not IL-10 results in acute severe hepatitis.

Acute Disease↗
PubMed1996

Assessment of hyperacute rejection in a rat-to-primate cardiac xenograft model.

We studied a rat-to-cynomolgous monkey model for xenotransplantation of vascularized organs and found that a rat heart was rejected in 5.5 +/- 1.4 min (n = 10). This hyperacute rejection (HAR) was consistent with kinetic experiments in vitro that showed damage to rat endothelial cells (ECs) after 3 min of incubation with primate serum. Histopathology and ultrastructural analysis of rejected hearts showed marked EC damage and early adherence of platelets and polymorphonuclear leukocytes to the endothelium. Immunohistochemical analysis revealed deposition along endothelial surfaces of IgG, IgM, and complement (C) components of the classical but not the alternative pathway, suggesting that, as in the pig-to-primate model, HAR is mediated by the binding of recipient xenogeneic natural antibodies and C activation. The effect of C depletion on xenograft survival was evaluated in two recipients that were treated with cobra venom factor (CVF). CVF caused complete C inactivation, demonstrated by lack of serum hemolytic activity and C-dependent EC cytotoxicity at engraftment and until the animals died. The rat cardiac transplants survived for at least 9 hr and 77 hr. Histology showed massive interstitial hemorrhage, edema, and cellular infiltration with scanty fibrin deposits. These results in CVF-treated recipient monkeys indicate that C activation mediates the development of HAR in this rat-to-primate model. We suggest that the model may be of interest as an alternative to the more expensive and time-consuming pig-to-primate model for testing the efficacy of transgenic modification of donor organs to prolong xenograft survival and for studying mechanisms of discordant xenograft rejection.

Animals↗
PubMed1996

Transgenesis in rats: technical aspects and models.

The production of transgenic rats by DNA-microinjection into fertilized ova has now become an established procedure, although fewer than 20 lines have been described during the last 5 years. Overall, transgenic rats remain more difficult to produce than transgenic mice, but satisfactory yields have been obtained by several laboratories. A review of the methods used to generate transgenic rats shows considerable variation between different laboratories, particularly in choice of strain, superovulation protocols and the use of embryo culture before reimplantation. In some instances, the production of transgenic rats has provided data that are new and relevant, compared to data obtained in mice bearing the same transgene. Models have been developed for human diseases such as hypertension and autoimmunity, and applications have been found in the study of carcinogenesis and in pharmacological research. Transgenic rat technology also opens up interesting perspectives for transplantation research, in which microsurgery is an essential procedure. Intensive research is in progress in several laboratories to produce rat embryonic stem (ES) cell lines, but existing lines have not participated in germ line formation a prerequisite for their use in gene knock out experiments.

Animals↗