The VPAC2 agonist peptide histidine isoleucine (PHI) up-regulates glutamate transport in the corpus callosum of a rat model of amyotrophic lateral sclerosis (hSOD1G93A) by inhibiting caspase-3 mediated inactivation of GLT-1a.

Degeneration of corpus callosum appears in patients with amyotrophic lateral sclerosis (ALS) before clinical signs of upper motor neuron death. Considering the ALS-associated impairment of astrocytic glutamate uptake, we have characterized the expression and activity of the glutamate transporter isoforms GLT-1a and GLT-1b in the corpus callosum of transgenic rats expressing a mutated form of the human superoxide dismutase 1 (hSOD1(G93A)). We have also studied the effect of peptide histidine isoleucine (PHI), a vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating polypeptide (PACAP) receptor 2 (VPAC(2)) agonist on glutamate transporters both in vivo and in callosal astrocytes. Before the onset of motor symptoms, the expression of both transporter isoforms was correlated with a constitutive activity of caspase-3. This enzyme participates in the down-regulation of GLT-1 in ALS, and here we demonstrated its involvement in the selective degradation of GLT-1a in the white matter. A single stereotactic injection of PHI into the corpus callosum of symptomatic rats decreased caspase-3 activity and promoted GLT-1a expression and uptake activity. Together, with evidence for a reduced expression of prepro-VIP/PHI mRNA in the corpus callosum of transgenic animals, these data shed light on the modulatory role of the VIP/PHI system on the glutamatergic transmission in ALS.

Differential regulation of C-terminal splice variants of the glutamate transporter GLT-1 by tumor necrosis factor-alpha in primary cultures of astrocytes.

The high-affinity glutamate transporter GLT-1 plays a key role in the control of the glutamate homeostasis in the central nervous system and protects neurons against excitotoxicity. Splice variants of the original transcript have been identified and their involvement in neurodegenerative disorders has been proposed. However, the functions and the regulations of these isoforms remain unclear. In this study, we focused our interest on the expression of two C-terminal splice variants of GLT-1 (GLT-1a and b) in primary astrocyte cultures exposed to distinct chemical environments. While GLT-1a and GLT-1b mRNAs were both increased in response to treatment with N(6),2'-O-dibutyryladenosine 3',5'-cyclic monophosphate (dBcAMP), the culture supplement G5 or tumor necrosis factor-alpha (TNF-α), the regulation of GLT-1b appeared quicker and was more pronounced. Besides, using validated antibodies, we evidenced a differential regulation of the two proteins in cells exposed to TNF-α. Thus, while dBcAMP and the G5 supplement stimulated the expression of both isoforms at 3 and 7 days, a transient upregulation of GLT-1a was induced by TNF-α, which contrasts with the sustained induction of the GLT-1b isoform. These results shed light on the complex influence of the pro-inflammatory cytokine TNF-α on GLT-1a mRNA and protein expression and on the necessity to distinctly consider the GLT-1 isoforms with appropriate tools in studies addressing the regulation of glutamate transporters.

Antibody production by injection of living cells expressing non self antigens as cell surface type II transmembrane fusion protein.

Antigen expression and purification are laborious, time consuming and frequently difficult steps in the process of antibody production. In the present study, we developed a method avoiding these two steps. This method relies on the injection of histocompatible living cells stably expressing the antigen as a cell surface type II transmembrane fusion protein. A vector, nicknamed pCD1-CD134L, was constructed to express the antigen fused at the carboxyterminal end of the human CD134 ligand (CD134L) type II transmembrane protein on the surface of eucaryotic cells. This vector was shown to induce cell surface expression of epitopes from human c-Myc (soluble protein), uterogloblin-related protein 1 (secreted protein) and CD94 (type II transmembrane protein). Using this vector, we developed a method to produce antibodies without antigen production. The flowchart of this method is as follows: (i) cloning of the antigen in the pCD1-CD134L vector; (ii) production of a histocompatible cell line stably expressing the CD134L-antigen fusion protein; (iii) testing for cell surface expression of the fusion protein by targeting the CD134L carrier; and (iv) prime-boost immunisation with living cells expressing the fusion protein. This method was successfully used for production of polyclonal antibodies raised against Ixodes ricinus calreticulin (secreted protein) in mice and for production of monoclonal antibodies raised against an epitope of Vaccinia virus A56 (type I transmembrane protein) protein in rat. The present study is the first to demonstrate the use of a type II transmembrane protein as a carrier for cell surface display of antigens.

Induction of autoantibodies against mouse soluble proteins after immunization with living cells presenting the autoantigen at the cell surface in fusion with a human type 2 transmembrane protein.

Induction of autoantibodies towards immune regulatory proteins, such as cytokines or their receptors, is a powerful strategy for functional studies on the role of these factors in vivo. Here we describe a new procedure to elicit autoantibodies by taking advantage of tumor cells as a vaccine against peptides presented at their surface in fusion with the human CD134L transmembrane protein. P1.HTR, an immunogenic variant of the P815 mastocytoma cell line, was used to generate stably transfected cell clones with expression vectors encoding the human CD134L transmembrane protein fused with either mouse IL-22BP or IL-9. Following repeated injections of living tumor cells expressing the mIL-22BP construct, mice developed autoantibodies that bind to mIL-22BP and inhibit its interaction with IL-22 in vitro. Mice similarly immunized against mIL-9 produced high titers of autoantibodies that block the activity of this cytokine in the TS1 bioassay. This procedure also inhibits IL-9 activity in vivo as no increase of serum MMCP-1 mast cell protease concentration was observed following IL-9 administration to immunized mice. As an alternative to the injection of living tumor cells expressing the CD134L-antigen fusion protein, intramuscular electrotransfer of the corresponding DNA construct also induced autoantibodies. These results validate this method as a simple and convenient approach to knock down the in vivo activity of soluble regulatory proteins, including cytokines and their receptors.

Two widely expressed plasma membrane H(+)-ATPase isoforms of Nicotiana tabacum are differentially regulated by phosphorylation of their penultimate threonine.

The plasma membrane H(+)-ATPases PMA2 and PMA4 are the most widely expressed in Nicotiana plumbaginifolia, and belong to two different subfamilies. Both are activated by phosphorylation of a Thr at the penultimate position and the subsequent binding of 14-3-3 proteins. Their expression in Saccharomyces cerevisiae revealed functional and regulatory differences. To determine whether different regulatory properties between PMA2 and PMA4 exist in plants, we generated two monoclonal antibodies able to detect phosphorylation of the penultimate Thr of either PMA2 or PMA4 in a total protein extract. We also raised Nicotiana tabacum transgenic plants expressing 6-His-tagged PMA2 or PMA4, enabling their individual purification. Using these tools we showed that phosphorylation of the penultimate Thr of both PMAs was high during the early exponential growth phase of an N. tabacum cell culture, and then progressively declined. This decline correlated with decreased 14-3-3 binding and decreased plasma membrane ATPase activity. However, the rate and extent of the decrease differed between the two isoforms. Cold stress of culture cells or leaf tissues reduced the Thr phosphorylation of PMA2, whereas no significant changes in Thr phosphorylation of PMA4 were seen. These results strongly suggest that PMA2 and PMA4 are differentially regulated by phosphorylation. Analysis of the H(+)-ATPase phosphorylation status in leaf tissues indicated that no more than 44% (PMA2) or 32% (PMA4) was in the activated state under normal growth conditions. Purification of either isoform showed that, when activated, the two isoforms did not form hetero-oligomers, which is further support for these two H(+)-ATPase subfamilies having different properties.

Different subcellular localization and glycosylation for a functional antibody expressed in Nicotiana tabacum plants and suspension cells.

Genes encoding the heavy and light chains of LO-BM2, a therapeutic IgG antibody, were assembled in the tandem or inverted convergent orientation and expressed in Nicotiana tabacum plants and BY-2 suspension cells. The tandem construct allowed higher expression in both expression systems. A similar degradation pattern was observed for the secreted antibody recovered from the leaf intercellular fluid and BY-2 culture medium. Degradation increased with leaf age or culture time. Antibodies purified from leaf tissues and BY-2 cells were both functional. However, MS analysis of the N-glycosylation showed complex plant-type glycans to be the major type in the antibody purified from plants, whereas, oligomannosidic was the major glycosylation type in that purified from BY-2 cells. LO-BM2 was observed mainly in the endoplasmic reticulum of BY-2 cells while, in leaf cells, it was localized mostly to vesicles resembling prevacuolar compartments. These results and those from endoglycosidase H studies suggest that LO-BM2 is secreted from BY-2 cells more readily than from leaf cells where it accumulates in a post-Golgi compartment.

Inhibition of humoral response to allogeneic porcine mesenchymal stem cell with 12 days of tacrolimus.

BACKGROUND: In vivo studies have highlighted allogeneic mesenchymal stem-cell (MSC) immunogenicity. We investigated in vitro MSC-immunosuppressive drugs interaction and further tested in vivo the humoral response to intracardiac allogeneic MSC transplantation in a mini-swine model receiving a short course of immunosuppression. METHODS: For in vitro experiments, long-term culture MSCs were used. Immunosuppressive drugs tested were mycophenolate mofetil, cyclosporin, tacrolimus (TAC), sirolimus (SIR), and everolimus. Cell proliferation/viability was assessed on day 7. For each drug, the C50 was determined, and the agonistic effect between immunosuppressive drugs and MSCs on alloreactivity was measured in proliferation assay of MSC-peripheral blood mononuclear cell cultures. For in vivo experiments, one-haplotype swine leukocyte antigen class I and II mismatch (n=11) were used. Allogeneic MSCs were transplanted into ischemic myocardium. TAC was administered 12 days. Donor-specific antibody response was assessed by flow cytometry and complement-mediated cytotoxicity assay. RESULTS: All drugs except TAC significantly decreased cell proliferation (from 17% to 62%). In MSC-peripheral blood mononuclear cell co-culture assay, MSCs' immunomodulatory properties were maintained when TAC or SIR were used. In vivo experiments showed that only 2 of 11 animals under TAC developed donor-specific antibodies. Importantly, sera from those two animals did not elicit a complement-mediated cytotoxic response. CONCLUSIONS: Immunosuppressive drugs significantly affect proliferation and viability of MSCs, but neither TAC nor SIR had a detrimental impact on MSCs' immunomodulatory properties. In this large-animal model, addition of short course of immunosuppression seems to overcome the immune response to intracardiac allogeneic MSCs, which was recently demonstrated to occur in the absence of immunosuppression.

Natural antibody--complement dependent neutralization of bovine herpesvirus 4 by human serum.

In contrast to most gammaherpesviruses, Bovine herpesvirus 4 (BoHV-4) has a broad range of host species both in vitro and in vivo. Several in vitro studies demonstrated that some human cell lines are sensitive or even permissive to BoHV-4. These observations led to the hypothesis that cross-species transmission of BoHV-4 could lead to human infections. In the present study, we investigate the sensitivity of BoHV-4 to neutralization by naïve human sera in order to determine if humans exhibit innate anti-viral activities against this virus. Our results demonstrate that human sera from naïve individuals, in contrast to the sera of naïve subjects from various animal species, neutralize BoHV-4 efficiently. A series of complementary experiments were performed to unravel the mechanism(s) of this neutralization. The data obtained in this study demonstrates that human serum neutralizes BoHV-4 in a complement dependent manner activated by natural antibodies raised against the Galalpha1-3Galbeta1-4GlcNAc-R epitope expressed by bovine cells.

Cell-mediated cytotoxicity to porcine aortic endothelial cells is not dependent on galactosyl residues when baboon peripheral blood lymphocytes are previously primed with pig xenoantigens.

BACKGROUND: In the pig-to-baboon model, acute vascular rejection remains the main hurdle for successful long-term xenograft survival. The production of galactosyl knockout pigs could solve concomitantly the problem of hyperacute and acute vascular rejection. This work studies in vitro the cell-mediated cytotoxicity of natural killer (NK) and T cells after priming of baboon peripheral blood lymphocytes (PBLs) with pig antigens to evaluate whether cytotoxicity is galactosyl-dependent. MATERIAL AND METHODS: PBLs from naive and primed baboons were used as effectors on primary porcine aortic endothelial cells (PAECs) to assess cytotoxicity. Untreated or galactosidase-digested PAECs were used to evidence the role of galactosyl residues on cell-mediated cytotoxicity. Two rat-anti baboon monoclonal antibodies were tested to inhibit either T+NK cells (LO-CD2b) or NK cells alone (LO-CD94). RESULTS: When using PBLs from naive animals, spontaneous lysis occurred and was inhibited by both LOCD-2b and LO-CD94. In comparison, lysis of PAECs was significantly higher when baboon PBLs were first primed in vivo with pig xenoantigens. In this case, cytotoxicity was completely inhibited by LO-CD2b but only partially by LO-CD94. Reduction of galactosyl residues by galactosidase digestion showed that PAEC lysis almost completely disappeared with naive baboon PBLs but not with primed baboon PBLs, thereby indicating that anti-pig T-cell response is not dependent on galactosyl residues. CONCLUSION: Galactosyl knockout pigs could solve hyperacute rejection and also prevent the activation of NK cells even after xenogeneic priming. T cells will then be the next hurdle for the success of xenografting.