RESUMO
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.
Assuntos
Anticorpos Monoclonais/biossíntese , Proteínas de Membrana/imunologia , Ligante OX40/imunologia , Proteínas Recombinantes de Fusão/imunologia , Animais , Feminino , Ixodes , Camundongos , Camundongos Endogâmicos BALB C , Coelhos , RatosRESUMO
The cholinergic anti-inflammatory pathway has been identified as playing a key role in the communication between the central nervous system and the immune system during inflammation. The potential beneficial role of vagus nerve stimulation (VNS) remains to be clarified in established sepsis. We hypothesized that VNS or nicotine administration would reduce lung injury and mortality in established sepsis. We conducted a prospective, randomized experimental study. Four hours after peritonitis induction by cecal ligation and puncture (CLP), rats were randomized into three groups of seven animals according to the intervention: control group, VNS group (15 V, 2 ms, 5 Hz during 20 min), and nicotine group (400 µg/kg intraperitoneal). Survival was determined as lung injury score 4 and 8 h after CLP. Tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6, IL-10, cytokine-induced neutrophil chemoattractant (CINC)-3 and thrombin-antithrombin complexes (TATc) were measured at baseline and at 4 and 8 h after CLP. Survival at 8 h was 71.4%, 100%, and 23.8% in the control, VNS, and nicotine groups, respectively (p < 0.05). All animals had lung damage but without significant difference between groups even if nicotine-treated animals tended to have a higher score than the controls (p = 0.09). Neutrophil polymorphonuclear (PMN) infiltration was more pronounced in the nicotine group compared with the VNS group (p = 0.015) but not with the controls. TNF-α, IL-6, IL-10, CINC-3, and TATc were elevated in all groups (NS). In this model of established sepsis, posttreatment by VNS was associated with increased survival, while nicotine administration increased lung PMN infiltration and mortality. Nicotine-induced bacterial clearance impairment and nicotine systemic effects may explain these observations.
Assuntos
Lesão Pulmonar Aguda/imunologia , Lesão Pulmonar Aguda/patologia , Terapia por Estimulação Elétrica , Nicotina/farmacologia , Peritonite/imunologia , Peritonite/patologia , Nervo Vago/fisiologia , Animais , Antitrombina III , Ceco , Quimiocina CXCL2/sangue , Inflamação/imunologia , Interleucina-10/sangue , Interleucina-6/sangue , Estimativa de Kaplan-Meier , Ligadura , Infiltração de Neutrófilos/efeitos dos fármacos , Peptídeo Hidrolases/sangue , Peritonite/complicações , Ratos , Fator de Necrose Tumoral alfa/sangueRESUMO
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.