| Hemoglobin-based oxygen carriers(HBOCs) is a kind of novel therapeutic biological medicine which is in development at present. The solution can be used as oxygen carrier delivering oxygen. Hemoglobin used in HBOCs so far is of either human or bovine origin. But there are serious disadvantages of these two Hb resources, the most concern is the potential negative effects of the increasing incidence of new variant Creutzfeldt-Jacob disease,HIV and hepatitis virus. Therefore, other sources of hemoglobin may offer solutions to the problems encountered in using human and bovine hemoglobin. In the past few years we studied porcine hemoglobin as Hb resources of HBOCs ,the results indicated that Porcine hemoglobin has high oxygen affinity, similar to that of human hemoglobin and much higher than that of bovine hemoglobin. Rich supply, the unique properties of oxygen affinity and relative stability of porcine hemoglobin could make this type of protein a useful material in molecular design and large-scale production of HBOCs.At present time,many HBOCs have been brought into clinical trial,but several of them have been discontinued because of problems of safety evaluation.So potential toxicity of HBOCs become the main barrier of the clinical application of HBOCs,Immunogenity and immunological toxicity are important parts of development of biological protein medicine and safety evaluation of HBOCs.So it is important to assess the immunogicity and the immunotoxicity of glutaradehyde polymerized porcine hemoglobin.of immunological properties .In this article, Using pig blood as the raw material, ultra-pure porcine hemoglobin was obtained through multiple purification steps; with glutaraldehyde as the cross-linker, polymerized porcine hemoglobin (pPolyHb) was then produced.The immunogenity of porcine hemoglobin and glutaradehyde polymerized porcine hemoglobin were investigated through detection of the serum specific IgG responses using indirect ELISA in animals after administration of the appropriated hemoglobin samples via different routes and under various conditions.In addition, the thesis was also concerned with the possible origin of enhanced immunogenicity of porcine glutaraldehyde-polymerized hemoglobin.The reasons why glutaraldehyde polymerized porcine hemoglobin were not antigenic when administrated via intravenous route repeatedly were also concerned in the thesis.The results indicated that in the absence of an adjuvant, porcine hemoglobin did not elicit detectable specific serum IgG antibody response in mice after repeatedly subcutaneous or intraperitoneal administration of the protein samples. However, in the presence of Freuand's adjuvant, a significant anti-porcine hemoglobin antibody response was observed even after the first boosting injection through intraperitoneal or subcutaneous route administrated into mice,rats or rabbits. glutaraldehyde-polymerized porcine hemoglobin stimulated apparent production of specific serum IgG antibodies in the animals after repeated subcutaneous or intraperitoneal administration of the protein sample even in the absence of an adjuvant. Subcutaneous administration elicited stronger specific serum IgG responses than intraperitoneal delivery of the protein samples. However, repeatedly intravenous administration of therapeutic dosages of pPolyHb failed to induce detectable serum specific IgG responses in rats. Further experiments showed that in a clear dose-dependent manner, intravenous infusion of therapeutic amounts of the pPolyHb markedly inhibited the specific serum IgG responses elicited by subsequent repeated intraperitoneal administrations of pPolyHb in the presence of the Freund's adjuvant, but did not affect the IgG responses elicited by the co-administrated bovine serum albumin (BSA) in rats. These results indicated that intravenous administration of therapeutic dosages of glutaraldehyde-polymerized porcine hemoglobin led to significant, specific immune tolerance to glutaraldehyde-polymerized porcine hemoglobin, but did not affect humoral IgG responses to the stimulation of other unrelated antigens in tested animals.The investigation of possible origin of glutaraldehyde polymerized porcine hemoglobin indicated that the repeated subcutaneous injections of glutaraldehyde polymerized rabbit hemoglobin back to rabbits in the presence of fruend's adjuvant didn't result in detective anti- glutaraldehyde polymerized rabbit hemoglobin IgG,.This indicated that there were no neo-antigens formed in polymerized hemoglobin,the rabbit's immune system was immunological torlerant to its own hemoglobin.The data of immunogenicity of different molecular weight of pPolyHb indicated that the immunogenicity of pPolyHb enhanced along with the increase of molecular weight from 67.4KD to 147.6kD .the immunogenicity of pPolyHb decreased with the increase of of molecular weight from 147.6kD-1150KD .The results of cross-binding reactions of mouse anti- hHb, bHb or pHb IgG or anti-hPolyHb, bPolyHb or pPolyHb IgG with hHb, bHb and pHb and their related polymers suggested that there were no neo-antigens induced in resultant glutaradehyde polymerized hb.The reason of enhanced immunogenicity of polymerized Hb was not glutaraldehyde,there was no production of specific anti-glutaraldehyde IgG.The above series experimental results suggested that the reasons of weak immnogenicity of native/unmodified porcine hemoglobin are as follows .1)Animals including human metabolize a fairly large amount of hemoglobin constantly leased from aged red blood cells mainly through the phagocytical function of phagocytic cells.The fact that hemoglobin of various species are very weekly immunogenic indicates that the fate of hemoglobin after taken up by phagocytic cells , is being mostly hydrolyzed, leaving little or none bound to MHC II complex for presentation to T cells. This pathway would have some leakage, resulting in a very small amount of peptides remained intact and bound to MHC II complex. In the early stage of development of the immune system, this leakage pathway would result in tolerance of peptides derived from homologous hemoglobin. 2)Hemoglobins are highly conserved in animals. heterologous hemoglobin may share the same cleavage sites for endosomal/lysosomal proteases as the hemoglobin of the host so that hetero-hemoglobin would have the same fate inside endocytic/lysomal system of phagocytes as homo-hemoglobin of the host, i.g. most being directed to complete hydrolyzing pathway,This would explain why hetero-hemoglobin exhibits very week immunogenicity, which normally can only be detected when immunization is done in the presence of an adjuvant. Adjuvant-induced local inflammation would facilitate phagocytosis of the protein by DCs so that access amount of hetero-hemoglobin would result in a larger flux of the leakage leading to enhance production of peptides derived from the hetero-hemoglobin and increase opportunities for DC to present the peptides on MHC II complex and induce specific immuno-reactions to the protein.The possible origin of enhanced immunogenicity of glutaraldehyde polymerized porcine hemoglobin is as followings.1) Polymerized hemoglobin may be able to remain partially hydrolyzed due to inaccessibility of some of protease cleavage sites because of local environmental change after polymerization. The final resultant peptides would be bound to MHC II complex and presented onto the cell surface to initiate subsequent specific T cell activation. 2)The polymerized Hb was more stable than native Hb,the T1/2 was prolonged in vivo,the stimulating time to lymphocytes was also prolonged,therefore, the immunogenicity was enhanced.3)The increase of molacular weight was also one of the reasons of the origin of enhanced immunogenicity of polymerized Hb. 4)To an extent, the larger the polymers, the more protease cleavage sites become inaccessible so that more peptides would remain intact in the endocytical pathway and be presented to the cell surface with MHC II.. Too large size of the protein polymers would reduce effectiveness of phagocysis of the protein, resulting in less specific immune response to the proteins.Many investigators reported that patients treated with HBOCs may have increased susceptibility to bacterial infection.The data of intravenous infusion of different doses of pPolyHb at various time of infection indicated that the host defense of mice were not decreased but enhanced after intravenous infusion of 130mg/kg body weight of pPolyHb 4,8 0r 20 hrs before infection of p. aeruginosa.So pPolyHb may enhance host defense and accelerate the phagocytosis function of reticulation endothelium system.But the host defense was impaired when mice were infused 130mg/kg body weight of pPolyHb after 4 hrs or simutanously infection of p. aeruginosa.We found that when low doses of 13 or 1.3mg/kg of pPolyHb were infued simutanous with p. aeruginosa, the host defense of mice were enhanced,furthermore,the enhance of host defense of mice were enhanced signicantly at doses of 1.3mg/kg.The microorganisms can be phagocytized by macrophages before the immune responses when invaded into the host,this is an important immunological defense.The data of effects of pPolyHb on phagocytosis of p. aeruginosa by TIB-67 indicated that at time of 0min TIB-67 were infected with p. aeruginosa when incubated with different concentrations of pPolyHb,the data indicated that high concentrations of pPolyHb (8 or 2mg/ml) can inhibit the phagocytosis of p. aeruginosa of TIB-67. On the contrary, the low concentration of pPolyHb (200,20μg/ml) can promote the phagocytosis function of TIB-67.Incubated with different concentrations of pPolyHb (8 mg/ml, 2mg/ml,200μg/ml,20μg/ml or 2μg/ml) 2 or 4 hrs later, the TIB-67 were infected with p. aeruginosa,the data showed that pPolyHb can promote the phagocytosis function of TIB-67,moreover, 200μg/ml pPolyHb could significantly enhance the phagocytosis function of TIB-67.At present,We are investigating the... |
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