Immunogenicity of allograft components: I. Assay for immunogenicity

We describe an assay for in vivo quantitation of the “immunogenicity” of isolated cell populations. The assay is based on the observation that if an AgB-incompatible recipient rat is primed with donor strain spleen cells 72 hr prior to transplantation, heart allograft survival is reduced from 6.2 to 3.0 days. The effect is independent of the priming cell dose at levels above 3 × 10⁵ cells, whereas doses lower than 10⁵ spleen cells are unable to reduce the survival. The effect is suboptimal if the priming-transplantation interval is less than 3 days, or is prolonged to 4–10 days. The effect is immunologically specific: priming with irrelevant AgB-incompatible spleen cells fails to reduce the survival. Priming with cell populations previously reported “less immunogenic,” such as ultrasonicated spleen cells, erythrocytes, spleen T cells, or spleen cells deriving from methotrexate or cyclophosphamide-treated rats, fails to reduce the survival, or reduces it only when given in 100-fold higher numbers than the minimal dose of intact spleen cells giving maximal reduction.     

Toxicity interactions and ways of reducing side effects of anticancer drugs

Side effects of cytostatics commonly used in the Haematology Clinic are analysed. The toxic action on the host's organs is discussed in L-asparaginase, azathioprine, bleomycine, busulfan, cyclophosphamide, cytosin-arabinoside, daunorubicine, fluorouracil, mercaptopurine, methotrexate, dichlorplatinum, procarbazine and the vinca alkaloids. In addition to toxic symptoms arising from single organs the most important 21 anticancer drugs are gathered in a table. Metabolism of activation and inactivation are mentioned to interprete symptoms of toxicity. Furthermore, the interactions between commonly administered drugs and carcinostatics which may enhance or suppress their carcinostatic efficacy are exposed. A final survey of possible pharmacological rescue measures, which may improve the tolerance of anticancer drugs by diminishing their toxicity is presented.     

Research on PEGylation of porcine prothrombin for improving biostability and reducing animal immunogenicity

Prothrombin is a vitamin K-dependent serine protease and plays pivotal roles in both procoagulant and anticoagulant pathway of hemostasis. In this study, prothrombin purified from porcine plasma was modified through PEGylation at N-terminal residue using 40 kDa PEG-phenyl-isothiocyanate (PIT-PEG40K). The monoPEGylated prothrombin enhanced biostability and remarkably prolonged circulating half-life in mice as compared with that of the nonmodified prothrombin. Moreover, the immunogenicity of PEGylated prothrombin in mice is significantly decreased compared to nonmodified prothrombin. These studies demonstrated the feasibility of PEGylating prothrombin as a promising way for the development of new prothrombin drugs.     

Mapping the antigenic determinants and reducing the immunogenicity of trichosanthin by site-directed mutagenesis

Summary Trichosanthin (TCS) is a type I ribosome-inactivating protein (RIP) possessing multiple pharmacological properties. One of its interesting properties is to inhibit human immunodeficiency virus (HIV) replication but its strong immunogenicity has limited the repeated clinical administration. To map the antigenic determinants and reduce the immunogenicity of TCS, two potential antigenic sites (YFF81–83 and KR173–174) were identified by computer modeling, and then three TCS mutants namely TCS_{YFF81–83ACS}, TCS_{KR173–174CG}, and TCS_YFF-KR were constructed by site-directed mutagenesis. The RI activity and DNase-like activity of the three constructed TCS mutants were similar to natural TCS but with much lower immunogenicity. Results suggested that the two selected sites are all located at or near the antigenic determinants of TCS. In toxicity studies, the LD₅₀ of the three TCS mutants was not different from natural TCS. These findings would be useful in designing a better therapeutic agent for AIDS.Qunxing An and Sanhua Wei equally contribute to this work.     

Homology modeling threedimensional structure of AnxB1 and reducing its immunogenicity by sequence-deleted mutagenesis

AnxB1,a novel annexin previously isolated from Cysticercus cellulose,shows high thrombi affinity and anticoagulant activity in vivo.In order to investigate the relationship between structure and biological function,a predicted three-dimensional(3D)model of AnxB1 was generated by homology modeling.This model contains four homologous internal-domains and the Cα trace of domain Ⅰ,Ⅱ and IV shows high similarity.Based on the structure characterization,four sequence-deleted mutants were constructed and expressed as GST fusion proteins in E.coli.Two of the mutants,GST-M3 and GST-M4 reserved high anticoagulant activity(p＜0.01 vs.GST).Furthermore,compared with the wild type GST-AnxB1,the immunogenicity of GST-M3 and GST-M4 was reduced significantly(p＜0.01)and the molecular weight was lowered to 27 kD and 34 kD,respectively.These observations laid a solid foundation for further study on developing new thrombolytic agents with higher efficiency and lower side effect.     

Function of lymphocytes and macrophages after cryopreservation by procedures for pancreatic islets: potential for reducing tissue immunogenicity

The survival of tissue allografts can be extended by pretreating the tissue to remove the stimulatory leucocytes that populate the graft; with this in mind, we have recently begun to explore a cryobiological approach to modulating tissue immunogenicity by using the differential susceptibility of different cells to freezing injury. The sensitivity of leucocytes to fast cooling rates, which were used in procedures that have been reported to yield viable pancreatic islets of Langerhans, was examined. The loss of both cell numbers and the ability of peripheral blood lymphocytes to undergo blastogenic transformation in response to the mitogen concanavalin A after freezing and thawing was determined over a range of cell concentrations using the "curve-shift" method. Lymphocytes frozen at 1 degree C/min by a control procedure that was designed to yield maximum survival of lymphocytes showed that although there was a decrease in the number of responding cells, there was no reduction in the ability of the recovered cells to undergo blastogenesis when compared with the response of nonfrozen cells. However, cooling at 1 degree C/min in the experimental procedures resulted in both the loss of cells as well as a marked reduction in the ability of recovered cells to incorporate 125I-deoxyuridine into nucleic acid. Cells cooled at either 20 or 75 degrees C/min by any of the procedures showed total inability to respond to stimulation. Lysozyme is produced continuously by all types of macrophages in culture. The large net increase in total lysozyme content of macrophage cultures is therefore a useful measure of the viability of these accessory cells. Cooling at 1 degree C/min by a control, optimized procedure yielded 91% survival of viable peritoneal exudate cells. Cooling at either 1 or 20 degrees C/min in the experimental procedures resulted in 72-75% survival of cells frozen by one method and 33% survival when frozen by an alternative procedure. Negligible recovery of viable cells was obtained after cooling at 75 degree C/min. The preservation protocols employed in this study differ significantly in the variables known to influence the survival of the cells; these include the concentration of cryoprotectant (CPA), the length and temperature of exposure to CPA, the dilution regimen, and the optimum cooling rate for survival of pancreatic islets. This study therefore defines clearly those conditions most likely to effect a depletion of "passenger" lymphoid cells by freezing during the cryopreservation of islets of Langerhans.     

Mechanism of thyroid allograft rejection.

Balb/c thyroids, held in organ culture for 26 days, survive and function as well as isografts for greater than 100 days in CBA recipients. Uncultured allografts are totally rejected by 20 days after transplantation. Prolonged allograft survival can also be achieved by the treatment of donor animals with cyclophosphamide prior to harvesting tissues for transplantation. These allografts do not survive as well as 26 day cultured allografts, but cyclophosphamide pretreatment reduces the culture time required to achieve indefinite survival to 7 days. The provision of an allogeneic (LD) stimulus by thyroid tissue that is I-region incompatible with the host does not facilitate the rejection of a tolerated cultured allograft. However, activation of the host immune system by an uncultured graft syngeneic to a tolerated cultured allograft leads to the chronic rejection of the cultured transplant. The transfer of a tolerated cultured allograft back to its strain of origin induces an acute inflammatory reaction that causes tissue damage within the transplant but does not lead to the total destruction of the tissue.     

Expression of increased immunogenicity by thiol-releasing tumor variants.

Even moderate variations of the extracellular cysteine concentration were previously shown to affect T cell functions in vitro despite high concentrations of cystine. We therefore analyzed the membrane transport activities of T cells for cysteine and cystine, and the role of low molecular weight thiol in T cell-mediated host responses against a T cell tumor in vivo. A series of T cell clones and tumors including the highly malignant lymphoma L5178Y ESb and its strongly immunogenic variant ESb-D was found to express extremely weak transport activity for cystine but strong transport activity for cysteine. However, not all cells showed the expected requirement for cysteine (or 2-mercaptoethanol (2-ME)) in the culture medium. One group of clones and tumors including the malignant ESb-lymphoma did not respond to changes of extracellular cystine concentrations and was strongly thiol dependent. This group released only little acid soluble thiol (cysteine) if grown in cystine-containing cultures. The other T cell lines, in contrast, were able to maintain high intracellular GSH levels and DNA synthesis activity in cystine-containing culture medium without cystein or 2-ME and released substantial amounts of thiol. This group included the immunogenic ESb-D line. Additional thiol-releasing ESb variants were obtained by culturing large numbers of L5178Y ESb tumor cells in cultures without cysteine or 2-ME. All of these ESb variants showed a significantly decreased tumorigenicity and some of them induced cytotoxic and protective host responses even against the malignant ESb parent tumor. Taken together, our experiments suggest that the host response against a tumor may be limited in certain cases by the failure of the stimulator (i.e., the tumor) cell to deliver sufficient amounts of cysteine to the responding T cells.     

Synthesis of tumor associated sialyl-T-glycopeptides and their immunogenicity.

Sialyl-T-glycopeptides were synthesized by solid-phase techniques, using a PEGA resin as the solid support. An appropriately protected building block containing alpha-Neu5Ac-(2 --> 3)-beta-Gal-(1 --> 3)-alpha-GalN3-(1-->) attached to Fmoc-Thr/Ser-OPfp was employed in a solid phase glycopeptide assembly of a 10-mer glycopeptide, using a general Fmoc/OPfp-ester strategy. Reduction of the azido group of the GalN3 residue was effected on solid-phase, using DTT and DBU. After acidolytic cleavage from the resin, the methyl ester of the sialic acid residue and acetyl groups were removed with 30% NaOMe/MeOH in MeOH and water pH 14, at -30 degrees C for 2 h. At this low temperature, the highly basic conditions did not result in any detectable beta-elimination. However, one O-acetyl group, located at the 2-position of the Gal was resistant to hydrolysis. To remove this remaining acetyl group, reaction with hydrazine hydrate in CHCl3 and MeOH at room temperature for 2.5 h was successful. The two target sequences of sialyl-T-glycopeptides were obtained in good yield. In contrast to the the analogs carrying the T-antigen, the Sial-T-glycopeptides were nonimmunogenic, supporting the idea that the sialylation is a method of circumventing the recognition by the immune system.     

Pyrimidine reducing enzymes of rat liver.

Dihydrouracil dehydrogenase (NADP+) (EC 1.3.1.2) was partially purified from the cytosol fraction of rat liver and fractionated by disc gel electrophoresis. A major and minor band were visualized by staining for enzyme activity. The substrate specificity of these bands was investigated. It was found that both bands were two to three times more active with dihydrothymine as substrate than with dihydrouracil in the presence of NADP+ and the optimum pH of 7.4. Mitochondrial fractions containing most of the NADH-dependent uracil reductase of rat liver cells were fractionated by centrifugation in sucrose density gradients. Two procedures involving linear or discontinuous gradients were used. By both, good separation of NADH- and NADPH- dependent reductases was achieved. Marker enzyme studies supported the view that the NADH-dependent enzyme is located principally in mitochondria whereas the NADPH-dependent enzyme is mainly in plasma and endoplasmic reticulum membranes. For the NADH-dependent reductase the apparent Km for thymine at pH 7.4 was 1.39 times that found for uracil whereas for the NADPH-dependent enzyme the apparent Km values were similar for the two substrates at this pH. Dihydrouracil was the principal product isolated by paper chromatography from the reaction mixture containing a partially purified fraction of mitochondria, uracil and NADH at pH 7.4. This fraction also catalyzed the formation of radioactive carbon dioxide from [2-14C]uracil. The proportion of CO2 formed by the mitochondria was about 10% of that formed by the original homogenate.     

Detection of renal allograft rejection by computer.

A computer program incorporating an adaptation of a statistical method, the multiprocess Kalman filter, was used to detect changes in trends of plasma creatinine and urea concentrations. In 28 recipients of renal allografts a definite deterioration in renal function was identified retrospectively on 32 occasions by an experienced renal physician independently of the statistical analysis. The computer identified 31 of these 32 episodes using creatinine and urea results, and 29 using creatinine alone. Dysfunction was identified by the computer significantly earlier (p less than 0.05) than by the clinician and a median of one day earlier (p less than 0.02) than treatment was actually initiated. The computer identified dysfunction on 11 out of 1259 days when the clinician did not suspect rejection. These 11 episodes may have had a pathological importance, though no clinical diagnosis was made. This computer method is useful for immediate analysis of incoming results and for timing events either prospectively or retrospectively.     

Blood transfusion and renal allograft survival.

Thirty-two first renal transplantations with cadaveric allografts were reviewed to see how many of the recipients had received blood transfusions preoperatively. There was a significant difference in transplant survival between patients who had and patients who had not received blood transfusion before transplantation; this difference was entirely due to acute rejection within three months after transplantation in patients who had not received transfusion. Other factors studied had no effect on survival.     

HLA-DRw6 and renal allograft rejection.

HLA-DRw6-positive patients are "high responders" to certain renal allograft antigens. A study was therefore conducted of the outcome of 247 first renal allografts in 74 DRw6-positive and 173 DRw6-negative recipients. The effectiveness of matching for HLA-DR determinants in both groups was also analysed. The one-year graft survival in DRw6-positive patients was 59% as compared with 75% in DRw6-negative recipients (p = 0.012). A striking difference between the two groups was that HLA-DR matching significantly improved renal allograft survival only in the DRw6-positive patients. In those patients the one-year survival of HLA-DR-identical grafts was 95% as compared with only 38% for 2-DR mismatched grafts (p = 0.009). In DRw6-negative patients only a slight beneficial effect of HLA-DR matching was observed (83% versus 72% at one year for the 0-DR and 2-DR mismatched grafts, respectively) (p greater than 0.05). These findings are clear evidence that DRw6-positive patients (about a quarter of the patients on the waiting list of Eurotransplant) should be given HLA-DR-identical kidney transplants only.