Plasmodium Yoelii-infected Mice Research Articles

Studies of murine malaria antigens using monoclonal antibodies. Production, selection, and characterization of antibodies.

A panel of ten monoclonal antibodies made against Plasmodium chabaudi and Plasmodium yoelii infected mouse erythrocytes were used for characterization of antigens present in murine malaria. Screening of the antibodies in ELISA with different fractions of infected erythrocytes revealed both species-specific and fraction-specific monoclonal antibodies (MAbs), but also MAbs cross-reacting between the species. Two MAbs bound normal erythrocyte components. Subcellular localization of the target antigens was studied by immunofluorescence and their molecular identity by immunoblotting after SDS-PAGE. Of the MAbs to P. yoelii, one reacted with a cytoplasmic granule component of 137 k and two others reacted with vacuole-associated antigens of 26 k and 25/70/73 k, respectively. The latter antibodies cross-reacted with P. chabaudi antigens. Of the MAbs to P. chabaudi, all were species specific, one reacting with parasite surface antigens of 79 and 250 k and two with a vacuole-associated antigen of 70 k.

Comparison of fast (one-step) and interrupted slow cooling methods using a range of intracellular and extracellular cryoprotectants for the freeze-preservation of Plasmodium yoelii-infected mouse erythrocytes

Several cryoprotectants were compared using two different cooling procedures for the cryopreservation of both normal and Plasmodium yoelii-infected mouse erythrocytes. Fast cooling to −196 °C by direct plunge into liquid nitrogen followed by rapid thawing in a 37 °C water bath protected uninfected and parasitized erythrocytes against freeze-thaw damage significantly better than an interrupted slow cooling procedure in which cells were frozen to −70 °C at a rate of −1 °C/min followed by storage in liquid nitrogen. Using the former procedure, the highest percentage erythrocyte recoveries (over 85%) and shortest pre-2% parasitaemia times (equivalent to unfrozen cells) in mice challenged with thawed parasitized blood were observed with the intracellular (penetrating) cryoprotectants glycerol or dimethylsulphoxide (Me 2S0). At the concentrations used in this study, the extracellular (non-penetrating) cryoprotectants, hydroxyethylstarch, dextran or polyvinyl pyrrolidone were significantly less effective at protecting against freeze-thaw damage. Some evidence of freeze-thaw damage was obtained in cells fast frozen at low cell density (⩽10 4/ml) in glycerol or Me 2SO. This effect was masked when higher cell densities (10 7 to 10 8/ml) were used. Addition of the anti-oxidant and membrane stabilizing molecules, vitamin E, sodium selenite or selenomethionine to infected erythrocytes just before and during cryopreservation, did not improve, and in one case inhibited, subsequent development in challenged mice.

Inhibitory effect of rhodamine 123 on the growth of the rodent malaria parasite, Plasmodium yoelii.

The effect of the cationic permeant fluorescent dye, rhodamine 123 (R123), on the in vivo growth of Plasmodium yoelii was examined. Plasmodium yoelii-infected mouse erythrocytes were incubated in vitro with R123 and injected intravenously into mice. Examination of daily parasitemias showed that R123 delayed parasite growth whereas rhodamine 110, a neutral compound, and fluorescein, a negatively charged fluorescent dye, did not. Infected erythrocytes treated with R123 were not cleared from the circulation even 7 h after injection. Quantitation of cell-associated R123 by spectrophotometry revealed that infected cells with increased levels of R123 considerably prolonged the 2% prepatent period, the time required for the parasite to develop a 2% parasitemia. Degenerating parasites within and outside the host erythrocytes were observed on day 1 of infection in the mice. Thus it follows that R123, which accumulated in infected erythrocytes, inhibits the growth of P. yoelii; moreover, when R123-labeled infected erythrocytes were treated with 1-10 microM carbonylcyanide m-chlorophenylhydrazone (CCCP), a proton ionophore, to release R123 from the cells, the inhibitory effect on the growth rate of P. yoelii was partially reversed.

Staining of Plasmodium yoelii-infected mouse erythrocytes with the fluorescent dye rhodamine 123.

The cationic permeant fluorescent dye rhodamine 123 (R123) was used to stain Plasmodium yoelii-infected mouse erythrocytes. Fluorescence microscopic observations demonstrated that the parasite, but not the matrix of the infected erythrocyte, accumulated the dye. Differences in fluorescence intensity could not be found at the various developmental stages of the parasite; however, quantitation of the cell-associated dye revealed an increase in R123 uptake with parasite development. The retention of the parasite-associated dye, as measured by fluorescence microscopy and spectrophotometry after extraction of R123 with butanol, was markedly reduced by treatment of the infected erythrocytes with a proton ionophore, carbonyl cyanide m-chlorophenylhydrazone (CCCP), and an inhibitor of proton ATPase, dicyclohexylcarbodiimide (DCCD). These results indicate that the accumulation and retention of R123 in P. yoelii reflect the parasite membrane potential and suggest that the parasite plasma membrane has a membrane potential-generating proton pump.

Hypergammaglobulinemia and Erythrocyte Autoantibody Complicate Enzyme Immunoassay of Antimalarial Antibody

Enzyme immunoassay of specific antimalarial antibody in sera from Plasmodium yoelii-infected mice was complicated by hypergammaglobulinemia and erythrocyte (RBC) autoantibody. Malarious serum had higher immunoglobulin levels which resulted in a substantial nonspecific adsorption of antibody to antigen uncoated microliter wells even in the presence of a surfactant. Since it was possible that some of the antibody binding to solid phase-bound malarial antigen was also due to nonspecific adsorption, the antibody binding of nonimmune serum was compared to that of malarial immune serum diluted to an equivalent immunoglobulin concentration. This allowed differentiation of specific antimalarial antibody binding from nonspecific adsorption of immunoglobulin. Although malarial antigen was partially purified from intraerythrocytic parasites, it bound a significant amount of rabbit anti-mouse RBC antibody, indicating the presence of residual RBC antigens. Serum from immune mice, but not nonimmune mouse serum, also showed substantial binding to RBC stroma antigen, suggesting that a component of the observed antibody binding to malarial antigen was anti-RBC antibody. Hypergammaglobulinemia and the induction of RBC autoantibody may both be related to the polyclonal activating property of the malaria infected mice necessitates a more conservative interpretation of specific antimalarial antibody levels until a more purified malarial antigen is available.

Antibody response in vitro of spleen cells from Plasmodium yoelii-infected mice

The primary antibody response to sheep erythrocytes and dinitrophenylated Ficoll of spleen cells from mice infected with Plasmodium yoelii was studied in vitro. The response to sheep erythrocytes was enhanced between 2 and 4 days after infection and depressed at later intervals. Cell fractionation experiments carried out at the time of immunosuppression indicated a defect of macrophage function. At the same time the response to dinitrophenylated Ficoll was normal.