We previously identified a Plasmodiumfalciparum trophozoite cysteine proteinase (TCP) and hypothesized that it is required for the degradation of host hemoglobin by intraerythrocytic malaria parasites. To test this hypothesis and to evaluate TCP as a chemotherapeutic target, we examined the antimalarial effects of a panel of peptide fluoromethyl ketone proteinase inhibitors. For each inhibitor, effectiveness at inhibiting the activity of TCP correlated with effectiveness at both blocking hemoglobin degradation and killing cultured parasites. Benzyloxycarbonyl (Z)-Phe-Arg-CH2F, the most potent inhibitor, inhibited TCP at picomolar concentrations and blocked hemoglobin degradation and killed parasites at nanomolar concentrations. Micromolar concentrations of the inhibitor were nontoxic to cultured mammalian cells. These results support the hypothesis that TCP is a necessary hemoglobinase and suggest that it is a promising chemotherapeutic target. (J. Clin. Invest.

Antigenic variation of infectious organisms is a major factor in evasion of the host immune response. However, there has been no definitive demonstration of this phenomenon in the malaria parasite Plasmodium falciparum. In this study, cloned parasites were examined serologically and biochemically for the expression of erythrocyte surface antigens. A cloned line of P. fakciparum gave rise to progeny that expressed antigenically distinct forms of an erythrocyte surface antigen but were otherwise identical. This demonstrates that antigenic differences on the surface of P. fakiparum-infected erythrocytes can arise by antigenic variation of clonal parasite populations. The antigenic differences were shown to result from antigenic variation of the parasite-encoded protein, the P. falciparum erythrocyte membrane protein 1.

Erythrocytes infected with trophozoites and schizonts ofthe human malaria parasite Plasmodium falciparum develop surface protrusions (knobs) (1) by which the infected erythrocytes (IRBCs)t adhere specifically to venular endothelium in vivo (2, 3) and to human endothelial cells (4) and some lines of melanoma cells (5) in vitro. Cytoadherence between IRBCs and venular endothelium has a critical role in the pathogenesis of falciparum malaria, since it permits the mature parasites to evade spleen-dependent immune mechanisms (6) and since the sequestered parasites may occlude blood flow, as seen in cerebral malaria (7). Antibody in immune serum reacts with a strain-specific parasite-determined antigen on IRBCs and inhibits cytoadherence in vitro (8) and in vivo (9). The inhibition of cytoadherence by antibody may protect the host from the clinical consequences of falciparum malaria.Both cytoadherence and the IRBC surface antigen were shown to be destroyed by incubating IRBCs with proteases (9, 10), suggesting that the two properties are determined by proteins on the IRBC surface. In addition, the cytoadherence phenotype ofP.falciparum parasites and the expression ofthe IRBC surface antigen were modulated together by the spleen in a monkey model of falciparum malaria (9, 10), suggesting that the two properties are linked and perhaps determined by the same protein. A family ofpotential cytoadherence proteins was identified in studies with IRBCs from Aotus monkeys (11) . The members ofthe protein family differed in antigenicity and molecular size among strains ofP.falciparum, but had in common several biochemical properties, including their accessibility to surface radioiodination, detergent solubility, and cleavage by the same concentration of trypsin that inhibited cytoadherence (11) .

We analyzed CD4+ T helper responses to wild-type (wt) and mutated (mut) p53 protein in normal and tumor-bearing mice. In normal mice, we observed that although some self-p53 determinants induced negative selection of p53-reactive CD4+ T cells, other p53 determinants (cryptic) were immunogenic. Next, BALB/c mice were inoculated with J774 syngeneic tumor cell line expressing mut p53. BALB/c tumor-bearing mice mounted potent CD4+ T cell responses to two formerly cryptic peptides on self-p53. This response was characterized by massive production of IL-5, a Th2-type lymphokine. Interestingly, we found that T cell response was induced by different p53 peptides depending upon the stage of cancer. Mut p53 gene was shown to contain a single mutation resulting in the substitution of a tyrosine by a histidine at position 231 of the protein. Two peptides corresponding to wt and mutated sequences of this region were synthesized. Both peptides bound to the MHC class II-presenting molecule (Ed) with similar affinities. However, only mut p53.225–239 induced T cell responses in normal BALB/c mice, a result strongly suggesting that high-affinity wt p53.225–239 autoreactive T cells had been eliminated in these mice. Surprisingly, CD4+ T cell responses to both mut and wt p53.225–239 peptides were recorded in J774 tumor-bearing mice, a phenomenon attributed to the recruitment of low-avidity p53.225–239 self-reactive T cells.

Female murine mammary tumor virus (MMTV)/neu transgenic mice, expressing a wild-type rat neu oncogene driven by an MMTV promoter, develop focal mammary adenocarcinomas that are pathologically very similar to human breast tumors. Two new cell lines were established from a mammary tumor that arose in a female MMTV/neu transgenic mouse. One of these lines, mammary carcinoma from Neu transgenic mouse A (MCNeuA), has an epithelial morphology, is cytokeratin positive, and expresses high levels of the neu transgene. Karyotyping and comparative genomic hybridization analyses demonstrated genomic alterations in the MCNeuA cell line. The other line, N202Fb3, has a fibroblast morphology, is cytokeratin negative, and expresses the neu transgene at a very low level. This cell line also expresses smooth muscle alpha-actin, suggesting that it is a myofibroblast line. The MCNeuA cell line is tumorigenic when injected into syngeneic MMTV/neu transgenic mice, with an in vivo doubling time of about 14 d. The rationale for establishing this tumor cell line was to provide a tumor transplantation system for rapidly assessing immunotherapeutic interventions before testing in the more cumbersome model of spontaneous tumor development in the MMTV/neu transgenic mice. Mice immunized with a Neu extracellular domain protein vaccine were protected against a subsequent inoculation of MCNeuA cells, indicating that this cell line will be useful for evaluating cancer vaccine strategies. This tumor cell line may also prove useful in studying the biological properties of the neu oncogene and its role in the malignant process. In addition, the tumor-derived fibroblast line may be useful for studying tumor-stromal cell interactions.