Maurice Marchand

A limiting dilution assay for quantifying Leishmania major in tissues of infected mice.

Summary A limiting dilution assay for the quantification of Leishmania major in infected mouse tissue was developed. The assay was found to be both sensitive and reliable, and, due to its design, could be scored either visually or following the incorporation of 3H‐thymidine by the growing parasites. Results are presented in which the assay was employed to enumerate L. major in the tissues of susceptible (BALB/c) and resistant (CBA) mice at intervals after infection with L. major. It was found that parasites could be detected at the site of injection with L. major as early as 3 days after infection. By day 8, a substantial increase in the number of parasites at the lesion site had occurred in both strains of mice. Subsequently, whereas the number of parasites decreased in the lesions of CBA mice, their number steadily increased in the lesions of BALB/c mice. Parasites were detected in lymph nodes draining the lesion site in both BALB/c and CBA mice by 28 days after infection. Interestingly, a low number of L. major was found in the lymph nodes of CBA mice at 100 days after infection, a time when no parasites could be detected at the lesion site. Previous results from this laboratory have demonstrated that the adoptive transfer of L. major‐specific L3T4‐positive T‐cell populations exacerbated cutaneous lesions induced by L. major in BALB/c mice. Experiments presented here indicate that the adoptive transfer of L. major‐specific T‐cells also exacerbated cutaneous leishmaniasis in CBA mice. Using the sensitive limiting dilution assay presently described, it was found that this unexpected exacerbative effect of L. major‐specific T‐cells on lesion development was accompanied by a substantial increase in the number of parasites in the lesions of the adoptively transferred mice.

Complete nucleotide sequence coding for rat liver 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase derived from a cDNA clone

cDNA clones for 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase were isolated from rat liver expression libraries in λgt11 by antibody, oligonucleotide, and cDNA screening. One 1860 bp long clone contained a full‐length nucleotide sequence coding for the 470 amino acids of each of the two identical subunits of the bifunctional enzyme. This clone also contained untranslated sequences, one 173 bp long upstream from the ATG start codon and one 271 bp long downstream from the TGA stop codon. The clone was terminated by a poly(A) tail of 29 nucleotides.

Trypanosoma cruzi variants with reduced virulence obtained by mutagenesis

Summary Previous reports have indicated that by mutagen treatment of mouse tumour cells in vitro it is possible to obtain at high frequency stable tumour cell variants that fail to form tumours in syngeneic mice because of increased immunogenicity. By analogy with these tumour cell variants, we examined whether variants with reduced virulence could be obtained by mutagen treatment of trypomastigotes derived from a Trypanosoma cruzi strain that was adapted to culture and produced lethal infections in DBA/2 mice at a dose of 5 × 104 parasites. A very large frequency of T. cruzi clones were obtained that failed to provoke an acute lethal infection after injection of 5 × 105 parasites. Most of these variants with reduced virulence (vir‐) multiplied actively in normal mice until day 8 after injection. After that time the parasitaemia decreased gradually. For most variants a low level of residual parasitaemia persisted for more than 100 days. Unlike the situation encountered with mouse tumour cell variants it was not possible to demonstrate the presence of new antigens on the T. cruzi vir‐ variants. However, these variants seemed to have acquired an increased immunogenicity since they provoked the rejection of virulent parasites injected concomitantly. Mice that had been immunized with living vir‐ clones were protected against a challenge with a virulent clone derived from the original parasite population.