L. Pereira Da Silva

Genetics and physiology of defective lysogeny in K12 (λ): Studies of early mutants

Abstract The properties of a number of strains of K12 (λ) which are defective in their ability to form lambda DNA after induction have been examined by genetic, physiological, and biochemical tests. The strains fall into four classes, and include mutants in cistrons N, O, and P, previously identified by Campbell (1961), and a fourth new class, called x, in a region between C I and C II . Mutants in each of the classes can be distinguished genetically, by their relative ability to form the λ-exonuclease, and by their tendency to show curing of their prophage after induction. Mutants in the P and O cistrons form normal levels of λ-exonuclease, whereas mutants in the N cistron form very low levels, and mutants in the x region, relatively high levels of this enzyme. Curing is very efficient for mutants in the x region, less efficient for mutants in the O and P cistrons, and is seldom observed with mutants in the N cistron. These patterns of curing are reflected in the curves for UV survival of the strains carrying defective mutations in the various cistrons. The significance of these findings in respect to the early events in phage development are discussed.

Protective antibodies against erythrocytic stages of Plasmodium falciparum in experimental infection of the squirrel monkey, Saimiri sciureus

Serum and ascitic fluid from squirrel monkeys (Saimiri sciureus) inoculated with erythrocytic stages of Plasmodium falciparum were collected at different periods of the infection. Protection against P. falciparum was achieved by passive transfer of the sera or fluid recovered from animals after spontaneous or drug‐induced cure. Purified immunoglobulins from the ascitic fluid also conferred protection. In contrast, protective antibodies directed against erythrocytic stages of P. falciparum could never be demonstrated during the acute phase of infection in spite of the high titres of malarial antibodies detected by immunofluorescence. The comparative immunochemical analysis of antigens recognized by protective and non‐protective antibodies revealed quantitative differences which may be of use for the identification of antigens inducing protection.

Cloning and expression of genomic DNA sequences coding for putative erythrocyte membrane-associated antigens of Plasmodium falciparum.

Genomic DNA fragments of Plasmodium falciparum generated by mung bean nuclease digestion were cloned in the lambda expression vector lambda JK2. The resulting library was screened with a rabbit antiserum raised against purified membranes of P. falciparum-infected erythrocytes and with a serum pool from immune humans from an endemic area of Liberia. Positive clones were rescreened with a series of human and monkey sera. Twelve selected clones were analysed in detail. Four of them corresponded to already described membrane-associated P. falciparum antigens. The other positive clones contained inserts which, according to the nucleotide sequence, Southern blot analysis and immunological characteristics, correspond to so far unknown antigens.

Glycolipid anchorage of plasmodium falciparum surface antigens

Human red blood cells (RBC) were infected with the malarial parasite Plasmodium falciparum, the anchoring of schizont proteins to RBC membranes by glycoinositol phospholipids was demonstrated by three criteria: (1) metabolic incorporation of 3H-ethanolamine and 3H-myristate into the protein; (2) release of 35S-methionine-labelled protein into the supernatant after incubation with phosphatidylinositol-specific phospholipase C; and (3) the exposure of a glycoinositol phosphate epitope on the methionine-labelled protein following phospholipase C cleavage. Labelled proteins were analysed by immunoprecipitation, polyacrylamide gel electrophoresis in sodium dodecylsulphate and gel fluorography. Several candidate proteins were observed when each criteria was investigated. Among these, 3 proteins which met all three criteria were identified by immunoprecipitation with monospecific sera or monoclonal antibodies. These included 3 possible vaccine candidates, the p190 major surface antigen, the p76 serine protease and the p71 protein which is thought to be a member of the family of heat-shock Hsp70 proteins.

Phenotypic suppression of morphogenetic mutants of Dictyostelium discoideum

Abstract The effects of cAMP pulses on the capacity of 15 aggregateless mutants to differentiate and construct fruiting bodies are compared to those obtained when mutant cells are starved with wild-type amoebae. Mutant strains are classified into three main groups depending upon the degree to which their phenotypic defects can be corrected. These data extend studies published earlier [Darmon, M., Brachet, P., and Pereira da Silva, L. (1975). Chemotactic signals induce cell differentiation in Dictyostelium discoideum. Proc. Nat. Acad. Sci. USA 72, 3163–3166; Pereira da Silva, L., Darmon, M., Brachet, P., Klein, C., and Barrand, P. (1975). Induction of cell differentiation by the chemotactic signal in Dictyostelium discoideum. In “Proceedings of the Tenth FEBS Meeting,” pp. 269–276]. (1) Only one mutant was unresponsive both to cAMP pulses and to the presence of wild-type amoebae and did not display any of the properties of differentiated cells. (2) Following treatment with cAMP pulses, 11 mutants developed certain properties of aggregation-competent amoebae. They increased their levels of cellular phosphodiesterase, showed an enhanced chemotactic sensitivity to cAMP, and established specific cell contacts. None of these amoebae could differentiate further. They did co-aggregate to some extent with wild-type cells, but failed to differentiate into spores. Rather, mutant cells were excluded from the pseudoplasmodium during the process of morphogenesis of the fruiting body. (3) In contrast, the aggregateless phenotype of three mutants was fully corrected by both cAMP pulses and the presence of wild-type cells. These findings are discussed on the basis of a relationship between the chemotactic signal and cell differentiation.

Flow cytometry detection of surface antigens on fresh, unfixed red blood cells infected by Plasmodium falciparum

The immunofluorescence detection of parasite-specific antigens on the surface of red blood cells infected by Plasmodium falciparum parasites is usually performed by visual detection under a fluorescence microscope. We describe here a technique permitting the analysis of surface immunofluorescence labelling by flow cytometry. Infected red blood cells are selected on the basis of their parasitic DNA and RNA content by Hoechst and Thiazole Orange vital dyes. Cytometric analysis of these labels, as well as general erythrocyte characteristics assessed by analysis of forward and side scatter allows the selection of viable intact infected erythrocytes from other blood cells. The integrity of these selected erythrocytes was confirmed by the absence of labelling with antibodies directed against internal components such as spectrin. This technique permits the detection of specific surface immunofluorescence staining on red blood cells infected with mature stages of P. falciparum by antibodies in sera from hyperimmune Saimiri monkeys. Using Thiazole Orange dye for detection of parasitised cells, this analysis was performed on a FACSscan apparatus equipped with a single laser.

Antibodies from immune African donors with a protective effect in Plasmodium falciparum human infection are also able to control asexual blood forms of the parasite in Saimiri monkeys

We have recently shown that an IgG preparation obtained from immune African donors was able to control Plasmodium falciparum multiplication in the blood of Thai patients, but had no inhibitory activity against the parasite in vitro. The same IgG preparation was passively transferred to Saimiri monkeys acutely infected with two different strains of P. falciparum, one of African origin and the other from French Guyana. A dose-dependent in vivo inhibition of parasite development was observed with both strains. The results justify the use of Saimiri monkeys for malaria vaccine trials and permit the introduction of new techniques for screening of candidate antigens for vaccines.

Malaria proteases and red blood cell invasion

Studies of malaria proteases have focused on two general groups, corresponding to activities specific to malaria parasites: (1) proteases involved in hemoglobin degradation which are active in the food vacuole and which exhibit optimal activity at low pH; and (2) proteases specific to schizonts and/or merozoites which are involved in merozoite maturation and red blood cell invasion and which exhibit optimal activity at neutral pH. In this paper, Catherine Braun Breton and Luis H. Pereira da Silva will focus on those activities necessary for the release of infectious merozoites and the entry of the parasite into its host cell.

Uptake and killing of Leishmania mexicana amazonensis amastigotes by human skin fibroblasts.

This paper describes the in vitro infection of human established fibroblast lines by Leishmania mexicana amazonensis amastigotes. Intracellular parasites were located within vacuoles. The proportion of infected cells reached a peak of about 50% on Days 2 or 3, and decreased to almost 0 on Days 6 or 8. Transmission electron microscopy was used to document different stages of the degeneration of L. m. amazonensis amastigotes on Days 4 and 5. The proportion of infected fibroblasts decreased in both the control and the irradiated cells, indicating that dilution of the parasites by cell multiplication was not the main factor in the observed decrease of the infection with time. This conclusion was also supported by studies with fibroblasts prelabelled with 3H thymidine. In these studies, total cell associated radioactivity as well as the total and TCA precipitable radioactivity of the culture medium were similar for infected and non-infected fibroblasts during the course of infection. The abortive infection of fibroblasts by L. m. amazonensis provides a potentially useful model for the study of the relationship between host cells and intracellular parasites.

Antibody response against Plasmodium falciparum exoantigens and somatic antigens: A longitudinal survey in a rural community in Rondônia, Western Brazilian Amazon

Three clinical and sero-epidemiological cross-sectional surveys involving 50 subjects were performed at six-month intervals in Urupá, a rural community characterized by unstable malaria transmission, situated in Rondônia State, Western Brazilian Amazon. Between the surveys, a clinically and parasitologically passive surveillance was established in this community and 48 malaria attacks (28 due to Plasmodium falciparum and 20 due to Plasmodium vivax) were recorded in this cohort of 50 subjects. Serum samples were collected at each survey and tested by enzyme immunoassay (ELISA) for IgG, IgG subclass and IgM antibodies against P. falciparum exoantigens isolated from culture supernatants and detergent-soluble somatic antigens. As expected, both anti-malarial IgG and IgM antibody titres were shown to rise after a malaria outbreak observed during the follow-up period. Nevertheless, in marked contrast with the profile of anti-malarial IgG subclasses described for semi-immune Africans, in this Amazonian community IgG2 antibodies (that are non-cytophilic) against both antigens were shown to predominate over other IgG subclasses. Such overall predominance of IgG2 subclass titres was statistically significant concerning exoantigens, but was of borderline significance in relation to IgG1 antibodies against somatic antigens (p = 0.052). Moreover, highly variable patterns of boosting were observed in antibody responses against both antigens among the patients who suffered P. falciparum malaria attack during the study.