Human immune response in Plasmodium falciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf155/RESA antigen induce production of parasite-specific antibodies in vitro.

Autologous cell mixtures containing T cells, B cells, and adherent accessory cells from individuals primed to the malaria parasite Plasmodium falciparum by repeated natural infections were investigated for induction of Ig and antibody secretion in vitro. In vitro activation of cell cultures with two synthetic peptides corresponding to immunodominant T cell epitopes of the merozoite Ag ring-infected erythrocyte surface Ag (Mr 155,000) (Pf155/RESA), one from its carboxyl-terminal repeat and one from its nonrepeated amino-terminal region, gave rise to significant IgG secretion. Supernatants from lymphocyte cultures activated with either one of these peptides contained antibodies reacting with P. falciparum Ag in immunofluorescence assays and with Pf155/RESA peptides in a slot blot assay. No anti-P. falciparum antibodies were induced in the medium controls by lymphocyte stimulation with either tetanus toxoid or PWM. Induction in vitro of anti-Pf155/RESA antibodies was correlated with the presence of such antibodies in the sera of the lymphocyte donors, suggesting that the induction of antibody secretion reflected a secondary response in vitro of in vivo primed cells. Inspection of antibody profiles in individual donors revealed that the peptide corresponding to a sequence in the 3' repeat region induced anti-Pf155/RESA peptide antibodies reacting with identical or related and cross-reacting sequences in the 3' or 5' repeat region of the molecule. In contrast, the peptide corresponding to a nonrepeated T cell epitope in the amino terminus of the molecule only induced antibodies to an immunodominant amino-terminal B cell epitope partly overlapping with the T cell reactive sequence. Similar findings were made in the lymphocyte donors' plasma, frequently displaying significant correlations between antibody reactivities to the repeat peptides but not between these reactivities and those to the amino-terminal peptide. The marked specificity of this antibody formation in vitro suggests an underlying process of cognate recognition involving Ag-specific T and B cells reacting with different segments of the inducer peptide. The present experimental system should be well suited for identification of Th epitopes capable of inducing the production of antibodies of defined specificity in the human system.     

T and B cell responses of Plasmodium falciparum malaria-immune individuals to synthetic peptides corresponding to sequences in different regions of the P. falciparum antigen Pf155/RESA

The C-terminal (3') amino acid repeat region of the Plasmodium falciparum Ag Pf155/RESA, a vaccine candidate, contains immunodominant T and B cell epitopes. In order to identify additional T cell epitopes in the molecule, synthetic peptides corresponding to the centrally (5') located repeat region, as well as to four nonrepeated regions, were synthesized. T cells from 46 P. falciparum-primed individuals living in a holoendemic area of The Gambia where malaria transmission is seasonal were tested for their responsiveness to the peptides by thymidine incorporation and IFN-gamma release. There was a considerable variation in the response to different peptides. Proliferation and IFN-gamma release were not correlated in individual donors, underlining the importance of measuring both activities when screening donor populations for total T cell responsiveness to a given Ag. Whereas 72% of the donors responded with proliferation and/or IFN-gamma release to the intact protein the mean % responders to the peptides was 40%. The most frequent responses (up to 60%) were induced with peptides from the 3'- and 5'-repeat region of the protein. Analysis of some closely related sequences in the 3'-repeat region indicated that they contained at least two epitopes that were either distinct or cross-reacting in different donors, suggesting difference in the genetic control of these responses. When the same peptides were investigated for reactivity with antibodies, the best T cell inducing sequences also displayed the best antibody reactivities. However, in individual donors, T and B cell responses were not correlated. T cell responses were shown to persist after a period with no P. falciparum transmission, whereas antibody concentrations tended to decrease, suggesting differences in the requirements of boosting at the T and B cell levels, respectively.     

By-products as an aid in residue identification during peptide sequence analysis with dimethylaminoazobenzene isothiocyanate

4-N,N-Dimethylaminoazobenzene 4-isothiocyanate degradations permit sensitive and fast manual sequence analysis, but assignments of some residues are difficult. Hydrophobic residues, especially leucine and isoleucine, are badly resolved on polyamide thin-layer chromatography. Differently colored by-products have been described before for a few labile residues, but it is now shown that most residues can give rise to characteristic by-products. These have different colors and chromatographic properties, depending on the nature of the parent residue. Thus, two to three sets of spots characterize each residue, giving multiple identification with increased reliability. Although variable and dependent on chemicals and conditions, by-products are often prominent after conversion with 50% trifluoroacetic acid, and can be utilized to improve the identifications.Abbreviations used are DABITC: 4-N,N-dimethylaminoazobenzene 4-isothiocyanate; DABTH: dimethylaminoazobenzene thiohydantoin; DABTC: dimethylaminoazobenzene thiocarbamoyl; DABTZ: dimethylaminoazobenzene thiazolinone; dansyl: 5-dimethylaminonaphthalene 1-sulfonyl; HPLC: high-performance liquid chromatography; PTH: phenylthiohydantoin; TFA: trifluoroacetic acid.     

Soluble and membrane-bound enzyme-active antigens of rat-liver lysosomes.

Secondary lysosomes were isolated from rat liver and separated into a soluble and a membrane fraction. Plasma membranes and microsomes were also isolated and antisera against the various fractions were prepared in rabbits. Lysosomal content and detergent-solubilized membrane fractions were analysed in two-dimensional immunoelectrophoresis (crossed immunoelectrophoresis). The immunoprecipitates were stained by histochemical procedures for different enzyme activities such as phosphatases, non-specific esterase, arylsulphatase, glycosidases and L-leucyl-beta-naphthylamidase. When lysosomal content was tested against its corresponding antiserum, 17 different precipitates could be seen. Most of the enzyme activities tested were shown to reside separately in one or a few precipitates each. In contrast, when the membrane extracts were investigated, a more polymorphic pattern of enzyme-active precipitates appeared. Thus, when lysosomal membrane extracts were reacted with homologous antiserum 11 precipitates with acid phosphatase activity were obtained. Several of the antigens were electrophoretically different and immunologically non-identical. As expected from the biology of secondary lysosomes, many of their antigens were also found in microsomes and/or plasma membranes, but several antigens unique for lysosomes were detected concomitantly. Closer analysis of these results indicated that several seemingly identical enzyme-active proteins occurred both in soluble and membrane-associated forms. However, while many of the membrane antigens expressed 2-4 different enzyme activities, only one activity was detected in individual precipitates of the lysosomal content. Thus, acid phosphatase activity was found together with esterase activity in three membrane-associated antigens. The precipitates formed by two of these also stained for arylsulphatase and nucleoside tri-, di- and monophosphatase activities. L-Leucyl-beta-naphthylamidase activity was found in one additional acid-phosphatase-active precipitate.     

Cytological observations on the cytotoxic interaction between lymphocytes and antibody-coated monolayer cells

Chang cells in monolayers were killed when exposed to highly purified human peripheral blood lymphocytes in the absence of complement and in the presence of a concentration of heat inactivated rabbit anti-Chang cell serum (ACS) chosen for inability by itself to cause cytolysis. As evident by plaque formation, the cytotoxic reaction was confined to areas of the cultures covered with lymphocytes. In the presence of ACS, lymphocytes attached to and infiltrated the monolayers. Uropod formation indicated activation of the lymphocytes. Close contact was established between effector and target cells. The cytotoxic reaction was not accompanied by protracted cytopathological changes but led to detachment and rapid lysis of the target cells. Horse anti-thymocyte globulin, added to the cultures, completely inhibited plaque formation and lymphocyte infiltration.     

Interactions between human lymphocytes and paramyxovirus-infected cells: adsorption and cytotoxicity.

The capacity of human lymphocytes to adhere to paramyxovirus-infected monolayers and their capacity to kill paramyxovirus-infected cells was investigated. A large fraction of human lymphocytes was found to adhere firmly to the paramyxovirus-infected monolayers. Predsorption of lymphocytes on mumps virus-infected cells impaired their adsorption to a second cell monolayer of the same type. The cytotoxic activity of lymphocytes against mumps virus-infected cells was also reduced after predsorption on mumps virus- or Newcastle disease virus-infected (NDV) cell monolayers. Exposure of lymphocytes to trypsin did not significantly decrease either adsorption or cytotoxicity. Pretreatment of lymphocytes with neuraminidase (NANase) partly inhibited adsorption whereas cytotoxicity was not decreased. Cell fractionation experiments after rosetting of the lymphocytes with sheep erythrocytes (E) indicated that T cells were equally or better adsorbed than "non-T" cells. Taken together with previous experiments which showed that the majority of T lymphocytes are not cytotoxic against mumps virus-infected cells these results suggest that adherence of lymphocytes to infected cells and cytotoxicity may be unrelated phenomena.     

Effect of different dietary fat sources on production traits, lipid peroxide status and on the glutathione redox system in African catfish [Clarias gariepinus (Burchell)] fingerlings. Short communication

Lipids are used to provide the energy to cover the metabolic needs and to provide essential fatty acids, which are important for membrane function [12]. Fats may contain high level of long chain polyunsaturated fatty acids, which are prone to peroxidation [8] and will interact with the antioxidant defense system [1]. There is contradiction in the literature about whether the intake of fish oil enhance [7] or deplete [4] tissue antioxidant defenses and the glutathione redox system in different organisms. The aim of the present study was to examine the effects of different dietary oils on parameters of the lipid peroxide state and the glutathione redox system in C. gariepinus fingerlings.     

Insertion of spliceosomal introns in proto-splice sites: the case of secretory signal peptides

Analysis of the exon-intron structures of 2208 human genes has revealed that there is a statistically highly significant excess of phase 1 introns in the vicinity of the signal peptide cleavage sites. It is suggested that amino acid sequences surrounding signal peptide cleavage sites are significantly enriched in phase 1 proto-splice sites and this has favored insertion of spliceosomal introns in these sites.