Biomedical subjects

P Perlmann

Publications and source records attributed to P Perlmann.

At least 91 records · Page 5Linked to original sources

PubMed1990

Pf155/RESA antigen is localized in dense granules of Plasmodium falciparum merozoites.

Immunoelectron microscopy demonstrated the presence of Pf155/RESA in dense granules of Plasmodium falciparum merozoites rather than in micronemes as previously suggested. Since the dense granules are released after the merozoite enters the parasitophorous vacuole, the role of Pf155/RESA in invasion and subsequent steps of parasite development may differ from that of a molecule located in the micronemes.

Animals↗

PubMed1990

Plasmodium chabaudi: polymorphic and nonpolymorphic epitopes of the antigen Pch105/RESA.

The localization in the erythrocyte membrane of Pch105/RESA, the ring stage-infected erythrocyte surface antigen of Plasmodium chabaudi, the proposed analog to the vaccine candidate Pf155/RESA in P. falciparum, is here confirmed by the use of the immunogold technique in electron microscopy. Furthermore, a number of monoclonal antibodies to other P. chabaudi erythrocyte membrane antigens in the same molecular weight range as Pch105 were compared in different test systems. Data from immunoblotting of native and recombinant antigen as well as an inhibition ELISA indicate that Pch105 is identical to Pc96 and two other described antigens of 105 and 110 kDa. Pch105 could also be shown to have polymorphic epitopes, varying between different strains of P. chabaudi, without impact on the molecular weight.

Animals↗

PubMed1990

Characterization of regulatory T cell responses to defined immunodominant T cell epitopes of the Plasmodium falciparum antigen Pf155/RESA.

Several immunodominant B and T cell epitopes of the P. falciparum blood stage antigen Pf155/RESA, a vaccine candidate, are located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. Here we have attempted to functionally analyze human T cell responses to some of the T cell epitopes. For this purpose short synthetic peptides corresponding to these epitopes were used to study the induction of in vitro expression of IL-4 mRNA, IFN-gamma secretion, proliferation and B cell help for antibody production. In individual malaria immune donors these different T cell activities were not correlated. The findings emphasize the importance of examining multiple parameters of T cell activation when estimating the total proportion of individuals responding to a defined antigen. IL-4 mRNA was expressed in activated T cells of donors who had elevated serum concentrations of antibodies to the peptide used for T cell activation. These results suggest the involvement of IL-4 producing T helper cells in the induction of Pf155/RESA specific antibody production in individuals in which immunity has been induced by natural infection. Taken together, these findings also suggest that functionally distinct CD4+ T cells occur in humans similarly to what has been described in mice. In further experiments, we have also attempted to establish MHC class II restriction of the immune response to these epitopes at the level of the donor populations. When studying monozygotic twins, antibody responses to Pf155/RESA derived peptides and some of the T cell responses could be paired within the twin pairs, indicating a genetic regulation of their B cell responses. Whether or not this regulation reflects MHC class II restriction, or other factors needs to be elucidated.

Amino Acid Sequence↗

PubMed1990

Production by activated human T cells of interleukin 4 but not interferon-gamma is associated with elevated levels of serum antibodies to activating malaria antigens.

T cells play a crucial role in antibody-mediated and antibody-independent immunity against Plasmodium falciparum malaria. Therefore, a vaccine immunogen should include parasite-derived B- and T-cell epitopes capable of giving rise to protective responses in both systems. The P. falciparum antigen Pf155/ring-infected erythrocyte surface antigen (RESA), a vaccine candidate, contains immunodominant T- and B-cell epitopes located in the central (5') and C-terminal (3') invariant repeat regions of the molecule. To relate Pf155/RESA-peptide-specific responses of T cells to function, T cells from P. falciparum immune donors were activated with peptides corresponding to these immunodominant regions. Activation was measured as induction of interferon-gamma secretion, T-cell proliferation (DNA synthesis), or transcription and translation of interleukin 4 (IL-4) mRNA. Peptides from both regions were shown to induce interferon-gamma, IL-4, proliferation, or any combination. In individual donors, there was no correlation between these different activities. Rather, they were negatively correlated, demonstrating the importance of examining multiple parameters of T-cell activation when estimating the proportion of individuals responding to a given epitope. However, IL-4 mRNA and intracellular IL-4 could be induced in T cells of donors who had elevated concentrations of serum antibodies to the same peptide that was used for T-cell activation. These results suggest that a causal relationship exists between the activation of IL-4-producing T-cell subsets and production of the anti-Pf155/RESA-specific antibodies in individuals in which immunity has been induced by natural infection. This finding has implications that should be considered for the selection of immunogens to be included in a future P. falciparum subunit vaccine and for vaccine development in general.

Adult↗

PubMed1990

Antibodies to a histidine-rich protein (PfHRP1) disrupt spontaneously formed Plasmodium falciparum erythrocyte rosettes.

Cerebral involvement in Plasmodium falciparum malaria is associated with sequestration of infected red blood cells and occlusion of cerebral vessels. Adhesion of infected erythrocytes along the vascular endothelium as well as binding of uninfected erythrocytes to cells infected with late-stage asexual parasites (rosetting) may be important in erythrocyte sequestration. We report that the recently discovered rosetting phenomenon shares characteristics with other human cell-cell interactions (heparin sensitivity, temperature independence, Ca2+/Mg2+ and pH dependence). Mono- and polyclonal antibodies specific for PfHRP1, a histidine-rich protein present in the membrane of P. falciparum-infected erythrocytes, disrupt rosettes but do not affect attachment of infected erythrocytes to endothelial cells. The inhibitory anti-PfHRP1 antibodies reacted with rosetting parasites in indirect immunofluorescence and with P. falciparum polypeptides of Mr 28,000 and Mr 90,000 in immunoprecipitation and immunoblotting, respectively. No inhibitory effects on erythrocyte rosetting were obtained with antibodies to related histidine-rich or other antigens of P. lophurae or P. falciparum. Whether the epitope that mediates rosetting, and is recognized by the anti-PfHRP1 antibodies, is located on PfHRP1 or on a crossreactive antigen remains to be established. The results suggest that endothelial cytoadherence and erythrocyte rosetting involve different molecular mechanisms.

Animals↗

PubMed1990

Consecutive determinations of seroreactivities to Pf 155/RESA antigen and to its different repetitive sequences in adult men from a holoendemic area of Liberia.

Sera from 32 adult men residing in a malaria holoendemic area of Liberia were investigated for seroreactivities to different asexual blood-stage malaria antigens on five consecutive occasions from 1984 to 1986. The seroreactivities to crude parasitic antigens and to Pf 155/RESA (EMIF) were determined by immunofluorescence and to repetitive sequences of Pf 155/RESA by enzyme-linked immunosorbent assay (ELISA). All sera were highly reactive against the crude parasitic antigens with reciprocal titres varying from 5000 to 100,000. The EMIF titres showed a wider variation from negative (less than 10) to 25,000, and when the same individuals were re-examined on subsequent surveys similar EMIF titres were found. The ELISA seroreactivities to three different repetitive sequences of Pf 155/RESA also showed different individual profiles which were rather consistent on consecutive surveys. High EMIF titres appeared to be correlated mainly to one of the peptide sequences, namely (EENV)2. The consistent individual profiles of the seroreactivities to Pf 155 and its repetitive sequences suggest genetic restriction of the humoral immune response. Although no significant correlation was found between EMIF titres and parasitic densities in the adult hyperimmune men the specific peptides, however, offer new possibilities of further investigating protective capacities of different immune responses to specific epitopes of the malaria parasite.

Adolescent↗

PubMed1990

Immunogenicity and antigenicity in rabbits of a repeated sequence of Plasmodium falciparum antigen Pf155/RESA fused to two immunoglobulin G-binding domains of staphylococcal protein A.

A synthetic gene encoding a tetramer of the repeated subunit EENVEHDA of the Plasmodium falciparum antigen Pf155/RESA was expressed in a dual-expression system. The resulting fusion proteins, designated ZZ-M1 and BB-M1, comprised the EENVEHDA repeats and either two immunoglobulin G-binding domains from staphylococcal protein A or the human serum albumin-binding domains from streptococcal protein G, respectively. The soluble fusion proteins were affinity purified to homogeneity in one-step procedures. ZZ-M1 was used for immunization of rabbits. The rabbit antisera reacted with BB-M1 in an enzyme-linked immunosorbent assay and with Pf155/RESA in immunofluorescence of infected erythrocytes and immunoblotting. Inhibition studies revealed that the antibodies mainly recognized epitopes formed by two or more EENVEHDA subunits and were remarkably specific for Pf155/RESA. Importantly, the antibodies also inhibited P. falciparum merozoite reinvasion in vitro efficiently, indicating that they reacted with biologically important epitopes exposed on the native antigen. Immunization with Freund complete adjuvant resulted in high levels of specific immunoglobulin G antibodies over a 1-year period, whereas the antibody response obtained after immunization without adjuvant was generally weaker, immunoglobulin G and M mediated, and not sustained for longer periods. However, these titers were restored after booster injection. Taken together, the results support the usefulness of recombinant gene constructs of this type as immunogens for malaria vaccines.

Amino Acid Sequence↗

PubMed1990

Anti-idiotypic antibodies counteract the invasion inhibition capacity of antibodies to major epitopes of the Plasmodium falciparum antigen Pf155/RESA.

Rabbits were immunized with the synthetic peptide EENVEHDA or (EENV)2, corresponding to a tandemly repeated sequence in the C-terminal part of the Plasmodium falciparum antigen Pf155/RESA, or with Escherichia coli-derived fusion proteins containing the corresponding repeats. For all sera, the capacity of the total immunoglobulin G fractions to inhibit P. falciparum merozoite invasion in vitro was similar and relatively low. Affinity purification of Pf155/RESA-specific antibodies on parasite-infected erythrocyte monolayers or on peptide columns increased the inhibitory capacity 50 to 5,000 times, whereas the immunofluorescence titers were increased only 10 times. The addition of small amounts of total immunoglobulin G to the affinity-purified antibodies gave a marked and dose-dependent reduction of the inhibitory capacity of the purified antibodies. However, this reduction was only seen in combinations where the immunoglobulin G fraction was from the same serum as the affinity-purified antibodies, suggesting that it was mediated by anti-idiotypic antibodies reacting with non-cross-reacting idiotopes of the invasion-inhibiting antibodies.

Amino Acid Sequence↗

PubMed1990

Number of cells from Plasmodium falciparum-immune donors that produce gamma interferon in vitro in response to Pf155/RESA, a malaria vaccine candidate antigen.

Secretion of gamma interferon (IFN-gamma) in response to stimulation of Plasmodium falciparum-primed T cells by specific antigens may be a useful indicator of cellular immunity to malaria. An enzyme-linked immunospot (ELISPOT) assay designed to detect IFN-gamma at the single-cell level was used to study IFN-gamma-producing cells from P. falciparum-primed donors from The Gambia after in vitro stimulation with various malarial antigens. IFN-gamma secreted into the culture supernatant was measured by conventional enzyme-linked immunosorbent assay (ELISA). There was a good correlation in individual donors between the level of IFN-gamma secreted into the culture supernatant and the number of IFN-gamma-secreting cells. However, the ELISPOT assay was apparently more sensitive in demonstrating low levels of IFN-gamma production than the ELISA was. Thus after stimulation with crude P. falciparum antigen from infected erythrocytes, 72% of the primed donors responded positively in the ELISPOT assay but only 55% responded positively in the ELISA. When stimulated with synthetic peptides representing immunodominant epitopes of the malarial antigen Pf155/RESA, a vaccine candidate, 31 to 55% responded in the ELISPOT assay and 21 to 36% responded in the ELISA. Unprimed Europeans did not respond positively to these antigens in either of the assays, and background in antigen-free controls was generally low. These results indicate that measurement of IFN-gamma by the ELISPOT assay or ELISA should have wide applications in large-scale epidemiological studies of malaria immunity. In addition, the ELISPOT assay makes it possible to analyze the T cells responding to malarial antigens in terms of both numbers and functional heterogeneity.

Adult↗

PubMed1989

Monoclonal antibody two-site ELISA for human IFN-gamma. Adaptation for determinations in human serum or plasma.

Mouse monoclonal antibodies (mAbs) against human interferon-gamma (IFN-gamma) were produced after immunization with recombinant IFN-gamma. Two mAbs (1-D1K and 7-B6-1) recognizing distinct epitopes on natural IFN-gamma were selected for the development of a two-site ELISA. The sensitivity was similar for IFN-gamma diluted in PBS with 1% bovine albumin, spent culture medium or fetal calf serum but reduced to approximately 50% when diluted in normal human serum. Individual normal human sera were tested and three of 14 gave false reactivities in the ELISA. One serum factor with major impact on the individual variation and the decreased sensitivity could be adsorbed to and eluted from protein A-Sepharose. Based on these observations we established a new ELISA protocol which made it possible to test for low levels of IFN-gamma in human serum and plasma samples. The modifications in this protocol are easy to apply with basic laboratory equipment.

Antibodies, Monoclonal↗

PubMed1989

A dual expression system for the generation, analysis and purification of antibodies to a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA.

A novel dual expression system for the generation and analysis of immune responses to recombinant protein is described. The two expression systems are based on the IgG-binding domains (ZZ) of staphylococcal protein A (SpA) and the human serum albumin (HSA) binding domains (BB) of streptococcal protein G, respectively. Products of fusions with the ZZ region are used to generate an immune response against the recombinant peptide and the corresponding peptide fused to the BB region is used for analysis and purification of the specific antibodies. The protein A and protein G expression systems were used to produce fusion proteins with the repeated C terminal octapeptide subunit EENVEHDA of the Plasmodium falciparum merozoite derived protein Pf155/RESA. Rabbits were immunized with the protein A-derived fusion protein (designated ZZ-M1) and the antibody response was analyzed using the protein G-derived fusion protein (designated BB-M1). The rabbit antisera reacted with BB-M1 in both ELISA and immunoblotting. In addition, BB-M1 proved to be an efficient ligand for affinity purification of antibodies specific for the malaria peptide. Furthermore, the rabbit antisera reacted with Pf155/RESA both in merozoite extracts and when deposited in the membrane of parasite infected erythrocytes.

Animals↗

PubMed1989

Plasmodium falciparum-infected erythrocytes form spontaneous erythrocyte rosettes.

Erythrocytes infected with trophozoites or schizonts of Plasmodium falciparum bind uninfected erythrocytes, leading to rosette formation. Both established laboratory strains and fresh isolates from patients form such rosettes, but at widely different frequencies. IgG preparations from the serum of some P. falciparum-immune donors and heparin inhibited rosette formation. The results indicate that cytoadherence of infected erythrocytes to endothelial cells and rosetting represent distinct genetic traits.

Animals↗

PubMed1989

Cytoadherence of knobless Plasmodium falciparum-infected erythrocytes and its inhibition by a human monoclonal antibody.

Red blood cells infected with mature stages of the malaria parasite Plasmodium falciparum bind to the endothelial lining of capillaries and venules. This sequestration is important for the survival of the parasite but may have severe consequences for the host. For example, it is involved in the causation of cerebral malaria which carries 25% mortality. Knob-like protrusions present on the surface of infected erythrocytes have been considered necessary but not sufficient for this cytoadherence. Here we describe the adhesion to endothelial cells of infected erythrocytes which do not have knobs. A human monoclonal antibody (33G2) which was specific for an epitope containing regularly spaced dimers of glutamic acid present in the repeated amino-acid sequences of some defined P. falciparum antigens was found to inhibit cyto-adherence and may therefore be an important reagent for elucidating the molecular basis of parasite sequestration.

Animals↗

PubMed1989

Enhancement or inhibition of Plasmodium falciparum erythrocyte reinvasion in vitro by antibodies to an asparagine rich protein.

A clone encoding a recombinant protein which reacted strongly with human antibodies from a donor clinically immune to malaria, was isolated from a genomic Plasmodium falciparum library. Mice injected with this protein, designated 10b, produced antibodies which reacted with all developmental stages of erythrocytic asexual parasites in indirect immunofluorescence. In immunoblotting, the same antibodies recognized two P. falciparum polypeptides of 36 kDa and 33 kDa. Of three monoclonal antibodies raised against the 10b recombinant protein, two inhibited parasite reinvasion of erythrocytes in an isolate specific manner. Surprisingly, however, the third was found to significantly enhance reinvasion of erythrocytes and also to induce a more rapid maturation of intraerythrocytic parasites in all isolates tested. Nucleotide sequence analysis of the 1124 bp insert revealed that it encodes a protein which consists of 30% asparagine and contains three asparagine rich, imperfect tandem repeats: Lys-Lys-Asn-Asn (3x), Met-Asn-His/Gln-Pro-Asn-Asn (14x), and Lys-Asn-Asn-Asn-Asn (7x).

Amino Acid Sequence↗

PubMed1989

Biochemical characteristics and partial amino acid sequence of the receptor-like human B cell and carcinoma antigen CDw40.

The Ag CDw40 (p50, Bp50) is a phosphoprotein expressed on the surface of both B lymphocytes and on certain malignant cell types of nonhemopoietic origin. Antibodies to this Ag have been shown to act as a potent co-mitogen for B cells. In order to elucidate the function of this Ag, we have now investigated some of its biochemical characteristics as well as the relationship of B cell derived CDw40 to that derived from urinary bladder carcinoma (transitional cell carcinoma, TCC) cells. CDw40 from normal B cells or from the Burkitt lymphoma line Raji showed a characteristic pattern of three bands when analyzed by SDS-PAGE and Western blotting: a main band of 47 kDa, a degradation product of 43 kDa, and a dimer of 85 kDa. The dimer was disrupted by reduction with 2-ME but was reformed spontaneously from the purified monomers under nonreducing conditions. CDw40 from two bladder cancer cell lines gave a similar pattern but formed little or no dimer. Thirty amino acids of the amino terminal end of CDw40 from Raji and 22 amino acids of that from TCC cells (HU549) were sequenced. The sequences were unusually rich in cysteines and differed only in that the cysteine in position 6 in Raji CDw40 had been replaced by glutamine in HU549. In addition there were two conservative changes in positions 15 and 19. Taken together these results show that CDw40 derived from B cells or from TCC cells are the same or closely related molecules. Comparisons of the amino acid sequence and biochemical characteristics of CDw40 with proteins having receptor functions indicated a close structural resemblance of CDw40 to the nerve growth factor-receptor.

Amino Acid Sequence↗

PubMed1989

The human B lymphocyte and carcinoma antigen, CDw40, is a phosphoprotein involved in growth signal transduction.

The human B lymphocyte and carcinoma-associated Ag, CDw40, (p50, Bp50) is a receptor candidate for normal growth regulation. Interaction of mAb with this pan-B Ag, together with preactivating agents such as 12-O-tetradecanoylphorbol-13-acetate or anti-mu, deliver strong growth-promoting signals to the cells. We here demonstrate that signaling through this Ag is dependent on its aggregation on the cell surface. Thus, monovalent antibody fragments were relatively inefficient in this respect but effectively blocked stimulation by intact antibody. By using affinity purified CDw40 protein we have also demonstrated that it is antigenically distinct from other B cell-associated Ag, including the six differentiation clusters CD19 to CD24. The mAb S2C6 and G28.5, prepared by immunizing mice with human bladder carcinoma cells or tonsillar B-cells, respectively, were the only antibodies giving detectable binding. Either of these antibodies could also completely block the binding of the other, suggesting an identity or structural proximity of the epitopes recognized. The CDw40 Ag was shown to be a phosphoprotein lacking intrinsic protein kinase activity. The results provide further evidence for CDw40 being an important B cell growth factor receptor which may also have growth regulatory functions in the development of certain human carcinomas.

Antigen-Antibody Reactions↗

PubMed1989

Why do Plasmodium falciparumm-infected erythrocytes form spontaneous erythrocyte rosettes?

Plasmodium falciparum malaria is one o f the most widespread o f human parasitic diseases and is responsible for the deaths of several million people in subtropical and tropical regions o f the world. The interaction o f malarial merozoites with erythrocytes and the adherence o f infected erythrocytes to the endothelium are among the cellular interactions extensively studied to define candidate antigens for a blood stage vaccine. However, the exact mechanisms underlying the invasion o f erythrocytes by P. falciparum merozoites and their subsequent binding to endothelium are not yet understood. Here Mats Wahlgren, Johan Carlson, Rachonee Udomsangpetch and Peter Perlmonn discuss a novel cytoodherence phenomenon which may be o f great importance in this context, that is, the spontaneous binding o f uninfected erythrocytes to those infected with late-stage parasites (trophozoites/schizonts).

Journal Article↗

PubMed1989

Cross-reactive antigenic determinants present on different Plasmodium falciparum blood-stage antigens.

A gene encoding a previously undescribed antigen of Plasmodium falciparum has been isolated from a genomic expression library by use of a pool of human immune sera. Northern blot analysis indicated that the gene is expressed at the late stages of the intra-erythrocytic cycle. This antigen, 332, contains a series of degenerated amino acid repeats. Human antibodies affinity-purified on the 332 recombinant antigen reacted with a family of parasite proteins that are products of different genes. We identified antigens 11.1 and Pf155-RESA as members of this family and confirmed, using a human monoclonal antibody, the presence of cross-reacting determinants. The sequences of these antigens also share some structural homologies. The significance of this family of blood-stage antigens is discussed.

Amino Acid Sequence↗