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A Sjölander

Publications and source records attributed to A Sjölander.

At least 55 records · Page 3Linked to original sources
PubMed1995

The regulation of leukotriene D4-induced calcium influx in human epithelial cells involves protein tyrosine phosphorylation.

Leukotriene D4 (LTD4) has been found to induce calcium signalling in the intestinal epithelial cell line Int 407, and this action involves the activation of both different GTP-binding proteins (G-proteins) and phospholipase C of the gamma-subtype (PLC-gamma). With this knowledge as the incentive, we investigated the possible regulatory role of protein tyrosine kinase activities in the calcium signalling system of the LTD4 receptor. The tyrosine kinase inhibitors genistein and herbimycin. A both reduced the LTD4-induced calcium signal by 70% when Int 407 cells were stimulated in the presence of extracellular calcium, but had no effect on the signal when the cells were stimulated in a calcium-free medium. In accordance with these findings, pretreatment with a tyrosine kinase inhibitor also blocked thapsigargin-induced cellular influx of calcium. These inhibitors had no effect on the intracellular mobilisation of calcium, which was supported by the findings that LTD4 was able to induce an increase in the tyrosine phosphorylation of PLC-gamma even when one of the tyrosine kinase inhibitors was present. Of possible interest regarding the effect of genistein on LTD4-induced calcium influx is that two major tyrosine phosphorylated protein bands were detected in immunoprecipitates obtained with PLC-gamma antibodies from LTD4-stimulated cells. These proteins, which associate with PLC-gamma, have estimated molecular weights of 84 and 97 kD. Preincubation with genistein completely abolished the LTD4-induced increase in tyrosine phosphorylation of the major 97 kD band, whereas the 84 kD protein band, like the PLC-gamma band, still exhibited an increased phosphorylation of tyrosine residues in response to LTD4. Neither this effect nor any of the other effects of genistein were induced when cells were preincubated with daidzein, an inactive analogue of genistein. The present results suggest that LTD4-induced calcium signalling in epithelial cells involves not only tyrosine phosphorylation of PLC-gamma, but also a tyrosine kinase-dependent step which occurs downstream of PLC-gamma activation and is directly implicated in the regulation of agonist-mediated calcium influx.

Benzoquinones↗
PubMed1995

Genetic restriction and specificity of the immune response in mice to fusion proteins containing repeated sequences of the Plasmodium falciparum antigen Pf155/RESA.

The genetic restriction and specificity of the immune response in mice to two fusion proteins, ZZ-M3 and ZZ-M5, were studied. These proteins contain two IgG-binding domains (ZZ) from staphylococcal protein A, and repeated sequences from the C-terminal [(VEHDAEEN)5 (VEEN)10] (M3) or central [(VEEPTVADDEH)3(VEEPTVAEEH)2] (M5) regions of the Plasmodium falciparum malaria blood stage antigen Pf155/RESA. Strong antibody and T-cell responses to M3 and M5 were linked to expression of the I-Ak allele, and T-cell responses to the bacterial fusion partner ZZ were restricted to mice of the H-2k haplotype. The response to M5 was less restricted than that to M3, giving intermediate responses in mice of H-2d haplotypes as well. However, ZZ-M5-primed lymph node (LN) cells from these mice were primarily induced to proliferate in vitro by the complete ZZ-M5 construct and not by synthetic peptides representing the repeated subunits in M5. The reactivity with intact Pf155/RESA in erythrocyte membrane immunofluorescence was weak of antisera from mice immunized with ZZ-M5, whereas the reactivity of antisera from mice immunized with ZZ-M3 roughly paralleled their reactivity with M3 in an enzyme-linked immunosorbent assay (ELISA). The antibody responses induced by immunization with ZZ-M3 or ZZ-M5 were specific for M3 or M5, respectively, while activated T cells displayed cross-reactivity between M3 and M5 in an in vitro proliferation assay. The results indicate that the assembly of repeated sequences in fusion proteins affects both the MHC class II restriction and the specificity of the induced antibody and T-cell responses.

Amino Acid Sequence↗
PubMed1994

Leukotriene D4-induced signal transduction.

In a human epithelial cell line LTD4 induces a calcium signal that is dependent on both intracellular mobilization and influx of calcium. This calcium signal is generated via the activation of dual G protein pathways. Whereas the intracellular mobilization of calcium is regulated by a pertussis toxin-insensitive G protein, the subsequent influx of calcium is regulated by a pertussis toxin-sensitive G protein. Furthermore, a LTD4-induced cellular elevation of cAMP also participates in the regulation of this calcium signal. The increase in cAMP is directly related to the LTD4-induced influx of calcium, perhaps by an activation of protein kinase A and a subsequent phosphorylation of a plasma membrane channel. This model of the LTD4-induced signaling pathway in epithelial cells is outlined in Figure 2.

Calcium↗
PubMed1994

Chemotactic factor receptor activation transiently impairs the Ca2+ signaling capacity of beta 2 integrins on human neutrophils.

Neutrophil motility involves a delicate interplay between intracellular signals elicited by adhesion and chemotactic factor receptors. To explore certain aspects of these complex receptor interactions in neutrophils, we studied how engagement of the chemotactic receptor for N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMet-Leu-Phe) and the beta 2 integrins affect the Ca2+ signaling properties of each other. Specific antibody engagement of the beta 2 integrins on suspended neutrophils generated both an intracellular mobilization and an influx of Ca2+, effects which could be temporarily impaired by preengaging the fMet-Leu-Phe receptors on the cells. In contrast to these findings, preengagement of the beta 2 integrins on suspended neutrophils affected neither a subsequent fMet-Leu-Phe-induced mobilization of Ca2+ nor an influx of Ca2+ from outside the cell. We also found that fMet-Leu-Phe could mobilize intracellular Ca2+ after a premobilization of Ca2+ via beta 2 integrins, despite the fact that both receptors mobilize Ca2+ from thapsigargin-sensitive intracellular stores. The findings that preactivation of beta 2 integrins did not affect the ability of fMet-Leu-Phe to induce an intracellular Ca2+ signal, whereas preactivation of fMet-Leu-Phe receptors transiently impaired the Ca2+ signaling ability of beta 2 integrins, suggest that the fMet-Leu-Phe-induced reduction is due to an interaction upstream of the release of Ca2+ from intracellular stores.

Antibodies, Monoclonal↗
PubMed1994

Propofol induces changes in the cytosolic free calcium concentration and the cytoskeletal organization of cultured human glial cells and primary embryonic rat brain cells.

BACKGROUND: The site of action of the intravenous anesthetic drug propofol is uncertain. Therefore, we examined the effects of propofol on the cytosolic free calcium levels of cultured primary embryonic rat brain cells (80-85% neurons), and on the organization of the cytoskeleton in these rat cells and in a model system of cultured human glial cells (astrocytes). METHODS: Propofol was added to the cells as the clinically available solution Diprivan. Cytosolic free calcium changes in neurons were studied using Fura2 and a single-cell microfluorometric method. Fluorescence microscopy was used to study the organization of actin filaments and tubulin in detergent-extracted cells. RESULTS: An increase in the cytosolic free calcium concentration of 116 +/- 39 nM was seen shortly after the addition of 0.3 microgram.ml-1 propofol, and a propofol concentration of 0.03 microgram.ml-1 resulted in an increase in cytosolic free calcium concentration of the same magnitude, 119 +/- 42 nM. Most of the calcium (60-75%) came from the extracellular environment, and the rest was from intracellular stores. When neurons were depleted of intracellular calcium by 1,2-bis-5-methyl-amino-phenoxylethane-N,N'-tetra-acetoxymethyla cetate (MAPT/AM), no changes were seen in the actin organization of the cytoskeleton. Actin organization was affected by all concentrations of propofol, 0.3-50 micrograms.ml-1 (1.7-280 microM), when exposure to the drug was achieved by a 30-min incubation. After the incubation, the exposed cells were more rounded and exhibited increased ruffling activity, both at the periphery and on the cellular surface, and ring-shaped actin structures were also seen. These effects were concentration dependent and reversible, and reached a maximum after 20 min of incubation. Propofol had no apparent effect on the organization of tubulin. CONCLUSIONS: Propofol induced changes in the cytoskeletal organization of actin in cultured rat neurons and human glial cells. These changes must have been due to the increase in intracellular calcium seen shortly after the addition of propofol, since no effects on actin organization were seen when intracellular calcium was depleted.

Actins↗
PubMed1993

Plasmodium falciparum: the immune response in rabbits to the clustered asparagine-rich protein (CARP) after immunization in Freund's adjuvant or immunostimulating complexes (ISCOMs).

The Plasmodium falciparum clustered asparagine-rich protein (CARP) is a merozoite-associated antigen which contains approximately 30% asparagine. Analysis of the DNA sequences located 5' of the cloned 1.4-kb CARP gene in the P. falciparum genome suggests that this gene fragment may encode the complete CARP and that the gene product is a protein of M(r) 50,000. To analyze the immunogenicity of CARP, the gene was expressed as a fusion protein with staphylococcal protein A (SpA-CARP). Immunization of rabbits with SpA-CARP in Freund's complete adjuvant (FCA) resulted in a strong antibody response against CARP as measured in ELISA. This response was efficiently boosted and sustained over a long time while that induced by two immunizations with SpA-CARP in ISCOMs was weak and of shorter duration. In both instances, the antibody levels against CARP were further increased by a second booster injection consisting of either SpA-CARP or CARP fused to the serum albumin-binding region (BB) of streptococcal protein G (BB-CARP) in PBS, indicating that immunizations with SpA-CARP in FCA or ISCOMs had induced a CARP-specific immunological memory. Boosting with BB-CARP in PBS was more efficient than boosting with SpA-CARP in PBS. In all rabbits, the antibodies obtained after the booster with CARP in PBS were the most efficient inhibitors of merozoite invasion in vitro. The antisera reacted with the intracellular parasite in immunofluorescence and with a band of M(r) 50,000 in immunoblotting while several high-molecular-weight components as well as the one of M(r) 50,000 were immunoprecipitated. The specificity of the antibody responses varied between the different rabbits as indicated in ELISA, with short synthetic peptides representing different CARP sequences. Taken together, the results suggest that a previously cloned genomic DNA fragment may encode the complete P. falciparum blood-stage antigen CARP and that CARP is immunogenic in rabbits both when administered in FCA or ISCOMs.

Amino Acid Sequence↗
PubMed1993

Engineered bacterial receptors in immunology.

Gram-positive surface receptors, such as staphylococcal protein A ans streptococcal protein G, have been genetically engineered for many applications in the field of immunology, including detection antigens, affinity purification of fusion proteins and display of heterologous epitopes on the surface of bacterial cells.

Antigens↗
PubMed1993

Beta 2 integrin engagement triggers actin polymerization and phosphatidylinositol trisphosphate formation in non-adherent human neutrophils.

Beta 2 integrins are involved in the adhesion of leukocytes to other cells and surfaces. Although adhesion is required for cell locomotion, little is known regarding the way beta 2 integrin-receptors affect the actin network in leukocytes. In the present study filamentous actin (F-actin) levels in non-adherent human neutrophils have been measured by phalloidin staining after antibody cross-linking of beta 2 integrins. Antibody engagement of beta 2 integrins resulted in a rapid and sustained (146 and 131% after 30 and 300 s, respectively) increase in the neutrophil F-actin content. This is in contrast to stimulation with N-formyl-l-methionyl-l-leucyl-l-phenylalanine (fMLP), which causes a prompt and pronounced but rapidly declining rise in F-actin (214 and 127% after 15 and 300 s, respectively). Priming neutrophils with 1 nM PMA, a low concentration that did not influence the F-actin content per se, increased the magnitude of the beta 2 integrin-induced response but had no effect on the kinetics (199% after 30 s and 169% after 300 s). Removal of extracellular Ca2+ only marginally affected the beta 2 integrin-induced F-actin response for cells that were pretreated with PMA whereas the response for nonprimed cells was reduced by half. This suggests that even though extracellular Ca2+ has a modulatory effect it is not an absolute requirement for beta 2 integrin-induced actin polymerization. beta 2 integrin engagement did not affect the resting cellular level of cAMP arguing against a role of cAMP in beta 2 integrin-induced actin assembly.(ABSTRACT TRUNCATED AT 250 WORDS)

1-Methyl-3-isobutylxanthine↗
PubMed1993

Immunogenicity in rabbits and monkeys of influenza ISCOMs conjugated with repeated sequences of the Plasmodium falciparum antigen Pf155/RESA.

Rabbits and monkeys were immunized with two fusion proteins, ZZ-M3 and ZZ-M5, coupled to pre-formed influenza virus membrane glycoprotein ISCOMs. The fusion proteins comprise two IgG-binding domains from staphylococcal protein A (ZZ) and repeated amino acid sequences from the C-terminal (M3) or central (M5) repeat regions of the Plasmodium falciparum antigen Pf155/RESA. The induced antibody responses were of long duration, could be efficiently boosted and were comparable to those obtained with Freund's Adjuvant. The produced antibodies reacted with M3, M5, protein A and the influenza glycoprotein, recognized Pf155/RESA and inhibited merozoite invasion in vitro. These results suggest that coupling of immunogens to pre-formed ISCOMs may be a basis for construction of multivalent subunit vaccines.

Amino Acid Sequence↗
PubMed1993

Intestinal tissue distribution and epithelial transport of the oral immunogen LTB, the B subunit of E. coli heat-labile enterotoxin.

LTB provokes a systemic immune response and exerts adjuvant effects on mucosal immune responses to unrelated antigens. The binding and uptake of fluorescein-labelled LTB in the normal villus epithelium was compared to that in Peyer's patch dome epithelium in mouse intestine. LTB was bound by the GM1-receptor and taken up extensively by both tissues, indicating that not only the Peyer's patches but also the normal villus epithelium play a significant role in the transport of orally administered antigens. These results were supported by transport studies in the human intestinal epithelial cell line Caco-2 using 125I-LTB. After 2 h incubation, 5.1 +/- 0.1% and 5.9 +/- 0.1% of the added radioactivity was transported in the apical to basolateral and basolateral to apical direction, respectively. Less than 1% of the transported radioactivity was immunoprecipitated with anti-LTB antiserum indicating that LTB was extensively degraded during the transport. The results suggest that normal enterocytes play a significant role in the binding, uptake and transport of orally administered LTB.

Animals↗
PubMed1992

Agonist-induced association of the p21ras GTPase-activating protein with phosphatidylinositol 3-kinase.

The signal transduction properties of the 21-kDa GTP-binding proteins, encoded by the ras genes, are only partly known. In a recent report, we demonstrated that the signaling pathway of p21ras, like that of several growth factors, is closely associated with phosphatidylinositol 3-kinase (PtdIns 3-kinase) activity. We showed that insulin-like growth factor-1 (IGF-1) and insulin increased the phosphatidylinositol 3-kinase activity in immunoprecipitates obtained with anti-phosphotyrosine and anti-ras antibodies in Ha-ras-transformed epithelial cells. Several findings in this previous study suggested that an additional protein was likely to be associated with the PtdIns 3-kinase. The suggestion that p21ras GTPase-activating protein (GAP) acts not only as a regulator of p21ras activity but also as a direct downstream target in the signaling pathway of p21ras led us to investigate the possible association of PtdIns 3-kinase with GAP. The stimulation of Ha-ras-transformed epithelial cells with IGF-1 caused an increased association of PtdIns 3-kinase activity with GAP, as seen by immunoprecipitation with anti-p21ras and anti-GAP antibodies. The 85-kDa regulatory subunit of PtdIns 3-kinase was present in immunoprecipitates obtained with antibodies against GAP and p21ras of IGF-1 stimulated cells. These data suggest that GAP acts as a downstream target for p21ras via its association with PtdIns 3-kinase.

Animals↗
PubMed1992

Involvement of Pf155/RESA and cross-reactive antigens in Plasmodium falciparum merozoite invasion in vitro.

Lines of Plasmodium falciparum FCR3 either expressing or not expressing the blood-stage antigen Pf155/RESA were used to analyze the possible involvement of this antigen in the merozoite invasion process in vitro. Antibodies from human sera, affinity purified on synthetic peptides corresponding to C-terminal repeated sequences in Pf155/RESA, were shown to inhibit merozoite invasion of both types of parasites with similar efficiency. Reversal of the invasion inhibition by fusion proteins containing repeated sequences of Pf155/RESA but not of the cross-reactive antigens Ag332 and Pf11.1 indicated that the inhibitory antibodies had similar target antigens in both Pf155/RESA+ and Pf155/RESA- parasites that involved cross-reacting epitopes present in Pf155/RESA. Rabbit antibodies specific for Pf155/RESA repeats inhibited merozoite invasion of Pf155/RESA expressing parasites efficiently but had no or very small effect on the invasion of Pf155/RESA-deficient parasites. In contrast, rabbit antibodies specific for Ag332 repeats as well as human antibodies affinity purified on synthetic Ag332 peptides inhibited merozoite invasion of both types of parasites with high efficiency. A similar inhibition pattern was seen with the human monoclonal antibody 33G2, which has specificity for Ag332 but also cross-reacts with Pf155/RESA and Pf11.1. Taken together, our data suggest that Pf155/RESA and related cross-reactive antigens as well as Ag332 are involved in the merozoite invasion process and may constitute targets for invasion inhibitory antibodies.

Animals↗
PubMed1992

Expression of recombinant proteins on the surface of the coagulase-negative bacterium Staphylococcus xylosus.

An expression system to allow targeting of heterologous proteins to the cell surface of Staphylococcus xylosus, a coagulase-negative gram-positive bacterium, is described. The expression of recombinant gene fragments, fused between gene fragments encoding the signal peptide and the cell surface-binding regions of staphylococcal protein A, targets the resulting fusion proteins to the outer bacterial cell surface via the membrane-anchoring region and the highly charged cell wall-spanning region of staphylococcal protein A. The expression system was used to secrete fusion proteins containing sequences from a malaria blood-stage antigen and a streptococcal albumin-binding receptor to the cell surface of S. xylosus. Analysis of the recombinant cells by immunogold staining and immunofluorescence revealed that both the receptor and the malaria peptide were properly processed and exposed on the surface of the host cells. However, only approximately 40 to 50% of the recombinant cells were strongly stained with antiserum reactive with the albumin-binding receptor, while approximately 10 to 15% of the cells were stained with antiserum reactive with the malaria peptide. The incomplete staining of some of the cells suggests steric effects that make the recombinant fusion proteins inaccessible to the reactive antibodies because of variable cell wall structures. However, the results demonstrate for the first time that recombinant techniques can be used to express heterologous receptors and immunogens on the surface of gram-positive cells.

Amino Acid Sequence↗
PubMed1991

Association of p21ras with phosphatidylinositol 3-kinase.

In mammalian cells, ras genes code for 21-kDa GTP-binding proteins. Increased expression and mutations in specific amino acids have been closely linked to alterations of normal cell morphology, growth, and differentiation and, in particular, to neoplastic transformation. The signal transduction induced by these p21ras proteins is largely unknown; however, the signaling pathways of several growth factors have been reported to involve phosphatidylinositol (PtdIns) 3-kinase. In the present study of a Ha-ras-transformed epithelial cell line, we demonstrated increased PtdIns 3-kinase activity in anti-phosphotyrosine and anti-receptor (insulin and hybrid insulin-like growth factor I) immunoprecipitates of cells that had been stimulated with insulin or insulin-like growth factor I. The PtdIns 3-kinase activity was also immunoprecipitated in these experiments by the anti-Ras monoclonal antibody Y13-259. The specificity of this association with p21ras was ascertained by the neutralizing effect of the antigen peptide and the absence of PtdIns 3-kinase activity in Y13-259 immunoprecipitates from cells in which the ras gene was turned off. These data indicate that PtdIns 3-kinase activity is an important step in the cascade of reactions in p21ras signal transduction, suggesting that the alterations of the cytoskeleton and growth in ras-transformed cells could be mediated by PtdIns 3-kinase activity.

1-Phosphatidylinositol 4-Kinase↗
PubMed1991

High antibody responses in rabbits immunized with influenza virus ISCOMs containing a repeated sequence of the Plasmodium falciparum antigen Pf155/RESA.

Immunostimulating complexes (ISCOMs) are spherical structures where immunogens are presented as multimers in a matrix of the adjuvant Quil A. ISCOMs have been shown to enhance the immunogenicity of several antigens important to both human and veterinary vaccine development. We have coupled a fusion protein, designated ZZ-M2, comprising eight copies of the C-terminal repeat subunit EENV of the Plasmodium falciparum blood-stage antigen Pf155/RESA and two IgG-binding domains of staphylococcal protein A (SpA), to preformed influenza virus envelope protein ISCOMs. Rabbits immunized with the conjugated ISCOMs produced high titres of antibodies even after the first injection. These antibodies reacted with the EENV repeat sequence in ELISA and with Pf155/RESA in immunofluorescence on infected erythrocytes. The antibody response, which was sustained for more than 20 weeks, was efficiently boosted and superior or equal to that obtained after immunization with ZZ-M2 in Freund's complete adjuvant. In contrast, the antibody response induced in rabbits immunized with ZZ-M2 in Syntex Adjuvant Formulation-MF (SAF-MF) was weak and of short duration. The antibodies produced after immunization with ZZ-M2 coupled to influenza virus ISCOMs mainly recognized epitopes formed by two or more EENV subunits and were highly specific for Pf155/RESA. Furthermore, the antibodies efficiently inhibited merozoite reinvasion of erythrocytes in vitro, indicating that they recognized epitopes exposed on the native antigen. In addition, the ZZ-M2-conjugated ISCOMs also induced high titres of antibodies reacting with SpA or the influenza virus envelope protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Adjuvants, Immunologic↗
PubMed1991

The adjuvant action of cholera toxin is associated with an increased intestinal permeability for luminal antigens.

This study addresses the question of whether cholera toxin (CT) increases gut permeability for molecules greater than 3000 Da and whether such an effect is associated with an adjuvant function by CT on the gut immune response. We found that CT after oral administration gives rise to strikingly increased gut permeability for Dextran (Mw 3000) concomitantly with a strong enhancing effect on the anti-keyhole limpet hemocyanin (KLH) specific immune response in the lamina propria after oral immunization with KLH plus Dextran and CT. In contrast, the B-subunit of the holotoxin, which lacks the adenylate cyclase/cAMP-activating property of CT, failed to increase gut permeability as well as local anti-KLH immune responses. These results might suggest a causal linkage between the ability of CT to increase gut permeability and its adjuvant property on gut mucosal immune responses. In addition this finding supports the notion that the adenylate cyclase/cAMP system plays a regulatory role in gut permeability and is important in enhancing mucosal immune responses. Based on previous studies and the present data we propose that the mechanism for CT's adjuvant function on mucosal immune responses is by affecting antigen-presenting cells, T and B cells in the gut to give a net enhancing effect on the stimulation of local immunity, and that the CT-induced increase in gut permeability might be part of the adjuvant mechanism by facilitating luminal antigens to access the gut mucosal immune system.

Adenylyl Cyclases↗