Biomedical subjects

A Sjölander

Publications and source records attributed to A Sjölander.

At least 37 records · Page 2Linked to original sources

PubMed1998

Characterization of immune responses to experimental polyvalent subunit vaccines assembled in iscoms.

Immune responses to experimental polyvalent subunit vaccines assembled in a particulate adjuvant/delivery system, iscoms, are described. The fusion protein ZZ-M5 comprises structures of staphylococcal protein A (ZZ) and the Plasmodium falciparum malaria antigen Pf155/RESA (M5). MHC congenic mice were immunized with ZZ-M5 conjugated to iscoms containing human influenza virus antigen (flu ag, M5-flu-isc) or to iscom matrix (iscom particles without flu ag, M5-isc). Comparison of antibody and T-cell responses to M5-isc and M5-flu-isc demonstrated that the flu ag in M5-flu-isc exhibits carrier-related helper functions and that the assembly of immunogens in M5-flu-isc did not result in any apparent antigenic competition. In addition, assembly of ZZ-M5 and flu ag in iscoms induced an alteration of the IgG subclass profile of the antibody response to M5. The results suggest that assembly of immunogens in iscoms may be a useful approach to the design of subunit vaccines but that both quantitative and qualitative aspects of the immunogenic properties of such constructs should be scrutinized.

Amino Acid Sequence↗

PubMed1998

The inflammatory mediator leukotriene D4 triggers a rapid reorganisation of the actin cytoskeleton in human intestinal epithelial cells.

Epithelial cells play an important role in maintaining the intestinal mucosa barrier, a barrier that is impaired in several inflammatory conditions. The mechanisms behind this impairment are not known, but it can be presumed that structural alterations of the epithelial cells are involved. In support of this notion, we here show the inflammatory mediator leukotriene D4 (LTD4) triggered first a rapid (10 s) increase and immediately thereafter (30 s) a sustained decrease in the cellular filamentous actin (F-actin) level in intestinal epithelial cells. The initial LTD4-induced increase in F-actin content was effectively blocked by preincubating the cells with either pertussis toxin or the tyrosine kinase inhibitor genistein. A possible involvement of the tyrosine kinase-dependent phosphatidylinositol-3-kinase (PI-3-kinase) in the polymerisation of actin was supported by the observations that LTD4 induced a translocation to a membrane fraction of PI-3-kinase and by the findings that wortmannin, a PI-3-kinase inhibitor, totally abolished both this translocation of PI-3-kinase as well as the initial LTD4-induced polymerisation of actin. In addition, pertussis toxin and genistein, both known to interfere with the LTD4-induced calcium signal, completely or partially reversed the actin-depolymerising effect of LTD4. The calcium ionophore ionomycin (30s) induced actin depolymerisation to the same extent as LTD4 (30 s) did, but lacked the initial effect on actin polymerisation. In cells loaded with the calcium chelator MAPT, LTD4 induced a normal actin polymerisation response but the subsequent depolymerisation was completely inhibited. Similar results were obtained when the cells were preincubated with the protein kinase A inhibitor Rp-cAMPS, which has been shown to impair the LTD4-induced calcium signal in these epithelial cells. The present results show that the inflammatory mediator LTD4 triggers a reorganisation of the actin network in intestinal epithelial cells that is likely to contribute to the impairment of the intestinal barrier function.

Actins↗

PubMed1998

Vaccination with recombinant Parasite Surface Antigen 2 from Leishmania major induces a Th1 type of immune response but does not protect against infection.

Vaccination with the native Parasite Surface Antigen 2 of Leishmania major with Corynebacterium parvum as adjuvant protects mice from leishmaniasis through a Th1 mediated response. Here we show that vaccination with a recombinant form of this protein, purified from Escherichia coli and administered in iscoms or with C. parvum as adjuvant, does not induce protective immunity despite the induction of Th1 responses. The results suggest that protective immunity depends on the ability of the vaccinating antigen to induce Th1-like T cells with ability to be recalled by infection. Therefore, the conformation of antigens may play a more major role for the induction of T cell mediated immunity than originally considered.

Animals↗

PubMed1998

Kinetics, localization and characteristics of B- and T-cell responses to iscoms containing human influenza virus glycoproteins.

B- and T-cell responses have been studied after primary and secondary immunizations of mice with iscoms containing influenza virus envelope glycoproteins. After primary immunization both B- and T-cell responses were initiated in the draining lymph nodes. T-cells showed peak activity with respect to proliferation and cytokine production after five to eight days and the highest number of IgG secreting cells (IgG-SC) was recorded at day seven. The responses in the spleen developed slowly but were of longer duration. Cytokines produced by spleen cells included high levels of IFN-gamma and IL-2. After a secondary immunization the frequencies of IgG-SC were drastically increased in both LN and spleen, but decreased rapidly with time. At day eight after the secondary immunization high numbers of IgG-SC were detected in the bone marrow. High titres of IgG1 and IgG2a and substantial titres of IgG2b and IgG3 were detected in serum.

Adjuvants, Immunologic↗

PubMed1997

Iscoms containing purified Quillaja saponins upregulate both Th1-like and Th2-like immune responses.

The immune stimulating complex (iscom) is built up by antigen, cholesterol, phospholipids, and adjuvant active Quillaja saponins. Previous studies have shown that iscoms containing Quil A (a semipurified preparation of saponins) efficiently induce antibody and cell-mediated immune responses. In this study, we demonstrate that iscoms containing a mixture of two purified low toxicity Quillaja saponin fractions (ISCOPREP 703) are able to upregulate both Th1-like and Th2-like immune responses. Thus, ovalbumin (OVA) iscoms induced higher levels of antigen-specific IgG1 and IgG2a antibodies and increased the production of both IFN-gamma and IL-4 compared with OVA administered without adjuvant. In contrast, OVA formulated in Al(OH)3 elicited IgG1 and IgE antibodies and primed spleen cells producing IL-4 and IL-10, suggesting the activation of primarily Th2-like cells. These findings underline that adjuvants are able to alter the character of immune responses and may be used to generate responses with desired properties.

Animals↗

PubMed1997

The serum albumin-binding region of streptococcal protein G: a bacterial fusion partner with carrier-related properties.

In this study, we have explored the use of the serum albumin-binding region (BB) from streptococcal protein G (SpG) as a bacterial fusion partner for production of peptide immunogens. The fusion protein BB-M3, containing BB and repeated structures from the Plasmodium falciparum malaria antigen Pf155/RESA, was efficiently purified from Escherichia coli culture supernatants by affinity chromatography using BB as an affinity tag. Rabbits immunized with BB-M3 in Freund's adjuvant produced high levels of antibodies which reacted with both M3 and BB in ELISA and stained intact Pf155/RESA in the membrane of infected erythrocytes. These antibody levels were sustained for more than 30 weeks. BB-M3 also induced antibody responses to M3, BB and intact Pf155/RESA in a number of mouse strains, including several strains which are non-responders to the malaria sequences. In the latter mice, however, BB-M3 only activated BB-specific T cells, suggesting that BB has ability to provide carrier-related T cell help for antibody production. Moreover, the minimal albumin-binding motif of SpG, containing only 46 amino acids, was immunogenic in both B10.BR, B10.D2 and C57BL/6 mice (H-2k, H-2d and H-2b, respectively). These results indicate that BB has both affinity tag and carrier-related properties and suggest that fusion proteins containing BB can be efficient tools for the generation of antibody responses to peptides which are weak immunogens.

Animals↗

PubMed1997

Leukotriene D4-induced signalling events in human epithelial cells: G alpha i3 activation and translocation.

Our model of LTD4-induced signal transduction in epithelial cells is summarised in Figure 2. Extending what is already known about LTD4 signalling in epithelial cells, we identified the Gi3-protein as the crucial PTX sensitive G-protein and found that it is translocated to what might be a cytoskeletal fraction. This finding suggests a subtle response to LTD4, mediated via the bifurcation at the alpha/beta gamma junction. Although little is known about the role of epithelial cells in inflammation, it has been shown that such cells produce the potent chemoattractant LTB4 and the proinflammatory 5-HETE in response to intracellular accumulation of Ca2+ 24. The target protein(s) and the effect(s) of the translocation of the activated G alpha i3-proteins, as well as the possible role of the beta/gamma-subunits of Gi3, remain to be elucidated.

Animals↗

PubMed1997

Kinetics, localization and cytokine profile of T cell responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins.

Immune stimulating complexes (iscoms) are 40 nm particles combining adjuvant-active Quillaja saponins and multimeric presentation of antigens. The distribution in mice of influenza virus iscoms and the resulting T cell responses in the lymph nodes (LN) and spleen were characterized. After a single subcutaneous injection, iscoms were delivered to the draining LN where they induced a transient population of LN cells which responded with proliferation and secretion of interleukin-2 (IL-2), gamma-interferon (IFN-gamma) and interleukin-4 (IL-4) after restimulation. The response in the spleen developed more slowly, sustained for 12 weeks and was characterized by cells producing in particular IL-2 and IFN-gamma but also IL-4. A booster resulted in a dramatic enhancement of the production of IFN-gamma, indicating that iscoms efficiently recruit cells with Th1 properties. Comparisons of T cell responses to iscoms and to influenza virus antigen in Freund's complete adjuvant demonstrate that these adjuvants affect both the localization and cytokine profile of T cell responses.

Animals↗

PubMed1996

Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells.

The present results show that stimulation of Intestine 407 epithelial cells with LTD4 (Leukotriene D4) triggers a rapid activation of the pertussis-toxin-sensitive Gi3-protein and a simultaneous translocation of its alpha-subunits to a crude cytoskeletal fraction. The activation of G alpha i3, which was measured as the GTP/ GDP exchange ratio, peaked about 15 s after the addition of LTD4. Western blot analyses of subcellular fractions showed that G alpha i3-subunits accumulated in the cytoskeletal fraction and decreased in the membrane fraction, and the decrease was most marked 15 s after the exposure to LTD4. None of the other pertussis-toxin-sensitive G-proteins (Gi3-Z and G alpha 0) were activated or translocated upon stimulation with LTD4. This agonist was also found to reduce the GTP/GDP exchange ratio of Gi-proteins without affecting the subcellular distribution of its alpha-subunits. These findings imply that the Gi3-protein is the pertussis-toxin-sensitive G-protein previously found to mediate several downstream LTD4-stimulated signalling events. Furthermore, the translocation of G alpha i3-subunits to the cytoskeleton and the simultaneous inhibition of G3-proteins indicate that the cytoskeleton might participate in the signalling process of human epithelial cells.

Biological Transport↗

PubMed1996

Antibody-induced engagement of beta 2 integrins on adherent human neutrophils triggers activation of p21ras through tyrosine phosphorylation of the protooncogene product Vav.

It is known that beta 2 integrins are crucial for leukocyte cell-cell and cell-matrix interactions, and accumulating evidence now suggests that integrins serve not only as a structural link but also as a signal-transducing unit that controls adhesion-induced changes in cell functions. In the present study, we plated human neutrophils on surface-bound anti-beta 2 (CD18) antibodies and found that the small GTP-binding protein p21ras is activated by beta 2 integrins. Pretreatment of the cells with genistein, a tyrosine kinase inhibitor, led to a complete block of p21ras activation, an effect that was not achieved with either U73122, which abolishes the beta 2 integrin-induced Ca2+ signal, or wortmannin, which totally inhibits the phosphatidylinositol 3-kinase activity. Western blot analysis revealed that antibody-induced engagement of beta 2 integrins causes tyrosine phosphorylation of several proteins in the cells. One of these tyrosine-phosphorylated proteins had an apparent molecular mass of 95 kDa and was identified as the protooncogene product Vav, a p21ras guanine nucleotide exchange factor that is specifically expressed in cells of hematopoietic lineage. A role for Vav in the activation of p21ras is supported by the observations that antibody-induced engagement of beta 2 integrins causes an association of Vav with p21ras and that the effect of genistein on p21ras activation coincided with its ability to inhibit both the tyrosine phosphorylation of Vav and the Vav-p21ras association. Taken together, these results indicate that antibody-induced engagement of beta 2 integrins on neutrophils triggers tyrosine phosphorylation of Vav and, possibly through its association, a downstream activation of p21ras.

Antigen-Antibody Reactions↗

PubMed1996

Leukotriene D4-induced mobilization of intracellular Ca2+ in epithelial cells is critically dependent on activation of the small GTP-binding protein Rho.

We have previously shown that the leukotriene D4 (LTD4)-induced mobilization of intracellular Ca2+ in epithelial cells is mediated by a G-protein that is distinctly different from the pertussis toxin-sensitive G-protein that regulates the subsequent influx of Ca2+. In the present study, we attempted to gain further knowledge about the mechanisms involved in the LTD4-induced mobilization of intracellular Ca2+ in epithelial cells by investigating the effects of compactin, an inhibitor of the isoprenylation pathway, on this signalling event. In cells preincubated with 10 microM compactin for 48 h, the LTD4-induced mobilization of intracellular Ca2+ was reduced by 75% in comparison with control cells. This reduction was reversed by co-administration of mevalonate (1 mM). The effect of compactin occurred regardless of whether or not Ca2+ was present in the extracellular medium, suggesting that isoprenylation must occur before Ca2+ is released from intracellular stores. In accordance with this, we also found that both the LTD4-induced formation of inositol 1,4,5-trisphosphate and the LTD4-induced phosphorylation of phospholipase C gamma 1 (PLC gamma 1) on tyrosine residues were significantly reduced in compactin-pretreated cells. These results open up the possibility that the activation of PLC gamma 1 is related to a molecule that is sensitive to impaired activity of the isoprenylation pathway, such as a small monomeric G-protein. This idea was supported by the observation that Clostridium botulinum C3 exoenzyme-induced inhibition of Rho proteins abolished the LTD4-induced intracellular mobilization of Ca2+. A regulatory role of Rho proteins in the LTD4-induced activation of PLC gamma 1 is unlikely to be indirectly mediated via an effect on the cytoskeleton, since cytochalasin D had no major effect on the LTD4-induced mobilization of Ca2+. Although the mechanism of interaction remains to be elucidated, the present findings indicate an important role of an isoprenylated protein such as Rho in the LTD4-induced Ca2+ signal.

ADP Ribose Transferases↗

PubMed1996

N-linked glycans in the CD4-binding domain of human immunodeficiency virus type 1 envelope glycoprotein gp160 are essential for the in vivo priming of T cells recognizing an epitope located in their vicinity.

Deglycosylation of viral glycoproteins has been suggested to influence the number of available T cell determinants and to increase T cell recognition of antigens. In this study, we have investigated whether T cell responses to the HIV-1 envelope glycoprotein gp160 were influenced by deletion of three N-glycans of the protein. Wild type (wt) and a mutated form of gp160 (gp160A123) lacking the three N-glycans in the C-terminal CD4-binding region efficiently induced antigen-specific T cell responses in mice of the H-2b, H-2d, and H-2k haplotypes. Further, T cells primed by either wt gp160 or gp160A123 were stimulated in vitro to a similar extent by the homologous and heterologous protein, indicating that deletion of the glycans did not affect the overall immunogenicity and antigenicity of gp160A123. Wild-type gp160 and gp160A123 induced comparable T cell responses to those of epitopes which with respect to the secondary structure of gp160 were distant from the deleted glycans. However, in mice of the H-2b haplotype, wt gp160 primed T cells which responded in vitro to a peptide containing one of the deleted N-glycosylation sites (Asn448), whereas T cells induced by gp160A123 were unable to recognize this peptide. Thus, deletion of the glycans abrogated the in vivo priming of T cells recognizing an epitope in close proximity to the deletion sites. Furthermore, enzymatically deglycosylated gp160 failed to induce a T cell response to this epitope. These results indicate that the in vivo generation of certain T cell determinants from glycoproteins is dependent on the glycosylation of the protein.

Amino Acid Sequence↗

PubMed1996

Iscoms in parasitological research.

During the history of vaccine development, a number of adjuvants and adjuvant formulations have been tested and evaluated for their ability to increase the immunogenicity of different antigens. In this review, Anna Lundén, Karin Lövgren Bengtsson, Anders Sjölander and Arvid Uggla focus on iscoms (immune stimulating complexes), their characteristics and applications to different types of parasitic antigens.

Journal Article↗

PubMed1996

Adjuvant activity of iscoms; effect of ratio and co-incorporation of antigen and adjuvant.

We have studied the importance of co-incorporation of antigen and adjuvant in iscoms and the effects of different ratios of adjuvant and antigen in the iscom particles. Immune responses to influenza virus antigens (flu-Ag) in iscoms were compared to those induced by flu-Ag mixed with iscom-matrix, i.e. antigen and adjuvant delivered in separate packages. Higher doses of Quil A were required with iscom-matrix to induce strong immune responses compared to iscoms containing the same amount of antigen. The immunogenic properties of iscoms were affected by the ratio between antigen and adjuvant in the particles. Both iscoms and flu-Ag mixed with iscom-matrix induced antigen-specific antibodies with similar IgG subclass distribution and activated spleen cells producing high levels of IL-2 and IFN-gamma in vitro.

Adjuvants, Immunologic↗

PubMed1996

Novel adjuvants and vaccine delivery systems.

Conventionally the efficiency of an adjuvant is measured by the capacity to induce enhanced antibody serum titres and cell mediated immunity (CMI) to a given antigen. Nowadays the capacity of an adjuvant is also measured by the quality as well as the magnitude of the induced immune response, guided by the protective immune response required. Quality includes isotype and IgG subclass responses, T-helper cell responses characterized by the cytokine profile and cytotoxic T cells (CTL). In the early phase of immunization some adjuvants influence the antigen administration and uptake by a so-called depot effect exemplified by aluminium hydroxide gel and oil adjuvants, which possibly is not as desired as alledged. A modern depot is exerted by slow release formulations continuously releasing the antigen over a period of time or by pulses at intervals aiming at 'single injection' vaccine. Great efforts are made to formulate efficient delivery formulations targeting the antigens from the site of administration, to draining lymph nodes or distant lymphatic tissue or to mucosal surfaces by parenteral or mucosal administrations. Nowadays, non-replicating carriers besides replicating vaccines are formulated to induce mucosal immune responses encompassing secretory IgA and CMI. Efforts to evoke immune responses on mucosal membranes distant from the site of administration have resulted mostly in little success. For a long time it was considered that CTL under the restriction of MHC Class I only could be evoked by replicating viruses or intracellular parasites. However, novel adjuvant delivery systems readily induce CTL by delivering the antigen to the APC resulting in intracellular transport to the cytosol for the MHC Class I presentation system, as well as to the endosomal pathway for the MHC Class II presentation.

Adjuvants, Immunologic↗

PubMed1996

Kinetics, localization and isotype profile of antibody responses to immune stimulating complexes (iscoms) containing human influenza virus envelope glycoproteins.

The immune stimulating complex (iscom) is a particulate adjuvant formulation combining multimeric presentation of antigen with a built-in adjuvant, Quillaja saponin. Iscoms induce strong serum antibody responses that are readily boosted. To further characterize this property of iscoms, the development and maturation of primary and secondary antibody responses to iscoms containing influenza virus antigen were investigated, in serum by ELISA and on single B-cell level by ELISPOT. After a single subcutaneous injection, B cells secreting antigen-specific IgG (IgG-SC) were primarily observed in the draining lymph nodes (LN), showing peak numbers at day 7 which then declined rapidly. Serum IgG levels, as well as IgG-SC in the spleen, persisted for several weeks and, with time, IgG-SC cells also appeared in the bone marrow (BM). These results suggest that the IgG response to iscoms initially is located to the LN but that IgG-SC are redistributed with time and may persist for a long time in other organs, including the spleen and BM. Moreover, a booster dramatically enhanced the frequency of IgG-SC in LN, spleen and BM suggesting that iscoms induce a potent B-cell memory. Comparisons of antibody responses to iscoms with those to influenza virus antigen in Freund's complete adjuvant, TiterMax or aluminium hydroxide suggest that the choice of adjuvant influences both the magnitude, kinetics, localization and isotype profile of antibody responses.

Adjuvants, Immunologic↗

PubMed1996

Transport of an influenza virus vaccine formulation (iscom) in Caco-2 cells.

The influenza virus envelope glycoproteins hemagglutinin and neuraminidase were administered to the apical or basolateral sides of Caco-2 monolayers either as native protein micelles (mic-ag) or after incorporation into the orally active adjuvant formulation, immune stimulating complexes (iscoms) (isc-ag). Biotin-conjugated isc-ag were localized in intracellular vesicles as early as 2 min after administration to the apical side at 37 degrees C. Ten minutes after administration, both intracellular vesicles and intercellular spaces were labeled, and extracellular labeling was observed on the basolateral side of the cells, indicating that isc-ag were transported across the epithelium within 10 min of exposure. Transport of 125I-labeled isc-ag and mic-ag in the apical-to-basolateral and basolateral-to-apical directions across Caco-2 monolayers was comparable at 37 degrees C. Gel chromatography analysis revealed that only 0.55-3.1% of transported isc-ag and mic-ag had a molecular weight of > 5,000, while 21.0-42.3% was eluted at a position corresponding to peptides of approximately 10 amino acids. Although isc-ag and mic-ag were transported and degraded by Caco-2 monolayers in comparable amounts, only transported isc-ag induced a dose-dependent proliferative response in vitro of T cells primed with influenza virus antigen. High-performance gel chromatography and reverse-phase high-performance liquid chromatography indicated that transported antigenic isc-ag consisted of hydrophobic peptides with a molecular weight of < or = 3,000. These results indicate that antigens incorporated into the orally active adjuvant formulation iscom are degraded to antigenic peptides during transport across the intestinal epithelium.

Adjuvants, Immunologic↗

PubMed1995

The Ca2+ signaling capacity of the beta 2-integrin on HL60-granulocytic cells is abrogated following phosphorylation of its CD18-chain: relation to impaired protein tyrosine phosphorylation.

The phosphorylation state of the CD18-chain of beta 2-integrins have been shown not to mediate changes in the avidity of these receptors (i.e., inside-out signaling); however, no alternative functional significance has been proposed. Our study focused on how changes in the phosphorylation state of beta 2-integrin-receptors on HL60-granulocytic cells are related to its intracellular signal transduction properties (i.e., outside-in signaling). Engagement of beta 2-integrins on differentiated HL60 cells induced a transient increase in the cytosolic free Ca2+ concentration and an increased tyrosine phosphorylation of three major protein bands (70, 115, and 140 kDa). These signaling events occurred without any detectable phosphorylation of the CD18-chain. However, a strong phosphorylation of the CD18-chain by preexposure to phorbol myristate acetate (PMA) coincided with an abolishment of both the beta 2-integrin-induced Ca2+ signal and the protein tyrosine phosphorylations. By comparison, none of these effects were exhibited by 4-alpha-PMA, an analogue that does not activate protein kinase C. Thus, phosphorylation of the CD18-chain of beta 2-integrins is not required for outside-in signal transduction by these receptors, but it could constitute an effective mechanism by which the signaling properties of beta 2-integrins can be modulated by exogenous factors and possibly also by intracellular signals induced by other receptors. The fact that both the cytosolic free Ca2+ signal and protein tyrosine phosphorylations were abrogated by PMA suggests an intimate relationship between these two intracellular signals. To explore this possible relationship, we chelated the beta 2-integrin-induced Ca2+ signal with BAPTA. The beta 2-integrin-induced protein tyrosine phosphorylations were blocked by BAPTA but not by abolishment of the Ca2+ signal due to chelation with MAPT or by pretreatment with thapsigargin. These findings and the observation that pretreatment of cells with methyl-2,5-dihydroxycinnamate (a tyrosine kinase inhibitor) blocked the beta 2-integrin- but not the fMet-Leu-Phe-induced Ca2+ signal suggest that beta 2-integrin-induced tyrosine kinase activation occurs prior to and is a prerequisite for the subsequent Ca2+ signal.

CD18 Antigens↗