Biomedical subjects

A Sjölander

Publications and source records attributed to A Sjölander.

At least 19 recordsLinked to original sources

PubMed2006

Endogenous production of leukotriene D4 mediates autocrine survival and proliferation via CysLT1 receptor signalling in intestinal epithelial cells.

The cysteinyl leukotriene1 (CysLT1) receptor (CysLT1R) enhances survival and proliferation of intestinal cells via distinct pathways. Here, we have demonstrated that there is significant endogenous production of CysLTs from both non-tumour- and tumour-derived intestinal epithelial cells. Treatment of two non-tumour cell lines, Int 407 and IEC-6, with CysLT1R antagonists led to shrinkage and detachment of cells, confirmed as apoptotic cell death, and a dose-dependent reduction in proliferation. However, in the tumour intestinal cell lines Caco-2, SW480, HCT-116 and HT-29, treatment with CysLT1R antagonists significantly reduced proliferation, but had no effect on apoptosis. A unique characteristic of intestinal cancer cells is the presence of nuclear CysLT1Rs, which are inaccessible to receptor antagonists. In these cells, inhibition of the endogenous production of CysLTs indirectly, by 5-lipoxygenase inhibition, impaired CysLT1R signalling throughout the cell, and resulted in apoptosis of the tumour cells. These data reveal the existence of constitutive CysLT1R signalling that mediates both survival and proliferation in intestinal cells. Importantly, we propose that tumour-derived intestinal cells are resistant to CysLT1R antagonist-induced apoptosis, a phenomena that could be explained by nuclear CysLT1R signalling.

Apoptosis↗

PubMed2005

Pro-inflammatory mediator leukotriene D4 induces transcriptional activity of potentially oncogenic genes.

The inflammatory mediator LTD4 (leukotriene D4) is present at high levels in many inflammatory conditions, and areas of chronic inflammation have an increased risk for subsequent cancer development. We demonstrate here that following LTD4 stimulation, beta-catenin is translocated to the nucleus, triggering the transcriptional activity of the TCF (T-cell factor)/LEF (lymphoid enhancer factor) family of transcription factors. These events are dependent on phosphoinositide-3 kinase activation and glycogen synthase kinase inhibition. Our data suggest that, similar to Wnt signalling, LTD4 increases free beta-catenin and targets it to the nucleus.

Gene Expression Regulation↗

PubMed2002

A tyrosine kinase regulates propofol-induced modulation of the beta-subunit of the GABA(A) receptor and release of intracellular calcium in cortical rat neurones.

Propofol, an intravenous anaesthetic, has been shown to interact with the beta-subunit of the gamma-amino butyric acid(A) (GABA(A)) receptor and also to cause changes in [Ca2+]i. The GABA(A) receptor, a suggested target for anaesthetics, is known to be regulated by kinases. We have investigated if tyrosine kinase is involved in the intracellular signal system used by propofol to cause anaesthesia. We used primary cell cultured neurones from newborn rats, pre-incubated with or without a tyrosine kinase inhibitor before propofol stimulation. The effect of propofol on tyrosine phosphorylation and changes in [Ca2+]i were investigated. Propofol (3 microg mL(-1), 16.8 microM) increased intracellular calcium levels by 122 +/- 34% (mean +/- SEM) when applied to neurones in calcium free medium. This rise in [Ca2+]i was lowered by 68% when the cells were pre-incubated with the tyrosine kinase inhibitor herbimycin A before exposure to propofol (P < 0.05). Propofol caused an increase (33 +/- 10%) in tyrosine phosphorylation, with maximum at 120 s, of the beta-subunit of the GABA(A)-receptor. This tyrosine phosphorylation was decreased after pre-treatment with herbimycin A (44 +/- 7%, P < 0.05), and was not affected by the absence of exogenous calcium in the medium. Tyrosine kinase participates in the propofol signalling system by inducing the release of calcium from intracellular stores and by modulating the beta-subunit of the GABA(A)-receptor.

Animals↗

PubMed2001

Local immune responses to influenza antigen are synergistically enhanced by the adjuvant ISCOMATRIX.

The peripheral (draining) lymph node, as the primary site of immune induction, determines the course of systemic responses to an injected antigen. Lymphatic duct cannulation procedures in sheep were used to investigate local immunoreactivity to human influenza virus antigen (Flu ag) admixed with the adjuvant ISCOMATRIX (IMX). Compared to Flu ag or IMX alone, the co-administration of Flu ag and IMX (Flu ag+IMX) synergistically enhanced a number of immunological responses (lymphocyte and blast migration from the node, antigen-specific antibody levels and IL6 output in efferent lymph, and antigen-induced proliferation in cultured efferent lymph cells). Together, these results demonstrate that IMX is an immune modulator, and that lymphatic duct cannulation procedures may be used to evaluate antigen/adjuvant combinations for vaccine development.

Adjuvants, Immunologic↗

PubMed2001

Immune responses to ISCOM formulations in animal and primate models.

ISCOMs are typically 40 nm cage-like structures comprising antigen, saponin, cholesterol and phospholipid. ISCOMs have been shown to induce antibody responses and activate T helper cells and cytolytic T lymphocytes in a number of animal species, including non-human primates. Recent clinical studies have demonstrated that ISCOMs are also able to induce antibody and cellular immune responses in humans. This review describes the current understanding of the ability of ISCOMs to induce immune responses and the mechanisms underlying this property. Recent progress in the characterisation and manufacture of ISCOMs will also be discussed.

Animals↗

PubMed2001

The epsilon isoform of protein kinase C is involved in regulation of the LTD(4)-induced calcium signal in human intestinal epithelial cells.

We investigated the potential roles of specific isoforms of protein kinase C (PKC) in the regulation of leukotriene D(4)-induced Ca(2+) signaling in the intestinal epithelial cell line Int 407. RT-PCR and Western blot analysis revealed that these cells express the PKC isoforms alpha, betaII, delta, epsilon, zeta, and mu, but not betaI, gamma, eta, or theta;. The inflammatory mediator leukotriene D(4) (LTD(4)) caused the TPA-sensitive PKC isoforms alpha, delta, and epsilon, but not betaII, to rapidly translocate to a membrane-enriched fraction. The PKC inhibitor GF109203X at 30 microM but not 2 microM significantly impaired the LTD(4)-induced Ca(2+) signal, indicating that the response involves a novel PKC isoform, such as delta or epsilon, but not alpha. LTD(4)-induced Ca(2+) signaling was significantly suppressed in cells pretreated with TPA for 15 min and was abolished when the pretreatment was prolonged to 2 h. Immunoblot analysis revealed that the reduction in the LTD(4)-induced calcium signal coincided with a reduction in the cellular content of PKCepsilon and, to a limited extent, PKCdelta. LTD(4)-induced Ca(2+) signaling was also markedly suppressed by microinjection of antibodies against PKCepsilon but not PKCdelta. These data suggest that PKCepsilon plays a unique role in regulation of the LTD(4)-dependent Ca(2+) signal in intestinal epithelial cells.

Antibodies↗

PubMed2001

Reorganization of actin in neurons after propofol exposure.

BACKGROUND: It has previously been shown that propofol in clinically relevant concentrations induces a calcium-dependent conformational change in the cytoskeleton. The aim of this study was to further clarify the effect of propofol on the actin cytoskeleton and to determine if this conformational change is mediated by the interaction between the GABA(A)-receptor and propofol. METHODS: Primary cultured cortical neurons from newborn rats were treated with propofol 3 microg x ml(-1) in a time-response titration, with and without preincubation with the GABA(A)-receptor antagonist, bicuculline. Actin-protein content was detected by Western blot analysis and the cellular content of F-actin measured by a spectrophotometric technique. RESULTS: Propofol triggers a relatively slow statistically significant increase in the intracellular F-actin content, maximum after 20-min incubation (160%+/-16.3) (mean+/-SEM) P<0.05. The propofol-induced increase in F-actin was effectively blocked by bicuculline. The increase in intracellular actin content after exposure to propofol as well as the effect of bicuculline were verified by Western blot analysis. CONCLUSION: The present study shows that propofol triggers a time-dependent change of actin. Since this reorganization can be blocked effectively by a GABA(A)-receptor antagonist, this suggests that the GABA(A)-receptor is involved in the pathway leading to cytoskeletal reorganization after propofol treatment. The actin polymerization reached its maximum after 20 min. Therefore, we believe that the propofol-induced changes might be connected with slower cellular responses such as cell-to-cell interaction and/or channel regulation.

Actins↗

PubMed2001

Leukotriene D(4) affects localisation of vinculin in intestinal epithelial cells via distinct tyrosine kinase and protein kinase C controlled events.

Local inflammatory reactions affect the integrity of intestinal epithelial cells, such as E-cadherin-mediated cell-cell interactions. To elucidate this event, we investigated the effects of an inflammatory mediator, leukotriene D(4 )(LTD(4)), on the phosphorylation status and properties of vinculin, a multi-binding protein known to interact with both the E-cadherin-catenin complex and the cytoskeleton. Treatment of an intestinal epithelial cell line with LTD(4 )induced rapid tyrosine phosphorylation of vinculin, which was blocked by the Src family tyrosine kinase inhibitor PP1. Simultaneously, LTD(4) caused an increased association between vinculin and actin, and that association was decreased by PP1. LTD(4) also induced dissociation of vinculin from alpha-catenin without affecting the catenin complex itself. This dissociation was not blocked by PP1 but was mimicked by the protein kinase C (PKC) activator 12-O-tetradecanoylphorbol 13-acetate (TPA). Also, the PKC inhibitor GF109203X abolished both the LTD(4)- and the TPA-induced dissociation of vinculin from alpha-catenin. Furthermore, LTD(4) caused a colocalisation of vinculin with PKC-alpha in focal adhesions. This accumulation of vinculin was blocked by transfection with a dominant negative inhibitor of PKC (PKC regulatory domain) and also by preincubation with either GF109203X or PP1. Thus, various LTD(4)-induced phosphorylations of vinculin affect the release of this protein from catenin complexes and its association with actin, two events that are necessary for accumulation of vinculin in focal adhesions. Functionally this LTD(4)-induced redistribution of vinculin was accompanied by a PKC-dependent upregulation of active beta1 integrins on the cell surface and an enhanced beta1 integrin-dependent adhesion of the cells to collagen IV.

Actins↗

PubMed2000

Induction of lymphocyte recruitment in the absence of a detectable immune response.

Lymphocyte recruitment from blood into the lymph node is thought to be initiated by the presence of antigen. In this study, we have used lymphatic cannulation in sheep to demonstrate that the adjuvant ISCOMATRIX can induce dramatic lymph node activation in the absence of antigen. Consistent patterns of node shutdown (decreased output) and cell recruitment (increased output) with minimal blast cell responses were observed indicating that an antigen-specific immune response is not required. Production of IL-6, IL-8 and IFN-gamma, and the transient presence of red blood cells and neutrophils in the efferent lymph were associated with changes in efferent lymph cell trafficking. These early events may facilitate the screening of low frequency antigen-specific cells for binding to antigen and the subsequent amplification of the immune response.

Adjuvants, Immunologic↗

PubMed2000

Therapy of murine cutaneous leishmaniasis by DNA vaccination.

Prophylactic DNA vaccination protects mice against infection with Leishmania major by inducing an exclusive Th1 immune response dominated by the production of IFN-gamma. Here we show that DNA vaccines, initially designed to prevent infection, can also have a significant therapeutic effect. In L. major infected mice, vaccination with DNA encoding the Parasite Surface Antigen/gp46/M2 causes reduction in lesion size and promotes healing in both genetically resistant C3H/He mice and susceptible BALB/c mice. The therapeutic effect is underpinned by a shift in the T cell-derived cytokine environment with an increase in the IFN-gamma producing Th1 type cells. Application of such immunotherapy in conjunction with antiparasite drugs may result in faster or more certain cure of the disease in humans.

Animals↗

PubMed2000

Leukotriene D(4) triggers an association between gbetagamma subunits and phospholipase C-gamma1 in intestinal epithelial cells.

The proinflammatory mediator leukotriene D(4) (LTD(4)) binds to the seven-transmembrane receptor CYSLT(1). Although this leukotriene plays an important biological role, its intracellular signaling pathways are only partly known. In previous experiments, we found that LTD(4) induced tyrosine phosphorylation and translocation of phospholipase (PLC)-gamma1 to a plasma membrane fraction in a human epithelial cell line (Int 407). In the present study, we further examined these signaling events and found that LTD(4) induced a rapid interaction between Gbetagamma subunits and PLC-gamma1; results obtained with GST fusion proteins of PLC-gamma1 suggest that this interaction is mediated via the pleckstrin homology domain of PLC-gamma1. Moreover, LTD(4) induced an increased association of c-Src with PLC-gamma1, and the selective Src family tyrosine kinase inhibitor PP1 blocked both LTD(4)-induced tyrosine phosphorylation of PLC-gamma1 and the association of PLC-gamma1 with Gbetagamma subunits. The relevance of these observations in intracellular calcium signaling was investigated by microinjecting cells with anti-Gbeta, anti-PLC-gamma1, or anti-c-Src antibodies and by pretreatment with PP1. LTD(4)-induced calcium mobilization was blocked by each of the indicated antibodies (but not isotype-matched control antibodies) and by PP1. Our data suggest that Gbetagamma subunits can, directly or indirectly, serve as membrane-bound partners for PLC-gamma1 and c-Src and that each of these proteins is essential for LTD(4)-induced downstream PLC-gamma1 signaling.

Calcium↗

PubMed2000

Leukotrienes induce cell-survival signaling in intestinal epithelial cells.

BACKGROUND & AIMS: Inflammatory bowel conditions, particularly ulcerative colitis, are associated with an increased incidence of neoplastic transformation. High levels of proinflammatory leukotrienes (LTs) and up-regulated expression of cyclooxygenase (COX)-2 are characteristic of inflammation. Moreover, COX-2 has been implicated in cell survival and early colon carcinogenesis. Other aspects of interest for intestinal cell viability are the levels of beta-catenin and the antiapoptotic protein Bcl-2. We investigated the possibility that LTs participate in the regulation of these survival factors. METHODS: We used the human intestinal epithelial cell line Int 407 and the rat intestinal epithelial cell line IEC-6. Immunoblotting was applied to ascertain protein expression and distribution, and enzyme immunoassay methodology was used to measure prostaglandin E(2) (PGE(2)) production. Apoptotic ability was assessed by trypan blue exclusion, Hoechst staining, DNA fragmentation, and a caspase-3 activity assay. RESULTS: LTD(4) and LTB(4), but not LTC(4), caused a time- and dose-dependent increase in expression and/or membrane accumulation of COX-2, beta-catenin, and Bcl-2, as well as PGE(2) production. Apoptosis assays showed that the effects of LTs on these transformation-associated proteins correlated well with the ability of these LTs to reduce programmed cell death. CONCLUSIONS: The results suggest that inflammatory conditions are associated with the expression and distribution of proteins that are characteristic of transformed cells; such conditions may involve a signaling mechanism comprising an altered rate of apoptosis.

Animals↗

PubMed1999

General expression vectors for production of hydrophobically tagged immunogens for direct iscom incorporation.

A new general strategy for the production of recombinant protein immunogens has been investigated. The rationale involves the production of a recombinant immunogen as fused to a composite tag comprising one domain suitable for affinity purification and a hydrophobic tag designed for direct incorporation through hydrophobic interaction of the affinity-purified immunogen into an adjuvant system, in this case immunostimulating complexes (iscoms). Three different hydrophobic tags were evaluated: (i) a tag denoted IW containing stretches of hydrophobic isoleucine (I) and tryptophan (W) residues; (ii) a tag denoted MI consisting of the transmembrane region of hemagglutinin from influenza A virus; and (iii) a tag denoted PD designed to be pH-dependent in such a way that an amphiphatic alpha-helix would be formed at low pH. As an affinity tag, an IgG-binding domain Z derived from Staphylococcus aureus protein A (SpA) was used, and a malaria peptide M5, derived from the central repeat region of the Plasmodium falciparum blood-stage antigen Pf155/RESA, served as a model immunogen in this study. Three different fusion proteins, IW-Z-M5, MI-Z-M5 and PD-Z-M5, were produced in Escherichia coli, and after affinity purification these were evaluated in iscom-incorporation experiments. Two of the fusion proteins, IW-Z-M5 and MI-Z-M5 were found in the iscom fraction following preparative ultracentrifugation, indicating iscom incorporation. This was further supported by electron microscopy analysis showing that iscoms were formed. Furthermore, these iscom preparations were demonstrated to induce efficient M5-specific antibody responses upon immunization of mice, confirming successful incorporation into iscoms. The implications of these results for the design and production of subunit vaccines are discussed.

Amino Acid Sequence↗

PubMed1998

Uptake and adjuvant activity of orally delivered saponin and ISCOM vaccines.

Saponins are a highly heterogenous group of glycosides which are common in plants and have been known to have adjuvant properties since the 1920s. The immunostimulating complex or ISCOM is a particulate adjuvant/antigen delivery system. ISCOMs are open cage-like complexes typically with a diameter of about 40 nm which are built up by cholesterol, lipid, immunogen and saponins from the bark of Quillaia saponaria Molina (soap bark tree). ISCOMs and saponins are used as adjuvants in some commercial veterinary vaccines and have been examined as adjuvants in a large number of human experimental vaccines. This review describes the current status and potential of saponin and ISCOMs as adjuvants for orally-administered vaccines with special reference to the induction of local and systemic immune responses and interactions with the intestinal epithelium. The structure and composition of saponins and ISCOMs will also be reviewed.

Journal Article↗

PubMed1998

ISCOMs and other saponin based adjuvants.

Saponins are chemically a heterogeneous group of sterol glycosides and triterpene glycosides which are common constituents of plants. One source of triterpenoid saponins obtained from the bark of Quillaia saponaria Molina (the soap bark tree) have been known to cause substantial enhancement of immune responses since the 1920s. Despite their use in animal vaccines, the development of saponin-based formulations for human vaccines has been impeded by their complexity and concerns about toxicity. This review briefly covers the use of saponins for animal vaccines but focuses mainly on the development of these adjuvants for use in man. Important aspects include preparation of purified or highly defined saponin fractions, improved understanding of the relationships between adjuvant activity, toxicity and structure of saponins and formulation of saponins into structures with reduced toxicity such as ISCOMs. Recent developments in the understanding of cellular interactions, cytokine induction and the in vivo localisation of saponin containing formulations will also be reviewed.

Journal Article↗

PubMed1998

Induction of a Th1 immune response and simultaneous lack of activation of a Th2 response are required for generation of immunity to leishmaniasis.

Experimental systems based on immunization with plasmid DNA or immune-stimulating complexes were used to delineate the requirements for generation of protective immunity against murine leishmaniasis. Vaccination with plasmid DNA encoding the host-protective Leishmania major parasite surface Ag-2 primed for an essentially exclusive Th1 response that protected mice against L. major infection. In contrast, parasite surface Ag-2 in immune-stimulating complexes generated an immune response with mixed Th1-like and Th2-like properties that was not protective despite the activation of large numbers of CD4+ T cells secreting IFN-gamma. These results indicate that a Th1 response is sufficient to protect against cutaneous leishmaniasis, but the induction of a simultaneous Th2 response abrogates the Th1 effector function. DNA vaccines may therefore have an advantage for diseases in which protection depends on the induction of Th1 responses.

Animals↗

PubMed1998

ISCOMs: an adjuvant with multiple functions.

Aluminum salts are currently the only widely used adjuvant for human vaccines. Over the past 10-15 years, a large research effort has attempted to find novel adjuvants with ability to induce a broad range of immune responses, including cell-mediated immunity. The immunostimulating complex or ISCOM is one adjuvant with multiple adjuvant properties. ISCOMs are open cage-like complexes typically with a diameter of about 40 nm that are built up by cholesterol, lipid, immunogen, and saponins from the bark of the tree Quillaia saponaria Molina. ISCOMs have been demonstrated to promote antibody responses and induce T helper cell as well as cytotoxic T lymphocyte responses in a variety of experimental animal models, and have now progressed to phase I and II human trials. This review describes recent developments in the understanding of the structure, composition, and preparation of ISCOMs and will cover important aspects of the understanding of the adjuvant functions of ISCOMs and how they act on the immune system.

Adjuvants, Immunologic↗

PubMed1998

Leukotriene D4 induces a rapid increase in cAMP in the human epithelial cell line, Int 407: a potential role for this signal in the regulation of calcium influx through the plasma membrane.

Although the LTD4-induced Ca2+ influx in human epithelial cells has been shown to be regulated by a pertussis toxin-sensitive heterotrimeric G-protein, most likely a G alpha i3 protein [Adolfsson J.L.P., Ohd J.F., Sjölander A. Leukotriene D4-induced activation and translocation of the G-protein alpha i3-subunit in human epithelial cells. Biochem Biophys Res Commun 1996; 226: 413-419], the signalling pathway further downstream is still unclear. In the present study, we investigated the possible involvement of cAMP and protein kinase A activity in the LTD4-induced Ca2+ influx in the epithelial cell line Int 407. Stimulation with LTD4, but not with the calcium ionophore ionomycin, triggered a rapid increase (peak at 7 s) in the cellular cAMP level, an effect that was totally abolished by pertussis toxin. Furthermore, the LTD4-induced Ca2+ signal was reduced by 60% when cells that had been pre-incubated with the protein kinase A inhibitor Rp-cAMPS (50 microM for 30 min) were stimulated in a calcium containing medium. In contrast, Rp-cAMPS had no apparent effect on the LTD4-induced Ca2+ signal when the cells were stimulated in a calcium-depleted medium. The immediate LTD4-induced protein tyrosine phosphorylation (15 s), previously shown to be necessary for the subsequent Ca2+ influx, was abolished not only by pretreatment with pertussis toxin but also by exposure to Rp-cAMPS. Furthermore, direct activation of the cellular adenylyl cyclase activity by treatment with forskolin alone induced a prompt Ca2+ signal in the presence, but not in the absence, of extracellular Ca2+, identical results were obtained with the cell permeable cAMP analogue 8-bromo-cAMP. In addition, forskolin induced protein tyrosine phosphorylation similar to that seen with LTD4. These results suggest that protein kinase A activity participates in the regulation of the LTD4-induced Ca2+ influx at a site that is downstream of the activation of the pertussis toxin-sensitive G-protein but upstream of a LTD4-stimulated tyrosine kinase(s).

Adenylyl Cyclases↗