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N Lycke

Publications and source records attributed to N Lycke.

At least 55 records · Page 3Linked to original sources
PubMed1996

Differential activation requirements of isotype-switched B cells.

In the present studies, we compared the activation requirements of sIgM+/sIgD+ B cells with those of isotype-switched sIgM-/sIgA+ B cells. We found that whereas sIgM+ B cells respond to T cell-independent (TI) and T cell-dependent (TD) Ag with no significant bias toward one stimulus, sIgA+ B cells were deficient in their ability to respond to antigen receptor cross-linking but responded remarkably well to TD stimuli. Thus, dextran-conjugated anti-IgA antibody (anti-IgA-dextran), anti-kappa-dextran, or various immobilized anti-IgA antibodies (Ab) induced only low-level IgA B cell proliferation and no IgA secretion in the presence of various lymphokines; in marked contrast, sIgA+ B cells responded to cognate and noncognate T cell stimulation as well as to stimulation by CD40 ligand-bearing fibroblasts by secreting large amounts of IgA (up to 240 000 ng/ml per 10(5) cells). This pattern of sIgA+ B cell responsiveness was noted with both germinal center peanut agglutininhi (PNAhi) and non-germinal center PNAlo B cells. In confirmation of these results, whole Peyer's patch or lamina propria cell populations containing less than 15% sIgA+ B cells stimulated with a noncognate T cell stimulus or T cell membranes secreted mainly IgA (68%-94% of the total Ig secreted) and relatively little IgM. The strict T cell dependence of IgA B cell activation and differentiation provides important insights into immune responses of mucosal tissues and must be considered in the development of vaccines, particularly those designed to stimulate mucosal tissues containing large numbers of isotype-switched B cells.

Animals↗
PubMed1996

CD8-deficient mice exhibit augmented mucosal immune responses and intact adjuvant effects to cholera toxin.

We used normal, CD4 and CD8 gene-targeted mice to investigate the role of CD4+ and CD8+ T cells in the regulation of gut mucosal immune responses following oral immunizations with cholera toxin (CT) adjuvant. Phenotypic analysis of mucosa-associated tissues revealed normal CD3+ T-cell frequencies in CD4-/- and CD8-/- mice such that in CD4-/- mice the CD8+ and double-negative (DN) T cells were increased. In CD8-/- mice the CD4+ T cells were increased, with the exception that in the intraepithelial compartment the CD3+ T cells were predominantly DN gamma delta T-cell receptor (TCR)+ T cells. All mice, normal and deficient, failed to respond to oral immunization with the antigen, keyhole limpet haemocyanin (KLH), alone. In the presence of CT adjuvant, however, CD8-/- mice consistently exhibited three- to fivefold stronger gut mucosal responses compared to normal C57B1/6 mice. By contrast, no difference was observed for systemic responses between CD8-/- and normal mice. Thus the up-regulation selectively affected mucosal responses, suggesting that, contrary to the CD8-/- mouse gut, the normal gut mucosa may host CD8+ T cells that exert a local suppressive effect on T- and B-cell responses. The magnitude of the enhancing effect of CT was comparable in CD8-/- and normal mice, clearly demonstrating that the adjuvant mechanism of CT does not require CD8+ T cells. On the other hand, the adjuvant effect of CT required CD4+ T cells, because no or poor anti-KLH responses were observed in CD4-/- mice. In both normal and CD8-/- mice CT adjuvant promoted KLH-specific CD4+ T-cell printing without any selective effect on the differentiation towards a T-helper type-1 (Th1) or Th2 dominance. Furthermore, CT adjuvant increased the frequency of CD4+ T cells expressing a memory phenotype, i.e. CD44high, LECAM-1low and CD45RBlow. We have shown, using gene-targeted mice, that CD8+ T cells are not required for the adjuvant effect of CT, and that CD8+ T cells may exert local mucosal down-regulation of intestinal immune responses.

Adjuvants, Immunologic↗
PubMed1996

Local intravaginal vaccination of the female genital tract.

In a clinical trial the authors tested whether local intravaginal or oral vaccination would stimulate a mucosal immune response in the female genital tract. The whole cell/B subunit (CTB) oral cholera vaccine was used. Two groups of previously unimmunized volunteers were given three doses of vaccine at 2-week intervals: a first group of seven women received oral immunizations and a second group of seven women were immunized locally in the genital tract by mixing the vaccine with a well defined gel, eldexomer, and applying it directly in the fornix of the vagina. The women were given the first vaccination on day 10 of the menstrual cycle. Sampling of peripheral blood and of cervical mucus (CM) using an Aspiglaire syringe was performed immediately prior to the first dose and at 8-10 days following the last immunization. The study showed that while only three of the seven orally immunized women responded with detectable IgA and IgG anti-CTB antibodies in the genital tract, six out of the seven women in the locally vaccinated group responded with genital tract antibodies. The responses were also generally stronger and CM contained higher specific IgA and secretory component containing anti-CTB titres in the locally vaccinated group. Of the orally vaccinated individuals all responded with increases in serum anti-CTB IgG and 4,7 also exhibited specific IgA serum titres. By contrast, only 3/7 in the intravaginal group responded with increases in serum IgG and IgA anti-CTB titers following immunization. The authors conclude that local intravaginal vaccination using a well-defined gel appears to be the route of choice to stimulate immunity in the female genital tract.

Administration, Intravaginal↗
PubMed1996

Lack of interferon-gamma receptor does not influence the outcome of infection in murine schistosomiasis mansoni.

Studies of vaccine-induced immunity in experimental schistosomiasis in mice have suggested that interferon-gamma (IFN-gamma) is an important factor for the induction of protective immunity against schistosomiasis. The present study compares some parameters during primary schistosome infection in IFN-gamma receptor deficient mice and wild type mice. No significant difference in worm burden between the two groups was found. Almost the same number of eggs in the liver as well as typical granulomas with numerous macrophages and eosinophils were observed in both groups of mice. Furthermore, IFN-gamma receptor deficient mice infected with S. mansoni displayed a significant reduction in the number of IgG2a secreting cells in the spleen and a significant enhancement of IgA secreting cells in the spleen and in the lungs. These findings suggest that the lack of IFN-gamma activity may result in an enhanced dominance of Th2 cells which, however, does not influence the development of a primary schistosome infection.

Animals↗
PubMed1995

Impaired mucosal immune responses in interleukin 4-targeted mice.

Interleukin 4-targeted (IL-4-/-) mice are defective in T helper (Th)2 cytokine production as determined after nematode infection. As Th2 cells appear to be selectively induced by oral immunization we investigated the ability of IL-4-/- mice to respond to perorally administered antigen. We found that IL-4-/- mice failed to respond to soluble protein antigens given perorally together with cholera toxin (CT) as a mucosal adjuvant. In contrast to wild-type mice no or poor anti-keyhole limpet hemocyanin (KLH) or anti-ovalbumin (OVA) B cell responses were observed in gut lamina propria, spleen, or serum of IL-4-/- mice after oral immunization. In addition, mucosal immunization failed to stimulate antigen-specific T cell responses in these mice. The lack of responsiveness was specific for mucosal administration of antigen and was not seen after intravenous injections with antigen and CT-adjuvant. The systemic adjuvant effect of CT was not impaired in IL-4-/- mice as evidenced by the strong enhancement of anti-KLH responses after intravenous immunization with KLH plus CT as opposed to KLH alone. However, CT as an immunogen, in contrast to KLH or OVA, stimulated significant mucosal and systemic immune responses in IL-4-/- mice after oral immunization. Both serum and intestinal IgA anti-CT antibodies were demonstrable in IL-4-/- mice as well as in wild-type mice. Total IgA levels in gut lavage and in serum of immunized IL-4-/- mice were of similar magnitude as in wild-type mice, suggesting that the ability of naive B cells to undergo isotype switch-differentiation from IgM to IgA in IL-4-/- mice did not appear to be impaired. Immunohistochemical analysis of Peyer's patches demonstrated a complete inability to form germinal centers in IL-4-/- mice in contrast to wild-type mice. Our data suggest that IL-4-/- mice are unable to respond to oral/mucosal immunization due to a failure to stimulate antigen-specific cells required to induce germinal center reactions in the Peyer's patches. Our findings demonstrate that IL-4 and probably functional Th2 cells are required for induction of gut mucosal antibody responses.

Adjuvants, Immunologic↗
PubMed1995

Cholera toxin increases T lymphocyte responses to unrelated antigens.

We have found that CT adjuvant greatly augmented oral priming immunizations. Limiting dilution analysis revealed strong increases in the frequency of primed antigen specific T lymphocytes subsequent to the use of CT adjuvant. Moreover, several fold stronger lymphokine production accompanied the strong proliferative responses seen with CT-treated and KLH-primed T cells. The lymphokine pattern secreted by these T cells suggested that TH1 as well as TH2 types of cells were effectively primed by KLH, and that CT adjuvant did not exert a selective effect on any of these T-cell subsets. These effects of CT were truly due to immunomodulation and not simply the result of a more efficient uptake of gut luminal antigens in the presence of CT, because CT enhanced equally well T cell priming by peroral as well as parenteral immunization with KLH. Moreover, the CT-induced enhanced responsiveness to antigen was also observed with CD4(+)-enriched T cells suggesting that CT primarily affected T cell priming rather than the balance between CD4+ and CD8+ T cell subsets.

Adjuvants, Immunologic↗
PubMed1995

Mucosal memory B cells retain the ability to produce IgM antibodies 2 years after oral immunization.

In recent studies we have demonstrated that immunological B- and T-cell memory may be stimulated effectively by oral immunization, simply by admixing protein antigens with cholera toxin (CT) adjuvant. Here we extend information by employing a hapten-carrier system allowing us to separate B- and T-cell memory and to evaluate the requirement of memory T cells for effective reactivation of mucosal memory B cells. We found that 2 weeks following oral priming immunizations with dinitrophenyl-keyhole limpet haemocyanin (DNP-KLH) plus CT adjuvant, significant serum anti-DNP antibodies of IgG, IgA and IgM immunoglobulin classes were demonstrated. However, after 2 years only IgM anti-DNP antibodies could still be detected in serum. When memory lymphocytes were isolated from these mice, from both systemic and gut-associated lymphoid tissues, and challenged with antigen in vitro, vigorous IgM, but no IgG or IgA, anti-DNP production was observed. By contrast, when the DNP-KHL-primed memory mice were challenged in vivo by an oral booster immunization with DNP-KLH plus CT adjuvant, strong systemic IgG and local mucosal IgA anti-DNP responses were recorded, while IgM anti-DNP production was poor. Moreover, the mucosal memory B cells from DNP-KHL-immunized mice were more responsive in vivo to an oral booster immunization with the carrier-specific antigen, DNP-KLH, compared to that provided by an unrelated carrier, DNP-human serum albumin (HSA), which gave only poor mucosal and systemic anti-DNP B-cell responses. Taken together our data suggest that mucosal memory B cells are recirculating cells that have retained their ability to produce IgM antibodies and, therefore, have not undergone switch differentiation involving gene rearrangements with constant mu-chain deletions. Furthermore, mucosal B-cell memory and CD4+ T-cell memory are closely interconnected phenomena, requiring both components for effective expression and probably also for maintenance of immunological memory in the mucosal immune system.

Administration, Oral↗
PubMed1994

Production of large amounts of recombinant interleukins by cDNA transfected mouse myeloma cells cultured in dialysis tubing.

Studies of interleukin function often require large quantities of these highly expensive substances. The available interleukins are generally recombinant proteins produced in bacteria or yeast and, less commonly, interleukins produced by mammalian cells, which provide appropriate glycosylation and other post-translational modifications. Due to differences in biosynthesis, difficulties in production and purification the quality of the interleukin preparations may vary. We have taken advantage of the recently developed constitutively interleukin-secreting mouse myeloma cell lines and the dialysis tubing culture technique, which permit cells to be grown at high densities, in order to establish a method for the production of large amounts of recombinant murine IL-2 and IL-4. We show that these interleukins can be produced at low cost and in concentrations 20-30-fold higher than in conventional culture flasks. A single dialysis tubing culture will produce more than 10(6) U of interleukin which may be compared with the available commercial preparations containing between 10- and a 100-fold less per vial. The IL-2 and IL-4 produced in this manner are biologically active molecules as demonstrated by the strong proliferative response of clonal T cells and the isotype-switching effect in LPS-stimulated splenic B cell cultures. The dialysis tubing culture technique is a simple and highly cost-effective means of generating large quantities of biologically active interleukins and is especially suitable for research laboratories interested in functional studies of these proteins.

Animals↗
PubMed1994

Fusion proteins with heterologous T helper epitopes. Recombinant E. coli heat-stable enterotoxin proteins.

Fusion proteins containing specific B cell and T cell epitopes were used to examine how the intramolecular arrangement of T and B cell epitopes within a chimeric protein influences antigen-specific B cell antibody responses as well as specific T cell activation. Chimeric proteins, containing single or multiple copies of the Th epitope ovalbumin 323-339 (ova) linked at different positions to STa, the heat-stable enterotoxin of E. coli, were compared with respect to their ability to induce STa-specific antibody production and to induce ova-specific T cell activation. Chimeric proteins induced ova-dependent antibody production against STa at the amino terminal end, irrespective of the positioning of ova. Multiple tandem copies of ova in any position led to increased levels of antibody production against this epitope. In contrast, T cell help for antibody production against a second B cell epitope at the carboxy terminus of the fusion proteins was more effective after insertion of multiple copies of ova in a distal than in an adjacent position. A fusion protein, containing four copies of ova effectively elicited T cell help for antibody production against both examined B cell determinants, showing that activated Th cells recognizing a single epitope could simultaneously provide help for distinct sets of B cells specific for widely separated epitopes within a protein. T cell recognition of ova in all chimeric peptides, independently of its position, following the same pattern of genetic restriction (i.e. immunodominant in H-2d and nonimmunogenic in H-2k) as in the native ovalbumin molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Strategies for the induction of immune responses at mucosal surfaces making use of cholera toxin B subunit as immunogen, carrier, and adjuvant.

The concept of a common mucosal immune system, through which specific antigen-activated lymphocytes from the gut can disseminate immunity both along the intestinal tract and to various other mucosal and glandular tissues, has generated much current interest in the possibility of developing oral vaccines, not only for enteric infections but also for infections in the respiratory and urogenital tracts. However, to date it has proven difficult in practice to stimulate strong mucosal IgA immune responses by either parenteral or oral-mucosal administration of most antigens, and experience with soluble protein antigens has, on the whole, been disappointing. A notable exception in this regard is cholera toxin (CT) and in humans more than in other species, its nontoxic B subunit pentamer moiety (CTB). Based on this, CTB has become an important component in recently developed oral vaccines against cholera as well as against diarrhea caused by enterotoxigenic Escherichia coli producing CT-like heat-labile enterotoxin(s). Since the strong immunogenicity of CT and CTB can, to a large extent, be explained by their ability to bind to receptors on the intestinal mucosal surface, there has recently been much interest in approaches using CTB as an oral delivery carrier system for other vaccine-relevant antigens, and much progress has been made in preparing immunogenic hybrid proteins by coupling various protein or peptide antigens chemically or genetically to CTB. Indeed, in several systems, oral administration of such hybrid antigens has been found to markedly potentiate both intestinal and extraintestinal IgA immune responses against the CTB-coupled antigens and also to elicit substantial circulating antibody responses. Besides the mucosal immunopotentiating effect of either CT or CTB owing to their similar capacity as oral antigen-delivery vehicles, CT, but in most systems tested not CTB, also has strong adjuvant properties for stimulating mucosal IgA immune responses to admixed (not coupled) unrelated antigens after oral immunization. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT (and specifically of its A subunit) leading to enhanced cyclic AMP formation in the affected cell, and efforts to eliminate the enterotoxic activity without losing adjuvanticity have so far not met with success.

Adjuvants, Immunologic↗
PubMed1993

A novel large granular lymphocyte-like cell isolated from IL-2-supplemented murine intestinal lamina propria lymphocyte cultures with potent inhibitory action on lymphocyte proliferation.

Regulation and control of the local immune system in the gut mucosa are poorly understood phenomena. Recently we observed in whole alpha-CD3-driven, IL-2-supplemented, lamina propria (LP) T cell cultures that pronounced anergy developed concomitant with the appearance of large granular lymphocyte (LGL)-like cells. The LGL-like cells did not appear in the cultures in the absence of alpha-CD3 activation. These cells expressed Thy 1.2, NK1.1, AsGM1, CD3, and most often CD8 but were always negative for CD4. They exclusively required IL-2 to proliferate and survive in culture and several cell lines were established. Analysis of the regulatory ability of these cells on immune responses revealed that the gut LGL-like cells strongly inhibited T as well as B cell proliferation by releasing a soluble factor(s). Subsequent detection in the culture supernatants of cytokines with reported inhibitory properties on lymphocyte proliferation, interferon-gamma, and transforming growth factor beta correlated poorly to the inhibitory action of the supernatants. Despite the NK-like nature of these cells no or weak cytotoxic activity could be detected against Yac-1 target cells. The LGL-like cells expressed Fc receptors for IgE and demonstrated properties in EM and cytochemical analysis which resembled mucosal mast cells, but unlike such cells the LGL-like cells did not contain histamine or serotonin. The lamina propria of normal mouse small intestine was found to contain cells with morphology and staining pattern similar to those of cultured LGL-like cells, i.e., double-positive for Thy 1.2 and NK 1.1. We propose that this novel intestinal LGL-like cell, probably of T cell origin, may act as a potent suppressive cell in mucosal immune responses exerting a regulatory function on T cell activities and preventing adverse inflammatory reactions in the intestinal mucosa. Therefore, we suggest that these cells may be referred to as granulated inhibitory lymphocytes (GIL) cells. The lamina propria GIL cells exhibit many similarities to the recently described immunosuppressive decidual LGL cells in the placenta.

Animals↗
PubMed1993

Cholera toxin adjuvant greatly promotes antigen priming of T cells.

Cholera toxin (CT) given perorally is a powerful mucosal immunogen and adjuvant. Information that explains the adjuvant effect of CT may be used for the development of more effective oral vaccines and might also contribute to our understanding of the mechanisms involved in regulating mucosal immunity. The present study was undertaken to investigate if CT administered together with keyhole limpet hemocyanin (KLH) would act to promote or inhibit priming of KLH-specific T cells and whether the adjuvant effect of CT is restricted to mucosal immune responses or is a generalized phenomenon due to direct immunomodulating effects of CT. We found that CT adjuvant greatly augmented the effectiveness of a single oral priming immunization with KLH: re-challenge with KLH in vitro 1 week following immunization gave several-fold stronger proliferation in KLH-specific spleen, mesenteric lymph node, Peyer's patch and gut lamina propria T cells from KLH + CT adjuvant as opposed to KLH only-treated mice. Moreover, several-fold stronger cytokine production, i.e. interleukin (IL)-2, IL-4, IL-5, IL-6, IL-10 and interferon-gamma accompanied the enhanced proliferative response of T cells from CT adjuvant-treated mice. The adjuvant effect of CT was not restricted to mucosal immune responses but was evident also following a single parenteral immunization with KLH + CT. Limiting dilution analysis revealed that CT adjuvant promoted a 20- to 40-fold increase in the frequency of primed KLH-specific T cells. Phenotypic and functional analyses clearly demonstrated that CT adjuvant primarily enhanced priming of CD4+ rather than CD8+ T cells and the pattern of lymphokine secretion disclosed that CT most probably promoted antigen priming of both Th1 and Th2 type of CD4+ T precursor cells.

Adjuvants, Immunologic↗
PubMed1993

Cholera toxin and cholera B subunit as oral-mucosal adjuvant and antigen vector systems.

Cholera toxin (CT) and the analogous heat-labile enterotoxin (LT) from Escherichia coli have several immunomodulating effects which alone or in combination might explain their strong adjuvant action in stimulating mucosal IgA and other immune responses to admixed unrelated antigens after oral immunization. These effects include, depending on animal species and experimental systems, enhanced antigen presentation by a variety of cell types; promotion of isotype differentiation in B cells leading to increased IgA formation; and complex stimulatory as well as inhibitory effects on T-cell proliferation and lymphokine production. This adjuvant activity appears to be closely linked to the ADP-ribosylating action of CT and LT associated with enhanced cyclic AMP formation in the affected cells, and thus it may prove difficult to eliminate the enterotoxic activity without loss of adjuvanticity. However, through a separate mechanism, as an antigen-carrier system providing specific binding to epithelium including the M cells of intestinal Peyer's patches, both CT and its non-toxic binding subunit moiety (CTB) have been shown to markedly enhance the mucosal immune response to various foreign antigens or epitopes covalently linked to these molecules. This gives promise for the future use of CTB or related non-toxic binding derivatives as vehicles to facilitate induction of mucosal immune responses to a broad range of antigens for human vaccination purposes.

Adjuvants, Immunologic↗
PubMed1993

Cholera toxin enhances alloantigen presentation by cultured intestinal epithelial cells.

In the present study we show that cholera toxin (CT) strongly potentiates antigen presentation by intestinal epithelial cells, probably by enhancing co-stimulation. This was demonstrated in an allogeneic system using cells from the IEC-17 rat epithelial cell line as antigen presenting cells (APC). These cells were induced by optimal concentrations of IFN-gamma to express good amounts of Ia antigen and cultured for 24-48 h in the presence or absence of CT. Thereafter the cells were thoroughly washed and added to cultures containing MHC-incompatible spleen cells as responder cells. Epithelial cells exposed to CT demonstrated greatly enhanced ability to trigger allogen-specific T-cell proliferation as compared with IEC-17 cells treated with IFN-gamma alone. The mechanism for the enhanced APC function was investigated by analysing CT-treated IEC-17 cells for increased class II MHC antigen expression or enhanced production of cytokines with known co-stimulatory function. We found no significant increase in class II MHC antigen expression. By contrast, CT strongly promoted, in a dose-dependent fashion, the production of both IL-1 and IL-6 cytokines by IEC-17 cells as compared with untreated epithelial cells. This effect of CT was specific and not due to contaminating endotoxin because excess amounts of soluble toxin receptor, ganglioside GM1, added to the IEC-17 cultures completely abrogated the cytokine response to CT. These results together with our previous findings of enhanced antigen presentation by macrophages stimulated by CT suggest that the potent adjuvant function of CT for induction of mucosal immune responses might be attributed to an enhanced co-stimulating ability of several putative APC in the mucosal immune system: macrophages, B cells and epithelial cells.

Animals↗
PubMed1993

Stimulation of antigen-specific T- and B-cell memory in local as well as systemic lymphoid tissues following oral immunization with cholera toxin adjuvant.

In the present study we investigated immunological memory at the cellular level following oral immunization using cholera toxin (CT) as the mucosal adjuvant. We found that memory cells, isolated from mice orally primed with keyhole limpet haemocyanin (KLH) admixed with CT adjuvant 8 months earlier, responded by increased proliferation to antigen-challenge in vitro. In contrast, unstimulated memory cells or KLH-stimulated cells from naive mice did not respond. Memory cells were isolated from different lymphoid tissues; spleen (SP), mesenteric lymph nodes (MLN), Peyer's patches (PP) as well as the intestinal lamina propria (LP). Thus, oral immunization using CT adjuvant promoted the generation of memory cells that were present in both systemic and local intestinal lymphoid tissues. The demonstration of lymphokine production in the KLH-responsive cultures indicated the presence of antigen-specific memory T cells. Lymphokine production early in culture was dominated by interleukin-2 (IL-2), which peaked on day 2-3, followed by IL-5 and, in particular, interferon-gamma (IFN-gamma) which increased over time. Lamina propria memory cells were found to proliferate poorly to recall antigen in vitro compared to lymphocytes from SP or MLN. In contrast, very significant production of IL-5 and, in particular, IFN-gamma was demonstrable in LP cell cultures. The use of CT adjuvant also stimulated the generation of antigen-specific memory B cells following oral immunization. This was evidenced by KLH-specific antibody production in antigen-challenged memory lymphocyte cultures. The memory B cells produced IgM anti-KLH, while no detectable antigen-specific IgG or IgA was found. Unstimulated memory cells or naive cells failed to produce anti-KLH antibodies. These in vitro findings provide evidence that oral immunization using CT adjuvant stimulates both antigen-specific memory T and B cells. Furthermore, our data suggest the existence of memory B cells following oral CT adjuvant immunization which have retained the ability to produce IgM and which therefore probably have not undergone terminal isotype switch differentiation to other isotypes and thus have not deleted the mu constant heavy-chain gene. Finally, our data also suggest that memory T and B cells, either sessile in the various lymphoid tissues or recirculating, can be activated by antigen in situ in, for example, lymph nodes and spleen and, more importantly, in the intestinal LP itself.

Adjuvants, Immunologic↗