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K Blaser

Publications and source records attributed to K Blaser.

At least 163 records · Page 9Linked to original sources
PubMed1991

T cell subsets and their soluble products regulate eosinophilia in allergic and nonallergic asthma.

Lymphokines derived from activated T cells regulate the proliferation and postmitotic differentiation of eosinophils in vitro. We investigated whether peripheral blood eosinophilia, which is a characteristic feature of both allergic and nonallergic asthma, correlates with T cell activation and lymphokine production in asthmatic patients. Flow cytometric analysis of T cell activation markers revealed that asthmatic individuals are characterized by increased numbers of IL-2R (CD25)-bearing T cell subsets. The absolute number of IL-2R+ T cells correlated with the eosinophilia observed in the asthmatic patients. Purified CD4+ and CD8+ T cells from allergic and nonallergic asthmatic individuals spontaneously secreted factors that extend the lifespan of eosinophils in vitro. T cells from normal donors displayed this effect only after polyclonal stimulation with anti-CD3 antibody. The eosinophil lifespan-extending factors were also found in sera of asthmatic patients. Identification of these factors was performed by using neutralizing antibodies against IL-3, IL-5, and granulocyte-macrophage CSF. In sera, mainly IL-5 and granulocyte-macrophage CSF were responsible for prolonged eosinophil survival, whereas granulocyte-macrophage CSF was dominant in T cell supernatants. These results indicate that T cells and secretion of lymphokines play an important regulatory function toward eosinophils, which are thought to represent major proinflammatory effector cells in certain types of asthma.

Adult↗
PubMed1991

Activated T cells and eosinophilia in bronchoalveolar lavages from subjects with asthma correlated with disease severity.

Activated T-lymphocytes may regulate the eosinophilic inflammation of bronchial asthma. In the present study, we investigated T cell activation and eosinophilia in blood and bronchoalveolar lavage (BAL) in 17 patients with asthma not receiving steroid treatment. Compared to normal individuals, BAL from patients with asthma contained significantly increased numbers of both lymphocytes and eosinophils (EOSs). The lymphocytosis consisted of increased numbers of both CD4+ and CD8+ T cells, and these T cell populations expressed elevated levels of T cell activation markers (interleukin-2 receptor [CD25], HLA-DR, and very late activation antigen 1). Close correlation was found between numbers of BAL CD4+ IL-2R+ T cells and numbers of EOSs. Moreover, the numbers of activated T cells and EOSs were related to the severity of asthma as measured by impairment of FEV1 and increased methacholine bronchial responsiveness. We demonstrate in both blood and BAL a close correlation between T cell activation, eosinophilia, and severity of asthma, suggesting that recruitment and activation of lymphocytes and EOSs are fundamental to the pathogenesis of bronchial asthma.

Adult↗
PubMed1991

Cellular cytotoxicity mediated by isotype-switch variants of a monoclonal antibody to human neuroblastoma.

The biological property of an antibody is determined by its antigen binding characteristics and its isotype-related effector functions. We have established monoclonal antibodies of different isotypes by stepwise selection and cloning of the hybridoma CE7. The original CE7 secretes an IgG1/kappa (CE7 gamma 1) antibody that recognises a 185 kD cell surface glycoprotein expressed on all human sympatho-adrenomedullary cells. Isotype-switch variants were isolated in the following sequence: from the original CE7 gamma 1, CE7 gamma 2b variants were isolated, and from a CE7 gamma 2b variant CE7 gamma 2a variants were isolated. The antibodies of three different isotype variant cell lines possess identical antigen binding characteristics, but display distinct effector functions as demonstrated by antibody dependent cell-mediated cytotoxicity (ADCC). ADCC was performed with the neuroblastoma line IMR-32 as the target cells, and different FcR gamma positive cells were either freshly isolated from human peripheral blood leukocytes or cultured for 6-10 days and tested as potential effector cells. Tumour lysis mediated by monocyte-derived macrophages depended on the presence of CE7 gamma 2a antibodies; antibodies from the CE7 hybridomas of gamma 2b and gamma 1 isotypes were virtually inactive in ADCC assay. Pre-exposure of macrophages to rIFN-gamma enhanced their ADCC activity, a result that is compatible with the notion that the high affinity Fc IgG receptor (FcR gamma I/CD64) is involved in the triggering of ADCC in macrophages. In contrast to macrophages, mononuclear cells, nonadherent cells and monocytes displayed considerable non-specific lytic activity, which was little influenced by the presence of antibody regardless of the isotype added.

Animals↗
PubMed1991

Sputum eosinophils from asthmatics express ICAM-1 and HLA-DR.

Sputum from symptomatic asthmatics is a rich source of eosinophils from the respiratory tract. Following liquefaction of sputum with dithioerythritol (DTE), a cell suspension for indirect double immunofluorescence with flow cytometry was obtained. Eosinophils were identified using anti-CD9 fluorescein conjugate, and particular surface markers measured with the relevant mouse MoAb followed by goat anti-mouse immunoglobulin phycoerythrin conjugate. Blood and sputum eosinophil surface markers were determined in parallel from asthmatics not receiving steroid therapy. Sputum eosinophils were found to have considerably elevated levels of CD11b, a reflection of eosinophil activation. Sputum but not blood eosinophils were found to express ICAM-1 (nine out of 11 cases) and HLA-DR (eight out of 11 cases). Furthermore, following culture of normal blood eosinophils with pooled T cell supernatants, ICAM-1 and HLA-DR could be induced in vitro. The induction of eosinophil adhesion molecules such as ICAM-1 and HLA-DR may influence eosinophil localization and function in asthma.

Antigens, CD↗
PubMed1991

T cells and asthma. I. Lymphocyte subpopulations and activation in allergic and nonallergic asthma.

Abnormalities of peripheral-blood lymphocyte subsets and activation markers were detected in patients with both allergic and nonallergic asthma. Most allergic asthmatics were characterized by increased numbers of IL-2R+ helper T cells and CD23+ B cells. In contrast, nonallergic asthmatics showed increased numbers of IL-2R+ and HLA-DR+ helper and cytotoxic T cells, and a clear redistribution from naive (CD45RA+) to memory (CD45RO+) cells. The number of IL-2R+ T cells correlated with the number of CD23+ B cells in allergic asthma. These changes in the distribution and activation state of T cells suggest an active role for T cells in the pathogenesis of both allergic and nonallergic asthma.

Antigens, CD↗
PubMed1991

T cells and asthma. II. Regulation of the eosinophilia of asthma by T cell cytokines.

Peripheral blood eosinophilia of both allergic and nonallergic asthmatics was found to correlate with blood T cell activation and lymphokine production. A close correlation was shown between the increase of IL-2 receptor expressing T cells and the number of eosinophils. These in vivo activated T cells spontaneously released factors that prolonged eosinophil survival in vitro. The T cell derived lymphokines IL-5, GM-CSF and IL-3 were demonstrated to be responsible for prolonged eosinophil survival in vitro, and were identified in T cell supernatants and sera from asthmatics. In summary, T cell derived cytokines play an important regulatory function towards eosinophils in asthma.

Asthma↗
PubMed1990

An in vitro murine model of a penicillin specific IgE anamnestic response.

A new murine model system was developed to study hapten specific IgE anamnestic antibody forming cell (AFC) responses induced in vitro. BALB/c mice injected intraperitoneally with BPO-KLH (10 micrograms) in aluminum hydroxide gel (alum) on day 0 and 21 were killed on day 42. Spleen cells (4 X 10(7] were cultured for 0-8 days in the presence of BPO-KLH (0.25-2500 ng/ml), after which the numbers of BPO specific IgE AFC were enumerated in an ELISPOT assay at 37 degrees C using BPO-BSA coated plates. Peak BPO specific IgE anamnestic AFC responses (approximately 40 AFC/10(7) cultured cells) occurred when cells were cultured with 100 ng/ml BPO-KLH for 5 days; peak BPO specific IgG1 and IgA responses (approximately 3000 and approximately 280 AFC/10(7) cultured cells) were detected one day earlier with the same antigen concentration. No ELISPOTs of any isotype were detected if: cells were cultured in the absence of specific antigen; cells from alum treated or unsensitized mice were used; assay wells were coated with BSA alone; isotype specific antibodies were omitted; or assays were performed in the presence of cycloheximide or at 4 degrees C. Cellular interactions in IgE anamnestic AFC responses were studied using the newly developed model system. We found that these responses were partially T cell independent, and that spleens from sensitized and unsensitized adult mice, as well as neonatal mice, contained cells which suppressed these responses.

Animals↗
PubMed1990

Fine specificity and heavy chain V gene usage of antibodies to the phosphorylcholine hapten.

In the course of the immune response to PC-KLH in mice, two major groups of anti-PC antibodies are expressed which differ in fine specificity, Id expression and isotype preference. Group I antibodies react predominantly with the PC moiety of the hapten and display mostly the T15 Id. On the other hand, Group II antibodies recognize the PC hapten including the azophenyl spacer to the carrier and are T15 Id-. In order to investigate the relationship between the two antibody populations, we analyzed the composition of 20 anti-PC hybridomas of all isotypes with respect to their fine specificity and VH gene segment utilization. Characterization of the antibodies by hapten inhibition analyses with PC and p-nitro-phenyl-PC indicated that eight hybridomas secreted Group I and 12 Group II antibodies. Southern blot analysis of total genomic DNA using a germ-line JH probe demonstrated that, with the exception of aPC-111-1, all Group II cells utilized gene segments distinct from those of Group I. In contrast to the restricted VH gene usage in Group I hybridomas, a more pronounced heterogeneity in H chain V gene rearrangements in Group II cells was observed. We found three groups of hybridomas with identical VH gene rearrangements. Two hybridomas used a VH gene segment of the 7183 VH gene family. Three lambda 1 bearing Group II hybridomas shared a common hybridizing band. One of these, aPC-56-1, is known to utilize the same VH gene segment as MOPC 141. Finally, three cell lines, one of which (aPC-104-8) utilizes the VH1-DH-JH2 rearrangement, showed fragments of identical sizes. These results emphasize the independent origin of Group I and Group II anti-PC antibodies and demonstrate a larger germ-line repertoire of Group II antibodies as well as a less restricted use of particular VH genes relative to that of Group I.

Animals↗
PubMed1990

Fine specificity and isotype expression of anti-PC antibodies in BALB/c, MRL/Mp-lpr/lpr, and -(+)/+ mice at different ages.

The MRL/Mp congenic mouse strains develop autoimmune disease with age. We have investigated age- and autoimmune-related changes in fine specificity, isotype spectra and T15 idiotype expression of the anti-phosphorylcholine (PC) response in BALB/c, MRL/Mp- + and -lpr congenic mice and in (BALB/c x MRL/Mp-lpr) F1 hybrids. Two groups of anti-PC antibodies with distinct fine specificity are elicited in the memory response. Group I antibodies recognize the PC moiety and express the T15 idiotype. Antibodies of group II are specific for phenyl-phosphorylcholine and are found predominantly in the memory response. In the MRL/Mp-lpr and - + strains only a minor population of antibodies expresses the T15 idiotype at all ages. However, a third group of antibodies was observed which binds to PC-coated proteins and to Diplococcus pneumoniae R-36A. This binding was not inhibited by PC-chloride and appeared mainly in the memory response at old age. The isotype distribution among anti-PC antibodies was similar in all strains analysed. In the initial response primarily mu, gamma 3 and gamma 1 isotypes were produced, while in the memory response gamma 1 was dominant. Thus autoimmune defects and ageing result in altered anti-PC antibody and idiotype profile, probably related to altered states in both the T- and B-cell compartments.

Age Factors↗
PubMed1989

Quantification of IgG and IgG4 antibodies to bee venom phospholipase A2 by competitive inhibition in ELISA.

Phospholipase A2 (PLA) is the major antigen of bee venom. Individuals often stung by bees, such as bee keepers, show a restricted immune response mainly of anti-PLA IgG4 antibodies. In contrast, patients allergic to bee venom produce high levels of PLA-specific IgE. This isotype restriction, the clinical relevance and the well defined structure of the PLA antigen, provide a useful model for the study of the principles regulating isotype expression in the human antibody response. A fundamental requirement for such studies is the availability of quantitative and sensitive assays to measure PLA-specific antibodies. Here we describe an ELISA method for direct isotype-specific quantification of anti-PLA IgG and IgG4 antibodies. Serum containing anti-PLA IgG antibodies was added at a predetermined dilution to PLA coated microtiter plates. Then mouse monoclonal antibodies to human IgG or IgG4 and different concentrations of purified human IgG and IgG4, respectively, were added simultaneously. The concentration of anti-PLA IgG and IgG4 antibodies in the serum was calculated from the resulting inhibition curve. Additionally, an analytical method to compare unknown antibody samples with a standard in ELISA - avoiding problems of different affinities - is described. Using the technique described here, isotype-specific quantification of anti-PLA antibodies can be performed at a sensitivity of approximately 70 pg/ml with a reproducibility range of 10-15%.

Adult↗
PubMed1989

Production of human antibodies to bee venom phospholipase A2 in vitro.

Phospholipase A2 (PLA) is the major antigen of bee venom. Whereas individuals frequently stung by bees, such as bee keepers, show high levels of IgG4 anti-PLA antibodies in serum, most patients sensitive to bee venom possess increased IgE anti-PLA. We have established a culture system by which anti-PLA antibodies can be induced in vitro. Peripheral blood mononuclear cells were stimulated in a first step with PLA and/or pokeweed mitogen (PWM). After 3 days of culture the cells were washed and further incubated with fresh medium. Anti-PLA antibodies were estimated by an enzyme-linked immunosorbent assay (ELISA) and anti-PLA antibody secreting cells were counted by means of an ELISA plaque assay. Cells from bee keepers, but not those from normal donors, produced anti-PLA IgG in vitro. The isotype pattern of anti-PLA antibodies produced in vitro was identical to that found in serum of the respective donor. Anti-PLA IgM antibody-secreting cells developed at low frequencies in both bee keepers and normal donors whereas PLA-specific IgE could not be detected. The formation of anti-PLA IgG was suppressed by high amounts of antigen, while the IgM response was not affected. We conclude that PWM-induced antibodies reflect the in vivo situation of the donors and that anti-PLA IgG and IgM are regulated by different pathways and mechanisms.

Bee Venoms↗
PubMed1989

Age-dependent changes of isotype and idiotype expression in the antibody response to the phosphorylcholine hapten in BALB/c mice.

The distribution of antibodies among different isotypes in an immune response to a given antigen reflects the immunoregulatory linkage of T and B cell compartments, the genetic background of an individual and the functional state of its immune system. In addition, idiotypes are markers for the clonal origin of antibodies and their genetic relationship. Therefore, we have analyzed the isotype patterns and development of the major idiotype (T15) in BALB/c mice of different ages, immunized with a T-cell-dependent phosphorylcholine conjugate. The response was dominated by mu, gamma 3 and gamma 1 isotypes. The proportion of these antibodies changed in the progress of immunization but not with age in the primary response. An age-dependent change of the isotype distribution was observed only in the memory response. The T15 idiotype was dominantly expressed in the primary response and decreased in the memory response by 80-95% independently of the age of the mice. The results demonstrate that 2 populations of anti-phosphorylcholine antibodies which both prefer particular isotypes are expressed according to the functional state of the T and B cell compartments with age.

Aging↗
PubMed1989

Establishment of a memory in vitro murine IgE response to benzylpenicillin and its resistance to suppression by anti-IL-4 antibody.

Regulation of a memory IgE antibody response may be different from the induction of a primary response and may, therefore, be more relevant to the study of allergic diseases and the therapeutic manipulation of IgE antibody formation. In this paper a murine hapten-specific in vitro memory IgE antibody response to benzylpenicilloyl(BPO)-KLH is described. The response was analyzed by determining the number of antibody-producing cells (APC) in an ELISA spot assay. Of the total number of BPO-specific APC (10,000 APC/10(6) cultured spleen cells), about 1% were IgE-producing cells (100/10(6) cultured cells), as detected on day 6 of culture. The level of the antibody response is antigen dose-dependent, and the detected APC are BPO specific. The memory IgE response is not inhibited by the addition of anti-IL-4 antibody (11B11), even at a high excess. In the presence of the mitogen lipopolysaccharide, it has been shown that switch of B cells to IgE is induced by IL-4, a process which can be inhibited by anti-IL-4 antibody. Because the antigen-induced IgE response cannot be inhibited by anti-IL-4 antibody, in vitro responding cells derived from BPO-KLH-preimmunized mice may, therefore, have already switched in vivo to IgE. On the other hand, B cells switching to IgE in a situation of cognate T-B cell interaction might receive IL-4 in a transsynaptical way from T cells which might not be accessible to inhibition by anti-IL-4 antibody. The identification of the two possibilities in situations of established allergic disorders will be decisive for determining whether pharmacological inhibition of IL-4 (or IL-4-induced switch)--e.g., by putative low molecular weight compounds--will ever be a meaningful approach to suppress allergic diseases.

Animals↗
PubMed1988

Antibody response and acquired tolerance of A/J mice: age- and immunogen-related isotype differences.

In earlier work we have observed strain- and age-related diversity of immunological ageing in inbred mice pretreated with tolerogen or left without pretreatment and then immunized. Different isotypes show different age- and strain-related changes. Here, we extend this isotype analysis from C57BL/6 and SJL to A/J. By comparison with the first two of these, animals of strain A/J show very little age-related change, as judged by indirect plaque-forming response. We have found that in A/J, as in SJL and C57BL/6 mice, age-related changes are isotype-dependent. The age-related changes in isotype predominance and magnitude differ for different determinants. They depend on the structural relation between the tolerance-inducing or -sensitizing macromolecule and the immunogen.

Age Factors↗
PubMed1988

Characterization of the group I and group II antibody response against PC-KLH in normal and T15 idiotype-suppressed BALB/c mice.

The memory response of BALB/c mice to phosphocholine-keyhole limpet haemocyanin (PC-KLH) consists of two antibody populations, designated Group I and Group II, that differ in their fine specificity, as determined by hapten inhibition using phosphocholine (PC) and p-nitrophenylphosphocholine (NPPC). It is known that Group I response is dominated by T15 idiotype-positive antibodies that utilize the VH1 heavy-chain gene in combination with V kappa 22 light-chain gene. In this paper we show that Group II serum antibodies of BALB/c mice are also highly restricted in their heterogeneity, as determined by N-terminal amino acid sequence analysis. Furthermore, we demonstrate that the Group II response is not affected by neonatally induced T15 suppression, whereas the Group I response in these mice consists of T15-negative antibodies. This suggests that the expression of the two antibody populations is regulated independently. Finally, we show that the isotype distributions within a fine specificity are the same in normal and T15-suppressed mice. Interestingly this is true not only for the Group II but also for the Group I antibodies. Because the isolated Group I antibodies from normal mice differ in structure from those of T15-suppressed mice, i.e. different light chains, our data indicate that the isotype distribution of these two populations is associated with their fine specificity in addition to their clonal origin.

Amino Acid Sequence↗
PubMed1987

[Quantitative detection and elimination of neuroblastoma cells in vitro. Comparative study of the efficacy of an immunomagnetic method and lymphokine-activated killer cells].

Much effort is devoted to eliminating residual tumor cells - which escape detection by conventional methods - from remission bone marrow harvested for autologous transplantation. The quantitative determination of the efficacy of these purging methods is generally difficult. This report describes an in vitro reproducible tumor model. Normal mononuclear blood cells were deliberately contaminated with cells from the human neuroblastoma cell line SK-N-AS. The frequency of clonogenic SK-N-AS cells was determined in limiting dilution culture before and after treatment. Two methods of purging these tumor cells from the mixed cell suspension were compared, 1) an immunomagnetic method taking advantage of the existence of a monoclonal antibody (mAb) specific for 90-95% of the tumor cells, and 2) a method based on cell mediated cytotoxicity testing the activity of previously lymphokine activated killer (LAK) cells. Immunomagnetic purging eliminated 90% (1 log) of all SK-N-AS cells and 99% (2 log) of the mAb-binding clonogenic tumor cells. In contrast, LAK cell treatment removed only between 0.1 to 0.3 log (23-50%) of the clonogenic SK-N-AS cells.

Antibodies, Monoclonal↗
PubMed1987

Estimation of heterokaryon formation and hybridoma growth in murine and human cell fusions.

Four mouse myelomas commonly used for cell fusions (X63.Ag8.653, SP2/0, NS1, P3U1), 3 human myeloma-like cell lines (ARH77, U-266, GM1500) and 3 human x mouse hybridomas (SPAZ4, SA2, SA3) were compared for their heterokaryon formation and successful hybridoma growth after cell fusion with polyethylene glycol. The cells were stained with different fluorescent dyes which do not alter hybridoma growth or antibody secretion. After fusion myeloma cells containing at least 1 nucleus from a lymphocyte (heterokaryons) were counted from fluorescence photomicrographs and the heterokaryon frequency was calculated. Mouse myelomas fused at a frequency of 1-7%, whereas human myeloma lines showed a higher heterokaryon frequency ranging from 3-25%. In mouse fusions almost every well contained growing hybridomas showing a minimum hybridoma frequency of 2/10(6) lymphocytes. In human fusions the SPAZ4 and SA2 lines showed a heterokaryon frequency nearly as good as mouse myelomas, whereas U-266 yielded no growing hybridomas despite 20% heterokaryon frequency. Furthermore, human cell lines showed a high tendency of multikaryon formation whereas this phenomenon was rarely observed with murine and murine x human heterohybrids. In individual fusion experiments no correlation was found between heterokaryon formation and the number of growing hybridomas. Thus, our study shows that defects in hybridoma growth may not always result from lack of a successful fusion and human hybridomas might be more sensitive to post-fusion conditions.

Animals↗