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Biomedical subjects

A Radbruch

Publications and source records attributed to A Radbruch.

At least 19 recordsLinked to original sources
PubMed1997

Organ culture of human lymphoid tissue. II. Marked differences in cytokine production and proliferation between slice and suspension cultures of human spleen.

Recently, we have established a method for the culture of human spleen slices in vitro. The procedure allows thin slices (200-350 microns) of human spleen to be cultured for up to 7 days. Using this method, we have previously established that unstimulated spleen slices spontaneously synthesize and secrete considerably higher levels of immunoglobulin than suspension cultures of the same tissue run in parallel. In this study, we report that there are also marked differences in the cytokine secretion profile between slices and suspensions and in their proliferative response. In brief, control and PHA-stimulated spleen slices secrete high levels of IL-1 beta, IL-6, IL-8 and IL-11 while the levels found in suspension supernatants are appreciably lower. By way of contrast, high levels of IL-2, IL-4, IL-10 and TNF alpha are found in suspension culture supernatants following PHA stimulation while the response in slice cultures is extremely low. These differences are also reflected in the results obtained at the cellular (intracellular cytokine) level. Additional studies reveal that spontaneous immunoglobulin production observed in spleen slices can be inhibited by the addition of specific antibodies to IL-1 beta, IL-6 and TNF alpha and that the bulk of the IL-6 and IL-1 beta detected in culture supernatants represents de novo synthesis. Finally, the background and mitogen-stimulated proliferative response of tissue slices is meagre compared with that observed in spleen suspensions suggesting that proliferation in the former is held under strict control. Collectively, we believe that the tissue slice procedure described provides us with a system for studying integrated events in lymphoid tissues in vitro and evaluating immunomodulatory substances of potential clinical importance.

Adolescent↗
PubMed1997

CD45RA-expressing memory/effector Th cells committed to production of interferon-gamma lack expression of CD31.

It has been considered before that human naive and memory/effector CD4+ T-cells cannot be subdivided solely according to the differential expression of CD45 isoforms. By the lack of expression of CD31 we have identified a subset of CD4+ CD45RA+ CD31- cells which show distinct features of antigen-experienced Th1 cells. Short term stimulation of highly purified human peripheral blood CD4+ T-cells with PMA/ionomycin, followed by the cytometric analysis of intracellular cytokines, showed that a minor subpopulation of CD4+ CD45RA+ CD45RO- cells is able to produce interferon-gamma (IFN-gamma) rapidly, a characteristic of antigen-experienced Th1 cells. Whereas among CD45RA+ CD4+ T-cells both CD31+ and CD31- subsets produce interleukin-2 (IL-2) upon PMA/ionomycin stimulation, only the CD31- subpopulation is able to produce IFN-gamma. Thus, our phenotypic and functional characterization of CD45RA+ CD45RO- Th cells shows that CD45RA+ CD45RO- cells do not represent a homogeneous population of antigen-unexperienced, naive T-cells. We speculate that a certain subset of human CD4+, CD45RO+ memory T-cells reverts to expression of the CD45RA isoform, and that this subset can be identified by the lack of CD31 expression.

Animals↗
PubMed1997

P- and E-selectin mediate recruitment of T-helper-1 but not T-helper-2 cells into inflammed tissues.

When activated, T helper cells differentiate into one of two subsets, Th1 and Th2, characterized by distinct profiles of cytokine production. Th1 cells activate pro-inflammatory effector mechanisms involved in protection and autoimmunity, whereas Th2 cells induce humoral and allergic responses and downregulate local inflammation. Apart from differences in the repertoire of cytokines, no phenotypic attributes are established that distinguish the two subsets. Here we show that Th1 cells, but not Th2 cells, are able to bind to P-selectin and E-selectin. Moreover, only Th1 cells can efficiently enter inflamed sites in Th1-dominated models, such as sensitized skin or arthritic joints, but not in a Th2-dominated allergic response. Immigration of Th1 cells into inflamed skin can be blocked by antibodies against P- and E-selectin. These results provide evidence for adhesion mechanisms to distinguish between the two T helper subsets and mediate their differential trafficking. They indicate that selective recruitment is an additional level of regulation for both effector function profile and character of a local immune response.

Animals↗
PubMed1996

Specific expression of surface interferon-gamma on interferon-gamma producing T cells from mouse and man.

Interferon (IFN)-gamma is a potent immunoregulatory protein secreted by CD4+ and CD8+ T cells and by natural killer cells. Here, we show that IFN-gamma is specifically displayed at a low concentration on the cell surface of those activated T cells from mouse and man which express IFN-gamma. It is transiently expressed on the cell surface with kinetics similar to those of intracellular IFN-gamma expression. Detectable surface IFN-gamma is not expressed by activated T helper (Th) cells producing other cytokines but which do not express IFN-gamma. Thus, surface IFN-gamma is the first available marker for live T lymphocytes expressing IFN-gamma, e.g. Th1 cells.

Animals↗
PubMed1995

Isolation of full-size mRNA from ethanol-fixed cells after cellular immunofluorescence staining and fluorescence-activated cell sorting (FACS).

Preparation of intact, full-size RNA from tissues or cells requires stringent precautions against ubiquitous and rather stable RNases. Fluorescence-activated cell sorting (FACS) usually aims at the isolation of cells according to cell surface markers on living cells, from which RNA can be obtained by standard protocols. The separation of cells according to intracellular immunofluorescence markers, such as intranuclear, intracytoplasmic, or secreted molecules, requires permeation of the cell membrane for the staining antibodies, which is usually achieved by fixation. However, commonly used fixatives such as ethanol, methanol, or formaldehyde do not inactivate RNases completely, thereby hampering the analysis of complete RNA molecules from fixed cells. We report isolation of intact, full-size RNA suitable for Northern blotting from cells that were fixed by 95% ethanol/5% acetic acid containing RNase inhibitors, stained intracellularly, and sorted by FACS.

Acetates↗
PubMed1995

Switch transcripts in immunoglobulin class switching.

B cells can exchange gene segments for the constant region of the immunoglobulin heavy chain, altering the class and effector function of the antibodies that they produce. Class switching is directed to distinct classes by cytokines, which induce transcription of the targeted DNA sequences. These transcripts are processed, resulting in spliced "switch" transcripts. Switch recombination can be directed to immunoglobulin G1 (IgG) by the heterologous human metallothionein IIA promoter in mutant mice. Induction of the structurally conserved, spliced switch transcripts is sufficient to target switch recombination to IgG1, whereas transcription alone is not.

Animals↗
PubMed1995

Analysis and sorting of live cells according to secreted molecules, relocated to a cell-surface affinity matrix.

We have developed a technology for analysis and sorting of live cells according to secreted molecules. An artificial affinity matrix, specific for the secreted product of interest, is created on the cell surface, and the cells are allowed to secrete for a defined time period. The secreted molecules bind to the affinity matrix on the secreting cell and are subsequently labeled with specific fluorescent or magnetic staining reagents for cytometric analysis and cell sorting. Crossfeeding of the secreted products to other cells is prevented by decreasing the permeability of the incubation medium. This approach will have a wide range of applications in biotechnology and biomedical research. Here, we describe analysis and sorting of hybridoma cells, according to secreted antibodies, and of activated T lymphocytes, according to secreted cytokines.

Animals↗
PubMed1995

Monoclonal equine IgM and IgG immunoglobulins.

In order to define equine immunoglobulins (Igs) and to produce monoclonal reference Igs we fused equine peripheral blood mononuclear cells with X63-Ag8.653 non Ig producing murine myeloma cells. A total of 29 equine Ig producing equi-murine heterohybridomas were obtained, of which ten expressed equine Ig for more than 3 months. One of these heterohybridoma lines produced monoclonal IgM, an equine isotype which has not been available in monoclonal form before. Four lines secreted equine IgG of two distinct Ig heavy chain types as assessed by the molecular weight (MW), while the remaining five lines expressed only equine Ig light chains. A sixth Ig light chain expressing variant was obtained by cloning of one of the IgG producing heterohybridoma lines. These monoclonal IgGs were compared with previously described equine IgG monoclonal antibodies (mabs) by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). According to their MW we identified five groups of gamma-chains and four groups of Ig light chains.

Animals↗
PubMed1995

Detection and isolation of rare cells.

Cells of biomedical interest are often present in very small numbers (i.e. they are rare), despite their functional significance. The analysis and isolation of previously inaccessible rare cells, such as peripheral hematopoietic stem cells, fetal cells in maternal blood, residual tumor cells or antigen-specific lymphocytes, has now become feasible through the development of new methods. Today, these techniques allow the detection, isolation and analysis of cells less frequent than one in a million. Not many problems in immunology would require higher resolution.

Animals↗
PubMed1995

Isolation and characterization of allergen-binding cells from normal and allergic donors.

BACKGROUND: Flow cytometry of the immune system so far has been limited to the analysis of subpopulations according to lineage markers. The cells involved in a particular immune response could not be assayed due to their low frequency. Here we show the potential of antigen-specific high gradient magnetic cell sorting to enrich cells for visualisation in multiparameter cytometry, functional studies and immortalization. OBJECTIVES: The aim of this study was the development of an efficient technology for staining and isolation of antigen-binding cells from human peripheral blood. In particular, allergen-specific cells from normal and allergic donors should be analysed and compared to develop a cellular diagnosis of allergy. STUDY DESIGN: The rare antigen-specific cells were sorted by high-gradient magnetic cell sorting with MACS. Haptenized phospholipase A2 (PLA2), the major allergen of bee venom, or haptenized ParoI, the major allergenic component of Parietaria officinalis, were used as antigens. The cells from normal and allergic donors, binding to the allergen were characterized phenotypically by immuno-fluorescence. Allergen-specific B-cells were immortalized by EBV transformation. RESULTS AND CONCLUSION: Allergen-specific cells can be enriched from blood of both allergic and normal donors to purities of up to 75%, by high gradient magnetic cell sorting. The specificity of labelling with allergen was confirmed by establishing allergen-specific EBV-transformed B-cell lines from the sorted cells. Clear differences exist in the cellular composition of allergen-binding cells from normal compared to allergic donors. In normal donors the allergen-binding cells are B-cells expressing CD19 and CD21. In allergic donors, in addition to allergen-binding B-cells, occurring in about equal absolute numbers as in normal donors, basophilic granulocytes are labeled by allergen. These cells express CD38, CD9 and CD25 on their surface, and stain for IgE.

Allergens↗
PubMed1995

Magnetofluorescent liposomes for increased sensitivity of immunofluorescence.

BACKGROUND: Immunofluorescence and immunomagnetism are important technologies for analysis and sorting of cells according to specific marker molecules. Due to the limited sensitivity at least several thousands of antigens per cell are required for optical detection. Molecules expressed at low copy numbers cannot be analysed, although they may be of considerable functional importance. OBJECTIVES: Development of a magnetic and fluorescent staining reagent for analysis and sorting of cells according to antigens expressed at low number. To this end, uniformly sized, antibody-conjugated liposomes loaded with large amounts of dye molecules and small magnetic particles were generated. STUDY DESIGN: A method for the preparation of homogeneously sized large unilamellar liposomes which contain carboxyfluorescein, magnetic particles and surface-bound antibodies had to be developed. These liposomes were then tested for their ability to enhance immunofluorescence compared to conventional staining in a model system and by staining of CD25 on resting B and T cells. RESULTS AND CONCLUSION: Large unilamellar liposomes, homogeneous in size and loaded with fluorescein and magnetic beads can be prepared by combining membrane extrusion and magnetic filtration. Hapten-specific antibodies conjugated to their surface make them a universal tool for immunofluorescence. With such liposomes, intensity of fluorescent staining can be increased 100-1000-fold without increased background fluorescence, compared to conventional fluorochrome-conjugated antibodies. Due to the simultaneous magnetic labelling, stained cells can easily be isolated by MACS. The magnetofluorescent liposomes proved to be useful for improvement of sensitivity of detection and physical separation in general and to visualize and sort cells according to antigens expressed at low levels. The high affinity IL2 receptor CD25 is expressed in low copy number on a significant fraction of resting B and T lymphocytes in human peripheral blood, as can be shown exclusively by the magnetofluorescent liposomes.

Antibodies↗
PubMed1995

How cytokines control immunoglobulin class switching.

B lymphocytes can alter the class of antibody they produce by immunoglobulin class switch recombination. This recombination is targeted by distinct cytokines to particular switch regions. Prior to switch recombination, the same cytokines induce transcription through the targeted switch regions and generate IH "switch" transcripts. To show whether the two events are functionally related, we have replaced the endogenous interleukin-4 (IL-4) dependent promoter of murine I gamma 1 switch transcripts by an heterologous promoter, the human metallothionein IIA (hMT) promoter. Indeed, switch recombination can be targeted to IgG1 by the hMT promoter. In mutant mice, which cannot generate processed switch transcripts, switch recombination cannot be targeted to IgG1 by the hMT promoter. Thus, IL-4 targets switch recombination to IgG1 by induction of processed switch transcripts.

Animals↗
PubMed1994

Frequency of immunoglobulin E class switching is autonomously determined and independent of prior switching to other classes.

Both, in humans and in mice, a major fraction of immunoglobulin E (IgE)-expressing B lymphocytes develops by sequential Ig class switching from IgM via IgG to IgE. This sequential class switch might have functional implications for the frequency and repertoire of IgE+ cells. Here we show that in mutant mice, in which sequential switching to IgE via IgG1 is blocked, the frequency of cells switching to IgE is not affected. Thus, sequential class switching to IgE merely reflects the simultaneous accessibility of two acceptor switch regions for switch recombination, induced by one cytokine, but with markedly distinct efficiency. Analysis of switch recombination on both IgH alleles of switched cells shows that the low frequency of switching to IgE is an inherent feature of the S epsilon switch region and its control elements.

Animals↗