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Biomedical subjects

S Ansorge

Publications and source records attributed to S Ansorge.

At least 127 records · Page 7Linked to original sources
PubMed1991

Induction of protein disulphide-isomerase and immunoglobulins by pokeweed mitogen in human lymphocytes.

The Protein disulphide-isomerase (PDI, EC 5.3.4.1, Thiol-proteindisulphide oxidoreductase, EC 1.8.4.2) is thought to regulate the sulfhydryl status of cells and to catalyze thiol/disulphide exchange reactions involved in the post-translational processing of disulphide containing secretory proteins. The aim of the present investigations was to study the possible function of this enzyme in differentiation of B lymphocytes and immunoglobulin synthesis. Non-adherent human mononuclear cells or purified T cells were cultured in presence and absence of Pokeweed mitogen over 3, 5 and 7 days. Monoclonal antibodies and a rabbit polyclonal antiserum specific for human liver PDI were produced to determine the concentration of PDI by an ELISA technique and cytoplasmic immunofluorescence. After PWM stimulation, both, the cellular content of PDI as well as that of immunoglobulin, particularly IgM, have been found to be induced in a time dependent manner with a 2-3 fold increase in comparison to unstimulated cells. The specific induction of PDI in human B lymphocytes was also confirmed in Western blotting. Our findings suggest that PDI plays a critical role in the final stages of B cell differentiation and immunoglobulin synthesis by activated B cells and plasma cells, respectively.

Antibodies, Monoclonal↗
PubMed1991

Dipeptidyl peptidase IV in the immune system. Effects of specific enzyme inhibitors on activity of dipeptidyl peptidase IV and proliferation of human lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase playing a significant role in the process of activation and proliferation of human thymus-derived lymphocytes. This conclusion is drawn from (1) the induction of this enzyme on mitogen-activated T lymphocytes (cf. Schön, E. & Ansorge, S. (1990) Biol. Chem. Hoppe-Seyler 371, 699-705) and (2) the impairment of different functions of activated T cells in the presence of specific inhibitors and antibodies against DP IV (Schön, E. & al. (1987) Eur. J. Immunol 17, 1821-1826). This paper is aimed at testing new active site-specific peptide inhibitors for their efficiency as inhibitors of lymphocyte DP IV and DNA synthesis of mitogen-stimulated lymphocytes. These inhibitors comprise (i) diacylhydroxylamine derivatives of Xaa-Pro or Xaa-Ala peptides, (ii) different oligopeptides with N-terminal Xaa-Pro-sequences, and (iii) amino-acid amides of the pyrrolidide and the thiazolidide type. The thiazolidides of epsilon-(4-nitrobenzyloxycarbonyl)-L-lysine and of L-isoleucine as well as Ala-Pro-nitrobenzoylhydroxylamine are the most effective inhibitors in both test systems, yielding half-maximal inhibitory concentrations in the micromolar range. Cell viability was not impaired in this effective concentration range. Other inhibitors of DP IV are one to two orders of magnitude less efficient in the suppression of lymphocyte proliferation.

Binding Sites↗
PubMed1991

Membrane-bound peptidases of lymphocytes: functional implications.

The paper is aimed towards the role of the membrane ecto-enzymes aminopeptidase N (CD13, AP-N) and dipeptidyl peptidase IV (CD26, DP IV) in the immune system. Both peptidases have been identified on T lymphocytes, AP-N also on monocytes and non-T cells. Using enzyme inhibitors and antibodies it has been shown by different groups that DP IV plays a key role in T cell activation and growth. Inhibition studies of our laboratory, using bestatin, actinonin and probestin, also demonstrated an essential role of AP-N in regulation of T cell growth. Moreover, the action of cytokines/lymphokines having DP IV susceptible bonds, as IL-1, IL-2 and IL-6, on lymphocyte proliferation, was found to be suppressed by specific inhibitors of DP IV as well as AP-N. These results are in favour of our hypothesis that DP IV and AP-N, possibly both in a concerted action, are involved in cytokine/lymphokine mediated signalling between immune cells.

Amino Acid Sequence↗
PubMed1991

[Measurement of cellular activity with the MTT test. Optimization of the method].

Since a few years activity of mitochondrial dehydrogenases has been determined by MTT-test as alternative method to measurement of the cellular activity and proliferation by incorporation of tritiated thymidine. In this test the tetrazolium salt MTT is converted into blue, insoluble formazan dye crystals, which have to be dissolved by a suitable extraction mixture. The present paper describes a modified extraction method using an isopropanol-dimethylformamide mixture acidified to pH 5.5. The modified method enables an optimal, easy to handle, less time and work consuming MTT-assay. A MTT concentration of 1 mg/ml was found to be optimal. The extinction maximum was identified at 566 nm.

Cell Line↗
PubMed1991

[Immunochemical determination of human thiol protein disulfide oxidoreductase in cell and tissue homogenates by competitive EIA].

Different monospecific antisera against thiol-protein disulfide oxidoreductase (TPO, EC 1.8.4.2, protein-disulfide isomerase, EC 5.3.4.1) were raised in rabbits by immunization with purified human TPO and characterized by means of Laurell and immunoblot techniques. A competitive anti-TPO-EIA with insolubilized TPO has been used to determine this enzyme in cells and tissue homogenates. The assay shows a sensitivity of 1.2 ng/ml and a specificity of about 99%. The TPO content in relation to the total protein was found to be: in pancreas 0.65%, liver 0.45%, spleen 0.12%, placenta 0.16%, tonsils 0.06% and lymph nodes 0.03%.

Blotting, Western↗
PubMed1991

[Biofreezer BF 80--a universally employed apparatus in preservation at very low temperatures].

A computer-controlled freezing apparatus is described which uses liquid nitrogen (LN2) as coolant. The freezing equipment BF 80 consists of a big cryochamber (volume 24 l), a LN2 tank and a computer for the control of the freezing procedure. The biofreezer functions in the range from 40 to -180 degrees C. A complete cooling curve can be composed of up 15 linear segments with freezing rates between 0 and 45 K.min-1. In the computer up to 80 different freezing curves can be stored and activated by simple operations. The reliability of the biofreezer BF 80 was successfully tested for the cryopreservation of lymphocytes.

Cell Survival↗
PubMed1990

Dipeptidyl peptidase IV in the immune system. Cytofluorometric evidence for induction of the enzyme on activated T lymphocytes.

Dipeptidyl peptidase IV (DP IV) is a membrane peptidase with essential functional significance in thymus derived lymphocytes. This conclusion is drawn from 1) the induction of this enzyme after stimulation of T lymphocytes in vitro and 2) the impairment of T cell functions in presence of active site-specific inhibitors of the enzyme. The first item will be addressed in this paper, whereas the second one will be treated in a forthcoming article. Using flow cytofluorometry we investigated the expression of dipeptidyl peptidase IV on activated lymphocytes and the phenotype of lymphocytes expressing this enzyme. After stimulation by mitogenic lectins the number of epitopes on the cell surface binding polyclonal antibodies against DP IV increases 4 to 6 times. By means of double fluorescence staining the enzyme has been shown to be restricted nearly exclusively to T lymphocytes even after mitogenic stimulation. The highest density of DP IV epitopes has been found in cells coexpressing activation markers like receptors for interleukin 2 or transferrin in a high density.

Dipeptidyl Peptidase 4↗
PubMed1990

[Characterization of T lymphocyte defects in patients with Hodgkin's disease using enzyme chemical markers].

Patients suffering from lymphogranulomatosis were studied with respect to cellular immune deficiencies. For this purpose, mononuclear cells from venous blood were separated and subjected to analysis of lymphocyte markers. T-lymphocytes were enumerated by means of the sheep erythrocyte (SE) rosette test. T cell subpopulations were determined using enzyme cytochemical staining for dipeptidyl peptidase IV (DP IV) and unspecific acid alphanaphthylacetate esterase (ANAE). In 18 patients with M. Hodgkin a significant reduction in the T lymphocyte count in peripheral blood was found. This T cell defect is due to a selective decrease in the TM-subpopulation as identified by enzyme cytochemical markers DP IV and ANAE (focal reaction). From these results it is concluded that patients with lymphogranulomatosis have characteristic abnormalities in the immune system in the sense of a disturbed equilibrium of immune regulatory cells.

Adult↗
PubMed1990

[Aminopeptidase activity in populations of human blood mononuclear cells].

The aminopeptidase N (AP N, EC 3.4.11.2) is an ectoenzyme of the plasma membrane, playing presumably an important role in the regulation of immunological processes. The specific activities of Ala-pNA and Leu-pNA cleavage (per cell) are distributed in monocytes T- and non-T-lymphocytes in a proportion of 1:0.2:0.25 and 1:0.17:0.18, respectively. The capacities of Ala-pNA and Leu-pNA hydrolysis in the total fraction T- and non-T-cells are distributed as 1:0.8 and 1:0.7, respectively. The main part of Ala-pNA cleavage was shown to be caused by AP N on the basis of the KM-value (0.5 mmol/l), the activation by CO2+ ions and the pH optimum (7.0-7.5). The Leu-pNA cleavage is dependent on CO2+ and DTT and distinct from the classical cytosolic leucyl aminopeptidase.

Aminopeptidases↗
PubMed1990

[IL-2 production by peripheral blood mononuclear cells after stimulation with pokeweed mitogen].

Peripheral blood mononuclear cells were cultured for 72 hours in presence of phytohaemagglutinin (PHA), phytohaemagglutinin/phorbolmyristate acetate (PHA/PMA), pokeweed mitogen (PWM) and in absence of these stimulators. The IL-2 concentrations of cultural supernatants were determined by an IL-2 dependent cell line. There was no IL-2 determined in absence of any mitogenic stimulators. Induction of IL-2 by PWM does not require the presence of PMA in contrast to stimulation by PHA/PMA, where PMA was found to be necessary for stimulation of production of IL-2. PHA/PMA induced IL-2 release shows only a weak maximum at 24 hours, whereas PWM induced IL-2 release was found to have a clear maximum at 48 hours. Stimulation by PWM enables comparative examination of DNA synthesis (lymphocyte transformation test) and IL-2 production in the same cell system using only one mitogen.

Humans↗
PubMed1990

[Staphylococcus aureus (STA), a B- and T-cell mitogen].

Mitogenesis in peripheral blood lymphocyte and separated T and non-T cells cultures activated with formalin-fixed and heated staphylococcus aureus strain 520 (STA) was studied. The peak of DNA-synthesis in presence of STA is on day 7. The maximum of proliferation was found for T cell on day 7 and for non-T cells on day 3 after stimulation with STA. We found a strong correlation between the expression of CD25 antigen and the 3H-thymidine incorporation. It is concluded that B and T cell proliferation can be studied without purifying the cells using the different proliferation kinetics in presence of STA.

B-Lymphocytes↗
PubMed1989

[Determination of cellular and humoral parameters of fatty liver].

Cellular and humoral immunological parameters were examined in mononuclear cells from peripheral blood of patients with alcoholic and nonalcoholic steatosis hepatis. The ratio of T4 and T8 positive lymphocytes and the number of monocytes of these patients were in the normal range. The percentage of NK-cells, B-lymphocytes and DR-antigen-positive cells was increased.

Antigens, Differentiation↗
PubMed1988

Direct immunogold staining technique based on monoclonal antibodies: an immediate approach to the immunostaining of lymphocytes.

The report provides a procedure for labelling of monoclonal antibodies (moab's) with colloidal gold particles (mean diameter 30 or 40 nm). This procedure was exemplified for the moab's BL-T2, BL-Ig-L/1, and BL-Ia/1, which label mature T (and some populations of B) lymphocytes, B lymphocytes, and HLA class II antigen bearing lymphocytes, respectively. Each moab was characterized by an individual pH optimum for stabilization of the gold sol. The pH optima were +/- 5 (BL-T2), +/- 7 (BL-Ia/1), and +/- 9 (BL-Ig-L/1). The immunogold staining reagents were used for a direct immunostaining protocol of lymphocytes, which needs less than 100 min. The intensity of this direct immunolabelling was comparable with this one resulting from the indirect IGS. Furthermore, the comparison of the direct IGS with the immunofluorescence technique does not revealed any significant difference in the mean cell counts. The presented immunolabelling method offers a straight cell tagging, which can be advantageously used for a double staining procedure.

Adult↗
PubMed1988

Immunohistochemical analysis of thiol: protein disulfide oxidoreductase in hypothalamic neurons of Brattleboro rat.

The immunolocalization of thiol: protein disulfide oxidoreductase (TPO) in CNS of Wistar rats and homozygous Brattleboro rats was investigated by use of monospecific antiserum and Sternberger's unlabelled immunoenzyme technique. It was revealed that TPO is present in hypothalamic neurons belonging to nucleus supraopticus and paraventricularis. The number of immunopositive nerve cells was reduced in Brattleboro rats as compared to Wistar rats. It was concluded that TPO must have tasks in CNS unrelated to the management of vasopressin production and/or processing.

Animals↗
PubMed1987

The role of dipeptidyl peptidase IV in human T lymphocyte activation. Inhibitors and antibodies against dipeptidyl peptidase IV suppress lymphocyte proliferation and immunoglobulin synthesis in vitro.

Dipeptidyl peptidase IV (DP IV), an ectoenzyme in the cell membrane of T lymphocytes, is an important constituent in the process of lymphocyte activation. This conclusion is drawn from the following observations: (a) Proliferation of peripheral blood lymphocytes induced by mitogenic lectins (phytohemagglutinin, concanavalin A, pokeweed mitogen) is suppressed in the presence of DP IV inhibitors. This effect is specific and applies to an irreversible suicide inhibitor as well as to a competitive one in a dose-dependent fashion. (b) Inhibition of DNA synthesis was also induced by a polyclonal anti-DP IV immunoglobulin. (c) As a consequence of impaired T cell function the production of immunoglobulins by pokeweed mitogen-stimulated lymphocytes is also markedly reduced in the presence of DP IV inhibitors.

Antibody Formation↗
PubMed1987

Immunolocalization of dipeptidyl aminopeptidase (DAP IV) in the developing human brain.

By means of immunohistochemical techniques we have investigated the presence of dipeptidyl aminopeptidase IV immunoreactivity in brain material derived from human fetuses, newborns and aged persons. It was revealed that the enzyme protein is abundantly present in the immature human CNS. On the contrary the adult human brain contains much less dipeptidyl aminopeptidase immunoreactivity. It is speculated that the enzyme might play an important role in neuronal proliferation and/or differentiation especially with regard to its possible action on certain neuronotrophic peptides (IGF II, growth hormone).

Aged↗
PubMed1987

Immunogold cell staining using monoclonal antibodies: application to lymphoid cells on coated slides.

The immunogold staining technique was presented for light microscopic detection of cell surface antigens with monoclonal antibodies. The lymphoid cells were adhered to glass slides precoated with the adhesive poly-(dimethyl-diallyl-)ammonium chloride. The staining with the monoclonal mouse antibodies followed by the gold labelled sheep anti-mouse antibody was carried out on the cells adhered to the slides. In spite of the adherence, the patching of the gold marker as the prerequisite for the light microscopic detection occurred and the positively stained cells were visualized. On basis of this method the content of T lymphocytes (BL-T2+), B lymphocytes (BL-Ig-L/1+) and monocytes (BL-M/G+) in the mononuclear cell fraction of peripheral blood was determined in a population of healthy donors and of patients. The content of T lymphocytes of 29 healthy donors was determined using the immunogold staining technique (66.7 +/- 10.4%) and the immunofluorescence technique (67.6 +/- 9.5%). The correlation between the results obtained with both methods was highly significant (p less than 0.001).

Adult↗