Biomedical subjects
Publications and source records attributed to S Ansorge.
The degradation of insulin and glucagon was investigated in rat brain, kidney, and liver during postnatal ontogenesis. It was found that a maximal activity around 13 d is unique for CNS and differs remarkably from time-course in liver and kidney. It is concluded that this result supports the hypothesis of a growth promoting role of insulin in developing rat brain.
Dipeptidyl peptidase IV (DP IV; E.C. 3.4.14.5), a plasma membrane structure of human T lymphocytes has been shown to be an important enzyme in the process of activation and proliferation of lymphocytes. In presence of specific inhibitors and antibodies against DP IV different functions of lymphocytes in vitro were found to be impaired. This holds true for mitogen and alloantigen induced DNA synthesis, immunoglobulin production and secretion, and interleukin-2 as well as interferon-gamma production. Studies of mitogen-induced expression of different activation markers (HLA class II antigen, 4F2, Tac) suggested that one of the functions of DP IV lies in overriding the cell cycle restriction point at G1. These data, together with other features of the DP IV, support the notion that this enzyme plays a key role in the modulation of lymphokine action by X-Pro- or X-Ala-directed limited proteolysis. Moreover the high frequency of DP IV susceptible bonds in different growth factors (e.g. IL-1, IL-2) and other biologically active peptides leads to the speculation that this peptidase is of more general significance to the regulation of cell growth.
Several methods for enumeration of Fc receptor bearing T lymphocytes and mononuclear cells from human peripheral blood were compared. The detection of Fc receptors is based on the formation of EA rosettes by using bovine erythrocytes and purified rabbit IgG or IgM antibodies. As alternative method the mixed rosette assay (EA rosettes plus sheep erythrocyte rosettes) (3) was applied for determining TG lymphocytes without the need of T cell separation. Independent of the method used for T cell separation (preparative rosetting with sheep erythrocytes stabilized by AET or HSA) the number of TG and TM lymphocytes was found to be identical. TG values obtained by use of the mixed rosette assay were significant lower (10 +/- 2%) than those obtained with the classical test (18 +/- 5%) (EA rosettes after T cell separation). Obviously this difference is due to a contamination of T lymphocyte preparations by non-T cells. On freshly isolated T lymphocytes without overnight culture we obtained 29% and 35%, respectively TM lymphocytes after separation of T cells using sheep erythrocyte rosettes stabilized with AET or HSA. The expression of FcIgM receptors was found to be strongly dependent on the composition and pH value of the culture medium. In the presence of human AB serum the maximum of FcIgM receptor expression on isolated T cells was obtained at pH 8.5. Under optimum conditions we found 63% and 66% respectively TM lymphocytes after T cell separation using AET or HSA stabilized sheep erythrocytes.
The in vivo function of the thiol-proteindisulfide oxidoreductase (TPO, EC 1.8.4.2; proteindisulfide isomerase, EC 5.3.4.1) in biosynthesis of immunoglobulin was investigated by studying the enzyme content in human lymphoid and other cells by an immunocytochemical method. In contrast to peripheral blood, B lymphocytes which showed no or no demonstrable TPO, normal as well as malignant bone marrow plasma cells (all Ig classes) were found to contain abundant amounts of this enzyme. TPO containing plasma cells were identified by double-staining techniques. This finding suggests that TPO is involved in the terminal step of B cell differentiation and immunoglobulin biosynthesis. Besides plasma cells, approximately 10% of mononuclear marrow cells as yet unidentified medium-sized and large cells, exhibited also strong anti-TPO reactivity. Furthermore, using surface-cytoplasmic double staining methods, monocytes from human peripheral blood could be identified to represent the only cytoplasmic TPO-containing normal mononuclear blood cells.
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The occurrence of DP IV-reactive T-lymphocytes in patients with aleukemic non-Hodgkin's lymphoma (NHL) was studied using mononuclear cells (MNC) obtained from peripheral blood, bone marrow aspirates and lymph nodes, as well as cryostat sections of lymph nodes. These patients were found to have a reduced percentage of DP IV-reactive T-cells in MNC from peripheral blood (32%) in comparison to a healthy control group (44%). Patients suffering from chronic lymphocytic leukemia (B-type CLL) also had significant reduced level of DP IV-reactive cells (5% DP IV-reactive cells in the MNC fraction corresponding to about 40% of the T-cell fraction) in peripheral blood, whereas in lymph nodes of these patients 72% of the T-cells expressed DP IV. Among the MNC from lymph nodes of patients with aleukemic NHL about 24% were DP IV positive corresponding to 57% of the T-cell fraction. MNC from bone marrow of these patients were shown to contain about 21% T-lymphocytes and 18% DP IV-reactive cells. In addition to that, cryostat sections of lymph nodes were analysed for the occurrence and distribution of DP IV. In normal lymph nodes and tonsils DP IV-positive cells have the same distribution pattern like T-cells in general. In lymph node sections from patients with NHL of B-cell origin, such as B-CLL, the DP IV-reactive cells are sparsely and irregularly distributed and displaced by the proliferating B-cells. It is concluded that DP IV is a useful marker not only for T-lymphocytes in proliferating lymphatic tissue but also an indicator of imbalances between regulatory T-cell subsets in peripheral blood.
Dipeptidyl peptidase IV (DP IV), an ectoenzyme in the cell membrane of human T lymphocytes, is an important constituent in the process of lymphocyte activation and proliferation. In the presence of specific inhibitors and antibodies against DP IV several in vitro functions of activated lymphocytes were found to be impaired. These include mitogen induced DNA synthesis, immunoglobulin secretion and production of interferon-gamma. The functional importance of DP IV during activation of lymphocytes is underlined by an increase in cellular DP IV activity upon mitogenic stimulation.
Monocytes were enumerated by three different methods, cytochemical staining for alpha-naphthyl-acetate esterase-activity, immunofluorescence test using the monoclonal antibody BL-M/G and immunochemical staining for the enzyme thiol-proteindisulfide-oxidoreductase (TPO, EC 1.8.4.2) using a polyclonal rabbit anti-rat TPO immunoglobulin. For the comparison of these methods mononuclear cells from peripheral blood of healthy volunteers, patients with different diseases and adherent cell populations were tested. Statistical analysis showed no differences between the markers within these groups (H-test) and a significant correlation between the numbers of monocytes. TPO was found to be also valid for enumeration of monocytes obtained by adherence methods.
Thiol: Protein-disulfide oxidoreductase (TPO) was localized in the brain of juvenile and adult rats by use of the unlabeled immunoperoxidase technique. In the brain of adult animals, TPO protein was revealed in neurons of Nucc. supraopticus and paraventricularis as well as in the subcommissural organ. In the developing rat, TPO seems to be a constituent of almost all neurons. The roles played by the enzyme remain to be elucidated, however.
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Mononuclear cells (MNC) of 17 patients suffering from B chronic lymphocytic leukemia (B-CLL) were analysed by various immunological methods. The B cell nature of CLL cell was determined by classical tests (MRBC-rosette-test, immunofluorescence test for detection of membrane bound immunoglobulins). The cytochemical detection of the new T-cell marker dipeptidyl peptidase IV (DP IV) was found to be suitable for the characterization of B-CLL. The B-CLL cells showed granular pattern of alpha-naphthylacetate esterase (ANAE) reaction and binding of the monoclonal pan T antibody BL-T2. These non typical reactions for normal B lymphocytes can be used for differential diagnosis of B-CLL in combination with other reliable T cell markers. Avoiding the separation of T cells, the mixed rosette assay was used to enumerate Fc-IgG-receptor bearing T(TG) and non T cells. Both cell populations were found to be significantly elevated in MNC of B-CLL.
Dipeptidyl peptidase IV can be demonstrated in 60% of T lymphocytes from human peripheral blood. As could be shown recently, this enzyme is irreversibly inhibited by N-Ala-Pro-O-(nitrobenzoyl-)hydroxylamine, a substrate analogous enzyme-activated inhibitor of X-Pro-specific peptidases. We found that this inhibitor was able to suppress the proliferation of human lymphocytes stimulated with mitogens or allogeneic cells. Furthermore, after stimulation with pokeweed mitogen the development of lymphocytes containing cytoplasmic immunoglobulins and thiol proteindisulfide oxidoreductase was reduced in the presence of this inhibitor. It is concluded that dipeptidyl peptidase IV is involved or might represent a limiting factor in the process of regulation of T lymphocyte proliferation.
The method for purification of monocytes using adherence to gelatin coated glass surfaces described by Chien et al. was optimized by drastic shortening the incubation time and modifying the culture media. After one adherence step we obtained monocytes with a purity of 73-78% and a recovery of 53%. Thiol-protein-disulfide-oxidoreductase (TPO), a new enzyme marker of monocytes, was found to be also valid for monocytes obtained by adherence methods. Comparing the number of TPO-containing monocytes with other markers (alpha-naphthylacetate esterase, peroxidase, phagocytosis of latex particles, acridine orange fluorescence, antigens detected by the monoclonal BL-M/G antibody) almost identical values were found.
Different methods for determination of T-lymphocytes in human peripheral blood were compared: rosetting with sheep erythrocytes (SRBC), AET-treated SRBC, immunofluorescence using a monoclonal antibody against T cells (BL-T2), complement dependent cytolysis with polyclonal antisera against thymocytes, cytochemical demonstration of unspecific acid alpha-naphthyl-acetate esterase (ANAE), and electrophoretic mobility using a cell electrophoresis system (PARMOQUANT 2). Depending on the method, mean values between 70 and 79% T cells among separated mononuclear cells (MNC) were found. All paired observations were subjected to statistical analysis using rank correlation and U-test. From this analysis it is concluded that rosetting with SRBC, immunofluorescence using the monoclonal T-cell antibody and cytochemical reactivity for ANAE are favored methods for determining the T cell content of human MNC. However, the monoclonal antibody BL-T2 and the ANAE are not generally applicable because both markers were also found on malignant B-lymphocytes (B-CLL).
Glycylproline p-nitroanilide is hydrolysed in lymphocytes from human blood exclusively by dipeptidyl peptidase IV. This was demonstrated by specific inhibition with N-alanylprolyl-O-(4-nitrobenzoyl)hydroxylamine and di-isopropyl phosphorofluoridate and by studying the membrane localization of dipeptidyl peptidase IV and determining specific dipeptidyl peptidase II activity. Additional evidence that dipeptidyl peptidase IV is a marker for T-lymphocytes, obtained from determinations of biochemical activity on intact lymphocyte preparations and correlation studies with other T-cell markers, is also presented.
Dipeptidyl peptidase IV (DP IV) activity of human lymphocytes was measured using biochemical assays of cell suspensions and enzyme cytochemical staining of smears from capillary blood and mononuclear cells (MNC). The hydrolysis rate of Gly-Pro-pNA in suspensions of MNC correlates well with the number of DP IV reactive cells as determined by cytochemical staining. MNC from healthy volunteers were shown to contain 44 +/- 10% lymphocytes reactive for DP IV. Using preparations of T lymphocytes and adherent MNC it was shown that DP IV is specific for T lymphocytes. About 60% of T lymphocytes contain DP IV and 94% of DP IV reactive cells form rosettes with sheep erythrocytes. Parallel staining for DP IV and unspecific acid alpha-naphthylacetate esterase (ANAE) yielded nearly the same figure of cells stained for ANAE in a dot-like pattern (50 +/- 10% MNC) as was observed for DP IV. Correlation of both markers indicates that DP IV expressing lymphocytes presumably represent that subpopulation of TM cells which is characterized by a dot-like reaction pattern of ANAE.
We investigated the possibility using Dextran M 70 instead of the common Ficoll 400, both in combination with Visotrast 370, for separation of lymphocytes from human peripheral blood by density gradient centrifugation. No significant differences were found with regard to parameters such as yield and vitality and numbers of B- (surface immunoglobulins) and T-lymphocytes (sheep red blood cells-rosette assay, and the monoclonal antibody BL-T2). From the data available as yet we can conclude--at least for the markers studied--that it is possible to use Dextran M 70 instead of Ficoll 400 for the separation of mononuclear cells of human peripheral blood.