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Biomedical subjects

H W Mansfeld

Publications and source records attributed to H W Mansfeld.

14 recordsLinked to original sources
PubMed1993

Detection of inhibition of HIV-1 protease activity by an enzyme-linked immunosorbent assay (ELISA).

An ELISA is described for the detection of HIV-1 protease activity using an immobilized gag-related polyprotein as substrate. Proteolytic activity was demonstrated with either bacterial lysates expressing HIV-1 protease or purified protease. No cleavage was observed with a protein preparation from control bacteria not expressing HIV-1 protease. Under these conditions the aspartyl-type protease inhibitor, pepstatin A, was found to inhibit HIV-1 protease cleavage by > 90% at a concentration of 0.1 mM. This assay may be a useful tool for the study of both synthetic and natural inhibitors of HIV-1 protease.

Enzyme-Linked Immunosorbent Assay↗
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↗
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↗
PubMed1987

[Fc-IgG and Fc-IgM receptor-carrying lymphocytes in human blood. Optimization of determining T(M) lymphocytes].

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.

Humans↗
PubMed1986

The thiol-proteindisulfide oxidoreductase in human mononuclear cells of blood and bone marrow.

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.

B-Lymphocytes↗
PubMed1986

The thiol-proteindisulfide-oxidoreductase--a new marker enzyme of monocytes from peripheral blood.

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.

Antibodies, Monoclonal↗
PubMed1985

Comparison of different markers on blood lymphocytes of chronic lymphocytic leukemia.

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.

Aged↗
PubMed1985

The dipeptidyl peptidase IV, a membrane enzyme involved in the proliferation of T lymphocytes.

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.

Cell Division↗
PubMed1985

[Isolation of monocytes by adherence to gelatin-layered surfaces].

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.

Acridine Orange↗
PubMed1985

[Comparative studies of the determination of T lymphocytes in human peripheral blood].

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).

Adult↗
PubMed1984

[Lymphocyte separation with dextran M 70].

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.

Cell Separation↗