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Biomedical subjects

H D Flad

Publications and source records attributed to H D Flad.

At least 199 records · Page 11Linked to original sources
PubMed1986

A phase I/II trial of recombinant interleukin-2 in AIDS/ARC: alterations of phenotypes of peripheral blood mononuclear cells.

In the context of a clinical phase I/II trial with recombinant interleukin 2 (rIL-2) surface phenotypes of peripheral blood leukocytes and delayed-type hypersensitivity skin reactions were monitored in four patients with acquired immunodeficiency syndrome (AIDS) and three patients with AIDS-related complex (ARC). The phenotypic alterations during the 14-day course of treatment were characterized by 1. a decrease in the proportion of OKT9 positive lymphocytes in AIDS patients, 2. an increase in the proportion of Leu8-/Leu3a+ lymphocytes in all patients and, in particular, 3. the transient significant decrease of the Leu7+/OKT3+ ratio, which was elevated pretherapeutically in AIDS patients (0.78 +/- 0.21) and ARC patients (0.48 +/- 0.06) compared to healthy controls (0.18 +/- 0.08). In contrast, the T4/T8 ratio of the patients' T-cells was not significantly affected by rIL-2 treatment. Delayed skin reactivity to recall antigens was improved in some patients. In one patient the surface phenotypes of peripheral blood mononuclear cells were determined on the first day of rIL-2 treatment in the morning before and in the evening after infusion of rIL-2. A rise of HLA-DR positive non-B-lymphocytes was observed. We conclude that the Leu7/OKT3 ratio, OKT9 and HLA-DR may be useful markers for monitoring the effects of biological response modifiers in patients with AIDS/ARC.

AIDS-Related Complex↗
PubMed1986

Binding characteristics of a monoclonal antibody against human IL-2 and its application for IL-2 measurement.

A murine monoclonal antibody, obtained by immunization with human recombinant interleukin 2 (r-IL-2), is presented. The antibody, termed BO-7, recognizes r-IL-2 as well as natural human IL-2 from different sources. As deduced from binding studies with synthetic IL-2 derived peptides, the epitope reactive with the antibody is associated with amino-acid residues 59-72 of the intact IL-2 molecule. The application of the antibody as a specific reagent for the quantitation of IL-2 from crude supernatants including samples from lectin-stimulated mononuclear cells in a sandwich-type ELISA is presented.

Animals↗
PubMed1985

Interleukin 2 production by human T lymphocytes identified by antibodies to dipeptidyl peptidase IV.

T-Cell subsets identified by polyclonal and monoclonal antibodies to dipeptidyl peptidase IV (DP IV) were investigated. Analysis in a cytofluorograf revealed 63 +/- 7% positive scatter-gated T lymphocytes. DP IV-positive cells were found to be T11+, 74-81% OKT4+, and 12-19% OKT8+. DP IV-negative cells were T11+ and comprise 16-40% OKT8+, and 10-30% OKT4+ T cells. Treatment of T lymphocytes with rabbit anti-DP IV and complement as well as the presence of rabbit anti-DP IV during culture resulted in a reduction of interleukin 2 (IL-2) production. This reduction was not observed with the mouse monoclonal anti-DP IV antibody II-19-4-7. Positive enrichment of DP IV-positive lymphocytes by cell sorting revealed excellent IL-2 production of DP IV-positive cells and very poor IL-2 activity in supernatants obtained from DP IV-negative lymphocytes. Thus, DP IV may serve as cell surface marker for IL-2-producing T lymphocytes.

Animals↗
PubMed1985

Response of human T lymphocytes to phytohemagglutinin (PHA) after sequential depletion of monocytes, HLA-DR+, Leu11a+, and Leu7+ cells.

The experiments presented in this paper deal with the question of whether there is an absolute requirement for alpha-naphtylacetate esterase (ANAE)-positive monocytes, HLA-DR+, Leu11a+, and/or Leu7+ cells to stimulate human peripheral blood T lymphocytes by phytohemagglutinin (PHA). Purified (p) T lymphocytes containing less than 0.1% ANAE-positive monocytes were isolated from human peripheral blood mononuclear cells (MNC) by sequential removal of carbonyl-iron phagocytic cells and of low-density cells by density gradient centrifugation and isolation of E-rosette-forming cells (E-RFC). These pT-cells were further depleted of HLA-DR+, Leu11a+, and/or Leu7+ cells using monoclonal antibodies and cell sorting. The T lymphocytes were stimulated by PHA in an ultra-micro culture in glass capillaries at a volume of 1 microliter or 2 microliters, containing 1000 cells per culture. With this method, the accessory cell requirement could be studied under limiting cell number conditions. The results show that pT-cells can be stimulated by PHA in the absence of ANAE-positive monocytes. No ANAE-positive monocytes were found in the culture after stimulation, indicating the lack of differentiation into ANAE-positive monocytes from ANAE-negative precursors. A rabbit antiserum against leukocytic pyrogen (LP, also containing anti-IL 1 activity) only reduced but did not abrogate the stimulation of pT-lymphocytes by PHA. Addition of adherent cells resulted in an enhancement or in an inhibition of the response of pT-lymphocytes to PHA, depending on cell concentration and culture time: The lower the number of cultured T lymphocytes and the shorter the culture time, the higher was the enhancing activity by additional adherent cells, and vice versa. Further purification of the pT-cells using monoclonal antibodies and cell sorting led to the finding that depletion of either HLA-DR+, Leu11a+, or Leu7+ from pT-cells only reduced but did not abrogate the stimulation of the pT-cells by PHA. However, in absence of HLA-DR+ and Leu7+ cells, the pT-lymphocytes totally failed to respond to PHA. This abrogation of the response was not observed when pT-cells were depleted of HLA-DR+ and Leu11a+ cells. In addition, T11+/HLA-DR- T lymphocytes isolated from E-RFC by positive selection in a cell sorter also responded to PHA.(ABSTRACT TRUNCATED AT 400 WORDS)

Antibodies, Monoclonal↗
PubMed1985

Binding of interleukin 2 to target cells: determination of high and low affinity binding sites on T-lymphoblasts by differential dissociation.

A new method for the determination of both low and high affinity binding sites on interleukin 2 (IL-2) target cells is described, which is based on differential dissociation of the ligand receptor complex. The technique requires highly purified but not radiolabelled IL-2. In the presence of activated charcoal the low affinity binding sites had a dissociation half time of less than 1 min, while that of the high affinity binding sites was 80 min. Human T-lymphocytes expressed both classes of binding sites within 48 h after PHA stimulation. During culture of PHA-stimulated T-lymphocytes, low affinity binding sites appeared on day 2 and remained high until day 8. High affinity binding sites appeared on day 1, were highest on day 2, and remained high until day 6. The changes in the concentration of the high affinity binding sites are considered as being the result of: receptor stimulation by PHA and/or IL-2 (days 1-2); receptor down regulation by extensive IL-2 production (day 3); increase of unoccupied IL-2 receptor after disappearance of IL-2 from the medium (days 4-6) and cessation of receptor synthesis and receptor breakdown (days 7-10).

Binding Sites↗
PubMed1984

A new ultra-microculture system. I. Stimulation of human T lymphocytes by phytohemagglutinin (PHA).

We have developed an ultra-microtechnique for culturing lymphocytes in glass capillary tubes at a final culture volume of 1 microliter or 2 microliter. The advantage of the method is that a substantially lower number of cells and minute amounts of culture medium are required. The cultures are premixed in microtubes, sucked into glass capillary tubes and incubated for an appropriate culture period. For determination of [3H]thymidine ([3H]TdR) incorporation, the cells are transferred into the wells of microtiter plates. Some special accessories have been developed which allow routine use of this system for large numbers of cultures. Optimal culture conditions for stimulation of human T lymphocytes by PHA are described.

Blood Physiological Phenomena↗
PubMed1984

Dissociation of responses measured by natural cytotoxicity and chemiluminescence.

Monocyte/macrophage-mediated cytotoxicity requires the generation of activated oxygen radicals, which can be measured by chemiluminescence (CL). To investigate whether natural killer (NK) cell activity required activated oxygen species, both cytotoxicity against K562 target cells and CL were measured in cell populations of human peripheral blood. The following results were obtained: (a) Peripheral blood mononuclear cells (MNC) showed NK activity and a response in CL, which could be induced by viable or paraformaldehyde-fixed K562 target cells as well as by latex particles. (b) Both T cells and non-T cells exhibited NK activity, but T cells gave no K562- or latex-induced CL responses. (c) Depletion of phagocytic cells from MNC abolished CL, but only marginally affected NK activity. (d) Reconstitution of phagocyte-depleted MNC with adherent cells revealed a superadditive enhanced CL response, but had no augmenting effect on NK activity. (e) Phagocyte-depleted cell populations, enriched for NK activity by density gradient centrifugation, did not respond in K562- and latex-induced CL. (f) MNC, highly enriched for NK activity by cell sorting with a cytofluorograf using the fluorescein isothiocyanate-labeled monoclonal antibody anti-Leu-11a, responded only with reduced CL, whereas the NK activity was enriched up to 45-fold. From these results it is concluded that NK cell-mediated cytolysis of K562 target cells and K562-induced CL are not functionally correlated, but represent properties of two distinct cell populations, namely NK cells and monocytes.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Dipeptidyl peptidase IV as a new surface marker for a subpopulation of human T-lymphocytes.

Plasma membranes were isolated from normal human lymphocytes as well as from cells of chronic lymphocytic leukemia of the T type. In both cases the bulk of the dipeptidyl peptidase IV activity paralleled the distribution of 5'-nucleosidase and, therefore, was localized in the plasmalemma. Immunofluorescence experiments with normal human lymphocytes and with antibodies against dipeptidyl peptidase IV revealed that this peptidase was accessible on the surface of viable cells. Further, it was demonstrated that the relative number of dipeptidyl peptidase IV-positive cells is much higher in lymphocytes reacting with the OKT4 antibody than in OKT8-positive cells. On the other hand, it has been reported that this peptidase is absent in B lymphocytes and is predominantly found in T cells bearing the Fc receptor for IgM (T mu lymphocytes). Thus, it is concluded that dipeptidyl peptidase IV represents an easily demonstrable surface marker of this lymphocyte subset.

Cell Membrane↗
PubMed1984

Isolation and subfractionation of human peripheral blood mononuclear cells (PBMC) by density gradient centrifugation on Percoll.

The use of Percoll for isolation and subfractionation of PBMC and T-lymphocytes by discontinuous and continuous density gradient centrifugation is described: PBMC were isolated from human peripheral blood by discontinuous density gradient centrifugation on Percoll. The use of Percoll instead of Ficoll-Isopaque has the advantage that Percoll, in contrast to Ficoll-Isopaque, does not alter the density of monocytes. Therefore, a better separation of lymphocytes and monocytes was achieved after subsequent continuous density gradient centrifugation on Percoll. E-RFC were isolated by discontinuous density gradient centrifugation after a first low speed centrifugation step banding lymphocytes and SRBC on a Percoll-Ficoll cushion, and a subsequent high speed centrifugation step separating high density rosettes and SRBC from low density non-E-RFC. The advantage of this procedure is the short time of performance and that there is no need to resuspend the lymphocyte/SRBC pellet. PBMC, nph.PBMC T-lymphocytes were further subfractionated by continuous density gradient centrifugation on Percoll. The method described here resulted in a good separation of lymphocytes and monocytes. However, to obtain lymphocyte fractions with minute numbers of contaminating monocytes, a depletion of monocytes prior to further subfractionation of the lymphocytes by continuous density gradient centrifugation is recommended. A marker analysis of T-lymphocytes subfractionated by continuous density gradient centrifugation on Percoll shows that high density T-lymphocytes are enriched in ANAE positive lymphocytes of type 1 and depleted of ANAE positive lymphocytes of type 2. Low density T-lymphocytes are enriched in ANAE type 2 cells and depleted of ANAE type 1 cells. On the other hand, no considerable differences were found when analyzing the T-cells from different fractions for differentiation antigens by means of monoclonal antibodies (anti Lyt 3, OKT4, and OKT8). The results may indicate that subfractionation of T-lymphocytes by continuous density gradient centrifugation on Percoll provided T-cells in different functional states rather than T-cells of distinct subclasses.

Antibodies, Monoclonal↗
PubMed1984

[The C3 activating capacity in human serum--measured as monocyte mediated chemiluminescence].

Phagocytosing monocytes form activated oxygen species which can be detected by the luminol-mediated chemiluminescence (CL). C3b-coated particles yield enhanced CL-signals due to their binding to the monocytes' C3b-receptor. Thus, the zymosan-induced monocyte-CL in a medium containing fresh or heat-inactivated serum provides a means of measuring in sera the capacity to activate C3.

Biological Assay↗
PubMed1984

[Circulating immune complexes and NK activity in systemic lupus erythematosus].

NK-activity of normal mononuclear cells is inhibited in vitro by addition of immune complexes or aggregated IgG. We therefore studied 20 sera of patients with systemic lupus erythematosus in order to investigate whether there is any correlation between the presence of circulating immune complexes and suppression of NK-activity. A correlation was not found.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1983

Functional relationship of human T lymphocytes, monocytes and interleukins. II. Helper requirements for phytohaemagglutinin-induced proliferation of human T-lymphocyte colony-forming cells.

The different requirements of human T lymphocytes of different densities for accessory cells and helper factors (Interleukin 1 (Il-1) and Interleukin 2 (Il-2) ) in the response to phytohaemagglutinin (PHA) were investigated. Human T lymphocytes were subfractionated by discontinuous density gradient centrifugation. The various T-cell subsets were stimulated by PHA to form colonies in an agar micro-culture in the presence or absence of additional adherent cells or crude preparations of Il-1 or Il-2. The results show that the higher the density of the fractionated T lymphocytes and the lower the number of cells cultured, the greater is the number of adherent cells or the amount of helper factors required for the stimulation of colony-forming T lymphocytes. The results are consistent with the assumption that monocytes provide positive modulating activity during mitogenic stimulation of colony-forming T lymphocytes. The number of monocytes necessary for exerting an optimal modulating activity depends on the number of T cells cultured and the density of the T-cell fraction. This may reflect a distinct susceptibility of T cells of different densities to monocyte-mediated helper effects.

Cell Adhesion↗
PubMed1982

Adjuvant intermittent chemoimmunotherapy for primary breast cancer: a prospective study with immunologic follow-up.

In an interdisciplinary prospective study 50 patients surgically treated for breast cancer were treated with six monthly courses of aggressive adjuvant chemotherapy (adriamycin and cyclophosphamide) and were randomized to receive either immunotherapy with levamisole or no additional therapy. Probability of disease-free survival for the whole group is 54.9% at 42 months. There was no noticeable difference in disease-free survival for either pre- and postmenopausal women or for patients treated with or without levamisole. The addition of levamisole had no effect on the depression of in vitro immunologic functions during chemotherapy.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1982

Disseminated mycobacterial histiocytosis due to M. Fortuitum associated with helper T-lymphocyte immune deficiency.

Mycobacterial histiocytosis is a rare disease usually associated with haematological or immunological disorders. We report a fatal case caused by M. fortuitum infection showing the typical disseminated histiocytosis. Immunological investigations revealed impaired cellular immunity demonstrated by negative skin tests with different "recall-antigens", and in vitro an isolated defect of helper T-lymphocytes in the peripheral blood which in combination with hypergammaglobulinemia suggests a "lymphocyte and distribution syndrome".

Adult↗
PubMed1982

Functional relationships of human T-lymphocytes and interleukins: I. Different accessory cell requirements of human T-lymphocyte subpopulations for generation of interleukin-2.

Interleukin-2 (Il-2) producing human T-lymphocyte subsets were isolated by discontinuous density gradient centrifugation and their requirements for helper monocytes were investigated. The results show that T-lymphocytes from fractions 2, 3, and 4 from the density gradient (high density lymphocytes) which contain up to 75% Fcmu receptor-bearing cells, and less than 0.2% monocytes could be stimulated by PHA for the production of Il-2 only in the presence of additional monocytes. T-lymphocytes from fractions 6 and 7 and in most experiments also from fraction 5 (low-density lymphocytes containing between 20% and 50% Fcmu receptor bearing cells, but still 0.2% up to 30% monocytes) could be stimulated by PHA for the production of Il-2 also without additional monocytes. Dose-response experiments with various numbers of monocytes added to the system showed the following: the higher the density of a cell population, the higher the number of monocytes required for help in the stimulation of Il-2 production. However, the production of Il-2 by high-density lymphocytes did not reach the Il-2 production by low-density T-lymphocytes even in the presence of high numbers of additional monocytes. Dose-response experiments indicated that the difference in Il-2 producing activity of T-lymphocytes of different density cannot be explained by the various numbers and/or activities of residual monocytes present in the various T-cell fractions. We suggest that the various T-lymphocyte populations differ with respect to a further cell population, presumably of lymphatic origin. This population appears to participate in the regulation of Il-2 production.

Cell Communication↗
PubMed1982

Stimulation of colony formation and growth factor production of human T lymphocytes by wheat germ lectin.

Wheat germ lectin (WGL) induced proliferation of T-lymphocyte colony-forming units (TL-CFU) from human peripheral blood mononuclear cells (PBMC) in a one stage agar culture needing higher cell numbers than phytohaemagglutinin (PHA). However, WGL preactivated TL-CFU in a first stage liquid culture for the growth of PHA-dependent T-lymphocyte colonies in a subsequent agar culture. These results suggested that WGL and PHA have biological activities in common. WGL and PHA both induced the expression of Interleukin 2 (IL-2) receptors by PBMC to become responsive to IL-2, although production of IL-2 by WGL-stimulated PBMC was low compared with PHA-stimulated PBMC. This might account for the low proliferative response of PBMC to WGL.

Agar↗
PubMed1981

T-lymphocyte colony formation of murine spleen cells in a one-stage micro agar culture.

A one-stage agar culture technique for stimulation of T-lymphocyte colony-forming units (TL-CFU) derived from murine spleen cells is described. The cultures are in glass capillaries in a volume of 30 microliter. Both, phytohemagglutinin (PHA) and concanavalin A (Con A) stimulate growth of TL-CFU. The technique does not require prestimulation of lymphocytes in liquid culture or addition of growth factor(s). The most important requirements for optimal colony growth are a low concentration of agar (less than 0.1%) and the appropriate number of seeded cells (60 X 10(3) per culture). This one-stage micro agar culture is economical and easily handled. Many cultures can be prepared and rapidly evaluated. The plating efficiency is about 10 colonies per 10(4) nucleated spleen cells. The T-cells character of the TL-CFU was demonstrated by treatment of the spleen cells with a monoclonal anti-Thy-1.2 antibody (anti-Thy-1.2) and complement before assaying for colony growth and also by staining the cells in the cultures with anti-Thy-1.2-FITC. Growth characteristics of TL-CFU indicate that cell interactions are operative during activation and proliferation. The nature of the cells cooperating in this system is discussed.

Agar↗