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H D Flad

Publications and source records attributed to H D Flad.

At least 181 records · Page 10Linked to original sources
PubMed1989

Immunoenzymatic assessment of IL-1 beta mRNA by in situ hybridization using sulphonated probes.

An immunoenzymatic detection method of in-situ hybridization reactions for interleukin 1 (IL-1) beta was established, using sulphonated probes. As model system we used unstimulated and lipopolysaccharide (LPS) stimulated peripheral blood mononuclear cells (PBMNC). After hybridization, sulphone groups were targeted with a monoclonal antibody, and bound antibody was visualized by the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. In unstimulated PBMNC both the control and the IL-1 beta specific c-DNA probes were negative, whilst a large proportion of LPS treated PBMNC was positive with the sulphonated IL-1 beta plasmid only. This method may be a powerful alternative to radio-isotopic labeling. Since the entire procedure can be performed within one day, it may be applied for routine diagnostic purposes.

DNA Probes↗
PubMed1989

Interleukin 1 and tumor necrosis factor: studies on the induction by lipopolysaccharide partial structures.

Minimal concentration ranges of lipopolysaccharides, natural and synthetic lipid As, and partial structures were established for the induction of release of IL-1 and TNF from human MNC. LPS was more potent than lipid A. Partial structures of lipid A lacking nonhydroxylated fatty acids were inactive, but in combination with LPS or lipid A exhibited time-dependent synergistic or antagonistic effects.

Glycolipids↗
PubMed1988

Application of a new ultra-microculture system. II. Stimulation of human B lymphocytes.

An ultra-microtechnique for culturing human B-lymphocytes in glass capillary tubes using a volume of 2 microliter is described. The advantage of this ultra-microculture system is that only a small number of lymphocytes and minute amounts of culture medium (or test factors) are required. Optimal culture conditions for the formation of Ig-secreting plaque-forming cells (PFC) after stimulation of mononuclear cells with pokeweed mitogen are given. Furthermore it is shown that immunoglobulin secreted into culture supernatants by purified B cells in the presence of T cell subsets can be measured in a microELISA.

Antibody Formation↗
PubMed1988

Influence of human T lymphocytes identified by antibodies to dipeptidyl peptidase IV on differentiation of human B lymphocytes stimulated with Staphylococcus aureus Cowan I and pokeweed mitogen.

The influence of human T lymphocytes expressing the enzyme dipeptidyl peptidase IV (DPP IV) was investigated with respect to human peripheral B-lymphocyte differentiation. B cells stimulated with pokeweed mitogen in the presence of DPP IV-positive T cells produced high amounts of immunoglobulin. Moderate amounts of immunoglobulin could be measured when B cells were cultured in the presence of DPP IV-negative T cells. DPP IV defines a T-cell subset partially overlapping the subsets characterized by the differentiation antigens Leu 3a (helper/inducer) and Leu 2a (suppressor/cytotoxic). DPP IV-positive T cells exert, in contrast to DPP IV-negative T cells, high interleukin-2 activity after stimulation with phytohemagglutinin and pokeweed mitogen. To further functionally characterize DPP IV-positive and DPP IV-negative T cells, the helper effects of Leu 3a-positive T-cell subsets, differing in DPP IV expression, were investigated in pokeweed mitogen- and Staphylococcus aureus-driven B-cell differentiation systems. After pokeweed mitogen stimulation, immunoglobulin production was markedly reduced when B cells were cultured in the presence of Leu 3a-positive T cells expressing DPP IV (DPP IV+/Leu 3a+). In contrast, high amounts of immunoglobulin were produced in cultures with Leu 3a-positive but DPP IV-negative T cells (DPP IV-/Leu 3a+). This difference in immunoglobulin production of B cells cultured with DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells could not be observed in Staphylococcus aureus-stimulated cultures. Here, both T-cell subsets supported terminal differentiation of B cells. We conclude that in the pokeweed mitogen-driven culture systems, DPP IV+/Leu 3a+ and DPP IV-/Leu 3a+ T cells may differ in the production of growth and/or differentiation factors distinct from interleukin-2.

Antibodies, Monoclonal↗
PubMed1988

Stimulation of human and murine adherent cells by bacterial lipoprotein and synthetic lipopeptide analogues.

Lipoprotein from the outer membrane of Escherichia coli and its synthetically prepared N-terminal lipopeptide segments Pam3Cys-Ser-Ser-Asn-Ala and Pam3Cys-Ser, as well as lipoprotein from other Enterobacteriaceae, constitute potent polyclonal B lymphocyte activators. Here, we demonstrate that these compounds were also able to stimulate human and murine leukocytes: in murine macrophages, we could show the induction of interleukin 1 release by the mitogens, as measured in the thymocyte proliferation assay. Moreover, murine peritoneal exudate cells were stimulated to secrete prostaglandins E2 (PGE2) and F2 alpha (PGF2 alpha). The effect of Pam3Cys-Ser on the murine macrophage cell line P388D1 was also tested: the compound induced an increase in proliferation, as measured by a thymidine incorporation assay. In addition, the cell line could be induced to release IL 1 into the supernatant. Correspondingly, induction of IL 1 release could also be demonstrated in human mononuclear cells. Our results demonstrate that the two novel synthetic lipopeptides are potent stimulators for human monocytes and murine macrophages. These findings may be important for the elucidation of the role of these bacterial surface components in the course of bacterial infections.

Animals↗
PubMed1987

Interleukin 2 induces appearance of LGL/Leu11+/K562 lytic cells in Leu11- low density peripheral blood mononuclear cell population.

Low density Percoll fraction cells cultured with interleukin 2 (IL-2) showed a higher proportion of large granular lymphocytes (LGL) and higher K562 cytolytic activity, as compared to a culture lacking IL-2. Furthermore, in a negatively selected Leu11- population, derived from low density cells, cultured for 7 days in medium supplemented with lymphocyte (L) or recombinant (R) IL-2, there appeared LGL and Leu11+ cells. Moreover, some level of K562 lytic activity and higher proportion of DR+ and Tac+ cells was found as compared to lacking IL-2 culture. Cytofluorograph analysis of cells labelled with propidium iodide revealed that a proportion of the low density Leu11- starting cell population entered the growth cycle while cultured with IL-2. In addition was found that Leu11+ cells evolve during culture with IL-2 into population lacking in part this phenotype marker. The present work shows that precursors of K562 cytolytic cells lacking Leu11 antigen reside in low density cell fraction, and that they may differentiate in LGL/Leu11+ cells.

Antigens, Surface↗
PubMed1987

Corticosteroid-interleukin 2 interactions: inhibition of binding of interleukin 2 to interleukin 2 receptors.

PHA activated human peripheral blood mononuclear cells (MNC) were incubated with human interleukin 2 (IL-2) in the absence or in the presence of 10(-6)-10(-9) M hydrocortisone (HC). HC suppressed the proliferative response of the activated MNC to highly purified human leucocyte IL-2 in a dose dependent manner. This suppression was in full accordance with an inhibition of the IL-2 binding capacity, whereby the high affinity binding sites were reduced by 85%, while the low affinity sites were less affected. The mechanism by which HC inhibits the binding of IL-2 is still unknown. Evidence is presented that HC binds only weakly to IL-2. We conclude that HC interferes with the IL-2 binding by modulating the binding and/or signal processing function of the IL-2 receptor.

Binding, Competitive↗
PubMed1987

Large granular lymphocytes are central cells in the interleukin-2-dependent differentiation pathway of natural killer cells.

Peripheral blood low-density cells were sorted, with respect to their ability to accumulate the lysosomotropic agent mepacrine (Mep), into lysosome-rich (Mep+) and lysosome-poor (Mep-) cell populations. Cells of large granular lymphocyte (LGL) morphology and phenotype were found in the Mep+ but not in the Mep- cell population. The latter cells lacked any natural killer (NK) activity. Cultures of the Mep- cells resulted in the appearance of cells showing K-562 lytic activity, LGL morphology and CD16 and/or Leu-7 positivity. This process was facilitated by the supplementation of the culture with recombinant human interleukin-2 (rIL-2). Mep+ cells retested after 7 days of culture showed a decline in the fraction of granular (LGL and Mep+) cells. This decrease was less pronounced but also seen in rIL-2-supplemented cultures. In spite of the lower number of typical LGL, Mep+ cells cultured with rIL-2 were mostly large but scarcely granular; rIL-2-activated K-562 killing (rIL-2 AK) of originally Mep+ cells was much higher than K-562 lytic activity of these cells at the beginning of the culture, and as compared to rIL-2 AK of Mep- cells. From this finding it is apparent that the most active rIL-2 AK cells originate from low-density granular (Mep+) cells (LGL) and, therefore, we propose to call them 'giant' NK cells. Furthermore, in the presence of rIL-2, LGL differentiate from agranular (Mep-) low-density cells. In view of these data, LGL appear to be resting cells on the differentiation pathway of NK cells.

Cell Differentiation↗
PubMed1986

Pokeweed mitogen activated suppressor T-cells preferentially reduce immunoglobulin secretion by differentiated B-lymphocytes.

In contrast to pokeweed mitogen (PWM), S. aureus Cowan I (SAC) does not activate suppressor T-cells in cultures of human peripheral blood mononuclear cells, although the SAC induced response leading to the appearance of immunoglobulin secreting cells (ISC) is suppressed by PWM activated suppressor T-cells. Therefore, cultures co-stimulated by SAC and PWM were chosen to find out which stage of the SAC triggered B-cell response is controlled by PWM activated suppressor T-cells. By incorporation of [3H]thymidine and determination of B-cell number in culture it was shown that neither PWM itself nor PWM induced suppressor T-cells interfere with SAC induced B-cell proliferation. The final stages of B-cell maturation were monitored by parallel evaluation of cells producing immunoglobulins (cells with intracytoplasmic immunoglobulins) and secreting them (plaque assay). It was shown that PWM activated suppressor T-cells reduce the number of ISC more effectively than the number of immunoglobulin producing cells, indicating that these two features of differentiated B-lymphocytes may be independently controlled.

Antibody-Producing Cells↗
PubMed1986

Induction of human interleukin 1 by bacterial and synthetic lipid A.

Lipopolysaccharides (LPS) are well known inducers of interleukin 1 (IL 1). Here we show that synthetic heptaacyl Salmonella minnesota (compound 516) and synthetic E. coli type (compound 506) lipid A, as well as monodephospho part structures thereof, are able to induce IL 1 production in human mononuclear cells. The 4'-monodephospho part structure (compound 505) was found to be the most active IL 1 inducer as compared to compound 506 and the 1-monodephospho part structure (compound 504). Synthetic lipid A precursor Ia, lacking nonhydroxylated fatty acids (compound 406), and its 1- or 4'-monodephospho part structures (compounds 404 and 405) did not induce IL 1 production. IL 2 is not produced during stimulation with the synthetic compounds. IL 1 activity was partially heat labile and could be inhibited by a rabbit antiserum against human leukocytic pyrogen. These results show that the lipid A component of LPS is active in inducing IL 1 and that the presence of 3-acyloxyacyl residues is necessary for IL 1 induction.

Blood Physiological Phenomena↗
PubMed1986

Effects of systemic in vivo interleukin-2 (IL-2) reconstitution in patients with acquired immune deficiency syndrome (AIDS) and AIDS-related complex (ARC) on phenotypes and functions of peripheral blood mononuclear cells (PBMC).

In the context of a clinical phase I/II therapy study with recombinant interleukin-2 (rIL-2), we monitored immunological alterations in four patients with acquired immune deficiency syndrome (AIDS) and three patients with AIDS-related complex (ARC). By determining the surface phenotypes and in vitro functions of peripheral blood mononuclear cells (PBMC) before, during, and after treatment with rIL-2, we observed transient changes in all important leukocyte subpopulations, a minor restoration of immune reactivity in vitro, and an improvement in skin reactivity in vivo. In particular, we found a transient increase in C3b receptor-mediated monocyte activation in ARC patients; no influence of therapy on the otherwise intact LPS-induced interleukin-1 production in vitro; in some patients a transient corrective influence on the high pretherapeutic immunoglobulin secretion of B cells and their nonresponsiveness to pokeweed mitogen; low T-cell responses to soluble antigens and alloantigens, which were partially restored during rIL-2 treatment in ARC patients and in one AIDS patient; defective NK activity in PBMC of two AIDS patients, which was found to be restored when measured at the end of rIL-2 therapy; and a rather constant phenotypic pattern of PBMC in each patient during therapy except for the decreasing proportion of OKT9-positive lymphocytes in AIDS patients, the increasing proportion of Leu8-Leu3a+ lymphocytes in all patients, and in particular, the transient significant decrease in the Leu7+/OKT3+ ratio, which pretherapeutically was very high in AIDS patients (0.78 +/- 0.21) and high in ARC patients (0.48 +/- 0.06) as compared to healthy controls (0.18 +/- 0.08).

Acquired Immunodeficiency Syndrome↗
PubMed1986

Effect of human recombinant interleukin 2 on natural killing of low density Percoll fraction cells.

Human recombinant IL-2 (R IL-2) was used to validate the ability of this lymphokine to increase NK cell activity. It was found that R IL-2 was able to augment the K562 lytic activity of phagocyte-depleted mononuclear cells of low density in a dose dependent manner. This phenomenon was detectable after 24 h of incubation. Low concentrations of R IL-2 were sufficient for the activation of natural killing when the incubation time was longer than 24 h. Furthermore, IL-1 exerted an additive effect on R IL-2 induced augmentation of natural killing after 24 h, but not after longer incubation periods. Lymphocyte culture derived (L) and R IL-2 had similar effects on the K562 cytolytic potential of cells contained in low density fractions. The highest cytolytic activity was observed when Leu11a+ cells were incubated with IL-2. IL-2 induced K562 cytolytic activity was also seen after incubation of low density Leu11- cells for 48 h, 72 h, or 7 days. In cultures of low density Leu11- cells incubated with IL-2 for 7 days, a proportion of cells became Leu11+. From these findings we conclude that low density Leu11- cells are pre-NK cells which acquire natural killing potential under the influence of IL-2.

Cell Differentiation↗
PubMed1986

Functional and molecular characterization of a monoclonal antibody against human interleukin 2.

Human recombinant interleukin 2 (r-IL2) was used as an immunizing antigen to yield a murine monoclonal antibody (mAb) termed BO-7. Although the antibody binds to r-IL2 more avidly, it also reacted strongly with IL2 from natural sources in an enzyme-linked immunosorbent assay (ELISA), allowing the detection of the purified lymphokine at sensitivity levels closely approaching those found with the IL2 biological assay. Binding to the antigen is specific, as deduced from the close correlation of ELISA immunoreactivity with IL2 biological activity and from immunoblot analysis of electrophoretically separated IL2 from various sources. Binding studies with synthetic IL2-derived peptides revealed the location of the epitope, which is recognized by mAb BO-7: A peptide representing amino acid residues 59-72 (peptide 84) is strongly reactive with the antibody, while an overlapping peptide (residues 48-69) is not. Peptide 84, moreover, can be applied for immunopurification of mAb BO-7 and competes for binding to the antibody with the intact IL2 molecule. In turn, another monoclonal anti-IL2 antibody (35H10), showing the same reactivity pattern with peptides, competes with mAb BO-7 for binding to IL2. The application of mAb BO-7 as a specific reagent for the quantitation of IL2 in a sandwich-type ELISA is demonstrated.

Animals↗
PubMed1986

Monocyte-T-cell interactions in the regulation of polyclonal B-cell response.

Human peripheral blood monocyte subsets with and without Fc receptor for human IgG are known to suppress (FcR+) and enhance (FcR-) pokeweed mitogen-induced polyclonal immunoglobulin synthesis in vitro. The ability of these subsets to modulate immunoglobulin production in the presence or absence of OKT8+ T cells and under conditions where suppressor T-cell activation was blocked by irradiation or mitomycin C was studied. It was shown that, regardless of the presence or absence of suppressor T cells, FcR+ monocytes can suppress immunoglobulin production if their number in culture exceeds 20%. However, at lower numbers this monocyte subset was suppressive only when suppressor T cells were activated. The suppressor T-cell activation was shown to be independent of the predominant presence of the FcR+ or FcR- monocyte subset. Moreover, the enhancing effect of FcR- monocytes was not caused by their interference with suppressor T-cell activation.

B-Lymphocytes↗
PubMed1986

Immunoglobulin production of human lymphocytes stimulated by Staphylococcus aureus Cowan I and pokeweed mitogen: differential effects of recombinant interleukin-2.

The number of immunoglobulin-secreting cells (ISC) upon stimulation with pokeweed mitogen (PWM) or Staphylococcus aureus Cowan I (SAC) in recombinant interleukin-2 (rIL-2)-supplemented cultures of human peripheral blood mononuclear cells (PBMC) and co-cultures of B and T cells was studied. It has been shown that the addition of rIL-2 to culture can enhance or depress the number of ISC depending on the polyclonal B-cell activator used for culture stimulation. The SAC-induced response was enhanced in the presence of rIL-2, while the number of ISC in PWM-stimulated cultures was depressed. Moreover, in cultures stimulated simultaneously by both activators, the suppressive effect of rIL-2 prevailed, indicating that the reported direct effect of the lymphokine on SAC-activated B cells cannot overcome the suppressive activity of PWM-induced suppressor T cells. rIL-2 could not activate suppressor T cells in the absence of PWM, and has no effect on the number of helper or suppressor cells in the culture.

Antibody-Producing Cells↗
PubMed1986

Suppression of Staphylococcus aureus Cowan I-induced immunoglobulin synthesis in vitro: discrimination between the presence of suppressor T cell precursors and effectors.

In co-cultures with control cells lymphocytes obtained from some patients with hypogammaglobulinaemia can suppress PWM but not S. aureus Cowan I-induced polyclonal immunoglobulin production. When such co-cultures were stimulated at the same time with both mitogens, the response was greatly suppressed. This phenomenon was further studied in cultures of lymphocyte populations isolated from healthy donors. It was found that suppressor T lymphocytes activated by PWM in cultures co-stimulated with Con A, with high T:B cell ratio, or with an increased proportion of OKT8+ T cells can suppress the S. aureus-induced response. In contrast, under the same conditions S. aureus did not activate suppressor cells. Moreover, in cultures stimulated with this polyclonal B-cell activator OKT8+ lymphocytes could serve as helper cells.

Agammaglobulinemia↗
PubMed1986

Interleukin 2 binding capacity of human mononuclear leukocytes.

Interleukin 2 (IL-2) receptors which are expressed in T-lymphocytes after antigenic or mitogenic stimulation have been measured with either 3H- or 125J-labelled IL-2 or with receptor antibodies (e.g. anti-Tac). We have developed a new method which allows the determination of endogenously bound IL-2, or of the IL-2 binding capacity, respectively. The method utilizes incubation of target cells with human natural highly purified IL-2. Total bound IL-2 is determined by a conventional 3H-thymidine incorporation assay. An additional step, differential dissociation, eliminates low affinity bound IL-2 so that the remaining high affinity bound IL-2 can be determined. In PHA stimulated mononuclear cells we have found that high affinity binding sites appear after one day at high concentration and disappear gradually after more than ten days of culture. Low affinity binding sites appeared first on day 2, but showed then similar kinetics. IL-2 binding capacity closely correlated with the ability of the cells to respond to exogenous highly purified human IL-2. Our results demonstrate that in vitro in healthy blood donors the expression of IL-2 binding sites is necessary to render the cells responsive to IL-2 and to induce polyclonal expansion. Pharmacological doses of hydrocortisone abolishes both, IL-2 binding and IL-2 response.

Binding Sites↗