Inhibitory and stimulatory effects of Pseudomonas aeruginosa pyocyanine on human T and B lymphocytes and human monocytes.

Pyocyanine, a pigment produced by Pseudomonas aeruginosa, has dual dose-dependent stimulatory as well as inhibitory effects on immune responses in vitro as measured by DNA synthesis of human T and B lymphocytes, interleukin-2 (IL-2) production by human T lymphocytes, immunoglobulin production by human B lymphocytes, and monokine production by human monocytes. In general, stimulatory activity was found at low concentrations of pyocyanine, whereas high concentrations of the pigment resulted in an inhibition of responses. At a pyocyanine concentration of 0.

Functional and molecular characterization of a monoclonal antibody against the 165-186 peptide of human IL-1 beta.

A synthetic peptide of human recombinant interleukin 1 beta (hrIL-1 beta) 165-186, which exhibits biological activity in the human fibroblast assay, was used as an immunizing antigen to obtain a murine monoclonal antibody (MoAb) termed FIB 1. This MoAb, an IgG1, reacts specifically with hrIL-1 beta, but not with hrIL-1 alpha, as measured in enzyme-linked immunosorbent assays (ELISA). The MoAb FIB 1 detects the characteristic 17 kDa IL-1 protein in Western blots.

Induction of tumor necrosis factor-alpha release by lipopolysaccharide and defined lipopolysaccharide partial structures.

We have investigated the release of tumor necrosis factor-alpha (TNF-alpha) by human mononuclear cells (MNC) and isolated human monocytes/macrophages stimulated with S- and R-form lipopolysaccharide (LPS), natural lipid A, and natural and synthetic partial structures thereof. We found that LPS of Salmonella minnesota (S. min.

Lissencephaly, abnormal lymph nodes, and T-cell deficiency in one patient.

We report on a child with lissencephaly type I, abnormal lymph nodes, and immunodeficiency, associated with recurrent infections, autoimmune disease, spastic tetraplegia, and psychomotor retardation. Diagnostic measures included cranial computer tomography (CT) and magnetic resonance imaging (MRI) scanning, several in vivo and in vitro immunological tests, and histology of skin, lymph nodes, and liver including electron microscopy and immunohistology. Despite medical supervision, the child died at age 4 years.

Detection of interleukin 1 with human dermal fibroblasts.

A fibroblast proliferation assay was developed for the detection of interleukin 1 (IL 1). Proliferation was measured by thymidine incorporation and by staining of cellular proteins with crystal violet. Response of fibroblasts was optimal at cell numbers of 4,000 to 9,000 cells/culture and an incubation period of four days.

Dipeptidyl peptidase IV in human T lymphocytes. Impaired induction of interleukin 2 and gamma interferon due to specific inhibition of dipeptidyl peptidase IV.

Specific inhibitors of the membrane-bound dipeptidyl peptidase IV (DP IV) and polyclonal antibodies against this enzyme were used to investigate the relationships between DP IV activity and the production and action of T cell-derived lymphokines. Production of interleukin 2 (IL-2) and gamma interferon by mitogen plus phorbol ester-stimulated mononuclear cells from human blood was found to be reduced in the presence of N-Ala-Pro-O-(nitrobenzoyl-)-hydroxylamine, epsilon-(4'-nitro) benzoxycarbonyl-Lys-Pro, and anti-(DP IV) immunoglobulin in a dose-dependent manner. Moreover, the proliferative response of mitogen-stimulated mononuclear cells to IL-2 is impaired in the presence of DP IV inhibitors.

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.

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.

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).

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.

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.

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.

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.

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.

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.

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.

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).

Prospective evaluation of chymopapain sensitivity in patients undergoing chemonucleolysis.

Immediate anaphylactic reactions after intradiscal chymopapain (CP) injection may occur in 1% of patients undergoing chemonucleolysis (CN). Skin prick testing to CP (10 mg/ml), a prescreening history, and CP serum-specific IgE determinations by the RAST method were performed in order to identify patients presensitized to CP before CN. Follow-up repeat CP skin testing and serum-specific IgE were done 2 to 6 weeks after CN to detect CP IgE-mediated sensitization resulting from the injection.

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+.

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.

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.

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.

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.