Inhibition of production of tumor necrosis factor by monoclonal antibodies to lipopolysaccharides.

Murine monoclonal antibodies to lipopolysaccharides (LPS) from rough J5 mutant of Escherichia coli O111:B4 protected D-galactosamine-treated mice against lethal effects of LPS and provided protection in an experimental infection model with live E. coli. Three previously prepared anti-LPS antibodies were evaluated for ability to inhibit LPS-induced tumor necrosis factor (TNF) secretion. In vivo, TNF production was induced in mice treated with D-galactosamine and challenged with LPS. Two antibodies (D6B3 and D6B4) decreased serum TNF levels and prevented lethal effects of LPS. Nonprotective anti-LPS antibody (D9A2) and an unrelated antibody to neomycin did not reduce circulating TNF levels after LPS challenge. Pretreatment of mice with D6B3 and D6B4 protected mice from infection with E. coli and prevented serum TNF increases induced by E. coli infection. In nonprotected animals, high levels of TNF were detected in serum 3-6 h after infection; animals died within 24 h. In vitro, addition of protective antibodies to macrophage cultures at initiation of LPS stimulation inhibited TNF production. Nonprotective antibody D9A2 failed to block LPS-induced TNF production.

Structure of the O-specific polysaccharide chain from Klebsiella pneumoniae O1K2 (NCTC 5055) lipopolysaccharide.

The structure of the O-specific polysaccharide of Klebsiella pneumoniae O1K2 lipopolysaccharide was investigated by use of methylation, periodate oxidation, partial hydrolysis, and 1H- and 13C-n.m.r. spectroscopy. It was shown to consist of a linear chain composed of two disaccharide repeating units, [----3)-alpha-D-Galp-(1----3)-beta-D-Galp-(1----] and [----3)-alpha-D-Galp-(1----3)-beta-D-Galf-(1----].

Toxicity and activity of purified trichosanthin.

Trichosanthin was purified from fresh Chinese root tubers of Trichosanthes kirilowii and evaluated for anti-HIV activity. Trichosanthin inhibited syncytium formation between infected H9 cells and uninfected Sup-T1 cells from 0.5 to 4 micrograms/ml. Trichosanthin also inhibited HIV replication in H9 and CEM-SS cells at 1 microgram/ml, but was toxic for MT-4 cells (HTLV-I-positive), at doses greater than 0.25 microgram/ml. This new purification procedure confirms the anti-HIV activity of trichosanthin on some cell lines in different biological assays.

Murine monoclonal antibodies to Klebsiella pneumoniae protect against lethal endotoxemia and experimental infection with capsulated K. pneumoniae.

To prepare monoclonal antibodies (MAbs) directed against the core-lipid A fractions of smooth lipopoly-saccharide (LPS) from Klebsiella pneumoniae O1:K2, we immunized BALB/c mice with the LPS-associated proteins plus LPS. This preparation exposed the core-lipid A moiety, which is normally hidden in the micellar structure of classical LPS preparations. Among 10 MAbs selected for their reactivity with LPS-associated proteins plus LPS from K. pneumoniae O1:K2, 6 (3A3, 3C2, 3C4, 7D2, 11C3, and 12B6) were directed against the core fraction and 2 (6C5 and 10A5) were directed against the lipid A fraction. Only one (2A4) recognized the O antigen, and one (6D5) had an undefined specificity. When injected before challenge with K. pneumoniae O1:K2 LPS in galactosamine-sensitized mice, five of the MAbs (3C4, 6D5, 7D2, 11C3, and 12B6) provided protection in this model of lethal endotoxemia. MAb 7D2 was also protective in an experimental infection with capsulated K. pneumoniae O1:K2.

Effect of RU 41740 on autologous hemopoietic reconstitution of sublethally irradiated mice.

RU 41740, a glyco-protein extract of Klebsiellae pneumoniae 01 K2 strain, is known to have immunomodulator activities. It acts on macrophages as well as on B and T-cells, and enhances their cytokine production, particularly interleukin 1 (IL-1). The fact that cytokines have an effect on hemopoiesis led us to suspect an effect of RU 41740 on hemopoietic reconstitution. In this study, a model of autologous reconstitution of a hemopoietic system after sublethal irradiation in mice was used. C57 BL/6 mice were treated orally with RU 41740 (10 mg/kg/day) before or after irradiation (6.5 Gy). LPS of Klebsiella pneumoniae was used as a positive control. The hemopoietic reconstitution occurred more rapidly in treated animals especially when RU 41740 was given before irradiation.

Protective effects of murine monoclonal antibodies in experimental septicemia: E. coli antibodies protect against different serotypes of E. coli.

Murine monoclonal antibodies that bind outer membrane antigens of the J5 mutant of Escherichia coli O111:B4 were derived from spleen cells of BALB/c mice immunized with killed whole cells and boosted with lipopolysaccharide (LPS) and LPS-associated proteins. Seven hybridomas were selected for their reactivity against the J5 LPS; they cross-reacted with O111, O55, O127, and O128 E. coli LPS. One (B7B3) also reacted with the Serratia marcescens LPS and Klebsiella pneumoniae lipid A. A protective effect was obtained with D6B4 antibody in a lethal endotoxemia model induced by LPS from O111, O127, and O128 E. coli serotypes in D-galactosamine-sensitized mice. D6B4 and D6B3 antibodies protected mice infected with E. coli O111:B4, when administered before infection. The D6B4 antibody was also protective when administered after infection. The antibodies D6B3 and D4B5 were protective in heterologous infection induced by E. coli O2:K1.

Immunological activities of RU-41740, a glycoprotein extract from Klebsiella pneumoniae. III. Role of LPS-like and LPS-non-related molecules.

RU-41740, a glycoprotein complex extracted from Klebsiella pneumoniae, is an immunomodulating agent which acts on B cells and macrophages. It has been shown that RU-41740 is composed mainly of two macromolecular fractions, F1, having an LPS-related structure, and P1, with a proteoglycan structure. In the present paper, the effects of these molecules on B cells and on IL-1 and tumour necrosis factor (TNF), production by macrophages were compared. Data reveal that both fractions were mitogenic for murine B cells and induced IL-1 and TNF production by macrophages. The LPS-like fraction (F1) was sensitive to polymyxin B and was unable to activate macrophages and spleen cells from LPS non-responder mice. The P1 fraction was mitogenic for B cells and induced the production of IL-1 and TNF activities by macrophages from LPS non-responder C3H/HeJ mice. The cytotoxic activity was due to TNF alpha, since treatment with anti-TNF alpha antiserum abrogated the lytic activity of supernatants from stimulated macrophages. The differences observed between P1 and F1 fractions in terms of sensitivity to polymyxin B and activity towards C3H/HeJ spleen cells and macrophages suggest that the two structurally distinct molecules isolated from RU-41740 could act at different sites on immunocompetent cells.

The effect of RU 41740 on the in vitro maturation of human B-cells.

We have tested the effect of a Klebsiella pneumoniae extract, RU 41740, and its lipopolysaccharidic fraction (LPS-LAP) on the in vitro activation of human B-cells. Two models have been used: the polyclonal activation induced by pokeweed mitogen and the specific antibody production to mannan, a polysaccharide extracted from the cell wall of Candida albicans. We have shown that RU 41740, and especially its lipopolysaccharidic fraction, significantly increases the production of immunoglobulins and specific antibodies. This effect is mediated by T-lymphocytes, since preincubation of isolated T-cells, but not of non-T-cells, resulted in the same effect. Together with the known enhancing effect of LPS-LAP on T-cell proliferation, these data strongly suggest that LPS-LAP increases the production of B-cell-activating lymphokines by T-cells.

Purification of the lipopolysaccharide fraction from Klebsiella pneumoniae O1 K2 by high-performance liquid chromatography.

An high-performance liquid chromatography technique was applied to purify the lipopolysaccharide fraction from a lysate of Klebsiella pneumoniae O1 K2 (NCTC 5055). The separation of the lipopolysaccharide fraction from the proteins was carried out with a reversed-phase column. By this method the lipopolysaccharide fraction was obtained in a pure state, devoid of proteins but possessing the same biological properties as the lipopolysaccharide fraction prepared by the classical phenol-water technique.

Enhancement of bronchoalveolar cell recovery and stimulation of alveolar macrophage chemiluminescence and resistance to influenza virus after treatment with RU 41821 aerosol.

Aerosol treatment with RU 41821, a glycoprotein extract from Klebsiella pneumoniae, was tested in mice for its effect on the kinetics of the induction of bronchoalveolar cells (i.e., alveolar macrophages, monocytes, lymphocytes, and polymorphonuclear leukocytes). RU 41821 led to an increase in the total number of bronchoalveolar cells. The largest increase was observed for polymorphonuclear leukocytes, and more moderate increases occurred in the numbers of alveolar macrophages, monocytes, and lymphocytes. The alveolar macrophages recruited in response to RU 41821 were activated, as indicated by luminol-dependent chemiluminescence in response to stimulation by opsonized zymosan. The effects of five RU 41821 aerosol treatments and those of a single treatment were further examined in vivo by aerosol infection of mice inoculated with a mouse-pathogenic influenza virus. The maximum protective effect was obtained after five once-a-day treatments and was correlated with the largest increase in the total number of bronchoalveolar cells.

RU-41740 (K. pneumoniae glycoprotein) enhances resistance to experimental candidiasis and stimulates phagocytic functions.

RU-41740, a purified glycoprotein extract from Klebsiella pneumoniae, (which is an efficient non-specific immune activator in a broad spectrum of in vitro and in vivo reactions) was administered either orally or parenterally in the mouse. It enhanced the resistance of mice to candidiasis, both in terms of survival rate and a decrease in viable yeast cell recovery in kidneys. The drug administered at 0.1 mg or 1 mg/kg augmented 4-fold the mean survival time (MST) of animals infected with 1 to 2 X 10(6) Candida albicans, both by the intraperitoneal and the intravenous route. The effect of the orally administered drug was less striking but nonetheless present. At 10 mg/kg, the MST of infected animals increased about 2-fold. In vitro, in the presence or absence of zymosan, the drug at 10 or 100 micrograms/ml was able to stimulate the phagocytic process of elicited mouse peritoneal cells (65% polymorphonuclear cells, 35% macrophages) and human peripheral blood cells (95% polymorphonuclear cells, 5% monocytes) in terms of activated oxygen species production. The involvement of polymorphonuclear cells in the mechanisms of natural resistance to C. albicans infection led us to discuss the role of these cells as targets for the drug.

Effects of RU 41740 aerosol treatment on mouse bronchoalveolar cells, and protection afforded against influenza virus infection.

RU 41740, an immunomodulating compound extracted from Klebsiella pneumoniae, was previously shown to enhance mice resistance to bacterial and viral lung infections. To explore lung defense mechanisms, we studied the influence of RU 41740 aerosol treatment on the bronchoalveolar cell populations. Five successive daily RU 41740 aerosol treatments induced a large accumulation of leukocytes in the lungs 4h after the last treatment. Polymorphonuclear leukocytes predominated. The numbers of lymphocytes and monocytes rose significantly. A single RU 41740 aerosol treatment significantly raised the number of polymorphonuclears only. A luminol-dependent chemiluminescence assay was used to test the effect of RU 41740 on the opsonized zymosan induced response of alveolar macrophages. In vitro, addition of RU 41740 enhanced this chemiluminescence. After a single RU 41740 aerosol treatment of mice, the chemiluminescence of purified alveolar macrophages from these mice increased significantly. The protective effect of five daily RU 41740 aerosol treatments against influenza virus infection was believed to be due to the great intensity of the cellular response and the polymorphonuclear influx. The alveolar macrophage activation observed might also explain the enhanced resistance of mice to influenza virus infection.

Enhanced resistance of mice against influenza virus infection after local administration of glycoprotein extracts from Klebsiella pneumoniae.

RU. 41 740, a glycoprotein extract from Klebsiella pneumoniae O1K2 strain was tested for its ability to enhance resistance of mice against influenza virus infection. Local (aerosol) and systemic (IP) routes of RU. 41 740 administration were compared for their effectiveness in protecting mice. When RU. 41 740 was administered prophylactically (10 mg/kg) via aerosol route (5 consecutive days before challenge), significant protection (P less than 0.0001) was conferred against lethal aerosol inoculation of influenza virus. Treated mice exhibited a reduced mortality, a decreased lung-to-body weight ratio and lower intrapulmonary virus titers. The main glycoprotein soluble fraction (RU. 41 821) was as active as the total glycoprotein extract (P less than 0.0001). Whereas the local (aerosol) route of administration was effective, the systemic (intraperitoneal) route of administration did not confer significant protection against an aerosol inoculum of virus. This finding suggests the important role of local immunity. The levels of interferon in the lavage fluids of immunized and infected mice suggest that interferon is not the main protective mechanism. The enhanced protection observed could be related to an augmented humoral or cell-mediated response within the lung.

Opposite effects of a glucocorticoid and an immunostimulating agent on prostaglandin production by two different cell types.

Studies were performed to investigate the effects of several agents known to modulate wound healing on prostaglandin production by mouse embryo fibroblasts and adult thymic phagocytic cells in culture. Dexamethasone (10(-6)M) induced in both cell types a significant inhibition of the production of PGE2 and 6 keto PGF1 alpha, and a moderate inhibition of PGF2 alpha. Using an antiglucocorticoid compound, RU 38486, we were able to demonstrate that the inhibition of PG secretion represents a classical receptor-mediated effect of the steroid. In contrast, LPS and RU 41740 (5 micrograms/ml) induced a significant stimulation of PGE2 and 6-keto-PGF1 alpha secretion in the two types of cells. These results suggest that agents which modulate in different ways the process of tissue repair have opposite effects on PG production by cells involved in inflammatory and/or immunological reactions.

Immunological activities of RU-41740, a glycoproteic extract from Klebsiella pneumoniae. I.--Activation of murine B cells and induction of interleukin-1 production by macrophages.

RU-41740, a glycoprotein extract from Klebsiella pneumoniae K2O1 strain, is an immunomodulating compound which has been shown to reduce infectious episodes in immunodeficient patients. Data from preliminary experimental designs suggested that RU-41740 could affect several target cells, such as T cells, B cells and macrophages. In the present report, we show that RU-41740 is a selective B-lymphocyte activator. It induces blast transformation in Nude mouse spleen cell cultures and in B-cell-enriched fractions obtained from normal mice. It does not activate T lymphocytes to proliferate. Activation of mouse B lymphocytes by RU-41740 is not affected by removal of adherent cells. RU-41740 also activates immunoglobulin secretion by murine B lymphocytes. Incubating spleen cells from C3H/HeJ mice with RU-41740 results in cell proliferation and activation of antibody-forming cells. This suggests that B-cell activation is not due to LPS contamination. Other experiments show that RU-41740 can also trigger mouse macrophages to produce interleukin-1 activity. Indeed, supernatants from peritoneal adherent cells incubated in the presence of RU-41740 can stimulate blastogenesis in thymocytes from C3H/HeJ mice. Thus, B-cell activation and IL-1 production by macrophages could constitute two additive mechanisms involved in immunomodulation induced by RU-41740.

Enhancement of delayed cutaneous hypersensitivity in untreated cancer patients given a short-term oral treatment with C 1821.

C 1821 is a purified glycoprotein extract of Klebsiella pneumoniae serotype 2 with immunomodulating properties in animals (in vivo and in vitro) and in humans (in vitro). The compound is devoid of any apparent toxicity when given orally. The aim of the present work was to evaluate the effects of a short term oral administration of C 1821 on delayed cutaneous hypersensitivity to recall antigens in untreated cancer patients (mostly lymphomas). Consecutive patients were alternately allocated to receive C 1821 or placebo for 14 days. C 1821 restored and significantly (P less than 0.02) enhanced skin reactions, as shown using the Multitest system.