Activation of tumor-infiltrating macrophages by a synthetic lipid A analog (ONO-4007) and its implication in antitumor effects

ONO-4007 is a novel synthetic analog of lipid A subunit and has been shown to exert antitumor activities on various experimental tumors with less toxicity than lipopolysaccharide. It remains unclear, however, what biological activities of this compound are relevant to its antitumor effects. We therefore investigated the activation of macrophages by ONO-4007 in vitro and in vivo and its implication in antitumor effects, using mouse MM46 mammary tumor as an experimental model. Intravenous injection of ONO-4007 produced significant therapeutic effects on this solid tumor. ONO-4007 could stimulate glycogen-elicited peritoneal macrophages in vitro, not only to produce tumor necrosis factor (TNF), but also to exert cytocidal activities against MM46 cells in vitro. Substantial TNF production was induced in tumor tissue by i. v. injection of ONO-4007, and its successive administration to tumor-bearing mice gave tumor-infiltrating macrophages a prominent in vitro tumoricidal activity and primed them for in vitro TNF secretion. These results suggest that activation of tumor-infiltrating macrophages to a direct tumoricidal state as well as to TNF secretion in tumor tissues may be at least some of the antitumor effects of this novel lipid A analog.     

S-form lipopolysaccharide (LPS), but not lipid A or R-chemo-type LPS, induces interleukin-6 production in vitamin D3-differentiated THP-1 cells

Bacterial lipopolysaccharide (LPS) induces the production of various inflammatory cytokines and the inducibility is considered attributable to the glycolipid part of LPS called lipid A. We report an in vitro model in which lipid A is not necessarily a minimal structure for the LPS activity. Vitamin D3-differentiated THP-1 cells, cultured human monocytic leukemia cells, produced a high level of interleukin-6 (IL-6) by stimulating LPS from Escherichia coli O111:B4, but not by stimulating synthetic E. coli-type lipid A (compound 506), E. coli Re mutant LPS (ReLPS), or alkali-treated LPS. The induction by LPS was inhibited by the anti-CD14 antibodies or by the synthetic lipid A precursor (compound 406). An alkali-treated LPS or compound 506 partially inhibited the LPS-induced IL-6 production. These facts suggest that lipid A alone is not sufficient for the IL-6-inducing activity, but the polysaccharide part in LPS contributes or acts as a co-factor for activation of differentiated THP-1 cells.     

Interferon Production by Nonviral Stimuli of Microbial Origin

An increasing number of nonviral materials of microbial origin has been reported to stimulate the production of interferon in cell cultures and (or) in animals. These materials include (a) gram-negative bacteria or the endotoxins prepared from their cell walls, (b) other microorganisms such as Rickettsiae, Bedsoniae, Protozoa, and (c) fungal products such as a mannan from Candida and various antibiotics which act as protein synthesis inhibitors, e.g., glutarimide antibiotics and tenuazonic acid. A summary is presented of the current state of knowledge about interferon production in animals by the most thoroughly studied nonviral substance of microbial origin, bacterial endotoxin. Further evidence is presented which clearly distinguishes the "endotoxin-type" of interferon response in animals from the response seen after the injection of virus. The data suggest that the release of preformed interferon from the tissues occurs in animals injected with endotoxin. On the other hand, interferon produced in response to the injection of virus is newly synthesized protein. While the exact chemical structure of the component of bacterial endotoxin responsible for interferon release has not yet been elucidated, it is clear that the lipid portion of the lipopolysaccharide, rather than the O-specific polysaccharide side chains or the core polysaccharide, is the active moiety.     

Chemokine-containing exosomes are released from heat-stressed tumor cells via lipid raft-dependent pathway and act as efficient tumor vaccine

Exosomes derived from dendritic cells or tumor cells are a population of nanometer-sized membrane vesicles that can induce specific antitumor immunity. During investigation of the effects of hyperthermia on antitumor immune response, we found that exosomes derived from heat-stressed tumor cells (HS-TEX) could chemoattract and activate dendritic cells (DC) and T cells more potently than that by conventional tumor-derived exosomes. We show that HS-TEX contain chemokines, such as CCL2, CCL3, CCL4, CCL5, and CCL20, and the chemokine-containing HS-TEX are functionally competent in chemoattracting CD11c(+) DC and CD4(+)/CD8(+) T cells both in vitro and in vivo. Moreover, the production of chemokine-containing HS-TEX could be inhibited by ATP inhibitor, calcium chelator, and cholesterol scavenger, indicating that the mobilization of chemokines into exosomes was ATP- and calcium-dependent and via a lipid raft-dependent pathway. We consistently found that the intracellular chemokines could be enriched in lipid rafts after heat stress. Accordingly, intratumoral injection of HS-TEX could induce specific antitumor immune response more efficiently than that by tumor-derived exosomes, thus inhibiting tumor growth and prolonging survival of tumor-bearing mice more significantly. Therefore, our results demonstrate that exosomes derived from HS-TEX represent a kind of efficient tumor vaccine and can chemoattract and activate DC and T cells, inducing more potent antitumor immune response. Release of chemokines through exosomes via lipid raft-dependent pathway may be a new method of chemokine exocytosis.     

Direct evidence that bacterial lipopolysaccharides elevate cyclic GMP levels in rat fetal liver cells

We have previously shown that crude bacterial lipopolysaccharide (LPS) preparations markedly increase cGMP levels in rat fetal liver cells in a time- and dose-dependent manner. To provide evidence that this effect was due to LPS and not an impurity in the preparations, three series of experiments were undertaken. First, LPS was prepared from Escherichia coli 055:B5 cells and its cGMP potency assessed at various stages of purification; second, the cGMP activity of three highly purified LPS preparations of known chemical structure was measured, and third, a well characterized LPS was broken into its lipid A and polysaccharide fractions and the cGMP activity of each fraction determined. The results showed that the cGMP stimulatory activity in E. coli 055:B5 cells co-purified in a parallel fashion with the LPS molecule derived from those cells, that the three chemically defined, highly purified LPS preparations were all very potent stimulators of cGMP levels, and that the ability to increase cGMP levels of lipid A prepared from a highly purified LPS was comparable in potency to the intact LPS, whereas the polysaccharide portion of the molecule was without activity. These findings indicate that the cGMP effect of LPS preparation is due to LPS and not a contaminant and that the activity resides within the lipid A moiety of the molecule.     

Antioxidant and antitumor activities of the polysaccharide from seed cake of Camellia oleifera Abel

To explore biomedical potential of the polysaccharide from seed cake of Camellia oleifera Abel, we investigated antioxidant and antitumor capacities of the polymer. The results showed that the polysaccharide is capable of scavenging both superoxide anion and hydroxyl radicals in vitro. The highest scavenging rate of superoxide anion and hydroxyl radicals is 85% and 76%, respectively. Using the model animal, Caenorhabditis elegans, we further show that the polysaccharide can increase antioxidant enzyme activity, decrease lipid peroxidation level, and reduce paraquat-induced oxidative damage at a polysaccharide concentration more than 50mg/l. We also revealed that the polysaccharide has some ferric chelating ability and strong in vivo antitumor activity. The antitumor rate against Sarcoma180 solid tumor grown in BALB/C mice reached 85.6% at the highest dose of 40×20mg/kgdays.     

Analysis of a common-antigen lipopolysaccharide from Pseudomonas aeruginosa.

Lipopolysaccharide isolated from Pseudomonas aeruginosa PAO1 (O5 serotype) was separated into two antigenically distinct fractions. A minor fraction, containing shorter polysaccharide chains, reacted with a monoclonal antibody to a P. aeruginosa common antigen but did not react with antibodies specific to O5-serotype lipopolysaccharide. In contrast, fractions containing long polysaccharide chains reacted only with the O5-specific monoclonal antibodies. The shorter, common-antigen fraction lacked phosphate and contained stoichiometric amounts of sulfate, and the fatty acid composition of this fraction was similar to that of the O-antigen-specific fraction. The lipid A derived from the serotype-specific lipopolysaccharide cross-reacted with monoclonal antibodies against lipid A from Escherichia coli, while the lipid A derived from the common antigen did not react. We propose that many serotypes of P. aeruginosa produce two chemically and antigenically distinct lipopolysaccharide molecules, one of which is a common antigen with a short polysaccharide and a unique core-lipid A structure.     

Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Autologous tumor killing and natural cytotoxic activity of tumor-associated macrophages in cancer patients

Summary Tumor-associated macrophages (TAM) isolated from pleural effusions and ascites fluids of cancer patients were tested for cytotoxicity against freshly isolated autologous tumor cells and K562 in a 4-h⁵¹ Cr-release assay, and in vitro effects of OK432 (a streptococcal preparation) and partially purified human leukocyte interferon (IFN) on their cytotoxicities were examined. Positive cytotoxicities against K562 were recorded for TAM samples from 2 of 23 pleural effusions and 3 of 10 ascites specimens. Tumor-associated macrophages were not cytotoxic to autologous tumor cells, while low but significant lysis was observed with tumor-associated lymphocytes (TAL) samples from 2 of 13 pleural effusions and 1 of 6 ascites specimens. In vitro treatment with OK432 resulted in an enhancement of natural cytotoxicity in 4 of 13 TAM and 10 of 15 TAL samples. An induction or augmentation of autologous tumor killing activity by OK432 was observed in 2 of 10 TAM and 8 of 11 TAL samples. In contrast, IFN failed to induce autologous tumor killing activity, although IFN-enhanced lysis of K562 was detected in 1 of 7 TAM and 2 of 9 TAL samples. These results indicated that autologous tumor killing and natural cytotoxic activities were defective in macrophages and lymphocytes at the site of the tumor growth, and both activities were strongly enhanced by OK432 rather than IFN.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.     

Protective effect of fungal growth product (6MFA), assessed therapeutically against Ehrlich's ascites tumor in Swiss mice

6MFA is a growth product of the fungus Aspergillus ochraceus (ATCC 28706) obtained by fermentation in stationary culture. It has both interferon inducing and antiviral properties, in vivo and in vitro, with a relatively high margin of safety (9, 17, 18). Ehrlich's ascites tumor bearing Swiss albino male mice were treated with 0.5 ml of acqueous preparation of 6MFA (0.75 mg total solids) i.p. in a therapeutic regimen schedule; the sham treated mice received only PBS. 6MFA treatment produced an increase in mean survival time over the untreated controls, restricted the body weight increase due to ascites and decreased the rate of mortality. As much as 100% of survival response was obtained in the group treated with 0.5 ml of 6MFA at the rate of one inoculation per week for 5 weeks. In general a dose-dependent response was seen in the antitumor effect of 6MFA against Ehrlich's ascites tumor in Swiss mice. Delay in administration of 6MFA to tumor bearing mice affected the survival rate.     

In vivo antitumor effects of murine interferon- γ -inducing factor/interleukin-18 in mice bearing syngeneic Meth A sarcoma malignant ascites

Interferon-γ-inducing factor/interleukin-18 is a novel cytokine that reportedly augments natural killer (NK) activity in human and mouse peripheral blood mononuclear cell cultures in vitro and has recently been designated IL-18. In this study, IL-18 exhibited significant antitumor effects in BALB/c mice challenged intraperitoneally (i.p.) with syngeneic Meth A sarcoma when administered i.p. on days 1, 2 and 3 after challenge. Intravenous (i.v.) administration also induced antitumor effects in the tumor-bearing mice; however, subcutaneous (s.c.) administration did not. When mice were twice pretreated with 1 μg IL-18 3 days and 6 h before tumor challenge, all mice survived whereas control mice died within 3 weeks of challenge. Inhibitory effects on Meth A cell growth in vitro were not observed with either IL-18 or interferon γ. The effects of IL-18 pretreatment were abrogated by abolition of NK activity after mice had been injected with anti-asialo GM1 antibody 48 h before and, 24 h and 72 h after tumor challenge. Mice pretreated with IL-18 and surviving tumor challenge resisted rechallenge with Meth A cells but could not reject Ehrlich ascites carcinoma, and spleen cells from the resistant mice, but not control mice, exhibited cytotoxic activity against Meth A cells in vitro after restimulation with mitomycin C-treated Meth A cells for 5 days. The effector cells in the spleen cell preparations from resistant mice appear to be CD4⁺ cells because cytolytic activity was significantly inhibited after depletion of this subset by monoclonal antibodies and complement. In conclusion, IL-18 exhibits in vivo immunologically (primarily NK) mediated antitumor effects in mice challenged with syngeneic Meth A sarcoma and induces immunological memory and the generation of cytotoxic CD4⁺ cells. Received: 17 September 1996 / Accepted: 8 November 1996     

In vivo generation of lymphokine activated killer cell activity by ABPP and interleukin-2 and their antitumor effects against immunogenic and nonimmunogenic tumors in murine tumor models

Summary The capacity of the interferon inducer ABPP and recombinant interleukin-2 (IL-2) to generate lymphokine activated killer (LAK) cell activity in vivo was examined and compared to the cytolysis of fresh tumor cells by in vitro generated LAK cells. Various tumors differing in histology and immunogenicity were used in in vitro and in vivo experiments. The i.p. administration of ABPP or IL-2 generated much higher levels of LAK cell activity in the peritoneal exudate than in the spleen. Administration of 2 injections of ABPP was as effective as a 3-day course of moderate doses of IL-2. Generation of LAK cell activity by IL-2 was dose dependent. ABPP had significant antitumor activity in vivo in both the i.p. tumor model and the pulmonary metastasis model when administered early (24–48 h after tumor inoculation), but was ineffective against established (day 3) tumor or advanced grossly visible i.p. (day 8) tumor. Treatment of established tumor with IL-2 and LAK cells was not more effective when ABPP was given concurrently. In contrast when ABPP preceded IL-2 and LAK treatment an additional antitumor effect was seen. Immunogenic tumors were more sensitive to treatment with ABPP than nonimmunogenic tumors. Only a marginal difference in lysability in vitro existed. The antitumor effects of ABPP in vivo may therefore be mediated by mechanisms other than cytolysis by activated killer cells alone. These data taken together suggest that ABPP and IL-2 induce discernable levels of LAK cell activity, but do not synergize when combined     

Antitumor resistance induced by zinostatin stimalamer (ZSS), a polymer-conjugated neocarzinostatin (NCS) derivative

Zinostatin stimalamer (ZSS) is a new anticancer agent derived from neocarzinostatin (NCS), which is synthesized by conjugation of one molecule of NCS and two molecules of poly(styrene-co-maleic acid). ZSS exhibited potent in vitro and in vivo antitumor activity in preclinical experiments, and a clinical trial of the intra-arterial administration of ZSS with iodized oil on hepatocellular carcinoma showed potent antitumor activity. We investigated the effect of ZSS and NCS on antitumor resistance and found that pretreatment with either drug suppressed the growth of MethA tumors in Balb/c mice and induced tumor eradication when given separately by single administration at therapeutic doses between 1 day and 4 weeks before tumor transplantation. The findings that the cytocidal activity of these drugs was not detected in vivo at the time of tumor transplantation and that tumor regression was preceded by a period of transient growth suggested that tumor regression was due to host-mediated antitumor activity induced by these drugs. Pretreatment with ZSS or NCS also suppressed the growth of Colon 26 carcinoma and Sarcoma 180. The finding that NCS showed the same effect as ZSS suggests that poly(styrene-comaleic acid) is not essential for the induction of hostmediated antitumor activity. Furthermore, apo-ZSS, which lacks cytocidal activity, did not induce antitumor activity. From this, it is suggested that the cytocidal effect of ZSS involves the induction of hostmediated antitumor resistance. In athymic Balb/cnu/nu mice, pretreatment with ZSS or NCS did not induce tumor eradication, suggesting that mature T lymphocytes play an important role in tumor eradication. Challenging MethA was rejected withot transient growth in mice that had been cured of MethA, but challenging Colon 26 was not, showing that anti-MethA resistance was augmented selectively in the MethaA-eradicated mice. Splenocytes from MethA-bearing mice pretreated with the drug showed tumorneutralizing activity beginning 14 days after tumor transplantation. Tumor-neutralizing activity was only induced after MethA transplantation. The effector cells of this tumor-neutralizing activity were Thy1.2⁺ T lymphocytes that had been passed through a nylonwool column, but no significant augmentation of cell-mediated cytotoxic activity of splenocytes from MethA-eradicated mice was observed in vitro.     

Iron uptake byDesulfovibrio vulgaris outer membrane components in artificial vesicles

Desulfovibrio vulgaris lipopolysaccharide and outer membrane proteins (OMPs) were incorporated into vesicles ofD. vulgaris phospholipid and studied for [⁵⁵Fe]binding activity. Both lipopolysaccharide and an extract of two major OMPs caused large increases in⁵⁵Fe uptake over control (phospholipid only) vesicles. CommercialSalmonella typhimurium lipopolysaccharide gave a similar result, but the effect was inhibited by calcium ions; this was not the case forDesulfovibrio. The lipid A portion ofS. typhimurium lipopolysaccharide had a high iron-binding ability, whereasDesulfovibrio lipid A iron binding was little different from control values;D. vulgaris lipopolysaccharide thus has a specific iron-binding site within its polysaccharide side chain.     

Separation of 6-deoxy-heptan [correction of 6-deoxy-heptane] from a smooth-type lipopolysaccharide preparation of Burkholderia pseudomallei

Smooth-type lipopolysaccharide (LPS) of Burkholderia pseudomallei has been reported to contain two kinds of O-antigenic polysaccharides, a 1,3-linked homopolymer of 6-deoxy-heptose and a polymer with a repeating unit of -->3)-glucose-(1-->3)-6-deoxy-talose-(1--> with O-acetyl or O-methyl modifications. A LPS preparation containing these two polysaccharides was separated by gel-permeation chromatography in this study. Chemical analysis of the separated fractions revealed the 6-deoxy-heptan [corrected] to be a polysaccharide without a lipid portion and the polymer of glucose and 6-deoxy-talose to be an O-antigenic polysaccharide of the LPS. This result was further supported by the assay of these polysaccharide molecules for macrophage activation activity. The 6-deoxy-heptan [corrected] showed no macrophage activation, indicating that this polysaccharide was not the LPS, but one of the capsular polysaccharides of B. pseudomallei.     

Comparative study of lipid A from pertussis microbes differing in the toxicity of their O-antigens. I. Chemical composition and stability of the bond between lipid A and the specific polysaccharide in B. pertussis lipopolysaccharide]

Data presented in this work indicated that antigens, contrastive by toxicity, obtained by Boiven's method and O'Neill and Tood's method from two strains of Bordetella pertussis differed by stability of lipid A binding with the specific polysaccharide. The influence of duration of the lipopolysaccharide hydrolysis on the fatty acid content in lipid A, and of heptose in the specific polysaccharide was demonstrated. Lipid A fatty acid composition was studied. It is supposed that bound fatty acids are presented as C14 and C19--C22. There was a correlation between the antigen toxicity and the stability of lipid A bond with the specific polysaccharide. Stability of the lipopolysaccharide complex bond depended on heptose and lipid A content and on the composition and the amount of fatty acids in the lipid A preparations.     

Effect of schisanhenol on the antitumor activity of adriamycin

Schisanhenol (Sal) did not diminish the antitumor activity of adriamycin in mice bearing P388 ascites tumor. Sal did not antagonize the suppressive effect of adriamycin on DNA synthesis and cell proliferation in an L1210 ascitic tumor cell culture. Furthermore, Sal at the concentration of 0.1, 0.25, or 1 mM accelerated adriamycin-dependent DNA damage in the presence of Fe3+ in vitro. It appears that Sal was able to protect against adriamycin induced heart mitochondrial toxicity, while it did not antagonize the antitumor activity of adriamycin.     

Radioprotective properties of detoxified lipid A from Salmonella minnesota R595

In the past, the toxicity of bacterial lipopolysaccharide (LPS) or its principal bioactive component, lipid A, has detracted from their potential use as radioprotectants. Recently, a relatively nontoxic monophosphoryl Lipid A (LAM) that retains many of the immunobiologic properties of LPS has been isolated from a polysaccharide deficient Re mutant strain of Salmonella minnesota (R595). The ability of the native endotoxic glycolipid (GL) from S. minnesota (R595) as well as diphosphoryl lipid A (LAD) and nontoxic monophosphoryl lipid A (LAM) derived from GL to protect LPS responsive (CD2F1 or C3H/HeN) and nonresponsive (C3H/HeJ) mice from 60Co gamma irradiation has been studied. Administration of GL, LAD, or LAM to CD2F1 or C3H/HeN mice (400 micrograms/kg) 24 h prior to exposure provided significant radioprotection. No protection was afforded to C3H/HeJ mice. Experiments were also conducted to determine the relative abilities of GL, LAD, and LAM to stimulate hematopoiesis as reflected by the endogenous spleen colony (E-CFU) assay. Protection was not correlated with the ability of these substances to increase E-CFUs or to induce colony-stimulating activity (CSA).     

Strong interaction of lipopolysaccharides possessing the mannose homopolysaccharides with complement and its relation to adjuvant action

LPS from Klebsiella pneumoniae O3 (KO3 LPS) exhibited an extremely high anticomplementary activity by the hemolysis assay using human sera. The free lipid A isolated from KO3 LPS by acid hydrolysis and R form LPS from a mutant lacking the O-specific polysaccharide portion possessed lower anticomplementary activity, and the O-specific polysaccharide fraction isolated from KO3 LPS alone did not activate the C system. It was suggested that the O-specific polysaccharide moiety enhanced the C activation by the lipid A portion. This was also supported by the finding that modification of the O-specific polysaccharide moiety with Con A or tyramine decreased anticomplementary activity of KO3 LPS, and that the other LPS preparations possessing the mannose homopolysaccharides as the O-specific polysaccharide portions such as KO3 LPS, such as LPS from Klebsiella O5, Escherichia coli O8 and O9, exhibited a high anticomplementary activity. KO3 LPS could activate the C system in either the classical or the alternative pathway, whereas the lipid A or R form LPS activated the classical pathway alone. The intensity of anticomplementary activity of LPS was parallel to that of their adjuvant action on antibody response to deaggregated BSA. The role of the anticomplementary activity in the expression of the adjuvant action of LPS is discussed.