INFECTION AND IMMUNITY, Dec. 1986, p. 673-6820019-9567/86/120673-10$02.00/0Copyright C 1986, American Society for Microbiology

Immunobiological Activities of Synthetic Lipid A Analogs withLow Endotoxicity

SHOZO KOTANI,1* HARUHIKO TAKADA,' ICHIRO TAKAHASHI,' TOMOHIKO OGAWA,' MASACHIKATSUJIMOTO,' HIDETOSHI SHIMAUCHI,' TAKAKO IKEDA,' HARUKI OKAMURA,' TOSHIHIDE TAMURA,2

KAZUHIRO HARADA,3 SHIGENORI TANAKA,4 TETSUO SHIBA,5 SHOICHI KUSUMOTO,5 AND

TETSUO SHIMAMOTO5

Department of Microbiology and Oral Microbiology, Osaka University Dental School, Suita, Osaka 5651; Department ofBacteriology, Hyogo College of Medicine, Nishinomiya, Hyogo 6632; Department of Microbiology, Osaka Prefectural

Institute for Public Health, Higashinari-ku, Osaka 5373; Tokyo Research Institute, Seikagaku Kogyo, Co. Ltd.,Higashiyamato, Tokyo 1894; and Faculty of Science, Osaka University, Toyonaka, Osaka 560,5 Japan

Received 28 May 1986/Accepted 5 August 1986

Synthetic lipid A analogs, j0(1-6)glucosamine disaccharide 1,4'-bisphosphates, which possesses fourtetradecanoyl groups at the 2- and 2'-amino, and 3- and 3'-hydroxyl groups (LA-17-PP), and each two of the(R)-3-hydroxytetradecanoyl and tetradecanoyl groups at the 2- and 2'-amino and 3- and 3'-hydroxyl groups,respectively (LA-18-PP), were far less endotoxic than synthetic (506, LA-15-PP) and bacterial Escherichia colitype lipid A's; neither compound showed any detectable lethal toxicity in chicken embryos or preparatoryactivity for the local Shwartzman reaction in rabbits. Also both compounds were only weakly pyrogenic andcomparably less lethally toxic in galactosamine-loaded mice than the reference synthetic and bacterial lipid A'sand a synthetic counterpart to biosynthetic lipid A precursor Ia (406, LA-14-PP). Nevertheless, LA-17-PP andLA-18-PP exhibited definite in vivo immunoadjuvant activity in mice, and the ability to induce a possible tumornecrosis factor and alpha/beta interferon in Mycobacterium bovis BCG and Propionibacterium acnes-primedmice, respectively, although these activities were weaker than those of the reference lipid A's. 4'-Monophosphate analogs of the above two test compounds exhibited neither endotoxic nor beneficial activities,but they showed remarkable in vitro bioactivities comparable to those of the corresponding bisphosphatecompounds; the ability to activate the human complement system and the clotting enzyme cascade of horseshoecrab amoebocyte lysate, stimulatory effects on guinea pig and murine peritoneal macrophages, and murinesplenocytes.

In this series of studies on synthesis of lipid A's andrelated compounds (11, 12), synthetic counterparts of Esch-erichia coli-type lipid A (compound 506, renamed LA-15-PP)and of Salmonella minnesota-type lipid A (compound 516,LA-16-PP) as well as the compound (compound 406, LA-14-PP) corresponding to biosynthetic lipid A precursor Ia (3, 7,8, 31) have been prepared (Fig. 1). Their endotoxic and otherbiological activities have been extensively investigated (4, 5,9, 13, 14, 17-19, 32, 34). One of the important findingsobtained is that ,B-acyloxyacyl groups which substitute thehydroxyl or amide groups of the P(1-6)-D-glucosamine disac-charide 1,4'-bisphosphate backbone play a prominent role inthe manifestation of endotoxic activities such as pyrogenic-ity, lethality, and preparative activity for the local Shwartz-man reaction. In addition, some of the bioactivities of lipid Ado not necessarily require the complete structure of lipid A(4, 13, 17-19, 32).

Several beneficial activities of bacterial endotoxin, forexample, immunoadjuvanticity, antitumor activities, en-hancing effects of nonspecific host defense mechanismsagainst microbial infections, and related activities, havebeen reported (21, 22, 27). These bioactivities have beeninvestigated for possible clinical use of lipopolysaccharide(LPS) derivatives with low toxicity (28, 29, 33). One of themost important projects of synthetic studies on lipid A is toprepare lipid A analogs that retain strong enough beneficialactivities of LPS, but have no or little toxicity. For thispurpose, four kinds of lipid A analogs (LA-17-PP, LA-17-

* Corresponding author.

PH, LA-18-PP, and LA-18-PH) resembling LA-14-PP, whichproved to have low endotoxicity but to still retain many oftheir immunobiological activities including beneficial ones(4, 13, 17-19, 32), were recently synthesized in Shiba'slaboratory (Fig. 1). We examined the endotoxicity andimmunobiological activities of these lipid A analogs ascompared with those of LA-15-PP (506), LA-14-PP (406),and bacterial lipid A's (F515 and R595 lipid A's) in a varietyof in vivo and in vitro assay systems.(A part of this study was presented by one of the authors

in the International Symposium on "Synthetic Immunomod-ulators and Vaccines" at Trebon, 14 to 18 October 1985 [20].The present study is a comprehensive extension of thisrecently reported study.)

MATERIALS AND METHODSAnimals. BALB/c, C57BL/6, DBA/2, C3H/HeN, and ICR

mice were purchased from Charles River Japan, Osaka,Japan. ICR mice were also supplied by Shizuoka Experi-mental Animal Co., Hamamatsu, Japan. BALB/c nulnu andC3H/HeJ mice were obtained from Clea Japan, Osaka, andJackson Laboratory, Bar Harbor, Maine, through the cour-tesy of Hajime Kawasaki (NRI Life Science, Kanagawa,Japan), respectively. Domestic Japanese white rabbits andHartley albino guinea pigs were obtained from Nihon RabbitCo., Osaka, Japan.

Test materials. (i) Synthetic lipid A analogs and referencesynthetic compounds. The synthetic lipid A analogs used inthis study (LA-17 and LA-18 series compounds) were pre-pared by a strategy similar to that described for an E. coli-

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674 KOTANI ET AL.

CH2 / CH2

R4-0 0 HO O

0 013 2 -R'1

- R~~~~~~~

Compound R3' R2 R3 R2 R4 R1

LA-17-PP C14 C14 C14 C14 P P

LA-17-PH C14 C14 C14 C14 P H

LA-18-PP C14 C14-0H C14 C14-OH P P

LA-18-PH C14 C14-OH C14 C14-OH P H

LA-14-PP C14-OH C14-OH C14-OH C14-OH P P(406)

LA-15-PP C14-0-(C14) C14-0-(C12) C14-OH C14-OH P P(506)

FIG. 1. Chemical structure of test synthetic lipid A analogs and those of reference synthetic compounds. Abbreviations: P, PO(OH)2; C14,tetradecanoyl; C14-OH, (R)-3-hydroxytetradecanoyl; C14-O-(C12), (R)-3-dodecanoyloxytetradecanoyl; C14-O-(CM4), (R)-3-tetradecanoyloxytet-radecanoyl.

type lipid A, LA-15-PP (11). A P(1-6)-linked glucosaminedisaccharide was used as a common synthetic intermediate,which was obtained by coupling of 2-deoxy-4-O-diphenyl-phospho - 3 -0 - tetradecanoyl - 6-0-(2,2,2-trichloroethoxycar-bonyl)2-(2,2,2-trichloroethoxycar-bon-ylamino)-ci-D-gluco-pyranoside. Deprotection after simultaneous acylation of thetwo amino groups of the disaccharide with eithertetradecanoic or (R)-3-benzyloxytetradecanoic acid gave the4'-monophosphate LA-17-PH or LA-18-PH, respectively.Deprotection after phosphorylation of the glycosidic posi-tion gave the corresponding 1,4'-bisphosphates (LA-17-PPand LA-18-PP). The structures of all key intermediates wereconfirmed by means of spectroscopic and elemental analy-ses. Each final product showed a single spot on thin-layerchromatography and gave a satisfactory result in elementalanalysis as follows. Found for LA-17-PP: C, 60.91; H, 9.65;N, 2.44% (calculated for C68H130N2019P2: C, 60.87; H, 9.77;N, 2.09%). Found for LA-17-PH: C, 64.46; H, 10.25; N,2.16% (calculated for C68H129N2016P: C, 64.73; H, 10.30; N,2.22%). Found for LA-18-PP: C, 57.99; H, 9.55; N, 2.11%(calculated for C68H130N2021P2 * 2H20: C, 57.94; H, 9.58;N, 1.99%). Found for LA-18-PH: C, 62.47; H, 10.03; N,2.43% (calculated for C68H129N2018P * 3/4H20: C, 62.48; H,10.06; N, 2.14%). LA-14-PP (406) and LA-15-PP (506),whose synthesis (11, 12) and biological activities were de-scribed previously (4, 5, 8, 13, 17-19, 31, 33), were used asreference synthetic compounds, except in a few cases inwhich the amount of the reference compounds was verylimited.

(ii) Reference natural products. Lipid A specimens fromthe E. coli 08:K27 Re mutant (strain F515) and S. minnesotaRe mutant (strain R595) were generously supplied by C.Galanos, Max-Planck-Institut fur Immunbiologie, Freiburg,Federal Republic of Germany, and N. Kasai, Showa Uni-versity, Tokyo, Japan, respectively. These specimens wereprepared by acid hydrolysis with 0.1 M acetic acid (in thecase of F515) or with 0.17 M acetic acid (R595) of the

respective glycolipids isolated by the phenol-chloroform-petroleum ether method (6). Their chemical analytical datahave been reported in brief by Imoto et al. (10) and Kotani etal. (17), respectively.

(iii) Preparation of test solution or suspension. Both syn-thetic and natural products were -dissolved at a concentrationof 2 mg/ml in a 0.1% (vol/vol) aqueous solution of triethyl-amine (Wako Pure Chemical, Ltd., Osaka, Japan), exceptfor the assay of pyrogenicity. All the test compounds weredissolved at least as judged macroscopically except LA-17-PH and LA-18-PH which, especially the former, were hardlydissolved at this concentration but gave a homogeneous finesuspension after ultrasonication. The stock solutions thusprepared were kept at 4°C and appropriately diluted withdistilled water, physiological saline (the saline), or cellculture medium before use for bioassays except in thefollowing tests. In the test for preparatory activity in thelocal Shwartzman reaction in rabbits, the stock solutionswere diluted with an equal volume of an aqueous solution (2mg/ml) of bovine serum albumin (BSA; A-7511; SigmaChemical Co., St. Louis, Mo.) and then diluted with thesaline. For the pyrogenicity test, test specimens (triethylam-monium salt form) were dissolved or suspended directly inthe saline.

Endotoxic activities. (i) Lethal toxicity. The assays werecarried out with galactosamine-loaded mice and chickenembryos by the methods of Galanos et al. (2) and Finkelstein(1), respectively, as described previously (19). In the former,C57BL/6 mice (male, 8 to 10 weeks old) were sensitized byintraperitoneal injection of 16 mg of D-galactosamine hydro-chloride (Wako) in 0.5 ml of 0.01 M phosphate-bufferedsaline (pH 7.5), followed immediately by intravenous injec-tion of the test material in 0.2 ml of distilled water. Thenumber of mice that died by intoxication was counted over a24-h period; the 50% lethal dose was calculated by themethod of Karber (15). In the latter, chicken embryos (11days old, White Leghorn, ISA Babcock-B300; Murayama

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BIOACTIVITIES OF LOW TOXIC SYNTHETIC LIPID A ANALOGS

Poultry Farm, Kagawa, Japan) were injected intravenouslywith test material in 0.2 ml of distilled water and observedfor 48 h to determine the 50% lethal dose.

(ii) Pyrogenicity. Rectal temperature of Japanese domesticrabbits (2 to 2.5 kg) after intravenous injection of the testspecimens was recorded continuously for three hours ac-cording to the Japanese Pharmacopeia, and a maximumincrease of rectal temperature of more than 0.6°C wasregarded as a positive febrile response.

(iii) Preparative activity for the local Shwartzman reaction.This was tested in Japanese white rabbits (female, weighingca. 3 kg) by a conventional method (17).Limulus test. The Limulus test was performed by colori-

metric methods (Toxicolor Test; Seikagaku Kogyo, Tokyo,Japan [23]; ES-Test; Seikagaku Kogyo [24]), and the con-ventional gelation method was performed with Pre Gel, anamoebocyte lysate of Tachypleus tridentatus (SeikagakuKogyo). An LPS specimen from E. coli O111:B4 (LPS-W;Difco Laboratories, Detroit, Mich.) was used as a referencestandard in these tests.

Activation of human complement. Pooled, fresh adult hu-man serum, kept at -70°C, was incubated with an equalvolume of test material solution or suspension appropriatelydiluted with distilled water. After incubation at 37°C for 1 h,the decrease in total hemolytic activity (median complementhemolytic dose) was determined by the one-point method(16). The blocking effect on the classical pathway by theaddition of 10 mM EGTA [ethyleneglycol-bis-(f3-amino-ethylether)-N,N,N', N'-tetraacetic acid] plus 5 mM MgCl2was also determined. In the latter assay, zymosan (Nutri-tional Biochemical Corp., Cleveland, Ohio) activated byboiling for 90 min was used as a positive reference thatactivated the human complement system exclusively via thealternative pathway.

Immunobiological activities in vivo. (i) Immunoadjuvantactivities. Groups of eight BALB/c (male, 5 to 6 weeks old)were immunized by subcutaneous injection of 100 ,ug of BSA(fraction V; Sigma) with 100 ,ug of a test adjuvant, both ofwhich were incorporated into liposomes (30) consisting ofL-a-phosphatidylcholine dimyristoyl (purity, approximately98%; Sigma), cholesterol (.99%; Sigma) and dicetyl phos-phate (Sigma) in a molar ratio of 2:1.5:0.22 (the dose ofphosphatidylcholine dimyristoyl was 1.36 mg per mouse).The mice received the second injection of the same lipo-somes (in the case of experiment I) or the liposomes con-taining BSA alone (experiment II) 28 days after the primaryimmunization. Five days after the booster, the antibodylevel in peripheral blood was determined by the passivehemagglutination method and enzyme-linked immunosor-bent assay (25).

(ii) Induction of serum factor cytocidal to L-929 cells inBCG-primed mice. Groups of ICR mice (female, 5 weeks old;Charles River Japan), which were primed by intravenousinoculation of 1.3 x 108 viable units (in 0.2 ml of saline) offreeze-dried, percutaneous (Mycobacterium bovis BCG vac-cine (Japan BCG Laboratory, Tokyo) 2 weeks earlier, werechallenged by an intravenous injection of test specimens in0.2 ml of saline. Ninety minutes after the elicitation, bloodsamples were drawn and inactivated by heating at 56°C for30 min, and then their cytocidal effect on actinomycinD-treated L-929 cells (NCTC clone 929) was examined (17).

(iii) Induction of IFN-a/13 in mice pretreated with Propioni-bacterium acnes. Groups of ICR mice (female, 10 weeks old;Charles River Japan) were treated by intraperitoneal injec-tion of 1 to 1.5 mg of heat-killed P. acnes cells (in 0.2 ml ofphosphate-buffered saline). Several days after the treatment,

primed mice were intravenously injected with appropriatedoses of test material in 0.2 ml of phosphate-buffered saline.Blood samples were drawn from the heart 2 h after theeliciting injection, and the serum interferon (IFN) levelswere assayed by measuring the capacity to inhibit thecytopathic effect of vesicular stomatiotis virus on L-929 cells(26). Titers were expressed as international reference unitsby using the National Institutes of Health reference IFN asa standard in these tests.

Immunobiological activities in vitro. (i) Stimulation ofguinea pig macrophages. The stimulation was measured interms of enhanced glucosamine incorporation and superox-ide anion (02-) generation by peritoneal macrophages (17).Oil-induced macrophages obtained from the peritoneal washof a Hartley albino guinea pig (female, weighing around 500g) that had received an intraperitoneal injection of 20 ml ofsterile liquid paraffin (Wako) 4 days before, were culturedwith test specimens (none in control) for 48 h in RPMI 1640medium supplemented with 10% fetal calf serum (FlowLaboratories Inc., McLean, Va.) at a density of 5 x 105 cellsper 0.5 ml per well of a 24-well microculture plate (Falcon3047; Becton Dickinson and Co., Paramus, N.J.). For theassay of glucosamine incorporation, during the final 6 h ofthe culture the cells were pulsed with 0.1 ,uCi of D-[14C]glucosamine hydrochloride (New England NuclearCorp., Boston, Mass.). After completion of the culture, theradioactivity in the trichloroacetic acid-insoluble fraction ofeach culture was measured by the liquid scintillationmethod.For the assay of 02- generation, cells prepared and

cultured with test specimens for 48 h as described abovewere incubated with 1 ml of 50 ,uM cytochrome c (from horseheart, type VI; Sigma) in N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer at 37°C for 10 min. Then, 10 RIeach of wheat germ agglutinin (4 mg/ml; Sigma) andcytochalasin E (500 ,ug/ml; Sigma) were added, and theculture was incubated at 37°C for 15 min. The rate ofreduction of cytochrome c in the reaction mixture wasmeasured by recording the absorption at 550 to 540 nm witha double-beam spectrophotometer (model 200-20; HitachiLtd., Tokyo) and expressed as nanomoles per minute per 106cells (molar absorption coefficient, 19.1 x 103).

(ii) Stimulation of murine macrophages. The enhancingeffects of test specimens on prostagrandin E2 (PGE2) andinterleukin 1 (IL-1) production was examined as describedpreviously (17). In brief, for the PGE2 assay, macrophagemonolayers prepared from resident peritoneal cells obtainedfrom normal ICR mice (male, 8 weeks old; Shizuoka Exper-imental Animal Co.) were cultured with the test specimen

TABLE 1. Lethal toxicity of LA-17 and LA-18 series lipid Aanalogs and reference synthetic and bacterial products in

galactosamine-loaded mice

No. of deaths/no. of mice testedCompound with the following dose (ng): 50%, Lethaldose (ng)

1,000 100 10 1

LA-17-PP 9/10 3/5 0/5 _100LA-17-PH 0/10 0/5 0/5 _aLA-18-PP 6/10 4/5 0/5 _126LA-18-PH 0/10 0/5 0/5 -

LA-14-PP 5/5 10/20 0/5 10LA-15-PP 5/5 4/10 0/5 13F515 lipid A 5/5 3/10 0/5 16R595 lipid A 5/5 2/5 0/5 13

a Not calculable.

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676 KOTANI ET AL.

TABLE 2. Comparison of the pyrogenicity of LA-17 and LA-18series of lipid A analogs with those of reference synthetic and

bacterial products

Compound Dose Avg of maximum No. of rabbits(jg/kg) increase of temp (°C) positive/no. tested

LA-17-PP 10 1.40 3/31 0.13 0/3

LA-17-PH 10 0.53 2/31 0.00 0/3

LA-18-PP 10 0.22 0/31 0.13 0/3

LA-18-PH 10 0.33 0/31 0.32 0/3

LA-14-PP 10 0.95 1/11 0.80 3/40.1 0.24 0/3

LA-15-PP 1 1.24 4/40.1 1.07 3/30.01 1.23 2/20.001 0.48 0/2

F515 lipid A 1 1.11 6/60.1 0.42 1/60.01 0.36 0/2

(none in control) for 24 h at a density of 2 x 106 cells per mlof RPMI 1640 medium with 10% fetal calf serum in Falconpetri dishes (35-mm diameter). Then the culture superna-

tants were collected, and the PGE2 content was measured byradioimmunoassay with the bicyclic PGE2 assay system(TRK 800; Amersham Japan, Tokyo).For IL-1 production, macrophage monolayers prepared

from peritoneal exudate cells from DBA/2 mice (female, 8 to10 weeks old) that were pretreated by intraperitoneal injec-tion of 1 ml of thioglycolate broth (Nissui PharmaceuticalCo., Tokyo) 3 days before, were cultured with test speci-mens for 24 h at a density of 1.5 x 106 cells per 3 ml in RPMI1640 medium without fetal calf serum in the 35-mm petridish. The culture supernatants were collected, and their IL-1activitiy was determined in terms of the enhancement of[3H]thymidine (Amersham International plc., Backingham-shire, England) uptake by thymocytes of C3H/HeJ mice

TABLE 3. Activity of LA-17 and LA-18 series lipid A analogs as

compared with those of reference synthetic and bacterial productsto prepare rabbit skin for the local Shwartzman reactiona

No. of hemorrhagic sites/no. of sites tested with the

Compound following dose (pLg/site):

80 20 10 5 2.5 1.25

LA-17-PP 0/3LA-18-PP 0/3LA-14-PP 1/3LA-15-PP 2/2 2/2 1/2 1/2 1/2F515 lipid A 2/2 2/2 2/2 2/2 2/2R595 lipid A 2/2 2/2 2/2 2/2 0/2

a Skin sites of three rabbits were prepared by intracutaneous injection of theindicated doses of test compounds (in 0.2 ml of saline). After 18 h, the rabbitsreceived an intravenous injection of 100 ,ug of E. coli 0127:B8 LPS (in 10 mlof saline) per kg of body weight for provocation of the local Shwartzmanreaction.

(male, 4 to 8 weeks old; nonresponders to LPS) coculturedwith a submitogenic concentration (1 ,g/ml) of phytohemag-glutinin (HA16; Wellcome Reagents, Beckenham, England)for 72 h.

(iii) Stimulation of murine splenocytes. For the assay of themitogenic effect, splenocytes (5 x 105 cells) of BALB/cnulnu (male, 8 weeks old) and C3H/HeJ (male, 6 to 8 weeksold) mice were cultured with graded doses of test materialsin 0.2 ml of RPMI medium supplemented with 10% fetal calfserum for 48 h. In the experiment with splenocytes ofC3H/HeJ mice, phytohemagglutinin and concanavalin A(type IV; Sigma) were used to check the viability or thereactivity of the cells. During the final 24 h of cultivation,cells were pulsed with 0.5 ,uCi of [3H]thymidine. Thymidineuptake was measured by the conventional scintillationmethod. For measurements of polyclonal B-cell activation,splenocytes (6 x 106 cells) of BALB/c mice (male, 8 weeksold) were cultured with graded doses of test materials inRPMI medium supplemented with 10% fetal calf serum.After 72 h of cultivation, the number of hemolytic plaque-forming cells against 2,4,6-trinitrophenylated sheep erythro-cytes was determined in a Cunningham chamber (32).Other procedures. Meticulous precaution was taken to

avoid external contamination of experimental ware andreagents with LPS. In most of the in vitro assays, eachdetermination was made in triplicate cultures to obtain themean and standard error of the stimulation index (ratio ofvalue in each test culture to that in the respective controlculture). Statistical significance of the difference betweeneach test and the respective control was examined by theStudent t test in most of the assays.

RESULTS

Endotoxic activities. (i) Lethal toxicity. LA-17-PP and LA-18-PP exhibited lethal toxicity in galactosamine-loaded mice(Table 1), but their 50% lethal doses were about 10 timeshigher than those of LA-14-PP, LA-15-PP, and referencebacterial lipid A's. The monophosphate compounds, LA-17-PH and LA-18-PH, did not show any detectable toxicity atthe highest dose examined. In chicken embryos, like LA-14-PP, neither LA-17-PP nor LA-18-PP exerted lethal toxicityat the highest dose examined (10 ,ug per embryo) under theexperimental conditions in which LA-15-PP and referencebacterial lipid As were definitely toxic at a dose of 0.1 ,g perembryo (data not shown).

(ii) Pyrogenicity. LA-17-PP and its monophosphate analog

TABLE 4. Activation of the clotting enzyme cascade of T.tridentatus amoebocyte lysate by LA-17 and LA-18 series lipid A

analogs and reference synthetic and bacterial products

Equivalent of referenceMinimum effective dose LPSa (mg/mg) in theCompound (ng/test), Pre Gel test following colorimetric test:

Toxicolor test ES-test

LA-17-PP 0.1 0.00084 0.00071LA-17-PH 0.1 0.070 0.068LA-18-PP 0.01 0.24 0.25LA-18-PH 1.0 0.037 0.041LA-14-PP 0.01 1.98 1.56LA-15-PP 0.01 5.52 5.18F515 lipid A 0.01 4.45 3.96R595 lipid A 0.01 3.48 3.06

a The reference LPS was prepared from E. coli O111:B4 (LPS-W; Difco).One nanogram of this LPS is equivalent to 2.9 endotoxin units of USPreference endotoxin prepared from E. coli 0113 in the Toxicolor test.

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80

0

4-,

aXE 6

0: 4

010 40 100 200 4-0 ' 600,ug / ml

FIG. 2. Activation of human serum complement by LA-17 and LA-18 series lipid A analogs and reference synthetic and bacterial products.

Symbols: 0, LA-17-PP;@*, LA-17-PH; A\, LA-18-PP; *, LA-18-PH; O, LA-14-PP; *, LA-15-PP; OJ, F515 lipid A.

LA-17-PH caused a significant febrile response in rabbitsonly when a higher dose (10 ,ug/kg of body weight) was used;thus the pyrogenicities of these compounds were about 10-and 1,000-fold weaker than those of LA-14-PP and thereference bacterial lipid A, and LA-15-PP, respectively(Table 2). Neither LA-18-PP nor its monophosphate analogLA-18-PH was pyrogenic at doses up to 10 ,ug/kg.

(iii) Preparative activity for the local Shwartzman reaction.LA-17-PP and LA-18-PP did not prepare the rabbit skin forthe local Shwartzman reaction at the highest dose tested,which was 80 ,ug per site (Table 3). In the same threeanimals, LA-15-PP and the reference bacterial lipid A'sdefinitely prepared the injected skin site at doses less than2.5 jig per site. LA-14-PP showed a marginal activity at adose of 80 ,ug per site.Limulus test. In the conventional Pre Gel test, the mini-

mum gelation dose of LA-18-PP was identical to those of

reference synthetic and bacterial products, whereas theother three test compounds were less effective (Table 4). Onthe other hand, in the Toxicolor test, compounds of theLA-17 and LA-18 series showed far less activity than thereference compounds; thus, remarkable discrepancy wasnoted in the assay results between Pre Gel and Toxicolortests (Table 4).

Activation of human complement cascade. All four testspecimens definitely consumed the complement in normalhuman serum. Dose-response curves of these compoundsshowed that the activity of LA-18-PH, which was thestrongest among the four tested specimens, was comparableto that of LA-15-PP, and those of LA-17-PH and LA-18-PPwere similar to that of LA-14-PP. LA-17-PP was much lessactive in complement activation than the other test speci-mens (Fig. 2). The complement activation by all of the testand reference compounds, except LA-17-PH, were com-

TABLE 5. Adjuvant activity of LA-17 and LA-18 series lipid A analogs as compared with reference synthetic and bacterial products onanti-BSA antibody production of BALB/c mice by subcutaneous injection with BSA in liposomesa

Passive Enzyme-linked immunosorbent assay (mean ± SE) for:Expt Compound hemagglutination IgG IgM IgA

titer (2n) (mean ± SE) (ratio)b (OD405, 102)C (OD405, 102)C

I LA-17-PP 10.86 ± 0.26d 78.75 ± 16.79d 279 ± 29e 142 ± 12dLA-17-PH 5.86 ± 0.67d 4.52 ± 1.44d 173 ± 34 106 ± 28dLA-18-PP 10.14 ± 0.46d 65.37 ± 20.22d 305 ± 37e 199 ± 12dPA-18-PH 5.58 ± 0.84d 3.19 ± 1.06e 229 ± 44 72 ± 23dF515 lipid A 11.57 ± 0.30" 268.58 ± 88.50d 468 ± 59d 197 ± 17dNone 3.25 ± 0.48 1.00 ± 0.30 208 ± 20 24 ± 8

II LA-17-PP 10.10 ± 0.35d 2.73 ± 0.65e 179 ± 8d 90 ± 4dLA-18-PP 7.30 ± 0.84 1.23 ± 0.34 107 ± 11 34 ± 9LA-15-PP 12.20 ± 0.42d 15.82 ± 3.06" 251 ± 24d 130 ± 12"None 5.58 ± 0.84 1.00 ± 0.28 122 ± 5 41 ± 9

a Groups of eight BALB/c mice were immunized by subcutaneous injection of 100 pLg of test adjuvant in liposomes, and 28 days later they received the secondinjection of the same mixture (in experiment I) or BSA alone (in experiment II) in liposomes. Five days after the booster, the serum anti-BSA antibody level wasdetermined by passive hemagglutination method and enzyme-linked immunosorbent assay.

b Ratio of enzyme-linked immunosorbent assay units in each test group to that in the respective control.c OD4o5, Optical density at 405 nm.d p < 0.01.e p < 0.05.

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678 KOTANI ET AL.

Dose Serum(pg/ dilution

mouse) (xlOO)

0.52.5

10 12.562.5

312.5

0.52.5

2 12.562.5

312.5

0.52.5

10 12.562.5

312.5

0.52.5

2 12.5

62.5312.5

0.52.5

2 12.5

62.5312.5

0.504 2.5

0.4 12.562.5

312.5

Cytotoxic effect (%) Compound

0 20 40 60 80 100

LA-17-PP

LA-17-PH

LA-18-PP

LA-18-PH

F515 lipid A

Dose(yg)

101

0.1

101

0.1

101

0.1

1010.1

101

0.1

Stimulation index (mean + SE)

FIG. 4. Effects of LA-17 and LA-18 series lipid A analogs and a

reference bacterial lipid A on glucosamine uptake by guinea pigperitoneal macrophages. Doses are shown in micrograms per 5 x 101cells. The counts of [14C]glucosamine uptake (mean + standarderror) in control cultures were 232 ± 13 dpm. **P < 0.01.

z*

FIG. 3. Cytocidal effects on L-929 cells of serum specimens fromBCG-primed mice, elicited by intravenous injection of LA-17 andLA-18 series lipid A analogs and a reference bacterial lipid A.Results significantly different from the control are indicated byasterisks (*P < 0.05, **P < 0.01).

pletely blocked by the addition of EGTA-Mg2+ to the testserum, whereas the same procedure did not block any of thecomplement activity by zymosan (data not shown). LA-17-PH seemed to activate the complement cascade via both thealternative and the classical pathways.

Immunobiological activities in vivo. (i) Adjuvant activities.The mice that were immunized twice by subcutaneousinjection of liposomes containing LA-17-PP or LA-18-PP andBSA showed much higher immune responses than the con-trol animals receiving BSA alone in liposomes, in terms ofenhanced formation of anti-BSA immunoglobulin G (IgG),IgM, and IgA class antibodies, although their adjuvant effect

seemed to be weaker than that of the F515 lipid A. Themonophosphoryl analogs LA-17-PH and LA-18-PH alsoshowed significant activity, but as far as comparison wasmade with the test dose of 100 ,ug per mouse, theiradjuvanticity was much weaker than that of bisphosphorylcompounds (Table 5, experiment I). Another comparativeexperiment, where the booster immunization was made withBSA alone without test specimens, revealed that the adju-vant activity of LA-17-PP and LA-18-PP, especially thelatter, was less obvious under the conditions where LA-15-PP exhibited powerful adjuvanticity (Table 5, experimentII). Thus, both LA-17-PP and LA-18-PP were definitelyactive in enhancing antibody production, but their activitywas less than that of synthetic and bacterial E. coli-type lipidA.

(ii) Induction of serum factor cytocidal to L-929 cells inBCG-primed mice. LA-17-PP and LA-18-PP showed power-ful activity to induce the serum factor cytocidal to L-929cells (possible tumor necrosis factor [TNF]) in BCG-primedICR mice. Their activity, however, was weaker than that ofthe reference bacterial lipid A, as judged from the effectivedose required to elicit the comparable serum level of TNF(Fig. 3). Both monophosphate analogs were inactive even ata dose of 50 jig per mouse (data not shown).

(iii) Induction of IFN-a$/ in P. acnes-primed mice. BothLA-17-PP and LA-18-PP were active in inducing serum

TABLE 6. Induction of IFN-cs/P in mice pretreated with P. acnes by LA-17 and LA-18 series lipid A analogs: comparison withreference synthetic and bacterial productSa

Serum IFN-a/o titer (IU/ml of serum) induced with following dose (,ug/mouse):Compound

10 3.16 1 0.316 0.1

LA-17-PP 800 (2)C ND 480 (2) ND 100 (2)LA-18-PP 640 (2) ND 480 (2) ND <20 (2)LA-14-PPb 3,413 (3) ND 4,267 (3) 2,133 (3) 226 (3)LA-15-PPb ND 2,560 (3) 800 (4) 1,920 (2) 360 (4)F515 lipid Ab 2,560 (2) 3,840 (2) 960 (4) 1,920 (2) 480 (4)

a ICR mice pretreated with P. acnes 1 week earlier were challenged intravenously with test specimens. The assay results obtained in the same experimentalconditions but by separate experiments were combined. Numbers within parentheses indicate numbers of mice tested. ND, Not determined.

b Data from the previous report (17).

ComPound

LA-17-PP

LA-18-PP

R595 lipid A

p* *

r

I

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BIOACTIVITIES OF LOW TOXIC SYNTHETIC LIPID A ANALOGS

Compound

LA-17-PP

LA-17-PH

LA-18-PP

LA-18-PH

F515 lipid A

Dose Stimulation index (mean + SE)(pg) 0 2 4 6

10

0.1

10

0.1

10

1

0.1

10

1

0.1

10

0.1

**

*

10

0.

10I.-i

I.

FIG. 5. Effects of pretreatment with LA-17 and LA-18 serieslipid A anlogs and a reference bacterial lipid A on superoxide aniongeneration in guinea pig peritoneal macrophages. Doses are shownin micrograms per 5 x 105 cells. Superoxide anion generation incontrol cultures (mean ± standard error) was 267 ± 29 nmol/min per

106 cells. **P < 0.01.

IFN-at/ in P. acnes-primed ICR mice. The inducing activitywas weaker than that of the reference synthetic and bacterialE. coli-type lipid A's (Table 6). The activity of LA-14-PPseemed to be stronger than that of the above reference lipidA's, although the assay results obtained under the same

experimental conditions but by separate experiments were

combined in Table 6.

Compound Dose(,Pg) 0

Stimulation index (meon + SE)

2 4 6 8

10 IHLA-17-PP I1 r

0.1

10LA-17-PH 1

0.1

10LA-18-PP 1

0.1 ,

10 !_LA-18-PH 1 |b

0.1

10 *LA-14-PP 1I *

0.1 ! *

LA-15-PP O .1 *

0.01

F515 liPid A 1O .1L' ._*

FIG. 6. PGE2 production by murine resident macrophages stim-ulated by LA-17 and LA-18 series lipid A analogs and referencesynthetic and bacterial products. Doses are shown in microgramsper 2 x 106 cells per ml. PGE2 production in control cultures (mean+ standard error) was 100 5 pg per culture. **P < 0.01.

Immunobiological activities in vitro. (i) Stimulation ofguinea pig macrophages. In both the glucosamine uptake(Fig. 4) and 02 generation (Fig. 5) assays, all four testedlipid A analogs, especially LA-18-PP, showed definite activ-ities to stimulate guinea pig peritoneal macrophages, al-though the extent of stimulation was considerably less thanthat of the F515 lipid A in both assays.

(ii) Stimulation of murine macrophages. All of the testsynthetic analogs activated murine resident peritoneal mac-rophages to increase PGE2 production (Fig. 6). The activityof bisphosphate analogs was stronger than that of the corre-sponding monophosphate analogs. The extent of stimulationby LA-17-PP and LA-18-PP was comparable to or slightlystronger than that of LA-14-PP but considerably weaker thanthat of the reference synthetic and bacterial lipid A's. Table7 shows representative data on the IL-1 production assay byusing thioglycolate-elicited murine peritoneal macrophages.A 1-,ug sample of each test specimen was added to themacrophage culture. All of the four test analogs as well asLA-14-PP were shown to activate definitely the macro-phages to enhance IL-1 production. The four test syntheticcompounds were less active than the reference E. coli-typelipid A's. The two other experiments with 10 and 0.1 p,g ofeach test specimen gave essentially the same results, exceptthat remarkably less activity of LA-17-PH and LA-18-PHwas noticed than those of the corresponding bisphosphateanalogs with 0.1 ,ug of the test dose (data not shown).

(iii) Activation of murine splenocytes. All four test analogsexhibited definite mitogenic activities on the splenocytes ofathymic nude mice (Fig. 7). The B-cell mitogenic activity ofLA-17-PP was the strongest among the four and was com-parable to that of LA-14-PP, but was weaker than that of thereference E. coli-type lipid A's, either synthetic or bacterial,in terms of the required dose to achieve about a fivefoldincrease of thymidine uptake. The splenocytes of LPS-nonresponder C3H/HeJ mice were not susceptible to themitogenic effects of the test synthetic analogs as were thereference materials (Fig. 7).Regarding polyclonal B-cell activation, a similar, but

clearer tendency to that shown with the above B-cellmitogenicity was observed in the relative potency of the testcompounds; LA-17-PP was the strongest among the four,although it was less active than a reference bacterial lipid A,and LA-17-PH and LA-18-PH were less active than thecorresponding bisphosphate analogs (Fig. 8).

TABLE 7. IL-1 production by murine peritoneal macrophagesstimulated by LA-17 and LA-18 series lipid A analogs as

compared with that by reference synthetic and bacterial products

Stimulation index (mean + SE) at the indicated dilution of

Compound macrophage culture supernatanta:1:1 1:2 1:4 1:8

LA-17-PP 38.5 5.2b 24.3 6.6b 11.0 1.2b 12.1 5.7cLA-17-PH 37.7 ± 3.0b 33.7 ± 3.0b 17.2 2.3b 14.5 ± 30bLA-18-PP 26.7 5.2b 31.5 2.1b 9.1 ± 0.4b 11.0 2.4bLA-18-PH 15.7 4.4b 10.6 3.5b 3.3 ± 0.1b 4.7 ± 2.0cLA-14-PP 28.2 3.6b 19.7 2.4b 12.4 1.4b 8.9 0.7bLA-15-PP 33.5 1.8b 32.8 1.6b 37.9 ± 7.0b 39.2 ± 15.3bF515 lipid A 26.3 3.7b 28.6 ± 20b 30.8 1.7b 33.2 ± 7.0bNone 0.7 ± 0.1 1.1 ± 0.2 0.5 ± 0.1 1.4 ± 0.2

a Macrophages (0.5 x 106/ml) from DBA/2 mice were cultured with 1.0 F±gof test specimen per ml for 24 h. The count in control C3H/HeJ thymocytecultures (with 1 p.g of phytohemagglutinin per ml alone) was 1,203 ± 313 dpm.

b p < 0.01.c P < 0.05.

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680 KOTANI ET AL.

Compound Dose(ygg)

10LA-17-PP

:

10LA-17-PH 1

8:6110

LA-18-PP 10.10.01

10LA-18-PH 10.1

0.0110

LA-14-PP 10.10.01

10LA-15-PP 1

0.10.01

10F515 lipid A 1

0.10.01

Stimulation index (mean + SE)BALB/c nu/nu mice C3H/HeJ mice

0 5 10 15 0 5

'* . l l

b** 1

** IND**hjN

,H** 10 *h** 1 **

I ** 0.1

INDa**IIH** lw10**10 1** 0.1

** s*** lo R**0.1

**H 10H** 1'IH** 0.1

FIG. 7. Thymidine uptake in BALB/c nulnu and C3H/HeJ mouse splenocytes by LA-17 and LA-18 series lipid A analogs and referenceand bacterial products. Doses are shown in micrograms per 5 x 105 cells. The stimulation index (mean ± standard error) ofphytohemagglutinin (1 ,ug) in the experiment with C3H/HeJ mouse splenocytes was 32.9 ± 0.4. The counts (mean ± standard error) in controlcultures were 7,218 ± 344 dpm (BALB/c nulnu mice) and 3,319 ± 112 dpm (C3H/HeJ mice). *P < 0.05, **P < 0.01.

DISCUSSIONWe have described the endotoxic and other biological

activities of four kinds of newly synthesized lipid A analogswith a ,3(1-6)-D-glucosamine disaccharide backbone, whichcarried four tetradecanoyl groups at the 2- and 2'-amino, and3- and 3'-hydroxyl groups (LA-17 compounds), or two eachof the (R)-3-hydroxytetradecanoyl and tetradecanoyl groupsat the 2- and 2'-amino and 3- and 3'-hydroxyl groups,respectively (LA-18 compounds), and had 1,4'-bisphosphate

Dose(jig)

1010.1

LA-17-PH 100.1

10LA-18-PP 1o0.1

10LA-18-PH 1

0.1

10F515 lipid A 1

0.1

Stimulation index (mean + SE)20 40 60 80

. * *

ZIF

FIG. 8. Polyclonal B-cell activation of BALB/c mouse spleno-cytes by LA-17 and LA-18 series lipid A analogs and referencesynthetic and bacterial products. Doses are shown in microgramsper 2.5 x 106 cells. **P < 0.01.

(LA-17-PP and LA-18-PP) or 4'-monophosphate (LA-17-PHand LA-18-PH). These four lipid A analogs were shown tohave lower endotoxicity not only compared with the refer-ence synthetic and bacterial lipid A's, but also comparedwith the synthetic counterpart of a lipid A precursor Ia(LA-14-PP) that has been demonstrated to have lowendotoxicity (4, 13, 18, 19, 32, 34). In galactosamine-loadedmice, LA-17-PP and LA-18-PP were about 10-fold less toxicthan LA-14-PP, and LA-17-PH and LA-18-PH were devoidof any detectable lethal toxicity. In chicken embryos, nodetectable toxicity was found with any of the above four testanalogs. LA-17-PP and LA-17-PH were also about 1,000-foldless pyrogenic than LA-15-PP and LA-18-PP and LA-18-PHwere nonpyrogenic in rabbits. Further, LA-17-PP and LA-18-PP did not prepare rabbit skin for the local Shwartzmanrection (no data for LA-17-PH or LA-18-PH by limitedavailability). Thus, these four synthetic lipid A analogs hadno or weak toxicity, which makes them possible candidatesfor use in clinical or preventive medicine.

Concerning the beneficial effects in animals, LA-17-PPand LA-18-PP were demonstrated to be definitely active instimulating the humoral anti-BSA antibody formation inmice and in induction (elicitation) of a possible TNF andIFN-a/P in primed mice. The monophosphate analogs LA-17-PH and LA-18-PH were considerably less effective in theabove bioassays. It should be pointed out, however, thatthere is possibility that at higher doses these monophos-phoryl compounds could exhibit strong activities compara-ble to those of bisphosphoryl compounds, since the limitedamount of test compounds did not allow us to performdose-response experiments where higher doses of LA-17-PHand LA-18-PH were examined. Further study on monophos-phoryl PH type analogs is needed in consideration of several

Compound

LA-17-PP

INFECT. IMMUN.

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BIOACTIVITIES OF LOW TOXIC SYNTHETIC LIPID A ANALOGS

reports that monophosphoryl lipid A's prepared from Sal-monella typhimurium Re-lipopolysaccharide are scarcelytoxic but still exhibit enough beneficial activities (28, 29, 33)and the fact that LA-17-PH and LA-18-PH can be synthe-sized more easily than the corresponding bisphosphate com-pounds. In this connection, LA-14-PP, a synthetic counter-part of the biosynthetic lipid A precursor Ia, is also apromising compound, in view of its definite useful bioactivi-ties such as the adjuvanticity and the ability to elicit IFN inprimed mice (19, 32, 34) and the ability to cause tumornecrosis (34). Since LA-14-PP is the direct parent moleculeof LA-17 and LA-18 series analogs, this compound should befurther investigated in more detail as one of the key struc-tures for production of useful compounds in the future.

In most in vitro assays the monophosphate analogs acti-vated immunocompetent cells as the correspondingbisphosphate compounds did, and there were no significantdifferences between the two groups. This indicates that theresults of in vitro assays do not necessarily reflect theoutcome of in vivo bioactivities and suggests the importanceof the in vivo system of the assay in screening out com-pounds having useful biomedical activities.Concerning the Limulus test, the assay results obtained

with the test and reference compounds in the conventionalPre Gel and colorimetric Toxicolor tests were markedlydissociated with each other. This type of dissociation wasalso observed with acyl derivatives of glucosamine mono-saccharide phosphate having part of the structure of lipid A's(I. Takahashi, S. Kotani, H. Takada, M. Tsujimoto, T.Ogawa, T. Shiba, S. Kusumoto, M. Yamamoto, A.Hasegawa, M. Kiso, M. Nishijima, K. Harada, S. Tanaka,H. Okamura, and T. Tamura, Infect. Immun., in press), andamphipathic compounds that were extracted from delipid-ated cells of members of the family Mycobacteriaceae by thephenol-water method and exhibited potent endotoxin-likebioactivities (T. Ikeda, S. Kotani, M. Tsujimoto, T. Ogawa,I. Takahashi, H. Shimauchi, S. Nagao, S. Kokeguchi, K.Kato, I. Yano, H. Okamura, K. Harada, H. Usami, A.Yamamoto, S. Tanaka, and Y. Kato, submitted for publica-tion). The question of which of the above two tests bettercorrelates with endotoxic activities represented bypyrogenicity remains to be elucidated.One notable finding is that LA-18-PH exhibited strong

complement-activating effects comparable to those of refer-ence synthetic and bacterial lipid A's. We observed similarstrong activities of LA-14-PH (compound 404) and LA-14-HP (405) (32). These findings suggest that appropriate hy-drophobicity is important for efficient manifestation of theinherent activity to activate the complement cascade of0(1-6)glucosamine disaccharide acyl derivatives, because 4'-and 1-monophosphate compounds (LA-14-PH and LA-14-HP), 2',3'-diacyloxyacyl compounds (LA-15-PP and LA-15-PH), and a 2,2',3'-triacyloxyacyl compound (LA-16-PP)were equally active in complement activation as the bacteriallipid A's (17, 19, 32).Very recently, Kanegasaki et al. (14) also reported several

bioactivities of LA-17-PP and LA-18-PP: lethal toxicity ingalactosamine-loaded mice, pyrogenicity, preparatory effectfor the local Shwartzman reaction, TNF induction in P.acnes-primed mice, Limulus test, and mitogenicity. Most oftheir findings were essentially consistent with ours, but thereare some discrepancies on the results on progenicity andinduction of TNF-like activity. They reported definitepyrogenicity of both LA-18-PP and LA-17-PP, whereas inour assay system LA-18-PP was not pyrogenic at the highestdose examined (10,ug/kg). The discrepancy might be due to

the fact that they injected test materials together with BSA,whereas we did not add BSA in the present test. Thepresence or absence of BSA in test materials used ininduction of serum TNF in primed mice may also result inthe difference in assay results between the two groups: theyfound LA-17-PP to be a stronger inducer than LA-18-PP,where we did not find any detectable difference in theactivity of the two compounds.

ACKNOWLEDGMENTS

We thank N. Murakami (Research Institute, Daiichi Seiyaku Co.,Ltd., Tokyo, Japan) for his cooperations in the assay of pyrogenicactivity. We also thank M. Nagaki and H. Kitamura (Center ofAdult Disease, Osaka) and T. Kubo and T. Fukunishi (KannonjiInstitute, Research Foundation for Microbial Diseases, Osaka Uni-versity) for their help in complement consumption assays and thelethality test on chicken embryos, respectively.

This work was supported in part by grants-in-aid for ScientificResearch (nos. 59116005, 60108003, 61770295, and 61771446) fromthe Ministry of Education, Science, and Culture of Japan.

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endotoxin in chick embryos. Proc. Soc. Exp. Biol. Med. 115:702-707.

2. Galanos, C., M. A. Freudenberg, and W. Reutter. 1979.Galactosamine-induced sensitization to the lethal effects ofendotoxin. Proc. Natl. Acad. Sci. USA 76:5939-5943.

3. Galanos, C., T. Hansen-Hagge, V. Lehmann, and 0. Luderitz.1985. Comparison of the capacity of two lipid A precursormolecules to express the local Shwartzman phenomenon. In-fect. Immun. 48:355-358.

4. Galanos, C., V. Lehmann, 0. Luderitz, E. T. Rietschel, 0.Westphal, H. Brade, L. Brade, M. A. Freudenberg, T. Hansen-Hagge, T. Luderitz, G. McKenzie, U. Schade, W. Strittmatter,K. Tanamoto, U. Zahringer, M. Imoto, H. Yoshimura, M.Yamamoto, T. Shimamoto, S. Kusumoto, and T. Shiba. 1984.Endotoxic properties of chemically synthesized lipid A partstructures. Comparison of synthetic lipid A precursor andsynthetic analogues with biosynthetic lipid A precursor and freelipid A. Eur. J. Biochem. 140:221-227.

5. Galanos, C., 0. Luderitz, E. T. Rietschel, 0. Westphal, H.Brade, L. Brade, M. Freudenberg, U. Schade, M. Imoto, H.Yoshimura, S. Kusumoto, and T. Shiba. 1985. Synthetic andnatural Escherichia coli free lipid A express identical endotoxicactivities. Eur. J. Biochem. 148:1-5.

6. Galanos, C., 0. Luderitz, and 0. Westphal. 1969. A new methodfor the extraction of R lipopolysaccharides. Eur. J. Biochem.9:245-249.

7. Hansen-Hagge, T., V. Lehmann, and 0. Luderitz. 1985. Freeflow electrophoresis as a tool for enrichment of mutants withtemperature-dependent lethal mutations in lipid A synthesis.Eur. J. Biochem. 148:21-27.

8. Hansen-Hagge, T., V. Lehmann, U. Seydel, B. Lindner, and U.Zahringer. 1985. Isolation and structural analysis of two lipid Aprecursors from a KDO deficient mutant of Salmonellatyphimurium differing in their hexadecanoic acid content. Arch.Microbiol. 141:353-358.

9. Homma, J. Y., M. Matsuura, S. Kanegasaki, Y. Kawakubo, Y.Kojima, N. Shibukawa, Y. Kumazawa, A. Yamamoto, K.Tanamoto, T. Yasuda, M. Imoto, H. Yoshimura, S. Kusumoto,and T. Shiba. 1985. Structural requirements of lipid A respon-sible for the functions: a study with chemically synthesized lipidA and its analogues. J. Biochem. (Tokyo) 98:395-406.

10. Imoto, M., S. Kusumoto, T. Shiba, H. Naoki, T. Iwashita, E. T.Rietschel, H.-W. Wolienweber, C. Galanos, and 0. Luderitz.1983. Chemical structure of E. coli lipid A: linkage site of acylgroups in the disaccharide backbone. Tetrahedron Lett.24:4017-4020.

11. Imoto, M., H. Yoshimura, N. Sakaguchi, S. Kusumoto, and T.

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Shiba. 1985. Total synthesis of Escherichia coli lipid A. Tetra-hedron Lett. 26:1545-1548.

12. Imoto, M., H. Yoshimura, M. Yamamoto, T. Shimamoto, S.Kusumoto, and T. Shiba. 1984. Chemical synthesis of phosphor-ylated tetraacyl disaccharide corresponding to a biosyntheticprecursor of lipid A. Tetrahedron Lett. 25:2667-2670.

13. Kanegasaki, S., Y. Kojima, M. Matsuura, J. Y. Homma, A.Yamamoto, Y. Kumazawa, K. Tanamoto, T. Yasuda, T. Tsu-mita, M. Imoto, H. Yoshimura, M. Yamamoto, T. Shimamoto, S.Kusumoto, and T. Shiba. 1984. Biological activities of analoguesof lipid A based chemically on the revised structural model.Comparison of mediator-inducing, immunomodulating andendotoxic activities. Eur. J. Biochem. 143:237-242.

14. Kanegasaki, S., K. Tanamoto, T. Yasuda, J. Y. Homma, M.Matsuura, M. Nakatsuka, Y. Kumazawa, A. Yamamoto, T.Shiba, S. Kusumoto, M. Imoto, H. Yoshimura, and T. Shima-moto. 1986. Structure-activity relationship of lipid A: compari-son of biological activities of natural and synthetic lipid A's withdifferent fatty acid compositions. J. Biochem. (Tokyo) 99:1203-1210.

15. Karber, G. 1931. Beitrag zur kollektiven Behandlung pharma-kologischer Reihenversuche. Arch. Exp. Pathol. Pharmakol.162:480-483.

16. Kitamura, H., S. Inai, and K. Nagaki. 1983. A simple procedurefor the titration of total hemolytic complement activity. One-point method. Jpn. J. Clin. Chem. 12:143-147.

17. Kotani, S., H. Takada, I. Takahashi, M. Tsujimoto, T. Ogawa,T. Ikeda, K. Harada, H. Okamura, T. Tamura, S. Tanaka, T.Shiba, S. Kusumoto, M. Imoto, H. Yoshimura, and N. Kasi.1986. Low endotoxic activities of synthetic Salmonella-typelipid A with an additional acyloxyacyl group on the 2-aminogroup of P(1-6)glucosamine disaccharide 1,4'-bisphosphate. In-fect. Immun. 52:872-884.

18. Kotani, S., H. Takada, M. Tsujimoto, T. Ogawa, K. Harada, Y.Mori, A. Kawasaki, A. Tanaka, S. Nagao, S. Tanaka, T. Shiba,S. Kusumoto, M. Imoto, H. Yoshimura, M. Yamamoto, and T.Shimamoto. 1984. Immunobiologically active lipid A analogssynthesized according to a revised structural model of naturallipid A. Infect. Immun. 45:293-296.

19. Kotani, S., H. Takada, M. Tsujimoto, T. Ogawa, I. Takahashi,T. Ikeda, K. Otsuka, H. Shimauchi, N. Kasai, J. Mashimo, S.Nagao, A. Tanaka, S. Tanaka, K. Harada, K. Nagaki, H.Kitamura, T. Shiba, S. Kusumoto, M. Imoto, and H. Yoshimura.1985. Synthetic lipid A with endotoxic and related biologicalactivities comparable to those of a natural lipid A from anEscherichia coli Re-mutant. Infect. Immun. 49:225-237.

20. Kotani, S., M. Tsujimoto, T. Ogawa, K. Nerome, A. Ooya, T.Takahashi, Y. Goto, T. Shiba, S. Kusumoto, and T. Shimamoto.1986. Synthetic immunomodulators mimicking bacterial cellsurface components. Acyl derivatives of muramylpeptides andlow toxic lipid A analogues as possible adjuvants for vaccines,p. 40-64. In M. Zaoral, Z. Havlas, 0. Mikes, and Z. Prochazka(ed.), Synthetic immunomodulators and vaccines. Institute ofOrganic Chemistry and Biochemistry, Czechoslovak Academyof Sciences, Prague.

21. Nowotny, A. 1985. Antitumor effects of endotoxin, p. 389-448.In L. J. Berry (ed.), Handbook of endotoxin, vol. 3: cellular

biology of endotoxin. Elsevier Science Publishers, Amsterdam.22. Nowotny, A., A. Abdelnoor, U. H. Behling, R. C. Butler, A. G.

Johnson, and A. M. Nowotny. 1982. Molecular aspects of theadjuvant and other beneficial effects of endotoxins, p. 103-116.In Y. Yamamura, S. Kotani, I. Azuma, A. Koda, and T. Shiba(ed.), Immunomodulation by microbial products and relatedsynthetic compounds. Excerpta Medica, Amsterdam.

23. Obayashi, T. 1984. Addition of perchloric acid to blood samplesfor colorimetric limulus test using chromogenic substrate: com-parison with conventional procedures and clinical applications.J. Lab. Clin. Med. 104:321-330.

24. Obayashi, T., H. Tamura, S. Tanaka, M. Ohki, S. Takahashi, M.Arai, M. Masuda, and T. Kawai. 1985. A new chromogenicendotoxin-specific assay using recombined limulus coagulationenzymes and its clinical applications. Clin. Chim. Acta149:55-65.

25. Ogawa, T., S. Kotani, and H. Shimauchi. 1986. Enhancement ofserum antibody production in mice by oral administration oflipophilic derivatives of muramylpeptides and bacterial lipo-polysaccharides with bovine serum albumin. Methods Find.Exp. Clin. Pharmacol. 8:117-125.

26. Okamura, H., K. Kawaguchi, K. Shoji, and Y. Kawade. 1982.High-level induction of gamma interferon with various mitogensin mice pretreated with Propionibacterium acnes. Infect. Im-mun. 38:440-443.

27. Parant, M. 1983. Effect of LPS on nonspecific resistance tobacterial infections, p. 179-196. In A. Nowotny (ed.), Beneficialeffects of endotoxins. Plenum Publishing Corp., New York.

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