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A chronic inflammatory response produced by cell walls of Mycobacterium bovis strain BCG associated with microscopic oil droplets (BCG CW-O) was studied with reference to the tumor-regressing ability of this preparation. When BCG CW-O was injected intradermally in the footpads of guinea pigs, intense inflammation developed at the site of injection and in the draining popliteal lymph node. This was characterized histologically as granulomatous inflammation. The intensity of the response was related to the dose of BCG CW-O. Cell walls without oil produced the same type of inflammation but much less in degree. Data are presented that demonstrate the immunological nature of this response and classify it as a cell-mediated immune reaction. Ramifications of this chronic inflammatory reaction occurring at the site of a tumor are discussed.

The monophosphoryl lipid A (MLA) obtained from the lipopolysaccharides of Salmonella minnesota R595 was fractionated on a silicic acid column to yield the heptaacyl, hexaacyl, and pentaacyl MLA. Each of these MLAs was methylated with diazomethane to yield the dimethyl derivative and purified to homogeneity by reverse-phase high performance liquid chromatography. The molecular ions obtained by positive ion fast atom bombardment mass spectrometry of purified dimethyl heptaacyl MLA allowed us to establish the molecular formula and Mr of C112H211N2O23P and 1983.3, respectively. Cleavage at the glycosidic linkage yielded an oxonium ion of mass 1115, which showed that the distal sugar unit contained one phosphate (dimethyl), two hydroxymyristates, one laurate, and one myristate, while the reducing sugar unit contained two hydroxymyristates and one palmitate. By utilizing two-dimensional NMR spectroscopy, we were able to assign all of the protons of dimethyl heptaacyl MLA. This assignment included the beta protons of the three acyloxyacyl groups. A substantial downfield shift of the protons at the 3- and 3' -carbons was observed, which indicated that these two positions are occupied by ester groups. Fast atom bombardment mass spectral analysis of the hexaacyl and pentaacyl MLAs showed that these structures were identical to the previously designated TLC-3 and TLC-5 fractions, respectively, from Salmonella typhimurium. From this study, the complete structures of the MLA series found in the LPS of S. minnesota can now be described.

Endotoxin extracted from the heptose-less mutant of Salmonella typhimurium was hydrolyzed in 0.1 N HCl in methanol/water (1:1, v/v) at 100 degrees C to yield lipid A, which was then fractionated on a Sephadex LH-20 column to yield a major monophosphoryl lipid A fraction. The monophosphoryl lipid A was further fractionated by preparative thin layer chromatography. This process yielded three major bands (TLC-1, -3, and -5) and two minor bands (TLC-7 and -9). The purity of these fractions was established by ion exchange and reverse phase high performance liquid chromatography. The thin layer fractions were analyzed by fast atom bombardment mass spectrometry. TLC-1 and -3 gave molecular ions (M-H)- at m/e 1730 and 1716, respectively. Both of these fractions contained beta-hydroxymyristic, lauric, and 3-myristoxymyristic acids in O-acyl linkages. The molecular formula and Mr of TLC-1 are C95H179O22N2P and 1731.16; those of TLC-3 are C94H177O22N2P and 1717.15. TLC-1 was a methyl homolog of TLC-3. The major component of TLC-5 (C80H151O22N2P and Mr = 1506.99) gave a molecular ion at m/e 1506 and contained two beta-hydroxymyristic acids and a lauric acid in the O-acyl linkages. The major component of TLC-7 (C66H125O19N2P and Mr = 1280.83) and the single component of TLC-9 gave molecular ions at m/e 1280 and 1098, respectively. TLC-7 contained lauric and beta-hydroxymyristic acids in the O-acyl linkages. TLC-9 (C54H103O18N2P and Mr = 1098.69) contained a single O-acylated beta-hydroxymyristate group. TLC-1 and -3 were nontoxic in the chick embryo lethality test and regressed established tumors in the syngeneic guinea pigs.

Various microbial products are known to influence the function of mouse peritoneal macrophages. Lipopolysaccharide (LPS) and certain lipid A-associated proteins are known to enhance the tumoricidal effects of macrophages. The purpose of this study was to determine whether porins (outer membrane proteins) of Salmonella typhimurium G30/C21 would influence the activity of macrophages from lipid A-responsive and -unresponsive mice. Porins, extracted by a combined sodium dodecyl sulfate-EDTA method from cell walls, were free of LPS as determined by Limulus amebocyte lysate assay and appeared as a band at approximately 36,000 molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In tumor cell killing assays done under LPS-free conditions, the porins in doses of 1 to 10 ng/ml enhanced the tumoricidal effect of macrophages from bacillus Calmette-Guérin-infected C3H/HeN or C3H/HeJ mice. Protein-free LPS enhanced the tumoricidal activity of macrophages from bacillus Calmette-Guérin-infected C3H/HeN but not C3H/HeJ mice. The tumoricidal-enhancing activity of protein-free LPS was blocked by the lipid A-binding antibiotic polymyxin B sulfate, but the effects of porins were not altered by the polymyxin B sulfate. These results suggest that porins, proteins known to alter membrane function, may alter macrophage function by interaction with macrophage membranes.

We examined the relationship between the fine structure of lipid A and the toxicity of endotoxin or lipopolysaccharides as measured by the Limulus amebocyte lysate (LAL), rabbit pyrogenicity, chicken embryo lethal dose, and dermal Shwartzman reaction tests. Lipid A and lipid A-like compounds obtained from deep-rough mutants of Salmonella spp. and Escherichia coli had a wide range of structural variations. These compounds included native lipopolysaccharides, diphosphoryl and monophosphoryl lipid A's, and lipid X (a monosaccharide). The LAL test was positive for all lipids tested with lysates from Travenol Laboratories and from Associates of Cape Cod (2.9 X 10(3) to 2.6 X 10(7) endotoxin units per mg), except for O-deacylated and dephosphorylated lipid X, which were negative. The Mallinckrodt lysate gave negative tests for lipid X. In the rabbit pyrogenicity and chicken embryo lethal dose tests, only native lipopolysaccharide and diphosphoryl lipid A's were judged toxic. The Shwartzman reaction was positive for a specific purified diphosphoryl lipid A (thin-layer chromatography-3 fraction) but negative for the purified monophosphoryl lipid A (also a thin-layer chromatography-3 fraction). These results show that the LAL test is not a valid measure of all parameters of toxicity of a lipid A or lipid A-like compound and can yield false-positive results. However, these findings are not in conflict with the widespread use of the LAL assay for pyrogens in the pharmaceutical industry since a good correlation exists between LAL results and pyrogenicity when undegraded endotoxin is evaluated in parallel assays.

After a sublethal exposure to lipopolysaccharide (LPS) or to lipid A, which is that portion of the LPS molecule associated with endotoxicity, a transient period ensues during which a normally responsive individual is rendered hyporesponsive to LPS-induced toxicity. This period has been defined as early-phase endotoxin tolerance. Recently, a nontoxic derivative of lipid A from Salmonella typhimurium, monophosphoryl lipid A (MPL), was isolated and purified. In this study, we assessed the ability of MPL to induce early endotoxin tolerance. Initial injection of MPL resulted in a dose-dependent stimulation of both serum colony-stimulating factor and serum interferon, indicators of in vivo LPS responsiveness. In contrast, MPL failed to induce the symptoms of endotoxicity which are normally seen after injection of even sublethal amounts of intact endotoxin or lipid A preparations. Injection of MPL on day 0 reduced significantly the amount of LPS-induced serum colony-stimulating factor and interferon produced upon challenge with Escherichia coli LPS 3 days later and also mitigated toxic manifestations, as evidenced by a marked increase in the 50% lethal dose. Like the early tolerance induced by wild-type (toxic) LPS, MPL-induced tolerance was characterized by an accompanying elevation in the number of bone marrow-derived macrophage progenitor cells and by an alteration in bone marrow cell sizing profiles. These results indicate that MPL is effective in inducing a state of LPS-hyporesponsiveness without the toxic side effects of endotoxin and that the structural component(s) necessary for induction of early-phase endotoxin tolerance is contained within MPL.

A modification of the apparatus previously described permits the preparation of cell walls in quantity. This consists of a heavy duty, double-acting hydraulic press with motor-driven pump, and a superstrength alloy steel pressure cell which is corrosion resistant. Liquid cooling of the jet is substituted for the previously used gas cooling to minimize aerosol formation and to facilitate subsequent treatment of the products. The device produces cell walls of excellent quality in good yield. The pressure cell has been used satisfactorily up to about 60,000 psi. Design details are given.

Endotoxin dissociated into subunits by sodium deoxycholate treatment exhibited diminished capacity to kill chick embryos, protect mice against the lethal effects of infection with Salmonella typhi, evoke hemorrhagic necrosis in skin inoculated with epinephrine, prepare for and provoke the dermal Shwartzman reaction, and induce pyrogenic tolerance. Surfactant-treated material which had been allowed to reaggregate displayed activity equivalent to that of untreated material. These findings were consistent with the working hypothesis that a macromolecular complex of critical size is required in order for endotoxin to elicit its characteristic effects in the host.

ProteusL forms were disrupted by osmotic shock, and the sedimentable material present in the homogenate was further fragmented in a Sorvall pressure cell. The pressure cell was also used for disrupting normal Proteuscells. The homogenates obtained were fractionated by differential centrifugation. Purified endotoxins were isolated from the major fractions by phenol extraction. Material extracted with phenol from the membrane fraction of the L forms was about as toxic and pyrogenic on a weight basis as the typical enterobacterial endotoxins isolated from cell walls of normal bacteria. The yield of extract from L forms was about one-third of that from an equal weight of normal bacteria. No differences in the gross chemical composition of the phenol extracts from the L forms and the normal cells could be ascertained. A close serological relationship existed between extracts obtained from two L forms and their respective parent bacteria, but no such relationship was found in the case of the third L form studied and its parent bacterium. Diaminopimelic acid was not detected in the membranes of the L forms, but these membranes contained most of the succinic dehydrogenase of the organisms. Only small amounts of this enzyme were present in the wall fraction of normal bacteria. The data obtained suggest that precursors of the Proteusendotoxins are formed either in the soluble protoplasm of normal cells and L forms or at sites on the membrane from which they are readily liberated into the protoplasm, whereas the final steps of the synthesis of these toxins take place at the cytoplasmic membrane. In normal cells, much endotoxin is transported to and concentrated in the walls.

Treatment of Boivin-type endotoxin from E. coliwith LiAlH4according to the method of Noll and Braude yielded a small amount (about 10%) of an essentially pure nonphosphorylated polysaccharide which was nontoxic and nonpyrogenic. In contrast to their RE fraction this product did not stimulate the production of antibodies in rabbits, precipitate antibody nor produce tolerance to endotoxin. The bulk of the product resulting from treatment with LiAlH4consisted of biologically unaltered endotoxin. It was isolated as a rapidly soluble, lipid-containing aluminum citrate-endotoxin complex, the infrared spectrum of which was nearly identical to that of an RE fraction with 100-fold reduced pyrogenicity. No correlation was found between fatty acid ester content, as estimated from infrared spectra, and toxic and pyrogenic properties of the endotoxin.

Suitability of the rhesus monkey (Macaca mulatta) as an experimental host for evaluation of vaccines against airborne infection with Mycobacterium tuberculosisstrain H37Rv was investigated. Nonvaccinated monkeys were exposed to estimated doses of 12, 25, or 49 units of H37Rv in a modified Henderson apparatus, and the course of the disease was followed by chest X rays, skin testing with purified protein derivative, body-weight determinations, and autopsy 8 weeks postinfection. These animals developed progressive and extensive tuberculosis with pathological changes proportional to the infecting dose. Four of seven monkeys vaccinated intravenously with 1 mg of live BCG 8 weeks prior to challenge with 40 units of H37Rv had no gross evidence of disease at autopsy 13 weeks postinfection; the other three monkeys had minimal disease. These data demonstrated that (i) reproducible and progressive infection could be induced in rhesus monkeys infected in a manner which simulated natural infection of man and (ii) a high level of resistance to infection could be induced by BCG vaccine in the rhesus monkey, which in nature is highly susceptible to tuberculous infection.

In an effort to evaluate the role of delayed hypersensitivity in acquired resistance of mice to airborne infection with Mycobacterium tuberculosisH37Rv, the ability of lung and peritoneal cells from mice vaccinated in various ways with mycobacterial fractions or with M. bovisBCG to inhibit, in the presence of purified protein derivative, in vitro migration of normal peritoneal cells was determined. The degree of inhibition induced by lung cells was correlated with immunity, but that induced by peritoneal cells could not be associated with enhanced resistance. Live BCG given intravenously to mice stimulated greater resistance to infection and inhibitory activity of lung cells than did live BCG given subcutaneously. Vaccines with a protective index greater than 1 also induced a significant increase in lung weight. Although a correlation between ability of lung cells to inhibit cell migration and acquired resistance of the host to airborne infection with H37Rv was demonstrated, the data do not exclude the possibility that the two phenomena are independent responses to the immunologically complex mycobacterial antigens.

Fukushi, K. (National Institute of Allergy and Infectious Diseases, Rocky Mountain Laboratory, Hamilton, Mont.), R. L. Anacker, W. T. Haskins, M. Landy, K. C. Milner, and E. Ribi. Extraction and purification of endotoxin from Enterobacteriaceae: a comparison of selected methods and sources. J. Bacteriol. 87:391–400. 1964.—Endotoxins containing only 0.2% N (accounted for by amino sugars) and 2% ester- and amide-linked fatty acids (calculated as palmitic acid) were prepared from Salmonella enteritidis. These products were of high biological potency, and were rapidly destroyed by acid hydrolysis. Equally potent acid-susceptible products were derived from different strains of Escherichia coliand Serratia marcescens. No correlation was evident between potency and content of nitrogen, fatty acids, and hexosamine; however, low values for carbohydrate were invariably associated with reduced endotoxic activity. Factors such as strain, method of cultivation, and extraction procedure markedly affected the chemical composition and activity of primary extracts. Results are given which demonstrate significant advantages in the use of cell walls for the isolation and purification of endotoxins.

Footpads of mice sensitized by oil-treated Bacillus Calmette-Guérin (BCG) cell walls given either intravenously, subcutaneously, intradermally, intraperitoneally, or intramuscularly became swollen and reddened after injection of purified protein derivative (PPD). This reaction, greatest after intradermal and subcutaneous sensitization, generally reached a maximum about 24 hr after challenge and was still marked at 48 hr. The histological response was characterized by infiltration with both polymorphonuclear and mononuclear cells. The proportion of mononuclear cells increased with time and they predominated at 48 hr. The footpad reaction could be detected as early as 1 week after sensitization and persisted for at least 37 weeks. Footpad sensitivity to PPD and acquired resistance to airborne infection with Mycobacterium tuberculosisH37Rv were correlated in that (i) both reached a peak approximately 1 month after sensitization of the mouse, and (ii) cell walls treated with NaOH or given without oil neither protected mice against challenge infection nor sensitized them to PPD. Although, as we reported previously, mice vaccinated subcutaneously or intradermally exhibited little or no enhanced resistance to experimental infection, mice given oil-treated cell walls by these routes were highly sensitive to footpad inoculation of PPD. Therefore, the footpad test cannot be used to determine immunity of the mouse to pulmonary infection with tubercle bacilli.

Treatment with alkali is one of several methods for removing fatty acids from bacterial endotoxins and, in the process, detoxifying the material. Saponification of fatty acid esters is the major detectable chemical change produced by alkali; however, kinetic studies of mild alkaline hydrolysis of endotoxin failed to correlate rates of detoxification with rates of loss of ketodeoxyoctonates, heptose, O-acetyl groups, or fatty acid esters. The alterations occurring during the critical stages of hydrolysis apparently changed the essential chemical conformation of endotoxic particles before cleavage of a significant amount of material took place. The rates of both saponification and detoxification were markedly increased by carrying out the reaction in media of ethyl alcohol or dimethylsulfoxide instead of water.

Ribi, E. (Rocky Mountain Laboratory, Hamilton, Mont.), R. L. Anacker, R. Brown, W. T. Haskins, B. Malmgren, K. C. Milner, and J. A. Rudbach. Reaction of endotoxin and surfactants. I. Physical and biological properties of endotoxin treated with sodium desoxycholate. J. Bacteriol. 92:1493–1509. 1966.—Endotoxins from three species of gram-negative bacteria were shown to be dissociated by the bile salt sodium deoxycholate (NaD) into nontoxic subunits with molecular weights of about 20,000. When the bile salt was removed by dialysis, the subunits reaggregated in an orderly manner to form a relatively uniform population of biologically active endotoxin particles with average molecular weights of 500,000 to 1,000,000. If a small amount of human plasma was added to the dissociated endotoxin before removal of the NaD, reassociation apparently did not occur and the preparation remained nonpyrogenic. However, the plasma protein could subsequently be removed from the endotoxin subunits, and reaggregation to the toxic form would then occur. The studies on the physical nature of endotoxin performed with biophysical solution techniques were supplemented and confirmed by direct examination of the endotoxin polymers by electron microscopy. The results of these studies were consonant with the theory that the biologically active endotoxic elements are composed of micellar aggregates of linear lipopolysaccharide subunits.