Nontoxic Lipopolysaccharide Research Articles

Rhodobacter capsulatus PG Lipopolysaccharide Blocks the Effects of a Lipoteichoic Acid, a Toll-Like Receptor 2 Agonist.

Lipopolysaccharides (LPS) and lipoteichoic acids (LTA) are the major inducers of the inflammatory response of blood cells caused by Gram-negative and some Gram-positive bacteria. CD14 is a common receptor for LPS and LTA that transfers the ligands to TLR4 and TLR2, respectively. In this work, we have demonstrated that the non-toxic LPS from Rhodobacter capsulatus PG blocks the synthesis of pro-inflammatory cytokines during the activation of blood cells by Streptococcus pyogenes LTA through binding to the CD14 receptor, resulting in the signal transduction to TLR2/TLR6 being blocked. The LPS from Rhodobacter capsulatus PG can be considered a prototype for developing preparations to protect blood cells against the LTA of gram-positive bacteria.

Effect of lipopolysaccharide structure on functional response of whole blood cells

Lipopolysaccharides (LPSs) induce a wide spectrum of functional activities after interaction with blood cells. Effect of structure of toxic LPS from S- and Re-chemotypes of E. coli and/or non-toxic LPS of Rhodobacter capsulatus PG (R. caps.) on activation of neutrophils and monocytes of human whole blood were studied, particularly, expression of TLR4, CD14 and CD11b receptors, phagocytosis of BioParticles Alexa Fluor 488, synthesis of cytokines and chemokines. A leading role of CD11b receptor in phagocytic activity of neutrophils primed by LPS from various E. coli chemotypes was shown. The non-toxic LPS of R. caps. does not affect the efficiency of phagocytosis activity of the neutrophils. The LPS of R. caps. was shown to induce production of TRIF-dependent cytokine IFN-β in human whole blood leukocytes selectively, without activating MyD88-dependent pathway of pro-inflammatory cytokine synthesis, displaying properties of patrial agonist of TLR4. Structure and biological activity of LPS R. caps. allows considering it as a promising immunity stimulating pharmacological agent.

Dissociation of TRIF bias and adjuvanticity

Toll-like receptors (TLRs), a family of “pattern recognition receptors,” bind microbial and host-derived molecules, leading to intracellular signaling and proinflammatory gene expression. TLR4 is unique in that ligand-mediated activation requires the co-receptor myeloid differentiation 2 (MD2) to initiate two signaling cascades: the MyD88-dependent pathway is initiated at the cell membrane, and elicits rapid MAP kinase and NF-κB activation, while the TIR-domain containing adaptor inducing interferon-β (TRIF)-dependent pathway is initiated from TLR4-containing endosomes and results in IRF3 activation. Previous studies associated inflammation with the MyD88 pathway and adjuvanticity with the TRIF pathway. Gram-negative lipopolysaccharide (LPS) is a potent TLR4 agonist, and structurally related molecules signal through TLR4 to differing extents. Herein, we compared monophosphoryl lipid A (sMPL) and E6020, two synthetic, non-toxic LPS lipid A analogs used as vaccine adjuvants, for their capacities to activate TLR4-mediated innate immune responses and to enhance antibody production. In mouse macrophages, high dose sMPL activates MyD88-dependent signaling equivalently to E6020, while E6020 exhibits significantly more activation of the TRIF pathway (a “TRIF bias”) than sMPL. Eritoran, a TLR4/MD2 antagonist, competitively inhibited sMPL more strongly than E6020. Despite these differences, sMPL and E6020 adjuvants enhanced antibody responses to comparable extents, with balanced immunoglobulin (Ig) isotypes in two immunization models. These data indicate that a TRIF bias is not necessarily predictive of superior adjuvanticity.

Herbal Formula Fo Shou San Attenuates Alzheimer's Disease-Related Pathologies via the Gut-Liver-Brain Axis in APP/PS1 Mouse Model of Alzheimer's Disease.

Fo Shou San (FSS) is an ancient paired-herb decoction, used in China to treat blood deficiency, blood stasis, stroke, and ischemic cerebral vascular disease for about one thousand years. The mechanisms associated with these properties, however, are not completely understood. Gut bacteria, gut bacterial lipopolysaccharides (LPS), alkaline phosphatase (AP), and lipid peroxidation are common biochemical signaling that takes place on gut-liver-brain axis. Growing evidences have revealed that gut bacterial lipopolysaccharides (LPS) enter the systemic circulation via the portal vein, and finally entering the brain tissue is an important cause of inflammatory degeneration of Alzheimer's disease (AD). Alkaline phosphatase (AP) dephosphorylates LPS forming a nontoxic LPS and reduces systemic inflammation via gut-liver-brain axis. In this study, to identify the differentially gut-liver-brain axis among APP/PS1 mice, FSS-treated APP/PS1 mice, and control mice, behavioral tests were performed to assess the cognitive ability and hematoxylin-eosin staining was used to assess neuronal damage in the hippocampus; immunohistochemistry, western blotting, a quantitative chromogenic end-point Tachypleus amebocyte lysate (TAL) assay kit, Malondialdehyde (MDA) assay kit, AP Assay Kit, and real-time quantitative PCR (qPCR) were used to assess the level of LPS, MDA, AP, and gut bacteria. We found that FSS regulates gut-liver-brain axis to regulate AP and gut bacteria and attenuate the LPS-related systemic inflammation, oxidative stress (MDA), and thereby AD-related pathology in APP/PS1 mice. This is the first study to provide a reference for FSS-treated AD mice to aid in understanding the underlying mechanisms of FSS. FSS may also improve gastrointestinal tract barrier and blood-brain barrier and thus ameliorates the symptoms of AD; this is subject to our further study.

The Effect of a Lipopolysaccharide from Rhodobacter capsulatus PG on Inflammation Caused by Various Influenza Strains.

The development of a specific inflammation in mice that had been infected by two influenza virus strains, A/chicken/Kurgan/5/2005 (H5N1) and A/Hamburg/2009 MA (H1N1), was studied. We investigated the effect of a non-toxic lipopolysaccharide from Rhodobacter capsulatus PG on the survival and body weight of the mice, production of IgG antibodies, and the induction of pro- and anti-inflammatory cytokines in blood serum. The administration of the R. capsulatus PG lipopolysaccharide was shown to induce interferon-β synthesis, both in healthy and influenza A virus-infected mice, and to promote production of antiviral antibodies in the blood of the influenza-infected animals.

In vivo Proinflammatory Cytokine Production by CD-1 Mice in Response to Equipotential Doses of Rhodobacter capsulatus PG and Salmonella enterica Lipopolysaccharides.

The capacities of relatively nontoxic lipopolysaccharide (LPS) from Rhodobacter capsulatus PG and highly potent LPS from Salmonella enterica serovar Typhimurium to evoke proinflammatory cytokine production have been compared in vivo. Intravenous administration of S. enterica LPS at a relatively low dose (1 mg/kg body weight) led to upregulation of TNF-α, IL-6, and IFN-γ production by non-sensitized CD-1 mice. LPS from R. capsulatus PG used at a four-times higher dose than that from S. enterica elicited production of almost the same amount of systemic TNF-α; therefore, the doses of 4 mg/kg LPS from R. capsulatus PG and 1 mg/kg LPS from S. enterica were considered to be approximately equipotential doses with respect to the LPS-dependent TNF-α production by CD-1 mice. Rhodobacter capsulatus PG LPS was a weaker inducer of the production of TNF-α, IL-6, and IFN-γ, as compared to the equipotential dose of S. enterica LPS. Administration of R. capsulatus PG LPS before S. enterica LPS decreased production of IFN-γ, but not of TNF-α and IL-6, induced by S. enterica LPS. Rhodobacter capsulatus PG LPS also suppressed IFN-γ production induced by S. enterica LPS when R. capsulatus PG LPS had been injected as little as 10 min after S. enterica LPS, but to a much lesser extent. Rhodobacter capsulatus PG LPS did not affect TNF-α and IL-6 production induced by the equipotential dose of S. enterica LPS. In order to draw conclusion on the endotoxic activity of particular LPSs, species-specific structure or arrangement of the animal or human immune systems should be considered.

Respiratory burst activated by Escherichia coli in human neutrophils primed with different lipopolysaccharides

Endotoxic shock is a dangerous complication of infection caused by gram-negative bacteria. Searching for agents capable to block the effects of endotoxins is a fundamental scientific and medical problem. Here we studied the effects of neutrophil priming with non-toxic lipopolysaccharide from Rhodobacter capsulatus (LPS Rb. caps. ) and toxic LPS from Escherichia coli (LPS E. coli ) on the respiratory burst evoked in neutrophils by opsonized E. coli. We found that preincubation of neutrophils with each of the LPS increased ROS production by neutrophils as compared to non-primed neutrophils. Subsequent incubation of neutrophils with LPS Rb. caps. and then with LPS E. coli practically abolished the effects of both endotoxins.

Immunobiological activities of a new nontoxic lipopolysaccharide fromAcidiphiliumGS18h/ATCC55963, a soil isolate from an Indian copper mine

A novel nontoxic lipopolysaccharide (LPS) was purified from Acidiphilium strain GS18h/ATCC55963. The chemical composition of the lipid A part of this LPS is distinctly different from that of known lipid A molecules. The LPS was investigated to determine its capacity to provide protection against toxic LPS or endotoxic shock, as has been reported for other nontoxic LPSs (Rhodobacter sphaeroides and Rhodobacter capsulatus), and also the extent and type of immunomodulatory response in terms of tumor necrosis factor alpha (TNF-alpha), interleukin-1beta (IL-beta), and IL-6 release as well as NO secretion by stimulated monocyte-macrophage systems. This study demonstrates clearly that mice immunized or primed with this LPS are fully protected against challenge with toxic Escherichia coli LPS. Unlike most of the extensively studied nontoxic LPSs, this LPS induced reactive nitrogen intermediates and released TNF-alpha, IL-beta and IL-6 in both mouse and human monocyte-macrophage systems. However, the extent of the cytokine and lymphokine releasing response was well below the range of the toxic LPS, for example that of E. coli. Owing to its capacity to provide immunostimulation of the host without causing any lethality to ensure protection against endotoxic shock, this LPS appears to have potential therapeutic value.

Characterization of Lipopolysaccharides Present in Settled House Dust

The 3-hydroxy fatty acids (3-OHFAs) in lipopolysaccharides (LPS) play an important role in determining endotoxin activity, and childhood exposure to endotoxin has recently been associated with reduced risk of atopic diseases. To characterize the 3-OHFAs in house dust (HD), we used gas chromatography-mass spectrometry to assay 190 HD samples. Dust from beds, bedroom floors, family rooms, and kitchen floors was collected as part of a birth cohort study of childhood asthma (study 1) and a longitudinal study of home allergen and endotoxin (study 2). We also measured endotoxin activity with a Limulus assay and computed specific activity (endotoxin activity per nanomole of LPS). Longer-chain (C(16:0) and C(18:0)) 3-OHFAs were predominant in HD compared with short-chain (C(10:0), C(12:0), and C(14:0)) acids. Endotoxin activity was positively correlated with short-chain 3-OHFAs in both studies. In study 2, 3-OH C(16:0) was negatively correlated and 3-OH C(18:0) was not correlated with endotoxin activity, consistent with previous findings that the Limulus assay responds preferentially to LPS containing short-chain 3-OHFAs. Kitchen dust contained the highest concentrations of 3-OH C(10:0), the highest endotoxin activities, and the highest specific activities (P < 0.03). Bed dust contained the largest amounts of long-chain 3-OHFAs, the highest concentrations of LPS, and the lowest specific activities. Apartments had significantly different types of LPS (P = 0.03) compared with single-family homes in study 2. These data suggest that the Limulus assay may underestimate exposure to certain types of LPS. Because nontoxic LPS may have immune modulating effects, analysis of 3-OHFAs may be useful in epidemiologic studies.

Nuclear translocation of NF-kappaB in lipopolysaccharide-treated macrophages fails to correspond to endotoxicity: evidence suggesting a requirement for a gamma interferon-like signal.

Elucidation of a signal transduction pathway essential to lipopolysaccharide (LPS)-induced macrophage activation has the capacity to provide new targets for the treatment of septic shock. In this regard, activation of the transcription factor NF-kappaB is commonly thought to be critical to LPS-stimulated macrophage inflammatory mediator production, although certain immunological, genetic, and molecular evidence suggests that other factors are involved. To address this issue, we hypothesized that the degree of LPS-induced NF-kappaB mobilization should correlate with the murine endotoxicity of the species of LPS used for in vitro study. Therefore, using D-galactosamine-sensitized mice, we assessed the lethal potencies of eight LPS preparations from Escherichia, Salmonella, Klebsiella, Bacteroides, Pseudomonas, Neisseria, and Rhodobacter species as well as that of the endotoxin substructure lipid X. The lethal potencies of these LPS preparations varied by > 160-fold. Treatment of RAW 264.7 cells with the same LPS preparations induced levels of tumor necrosis factor alpha (TNF-alpha) and NO production that correlated with the LPS 50% lethal dose. The combined analysis of the levels of these two mediators produced in response to LPS in RAW cells was found to be a strong predictor of murine endotoxic lethality. Interestingly, while relatively nontoxic in mice, Rhodobacter capsulatus LPS stimulated RAW cell NF-kappaB-like DNA binding protein mobilization and TNF-alpha production to levels comparable to those of more toxic species of LPS but was unable to induce NO generation in RAW cells. These data indicate that neither NF-kappaB activation nor TNF-alpha production alone is a dependable predictor of LPS lethality. Additionally, cotreatment of RAW cells with the potent inflammatory mediator ADP had no effect on the ability of R. capsulatus LPS to stimulate NO production but significantly enhanced induction of NO production by the toxic species of LPS. In contrast, cotreatment of RAW cells or peritoneal macrophages with gamma interferon (IFN-gamma) normalized the abilities of both toxic and nontoxic LPS preparations to induce NO production, suggesting that selected preparations of LPS may preferentially generate an IFN-gamma-like signal that accounts for enhanced toxicity. In sum, the activation of NF-kappaB does not correspond to LPS lethality, thereby complicating models of macrophage activation that highlight NF-kappaB alone as a signal transduction factor necessary for LPS-mediated toxicity.

Diphosphoryl lipid A derived from the lipopolysaccharide (LPS) ofRhodobacter sphaeroidesATCC 17023 is a potent competitive LPS inhibitor in murine macrophage-like J774.1 cells

Pentaacyl diphosphoryl lipid A derived from the nontoxic lipopolysaccharide (LPS) of Rhodobacter sphaeroides ATCC 17023 (RsDPLA) did not induce tumour necrosis factor-alpha nor interleukin-6 release in the murine macrophage-like cell line J774.1. However, it effectively inhibited the induction of these two cytokines by LPS of Salmonella minnesota Re mutant R595 (ReLPS) in a concentration-dependent manner. Maximal inhibition and half-maximal inhibition occurred when the ReLPS to RsDPLA mass ratio was 1:30 and 1:1, respectively. A binding study was performed in the presence of serum to determine whether RsDPLA is competing with ReLPS for LPS binding sites on J774.1 cells. This assay allows the determination of LPS binding to J774.1 cells via a mechanism involving CD14, a receptor for complexes of LPS with LPS binding protein (LBP), and its possible inhibition. The results show that RsDPLA strongly inhibits the binding of 125I-labelled ReLPS to J774.1 cells. Maximal and one-half maximal inhibition of binding occurred when the ReLPS to RsDPLA mass ratios were 1:2.5 and 1:0.5, respectively. It was found that the inhibition of binding by RsDPLA was much stronger than that by unlabelled ReLPS. These results suggest that RsDPLA is competing with ReLPS for CD14-dependent recognition of LPS on J774.1 cells.

Lipopolysaccharidelike immunological properties of cell wall glycoproteins isolated from Cytophaga johnsonae

Glycoproteins (GP) previously shown to be involved in the gliding motility of Cytophaga johnsonae were examined for biological activities characteristic of lipopolysaccharide (LPS). These integral membrane proteins activated 70Z/3 pre-B cells to synthesize immunoglobulin M, induced B cells to synthesize non-antigen-specific polyclonal immunoglobulin, induced macrophages to produce tumor necrosis factor, and modulated the antibody response to type III pneumococcal polysaccharide in the absence of thymus-derived (T) lymphocytes. Except for the GP activity in the 70Z/3 assay, all activities of the GP were comparable to or greater than those of LPS. No LPS was detected in the preparations of GP used or in the phenol-water extracts of C. johnsonae. The mechanism by which these GP exerted their biological activities was distinct from that of LPS, since LPS-resistant C3H/HeJ mice responded to GP. Furthermore, biologically inactive diphosphoryl lipid A obtained from nontoxic LPS of Rhodopseudomonas sphaeroides (an analog of toxic lipid A), which is an antagonist of LPS, did not block the induction of tumor necrosis factor by GP in macrophages. These results showed that the cell surface GP from C. johnsonae are potent LPS-like activators of B cells and macrophages. We suggest that these GP might be good candidates for use in developing an effective adjuvant system.

Chemical reduction of 3-oxo and unsaturated groups in fatty acids of diphosphoryl lipid A from the lipopolysaccharide of Rhodopseudomonas sphaeroides. Comparison of biological properties before and after reduction.

Unlike the diphosphoryl lipid A (DPLA) derived from toxic lipopolysaccharide of Escherichia coli and Salmonella strains, the DPLA from nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 is biologically inactive. This could be due to the presence of 3-oxotetradecanoic and delta 7-tetradecenoic acids. These two fatty acids in R. sphaeroides DPLA were catalytically reduced in platinum oxide/H2 to the 3-hydroxy and saturated fatty acids, respectively. The biologically active E. coli DPLA was also treated with platinum oxide/H2, but as expected, the reduction step did not change the structure. These two preparations were then compared with the untreated samples for biological activity in three select in vitro assays. Over a range of 0.01-100 ng/ml, both normal and reduced DPLA from R. sphaeroides were inactive in priming phorbol myristate acetate-stimulated superoxide anion release in human alveolar macrophages. Over a range of 10-10(3) ng/ml, both samples failed to induce tumor necrosis factor in the RAW 264.7 murine macrophage cell line. The reduced DPLA marginally activated 70Z/3 pre-B cells at concentrations of 0.1-30 micrograms/ml. In every case, both normal and platinum oxide/H2-treated E. coli DPLA were biologically active. These results indicate that the lack of biological activity of R. sphaeroides DPLA is not due to the presence of 3-oxo and unsaturated fatty acids, but rather to one or more of the following: (i) presence of only five fatty acyl groups (compared to six in active lipid A); (ii) presence of 3-hydroxydecanoic acids (rather than 3-hydroxytetradecanoic, in active lipid A); (iii) greater variation in size of the fatty acids.

Diphosphoryl lipid A obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides is an endotoxin antagonist in mice

Diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not induce interleukin-1 release by murine peritoneal macrophages. However, it blocked this induction by toxic deep-rough chemotype LPS (ReLPS) from Escherichia coli D31m4. Previously, we obtained similar results on the induction of tumor necrosis factor (TNF) by macrophages. These results showed that DPLA is able to block in vitro the induction of two important mediators of gram-negative bacterial sepsis. We then wanted to determine whether DPLA could also block the induction of TNF by LPS in animals. Mice were treated with 100 micrograms of R. sphaeroides DPLA and challenged 60 min later with 1.0 micrograms of ReLPS from E. coli. The serum TNF level was measured after 60 min. Treatment of mice with this DPLA blocked the rapid and transient rise of TNF caused by ReLPS. This result suggested that R. sphaeroides DPLA might be able to protect animals against endotoxin shock caused by gram-negative bacterial infection.

Diphosphoryl lipid A derived from lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides inhibits activation of 70Z/3 cells by LPS

Diphosphoryl lipid A derived from nontoxic lipopolysaccharide (LPS) of Rhodopseudomonas sphaeroides ATCC 17023 did not stimulate the murine pre-B cell line 70Z/3 to synthesize surface immunoglobulin or kappa mRNA. However, it effectively blocked Escherichia coli LPS-induced activation of 70Z/3 cells in a concentration-dependent manner. This inhibition was specific only to cells activated by LPS, since it did not inhibit activation of 70Z/3 cells by gamma interferon. Maximal inhibitory effect occurred when the antagonist was added within 2 h before adding the LPS. These results strongly suggested that R. sphaeroides diphosphoryl lipid A is competing with E. coli LPS for physiological lipid A receptors on the 70Z/3 cells.

Inactivation of suppressor T cell activity by the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides

Antibody responses of mice immunized with type III pneumococcal polysaccharide were examined with and without treatment with nontoxic lipopolysaccharide from Rhodopseudomonas sphaeroides (Rs-LPS). The results obtained were similar to those described previously for mice treated with monophosphoryl lipid A (MPL) except that lower amounts of Rs-LPS were needed. Both were without effect when given at the time of immunization with type III pneumococcal polysaccharide but elicited significant enhancement when given 2 to 3 days later. Such enhancement was T cell dependent and not due to polyclonal activation of immunoglobulin M synthesis by B cells. Treatment with either Rs-LPS or MPL abolished the expression but not induction of low-dose paralysis, a form of immunological unresponsiveness known to be mediated by suppressor T cells (Ts). The in vitro treatment of cell suspensions containing Ts with extremely small amounts of Rs-LPS or MPI completely eliminated the capacity of such cells to transfer suppression to other mice. These findings indicate that the immunomodulatory effects of both MPL and Rs-LPS are mainly the result of eliminating the inhibitors effects of Ts; this permits the positive effects of amplifier T cells to be more fully expressed, thereby resulting in an increased antibody response. The significance of these and other findings to the use of Rs-LPS as a pharmacotherapeutic agent for gram-negative bacterial sepsis is discussed.

Differential cytokine induction by doses of lipopolysaccharide and monophosphoryl lipid A that result in equivalent early endotoxin tolerance

The phenomenon of early endotoxin tolerance, which is induced by sublethal injection of lipopolysaccharide (LPS), results in a protracted period of hyporesponsiveness that is most profound at 3 to 4 days after injection and is marked by reduced cytokine production after a challenge injection of LPS. Early endotoxin tolerance is also induced by the nontoxic LPS derivative monophosphoryl lipid A (MPL), although much more of the monophosphoryl derivative is required to produce a state of tolerance equivalent to that evoked by LPS. In this study, equivalent tolerance-inducing doses of LPS and MPL were tested, and the levels of cytokines induced by LPS and MPL were compared. Although induced levels of colony-stimulating factor were comparable following doses of LPS and MPL that elicited an equivalent state of early endotoxin tolerance, levels of tumor necrosis factor, interleukin-6, and interferon were significantly lower in MPL-injected animals. These results suggest that the lowered toxicity of MPL may be related to its elicitation of significantly lower levels of potentially toxic intermediaries such as tumor necrosis factor, interferon, and interleukin-6.

Diphosphoryl lipid A from Rhodopseudomonas sphaeroides ATCC 17023 blocks induction of cachectin in macrophages by lipopolysaccharide.

Purified diphosphoryl lipid A (DPLA) obtained from the nontoxic lipopolysaccharide of Rhodopseudomonas sphaeroides ATCC 17023 was shown to block the induction of cachectin (tumor necrosis factor) in the RAW 264.7 macrophage cell line by toxic deep-rough-chemotype lipopolysaccharide (ReLPS) of Escherichia coli in a concentration-dependent manner. The ReLPS-to-DPLA mass ratios of 1:10 and 1:100 (when 1.0 ng of ReLPS per ml was used) gave 55 and 95% inhibitions, respectively, of the induction of cachectin. Since the structure of the DPLA from R. sphaeroides is so similar to that of the lipid A moiety of the toxic ReLPS from E. coli, we suggest that this inhibition could have been due to competitive binding by DPLA to the active sites on the macrophage. This DPLA could become a useful reagent to study the nature of lipopolysaccharide/lipid A binding in macrophages and perhaps other responding cells.

Interaction of lipopolysaccharides and lipid A with complement in rats and its relation to endotoxicity.

Uniform salt forms of endotoxic lipopolysaccharides (LPS) and free lipid A showing distinct differences in their anticomplementary activity, as well as a nontoxic LPS, are used in a new approach of studying the role of complement in endotoxin shock. The use of these defined LPS forms led to the identification of two timely, distinct depressions in complement hemolytic activity after administration of endotoxin in rats. An early depression occurred within 10 min after injection, and a late one developed more gradually, with lowest values at 6 to 9 h. The early depression represents a direct interaction of LPS with complement. It was obtained by toxic and nontoxic preparations that exhibit a high molecular weight and anticomplementary activity in vitro. The early depression was not related to the toxic properties of the LPS. The late depression was obtained only with endotoxically active LPS in lethal and 100-fold-lower concentrations, regardless of their molecular weight and of their in vitro anticomplementary activity.

Biological activities of lipopolysaccharides and lipid A from Rhodospirillaceae.

The lipopolysaccharides and free lipid A from several strains of Rhodospirillaceae were assayed comparatively with those of Enterobacteriaceae in a number of biological tests. Free lipid A's from Rhodopseudomonas gelatinosa and Rhodospirillum tenue exhibited strong serological cross-reactions with each other and with free lipid A from Salmonella. Lipid A's from Rhodopseudomonas viridis and Rhodopseudomonas palustris, although cross-reacting with each other, did not do so with either the lipid A of R. gelatinosa or R. tenue or with that of Salmonella. The presence or absence of the above cross-reactions agreed with corresponding similarities or differences in the chemical structure of the lipid A preparations. The lipopolysaccharide of R. gelatinosa was highly toxic for adrenalectomized mice and pyrogenic for rabbits; however, it exhibited no anti-complementary activity. The activity of the R. tenue lipopolysaccharide was very low in both the lethality and pyrogenicity tests. Its corresponding free lipid A also exhibited low pyrogenic activity; however, its lethal toxicity for adrenalectomized mice was considerably higher than that of the intact parent lipopolysaccharide. Both intact lipopolysaccharide and, unexpectedly, the free lipid A exhibited no anti-complementary activity. The lipopolysaccharides of R. viridis and R. palustris were virtually nontoxic for mice and nonpyrogenic for rabbits. Both lipopolysaccharides were highly potent in their interaction with complement. They therefore represent the first example of nontoxic lipopolysaccharides exhibiting high anti-complementary activity.