Sepsis increases endocytosis of endotoxin into hepatocytes

BACKGROUND: Chylomicrons bind endotoxins and accelerate their clearance from plasma to the liver. This results in reduced mortality from septic shock in a rodent model. We hypothesized that the clearance of the LPS-chylomicron (LPS-CM) complex by hepatocytes is due to receptor-mediated endocytosis and that sepsis up-regulates this process. METHODS: Three groups of Sprague-Dawley rats; (1) control; (2) pretreated with 10 micrograms/kg LPS 24 hours before treatment; and (3) pretreated with 17-alpha-ethinyl estradiol (EE, 5 mg/kg subcutaneously for 3 days), were infused with labeled I125-LPS alone or with I125-LPS bound to chylomicron. Livers were removed 2.5, 15, and 30 minutes after LPS injection, and hepatic endosomes were isolated from the liver homogenates by serial ultracentrifugation in sucrose gradients. RESULTS: The injection of I125-LPS-CM complexes resulted in higher levels of endosomal I125-LPS in all groups, as compared with I125-LPS alone. In addition, the endosomal uptake of I125-LPS was markedly increased by both LPS and EE pretreatments. CONCLUSIONS: These data strongly suggest a primary role for receptor-mediated endocytosis in the increased clearance of LPS when bound to chylomicron. In addition, exposure to LPS appears to increase the accumulation of LPS in endosomes by a mechanism similar to that of EE, which is known to up-regulate receptor-mediated lipoprotein uptake. This endogenous pathway for the catabolism of endotoxins may provide a teleological explanation for the hypertriglyceridemia observed during sepsis.     

Immunological properties of Weil-Felix test negative sera from patients with Japanese spotted fever

Sera from 4 out of 19 patients with the Japanese spotted fever were negative to OX2 antigen of Weil-Felix (WF) test. These WF test negative sera were analyzed by ELISA and immunoblot used whole cells and lipopolysaccharides (LPS) of rickettsiae and Proteus strains as antigens. These acute-phase sera have already possessed the IgG antibodies against LPS of Proteus OX2 strain, whereas IgM antibodies in these acute- and convalescent-phase sera did not react with this LPS. On the other hand, the reactivity of IgM antibodies of the convalescent-phase sera in the 2 patients with LPS of Proteus OX19 strain increased as compared with that of the acute-phase sera by ELISA, and these IgM antibodies also showed the reactivity with bands of OX19-LPS in the immunoblot. On the basis of these results, it is interpreted that the WF test negative sera from patients with Japanese spotted fever are due to the presence of IgG antibodies against OX2-LPS in the sera.     

Synthetic lipid A-induced uveitis and endotoxin-induced uveitis--a comparative study

Endotoxin-induced uveitis (EIU) is an animal model of ocular inflammation induced by lipopolysaccharide (LPS). The lipid A (LA) region of the LPS chemical structure is believed to be responsible for virtually all the biological activities induced by LPS. The aim of this study was to perform a more detailed investigation of the potency of LA in reproducing EIU. Various doses of either LPS or LA were injected into the footpad of an inbred strain of Lewis rat and the inflammation patterns were compared by assessing the protein concentration, by cytological study, and by determining the inflammatory cell content in samples of aqueous humor obtained during 96-hour follow-up. Evaluation of the cell number and protein concentration ratio of both groups showed the LA-stimulated group presented a higher ratio than the LPS group (Welch's t-test, (P < 0.00001). It was noteworthy that even the injection of high doses of LPS could not reproduce the level of cellular infiltration induced by LA. Histological study confirmed the enhanced cellularity in the LA group, neutrophils being predominant in both the LPS- and the LA-stimulated groups. The divergent findings in these two models of uveitis may be valuable to further investigations of the process of inflammatory cell migration into the anterior chamber of the eye.     

The polymerase chain reaction in pathology

Membrane-fixed CD14 acts as a receptor for the protein-bound endotoxin (LPS) complex and mediates the cellular effects of endotoxin. Soluble CD14 (sCD14) is suggested to neutralize circulating LPS, i.e., acting as an endotoxin antagonist. The aim of this study was to elucidate the release of both sCD14 and endotoxin in traumatized patients, starting from the earliest phase after trauma. A total of 15 patients (O ISS = 19, 9-75) suffering major trauma were enrolled in this prospective study. Blood samples were collected as early as immediately at the site of accident, on hospital admission, and thereafter hourly, then daily. For patients (O ISS = 47) died within 24 h because of their severe injuries. Immediately after the accident as well as during the first 2 h after hospital admission, the mean sCD14 levels of surviving patients did not differ from those of healthy volunteers (n = 53). Thereafter, however, sCD14 increased continuously in the trauma group. The concentrations remained elevated throughout the entire observation period. There was, however, no relation between the sCD14 release and the pattern or the severity of injury. In contrast, endotoxin levels revealed a pattern-specific release. The highest plasma concentrations of LPS were observed in patients suffering from (additional) thoracic injury. On the basis of these results we conclude that the release of sCD14 after trauma does not reflect a strict principle such as action/reaction caused by the appearance of endotoxin immediately after the injury. Soluble CD14 is more likely release by an endotoxin-independent mechanism.     

Biophysical characterisation of lysozyme binding to LPS Re and lipid A

The binding of lysozyme to bacterial deep rough mutant lipopolysaccharide (LPS) Re and to its lipid moiety lipid A, the 'endotoxic principle' of LPS, was investigated using biophysical techniques. The beta<-->alpha gel to liquid crystalline phase transition, the nature of the functional groups of the endotoxins, the secondary structure of lysozyme, and competition with polymyxin B were studied by Fourier-transform infrared spectroscopy (FTIR); the supramolecular aggregate structure of the endotoxins was determined with synchrotron radiation X-ray diffraction and the binding stoichiometry with microcalorimetry. The results were compared with those found with zwitterionic and negatively charged phospholipids. It can clearly be shown that lysozyme binds electrostatically to charged groups of the endotoxin molecules with the consequence of acyl-chain rigidification and an initiation of a transition from inverted cubic to multilamellar structures. The binding stoichiometry of endotoxin and lysozyme is a 3:1 molar ratio for both LPS Re and lipid A, indicating a dominant binding of lysozyme to the lipid A-phosphates. This could be confirmed by the analysis of a phosphate vibration and by the use of a dephospho LPS. Parallel to lysozyme binding to endotoxin, a conformational change of the secondary structure in the protein from mainly alpha helix to more unordered structures takes place, while the residual beta-sheet substructure does not exhibit a clear concentration dependence. Binding is found to be specific for the endotoxins since, for the zwitterionic phosphatidylcholine, no binding is observed and, for the negatively charged phosphatidylglycerol, only very weak binding is found. The results are discussed in the context of the ability of lysozyme to reduce endotoxicity.     

Correlation between structural transformation and cleavage of the major head protein of T4 bacteriophage

Human infections associated with Yersinia enterocolitica in Hungary in the years 1969-1974 have been surveyed. During this period the public health laboratory network isolated 1355 strains from 1096 persons. The number of isolates according to sero-groups was: 1343 O3, 6 O9, 2 O1, 2a, 3 and 1 strain for O5, O6, O1O and O15 each. (ii) A total of 2192 serum specimens from patients gave positive agglutination reaction with antigen O3 in 26.8%, with antigen O9 in 3.2%. (iii) Fifty-six per cent of bacteriologically positive persons had enteritis. Other clinical forms (pseudoappendicitis and other abdominal complaints, erythema nodosum, rheumatoid arthritis) were encountered in O 1-2.7%. Symptomless excreters of Y. enterocolitica amounted to 23.1% of all positive persons. (iv) Patients with enteritis and symptomless excreters were rather evenly distributed between 10 and 60 years of age; 1--9-year-old children were affected more frequently (47.7% of all positive persons). Six distribution was, males: females = 1.5 : 1. In seasonal incidence yersiniosis differed from other enteric diseases: it showed a peak in the autumn-winter months. Sporadic cases and family outbreaks were the most frequent; epidemic infections in nurseries were also recorded. (v) Out of 59 animal strains 39 group O3 cultures were isolated from pigs, which may be assumed as the most important reservoir of yersinosis in Hungary.     

Role of YadA-mediated collagen binding in arthritogenicity of Yersinia enterocolitica serotype O:8: experimental studies with rats

Outer membrane protein YadA, Yersinia adhesin, is one of the plasmid-encoded virulence factors of yersiniae. YadA protects bacteria against host defense through several different mechanisms. One important role of YadA is to mediate binding to several collagen types. Our recent study revealed that a yadA null mutant of Yersinia enterocolitica serotype O:8 has a drastically reduced arthritogenic capacity when injected intravenously into Lewis rats. To further characterize the arthritogenic role of YadA, we repeated the rat experiments with strain Y. enterocolitica O:8/pYV082; this strain expresses a YadA deletion derivative lacking 22 amino acids from the amino-terminal hydrophobic region and does not bind to collagen. Y. enterocolitica O:8/pYV082 induced arthritis in 5 to 14% of rats inoculated with arthritogenic doses, whereas the arthritis incidence with the wild-type parent strain was 65%. The parent strain was slightly more virulent than Y. enterocolitica O:8/pYV082, as determined by rat mortality. It also frequently induced skin abscesses, whereas Y. enterocolitica O:8/pYV082 did not. Infection kinetics in spleen and mesenteric lymph nodes were about the same with both of the bacterial strains used, and the same was true of the Yersinia-specific antibody response. Altogether, these results suggest that YadA-mediated collagen binding contributes to the arthritogenicity of Y. enterocolitica O:8.     

Yersinia enterocolitica: the charisma continues

Yersinia enterocolitica, a gram-negative coccobacillus, comprises a heterogeneous group of bacterial strains recovered from animal and environmental reservoirs. The majority of human pathogenic strains are found among distinct serogroups (e.g. O:3, O:5,27, O:8, O:9) and contain both chromosome- and plasmid (60 to 75 kb)-mediated virulence factors that are absent in "avirulent" strains. While Y. enterocolitica is primarily a gastrointestinal tract pathogen, it may produce extraintestinal infections in hosts with underlying predisposing factors. Postinfection sequelae include arthritis and erythema nodosum, which are seen mainly in Europe among patients with serogroups O:3 and O:9 infection and HLA-B27 antigen. Y. enterocolitica is acquired through the oral route and is epidemiologically linked to porcine sources. Bacteremia is prominent in the setting of immunosuppression or in patients with iron overload or those being treated with desferrioxamine. metastatic foci following bacteremia are common and often involve the liver and spleen. Of particular concern is blood transfusion-related bacteremia. Evidence has accumulated substantiating the role of Y. enterocolitica as a food-borne pathogen that has caused six major outbreaks in the United States. The diagnosis of Y. enterocolitica gastroenteritis is best achieved through isolation of the bacterium on routine or selective bacteriologic media. When necessary, serogrouping, biogrouping, and assessment for plasmid-encoded virulence traits may aid in distinguishing virulent from "avirulent" strains. Epidemiologically, outside of identified food-borne outbreaks, the source (reservoir) of Y. enterocolitica in sporadic cases is speculative. Therefore, prevention and control measures are difficult to institute.     

Induction of early gene expression in murine macrophages by synthetic lipid A analogs with differing endotoxic potentials

Numerous lipid A analogs have been synthesized in an attempt to dissociate endotoxic activities from beneficial immunomodulatory activities. In the present study, we have evaluated select lipid A analogs in macrophages for their ability to induce a panel of lipopolysaccharide (LPS)-inducible genes to gain insights into the molecular mechanisms which underlie endotoxicity. We evaluated three monosaccharide lipid A analogs: SDZ MRL 953, an agonist with an improved therapeutic margin over endotoxin; SDZ 281.288, a more toxic analog; and SDZ 880.431, an analog with proven LPS-inhibitory activity. In addition, three disaccharide lipid A analogs (i.e., lipid IVA, SDZ 880.611, and SDZ 880.924) that differ in acylation and phosphorylation patterns were also examined and compared with synthetic lipid A. With the exception of SDZ 880.431, each of these structurally diverse analogs was able to induce the complete panel of LPS-inducible genes, specifically genes which encode tumor necrosis factor alpha (TNF-alpha), interleukin-1 beta, 75-kDa type 2 TNF receptor (D7), IP-10, D3, and D8. These results underscore that macrophage stimulation by lipid A analogs is permissive to considerable structural diversity. Structures with favorable therapeutic indices (SDZ MRL 953, SDZ 880.611, and SDZ 880.924) were not different from structures with poor therapeutic indices (lipid A, lipid IVA, and SDZ 281.288) with regard to gene induction. Nonetheless, the nontoxic SDZ MRL 953 was approximately 1,000-fold less potent than synthetic lipid A at inducing TNF-alpha secretion, and perhaps this contributes to the lack of toxicity exhibited by this compound. The ability of compound SDZ 880.431 to inhibit TNF-alpha secretion induced by both SDZ MRL 953 and smooth LPS suggests that the monosaccharide and smooth LPS share a receptor or a portion thereof. A pattern of protein tyrosine phosphorylation similar to that induced by LPS was stimulated by the monosaccharide SDZ MRL 953 and SDZ 281.288 and disaccharides lipid IVA, SDZ 880.924, and SDZ 880.611, providing evidence for a common signalling pathway.     

Diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA): optimal conditions for quantitation of antibodies

In the diffusion-in-gel enzyme-linked immunosorbent assay (DIG-ELISA) the quantitation of antibodies is based on their ability to form a diffusion gradient over an antigen-coated polystyrene surface. The antigen-antibody reaction is then visualized by an enzyme-conjugated anti-immunoglobulin. The enzyme-substrate reaction is finally performed by pouring a substrate-containing gel over the polystyrene surface. In this study with bovine serum albumin as antigen and a corresponding rabbit antiserum, the diffusion time of antiserum was shown to be the most critical variable of the method, while the antigen concentration used for coating, the conjugate binding time and the enzyme-substrate reaction time had a minor influence on the quantitation of antibodies. High antibody levels were measured with greater accuracy than low levels, but the standard deviation was below 10%. It was also shown that different sera containing antibodies to Salmonella typhi O LPS, Klebsiella pneumoniae K1 and O4 LPS, Escherichia coli O2 LPS, Yersinia enterocolitica Y3 LPS, cardiolipin and pneumococcus could be quantitated with the same accuracy.     

Structural elucidation of the lipopolysaccharide core regions of the wild-type strain PAO1 and O-chain-deficient mutant strains AK1401 and AK1012 from Pseudomonas aeruginosa serotype O5

Lipopolysaccharide (LPS) of the Pseudomonas aeruginosa serotype O5 wild-type strain PAO1 and derived rough-type mutant strains AK1401 and AK1012 was isolated by a modified phenol/chloroform/petroleum-ether extraction method. Deoxycholate/PAGE of the LPS from the rough mutant AK1401 indicated two bands near the dye front with mobilities similar to those of the parent strain, indicating that both LPS contain a complete core and a species comprising a core and one repeating unit. Composition analysis of the LPS from strains PAO1 and AK1401 indicated that the complete core oligosaccharide was composed of D-glucose (four units), L-rhamnose (one unit), 2-amino-2-deoxy-D-galactose (one unit), L-glycero-D-manno-heptose (Hep; two units), 3-deoxy-D-manno-octulosonic acid (Kdo; two units), L-alanine (one unit) and phosphate (three units). The glycan structure of the LPS was determined by one-dimensional and two-dimensional (2D) NMR techniques in combination with MS-based methods on oligosaccharide samples obtained from the LPS by delipidation procedures. The locations of three phosphomonoester groups on the first heptose residue were established by a two-dimensional 31P (omega1)-half-filtered COSY experiment on the reduced core oligosaccharide sample of the LPS from the wild-type strain. The presence of a 7-O-carbamoyl substituent was observed on the second heptose. The structure of the core region of the O-chain-deficient LPS from P. aeruginosa serotype 05 is as follows: [structure: see text] where R1 is beta-D-Glcp-(1-->2)-alpha-L-Rhap-(1-->6)-alpha-D-Glcp-(1--> and R2 is alpha-D-Glcp-(1-->6)-beta-D-Glcp-(1->. A structural model is presented that is also representative of that for P. aeruginosa serotype O6 LPS. A revised structure for the serotype O6 mutant strain A28 is presented.     

Comparison of siderophore production and utilization in pathogenic and environmental isolates of Yersinia enterocolitica

Yersinia enterocolitica strains of serotypes lethal to mice have been reported previously to produce an endogenous siderophore. In this study, an ethyl acetate-extractable siderophore was characterized and given the name yersiniophore. Yersiniophore was produced by 16 of 16 human isolates of serotypes O:4, O:4,32, O:8, O:21, and one nonhuman isolate of serotype O:21. It was not produced by isolates of serotype O:3, O:5, or O:9. One strain of Yersinia pseudotuberculosis produced yersiniophore, but strains of Yersinia kristensenii, Yersinia frederiksenii, and Yersinia intermedia did not produce or utilize yersiniophore. Food and water isolates of Y. enterocolitica produced a water-soluble siderophore but not yersiniophore. Sixty-two strains of Y. enterocolitica including 42 isolates from human infections, 2 animal isolates, and 18 water and food isolates were examined for utilization of yersiniophore, the water-soluble siderophore, and ferrioxamine. Yersiniophore promoted growth rate, iron binding, and uptake in 17 of 62 strains, all of which produced yersiniophore. Ten of 17 food and water isolates and one human isolate were capable of utilizing the water-soluble siderophore. Utilization studies suggest that at least one additional water-soluble siderophore may be produced. Ferrioxamine promoted the growth of 60 of 62 strains examined; however, only the 17 strains which produced yersiniophore actively accumulated [59Fe]ferrioxamine. Yersiniophore production and utilization may be important in clinical infections since all human strains belonging to serotype O:8 produced yersinophore. The water-soluble siderophore was not detected in human isolates.     

An ectopic parathyroid adenoma revealed by L-thyroxine side effect on bone. Case report

Yersinia was isolated from imported raw meat and fowl products by HeLa cell treatment and conventional KOH-treatment, to obtain information on the origin of pathogenic Yersinia in Japan. Forty-one strains of Yersinia enterocolitica and one strain of Yersinia pseudotuberculosis, serotype 4b were isolated from 38 (3.0%) of 1278 samples of pork, two (0.3%) of 612 samples of beef and two (0.3%) of 615 samples of chicken. Y. enterocolitica isolates belonged to B:4/O:3 (biotype/serotype, 15 strains), B:3/O:3 (two strains) and B:3 variant/O:3 (17 strains) and B:3/O:5.27 (seven strains). The B:4/O:3 which is globally prevalent among humans and animals was isolated from pork samples from Denmark and the US and from beef samples from Australia, the B:3/O:3 from pork samples from Canada, the B:3 variant/O:3 from pork samples from Taiwan and from chicken samples from Thailand, the B:3/O:5.27 from pork samples from the US and Taiwan and Y. pseudotuberculosis, serotype 4b from pork samples from Canada. These findings suggest that pathogenic Y. enterocolitica strains can be introduced into Japan by the import of pork from pig producing countries. The HeLa cell treatment was found to be superior to the conventional method.     

Effects of pretreatment with SDZ MRL 953, a novel immunostimulatory lipid A analog, on endotoxin-induced acute lung injury in guinea pigs

SDZ MRL 953 (SDZ), a novel immunostimulatory lipid A analog, has been reported to have immunopharmacological activities similar to those of lipopolysaccharide (LPS) but to have little of the toxicity of LPS. We investigated the effects of pretreatment with SDZ on Escherichia coli endotoxin-induced acute lung injury in guinea pigs. Four experimental groups consisted of saline control (n = 16), SDZ (-12 h) plus LPS (2 mg/kg of SDZ per kg of body weight injected intravenously 12 h before intravenous injection of 2 mg of LPS per kg; n = 15), SDZ (-10 min) plus LPS (SDZ injected 10 min before LPS injection; n = 10), and LPS alone (n = 16). The animals were sacrificed, and lung tissue was sampled 4 h after LPS or saline infusion. Lung injury was assessed by measuring the wet weight-to-dry weight ratio and the level of 125I-labeled albumin accumulation in bronchoalveolar lavage fluid relative to that in plasma. In the SDZ (-12 h) plus LPS group, these two parameters of acute lung injury were decreased compared with those in the LPS alone group. However, they were not decreased in the SDZ (-10 min) plus LPS group. We conclude that SDZ attenuates endotoxin-induced acute lung injury when it is administered 12 h before LPS injection. The attenuating effects of SDZ are speculated to be due to down regulation of the response to endotoxin rather than to receptor blocking.     

Characterization of lipopolysaccharide-deficient mutants of Pseudomonas aeruginosa derived from serotypes O3, O5, and O6

Well-characterized rough mutants are important for the understanding of structures, functions, and biosynthesis of lipopolysaccharide (LPS) in gram-negative organisms. In this study, three series of Pseudomonas aeruginosa LPS-deficient mutants, namely PAC strains derived from serotype O3, AK strains derived from strain PAO1 (serotype O5), and serotype O6-derived mutants were subjected to biochemical analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and silver staining as well as immunochemical characterization using LPS-specific monoclonal antibodies. The O-side-chain deficiency among the O6-derived mutants was also examined, and three mutants, A28, R5, and H4, were subsequently chosen for the elucidation of component sugars of the core structure of serotype O6 LPS. LPS of strain A28 has L-rhamnose and proportionally higher amounts of D-glucose, a feature shared by the O5-derived mutant, strain AK1401 (previously demonstrated as a mutant with a core-plus-one O repeat). In contrast strains R5 and H4 were shown to be devoid of L-rhamnose and have low and undetectable amounts of D-glucose, respectively, which indicated their core deficiency. The LPS-deficient or -sufficient characteristics of the P. aeruginosa strains examined correlated will with serum sensitivity data. This report represents a comprehensive analysis of rough mutants derived from O3 and O5 strains that have been used by others in many studies and a first look at the core oligosaccharide region of serotype O6 LPS obtained with the O6-derived mutants generated in this study.     

Relationship of structure and biological activity of monosaccharide lipid A analogues to induction of nitric oxide production by murine macrophage RAW264.7 cells

Lipid A is the active center of bacterial lipopolysaccharide (LPS), which exhibits diverse biological activities via the production of various mediators. We investigated the production of nitric oxide (NO), one of the mediators, by a murine macrophage cell line, RAW264. 7, upon stimulation with a series of monosaccharide lipid A analogues to elucidate the relationship of structure and activity in NO production. The production of other representative mediators, such as tumor necrosis factor alpha (TNF-alpha) and interleukin-6 (IL-6), was also investigated to compare the structural requirements for the production of these cytokines with those for the production of NO. Structure-activity relationships in NO production correlated well with those in the production of TNF-alpha and IL-6. Among the lipid A analogues possessing different numbers of acyl groups on a 4-O-phosphono-D-glucosamine backbone, compounds like GLA-60 that possess three tetradecanoyl (C14) groups exhibited stronger activities in the production of the mediators than compounds possessing four or two C14 groups. Time course study of the production of these mediators showed that production of NO started and peaked later than those of TNF-alpha and IL-6. Neither neutralization of TNF-alpha activity by antibody nor suppression of TNF-alpha production by pentoxifylline showed a significant suppressive effect on production of NO and IL-6 upon stimulation with LPS or lipid A analogues. Neutralization of IL-6 activity by antibody showed no significant suppressive effect on production of NO and TNF-alpha. A monosaccharide lipid A analogue (GLA-58) which exhibited no detectable agonistic activity showed a suppressive effect on the production of all three mediators upon stimulation with LPS or lipid A analogues. These results indicate that signals for NO production by LPS agonists in murine macrophages are transduced in good correlation with those for production of TNF-alpha and IL-6, although they are not transduced via production of those cytokines.     

Suppression of murine endotoxin response by E5531, a novel synthetic lipid A antagonist

As a consequence of blood-borne bacterial sepsis, endotoxin or lipopolysaccharide (LPS) from the cell walls of gram-negative bacteria can trigger an acute inflammatory response, leading to a series of pathological events and often resulting in death. To block this inflammatory response to endotoxin, a novel lipid A analogue, E5531, was designed and synthesized as an LPS antagonist, and its biological properties were examined in vitro and in vivo. In murine peritoneal macrophages, E5531 inhibited the release of tumor necrosis factor alpha (TNF-alpha) by Escherichia coli LPS with a 50% inhibitory concentration (IC50) of 2.2 nM, while E5531 elicited no significant increases in TNF-alpha on its own. In support of a mechanism consistent with antagonism of binding to a cell surface receptor for LPS, E5531 inhibited equilibrium binding of radioiodinated LPS ([125I]2-(r-azidosalicylamido)-1, 3'-dithiopropionate-LPS) to mouse macrophages with an IC50 of 0.50 microM. E5531 inhibited LPS-induced increases in TNF-alpha in vivo when it was coinjected with LPS into C57BL/6 mice primed with Mycobacterium bovis bacillus Calmette-Guérin (BCG). In this model, the efficacy of E5531 was inversely correlated to the LPS challenge dose, consistent with a competitive antagonist-like mechanism of action. Blockade of the inflammatory response by E5531 could further be demonstrated in other in vivo models: E5531 protected BCG-primed mice from LPS-induced lethality in a dose-dependent manner and suppressed LPS-induced hepatic injury in Propionibacterium acnes-primed or galactosamine-sensitized mice. These results argue that the novel synthetic lipid A analogue E5531 can antagonize the action of LPS in in vitro and suppress the pathological effects of LPS in vivo in mice.     

Structural studies on the short-chain lipopolysaccharide of Vibrio cholerae O139 Bengal

A Vibrio cholerae O139 strain, MO10-T4, lacking capsular polysaccharide, produces a short-chain lipopolysaccharide (LPS), similar to enterobacterial SR strains. It was studied by acidic and alkaline degradation, dephosphorylation, sugar and methylation analysis, high-performance anion-exchange chromatography, one- and two-dimensional 1H-, 13C-, and 31P-NMR spectroscopy, and electrospray ionization mass spectrometry. The following structure was proposed for the core region of the LPS: [structure: see text] where PEtn stands for 2-aminoethyl phosphate, Fru for fructose, Hep for L-glycero-D-manno-heptose, and Kdo for 3-deoxy-D-manno-octulosonic acid; unless otherwise stated, the monosaccharide residues are D and present in the pyranose form. An O-acetyl group is present on a secondary position, tentatively O4 of the alpha-linked glucosyl group. Some LPS species contain an additional putative fructose residue whose location remains unknown. An O139-negative mutant strain, Bengal-2R, derived from V. cholerae O139, has also been investigated and shown to produce an O-antigen-lacking LPS similar to those from enterobacterial R strains, some of the LPS species containing the same core region as the strain MO10-T4 LPS and the other lacking the lateral heptose residue. The carbohydrate backbone core structure is the same for the V. cholerae O139 and V. cholerae O1 LPS, thus confirming the close relation between these bacteria; however, the 2-aminoethyl phosphate, the O-acetyl group, and the second fructose residue have not been reported for the O1 LPS. In the V. cholerae O139 strain MO10-T4 LPS, a short O-side chain is attached at position 3 of the 7-substituted heptose residue and has the same structure as one repeating unit of the V. cholerae O139 capsular polysaccharide. Some details of the structure proposed are at variance with those recently published for another V. cholerae O139 strain [Cox, A. D., Brisson, J.-R., Varma, V. & Perry, M. B. (1996) Carbohydr. Res. 290, 43-58; Cox, A. D. & Perry, M. B. (1996) Carbohydr. Res. 290, 59-65.]     

Chromosome-mediated resistance of Yersinia enterocolitica serotype O9 to intracellular killing by mouse peritoneal macrophages

The survival of Yersinia enterocolitica serotype O9 within mouse peritoneal macrophages was investigated. To evaluate the role of the virulence plasmid in the resistance to intracellular killing, an isogenic pair of virulent (plasmid-bearing) and avirulent (plasmid-less) O9 strains was used. The virulent strain was able to express plasmid-encoded outer membrane proteins and to colonize the Peyer's patches of orally infected mice. When mice were infected intraperitoneally, both strains were recovered at similar rates and over the same time from the peritoneal cavity. When in vitro assays were performed, both strains showed similar resistance to intracellular killing by monolayers of resident and inflammatory peritoneal macrophages. Previous opsonization of bacteria did not modify their survival within macrophage monolayers. We concluded that serotype O9 strains display a chromosome-mediated resistance to intracellular killing by mouse peritoneal macrophages. Moreover, macrophage resistance does not seem to be of importance for virulence of serotype O9 strains in mice.     

Molecular cloning of bruchid (Zabrotes subfasciatus) alpha-amylase cDNA and interactions of the expressed enzyme with bean amylase inhibitors

Our previous study has reported that ethanol (ETOH) partially inhibited the endotoxin (LPS)-induced tissue factor (TF)-activation in monocytes including blood peripheral monocytes as well as cultured leukemic U937 and THP-1 cells. The present study shows a strong correlation (r = 0.92; p < 0.01) between TF-activation and depression in LPS binding blocked by ETOH in U937 cells. The antagonism by ETOH of LPS binding was not due to a direct extracellular blockade, since ETOH did not affect the affinity of fluorescein isothiocyanate (FITC)-LPS or -anti CD14 mAb on U937 cells. After U937 cells were treated with 2 per cent (v/v) ETOH for 3 h, LPS binding was however drastically inhibited as shown by immunostaining with FITC-LPS which was viewed on a confocal laser scanning microscope. The results imply that cellular events of the ETOH effect mediate this inhibition of LPS binding. Anti-CD14 mAb (UCHM-1) inhibited LPS binding in a dose-dependent fashion, revealing a competitive specific binding to the LPS receptor. The results suggest that CD14 plays an important role in the recognition of LPS. FITC-UCHM-1 binding was significantly reduced in the cells pretreated with 2 per cent (v/v) ETOH for 3 h, indicating that ETOH modulates the ability to express CD14. CD14 expression was upregulated by priming with LPS which was offset by ETOH. Acetaldehyde, a possible metabolite of ETOH, was tested with no effect on CD14 expression. Taken together, our results show that ETOH downregulates the recognition of LPS, and suggest that the inhibitory action is likely to be mediated by the depression in CD14 expression which was also accompanied by a significantly altered membrane fluidity. Thus, the antagonism by ETOH of the binding of LPS results in a depression in the LPS-induced TF-activation.