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Detection and quantification of bacterial endotoxins is important in a range of health-related contexts, including during pharmaceutical manufacturing of therapeutic proteins and vaccines. Here we combine experimental measurements based on nematic liquid crystalline droplets and machine learning methods to show that it is possible to classify bacterial sources (Escherichia coli, Pseudomonas aeruginosa, Salmonella minnesota) and quantify concentration of endotoxin derived from all three bacterial species present in aqueous solution. The approach uses flow cytometry to quantify, in a high-throughput manner, changes in the internal ordering of micrometer-sized droplets of nematic 4-cyano-4'-pentylbiphenyl triggered by the endotoxins. The changes in internal ordering alter the intensities of light side-scattered (SSC, large-angle) and forward-scattered (FSC, small-angle) by the liquid crystal droplets. A convolutional neural network (Endonet) is trained using the large data sets generated by flow cytometry and shown to predict endotoxin source and concentration directly from the FSC/SSC scatter plots. By using saliency maps, we reveal how EndoNet captures subtle differences in scatter fields to enable classification of bacterial source and quantification of endotoxin concentration over a range that spans eight orders of magnitude (0.01 pg mL-1 to 1 μg mL-1). We attribute changes in scatter fields with bacterial origin of endotoxin, as detected by EndoNet, to the distinct molecular structures of the lipid A domains of the endotoxins derived from the three bacteria. Overall, we conclude that the combination of liquid crystal droplets and EndoNet provides the basis of a promising analytical approach for endotoxins that does not require use of complex biologically-derived reagents (e.g., Limulus amoebocyte lysate).

Purpose: The aim of this study was to analyze in S. Minnesota the presence of virulence genes, phylogenetic relationship, biofilm formation index (BFI) and its ultrastructure at temperatures of 4 °C, 25 °C and 36 °C and also the ability of biocidal agents to promote inhibition of the sessile structure formed by 20 strains isolated in the years 2009, 2010 and 2014 in broiler slaughter plants of two Brazilian companies.Methods: The presence of specific genes was evaluated by PCR and the phylogeny between the strains by PFGE, while BFI was performed by the classical method using TSB broth supplemented with 5% of chiken juice and its structure observed by SEM Chlorhexidine 1%, sodium hypochlorite 1% and peracetic acid 0.8% were tested for biofilm inhibition.Results: The presence of specific genes indicates that S. Minnesota has the potential to cause disease in humans and to adapt to adverse conditions. In general, temperatures of 25 °C and 36 °C favored more biofilm formation, although at 4 °C there is still biomass with potential contaminant from the final product. Tolerant strains (12/20–60%) to all biocides pose a real risk for the development of adaptation mechanisms, especially for resistance to sodium hypochlorite. Phylogenetic analysis indicated the existence of cross contamination problems and the spread among companies, probably related to the formation of biofilms.Conclusion: The results show the necessity of attention to this serovar considering its resistance to sodium hypochlorite in addition to the need for rigorous monitoring and control measures based on the adoption of low temperatures in order to prevent the formation of biofilms in the poultry industry.

Mango has been implicated as food vehicle in several Salmonella-causing foodborne outbreaks. Here, Salmonella enterica subsp. enterica serovar Minnesota was isolated from fresh mango fruit imported from Mexico in 2014. The complete genome sequence of S. Minnesota CFSAN017963 was sequenced using single-molecule real-time DNA sequencing. Distinct prophage regions, Salmonella pathogenicity islands, and fimbrial gene clusters were observed in comparative genomic analysis on S. Minnesota CFSAN017963 with other phylogenetically closely related Salmonella serovars. Core genome multilocus sequencing typing analysis of all the S. Minnesota isolates in the Genbank and Enterobase also revealed a high genomic diversity among the genomes analyzed.

The present study investigated the effectiveness of a single Salmonella prevention and control program applied in two different processing plants, located in the states of Mato Grosso do Sul (plant A) and Santa Catarina (Plant B), belonging to the same company, and identified Salmonella strain subtypes isolated from broilers, carcasses before and after chilling, and frozen chicken breasts. The Salmonella prevention and control program was 90% effective in plant A and 100% in plant B, considering a level of 10% positive samples per frozen chicken breast batch acceptable. A total of 128 strains were serotyped, being 10 from drag swabs, 31 from cloacal swabs, 83 from carcasses, and 4 from frozen chicken breasts. After serotyping analysis, 30 strains isolated at different processing steps and drag swabs, and three Salmonella Minnesota strains isolated in 2012 in plant A, were genotyped by PFGE. In plant A, the most frequently strain isolated was Salmonella Minnesota (90.35%), followed by Salmonella Newport (8.77%), and in Plant B, Salmonella Senftenberg (80%). Salmonella Minnesota strains were differentiated by PFGE into 19 pulsotypes distributed in three clusters. The phenotypic identification by serotyping of four strains diverged from their PFGE genotypic results. Most Salmonella Minnesota strains genotyped in plant A and the strains isolated from environmental samples in 2012 in the same broiler processing plant belong to a single cluster, confirming the dominance and persistence of this clone over time.

The aim of this study was to determine whether probiotic-feeding affected the expression of cathelicidins (CATHs), a major family of antimicrobial peptides, in response to lipopolysaccharides (LPS) challenge in the proventriculus and cecum of broiler chicks. One-day-old male Chunky broiler chicks were fed with or without 0.4% probiotics for 7 days (P-group and non-P-group, respectively). Then, they were orally challenged with no LPS (0-LPS), 1 µg LPS (1-LPS), or 100 µg LPS (100-LPS) (n=5 in all groups) in Experiment 1, and with no LPS or 1 µg LPS (n=6 in all groups) in Experiment 2. Five hours after LPS challenge, the proventriculi and ceca were collected to analyze CATHs expression. Expression of CATHs was examined at first by reverse transcription-polymerase chain reaction (RT-PCR) using the 0-LPS chicks of non-P-group. The differences in CATHs expression upon probiotics-feeding and LPS were analyzed by real time-PCR. All four CATHs (CATH1, 2, 3 and 4) were expressed in the proventriculus and cecum of chicks. In the proventriculus, the expression of CATHs after LPS challenge did not show significant differences between non-P and P-groups in Experiment 1 and 2. In the cecum, the interactions of the effects of probiotics and LPS on the expression of CATH2 in Experiment 1 and CATH1 and 2 in Experiment 2 were significant, and their expression in 1-LPS chicks was higher in P-group than in non-P-group. However, CATH3 and 4 did not show any significant differences between non-P- and P-groups challenged with LPS. These results suggest that probiotics-feeding may stimulate the immunodefense system mediated by CATH2 and possibly CATH1 against infection by Gram-negative bacteria in the cecum.

Cationic antimicrobial cationic peptides (CAMP) have been found in recent years to play a decisive role in hosts’ defense against microbial infection. They have also been investigated as a new therapeutic tool, necessary in particular due to the increasing resistance of microbiological populations to antibiotics. The structural basis of the activity of CAMPs has only partly been elucidated and may comprise quite different mechanism at the site of the bacterial cell membranes or in their cytoplasm. Polymyxin B (PMB) is a CAMP which is effective in particular against Gram-negative bacteria and has been well studied with the aim to understand its interaction with the outer membrane or isolated membrane components such as lipopolysaccharide (LPS) and to define the mechanism by which the peptides kill bacteria or neutralize LPS. Since PMB resistance of bacteria is a long-known phenomenon and is attributed to structural changes in the LPS moiety of the respective bacteria, we have performed a thermodynamic and biophysical analysis to get insights into the mechanisms of various LPS/PMB interactions in comparison to LPS from sensitive strains. In isothermal titration calorimetric (ITC) experiments considerable differences of PMB binding to sensitive and resistant LPS were found. For sensitive LPS the endothermic enthalpy change in the gel phase of the hydrocarbon chains converts into an exothermic reaction in the liquid crystalline phase. In contrast, for resistant LPS the binding enthalpy change remains endothermic in both phases. As infrared data show, these differences can be explained by steric changes in the headgroup region of the respective LPS.

Salmonella minnesota R 595 (Re) and other Salmonella strains incorporate cis-delta 9-16:1 (palmitoleic acid) at the expense of mainly dodecanoic acid into the lipid-A portion of lipopolysaccharides, when the cells are grown at low temperature (12 degrees C). It has recently been shown, that in S. minnesota R 595 grown at 37 degrees C dodecanoic acid is linked to the 3-hydroxyl group of an amide-bound 3-hydroxytetradecanoic acid. The present data suggest, that cis-delta 9-16:1 occupies the same position in lipid A (12 degrees C).

International audience; In this study, we aimed to evaluate the antioxidant and anti-inflammatory properties of Opuntia ficus-indica cactus cladode extracts in microglia BV-2 cells. Inflammation associated with microglia activation in neuronal injury can be achieved by LPS exposure. Using four different structurally and biologically well-characterized LPS serotypes, we revealed a structure-related differential effect of LPS on fatty acid β-oxidation and antioxidant enzymes in peroxisomes: Escherichia coli-LPS decreased ACOX1 activity while Salmonella minnesota-LPS reduced only catalase activity. Different cactus cladode extracts showed an antioxidant effect through microglial catalase activity activation and an anti-inflammatory effect by reducing nitric oxide (NO) LPS-dependent production. These results suggest that cactus extracts may possess a neuroprotective activity through the induction of peroxisomal antioxidant activity and the inhibition of NO production by activated microglial cells.

After about 70 years two new adjuvants have been approved for human vaccines. The first is MF59 developed by the ex-Chiron now Novartis Vaccines and it consists in an oil-in-water emulsion, comprising a low content of biodegradable squalene oil (4.3%) as the dispersed phase, which is stabilized by two non-ionic surfactants (Tween 80 and Span 85), and a low ionic strength citrate buffer as the continuous phase. The second one, defined as AS04, has been developed by GSK Biologics and consists in 3-0-desacyl-4'-monophosphoryl lipid A (MPL) that comes from the cell wall LPS of Gram-negative Salmonella minnesota R595 and is detoxified by mild hydrolytic treatment and purification. It is absorbed on aluminum hydroxide or aluminum phosphate. Thus, new molecules are available to improve the immune response to vaccines also in humans: this is the beginning of a new era in vaccinology.

Structural investigations have shown that lipid A constitutes the active principle of lipopoly saccharide (LPS, endotoxin), a complex amphipathic molecule located on the cell surface of Gram-negative bacteria.1 Lipid A elicits not only the typical endotoxic reactions such as fever and lethal shock but also adjuvant, antitumor and other beneficial effects.1 As a result, there has been a great deal of interest in the synthesis of lipid A derivatives possessing low toxicity.1,2

Recently published studies showed that human hemoglobin (Hb) forms stable complexes with bacterial endotoxin (lipopolysaccharide, LPS), and that these complexes have much greater biological activity than LPS alone. LPS in LPS-Hb complexes has greater filterability, is less turbid, and has a lower sedimentation velocity compared to LPS alone, changes consistent with smaller LPS size. To further characterize the physical changes in LPS induced by Hb, we examined the ultrastructure of LPS alone and LPS-Hb complexes by negative staining. We studied two rough (Re) LPS and one smooth LPS, before and after incubation with purified human HbA0. Salmonella minnesota (Re) 595 LPS was visibly turbid, and was composed mostly of ribbon-like and mesh-like structures. In addition, this LPS contained large irregular membrane-like masses, and a few smaller discoidal particles. After incubation with Hb, none of the large mesh-like, ribbon-like or membrane-like structures remained; the resultant LPS particles were discoidal particles of 5-20 nm. Escherichia coli (Re) LPS alone was comprised predominantly of ribbon-like structures; after incubation with Hb, this LPS was comprised of circular membrane-like sheets (predominantly 40-200 nm). Smooth E. coli 055:B5 LPS alone contained heterogeneous particles of 10-100 nm. Most of the largest of these particles were disintegrated by the addition of Hb. The time course of Hb-induced LPS disaggregation was studied with S. minnesota LPS because the great turbidity of this LPS allowed us to monitor its disaggregation spectroscopically. Turbidity was decreased by 50% within approximately 5 h, and was not measurable by 25 h. We propose that the toxic domains of LPS (i.e. lipid A) are cryptic in native LPS aggregates and become exposed upon binding with Hb, thereby resulting in increased biological activity of LPS-Hb complexes compared to LPS alone.

The protein product of the early response gene, Egr-1, is required for macrophage differentiation. Egr-1 mRNA is induced by lipopolysaccharide within minutes in murine peritoneal macrophages through an effect on the rate of transcription of Egr-1. Therefore, we undertook a series of studies to characterize Egr-1 protein induced by LPS and to define enhancer elements in the Egr-1 promoter which mediate the effect of LPS in murine macrophages. Salmonella minnesota Re 595 LPS (10 ng/ml or higher) induced Egr-1 protein of 80 kDa within 45 min in thioglycollate-elicited peritoneal macrophages. The promoter region of Egr-1 contains six serum response elements (SRE) which each contain a core CArG box. CAT activity in LPStreated macrophages transfected with Egr-1-CAT constructs containing the two most upstream 5' SREs was increased 4.7-fold whereas CAT activity in macrophages transfected with constructs lacking these two most upstream SREs was not affected by LPS. LPS induced CAT activity in macrophages transfected with constructs containing tetramers of each of the two most 5' SREs upstream of a heterologous promoter linked to CAT. Mutation of the CArG box abolished the effect of LPS. These findings suggest that the CArG box elements within the two most upstream SREs may mediate the effect of LPS on transcription of Egr-1.

The conservation of opr F gene among 25 clinical Pseudomonas aeruginosa strains and a set of 17 serotype-specific representative strains of the international antigen typing scheme (IATS) was analysed by dot-blotting using five specific oligonucleotide probes. The oligo 1, 2, 3, 4, 5 correspond to five different regions of the opr F gene and hybridized strongly with respectively 88%, 88%, 76%, 94% and 71% of the IATS strains and 88%, 96%, 92%, 88% and 92% of the clinical strains. A parallel study performed with the whole opr F gene showed a lack of specificity of this probe: indeed, the probe hybridized not only with the 42 Pseudomonas aeruginosa strains but also with Escherichia coli and Salmonella minnesota . This study suggests that the gene sequence encoding the protein F is not totally conserved among Pseudomonas aeruginosa strains.

The phenol-chloroform-petroleum ether (PCP) extraction is the method of choice for isolating LPS chemotypes from rough mutant bacteria. However, we have observed that a high percentage of PCP-purified LPS preparations contain trace levels of protein contaminants, and these protein contaminants may exhibit endotoxin-like activity. To obtain protein-free rough LPS, a modified phenol-water extraction procedure was developed for use as a final step to follow PCP extraction. Using this procedure, Salmonella minnesota Ra, Rc, Rd and Re LPS were recovered in yields of 82-100% from the original PCP-extracted preparations. Yields were determined by recovery of KDO and supported by analysis of silver stained SDS-PAGE gels. Although the original PCP-purified chemotypes were contaminated by major 41, 38.5, 29.5, 23, 17 and 14 kDa proteins (detected by Western blotting and gold staining), the repurified LPS contained no detectable protein. The repurified LPS retained all of its original bioactivity as defined by the ability to stimulate normal C3H/OuJ macrophages to secrete TNF. The importance of utilizing repurified LPS in biologic studies was illustrated by the loss of > 99% of activity when repurified LPS was used to stimulate TNF secretion by LPS 'hyporesponsive' C3H/HeJ macrophages.

Structural investigations have shown that lipid A constitutes the active principle of lipopoly saccharide (LPS, endotoxin), a complex amphipathic molecule located on the cell surface of Gram-negative bacteria.1 Lipid A elicits not only the typical endotoxic reactions such as fever and lethal shock but also adjuvant, antitumor and other beneficial effects.1 As a result, there has been a great deal of interest in the synthesis of lipid A derivatives possessing low toxicity.1,2

Free GSL molecules are poorly immunogenic using conventional immunization procedures (1,2). Although immunogenicity has been increased by immunizing with purified glycolipids coated onto the surface of Salmonella minnesota organisms (3,4), this has not always been successful (5). Theoretically, one can take advantage of GSLs absorbed on silica as occurs in thin-layer chromatography (t. l. c.) separation (6), and then to use the silica-absorbed GSL spots scraped from the plate as immunogen. This proposition is based on the principle that aggregation and/or presentation on solid surfaces often leads to increased immune responses (7), and is supported by the demonstration that silica enhanced the response to poorly immunogenic proteins in an in vitro system (8).

The patterns of susceptibility to hydrophobic and hydrophilic drugs and the uptake of the fluorescent probe N-phenyl-naphthylamine in Brucella spp., Haemophilus influenzae, Escherichia coli, and deep rough Salmonella minnesota mutants were compared. The results show that the outer membranes of smooth and naturally rough Brucella spp. do not represent barriers to hydrophobic permeants and that this absence of a barrier relates at least in part to the properties of Brucella lipopolysaccharide.

Endotoxin is one of the principal mediators of Gram-negative septic shock. Pretreatment with monophosphoryl lipid A, a hydrolyzed derivative of endotoxin from Salmonella minnesota R595, induces endotoxin tolerance and nonspecific resistance to infection in experimental animals. The present clinical trial was undertaken to test the response to monophosphoryl lipid A in humans and the ability of monophosphoryl lipid A to attenuate the response of normal human volunteers to U.S. Reference Ec-5 endotoxin.Prospective, double-blind, randomized, controlled trial.Clinical research center.Forty-four healthy volunteers.In part 1 of the study, 29 volunteers were randomized in varying ratios to receive vehicle control or monophosphoryl lipid A intravenously in a double-blind dose escalation trial. In part 2 of the study, 12 volunteers were randomized to receive either monophosphoryl lipid A (20 micrograms/kg) or vehicle control and, 24 hrs later, all 12 volunteers were challenged with U.S. Reference Ec-5 endotoxin (20 units/kg intravenous, bolus injection). Systemic response to endotoxin challenge was evaluated and compared between the monophosphoryl lipid A and vehicle control-pretreated subjects.In part 1 of the study, subjective effects and increases in cytokine levels were not observed until a dose of 10 micrograms/kg of monophosphoryl lipid A was administered. Six volunteers receiving a maximum dose of 20 micrograms/kg experienced mild-to-moderate symptoms that did not require therapy. Moderate increases in temperature, heart rate, and tumor necrosis factor (TNF)-alpha, interleukin (IL)-6, and IL-8 release were observed. IL-1 alpha and IL-1 beta were not detected but a significant increase in IL-1 receptor antagonist was observed. In part 2 of the study, monophosphoryl lipid A pretreatment reduced the number of volunteers who experienced one or more subjective complaints after endotoxin administration (3/6 vs. 6/6; p = .09). The febrile response and tachycardic response to endotoxin were significantly reduced by pretreatment with monophosphoryl lipid A. Monophosphoryl lipid A-pretreated volunteers demonstrated significantly reduced concentrations of TNF-alpha after endotoxin challenge, as compared with subjects treated with vehicle control (84 +/- 76 vs. 244 +/- 128 pg/mL; p.05). IL-6 concentrations (100 +/- 91 vs. 268 +/- 171 pg/ml; p.05) and IL-8 concentrations (136 +/- 86 vs. 632 +/- 323 pg/mL; p.05) elicited by endotoxin challenge were also significantly reduced by monophosphoryl lipid A pretreatment.Data indicate that monophosphoryl lipid A, in a dose 10,000 times that of endotoxin, used in experimental pyrogenicity trials, is well tolerated in human volunteers. Pretreatment of normal human volunteers with monophosphoryl lipid A attenuated the systemic response to bacterial endotoxin. These data support further clinical testing of monophosphoryl lipid A for the prevention or amelioration of the severe sequelae of sepsis.

FTIR spectroscopy has been used to study the thermotropic phase behaviour of mixed vesicles made up by lipopolysaccharides (LPS) and phospholipids. “Deep rough” LPS-mutants from Salmonella minnesota and deuterated phosphatidylethanolamines (DMPEd54) have been used as model lipids. Membrane-active agents like e.g. the polycationic, highmolecular peptide poly-L-lysin500 (PLL500) were used to influence the polymorphism of the lipid phase and to induce phase separations. Specific LPS/DMPEd54, LPS/LPS, and LPS/Peptide interactions have been studied using the number, the temperature, and the half-widths of the gel to liquid-crystal phase transition detected by the IR-technique.

The increasing threat to the human population posed by new or resurgent infectious diseases, coupled with an alarming rise in the incidence of antibiotic-resistant microbes, has created a tremendous need for new vaccines. A critical element in the development of these new vaccines is the ability, via adjuvants, to potentiate and focus the immune response to the vaccine in beneficial ways so that optimal protection can be achieved. One promising candidate adjuvant in this regard is MPL® immunostimulant, a monophosphoryl lipid A preparation derived from the lipopolysaccharide (LPS) of Salmonella minnesota R595. MPL® is being considered as an adjuvant for a number of human vaccines, and experience to date has shown that it is safe, well tolerated, and able to provide a heightened immune response to coadministered antigens. In this chapter, various topics related to the preparation, formulation, and use of MPL® as an adjuvant will be discussed. In addition, our current knowledge of the mechanisms of action of MPL® will be reviewed.

This Midterm report details new analytical approaches developed for the determination of endotoxin structures at much improved sensitivity and specificity, and an application of those techniques to Coxiella burnetti LPS. Foremost in this methodology is a reducing-end reagent synthesized to provide femtomole (10-15) detection for 0-antigen oligosaccharides. A second development focuses on methodology to extract the details of linkage, branching, and sequence, by periodate oxidation, supercritical fluid chromatography, and mass spectrometry. These techniques combine to provide the elements of a global approach to oligosaccharide structure. The utility of supercritical fluid chromatography for a determination of Lipid-A from Salmonella minnesota has been demonstrated. Finally, the structural features of Lipid-A from Coxiella burnetii is described using a combination of these newly developed analytical techniques. Keywords: Lipopolysaccharide; Immunogen; Coxiella burnetii.

Previously we showed that Salmonella minnesota Re and Ra lipopolysaccharides (LPSs) and Escherichia coli K-12 LPS formed three-dimensional crystals, either hexagonal plates or solid columns, when they were precipitated by the addition of 2 volumes of 95% ethanol containing 375 mM MgCl2 and incubated in 70% ethanol containing 250 mM MgCl2, and stored at 4 C for 10 days. Later, Escherichia coli K-12 LPS thus treated was found to form discoid crystals as well as hexagonal plate crystals and solid column crystals. Analysis by electron diffraction of the discoid crystals from the direction perpendicular to the basal plane showed that they consisted cf hexagonal lattices with the a axis of 4.62 A. This result was quite the same as that of the hexagonal plate crystals. Electron micrographs of the edges of the discoid crystals revealed stacked sheets of the hexagonal plate crystals. From these results it was concluded that formation of the discoid crystals results from irregular overlapping of the hexagonal plate crystals.

The lipolysaccharides of two mutants of Salmonella minnesota with known chemical structure were investigated by X-ray methods. Both thickness of the lipopolysaccharide layer and arrangement of the molecular components could be estimated. Using these informations a space-filling model of the structure was built.

The ability of rat serum to inactivate endotoxin (LPS) was assessed with the aid of the limulus amebocyte lysate assay. Following the addition of various amounts of endotoxin to normal serum the mixture was incubated for 1 hr at 37 degrees C and the residual endotoxin activity determined. One milliliter of rat serum inactivated between 5 and 10 micrograms Escherichia coli LPS per hour. Heating serum for 45 min at 56 degrees C resulted in loss of 80-90% of the LPS inhibitor (LPSI) activity. Serum from cobra venom factor (CVF)-treated rats inactivated between 0.5 and 2.5 micrograms LPS/ml serum. Serum from tolerant rats, even after heating for 45 min at 56 degrees C, inactivates between 10 and 15 micrograms LPS/ml serum/hr; decomplemented tolerant rat serum neutralizes between 5 and 10 micrograms LPS/ml serum/hr. Clearly, the tolerant rat has large quantities of LPSI activity, which does not appear to be complement. The inhibitor found in tolerant rat serum is not species specific since it inactivates Salmonella minnesota and Salmonella typhimurium endotoxins to the same degree and in the same amount as E. coli endotoxin, the agent used to induce tolerance. Both heating serum (56 degrees C) and lead acetate reduce LPSI activity.

It is not clear whether baboons develop fever in response to endotoxin or other pyrogens. We injected various pyrogens intravenously in 12 unrestrained baboons (Papio ursinus) and measured their body temperature using intra-abdominal radio-telemeters. Serum iron concentration was also measured. The baboons developed fever after injection of killed Staphylococcus aureus (5 × 107 organisms/kg). No significant fever was measured after injection of lipopolysaccharide (Salmonella typhosa) (0.1, 8, 40, and 100 μg/kg), bovine serum albumin (4 mg/kg), killed Salmonella minnesota (5 × 107 organisms/kg), and killed Salmonella typhi (5 × 107 organisms/kg). A significant decrease in serum iron concentration was found only after injection of S. aureus and lipopolysaccharide, 100 μg/kg. The phagocytic synthesis of interleukin-1 following pyrogen stimulation in baboons and some other primates appears to differ from that in man and in nonprimates.

An IgG1 monoclonal antibody, Sulph I, reacting with sulphatide (3′-sulphogalactosylceramide), was produced by immunizing Balb/c mice with that glycolipid coated on Salmonella minnesota bacterial membrane. Radioimmunodetection of the binding of the monoclonal antibody to structurally related glycolipids adsorbed to microtitre plates or chromatographed on thin-layer plates was used to determine its binding epitope. The antibody showed similar binding avidity to three sulphated glycolipids: sulphatide, sulpholactosylceramide and seminolipid. Lysosulphatide did bind the antibody, but, compared with sulphatide, 30 times more antigen was needed for half-maximal binding. Bis(sulphogangliotriosyl)ceramide and bis-sulphogangliotetraosylceramide did not bind the antibody. These results suggest that terminal galactose-3-O-sulphate and part of the hydrophobic region of the glycolipid are recognized by the Sulph I antibody.

Spectroscopic data (1H- and 13C-N.m.r.) indicate that the tetrasaccharide αdOclAp(2→4)αdOclAp(2→6)βGIcNp(1→6)GlcN is formed upon hydrazinolysis of the lipopolysaccharide from Salmonella minnesota Re 595.

The specificity of monoclonal antibodies directed against the Salmonella minnesota R595 lipid A and the structural requirements of lipid A epitopes were studied with chemically synthesized lipid A analogues by enzyme-linked immunosorbent assay and its inhibition test. The results suggest that lipid A has specific and common epitopes, in which the specificities are derived from the chemical and conformational structures of the backbone and/or acyl groups.

Smooth strains of Salmonella typhimurium and S. minnesota, and chemotypes Ra, Rb, and Rc, which are deficient in lipopolysaccharide components of the somatic side chains and outer core region, grow normally on nutrient agar and nutrient broth up to 45 degrees C. However, most mutants with defects in the heptose region of the LPS (chemotypes Rd2 and Re) do not grow on this medium at 42 degrees C or above; a few grow at 42 degrees C but not at 45 degrees C. In liquid medium (nutrient broth, or phosphate minimal medium), growth, measured as turbidity or as colony-forming units, stops 60 to 90 min after shift from 30 to 42 degrees C; DNA and protein synthesis cease at the same time. Growth does not reoccur at 42 degrees C; protein synthesis and growth reinitiate upon shift to 30 or 37 degrees C. Growth cessation does not alter cell morphology in the phase-contrast microscope. Growth of heptose-deficient strains at 42 degrees C in nutrient broth is restored by MgCl2 (0.5 mM), NaCl (50 mM), or sucrose (100 mM). Sensitivity to smooth-specific and rough-specific phages, and analysis of LPS composition, indicate that heptose-deficient mutants grown at temperatures from 30 to 45 degrees C, and in the presence or absence of high salt, do not contain heptose or O-specific sugars in their LPS.

Benzyl-3- O -benzyl-2-benzyloxycarbonylamino-6- O -[2-benzyloxycarbonyl-amino-2-deoxy-3,4- O -(tetraisopropyldisiloxane-1,3-diyl)- β- d -glucopyranosyl]-2-deoxy-α- d -glucopyranoside was coupled with methyl (4,5,7,8-tetra- O -acetyl-3-deoxy-α- d - manno -2-octulopyranosyl bromide)onate ( 13 ) to yield the α-glycosidically linked trisaccharide. After deacetylation and selective introduction of a second 7′,8′- O -tetraisopropyldisiloxane group, a further glycosidation reaction with 13 led regioselectively to the tetrasaccharide benzyl O -[methyl (4,5,7,8-tetra- O -acetyl-3-deoxy-α- d - manno -2-octulopyranosyl)onate]-(2→4)- O -{methyl [3-deoxy-7,8- O -(tetraisopropyldisiloxane-1,3-diyl)-α- d - manno -2-octulopyranosyl]-onate}-(2→6)- O - [2-benzyloxycarbonylamino-2-deoxy-3,4- O -(tetraisopropyldisiloxane-1,3-diyl)-β- d -glucopyranosyl]- (1→6)-3- O -benzyl-2-benzyloxycarbonyl-amino-2-deoxy-α- d -glucopyranoside. A series of deblocking steps gave O -(3-deoxy-α- d - manno -2-octulopyranosylonic acid)-(2→4)- O -(3-deoxy-α- d - manno -2-octulopyranosylonic acid)- (2→6)- O -(2-amino-2-deoxy-β- d -glucopyranosyl)-(1→6)-2-amino-2-deoxy- d -glucopyranose which was identical with a tetrasaccharide that had been isolated by hydrazinolysis of the lipopolysaccharide from Salmonella minnesota R 595. Hence, synthetic proof is provided for the linkages in this part of the inner core region of lipopolysaccharides.

Summary In the present work, the capacity of Lyme disease (LD) Spirochetes to spontaneously adhere to rat lymphocytes has been evaluated. Borrelia organisms adhere to thymus, spleen, Payer's patches and peripheral blood lymphocytes in a higher frequency than that observed with Salmonella minnesota R345 (Rb) used as reference strain. Employing enriched splenic T and B cell populations, Spirochete binding to B lymphocytes is more elevated than that obtained with Salmonella, while similar percentage of T cells forms rosettes with both microorganisms. Taken together, these findings provide evidence for a novel mode of interaction between LD Spirochetes and host immune system.

Zusammenfassung The tetrasaccharide α-KDO-(2-4)-α-KDO-(2-6)-β- d -GlcN(1-6)- d -GlcN 18 has been synthesized in a sequencial oligosaccharide synthesis. This compound is identical with the degradation product which has been isolated from a R-mutant of Salmonella minnesota . Thus, the kind of linkage of KDO to each other with the lipid A has been proved in a systematic way.

An enzyme-linked immunosorbent assay was developed which allowed the measurement of rabbit immunoglobulin G antibodies directed to Salmonella minnesota Re glycolipid. Efficient adsorption of the antigen to polystyrene could only be effected provided it had been previously dialyzed against 0.2 M EDTA (pH 7.0) and subsequently treated with 0.2% sodium deoxycholate (15 min at 56 degrees C) in 0.05 M diethanolamine buffer (pH 9.6). The method is by far more sensitive than quantitative precipitation in the determination of IgG antibodies. Inhibition by glycolipid of binding S. minnesota Re antibodies to immobilized glycolipid attests to the specificity of the assay and permits the detection of as little as 100 ng of glycolipid.

The role of the lipopolysaccharide (LPS) structure in the recipient cell during bacterial conjugation was studied using a series of well-defined LPS mutations in Salmonella minnesota. The plasmids Flac (IncFI) and R1drd19 (IncFII) transferred at a high frequency to the smooth S218 parent strain and the rough LPS mutants. However, R64drd11 (IncIα) transferred poorly to the LPS mutants compared with transfer to the smooth LPS parent strain. The decrease in R64drd11 transfer frequency correlated with the extent of the defect in LPS structure, suggesting that intact LPS on the recipient is a major requirement for R64drd11 mating. Transfer of the P-group plasmid, RK2, was not affected by changes in LPS structure. These results show that plasmids use different cell surface structures during conjugation, and that LPS is particularly important for R64drd11 transfer.

The bactericidal effect of 25 serum samples from 23 patients with chronic lymphocytic leukaemia (CLL) against the Salmonella minnesota S form and Re mutant has been studied. No killing of the S form was observed in any of the sera tested, whereas a normal bactericidal effect was found against the Re mutant (group 1) in 17/25 sera of CLL patients. By contrast, in 8 serum samples from 6 CLL patients no killing or a markedly delayed killing of the Re strain was observed (group 2). Significant differences in the complement levels were found between the sera of group 1 and group 2. In group 1 mostly normal or elevated complement levels were observed, while in group 2 levels of C4 and C2 were found to be strongly decreased. A significant correlation was found between the bactericidal effect on the one hand and the C4 and C2 levels on the other.

The clinical toxicity of acetic acid-treated « Salmonella minnesota » R595 (Re) organisms was evaluated in 24 cancer patients. Bacteria were injected i.v. four times at increasing doses for a total of 6.5 μg. This therapeutic regimen was free of major side effects (one patient had fever higher than 38 °C and 10 patients complained of pruritus). Furthermore, this bacterial preparation which possesses a more exposed lipid A on its surface, exhibited immunomodulating capacities in that it normalized the inverted T helper/T suppressor ratio and enhanced natural killer activity in tumor patients. The mechanisms of the lower toxicity and immunomodulating activities of these bacteria compared to other lipid A preparations are discussed.

SUMMARY: Isolated lipopolysaccharide from a rough mutant of Salmonella minnesota was shown to act as a receptor for Felix 0-1 bacteriophage. The isolated receptor structure could be dissociated by sodium deoxycholate with subsequent loss of receptor activity. When the deoxycholate was removed by dialysis the receptor activity was restored. Ultrasonic treatment destroyed the receptor activity and electron micrographs indicated that the ultrasonic treatment had altered the structure of the lipopolysaccharide. The experiments suggest that the inactivation of phages can be used as a test of the biological activity of a lipopolysaccharide. The interaction between the 0-1 phage and the receptor structure was also studied with the aid of electron microscopy.

Lipid A preparations from the lipopolysaccharides of four Salmonella minnesota R mutants and one Escherichia coli 0100 R mutant were assayed as soluble complexes with bovine serum albumin for lethality in mice, pyrogenicity in rabbits and complement inactivation. Although generally less active than endotoxic lipopolysaccharides, these lipid · albumin complexes nevertheless exhibited strong biological activity. These results indicate that lipid A contains the endotoxic principle of bacterial lipopolysaccharides.

Based on chemical alterations in the cell surface of normal cells following transformation and the fixation of bacterial glycolipid mR595 by normal, transformed and trypsinized cells, theoretical models for the insertion of glycolipid mR595 in the lipid bilayer of normal, trypsinized and transformed cells are presented.

About 30 R mutants derived from Salmonella minnesota and Salmonella ruiru and the corresponding wild-type strains were analysed comparatively for the presence of enzymes catalyzing the synthesis of UDP-glucose, UDP-galactose, UDP-N-acetylglucosamine and UDP-N-acetylgalactosamine.

Effective serum-mediated killing of sensitive gram-negative bacteria requires all the complement components. In the preimmune phase the antibody-independent interaction of the first component of complement, C1, with the bacteria might be especially important. Electron microscopic studies showed that the C1 subcomponent C1q binds only to the serum-sensitive R form of Salmonella minnesota and not to the serum-resistant S form.

From heptose transferase-less Re mutants of Salmonella minnesota and Shigella sonnei, a mixture of nucleotide-linked heptoses was isolated. After paper chromatography in different solvent systems, ADP derivatives of D-glycero-D-mannoheptose and L-glycero-D-mannoheptose could be isolated in pure form. The structure of ADP-L-glycero-D-mannoheptose was verified by analytical methods and by transformation of ADP-D-glycero-D-mannoheptose with ADP-D-glycero-D-mannoheptose-6-epimerase.