Zwitterionic Phosphodiester-Substituted Neoglycoconjugates as Ligands for Antibodies and Acute Phase Proteins.

Zwitterionic modifications of glycans, such as phosphorylcholine and phosphoethanolamine, are known from a range of prokaryotic and eukaryotic species and are recognized by mammalian antibodies and pentraxins; however, defined saccharide ligands modified with these zwitterionic moieties for high-throughput studies are lacking. In this study, we prepared and tested example mono- and disaccharides 6-substituted with either phosphorylcholine or phosphoethanolamine as bovine serum albumin neoglycoconjugates or printed in a microarray format for subsequent assessment of their binding to lectins, pentraxins, and antibodies. C-Reactive protein and anti-phosphorylcholine antibodies bound specifically to ligands with phosphorylcholine, but recognition by concanavalin A was abolished or decreased as compared with that to the corresponding nonzwitterionic compounds. Furthermore, in array format, the phosphorylcholine-modified ligands were recognized by IgG and IgM in sera of either non-infected or nematode-infected dogs and pigs. Thereby, these new compounds are defined ligands which allow the assessment of glycan-bound phosphorylcholine as a target of both the innate and adaptive immune systems in mammals.

Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Autacoid local injury antagonist amides (ALIAmides) are a family of endogenous bioactive acyl ethanolamides that include the renowned palmitoyl ethanolamide (PEA), oleoyl ethanolamide (OEA), and stearoyl ethanolamide (SEA), and that are involved in several biologic processes such as nociception, lipid metabolism, and inflammation. The role of ALIAmides in the control of inflammatory processes has recently gained much attention and prompted the use of these molecules or their analogs, and the pharmacologic manipulation of their endogenous levels, as plausible therapeutic strategies in the treatment of several chronic inflammatory conditions. Since chronic inflammation is mainly driven by cells of adaptive immunity, particularly T lymphocytes, we aimed at investigating whether such bioactive lipids could directly modulate T-cell responses. We found that OEA, PEA, and eicosatrienoyl ethanolamide (ETEA) could directly inhibit both T-cell responses by reducing their production of TNF-α and IFN-γ from CD8 T cells and TNF-α, IFN-γ and IL-17 from CD4 T cells. Furthermore, neither SEA nor docosatrienoyl ethanolamide (DTEA) could affect cytokine production from both T cell subsets. Interestingly, unlike OEA and ETEA, PEA was also able to enhance de novo generation of forkhead box P3 (FoxP3)-expressing regulatory T cells from CD4-naive T cells. Our findings show for the first time that specific ALIAmides can directly affect different T-cell subsets, and provide proof of their anti-inflammatory role in chronic inflammation, ultimately suggesting that these bioactive lipids could offer novel tools for the management of T-cell dependent chronic inflammatory diseases.-Chiurchiù, V., Leuti, A., Smoum, R., Mechoulam, R., Maccarrone, M. Bioactive lipids ALIAmides differentially modulate inflammatory responses of distinct subsets of primary human T lymphocytes.

Cutaneous Delayed-Type Hypersensitivity to Surfactants.

BACKGROUND: Repeated and prolonged use of surfactants can cause irritant as well as allergic contact dermatitis. OBJECTIVE: This study reports the frequency of positive patch test results to surfactants tested on the North American Contact Dermatitis Group screening series including cocamidopropyl betaine (CAPB), amidoamine (AA), dimethylaminopropylamine (DMAPA), oleamidopropyl dimethylamine (OPD), and cocamide diethanolamide (CDEA), and correlations of positive reactions between CAPB and the other surfactants. METHODS: This was a retrospective analysis of 10 877 patients patch tested between 2009 and 2014 to the surfactants CAPB, AA, DMAPA, OPD, and CDEA. Frequencies of positive reactions to these surfactants were calculated, and trends of reactivity between the surfactants analyzed. CONCLUSIONS: The OPD had the highest rate of positive patch reactions (2.3%) followed by DMAPA (1.7%), and CAPB (1.4%). The AA and CDEA had the lowest rate of positive reactions (0.8%). There was a high degree of overlap in positive patch tests between the surfactants. The CDEA was the least likely to coreact with another surfactant.

Roles of fatty acid ethanolamides (FAE) in traumatic and ischemic brain injury.

Ethanolamides of long-chain fatty acids are a class of endogenous lipid mediators generally referred to as N-acylethanolamines (NAEs). NAEs include anti-inflammatory and analgesic palmitoylethanolamide, anorexic oleoylethanolamide, stearoylethanolamide, and the endocannabinoid anandamide. Traumatic brain injury (TBI), associated with a high morbidity and mortality and no specific therapeutic treatment, has become a pressing public health and medical problem. TBI is a complex process evoking systemic immune responses as well as direct local responses in the brain tissues. The direct (primary) damage disrupts the blood-brain barrier (BBB), injures the neurons and initiates a cascade of inflammatory reactions including chemokine production and activation of resident immune cells. The effect of TBI is not restricted to the brain; it can cause multi-organ damage and evoke systemic immune response with cytokine and chemokine production. This facilitates the recruitment of immune cells to the site of injury and progression of the inflammatory reaction. Depending on severity, TBI induces immediate neuropathologic effects that, for the mildest form, may be transient; however, with increasing severity, these injuries cause cumulative neural damage and degeneration. Moreover, TBI leads to increased catabolism of phospholipids, resulting in a series of phospholipid breakdown products, some of which have potent biological activity. Ischemia-reperfusion (I/R) injury resulting from stroke leads to metabolic distress, oxidative stress and neuroinflammation, making it likely that multiple therapeutic intervention strategies may be needed for successful treatment. Current therapeutic strategies for stroke need complimentary neuroprotective treatments to provide a better outcome. Prior studies on NAEs have demonstrated neurotrophic/neuroprotective activities across a broad spectrum of cellular and animal models of neurodegenerative and acute cerebrovascular disorders. The present review will summarize our knowledge of the biological role of these lipid signaling molecules in brain and highlights their therapeutic effect from multipotential actions on neuronal cell death and neuroinflammatory pathways.

Palmitoylethanolamide in CNS health and disease.

The existence of acylethanolamides (AEs) in the mammalian brain has been known for decades. Among AEs, palmitoylethanolamide (PEA) is abundant in the central nervous system (CNS) and conspicuously produced by neurons and glial cells. Antihyperalgesic and neuroprotective properties of PEA have been mainly related to the reduction of neuronal firing and to control of inflammation. Growing evidence suggest that PEA may be neuroprotective during CNS neurodegenerative diseases. Advances in the understanding of the physiology and pharmacology of PEA have potentiated its interest as useful biological tool for disease management. Several rapid non-genomic and delayed genomic mechanisms of action have been identified for PEA as peroxisome proliferator-activated receptor (PPAR)-α dependent. First, an early molecular control, through Ca(+2)-activated intermediate- and/or big-conductance K(+) channels opening, drives to rapid neuronal hyperpolarization. This is reinforced by the increase of the inward Cl(-) currents due to the modulation of the gamma aminobutyric acid A receptor and by the desensitization of the transient receptor potential channel type V1. Moreover, the gene transcription-mediated mechanism sustains the long-term anti-inflammatory effects, by reducing pro-inflammatory enzyme expression and increasing neurosteroid synthesis. Overall, the integration of these different modes of action allows PEA to exert an immediate and prolonged efficacious control in neuron signaling either on inflammatory process or neuronal excitability, maintaining cellular homeostasis. In this review, we will discuss the effect of PEA on metabolism, behavior, inflammation and pain perception, related to the control of central functions and the emerging evidence demonstrating its therapeutic efficacy in several neurodegenerative diseases.

Improving the immunostimulatory potency of diethanolamine-containing lipid A mimics.

Lipid A is the active principal of gram negative bacterial lipopolysaccharide (LPS) in the activation of Toll-like receptor 4 (TLR4). Given the important role TLR4 plays in innate immunity and the development of adaptive immune responses, ligands that can modulate TLR4-mediated signaling have great therapeutic potential. Recently, we have reported a series of monophosphorylated lipid A mimics as potential ligands of TLR4, in which a diethanolamine moiety is employed to replace the reducing end (d-glucosamine). In this paper, we describe the synthesis of two further diethanolamine-containing lipid A mimics, 3 and 4, in an effort to mimic more closely the di-phosphate nature of natural lipid A. Both mimic 3, with an additional phosphate on the diethanolamine acyclic scaffold, and mimic 4, with a terminal carboxylic acid moiety as a phosphate bioisostere, serve to increase the potency of the immunostimulatory response induced, as measured by the induction of the cytokines TNF-α, IL-6, and IL-1ß in the human monocytic cell line THP-1. In addition, mechanistic studies involving the known TLR4 antagonist lipid IVa confirm TLR4 as the target of the diethanolamine-containing lipid A mimics.

Phosphoethanolamine residues on the lipid A moiety of Neisseria gonorrhoeae lipooligosaccharide modulate binding of complement inhibitors and resistance to complement killing.

Loss of phosphoethanolamine (PEA) from the lipid A of gonococcal strain FA19 results in increased sensitivity to killing by the classical pathway of complement. Here we demonstrate that loss of PEA from lipid A diminishes binding of the complement regulatory protein C4b binding protein (C4BP) to the FA19 porin B (PorB), providing a molecular basis to explain the susceptibility of an lptA null strain of FA19 to killing by normal human serum (NHS). Loss of PEA from lipid A in three additional gonococcal strains that expressed diverse PorB molecules also resulted in decreased C4BP binding, increased deposition of C4b, and increased susceptibility to killing by NHS. Complementation of lptA null strains with lptA restored C4BP binding, decreased C4b deposition, and increased resistance to killing by NHS. These effects of lipid A PEA on C4BP binding to gonococcal PorB and serum resistance were simulated when gonococcal PorB was expressed in a meningococcal background. Loss of PEA from lipid A also affected binding of the alternative pathway regulator factor H (fH) to PorB of some strains. For instance, PorB molecules of lptA null mutants of strains 252 and 1291 bound less fH than those of their parent strains when lipooligosaccharide (LOS) was sialylated, whereas PorB molecules of lptA null mutants of strains FA1090 and 273 retained the ability to bind fH when LOS was sialylated. These data indicate that replacement of lipid A with PEA alters binding of C4BP and fH to PorB and contributes to the ability of gonococci to resist complement-mediated killing.

Phosphoethanolamine substitution of lipid A and resistance of Neisseria gonorrhoeae to cationic antimicrobial peptides and complement-mediated killing by normal human serum.

The capacity of Neisseria gonorrhoeae to cause disseminated gonococcal infection requires that such strains resist the bactericidal action of normal human serum. The bactericidal action of normal human serum against N. gonorrhoeae is mediated by the classical complement pathway through an antibody-dependent mechanism. The mechanism(s) by which certain strains of gonococci resist normal human serum is not fully understood, but alterations in lipooligosaccharide structure can affect such resistance. During an investigation of the biological significance of phosphoethanolamine extensions from lipooligosaccharide, we found that phosphoethanolamine substitutions from the heptose II group of the lipooligosaccharide beta-chain did not impact levels of gonococcal (strain FA19) resistance to normal human serum or polymyxin B. However, loss of phosphoethanolamine substitution from the lipid A component of lipooligosaccharide, due to insertional inactivation of lptA, resulted in increased gonococcal susceptibility to polymyxin B, as reported previously for Neisseria meningitidis. In contrast to previous reports with N. meningitidis, loss of phosphoethanolamine attached to lipid A rendered strain FA19 susceptible to complement killing. Serum killing of the lptA mutant occurred through the classical complement pathway. Both serum and polymyxin B resistance as well as phosphoethanolamine decoration of lipid A were restored in the lptA-null mutant by complementation with wild-type lptA. Our results support a role for lipid A phosphoethanolamine substitutions in resistance of this strict human pathogen to innate host defenses.

Sympathomimetic drug allergy: cross-reactivity study by patch test.

INTRODUCTION: Sympathomimetic (alpha-adrenergic) drugs are mainly used because of their vasoconstrictor properties, for nasal congestion, or as mydriatics. Although sympathomimetic drugs are used often, allergic reactions are rare, especially when the drugs are administered systemically. Cross-reactivity may exist among catecholamine derivatives, although reported data on this are contradictory. In this study, we investigate if there is cross-reactivity in patch tests among these drugs. MATERIAL AND METHODS: Patch tests with 10% phenylephrine and 10% pseudoephedrine in petrolatum, and 10% and 20% ephedrine, 10% phenylpropanolamine, 5% fepradinol, 1% methoxamine, and 10% oxymetazoline, all administered in dimethyl sulfoxide (DMSO), were carried out in 14 patients with a history of allergy to any of these drugs. DMSO was used as the negative control. RESULTS: All patients except one (patient number five) showed positive patch-test reactions to at least two different drugs. Nine patients (64.3%) were cross-sensitized to three or more different drugs, and 57.1% of patients were sensitized to four or more sympathomimetic drugs. Patients who experienced generalized rashes caused by orally administered pseudoephedrine had a stronger response and more cross-reactivity with other sympathomimetic drugs in patch tests than those who experienced local contact dermatitis. CONCLUSIONS: We conclude that there is cross-reactivity among the different sympathomimetic drugs tested, especially if the drug is administered systemically.

Structural analysis of the lipopolysaccharide from Neisseria meningitidis strain BZ157 galE: localisation of two phosphoethanolamine residues in the inner core oligosaccharide.

The structure of the phase-variable lipopolysaccharide (LPS) from the group B Neisseria meningitidis strain BZ157 galE was elucidated. The structural basis for the LPS's variation in reactivity with a monoclonal antibody (MAb) B5 that has specificity for the presence of phosphoethanolamine (PEtn) at the 3-position of the distal heptose residue (HepII) was established. The structure of the O-deacylated LPS was deduced by a combination of monosaccharide analyses, nuclear magnetic resonance (NMR) spectroscopy and mass spectrometry. These analyses revealed the presence of a novel inner core oligosaccharide (OS) structure in the MAb B5 reactive (B5+) LPS that contained two PEtn residues simultaneously substituting the 3- and 6-positions of the HepII residue. The determination of this structure has identified a further degree of variability within the inner core OS of meningococcal LPS that could contribute to the interaction of meningococcal strains with their host.

Genetic linkage analysis to identify a gene required for the addition of phosphoethanolamine to meningococcal lipopolysaccharide.

Lipopolysaccharide (LPS) is important for the virulence of Neisseria meningitidis, and is the target of immune responses. We took advantage of a monoclonal antibody (Mab B5) that recognises phosphoethanolamine (PEtn) attached to the inner core of meningococcal LPS to identify genes required for the addition of PEtn to LPS. Insertional mutants that lost Mab B5 reactivity were isolated and characterised, but failed to yield genes directly responsible for PEtn substitution. Subsequent genetic linkage analysis was used to define a region of DNA containing a single intact open reading frame which is sufficient to confer B5 reactivity to a B5 negative meningococcal isolate. The results provide an initial characterisation of the genetic basis of a key, immunodominant epitope of meningococcal LPS.

Cross-reactive polyclonal antibodies to the inner core of lipopolysaccharide from Neisseria meningitidis.

Sera from mice immunized with native or detergent-extracted outer membrane vesicles derived from lipopolysaccharide (LPS) mutant 44/76(Mu-4) of Neisseria meningitidis were analyzed for antibodies to LPS. The carbohydrate portion of 44/76(Mu-4) LPS consists of the complete inner core, Glc beta 1-->4[GlcNAc alpha 1-->2Hep alpha 1-->3]Hep alpha 1-->5KDO[4-->2 alpha KDO]. Immunoblot analysis revealed that some sera contained antibodies to wild-type LPS which has a fully extended carbohydrate chain of immunotype L3,7, as well as to the homologous LPS. Sera reacted only weakly to LPS from 44/76(Mu-3), which lacks the terminal glucose of the inner core. No binding to more truncated LPS was observed. Consequently, the cross-reactive epitopes are expressed mainly by the complete inner core. Dephosphorylation of wild-type LPS abolished antibody binding to LPS in all but one serum. Thus, at least two specificities of cross-reactive antibodies exist: one is dependent on phosphoethanolamine groups in LPS, and one is not. Detection of these cross-reactive antibodies strongly supports the notion that epitopes expressed by meningococcal LPS inner core are also accessible to antibodies when the carbohydrate chain is fully extended. Also, these inner core epitopes are sufficiently immunogenic to induce antibody levels detectable in polyclonal antibody responses. Meningococci can escape being killed by antibodies to LPS that bind only to a specific LPS variant, by altering the carbohydrate chain length. Cross-reactive antibodies may prevent such escape. Therefore, inner core LPS structures may be important antigens in future vaccines against meningococcal disease.

Regulation of calcium homeostasis in activated human neutrophils--potential targets for anti-inflammatory therapeutic strategies.

OBJECTIVES: The objectives of the current study were to: (i) present an integrated model for the restoration of calcium homeostasis in activated human neutrophils based on current knowledge and recent research; and (ii) identify potential targets for the modulation of calcium fluxes in activated neutrophils based on this model and to investigate the effects of intracellular probes which target key processes involved in calcium homeostasis and pro-inflammatory activity in these cells. DESIGN AND SETTING: Laboratory-based experimental research using purified human neutrophils from healthy, adult human volunteers. OUTCOME MEASURES: Calcium metabolism and pro-inflammatory activity of neutrophils. RESULTS: Modulation of calcium fluxes in activated human neutrophils can be achieved by cAMP-dependent upregulation of the activity of the endomembrane Ca(2+)-ATPase which resequesters cytosolic Ca2+. Formoterol, a long-acting beta 2-agonist, elevates intracellular cAMP levels, accelerates Ca2+ restoration in activated neutrophils and downregulates the pro-inflammatory responses of these cells. Alterations in the membrane potential of activated neutrophils may play a role in regulating calcium reuptake into the cells as attenuation of the membrane depolarisation response is associated with accelerated calcium influx. CONCLUSIONS: Modulation of the activity of the endomembrane Ca(2+)-ATPase in human neutrophils represents an important target for anti-inflammatory

Conservation and accessibility of an inner core lipopolysaccharide epitope of Neisseria meningitidis.

We investigated the conservation and antibody accessibility of inner core epitopes of Neisseria meningitidis lipopolysaccharide (LPS) because of their potential as vaccine candidates. An immunoglobulin G3 murine monoclonal antibody (MAb), designated MAb B5, was obtained by immunizing mice with a galE mutant of N. meningitidis H44/76 (B. 15.P1.7,16 immunotype L3). We have shown that MAb B5 can bind to the core LPS of wild-type encapsulated MC58 (B.15.P1.7,16 immunotype L3) organisms in vitro and ex vivo. An inner core structure recognized by MAb B5 is conserved and accessible in 26 of 34 (76%) of group B and 78 of 112 (70%) of groups A, C, W, X, Y, and Z strains. N. meningitidis strains which possess this epitope are immunotypes in which phosphoethanolamine (PEtn) is linked to the 3-position of the beta-chain heptose (HepII) of the inner core. In contrast, N. meningitidis strains lacking reactivity with MAb B5 have an alternative core structure in which PEtn is linked to an exocyclic position (i.e., position 6 or 7) of HepII (immunotypes L2, L4, and L6) or is absent (immunotype L5). We conclude that MAb B5 defines one or more of the major inner core glycoforms of N. meningitidis LPS. These findings support the possibility that immunogens capable of eliciting functional antibodies specific to inner core structures could be the basis of a vaccine against invasive infections caused by N. meningitidis.

Enantioselective immunoaffinity extraction for simultaneous determination of optically active bufuralol and its metabolites in human plasma by HPLC.

A combined method of immunoaffinity extraction with high-performance liquid chromatography has been developed for the enantioselective determination of bufuralol and its metabolites in human plasma. The antibodies having high affinity toward the asymmetric center at the C-1 position of bufuralol and its 1'-oxidized metabolites and low affinity to their antipodes were elicited by immunization of rabbits with immunogens, (1R)- and (1S)-1'-oxobufuralol O-carboxymethyloxime-bovine serum albumin conjugates, respectively. 0.5 ml Of the immunoaffinity adsorbent (7.6 mg.ml-1 for anti-(1S)-antibody and 28.8 mg.ml-1 for anti-(1R)-antibody) prepared by immobilization of an antibody was capable of retaining up to 1 microgram of (R)- and (S)-bufuralol and up to 500 ng of other metabolites. The adsorbates were recovered quantitatively by elution with methanol-10 mM ammonium acetate buffer (pH 5) (95:5, v/v) without any interfering peaks on the high-performance liquid chromatogram. The proposed method was evaluated to be useful for the simultaneous determination of optically active bufuralol and its metabolite in plasma with acceptable recovery and precision.

Diethanolamine-induced occupational asthma, a case report.

BACKGROUND: Amino alcohols are low molecular weight chemicals used widely in industrial processes, often as minor constituents. They have been found to cause allergic contact dermatitis. Marked exposure through airways is uncommon in other than occupational settings where chemicals containing amino alcohols may be heated or vaporized, liberating free amino alcohols into the ambient air. A few cases of asthma and allergic rhinitis have been reported, but the amounts inducing the airway reactions have not been defined. OBJECTIVE: To further characterize ethanolamine-induced asthma and define the concentration inducing the asthmatic reaction, a case of diethanolamine-induced occupational asthma in a patient handling diethanolamine containing cutting fluid is reported. METHODS: Suspicion of work related asthma was raised by symptoms and peak expiratory flow monitorings at work and at home. Specific bronchial provocation tests with the cutting fluid containing DEA and with DEA aerosol at two different concentration below the American Conference of Governmental Industrial Hygienists threshold limit value of DEA (2.0 mg/m3) were done. RESULTS: DEA caused asthmatic airway obstruction at two different concentrations below the ACGIH TLV. A slight dose-response relationship was observed. Specific IgE-antibodies against DEA could not be found. CONCLUSIONS: DEA is able to induce occupational asthma by a sensitization mechanism, the exact pathophysiological mechanism of which is not known.

The influence of the adjuvant Quil A on the epitope specificity of meningococcal lipopolysaccharide anti-carbohydrate antibodies.

Rabbits were immunized with immunotype L3,7,9 phosphoethanolamine (PEA) group containing oligosaccharide-tetanus toxoid conjugates both with and without the addition of the adjuvant Quil A. The epitope specificity of the antibodies present in these antisera was analysed in an immunotype L2 and L3,7,9 specific inhibition ELISA using the homologous and heterologous lipopolysaccharide, oligosaccharide and partial dephosphorylated oligosaccharide as inhibitors. Two groups of antisera could be identified. In one group of antisera, at least two antibody populations are present, namely directed against the PEA group containing determinants on immunotype L3,7,9 lipopolysaccharide and against immunotype L2 specific epitopes in which no PEA group is present. In the second group of antisera, one but probably more antibody populations are detected with a similar specificity towards the conserved epitopes of both immunotypes. In general, immunization with the conjugates only resulted in the induction of antibodies against the PEA group containing epitopes on the L3,7,9 lipopolysaccharide (80%). Antibodies directed against the conserved epitopes of both immunotypes are mainly evoked with the conjugates in combination with the adjuvant Quil A (80%). Although these results suggest that the epitope specificity of the antibodies induced depends on the use of Quil A, the influence of genetic factors cannot be excluded. At the moment it is not known whether the differences in epitope specificities are reflected in biological function of these antibodies. However, the induction of antibodies with clearly different epitope specificities after immunization of different rabbits with the same antigen stresses the importance of this kind of analysis when developing a vaccine based on oligosaccharide-protein conjugates.

Preparation, characterization, and immunogenicity of meningococcal immunotype L2 and L3,7,9 phosphoethanolamine group-containing oligosaccharide-protein conjugates.

A method was developed for the well-defined coupling of phosphoethanolamine group (PEA)- and carboxylic acid group-containing polysaccharides and oligosaccharides to proteins without the need for extensive modification of the carbohydrate antigens. The carboxylic acid group of the terminal 2-keto-3-deoxyoctulosonic acid moiety was utilized to introduce a thiol function in meningococcal immunotype L2 and L3,7,9 lipopolysaccharide-derived oligosaccharides. The thiol group-containing oligosaccharides were subsequently coupled to bromoacetylated proteins. Immunotype L2 and L3,7,9 PEA group-containing oligosaccharide-tetanus toxoid conjugates were prepared, and their immunogenicities were studied in rabbits. Both the immunotype L2 and immunotype L3,7,9 conjugates evoked high immunoglobulin G (IgG) antibody titers after the first booster injection. These conjugates also displayed an ability to induce long-lasting IgG antibody levels which could be detected until 9 months after one booster injection at week 3. The adjuvant Quil A enhanced the immune response to all the conjugates to a minor extent, which is in contrast with reported adjuvant effects of Quil A on these types of antigens in mice. A conjugate prepared from the dephosphorylated L3,7,9 oligosaccharides evoked a significantly lower IgG response than a similar PEA-containing conjugate, and enzyme-linked immunosorbent assay inhibition studies indicated a different epitope specificity. Furthermore, antisera elicited with the complete bacteria contained antibodies directed against PEA-containing epitopes, which stresses the importance of the presence of unmodified PEA groups in meningococcal lipopolysaccharide-derived oligosaccharide-protein conjugates. The procedure developed offers an elegant solution for the specific coupling of meningococcal PEA-containing oligosaccharides to proteins and may therefore be a very useful tool in the development of a vaccine against group B meningococci.

Characterization of antibodies to the glycosyl-phosphatidylinositol membrane anchors of mammalian proteins.

Two polyclonal antisera were raised in rabbits to the phospholipase C-solubilized forms of pig renal dipeptidase (EC 3.4.13.11) and pig aminopeptidase P (EC 3.4.11.9). These antisera were purified and shown to cross-react with other glycosyl-phosphatidylinositol (G-PI)-anchored proteins isolated from pig, human and trypanosomes. The epitopes involved in this cross-reactivity were characterized by Western-blot analysis after mild acid or nitrous acid treatment of the G-PI-anchored proteins and by a competitive e.l.i.s.a. with other G-PI-anchored proteins and individual components of the anchor structure. These studies revealed that the primary epitope for both antisera is the inositol 1.2-(cyclic)monophosphate that is formed on phospholipase C cleavage of the intact G-PI anchor. Other minor epitopes, such as phosphoethanolamine, probably involve side-chain modifications to the core anchor structure that may be species-specific.