Antibiotic susceptibility of attached and free-floating Helicobacter pylori.

Helicobacter pylori are found attached to mucous cells of the human stomach or under the mucous layer. Models mimicking the in vivo situation may be more suitable for H. pylori MIC determinations than traditional agar dilution methods. Megraud et al. (Antimicrobial Agents and Chemotherapy 1991, 35, 869-72) developed a model for measuring the susceptibility of attached and free-floating H. pylori. We have modified this model so that free-floating and attached H. pylori are treated in a more similar manner, before and after incubation with antibiotic, and performed additional controls to ensure H. pylori and tissue culture cells are not detrimentally affected and maintain their viability during the course of the experiment. We found only 10% of plate-grown H. pylori were competent for attachment to HEp-2 cells; however, all progeny of attached bacteria remained adherent. Killing curves were performed using 0, 0.001, 0.01, 0.1 and 1 mg/L amoxycillin, and 0, 0.0025, 0.0075 and 0.01 mg/L clarithromycin. H. pylori divided at concentrations

Treatment of acute otitis media with an antiadhesive oligosaccharide: a randomised, double-blind, placebo-controlled trial.

BACKGROUND: Antiadhesive compounds are promising candidates for prevention or treatment of infections. We have investigated the efficacy of such an agent, 3'-sialyllacto-N-neotetraose (NE-1530), given intranasally for prophylaxis of acute otitis media and for effect on nasopharyngeal carriage of bacteria. METHODS: We did a randomised, double-blind placebo-controlled study at one study site. 507 healthy children were randomly assigned either NE-1530 (n=254) or placebo (253) as intranasal sprays twice daily during 3 months. The children were examined by the study physicians once a month and during illness. Treatment efficacy was estimated from Cox proportional hazards model. A sample of nasopharyngeal secretion was taken at every visit for culture of Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. Adverse events were recorded in study diaries. FINDINGS: At least one event of acute otitis media was diagnosed in 108 (43%) of 254 children in the NE-1530 group and in 86 (34%) of 253 children in the placebo group. The efficacy of treatment was negative, -27% (95% CI -68 to 5; p=0.10). The nasopharyngeal carriage of S pneumoniae, H. influenzae, and M. catarrhalis was not affected by treatment, and the adverse event profiles were almost identical for NE-1530 and placebo. INTERPRETATION: NE-1530 did not have a beneficial effect on the occurrence of acute otitis media or on the nasopharyngeal carriage of bacteria in children.

Lewis antigen expression and stability in Helicobacter pylori isolated from serial gastric biopsies.

The expression of Lewis antigens by the gastric pathogen Helicobacter pylori in serial biopsy isolates was investigated to assess antigen distribution and stability. A total of 26 asymptomatic subjects were given various doses of 3' sialyllactose for up to 56 days. Gastric biopsies were performed during the dosing period, as well as 30 days after dosing, which provided 127 H. pylori isolates that were examined by use of ELISA and immunoblot. A large proportion of subjects (14/26) yielded sequential H. pylori isolates, which appeared genetically identical but had variable Lewis antigen expression. The proportion of subjects with H. pylori isolates not expressing any Lewis antigens was greater than that previously reported (10/26). Thus, the expression of the Lewis antigens by H. pylori does not appear to be a requirement for colonization, whereas the antigen expression after human infection is more variable than the previously reported rate observed with in vitro cultures.

Treatment of Helicobacter pylori infection in rhesus monkeys using a novel antiadhesion compound.

BACKGROUND & AIMS: Helicobacter pylori can be eradicated by administration of antimicrobials, but resistant strains have emerged, and there is a need for novel therapeutic approaches against this infection. This study aimed to determine the safety and efficacy of 3'-sialyllactose sodium salt (3'SL), an oligosaccharide that occurs naturally in human and bovine milk and that can inhibit the adhesion of H. pylori to human epithelial cells in vitro. METHODS: Twelve H. pylori-positive rhesus monkeys were given 3'SL, either alone (regimens 1 and 2; n = 6) or in combination with omeprazole (regimen 3; n = 4), or bismuth subsalicylate (regimen 4; n = 6). Videogastroscopies were performed before, during, and after treatment, and gastric biopsy specimens were obtained for quantitative cultures and histology. The H. pylori strains colonizing the animals were genotyped. RESULTS: After regimen 1 or 2, 2 of 6 animals were cured permanently, and a third animal was transiently cleared. The 3 other animals remained persistently colonized and did not respond to regimen 3. Regimen 4 resulted in transient decreases in colony counts in 3 of 6 other animals. Gastritis was suppressed only in the 2 animals who became persistently H. pylori negative. There was no apparent relation between 3'SL efficacy and any of the H. pylori tested genotypes. No side effects were observed in any of the animals receiving 3'SL. CONCLUSIONS: Antiadhesive therapy is safe; it can cure or decrease H. pylori colonization in some rhesus monkeys, but the addition of a proton pump inhibitor or bismuth subsalicylate does not increase cure rate.

Analysis of sialyllactoses in blood and urine by high-performance liquid chromatography.

A sensitive and highly selective high-performance liquid chromatography (HPLC)-based method has been developed for the analysis of oligosaccharides in biological fluids. In this method, a sample of biological fluid, such as blood serum or urine, is filtered through a 10,000 molecular weight cutoff filter cartridge to remove large molecules such as proteins and lipids. The carbohydrates in the filtrate are then derivatized with 1-phenyl-3-methyl-5-pyrazolone (PMP) as described previously [Anal. Biochem. 180, 351-357, (1989)]. The derivatized carbohydrates are separated by reverse-phase HPLC and monitored by UV absorbance at 245 nm. Quantitative analysis of the carbohydrates can be achieved based on their integration values relative to a standard calibration curve. Since neutral and acidic carbohydrates can be separated by using Dowex 1-X8 anion exchange resin, this method can be used specifically to analyze neutral, acidic, and total carbohydrates in the biological fluids. Because PMP specifically reacts with reducing aldoses, interference from noncarbohydrate components present in the biological fluids is essentially eliminated. This method has proven to be highly sensitive, requiring as little as 5 pmol of analyte for reliable analysis. It has also been used successfully for pharmacokinetic analysis of carbohydrate drugs in human blood and urine samples.

Neutral oligosaccharide content of preterm human milk.

Human milk oligosaccharides are known to play a role in protection against certain infectious diseases. Previous reports indicate that the content of human milk oligosaccharides varies widely among individuals at term but such information on preterm milk is lacking. After removal of the fat, protein and most of the lactose from non-pooled human milk samples, a total neutral oligosaccharide fraction was isolated by ion-exchange chromatography followed by gel filtration. A Dionex high-performance anion-exchange chromatography system equipped with a pulsed electrometric detector was then employed to measure the levels of ten neutral oligosaccharides in the individual milk samples. Twenty-three milk samples from thirteen mothers who delivered at a mean gestational age of 29.5 (SD 3.1) weeks were collected between days 0 and 33 of lactation, and compared with three samples of term milk from two mothers. The ranges of the total and individual levels of the ten neutral oligosaccharides in preterm milk were similar to those in term milk. Further, as previously described in term milk, preterm milk exhibited a quantitative individual variation. This variation was independent of the gestational age, day of lactation, and postconceptional age. In conclusion, levels of ten neutral oligosaccharides did not differ between preterm and term human milk.

Weak affinity chromatography of small saccharides with immobilised wheat germ agglutinin and its application to monitoring of carbohydrate transferase activity.

In this work we have evaluated the potential to use wheat germ agglutinin (WGA) for weak affinity chromatography (WAC) of N-acetyl derivatives of mono-, di-, tri- and tetrasaccharides. WGA was used as a ligand in a high performance liquid affinity chromatography (HPLAC) system. Isocratic affinity chromatography was conducted where similar N-acetyl saccharides were separated according to their binding strength to WGA. Affinities are weak and lie typically in the mM range. For example, for 3'sialyllactose, the dissociation constant (Kd) was found to be 2.4 mM at 8 degrees C. It was interesting to note that the WGA-HPLC column can distinguish between the anomeric forms of N-acetylglucosamine. Weak affinity chromatography with immobilised WGA was used in an enzyme assay to detect the activity of GlcNAc-transferases.

Heterodimer SRP9/14 is an integral part of the neural BC200 RNP in primate brain.

BC200 RNA is a brain-specific, small non-messenger RNA with a somatodendritic localization in primate neurons and a constituent of a ribonucleoprotein (RNP) complex. The primary and secondary structure of the 5' domain of BC200 RNA resembles that of the Alu domain of 7SL RNA, which is an integral part of the signal recognition particle (SRP). This would predict that similar proteins bind to this defined domain of both RNA species in vitro and in vivo. The data presented in this paper reveal that a protein that binds BC200 RNA in vivo is immunoreactive with antibodies against SRP9. This further supports the notion that the 5' domain of the BC200 RNA can fold into structures similar to the SRP Alu domain and, as a result, bind identical or similar proteins in vivo. The SRP9 protein binds only as dimer with SRP14 protein to the Alu domain of 7SL RNA to form a subdomain that, in SRP, is functional in translation arrest. Therefore, our data also indicate that the neuronal BC200 RNP is a candidate for regulating decentralized protein biosynthesis in dendrites, possibly with a mechanism that resembles translation arrest of the SRP.

Adherence of Streptococcus pneumoniae to respiratory epithelial cells is inhibited by sialylated oligosaccharides.

To study carbohydrate-mediated adherence of Streptococcus pneumoniae to the human airway, we measured binding of live S. pneumoniae organisms to a cultured cell line derived from the lining of the conjunctiva and to primary monolayers of human bronchial epithelial cells in the presence and absence of oligosaccharide inhibitors. Both encapsulated and nonencapsulated strains of S. pneumoniae grown to mid-logarithmic phase in suspension culture adhered to cultured primary respiratory epithelial cells and the conjunctival cell line. Adherence of nine clinically prevalent S. pneumoniae capsular types studied was inhibited preferentially by sialylated oligosaccharides that terminate with the disaccharide NeuAc alpha2-3(or 6)Galbeta1. Adherence of some strains also was weakly inhibited by oligosaccharides that terminate with lactosamine (Galbeta1-4GlcNAcbeta1). When sialylated oligosaccharides were covalently coupled to human serum albumin at a density of approximately 20 oligosaccharides per molecule of protein, the molar inhibitory potency of the oligosaccharide inhibitor was enhanced 500-fold. The above-mentioned experiments reveal a previously unreported dependence upon sialylated carbohydrate ligands for adherence of S. pneumoniae to human upper airway epithelial cells. Enhanced inhibitory potencies of polyvalent over monovalent forms of oligosaccharide inhibitors of adherence suggest that the putative adhesin(s) that recognizes the structure NeuAc alpha2-3(or 6)Galbeta1 is arranged on the bacterial surface in such a manner that it may be cross-linked by oligosaccharides covalently linked to human serum albumin.

Intravenous infusion of Galalpha1-3Gal oligosaccharides in baboons delays hyperacute rejection of porcine heart xenografts.

BACKGROUND: Hyperacute rejection (HAR) of pig-to-primate discordant xenografts is caused by the deposition of preexisting natural antibodies that recognize Galalpha1-3Gal (alphaGal)-terminating oligosaccharides on glycoproteins and glycolipids, followed by complement-mediated lysis of the graft's endothelium. In vitro, these natural xenoantibodies can be blocked by alphaGal-containing oligosaccharides. We undertook in vivo pig-to-baboon cardiac xenotransplantation experiments to evaluate free oligosaccharides as inhibitors of HAR. METHODS: Initial 15-min intravenous infusions of alphaGal-oligosaccharides into baboons were used to measure pharmacokinetic parameters, and to assess the extent of neutralization of anti-alphaGal antibody activity. AlphaGal trisaccharide (Galalpha1-3Galbeta1-4GlcNAc) or pentasaccharide (Galalpha1-3Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc ) was administered at 0.5 mmol/kg into baboons. Next, two baboons that received porcine heterotopic heart xenografts were continuously infused with alphaGal pentasaccharide for 4-5 hr, maintaining the serum oligosaccharide concentration in the millimolar range. RESULTS: Pharmacokinetic analysis indicated that the oligosaccharides were rapidly cleared from the blood, with a serum half-life of 50 min. In the period during which blood oligosaccharide concentration was above 1 mM, as determined by high-pressure liquid chromatography, the serum cytotoxic activity against porcine cells was completely abolished. HAR of the xenograft was inhibited during the infusions, although there was some histological and immunohistological evidence of antibody-mediated injury on biopsies taken at the end of this period. CONCLUSIONS: Intravenous alphaGal oligosaccharides, by inhibiting anti-alphaGal antibody activity, delay but do not abolish the onset of HAR.

Oligosaccharides interfere with the establishment and progression of experimental pneumococcal pneumonia.

Oligosaccharides that block the adherence of bacteria to epithelial cells in vitro--lacto-N-neotetraose (LNnT) and its alpha2-3- and alpha2-6-sialylated derivatives--were tested for their abilities to attenuate the course of pneumococcal pneumonia and to prevent colonization of the nasopharynx in animal models. Intratracheal administration of these agents concurrently with bacteria dramatically decreased pneumococcal load in the lungs of rabbits and conferred protection from bacteremia. The oligosaccharides ameliorated pneumonia and bacteremia when given therapeutically 24 h after infection was established. When administered intranasally, neoglycoconjugates of the active oligosaccharides prevented colonization of the nasopharynx of infant rats. In addition to in vitro anti-adherence properties, LNnT acted directly on cultured lung epithelial cell lines to induce changes such that pneumococcal adherence was prevented for prolonged periods. These activities encourage continued development of oligosaccharides as a class of potentially preventive and therapeutic agents for infectious diseases.

Inhibition of Helicobacter pylori binding to gastrointestinal epithelial cells by sialic acid-containing oligosaccharides.

Helicobacterpylori, the ulcer pathogen residing in the human stomach, binds to epithelial cells of the gastric antrum. We have examined binding of 13 bacterial isolates to epithelial cell lines by use of a sensitive microtiter plate method in which measurement of bacterial urease activity provides the means for quantitation of bound organisms. Several established human gastrointestinal carcinoma cell lines grown as monolayers were compared for suitability in these assays, and the duodenum-derived cell line HuTu-80 was selected for testing bacterial binding inhibitors. When bacteria are pretreated with oligosaccharides, glycoproteins, and glycolipids, a complex picture of bacterial-epithelial adherence specificities emerges. Among the monovalent inhibitors tested, 3'-sialyllactose (NeuAc alpha2-3Gal beta1-4Glc; 3'SL) was the most active oligosaccharide, inhibiting adherence for recent clinical isolates of H. pylori with a millimolar 50% inhibitory concentration (IC50). Its alpha2-6 isomer (6'SL) was less active. Most of the recent clinical isolates examined were inhibited by sialyllactose, whereas long-passaged isolates were insensitive. Among the long-passaged bacterial strains whose binding was not inhibited by 3'SL was the strain ATCC 43504, also known as NCTC 11637 and CCUG 17874, in which the proposed sialyllactose adhesin was recently reported to lack surface expression (P. G. O'Toole, L. Janzon, P. Doig, J. Huang, M. Kostrzynska, and T. H. Trust, J. Bacteriol. 177:6049-6057, 1995). Pretreatment of the epithelial monolayer with neuraminidase reduced the extent of binding by those bacteria that are sensitive to inhibition by 3'SL. Other potent inhibitors of bacterial binding are the glycoproteins alpha1-acid glycoprotein, fetuin, porcine gastric and bovine submaxillary mucins, and the glycolipid sulfatide, all of which present multivalent sialylated and/or sulfated galactosyl residues under the conditions of the binding assay. Consistent with this pattern, a multivalent neoglycoconjugate containing 20 mol of 3'SL per mol of human serum albumin inhibited bacterial binding with micromolar IC50. The H. pylori isolate most sensitive to inhibition by 3'SL was least sensitive to inhibition by sulfatide, gastric mucin, and other sulfated oligosaccharides. Bacteria that have been allowed to bind epithelial cells are also effectively detached by 3'SL. These results describe a heterogeneous adherence repertoire for these bacteria, but they also confirm the critical role of the 3'SL structure on human gastric epithelial cells as an adherence ligand for recent isolates of H. pylori.

Functional substitution of the signal recognition particle 54-kDa subunit by its Escherichia coli homolog.

The 54-kDa subunit of the mammalian signal recognition particle (SRP54) binds to the signal sequences of nascent secretory and transmembrane proteins and facilitates their cotranslational targeting to the membrane translocation apparatus in the endoplasmic reticulum (ER). A 48-kDa Escherichia coli protein that shares extensive sequence similarity with SRP54 was identified in homology searches. Recent genetic experiments by Phillips and Silhavy [Phillips, G. J. & Silhavy, T. J. (1992) Nature (London) 359, 744-746] have shown that depletion of this protein, designated Ffh (fifty-four homolog), leads to a significant secretory defect in vivo. We demonstrate here that Ffh is structurally and functionally related to SRP54 by virtue of its ability to mimic closely its mammalian counterpart in several established biochemical assays, thereby suggesting that it plays a direct role in protein export. Ffh assembled efficiently with mammalian SRP components into a chimeric ribonucleoprotein ["SRP(Ffh)"] and bound at the site normally occupied by SRP54. Like SRP54, the Ffh moiety of the chimeric particle specifically recognized the signal sequence of preprolactin in a photocrosslinking assay. Moreover, Ffh could also act in concert with other SRP components to arrest elongation of preprolactin upon recognition of the signal sequence. In all of these assays, Ffh had approximately the same specific activity as SRP54. In contrast, SRP(Ffh) did not promote the translocation of preprolactin across the membrane of microsomal vesicles, suggesting that Ffh cannot mediate an interaction with a membrane component that is required for the translocation of nascent chains.

GTPase domain of the 54-kD subunit of the mammalian signal recognition particle is required for protein translocation but not for signal sequence binding.

The 54-kD subunit of the signal recognition particle (SRP54) binds to signal sequences of nascent secretory and transmembrane proteins. SRP54 consists of two separable domains, a 33-kD amino-terminal domain that contains a GTP-binding site (SRP54G) and a 22-kD carboxy-terminal domain (SRP54M) containing binding sites for both the signal sequence and SRP RNA. To examine the function of the two domains in more detail, we have purified SRP54M and used it to assemble a partial SRP that lacks the amino-terminal domain of SRP54 [SRP(-54G)]. This particle recognized signal sequences in two independent assays, albeit less efficiently than intact SRP. Analysis of the signal sequence binding activity of free SRP54 and SRP54M supports the conclusion that SRP54M binds signal sequences with lower affinity than the intact protein. In contrast, when SRP(-54G) was assayed for its ability to promote the translocation of preprolactin across microsomal membranes, it was completely inactive, apparently because it was unable to interact normally with the SRP receptor. These results imply that SRP54G plays an essential role in SRP-mediated targeting of nascent chain-ribosome complexes to the ER membrane and also influences signal sequence recognition, possibly by promoting a tighter association between signal sequences and SRP54M.

VILIP, a cognate protein of the retinal calcium binding proteins visinin and recoverin, is expressed in the developing chicken brain.

Using a selective cloning approach we previously isolated a number of cDNAs of transcripts that are newly expressed during terminal differentiation of the chicken optic tectum. Here, we have characterized one of these cDNAs (OZ1) by Northern analysis and in situ hybridization. The OZ1 cDNA hybridizes to two transcripts of 1.6 kb and 2.9 kb which are widely expressed in the brain but not detectable in liver, heart or skeletal muscle. Cloning of overlapping cDNAs revealed that both transcripts encode the same open reading frame for a polypeptide of 191 amino acids. The deduced protein contains 4 EF-hand consensus motifs characteristic of calmodulin-like Ca(2+)-binding proteins. It displays 40% and 46% sequence identity with the retinal photoreceptor-specific Ca(2+)-binding proteins visinin and recoverin, respectively, and was termed VILIP (visinin-like protein). VILIP transcripts are also expressed in the retina. However, the expression pattern does not overlap with that of visinin or recoverin. The possible functional implications of the similarity to recoverin, which regulates guanylate cyclase activity of retinal rod cells in a Ca(2+)-dependent manner, are discussed.

Assay of alpha 1,3 N-acetyl-D-galactosaminyl transferase by affinity chromatography.

A high-performance liquid affinity chromatography column that contains immobilized anti-A monoclonal antibody specifically retards blood group A-active oligosaccharides and can be used to detect the product(s) of the reaction catalyzed by alpha-1,3-N-acetyl-D-galactosaminyltransferase: [formula: see text] After a brief incubation (15 min) of an assay mixture containing 1-100 microliters human serum, the sugar nucleotide donor UDP-GalNAc, and radiolabeled oligosaccharide acceptors 2'-fucosyllactose and/or lacto-N-fucpentaose I blood group A-active products are isolated and quantitated in a single affinity chromatographic step that takes less than 30 min. Kinetic studies to determine the pH optima for serum alpha-3-GalNAc transferase from individuals of blood groups A1 and A2 and the Km value for UDP-GalNAc for the A1 transferase agree with previous determinations. As monoclonal antibodies against many different complex carbohydrate antigens are now available, the method described could be adapted to give rapid, inexpensive assays for a variety of glycosyltransferases.

Isolation of two novel sialyl-Lewis X-active oligosaccharides by high-performance liquid affinity chromatography using monoclonal antibody Onc-M26.

A monoclonal antibody, Onc-M26, that recognizes a cancer-associated antigen expressed by most human adenocarcinomas of the breast was shown previously to recognize a carbohydrate epitope carried on a hexaglycosyl ganglioside carrying the sialyl-Lewis X (SLex) antigen (P.S. Linsley et al., 1988, Cancer Res. 48, 2138-2148). Evidence that the antibody binds even more avidly to minor gangliosides containing more complex carbohydrate chains prompted us to search for a higher affinity epitope among sialylated oligosaccharides from pooled human milk. Affinity chromatography of a partially purified fraction of monosialylated milk oligosaccharides on a column containing monoclonal antibody Onc-M26 bound to a macroporous silica matrix gave a peak with a retention volume significantly greater than that of a standard SLex-active hexasaccharide. The retained material consisted of two nonasaccharides, each containing the SLex tetrasaccharide sequence, Neu5Ac alpha 2-3Gal beta 1-4(Fuc alpha 1-3) GlcNAc, linked beta 1-6 to a 3,6-disubstituted galactosyl residue.