Serological Studies of an Acid-Labile O-Polysaccharide of Proteus vulgaris OX19 Lipopolysaccharide Using Human and Rabbit Antibodies

In a Weil-Felix test, sera from patients infected with Rickettsia sp. agglutinate Proteus OX types of bacteria and Proteus lipopolysaccharide (LPS) are responsible for the cross-reaction. Data on the character of LPS of one of the OX group strains, Proteus vulgaris OX19, are contradictory, and it remained unclear whether it has an O-polysaccharide (OPS) and is thus LPS of the smooth type (S) or not (rough-type LPS). Our studies showed that P. vulgaris OX19 (strain PZH-24) produces a smooth-type LPS that contains a long-chain OPS, but it undergoes depolymerization during mild acid hydrolysis conventionally used for LPS delipidation and loses the serological activity. An elucidation of the complete structure of OPS demonstrated the presence of a glycosyl phosphate linkage responsible for the acid-lability of the polysaccharide chain. In ELISA, both IgM type antibodies in a Weil-Felix test with human anti-Rickettsia typhi sera and rabbit anti-P. vulgaris OX19 antibodies reacted with OPS. Rabbit antibodies did not inhibit the cross-reaction with human antibodies and thus bind to different epitopes.     

Evasion of killing by human antibody and complement through multiple variations in the surface oligosaccharide of Haemophilus influenzae

The lipopolysaccharide (LPS) of H. influenzae is highly variable. Much of the structural diversity is derived from phase variation, or high frequency on‐off switching, of molecules attached during LPS biosynthesis. In this study, we examined the dynamics of LPS phase variation following exposure to human serum as a source of antibody and complement in multiple H. influenzae isolates. We show that lic2A, lgtC and lex2A switch from phase‐off to phase‐on following serial passage in human serum. These genes, which control attachment of a galα1–4gal di‐galactoside structure (lic2A and lgtC phase‐on) or an alternative glucose extension (lex2A phase‐on) from the same hexose moiety, reduce binding of bactericidal antibody to conserved inner core LPS structures. The effects of the di‐galactoside and alternative glucose extension were also examined in the context of the additional LPS phase variable structures phosphorylcholine (ChoP) and sialic acid. We found that di‐galactoside, the alternative glucose extension, ChoP, and sialic acid each contribute independently to bacterial survival in the presence of human complement, and have an additive effect in combination. We propose that LPS phase variable extensions serve to shield conserved inner core structures from recognition by host immune components encountered during infection.     

Serotype‐dependent expression patterns of stabilized lipopolysaccharide aggregates in Aggregatibacter actinomycetemcomitans strains

Above a critical concentration, amphiphilic lipopolysaccharide (LPS) molecules in an aqueous environment form aggregate structures, probably because of interactions involving hydrophobic bonds. Ionic bonds involving divalent cations stabilize these aggregate structures, making them resistant to breakdown by detergents. The aim of this study was to examine expression patterns of stabilized LPS aggregates in Aggregatibacter actinomycetemcomitans, a microorganism that causes periodontitis. A. actinomycetemcomitans strains of various serotypes and truncated LPS mutants were prepared for this study. Following treatment with a two‐phase separation system using the detergent Triton X‐114, crude LPS extracts of the study strains were separated into detergent‐phase LPS (DP‐LPS) and aqueous‐phase LPS (AP‐LPS). Repeated treatment of the aqueous phase with the two‐phase separation system produced only a slight decrease in AP‐LPS, suggesting that AP‐LPS was resistant to the detergent and thus distinguishable from DP‐LPS. The presence of divalent cations increased the yield of AP‐LPS. AP‐LPS expression patterns were serotype‐dependent; serotypes b and f showing early expression, and serotypes a and c late expression. In addition, highly truncated LPS from a waaD (rfaD) mutant were unable to generate AP‐LPS, suggesting involvement of the LPS structure in the generation of AP‐LPS. The two‐phase separation was able to distinguish two types of LPS with different physical states at the supramolecular structure level. Hence, AP‐LPS likely represents stabilized LPS aggregates, whereas DP‐LPS might be derived from non‐stabilized aggregates. Furthermore, time‐dependent expression of stabilized LPS aggregates was found to be serotype‐dependent in A. actinomycetemcomitans.     

Characterization of Immunological Activity of a Low Toxicity Antitumor Lipopolysaccharide from Bordetella pertussis

Immunological properties of a low toxicity lipopolysaccharide (BP-LPS) extracted from Bordetella pertussis (Tohama strain) which was reported to have high antitumor activity against murine tumors were examined and compared with those of LPS extracted from other enterobacteria. The activation or stimulation of murine macrophages and lymphocytes by these LPS, including TNF induction, was found to be similar. However, BP-LPS was clearly less active in its stimulation of murine and human neutrophils as estimated by neutrophil-adherence assay and by their TNF production than E. coli LPS. Furthermore, BP-LPS also suppressed the activation of human neutrophils by Escherichia coli LPS. A comparative study with 7 LPS preparations indicated that their toxicity in terms of animal body weight loss correlated with their ability to induce human neutrophil adherence. The inability of BP-LPS to activate neutrophils may thus have some bearing on its low toxicity.     

Characterization of lipopolysaccharides from four Pasteurella haemolytica serotype strains: evidence for presence of sialic acid in serotypes 1 and 5

Highly purified lipopolysaccharides (LPS) obtained from four strains of Pasteurella haemolytica representative of four different serotypes were studied to ascertain their overall structural elements and sugar and fatty acid compositions. SDS-PAGE analysis revealed that each LPS was of the smooth-type although they differed in migration patterns. Somewhat unusual features of these LPS included the presence of: (a) rhamnose in the core oligosaccharides of serotypes 2 and 3; and (b) sialic acid in the LPS of serotypes 1 and 5. The fatty acids, myristic, hydroxymyristic and palmitic occur in essentially equivalent amounts in each of these LPS. In addition, stearic acid was present in small amounts of serotypes 1 and 5.     

Separation and characterization of two chemically distinct lipopolysaccharides in two Pectinatus species.

Lipopolysaccharides (LPS) from the type strains of the anaerobic beer spoilage bacteria Pectinatus cerevisiiphilus and P. frisingensis were extracted with the 5:5:8 volume ratio modification of the phenolchloroform-petroleum ether method (H. Brade and C. Galanos, Eur. J. Biochem. 122:233-237, 1982). Sequential precipitations of LPS with water and acetone from the phenol phase yielded LPS which differed in that water-precipitable material (LPS-H2O; 0.1 to 0.4% of the dry weight of the cells) was rough-type LPS, whereas acetone-precipitable material (LPS-Ac; 4.6 to 5.8% of the dry weight) contained both rough-type LPS and high-molecular-weight material resembling smooth LPS. The LPS were chemically characterized, and they contained D-glucosamine, 4-amino-4-deoxy-L-arabinose, 3-deoxy-D-manno-2-octulosonic acid, D-fucose, D-galactose, D-glucose, D-mannose, and phosphate. D-Fucose was present mostly in LPS-Ac, suggesting that it is a constituent of the O antigen. The major fatty acids were ester- and amide-linked (R)-3-hydroxytridecanoic and ester-linked undecanoic acids, with minor amounts of ester-linked tridecanoic and (R)-3-hydroxyundecanoic acids. The chemical compositions of LPS-H2O and LPS-Ac suggested that they differ not only in their smooth or rough nature but also in the structure of their core regions. This may explain their different precipitabilities from the extraction mixture. The extraction method was also shown to be applicable to the isolation of smooth-type LPS from Salmonella enterica serovar Typhimurium. Extraction of two Typhimurium strains carrying chemically different O antigens resulted in high yields (8% of the dry weight) of LPS. Strain SH2183, which contains the relatively hydrophobic O-4,5,12 antigen yielded almost exclusively LPS-Ac, whereas the LPS of strain SH5770, which has a hydrophilic O-6,7 antigen, was exclusively LPS-H2O. No fractionation to smooth and rough LPS occurred with the Typhimurium strains.     

Preparation and characterization of truncated human lipopolysaccharide-binding protein in Escherichia coli

Lipopolysaccharide (LPS) is a component of the outer membrane of Gram-negative bacteria, and is the causative agent of endotoxin shock. LPS induces signal transduction in immune cells when it is recognized by the cell surface complex of toll-like receptor 4 (TLR4) and MD-2. The complex recognizes the lipid A structure in LPS, which is buried in the membrane of the outer envelope. To present the Lipid A structure to the TLR4/MD-2, processing of LPS by LPS-binding protein (LBP) and CD14 is required. In previous studies, we expressed recombinant proteins of human MD-2 and CD14 as fusion proteins with thioredoxin in Escherichia coli, and demonstrated their specific binding abilities to LPS. In this study, we prepared a recombinant fusion protein containing 212 amino terminal residues of human LBP (HLB212) by using the same expression system. The recombinant protein expressed in E. coli was purified as a complex form with host LPS. The binding was not affected by high concentrations of salt, but was prevented by low concentrations of various detergents. Both rough-type LPS lacking the O antigen and smooth-type LPS with the antigen bound to HLBP212. Therefore, oligosaccharide repeats appeared to be unnecessary for the binding. A nonpathogenic penta-acylated LPS also bound to HLBP212, but the binding was weaker than that of the wild type. The hydrophobic interaction between the LBP and acyl chains of lipid A appears to be important for the binding. The recombinant proteins of LPS-binding molecules would be useful for analyzing the defense mechanism against infections.     

Cytopathic effects of the pathogenic Neisseria. Studies using human fallopian tube organ cultures and human nasopharyngeal organ cultures

Infection of mucosal surfaces by N. gonorrhoeae and N. meningitidis may result in inflammation indicating potential injury to host cells. We used human fallopian tube organ cultures (FTOC) and human nasopharyngeal organ cultures (NPOC) to study the mechanisms by which gonococci and meningococci damage human mucosal surfaces. Early in the course of FTOC infected with gonococci and NPOC infected with meningococci, damage was most apparent to ciliary activity. Loss of ciliary activity was accompanied by sloughing of ciliated cells. The damage to ciliated cells was not associated with attachment of gonococci or meningococci to these cells or the presence of organisms within ciliated cells. Infection with the commensal N. subflava did not result in significant damage to human FTOC or NPOC ciliary activity. LPS appears to be a major toxin of gonococci for human FTOC ciliated cells. Gonococcal peptidoglycan fragments also damage FTOC ciliary activity. Both piliated (P+) and nonpiliated (P-) gonococci and meningococci damage FTOC and NPOC ciliary activity, but P+ organisms damage ciliary activity more rapidly than P- organisms. Damage to FTOC ciliated cells was produced by <10 g/ml of purified gonococcal and meningococcal LPS. By 1–2h after exposure to LPS, vesicles containing LPS were distributed throughout the cytoplasm of ciliated cells. Polymyxin B neutralized LPS-induced damage, suggesting that the lipid A portion of LPS was the toxic moiety. In contrast, purified gonococcal and meningococcal LPS at 100 g/ml did not damage human NPOC or FTOC from rabbits, pigs and cows. These studies indicate that N. gonorrhoeae and possibly N. meningitidis damage ciliated epithelial celsl indirectly by release of toxins from the organisms. The differences in susceptibility of FTOC and NPOC to LPS may suggest changes in density of receptors for LPS and may help explain variation in severity of gonococcal and meningococcal interactions at different human mucosal surfaces.     

The Effect of the Cultivation Method and the Growth Phase on the Lipopolysaccharide Composition of Yersinia pseudotuberculosis

Effects of the cultivation method (suspension cultures in a liquid nutrient broth or colonies on a solid agarized medium) and the growth phase on the lipopolysaccharide (LPS) composition of Yersinia pseudotuberculosis(O : Ib serovar, strain KS 3058) grown in cold (5°C) were studied. The amount of the LPS synthesized by cells depended on the bacteria growth phase for both media. The LPS acylation degree was constant, whereas the length of the O-specific polysaccharide chain varied with the culture age and for both media achieved maximum in the stationary growth phase. The bacteria cultivation on the nutrient agar stimulated more intensive synthesis of LPS, which were extracted more easily, had longer polysaccharide O-chains, and were more toxic than LPS of the bacteria cultivated in the liquid medium. It was proposed that the cultivation of Yersinia pseudotuberculosisin cold as colonies on the agar surface increases the bacterial virulence.     

Isolation and characterization of serum-resistant strains ofPseudomonas aeruginosa derived from serum-sensitive parental strains

Six serum-resistant (serR) mutantPseudomonas aeruginosa strains were isolated from six serum-sensitive (serS) parental strains by subculturing the sensitive strains in increasing concentrations of normal pooled fresh human serum (FHS). Although the colonial type of the mutant was similar to that of the parental strains, each of the serR mutants had an altered serotype when compared to its parental counterpart. Three mutant strains and their corresponding parental strains were chosen for further examination. The lipopolysaccharide (LPS) preparations from the serR strains were found to be heterogeneous, containing LPS with varying degrees of O-side-chain substitution, whereas the LPS of the serS strains contained primarily lipid A-core polysaccharide components. Although two of the serR mutant strains had an outer membrane protein (OMP) profile analogous to their serS parental counterparts, one serR strain differed from its parental strain by the absence of a 32,000 dalton major OMP. These studies suggest that the susceptibility ofP. aeruginosa to the bactericidal activity of FHS may be related to either or both LPS structure or OMP content.     

Molecular mechanisms and implications for infection of lipopolysaccharide variation in Neisseria

The lipopolysaccharides of the pathogenic Neisseria species are subject to structural variation owing to a combination of intrinsic changes in lipopolysaccharide (LPS) biosynthesis and external modification of the LPS molecule with sialic acid. This variation appears to control bacterial behaviour by altering their ability to interact with human cells and to evade host Immune defences. This interconversion of LPS phenotypes, which is also observed during the natural infection, is probably due to environmental regulation of LPS biosynthesis superimposed on spontaneous changes in the DNA of distinct LPS loci. LPS variation may be a common strategy of mucosal pathogens to colonize and persist within the human host.     

Binding of hemolin to bacterial lipopolysaccharide and lipoteichoic acid. An immunoglobulin superfamily member from insects as a pattern-recognition receptor.

Hemolin, a plasma protein from lepidopteran insects, is composed of four immunoglobulin domains. Its synthesis is induced by microbial challenge. We investigated the biological functions of hemolin in Manduca sexta. It was found to bind to the surface of bacteria and yeast, and caused these micro-organisms to aggregate. Hemolin was demonstrated to bind to lipopolysaccharide (LPS) from Gram-negative bacteria and to lipoteichoic acid from Gram-positive bacteria. Binding of hemolin to smooth-type forms of LPS was competed for efficiently by lipoteichoic acid and by rough mutant (Ra and Rc) forms of LPS, which differ in polysaccharide length. Binding of hemolin to LPS was partially inhibited by calcium and phosphate. Hemolin bound to the lipid A component of LPS, and this binding was completely blocked by free phosphate. Our results suggest that hemolin has two binding sites for LPS, one that interacts with the phosphate groups of lipid A and one that interacts with the O-specific antigen and the outer-core carbohydrates of LPS. The binding properties of M. sexta hemolin suggest that it functions as a pattern-recognition protein with broad specificity in the defense against micro-organisms.     

Ecabet sodium attenuates reactive oxygen species produced by neutrophils after priming with bacterial lipopolysaccharides

The pathogenic roles of reactive oxygen species (ROS) have been implicated in ulcerative colitis (UC). The aim of this study was to examine the effects of ecabet sodium on ROS produced by human neutrophils, particularly after being primed by bacterial lipopolysaccharides (LPS). Neutrophils were isolated from six healthy volunteers. Each well of a 96‐well microplate received neutrophil suspension (1.0 × 10⁵ cells) and the plates were incubated at 37°C for 30 min with or without E. coli LPS (f.c. 0.001 ng/µL). Ecabet sodium (f.c. 0–5.0 mg/mL) was added before starting or after finishing the incubation. Neutrophils were stimulated by opsonized zymosan (OZ; 1.0 mg/mL) or calcium ionophore (A21837; 0.3 µmol/L) and luminol‐dependent chemiluminescence response was measured using a Lumi Box H‐1000. Ecabet sodium attenuated ROS production at a concentration of 5.0 mg/mL (p < 0.05) in LPS‐primed neutrophils. However, attenuating effects were not significantly different when ecabet sodium was added before or after the incubation with E. coli LPS. Ecabet sodium may have some attenuating effects on ROS produced by human neutrophils even after neutrophils are primed by bacterial LPS. These results may explain, in part, the therapeutic effects of ecabet sodium for UC. Copyright © 2003 John Wiley & Sons, Ltd.     

Sialic Acid-Containing Lipopolysaccharides of Salmonella O48 Strains—Potential Role in Camouflage and Susceptibility to the Bactericidal Effect of Normal Human Serum

Sialic acid (N-acetylneuraminic acid, NeuAc) plays an essential role in protecting gram-negative bacteria against the bactericidal activity of serum and may contribute to the pathogenicity of bacteria by mimicking epitopes that resemble host tissue components (molecular mimicry). The role of sialic acid (NeuAc)-containing lipopolysaccharides (LPS) of Salmonella O48 strains in the complement activation of normal human serum (NHS) was investigated. NeuAc-containing lipooligosaccharides cause a downregulation of complement activation and may serve to camouflage the bacterial surface from the immunological response of the host. Serotype O48 Salmonella strains have the O-antigen structure containing NeuAc while its serovars differ in outer membrane protein composition. In this study, the mechanisms of complement activation responsible for killing Salmonella O48 serum-sensitive rods by NHS were established. Four of such mechanisms involving pathways, which are important in the bactericidal mechanism of complement activation, were distinguished: only the classical/lectin pathways, independent activation of the classical/lectin or alternative pathway, parallel activation of the classical/lectin and alternative pathways, and only the alternative pathway important in the bactericidal action of human serum. To further study the role of NeuAc, its content in bacterial cells was determined by gas-liquid chromatography-mass spectrometry in relation to 3-deoxy-D-manno-2-octulosonic acid (Kdo), an inherent constituent of LPS. The results indicate that neither the presence of sialic acid in LPS nor the length of the O-specific part of LPS containing NeuAc plays a decisive role in determining bacterial resistance to the bactericidal activity of complement and that the presence of sialic acid in the structure of LPS is not sufficient to block the activation of the alternative pathway of complement. We observed that for three strains with a very high NeuAc/Kdo ratio the alternative pathways were decisive in the bactericidal action of human serum. The results indicated that those strains are not capable of inhibiting the alternative pathway very effectively. As the pathogenicity of most Salmonella serotypes remains undefined, research into the interactions between these bacterial cells and host organisms is indispensable.     

Separation of two lipopolysaccharide populations with different contents of O-antigen factor 122 in Salmonella enterica serovar Typhimurium

Lipopolysaccharide (LPS) extraction from smooth-type Salmonella enterica sv. Typhimurium was carried out with the modified phenol/chloroform/petroleum ether method (volume ratio 5:5:8). In this procedure, LPS was precipitated from 90% phenol sequentially with water and acetone to yield LPS-H2O (minute amounts) and LPS-Ac (major amounts), respectively. Chemical analyses of the LPS fractions revealed that in the O antigen of LPS-H2O position C4 of the D-galactose was extensively glucosylated, corresponding corresponding to the O-antigen factor 122. In LPS-Ac, this glucosylation was negligible. Inspection of the LPS fractions by sodium dodecyl sulphate/polyacrylamide gel electrophoresis and silver staining suggested that the glucosylation in LPS-H2O was present only in LPS species with a chain length higher than six repeating units.     

Material structure, stiffness, and adhesion: why attachment pads of the grasshopper (Tettigonia viridissima) adhere more strongly than those of the locust (Locusta migratoria) (Insecta: Orthoptera)

The morphology, ultrastrucure, effective elastic modulus, and adhesive properties of two different smooth-type attachment pads were studied in two orthopteran species. Tettigonia viridissima (Ensifera) and Locusta migratoria (Caelifera) have a similar structural organization of their attachment pads. They both possess a flexible exocuticle, where the cuticular fibrils are fused into relatively large rods oriented at an angle to the surface. The compliant material of the pad contributes to the contact formation with the substrate. However, the pad material structure was found to be different in these two species. L. migratoria pads bear a thick sub-superficial layer, as well as a higher density of rods. The indentation experiments showed a higher effective elastic modulus and a lower work of adhesion for L. migratoria pads. When the indentations were made at different depths, a higher effective elastic modulus was revealed at lower indentation depths in both species. This effect is explained by the higher stiffness of the superficial pad layer. The obtained results demonstrate a clear correlation between density of the fibres, thickness of the superficial layer, compliance of the pad, and its adhesive properties. Such material structures and properties may be dependent on the preferred environment of each species.     

Examination of AsmA and its effect on the assembly of Escherichia coli outer membrane proteins

asmA mutations were isolated as extragenic suppressors of an OmpF assembly mutant, OmpF315. This suppressor locus produced a protein that was present in extremely low levels and could only be visualized by Western blotting in cells where AsmA expression was induced from a plasmid. Detailed fractionation analyses showed that AsmA localized with the inner membrane. Curiously, however, the mutant OmpF assembly step influenced by AsmA occurred in the outer membrane, perhaps indicating an indirect involvement of AsmA in the assembly of outer membrane proteins. Biochemical examination of the outer membrane showed that asmA null mutations reduce lipo-polysaccharide (LPS) levels, thereby lowering the ratios of glycerolphospholipids to LPS and envelope proteins to LPS in the outer membrane. Despite these quantitative alterations, no apparent structural changes in LPS or major phospholipids were noted. Reduced LPS levels in asmA mutants indicate a possible role of AsmA in LPS biogenesis. Data presented in this study suggest that asmA-mediated OmpF assembly suppression may have been achieved by altering the outer membrane fluidity, thus making it more amenable for the assembly of mutant proteins.     

Efficient production of IGG human monoclonal antibodies by lymphocytes stimulated by lipopolysaccharide,pokeweed mitogen,and interleukin 4

Summary Extensive screening of the mitogens lipopolysaccharide (LPS), pokeweed mitogen (PWM), andStaphylococcus aureus Cowan I (SAC I), alone and in combination and with and without interleukin (IL) was performed forin vitro activation of regional lymph node lymphocytes from lung cancer patients for the production of human IgG, IgM, and IgA. As assessed by electrofusion of the lymphocytes following their exposure to these agents with mouse myeloma cells and incubation of the fused hybridoma, a remarkable stimulatory effect was shown by LPS and particularly by LPS plus IL-4, which was substantially greater than that of either SAC I or PWM with or without various IL. Optimization studies indicated that the addition of PWM to LPS and IL-4 in the culture medium further stimulated the human antibody (Ab) production, and that the optimal formulation for stimulations of human IgG production was a culture medium containing 20 μg/ml of LPS, 1/500 of PWM, and 100 u/ml of IL-4.     

Effects of 47C allele (rs4880) of the SOD2 gene in the production of intracellular reactive species in peripheral blood mononuclear cells with and without lipopolysaccharides induction

Abstract

Challenging of peripheral blood mononuclear cells (PBMCs) with lipopolysaccharides (LPS) has been shown to activate monocytes and macrophages, leading to the production of pro-inflammatory cytokines and reactive oxygen species (ROS). Manganese superoxide dismutase (MnSOD) is an important enzyme that may play a central role in the response to oxidative stress. 47C> T SNP of the SOD2 gene, the -9Val MnSOD is less efficient than the -9Ala version. We have previously characterized the cellular redox status of human PBMCs expressing either -9Ala (CC) or -9Val (TT) SOD2 and analyzed the responses of these cells to oxidative stress induced by LPS. Due to the observed alterations in the activities of these antioxidant enzymes, we decided to investigate their immunocontent and analyze the production of intracellular oxidants, as well as any resulting DNA damage. PBMCs were isolated from the blood of 30 healthy human volunteers (15 volunteers per allele). We then analyzed levels of nitrite, DNA damage by comet assay, TNF-α, carboxymethyl lysine and nitrotyrosine and assessed production of intracellular reactive species by the DCFH-DA-based assay and western blots were used to analyze protein levels. Our results show that there occurs an increase in nitric oxide production in both allele groups after challenge with LPS. A significant increase in DNA damage was observed in PBMCs after an 8-h LPS challenge. Cells expressing the SOD2 47C allele quickly adapt to a more intense metabolism by upregulating cellular detoxification mechanisms. However, when these cells are stressed over a long period, they accumulate a large quantity of toxic metabolic byproducts.     

Vitamin E, γ-tocopherol,reduces airway neutrophil recruitment after inhaled endotoxin challenge in rats and in healthy volunteers

Epidemiologic studies suggest that dietary vitamin E is an important candidate intervention for asthma. Our group has shown that daily consumption of vitamin E (γ-tocopherol, γT) has anti-inflammatory actions in both rodent and human phase I studies. The objective of this study was to test whether γT supplementation could mitigate a model of neutrophilic airway inflammation in rats and in healthy human volunteers. F344/N rats were randomized to oral gavage with γT versus placebo, followed by intranasal LPS (20 μg) challenge. Bronchoalveolar lavage fluid and lung histology were used to assess airway neutrophil recruitment. In a phase IIa clinical study, 13 nonasthmatic subjects completed a double-blinded, placebo-controlled crossover study in which they consumed either a γT-enriched capsule or a sunflower oil placebo capsule. After 7 days of daily supplementation, they underwent an inhaled LPS challenge. Induced sputum was assessed for neutrophils 6 h after inhaled LPS. The effect of γT compared to placebo on airway neutrophils post-LPS was compared using a repeated-measures analysis of variance. In rats, oral γT supplementation significantly reduced tissue infiltration (p<0.05) and accumulation of airway neutrophils (p<0.05) that are elicited by intranasal LPS challenge compared to control rats. In human volunteers, γT treatment significantly decreased induced sputum neutrophils (p=0.03) compared to placebo. Oral supplementation with γT reduced airway neutrophil recruitment in both rat and human models of inhaled LPS challenge. These results suggest that γT is a potential therapeutic candidate for prevention or treatment of neutrophilic airway inflammation in diseased populations.