Effects of lactoferrin on the viability and the encystment of Acanthamoeba trophozoites.

Lactoferrin (LF) is an iron-binding basic glycoprotein that has an antimicrobial effect against certain microbes. The purpose of this study is to evaluate the amoebicidal effect of bovine milk LF (bLF) against Acanthamoeba clinical-isolate trophozoites, which cause severe keratitis. Most of the risk factor for Acanthamoeba keratitis is from wearing soft contact lenses (SCLs). Acanthamoeba trophozoites were incubated in bovine LF (bLF) solution, and the ratios of viability and encystment were determined with microscopic analysis of cyst formation. The amoebicidal effect of bLF was assessed by Trypan blue assay. The ratios of viable cells in the presence of iron-free bLF (apo-bLF), native-bLF, and iron-saturated bLF (Fe-bLF) at the concentration of 10 µmol/L for 60 min were 7.7% ± 4.6%, 80.7% ± 10.1%, and 97.3% ± 1.5%, respectively. Apo-bLF showed potent amoebicidal effect against Acanthamoeba trophozoites, but Fe-bLF did not have this effect. After treating with apo-bLF, most dead cells were nonglobular forms of trophozoites but not cystic forms. Encystment of Acanthamoeba was assessed by the sarkosyl-calcofluor white assay. The encystment ratios treated with 0.5% propylene glycol (positive control) and 10 µmol/L apo-bLF for 24 h were 96.12% ± 10.6% and 0.47% ± 0.5%, respectively. These results suggest that the amoebicidal effect of apo-bLF without encystment might lead to the prevention of contamination of Acanthamoeba in SCL stock cases.

Effects of Lactoferrin on Oral and Throat Conditions under Low Humidity Environments: A Randomized, Double-Blind, and Placebo-Controlled Crossover Trial.

To evaluate the effects of a single ingestion of bovine lactoferrin (bLF) on oral and throat conditions under a low-humidity environment. A randomized, double-blind, 2-sequence, 2-treatment, and 2-period placebo-controlled crossover trial was conducted. Healthy adult subjects orally ingested bLF dissolved in water, or placebo water, followed by exposure to low humidity (20 °C, 20% relative humidity (RH)) for 2 h. The primary endpoint was subjective oral and throat discomfort assessed by a visual analog scale (VAS), which positively correlated with the discomfort. Secondary endpoints were unstimulated whole salivary flow rate (UWSFR) and salivary immunoglobulin A (IgA) secretion rate. Overall, 40 subjects were randomly assigned to two sequences (20 each) and 34 were analyzed. The VAS values for oral and throat discomfort in the bLF treatment were significantly lower than in the placebo treatment, whereas UWSFR and IgA secretion rates were comparable between the two treatments. Adverse drug reactions were not observed. Subjective oral and throat discomfort associated with low humidity is suppressed by a single ingestion of bLF. Our findings demonstrate the novel use of bLF in a clinical situation that leverages its unique characteristics.

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody 92-2.

A human-mouse hybridoma clone 92-2 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was established. To detect antibody-binding sequences on Ara h1, we synthesized a series of peptides of the Ara h1 protein on a multi-pin apparatus for the pin-peptide ELISA. The 92-2 human monoclonal antibody was found to recognize a sequence of GREGEQEWGTPGSHVREETS. Further analysis with shorter pin-peptides with eight amino acid-long showed that the sequence of QEWGTPGS was an essential linear sequence of this epitope. When the QEW part of the sequence was replaced by alanine, the 92-2 monoclonal antibody did not bind to the substituted peptide, showing that those amino acids play an important role in the binding of the 92-2 monoclonal antibody.

Epitope analysis of peanut allergen Ara h1 with human monoclonal IgM antibody clone #86.

A human-mouse hybridoma clone #86 secreting IgM-class human monoclonal antibody to peanut allergen protein Ara h1 was newly established. To detect an antibody-binding sequence (epitope) on Ara h1, the monoclonal antibody #86 was reacted with multi-pin apparatus with a series of peptides synthesized from the amino acid sequence of Ara h1. The antibody #86 was found to bind to a peptide with amino acid sequence of 481EEEEDEDEEEEGSNREVRRY500. Further analysis with shorter pin-peptides with ten amino acid-long showed that the peptides reacted with the antibody #86 contained a sequence of 485DEDEEEE491. This might be an essential linear sequence of this epitope. When the 485DED487 part of the peptide was replaced by alanine, decreased binding of antibody #86 was observed.

Bacterial population moves toward a colon-like community in the pouch after total proctocolectomy.

BACKGROUND: Colonic transformation is defined by phenotypic alterations in the ileum after total proctocolectomy. Changes in microbiota of the ileal pouch and the roles of these microbes in colonic transformation, however, have not been addressed. METHODS: A total of 151 stool samples were collected from patients with ulcerative colitis patients and an ileostomy, those with an ileal pouch, and healthy control volunteers. Bacterial DNA was extracted from stool, and the diversity of complex bacteria was assessed by terminal restriction fragment length polymorphism (T-RFLP) analysis, a novel DNA-based approach that enables us to investigate the presence of nonculturable bacteria. To determine whether ileal pouch bacterial communities shift to a more colon-like distribution, the relative abundance of terminal restriction fragments that could be classified as "colonic," "ileal," or "common" was investigated. RESULTS: Cluster analysis demonstrated that most of the ileostomy samples were categorized into Cluster I or II and that less than 10% of ileostomy samples were classified into Cluster IV. In contrast, more than 90% of control samples were grouped in Cluster IV. In further analyses, the median lifetimes of pouches in Clusters I, II, III, and IV were significantly different at 11, 56, 265, and 310 days, respectively. T-RFLP patterns of the ileal pouch were characterized by a time-dependent decrease in "ileal" and increase in a part of "colonic" fragments, which represented mainly nonculturable bacteria such as the Clostridium coccoides group. CONCLUSION: T-RFLP analysis demonstrated that a time-dependent shift to a "colon-like" bacterial community, including nonculturable bacteria, in the ileal pouch after total proctocolectomy.

Prevotella copri sp. nov. and Prevotella stercorea sp. nov., isolated from human faeces.

Six strains (CB7(T), CB18, CB23, CB26, CB28 and CB35(T)) were isolated from human faeces. Based on phylogenetic analysis, phenotypic characteristics, cellular fatty acid profiles and menaquinone profiles, these strains could be included within the genus Prevotella and made up two clusters. 16S rRNA gene sequence analysis indicated that five strains were most closely related to Prevotella veroralis, sharing about 92 % sequence similarity; the remaining strain was most closely related to Prevotella shahii, sharing about 90 % sequence similarity. All six strains were obligately anaerobic, non-pigmented, non-spore-forming, non-motile, Gram-negative rods. The cellular fatty acid compositions of the six strains differed significantly from those of other Prevotella species. Five strains (CB7(T), CB18, CB23, CB26 and CB28) contained dimethyl acetals and the major menaquinones of these strains were MK-11, MK-12 and MK-13. The major menaquinones of CB35(T) were MK-12 and MK-13. Based on phenotypic and phylogenetic findings, two novel species, Prevotella copri sp. nov. and Prevotella stercorea sp. nov., are proposed, representing the two different strain clusters. The DNA G+C contents of strains CB7(T) and CB35(T) were 45.3 and 48.2 mol%, respectively. The type strains of P. copri and P. stercorea are CB7(T) (=JCM 13464(T)=DSM 18205(T)) and CB35(T) (=JCM 13469(T)=DSM 18206(T)), respectively.

Bacteroides coprophilus sp. nov., isolated from human faeces.

Three Gram-negative, anaerobic, rod-shaped bacteria (strains CB40, CB41 and CB42(T)) were isolated from human faeces. Based on phylogenetic analysis and specific phenotypic characteristics, these strains were included in the genus Bacteroides, and 16S rRNA gene sequence analysis indicated that these strains represented a novel species. The strains were most closely related to the type strains of Bacteroides barnesiae and Bacteroides salanitronis, with sequence similarities of 93.4 and 89.8 %, respectively. The G+C content of strain CB42(T) is 44.7 mol%. Major fatty acids were anteiso-C(15 : 0), C(16 : 0), iso-C(17 : 0) 3-OH and C(18 : 1)omega9c. On the basis of the data presented, a novel Bacteroides species, Bacteroides coprophilus sp. nov., is proposed, with CB42(T) (=JCM 13818(T)=DSM 18228(T)) as the type strain.