Esophageal Microbiome in Eosinophilic Esophagitis

Objective

The microbiome has been implicated in the pathogenesis of a number of allergic and inflammatory diseases. The mucosa affected by eosinophilic esophagitis (EoE) is composed of a stratified squamous epithelia and contains intraepithelial eosinophils. To date, no studies have identified the esophageal microbiome in patients with EoE or the impact of treatment on these organisms. The aim of this study was to identify the esophageal microbiome in EoE and determine whether treatments change this profile. We hypothesized that clinically relevant alterations in bacterial populations are present in different forms of esophagitis.

Design

In this prospective study, secretions from the esophageal mucosa were collected from children and adults with EoE, Gastroesophageal Reflux Disease (GERD) and normal mucosa using the Esophageal String Test (EST). Bacterial load was determined using quantitative PCR. Bacterial communities, determined by 16S rRNA gene amplification and 454 pyrosequencing, were compared between health and disease.

Results

Samples from a total of 70 children and adult subjects were examined. Bacterial load was increased in both EoE and GERD relative to normal subjects. In subjects with EoE, load was increased regardless of treatment status or degree of mucosal eosinophilia compared with normal. Haemophilus was significantly increased in untreated EoE subjects as compared with normal subjects. Streptococcus was decreased in GERD subjects on proton pump inhibition as compared with normal subjects.

Conclusions

Diseases associated with mucosal eosinophilia are characterized by a different microbiome from that found in the normal mucosa. Microbiota may contribute to esophageal inflammation in EoE and GERD.     

Restriction-modification system with methyl-inhibited base excision and abasic-site cleavage activities

The restriction-modification systems use epigenetic modification to distinguish between self and nonself DNA. A modification enzyme transfers a methyl group to a base in a specific DNA sequence while its cognate restriction enzyme introduces breaks in DNA lacking this methyl group. So far, all the restriction enzymes hydrolyze phosphodiester bonds linking the monomer units of DNA. We recently reported that a restriction enzyme (R.PabI) of the PabI superfamily with half-pipe fold has DNA glycosylase activity that excises an adenine base in the recognition sequence (5′-GTAC). We now found a second activity in this enzyme: at the resulting apurinic/apyrimidinic (AP) (abasic) site (5′-GT#C, # = AP), its AP lyase activity generates an atypical strand break. Although the lyase activity is weak and lacks sequence specificity, its covalent DNA–R.PabI reaction intermediates can be trapped by NaBH₄ reduction. The base excision is not coupled with the strand breakage and yet causes restriction because the restriction enzyme action can impair transformation ability of unmethylated DNA even in the absence of strand breaks in vitro. The base excision of R.PabI is inhibited by methylation of the target adenine base. These findings expand our understanding of genetic and epigenetic processes linking those in prokaryotes and eukaryotes.     

Effects of color CRT display on pupil size in color-blind subjects.

The effects of color cathode ray tube (CRT) display on the pupil size in color-blind subjects were studied experimentally. Red, magenta, green, cyan, yellow, and white were presented on the CRT. Five protan subjects, five deutans, and five normal subjects were tested using infrared pupillography. In the protan group, the pupils were significantly less sensitive to red (wavelength: 600 nm) targets compared to the other colors. In the deutans and the control group, there were non-significant changes in pupil size in response to the colors. Dominant wavelengths above 600 nm in color CRTs should be avoided in routine work presentation because of less sensitivity in protanopias.     

Purification and characterization of glucosyltransferases from Streptococcus mutans 6715

A water-soluble glucan-synthesizing glucosyltransferase (GTase-S) and a water-insoluble glucan-synthesizing glucosyltransferase (GTase-I) were purified from culture supernatant of Streptococcus mutans 6715 (serotype g) by ammonium sulphate precipitation, chromatofocusing on a Polybuffer exchanger PBE 94 column, and subsequent phenyl-Sepharose CL-4B or hydroxyapatite column chromatography. The GTase-S and GTase-I activities were purified 4019- and 4714-fold, respectively, and the molecular weights were calculated to be 160000 and 165000, respectively. GTase-S had a pH optimum of 5.0, a Km of 8.8 mM for sucrose in the presence of 20 microM-dextran T10, and an isoelectric point of pH 4.3. GTase-I had two pH optima of 5.0 and 7.0, Km values of 4.9 mM (at pH 5.0) and 7.0 mM (at pH 7.0), mM (at pH 7.0), and an isoelectric point of pH 4.9. Methylation analysis indicated that the water-soluble glucan produced by GTase-S was a highly branched 1,6-alpha-linked D-glucan with 1,3-linked glucose residues, and that the water-insoluble glucan synthesized by GTase-I was composed of 1,3-alpha-linked glucose units.     

Improved analytical method for the hexuronic acids at the reducing terminals of glycosaminoglycans

When endo-uronidases act on glycosaminoglycans, the reaction products have hexuronic acid residues at the reducing terminals. An analytical method for hexuronic acids at the reducing terminals was devised for hyaluronate oligosaccharides having hexuronic acid residues at the reducing terminals.The procedure is as follows: Hexuronic acid residues at the reducing terminals of hyaluronate oligosaccharides were tritiated with reduction using NaB[³H]₄ and the products were hydrolyzed with trifluoroacetic acid and nitrous acid. As a result, the tritiated and reduced hexuronic acid residues, that is aldonic acids, were liberated from the reducing terminals. After passing them through anion and cation ion-exchange resins, the aldonic acids were lactonized. The lactones were developed on paper chromatography, and their radioactivities determined on the paper.The method is also useful for discrimination between glucoronic acid and iduronic acid at the reducing terminals of glycosaminoglycans.     

Simultaneous determination of stable isotopically labelled l-histidine and urocanic acid in human plasma by stable isotope dilution mass spectrometry

A capillary gas chromatographic—mass spectrometric method for the simultaneous determination of stable isotopically labelled l-histidine (l-[3,3-²H₂,1′,3′-¹⁵N₂]histidine, l-His-[M + 4]) and urocanic acid ([3-²H,1′,3′-¹⁵N₂]urocanic acid, UA-[M + 3]) in human plasma was developed using dl-[2,3,3,5′-²H₄,2′-¹³C,1′,3′-¹⁵N₂]histidine (dl-His-[M + 7]) and [2,3,5′-²H₃,2′-¹³C,1′,3′-¹⁵N₂]urocanic acid (UA-[M + 6]) as internal standards. l-Histidine and urocanic acid were derivatized to ^αN-(trifluoroacetyl)-^imN-(ethoxycarbonyl)-l-histidine n-butyl ester and ^imN-(ethoxycarbonyl)urocanic acid n-butyl ester. Quantification was carried out by selected ion monitoring of the molecular ions of the respective derivatives of l-His-[M + 4], dl-His-[M + 7], UA-[M + 3] and UA-[M + 6]. The sensitivity, specificity, precision and accuracy of the method were demonstrated to be satisfactory for measuring plasma concentrations of l-His-[M + 4] and UA-[M + 3] following administration of trace amounts of l-His-[M + 4] to humans.     

Stimulatory effect of gangliosides on phagocytosis, phagosome–lysosome fusion, and intracellular signal transduction system by human polymorphonuclear leukocytes

Gangliosides are known to be differentiation-inducing molecules in mammalian stem cells. We studied the interaction between the molecular structure of glycosphingolipids (GSLs) and their promoting mechanisms of the phagocytic processes in human polymorphonuclear leukocytes (PMN). The effect of various gangliosides from mammalian tissues on adhesion, phagocytosis, phagosome–lysosome (P–L) fusion and superoxide anion production was examined by human PMN using heat-killed cells of Staphylococcus aureus coated with GSLs. Gangliosides GM3, GD1a, GD3 and GT1b showed a marked stimulatory effect on the phagocytosis and P–L fusion in a dose-dependent manner, while ganglioside GM1, asialo GM1 and neutral GSLs did not. The relative phagocytic rate of ganglioside GM3-coated S. aureus was the highest among the tested GSLs. Both P–L fusion rate and phagocytosis of S. aureus were elevated significantly when coated with ganglioside GD1a, GD3 or GT1b, and GT1b gave a five times higher rate than that of the non-coated control. These results suggest that the terminal sialic acid moiety is essential for the enhancement of phagocytosis and that the number of sialic acid molecules in the ganglioside is related to the enhancement of the P–L fusion process. On the other hand, the superoxide anion release from PMN was not affected by ganglioside GM2, GM3, GD1a or GT1b. Furthermore, to clarify the trigger or the signal transduction mechanism of phagocytic processes, we examined the effect of protein kinase inhibitors such as H-7, staurosporine (protein kinase C inhibitor), H-89 (protein kinase A inhibitor), genistein (tyrosine kinase inhibitor), ML-7 (myosin light chain kinase inhibitor), and KN-62 (Ca2+/calmodulin-dependent protein kinase II inhibitor) on ganglioside-induced phagocytosis. H-7, staurosporine and KN-62 inhibited ganglioside-induced phagocytosis in the range of concentration without cell damage, while H-89, genistein and ML-7 did not. Moreover, H-7 and KN-62 inhibited ganglioside-induced P–L fusion. These results suggest that protein kinase C and Ca2+/calmodulin-dependent protein kinase II may be involved in the induction of phagocytosis and P–L fusion stimulated by gangliosides. This revised version was published online in November 2006 with corrections to the Cover Date.     

Cell-free synthesis of the enzymes of peroxisomal beta-oxidation

Three enzymes of peroxisomal β-oxidation of rat liver were synthesized in a cell-free protein-synthesizing system derived from rabbit reticulocyte lysate. The $\text{in}\text{vitro}$ products of acyl-CoA oxidase and enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase multifunctional protein were similar in size to or slightly larger than the subunit of the respective mature enzymes. The $\text{in}\text{vitro}$ product of peroxisomal 3-ketoacyl-CoA thiolase was about 3,000 daltons larger than the mature subunit. The hepatic levels of translatable mRNAs coding for these three enzymes were about 10 times higher in rats fed a di(2-ethylhexyl)phthalate-containing diet than in control animals.