Spatial variation of the colonic microbiota in patients with ulcerative colitis and control volunteers.

OBJECTIVES: The relevance of spatial composition in the microbial changes associated with UC is unclear. We coupled luminal brush samples, mucosal biopsies and laser capture microdissection with deep sequencing of the gut microbiota to develop an integrated spatial assessment of the microbial community in controls and UC. DESIGN: A total of 98 samples were sequenced to a mean depth of 31,642 reads from nine individuals, four control volunteers undergoing routine colonoscopy and five patients undergoing surgical colectomy for medically-refractory UC. Samples were retrieved at four colorectal locations, incorporating the luminal microbiota, mucus gel layer and whole mucosal biopsies. RESULTS: Interpersonal variability accounted for approximately half of the total variance. Surprisingly, within individuals, asymmetric Eigenvector map analysis demonstrated differentiation between the luminal and mucus gel microbiota, in both controls and UC, with no differentiation between colorectal regions. At a taxonomic level, differentiation was evident between both cohorts, as well as between the luminal and mucosal compartments, with a small group of taxa uniquely discriminating the luminal and mucosal microbiota in colitis. There was no correlation between regional inflammation and a breakdown in this spatial differentiation or bacterial diversity. CONCLUSIONS: Our study demonstrates a conserved spatial structure to the colonic microbiota, differentiating the luminal and mucosal communities, within the context of marked interpersonal variability. While elements of this structure overlap between UC and control volunteers, there are differences between the two groups, both in terms of the overall taxonomic composition and how spatial structure is ascribable to distinct taxa.

Correlations between colonic crypt mucin chemotype, inflammatory grade and Desulfovibrio species in ulcerative colitis.

AIM: The colonic mucus gel layer is composed of mucins that may be sulphated or sialyated. Sulphated mucins predominate in health while in ulcerative colitis (UC) sulphation is reduced. These differences result directly from inflammatory events. It may also be hypothesized that they arise in part from alterations in the colonic microbiota, particularly changes in the burden of sulphated mucin-metabolizing species, such as Desulfovibrio (DSV) bacteria. The aim of this study was to correlate colonic mucin chemotypes and inflammatory scores in health and UC and relate these changes to changes in the colonization of colonic crypts by DSV. METHOD: Paired colonic biopsies from 34 healthy controls (HC) and 19 patients with active UC were collected for the purpose of parallel histological and microbiological assessment. High-iron diamine and Alcian blue staining and haematoxylin and eosin of mucosal biopsy specimens were used to assess histological changes within the clinical spectrum of UC. Quantitative real-time polymerase chain reaction analysis was employed to determine the total and DSV copy number within the colonic crypts. RESULTS: Compared with HC, the mucin chemotype in UC was less sulphated and inversely correlated with the degree of mucosal inflammation. A weak but significant negative correlation was found between the abundance of sulphated mucins and DSV burden. CONCLUSION: Mucin composition strongly correlates with the degree of mucosal inflammation, and to a lesser extent with DSV burden. These data suggest that mucin chemotype and DSV burden are linked phenomena and highlight the need to consider changes in mucin chemotype in the setting of microbial dysbiosis occurring within the colitic colon. What does this paper add to the literature? Decreased sulphation of mucins has been associated with inflammation in ulcerative colitis. Currently there are few data describing the relationship between microbial species and changes in mucin chemotype. This study validates previous findings and presents evidence of changes in mucin chemotype occurring in tandem with coherent changes in the microbiota within crypt niches.

Evaluation of serum transferrin receptor assay in a centralized iron screening service.

This study assesses the impact of permitting unrestricted access to requests for soluble transferrin receptor (sTfR) analysis in screening for iron deficiency (ID). Biochemical data including sTfR, serum ferritin (sFn), transferrin saturation, zinc protoporphyrins (ZPP) and also erythrocyte indices are used to highlight the differences between hospital (H) and general practitioner (GP) patient groups. A significantly higher number of abnormal sFn values (40%) over abnormal sTfR values (25%) occurred in GP patients. This trend was reversed in the H patient group where high sTfR values predominated. Consequently, screening with sFn, exclusively, missed ID (sTfR > 28.1 nmol/l) in 5% of GP patients and in 20% of H patients. Some 40% of H patients had elevated CRP values (CRP > 10 mg/l) indicating inflammatory disease, however, ZPP was more efficient than CRP at screening the validity of normal sFn values in the group. Unrestricted access to sTfR, sFn and ZPP analyses should expedite diagnosis in all patients, particularly H patients, but may be costly. The high specificity (>90%) of the mean cell haemoglobin for ID may be under-utilized diagnostically.

Import of human bifunctional enzyme into peroxisomes of human hepatoma cells in vitro.

A polypeptide containing the carboxyl-terminal fragment of human peroxisomal enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme was synthesized in vitro from its cDNA clone. This expression polypeptide was transported into purified rat liver peroxisomes. When the expression polypeptide was incubated with postnuclear supernatant fractions of human hepatoma cells and analyzed by Nycodenz gradient SDS-PAGE and fluorography, it was imported specifically into peroxisomes as indicated by its resistance to proteinase K degradation. A deletion of the last nine amino acid residues at the carboxyl-terminus of this polypeptide prevents its peroxisomal import. A tripeptide sequence, SKL, located at the carboxyl-terminus of human bifunctional enzyme appears to be the targeting signal for the peroxisomal importation of bifunctional enzyme in human cells.

Aberrant subcellular localization of peroxisomal 3-ketoacyl-CoA thiolase in the Zellweger syndrome and rhizomelic chondrodysplasia punctata.

Fibroblasts from patients with the inherited disorder Zellweger syndrome have few or no peroxisomes; multiple biochemical processes that normally occur in this organelle are defective. Rhizomelic chondrodysplasia punctata (RCDP) is another inherited disorder in which two unrelated peroxisomal metabolic processes, plasmalogen synthesis and phytanic acid oxidation, are impaired despite the normal appearance of peroxisomal structure. It was previously reported that one of the enzymes of peroxisomal fatty acid beta-oxidation, 3-ketoacyl-CoA thiolase (beta-keto-thiolase), was present in precursor rather than mature form in both of these diseases. Immunofluorescent staining for peroxisomal beta-ketothiolase showed the immunoreactivity to be localized in subcellular particles in fibroblasts from both Zellweger syndrome and RCDP patients, even though the former lack normal peroxisomes. Immunoblot studies were performed to determine the subcellular location of the thiolase precursor in fractionated fibroblasts from Zellweger and RCDP patients. In both disorders, thiolase immunoreactivity was detected in subcellular fractions having a lower density than normal peroxisomes and mitochondria, and was resistant to digestion by proteinase K. The density of the thiolase precursor-containing fractions was similar to that of peroxisomal membrane "ghost" fractions recently described by Santos et al. (J Biol Chem 263:10502-10509, 1988). Our results suggest that these are not empty membrane vesicles but contain at least one peroxisomal matrix protein. Furthermore, they exist not only in cells in which normal peroxisomes fail to form (Zellweger syndrome), but also in some cells which have catalase-containing peroxisomes (RCDP).

Peroxisomal bifunctional enzyme deficiency.

Peroxisomal function was evaluated in a male infant with clinical features of neonatal adrenoleukodystrophy. Very long chain fatty acid levels were elevated in both plasma and fibroblasts, and beta-oxidation of very long chain fatty acids in cultured fibroblasts was significantly impaired. Although the level of the bile acid intermediate trihydroxycoprostanoic acid was slightly elevated in plasma, phytanic acid and L-pipecolic acid levels were normal, as was plasmalogen synthesis in cultured fibroblasts. The latter three parameters distinguish this case from classical neonatal adrenoleukodystrophy. In addition, electron microscopy and catalase subcellular distribution studies revealed that, in contrast to neonatal adrenoleukodystrophy, peroxisomes were present in the patient's tissues. Immunoblot studies of peroxisomal beta-oxidation enzymes revealed that the bifunctional enzyme (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase) was deficient in postmortem liver samples, whereas acyl-CoA oxidase and the mature form of beta-ketothiolase were present. Density gradient centrifugation of fibroblast homogenates confirmed that intact peroxisomes were present. Immunoblots of fibroblasts peroxisomal fractions showed that they contained acyl-CoA oxidase and beta-ketothiolase, but bifunctional enzyme was not detected. Northern analysis, however, revealed that mRNA coding for the bifunctional enzyme was present in the patient's fibroblasts. These results indicate that the primary biochemical defect in this patient is a deficiency of peroxisomal bifunctional enzyme. It is of interest that the phenotype of this patient resembled neonatal adrenoleukodystrophy and would not have been distinguished from this disorder by clinical study alone.

Substance P-like immunoreactivity in the nervous system of hydra.

Using immunocytochemistry we find substance P-like material in nerve cells of hydra. These nerve cells are situated in the ectoderm of the basal disk and tentacles. Radioimmunoassay of hydra extracts gives dilution curves parallel to that of synthetic substance P, from which it can be calculated that one animal contains at least 0.6 fmol substance P-like immunoreactivity. After chromatography on Biogel P-100, the substance P-like immunoreactivity elutes as a peak in the void volume and a peak at the position of synthetic substance P.

Substance P in human cord blood and its degradation by placenta in vitro.

Plasma immunoreactive substance P (iSP) was measured by radioimmunoassay in 12 paired samples of maternal and cord blood, and in placental extracts. The mean plasma iSP concentration in the cord vein was 42.5 pg/ml, as compared to 77.3 pg/ml in the maternal peripheral vein plasma. There was no difference in the mean iSP concentration in the cord vein and artery. Although no iSP was detected in placental tissue, the placenta could not be excluded as the source of iSP in cord blood in view of the rapid degradation of synthetic SP by the placental in vitro.

Lower limb amputation in a general hospital: a comparative review.

This paper presents the results of a recent review of lower limb amputations carried out in a general hospital, and compares them with those of previous study of similar amputations. Particular attention is paid to the type of amputation-below-knee, through-knee or above-knee--and the associated morbidity, mortality and rehabilitation prospects. There is a need for an active approach to the problems of amputation with emphasis on preoperative preparation of the patient, the operation itself and rehabilitation follow-up in an amputation clinic.