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We previously identified two conjugated bile acid transporters, CbsT1 and CbsT2, in Lactobacillus johnsonii 100-100 and Lactobacillus acidophilus KS-13 that are gene duplicates encoded in tandem with a conjugated bile salt hydrolase (BSH) [Elkins and Savage, J. Bacteriol. 180:4344-4349, 1998; Elkins et al., Microbiology 147: 3403-3412, 2001]. CbsT2 from 100-100 was shown to increase taurocholic acid (TCA) uptake in Escherichia coli; however, higher levels were achieved when an extracellular factor (EF) from 100-100 was present in the assay medium (spent medium from 100-100, pH 4.2). We continued this study here to determine the role of EF in this transport system. Kinetic studies revealed that the previously observed CbsT2- and EF-mediated TCA accumulation is rapid (< 15 s) but not saturable, suggesting that EF is limiting. In addition, uptake of TCA by E. coli expressing CbsT2 was insensitive to ionophores, 2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone, and thus, is independent of the proton motive force. Since BSH converts [24-(14)C]TCA to [24-(14)C]cholic acid (CA), we measured net radiolabel uptake in E. coli cells expressing transporter(s) and BSH. Interestingly, such cells accumulated less 14C radiolabel (by approximately half) than cells expressing CbsT2 alone. These data can be explained if CA diffuses out of E. coli through the transporter(s). We, therefore, added exogenous, unlabeled CA to EF-spent media, which under our assay conditions, performed similarly to EF+ culture supernatant in TCA and CA uptake assays. Thus, unlabeled CA (a protonated, neutral lipophile) can partition directly into E. coli cells especially at low pH. These findings were validated in uptake assays with [3H]TCA, which yields [3H]taurine (a hydrophilic moiety) upon hydrolysis by the BSH. Amounts of cell-associated 3H radiolabel remained similar in cells expressing CbsT2 and BSH versus cells expressing only CbsT2, both of which were higher than in cells expressing BSH alone. Our data support a hypothesis that these transporters, which comprise a new subfamily of the major facilitator superfamily, facilitate antiport of TCA and CA., (Copyright 2003 S. Karger AG, Basel)

Lactobacillus johnsonii strain 100-100 expresses two antigenically distinct conjugated bile salt hydrolases (BSH), alpha and beta, that combine to form native homo- and heterotrimers. This paper reports characterization of loci within the genome that encode this capacity. A locus that encodes BSH beta (cbsH beta), a partial (cbsT1) and a complete conjugated bile salt transporter (cbsT2) was identified previously. DNA sequence analysis at this locus was extended and revealed a complete ORF for cbsT1 and no other ORFs in tandem. The three genes, cbsT1, cbsT2 and cbsH beta, probably constitute an operon; a putative promoter was identified upstream of cbsT1. A second locus that expresses BSH activity in strain 100-100 was identified. Sequence analysis of the clone predicted a 978 nt ORF that did not share tandem organization with other ORFs, was similar in sequence to other BSH genes, and matched, in predicted protein sequence, the first 25 amino acids of BSH alpha. A phenotypic screen for BSH activity and a genetic screen for the cbsH beta locus were performed on 50 Lactobacillus isolates from humans or dairy products. Nearly all of the isolates that were positive for cbsH beta were from human sources. Variability in the BSH phenotype and cbsH beta genotype was identified in isolates of the same species. DNA sequence was obtained and analysed from the cbsH beta locus of one human isolate, L. acidophilus strain KS-13. This organism has cbsT1, cbsT2 and cbs beta genes that are 84, 87 and 85% identical in DNA sequence to those of strain 100-100. DNA sequence identity to strain 100-100 ends in regions flanking this locus. The findings of this study suggest that BSH genes have been acquired horizontally and that BSH activity is important at some level for lactobacilli to colonize the lower gastrointestinal tract.

Bacteria of numerous species isolated from the human gastrointestinal tract express bile salt hydrolase (BSH) activity. How this activity contributes to functions of the microorganisms in the gastrointestinal tract is not known. We tested the hypothesis that a BSH protects the cells that produce it from the toxicity of conjugated bile salts. Forty-nine strains of numerous Lactobacillus spp. were assayed to determine their capacities to express BSH activities (taurodeoxycholic acid [TDCA] hydrolase and taurocholic acid [TCA] hydrolase activities) and their capacities to resist the toxicity of a conjugated bile acid (TDCA). Thirty of these strains had been isolated from the human intestine, 15 had been recovered from dairy products, and 4 had originated from other sources. Twenty-six of the strains expressed both TDCA hydrolase and TCA hydrolase activities. One strain that expressed TDCA hydrolase activity did not express TCA hydrolase activity. Conversely, in one strain for which the assay for TDCA hydrolase activity gave a negative result there was evidence of TCA hydrolase activity. Twenty-five of the strains were found to resist the toxicity of TDCA. Fourteen of these strains were of human origin, nine were from dairy products, and two were from other sources. Of the 26 strains expressing both TDCA hydrolase and TCA hydrolase activities, 15 were resistant to TDCA toxicity, 6 were susceptible, and 5 gave inconclusive results. Of the 17 strains that gave negative results for either of the enzymes, 7 were resistant to the toxicity, 9 were susceptible, and 1 gave inconclusive results. These findings do not support the hypothesis tested. They suggest, however, that BSH activity is important at some level for lactobacillus colonization of the human intestine.

Research on the indigenous intestinal microbiota of man was initiated well before the end of the 19th Century. The work continued at a slow but steady pace throughout the first half of the 20th Century. Findings from the effort had little impact on medicine and other aspects of human biology, however, until the 6th decade of the 20th Century. During that decade, research in the area was begun by eight groups of investigators, each of which was led by one or two senior scientists with great experimental talent, creativity and foresight. Their findings added new dimension to knowledge of the microbiota and initiated an explosion of interest in research in the field that has continued to the present day. The research of the groups during the 1960's is described in this review as a tribute to the senior scientists who had such critical impact on this important field of study.

The potential for biodegradation of aromatic hydrocarbons was evaluated in soil samples recovered along gradients of both contaminant levels and pH values existing downstream of a long-term coal pile storage basin. pH values for areas greatly impacted by runoff from the storage basin were 2.0. Even at such a reduced pH, the indigenous microbial community was metabolically active, showing the ability to oxidize more than 40% of the parent hydrocarbons, naphthalene and toluene, to carbon dioxide and water. Treatment of the soil samples with cycloheximide inhibited mineralization of the aromatic substrates. DNA hybridization analysis indicated that whole-community nucleic acids recovered from these samples did not hybridize with genes, such as nahA, nahG, nahH, todC1C2, and tomA, that encode common enzymes from neutrophilic bacteria. Since these data suggested that the degradation of aromatic compounds may involve a microbial consortium instead of individual acidophilic bacteria, experiments using microorganisms isolated from these samples were initiated. While no defined mixed cultures were able to evolve 14CO2 from labeled substrates in these mineralization experiments, an undefined mixed culture including a fungus, a yeast, and several bacteria successfully metabolized approximately 27% of supplied naphthalene after 1 week. This study shows that biodegradation of aromatic hydrocarbons can occur in environments with extremely low pH values.

Cytosolic extracts of Lactobacillus johnsonii 100-100 (previously reported as Lactobacillus sp. strain 100-100) contain four heterotrimeric isozymes composed of two peptides, alpha and beta, with conjugated bile salt hydrolase (BSH) activity. We now report cloning, from the genome of strain 100-100, a 2,977-bp DNA segment that expresses BSH activity in Escherichia coli. The sequencing of this segment showed that it contained one complete and two partial open reading frames (ORFs). The 3' partial ORF (927 nucleotides) was predicted by BLAST and confirmed with 5' and 3' deletions to be a BSH gene. Thermal asymmetric interlaced PCR was used to extend and complete the 948-nucleotide sequence of the BSH gene 3' of the cloned segment. The predicted amino acid sequence of the 5' partial ORF (651 nucleotides) was about 80% similar to the C-terminal half of the largest, complete ORF (1,353 nucleotides), and these two putative proteins were similar to several amine, multidrug resistance, and sugar transport proteins of the major facilitator superfamily. E. coli DH5alpha cells transformed with a construct containing these ORFs, in concert with an extracellular factor produced by strain 100-100, demonstrated levels of uptake of [14C]taurocholic acid that were increased as much as threefold over control levels. [14C]Cholic acid was taken up in similar amounts by strain DH5alpha pSportI (control) and DH5alpha p2000 (transport clones). These findings support a hypothesis that the ORFs are conjugated bile salt transport genes which may be arranged in an operon with BSH genes.

The use of filter paper four point 24 hour cortisol profiles in the diagnosis and management of secondary adrenocortical deficiency was examined. Eighteen children with familial short stature and isolated growth hormone deficiency had normal 24 hour and morning plasma cortisol concentrations measured. Eight of 11 children with multiple pituitary hormone deficiencies had evidence of hypocorticalism despite previously normal baseline cortisols or responses to insulin hypoglycaemia or tetracosactrin. Nine of 11 children with hypopituitarism on replacement treatment (4.9-12.5 mg/m2/day) had satisfactory concentrations of cortisol, though morning cortisols were often low. Filter paper profiles are a simple, inexpensive, and relatively non-invasive way of managing children with suspected hypocorticalism and of monitoring their treatment.

The presentation, pattern of acute illness, and incidence of learning difficulties are described in 63 (33 boys, 30 girls) children with salt wasting 21-hydroxylase deficiency, drawn from a cohort study of congenital adrenal hyperplasia in the South West Region of England between 1968 and 1988. Thirty boys presented with a salt losing crisis from birth whereas the other three boys presented between 2 and 14 months of age with failure to thrive and hyponatraemia. Diagnostic uncertainty led to 13 (43%) of 30 girls developing a salt losing crisis. Five girls were misassigned as boys at birth. There were four deaths in the group, two due to salt losing crisis, one to complications of prematurity possibly compounded by 21-hydroxylase deficiency, and one from heart failure probably related to an excess of steroids. Acute admissions were common, especially during the first year of life, with convulsions in 7% of admissions. The 9% incidence of hypoglycaemia was considered to be an underestimate as blood glucose was measured in only 56 (22%) of 254 admissions. No convulsions occurred in the 38 (15%) admissions where the parents had given intramuscular hydrocortisone before bringing the child to hospital. A high incidence of learning difficulties was found among the 59 surviving children (9/30 (30%) boys and 6/29 (21%) girls), and in only two children could any factor other than 21-hydroxylase deficiency be invoked. Analysis of the subgroup with learning difficulties indicated that they were more ill at presentation with a significantly higher incidence of hypoglycaemia, and that growth in the first year was significantly worse. It is concluded that congenital adrenal hyperplasia remains a formidable disorder with an appreciable mortality and morbidity. The high incidence of learning difficulties seen in salt wasting 21-hydroxylase deficiency needs further attention. A prospective study is indicated to examine the effect of neonatal screening on morbidity from congenital adrenal hyperplasia, particularly the intellectual impairment seen in this study.

Roseburia cecicola is an obligately anaerobic bacterium that is extremely sensitive to oxygen. Genomic DNA isolated from cells exposed to air for even a brief period (< 5 min) is partially degraded, while DNA extracted from cells maintained in an anaerobic environment remains intact. Cells exposed to air for longer and longer periods yield DNA which is progressively degraded into fragments with decreasing sizes. Oxygen toxicity for this anaerobe appears to result, at least in part, from degradation of its genomic DNA. Cell lysates of the organism exhibited a similar ability to degrade exogenous sources of DNA when assayed in vitro under aerobic conditions. A substance that degrades both DNA and RNA when incubated aerobically was partially purified from such lysates. It has an approximate molecular weight of 2,800 and is unlikely to be a protein. It requires a reducing agent for activity and can be inhibited by catalase and peroxidase but not superoxide dismutase. The rate at which it degrades DNA in vitro can be enhanced by temperatures above 37 degrees C or by oxygen at partial pressures above atmospheric pressure. These results suggest that this substance degrades nucleic acids by a mechanism involving oxygen radicals.

Four isozymes of bile salt hydrolase (BSH) have been purified from the cytosol of cells of Lactobacillus sp. strain 100-100. The four proteins were designated BSH A, B, C, and D. They eluted from anion-exchange high-pressure liquid chromatography columns at 0.15, 0.18, 0.21, and 0.25 M NaCl, respectively. They are catalytically similar, except that the Vmax of BSH D is about 10-fold lower than those of the other three isozymes. All four proteins consist of one or two polypeptides. The peptides have molecular weights of 42,000 and 38,000 and are designated alpha and beta, respectively. The approximate native molecular weights of BSH A, B, C, and D are 115,000, 105,000, 95,000, and 80,000, respectively. The native proteins are probably trimers; the four isozymes are the array of possible subunit combinations alpha 3, alpha 2 beta 1, alpha 1 beta 2, and beta 3 for A, B, C, and D, respectively. The two subunits are antigenically distinct. Polyclonal antibodies raised against BSH A (all alpha peptide) react in Western blots (immunoblots) only with proteins containing the alpha peptide; such antibodies raised against BSH D (all beta peptide) react only with proteins containing the beta peptide. The amino acid compositions of the two peptides differ. This is the first report of a bacterium that makes four BSH isozymes.

Bile salt hydrolase activity in Lactobacillus sp. strain 100-100 is strictly intracellular. The strain produces an extracellular factor that stimulates the intracellular hydrolase activity. The factor is inducible by conjugated bile salts, has an apparent molecular mass over 12 kDa but less than 25 kDa, is stable in air, and resistant to pronase and heat. It is partially extractable into organic solvents and inactivated by a sulphydryl group inhibitor. We postulate that the factor functions by a novel mechanism to facilitate entry of conjugated bile salts into the bacterial cells.

Lactobacillus strains of numerous species isolated from several animal sources exhibited cellular hydrophobicities that differed from those expected on the basis of their abilities to colonize the keratinizing stratified squamous epithelium in the mouse stomach. Cells of Lactobacillus fermentum 100-33, grown to either exponential or stationary phase, were strongly hydrophilic. By contrast, cells of L. fermentum RI and six transformant derivatives of strain RI and 100-33, strains DM101 through DM106, were hydrophobic to various degrees in either growth phase. Most of them were less hydrophobic, however, when in the stationary phase than in the exponential phase. Cells of strains RI and 100-33 in the exponential phase adhered in the same number in vitro to disks of keratinized mouse gastric mucosa. By contrast, when in stationary phase, strain RI and two transformants, DM103 and DM104, adhered to the surface in higher numbers than 100-33. In contrast to their cellular progenitor, 100-33, the transformant strains share with their DNA donor, RI, the capacity to colonize the keratinizing gastric epithelium in mice. These findings indicate that lactobacilli able to colonize the surface of the keratinocytes in the murine stomach can adhere to that surface by either hydrophilic or hydrophobic molecules.

Data from our previous studies suggested that the fungal cell wall component, chitin, is involved in the adhesion of Candida albicans to mucosal surfaces. In the present study, we investigated the effect of polyoxin D, an inhibitor of chitin synthase, on the interaction of the fungus with epithelial cells. The effect of polyoxin D on Candida was evaluated in in vitro assays for its capacity to adhere to buccal epithelial cells (BEC), and by fluorescent-microscopy photometry and flow cytometry using cells stained with cellufluor (CF), a fluorochrome with affinity for chitin. C. albicans grown with and without polyoxin D was stained with CF and examined in a fluorescent microscope equipped with a photometer. Measurements of fluorescence revealed a wide range of intensity among C. albicans cells and a decreased intensity in polyoxin D treated cultures. Flow cytometry analyses of yeasts revealed 2 peaks of fluorescence intensity, and pointed to differences between polyoxin D treated and non-treated microorganisms. C. albicans stained with CF were separated into 2 subpopulations by flow cytometry according to fluorescence intensity. In vitro adhesion of each subpopulation to BEC was similar. Polyoxin D treated fungi showed significantly reduced adherence to BEC, as evaluated by a radioactivity assay with radiolabelled yeasts and by microscopic readings. The reduction in adhesion was Polyoxin D concentration dependent. These observations support our previous findings suggesting involvement of chitin in the attachment process of C. albicans (CBS562) to epithelial cells.

We describe three children, now aged between 5 and 6 years, with a persistent mild hyperthyrotropinaemia since the neonatal period and normal levels of thyroid hormones. The increased thyroid stimulating hormone concentration is not artefactual and is not caused by antibody interferences. Their growth and development is normal and none has received thyroid hormone replacement. We believe that they have compensated hypothyroidism, and that before the advent of screening for congenital hypothyroidism these children would have presented in mid-childhood with juvenile hypothyroidism.

We have characterized and purified the bile salt hydrolase from Lactobacillus sp. strain 100-100. Bile salt hydrolase from cells of the strain was purified with column and high-performance liquid chromatography. The activity was assayed in whole cells and cell-free extracts with either a radiochemical assay involving [14C]taurocholic acid or a nonradioactive assay involving trinitrobenzene sulfonate. The activity was detectable only in stationary-phase cells. Within 20 min after conjugated bile acids were added to stationary-phase cultures of strain 100-100, the activity in whole cells increased to levels three- to fivefold higher than in cells from cultures grown in medium free of bile salts. In cell-free extracts, however, the activity was about equal, 1.41 and 1.53 mumol/min per mg of protein, respectively, whether or not the cells have been grown with bile salts present. When supernatant solutions from cultures grown in medium containing taurocholic acid were used to suspend cells grown in medium free of the bile salt, the bile salt hydrolase activity detected in whole cells increased two- to threefold. Two forms of the hydrolase were purified from the cells and designated hydrolases A and B. They eluted from anion-exchange high-performance liquid chromatography in two sets of fractions, A at 0.15 M NaCl and B at 0.18 M NaCl. Their apparent molecular weights in nondenaturing polyacrylamide gel electrophoresis were 115,000 and 105,000, respectively. However, discrepancies existed in the apparent molecular weights and number of peptides detected in sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the two forms. Both had similar substrate specificities, highest on taurodeoxycholic and glycocholic acid, and pH optima between 3.8 and 4.5. The kinetic properties were also similar, with Vmaxs of 17 and 53 micromoles/min per mg of protein and Kms of 0.76 and 0.95 mM taurocholic acid for A and B, respectively. Therefore, whether the enzyme exists in two forms in the cells remains to be determined.

Eighteen diabetic children ate three different snacks (free sucrose, sucrose plus fibre, fructose plus fibre) or had no snack on each of 4 mornings. Subsequently 10 children from this group took a standard snack (free sucrose) or no snack on two afternoons. In other respects the day of testing was standardised, the children going to school as normal and collecting their blood spots on filter paper for glucose analysis. There was no difference in the blood glucose profiles between different snacks or when no snack was taken either in the mornings or afternoon. This suggests both that moderate amounts of simple sugar do not have a detrimental effect on glycaemic control and that snacks can generally be omitted without serious hypoglycaemic problems.

Opsonized Rhodococcus equi activated the respiratory burst of resident alveolar macrophages (AM) from adult horses in a logarithmic-linear, mass-related manner. The effect of R equi was not significantly different from that of equal masses of opsonized zymosan A. Therefore, R equi does not appear to attenuate the respiratory burst of equine AM. The stimulatory effect of R equi was not reflected by increased production of superoxide anion (O2-), but increased activity of the hexose monophosphate shunt was observed. These results suggest a similarity between the respiratory burst of AM from horses and that of AM from rabbits. We concluded that resident AM from adult horses do not produce O2- concurrently with an increase in activity of the hexose monophosphate shunt when stimulated with either opsonized zymosan A or opsonized R equi. This suggests that O2- is not an important component of the antibacterial defenses of equine AM. Whether equine AM are incapable of producing O2- or require different stimuli to produce it was not determined.

Lactobacillus sp. strain 100-33 is resistant to macrolides, lincosamides, and streptogramin B-type antibiotics (MLSR) and appears to contain several major and minor plasmids. One of these plasmids, pLAR33, is approximately 18 kbp in size. When cells of strain 100-33 were protoplasted and regenerated, an MLSS isolate was derived. The derivative, designated strain ES1, contained a unique plasmid complement in which it had apparently lost the major plasmids of the parental strain, including pLAR33, and retained only a minor plasmid seen in low concentrations in strain 100-33. The MLSR determinant was cloned from plasmid DNA of strain 100-33 on a 3-kbp EcoRV fragment into pBR322 and localized to pLAR33. The determinant expressed macrolide and lincosamide resistance in Escherichia coli HB101, was localized to approximately 1 kbp on the cloned sequence, and is apparently under the control of its own promoter. MLSR electroporants were derived from strain ES1 electroporated with plasmid DNA from strain 100-33; these MLSR isolates had acquired a plasmid complement similar to that of strain 100-33, including pLAR33. Endonuclease digestion and Southern analysis of plasmid DNA from both strains indicated that the major plasmids are multimeric and deleted forms of one archetypal extrachromosomal element.

Strains of indigenous Escherichia coli, Bacteroides, and Lactobacillus were isolated from the gastrointestinal tracts of specific pathogen-free (SPF) mice. Nonvaccinated SPF mice exhibited in their spleens low numbers of plaque-forming cells (PFC) and rosette-forming cells reacting with antigens of these andigenous bacteria. PFC reacting with these bacterial antigens were not detected in infant SPF mice until 7 days after birth. Compared with nonvaccinated controls, SPF mice vaccinated parenterally with indigenous E. coli or Bacteroides produced a moderate increase in the numbers of specific PFC. Thus, the SPF mouse is capable of responding immunologically after vaccination with microbes indigenous to its intestinal tract. However, more PFC reacting with homologous vaccine antigens were detected after parenteral vaccination of SPF mice with nonindigenous E. coli O127:B8, E.coli O14, or B. fragilis than after parenteral vaccination with indigenous E. coli or Bacteroides. Gnotobiotic mice orally monoassociated with these nonindigenous bacteria exhibited greater immune responses to antigens of the bacteria used to monoassociation than did gnotobiotes monoassociated with the indigenous microbes. The results are consistent with the hypothesis that mice are more responsive immunologically to antigens of nonindigenous bacteria than they are to antigens of certain microbes indigenous to their gastrointestinal tracts.

Some indigenous microorganisms localize on epithelial surfaces in various areas of the digestive tracts of animals. One of these, a segmented, filamentous microbe, localizes on the epithelium in the small bowels of mice and rats. These filamentous microbes colonize mice at weaning time and persist in adult animals for at least 2 months. Results of the study of light and electron micrographs suggest that the microorganisms are procaryotic, and that they interact with small bowel epithelial cells to form an attachment site. This site consists of modified epithelial cell membrane and apical cytoplasm adjacent to the attached bacterium. The microbe fills the site with part of its first segment. This segment has a nipple-like appendage on the end inserted into the epithelial cell. The other segments, which compose the rest of the filament, are usually separated by septa. Many of the individual segments contain intrasegmental bodies that appear to be procaryotic cells. Some of these intrasegmental bodies are similar in morphology to the first segment of each filament inserted into an epithelial cell. These intracellular bodies may be components in the life cycle of the microorganism. The organism has not yet been cultured in recognizable form. Therefore, such a hypothesis cannot be proved as yet, nor can the microbe be classified with certainty. Because it localizes in an epithelial habitat in the small bowel, however, it may be a particularly important microbial type in the gastrointestinal ecosystem of laboratory rodents.

Congenital adrenal hypoplasia is reported in two siblings. The first died at 16 months of purulent bronchopneumonia after a history of adrenal insufficiency. No gross adrenal tissue was found at autopsy and urinary steroids were not excreted in detectable amounts before death. In a subsequent uncomplicated pregnancy, extremely low estrogens were recorded in the last trimester. Analysis of steroids in the urine of the neontate by gas chromatography revealed virtual absence of 3 beta-hydroxy-5-ene steroids. These suggest hypoplasia of the fetal adrenal cortex. Metabolites of cortisol were excreted in normal amounts and responded adequately to ACTH stimulation. Neonatal hyponatremia was associated with subnormal excretion of corticosterone and aldosterone metabolites. It is proposed that in the perinatal period, the fetal zone is required for mineralocorticoid synthesis, possibly by providing essential precursor steroids, e.g. 21-hydroxypregnenolone.

Germfree CD-1 mice challenged by the oral route with Salmonella typhimurium had ceca that were abnormal in appearance and reduced in size compared to those of germfree controls. Similarly, germfree mice injected with heat-killed S. typhimurium or gnotobiotes associated with three indigenous microbes (Lactobacillus, Bacteroides, Clostridium), and subsequently challenged with S. typhimurium also had small ceca. By contrast, gnotobiotic mice that had been both injected with the heat-killed S. typhimurium and associated with the three indigenous microbes before challenge with S. typhimurium had ceca similar in size and appearance to germfree mice. Thus, indigenous microorganisms could interfere with the mechanism by which the pathogen induced the decrease in cecal size, but could do so only in mice injected with heat-killed bacteria. This phenomenon suggests synergism between the interference effected by the indigenous bacteria and the resistance mechanisms of the animal.

Children with medulloblastoma in Bristol are treated surgically and with craniospinal irradiation, and in some cases chemotherapy. Thirteen medium or long term survivors were investigated to determine their growth and endocrine function. Their rate of growth was considerably reduced through the first year of their illness and after spinal irradiation spinal growth was poor. Nine children developed growth hormone deficiency. They were clinically euthyroid but 7 had raised basal thyroid stimulating hormone values. Gonadal function was abnormal in all but the youngest child. The rate of survival is increasing in children with medulloblastoma but this is associated with appreciable endocrine abnormalities. Some of these problems are present shortly after treatment ends but others may develop later and long term surveillance is therefore essential.

Flagella from Roseburia cecicola, an obligately anaerobic bacterium originally isolated from murine caecal mucosa, were purified by mechanical shearing followed by differential centrifugation. Purity of the flagellar preparation was determined by polyacrylamide gel electrophoresis, electron microscopy and chemical analysis. The flagella were composed of a single protein subunit (flagellin) with an estimated molecular weight of 42 000. The amino acid composition of the flagellin was similar to that of some facultatively anaerobic and aerobic bacteria.

Ninety diabetic children each provided at least one 24-hour blood glucose profile at home using an impregnated filter paper strip. The mean 24-hour blood glucose level correlated significantly with urine control, height velocity, and Hb A1. The correlation coefficient for individual blood glucose values (r = 0.61) and for mean 24-hour blood glucose values (r = 0.73) repeated within 14 days showed an acceptable degree of reproducibility for the blood glucose profiles. Mean 24-hour blood glucose values fell significantly overall (11.4 to 9.8 mmol/l; 205 to 176 mg/100 ml) in 47 children who had repeated profiles more than 2 weeks apart. Unrecognised nocturnal hypoglycaemia (less than 3.0 mmol/l; 54 mg/100 ml) was found in 19% of children on twice-daily Semitard insulin. The study shows that children over age 7 years manage home blood glucose monitoring without difficulty. It shows that the results are reproducible and correlate with other indices of control, and that it provides a practical basis for the improvement of diabetic control.

A 7 year old girl with precocious puberty was treated with buserelin, a long acting analogue of gonadotrophin releasing hormone. Spontaneous and stimulated gonadotrophin secretion became prepubertal but returned to pubertal values when buserelin was withdrawn, suggesting that normal sexual maturation should follow cessation of treatment.

This study describes an in-vitro assay for estimating numbers of Candida albicans adherent to mouse intestinal mucosa. No significant differences were observed in the capacity of C. albicans to adhere to genetically distinct mouse strains nor in the binding ability of three C. albicans strains. Chitin soluble extracts, prepared from chitin isolated from C. albicans or from chitin obtained commercially, inhibited adhesion of the yeasts to duodenal tissue by 48% and 43%, respectively.

Twenty-one insulin-dependent diabetic children completed a trial of 4 different breakfasts, given in random order. Three diets differed in fibre content. The fourth diet contained soya beans as part (38%) of the dietary fibre source. Children collected capillary blood samples on to filter paper strips which were analysed for blood glucose content. Each morning the children were asked to exercise vigorously for an hour and to rest for an hour, resulting in comparable rest and exercise periods for each child. Mean initial blood glucose levels on the 4 diets were not significantly different. The low-fibre diet resulted in the highest blood glucose concentrations after breakfast. Blood glucose levels on the high-fibre diet did not differ from those on the medium-fibre diet. The bean diet produced the lowest mean blood glucose level and the smallest reduction in blood glucose level in the hour before lunch. All the children found the bean diet unacceptable but liked the high- and medium-fibre diets, which were as popular as the low-fibre diet. The level of prescribed exercise had no effect on the level of blood glucose. It appears that the potentially major benefits from beans are limited by their unpalatability. The more acceptable cereal fibre produces a smaller but important benefit on morning hyperglycaemia after breakfast.

The protein content and the activities of alkaline phosphatase, maltase, and sucrase were measured at 0800, 1000, 1200, 1400, and 1600 in saline extracts of the proximal small bowels of germfree and of ex-germfree mice colonized with an indigenous microbiota. In extracts prepared from germfree mice, the total activities of all of the enzymes were relatively constant throughout the sampling period. Likewise, the total activity of alkaline phosphatase in extracts prepared from associated mice varied little as a function of time. By contrast, the total activities of maltase and sucrase in the extracts from these latter animals varied significantly from sample to sample. The total activity levels in extracts from germfree mice were approximately twofold greater than the levels in extracts from associated mice. The specific activities of alkaline phosphatase and sucrase did not vary from sample to sample in extracts prepared from either type of mouse. In contrast, the specific activity of maltase in extracts prepared from both germfree and associated mice differed significantly from sample to sample. The specific activities of all three enzymes were greater in extracts from germfree animals than in those from associated animals. The protein content of extracts prepared from germfree mice also was greater than that of extracts prepared from associated animals at every sampling time. The amount of protein extractable from the mucosa of the small bowels of the former animals varied significantly at different sampling times during the day, whereas the amount of protein extractable from the tracts of associated animals remained relatively constant throughout the day. The indigenous microbiota apparently stabilizes in some way the amount of protein extractable from the mucosa of the mouse small bowel.

Segmented, filamentous prokaryotic microorganisms colonize and attach to the cells in the epithelium of the mucosa of the small bowels of mice and rats. Scanning electron micrographs, derived from specimens of mouse small intestine, reveal microbial filaments of at least two types. One type is thin (0.8μm) with only faint lines suggesting septa; the other is thicker (1.4μm) and has distinct segments with pronounced septa. Most of the segments are rounded; a few are thin and elongated. Immediately surrounding the attachment site of these organisms, the surface of the epithelial cells appears roughened and occasionally stringy. The filaments may differ morphologically because they represent different phases in the life cycle of a single microbial type. Alternatively, however, they may differ because they are the cells of different microbial types colonizing the same epithelial habitat.

Germfree mice housed in isolators under controlled environmental and nutritional conditions were associated with an intestinal microflora. These associated animals and germfree mice drawn from the same population were tested for the rate at which the epithelial cells transited from the crypts of Lieberkuhn to the tips of the villi in their small intestines. The method for estimating the rate of transit of epithelial cells involved the use of liquid scintillation counting to determine the amount of radioactivity entering the cells while the animals were being injected with [3H]thymidine and statistical analysis of th data with a computer program developed for the purpose. As estimated by that method, the cells transited from the crypts to the villous tips in germfree mice in about 115 h and in the associated animals in about 53 h. In monoassociated mice, a strain of a Lactobacillus sp. had no effect on the transit time of the epithelial cells. A strain of Torulopsis pintolopesii stimulated uptake of 3[H]thymidine by the small bowel mucosae in mice monoassociated with the organisms for 5 weeks. In animals monoassociated with the yeast fo 3, 4, and 6 weeks, however, the radioactive compound was incorporated into the bowel mucosae to the same extent as the mucosae of germfree mice. Therefore, similarly to the Lactobacillus strain, T. pintolopesii has no obvious influence on the transit rate of small bowel epithelial cells.

A freeze-fracture study has provided new information about the filamentous, segmented microorganism known to live in the murine small bowel. The intracellular bodies produced by this microbe appear to arise by a modified sporogenesis so that they are enclosed in an envelopment membrane at least prior to release by the filament mother cell. At least some of the intracellular bodies divide while still within the mother cell, suggesting a reproductive role for these structures. The host epithelial membrane remains intact at the site of attachment, but does appear to have a reduced concentration of intramembrane particles. Changes in the host cytoplasm adjacent to the attachment site are documented and interpreted to be a sol-gel transformation which may stabilize the attachment socket.

Aerobic and anaerobic cultural techniques and histological methods were used in a study of the effects of environmental and dietary stress on the indigenous microbiota of the gastrointestinal tract of mice. Mice previously inoculated with Salmonella typhimurium were examined in a similar manner. Three strains of mice (CD-1, Ha/ICr, and C57BL) were used. Control animals previously inoculated with S. typhimurium had low population levels of Salmonella bacteria in the small and large bowel. Mice previously inoculated with Salmonella and then deprived of food, water, and bedding for 48 h harbored high population levels of these bacteria in their small and large bowels. Coliforms increased in numbers in the large bowel of stressed mice inoculated with Salmonella and in the jejunum-ileum and cecum of stressed mice not previously inoculated with Salmonella. Control mice had high population levels of lactobacilli inhabiting the keratinized squamous epithelium of the stomach. Stressed mice showed dramatic reductions in these populations of lactobacilli. Populations of fusiform-shaped bacteria associated with the mucosal epithelium of the cecum and colon in control mice were reduced in stressed mice as determined by microscope examination of histological sections. Total anaerobic counts were similar, however, in both stressed and control animals. Environmental and dietary stress markedly alter the gastrointestinal microbiota in mice. Therefore, such stressful conditions profoundly affect the factors that regulate the localization and population levels of microorganisms in the stomach and intestines.

The excretion of 7 individual 17-oxosteroids and 7 individual corticosteroids in 24 h urine samples from 62 normal infants, children and adolescents, based on an accurate and specific paper chromatographic method for their separation and quantitation, is reported. The excretion of the 11-deoxy-17-oxosteroids gradually increases from 7 years of age and the increase becomes more rapid 2 or 3 years before the clinical signs of puberty appear. The rise continues throughout puberty and beyond it until the adult level is reached. The increase far exceeds that which would be accounted for by the growth of the individual. The increase in the excretion of the 11-oxy-17-oxosteroids with age is much more gradual. Androgens favour the formation of 5alpha metabolites and the 5alpha:5beta ratio of the total 5alpha 17-oxosteroids and the total 5beta 17-oxosteroids shows a statistically significant increase with age. In addition, a relatively high 5alpha:5beta ratio is noted in male infants, which is likely to be related to their relatively high plasma testosterone levels. The excretion of the 17-hydroxycorticosteroids and the alpha-ketolic metabolites of cortisol gradually rises with age and correlates with body weight. The alpha-ketolic metabolites of corticosterone are relatively high in infancy, but after the age of 4 years their excretion also correlates with body weight. An increase in the 5alpha:5beta ratio of allo-THF to THF is noted at puberty similar to that found with the 5alpha:5beta ratios of the 17-oxosteroids.

Torulopsis pintolopesii is a yeast indigenous to the gastrointestinal tracts of conventional mice and rats from many colonies. In such natively colonized animals, the organism forms layers on the surface of the epithelium in the secreting portion of the stomach and can be cultured from all areas of the gastrointestinal tract. When given in water or food to germfree mice or specific pathogen-free mice possessing an indigenous microbiota free of yeast, T. pintolopesii also can be cultured from all areas of the tract at population levels ranging from 10(5) to 10(8) cells per g (wet weight). Likewise, as in its native hosts, the organism forms layers on gastric surfaces in the associated animals. The layers form on the secreting surface in both the specific pathogen-free and monoassociated ex-germfree mice. In the latter animal, however, a layer of yeast also forms on the nonsecreting gastric surface. In tests of its capacity to adhere to gastrointestinal surfaces in vitro, the organism adheres to epithelia from all areas of the mouse tract. These findings support an hypothesis that the capacity of T. pintolopesii to adhere to epithelial surfaces may be only one determinant influencing it to form layers on the gastric secreting surface in its native hosts.

Two assays were designed with which isogenic laboratory strains of Escherichia coli K-12 with and without ColV plasmids were compared for their ability to adhere in vitro to mouse intestinal epithelium. In both assays, disks of intestinal tissue were exposed to bacteria. In the first, disks were homogenized and the numbers of viable bacteria adherent to them were estimated from colony counts of plates inoculated with dilutions of the homogenates. In the second, bacteria were labeled with [(14)C]aspartic acid; the number of adherent cells per disk was estimated by liquid scintillation spectrometry. Data from each assay were compared by analysis of variance. In both assays, strains bearing the ColV plasmid adhered in two- to threefold-greater numbers than isogenic strains without the plasmid. These differences were highly significant statistically. A non-colicinogenic strain free of the ColV plasmid was selected by treatment of a ColV strain with sodium dodecyl sulfate. In the radioisotopic assay, the ColV strain associated with the epithelium in significantly greater numbers than the cured derivative. A ColV strain was created by conjugation; in the radioisotopic assay this strain bound to epithelium in significantly greater numbers than the recipient strain without the plasmid. The original ColV strain, when negatively stained and examined by electron microscopy, had pili that adsorbed male-specific bacteriophage, whereas its isogenic variant without ColV did not. Some such properties, coded by the plasmid, may increase the virulence of the bacteria.

Lactobacillus isolates able to colonize the surfaces of the nonsecreting epithelia in the stomachs of monoassociated ex-germfree mice were derived from Lactobacillus acidophilus 100-33. Strain 100-33 was originally isolated from pig feces and is unable to colonize the murine gastric epithelium. In experiments involving attempts genetically to transform the capacity to colonize the epithelium, cells of strain 100-33 were treated with muralytic enzymes and mixed with polyethylene glycol and genomic or plasmid DNA extracted from Lactobacillus fermentum RI. Strain RI was originally isolated from a conventional mouse and has the capacity to colonize the nonsecreting gastric epithelium. The mixtures containing cells, polyethylene glycol, and DNA were plated on a regeneration medium. After overnight incubation, the cells were washed from the plates and introduced by gastric gavage into germfree mice. Only mice that received regenerated 100-33 cells previously mixed with genomic DNA from strain RI had layers of gram-positive bacteria on the keratinized epithelia of their stomachs. Six isolates cultured from the washed gastric tissues of these animals were characterized. When a culture of each or a pool of cultures of the six were orally administered to germfree mice, layers of gram-positive bacterial cells were visible on the keratinized gastric epithelia of the animals within 1 to 3 weeks. Cells of all six, but not of strain 100-33, reacted with antibody made in rabbits to L. fermentum RI cells, as determined by an enzyme-linked immunosorbent assay. Nevertheless, all six had fermentation profiles identical to that of strain 100-33.(ABSTRACT TRUNCATED AT 250 WORDS)

Growth within the pH range 2 to 8 of a strain of the yeast Torulopsis pintolopesii was tested in media containing various sugars as carbon and energy sources. Of the sugars tested, only D-glucose, D-fructose, and D-mannose supported growth of the yeast. In media containing those sugars, the organism grew over the entire pH range tested.

Thyroid function was assessed in three selected groups of children who had survived cancer. Children in group 1 had received radiotherapy to the thyroid area, group 2 had radiotherapy to the thyroid area and adjuvant chemotherapy, and group 3 had chemotherapy with or without radiotherapy away from the thyroid area. There were 75 survivors and 63 (40 boys, 23 girls) were available for study. Eighteen (29%) were found to have thyroid dysfunction, and these included all those who had had lymphangiograms or received a radiation dose greater than 40 Gy to the thyroid area. Only nine of the 18 children were already known to have thyroid dysfunction, and only 15 of 44 children who had had irradiation to the thyroid area had had their thyroid function examined. This study shows that children who have received radiotherapy to the thyroid area should have their thyroid function assessed regularly. Chemotherapy does not appear to be a risk factor but longer follow up of these children is necessary.

Indigenous segmented filamentous microbes attach to murine ileal epithelial cells. These microbes can be seen on the epithelial surface with a scanning electron microscope. They colonize preferentially the distal ileum in mice. Penicillin, placed in the animal's drinking water, eliminates the microbes from the mouse ileum, but recolinization of the ileum is observed 4 to 5 weeks after the penicillin treatment is stopped. Within 3 to 5 h after rats are given penicillin, the morphology of the microbes is changed. Their external surfaces are wrinkled or broken. Vacated and partially vacated attachment sites are observed. Almost all of the organisms disappear from murine ilea after the animals are exposed to penicillin for 10 h. These observations are discussed in relation to the microbe itself and in its interaction with ileal epithelial cells.