Faecal microbiota transplant for recurrent Clostridium difficile infection using long-term frozen stool is effective: clinical efficacy and bacterial viability data.

BACKGROUND: Faecal microbial transplant (FMT) for recurrent Clostridium difficile infection (rCDI) is greatly facilitated by frozen stool banks. However, the effect of frozen storage of stool for greater than 2 months on the viability of stool bacteria is unknown and the efficacy of FMT is not clear. AIM: To evaluate the viability of bacteria in stool frozen for up to 6 months, and the clinical efficacy of FMT with stool frozen for 2-10 months, for the treatment of rCDI. METHODS: Viability of six representative groups of faecal bacteria after 2 and 6 months of storage at -80 °C, in normal saline (NS) or 10% glycerol were assessed by culture on plate media. The clinical outcomes of 16 consecutive patients with rCDI treated with aliquots of stool frozen in 10% glycerol and stored for 2-10 months were also examined. RESULTS: Viability at both 2 and 6 months was similar to baseline, in specimens stored in 10% glycerol and at 2 months in stool stored in NS, but was reduced by >1 log at 6 months for Aerobes (P < 0.01), total Coliforms (P < 0.01) and Lactobacilli (P < 0.01) in NS. Using stool frozen for 2-10 months in 10% glycerol, the cure rate for rCDI was 88% with one FMT and 100% after repeat FMT in those who relapsed. CONCLUSION: Stool for faecal microbial transplant to treat rCDI can be safely stored frozen in 10% glycerol for at least 6 months without loss of clinical efficacy or viability in the six bacterial groups tested.

Overestimation of the abundance of sulfate-reducing bacteria in human feces by quantitative PCR targeting the Desulfovibrio 16S rRNA gene.

The dominant genus of sulfate-reducing bacteria (SRB) in humans is Desulfovibrio, and quantitative PCR (QPCR) targeting the 16S rRNA gene is often used in assays. We show that the 16S rRNA gene assay overestimated SRB abundance in feces from 24 adults compared to QPCR assays using primers targeting two genes involved in SRB energy metabolism.

Resistant starch, large bowel fermentation and a broader perspective of prebiotics and probiotics.

The metabolic end products of the large bowel microbiota contribute significantly to human health. After weaning to solid foods, some of the most important of these are the short chain fatty acids (SCFA) produced by the fermentation of undigested dietary components and endogenous secretions. The main SCFA are acetate, propionate and butyrate which have numerous documented effects promoting large bowel function. Of the major acids, butyrate seems especially important. It is a major metabolic fuel for colonocytes and promotes a normal phenotype in these cells, potentially lowering the risk of diseases such as colo-rectal cancer. Imbalances in the microbiota are thought to predispose to large bowel dysfunction and probiotics are being developed to correct this. However, most commercial products contain bacteria (lactobacilli and bifidobacteria) which are dominant species in milk-fed infants but have limited roles in adults. Prebiosis is defined usually by the specific stimulation of these bacteria. However, the end products of most probiotics do not include butyrate or propionate which raises questions about their effectiveness in promoting bowel health in adults. Resistant starch (RS) is a dietary fibre component and its fermentation generally favours butyrate production. Dietary RS intakes and faecal butyrate levels are high in populations at low risk of diet-related large bowel diseases. Conversely, RS intakes and faecal butyrate levels are very low in high risk groups. This raises the possibility that greater RS consumption could be of health benefit. RS is not regarded widely as a prebiotic but (according to the accepted definition) most forms show the requisite features in stimulating specific bacteria, giving raised total SCFA and butyrate levels and a consequent benefit to the host. Current efforts to improve public health through increasing RS consumption could be facilitated by greater recognition of its prebiotic role.

Muscle protein synthesis rate is altered in response to a single injection of insulin-like growth factor-I in seven-day-old Leghorn chicks.

To determine if a single injection of insulin-like growth factor-I (IGF-I) can affect muscle protein synthesis in chickens, 7-d-old male Single Comb White Leghorn chicks were injected s.c. with physiological saline (control) or 35 microg of recombinant human IGF-I. After 2 h 30 min, or 6, 12, or 24 h the chicks were injected with 3H-phenylalanine and killed, and the fractional synthesis rate (Ks) of breast muscle protein was measured. The Ks of IGF-I-treated birds were lower (P = 0.03) than controls at 2 h 30 min post-injection, higher (P = 0.07) than controls at 6 h post-injection, but not different from controls at later times. A second experiment examined serum changes during the 6 h after chicks were injected with IGF-I or saline as in the first experiment. Serum IGF-I concentration increased relative to almost undetectable levels (1 ng/mL) of controls to 216 +/- 59 ng/mL at 20 min after IGF-I injection (P < 0.001) and decreased to 12 +/- 6 ng/ mL by 6 h. Serum glucose and nonprotein nitrogen concentrations were significantly decreased for all or most of the 3 h after IGF-I injection, respectively, but only glucose concentration was the same as controls by 6 h. Low serum glucose and nonprotein nitrogen during the first few hours after IGF-I injection may contribute to the inhibition of Ks at 2.5 h, but the mechanisms behind the increased Ks at 6 h are not clear. These results support a role for IGF-I in the posthatching muscle development of chicks.

Porcine growth hormone and LongR(3)IGF-I can improve recovery from surgery-induced weight loss in guinea pigs.

In the present study, porcine growth hormone (pGH) and LongR(3)IGF-I (LR(3)IGF-I), a potent analogue of IGF-I, were infused continuously into 430-g guinea pigs for 7 days, either alone or in combination, to examine whether pGH can counteract the reduction in circulating IGF-I concentrations induced by LR(3)IGF-I administration. The pGH and LR(3)IGF-I were infused at rates of 400 microg/day (0.93 mg/kg/day) and 120 microg/day (0.28 mg/kg/day), respectively, by miniosmotic pumps. The same doses were infused in the combination treatment. During the first day of treatment, animals lost between 2 and 3% of body weight. Cumulative body weight gains as a percentage of initial body weight were significantly (P < 0.001) increased relative to vehicle-treated controls by the LR(3)IGF-I, pGH, and combination treatments when effects were analyzed across the whole 7-day treatment period. The increased weight gains relative to controls were largely made on day 2, but these gains were not associated with increased water or feed intakes, indicating that pGH and LR(3)IGF-I improved feed conversion efficiency. LR(3)IGF-I alone or in combination with pGH significantly increased the fractional weight of kidneys at the end of the 7-day treatment period, whereas LR(3)IGF-I alone increased the fractional weight of spleens. Concentrations of IGF-I in serum collected after 7 days of treatment were decreased by LR(3)IGF-I, but this decrease was not ameliorated by coinfusion with pGH. GH alone did not have any effects on IGF-I concentration. This study suggests that pGH does not have a strong influence on circulating IGF-I concentrations in the guinea pig. We have also demonstrated that pGH and LR(3)IGF-I are capable of enhancing the recovery of body weight lost in response to surgery in the guinea pig.

Refeeding increases hepatic insulin-like growth factor-I (IGF-I) gene expression and plasma IGF-I concentration in fasted chicks.

1. We examined the influence of refeeding after 2 d of fasting on plasma insulin-like growth factor-I (IGF-I) concentration and hepatic IGF-I gene expression in chickens at 6 weeks of age. 2. Hepatic IGF-I mRNA was measured by ribonuclease protection assay and plasma IGF-I concentration was determined by radioimmunoassay. 3. Plasma IGF-I concentration decreased following fasting, increased to the level of fed controls after 2 h of refeeding but then fell back to the level of fasted chickens after 6 h of refeeding. 4. Fasting reduced hepatic IGF-I mRNA concentrations to less than half of those in the fed controls. Refeeding increased IGF-I mRNA sharply at 2 h after refeeding, but by 6 h after refeeding they had taller back again to levels significantly lower than at 2 h. 5. A significant correlation between plasma IGF-I concentration and hepatic IGF-I gene expression was found, suggesting that when chicks are refed after 2 d of fasting, the short-term increase in plasma IGF-I concentration may be partly regulated by the alteration in hepatic IGF-I mRNA.

Long R3 insulin-like growth factor-I (IGF-I) infusion stimulates organ growth but reduces plasma IGF-I, IGF-II and IGF binding protein concentrations in the guinea pig.

We have tested whether an animal with substantial amounts of both IGF-I and IGF-II in circulation, such as the guinea pig, would respond to chronic IGF infusion in the same manner as the adult rat, which has negligible amounts of IGF-II in blood. Female guinea pigs of 350 g body weight were continuously infused for 7 days with recombinant guinea pig IGF-I or -II (120 or 360 micrograms/day) or long R3 IGF-I (LR3IGF-I) (120 micrograms/day), an analogue which has much reduced affinities for IGF binding proteins. IGF-I or IGF-II infusion led to substantial increases in plasma IGF-I or IGF-II respectively in comparison with vehicle-infused animals. Nevertheless, body weight gain, feed intake, feed conversion efficiency and carcass composition were not significantly affected by any treatment (significance was deemed to be P < 0.05). Amongst the tissues examined only the fractional weight (g/kg body weight) of the adrenals was increased, and that only by the higher dose (360 micrograms/day) of IGF-I. However, the fractional weight of adrenals, gut, kidneys and spleen were significantly increased by LR3IGF-I, but again overall growth was not stimulated. A possible explanation for the lack of IGF-I effects is that total circulating IGF concentrations were not increased by these treatments. IGF-II significantly raised total IGF concentrations at the higher dose only. Plasma IGF-I was reduced by IGF-II infusion, as was plasma IGF-II by IGF-I infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

Continuous 14 day infusion of IGF-II increases the growth of normal female rats, but exhibits a lower potency than IGF-I.

The effects of continuous 14 day infusion of recombinant human IGF-I (104 or 260 micrograms/day) or IGF-II (104, 260 or 650 micrograms/day) via s.c. implanted osmotic pumps were compared in young female rats in order to establish the relative efficacies of these two growth factors. Significant increase in body weight gain and feed conversion efficiency were achieved by 260 micrograms/day of IGF-I or 650 micrograms/day of IGF-II. These treatments were associated with increased nitrogen retention and increases in the fractional weights of kidneys, spleen, total gut and individual gut regions. There was an increase in the size of villi and muscularis lining the jejunum, suggesting an increased absorptive capacity of the gut. However there was no significant change in the amount of faecal nitrogen excretion when expressed as a percentage of nitrogen intake. Interestingly, IGF-II was at least as potent as IGF-I in increasing the depth of jejunal crypts. Infusion of equivalent doses of either IGF-I or IGF-II resulted in similar increases in circulating concentrations of the respective peptides, though IGF-II infusion dose-dependently decreased plasma IGF-I concentrations from those of the controls. Plasma IGF-binding protein levels were increased by both IGF-I and IGF-II treatments, though IGF-I elicited greater responses. In summary, IGF-II can promote the growth of young female rats, although generally less potently than IGF-I.

Immunoreactive and receptor-active insulin-like growth factor-I (IGF-I) and IGF-binding protein in blood plasma from the freshwater fish Macquaria ambigua (golden perch).

The presence of insulin-like growth factor-I (IGF-I)-related molecules and IGF-binding factors in blood from golden perch, Macquaria ambigua, an Australian native freshwater fish, was investigated. Serum was acidified to dissociate IGF and IGF-binding protein complexes that might be present, and fractionated by size-exclusion high-performance liquid chromatography at pH 2.8. Fractions were neutralized and their activities assessed by (i) an immunoassay for mammalian IGF-I which also detects chicken IGF-I but in which all known forms of IGF-II react very poorly, (ii) a receptor assay for IGF-II in which all known forms of IGF-I react poorly, and (iii) a type-I IGF receptor assay in which mammalian IGF-I and IGF-II polypeptides are amost equivalent. No IGF-II-like activity was detected. Three peaks of IGF-I-like activity were detected by IGF-I immunoassay and type-I IGF receptor assay. The major peak of activity was similar in molecular size to human IGF-binding protein-3, 45-55 kDa ('large IGF'), and a minor peak of activity which was similar in size to mammalian IGFs, 7.5 kDa. A third peak of activity was observed eluting at a time which indicates that it is a smaller molecule than any previously described IGF. The large IGF was temperature-sensitive, but was not a binding protein for 125I-labelled human IGF-I (hIGF-I). This material therefore was able to bind to anti-hIGF-I antibodies and to human type-I IGF receptors, and may represent the fish equivalent of mammalian prepro-IGFs.(ABSTRACT TRUNCATED AT 250 WORDS)

Developmental changes in growth hormone, insulin-like growth factors (IGF-I and IGF-II) and IGF-binding proteins in plasma of young growing pigs.

The relationship between plasma concentrations of normally secreted GH and insulin-like growth factor-I (IGF-I) was investigated in pigs after weaning. Frequent blood sampling for between 12 and 24 h showed that plasma GH was pulsatile in pigs of 10, 20 and 35 kg liveweight. Pulses were brief in duration, low in amplitude and variable in frequency. Basal and average daily plasma concentrations of GH changed significantly with development, increasing by about 50% between 10 and 20 kg liveweight. Concentrations of IGF-I in plasma showed little or no evidence of diurnal periodicity and were not increased by GH pulses. Average daily concentrations of both IGF-I and IGF-II in plasma progressively increased between 10 and 35 kg liveweight, as did the total desaturated IGF-binding protein (IGFBP) activity of plasma. A strong positive correlation was observed between the total concentration of IGFs (IGF-I plus IGF-II) in the circulation and plasma IGFBP activity. The developmental rise in IGFBP activity of plasma was associated with increased labelling with 125I-labelled human IGF-II in ligand blots of binding proteins of apparent molecular masses greater than 200, 50, 43 and 29 kDa. One class of binding proteins of 34.5 kDa decreased with development. This study of young growing pigs shows that normally secreted endogenous GH exerts no significant immediate control over plasma IGF-I concentrations, and that plasma levels of IGF-I and IGF-II increase with maturation in this species.(ABSTRACT TRUNCATED AT 250 WORDS)