Genome-wide DNA methylation analysis identifies promoter hypermethylation in canine malignant melanoma.

Canine malignant melanoma is a common cancer with a high mortality rate. Although previous studies have evaluated various aspects of this tumour, the exact mechanism of tumourigenesis remains unknown. Epigenetic mechanisms, such as DNA methylation, have recently gained attention as aetiological factors for neoplasia in humans. This study aimed to analyse genome-wide DNA methylation patterns in canine malignant melanoma based on next-generation sequencing data. A total of 76,213 CpG sites, including 29,482 sites in CpG islands (CGIs), were analysed using next-generation sequencing of methylation-specific signatures, obtained by sequential digestion with enzymes, to compare normal oral mucosal samples from four healthy dogs, four canine melanoma cell lines (3 oral cavity and 1 skin), and five clinical samples of oral canine melanoma. Malignant melanoma showed increased methylation at thousands of normally unmethylated CpG sites in CGIs and decreased methylation at normally methylated CpG sites in non-CGIs. Interestingly, the promoter regions of 81-393 genes were hypermethylated; 23 of these genes were present in all melanoma cell lines and melanoma clinical samples. Among these 23 genes, six genes with "sequence-specific DNA binding" annotation were significantly enriched, including three Homeobox genes-HMX2, TLX2, and HOXA9-that may be involved in the tumourigenesis of canine malignant melanoma. This study revealed widespread alterations in DNA methylation and a large number of hypermethylated genes in canine malignant melanoma.

Imaging of spindle cell lipoma.

AIM: To review the evaluation, diagnosis, and treatment of spindle cell lipoma (SCL) with emphasis on the location of these tumours and the spectrum of magnetic resonance imaging (MRI) and computed tomography (CT) appearances. MATERIALS AND METHODS: The MRI and CT findings of 27 histopathologically proven SCLs were evaluated retrospectively. Imaging features evaluated included margins, percentage visible fat, MRI signal characteristics, oedema, and contrast enhancement patterns. RESULTS: Patient ages ranged from 18 to 80 years with an average age of 56.5 years. Men were affected twice as frequently as women (M=18, F=9). SCLs ranged in size from 2 to 10 cm, with an average greatest dimension of 5.5 cm. Five lesions (19%) contained no visible fat on CT or MRI, and the leading differential diagnosis of high-grade soft-tissue sarcoma diagnosis was suggested by referring surgeons. Five lesions (19%) had <50% fatty areas, nine lesions (52%) demonstrated >50% but <90% fat at MRI or CT. Only three of 25 lesions (12%) had an appearance of a typical lipoma on unenhanced MRI sequences. All SCLs that were imaged with contrast medium (n = 18) demonstrated some degree of enhancement, with eight (44%) showing marked enhancement, four (22%) showing moderate, and six (33%) minimal enhancement. CONCLUSION: SCLs have considerably variable imaging appearances and may have minimal or no visible fat at MRI or CT. Imaging features may make it difficult to distinguish this benign tumour from a potentially higher-grade malignant tumour.

Dynamic changes in DNA methylation patterns in canine lymphoma cell lines demonstrated by genome-wide quantitative DNA methylation analysis.

DNA methylation is the conversion of cytosine to 5-methylcytosine, leading to changes in the interactions between DNA and proteins. Methylation of cytosine-guanine (CpG) islands (CGIs) is associated with gene expression silencing of the involved promoter. Although studies focussing on global changes or a few single loci in DNA methylation have been performed in dogs with certain diseases, genome-wide analysis of DNA methylation is required to prospectively identify specific regions with DNA methylation change. The hypothesis of this study was that next-generation sequencing with methylation-specific signatures created by sequential digestion of genomic DNA with SmaI and XmaI enzymes can provide quantitative information on methylation levels. Using blood from healthy dogs and cells obtained from canine lymphoma cell lines, approximately 100,000CpG sites across the dog genome were analysed with the novel method established in this study. CpG sites in CGIs broadly were shown to be either methylated or unmethylated in normal blood, while CpG sites not within CpG islands (NCGIs) were largely methylated. Thousands of CpG sites in lymphoma cell lines were found to gain methylation at normally unmethylated CGI sites and lose methylation at normally methylated NCGI sites. These hypermethylated CpG sites are located at promoter regions of hundreds of genes, such as TWIST2 and TLX3. In addition, genes annotated with 'Homeobox' and 'DNA-binding' characteristics have hypermethylated CpG sites in their promoter CGIs. Genome-wide quantitative DNA methylation analysis is a sensitive method that is likely to be suitable for studies of DNA methylation changes in cancer, as well as other common diseases in dogs.

A CpG island methylator phenotype in acute myeloid leukemia independent of IDH mutations and associated with a favorable outcome.

Genetic changes are infrequent in acute myeloid leukemia (AML) compared with other malignancies and often involve epigenetic regulators, suggesting that an altered epigenome may underlie AML biology and outcomes. In 96 AML cases including 65 pilot samples selected for cured/not-cured, we found higher CpG island (CGI) promoter methylation in cured patients. Expanded genome-wide digital restriction enzyme analysis of methylation data revealed a CGI methylator phenotype independent of IDH1/2 mutations we term AML-CGI methylator phenotype (CIMP) (A-CIMP+). A-CIMP was associated with longer overall survival (OS) in this data set (median OS, years: A-CIMP+=not reached, CIMP-=1.17; P=0.08). For validation we used 194 samples from The Cancer Genome Atlas interrogated with Illumina 450k methylation arrays where we confirmed longer OS in A-CIMP (median OS, years: A-CIMP+=2.34, A-CIMP-=1.00; P=0.01). Hypermethylation in A-CIMP+ favored CGIs (OR: CGI/non-CGI=5.21), and while A-CIMP+ was enriched in CEBPA (P=0.002) and WT1 mutations (P=0.02), 70% of cases lacked either mutation. Hypermethylated genes in A-CIMP+ function in pluripotency maintenance, and a gene expression signature of A-CIMP was associated with outcomes in multiple data sets. We conclude that CIMP in AML cannot be explained solely by gene mutations (for example, IDH1/2, TET2), and that curability in A-CIMP+ AML should be validated prospectively.

Three-dimensional analysis versus goniometric measurement of total active elevation in normal subjects.

BACKGROUND: Multiple planes of motion have been reported for shoulder elevation performed by visual inspection with a goniometer. It is typically measured by a clinician who is standing or sitting at the side of the patient. Instead, accurate assessment of shoulder elevation must be performed by using a plane of reference that is perpendicular to the plane of motion being measured. METHODS: Three repetitions of humeral elevation in the sagittal, scapular, and coronal planes were performed in a random order and measured by goniometry and three-dimensional (3D) electromagnetic sensors. A guide bar was used to control the initial plane of motion for the sagittal and coronal planes. The plane of motion at 90° and at peak elevation was recorded for each of the 3 defined planes. A goniometer was used to measure the range of maximal elevation performed in each plane, for each subject, by visual inspection. RESULTS: The 3D data revealed that subjects consistently moved toward scaption as the extremity moved above 90° of elevation, regardless of the initial plane of motion. Significant differences were seen in the goniometric data for the plane of motion at 90° (P = .00) in flexion, abduction, and scaption. Goniometric measurements revealed greater maximum elevation angles in comparison to the 3D kinematic measurements. CONCLUSIONS: Maximal glenohumeral elevation occurred near the plane of the scapula in all subjects, regardless of the plane in which elevation was initiated. Goniometric measurement of total elevation resulted in greater range of motion measurements than actually occurred because the observer was not routinely positioned in a plane perpendicular to the plane of actual elevation of the upper extremity.

Liver DNA methylation analysis in adult female C57BL/6JxFVB mice following perinatal exposure to bisphenol A.

Bisphenol A (BPA) is a compound released from plastics and other consumer products used in everyday life. BPA exposure early in fetal development is proposed to contribute to programming of chronic diseases like obesity and diabetes, by affecting DNA methylation levels. Previously, we showed that in utero and lactational exposure of C57BL/6JxFVB hybrid mice via maternal feed using a dose range of 0-3000µg/kg body weight/day resulted in a sex-dependent altered metabolic phenotype in offspring at 23 weeks of age. The most univocal effects were observed in females, with reduced body weights and related metabolic effects associated with perinatal BPA exposure. To identify whether the effects of BPA in females are associated with changes in DNA methylation, this was analyzed in liver, which is important in energy homeostasis. Measurement of global DNA methylation did not show any changes. Genome-wide DNA methylation analysis at specific CpG sites in control and 3000µg/kg body weight/day females with the digital restriction enzyme analysis of methylation (DREAM) assay revealed potential differences, that could, however, not be confirmed by bisulfite pyrosequencing. Overall, we demonstrated that the observed altered metabolic phenotype in female offspring after maternal exposure to BPA was not detectably associated with liver DNA methylation changes. Still, other tissues may be more informative.

A phase I study of decitabine with pegylated interferon α-2b in advanced melanoma: impact on DNA methylation and lymphocyte populations.

BACKGROUND: Melanoma cell lines treated with decitabine show upregulation of cancer antigens, and interferon-α upregulates MHC Class I antigens in cancer cells, leading to enhanced T-cell recognition and T-cell mediated tumor apoptosis. We evaluated the synergy between the hypomethylating effects of decitabine and the immunomodulatory effects of interferon in a combination regimen administered to advanced melanoma patients in a phase 1 trial. METHODS: Patients with one prior systemic therapy were eligible. Using a modified 3 + 3 design, patients received escalating doses of decitabine and pegylated interferon α-2b (PEG-IFN) during every 28-day treatment cycle. Global DNA methylation was measured on days 1 and 5 of cycles 1 and 3. Cytokine profiling and quantification of T-cell subpopulations by FACS were performed at baseline and cycle 3. RESULTS: Seventeen patients were assigned to one of four dose levels. Decitabine 15 mg/m2/d + PEG-IFN 3 µg/kg was the maximum tolerated dose (MTD). Grade 3/4 cytopenias were seen across all dose levels: anemia (1), neutropenia (7), and thrombocytopenia (2). One patient remained progression-free for 37 weeks. The other 16 patients progressed at or before 12 weeks. Median overall survival was 39 weeks. Hypomethylation was seen at all dose levels. Due to treatment-induced lymphocytopenia, absolute changes in T-cell populations post-treatment were too small to be meaningfully interpreted. CONCLUSIONS: The response to this combination regimen was characterized by significant myelosuppression, particularly neutropenia. Although disappointing efficacy and slow accrual led to early closure of the trial, hypomethylation showed pharmacodynamic evidence of a therapeutic effect of decitabine at all dose levels.

Phase I trial of dasatinib and ixabepilone in patients with solid tumors.

PURPOSE: Dasatinib is an oral tyrosine kinase inhibitor (TKI) of BCR-ABL and SRC family and ixabepilone is an epothilone B analog. Synergistic activity has been reported when combining dasatinib with chemotherapy. This study was conducted to determine the dose-limiting toxicities (DLTs) and the maximum tolerated doses (MTDs) for this combination. PATIENTS AND METHODS: Patients with metastatic solid tumors who progressed on standard therapy received dasatinib orally daily and ixabepilone IV every 3 weeks at escalating doses using 3 + 3 design. An expansion cohort was studied after reaching the MTD. Pharmacokinetic studies were performed. RESULTS: Nineteen patients were enrolled. No DLTs were observed at dose level (DL) 1 (dasatinib 100 mg and ixabepilone 30 mg/m(2)). At DL 2 (dasatinib 100 mg and ixabepilone 40 mg/m(2)), one patient had multiple DLTs. At DL 3 (dasatinib 150 mg and ixabepilone 40 mg/m(2)), the first patient developed grade 3 AE during cycle 2, the second patient had a DLT and a grade 3 AE during cycle 2. The accrual to DL 3 was halted without reaching the maximally administered dose (MAD) and MTDs were determined to be dasatinib 100 mg and ixabepilone 40 mg/m(2) (DL 2). One patient had a partial response and 12 patients stable disease as their best response. Fourteen patients came off study due to toxicities. CONCLUSION: The combination of dasatinib and ixabepilone showed modest clinical activity with doses 100 mg orally daily and 40 mg/m(2) IV every 3 weeks, respectively. Treatment related toxicities were seen frequently.

In vitro activation of CMV-specific human CD8(+) T cells by adenylate cyclase toxoids delivering pp65 epitopes.

Human CMV infects between 50-85% of healthy individuals and can cause live-threatening infections in immunocompromised patients. Therefore, peptide vaccination is being developed as a promising immunotherapeutic approach for treatment of patients at risk of CMV disease. The enzymatically inactive toxoid of Bordetella adenylate cyclase (CyaA-AC(-)) was shown to be an efficient tool for delivery of peptide epitopes and stimulation of Ag-specific T-cell immune responses. We investigated here the capacity of two CyaA-AC(-) constructs to deliver epitopes derived from the CMV phosphoprotein pp65 for activation of human T cells in vitro. Expansion of γ-IFN-secreting CMV-specific CD8(+) T cells, as well as increase of total IFN-γ and TNF-α production by PBMCs from CMV-seropositive donors were observed after in vitro stimulation with CyaA-AC(-) constructs carrying CMV epitopes, whereas limited activation of immune response occurred with free peptides. The activation of immune response was confirmed by expansion of CMV-specific T-cell clones and anti-CMV cytotoxic effect of stimulated PBMCs. These data open the way to clinical evaluation of CyaA-AC(-) constructs as tools for detection and expansion of CMV-specific T-cell immune responses for diagnostic and immunotherapeutic applications against CMV-associated diseases.

Management of unstable ankle fractures and syndesmosis injuries in athletes.

Athletes with unstable ankle injuries treated with rigid and anatomic internal fixation with concomitant repair of indicated ligaments followed by an accelerated rehabilitation program consisting of early weight bearing and near-immediate range of motion (ROM) can obtain excellent outcomes. Early ROM and weight bearing, if indicated depending on the specific injury pattern, can be effective with low morbidity. Return to sports can be expected as early as 4 weeks after rigid fixation of an isolated fibula fracture and up to 8 to 10 weeks after stabilization of a bimalleolar equivalent fracture with deltoid repair. Syndesmosis fixation can take up to 4 to 6 months before successful return to sport.

Genome-wide identification of aberrantly methylated promoter associated CpG islands in acute lymphocytic leukemia.

We performed a genome-wide analysis of promoter associated CpG island methylation using methylated CpG island amplification (MCA) coupled to representational differential analysis (RDA) or a DNA promoter microarray in acute lymphoblastic leukemia (ALL). We identified 65 potential targets of methylation with the MCA/RDA approach, and 404 with the MCA/array. Thirty-six (77%) of the genes identified by MCA/RDA were shared by the MCA/array approach. Chromosomal location of these genes was evenly distributed in all autosomes. Functionally, 303 of these genes clustered in 18 molecular pathways. Of the 36 shared genes, 31 were validated and 26 were confirmed as being hypermethylated in leukemia cell lines. Expression analysis of eight of these genes was epigenetically modulated by hypomethylating agents and/or HDAC inhibitors in leukemia cell lines. Subsequently, DNA methylation of 15 of these genes (GIPC2, RSPO1, MAGI1, CAST1, ADCY5, HSPA4L, OCLN, EFNA5, MSX2, GFPT2, GNA14, SALL1, MYO5B, ZNF382 and MN1) was validated in primary ALL samples. Patients with methylation of multiple CpG islands had a worse overall survival. This is the largest published list of potential methylation target genes in human leukemia offering the possibility of performing rational unbiased methylation studies in ALL.

A mixture of prebiotic oligosaccharides reduces the incidence of atopic dermatitis during the first six months of age.

BACKGROUND: Oligosaccharides may alter postnatal immune development by influencing the constitution of gastrointestinal bacterial flora. AIMS: To investigate the effect of a prebiotic mixture of galacto- and long chain fructo-oligosaccharides on the incidence of atopic dermatitis (AD) during the first six months of life in formula fed infants at high risk of atopy. METHODS: Prospective, double-blind, randomised, placebo controlled trial; 259 infants at risk for atopy were enrolled. A total of 102 infants in the prebiotic group and 104 infants in the placebo group completed the study. If bottle feeding was started, the infant was randomly assigned to one of two hydrolysed protein formula groups (0.8 g/100 ml prebiotics or maltodextrine as placebo). All infants were examined for clinical evidence of atopic dermatitis. In a subgroup of 98 infants, faecal flora was analysed. RESULTS: Ten infants (9.8%; 95 CI 5.4-17.1%) in the intervention group and 24 infants (23.1%; 95 CI 16.0-32.1%) in the control group developed AD. The severity of the dermatitis was not affected by diet. Prebiotic supplements were associated with a significantly higher number of faecal bifidobacteria compared with controls but there was no significant difference in lactobacilli counts. CONCLUSION: Results show for the first time a beneficial effect of prebiotics on the development of atopic dermatitis in a high risk population of infants. Although the mechanism of this effect requires further investigation, it appears likely that oligosaccharides modulate postnatal immune development by altering bowel flora and have a potential role in primary allergy prevention during infancy.

Acidic oligosaccharides from pectin hydrolysate as new component for infant formulae: effect on intestinal flora, stool characteristics, and pH.

OBJECTIVES: To come even closer to the functional composition of human milk, acidic oligosaccharides (AOS) from pectin were added to well known neutral prebiotics (galacto-oligosaccharides (GOS) and long-chain fructo-oligosaccharides (FOS)). The effect of AOS and GOS/FOS/AOS on intestinal flora, stool characteristics as well as acceptance and tolerance was investigated. METHODS: Human milk contains 75% to 85% neutral and 15% to 25% acidic oligosaccharides. In this prospective, randomized, double blind study, a mixture of 80% neutral oligosaccharides (from long-chain galacto- and long-chain fructo-oligosaccharides) with 20% acidic oligosaccharides derived from pectin hydrolysis was investigated. Forty-six term infants were fed a standard formula supplemented with either maltodextrin as control (n=15), or with 0.2 g acidic oligosaccharides (n=16), or with the latter plus 0.6 g neutral oligosaccharides (mixture of galacto- and fructo-oligosaccharides; n=15). Fecal flora using plating technique and pH were measured. Stool characteristics and possible side effects (crying, vomiting, and regurgitation) were recorded. RESULTS: There was no difference in the bifidobacteria counts between the control and the group supplemented with acidic oligosaccharides alone (8.75+/-0.50 vs. 8.58+/-0.94 log colony forming units [CFU]/g stool). In infants fed the combination of acidic and neutral oligosaccharides, bifidobacteria were increased (9.61+/-0.70 log CFU/g stool; P<0.01). The same pattern was observed with lactobacilli. Stool consistency was softest in infants fed the complete oligosaccharide mixture, but also in those fed formula supplemented with acidic oligosaccharides alone, the stool consistency was significantly softer compared with the control group. Fecal pH increased in the controls, remained constant in acidic oligosaccharides alone, and decreased in the complete mixture of oligosaccharides group. CONCLUSION: There was no difference in growth, crying, vomiting, and regurgitation patterns between the groups. In summary, acidic oligosaccharides from pectin hydrolysate are well tolerated as ingredient in infant formulae but do not affect intestinal microecology.

Prebiotics in infant formulas.

BACKGROUND: The intestinal flora of breast-fed infants is an important physiologic factor in the function of the gut and in the development of the immune system. The current research is part of a group of studies performed to answer the question whether a bovine milk formula supplemented with a prebiotic mixture from galactooligosaccharides and fructooligosaccharides can stimulate an intestinal flora similar to that of breast-fed infants. METHODS: The prebiotic effect of the oligosaccharide mixture was tested in preterm and term infants by measuring fecal flora using plating as well as fluorescent in situ hybridization techniques. The effect of the oligosaccharides on the bacterial metabolism was studied by measuring short-chain fatty acid production in vitro and the short-chain fatty acid pattern in the stools of a group of term infants. RESULTS: The oligosaccharide mixture increases significantly the number of bifidobacteria and reduces the number of pathogens in term as well as in preterm infants when compared with a group of infants fed an unsupplemented formula. Using a concentration of 0.8 g oligosacchrides/100 mL formula, the amount of bifidobacteria is similar to that typical of breast-fed infants. In vitro, the short-chain fatty acids produced by the mixture of oligosaccharides under study were similar to those produced by the human milk oligosaccharides fraction. In clinical trials the pattern of fecal short-chain fatty acids in infants fed the oligosaccharide mixture was similar to that of breast-fed infants but was significantly different from that of a group of infants fed with an unsupplemented formula. Additionally, the fecal pH was significantly higher in the group fed an unsupplemented formula than in the groups fed either breast milk or a supplemented formula. CONCLUSION: The data obtained indicate that the prebiotic mixture under study is able to stimulate the development of a microbial flora similar to that of breast-fed infants. Several biota, whose growth is enhanced by this prebiotic mixture, represent important factors in the postnatal development of the immune system. On this evidence it can be suggested that prebiotics may play a role as modulators of the postnatal development of the immune system.

The role of chloride in deoxycorticosterone hypertension: selective sodium loading by diet or drinking fluid.

To evaluate the role of chloride in the pathogenesis of salt-dependent deoxycorticosterone (DOC) hypertension, we studied young Wistar rats chronically loaded with sodium bicarbonate (NaHCO(3)) or sodium chloride (NaCl) which were administered either in the diet or in the drinking fluid. Selective sodium loading (without chloride) increased blood pressure (BP) in DOC-treated animals only if NaHCO(3) was provided in the diet. In contrast, no significant blood pressure changes were induced by DOC treatment in rats drinking NaHCO(3) solution. Hypernatremia and high plasma osmolality occurred only in rats drinking NaCl or NaHCO(3) solutions. Compared to great volume expansion in NaCl-loaded DOC-treated rats, the degree of extracellular fluid volume expansion (namely of its interstitial fraction) was substantially lower in both NaHCO(3)-loaded groups in which significant hypokalemia was observed. NaHCO(3)-drinking rats without significant blood pressure response to DOC treatment represented the only experimental group in which blood volume was not expanded. In conclusion, our data confirm previous observations that NaHCO(3) loading is less potent in eliciting DOC hypertension than NaCl loading, but blood pressure rise in rats fed NaHCO(3) diet clearly demonstrated that selective sodium loading could potentiate the development of DOC hypertension if NaHCO(3) is offered within the appropriate dietary regimen. The reasons for the failure of NaHCO(3)-drinking rats to elevate blood pressure in response to chronic mineralocorticoid treatment are not obvious. However, the absence of a significant plasma volume expansion together with hypernatremia and increased plasma osmolality suggest a considerable degree of dehydration in these animals which fail to increase their fluid consumption compared to water drinking rats.

Short term effects of dietary medium-chain fatty acids and n-3 long-chain polyunsaturated fatty acids on the fat metabolism of healthy volunteers.

BACKGROUND: The amount and quality of dietary fatty acids can modulate the fat metabolism. OBJECTIVE: This dietary intervention is based on the different metabolic pathways of long-chain saturated fatty acids (LCFA), which are mostly stored in adipocytic triacylglycerols, medium-chain fatty acids (MCFA) which are preferentially available for hepatic mitochondrial beta-oxidation and n-3 long-chain polyunsaturated fatty acids (n-3 LCPUFA) suggested to modulate fat oxidation and storage by stimulating the peroxisomal beta-oxidation. Combined dietary MCFA and n-3 LCPUFA without LCFA may synergistically stimulate fatty acid oxidation resulting in blood lipid clearance and LCFA release from adipocytes. DESIGN: In a short term, parallel, randomized, double-blind trial effects on the fatty acid metabolism of 10 healthy volunteers (Body Mass Index 25-30) of a formula containing 72% MCFA and 22% n-3 LCPUFA without LCFA (intake: 1.500 kcal/day; fat: 55.5% of energy) were measured in comparison to an isoenergetic formula with equal fat amount and LCFA dominated lipid profile. RESULTS: The plasma triacylglycerol (p < 0.1) and cholesterol (p < 0.05) content decreased in the test group. The n-3/n-6 LCPUFA (> or = C 20) ratio increased (p < 0.0001) after 4 days treatment. The LCFA content was similar in both groups despite missing LCFA in the test formula indicating LCFA release from adipocytes into the plasma. Both groups significantly reduced body weight considerably 4 kg (p < 0.01) and fat mass up to 50% of weight loss (p < 0.05). CONCLUSION: Combined dietary 72% MCFA and 22% n-3 LCPUFA without LCFA stimulate the fatty acid oxidation and release from adipocytes without affecting any safety parameters measured.

Prebiotic concept for infant nutrition.

In the neonatal period, the intestine is colonised in a stepwise process that depends on mode of delivery, environmental factors, bacterial interactions, and the host itself resulting in a colonisation with a complex heterogeneous bacterial flora. Oligosaccharides have been identified as an important prebiotic factor of human milk As long as analogues of human milk oligosaccharides are not available now and in the near future it is aimed to resemble the prebiotic effect of human milk by oligosaccharides from available sources. In the present study in preterm infants, a mixture of 90% galacto-oligosaccharides and 10% fructo-oligosaccharides has been tested. The mixture of GOS/FOS was composed to mimic the molecule size distribution of human milk oligosaccharides. Microbiological analysis of the faces was performed before and 7, 14, and 28 days after start of supplementation and stool characteristics have been recorded. Maltodextrin was used as placebo and infants fed human milk have been used as reference. After a 28 days feeding period, the number of bifidobacteria of the group fed the oligosaccharide supplemented formula was in the upper range of the reference group whereas the numbers of the group fed the formula supplemented with the placebo were in the lower range of the reference group (placebo: 7.9 +/- 0.83 and GOS/FOS mixture: 10,0 +/- 2.05 log 10 CFU/g wet stool; reference (M +/- SD): 7.14-10.7 log 10 CFU/g wet stool). Stool characteristics in the group fed the supplemented formula were close to those found in the human milk fed infants. In summary, supplementation of a preterm formula with a mixture of galacto- and fructo-oligosaccharides has a stimulating effect on the growth of bifidobacteria in the intestine and results in more frequent produced and softer stools. Thus, prebiotic mixtures such like the studied oligosaccharide mixture might help in improving intestinal tolerance to enteral feeding in preterm infants.