The establishment and succession of bacterial communities in infants may have a profound impact in their health, but information about the composition of meconium microbiota and its evolution in hospitalized preterm infants is scarce. In this context, the objective of this work was to characterize the microbiota of meconium and fecal samples obtained during the first 3 weeks of life from 14 donors using culture and molecular techniques, including DGGE and the Human Intestinal Tract Chip (HITChip) analysis of 16S rRNA amplicons. Culture techniques offer a quantification of cultivable bacteria and allow further study of the isolate, while molecular techniques provide deeper information on bacterial diversity. Culture and HITChip results were very similar but the former showed lower sensitivity. Inter-individual differences were detected in the microbiota profiles although the meconium microbiota was peculiar and distinct from that of fecal samples. Bacilli and other Firmicutes were the main bacteria groups detected in meconium while Proteobacteria dominated in the fecal samples. Culture technique showed that Staphylococcus predominated in meconium and that Enterococcus, together with Gram-negative bacteria such as Escherichia coli, Escherichia fergusonii, Klebsiella pneumoniae and Serratia marcescens, was more abundant in fecal samples. In addition, HITChip results showed the prevalence of bacteria related to Lactobacillus plantarum and Streptococcus mitis in meconium samples whereas those related to Enterococcus, Escherichia coli, Klebsiella pneumoniae and Yersinia predominated in the 3rd week feces. This study highlights that spontaneously-released meconium of preterm neonates contains a specific microbiota that differs from that of feces obtained after the first week of life. Our findings indicate that the presence of Serratia was strongly associated with a higher degree of immaturity and other hospital-related parameters, including antibiotherapy and mechanical ventilation.

Wilms tumor protein 1 (WT1) is a transcription factor overexpressed in several types of leukemia and solid tumors. For this reason, WT1 is an attractive target for immunotherapy. Four peptide nonamers from WT1 have been identified by others to generate a WT1-specific cytotoxic response in the context of human leukocyte antigen (HLA)-A0201 and A2402. However, as WT1 is a self-antigen, breaking tolerance is a potential obstacle to vaccination. Here, we use a strategy to circumvent tolerance by designing synthetic immunogenic analog peptides that could crossreact to the native peptides (a heteroclitic response). A number of synthetic peptides derived from nonamer sequences of the WT1 protein were designed in which single amino-acid substitutions were introduced at HLA-A0201 major histocompatibility complex (MHC)-binding positions. Several of new peptides could stabilize MHC class I A0201 molecules better than native sequences. Some analogs were also able to elicit WT1-specific T-cell recognition and cytotoxic T-cell lymphocytes more effectively than native sequences. Importantly, T cells stimulated with the new analogs crossreacted with the native WT1 peptide sequence and were able to kill HLA-matched chronic myeloid leukemia cell lines. In conclusion, analog heteroclitic WT1 peptides with increased immunogenicity can be synthesized and are potential cancer vaccine candidates.

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Chronic lymphocytic leukemia (CLL), the most frequent form of adult leukemia in Western countries, is characterized by a highly variable clinical course. Expression profiling of a series of 160 CLL patients allowed interrogating the genes presumably playing a role in pathogenesis, relating the expression of functionally relevant signatures with the time to treatment. First, we identified genes relevant to the biology and prognosis of CLL to build a CLL disease-specific oligonucleotide microarray. Second, we hybridized a training series on the CLLspecific chip, generating a biology-based predictive model. Finally, this model was validated in a new CLL series. Clinical variability in CLL is related with the expression of two gene clusters, associated with B-cell receptor (BCR) signaling and mitogen-activated protein kinase (MAPK) activation, including nuclear factor-jB1 (NF-jB1). The expression of these clusters identifies three risk-score groups with treatment-free survival probabilities at 5 years of 83, 50 and 17%. This molecular predictor can be applied to early clinical stages of CLL. This signature is related to immunoglobulin variable region somatic hypermutation and surrogate markers. There is a molecular heterogeneity in CLL, dependent on the expression of genes defining BCR and MAPK/NF-jB clusters, which can be used to predict time to treatment in early clinical stages.

There is a sharing of bacterial strains between the neonates' gastrointestinal microbiota and the feeding tubes used to feed them.

Preterm infants in a neonatal intensive care unit (NICU) are exposed to multidrug-resistant bacteria previously adapted to the hospital environment. The aim of the present study was to characterize the bacterial antibiotic-resistant high-risk lineages colonizing preterm infants during their NICU stay and their persistence in faeces after 2 years. A total of 26 preterm neonates were recruited between October 2009 and June 2010 and provided 144 faecal samples. Milk samples (86 mother's milk, 35 human donor milk and 15 formula milk) were collected at the same time as faecal samples. An additional faecal sample was recovered in 16 infants at the age of 2 years. Samples were plated onto different selective media, and one colony per morphology was selected. Isolates were identified by 16S rDNA nucleotide sequence and MALDI-TOF. Antibiotic susceptibility (agar dilution), genetic diversity (RAPD, PFGE and MLST) and virulence factors (only in enterococcal and staphylococcal isolates) were determined by PCR. A high proportion of antibiotic-resistant high-risk clones was detected in both faecal and milk samples during the NICU admittance. Almost all infants were colonized by Enterococcus faecalis ST64 and Enterococcus faecium ST18 clones, while a wider genetic diversity was observed for the Gram-negative isolates. Multidrug-resistant high-risk clones were not recovered from the faecal samples of the 2-year-olds. In conclusion, the gut of preterm infants admitted to the NICU might be initially colonized by antibiotic-resistant and virulent high-risk lineages, which are later replaced by antibiotic-susceptible community ones.

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