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We previously described the isolation and characterization of three probiotic strains from the feces of exclusively breast-fed newborn infants: Lactobacillus paracasei CNCM I-4034, Bifidobacterium breve CNCM I-4035 and Lactobacillus rhamnosus CNCM I-4036. These strains were shown to adhere to intestinal mucus in vitro, to be sensitive to antibiotics and to resist biliary salts and low pH. In the present study, a multicenter, randomized, double-blind, placebo-controlled trial with 100 healthy volunteers in three Spanish cities was carried out to evaluate the tolerance, safety, gut colonization and immunomodulatory effects of these three probiotics. Volunteers underwent a 15-day washout period, after which they were randomly divided into 5 groups that received daily a placebo, a capsule containing one of the 3 strains or a capsule containing a mixture of two strains for 30 days. The intervention was followed by another 15-day washout period. Patients did not consume fermented milk for the entire duration of the study. Gastrointestinal symptoms, defecation frequency and stool consistency were not altered by probiotic intake. No relevant changes in blood and serum, as well as no adverse events occurred during or after treatment. Probiotic administration slightly modified bacterial populations in the volunteers’ feces. Intestinal persistence occurred in volunteers who received L. rhamnosus CNCM I-4036. Administration of B. breve CNCM I-4035 resulted in a significant increase in fecal secretory IgA content. IL-4 and IL-10 increased, whereas IL-12 decreased in the serum of volunteers treated with any of the three strains. These results demonstrate that the consumption of these three bacterial strains was safe and exerted varying degrees of immunomodulatory effects., Part of the research currently in progress in the authors' laboratory is funded by the company Hero Spain, S. A. through the grant #3582 managed by the Fundacion General Empresa-Universidad de Granada.

Background Estrogen receptor-positive breast cancer tumors depend on estrogen signaling for their growth and replication and can be treated by anti-estrogen therapy with tamoxifen. Polymorphisms of the CYP2D6 and CYP2C19 genes are associated with an impaired response to tamoxifen. The study objective was to investigate the impact of genetic polymorphisms in CYP2D6 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Spanish women with estrogen receptor-positive breast cancer who were candidates for tamoxifen therapy. Methods: We studied 90 women with estrogen receptor-positive breast cancer, using the AmpliChip CYP450 test to determine CYP2D6 and CYP2C19 gene variants. Plasma levels of tamoxifen and its metabolites were quantified by high-performance liquid chromatography. Results The CYP2D6 phenotype was extensive metabolizer in 80%, intermediate metabolizer in 12.2%, ultra-rapid metabolizer in 2.2%, and poor metabolizer in 5.6% of patients, and the allele frequency was 35.0% for allele *1, 21.0% for *2, and 18.9% for *4. All poor metabolizers in this series were *4/*4, and their endoxifen and 4-hydroxy tamoxifen levels were 25% lower than those of extensive metabolizers. CYP2C19*2 allele, which has been related to breast cancer outcomes, was detected in 15.6% of the studied alleles. Conclusion CYP2D6*4/*4 genotype was inversely associated with 4-hydroxy tamoxifen and endoxifen levels. According to these results, CYP2D6 and CYP2C19 genotyping appears advisable before the prescription of tamoxifen therapy.

Probiotic bacteria have been shown to modulate immune responses and could have therapeutic effects in allergic and inflammatory disorders. However, little is known about the signalling pathways that are engaged by probiotics. Dendritic cells (DCs) are antigen-presenting cells that are involved in immunity and tolerance. Monocyte-derived dendritic cells (MoDCs) and murine DCs are different from human gut DCs; therefore, in this study, we used human DCs generated from CD34+ progenitor cells (hematopoietic stem cells) harvested from umbilical cord blood; those DCs exhibited surface antigens of dendritic Langerhans cells, similar to the lamina propria DCs in the gut. We report that both a novel probiotic strain isolated from faeces of exclusively breast-fed newborn infants, Lactobacillus paracasei CNCM I-4034, and its cell-free culture supernatant (CFS) decreased pro-inflammatory cytokines and chemokines in human intestinal DCs challenged with Salmonella. Interestingly, the supernatant was as effective as the bacteria in reducing pro-inflammatory cytokine expression. In contrast, the bacterium was a potent inducer of TGF-β2 secretion, whereas the supernatant increased the secretion of TGF-β1 in response to Salmonella. We also showed that both the bacteria and its supernatant enhanced innate immunity through the activation of Toll-like receptor (TLR) signalling. These treatments strongly induced the transcription of the TLR9 gene. In addition, upregulation of the CASP8 and TOLLIP genes was observed. This work demonstrates that L. paracasei CNCM I-4034 enhanced innate immune responses, as evidenced by the activation of TLR signalling and the downregulation of a broad array of pro-inflammatory cytokines. The use of supernatants like the one described in this paper could be an effective and safe alternative to using live bacteria in functional foods., This work was funded by Hero Spain S.A. through the contract n° 3143 signed with the Fundación General Universidad de Granada Empresa and co-sponsored by the CDTI, a public entity of the Ministry of Economy and Competitiveness of the Spanish Government. Carolina Gomez-Llorente received a postdoctoral fellowship from Plan Propio of University of Granada.

Huntington disease (HD) is a neurodegenerative disorder associated with the expansion of a polymorphic trinucleotide CAG repeat in the HD gene. We have developed an assay to accurately determine CAG repeats that combines a novel oligonucleotide design and the resolution of capillary electrophoresis. A mismatch in the second nucleotide from the 3' end enhanced specificity by avoiding mispriming and diminishing shadow bands and artifactual PCR products. The coupling of capillary electrophoresis analysis with the assay added the advantages of accuracy, high resolution, semi-automation, rapid analysis and low sample consumption. Analysis of 200 chromosomes in the Spanish population sample studied (control group) gave a peak frequency for 16 CAG repeats and of 7 triplets for CCG repeats. Diagnosis of HD was confirmed in 22 of 34 individuals with a range of CAG repeats from 39 to 52. Predictive testing was also carried out for 19 relatives of the HD families diagnosed at our laboratory. The method proposed in this article provides an accurate sizing of DNA repeats that can be applied to the analysis of DNA size-related disorders.

Clinical manifestations of cystic fibrosis (CF) are variable. Genetic and environmental factors that determine whether an individual will develop associated complications are still under investigation. The present study reports the genetic analysis of a family with different clinical forms of CF and addresses the difficulty of CF diagnosis in an individual with mutant alleles G542X and R117H because of the variable phenotype associated with R117H mutation. Both children in this family were heterozygous for G542X/R117H with the same thymine sequence (7T/9T) in intron 8 of CF transmembrane conductance regulator. The girl was diagnosed with CF, whereas the boy was diagnosed with azoospermia as the sole clinical manifestation. The possible implication of the hemochromatosis gene as a CF modifier locus was analyzed because the 2 children had the same genotype. No genetic differences were detected between brother and sister that explained the different clinical manifestations of CF.

Three HFE gene mutations (HFE 845 G--A, 187 C--G and 193 A--T) are the most common mutations related to hereditary haemochromatosis (HH). The genotype for these mutations was analysed in 359 Spanish individuals with altered iron metabolism and iron overload. Various biochemical parameters were measured in serum samples from 96 of these individuals, and the effect of the genotype on these parameters was studied. Allele frequencies were 12.95% for the HFE C282Y variant, 28.97% for the HFE H63D variant and 0.69% for the HFE S65C variant, calculated in a total of 718 chromosomes. Multiple comparisons analysis showed very significant differences (p=0.001) in transferrin saturation index (TSI) between the HFE C282Y variant homozygous and control (ten healthy volunteers) groups. Highly significant (p=0.0001) and significant (p=0.005) differences in serum ferritin values were found between the HFE C282Y variant homozygous and control groups and between compound (HFE C282Y/H63D variant) heterozygous and control groups, respectively. Very significant differences (p=0.001) in serum iron values were observed between the HFE C282Y variant homozygous and control groups. TSI and serum ferritin values detected most HFE C282Y variant homozygotes and are recommended to facilitate the clinical diagnosis of HH.

Since the initial characterization of the predominant mutation (ΔF508) in cystic fibrosis (CF), more than 1000 pathogenic mutations and numerous polymorphisms have been identified in the CFTR gene (1). The frequencies and types of CFTR mutations vary according to the geographic and ethnic origins of the population under study. A recent worldwide survey revealed great mutational heterogeneity for CF in the Mediterranean region (2). Spain may have the highest heterogeneity of CF mutations among Mediterranean countries, with more than 75 different mutations detected, representing 90.2% of the CF chromosomes (3). On the other hand, only 10 mutations had a frequency higher than 1%, and these accounted for 74.2% of the CF chromosomes studied. Among the 75 different CFTR mutations detected to date in Spain, 56 are not included in the commercial Applied Biosystems assay, which may account for the numerous uncharacterized CFTR alleles in a previous study in Southern Spain (4). We attempted to solve this analytical problem by developing a PCR method that uses fluorescent detection and capillary electrophoresis to detect 11 of the most frequent CFTR mutations not included in the Applied Biosystems assay. If used in conjunction with the Applied Biosystems assay, our novel PCR technique could increase the rate of CF allele detection among European populations. Mutations in the CFTR gene were studied in 140 samples from patients diagnosed with CF at the 12 de Octubre Hospital (Madrid, Spain) or the IRO, Hospital Duran I Reynals (Barcelona, Spain), and from patients with suspicion of CF referred to the Hospital Universitario “San Cecilio” (Granada, Spain). Human genomic DNA was extracted from whole-blood samples by use of a QIAamp® Blood Mini Kit (Qiagen). The DNA concentrations in samples were determined spectrophotometrically by use of a UV-1603 Shimadzu spectrophotometer. Oligonucleotide primers were designed to avoid the formation of primer-dimers, …