Nieves Fernández-Arcás

Genetic selection and folate intake during pregnancy

The Ala225Val mutation (677C➝T) for the methylenetetrahydrofolate reductase (MTHFR) gene has been associated with a thermolabile enzyme with a decreased activity that can provide an increase in plasma homocysteine concentrations. Several reports have studied the relation of this polymorphism with cardiovascular diseases as well as schizophrenia, depression, and cancer. The frequency of the homozygous VV genotype, however, varies geographically in Europe from 6–10% in northern countries to 13–18% in the Mediterranean population. We have studied the evolution with age of the allele and genotype frequencies of this polymorphism in a healthy population from southern Spain (n=695) that was homogeneously distributed by age. We excluded people older than 40 years to prevent changes in frequency due to the possible implication of this gene in different pathologies and to nutritional habits. All the individuals were genotyped for the insertion/deletion polymorphism of the angiotensinconverting-enzyme gene, a locus different to the one studied, to know the genetic homogeneity of the adult and young populations. The allele and genotype frequencies did not differ significantly when both populations were compared. Unexpectedly, we found a substantial increase in frequency of the VV homozygous genotype in individuals younger than 20 years (figure). In a more detailed study, we found a shift in the VV genotype frequency, from 13% to 26%, that started in people born between 1977 and 1982 and that remained at this high proportion (figure). We found also that the population from which these individuals were derived was in Hardy-Weinberg equilibrium. In 1982 early folate treatment for all pregnant women was recommended by the Spanish national health service to

Both alleles of the M235T polymorphism of the angiotensinogen gene can be a risk factor for myocardial infarction

We have studied the role of three polymorphic genes of the renin‐angiotensin system (RAS) as independent risk factors for myocardial infarction (MI) and their correlation with three of the major coronary risk factors: serum cholesterol (CH), hypertension (HT) and smoking (SM). A population of 392 men was genotyped for the M235T polymorphism of the angiotensinogen (AGT) gene, the insertion/deletion of the angiotensin‐converting enzyme (ACE) and the all66c of the angiotensin‐II type 1 receptor (AT1R), by means of polymerase chain reaction (PCR) and restriction enzyme analysis. It was observed that the T allele frequency increased significantly in the MI with HT, CH, and SM subgroup (0.58 vs 0.31) (p<0.01). In contrast, the M allele frequency was higher in the MI without HT, CH, and SM (0.69 vs 0.42) (p<0.01). A strong association between the MM genotype and MI (p<0.001, odds ratio=4.29, confidence interval=1.95–9.42) was found when age‐matched MM control subjects were compared to MI individuals with none of the other known major coronary risk factors. Futhermore, subjects with the MM genotype showed a significantly higher plasma renin activity (PRA) profile than those with the TT genotype (p<0.001). It can be concluded that the M allele is an independent risk factor for MI and the T allele modified the risk when other major risk factors are present.

The genotype interactions of methylenetetrahydrofolate reductase and renin-angiotensin system genes are associated with myocardial infarction

We analyzed the evolution with age of the frequencies of the I/D polymorphism of the angiotensin I-converting enzyme (ACE), a1166c of the angiotensin II AT1 receptor (AT1R), M235T of the angiotensinogen (AGT) and A225V of their methylenetetrahydrofolate reductase (MTHFR) gene in a healthy (H) population and the subsequent comparison to age- and sex-matched groups of myocardial infarction (MI) subjects. A total of 472 H subjects were divided into three groups < 30, 30-55 and > 55 years old and 277 individuals with MI into two groups 30-55 and > 55 years old. The evolution with age showed that the AGT M allele (P < 0.001) and the MTHFR V allele (P < 0.05) frequency decreased with age in H men. The comparison between healthy and MI groups showed that the MM genotype frequency increased in MI men > 55 years (OR =4.16; 95% CI; 1.72-10.1) The cc genotype showed a similar behaviour (OR = 3.96; 95% CI; 1.21-12.9). In men, all the combinations with MM genotype presented a high risk, with OR values between 1.10 and 7.22. In women, the cc genotype increased in the MI > 55 group (OR = 6.66; 95% CI; 2.02-21.9). All the combinations with the cc genotype showed OR values between 1.71 and 13.3. The MM genotype in men and cc genotype in men and women, are independent risk factors for MI. We propose that the study of the allele frequency evolution in an H population at different ages is essential to determine risk factors for MI in case-control studies, since data from isolated age-matched groups can be misinterpreted.

Lifespan of human amniotic fluid-derived multipotent mesenchymal stromal cells

BACKGROUND AIMS Human multipotent mesenchymal stromal cells (hMSC) have become one of the main interests in regenerative medicine because of their ability to differentiate into different lineages. Human amniotic fluid is reported to contain MSC (hAMSC) and therefore may be a useful source of cells for clinical applications. However, our understanding of the behavior of these cells in indefinite in vitro culture conditions is very limited. METHODS We systematically evaluated and characterized, throughout their whole lifespan, the expansion potential, chromosomal stability, surface and intracellular phenotype and differentiation potential of fibroblastoid hAMSC (F-type hAMSC). RESULTS Nine F-type hAMSC cultures could be expanded in in vitro culture conditions for 223.25 ± 24.44 days (mean ± SD), during which time 28.96 ± 1.5 passages were made giving rise to 54.95 ± 3.17 population doublings (PD) and an estimated number of accumulated cells of between 1.0 × 10(22) and 9.7 × 10(23), with no visible alterations in the chromosome during their lifespan. All the cultures showed unchanged percentages of strongly positive expressions of the surface markers CD29, CD44, CD73, CD90, CD95, CD105 and HLA-ABC, as well as the embryonic intracellular markers Nanog and Sox2, during their lifespan, whereas the expression of the embryonic surface markers SSEA3, SSEA4, TRA-1-60 and TRA-1-81 fell until it disappeared with progression of the culture. These cells retained their differentiation capacities to adipogenic, chondrogenic and osteogenic lineages throughout their lifespan. CONCLUSIONS F-type hAMSC exhibit reproducible biologic characteristics, confirming that these cells are ideal candidates for use in regenerative medicine.

Differential transcriptional expresión of the polymorphic myxovirus resistance protein A in response to interferon-alpha treatment.

Levels of myxovirus resistance protein A (MxA) mRNA were studied for a single nucleotide polymorphism in the promoter region at nucleotide position -88 of the gene to identify individual-specific responses to interferon (IFN)-alpha2 that might predict responsiveness to IFN-alpha therapy. We quantified MxA expression by reverse transcription and real-time polymerase chain reaction in peripheral blood mononuclear cells (PBMC) in vitro, induced by IFN-alpha2, from 22 healthy donors, in relation with G/T polymorphism located in the promoter of the MxA. MxA mRNA was significantly upregulated in all subjects (mean of 53-fold) in response to IFN-alpha2 in vitro (P < 0.01). Comparison of the inducibility of MxA mRNA expression in relation with G/T polymorphism showed a 4.26-fold higher induction of MxA mRNA levels in PBMC from carriers of the mutant allele (GT or TT) than homozygotes with the wild-type allele (GG) (P < 0.001). We propose that expression of the IFN-inducible MxA is affected by a single nucleotide polymorphism in the MxA promoter which can identify an individual response to IFN-alpha2.

Evaluation of a low cost cryopreservation system on the biology of human amniotic fluid-derived mesenchymal stromal cells ☆

BACKGROUND Human amniotic-derived mesenchymal stromal cells (hAMSC) are a novel population of multipotent stem cells that have been shown to have great potential for use in regenerative medicine. However, procedures to store and preserve hAMSC for future clinical applications have not been explored extensively. METHODS In this study, we analyzed the influence of cryopreservation, using a protocol based on freezing rate of 1 °C/min, 10% dimethyl sulfoxide as cryoprotectant and a thawing rate >100 °C/min, on hAMSC morphology, proliferation rates, viability, cell cycle, karyotype, immune phenotype and multilineage differentiation potential. RESULTS This study found that this cryopreservation protocol does not affect the biological properties of hAMSC. DISCUSSION This shows that this protocol is a viable system for banking hAMSC, with the associated advantages that has a low cost in terms of expense, time and personnel involved and is easy to implement.

Changes in CDKN2D , TP53, and miR125a expression: potential role in the evaluation of human amniotic fluid-derived mesenchymal stromal cell fitness.

Human amniotic fluid‐derived mesenchymal stromal cells (hAMSC) have become one of the main cell populations used in regenerative medicine and for the study of various clinical disorders. These cells have a great capacity for proliferation and differentiation and do not form teratomas when transplanted into animal models, and their stemness seems to be between embryonic cells and adult mesenchymal cells. Before their use in cell therapy, they must be cultured and expanded in vitro, but the effect this process has on their fitness, a determining factor for the success or failure of cell therapy, is unknown. We undertook a follow‐up of gene and microRNAs (miRNAs) expression using microarray of hAMSC for the first 15 passages. Significant changes were noted in the expression of various mRNAs and miRNAs, particularly down‐regulation of TP53, increased expression of hsa‐miR‐125a and up‐regulation of CDKN2D . The variations in TP53 and hsa‐miR‐125a may act as an indicator of the stemness of the hAMSC, whereas CDKN2D may indicate the begging of early senescence process in a p53‐independent mechanism. The genes described in this study will help evaluate the fitness of hAMSC, thus guaranteeing their biological quality for use in regenerative medicine.

Direct Quantification of HIV-1 RNA in Human Plasma by Free Solution Capillary Electrophoresis (FSCE)

SUMMARY The levels of human immunodeficiency virus type 1 (HIV-1) RNA have been directly quantitated, after an isolation step, in plasma from patients with primary HIV-1 infection by free solution capillary electrophoresis (FSCE) with ultraviolet detection. HIV-1 RNA was detected and quantified at physiological levels by measuring the absorbance by FSCE. All the patients with primary infection showed concentrations in a range of 1.08-1.71 x 10(8) virions/ml of plasma. No signals were observed in seronegative donors. This procedure represents a practical alternative to other methods to quantify HIV-1 RNA and may be useful in assessing the efficiency of antiretroviral agents, especially during the early stage when other conventional viral markers are often negative.