Patricia Esperón

Biological studies in animal models using [99mTc](CO)3 recombinant annexin V as diagnostic agent of apoptotic processes.

INTRODUCTION There are many diseases associated with variations in the expression of apoptosis such as organ rejection after transplantation, myocardial ischemia or infarct and neurodegenerative diseases. For this reason, the early visualization of this process is relevant to set fast and effective therapeutic strategies. METHODS The precursor was prepared according to the procedure reported by R. Alberto, R. Schibli, P. Schubiger, U. Abram, and T. Kaden [Reactions with the technetium and rhenium carbonyl complexes (NEt(4))[MX(3)(CO)(3)]. Synthesis and structure of Tc(CN-But)(3)(CO)(3)](NO(3)) and (Net(4))[Tc(2)(μ-SCH(2)CH(2)OH)(3)(CO)(3)], Polyhedron 1996;15: 1079-89]. Recombinant annexin V was incubated with [(99m)Tc](H(2)O)3(CO)(3)(+) solution, previously neutralized with buffer. Biodistribution studies were performed in 8-week-old female Wistar rats. Animals were housed and treated in compliance with institutional guidelines related to animal experimentation. Work protocol was previously approved by the Animal Ethics Committee of the university. Two groups of rats were defined. One was used as control and the other group was previously injected with 150 mg/kg ip of cyclophosphamide to induce apoptosis. RESULTS The synthesis of carbonyl precursor achieved yields higher than 90%, and the radiolabeled protein was obtained with 92% of radiochemical purity and high stability in vitro. An important uptake in apoptotic tissues was confirmed by biodistributions, scintigraphic images and histological studies. CONCLUSIONS Biodistribution studies revealed hepatobiliary elimination, high stability in vivo and important uptake in the reticuloendothelial system. In the pathologic model, higher uptake values correspond to the liver, spleen, lungs and femur. Histological studies confirmed the development of apoptosis at 8 and 24 h postinduction in the spleen and lymphocyte bulks in the peribronchial area. Scintigraphic images confirmed high uptake both the spleen and the lungs.

Evaluation of a Labelled Bacteriophage with 99mTc as a Potential Agent for Infection Diagnosis.

BACKGROUND AND OBJECTIVE The design of target-specific molecular imaging probes to determine infection sites are mainly based on the biochemistry of the inflammatory response that may lead to an ideal agent for infection imaging. Infectious diseases timely and specifically diagnosed can be clinically challenging but essential for the patients recovery. Laboratory tests can detect the responsible microorganism but cannot discriminate between sterile inflammatory disease and truly infectious disease. On the other hand, scintigraphic images, can pinpoint the infection in the body. METHODS Bacteriophages (phages) are viruses that infect specific bacterial strains. Given the composition of the protein capsid, they could be used as radiopharmaceuticals to diagnose bacterial infection. In this case, PP7 phage was labelled and evaluated as a specific tracer for Pseudomonas aeruginosa infections. 99mTc-Phage synthesis used HYNIC as a bifunctional agent. Physicochemical evaluation included studies such as stability in time, ligand exchange, lipophilicity and bacterial binding assay. Three groups of animals namely; healthy, infected with Pseudomonas aeruginosa and induced sterile inflammation were used to conduct biological evaluation Results: The radiolabelling process required size exclusion purification of the 99mTc-Phage, which was obtained with a radiochemical purity higher than 90%, during 18 hours post labelling. The collective accumulation in the stomach, small intestine and large intestine and thyroid of 99mTc-Phage was negligible, indicating no in vivo reoxidation. The complex presented urinary elimination. Target/ non-target ratio (T/NT) was determined both for sterile inflammation and for infection. Values were 2.5 ± 0.4 and 4.2 ± 0.3 respectively. These values indicate significant differences between sterile inflammation and infection by Pseudomonas aeruginosa (p<0.05 unpaired two sided t-test). CONCLUSION Targeted biodistribution profile and good T/NT ratios, indicate that this complex presents enough specificity to discriminate between infection caused by Pseudomonas aeruginosa and sterile inflammation.

Molecular characterization of genes modifying the age at onset in Huntington's disease in Uruguayan patients

Huntingtons disease (HD) is a hereditary neurodegenerative disorder. The genetic cause is an expansion of CAG repeats located in the IT15 gene. Though the number of CAG repeats ((CAG)n) can largely explain the age at onset (AAO) of symptoms, a percentage of its variation could be attributed to modifier genes and to environmental factors. The study aimed to evaluate the influence of genetic modifiers of the AAO of HD including: (CAG)n and del2642 in the IT15 gene, ADORA2A rs5751876, HAP1 rs4523977, PGC1-α rs7665116 and UCH-L1 rs5030732. Eighteen patients with positive family history and HD-suggestive symptoms were recruited. The (CAG)n and gene polymorphisms were determined by different molecular biology techniques. We observed that the (CAG)n influenced in a 64.5% of the variability in the AAO. We also showed that the rs5751876 variant significantly affected this variability. However, the influence of UCH-L1, del2642, HAP1 and PGC1-α gene polymorphisms could not be replicated, perhaps due to small sample size. Genetic studies including the molecular determination of (CAG)n, in addition to other genetic modifiers involved in the variability of the AAO, were first performed in Uruguay. We could replicate in our cohort the anticipation effect on the AAO by the ADORA2A rs5751876. Our results confirm the usefulness of an expanded molecular characterization in HD patients.

Methotrexate pharmacogenetics in Uruguayan adults with hematological malignant diseases

Background: Individual variability is among the causes of toxicity and interruption of treatment in acute lymphoblastic leukemia (ALL) and severe non‐Hodgkin lymphoma (NHL) patients under protocols including Methotrexate (MTX): 2,4‐diamino‐N10‐methyl propyl‐glutamic acid. Methods: 41 Uruguayan patients were recruited. Gene polymorphisms involved in MTX pathway were analyzed and their association with treatment toxicities and outcome was evaluated. Results: Genotype distribution and allele frequency were determined for SLC19A1 G80A, MTHFR C677T and A1298C, TYMS 28 bp copy number variation, SLCO1B1 T521C, DHFR C−1610G/T, DHFR C‐680A, DHFR A‐317G and DHFR 19 bp indel. Multivariate analysis showed that DHFR‐1610G/T (OR = 0.107, p = 0.018) and MTHFR677T alleles (OR = 0.12, p = 0.026) had a strong protective effect against hematologic toxicity, while DHFR‐1610CC genotype increased this toxicity (OR = 9, p = 0.045). No more associations were found. Conclusions: The associations found between gene polymorphisms and toxicities in this small cohort are encouraging for a more extensive research to gain a better dose individualization in adult ALL and NHL patients. Besides, genotype distribution showed to be different from other populations, reinforcing the idea that genotype data from other populations should not be extrapolated to ours. Graphical Abstract Figure. No caption available.

Una nueva mutación en el promotor del gen del receptor de las lipoproteínas de baja densidad asociada a hipercolesterolemia familiar en homocigosis y heterocigosis

La hipercolesterolemia familiar (HF) es una enfermedad hereditaria, relativamente frecuente y asociada al desarrollo de ateroesclerosis y enfermedad coronaria prematuras. En este trabajo, describimos el hallazgo de una nueva mutacion (−47C>A) en la region promotora del gen del receptor de las lipoproteinas de baja densidad (rLDL), principal encargado de este fenotipo. Esta mutacion se identifico en una familia uruguaya con un fenotipo tipico de HF, en la cual aparecen individuos heterocigotos y homocigotos para la mutacion debido a la existencia de consanguinidad. El cambio nucleotidico ocurre en un sitio conservado y funcionalmente relevante del promotor; region en la que anteriormente se han descrito diversas mutaciones que provocan descensos drasticos en la actividad transcripcional del gen. No se han detectado otras variantes en las regiones analizadas del gen. Los analisis geneticos del rLDL asociados a otros genes de susceptibilidad permiten establecer un perfil genomico de riesgo cardiovascular, de aplicacion muy necesaria en el tratamiento clinico de familias con HF.

Genetic markers in methotrexate treatments

Methotrexate (MTX), a structural analog of folic acid, is widely employed in the treatment of different cancers and autoimmune diseases. Despite the successful results observed, the main disadvantage lies in interpatient variability in the pharmacokinetic and pharmacodynamic parameters. In particular, adverse events and toxicities induced by MTX are a matter of concern and can be the cause of dose reduction or treatment discontinuation. Among the different approaches to reduce MTX therapeutic limitations, pharmacogenomics contributes by considering the effect of inherited genetic differences on those parameters. This review provides an update on MTX pharmacogenomics. It reports the contribution of main gene polymorphisms involved in the influx, efflux, cellular effect, and elimination on MTX toxicity and efficacy, on all the diseases treated with this drug. From the analysis of the data presented in this review, we concluded that only gene polymorphisms MTHFR rs1801133, SLC19A1 rs1051266, and TYMS rs34743033 could influence clinical decision-making.

Activity of the Calcineurin Pathway in Patients on the Liver Transplantation Waiting List: Factors of Variability and Response to Tacrolimus Inhibition

BACKGROUND We sought to evaluate, in patients on a liver transplantation waiting list, potential biomarkers of the base calcineurin pathway activity with use of a new model of nonstimulated peripheral blood mononuclear cells (PBMC) and ex vivo response to tacrolimus (TAC). METHODS The calcineurin pathway activity was explored ex vivo in stimulated and nonstimulated PBMC from 19 patients. The inhibition of NFAT1 translocation to PBMC nuclei, expression of intracellular IL-2, and membrane CD25 in different T-cell subsets were measured by multiparametric flow cytometry before and after exposure to TAC. We also studied the influence on the individual response of polymorphisms in 3 key genes of the calcineurin pathway: PPIA, PPP3CA, and IL2RA. RESULTS All pharmacodynamics profiles closely fitted an I/Imax sigmoid model. Interindividual variability was higher in nonstimulated than in stimulated conditions, as well as in the presence of TAC. IL-2+CD8+ cells at TAC Imax showed the highest interindividual variability, suggesting its usefulness as a biomarker of individual TAC effects integrating many different sources of regulation and variability. Moreover, in the absence of TAC, patients with end-stage liver disease exhibited lower NFAT1 translocation and T-cell activation than healthy volunteers from a previous study under similar conditions. Multivariate statistical analysis showed strong and significant associations between TAC pharmacodynamic parameters and 2 polymorphisms in the gene-coding cyclophilin A (rs8177826 and rs6850). CONCLUSIONS We show the feasibility of using nonstimulated PBMCs to explore the calcineurin pathway under more physiologic conditions and point toward potential biomarkers for TAC pharmacodynamic monitoring. ClinicalTrials.gov Identifier: NCT01760356.

Different methodological approaches for interleukin 28B genotyping

Background and Objectives: Treatment responsiveness to pegylated interferon-α and ribavirin against hepatitis C virus genotype 1 has strongly been associated with two single nucleotide polymorphisms (rs8099917 and rs12979860) in the region of the interleukin 28B (IL28B) gene. The aim was to perform three genotyping methods and evaluate their specificity, technical characteristics, and costs. In addition, the distribution of both polymorphisms in an Uruguayan population was assessed. Materials and Methods: One hundred DNA samples were genotyped by allele-specific polymerase chain reaction (AS-PCR), real-time PCR high resolution melting (RT-HRM), and Sanger sequencing methods. Results: The rs12979860 CC genotype, followed by the CT, was the most prevalent (52% and 39%, respectively). For rs8099917, the TT genotype was the most common (61%) followed by the GT (34%). AS-PCR and RT-HRM assays were specific for both IL28B genotypes determinations and showed a total concordance with Sanger sequencing results. Conclusions: Any of three genotyping methods is suitable for IL28B genotyping. The choice of the assay will depend on costs, special equipment availability, turnaround time, and specialized human resources.

DNA–protein interactions detected by silver staining

▼The mobility shift assay (EMSA) is one of the most powerful methods for the analysis of DNA–protein or RNA–protein complexes. These specific interactions are essential to many basic processes including the control of gene expression, site-specific recombination, replication and the repair of DNA damage. The most important applications of this technique are the kinetic and thermodynamic characterization of complex formation, the definition of binding sites, and conformational analysis. In order to perform a gel shift assay, the DNA fragments and the proteins (crude extracts or purified) are mixed together. The reaction mixtures are then run on nondenaturing polyacrylamide or agarose gels. Protein binding to the DNA fragments can result in a complex with reduced electrophoretic mobility relative to the free DNA. Various techniques have been reported for detecting DNA–protein complexes. The complexes can be visualized using either 32Por 33P-dNTP-labelled DNA fragments (Ref. 1, 2) or fluorescently labelled oligonucleotides (Ref. 3). Alternatively, the complexes can be detected by ethidium bromide staining (Ref. 4). We report here an innovative technique to detect DNA–protein complexes easily in polyacrylamide gels. The technique is performed using purified protein, and complexes are developed by staining the gel with silver nitrate. The main advantages of this technique are