Bioquímica de la función tiroidea

Peer Reviewe

A novel interplay between membrane and nuclear melatonin receptors in human lymphocytes: significance in IL-2 production

10 páginas, 7 figuras, 1 tabla.Human lymphocyte melatonin, through membrane and nuclear receptors binding, acts as an activator in IL-2 production. Antagonism of membrane melatonin receptors using luzindole exacerbates the drop of the IL-2 production induced by PGE2 in peripheral blood mononuclear and Jurkat cells. This paper studies the melatonin membrane and nuclear receptors interplay in PGE2-diminished IL-2 production. The decrease in IL-2 production after PGE2 and/or luzindole administration correlated with downregulation in the nuclear receptor RORα. We also highlighted a role of cAMP in the pathway, because forskolin mimicked the effects of luzindole and/or PGE2 in the RORα expression. Finally, a significant RORα downregulation was observed in T cells permanently transfected with inducible MT1 antisense. In conclusion, we show a novel connection between melatonin membrane receptor signalling and RORα expression, opening a new way to understand melatonin regulation in lymphocyte physiology.This work was supported by a grant from the Ministerio de Sanidad FIS (06/0091) and Red-FIS (RD06/0013/0001) and Grupo Excelencia del Plan Andaluz de Investigación (P06-CTS-01604).Peer reviewe

Non-invasive Prenatal Diagnosis of Feto-Maternal Platelet Incompatibility by Cold High Resolution Melting Analysis

Gahan P., Fleischhacker M., Schmidt B. (eds).Fetal and Neonatal alloimmune thrombocytopenia (FNAIT) is a condition which could occur when pregnant women develop an alloimmunization against paternally inherited antigens of the fetal platelets. Approximately 80 % of FNAIT cases are caused by anti-HPA-1a, about 15 % by anti-HPA-5b and 5 % by other HPA antibodies. Only 2 % of the total population is HPA-1a negative (HPA-1b1b). The HPA-1a allele differs by one single nucleotide from HPA-1b allele, yet it represents around 27 % of total severe thrombocytopenias. HPA-1 was studied in serum cDNA from 12 volunteer pregnant women to determine their HPA-1 genotype by HRM (high resolution melting) PCR. When an homozygous HPA-1 gene was detected in a mother, a COLD HRM was performed to determine whether or not the fetal genotype differs from the mother’s. The differences in the melting curve shapes allow us to accurately distinguish the three pregnants genotypes. The fetal heterozygous genotype of homozygous pregnant women was correctly detected by COLD PCR HRM in maternal serum. HPA-1 genotyping by HRM may be a useful aproach for genotyping all pregnant women in inexpensively. Moreover, when HPA-1 homozygosis was detected in a pregnant woman, fetal heterozygosis may be diagnosed by COLD HRM to select pregnancies for preventive monitoring.Peer reviewe

Screening of KRAS Mutation in Pre- and Post-Surgery Serum of Patients Suffering from Colon Cancer by COLD-PCR HRM

Gahan P., Fleischhacker M., Schmidt B. (eds).Genomic characterization of cell-free circulating tumour DNA (ctDNA) may offer an opportunity to assess clonal dynamics throughout the course of a patient’s illness. The existence of KRAS driver mutations in colon cancer patients is determinant to decide their treatment and to predict their outcome. DNA is extracted automatically from 400 μL of serum using the MagNa Pure Compact with the Nucleic Acid Isolation Kit I. DNA amplification, COLD-PCR and HRM were performed in the same run in the Light Cycler 480. We found three different situations: pre- and post-surgical samples grouped with the negative control, pre-surgical samples appear to group with the positive control and the post-surgical samples appear to group with the negative control and finally both samples, pre- and post-surgical ones, appear to be grouped with the positive control. COLD-PCR HRM is a cost-effective way for screening one of the most common driver mutations to predict the worst prognosis in colorectal cancer.Peer reviewe

Temporal expression patterns of the melatoninergic system in the human thymus of children

Objectives: To obtain greater knowledge of the extra-pineal sources of melatonin during development, the amount of indolamine and the expression levels of the last two enzymes involved in its biosynthesis, Arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin Omethyltransferase (ASMT), were analyzed in the human thymus from children from three different age groups (from days to years). The melatonin membrane and nuclear receptor expression levels also were studied. Methods: Quantitative reverse transcriptase PCR and western blot were performed to investigate the receptor and enzyme expression levels. The results were examined and correlated with the ages of the thymuses. Results: We found high levels of indolamine in the thymuses of newborns (younger than 1 month), which decreased during development; thymuses from the months (from 2 to 11 months) and years (from 1 to 12 years) groups showed lower levels. A similar decline was also observed in the mRNA of the AANAT enzyme and the expression levels of melatonin receptors. However, ASMT expression was exactly the opposite, with low levels in the newborn group and higher levels in the years group. Our results show that the thymic synthesis of melatonin occurs very early in childhood. Additionally, this is the first report that is focused on melatonin receptors expression in the human thymus. Conclusion: Considering the limited melatonin synthesis performed by the newborn pineal gland, we suggest that the high levels of melatonin found in human thymus in this experimental group arise from synthesis in the tissue itself, which could be contributing to the immune efficiency at the thymic level.Regional Government Ministry of Health PI-0209-2010PAIDI Program from the Andalusian Government CTS160Andalusian Public Foundation Progress and Health PI-0485-2014FPU programSpanish Government FPU13/01210Instituto de Salud Carlos III, Ministerio de Economía y Competitividad RD06/ 0013/0001 and RD12/0043/001

Donor-Specific Cell-Free DNA qPCR Quantification as a Noninvasive Accurate Biomarker for Early Rejection Detection in Liver Transplantation

(1) Background: Graft-cell-free DNA (cfDNA) in the circulation of liver transplant recipients has been proposed as a noninvasive biomarker of organ rejection. The aim of this study was to detect donor-specific cfDNA (ds-cfDNA) in the recipient’s serum after either liver damage or rejection using a qPCR-based method. (2) Methods: We proposed a qPCR method based on the amplification of 10 specific insertion–deletion (InDel) polymorphisms to detect donor-specific circulating DNA diluted in the recipient cfDNA. ds-cfDNA from 67 patients was evaluated during the first month post-transplantation. (3) Results: Graft rejection in the first month post-transplantation was reported in 13 patients. Patients without liver complications showed a transitory increase in ds-cfDNA levels at transplantation. Patients with rejection showed significant differences in ds-cfDNA increase over basal levels at both the rejection time point and several days before rejection. Receiver operator characteristic (ROC) analysis showed that ds-cfDNA levels discriminated rejection, with an AUC of 0.96. Maximizing both sensitivity and specificity, a threshold cutoff of 8.6% provided an estimated positive and negative predictive value of 99% and 60%, respectively. (4) Conclusions: These results suggest that ds-cfDNA may be a useful marker of graft integrity in liver transplant patients to screen for rejection and liver damage

Detection of p53 Mutations in Circulating DNA of Transplanted Hepatocellular Carcinoma Patients as a Biomarker of Tumor Recurrence

Gahan P., Fleischhacker M., Schmidt B. (eds).p53 is the most commonly mutated gene in malignant human cancers. To detect p53 mutations in circulating DNA (cirDNA) of transplanted hepatocellular carcinoma (HCC) patients could be an interesting approach to know of any tumor recurrence. In this study, our objective was to determine the utility of this method in the diagnosis and the prognosis of HCC tumor recurrence. Twenty four liver transplanted HCC patients were included in the study together with a group of healthy controls. Detection of the specific p53 mutation in cirDNA was performed by high-resolution melting PCR (HRM-PCR) and COLD-PCR immediately before the transplantation. Serum anti-p53 was also determined using a p53-autoantibody ELISA kit. The results of the HRM-PCR and COLD-PCR showed two well-differentiated groups of transplanted patients after normalization by healthy controls. These data allow us to distinguish between patients with p53 mutated cirDNA and those with wild type cirDNA. Moreover, we have found that most of p53 mutated patients also presented elevated anti-p53 antibodies. The present results indicate that it is possible to detect mutated p53 genes with the cirDNA and that this could be used as a biomarker of tumor recurrence during the clinical evolution of the transplanted patients.Peer reviewe

Evaluation of the State of Transplanted Liver Health by Monitoring of Organ-Specific Genomic Marker in Circulating DNA from Receptor

Gahan P., Fleischhacker M., Schmidt B. (eds).The evaluation of the transplanted liver health by non-invasive approaches may offer an improvement in early clinical intervention. As transplanted organs have genomes that are distinct from the host’s genome, the quantification of the specific DNA of the donated liver in the patient serum will allow us to obtain information about its damage. We evaluated the state of transplanted liver health by monitoring the RH gene in serum circulating DNA (cirDNA) from 17 recipient and donor mismatched for this gene. cirDNA RH gene was quantified by RT- PCR before, at the moment of transplantation (day 0) and during the stay at the intensive care unit. Beta-globin cirDNA was quantified as a general cellular damage marker. Patients were grouped based on clinical outcomes: (A) patients with no complication; (B) patients that accepted the organ but suffered other complications; (C) patients that suffered organ rejection. All patients showed an increased cirDNA levels at day 0 that decreased until patient stabilization. Patients from groups A and B showed low levels of the RH gene cDNA during the follow-up, with an increase of beta-globin gene at the moment of any clinical complication. Patients from group C showed an increase in the RH gene during rejection.Peer reviewe

Donor-specific circulating cell free DNA as a noninvasive biomarker of graft injury in heart transplantation

[Background] Considerable effort has been exerted to develop noninvasive diagnostic biomarkers that might replace or reduce the need to perform endomyocardial biopsies. In this context, graft DNA circulating on transplant recipients has been proposed as a potential biomarker of organ rejection or cellular graft injury. [Methods] We propose a digital PCR (dPCR) method based on the amplification of ten specific InDels sufficiently sensitive to detect small amounts of specific donor circulating DNA diluted on the host cell free DNA (cfDNA). We obtained 23 informative mismatches from 30 host and donor organ biopsy pairs. [Results] Patients without heart-related complications showed a high increase in the specific genomic marker levels during the first 24 h after transplantation that dropped to the basal levels on days 3–4 post-surgery. In contrast, patients with complications presented a significantly lagged decay pattern from day one after transplantation. A specific donor cfDNA increase was detected in one patient two days before rejection diagnosis, diminishing the basal levels after successful immunotherapy. A cfDNA increase was also observed during graft injury due to heart damage. [Conclusion] These results suggest that cfDNA monitoring of transplanted patients may be a useful tool to detect and probably anticipate graft rejection.This work was supported by a grant from the Instituto de Salud Carlos III [FIS: PI15/00939]-Fondos FEDER