A Reliable Method for Quantifying Plasma Cell-Free DNA Using an Internal Standard Strategy: Evaluation in a Cohort of Non-Pregnant and Pregnant Women.

The use of plasma cell-free DNA (cfDNA) as a useful biomarker in obstetric clinical practice has been delayed due to the lack of reliable quantification protocols. We developed a protocol to quantify plasma cfDNA using an internal standard strategy to overcome difficulties posed by low levels and high fragmentation of cfDNA. cfDNA was isolated from plasma samples of non-pregnant (NP, n = 26) and pregnant (P, n = 26) women using a commercial kit and several elution volumes were evaluated. qPCR parameters were optimized for cfDNA quantification, and several quantities of a recombinant standard were evaluated as internal standard. Absolute quantification was performed using a standard curve and the quality of the complete method was evaluated. cfDNA was eluted in a 50-µl volume, actin-ß (ACTB) was selected as the target gene, and qPCR parameters were optimized. The ACTB standard was constructed and 1000 copies were selected as internal standard. The standard curve showed R2 = 0.993 and E = 109.7%, and the linear dynamic range was defined between 102 and 106 ACTB copies/tube. Repeatability and reproducibility in terms of CV were 19% and up to 49.5% for ACTB copies per milliliter of plasma, respectively. The range of cfDNA levels was 428-18,851 copies/mL in NP women and 4031-2,019,363 copies/mL in P women, showing significant differences between the groups. We recommend the application of internal standard strategy for a reliable plasma cfDNA quantification. This methodology holds great potential for a future application in the obstetric field.

Insulin resistance induced by long-term hyperinsulinemia abolishes the effects of acute insulin exposure on cell-surface nicotinic acetylcholine receptor levels and actin cytoskeleton morphology.

Using CHO-K1/A5 cells, a clonal cell line that robustly expresses adult muscle-type nicotinic acetylcholine receptor (nAChR), we explored whether insulin resistance in these mammalian cells affects cell-surface expression of the nAChR, its endocytic internalization, and actin cytoskeleton integrity. Acute nanomolar insulin stimulation resulted in a slow increase in nAChR cell-surface levels, reaching maximum levels at ∼1 h. Long periods of insulin incubation caused CHO-K1/A5 cells to become insulin resistant, as previously observed with several other cell types. Furthermore, long-term insulin treatment abolished the effects of short-term insulin exposure on cell-surface nAChR levels, suggestive of a desensitization phenomenon. It also affected the kinetics of ligand-induced nAChR internalization. Since the integrity of the cortical actin cytoskeleton affects nAChR endocytosis, we also studied the effects of long-term insulin treatment on this meshwork. We found that it significantly affected the cortical actin morphology of CHO-K1/A5 cells and the response of the actin cytoskeleton to a subsequent short-term insulin stimulus. Overall, the present results show for the first time the effects of insulin signaling on cell-surface nAChR expression and actin cytoskeleton-associated internalization.

Neonatal overfeeding alters the functioning of the mesolimbic dopaminergic circuitry involving changes in DNA methylation and effects on feeding behavior.

Mesolimbic dopaminergic circuit is essential for food reward and motivational behaviors and can contribute to weight gain and obesity. Litter reduction is a classical model for studying the effects of neonatal overfeeding and overweight. Litters of Wistar rats were reduced to 4 pups/dam for small litter (SL) and 10 pups/dam for normal litter at postnatal day (PND) 4. Immediately after performing the feeding behavior tests, the animals were sacrificed in PND21 and PND90. The ventral tegmental area (VTA), Nucleus Accumbens Core (NAcC) and Shell (NAcSh) were isolated from frozen brain sections using the Palkovits micropunch technique. RNA and DNA were extracted from these areas, gene expression was measured by RT-qPCR and DNA methylation levels were measured by MSRM-qPCR technique. SL-PND21 animals presented increased expression levels of Tyrosine Hydroxylase and Dopamine Receptor D2 in VTA, decreased expression levels of dopamine active transporter (DAT) in VTA, and higher expression levels of DAT in NAcC. On the other hand, SL-PND90 animals showed decreased expression levels of Dopamine Receptor D1 and higher expression of DAT in NAcSh. These animals also evidenced impaired sensory-specific satiety. In addition, altered promoter methylation was observed at weaning, and remained in adulthood. This work demonstrates that neonatal overfeeding induces disruptions in the mesolimbic dopaminergic circuitry and causes alterations in feeding behavior from weaning to adulthood, suggesting that the neonatal period is critical for the normal development of dopaminergic circuit that impact on feeding behavior.

Epigenetic regulation of steroidogenic enzymes expressed in peripheral blood mononuclear cells from healthy individuals and from patients with chronic lymphocytic leukemia.

Sex hormone synthesis occurs in various organs and tissues besides the gonads, such as adrenal glands, brain, intestines, skin, fat, bone, and cells of the immune system. Regarding the latter, it is still not clear which pathways are active, and if they are modified in case of illness of the immune system. Our goal in this study was to determine mRNA expression of different steroidogenic enzymes in peripheral blood mononuclear cells (PBMCs) from healthy individuals of both sexes and of different ages, and then to compare their expression between healthy individuals and patients with Chronic Lymphocytic Leukemia (CLL). Furthermore, to elucidate possible mechanisms that regulate enzyme expression, we analyzed epigenetic events like promoter methylation. We determined that normal cells of the immune system, regardless of sex and age, expressed P450 side chain cleavage (P450scc), cytochrome P450 17α-hydroxylase/c17,20-lyase (P45017α), 3ß-hydroxysteroid dehydrogenase/Δ5-Δ4-isomerase (3ß-HSD), steroid 5 α reductase (5α-R) types 1, 2 and 3, 3α-hydroxysteroid dehydrogenase (3α-HSD) type 3, and 17ß-hydroxysteroid dehydrogenase (17ß-HSD) types 1, 3 and 5. We also established that 5α-R 1, 5α-R 3, 3α-HSD 3, 17ß-HSD 1 and 17ß-HSD 5 expression was altered in CLL patients, and that promoter regions of 5α-R 1, 17ß-HSD 1 and 17ß-HSD 5 were diferentially methylated. These results suggest that steroidogenic pathways may be affected in CLL cells, and this could be related to disease pathogenesis.

La sepsis como estresor: asociación con los niveles séricos de cortisol, proteína C reactiva e interleuquina1beta / Sepsis as a stressor: association with serum levels of cortisol, C reactive protein and interleukin 1beta

El estrés tiene alta prevalencia en el mundo; una de sus causas es la sepsis. Durante la misma, se liberan citoquinas que activan el eje hipotálamo-hipofiso suprarrenal (HHS) elevándose el cortisol y proteínas de fase aguda. El objetivo de este trabajo fue analizar el efecto de la sepsis sobre el eje HHS a través del cortisol, y asociarlo con interleuquina 1 beta (IL1-beta) y proteína C reactiva (PCR). Se dosó cortisol por electroquimioluminscencia, PCR por inmunoturbidimetría e IL1-beta por ELISA en sueros de pacientes sépticos (S, n=40) y no sépticos (NS, n=21). El cortisol fue significativamente mayor al valor de corte (VC), establecido mediante curva ROC, en el 63% de pacientes S, y sólo en el 14% de NS (OR: 10,0; IC: 2,5 - 39,7; p<0,05). La PCR estuvo elevada en el 55% de S, y en el 43% de NS (p>0,05). En algunos pacientes S se evidenciaron niveles muy aumentados de IL1-beta en el día de admisión. La elevación conjunta de PCR y cortisol fue del 40% en S y del 5% en NS, mientras que el aumento de los tres parámetros sólo se vio en S (8%). En conclusión, en el grupo de pacientes estudiado la sepsis actuó como activador del eje HHS, evidenciado por el incremento en el cortisol.

Stress has a high prevalence in the world; one of its causes is sepsis. During this syndrome, cytokines are released, which activate the hypothalamic - pituitary - adrenal (HPA) axis. This raises cortisol levels and acute phase proteins. The goal of this work was to analize the effects of sepsis on the HPA axis, through cortisol measurements, and to associate it with interleukin 1 beta (IL1-beta) and C reactive protein (CRP). Cortisol was measured by electrochemoluminiscence, CRP by immunoturbidimetric method and IL1-beta by ELISA in sera of septic (S, n=40) and non septic (NS, n=21) patients. Cortisol was significantly higher than its cut-off (CO), established by ROC curves, in 63% of S patients, and only in 14% of NS (OR: 10,0; CI: 2.5 - 39.7; p<0.05). CRP was elevated in 55% of S, and in 43% of NS (p>0.05). In some S patients, very high levels of IL1-beta were observed on the day of admission. The joint elevation of CRP and cortisol was of 40% in S and 5% in NS, while rises in all parameters were only seen in S (8%). It can be concluded that in the group of patients studied sepsis acted as an activator of the HPA axis, as evidenced by the elevated cortisol levels.