Melatonin reduces inflammatory response in peripheral T helper lymphocytes from relapsing-remitting multiple sclerosis patients.

Multiple sclerosis (MS) is a neuroinflammatory disease of the central nervous system in which the immune system plays a central role. In particular, effector populations such as T helper (Th) 1, Th9, Th17, and Th22 cells are involved in disease development, whereas T regulatory cells (Tregs) are associated with the resolution of the disease. Melatonin levels are impaired in patients with MS, and exogenous melatonin ameliorates the disease in MS animal models by modulating the Th1/Th17/Treg responses and also improves quality of life and several symptoms in patients with MS. However, no study has examined melatonin's effect on T cells from relapsing-remitting MS (RR-MS) patients. Therefore, the objectives of the present study were to evaluate the effects of the in vitro administration of melatonin to peripheral blood mononuclear cells (PBMCs) from 64 RR-MS patients and 64 sex- and age-matched healthy subjects on Th1, Th9, Th17, Th22, and Treg responses and to analyze the expression of the melatonin effector/receptor system in these cells. Melatonin decreased Th1 and Th22 responses in patients, whereas it did not affect the Th17 and Treg subsets. Melatonin also promoted skewing toward a more protective cytokine microenvironment, as shown by an increased anti-inflammatory/Th1 ratio. Furthermore, for the first time, we describe the overexpression of the melatonin effector/receptor system in PBMCs from patients with MS; this alteration might be relevant to the disease because acetylserotonin O-methyltransferase expression significantly correlates with disease progression and T effector/regulatory responses in patients. Therefore, our data suggest that melatonin may be an effective treatment for MS.

Evaluation of the immunomodulatory effect of melatonin on the T-cell response in peripheral blood from systemic lupus erythematosus patients.

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by the production of antinuclear autoantibodies. In addition, the involvement of CD4+ T-helper (Th) cells in SLE has become increasingly evident. Although the role of melatonin has been tested in some experimental models of lupus with inconclusive results, there are no studies evaluating the melatonin effect on cells from patients with SLE. Therefore, the aim of this study was to analyse the role of in vitro administered melatonin in the immune response of peripheral leukocytes from treated patients with SLE (n = 20) and age- and sex-matched healthy controls. Melatonin was tested for its effect on the production of key Th1, Th2, Th9, Th17 and innate cytokines. The frequency of T regulatory (Treg) cells and the expression of FOXP3 and BAFF were also explored. Our results are the first to show that melatonin decreased the production of IL-5 and to describe the novel role of melatonin in IL-9 production by human circulating cells. Additionally, we highlighted a two-faceted melatonin effect. Although it acted as a prototypical anti-inflammatory compound, reducing exacerbated Th1 and innate responses in PHA-stimulated cells from healthy subjects, it caused the opposite actions in immune-depressed cells from patients with SLE. Melatonin also increased the number of Treg cells expressing FOXP3 and offset BAFF overexpression in SLE patient cells. These findings open a new field of research in lupus that could lead to the use of melatonin as treatment or cotreatment for SLE.

Evidence of immune system melatonin production by two pineal melatonin deficient mice, C57BL/6 and Swiss strains.

We evaluated two pineal melatonin deficient mice described in the literature, i.e., C57BL/6 and Swiss mice, as animal models for studying the immunomodulatory action of melatonin. Plasma melatonin levels in C57BL/6 and Swiss strains were detectable, but lower than levels in control C3H/HENHSD mice. Since these strains are suppose to be pineal melatonin deficient an extrapineal melatonin synthesis may contribute to plasma levels. Regarding cells and tissues from the immune system, all of them were found to synthesize melatonin although at low levels. N-acetyltransferase (AANAT) mRNA was also amplified in order to analyze the alternative splicing between exons 3-4 described for pineal C57BL/6 mice which generates an inclusion of a pseudoexon of 102 bp. For the pineal gland, both the wild type and the mutant isoforms were present in all mice strains although in different proportions. We observed a predominant wild type AANAT mature RNA in thymus, spleen and bone marrow cells. Peripheral blood mononuclear cells (PBMC) culture shown an evident AANAT amplification in all strains studied. Although the bands detected were less intense in melatonin deficient mice, the amplification almost reached the control cell intensity after stimulation with phytohemaglutinin (PHA). In summary, melatonin detection and AANAT mRNA expression in inbred and outbred mice clearly indicate that different cells and tissues from the immune system are able to synthesize melatonin. Thus, the pineal defect seems not to be generalized to all tissues, suggesting that other cells may compensate the low pineal melatonin production contributing to the measurable plasma melatonin level.

Total and Fetal Circulating Cell-Free DNA, Angiogenic, and Antiangiogenic Factors in Preeclampsia and HELLP Syndrome.

BACKGROUND: Preeclampsia (PE) is a hypertensive disorder of pregnancy characterized by hypertension and proteinuria. The HELLP syndrome is the most severe form of PE. The aim of the present study was to determine different potential biomarkers that may help us perform an early diagnosis of the disease, assess on the severity of the disease, and/or predict maternal or fetal adverse outcomes. METHODS: We measured serum levels of total and fetal circulating cell-free DNA (cfDNA), soluble endoglin, soluble form of vascular endothelial growth factor receptor, and placental growth factor in a healthy control group of pregnant women (n = 26), patients with mild (n = 37) and severe PE (n = 25), and patients with HELLP syndrome (n = 16). RESULTS: We observed a gradual and strong relationship between all the biomarkers mentioned and the range of severity of PE, with the highest levels in patients with HELLP syndrome. Nevertheless, only the values of total cfDNA were able to significantly differentiate severe PE and HELLP syndrome (20957 ± 2784 vs. 43184 ± 8647 GE/ml, P = 0.01). Receiver operating characteristic (ROC) curves were constructed (i) for the healthy group with respect to the groups with PE and (ii) for patients with PE with respect to the group with HELLP syndrome; sensitivity and specificity values at different cutoff levels were calculated in each case. The maximum ROC area under the curve value for PE and HELLP syndrome (with respect to controls) was 0.91 (P < 0.001). CONCLUSIONS: The measured biomarkers of cell damage, angiogenesis, and antiangiogenesis may reflect the severity of PE, with higher levels in patients who develop HELLP syndrome. In addition, these biomarkers may also help predict adverse fetal and maternal outcomes.

Associations between frailty and serum N-terminal propeptide of type I procollagen and 25-hydroxyvitamin D in older Spanish women: The Toledo Study for Healthy Aging.

Frail older people are at high risk for fractures and falls increasing the rates of institutionalization and mortality. Bone markers have been related to both aging and fractures. However, no previous reports have shown a potential relationship between serum bone markers such as N-terminal propeptide of type I procollagen (PINP), C-terminal telopeptide of type I collagen (ß-CTX) and parathyroid hormone (PTH) with frailty in elderly female populations. This study is aimed at examining the associations of bone metabolism markers and frailty in older Spanish women through a descriptive cross-sectional analysis based on a cohort of the Toledo Study for Healthy Aging (TSHA). The levels of serum PINP, ß-CTX, PTH and 25-hydroxyvitamin D (25(OH)D) were assessed in 592 participants (median age 74years) who were defined as robust, prefrail and frail according to Fried's approach. Frail subjects had significantly high levels of PINP, ß-CTX and PTH and low production of 25(OH)D. After adjustment for confounders, high PINP levels (defined by the upper quartile) and low levels of 25(OH)D (lower quartile) remained significantly associated to frailty [OR for PINP: 2.19 (95% CI, 1.15-4.18; P=0.017); OR for 25(OH)D: 1.65 (95% CI, 1.02-2.67; P=0.042)]. Women with both high PINP levels and low 25(OH)D levels presented a 5.85-fold increased frailty risk (95% CI, 1.64-20.93; P=0.007). The main contribution of this paper is the novel definition of PINP and 25(OH)D markers as potential biomarkers of frailty and targets for intervention.

Standardization non-invasive fetal RHD and SRY determination into clinical routine using a new multiplex RT-PCR assay for fetal cell-free DNA in pregnant women plasma: results in clinical benefits and cost saving.

INTRODUCTION: Among negative RhD mothers it is essential to know the fetal RhD status in order to avoid the possibility of hemolytic disease of the newborn. In this regard, the detection of fetal DNA in maternal plasma might become a new diagnostic tool. In the current study, we have evaluated the standardization of a Multiplex-PCR targeted towards two exons of the RHD and one SRY gene to monitor RhD negative women. The current study addresses questions concerning feasibility and applicability of this approach into the clinical practice. MATERIALS AND METHODS: Both single and multiplex real-time PCRs targeting RHD exons 5 and 7 and SRY were applied for the detection of fetal-specific RHD sequences and sex in maternal plasma. A large cohort of 2127 women was studied between 10 and 28 weeks of pregnancy. 134 of them were used for single TaqMan PCR studies and 1993 were evaluated using Multiplex TaqMan PCR studies. All of them were serologically typed as RhD negative according to Spanish guidelines. Single and multiplex real-time PCR results were compared with postnatal serology and sex identification. RESULTS: There was a 100% concordance between results obtained with single and multiplex real-time PCR assays. At present, 1012 of the 1993 pregnant women studied gave birth and the results of RHD status obtained with the multiplex TaqMan PCR assay were confirmed postpartum by serological methods showing that sensitivity, specificity, and accuracy of the multiplex assay were 100, 98.6, and 99.3%, respectively. This procedure improved the speed of the assay, avoided over-treatment among RhD negative pregnant women bearing RhD negative fetus, and reduced the requirements for clinical and biological monitoring, resulting in a clinical benefit and cost saving. CONCLUSIONS: The routine determination of fetal RHD status and SRY in maternal plasma, using multiplex real-time PCR, is feasible. The use of multiplex real-time PCR allows improving the response of the laboratory, saving time and reagent costs, opening the door to a complete automatization of the process.

Autophagy upregulation and loss of NF-kappaB in oxidative stress-related immunodeficient SAMP8 mice.

Aged spleens from senescence-accelerated prone mice 8 (SAMP8) and senescence-accelerated resistant mice 1 (SAMR1) were examined to determine whether sex or melatonin had an effect on oxidative stress-related immune impairments. We observed that the immunosenescence of SAMP8 mice was associated with a redox imbalance, leading to an age-related increase in oxidative damage, resulting from a decrease in antioxidant defense and protease activity. Moreover, increased apoptotic cell death, a decrease in proliferative activity and the loss of NF-kappaB activation were also related to the immunodeficiency seen in SAMP8 compared to SAMR1 mice. Females demonstrated higher oxidative stress-related alterations in the immune response, and subsequent, melatonin treatment provided the best protective effects. Pathways involved in autophagy were upregulated in SAMP8 as an adaptive response to oxidative stress, in an attempt to rescue the cell from increased apoptosis and age-related immunodeficiency. However, the NF-kappaB signaling and autophagic processes were unaffected by treatment with melatonin. Therefore, we propose a key role for NF-kappaB signaling and autophagy in the oxidative stress-related immunosenescent spleens of SAMP8 mice.