Effects of peripartal energy supply and nicotinic acid supplementation on functional activity and gene expression of blood leukocytes and on serum antioxidant variables of periparturient dairy cows : [kumulative Dissertation

The timing of clock gene expression in human leukocytes was investigated following a phase-advancing light stimulus to determine whether the response is wavelength- and/or age-dependent. PERIOD3 (PER3) clock gene expression in leukocytes and plasma melatonin were analyzed before and after monochromatic blue and green light exposure. Significant phase advances were observed in the peak timing of both PER3 expression and melatonin following blue but not green light. The amplitude of the PER3 rhythm at baseline was significantly reduced with age. However, age did not affect the response of the PER3 rhythm to light. (Author correspondence: d.skene@surrey.ac.uk)

Inflammation contributes to the development of atherosclerosis and cardiovascular events. Counteracting pro- and anti-inflammatory responses of serum cytokines have been reported, but the relevance of TNF-alpha, TGF-beta and IL-6 gene expression in peripheral blood leukocytes and their contribution to systemic inflammation in atherosclerosis, especially after acute myocardial infarction (AMI), has not been investigated yet. Using quantitative RT-PCR, we determined temporal cytokine mRNA expression alterations in blood cells from patients with AMI (n = 51). Serum cytokine concentrations were analyzed in parallel using the ELISA technique. TNF-alpha mRNA expression rates and serum concentrations were significantly elevated in AMI patients compared to controls (n = 77), while mRNA expression and serum content of TGF-beta were decreased. Interestingly, we found no statistically significant correlation between transcript and protein levels, indicating that gene expression in leukocytes may be an independent sign for systemic inflammation. While IL-6 was significantly increased in serum from AMI patients with positive correlation to left ventricular dysfunction and negative correlation to ejection fraction, IL-6 mRNA levels did not differ between patients and controls. Gene expression alterations indicate a sophisticated regulation of counteracting TNF-alpha and TGF-beta cytokine expression in peripheral blood leukocytes after AMI with bias towards a pro-inflammatory situation.

Mammals have circadian clocks, which consist of the central clock in the suprachiasmatic nucleus and the peripheral clocks in the peripheral tissues. The effect of exercise on phase of peripheral clocks have been reported in rodents but not in humans. Continuous sampling is necessary to assess the phase of the circadian rhythm of peripheral clock gene expressions. It has been assumed that the expression of the genes in leukocyte may be "an accessible window to the multiorgan transcriptome." The present study aimed to examine whether exercise affects the level and phase of clock gene expression in human leukocytes. Eleven young men participated in three trials, in which they performed a single bout of exercise at 60% V̇o2max for 1 h beginning either at 0700 (morning exercise) or 1600 (afternoon exercise) or no exercise (control). Blood samples were collected at 0600, 0900, 1200, 1500, 1800, 2100, and 2300 and at 0600 the next morning, to assess diurnal changes of clock gene expression in leukocytes. Brain and muscle ARNT-like protein 1 (Bmal1) expression level increased after morning and afternoon exercise, and Cryptochrome 1 (Cry1) expression level increased after morning exercise. Compared with control trial, acrophase of Bmal1 expression tended to be earlier in morning exercise trial and later in afternoon exercise trial. Acrophase of Cry1 expression was earlier in morning exercise trial but not affected by afternoon exercise. Circadian locomotor output cycles kaput (Clock), Period 1-3 (Per1-3), and Cry2 expression levels and those acrophases were not affected by exercise. The present results suggest a potential role of a single bout of exercise to modify peripheral clocks in humans.NEW & NOTEWORTHY The present study showed that a single bout of exercise affected peripheral clock gene expression in human leukocytes and the effect of exercise depended on when it was performed. Brain and muscle ARNT-like protein 1 (Bmal1) expression was increased after exercises performed in the morning and afternoon. Cryptochrome 1 (Cry1) expression was also increased after the morning exercise. The effect of exercise on acrophase of Bmal1 depended on the time of the exercise: advanced after morning exercise and delayed after afternoon exercise.

Impaired balance of T regulatory and T effector lymphocytes has recently been considered as an important pathogenetic link in arterial hypertension (AH). There are, however, contradictory literature data about contents of these cells in the patients with hypertension, or obtained in experimental animal models of induced hypertension. Most results about changed patterns of immune cells in cardiovascular diseases were obtained by means of flow cytometry. There are also some works on expression of genes encoding surface and cytoplasmic differentiation antigens of immune cells in the patients with cardiovascular pathologies. These results coincide with the data obtained with flow cytometric techniques. Purpose of the present study was to analyze of the levels of gene transcripts encoding differentiation markers of regulatory (FOXP3, IL2R) T cells, effector T subpopulations (T helpers 17 (RORγ), and CD8 lymphocytes (CD8A) in healthy subjects and the patients with arterial hypertension (stages I-II). We examined healthy individuals (40 people, 20 men and 20 women), 27 patients with hypertension who did not receive antihypertensive therapy (14 men and 13 women), 26 hypertensive patients taking β-adrenergic receptor blockers (metoprolol or bisoprolol), including 12 men and 14 women. The relative levels of transcripts in peripheral blood leukocytes were assessed by real-time RT-PCR. It was shown that the transcriptional activity of FOXP3, IL2R, RORγ, and CD8A genes in peripheral blood leukocytes of the diseased people was significantly higher than in healthy individuals (p < 0.01). This finding may indicate an increased number of circulating T regulatory lymphocytes, CD8+ cells and T helpers 17 in hypertensive patients, and activation of T cell immunity in these patients. There were no statistically significant gender differences in FOXP3, IL2R, RORγ and CD8A gene expression in leukocytes, both in the group of healthy people and in hypertensive patients. The patients receiving cardioselective β-adrenergic receptor blockers (metoprolol and bisoprolol) exhibited lower expression of these genes, thus, probably, indicating antiinflammatory and immunomodulatory properties of these drugs.

Adrenergic receptor gene expression in bovine leukocytes

Immunostimulant effects and potential application of β-glucans derived from marine yeast Debaryomyces hansenii in goat peripheral blood leucocytes

Effects of energy supply and nicotinic acid supplementation on phagocytosis and ROS production of blood immune cells of periparturient primi- and pluriparous dairy cows

The constant intensification of aquaculture has considerable increased the stress levels of farmed fish and, consequently, the number and intensity of diseases outbreaks. Thus, studies on fish immune response, especially regarding the interaction of fish leukocytes with potential pathogens and xenobiotics are of great importance in order to develop new prophylactic and curative strategies. We isolated leukocytes from the head kidney of Astyanax lacustris-an important Neotropical fish species for aquaculture and a potential model for Neotropical aquaculture research-using a Percoll centrifugation protocol. The isolated leukocytes were incubated with lipopolysaccharide (LPS), and the expression of genes IL-1β, IL-8, LysC, and LysG were measured. We assessed the phagocytotic activity of leukocytes using Congo red-dyed yeast, a novel and cost-effective protocol that has been developed in this study. The isolated leukocytes responded to LPS induction, exhibiting strong IL-1β and IL-8 upregulation, two of the most important pro-inflammatory interleukins for vertebrates immune reponse. The optimal concentration of yeast for the phagocytic assay was 106 cells mL-1, resulting in acceptable phagocytic capacity (PC) but without excess of yeasts during the counting process, ensuring a high precision and accuracy of the method. To the best of our knowledge, the present study is the first to investigate the in vitro gene expression and phagocytic activity of leukocytes isolated from A. lacustris. Our findings will serve as a reference for future studies on the immunology and toxicology of Neotropical fish.

The periparturient period is accompanied by metabolic and oxidative stress. Niacin is known to decrease lipolysis but is also reported to have anti-oxidative effects. Therefore, we examined the effects of energy supply and a nicotinic acid (NA) supplementation on anti-oxidative serum parameters and on the expression of oxidative stress-related genes in blood leucocytes of periparturient dairy cows, differing in parity. Twenty-nine pluriparous and 18 primiparous cows were allocated to four different feeding groups 42days before expected parturition until 100days postpartum and fed a ration with either a low concentrate proportion of 30% (LC) or a high concentrate proportion of 60% (HC). After parturition, all animals received 30% concentrate which was increased to 50% either within 16 (LC group) or 24days (HC group). Half of the animals per group were supplemented with 24g NA per day from 42days prepartum until 24days postpartum. All investigated parameters varied significantly over time compared to parturition (p<.05). Ferric reducing ability (FRA) exhibited a nadir before parturition, and the antioxidant enzymes glutathione peroxidase (GPX) and superoxide dismutase (SOD) showed peak activities around parturition. Expression levels of GPX1, SOD2, xanthine dehydrogenase (XDH) and nuclear factor (erythroid-derived 2)-like 2 (NRF2) peaked before calving. The concentrate level influenced GPX activity and mRNA abundance of SOD2, XDH and poly (ADP-ribose) polymerase 1 (PARP1). Pluriparous animals exhibited higher serum GPX activities, a more distinct nadir for FRA and higher expression levels for GPX1, SOD2 and XDH. Primiparous cows displayed higher serum SOD activities. NA supplementation increased serum SOD activity antepartum in LC animals. Parturition was characterised by an increased need for antioxidants and an increased expression of oxidative stress-related genes that clearly differed with parity and was influenced by energy supply while NA exerted only minor effects on the investigated parameters.

Nicotinic acid and nicotinamide are soluble compounds of the vitamin B group, widely used to regulate the lipid profile in hyperlipidemic individuals. Higher doses of nicotinic acid are associated with adverse effects, especially flushing. A unique tolerable upper intake level (UL) of nicotinic acid has not been defined. This meta-analysis aims to evaluate adverse effects and their incidence after supplementation with different doses of nicotinic acid and nicotinamide, comparing results with current ULs in Europe and the United States. PubMed was searched for articles providing detailed information about nicotinic acid or nicotinamide supplementation and related outcomes. A total of 2670 citations were selected for screening. Two primary outcomes were considered: occurrence of adverse effects following nicotinic acid or nicotinamide supplementation, and dose at which adverse effects occurred. Details on study population, type and duration of treatment, dosage of vitamins, association with lipid-influencing drugs, length of follow-up, and incidence and type of adverse events were extracted. After screening, 47 articles involving 11 741 individuals were included. Meta-analysis was based on estimation of benchmark doses for the probability of adverse effects after supplementation. In individuals with dyslipidemia or cardiovascular disease, nicotinic acid monotherapy seems to be protective against any adverse effects considered, as adverse events occurred at doses above those used with other treatments. In healthy individuals treated with nicotinic acid alone, major adverse effects occurred at doses below 1000 mg/d. Results may indicate a high degree of conservativeness in the UL of nicotinic acid, fixed at 35 mg/d in United States and 10 mg/d in Europe. Reconsideration of the UL of nicotinic acid for nutritional supplements, possibly differentiating between ULs in healthy and unhealthy individuals, may be warranted.

BackgroundHuman labor is associated with an inflammatory process that takes place at the maternal–fetal interface, where leukocytes infiltrate and contribute to the local production of effector molecules such as cytokines, chemokines, MMPs, etc. This process may be altered by a low‐grade chronic inflammation, characteristic of obesity, resulting in adverse pregnancy outcomes. In this cross‐sectional pilot study, we analyzed the relationship between maternal adiposity and inflammation‐related gene expression in leukocytes from six healthy women with term pregnancies without labor.MethodsWe estimated maternal adiposity and examined the relative expression of 211 inflammation‐related genes in maternal peripheral blood leukocytes (MAT), placental intervillous blood leukocytes (PLA), and choriodecidual leukocytes (CHD) by real‐time qPCR. Finally, we analyzed the correlation between maternal adiposity and gene expression.ResultsParticipants’ adiposity ranged from 27.6% to 61.1% (n = 6). The expression of 23 genes significantly differed (p < 0.05) in MAT, PLA, and CHD leukocytes, most of which code for chemokines and proinflammatory cytokines. Importantly, increasing maternal adiposity correlated (r > 0.7) mostly positively with the expression of genes related to activation, migration, infiltration, and proinflammation in MAT (36 genes) and PLA (31 genes). In contrast, in CHD leukocytes maternal adiposity correlated only negatively with seven genes, involved in migration and infiltration.ConclusionOur findings suggest that during term pregnancy, increased maternal adiposity may enhance the priming of peripheral leukocytes, while in choriodecidua it may alter leukocyte recruitment and proinflammatory activity. Maternal adiposity must be considered an important variable in further studies that analyze inflammation‐related gene expression in pregnant women.

Na+, K+‐ATPase (NKA) activity, which establishes the sodium and potassium gradient across the cell membrane and is instrumental in the propagation of the nerve impulses, is altered in a number of neurological and neuropsychiatric disorders, including autism spectrum disorders (ASD). In the present work, we examined a wide range of biochemical and cellular parameters in the attempt to understand the reason(s) for the severe decrease in NKA activity in erythrocytes of ASD children that we reported previously. NKA activity in leukocytes was found to be decreased independently from alteration in plasma membrane fluidity. The different subunits were evaluated for gene expression in leukocytes and for protein expression in erythrocytes: small differences in gene expression between ASD and typically developing children were not apparently paralleled by differences in protein expression. Moreover, no gross difference in erythrocyte plasma membrane oxidative modifications was detectable, although oxidative stress in blood samples from ASD children was confirmed by increased expression of NRF2 mRNA. Interestingly, gene expression of some NKA subunits correlated with clinical features. Excess inhibitory metals or ouabain‐like activities, which might account for NKA activity decrease, were ruled out. Plasma membrane cholesterol, but not phosphatidylcholine and phosphatidlserine, was slighty decreased in erythrocytes from ASD children. Although no compelling results were obtained, our data suggest that alteration in the erytrocyte lipid moiety or subtle oxidative modifications in NKA structure are likely candidates for the observed decrease in NKA activity. These findings are discussed in the light of the relevance of NKA in ASD. Autism Res 2018, 11: 1388–1403. © 2018 International Society for Autism Research, Wiley Periodicals, Inc.Lay SummaryThe activity of the cell membrane enzyme NKA, which is instrumental in the propagation of the nerve impulses, is severely decreased in erythrocytes from ASD children and in other brain disorders, yet no explanation has been provided for this observation. We strived to find a biological/biochemical cause of such alteration, but most queries went unsolved because of the complexity of NKA regulation. As NKA activity is altered in many brain disorders, we stress the relevance of studies aimed at understanding its regulation in ASD.

The intravenous administration of a bolus dose of endotoxin to healthy human subjects triggers acute systemic inflammatory responses that include cytokine production and dynamic changes in gene expression in peripheral blood leukocytes. This study sought to determine the state of clock gene expression in human peripheral blood leukocytes, and leukocyte subpopulations, challenged with in vivo endotoxin at two circadian/diurnal phases of the clock. Clinical and laboratory investigation. University-based research laboratory and clinical research center. Human volunteers. Human subjects were administered a standard dose of endotoxin (2 ng/kg) or saline at either 0900 or 2100 hrs. Blood samples were collected at selected time points pre- and postinfusion. Clock gene expression was determined in human peripheral blood leukocytes, neutrophils, and monocytes by quantitative real-time polymerase chain reaction. The fold change for each gene was determined by use of the 2 method. We show that endotoxin causes profound suppression of circadian clock gene expression, clearly manifested in human peripheral blood leukocytes, neutrophils, and monocytes. Clock, Cry1-2, Per3, CSNK1epsilon, Rora, and Rev-erb gene expression were all reduced by 80% to 90% with the nadir between 3 and 6 hrs postinfusion. Per1 and Per2 reached an expression nadir between 13 and 17 hrs postinfusion. The levels of plasma interleukin-6 and tumor necrosis factor peaked and then returned to baseline within 6 hrs. In contrast, clock gene expression remained suppressed for up to 17 hrs irrespective of the phase of the clock at the time of the endotoxin challenge. Endotoxin did not perturb the melatonin secretory rhythm. Circadian clock gene expression in peripheral blood leukocytes is dramatically altered and possibly uncoupled from the activity of the central clock during periods of acute systemic inflammation. The realignment of the central and peripheral clocks may constitute a previously unappreciated key factor affecting recovery from disease in humans.

Effects of supplementing rumen-protected niacin on fiber composition and metabolism of skeletal muscle in dairy cows during early lactation

LPS-induced TLR4 activation alters cellular bioenergetics and triggers proteolytic cleavage of AMPKα and HIF-1α expression in leukocytes. In human leukocytes, and more specifically neutrophils, AMPKα cleavage yields 55- and 35-kDa protein fragments. In this study, we address the mechanism by which AMPKα is cleaved and its relevance to human health. Our data indicate that AMPKα cleavage is linked to MMP9 expression and that both are required for mammalian target of rapamycin complex-1 and S6K1 activation and HIF-1α expression in LPS-stimulated human and mice leukocytes. Three key observations support this conclusion. First, no changes in AMPKα and TLR4 signaling intermediates (mammalian target of rapamycin complex-1/S6 kinase 1/HIF-1α) were detected in LPS-stimulated MMP9-deficient mice leukocytes. Second, rMMP9 cleaved human AMPKα ex vivo, producing degradation products similar in size to those detected following LPS stimulation. Third, MMP9 inhibitors prevented AMPKα degradation and HIF-1α expression in LPS-activated human leukocytes, whereas AMPK activators blocked MMP9 and HIF-1α expression. Significantly, AMPKα degradation, MMP9, and TLR4 signaling intermediates were all detected in leukocytes from patients with type 2 diabetes mellitus and patients following cardiopulmonary bypass surgery. Plasma from these two patient cohorts induced AMPKα cleavage and TLR4 signaling intermediates in healthy donor leukocytes and either a TLR4 inhibitor or polymyxin prevented these outcomes. Detection of AMPKα degradation, MMP9 expression, and TLR4 signaling intermediates described in this study in leukocytes, the most readily available human cells for clinical investigation, may provide a powerful tool for further exploring the role of TLR4 signaling in human diseases and lead to identification of new, context-specific therapeutic modalities for precision medicine.