Iron increases the profibrotic properties of the senescent peritoneal mesothelial cells.

BACKGROUND: Treatment of anemia in peritoneal dialysis patients often requires intravenous iron supplementation. Iron diffuses into the peritoneal cavity and is injurious to the peritoneum. We studied how intermittent exposure to iron changes the properties of the senescent peritoneal mesothelial cells (MC). METHODS: Replicative senescence was induced in MC in control medium (Con) or in control medium with intermittent exposure to iron isomaltoside 15 µg/dL (Con-IIS). After 10 passages properties of MC from both groups were compared to MC not exposed to replicative senescence. RESULTS: In senescent MC population doubling time was elongated, intracellular generation of free radicals and staining for ß-galactosidase was stronger than in MC not exposed to replicative senescence. All these effects were stronger in MC intermittently exposed to IIS. In these cells intracellular iron content was also higher. Also expression of genes p21 and p53 was stronger in MC intermittently treated with IIS. In senescent cells higher release and expression of IL6 and TGFß1 was observed and that effect was stronger in MC treated with iron. Senescent MC had reduced fibrinolytic activity, what may predispose to the peritoneal fibrosis. Synthesis of collagen was higher in senescent cells, more in MC treated with iron. CONCLUSION: MC aging results in change of their genotype and phenotype which lead to their profibrotic effect. Exposure to iron enhances these changes.

Endogenous retrotransposons cause catastrophic deoxyribonucleic acid damage in human trophoblasts.

OBJECTIVE: To determine the mechanistic role of mobile genetic elements in causing widespread DNA damage in primary human trophoblasts. DESIGN: Experimental ex vivo study. SETTING: Hospital-affiliated University. PATIENT(S): Trophoblasts from a patient with unexplained recurrent pregnancy loss and patients with spontaneous and elective abortions (n = 10). INTERVENTION(S): Biochemical and genetic analysis and modification of primary human trophoblasts. MAIN OUTCOME MEASURE(S): To phenotype and systematically evaluate the underlying pathogenic mechanism for elevated DNA damage observed in trophoblasts derived from a patient with unexplained recurrent pregnancy loss, transcervical embryoscopy, G-band karyotyping, RNA sequencing, quantitative polymerase chain reaction, immunoblotting, biochemical and siRNA assays, and whole-genome sequencing were performed. RESULT(S): Transcervical embryoscopy revealed a severely dysmorphic embryo that was euploid on G-band karyotyping. RNA sequencing was notable for markedly elevated LINE-1 expression, confirmed with quantitative polymerase chain reaction, and that resulted in elevated expression of LINE-1-encoded proteins, as shown by immunoblotting. Immunofluorescence, biochemical and genetic approaches demonstrated that overexpression of LINE-1 caused reversible widespread genomic damage and apoptosis. CONCLUSION(S): Derepression of LINE-1 elements in early trophoblasts results in reversible but widespread DNA damage.

Effect of the one-day fasting on cortisol and DHEA daily rhythm regarding sex, chronotype, and age among obese adults.

Introduction: Physiological and biochemical processes in the human body occur in a specific order and show rhythmic variability. Time dependence characterizes the secretion of cortisol and dehydroepiandrosterone (DHEA). One-day fasting implies alternating fasting days and eating days. The study aimed to determine how 24-h fasting affects the daily rhythm of cortisol and DHEA levels in obese people while taking into account gender and chronotype. Methods: Forty-nine obese patients (BMI 32.2-67.1 kg/m2; 25 women and 24 men) underwent a 3-week hospital-controlled calorie restriction diet to reduce body weight. During hospitalization, patients fasted for 1 day, during which only water could be consumed. Samples of whole mixed unstimulated saliva were collected at 2-3-h intervals over a 64-h period and analyzed for cortisol and DHEA by immunoassays. The individual chronotypes were assessed by the morning and evening questionnaire, according to Horne and Östberg. Three components of daily rhythm were evaluated: amplitude, acrophase, and the so-called MESOR. Results: Cortisol rhythm showed differences in amplitude (p = 0.0127) and acrophase (p = 0.0005). The amplitude on the fasting day was 11% higher (p = 0.224) than the day after. The acrophase advanced on the day of fasting, 48 min earlier than the day before (p = 0.0064), and by 39 min to the day after fasting (p = 0.0005). In the rhythm of DHEA, differences were found in the MESOR (p = 0.0381). The MESOR on the fasting day increased. Discussion: Our results obtained during 64 consecutive hours of saliva sampling suggest that one-day fasting may affect three components of cortisol and DHEA daily rhythm. Additionally, no differences were found in the daily rhythm between the morning and evening chronotypes and between females and males. Although aging did not influence daily cortisol rhythm, DHEA amplitude, MESOR, and acrophase changed with age. To the best of our knowledge, this is the first presentation of changes in DHEA rhythm during one-day fasting.

Sulodexide Prevents Hyperglycemia-Induced Endothelial Dysfunction and Oxidative Stress in Porcine Retinal Arterioles.

Diabetes mellitus may cause severe damage to retinal blood vessels. The central aim of this study was to test the hypothesis that sulodexide, a mixture of glycosaminoglycans, has a protective effect against hyperglycemia-induced endothelial dysfunction in the retina. Functional studies were performed in isolated porcine retinal arterioles. Vessels were cannulated and incubated with highly concentrated glucose solution (HG, 25 mM D-glucose) +/- sulodexide (50/5/0.5 µg/mL) or normally concentrated glucose solution (NG, 5.5 mM D-glucose) +/- sulodexide for two hours. Endothelium-dependent and endothelium-independent vasodilatation were measured by videomicroscopy. Reactive oxygen species (ROS) were quantified by dihydroethidium (DHE) fluorescence. Using high-pressure liquid chromatography (HPLC), the intrinsic antioxidant properties of sulodexide were investigated. Quantitative PCR was used to determine mRNA expression of regulatory, inflammatory, and redox genes in retinal arterioles, some of which were subsequently quantified at the protein level by immunofluorescence microscopy. Incubation of retinal arterioles with HG caused significant impairment of endothelium-dependent vasodilation, whereas endothelium-independent responses were not affected. In the HG group, ROS formation was markedly increased in the vascular wall. Strikingly, sulodexide had a protective effect against hyperglycemia-induced ROS formation in the vascular wall and had a concentration-dependent protective effect against endothelial dysfunction. Although sulodexide itself had only negligible antioxidant properties, it prevented hyperglycemia-induced overexpression of the pro-oxidant redox enzymes, NOX4 and NOX5. The data of the present study provide evidence that sulodexide has a protective effect against hyperglycemia-induced oxidative stress and endothelial dysfunction in porcine retinal arterioles, possibly by modulation of redox enzyme expression.

Protective role of N-acetylcysteine and Sulodexide on endothelial cells exposed on patients' serum after SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus-2 causes hyperinflammation and activation of coagulation cascade and, as a result, aggravates endothelial cell dysfunction. N-acetylcysteine and Sulodexide have been found to mitigate endothelial damage. The influence on coronary artery endothelial cells of serum collected after 4 ± 1 months from coronavirus infection was studied. The concentrations of serum samples of interleukin 6, von Willebrand Factor, tissue Plasminogen Activator, and Plasminogen Activator Inhibitor-1 were studied. The cultures with serum of patients after coronavirus infection were incubated with N-acetylcysteine and Sulodexide to estimate their potential protective role. The blood inflammatory parameters were increased in the group of cultures incubated with serum from patients after coronavirus infection. Supplementation of the serum from patients after coronavirus infection with N-acetylcysteine or Sulodexide reduced the synthesis of interleukin 6 and von Willebrand Factor. No changes in the synthesis of tissue Plasminogen Activator were observed. N-acetylcysteine reduced the synthesis of Plasminogen Activator Inhibitor-1. N-acetylcysteine and Sulodexide increased the tPA/PAI-1 ratio. N-acetylcysteine may have a role in reducing the myocardial injury occurring in the post-COVID-19 syndrome. Sulodexide can also play a protective role in post-COVID-19 patients.

Effect of Flaxseed (Linum usitatissimum L.) Supplementation on Vascular Endothelial Cell Morphology and Function in Patients with Dyslipidaemia-A Preliminary Observation.

CONTEXT: Flaxseed has a characteristic fatty acids composition and unique phytonutrient profile that may have health-promoting properties. OBJECTIVE: This study aimed to determine the effects of 10 weeks of supplementation with the flaxseed (28 g/day) on endothelial cells (EC) function, serum lipids and proinflammatory mediators in patients with mild and severe dyslipidaemia. MATERIALS AND METHODS: Eleven lean patients with severe dyslipidaemia treated with apheresis (group 1; 10 weeks treated in four phases: (i) ordinary diet, (ii) ordinary diet + flaxseed, (iii) ordinary diet (wash out), (iv) ordinary diet + placebo) and eleven obese patients with mild dyslipidaemia-not treated with apheresis (group 2; 10 weeks treated in two phases: (i) ordinary diet, (ii) low fat diet + flaxseed). Flaxseed was given blindly. Serum was collected at the end of each phase of the study. ECs were exposed in vitro to the medium supplemented with pooled serum taken from patients from both groups to detect their morphological changes using light and electron microscopy. ECs proliferation was also measured at the end of each study phase. RESULTS: Serum vascular endothelial growth factor was decreased after flaxseed supplementation but only in group 1. ECs proliferation was increased after flaxseed supplementation only in obese patients. ECs exposed to medium supplemented with obese patients' serum revealed the following cellular abnormalities: accumulation of lipid droplets, changes of rough endoplasmic reticulum and mitochondria, and flaxseed did not reverse observed changes. At the same time, flaxseed supplementation decreases total cholesterol in both tested groups, low-density lipoprotein cholesterol in group 1 and triglycerides in group 2. CONCLUSIONS: Our findings support the potential role of flaxseed in treating dyslipidaemia but indicate only a slight impact on endothelial cell function.

Secretory activity of the coronary artery endothelial cells in conditions of the peritoneal dialysis.

INTRODUCTION: Endothelial dysfunction is frequent in patients treated with peritoneal dialysis and may lead to cardiac complications. We evaluated the effect of effluent dialysates and serum on the function of coronary artery endothelial cells (CAEC). METHODS: Human CAEC in in vitro culture were exposed to serum and dialysates from 24 patients treated with continuous ambulatory peritoneal dialysis (CAPD) and secretion of interleukin-6 (IL6), von Willebrand factor (vWF), tissue plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured. Modulation of the secretory activity of CAEC by Sulodexide, mixture of glycosaminoglycans: heparin sulfate and dermatan sulfate, was studied. RESULTS: Serum from CAPD patients stimulated synthesis of IL6 (+93%), vWF (+18%), and PAI-1 (+20%) and did not change t-PA secretion in CAEC. Dialysates stimulated secretion of IL6 (+89%), vWF (+29%), and PAI-1 (+31%) and did not change t-PA synthesis. Dialysates collected in 12 patients after 6 months more strongly stimulated synthesis of IL6 (+37%) and PAI-1 (+7%). Sulodexide suppressed the secretory activity of CAEC stimulated by the studied sera: IL6 (-38%), vWF (-19%), t-PA (-13%), and PAI-1 (-12%). CONCLUSIONS: Serum and the dialysate from CAPD patients induce inflammatory and prothrombotic reaction in coronary arterial endothelial cells. The general pattern of the observed effects for serum and dialysates was similar but the intensity of the effects was not identical. Sulodexide reduced these effects.

Adverse effects of iron toward the peritoneal mesothelial cells are reversible.

INTRODUCTION: Evaluation of the effects of the drugs used for iron supplementation on the peritoneal mesothelial cells. METHODS: Acute effects during18-h incubation of iron sucrose, ferric carboxymaltose, and iron isomaltoside in concentrations 1, 5, and 20 ug/dl on properties of the human peritoneal mesothelial cells in in vitro culture were evaluated, and their reversibility after the following culture for 14 days in the iron-free medium was studied. RESULTS: All studied compounds reduced viability of mesothelial cells and proliferation rate and increased intracellular oxidative stress and iron content in the cytosol. Secretion of monocyte chemoattractant protein-1 was increased and tissue plasminogen activator (t-PA) decreased. After 14 days of culture in iron-free medium, reversibility of all these effects was observed. CONCLUSION: Iron compounds impair the functional properties of mesothelial cells in a dose-dependent manner, but these effects are reversible after the following culture of the cells in an iron-free medium.

No Significant Effect of the Individual Chronotype on the Result of Moderate Calorie Restriction for Obesity-A Pilot Study.

BACKGROUND: Chronotype is the pattern of the circadian rhythm that allows an individual to optimize times of sleep and activity. It has been observed that chronotypes may associate with some conditions and diseases, including obesity. It is not known, however, whether chronotypes determine the effectiveness of weight loss regimens. Therefore, in the present study, we compared the outcomes of a 3-week moderate calorie restriction undertaken by individuals with obesity under the same controlled hospital conditions. METHODS: A total of 131 participants with obesity (median BMI 40.0) were studied. The subjects underwent the same dietary intervention over 3 weeks, with a 30% reduction in daily caloric intake. The individual chronotypes were assessed by the morning and evening questionnaire (MEQ) according to Horne and Östberg. Anthropometric and biochemical parameters were assessed by routine methods. RESULTS: Of all patients examined, 75% had the morning (lark) chronotype and 25% had the evening (owl) chronotype. These patient sub-groups did not differ in terms of demographic, anthropometric and biochemical characteristics at baseline. After 3 weeks of calorie restriction, both groups experienced a similar loss of weight and BMI (Body Mass Index) (3.4 ± 0.38% for larks vs. 4.1 ± 0.47% for owls, p = 0.45), with owls exhibiting a marginally greater loss of body fat (3.1 ± 0.79%) compared with larks (2.6 ± 0.64%), p = 0.02. On the other hand, the larks had a more discernable, but not statistically significant from owls, decrease in glycated haemoglobin and CRP (C Reactive Protein). CONCLUSIONS: The chronotype of individuals with obesity does not have a significant effect on the magnitude of the body weight loss, but there is a tendency observed towards the reduction in body fat content in owls through changing their meal and sleep timing to earlier hours, in response to moderate calorie restriction applied under the same controlled conditions.

Moderate Caloric Restriction Partially Improved Oxidative Stress Markers in Obese Humans.

Oxidative stress and inflammation are implicated in obesity. Therefore, we investigated whether moderate and short-term calorie restriction (CR) reflects a real-life situation, mediates weight loss, and improves oxidative stress markers. We analyzed oxidative stress markers in patients with obesity undergoing moderate CR. Serum oxidative stress markers (myeloperoxidase (MPO), superoxide dismutase (SOD), catalase, total antioxidant status (TAS), and reactive oxygen species (ROS) (generation by endothelial cells in vitro)) were measured in 53 subjects (mean BMI 37.8 ± 5.9 kg/m2) who underwent 8 weeks of CR, which included a reduction of 300-500 kcal/day. MPO was the most CR-sensitive parameter. The mean level of serum MPO in patients with obesity was 20% higher than that in post CR intervention (p < 0.001). SOD increased by 12% after CR (p < 0.05), which was largely due to the improvement in glucose tolerance and the reduction in insulin resistance after CR. Other tested parameters were not modified during the treatment. CR resulted in an expected decrease in body weight (by 5.9 ± 4.6 kg, p < 0.0001) and other anthropometric parameters. Additionally, it was accompanied by a significant change in hsCRP, hsTNF alpha, hsIL-6, leptin (all p < 0.0001), and HOMA-IR (p < 0.05). Cardiovascular and metabolic parameters were also partially improved. Short-term, moderate CR partially improves antioxidant capacity but is enough to substantially change anthropometric parameters in obese patients. Our observations indicate that mimicking real-life situations and low-cost dietary intervention can be successfully implemented in obesity treatment with a simultaneous moderate effect on antioxidant status.

Uremic serum induces prothrombotic changes in venous endothelial cells and inflammatory changes in aortic endothelial cells.

BACKGROUND: Uremia induces various pathologic changes in the endothelium. However, there is limited information about the differences of these effects in endothelial cells originating from different parts of the vascular tree. METHODS: The effect of uremic serum obtained from patients with end stage renal failure on the gene expression and secretory activity of venous endothelial cells (VEC) and aortic endothelial cells (AEC) was studied in in vitro culture. RESULTS: In VEC, the expression of genes regulating the synthesis of von Willebrand factor (vWF) was increased by 254% (p<.005), vascular endothelial growth factor (VEGF) synthesis by 150% (p<.001), tissue plasminogen activator (t-PA) synthesis by 62% (p<.005), platelet endothelial cell adhesion molecule by 89% (p<.005), and the expression of gene regulating interleukin-6 (IL-6) synthesis was reduced. In AEC, the expression of the gene regulating synthesis of IL-6 was increased by 174% (p<.001), and the expression of the other genes was reduced. The secretion of IL-6 was reduced in VEC by 38% (p<.01) and increased in AEC by 55% (p<.005). In VEC, increased synthesis of VEGF 64% (p<.001) vWF (+34%, p<.01), and t-PA (+53%, p<.002) was observed, and in AEC it was reduced. CONCLUSIONS: VEC and AEC respond in different ways after exposure to uremic serum. VEC acquires the prothrombotic phenotype, whereas in AEC the inflammatory phenotype appears.

Anticancer Activity of Novel NF-kappa B Inhibitor DHMEQ by Intraperitoneal Administration.

There have been great advances in the therapy of cancer and leukemia. However, there are still many neoplastic diseases that are difficult to treat. For example, it is often difficult to find effective therapies for aggressive cancer and leukemia. An NF-κB inhibitor named dehydroxymethylepoxyquinomicin (DHMEQ) was discovered in 2000. This compound was designed based on the structure of epoxyquinomicin isolated from a microorganism. It was shown to be a specific inhibitor that directly binds to and inactivates NF-κB components. Until now, DHMEQ has been used by many scientists in the world to suppress animal models of cancer and inflammation. Especially, it was shown to suppress difficult cancer models, such as hormone-insensitive breast cancer and prostate cancer, cholangiocarcinoma, and multiple myeloma. No toxicity has been reported so far. DHMEQ was administered via the intraperitoneal (IP) route in most of the animal experiments because of its simplicity. In the course of developmental studies, it was found that IP administration never increased the blood concentration of DHMEQ because of the instability of DHMEQ in the blood. It is suggested that inflammatory cells in the peritoneal cavity would be important for cancer progression, and that IP administration, itself, is important for the effectiveness and safety of DHMEQ. In the present review, we describe mechanism of action, its in vivo anticancer activity, and future clinical use of DHMEQ IP therapy.

Flaxseed (Linum Usitatissimum L.) Supplementation in Patients Undergoing Lipoprotein Apheresis for Severe Hyperlipidemia-A Pilot Study.

Being rich in polyunsaturated fatty acids, flaxseed (Linum usitatissimum L.) is thought to be able to decrease lipid levels and dampen inflammation. In this pilot study, we aimed to determine whether flaxseed supplementation could improve the profiles of lipids and inflammatory mediators in patients with severe hyperlipidemia resistant to conventional lipid-lowering pharmacotherapy and requiring lipoprotein apheresis. To this end, six patients received, blindly-in addition to their normal lipoprotein apheresis regimen-a 10-week dietary supplementation with flaxseed (28 g/d) administered in biscuits. This was followed by a 10-week washed out-period and a 10-week supplementation phase with whole wheat placebo. Blood samples were collected at the end of each phase, before the lipoprotein apheresis session. The primary endpoint was the lipid profile and the secondary endpoints were the concentrations of inflammatory mediators and tolerability. Flaxseed supplementation was well-tolerated and resulted in a consistent and significant decrease in total cholesterol and low-density lipoprotein (LDL) levels. The median (and range) percentage decrease was 11.5% (0-18.8) and 7.3% (4.4-26.6), for cholesterol (p = 0.015) and LDL-C (p = 0.003), respectively. On the other hand, there was no significant effect of flaxseed on lipoprotein(a) (Lp(a)), C-reactive protein (CRP), and interleukin 6 (IL-6) concentrations. These observations indicate that flaxseed can produce a cholesterol- and LDL-lowering effect in patients treated with lipoprotein apheresis. Thus, flaxseed supplementation may help to control cholesterol in this patient population. The flaxseed supplementation protocol applied may be of use for further adequately-powered studies to validate and extend our findings.

Quality of design and reporting of animal research in peritoneal dialysis: A scoping review.

The concerns about reproducibility and validity of animal studies are partly related to poor experimental design and reporting. Here, we undertook a scoping review of the literature to determine the extent and quality of reporting of animal studies on peritoneal dialysis (PD). Online databases were searched to identify 567 relevant original articles published between 1979 and 2018. These were analyzed with respect to bibliographic parameters and general aspects of animal experimentation. A subgroup of 120 studies was analyzed in detail in terms of the impact on the reporting quality of the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines for animal studies. The number of animal studies on PD increased continuously over the years with a thematic shift toward long-term preservation of the peritoneum as a dialyzing organ. There were significant deficiencies in research design with the lack of sample size estimation, randomization, and blinding being the commonest shortcomings. The description of animal numbers, housing conditions, use of medication, and statistical analysis was incomplete. The introduction in 2010 of the ARRIVE guidelines produced very little improvement in the completeness of reporting regardless of journal impact factor. The animal studies on PD suffer from deficits in experimental protocols and transparent reporting. These drawbacks need to be corrected to ensure high-quality and much-needed animal research in PD.

N-Acetylcysteine and Sulodexide Reduce the Prothrombotic Effect of Uremic Serum on the Venous Endothelial Cells.

BACKGROUND/AIMS: Thromboembolic episodes are a frequent problem in end stage renal failure patients. The pathomechanism of the disorder is complex, including bioincompatibility of renal replacement therapy, endothelial dysfunction, increased blood level of procoagulant factors and uremic toxins. We studied changes in the functional properties of venous endothelial cells (VEC) in the presence of uremic serum and evaluated their possible modulation by N-acetylcysteine (NAC) or sulodexide (SUL). METHODS: Serum samples from 12 uremic patients treated with hemodialysis were studied ex vivo on in vitro cultured VEC. In separate experiments, NAC 1 mmol/L or SUL 0.5 LRU/mL were added to uremic serum samples. Both changes in the gene expression and secretory activity of VEC were studied. RESULTS: Uremic serum increased the expression of the following genes: IL6 +97%, p < 0.002; VEGF +28%, p < 0.002; vWF +47%, p < 0.002; PECAM +76%, p < 0.002; ICAM-1 +275%, p < 0.002; t-PA +96%, p < 0.002. Changes in gene expression were reflected by the increased secretory activity of VEC treated with the uremic serum. Exposure of VEC to uremic serum supplemented with NAC or SUL resulted in weaker stimulation of the studied genes' expression. Also, secretion of the studied solutes, with the exception of ICAM-1, was reduced in the presence of NAC: IL6 -34%, p < 0.01; VEGF -40%, p < 0.005; vWF -25%, p < 0.001; t-PA -47%, p < 0.01, and MMP9 -37%, p < 0.001. SUL reduced the uremic serum-induced secretion of all solutes: IL6 -24%, p < 0.05; ICAM-1 -43%, p < 0.01; VEGF -38%, p < 0.01; vWF -23%, p < 0.01; t-PA -49%, p < 0.01, and MMP9 -25%, p < 0.05. CONCLUSIONS: Uremic serum induces prothrombotic changes in VEC, which may cause a predisposition to thrombotic disorders in patients with renal failure. NAC and SUL reduce the effects of the uremic serum in VEC, which suggests their potential therapeutic application in uremic patients.

Amaranth (Amaranthus cruentus L.) and canola (Brassica napus L.) oil impact on the oxidative metabolism of neutrophils in the obese patients.

CONTEXT: Amaranth and canola oils have been used traditionally. Amaranth has been identified as being of interest because of its outstanding nutritive value. Amaranth oil is a rich source of highly unsaturated fats and so could be a valuable dietary alternative for individuals affected with obesity. Reactive oxygen species (ROS) are postulated to be involved in systemic inflammation and oxidative stress. Activated polymorphonuclear neutrophils (PMNs) generate high amounts of reactive oxygen species. OBJECTIVE: Our study investigates the impact of amaranth and canola oils supplementation on oxidative metabolism in patients with obesity. We hypothesized that, due to its lipid-lowering and antioxidant properties, amaranth and canola oil would protect against oxidative stress. MATERIALS AND METHODS: We tested 19 obese patients [body mass index (BMI) = 41.1 ± 7.8 kg/m2, (mean ± SD)]. The protocol consisted of two stages: a run-in phase of 2 weeks and an experimental stage - canola or amaranth oil supplementation (20 mL/d) with calorie restriction diet for 3 weeks. The neutrophil oxidative burst was expressed by fluorescence intensity (IF). RESULTS: The oxidative burst had increased significantly at the end of treatment in both groups IF: (21.4 ± 11.15 vs. 35.9 ± 20.3; mean ± SD) p < 0.05. The levels of IF were significantly higher in neutrophils of patients who received canola oil (41.05 ± 25.3) compared to those who received amaranth oil (28.4 ± 11.8) p < 0.05. CONCLUSIONS: Canola oil exerts possible effects on oxidative burst activity in neutrophils in vivo conditions.

Seasonal differences in rhythmicity of salivary cortisol in healthy adults.

The existence of seasonal changes in secretion of stress hormones and inflammatory mediators by humans is not certain. Here, we aimed to determine whether concentrations of cortisol and IL-6 displayed seasonal rhythmicity. The study was performed in Poznan, Poland (52°N, 16°E) in 7 healthy female volunteers (age 22.6 ± 0.8 yr). Samples of whole mixed unstimulated saliva were collected in winter (February) and summer (June) at 2-h intervals over a 24-h period and analyzed for cortisol and IL-6 by immunoassays. During each season, the subjects answered questionnaires related to their sleeping habits, food intake, physical activity, and perceived seasonality. It turned out that salivary concentrations of cortisol followed a daily rhythm both in winter and summer, as determined by a cosine analysis. However, compared with the winter season, a midline-estimating statistic of rhythm in the summer was significantly higher. Moreover, the rhythm acrophase occurred ~4 h later in the summer than in the winter, whereas the amplitudes did not differ. These fluctuations did not correspond to sleeping habits, food and fluid intake, physical exercise, and the self-assessed chronotype. However, the individuals with higher scores on the seasonal affective disorder scale showed a tendency toward lower relative cortisol amplitude in the summer. In contrast to cortisol, salivary IL-6 concentration did not display daily rhythmicity, and its concentrations did not differ significantly between the seasons. In conclusion, in the summer, cortisol level in saliva is elevated, and its circadian pattern of secretion is shifted. The causes for these alterations do not seem to be related to lifestyle and thus remain to be established.

'Heart development and morphogenesis' is a novel pathway for human ovarian granulosa cell differentiation during long­term in vitro cultivation­a microarray approach.

Granulosa cells (GCs) have many functions in the endocrine system. Most notably, they produce progesterone following ovulation. However, it has recently been proven that GCs can change their properties when subjected to long­term culture. In the present study, GCs were collected from hyper­stimulated ovarian follicles during in vitro fertilization procedures. They were grown in vitro, in a long­term manner. RNA was collected following 1, 7, 15 and 30 days of culture. Expression microarrays were used for analysis, which allowed to identify groups of genes characteristic for particular cellular processes. In addition, reverse transcription­quantitative polymerase chain reaction (RT­qPCR) was performed to validate the obtained results. Two ontological groups characteristic for processes associated with the development and morphogenesis of the heart were identified during the analyses: 'Heart development' and 'heart morphogenesis'. The results of the microarrays revealed that the highest change in expression was demonstrated by the lysyl Oxidase, oxytocin receptor, nexilin F­actin binding protein, and cysteine­rich protein 3 genes. The lowest change was exhibited by odd­skipped related transcription factor 1, plakophilin 2, transcription growth factor­ß receptor 1, and kinesin family member 3A. The direction of changes was confirmed by RT­qPCR results. In the present study, it was suggested that GCs may have the potential to differentiate towards other cell types under long­term in vitro culture conditions. Thus, genes belonging to the presented ontological groups can be considered as novel markers of proliferation and differentiation of GCs towards the heart muscle cells.

Genes responsible for proliferation, differentiation, and junction adhesion are significantly up-regulated in human ovarian granulosa cells during a long-term primary in vitro culture.

The human ovarian granulosa cells (GCs) surround the oocyte and form the proper architecture of the ovarian follicle. The ability of GCs to proliferate and differentiate in the conditions of in vitro culture has been proven. However, there is still a large field for extensive investigation of molecular basics, as well as marker genes, responsible for these processes. This study aimed to find the new marker genes, encoding proteins that regulate human GCs in vitro capability for proliferation and differentiation during long-term primary culture. The human follicular GCs were collected from hyper-stimulated ovarian follicles during IVF procedures and transferred to a long-term in vitro culture. The culture lasted for 30 days, with RNA samples isolated at days 1, 7, 15, 30. Transcriptomic analysis was then performed with the use of Affymetrix microarray. Obtained results were then subjected to bioinformatical evaluation and sorting. After subjecting the datasets to KEGG analysis, three differentially expressed ontology groups "cell differentiation" (GO:0030154), "cell proliferation" (GO:0008283) and "cell-cell junction organization" (GO:0045216) were chosen for further investigation. All three of those ontology groups are involved in human GCs' in vitro lifespan, proliferation potential, and survival capability. Changes in expression of genes of interest belonging to the chosen GOs were validated with the use of RT-qPCR. In this manuscript, we suggest that VCL, PARVA, FZD2, NCS1, and COL5A1 may be recognized as new markers of GC in vitro differentiation, while KAT2B may be a new marker of their proliferation. Additionally, SKI, GLI2, FERMT2, and CDH2 could also be involved in GC in vitro proliferation and differentiation processes. We demonstrated that, in long-term in vitro culture, GCs exhibit markers that suggest their ability to differentiate into different cells types. Therefore, the higher expression profile of these genes may also be associated with the induction of cellular differentiation processes that take place beyond the long-term primary in vitro culture.