Comparison of the immunomodulatory effect of single MSC batches versus pooled MSC products.

Severe corticosteroid-refractory graft-versus-host-disease (GVHD) is a major non-relapse cause of mortality and morbidity after an allogeneic hematopoietic stem cell transplantation (allo-HSCT). One of the most promising treatment options is using advanced therapy medicinal products based on mesenchymal stem cells (MSCs) immunomodulation ability. The protocols of MSC application differ in many parameters including a source of MSC, a dose, a number of doses or way of preparation of the medicinal product. The process is limited by the need for laborious and expensive manufacturing processes fraught with batch-to-batch variability. In our study, we compared the immunomodulatory effects of different MSC batches versus pooled MSC, specifically the influence on lymphocyte proliferation, the metabolic activity, and the expression of activation markers on T cells. Our goal was to determine whether the effect depends on donor-to-donor heterogeneity and if pooling of MSCs could increase their immunomodulatory ability. All tested batches showed an immunomodulatory effect, with no significant differences between the groups. Our study suggests that immunosuppressive potential is comparable in single batches and pooled products, and the use of products got from individual donors is suitable to treat corticosteroid-refractory GVHD.

Mitochondrial respiration of adipocytes differentiating from human mesenchymal stem cells derived from adipose tissue.

Burden of obesity is increasing in the contemporary world. Although multifactorial in origin, appropriate mitochondrial function of adipocytes emerges as a factor essential for healthy adipocyte differentiation and adipose tissue function. Our study aimed to evaluate mitochondrial functions of human adipose-derived mesenchymal stem cells committed to adipogenesis. On days 0, 4, 10, and 21 of adipogenesis, we have characterized adipocyte proliferation and viability, quantified lipid accumulation in maturing cells, performed qualitative and quantitative analysis of mitochondria, determined mitochondrial respiration of cells using high-resolution respirometry, and evaluated mitochondrial membrane potential. In the course of adipogenesis, mitochondrial oxygen consumption progressively increased in states ROUTINE and E (capacity of the electron transfer system). State LEAK remained constant during first days of adipogenesis and then increased probably reflecting uncoupling ability of maturing adipocytes. Citrate synthase activity and volume of mitochondrial networks increased during differentiation, particularly between days 10 and 21. In addition, lipid accumulation remained low until day 10 and then significantly increased. In conclusion, during first days of adipogenesis, increased mitochondrial respiration is needed for transition of differentiating cells from glycolytic to oxidative metabolism and clonal expansion of preadipocytes and then more energy is needed to acquire typical metabolic phenotype of mature adipocyte.

Beneficial effects of mesenchymal stem cells on adult porcine cardiomyocytes in non-contact co-culture.

Mesenchymal stem cells (MSCs) have been reported to improve survival of cardiomyocytes (CMCs) and overall regeneration of cardiac tissue. Despite promising preclinical results, interactions of MSCs and CMCs, both direct and indirect, remain unclear. In this study, porcine bone marrow MSCs and freshly isolated porcine primary adult CMCs were used for non-contact co-culture experiments. Morphology, viability and functional parameters of CMCs were measured over time and compared between CMCs cultured alone and CMCs co-cultured with MSCs. In non-contact co-culture, MSCs improved survival of CMCs. CMCs co-cultured with MSCs maintained CMCs morphology and viability in significantly higher percentage than CMCs cultured alone. In viable CMCs, mitochondrial respiration was preserved in both CMCs cultured alone and in CMCs co-cultured with MSCs. Comparison of cellular contractility and calcium handling, measured in single CMCs, revealed no significant differences between viable CMCs from co-culture and CMCs cultured alone. In conclusion, non-contact co-culture of porcine MSCs and CMCs improved survival of CMCs with a sufficient preservation of functional and mitochondrial parameters.

Repeated exposure to hyperbaric hyperoxia affects mitochondrial functions of the lung fibroblasts.

Hyperbaric oxygen (HBO) therapy, i.e. breathing pure oxygen under increased environmental pressures serves as a treatment for diverse medical conditions. However, elevated oxygen concentration can be detrimental to central nervous system or lungs. Our study aimed to evaluate the effects of repeated exposure to HBO on mitochondrial respiration assessed by high-resolution respirometry (HRR), cell viability estimated by PrestoBlue® reaction, morphology analyzed by routine phase contrast and fluorescent microscopy, and superoxide dismutase (SOD) and citrate synthase (CS) activities using human lung fibroblasts. The cells were exposed to HBO for 2 h per day for 5 consecutive days. One day after the last exposure, HBO cells displayed significantly smaller area and perimeter, compromised viability and elevated SOD activity. No changes were detected in CS activity or quality of mitochondrial network. HRR revealed impaired mitochondrial oxygen consumption manifested by increased leak respiration, decreased activity of complex II and compromised ATP-related oxygen consumption when fatty acids were oxidized. Our findings document that in conditions mimicking chronic intermittent exposure to HBO, lung fibroblasts suffer from compromised mitochondrial respiration linked to complex II and impaired cellular growth in spite of increased antioxidant defense. Underlying mechanism of this HBO-induced mitochondrial dysfunction should be further explored.

Risk of anal incontinence in women with inflammatory bowel diseases after delivery.

AIM: The aim of our prospective study was to evaluate the development of postpartum anal incontinence in patients with inflammatory bowel disease (IBD) compared to healthy women. MATERIAL AND METHODS: Patients with IBD and healthy controls enrolled in the study from January 1st 2013 to November 30th 2016 and filled in the anal incontinence questionnaire in the beginning of pregnancy and after vaginal delivery. The results were statistically processed using suitable tests. RESULTS: A total of 57 women were enrolled, 17 (29.8 %) with ulcerative colitis, 23 (40.4 %) with Crohn's disease, and 17 (29.8 %) healthy controls. Incidence of postpartum anal incontinence is comparable across all groups; there was no statistically significant difference between the IBD and control groups (Kruskal-Wallis test by ranks with Dunn correction, non-significant). Postpartum anal incontinence was strongly correlated with the extent of perineal injury (r = 0.80; p < 0.0001; Pearson's linear correlation). CONCLUSIONS: Women with inflammatory bowel disease in remission do not exhibit higher incidence of postpartum anal incontinence (PPAI) compared to healthy controls; the key correlate of PPAI appears to be the extent of obstetric injury, consistently across all study groups. These results suggest that concerns about postpartum anal incontinence development should not be an indication for Caesarean section in IBD patients (Tab. 6, Fig. 1, Ref. 34).

Polychlorinated biphenyl 153 in lipid medium modulates differentiation of human adipocytes.

Emerging evidence indicates that polychlorinated biphenyls (PCBs) are involved in the development of diabetes mellitus in the obese. The purpose of this study was to determine mechanisms by which PCB 153 (2,2´,4,4´,5,5´-hexachloro-biphenyl) could influence diet-induced obesity and insulin resistance during adipogenesis. Lineage of h-ADMSCs was differentiated either as control (differentiation medium only), or with lipid vehicle modeling high fat nutrition (NuTRIflex) or lipid free vehicle (dimethylsulfoxide) for 28 days with or without PCB 153 daily co-exposure (in three concentrations 0.1, 1, and 10 microM). Gene expression analyses were performed using RT-qPCR at days 4, 10, 21, 24, 28; protein levels Akt and phosphorylated Akt (Phospho-Akt) by Western blot at days 4, and 21. PCB 153 treatment of h-ADMSCs only in lipid vehicle was associated with down regulation of key master genes of adipogenesis: PPARgamma, SREBP-1, PPARGC1B, and PLIN2 during the whole process of differentiation; and with increased Akt and decreased Phospho-Akt protein level at day 21. We have shown that PCB 153, in concentration 0.1 microM, has a potential in lipid rich environment to modulate differentiation of adipocytes. Because European and U.S. adults have been exposed to PCB 153, this particular nutrient-toxicant interaction potentially impacts human obesity and insulin sensitivity.

Multiple roles of mitochondria in aging processes.

Aging is a multifactorial process influenced by genetic factors, nutrition, and lifestyle. According to mitochondrial theory of aging, mitochondrial dysfunction is widely considered a major contributor to age-related processes. Mitochondria are both the main source and targets of detrimental reactions initiated in association with age-dependent deterioration of the cellular functions. Reactions leading to increased reactive oxygen species generation, mtDNA mutations, and oxidation of mitochondrial proteins result in subsequent induction of apoptotic events, impaired oxidative phosphorylation capacity, mitochondrial dynamics, biogenesis and autophagy. This review summarizes the major changes of mitochondria related to aging, with emphasis on mitochondrial DNA mutations, the role of the reactive oxygen species, and structural and functional changes of mitochondria.

Propofol-induced mitochondrial and contractile dysfunction of the rat ventricular myocardium.

Propofol is a short-acting hypnotic agent used in human medicine for sedation and general anesthesia. Its administration can be associated with serious cardiovascular side-effects that include decrease in arterial blood pressure and cardiac output. The aim of the present study was to evaluate propofol effects on mitochondrial respiration, myocardial contractility and electrophysiology in the same samples isolated from the heart ventricles of adult rats. Mitochondrial oxygen consumption was measured in permeabilized samples dissected from free walls of both ventricles using high-resolution respirometry. State LEAK was determined with malate and glutamate. Active respiration was induced by ADP (state PI) and further by succinate, a Complex II substrate (PI+II). Rotenone was injected to measure state PII. Antimycin A, a Complex III inhibitor was used to determine residual oxygen consumption (ROX). N,N,N',N'-tetramethyl-p-phenylenediamine dihydrochloride and ascorbate were injected simultaneously for respirometric assay of cytochrome c oxidase activity (CIV). Isometric contractions and membrane potentials were determined on multicellular preparations isolated from right and left ventricles. Propofol concentrations used ranged from 0.005 to 0.5 mmol/l. All respiratory parameters were significantly higher in the left control ventricles compared to the right ones. Propofol significantly decreased Complex I activity at concentration 0.025 mmol/l and papillary muscle contraction force at 0.1 mmol/l. Propofol did not affect action potential duration at any concentration studied. Our study suggests that mechanisms contributing to the impaired myocardial contraction during propofol anesthesia might include also mitochondrial dysfunction manifested by compromised activity of the respiratory Complex I.

Neonatal capsaicin administration impairs postnatal development of the cardiac chronotropy and inotropy in rats.

The present study evaluated the impact of neonatal administration of capsaicin (neurotoxin from red hot pepper used for sensory denervation) on postnatal development of the heart rate and ventricular contractility. In the rats subjected to capsaicin administration (100 mg/kg) on postnatal days 2 and 3 and their vehicle-treated controls at the ages of 10 to 90 days, function of the sympathetic innervation of the developing heart was characterized by evaluation of chronotropic responses to metipranolol and atropine, norepinephrine concentrations in the heart, and norepinephrine release from the heart atria. Sensory denervation was verified by determination of calcitonin gene-related peptide levels in the heart. Direct cytotoxic effects of capsaicin were assessed on cultured neonatal cardiomyocytes. Capsaicin-treated rats displayed higher resting heart rates, lower atropine effect, but no difference in the effect of metipranolol. Norepinephrine tissue levels and release did not differ from controls. Contraction force of the right ventricular papillary muscle was lower till the age of 60 days. Significantly reduced viability of neonatal cardiomyocytes was demonstrated at capsaicin concentration 100 micromol/l. Our study suggests that neonatal capsaicin treatment leads to impaired maturation of the developing cardiomyocytes. This effect cannot be attributed exclusively to sensory denervation of the rat heart since capsaicin acts also directly on the cardiac cells.

[Sperm mitochondrial function in men with normozoospermia and asthenozoospermia]. / Funkce mitochondrií ve spermii u muzu s normozoospermií a astenozoospermií

OBJECTIVE: One of causes of male infertility is reduced sperm motility. It turns out that the reduced efficiency of the mitochondrial respiratory activity may play a role in the development of this disorder. The aim of our study was to comprehensively determine mitochondrial respiratory activity of sperm with normal and reduced motility. DESIGN: Prospective study. SETTING: Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague; Department of Physiology, Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Centers Prof. Zech, Plzen. METHODS: Ejaculates of 14 men were obtained from IVF Center Prof. Zech, Pilsen. According to the World Health Organization classification, samples were divided into normozoospermatic (n = 7) and asthenozoospermatic(n = 7) groups. Respiratory activity of sperm was measured on two-chamber oxygraph Oroboros. RESULTS: In asthenozoospermatic samples, significantly reduced activity of complex I (p = 0.007) and increased respiration after application of ATP-synthase inhibitor oligomycin (showing increased uncoupled oxidation and phosphorylation, p = 0.046) were found. Inhibition of complex I by rotenone showed that complex I contribution to the total capacity of oxidative phosphorylation of healthy sperm was relatively lower than it is typical for somatic cells. CONCLUSION: In our study, we measured mitochondrial respiratory activity of human sperm, permeabilized by digitonin, by high-resolution oxygraphy, which allows the determination of oxygen consumption from the smallest possible number of germ cells. The study results confirm reduced activity of complex I in asthenozoospermatics and suggest that increased leakage of protons from the mitochondrial matrix, which leads to reduced efficiency of phosphorylating process, could participate in the reduced sperm motility. Better characterization of male germ cells, either completely healthy or with affected motility, will help us to understand better the physiological process of fertilization and also to choose the most viable sperm for infertility treatment by methods of assisted reproduction.

[Effect of overweight on the outcome of assisted reproductive technology in infertile women]. / Vliv nadváhy na úspesnost technik asistované reprodukce u neplodných zen.

OBJECTIVE: Obesity and overweight negatively affect the ability of women to conceive naturally, contributes to the increased incidence of obstetric complications during pregnancy and affect the outcome of assisted reproduction techniques (ART). The aim of our study was to compare the results of treatment of infertilityin a group of infertile women undergoing ART, depending on the values of BMI and changes in levels of selected hormones and markers of oxidative stress in follicular fluid. DESIGN: Retrospective comparative study. SETTING: Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Centers - Prof. Zech, Plzen. METHODS: The studied group consisted of 44 women (mean age of 31.9 years, SD = 4.35) treated for infertility at the Institute of Reproductive Medicine and Endocrinology - IVF Centers Prof. Zech. Women were divided into 2 groups according to BMI (37 women had normal BMI, 7 women were overweight). Prolactin, free T3 and T4 hormone, homocysteine, malondialdehyde, glutathione peroxidase, total antioxidant capacity and total protein were analyzed in the follicular fluid both groups. Only blood free samples were studied after pooling of all FF samples of each patient. RESULTS: We observed significantly lower levels of glutathion peroxidase in the group of overweight women(p = 0.0044). The pregnancy success rate with women with normal BMI and overweight women did not differ significantly from each other (p = 0.4430). CONCLUSION: Our study did not confirm the negative effect of obesity on the results of treatment of infertility, specifical-ly pregnancy rate in a group of infertile women undergoing treatment with assisted reproduction techniques.

[Analysis of point mutations in interleukin-11 gene in the population of infertile patients and fertile control women]. / Vysetrení bodových mutací v genu pro interleukin-11 v populaci neplodných zen a plodných kontrol.

OBJECTIVE: The interleukin-11 (IL-11) is one of the most important signaling factors in the process of decidua-lization and embryo-maternal cross talk during the embryo implantation. We investigated the prevalence of the IL-11 gene point variants in the population of infertile women and fertile control subjects. DESIGN: A cohort study. SETTING: Department of Obstetrics and Gynecology, Faculty of Medicine and University Hospital of Charles University, Pilsen. SUBJECTS AND METHODS: The population to screen consisted of 354 infertile women. The control population was comprised of 195 healthy fertile subjects. For the mutational analysis, the temperature gradient gel electrophoresis (TGGE) and direct sequencing had been used. The groups of fertile controls and infertile patients were compared for statistically significant difference using the Fisher´s 2 by 2 Exact test. RESULTS: Thirteen potentially functional IL-11 gene variants, the G to A transversions at the position 3651 (G3651A) leading to the arginin to histidin exchange on the position 113 (R113H) were detected in the group of infertile women. No mutations were identified in the control group, which means that the frequency of functionally relevant mutations of the IL-11 gene in infertile women is significantly enhanced in comparison with controls (p = 0.01, Fisher´s 2 by 2 Exact test). CONCLUSION: The results prove the presence of the potentially functionally relevant IL-11 gene variants in the population of infertile women. The presence of variants does not have to lead directly to decrease fertility but may contribute to the dysbalance of cytokine milieu which is needed for the decidualization as well as subsequent embryo implantation. Even thought IL-11 gene variants occur infrequently, their impact on molecular events during early phases of pregnancy should be further elucidated.

[The incidence of latent asymptomatic celiac disease in women with decreased fertility]. / Výskyt latentní asymptomatické celiakie u zen se snízenou plodností

OBJECTIVE: To determine whether patients with recurrent spontaneous abortions and IVF suffer from latent celiac disease. DESIGN: Perspective study. SETTINGS: Department of Gynecology and Obstetrics and Faculty Hospital, Charles University, Pilsen. MATERIAL AND METHODS: Sera from 442 infertile patients aged 23-47 years and 86 healthy fertile controls aged 22-38 years were examined by commercial ELISA sets for the presence (absence) of antibodies against gliadin IgG, IgA and tissue transglutaminase in IgG and IgA. RESULTS: We found a statistically significant difference between the control group and patients with decreased fertility. They have a positive values in anti-tissue transglutaminase IgA (p = 0.0096). Frequent, very low positive reaction of antigliadin IgG was also found in the control group of women. CONCLUSION: Patients with unexplained repeated miscarriages and repeated unsuccessful IVF with IgA positive tissue transglutaminase have new complement of other tests for autoimmunity and examination of a total IgA, and gastroenterological examination. Eleven patients with significant positive anti-tissue transglutaminase IgA were successfully conceived only under strict gluten-free diet.

[Comparison of prolactin, free T3 and free T4 levels in the follicular fluid of infertile women and healthy fertile oocyte donors]. / Srovnání hladin prolaktinu, fT3 a fT4 ve folikulární tekutine zen s poruchou plodnosti a zdravých plodných dárkyn oocytu

OBJECTIVE: Follicular fluid (FF) provides an important microenvironment for the development of oocytes. The biochemical composition of the FF plays critical role in the oocyte competence. The aim of our study was to compare the levels of selected hormones in FF of infertile women and healthy fertile oocyte donors. DESIGN: Retrospective comparative study. SETTING: Department of Histology and Embryology, Faculty of Medicine in Pilsen, Charles University in Prague; Department of Gynecology and Obstetrics, Charles University and University Hospital in Pilsen; Institute of Reproductive Medicine and Endocrinology, IVF Centers Prof. Zech, Plzen. METHODS: Levels of prolactin, free T3 and free T4 hormones in the FF of 146 women were analyzed. We have analysed FF of 74 infertile patients (mean age 31 years, SD = 4.65) and 72 healthy fertile oocyte donors (mean age 26 years, SD = 4.44). Only blood free samples were studied after pooling of all FF samples each patient. Levels of hormones were determined using ECLIA method (Electro-Chemi-Luminiscent Immunoassay) on the Cobas e411. RESULTS: RESULTS showed statistically significantly higher levels of prolactin (p=0.0006) and free T4 hormone (p=0.0246) in FF of infertile women in comparison to the group of healthy fertile oocyte donors. CONCLUSION: Our study confirms the presence of prolactin and thyroid hormones in FF and it can be suggested that they play a key role in the regulation of reproductive processes. The study of FF from donors and their detailed comparison with infertile patients with various gynaecological causes of infertility has great value for better understanding of regulatory mechanisms of fertility.

[Comparison of selective oxidative stress parameters in the follicular fluid of infertile women and healthy fertile oocyte donors]. / Srovnání vybraných parametru oxidacního stresu ve folikulární tekutine u zen s poruchou plodnosti a u zdravých fertilních dárkyn oocytu

OBJECTIVE: Follicular fluid (FF) affects oocyte development and disruption of its homeostasis has a crucial effect on egg developmental potential. The aim of this study was to compare the levels of selected oxidative stress markers in the FF of women with impaired fertility and healthy fertile oocytes donors. DESIGN: A retrospective comparative study. SETTING: Faculty of Medicine in Pilsen, Charles University in Prague; Institute of Reproductive Medicine and Endocrinology, IVF Center Prof. Zech, Pilsen. METHODS: Levels of homocysteine (Hcy), malondialdehyde (MDA), glutathione peroxidase (GPx), total antioxidant capacity (AOK) and total protein (CB) were analyzed in the FF. We have analysed FF of 146 women - 74 infertile patients (mean age 31 years, SD = 4.65) and 72 healthy fertile oocyte donors (mean age 26 years, SD = 4.44). Only blood free samples were studied after pooling of all FF samples each patient. RESULTS: The study showed a statistically significantly higher Hcy levels (p < 0.0001) in the FF of healthy fertile women compared with impaired fertility group both - comparing the two groups regardless the age and in groups of the same age range (for the age group between 20 to 29 years isp = 0.0002, for the age group between 30 to 39 years is p < 0.0001). When divided into above age ranges we found statistically significantly higher levels of MDA in the control group aged 20 to 29 years compared to same age infertile patients (p = 0.0374) and statistically significantly higher AOK in infertile women between 30 to 39 years of age compared to same age control group (p = 0.0458). CONCLUSION: The presence or on the contrary the absence of prooxidant parameters in the FF has an important role in the ability of conception and subsequent embryo development.

[Contribution of molecular biology to the diagnosis and therapy of colorectal carcinoma--the present and future]. / Prínos molekulární biologie v diagnostice a lécbe kolorektálního karcinomu--soucasnost a budoucnost.

Colorectal cancer is one of the most frequent malignant disease and despite of the development of modern surgical and oncological treatment, it is still a very severe diagnosis for the patient. The survival of the patient after the radical surgery is mostly affected by the time of detection of the disease and by the selection of the appropriate oncological treatment. The effectivity of the oncological treatment depends mainly on the features of the malignant tissue. During the last decade, the importance of the molecular biology and it's methodology have been growing for both detection of the disease and the selection of the best treatment for the individual patient. Genetic and epigenetic characteristics of the tumours helps to predict the prognosis of the disease and also select the best treatment, which extends the disease-free and overall survival of the patient. The presented review describes the most important molecular-biological characteristics with the prognostic or predictive function, which are used in the clinical practice or are in the later phase of clinical study.

[The lifestyle of Pilsner University female students and its effect on vaginal mycosis]. / Zivotní styl plzenských vysokoskolacek a jeho vliv na mykotická onemocnení.

BACKGROUND: Up to now, it does not exit whole look at the life quality of the Pilsner female University students, especially in connection with gynecological history. It is reality that in last years the number of gynecological problems among young women increases such as the number of diseases with immunological background. METHOD: We prepared questionnaire with 36 questions directed to life style, gynecological history, partner's life, food and hygienic habits, and nicotinism. RESULTS: 28 female students from the total group of 141 suffered from vaginal mycosis--22 (78.6%) use pills, 6 (21.4%) no hormonal contraception. 17 (60.7%) prefer flyaway cloth and 9 (32.1%) tight cloth. 141 students mentioned various number (0-25) of their sexual partners: 1-3 (79 students) in 15 cases (19%) mycoses, more than 4 partners (49 students) in 13 (26.5%) mycoses appeared. The first menstruation appeared in o 13 years (10-17 years). High incidence of vaginal mycosis was proven in students using only tampons than hygienic towels. CONCLUSION: Vaginal mycosis is affected by many endogenous and exogenous factors. The main factors in pilsner university female students are hormonal contraception, promiscuity and hygienic habits.