From fish to cells: Establishment of continuous cell lines from embryos of annual killifish Nothobranchius furzeri and N. kadleci.

There is a growing need of alternative experimental models that avoid or minimize the use of animals due to ethical, economical, and scientific reasons. Surprisingly, the stable embryonic cell lines representing Nothobranchius spp., emerging vertebrate models in aging research, regenerative medicine, ecotoxicology, or genomics, have been not derived so far. This paper reports establishment and deep characterization of ten continuous cell lines from annual killifish embryos of N. furzeri and N. kadleci. The established cell lines exhibited mostly fibroblast- and epithelial-like morphology and steady growth rates with cell doubling time ranging from 27 to 40 h. All cell lines retained very similar characteristics even after continuous subcultivation (more than 100 passages) and extended storage in liquid nitrogen (∼3 years). The cytogenetic analysis of the cell lines revealed a diploid chromosome number mostly equal to 38 elements (i.e., the native chromosome count for both killifish species), with minor but diverse line/passage-specific karyotype changes compared to the patterns observed in non-cultured N. furzeri and N. kadleci somatic cells. Based on transcriptional analysis of marker genes, the cell lines displayed features of an undifferentiated state without signs of senescence even in advanced passages. We confirmed that the cell lines are transfectable and can form viable 3-D spheroids. The applicability of the cell lines for (eco)toxicological surveys was confirmed by assessing the effect of cytotoxic and growth inhibitory agents. Properties of established Nothobranchius embryonic cell lines open new possibilities for the application of this model in various fields of life sciences including molecular mechanisms of aging, karyotype (in)stability or differences in lifespan.

Procalcitonin kinetics following abdominal aortic surgery and its value for postoperative intestinal ischaemia detection.

OBJECTIVE: The purpose of our study was to describe perioperative kinetics of procalcitonin (PCT) in patients undergoing aortic surgery, to compare the kinetics in the open abdominal aortic aneurysm (AAA) repair and aortobifemoral bypass for aortoiliac occlusive disease (AIOD), and to evaluate the ability of PCT to detect intestinal ischaemia. METHODS: A prospective non-randomized observational cohort study in 80 patients (62 men and 18 women) undergoing elective aortic surgery was performed. Serum PCT was measured at baseline and defined intraoperative and postoperative timepoints up to postoperative day 7. MRI contrast-enhanced imaging was used to detect intestinal ischaemia. RESULTS: The comparison of the AAA and AIOD cohort did not show any significant difference in PCT levels. Patients with intestinal ischaemia had higher serum PCT at multiple timepoints postoperatively. The most accurate timepoints for early diagnosis were postoperative day 3, followed by 24 h after declamping of the vascular reconstruction, and postoperative day 7. The sensitivity and negative predictive values were 100% in all mentioned timepoints. However, event at the best timepoint the specificity was 89% and the positive predictive value 43%. CONCLUSIONS: Procalcitonin levels in the postoperative period at proper timepoints might help to detect postoperative intestinal ischaemia. The limitation of this marker is its low specificity for intestinal ischaemia and low positive predictive value. The highest value of this marker is that it can rule out this complication because normal PCT levels mean that intestinal ischaemia is very unlikely.

l-lactate kinetics after abdominal aortic surgery and intestinal ischemia - An observational cohort study.

BACKGROUND: Postoperative intestinal ischemia is a severe complication in abdominal aortic surgery. Early diagnosis is needed for adequate and timely treatment. We studied the postoperative kinetics of l-lactate in vascular patients to assess its value as a marker for early postoperative intestinal ischemia detection. MATERIAL AND METHODS: We performed a prospective non-randomized single-center observational cohort study in eighty elective patients, fifty operated on for abdominal aortic aneurysm (AAA) and thirty for aortoiliac occlusive disease (AIOD). Serum l-lactate was measured preoperatively, intraoperatively, and postoperatively at defined timepoints up to postoperative day 7. Intestinal ischemia was detected using MRI enterocolography. We have used univariate logistic regression and receiver operating characteristics curves for the evaluation of marker accuracy. RESULTS: We recorded 6 cases of postoperative intestinal ischemia (7.5%), five non-transmural and one transmural. Two patients died because of this complication (mortality 33%). The comparison of AAA and AIOD cohorts showed a significant difference in l-lactate levels at one intraoperative timepoint, which was attributable to procedure differences. The only preoperative factor associated with higher l-lactate levels at some timepoints was chronic kidney disease. Patients suffering postoperative intestinal ischemia had elevated serum l-lactate levels at multiple timepoints. The most accurate timepoint for diagnosis was 24 h after the declamping of the vascular reconstruction (DC24H), the second was 10 min after declamping. Sensitivity, specificity, positive and negative predictive values at timepoint DC24H were 100%, 82%, 32%, and 100%, respectively. CONCLUSION: Serum l-lactate levels might help in the early detection of postoperative intestinal ischemia after aortic surgery if proper timepoints are used. Cutoff values need to be established in large-scale prospective studies.

Pro-Inflammatory and Neurotrophic Factor Responses of Cells Derived from Degenerative Human Intervertebral Discs to the Opportunistic Pathogen Cutibacterium acnes.

Previously, we proposed the hypothesis that similarities in the inflammatory response observed in acne vulgaris and degenerative disc disease (DDD), especially the central role of interleukin (IL)-1ß, may be further evidence of the role of the anaerobic bacterium Cutibacterium (previously Propionibacterium) acnes in the underlying aetiology of disc degeneration. To investigate this, we examined the upregulation of IL-1ß, and other known IL-1ß-induced inflammatory markers and neurotrophic factors, from nucleus-pulposus-derived disc cells infected in vitro with C. acnes for up to 48 h. Upon infection, significant upregulation of IL-1ß, alongside IL-6, IL-8, chemokine (C-C motif) ligand 3 (CCL3), chemokine (C-C motif) ligand 4 (CCL4), nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF), was observed with cells isolated from the degenerative discs of eight patients versus non-infected controls. Expression levels did, however, depend on gene target, multiplicity and period of infection and, notably, donor response. Pre-treatment of cells with clindamycin prior to infection significantly reduced the production of pro-inflammatory mediators. This study confirms that C. acnes can stimulate the expression of IL-1ß and other host molecules previously associated with pathological changes in disc tissue, including neo-innervation. While still controversial, the role of C. acnes in DDD remains biologically credible, and its ability to cause disease likely reflects a combination of factors, particularly individualised response to infection.

Changes in blood morphology and chosen biochemical parameters in ultra-marathon runners during a 100-km run in relation to the age and speed of runners.

OBJECTIVES: The objective of the study was to reveal morphology, electrolyte and chosen biochemical parameters in terms of health risk in runners in reference to their age and running speed in the case of running a distance of 100 km, which occur after 12 h or 24 h of recovery. MATERIAL AND METHODS: Fourteen experienced, male, amateur, ultra-marathon runners, divided into two age and two speed groups took part in the 100-km run. Blood samples for analyses indexes were collected from the ulnar vein just before the run, after 25 km, 50 km, 75 km and 100 km, as well as 12 h and 24 h after termination of the run. RESULTS: The sustained ultramarathon run along with the distance covered (p < 0.05) caused an increase in myoglobin (max 90-fold), bilirubin (max 2.8-fold) and total antioxidant status (max 1.15-fold), which also continued during the recovery. Significant changes in the number of white blood cells were observed with each sequential course and could be associated with muscle damage. The electrolyte showed changes towards slight hyperkalemia, but no changes in natrium and calcium concentrations. There were no significant differences between the age and speed groups for all the parameters after completing the 100-km run as well as after 12 h and 24 h of recovery. CONCLUSIONS: Considering changes in blood morphology and chosen biochemical parameters in ultra-marathon runners during a 100-km run it can be stated that such an exhausting effort may be dangerous for human health due to metabolic changes and large damage to the organs. Negative metabolic changes are independent of age of an ultramarathon runner and occur both in younger (32±5.33 years) and older participants (50.56±9.7 years). It can be concluded that organ damage and negative metabolic changes during a 100-km run occur similarly in participants less experienced as well as in well trained runners. Int J Occup Med Environ Health 2016;29(5):801-814.

Damage to Liver and Skeletal Muscles in Marathon Runners During a 100 km Run With Regard to Age and Running Speed.

The purpose of this study was to determine: (1) whether damage to liver and skeletal muscles occurs during a 100 km run; (2) whether the metabolic response to extreme exertion is related to the age or running speed of the participant; (3) whether it is possible to determine the optimal running speed and distance for long-distance runners' health by examining biochemical parameters in venous blood. Fourteen experienced male amateur ultra-marathon runners, divided into two age groups, took part in a 100 km run. Blood samples for liver and skeletal muscle damage indexes were collected from the ulnar vein just before the run, after 25, 50, 75 and 100 km, and 24 hours after termination of the run. A considerable increase in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was observed with the distance covered (p < 0.05), which continued during recovery. An increase in the mean values of lactate dehydrogenase (LDH), creatine kinase (CK) and C-reactive protein (CRP) (p < 0.05) was observed with each sequential course. The biggest differences between the age groups were found for the activity of liver enzymes and LDH after completing 75 km as well as after 24 hours of recovery. It can be concluded that the response to extreme exertion deteriorates with age in terms of the active movement apparatus.

[Pulmonary hypertension - disease mechanisms]. / Plicní hypertenze - patofyziologické mechanizmy.

Pulmonary hypertension (PH) is known for its variable etiology. PH pathophysiology is very complex and our therapeutic options are limited. Most of known underlying disease mechanisms play a role across all etiological groups of PH, and they are followed by the same morphological and functional changes of pulmonary vasculature. Mostly, we are not able to determine whether one particular mechanism works as a cause or consequence in the chain of events. An imbalance between vasoconstriction and vasodilation becomes the major functional change of pulmonary vasculature in PH. The main morphological changes (termed together as "remodeling") include cell hyperplasia of pulmonary artery leading to its thickening and narrowing, and impaired regulation of extracellular matrix production leading to reduction in its elasticity. As a result of all these changes, the peripheral vascular resistance in pulmonary vascular bed rises, thus increasing afterload of the right ventricle and finally progressing to its failure. This review aims to summarize and explain the nature of the functional and histological changes in pulmonary arteries which occur in pulmonary hypertension, separately define the role of endothelium and pulmonary artery myocytes, and discuss the most important known pathophysiological mechanisms that lead to these changes.

Time-dependent expression of cytochrome p450 epoxygenases during human prenatal development.

There is growing evidence that some members of cytochrome P450 enzymes contribute to regulation of normal prenatal development. CYP epoxygenases (CYP2C and CYP2J subfamilies) convert arachidonic acid into four regioisomeric epoxyeicosatrienoic acids (EETs), biologically active molecules involved in mitogenesis and cell signaling. Almost nothing is known about localization of their expression in tissues during human prenatal development. The spatio-temporal expression pattern of CYP2C8, CYP2C9, CYP2C19 and CYP2J2 in human embryonic/fetal intestines, liver, and kidney was investigated by immunohistochemical method. CYP epoxygenases are expressed already in early stages of development in these embryonic/fetal tissues (as early as 7th week of IUD in the intestines, 5th week of IUD in the liver, and 6th week of IUD in the kidney). In kidney, CYP epoxygenases are expressed in the metanephrogenic blastema (but not in the uninduced mesenchyme) and in the tubular system. In the intestines, diverse CYP epoxygenases distribution along crypt-villus axis could suggest role in cell differentiation. Moreover, we detected higher CYP2J2 level in these organs than in adult tissue samples.

Peroxisome proliferator-activated receptors in regulation of cytochromes P450: new way to overcome multidrug resistance?

Embryonic and tumour cells are able to protect themselves against various harmful compounds. In human pathology, this phenomenon exists in the form of multidrug resistance (MDR) that significantly deteriorates success of anticancer treatment. Cytochromes P450 (CYPs) play one of the key roles in the xenobiotic metabolism. CYP expression could contribute to resistance of cancer cells to chemotherapy. CYP epoxygenases (CYP2C and CYP2J) metabolize about 20% of clinically important drugs. Besides of drug metabolism, CYP epoxygenases and their metabolites play important role in embryos, normal body function, and tumors. They participate in angiogenesis, mitogenesis, and cell signaling. It was found that CYP epoxygenases are affected by peroxisome proliferator-activated receptor α (PPARα). Based on the results of current studies, we assume that PPARs ligands may regulate CYP2C and CYP2J and in some extent they may contribute to overcoming of MDR in patients with different types of tumours.