Katarina Ondicova

Human adipose tissue‐derived mesenchymal stem cells expressing yeast cytosinedeaminase::uracil phosphoribosyltransferase inhibit intracerebral rat glioblastoma

Prodrug cancer gene therapy by mesenchymal stem cells (MSCs) targeted to tumors represents an attractive tool to activate prodrugs directly within the tumor mass, thus avoiding systemic toxicity. In this study, we tested the feasibility and efficacy of human adipose tissue‐derived MSCs, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracranial rat C6 glioblastoma. Experiments were designed to simulate conditions of future clinical application for high‐grade glioblastoma therapy by direct injections of therapeutic stem cells into tumor. We demonstrated that genetically modified therapeutic stem cells still have the tumor tropism when injected to a distant intracranial site and effectively inhibited glioblastoma growth after 5‐fluorocytosine (5‐FC) therapy. Coadministration of C6 cells and therapeutic stem cells with delayed 5‐FC therapy improved the survival in a therapeutic stem cell dose‐dependent manner and induced complete tumor regression in a significant number of animals. Continuous intracerebroventricular delivery of 5‐FC using osmotic pump reduced the dose of prodrug required for the same therapeutic effect, and along with repeated administration of therapeutic stem cells increased the survival time. Intracerebral injection of therapeutic stem cells and treatment with 5‐FC did not show any detectable adverse effects. Results support the arguments to begin clinical studies for treatment of high‐grade brain tumors.

Complete regression of glioblastoma by mesenchymal stem cells mediated prodrug gene therapy simulating clinical therapeutic scenario

Suicide gene therapy mediated by mesenchymal stem cells with their ability to engraft into tumors makes these therapeutic stem cells an attractive tool to activate prodrugs directly within the tumor mass. In this study, we evaluated the therapeutic efficacy of human mesenchymal stem cells derived from bone marrow and from adipose tissue, engineered to express the suicide gene cytosine deaminase::uracil phosphoribosyltransferase to treat intracerebral rat C6 glioblastoma in a simulated clinical therapeutic scenario. Intracerebrally grown glioblastoma was treated by resection and subsequently with single or repeated intracerebral inoculations of therapeutic stem cells followed by a continuous intracerebroventricular delivery of 5‐fluorocytosine using an osmotic pump. Kaplan–Meier survival curves revealed that surgical resection of the tumor increased the survival time of the resected animals depending on the extent of surgical intervention. However, direct injections of therapeutic stem cells into the brain tissue surrounding the postoperative resection cavity led to a curative outcome in a significant number of treated animals. Moreover, the continuous supply of therapeutic stem cells into the brain with growing glioblastoma by osmotic pumps together with continuous prodrug delivery also proved to be therapeutically efficient. We assume that observed curative therapy of glioblastoma by stem cell‐mediated prodrug gene therapy might be caused by the destruction of both tumor cells and the niche where glioblastoma initiating cells reside.

Role of nervous system in cancer aetiopathogenesis

There have been several reports on tumour tissue innervation, the effect of neurotransmitters on tumour growth, the development of metastases, and the effect of altered nervous-system activity on tumour cell proliferation. In this personal view, we summarise recent findings related to the interactions between the nervous system and tumour cells and suggest further research into the role of the nervous system in the aetiopathogenesis of cancer. Data showing the transmission of signals between the brain and tumour tissue create a complex view of the nervous system in the aetiopathogenesis of cancer. This neurobiological view of cancer aetiopathogenesis suggests that humoral and nervous pathways convey signals from tumour cells to the brain, and that the brain might consequently modulate the neuroendocrine-immune system to regulate tumour growth in peripheral tissues.

Tauopathy in transgenic (SHR72) rats impairs function of central noradrenergic system and promotes neuroinflammation.

BackgroundBrain norepinephrine (NE) plays an important role in the modulation of stress response and neuroinflammation. Recent studies indicate that in Alzheimer’s disease (AD), the tau neuropathology begins in the locus coeruleus (LC) which is the main source of brain NE. Therefore, we investigated the changes in brain NE system and also the immune status under basal and stress conditions in transgenic rats over-expressing the human truncated tau protein.MethodsBrainstem catecholaminergic cell groups (LC, A1, and A2) and forebrain subcortical (nucleus basalis of Meynert), hippocampal (cornu ammonis, dentate gyrus), and neocortical areas (frontal and temporal association cortices) were analyzed for NE and interleukin 6 (IL-6) mRNA levels in unstressed rats and also in rats exposed to single or repeated immobilization. Moreover, gene expression of NE-biosynthetic enzyme, tyrosine hydroxylase (TH), and several pro- and anti-inflammatory mediators were determined in the LC.ResultsIt was found that tauopathy reduced basal NE levels in forebrain areas, while the gene expression of IL-6 was increased in all selected areas at the same time. The differences between wild-type and transgenic rats in brain NE and IL-6 mRNA levels were observed in stressed animals as well. Tauopathy increased also the gene expression of TH in the LC. In addition, the LC exhibited exaggerated expression of pro- and anti-inflammatory mediators (IL-6, TNFα, inducible nitric oxide synthases 2 (iNOS2), and interleukin 10 (IL-10)) in transgenic rats suggesting that tauopathy affects also the immune background in LC. Positive correlation between NE and IL-6 mRNA levels in cornu ammonis in stressed transgenic animals indicated the reduction of anti-inflammatory effect of NE.ConclusionsOur data thus showed that tauopathy alters the functions of LC further leading to the reduction of NE levels and exaggeration of neuroinflammation in forebrain. These findings support the assumption that tau-related dysfunction of LC activates the vicious circle perpetuating neurodegeneration leading to the development of AD.

Exaggerated phosphorylation of brain tau protein in CRH KO mice exposed to repeated immobilization stress.

Abstract Neuroendocrine and behavioral stress responses are orchestrated by corticotropin-releasing hormone (CRH) and norepinephrine (NE) synthesizing neurons. Recent findings indicate that stress may promote development of neurofibrillary pathology in Alzheimers disease. Therefore, we investigated relationships among stress, tau protein phosphorylation, and brain NE using wild-type (WT) and CRH-knockout (CRH KO) mice. We assessed expression of phosphorylated tau (p-tau) at the PHF-1 epitope and NE concentrations in the locus coeruleus (LC), A1/C1 and A2/C2 catecholaminergic cell groups, hippocampus, amygdala, nucleus basalis magnocellularis, and frontal cortex of unstressed, singly stressed or repeatedly stressed mice. Moreover, gene expression and protein levels of tyrosine hydroxylase (TH) and CRH receptor mRNA were determined in the LC. Plasma corticosterone levels were also measured. Exposure to a single stress increases tau phosphorylation throughout the brain in WT mice when compared to singly stressed CRH KO animals. In contrast, repeatedly stressed CRH KO mice showed exaggerated tau phosphorylation relative to WT controls. We also observed differences in extent of tau phosphorylation between investigated structures, e.g. the LC and hippocampus. Moreover, CRH deficiency leads to different responses to stress in gene expression of TH, NE concentrations, CRH receptor mRNA, and plasma corticosterone levels. Our data indicate that CRH effects on tau phosphorylation are dependent on whether stress is single or repeated, and differs between brain regions. Our findings indicate that CRH attenuates mechanisms responsible for development of stress-induced tau neuropathology, particularly in conditions of chronic stress. However, the involvement of central catecholaminergic neurons in these mechanisms remains unclear and is in need of further investigation.

Stress-Induced Activation of the Sympathoadrenal System is Determined by Genetic Background in Rat Models of Tauopathy

Stress may accelerate onset of neurodegenerative diseases in vulnerable subjects and, vice versa, neurodegeneration affects the responsiveness to stressors. We investigated the neuroendocrine response to immobilization stress in normotensive Wistar-Kyoto rats (WKY), spontaneously hypertensive rats (SHR), and transgenic rats of respective WKY and SHR strains overexpressing human truncated tau protein. Plasma levels of epinephrine, norepinephrine, and corticosterone were determined. An immobilization-induced elevation of epinephrine and norepinephrine was significantly reduced in WKY transgenic rats compared to WKY wild-type rats, while no differences were seen between SHR transgenic and SHR wild-type animals. Our data have shown that sympathoadrenal system response to stress strongly depends on both tau protein-induced neurodegeneration and genetic background of experimental animals.

Subdiaphragmatic vagotomy enhances stress-induced epinephrine release in rats

Neuroendocrine stress response is regulated by several feedback loops. Since it has been suggested that afferent vagal pathways contribute to these feedback loops, we examined the effect of surgical subdiaphragmatic vagotomy on both baseline and stress-induced increases in plasma epinephrine, norepinephrine, and corticosterone levels in vagotomized and sham-operated Sprague Dawley rats. On either the 3rd or 14th day following vagotomy, the animals were exposed to acute immobilization stress and blood from the jugular vein was collected both before and during stress exposure. We found that vagotomy significantly enhanced immobilization-induced increases of plasma epinephrine, norepinephrine, and corticosterone levels on the 3rd day following surgery. However, on the 14th day following surgery, vagotomy enhanced only increase of plasma epinephrine levels in stressed rats. Our data indicate that afferent pathways of the vagus nerve are involved in negative feedback regulation of epinephrine secretion from the adrenal medulla during stressful conditions. We hypothesize that this feedback mechanism might be mediated by the binding of circulating epinephrine on β2-adrenergic receptors localized on sensory endings of the vagus nerve.

Increased ubiquinone concentration after intracerebroventricularly-administered ubiquinol to selected rat brain regions.

Brain coenzyme Q10 (CoQ10) concentration can influence the activity of several brain regions, including those which participate in the regulation of cardiovascular circadian rhythms, food intake, neuroendocrine stress response, activity and sleep regulation. However, the effect of supplemented ubiquinol (reduced CoQ) into brain regions is not known. This study determined baseline levels of ubiquinone (oxidized CoQ) in various rat brain regions and proved the bioavailability of the liposomal ubiquinol to selected brain regions after its administration into right brain ventricle. Our data indicate that administration of ubiquinol may create the basis for modulation of neuronal activities in specific brain regions.

EFFECTS OF AEROBIC-STRENGTH TRAINING ON SELECTED MOLECULAR TARGETS IN CEREBROSPINAL FLUID OF SENIORS WITH MILD COGNITIVE IMPAIRMENT

Background:Cerebrospinal fluid (CSF) levels of total tau protein (hTau), phosphorylated tau (pTau181P), and amyloid-beta of 42 amino acids (Ab1-42) are established biomarkers for the diagnosis of Alzheimer’s disease (AD), but the discriminatory power for differential dementia diagnosis remains suboptimal. Also, current laboratory measures of CSF pTau181P are thought to underestimate the total levels of phosphorylated tau. The goal of this study is to investigate if the non-phosphorylated tau fraction (pTau rel) would improve the diagnostic performance of the routine AD biomarker panel for differential dementia diagnosis. Methods:The study population consisted of clinically diagnosed AD patients (n1⁄445), definite frontotemporal lobar degeneration (FTLD) patients (n1⁄445), definite Creutzfeldt-Jakob disease (CJD) patients (n1⁄420) and cognitively healthy controls (n1⁄420). CSF levels of Ab1-42, hTau, pTau181P and pTau rel were determined with commercially available single-analyte ELISA kits (INNOTEST b-Amyloid(142), INNOTEST hTau-Ag and INNOTEST Phospho-Tau(181P) from Fujirebio Europe, Belgium; pTAU rel ELISA Kit from AJ Roboscreen, IBL International GmbH, Germany). Receiver operating characteristic (ROC) curve analyses were used to obtain area under the curve (AUC) values. AUC values were compared using DeLong tests. Results:Diagnostic performance of single markers as well as biomarker ratios was determined for each pairwise comparison of different dementia groups and controls. To evaluate the diagnostic power of pTau rel in the routine AD biomarker panel, the single (routine) marker with the highest AUC value was compared with that of pTau rel. Additionally, the (routine) biomarker ratio with the highest AUC value was compared with its equivalent using pTau rel. An overview of the results is listed in Table 1. Conclusions: The diagnostic performance of pTau rel for differential dementia diagnosis (comparing AD, FTLD, CJD patients and healthy controls) is not better than the diagnostic performance of the routine AD CSF biomarkers.

Ambiguous effect of signals transmitted by the vagus nerve on fibrosarcoma incidence and survival of tumor-bearing rats

While the parasympathetic nervous system appears to be involved in the regulation of tumor progression, its exact role is still unclear. Therefore, using a rat BP6-TU2 fibrosarcoma tumor model, we investigated the effect of (1) reduction of vagal activity produced by subdiaphragmatic vagotomy; and (2) enhancement of vagal activity produced by continuous delivery of electric impulses to the cervical part of the vagus nerve on tumor development and survival of tumor-bearing rats. We also evaluated the expression of cholinergic receptors within in vitro cultivated BP6-TU2 cells. Interestingly, we found that both, vagal stimulation and subdiaphragmatic vagotomy slightly reduced tumor incidence. However, survival of tumor-bearing rats was not affected by any of the experimental approaches. Additionally, we detected mRNA expression of the α1, α2, α5, α7, and α10 subunits of nicotinic receptors and the M1, M3, M4, and M5 subtypes of muscarinic receptors within in vitro cultivated BP6-TU2 cells. Our data indicate that the role of the vagus nerve in modulation of fibrosarcoma development is ambiguous and uncertain and requires further investigation.