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Chromaffin cells from adrenal medulla are the primary source of epinephrine and norepinephrine secretion. Both of chromaffin cells and the closely related sympathetic neurons have been assumed to derive from a common sympathoadrenal progenitor cell which develops from the neural crest during early embryogenesis. But unlike the sympathetic neurons, they are able to proliferate throughout the life span. The aim of this study is to isolate and to induce the differentiation from the chromaffin progenitor cells of adult bovine adrenal medulla. Chromaffin progenitor cells isolated in low-attachment condition grew into spheres and showed self-renewal ability. In molecular expression analysis, these chromospheres not only showed progenitor markers including Nestin and Sox1, they also expressed chromaffin cell markers such as TH and PNMT. The adrenal androgens DHEA and dehydroepiandrosterone (DHEAS) are the most abundant androgens in human body. They are produced by the inner adrenocortical zone, zona reticularis, which in direct contacts with the adrenomedullary chromaffin cells. Our previous research showed that these steroids influence chromaffin cell proliferation caused by growth factors. We therefore addressed the question of a potential influence of these hormones on chromospheres differentiation. The expression of Nestin mRNA was reduced by treatment with DHEAS, and PNMT expression was induced by treatment with DHEA as well as dexamethasone. These data suggest that DHEA and DHEAS might play an important role in chromaffin cell development. [ABSTRACT FROM AUTHOR]

Background and Objectives : Prehypertension and hypertension are the most common cardiovascular disorders worldwide and are increasingly considered one of the most serious public health problems, particularly in developing countries. The objective of this study was to determine the frequency and demographic and socioeconomic predictors of prehypertension and hypertension in the adults in Serbia, and to examine the relationship between prehypertension and hypertension and health behavior determinants (smoking, alcohol use, physical activity) and individual aspects of health (a health self-assessment, multimorbidity, BMI, depressive symptoms). Materials and Methods : The research is part of the fourth National Population Health Survey conducted in 2019, which was conducted by the Republic Institute of Statistics, in cooperation with the Institute of Public Health of Serbia and the Ministry of Health of the Republic of Serbia. As a research instrument, questionnaires were used in accordance with the methodology of the European Health Survey. For the purposes of this research, data on the adult population aged 20 and over were used. Results : Women are at a reduced risk for both prehypertension (OR  =  0.328) and hypertension (OR = 0.349) by nearly 70%. Similarly, those aged below 60 years have a lower risk for prehypertension and those younger than 40 years have a lower risk for hypertension (OR = 0.995), whereas people with a lower education have a 4.3 times higher risk of prehypertension (OR = 4.323) and a 1.6 times higher risk of hypertension (OR =  1.614). The poor have a 1.4 times higher risk of prehypertension (OR = 1.413) and a 1 times higher risk of hypertension (OR = 1.035). People with multimorbidity have a 1.2 times higher risk of both prehypertension (OR = 1.218) and a 4.8 times higher risk of hypertension (OR = 4.867). Conclusions : Male gender, lower education, poverty, age and the presence of multimorbidity are significant predictors of prehypertension and hypertension in the Serbian adult population, so preventive strategies should be aimed at these sensitive population groups., Competing Interests: The authors declare no conflicts of interest.

Introduction: Peptide receptor radionuclide therapy (PRRT) is a treatment option for well-differentiated, somatostatin receptor positive, unresectable or/and metastatic neuroendocrine tumors (NETs). Although high disease control rates seen with PRRT a significant number NET patients have a short progression-free interval, and currently, there is a deficiency of effective biomarkers to pre-identify these patients. This study is aimed at determining the prognostic significance of biomarkers on survival of patients with NETs in initial PRRT treatment., Methodology: We retrospectively analyzed 51 patients with NETs treated with PRRT at the Department for nuclear medicine, University Clinical Center Kragujevac, Serbia, with a five-year follow-up. Eligible patients with confirmed inoperable NETs, were retrospectively evaluated hematological, blood-based inflammatory markers, biochemical markers and clinical characteristics on disease progression. In accordance with the progression og the disease, the patients were divided into two groups: progression group (n=18) and a non-progression group (n=33). Clinical data were compared between the two groups., Results: A total of 51 patients (Md=60, age 25-75 years) were treated with PRRT, of whom 29 (56.86%) demonstrated stable disease, 4 (7.84%) demonstrated a partial response, and 14 (27.46%) demonstrated progressive disease and death was recorded in 4 (7.84%) patients. The mean PFS was a 36.22 months (95% CI 30.14-42.29) and the mean OS was 44.68 months (95% CI 37.40-51.97). Univariate logistic regression analysis displayed that age (p<0.05), functional tumors (p<0.05), absolute neutrophil count (p<0.05), neutrophil-lymphocyte ratio-NLR (p<0.05), C-reactive protein-CRP (p<0.05), CRP/Albumin (p<0.05), alanine aminotransferase-ALT (p<0.05), were risk factors for disease progression. Multivariate logistic regression analysis exhibited that functional tumors (p<0.001), age (p<0.05), CRP (p<0.05), and ALT (p<0.05), were independent risk factors for the disease progression in patients with NETs. Tumor functionality was the most powerful prognostic factor. The median PFS (11.86 ± 1.41 vs. 43.38 ± 3.16 months; p=0.001) and OS (21.81 ± 2.70 vs 53.86 ± 3.70, p=0.001) were significantly shorter in patients with functional than non-functional NETs respectively., Conclusion: The study's results suggest that tumor functionality, and certain biomarkers may serve as prognostic survival indicators for patients with NETs undergoing PRRT. The findings can potentially help to identify patients who are at higher risk of disease progression and tailor treatment strategies accordingly., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Vukomanovic, Nedic, Radojevic, Dagovic, Milosavljevic, Markovic, Ignjatovic, Simic Vukomanovic, Djukic, Sreckovic, Backovic, Vuleta, Djukic, Vukicevic and Ignjatovic.)

Despite the pivotal role of IFN-λs in the innate immune response, the data on its genetic polymorphism in relation to COVID-19 severity are scarce and contradictory. In the present study, we aimed to determine if the presence of the most frequent functional single nucleotide polymorphisms (SNPs) of the two most important IFN-λs coding genes, namely IFNL3 and IFNL4, alters the likelihood of SARS-CoV-2-infected patients to develop more severe form of the disease. This observational cohort study involved 178 COVID-19 patients hospitalized at the University Clinical Centre Kragujevac, Serbia. Patients' demographics, clinical characteristics, and laboratory parameters were collected at admission. COVID-19 signs and symptoms were assessed during the hospital stay, with the worst condition determining the disease severity. Genotyping for IFNL3 (rs12980275 and rs8099917) and IFNL4 (rs12979860 and rs368234815) SNPs was conducted using TaqMan assays. Our study revealed carriers of IFNL3 and IFNL4 minor alleles to be less likely to progress from mild to moderate COVID-19, that is, to develop COVID-19-related pneumonia. After adjustment for other factors of influence, such as age, sex, and comorbidities, the likelihood of pneumonia development remained significantly associated with IFNL4 polymorphism (odds ratios [ORs] [95% confidence interval (95% CI)]: 0.233 [0.071; 0.761]). When the patients were stratified according to sex, the protective role of IFNL4 minor alleles, controlled for the effect of comorbidities, remained significant only in females (OR [95% CI]: 0.035 [0.003; 0.408]). Our results strongly suggest that IFNL4 rs12979860 and rs368234815 polymorphisms independently predict the risk of COVID-19-related pneumonia development in females., (© 2023 Wiley Periodicals LLC.)

Background and Objectives: The physiological oxygen tension in fetal brains (∼3%, physioxia) is beneficial for the maintenance of neural stem cells (NSCs). Sensitivity to oxygen varies between NSCs from different fetal brain regions, with midbrain NSCs showing selective susceptibility. Data on Hif-1α/Notch regulatory interactions as well as our observations that Hif-1α and oxygen affect midbrain NSCs survival and proliferation prompted our investigations on involvement of Notch signalling in physioxia-dependent midbrain NSCs performance., Methods and Results: Here we found that physioxia (3% O 2 ) compared to normoxia (21% O 2 ) increased proliferation, maintained stemness by suppression of spontaneous differentiation and supported cell cycle progression. Microarray and qRT-PCR analyses identified significant changes of Notch related genes in midbrain NSCs after long-term (13 days), but not after short-term physioxia (48 hours). Consistently, inhibition of Notch signalling with DAPT increased, but its stimulation with Dll4 decreased spontaneous differentiation into neurons solely under normoxic but not under physioxic conditions., Conclusions: Notch signalling does not influence the fate decision of midbrain NSCs cultured in vitro in physioxia, where other factors like Hif-1α might be involved. Our findings on how physioxia effects in midbrain NSCs are transduced by alternative signalling might, at least in part, explain their selective susceptibility to oxygen.

Coronavirus Disease 2019 (COVID-19) has been ranked among the most fatal infectious diseases worldwide, with host's immune response significantly affecting the prognosis. With an aim to timely predict the most likely outcome of SARS-CoV-2 infection, we investigated the association of IFNL3 and IFNL4 polymorphisms, as well as other potentially relevant factors, with the COVID-19 mortality. This prospective observational case-control study involved 178 COVID-19 patients, hospitalized at Corona Center or Clinic for Infectious Diseases of University Clinical Centre Kragujevac, Serbia, followed up until hospital discharge or in-hospital death. Demographic and clinical data on all participants were retrieved from the electronic medical records, and TaqMan assays were employed in genotyping for IFNL3 and IFNL4 single nucleotide polymorphisms (SNPs), namely rs12980275, rs8099917, rs12979860, and rs368234815. 21.9% and 65.0% of hospitalized and critically ill COVID-19 patients, respectively, died in-hospital. Multivariable logistic regression analysis revealed increased Charlson Comorbidity Index (CCI), N/L, and lactate dehydrogenase (LDH) level to be associated with an increased likelihood of a lethal outcome. Similarly, females and the carriers of at least one variant allele of IFNL3 rs8099917 were almost 36-fold more likely not to survive SARS-CoV-2 infection. On the other hand, the presence of at least one ancestral allele of IFNL4 rs368234815 decreased more than 15-fold the likelihood of mortality from COVID-19. Our results suggest that, in addition to LDH level, N/L ratio, and CCI, IFNL4 rs368234815 and IFNL3 rs8099917 polymorphisms, but also patients' gender, significantly affect the outcome of COVID-19., (© 2023 Wiley Periodicals LLC.)

Objective: The increased level of interleukin (IL)-33 is considered as a predictor of severe coronavirus disease 2019 (COVID-19) infection, but its role at different stages of the disease is still unclear. Our goal was to analyze the correlation of IL-33 and other innate immunity cytokines with disease severity. Methods: In this study, 220 patients with COVID-19 were included and divided into two groups, mild/moderate and severe/critical. The value of the cytokines, clinical, biochemical, radiographic data was collected and their correlation with disease severity was analyzed. Results: Most patients in the severe/critical group were male (81.8%) and older (over 64.5 years). We found a statistically significant difference ( p < 0.05) in these two groups between clinical features (dyspnea, dry cough, fatigue, and auscultatory findings); laboratory [(neutrophil count, lymphocyte count, monocyte count, hemoglobin, plasma glucose, urea, creatinine, total bilirubin (TBIL), direct bilirubin (DBIL), aspartate aminotransferase (AST), albumin (ALB), lactate dehydrogenase (LDH), creatinine kinase (CK), D-dimer, C-reactive protein (CRP), procalcitonin (PCT), Fe, and Ferritin)], arterial blood gases (oxygen saturation-Sa0 2 , partial pressure of oxygen -p0 2 ), and chest X-rays (CXR) lung findings ( p = 0 .000). We found a significantly higher serum concentration ( p < 0.05) of TNF-α, IL-1β, IL-6, IL-12, IL-23, and IL-33 in patients with COVID-19 with severe disease. In the milder stage of COVID-19, a positive correlation was detected between IL-33 and IL-1β, IL-12 and IL-23, while a stronger positive correlation between the serum values of IL-33 and TNF-α, IL-1β, IL-6, and IL-12 and IL-23 was detected in patients with COVID-19 with severe disease. A weak negative correlation ( p < 0.05) between pO 2 and serum IL-1β, IL-12, and IL-33 and between SaO 2 and serum IL-33 was noted. The positive relation ( p < 0.05) between the serum values of IL-33 and IL-12, IL-33 and IL-6, and IL-6 and IL-12 is proven. Conclusion: In a more progressive stage of COVID-19, increased IL-33 facilitates lung inflammation by inducing the production of various innate proinflammatory cytokines (IL-1β, IL-6, TNF-α, IL-12, and IL-23) in several target cells leading to the most severe forms of the disease. IL-33 correlates with clinical parameters of COVID-19 and might represent a promising marker as well as a therapeutic target in COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Markovic, Jovanovic, Gajovic, Jurisevic, Arsenijevic, Jovanovic, Jovanovic, Mijailovic, Lukic, Zornic, Vukicevic, Stojanovic, Maric, Jocic and Jovanovic.)

SARS-CoV-2 virus causes infection which led to a global pandemic in 2020 with the development of severe acute respiratory syndrome. Therefore, this study was aimed at examining its possible role in predicting severity and intrahospital mortality of COVID-19, alongside with other laboratory and biochemical procedures, clinical signs, symptoms, and comorbidity. This study, approved by the Ethical Committee of Clinical Center Kragujevac, was designed as an observational prospective cross-sectional clinical study which was conducted on 127 patients with diagnosed respiratory COVID-19 viral infection from April to August 2020. The primary goals were to determine the predictors of COVID-19 severity and to determine the predictors of the negative outcome of COVID-19 infection. All patients were divided into three categories: patients with a mild form, moderate form, and severe form of COVID-19 infection. All biochemical and laboratory procedures were done on the first day of the hospital admission. Respiratory ( p < 0.001) and heart ( p = 0.002) rates at admission were significantly higher in patients with a severe form of COVID-19. From all observed hematological and inflammatory markers, only white blood cell count (9.43 ± 4.62, p = 0.001) and LDH (643.13 ± 313.3, p = 0.002) were significantly higher in the severe COVID-19 group. We have observed that in the severe form of SARS-CoV-2, the levels of superoxide anion radicals were substantially higher than those in two other groups (11.3 ± 5.66, p < 0.001) and the nitric oxide level was significantly lower in patients with the severe disease (2.66 ± 0.45, p < 0.001). Using a linear regression model, TA, anosmia, ageusia, O 2 - , and the duration at the ICU are estimated as predictors of severity of SARS-CoV-2 disease. The presence of dyspnea and a higher heart rate were confirmed as predictors of a negative, fatal outcome. Results from our study show that presence of hypertension, anosmia, and ageusia, as well as the duration of ICU stay, and serum levels of O 2 - are predictors of COVID-19 severity, while the presence of dyspnea and an increased heart rate on admission were predictors of COVID-19 mortality., Competing Interests: The authors declare that there is no conflict of interest., (Copyright © 2021 Ivan Cekerevac et al.)

Clinical manifestations of SARS-CoV-2 infection range from mild to critically severe. The aim of the study was to highlight the immunological events associated with the severity of SARS-CoV-2 infection, with an emphasis on cells of innate immunity. Thirty COVID-19 patients with mild/moderate symptoms and 27 patients with severe/critically severe symptoms were recruited from the Clinical Center of Kragujevac during April 2020. Flow cytometric analysis was performed to reveal phenotypic and functional alterations of peripheral blood cells and to correlate them with the severity of the disease. In severe cases, the number of T and B lymphocytes, dendritic cells, NK cells, and HLA-DR-expressing cells was drastically decreased. In the monocyte population proportion between certain subsets was disturbed and cells coexpressing markers of M1 and M2 monocytes were found in intermediate and non-classical subsets. In mild cases decline in lymphocyte number was less pronounced and innate immunity was preserved as indicated by an increased number of myeloid and activated dendritic cells, NK cells that expressed activation marker at the same level as in control and by low expression of M2 marker in monocyte population. In patients with severe disease, both innate and adoptive immunity are devastated, while in patients with mild symptoms decline in lymphocyte number is lesser, and the innate immunity is preserved., Competing Interests: The authors have declared that no competing interests exists.

Osteoporosis and obesity result from disturbed osteogenic and adipogenic differentiation and present emerging challenges for our aging society. Because of the regulatory role of Thy-1 in mesenchyme-derived fibroblasts, we investigated the impact of Thy-1 expression on mesenchymal stem cell (MSC) fate between osteogenic and adipogenic differentiation and consequences for bone formation and adipose tissue development in vivo. MSCs from Thy-1-deficient mice have decreased osteoblast differentiation and increased adipogenic differentiation compared to MSCs from wild-type mice. Consistently, Thy-1-deficient mice exhibited decreased bone volume and bone formation rate with elevated cortical porosity, resulting in lower bone strength. In parallel, body weight, subcutaneous/epigonadal fat mass, and bone fat volume were increased. Thy-1 deficiency was accompanied by reduced expression of specific Wnt ligands with simultaneous increase of the Wnt inhibitors sclerostin and dickkopf-1 and an altered responsiveness to Wnt. We demonstrated that disturbed bone remodeling in osteoporosis and dysregulated adipose tissue accumulation in patients with obesity were mirrored by reduced serum Thy-1 concentrations. Our findings provide new insights into the mutual regulation of bone formation and obesity and open new perspectives to monitor and to interfere with the dysregulated balance of adipogenesis and osteogenesis in obesity and osteoporosis., (Copyright © 2018 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Objectives: To assess the prevalence of heparin-induced thrombocytopenia and to study platelet count trends potentially suggestive of heparin-induced thrombocytopenia in a population of extracorporeal membrane oxygenator patients., Design: Retrospective cohort study., Setting: A total of 926-bed teaching hospital., Patients: Extracorporeal membrane oxygenator patients who survived longer than 48 hours from extracorporeal membrane oxygenator initiation between January 1, 2009, and December 31, 2013., Interventions: None., Measurements and Main Results: Demographic and clinical data were collected prospectively on all extracorporeal membrane oxygenator patients. Heparin-induced thrombocytopenia testing results and platelet count variables were obtained from the electronic medical record. We used our institutional algorithm to interpret the results of heparin-induced thrombocytopenia testing. Ninety-six extracorporeal membrane oxygenator patients met the inclusion criteria. Eight patients met the algorithm criteria for heparin-induced thrombocytopenia diagnosis and seven of those had documented thromboembolic event while on extracorporeal membrane oxygenator (prevalence of heparin-induced thrombocytopenia and heparin-induced thrombocytopenia related thrombosis, 8.3 and 7.3, respectively). Heparin-induced thrombocytopenia positive patients were younger; all underwent venoarterial extracorporeal membrane oxygenator; spent more hours on extracorporeal membrane oxygenator; had significantly higher heparin-induced thrombocytopenia enzyme-linked immunosorbent assays optical density; had a higher prevalence of thromboembolic events and reached platelet count nadir later. There was no difference in mortality between heparin-induced thrombocytopenia positive and negative patients. Comparison of platelet count trends revealed that there was no statistically significant difference between the predefined study groups., Conclusions: Prevalence of heparin-induced thrombocytopenia and heparin-induced thrombocytopenia-related thrombosis among extracorporeal membrane oxygenator patients at our institution is relatively high. Using platelet count trends to guide decision to test for heparin-induced thrombocytopenia is not an optimal strategy in extracorporeal membrane oxygenator patients. Without a validated pretest probability clinical score, serosurveillance in a defined high-risk group of extracorporeal membrane oxygenator patients may be needed.

Introduction: Head posture in relation to the cervical spine is correlated with the morphology of the face and jaw, the position and the mutual relationship of the maxilla and mandible, their length and inclination. The aim of this study is to examine the relationship between the head posture and parameters of the sagittal position and length of the jaws., Material and Methods: The study included 90 subjects (30 for I, II and II skeletal class each) between the ages of 8 and 14 years, who were examined at the Department of Dentistry of Vojvodina inNovi Sad. Each subject had the lateral cephalogram made, and the analysis was performed by means of the computer program "Onyx Ceph." The following parameters were analyzed: craniocervical angle, angle of maxillary prognathism, angle of mandibulary prognathism, the difference angles of maxillary and mandibulary prognathism, length of maxillae, and length of mandible., Results: The angle of maxillary prognathism was in positive correlation with the craniocervical angle in the patients with class I and II, and they were negatively correlated in the patients with class III. The angle of mandibulary prognathism was in positive correlation with the craniocervical angle in the patients with class I and III, but they were negatively correlated in the patients with class II. The patients wi th class II had a statistically significant positive correlation between the craniocervical angle and length of the maxilla, and a significant, but negative correlation between the craniocervical angle and length of the mandible., Conclusion: Increased extension of the head in relation to the cervical spine can be a contributing factor to the formation of class II malocclusion.

The adrenal is a highly plastic organ with the ability to adjust to physiological needs by adapting hormone production but also by generating and regenerating both adrenocortical and adrenomedullary tissue. It is now apparent that many adult tissues maintain stem and progenitor cells that contribute to their maintenance and adaptation. Research from the last years has proven the existence of stem and progenitor cells also in the adult adrenal medulla throughout life. These cells maintain some neural crest properties and have the potential to differentiate to the endocrine and neural lineages. In this article, we discuss the evidence for the existence of adrenomedullary multi potent progenitor cells, their isolation and characterization, their differentiation potential as well as their clinical potential in transplantation therapies but also in pathophysiology., (Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.)

We present a method to efficiently culture primary chromaffin progenitors from the adult bovine adrenal medulla in a defined, serum-free monolayer system. Tissue is dissociated and plated for expansion under support by the mitogen basic fibroblast growth factor (bFGF). The cultures, although not homogenous, contain a subpopulation of cells expressing the neural stem cell marker Hes3 that also propagate. In addition, Hes3 is also expressed in the adult adrenal medulla from where the tissue is taken. Differentiation is induced by bFGF withdrawal and switching to Neurobasal medium containing B27. Following differentiation, Hes3 expression is lost, and cells acquire morphologies and biomarker expression patterns of chromaffin cells and dopaminergic neurons. We tested the effect of different treatments that we previously showed regulate Hes3 expression and cell number in cultures of fetal and adult rodent neural stem cells. Treatment of the cultures with a combination of Delta4, Angiopoietin2, and a Janus kinase inhibitor increases cell number during the expansion phase without significantly affecting catecholamine content levels. Treatment with cholera toxin does not significantly affect cell number but reduces the ratio of epinephrine to norepinephrine content and increases the dopamine content relative to total catecholamines. These data suggest that this defined culture system can be used for target identification in drug discovery programs and that the transcription factor Hes3 may serve as a new biomarker of putative adrenomedullary chromaffin progenitor cells., (©AlphaMed Press.)

Chromaffin cells, sympathetic neurons of the dorsal ganglia, and the intermediate small intensely fluorescent cells derive from a common neural crest progenitor cell. Contrary to the closely related sympathetic nervous system, within the adult adrenal medulla a subpopulation of undifferentiated progenitor cells persists, and recently, we established a method to isolate and differentiate these progenitor cells from adult bovine adrenals. However, no studies have elucidated the existence of adrenal progenitor cells within the human adrenal medulla. Here we describe the isolation, characterization, and differentiation of chromaffin progenitor cells obtained from adult human adrenals. Human chromaffin progenitor cells were cultured in low-attachment conditions for 10-12 days as free-floating spheres in the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor. These primary human chromosphere cultures were characterized by the expression of several progenitor markers, including nestin, CD133, Notch1, nerve growth factor receptor, Snai2, Sox9, Sox10, Phox2b, and Ascl1 on the molecular level and of Sox9 on the immunohistochemical level. In opposition, phenylethanolamine N-methyltransferase (PNMT), a marker for differentiated chromaffin cells, significantly decreased after 12 days in culture. Moreover, when plated on poly-l-lysine/laminin-coated slides in the presence of FGF-2, human chromaffin progenitor cells were able to differentiate into two distinct neuron-like cell types, tyrosine hydroxylase (TH)(+)/β-3-tubulin(+) cells and TH(-)/β-3-tubulin(+) cells, and into chromaffin cells (TH(+)/PNMT(+)). This study demonstrates the presence of progenitor cells in the human adrenal medulla and reveals their potential use in regenerative medicine, especially in the treatment of neuroendocrine and neurodegenerative diseases.

The capacity of sympathoadrenal progenitors from adrenal medulla to generate dopaminergic neurons in vitro makes them an attractive source for replacement therapies of neurodegenerative diseases such as Parkinson's disease. Dopaminergic cells constitute one percent of the adult adrenal medulla only. Thus, isolation of sympathoadrenal progenitors and enhancement of their capacity to derive dopaminergic neurons is a strategy to be considered. Here, we summarize data on the characterization and isolation of sympathoadrenal progenitors from adult adrenal medulla capable to give rise to functional dopaminergic neurons, in vitro. Pretransplantation treatment of these cells with pharmacological means is an important prerequisite to improve dopaminergic differentiation and efficient engraftment of sympathoadrenal progenitors. Treatment of these cells with retinoic and ascorbic acids significantly increased dopamine secretion from derived neurons. Furthermore, inhibition of Notch signaling activated molecular mechanisms involved in the determination of dopaminergic neuronal subtype. Taken together, somatic adrenomedullary sympathoadrenal progenitor cells are a valid cell source for replacement therapies with a high potential for dopaminergic neuronal differentiation.

The differentiation of dopamine-producing neurons from chromaffin progenitors might represent a new valuable source for replacement therapies in Parkinson's disease. However, characterization of their differentiation potential is an important prerequisite for efficient engraftment. Based on our previous studies on isolation and characterization of chromaffin progenitors from adult adrenals, this study investigates their potential to produce dopaminergic neurons and means to enhance their dopaminergic differentiation. Chromaffin progenitors grown in sphere culture showed an increased expression of nestin and Mash1, indicating an increase of the progenitor subset. Proneurogenic culture conditions induced the differentiation into neurons positive for neural markers β-III-tubulin, MAP2, and TH accompanied by a decrease of Mash1 and nestin. Furthermore, Notch2 expression decreased concomitantly with a downregulation of downstream effectors Hes1 and Hes5 responsible for self-renewal and proliferation maintenance of progenitor cells. Chromaffin progenitor-derived neurons secreted dopamine upon stimulation by potassium. Strikingly, treatment of differentiating cells with retinoic and ascorbic acid resulted in a twofold increase of dopamine secretion while norepinephrine and epinephrine were decreased. Initiation of dopamine synthesis and neural maturation is controlled by Pitx3 and Nurr1. Both Pitx3 and Nurr1 were identified in differentiating chromaffin progenitors. Along with the gained dopaminergic function, electrophysiology revealed features of mature neurons, such as sodium channels and the capability to fire multiple action potentials. In summary, this study elucidates the capacity of chromaffin progenitor cells to generate functional dopaminergic neurons, indicating their potential use in cell replacement therapies.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Different lines of evidence suggest the existence of a subpopulation of proliferation-competent progenitor cells even in the adult state. The identification of sympathoadrenal progenitors in the adrenal would greatly enhance the understanding of adrenal physiology and their potential role in adrenal pathogenesis. Isolation and differentiation of these progenitor cells in culture would provide a tool to understand their development in vitro. Furthermore, due to the close relation to sympathetic neurons, these cells might provide an expandable source of cells for cell therapy in the treatment of neurodegenerative diseases. We therefore aim to establish protocols for the efficient isolation, enrichment and differentiation of chromaffin progenitor cells to dopaminergic neurons in culture.

The potential use of neural stem cells in basic research, drug testing and for development of therapeutic strategies requires large scale in vitro amplification, increasing the probability of genetic instability and transformation. Little is known, however, about potential correlations between long-term culture of neural stem and progenitor cells (NSPCs), changed differentiation and self-renewal capacities, and the occurrence of chromosomal instability. This study investigates the effect of extended culture time on self-renewal, differentiation capacity, cell cycle phase distribution, telomere length, telomerase activity and chromosomal stability on fetal brain-derived cells that form floating sphere colonies (neurospheres). We observed that increased sphere-forming capacity indicative of increased proliferation was accompanied by a decreased ability to differentiate into neural lineages. The high mobility group A (Hmga2) gene positively regulates self-renewal via repression of p16(Ink4a) and p19(ARF) gene expression. This study discerned an upregulation of Hmga2 gene and protein expression and decreased p16(Ink4a) and p19(ARF) gene expression, suggesting that Hmga2 might promote the proliferation of neurosphere cells in long-term culture. Further, our analyses revealed a significant decrease in telomere length after 4 weeks of culturing that is paralleled by a moderate upregulation of telomerase activity. Importantly, regular gain of chromosome 1 with random structural chromosomal aberrations was observed within 16 weeks of neurosphere cell culture. Genetic instability and diminished differentiation capacity seem to be a consequence of long-term culture of neurosphere cells. These data indicate the necessity to analyze self-renewal, differentiation capacity, telomere length, tumor suppressor genes and chromosomal stability in neurosphere cultures prior to their usage in basic research, drug testing or the development of therapeutic strategies., (2010 Elsevier Ireland Ltd. All rights reserved.)

Background Aims: Human adult bone marrow (BM)-derived mesenchymal stromal cells (hMSC) are reported to break germ layer commitment and differentiate into cells expressing neuroectodermal properties. Although it is of pivotal interest for cell replacement therapies for neurologic disorders, no data exist on the influence of the donor's age on this multipotent differentiation behavior., Methods: We evaluated various epigenetic neuroectodermal conversion protocols in adult hMSC derived from older donors (>45 versus 18-35 years of age) using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and immunocytochemistry. The protocols included single- and multi-step conversion-differentiation protocols combined with co-culture techniques. Furthermore, the age dependency of mesodermal differentiation potential and cell senescence were investigated., Results: The neuroectodermal differentiation potential of hMSC derived from old donors was completely lost, with no cells showing mature neuroectodermal phenotypes using single- and multi-step conversion-differentiation protocols and no improvement of neurogenesis by various co-culture conditions. Comparison of young versus old donor-derived hMSC showed fewer cells expressing early neuroectodermal marker proteins in the latter samples. qRT-PCR showed reduced expression of the proliferation marker KI67 and the neuroectodermal gene NES (nestin) in old donor-derived cells compared with young donor hMSC. Telomere length analysis showed no general cell aging., Conclusions: Our data provide evidence that only young donor-derived hMSC can be epigenetically differentiated in vitro into neuroectodermal cells, pointing towards senescence of multipotentiality of old donor-derived hMSC. There is thus an urgent need to develop better protocols for successful neuroectodermal differentiation of hMSC from old individuals as a prerequisite for autologous cell replacement strategies for neurologic diseases in elderly patients.

Chromaffin cells of the adrenal medulla are neural crest-derived cells of the sympathoadrenal lineage. Unlike the closely-related sympathetic neurons, a subpopulation of proliferation-competent cells exists even in the adult. Here, we describe the isolation, expansion, and in vitro characterization of proliferation-competent progenitor cells from the bovine adrenal medulla. Similar to neurospheres, these cells, when prevented from adherence to the culture dish, grew in spheres, which we named chromospheres. These chromospheres were devoid of mRNA specific for smooth muscle cells (MYH11) or endothelial cells (PECAM1). During sphere formation, markers for differentiated chromaffin cells, such as phenylethanolamine-N-methyl transferase, were downregulated while neural progenitor markers nestin, vimentin, musashi 1, and nerve growth factor receptor, as well as markers of neural crest progenitor cells such as Sox1 and Sox9, were upregulated. Clonal analysis and bromo-2'-deoxyuridine-incorporation analysis demonstrated the self-renewing capacity of chromosphere cells. Differentiation protocols using NGF and BMP4 or dexamethasone induced neuronal or endocrine differentiation, respectively. Electrophysiological analyses of neural cells derived from chromospheres revealed functional properties of mature nerve cells, such as tetrodotoxin-sensitive sodium channels and action potentials. Our study provides evidence that proliferation and differentiation competent chromaffin progenitor cells can be isolated from adult adrenal medulla and that these cells might harbor the potential for the treatment of neurodegenerative diseases, such as Parkinson's disease.

In the case of accidental radiation exposure, biological dosimetry has an important role. Previous studies have indicated that the flow cytometric micronucleus (MN) assay in human transferrin receptor positive reticulocytes (Tf-Ret) in blood could be a sensitive biomarker for chromosome damage. In the present investigation, the utility and sensitivity of this method was studied in 44 young patients from Belarus, who were treated with 131I for thyroid cancer. Red marrow (RM) is the critical organ in radioiodine therapy (RIT). In our patients, it was exposed to 100-700 mSv low-dose rate irradiation within 2-4 days. About 3 days after 131I administration, the frequency of micronucleated-Tf-Ret (f(MN-Tf-Ret)) increases within 1 day to a maximum and declines in the following 2-5 days to its value before treatment. A total dose of 100 mSv was easily detectable. The sensitivity of the assay after acute irradiation may be 50 mSv. The method should be useful for monitoring individuals after a radiation accident, provided blood samples can be obtained within a few days after exposure. The time-course of f(MN-Tf-Ret) is interpreted using a model, which considers the exponential exposure of red marrow in RIT as well as the kinetics of erythroblast maturation and reticulocyte migration into the peripheral circulation. Similar modelling was done on published data of MN in immature mouse erythrocytes. Striking similarities in the kinetic and in the yield of MN-induction were found between these two species. This lends support for the use of the mouse as a model for the MN-induction in humans.

Defects in apoptosis play a decisive role in both tumorigenesis and drug resistance in tumor treatment. The purpose of this study was to investigate the balance between formation of genomic damage and induction of apoptosis upon genotoxic stress. For this, we influenced the apoptotic response and measured the amount of genomic damage expressed as micronucleus formation after treatment with the topoisomerase II inhibitor etoposide. Apoptosis was reduced by the addition of pifithrin (PFT) alpha and enhanced by transient transfection with bcl-2 antisense-oligonucleotide in Bcl-2-overexpressing cells. We used three human lymphoblastoid cell lines with different p53 status (TK6, wild-type p53; WTK1, mutated p53; NH32, p53 double knockout). Under conditions of reduced apoptosis, micronucleus formation was also reduced. When apoptosis was increased, micronucleus formation remained unchanged or was also increased. Overall, we did not find an expected inverse correlation between induction of apoptosis and genomic damage.