Attila Tárnok

Increase of Circulating Endothelial Progenitor Cells in Patients with Coronary Artery Disease After Exercise-Induced Ischemia

Objectives—The concept of neovascularization in response to tissue ischemia has been extended by the finding of postnatal vasculogenesis initiated by endothelial progenitor cells (EPCs). The aim of this study was to analyze whether a maximal stress test in patients with coronary artery disease (CAD) increases the number of circulating EPCs. Methods and Results—Blood concentration of EPCs was analyzed by FACS and cell culture assay in CAD patients with (n=16) or without (n=12) exercise-induced myocardial ischemia and in healthy subjects (n=11) for up to 144 hours after maximal stress test. Plasma concentrations of vascular endothelial growth factor (VEGF), basic fibroblast growth factor, tumor necrosis factor-&agr;, and granulocyte macrophage-colony stimulating factor were determined by ELISA. EPCs increased significantly in ischemic patients, with a maximum after 24 to 48 hours (cell culture: 3.3±0.5-fold increase; FACS: 3.1±0.6-fold increase) and returned to baseline within 72 hour. In nonischemic patients and healthy subjects, no EPC increase was detectable. VEGF levels in ischemic patients increased significantly after 2 to 6 hours (maximum after 2 hours; 4.0±1.1-fold increase) and no change was observed in nonischemic patients and healthy subjects; &Dgr;VEGF and &Dgr;EPC correlated significantly (r =0.66). Conclusions—Patients with symptomatic CAD respond to a single episode of exercise-induced myocardial ischemia with a time-dependent increase in circulating EPCs. This increase may be related to and preceded by an increase in plasma VEGF.

Aneuploidy and DNA Replication in the Normal Human Brain and Alzheimer's Disease

Reactivation of the cell cycle, including DNA replication, might play a major role in Alzheimers disease (AD). A more than diploid DNA content in differentiated neurons might alternatively result from chromosome mis-segregation during mitosis in neuronal progenitor cells. It was our objective to distinguish between these two mechanisms for aneuploidy and to provide evidence for a functional cell cycle in AD. Using slide-based cytometry, chromogenic in situ hybridization, and PCR amplification of alu-repeats, we quantified the DNA amount of identified cortical neurons in normal human brain and AD and analyzed the link between a tetraploid DNA content and expression of the early mitotic marker cyclin B1. In the normal brain, the number of neurons with a more than diploid content amounts to ∼10%. Less than 1% of neurons contains a tetraploid DNA content. These neurons do not express cyclin B1, most likely representing constitutional tetraploidy. This population of cyclin B1-negative tetraploid neurons, at a reduced number, is also present in AD. In addition, a population of cyclin B1-positive tetraploid neurons of ∼2% of all neurons was observed in AD. Our results indicate that at least two different mechanisms need to be distinguished giving rise to a tetraploid DNA content in the adult brain. Constitutional aneuploidy in differentiated neurons might be more frequent than previously thought. It is, however, not elevated in AD. In addition, in AD some neurons have re-entered the cell cycle and entirely passed through a functional interphase with a complete DNA replication.

Microfluidic impedance‐based flow cytometry

Microfabricated flow cytometers can detect, count, and analyze cells or particles using microfluidics and electronics to give impedance‐based characterization. Such systems are being developed to provide simple, low‐cost, label‐free, and portable solutions for cell analysis. Recent work using microfabricated systems has demonstrated the capability to analyze micro‐organisms, erythrocytes, leukocytes, and animal and human cell lines. Multifrequency impedance measurements can give multiparametric, high‐content data that can be used to distinguish cell types. New combinations of microfluidic sample handling design and microscale flow phenomena have been used to focus and position cells within the channel for improved sensitivity. Robust designs will enable focusing at high flowrates while reducing requirements for control over multiple sample and sheath flows. Although microfluidic impedance‐based flow cytometers have not yet or may never reach the extremely high throughput of conventional flow cytometers, the advantages of portability, simplicity, and ability to analyze single cells in small populations are, nevertheless, where chip‐based cytometry can make a large impact.

Phenotypes of stem cells from diverse origin

Stem cells have turned into promising tools for studying the mechanisms of development, regeneration, and for cell therapy of various disorders. Stem cells are found in the embryo and in most adult tissues participating in endogenous tissue regeneration. They are capable of autorenovation, often maintain their multipotency of differentiation into various tissues of their germ line and are, therefore, ideal candidates for cellular therapy taken that they can be unequivocally identified and isolated. In this review, we report stem cell marker expression used for identification of various stem cell lineages, including very small embryonic stem cells, neural, hematopoietic, mesenchymal, epithelial and limbal epithelial stem cells, endothelial progenitor cells, supra‐adventitial adipose stromal cells, adipose pericytes, and cancer stem cells. These cells usually cannot be distinguished by a single stem cell marker, because their expression partially overlaps between lineages. Recent advances in flow cytometry allowing the simultaneous detection of various markers have facilitated stem cell identification for clinical diagnosis and research. So far experimental evidence suggests the existence of cells with different properties, i.e., the capability to different in various cell types. Several studies indicate that expression of classical markers for stem cell classification, such as CD34, CD45, and CD133, may differ between the virtually same stem and progenitor cells, i.e., endothelial progenitor or mesenchymal stem cells, when they were obtained from different tissues. This finding raises questions whether phenotypic differences are due to the source or if it is only caused by different isolation and experimental conditions.

Approaching clinical proteomics: current state and future fields of application in fluid proteomics

Recent developments in proteomics technology offer new opportunities for clinical applications in hospital or specialized laboratories including the identification of novel biomarkers, monitoring of disease, detecting adverse effects of drugs, and environmental hazards. Advanced spectrometry technologies and the development of new protein array formats have brought these analyses to a standard, which now has the potential to be used in clinical diagnostics. Besides standardization of methodologies and distribution of proteomic data into public databases, the nature of the human body fluid proteome with its high dynamic range in protein concentrations, its quantitation problems, and its extreme complexity present enormous challenges. Molecular cell biology (cytomics) with its link to proteomics is a new fast moving scientific field, which addresses functional cell analysis and bioinformatic approaches to search for novel cellular proteomic biomarkers or their release products into body fluids that provide better insight into the enormous biocomplexity of disease processes and are suitable for patient stratification, therapeutic monitoring, and prediction of prognosis. Experience from studies of in vitro diagnostics and especially in clinical chemistry showed that the majority of errors occurs in the preanalytical phase and the setup of the diagnostic strategy. This is also true for clinical proteomics where similar preanalytical variables such as inter‐ and intra‐assay variability due to biological variations or proteolytical activities in the sample will most likely also influence the results of proteomics studies. However, before complex proteomic analysis can be introduced at a broader level into the clinic, standardization of the preanalytical phase including patient preparation, sample collection, sample preparation, sample storage, measurement, and data analysis is another issue which has to be improved. In this report, we discuss the recent advances and applications that fulfill the criteria for clinical proteomics with the focus on cellular proteomics (cytoproteomics) as related to preanalytical and analytical standardization and to quality control measures required for effective implementation of these technologies and analytes into routine laboratory testing to generate novel actionable health information. It will then be crucial to design and carry out clinical studies that can eventually identify novel clinical diagnostic strategies based on these techniques and validate their impact on clinical decision making.

Cytometric Bead Array to Measure Six Cytokines in Twenty-Five Microliters of Serum

Infections and sepsis are among the most common reasons for neonatal morbidity and mortality. Early diagnosis is difficult because clinical presentation is highly variable and signs are often subtle and common to a variety of conditions. Among the proposed early indicators of infection and sepsis are serum concentrations of interleukin (IL)-6, IL-8, and IL-10. It is believed that IL-8 is a sensitive indicator of infection and that high concentrations of IL-6 and IL-10 are indicators of sepsis and predictors of mortality (1)(2)(3). The concentrations of each of these cytokines in serum vary by several orders of magnitude (1)(2)(3). Literature-reported cutoff values for IL-8 are >70 ng/L (2) or >18 ng/L (1) for infection, and values >10 000 ng/L have been reported (1). IL-6 >175 ng/L is predictive of sepsis, and values >747 ng/L are predictive for pneumonia (3). IL-6 is also believed to be predictive of necrotizing enterocolitis (3). IL-10 >420 ng/L correlates with neonatal death (3). The ELISAs commonly used for cytokine detection require 50–100 μL of serum (∼100–200 μL of peripheral blood in the neonate) per cytokine. To determine the stage of an infection, measurement of several cytokines at multiple time points can be of importance (3). Combining pro- and antiinflammatory cytokines in a single assay yields an overall view on the patient’s inflammatory status; may allow differentiation among infection, sepsis, and enterocolitis; and thus may improve diagnostic accuracy. In neonates, however, particularly preterm neonates, such combined measurements are often hampered by lack of sufficient obtainable blood (1). Furthermore, although the ELISAs are adequate for measuring these cytokines, they often require multiple dilutions to cover a wide concentration range because …

Polychromatic (eight-color) slide-based cytometry for the phenotyping of leukocyte, NK, and NKT subsets.

Natural killer (NK) and NK T (NKT) cells are important in innate immune defense. Their unequivocal identification requires at least four antigens. Based on the expression of additional antigens, they can be further divided into functional subsets. For more accurate immunophenotyping and to describe multiple expression patterns of leukocyte subsets, an increased number of measurable colors is necessary. To take advantage of the technologic features offered by slide‐based cytometry, repeated analysis was combined with sequential optical‐filter changing.

In Vivo Flow Cytometry: A Horizon of Opportunities

Flow cytometry (FCM) has been a fundamental tool of biological discovery for many years. Invasive extraction of cells from a living organism, however, may lead to changes in cell properties and prevents studying cells in their native environment. These problems can be overcome by use of in vivo FCM, which provides detection and imaging of circulating normal and abnormal cells directly in blood or lymph flow. The goal of this review is to provide a brief history, features, and challenges of this new generation of FCM methods and instruments. Spectrum of possibilities of in vivo FCM in biological science (e.g., cell metabolism, immune function, or apoptosis) and medical fields (e.g., cancer, infection, and cardiovascular disorder) including integrated photoacoustic‐photothermal theranostics of circulating abnormal cells are discussed with focus on recent advances of this new platform.

Hyperchromatic cytometry principles for cytomics using slide based cytometry

Polychromatic analysis of biological specimens has become increasingly important because of the emerging new fields of high‐content and high‐throughput single cell analysis for systems biology and cytomics. Combining different technologies and staining methods, multicolor analysis can be pushed forward to measure anything stainable in a cell. We term this approach hyperchromatic cytometry and present different components suitable for achieving this task. For cell analysis, slide based cytometry (SBC) technologies are ideal as, unlike flow cytometry, they are non‐consumptive, i.e. the analyzed sample is fixed on the slide and can be reanalyzed following restaining of the object.

Pediatric cardiac surgery with cardiopulmonary bypass: pathways contributing to transient systemic immune suppression.

Cardiovascular surgery with cardiopulmonary bypass (CPB) can lead to postoperative complications like postpericardiotomy syndrome (PPS), capillary leak syndrome, or multiple organ failure. In children, PPS morbidity is up to 30%, and intra- and immediate postoperative mortality is up to 4%. For these complications, the CPB is made responsible. Its etiology is not yet clarified in detail, but is thought to be of immunologic origin. The exact knowledge of these reactions is crucial for the selection of treatment strategies. The immune response to CPB surgery in children comprises of a cascade of pro- and anti-inflammatory events. Proinflammatory responses are indicated by the release of interleukin (IL)-6 and IL-8, and the activation of alternative complement pathway. This reaction is mainly a response to surgical trauma and medication and only activation of the alternative complement pathway is CPB specific. Antiinflammatory response during CPB surgery is serologically indicated by the systemic release of the immunosuppressive cytokine IL-10 already before that of proinflammatory cytokines. CPB surgery induces population shifts of the leukocyte subsets, changes their degree of activation, and contributes to the phenotype of a peripheral immune suppression. Circulating neutrophils are selectively filtered and inactivated. T-helper (Th) cells shift transiently to the Th2 phenotype, indicating the prevalence for a humoral immune response. These alterations start immediately after the onset of the CPB. Increased immunosuppression may be involved in PPS development and may be linked to an allergic/atopic predisposition. A generalized model of the immune sequela to pediatric cardiovascular surgery with CPB is drawn. CPB induces a systemic transient anti-inflammatory response by elimination of activated cells, by compensatory reaction to local, systemically not observable, proinflammatory responses, by IL-10 release, by anesthetics and medication, and by leukocyte extravasation. The subsequent proinflammatory reaction is the reaction to surgical trauma modulating the anti-inflammatory reaction. Possible therapeutic consequences of these findings may include treatment strategies that modulate the anti-inflammatory response. More studies are needed to test this hypothesis.