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Annulling gravity during cell culturing triggers various types of cells to change their protein expression in a time dependent manner. We therefore decided to determine gravity sensitive proteins and their period of sensitivity to the effects of gravity. In this study, thyroid cancer cells of the ML-1 cell line were cultured under normal gravity (1 g) or in a random positioning machine (RPM), which simulated near weightlessness for 7 and 11 days. Cells were then sonicated and proteins released into the supernatant were separated from those that remained attached to the cell fragments. Subsequently, both types of proteins were fractionated by free-flow isoelectric focussing (FF-IEF). The fractions obtained were further separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to which comparable FF-IEF fractions derived from cells cultured either under 1 g or on the RPM had been applied side by side. The separation resulted in pairs of lanes, on which a number of identical bands were observed. Selected gel pieces were excised and their proteins determined by mass spectrometry. Equal proteins from cells cultured under normal gravity and the RPM, respectively, were detected in comparable gel pieces. However, many of these proteins had received different Mascot scores. Quantifying heat shock cognate 71 kDa protein, glutathione S-transferase P, nucleoside diphosphate kinase A and annexin-2 by Western blotting using whole cell lysates indicated usefulness of Mascot scores for selecting the most efficient antibodies.

In the diagnosis of coronary artery disease (CAD) with Dobutamine Stress Echocardiography (DSE), regional wall motion abnormalities (RWMA) are assumed to indicate a perfusion deficit.For a more particular examination of RWMAs, we compared simultaneous echo-contrast (Optisone)-enhanced DSE (0-40 microg/kg Dobutamine, 16-segment- model) and MiBi-SPECT in a prospective double-blinded study design in 69 non-selected consecutive patients (44 male, 25 female, age 64+/-12 years). Additionally, all patients were examined by coronary-angiography. The prevalence of significant CAD (stenosis50% lumen diameter) was 52%. DSE had a sensitivity of 78% and a specificity of 66% for the detection of significant CAD with a positive and negative predictive value of 72 and 73%, respectively. Among 28 patients with significant CAD and positive DSE study (true positive), 78% displayed a corresponding perfusion deficit in MiBi-SPECT. Among 11 patients with a positive DSE study but no current significant coronary stenosis (false positive), 82% showed stress-induced RWMAs in the inferior/posterior region, 73% displayed left ventricular hypertrophy, 54% resting-ECG abnormalities and 45% resting-RWMA (3 previous MI, 2 previous CABG surgery). Among 8 patients with negative DSE study but significant coronary stenosis (false negative), 75% had a stenosis of the LCX, 63% displayed resting- WMA, 63% displayed left bundle branch block or ST-segment depression, 50% displayed only peripheral coronary stenosis, and DSE visualization was suboptimal in 38%.This prospective study in non-selected patients shows that the majority of RWMAs in DSE are matched to a perfusion deficit detectable by nuclear imaging. Nevertheless, pre-existing cardiac abnormalities may also lead to stress-induced RWMA not associated with a perfusion deficit or mask a perfusion deficit upon DSE. Particularly in patients with LV hypertrophy, resting-RWMA, bundle branch block or ST segment depression, the predictive value of DSE may, therefore, be limited.

ZusammenfassungZiel: Die Entwicklung neuer Diagnoseverfahren und Implementierung eines modernen Qualitätsmanagements bedingen die permanente Anpassung vorhandener EDVStrukturen an den Arbeitsablauf in einer nuklearmedizinischen Abteilung. Die gesetzliche Pflicht zur Patientendatensicherung und ein schneller Datenzugriff mittels Vernetzung, wird durch ein PACS mit Anschluss an das KIS ermöglicht bzw. wesentlich erleichtert. Ziel dieser Arbeit ist deshalb den Aufbau, die Struktur und Ergebnisse eines derartigen Systems darzustellen und zu bewerten. Methode: Initial wurde der Workflow der Nuklearmedizin analysiert und den einzelnen Systemkomponenten der Abteilung zugeordnet. Die im Klinikum eingesetzte, auf den Standardverfahren SAP R/3 und IS-H bzw. IS-H*med basierende Software zur Patientenverwaltung, wurde entsprechend den Bedürfnissen der Nuklearmedizin angepasst. Die Vernetzung der bildgebenden Systeme erfolgte durch die Integration eines PACS. Im letzten Schritt erfolgte die Anbindung des PACS an das KIS mit der Möglichkeit dem schriftlichen Befund die entsprechenden Bildbefunde anzuhängen. Ergebnisse, Schlussfolgerung: Durch Vernetzung des KIS mit dem nuklearmedizinischen PACS konnte der Workflow wesentlich verbessert werden. Der Datenfluss kann von der Anmeldung, über die Akquisition, bis zum Abruf von schriftlichem und bildlichem Befund transparent nachvollzogen werden. Eine wesentliche Kostenreduktion, sowie ein schnellerer Zugang zu schriftlichem Befund und Bilddaten von Seiten der anfordernden Abteilungen ist festzuhalten. Die Integration von PACS und KIS in den Workflow der Nuklearmedizin dient auch unter Berücksichtigung der kritischen Aspekte, wie permanenter Systempflege oder regelmäßiger Aktualisierung, trotzdem der Effizienzsteigerung und Kostenersparnis. Das Patientenmanagement und die Nachvollziehbarkeit des Datenflusses werden erheblich verbessert.

Here, we report the case of fever of unknown origin (FUO) in a 77-year-old white man. The patient presented with a 3-week history of fever (between 38.5 and 39 degrees C) and general malaise. These symptoms had occurred about five to seven times during the past 30 years, and despite repeated hospitalizations, no diagnosis was made. Physical examination did not reveal any specific signs of infection nor did the patient fulfill the criteria for any rheumatic disease including vasculitides. Blood chemistry showed a greatly elevated C-reactive protein (CRP; 158.2 mg/l) and an erythrocyte sedimentation rate100 mm, indicating an active inflammatory process, and leukocytes were significantly elevated (20,000/mul). Rheumatological parameters showed only nonspecific changes. Finally, a 2-[(18)F]-fluoro-2-deoxy-D: -glucose-positron emission tomography was performed, revealing a markedly enhanced glucose uptake in the ascending aorta and the cardiac valves, indicating vasculitis as the cause of FUO in this patient. Based on this finding, treatment was started with corticosteroids, and 2 days after the initiation of treatment, the patient had normal body temperature, and after 5 days, CRP values had returned to normal. After tapering and final complete removal of steroid treatment, the patient was still free of symptoms, hence no disease-modifying antirheumatic drug therapy was necessary.

SummaryIn this paper we present investigations on a simplified method of quantitative whole body scintigraphy by using a dual head LFOV-gamma camera and a calibration algorithm without the need of additional attenuation or scatter correction. Validation of this approach to the anthropomorphic phantom as well as in patient studies showed a high accuracy concerning quantification of whole body activity (102.8% and 97.72%, resp.), by contrast organ activities were recovered with an error range up to 12%. The described method can be easily performed using commercially available software packages and is recommendable especially for quantitative whole body scintigraphy in a clinical setting.

Using antibody-related methods, we recently found that human thyroid cells express various proteins differently depending on whether they are cultured under normal gravity (1g) or simulated microgravity (s-microg). In this study, we performed proteome analysis in order to identify more gravity-sensitive thyroid proteins. Cells cultured under 1g or s-microg conditions were sonicated. Proteins released into the supernatant and those remaining in the cell fragments were fractionated by free-flow IEF. The fractions obtained were further separated by SDS-gel electrophoresis. Selected gel pieces were excised and their proteins were determined by MS. A total of 235 different proteins were found. Out of 235 proteins, 37 appeared to be first identifications in human thyroid cells. Comparing SDS gel lanes of equally numbered free-flow IEF fractions revealed similar patterns with a number of identical bands if proteins of a distinct cell line had been applied, irrespective of whether the cells had been cultured under 1g or s-microg. Most of the identical band pairs contained identical proteins. However, the concentrations of some types of proteins were different within the two pieces of gel. Proteins that concentrated differently in such pieces of gel are considered as candidates for further investigations of gravitational sensitivity.

Following a severe ischemic injury or myocardial infarction, the extracellular matrix (ECM) of the heart is involved in pathophysiological conditions such as dilatation and cardiac dysfunction. Osteopontin (OPN) has been shown to interact with fibronectin suggesting its possible role in matrix organization, stability and wound healing. There is increased expression of OPN in several tissues in response to injury. Therefore, we tested the hypothesis that acute ischemia (2 h), followed by reperfusion (4 h) may induce early OPN and fibronectin in an isolated hemoperfused working porcine heart model. Twenty hearts were prepared and connected to a perfusion system. After 1 h of perfusion, these hearts were randomized to two groups: ten infarcted (MI, ramus circumflexus) and ten non-infarcted hearts (C). In addition, cardiac fibroblasts derived from infarcted, remote and control myocardium were investigated. In both groups, the heart rate, electrolytes, pH, blood gases, and lactate remained similar. The LVEDP and perfusion pressure of MI hearts increased significantly (P

Apoptosis plays a pivotal role in development, tissue homeostasis, cancer, immune defense, and response to weightlessness. It can be initiated by external signals via death receptors, but may also emerge from mitochondria. We exposed mitochondria-rich thyroid carcinoma cells (ONCO-DG1 cell line) and normal thyroid cells (HTU-5) to conditions of simulated microgravity. After 24 h, 10% of the cancer cells had entered a Fas-dependent apoptotic pathway, but destruction and redistribution of mitochondria, microtubuli disruption, and caspase-3 activation were also detected, demonstrating the activation of extrinsic as well as intrinsic pathways. Furthermore, ONCO-DG1 cells grown on the clinostat showed elevated amounts of Bax, but reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4',6-diamidino-2-phenylindole staining, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase activity and apoptosis. Apoptosis was also detected in normal HTU-5 cells, as demonstrated by electron microscopy, activation of caspase-3, increases in Fas and Bax, and elevation of 85-kDa apoptosis-related cleavage fragments resulting from enhanced poly(ADP-ribose) polymerase activity. Gravitational unloading affects the mitochondria and thereby may trigger apoptosis in thyroid cells subjected to weightlessness by clinorotation.

PURPOSE: An accelerated rate of glucose metabolism mediated by overexpression of key regulatory glycolytic enzymes and glucose transporters is among the most characteristic biochemical marker of malignant transformed cells. In thyroid neoplasms, however, an increased uptake of glucose [measured by 2-[18F]-fluoro-2-deoxy-D-glucose (FDG) and positron emission tomography (PET)] seems to be restricted to more aggressive and high-grade tumors, whereas tumors with favorable prognosis demonstrate no significant tracer uptake. We therefore studied the expression of glucose transporters in thyroid carcinomas with different grades of malignancy. METHODS: Sections of formalin-fixed and paraffin-embedded tissue obtained from 45 patients with thyroid cancer (5 anaplastic, 20 papillary and 20 follicular tumors) were investigated. Polyclonal rabbit antiglucose transporter antibodies, reactive with glucose transporters 1-5 (GLUT1-5), were used after heat pretreatment of the sections. Staining was performed by the avidin-biotin conjugate immunoperoxidase reaction and evaluated semiquantitatively. RESULTS: Expression of GLUT1 transporter on the cell membrane was closely related to the grade of malignancy in thyroid neoplasms (Fisher exact test p < 0.05). All anaplastic tumors showed a high level of GLUT1 expression in the cytoplasm and on the cell membrane. Positive membranous staining in differentiated tumors was detected predominantly in neoplasms with unfavorable prognosis, e.g., in widely invasive follicular or metastatic tumors, whereas low or no immunoreactivity could be seen in well-differentiated tumors or in normal thyroid epithelium. CONCLUSIONS: These data indicate that overexpression of GLUT1 on the cell membrane of thyroid neoplasms is closely related to tumors demonstrating a more aggressive biological behavior. Therefore, determination of GLUT1 expression in thyroid cancer tissue may be a prognostic marker, and FDG-PET may be a helpful technique in identifying patients at a higher risk.

This study focuses on the effects of simulated microgravity (0g) on the human follicular thyroid carcinoma cell line ML-1. Cultured on a three-dimensional clinostat, ML-1 cells formed three-dimensional MCTSs (MCTS diameter: 0.3 +/- 0.01 mm). After 24 and 48 h of clinorotation, the cells significantly decreased fT3 and fT4 secretion but up-regulated the thyroid-stimulating hormone-receptor expression as well as the production of vimentin, vinculin, and extracellular matrix proteins (collagen I and III, laminin, fibronectin, chondroitin sulfate) compared with controls. Furthermore, ML-1 cells grown on the clinostat showed elevated amounts of the apoptosis-associated Fas protein, of p53, and of bax but showed reduced quantities of bcl-2. In addition, signs of apoptosis became detectable, as assessed by terminal deoxynucleotidyl transferase-mediated dUTP digoxigenin nick end labeling, 4', 6-diamidino-2-phenylindole staining, DNA laddering, and 85-kDa apoptosis-related cleavage fragments. These fragments resulted from enhanced 116-kDa poly(ADP-ribose)polymerase (PARP) activity and apoptosis. These observations suggest that clinorotation elevates intermediate filaments, cell adhesion molecules, and extracellular matrix proteins and simultaneously induces apoptosis in follicular thyroid cancer cells. In conclusion, our experiments could provide a regulatory basis for the finding that astronauts show low thyroid hormone levels after space flight, which may be explained by the increase of apoptosis in thyrocytes as a result of simulated 0g.

The present study focuses on the establishment and characterization of a new follicular thyroid carcinoma cell line. The human cell line ML-1 was derived from a dedifferentiated follicular thyroid carcinoma relapse, which progressed despite preceding surgery followed by two radioiodine therapies. More than 90% of the cells of this line express thyroglobulin, chondroitin sulfate, and vimentin antigens, but only about 70% show cytokeratin filaments and a negative surface charge density such as human erythrocytes. More importantly, cells of this line are able to take up iodine and/or glucose both in vitro and in vivo and to secrete thyroglobulin, chondroitin sulfate, and fibronectin into the interstitial space. In addition, triiodothyronine is released constitutively into culture supernatants. Moreover, it is also suitable for xenotransplantation studies because it is tumorigenic in NMRI nude mice in vivo. The cell line forms tumors with follicular structures when transplanted to nude mice. Due to these unique features the ML-1 cell line can be considered as a very suitable test model for pharmacological and cell biological studies. Since chemicals may interfere with the production of thyroid hormones, this cell line represents also a tool for toxicological investigations.

Human tumors frequently present heat shock protein 70 (Hsp70) on their cell membranes, whereas corresponding normal tissues fail to do so. Therefore, an Hsp70 membrane-positive phenotype provided a tumor-specific marker. Moreover, membrane-bound Hsp70 provides a target structure for the cytolytic attack mediated by natural killer (NK) cells. Vitamin A derivatives 13-cis retinoic acid (13-RA) and all-trans retinoic acid (ATRA) and sodium-butyrate (SBU) are known for their redifferentiating capacity. Therefore, we asked the question whether loss in tumorigenicity might be associated with a reduced Hsp70 membrane expression. For our studies we used epithelial colon (CX+/CX-) and thyroid (ML-1) cancer cells, with initially different Hsp70 cell surface expression pattern. After treatment up to 7 weeks with freshly prepared 13-RA, ATRA, and SBU at nonlethal concentrations of 10 microM, 1 microM, and 0.5 mM, respectively, growth morphology, Hsp70 levels, and sensitivity toward Hsp70-specific NK cells were compared with that of untreated tumor cells. Significant growth delay was determined in CX+ tumor cells after 6 weeks treatment with 13-RA. Concomitantly, growth morphology changed from spheroid cell clusters to monolayers. Despite a weak increase in cytosolic Hsp70, the percentage of Hsp70 membrane-positive cells dropped significantly after repeated treatments with 13-RA and ATRA in CX+ and ML-1 but not in CX- tumor cells. Similar results were observed with SBU. Functionally, the decrease in Hsp70 membrane-positive CX+ and ML-1 cells correlated with a reduced sensitivity to lysis mediated by NK cells. In summary, redifferentiating agents predominantly affected Hsp70 membrane-positive tumors. The decrease in Hsp70 membrane positivity correlated with a lower sensitivity to NK lysis, growth delay, and altered growth morphology.