Paul Debbage

Imaging with a Terahertz quantum cascade laser.

We demonstrate bio-medical imaging using a Terahertz quantum cascade laser. This new optoelectronic source of coherent Terahertz radiation allows building a compact imaging system with a large dynamic range and high spatial resolution. We obtain images of a rat brain section at 3.4 THz. Distinct regions of brain tissue rich in fat, proteins, and fluid-filled cavities are resolved showing the high contrast of Terahertz radiation for biological tissue. These results suggest that continuous-wave Terahertz imaging with a carefully chosen wavelength can provide valuable data on samples of biological origin; these data appear complementary to those obtained from white-light images.

Lectin Intravital Perfusion Studies in Tumor-bearing Mice: Micrometer-resolution, Wide-area Mapping of Microvascular Labeling, Distinguishing Efficiently and Inefficiently Perfused Microregions in the Tumor

Intravital lectin perfusion was combined with computer-guided scanning digital microscopy to map the perfused elements of the vasculature in tumor-bearing mice. High-precision composite images (spatial precision 1.3 μm and optical resolution 1.5 μm) were generated to permit exact positioning, reconstruction, analysis, and mapping of entire tumor cross-sections (c. 1 cm in diameter). Collation of these mosaics with nuclear magnetic resonance maps in the same tumor plane identified sites of rapid contrast medium uptake as tumor blood vessels. Digitized imaging after intravital double labeling allowed polychromatic visualization of two different types of mismatched staining. First, simultaneous application of two lectins, each bearing a different fluorochrome, revealed organ-specific differential processing in the microvascular wall. Second, sequential application of two boluses of one lectin, bearing different fluorochromes successively, distinguished between double-labeled microvessels, representing efficiently perfused vascular segments, and single-labeled microvessels, with inefficient or intermittent perfusion. Intravital lectin perfusion images of blood vessels in the vital functional state thus highlighted biologically significant differences in vessel function and served as high-resolution adjuncts to MR imaging.

Hyperphosphorylation of histone H2A.X and dephosphorylation of histone H1 subtypes in the course of apoptosis.

Chromatin condensation paralleled by DNA fragmentation is one of the most important nuclear events occurring during apoptosis. Histone modifications, and in particular phosphorylation, have been suggested to affect chromatin function and structure during both cell cycle and cell death. We report here that phosphate incorporation into all H1 subtypes decreased rapidly after induction of apoptosis, evidently causing a strong reduction in phosphorylated forms of main H1 histone subtypes. H1 dephosphorylation is accompanied by chromatin condensation preceding the onset of typical chromatin oligonucleosomal fragmentation, whereas H2A.X hyperphosphorylation is strongly correlated to apoptotic chromatin fragmentation. Using various kinase inhibitors we were able to exclude some of the possible kinases which can be involved directly or indirectly in phosphorylation of histone H2A.X. Neither DNA-dependent protein kinase, protein kinase A, protein kinase G, nor the kinases driven by the mitogen-activated protein kinase (MAP) pathway appear to be responsible for H2A.X phosphorylation. The protein kinase C activator phorbol 12-myristate 13-acetate (PMA), however, markedly reduced the induction of apoptosis in TNFα-treated cells with a simultaneous change in the phosphorylation pattern of histone H2A.X. Hyperphosphorylation of H2A.X in apoptotic cells depends indirectly on activation of caspases and nuclear scaffold proteases as shown in zVAD-(OMe)-fmk- or zAPF-cmk-treated cells, whereas the dephosphorylation of H1 subtypes seems to be influenced solely by caspase inhibitors. Together, these results illustrate that H1 dephosphorylation and H2A.X hyperphosphorylation are necessary steps on the apoptotic pathway.

Endocytic routes to the Golgi apparatus.

Abstract The endocytic routes of labelled lectins as well as cationic ferritin were studied in cells with a regulated secretion, i.e. pancreatic beta cells, and in constitutively secreting cells, i.e. fibroblasts and HepG2 hepatoma cells, paying particular attention to routes into the Golgi apparatus. Considerable amounts of internalised molecules were taken up into the trans Golgi network (TGN) and into Golgi subcompartments in all three cell types as well as in secretory granules of the pancreatic beta cells. The internalised materials did not pass rapidly the TGN and Golgi stacks, but were still present hours after internalisation, being then particularly concentrated in TGN-elements and in the transmost Golgi cisterna. Endocytosed materials reached forming secretory granules present in the TGN. Further, direct fusion between endocytotic vesicles and mature secretory granules was observed. Golgi subcompartments as well as endocytic TGN containing endocytosed materials were in close apposition to specialised regions of the endoplasmic reticulum. The Golgi apparatus including its parts containing endocytosed materials were transformed into a tubular reticulum upon treatment with the fungal metabolite Brefeldin A. Rarely, internalised material was observed in the lumen of the endoplasmic reticulum, thus providing evidence for an endocytic plasma membrane to endoplasmic reticulum route.

Ossification in the human calcaneus: a model for spatial bone development and ossification

Perichondral bone, the circumferential grooves of Ranvier and cartilage canals are features of endochondral bone development. Cartilage canals containing connective tissue and blood vessels are found in the epiphysis of long bones and in cartilaginous anlagen of small and irregular bones. The pattern of cartilage canals seems to be integral to bone development and ossification. The canals may be concerned with the nourishment of large masses of cartilage, but neither their role in the formation of ossification centres nor their interaction with the circumferential grooves of Ranvier has been established. The relationships between cartilage canals, perichondral bone and the ossification centre were studied in the calcaneus of 9 to 38‐wk‐old human fetuses, by use of epoxy resin embedding, three‐dimensional computer reconstructions and immunhistochemistry on paraffin sections. We found that cartilage canals are regularly arranged in shells surrounding the ossification centre. Whereas most of the shell canals might be involved in the nourishment of the cartilage, the inner shell is directly connected with the perichondral ossification groove of Ranvier and with large vessels from outside. In this way the inner shell canal imports extracellular matrix, cells and vessels into the cartilage. With the so‐called communicating canals it is also connected to the endochondral ossification centre to which it delivers extracellular matrix, cells and vessels. The communicating canals can be considered as inverted ‘internal’ ossification grooves. They seem to be responsible for both build up intramembranous osteoid and for the direction of growth and thereby for orientation of the ossication centre.

Entwicklung und Anwendung dynamischer MRT-Messungen zur Evaluierung von Perfusionsveränderungen bei Rektumkarzinomen unter Bestrahlung in der klinischen Routine Erste Ergebnisse

Ziel: Entwicklung und Anwendung dynamischer Magnetresonanztomographiemessungen zur Erhebung von Perfusionsparametern bei Rektumkarzinomen unter Bestrahlung in der klinischen Routine. Patienten und Methode: Bei Rektumkarzinompatienten (n = 8), die sich einer präoperativen kombinierten Radiochemotherapie unterzogen, wurden Perfusionsdaten erhoben. An einem 1,5-Tesla-Ganzkörperkernspintomographen wurden ultraschnelle T1-Mapping-Sequenzen zum Erhalt von T1-Maps mit Intervallen von 14 und 120 Sekunden implementiert. Die Meßzeit der dynamischen Messungen betrug 40 Minuten. Die Maßschicht (Schichtdicke 5mm) wurde so gewählt, daß sowohl Tumor als auch arterielle Gefäße dargestellt wurden. Gadolinium-DTPA-(Gd-DTPA-)Konzentrations-Zeit-Kurven wurden nach einem prolongierten Bolus im arteriellen Blut und im Tumor berechnet. Die angewendete Methode erlaubte eine räumliche Auflösung von 2 × 2 × 5 mm und eine zeitliche Auflösung von 14 Sekunden. Die Meßdaten wurden vor und in konstanten Intervallen während Therapie erhoben. Ergebnisse: Die räumliche und zeitliche Auflösung der T1-Maps war ausreichend, um Areale mit unterschiedlicher Kontrastmittelkinetik innerhalb des Tumors zu erfassen sowie die großen Beckenarterien sicher zu identifizieren. Bei sechs Patienten konnten Gd-DTPA-Konzentrationskurven im Tumor unter Therapie erhoben werden. Der Perfusionsindex (Pi) versus Strahlendosis zeigte eine signifikante Zunahme in der ersten oder zweiten Woche der Bestrahlung, bevor er entweder kontinuierlich absank oder nach anfänglichem Abfall einen erneuten Anstieg aufwies. Der durchschnittliche Pi-Ausgangswert betrug 0,16 (±0,049), das durchschnittliche Pi-Maximum war 0,23 (± 0,058). Die relativen Perfusionsveränderungen betrugen zwischen 20 und 83%. Schlußfolgerung: Unsere Ergebnisse zeigen, daß sich die verwendete Methode zur Erfassung von Perfusionsparametern unter Bestrahlung eignet und in der klinischen Routine anwendbar ist. In der Zukunft könnte mittels der gewonnenen Daten eine individualisierte tumor- und perfusionsangepaßte Therapieoptimierung bei kombinierter Radiochemotherapie durchgeführt werden.Purpose: This study was aimed at measuring microcirculatory parameters and contrast medium accumulation within the rectal carcinoma during fractionated radiotherapy in the clinical setting. Materials and Methods: Perfusion data were observed in patients with rectal carcinoma (n = 8) who underwent a properative combined chemo/radiotherapy. To acquire perfusion data, an ultrafast T1 mapping sequence was carried out on a 1.5-Tesla whole body imager to obtain T1 maps at intervals of 14 or 120 seconds. The overall measurement time was 40 minutes. The transaxial slice thickness (5mm) was chosen in such a way that both arterial vessels and the tumor could be clearly identified. The gadolinium-DTPA (Gd-DTPA) concentration time curve was evaluated for arterial blood and tumor after intravenous constant rate infusion. The method allows a spatial resolution of 2 × 2 × 5 mm and a temporal resolution of 14 seconds. Patients underwent MR imaging before and at constant intervals during fractionated radiotherapy. Results: Spatial and temporal resolution of dynamic T1 mapping was sufficient to reveal varying CM accumulation levels within the tumor and to identify the great arteries in the pelvis. In 6 patients Gd-DTPA concentration-time-curves were evaluated within the tumor during radiation. Pi index of Gd-DTPA versus radiation dose showed a significant increase in the first or second week of treatment, then either returned slowly to pretreatment level or a renewed increase was observed. The average Pi-value at the beginning was 0.16 (±0.049), reaching highest level of 0.23 (±0.058). In all groups the rise from the Pi-value to the Pi-maximum was statistically significant. The relative increase in perfusion ranged between 20 to 83%. Conclusion: The results show, that the ultrafast MR-technique described above provide a suitable tool for monitoring tumor microcirculation during therapeutic interventions and offers the potential for an individualized optimization of therapeutic procedures.

5000-year-old myelin: uniquely intact in molecular configuration and fine structure

We are indebted to W. Platzer, O. Gaber and K.H. Kunzel (Innsbruck) for tissue sampling, B. Afzelius and B. Fundin (Stockholm), H. Lassmann and R. Valenta (Vienna), G. Rutter (Hamburg), M. Pavelka, W. Pfaller, W. Schmidt and G. Sperk (Innsbruck) for helpful discussions, and C. Huckhagel, K. Gutleben and R. Haring for technical assistance.