The influence of cardiopulmonary bypass operation on the biodistribution of 99mTc-HMPAO-labelled granulocytes – Evaluation in pigs by planar scintigraphy and section-analyses

Aim of the study was to evaluate the influence of an extra corporal perfusion (cardiopulmonary bypass operation - cpb) on activation and biodistribution of Tc-99m labelled granulocytes in pigs with and without inhibition of the granulocytes by a leukocyte inhibition module (LIM). The cpb is often related to an activation of granulocytes resulting in an inflammatory answer. The biological mechanisms are unsolved yet. First trials of our group showed that LIM may inhibit the activation of neutrophils and therefore antagonize a cpb-caused impairment of cardiac function. This study is the continuation of these experiments with a higher number of animals and the focus on scintigraphic imaging. Animals, material, methods: 39 German landrace pigs were subdivided into three groups: group A (control) median sternotomy without cpb, group B with cpb, group C with LIM in addition to cpb. After labelling with Tc-99m-HMPAO autologues granulocytes were reinjected. Subsequently to cpb, the animals underwent scintigraphic imaging. Quantification was performed with ROI evaluation and with tissue samples (section analysis) examined in a well counter. Results:A high uptake of Tc-99m-HMPAO was found in the liver. The count rates in brain, heart, lung, spleen and kidneys were far below. The amount of Tc-99m-activity in the organ related to the half life corrected administered activity [%] was for the tissue samples (group A/B/C): brain 0.01/0.02/0.03; lung 12.1/8.3/11.5; heart 0.35/0.54/0.42; kidney 1.24/0.87/1.02; spleen 4.0/4.0/4.5, liver 16.8/20.9/19.6. The count rates determined by ROI-evaluation of the scintigraphic images related to the total count rate in the image [%] were (group A/B/C): brain 1.1/0.9/1.0; lung 15.6/10.4/12.2; heart 4.0/3.5/3.4; kidney 4.0/2.9/3.2; spleen 7.6/7.7/9.5, liver 23.1/36.7/31.4. A significant difference in the tracer uptake between the groups could neither be detected by scintigraphic imaging nor evaluation of tissue samples. Conclusion: Scintigraphic imaging as well as section analysis showed a comparable biodistribution of the tracer. Therefore, the initial results of our group were not confirmed with a considerably higher number of animals. Neither cpb nor the use of the LIM influenced distribution of Tc-99m-labelled granulocytes in pigs significantly.

Embryonic stem cells express multiple Eph-subfamily receptor tyrosine kinases.

Eph and its homologues form the largest subfamily of receptor tyrosine kinases. Normal expression patterns of this subfamily indicate roles in differentiation and development, whereas their overexpression has been linked to oncogenesis. This study investigated the potential role of Eph-related molecules during very early embryonic development by examining their expression in embryonic stem (ES) cells and embryoid bodies differentiated from ES cells in vitro. By use of a strategy based on reverse transcriptase-mediated PCR, nine clones containing Eph-subfamily sequence were isolated from ES cells. Of these, eight were almost identical to one of four previously identified molecules (Sek, Nuk, Eck, and Mek4). However, one clone contained sequence from a novel Eph-subfamily member, which was termed embryonic stem-cell kinase or Esk. Northern analysis showed expression of Esk in ES cells, embryoid bodies, day 12 mouse embryos, and some tissues of the adult animal. Levels of expression were similar in ES cells and embryoid bodies. By comparison, Mek4 showed no significant transcription in the ES cell cultures by Northern analysis, whereas Eck displayed stronger signals in ES cells than in the embryoid bodies. These results suggest that Eph-subfamily molecules may play roles during the earliest phases of embryogenesis. Furthermore, the relative importance of different members of this subfamily appears to change as development proceeds.

Věstník

Issued by the Třída Filosoficko-historicko-jazykozpytná, 1885-1931; Třída Filosoficko-Historická, 1932-1935; Třída Filosoficko-historicko-filologická, 1936-1952

Repelling class discrimination: ephrin-A5 binds to and activates EphB2 receptor signaling

The interactions between Eph receptor tyrosine kinases and their ephrin ligands regulate cell migration and axon pathfinding. The EphA receptors are generally thought to become activated by ephrin-A ligands, whereas the EphB receptors interact with ephrin-B ligands. Here we show that two of the most widely studied of these molecules, EphB2 and ephrin-A5, which have never been described to interact with each other, do in fact bind one another with high affinity. Exposure of EphB2-expressing cells to ephrin-A5 leads to receptor clustering, autophosphorylation and initiation of downstream signaling. Ephrin-A5 induces EphB2-mediated growth cone collapse and neurite retraction in a model system. We further show, using X-ray crystallography, that the ephrin-A5-EphB2 complex is a heterodimer and is architecturally distinct from the tetrameric EphB2-ephrin-B2 structure. The structural data reveal the molecular basis for EphB2-ephrin-A5 signaling and provide a framework for understanding the complexities of functional interactions and crosstalk between A- and B-subclass Eph receptors and ephrins.