Volumetric measurements of the brachial and carotid arteries in dialysis grafts: is there evidence of a subclavian-carotid artery steal syndrome?

Dialysis grafts may lead to major hyperperfusion in the graft arm and to hypertrophic, hypervolemic cardiomyopathy. No data have been published concerning the impact of dialysis grafts on the cerebral perfusion in relation to a potential carotid steal syndrome, possibly causing neurologic or neuropsychologic symptoms. In 30 patients (32 to 74 years old) with dialysis grafts we studied the following hemodynamic parameters in the brachial and common carotid arteries bilaterally: Flow velocities (spectral Doppler sonography), diameter (B-mode) and volume flow (color M-mode) with a color duplex system (Philips P700). Volume flow in the brachial arteries of the graft arm averaged 1032 ml/min (range, 158 to 2854 ml/min) as compared to 42 ml/min (range, 15 to 108 ml/min) in the nongraft arm. Almost identical volume flow data could be seen in both common carotid arteries (418 versus 421 ml/min) with no evidence of reduced flow in the carotid arteries in patients with high flow in the graft arm. A tendency toward higher volume flow in both carotid arteries in patients with high volume flow in the graft arm was noted. We found no evidence of shunt-induced cerebral hypoperfusion. Cerebral autoregulation appears to be patent even with high brachial artery shunt volume.     

Analogs of GnRH-I and GnRH-II inhibit epidermal growth factor-induced signal transduction and resensitize resistant human breast cancer cells to 4OH-tamoxifen

About 50-64% of human breast cancers express receptors for GnRH-I. Direct antiproliferative effects of analogs of GnRH-I on human breast cancer cell lines have been shown. They are at least in part mediated by antagonizing growth promoting effects of estradiol, epidermal growth factor (EGF) or insulin-like growth factor. Recently, expression of a putative receptor for GnRH-II in human tissues was demonstrated. Antiproliferative effects of GnRH-II in human endometrial and ovarian cancer cells were shown not to be mediated through the GnRH-I receptor. Now we demonstrate direct anti-proliferative effects of the GnRH-I analog Triptorelin and the GnRH-II analog [d-Lys(6)]GnRH-II in MCF-7 and T47D human breast cancer cells expressing GnRH-I receptors and putative GnRH-II receptors. Pretreatment with Triptorelin or [d-Lys(6)]GnRH-II blocked EGF-induced autophosphoryla-tion of EGF receptor and activation of mitogen-activated protein kinase (extracellular-signal-regulated kinase 1/2 (ERK1/2)) in these cells. In sublines of MCF-7 and T47D cells, which were developed to be resistant to 4OH-tamoxifen, HER-2/p185 was overexpressed. Pretreatment of these cell lines with Triptorelin or [d-Lys(6)]GnRH-II completely abolished resistance to 4OH-tamoxifen, assessed by 4OH-tamoxifen-induced apoptosis. Analogs of GnRH-I and GnRH-II counteract EGF-dependent signal transduction in human breast cancer cells with expression of receptors for GnRH-I and GnRH-II. Through this mechanism, they probably reverse acquired resistance to 4OH-tamoxifen mediated through overexpression or activation of receptors of the c-erbB family.     

Cell distributions and functions of Toll-like receptor 4 studied by fluorescent gene constructs

Bacterial lipopolysaccharide (LPS) is recognized in mammals by a receptor complex composed of CD14, Toll-like receptor 4 (TLR4), and MD-2. The detailed mechanisms of how TLR4 transmits the signal from the outside to the inside of the cell remain to be elucidated. One way of studying TLR4 signaling mechanisms is to construct chimeras of TLR molecules C-terminally fused to fluorescent proteins and stably express these constructs in cells. Such constructs are functional when transfected into HEK293 epithelial cells. Confocal microscopy of TLR4 expression in live cells demonstrated pronounced expression on the plasma membrane as well in the Golgi apparatus. Studies were performed to clarify whether expression of TLR4 in the Golgi was necessary for LPS stimulation. Rapid recycling of TLR4/CD14/MD-2 complexes between the Golgi and the plasma membrane was a prominent phenomenon. In agreement with other types of plasma membrane receptors, aggregation of TLR4 by immobilized TLR4 antibodies was sufficient to induce signaling. Also, pharmacological disruption of the Golgi did not inhibit LPS induced NF-kappaB activation. Furthermore, LPS stimulation recruited the adapter molecule, MyD88, to the inside of the plasma membrane. Thus, LPS signaling commences on the plasma membrane and is independent of trafficking to the Golgi.     

OutKnocker: a web tool for rapid and simple genotyping of designer nuclease edited cell lines

The application of designer nucleases allows the induction of DNA double-strand breaks (DSBs) at user-defined genomic loci. Due to imperfect DNA repair mechanisms, DSBs can lead to alterations in the genomic architecture, such as the disruption of the reading frame of a critical exon. This can be exploited to generate somatic knockout cell lines. While high genome editing activities can be achieved in various cellular systems, obtaining cell clones that contain all-allelic frameshift mutations at the target locus of interest remains a laborious task. To this end, we have developed an easy-to-follow deep sequencing workflow and the evaluation tool OutKnocker (www.OutKnocker.org), which allows convenient, reliable, and cost-effective identification of knockout cell lines.Advances in targeted genome editing technologies have opened new avenues for addressing challenging questions in the field of life sciences. The recent introduction of designer nucleases such as ZFNs (Carroll 2011), TALENs (Miller et al. 2011), or CRISPR/Cas systems (Jinek et al. 2012; Cong et al. 2013; Mali et al. 2013) allows for highly efficient, flexible, and specific induction of DNA double-strand breaks (DSB) in eukaryotic genomes. DSBs trigger two distinct repair pathways that can be exploited to specifically modify gene architecture (Carroll 2011). While the process of homologous recombination (HR) accurately repairs DSBs using the sister chromatid as a template, nonhomologous end-joining (NHEJ) repair is an error-prone end-joining mismatch repair pathway that frequently leads to genetic alterations (Lieber 2010; Chiruvella et al. 2013). Providing a donor construct with appropriate homology arms as a template, the pathway of DSB-triggered HR can be used to site-specifically introduce heterologous genetic material into cells (Carroll 2011). For example, it is possible to generate gene knockouts in somatic cell lines by introducing marker cassettes with premature stop codons. However, this strategy is time consuming and laborious and therefore not optimal for high-throughput approaches. The DSB-induced NHEJ repair pathway, on the other hand, leads to insertions or deletions (indels) (Lieber 2010) that can result in frameshift mutations and thus loss-of-function phenotypes if located within early coding exons.While in HR-based genome editing approaches marker genes can be introduced to select for the desired genotype starting from a polyclonal cell culture, frameshift mutations induced by NHEJ are difficult to select for unless the editing event provides a survival benefit. To this end, single-cell cloning and subsequent sequencing of the genetic locus is required to obtain cells with the desired gene disruption. Sanger sequencing is most commonly used to identify modified alleles. However, in addition to being costly, this method requires a locus-specific PCR to be subcloned in order to sequence single alleles, and thus is not practical for large-scale projects. Moreover, the ploidy of the genome may vary between cell lines and even between loci, which may require the sequencing of a considerable number of PCR subclones to reliably identify cell clones with all-allelic frameshift mutations. Small benchtop deep sequencing machines can achieve a far greater throughput. Theoretically, even low sequencing capacities are sufficient to analyze hundreds of clones in parallel, without the need to subclone PCR products. However, analysis of deep sequencing data remains challenging and no streamlined workflow has been described that would allow full exploitation of deep sequencing capacities in gene disruption projects.Here we describe OutKnocker, a web-based application that facilitates the analysis of deep sequencing data to identify knockout cells obtained from designer nuclease-mediated genome editing. We aimed at developing an evaluation tool to genotype single-cell clones at a confined genomic region for indel mutations, as they are typically induced by designer nuclease targeting. As such, we established an algorithm that focuses on identifying a single indel event per sequencing read around a predefined target site, while ignoring SNPs or point mutations originated during sequencing. Optionally, our software also allows the detection of specific point mutations introduced by targeted mutagenesis. To fully exploit sequencing capacities, OutKnocker was designed to analyze data of sequencing runs that have been multiplexed to evaluate the same or different genomic target regions in parallel, while only requiring a limited number of unidirectional sequencing reads. OutKnocker is operated from a web browser making it conveniently accessible to any user.     

Results of psychodynamic inpatient psychotherapy in relation to diagnosis

Therapy outcome is analysed according to the main diagnoses in a 5 years' sample of psychodynamic inpatient psychotherapy (n=461) and controlled in a 6-9 months follow-up (n=312). Therapy effects, as measured in a pre-post comparison of the patients' self-rating, are generally good and especially noticeable for affective and anxiety disorders, whereas the effects for somatoform disorders are relatively low. The opposite tendencies are seen in follow-up, i.e. relapses in anxiety and affective disorders, and further improvement in somatoform disorders. A surprisingly positive outcome is shown for personality disorders and especially for severe personality disorders (e.g. Borderline). Regardless of the diagnoses, 80% of the patients show a high degree of satisfaction with their treatment and its outcome.     

Pentoxifyllin Attenuates the Systemic Inflammatory Response Induced During Isolated Limb Perfusion With Recombinant Human Tumor Necrosis Factor-α and Melphalan

Background: Isolated limb perfusion (ILP) with recombinant human tumor necrosis factor- (rhTNF-) and melphalan harbors the risk of septic shock–like syndrome. Pentoxifyllin (PTX) produced a beneficial effect on cytokine response and survival in animal experiments of septic shock, and we were interested to explore its effect during TNF-ILP in humans.Methods: Eighteen consecutive patients underwent TNF-ILP and received PTX (30 mg/kg/day), whereas another 13 consecutive patients did not. PTX was given systemically after the limb extracorporeal circulation was started. Cardiac index, systemic vascular resistance (SVR), and pulmonary vascular resistance were recorded via a Swan-Ganz catheter. Blood levels of TNF-, interleukin-6, procalcitonin, and lipopolysaccharide-binding protein were determined before, during, and after ILP.Results: After reperfusion, systemic levels of TNF- were significantly less increased in the PTX group (peak, 2.8 vs. 1.3 ng/mL; P < .05), as were interleukin-6 values (peak, 68 vs. 22 pg/mL; P < .02) and lipopolysaccharide-binding protein plasma levels (peak, 215 vs. 105 g/mL; P < .03). Differences in cardiac index, SVR, and mean arterial blood pressure were not significantly different. Norepinephrine or dobutamine to maintain SVR was less required in the PTX group.Conclusions: PTX attenuates systemic cytokine production and influences components of the systemic inflammatory response after TNF-ILP. PTX may play a beneficial role in the management of septic shock–like syndrome, particularly in patients with leakage from the ILP circuit.Presented at the 55th Annual Meeting of the Society of Surgical Oncology, Denver, Colorado, March 14–17, 2002.     

Population based quantification of dendrites: evidence for the lack of microtubule-associate protein 2a,b in Purkinje cell spiny dendrites

The high molecular weight isoforms (a and b) of microtubule-associate protein 2 (MAP2a,b) are widely believed to be specific markers for neuronal somata and dendrites. We analyzed and quantified MAP2a,b stained dendrites of the cerebellar molecular layer using a novel approach that segmented and 3D reconstructed them, and the results have been compared with those obtained by other methods, including single-cell reconstruction and analysis of electron micrographs. Our results show that the molecular layer dendritic volume fraction is lower than in the neocortex (10% compared to neocortical 29%). The low total volume fraction of dendrites in the molecular layer is best explained by the majority of the afferents to the dendrites being from the very densely packed parallel fibers, which allows the dendritic fields of individual neurons to be smaller and more compact than in the cerebral cortex. However, the MAP2a,b dendritic volume fraction is even lower (5.2%) than the total volume fraction of dendrites in the molecular layer (10%). Analysis of the material shows that this difference between the two results is due to the unexpected finding that there were few MAP2a,b stained Purkinje cell spiny dendrites.     

Cytogenetisch-karyologische Studien an klinisch behandelten gynäkologischen Tumoren

Zusammenfassung Tumorzellen von acht menschlichen Ovarialcarcinomen und einem Collumcarcinom mit Ascitesbildung wurden nach Behandlung der Patienten mit Hormonen, Cytostatica und Bestrahlung cytogenetisch und karyologisch untersucht. Von den neun Tumoren lagen die Stammzellen von sechs Tumoren im diploiden Bereich der Chromosomenzahlen (37–46). Dabei war die Streuung der Chromosomenzahlen und der Prozentsatz der polyploiden Zellen sehr unterschiedlich. Einzelne Tumoren wurden mehrfach analysiert. Die sich dabei ergebenden Veränderungen waren uncharakteristisch. Die Analyse der Karyogramme der jeweiligen Stammzellen verschiedener Tumoren ergab charakteristische Verschiebungen innerhalb der Chromosomengruppen, die statistisch in gynäkologischen Tumoren offenbar miteinander vergleichbar sind.

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Summary Tumor cells of eight human ovarian carcinomas and a cervical carcinoma with ascites were studied cytogenetically and karyologically after the patients had been treated with hormones, cytostatic agents, and X-irradiation. The stem-cells of six of the tumors were in the diploid range of the chromosome numbers (37–46). In these 6, the distribution of the chromosome numbers and the percentage of polyploid cells were very different. Some tumors were studied several times; the variations obtained thereby were not significant. The analyses of the karyogram of the stem-cells of the various tumors showed characteristic displacements within the groups of chromosomes which in gynecologial tumors apparently are statistically comparable with one another.

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Mit 3 Textabbildungen

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Herrn Prof. K. H. Bauer zum 75. Geburtstag gewidmet.