Reflection of neuroblastoma intratumor heterogeneity in the new OHC-NB1 disease model

Accurate modeling of intratumor heterogeneity presents a bottleneck against drug testing. Flexibility in a preclinical platform is also desirable to support assessment of different endpoints. We established the model system, OHC-NB1, from a bone marrow metastasis from a patient diagnosed with MYCN-amplified neuroblastoma and performed whole-exome sequencing on the source metastasis and the different models and passages during model development (monolayer cell line, 3D spheroid culture and subcutaneous xenograft tumors propagated in mice). OHC-NB1 harbors a MYCN amplification in double minutes, 1p deletion, 17q gain and diploid karyotype, which persisted in all models. A total of 80–540 single-nucleotide variants (SNVs) was detected in each sample, and comparisons between the source metastasis and models identified 34 of 80 somatic SNVs to be propagated in the models. Clonal reconstruction using the combined copy number and SNV data revealed marked clonal heterogeneity in the originating metastasis, with four clones being reflected in the model systems. The set of OHC-NB1 models represents 43% of somatic SNVs and 23% of the cellularity in the originating metastasis with varying clonal compositions, indicating that heterogeneity is partially preserved in our model system.     

Riluzole therapy in cervical dystonia.

We conducted a 6-week open-label pilot study with blinded video rating of riluzole (50 mg twice a day) in six patients with cervical dystonia (CD) refractory to botulinum toxin A and oral pharmacological treatment. The Tsui rating scale served as primary efficacy measure and improved significantly under riluzole (P = 0.002). In three of six patients, the Tsui score improved by more than 30% with a greater 50% reduction in the head tremor/jerk subscore of the Tsui scale. These data suggest that riluzole may be helpful in a subgroup of patients with disabling CD refractory to other therapies.     

The Sperm Acrosome: Immunological Analysis using Specific Polyclonal and Monoclonal Antibodies Directed Against the Outer Acrosomal Membrane of Boar Spermatozoa

An antiserum to the purified porcine outer acrosomal membrane (OAM) was raised in female Balb/c mice and was characterized by means of an indirect ELISA. The hyperimmune serum reacted selectively with the acrosomal cap of the sperm head and showed an extremely good cross reactivity with bull and human spermatozoa when assayed by indirect immunofluorescence. Immunoelectron microscopy using the protein A-gold method further confirmed the specificity of the anti-OAM-antiserum for the OAM. In an effort to identify the OAM antigens recognized by the hyperimmune serum and to analyse the extent of cross reactivity on a molecular level, the SDS-extractable proteins were separated by SDS-PAGE, transblotted and immunoprinted using an 125J-conjugated anti-mouse-antibody. To facilitate functional and structural analysis of distinct OAM-proteins monoclonal antibodies were generated by hybridization of mouse myeloma cells with the splenocytes of female Balb/c mice immunized with the purified OAM. One fusion resulted in about 100 anti-OAM-antibodies secreting hybridoma cultures, of which about 30% showed cross reaction with human and bull spermatozoa. Four stable cell lines were selected for this study secreting antibodies directed against the outer acrosomal membrane of boar spermatozoa. Whereas the polyclonal immune mouse serum stained the entire acrosomal cap, the four hybridoma antibodies generated a patch-work-like immunofluorescence pattern over the acrosome. HPLC-ELISA of the solubilized OAM revealed first information on the nature of the corresponding membrane antigen.     

Transjugular intrahepatic portosystemic shunting (TIPS) with balloon-expandable and self-expanding stents: Technical and clinical aspects after 3 1/2 years’ experience

Purpose To evaluate prospectively our experience with transjugular intrahepatic portosystemic shunt (TIPS) using four different metallic stents. Methods Between November 1991 and April 1995, 57 patients (41 men and 16 women; age 35–72 years, mean 54 years) underwent the TIPS procedure. Techniques for portal vein localization before and during TIPS were fluoroscopy, computed tomography (CT) studies, wedged hepatic venography, arterial portography, and ultrasound. After predilation we deployed balloon-expandable (n=48) and self-expanding (n=45) metallic stents. Fifteen patients underwent variceal embolization. Initial follow-up angiograms (mean 6.9 months, range 3–24 months) were obtained in 39 of these patients. Results Fifty-three patients (93%) had successful TIPS placement. The mean decrease in portal pressure was 42.7%. Besides fluoroscopy, the most helpful techniques for portal vein localization were venography and CT. Residual stenosis (n=1) and late shortening (n=4) of Wallstents resulted in shunt dysfunction. The technical problems encountered with the Palmaz stent resulted from its lack of flexibility. We combined balloon-expandable and self-expanding stents in 12 patients. The 30-day and late follow-up (mean 11.9 months) percutaneous reintervention rates were 11.3% and 64.2%, respectively. There were no clinically significant complications related to the TIPS insertions. Conclusion An ideal stent does not exist for TIPS, and the authors recommend combining a Palmaz stent with a flexible self-expanding stent.     

The influx of Ca and the release of noradrenaline evoked by the stimulation of presynaptic nicotinic receptors of chick sympathetic neurons in culture are not mediated via L-, N-, or P-type calcium channels

We have shown earlier that nicotinic agonists induce the release of noradrenaline from chick sympathetic neurons in culture in two ways: (a) by activating the postsynaptic nicotinic receptors on nerve cell bodies, giving rise to spreading electrical activity and opening of voltage operated calcium channels in neuronal processes; (b) by activating the presynaptic nicotinic receptors on neuronal processes. In the present work, we investigated the contribution of various pathways to the observed Ca²⁺ influx and subsequent noradrenaline release. Sympathetic neurons in culture were stimulated either by the nicotinic agonist dimethylphenylpiperazinium or electrically, in the presence or absence of tetrodotoxin and of specific blockers of calcium or nicotinic channels, and the effects on [Ca²⁺]_i in the area of neuronal processes and on noradrenaline release were measured. Under control conditions, the N-type channel blocker ω-conotoxin (0.1 μmol/1) diminished the release of noradrenaline and the increase of intraterminal Ca²⁺ by 48% and 55%, respectively, whereas the L-type channel blocker (+)Bay k 8644 (1 μmol/1) diminished the release of noradrenaline by 25% and the increase of [Ca²⁺]_i by 39%. The P-type channel blocker ω-agatoxin (0.3 μmol/1) had no effect. The effects of the L-type channel ligands were complex and could only be explained on the assumption that, at high concentrations, these drugs also act as nicotinic antagonists. Tetrodotoxin blocked the Ca²⁺ response evoked by electrical stimulation whereas DMPP applied in the presence of tetrodotoxin still evoked an increase of [Ca²⁺]_i and the release of noradrenaline (27% and 30% of control without tetrodotoxin, respectively). These residual responses were not blocked by any of the calcium channel blockers used or by their combination. Apparently, a substantial part of the influx of Ca²⁺ induced by the activation of presynaptic nicotinic receptors is not carried by the N-, L- or P-type channels and probably occurs directly via the open channels of nicotinic receptors.     

Aktuelles zum Thema Latex-Allergie

Zusammenfassung Die Typ I-Allergien gegen Latex sind in den vergangenen Jahren zu einem zunehmenden berufsdermatologischen Problem geworden, zumal mindestens 10% der Angestellten im Gesundheitswesen betroffen sind. In der Dermatologischen Klinik der Universit?t Erlangen-Nürnberg stieg die Anzahl der j?hrlich diagnostizierten Patienten mit Latexallergien von 1989 bis 1995 auf das 12fache, wobei der Anteil der schweren, generalisierten Formen der Erkrankung von 10,7% (1989/1990) auf 44% (1994/1995) zunahm. Unter den m?glichen Ausl?sern der Latexallergie (wasserl?sliche Proteine mit Molekulargewichten von 2 bis 200 kD) sind mindestens 5 Hauptproteine mit bereits bekannter Prim?rstruktur zu berücksichtigen. Zus?tzlich gibt es Hinweise für Markerproteine, die in bestimmten Risikogruppen geh?uft zur Ausl?sung spezifischer IgE-Antik?rper führen (z.B. 46 kD-Protein in medizinischen Berufen, 14,6 kD- und 27 kD-Proteine bei Kindern mit Spina bifida). Das Vorkommen von Kreuzreaktionen zwischen Latex und unterschiedlichen Früchten (besonders Avocado, Kiwi, Banane, E?kastanie) bei 60 bis 70% der Latexallergiker ist bei der allergologischen Abkl?rung und Beratung dieser Patienten zu beachten. Wesentliche Aspekte der Prophylaxe umfassen die konsequente Umstellung medizinischer Einrichtungen auf ungepuderte Latexhandschuhe mit niedrigem Proteingehalt. Eine Zusammenstellung von OP- und Untersuchungshandschuhen, welche Angaben über die von uns ermittelten Proteinkonzentrationen (modifizierte Lowry-Methode und Hochdruck-Flüssigkeits-Chromatographie, HPLC) enth?lt, soll ein Leitfaden bei der Auswahl allergologisch geeigneter Handschuhe sein. Eingegangen am 10. August 1996 Angenommen am 21. August 1996     

Olfactory Absorption of Insulin to the Brain

A vast number of potent neuropharmaceuticals, many of which are peptides, are excluded from entry into the brain because of the highly selective blood-brain barrier. The fact that a number of drugs have been shown to be transported directly to the central nervous system following application to the olfactory region of the nose is therefore of major interest. In the present study, the feasibility of delivering peptides to the brain via the olfactory route was assessed using insulin as a model peptide. Systemic hyperinsulinemia induced by subcutaneous injection did not significantly reduce the amount of ¹²⁵I-insulin transported from the nose to the brain in vivo, which suggests that the impact of systemic absorption on drug transport is minimal. A linear relationship was seen between insulin accumulation in the brain and the dose applied, without any relevant saturation. Contrary to what was expected, both systemic and olfactory absorption of insulin was enhanced when the pH of the medium was near the isoelectric point. The amount absorbed to the brain was found to be linearly related to the net charge of the molecule (r = -0.61; n = 20). It was concluded that insulin gains access to the central nervous system from the olfactory region of the nose by a nonspecific pathway. The olfactory route may therefore become an important means to deliver peptides to the brain.     

Immediate saccade amplitude disconjugacy induced by unequal images

We tested the ability of normal subjects to make changes in the conjugacy of their saccades. Subjects dichoptically viewed a grid the size of which was 10% larger in one eye. The grids were centred onto a flat screen at 57 cm or 1 m from the subject. Horizontal saccades immediately became larger in the eye viewing the larger grid. For some subjects this disconjugacy persisted even under subsequent monocular viewing. Such persistent changes occurred mainly in the field where the required disconjugacy was divergent for centrifugal saccades, convergent for centripetal saccades. Vertical saccades also developed compensatory disconjugacy; its amplitude was smaller but less variable. To explain these results we propose a fast associative learning mechanism that pairs peripheral disparity with saccades and is capable of producing saccade disconjugacy even in the absence of disparity. For horizontal saccades a secondary conditioning of monocular depth cues by the disparity would also be involved.