Increasing the throughput of label-free cell assays to study the activation of G-protein-coupled receptors by using a serial agonist exposure protocol.

Label-free, holistic assays, monitoring, for example, the impedance of cells on electrodes, are gaining increasing popularity in the evaluation of G-protein-coupled receptor (GPCR) ligands. It is the strength of these approaches to provide the integrated cellular response non-invasively, highly automated and with a device-dependent time resolution down to several milliseconds. With an increasing number of samples to be studied in parallel, the available time resolution is, however, reduced and the cost for the disposable sensor arrays may become limiting. Inspired by protocols from organ pharmacology, we investigated a simple serial agonist addition assay that circumvents these limitations in impedance-based cellular assays. Using a serial addition of increasing concentrations of a GPCR agonist while continuously monitoring the sample's impedance, we were able to establish a full concentration-response curve for the endogenous agonist histamine on a single layer of U-373 MG cells endogenously expressing the histamine 1 receptor (H1R). This approach is validated with respect to conventional, parallel agonist addition protocols and studies using H1R antagonists such as mepyramine. Applicability of the serial agonist addition assay was shown for other GPCRs known for their signaling via one of the canonical G-protein pathways, Gq, Gi/0 or Gs as well. The serial agonist addition protocol has the potential to further strengthen the output of label-free analysis of GPCR activation.

[TILGen study-immunological targets in patients with breast cancer : Influence of tumor-infiltrating lymphocytes]. / TILGen-Studie ­ Immunologische Angriffspunkte bei Patientinnen mit Brustkrebs : Einfluss der tumorinfiltrierenden Lymphozyten.

BACKGROUND: The interaction of our immune system with breast cancer (BC) cells prompted the investigation of tumor-infiltrating lymphocytes (TILs) and targeted, tumor antigen-specific immunotherapy. OBJECTIVES: Correlation between TILs and pathological complete response (pCR) after neoadjuvant systemic therapy (NACT). Tumor-specific antigens (TSAs) in HER2+ and triple negative BC and establishment of TSA-specific therapies within the interdisciplinary TILGen study. METHODS: Illustration of the TILGen study design. Assessment of TILs and correlation with pCR within this BC study. RESULTS: pCR was achieved in 38.4% (56/146) and associated with estrogen receptor/progesterone receptor negative (ER-/PR-) and HER2+ tumors. Lymphocytic predominant BC (LPBC) was found in 16.4% (24/146), particularly in ER-/PR- (ER-: 27.3% vs. ER+: 9.9%, PR-: 22.3% vs. PR+: 8.2%), large, and poorly differentiated BC. TILs were significantly correlated with pCR in multivariate analysis. In LPBC, pCR was achieved in 66.7%, whereas it was 32.8% in non-LPBC. CONCLUSIONS: First results confirm the influence of the human immune system on the response to NACT in HER2+ and triple negative BC. TSA-specific immunotherapy might improve the outcome in BC patients but there is an urgent need for comprehensive studies to further investigate this issue.

Cell retention by encapsulation for the cultivation of Jurkat cells in fixed and fluidized bed reactors.

Jurkat cells are accepted model cells for primary human T lymphocytes, for example, in medical research. Their growth to tissue-like cell densities (up to 100 × 10(6) cells/mLcapsule ) in semi-permeable (molecular weight cut off <10,000 Da) sodium cellulose sulfate/poly(diallyldimethylammonium chloride) polyelectrolyte capsules has previously been shown by us [Werner et al. (2013). Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells. Biotechnol Prog 29(4): 986-993]. Herein, we demonstrate that encapsulation can be used to retain the cells in continuously operated bioreactors, which opens new possibilities for research, for example, the use of Jurkat cells in pulse response experiments under steady state conditions. Two reactor concepts are presented, a fluidized and a fixed bed reactor. A direct comparison of the growth kinetics in batch and repeated batch spinner cultivations, that is, under conditions where both encapsulated and non-encapsulated cells can be cultivated under otherwise identical conditions, showed that maximum specific growth rates were higher for the encapsulated than for the non-encapsulated cells. In the subsequent batch and repeated batch bioreactor experiments (only encapsulated cells), growth rates were similar, with the exception of the fixed bed batch reactor, where growth kinetics were significantly slower. Concomitantly, a significant fraction of the cells towards the bottom of the bed were no longer metabolically active, though apparently not dead. In the repeated batch fluidized bed reactor cellular division could be maintained for more than two weeks, albeit with a specific growth rate below the maximum one, leading to final cell densities of approximately 180 × 10(6) cell/gcapsule . At the same time, the cell cycle distribution of the cells was shifted to the S and G2/M phases.

[Lid reconstruction: functional and aesthetic aspects]. / Lidrekonstruktion: funktionelle und ästhetische Aspekte.

There are numerous different procedures for eyelid reconstruction, one of them being the transfer of pedicled full eyelid flaps. With regard to the increasing demands of the patients the so-called sandwich techniques in most cases offer the chance to get the best functional and aesthetic result in a individual situation. Some of these techniques are presented here.

Pharmacological profile of 2-bromoterguride at human dopamine D2, porcine serotonin 5-hydroxytryptamine 2A, and α2C-adrenergic receptors, and its antipsychotic-like effects in rats.

Dopaminergic, serotonergic, and adrenergic receptors are targets for therapeutic actions in schizophrenia. Dopamine D2 receptor partial agonists such as aripiprazole represent a treatment option for patients with this severe disorder. The ineffectiveness of terguride, another D2 receptor partial agonist, in treating schizophrenia was recently attributed to its considerably high intrinsic activity at D2 receptors. In this study, we used functional assays for recombinant D2 receptors and native 5-hydroxytryptamine 2A (5-HT2A), α2C-adrenergic, and histamine H1 receptors to compare the pharmacological properties of terguride and three of its halogenated derivatives (2-chloro-, 2-bromo-, 2-iodoterguride) with those of aripiprazole. Subsequently, we studied the antidopaminergic effects of 2-bromoterguride using amphetamine-induced locomotion (AIL). Its influence on spontaneous behavior was tested in the open field. Extrapyramidal side effect (EPS) liability was evaluated by catalepsy test. In a guanosine 5'-O-(3-[(35)S]thio)triphosphate ([(35)S]GTPγS) binding assay, 2-chloro-, 2-bromo-, and 2-iodoterguride produced intrinsic activities at human D2short (hD2S) receptors that were half as high as the intrinsic activity for terguride; aripiprazole lacked agonist activity. 2-Bromoterguride and aripiprazole activated D2S receptor-mediated inhibition of cAMP accumulation to the same extent; intrinsic activity was half as high as that of terguride. All compounds tested behaved as antagonists at human D2long/Gαo (hD2L/Gαo) receptors. Compared with aripiprazole, terguride and its derivatives displayed higher affinity at porcine 5-HT2A receptors and α2C-adrenoceptors and lower affinity at H1 receptors. 2-Bromoterguride inhibited AIL and did not induce catalepsy in rats. Because of its in vitro and in vivo properties, 2-bromoterguride may be a strong candidate for the treatment of schizophrenia with a lower risk to induce EPS.

Use of the mitochondria toxicity assay for quantifying the viable cell density of microencapsulated jurkat cells.

The mitochondria toxicity assay (MTT assay) is an established method for monitoring cell viability based on mitochondrial activity. Here the MTT assay is proposed for the in situ quantification of the living cell density of microencapsulated Jurkat cells. Three systems were used to encapsulate the cells, namely a membrane consisting of an interpenetrating polyelectrolyte network of sodium cellulose sulphate/poly(diallyldimethylammonium chloride) (NaCS/PDADMAC), a calcium alginate hydrogel covered with poly(L-lysine) (Ca-alg-PLL), and a novel calcium alginate-poly(ethylene glycol) hybrid material (Ca-alg-PEG). MTT results were correlated to data obtained by the trypan blue exclusion assay after release of the cells from the NaCS/PDADMAC and Ca-alg-PLL capsules, while a resazurin-based assay was used for comparison in case of the Ca-alg-PEG material. Analysis by MTT assay allows quick and reliable determination of viable cell densities of encapsulated cells independent of the capsule material. The assay is highly reproducible with inter-assay relative standard deviations below 10%.

[Congenital ptosis]. / Kongenitale Ptosis.

Simple congenital ptosis is the most common form, characterised by fatty dystrophy and fibrosis of the levator muscle, which leads to a restricted elevation in upgaze and a lid lag in downgaze. In the complicated form additional changes will be found: fibrosis of extraocular muscles, synkinesia or anomalies of the lids. Moreover, congenital ptosis is relatively often associated with refractive errors and/or disturbance of the binocular vision. Therefore a careful examination is necessary. Therapeutic management includes first prevention of amblyopia either by occlusion or by early surgical intervention. The choice of operation depends on the grade of ptosis, the actual levator function and the presence of lid anomalies as well.

Synthesis, biological evaluation and radiolabelling by 18F-fluoroarylation of a dopamine D3-selective ligand as prospective imaging probe for PET.

Radical (18)F-fluoroarylation with fluorine-18-labelled arenediazonium chlorides has been successfully applied to the radiochemical synthesis of the dopamine D(3)-selective ligand SH 317 ([(18)F]8). SH 317 has been evaluated as a new PET ligand candidate by in vivo experiments.

Tissue distribution of radioiodinated FAUC113: assessment of a pyrazolo(1,5-a) pyridine based dopamine D4 receptor radioligand candidate.

AIM: Disturbances of the D4 receptor subtype have been implicated in the genesis of a broad range of psychiatric disorders. In order to assess the suitability of a radioiodinated analogue of the D4-selective ligand FAUC 113 for tracer studies in vivo, we investigated the in-vivo stability, biodistribution and brain-uptake of 7-(131)I-FAUC 113 in Sprague-Dawley rats. METHODS: Radiolabelling was carried out with high radiochemical yield and specific activity. After intravenous injection, blood and tissue samples, taken at designated time intervals, were collected for analysis. Analyses of metabolites were performed by radio-hplc and radio-tlc. For in-vivo evaluation, sagittal cryo-sections of the rat brain were investigated by in-vitro and ex-vivo autoradiography on a mu-Imager system. RESULTS: 7-(131)I-FAUC 113 was rapidly cleared from blood. Highest uptake was observed in kidney (0.603 +/- 0.047% ID/g, n = 4) and liver (0.357 +/- 0.070% ID/g, n = 4) at 10 min p.i.; 7-(131)I-FAUC 113 displayed rapid uptake (0.21-0.26% ID/g) and fast clearance in various brain regions consistent with the determined logP-value of 2.36 +/- 0.15 (n = 4). In-vivo stability of 7-(131)I-FAUC 113 was confirmed in the frontal cortex (>95%). Ex-vivo autoradiography revealed a frontal cortex-to-cerebellum ratio of 1.57 +/- 0.13 at 10 min p.i. (n = 6). Coinjection with L-750667 could not suppress any putative specific binding of 7-(131)I-FAUC 113. In-vitro autoradiography using authentic 7-iodo-FAUC 113 or L-750667 failed to cause significant displacement of the radioligand. CONCLUSIONS: Radioiodinated FAUC 113 does not allow imaging of D4 receptors in the rat brain in vivo nor in vitro. Further work should aim at the development of selective dopamine D4 radioligands with improved tracer characteristics, such as receptor affinity and subtype selectivity, specific activity or blood-brain barrier permeability.

Dopaminergic 7-aminotetrahydroindolizines: ex-chiral pool synthesis and preferential D3 receptor binding.

Starting from both isomers of enantiopure asparagine, heterocyclic bioisosteres of the preferential dopamine D3 receptor agonist (R)-7-OH-DPAT were investigated when SAR studies led to the 3-formyl substituted aminoindolizine (S)-1e (FAUC 54) displaying a K(i) value of 6.0 nM for the high affinity D3 binding site. In contrast, D3 affinity of the enantiomer (R)-1e was 300 fold lower.

Benzamide bioisosteres incorporating dihydroheteroazole substructures: EPC synthesis and SAR leading to a selective dopamine D4 receptor partial agonist (FAUC 179).

Conformationally restricted benzamide bioisosteres were investigated when the chiral phenyldihydroimidazole derivative 4e (FAUC 179) showed strong and highly selective dopamine D4 receptor binding (K(i)high=0.95nM). Mitogenesis experiments indicated partial agonist properties (42%). EPC syntheses of the target compounds of type 4 were performed starting from alpha-amino acids.

[Transplantation of autologous oral mucosa in the treatment of a symblepharon in Wegener's disease--a case report]. / Schleimhautplastik mit autologer oraler Mukosa zur Therapie des Symblepharon bei Morbus Wegener--eine Kasuistik.

BACKGROUND: Wegener's disease, an immune vasculitis, is characterized by granulomata and vasculitis of small and large vessels. Ocular manifestations are observed in approximately half of all cases. PATIENT: We report on a 58-year-old male with severe generalized Wegener's disease which was first diagnosed in 1986. The involvement of the central nervous system became clinically manifest by a stroke, of the kidneys by incipient renal insufficiency, and of the larynx by recurrent subglottic stenosis of the trachea. The first ocular involvement, a conjunctivitis, was observed in 1988. Despite systemic immuno-suppressive therapy, local conservative therapy and repeated surgical procedures, his vision decreased bilaterally from 60/60 to hand movement (OD) and light perception only (OS) during the following years because of a massive bilateral symblepharon. Transplantation of autologous oral mucosa to the conjunctiva led to a persistent increase of vision and a good cosmetic result. CONCLUSIONS: We conclude that in patients with Wegener's disease, who have developed a symblepharon despite intensive therapy, the transplantation of oral mucosa can produce a sufficient functional and cosmetic result.

Rationally based efficacy tuning of selective dopamine d4 receptor ligands leading to the complete antagonist 2-[4-(4-chlorophenyl)piperazin-1-ylmethyl]pyrazolo[1,5-a]pyridine (FAUC 213).

Structure dependent efficacy studies in the field of selective D4 ligands led to the 2-aminomethyl substituted azaindole 2 (FAUC 213) that displayed strong D4 binding, high subtype selectivity, and complete antagonist properties in ligand-induced mitogenesis experiments. According to our schematic molecular model, the intrinsic activity of the regioisomers investigated is controlled by the ability of the heterocyclic unit to interact with both elements of the D4 binding-site crevice, the aromatic microdomain in TM6, and a serine residue in TM5.

Minor constituents of Spigelia anthelmia and their cardiac activities.

A more detailed phytochemical analysis of extracts of the aerial parts of Spigelia anthelmia L. (Loganiaceae) yielded 20 structurally related new compounds besides spiganthine and ryanodine. Structure elucidation was achieved mainly by spectroscopic methods. The compounds were tested on their cardiac and on their insect antifeedant activities.

Cyanoindole derivatives as highly selective dopamine D(4) receptor partial agonists: solid-phase synthesis, binding assays, and functional experiments.

Traceless linking of diethoxymethyl (DEM)-protected 5- and 6-cyanoindoles and subsequent incorporation of phenylpiperazine derivatives led to the 2- and 3-piperazinylmethyl-substituted cyanoindoles 3a-m. Dopamine receptor binding studies on the final products 3a-m clearly indicated strong and selective recognition of the D(4) subtype which is known as a promising target for the treatment of neuropsychiatric disorders. The most interesting binding properties were observed for the 2-aminomethyl-5-cyanoindoles FAUC 299 (3f) and FAUC 316 (3j) (K(i) = 0.52 and 1.0 nM, respectively) when the fluoro derivative 3j proved extraordinary selectivity over D(1), D(2long), D(2short), and D(3) (>8600). To determine ligand efficacy, mitogenesis experiments were performed indicating partial agonist effects for the test compounds 3f,j (35% and 30%, when compared to the full agonist quinpirole).

Azepino- and diazepinoindoles: synthesis and dopamine receptor binding profiles.

Starting from the readily available building blocks 7, 10, 11, and 15, the synthesis of the fused indoles 1, 2, 5, and 6, respectively, is reported. The syntheses involved Pictet-Spengler cyclizations, Michael addition reactions, lactamization, directed metallation, and reductive amination as the key reaction steps. Radioligand displacement studies comprising the dopamine receptor subtypes D1, D2long D2short, D3, and D4.4 were performed when the diazepinoindole 6 revealed D1 and D4 affinities (Ki = 0.11 microM and 1.7 microM, respectively) which are comparable to the partial D1 agonist SKF 38393 (3b). In contrast to the benzazepine 3b, the indole based test compounds turned out less selective over the D2 and D3 receptor subtype.

Phenyloxazoles and phenylthiazoles as benzamide bioisosteres: synthesis and dopamine receptor binding profiles.

Conformationally restricted benzamide bioisosteres were investigated when the aminomethylpyrrolidine derivative 4o proved D3 as well as D4 binding properties which were comparable to those of the atypical neuroleptics sulpiride and clozapine, respectively.

Conjugated enynes as nonaromatic catechol bioisosteres: synthesis, binding experiments, and computational studies of novel dopamine receptor agonists recognizing preferentially the D(3) subtype.

To evaluate nonaromatic catechol bioisosteres, the conformationally restrained enynes 1 and enediynes 2 were synthesized via palladium-catalyzed coupling as the key reaction step. Subsequent receptor binding studies at the dopamine receptor subtypes D(1), D(2 long), D(2 short), D(3), and D(4) showed highly interesting binding profiles for the enynes 1a and 1b when compared to dopamine. At the guanine nucleotide-sensitive high-affinity binding site of the D(3) receptor, the target compound 1b (K(i) = 5.2 nM) was 10-fold more potent than dopamine but less potent at the D(2) and D(4) subtypes. In contrast to dopamine the agonists 1a and 1b showed strong selectivity for the receptors of the D(2) family (D(2)-D(4)). As far as we know, this study represents the first report on nonaromatic dopamine agonists. Comparison of molecular electrostatic potentials, derived from semiempirical molecular orbital calculations, and lipophilicity maps was performed.

Selective insect antifeedant and toxic action of ryanoid diterpenes.

In this work, we have studied the antifeedant and insecticidal effects of several natural ryanoid diterpenes. These compounds can be classified in two groups according to their chemical structures: ryanodol/isoryanodol-type (nonalkaloidal type) and ryanodine-type (alkaloidal type) ryanoids. The nonalkaloidal ryanoids were isolated from Persea indica (Lauraceae) while the alkaloidal ryanoids (ryanodines and spiganthines) were isolated from Spigelia anthelmia (Loganiaceae). The effects of these compounds on the feeding behavior and performance (with and without piperonyl butoxide pretreatment) of Spodoptera littoralis larvae and Leptinotarsa decemlineata adults indicate that some strongly deterred these insects, L. decemlineata being less sensitive than S. littoralis. Their antifeedant effects did not parallel their toxic action. Additionally, more than 60% of the nonalkaloidal ryanoids were antifeedants and/or toxic in contrast to 30% of active alkaloidal ones, supporting the hypothesis of a ryanodol-specific mode of action in insects.

Piperidinylpyrroles: design, synthesis and binding properties of novel and selective dopamine D4 receptor ligands.

Piperidinylpyrroles of type 3 were synthesized through a modified Paal-Knorr reaction. For the introduction of pyrrole-substituents high yielding transformations including Sonogashira cross-coupling reactions were utilized. Employment of the reagent TosMIC gave access to the regioisomeric oxazolyl derivatives 7 and 11 which showed the highest dopamine D4 receptor binding of the series investigated.